Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: an in vivo and in vitro evaluation.
BACKGROUND
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.


OBJECTIVE
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.


METHODS
Neutralization of CCl4-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 H2O2-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<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl4-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H2O2-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes.


CONCLUSIONS
Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl4-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H2O2-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.
Department of Studies in Biotechnology, University of Mysore, Mysore, Karnataka, India.
Hadi
H
Ghaffari
M
M
Venkataramana
S Chandra
SC
Nayaka
Behrouz Jalali
BJ
Ghassam
Nataraju
N
Angaswamy
Shailashree
S
Shekar
K K
KK
Sampath Kumara
H S
HS
Prakash
D000818:Animals; D000975:Antioxidants / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use*; D002251:Carbon Tetrachloride / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D004195:Disease Models, Animal; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008297:Male; D000432:Methanol / Q000737:chemistry*; D031342:Orthosiphon / Q000737:chemistry*; D018384:Oxidative Stress / Q000187:drug effects; D035261:Plant Components, Aerial / Q000737:chemistry; D010936:Plant Extracts / Q000302:isolation & purification / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar
D000975:Antioxidants; D010936:Plant Extracts; D002251:Carbon Tetrachloride; D000432:Methanol
ALP; Alkaline phosphatase; CAT; CCl(4); CCl(4)-induced hepatotoxicity; Carbon tetrachloride; Catalase; GPx; GSH; GST; Glutathione; Glutathione peroxidase; Glutathione-s-transferase; H(2)O(2)-induced oxidative stress; HepG2; Hepatoprotective; Human hepatocellular carcinoma cells; LDH; Lactate dehydrogenase; MAF; MD; Methanol active fraction; Multiple dose; Orthosiphon diffuses; Orthosiphon diffusus; SD; SOD; Silymarin; Single dose; Superoxide dismutase