hepatoproctive activity of leaves extract of carrisa crandas lin

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Indo American Journal of Pharmaceutical Research, 2014 ISSN NO: 2231-6876

HEPATOPROTECTIVE ACTIVITY OF LEAVES EXTRACTS OF CARISSA CARANDAS LINN

Pooja Bhati, Ajay Shukla*, Maya Sharma, Pramod mourya Guru Ramdas Khalsa Institute of Science and Technology (Pharmacy) Jabalpur M.P.Mohan Lal Sukhadiya University, Udaipur,

Rajasthan,NRI Institutes of Pharmacy Bhopal, M.P.

Corresponding author

Ajay Shukla

Research scholar

Mohan Lal Sukhadiya University, Udaipur, Rajasthan

House no. 1853/A Shakti nagar Gupteshwar Chowk Madan Mahal Jabalpur M.P. 482001

[email protected]

919893735320

Copy right © 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research,

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ARTICLE INFO ABSTRACT

Article history

Received 16/10/2014

Available online

30/11/2014

Keywords

Carissa Carandas,

Hepatoprotective,

Carbon Tetra Chloride.

The Hepatoprotective activity of leaves of Carissa carandas linn against carbon tetra chloride

induced hepatotoxicity in albino rats was determined. The leaves were collected and dried

under shade followed by crushing. The extraction of crushed powder involve the Maceration

by using methanol and acetone (w/v 1:3) solvent and concentrated to dry mass by using

rotary vacuum evaporator. Dark green waxy residues (leaves) were collected separately. The

different parameter of evaluation were taken such as histopathological studies, Total

Bilirubin, Direct Bilirubin, SGPT and SGOT etc. The Liver section of animal which was

treated with methanol and acetone (w/v 1:3) extract clearly shows the normal hepatic cells

and compared with Silymarin treated group of animals. The result suggests that the

methonolic extract of leaves of Carissa carandas linn possess a significant role as

hepatoprotective activity.

Please cite this article in press as Pooja Bhati et al. Hepatoprotective Activity of Leaves Extracts of Carissa carandas linn. Indo

American Journal of Pharm Research.2014:4(11).

mailto:[email protected]

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Vol 4, Issue 11, 2014. Pooja Bhati, et al. ISSN NO: 2231-6876

INTRODUCTION

Herbal medicine

Herbal medicine includes herbs, herbal materials, and herbal preparations and finished herbal products. In some countries herbal

medicines may contain, by tradition, natural organic or inorganic active ingredients that are not of plant origin (e.g. animal and mineral

material [1]. Herbal medicine, or phytotherapy, is the science of using herbal remedies to treat the sick. It therefore covers everything

from medicinal plants with powerful actions, such as Digitalis and Belladonna, to those with very gentle action, such as chamomile, mint

and many others. It should be noted that „very gentle‟ action, when referring to chamomile or mint, does not mean they are more or less

ineffective, but rather that one would not expect these plants to produce instant and powerful effects like those seen, for instance, after an

injection of digitalis or strophanthin. „Gentle‟ action also means that these „simple‟ medicinal plants do not as a rule have any appreciable

toxic effects, and may therefore be safely taken over an extended period of time. Traditional medicine is the sum total of the knowledge,

skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the

maintenance of health and in the prevention, diagnosis, improvement or treatment of physical and mental illness. The terms

“complementary medicine”, “alternative medicine” and “non-conventional medicine” are used interchangeably with “traditional

medicine” in some countries[2] Liver are responsible for metabolism of chemicals and foods for the regulation of internal environment.

The vital role of the liver is carbohydrate, protein, and fat metabolism, and detoxification, secretion of bile and storage of

vitamins. Excess consumption of certain toxic chemicals such as antibiotics, chemotherapeutics, peroxidised oils, acetaminophen,

aflatoxin, carbon tetrachloride, chlorinated hydrocarbons, alcohol etc leads to infections and autoimmune disorder. Modern drugs have

little to offer for alleviation of hepatic diseases and it is chiefly the herbal based preparations which are employed for the treatment of

liver disorders. For a long time, medicinal plants and their extracts were widely used in the treatment of liver diseases like hepatitis, and

liver cirrhosis. There are numerous herbal plants and polyherbal formulations claimed to have hepatoprotective activity. Nearly about 150

phytoconstituents from 101 plants have been claimed to possess liver protecting activity [3, 4], in the traditional herbal system of

medicine, many medicinal plants have been reported to possess the potential to treat liver diseases. Carissa carandas Linn. Is extensively

used in the indigenous and herbal folklore medicine systems to treat hepatotoxicity. It is used as an antidote of poisons, blood purifier,

expectorant, fever, erache, soreness and syphilitic pain of the mouth. A tincture preparation of fruits is used in infections and skin

infections and a decoction of wood is employed as a tonic to strengthen the tendons of slim patient. The ripe fruit is taken as an

antiscorbutic and remedy for biliousness. Additionally the C.congesta has shown wide range of evidences for its cardiotonic,

hepatoprotective, free radical scavenging and xanthine oxidase inhibitory, histamine releasing, antirhumatic, antibacterial, antiviral and

anticonvulsant activity. A higher gross heat value of this species indicates its higher potential to be used as good fuel source. [5-11]

MATERIALS AND METHODS

Biochemical Estimations

Aspartate transferase, alanine aminotransferase, acid phosphatase and alkaline phosphatase kits and the drugs and fine chemicals

were purchased from Sigma Aldrich (USA) and Ranbaxy (India). All other chemicals and solvents were obtained from local firms and

were of analytical grade. The activities of superoxide dismutase (SOD) [12], catalase (CAT) [13], glutathione peroxidase (GPx) [14],

reduced glutathione [15], α–tocopherol (vitamin E) [17], ascorbic acid (vitamin C) [16] were estimated. For histopathological

examination liver sections were stained with haematoxylin and eosin using standard protocol and then analyzed by light microscope.

Extraction, separation and purification of the compound

Plant material was collected locally from Bhopal. Herbarium was prepared and submitted at botany department of Safia

College, Bhopal for authentication. The plant was authenticated by Dr. Ziaul Hasan, Department of Botany, Safia Science College, and

Bhopal. A voucher specimen no.256 /Bot/Safia/2011 of the plant. The leaves were shade dried and coarsely powdered for extraction. The

dried leaf material (1kg) was extracted with methanol and acetone (w/v 3:1) three times at room temperature for a week. The methanol

crude extract was combined and concentrated to yield a residue (140 g) which was subjected to successive solvent partitioning to give

chloroform (26g), methanol (114g) soluble fractions. The methanol fraction have selected for study of hypatoprotective activity.

Extraction of Dried Leaf

Leaves were washed and air dried under shade. Dried leaves were crushed to fine powder using grinder at Pinnacle Biomedical

Research institute (PBRI), Bhopal and stored in an air tight container for further studies. Plant material (1 Kg) was weighed and packed

with Petroleum Ether (40:60) in air tight container for maceration. Sample was regularly shaked in between. After 15 days solvent was

filtered under vacuum. Marc was dried under shade and further packed with Methanol: Acetone (70: 30) solvent system for fifteen days

with regular shaking. Solvent was filtered and evaporated in rotary vacuum evaporator at 40oC. Extract (CCE) was packed in air tight

container and kept in cool place for further studies [18-22,]

Preliminary Phytochemical Screening

The plant may be considered as biosynthetic laboratory for multitude of compounds like alkaloid, glycoside, volatile oils,

tannins, saponins, flavonoids etc. These compounds are termed as secondary metabolites and are responsible for therapeutic effects [23,

24].To check the presence or absence of primary and secondary metabolites, the extract were subjected to battery of chemical tests.

1. Test for Carbohydrates

2. Test for alkaloids

3. Test for Phenolic compounds

4. Test for Cardiac glycoside

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5. Test for Glycosides

6. Test for Steroids and Triterpenoids

7. Test for Tanins

8. Test for Proteins

9. Test for Saponins

In vivo Hepatoprotective Activity

Animals and their housing conditions

In vivo hepatoprotective activity of Carrisa Carandas Leaves Extract was done by using CCl4-induced hepatotoxicity in Albino

rat model. The animal handling and in vivo experiments were carried out in accordance with the guidelines of the “Committee for the

purpose of control and supervision on experiments on animals (CPCSEA), Ministry of Environment and Forests, Govt. of India”. An

experimental protocol was approved by Institutional Animal Ethics Committee (IAEC) of Pinnacle Biomedical Research Institute, Bhopal

(Regd. No. 1283/c/09/CPCSEA).Swiss albino rats of either sex (100–150gm) were collected at random from the animal house of Pinnacle

Biomedical Research Institute, Bhopal. All animals were kept in a propylene cage with sterilized husk as bedding material in a group of

four animals per cage at 22±2 °C in 12:12 light: dark cycle. Standard feeding pellets (Golden feeds, New Delhi) was used for animal

feeding and water was available ad libitum.

Method: CCl4 induced hepatotoxicity: Animals were divided into 5 groups (n=6/group).

Group A- Normal control: The animals received saline for 7 days.

Group B- Induction of hepatotoxicity by using CCl4: The animals received distilled water for days and given CCl4 (Themis Pharma,

Mumbai) single dose, 500 mg/kg BW Orally on day 8.

Group C- Pre-treatment with standard drug sillymarin (100mg/kg) bw/day for 7 days (p.o) Followed by a single dose of CCl4 on day 8.

Group D- Pre-treatment with Acetone: Methanol (70:30) extract of Carissa carandas Leaves (50mg/kg bw/day for 7 days (p.o) followed

by a single dose of CCl4 on day 8.

Group E- Pre-treatment with Acetone:Methanol (70:30) extract of Carissa carandas leaves (100mg/kg) bw/day for 7 days (p.o) followed

by a single dose of CCl4 on day 8.

Histological interpretation

All histological slides of 50 µm from treated animals were investigated at 40x and 100x for presence of any damage. Structural

damage with integrity and presence of any type of necrosis or inflammation were prime consideration. Observations were as follow –

Group A: vehicle treated animal

In vehicle treated group histological examination revealed normal architect of cellular constitution. All integral part of

hepatocellular arrangement were found to be properly arranged and without any type of damage. Hepatic lobule, synosoids, hepatocytes,

portal triad (constituting portal vein, portal artery and bile duct) general endothelial lining, central vein, vascular endothelium,

prominency of nucleus, kupffer cells were observed. All of them were found to be properly arranged and without any sign of necrosis or

inflammation.

Group B: CCl4 + Vehicle treated group

The customary architecture of liver was utterly lost in rats treated with CCl4 with the manifestation of vacuolated Hepatocytes

and degenerated nuclei. Vacuolization, fatty changes and necrosis of hepatocytes were stern in the centrilobular region. CCl4 poisoning

led to disproportionate pattern of deposition of connective tissue and development of scars and such liver sections were thus assigned as

damaged. Livers of animals treated with CCl4 on gross examination seen with scattered yellow and white areas attributed to fatty and

necrotic changes.

Group C: CCl4 + Silymarin treated group

All histological examination revealed that Silymarin provided protection even at cellular level. All arrangement were normal and

sign of damage was not present.

Group D: CCl4 + Extract 50 mg/kg treated group

In extract treated group at 50 mg/kg protection was present but it was not as much significant. Sign of necrosis and inflammation were

present.

Group E: CCl4 + Extract 100 mg/kg treated group

In Extract 100 mg/kg treated group protection was quite significant. Sign of intoxication was almost absent. Although few

inflammation signs were present but necrosis and damage was absent. Thus it can be concluded that the extract at selected dose also

provide protection at cellular level.

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Histological interpretation

Group A: vehicle treated animal

Fig 1– Histology of vehicle treated animal.

Portal artery

Bile duct

Portal vein

Portal triad

Central vein

Sinusoids

Vascular endothelium

Central vein

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Group B: CCl4 + Vehicle treated group

Group C: 1CCl4 + Silymarin treated group

Group D: CCl4 + Extract 50 mg/kg treated group

Fig 3.1CCl4 + Silymarin treated

group

Fig 3.2 CCl4 + Silymarin treated

group

Fig 2.1CCl4 + Vehicle treated group Fig 2.2 CCl4 + Vehicle treated group

Fig 4.1 CCl4 + Extract 50 mg/kg

treated group


treated group

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Group E: CCl4 + Extract 100 mg/kg treated group

Table 1: Results of Preliminary Phytochemical Screening.

Result of hepatoprotective activity

CCl4 Induced Hepatotoxicity.

Table No. 2:- Effect of extract of Carissa carandas on CCl4 induce Hepatotoxicity in Rat

*Significant elevated level as compared to vehicle treated group (P<0.05)

**Significant protection as compared to CCL4treated group (P<0.05)

# Significant difference as compared to lower dose i.e. 50 mg/kg (P<0.05)

Sr. No Phyto-Constituent Category Inference

1 Carbohydrate Positive

2. Cardiac glycoside Negative

3 Flavonoids Positive

4 Alkaloids Positive

5. Tannin and phenolic component Positive

6. Protein Positive

7. Amino acid Positive

8. Saponin Positive

Groups Treatment AST(U/L) SGPT(U/L) ALP (lU/L) Total Bilirubin Direct Bilirubin

A Vehicle 75.08±4.206 53.055±3.612 97.77±2.324 1.565±0.0403 0.473±0.045

B CCl4 273.906±6.353* 227.09±4.99* 298.51±12.73 4.26±0.335 1.283±0.098

C Silimarin+

CCl4

85.84±2.414** 59.16±3.506** 98.275±3.335 1.638±0.1222 0.505±0.052

D Extract 50mg/kg+

CCl4

109.23±3.579** 93.421±1.005** 146.39±1.058 3.736±0.471 0.8533±0.0225

E Extract 100mg/kg+

CCl4

130.41±1.343**, # 84.87±0.899**,

# 122.41±5.8055 2.015±0.078 0.7133±0.0467


treated group


treated group

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RESULTS AND DISCUSSION

The methanolic and acetonic(3:1) extract of leaves Carissa carandas at a dose 50 mg/kg showed decrease in Total Bilirubin,

Direct Bilirubin, SGPT and SGOT levels compared to CCl4 induced animals. However methanolic and acetonic(3:1) extract of leaves of

Carissa carandas at a dose 100mg/Kg shown better protection than (50mg/kg), It was further confirmed that methanolic and

acetonic(3:1) extracts of leaves (100mg/Kg) and (50mg/kg) of Carissa carandas showed comparable results with Silymarin (100mg/Kg)

(Table. 2.). Histopathology of liver section of control group (Figure. 1.) Showed normal arrangement of hepatocytes with clear board of

central vein at portal layer. Microscopical examination of liver section of CCl4 treatment (Figure.2.1 & 2.2) showed various pathological

changes, centrilobular fatty degeneration, cloudy swelling and necrosis of hepatic cells. Liver section of methanolic and acetonic(3:1)

extract of Carissa carandas Linn of leaves, treated groups (Figure. 4.1, 4.2 & 5.1, 5.2) Clearly showed normal hepatic cells and central

veins, which are comparable with Silymarin treated animals (Figure. 1).

CONCLUSION

The potent hepatoprotective activity of leaves of Carissa carandas Linn was found to reduce the level of Total Bilirubin, Direct

Bilirubin, SGPT and SGOT in CCl4 treated animals.

ACKNOWLEDGEMENTS

Authors Mr.Ajay Kumar Shukla and Mrs Pooja Bhati are thankful to Dr .Ritu Charde and Pinnacle Biomedical Research

Institute, Bhopal for good guidance to carry out this research work.

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hepatoproctive activity of leaves extract of carrisa crandas lin

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