Chapter II

REVIEW OF LITERATURE

10

REVIEW OF LITERATURE

Free radicals are shown to be deleterious to the body causing cellular

damage, aging and certain diseases like cancer, atherosclerosis, liver

disorders etc.

Although molecular oxygen is found to be highly essential for

subsistence of living organisms, some of the oxygen species such as singlet

oxygen (1 0 2), super oxide radical (02") hydroxyl radical (OR), hydrogen

peroxidetfl-O,") and other hydroperoxides including lipid peroxides (LaO)

generated in the body have been found to be highly reactive.63,64 Also,

reactive oxygen species (ROS) are constantly produced during the normal

oxidation of foodstuffs, due to leaks in the electron transport chain in

mirochondria" About 1-4% of oxygen taken up in the body is converted as

free radicals. Dionsi et al66 demonstrated the formation of superoxide

dismutase (SOD) and H202 in the organelle from normal and neoplastic

tissues. The reactions of oxygen radicals in biology are of special interest

because of their involvement in tissue damage.

Normal cellular mechanisms such as escape of partially reduced

oxygen from electron transport chain, purine metabolism, phagocytosis,

nitric oxide synthesis, release of transition metal ions, microsomal and

11

nuclear membrane electron transport system involved in drug metabolism

(via cytochrome P4SO and b, systems) produce reactive oxygen species

(ROS). The action of many compounds provokes tissue damage through the

free radical mediated mechanism. These compounds include a large number

of drugs, anticancer agents, halogenated compounds and environmental

pollutants. Physical sources that produce free radicals are UV rays, X-rays

and y-rays, high-energy particles, neutron, proton, electron and a-particles.

Oxidative stress occurs in a cell or tissue when the concentration of

ROS exceeds the antioxidant capability of that cell.67 Almost all the

macromolecules are damaged by the free radicals. Eg. peroxidation of

polyunsaturated fatty acid (PUFA) in plasma membrane, oxidative

inactivation of sulfhydryl containing enzyme etc. But the human body has

mechanisms to scavenge these free radicals and reduce these injuries by the

action of enzymes such as superoxide dismutase (SOD), catalase,

glutathione peroxidase (GPx) etc. In addition, there are compounds, which

can either inhibit the production of free radicals or scavenge free radicals or

both and control the disease process. These are the antioxidants. They may

be classified as (l) endogenous antioxidants: those, which are physiological

in origin (eg. SOD, catalase and glutathione peroxidase and thiols (2)

exogenous antioxidants: those, which cannot be produced by human body,

but may protect it against pro-oxidant forces when administered as

12

supplement. Eg. Vit. E and Vit. C, carotenoids'V"etc. Exogenous

antioxidants are further classified as natural and synthetic antioxidants like

butylated hydroxy toluene(BHT) and butylated hydroxy anisole(BHA)

which are used in food industry. Among the natural antioxidants vitamins

have received the most careful scrutiny as chemopreventers.

Lipid Peroxidation and Tissue damage

Lipid peroxidation has been broadly defined as the "oxidative

deterioration of polyunsaturated lipids", ie, lipids that contain more than

two carbon-carbon double bonds (>C=C<). The major constituents of

biological membranes are lipid and protein, the amounts of protein

increasing with the number of functions the membrane performs. Damage

to PUFA tends to reduce membrane fluidity, which is known to be essential

for the proper functioning of biological membranes. Peroxidation sequence

in a membrane of PUFA is initiated by any species that has sufficient

reactivity to abstract a hydrogen atom from methylene (-CHT ) group

generating a carbon radical,-CH-,which is stabilized by a molecular

rearrangement to produce a conjugated diene, which then easily reacts with

an oxygen molecule to give a peroxy radical, R-OO. Peroxy radical can

abstract a hydrogen atom from another lipid molecule, thereby propagating

the damage." One free radical generates another free radical in the

neighbouring molecule; this is sometimes called "death kiss" by free

13

radicals. The lipid peroxy radical can also cyclize to form a five membered

endoperoxide radical. The breakdown of R-OOH and endoperoxides leads

to the formation of numerous products including malonaldehyde, MDA, 2­

alkene and hydroxy alkenals". Aldehydes are long lived and diffuse from

the site of their origin and attack target intracellularly and extracellularly.

Lipid peroxidation is one of the several types of microsomal

oxidations with important biomedical implications especially in the

pathogenesis of many types of tissue injury caused by ionizing radiation,

xenobiotics and therapeutic agents.7 1•72

Endogenous antioxidants

The important antioxidant enzymes within the body are superoxide

dismutase (SOD), catalase and glutathione peroxidase (GPx). Superoxide

dismutase has been found to be the first line of defense against superoxide

radical- mediated injury by catalyzing its conversion to H202

In mammalian tissues, two types of SOD have been described (1)

Cytosolic cuprozinc-SOD (Cu Zn SOD) and (ii) mitochondrial mangano­

SOD (MnSOD). H202 thus produced is detoxified either by catalase or

reduced by glutathione dependent reactions. SOD has an important role in

scavenging the superoxide O2 generated by redox cycling chemicals8.73

.

14

Two possible mechanisms are proposed (i). It is likely to act at the level of

the cell membrane and remove or prevent radical formation. (ii) Removal of

oxygen radicals in the growth medium.

Catalase is present virtually in all mammalian cells and is suggested

to playa dual role. (i) a catalytic role in the decomposition of HzOz. (ii) a

peroxidic role in which the peroxide is utilized to oxidize a range of

hydrogen donors (AHz) such as methanol, ethanol and formate. It is mostly

localized in the peroxisomes (microbodies) of liver and kidney. The catalase

reaction mechanism may be written as follows.

(1)

(2)

Catalase - Fe (iii)+ HzOz

Compound I + HZ02

----l~~ Compound I

--.~ Catalase - Fe (iii) +

2HzO+Oz

--.~ A +2HzO

At particular conditions, the protective action of superoxide

dismutase and catalase compliment each other in a sequential fashion.

Glutathione (L-y-glutamyl-L-Cysteinyl glycine) is important in the

circumvention of cellular oxidative stress, detoxification of electrophiles

and maintenance of intracellular thiol redox status74. Glutathione peroxidase

(GPx),a Selenium (Se) containing enzyme, catalyses the oxidation of GSH

to GSSG at the expense of HzOz.

15

This enzyme has high activity in liver, moderate activity in heart,

lung and brain and low activity in muscle. Glutathione reductase (GR)

catalyses the regeneration of GSH by the following reaction.

+GSSG+ NADPH+H

OR +---.~ 2GSH+ NADP ,

The oxidative stress in tissues is often reflected as high GSSG level

in the serum. GSH also plays a central role in co-ordinating the synergism

of various crucial antioxidants. Several thiols, dithiolthiones, disulfiram

analogs and Selenium compounds are GSH enhancers.

Xenobiotics

There are several xenobiotics that show adverse effects on the body.

They include acetaminophen." phenobarhital.f rifampicine," carbon

tetrachloride," cyclophosphamide.f thioacetamide'" etc. The effects of

each one are different. But all of them are found to produce toxicity on the

liver.

Carbon tetrachloride on Liver function.

Carbon tetrachloride (CC14) is a direct hepatotoxicant and liver of

most of the higher species of mammals is susceptible to CC14

damage.9, lO,l l ,80 Hepatic lipid accumulation was proved as a consistent

feature of CC14 toxicity as early as 1944.

16

According to lipid peroxidation hypothesis, CCl4 poisoning initiates

an intrahepatic process of destructive lipid peroxidation. Earlier efforts to

detect malondialdehyde (MDA), one of the products of lipid peroxidation,

after CCl4 poisoning, have not been successful.Y'" Placer et al85 have

noticed a rapid disappearance of MDA in vivo after intraperitoneal

administration of CCl4in rats. Recknagel and Ghoshal83-84 have shown that

MDA could be metabolized via mitochondrial pathway. Selective toxicity

of CCl4 has been shown to depend on its hepatic metabolism. Butler86

showed that CCl4 was reduced to CHCl3 both in vitro and invivo. He

suggested that carbon-chlorine bond in CCl4 and CHCl3 was subjected to

homolytic cleavage yielding the corresponding free radicals which then

alkylate SH groups of enzymes.

Alcohol pretreatment remarkably stimulates the toxicity of CCl4 due

to increased production of toxic reactive metabolites of CCI4, namely

trichloro methyl radical (CCI3) by the microsomal mixed function oxidase

system'" (MFOS). This activated radical binds co-valently to' the

macromolecules and induce peroxidative degeneration of PUFA.. This lipid

peroxidative degradation of biomembranes is one of the principal causes of

the hepatotoxicity.81,88-91

17

Time sequence of biochemical events in CCl4 toxicity

Within 30 minutes of CC14 administration, lipid metabolism IS

disturbed; at the end of 24 hrs the blood levels of hepatic enzymes are

maximum and there is marked centrilobular necrosis affecting up to half the

lobules'". Aspartate aminotransferase (AST) showed a decline after 1 day of

CC14 poisoning ; the decline became significant after 3 days and became

maximum after 5 days. However the level returned to normal by 10 days.

Protein synthesis is depressed after one hour of CCl4 poisoning with

dislocation of RNA particles." Recknagel and Ghoshal'" pointed out that

the endoplasmic reticulum of the hepatic parenchyma cells was the primary

subcellular organelle involved.

Pro-oxidant effect of CCl4 has been reported by Comporti et a194,

Dianzani et a195 and Glende et a196. Irreversible binding of CC14 to

microsomal phospholipids has also been reported by Vellarruel et a1.97

CC14 is used to induce liver injury in animal experiments to study

impaired hepatic function and the elevation of serum transaminases is taken

as evidence of impaired hepatic function. 98 Drotman et ae9 reported that

elevated levels of serum enzymes are indicative of cellular leakage and loss

of functional integrity of the cell membrane in liver' There are reports that

suggest that CCl4 cause liver damage due to liberation of free radicals. 100-101

Recknagel et a19 1 found that CC14 causes accumulation of fat in the liver

18

especially by interfering with the transfer of triglycerides from the liver into

the plasma. Blockage of the secretion of hepatic triglycerides into the

plasma is the basic mechanism underlying fatty liver induced by CC14 in the

rat. This causes elevated amounts of fats predominantly triglycerides in the

parenchymal cells.!" Bahar Ahmed et al l 02 observed an increase in plasma

aspartate aminotransferase (AST), alanine aminotransfrase (ALT), alkaline

phosphatase (ALKP) and decrease in total protein and albumin after CC14

administration in rats. Dhumal et al l03 reported that a single i.p injection of

CC14 caused an elevation in the activity of serum transaminases in rabbits.

Also Soni et al l 04 observed an increase in plasma bilirubin caused by single

i.p. administration of CC14 (100 111/ kg body weight). Agostini and Alfisi 105

found that CC14 induced a decline in albumin level related to the dose in

rats.

The aetiology of liver disorder caused by CC14 ingestion highlights

induced lipid peroxidation. The progression of liver injury after a single i.p

injection of CC14 (1.0 ml/kg body wt) was observed by Ohta et al. 106 Ashok

Shenoy et al l07 noted a decrease in the activity of hepatic SOD, catalase and

glutathione reductase after 24 hrs of CC14 intoxication ; hepatic glutathione

(GSH) and ascorbic acid was reduced and lipid peroxide content was

increased. Dhawan et al l 08 observed a significant depression of glutathione

concentration following long term treatment of CC14 to male albino rats.

19

Wang et al109 reported a decrease in catalase activity resulting from single

i.p. injection of 20% CCl4 in olive oil/g body weight. Carbon tetrachloride

plays a significant role in inducing liver damage by increasing lipid

peroxidation in membranes whose structural integrity is necessary for

lipoprotein release.IID-113

Paracetamol and Liver function

Paracetamol or acetaminophen is a commonly used safe analgesic

drug, which is known to cause centrilobular hepatic necrosis upon

overdose.!" Its toxicity also accounts for many emergency hospital

admissions and continues to be associated with high mortality. I 15 The

hepatotoxicity has been related to the production of a highly reactive

intermediate metabolite, N-acetyl-p-benzoquinone- imine (NAPQI), formed

by Cytochrome P450 mediated oxidation. 1I3 Following an overdose, hepatic

glucuronide and sulphate become depleted with a consequent increase in

P450 catalysed oxidation. The increased production of NAPQI coupled with

a decreased capacity to render the substance non-toxic, results in its

intracellular accumulation I 16, NAPQI with electrophilic and oxidant

characteristics consequently can deplete intracellular glutathione and

protein thiol groups, by alkylation and oxidation, and lead to the formation

of mixed disulfide. These events subsequently give rise to changes in the

cellular calcium homeostasis, lipid peroxidation and loss of mitochondrial

20

respiratory function 117-119. The damaged hepatocytes release factors that

both attract and activate hepatic macrophages, causing cell necrosis by

I d reacti . 120release of proteolytic lysosoma enzymes an reactive oxygen species.

Ryu et al121 has .described the elevation of AST and ALT, hepatic

lipid peroxidation and depletion of glutathione content after 4 hours of

paracetamol intoxication. Padma GM Rao l 22 has found that liver is capable

of spontaneous recovery a few days after paracetamol intoxication.

Experimental Models for the evaluation of antihepatotoxic activity

Experimental hepatotoxic states provide essential models for the

study of the physiologic and biochemical reflections of hepatic disease. The

classical agent for the study of the effects of hepatic necrosis is CCI/,IO

although many other necrogenic substances have also been used,

Galactosamine has been described to produce a lesion in experimental

'I hi h bl h f' I h ,,123-125 Thi idamma s, w IC resem es t at 0 vira epatins. ioacetami e-

induced liver injury has also been used, though less frequently, to evaluate

the hepatoprotective activity. It is reported to cause inhibition of the

respiratory metabolism of the liver due to the uncontrolled entry of ca"

ions, resulting in inhibition of oxidative phosphorylation. 126-127 .

Acetaminophen (paracetamol) is a non-toxic drug in the usual therapeutic

dose. In overdose, however, as a suicide attempt, it causes severe hepatic

necrosis. 128

21

Whole animals

The studies of the effects of toxic agents in the liver have utilized

whole animals for various in vitro preparations. The use of the whole

animal is essential for the demonstration that an exogenous agent has a true

adverse effect on the liver in a setting of physiologic significance. Whole

animal also elucidate the effect of various factors and manipulation on the

mechanisms of injury and the pathophysiologic impact of the hepatic injury.

The in vitro models, however, may be employed to elucidate specific

aspects of the mechanism of injury.

Parameters of injury

The utilization of the whole animal has involved selection of

measures of hepatic injury. These include lethality, histological changes

seen by light and electron microscopy, chemical changes in the liver- lipids,

hepatic contents of dienes and malondialdehyde, glutathione, superoxide

dismutase and physiologic and biochemical tests like AST, ALT, alkaline

phosphatase (ALKP), total bilirubin, total protein and albumin, that measure

the functional status or that reflect the type of intensity of hepatic injury.

Plants as Hepatoprotective Agents

Despite tremendous strides in modern medicine, there are hardly any

drugs that stimulate liver function, offer protection to the liver from damage

or help the regeneration of the parenchyma cells. Liver injury caused by

22

toxic chemicals and certain drugs has been recognized as a toxicological

problem. Herbal drugs are playing an important role in health care

programmes worldwide, and there is a resurgence of interest in herbal

medicines for treatment of various ailments including hepatopathy I29.

India, the abode of Ayurvedic system of medicine, assigns much

importance to the pharmacological aspects of many plants. There are

numerous plants and polyherbal formulations claimed to have

hepatoprotective activities.130-131 Two reviews have been published which

cover most of the works carried out in this field. Vaidya et al132 have

reviewed the experimental and clinical research work related to

heptoprotective effects of various formulations available in the Indian

market. Bhatt and Bhatt l33 have not only compiled the information

available regarding the studies on various promising plant drugs from India,

but also have discussed the problems and pitfalls pertaining to this research.

However, we do not have readily available satisfactory plant drugs /

formulations to treat severe liver diseases. Most of the studies on

hepatoprotective plants were carried out using chemical-induced liver

damage in rodents. A few excellent reviews have appeared on this subject

in the recent past. 134-135 These reviews attempt to focus on a more practical

and systematic approach towards the development of standardized

phytomedicine / herbal formulations for liver disorders and to update the

literature in this area.

23

In India, more than 87 medicinal plants are used in different

combinations III the preparation of about 33 patented herbal

formulations.136-137 Most commonly used 12 plants in herbal formulations

are given in Table 2.1. Several plants were reported as hepatoprotectives

against induced liver toxicity in animals by Indian investigators during the

last decades.138-174 (Table 2.2) Some of the polyherbal formulations are

verified for their hepatoprotective actions against chemical-induced liver

damage in experimental animals,175-184 (Table 2.3) Studies carried out in

foreign countries also show a good number of hepatoprotective plants.

(Table 2.4)

Table 2.1 Most commonly used plants in herbal formulations inIndia 137

No. Name of Plant Number of formulations reported1. Andrographis paniculata 28*2. Boerhavia diffusa 10*

I--

3. Eclipta alba 10*4. Oldenlandia corymbosa 10*5. Asteracantha longifolia 86. Apium graveolens 87. Cassia occidentalis 88. Cichorium intvbus 8*9. Embelia ribes 810. Picrorrhiza kurroa 10*11. Tinospora cordifolia 8*12. Tachyspermum ammi 8

* Scientifically validated in experimental animals.

The antihepatitis viral activities of the traditional plants are not

24

studied in experimental animals except a few plants. This is mainly due to

the lack of ideal in vivo test systems. Picrorrhiza kurroa, Glycyrrhiza

glabra, Eclipta alba and Andrographis paniculata are reported to have

activity against jaundice-producing Hepatitis B virus. Phyllanthus amarus

also appears to be very effective against hepatitis.

Studies carried out in Tropical Botanical Garden and Research Institue,

Thiruvananthapuram, have shown that Tricopus zeylanicus, Phyllanthus

madaraspatensis and P. kozhikodianus are extremely active against

paracetamol- induced liver damage in rats. 137

Table 2.2 Plants having liver protective property againstChemical- induced damage in experimental animals.

Sl. No. Plants Year of publication Ref. No.

1 Boerhavia diffusa 1989 138

2 Wedelia calendulacea 1989 139

3. Andrographis paniculata 1990 140·

4. Gracinia kola 1990 141

5. Withania somnifera 1991 142

6. Tridax procumbens 1991 143

7. Ocimum sanctum 1992 144

8. Gymnema sylvester 1992 145

9. Eclipta alba 1993 146

10. Mikania cordata 1993 147

11. Phyllanthus niruri 1993 148

12. Ricinus communis 1993 148

25

13. Tephrosia purpurea 1993 126

14 Phyllanthus emblica 1994 149-

15 Achillea mellifolium 1995 150

16. Cichorium intybus 1995 150

17. Capparis spinosa 1995 150

18. Artemisia maritima 1995 151

19. Geophila reniformis 1996 152

20 Acacia catechu 1997 153

21. Glycosmis pentaphylla 1997 154

22. Swertia chirata 1997 155

23. Sida rhombifolia 1997 156

24. Verbena officinalis 1998 157

25. Picrorrhiza kurroa 1998 158

25. -do- 1999 159

26 Moringa oleifera Lam 1998 160

27. Tinospora cordifolia 1998 161

28. Curcuma longa 1998 162

29 Tricopus zeylanicus 1998 163

30 Rosmarinus officinalis 1999 164

31 Phyllanthus fraternus 1999 165

32. Arctium lappa 2000 166

33. Ambrosia maritma 2001 167

34. Hedychium spicatum 2002 168

35 Polygala elongata 2002 169

36. Nigella sativa 2002 170

36. do- 2003 171

37. Foeniculum vulgare 2003 172

38. Azadiracta indica 2003 173

39. do- 2003 174

26

A recent report indicates that fumaric acid obtained from Sida

cordifolia has significant antihepatotoxic activity in rats 185. Ursolic acid,

occuring in many plants, also showed promising hepatoprotection against

paracetamol and carbon tetrachloride-induced liver damage in rats I K6-87.

Some of the plant constituents reported to have antihepatotoxic activity are

given in Table 2.5

Table 2.3 Some of the polyherbal formulations verified for their

antihepatotoxicity against toxic chemical induced liver

damage in experimental anlmals'r"

SI.No Formulation Reference No.

1. Liv.52 175,176,177,178

2. Liv. 100 178, 179

3. Jigrine 180, 181

4. Hepatomed 182

5. Koflet 183

6. Hepatogard 122

7. Liver cure 184

8. Livol 184

9. B. Liv. 184

10. HD-03 128

27

Table-2.4: Plants showing antihepatotoxicity in experimental animals

by investigators in foreign countries 137

Acacia catechu Acuba japonica Anacardium Aralia elataoccidentalis

Artemisia Arnica montana Atracylodes Baeckeacappillaris macrocephala [rutescensBunium persicum Bupleurum falcatum Curcuma longa Cucurbita pepo

Delphinium Dianthus superbus Ganoderma Ganodermadenudatum iaponicum lucidumGlycyrrhiza Lindera strychinifolia Linum Panax ginsengglabra usitatissimumPeumus boldus Plantago asiatica Rauwolfia Schizandra

species. chinensisSilybium Thujopsis dolabrata Withania Withaniamartanum frutescens somnifera

Table 205: Some of the plant constituents possessing hepatoprotective

t o it 137ac IVI y

Name of plant Name of active constituentAndrographis paniculata Andrographolide

Anacardium occidentalis Catechin

Curcuma longa Curcumin

Sida cordifolia Fumaric acid

Schizandra chinensis Gomishins

Glycyrrhiza glabra Glycyrrhyzin

Glycyrrhiza glabra Glycyrrhetinic acid

Picrorrhiza kurroa Kutkoside--

Picrorrhiza kurroa Picroside I

Picrorrhiza kurroa Picroside II

Schizandra chinensis Schizandrin A

Bupleurum falcatum Saikosaponins

Sedum sarmentosum Sarmentosins

Silybum marianum Silibin..-

Eucalyptus species Ursolic acid

Schizandra chinensis Wuweizisu C

28

There are reports establishing the fact that many of the plants exert

h . h . ff h h . id 188-197 This ff tt eir epatoprotectlve e ect t roug antroxi ant property. IS e ec

b .. Vi . . d bl 198-201can e seen even m certam itarruns, spices an vegeta es .

More than 60 medicinal plants have been shown to stimulate

secretion of bile fluid (choleretic) and salts (cholagogue) in experimental

animals. Most of them were done on normal anesthetized rodents.1.32,137

Chaudhury et ae02. , Subramaniam et a1 163, and Ansari et ae03 have

established the anticholestatic activity of Andrographis paniculata,

Tricopus zeylanicus and Picrorrhiza kurroa respectively in normal rats.

Regeneration

It is closely related to proper nutrition, including trace elements

intake such as Zinc and Strontium and Germanium. Astragali Radix,

Atractylodes Rhizoma, Codonopsis pilosulae Radix, Ginseng Radix,

Bupleuri Radix, Lycli fructus etc are rich in Sinidium, Zinc and

Germanium. Clinical and animal studies found that these herbs can promote

liver cell regeneratlon.F" Srivastava et ae05 demonstrated the stimulant

effect of picroliv, in liver regeneration, on partially hepatectomized rats by

enhancing macromolecular synthesis. According to them, this effect

occurred in the early phase of regeneration and significantly picroliv was

found to be more potent than silymarin. Later, Srivastava et ae06

29

investigated the effect of picroliv on DNA, RNA, protein and cholesterol

contents in livers of partially hepatectomised rats. Singh et ae07

demonstrated the stimulation of nucleic acid and protein synthesis in rat

liver with oral administration of Picrorrhiza which was comparable to

silymarin. Sonnenbichler et aeo8,209found that silymarin, a potent

hepatoprotective, stimulated the regeneration of hepatic tissue causing

increase in protein synthesis in damaged livers. In both in vivo and in vitro

experiments, significant increase in the formation of ribosomes and DNA

synthesis were measured in addition to the protein synthesis. The increased

protein synthesis was observed only in damaged livers (partial

hepatectomy), not in controls.

Hepatitis (Viral)

There are plants having specific activity against viral hepatitis.

According to Susuki et ae lO, a double blind study against viral hepatitis

with intravenous administration of glycyrrhizin was found to be effective,

particularly, in chronic viral hepatitis. In another study with (+)- catechin (a

polyphenol, obtained from green tea (Camellia sinensis), Susuki et al21l

found a significant drop in antibodies to hepatitis Be antigen (HBeAg) in

patients with HBeAg positive Hepatitis B. In yet another double blind

study Kanai et ae l2 observed the combined effect of (+) - catechin and

recombinant human alpha interferon in HBeAg positive patients. Mehrotra

30

et al,213 (1990) evaluated the anti- HBs-like activity of Picrorrhiza and

other compounds on serum samples obtained from patients of Hepatitis B

virus (HBV) associated acute and chronic liver diseases and healthy

HBsAg carriers. Promising anti HBsAg activity was noticed for

Picrorrhiza. Also it inhibited purified HBV antigen prepared from healthy

HBsAg carriers.

Although initial studies by Thyagarajan et ae14, in 1988, with

Phyllanthus amarus, showed promising results III Hepatitis B carriers,

further studies by Doshi et ae15 demonstrated that the plant could not clean

the hepatitis B surface antigen (HBsAg) in asymptomatic carriers of the

antigen.

Meanwhile in 1990, Crance et ae16could prove that Glycyrrhiza was

able to exert antiviral activity in vitro against a number of viruses including

hepatitis A .

In 1995, studies by Wang et al,217 on Phyllanthus niruri, have

revealed that it blocked DNA polymerase, the enzyme needed for the

Hepatitis B virus to replicate. Fiftynine percentage of those infected with

chronic viral hepatitis B lost one of the major blood markers of HBV

infection (HBsAg) after using Phyllanthus for 30 days. While clinical

studies on the outcome of Phyllanthus and HBV have been mixed, the

species P. urinaria and P. niruri seem to work far better than P. amarus.

31

Many previous studies on the hepatoprotective effects of P. niruri

corroborated traditional knowledge of its role in liver disorders. However,

in 1996, in an in vitro study, the aqueous extract of Phyllanthus amarus was

found to be effective in inhibiting the production of HBsAg for 48 hours

(in Alexander cell line) proving its anti-hepatitis B virus property at the

cellular level. 2 18 Again in 1996, Vaidya et al2 19, demonstrated a clinical

study with Picrorrhiza, on 32 patients diagnosed with acute viral hepatitis,

whose bilirubin level was normalized in an average of 27.4 days

Recently in 2000, Wang et ae20 observed that Glycyrrhizin

administered in a physiologic saline solution in combination with cysteine

and glycine (a product called stronger Neo Minophagen-C, or SNMC)

Glycyrrhiza stimulated endogenous interferon production in addition to its

antioxidant and detoxifying effects.

Review of Literature of Acalypha indica.

Acalypha indica Linn. belongs to the family Euphorbiaceae. It is the

second largest family with about 7500 species of plants distributed in 300

genera of which 374 species are found in India. They are herbs, shrubs and

trees often with an acrid milky juice. The plants in this family are very

diverse in their morphology and chemical constituents and thus much

disagreement remains with respect to their ranks, delimitation and systemic

relationships. 221

32

Genus Acalypha: Acalypha with 450-500 species, is one of the diversified

sorts more of the Euphorbiaceae family (after Euphorbia, Croton and

Phyllanthus). It has mainly a tropical and subtropical distribution- except in

Hawai and a few archipelagos of the Pacific with some representatives in

tempered zones.222, 223

The American continent lodges approximately two-third of the

species of known Acalypha from the south of United State to Uruguay and

north of Argentina. The dominant centers are located in Mexico, Bolivia

and Peru.224 About 27 species are found in India, of which 17 exotics have

been introduced as ornamentals.

Acalypha species known as copper leaf (Acalypha indica) or three

seeded mercury, are hardy and colorful plants with variously colored leaves

which have produced colorful varieties through mutation; they are the best

for adding color in the absence of flowers. Growth is more vigorous in hot

moist areas than in cooler regions. The plants may be pruned once in a year

to the desired height.

Occurrence and Distribution 225,226,227

Acalypha indica occurs as a weed in gardens, waste places and along

the roadsides, throughout the plains and hotter parts of India, Pakistan and

Srilanka. It extends to the Philippine Islands and Tropical Africa, from

33

Bihar eastward to Assam and Southward to Kerala, ascending the hills in

Orissa up to 210m.

The plant grows as an obnoxious weed and can be controlled by

spraying 2,4-D at the rate of 2.2 kg/ha.227

Acalypha indica Linn. (Euphorbiaceae)

The common names of the plant are:

English

Sanskrit

Hindi

Bengali

Gujarati

Telugu

Tamil

Malayalam

Kannada

Sinhala

Marathi

Oriya

Arabic

Chineese

French

Acalypha indica, Three seeded mercuryIndian Acalypha, Indian mercury.

Harita manjari, Arittanunjayrie, Muktavarcha, Rudra.

Kuppi khokli, Khokali,

Muktajhuri, Shwetbusunta

Dadano, Vanchhi-Kanto

Kuppichettu, Morrkondachettu, Mulakandachettu,Kuppaichettu, Pappantichettu.

Kuppaimeni, Kuppameni, Kuppamenia, Poonamayakki.

Kuppamani

Kuppi gida, Chalmari, Tuppakire.

Kuppameniya

Khajoti, Khokla, Kupi.

Indramaris

Harram-ed-d hibbel

T'ie Hau Ts'ai

Bois queue de rat.

34

Acalypha indica Linn (Euphorbiaceae)

The herb is a favuorite remedy in chronic bronchitis, asthma and

pneumonia; the plant is said to increase the secretions of the pulmonary

organ. It is an expectorant and a substitute for Senega. It is a substitute for

Ipecac also. As an emetic the herb is of special value in croup. It is said to

possess diuretic and carminative properties, but it causes gastrointestinal

irritation. A decoction of the plant is used as a safe and speedy laxative and

also to cure tooth and ear ache. It is used in congestive headaches. A piece

of cotton saturated with the expressed juice of the plant when inserted into

each nostril relieves symptoms by causing haemorrhage from the nose.226

Leaf. A paste of the leaves is applied to burns; with lime juice it is useful

in early stages of ringworm infestation.

35

Fresh leaf juice is applied with oil, salt or lime in rheumatoid arthritis

and to cure scabies, eczema and other skin infections.

The powdered leaves are used for bedsores and maggot infested

wounds. The leaf extract is said to possess anti-fungal properties. It is also

used in fractures.

The leaf juice or its decoction with addition of a little garlic is used

as an anthelmintic.

A suppository made of fresh leaves IS useful for small children

suffering from constipation.

The leaf juice together with tender shoots is occasionally mixed with

a small portion of margosa oil and rubbed on the tongues of infants for the

purposes of sickening and clearing their stomach of viscid phlegm.

In Malaysia, an infusion of the dried leaves is used as an expectorant,

. . d . 228antitussive an purgatrve,

Root:- An infusion of the root acts as a cathartic.r" The root in small doses

is an expectorant and nauseant; in large doses it is an emetic.23o A decoction

of the dried root with ginger and pepper is given orally to expel hookworm

in children.r'" The fresh root is used as a narcotic for cats.

In Ayurvedic medicine the hot aqueous extract of the plant is used

externally for the treatment of scabies, for relieving the pain of snakebite

36

and the irritation caused by the bite of centipeder'" and orally as a laxative

and for mania. In Ethnomedicine, the plant is recommended for the

f . 231treatment 0 acute mama.

In Homeopathy, a tincture of the plant is used as a remedy for severe

cough associated with bleeding from the lungs, haemoptysis and acute

epitaxis and incipient phthisis. 232 In 1986 Vaidyar'" made a note on role of

kuppamani in Sidha Vaidhya.

Biological Studies

The ethanolic (95%) extract of the entire plant was found to be

inactive, rarely,when tested for anti-anchylostomiasis activity, in human

adults.226

In 1979, Ganapathi Raman et at234 while doing a pilot study of A.

indica in bronchial asthma on 38 patients suffering from wheezing, cough,

dyspnoea etc. found relief in 60% cases and 90% got relief from severe

cough and eosinophilia within 2 weeks. Finally they got normalized.

In 1981, Bhowmick et at235 studied anti-fungal activity of Acalypha

indica on Curvularia lunata. . In 1982, they also studied antimycotic

activity of leaf extract on Drechsclera turcica that causes leaf blight of

maize.236

In 1982, Nisteswar et at237 made preliminary studies of A. indica on

isolated frog heart and skeletal muscles where the former exhibited positive

37

inotropic and positive chronotropic effects while the latter showed no

significant effect at all.

In 1983, Shanmughasundaram et ae38 reported the effect of Anna

Pavala Sindhooram, a herbo-mineral preparation containing leaves of A.

indica, used in the Sidha system of Indian medicine, for the prevention and

reversal of atherosclerosis.

Ethanolic (50%) extract of the dried entire plant tested for cytotoxic

activity at a concentration of 25 mcg/ml was found to be inactive.239

In 1994, Lamabadusuriya et ae40 reported about a clinical study

using A. indica (sinhala - kuppamaniya) where all the patients (four out of

four) developed acute intravascular haemolysis after ingestion of a broth

containing the drug. In the same year reports about the same effect came

from Sellahewai'" also. In 1996, Senanayaka et ae42 reported about the

above said effect from their clinical study.

Perumal Samy et ae43 in 1999 August reported the antibacterial

activity of aqueous residues of 16 different ethnomedicinal plants.

According to them, one among the most effective against Aeromonas

hydrophilla and Bacillus cerues was A. indica. In November 1999,

Hiremath et a1244, reported about the post coital anti-fertility effect of

different solvent extracts of A. indica where the petroleum ether and ethanol

extracts showed maximum effect.

3S

In 2000, Gopalakrishnan et all41S reported the anti-microbial activity

of chloroform and methanolic extracts of dried leaves of A. indica. They

found that the methanol extract showed maximum effect against Salmonella

typhosa.

In 2002, Reddy et ae46 reported about the wound healing property

of three drugs in rats of which one was Acalyplla indica.

Phytochemical Studies

Many phytoconstituents had been isolated from the plant so far.

I. In 1981, Bani Talapatra et all-1171 isolated a new amide designated

acalyphamide as its acetate C-II1H71SNo3b M.P. 1260lDlC along with

modified dipeptide aurantiamide and its acetate, snccinimide, 2­

methyl anthraquinone, tri-Osmerhyl-ellagic acid, fJ -sitosterol and B­

sitosterol- P -Dsglucoside from the leaves and twigs of A. indica.

Acalyphamide had been characterized as an amide of tyramine and

dotriacontanoic acid (C.uH6J,COOH). Aurantiamide and its acetate

are the first modified dipeptides reported from an Euphorbiaceae

plant and acalyphamide is one of the few natural tyramine arnides.

The occurrence of the amides and modified dipeptides in this plant is

of considerable biogenetic and chemotaxonomic significance.

2. Adolf Nahrstedt et aj1.II$; have reported about the isolation of a new

cyanogenetic glucoside, Acalyphin, from aerial parts of Acalypha

indica and its structure was identified mainly by 'HNMR and

39

13CNMR methods as 3-cyano-3- ~ -D- gluco pyranosyloxy -2

hydroxy-4 - methoxy-l- methyl -6 (2,3 dihydro) pyridone. The new

compound represents a new biogenetic type of cyanogenetic

glycoside, which is probably derived from nicotinic acid metabolism.

Acalyphin occurs in all parts of the plant together with a ~ ­

glucosidase capable of hydrolyzing 50 times as fast as prunasin and

13 times faster than coniferin. It is noteworthy that during the last

few years of continuous interest in the study of cyanogenetic

glycoside, only one totally new compound had been detected.

3. A cyanoglucoside of Acalypha was isolated and characterized III

1985.249 In addition to HCN, the plant contains other substances

which cause' intense dark chocolate brown discolouration of blood

and gastro intestinal irritation in rabbits.252

4. In 1993, studies on the isolation of Acalyphin from aerial parts and

its structure elucidation had been carried OUt,250

5. In 1994, stigmasterol had been isolated from the roots and leaves and

Acalyphol acetate from the leaves.229

6. Asima and Satyeshf" in 1997, reported about the presence of

aurantiamide and its acetate, succinimide, acalyphol acceatae, 2­

methyl anthraquinone, tri-o-methyl - ellagic acid as its acetate and ~­

D glucoside in the leaves.