Cassia Fistula Pod Extract Induces IFIT1, Antiviral Protein

Indian Journal of Traditional Knowledge Vol. 17(3), July 2018, pp. 474-479

Anthraquinone rich Cassia fistula pod extract induces IFIT1, antiviral protein

Damuka Naresh1, Dhammapal Bharne1, Paramananda Saikia2 & Vaibhav Vindal1,* 1Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, India; 2Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, USA E-mail: [email protected]

Received 16 November 2017, revised 27 April 2018

Cassia fistula L., an Indian Laburnum has therapeutics significance in human healthcare since ancient days. Its pod is known to rich sources of anthraquinones. Anthraquinone compounds have antiviral as well as immune modulatory properties. Human IFIT1 protein (Interferon induced protein with tetratricopeptide repeats 1) is a crucial component of innate immunity. The expression of IFIT1 protein normally remains silent but strongly induced after interferon’s (IFNs) treatment or viral infections. IFIT1 protein indeed has broad spectrum of antiviral activity by blocking different stages of viral replication, translation and assembly of new viral proteins. Modulation of IFIT1 protein expression may be useful in the treatment of viral diseases. The aim of present study is to identify Cassia fistula pod rich with anthraquinones extract (CF) to induce IFIT1 antiviral protein. Therefore, CF is extracted by soxhlet extraction process. HT1080 cells were used to study the expression of IFIT1 with CF extract. The IFIT1 protein induction in cells was assessed by Western blot and RT-PCR. The results showed that, CF extract significantly induced IFIT1 antiviral protein expression suggesting anthraquinones as potential agonistic compounds for inducing innate immune system to treat viral infections.

Keywords: Medicinal plant, Innate immunity, Interferon, Viral infections

IPC Int. Cl.8: A61K 36/00, G01 33/537, C07K 14/52, A61K, C12M, C12N, C12Q, C07C 50/18, C07C 225/34, C09B 1/16

Cassia fistula L., a semi-wild Indian labrum and laxatives12. In some studies, the ethanol extract of furthermore recognized as the Golden Shower has Cassia fistula pod showed inhibition against therapeutics significance in human healthcare since (Ranikhet disease virus (RDV), Vaccinia virus13. old circumstances1. Charaka and Sushruta Samhita Moreover, an aqueous extract of Cassia fistula pod has also reported the usages of Cassia fistula as a part inhibits dose-dependent anti-bovine rhinotracheitis of the treatment for different diseases in ancient virus activity14. The active principles of Cassia fistula India2. It has been broadly utilized as a part of various pod are anthraquinones, in which rhein, aloin, conventional prescriptions including Ayurveda, emodin, sennosides, fistulic acid, aolin, 3-formyl-1- Chinese and Unani for the treatment and avoidance of hydroxy-8-methoxyanthraquinone, chysophanol and diseases3. In Ayurvedic medicine, Cassia fistula is aloe-emodin are significant components15-19. These used against various diseases such as leucoderma, are in aglycone and glycoside frames. Many studies pruritus, and haematemesis4. The antifungal and have indicated that the anthraquinone compounds antibacterial effect of Cassia fistula has also been have antiviral and immune modulation properties, 20 reported5-7. Some parts of plants, for example, stem, such as modulation of humoral immunity , induces + leaf, root and pod contain distinctive chemical proliferation of CD4 T lymphocytes of human and 21 constituents, and such constituents have demonstrated secretion of IL10 , inactivation of enveloped 22 23 therapeutic potential in human healthcare8 such as viruses , antiretroviral activities , antiviral activity 24 25 antitumor9, antioxidant7, hypoglycemic10, inflammatory against influenza A virus , polio virus and hepatitis 26 diseases, rheumatism, ulcers, skin diseases and B virus replication . anorexia activities11. In the recent years, several groups have recognized Cassia fistula pods were broadly used as a novel genes downstream signaling of type I interferon 27 traditional medicine for antipyretic, diuretics and to inhibit infection of viruses . These genes are —————— named interferon-stimulated genes (ISGs) or *Corresponding author interferon-induced protein with tetratricopeptide NARESH et al.: ANTHRAQUINONE RICH CASSIA FISTULA POD EXTRACT INDUCES IFIT1, ANTIVIRAL PROTEIN 475

repeats (IFIT)28. This ISGs gene shows a broad Plant material spectrum of antiviral activity29. Among the ISGs, Cassia fistula pods were collected from University IFIT1 (ISG56) protein has gained enormous attention of Hyderabad campus, Telangana, India. The plant in antiviral activity. IFIT1 protein rapidly induced by was authenticated by Prof. Ajmeera Ragan, virus-infected cells with interferon-dependent or Department of Botany, Kakatiya University, India. interferon independent pathways30. IFIT1 protein has The voucher specimen (KUW: 1333) deposited the tetratricopeptide repeats (TPRs) with distinctive helix- herbarium of Kakatiya University, India. turn-helix motifs, which play important role in protein-protein and protein-RNA interactions. Preparation of anthraquinone crude extract Because of structural adaptability of IFIT1 protein Cassia fistula pod along with seed was powdered shows a diverse biological effect that inhibits different (100 g) and extracted with methanol (500 mL) for phases of the viral lifecycle including virus entry, 24 h using Soxhlet apparatus37. The extract was assembly, translation and viral replication31. A couple filtered and evaporated to dryness under reduced of examples are, inhibition of replication of Human pressure. To separate anthraquinone from the Papillomavirus (HPV) by binding to its helicase E1 methanol extract, method established by Stephen protein32, restraint of replication of the Japanese Chao Yung Su was used38. In this method, chloroform encephalitis (JEV) infection by binding to 5' capped and ethyl acetate solvents were used to separate 2'-O unmethylated RNA33, Inhibition of Hepatitis C hydroxyanthraquinones. The methanol crude extracts virus (HCV) translation initiation by binding to obtained above was macerated with ethyl acetate elongation initiation factor34, inhibition of viral solvent at 24 h. Ethyl acetate solvent liberated mRNAs translation which lacks N7- and 2′-O- anthraquinones from the extract. The maceration methyltransferases35, and also exhibits antiviral mixture was air dried and extracted with chloroform activity against influenza A virus and vesicular until free of anthraquinones. The anthraquinone stomatitis virus36. extraction monitored by Borntrager‘s reaction. The Thus, IFIT1 protein indeed has abroad spectrum of anthraquinone extracted chloroform layer was antiviral activity. Modulation of IFIT1 expression concentrated under reduced pressure using rotary may offer novel approaches for the treatment of viral evaporator. The extract was stored at 4 °C. Methanol diseases. Several medicinal plants could offer a rich and chloroform extracts were weighed and their asset for the medication disclosure against viral percentage yield was calculated. infections. Cassia fistula pod is rich sources of anthraquinone secondary metabolites. Many studies Identification of Anthraquinones have indicated that the anthraquinone secondary Borntrager‘s reaction was used to identify the metabolites have antiviral and immune modulation presence of anthraquinone in the extract. Briefly, 2M properties. In the current study, we aimed to test the HCl was added to the extract and boiled in a hot water ability of Cassia fistula pod extract rich with bath for 15 min, later the test tube containing boiled anthraquinones toinduce IFIT1 antiviral protein. extract was cooled and filtered. The filtrate mixture was extracted with chloroform, further separated Material and methods and mixed with dilute ammonia solution. The upper Chemicals layer becomes pink in color. The procedure for Dulbecco's modified Eagle's minimum essential quantitative analysis of total anthraquinone in the medium (DMEM), pen-strep solution and fetal bovine chloroform extract was measured by UV-visible serum (FBS), were obtained from Gibco-Thermo spectrophotometric as described earlier15. fisher scientific. Monoclonal antibodies of IFIT1 (Sc- 134948), β-actin (Sc-47778), and peroxidase- Thin layer chromatography (TLC) and LC-MS conjugated secondary antibody (Sc-2004) were analysis of anthraquinone extract bought from Santa Cruz Biotechnology, Inc. MTT, Chromatographic analysis of anthraquinone extract rhein obtained from Sigma–Aldrich. ECL plus was performed by TLC on a pre-coated aluminum detection kit procured from GE Healthcare plate of silica gel (12 x 6 cm) using petroleum ether : Biosciences. RT-PCR kit was obtained from ethyl acetate : glacial acetic acid (90:10:6) as the Affymetrix, USB. IFIT1 and β-actin primers were mobile phase. 10 % alcoholic potassium hydroxide synthesized from Integrated DNA Technologies. (KOH) solution was used to visualize the spots of 476 INDIAN J TRADIT KNOWLE, VOL. 17, NO 3, JULY 2018

anthraquinones. Anthraquinone show pink-red Nitrocellulose membrane blots were developed by spots. The hRf of anthraquinone components were ECL plus Western blot detection solutions. Fiji Image determined by comparing with the standard hRf value analysis software was used to determine the band of rhein38. Further, the extract was evaluated by LC- intensities. MS (Liquid chromatography-mass spectrometry). RT-PCR for IFIT1 mRNA Cell culture Total CF extract treated and untreated cellular HT1080 cells (Human fibrosarcoma cell line) were RNA was isolated using Trizol RNA isolation procured from National Center for Cell Science method. Total cDNA was prepared from the isolated (NCCS), Pune, India. HT1080 cells were maintained RNA sample using two steps RT-PCR kit. PCR in complete DMEM media added with FBS (10 %) experiment was performed to detect IFIT1 mRNA and and pen-strep solution (100 IU/mL), at 37 °C in an β-actin mRNA as an internal control. The volume of atmosphere of CO2 (5 % ). PCR reaction was 25 µL containing; 1.25 unit of TaqDNA polymerase, 0.2 mM of dNTPs, 1X reaction MTT assay to measure the cytotoxic dose of buffer, and 0.4 µmol of forward and reverse primers. extract The PCR amplification cycles were performed for The in vitro cytotoxicity assay was carried with the IFIT1 and βactin (initial denaturation at 95 °C for 3 CF extract to find out the MNTD (maximum-non min, denaturation 95 °C for 30 sec, annealing at toxic dose) of HT1080 cells. Briefly, these cells 56 °C for 1 min, and extension at 72 °C for 30 sec). cultured in 96-well cell culture plate. After 12 h The PCR primers of IFIT1 (Forward 5’ – different concentration of the CF extract prepared in TAGCCAACATGTCCTCACAGAC-3’; Reverse 5’ – complete media was added to the cultured cells. The TCTTCTACCACTGGTTTCATGC-3) and β -actin 96 microtitreplates were incubated at 37 °C with 5 % (Forward 5’ –GCTCCGGCATGTGCAA-3’; Reverse CO2 in an incubator for a period of 24 h and 48 h. 5’ –AGGATCTTCATGAGGTAGT-3’) was used in After the CF extract treatment, the media was this study. The PCR reaction samples were replaced with 100 µL new complete DMEM media. electrophoresed on agarose gel. The PCR amplified 20 µL of MTT (5 mg/mL) solution were added to bands visualized by EtBr (ethidium bromide) staining each microtitre-96 well, further incubated at 37 °C for under UV gel doc. Fiji Image analysis software was 4 h. After 4 h incubation media was removed used to determine the band intensities. carefully and added 150 µL of DMSO. MTT formazan product absorbance was determined by Statistical evaluation ELISA plate reader at 590 nm with the reference filter Simple linear regression analysis was performed to set to 620 nm. calculate the mean values of anthraquinones content in Cassia fistula pod by using GraphPad Prism Western blotting for IFIT1 protein software v. 7. The method of relative quantification To test the effect of CF extract to induce IFIT1 was used for analyzing the results of western blot and expression in HT1080, CF extract treated and PCR with respect to their blots. untreated cells were collected by centrifugation. The cell pellets were re-suspended in RIPA lysis buffer Results and incubated for 30 min. The protein concentration Extraction and identification of Anthraquinones was ascertained by BCA Protein assay kit. 50 µg of In order to identify CF inducing IFIT1 protein, we CF extract treated and untreated protein was have initially extracted Cassia fistula pod with electroblotted onto a nitrocellulose membrane, further MeOH. The yield of methanol crude extract from 100 separated on 10 % SDS–PAGE. The nitrocellulose g Cassia fistula pod was 15 g or 15 %. Further, membrane was blocked by 1X TBST containing 5 % MeOH extract was further subjected to ethyl acetate non-fat dry milk at RT for 1 h. The membrane was and chloroform extraction. The yield of chloroform incubated with IFIT1 antibody with 1:4000 dilutions extract from 15 g of methanol crude extract was 1 g or and for β-actin 1:5000 dilution at room temperature 6.6 %. Borntrager‘s reaction was used to monitor for 2 h. The later nitrocellulose membrane was anthraquinone extraction process. The extract of CF washed with TBST (3 times) followed by incubation gave a positive result for anthraquinones in Bontrager with secondary antibody at room temperature for 1 h. reaction and showed four spots on the TLC NARESH et al.: ANTHRAQUINONE RICH CASSIA FISTULA POD EXTRACT INDUCES IFIT1, ANTIVIRAL PROTEIN 477

plate. The UV-visible spectrophotometric method Fig. 3 shows that CF extract induces IFIT mRNA used to calculate the total content of anthraquinones expression significantly as similar to that of Poly in CF extract. The relationship was taken within the (I:C). While the extract had no effect on the concentration range of 10- 50 µg/mL of rhein expression of β-actin. standard with correlation coefficient r2 = 0.9932. The representative linear equation for rhein was y = Discussion 0.0434x - 0.0025. The total content of anthraquinones In the present study, we tested Cassia fistula pod calculated as compared to rhein was 36 µg/mL. In for induction of a potential antiviral protein, IFIT1. LC-MS analysis, identified anthraquinones are rhein, Cassia fistula pod is known to have high aloe-emodin, chrysophanol, emodin, physcion and anthraquinone compounds which are known to have cassialoin. In LC-MS analysis indicating RT anti-inflammatory as well as immune modulatory (retention time) of the rheinis 12.8 and area properties but a scientific evaluation was not carried percentage 15, alo-emodin RT is 9.9 and area out yet. Research work from many laboratories shown percentage 5.42, chrysophanol RT is 9.1 and area that IFIT has a direct antiviral property where it percentage 50.16, cassialoin RT is 6.2 and area interferes with different stages of the viral replication percentage 2.9, physcion RT is 10.4 and area percentage 8.32, emodin RT is 12.6 and area percentage 1.07..

Maximum nontoxic dose (MNTD) of extracts The maximum non-toxic dose of extracts in HT1080 cells was determined prior to IFIT1 expression analysis. MTT assay showed that treatment of HT1080 cells with CF extract at different concentrations (25 µg/mL to 200 µg/mL) for 24 and 48 h. In 24 h treatment, CF extract did not show significant cytotoxicity (Fig. 1). The cytotoxicity results indicate that the concentrations used for IFIT1 expression analysis could not be attributable to non- Fig. 1 — MTT Assay with CF extract in HT1080 cells. MTT Assay representing the toxicity plot of CF extractin HT1080 specific cytotoxicity. Cells: HT1080 Cells were mixed with different concentration (25- 200µg/mL) of extract for 24 and 48 h. The experiment was Effects of CF extract on IFIT1 protein expression prepared in triplicates. The data represent the percentage of Western blot analysis was carried out to check the viability cells (means ± SD). result of CF extract on HT1080 cells for IFIT1 protein expression. HT1080 cells were mixed with various concentrations (0, 10, 40, 80 µg/mL) of CF extract. Interestingly, CF extract significantly expressed IFIT1 in HT1080 cells in a concentration of 40 and 80 µg/mL (Fig. 2).

Effect of CF extract on IFIT1 mRNA expression

To examine the effects of CF extract on the expression of IFIT1 mRNA level, the RT-PCR was Fig. 2 — Effects of CF extract on IFIT protein expressions in performed. Poly I:C used as positive control for HT1080 cells.HT1080 cells were mixed with CF extract with various concentrations (10, 40 and 80 µg/mL) for 18 h. Poly I: C expression of IFIT1 mRNA. HT1080 Cells were (50 µg/mL) is added to culture media as a positive control for treated with poly I:C (50 µg/mL) and CF extract (80 induction of IFIT1 protein. Then cells were harvested, and µg/mL) for 10 h to check induction of IFIT1 mRNA. Western blotting was performed with IFIT1 antibody to detect After 10 h incubation, total cellular RNA was endogenous IFIT1. β-actin served as an internal control. The extracted from treated, untreated HT1080 Cells; results demonstrated CF extract (panel B) induces IFIT1 protein at a concentration of 40 and 80 µg/mL. The expression level of prepared cDNA was amplified by PCR using IFIT panel A blot was quantified by ImageJ software and the results are primers, β-actin mRNA used as an internal control. presented in B. 478 INDIAN J TRADIT KNOWLE, VOL. 17, NO 3, JULY 2018

Conclusion Cassia fistula broadly used as a tradition for antipyretic and antiviral activity. The mode of action of antiviral activity has been studied through inactivating viruses. The current study revealed that the Cassia fistula pod rich with anthraquinones extract (CF) significantly induced IFIT1 antiviral protein. The IFIT1 induction of the CF extract as showed by the Western blot and RT-PCR. Therefore, the present study confirms CF extract to have IFIT1 protein induction might give a very important insight into the development of anti-viral therapeutic from the Cassia fistula anthraquinones.

Acknowledgment Fig. 3 — Effects of CF extract on IFIT1 mRNA expression in This work was supported in part by UGC-UPE-II. HT1080 cells: HT1080 cells were mixed with CF extract DN is supported by DBT fellowship. The authors (80 µg/mL) and Poly I: C (50 µg/mL) as a positive control for gratefully acknowledge the metabolomics facility of 10h. Total RNA was extracted and cDNA was synthesized using the University of Hyderabad for facilitating LC-MS IFIT1and β-actin primer. The PCR reaction was analyzed by 1% agarose gel electrophoresis. 394 bp of IFIT-specific band was analysis. detected in PolyI:C (lane-2), and CF (lane-4). β-actin (500bp) was utilized as a positive loading control. The mRNA expression References levels were quantified by ImageJ software; the results are 1 Chatterjee A & Pakrashi SC, The treatise on Indian indicated in B. medicinal plants, Vol 1, (New Delhi: Publications and Information Directorate, CSIR),1991, 172. process. The expression of IFIT is not detected in 2 Nadkarni KM, The Indian Materia Medica, The Indian Materia Medica,(Popular Book Depot, Bombay & resting cells but induces > 200 folds upon virus Dhootapapeshwar Limited, Panvel), 1927, 962. infection or interferon treatment. So induction of IFIT 3 Rahmani & Arshad H, Cassia fistula L.: Potential candidate protein is a positive feedback towards antiviral state in the health management, Pharmacogn Res,7 (3) (2015) for cells. In this notion, we started our work in 217. 4 Rizvi MMA, Hassadi IMG & Younis SB, Bioefficacies of evaluating CF extract for IFIT1 gene induction Cassia fistula: an Indian labrum, Afr J Pharm Pharmacol, through PCR as well as western immunoblotting. The 3 (6) (2009) 287-292. CF extract induces the expression of IFITI in a dose- 5 Kumar VP, Chauhan NS, Padh H & Rajani M, Search dependent manner at the protein level using specific for antibacterial and antifungal agents from selected IFIT1 antibody. The protein expression data was Indian medicinal plants, J Ethnopharmacol,107 (2) (2006) 182-188. further supported by mRNA expression data where 6 Rajeswari R, Thejomoorthy P, Mathuram L & Narayana CF extract induces IFIT1 expression was comparable Raju K, Anti-inflammatory activity of Cassia fistula L., bark to poly I:C induction. Further, we confirmed the extracts in sub-acute models of inflammation in rats, Tamil presence of anthraquinone compounds in the CF Nadu J Vet Anim Sci, 2 (5) (2006) 193-199. 7 Luximon RA, Bahorun T, Soobrattee MA & Aruoma OI, extract through thin layer chromatography and LC- Antioxidant activities of phenolic, proanthocyanidin, and MS analysis. Anthraquinone compounds have been flavonoid components in extracts of Cassia fistula, J Agric shown to have direct antiviral properties against many Food Chem, 50 (18) (2002) 5042-5047. viruses; for example, human immunodeficiency 8 Dave H & Ledwani L, A review on anthraquinones isolated virus39. Where anthraquinones inhibit viral reverse from Cassia species and their applications, Indian J Nat Prod 40 Resour, 3 (3) (2012) 291-319. transcriptase activity and human cytomegalovirus . 9 Gupta M, Mazumder U, Rath N & Mukhopadhyay D, Overall, our present study confirms CF extract to have Antitumor activity of methanolic extract of Cassia fistula L. IFIT1 induction ability which predicts and confirms seed against Ehrlich ascites carcinoma, J Ethnopharmacol, ethnically known medicinal properties of CF as 72 (1-2) (2000) 151-156. 10 Bhakta T, Mukherjee PK, Saha K, Pal M, & Saha BP, antiviral. Further studies are needed to delineate the Hypoglycemic activity of Cassia fistula Linn.(Leguminosae) specific actions of anthraquinone found in CF to leaf (Methanol extract) in alloxan-induced diabetic rats, inhibit a specific viral replication/multiplication. J Ethnobot, 9 (1997) 35-38. NARESH et al.: ANTHRAQUINONE RICH CASSIA FISTULA POD EXTRACT INDUCES IFIT1, ANTIVIRAL PROTEIN 479

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