Improvement on the Carbon Tetrachloride inducing Hepatotoxicity in rats using Ipomoea turpethum Root

 

Vrunda V. Shah¹*, Vipul K. Shah², Krishnkant N. Patel³, Navin R. Sheth⁴, M. M. Patel¹

¹Shree Swaminarayan Sanskar Pharmacy College, Zundal, Gandhinagar-382421, Gujarat, India

²Troikaa Pharmaceuticals Ltd., Ahmedabad, Gujarat, India

³SAL Institute of Pharmaceutical Education and Research, Ahmedabad, Gujarat, India.

⁴Vice Chancellor of Gujarat Technological University, Ahmedabad, Gujarat, India.

 

ABSTRACT:

Liver toxicity is a major health problem of worldwide proportions. Herbal medicines derived from plant extracts are being increasingly utilized to treat a wide variety of clinical diseases.  Objective of the present work was to evaluate Hepatoprotective potential of Hydro alcoholic extract of Ipomoea turpethum root. This in vivo efficacy was reinforced by a significant dose dependent hepatoprotection (at 100 mg/kg and 200mg/kg dose) in CCl₄ induced Hepatotoxicity using wistar rats five in each groups. In this study extract of plant (100mg/kg and 200mg/kg, orally) was given for 6 days daily and CCl₄ (1 ml/kg, 1:1 with liquid paraffin, s.c.) is given at 6th day after 2 hours from dosing. The Hepatoprotective activity is confirmed by decreasing the activity of serum enzymes, SGOT, SGPT, ALKP, Bilirubin, Cholesterol, while it significantly increased the reduced Protein levels in dose dependant manner. The study was also supported by histopathology of liver sections. The Hepatoprotective activity of the extract is being comparable to standards Silymarin (200mg/kg). The results obtained in the present study indicate that Ipomoea turpethum is a potential source of natural Hepatoprotectives.

 

 

 

1.      INTRODUCTION:

Herbal drugs are prescribed widely even when their biologically active components are unknown because of their effectiveness, fewer side effects and relatively low cost¹. Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects².

 

In the absence of a reliable liver protective drug in modern medicine there are a number of medicinal preparations in Ayurveda recommended for the treatment of liver disorders³. In view of severe undesirable side effects of synthetic agents, there is growing focus to follow systematic research methodology and to evaluate scientific basis for the traditional herbal medicines that are claimed to possess hepatoprotective activity.

 

Ipomoea turpethum syn. Operculina turpethum which is commonly known as trivit is a large stout perennial twinner with milky juice and fleshy branched roots¹. The Root is bitter, sweet, with a sharp taste; heating, anthelmintic, purgative, anti pyretic, alexiteric; useful in ascites, leucoderma, itch, ulcers, constipation, abdominal troubles, inflammation, anaemia, fevers, biliousness, piles, erysipelas, tumors, jaundice, ophthalmia, bites from insects; Good in diseases of the liver, the heart, the eye; causes “Vata”. The black variety is a powerful drastic; useful in loss of consciousness, burning sensation, intoxication.-The white variety is a moderately mild cathartic; useful in bilious fevers, inflammation and diseases of the abdomen².

 

It is one of the plants mentioned in the literature having claims of activity against liver Disorders³. It contains a wide variety of phyto constituents, which are useful in treatment of different ailments and includes glycosidic resin, coumarins, beta-sitosterol, and essential oils^3-4.

 

Upon literature review, it was found that the active principle of the plant is glycosidic resin⁵. The scopoletin, a coumarin derivative, turpethinic acid and its derivatives were isolated from the plant. Previous phytochemical studies with Ipomoea turpethum reported the isolation of β-sitosterol, betulin and lupeol⁶; some terpenoid cycloartenol, lanosta-5-ene and 24-methylene-δ 5-lanosterol ⁷.

 

2.   MATERIAL AND METHODS:

2.1 Collection and Authentication of plant:

The Roots of Ipomoea turpethum were collected from Saurashtra region, Junagadh during the month of October-November. The plant was authentified by Botanical Survey of India, Ministry of India, Jodhpur and a voucher specimen no. SU/DPS/Herb/09 was deposited. Further, the plant was identified by comparing it morphologically and microscopically with the description given in different standard texts and floras ^{2, 8}. Fresh Roots of plant were cleaned, dried at room temperature and powdered.

 

2.2 Preparation of plant extract:

The shade dried Root of Ipomoea turpethum about 800 gm was subjected for size reduction to fine powder. The powder was macerate with hydroalcohol (50%) solution for 6-7 days on rotary shaker. Filter and concentrated under vacuum to get the residues. The extracts were suspended in 7% acacia solution for pharmacological study.

 

2.3 Drugs and Chemicals:

Silymarin (Cadila Healthcare Pvt. Ltd.), Carbon tetrachloride, Liquid paraffin (E.Merck (India) Ltd., Mumbai). SGOT, SGPT, ALKP, Total cholesterol, Total bilirubin, Albumin, Total protein

Estimation kit ( Span diagnostic, Surat ).

 

2.4 Acute toxicity study:

Different groups of five mice each were given graded doses of the plant extract (0.5-3.0 g/kg, orally) and were kept under constant observation for 6 h to note any behavioral changes and mortality was recorded after 24 h of the drug administration⁹.

 

2.5 Animal selection and Ethical clearance:

Wistar albino male rats weighing 200-250 gm were acclimatized to the experimental room having temperature 23 ± 2°C, controlled humidity conditions (55±10 %), and 12:12 hour light and dark cycle. Animals were caged in polypropylene cages with maximum of five animals per cage. The rats were fed with standard food pellets and water ad libitum. Study was conducted after obtaining the approval from the Institutional Animal ethical committee as per the guidance of the committee for the purpose of control and supervision of experiments on animals (CPCSEA).

 

2.6 Assessment of Hepatoprotective activity:

Animals were divided into five groups of five rats in each for all the experiment. The first group served as vehicle control and received 7% gum acacia in distilled water (2ml/kg) only. The second group served as carbon tetra chloride (CCl4) intoxicated control and received by gavage vehicle (7% gum acacia in distilled water) and CCl4 diluted with liquid paraffin (1:1).

 

Third group was given standard drug Silymarin at the dose of 100 mg/kg body weight^10-11 and the remaining two groups were given ITHAE (Ipomoea turpethum Hydroalcoholic extract) at the dose of 100 mg/kg and 200 mg/kg body weight respectively and CCl4. The vehicle (7% gum acacia in distilled water) or test drugs were administered orally for 6 days. CCl4 diluted with liquid paraffin (1:1) was administered in a dose of 1 ml/kg subcutaneously (s.c) on 6th day (After 2 hr. of previous administration).

 

Group I: Vehicle (7% gum acacia in distilled water, 2ml/kg. p.o.)

Group II: Vehicle for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

Group III: Silymarin (100mg/kg, p.o.) for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

Group IV: ITHAE (100 mg/kg, p.o.) for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

 Group V: ITHAE (200 mg/kg, p.o.) for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

 

Food was withdrawn 12 hr. before CCl4 administration to enhance liver damage in animals of groups II, III, IV and V. Twenty four hours after CCl4 administration, blood was obtained from all groups of rats by puncturing retro-orbital plexus. The blood samples were allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2500 rpm at 30°C for 15 min and analyzed for various biochemical parameters¹².

 

2.7 Parameters assessed for Hepatoprotective activity:

On the 7th day blood was collected from all groups of rats by puncturing retro-orbital plexus and animals were sacrificed. The  parameters were assessed are Serum glutamate oxaloacetae transaminase (SGOT) Serum glutamate pyruvate transaminase (SGPT) Alkaline phosphatase (ALKP) Total Bilirubin (TB) and Direct Bilirubin (DB) Total Cholesterol (CHL) Total Protein (TP) Albumin (ALB) .

 

2.8 Histopathological Studies:

One animal from each of the treated groups, showing maximum activity as indicated by improved biochemical parameters was used for this purpose. The animals were sacrificed and the abdomen was cut open to remove the liver. The liver was fixed in 10 % formalin solution. After 12 hours, liver was embedded in paraffin using conventional methods ¹³ and cut in to 5μm thick section and stained using haematoxylin-eosin dye and finally mounted in di-phenyl xylene. Then the sections were observed under microscope for histopathological changes in liver architecture and their photomicrographs were taken.

 

2.9 Photomicrography:

The photomicrography of the sections at different magnifications as demanded by the anatomical details to be studied. The microphotographs were taken using Olympus CH20i microscope attached with Magnus MIPS camera.

 

2.10 Statistical analysis:

The mean values ± S.E.M. are calculated for each parameter. For determining the significant inter group difference, each parameter was analyzed separately and one way analysis of variance (ANOVA) ¹⁴ was carried out.

 

3.      RESULT AND DISCUSSION:

3.1 Acute Toxicity Study: LD50 was found to be 2000 mg/kg.

3.2 In vivo Hepatoprotective Study: The results of hepatoprotective effects of Ipomoea turpethum extracts on CCl4-intoxicated mice are shown in Table 1.

 

Administration of CCl4 in normal rat elevated the serum level of SGOT, SGPT, ALKP, TB, DB and CHL; quite decreased in TP and ALB were observed, significantly indicate Hepatocellular damage. It may be due to enzymatic activation CCl3 free radical which alters the structure and function of liver cell. The rat treated with Ipomoea turpethum Hydroalcoholic (50%) extract showed decreased in all elevated level of SGOT, SGPT, ALKP, TB, DB and CHL level and increased in TP and ALB levels. Pretreatment with Ipomoea turpethum Hydroalcoholic (50%) extract showed dose dependent protection against injurious effect of CCl4 that may result from interfere with cytochrome P450 resulting in the hindrance of the formation of Hepatotoxic free radical. This biochemical restoration may be due to the inhibitory effect on cytochrome P450 and/or promotion of its glucuronidation. It also indicates stabilization of plasma membrane as well as repair of Hepatic tissue damage, caused by CCl4.

 

3.3 Histopathology of liver:

HC

Histopathological studies Shown in the fig.1 also provided supportive evidence for biochemical analysis. Histology of the liver section of Normal control group showed in Fig. 1A showed distinct hepatic cells, sinusoidal spaces HC: Hepatocyte, SS: Sinusoidal space, VC: Vacuoles. Histology of the liver section of Standard group showed in Fig. 1B showed regeneration of Hepatocyte, less vacuoles, reduced sinusoidal space compared to hepatotoxin. Histology of the liver section of hepatotoxic group showed in Fig. 1C showed degeneration of normal hepatic cells with lobular necrosis, vacuole formation. Histology of the liver section of ITHAE (100mg/kg) showed in Fig. 1D showed less degeneration of Hepatocyte, less vacuoles, less sinusoidal space compared to hepatotoxin. Histology of the liver section of ITHAE (200mg/kg) showed in Fig. 1E showed regeneration of Hepatocyte, less vacuoles, reduced sinusoidal space compared to hepatotoxin.

 

Table 1: Hepatoprotective Effect of the Hydroalcoholic extract of Ipomoea turpethum root on CCL₄ induced hepatotoxicity. 

Treatment	Dose	SGOT  (IU/l)	SGPT (IU/l)	TB (mg/dl)	ALKP (IU/l)	DB (mg/dl)	CHL (mg/dl)	TP (mg/dl)	ALB  (g/dl)
Control (Vehicle)	2ml/kg	99.2± 16.608	42± 4.658	0.302± 0.0332	5.878± 0.0153	0.358± 0.0248	114.932± 9.218	5.810± 0.376	3.202± 0.238
Hepatotoxin (CCl4)	1ml/kg	178.8± 34.852	109.6± 9.042	1.418± 0.0427	51.112± 3.996	0.636± 0.0339	210.130± 7.808	3.120± 0.397	1.908± 0.0969
Standard (Silymarin)	100mg/kg	116.6± 7.58	68± 7.273	0.296± 0.0178	5.282± 0.585	0.404± 0.0293	117.6± 10.402	5.300± 0.414	2.720± 0.116
ITHAE (100mg/kg)	100mg/kg	153.8± 7.151	76± 15.492	0.672± 0.0475	31.524± 4.950	0.4± 0.0164	129.064± 3.283	3.858± 0.177	2.300± 0.0957
ITHAE (200mg/kg)	200mg/kg	111± 9.105	47.6± 12.106	0.434± 0.0256	23.01± 2.318	0.32± 0.0513	92.128± 10.553	4.560± 0.0927	2.760± 0.103

Value are mean ±SEM; n=5. Compared to Control (P=0.001)

 

Fig. 1 Histopathological study of Liver.

 

 

4.      CONCLUSION:

From present study, results show that the roots of Ipomoea turpethum have a strong Hepatoprotective activity.

 

HC

SS

SS

VC

HC

HC

5. ABBREVIATIONS:

(IL) Interleukins

(ITHAE) Ipomoea turpethum Hydroalcoholic extract

(CCl₄ )  Carbon tetrachloride

(SGOT) Serum glutamate oxaloacetae transaminase

(SGPT) Serum glutamate pyruvate transaminase

(ALKP) Alkaline phosphatase

(TB) Total Bilirubin and (DB) Direct Billirubin

(CHL) Total Cholesterol

(TP) Total Protein

(ALB) Albumin

(AST) Aspartate Transaminase

(ALT) Alanine Transaminase

(S.E.M.) Standard Error Mean

(CE) Cholesterol Esterase

(POD) Peroxidase

(CHOD) Cholesterol Oxidase

 

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3.     Vasudesan N. V., In Indian Medicinal Plants, Vol. IV, Orient Longman Ltd, Chennai. 1995: 172.

4.     Deeaph G., Malti G., Indian Drugs. 1994; 31: 294.

5.     Ambasta S.P., The Useful Plants of India, Publications and Information directorate, New Delhi CSIR. 1986: 409.

6.     Nasar E.L.S.M.M., Comparisons of Convolvulus lantus and C.arvensis. Fitoterapia. 1982; 53: 189-191.

7.     Sahabuddin, Triterpenois from the stem of Operculina turpethum L. J. Biol. Sci.1999; 8: 157-163.

8.     The Ayuyrvedic Pharmacopoiea, Govt. of India, Ministry of health and family welfare, New Delhi, 1(2): 133.

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Received on 29.07.2017       Modified on 11.09.2017

Accepted on 21.10.2017      ©A&V Publications All right reserved