Hepatoprotective Potential of Trichosanthes dioica Roxb in Hepatotoxicity Induced by Simvastatin and its Consequences on Biochemical and Haematological Indices

Gupta, Swain, Sahoo, Gupta, and Chaudhary: Hepatoprotective Potential of Trichosanthes dioica Roxb in Hepatotoxicity Induced by Simvastatin and its Consequences on Biochemical and Haematological Indices

Authors

INTRODUCTION

Trichosanthes dioica Roxb, commonly known as parwal, belongs to cucurbitaceae family and is an annual or perennial herb distributed throughout India. Out of 20 species in India, two are cultivated as vegetable (T. anguina and T. dioica). In Charaka Samhita, leaves and fruits are enlisted for treatment of alcoholism and jaundice. In Ayurveda, leaves are utilized as antipyretic, diuretic, cardiotonic, laxative, antiulcer. T. dioica accomodate numerous chemical constituents like vitamin A, saponins, tannins, alkaloids, mixture of novel peptides, proteins and vitamin C.12 Thevarious scientific research revealed that T. dioica exhibited antidiabetic,3 anti-hypercholesteraemic,4 hepatoprotective,5 anti-ulcer,6 immunomodulatory,7 antimicrobial,8 antioxidant,9 anti-diarrheal,10 nephroprotective,11 and wound healing activity.12

HMG CoA reductase inhibitors (Simvastatin) are efliective in reducing cardiovascular mortality and are widely prescribed around the globe. More than 145 million patients were prescribed with statins in United States in 2005. The use of statins is increasing day by day, although liver toxicity has been a concern since their initial introduction.13 No scientific results are available affirming hepatoprotective potential of Trichosanthes dioica Roxb against hepatotoxicity induced by simvastatin. Hence, present investigation was designed to demonstrate hepatoprotective activity of Trichosanthes dioica Roxb against simvastatin induced liver toxicity.

MATERIALS AND METHODS

Drugs and Chemicals

Simvastatin (Merck Pharmaceutical, India). All chemicals used were of analytical grade and procured from Sigma Chemicals Co., USA and Qualigens fine chemicals, Mumbai, India.

Collection and authentication of plant

Fresh and matured fruits of T. dioica were purchased from local market of Lucknow, India in August 2016. The plant material was identified and authenticated ?by National Botanical Research Institute, Lucknow, India.

Extraction of plant material

The fruits of T. dioica were dried and powdered. The powdered plant material was macerated with petroleum ether; the marc was exhaustively extracted with of 50% methanol for three days. The extract was dried by rotator evaporator (IKA, Germany) under reduced pressure and procured in desiccator. The % yield was discovered to be 0.75%. 1% Tween-80 was used to prepare extract suspension of desirable concentration needed for pharmacological studies.

Phytochemical investigation

The methanolic extract of T. dioica fruits were subjected to preliminary phytochemical screening for detecting the presence or absence of active phytochemical constituents.14,15

Animals

Wistar rats weighing (150-170 g) of either sex were procured from Animal house of College of Pharmacy, Shri Ram Murti Smarak College of Engineering and Technology, Bareilly, India. They were kept in departmental animal house in well cross ventilated room at 22±2°C with light and dark cycles of 12 h for 1 week before and during the experiments. The experimental protocols were approved by Institutional Animal Ethical Committee, India (Reg. No. 715/02/CPCSEA).

Acute toxicity study

Acute toxicity study was performed according to OECD guidelines 423. Albino mice (20-25 g) were divided into five groups with 5 mice in each. Group-I numbered as control received distilled water orally. Group-II, III, IV, and V were administered T. dioica extract at a dose of 5, 50, 300, 2000 mg/kg, orally, respectively. The animals were noticed for toxicity sign or mortality every 24 h, daily for 2 weeks.16

Experimental design

Wistar rats were divided into five different groups, each group having 6 rats. Group I received distilled water only for 30 days. Group II rats charged with simvastatin (20 mg/kg, p.o.) alone for 30 days orally. Group III and IV rats received simvastatin along with T. dioica fruits extracts(200 mg/kg and 400 mg/kg, p.o. respectively) for 30 day and Group V rats received simvastatin along with silymarin (20mg/kg,p.o.) for 30 days. On the 31thday, blood samples were collected, and all the animals were sacrificed by cervical dislocation under mild ether anesthesia and liver sample were harvested, rinsed in saline and stored at -80°C for further biochemical analysis.17

Evaluation of liver protective activity

The collected blood was allowed to clot and serum was separated by centrifugation in a refrigerated tabletop centrifuge at 2500 rpm for 15 min and the biochemical parameters like serum enzymes: Serum glutamic oxaloacetic transaminase (SGOT, U/L), serum glutamic-pyruvic transaminase (SGPT, U/L),18 alkaline phosphatase (ALP, U/L),19 total bilirubin (mg/dL),20 total protein and albumin were evaluated.21,22

Evaluation of hematological parameter

Red blood cell (RBC) count, haemoglobin (Hb), white blood cell (WBC) count, platelet (PLT) and lymphocytes were determined by the fully automated hematology analyzer (XP 100 Hematology Analyzer, Transasia Bio-Medicals Ltd., India).

Histopathological studies

For histopathological inspection, the liver tissues were affixed with 10% phosphate buffered neutral formalin, dehydrated in graded (50-100%) alcohol and embedded in paraffin. fine sections (5 M) were cut and stained with routine hematoxylin and eosin stain for photo microscopic analysis. All the slides were studied under a light microscope for any histological destruction and protection.

Statistical analysis

The statistical comparison between the groups were made by One Way Analysis of Variance (ANOVA) and followed by Student-Newman-Keuls test. The value p<0.05 was considered statistically using, Graph Pad Prism 5.03 Software. The values were represented as mean ± SEM for six rats.

RESULTS

Phytochemical screening

Phytochemical screening showed the presence of tannins, alkaloids, carbohydrates, flavonoids, glycosides and steroids as documented in Table 1.

Table 1

Preliminary phytochemical analysis of T. dioica fruit extract.

S.NoName of testConstituentsInference
1Ferric Chloride & Gelatin test.Tannin+
2Mayers&Wagners Test.Alkaloids+
3Benedict’s test.Carbohydrates++
4Ferric Chloride &Shinoda Test.Flavonoids+
5Keller Killiani& Bromine water test.Glycosides-
6Salkowaski Test.Steroids+

[i] “+” means present.

Acute toxicological outcome

The methanolic extract of T. dioica at doses of 200 and 400 mg/kg body weight does not produce any toxic outcome. Therefore, these doses were selected for hepatoprotective studies.

Effect of TME on serum hepatic parameters

The outcome of T. dioica fruits extract dose was investigated on liver serum markers like SGOT, SGPT, ALP, bilirubin (BLB), total protein (TP) and albumin (ALB) level. Hepatic abrasion due to dose of simv-astatin generate significant elevation in marker enzymes as SGOT by 336.5%, SGPT by 135.7%, ALP by 144%, BLB by 39%, and demotion in level of TP by 48.3% and ALB by 32.5% when compared to control (Group I). The dose of extract, TME 200 (Group III) and TME 400 (Group IV) declined the elevated level of SGOT (10.19%, P<0.01, 66.3%, P<0.001), SGPT (15.41%, P<0.01, 73.2%, P<0.001), ALP (7.30%, P<0.05, 29.68%, P<0.001), BLB (12.64%, P<0.01, 53.12%, P<0.001) and promote the level of TP (17.94%, ns, 37.25%, P<0.01), ALB (21.61%, P<0.001, 26.36%, P>0.001) respectively as compared to group II. Similarly, silymarin decreased SGOT by (120.6%, P<0.001), SGPT (110.1%, P<0.001), ALP (105%, P<0.001), BLB (216%, P<0.001) and increased TP by (42.85%, P<0.001), ALB (30.57, P<0.001) as compared to group II. The results are tabulated in Table 2.

Table 2

Effect of TME on serum SGOT, SGPT, ALP, BLB, TP and ALBagainst simvastatin induced liver toxicity in rats.

GroupsSGOTSGPTALPBLBTPALB
Control31.2 ± 1.541.3 ± 2.187.32 ± 3.20.2 ± 0.016.2 ± 0.63.6 ± 0.01
SIM136.2 ± 4.3’97.35 ± 3.4’213.1 ± 4.2’0.98 ± 0.02’3.2± 0.4’2.43± 0.02’
TME200123.6 ± 3.4b84.35 ± 3.1b198.6 ± 4.1a0.87± 0.04b3.9 ±0.3ns3.1±0.01c
TME40081.9 ± 3.1c56.2 ± 2.9c164.32 ± 3.2c0.64 ± 0.03c5.1 ± 0.2b3.3 ± 0.02c
SYL 2061.74 ± 2.9c46.32 ± 2.1c103.5 ± 3.6c0.31 ± 0.02c5.6 ± 0.4c3.5 ± 0.02c

[i] Values are mean ± S.E.M. of 6 rats in each group, ns: non-significant.

[ii] P values:<0.001 compared with respective control group I.

[iii] P values: a<0.05, b<0.01, c<0.001 compared with group II.

Effect of TME on body weight and liver weight

The effect of different doses of TME on body weight and liver weight were studied (Table 3). In Group II body weight decreased by 5.845% while liver weight increased by 26.36%. Animal Treated with TME at the doses of 200 and 400 mg/kg (Group III and IV) significantly increased in body weight by 2.15%, 3.81% respectively while liver weight was decreased by 1.01% and 8.56% respectively.

Table 3

Effect of TME on body weight and liver weightagainst simvastatin induced liver toxicity in rats.

‘ S.NoTreatmentBody weight (g)Liver weight (g)
1Control172.8±1.26.42±0.01
2SIM162.7±1.37.92±0.02
3TME200166.2±1.3ns7.84±0.03b
4TME400168.9±1.4b7.24±0.02c
5SYL20170.8±1.1c6.98±0.01c

[i] Values are mean ± S.E.M. of 6 rats in each group, ns: non-significant.

[ii] P values:<0.001 compared with respective control group I.

[iii] P values: a<0.05, b<0.01, c<0.001 compared with group II.

Effect of TME on blood parameters

The outcome in (Table 4) showed a significant change in RBC, Hb, PLT, WBC, and % Lymphocytes counts. RBC PLT, WBC, and % Lymphocytes counts significantly increased at the dose of 1000 and 2000 mg/kg, while Hb count non-significantly increases at the dose of 1000mg/kg as compared to control group. Hb, PLT, WBC and % Lymphocytes counts significantly increased at the dose of 4000 mg/kg, whereas RBC’s count non-significantly increases at the dose of 4000mg/kg when compared to the control group.

Table 4

Effect of TME on blood parameters against simvastatin induced liver toxicity in rats.

ParameterControl1000 mg/kg2000 mg/kg4000 mg/kg
RBC (x1012)7.6 ± 0.18.1 ± 0.12 a8.4 ± 0.13 c7.9 ± 0.16 ns
Hb (g/dL)13.01 ± 0.213.91 ± 0.3ns14.6 ± 0.4a14.4 ± 0.5a
PLT (x109/L)613.2 ± 5.6634.6 ± 6.2a645.2 ± 4.6b653.5± 5.1c
WBC (x109)8.1 ± 0.129.2 ± 0.2a11.5 ± 0.6c10.01 ± 0.3b
Lymphocytes (%)41.2 ± 2.150.3 ± 3.2a54.2 ± 3.1a58.6 ± 3.5b

[i] Values are mean ± S.E.M. of 6 rats in each group, ns: non-significant.

[ii] P values:<0.001 compared with respective control group I.

[iii] P values: a<0.05, b<0.01, c<0.001 compared with group II.

Histopathological observations

The histological explanation (Figure 1) support the results obtained from serum enzyme assays. Liver sections of control rats showed normal hepatic cells with well-preserved cytoplasm and well brought out central vein. Simvastatin (20mg/kg) treated rats (Group II), displayed the massive fatty changes, necrosis, central vein congestion, ballooning degeneration, and the loss of cellular boundaries, whereas TME 200mg/kg treated groups (Group III) showed mild congestion in central vein with less fatty changes, mild necrotic cells, with minimal inflammatory conditions and less infiltration of the leucocytes while TME 400mg/kg treated group (Group IV) showed regeneration of hepatocyte around central vein with near normal liver architecture, prominent nucleus and possessing maximum hepatoprotective action. Rats in (Group V) exhibited well brought out central vein, hepatic cell with well-preserved cytoplasm, prominent nucleus.

Figure 1

Histopathology of H&E stained sections of liver at 250X.

  • (a) Liver sections of normal control rats showed the normal hepatic cells with well-preserved cytoplasm; well brought out central vein.

  • (b) Liver section of SIM20 showed the massive fatty changes, necrosis, central vein congestion, ballooning degeneration, and the loss of cellular boundaries.

  • (c) Liver section of TME 200 showed mild congestion in central vein with less fatty changes, mild necrotic cells, with minimal inflammatory conditions, less infiltration of the leucocytes.

  • (d) Liver section of TME400 showed regeneration of hepatocyte around central vein with near normal liver architecture, prominent nucleus and nucleolus and possessing maximum hepatoprotective action.

  • (e) Liver section of rats SYL20 showed well brought out central vein, hepatic cell with well-preserved cytoplasm, prominent nucleus and nucleolus.

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DISCUSSION

The WHO survey confirmed that 70-80% of the world population rely on noncommercial medicine from herbal sources in primary health care units.23 The results of the present study clearly indicated hepatoprotective effects of the methanolic fruits extract of Tricoxanthes dioca against simvastatin induced liver toxicity in rats. Liver is a vital organ within the body, playing essential role in metabolic homeostasis and detoxification of variety of drugs and xenobiotic.24 Assessment of liver function can be performed by estimating the activities of serum SGOT, SGPT, ALP, total bilirubin, albumin and total proteins, which are originally present in higher concentrations in hepatocytes. During liver disease, these enzymes leak into the bloodstream in conformity with the extent of liver damage.25 Bilirubin is an index of liver function and its elevated level indicate damage to the liver and bile duct.26 Liver damage induced by simvastatin represents disturbances of metabolism of liver cells that leads to distinctive changes in the liver serum markers. The increased levels of hepatic serum markers like SGOT, SGPT, ALP, total biliru-bin and decreased in the albumin, was observed in simvastatin treated animals; this may bedue to the changes in the cell membrane permeability indicating severity of hepatocellular damage.2728 The animals treated with methanolic extract of T. dioica significantly reduced the levels of SGOT, SGPT, ALP, total bilirubin while increase total protein and albumin levels in dose dependent manner as compared with simvastatin as well as silymarin treated animals. Blood parameters dispense valuable data regarding health of animals.29

Administration of the plant extract resulted in significant increment inHb, PLT, WBC and % Lymphocytes counts at the dose of 4000 mg/kg. RBC and Hb are vital in transporting respiratory gases. The increment in levels of RBC and Hb implies that extract did not adversely afliect oxygen carrying capacity of the blood and the amount of oxygen delivered to tissues, therefore can be used in anaemia.30 The significant increment in the platelet count following administration of plant extract is the indication of stimulation of thrombopoietin creation as it has hemostatic capability of the blood and upholding blood clotting mechanism.31 Inflammatory response is characterized by the involvement of WBC. In this study, the incrementin level of WBC indicates the stimulation of immune system, in retort to toxic environment.32 Lymphocytes are the key cells of the immune system and elevation in its level indicates pathogenic attack and play the chief role in body defense mechanisms.33,34 Increased level of WBC’s and Lymphocytes suggesting that the TME extract challenge the immune system of the animals. Liver protective outcome of TME was further investigated by histopathological study. TME at diflierent dose levels ofliers liver protection, but 400 mg/kg is more efliective than all other inferior doses. As demonstrated in the present study, administration of simvastatin significantly elevated serum levels of hepatic enzymes, and that representing significant hepatocellular harm. Thus, our study confirmed the hepatoprotective potential of Trichosanthes dioica Roxb against simvastatin induced liver toxicity in rats. In fact, activity of Trichosanthes dioica Roxb is quite like silymarin, as reference hepato-protective agent.

ACKNOWLEDGEMENT

The authors are thankful to Shri Dev Murti, Chairman, Shri Ram Murti Smarak College of Engineering and Technology, Bareilly, Uttar Pradesh, India for providing facilities to carry out the research.

CONFLICT OF INTEREST

No conflict of interest associated with this research work.

ABBREVIATIONS

SGOT:

Serum glutamic oxaloacetic transaminase

SGPT:

Serum glutamic pyruvic transaminase

ALP:

Alkaline phosphatase

SEM:

structural equation modeling

SIM:

Simvastatin

SLY:

Silymarin.

GRAPHICAL ABSTRACT

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SUMMARY

  • Hepatoprotective potential methanolic fruit extract of TrichosanthesdioicaRoxb was estimated along with its effect on biochemical and haematological indices in hepatotoxicity induced by simvastatin in rats. The results demonstrate the alterations in elevated levels ofSGOT, SGPT, ALP, total bilirubin and restored the total protein and albumin level. The histological study showed regeneration of hepatocytes around central vein with near normal liver architecture revealing hepatoprotective effect of fruit extract.

ABOUT AUTHORS

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Ramesh Kumar Gupta: Asst. Professor, Sherwood College of Pharmacy, Barabanki.

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