Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: An in vivo and in vitro evaluation Hadi Ghaffari a , V. Venkataramana b , S.Chandra Nayaka a , Behrouz Jalali Ghassam a , Nataraju Angaswamy c , Shailashree Shekar a , K.K. Sampath Kumara a , H.S. Prakash a,n Q1 a Department of Studies in Biotechnology, University of Mysore, Mysore, Karnataka, India b Department of Food Microbiology, Defense Food Research Laboratory, Mysore, Karnataka, India c Department of Surgery, Washington University School of Medicine, Saint Louis, MO, USA article info Article history: Received 25 February 2013 Received in revised form 24 June 2013 Accepted 27 July 2013 Keywords: Orthosiphon diffusus CCl 4 -induced hepatotoxicity H 2 O 2 -induced oxidative stress Silymarin Hepatoprotective abstract Ethnopharmacological relevance: Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities. Aim of the study: To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models. Materials and methods: Neutralization of CCl 4 -induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H 2 O 2 -induced toxicity by MAF using MTT, Comet assay and up- regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells. Results: Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p o0.05) and ALP (1.6-fold, p o0.05). Similarly, multiple dose MAF administration demon- strated significantly enhanced levels (p o0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl 4 -induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H 2 O 2 -induced oxidative stress, genotoxicity and significantly enhanced ( 6-fold, p o0.01) expression of genes for antioxidant enzymes. Conclusions: Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl 4 -induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H 2 O 2 -induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents. & 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Oxidative stress is attributed to either an increase in reactive oxygen species (ROS) generation and/or a decrease in the anti- oxidant defense mechanisms which lead to many degenerative diseases including a variety of hepatopathies (Hensley et al., 2000). However, the overproduction of ROS and therefore associated oxidative stress can be attributed to exposure to xenobiotics such as carbon tetrachloride (CCl 4 ), acetaminophen, polychlorinated biphenyls, polychlorinated dibenzodioxins etc. (Fenichel, 1996; Goeptar et al., 1995; Jaeschke et al., 2012; Jaiprakash et al., 2012). These xenobiotics, introduced into the water mainly as industrial wastes from their primary use in manufacture of chlorofluorocar- bons, dry cleaning fluids, fire extinguishing agents etc. produce hepatotoxicity in human beings and animals (Borzelleca et al., 1990). CCl 4 —induced hepatotoxicity in a rodent model closely resembles humans and therefore is commonly used for the screening of hepatoprotective activities of drugs (Deng et al., 2012; Khan et al., 2012). The hepatotoxic effects of CCl 4 are through generation of highly reactive trichloromethylperoxyl radicals (  OOCCl 3 and  CCl 3 ), which in the presence of oxygen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.07.034 Abbreviations: ALP, Alkaline phosphatase; CCl 4 , Carbon tetrachloride; CAT, Catalase; GPx, Glutathione peroxidase; GSH, Glutathione; GST, Glutathione-s- transferase; HepG2, Human hepatocellular carcinoma cells; LDH, Lactate dehy- drogenase; MAF, Methanol active fraction; MD, Multiple dose; Orthosiphon diffuses, Orthosiphon diffuses; SD, Single dose; SOD, Superoxide dismutase n Corresponding author. Tel.: +91 821 2419877, +91 9886640778; fax: +91 821 2410080. E-mail address: moonnayak@gmail.com (H.S. Prakash). Please cite this article as: Ghaffari, H., et al., Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: An in vivo and in.... Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.07.034i Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎