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31
CHAPTER II
EXTRACTION, ISOLATION AND CHARACTERIZATION OF
NATURALLY OCCURRING ANTIOXIDANTS
1. Introduction
Traditional medicines are used by approximately 60 per cent of the world's
population. These are used for primary health care not just in rural areas of developing
countries, but also in developed countries where modern medicines are predominantly
used. While traditional medicines are derived from plants, minerals, and organic matter,
the herbal drugs are prepared exclusively from medicinal plants [1, 2]. Adaptation to
environmental change is crucial for plant growth and survival. Environmental stress
enhances the generation of reactive oxygen species (ROS) such as superoxide anion,
hydrogen peroxide and hydroxyl radicals [3, 4]. ROS have been proposed as a central
component of plant adaptation to both biotic and abiotic stresses. ROS are produced by
leakage of electrons on to molecular oxygen from chloroplasts, mitochondria and plasma
membrane linked electron transport [5]. Different types of ROS based on their in vivo
concentration can perform beneficial or deleterious functions in the cell [6].
Many antioxidants such as vitamin C, vitamin E and carotenoids, occur as dietary
constituents. There are a lot of strong antioxidant compounds found in fruits, vegetables
[7-9] and in different beverages [10-16]. For example, fair antioxidants have been found
in berries [17-21], apples [22- 25], citrus fruits [26] and in fruit juices [27-29]. Highly
active antioxidants were found in olives [30-32] and olive oil [33-39]. Activity changes
during the processing of olive oil have also been evaluated [40, 41]. Many research
studies were carried out on antioxidants in fruits, and changes of antioxidants during fruit
processing [27, 42, 43]. The effects of processing have been evaluated also on the
changes of antioxidant activity in some roasted cereal products [44]. Red wines contain a
variety of polyphenolic compounds, the most abundant being anthocyanins, and they
have been shown to have high antioxidant activity [45-49].
However, not all polyphenols are extracted from grapes during the wine
production process. Among the best known and most biologically active are resveratrol,
32
quercetin and the catechins. It has been reported that grape seeds [50] and grape pomace
peels [51] continue to contain antioxidants, so wine production can be considered as a
source of antioxidants. Antioxidant activity was also reported in whiskys [52, 53]. Green
and black teas have been extensively studied for antioxidant properties [12, 14, 54-56].
The main compounds responsible for antioxidant activity were found to be catechins [16].
(–)- Epigallocatechin 3-gallate, (–)-epigallocatechin, (–)-epicatechin 3-gallate, (–)-
epicatechin, (+)-gallocatechin and (+)-catechin were identified and their antioxidant
activities have been studied [57- 59]. Herbs and spices are also good sources of
antioxidants [60-64]. Extensive research has been performed in this area, but only some
extracts from rosemary and sage are available as commercial antioxidants [65, 66].
The main problem in the application of such extracts is that usually they have a
characteristic odour, taste or colour, which in most cases is undesirable in the final
product [67]. Therefore there have been attempts to obtain odourless extracts having
antioxidative properties [68, 69]. A great number of different spices and aromatic herbs have
been tested for their antioxidant activity, with rosemary and sage being the most investigated
[70-75]. However many more herbs and spices have never been examined in this respect.
Nowadays, there is an increasing demand for natural products and therefore
research in the area of natural compounds is also growing. It should be noted however,
that natural is not always synonymous with safety. Therefore natural compounds must
also be tested for safety aspects before applying them in foods for human consumption.
There are various methods available to measure lipid oxidation in foods. Changes in
chemical, physical, or organoleptic properties of fats and oils during oxidation can be
monitored; however, there is no single method for assessing all oxidative changes in
different food systems. To determine primary oxidation of fats, various parameters like
changes of fatty acid composition [81], weight gain at different time intervals [82, 83],
amount of hydroperoxides [84], or conjugated dienes, which correlate well with peroxide
values [85], can be monitored. Addition of antioxidants decreases oxidation rates of
samples and the decrease can be expressed as the antioxidant activity. These methods
33
require a lot of time and therefore are not convenient for screening purposes. Nowadays
accelerated methods, such as Rancimat, active oxygen method (AOM), or OXIPRES
method, are used for assessing the oxidative stability of fats and oils [86].
This chapter describes the collection, identification and certification of plant
sources for antioxidant isolation. It also gives details of experimental procedures for
extraction, isolation, purification and spectral investigation of natural antioxidants. Use of
soxhlet extraction procedure with solvents of various polarities is also explained. This chapter
also deals with the purification protocol of isolated compounds. The characterization of the
isolated compounds using various techniques and structural elucidation based on spectral
investigation are described in this chapter. This chapter presents the details of
investigations using techniques such as TLC, UV-Vis spectrophotometery, FT-IR, 1H-
NMR, 13C-NMR, GC, GC-MS, HPLC and MS. All the plant selected were commonly
known and easily available. Out of the six plant selected four were edible and two were
non edible.
Sl.No Plant Name Identified Name Identification
Number Herbarium
Acc. Number
1 Curry Leaf Murraya koenigii L. BSI/SRC/5/23/2011-
12/Tech. -100 006152
2 Coriander Leaf Coriandrum sativum L. BSI/SRC/5/23/2011-
12/Tech. -101 006151
3 Mint Leaf Mentha arvensis L. BSI/SRC/5/23/2011-
12/Tech – 99 006150
4 Turmeric Curcuma lunga BSI/SRC/5/23/2011-
12/Tech-1330 006158
5 Bitter apple or Bitter cucumber
Citrullus colocynthis
(L.)
BSI/SRC/5/23/2011-12/Tech. -500
006157
6 Water hyacinth Eichhornia crassipes
(Mart.) Solms. – Laub. BSI/SRC/5/23/2011-
12/Tech. -145 006149
Table 2.1: Identification and certification of plant sources.
The six plants were collected from different places in Tamilnadu, and identified and
certified by the Botanical Survey of India, TNAU branch, Coimbatore, and were assigned
34
identification number and the herbarium account number (Table 2.1). The identified plants are
stored at the herbarium at Botany Department, Bharathiyar University, Coimbatore.
2. Murraya Koenigii L. (curry leaf)
The plant Murraya koenigii (L) Spreng, belonging to the family Rutaceae, is
native to India and now distributed in most of southern Asia. The Murraya species has
also been used in traditional medicine in eastern Asia. Previous studies on the Murraya
species include reports of coumarins, terpenoids and many investigations on carbazole
alkaloids [87 - 90]. The leaves of Murraya koenigii are also used as an ingredient in
ayurvedic medicine. Their properties include much value as an antidiabetic [91] and
hepatoprotectant [92]. Studies on carbazoles isolated from Murraya koenigii leaves have
been shown to possess antioxidant and antimicrobial activity [93 - 95].
It is a small tree, growing 4-6 m tall, with a trunk up to 40 cm in diameter.
The leaves are pinnate, with 11-21 leaflets, each leaflet 2-4 cm long and 1-2 cm broad.
The flowers are small white, and fragrant. The small black, shiny berries are edible, but
their seeds are poisonous. The wood is greyish white, hard, even, close grained and
durable. It has been used as timber for manufacture of many types of products. The curry
plant therefore is a multi-product source. If it can be utilised in a planned manner a chain
of rural industrial units can be planned and implemented [96].
The free amino acids present in the curry leaves are asparagine, serine, aspartic
acid, glutamic acid, threonine, proline, alanine, tyrosine, tryptophan, histidine, etc [97].
The leaves also contain a crystalline glucoside, koenigin and a resin. The twigs and leaves
contain 0.8% potash on dry matter basis. Fresh leaves, on steam distillation under pressure
yield 2.6% of a volatile oil, which may find use as a fixative for heavy type of soap perfume;
distillation at ordinary pressure gives very poor yield of oil, while distillation with
superheated steam yields dark colored foul-smelling oil. Rectified curry leaf oil is deep
yellow in colour with a strong spicy odour and pungent clove like taste [97, 98].
The use of essential oil as flavor or perfume is not yet popularised. There is a
good scope to create demand considering its virtues. In fact, the oil also has medicinal
virtues. The leaves, bark and the root are used in indigenous medicine as a tonic,
35
stomachic, stimulant and carminative. The leaves when taken with pepper early in the
morning on an empty stomach are known to reduce blood sugar. An infusion of the
roasted leaves is used to stop vomiting. The dry leaves of are ingredients in many herbal
medicines. The juice of the root is taken to relieve pain associated with the kidney.
The leaves are highly valued as seasoning in South Indian and Sri Lankan cooking, much
like bay leaves and especially in curries with fish or coconut milk. They are also available
dried, though the aroma is much inferior [98, 99].
Curry leaves are also known to be good for hair, in keeping them healthy and
long. Although most commonly used in curries, it is also used in many other dishes to
add spice [99 - 101]. The Murraya koenigii leaf contains nearly 42 compounds according
to several reports [101]. Several researchers reported the isolation of carbazole alkaloids
from curry leaves. This study reports the isolation of simple compounds like cymene,
caryophylene, carvone and phellendrene from the leaves of Murraya koenigii. These four
bioactive compounds are simple in structure and were characterized using FT-IR, 1H-NMR, GC, and GC-MS.
2.1. Materials and Method
Fresh curry leaf was collected from Siruvani crop field, Coimbaotre, India.
Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform (Merck,
Germany), absolute alcohol (Jiangsu Huaxi, China), sodium sulphate (Na2SO4)
(Qualigens, India), TLC plates (Merck, Germany). Acetone (Merck, Germany) Tris.Hcl
(Loba, India) were used.
2.2. Extraction with Soxhlet apparatus
A Soxhlet extractor is laboratory apparatus invented in 1879 by Franz von
Soxhlet. It was originally designed for the extraction of a lipid from a solid material,
thought it is not limited to the extraction of lipids. Typically, a Soxhlet extraction is
required only when the desired compound has high solubility in a solvent, and the
impurity is insoluble in the solvent. If the desired compound has a high solubility in a
solvent then a simple filtration can be used to separate the compound from the insoluble
substance. Normally a solid material containing some of the desired compound is placed
inside a thimble made from thick filter paper, which is loaded into the main chamber of
36
the Soxhlet extractor. The Soxhlet extractor is positioned into a flask containing the
extraction solvent. The Soxhlet is then fitted with a condenser [88, 94].
The solvent is heated to reflux. The solvent vapour travels up a distillation tube
and floods into the chamber housing the thimble holding the solid. The condenser ensures
that solvent vapour condenses and drips back down into the chamber housing the solid
material. The chamber containing the solid material slowly fills with warm solvent. Some
of the desired compound then dissolves in the warm solvent. When the Soxhlet chamber
is almost full, the chamber is automatically emptied by a siphon side arm, with the
solvent running back down to the distillation flask. This cycle may be allowed to repeat
many times, over hours or days.
During each cycle, a portion of the non-volatile compound dissolves in the
solvent. After many cycles the desired compound is concentrated in the distillation flask.
The advantage of this system is that instead of many portions of warm solvent being
passed through the sample just one batch of solvent is recycled. After extraction the
solvent is removed typically by means of a rotary evaporator yielding the extracted
compound. The non-soluble portion of the extracted solid remains in the thimble,
and is usually discarded.
Fresh curry leaves obtained from the Siruvani crop field, Coimbatore, India were
washed and cleaned thoroughly under running water. The excess water was drained and
the leaves were dried in vacuum for one week. Dried curry leaves were powdered in a
multi-mill fitted with sieve to obtain a coarse powder.
Figure.2.1: Rotary evaporator setup
37
Two solvents namely chloroform and ethanol was used for the extraction.
The extraction from 35 g curry leaf powder with 250 ml of the solvent was carried out at
60°C for a period of 10 h. The solvent was evaporated using a rotator evaporator.
The evaporator was maintained below 50oC. 7 gm of chloroform extract of crude and 5
gm of ethanol extract of crude were obtained. Totally 8 such fractions were analyzed by
TLC.
2.3. Isolation of compounds from the extract
Column chromatography (CC) is another common and useful separation
technique in organic chemistry. This separation method involves the same principle as
TLC, but can be applied to separate larger quantities of compounds compared to TLC.
Column chromatography can be used on both large and small scales. TLC is useful in
determining the type and number of ingredients in the mixture, but isolating individual
components at preparative scale is difficult. However, column chromatography allows
separation and isolation of individual compounds in bulk.
Solvent systems for use as mobile phases in CC can be determined from previous
TLC experiments, literature, or experimentally. Normally, the separation process begins
by using nonpolar or low polarity solvent, allowing the compounds to adsorb to the
stationary phase. The polarity of the solvent is then gradually increased which desorbs the
compounds and allows them to travel along with the mobile phase. On a macroscale, the
mixing of two solvents can create heat and crack the column leading to poor separation.
There are several methods available to pack a column. The slurry method which
normally achieves the best packing results is often used for macroscale separations.
The solid stationary phase is thoroughly mixed with a small amount of nonpolar solvent
taken in a beaker until a consistent paste is formed, capable of flowing. This homogeneous
mixture is poured into the column carefully using a spatula to scrape out the solid as the
liquid is poured. Care is taken in order to create an evenly distributed and tightly packed
stationary phase. Cracks, air bubbles and channelling in the stationary phase eventually
leads to a poor separation of the compound.
38
After the column is packed, the sample mixture is loaded directly to the top of the
column. Normally, a minimum amount of a polar solvent, about 5-10 drops, is used to
dissolve the mixture. The mixture is then carefully added to the top of the column using
a pipette without disrupting the surface of the column. A thin horizontal band of sample
over the packing material is best for an optimal separation. After the sample is loaded, a
small layer of white sand is added to the top of the column which helps to keep the top of
the column level when adding the eluent. Once the mixture is added and the protective
layer of sand is in place, solvent eluent is continuously added while collecting small
fractions at the bottom of the column. Using a pipette to add the first bit of solvent on top
of the packing material, and the addition of white sand minimizes the disturbance of the
column and dilution of the sample [23 - 25].
Figure. 2.1: Schematic diagram of extraction and isolation of antioxidants from plants.
39
2.4. Results and Discussion
The isolation and study of antioxidant activity of the four compounds -
caryophyllene, cymene, carvone and phellandrene isolated from Murraya koenigii are
given in the following text. These four bioactive compounds are simple in structure and
were characterized using FT-IR, 1H-NMR, GC, and GC-MS. These simple bioactive
compounds can be used as food supplements and as natural antioxidants.
2.4.1. Structural elucidation of compounds
2.4.1.1. Chloroform extract from Murrya koenigii
The chloroform extract was separated using column chromatography with silica
gel as packing material, and hexane as a solvent. The polarity of the solvent was
increased step up step using hexane and acetone. The first two fractions of the extract
were obtained using difference in polarity of the eluent. The first compound was isolated
using hexane and the second compound was isolated using a mixture of 99.5% hexane
and 0.5% acetone. Both compounds were analysed by FTIR, GC, GC MS, HPLC, and 1H-NMR as described below.
2.4.1.2. Compound 1 from chloroform extract
IR spectrum (ν, cm-1) of compound 1 revealed bands at 1259 cm-1, 1126 cm-1 and
1301 cm-1 (-C-C- str), 1058 cm-1, 1028 cm-1 (-C=C-), 873 cm-1 (-C=C- mono subs), 538,
718, 1514 (H-C-H bend), 1893, 2869, 2925, 2978, 3016 and 3049 cm-1 (H-C-H Asy str).
Compound 1 was tested by GC and this compound gave a single peak confirming
the presence of only one compound. The retention time of the compound was 6.33 min
with purity of 99.4%.
The spectral data of compound 1 was found to be in good agreement with the
reported value for cymene [102]. In the 1H-NMR spectrum, characteristic signals for
isopropyl group was observed at 2.85 δ, and 1.23 δ (singlet, 6 proton) while the benzylic
methyl group appeared at 2.32 δ.
40
GC-MS studies revealed a sharp peak with m/z value of 134.20, which
corresponds to the molecular weight of cymene (134.22). From the NMR and GC-MS
data (Figure 2.3) we conclude that the isolated compound is cymene (Figure 2.2).
H3C CH3
CH3
Figure. 2.2: Structure of cymene
a
41
c
b
42
Figure. 2.3: Characterization of cymene isolated from murrya koenigii leaf using
chloroform as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS
2.4.1.3. Compound 2 from chloroform extract
IR spectrum (ν, cm-1) of compound 2 revealed bands at 719, 813, 927.79 cm-1,
1020, 1055, 1107 cm-1 (-C=C-mono subs), 1278, 1303, 1363, 1380 cm-1 (-C-C- str),
1460, 1514 cm-1 (H-C-H bend), 2869, 2925, 2960, 3016, 3049 cm-1 (H-C-H Asy str).
The chloroform extract of compound 2 was tested using a GC and the compound
yielded a single peak confirming the presence of only one compound. The retention time
was 22.5 min., with 98.74% purity.
The spectral data of compound 2 was found to be in good agreement with the
reported value for caryophyllene [103]. In the 1H-NMR spectrum of compound 2, the
peaks appearing at 1.29 δ and 1.96 δ indicate the presence of the CH2 group in the
molecule. The peak appearing at 2.73 δ shows the presence of a cyclobutane linkage
between the five-membered rings and the six-membered rings. The peaks appearing in
the range of 4.75 and 5.25 δ are due to presence of ethylene linkage.
d
43
GC-MS studies revealed a sharp peak with a retention time of 8.4 minutes, with
m/z value of 221 which corresponds to the molecular weight of caryophyllene (220.35).
From the GC-MS and 1H-NMR data (given below) we conclude that the isolated
compound is caryophyllene, whose structure is given below in Figure 2.4.
Figure. 2.4: Chemical structure of caryophyllene
H3CCH3
H2C
CH3
HH
a
44
c
b
45
Figure 2.5: Characterization of caryophyllene isolated from Murrya koenigii leaf using
chloroform as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS
2.4.2. Ethanol extract from Murrya koenigii
UV spectrum of the ethanol extract of the Murraya koenigii leaf revealed the
presence of eight peaks at 671, 610, 510, 490, 417, 298, 263.4 and 223.8 nm,
corresponding to eight different compounds. Of these, we were interested in studying
only the two major compounds whose absorption peaks were at 223.8 nm and 263.4 nm.
The extract was separated using a silica gel column, employing hexane as the
eluent initially. The polarity of the solvent was increased gradually using a mixture of
hexane and acetone. The first two fractions of the extract were obtained using difference
in polarity of the eluent. The first compound isolated (using 100% hexane) was analyzed
by UV spectrophotometer, which gave an absorption maximum of 223 nm. Column
chromatography was continued using hexane:acetone (95:5 v/v) mixture for the isolation
of the second compound. On UV spectrophotometery, the second compound exhibited an
absorption maximum of 263.4 nm.
d
46
2.4.2.1. Compound 1 from ethanol extract
IR spectrum (ν, cm-1) of compound 1 revealed bands at 1690 - 1760 cm-1 (-C=O-),
1259 cm-1, 1126 cm-1 (-C-C- str), 1058 cm-1, 1028 cm-1 (-C=C-)
GC analysis of the ethanol extract of compound 1 revealed a single peak with a
retention time of 8.53 min and 99.465% purity.
1H-NMR spectra of the extracted compound was found to be in good agreement
with the reported data [104]. In the 1H-NMR spectrum of compound 1, the signal
appearing at 1.37 δ and 1.6 δ indicate the presence of the CH2 group in the compound.
The signal appearing at 3.93 δ shows the presence of a C-C linkage between the
five-membered rings and the six-membered rings. The spectrum also showed a signal at
6.75 δ corresponding to the α, β – unsaturated olefin while the terminal olefin displays
signals at 4.85 – 4.7 δ. Two methyl groups appeared as singlets at 1.8 δ and 1.75 δ.
GC-MS studies revealed a sharp peak with m/z value of 150, which corresponds
to the molecular weight of carvone (150.2). From the NMR and GC-MS data (Figure 2.6)
we conclude that the isolated compound is carvone.
O
H
Figure. 2.6: Chemical structure of carvone
47
a
b
48
Figure. 2.7: Characterization of carvone isolated from Murrya koenigii leaf using ethanol
as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS
d
c
49
2.4.2.2. Compound 2 from ethanol extract
The ethanol extract of compound 2 gave a single peak on analysis with GC,
confirming the presence of a single compound with a retention time of 5.916 minuntes
and 97.44% purity.
IR spectrum (ν, cm-1) of compound 2 revealed bands at: 927.79 cm-1, 1058 cm-1
(-C=C-mono subs), and 1126 cm-1 (-C-C- str).
In 1H-NMR spectrum of compound 2, the peaks at 1.50 δ clearly showed the
presence of the CH group in the molecule. The peak around 2.15 δ indicated the presence of
the CH group of an aliphatic molecule. The peak around 3.93 δ in the spectrum indicated the
presence of a C-C linkage between the five-membered ring and a six-membered ring.
The peak at 7.32 δ was due to the presence of an aromatic ring in the compound.
GC-MS studies revealed a sharp peak with a m/z value of 136.2 which
corresponds to the molecular weightt of phellandrene (136.2). The NMR and GC-MS
data taken together confirms that the compound is phellendrene (Figure. 2.8). The GC,
FTIR, NMR and GCMS spectra are shown in Figure 2.9.
Figure. 2.8: Chemical structure phellendrene
50
a
b
51
Figure. 2.9: Characterization of phellendrene isolated from Murrya koenigii leaf using
ethanol as solvent a) FTIR, b) GC, c) 1H NMR, d)GC MS
d
c
52
3. Mentha arvensis L. (mint leaves)
‘Mint’...just say the word and cool, refreshing images come to mind: frosty
glasses of lemonade garnished with curly springs of spearmint; the clean, chilling taste of
a mint candy cane. Even chewing gum, mouthwash, and toothpaste companies use
images of crisp, clean snowy slopes to let us know how refreshing their mint flavoured
products are. Delicious recipes for soups and deserts have Mint as an ingredient. Mint
based remedies are available for various health troubles and countless other uses [105].
The plant Mentha arvensis L. belonging to the family Rutaceae, is native to India
and now distributed in most of southern Asia and European countries. It is commonly
called ‘Pudina’ in most Indian vernacular languages.
3.1. Main constituents
The essential oil of mint (up to 2.5% in the dried leaves) is mostly made up from
menthol (ca. 50%), menthone (10 to 30%), menthyl esters (up to 10%) and monoterpene
derivatives (pulegone, piperitone, menthofurane). Traces of jasmone (0.1%) improve the
oil's quality remarkably. Menthol and menthyl acetate are responsible for the pungent and
refreshing odour; they are mostly found in older leaves and are preferentially formed
during long daily sunlight periods [106].
3.2. Materials and Methods
Fresh Mint leaf was collected from Siruvani crop fields, Coimbatore, India.
Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform (Merck,
Germany), absolute alcohol (Jiangsu Huaxi, China), sodium sulphate (Na2SO4;
Qualigens, India), TLC plates (Merck, Germany), tris.HCl(Loba, India) were used. All
the other materials and experimental techniques are as described in the previous section.
3.3. Results and Discussion
3.3.1. Chloroform extract of mint leaf
Mint leaf extracted with chloroform contained three compounds, as confirmed by
TLC and UV-Vis spectrophotometry. The major peak with a λmax of 223.3 nm indicated
53
the presence of carvone as one of the major constituents in the extract. This was further
confirmed by IR, NMR, GC and GC-MS analyses. The second peak observed at λmax of
263.4 nm was that of phellendrene.
3.3.2. Ethanol extract of mint leaf
TLC analysis of the ethanol extract exhibited 5 spots and UV-Vis spectrophotometry
revealed 5 peaks. However, carvone peak was not seen.
The extract was separated using column chromatograpy using a mixture of hexane
and acetone (9.6:0.4). The eluent had one compound which did not show absorbance
under UV. This compound was solid, colourless and had a sweet odour. According to
literature, mint leaves have 76% menthol which is solid in nature, indicating that this
compound could be menthol.
The IR spectrum revealed sharp bands at 3580 cm-1 to 3650 cm-1 due to the
presence of phenol or alcohol group. The band at 1126.47 cm-1 was due to -C-C-
stretching. The band at 2931.90 cm-1 was due to -C-H stretching. The band at 1259.96
cm-1 was due to-C-C- stretching.
The 1H-NMR data of extracted menthol was found to be in good agreement with
the reported data [107]. In the 1H-NMR spectrum, the characteristic signals for isopropyl
group appeared as multiplet at δ 2.15 and as doublet at δ 0.92. The -CH- proton attached
to –OH group was deshielded and was observed in the downfield at δ 3.4.
GC-MS analysis revealed a sharp peak with a retention time of 13.48 minutes,
with m/z value of 156.2, which corresponds to the molecular weight of menthol (155.6).
GCMS data and other spectral information confirm the isolated compound to be menthol
(Figure 2.10). The HPLC, FTIR, NMR and GCMS spectrum is shown in Figure 2.11.
54
OH
Figure. 2.10: Chemical structure of menthol
0.0 2.5 5.0 7.5 10.0 min
-100
0
100
200
300
400
500
600
700
800
mAU
/8.509
a
b
55
Figure. 2.11: Characterization of menthol isolated from mint leaf using ethanol as solvent
a) FTIR, b) HPLC, c) 1H NMR, d)GC MS
c
d
56
4. Curcuma lunga (Turmeric)
India has a rich history of using plants for medicinal purposes. Turmeric (Curcuma
longa L.) is a medicinal plant extensively used in ayurveda, unani and siddha medicine,
and as home remedy for various diseases [108]. C. longa L., botanically related to ginger
(Zingiberaceae family), is a perennial plant having a short stem with large oblong leaves
and bears ovate, pyriform or oblong rhizomes, which are often branched and brownish-
yellow in colour. Turmeric is used as a food additive (spice), preservative and colouring
agent in Asian countries, including China and South East Asia. It is also considered as
auspicious and is a part of religious rituals. In ancient Hindu medicine, it was extensively
used for the treatment of sprains and swelling caused by injury [109].
In recent times, traditional Indian medicine uses turmeric powder for the
treatment of biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic
disorders, rheumatism and sinusitis. In China, C. longa is used for diseases associated
with abdominal pains. The colouring principle of turmeric is the main component of this
plant and is responsible for the anti-inflammatory property. For the last few decades,
extensive work has been done to establish the biological activities and pharmacological
actions of turmeric and its extracts. Curcumin (diferuloylmethane), the main yellow
bioactive component of turmeric has been shown to have a wide spectrum of biological
actions. These include its, antioxidant, antibacterial, and antifungal activity. Clinically,
curcumin has already been used to reduce post-operative inflammation. Safety evaluation
studies indicate that both turmeric and curcumin are well tolerated at a very high dose
without any toxic effects. Thus, both turmeric and curcumin have the potential for the
development of modern medicine for the treatment of various diseases [108].
Turmeric is the rhizome or underground stem of a ginger-like plant. It is usually
available ground, as a bright yellow, fine powder. The whole turmeric is a tuberous
rhizome, with a rough, segmented skin. The rhizome is yellowish-brown with a dull
orange interior that looks bright yellow when powdered. The main rhizome measures
2.5 - 7 cm in length with a diameter of 2.5 cm with smaller tubers branching off.
57
4.1. Materials and Methods
Fresh turmeric plant was collected from Siruvani crop fields, Coimbatore, India.
Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform (Merck,
Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC
plates (Merck, Germany), acetone (Merck- Germany) Tris.HCl (Loba, India) were used.
All experimental techniques were as described the previous sections.
4.2. Results and Discussion
4.2.1. Petroleum ether extract of turmeric
Turmeric was first extracted with petroleum ether at 40 – 60 °C and the extract
was subjected to TLC and UV-Visible spectrometry. The petroleum ether extract gave
only one spot on TLC and only one peak in UV-Visible spectrometry. The λmax of this
peak was 420.1 nm.
Figure 2.12: UV spectrum of petroleum ether extract of C. longa containing curcumin.
According to literature turmeric is a yellow solid that contains curcumin and has a
λmax of 420 nm. The petroleum ether extract on drying resulted in a yellow coloured solid
Indicating that this compound could be curcumin.
IR spectrum of the extract revealed the presence of a broad peak from 3580 cm-1 to 3
650 cm-1 due to the presence of phenols or alcohol groups. The band at 1710 cm-1 to 1720 cm-1
58
could be due to -C=O stretching, and the band at 1900 cm-1 to 2000 cm-1 may be due to alkenes
stretching and the band at 1259.96 cm-1 could be due to -C-C- stretching band.
HPLC analysis of the chloroform extract exhibited a single peak confirming the
presence of a single compound with a retention time of 2.62 minutes and with 99.4% purity.
The 1H-NMR data of extracted compound was found to be in good agreement
with the reported data [110]. In the 1H-NMR spectrum, the signal at δ 3.82 corresponds to
the methyl (-O-CH3) protons while the two signals at δ 6.3 (-C=CH-OH) and at 9.64
(-CH=CH-OH) corresponds to the enol form. The α, β unsaturated olefins (-CH=CH-
C=O) displayed two signals at δ 6.74 and δ 7.52 with high coupling constant (J=16 Hz),
while the aromatic protons gave rise to three signals at δ 6.82 (d), 7.14 (d), and 7.31 (s).
MS analysis revealed a sharp peak with a m/z value of 368.97 corresponding to
the molecular weight of curcumin (368.38). All the results taken together confirm that the
compound isolated was curcumin, whose structure is given below (Figure 2.13). The GC,
FTIR, NMR and MS spectra of curcumin is shown in Figure 2.14.
O O
H3CO
HO
OCH3
OH
H
Figure 2.13: Chemical structure of curcumin
59
a
b
60
Figure. 2.14: Characterization of curcumin isolated from Curcuma longa using
chloroform as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS
d
c
61
4.3. Ethanol extract of turmeric
Turmeric was extracted with ethanol and analyzed by TLC and UV-Visible
spectrophotometry. The extract gave two spots on TLC and two peaks when analyzed by
UV-Visible spectrophotometry. The λmax of the two peaks were 420.1 nm (corresponding
to curcumin) and 221 nm (Figure 2.15).
Figure 2.15: UV spectrum of ethanol extract of Curcuma longa.
The peak at 221 nm could be another compound or impurity. The extract was
dried to yield a yellow powder, which was subsequently washed with chloroform to
finally yield pure curcumin powder.
5. Citrullus colocynthis (L.)
Citrullus colocynthis is the plant which produces colocynth apples and is very
similar to the common watermelon vine. The colocynth apples are small, hard fruit with a
bitter pulp. The plant bears solitary sterile flowers and branched tendrils. The colocynth
plant is a native of arid soils. It has a large, fleshy perennial root, which sends out
slender, tough, angular, scabrid vine-like stems. These usually lie on the ground for want
of something to climb over, but which, if opportunity presents, climb over shrubs and
herbs by means of axiliary branching tendrils. The leaves are angular, lobed and, as
already stated, almost the exact duplicate of watermelon leaves. The flowers are yellow,
long-peduncled, solitary in the axils of the leaves. They are monecious, the stamens and
pistils being borne in different flowers on the same plant. Each has a yellow campanulate,
five-lobed corolla and a five-parted calyx. The female flowers are readily distinguished
62
by a globose, hairy, inferior ovary. The fruit is globular, smooth, with a hard but thin
rind, something like a gourd. It is filled with a soft, white pulp, in which are imbedded
numerous seed. This pulp is the article used in medicine [111].
The colocynth, or bitter-apple, of commerce, when deprived of its rind, as is
mostly the case, presents a white, light and spongy pulp that readily breaks into three
wedge-shaped pieces, each holding imbedded near its outer rounded surface a number of
flat, ovate seeds. The proportion between pulp and seed varies according to different
authors, from 23 to 33 per cent of pulp and 67 to 77 per cent of seed. The intensely bitter
taste of colocynth resides in the pulp only, while the seeds at best contain only traces of it;
hence the inert seeds are removed before making pharmaceutical preparations of colocynth.
The bitter taste and the powerful medicinal virtues of the pulp are due to the presence of a
probably amorphous glucoside colocynthin, first identified and named by Meissner and by
Vauquelin (1818), and later investigated and obtained in a much purer form [112].
Colocynthin is soluble in water and alcohol, but insoluble in benzol, benzin,
carbon disulfide and ether. Dilute acids resolve it into dextrose and tasteless colocynthein,
acetic acid being likewise formed [113]. Walz obtained from an alcoholic extract of
colocynth an ether-soluble crystalline and tasteless substance insoluble in water, which he
called colocynthin [114]. The ash of the pulp varies from 8.6 to 14 per cent while that of
the seeds amounts to about 2.5 percent [112].
Citrullus colocynthis (L.) has been the object of innumerable investigations.
Citrullus colocynthis has been used for its medicinal properties since ancient times. The
oldest record of Citrullus colocynthis is supposed to be found in connection with Prophet
Elisha’s miracle. During the second half of the nineteenth century [115]. While Hexke
could not establish the glycosidic character of colocynthin, Johannson described its
hydrolysis products and reported the isolation of colocynthein and α-elaterin together
with other substances [116]. Later Naylor and Chappe obtained colocynthin in a
crystalline form [117]. Power and Moore, who doubted the purity of the substances obtained
by their predecessors, reinvestigated the plant and isolated citrullol, α-elaterin, an alkaloid
and various other components [118]. While Hamilton and Kermac could not repeat the
63
previous work and did not isolate aelaterin, Siddiqui et al., obtained a series of crystalline
compounds including α-elaterin [119]. A recent publication by Khadem and Rahman’O
reported the isolation of a glycoside using a procedure that is followed in this work [120].
The distribution of cucurbitacins among the various species of the Cucurbitaceae
has been studied extensively by Enslin and a group of South African workers [121]. They
have found that these substances occur in nature as glycosides or as aglycones according
to the presence or absence of an enzyme named elaterase, a glycoside hydrolase, of
undetermined specificity which is capable of rapidly hydrolysing the glycosides.
A comparative study of several species showed that elaterase is present in high
concentration in genera such as Cucumis and Lugenaria, whereas it is absent in Citrulius
and Cucurbita. The complicated structure of the cucurbitacins and their high sensitivity
to hydrolytic agents accounts for the difficulties encountered by several authors in the
identification and the study of the nature of the aglycones and their glycosides. This
identification could best be done when they were extracted and isolated from plants in
which the enzyme was present, as for instance in Ecbaliium elaterium or other species.
5.2. Materials and methods
Fresh Citrulus colocynthis plants were collected from coastal areas of Tuticorin,
India. Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform
(Merck, Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC
plates (Merck, Germany)., acetone (Merck, Germany), Tris.HCl (Loba, India) were used as
obtained. Experimental techniques followed were as described in the previous sections.
5.3. Results and Discussion
The Citrullus colocynthis fruit pulp was taken for extraction using ethanol as
solvent. A paste like compound was obtained which was washed with chloroform and the
solvent was carefully removed in nitrogen atmosphere. TLC and UV-Visible
spectrophotometer analysis of this extract revealed the presence of a single compound
which was further analysed by FTIR, HPLC, NMR, MS.
The FTIR analysis of the extract gave a broad band at 3477 cm-1 which was due to
OH stretching, bands at 2974, 2931, 2879 cm-1 can be attributed to H-C-H asymmetric
64
stretching, bands at 1707 and 1637 cm-1 are attributed to C=O stretching, bands at 1512,
1458, 1429, 1379 cm-1 can be assigned to -C-C- stretching, bands at 1265, 1222 cm-1 could
be because of -C-C- stretching, and bands at 1039, 927, 615cm-1 due to -C=C- mono subs.
The ethanol extract of compound was analyzed by HPLC and the extract gave a
single peak confirming the presence of a single compound with a retention time of 5.68
min and 99.6% purity.
The 1H-NMR data of extracted compound was found to be in good agreement
with the reported data [122]. In the 1H-NMR spectrum, the signals at δ 2.232 (3, 1H, d,
J=5.590), 1.443 (10, 3H), 1.443 (11, 3H), 1.423 (14, 3H), 3.825 (15, 1H, ddd, J=7.320,
J=5.590, J=2.190), 1.878 (16, 1H, dd, J=10.698, J=7.320), 1.833 (16, 1H, dd, J=10.698,
J=2.190), 2.474 (18, 1H, d, J=16.680), 2.447 (18, 1H, d, J=16.680), 1.742 (21, 1H, dd,
J=10.190, J=3.380), 2.245 (22, 1H, ddd, J=13.283, J=10.190, J=7.129), 2.249 (22, 1H, ddd,
J=13.283, J=5.229, J=3.380), 5.580 (23, 1H, dd, J=7.129, J=5.229), 3.039 (30, 1H, d, J=6.777).
Mass spectra studies reveal a single sharp peak with a m/z value of 515.41 which
corresponds to the molecular weight of cucurbitacin I (514.70). The HPLC, FTIR, NMR
and MS spectra are shown in Figure 2.16.
a
65
9 8 7 6 5 4 3 2 1 0 ppm
0.8035
0.8208
0.8297
0.8325
0.8534
0.8556
0.8620
0.8852
0.8928
0.9061
0.9218
0.9304
0.9387
0.9541
0.9626
0.9665
0.9858
0.9940
0.9961
1.0175
1.0767
1.0841
1.0917
1.1017
1.1090
1.1149
1.1395
1.3474
1.3592
1.4165
1.4248
1.4329
1.5905
1.5985
1.6067
1.6225
1.6304
1.6380
1.6473
1.6550
1.6644
1.6731
1.6781
1.6862
1.9438
1.9489
1.9526
1.9577
1.9790
1.9827
1.9878
2.1503
2.1573
2.1678
2.1747
2.1852
2.1922
3.3987
3.4108
3.4229
6.90
0.90
13.56
3.00
2.08
1.91
2.04
4.85
2.03
1.99
2.00
Current Data ParametersNAME CRR-SMR-I-43EXPNO 1PROCNO 1
F2 - Acquisition ParametersDate_ 20110910Time 17.27INSTRUM spectPROBHD 5 mm BBO BB-1HPULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8223.685 HzFIDRES 0.125483 HzAQ 3.9846387 secRG 128DW 60.800 usecDE 6.00 usecTE 294.4 KD1 1.00000000 secTD0 1
======== CHANNEL f1 ========NUC1 1HP1 14.00 usecPL1 -0.90 dBSFO1 400.1324710 MHz
F2 - Processing parametersSI 32768SF 400.1300012 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.003.353.403.45 ppm
3.374
3.385
3.399
3.411
3.423
3.437
3.448
c
b
66
Figure. 2.16: Characterization of cucurbitacin I isolated from Citrullus colocynthys using
ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS
Based on all the spectral analysis, the isolated compound was identified as
cucurbitacin I whose structure is given below (Figure 2.17).
Figure 2.17: Chemical structure of cucurbitacin I
O
OOH
OH
OH
O
HO
d
67
6. Coriandrum sativum L.
Coriander (Coriandrum sativum) also known as cilantro, Chinese parsley or dhania, is
an annual herb belonging to the family Apiaceae. Coriander is native to regions spanning
from southern Europe and North Africa to southwestern Asia. It is a soft, hairless plant
growing to 50 cm tall. The leaves are variable in shape, broadly lobed at the base of the
plant, and slender and feathery higher on the flowering stems. The flowers are borne in
small umbels, white or very pale pink, asymmetrical, with the petals pointing away from
the centre of the umbel longer (5–6 mm) than those pointing towards it (only 1–3 mm
long). The fruit is a globular, dry schizocarp 3–5 mm (0.12–0.20 in) in diameter [123].
The plant is grown widely all over the world for seed, as a spice, or for essential
oil production. At one time, Coriander was one among the world’s leading essential oil
plants [124, 125]. The composition of the essential oil of Coriander fruits in some of the
world has been studied and found to differ from each other. It has been reported that
Coriander seed oil contains linalool (60-70%) and 20% hydrocarbons and the
composition of the herb oil completely differs from the seed oil [126]. Rastogi and
Mehrotra reported detection of α-pinene, limonene, β-phellandrene, eucalyptol, linalool,
borneol, β-caryophyllene, citronellol, geraniol, thymol, linalyl acetate, geranyl acetate,
caryophyllene oxide, elemol and methyl heptenol in seed oil by TLC [127]. Telci et al.,
reported that in the ripe fruits, the content of essential oil is comparably low (typically,
less than 1%); the oil consists mainly of linalool (50 to 60%) and about 20% terpenes
(pinenes, γ-terpinene, myrcene, camphene, phellandrenes, α-terpinene, limonene,
cymene) [128]. Asolkar et al., reported a type from Mysore that contained high geranyl
acetate [129]. Coriander oil is a valuable ingredient in perfumes. Its soft, pleasant,
slightly spicy note blends into scents of oriental character. Ghani reported the presence of
linalool, pinene, cymene, phellandrene, geraniol and borneol [130].
6.2. Materials and Methods
Fresh Coriander plant was collected from Siruvani crop fields, Coimbatore, India.
Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform (Merck,
Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC
68
plates (Merck, Germany),, acetone (Merck, Germany) Tris.HCl (Loba, India) were used
as received. All the other techniques adopted were as described in the previous sections.
6.3. Results and Discussion
The chloroform extract was separated by silica gel column chromatography with
hexane as the solvent. The polarity of the solvent was increased step by step using
acetone. The first two fractions of the extract were obtained by the difference in polarity
of the eluent. The first compound was isolated using hexane (100%) and analysed by
FTIR, GC, GC MS, HPLC, 1H-NMR. The second compound was isolated using a
mixture of 99% hexane and 1% acetone. This compound too was analysed by FTIR, GC,
GC MS, HPLC, 1H NMR.
6.3.1. Chloroform extract of Coriander leaves
6.3.1.1. Compound 1
Compound 1 isolated using chloroform, gave the same characteristic peak as
cymene in Gas chromatography. Cymene is explained in detail under the curry leaf
section. The structure of compound 1 was confirmed by mass spectrophotometry and
GC-MS which gave values that were identical to that of cymene. The FTIR, GC, GC MS
and 1H-NMR characterization data is already presented in the section explaining the
extract analysis of curry leaf.
6.3.1.2. Compound 2
The FTIR analysis of the extract gave a broad band at 3477cm-1 to 3000 cm-1
which was due to OH stretching, bands at 2974, 2931, 2879 cm-1 can be attributed to H-
C-H asymmetrical stretching, bands at 1707 and 1728 cm-1 are due to C=O stretching,
bands at 1512, 1458, 1429, 1379 and 1599 cm-1 can be assigned to -C-C- stretching,
bands at 1265, 1222 cm-1 is because of -C-C- stretching, and bands at 1039, 927, 615cm-
1 is due to -C=C- mono subs.
The chloroform extract of compound 2 was tested by HPLC and it gave a single
peak with a retention time of 3.822 minutes, with 99.2% purity.
69
The 13C-NMR analysis of compound 2 displayed the characteristic signal of 1,4
dihydroquinone at 152.5 δ, 150.2 δ, 118.5 δ and 117.2 δ. The methane carbon groups
(-O CH-O-) appeared at 101.2 δ while all other methane carbons ( ) appeared
between 77.1 δ and 68.5 δ.
Mass spectral analysis revealed a sharp peak with a m/z value of 272.39 which
corresponded to the molecular weight of arbutin (272.25), confirming its presence.
The HPLC, FTIR, NMR and GCMS spectra are shown in Figure 2.18.
a
70
c
b
71
Figure. 2.18: Characterization of arbutin isolated from Coriandrum sativum using
chloroform as solvent a) FTIR, b) HPLC, c) C13 NMR, d) MS
Thus based on spectral analysis, the isolated compound was identified as Arbutin,
whose structure is given below (Figure 2.19).
Figure. 2.19: Chemical structure of arbutin
OH
OO
OH
HO
HO
OH
d
72
6.3.2. Ethanol extract of Coriander leaves
The ethanol extract was separated by silica gel column chromatography using
hexane as a solvent. The polarity of the solvent was increased step by step using acetone.
The first two fractions of the extract were obtained using difference in polarity of the
eluent. The first compound was isolated using hexane (100%) and analysed by FTIR, GC,
GC MS, HPLC, 1H NMR. The second compound was isolated using a mixture of 99%
hexane and 1% acetone, and analysed by FTIR, GC, GC MS, HPLC, 1H-NMR techniques.
6.3.2.1. Compound 1
TLC and UV-Vis spectroscopy data of compound 1 isolated using ethanol exactly
matched the results obtained for phellendrene (Section 2.4.2.2), thereby confirming
its presence.
6.3.2.2. Compound 2
The FTIR analysis of the second compound gave a broad band at 3482 cm-1 to
3000 cm-1 which was due to OH stretching, bands at 2974, 2931, 2879 and 2788 cm-1 can
be attributed to H-C-H asymmetrical stretching, bands at 1707, and 1728 cm-1 are due to
C=O stretching, bands at 1512, 1228, 1280, 1458, 1429, 1379 and 1599 cm-1 can be
assigned to -C-C- stretching, bands at 1265 and 1222 cm-1 is because of -C-C- streching,
and bands at 1039, 927, 615 cm-1 is due to -C=C- mono subs.
Analysis by HPLC revealed that compound 2 had a retention time of 8.509
minutes, with 98.2% purity. The HPLC analysis confirmed the presence of this
compound as the sole compound.
The 1H-NMR spectrum (Figure 2.20) displayed the two aromatic protons of the
fused rings of rutin and appeared as singlets at 6.13 δ and 6.35 δ, while that of attached
aromatic ring showed signals at 6.79 δ (d), 7.52 δ (d), 7.5 δ (s). The acetal protons
displayed two signals at 6.32, 6.37 and 6.31 δ while methine protons of carbohydrate unit
gave signals at 6.18 δ and 6.39 δ.
Mass spectral studies (Figure 2.20) revealed a sharp peak with a m/z value of
664.24 which corresponds to the molecular weight of of Rutin (664.57). Based on MS,
73
the major peak was confirmed to be that of Rutin. The HPLC, FTIR, NMR and MS
spectra are shown in Figure 2.20.
a
b
74
Figure. 2.20: Characterization of rutin isolated from Coriandrum sativum using ethanol
as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS
Thus based on spectral analysis, the isolated compound was identified as rutin
whose structure is given below (Figure 2.21).
d
c
75
Figure. 2.21: Chemical structure of rutin
7. Eichhornia crassipes (Mart.) Solms. – Laub.
Its habitat ranges from tropical desert to subtropical or warm temperate desert to
rainforest zones. It tolerates annual precipitations of 8.2 dm to 27.0 dm (mean of
8 cases = 15.8 dm), annual temperatures from 21.1°C to 27.2°C (mean of 5 cases = 24.9°C),
and its pH tolerance is estimated at 5.0 to 7.5. It does not tolerate water temperatures
>34°C. Leaves are killed by frost and salt water, the latter trait being used to kill some of
it by floating rafts of the cut weed in the sea. Water hyacinths do not grow when the
average salinity is greater than 15% of sea water. In brackish water, its leaves show
epinasty and chlorosis, and eventually die [131].
7.1. Phytoremediation, waste water treatment
The roots of Eichhornia crassipes naturally absorb pollutants, including lead,
mercury, and strontium-90, as well as some organic compounds believed to be carcinogenic, in
concentrations 10,000 times that in the surrounding water [132]
There are two types of water hyacinth available in the region
1) Long type 2) Dwarf type.
O
OH
OH
O
OH
HO
O
O
OH
O
HO OH
OOH
OH
HO
HO
76
The former type is mostly available in stagnant water polluted with high effluents
while the later is available mostly in the paddy field. Analysis indicated that these plants
contain lot of potassium and sodium salts which hamper the balances of magnesium and
causes the symptoms of diuretics. The other anti-metabolites are due to the presence of
oxalates which affects its utilization.
Tannin content was also recorded to be a little lower in long type compared to
dwarf type of water hyacinth. There was much difference in tannin content of leaf, stem.
The tannin content of fresh water hyacinth was found to be a little higher than the
naturally dried plants. Alkaloids and glycosides were not present.
7.2. Bioenergy
Because of its extremely fast growth rate, Eichhornia crassipes is an excellent source
of biomass. One hectare of standing crop can thus produce more than 70,000 m3 of biogas
[131]. According to Curtis and Duke, one kg of dry matter can yield 370 liters of biogas,
giving a heating value of 22,000 kJ/m3 compared to pure methane. Wolverton and
McDonald report only 0.2 m3 methane per kg, indicating requirements of 350 MT
biomass/ha to attain the 70,000 m3 yield projected by the National Academy of Sciences
[133]. Ueki and Kobayashi mention more than 200 MT/ha/yr [134]. Reddy and Tucker
found an experimental maximum of more than a half ton per day.[13] Bengali farmers collect
and pile up these plants to dry at the onset of the cold season; they then use the dry water
hyacinths as fuel [135].
In India, a ton of dried water hyacinth yield circa 50 liters ethanol and 200 kg
residual fiber. Bacterial fermentation of one ton yields 26,500 cu ft gas (600 Btu) with
51.6% methane, 25.4% hydrogen, 22.1% CO2, and 1.2% oxygen. Gasification of one ton
dry matter by air and steam at high temperatures (800°) gives circa 40,000 ft3 (circa 1,100 m3)
natural gas (143 Btu/cu ft) containing 16.6% H3, 4.8% methane, 21.7% CO, 4.1% CO2,
and 52.8% N. The high moisture content of water hyacinth, adding so much to handling
costs, tends to limit commercial ventures [136]
77
7.3. Materials and methods
Fresh Eichhornia crassipes plant was collected from Ukkadam pond, Coimbatore,
India. Petroleum ether (Merck, Germany), hexane (Merck, Germany), chloroform
(Merck, Germany), absolute alcohol (Jiangsu Huaxi, China), Na2SO4 (Qualigens, India), TLC
plates (Merck, Germany), Acetone (Merck, Germany), Tris.HCl (Loba, India) were used as
received. The experimental techniques were adopted as described in previous sections.
7.4. Results and Discussion
The ethanol extract was separated by silica gel column chromatography using hexane
and acetone as solvents. The polarity of the solvent was increased step by step using acetone.
Three fractions of the extract were obtained using difference in polarity of the eluent. The first
compound was isolated employing a mixture of hexane and acetone in the ratio of 99.5:0.5.
The extract was analyzed by FTIR, GC, GC-MS, HPLC, 1H-NMR. The second compound was
isolated using a mixture of hexane and acetone in the ratio 98.5: 1.5. The third compound was
isolated using a mixture of hexane and acetone in the ratio 95:5. The second and third
compounds were again analysed by FTIR, GC, GC-MS, HPLC and 1H-NMR.
7.4.1. Compound 1
IR spectrum (ν, cm-1) of compound 1 revealed a broad band at 3100 cm-1 to 3000 cm-1
which was due to OH stretching. Bands at 1721 and 1681 cm-1 can attributed to C=O
stretching, bands at 1259 cm-1, 1126 cm-1 and 1301 cm-1 can be attributed to
-C-C- stretching, bands at 1058 cm-1, and 1028 cm-1 can be because of -C=C- stretching,
band at 873 cm-1 may be due to -C=C- mono subs, and those at 538, 718, 1514 cm-1 may
be due to H-C-H bending.
HPLC analysis of the ethanol extract revealed a single peak with a retention time
of 2.38 minutes and 99.74% purity, confirming the presence of a single compound.
The 1H-NMR can be viewed better as pyranone fused with aromatic ring and
attached with another aryl group. In the 1H-NMR spectrum, the protons of fused ring
appeared as two singlets at 6.18 and 6.39 δ while the protons of the attached ring
appeared at 7.69 δ (s), 7.52 δ (d) and 6.85 δ (d).
78
Mass spectal studies (Figure 2.22) exhibited m/z value of 176.17, which
corresponds to the molecular weight of 7-hydroxyl-4-methylcoumarin (177.25),
confirming the presence of methyl hydroxyl coumarin.
a
b
79
Figure. 2.22: Characterization of methyl hydroxyl coumarin isolated from Eichhornia
crasipus leaf using ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS
c
d
80
7.4.2. Compound 2
IR spectrum (ν, cm-1) of compound 2 revealed a broad band at 3400 cm-1 to 3358 cm-1
which was due to OH stretching. Bands at 2721 and 2681 cm-1 can attributed to C=O
stretching, those at 1259 cm-1, 1126 cm-1 and 1301 cm-1 can be attributed to
-C-C- stretching, the ones at 1058 cm-1and 1028 cm-1 to -C=C- stretching, the one at
873 cm-1 to -C=C- mono subs, and those at 538, 718, 1514 to H-C-H bending.
HPLC analysis of the second ethanol extract fraction resulted in a single peak
with a retention time of 3.506 minutes and a purity of 98.4%, confirming the presence of
a single compound.
The 1H-NMR spectrum (Figure 2.23) displayed signals at 7.92 δ (dd, J = 8.0Hz,
3.2Hz, 2H), 7.56-7.60 δ (m, 2H), 7.52 δ (dd, J = 2.1Hz, 1.2Hz, 2H), 7.32 δ (dd, J =4.0Hz,
1.2Hz, 2H), 3.78 δ (s, 2H).
Mass spectra (Figure 2.23) of the second compound exhibited a m/z value of
338.26 which corresponds to the molecular weight of quercetin dehydrate (338.72).
The HPLC, FTIR, NMR and MS spectra are shown in Figure 2.23.
a
81
b
c
82
Figure 2.23: Characterization of quercetin dehydrate isolated from Eichhornia crasipus
leaf using ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS
7.4.3. Compound III
IR spectrum (ν, cm-1) of compound 3 revealed a broad band at 3500 cm-1 to
2950 cm-1 which was due to OH stretching. Bands at 1705 and 1681 cm-1 can be
attributed to C=O stretching, those at 1893, 2869, 2925, 2978, 3016 and 3049 cm-1 to
H-C-H asymmetrical stretching, those at 1259 cm-1, 1126 cm-1 and 1301 cm-1 to
-C-C- stretching, the ones at 1058 cm-1 and 1028 cm-1 to -C=C- stretching, the one at
873 cm-1 to -C=C- mono subs, and those at 538, 718 and 1514 cm-1 to H-C-H bending.
The 1H-NMR spectrum (Figure 2.24) displayed signals at 8.7 to 9.6 ppm broad
singlet seems to be presence of phenolic OH, 9.8 to 10.2 ppm broad singlet is
also phenolic OH, 6.8 ppm to 7.2 ppm corresponding peaks indicates presence of
aromatic ring, 5.3 ppm and 5.5 ppm which peaks corresponds to O-CH2 protons. All the
δ values are given below, 4.443 δ (4, 2H, d, J=3.764), 3.337 δ (5, 1H, td, J=3.764,
J=2.690), 5.30 δ (7, 1H, d, J=2.680), 7.385 δ (12, 1H, d, J=2.033), 7.213 (23, 1H, d,
J=0.000), 7.204 δ (30, 1H, d, J=0.000), 5.283 δ (31, 1H, dd, J=3.470, J=2.680), 7.390 δ
d
83
(36, 1H, d, J=2.044), 7.106 δ (47, 1H, d, J=0.000), , 4.498 δ (55, 1H, dd, J=3.470,
J=3.460), 7.114 δ (71, 1H, d, J=0.000), 7.098 δ (78, 1H, d, J=0.000), 5.280 δ (79, 1H, dd,
J=3.460, J=2.690), 7.274 δ (95, 1H, d, J=0.000), 2.495 (103, 3H).
HPLC analysis of the third fraction of ethanol extract resulted in a single peak
with a retention time of 4.58 minutes and a purity of 99.4%, confirming the presence of a
single compound.
Mass spectra (Figure 2.24), showed that the third compound had a m/z value of
1699, which corresponds to the molecular weight of tannic acid (1701). The HPLC,
FTIR, NMR and MS spectra are shown in Figure 2.24.
a
84
b
c
85
Figure. 2.24: Characterization of methyl hydroxyl coumarin isolated from Eichhornia
crasipus leaf using ethanol as solvent a) FTIR, b) HPLC, c) 1H NMR, d) MS.
Based on spectral analyses compound 1 was identified as coumarin, compound 2 was
identified as quercetin dehydrate and compound 3 was identified as tannic acid.
The structure of coumarin, quercetin and tannic acid are given in Figure 2.25.
.
Quercetin Methylene hydroxyl coumarine
O OO O
OHOH
O
OH
OH
OH
OH
HO
O
d
86
Tannic acid
Figure. 2.25 : Chemical structure of quercetin, coumarin and tannic acid.
4. Conclusion
This chapter describes the collection, identification and certification of plant
sources. Details of experimental procedures for extraction, isolation, purification and
spectral investigation of natural antioxidants are also presented. Use of Soxhlet extraction
procedure with solvents of various polarities is explained. This chapter also deals with the
purification protocol of isolated compounds. Six different plants were selected from
which twelve compounds were isolated.
The characterization of isolated compounds using various techniques and
structure elucidation based on spectral investigation are described in this chapter. This
chapter presents the details of spectral/chromatographic investigations such as TLC, UV-
Vis spectrophotometery, FT-IR, 1H-NMR, 13C-NMR, GC, GC-MS, HPLC and MS. Table
2.2 summarizes the details of plant sources, the isolated natural antioxidants and the
investigation carried out to confirm their chemical structures.
O
OO
OO
O
O H
O H
H O
H O
O
O
H O
H O O H
O
O
H O O H
O
O
O H
O H
O H
O
OH
H O
O H
O
O
O H
O HO
O
H O O H
H O
O
O
O HH O
O
O
O H
O H
O H
87
Sl. No
Plant Sources Isolated Natural
Antioxidant FTIR GC
GC MS
HPLC MS 1H
NMR
13C NMR
1 Curry Leaf & Mint leaf
Carvone √ √ √ - - √ -
2 Curry, Mint & Coriander leaf
Phellandrene √ √ √ - - √ -
3 Curry, Mint & Coriander leaf
Cymene √ √ √ - - √ -
4 Curry leaf Caryophyllene √ √ √ - - √ -
5 Mint leaf Menthol √ - √ √ - √ √
6 Turmuric Curcumin √ - - √ √ √ -
7 Citrulus
colocynthis Cucurbitacin I √ - - √ √ √ -
8 Coriander Arbutin √ - - √ √ - √
9 Coriander, Citrulus
colocynthis Rutin √ - - √ √ √ -
10 Eichhornia
crasipes
3,3-methylene-bis (4-hydroxy
coumarin √ - - √ √ √ -
11 Eichhornia
crasipes Quercetin √ - - √ √ √ -
12 Eichhornia
crasipes Tannic acid √ - - √ √ √ -
Table 2.2: Isolated antioxidants and the charecterization investigation carried out.
88
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