i

Physiological Responses of Field Grown Lemongrass (Cymbopogon citratus) to Seasonal Changes

BY ABIDA AZIZ

M. Phil (UAF)

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

In

Botany

DEPARTMENT OF BOTANY FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE FAISALABAD

PAKISTAN

2014

ii

The Controller of Examinations, University of Agriculture, Faisalabad

We, the Supervisory Committee, certify that the contents and form of t h e s i s

submitted by Abida Aziz, 2002-ag-1392 have been found satisfactory. The

suggestions by external examiners have been incorporated, checked and found

satisfactory. It is submitted with the recommendations for further necessary action

and final award of the Ph.D. Degree.

Supervisory Committee

----------------------------------------------

1. Chairman (Prof. Dr. Abdul Wahid)

--------------------------------------------

2. Member (Dr. Farrukh Javed)

------------------------------------------ 3. Member (Dr. Muhammad Farooq)

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Declaration

I hereby declare that contents of the thesis, “Physiological responses of field grown

Lemongrass (Cymbopogon citratus) to seasonal changes” are product of my own research and

no part has been copied from any published source (except the references, standard mathematical

/formulae/ protocols etc.). I further declare that this work has not been submitted for award of

any other diploma/degree. The university may take action if the information provided is found

inaccurate at any stage. (In case of any default, the scholar will be proceeded against as per HEC

plagiarism policy).

Abida Aziz 2002-ag-1392

iv

Dedicated

TO

My Father

v

Acknowledgements

Thanks to my Supervisor Prof. Dr. Abdul Wahid, Chairman, Department of Botany, University

of Agriculture, Faisalabad, for his kindred spirit, his belief in me and his scholastic approach, insight,

excellence cooperation and write up guidance throughout my study. I am really obliged and thankful to

other committee members Dr. Farukh Javeed Associate professor Department of Botany and Dr.

Muhammad Farooq, Associate professor, Department of Agronomy for their valuable advice and

practical help during the conduct of this research work.

I am highly indebted to Dr. Shahzad Maqsood Ahmad Basra, Professor, Department of Crop

Physiology, University of Agriculture, Faisalabad for his utmost help, guidance, advice and facilitation

during the whole study I am grateful and feel highly obliged to Prof. Dr. Mohammad Ashfaq

Director, Institute of Agricultural and Resource Economics, University of Agriculture, Faisalabad for

moral support and encouragement during my PhD.

Special thanks to my dear friends Asima Batool, Rumana Sadiq, Durr-i-Ashian, Bushra

Munir, Shumaila Firdos, Rabia Qadri, Madiha Adeel and Sara for their best wishes for my success

in carrier. Really there are too many names to list of the multitude that have helped make this a reality,

apologies to those whose names I have neglected or omitted here, please forgive. You are not forgotten

and your aid and friendship are most appreciated.

I am indebted to my Mother and brother Wajid Aziz who instilled the desire to explore creation,

till the soil and forage for foods. Thanks to my brothers (Abid, Majid and Adil) and sisters (Musrat,

Naila, Nusrat, Farhat, Samina, Ishrat and Aliya) for your love.

Last but not the least I am highly obliged to Higher Education Commission (HEC),

Islamabad for sponsoring this research project under Indigenous Scholarship Scheme 5000.

Abida Aziz

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C O N T E N T S

Chapter Title Page 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 3 MATERIALS AND METHODS 22 4 RESULTS AND DISCUSSION 37 5 SUMMARY 115 LITERATURE CITED 117

vii

ABSTRACT

A population of lemongrass (Cymbopogon citratus) was investigated in this research for

changes in metabolite profiles in the leaves of different ages i.e. penultimate (second fully

expanded leaf from the top), middle (a leaf from the central position of a tiller) and bottom (a

lowermost green leaf on a tiller) with changing seasons round the year for two consecutive years

(2010-2011). Measurements were made for some primary and secondary metabolites while the

essential oils profile of leaves was analyzed using GC-MS. The photosynthetic pigments

displayed sharp variations in the leaves of different ages with changes in the environmental

conditions. Higher chlorophyll a, b and carotenoid were recorded during summer months. The

accumulation trends of osmolytes in lemongrass showed notable seasonal variation. The

accumulation of total free amino acids, free proline and GB were higher during summer months.

The antioxidant enzymes minimized the effect of oxidative damage by scavenging H2O2 and

reuced MDA contents in extreme environmental conditions especially during summer. The

younger leaves of lemongrass exhibited much higher concentrations of vitamins as compared to

bottom leaves. The secondary metabolites i.e. alkaloid, phenolic and flavonoid contents in

lemongrass leaves were markedly increased with increased temperature. Seasonal variation

affected the nutritional profile of lemongrass substantially. The production of NDF ADF, ADL,

cellulose and starch was greater during summer. Cellulose and silica contents were higher in

bottom leaf possibly due to plant aging. Starch content was higher in penultimate leaf, which

declined with leaf age. Mineral concentration of lemongrass appears to be largely dependent on

temperature and evapotranspiration. The GC-MS analysis of lemongrass oil carried out in the

leaf samples collected during January, April, July and October revealed that out of 54

compounds analyzed, neral, citral, geranial, allerthin, caryophyllene oxide were major ones

synthesized in various sesasons.

1

CHAPTER-1

INTRODUCTION

Pakistan has diverse range of climates and biodiversity in four distinct phytogeographical

regions, Irano-Turanian (45% species), Sino-Himalayan (10%), Saharo-Sindian (9.5%) and

Indian element (6%) (Shinwari, 2010). Total flora of Pakistan comprises 6000 species (Shinwari

et al., 2000) out of which about 600 are medicinally important. These medicinal plants were

explored in different areas. For example, 70% species were reported in Mansehra (Haq and

Hussain, 1993), 83 taxa used locally in Chitral (Ali and Qaiser, 2009), 114 in Baluchistan

(Goodman and Gafoor, 1992), 171 species in Kharan (Shah and Shinwari, 1996), throughout

Lahore-Islamabad motorway (Ahmad, 2007) and salt range (Ahmad and Hussain, 2008). With

this exclusive biodiversity of medicinal plants in Pakistan, there is a need for precise research on

these plants. According to National Institute of Health (NIH), Tibbi Pharmacopoeia listed 900

single drugs and 500 compound drugs obtained from medicinal plants. Some indigenous

medicinal plants of Pakistan are Artemisia sp., Ephedra sp., Bunum persicum, Emblica

officinalis, Glycyrrhiza glabra, Atropa acuminate, Commiphora wightii (Reddy et al., 2012) and

Cymbopogon citratus (Duke, 1990), which belong to various plant families i.e. Poeaceae

Asteraceae, Solanaceae, Liliaceae, Apocynaceae, Caesalpinaceae, Sapotaceae, Rutaceae,

Piperaceae, etc.

Influences of seasonal changes on ecosystems are impinging tremendously and transition

in weather conditions, temperature variation, rainfall trends and concomitant phenomena are

associated to environmental changes (Root et al., 2003). Researchers opine that chemical

constituent and endurance of medicinal plants are affected greatly by the environmental changes.

Normally plants under stress conditions can accumulate more secondary metabolites due to

inhibition of growth and diversion of fixed carbon in the biosynthesis of phenolics and glycosidic

compounds instead of photosynthesis (Gairola et al., 2010). Depending upon environmental

variations, photosynthesis fluctuate among species and decrease steadily with increasing leaf age

(Herath and Ormrod, 1979). Many studies have demonstrated that rise in temperature enhanced

the secondary metabolites production (Litvak et al., 2002), although others think that these

compounds declined (Snow et al., 2003). Many researchers believed that increase in temperature

2

only enhances the streaming of chemical compounds in plants (Loreto et al., 2006) and the

effects of high temperature are still under consideration (Wahid et al., 2007).

Enzyme activities are an important index to foretell the plant responses to the changing

environments (Sen and Mukherji, 2009). Antioxidants in plants act as a line of defense against

unfavorable conditions (Lohrmann et al., 2004). Defense mechanisms of antioxidant in plants

comprise various enzymes such as induction of glutathione reductase, peroxidase, superoxide

dismutase, catalase and ascorbate peroxidase (Keles and Oncel, 2002). Lipids and fatty acids

composition in medicinal plants is also influenced by seasonal variations. Similarly fluctuations

in plant lipid levels are associated with uncertain weather conditions. Essential oils, being

complex mixtures, primarily comprise of lower classes of terpenes (Langenheim, 1994).

Silvestre et al. (1997) studied the influence of seasonal variations in the composition and

accumulation of sequestered oils. There are several reports on terpenoids accumulation in

aromatic plants, which vary with seasons seasons (Hendriks et al., 1997). Essential oils

accumulation in plant might be due to influence of stress on plant metabolism rather than

structural adaptation (Emara and Shalaby, 2011). Phenolics like flavonoids, anthocyanins and

lignin are important secondary metabolites and are well known for their role in the adaptation to

abiotic stress tolerance (Wahid and Tariq, 2008).

Medicinal plants are potential natural source of new drugs and bioactive compounds for

researchers and medicinal manufactures (Gangwar et al., 2010). Herbal remedies are as old as

human life. Even presently 80% world population of the world believes in traditional herbal

health care (Ahmad, 2007). Out of 250,000 higher plants species worldwide, more than 70,000

synthesize the phytochemicals used as drugs to treat various diseases (Farnsworth, 1988).

Modern pharmacopoeia has been using 280 chemical compounds extracted from various plant

tissues like leaves, root, stem, bark, seed, and from various plant liquid components such as sap,

latex, mucilage, gums etc. Such biologically active ingredients of plants are very vital and

important sources of new medicines that are beneficial in the treatment of several diseases (Dev,

1997). In the past, aspirin, artimesinin, atropine, reserpine digoxin, colchicine, morphine

ephedrine, pilocarpine physostigmine, quinine, quinidine, taxol, tubocurarine, vincristine, and

vinblastine have been obtained from medicinal plants (Ramawat and Goyal, 2008). These drugs

are good therapeutic agents and used to cure diabetes, mental sickness, skin infections,

tuberculosis, jaundice, hypertension and cancer. Many plants possess antibacterial, antidiabetic

3

(Arcamone et al., 1980), antimicrobial activites (Perumal-Samy et al., 2006). Similarly,

Lagenaria breviflora is known for its broad spectrum medicinal uses, since many phytochemical

assays exposed the presence of saponins, phenolic acids and cucurbitacins in it (Wakimoto et al.,

2008).

Lemongrass [Cymbopogon citratus (D.C.) Stapf] belongs to the family Poaceae, and is a

commercially important aromatic C4 grass. In tropical and subtropical areas about 140 species of

Cymbopogon are found (Chase and Niles, 1962). Among these, 52 species are scattered in

Africa, 45 in the subcontinent (out of which eight species grow in Pakistan), six in Australia and

South America, four in European countries, and two in western hemisphere (Kak and Kaul,

1997). It is endemic to India and also grows in West Indies, Guatemala, Haiti and Pakistan

(Hassan et al., 2007). It can grow in diverse environments but hot and humid climate with

enough sunshine is ideal for its growth. Total age of lemongrass is 548 to 730 days, while it is

requisite to revive its cultivation after 6 to 8 years (Atal and Kapur, 1982). Lemongrass is rich in

such compounds which are greatly demanded due to their use in drugs, flavors, perfumes and

pharmaceutical industry. Aromatic drinks, decoctions or infusions from dry leaves of lemongrass

have been used as antispasmodic, stomachic, carminative and antifungal agents (Borrelli and

Izzo, 2000). Leaves extracts of lemongrass have protection against oxidative stress (Melo et al.,

2002), hypotensive, vasorelaxating and cancer chemo-prevention (Puatanachokchai et al., 2002).

Lemongrass oil has a strong lemon-like aroma and is yellow or reddish-brown in color.

Its chemical composition differs due to genetic diversity, habitat and agronomic practices.

Lemongrass essential oil consists of 41% citral, 0.3% - 4.5% neral and 0.5%-40.2% geraninal

contents (Khanuja et al., 2005; Negrelle and Gomes, 2007), which is used in the preparation of

many aromatic compounds, pharmaceuticals, vitamin A and E, and in various scents (Abello et

al., 2007). In addition to citral, lemongrass contains variety of compounds like flavonoids,

alkaloids, saponins and terpenes according to habitats (Crowford et al., 1975). Oil extracted from

lemongrass leaves have diuretic, tranquilizing and anti-inflammatory properties. It possesses

antioxidant activities, which is beneficial for human health. The lemongrass juice extraction

shows inhibition activity against carcinogenesis promotion, which is induced by cotton oil. In

oral form it is administered as anti-tumor drug for cancer and for lengthening survival time in

combination with cyclodextrin (Zheng et al., 1993). It contains high vitamin C contents, which is

used most often in the treatment of many diseases. Lemongrass oil is used for dissolving

4

gallstone (Igimi et al., 1991) and shows activity against phytopathogenic fungi. In combination,

it has uses in human and domestic animal pathogens (Kisaki and Yama, 1998).

In view of all this information available in literature, the production of chemical

substances in lemongrass does not remain the same throughout the year. It depends upon

lemongrass type and climates where it grows. The aim of the current research was to evaluate the

consequences of seasonal changes on leaf physiology and its role in the synthesis of primary and

secondary metabolites and their association to ambient meteorological conditions. Following

were the specific objectives of the present project.

1. Determination of the consequence of seasonal variation in the leaf physiological

attributes and repercussion to climatic aspects on lemongrass

2. Determination of the consequence of seasonal variation in the primary and secondary

metabolites accumulation, phytochemical relations and nutritional constituents on

lemongrass over the various seasons

3. Studying the possible associations of various meteorological factors and phytochemical

constituents and their role in climatic response

5

CHAPTER-2

REVIEW OF LITERATURE

2.1 Preliminary

Environmental perturbations are such changes that may induce measurable changes in the

metabolic phenomena in the living organisms. Land plants as immobile living organisms on

the earth are always subjected to environmental perturbations and thus have developed

adaptive features to cope with these adversaries. These features are of multifarious nature.

The plants capable of synthesizing excessive amounts of secondary phytochemicals, mainly

referred to as medicinal plants, show the physiological adaptive features by showing

modulations in their metabolites levels parallel with the changing climatic conditions.

Lemongrass (Cymbopogon citratus) is a C4 grass, capable of growing at supra-optimal

temperatures, and shows the synthesis of medicinally important phytochemicals. The

determination of physiological and biochemical characteristics enabling lemongrass to adjust

to prevailing environmental conditions is important in view of being a high-value plant. An

account of relevant literature on the environmental changes and their influences on the

changes in plants with particular reference to medicinal plants is given below.

2.2 Plants Interaction with Seasonal Variations

Biosynthetic and metabolic pathways in plants are greatly influenced by the seasonal

changes, wherein the synthesis of some compounds is induced while those of others are curtailed

(Salminen et al., 2001). Low seasonal rainfall during winter is associated with tannin

accumulation in the aerial parts of the plants (Wang et al., 2007). In many plants, seasonal

variations in cuticle thickness occur, which increase in spring and summer and decrease in winter

(Emara and Shalaby, 2011). Mostly higher levels of phytochemical compounds were extracted in

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autumn leaf tissues than spring samples where equal distribution of the phytochemicals was

observed (Chokoe et al., 2008). In shoots and leaves of bamboo secondary metabolites contents

were lower at the initial stages of growth in spring and summer but in winter and autumn when

metabolism is slow the greater accumulation of flavonoids, phenolics and triterpenes get started.

This is parallel to general law of accumulation and transition of secondary metabolites in plants

(Raffo et al., 2006). Moreover, biosynthesis of phenolic is peculiarly susceptible to different

abiotic and biotic stresses (Dixon and Paiva, 1995). Various other studies explain chemical

profile of aromatic compounds and secondary metabolites synthesis in different plants in various

seasons for measuring seasonal changes (Celiktas et al., 2007; Hussain et al., 2008). Wildy et al.

(2000) in Australia observed 0.01 to 13.0% in Australia, while Zafar et al. (2003) in Pakistan

noted 0.58 to 1.47% variations in oil production in various Eucalyptus species, which was

associated with soil composition and various agro-climatic regions.

Plants respond to stress by showing resistance, avoidance and acclimation. However,

under harsh environmental conditions plant cellular organization collapses, which results in

altered geographical distribution and early crop maturity (Schoffl et al., 1999; Porter, 2005;

Howarth, 2005). Changed climates result in reduction in crop production worldwide (Hall,

2001). The acclimation approaches adopted by common plants vary from morpho-anatomical

to physiochemical (Zhu, 2001). Plants make use of special acclimation mode by the

collaboration of genetic characters and stress type (Bray et al., 2000). Plants contain many

compounds of undetermined biological activity, and the quality and quantity of these compounds

can vary substantially based on the region where they are grown, the season in which they are

harvested and the genotypes cultivated (Currier et al., 2000; Ma et al., 2003). The seasonality in

environmental condition fluctuates in time and space so it is challenging to anticipate the plant

responses to the changing environments. Tolerance to such seasonal fluctuations in plants is due

to their physiological plasticity as different enzyme actions and physiological processes are

important plant response indices (Sen and Mukherji, 2007).

Mediterranean climate is characterized by the incidence of two stress periods; summer

drought and winter cold (Nahal, 1981). It has been argued that the prevailing stressful conditions

are helpful in the optimization of the growing and harvesting times in the medicinal plant species

(Lee et al., 2005). Saliminen et al. (2001) reported that seasonal and meteorological changes

bring about variations in the metabolic phenomena of the plants leading to variation in the

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contents and fluxes of the bioactive compounds. Wang et al. (2007) argued that changed pattern

of rainfall in winter season led to tannins accumulation in the aboveground part of Toona

sinensis. According to Emara and Shalaby (2011), the cuticle thickness in four Eucalyptus spp.

was strongly dependent upon seasonal changes. It increased from spring to summer but

decreased in the winter season. In Carpobrotuse dulis, during spring harvest, equal distribution

of the phytochemicals was observed within the leaf debris and the filtrate, but high

phytochemicals were found in the extracts of the samples collected during autumn season

(Chokoe et al., 2008). The phenolics biosynthesis in plants is particularly sensitive to induction

due to various biotic and abiotic stresses (Achakzai et al., 2009).

2.2.1 Plant morphology, water and osmotic relations

Physiological, morphological and biochemical responses vary from crop to crop, in

different growing seasons and in the environments in which they are grown. Seasonal

variations help the plants to acclimate and adapt to new environments and in stress

avoidance. Most useful strategies to surmount seasonal variation ranges from morpho-

anatomical to physiological and biochemical during the plant life cycle (Zhu, 2001). Not a

single factor controls the physiological processes in plants. Environmental conditions may

change biochemical and physiological processes along with low uptake and absorption of

nutrients in plants (Garg, 2003). Research on medicinal plants under field conditions suggested

that high soil water contents enhanced the growth performance of plants by increasing number of

leaves, plant height (Bargali, 1997), shoot length, leaf weight (Van Schaik et al., 1997) and leaf

width (Pandey et al., 1998). Carrot (Daucus carota) indicated unpredicted seasonal changes,

which affect the leaf surface (Brooks et al., 1996). A substantial decline in the processes like

transpiration rate, active transport, membrane accessibility and photoassimilate quality has

been reported in response to environmental stresses (Gunes et al., 2006).

Disturbances in water relations due to environmental effects are counteracted with the

accumulation of compatible solutes. Not only the nature of the osmolytes, but also their

concentrations exhibit specific seasonal pattern in plants. Compatible solutes are liable for

osmotic adjustment since they are also compatible with the cells metabolism (Wahid et al.,

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2007). In addition to their main function of osmotic adjustment, compatible solutes can either

help in stabilizing macromolecules under adverse conditions or protect against oxidative

damage (Flowers and Yeo, 1988; Wahid and Shabbir, 2005). Bhowmik and Matsui (2003)

examined changes in carbohydrate contents in changing seasons in many plants. Chinnasamy

and Bal (2003) and Schaberg et al. (2000) reported high concentrations of total soluble sugar in

red spruce seedlings during mid-winter. In another study, it was reported that high concentration

of soluble sugars act as cryoprotectants during cold acclimation (Taulavuori et al., 2001).

Glycinebetaine (GB) is a derived from glycine and acts as an excellent

osmoprotectant (Flowers and Yeo, 1988). It is naturally found in many living organisms in a

sufficient quantity (Rhodes and Hanson, 1993). It is a dipolar molecule, soluble in water at

physiological pH and acts as neutral molecule (Sakamoto and Murata, 2002). Such dynamic

features of glycinebetaine are helpful in stabilization of three dimensional structures of

protein and enzymes as a result of their hydrophilic and hydrophobic domains interaction

(Sakamoto and Murata, 2002). GB plays an important role against stress defense in many

plants (Ashraf and Foolad, 2007; Chen and Murata, 2008; Wang et al., 2010). Boscaiu et al.

(2011) reported changes in GB contents due to seasonal variation, which were higher in

extreme dry summer conditions.

Proline acts as a compatible solute (not toxic under high concentration) and plays

vital role in osmotic adjustment (Yamada et al., 2005), removes the harmful effect of

reactive oxygen species, helps to maintain the three dimensional structure of proteins and

also protects chloroplast and mitochondrion under fatal environmental situation when

accumulated in free form (Ashraf and Foolad, 2007; Wahid et al., 2007). When the stress is

alleviated, it is converted into reducing agents, which plays important role in oxidative

phosphorylation (Hare et al., 1998). Proline also plays role in the induction of stress

responsive genes (Johari-pireivatlou et al., 2010). Accumulation of proline as a result of

abiotic stresses occurs in many species of plant families, but its quantity is based on species

type and severity of stress (Wahid et al., 2007; Lotfi et al., 2010). In Camphorosma annua

and Limonium gmelini leaves, proline levels were higher in early spring which decreased

subsequently (Murakeozy et al., 2003). In the autumn, a decrease in proline content occurred in

L. anatolicum and L. lilacinum, while an increase in L. iconicum (Furtana et al., 2013).

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2.2.2 Oxidative stress and antioxidant activities

The most deteriorating factor under stress at molecular level is overproduction of reactive

oxygen species (Xu et al., 2006). During the partial reduction of light-induced respiration, ROS

reduced oxygen in the atmosphere, which affects photosynthesis and respiration (Xu et al.,

2006). They include superoxide -O2-, OH- and H2O2 (Mittler, 2002). Reactions of active oxygen,

followed by the peroxidation products (Scandalios, 1993) eventually lead to imbalance in the

normal metabolism. Evidences show that membrane damage is due to the ROS generation,

which results in lipid peroxidation of plasmalemma or intracellular organelles (Stewart and

Bewley, 1980; Farooq et al., 2009). Similarly, denaturation of proteins, nucleic acid and

hormonal damage, ultimately distresses the plant homeostasis (Mittler, 2002). Xu et al. (2006)

showed that heat resistance in different turfgrass species was based on the increased membrane

thermostability.

Hydrogen peroxide (H2O2) is most dominant and stable ROS with ability to regulate the

basic acclimatory, developmental and defensive processes in plants (Ślesak et al., 2007). It is

fatal for cell when it reacts with lipids, nucleic acid and proteins (Mittler, 2002). In leaves

photochemical quenching of excess light by photorespiration is involved in H2O2 production. In

leaves of wheat, H2O2 production during photorespiration is 70% of the total H2O2 produced and

it gradually increased with a decrease in CO2 concentration. Production of photorespiratory H2O2

escalates in high light and low CO2 concentrations (Noctor et al., 2002). H2O2 and other ROS are

ineluctably produced with the electron leakage to O2 during aerobic metabolic processes like

photosynthesis and respiration. Higher H2O2 concentrations were recorded for Aesculus glabra,

Plantago major, Glechoma hederacea and Viola soraria in the warm days while there were no

significant change in H2O2 production in Quercus macrocarpa (Cheeseman, 2006).

In order to reduce the intensity of damage or to reduce ROS generation under stress

condition, plants widely adapt and develop defense strategies. These strategies include the

removal of ROS through enzymatic and non-enzymatic defenses, which are important to cope

with ROS (Allen, 1995). In harsh environmental conditions, the ROS attack on biologically

important molecules, interfere with cell metabolism (Liu and Huang, 2000), cause ionic

imbalance (Taiz and Zeiger, 2010), self-catalytic membrane lipid peroxidation and loss of

10

membrane integrity. Hence, MDA concentration and relative electrolyte leakage is important

tool to evaluate heat damage in a variety of crops as trufgrass (Xu et al., 2006) and in

Phalaenopsis (Ali et al., 2005).

During environmental stress, superoxide dismutase (SOD) provides protection from

activated oxygen (Bowler et al., 1992; Scandalios, 1993; Smirnoff, 1993; Foyer et al., 1997). In

Retama raetam and Atriplex halimus, the SOD activity was much higher under harsh desert sites

than in non-desert areas (Streb et al., 1997). Peroxidase (POD) comprises of multifunctional

enzymes family that regulates ROS production (Passardi et al., 2005) and stimulates the

breakdown of H2O2 by utilizing different substrates (phenolic, lignin, auxins) within plant cells

(Hiraga et al., 2001; De Gara 2004; Passardi et al., 2005). POD plays important protective role in

plants against different abiotic stresses. CAT is a mono-functional protein which is especially

localized in peroxisomes (Engel et al., 2006) and has ability to scavenge the huge amounts of

H2O2 into water and O2 (Sofo et al., 2004).

Medicinal plants are potential source of antioxidant compounds, it is necessary to study

their constituent and mode of action, and thus validate their utilization. According to Korotkova

et al. (2003), flavonoids, phenolic acids, tannins are many of the compounds that have

antioxidant activity and frequently found in medicinal plants. Antioxidants can be classified as

enzymatic and non-enzymatic present in almost all plants (Mittler, 2002), and help in ROS

detoxification; the enzymatic ones including SOD, POD and CAT while non-enzymatic ones

include AsA, carotenoide, tcopherols, flavonoids, and phenolic compounds (Ashraf et al., 2010).

Swanberg and Verhoeven (2002) found that yew leaves had capability to respond to

changing seasonal variations with the induction of antioxidants. During winter in leaves of

different plants antioxidant systems were increased (Logan et al., 1998). According to Vuleta et

al. (2010), greater activity of POD in leaves of Iris pumila was observed during spring and

summer seasons. POD activity was declined during spring through winter while CAT activity

increased before rainfall and then decreased (Kashefi et al., 2010). In dry conditions, these

antioxidant enzyme activities increased in different plant species, amongst the species and within

a species (Csiszar et al., 2007; Nikolaeva et al., 2010). Extended stress situations may lead to a

boost in antioxidant ability in different parts of plants (Pastori et al., 2000).

11

2.2.3 Reducing powers and vitamins

The reducing power is a measure of electron transfer capacity of the compounds, which

acts as a powerful tool of its antioxidant activity. Raja et al. (2012) reported that nut extract

showed high reducing power activity. Shimada et al. (1992) reported that in mushroom high

reducing power activity was due to its electron donating ability. Huang (2000) reported that the

methanolic extract of Antrodia camphorata exhibited greater reducing power activity than

Brazilian mushrooms.

Vitamins are exclusive sources of reducing powers in plants. Ascorbic acid (vitamin C;

AsA) is evidenced for its role as antioxidant in plants, acts as cofactor, involved in cell

signaling, regulator of cell wall biosynthesis and provides photoprotection to the cells and

tissues (Vaidyanathan et al., 2003). AsA reduced the lipid peroxidation (Sairam et al., 2000)

and decreased the harmful effect of stress and increased stress resistance (Iriti and Faoro,

2007). AsA showed sensitivity towards air, water and temperature, acted as an important

antioxidant source and provided protection against free radicals (Demmig-Adams and Adams

2002). In greenhouse grown tomato, AsA content increased as the temperature increased (Liptay

et al., 1986; Vanderslice et al., 1990).

Riboflavin, also called a growth vitamin, is formed in the plastids (Sandoval et al., 2008). It

is modified to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the cytosol

(Sinclair et al., 2000). Riboflavin assists in regulating the cellular redox state and thus controls the

plant antioxidant defense system (Sandoval et al., 2008). Niacin is yet another vitamin of great

physiological significance. Temperature, humidity, photoperiod and rainfall of the growing season

affect the niacin biosynthesis. Niacin increased in plants with increase in the day length. In maize,

an increase in niacin content with increase in temperature was observed (Mahmood et al., 2012).

2.2.4 Secondary metabolites synthesis

Phytochemicals extracted from plants are classified as primary or secondary

metabolites. Sugars, purines, proteins, amino acids and pyrimidines of nucleic acids and

chlorophylls that are essential for growth are categorize as primary metabolite (PM) while

12

plant secondary metabolites (SM) included alkaloids, cyanogenic glycosides, phenolics,

terpenes, saponins, glucosinolates, anthraquinones and polyacetylenes (Wink, 2003). Secondary

metabolites are found in all higher plants and protect them attack of bacteria, fungi and viruses

and herbivore. Limonene (monoterpene) exhibited insecticidal and preventative role in plants

while carvoneis is a good sprouting inhibitors (Aflatuni, 2003).

Plants are well known dynamic organisms, therefore it is predicted that concentrations of

secondary metabolites in plants change within a plant both by spatially and temporally over

successive years (Brooks and Feeny, 2004). However, changes in levels of plant secondary

metabolites interfere with the effectiveness of phytomedicines (Gurib-Fakim, 2006). Scientific

studies revealed that changes in active compounds as a consequence of biotic and abiotic factors

can help to optimize the harvest time of medicinal raw material or to obtain the largest amounts

of active compounds (Lee et al., 2005). Seasonal changes in the composition and level of active

compounds of sequestered oils have been observed (Silvestre et al., 1997). Similarly in aromatic

plants the levels of active compounds such as terpenoids change due to internal biological clock

and also around the seasons (Hendriks et al., 1997).

Phenolic compounds are very essential for plants and play important role for tolerance

against different stresses (Sgherri et al., 2004; Wahid and Tariq, 2008). Phenolics compound

are good electron carriers, involved in energy transfer with the help of their own electronic

configuration and, in this way, enhance the absorbance of light in visible and UV range

(Cockell, 1997). They also play active role in programmed cell death of plants (Beckman,

2000). In Polygonum acre concentrations of total phenolics remain the same (approximately

1.57%) around the year (Lima et al., 2010). The spatial, seasonal and inter-specific variation

trend of concentrations of total phenolics in the Apocynum venetum and Poacynum

hendersonii were observed maximum in April to July (Ma et al., 2003) and then an obvious

decline after July. Concentrations of carotenoids were changed due to seasonal variation

(Lamare and Hoffman, 2004). In leaves of Carpobrotus edulis, the phytochemicals showed a

similar ratio in spring season but it increased in autumn. However, antioxidants showed

different trend; a high activity was detected in autumn (Chokoe et al., 2008).

Flavonoids belong to a large group of phenolic secondary metabolites; the major ones

are flavanols, flavones, anthocyanins and isoflavonoids. In nature, all plants contain these

13

compounds present mainly as glycoside structures. They provide protection by absorbing

ultraviolet (UV) radiations (Harborne and Williams, 2000) and their production was

restricted to UV exposed tissues (Chalker-Scott, 1999; Winkel-Shirley, 2002). In low

temperature months leaf flavonoid content production increases (Christie et al., 1994).

Luengas-Caicedo et al. (2007) reported concentrations of flavonoid in Cecropia glaziovi

young and mature leaves did not significantly differ in the same season whereas both young

and mature leaves exhibited higher concentration in rainy season as compared to dry period.

Anthocyanins are a subclass of flavonoids and highly soluble in water. They may work

with other antioxidants to protect vegetative tissues against photo-oxidative stress (Chalker-

Scott, 1999; Grace, 2005). They are good photo-protector having ability to absorb high energy

light, which harms the Chlorophyll b (Hughes and Smith 2007). They also act as antioxidants for

by scavenging the free radicals, which cause structural and functional damage to thylakoid

membranes (Tattini et al., 2005). Edreva et al. (2007) suggested that in cotton much

physiological chaos due to stress was reduced by antioxidant capacity of anthocyanins.

Wahid (2007) has reported the exclusive role of anthocyanins in heat tolerance of sugarcane

sprouts. In leaves of Iris pumila anthocyanin contents depended on course of growth season and

were higher when oxidative load on leaf was at peak in hot season (Vuleta et al., 2010).

Tannins are common polyphenols in all plants and synthesized approximately equal to 50%

of the dry weight of leaf. Tannins are widely used in industries and also provide protection against

herbivores. Wahid and Tariq (2008) observed that plants grown in acidic soil with low mineral

nutrients show high accumulation of tannin. In Polygonum acre the contents of condensed tannins

(CT) varied significantly; the amount increased from April to August, and declined from August to

November (Lima et al., 2010). In start of hot season a higher content of hydrolysable tannins (HT)

has been observed in Quercus robur (Tikkanen and Julkunen-Tiitto, 2003) and birch tree (Riipi et

al., 2002). Vaithiyanathan and Singh (1989) observed that different species of tall plant leaves

responded differently to seasonal variation, with respect to tannin concentrations. Some species

showed increased tannins in hot months while the others in winter. Gupta et al. (1992) reported an

increase in tannin concentration in hot season. Salaj and Karmutak (1995) found higher tannin

concentration in cooler months.

14

Studied have been made to explore the medicinal value of alkaloids in many plants

(Robber and Tyler, 1996). More than three thousand alkaloids have been recognized in 4000

plant species and most of them were present in herbaceous dicots and some in fungi. The

alkaloids level was higher in leaves in hot weather than winter or in monsoon in Sesbania

bispinosa (Momin and Kadam, 2011). In Atropa belladonna, the accumulation of alkaloid was

two times higher in July as compared to June (Sporer et al., 1993). Their concentration in roots

of Macleaya microcarpa varied significantly during vegetative growth and was negligible during

the month of June and July (Pencikova et al., 2011).

The temperature, photoperiod, relative humidity, irradiance (environmental factors) and

management practices effect the secondary metabolites composition. The nature of essential oils

in different seasons varied from winter to during summer seasons. Best essential oils percentages

were estimated in E. camaldulensis and E. cinerea, which ranged from 2.5 to 1.95%, respectively

in hot months. This might be due to the physical and chemical stresses on the plants especially

during summer drought (Emara and Shalaby, 2011). The incidence of stress resulted in

production of different protective compounds especially terpenoid in plants (Samuelsson, 1999).

2.2.5 Leaf pigments

Leaf chlorophyll is an important biochemical attribute of plants. It is related to

seasonal water availability and nutritional level of plants and reflects plant health (Epstein

and Bloom, 2005). Normally, light-harvesting ability of C3 plants is inflated as compare to

the C4 plant species (Salvucci and Crafts-Brandner, 2004). Harsh seasonal changes have a

great influence on PS-II leading to inhibition of photochemical efficiency (Barber and

Andersson, 1991). A significant decrease in phytochemical efficiency of PS-II during the month

of October was due to decline of chlorophyll a content (Polle et al., 1999). Different accession

of sugarcane exhibited an increase in chlorophyll a/b ratio and decrease in “chlorophyll b”

under stress (Wahid and Ghazanfar, 2006).

Carotenoids are very important accessory photosynthetic pigments and act as receiver of

extra light and act as cellular protector during different abiotic and biotic stress periods (Iriti and

15

Faoro, 2007; Wahid, 2007). Carotenoids not only provides a mechanism of photoprotection but

also act as antioxidants under different seasonal conditions (Krinsky, 1998). They magnify the

stability of membrane by lowering peroxidation of membrane lipids in stress conditions (Havaux,

1998). Carotenoids are hydrophobic in nature and defend lipophilic membrane surfaces of

macromolecules (Vasconsuelo and Boland, 2007). This property of carotenoids is due to their

efficiency to scavenge reactive oxygen species (Rodrigues et al., 2003).

Seasonal variations affected the carotenoid concentration in Piceasit chensis in winter

period (Lewandowska and Jarvis, 1977). Tomato plant when grown in the rainy season exhibited

lower carotenoid content, which were generally liable to oxidative damage due to low

scavenging and detoxification activity of the ROS (Sen and Mukherji, 2000). Ollykainen (1969)

found that the carotenoid content increased during the vegetative growth periods during summer

but decreased during the winter. Under high light intensity, accumulation of carotenoid increased

lipid droplets (Ben-Amotz et al., 1982). While studying seasonal variations in carotenoid

concentration, Raffo et al. (2006) reported that carotenoid did not show specific seasonal

patterns in relation to solar radiation and average temperature.

2.2.6 Nutrient concentrations and nutritional relations

Various environmental conditions affect the availability of soil nutrients (Fageria et

al., 2003). Normally a decline in the absorption of plant nutrients results in a reduction in

transpiration rate, which reduces the membrane accessibility, active transport (Baligar et al.,

2001; Ganesh et al., 2008) and reduced tissue nutrient contents (McWilliams, 2003).

Different macro- and micronutrients like potassium, calcium, nitrogen, phosphorus and sulfur

play a prominent role in plant growth, osmotic adjustment, enzyme activation (respiration

and photosynthesis), stomatal opening/closing, phloem transport and so many other

metabolic processes (Hepler, 2005; Lopez et al., 2008).

Mineral composition of plants depends upon various environmental factors such as

seasonal changes, geography, climate, soil minerals and plant capacity to uptake soil

nutrients (Ganskopp and Bohnert, 2003; Khan et al., 2006).). Huston et al. (1981) found

lower values of calcium and phosphorus (1.4 and 1.0 g/kg, respectively) in early summer in

16

Panicum hallii. Humphreys (1984) reported that, K+ concentration in tropical grass ranged

up to 12 g/kg. Abiotic stresses induced a change in Ca2+ concentration which brought in

different physiological responses (Reddy and Reddy, 2004). Mirza et al. (2004) found

significant changes in trace elements including copper (Cu), zinc (Zn), manganese (Mn), iron

(Fe), cobalt (Co), nickel (Ni), cadmium (Cd), lead (Pb), chromium (Cr), silver (Ag), sodium

(Na) and potassium (K) in indigenous medicinal diuretic plants.

Plants absorb nitrogen in the form of nitrate (NO3-), which is influenced by various

environmental factors like drought (Younis et al., 1965; Huffaker et al., 1970), light (Schuphan et

al., 1967; Cantliffe, 1972a, b; Scaife and Schloemer, 1994), temperature (Cantliffe, 1972c) and soil

type (Raikova and Petkov, 1996). P, a macronutrient for plants, is a key component of nucleic

acids, phospholipids, ATP and regularizes metabolic pathways (Theodorou and Plaxton, 1993). In

Pennisetum pedicellatum the P level in wet season was much higher than in the dry season (Ziblim,

2012). K+ is important plant cation, and its concentrations ranges from 50–150 mM in plants

(Leigh and Wyn Jones, 1984). High K+ concentration can temper intense environmental condition

like cold, late season rains and heat waves through stomatal movement and osmotic adjustment

(Marschner, 1995). In Ceratonia siliqua leaf K+ contents decreased during winter and increased

from spring until autumn (Correia and Martins-Loucao, 1997). On the other hand in clover

monthly fluctuations were observed; high levels were observed in late winters and early spring

while the lowest levels were recorded during early summer (Metson, 1978). Seasonal variation had

a significant impact on the nutritive value of plants (Snyman, 2006). In plants production of

different nutritive constituents like nutrient detergent fiber (NDF) and acid-detergent fiber (ADF),

acid detergent lignin (ADL), silica, cellulose, starch and protein content, showed changes within

years and in various plant growth phases within a growing season (Ball et al., 2001).

2.2.7 Essential oils composition

Essential oils are a mixture of fragrant compounds including terpenoids, aldehydes

(geranial, citronellal), alcohols (geraniol, citronellol, nerol), esters (linalyl acetate, citronellyl

acetate, isobornyl acetate) (Burt, 2004), which accumulate in different organ like leaves, barks,

woods, roots, rhizomes, fruits, and seeds. Essential oils are synthesized and accumulated in

17

specialized organs (trichomes, secretory cavities or canals) located near surfaces (Bruneton,

1995). So any change in seasons has direct influence on essential oil concentration and

component (Silvestre et al., 1997). Environmental factors (temperature, irradiance, relative

humidity, photoperiod etc.) and cultivation practices have great effect on the composition and

concentration of essential oils. Many authors have reported seasonal variation in essential oil of

aromatic plants (Hendriks et al., 1997). Such seasonal changes in essential oil component were

observed in many plants like Santolina rosmarinifolia (Pala-Paul et al., 2001), Thymus vulgaris

(McGimpsey et al., 2006) and Abies sachalinensis (Satou et al., 2009).

Many environmental, physiological and genetic factors influenced the chemical profile

and make up of essential oils in plants (Angioni et al., 2006). Microenvironments like sun and

shade also affect the oil yield from month to month in growing season (Juliani et al., 2002).

Moreover, this variation in oil depends on precipitation and temperature (Pala-Paul et al., 2001).

Atti-Santos et al. (2004) observed higher essential oil concentration in spring in Thymus

vulgaris. In leaves of Pilocarpus microphyllus higher percentage of essential oil was observed in

the rainy season (Taveira et al., 2003). The α-pinene content in leaves of Abies sachalinensis oil

was higher in April to June, while a steady decline observed in November to December and this

increase was attributed to high precipitation, temperature, sunshine (Satou et al., 2009).

In Eucalyptus the contents of essential oils in different seasons varied from 0.05% during

winter to 2.5% during summer season. Best essential oils percentages were estimated in E.

camaldulensis and E. cinerea (2.5 and 1.95% respectively) in the summer season (Emara and

Shalaby, 2011). Similarly the level of these components reached its maximum in all studied plant

species during the summer season and had the lowest value during winter, and this may be due to

the physical and chemical stress on plant especially during summer drought. This stress led to

plant secretion to different defense components called secondary metabolites, as protecting

agents, especially terpenoid compounds (Samuelsson, 1999). With regard to significance

estimates, more consistent trends in percentages variation in Eucalyptus spp. during different

seasons were present for essential oils than for lipids, and this further confirm that the former are

mainly due to the effect of environmental condition on plant synthesis. Wildy et al. (2000)

investigated four promising Eucalyptus species and reported 0.01 to 13.0% oil production from

Western Australia from six locations. Zafar et al. (2003) also reported 0.58 to 1.47% oil potential

18

for the different Eucalyptus species. These variations might be attributed to different agro-

climatic regions and soil composition in the districts of Punjab.

2.3 Medicinal Plants

Since ancient times medicinal plants have been used in almost all cultures as a source of

medicine. Many archaeological records have revealed that medicinal plants were used by

humans in Paleolithic period, approximately 60,000 years ago (Sumner, 2000). These plants

occupied different habit and habitat covering wild altitudinal range, desert and seacoast including

aquatic and lower plants like algae (Solecki, 1975). Only 10% information about herbal remedy

of medicinal plant is available. Thus a lot of research is required to explore the roles of leaves,

roots, stem, flowers and whole plants, which are commonly used in phytoextraction (Shanker,

1998). A huge range of chemicals such as flavonoids, alkaloids, phenolic and tannins are present

in medicinal plants; Almost 12,000 active compounds have been extracted from medicinal plants

until now, which is approximately less than 10% of the total compounds (Lai and Roy, 2004).

All parts of Aegle marmelosis i.e., leaves, roots, fruits and barks have medicinal

importance (Duke and Jo, 2002; Das et al., 2006). Adesanwo et al. (2009) observed that leaves

of Melaleuca bracteata (family Myrtaceae), is also famous for curing wounds and skin disorders.

It stimulates glandular secretions and reduces vein congestion. Numerous medicinal herbs and

spices, which are part of our everyday food intake help keep away food borne pathogens

(Tapsell et al., 2006). Medicinal value of myrrh and opium were reported five thousands year

ago (Sumner, 2000). Ayurveda medicines, combination of herbs, including turmeric has been

used perhaps as early as 1900 BC in subcontinent. Ibn Sina described medicinal healing system

in his Medical Encyclopedia “The Canon of Medicine”. In his book Pen Tsao, Shen Nung

described 365 plants including hemp, chaulmoogra and ephedra for treatments of various

ailments (Sumner, 2000). Medicinal plants can be demarcated in two important sectors: (a)

modern medicines that utilize about 30 to 35 medicinal plants; (b) traditional medicines that are

organized and codified with written treatise texts such as Ayurveda, Siddha, Unani, Amchi, and

Tibetan systems of medicine that used about 1,200 to 2,000 medicinal plant species (Dahanukar

et al., 2000; Rajasekharan and Ganeshan, 2002).

19

Conventional and traditional medicines are used since geneses in domestic recipes and also

as source of minerals (Meskin, 2002). Ayurveda is one the world’s oldest medicinal system. In

China, about 5000 traditional remedies have been reported, which make up one fifth of the whole

Chinese pharmaceutical market (Lee, 2000). Just like other parts of the world, local communities

in Pakistan also use medicinal plants to cure various diseases (Morgan, 2002). In traditional health

care system, drug production methods involved chopping and boiling of particular bark, roots and

leaf parts. For stress relief tea decoction orally or sometimes mixed with milk and sugar was used.

Elixirs wine and herbal extracts of alcohol, usually with 12-38% alcohol content can also be used.

Saba et al. (2009) revealed that Lagenaria breviflora fruit ethanolic extract has toxicological

effects and commonly used in the West African folk medicine. In Ayurvedic system, Hemidesmus

indicus is regarded good for rheumatism, leprosy, impotence, skin infections, anti-ulcerogenic

(Jegadeesan, 2009), anti-thrombotic and anti-oxidation activity (Mary et al., 2003). L. breviflora is

used in a wide range of gastrointestinal problems and measles in West Africa (Tomori et al., 2007).

About 120 phytochemicals have been derived from plants, which are being used in

medicines (Fabricant and Farnsworth, 2001). Among these morphine, deserpidine, camphor,

vincristine atropine, vinblastine and yohimbine are derived from higher plants. Many new drugs in

era of 1971-1995 such as phyllodulcin, emetine, teniposide, pinitol, lectinan, ouabain and

ginkgolides rose all over the world. These plants derived drugs had eminent contribution in modern

therapies for example: morphine isolated from the Papaver somniferum was a famous analgesic.

Vinblastine extracted from the Catharanthus rosesus is good in curing choriocarcinoma, Hodgkins,

neck cancer (Farnsworth et al., 1967), acute cervical cancer and lymphocytic leukemia

(Farnsworth and Bingel, 1977).

Lack of herbal medicines safety and efficacy standards is a big issue now-a-days

(Ramawat and Goyal, 2008). In 1991, the World Health Organization (WHO) developed

guidelines in the 6th International Conference in Ottawa for the evaluation of medicinal herbs.

Same year, the Drug Regulatory Authorities approved the same guidelines. It has been estimated

that 80% of the population in Asian and African countries used medicinal plants for health care

having income less than $2 a day because pharmaceuticals are very expensive (Da Silva et al.,

2002). In fact, WHO reported that in United States, about 25% of the drugs are derived from

medicinal plants. In European countries, the sale of medicinal plants is varied and estimated as

the $1.5 billion (Germany), $1.6 billion (France), $ 0.6 (Italy) and 1.5 billion (Japan) in 1996.

20

Currently, the world market of medicinal plants is increasing and observed only in the United

States to US$ 250 billion (Brower et al., 1998).

2.4 Lemongrass: Origin and Nature

Lemongrass is a famous Graminaceous perennial plant. Morphologically, the plant grows

in thick cluster up to 2 meters in diameter, 1.2 to 1.8 meter in height with long narrow leaves up

to one meter long and mostly cultivated in tropic and subtropics regions (Simon et al., 1984). It

has very pleasant taste, like lemon juice with a hint of ginger flavor. Lemongrass is marvelous

medicinal plant with sweet flavor. East Indian lemongrass (Cymbopogon flexuosus) is endemic

to India, Burma, Sri Lanka and Thailand. Its leaves are excessively consumed in Brazilian

traditional medicine as infusions due to its analgesic, anti-inflammatory, anti-spasmodic,

sedative, antipyretic properties and as diuretic (Blanco et al., 2009).

Essential oils in lemongrass are of tremendous economic importance as good food

preservative, tonic and flavoring ingredient in cosmetics and perfumes (Ganjewala and Luthra,

2007). Many pharmacologic activities of essential oil have been illustrated, like anticonvulsant

and anxiolytic activities (Silva et al., 2010) and antibacterial, antifungal and anti protozoal

properties (Oliveira et al., 2009). Lemongrass essential oil is recognized by its high citral content

(>45%) (Khanuja et al., 2005) which is raw material for the production of vitamin A, ionone and

beta carotene (Paviani et al., 2006). Citral is a mixture of trans-citral (geranial) and cis-citral

(neral) (Negrelle and Gomes, 2007). Many scientist reported antifungal properties of lemon grass

(Fiori et al., 2000; Pedroso et al., 2006). Caccioni et al. (1998) and Wang et al. (2007) also

reported lemongrass juice to possess antifungal ability, which assists the idea that lemongrass

and its juice are better in treating oral thrash than 0.5% aqueous solution of gentian violet in

HIV/AIDS patients (Wright et al., 2009).

2.4.1 Response of lemongrass to seasonal variation

Lemongrass has C4 photosynthesis with low CO2 compensation values like other C4

grasses. Photosynthetic rate in lemongrass leaves was significantly affected by leaf age and

21

photosynthetic activity declined with an increment in leaf age, and from the apex to the bottom

of the leaf blade (Maffei et al., 1988). Lemongrass shows optimum growth at 27/21°C day/night

temperatures respectively. It required 8 h photoperiod, which is appropriate for maximum

photosynthetic activity. Herath and Ormrod (1979) measured highest photosynthetic activity in

newly developed fully expanded leaves in both strains of C. flexuosus and C. citrates

(lemongrass). Keeping in view the ecological conditions, lemongrass cultivation has very high

values of accumulation, even when the summer temperature remains near or below 18-21/30-

35°C. The fastidious effectiveness of the carboxylating reserves also in accordance with higher

chlorophyll a/b ratio (5.49) showed a plausible enrichment of the light-harvesting reaction

centers (Maffei et al., 1988).

In Thymus vulgaris oil composition and yield both were critical to the time of harvest

(Badi et al., 2004). Although lemongrass (Cymbopogon citratus) is mostly being examined for

its phytochemical components, insufficient literature is published for its growth variation in

different seasons. In lemongrass tiller production is sensitive to seasonal variation and its

production varies with the level of irrigation and season of harvest (Singh at al., 2000).

Lemongrass oil yield affected when grown in different season. Generally, lemongrass yielded

0.2% and 0.35% essential oil in rainy and dry seasons respectively. In Pinienta racemosa

essential oil content varied between 1.32 to 3.40% and this yield decreased with an increase in

rainfall (Childers et al., 2006). Lemongrass oil showed great variations in chemical composition

in different seasons (Kulkarni et al., 1997). Sarma et al. (2011) recorded higher oil contents in

lemongrass leaves at the onset of monsoon as compare to post monsoon period.

22

CHAPTER-3

MATERIALS AND METHODS

3.1 Experimental Plan

The current work was carried out to appraise the variations in physiological and

biochemical characteristics of leaves of lemongrass (Cymbopogon citratus) in spring, summer

winter and autumn seasons. Experimental field was selected from New Botanical Garden,

University of Agriculture, and Faisalabad. Propagules of a selected lemongrass population were

transplanted in plots measuring 2 × 2 m in the month of October, 2010 and 2011. In both the

years, after seed germination, 100 plants were retained in each plot with a plant to plant distance

of 20 cm (Fig. 3.1). The experimental design was randomized complete block design (RCBD).

All the plots were watered using irrigation water at fortnightly intervals during summer season

with at three weeks interval during winter season. There were three blocks; each block with three

replicates. From each replicates 500 g of the leaves were taken from three positions i.e.,

penultimate, middle and bottom (Fig. 3.2) on 10th of each month during the years 2010 and 2011.

Half of the samples of the leaves of three ages were oven dried, while the other half samples

were frozen for fresh analysis at -50ºC. Physiological parameters were analyzed using frozen

fresh material. Nutrient composition and Oil composition, Na, K and Ca, nitrate and phosphate

were estimated from dry material.

3.2 Meteorological Data

Meteorological data was obtained from the Weather Observatory of the Department of

Crop Physiology, University of Agriculture Faisalabad. These data were used to draw the

correlations of various physiological and biochemical attributes with the environmental

conditions prevailing during the course of experiment.

23

Fig. 3.1: Field sown lemongrass population at early stage

Fig. 3.2: Leaves of various ages/positions used to perform the analytical work

24

3.3 Analytical Procedures

The determinations were made for oxidative stress and antioxidants characteristics,

osmoprotectants, some important secondary metabolites, vitamins photosynthetic pigments,

nutrients, nutritional and fiber characteristics during both the years.

3.3.1 Determination of oxidative stress parameters

a. Hydrogen peroxide estimation

Hydrogen peroxide (H2O2) contents were determined by using method of Velikova et al.

(2000). Fresh leaf tissues (0.1 g) were extracted in 0.1% trichloroacetic acid (TCA) in ice bath.

Then this extract was centrifuged at 12,000 × g and supernatant was collected. A 0.5 mL of 10

mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide was added to 0.5

mL of supernatant. This mixture was vortexed and absorbance was noted at 390 nm.

b. Malondialdehyde (MDA) Determination

The (MDA) was measured with the method given by Heath and Packer (1968). For the

determination of MDA, 0.1 g of fresh leaf tissue was extracted in 1 mL of 5% (w/v) TCA and

centrifuged at 12000 × g and supernatant was taken. To 1 mL of supernatant equal volume of

thiobarbarturic acid [(TBA) 0.5% in 20% (w/v) in TCA] was added followed by heating at

100oC. Reaction mixture was centrifuged at 7500 × g for 5 min. Absorbance was noted at 532

and 600 nm. MDA was calculated using extinction coefficient of 155 mmol mL-1 as:

MDA equivalents (nmol mL-1) = [(A532-A600) / 155000] × 106

25

3.3.2 Determination of enzymatic antioxidants

a. Enzyme extraction

Fresh plant material (0.1 g) was extracted in 0.9 mL of 50 mM cooled phosphate buffer

(pH 7.8) in the presence of protease inhibitors (Cocktail protease inhibitors, Sigma, USA). This

extract was centrifuged at 15000 × g at 4oC and supernatant was collected and stored at -30oC for

enzyme assay.

Prior to the determination of the activities of enzymatic antioxidants, the proteins

concentration of the extract was determined with dye-binding method of Bradford (1976), as

described below (section 3.3.7 g). The activities of the enzymes were expressed as U/g

protein/min.

b. Superoxide dismutase (SOD) activity

Activity of SOD was assayed by following the method of Giannopolitis and Ries (1977).

Enzyme extract (50 μL) from the above was taken and mixed with 50 mM phosphate buffer (pH

7.8). To this extract, 50 μM NBT (Nitro blue tetrazolium dissolved in ethanol) was added

followed by 1.3 μM riboflavin and 13 mM methionine and 75 mM EDTA were added (total

reaction solution including enzyme extract 1 mL) and this mixture was kept in dark chamber

coated with aluminum foil. Then the reaction mixture was illuminated under fluorescent lamps of

30 (W FPL30EX-D) for 5 min. The SOD activity was estimated by monitoring the inhibition of

photochemical reduction of NBT at 560 nm by using a UV-visible spectrophotometer (UV-

4000, ORI). One unit of SOD was defined as the amount of enzyme required to cause 50%

inhibition of the rate of NBT reduction at 560 nm. Blank was used for comparison.

c. Catalase (CAT) and peroxidase (POD) activities

The activities of CAT and POD were estimated by following the method of Chance and

Maehly (1955) with some modification. Enzyme extract (0.1 mL) was taken and mixed with 50

mM phosphate buffer (pH 7.8) and 5.9 m M H2O2 and diluted up to 3 mL. The absorbance was

26

noted at 240 nm after each 20 sec. CAT activity was expressed in units as μM of H2O2

decomposed per min. One unit was defined as an absorbance change of 0.01 units per min.

For POD activity, reaction solution containing 50 mM phosphate buffer (pH 7.0), 20 mM

guaiacol, 40 mM H2O2.was taken. Enzyme extract (0.1 mL) was added and change in the

absorbance was noted at 470 nm after each 20 sec. One unit POD activity was defined as the

change of 0.01 absorbance unit per min per mg of protein.

3.3.3 Osmoprotectants

a. Free proline determination

Free proline was determined by using the protocol of Bates et al. (1973). Fresh plant

material was extracted in 3% aqueous sulphosalicylic acid. One mL of the extract was dissolved

with 1 mL of glacial acetic acid and 1 mL of acid-ninhydrin (1.25 g ninhydrin in 30 mL glacial

acetic acid) in a test tube and was go through vortex. This mixture was heated at 100oC in a water

bath. After heating it was placed in an ice bath. After cooling in an ice bath, toluene was added

followed by vortexing the mixture for 5 sec. After warming to room temperature, the chromophore

was aspirated and its absorbance was taken at 520 nm. For preparation of standard curve, proline

standard from 5 to 25 µg/mL was used. Amount of free proline was calculated with the formula:

µg proline/mL × mL of toluene µmoles proline/g fresh weight = -------------------------------------------------

(115.5 µg/mole)/g sample/5

b. Glycinebetaine

Glycinebetaine contents were estimated according to the method of Grieve and Grattan

(1983). Dry plant material (0.5 g) was extracted in 20 mL of deionized water. This extract was

filtered and diluted with 2N H2SO4 followed by cooling on ice for 1 h. This mixture was added

with 0.5 mL of cold IK-I2 (periodide reagent) and stored at 4oC for 16 h. The reaction mixture

was centrifuged at 10,000 × g and periodide crystals were collected. These crystals were

dissolved in 1, 2-dichloroethane and left at room temperature for 2 h. The absorbance of the

27

colored complex was noted at 365 nm. For construction of standard curve, standard series of

glycinebetaine (50, 100, 150 and 200 µg/mL) was run along with samples.

c. Total free amino acids (TFAA)

TFAA were determined by using the method given by Hamilton and Van Slyke (1943).

Fresh plant material (0.1 g) was extracted in phosphate buffer (pH 7.0) and extract was mixed

with 1 mL of 10% pyridine solution and 1 mL of 2% ninhydrin solution followed by heating for

30 min. After heating, the mixture was diluted up to 50 mL. Absorbance of this mixture was

taken at 570 nm using a spectrophotometer. For standard curve, leucine was used and total free

amino acids were calculated by using following formula:

TFAA (mg/g fresh weight) = Graph reading of sample × volume of the sample × dilution factor Weight of the tissue × 1000

d. Soluble sugars

Soluble sugars were determined by the method of Yoshida et al. (1976). Fresh plant

material (0.1 g) was taken and boiled in 5 mL distilled water, filtered and diluted up to 50 mL

with distilled water. To 1 mL of the diluted filtrate, 5 mL of anthrone reagent (prepared by

dissolving 1 g anthrone in 1 L conc. H2SO4) was added followed by heating at 90oC for 20 min

and absorbance was noted at 620 nm using a spectrophotometer. Glucose series (0, 20, 40, 60, 80

and 100 µM) was used for the preparation of standard curve.

3.3.4 Secondary Metabolites

For the determination of soluble phenolics, anthocyanins and flavonoids, frozen fresh material

was used. For the estimation of alkaloids, saponins and tannins, powdered dry material was

defatted following the method of AOAC (1990). For this purpose, plant sample (3 g) was mixed

with 30 mL hexane. This mixture was left on a shaker at 100 rpm for 24 h. Then this mixture was

centrifuged at 3500×g for 18 min. The residue was taken and again mixed with hexane followed

by centrifugation and allowed to stand for 24 h. After centrifuging, the supernatant and residue

were separated. This residue was oven dried and used as fat free sample for further analysis.

28

a. Alkaloids

Alkaloids were determined by using the method of Harborne (1973). Fat free sample (0.5

g) was added in 20 mL of 10% acetic acid (in ethanol) in 250 mL beaker and taken in test tubes.

The test tubes were covered and allowed to stand for 4 h. The mixture was filtered and filtrate was

heated on a water bath at 90oC till one quarter of the original volume of extract was obtained. After

heating, conc. NH4OH was added drop-wise until the formation of precipitates, which were

collected, washed with NH4OH and re-filtered. The residue was oven dried and weighed.

b. Saponins

The total saponin contents were evaluated according to the method of Chapagain and

Wiesman (2005). Fat free sample (0.1 g) was taken after drying for 24 h and added 30 mL of

methanol. The mixture was shaken on a shaker at 100 rpm for two days and centrifuged. After

that three consecutive extractions were carried out by using solvent methanol. The solvent was

evaporated and a yellowish crystalline residue was obtained, which was carefully weighed and

saponins were estimated.

c. Tannins

The extraction of tannins was carried out by following the procedure of Van-Burden and

Robinson (1981) with some modifications. Fat free sample was taken and mixed with 50 mL

distilled water. This mixture was shaken for 1 h on a shaker at 100 rpm, filtered and volume

made up to 50 mL. A 5 mL of the filtrate was taken and mixed with 0.1 M FeCl3 (in 0.1 N HCl)

and 0.008 M potassium ferrocyanide. Absorbance of the mixture was taken at 605 nm. For

construction of standard curve, 10, 20, 30, 40 and 50 µg/mL concentrations of tannic acid were

used and run along with the unknown samples.

d. Anthocyanins

Anthocyanins were determined according to the method of Stark and Wray (1989). For

this, fresh leaf material (0.1 g) was extracted in 1 mL acidified methanol (1% HCl v/v). The

29

mixture was heated at 50oC for 1 h and extract was filtered. The absorbance of the filtrate was

measured at 535 nm by using a spectrophotometer.

e. Total soluble phenolics

Total soluble phenolics were evaluated by using the method of Julkunen-Tiitto (1985)

using Folin-Ciocalteu reagent. Fresh plant material (0.5 g) was extracted in 80% acetone; extract

centrifuged at 12,000 and supernatant collected. A 100 µL aliquot of the extract was mixed with

0.5 mL of Folin-Ciocalteu’s phenol reagent and 2.5 mL of 20% Na2CO3. The volume of mixture

was made up to 5 mL and vortexed. Absorbance of the reaction mixture was noted at 750 nm.

f. Flavonoids

Flavonoids contents were determined by the method of Zhishen et al. (1999). Plant

material (0.1g) was extracted in 80% acetone and 1 mL of extract was added in a 10 mL

volumetric flask containing 4 mL of distilled water. The reaction mixture was added with 0.6 mL

of 5% NaNO2, 0.5 mL of 10% AlCl3 after 5 min, and 2 mL of 1 M NaOH after 1 min. The

reaction mixture was diluted with 2.4 mL of distilled water and mixed. The absorbance was

taken at 510 nm. The quercetin was used as a standard for the calibration curve.

3.3.5 Vitamins

a. Niacin

Niacin was estimated by the method of Okwu and Josiah (2006). Fat free sample (0.5 g)

as given above (section 3.2.4.) was taken and mixed with 5 mL of 1 N H2SO4 with shaking. Then

three drops of NH3 solution were mixed and filtered. Filtrate (1 mL) was taken and mixed with

0.5 mL potassium cyanide solution and 0.5 mL of 0.02 N H2SO4. The absorbance was noted at

470 nm by using a spectrophotometer. Final quantities of unknown samples were determined

from a standard curve constructed from the niacin.

30

b. Riboflavin

Riboflavin was determined by the method of Okwu and Josiah (2006). For this, 0.5 g fat

free sample was extracted in 10 mL of 50% ethanol for 1 h and filtered. To 1 mL of the filtrate, 1

mL of 5% potassium permanganate and 1 mL of 30% H2O2 was added followed by heating in a

water bath at 50oC for 30 min. After cooling, 0.2 mL of 40% sodium sulfate was added to it and

diluted the reaction mixture up to 5 mL. After 5 min, the absorbance of the colored complex was

measured at 510 nm. Standard curve was constructed by using riboflavin.

c. Ascorbic acid

Ascorbic acid was determined by the method described by Mukherjee and Choudhuri

(1983). Fresh plant material (0.25 g) was extracted in 10 mL solution of 6% TCA. To 4 mL

extract, 2 mL of 2% dinitrophenyl hydrazine solution (in acidic medium by dissolving 2 g of

compound in 100 mL of HCl (37%) was added followed by addition of one drop of 10%

thiourea solution. The reaction mixture was heated in a water bath for 20 min. After cooling to

room temperature, 5 mL of 80% H2SO4 (v/v) was added. The absorbance was taken at 530 nm.

Standard curve was constructed by using the ascorbic acid.

d. Reducing powers assay (RPA)

For RPA, the leaf samples were prepared following the method of Sofowora (1993). The

samples were air-dried at room temperature and blended to a mesh size of 1 mm. The blended

samples (5 g) were soaked in 20 mL of 98% methanol for 48 h, filtered and filtrate was

concentrated to dryness using rotary evaporator and refrigerated. The reducing power (RPA) of

leaves was quantified with the method of Perumal and Becker (2003) with some modification.

The refrigerated powder was re-dissolved in 80% methanol. One mL of this extract was mixed

with phosphate buffer (5.0 mL of 2.0 M, pH 6.6) and potassium ferricyanide (5.0 mL of 1%

solution) mixed and incubated at 50oC for 20 min. On cooling 5 mL of TCA (10%) was added

and the mixture was centrifuged at 3000 × g for 10 min. The upper 5 mL layer of the solution

was aspirated and was mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride. The

31

absorbance of the pink color mixture was measured spectrophotometrically at 700 nm. Increased

absorbance of the mixture indicated increased reducing power.

3.3.6. Photosynthetic pigments

Chlorophyll a and b was determined by using the method of Arnon (1949) and

carotenoids was determined by the method of Davis (1976). For this, 0.5 g fresh plant material

was completely homogenized with pestle and mortar in 80% acetone in the darkness, filtered and

made the volume of filtrate up to 10 mL by adding 80% acetone. For the chlorophylls estimation,

the absorbance was taken at 645 nm for chlorophyll a; at 663 nm for chlorophyll b and at 480 nm

for carotenoids using spectrophotometer. The quantities of chlorophyll a, b and total chlorophyll

were calculated by using following formulas given by Arnon (1949).

Chl. a (mg/g) = [12.7(OD663)-2.69(OD645) × V/1000 × W

Chl. b (mg/g) = [22.9(OD645)-4.68(OD663) × V/1000 × W

Total Chl. (mg/g) = [20.2 (OD645) + 8.02 (OD663) × V/1000 × W

Where V= Volume of acetone used in extract (mL)

W= Fresh weight of plant in g

For the estimation of carotenoids, the following formula given by Davis (1976) was used.

Carotenoids (mg/mL) = (Acar/Em × 100)

where Em × 100 = 2500 and ACar = [(OD480) +114(OD663)-0.638(OD645)]/2500

3.3.7 Nutrients, nutritional and fiber characteristics

a. Determination of K+ and Ca2+

Determination of K+ and Ca2+ was done by using the method of Yoshida et al. (1976).

Dry plant material (0.2 g) was digested in 2.5 mL of mixture of concentrated nitric acid and

perchloric acid (2:1 ratio) at 250oC on a heating block till the samples became clear. The volume

of the digest was diluted up to 50 mL with distilled water. Ca2+ and K+ were estimated by using

32

flame photometer (Sherwood model, 410 UK). Standard curves were constructed by running the

different grade series (10, 20, 30, 40, and 50 ppm) of Ca2+ and K+.

b. Soluble nitrate (NO3--N)

Soluble Nitrate (NO3--N) was determined by using the method of Kowalenko and Lowe

(1973). Dried ground material (0.5 g) was put in a test tube containing 5 mL distilled water and

autoclaved for 15 min. Then solution was filtered and diluted up to 50 mL with distilled water.

To 3 mL of the extract, 7 mL of the chromotropic acid (CTA) solution (prepared by dissolving 1

g CTA in 100 mL of conc. HNO3) was added and let stand for 20 min. The absorbance was taken

at 430 nm on a spectrophotometer. Distilled water was used as blank. Standard curve was drawn

by using the KNO3 grade series 10, to 50 mg L-1 NO3- prepared by diluting 100 mg L-1 NO3

-

stock solution (prepared by dissolving 0.7216 g of pure dried KNO3 in 1 L of distilled water).

c. Soluble phosphate (PO43--P)

For soluble PO43-‒P determination, to 1 mL of extract from the above (section 3.2.7 b), 2

mL of 2N HNO3 was added followed by dilution up to 8 mL with distilled water in a test tube.

Then 1 mL of the molybdate-vanadate reagent was added and volume was made 10 mL with

distilled water and stand for 20 min. The absorbance was taken at 420 nm. Distilled water was

used as blank and standard curve was made using the phosphate grade series (2.5‒15 mg L-1).

For preparation of this series stock solution (25 mg L-1 PO43- ) was prepared by dissolving 0.110

g of dried monobasic phosphate (KH2PO4) in 1000 mL of distilled water then diluted to 2.5, 5.0,

7.5, 10.0, 12.5 and 15.0 mg L-1 PO43- standard series (Yoshida et al., 1976).

d. Sulfate (SO42--S)

Sulfate was determined following the method described by Tendon (1993). To 10 mL of

the HNO3-HClO4 extract in a conical flask, 1 mL of 6N HCl and 1 mL of 0.5% (w/v) gum-acacia

solution were added. The solution was swirled for 10‒15 sec followed by the addition of 0.5 g

barium chloride crystals and swirled again and again until the solution became clear with

dissolved crystals. Sample transmittance was read at 340 nm. For constructing the standard

33

curve, 100 mg L-1 stock solution was prepared by dissolving 0.543 g of K2SO4 in distilled water.

The stock was diluted in 250 mL volumetric flasks, to make 0, 4, 8, 12, 16 and 20 mg L-1 sulfate

solution. To each flask, 25 mL of salt-buffer solution (prepared as 50 g MgCl2.6H2O, 4.1 g

KNO3 and 28 mL ethanol in a final volume of 1 L) was added. The remaining procedure was the

same as described above.

e. Starch contents

Starch contents were estimated by following the method of Malik and Srivastava (1985).

Methanolic extract of fresh plant material was taken and filtered. Then residue was dried and re

extracted in 5 mL of distilled water. To this extract, 52% HCL (1:1 v/v) was added, centrifuged

and supernatant collected. To 0.5 mL of this supernatant 2 mL of anthrone reagent was added

and heated for 30 min. After cooling, the absorbance was taken at 625 nm. The starch content

was calculated by formula:

Starch (mg/g dry weight) = standard reading (absorbance/mg) × sample reading × dilution factor

g. Total soluble proteins

Total soluble proteins were analyzed by the method of Bradford (1976). For extraction of

total soluble proteins, 0.5 g fresh plant material was extracted in phosphate buffer saline (PBS;

2.7 mM KCl, 10 mM Na2HPO4, 1.37 mM NaCl and 2 mM KH2 PO4, pH 7.2 adjusted with

HCl), centrifuged and supernatant collected. For this purpose, 1.0 mL of the extract was

centrifuged and the supernatant from each sample was mixed with 200 µL of Coommassie

Brilliant Blue (CBB) dye reagent (Bio-Rad, USA) in the Appendorf tube (1.5 mL). After waiting

for 20 min at room temperature, the absorbance of the samples was taken at 595 nm using a

spectrophotometer. Bovine Serum Albumin (BSA) was used as standard (10-50 µg/mL) for the

construction of standard curve. Final amount of proteins present in the extract was computed

from the unknown samples.

34

h. Neutral detergent fiber (NDF)

NDF was estimated by the method of Van Soest (1963). For this, dried material (1.0 g)

and 0.25 g of Na2SO3 was added in each flask. Fifty mL of NDF reagent (EDTA, Na2HPO4,

Lauryl sulfate, Sodium borate decahydrate and ethylene glycol monoethyl ether) was added with

water cooling arrangement with it. Then heated it slowly (to avoid foaming) on heating plate for

1 h then allowed to cool and filtered the solution with the help of suction pump. Then washed the

residue with hot water (60‒70ºC) for 4‒5 times using 5‒7 mL warm water. The residue was

washed in crucible with 5 mL acetone twice on a filter paper and placed in the hot air oven at

105ºC for overnight. Percentage of NDF was calculated using following formula.

% NDF = [(crucible wt. with fiber wt.) – (crucible wt. without fiber)/ sample wt.] x 100

i. Acid detergent fiber (ADF)

ADF was determined by the method of Van Soest and Wine (1963). For this, 1.0 g of

dried plant material was added with 50.0 mL of ADF solution (Hexadecyl trimethyl ammonium

bromide, sulfuric acid and distilled water) and heated slowly on heating plate in flask and air

condenser fixed on it and reflux for 1 h. Washed the residue with hot water for 3‒4 times then

washed with 5.0 mL of acetone twice. Residue were transferred to dried crucible and kept in an

oven at a temperature 70 oC for 3‒4 h and then put in desiccator for cooling. The weight of the

residue was taken and amount of ADF was computed with the formula.

% ADF = [(crucible wt. + with fiber) – (crucible weight without fiber) / sample weight] × 100

j. Determination of acid detergent lignin (ADL)

For the measurement of ADL, 1 g of dry sample was taken in a conical flask and added

50 mL of 1 N H2SO4 and added ADF solution. The flask was fixed with air condenser and

reflexed for 1 h. The residue was taken and washed with boiling water for 3‒4 times and again

washed with 5.0 mL acetone twice. The washed residue was put in an oven at 105oC for 3 h. The

residue from crucible was transferred in 250 mL beaker, and add 25 mL 72% H2SO4 and stirred

for 3 h. Contents were diluted up to 200 mL. Residue was washed with distilled water to remove

acid and again washed with 95% ethanol to remove excess water. Then residue was transferred

to crucible and kept in oven for 3 h and weighed. This contains lignin, cutin and silica; then

35

heated the crucible in muffle furnace at 600oC for 1 h. Then weigh the crucibles. ADL was

calculated using following formula (Goring and Van Soest, 1970).

ADL (%) = (Crucible weight after acid soak – Crucible weight after ignition) / (Sample weight)

f. Cellulose

The ADL was used to determine the cellulose content by further extraction with acid

detergent, permanganate and 72% H2SO4. The cellulose content was calculated as loss of weight

from the ADF as described by Goring and Van Soest (1970).

g. Silica

Silica was determined as the crude, insoluble residue remaining after dissolving the ash

(from ignition at 500oC) in 6N-HC1 followed by evaporation to dryness on a steam bath and re-

dissolving the soluble residue in boiling 6N-HC1 (AOAC, 1955).

3.3.8 Leaf essential oil determination

Finely air dried grounded lemongrass leaves samples (100 g) collected during four

seasons in a year were hydro-distilled for 3 h in Clevenger-type apparatus. The lemongrass

essential oil layer was dissolved in diethyl ether and then detached through distilled water in

separating funnel by solvent extraction. Then extracted lemongrass essential oil was stored at -

4°C for Gas Chromatograph-Mass spectrometer (GC-MS) analysis.

GC analysis of lemongrass leaf essential oils was done using GC-17A Model (Data Apex

Ltd. CSW32-Chromatography station, Shimadzu, Japan) equipped with DB-wax column (30 m ×

0.25 mm), flame ionization detector (FID). The temperature of Injector and detector were set at

250 and 260°C, respectively. Column temperature was prearranged from 90°C for 2 min to

180°C at 2°C/min to 240°C/min. Helium gas at flow rate of 30 mL/min at 150 psi was used as a

carrier gas. Oil sample (1 µL) was injected through the injector. The lemongrass oil percentage

composition was calculated as a relative percentage of the total peak area.

To recognize the oil components a comparison of their mass spectra was made with NIST

mass spectral library compounds (Mass Spectral Library, 2002) and through the comparison of

36

their retention indices either with those of authentic compounds or with literature values (Adam,

2001; Mimica-Dukic et al., 2003; Vagionas et al., 2007).

3.4 Statistical Analysis

The design of the experiments in both the years was factorial randomized complete block,

with three blocks and three replications in each block for all the determinations. The data

presented here is the average of both the years. The differences between factors and their

interactions were ascertained with analysis of variance (ANOVA) using computer software

Statistix 8.1. Pearson’s correlation coefficients were derived among different attributes were

made using MS-Excel. Data was presented graphically by using MS-Excel.

3.5 Chemicals

All chemicals used in the analytical work were purchased from Sigma, Merck, Bio-Rad,

Reidle and BDH grade.

37

CHAPTER-4

RESULTS AND DISCUSSION

4.1 Preliminary

In nature the environmental factors are the key drivers in the production of an array of

changes in plants starting from gene expression to the appearance of visible features. Among the

environmental conditions, the changes in light, temperature and humidity are the most important

ones. Being a C4 plant, lemongrass (Cymbopogon citratus) responds fairly well to the prevailing

conditions and shows the synthesis of a wide range of plant secondary products, which enable it

to withstand the changing conditions in all the seasons. In the current work, the response of a

selected vegetatively propagated population of lemongrass was studied in primary and secondary

metabolites and oil composition of leaves of three ages (penultimate, middle and bottom) for two

consecutive years for seasonal variations on monthly basis.

4.2 Meteorological Attributes

Fig. 4.1 indicates average meteorological data for the years 2010 and 2011. It is evident

from the data that respective average minimum and maximum temperatures were 4 and 16oC in

January, which begun to increase from February onwards and was maximum in the month of

June. From July onwards, the temperature indicated a decline and in December, the temperature

was quite low. During both the experimental years, the maximum average RH was noted in the

August and September (~75%) when precipitation was maximum as well as in cool and humid

months of winter season (~73%). However, hot and dry months (May) exhibited the lowest RH

(~43%). The pattern of rainfall was very erratic and sporadic; highest in September, July and

August (155, 118 and 93 mm, respectively) of both the years but with great variations (Fig. 4.1).

Contrarily, months of January, November and December in both the years did not receive any

precipitation. Trend of evapotranspiration was closely associated with seasonal changes in the

38

temperature ranges. The evapotranspiration was more (~6 mm) in hot and dry months (May and

June) while lower (1.2 mm) in cool months (January and February).

The trend of seasonal changes was similar in both the years, but the changes in

temperature were more crucial. The changes in climatic conditions are mainly dependent upon

prevailing temperatures, which also trigger the survival of plants with the induction of requisite

survival mechanisms (IPCC, 2007). In response to changes in temperature, plant responses are

usually noticed in terms of biosynthesis of various primary and secondary compounds (Chalker-

Scott, 1999; Wahid, 2007; Wahid and Tariq, 2008). Such diversions from normal metabolism

lead to growth and yield decline especially in medicinally important plants including lemongrass

(Hussain et al., 2012). Lemongrass showed conspicuous fluctuations in its active compounds

during different seasons, which might be ascribed to variations in seasonal variables such as

temperature, rainfall, humidity and soil moisture etc. Being a tropical and subtropical plant,

lemongrass displays wide range of variations in physiological and biochemical attributes in

different seasons. The present study provides the most elaborated description of seasonal

variation in the biochemical and physiological contents of lemongrass leaves.

Fig. 4.1: Average monthly data of meteorological conditions during the year 2010 and 2011. The error bars indicate the average variation in rainfall during both the years

0

20

40

60

80

100

120

140

160

180

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Rainfall (mm)

Max temp

Min temp

RH (%)

Evapotranspiration (mm)

Months 2010-11

39

4.3 Membrane characteristic and antioxidants

Antioxidants have seasonality in their formation and mode of action in different seasons.

Excessive production of ROS is causing progressive oxidative damage to the cellular machinery.

In lemongrass, the adverse seasonal changes induce the formation of reactive oxygen species. In

this part of the manuscript, the influence of seasonal changes on the levels of hydrogen peroxide

(H2O2), MDA, CAT, POD and SOD activities of lemongrass penultimate, middle and bottom

leaves was studied.

4.3.1 Results

The statistical analysis of data and significance of variance sources of the oxidative stress and

pattern of antioxidants expression in lemongrass leaves over a measured in the present studies 12

months period are given in Table 4.1

Table 4.1: Analysis of variance (mean squares) of oxidative stress parameters and antioxidants activities in three leaves of lemongrass over a 12 months period

SOV df H2O2 MDA CAT POD SOD

Block (B) 2 2.50 1.90 14.49 263.53 0.81

Leaves (L) 2 44845.61** 1948.81** 15471.41** 39741.11** 112.03**

Months (M) 11 238.52** 43.18** 878.09** 17778.57** 5.15**

L × M 22 76.90** 4.58** 184.72** 1306.81** 1.53**

Error 70 14.53 0.76 16.38 42.30 0.71

Significant at: *, significant at P = 5%; **, P = 1% levels of probability, respectively

40

a. Hydrogen peroxide (H2O2)

There was significant difference in the leaves and months with significant interaction of

these factors for H2O2 concentrations (Table 4.1). In penultimate leaf H2O2 remained fairly

constant during spring (Feb-Apr) season. It increased slightly in summer (May-Jul) months but

declined markedly in autumn (Aug-Oct) months. It increased again in winter (Nov-Jan) months.

In middle leaf, with the onset of spring season, H2O2 consistently declined but then attained the

greatest value in summer (maximum in July) and a decline was observed in autumn, and then a

rise in winter. In bottom leaf, except for late winter months, the tend of H2O2 accumulation was

similar to penultimate and bottom leaves. The highest H2O2 concentrations was observed in

bottom, moderate in middle and lowest in penultimate leaf (Fig. 4.2).

Fig. 4.2: Effect of seasonal variation on hydrogen peroxide accumulation in the penultimate,

middle and bottom leaves of lemongrass

bc c cd dab ab a

cde e

cdb

abc

bcde

ede

cde ab

c aab

cdcd

ecd

e abc ab

de cde

efcd

ede

fbc

a acd

ef fab

0

20

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120

Jan

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Penultimate Middle Bottom

Hyd

roge

n pe

roxi

de (

nmol

/g f

resh

wei

ght)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

41

b. Malondialdehyde (MDA)

The MDA content in leaves of lemongrass over months varied significantly with a

significant interaction of these factors (Table 4.1). Both penultimate and middle leaves exhibited

similar contents and trend of MDA accumulation. In both these leaves, MDA contents were

lower in spring season, which indicated a sharp increase at the onset of summer and the

maximum values were noted in Jun and Jul. The MDA contents began to decrease sharply at the

start of autumn but again increased in winter season. Bottom leaf showed increased contents of

MDA contents from May and achieved an apical value in July, while a drop was detected in

August, which continued till October. However, a steady-state rise was observed from Oct to Jan

(winter season). In all leaves, the value of MDA increased during summer and winter seasons

while minimum value was found in spring and autumn seasons (Fig. 4.3).

Fig. 4.3: Effect of seasonal variation on MDA concentration in the penultimate, middle and

bottom leaf of lemongrass

bcfg fg

hgh

dea ab

efh gh

cd cd

bde cdde

bca a

bcde e

b b

bcgh

fgh

efb

acd

egh

hde

bcd

0

5

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Penultimate Middle Bottom

Mal

ondi

alde

hyde

(nm

ol/g

fre

sh w

eigh

t)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

42

c. Superoxide dismutase (SOD) activity

Data indicated significant differences in the leaves of various ages and sampling months

on SOD activity with a significant interaction of leaves and sampling months (Table 4.1).

Seasonal changes produced a great impact on the SOD activity of lemongrass leaves. The SOD

activity of the penultimate was the lowest SOD in spring season, which increased greatly in

summer season, attained the highest value in June (21.66 U/g protein/min), while a decline was

observed in July and onward which continued till October, while a gradual increase was

observed from November to December. SOD activity of middle leaf was more or less similar to

the penultimate leaf. However, bottom leaf indicated quite reduced SOD activity in most of the

months except a rise was noted in May (Fig. 4.4).

Fig. 4.4: Effect of seasonal variation on SOD activity in the penultimate, middle and bottom

leaves of lemongrass

bef

bcde bc bc

da

bef cd

efde

ff ef

cdf

de decd

aab

efcd

deef

bc

bccd

ede e de

abc

de

bbc

cde bc

d0

5

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Penultimate Middle Bottom

Sup

erox

ide

dism

utas

e (U

/g p

rote

in/m

in)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

43

d. Catalase (CAT) activity

The CAT activity of lemongrass leaves of three ages varied significantly in different

seasons with a significant interaction of leaf ages and sampling months (Table 4.1). All leaves

indicated individualistic behavior of CAT activity. The penultimate leaf showed the highest CAT

activity in July (113.14 U/g protein/min). However, a reduction in CAT activity was noticed

from August which attained a steady-state level up to December. In the middle leaf, CAT in

activity increased from January to March (winter to spring seasons) and attained the maximum

value in July (87.84 U/g protein/min), while a decline was observed from August to December.

On the other hand, bottom leaf over the seasons (sampling months) indicated lower CAT activity

winter months, declined in summer months, again increased in autumn and declined thereafter.

Overall, CAT activity was the highest in penultimate leaf followed by middle leaf (Fig. 4.5).

Fig. 4.5: Effect of seasonal variation on CAT activity in the penultimate, middle and bottom

leaves of lemongrass

cde

de decd

ba

cd dde

cdc

fef

ccd c

ba

c cde

ef f

fef

ab abde

bca a ab

cdef f

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Jan

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Penultimate Middle Bottom

Cat

alas

e ac

tivity

(U

/g p

rote

in/m

in)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

44

e. Peroxidase (POD) activity

The data indicated significant differences in the leaves and months as well as there was

significant interaction of these factors for POD activity (Table 4.1). Different lemongrass leaves

presented peroxidase (POD) activity with value ranging from as low as 17.46 protein/min in

bottom leaf to as high as 157.06 U/g protein/min in penultimate leaf. The POD activity of

penultimate and middle leaves was lower in winter and autumn seasons but higher in summer

season, which sequentially declined from August and attained steady-state level thereafter.

However, bottom leaf exhibited differential behavior of POD activity; three distinct peaks were

observed in Feb, Jul and Nov. Overall, penultimate leaf indicated the highest POD activity

followed by middle leaf (Fig. 4.6).

Fig. 4.6: Effect of seasonal variation on POD activity in the penultimate, middle and bottom

leaves of lemongrass

deef

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45

f. Correlation

Parallels drawn of oxidative stress parameters with antioxidants activities revealed that

H2O2 accumulation was positively related to MDA contents of all leaves. Among the

antioxidants, SOD activity was positively correlated with H2O2 accumulation in penultimate and

middle leaves. MDA contents indicated a positive relationship in case of penultimate and middle

leaves with the activities of SOD and POD while only with penultimate leaf in case of CAT

activity. Bottom leaf did not exhibit any association with the antioxidants activities (Table 4.2).

Table 4.2: Correlation coefficient (r) of metrological attributes with the Secondary metabolites of lemongrass at three leaves positions penultimate, middle, bottom X-Variable Y-Variable Penultimate Middle Bottom a. Correlations of oxidative stress parameters with antioxidants H2O2 MDA 0.777** 0.761** 0.610* SOD 0.581* 0.594* -0.193ns CAT 0.554ns 0.014ns 0.347ns POD 0.548ns 0.309ns -0.355ns MDA SOD 0.753** 0.711** -0.009ns CAT 0.774** 0.430ns -0.083ns POD 0.610* 0.622* -0.034ns b. Correlations of environmental variable with oxidative stress parameters and antioxidant Max. temperature H2O2 -0.062ns -0.188ns 0.205ns MDA 0.023ns 0.107ns 0.038ns SOD 0.173ns 0.371ns 0.021ns CAT 0.424ns 0.759** 0.632* POD 0.707* 0.759** -0.474ns Min. temperature H2O2 -0.069ns -0.100ns 0.346ns MDA 0.043ns 0.163ns 0.136ns SOD 0.120ns 0.367ns 0.043ns CAT 0.488ns 0.831** 0.729** POD 0.756** 0.823** -0.470ns Relative humidity H2O2 -0.210ns 0.377ns 0.346ns MDA -0.019ns 0.027ns 0.187ns SOD -0.281ns -0.091ns 0.331ns CAT 0.002ns -0.145ns 0.273ns POD -0.205ns -0.145ns 0.273ns Evapotranspiration H2O2 0.158ns -0.169ns 0.146ns MDA 0.169ns 0.203ns 0.101ns SOD 0.163ns 0.241ns -0.178ns CAT 0.454ns 0.721** 0.474ns POD 0.758** 0.760** -0.513ns Average rainfall H2O2 -0.136ns 0.218ns 0.616* MDA 0.050ns 0.206ns 0.246ns SOD -0.031ns 0.265ns 0.315ns CAT 0.495ns 0.649* 0.671* POD 0.492ns 0.566ns -0.253ns

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

46

As for the seasonal changes, POD activity of penultimate leaf was positively correlated

with maximum and minimum temperatures, and evapotranspiration. CAT and POD activities of

middle leaf were positively correlated with maximum and minimum temperatures,

evapotranspiration and average rainfall. In case of bottom leaf, the CAT activity was correlated

with maximum and minimum temperatures and average rain fall while H2O2 accumulation was

related to average rainfall (Table 4.2).

4.3.2 Discussion

Plants respond to the seasonal changes by showing adjustments in their metabolism. One

of the most common consequences of heat stress is the generation of ROS leading to membrane

damage and disruption of cellular phenomena (Wahid et al., 2013). In this experiment,

monitoring the leaf H2O2 contents indicated that its greater production took place in summer and

winter season in all leaves, although bottom leaf indicated the highest while penultimate leaf the

lowest amounts of H2O2 (Fig. 4.2). With the induction of oxidative stress, MDA is produced due

to β-oxidation of membrane lipids (Lu et al., 2009), which perturbs the plant phenomena

including photosynthesis and respiration (Malencic et al., 2004). In the present study, there was a

substantial increase in the MDA contents of all the studies leaves in the harsh conditions of

summer and winter, although leaf age had a large effect. The MDA contents indicated the pattern

more or less similar to H2O2 production (Fig. 4.2, 4.3). This was also evident from the positive

correlation of H2O2 and MDA for all leaves (Table 4.2). Savicka and Skute (2010) observed that

high temperature accelerates MDA production (up to 58%) at later growth stages as compared

with early seedling stage (by 27%). Enhanced lipid peroxidation during hot months could be

associated with the damage on cell membranes (Huang et al., 2004) and in rice it reduced

antioxidant enzyme activities (Cao et al., 2009). It was important to note here that both seasonal

changes and leaf age have great impacts on the production of MDA, where cooler months and

younger (penultimate) leaf exhibited lower MDA production.

Both plant age and seasonal variation influence antioxidant enzyme activities of plants,

which are important indices of plant responses (Sen and Mukherji, 2007). Temperature above

33°C induced oxidative stress, which damage cell membrane due to degradation of protein and

also decrease in enzyme activities in wheat (Bavita et al., 2012). To circumvent enhanced ROS

47

produced by seasonal variation, plant cells activate the key antioxidant enzyme like SOD, CAT

and POD (Mittler, 2002; Ozden et al., 2009). These antioxidants are the first line of defense

against ROS, which catalyzes the dismutation of O2- and dousing of other activated ions

(Takahashi and Asada 1983; Scandalios, 1993; Wahid et al., 2013). In the present study, the

SOD, CAT and POD activities in lemongrass leaves were increased markedly in summer months

(Figs. 4.4-4.6). However, leaves of different ages indicated differential response to the induction

of antioxidant defense. Correlation data indicated that there was a close association of the MDA

and H2O2 accumulation with the SOD and CAT activities in penultimate and middle leaves

(Table 4.2). A conspicuous increase in ROS scavenging activity with the induction of antioxidant

systems was of great advantage to these leaves. Under higher temperatures, plant respiratory

rates are higher which enhance antioxidant response due to subsequent higher ROS level in the

mitochondria. According to Dizengremel (2001), SOD formation increases with increase in

NADH synthesis during higher respiration. Similarly, high temperature stress enhanced the

respiration rate along with a significant increase in SOD-manganese activity in Nicotiana

plumbagifolia (Bowler et al., 1992).

Among the environmental variables, changes in the ambient temperature and

evapotranspiration were of greater significance. These factors cause osmotic strain on the leaves

and induction of enzymatic antioxidants has been well reported under abiotic stresses including

cold stress (Wu et al., 1999; McKersie et al., 1999), heat stress (Wahid et al., 2007), high light

intensity (Sen Gupta et al., 1993) and drought stress (Farooq et al., 2009). In the present study,

CAT and POD activities increased in lemongrass leaves in summer period during the months of

June and July (Fig. 4.5-4.6), which has close association with high and low temperatures and

evapotranspiration (Table 4.2). Increased CAT under adverse conditions has been considered as

a prerequisite for plants against fatal H2O2 accumulation (Streb et al., 1997a, b; Engel et al.,

2006). This notion stands fast in the current study too because high and low temperatures and

evapotranspiration are great stressing factor for plant growth, while POD protects the membrane

from damage due to H2O2 (Farooq et al., 2009).

To conclude, seasonal variation has a great impact on lemongrass antioxidant activity.

These antioxidant enzymes are capable of minimizing the effect of oxidative damage by

scavenging H2O2 and reducing MDA production in extreme environmental conditions.

48

4.4 Osmoprotectants

In osmotic adjustment of plant cell organic solutes play an important role. Seasonal

variations occur not only in the type of the organic solutes, but also in the pattern of their

accumulation. Seasonal variation in soluble sugars, free proline, glycinebetaine and total free

amino acids content were measured in penultimate, middle and bottom leaves of lemongrass.

The results are interpreted and discussed below.

4.4.1 Results

Results regarding statistical analysis of data for the changes in the osmoprotectants

accumulation in the leaves of lemongrass in various seasons are given in Table 4.3

Table 4.3: Analysis of variance of data (mean squares) for soluble sugar, free proline, total free amino acid, and glycinebetaine contents of lemongrass under influence of seasonal condition

SOV df Soluble sugar TFAA Free proline Glycinebetaine

Block (B) 2 4.80 18.49 2.91 0.54

Leaves (L) 2 3267.95** 57666.30** 8769.98** 2323.35**

Months (M) 11 46.32** 1857.01** 129.85** 30.17**

L × M 22 5.92ns 1627.28** 70.41** 11.74**

Error 70 4.32 26.47 2.17 1.44

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

49

a. Soluble sugars

The soluble sugars contents in different leaf positions of lemon grass varied significantly

in different sampling months with a non-significant interaction of these factors (Table 4.3).

Although there were substantial differences in the accumulation of soluble sugars in the leaves of

different ages and seasons, the trend of their accumulation in all leaves was similar. The soluble

sugars accumulation was lower in the spring (Feb-Apr), which increased reasonably in the

summer and autumn seasons (May-Sep). However, this accumulation was the greatest especially

in the month of Jan. (34.47 mg/g fresh weight) in winter season. Nonetheless, penultimate leaf

displayed more explicit variations in the soluble sugars accumulation than middle and bottom

leaves (Fig. 4.7).

Fig. 4.7: Effect of seasonal variation on soluble sugar concentration in the penultimate, middle

and bottom leaves of lemongrass

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Penultimate leaf = **Middle leaf = **Bottom leaf = **

50

b. Total free amino acids (TFAA)

Statistical analysis of data indicated significant difference in the leaves of various ages

and sampling months with a significant interaction of both the factors for TFAA levels (Table

4.3). The trend of TFAA accumulation was similar over the seasons in the leaves of all ages,

although there were great differences in their contents. In all leaves, TFAA accumulation was the

lowest in the spring season, which increased with the onset of summer season; however, this

increase was the highest in penultimate leaf followed by middle leaf. With the start of autumn

season, the TFAA levels again declined and indicated an increase in the winter season. These

changes were more explicit in the penultimate leaf as compared to middle leaf and bottom leaf

indicated fewer changes (Fig. 4.8).

Fig. 4.8: Effect of seasonal variation on total free amino acid in the penultimate, middle and

bottom leaves of lemongrass

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Penultimate leaf = **Middle leaf = **Bottom leaf = **

51

c. Free proline

Analysis of variance in the free proline concentration revealed highly significant (P<0.01)

difference in the leaves of different ages and sampling months with significant interaction of

both these factors (Table 4.3). Data on free proline accumulation in penultimate, middle and

bottom leaves exhibited individualistic trends. In penultimate leaf, free proline content was

lowest in spring season, which sharply increased in the summer season, reaching highest value in

July (44.89 µg/g fresh weight). In the autumn season, free proline levels again declined and then

showed an increase in the winter months. In middle leaf, free proline level was low in spring

season, which increased in Jun and Jul (~19 µg/g fresh weight). In autumn the free proline level

decreased and became steady in winter season. In bottom leaf free line level was kept low and

showed virtually no change over the seasons (Fig. 4.9).

Fig. 4.9: Effect of seasonal variations on free proline accumulation in the penultimate, middle

and bottom leaves of lemongrass

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52

d. Glycinebetaine (GB)

The amount of GB in leaves of various ages differed significantly in various sampling

months with a significant interaction of leaves and months (Table 4.3). Among the leaves of

three ages, penultimate leaf indicated the highest and more explicit trend of GB accumulation

followed by middle leaf, while bottom leaf indicated no specific change in GB accumulation. For

sampling months, both penultimate and middle leaves in autumn season (especially in Sep-Oct

months) indicated the lowest GB accumulation followed by spring season. The GB accumulation

in these leaves was the highest in both summer (especially in Jun) and winter (especially in Jan)

seasons (Fig. 4.10).

Fig. 4.10: Effect of seasonal variation on Glycinebetains concentration in the penultimate,

middle and bottom leaves of lemongrass

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Penultimate leaf = **Middle leaf = **Bottom leaf = ns

53

e. Correlation

In penultimate and middle leaves soluble sugars indicated positive association with free

proline in penultimate leaf and with TFAA and free proline in middle leaf. TFAA was positively

correlated with free proline in middle leaf, with GB in penultimate and middle leaves and free

proline was positively paralleled with GB in penultimate leaf. Among the environmental

variables, TFAA, free proline and GB of penultimate and middle leaves were positively related

to maximum temperature; evapotranspiration with TFAA in penultimate leaf and free proline

accumulation with average rainfall in middle leaf.

Table 4.4: Correlation coefficient (r) of mutual relationships of osmoprotectants and those of metrological attributes with the Osmoprotectant of lemongrass leaves

X-Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of osmoprotectants Soluble sugars TFAA -0.014ns 0.665* 0.485ns Proline 0.589* 0.602* 0.249ns GB 0.381ns 0.362ns 0.273ns TFAA Proline 0.284ns 0.688* 0.400ns GB 0.621* 0.810* 0.025ns Proline GB 0.795** 0.412ns -0.399ns b. Correlations environmental variable with osmoprotectants Max. temperature Soluble sugars -0.379ns -0.402ns -0.315ns TFAA 0.583* -0.583* -0.543ns Proline -0.685* 0.637* -0.114ns GB -0.673* -0.584* -0.387ns Min. temperature Soluble sugars -0.259ns -0.274ns -0.281ns TFAA 0.382ns -0.515ns -0.515ns Proline 0.005ns 0.371ns -0.200ns GB -0.289ns -0.182ns -0.327ns Relative humidity Soluble sugars 0.565ns 0.547ns 0.385ns TFAA -0.472ns 0.308ns 0.308ns Proline 0.202ns 0.305ns -0.224ns GB -0.110ns -0.204ns 0.309ns Evapotranspiration Soluble sugars -0.391ns -0.424ns -0.400ns TFAA 0.767** -0.436ns -0.436ns Proline 0.026ns 0.582* 0.068ns GB -0.029ns 0.138ns -0.461ns Average rainfall Soluble sugars 0.179ns 0.195ns -0.146ns TFAA 0.022ns -0.223ns -0.223ns Proline 0.181ns 0.583* -0.081ns GB -0.279ns -0.197ns -0.238ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

54

4.4.2 Discussion

In response to seasonal changes, a number of low molecular weight compounds can

accumulate in lemongrass leaves like amino acids, soluble sugars and quaternary ammonium

compounds (QAC). The osmolytes accumulation has been very well related to changes in the

prevailing environmental conditions (Bhowmik and Matsui, 2003; Wahid et al., 2007). In the

present study, among the various osmoprotectants studied, soluble sugars accumulation

displayed seasonal variations, being higher in winter followed by summer conditions while lower

in spring and autumn seasons (Fig 4.7). The harsh conditions during winter and summer produce

an osmotic stress, which is counterbalanced by the accumulation of osmoprotectant; the soluble

sugars appear to be major amongst those in the lemongrass. This is due to their property of being

hydrophilic in nature and having ability to replace water on the surfaces of proteins and

membranes, and thus ensuring their biological functions (Hasegawa et al., 2000). According to

Chinnasamy and Bal (2003) and Schaberg et al. (2000) total soluble sugar content showed

maximum amount in mid-winter. In another study, it was observed that high concentration of

soluble sugars act as cryoprotectants during cold acclimation (Taulavuori et al., 2001).

Amino acids are building blocks of proteins but their accumulation in free form has great

implications in the stress tolerance (Mahmood et al., 2012). In the present case, all the relatively

younger (penultimate and middle) leaves of lemongrass indicated great seasonality; their contents

were specifically higher in summer season. However, they were accumulated the least in bottom

leaves despite harsh condition of summer and winter (Fig. 4.8). Main function of the accumulation

of amino acids in free form appears to protect the younger tissues from stress damage by

associating with the cytoplasmic membranes (Simon-Sarkadi and Galiba, 1996).

Proline plays a role in osmotic adjustment (Yamada et al., 2005) and protects the sub-

cellular structures under adverse conditions (Ashraf and Foolad, 2007). Free proline

accumulation in response to stressful conditions occurs in a number of plant species (Ashraf

and Foolad, 2007; Farooq et al., 2009; Nasir et al., 2010). In present study, free proline

accumulation was significantly different in penultimate, middle and bottom leaves of

lemongrass. Among these leaves, penultimate leaf had higher proline concentration followed

by middle leaf (Fig. 4.9). Khan and Beena (2002) reported that in the leaves of Calotropis

procera, Senna holosericea and Aerva javanica, proline accumulation was greater in the

55

young leaves, as noted in the present case too. Free proline accumulation in various plants

has been reported to be under severity of the prevailing stress (Zhu, 2001; Kavi-Kishore et

al., 2005; Wie-Tao et al., 2011). In autumn a decrease was observed in proline content of

lemongrass. In lemongrass leaves, greater free proline levels in summer months and its drop

to normal levels in spring and autumn season strongly witnessed its accumulation as a

protective response. Upon relief from stressful conditions, it readily disappeared, which has

been assigned to its breakdown in oxidative phosphorylation (Hare et al., 1998) and use as a

reserve substance in chlorophyll synthesis (William and Sharon, 1981).

Glycinebetaine is another osmoprotectant of great biological significance to plants,

since it has been reported to improve the cell water balance during adverse environmental

conditions (Ashraf and Foolad, 2007; Wahid et al., 2007) and it stabilizes the enzyme and

proteins at higher temperature (Kishitani et al., 1994; Allard et al., 1998). In the present study,

seasonal changes had great impact on GB content of lemongrass. Higher concentration of GB

was detected in summer (May-June) and winter periods (Dec-Jan) preferentially in penultimate

leaf and then in middle leaf while no such seasonal changes were notable in the bottom leaf (Fig.

4.10). Fluctuations in the levels of GB with the changing environmental conditions showed that

its accumulation is predominantly in response to stress. Such changes make the GB as an

osmoprotectant of great value in the tissues showing its accumulation (Boscaiu et al., 2011).

The validity of above changes in the osmoprotectants accumulation was ascertained by

drawing their mutual correlations and with the meteorological attributes (Table 4.4). Close

correlations GB with TFAA and free proline indicated that these nitrogenous compounds co-

occurred to perform a concerted action in younger leaves. However, positive correlations of

TFAA, free proline and GB with high temperature indicated their specific implication. The

summer season in Pakistan is too harsh; sometimes shooting the temperatures to over 50oC,

while the winter months are relatively mild (winter temperature 2-4oC). So the accumulation of

these osmoprotectants is important to maintain the cell water balance and metabolic functions at

the required pace, especially in actively metabolizing tissues (Rasheed et al., 2011).

In crux, prevailing environmental conditions appear to be real determinant of

osmoprotectants accumulation in leaves of different ages. The patterns of osmolytes

accumulation in the younger leaves are of great physiological significance in the survival of

lemongrass in different seasons.

56

4.5 Secondary metabolites

In plants the relative proportion and total content of secondary metabolites can vary due

to seasonal and daily variation. Seasonal variation may lead to the synthesis of specific

secondary metabolites. In this part of the manuscript, lemongrass leaves were studied for

modulation in the level of soluble alkaloids, saponins, tannins, soluble phenolics, flavonoids and

anthocyanins, and their possible associations with seasonal variations.

4.5.1 Results

Statistical significance for the changes in the accumulation of various secondary

metabolites determined in the leaves of three ages of lemongrass over months is presented in

Table 4.5

Table 4.5: Analysis of variance of data (mean squares) for secondary metabolites accumulation in the lemongrass leaves under the influence of seasonal condition

SOV df Alkaloids Saponin Tannin Phenolics Flavonoid Anthocyanins

Block (B) 2 476.67 0.31 7.61 25.96 0.08 0.001

Leaves (L) 2 79275.51** 872.32** 29974.02** 2343.09** 78.48** 0.253**

Months (M) 11 205.23ns 4.90** 451.63** 1132.26** 8.71** 0.193**

L × M 22 168.61ns 2.46** 307.31** 973.40** 1.14** 0.102**

Error 70 119.00 0.36 7.62 598.97 0.10 0.005

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels, respectively

57

a. Alkaloids

The data revealed significant differences in the leaves but a non-significant difference in

the months as well as there was no interaction of these factors for alkaloids concentration (Table

4.5). The alkaloids contents were lowest in the penultimate leaf while the bottom leaf indicated

their highest synthesis. A comparison of data revealed that for penultimate and middle leaves

although there were not much fluctuations in the alkaloids accumulation across the seasons,

nevertheless summer season (May-Jul) induced the alkaloids accumulation. The bottom leaves

indicated greater changes in the alkaloids accumulation with the changes in the seasonal

conditions (Fig. 4.11).

Fig. 4.11: Effect of seasonal variation on alkaloids concentration in the penultimate, middle and

bottom leaves of lemongrass

bc c abc aba

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58

b. Saponins

Analysis of variance revealed significant differences in the leaves of three ages and

sampling months along with a significant interaction of these factors for the saponin contents

(Table 4.5). A comparison of leaves indicated not much difference in the saponin contents in the

penultimate and middle leaves but there was a large difference in the bottom leaf, which

displayed 2-3 times higher saponin contents. A comparison of sampling months revealed that

penultimate and middle leaves indicated slightly increased saponin contents in summer months,

which declined in the autumn season. Contrarily, bottom leaf showed much of the saponins

accumulation in spring and autumn seasons (Fig. 4.12).

Fig. 4.12: Effect of seasonal variation on saponins concentration in the penultimate, middle and

bottom leaves of lemongrass

cab bc bc ab a abc

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59

c. Tannins

Statistical analysis of results indicated significant difference in the leaves of different

ages and sampling months with significant interactions of these factors for tannins content (Table

4.5). The leaves indicated differential accumulation of tannins over various sampling months. In

penultimate leaf, the tannins contents were higher in the spring and autumn months while

reduced in the summer months. In middle leaf, both spring and summer seasons indicated greater

tannins accumulation, which declined in the autumn and winter seasons. In bottom leaf, there

was no clear trend of tannins accumulation over the seasons since large fluctuations were

observed in their contents. Overall penultimate leaf indicated the lowest while the bottom leaf

the highest tannins accumulation (Fig. 4.13).

Fig. 4.13: Effect of seasonal variation on tannins concentration in the penultimate, middle and

bottom leaves of lemongrass

fg fgab

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May Jun

Jul

Aug Sep Oct

Nov

Dec Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec

Penultimate Middle Bottom

Tan

nins

(μg

/g f

resh

wei

ght)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

60

d. Soluble phenolic

The soluble phenolic contents in different leaf positions of lemon grass varied

significantly (P<0.001) in different seasons. Also there was significant interaction of leaves and

sampling months for soluble phenolics accumulation (Table 4.5). In both penultimate and middle

leaves, the soluble phenolics were low in the spring season, which showed a substantial increase

in the summer season. In the autumn season, there was a decline in the contents of soluble

phenolics, which again showed an increase at the onset of winter season, although this increase

was much lesser than that observed in summer season. In bottom leaf the soluble phenolics

accumulation was substantially lower than the penultimate and middle leaf, although this leaf

also showed their marked accumulation in the summer and autumn season (Fig. 4.14).

Fig. 4.14: Effect of seasonal variation on soluble phenolics content in the penultimate, middle

and bottom leaves of lemongrass

bc cdcd

cda a

ab

bcd d d

bc

bcd

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a aba

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Penultimate Middle Bottom

Sol

uble

phe

nolic

s (μ

g/g

fres

h w

eigh

t)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

61

e. Flavonoids

Data indicated significant difference in the leaves and sampling months of lemongrass

with a significant interaction of these factors for the flavonoid contents (Table 4.5). Seasonal

variation in the concentration of flavonoids irrespective of leaf age were quite significant. The

highest (p<0.001) concentration of flavonoids were observed in the summer season i.e. in May-

Jul in case of penultimate and middle leaves while in Jun in bottom leaves. However, the lowest

values were observed in spring and autumn seasons. Overall, penultimate leaf exhibited the

greatest flavonoids accumulation followed by middle leaf (Fig. 4.15).

Fig. 4.15: Effect of seasonal variation on flavonoids content in the penultimate, middle and

bottom leaves of lemongrass

aab

cdd

abc ab ab

ce e

ded

abc

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cef

da

b

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Dec

Penultimate Middle Bottom

Fla

vono

ids

(μg/

g fr

esh

wei

ght)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

62

f. Anthocyanins

The anthocyanin contents in different leaves of lemon grass varied significantly in

different sampling times with a significant interaction of these factors (Table 4.5). Pattern of

anthocyanins synthesis was more or less similar in the penultimate and middle leaves, while

bottom leaf deviated considerably. The anthocyanins concentration showed a decrease from the

spring (Mar-Apr) season and reached the lowest value in summer (May-Jun) season. The

anthocyanins showed an increase with the onset of autumn (Sep-Oct) season and attained the

highest value in Oct (1.194 A535). On the other hand, bottom leaf although showed much lower

anthocyanins than the penultimate and middle leaves (Fig. 4.16).

Fig. 4.16: Effect of seasonal variation on anthocyanin’s concentration in the penultimate, middle

and bottom leaves of lemongrass

bcbc

dde

efe

f efc

ab

bc

cd cef

deg

hef

de

ab b

c

cbc bc

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cdc

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bcc

0

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Dec Jan

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Mar

Apr

May Jun

Jul

Aug Sep Oct

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Dec

Penultimate Middle Bottom

Ant

hocy

anin

s (A

535)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

63

g. Correlation

The interrelationships drawn of the secondary metabolites in three lemongrass leaves

indicated that in penultimate leaf, the saponins were positively related with phenolics but

negatively with anthocyanins. Tannins were negatively correlated with flavonoids, while soluble

phenolics were negatively correlated with flavonoids and anthocyanins. The phenolics were

positively correlated with flavonoids but negatively with tannins. For middle leaf, the saponins

were negatively related to anthocyanins while for bottom leaf alkaloids were negatively

associated with anthocyanins. In case of environmental attributes, for penultimate leaf,

anthocyanins indicated negative correlation with maximum and minimum temperature, relative

humidity was negatively related to alkaloids, while evapotranspiration was positively correlated

with alkaloids and soluble phenolics but negatively with anthocyanins. For middle leaf, relative

humidity was negatively correlated with alkaloids and evapotranspiration was positively

correlated with tannins and negatively with anthocyanins. In bottom leaf, soluble phenolics

contents were positively correlated with maximum and minimum temperature and

evapotranspiration (Table 4.6).

4.5.2 Discussion

Plant secondary products are formed via distinctive metabolic pathways and act as line of

defense against biotic and abiotic adversaries (Winkel-Shirley, 2002; Aflatuni, 2003; Wahid

and Tariq, 2008). However, thorough studies are scarce with respect to seasonal changes in the

concentrations of plant secondary products in plants, although sporadic reports are available (Ma

et al., 2003; Maknickiene and Asakaviciute, 2008). In the present research, lemongrass leaves of

three ages were investigated for nitrogen-containing and non-nitrogen containing secondary

metabolites on monthly intervals.

Alkaloids are a diverse group of nitrogen-containing compounds, which play role against

herbivores. Pencikova et al. (2011) reported a rise in the alkaloids contents in the leaves of

Maccleaya microcorpa during spring season as compared to autumn season. In the present

research the penultimate and middle leaves indicated a substantially reduced alkaloids and

saponins contents over the seasons (sampling months), whilst the bottom leaf indicated the

greatest accumulation. Despite that, leaves of all the ages exhibited higher contents of both

64

Table 4.6: Correlation coefficient (r) of meteorological attributes with the secondary metabolites of lemongrass at three leaves positions penultimate, middle, bottom X –Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of secondary metabolites Alkaloid Saponin -0.474ns -0.311ns 0.056ns Tannin 0.046ns 0.039ns 0.190ns Phenolics 0.449ns -0.114ns 0.026ns Flavonoid -0.077ns -0.140ns -0.468ns Anthocyanins -0.217ns -0.283ns -0.890** Saponin Tannin -0.140ns 0.107ns -0.244ns Phenolics 0.742** 0.224ns 0.304ns Flavonoid 0.178ns 0.192ns 0.046ns Anthocyanins -0.776** -0.723** 0.182ns Tannin Phenolics -0.152ns -0.390ns -0.126ns Flavonoid -0.659* -0.152ns -0.300ns Anthocyanins -0.257ns 0.102ns -0.405ns Phenolics Flavonoid 0.610* 0.562ns -0.176ns Anthocyanins -0.850** -0.365ns 0.667ns Flavonoid Anthocyanins -0.289ns -0.412ns -0.159ns b. Correlations environmental variable with secondary metabolites Max. temprature Alkaloid 0.473ns 0.468ns 0.062ns Saponin -0.214ns 0.266ns 0.242ns Tannin 0.557ns -0.252ns -0.286ns Phenolics 0.343ns 0.148ns 0.873** Flavonoid -0.368ns -0.447ns -0.367ns Anthocyanins -0.642* -0.433ns -0.132ns Min. temprature Alkaloid 0.227ns 0.219ns 0.055ns Saponin -0.114ns 0.389ns 0.404ns Tannin 0.546ns -0.354ns -0.424ns Phenolics 0.291ns 0.173ns 0.852** Flavonoid -0.311ns -0.444ns -0.171ns Anthocyanins -0.691* -0.359ns -0.018ns Relative humidity Alkaloid -0.871** -0.773** -0.034ns Saponin 0.210ns -0.046ns 0.256ns Tannin -0.151ns -0.323ns -0.293ns Phenolics -0.460ns 0.031ns -0.277ns Flavonoid 0.091ns 0.021ns 0.012ns Anthocyanins 0.286ns 0.563ns -0.035ns Evapotranpiration Alkaloid 0.656* 0.511ns -0.289ns Saponin -0.301ns 0.424ns 0.109ns Tannin 0.411ns 0.626* -0.192ns Phenolics 0.698* 0.210ns 0.735** Flavonoid -0.138ns -0.207ns 0.009ns Anthocyanins -0.724** -0.670* -0.422ns Average rainfall Alkaloid -0.336ns -0.297ns -0.229ns Saponin 0.065ns 0.416ns 0.369ns Tannin 0.271ns -0.241ns -0.379ns Phenolics -0.026ns 0.133ns 0.520ns Flavonoid -0.090ns -0.312ns -0.089ns Anthocyanins -0.438ns 0.101ns 0.555ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

65

alkaloids and saponins during summer (Fig. 4.11-4.12). The differences seen in the accumulation

of these nitrogen-containing products revealed that the younger leaves of lemongrass do not

preferentially induce the synthesis of alkaloids and saponins because of their no special

contribution in the primary growth responses. On the other hand, old (bottom) leaf indicated

quite higher accumulation of both these metabolites, which might be related to their age, since

aging induced saponins synthesis in Tamarix aphylla (Achakzai et al., 2009).

Phenolics are a large class of compounds, and are synthesized via the shikimic acid

pathway. Major classes of phenolics include lignins, tannins, flavonoids and anthocyanins. Being

water soluble, the synthesis and accumulation of phenolics is of great physiological significance

(Taiz and Zeiger, 2010). Prevailing growth conditions have great influence on their synthesis

(Sgherri et al., 2004; Wahid and Tariq, 2008), while the accumulation in various plants are not

well studied. Determinations made for tannins, soluble phenolics, flavonoids and anthocyanins

on the leaves of three ages of lemongrass revealed that seasonal changes had great influence on

the patterns and levels of their accumulation. Summer season did not induced the synthesis of

tannins in young leaves, while their synthesis increased with advancing leaf age (Fig. 4.13),

while soluble phenolics (Fig. 4.14) and flavonoids (Fig. 4.15) indicated greater synthesis in the

summer season, which declined to normal levels in spring and autumn season. Contrarily, the

anthocyanins synthesis took place only in the winter season, but severely declined in the summer

months (Fig. 4.16). The phenolics are synthesized after the elimination of ammonia with the

phenylalanine ammonia lyase (PAL) enzyme, the activity of which is much dependent on the

biotic and abiotic stress conditions (Collinge and Slusarenko, 1987; Wu and Lin, 2002). The

synthesis of various phenolics compounds takes place with the branching of pathways from the

trans-cinnamic acid for the synthesis of a variety of phenolics compounds (Adeyemi, 2011). In

this research, the synthesis of higher amounts of soluble phenolics and flavonoids in the younger

leaves in the summer and winter seasons suggested that these metabolites have definitive

physiological roles under adverse conditions, and the anticipated roles might be the osmotic or

antioxidative. However, a higher synthesis of tannins (with complex structure and water

insoluble) in the lemongrass leaves with the advancing age indicated that they have no-specific

physiological roles and plausibly leaf aging induces their synthesis. The anthocyanins are

reported for their greater roles in the reddening during winter season (Chalker-Scott, 1999),

66

Metabolic pathways in plants are regulated in a coordinated manner to protect plants

when adverse conditions occur during the year. To look into the possibility of their synthesis in

various seasons, mutual correlations of secondary metabolites and their association with the

seasonal conditions prevailing during various months of the year were established. It was noted

that for penultimate leaf most of the secondary metabolites were co-accumulated, which was not

the case for middle or bottom leaf, thereby showing that (actively metabolizing leaves

strategically synthesize the metabolites to ensure survival as a coordinated way. A greater

synthesis of the physiologically active phenolic compounds in summer seasons indicated that

accumulation of soluble phenolics and flavonoids is more important in dehydrating summer

conditions while anthocyanins synthesis was mainly related to the winter season.

In conclusion, seasonal variation has great impact on qualitative and quantitative contents

of secondary metabolites in lemongrass. The alkaloid, phenolic and flavonoid contents in

lemongrass leaves exhibited a substantial increase with increasing temperature while the

synthesis of anthocyanins was relatively better in winter season. These trends of secondary

metabolites accumulation have great implications in the survival of actively metabolizing tissues

under the adverse seasonal conditions.

67

4.6 Vitamins

The seasonal variation is related to changes in rain fall, light and temperature that affect

vitamins metabolism in leaves of lemongrass. Vitamins are organic substances which act as

coenzyme (NAD, NADP and FAD) in many oxidation-reduction reactions. They also act as

antioxidant and protect membrane and other hydrophobic compartments from damage. In this

part of the thesis, the seasonal changes were studied in niacin, ascorbic acid, riboflavin and

reducing powers assay (RPA), in the lemongrass leaves of three ages, the results of which are

given below.

4.6.1 Results

Leaf age and seasonal variations indicated significant differences in the concentrations of

vitamins and reducing powers assay. Statistical analyses of results of these parameters are given

in Table 4.7.

Table 4.7: Analysis of variance of data (mean squares) for niacin, ascorbic acid, riboflavin and reducing power assay of lemongrass leaves under the influence of seasonal condition

SOV df Niacin ASA Riboflavin RPA

Block (B) 2 0.001 0.023 0.018 0.018

Leaves (L) 2 32.753** 84.161** 24.940** 6.867**

Months (M) 11 0.766** 0.297** 0.329** 1.012**

L × M 22 0.505** 0.526** 0.111** 0.152**

Error 70 0.005 0.062 0.021 0.027

Significant at: ns, non-significant and **, significant at P<0.01 levels

68

a. Niacin

The data presented that there was a tendency of lemongrass niacin concentrations to

display changes in its levels during the sampling months and leaves of three ages with a

significant interaction of these factors (Table 4.7). The niacin exhibit that penultimate leaf had

higher levels (3.22 µg g-1) among the leaves. The niacin contents in penultimate leaf were

maximum in summer months (June and July, 3.11 and 3.22 µg/g, respectively) while remained

mimum in September (1.31 µg/g) and October (1.24 µg/g). In the middle leaf the highest value

of niacin was noticed in June and July whereas comparable to each other in the remaining

months (11.39 µg g-1). The bottom leaf exhibited entirly different behavior. Net accumulation of

niacin was very low and did not change much over the months. Overall, the niacin contents were

the greatest in penultimate leaf follwed by middle leaf (Fig. 4.17).

Fig. 4.17: Effect of seasonal variation on Niacin concentration in the penultimate, middle and

bottom leaves of lemongrass

bcc

ffg

ba a

fg g g

ed

cd

ed

bca

ab

ede

c bc

b b bc bcd

ab a ab bc bcd

bcd

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0

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3.5

Jan

Feb

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Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec

Penultimate Middle Bottom

Nia

cin

(μg/

g fr

esh

wei

ght)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = ns

69

b. Ascorbic acid (AsA)

There was significant difference in the leaves of different ages and sampling months with

significant interaction of these factors for the ascorbic acid contents (Table 4.7). The AsA

contents in penultimate leaves varied throughout the year. Penultimate leaf AsA contents

decreased from Jan to Apr and then sharply increased in May and remained same till Jul, while

gradually decreased during the rainy season and trend was continued till Oct followed by further

increased Nov and Dec. A similar trend was observed in middle leaf but the contents was quite

low throughout the years. As for bottom leaves, the AsA content was low and there were no

marked changes in AsA except a decline in the summer months (Fig. 4.18).

Fig. 4.18: Influence of seasonal condition on Ascorbic acid concentration in the penultimate,

middle and bottom leaves of lemongrass

ab bccd

cd

a aa

bcd

cd dbc

ab

abc

bccd

abc ab a

bccd cd

abc ab

bcd

bcd

edc

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ef fde

a abc

abcd

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Penultimate Middle Bottom

Asc

orbi

c ac

id (

μg/g

fre

sh w

eigh

t)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

70

c. Riboflavin

The leaf age and prevailing seasonal conditions indicated significant differences for the

contents of riboflavin with a significant interaction of these factors (Table 4.7). The riboflavin

contents indicated sharp changes during the entire year in penultimate leaf, being highest in

summer (May, Jun and Jul) and serially reduced thereafter and were the lowest in Oct. The

winter conditions again caused them to accumulate. The middle leaf also showed the changes

more or less similar to the penultimate leaf but the riboflavin contents were substantially low.

The bottom leaf indicated no marked changes in the riboflavin contents throughout the years

some increase in the spring and autumn months. Overall, the penultimate leaf synthesized the

highest amount of riboflavin followed by middle leaf (Fig. 4.19).

Fig. 4.19: Effect of seasonal variation on Riboflavin concentration in the penultimate, middle and

bottom leaves of lemongrass

bde

eff

da

bccd de

fde

ab

cde

f f def

ba

def

def

def

efcd

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cd def bc ab

f0

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Penultimate Middle Bottom

Rib

ofla

vin

(μg/

g fr

esh

wei

ght)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

71

d. Reducing power assay (RPA)

Results for the RPA indicated significant differences in the leaves of various ages and the

sampling months with a significant interaction of leaves and months (Table 4.7). For penultimate

and middle leaves, the value of RPA was relatively lower in the spring season (Mar-Apr), which

increased appreciably in the summer months and reached its maximum in Jun. This value

declined subsequently and was the lowest again in autumn season and exhibited a decline in the

winter months also. However, the middle leaf indicated relatively sharper changes in RPA value

than the penultimate leaf. In case of bottom leaf, no specific pattern of RPA value was evident

although it was relatively higher in spring season and lower in summer months. Overall, the

middle leaf indicated a higher RPA value followed by penultimate leaf (Fig. 4.20).

Fig. 4.20: Effect of seasonal variation on reducing power assay concentration in the penultimate,

middle and bottom leaves of lemongrass

bcbc

a

a

bbc

dcd

bc b bccd

cbc bc

aab

ce e

d

ab bce e

de

abb

acd

cde

e ecd c c

e0

0.5

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Penultimate Middle Bottom

Red

ucin

g po

wer

s as

say

(A70

0)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

72

e. Correlation

To find the implication of the changes in vitamin attributes some correlations were

established at different leaf positions of lemongrass. In the penultimate and middle leaves,

contents of all the vitamins were correlated with each other while for bottom leaf these

relationships were absent altogether. As for meteorological variables, in penultimate leaf, RPA

was positively related with maximum and minimum temperatures and evapotranspiration, while

the vitamins or RPA of middle and bottom leaves were correlated to none of the meteorological

attributes.

Table 4.8: Correlation coefficient (r) of meteorological attributes with the Vitamin content of lemongrass at three leaves positions penultimate, middle, bottom

X-Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of Vitamins Niacin AsA 0.909** -0.756** -0.404ns Riboflavin 0.804** 0.865** -0.516ns RPA 0.601* -0.706* 0.064ns AsA Riboflavin, 0.892** -0.898** 0.348ns RPA 0.586* -0.571* -0.133ns Riboflavin RPA 0.657* -0.750** 0.317ns b. Correlations environmental variable with Vitamins Max. temperature Niacin -0.007ns 0.378ns 0.160ns AsA 0.215ns -0.063ns -0.333ns Riboflavin 0.145ns 0.186ns -0.514ns RPA 0.656* 0.671* -0.236ns Min. temperature Niacin 0.026ns 0.376ns 0.327ns AsA 0.202ns -0.037ns -0.227ns Riboflavin 0.219ns 0.193ns -0.519ns RPA 0.587* 0.162ns -0.282ns Relative humidity Niacin -0.064ns -0.166ns 0.353ns AsA -0.249ns -0.039ns 0.543ns Riboflavin 0.073ns -0.134ns 0.009ns RPA 0.225ns -0.540ns -0.186ns Evapotranspiration Niacin 0.021ns 0.181ns 0.479ns AsA 0.058ns -0.037ns 0.075ns Riboflavin 0.297ns 0.165ns -0.531ns RPA 0.655* 0.005ns -0.330ns Average rainfall Niacin 0.210ns 0.448ns 0.208ns AsA 0.417ns 0.128ns -0.610ns Riboflavin 0.251ns 0.374ns -0.558ns RPA 0.438ns 0.488ns -0.102ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

73

4.6.2 Discussion

Vitamins are important enzymatic cofactors play important role in plant metabolism

because of their redox chemistry. However, there are various factors such as plant age and

prevailing conditions that greatly influence the vitamins contents and metabolic roles (Denslow

et al., 2007; Mahmood et al., 2012). In the current research, the determinations made on the

lemongrass leaves in various seasons revealed that young leaves showed greater contents of

niacin (Fig. 4.17), AsA (Fig. 4.18) and riboflavin (Fig. 4.19), while old (bottom) leaf did not

show their accumulation with the seasonal changes during two years. Furthermore, RPA

increased highly in the summer season followed by winter season while decreased markedly in

spring and autumn seasons (Fig. 4.20).

The vitamins play various metabolic roles such as coenzymes in enzyme activities (Taiz

and Zeiger, 2010) and alleviation of oxidative stress (Mahmood et al., 2012). The role of niacin

is to function as coenzyme in the regulation of carbohydrate metabolism. Riboflavin and its

derivatives FMN and FAD are indispensable components of photosynthesis, energy generation

and redox metabolism in plants under normal conditions (Sandoval et al., 2008). Under abiotic

stresses too, different vitamins are involved in alleviating the stress effects and better plant

survival (Demmig-Adams and Adams, 2002; Rapala-Kozik et al., 2008; Leuendorf et al., 2010).

A higher contents of these vitamins in the penultimate and to some extent in middle leaf of

lemongrass in the summer and winter seasons strongly suggests the maintenance of metabolic

activities under the changing seasons. Secondly, leaf age is a factor that also determines the

contents of vitamins and thus the operation of metabolic activities. The contents of all the

vitamins were substantially low in aged bottom leaf, which further suggested that as a metabolic

requirement, younger tissues have a greater demand for the vitamins than the aged tissues.

Generation of reducing powers in appropriate concentrations is important for regulation

of cellular metabolic functions. Therefore, availability of reducing powers under adverse

conditions is critical. A comparison of data drawing the correlations of the vitamins and RPA

values indicated that all were positively and tightly interrelated in penultimate and middle leaves

but not in bottom leaf (Table 4.8). This is important in view of the fact that younger tissues are

74

the sites of major metabolic activities while bottom (aged) leaves have lived their life and are

heading towards senescence, where there is least requirement for these metabolites.

Among the environmental variables, a positive relationship of RPA with maximum and

minimum temperatures and evapotranspiration in case of penultimate leaf and a positive

relationship of RPA with maximum temperature in middle leaf (Table 4.8) revealed that

generation of reducing powers is much important under high temperature and evapotranspiration

load (again related to high temperature) in young actively metabolizing leaves of lemongrass. Its

plausible role may be the provision of energy for the enzymatic activities triggered by the

vitamins (Rapala-Kozik et al., 2008; Leuendorf et al., 2010).

It summarized that an increase in vitamin contents in the lemongrass leaves is strongly

dependent upon the prevailing conditions while the hot and dry conditions are more conducive to

their synthesis. This might also be possibly involved in the alleviation of oxidative load on the

lemongrass leaves especially on the younger leaves.

75

4.7 Photosynthetic Pigment

Photosynthetic pigments are integral compound of light harvesting systems in plants. Any

distortion induced in the light harvesting ability due to ambient factors declines the

photosynthetic activity of plants. Results indicated considerable changes in the photosynthetic

pigments of lemongrass in various seasons. The results of chl a, chl b, their total chl and

carotenoids are described below:

4.7.1 Results

The results regarding the photosynthetic pigments contents in the leaves of three ages and

in different seasons have been presented in Table 4.9.

Table 4.9: Analysis of variance of data (mean squares) for chlorophyll a and chlorophyll b, total chlorophyll and carotenoid contents of lemongrass under influence of seasons SoV df Chl a Chl b Total Chl Car

Block (B) 2 0.02 0.01 0.38 0.010

Leaf (L) 2 20.92** 2.89** 16.62** 1.693**

Month (M) 11 1.45** 0.26** 3.89** 0.121**

L × M 22 0.28** 0.03* 0.90** 0.021**

Error 70 0.10 0.02 0.24 0.004

ns = non-significant; *, **, *** = significant at 0.05, 0.01 and 0.001 levels, respectively

76

a. Chlorophyll (Chl) a

Significant differences were noted in the sampling months and leaves of lemongrass with

a significant interaction of these factors for Chl-a contents (Table 4.9). Penultimate and middle

leaves indicated a lower contents of Chl-a in the spring (Feb-Apr) and autumn season (Aug-Oct),

while it was relatively higher in summer and winter seasons. On the other hand, bottom leaf

indicated higher Chl-a in the cool season (Nov-Feb), which declined in spring season and again

slightly increased in the summer season. Overall, Chl-a content was substantially higher in

penultimate and middle leave while it was much lower in the bottom leaf (Fig. 4.21)

Fig. 4.21: Effect of seasonal variation on chlorophyll a concentration in the penultimate, middle

and bottom leaves of lemongrass

vcxz

cdab

acd

d dc

aa ab ab

c

a ab b ccd cd cd

aba a

cb

ab aab ab

cbc

dcd d cd

abc ab a

ab

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Aug Sep Oct

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Penultimate Middle Bottom

Chl

orop

hyll

a (m

g/g

fres

h w

eigh

t)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

77

b. Chlorophyll b (point 46 of 2nd reviewer?)

Results indicated significant difference in the leaves of various ages and sampling months

with significant interaction of the factors for leaf Chl-b contents (Table 4.9). In penultimate leaf,

the Chl-b contents was low in winter season (Nov-Jan), which indicated a slight increase in the

summer months (May-July) but declined in the autumn months (Aug-Sep). A trend more or less

similar to the penultimate leaf was noted in middle leaf. The bottom leaf indicated no significant

fluctuations in the Chl-b contents over all seasons. Overall, penultimate and middle leaf

exhibited similar Chl-b contents while it was substantially lower in bottom leaf (Fig. 4.22)

Fig. 4.22: Effect of seasonal variation on chlorophyll b concentration in the penultimate, middle

and bottom leaves of lemongrass

dbc ab

bcab

cd cda

abc

cd cd

bc bb

c cab

aab

bc c c

aab

bcc

cb

c c c c c c

0

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Jan

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Feb

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Aug Sep Oct

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Penultimate Middle Bottom

Chl

orop

hyll

b (m

g/g

fres

h w

eigh

t)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

78

c. Total chlorophyll (Chl-T)

The two-way analysis of variance of the data indicated significant differences in the

leaves of various ages and sampling months with significant interaction of these factors for total

chlorophyll contents of leaves (Table 4.9). The Chl-T of the penultimate leaf decreased from Jan

to Apr but increased thereafter up to Jul. It again decreased in Aug-Sep and displayed a gradual

increase up to Dec. The middle leaf indicated the changes in Chl-T similar to those of

penultimate leaf except the Chl-T was relatively lesser in Dec-Jan. The bottom leaf indicated a

higher Chl-T in Jan-Feb, which declined in March and was steadier up to Jul. It showed a slight

increase in Aug but reduced in Sep and remained steady up to Dec. Overall, the Chl-T was

comparatively higher in middle leaf followed by penultimate leaf but the lower in bottom leaf

(Fig. 4.23).

Fig. 4.23: Effect of seasonal variation on total chlorophyll contents in the penultimate, middle

and bottom leaves of lemongrass

cab

ac cd cdc

aab

bc bcd

bcd

abc

bcd

cdd cd

bcd

ab aab

cdbc

d

a abcd

efde

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gef

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Penultimate Middle Bottom

Tot

asl c

hlor

ophh

yll (

mg/

g fr

esh

wei

ght)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

79

d. Carotenoids (Car)

Lemongrass leaves of different ages and sampling months showed significant differences,

also with a significant interaction of these factors for Car contents (Table 4.9). In penultimate

leaf the Car contents were lower from Jan up to Mar, which increased thereafter and attained the

highest value in June. Car showed a decreasing trend thereafter and was the lowest in Oct, and

then an increase in Nov and Dec. A more or less trend similar to penultimate leaf was observed

in middle leaf for changes in Car. On the other hand, bottom leaf indicated higher Car in Jan,

which declined up to March. It showed a steady Car contents up to Oct but a slight increase in

Nov-Dec. Overall, Car contents were relatively higher in penultimate and middle leaf while it

was quite low in the bottom leaf (Fig.4.24).

Fig. 4.24: Effect of seasonal variation on carotenoid concentration in the penultimate, middle and

bottom leaves of lemongrass

dbc

a abc

dcd

dbc

bcd

cd

decd

ba

cde e

decd

bcde de

cdb

acd

ef

def

efcd bc

def

ef f0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec

Penultimate Middle Bottom

Car

oten

oids

(m

g/g

fres

h w

eigh

t)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

80

e. Correlation

The correlations of leaf pigments showed that in penultimate leaf, the Chl-a, Chl-T and

Car were positively interrelated. For middle leaf, Chl-a was positively related to Chl-T, while

Chl-b and Chl-T were positively associated to Car. For bottom leaf, no pigments were

interrelated. As for meteorological attributes, in penultimate leaf the maximum temperature and

evapotrasnspiration were positively related to Chl-a, Chl-T and Car. For middle and bottom

leaves, none of the meteorological attributes were correlated with pigment contents (Table 4.10).

Table 4.10: Correlation coefficient (r) of meteorological attributes with the Pigment content of lemongrass at three leaves positions penultimate, middle, bottom X-Variable Y-Variable Penultimate Middle Bottom a. Mutual correlations of pigments Chl-a Chl-b 0.274ns -0.079ns 0.451ns Chl-T 0.976** 0.910** -0.002ns Car 0.721** 0.421ns -0.126ns Chl-b Chl-T 0.476ns 0.341ns 0.212ns Car 0.418ns 0.580* -0.329ns Chl-T Car 0.753** 0.614* 0.208ns b. Correlations environmental variable with pigments Max. temperature Chl-a 0.593* -0.259ns -0.300ns Chl-b 0.223ns 0.237ns -0.248ns Chl-T 0.593* -0.145ns -0.358ns Car 0.622* 0.145ns -0.058ns Min. temperature Chl-a 0.553ns -0.264ns -0.179ns Chl-b 0.092ns 0.076ns -0.202ns Chl-T 0.527ns -0.218ns 0.227ns Car 0.650ns 0.063ns -0.183ns Relative humidity Chl-a -0.280ns 0.061ns -0.427ns Chl-b -0.524ns 0.549ns -0.289ns Chl-T -0.374ns -0.227ns 0.493ns Car -0.246ns -0.280ns -0.244ns Evapotranspiration Chl-a 0.590* 0.114ns -0.297ns Chl-b 0.213ns -0.514ns -0.412ns Chl-T 0.648* -0.106ns -0.397ns Car 0.691* 0.416ns -0.010ns Average rainfall Chl-a 0.302ns 0.076ns -0.059ns Chl-b -0.213ns 0.418ns -0.262ns Chl-T 0.230ns -0.246ns -0.125ns Car 0.504ns -0.030ns -0.131ns

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

81

4.7.2 Discussion

Lemongrass is a C4 plant and can grow well under relatively higher ambient

temperatures. Although no report is available which may show any direct relationship with a

reduction in plant dry matter yield, this fact also cannot be delineated. This is because

photosynthetic rate of the plant is dependent upon the light harvesting efficiency (Ruban, 2009).

The leaves of different ages show differential photochemical efficiency and thus likely to have

differential contribution to the plant performance (Kitajima et al., 2002). In this research, the

determinations made the photosynthetic pigment concentration in the leaves of three ages

indicated that relatively younger (penultimate and middle) leaves indicated substantially greater

pigments of Chl-a (Fig. 4.21), Chl-b (Fig. 4.22), Chl-T (Fig. 4.23) and Car (Fig. 4.24) all round

the year, while older leaf was on a disadvantage. However, it is important to note that during

summer season there was an increase in the contents of all the photosynthetic pigments. The

results of this research are contrary to the earlier reports, wherein it has been reported that high

light intensity induced photo-oxidative damage is a major deterrent of the chlorophyll species

while the carotenoids are able to sustain their contents (Ramel et al., 2013). This appears to be a

better adaptation to the supra-optimal ambient temperatures in the summer month.

It is important to note that the Car contents of the young leaves remained fairly high

during the summer season while they were in the normal range during the autumn, winter and

spring seasons (Fig. 4.24). The Car in addition to their role as accessory light harvesting

pigments, are also effective scavenger of the ROS produced during adverse conditions (Ramel et

al., 2012). It is plausible that production of ROS is efficiently doused by the high contents of Car

especially in the chloroplast of younger (penultimate and middle) leaves, and protecting the

photosynthetic pigments in the photosystems. Furthermore, the association of both the

chlorophyll may be differential.

To appraise the validity of the above observations, some correlations were established of

the photosynthetic pigments mutually as well as with the prevailing meteorological conditions

round the year (Table 4.10). These correlation indicated that the contents of both the chlorophyll

species (Chl-a and b) were independent of each, rather Chl-a and Chl-T were closely associated

to the Car contents. These data further strengthened the notion that the Car was the important

element in rescuing the younger leaves from the harsh conditions of summer. Likewise, in the

82

penultimate leaf the Chl-a, Chl-T and the Car were positively correlated with the maximum

temperature and evapotranspiration throughout the year, which further showed that the

maintenance of greater chlorophyll contents was mainly related to the maintenance of Car, while

the older (bottom) leaf was on a disadvantage due to being having low photosynthetic efficiency

and probably heading towards senescence.

In conclusion, seasonal variation in meteorological attributes affects the photosynthetic

pigments levels in lemongrass leaves. Greater contents of Car and Chl-T suggested that the Car

played a major role in encountering the photo-oxidative damage and rescuing the photosystems

from the adverse conditions like those of heat stress and high evapotranspiration load.

83

4.8 Nutritional quality characteristics

Lemongrass shows a marked variation in nutritional profile during different seasons

which have been widely associated to variations in environmental variables. The variations in

such attributes usually appears to be greatly affected by rise in plant age as well as changes in

seasonal variation during different seasons. In this part of the dissertation, the seasonal changes

were studied in NDF, ADF, ADL, cellulose, silica, starch and protein contents in penultimate,

middle and bottom leafs of lemongrass.

4.8.1 Results

Various nutritional quality characteristics indicated significant changes in all the leaves

analyzed during the sampling months. The results regarding statistical analysis are given in Table

4.9.

Table 4.11: Analysis of variance of data (mean squares) for nutrient detergent fiber, acid detergent fiber, acid detergent lignin, cellulose, silica, starch and protein content of lemongrass under influence of seasonal condition

SoV df NDF ADL ADF Cellulose Silica Starch Protein

Block (B) 2 0.08 0.06 0.50 3.01 0.01 0.18 0.03

Leaf (L) 2 193.30** 67.42** 114.42** 4919.24** 21.70** 127.36** 381.17**

Month (M) 11 2.33** 1.09** 30.94** 46.68** 0.18** 9.69** 2.85**

L × M 22 2.01** 0.53* 7.43** 6.28** 0.04ns 2.66** 2.48**

Error 70 0.60 0.28 0.96 2.01 0.02 0.578 0.716

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

84

a. Nutrient detergent fiber (NDF)

Stsatistical analysis of results showed significant difference in the lemongrass leaves of

three ages and the sampling months for NDF contents with a significant interaction of these

factors (Table 4.11). In penultimate and middle leaf the NDF decreased from Jan and was the

lowest in May. It showed an increase from Jun and reached its highest in Aug, declined in Sep-

Nov and then increased in Dec to almost the level of Jan. On the other hand, in bottom leaf, the

NDF remained steady up to Aug except a small decline in Jun. However, it was the lowest in

winter months (Oct-Nov) but increased in Dec. Overall, NDF was higher in middle leaf, while

penultimate leaf values more or less comparable to bottom leaf (Fig. 4.25)..

Fig. 4.25: Effect of seasonal variation on Nutrient detergent fiber content in the penultimate,

middle and bottom leaves of lemongrass

bccd d cd

ed

ba

abc cd

bc

b bccd

d dc

ba b

cd cd c

abc

c abc abc

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ca a a

c cab

0

2

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Nov

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Feb

Mar

Apr

May Jun

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Nov

Dec

Penultimate Middle Bottom

ND

F (

%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = *

85

b. Acid detergent fiber (ADF)

Analysis of variance of the data for ADF indicated significant difference in lemongrass

leaves and sampling months with a significant difference of these factors (Table 4.11). In the

acid detergent fiber (ADF) contents between the evaluated seasons for penultimate middle and

bottom leaves indicated highly significant results (Table 4.11). With small differences, the trend

of ADF contents of penultimate, middle and bottom leaves were similar. ADF was generally low

from Jan to Apr. The ADF substantially increased in May-Jul (summer season) but declined

steadily in autumn and winter months. Overall, middle leaf indicated a greater ADF followed by

penultimate leaf (Fig. 4.26).

Fig. 4.26: Effect of seasonal variation on Acid detergent fiber content in the penultimate, middle

and bottom leaves of lemongrass

d dcd

db

a abbc

c cd cdd e

fe e

ba ab

c bc bc

d

debc

ede

a a abc c cd cd cd

0

5

10

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25

Jan

Feb

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Aug Sep Oct

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Apr

May Jun

Jul

Aug Sep Oct

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Dec Jan

Feb

Mar

Apr

May Jun

Jul

Aug Sep Oct

Nov

Dec

Penultimate Middle Bottom

AD

F (

%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

86

c. Acid detergent lignin (ADL)

Results revealed significant differnce in the leaves of lemongrass as well as sampling

months with significant interaction of both these factors for ADL (Table 4.11). It is evident from

the results that in all leaves, the ADL was low during winter season, which furher declined in the

spring season (Feb-Apr). The ADL increased from May and onward and was highest in Jul but

declined afterwards and was the lowest in Oct-Nov and increased again to the level dectected in

Jan. Overall, the ADL was the highest in bottom leaf but the lowest in penultimate leaf (Fig.

4.27).

Fig. 4.27: Effect of seasonal variation on Acid detergent lignin content in the penultimate,

middle and bottom leaves of lemongrass

c cd cd d cdbc

aab ab

cbc

dc

bc bc bcc

bca a a

bc bcb b

bcd

bcd

cdcd

bcd

ab abc

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dbc bc

d

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Dec

Penultimate Middle Bottom

AD

L (

%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

87

d. Cellulose

Statistical analysis of data indicated significant differences in the leaves of three ages and

sampling months with a significant interaction of both these factors for cellulose contents (Table

4.11). The penultimate leaf indicated the lowest contents of cellulose, which, with some

fluctuations remained steady throughout the year. The middle leaf showed lower cellulose

content during Jan, which declined up to Mar (spring season) but again showed an increase in

May and was the highest in Jul-Aug, but declined steadily during rest of the months/seasons. The

bottom leaf manifested the trend of cellulose synthesis similar to the middle leaf but the contents

were too low. Overall cellulose was highest in middle leaf followed by bottom leaf (Fig. 4.28).

Fig. 4.28: Effect of seasonal variation on Cellulose content in the penultimate, middle and

bottom leaves of lemongrass

cd cdd de

b b b bc bca ab

cd

dde e

cdab ab

a abb c c cd ab

cab

ccd c

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Penultimate Middle Bottom

Cel

lulo

se (

%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

88

e. Silica

Significant differences were observed in the lemongrass leaves of three ages and

sampling months with a non-significant interaction of these factors for the silica contents (Table

4.11). The silica content of bottom leaf was higher than penultimate and middle leaves, although

the trend of its accumulation was more or less similar in all leaves. A highest silica content in the

leaves of different ages was observed in May-Jul (summer season), which decreased steadily in

the autumn and winter seasons being the lowest in Dec. Overall, the silica content was in the

order: bottom leaf > middle leaf > penultimate leaf (Fig. 4.29)

Fig. 4.29: Influence of seasonal condition on silica content in the penultimate, middle and bottom

leaves of lemongrass

bcd

e de e

ba

aa

bcd

bcd

bc bc

cdbc

dd d

bcd

a ab

bc bc bc bcd

bcd

bcf ef

abc

aa

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dcb

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Dec

Penultimate Middle Bottom

Sil

ica

(%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

89

f. Starch

The starch contents showed statistically significant results in lemongrass leaves of three

ages and the sampling months with a significant interaction of these factors for starch contents

(Table 4.11). All the leaves indicated distinctive behavior across the seasons for starch contents

The penultimate leaf showed higher starch contents in winter and summer months; the highest

being in Jun-Aug and in Jan, while the lowest in Nov. The middle leaf indicated the trend of

starch contents similar to the penultimate leaf but there was large variation in the trend of

accumulation across the seasons. In bottom leaf, the starch contents were low at the end of

autumn to the end of winter season while it was higher in spring and summer season. Overall,

starch content was greater in bottom and middle leaves while it was lower was observed in the

penultimate leaf (Fig. 4.30).

Fig. 4.30: Influence of seasonal condition on Starch content in the penultimate, middle and

bottom leaves of lemongrass

ab bc bab

a a abc

cd dc

ba

cd d cda

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dcd

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Penultimate Middle Bottom

Sta

rch

(%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

90

g. Total soluble proteins

The total protein contents in the leaves of different ages of lemongrass and sampling

months varied significantly with a significant interaction of both the factors (Table 4.11). In

penultimate leaf total protein contents were higher than in middle and bottom leaves. In

penultimate leaf, the total soluble proteins were higher in winter and summer season while

reduced in spring and autumn seasons. The middle leaf showed a lower total soluble proteins in

Jan-Mar, which were greater in summer and autumn seasons and again showed a little decline in

the winter season. The bottom leaf indicated virtually no change in the soluble proteins across

the seasons (Fig. 4.31).

Fig. 4.31: Effect of seasonal variation on Total Proteins content in the penultimate, middle and

bottom leaves of lemongrass.

b abd

bcd

bca a bc ab a

b ab

cde

e deab ab

cab

cbc ab

abc

dab bc

d

bcd a ab a abc

bcd

abc

a d bc bc bc

0

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Aug Sep Oct

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Dec

Penultimate Middle Bottom

Tot

al p

rote

ins

(%)

Leaf position/sampling months

Probability level:

Penultimate leaf = **Middle leaf = *Bottom leaf = ns

91

h. Correlation

Possible relationships between different seasons and nutritional attributes indicated that

in penultimate leaf ADF was positively related to ADL, cellulose and Silica while ADL, silica

and cellulose was negatively related to starch. In middle leaf, ADF was positively related to

ADL, cellulose and silica. ADL was positively related to cellulose, silica and starch, while

cellulose was positively associated with silica. In bottom leaf, ADF was positively related to

ADL, cellulose and silica. ADL showed positive correlation with cellulose silica and starch;

cellulose was positively associated to starch and protein (Table 4.12).

The correlation of meteorological attributes with the nutritional attributes revealed that

maximum temperatures was positively related to ADF, ADL and silica; minimum temperature

was associated positively to ADF, ADL and starch contents; relative humidity to NDF;

evapotranspiration was positively correlated with ADF and starch and average rainfall with

ADL. For middle leaf, maximum temperature was positively correlated with ADF and proteins,

minimum temperature with ADF, cellulose, silica and proteins; relative humidity with NDF and

average rainfall with NDF, ADF, silica and proteins. For bottom leaf, maximum temperature was

positively correlated with ADF and cellulose; minimum temperature was positively related to

ADF, cellulose and silica; evapotranspiration was positively correlated with ADF, cellulose and

silica while average rainfall was associated to silica contents.

4.8.2 Discussion

Seasonal variations had a significant impact on the nutritional quality attributes of plants

(Snyman, 2006). Nutritive constituents such as NDF, ADF, ADL, cellulose, silica, starch and

protein vary between years and between plant growth stages within growing season (Ball et al.,

2001). Results of this study revealed a lot of variations in the nutritional quality attributes of

lemongrass leaves during the whole year (to year study). The most distinctive changes were

observed in the leaf fiber attributes, wherein the NDF was greater in middle leaf; ADF was

similar in penultimate and middle leaves while bottom leaf displayed lowest contents of these

attributes (Fig. 4.25-4.26). A higher fiber content is a quality attribute of nutrition for medical

purpose and animal feed, since this provides a rich source of energy (Turner and Lupton, 2011).

92

Table 4.12: Correlation coefficient (r) of meteorological attributes with the Nutritive content of lemongrass at three leaves positions penultimate, middle, bottom

Y-Variable X-Variable Penultimate Middle Bottom a. Mutual correlations of nutritional attributes NDF ADF -0.077na 0.214ns 0.082ns ADL 0.345ns 0.458ns 0.315ns Cellulose 0.220ns 0.209ns 0.081ns Silica 0.318ns 0.501ns 0.275ns Starch -0.375ns 0.366ns 0.107ns Protein -0.468ns 0.123ns -0.044ns ADF ADL 0.812** 0.670* 0.630* Cellulose 0.642* 0.873** 0.904** Silica 0.863** 0.871** 0.885** Starch 0.511ns 0.367ns 0.528ns Protein -0.231ns 0.536ns -0.193ns ADL Cellulose 0.040ns 0.684* 0.609* Silica 0.887** 0.789** 0.762** Starch 0.400ns 0.618* 0.709** Protein -0.487ns 0.049ns -0.170ns Cellulose Silica 0.221ns 0.826** 0.877** Starch -0.600* 0.473ns 0.769** Protein -0.273ns 0.264ns -0.229ns Silica Starch 0.314ns 0.510ns 0.746* Protein -0.544ns 0.372ns -0.276ns Starch Protein -0.047ns -0.385ns 0.137ns b. Correlations environmental variable with nutritional attributes Max. temperature NDF -0.161ns -0.138ns 0.104ns ADF 0.786** 0.628* 0.605* ADL 0.594* 0.240ns 0.196ns Cellulose -0.099ns 0.519ns 0.554ns Silica 0.578* 0.536ns 0.620* Starch 0.380ns -0.181ns 0.040ns Protein 0.298ns 0.664* -0.062ns Min. temperature NDF 0.064ns 0.140ns 0.252ns ADF 0.807** 0.667* 0.602* ADL 0.685* 0.382ns 0.312ns Cellulose -0.093ns 0.592* 0.595* Silica 0.325ns 0.666* 0.707** Starch 0.659* -0.051ns 0.217ns Protein 0.164ns 0.655* -0.053ns Relative humidity NDF 0.785** 0.844** 0.299ns ADF -0.166ns 0.046ns -0.209ns ADL 0.086ns 0.315ns 0.223ns Cellulose 0.350ns 0.138ns -0.153ns Silica 0.152ns 0.302ns 0.066ns Starch -0.481ns 0.355ns 0.439ns Protein -0.459ns -0.109ns -0.074ns Evapotranspiration NDF -0.399ns -0.311ns 0.066ns ADF 0.745** 0.524ns 0.658* ADL 0.551ns 0.159ns 0.160ns Cellulose -0.311ns 0.442ns 0.659* Silica 0.463ns 0.389ns 0.597* Starch 0.602* -0.105ns 0.014ns Protein 0.256ns 0.476ns -0.065ns Average rainfall NDF 0.591* 0.619* 0.541ns ADF 0.537ns 0.584* 0.353ns ADL 0.656* 0.462ns 0.424ns Cellulose -0.070ns 0.532ns 0.335ns Silica 0.561ns 0.689* 0.612* Starch -0.068ns 0.158ns 0.419ns Protein -0.248ns 0.599* -0.238ns Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 level

93

In addition, these attributes were detected in substantially greater amounts in the summer

months. It is important to note that lemongrass, as a C4 plant, has a higher tendency to grow and

sustain under relatively higher temperature (Henry et al., 2000). The maintenance of greater fiber

provides a great opportunity of using lemongrass when the availability of other plant sources is

meager in the summer season.

In addition to the fiber contents, ADL, cellulose, silica and proteins are among the other

important nutritionally important attributes. It has been reported that the ADL and cellulose

synthesis takes place as a stress reaction leading to wall thickening (Zhong et al., 2001). Silica

has been implicated in providing tolerance against biotic and abiotic factors and improving plant

health (Epstein, 1999), while starch is also accumulated as a long term storage product

(Geigenberger, 2011). These data indicated that young penultimate leaf indicated a reduced

amount of ADL, cellulose, silica and starch, while enhanced contents of soluble proteins as

compared to middle and bottom leaves. However, the contents of all these attributes were

substantially greater in the summer followed by winter months (Fig. 4.27-4.31). Such an

accumulation has great implications with physiological standpoint and is important to plant

survival during the harsh growth periods. The ADL, cellulose and silica appeared to provide the

structural support while soluble proteins are expected either to act as osmolyte or provide amino

acids for this purpose. The older leaf showing a differential trend for the accumulation of these

nutritional attributes plausibly due to ageing factor.

The correlations were drawn to validate the current findings. The interrelationships of the

nutritional quality variables revealed that ADL, cellulose and silica showed the most

conspicuous association in all the leaves, although these correlations were much stronger in the

case of older leaves. This indicated that older leaves have higher capacity to synthesize and

accumulate these metabolites since this trend is clearer and typical of leaf ageing (Bassey et al.,

2001).

Drawing the correlation of meteorological attributes with the nutritional quality attributes

provided useful information. The positive association of changes in minimum and maximum

temperatures, evapotranspiration and rainfall patterns with ADF, ADL, silica and starch in the

penultimate leaf indicated that their enhanced synthesis under higher temperatures or

dehydrating conditions is helpful in the plants survival. This survival appears to improve the cell

wall properties under such adverse conditions. Moreover, all these changes appear to be

94

transitory since the contents of these metabolites diminished when the adverse conditions were

relieved, and the young leaves indicated the normal growth patterns. Such changes appeared to

be lacking in the older leaves.

In conclusion, although the nutritional attributes are important with respect to animal feed

point of view and makes lemongrass an important nutritional source especially during the

summer months, such an accumulation is important with respect to changes in the wall properties

and acting as osmoprotectants, thus making it better able to survive under adverse conditions.

95

4.9 Ionic determination

Leaf mineral profile is good way to assess nutritional status of plants. Climatic changes in

temperature, rainfall and light intensity have great influence on mineral accumulation in

lemongrass. To understand the physiology of lemongrass, the knowledge of seasonal variation in

leaf nutrient concentrations is necessary. The results of soluble nitrate, soluble phosphate, sulfur,

potassium and calcium are given below:

4.9.1 Results

The statistical results of differences in the significance of variance sources i.e. leaves of

three ages and seasonal conditions, with respect to leaf minerals are presented in Table 4.13.

Table 4.13: Analysis of variance of data (mean squares) for soluble nitrate, soluble phosphate, sulfur, potassium and calcium contents of lemongrass under influence of seasonal condition

Source of Variation

Df Nitrate-N Phosphate-P Sulfate-S K Ca

Block (B) 2 0.03 0.12 0.59 2.56 0.67

Leaf (L) 2 312.86** 21.25** 127.55** 240.74** 55.07**

Month (M) 11 7.22** 1.96** 3.00** 26.40** 6.77**

L × M 22 4.05** 0.22ns 0.73** 3.83ns 4.57**

Error 70 0.67 0.22 0.24 4.90 0.52

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

96

a. Soluble nitrate-N

For soluble nitrate-N, the experimental results showed significant difference in the leaves

of various ages and sampling months with a significant interaction of these factors (Table 4.13).

In penultimate and middle leaves, the soluble nitrate-N contents decreased from Jan and attained

the lowest value in Feb-Mar (spring season). It attained the greatest amount in summer sesason

and again showed a decline in the autumn season followed by a rise in the winter season. In

bottom leaf, there were no variation in their contents along the changing seasons. Overall, the

soluble nitrate-N was much lower than those observed in the penultimate and middle leaves (Fig.

4.32).

Fig. 4.32: Effect of seasonal variation on Soluble Nitrate ion concentration in the penultimate,

middle and bottom leaves of lemongrass

bccd

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Penultimate leaf = **Middle leaf = **Bottom leaf = **

97

b. Soluble phosphate-P

Data revealed significant difference in the leaves of different ages, and sampling months

but with a non-significant interaction of these factors for soluble phosphate-P contents (Table

4.13). In case of penultimate leaf, the soluble phosphate-P was relatively lower in Jan, which

increased in Feb, and displayed a seady state level up to Oct, followed by a decline in Nov-Dec.

For middle leaf the soluble phosphate-P increased from Jan and was the highest in Apr, which

showed a decline thereafter up to Dec. For bottom leaf there was no specific trend of the

accumulation of soluble phosphate-P. Overall, the soluble phosphate-P accumulation was the

lowest in bottom leaf and highest in the middle leaf (Fig. 4.33).

Fig. 4.33: Effect of seasonal variation on soluble phosphate ion concentration in the penultimate,

middle and bottom leaves of lemongrass

cab ab ab

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Penultimate leaf = **Middle leaf = **Bottom leaf = **

98

c. Sulfate-S

There were highly significant differences in the leaves of different ages of lemongrass

and sampling months for the changes in the sulfate-S contents with a significant interaction of

leaves and sampling months (Table 4.13). Being relatively higher in Jan in penultimate leaf, the

sulfate-S declined to its lowest value in Mar, which indicated a gradual increase and attained a

highest value in summer months (Jun-Aug). Thereafter, it indicated a decline up to Nov and an

increase in Dec. In middle leaf, the sulfate-S indicated a steady state value up to Mar, but a rise

thereafter attaining the highest value in Jun-Aug and a decrease afterwards. The bottom leaf

indicated no significant change in the sulfate-S contents throughout the sampling months. The

penultimate leaf exhibited the highest sulfate-S contents while bottom leaf the lowest (Fig. 4.34).

Fig. 4.34: Effect of seasonal variation on Sulfate ion concentration in the penultimate, middle

and bottom leaves of lemongrass

cdd

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99

d. Potassium (K)

Results indicated significant differences in the leaves and sampling months with a

significant interaction of both these factors for K contents (Table 4.13). All the three selected

leaves showed significant seasonal changes for K contents. In penultimate leaf, the K content

was the highest in Jan which declined in Feb-Apr. It increased in the summer season (May-Aug)

and then indicated a decline in autumn season followed by an increase in the winter months. The

middle leaf showed a steady state level of K in the spring season. It increased in the summer

months and was the highest in July. From Aug and onward K content decreased up to Nov but

again increased in Dec. For bottom leaf, the pattern of K accumulation was similar to the

penultimate leaf but the content was lower than penultimate and middle leaf (Fig. 4.35).

Fig. 4.35: Influence of seasonal condition on Potassium ion concentration in the penultimate,

middle and bottom leaves of lemongrass

bccd

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Penultimate leaf = **Middle leaf = **Bottom leaf = **

100

e. Calcium (Ca)

It was evident from the results that leaves of three ages and months of sampling differed

significantly but there was no interaction of both these factors for Ca content (Table 4.13). The

Ca contents of penultimate leaf were low during winter season, which decreased further during

spring season. In summer season, the Ca content increased and was at its highest in May-Jul, but

decreased in the autumn season, and then showed an increase. In middle leaf too, the Ca content

was quite low in spring and autumn season but markedly higher in the summer and winter

seasons. The bottom leaf, however, indicated a reduced Ca content in Jan-Feb but a steady Ca

content during rest of the sampling months. Overall, the Ca content was higher in penultimate

leaf but lower in the bottom leaf (Fig. 4.36).

Fig. 4.36: Influence of seasonal condition on calcium ion concentration in the penultimate,

middle and bottom leaves of lemongrass

abc

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Probability level:

Penultimate leaf = **Middle leaf = **Bottom leaf = **

101

f. Correlation

Interrelationships of nutrients revealed that in both penultimate and middle leaf, nitrate-N

was positively correlated with sulfate-S, K and Ca; sulfate-S with K and Ca while K was

correlated with Ca (Table 4.14). The relationships of nutrients with meteorological variable

showed that maximum temperature was positively correlated with phosphate-P and sulfate-S,

minimum temperature and relative humidity were positively correlated with all nutrients except

K, while evapotranspiration and rainfall were positive correlated with sulfate-S and Ca. In case

of middle leaf, sulfate-S was correlated with maximum temperature; sulfate-S and Ca with

minimum temperature, evapotranspiration with nitrate-N, sulfate-S and Ca, while rainfall was

associated positively to sulfate-S and Ca. In case of bottom leaf, no other environmental variable

was correlated with any nutrient except sulfate-S and K (Table 4.14).

4.9.2 Discussion

Mineral nutrients are essential for plant growth and development. Seasonal variations in

tissue mineral concentration appear to be largely dependent on soil, light, temperature and

rainfall (Wells, 1996; Gent, 2002). As a part of present study, seasonal variations in chemical

composition of lemongrass leaves P, S, N, K, and Ca concentrations were determined on

monthly basis throughout the years in the penultimate, middle and bottom leaves of lemongrass.

The plant nutrients play both structural and functional roles in the plants, since they are the part

of macromolecules (Epstein and Bloom, 2005).

The fact remains that under relatively adverse conditions, the plants need greater

resources for growth and development; the availability of nutrients is pivotal in this regard. On

whole body basis, the plants generally show insufficient concentration of important nutrients in

different seasons (Epstein and Bloom, 2005). However, there may be differences in the leaves of

various ages for the acquisition of various nutrients. Results revealed that seasonal changes had

great impact on the concentration of soluble nitrate-N (Fig. 4.32), soluble phosphate-P (Fig.

4.33), sulfate-S (4.34), K (Fig. 4.35) and Ca (Fig. 4.36) in the leaves of all ages. Overall the

results indicated that generally the young (penultimate and middle) leaves had greater contents of

102

Table 4.14: Correlation coefficient (r) of meteorological attributes with the ion content of lemongrass at three leaves positions penultimate, middle and bottom

Significant at: ns, non-significant; * and **, significant at P<0.05 and P<0.01 levels

Y-Variable X-Variable Penultimate Middle Bottom a. Mutual correlations of nutrient elements Nitrate-N Phosphate-P 0.130ns -0.207ns -0.138ns Sulfate-S 0.904** 0.676* -0.560ns K 0.906** 0.731** 0.392ns Ca 0.823** 0.892** 0.501ns Phosphate-P Sulfate-S 0.441ns -0.312ns -0.027ns K -0.058ns 0.359ns -0.188ns Ca -0.175ns -0.330ns -0.268ns Sulfate-S K 0.743** 0.584* -0.219ns Ca 0.580* 0.739** 0.004ns K Ca 0.820** 0.682* 0.413ns b. Correlations environmental variable with nutrient elements Max. temperature Nitrate 0.521ns 0.504ns -0.012ns Phosphate 0.663* 0.052ns 0.314ns Sulfur 0.683* 0.693* -0.134ns K 0.302ns 0.302ns 0.180ns Ca 0.258ns 0.353ns 0.232ns Min. temperature Nitrate 0.612* 0.575ns -0.243ns Phosphate 0.749** 0.016ns 0.351ns Sulfur 0.793** 0.810** 0.068ns K 0.424ns 0.419ns 0.007ns Ca 0.823** 0.892** 0.412ns Relative humidity Nitrate 0.612* -0.086ns -0.486ns Phosphate 0.749** -0.393ns 0.111ns Sulfur 0.793** 0.152ns 0.611* K 0.424ns -0.064ns -0.686* Ca 0.823** -0.330ns -0.065ns Evapotranspiration Nitrate 0.533ns 0.578* 0.126ns Phosphate 0.490ns 0.203ns 0.139ns Sulfur 0.588** 0.578* -0.336ns K 0.357ns 0.462ns 0.449ns Ca 0.590* 0.739** -0.042ns Average rainfall Nitrate 0.553ns 0.467ns -0.392ns Phosphate 0.533ns -0.156ns 0.084ns Sulfur 0.725** 0.769** 0.416ns K 0.498ns 0.430ns -0.246ns Ca 0.820** 0.682* 0.197ns

103

all the nutrients while the older (bottom) were deprived of this characteristic. In addition to the

leaf age or position on the plant, prevailing weather conditions appeared to be the main

determinant of the nutrient concentrations.

As is evident from the results, a greater nutrient content in the younger leaves is of great

physiological significance. These nutrients being present in the soluble phase are available for

the maintenance of water balance by acting as osmotica during the summer season, when there is

excessive water loss by evapotranspiration, while in winter season essential nutrients may act as

the cryoprotectants of cellular membranes (Fuller, 2004). In addition to these roles, they are

assimilated in the synthesis of macromolecules and cellular structures at large. For all these

practical purposes, the younger leaves were on an advantage to show normal metabolism and

greater growth even under sub- or supra-optimal temperatures.

To substantiate the above findings, mutual correlations of the nutrients and those of

meteorological attributes with the nutrient contents separately for three leaves were established

(Table 4.14). These correlations were quite strongly evident with most of the environmental

variable in case of penultimate and middle leaves but not in the bottom leaf. These data

suggested that most of these nutrients were correlated tightly with the prevailing temperature and

evapotranspiration. Furthermore, quicker response of younger tissues to the nutrients

accumulation in making metabolic adjustments as compared to the older leaf is of great

significance for growth and survival standpoints.

In conclusion, greater amounts of soluble nutrient in the younger leaves are of advantage

to lemongrass for its sustainable growth in the stress environments. Moreover, seasonal patterns

of variation in mineral concentration of lemongrass appear to be largely dependent on

temperature and evapotranspiration.

104

4.10 Essential oil composition

The essential oils were extracted from the lemongrass leaves samples (as composite

samples) four times in Jan (winter season), Apr (spring season), Jul (summer season) and Oct

(autumn season). The results are given below.

4.10.1 Results

The GC-MS chromatograms for the chemical composition of lemongrass essential oils

are presented in Figs. 4.37-Fig. 4.40 and quantitative estimates are given in Table 4.15. The yield

of essential oil (% v/w) during different seasons varied from 0.6 to 1% with the highest in rainy

season. It is evident from the results that seasons have marked effect on quality and quantity of

lemongrass leaf essential oil constituents. Essential oil content was higher in summer season

(1%) followed by winter (0.9%) and autumn season (0.7%), whereas it was 0.6% during spring

season. In this study, a total of 54 essential oil components were identified in four different

seasons. There were 33 compounds representing 88.58% of lemongrass essential oil when

sampling was done in month of January. Lemongrass leaves sampled in Apr manifested 32

chemical compounds, representing 69.30% of essential oil. However, in July and October, 26

and 30 chemical compounds were detected representing 89.67 and 74.21%, respectively of the

essential oil were detected (Table 4.15). The principal components of lemongrass essential oils

were neral (11.22 – 17.28%), geranial (8.29% – 15.64%) and citral (13.86%). In addition,

lemongrass essential oils also contained a wide range of minor compounds e.g., Nerolidol (1.3 –

2%), Caryophyllene oxide (2.2 – 5.5%), Epoxy-linalooloxide (1.7 – 3.9%), Epiglobulol (1.0 –

3.4%) and 1-Heptatriacotanol (0.7% – 1.1%) in four different seasons (Fig. 4.37-4.40).

4.10.2 Discussion

As evident from the results, there was a great diversity of essential oils biosynthesized in

the leaves of lemongrass in four seasons, with great differences (Figs. 4.37-4.40 and Table 4.15).

105

Fig. 4.37: Typical GC-MS chromatogram of lemongrass essential oil collected during January

Fig. 4.38: Typical GC-MS chromatogram of lemongrass essential oil collected during Apr

106

Fig. 4.39: Typical GC-MS chromatogram of lemongrass essential oil collected during July

Fig. 4.40: Typical GC-MS chromatogram of lemongrass essential oil collected during October

107

Table 4.15: Chemical composition (%) of lemongrass essential oil in different seasons Probable match January April July October (−)-Myrtenal 2.296 0.784 1.93 1.249 (+)-α-Longipinene 3.337 3.03 ‒ 2.184 1,1 Bipheny 1,3,4-diethyl- ‒ 0.789 ‒ ‒ 1,4-Cineolep ‒ 0.272 ‒ ‒ 1-Heptatriacotanol 0.948 0.878 1.109 0.738 2-2,4-Trimethy 1-3-[3,8,12,16 tetramethyl-heptadeca-3,7,11,15-tetraenyl]- 0.561 2.171 0.36 2.385 23-Dimethoxy-5-methyl-6-dekaisoprenyl-chinon ‒ 0.863 ‒ ‒ 2-Tridecanon 2.293 ‒ 1.921 ‒ 3,7-Nonadien-2-ol,4,8-dimethyl- ‒ ‒ 1.663 ‒ 3-[5-benzyloxy-3-methylpent-3-enyl]-2,2 dimethyloxirane 0.757 ‒ ‒ ‒ 3-Methoxymethoxy-1,5,5-trimethyl-cyclohexene ‒ ‒ 1.18 ‒ 4-[2,2-dimethyl-6-methylenecychohexyl] butanol ‒ 2.3 1.229 1.213 9-Hexadecanoic acid ‒ 4.563 ‒ ‒ Allethrin 4.115 0 5.441 ‒ Alloaromadendrene oxide-[1]- 1.024 0 ‒ ‒ Arisol, p-aallyl- ‒ 1.055 ‒ ‒ Aromadendrene ‒ 1.592 ‒ ‒ Ascaridole epoxide 1.858 ‒ ‒ ‒ Aspidoalbine ‒ ‒ ‒ 4.719 Caryophyllene oxide 5.517 4.47 2.255 3.876 Cholestan-3-ol,2-methylene-,[3β5a]- 1.499 1.431 ‒ 1.107 Cis-Z- a – Bisabolene epoxide ‒ 0.851 1.45 ‒ Citral 13.846 ‒ ‒ ‒ DCP-LA ‒ 2.034 ‒ 1.317 Egrosteryl acetate ‒ 1.106 ‒ 0.88 Epiglobulol 3.411 1.031 3.015 3.294 Ethyliso-allocholate ‒ 2.035 2.911 ‒ Expoxy-Linaloolooxide 1.772 3.745 3.971 2.099 Farnesyl bromide 2.372 1.017 ‒ 3.066 Geranial ‒ ‒ 15.647 8.293 Globulol 1.534 1.464 6.418 ‒ Himachalol 1.291 ‒ ‒ 3.42 Ilicicolin F 1.113 ‒ ‒ 1.474 Ingol 12-acetate 0.65 ‒ ‒ ‒ Ingol 12-acetate ‒ 1.275 1.011 ‒ Isophorol ‒ ‒ 0.908 ‒ Linoleic acid ‒ ‒ 4.252 1.246 Longiborneol ‒ 4.188 ‒ 3.1 Mesityl oxide ‒ ‒ ‒ 0.656 Myristoleic acid ‒ ‒ 2.668 ‒ Neral 17.286 14.525 16.525 11.223 Neric acid 2.283 0.918 2.883 1.566 Nerol 3.401 ‒ ‒ ‒ Nerolidol 2.015 1.651 ‒ 1.331 Neryl acetate 1.343 ‒ 4.48 ‒ Oleic acid 2.058 ‒ ‒ 0.543 Viridiflorol ‒ ‒ ‒ 4.365 α- Muurolol ‒ 2.013 1.236 ‒ α-Bergamotene, cis- 3.01 ‒ ‒ ‒ α-Cadinol, 1.559 ‒ ‒ 3.05 α-Terpineol 3.253 ‒ 2.163 ‒ β-Acoradiene 0.433 2.337 1.196 1.063 β-Estradiol 1.105 3.484 2.211 ‒ γ-Cadinene 0.633 ‒ ‒ 1.392 ‒ indicates the detection of no oil

108

Among the oils, neral (17.28 - 11.22 %), citral (13.86%) and geranial (8.29-15.64%). Khanuja et

al. (2005); Negrelle and Gomes, (2007) reported that the composition of essential oil obtained

from lemongrass leaves varies markedly in different season. Tajidin et al. (2012) reported that

compounds (neral, geranial, nerol, and geranyl acetate) had concentrations greater than 1%.

These oils have high anti-inflammatory and antioxidant components.

This pattern of oil accumulation in lemongrass showed that the dynamics of the essential

oil content appears to be metabolically regulated during different seasons. The variations in the

chemical compositions of lemongrass essential oil across the year can be attributed to the varied

climatic and seasonal conditions of the region and adaptive metabolism of plant. The content of

the essential oils of lemongrass was distributed unevenly among seasons. The highest yield of

the oil was found during rainy season (1%) and winter season (0.9%) which decreased

significantly in autumn to 0.7%. Photoperiod, temperature, intensity of light (Clark and Menary,

1980) and seasons had great impact on oil composition in aromatic plants (McGimpsey et al.,

2006). Neral, a very important compound, was present in high quantity (17.28%, 16.52%) in the

lemongrass essential oil collected during winter and summer (Table 4.15). Sarma et al. (2011)

reported that in lemongrass leaves oil content was higher at the onset of monsoon as compare to

post-monsoon period. In the present investigation, lemongrass oil yield also increased in July up

to 1% and then decreased in October 0.7%. Citral and geranial are also commercially important

compounds, especially used in Vitamin A and ionone synthesis (Efraim et al., 1998). Citral,

geranial, Nerolidol, and Allethrin showed same seasonal pattern like neral, higher in summer and

winter period. The quality of the oil in Jamrosa ‘RL‐931 (Cymbopogon nardus var. confertiflorus

× C. jwarancusa) was high in July due to high temperature and low humidity, which enhanced

the accumulation of geraniol, and geranyl acetate (Bhan et al., 2003). Guenther (1961) reported

that lemongrass oil content is lower during the month of heavy rainfall compared to the dry

months.

Seasonal variation play a great role in changing the essential oil composition (both

quality and quantity). A higher essential oil yield was observed in the month of January and

October. Thus increased oil content in these months can be taken as a criterion for good harvest

time of lemongrass essential oils for commercial pursuits.

109

GENERAL DISCUSSION

Lemongrass is a plant of great economic importance by virtue of the oil contents in the

leaf. The oil has a number of uses with medical standpoint. The lemongrass possesses C4

pathway of photosynthesis and can withstand relatively higher ambient temperatures. It also has

the ability to grow in the cool season, which shows quite a high flexibility of this grass to adjust

in the changing climatic conditions. However, no report could be found showing the

physiological basis of success of this important plant species to thrive in different seasons. These

studies were therefore aimed at exploring the comparative physiological, biochemical and

nutritional mechanisms of lemongrass leaves of three ages over two years.

The adverse climatic conditions lead to the mounting of oxidative load with the enhanced

production of ROS (Mittler, 2000; Bavita et al., 2012). Although ROS production and quenching

is a physiological need of the plants under normal conditions (Taiz and Zeiger, 2010; Wahid et

al., 2013), their enhanced production versus low dousing under adverse conditions causes

damage to the cellular and organelle membranes (Huang et al., 2004; Lu et al., 2009). To

counteract such effects, the cells deploy the antioxidant system, which may be both enzymatic

and non-enzymatic in nature (Sairam et al., 2000; Sofo et al., 2004; Farooq et al., 2009). In the

lemongrass leaves of three different ages, it was found in this study that although there was

enhanced oxidative stress on the leaves as determined in terms of H2O2 and MDA generation, the

younger (penultimate and middle) leaves indicated low levels of ROS and MDA than the older

(bottom) leaf (Fig. 4.2-4.3). Furthermore, these effects were more pronounced in summer and

winter seasons. Determination of activities of the antioxidant enzyme in these leaves indicated

that younger leaves had substantially higher activities of SOD, CAT and POD especially in the

summer season (Fig. 4.4-4.6). Furthermore, the extent of oxidative stress and the activities of

three antioxidant enzymes were tightly associated in younger leaves (Table 4.2), which showed

that deployment of the antioxidant system was more efficient in these leaves.

In addition to the antioxidant enzymes, the plants synthesize a great variety of

compounds of primary and secondary nature, which show accumulation under stressful

conditions and are active in the protection of cytoplasmic and organelle membranes (Wahid et

110

al., 2007; Farooq et al., 2009). The osmoprotectants synthesis is important in the maintenance of

cell water balance and protection of biological membranes from being damaged by the

dehydrating forces such as salinity, drought and temperature extremes (Zhu, 2001; Ashraf and

Foolad, 2007; Furtana et al., 2013). Commonly studies osmoprotectants are soluble sugars,

amino acids and quaternary ammonium compounds (QACs) including betains (Cayley and

Record, 2003; Wahid et al., 2007). In this study, the determinations made in the leaves of various

ages indicated that all the osmoprotectants showed greater accumulation in the winter and

summer season while their levels were quite low in spring and autumn season. Moreover,

younger (penultimate and middle) leaves indicated their greater accumulation (Fig. 4.7- 4.10).

The correlations of these osmolytes indicated their co-accumulation as well as with the high

temperature and evapotranspiration (Table 4.4). This suggested their greater roles in rescuing the

younger tissues from the adverse effects of high temperature and dehydration being low

molecular weight and highly water soluble (Rinne et al., 1994; Bhowmik and Matsui, 2003).

Seasonal variations in lemongrass biochemical constituent appeared to be associated with

plant maturity, temperature and soil moisture contents throughout different seasons. Variation in

environmental condition enhanced the biosynthesis of secondary metabolites, presumably due to

preferred reversal of photosynthates to secondary metabolites as related to primary one (Morales

et al., 1993). All the biochemical compounds in lemongrass analyzed in the present experiments

varied significantly during different seasons. Photosynthetic pigments, secondary metabolites,

antioxidants, total minerals contents, and micro-nutrients were the highest in active growth

period, decreased in winter the period of languid growth. Such seasonal effects on biochemical

compounds have been recorded in different plant species such as Cymbopogon citatus (Siribel et

al., 2001), Ocimum basilicum (Hussain et al., 2008), Spinacia oleracea (Howard et el., 2002),

Mentha spicata (Kofidis et al., 2004), Adiantum capillus-veneris (Ahmad and Husain, 2008), and

Toona sinensis (Wang et al., 2007).

It is well recognized that quantitative and qualitative contents of secondary metabolites

has great variation, which is controlled by intrinsic and a biotic factors (moisture, light, nutrient

availability). Secondary metabolites implicated in stress tolerance include flavonoides (Winkel-

Shirley, 2002) anthocyanin (Chalker-Scott, 1999), tannin (Lees et al., 1994). In this study the

determinations made for the accumulation of alkaloids, saponins, tannins, soluble phenolics,

flavonoids and anthocyanins (Fig. 4.11-4.16). It was found that the synthesis of alkaloids and

111

saponins was the lowest in the young (penultimate and middle) leaves but higher in the older

(bottom leaf), whilst the synthesis of tannins was relatively greater in the middle leaf but surely

lower than the bottom leaf in different months. More notably, the accumulation of soluble

phenolics and flavonoids was much higher in the young tissues especially during the summer

season, while anthocyanins were accumulated more in the winter season. Such an accumulation

of these metabolites has great implication in the physiological terms. The accumulation of

phenolics, flavonoids and anthocyanins are helpful to plants in a number of way e.g., osmotic

and antioxidative roles under stressful conditions (Chalker-Scott, 1999; Wahid and Ghazanfar,

2006; Wahid, 2007), which was also substantiated in this study by the correlations of these

metabolites with the environmental attributes (Table 4.6). A lower accumulation of alkaloids and

saponins in the younger tissues but their greater accumulation appears to be due to their lesser

physiological role in the primary metabolic phenomena of plants (Wahid and Tariq, 2008).

Operation of normal metabolism in plants under the varying seasonal conditions is of

primary importance for plant survival. The regulation of activities of enzymes is carried out with

the help of vitamins, which act as enzymatic cofactors because of their redox chemistry. Climate

and seasons have great impact on vitamin constituents of green plants. Studies revealed that light

and temperature are major forces which involve in vitamin synthesis (Asensi-Fabado and

Munne-Bosch. 2010; Mahmood et al., 2012). The results from the present study exhibited a

notable seasonality in niacin (Fig. 4.17), ascorbic acid (Fig. 4.18) and riboflavin (Fig. 4.19)

contents of lemongrass leaves growing throughout the year. Moreover, the generation of

reducing powers, reflected by RPA was also substantially changed under the prevailing

conditions; in fact improved in summer season (Fig. 4.20). These fluctuating responses can be

assigned to seasonal changes imposed mainly by temperature stress and evapotranspiration

(Table 4.8). A correlation of vitamins contents and RPA in the younger leaves supports the

notion that vitamins are important in the normalization of the cellular functions under adverse

conditions.

Changes in the photosynthetic pigments are among the other important factors, which

determine the photochemical efficiency of leaves (Ruban, 2009; Taiz and Zeiger, 2010).

Stressful conditions are usually damaging to the photosynthetic pigments due to photo-oxidative

stress, but this tendency is relatively greater in the stress susceptible plants (Ramel et al., 2013).

Younger leaves of lemongrass, in this research, incurred a minimal loss of Chl-a (Fig. 4.21), Chl-

112

b (Fig. 4.22) and their total (Fig. 4.23), while there was generally an increase in the carotenoids

(Car) contents (Fig. 4.24). The Car have dual functions in plants; they act as accessory light

harvesting pigments (Taiz and Zeiger, 2010) and also are active in quenching the ROS produced

in the chloroplast as a result of photo-oxidative stress (Krinsky, 1998). Close association of Car

with the photosynthetic pigments and with the high temperature and evapotranspiration load

indicated that Car are mainly involved in alleviating the effect of heat stress on metabolically

active leaves of lemongrass.

There are a number of advantages of growing lemongrass. This grass can be found

growing round the year, can sprout from cuttings in any season and produces a large biomass

(Shylaraj et al., 1993). It has been very much exploited for the medicinal purpose, while its

capability as forage for animal rearing has been rarely explored. Generally the seasonal

conditions have a significant impact on the nutritive value of plants (Ball et al., 2001; Snyman,

2006). These studies were also focused to explore the possibility of using this grass for

nutritional purpose. The determinations were made for NDF (Fig. 4.25), ADF (Fig. 4.26), ADL

(Fig. 4.27), cellulose (Fig. 4.28), silica (Fig. 4.29), starch (Fig. 4.30) and protein content (Fig.

4.31). Related to the nutritional attributes are mineral nutrients, which are vital for performing

structural and functional roles in plants. Seasonal patterns of variation in mineral concentration

appear to be largely dependent on soil, light, temperature and rainfall (Wells, 1996; Gent, 2002).

As a part of present study, influence of seasonal variations on concentrations of lemongrass

leaves were studied for soluble nitrate-N (Fig. 4.32) soluble phosphate-P (Fig. 4.33), sulfate-S

(Fig. 4.34), K (Fig. 4.35) and Ca (Fig. 4.36). The concentrations of all these nutrients were in

greater quantities in the summer season followed by winter season, except soluble phosphate-P

which remained steady throughout the years. These data revealed that fiber contents (NDF and

ADF), starch and protein contents of the young leaves were higher while lignin cellulose and

silica contents were low, while the tissue nutrient contents were in the range as reported for the

traditional forage species. Taken together, all these attributes make the younger leaves of

lemongrass as a good source of nutrition. However, nutrient digestibility trials have to be made

before recommending lemongrass for the animal feed purposes.

The most important attribute of the lemongrass is the essential oil extracted from leaves,

which is commercially used for various purposes. The studies pertaining to the profile of

essential oils in different seasons are not available in literature. As regards commercial value of

113

lemongrass oil, Tajidin et al. (2012) reported that at different growth stages the concentrations of

essential oils including neral, geranial, nerol and geranyl acetate, having high anti-inflammatory

and antioxidant properties, were greater than 1%. The GC-MS analysis of essential oils in leaves

of lemongrass in four seasons revealed that out of 54 compounds detected, only seven were

detected in all four seasons (Fig. 4.37-4.40; Table 4.15). Some compounds present in quite high

amounts in one season were absent in the other seasons. The most predominant compounds

(having concentrations more than 4%) were neral, citral, caryophyllene oxide and allethrin in

winter (January) season; neral and 9-hexadecanoic acid in spring (April) season; neral and

geranial in summer (July) season and neral, geranial and caryophyllene oxide in autumn season.

These data showed that lemongrass provides a great opportunity for use in the herbal and

medicinal preparations. Furthermore, determination of oil constituents provide clues about

cultivating and harvesting of the lemongrass in order to extract their maximum oil contents for

medicinal purposes.

Lemongrass deploys quite a few physiological phenomena, which enables it to thrive

under relatively more stressful conditions, the younger leaves of lemongrass present a valuable

resource for being used as antioxidant, vitamins, plant secondary products, nutrition for the

ruminant animals. A great diversity of essential oils present in the leaves make lemongrass

ethnobotanically important plant, which has a great opportunity of its use in the medicinal

industry. However, more studies are required to find out its stress tolerance potential when

challenged with the stress factor such as salinity, drought, heat and chilling.

114

FUTURE PROSPECTS OF RESEARCH

The present studies on lemongrass have provided fundamental clues of the physiological

mechanisms of its survival in various seasons, together with its antioxidative and nutritional

values. There is a need to carry out concerted efforts on the seasonal variations in the gene

expression patterns in various plant parts under stressful conditions whilst comparing with the

control sets. These studies will further enhance our understanding of the functional analysis of

various genes and proteins conferring abiotic stress tolerance in this economically important

plant species.

115

CHAPTER-5

SUMMARY

Lemongrass [Cymbopogon citratus (D.C.) Stapf] is a C4 grass species which belongs to

family Poaceae. It can grow round the year and has a lot of economic benefits. However, no

studies are available on the seasonal physiological responses of this grass species for its survival

in the changing climatic conditions. During this two years study, the seasonal changes in the

antioxidative properties, vitamins biosynthesis, osmoprotectants accumulation, production and

accumulation of plant secondary products, nutritional attributes and essential oil profiles were

studied in the penultimate, middle and bottom leaves of lemongrass. This study elaborated the

suitable season for maximum oil contents which could be exploited for commercial purposes by

the lemongrass growers.

Results revealed that there was enhanced oxidative stress on the leaves as determined in

terms of H2O2 and MDA generation; the younger (penultimate and middle) leaves indicated their

low levels than the bottom leaf. The H2O2 and MDA synthesis was greater in summer and winter

seasons. The younger leaves indicated higher enzymatic antioxidant property as determined in

terms of SOD, CAT and POD activities in the summer season. Close association of H2O2 and

MDA with the antioxidant system in younger leaves indicated an effective ROS scavenging

system in them. The determination of soluble sugars, total free amino acids, free proline

accumulation and glycinebetaine in the leaves of various ages indicated that all the

osmoprotectants showed greater accumulation in the winter and summer seasons while their

levels were quite low in spring and autumn seasons. The correlations of these osmolytes

indicated their co-accumulation with high temperature and evapotranspiration. In this study the

determinations made for the accumulation of secondary metabolites revealed that the synthesis of

alkaloids and saponins was the lowest in the young (penultimate and middle) leaves but higher in

the older (bottom leaf), whilst the synthesis of tannins was relatively greater in the middle leaf

but surely lower than the bottom leaf in different months. More notably, the accumulation of

soluble phenolics, flavonoids was much higher in the young tissues especially during the summer

season, while anthocyanins were accumulated more in the winter season. The results from the

116

present study exhibited a notable seasonality in niacin, ascorbic acid and riboflavin. The

generation of reducing powers, was also substantially changed under the prevailing conditions

especially in the summer season. As for photosynthetic pigments, the younger leaves incurred a

minimal loss of Chl-a, Chl-b and their total, while there was generally an increase in the

carotenoids contents. Among the nutritional attributes of leaves, NDF, ADF, ADL, cellulose,

silica, starch and protein content were determined. Likewise, essential nutrient contents of the

leaves were also determined. The concentrations of all these nutrients were in greater quantities

in the summer season followed by winter season, except soluble phosphate-P the contents of

which remained steady throughout the years. The data revealed that fiber contents (NDF and

ADF), starch and protein contents of the young leaves were higher; lignin cellulose and silica

contents were low, while the tissue nutrient contents were in the range as reported for the

traditional forage species. Regarding medicinal properties, the GC-MS analysis of essential oils

in leaves of lemongrass in four season revealed that out of 54 compounds detected, only seven

were detected in all four seasons. Some compounds present in quite high amounts in one season

were absent in the other seasons. Among the compounds, neral, citral, caryophyllene oxide,

allethrin, 9-hexadecanoic acid and geranial in different seasons were the most distinct essential

oils. These data proved that the lemongrass provides a great opportunity for use in the herbal and

medicinal preparations because of a great variety of compounds synthesized in its leaves.

In short, while growing in different seasons, lemongrass exhibited the operation of quite a

few physiological phenomena, which appeared to enable it adapt to seasonal changes. More

notably, the younger leaves of lemongrass presented a valuable resource for its use as

antioxidant, vitamins, plant secondary products, nutrition for the ruminant animals. A great

diversity of essential oils present in the leaves make lemongrass ethnobotanically important

plant, which has the great opportunity of it use in the medicinal industry.

117

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