Anti-HSV-2 Activity of Terminalia Chebula Retz Extract and Its
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Kesharwani et al. BMC Complementary and Alternative Medicine (2017) 17:110 DOI 10.1186/s12906-017-1620-8 RESEARCH ARTICLE Open Access Anti-HSV-2 activity of Terminalia chebula Retz extract and its constituents, chebulagic and chebulinic acids Ajay Kesharwani1,3, Suja Kizhiyedath Polachira2, Reshmi Nair2, Aakanksha Agarwal1, Nripendra Nath Mishra1 and Satish Kumar Gupta1* Abstract Background: Development of new and effective therapeutics for sexually transmitted herpes simplex virus-2 (HSV- 2) infection is important from public health perspective. With an aim to identify natural products from medicinal plants, in the present study, the potential of Terminalia chebula Retz was investigated for its activity against HSV-2. Methods: Fruits of Terminalia chebula Retz were used to prepare 50% ethanolic extract. In addition, chebulagic acid and chebulinic acid both purified from T. chebula were also used. The extract as well as purified compounds were first used to determine their in vitro cytotoxicity on Vero cells by MTT assay. T. chebula extract, chebulagic acid, chebulinic acid along with acyclovir were subsequently assessed for direct anti-viral activity, and their ability to inhibit attachment and penetration of HSV-2 to the Vero cells. In addition, their anti-HSV-2 activity was also determined by in vitro post-infection plaque reduction assay. Results: Cytotoxicity assay using Vero cells revealed CC50 = 409.71 ± 47.70 μg/ml for the extract whereas chebulagic acid and chebulinic acid showed more than 95% cell viability up to 200 μg/ml. The extract from T. chebula (IC50 = 0.01 ± 0.0002 μg/ml), chebulagic (IC50 = 1.41 ± 0.51 μg/ml) and chebulinic acids (IC50 = 0.06 ± 0.002 μg/ml) showed dose dependent potent in vitro direct anti-viral activity against HSV-2. These also effectively prevented the attachment as well as penetration of the HSV-2 to Vero cells. In comparison, acyclovir showed poor direct anti-viral activity and failed to significantly (p > 0.05) prevent the attachment as well as penetration of HSV-2 to Vero cells when tested upto 50 μg/ml. However, in post-infection plaque reduction assay, T. chebula extract, chebulagic and chebulinic acids showed IC50 values of 50.06 ± 6.12, 31.84 ± 2.64, and 8.69 ± 2.09 μg/ml, respectively, which were much lower than acyclovir (71.80 ± 19.95 ng/ml). Conclusions: The results presented herein suggest that T. chebula extract, chebulagic and chebulinic acids have higher direct antiviral activity against HSV-2 and efficacy to inhibit virus attachment and penetration to the host cells as compared to acyclovir. However, acyclovir is more potent to inhibit post-infection virus replication. Hence, T. chebula may be a useful candidate for developing alternative therapy for prevention of sexually transmitted HSV-2 infection. Keywords: Anti-HSV-2 activity, Attachment assay, Penetration assay, Post-infection plaque reduction assay, Terminalia chebula * Correspondence: [email protected] 1Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kesharwani et al. BMC Complementary and Alternative Medicine (2017) 17:110 Page 2 of 11 Background Poojappura, Kerala and the identity was confirmed at Ayur- Herpes simplex virus-2 (HSV-2) is a double stranded veda Research Institute, Trivandrum, Kerala. The voucher DNA virus belonging to the family Herpesviridae.It specimen (HLL/04/2013) has been kept at Natural Prod- causes genital herpes, contributing to the risk of HIV in- ucts Division of HLL Lifecare Limited, Trivandrum, Kerala, fection through sexual route [1, 2]. HSV-2 is sexually India. Air and shade dried fruits were grinded and strained transmitted virus and infect 500 million people worldwide through a 30 mesh (0.5 mm). The finely grinded powder and 20–23 million new infections are reported annually (24 g) was subjected to pressurized sequential extraction [3], http://www.who.int/mediacentre/factsheets/fs400/en/ using Accelerated Solvent Extractor (ASE 150, Dionex Inc., #hsv2. Since virus is an intracellular parasite in neural Sunnyvale, CA, USA) essentially as described previously ganglia, it is difficult to completely eliminate it [1, 4]. Nu- [22, 23]. In brief, the material was mixed with diatomaceous cleoside derivatives such as acyclovir, valaciclovir, famci- earth at 4:1 ratio. The mixture was placed into a sample cell clovir and cidafovir have been widely used for the (100 ml) and loaded onto the ASE 150 system. Extraction treatment of HSV infection [5]. In addition to high cost, was performed using 50% ethanol under pressure treatment with acyclovir and other related drugs is also as- (1500 psi) at 60 °C with a flush volume of 60% using 2 static sociated with the emergence of drug resistant strains [6], cycles. The solvent was then evaporated in a rotary evapor- which is a major hurdle in the immunocompromised indi- ator (Büchi Labortechnik AG, 9230 Flawil, Switzerland). viduals, co-infected with other opportunistic Sexually The dried extract was weighed andusedforfurtherstudies. Transmitted Infections (STIs) such as HIV-1 [7, 8]. Highly pure commercial chebulagic acid and chebuli- Natural products from medicinal plants have been an nic acid, both purified from T. chebula, were obtained important source of new biologically effective compounds from MP Biomedicals, Ohio, Solon, USA. These com- exhibiting different modes of action against viral infection pounds were used as reference standard to identify the [9–11]. Terminalia chebula Retz. (T. chebula) is an ever- corresponding peaks in 50% ethanolic extract prepared green flowering tree of the Combretaceae family and ex- from fruits of T. chebula as well as to determine their tensively used in traditional medicine. Pharmacological anti-HSV-2 activity in various assays as described below. studies revealed the inhibitory activity of T. chebula on Both the compounds were dissolved in absolute ethanol viral infections, such as human immunodeficiency virus (10 mg/ml) and further dilutions were made in the culture type 1 (HIV-1), herpes simplex virus-1 (HSV-1), cyto- medium before use. Acyclovir (commercial name acyclo- megalovirus (CMV) and influenza [12–16]. In addition, guanosin) was purchased from Sigma-Aldrich Inc., St. the extract from T. chebula also has anti-bacterial, anti- Louis, MO, USA. It was dissolved in dimethyl sulphoxide fungal, anti-carcinogenic, anti-oxidant, anti-diabetic and (DMSO) to make stock solution of 7 mg/ml and further anti-aging activities [17]. T. chebula has various phytocon- diluted in culture medium to prepare working concentra- stituents like tannins, flavonoids, sterols, amino acids, tion (s) prior to use. The final concentration of DMSO fructose, resins and fixed oil etc. However, tannins such as was < 0.1% in various assays where acyclovir was used. chebulinic acid, chebulagic acid, gallic acid, chebulic acid, corilagin and ellagic acid may constitute 30% of various High performance liquid chromatography analysis phytochemicals [18, 19]. Though there are several studies The Reverse Phase High Performance Liquid Chroma- on the anti-HSV-1 activity of T. chebula extract and its tography (HPLC; LC Agilent, Agilent Technologies, constituents namely chebulagic acid [15, 20, 21], but their Boblingen, Germany) of the plant extract was performed activity against HSV-2 needs to be addressed. using a Reverse phase XTerra RP 18 column (4.6 × Keeping in view of the above, the 50% ethanolic extract 250 mm, 5 μm; Waters Corporation, Milford, USA) and prepared from the fruits of T. chebula was evaluated for pure compounds (chebulagic and chebulinic acids) were direct anti-viral activity and its impact on the attachment used as reference standards. HPLC grade solvents were and penetration of the HSV-2 to the Vero cells. Chebula- purchased from Merck, Mumbai, India. The solvent sys- gic and chebulinic acids from T. chebula were also tested tem used was 0.01% orthophosphoric acid in water : in these assays and their activity compared with well acetonitrile (80 : 20) with a flow rate of 1 ml/min. The known drug, acyclovir. Additionally, the extract and pure peaks were detected at 272 nm. The extract was pre- compounds were also evaluated for in vitro anti-HSV-2 pared at a concentration of 5 mg/ml and the standards activity in post-infection plaque reduction assay. at 1 mg/ml in the mobile phase solvent system and 20 μl of each was injected for analysis. The data was processed Methods using open lab software (Agilent Technologies). Plant material, preparation of 50% ethanolic extract, and compounds Cells and viruses The fruits of Terminalia chebula were collected from the African green monkey kidney cells (Vero cells) were ob- medicinal plants garden of Ayurveda Research Institute, tained from National Centre of Cell Science, Pune, India Kesharwani et al. BMC Complementary and Alternative Medicine (2017) 17:110 Page 3 of 11 and maintained in Dulbecco’s modified Eagle’s medium supernatant was aspirated and infected cells were (DMEM; Sigma-Aldrich Inc.) supplemented with 10% washed twice with serum free DMEM and overlaid with fetal bovine serum (FBS; Biological Industries, Kibbutz 1% low melting point (LMP) agarose overlay medium. beit HaemeK, Israel) and an antibiotic-antimycotic cock- After 48 h incubation, cells were fixed with 10% formal- tail [Penicillin (100 units/ml), Streptomycin (100 μg/ml) dehyde (in 50 mM PBS), stained with 0.2% crystal violet and Amphotericin B (250 ng/ml); Pen-Strep-Ampho sol, and number of plaques counted.