|Ahead of print publication
In vitro hepatoprotective activity of a polyherbal formulation on HepG2 cell line
Bibhuti Nath Bhatt1, Amitabha Dey1, Satyajyoti Kanjilal1, HS Srikanth2, Rajarshi Biswas1, Pallabi Chakraborty1, Deepa Gandhi1, Avinash Narwaria1, Chandra Kant Katiyar1
1 Research and Development Centre, Healthcare Division, Emami Limited, Kolkata, West Bengal, India
2 Department of Biology, Research and Development, Natural Remedies Pvt. Ltd., Bengaluru, Karnataka, India
Research and Development Centre, Healthcare Division, Emami Limited, 13, BT Road, Kolkata - 700 056, West Bengal
Source of Support: None, Conflict of Interest: None
Background: In Ayurveda, use of multiple herbs in a single formulation is popular and polyherbal Ayurvedic formulations are widely used as growth promoters, gastrointestinal and hepatic regulators, hepatic tonics and so on. Despite the widespread use, there is a lack of scientific evidence on their efficacy and safety. Aim: This study was undertaken to validate the efficacy by evaluating the hepatoprotective activity of one such polyherbal blend (LIVT) on HepG2 cell line. Materials and Methods: Four doses of test formulation (62.5, 125, 250 and 500 μg/ml) were tested on D-galactosamine induced HepG2 cell toxicity model. MTT assay was performed to determine the dose range for the hepatoprotective study. Results: The test formulation exhibited significant (P < 0.05) cytoprotective activity with a maximum protection of 37% at dose 62.5 μg/ml, and the activity was comparable with that of the standard, silymarin. Conclusion: The results of the present study indicate that the polyherbal blend demonstrated a significant hepatoprotective activity and could be used as an active herbal alternative for the treatment of liver ailments.
Keywords: Antioxidant, cytotoxicity, flavonoids, hepatoprotection, HepG2, polyherbal
|How to cite this URL:|
Bhatt BN, Dey A, Kanjilal S, Srikanth H S, Biswas R, Chakraborty P, Gandhi D, Narwaria A, Katiyar CK. In vitro hepatoprotective activity of a polyherbal formulation on HepG2 cell line. Ancient Sci Life [Epub ahead of print] [cited 2019 Mar 22]. Available from: http://www.ancientscienceoflife.org/preprintarticle.asp?id=252118
| Introduction|| |
The liver is an important organ actively involved in regulating metabolic functions and involved in detoxification of a large number of toxicants., The liver is vulnerable to exogenous toxicants and organic substances. Exposure to exogenous toxicants such as drugs, alcohol and environmental pollutants can cause hepatic damage by metabolic activation of those compounds to form highly reactive substances.
D-galactosamine is a well-established hepatotoxin, it induces a diffuse type of liver injury closely resembling human viral hepatitis and acute self-limiting hepatitis with necrosis, inflammation and regeneration, resembling drug-induced liver diseases in humans. The toxicity of D-galactosamine is mainly related to the depletion of uridine pools that are associated with limited ribonucleic acid (RNA) and protein synthesis, thus altering hepatocellular function. In spite of modern advances in medical sciences, pharmaco-therapeutic treatment with synthetic drugs is not yet realized for liver protection. Therefore, alternative therapeutic strategies using polyherbal formulations for liver protection are of great interest. In Ayurveda, a number of herbs and herbo-mineral formulations are mentioned for the treatment of liver disorders. Based on the Ayurvedic wisdom, one such polyherbal formulation was prepared by using extracts of nine medicinally important Ayurvedic herbs known for their hepatoprotective activity. Information available on medicinal phytochemistry and pharmacology has revealed the hepatoprotective potential of each of these plants on chemical induced liver injury models., Though the herbal formulation is marketed in India since many years providing its therapeutic benefits in protecting liver health, only one systematic scientific report has been published till date revealing its antioxidant potential on different biomarkers. Hence in order to scientifically validate and authenticate the long term therapeutic claims of the herbal blend, the present study was undertaken to evaluate the effect of polyherbal formulation on D-Galactosamine induced HepG2 cell toxicity model.
| Materials and Methods|| |
The Ayurvedic polyherbal formulation is being manufactured and marketed by Emami Limited under the brand name of Zandu Livotrit Forte in tablet form. The polyherbal blend (Coded as LIVT) used in this study was obtained from Research and Development Centre, Emami Ltd, Kolkata.
The polyherbal LIVT tablet was prepared by using nine herbal extracts such as, Śophaghnī [Boerhavia diffusa] (40 mg), Guduci [Tinospora cordifolia] (40 mg), Bhṛṅgarāja [Eclipta alba] (20 mg), Kirātatikta [Andrographis paniculata] (20 mg), Tikta [Picrorhiza kurroa](20 mg), Bhūmyāmalakī [Phyllanthus amarus] (20 mg), Jantughna [Embelia ribes] (12 mg), Kāsanī [Cichorium intybus] (10 mg) and Rohitaka [Tecomella undulata] (10 mg).
HepG2 cell culture and dose determination
HepG2 cells were obtained from ATCC (ATCC® HB-8065™). Cells were grown in Minimum Essential Medium (MEM, Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were grown in a humidified atmosphere containing 5% CO2 at 37°C.
Prior to commencing testing, the solubility of the test formulation was assessed in MEM. 10 mg of test substance was weighed and suspended in 1 ml of MEM. The stock solution was then filtered using a 0.22 μm syringe filter and used immediately.
MTT assay was performed to determine the dose range for the Hepatoprotective study. Five doses of test formulation i.e. 62.5, 125, 250, 500 and maximum 1000 μg/ml were tested. Cytotoxicity was determined by a decrease in the optical density (570 nm) compared to the control.
D-Galactosamine induced HepG2 cell toxicity
HepG2 cells were adjusted to be 4 × 104 cells/well in MEM supplemented with 10% FBS, and 200 μl of the cell suspension were plated into 96 – well culture plate and incubated at 37°C in a humidified atmosphere containing 5% CO2 for 16 h. Post incubation, the cells were treated with different concentrations (62.5, 125, 250 and 500 μg/ml) of test substance and incubated for 2 h. Silymarin (12.5 μg/ml) was used as a reference standard. After incubation, the cells were treated with 20 mM of D-galactosamine for 24 h. Thereafter, the supernatant was discarded and the cells were washed with Dulbecco's phosphate buffer solution, and the fresh growth medium along with MTT (5 mg/ml) was added and incubated for 1 h to allow the formation of formazan crystals. Finally, the medium was removed, and the formazan crystals were dissolved using DMSO, the absorbance was measured at 570 nm.
Data obtained are represented as mean ± SD from three replicates per treatment group. Statistical analysis was done by using one-way analysis of variance using GraphPad Prism statistical software (GraphPad software Inc., La Jolla, California, USA). A P value less than 0.05 was considered as statistically significant.
| Results|| |
The results are summarized in the [Figure 1]. HepG2 cells treated with test formulation at concentration ranging from 62.5 – 1000 μg/ml for 24 h showed cell viability similar to untreated control cells [Figure 1]a. Thus, it is revealed that the polyherbal LIVT did not exhibit any significant cytotoxicity and was non-toxic at dose 1000 μg/ml.
|Figure 1: (a) Effect of Polyherbal LIVT on cell viability and (b) D-galactosamine induced toxicity in HepG2 cells at 24 h of treatment. Values are expressed as mean ± standard deviation (n = 3). Statistical analysis was done by using one-way analysis of variance (Dunnet's test). Differences with P < 0.05 were considered to be statistically significant|
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On treatment with the different test concentrations (62.5 – 500 μg/ml) for 24 h, LIVT exhibited significant (P < 0.05) protection against D-galactosamine induced cytotoxicity and the activity was comparable with that of the standard Silymarin. The maximum protection was observed to be 37% at dose 62.5 μg/ml [Figure 1]b.
| Discussion|| |
In this study, we investigated the hepatoprotective effects of test formulation (LIVT) in HepG2 cell line. The results demonstrated that pre-treatment with the test formulation exhibit significant protection against D-galactosamine induced cytotoxicity. The polyherbal blend probably prevented apoptosis of HepG2 cells through suppression of reactive oxygen species production., The present study is in agreement with the published report, of increased antioxidant enzyme system.
The polyherbal blend used in this study is being currently commercialized for the treatment of liver disorders. The present study revealed that the polyherbal blend (LIVT) to be safe for liver tissues and did not induce any cell death even after its highest tested concentration (1000 μg/ml) up to 24 hrs.
Phytoconstituents such as flavonoids, terpenoids and steroids, etc., have received considerable attention in recent years due to their diverse pharmacological properties including hepatoprotective and antioxidant activity. The presence of such phytoconstituents in the polyherbal active blend may be responsible for the observed hepatoprotective activity. The polyherbal blend has eight different extracts of Ayurvedic herbs, and the active metabolites present in them gives nutrition to the hepatocytes., In addition, the diverse group of phytochemicals may inhibit lipid peroxidation process, stabilize the hepatocellular membrane and improve the detoxification or excretion capacities. Collectively all these effects may reduce the allostatic load on the liver. However, there is a scope to further quantify the active constituents responsible for this activity.
| Conclusion|| |
Based on the results the polyherbal blend demonstrated a significant hepatoprotective activity against D-galactosamine induced cytotoxicity. Thus, it can be concluded that the polyherbal formulation is a potential alternative for the management of various liver ailments with a special emphasis on alcohol or drug induced hepatic damages.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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