SEARCH FOR NATURAL ANTIMICROBIAL AND BIOLOGICALLY ACTIVE AGENTS

ABSTRACT : OF THE

THESIS " ^ SUBMITTED FOR THE AWARD OF THE OEGREf OP

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SARFARAZ A H M E D

DEPARTMENT OF CHEMfSTRY AUGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2010

ABSTRACT

Abstract

The thesis entitled "Search for natural antimicrobial and biologically

active agents" is comprised of four chapters dealing with general introduction,

antimicrobial screening of some compound Unani formulations, antimicrobial and

biological activity of plants used as ingredients in Unani medicines and isolation and

characterization of their phytoconstituents.

Growing concern about health and physical fitness has booming impact on

renaissance of the traditionally used medicines as fast acting synthetic drugs, often

have side effects on human body. These old miracle plants have their existence as

herbal drugs despite considerable advancements in medical sciences and have the

key root of traditional medicine system.

Chapter 1 deals the importance of medicinal plants and how they come in

prominence in medicines through out the world. Impact of infectious diseases on

human health, use of medicinal plants/phytoconstituents in infectious diseases and

synergic action of phytomedicines has also been discussed in this chapter.

Chapter 2 describes antimicrobial activity of some Unani drugs used for the

treatment of various infectious diseases. These Unani drugs are manufactured and

marketed by Dawakhana Tibbiya College, Aligarh Muslim University, Aligarh.

The ethanolic extract of five Unani drugs (Qurs-e-Sartaan Kafoori, Qurs-e-

Suzak, Dawa-e-Dibba, Safoof-e-Bars and Safoof-e-Kharish) having multiple

botanical ingredients with some chemical substances and substances of animal

origin was studied for scientific evaluation for their antibacterial and antifungal

activity by agar well diffusion method against the Escherichia coli, Salmonella

typhimuriun (clinical isolates), Staphylococcus aureus, Brucella abortus S-19

(standard strains) and a yeast Candida albicans, a clinical isolate.

The antibacterial and antifungal activity was assessed by presence or absence

of zone inhibition or zone diameter. The antibacterial and antifungal activity was

observed against all the tested bacteria and yeast for all the tested drugs, except

Safoof-e-Kharish which is not active against Escherichia coli and Salmonella

typhimurium. Escherichia coli seem to be least sensitive towards these drugs as

revealed by its zone diameter.

In chapter 3 the antimicrobial activity and hepatoprotective effect of

Operculina turpethum (roots) aqueous extract (OTE) have been discussed.

Operculina turpethum, commonly known as trivrit or nishot, belongs to the Family

Convolvulaceae, is of tremendous ethno-medicinal value. Mainly, roots or stem bark

of this plant are traditionally used for medicinal purposes.

The antimicrobial activity of the Operculina turpethum extract (OTE) were

tested against some pathogenic microbes includes Salmonella typhimurium. Listeria

monocytogenes, Candida albicans and Cryptococcus neoformans by micro dilution

method. The results of the antimicrobial screening are highly encouraging. The

Operculina turpethum extract (OTE) of the plant inhibits the growth of Candida

albicans at the lowest concentration of 6.25 |ig/ml.

The therapeutic effect of Operculina turpethum extract (OTE) against

NDMA induced hepatotoxicity in rats was assessed by histological observations and

biochemical parameters.

Hepatic fibrosis was induced in adult male albino rats through serial

intraperitoneal administrations of NDMA at a concentration of 10 mg/kg body

weight on three consecutive days of each week over a period of three weeks. A

3

group of rats received Operculina turpethum extract (OTE) orally in doses of 75,

150 and 200 mg/kg body weight at 5 hours after the administration of NOMA. The

controls and treated animals were sacrificed on days-7, 14 and 21 after the start of

the administration of NOMA. The progression of hepatic fibrosis as well as the

amelioration effect of Operculina turpethum extract (OTE) was evaluated

histopathologically as well as by immunohistochemical staining for the activation of

hepatic stellate cells. Alterations in serum and liver biochemical parameters and

LDH isoenzymes were also studied. Serial administration of NDMA resulted in well

formed fibrosis in the liver. Staining of a-SMA demonstrated activated stellate cells

from day-7 onwards which was dramatically increased on day-21. An elevation of

liver function enzymes, serum hydroxyproline levels and LDH isoenzymes 4 and 5

were also observed. All these changes were remarkably reduced in Operculina

turpethum extract (OTE) administered animals and fibrogenesis was completely

absent. Our results suggest that Operculina turpethum extract (OTE) has

hepatoprotective effects against NDMA-induced hepatic fibrosis.

In chapter 4 antimicrobial activity and isolation and characterization of the

constituent of Acacia leucophloea Willd. (stem bark) have been discussed. The

antimicrobial activity of the fractions of ethanolic extract of the stem bark of Acacia

leucophloea Willd. were tested by micro dilution method against the two bacteria

and two fungi. Acetone fraction showed significant activity at 6.25 ng/ml against

Candida albicans while the minimum inhibitory concentration of ethyl acetate

fraction in Salmonella typhimurium and Candida albicans was 12.5 ^g/ml.

The ethyl acetate fraction of ethanolic extract was adsorbed on silica gel and

passed over a column of silica gel set with petroleum ether. The column was eluted

with mixture of solvents increasing order of polarity and the fractions were mixed

together on the basis of TLC pattern. The antimicrobial activity of these fractions

was tested also by the same protocol against the same microorganisms described

above. These fractions showed activity against all the tested microorganisms.

The ethyl acetate eluent of the column chromatography affords a compound

which is identified as a phenolic trimer (A-4) on the basis of spectral analysis

(MASS, 'H-NMR '^C-NMR). 'H-NMR spectra of the compound was complex

therefore the compound was further purified by preparative HPLC using C-18

column. This pre-purified compound was analyzed by UPLC-MS for purity using

gradient system.

This chapter also includes comprehensive review of constituents isolated

from the different part of the plant.

SEARCH FOR NATURAL ANTIMICROBIAL AND BIOLOGICALLY ACTIVE AGENTS

THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OP

B o t t o r of ^I)tlQ£iQpbp iN

CHEMISTRY

BY

SARFARAZ A H M E D

DEPARTMENT OF CHEMfSTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2010

T6435

v_y

DEDICATED

MY PARENT:

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALlGARH-202002, INDIA

Phones / Ext.

• \ ln t . (0571)2703515 3350.3351

Dated ...IU.1-. h

This is to certify that the v^oxk discussed in this thesis is the original

contribution of the candidate and is suitable for the submission for the

degree of Doctor of Philosophy.

(Nizam U. Khan) Ph.D.

Professor of Chemistry

ACKNOWLEDGEMENTS

It my pkasure to express my sincere than^ and deep sense of gratitude to my supervisor

^Professor !Nizam V. %fian for his patient guidance, continued interest and encouragement and

support tHrough out this wor^ I am indebted to him for Seing toCerant of my personal shortcomings

andprofessionaC[imitations, afways helping me to remove my douSts anddifficuCties through many

iCCuminating conversation andaCCthe inspiration, I required to ta^ this wor^to completion, Infact

to wor^with him was a most pleasurable, enriching and memorable ej(perience of my life and I ta^

this opportunity to pay my sincere than^andSest regard to him.

I am than^C to (professor % S. Siddiqui Chairman (Department of Qf^mistry, JlCigarh

MusCim University JlCigarh, for providing the required infrastructure in the (Department of

Chemistry Migarh 'MusCim University J^Cigarh to carry out my wor^without which nothing would

have Been feasible. Jle has always have been source of inspiration and great support to me whenever

I needed from the department.

I feel obliged to (Dr. M. Owais, Interdisciplinary (Biotechnology Unit. Jlligarh ^Muslim

University Jiligarh and (Dr (Rjaz Jlhmad, (Department of Zoology, jAligarh Muslim University

Aligcirhfor his valuable suggestions, comments and encouragement through out this wor^ which

have helped me in improving the quality of this work^ Than^ are also due to Cl^irtnan, (Department

of Zoology, JLMU, Migarh for providing the necessary facility.

I would li^ to ey^ress my gratitude to (Professor Jamalji. %han and (Dr. yififullah %han,

(Department of Wild life Science J4^ligarh Muslim University Jiligarh in helping to collect the plant

material Than^ are also due to (Dr. Jithar Jili %han (Department of (Botany, JAligarh Muslim

University Jiligarh and (Professor S-^- Afa<], (Department of Ilmul Jidvia Jljmal ^ n TiSbiya

College, JAligarh Muslim University JAligarh for identification the plant material

I would liks to e^^endmy special thanks to (Dr. ^adeem JAhmad "Kjian for his committed

and loving support during last year of my (ph.(D wor^

Than^ are also due to all my respected teachers (Department of Chemistry JAligarh Muslim

University JAligarh for their moral support and various suggestions during this period.

Thanh^ are also indebt to the office and seminar staffs of the (Department of Chemistry,

JAligarh Muslim University, JAligarh, for the cooperation and providing the required official

assistance andvaluable literature.

/ am itufeStecfto all my good friends for the very Beginning of this Cong, Cong process who

aCways shared my joys and sorrows with equaC widingness and made my stay atJlCigarh, despite not

very happy circumstances, pCeasuraSCe than^ou for keeping me for quitting.

TinanciaCassistance provided6y Vgc isgratefuCCy ac^owkdged.

'Finally, I wouCdnot Be who I am without my parents and indeed I wouCdnot Be at aCCwith

out my parents. Trom an earCy age my parents encouraged me to reach higher and satisfy my

curiosity. 'Words can not express my indeBtness to my parents who through their Cove and affection,

their wishes and prayers have given me the inspirations to do the wor^ofmy choice. I can not than^

you enough for the support (emotionaCandfinanciaC) and Cove that you have given without question

over the years.

SARIFARAZ^HMED)

Abbreviations used

LB

SBCB

SB

QSK

SK

QS

DD

ST

EC

BA

SA

CA

YPD

NB

MIC

p-NAD

NBT

PMS

FBS

OTE

NDMA

ALP

GOT

Luria Bertani

Soyabean Casein Broth

Safoof-e-Bars

Qurs-e-Sartaan Kafoori

Safoof-e-Kharish

Qurs-e-Suzak

Dawa-e-Dibba

Salmonella typhimuriun

Escherichia coli

Brucella abortus S-19

Staphylococcus aureus

Candida albicans

Yeast extract, peptone, dextrose

Nutrient broth

Minimum inhibitory concentration

Nicotinamide adenine dinucleotide

Nitro blue tetrazolium

Phenazine methosuiphate

Fetal bovine serum

Operculina turpethum extract

jV-Nitrosodimethylamine

Alkaline phosphatase

Glutamic oxaloacetic transaminase

GPT

LDH

PBS

DAB

ml

m/z

NMR

HPLC

UPLC-MS

TLC

ESI-MS

CPU

nm

min

°C

Glutamic pyruvic transaminase

Lactate dehydrogenase

Phosphate buffer saline

3, 3'-Diaminobenzidine tetrahydrochloride hydrate

Microgram

Milliliter

Mass per unit charge

Nuclear magnetic resonance

High performance liquid chromatography

Ultra pjjrifTed liquid chromatography-Mass spectrometry

Thin layer chromatography

Electrospray ionization- Mass spectrometry

Colony forming unit

Nanometer

Minutes

Degree Celsius

Ill

Contents

Page No.

Chapter 1, General Introduction 1-18

1. Introduction 1-3 1.1. Chinese medicine 3-4 1.2. Indian medicin^yurveda) 4-4 1.3. Egyptian medicine 4-5 1.4. Greek medicine 5-5 1.5. Arab medicine 6-6 1.6. Greco - Arab system of medicine (Unani) 6-7 1.7. Indian medical trinity of traditional medicines 7-7 1.8. Infectious diseases 8-9 1.9. Medicinal plants as antimicrobials 9-13 1.10. Synergistic action of phytomedicines 13-13 1.11. Objectives of the thesis 14-14

References 15-18

Chapter 2. Scientific evaluation of some herbal drugs of Unani origin for antibacterial and antifungal activity 19-36

2. Introduction 19-22 2.1. Materials and Methods 22-22

2.1.1. Preparation of drugs extracts 22-22 2.1.2. Microorganisms used 22-22 2.1.3. Culture media 23-23 2.1.4. Inoculum 23-23 2.1.5. Agar diffiision assay 23-24

2.2 Results 24-24 2.3 Discussion 24-26 2.4 Conclusion 26-26

Tables (2.1-2.8) 27-34

References 35-36

Chapter 3. Antimicrobial activity and hepatoprotective effects ofOpercu/ina turpethum 3 7-65

3. Introduction 3 7-40 3.1. Materials and Methods 40-40

3.1.1. Microbial studies 40-40 3.1.2. Preparation of phytoextract 40-40 3.1.3. Microorganisms used 41-41 3.1.4. Culture media 41-41 3.1.5. Inoculum 41-41

IV

3.1.6. Antimicrobial susceptibility testing 41 -42 3.2. Biochemical Toxicology Studies 42-42

3.2.1. Chemicals 42-42 3.2.2. Animals 42-42 3.2.3. Induction of hepatic fibrosis 42-43 3.2.4. Administration of Operculina turpethum extract (OTE)..43-43 3.2.5. Assessment of liver injury 43-43 3.2.6. Staining of a-smooth muscle actin (a-SMA) 43-44 3.2.7. Biochemical parameters 44-44 3.2.8. Densitometry 45-45 3.2.9. Statistical analysis 45-45

3.3. Results 45-45 3.3.1. Microbial 45-45 3.3.2. Hematoxylin and eosin staining 45-45

3.3.2.1. Histological observations during NDMA treatment and progression of hepatic fibrosis 45-46 3.3.3.2. OTE treatment 46-46

3.3.3. Staining of a-smooth muscle actin (a-SMA) 46-46 3.3.3.1. Histological observations during NDMA treatment and progression of hepatic fibrosis 46-47 3.3.3.2. OTE treatment 47-47

3.3.4. Sera ALP, GOT, GPT and bilirubin levels 47-48 3.3.5. Hydroxyproline levels in the sera and urine 48-48 3.3.6. Total and fractional activity of LDH isoenzymes 48-49

3.4. Discussion 49-52 3.5. Conclusion 52-52

Tables (3.1-3.4) 53-56 Figures (3.1-3.3) 57-59

References 60-65

Chapter 4 Investigations on Acacia leucophloea Willd 66-97

4. Introduction 66-78 4.1. Materials and Methods 78-78

4.1.1. Plant material 78-78 4.1.2. Preparation of plant extract and column

chromatography 78-79 4.2. Microbial Study 79-79

4.2.1. Microorganisms used 79-79 4.2.2. Culture media 79-80 4.2.3. Inoculum 80-80 4.2.4. Antimicrobial susceptibility testing 80-80

4.3. Results 80-80 4.3.1. Microbial 80-81 4.3.2. Spectral analysis 81-82

4.4. Discussion 83-85 4.5. Conclusion 85-85

Tables (4.1 & 4.2) 86-87

Figures (4.1-4.6) 88-93

References 94-97

Publications

CHAPTER-1

GENERAL INTRODUCTION

1. Introduction

Medicinal plants have always been crucial in sustaining the health and well

being of mankind. Since time immemorial, traditional systems of medicine have

been practiced by man to cure various ailments. These traditional system of

medicine largely depended on medicinal and aromatic plants, a fact that can be

traced back from Vedas and Charak Samhita. Even today, many people rely on the

use of these plants for treating different diseases especially in the rural areas where

health facility is scarce. Moreover, growing concern about health and physical

fitness has provided a booming impact on renaissance of traditional medicines as

fast acting synthetic drugs, often have side effects on human body.

Population rise, inadequate supply of drugs, prohibitive cost of treatments,

side effects of several allopathic drugs and development of resistance to currently

used drugs for infectious diseases have led to increased emphasis on the use of plant

materials as a source of medicines for a wide variety of human ailments [Joy et al.,

2001]. There is no exact data available to assess the value and extent of the use of

plants or of active constituents derived from them in the health care systems. It has

been estimated by the World Health Organization (WHO) that approximately 80 %

of the more than 4000 million inhabitants of the world rely mainly on traditional

medicines for their primary health care [Famsworth, 1985].

In spite of the overwhelming influences and our dependence on modem

medicine and tremendous advances in synthetic drugs, a large segment of the world

population still like drugs from plants. In many of the developing countries the use

of plant drugs Is increasing because modem life saving drugs are beyond the reach

of three quarters of the third world's population although many such countries spend

40-50% of their total wealth on drugs and health care. As a part of the strategy to

reduce the financial burden on developing countries, it is obvious that an increased

use of plant drugs will be followed in the future [Joy et al., 2001].

Estimates indicate that approximately one quarter of prescribed drugs contain

plant extracts or active ingredients obtained from or modeled on plant substances.

According to UNDP report, the annual value of medicinal plants derived from

developing countries is approximately 32 billion US dollars. There are 47 major

modem pharmaceutical plant based drugs already present in the world market and

the predicted 328 drugs are yet to be discovered, having market potential of 147

billion US dollars. The most popular analgesic, aspirin, was originally derived from

species of Salix and Spiraea and some of valuable anti-cancer agents such as

paclitaxel and vinblastine are derived solely from plant sources [Tripathi and

Tripathi, 2003].

It is estimated that there are 250,000 to 500,000 species of plants on Earth

[Borris, 1996]. A relatively small percentage (1 to 10%) of these are used as food by

humans and also eaten by other animal species. It is possible that even more are used

for medicinal purposes [Moerman, 1996]. Hippocrates (in the late fifth century B.C.)

mentioned 300 to 400 medicinal plants [Schultes, 1978]. In the first century A.D.,

Dioscorides wrote De Materia Medica, a medicinal plant catalog which became the

prototype for modem pharmacopoeias. The Bible offers descriptions of

approximately 30 healing plants. Indeed, frankincense and myrrh probably enjoyed

their status of great worth due to their medicinal properties. Reported to have

antiseptic properties, they were even employed as mouthwashes. The fall of ancient

civilizations forestalled Westem advances in the understanding of medicinal plants,

with much of the documentation of plant pharmaceuticals being destroyed or lost

[Stockwell, 1988]. During the Dark Ages, the Arab world continued to excavate

their own older works and to build upon them. Of course, Asian cultures were also

busy compiling their own pharmacopoeia. In the West, the Renaissance years saw a

revival of ancient medicine, which was built largely on plant medicinals.

To understand exactly the usage of drugs of plant origin and other origins, as

to how they come into prominence in medicine through out the world. It is necessary

to go through the developmental history of these drugs. This will help not only to the

students, research scholars, and the workers engaged in this field but also physicians

as well.

The history of drug usage goes back to the prehistoric era. Basically there are

three schools of medicine in the world. These schools can be assigned to utilize the

drugs of the plants, mineral and animal origin for curing different type of ailments in

different ways with a difference of few hundred years of development of this

knowledge, simultaneously. These three schools are Chinese School of medicine

(2,735 B.C.) in East, Indian school of medicine (4,500- 1,600 and 2,500 - 600 B.C.)

and Greek School of medicine (460 B.C.) in West respectively [Khan, 1981]. Early

records show that the interaction of Greeks, Egyptian and Arabs led to the

emergence of Egyptian medicines, Arab medicines and Unani medicines [Khan,

1981].

1.1. Chinese medicine

The Chinese material medica has been extensively documented over the

centuries with the first record dating from about 1100 B.C. (Wu Shi Er Bing Fang,

containing 52 description) followed by work such as the Shennong herbal (~ 100

B.C.; 365 drugs), and the Tang herbal (~ 659 A.D.; 850 drugs) [Crag and Newman,

2002]. The Chinese medicine are compiled in the form of pharmacopoeia called

"Pun Tsao" in Chinese or the "Great Herbal" having 40 volumes describing

thousands of preparations [Khan, 1981].

1.2. Indian medicine (Ayurveda)

The Vedas are the earliest sacred book of the Hindu mythology. There are

four Vedas; Rigveda, Saumveda, Yajurveda and Atharvaveda. [Khan, 1981]. The

origin of Ayurveda is generally traced to the Atharavaveda (fc 1000 BC) which has

details of what may be called religious or priestly medicine similar to early Egyptian

medicine. An important aspect of Vedic medicine was the use of certain plants as

amulets, apart from their use either in the form of decoction or powder or fumigant

as medicines [Subbarayappa, 2001].

About 8,000 herbal remedies have been codified in Ayurveda. The Rigveda

(5000 BC) has recorded 67 medicinal plants, Yajurveda 81 species, Atharvaveda

(4500-2500 BC) 290 species, Charak Samhita (700 BC) and Sushrut Samhita (200

BC) had described properties and uses of 1100 and 1270 species respectively, in

compounding of drugs and these are still used in the classical formulations, in the

Ayurvedic system of medicine [Joy et al., 2001].

1.3. Egyptian medicine

In the west, before the advent of Greek medicine records, the drugs usages

are also available in the form of Egyptian Materia-Medica [Khan, 1981]. Egyptians

used medicinal herbs from nearly 2900 B.C and the best knowledge of Egyptian

Materia-Medica comes from two papyri, the Edwin Smith Papyrus of ca. 1600 B.C.

and the Ebers Papyrus of ca. 1500 B.C. [Bryan, 1930., Crag and Newman, 2002].

Egyptian medicine was essentially a belief system. It was believed that there

were thirty six gods of the atmosphere and thirty-six 'demons', and the human body

was conceptually divided into as many parts. If a part of the body was affected, the

5

concerned 'demon' had to be invoked for its cure. The concept of disease-demons

was very strong among Egyptians. Though plant medicaments were in use, they

were not considered to be efficacious without the appeasement of 'demons' through

magical rites [Subbarayappa, 2001]. Medicine and superstitious teleology were as a

two-in-one pair among Egyptian priests [Guthrie, 1920].

1.4. Greek medicine

The Greek system of medicine originated in ancient Greece around 1250-285

B.C. [Mouhajir, 2002]. In the early days, Greek medicine was closely linked with

religion. Apollo, the Sun- God was considered to be the protector of mankind from

the attack of epidemics such as plague. The practice of the sick visiting temples of

Aesculapius became popular and deep-rooted in Greek society around 1000 BC

[Subbayarapa, 2001]. It was in essence a faith cure and sick person offering prayers

and animal sacrifices in the temple were a common feature.

However, by about 500 B.C. medicine began to be separated from the

magical and spiritual world. Hippocratus (460-377 B.C.), the Greek "Father of

medicine" considered illness to be a natural rather than a supernatural phenomenon

and he felt that medicine should be given without ritual ceremonies or magic

[Andrew, 1996]. Hippocratic collection shows evidence of new medical ethics and a

scientific sprit [Chadwick and Mann, 1950]. More than 4000 drugs are mentioned in

his work. Important work by Theophrastus (370-285 B.C.) includes "Historia

plantarum" or "Enquiry into plants" which contain information about the collection

and preparation of vegetable drugs such as peony roots, food stuffs, perfumes, plant

diseases and weather sign [Mouhajir, 2002].

1.5. Arab medicine

In the period of golden age of the Muslims (A.D. 632-1150) the Arabs

having conquered Egypt, Syria, Iraq and Persia extended their empire from the

border of India to Spain [Mouhajir, 2002]. Several treatises dealing with what is

called Prophetic medicine were compiled by clerics engaged in the traditional

medical practices during the time of Prophet Mohammad [Subbayarappa, 2001]. The

concepts and contents relating to material medica and therapeutics of the Muslims in

the period from 750 A.D. to about 1350 were qualitatively and quantitatively far

ahead of those of preceding cultures. In quantity, the number of simple and

compound remedies rose to about 4,000 which is a fantastic number compared with

the mere hundreds in the Greek words [Mouhajir, 2002]. In addition, there emerged

a school of medicine followed by foundation of centers of learning where scholars

were encouraged to translate and incorporate other influential works of the time into

their own knowledge, findings and experience.

Apart from the systematizing the pharmacogical knowledge of Greeks,

Persian and Babylonians, many highly specialized treatises were compiled by the

Arabs where in genuinely original contributions to enhance the knowledge of the

medical Materia-Medica were made. The Arabic pharmacology lasted into the

nineteenth century as the strongest fact- based biological sciences [Mouhajir, 2002].

1.6. Greco - Arab system of medicine (Unani)

The system which originated in Greece and developed by Arabs into an

elaborate medical science on the basis of teaching of Hippocrates, Descroids and

Gallenics called Greco- Arab system of medicine which later on after centuries came

down to India with Muslim advents and flourished with the name Unani system of

medicine [Khan, 1981]. With the patronage of Muslim kings, the Unani medicine

flourished specially under the Mughal Empire. Its Materia-Medica includes mostly

herbal drugs, besides some of animal and mineral origin [Subbayarappa 2001].

1.7. Indian medical trinity of traditional medicines

Indian subcontinent has been reputed as the treasure of valuable medicinal

plants of the world on account of vast diversity of climatic conditions and India

enjoys the privilege of having time tested traditional systems of medicines based on

the natural products. Plant based products have been in use for medicinal,

therapeutic or other purposes right from the dawn of history. Additionally, India is

one of the 12-mega biodiversity centers having about 10% of the world's

biodiversity wealth, which is distributed across 16 agro-climatic zones. Out of

17,000 species of higher plants reported to occur within India, 7500 are known to

have medicinal uses. This proportion of medicinal plants is the highest known in any

other country against the existing flora of the country. Currently, approximately

25% of drugs are derived from plants, and many others are synthetic analogue buih

on prototype compounds isolated from plant species in modem pharmacopoeia

[Ahmad, 2008].

Of the Indian medical trinity - Ayurveda, Unani and Siddha - Ayurveda has

its roots in Vedic literature, while Unani, which is Greco-Arabic medicine, owes its

origin to Central and West Asia. Neither of them, however had any alchemical

undertone. The Siddha system was regionally confined to Tamil Nadu and its

adjoining areas among Tamil-speaking people [Subbarayappa, 1997]. These systems

of medicines (Ayurveda, Unani and Siddha) have a number of drugs in common,

though they are of different origin, and source. These drugs even now are identified

with different vernaculars and common names though the botanical identity is one

and the same. Similar is the case with mineral and animal origin [Khan, 1981].

1.8. Infectious Diseases

Infectious diseases are the leading cause of premature death, killing almost

50,000 people every day through out the world [Ahmad and Beg, 2001]. Infectious

diseases claim almost half of the deaths in tropical countries. In recent years, this

trend seems to be increasing even in developed countries [Iwu et al., 1999]. Not

surprising death from infectious disease, ranked 5th in 1981, has become the 3rd

leading cause of death in 1992, an increase of 58% [Pinner et al., 1996].

This is alarming given that it was once believed that we would eliminate

infectious disease by the end of the millenium. The increases are attributed to

increases in respiratory tract infections and HIV/AIDS. Other contributing factors

are an increase in antibiotic resistance in nosicomial and community acquired

infections. Furthermore, the most dramatic increases are occurring in the 25-44 year

old age group [Pinner et al., 1996].

These negative health trends call for a renewed interest in infectious disease in the

medical and public health communities and renewed strategies on treatment and

prevention. Proposed solutions are outlined by the CDC as a multi-pronged

approach that includes: prevention, (such as vaccination); improved monitoring; and

the development of new treatments. It is this last solution that would encompass the

development of new antimicrobials [Fauci, 1998].

Indiscriminate use of antibiotics and drug resistant of human pathogenic

microorganisms continue to be an area of concern [Ahmad and Beg, 2001].

The substance that can either inhibit the growth of pathogen or kill them and

have no or least toxicity to host cell are considered candidates for developing new

anti microbial drugs. (In recent years antimicrobial property of medicinal plants are

being increasingly reported from different parts of the world [Grosvener et al., 1995;

Ratnakar and Murthy, 1995; Silva, 1996; David, 1997; Saxena, 1997; Nimri et al.,

1999; Saxena and Sharma, 1999].

1.9. Medicinal plants as Antimicrobials

Plants have provided a good source of inspiration for novel drug compounds.

New drugs are still being derived from a wide variety of medicinal plants. These

drugs may serve as antimicrobial agents and inhibit disease causing pathogens and

have made large contributions to human health and well-being. The role of these

medicinal plants in the development of new drugs is two fold VIZ. (1) they may

become the base for the development of a medicine, a natural blueprint for the

development of new drugs, or; (2) a phytomedicine to be used for the treatment of

disease. There are numerous illustrations of plant derived drugs [Iwu et al., 1999].

Some selected examples, including those classified as anti-infective, are described

below.

The isoquinoline alkaloid emetine obtained from the underground part of

Cephaelis ipecacuanha, and related species, has been used for many years as an

amoebicidal drug as well as for the treatment of abscesses due to the spread of

Escherichia histolytica infections. Another important drug of plant origin with a

long history of use is quinine. This alkaloid occurs naturally in the bark of Cinchona

tree. Apart from its continued usefiilness in the treatment of malaria, it can be also

used to relieve nocturnal leg cramps. Currently, the widely prescribed drugs are

analogs of quinine such as chloroquine. Some strains of malarial parasites have

become resistant to the quinines; therefore, antimalarial drugs with novel mode of

action are required.

Similarly, higher plants have made important contributions in the areas

beyond antiinfectives, such as cancer therapies. Early examples include the

10

antileukaemic alkaloids, vinblastine and vincristine, which were both obtained from

the Madagascan periwinkle {Catharanthus roseus syn. Finca roseus) [Nelson,

1982]. Other cancer therapeutic agents include taxol, homoharringtonine and several

derivatives of camptothein. For example, a well-known benzylisoquinoline alkaliod,

papaverine, has been shown to have a potent inhibitory effect on the replication of

several viruses including cytomegalovirus, measles and HIV [Turano et al., 1989].

Most recently, three new atropisomeric naphthylisoquinoline alkaloid dimers,

michellamines A, B, and C were isolated from a newly described species tropical

liana Ancistrocladus korupensis from the rainforest of Cameroon. The three

compounds showed potential anti-HIV with michellamine B being the most potent

and abundant member of the series. These compounds were capable of complete

inhibition of the cytopathic effects of HIV-1 and HIV-2 on human lymphoblastoid

target cell in vitro [Boyd et al., 1994].

Plants have an almost limitless ability to synthesize aromatic substances,

most of which are phenols or their oxygen-substituted derivatives [Geissman, 1963].

Most are secondary metabolites, of which at least 12,000 have been isolated, a

number estimated to be less than 10% of the total [Schultes, 1978]. In many cases,

these substances serve as plant defense mechanisms against predation by

microorganisms, insects, and herbivores. Some, such as terpenoids, give plants their

odors; others (quinones and tannins) are responsible for plant pigment. Many

compounds are responsible for plant flavor (e.g., the terpenoid capsaicin from chili

peppers).

11

There are still some plant based drugs for which no synthetic alternative is

available listed in table below [Kumar et a!., 1997].

Drug

Vinblastine

Ajmalacine

Rescinnamine

Reserpine

Quinine

Pilocarpine

Cocaine

Morphine

Codeine

Atropine

Atropine

Cardiac glycosides

Artemisinin

Taxol

Berberine

Pristimerin

Quassinoids

Plumbagin

Diospyrin

Gossypol

Plant

Catharanthus roseus

Catharanthus roseus

Rauvolfia serpentina

Rauvolfia serpentina

Cinchona sp.

Pilocarpus jaborandi

Erythroxylum coca

Papaver somniferum

Papaver somniferum

Atropa belladonna

Hyoscyamus niger

Digitalis sp.

Artemesia annua

Taxus baccata T. Brevifolia

Berberis

Celastrus paniculata

Ailanthus

Plumbago indica

Diospyros montana

Gossypium sp.

Use

Anticancer

Anticancer, hypotensive

Tranquilizer

Tranquilizer

Antimalarial, amoebic dysentery

Antiglucoma

Topical anaesthetic

Painkiller

Anticough

Spasmolytic, cold

Spasmolytic, cold

For congestive heart failure

Antimalarial

Breast and ovary cancer Antitumour

For leishmaniasis

Antimalarial

Antiprotozoal

Antibacterial, antifungal

-O <

Antispermatogenic

12

Allicin

Ricin

Emetine

Glycyrrhizin

Nimbidin

Catechin

Sophoradin

Magnolol

Forskolin

Digitoxin,

Thevenerin,

Nerrifolin

Podophyllin

Indicine N-oxide

Elipticine

Homoharringtonine

Camptothecine

Allium sativum

Ricinus communis

Cephaelis ipecacuanha

Glycyrrhizia glabra

Azadirachta indica

Acacia catechu

Sophora subprostrata

Magnolia bark

Coleus forskohlii

Digoxin Digitalis, Thevetia Thevetia

Thevetia

Podophyllum emodi

Heliotropium indicum

Ochrosia

Cephalotaxus

Camptotheca acuminate

Antifungal, amoebiasis

•Yf^y\ I

Amoebiasis

Antiulcer

Antiulcer

Antiulcer

'.Antiulcer /

Peptic ulcer

Hypotensive, cardiotonic

Cardio tonic

Cardio tonic

Cardio tonic

Anticancer

Anticancer

Anticancer

Anticancer

Anticancer

13

BasicEilly the drugs of the Indian systems of medicines (Ayurveda, Unani and

Siddha) are used either singly or in combination of various formulations. A number

of plants used medicinally in our systems have been chemically analyzed as evident

from the published work in our country as well as abroad. We should now try them

pharmacologically and clinically to prove the efficacy of the exact, effective and

active chemical constituents. The pharmacological testing of the crude drugs (used

in various formulations) can not be done unless each and every single drug yielding

plant is identified botanically, pharmacogonostically and chemically and then put in

pharmacological and clinical trials [Khan, 1981].

1.10. Synergistic action of Phytomedicines

In comparison to synthetic drugs based upon single chemicals, many

phytomedicines exert their beneficial effects through the additive or synergistic

action of several chemical compounds acting at single or multiple target sites

associated with a physiological process [Briskin, 2000]. Synergistic or additive

pharmacological effect can be beneficial by eliminating the problematic side effects

associated with the predominance of a single xenobiotic compound in the body

[Tyler, 1999]. How synergistic interactions underlie the effectiveness of a number of

phytomedicines are extensively documented [Kaufman et al., 1999]. This theme of

multiple chemicals acting in an additive or synergistic manner likely has its origin in

the functional role of secondary products in promoting plant survival. For example,

in the role of secondary products as defense chemicals, a mixture of chemicals

having additive or synergistic effects at multiple target sites would not only ensure

effectiveness against a wide range of herbivores or pathogens but would also

decrease the chances of these organisms developing resistance or adaptive responses

[Kaufman et al., 1999; Wink, 1999].

14

1.11. Objectives of the thesis

The indiscriminate use of allopathic daigs has developed resistant pathogens.

Therefore, there is revival of interest in drugs of herbal origin. The aim of the

present investigations is as follows:

1. Scientific evaluation of some herbal drugs of Unani origin for

antibacterial and antifungal activity.

2. Antimicrobial activity and hepatoprotective effects of Operciilina

turpethum.

3. Investigations on Acacia leiicophloea Willd.

15

References

> Ahmad, I., Beg, A.Z., (2001). Antimicrobial and phytochemical studies on 45

Indian medicinal plants against multi-drug resistant human pathogens, J.

Ethnopharmacol. 74, 113-123.

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negundo L. andRuta graveolens L. Ph. D. thesis, Department of Botany, Aligarh

Muslim University, Aligarh, India.

> Andrew, C. MNIMH., (1996). The Encyclopedia of Medicinal Plants. Dorling

Kindersley Limited, London.

> Borris, R.P., (1996). Natural products research: perspectives from a major

pharmaceutical company, J. Ethnopharmacol. 51, 29-38.

> Boyd, M., Hallock, Y., Cardellina II, J., Manfredi, K., Blunt, J., McMahon, J.,

Buckheit, R., Bringmann, G., Schaffer, M., Cragg, G., Thomas, D., Jato, J.,

(1994). Anti-HIV michellamines from Ancistrocladus korupensis, Med. Chem.

37, 1740-1745.

> Briskin, Donald P., (2000). Medicinal plants and phytomedicines. Linking plant

biochemistry and physiology to human health. Plant Physiol. 124, 507-514.

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(Berlin).

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Chadwick, London.

> Cragg G.M., Newman D.J., (2002). Biodiversity medicinal for the millennia, Sci.

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> David, M., (1997). Antimicrobial activity of garlic, Antimicro. agents and

Chemother. 41, 2286.

16

> Famsworth, N.R., Akrele, O., Bingel, A. S., Soejarto, D.D., Guo, Z., (1985).

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compounds, p. 265. In M. Florkin, and E. H. Stotz (ed.). Pyrrole Pigments,

Isoprenoid Compounds and Phenolic Plant Constituents, vol. 9. Elsevier, New

York, N.Y.

> Grosvener, P.W., Supriono, A., Gray, D.O., (1995). Medicinal plants from Riau

Province, Sumatra, Indonesia. Part 2. Antibacterial and antifungal activity, J.

Ethnopharmacol. 45, 97-111.

> Guthrie, D., (1920). History of Medicine, London.

> Iwu, M.W., Duncan, A.R., Okunji, CO., (1999). New antimicrobials of plant

origin p 457-462. In: J. Janick (ed.). Perspectives on new crops and new uses.

ASHS Press, Alexandria, VA.

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Tropical Horticulture vol. 2. (eds. Bose, T.K., Kabir, J., Das, P. and Joy, P.P.).

Naya Prokash, Calcutta.

> Kaufman, P.B., Cseke L.J., Warber, S., Duke, J.A., Brielmann,H.L., (1999)

Natural Products from Plants. CRC Press, Boca Raton, FL.

> Khan, R., (1981). Pharmacognostical and Botanical Identification of Some

Important Bark Drugs Used in Indian System of Medicine (Unani), M. Phil,

dissertation. Department of Botany, Aligarh Muslim University Aligarh, India.

17

> Kumar, N., M. Abdul Khader, J. B. M., Rangaswami, P., Irulappan, I., (1997).

Introduction to Spices, Plantation Crops, Medicinal and Aromatic Plants,

Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi.

> Moeiman, D.E., (1996). An analysis of the food plants and drug plants of native

North America, J. Ethnopharmacol. 52, 1-22.

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Morocco: Ethnopharmacology and Phytochemistry. Ph. D thesis. Faculty of

Graduate Studies, Department of Botany, University of British Columbia.

> Nelson, R., (1982). The comparative clinical pharmacology and

pharmacokinetics of vindesine, vincristine and vinblastine in human patients

with cancer, Med. Pediatr. Oncol. 10, 115-127.

> Nimri, L.F., Meqdam, M.M., Alkofahi, A., (1999). Antimicrobial activity of

Jordanian medicinal plants, Pharmaceut. Biol. 37, 96-201.

> Pinner, R., Teutsch,S., Simonsen, L., Klug, L., Graber, J., Clarke, M.,

Berkelman, R., (1996). Trends in infectious diseases mortality in the United

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> Ratnakar, P., Murthy P.S., (1995). Purification and mechanism of action of

antitubercular principle from garlic {Allium sativum) active against isoniazid

susceptible and resistant Mycobacterium tuberculate H37RV, Ind. J. Clin.

Biochem. 10, 35-44.

> Saxena, K., (1997). Antimicrobial screening of selected medicinal plants from

India. J. Ethnopharmacol. 58, 75-83.

> Saxena, V.K., Sharma, R.N., (1999). Antimicrobial activity of essential oil of

Lantana aculeate, Fitoterapia 70, 59-60.

18

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(ed.), Medicines from the earth. McGraw-Hill Book Co., New York, N.Y.

> Silva, O., Duarte, A., Cabrita, J., Gomes, E. (1996). Antimicrobial activity of

guinea- Bissau traditional remedies, J. Ethnopharmacol. 50, 55-59.

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y.5/o^a. 26, 135-144.

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Trop. Pharm. Res. 2, 243-253.

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N., (1989). Inhibitory effect of papaverine on HIV replication in vitro, AIDS Res.

Hum. Retrovir. 5, 183-191.

> Tyler, V.E., (1999). Phytomedicines: Back to the future, J. Nat. Prod. 62, 1589-

1592.

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metabolites. In M Wink, ed. Functions of Plant Secondary Metabolites and Their

Exploitation in Biotechnology. CRC Press, Boca Raton, FL.

v_y

r\

CHAPTER-2 SCIENTIFIC EVALUATION OF SOME HERBAL DRUGS

OF UNANI ORIGIN =OR ANTIBACTERIAL ANE ANTIFUNGAL ACTIVITY

19

2. Introduction

Medicinal plants were extensively used by the old civilizations through out

the world. Chapter one describes that Egyptians used medicinal herbs from nearly

2900 B.C. and the best known Egyptian Pharmaceutical record is the "Edwin Smith

papyrus" and "Ebers Papyrus" dating from ca. 1600 B.C and ca. 1500 B.C.

respectively [Bryan, 1930; Cragg and Newman, 2002] which documents 700 drugs

(mostly of plants), and include formula such as, gargles, snuffs, poultices, infusions,

pills and ointments with beer, milk, wine and honey being commonly used as

vehicle. These ethno-medicinally important botanicals and their products now exist

as herbal drugs despite considerable advancement in medical sciences.

Increasing awareness on health and physical fitness has been putting pressure

on herbal foimiulations as fast acting synthetic drugs have many side effects on

human body. Keeping in view the growing demand of herbal drugs, industries are

now engaged in developing new products prepared from natural products or

substances of animal origin. As an alternative to synthetic drugs which show

resistance to pathogens, herbal formulations are globally acceptable and hence have

set stage for revival of traditional medicine. Recently, it has been demonstrated that

many human pathogenic bacteria have developed resistance against several synthetic

drugs [Piddock and Wise, 1989; Singh et al., 1992; Mulligen et al., 1993; Davis

1994; Robin etal., 1998].

Available reports on lesser efficacy and more side effects of synthetic drugs

have mobilized the masses to search alternative methods [Jain, 1991; Kumar et al.,

1997]. Conversely, the use of herbal drugs have proven promising in treating many

genetic, biochemical or physiological alterations of human body with least or no side

effects [Ahmad et al., 2008]. Moreover, recent researches on the prolong use of

20

ethno medicinal botanicals in crude form or their purified compounds have also

shown certain side effects and suggested for their limited use [Kumar et al., 1997;

Ahmad et al., 2008]. Therefore, it is necessary to re-evaluate the action and efficacy

of the commercially available formulations which are in use for centuries.

As mention in chapter 1, the Unani system of medicine has its roots in the

Greek system of medicine, developed and propagated by Arabs. It came to India

with the advent of Muslim rulers flourishing specially under the Mughal Empire.

According to Unani, the human body and its health are constituted of seven

components: elements (Al-Arkan), temperament (Al-Mizaj), four humours (Al-

Akhlat), organs (Al-A 'da), vital spirit (Al-Arwah), faculties (Al-Quwa) and functions

(Al- Afal). The Unani concept of elements is that of Empedocles (5th century BC) -

earth, fire, water and air with their four primary qualities: hot, cold, dry and moist

[Subbarayappa, 2001]. Its materia medica includes mostly herbal drugs, besides

some of animal and mineral origin. The Unani concept of temperament has its own

originality and even drugs are supposed to have their temperament according to

which they are classified.

In the present study efforts are made for scientific evaluation of five Unani

drugs which are used commonly against bacterial and fungal infections in humans.

These Unani drugs are Qurs-e-Sartaan Kafoori, Qurs-e-Suzak, Dawa-e-Dibba,

Safoof-e-Bars and Safoof-e-Kharish. These Unani drugs manufactured and marketed

by Dawakhana Tibbiya College, Aligarh Muslim University, Aligarh, have multiple

botanical ingredients in addition to some chemical substances and substances of

animal origin. The constitution of the selected drugs is summarized in tables 2.1 to

2.5, while other details are given below:

21

Qurs-e-Sartaan Kafoori [Hakim Kabiruddin, 1932]

Ingredients: Table-2.1

Action: Styptic, Antipyretic

Therapeutic use: Phthisis, Tuberculosis, Hectic fever

Dose: 2 to 4 gm

Qurs-e-Suzak [Patent]

Ingredients: Table-2.2

Action: Cicatrizant, Diuretic

Therapeutic use: Gonorrhea

Dose: Two tablets daily

Dawa-e-Dibha [Patent]

Ingredients: Table-2.3

Action: Purgative

Therapeutic use: Infantile Bronchopneumonia, Constipation

Dose: 125 mg

Safoof-e-Bars [Hakim Kabiruddin, 1932]

Ingredients: Table-2.4

Action: Blood purifier

Therapeutic use: Vitiligo/Leucoderma, Pruritis/Itching

Method of application: 10 gm of powder soaked in 50 ml of water over

night. The infusion is decanted and orally administered in the morning. The

sediment is mixed with sirka naishakar to prepare a paste and applied on the

affected part, which are exposed to the sunrays.

22

Safoof-e-Kharish [Patent]

Ingredients: Table -2. 5

Action: Soothing agent

Therapeutic use: Itching (For external use only)

Method of application: Mix with Jasmine oil and apply on the affected

portion.

2.1. Materials and Methods

The selected Unani drugs were purchased from the Dawakhana Tibbiya

College, Aligarh Muslim University, Aligarh. The constitution of these drugs is

summarized in Tables 1-5 while their dosage details and method of application have

been described in the Introduction.

2.1.1. Preparation of Drug extract

5 gm of each drug were soaked in 50 ml of absolute alcohol for 70 to 80 hrs.

Each extract was stirred every 10 to 12 hrs using a sterile glass rod and filtered

through Whatman filter paper No. 1. The alcoholic extract obtained was

concentrated on water bath at 40 "C. The extracts were stored at 4 °C for further use.

The amount of the extract obtained from 5 gm of each drug is given in Table-2.6.

2.1.2. Microorganisms used

The test organisms Escherichia coli, Salmonella typhimurium, Candida

albicans (Clinical isolates) and Staphylococcus aureus (Standard strain) were kindly

gifted by Prof Abida Malik, Department of Microbiology, Jawaharlal Nehru

Medical College and Hospital, A.M.U, Aligarh, India. Brucella abortus S-19

(Standard strain) was procured from IVRI Bareilly.

23

2.1.3. Culture media

The medium used for the activation of microorganisms was soyabean casein

broth. For antimicrobial test the microbes were grown on different agar media.

Escherichia coli and Salmonella typhimurium were grown on Mc Conky's medium

while S. aureus, B. abortus S-19 and the fungus C. albicans on Nutrient agar, Luria

Bertani (LB) and Yeast extract, peptone, dextrose (YPD) medium respectively. All

culture media were prepared and treated according to the manufacturer's guidelines

(Hi-media Pvt. Ltd. India).

2.1.4. Inoculum

The microorganisms were inoculated into Soyabean Casein Broth (SBCB)

and incubated at 35 ± 2 "C for 4 hours. The turbidity of the resulting suspension was

diluted with SBCB to obtain a transmittance of 25% at 580 nm. That percentage was

found spectrophotometrically comparable to I Mc Farland turbidity standard. The

level of turbidity is equivalent to approximately 3.0x10* CFU/ml.

2.1.5. Agar diffusion assay

The modified agar well diffusion method of Perez et al. [Perez et al., 1990]

was employed. Microorganisms suspended in SBCB were inoculated in selective

media. Once the agar was solidified, it was pinched for eight-millimeter diameter

wells and filled with 100 jil (~ 10 mg crude extract) and 200 ]i\ (~ 20 mg crude

extract) drugs extracts and blanks (ethanol 98% and distilled water) in separate

wells. The concentration of extracts employed was 100 mg/ml. For positive control

Gentamycin Sulphate (Brucella abortus S-19, Escherchia coli) Chloramphenicol

{Salmonella typhimuriun, Staphylococcus aureus) and Amphotericin B {Candida

albicans) were used. The tests were carried out in triplicate. The plates were

24

incubated at 35 ± 2 ''C for 24 hrs, except Candida albicans, which was incubated at

29 ± 2 °C.

2.2. Results

The results of the antibacterial and antifungal screening of the tested drugs

are shown in table- 2.7 and 2.8. The potency of the drugs was assessed by the

presence or absence of zone inhibition or zone diameter. Among the tested drugs

Safoof-e-Bars, Qurs-e-Suzak and Dawa-e-Dibba showed activity against all the

tested bacteria and yeast, Candida albicans.

While Safoof-e-Kharish did not show activity against S. typhimurium and E.

coli and Qurs-e-Sartaan Kafoori against S. aureus respectively.

Among the five tested microorganisms i.e. E. coli, S. typhimuriun, S. aureus,

B. abortus S-19 and yeast Candida albicans. E. coli seems to be the least sensitive

towards theses drugs as revealed by its zone diameter.

Safoof-e-Bars showed good inhibition zone against B. abortus S-19, S.

aureus, and Candida albicans. While Qurs-e-Suzak showed good inhibition zone

against S. aureus,. B. abortus S-19, S. typhimurium and S. aureus. Qurs-e-Sartaan

Kafoori showed good inhibition zone against B. abortus S-19 and S. aureus. Dawa-

e-Dibba showed good inhibition zone against S. aureus, B. abortus S-19 and

Candida albicans while Safoof-e-Kharish shows good activity against B. abortus S-

19 and S. aureus.

2.3. Discussion

With the changing perception and attitude towards the use of synthetic drugs,

industries are encouraged to replace them with some suitable alternative and the

Unani formulations are witnessing this change. Compared with various methods

being used to ameliorate the common ailments, method of treating with some

25

friendly and effective natural compounds is preferred [Ahmad et al., 2008]. The

present study on antimicrobial activity of five Unani drugs (ethanolic extract) having

multiple botanical ingredients along with some chemical substances and molecules

of animal origin and commonly used for the treatment of various infectious diseases

revealed good results. We have tested their activity against four pathogenic bacteria

Salmonella typhimuhum, Brucella abortus S-19, Escherchia coli. Staphylococcus

aureus and one pathogenic yeast Candida albicans. This is the first report describing

antimicrobial activity of Unani formulations involved in the treatment of infectious

disease.

We have taken the ethanolic extract of the drugs, as published literature

suggests suitability of alcohol as the better solvent for the extraction of antimicrobial

substances as compared to water or hexane [Ahmad and Beg 2001]. The

antibacterial and antifungal efficacy as well as the potency of the extracts of the

drugs was quantitatively assessed by the presence or absence of zone of inhibition or

zone diameter (Table- 2.7 and 2.8). Our results are highly encouraging and showed

activity of all the five extracts of the drugs against the tested bacteria and selected

yeast, C. albicans. The only exception was the ethanolic extract of Safoof-e-Kharish

which did not show any activity against S. typhimurium and E. coli. Qurs-e-Sartaan

Kafoor against S. aureus.

Among the microorganisms used, the only exception is E. coli which showed

least sensitivity against the drugs tested. Therefore, it can be inferred that the

importance of the selected Unani drugs becomes least while treating disease

occurred by E. coli infection. As indicated in table 2.7- 2.8, Safoof-e-Bars and Qurs-

e-Suzak showed very good inhibition zone against B. abortus S-19, S. aureus and

yeast C. albicans while Qurs-e-Sartaan Kafoori shows against S. typhimurium, B.

26

abortus S-19 and yeast, C. albicans respectively. Safoof-e-Kharish showed good

inhibition zone against B. abortus S-19 followed by S. aureus and Dawa-e-Dibba

displayed vtry good inhibition zone against S. aureus B. abortus S-19 and yeast, C.

albicans. Hence, it may be suggested that the fungal infections exists mainly due to

C. albicans, the patients may be advised to depend on Safoof-e-Bars, Qurs-e-Suzak

and Dawa-e-Dibba along with other supplements. Those infected with B. abortus S-

19, any of the four Unani formulations except Qurs-e-Sartaan Kafoori may be

recommended to eliminate the infection. However, the inhibition due to Qurs-e-

Sartaan Kafoori was observed very restricted during the present study, thus its

limited and confined use against the S. typhimurium stimulated infection is

advisable.

2.4. Conclusion

The tested drugs may be recommended for use against investigated

pathogens borne diseases wherein considerable inhibition has been noticed such as

Safoof-e-Kharish, Qurs-e-Sartaan Kafoori and Dawa-e-Dibba. The drugs have

traditional recommended doses, but their proper intake in evaluated dosages is

advisable along with other supplementary compounds for complete recovery.

27

Table 2.1 Ingredients of Unani drug Qurs-e-Sartaan Kafoori.

Unani Name

Kafoor

Sandal Safaid

Sandal Surkh

Sandal Zard

Tukhm-e-Kahu

Samagh-e-Arbi

Kateera

Tabasheer

Gule Surkh

Mu!athee(Uslussoos)

Rubussoos

Nishahta

Khurfa Siyah

Magz-e-Tukhm-e- Khairain

Maghz-e-Tukhm-e-

Kharbooza

Maghz-e-Tukhm-e- Kaddu

Tukhme Khaskhas

Sartaan

Shakar

English Name

Camphor

Sandal Wood

Red Sandal Wood

-

Lettuce

Babool Gum

Gum Tragacanth

Bamboo Manna

Red Rose

Sweet Wood

-

Wheat Exract

Purslane

Seeds of Cucumber

Seeds of Muskmelon

Seeds of Pumpkin

Popy Seeds

Crab

Sugar

Scientific Name

Cinnamomum camphora

Santalum album Linn

Pterocarpus santalinus

-

Lactuca sativa Linn.

Acacia nilotica

Cochlospermum reliogiosum

Bambusa bambos Druce

Rosa dotnascena Mill.

Glycyrrhiza glabra Linn.

-

Triticum aestivum

Portolae oleracea

Cucumis sativus

Cucumis melo

Cucurbita moschata

Papaver somniferum

Callinectes sapidus

-

Table 2.2 Ingredients of Unani drug Qurs-e-Suzak.

28

Unani Name

Shoora Qalmi

Hajrul Yahood

Sat Barzad

Raal Safaid

Sang Jarahat

Alu-Balu

Katha Safaid

Dammul Akhwain

Kateera

Bisbasa

Habbe Kaknaj

Khar-e-Khask Khurd

Tabasheer

Shakh Marjan Sokhta

Busad Ahmer Sokhta

Zohar Mohra

Samagiie Arbi

Arq Badyan

English Name

Lapis Judaicus

-

Pinus

White Damar

-

-

White Catechu

Dragons Blood

Gum Tragacanth

Mace

White Cherry

Small Caltrops

Bamboo Manna

Coral Branches

Coral Roots

Mineral Stone(Serpentine)

Babool Gum

Fennel

Scientific Name

Potassium Nitrate

Silicate of Lime

Pinus longifolia Roxb.

Valeria indica

Hydrated magnesium silicate

Prunus carasus Linn.

Acacia leucophloea Willd.

Dracaena cinnabari Balf

Cochlospermum religiosum

Myristicafragrance Houtt.

Physalis alkekenji Linn.

Tribulus terristeris Linn.

Bambusa bambos Druce

Corallium rubrum

Corallium rubrum

-

Acacia nilotica

Foeniculum vulgare Mill.

29

Table 2.3 Ingredients of Unani drug Dawa-e-Dibba.

Unani Name

Sibre Zard (Leaves gel)

Aabe Limu

English Name

Aloe Plant

Lemon

Scientific Name

Aloe barbadensis

Citrus aurentifolia

30

Table 2.4 Ingredients of Unani drug Safoof-e-Bars.

Unani Name

Ajmood

Gairoo

Surpukha

Fulfil Siyah

English Name

Celery

Red Ochre

Purple Teprisia

Pepper

Scientific Name

Apium graveolans Linn.

Silicate of Aluminium & Iron oxide

Tephrozia purpuria

Piper nigrum Linn.

Table 2.5 Ingredients of Unani drug Safoof-e-Kharish.

31

Unani name

Kameela

Murdar Sang

Safaida Khasgari

Gandhak

English name

Monkey Face Tree

Massicot

Flowers of Zinc

Sulpliur

Scientific name

Mallotus philippinemis

Monoxide of Lead

Zinc Oxide

-

32

Table 2.6 Amount of crude extracts obtained from each 5 gm of Unani drug.

Drugs

Qurs-e-Sartaan Kafoori

Qurs-e-Suzak

Dawa-e-Dibba

Safoof-e-Bars

Safoof-e-Kharish

Extract obtained (mg)

907.5

644.3

183.7

410.8

175.7

33

Table 2.7 Antibacterial and antifungal activity of the crude extracts of the tested

drugs*.

Drugs

SB

QSK

SK

QS

DD

PC

Blank

Crude extract

employed/well

10 mg

10 mg

10 mg

10 mg

10 mg

<

Antibacterial and antifungal activity in terms of

zone inhibition (in mm) against test

microorganisms

ST

2+

3+

-

3+

2+

4+

-

EC

2+

2+

-

2+

2+

3+

-

BA

3+

3+

4+

3+

3+

5+

-

SA

3+

-

3+

4+

4+

4+

-

CA

3+

3+

2+

3+

3+

2+

-

* Drugs key: SB, Safoof-e-Bars; QSK, Qurs-e-Sartaan Kafoori; SK, Safoof-e-

Kharish; QS, Qurs-e-Suzak; DD, Dawa-e-Dibba. Activity key: -, no inhibition; 1+,

<10 mm zone of inhibition; 2+, 10-15 mm; 3+, 16-20 mm; 4+, 21-25 mm; 5+, 26-

30 mm. Microorganisms key: ST, Salmonella typhimuriun; EC, Escherichia coli;

BA, Brucella abortus S-19; SA, Staphylococcus aureus; CA, Candida albicans.

Positive control key: PC, positive control employed/well; Chloramphenicol 20 \ig,

Salmonella typhimurium; Gentamycin sulphate 100 \xg, Escherichia coli;

Gentamycin sulphate 30 [ig. Brucella abortus S-19; Chloramphenicol 30 \ig,

Staphylococcus aureus; Amphotericin B 10 ig, Candida albicans.

34

Table 2.8 Antibacterial and antifungal activity of the crude extracts of the tested

drugs^

Drugs

SB

QSK

SK

QS

DD

PC

Blank

Crude extract

employed/well

20 mg

20 mg

20 mg

20 mg

20 mg

-7 <

Antibacterial and antifungal activity in terms of

zone inhibition (in mm) against test microorganisms

ST

3+

4+

-

3+

3+

4+

-

EC

2+

2+

-

3+

3+

4+

-

BA

4+

3+

4+

3+

3+

5+

-

SA

NT

NT

NT

NT

NT

NT

NT

CA

3+

3+

2+

3+

4+

3+

-

^Drugs key: SB, Safoof-e-Bars; QSK, Qurs-e-Sartaan Kafoori; SK, Safoof-e-

Kharish; QS, Qurs-e-Siizak; DD, Dawa-e-Dibba. Activity key: -, no inhibition; NT,

not tested; 1+, <10 mm zone of inhibition; 2+, 10-15 mm; 3+, 16-20 mm; 4+, 21-25

mm; 5+, 26-30 mm. Microorganisms key: ST, Salmonella typhimuriun; EC,

Escherichia coll; BA, Brucella abortus S-19; SA, Staphylococcus aureus; CA,

Candida albicans. Positive control key: PC, positive control employed/well;

Chloramphenicol 20 ^g. Salmonella typhimurium; Gentamycin sulphate 100 pg,

Escherichia coli; Gentamycin sulphate 30 jig, Brucella abortus S-19; Amphotericin B

10 ng, Candida albicans.

35

References

> Ahmad, ]., Beg, A.Z., (2001). Antimicrobial and phytochemical studies on 45

Indian medicinal plants against multi-drug resistant human pathogens, J.

Ethnopharmacol. 74, 113-123.

> Ahmad, R., Khan, A.V., Siddiqi, M.F., Hasnain, A., (2008). Effects of an

aqueous extract of Croton bonplandianum Bail!, in rats, Environ. Toxicol.

Pharmacol. 26 336-341.

> Bryan, C.P., (1930). The Papyrus Ebers. (Written ca. 1600 B.C.) Editor (Berlin).

> Cragg, G.M., Newman, D.J., (2002). Biodiversity Medicinal for the Millennia,

Sci. and Cult. 6S,3-\0.

> Davis, J., (1994). Inactivation of the antibiotic and the dissemination of

resistance genes. Science 264, 375-382.

> Hakim Kabiruddin., (1932). Beyaz-e-Kabir, vol. 2, Daftarul masih Karolbagh,

Delhi.

> Jain, S.K., (1991). Dictionary of Indian Folk Medicine and Ethnobotany, Deep

Publication, New Delhi.

> Kumar, S., Bagchi, G.D., Darokar, M.P., (1997). Antibacterial activity observed

in the seeds of some Coprophilous plants, Int. J. Pharmacog. 35, 179-184.

> Mulligen, M.E., Murry-Leisure, K.A., Ribner, B.S., Standiford, H.C., John, J.F.,

Karvick, J.A., Kaufmann, C.A., Yu, V.L., (1993), Methilicin resistant

Staphylococcus aureus, Am. J. Med. 94, 313-328.

> Patent formulation prescribed, manufactured and marketed by Dawakhana

Tibbiya College, A.M.U. Aligarh.

> Perez, C, Pauli, M., Bazerque, P., (1990). An antimicrobial assay by the well

agar method. Acta Biol. Med. Experimental. 15, 113-115.

36

> Piddock, K.J.v., Wise, R., (1989). Mechanism to resistant to quinilones and

clinical perspective, J. Antimicrob. Chemother. 23, 475-483.

> Robin, E.H., Anril, W., Alexander, M., Loeto, M., Keith, K., (1998).

Nasopharyngeal carriage and antimicrobial resistance in isolates of

Streptococcus pneumoniae and Hemophilus influenzae. Type b in children under

5 years of age in Botswana, Int. J. Infect. Dis. 3, 18-25.

> Singh, M., Chaudhry, M.A., Yadava, J.N.S., Sanyal, S.C, (1992).The spectrum

of antibiotic resistance in human and veterinary isolates of Escherichia coli

collected from 1984-1986 in Northern India, J. Antimicrob. Chemother. 29,

159-168.

> Subbarayappa, B.V., (2001).The roots of ancient medicine: an historical outline,

J. Biosci. 26, 135-144.

v_^

r^

CHAPTER-3 ANTIMICROBIAL ACTIVIT

HEPATOPROTECTIVE EFFECTS OF

37

3. Introduction

Operculina turpethum (Family: Convolvulaceae), commonly known as

trivrit or nishot in the Western part of India and adjoining Pakistan, is a plant with

immense ethno-medicinal value. This plant is widely grown throughout India and it

is occasionally cultivated in gardens as an ornament. This plant appears to be rarely

cultivated in non-Indian regions of Asia [Ooststroom, 1953; Verdcourt, 1963;

Fosberg and Sachet, 1977; Chang 1978] however, it is expected that the medicinal

use of this plant continued by Indian immigrants.

O. turpethum is a perennial climber with slender, fleshy and branched roots,

hard and twisted cord like stem with small ovate leaves [The Wealth of India, 2001].

Mainly, roots or stem bark of this plant are traditionally used for medicinal purpose.

The plant produces two types of roots: sufed or sveta or white roots that are mild,

and kala or krishna or black roots that give drastic, often poisonous effects [Watt,

1889]. The root, bark and seeds are reported to contain cardio-active glycosides,

formerly designated as neriodoprin, neriodorein and karabin which are anti-

inflammator)', stimulant and good pain relievers. Active constituents like P-

sitosterol, a- and P-turpethins, coumarin, scopoletin, lupeol and betulin [Husain et

al., 1992; Yoganarasimhan, 2000] glycosides, saponins flavonoids, steroids and

carbohydrates [^^^^ Kumar et al., 2009] have been reported to constitute bulk in

the plant. O. turpethum extract is used to treat wide range of ailments. For instance,

it is used to relieve periodic fevers, constipation, flatulence and colic obesity, to treat

anaemia, splenomegaly, raised lipid levels and obesity [Austin, 1982; Vasudevan,

1995; Suresh Kumar et al., 2006]. The oil extracted from the root bark is used in

skin diseases of a scaly nature. The fresh juice of leaves is dropped into the eyes for

38

inducing lachrymation in ophthalmia. It is also reported to use in the treatment of

piles, tumors and jaundice [Kirtikar and Basu, 1987].

In traditional medicine, natural or crude phytoextracts are considered as

alternative medicines, because some natural constituents present in them

counterbalance the side effects of synthetic medicines [Ahmad et al., 2008 a]. It is

therefore obvious that the therapeutic potential and risk efficiency of traditional

medicinal plants is based on the direct assessment of phytoextracts as well as effects

of their purified compounds. In case of botanicals also, the benign nature of the

constituents or their anti-mutagenic potential has to be ensured, since several

compounds produced within or derived from plants may possess mutagenic potential

[Ames, 1983; Agner et al., 2001]. Once their benign nature is ensured, anti-

mutagenic/ anti-clastogenic potential of phytoconstituents may also be exploited to

treat genetic and biochemical ailments. The approach will be at par with that

employed in cancer treatment, where the protective phytoconstituents were

consumed as part of the diet or designed remedies [Newman et al., 2003]. Reports

on anti-mutagenic or anti-clastogenic potential of a number of compounds extracted

from different plant products are available [Bruni et al., 2006; Barcelos et al., 2007 ].

Data are scarce on therapeutic potential of synthetic drugs to treat liver cirrhosis and

the toxic side effects may remain a persistent risk [Kang et al., 2002; Lotersztajn et

al., 2005; Kisseleva and Brenner, 2006]. Conversely, the use of a number of natural

products and phytoextracts has been reported to show negligible or no side effects

[Austin, 1982; Newman et al., 2003; Colvard et al., 2006; George et al., 2006; Arif

et al., 2007] against the ill effects produced by many synthetic compounds.

Therefore, in our opinion, it will be an interesting aspect to evaluate the therapeutic

potential of 0. turpethum (roots) against those chemical compounds which are of

39

routine use by industrial workers and cause serious damage. One such compound

selected during the present investigations is A -Nitrosodimethylamine (NDMA). The

present study was designed to evaluate whether O. turpethum (roots) aqueous extract

(OTE) exerts hepatoprotective effects in rats against NDMA induced liver toxicity.

TV -Nitrosodimethylamine (NDMA) is a potent hepatotoxin, carcinogen and

mutagen and it induces fibrosis and cirrhosis of the liver [George and Chandrakasan,

1996 a; George et al., 2001]. The toxicity produced by NDMA is mediated by its

reactive metabolites and not by the parent compound. NDMA is used as a softener

for copolymers production in industries, in addition to synthesis as a chemical

intermediate in the production of 1, 1-dimethylhydrazine and nematocide [lARC,

1971]. As a component of tobacco smoke condensate and certain alcoholic

beverages, NDMA can induce lung, liver or renal cancers [Magee and Barnes, 1967;

Lijinsky and Epstein, 1970].

It has been shown that NDMA induced hepatic fibrosis in rats is a suitable

and appropriate animal model to study biochemical and pathophysiological

alterations associated with the development of hepatic fibrosis and alcoholic

cirrhosis of human [Jenkins et al., 1985; Jezequel et al., 1987; Jezequel et al., 1990;

George and Chandrakasan, 1996 b; George, 2003]. Hepatic fibrosis is characterized

by excessive accumulation of connective fissue components, especially matured

collagen fibers in the extracellular matrix (ECM) of the liver. It is a complex

dynamic process which reflects the balance between ECM synthesis and degradation

[Friedman and Bansal, 2006]. The activation of hepatic stellate cells (HSCs) has

been associated with the pathogenesis of liver fibrosis [Friedman, 2003; Dataller and

Brenner, 2005]. Activated HSCs are proliferative and fibrogenic and expresses a-

smooth muscle actin (a-SMA) and various connective tissue proteins including

40

collagen type I, III and IV [Pinzani and Marra, 2001; Lotersztajn et al., 2005].

Moreover, activated HSCs have been implicated in hepatic inflammation through

their ability to secrete cytokines, including transforming growth factor-pi (TGF-pi)

during liver fibrogenesis [Friedman, 2003]. Hepatic fibrosis may be induced by

various chronic liver injuries including viral and autoimmune hepatitis, alcoholism

or biliary obstruction [Kisseleva and Brenner, 2006]. In general, fibrosis requires

years of exposure or sometimes decades to be visible clinically, but few notable

exceptions are there in which cirrhosis develops in months [Das and Vasudevan,

2007].

In the present study OpercuUna turpethum (roots) aqueous extract (OTE)

was used for hepatoprotective, antibacterial and antifungal activity.

3.1. Materials and Methods

3.1.1. Microbial studies

3.1.2. Preparation of phytoextract

Authentic plant material, OpercuUna turpethum, was procured from

Dawakhana (Pharmacy of Herbal Medicines), Ajmal Khan Tibbiya College, Aligarh

Muslim University. Following verification of the plant material by an established

plant taxonomist, some O. turpethum Linn root specimens were stored in the

Herbarium (Id. No. Cl/94) of Department of Pharmacology (Ilm-ul Advia), Ajmal

Khan Tibbiya College. Finely ground powder of the dried roots of O. turpethum L.

(~ 250g) was extracted in distilled water under reflux and filtered on Whatman #1.

The filtrate was dried in a rotary evaporator at a temperature of 40±1 °C under

reduced pressure [Harbone, 1973]. The yield of O. turpethum powder from 250g of

dried roots was ~10-12g. The powder was stored at -2 °C in sterilized and labeled

screw capped bottles.

41

3.1.3. Microorganisms used

The test microorganisms included one gram -ve bacterium. Salmonella

typhimurium (Clinical isolate) and one gram +ve bacterium, Listeria monocytogenes

(Standard strain) and two fungi, Candida albicans and Cryptococcus neoformans

(Clinical isolates).

3.1.4. Culture media

The media used to grow Candida albicans and Cryptococcus neoformans

was YPD (Yeast extract, peptone, dextrose) and to culture Salmonella typhimurium

nutrient broth (NB) was used. Listeria monocytogenes was cultured using brain heart

infusion agar. All culture media were prepared and treated according to the

manufacturer's guidelines (Hi-media Pvt. Ltd. India).

3.1.5. Inoculum

The microorganisms were inoculated into their respective medium as given

above. Inoculated broths were kept at 37°C for 12 hours. After 12 hours of

incubation absorbance was read against sterilized medium (used as Blank) at 580

nm. Culture was diluted to obtain 1x10^ CFU/ml.

3.1.6. Antimicrobial susceptibility testing

The MICs of the Operculina turpethum extract (OTE) for different

organisms were determined by broth micro dilution method described by National

committee for clinical laboratory standards [NCCLS, 1995]. The antimicrobial

agents were tested over the final concentration range of 50 to 0.048 |xg/ml. Tests

were perfonned in 96 well round bottom microtitre plates. Cell suspensions of

organisms were prepared to give final inoculums concentration of IxIO^ cells/ml.

The wells containing microbial inoculum with different concentrations of

42

compounds were incubated for 48 hours. The MIC was defined, as the lowest

concentration of compound at which there was complete inhibition of growth.

3.2. Biochemical Toxicology Studies

3.2.1. Chemicals

Acrylamide, 3, 3'-Diaminobennzidine tetrahydrochloride hydrate, A -

Dimethylnitrosamine, Nicotinamide adenine dinucleotide (P-NAD), Nitro blue

tetrazolium (NBT), Phenazine methosulphate (PMS) and Trizma base were

purchased from Sigma-Aldrich, India. Biochemical kits were from Span diagnostics

Ltd (India), Cyclophosphamide (500 mg Endoxan-N)''"' of Baxter Oncology GmbH

(Germany), Fetal Bovine Serum (FBS) was of Hi-Media, India, Goat anti-mouse

IgG-HRP conjugated was purchased from CALTAG laboratories, Bangkok, a-

Smooth muscle actin (a-SMA) antibodies were obtained from Trend Bio-products

Pvt. Ltd., India, All other chemicals used were of analytical grade.

3.2.2. Animals

Aduh male albino rats of Wistar strain were kept in well-aerated cages at the

departmental facility of Zoology department with equal interval of light-dark

exposure. The rats were acclimatized for a week and fed regularly with sterilized

diet and water ad libitum. Healthy, 8-10 week old rats weighing around 162 ± 10 g

were selected for the experiments.

3.2.3. Induction of hepatic fibrosis

The animals were divided into 4 groups of 5 each. One group served as the

negative control and the second group received intraperitoneal injections of normal

saline. The third group served as the positive control and received intraperitoneal

injections of cyclophosphamide in their recommended doses. The fourth group was

administered NDMA intraperitoneally in doses of 10 mg/kg body weight as

43

described previously [George and Chandralcasan, 2000]. The injections were given

on three consecutive days of each weelc for over 21 days.

3.2.4. Administration of Operculina turpethum extract (OTE)

Here also, the experimental animals were divided into 4 groups of 5 each.

One group served as the OTE control and received OTE alone. The remaining three

groups received NDMA injections as described above followed by OTE in

concentrations of 75, 150 and 200 mg/kg body weight OTE was administered after 5

hours of NDMA administration. The dose of OTE and its administration time was

selected on the basis of pilot studies [Suresh Kumar et al., 2006]. A set of animals

from each group were anesthetized with diethyl ether and sacrifice on days 7, 14 and

21 after the start of NDMA and OTE administrations. Blood was collected from a

deep cut made on the right jugular vein with a scalpel. Urine was collected under a

layer of toluene beginning from 24 hours prior to sacrifice.

3.2.5. Assessment of liver injury

The process of liver injury and toxicity was assessed through the progression

of hepatic fibrosis histochemically following staining of serial sections of liver with

hematoxylin and eosin (H&E). Stained slides were examined under Nikon

microscope with an LCD attachment (Model: 80/) and photographed.

3.2.6. Staining of a-smooth muscle actin (a-SMA)

The degree of hepatic fibrosis was monitored by following activation of

hepatic stellate cells as indicated by immunohistochemical (IHC) staining of a-

smooth muscle actin (a-SMA) filaments. For detection of a-smooth muscle actin (a-

SMA), serial liver sections (S^m) were stained immunohistochemically using a-

SMA monoclonal antibodies. Peroxidase activity was quenched by incubating the

sections with 3% hydrogen peroxide for 15-20 min. The sections were washed with

44

Phosphate Buffer Saline (PBS), layered with prediluted monoclonal a-SMA

antibody and incubated overnight at 2-8 °C in the moist chamber. After washing off

unbound antibody with PBS for 5 min, the sections were reacted with HRP

conjugated goat anti-mouse IgG immunoglobulins (secondary antibody) and

incubated for 30-45 min at room temperature. The slides were washed with cold

PBS and developed using 3% 3, 3'-diaminobenzidine tetrahydrochloride hydrate

(DAB) solution for 10-20 min. Developed slides were rinsed with PBS, counter

stained with Mayer's hematoxylin and mounted with DPX.

3.2.7, Biochemical parameters

The commercial kits of Span diagnostics Ltd. (India) were used for

determination of liver enzymes like alkaline phosphatase (ALP), glutamic

oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (OPT) and bilirubin

levels in rat sera. The enzyme activity levels were again verified by using other

routine laboratory techniques. Hydroxyproline (HP) levels were also estimated in

sera and urine samples of control and treated group of rats as per previously

described protocols [Woessner, 1961].

Levels of Lactate dehydrogenase (LDH) isoenzymes were analyzed in sera

and liver homogenates of experimental rats. Livers were homogenized in ice cold

Tris-HCl buffer (pH, 7.5), centrifuged at 12,000 rpm and -10-15 nl of clear

supematants were loaded and resolved on native 7.5% vertical slab polyacrylamide

gel electrophoresis [Ahmad and Hasnain, 2005]. LDH isoenzymes were visualized

by histochemical staining using L-lactate-PNAD and phenazine methosulphate

(PMS)-tetrazolium (NBT) protocol [Ahmad et al., 2009]. LDH isoenzyme bands

developed as purple to dark blue bands at 37°C within 25-30 min.

45

3.2.8. Densitometry

Quantitative estimates of LDH isoenzyme were made by densitometry of

enzyme gel-scans using Scion Imaging (Scion Corporation: Beta release-4.0)

software programs and presented as fractions in arbitrary units (AU) per gram wet

weight of the tissue.

3.2.9. Statistical analysis

The values of selected biochemical markers, hydroxyproline and LDH

isoenzymes were recorded in their corresponding units and presented as mean ± SD

(n=5). Analysis of variance (ANOVA) was applied to demonstrate significant

differences among levels of various liver enzymes.

3.3. Results

3.3.1. Microbial

The results obtained for antimicrobial screening of Operculina turpethum

extract (OTE) are shown in table-3.1. The antimicrobial activity has been tested

against one gram-negative bacterium, one gram-positive bacterium and two fungi.

The results of the screening indicate that OTE possessed good antimicrobial activity

against the entire tested microorganism. The Operculina turpethum extract (OTE)

was also observed to inhibit the growth of fungus, Candida albicans at the lowest

concentration of 6.25(ig/ml.

3.3.2. Hematoxylin and eosin staining

3.3.2.1. Histological observations during NDMA treatment and progression of

hepatic fibrosis

The hematoxylin and eosin stained slide of liver specimens during NDMA

administration and progression of hepatic fibrosis is shov.'n in Fig. 3.1. The control

liver sections showed normal lobular architecture (Fig. 3.1 A). On day-7

46

disintegration of liver parenciiyma, hepatic necrosis and severe centrilobular

congestion were observed (Fig. 3.1C). Massive hepatic necrosis along with

neutrophilic infiltration and multifocal collapse of parenchyma were the prominent

features in liver specimens (sections) of day-14 (Fig. 3.IE). On day-21, NDMA

treated liver specimens demonstrated disruption of normal liver architecture,

inflammation, hemorrhage and intensive fibrosis with deposition of thick collagen

fibers (Fig. 3. IG). At certain places sign of early cirrhosis was also visible.

3.3.2.2. OTE treatment

The hematoxylin and eosin staining of liver sections during amelioration by

OTE is also displayed in Fig. 3.1. The control liver specimens showed normal

cellular and lobular architecture with radiating hepatic cords (Fig. 3.1 B). A sort of

fatty change and a low level of Kupffer cells hyperplasia were the peculiar features

of OTE treated rats on the day-7 (Fig. 3.ID). On day-14, moderate infiltration of

mononuclear cells and regeneration of hepatocytes was observed in liver sections of

OTE treated rats (Fig. 3.IF). Remarkable decrease in the hepatic fibrosis was

visualized on day-21 of OTE treatment (Fig. 3.1H). The liver architecture restored to

normalize with more number of regenerating hepatocytes.

3.3.3. Staining of a-smooth muscle actin (a-SMA)

3.3.3.1. Histological observations during NDMA treatment and progression of

hepatic fibrosis

The staining of a-SMA for the visualization of activated hepatic stellate cells

is demonstrated in Fig. 3.2. In control liver specimens, the a-SMA staining was

absent (Fig. 3.2A). A large number of a-SMA positive stained stellate cells were

observed in the necrotic zone on day-7 of treatment with NDMA (Fig. 3.2C). From

day-7 onwards, the feature of staining of a-SMA was quite persistent. The staining

47

was quite deep (intense) and confined to the fibrotic area in liver specimens on day-

14 (Fig. 3.2E). The count of positively stained hepatic stellate cells further increased

in the fibrotic zone. On day-21, the a-SMA staining was almost restricted to the

fibrotic zone (Fig. 3.2G). The number of positive stained cells in the fibrotic area

was much higher when compared with normal areas of the liver sections.

3.3.3.2. OTE treatment

The a-SMA staining in liver specimens of rats treated with NDMA+OTE is

also shown in Fig. 3.2. Similar to the saline control, the a-SMA staining was also

absent in OTE control liver samples (Fig. 3.2B). On day-7, focal staining of a-SMA

around the central vein indicated the ameliorative action of OTE (Fig. 3.2D). In

liver specimens of day-14, the a-SMA staining was of very low intensity with a few

numbers of activated stellate cells distributed sporadically (Fig. 3.2F). On day-21,

the a-SMA staining was almost absent in liver sections of OTE treated rats. This

indicated restoration of liver parenchyma and regeneration of hepatocytes (Fig.

3.2H).

3.3.4. Sera ALP, GOT, GPT and bilirubin levels

Hepatotoxicity due to MDMA in rats was assessed by the levels of liver

function enzymes (SALP, SCOT, SGPT) and bilirubin in the sera. As compared to

their respective control group, significant increase in the activities of SALP, SCOT,

SGPT (/'<0.05) and bilirubin {P<0.00\) was recorded in rats treated with the

evaluated doses of NDMA (Table-3.2). Time and dose dependent protection' was

shown by OTE against NDMA induced hepatotoxicity in rats. Hepatoprotective

effect of OTE that was evaluated at three different concentrations: 75, 150 and 200

mg kg"' body weight day"' showed outstanding recovery in LFT values of rats

receiving 200 mg kg"' body weight day'' of OTE along with NDMA regimen. In

48

comparison with NOMA infused group at this dosage of OTE, significant reduction

in the levels of SALP, SCOT, SGPT (45.25, 28.94,10.6%) and bilirubin (65.77%)

was noted (Table-3.2).

3.3.5. Hydroxyproline levels in the sera and urine

Hydroxyproline levels were estimated in the sera and urine of NDMA treated

rats on day-21 of NDMA treatment. The increase of about 20.28 and 57.84% was

noted in sera and urine hydroxyproline levels, respectively (Table-3.2). Noticeable

decline in the activity of sera and urine hydroxyproline was observed in rats which

received OTE regimen (200 mg kg"' body weight day'') for 21 days. In sera and

urine samples of OTE receiving rats, the decrease in hydroxyproline levels was of

the magnitude of 16.65 and 47.29%, respectively.

3.3.6. Total and fractional activity of LDH isoenzymes

Lactate dehydrogenase (LDH) isoenzymes activity was estimated in the sera

and liver samples of treated animals and compared with control. The increase in

total LDH activity was noticed on day-7 onwards in sera samples of NDMA treated

rats. Particularly, the rise in the activity of LDH-4 and LDH-5 was observed in the

sera (Fig. 3.3A). OTE treated groups showed significantly decreased levels of sera

LDH-4 and LDH-5 within 21 days and the ranking of the LDH isoenzymes was

comparable to the control values (Table-3.3).

In liver samples of NDMA injected animals, an increase in the levels of

LDH-4 was observed on day-7, while LDH-5 was detected in significantly higher

levels at all the selected durations (Fig. 3.3B). On day-21, the ranking of LDH

isoenzymes was LDH-5>-4>-3>-l>-2 as compared with the control (LDH-5>-4>-

3>-2>-l). Treatment with OTE remarkably reduced the levels of LDH-4 and -5 in

liver samples of rats at all the durations. On day-21 of OTE treatment, LDH

49

isoenzymes ranking in liver samples was observed LDH-5>-4>-3>-2>-l which was

exactly similar to control values (Table-3.4).

3.4. Discussion

We obtained encouraging results of antimicrobial activity of Operculina

turpethum extract (OTE), with its activity against all the tested bacteria and fungi.

Evidence suggests that even in the alcoholic extracts of fresh roots of O. turpethum

antibacterial activity is present [The Wealth of India, 2001]. The extract was

observed to inhibit the growth of S. typhimurium and L. monocytogenes at a

concentration of 50ng/ml, while that of C. albicans successful inhibition was

obtained at 6.25ng/ml. The growth of C. neoformans was inhibited at a higher

concentration (>100 ng/ml) of OTE. It is presumed that compared with other routine

methods being used to ameliorate the common ailments, method of treating with

effective natural compounds is preferred [Ahmad et al., 2008 a].

The biochemical and molecular data obtained during the present study

strongly support to hepatic fibrosis and cirrhosis-inducing potential of JV-

Nitrosodimethylamine (NDMA) in rats [George and Chandrakasan, 1996 a; George

and Chandrakasan, 1996 b; George and Chandrakasan, 2000; George et al., 2001;

George, 2003; Das and Vasudevan, 2007]. The consequent histological and

biochemical alterations include increased synthesis and deposition of connective

tissue proteins, changes in hepatic architecture and concomitant impairment of

several biochemical processes [Jenkins et al., 1985; Savolainen et al., 1988] leading

to shrunken liver. For the first time we report that aqueous extract of O. turpethum

root (OTE) can protect NDMA-induced toxic effects on liver. OTE treatment

restored normal lobular architecture of the liver through extensive regeneration of

hepatocytes and recovery of biochemical processes.

50

We have taken up two aspects of histopathological changes in liver tissue: (i)

the changes which can be observed after hematoxyHn and eosin (H&E) staining and,

(ii) those detectable by immunohistochemical (IHC) staining for the expression of a-

smooth muscle actin (a-SMA). H&E stained liver sections show synthesis as well as

continued accumulation of collagen in liver from the day-7 onwards. In fact, by day-

21 post-treatment with NDMA, complete architecture of the liver deteriorated.

During hepatic fibrosis, the role of activated stellate cells in excessive collagen

synthesis is well established [Brenner et al., 2000; Das and Vasudevan, 2007;

George, 2008]. OTE administration in rats significantly reduced the inflammation,

necrosis and fibrotic area produced during NDMA induced hepatic fibrosis. The

reversal in necrosis and hepatic fibrosis due to the treatment with OTE was most

remarkable on day-14 and 21.

The expression of a-smooth muscle actin (a-SMA) by the stellate cells (SCs)

is considered as the most reliable marker for activated hepatic stellate cells which

add to collagenous connective tissue [Rockey et al., 1992]. The presence of high

number of activated SCs on day-7 of NDMA administration indicates that the

fibrosis had started much before day-7 [George and Chandrakasan, 1996 b; George

and Chandrakasan, 2000; George et al., 2001]. The late stage of hepatic fibrosis and

cirrhosis was characterized by more enhanced expression of a-SMA in the activated

SCs in the fibrotic area on day-14 and 21, respectively. OTE treatment caused

constant decline in the density of activated SCs on day-7 and 14. Antifibrotic and

hepatoprotective potential of OTE is evident by a decline in the a-SMA staining and

increase hepatocytes regeneration on day-21 indicating the elimination of activated

hepatic SCs.

51

Similar to previously published reports [George and Chandraicasan, 1997; El-

Zayat, 2007], we also recorded significant increases in total LDH activity in sera

with major contribution of LDH-4 and LDH-5 isoenzymes. These changes begin on

day-7 and become more intense on day-14 and 21. The initial increase has been

attributed to an increase in the synthesis of LDH due to the presence of higher

number of fiinctional hepatocytes during early fibrosis [George and Chandrakasan,

1997]. However, the increase in LDH levels at advanced stage of fibrosis appears to

be the consequence of tissue injury and necrosis leading to leakage of the enzymes

into blood stream [George and Chandrakasan, 1997; Ahmad et al., 2008 b; Ahmad et

al., 2009]. That the enzymatic changes may actually be the result of cellular injury is

supported by our histopathological data with the changes in enzyme and isoenzyme

levels. While the induction of fibrosis by NDMA disrupts cellular architecture of

liver tissue, which is also collateral to elevated enzyme levels, OTE significantly

reverses all of these changes.

No report is available on the hepatoprotective effect of OTE against NDMA

induced liver injury in rats, though its use as hepatoprotectant against paracetamol-

induced toxicity [Suresh Kumar et al., 2006] and in other clinical and pathological

conditions has been recognized [Austin, 1982; The Wealth of India, 2001].

According to the present data, the protection provided by OTE against NDMA-

induced hepatotoxicity was time as well as dose dependent. OTE at 200 mg kg''

body weight of rat for 21 days reduced the levels of hydroxyproline (sera/urine),

SALP, SGPT, SGOT and total bilirubin up to 16.65, 47.29 (P<0.001), 45.25, 10.6,

28.94 (P<0.05) and 65.77% (/'<0.001), respectively. The values in NDMA treated

rats on day-21 showed an increase of 20.28, 57.84, 55.72, 13.84, 33.68, 78.79 % in

sera, urine hydroxyproline, SALP, SGPT, SGOT levels and bilirubin, respectively.

52

This trend of increase is in agreement with the observations made earlier [George

and Chandrakasan, 2000; George et al., 2001].

The present study demonstrates that liver detoxification and the prevention

of collagen accumulation are simultaneous processes. This is evident from the

correspondence between histopathological observations of OTE treated rat livers and

the shift in sera ALP, GPT, GOT, LDH isoenzymes and bilirubin levels towards

control values. That there occurs lesser hydrolysis of collagen in OTE injected rats

on day-21 Is evident by lower hydroxyproline levels, which indicates reduced spill

of this non-essential amino acid in the sera/urine. Obviously, OTE restores

hydroxyproline levels by blocking the pathways to collagenesis. An increase in

hepatic collagenolytic enzymes and liver function test enzymes has been recorded in

cases of carbon tetrachloride-induced early fibrosis [Murawaki et al., 1990; Hall et

al., 1991] and the patients with chronic liver injuries [Murawaki and Hirayama,

1980].

3.5. Conclusion

The present study thus, suggests that 0. turpethum (roots) aqueous extract

(OTE) shows hepatoprotective effects by decreasing collagen contents, restoring a-

SMA activity and enzyme markers of the liver injury. Therefore it is advisable that

OTE in its maximum evaluated dose may be potentially useful in preventing liver

injuries in particular NDMA induced fibrosis.

Moreover, the extract also shows remedial effect against Salmonella

typhimurium, Listeria monocytogenes, Candida albicans and Cryptococcus

neoformans. Therefore may also be used to combat tested pathogenic borne

infection.

53

Table 3.1 Antimicrobial activity of Operculina turpethum.

Microorganisms

Salmonella typhimurium Listeria monocytogenes

Candida albicans Cryptococcus neoformans

Minimum inhibitory concentration in fig/ml

OTE 50 50

6.25 >]00

Positive control 1.25 2.0

0.25 0.06

OTE = Operculina turpethum extract, positive control ; Gentamicin (Salmonella

typhimurium. Listeria monocytogenes) and Amphotericin B {Candida albicans,

Cryptococcus neoformans)

54

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57

NDMA+OTE

Fig. 3.1 Hematoxylin and Eosin (H&E) staining of rat liver sections during the pathogenesis of NDMA induced hepatic fibrosis and concurrent administration of OTE. (A) Control liver (xI25). (B) OTE control (xl25). OTE was administered 200 mg/kg body weight (C) NDMA, Day 7 (xI25). Severe centrilobular congestion (arrow) and hemorrhagic necrosis. (D) NDMA + OTE, Day 7 (x250). Kupifer cells hyperplasia and fatty changes. (E). NDMA, Day 14 (xl25). Massive hepatic necrosis (arrow), severe neutrophilic infiltration and multifocal collapse of liver parenchyma. (F) NDMA + OTE, Day 14 (^125). Moderate infiltrafion of mononuclear cells and regenerafion of hepatocytes. (G) NDMA, Day 21 (xl25). Marked hepatic fibrosis (arrow) and deposition of collagen fibers. (H) NDMA + OTE, Day 21 (x250). Restoration of normal liver architecture with increased number of regenerating hepatocytes.

58

§1^4-*,. NOMA NDMA+OTE

Fig. 3.2 Immunohistochemical staining of a-smooth muscle actin (a-SMA) demonstrating activated hepatic stellate cells during the pathogenesis of NDMA induced hepatic fibrosis and ameliorating effects of OTE (A) Control liver (xlOO). Absence of a-SMA staining. (B) OTE control (xlOO). OTE was administered 200 mg/kg body weight. a-SMA staining is absent. (C) NDMA Day 7 (xlOO). Staining of a-SMA demonstrates activated hepatic stellate cells in the necrotic zone. (D) NDMA + OTE, Day 7 (x250). Focal staining of a-SMA around central vein indicating ameliorative effects of OTE. (E). NDMA, Day 14 (xlOO). Intense staining of a-SMA indicating extensive activation of hepatic stellate cells in the fibrotic zone. (F) NDMA + OTE, Day 14 (xlOO). Mild staining of a-SMA (G) NDMA, Day 21 (x200). Remarkable staining of a-SMA demonstrating enormous number of activated stellate cells in fibrotic zone. (H) NDMA + OTE, Day 21 (x250). Absence of a-SMA staining and restoration of normal liver parenchyma with regeneration of hepatocytes.

59

C D-- D-14 D-21 C D - Dl-1 D-21

# • • A

B

NOMA

_ LDH-I

- LDH-2 H - LDH-3

- LDH4 -

- LDH-5 -

U LDH-1

LDH-2 -\ LDH-3

- LDH4 -

- LDH-5 -

-*.--vr- SK;-^,!

• » 4 l

NDMA+OTE

Fig. 3.3 Polyacrylamide gel electrophoretic (PAGE) pattern of lactate dehydrogenase (LDH) isoenzymes in the serum (A) and liver homogenate (B) during the pathogenesis of NDMA induced hepatic fibrosis and concurrent administration of OTE in rats.

60

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> Harbone, J.B., (1973). Phytochemical methods-A guide to modern technique of

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(1985). A dimethylnitrosamine induced model of cirrhosis and portal hypertension

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F., (1990). Modulation of extracellular matrix components during

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F., (1987). A morphological study of the early stages of hepatic fibrosis induced

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r~\

INVESTIGATIONS ON

WILLD.

66

4. Introduction

Acacia leucophloea Willd. (Syn. A. alba Willd., Family Mimosaceae)

commonly kjiown as Safed Kikar is a moderate- sized deciduous tree, found in the

plains of Punjab, central India, Andhra Pradesh, Karnataka, Rajasthan and dry region

of peninsular India and is also found in the dry zones of Burma [Maheswari, 1963;

Anonymous, 1985].

The plant has been described for astringent, demulcent, aphrodisiac and other

medicinal uses, like treatment of traumatic ulcer and antisyphilitic agent [Chopra et

al., 1965; Nadkami and Nadkami, 1966; Anonymous, 1985; Khan et al., 1990; Khan

et al., 1991]. The bark extract is use as contraceptive and for menstrual problems

[Anita et al., 2004] and alcoholic extract of air dried heart wood powder possesses

antibacterial activity [Javed, 1990]. The plant has been reported for juvenomimetic

activity [Javed, 1990].

Isoprene, a - pinene and many other volatile organic compounds (VOC),

moderately emitted from the plant. [Padhey and Varshney, 2005]. The volatile organic

compounds have a great deal in atmospheric chemistry [Haagen-Smit et al., 1953;

Dodge 1984; Derwent 1999]. Among the volatile organic compounds Isoprene is ?

highly reactive species and has a great tendency to ozone formation [Dollard et al.,

1997].

Devi and Prasad (1991) quantitatively determined the presence of tannins and

related polyphenols from the leaf, bark and pods of the plant.

Tannins and polyphenols are the very important class of natural products

having great variation in their structure and phylogenetic distribution. Tannins are

very much important for leather industries as raw material to tan the hides and for

many other industrial applications [Edwards et al., 1952; Howes, 1953; McBride, 1983].

67

Kapoor et al., (1971) have reported the following seed analysis: ^ oister 71%,

protein 27.4%, pentosan 12.8%), water soluble mucilage 7.1%) and protein in mucilage

29.0% and the oil composition of seeds was reported by [Javed, 1990] with the

characteristics saponification value 193.4-194.9%), iodine value 110.3-111.2%),

percentage of oil 12.61, acid value 1.0-1.1, ester value 193.8-194.4%), specific gravity

d/''=1.108; viscosity E at 32°C - 64.5, moister 6.04%, ash content 1.04%), optical

activity - no rotation in 2%o solution of CHCI3, soluble in organic solvents and

charring with cone. H2SO4. The effect of temperature on viscosity of the oil was also

studied. The soap prepared from its oil was found to be similar to the soaps prepared

from edible oils.

Literature survey revealed that the plant has been examined for chemical

constituents and the constituents isolated from the different part of the plant listed

below.

68

Part of the plant

Flowers

Flowers

Flowers

Pods

Leaves and pods Stem Bark

Stem Bark

Heart wood

Heart wood Root bark

Roots

Roots

Compounds isolated

Behenic ester, p- Sitosterol, Quercetin-3-glucoside, Mannitol

Myricitin, Quercetin, 3'-Hydroxy-7-methoxyisoflavone, Apegenin, 8-c-Glucoside of Apegenin.

Octacosane, Trloacontane, Triacontanol, (+)- Pinitol, Gallic and p-Hydroxybenzoic acid, Methyl gallate, Kaempferol, Quercetin, Quercetrin, Rutin. P" Sitosterol, Luteolin, Mannitol

Hydrocyanic acid

n-Hexacosanol, P- Sitosterol, p-Amyrin

Isookanin, Cyanin, Leucodelphinidin -3-0- a-L-rhamnopyranoside, Delphinidin

P" Sitosterol, n-Octacosanol

Octacosanol, (+)- Pinitol Leucophleol, Leucophleoxol, Leucoxo!

l,3-Dihydroxy-5-methoxy-2-methylanthraquinone-8-0- a -L-rhamnopyranoside, l-Hydroxy-8-methoxy-2-methylanthraquinone-3-O-a-L- rhamnopyranoside, 1,5-Dihydroxy-8-methoxy-2-methylanthraquinone-3-0-a-L rhamnopyranoside, Galangin-3-O-a-L- rhamnopyranoside Betulinic acid-3-O-P-D-maltoside

References

Khan etal., 1991 Valsakumari and Suluchana, 1991 Rajasekhara etal., 1991

Khan et al., 1990 Bhadoria and Gupta, 1981 Joshi and Sharma, 1977 Clark-Lewis and porter, 1972Trivedi and Mishra,I984 Joshi and Sharma, 1977 Javed,1990 Bansal et al., 1980,Rojas, etal., 2001, Perales et al., 1980. Saxena and Srivastava, 1986

Srivastava and Mishra, 1985

69

H.,C- ,C00. -CH,

Behenic ester

p- Sitosterol

Quercetin-3- glucoside

HO-HO-

H-H-

CH2OH -H -H -OH -OH

CH2OH

Mannitol

70

Quercetin

H3CO

3'-Hydroxy-7-methoxy isoflavone

71

OH 0

8-c-Glucoside of Apegenin

H,C-

H,C

H,C.

Octacosane

Triacontane

Triacontanol

coh

(+)- Pinitol

72

COOH

HO' T OH OH

Gallic acid

COOH

OH

Para hydroxy! benzoic acid

HO' " T 'OH OH

Methyl gallate

73

OH OH

Rutin

Luteolin

H,C 3 " - \

Hexacosanol ^CHzOH

74

(3- Amyrin

Cyanin

OH OH Leucodelphinidin-3 -o- a-L-rhamnopyranoside

Delphinidin

75

H,C' XH2OH

Octacosanol

OH - J<'^

OH r T^CHzOH

Leucophleol

Leucophleoxol

76

J'"OH

Leucoxol

OCHP

l,3-Dihydroxy-5-methoxy-2-methylanthraquinone-3-o-a-L-rhamnopyranoside

OCHp OH

OH OH

1- Hydroxy-8 -methoxy-2-methylanthraquinone-3-o- a-L-rhamnopyranoside

OCHP OH

OH OH

1,5-Hihydroxy-8-methoxy-2-methylanthraquinone-3-o-a-L-rhamnopyranoside

77

OH OH

Galangin-3-o-a-L-rhamnopyranoside

COOH

Betulinic acid 3-o-P-D-maltoside

78

The bark of the plant is used as one of the ingredients in Unani medicine for

Gonorrhoea "Qurse-e-suzaPc" manufactured and mariceted by Dawalchana Ajmai Khan

Tibbiya College Aligarh Muslim University, Aligarh, India [Patent]. The use of bark

in herbal preparations prompted us to investigate the antimicrobial activity as well as

their phytoconstituents.

4.1. Materials and Methods

4.1.1. Plant material

The stem bark of the Acacia leucophloea Willd. was collected from the Gir

forest Gujarat, India in the month of August 2005 and identified by Dr. Athar Ali

Khan (Taxonomist) and logged in Department of Botany, Aligarh Muslim University,

Aligarh with the voucher specimen NO. 943.

4.1.2. Preparation of plant extract and column chromatography

The air dried bark 1.64 Kg was exhaustively extracted with ethanol. The crude

extract was evaporated in vacuum to yield dark brown crude product. The crude

product was successively fractionated with chloroform, ethyl acetate, acetone and

ethanol till the solvent in each case was almost colorless. Each fraction was

concentrated to dryness under reduced pressure and leveled as AL-2, AL-3, AL-4 and

AL-5 and kept at 4 °C in screw capped bottled for further use.

The ethyl acetate fraction (AL-3) 54.9 gm was adsorbed on silica gel and

passed over a column of silica gel (3 cm d x 120 cm.) set with petroleum ether. The

column was eluted with petroleum ether, chloroform, chloroform: ethyl acetate

(20:80, 50:50), ethyl acetate, ethyl acetate: acetone (50:50), acetone and methanol.

The fractions were monitored by TLC examination using different solvents

system and the same fractions were mixed together on the basis of TLC pattern. The

fractions obtained from petroleum ether and chloroform were found waxy so further

79

studies on these fractions were abandoned. The fractions obtained from chloroform :

ethyl acetate (20:80, 50:50), ethyl acetate, ethyl acetate : acetone (50:50), acetone and

methanol were taken under consideration for antimicrobial activity and phytochemical

investigation and labeled as A-1 (chloroform : ethyl acetate, 20:80) A-2 (chloroform :

ethyl acetate, 50:50), A-3, A-4, A-5 and A-6 (ethyl acetate), A-7 (ethyl acetate :

acetone 50:50), A-8 (acetone), A-9 (methanol).

The column chromatography of ethyl acetate fraction of Acacia leucophloea

Willd. bark extract afforded a fairly pure fraction, A-4. The 'H-NMR spectra of the

A-4 was very complex therefore the fraction A-4 was subjected to further purification

by preparative HPLC using C-18 column (30 X 250 mm). The pre-purified compound

was analyzed by UPLC-MS (Fig 4.1) for purity using gradient system: Column BEH

C-18 (2.1 X 100 mm) 1.7 , Mobile phase A 0.1% HCOOH (Aq.) and B Acetonitrile

(0.1% HCOOH), T% B: 0/03, 0.1/03, 1.4/100.0, 1.9/100.2, Diluent: methanol.

The purified fraction was subjected to mass spectrometry and NMR ( 'H-NMR

and ' C-NMR.

4.2. Microbial study

4.2.1. Microorganisms used

The test microorganisms included one gram -ve bacterium Salmonella

typhimurium (Clinical isolate) and one gram +ve bacterium Listeria monocytogenes

(standard strain) and two fungi, Candida albicans and Cryptococcus neoformans

(Clinical isolates).

4.2.2. Culture media

The medium used to grow Candida albicans and Cryptococcus neoformans

was YPD (Yeast extract, peptone, dextrose) and to culture Salmonella typhimurium

nutrient broth was used. Listeria monocytogenes was cultured using brain heart

80

infusion agar. All culture media was prepared and treated according to the

manufacturer's guidelines. (Hi-media Pvt.Ltd. India).

4.2.3. Inoculum

The microorganisms were inoculated into their respective medium as given

above. Inoculated broths were kept at 37°C for 12 hours. After 12 hours of incubation

absorbance was read against sterilized medium at 580 nm. Culture was diluted to

obtain IX 10 CFU/ml.

4.2.4. Antimicrobial susceptibility testing

The MICs of the fractions for different organisms were determined by broth

micro dilution method described by National Committee for Clinical Laboratory

Standards (NCCLS, 1995). The antimicrobial agents were tested over the final

concentration range of 50 to 0.048 fig/ml. Testing was performed in 96 well round

bottom microtitre plates. Cell suspensions of organisms were prepared to give a final

inoculum concentration of 1 X 10 cells/ml. The wells containing microbial inoculum

with different concentrations of compounds were incubated for 48 hours. The MIC

was defined, as the lowest concentration of compound at which there was complete

inhibition of growth.

4.3. Results

4,3.1. Microbial

The results of the antimicrobial screening of the fractions obtained from

ethanolic extract as well as the fractions obtained from column chromatography are

shown in table- 4.1 and 4.2 respectively. The results obtained are highly encouraging.

From the table 4.1 it seems very clear that ethyl acetate fraction (AL -3) inhibit the

growth of all the tested microorganisms.

Among the microorganisms used Listeria monocytogenes and Cryptococcus

neoformans shows good sensitivity against all the fractions. The chloroform fraction

does not inhibit the growth of Candida albicans while ethanol and acetone fractions

does not show activity towards Salmonella typhimurium.

From the table-4.2 we can observed that the fractions obtained from column

(Al to A9) shows activity against all the tested microorganism.

4.3.2. Spectral analysis

The mass spectrum of A-4 is given in Fig. 4.2 and Fig. 4.3 and ' H - N M R is

given in Fig.4.4 and Fig. 4.5. The '''C-NMR is given in Fig.4.6. The results are

summarized below:

ESl-MS: Positive ion mode [M+Hf m/z 835.5, 683.8, 563.7, 545.6

ESI-MS: Negative ion mode [M-H]' m/z 833.3, 561.1, 152.7

' H - N M R : Protons 5 6.92-6.08, 13H (m, br), 4.85-4.32, 6H (m, br), 4.23-4.04 3H

(m), 2.94-2.45 3H (m).

'^C-NMR: 5 157.8, 156.8, 156.4, 155.5, 146.0, 145.6, 145.3, 132.3, 132.0, 129.8,

121.0, 120.7, 19.4, 115.9, 113.3, 109.2, 103.6,84.5,84.2,82.1,72.1,71.5,68.5,42.5,

30.6, 30.4

The above spectral data support the following structure for A-4.

A-4

82

562,1

Chart 4.1

83

4.4. Discussion

Ethnopharmacologists, botanists, microbiologists, and natural-products

chemists are collaborating with each other for phytochemicals and engaged to develop

the safer alternatives for the treatment of infectious diseases [Cowan, 1999].

Historical evidences suggested that plants have provided a natural source of

antiinfective agents; emetine, quinine, and berberine remain highly effective

phytoproducts towards microbial infections. Phytomedicines derived from plants have

shown great promise in the treatment of intractable infectious diseases including

opportunistic AIDS infections [Iwu et al., 1999].

In the present study efforts are made to evaluate the antimicrobial activity and

phytochemical investigation of the Acacia leucophloea Willd. stem bark as it is used

in the herbal preparations. The antimicrobial activity of fractions obtained from the

ethanolic extract and the fractions obtained from column chromatography in terms of

minimum inhibitory concentration was assessed by micro dilution method as

suggested by National Committee for Clinical Laboratory Standards [NCCLS, 1995].

Among the fractions obtained from ethanolic extract the ethyl acetate fraction

shows remarkable activity against entire tested microorganisms.

Among the microorganisms used Salmonella typhimurium does not sensitive

against acetone and ethanol fraction but shows sensitivity against ethyl acetate

fraction and it inhibit the growth at a very low concentration of 12.5ng/ml. All the

fractions inhibit the fungal growth except chloroform fraction which does not show

activity against Candida albicans. All the fractions are active against Listeria

monocytogenes and Cryptococcus neoformans. The acetone fraction inhibits the

growth oi Candida albicans at a very low concentration of 6.25 |ig/ml as compared to

all other tested microorganisms.

84

Since ethyl acetate fraction showed activity against all the microorganisms

used, it was further fractionated by column chromatography.

The results of the screening of antimicrobial activity of fractions obtained

from column chromatography also displayed good activity against all the tested

microorganisms. All these fractions inhibit the growth of tested microorganisms.The

fraction A-1 inhibit the growth of yeast Candida albicans at the lowest concentration

of 25 ng/ml. Fractions A-3 and A-8 inhibit the growth of Salmonella typhimurium and

Listeria monocytogenes at the concentration of 25 ng/ml. while other fractions inhibit

the growth of microorganisms at the minimum concentration of 50 ng/ml and 100

Jig/ml.

The partially purified fractions (A1-A9) obtained from ethyl acetate fraction of

crude extract show inhibition at higher concentration compared to ethyl acetate

fraction itself. For example ethyl acetate extract (AL-3) inhibit the growth of

Salmonella typhimurium and Listeria monocytogenes at a concentration of 12.5 ng/ml

while partially purified fraction A-3 and A-8 inhibit at lowest concentration of 25

Hg/ml. Similarly A-1 inhibit the growth of Candida albicans at concentration of 25

^g/ml. Therefore the more inhibitory activity was observed in ethyl acetate fraction of

crude extract compared to partially purified compound may be attributed to synergic

effect.

As the literature survey revealed that plant is rich in tannins and related poly

phenols [Devi and Prasad 1991]. The compound isolated (A-4) from the stem bark of the

plant is very much related to condensed tannins. The condensed tannins are the

oligomeric and polymeric proanthocyanidins formed by the linkage of C-4 of one

catechin unit with C-8 or C-6 of the next monomeric catechin unit. [Khanbabaee and Ree

2001]. However, the structure of trimeric, tetrameric and higher oligimeric procyanidins

85

not yet fully known [Nonaka et al., 1981]. In the compound A-4, a new linkage between

monomeric catechin units is observe that is C-5 of one catechin unit linked with C-8 of

next catechin unit. This linkage was confirmed by the mass fragmentation (Chart 4.1).

The molecular ion peak of exact mass 834, appears in both positive and

negative ion mode so it confirms the molecular weight of the molecule. The m/z ion

appearing at 835.5, 683.8, 563.7, 545.6 in positive ion mode and 833.3, 561.1, 152.7

in negative ion mode suggest the trimeric structure of the compound, A-4.

The 'H N M R and ' C-NMR were run on a Varian 400 MHz instrument;

spectra were taken in methanol and tetramethylsilane as the internal standard.

Proton NMR spectra showed the phenylic protons at 5 6.92-6.08 as a multiplet

while the benzylic protons of C-4 carbons and C-2 carbons appeared at 5 4.85-4.32 as

a multiplet another set of benzylic protons of C-4 carbons appeared at 5 2.94-2.45 as

multiplet. The remaining protons of C-3 carbons appeared along with benzylic

protons at 5 4.23-4.04 as multiplet [Nonaka et al, 1981; Stafford et al, 1985; Ozawa et

al, 1990; Khanbabaee and Ree, 2001].

The structure was further supported by the appropriate resonance in the C-

NMR spectrum (5 157.8, 156.8,156.4,155.5, 146.0, 145.6, 145.3, 132.3, 132.0, 129.8,

121.0, 120.7, 19.4, 115.9, 113.3, 109.2, 103.6,84.5,84.2,82.1,72.1,71.5,68.5,42.5,

30.6, 30.4).

4.5. Conclusion

It may be concluded that the crude extracts rich in plant polyphenolics show

better antibacterial and antifungal activity compared to purified fractions. Therefore,

crude extract may be advisable for the treatment of tested pathogenic microorganisms

borne diseases.

86

Table 4.1 Antimicrobial activity of the fractions obtained from ethanolic extract of

Acacia leucophloea Wiild. stem bark.

Microorganisms

Salmonella typhimurium

Listeria monocytogenes

Candida albicans

Cryptococcus neoformans

Minimum inhibitory concentration in .g

AL-2

25

25

-

>100

Fractions*

AL-3

12.5

100

12.5

>50

AL-4

-

50

6.25

>25

AL-5

-

25

25

>100

/ml

Positive control

1.25

2.0

0.25

0.06

"Fractions key: AL-2 = Chloroform fraction, AL-3 = Ethyl acetate fraction,

AL-4 = Acetone fraction, AL-5 = Ethanol fraction, - = No activity, positive control;

Gentamicin {Salmonella typhimurium. Listeria monocytogenes) and Amphotericin

B {Candida albicans, Cryptococcus neoformans)

87

Table 4.2 Antimicrobial activity of the fractions obtained from column chromatography of ethyl

acetate fraction of Acacia leucophloea Willd. stem bark extract.

Microorganisms

Salmonella typhimuhum

Listeria monocytogenes

Candida albicans

Cryptococcus neoformans

Minimum inhibitory concentration in ;ig/ml

Fractions*

A-1

50

50

25

50

A-2

50

100

100

100

A-3

25

25

50

50

A-4

50

50

50

50

A-5

50

50

50

50

A-6

50

50

100

100

A-7

50

50

50

50

A-8

25

25

50

50

A-9

50

100

50

50

Positive control

1.25

2.0

0.25

0.06

'Fractions key: A-1 (CHCI3: Ethyl acetate, 20:80) A-2 (.CHCI3 : Ethyl acetate, 50:50), A-3, A-4,

A-5 and A-6 (Ethyl acetate), A-7 (Ethyl acetate : Acetone 50:50), A-8 (Acetone ), A-9

(Methanol). Positive control; Gentamicin (Salmonella typhimurium. Listeria monocytogenes) and

Amphotericin B {Candida albicans. Cryptococcus neoformans).

88

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References

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Publications

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Journal of Herbal Medicine and Toxicology 3 (1) 47-52 (2009).

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Chemico-Biological Interactions 181,145-153 (2009).