shodhganga.inflibnet.ac.inshodhganga.inflibnet.ac.in/bitstream/10603/35125/10... · punamavoside...

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2 Review of Literature PgNo

2.1 Ayurvedic Medicine in Treating Liver Disease 7-11

2.2 Review of plants mention in formulation. 12-32

2.3 Antioxidant activity 33

2.4 In vivo hepatoprotective activity 34-37

2.4.1 Hepatotoxins for experimental models

2.4.2 Biochemical parameters for evaluating hepatic function:

2.4.3 Histopathological study

2.5 In vitro hepatoprotective activity 38

1. Studies on isolated hepatocyte

2. Studies on HepG2 cell line

2.6 Clinical study 39

Review of Literature

2. REVIEW OF LITERATURE

2.1 AYURVEDIC MEDICINE IN TREATING LIVER DISEASE

Use of herbal drugs in the treatment of liver diseases has a long tradition,

especially in Eastern medicine. There are numerous plants and polyherbal formulations

claimed to have hepatoprotective activities. Polyherbal formulations have synergistic

potentiative agonistic/antagonistic agent within themselves that work together in a

dynamic way to produce therapeutic efficacy with minimum side effects. Nearly 150

phytoconstituents from 101 plants have been claimed to possess liver protecting activity

(Doreswamy & Sharma, 1995; Handa & Sharma, 1989). At the same time, surprisingly,

we do not have readily available satisfactory plant drugs/formulations to treat severe

liver disease. Most of the studies on hepatoprotective plants were carried out using

chemical-induced liver damage in rodents as models (Doreswamy & Sharma, 1995;

Handa & Sharma, 1989; Hikino & Kiso, 1983; Evans, 1996). In India, more than 87

medicinal plants are used in different combinations in the preparation of 33 patented

herbal formulations (Sharma et al., 1991). Most commonly used 12 plants in herbal

formulations are given in Table 1.

Table 1: Most commonly used plants in herbal formulations in India

Boerhaavia diffitsa (1 OJ v Apium graveolens (8J

Andrographis paniculata (28J--./ Cassia occidentalis (8)

Eclipta alba (1 OJ --./ Cichorium intybus (8)

Picrorrhiza kurroa (1 OJ --./ Embelia ribes (8J

Oldenlandi a corymbasa (1 OJ Tinospora cordifolia (8) V

Asteracantha longifolia (8) Trachyspermum ammi(8J

'v' Scientificall validated i ( ) y n expe mental ammals. n

( ) Indicates the number of formulations in which plant is used.

Only a small portion of the hepatoprotective plants as well as formulations used in

traditional medicine are pharmacologically evaluated for their efficacy. During the last

decade several plants were reported as hepatoprotective against hepatotoxicity in animals

by Indian investigators (Table 2).

Page 7


Table 2: Plants with hepatoprotective property against toxic chemical induced liver

damage in experimental animals.

Plants

Acacia catechu (Jayasekhar et al., 1997)

Achillea millefolium (Gadgoli & Mishra, 1995)

Azadirachta indica (Chattopadhyay et al., 1994)

Andrographis paniculata (Visen et al., 1993; Handa & Sharma 1990)

Boerhaavia diffitsa (Rawat et al., 1997; Chakorbatti & Handa, 1989a,b)

Berberis aristata (Janbaz, 1995)

Capparis spinosa (Gadgoli & Mishra et al., 1995)

Chelidonium majus (Doreswamy & Sharma 1995)

Cichorium intybus ( Gadgoli & Mishra, 1995 & 1997)

Daucus carota (Bishayee et aJ., 1995)

Eclipta alba (Saxena et al., 1993)

Geophila reniformis (Subramoniam et al., 1996)

Glycomis pentaphylla (Mitra & Sur, 1997)

Gingiber officina/is (Ajith et al., 2007)

Mikania cordata (Mandai et al., 1993)

Moringa oleifera (Ruckmani et al., 1998)

Ocimum sanctum (Chattopadhyay et al., 1992)

Phyllanthus emblica (Gulati et al., 1994)

Phyllanthus debilis (Sane et al., 1995)

Phyllanthus kozhikodianus ( Asha & Pushpangadan, 1998)

Phyllanthus maderaspatensis( Asha & Pushpangadan, 1998)

Phyllanthus niruri (Reddy et al., 1993)

Picrorrhiza kurroa (Ansari et al., 1998; Saraswat et al., 1997; Dwivedi, 1991)

Ricinus communis (Reddy et al., 1993)

Sida cordifolia (Kumar & Mishra, 1997a)

Sid a rhombifolia (Kumar & Mishra, 1997b)

Swertia chirata (Mukheijee et al., 1997)

Page 8


Tephrosia purpuria (Ramamurthy & Srinivasan 1993)

Terminalia chebula (Tasduq et al., 2006)

Tinospaora cordifolia (Singh et al., 2003)

Trichopus zeylanicus (Subramoniam & Evans, 1998)

Tricosanthes dioica (Ghaisas et al., 2008)

Verbena officina/is (Singh et al., 1998)

Wedelia calendulacea (Sharma et al., 1989)

Withania somnifera (Sudhir & Budhiraja,1991)

Some of the Polyherbal formulation like Liv.52 (Katuria & Singh, 1997 ),

Hepatomed (Sharma et al., 1995), Jigrine (Kapur et al.,1994; Karunakar et al., 1997),

Koflet (Farooq et al., 1997), Stimuliv (Asha, 1996) and classical Ayurvedic formulations

like Amalkadi Ghirta (Achliya et al., 2005), Arogyavardhini (Achliya et al., 2003),

Panchgaya Ghirta (Dange et al., 1987) were verified for their hepatoprotective action

against chemical induced liver damage in experimental animals. Studies carried out in

foreign countries also show a good number of hepatoprotective plants (Table 3)

(Subramoniam & Pushpangadan, 1999)

The antihepatitis virus activities of the traditional plants were not studied in

experimental animals except in a few plants. This is ~ainly due to the lack of ideal in

vivo test systems. Picrorhiza kurroa, Glycyrrhiza glabra, Eclipta alba and Andrographis

paniculata were reported to have activity against jaundice producing Hepatitis B virus

(Doreswamy & Sharma, 1995; Handa & Sharma, 1990). Phyllanthus amarus also appears

to be very effective against Hepatitis B (Ott et al., 1997; Thyagajaran et al, 1988). Only a

few plants are really very promising hepatoprotective agents based on the available data.

These include P. kurroa (Picroliv) (Dwivedi et al., 1991), A. paniculata

(Andrographolide) (Visen et al., 1993), Silibum marianwn (Silymarin) (Wang et al.,

1996; Chrungo et al., 1997 a,b) and Eclipta alba (fhyagarajan et al.,1982). Studies

carried out at Tropical Botanical Garden and Research Institute (TBGRI) had shown that

Trichopus zeylanicus (Subramoniam et al., 1998), Phyllanthus maderaspatensis, and P.

kozhikodianus (Asha & Pushpangadan, 1998) were extremely active against paracetamol

induced liver damage in rat. A recent report indicates that fumaric acid obtained from

Page 9


Sida cordifolia has significant anti-hepatotoxic activity in rats (Kumar & Mishra, 1997).

Ursolic acid which occurs in many plants also showed promising hepatoprotection

against paracetamol and carbon-tetrachloride induced liver damage in rats (Shukla et al.,

1992; Saraswat et al., 1996). Some of the plant constituents reported to have

antihepatotoxic activity is given in Table 4. Antioxidants can protect experimental

animals and human from oxidant mediated liver damages. This effect could be seen even

in certain common vitamins, spices and vegetables (e.g. Vitamin-E and turmeric)

(Subramoniam & Pushpangadan, 1999).

Table 3: Plants with antihepatotoxic activity researched abroad

Acacia catechu Atracylodes macrocephata

Ganoderma japonicum Plantago asiatica

A cuba japonica Baeckea frutescens

Ganoderma lucidum Rauwolfia spp

Anacordium oxidentalis Bunium persicum

Glycyrrhiza glabra Schizandra chinensis

Aralia elata Bupleurum falcatum

Lindera strychinifolia Silybum marianum

Arnica montana Curcuma longa

Linum usitatissimum Thujopsis dolabrata

Artemisia capillaries Cucurbita pepo

Panax ginseng Withania fnttescens

Atracylodes lanceae Delphinium denudatum

Peumus boldus Withania somnifera

Dianthus superbus

Page 10


Table 4: Plant constituents possessing hepatoprotective activity (Hikino & Kiso, .

1988; Evans, 1996; Asha, 1996; Saraswat et al., 1996)

Andrographolide Andrographis paniculata

Silybin Silybum marianum

Picroside I Picrorhiza kurroa

Picroside II Picrorhiza kurroa

Kutkoside Picrorhiza kurroa

Gomishins Schizandra chinensis

Schisandrin A Schizandra chinensis

Glycyrrhizn Glycyrrhiza glabra

Glycyrrhetinic acid Glycyrrhiza glabra

Saikosaponins Bupleurum falcatum

Sarmentosins Sedum sarmentosum

Wuweizisu C Schisandra chinensis

Catechin Anacardium occidentalis

Ursolic acid Eucalyptus spp.

Curcumin Curcuma longa

Fumaric acid Sida cordifolia

Page 11


2.2 PLANTS USED IN PUNARNAVASHATAK KWATH

FORMULATION

(Boerhaavia diffusa Linn, Picrorhiza Kurroa Royle ex Benth, Berberis aristata DC,

Tinospora cordifolia (Willd.) Miers, Terminalia chebula Retz, Azadirachta indica A.

Juss, Zingiber officina/is Rose, Tricosanthes dioica Roxb)

BOERHAA VIA DIFFUSA LINN

Synonym: B. repens Linn. B. procumbens Roxb.

Family: Nyctaginaceae.

Habitat: Throughout India as a weed.

English: Horse-purslane, Hogweed.

Botanical description of plant

Boerhaavia diffitsa is a perennial creeping weed, prostrate or ascending herb, up to 1 m

long or more, having spreading branches. The roots are stout and fusiform with a woody

root stock. The stem is prostrate, woody or succulent, cylindrical, often purplish, hairy,

and thickened at the nodes. Leaves are simple, thick, fleshy and hairy, arranged in

unequal pairs, green and glabrous above and usually white underneath. Flowers are

minute, subcapitate and present in a group of 4-10 together in small bracteolate umbels,

forming axillary and terminal panicles. These are hermaphrodite, pedicellate and white,

pink or pinkish red in color. Two or three stamens are present and are slightly exerted.

The stigma is peltate. The achene fruit is detachable, ovate, oblong, pubescent, five

ribbed and glandular, anthocarpous and viscid on the ribs (Thakur et al., 1989).

Traditional uses

In old Indian books of medicine such as the Charaka Samhita and Sushrita Samhita, it is

mentioned that the Ayurvedic preparations made from punarnava - namely,

pzmarnavastaka kvath, punarnavakshar and pzmarnava taila - were used for the

treatment of various ailments. The whole plant of B. diffitsa is a very useful source of the

drug pzmarnava, which is documented in Indian Pharmacopoeia as a diuretic (Chopra,

1969). The active principle contained in the herb is an alkaloid, known as punamavine.

The roots and leaves with flowers have been found to be highly potent (Anonymous,

Page 12

------------------------------------~-----~~-~


1988). In Ayurvedic medicine, different parts of this plant were reported to have various

medicinal properties. Its root has been widely used for the treatment of dyspepsia,

jaundice, enlargement of spleen and abdominal pain (Kirtikar & Basu, 1999e) and as an

antistress agent. It was used in renal ailments as diuretic (Anand, 1995) and to treat

seminal weakness and blood pressure (Gaitonde et a[;, 1974). It is also used in the

treatment of stomachache, anemia, cough, cold, as a diaphoretic, laxative, expectorant

and a potent antidote for snake and rat bites in the treatment of nephrotic syndrome

(Singh & Udupa, 1972), hepatitis, gall bladder abnormalities and urinary disorders

(Mudgal, 1975; Cruz, 1995). The flowers and seeds are used as contraceptive (Chopra et

al., 1956).

Chemical constituents

The B. diffusa plant contains a large number of compounds such as flavonoids, alkaloids,

steroids, triterpenoids, lipids, lignins, carbohydrates, proteins, and glycoproteins.

Punamavine (Agarwal & Dutt, 1936; Basu et al., 1947; Surange and Pendse, 1972),

boeravinone A-F (Kadota et al., 1989; Lami et al., 1990; 1992), hypoxanthine 9-L

arabinofuranoside (Ahmad & Hossain, 1968), ursolic acid (Mishra & Tiwari, 1971 ),

punamavoside (Jain & Khanna, 1989), liirodendrin (Aftab et al., 1996) and a

glycoprotein having a molecular weight of 16-20 k Da (Verma et al., 1979) were isolated

and studied in detail for their biological activity. The herb and roots were rich in proteins

and fats. The herb contains 15 amino acids, including 6 essential amino acids, while the

root contains 14 amino acids, including 7 essential amino acids. A new antifibrinolytic

compound 'punamavoside' was also reported from the roots of B. diffitsa (Seth et al.

1986).

Page 13

OH lj \·.

c~H A ./ooc~c~- -/:\~ _) r/ ~ -~

01~> 0~ OH- 1 ~C~~S

OH

Punarnavoside

Hepatoprotective activity

OH HO

Me


0

OH 0

Boeravinone A

An aqueous extract of thinner roots of B. diffitsa at a dose of 2 ml/kg exhibited marked

protection of various enzymes such as serum glutamic oxaloacetic transaminase, serum

glutamic-pyruvic transaminase and bilirubin in serum against hepatic injury in rats

(Rawat et al., 1997). The methanolic and chloroform extract of roots and of the aerial part

of B diffusa exhibited antihepatotoxic activity against CC14 intoxication in rats

(Chakorbatti & Handa, 1989a,b). An alcoholic extract of whole plant given orally

exhibited hepatoprotective activity against experimentally induced carbon tetrachloride

hepatotoxicity in rats and mice (Chandan et al., 1991 ). The effect of 50% ethanolic

extract of roots of Boerhaavia diffitsa on country made liquor (C. M. L.) induced

hepatotoxicity was studied in albino rats. B. diffusa (1 00 mg/1 00 g body weight/day)

protected the rats from hepatotoxic ·action of C. M. L. as evidenced by changes in serum

alanine aminotransferase (ALT), Triglycerides (TG), Cholesterol and total lipid levels in

both serum and tissues (Gulati et al., 1991).

Other pharmacological actions

Pharmacological studies had demonstrated that punarnava possesses punarnavoside,

which exhibits a wide range of properties, diuretic (Gaitonde et al., 1974); anti

inflammatory (Bhalla et a!., 1968); antifibrinolytic (Jain & Khanna, 1989);

anticonvulsant (Adesina, 1979); antibacterial (Olukoya eta!., 1993); antistress agent;

antihepatotoxic (Mishra, 1980; Chandan et al., 1991; Rawat et al., 1997); anthelmintic

febrifuge, antileprosy, anti-asthmatic, antiscabies, and anti-urethritis (Nadkami, 1976a);

and antinematodal activity (Vijayalakshmi et a!., 1979). The plant was reported to be

Page 14

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


efficacious in abdominal tumors and cancers (Leyon et al., 2005). Plant also showed

analgesic activity (Hiruma-Lima, 2000). Dried root powder of the plant showed curative

efficiency when administered orally for one month to children or adults suffering from

helminth infection (Singh & Udupa, 1972). The purified glycoprotein from B. diffusa

exhibited strong antimicrobial activity against RNA (ribonucleic acid) bacteriophages

(Awasthi and Menzel, 1986). An antifibrinolytic agent, punarnavoside, was found to stop

IUCD-induced bleeding in monkeys (Barthwal & Srivastava., 1994).

Ayurvedic formulations

Punarnavastaka kwatha curna; Punarnavasava; Punarnavadimandura; Sukhmara

ghrata; Sothagna !epa (Anonymous, 1990c)

PICRORHIZA KURROA ROYLE EX BENTH.

Other names: Kutki, Kuru (Beng & Hindi); Kadu (Guj.); Katuka (Sansk.)

Family: Scrophulariaceae.

Habitat: The alpine Himalayas from Kashmir to Sikkim.

English: Picrorhiza.

~otanical description of plant

Picrorhiza kurroa has a long, creeping rootstock that is bitter in taste and grows in rock

crevices and moist, sandy soil. The leaves of the plant are flat, oval and sharply serrated.

The flowers are white or pale purple and borne on a tall spike. The active constituents are

obtained from the root and rhizomes. The plant is self-regenerating but unregulated over

harvesting has caused it to be threatened to near extinction (Atal et al., 1986; Subedi,

2000).

Traditional uses

The rhizome of Picrorhiza has been traditionally used to treat worms, constipation, low

fever, scorpion sting, asthma and ailments affecting the liver.

Page 15



Kutkin was the active principal of Picrorhiza kurroa and comprised of kutkoside and the

iridoid glycoside picrosides I, II, and III. Other identified active constituents were

apocynin, drosin, and nine cucurbitacin glycosides (Weinges et al., 1972; Stuppner &

wagner, 1989). Apocynin is a catechol found to inhibit neutrophil oxidative burst in

addition to being a powerful anti-inflammatory agent (Simons et al., 1990) while the

cucurbitacins was highly cytotoxic and possess antitumor effects (Stuppner & wagner,

1989).

0

1:------v /--'\ ( \ -

AI ' HO/ \~--~

I /

Structure of Apocynin


The hepatoprotective action of Picrorhiza kurroa is not fully understood but may be

attributed to Picrorhiza's ability to inhibit the generation of oxygen anions and to

scavenge free radicals (Russo et al., 2001). Picrorhiza's antioxidant effect has been shown

to be similar to that of superoxide dismutase, metal-ion chelators and xanthine oxidase

inhibitors (Chander et al., 1992a). In rats infected, with malaria, Picrorhiza restored

depleted glutathione levels, thereby enhancing detoxification and antioxidation (Chander

et al., 1992b). Picrorhiza also demonstrated an anti-lipid peroxidative effect (Chander et

al., 1998). Like silymarin, Picrorhiza has been shown to stimulate liver regeneration in

rats, possibly via stimulation of nucleic acid and protein synthesis (Singh et al., 1992). Its

anti-inflammatory action is attributed to the apocynin constituent, which has been shown

to have potent anti-inflammatory properties in addition to inhibiting oxidative burst in

neutrophils (Simons et al., 1990). Although the mechanism is unclear, animal studies

Page 16


indicate Picrorhiza's constituents exhibit a strong anticholestatic activity against a variety

of liver-toxic substances, appearing to be even more potent than silymarin (Shukla et aL,

1991; Saraswat et al., 1999b). The active constituent of Picrorhiza kurroa, showed a dose

dependent hepatoprotective activity against oxytetracycline induced hepatic damage in

rats. Numerous animal studies, primarily in rats, have demonstrated that the active

constituents of Picrorhiza kurroa were effective in preventing liver toxicity and the

subsequent biochemical changes caused by numerous toxic agents. Hepatocytes damaged

by exposure to galactosamine, thiocetamide and carbon tetrachloride were incubated with

Picrorhiza constituents. A concentration-dependent restorative effect was observed in

regard to normal hepatocyte function (Visen et al., 1998). A similar effect was seen when

25 mg/kg/day oral Picrorhiza extract was administered to rats poisoned by aflatoxin B 1

exposure. Picrorhiza kurroa significantly prevented the biochemical changes induced by

aflatoxin B1 (Rastogi et al., 2001). Picrorhiza extract, when given at a dose of 3-12

mg/kg orally for 45 days, was also shown to be effective in reversing ethanol-induced

liver damage in rats (Saraswat et al., 1999b ). In an animal model of hepatic ischemia, rats

were given Picrorhiza orally at 12 mg/kg daily for 7 days, prior to induced ischemia,

demonstrated improved hepatocyte glycogen preservation and reduced apoptosis,

compared to control animals (Singh et al., 2000). Picrorhiza principals have also shown

to be effective in treating Amanita mushroom poisoning in an in vivo animal model

(Dwivedi et al., 1992). An in vitro study demonstrated Picrorhiza's antioxidant activity

by subjecting human Glioma and Hep 3~ cells to a hypoxic state. Picrorhiza treatment

reduced the cellular damage cause by hypoxia, indicating that its constituents may protect

against hypoxia/reoxygenation-induced injuries (Gaddipati et al., 1999). Studies indicate

that Picrorhiza extracts showed therapeutic value in treating viral hepatitis. An in vitro

study investigated anti-hepatitis B-like activity of Picrorhiza and found to have promising

anti-hepatitis B surface antigen activity (Mehrotra et al., 1990). In a randomized, double

blind, placebo-controlled trial of 33 patients diagnosed with acute viral hepatitis, 375 mg

Picrorhiza root powder was given three times daily for two weeks. The treatment group

comprised of 15 patients; the remaining 18 subjects acted as controls and received

placebo. Bilirubin, SGOT and SGPT values were significantly lower in the treatment

group (V aidya et al., 1996)

Page 17



In vivo studies of bronchial obstruction indicate that the drosin constituents of Picrorhiza

kurroa prevented allergen and platelet activating factor-induced bronchial obstruction

when given to guinea pigs via inhalant and oral routes. In vitro histamine release was also

inhibited by the plant extract (Dorsch & Wagner, 1991). Picrorhiza extract given orally at

25 mg/kg to mice and rats resulted in a concentration-dependent decrease in mast cell

degranulation. However, induced bronchospasm was not prevented, indicating a lack of

direct post-synaptic histamine receptor blocking activity (Baruah et al., 1998).


Aarogyavardhini vati; Katukadya loha; Tiktadi kwatha; Tiktadi ghirta (Anonymous,

1990b).

BERBERIS ARISTATA DC.

Family: Berberidaceae.

Habitat: North western Himalayas, Nilgiris, Kulu and Kumaon.

Common Name: Tree Turmeric, Indian Barberry, Ophthalmic Barberry

English: Indian Barberry.

Indian names: daruhaldi (Bengal), daruharidra, darvi, kata, pitadaru, suvarnavarna

(Sanskrit)


A large deciduous shrub usuallyl.8-3.6 min height. Twigs are whitish or pale yellowish

brown. Bark is pale brown, deeply furrowed and rough. Leaves are obovate or elliptic,

entire or spinous-toothed, base gradually narrowed with prominent reticulate nerves,

glossy dark green above, glossy pale green but not glaucous beneath. Inflorescence is

simple drooping raceme, 2.5-7.5 em long, dense flowered. Pedicel is stout, 4-6 mm long,

ovoid, blue black with a thick pale bloom. Wood and roots are yellow inside (Chauhan,

1999).

Page 18


Traditional uses

Berberis aristata has a long history of medicinal use in both Ayurvedic and Chinese

medicine (Sack & Froehlich, 1982). The plant has anti-bacterial, anti-inflammatory,

astringent, alternative, antipyretic, antiperiodic, anti-septic, anti-cancer, bitter,

cholagogue, diaphoretic, emmenagogue, laxative, stomachic and sweat-inducing

activities. It is mainly used in eye diseases, haemorrhoids, amenorrhoea, leucorrhoea,

piles, sores, peptic ulcers, dysentery, heartburn, indigestion, hepatitis, intermittent fever,

and chronic ophthalmia. An infusion of root is useful in treatment of malaria, skin

diseases, diarrhoea and jaundice. It is also used to treat infections, eczema, parasites,

psoriasis and vaginitis.


The major bioactive chemical constituents of B.aristata are Berberine, oxyberberine,

berbamine, aromoline, karachine, palmatine, oxycanthine and taxilamine

0 0

MeO

OMe

Berberine


Leaves of this plant showed hepatoprotective activity against acetaminophen induced

liver damage (Gilani & Janbaz 1992). Berberis aristata (shoot and fruits) and berberine

(an alkaloid from Berberis aristata) were found to be protective against both paracetamol

and CCl4-induced liver damage and also showed MDME (microsomal drug metabolizing

Page 19


enzymes) inhibitory activity (Janbaz, 1995). Berberine, a known compound from. this

plant, was studied for its possible antihepatotoxic action in rats against different toxicant

like acetaminophen or CC14, suggesting a selective curative effect against acetaminophen

(Janbaz & Gilani, 2003). Berberine also showed inhibitory effects on potassium and

calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection

(Wang et al., 2004).


Berberis aristata has been reported to possess antibacterial, antiamoebic, antifungal,

antihelminthic, and tuberculostatic properties (Soffar et al., 2001). It has also reported to

have immunomodulatory (Sohni et al., 1996), anti-carcinogenic (Anis et al., 2000), anti

hyperglycemic and antioxidant (Singh, 2009) activities

Toxicity

In higher doses, it gives rise to symptoms like vomiting, severe diarrhoea, excessive

sweating etc.

Ayurvedic formulations (Anonymous, 1978)

Darvyadi kwatha, Darvyadi leha, Darvyadi taila, Rasanjana, Dasanga !epa

TINOSPORA CORD/FOLIA (WILLD.) MIERS EX HOOK.

Common Name(s): Guduchi, amrita (Sanskrit), giloe, gulancha (Bengali), giloya

(Hindi), gado, galo (Gujarati), heartleafmoonseed (English)

Family: Menispermaceae.

Habitat: Tropical India and the Andamans.

Ayurvedic: Guduuchi, Guduuchikaa, Guluuchi, Amrita, Amritaa, Amritalataa,

Amritavalli, Chinnaruuhaa, Chinnodbhavaa, Madhupami, Vatsaadani, Tantrikaa,

Kundalini, Guduchi satva (starch).

Page 20



Tinospora cordifolia is a glabrous, succulent, climbing shrub native to India. It also

found in Mynamar and Sri Lanka. It thrives easily in the tropical region, often attains a

great height and seems to be particularly fond of climbing up the trunks of large neem

trees. The bark is gray or creamy-white in color, deeply cleft spirally and longitudinally,

with the space in between spotted with large, rosette-like lenticels. The wood is white,

soft, and porous. Freshly cut surface quickly assumes a yellow tint on exposure to air.

The branches bear smooth heart-shaped leaves, unisexual greenish flowers (summer), and

red berries (winter). Long thread-like aerial roots arise from the branches as well. The

viscous sap has a light yellow color, odor, and nauseating bitter taste (Chopra et al.,

1982).

Traditional Uses

It is an Antiperiodic, Antipyretic, Diuretic, Anti-inflammatory. It is a constituent of

several compound preparations. It is used in fever, urinary disorders, dyspepsia, general

debility and urinary diseases. It is also used in treatment of rheumatism and jaundice

(Kirtikar & Basu, 1999a; Nadkarni 1976c)


More recently, a wide variety of sesquiterpenes and diterpenes have been isolated from

the stems of the plant. The major isolated compounds include the following:

Cordiofolisides A, B, and C (new norditerpene furan glycosides) (Gangan et al., 1994)

,tinocordifolin and tinocordifolioside (daucane-type sesquiterpenes); (Maurya et al 1998;

Maurya et al 1997), palmatosides C and F (furanoid diterpene glucosides) (Gangan et al.,

1996) cordioside, tinosponone, and tinocordioside (clerodane diterpenoids); (Maurya et

al., 1995; W azir et al., 1995) tinosporaside (a novel 18-norclerodane diterpene glucoside),

(Khan et al., 1988) and tinocordiside (a new cadinane sesquiterpene glycoside) (Ghosal et

al., 1997). In addition, syringin, cordiol, cordioside, cordifoliosides A and B (new

phenylpropene disaccharides) were identified as the active principles with

anticomplement and immunomodulatory activities (Kapil et al., 1997; Maurya et al.,

1996). It has been shown that the stem of the plant contains the alkaloid berberine, and

Page 21


cultures of stem callus from this plant have been shown to have the capability of

synthesizing this compound (Padhya et al., 1986). Ecdysterone, makisterone A, and 20P

hydroxyecdysone are the phytoecdysones isolated from the aerial parts of the plant

(Pradhan et al. 1997; Gangan et al., 1997; Pathak et al., 1995). Other constituents

reported from T. cordifolia include a new phenolic lignan, Octacosanol, nonacosan-15-

one, heptacosanol, P-sitosterol, tinosporidine, cordifol, cordifolone, (Hanuman et al.,

1986; Khaleque et al., 1971) magnoflorine, tembetarine (Pachaly et al., 1981), syringine

and syringine apiosylglycoside (Sipahimalani et al., 1994).

H0, 0 () "" ' / /.

I /' --(

HO \ OH

OMe

Cordifolioside A


OMe

OH

Its hepatoprotective action was reported in one of the experiment in which goats treated

with this plant showed significant clinical and hemato-biochemical improvement in CCl4

induced hepatopathy. It also exhibited in vitro inactivating property against Hepatitis B

and E surface antigen in 48-72 h (Mehrotra et al., 2000). Effect of its extract on

modulation of hepatoprotective and immunostimulatory functions in carbon tetrachloride

(CC14) intoxicated mature rats were reported (Bishayi et al., 2002). Kupffer cells are

major determinants of outcome of liver injury. T. cordifolia showed significant

improvement in Kupffer cell function and a trend towards normalization (Nagarkatti et

al., 1994). Extract of T. cordifolia improved the cellular immune functions in

management of obstructive jaundice in the clinical study of 16 patients (Rege et al.,

1989).

Page 22


Other medicinal properties

The notable other medicinal properties reported are antipyretic, anti-malarial (Ikram et

al., 1987), anti-diabetic (Stanely et al., 2003), anti-allergy (Badar 2004), anti-spasmodic,

anti-inflammatory, anti-arthritic, anti-oxidant (Subramanian et al., 2002; Stanely et al.,

200 I), anti-allergic (Nayampalli et al., 1986), anti-stress, anti-leprotic (Asthana et al.,

2001), mmunomodulatory (Nair et al., 2004), antifertility (Gupta & Sharma, 2004) and

anti-neoplastic (Singh et al., 2005) activities.

Ayurvedic formulations (Anonymous, 1989; Anonymous 1990a)

Gaducyadi curna; Gadusyadi kvatha; Amrutarista; Dasmularista, Gaduci ghrta; Gaduci

tail a

TERMINAL/A CHEBULA Retz.

Synonym(s): Terminalia panJiflora Thwaites, Terminalia tomentella kurz

Common Name: Haritaki (Sanskrit and Bengali), Harad (Hindi), Karkchettu (Telugu),

Kadukkaya (Tamil), Harada (Marathi & Gujrati).

Family: Combretaceae.

Habitat: Abundant in Northern India. Also occurs in the forests of Assam, West Bengal,

Bihar, Assam, especially in Konkan.

English: Chebulic Myrobal~, Black Myrobalan.


Terminalia chebzt!a is a medium to large deciduous tree attaining a height of upto 30 m,

with widely spreading branches and a broad roundish crown. The leaves are elliptic

oblong, with an acute tip, cordate at the base, margins entire, glabrous above with a

yellowish pubescence below. The flowers are monoecious, dull white to yellow, with a

strong unpleasant odour, borne in terminal spikes or short panicles. The fruits are

glabrous, ellipsoid to ovoid drupes, yellow to orange brown in colour, containing a single

angle stone. Terminalia chebula is found throughout deciduous forests of the Indian

subcontinent, on dry slopes up to 900 meters in elevation (Nadkarni 1976b; Lemmens et

al., 1995)

Page 23


Traditional uses

The fruit is tonic, stomachic, carminative, expectorant, anthelmintic, antidysentric, useful

in asthma, sore throat, thirst, vomiting, hiccough, eye diseases, disease of heart and the

bladder, vesicular calculi, urinary discharge, ascites, biliousness, inflammation, tumours,

bleeding piles, typhoid fever, leucoderma, dysponea, constipation, anaemia, gout,

elephantiasis (kirtikar & basu, 1999c ).


Terminalia chebula is rich in tannin. The chief constituents of tannin are chebulic acid,

chebulagic acid, corilagin and gallic acid (Bruneton, 1995; Chevallier 1996). Tannin of

Terminalia chebula was pyrogallol (hydrolyzable) type. A group of researchers found 14

components of hydrolyzable tannins (gallic acid, chebulic acid, punicalagin, chebulanin,

corilagin, neochebulinic acid, ellagic acid, chebulegic acid, chebulinic acid, 1,2,3,4,6-

penta-0-galloyl-H-D-glucose, 1 ,6,-di-0-galloyl-D-glucose, casuarmm, 3,4,6-tri-0-

galloyl-D- glucose, terchebulin) from fruits (Juang et al., 2004). It should contain 32%

tannin content (Evans, 1996). The tannin content of Terminalia chebula varies with

geographical variation (Jayaramkumar, 2006). Besides, fructose, amino acids, succinic

acid, ~ sitosterol and resin, purgative principle of anthroquinone and sennoside was also

found to be present (Chevallier 1996; Creencia et al 1996). Flavonol glycosides,

triterpenoids, coumarin conjugated with gallic acids called chebulin as well as other

phenolic compounds were also isolated (Kapoor, 1990; Asish & Sashi 1993; Williumson,

2002).

COOH ID o.<-0

HO OH 0 0

OH

Gallic acid Ellagic acid

Page 24



Its extract was found to prevent hepatotoxicity caused by the administration of rifampicin

(RIF), isoniazid (INH) and pyrazinamide (PZA) (in combination) in a sub-chronic model

due to its prominent anti-oxidative and membrane stabilizing activity (Tasduq et al.,

2006). Six extracts and four compounds of its fruit exhibited antioxidant activity at

different magnitudes of potency (Cheng et al., 2003). Its fruit also showed antioxidant

and radioprotective activity in rats (Naik et al., 2004). In vitro and in vivo protective

effects of an aqueous extract of fruit on the tert-butyl hydroperoxide (t-BHP)- induced

oxidative injury in rats has also been documented (Lee et al., 2005; 2007). It also showed

stronger antioxidant activity than alpha-tocopherol (Saleem et al., 2001).


Other pharmacological activity includes antibacterial activity (Malckzadeh et al., 2000;

Kim et al., 2006), antifungal activity (Vonshak, et al., 2003), anti viral (Kurowa et al.,

1995; Jeong et al., 2002;), anticancer (Saleem et al., 2002). adaptogenic (Rege et al.,

1999), hypolipidemic (Shaila et al., 1998), cardioprotective activity (Reddy., 1990).

cytoprotective activity (Hamada etO al., 1997; Minkyun et al; 2004), radioprotective

activity (Gandhi & Nayar, 2005), antidiabetic (Rao & Srinavas, 2006), wound healing

activity (Sugana et al., 2002), Purgative property (Miglani et al., 1971 ).

immunomodulatory activity (Shivaprasad et al., 2006) were reported.


Abhayarista; Agastya haritaki rasayana; Triphala curna; Triphaladi taila; Danti

haritaki; Dashmula haritaki (Anonymous; 1990b)

AZADIRACHTA INDICA LINN

Synonym: Melia azadirar.hta Linn.

FamiJy : Meliaceae.

Habitat : Native to Burma; found all over India.

English : Neem tree, Margosa tree.

Page 25

-~~~~~~~~~~~~-----



Neem is a fast-growing tree that can reach a height of 15-20 m, rarely to 35-40 m.,

Leaves alternate, imparpinnate; leaflets subopposite serrate, very unequal at base;

Flowers hermaphrodite, in axillary panicles; calyx 5-lobed; Petals 5, much exceeding the

calyx, free, imbricate; Staminal tube a little shorter than the petals, cylindric, widening

above, 9-10 lobed at the apex, the lobes truncate again slightly toothed; anthers within the

tubes opposite to and shorter than the lobes. Ovary 3-celled; style elongate, slender;

stigma shortly cylindric, 3 lobed; Ovules 2 in each cell, collateral; Fruit a 1-seeded drupe,

endocarp woody; Seed ellipsoid; albumen 0; cotyledons thick, flesh

Traditional uses

Plant is bitter, astringent and gives bad taste in the mouth. It acts as a refrigerant. Bark

extract relieves from "kapha" and "pitta dosha including cough and ulcers. Bark extract

acts as anthelmintic, antiemetic, antacid, antileprotic, antipyretic, analgesic and anti

inflammatory agent. Bark extract is also useful in blood purification, amenorrhoea,

fatigue, thirst, and urinary tract infections. The young branches of neem plant are useful

in asthma, piles, tumors and urinary discharges. The root bark is more active and speedy

in its action than the bark (Kirtikar and Basu, 1999b ).

Chemical compositions

Condensed tannins from the bark contain gallic acid, (+) gallocatechin, (-)

epicatechin,(+), catechin and epigallocatechin, of which gallic acid (Chattetjee &

Pakrashi, 1994), (-) epicatechin and catechin are primarily responsible for inhibiting the

generation of chemiluminescence, by activated human polymorphonuclear neutrophil

(PMN) (Vander et al., 1991) indicating that these compounds inhibit oxidative burst of

PMN during inflammation. Three tricyclicditerpenoids, margolone margolonone

(Pennington & Styles., 1975) and isomargolonone isolated from neem stem bark are

active against Klebsiella, Staphylococcus and Serratia species (Ara., 1989). A

polysaccharide extracted from bark inhibits carrageenin-induced inflammation in mouse

. Two polymers isolated from an aqueous extract of neem bark possess anticomplement

Page 26


activity, amongst which the compound NB-11, a peptidoglycan of lower molecular

weight was found to be more potent (Vander et al., 1989).

I OH

Epicatechin

Margolone

Margolonone


Catechin

Iso margolonone

The aqueous extract of Neem leaf was found to offer protection against paracetamol

induced liver necrosis in rats (Bhanwra et al., 2000). Strong antioxidant potential of leaf,

flower and stem bark of the Neem tree have also been reported (Sithisam et al., 2005).


The chloroform extract of stem bark is effective against carrageenin-induced paw oedema

in rat and mouse ear inflammation {Tidjani, et al., 1989). Inflammatory stomatitis in

children is cured by the bark extract (Lorenz., 1976). Recently, an aqueous extract of

Page 27


stem bark has been shown to enhance the immune response of Balb-c mice to sheep red

blood cells in vivo (Njiro & Kafi-Tsekpo, 1999). The antisecretory and antiulcer effects

of aqueous extract of Neem (Azadirachta indica) bark have been reported

(Bandyopadhyay et al., 2003). Oil from the leaves, seeds and bark possesses a wide

spectrum of antibacterial action against Gram-negative and Gram-positive

microorganisms, including M. tuberculosis and streptomycin resistant strains (Chopra et

al., 1952). The crude ethanolic extract of stem bark and root bark showed hypotensive,

spasmolytic and diuretic activities (Abraham et al., 1986). Stem bark also showed anti

inflammatory and immunomodulatory activity (Sithisarn et al., 2005).


Nimbadi Kvatha Curna; Nimbadi Curna; Pathyadikwatha Curna; Sudersana Curna.

(Anonymous; 1999)

ZINGIBER OFFICINAL ROSCOE

Family: Zingiberaceae.

Habitat: Native to Southeast Asia; now cultivated mainly in Kerala, Andhra Pradesh,

Uttar Pradesh, West Bengal, Maharashtra.

Sanskrit : Adrakam, Ardraka, Hindi: Adrak, Sunthi, Sonth

Gujarati : Sunth

English: Ginger


A herbaceous rhizomatous perennial, reaching up to 90 em in height. Rhizomes are

aromatic, thick lobed, pale yellowish, bearing simple alternate distichous narrow oblong

lanceolate leaves. The herb develops several lateral shoots in clumps, which begin to dry

when the plant matures. Leaves are long and 2-3 em broad with sheathing bases, the

blade gradually tapering to a point. Inflorescence solitary, lateral radical pedunculate

oblong cylindrical spikes. Flowers are rare, rather small, calyx superior, gamosepalous,

three toothed, open splitting on one side, corolla of three subequal oblong to lanceolate

connate greenish segments (Schauenberg & paris 1977).

Page 28


Traditional uses

Ginger is carminative, pungent, stimulant, used widely for indigestion, stomachache,

malaria and fevers. It is chiefly used to cure diseases due to morbidity ofKapha and Vata.

Ginger with lime juice and rock salt increases appetite and stimulates the secretion of

gastric juices. It is said to be used for abdominal pain, anorexia, arthritis, atonic

dyspepsia, bleeding, cancer, chest congestion, chicken pox, cholera, chronic bronchitis,

cold extremities, colic, colitis, common cold, cough, cystic fibrosis, diarrhoea, difficulty

in breathing, dropsy, fever, flatulent, indigestion, disorders of gallbladder, hyperacidity,

hypercholesterolemia, hyperglycemia, indigestion, mommg sickness, nausea,

rheumatism, sore throat, throat ache, stomach ache and vomiting. Ginger forms an

important constituent of many Ayurvedic formulations (Nadkami, 1976e).


Ginger.. contains a number of pungent constituents and active ingredients. Steam

distillation of powdered ginger produces ginger oil, which contains a high proportion of

sesquiterpene hydrocarbons, predominantly zingiberene. The major pungent compounds

in ginger, from studies of the lipophilic rhizome extracts, have yielded potentially active

gingerols, which can be converted to shogaols, zingerone, and paradol. (Govindarajan,

1982) The compound 6-gingerol appears to be responsible for its characteristic taste.

Zingerone and shogaols are found in small amounts in fresh ginger and in larger amounts

in dried or extracted products. Shogaols have recently been found to be twice as pungent

as gingerols (Gopalam & Ratnambal, 1989).

0 ~H 0 ~0 ~0

•/

CCIJtn~ CCIJtn~ ..•

HO HO '-.~::/

Gingerols Shogaols [6]-Gingerol n=4 [6]-Shogaol n=4 [8]-Gingerol n=6 [8]-Shogaol n=6 [1 0]-Gingerol n=8 [ 1 0]-Shogaol n=8

Page 29



Hepatoprotective effect of aqueous ethanol extract of Z. officinale against

acetaminophen-induced acute toxicity was mediated either by preventing the decline of

hepatic antioxidant status or due to its direct radical scavenging capacity (Ajith et al.,

2007). Treatment with 1% dietary ginger for 4 weeks in rats improved antioxidant status

in ethanol treated rats, which suggest that treatment of ginger may have protective role

against ethanol induced hepatotoxicity (Mallikarjuna et al., 2008). Antihepatotoxic

activity of ethanolic extract of ginger was also reported (Bhandari et al., 2003).


Ginger has been reported to decrease nausea and vomiting associated with several

conditions including pregnancy (Blumenthal, 2003; Vutyavanich et al.,2001; Sharma et

al., 1997). The compounds 6-gingerol and 6-shogaol reported for a number of

pharmacological activities, including antipyretic, analgesic, antitussive, and hypotensive

effects (Suekawa et al., 1984). Ginger extracts showed inhibition of platelet aggregation

and thromboxane synthesis in vitro, (Srivastava., 1989; Kiuchi et al., 1992) which has led

to concerns that ginger extracts may prolong bleeding; however, several European studies

using ginger orally did not find any significant anticoagulant effects in vivo (Bordia.,

1997). In vitro studies suggested that ginger may produce anti-inflammatory effects by

inhibiting arachidonic acid metabolism in both the cyclooxygenase and lipoxygenase

pathways. (Srivastava, 1984; Srivastava & Mustafa 1992). Ginger extract has been

reported as an alternative to NSAID therapy for arthritic conditions (Bliddal., 2000)

Side Effects and Toxicity

Ginger is on the U.S. Food and Drug Administration's GRAS (Generally Recognized

As Sale) list. The British Herbal Compendium documents no adverse effects of ginger

(Bradley, 1990).

·Page 30


Warnings and Contraindications

Despite widespread use of ginger by pregnant women, the safety of this herb has not

been formally established. The Complete German Commission E Monographs

recommend against the use of ginger root for nausea and vomiting of pregnancy:

however, American editors, citjng thousands of years of use and no pertinent scientific

validity for this contraindication, refute this recommendation. The German Commission

E also mentions gallstones as a relative contraindication for ginger, without citing a

rational (Blumenthal, 1998).


Saubhagyaszmthi; Trikatu cuma; Saublzagyavati; Vaisvanara curna (Anonymous,

1990d)

TRICHOSANTHES DIOICA ROXB.

Family Name: Cucurbitaceae

Common Name: Pointed gourd

Sans: Patola; Hindi: Palwal; Mah: Kadupalval

Eng: Wild snake Gourd

Habitat: Common in Bengal and cultivated in Northen India, Punjab and Baroda

Part Used: Fruits and roots


Stem was slender, extensively climbing, more or less scabrous and woolly;. Leaves 7.5

by 5 em, ovate, oblong cordate, acute, sinuate, dentate, not lobed rigid, rough on both

surface; petiole 2 em. Flower: dioecious. Male flower not racemed, woolly outside,

calyx-tube 4.5 em, narrow; teeth linear erect, anthers are free. Fruits were 5-9 em oblong

or nearly spherical acute, smooth and orange red when ripe. Seed was Y2 ellipsoid,

compressed, corrugated on the margin. (Nadkami, 1976d)

Traditional uses

Triclzosanthes dioica Roxb. {TO) commonly known as Kadu-padval. According to

ayurveda the plant is used for bronchitis, biliousness, cancer, jaundice, liver enlargement,

Page 31


cough and blood diseases. It is also used as antipyretic diuretic, cardiotonic and laxative.

It has been used for overcoming problems like constipation, fever, skin infection,

wounds. The vegetable is provided with an intention to improve appetite and digestion.

The unripe fruit and the tender shoots and capsules are laxative (Kirtikar & Basu, 1999d)


Phytochemical evaluations of Aqueous and Ethanolic extracts showed the presence of

saponins, tannins and a non-nitrogenous bitter glycoside trichosanthin. Trichosanthes

dioica Roxb plant are very rich in protein, vitamin A and vitamin C. (Anonymous, 2003)


Trichosanthes dioica Roxb (TD) showed hepatoprotective activity in ferrous sulphate

(FeS04) intoxicated rats (Ghaisas et al, 2008).

Other pharmacological action

The fruits are easily digestible and diuretic in nature. They are also known to have

antiulcerous effects. The fruits and seeds have some prospects in the control of some

cancer- like conditions and haemagglutinating activities (Sharmila et al., 2007) 6).

Trichosanthes dioica w?..s found to possess anti-inflammatory activity (Fulzule., 2001) 3),

Blood sugar, serum cholesterol, high density lipoprotein, phospholipids and triglyceride

lowering activity (Sharma & Pant et al., 1988a; Chandrasekhar et al., 1988., Sharmila et

al., 2007; Sharma & Pant, 1988b).


Vajraka ghrta, Patoladi kvatha curna (Handa, 2008a)

Page 32


2.3 ANTIOXIDANT ACTIVITY

Majority of the diseases/disorders are mainly linked to oxidative stress due to free

radicals. Free radicals are fundamental to any biochemical process and represent an

essential part of aerobic life and metabolism. The most common reactive oxygen species

(ROS) include superoxide (02.) anion, hydrogen peroxide (H202), peroxyl (Roo·)

radicals and reactive hydroxyl (OH) radicals. The nitrogen derived free radicals are nitric

oxide (NO.) and peroxynitrite anion (ONOO "). ROS have been implicated in numerous

disease states which range . from arthritis and connective tissue disorders to

carcinogenesis, agmg, physical injury, infection and acquired immunodeficiency

syndrome. In treatment of these diseases, antioxidant therapy has gained immense

importance. Current research is directed towards finding naturally occurring antioxidants

of plant origin. Antioxidants have been reported to prevent oxidative damage by free

radical and ROS, and may prevent the occurrence of diseases. It can interfere with the

oxidation process by reacting with free radicals, chelating catalytic metals, and also by

acting as oxygen scavengers. The medicinal properties of plants have been investigated in

the recent scientific developments throughout the world, due to their potent antioxidant

activities, no side effects and economic viability. Flavonoids and phenolic compounds

widely distributed in plants have been reported to exert multiple biological effect,

including antioxidant, free radical scavengmg abilities, anti-inflammatory,

anticarcinogenic etc. They are also speculated to be potential iron chelators. Novel

natural antioxidants from some plants have been extensively studied in the past few years

for their antioxidant and radical scavenging properties (Velavan et al., 2007). Liver

diseases remain a serious health problem. It is well known that free radicals cause cell

damage through mechanisms of covalent binding and lipid peroxidation with subsequent

tissue injury. Antioxidant agents of natural origin have attracted special interest because

they can protect human body from free radicals (Osawa et al., 1990). Hence the present

study incorporates investigating in vitro and in vivo antioxidant study of Punamavashtak

kwath.

Page 33

2.4 IN VIVO HEPATOPROTECTIVE ACTIVITY

2.4.1 Hepatotoxins for experimental models


Advancement in the search for finding an effective hepatoprotective owe much to the

identification of the pathogenesis of the liver disorder and elaboration of suitable models

for hepatic injury comparable to those encountered in the clinical practice. Chemical and

drugs employed for inducing the experimental liver lesion in animals have been

reviewed. Given below are some of the important hepatotoxins employed in experimental

models for inducing different types of liver damage.

1) Carbon tetrachloride

Carbon tetrachloride (CC14) is the most investigated of all hepatotoxins. The cytotoxic

specificity of CC14, once attributed to its ability to solublize phospholipids and to the

primary effect on mitochondria, is now considered to be due to enzymatic activation of

CCl4 to CCh within the membrane of endoplasmic reticulum. This free radical attacks

and disrupts the structure and functions of lipid and protein macromolecule in the

organelles inducing microsomal lipid peroxidation. The hepatocytes thus show fatty

changes or are irreversibly injured. For inducing hepatotoxicity in rats carbon

tetrachloride is given by intraperitoneal route and animals are sacrificed two days after

the hepatotoxin.

2) Paracetamol

Hepatotoxicity of paracetamol (PCM), which is a commonly used antipyretic- analgesic,

sold over the counter has been reviewed. PCM is safe at therapeutic dose level but in

large doses it induces liver injury, in both human and experimental animal. PCM induced

liver damage is characterized by fulminating hepatic necrosis primarily centrilobular

which extend through the midzone to periportal area. Hepatotoxocity of PCM is

attributed to the formation of toxic metabolite, N-acetyl-p-benzoquinoneimine which is

formed via cytochrome P 450· This electrophilic toxic metabolite depletes the cellular

reservoir of glutathione as a result of which the reactive metabolite binds covalently to

the thiol group of cysteine residues in proteins leading to hepatocellular toxicity. PCM 3

Page 34


g/kg oral dose induce hepatic damage in rats where the animal are sacrificed two days

after the challenge.

3) Ethanol

Liver toxicity due to ethanol (EtOH), a widely abused compound has been extensively

reviewed. Development of ethanol induced hepatic damage in human and experimental

animals is dependent on the duration of use as well as dose of ethanol. Chronic

alcoholism causes fatty liver which progresses to hepatitis and cirrhosis. This effect is

produced indirectly due to nutritional imbalance and also due to direct toxic effect of

ethanol. The deleterious effect of ethanol is attributed to the accumulation, in the liver, of

acetaldehyde, one of the catabolic intermediate of EtOH, which is toxic. EtOH induced

fatty liver is mainly produced by peripheral metabolization of free fatty acids. Ethanol

increases triglyceride formation by reducing mitochondrial oxidation of fatty acids in the

liver. Progression of alcoholic hepatitis to cirrhosis is reported to be due to lipocyte

stimulated fibrogenesis and lactic acidosis induced collagenosis.

4) Other hepatotoxicants

Apart from the above mentioned hepatotoxins many other compounds like d

galactosamine, thiocetamide, a napthylisothiocynate, dimethyle nitrosamine, amatoxins,

lanthanum, antitubercular drugs, aflatoxins B. ethionine, yellow phosphorus,

pyrrolizidine alkaloids are used for producing liver lesions in animals (Handa et al.,

2008b)

2.4.2 Biochemical parameters for evaluating hepatic functions

(Sherlock, 1981; Sachdev 1999).

The governing principle in all the experimental models for evaluating antihepatotoxic

activity of a substance, a known hepatotoxin which produces marked and measurable

effect is administered to animals. The test substance is then administered along with the

toxic dose of the hepatotoxin and the toxic effect is blocked, then the substance under

investigation is considered to be effective. The magnitude of toxic effect can be measured

by estimating suitable liver function parameters and by direct histological examination of

Page 35


liver slices. Number of liver function tests has been described to determine the pathology

of liver disorders.

Serum aspartate transaminase (AST) [Serum glutamic oxaloacetic

transaminase (SGOT)]

It is a mitochondrial enzyme abundantly found in liver followed by lesser amount in heart

and skeletal muscle. The activity of this enzyme in red blood cell is about I 0 times the

normal serum levels. Whenever there is tissue damage, AST level increases due to its

release from the damaged cells. Very high value is indicative of hepatocellular damage,

myocardial infarction or haemolytic anemia.

Serum alanine transaminase(ALT) [Serum glutamic pyuruvic transaminase

(SGPT)]

This enzyme is present in the cytosol. Maximum concentration is present in the liver.

Although the absolute amount of SGPT in the liver is less than SOOT, a greater

proportion is present in the liver as compared to heart and skeletal muscle. Increase in the

level of AL Tis, therefore, more specific for liver damage.

Serum alkaline phosphatase (ALP)

It is a microsomal enzyme ubiquitous in distribution, largest amount being in liver and

gastrointestinal mucosa. The ALP is tightly bound to lipid membranes particularly those

in the canalicular area. Any disorder interfering with bile flow increases the synthesis of

ALP. The level of SALP rises considerably in cholestatis and to a lesser extent in

hepatocytic damage.

Serum bilirubin (SBRN)

Bilirubin, the end product of haem, is excreted in bile. In hepatocytic lesion and in

obstructive jaundice, excretion of bilirubin in the bile is hampered and hence, its level in

the blood increases. A rise in SBRN is, thus, indicative of jaundice or liver damage due to

toxin.

Page 36


Serum sorbitol dehydrogenase (SSDH)

It is mitochondrial and cytoplasmic enzymes primarily located in the liver. The

importance of assay of SDH is based on the fact that it is virtually absent in the serum of

healthy subjects and is detectable during infections, toxic and hypoxic liver damage.

Thus, SDH activity in the serum serves as an organ specific indicator of liver damage.

Hepatic triglyceraldehyde (HTG)

Triglyceraldehyde act as a store of energy and also as a mean for transport of energy from

the gut and the liver to peripheral tissue. Increased HTG level is indicative of disturbance

in lipid metabolism arising out of liver energy induced by some toxin, or obstructive

jaundice

Hepatic glycogen (HGN)

Glycogen a glucopoly saccharide is present exclusively in liver. Its level in the liver has

been reported to decrease in animal treated with CC14, PCM, or galactosamimne. A

decrease in HGN level is thus indicative of liver damage.

2.4.3 Histopathological study

To study histopathology several sections of the liver are taken and fixed in 10% buffered

formalin. After routine processing and paraffin embedding, four micron serial sections

are cut and stained by haematoxyline eosin. Histopathological studies usefully

complement results from the above biochemical studies described above and should be

best used in conjunction with these studies. Histopathological study is very useful for

studying drug effect on liver generation.

Page 37


2.5 IN VITRO HEPATOPROTECTIVE ACTIVITY

To estimate the risk of specific compounds to cause cancer in humans, many toxicity

studies were conducted on animals. However, because of species differences, there is a

need for a reliable human test system. Therefore, to reduce the use of animals for toxicity

assays, human cell culture models have been established. The preferred human in vitro

models are primary hepatocytes and hepatoma cell line (HepG2).

1) Studies on isolated hepatocyte

Freshly isol~ted hepatocytes from various animals are increasingly used worldwide to

understand the pharmaco-toxicology of hepatotoxicants. The cell offer advantages for

investigating the initial interactions of toxicants with hepatocyte because cell express

differentiated functions in terms of expression of liver specific cytochrome P450 (CYP)

forms. Several CYPs are involved in the biotransformation of large number of drugs and

chemical entering the body. While some of them are bioactivated by the specific CYP

isoform to reactivate intermediate which consequently bind with critical molecule of the

cells resulting in hepatotoxicity. The sensitivity of hepatocytes to different

hepatotoxicants appears to depend on the qualitative and quantitative prevalence of liver

specific CYPs and conjugates together with the pool of cofactor involved in

biotransformation. Differential biochemical response of freshly isolated rat hepatocyte to

paracetamol, CCl4, d-galactosamine toxicity, has proved useful for the rapid in vitro

screening of medicinal plant extracts and natural products for evaluating their

hepatoprotective potential (Handa et al., 2008b ).

2) Studies on HepG2 cell line

HepG2 cells, a human hepatoma cell line, are considered a good model to study in

vitroxenobiotic metabolism and toxicity to the liver, since they retain many of the

specialized functions which characterize normal human hepatocytes (Knasmuller et al.,

1998). In particular, HepG2 cells retain the activity of many phase I, phase II and

antioxidant enzymes ensuring that they constitute a good tool to study cytoprotective,

genotoxic and antigenotoxic effects of compounds (Knasmuller et al., 2004; Mersch

Sundermann et al., 2004).

Page 38


2.6 CLINJCAL STUDY

Only a well designed clinical study on a defined population can give meaningful results

(positive or negative) about any therapeutic intervention and its safety and efficacy.

Clinical study of many individual plant of this formulation showed hepatoprotective

activityO. but there are no reports regarding the clinical study of this formulation. So the

clinical study of this formulation was undertaken to see the efficacy of this formulation.

The Basic Principles of Good Clinical Practices involves the Current Standard

Operating Procedures, Informed Consent process, Institutional Review Board approval,

Compliance study protocol and related procedures, Controls of investigational supplies,

Sponsor /Monitor /Investigator's responsibilities, adequate safety surveillance and quality

assurance.

Composition of Institutional ethics committee

(www.icmr.nic.in/ethical_guide/ines.pdf)

IECs should be multidisciplinary and multisectorial in composition. Independence and

competence are the two hallmarks of an IEC. The number of persons in an ethical

committee be kept fairly small (5-7 members). It is generally accepted that a minimum of

five persons is required to compose a quorum. There is no specific recommendation for a

widely acceptable maximum number of persons but it should be kept in mind that too

large a Committee will make it difficult in reaching consensus opinion. 12 to 15 is the

maximum recommended number. The Chairperson of the Committee should preferably

be from outside the Institution and not head of the same Institution to maintain the

independence of the Committee. The Member Secretary who generally belongs to the

same Institution should conduct the business of the Committee. Other members should be

a mix of medical I non-medical, scientific and non-scientific persons including lay public

to reflect the differed viewpoints. The composition may be as follows:-

1. Chairperson

2. 1-2 basic medical scientists.

3. 1-2 clinicians from various Institutes

4. One legal expert or retired judge

5. One social scientist I representative of non-governmental voluntary agency

Page 39


6. On.e philosopher I ethicist I theologian

7. One lay person from the community

8. Member Secretary

The ethical committee at any institution can have as its members, individuals from other

institutions or communities if required. There should be adequate representation of age,

gender, community, etc. in the Committee to safeguard the interests and welfare of all

sections of the community/society. Members should be aware of local, social and cultural

norms, as this is the most important social control mechanism. If required subject experts

could be invited to offer their views, for example for drug trials a pharmacologist,

preferably a clinical pharmacologist, should be included. Similarly, based on the

requirement of research area, for example HIV, genetic disorders etc., specific patient

groups may also be represented in the Committee. Clinical study flow chart as follows

As per ICMR guide line.

Page 40

Planni11g

! Stt:dy design and Case rcponiom1 {CRF) Preparation

! Regulatory and Institutional ethical commi".tce approval

DocUJncnt Prcparatiot and collection o-: materials

! Select i nvestig;ttor:;

S. A~ ! d • • "al .. 1te .ni>Scssmcnt an mm VlSlts

! Pat:ents Recmitment and infom1ed consent form proces5

! '1 eaSUTmtlTI l 0 f ph )"Sl L:a[ paramelt'~

Biochc:nical parameters

! Drug treatment

! Periodic monito:-ing

/ ! "' Data entry DaLa ;;hock Stmistical analysis

. ~ f mal rcp011

~ Stuo\· Lerminalion


Flow chart for clinic~tl study

Page 41

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