I.

INTRODUCTION
1.1 HISTORY
Since disease, decay and death have always co-existed with life the study of disease
and their treatment also developed contemporaneously with the dawn of human
intellect. The primitive man used those therapeutically agents and remedial measures
which he was able procure with most ease. Rig-Veda, which is one of the oldest
books in library of man supplies curious information on the subject. The knowledge
of medical plants accumulated in the course of many centuries. Since time
immemorial, the extracts of plants have been recognized to possess important
medicinal activity. The people of India were acquainted with a far larger number of
medicinal plants than the native of any other country. Ayurveda, the most ancient
medicinal system contributed by our country includes Materia Medica drugs from
plant and animal sources. The records of the Susruta Samhita list some 760 medicinal
plants. In china, the recorded observation of the emperor Shennung of 2700 B.C.lists
about 365 drugs in the Pen Tsao herbs.

Enormous wealth of drugs is available from natural sources. Many of the currently
used drugs like Morphine, Digoxin, Atropine, Reserpine, Ephedrine, Emetine etc., are
of Plant origin. The drugs obtained from National Sources are helping in the cure of
wide range of disorders involving digestive, respiratory, renal and cardiovascular
system.

The study of medicinal plants has been neglected. The ease and cheapness with which
these are procurable, the marvelous powers that are attributed to them in the cure of
different maladies, induces us to investigate their properties and settle their claims.
The indigenous drugs have not been carefully and systematically studied. It is highly
desirable to go through the subject with great care, with the view of weeding out
worthless drugs and preparing the better class of native drugs. The importance of
herbal medicine is increasing, even in western countries.

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1.2 Renewed interest in traditional medicines:
During the last decade, there has been a growing interest in traditional and alternative
system of medicine in many developed countries. One third of American adults have
used alternative treatment, 60% of the public in the Netherlands and Belgium, and
74% in the United Kingdom are in favor of complimentary medicine being available
within the framework of National Health Service. The reasons for the inclusion of
traditional healers in primary health care are numerous. The healers know the socio-
cultural background of the people, they are highly respected and experienced in their
work, the strength of traditional believes as well as the shortage of health
professionals particularly in rural areas.

Traditional systems may not pass all the criteria of scientific and rational
behaviour.They will probably not pass in terms of standardization of treatment,
replicability of regimen or systematization of experimental procedures. Rational
skepticism, which is the basis of scientific thinking, is indeed necessary in evaluating
traditional, indigenous and natural therapies. Scientific and systematic evaluation of
various herbs is necessary to substantiate their use.

Hepatic disorders are one to the major disorders caused by various factors like
microbial organisms, chemicals, drugs etc., even today effective treatment is not
available with allopathic drugs. A number of hepatoprotective drugs like
tephrosiapurpurea pers, silymarin, green tea, artichoke etc., are used in practice.
Many indigenous drugs have been claimed to have hepatoprotective activity in
ayurvedic system of medicine, but they are not probably investigated. So the present
study is aimed at comparatively evaluating sum herbal formulations against a
standard drug and to substantiate their use as safe and effective hepatoprotectives. (1-3)

1.3 Oxidative Stress:

It has been implicated in the pathology of many diseases such as inflammatory
conditions, cancer, diabetes and aging. Free radicals induced by peroxidation have
gained much importance because of their use in several pathological conditions such

2
as atherosclerosis, ischemia, liver disorder, neural disorder, metal toxicity and
pesticide toxicity. Together with other derivatives of oxygen, they are inevitable
byproducts of biological redox reactions. Reactive oxygen species (ROS) such as
superoxide anions (O–2), hydroxyl radical (OH–) and nitric oxide (NO) inactivate
enzymes and damage important cellular components causing injury through covalent
binding and lipid peroxidation. Antioxidants may offer resistance against the
oxidative stress by scavenging the free radicals, inhibiting the lipid peroxidation and
by other mechanisms and thus prevent diseases. Foods rich in antioxidants have been
shown to play an essential role in the prevention of cardiovascular diseases, cancer,
neurodegenerative diseases, inflammation and problems caused by cell and cutaneous
aging.

Free radicals are chemical entities that can exist separately with one or more unpaired
electrons. The generation of free radicals can bring about thousands of reactions and
thus cause extensive tissue damage. Lipids, proteins and DNA are all susceptible to
attack free radicals. Antioxidants may offer resistance oxidative stress by scavenging
the free radicals.

Free radical or reactive oxygen species (ROS) are produced during biochemical redox
reactions as part of normal physiological cell metabolism (protection from infectious
organism) and as a response to environmental factors such as UV light, cigarette
smoke, environmental pollutants and gamma radiations. Once formed, ROS attack
cellular components causing damage to lipids, proteins and DNA, which can initiate
numerous diseases, including cancer, atherosclerosis, rheumatoid arthritis, diabetes,
liver damage and central nervous system disorders.

Living organisms have a large number of antioxidants, including macro and micro
molecules, and enzymes, which represent the total antioxidant activity of the system
and play a central role in preventing oxidative stress. Therefore quantitative
measurement of the cumulative antioxidant activity of body fluids, tissues and cells,
following different stimuli may provide important biological information. (4-8)

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1.4 Physiology of Liver

The liver is the largest glandular organ of the body. It weighs about 3lb (1.36kg).It is
reddish brown in color and is divided into four lobes of unequal size and shape. The
liver lies on the right side of the abdominal cavity beneath the diaphragm. Blood is
carried to the liver via two large vessels called the hepatic artery and the portal vein.
The portal vein carries oxygen-rich blood from the small intestine. These blood
vessels subdivide in the liver repeatedly, terminating in very small capillaries. Each
capillary leads to a lobule. Liver tissue is composed of thousands of lobules, and each
lobule is made up of hepatic cells, the basic metabolic cells of the liver.

The liver has many functions. Some of the functions are: to produce substances that
break down fats, convert glucose to glycogen, produce urea (the main substance of
urine), make certain amino acids (the building blocks of proteins), filter harmful
substances from the blood (such as alcohol), storage of vitamins and minerals
(vitamins A, D, K and B12) and maintain a proper level or glucose in the blood. The
liver is also responsible for producing cholesterol. It produces about 80% of the
cholesterol in the body.

Other functions of the liver include: Homeostasis of Glucose (sugar), Proteins, Fat,
Cholesterol, Hormones, Vitamins in particular the fat soluble ones (A, D, E and K).
Synthesis of proteins including the clotting factors, Bile acids (products of
cholesterol, important in fat digestion) and cholesterol. Excretion cholesterol, bile
acids, phospholipids, bilirubin, Drugs, Poisons (e.g. pesticides, insecticides, heavy
metals).

Filters poisons from the gut and nutrients such as amino acids, sugar and
fat.Bilirubin,bile acids, IgA, Drugs. Helps in Excretion of IgA(defence against
bacteria in the gut) Special macrophages(Kupffer cells) gobble up bacteria which
have crossed from the gut into the blood.

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1.5 Disease of the Liver
Several diseases states can affect the liver. Some of the diseases are Wilson’s disease,
Hepatitis (an inflammation of the liver), liver cancer, and cirrhosis (a chronic
inflammation that progresses ultimately to organ failure).Alcohol alters the
metabolism of the liver, which can have overall detrimental effects if alcohol is taken
over long periods of time. Hemochromatosis can cause liver problems.

Medications have side effects that may harm liver. Some of the medications that can
damage liver are: serzone, anti-cancer drugs (tagfur, MTX and cryptogam), and
medications used to treat diabetes. (9-17)

1.6 Silymarin
Name: Silymarin
Synonym: - Marian Thisle, Milk Thisle, the Wild Artichoke
Family: - Compositae
Parts used: - Ripe seeds, leaves, milk Thistle, seeds, Flowers, herbs, etc..,
Chemical Constituents: - Silymarin is present in ripe seeds from 4 – 6 %
It is composed of 3 isometric flavonoliganans called silybin, silycrystin and
silydianinn.
Milk Thisle seeds also contains flavonoliganans namely dehydrosilybin,
descosilycristin, desocrcy silydianin, silyhermin, neosilyhermin, silybinome and
silandrin.
The other constituents presents are betaine, apigenin and silybonol.
Uses : - Silymarin is mainly used in liver diseases.
It is used as hepatoprotective and in chronic inflammatory hepatic disorder including
hepatitis, cirrhosis and fatty infiltration.
It is effective against liver poisoning due to alpha – amanitin and palloidin.
Silymarin counteracts the tonic effects of a wide variety of poisons including alcohol,
CCl4, Acetaminophen overdose and death cap mushroom
It is used as bitter tonic, antidepressant.
In treatment of jaundice, bronchitis, etc..,

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Mechanism of Action: -
It involves altering the membranes of hepatic cells to decrease of flavnoids increasing
cellular regeneration by increasing protein synthesis, antioxidant activity including
use of inflammatory enzymes.

1.7 Unani drugs used as hepatoprotectives:

1.7.1) Sumbultib
Name: - Sumbultib (Unani)
Synonym: - Jatamansi, Muskroot, Balacharea
Family: - Valerianaceae
Parts used: - rhizome and oil from rhizome
Chemical constituents:-
• A volatile essential oil 0.5%
• The active principle is oleum jatamansi
• It also contains resin, sugar, starch, bitter extractive matter and gum
Mechanism of action:-Hot primary, dry secondary, tonic for heart, liver and brain
-It removes obstructions, diuretic and emmeragogue, jaundice and stone in kidney

1.7.2) Sharbat-e-kasni
Name:-Sharbat-e-kasni
Synonym:-Kacahni, kasini-virai, Tukhm-e-kasani
Family:-compositae
Parts used:-Seeds, root and flowers
Chemical constituents:-seeds contain bland oil
• Burnt chicory contains sugar, free extractive, cellulose, ash nitrogenous matter, fat,
etc.
• Roots contains nitrate and sulphate of potash, mucilage, some bitter extractive
principle and enulin-36%
• Flowers contain a colorless crystalline glucoside soluble in alkalies, hot water and
Alcohol, glucoside, cichorin, bitter substances lactucin, intybin present in root.

6
Uses:-
• A strong infusion of powder seeds is useful in obstructions or torpor of the liver and
in checking bilious enlargement of the spleen with general
• Roots are used as a substitute for coffee with other vegetable bitters it is given in
dyspepsia and fever.
• Flowers are made into sharbath given in liver disorders
Dose:- 3 to 4grains
The plant is applied externally on account of its cooling properties.

1.7.3) Bhangra
Name:-Bhangra
Synonym:-mako, bhringaraj, Radiem-el-bint, babri
Family:-Compositae
Parts used:-Herb roots and leaves.
Chemical constituents:-
A large amount of resin and an alkaloid principle eclipitine/resin does not yield the
reaction of podophyllin.
Uses:-
• Root is used as an application in the form of powder in hepatic and splenic
enlargement and in various chronic skin diseases
• Juices of the leaves is hepatic tonic
• Leaf juice of the yellow variety is used as a snuff in cephalagia
In combination with aromatics such as ajawan seeds. It is used in liver diseases. in
catarrhal jaundice fresh leaves(20gms). (18-22)

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 II. TAXONOMY AND ETHNOMEDICINE

PRUNELLA VULGARIS
Prunella vulgaris (Common Self heal, Heal-All or Heart-of-the-earth) is a medicinal
plant in the genus Prunella. Heal-All is both edible and medicinal. It can be used in
salads, soups, stews, or boiled as a pot herb. Used as an alternative medicine for
centuries on just about every continent in the world, and for just about every ailment
known to man, Heal-All is something of a panacea, it does seem to have some
medicinal uses that are constant. For medicinal purposes, the whole plant is gathered
when the flowers bloom, and dried. The leaves and small flowers of heal-all are
edible.

HABITAT & CULTIVATION

Heal-all is a perennial herb found throughout Europe, Asia, Japan and the U. S., as
well as most temperate climates. Its origin seems to be European, though it has been
documented in other countries since before any history of travel. Prunella vulgaris is
often found growing in waste ground, grassland, woodland edges, usually on basic
and neutral soils.(23) Heal-all is grown in any damp soil in full sun or in light shade.
Seeds are sewn in very early spring in a flat outdoor area.

Prunella vulgaris in Folklore

Heal all was once proclaimed to be a holy herb and was thought to be sent by God to
cure all ailments of man or beast. It was said to drive away the devil, which lead to
the belief that Heal-All was grown in the Witches garden as a disguise. The root was
also used to make a tea to drink in ceremonies before going hunting by one Native
American tribe to sharpened the powers of observation. (24)

FAMILY: Mint (Lamiaceae)

FLOWERING: May-September.

FIELD MARKS: Distinguishing features of this species are the crowded leaves
consisting of many flowers subtended by many overlapping bracts.

HABITAT AND CULTIVATION: Heal-all is a perennial herb found throughout

8
Europe, Asia, Japan and the U. S., as well as most temperate climates. Its origin
seems to be European, though it has been documented in other countries since before
any history of travel. Prunella vulgaris is often found growing in waste ground,
grassland, woodland edges, usually on basic and neutral soils.(25) Heal-all is grown in
any damp soil in full sun or in light shade. Seeds are sewn in very early spring in a
flat outdoor area.

STEMS: Spreading to erect, 4-sided, hairy, up to 2 feet tall.

LEAVES: Opposite, simple, lanceolate to elliptic to narrowly ovate, rounded or

pointed at the tip, rounded or tapering to the base, with or without teeth, usually
hairy, up to 3 1/2 inches long, up to 1 1/2 inches broad; leaf stalks present.

FLOWERS: Several crowded into cylindrical leaves; each flower 1/2 to 1 inch long,
subtended by a ciliate bract.
Sepals: 2-lipped, green or purple, hairy; the upper lip 3-toothed; the lower lip 2-lobed.
Petals: 2-lipped, purple or white, up to 1 inch long; the upper lip unlobed; the lower
lip 2-lobed.
Stamens: 4, curved under the upper lip of the corolla.
Pistils: Ovary superior, 4-parted; stigmas 2-cleft.
FRUITS: Nut lets 4, dark brown, ribbed, shiny, about 1/10 inch long.
Notes: This plant is also known as heal-all. (26-27)

While most of the traditional uses are of unknown (and clinically untested) efficacy,
Prunella vulgaris has been shown to be an antioxidant, immune stimulant, viral
replication inhibitor and an anti-inflammatory agent.(28-33)

CHEMICAL COMPONENTS:
Ursolic acid and caryophyllin (they are the saponin).

Contains prunellin, a kind of saponin, it's aglycon just Ursolic acid, other kinds of
content such as Ursolicacid, Oleanolic Acid, Rutin, hyperin, alpha-anisylacetone,
delphinidin, cyanidin. Seed contain coating oil and lipase. Herb contains naphtha,
composed by D-Camphor and fenchone, Vitamine B1, etc.

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Prunella vulgaris widely used as herb medicine to treat and prevent many disease,
also used widely recent years for it's good taste and rich nutrition, scientific data
proved that the fresh leaves and stem of Prunella vulgaris rich in protein, plant fat,
carbohydrate, Carotene, Vitamine B, and nicotinic acid (niacin, Vitamine B3).

FUNCTIONS OF PRUNELLA LEAVES

• Clear away liver-fire.
• Stop dizziness, headache, and fever due to wind-heat.
• Soothed the liver energy.
• Calms and reduces excitement, nervousness, and irritation.
• Eliminates phlegm and disperse stagnation.
• Reduces swelling of lymph glands.
• Relieves jaundice.

PHARMACEUTICAL ACTIONS:

1. Lower blood pressure: Action to the Cardiovascular System

Prunella vulgaris decoction proved to be used to reduce the high blood pressure for
animals.
Water decoction, water-alcohol decoction and 30% alcohol solution decoction of
Prunella vulgaris show function of lower cholesterol and decrease blood pressure for
the laboratory narcotic animal experiment.
For example, mainline decoction of Prunella vulgaris at dosage of 100mg/kg to the
dog, result showing good effect on lowering blood pressure, mainline to dog at
dosage 1-1.5G and make blood pressure decreased 40-60 mmHg, and recovered back
in 2-5 minutes. Ip 3-4 grams make blood pressure decreased 30-40 mmHg in 15-30
minutes, and lasting 1-2 hours. Pour 2-5 grams to dogs could show slight blood
pressure action in 30-60 minutes and continued 2 hours around.
Heart Perfusion experiment for toad heart and rabbit heart showing that low
concentration decoction of Prunella vulgaris cause exiting and make swing of heart
increase, and higher concentration the opposite effect.

2. Other Functions Good diuretic. Prunella vulgaris rich in Potassium salt so could be
used as good diuretic.
Decoction of Prunella vulgaris showing diuretic effect, 50% concentration of water

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decoction make rabbit uterus strongly shrinking, make rabbit gut wriggle strongly.

3. Anti-bacteria: Bacteria control Prunella vulgaris decoction diluted in tube as

1:1280 ratio proved to be strong inhibition and control on diarrhea bacilli, dilution as
1:640 show strong control on tuberculin Test proved that the decoction of Prunella
vulgaris shows inhibition on many bacteria, such as diarrhea, salmonella typhi, Vibrio
cholerae, E. Coli, Proteus valgaris, Staphylococcus aureus and Mycobacterium
tuberculosis.
Alcohol decoction of Prunella vulgaris shows inhibition on Pseudomonas aeruginosa,
water decoction of Prunella vulgaris show inhibition on fungi in test tube at rate 1:4.

4. Prunella vulgaris experiment on cardiac muscle: exciting action when low dosage,
and make swing increase, on the opposite side, high dosage makes swing decrease
and show inhibiting effects.

5. Prunella vulgaris decoction show exciting effects on the uterus and gut of rabbit.

6. Anti-inflammation: Prunella vulgaris show good function of anti-inflammation,

this character comes from and related with the synthesis and higher secretion of
adrenocortical hormones, especially of the glucocorticoid (GCS).
7. Applying Injection of Prunella Vulgaris could make thymus gland and spleen
atrophy, increase size of adrenal gland, also make the level of hydrocortisone
(cortisol increase high, all these proved that Prunella vulgaris is a kind of good
natural Immunosuppressant.

8. It is showing promise in research for cancer, AIDS, diabetes, and many other
maladies. (34-35)

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 III. REVIEW OF LITERATURE
1) It was reported that Phenolic acids (caffeic, chlorogenic, rosmarinic, and
ferulic) and extracts from Smallanthus sonchifolius and Prunella vulgaris
lowered glucose production via both gluconeogenesis (10 mM alanine or
dihydroxyacetone as precursors) and glycogenolysis (36).

2) It was reported that Plasma samples of pigs that were exposed to a 91-day oral
intake of rosmarinic acid (RA) via feed enriched by aerial parts of Prunella
vulgaris was directly analyzed using the sensitive isocratic HPLC/ECD
method as well as after enzymatic hydrolysis. In hydrolyzed plasma samples,
several other metabolites were determined, including dihydrocaffeic, ferulic,
and dihydroferulic acid. (37)

3) It was reported that SKI306X, which consists of biologically active ingredients

from Clematis mandsburica, Trichosanthes kirilowii, and Prunella vulgaris,
was developed and tested in preclinical trials in Korea. Those studies found
that SKI306X was associated with an anti-inflammatory and analgesic effect,
and that it can delay the destruction of cartilage in rheumatoid arthritis (RA).
(38)

4) It was reported that seven compounds were isolated from the leaves of
P.vulgaris. Their structures were established as autantiamide acetate (1), rhein
(2), tanshinone I (3).(The polysaccharide from Prunella vulgaris was isolated
by ethanol precipitation, dialysis, CTAB precipitation, and gel exclusion
chromatography. The isolated compound (PPS-2b) was a lignin-carbohydrate
complex with a molecular weight of 8500. The carbohydrate moiety was
composed of glucose, galactose), danshensu (4), stigmast-7, 22-dien-3-one (5),
3, 4, alpha-trihydroxy-methyl phenylpropionate (6), butyl rosmarinate (7). (39)

5) It was reported that the communities of AMF colonizing the roots of two plant
species, Prunella vulgaris and Antennaria dioica, in a Swedish seminatural

12
 grassland at different times of the year was characterized. P.vulgaris hosted a
rich AMF community throughout the entire growing season. The presence of
AMF in A. dioica decreased dramatically in autumn, while an increased
presence of Ascomycetes species was detected. (40)

6) It was reported that Prunella vulgaris (Labiatae; PVAE) 0.001-0.1 g/kg) dose
dependently inhibited compound 48/80-induced systemic anaphylaxis and
serum histamine release in mice. PVAE decreased the IgE-mediated local
allergic reaction, passive cutaneous anaphylaxis. (41)

7) It was identified that rosmarinic acid (alpha-o-caffeoyl-3,4-dihydroxyphenyl-

lactic acid; RosA) from Prunella vulgaris is an antagonist for the p56lck SH2
domain by screening natural products(42)

8) It was reported that in four doses of extracts from the leaves of P.vulgaris,
extract at dose of 100 mg/kg significantly suppressed the rise in blood glucose
after 30 min in the acute glucose tolerance test. (43)

9) It was reported that Wounds and injuries are treated with species of Usnea
longissima, Calendula officinalis, Arnica sp., Malva sp., Prunella vulgaris,
Echinacea purpurea, Berberis aquifolium/Mahonia aquifolium, Achillea
millefolium, Capsella bursa-pastoris, Hypericum perforatum, Lavandula
officinalis, Symphytum officinale and Curcuma longa. (44)

10) It was reported that Prunella vulgaris L. can suppress the proliferation of Raji
cells and may be a new anti-lymphoma drug. Inducing the apoptosis of Raji
cells maybe one of anti-lymphoma mechanisms. (45)

11) It was reported that Aqueous extracts from species of the Lamiaceae family
lemon balm (Melissa officinalis), peppermint (Mentha piperita), prunella
(Prunella vulgaris), rosemary (Rosmarinus officinalis), sage (Salvia
officinalis) and thyme (Thymus vulgaris) exert their antiviral effect on free
HSV and offer a chance to use them for topical therapeutic application against

13
 recurrent HERPES infections. (46)

12) It was reported that the optimal germination condition of P. vulgaris seeds is
12 hrs of marinating time at the temperature of 200C under illumination. (47)

13) It was reported that P.vulgaris and rosmarinic acid used in skin care cosmetics,
may offer protection against UVA-induced oxidative stress and may be
beneficial as a supplement in photoprotective dermatological preparations. (48)

14) It was reported that the therapeutic effects of Prunella stica inhibit the
proliferation of the epithelial cells derived from human endometrium. (49)

15) Water extracts of Prunella vulgaris and P. laciniata stimulated the

proliferation of T-lymphocytes and suppressed NO production in
lipopolysaccharide-stimulated macrophages dose dependently without any
(50)
cytotoxicity.

16) It was reported that P. vulgaris exhibited both immune stimulatory and anti-
inflammatory effects against microbial invasion. (51)

17) It was reported that the cardioprotective effect of Prunella vulgaris

ethylacetate fraction (PVEF) and its constituent rosmarinic acid (RA) was
evaluated on isolated rat cardiomyocytes subjected to doxorubicin-induced
oxidative stress. The cytoprotective activities of PVEF and RA were
concentration-dependent in the range of 0.005 to 0.05 mg/ml and the effect of
PVEF correlated with the RA content. Dexrazoxan (DE), used as positive
control, was less effective than PVEF or RA. (53)

18) It was reported that SKI 306X, a purified extract from the mixture of three
herbs, i.e. Clematis mandshurica, Trichosanthes kirilowii and Prunella
vulgaris protect osteoarthritis in part may result from the inhibition of
apoptosis in chondrocytes by Clematis mandshurica. (54)

14
19) It was reported that the RANTES gene was integrated in transgenic P.vulgaris
cells, and RANTES gene-stably expressed cell clones were available, which
could pave the way to obtain transgenic P.vulgaris plants demonstrating
specific pharmacological activities. (55)

20) It was reported that Prunella vulgaris fruiting spikes (EESP) consisting of a
mixture of triterpenoids, flavonoids, tannins and polysaccharide at 0.25, 0.5,
and 1.0 mg at intervals of 7 days for a total of five doses. The results suggest
that EESP could suppress the cellular and humoral response in mice. (56)

21) It was reported that the polysaccharides isolated from P.vulgaris have marked
immune stimulatory effects, which may bring about the anti-microbial effects
of P.vulgaris. (57)

22) The study show that Prunella vulgaris is effective against both the HSV-1 and
HSV-2 infections, and flow cytometry offers a quantitative and highly
reproducible anti-HSV drug-susceptibility assay. (58)

23) It was reported that Phenolic-rich extracts from the plants Silybum marianum
(silymarin) and Prunella vulgaris (PVE) improve antioxidant status in blood
and liver and positively affect plasma lipoprotein profile in an experimental
model of dietary induced hypertriglyceridemia. (59)

24) It was reported that the antioxidative, antimicrobial, together with antiviral
effects offer good prospects for the medicinal applications of P.vulgaris. (60)

25) It was reported that Rosmarinic acid from the Methanolic extract of Prunella
vulgaris, showed specific inhibitory activity against lymphocyte cell-specific
kinase (LCK) Src -homology 2 (SH2) by binding to a synthetic
phosphotyrosine-containing peptide (phosphopeptide). (61)

26) It was reported that Triterpenoid compounds were isolated from the plant of
Prunella genus, of which 20 were in Free State and 8 were triterpenoid

15
 saponins. The 13C NMR spectra of different triterpenoids showed different
features in respects such as skeletons, substituent groups and positions. (62)

27) It was reported that SKI 306X is a purified extract from a mixture of three
oriental herbal medicines (Clematis mandshurica, Trichosanthes kirilowii and
Prunella vulgaris) is a good OA agent with some cartilage protection activity.
(63)

28) The study demonstrated that SKI 306X, a new herbal anti-arthritic agent
provided clinical efficacy in patients with osteoarthritis. (64)

29) It was reported that aqueous extract of Prunella vulgaris (Labiatae) (PVAE)
PVAE inhibits immediate-type allergic reactions in rats. (65)

30) It was reported that the methanol extract of Paleonia suffruticosa still exerted
potent inhibition of HIV-1 integrase (EC50 = 15 = µg/ml) and the aqueous
extract of Prunella vulgaris caused moderate inhibition (EC50 = 45 µg/ml).
The results support the view that herbs represent a rich source of anti-HIV
compounds. (66)

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 IV. OBJECTIVE
Liver diseases are a serious health problem. In the absence of reliable liver protective
drugs in allopathic medical practices, herbs play a major role in the management of
various liver disorders. Numerous medicinal plants and their formulations are used for
liver disorders in ethno medical practices and in traditional system of medicines. In
India, However we do not have satisfactory remedy for serious liver disease, most of
the herbal drugs speed up the natural healing process of liver. So the search for
effective hepatoprotective drug continues.

Prunella vulgaris widely used as herb medicine to treat and prevent many diseases like
Clear away liver-fire.
Stop dizziness, headache, and fever due to wind-heat.
Soothed the liver energy.
Calms and reduces excitement, nervousness, and irritation.
Eliminates phlegm and disperse stagnation.
Reduces swelling of lymph glands.
Relieves jaundice.

The present study was made to evaluate the effect of ethanolic extract of leaves of
Prunella vulgaris against CCl4 induced hepatic damage in rats.

17
 V. PLAN OF WORK
The Leaves of the Prunella vulgaris was selected to screen for its Hepatoprotective
activity. Hence the plan of work was

• Extraction by using Soxhlet Apparatus.

• Ethanolic extract.

• Phytochemical tests for identifying chemical constituents.

• Screening of hepatoprotective activity.

• Method – CCl4 induced hepatotoxicity.

• Preparation of vehicle (2% Gum acacia).

• Dose administration orally.

• Dosage 50, 100 mg/kg body weight.

• Duration 7 days.

• Determination of biochemical parameters - SGOT, SGPT, ALKP, Direct and

Total BILIRUBIN.

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 VI. EXPERIMENTAL SECTION
The Leaves of Prunella vulgaris were collected from Munnalal Dawasaz (Mfg of
Ayurvedic medicine) Hyderabad. It was then completely air dried at room
temperature and powdered.

6.1 PREPARATION OF THE EXTRACT:

The coarse powder of shade dried leaves of Prunella vulgaris was extracted with
95% ethanol using Soxhlet Apparatus and concentrated in vacuum.

6.2 PRELIMINARY PHYTOCHEMICAL STUDIES:

6.2.1. TEST FOR CARBOHYDRATES

Molisch’s test: To 2-3ml aqueous extract, add few drops of alpha-naphthol solution
in alcohol and add conc. sulphuric acid from the sides of the test tube. Violet ring is
formed at the junction of the two liquids.

6.2.1.2. Tests for Reducing Sugars

a) Fehling’s test: Mix 1ml of Fehling’s A and B solutions. Boil for 1min.Add equal
volume of test solution. Heat in boiling water bath for 5-10 min. First a yellow and
then brick red ppt is observed.
b) Benedict’s test: Mix equal volumes of Benedict’s reagent and test solution in a test
tube. Heat in boiling water bath for 10 min. Solution appears green, yellow, or red
depending on the amount of reducing sugar present in the test solution.

1.3. Test for Monosaccharides

Barfoed’s test: Mix equal volumes of Barfoed’s reagents and test solution. Heat for
1-2 min in a boiling water bath and cool. Red ppt is observed.
1.4. Test for Pentose Sugars
Pentoses are components of certain gums.

a) Bial’s orcinol test: To boiling Bial’s reagent add few drops of test solution. Green
or purple color appears.

19
 b) Aniline acetate test: Boil test solution in test tube. Hold filter paper soaked in
aniline acetate in the vapour. Filter paper turns pink.
c) Mix equal amount of test solution and HCl heat. Add a crystal of phloroglucinol.
Red color appears.

1.5. Test for Hexose Sugars

a) Selinhoff’s test: Heat 3ml Selinhoff’s reagent and 1ml test solution in bearing
water bath for 1-2 min. Red color is formed.
b) Tollen’s phloroglucinol tyest for galactose: Mix 2.5ml conc. HCl and 4 ml. 0.5%
phloro- glucinol. Add 1-2ml test solution and heat. Yellow to red color
appears.
c) Cobalt-chloride test: Mix 3ml test solution with 2ml cobalt-chloride. Boil and
cool. Add few drops of NaOH solution. Solution appears greenish blue or
purple or upper layer greenish blue and lower layer purplish.

1.6. Test for Non-reducing Sugars

a) Test solution does not give response to Fehling’s and Benedict’s tests.
b) Hydrolyse test solution. Fehling’s and Benedict’s are positive.

1.7. Test for Non-reducing Polysaccharides

a) Iodine test: Mix 3ml test solution and few drops of dilute iodine solution. Blue
color appears; it disappears on boiling and reappears on cooling.
b) Tannic acid test for starch: With 20% tannic acid, test solution gives ppt.

6.2.2. TEST FOR GUMS

Hydrolyse test solution using dilute HCl. Perform Fehling’s and Benedict’s test. Red
color is developed.

6.2.3. TEST FOR MUCILAGES

a) Powdered drug material shows red color ruthenium red.
b) Powdered drug swells in water or aqueous KOH.

20
 6.2.4. TEST FOR ALKALOIDS
a) Dragendroff’s test: To 2-3ml of filtrate add few drops of Dragendroff’s
reagent. Orange brown ppt is formed.
b) Mayer’s test: 2-3 ml filtrate with few drops Mayer’s reagent gives ppt.
c) Hager’s test: 2-3 ml filtrate with Hager’s reagent gives yellow ppt.
d) Wagner’s test: 2-3 ml filtrate with few drops of Wagner’s reagent gives reddish
brown ppt.
e) Murexide test for purine alkaloids: To 3-4ml test solution add 3-4 drops or
conc. HNO3. Evaporate to dryness. Cool and add 2 drops of NH4OH. Purple
color is observed.

6.2.5. TEST FOR TANNINS AND PHENOLIC COMPOUNDS

To 2-3ml of aqueous or alcoholic extract, add few drops of following reagents:
a) 5% FeCl3 solution: deep blue-black color.
b) Lead acetate solution: White ppt.
c) Gelatin solution: white ppt.
d) Bromine water: decoloration of bromine water.
e) Acetic acid solution: red color solution.
f) Potassium dichromate: red ppt.
g) Dilute iodine solution: transient yellow color.
h) Dilute HNO3: reddish to yellow color.
i) Dilute NH4OH and potassium ferricyanide solution: red color solution.
One drop of NH4OH, excess 10% AgNO3 solution. Heat for 20 min. in boiling
water bath. White ppt observed then dark silver mirror deposits on wall of test
tube.
j) Dilute potassium permanganate solution: decoloration.
6.2.6. TEST FOR GLYCOSIDES
6.2.6.1Tests for Cardiac Glycosides
a) Baljet’s test: A thick section shows yellow to orange color with sodium picrate.
b) Legal’s test (Test for cardenoloids): To aqueous or alcoholic extract, add 1ml,
pyridine and 1ml sodium nitroprusside. Pink to red color appears.

21
 c) Test for deoxysugars (Kellar Killani test): To 2ml extract, add glacial acetic
acid, on drop 5% FeCl3 and conc.H2SO4. Reddish brown color appears at the
junction of the liquid layers and upper layer appears bluish green.

6.2.6.2 Test for Anthraquinone Glycosides

a) Borntrager’s test for anthraquinone glycosides: To 3ml extract add dil.
H2SO4, boil and filter.To filtrate, add equal volume of benzene or chloroform.
Shake well. Separate the organic solvent. Add ammonia. Ammonical layer
turns pink or red.
b) Modified Borntrager’s test for C- glycosides: To 5ml extract, add 5% FeCl3
and 5ml dil. HCl. Heat for 5 min in boiling water bath. Cool and add benzene
or any organic solvent. Shake well. Separate organic leyer, add equal volume
dilute ammonia. Ammonical layer shows pinkish red color.

6.2.6.3 Test for Saponin Glycosides

a) Foam test: Shake the drug extract or dry powder vigorously with water.
Persistent foam obsevered.
b) Haemolytic test: Add drug extract or dry powder to one drop of blood placed
on glass slide. Haemolytic zone appears.

6.2.6.4 Test for Cyanogenetic Glycosides

a) Guignard reaction or sodium picrate test: Soak a filter paper strip first in
10% picric acid, then in 10% sodium carbonate, dry. In a conical flask place
moistened powdered drug. Cork it; place the above filter paper strip in the slit in
cork. Filter paper turns brick red or maroon.
b) To dry powder or extract, add 3% aqueous mercurous nitrate solution. Metallic
mercury forms.
c) Dip a piece of filter paper in guaiacum resin and moist it with dilute copper sulphated
solution. Expose it to freshly cut surface of drug, blue stain is produced.

22
 6.2.6.5 Test for Coumarin Glycosides
a) Coumarin glycosides have aromatic odour.
b) Alcholic extract when made alkaline, shows blue fluorescence.
c) Take moistened dry powder in the test tube. Cover test tube with paper soaked
in dilute NaOH. Keep in water bath. After sometimes expose filter paper to U.V light.
It shows yellowish-green fluorescence.

6.2.7 TEST FOR FLAVONOIDS

a) Shinoda test: To dry powder or extract, add 5ml 95% ethanol, few drops
conc. HCl and 0.5g magnesium turnings. Pink color observed.
b) To small quantity of residue, add lead acetate solution. Yellow colored ppt is
formed.
c) Addition of increasing amount of sodium hydroxide to the residue shows
yellow coloration, which decolorizes after addition of acid.

6.2.8 TEST FOR PROTEINS

a)Biuret test (General test): To 3ml test solution add 4% NaOH and few drops of
1% CuSO4 solution. Violet or pink color appears.
b) Millon’s test (for proteins): Mix 3ml test solution with 5ml. Millon’s
reagent. White ppt. Warm ppt turns brick red or the ppt dissolves giving red
colored solution.
c) Xanthoprotein test (for protein containing tyrosine or tryptophan): Mix 3ml
test solution with 1ml conc. H2SO4. White ppt is formed. Boil, ppt turns
yellow. Add NH4OH, ppt turns orange.
d) Test for Protein containing sulphur: Mix 5ml test solution with 2ml 40%
NaOH and 2 drops 10% lead acetate solution. Boil solution turns black or
brownish due to PbS formation.
e)Precipitation test: The test solution gives white colloidal ppt with following
reagents:
i) Absolute alcohol
ii) 5% HgCl2 solution

23
 iii) 5 %CuSO4 solution
iv) Lead acetate
v) 5% ammonium sulphate

6.2.9 TEST FOR AMINO ACIDS

a) Ninhydrin test (General test): Heat 3ml test solution and 3 drops 5% Ninhydrin
solution in boiling water bath 10 min. Purple or bluish color appears.
b) Test for tyrosine: Heat 3ml test solution and drops Millon’s reagent. Solution shows
dark red color.
c) Test for tryptophan: To 3ml test solution and drops glyoxalic acid and conc.H2SO4.
Reddish violet ring appears at the junction of the two layers.
d) Test for cysteine: To 5ml test solution add few drops of 40% NaOH and 10% lead
acetate solution. Boil Black ppt of lead sulphate is formed.

6.2.10 TESTS FOR FATS AND OILS

a)Place a thick section of drug on the glass slide. Add a drop of Sudan red III reagent.
After 2 min, wash with 50% alcohol. Mount in glycerin. Observe under
microscope. Oil globules appear red.
b) To thin section, add a drop of 1% osmic acid. After one minute. Observe under
microscope. Oil drops appear black.
c) Solubility test: Oils are soluble in ether, benzene and chloroform, but insoluble in
90% ethanol and in water.
d) Filter paper gets permanently stained with oils.
e) Extract gives red color with 2-3 drops of tincture alkana.
f) Saponification test: Evaporate extract to get 10ml oil. To oil add 25ml 10%
NaOH. Boil in boiling water bath for 30 min cool. Add excess Na2SO4 solution.
Soforms and rise to the top. Filter. To filtrate add H2SO4. Evaporate. Collect
residue, it contains glycerol. Dissolve residue in ethanol. With ethanolic
solution.Perform following test:
i) To ethanolic solution, add few crystals of KHSO4. Heat vigorously. Pungent
odour of acrylic aldehyde is produced.

24
 ii) To ethanolic solution add few drops of CuSO4 and NaOH solutions. Clear blue
solution is absorbed.

6.2.11 TEST FOR STEROIDS

a) Salkowaski reaction: To 2ml of extract add 2ml of chloroform and 2ml of
conc.H2SO4. Shake well. Chloroform layer appears red and acid layer shows greenish
yellow fluorescence.
b) Libermann Burchard reaction: Mix 2ml of extract with chloroform add 1-2ml of
acetic anhydride and add 2 drops of conc.H2SO4 from the side of test tubes. First red,
then blue and finally green color appears.
c) Libermann’s reaction: Mix 3ml of extract with 3ml of acetic anhydride. Heat and
cool. Add few ml of conc.H2SO4 blue color appears.

6.2.12.TESTS FOR VOLATILE OILS

Hydrodistillate material separate volatile oil from distillate and perform following
tests.
a) Volatile oils have characteristic odour.
b) Filter paper is not permanently stained with volatile oil.
c) Solubility test: Volatile oils are soluble 90% alcohol

6.3 ANIMALS:
Albino rats (160-180 g) of either sex were used. They are kept in standard plastic
animal cages in groups of 6-8 animals, with 12 hr of light and dark cycle in the
institutional animal house. The animals were fed with standard rodent diet and
provided water ad libitum. After one week of acclimatization the animals were used
for further experiments. Approval from the institutional animal ethical committee for
the usage of animals in the experiments was obtained as per the Indian CPCSEA
guidelines.

25
 6.4 EXPERIMENTAL WORK
Carbon tetrachloride Induced Liver Fibrosis. (67-71)
This method was followed in the present study.
Assessment of hepaprotective activity is carried out in Albino rats. The animals were
segregated into five groups of six animals of each
Group A served as normal control and received subcutaneous administration of
liquid paraffin (L.P) only 3ml/kg on alternate days for one week.
All other groups B, C, D and E received Carbon tetrachloride (1ml/Kg)
subcutaneously in the lower abdomen in a suspension of L.P in the ratio 1:2 v/v on
alternate days for a week.
Group B animals were maintained as Carbon tetrachloride group.
Group C animals were treated with Silymarin 100mg/kg orally for 7 days.
Group D animals were treated with Ethanolic extract of Prunella vulgaris 50 mg/kg
orally for 7 days
Group E animals were treated with Ethanolic extract of Prunella vulgaris 100 mg/kg
orally for 7 days
After drug treatment all the animals were sacrificed, blood was collected by
puncturing the retro orbital plexus and was allowed to clot for 45 min at room
temperature, serum was collected by centrifugation at 2500 rpm for 15 min, used for
estimation of various bio-chemical parameters.

6.4.1 Assessment of Liver function:-

Bio-chemical parameters such as Serum Glutamate Oxalaocetate Transaminase
(SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase
(ALP) and Serum Bilirubin were determined.

26
 6.5. METHODS OF DETERMINATION OF BIOCHEMICAL PARAMETERS

6.5.1 Determination of Serum Glutamate oxaloacetate Transaminase (SGOT)

Method: Reitman and Frankel

Principle:
SGOT catalyses the following reaction:
α-ketoglutarate + L-Aspartate ↔ L-Glutamate + Oxaloacetate
Oxaloacetate so formed is coupled with 2, 4 – Dinitrophenyl hydrazine to give
corresponding hydrozone, which gives brown colour in alkaline medium and this can
be measured colorimetrically.

Reagents
Reagent 1: Buffered Aspartate α-Ketoglutarate Substrate, pH 7.4
Reagent 2: 2,4 – Dinitrophenyl hydrazine (DNPH) colour reagent
Reagent 3: Sodium Hydroxide, 4 N
Reagent 4: Working Pyruvate Standard, 2mM

Procedure:
Pipette into tube marked Test (T)
Reagent 1 0.25mL
Incubate at 37º C for 5 min
Serum 0.05mL
Mix well incubate at 37º C for 60min
Reagent 2 0.25mL
Mix well and allow to stand at room temperature (15 - 30º C)for 20 min
Dilute 1 ml of NaOH to 10ml with purified water 2.5mL

Mix well and allow standing at room temperature (15-30ºC) for 10 min and read the
optical density against purified water on colorimeter at 505nm.

27
Calculations: - Mark the optical density of test T on Y-axis of the standard curve and
extrapolate it to the corresponding enzyme activity on X-axis. (72-74)

6.5.2 Determination of Serum Glutamate pyruvate Transaminase (SGPT)

Method: Reitman and Frankel

Principle:
SGPT (ALT) catalyses the following reaction:
α-ketoglutarate + L-Alanine ↔ L-Glutamate + pyruvate
Pyruvate so formed is coupled with 2,4 –Dinitrophenyl hydrazine( 2,4 DNPH) to give
corresponding hydrazone, which gives brown colour in alkaline medium and this can
be measured colorimetrically.
Reagents
Reagent 1: Buffered Alanine α-Ketoglutarate Substrate, pH 7.4
Reagent 2: 2, 4 – Dinitrophenyl hydrazine (DNPH) colour reagent
Reagent 3: Sodium Hydroxide, 4N
Reagent 4: Working Pyruvate Standard, 2mM
Procedure:-
Pipette into tube marked Test (T)
Reagent 1 0.25mL
Incubate at 37º C for 5 min
Serum 0.05mL
Mix well incubate at 37º C for 30min
Reagent 2 0.25mL
Mix well and allow to stand at room temperature(15-30º C) for 20 min
Dilute 1 ml of NaOH to 10ml with purified water 2.5mL

Mix well and allow standing at room temperature (15-30 º C) for 10 min and reading
the optical density against purified water on colorimeter at 505nm.

Calculation: - Mark the optical density of Test on Y-axis of the standard curve and
extrapolate it to the corresponding enzyme activity on X-axis. (75-77)

6.5.3 Determination of direct & Total Bilirubin in serum

28
Method: - Mod.Jendrassik & Grof’s method
Bilirubin is mainly formed from the heme portion of Aged or damaged RBC’s. It then
combines with Albumin to form a complex which is not water soluble. This is
referred to as unconjugated Bilirubin. In the liver this Bilirubin complex is combined
with glucoronic acid into water soluble conjugate. This is referred to as conjugated
Bilirubin. Elevated levels of bilirubin are found in liver diseases (Hepatitis, cirrhosis),
excessive hemolysis / destruction of RBC (hemolytic jaundice) obstruction of the
biliary tract (obstructive jaundice) and in drug induced reactions. The differentiation
between the direct & indirect bilirubin is important in diagnosing the cause of
hyperbilirubinemia.

Principle: - Bilirubin reacts with diazotized sulphanilic acid to form a colored

azobilirubin compound. The unconjugated bilirubin couples with the sulphanilic acid
in the presence of a caffeine-benzoate accelerator. The intensity of the colour formed
is directly proportional to the amount of bilirubin present in the sample.
Bilirubin + Diazotized Sulphanilic acid→ Azobilirubin compound

Procedure
T1 T2 D1 D2
Diazo A(1) 1.0ml 1.0ml 1.0ml 1.0ml
Diazo B(2) 0.1ml - 0.1ml -
Activator(3) 1.0ml 1.0ml - -
Distilled water 2.5ml 2.6ml 3.5ml 3.6ml
Serum/plasma 0.2ml 0.2ml 0.2ml 0.2ml

Mix well and read the absorbance of D1 and D2 exactly after 1 min. on
photocolorimeter at 540 nm against distilled water. Mix well and keep the tubes T 1
and T2 dark at room temperature for 5 min. then read absorbance of T 1 and T2 and of
artificial standard (4) at 540 nm within 30 min.
Reagents
Diazo A 2 X 100ml
Diazo B 1 X 10ml
Activator 1 X 100ml

29
Artificial standard 2 X 10ml

Calculations:
a) Total bilirubin in mg % = Absorbance of T1-T2 x 10 (std. Conc)
Absorbance of Standard

(78-79)
b) Direct bilirubin in mg % = Absorbance of D1-D2 x 10 (Std. Conc)
Absorbance of Standard

6.5.4 Determination of Alkaline Phosphatase

Method: - Mod kind & king’s method

Alkaline phosphatase (ALP) is an enzyme of the hydrolase class of the enzymes and
acts in alkaline medium. It is found in high concentrations in the liver, biliary tract,
epithelium and in the bones. Normal levels are associated mainly with liver and bone
disease. Moderate increases are seen in Hodgkin’s disease and congestive heart
failure.
Principle:-Alkaline phosphate from serum converts Phenyl phosphatase to inorganic
Phosphatase with 4-Aminoantipyrine in presence of the oxidizing agent. Potassium
Ferricyanide and forms Orange-Red coloured complex, which can be measured
colorimetrically. The color intensity is proportional to the enzyme activity.
The Reaction can be represented by

Alk. Phosphatase
‘

Phenyl Phosphate  Phenol + Phosphate

pH 10.0

Pot-Ferricyanide
Phenol + 4-Aminoantipyrine ‘

 Orange Red Complex

OH

30
Reagents
Reagent 1: Buffered substrate, pH 10
Reagent 2: Chromogen Reagent
Reagent 3: Phenol Standard, 10mg%

Procedure:-
Pipette into four clean dry test tubes labeled as Blank (B), Standard (S), Control (C),
Test (T)
Addition Sequence B(mL) S(mL) C(mL) T(mL)
Working Buffered Substrate 0.05 0.5 0.5 0.5
Purified Water 1.5 1.5 1.5 1.5
Mix well and allow to stand at 37º C for 3 min
Serum - - - 0.05
Reagent 3 - 0.05 - -
Mix well and incubate at 37º C for 15 min
Reagent 2 1.0 1.0 1.0 1.0
Serum - - 0.05 -

Mix well after each addition. Measure the absorbance of the blank (Abs. B), standard
(Abs. S), control (Abs. C), and test (Abs.T) against distilled water.
Calculations:-

Serum Alkaline Phosphatase

(80-83)
Activity in K.A Units = O.D. Test – O.D. Control x 10
O.D Standard – O.D. Blank

31
 VII. OBSERVATIONS AND
CALCULATIONS
Table – 1 Results of Preliminary Phytochemical Tests
S.NO CHEMICAL TEST INFERENCE
1 Test for Alkaloids
a) Dragendroff’s test +
b) Mayer’s test +
c) Hager’s test +
d) Wagner’s test +
2 Test for Carbohydrates
a) Molisch’s test +
3 Test for Glycosides
Anthraquinone glycosides
a) _
Borntrager’s test
Cardiac glycosides
b)
i)Legal test _
ii)Baljet test _
iii) Killar killani test _
4 Test for Sugars
a) Fehling’s test +
b) Benedict’s test +
5 Test for Steroids
a) Libermann burchard test +
b) Salkowaski test +
6 Test for Fixed oils and Fats
a) Spot test _
b) Saponification test _
7 Test for Saponins
a) Foam test _
8 Test forTanins
a) Extract + lead acetate +
b) Extract + dil.Ferric chloride solution +
c) Extract + 1% gelatin + NaOH +
9 Test for phenols
a) Dil. 1ml of extract + ferric chloride solution +
10 Test for Proteins
a) Millon’s test _
b) Ninhydrin test
c) Biuret test _
d) Tannic acid test _

32
 e) Xanthoprotein test _
f) Picric acid test _
11 Test for gums and Mucilages _
12 Test for Volatile oils _
13 Test for Terpenoids
a) Noller’s test _
14 Test for Flavonoids
a) Extract + Ammonia seen in U.V +
b) Shinoda’s test +
d) Extract + NaOH +
e) Extract + conc.sulphuric acid +

Table – 2 Group A- Control

S.NO SGPT SGOT ALKP BILIRUBIN BILIRUBIN
(IU/L) (IU/L) (KA TOTAL (mg DIRECT (mg
Units) %) %)
1 35 48 31.8 0.96 0.20
2 37 50 29 0.99 0.18
3 39 46 30.2 0.99 0.17
4 35 51 32 0.97 0.21
5 36 50 29.84 0.99 0.22
6 34 49 31 0.99 0.16
Table – 3 Group B- Carbon tetrachloride treated

SGOT ALKP (KA BILIRUBIN BILIRUBIN

S.NO SGPT (IU/L)
(IU/L) Units) TOTAL (mg %) DIRECT (mg %)
1 120 120 73.5 2.07 0.25
2 115 140 73.3 1.07 0.31
3 125 142 76.6 3.07 0.26
4 135 118 70.2 2.10 0.30
5 105 135 78.4 2.04 0.27
6 120 125 75.6 2.07 0.29

Table – 4 Group C -- Carbon tetrachloride + Silymarin

ALKP (KA BILIRUBIN BILIRUBIN

S.NO SGPT(IU/L) SGOT(IU/L)
Units) TOTAL (mg %) DIRECT (mg %)
1 67 65 46.30 1.34 0.19
2 69 69 47.28 1.24 0.17
3 68 67 49.10 1.44 0.22
4 71 66 46.20 1.17 0.14
5 73 70 50.18 1.51 0.20

33
 6 72 71 51.08 1.34 0.16

Table – 5 Group D -- Carbon tetrachloride + Ethanolic extract of

Prunella vulgaris 50 mg/kg

S.NO SGPT(IU/L) SGOT(IU/L) ALKP BILIRUBIN BILIRUBIN

(KA Units) TOTAL (mg %) DIRECT (mg %)
1 105 87 63.4 1.79 0.21
2 113 86 60.2 1.59 0.25
3 108 85 66.6 1.63 0.22
4 103 89 62.3 1.64 0.24
5 110 90 64.5 1.75 0.21
6 111 91 63.0 1.76 0.25

34
Table – 6 Group E -- Carbon tetrachloride + Ethanolic extract of Prunella
vulgaris 100 mg/kg

S.NO SGPT(IU/L) SGOT(IU/L) ALKP BILIRUBIN BILIRUBIN

(KA Units) TOTAL (mg %) DIRECT (mg
%)
1 90 80 51.7 1.19 0.20
2 98 77 50.3 1.39 0.18
3 92 79 52.4 1.29 0.19
4 93 75 49.2 1.09 0.22
5 95 76 53.5 1.59 0.23
6 96 81 51.7 1.29 0.24

35
 Table – 7 Effect of Ethanolic extract of Prunella vulgaris on Carbon
tetrachloride induced hepatotoxicity in Rats.

Group Dose SGPT SGOT ALKP BILIRUBIN BILIRUBI

(IU/L) (IU/L) (KA TOTAL N DIRECT
Units) (mg %) (mg %)

A-Control Liquid paraffin 3 ml/kg

on alternate days 36 49 30.64 0.98 0.19

B-Carbon Carbon tetrachloride :

tetrachloride Liquid paraffin, 1: 2 s.c 2.07
120 130 74.6 0.28
1ml/kg on alternate
days
C-Carbon Carbon tetrachloride :
tetrachloride Liquid paraffin, 1: 2 s.c
+ Silymarin 1ml/kg on alternate
days + Silymarin 70 68 48.38 1.34 0.18
100mg/kg orally daily

D-Carbon Carbon tetrachloride :

tetrachloride Liquid paraffin, 1: 2 s.c
+ 50 mg/kg 1ml/kg on alternate
of Plant days + Ethanolic 108 88 63.4 1.69 0.23
Extract extract of Prunella
vulgaris 50 mg/kg
orally daily for 7 days
E-Carbon Carbon tetrachloride :
tetrachloride Liquid paraffin, 1: 2 s.c
+ 100 mg/kg 1ml/kg on alternate
of Plant days + Ethanolic 94 78 51.7 1.29 0.21
Extract extract of Prunella
vulgaris 100 mg/kg
orally daily for 7 days

36
 SGPT (IU/L)
120
120 108
94
100
70
80

60
36
40

20

0
GROUP A GROUP B GROUP C GROUP D GROUP E

Figure – 1

SGOT (IU/L)

130
140
120 88
100 78
68
80
49
60
40
20
0
GROUP A GROUP B GROUP C GROUP D GROUP E

Figure – 2

37
 ALKP (KA Units)

80
70 74.6

60
63.4
50
48.38 51.7
40
30 30.64

20
10
0
GROUP A GROUP B GROUP C GROUP D GROUP E

Figure – 3

BILURUBIN TOTAL (mg % )

2.5

1.5 2.07
1.69
1 1.34 1.29
0.98
0.5

0
GROUP A GROUP B GROUP C GROUP D GROUP E

Figure – 4

38
 BILURUBIN DIRECT mg%
0.28
0.3
0.23 0.21
0.25
0.19 0.18
0.2

0.15

0.1

0.05

0
GROUP A GROUP B GROUP C GROUP D GROUP E

Figure – 5

Group A – Control
Group B – Carbon tetrachloride
Group C – Carbon tetrachloride + Silymarin
Group D – Carbon tetrachloride + Ethanolic extract of Prunella vulgaris 50 mg/kg
Group E – Carbon tetrachloride + Ethanolic extract of Prunella vulgaris
100 mg/kg

39
 VIII. RESULTS AND DISCUSSION
Preliminary phytochemical test indicate that the following chemical constituents
alkaloids, carbohydrates, sugars, steroids, tannins, phenols, flavanoids are found to be
present in the Ethanolic extract of leaves of Prunella vulgaris indicated in Table 1.
Results indicated that the extract provides significant protection against the toxic
effect of CCl4 on liver. In CCl4 induced toxic hepatitis, toxicity begins with the
changes in endoplasmic reticulum, which results in the loss of metabolic enzymes
located in the intra cellular structures. The toxic metabolite CCl3 radical produced by
micrsomal oxidase system, binds covalently to macromolecule and causes
peroxidative degradation of lipid membranes of the adipose tissues. The blood
samples of the Group B animals showed drastic increase of Serum Glutamate
Oxaloacetate Transaminase (120 IU/L), Serum Glutamate pyruvate Transaminase
(130 IU/L) , Alkaline phosphatase (74.6 KA Units) , Direct Bilirubin (2.07 mg %) ,
Total Bilirubin (0.28 mg %).indicated in Table 3 depicted in Figure 2.Accumulation
of higher concentrations of bilirubin confirms the depth and intensity of the liver
damage.

Administration of Ethanolic extract of leaves of Prunella vulgaris showed recovery

against the toxic effects of CCl4 which was shown in the Table 5 & 6 depicted in
Figure 4 & 5 respectively whereas standard drug Silymarin 100mg/kg decreases the
elevated serum enzyme levels which was shown in Table 4 and depicted in Figure 3.
Ethanolic extract of Prunella vulgaris 100mg/kg showed hepatoprotective activity
and the values are closer to standard drug.

Hepatocellular necrosis leads to a very high level of Serum Glutamate Oxaloacetate

Transaminase, Serum Glutamate pyruvate Transaminase released from liver into the
blood. Among the two, GPT is a better index of liver injury, as liver GPT activity
represents 90% of total enzyme present in the body. ALP activities on the other hand
are related to the functioning of hepatocytes, increase in its activity is due to

40
increased synthesis in presence of increased biliary pressure. Reduction in the levels
of SGOT, SGPT towards the respective normal value is an indication of stabilization
of plasma membrane as well as repair of hepatic tissue damage caused by carbon
tetrachloride. This effect is an agreement with view that serum levels of
transaminases return to normal with healing of hepatic parenchyma and regeneration
of hepatocytes. Suppression of increased ALP activity with no current depletion of
raised bilirubin level suggests the stability of the biliary function in rat liver during
chronic hepatic injury with CCl4.

The reduction in the elevated enzyme levels in figures suggests the protection of
structural integrity of hepatocyte cell membrane or regeneration of damaged liver
cells by Ethanolic extract of leaves of Prunella vulgaris. The effectiveness of the
normal functional condition of the liver is indicated by the decreased level of Serum
Bilirubin. Our results suggests that the Ethanolic extract of leaves of Prunella
vulgaris protects the liver from severe damage caused by carbon tetrachloride.

41
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