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International Journal of Pharmacy & Life Sciences Research Article [Dharsana et al., 5(12): Dec., 2014:4049-4054] CODEN (USA): IJPLCP ISSN: 0976-7126 INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES (Int. J. of Pharm. Life Sci.) Antioxidant and Hepatoprotective activity of Anaphyllum wightii Schott. J.N.Dharsana1*, Sr. Molly Mathew2, P.N. Baby1, Sajith Kumar1 and Diljit S. Kuttoor1 1, Department of Pharmaceutical Chemistry, Academy of Pharmaceutical Sciences, Pariyaram Medical College, Kannur, Kerala - India 2, Malik Deenar College of Pharmacy, Kasargode, Kerala - India Abstract Herbal medicines have traditionally been used worldwide for the prevention and treatmentof liver disease with fewer adverse effects. The tubersof Anaphyllum wightiiplant were chosen andstudied for their antioxidant activity in vitro and hepatoprotective activity in vivo. The antioxidant activity of the chloroform extract was examined in vitro using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) freeradical scavenging assay and super oxide scavenging assay. The extract had significant dose-dependent antioxidant activity in all in vitro experiments. The percentage of free radical scavenging by DPPH and super oxide scavenging assay values of Anaphyllum wightiiand ascorbic acid (standard) were found to be 49.02±4.11, 60.20±3.43 and 72.78±3.97 respectively. Hepatoprotective activity of the plant extract was evaluated in a rat model of carbon tetrachloride (CCl4)induced liver damage. CCl4 significantly altered serum marker enzymes, total bilirubin, total protein, and liver weight. The extract caused these values to return to normal in rats with CCl4induced liver damage. This indicated the hepatoprotective potential ofAnaphyllum wightiiand was comparable to use of the standard drug silymarin. Thus, the present study revealed that Anaphyllum wightii have significant antioxidant and hepatoprotective activity. Key-Words: Anaphyllum wightii, antioxidant, hepatoprotective,DPPH Introduction Plant dugs have been a major source for treatment of Oxygenradicals have crucial action such as signal diseases for a long time. They have been used in the transduction,gene transcription and regulation of traditional medicine on the basis of experiences and synthesis of cyclicGuanosine monophosphate (cGMP) practice. With the advent of modern systems of in cells3,4. medicine need has been felt to investigate the active Nitric oxide (NO) is a common signaling moleculeand constituents present in these plants. Various molecules participates in virtually every one of the body'scellular have been isolated, characterized and tested for their and organ functions. Optimum amountsof NO related pharmacological activities. produced by endothelial cells are essential Reactive oxygen species (ROS) and reactive forregulating the relaxation and proliferation of nitrogenspecies (RNS) are produced by normal vascularsmooth muscle cells, leukocyte adhesion, physiologicprocesses and fulfill important functions in plateletaggregation, angiogenesis, thrombosis, vascular the body atminute or moderate concentrations. ROS tone andhemodynamics5. In addition, NO produced by and RNS arenecessary for the maturation process of neurons serves as a neurotransmitter6. cellular structuresand can act as weapons for the host's However, the generation of even slightly largeramounts defence system.The importance of ROS production by of these essential compounds during metabolismor in the immunesystem is clearly evident in patients with response to environmental pollutants, granulomatousdisease. These patients have a defective radiation,chemicals, toxins, deep fried and spicy foods, membrane-boundnicotinamide adenine dinucleotide andphysical stress can cause massive physiologic phosphate (NADPH)oxidase system that precludes problemsby oxidation of bio-molecules (protein, amino them from producingthe superoxide anion radical acids,lipid, and DNA). Free radical presumably plays a (O2ˉ), thereby resultingin multiple and persistent majorrole in all pathologies. Free radicals are believed infections1,2. to beresponsible for more than one hundred conditions likecancer, diabetes, atherosclerosis, arthritis, * Corresponding Author neuropathy, nephropathy, retinopathy, aging, E.Mail: [email protected] compromised immunity, and cardiovascular diseases7,8,9,10. © Sakun Publishing House (SPH): IJPLS 4049 Research Article [Dharsana et al., 5(12): Dec., 2014:4049-4054] CODEN (USA): IJPLCP ISSN: 0976-7126 The liver has a central role in transforming andclearing similar in appearance to those in the genus chemicals and is closely related to thegastrointestinal Anaphyllopsis.GenusAnaphyllum wightii is listed as an tract, which makes it susceptible to drugtoxicity, endemic and threatened genus of South India19. The xenobiotic, and oxidative stress. Dysfunctionof this species of the genus Anaphyllum are found in organ results in impairment of energy metabolismand marshes.They are characterized by pinnate leaves and intracellular oxidant stress with excessive formationof twisted spathe for the spadix.Anaphyllum is a genus of ROS. CYP2E1 is a cytochrome P450 flowering plants in the Araceae family. The tribal isoenzymeproduced by the liver that also facilitates communities (Kani Tribes, Malasars,Kadars, Pulayars, oxidativestress and cell injury11, 12.Although Kupffer Madhuvarsetc) use these plants as food and as an cellsand recruited neutrophils in the liver are part of antidote to snake bite.Leaves of Anaphyllum beddomei thehost-defence system, these inflammatory cells form a part of tribal diet.Arun et al. reported the use of initiateliver injury under certain circumstances,such as the corms of Anaphyllum wightii (Keerikizhangu), as when excess free radicals are present 13,14,15,16. an antidote to snake bite along with some medicinal Although the body has mechanisms to defend plants20. againstthe damaging properties of ROS17,18,its capacity Plant material tocontrolROS can be overwhelmed, causing Plant tubers were collected from Wynad hills, Wynad irreversibleoxidative damage and various conditions district, Kerala, India. The taxonomical identification that threaten theliver and other parts of the body. of the plant was done by Dr. N. Sasidharan, Scientist-F, Several synthetic andsemi-synthetic chemicals are Programme co-ordinator, FE& BC division, Kerala often used to protect theliver from the detrimental Forest Research Institute, Peachi, and Trissur. The effects of free radicals but they are not sufficiently voucher specimen was preserved in Academy of effective and cause adverse reactions.Modern medicine Pharmaceutical sciences,Pariyaram Medical College, includes many important bioactivemolecules with Kannur, and Kerala. antioxidant and hepatoprotective properties that were Preparation of plant extract derived from plant. Hence, recent research has The collected plant tubers was dried at room hastened to identify notablehepatoprotective agents temperature, pulverized by a mechanical grinder, from plant products that willreduce the harmful effects sieved through 40 mesh. About 200g of powdered of and problems associatedwith free radicals while materials were extracted with chloroform. The causing minimal adversereactions. extracted solution was filtered using a clean cloth and As part of ongoing research, the current study the filter paper. The extract wasconcentrated first in a examined the chloroform extract ofAnaphyllum rotary vacuum evaporator and then in a water bath. The wightiifor its use as a hepatoprotective agent to reduce extracted residue was weighed and the percentyield thedamage caused by ROS and RNS.No reports have was 9.21% w/w. The extract was then frozen prior to described the plant's hepatoprotectiveactivity. Thus, the examination of its potentialantioxidant and present study sought to investigate the chloroform hepatoprotective properties. extract of Anaphyllum wightiifor its Chemicals hepatoprotectiveactivity using a rat model of DPPH(2, 2-diphenyl-1-picrylhydrazyl), ascorbic acid, CCl4induced liver damageand different in vitro carbon tetra chloride,phosphate buffer saline (pH antioxidation experiments. 6.3),EDTA, nitro blue tetrazolium (NBT), riboflavin Material and Methods andsilymarin were purchased from Merck Pharmaceuticals. Experimental animals 21,22,23 Healthy albino rats weighing 150-200g of either sex DPPH Free Radical Scavenging Assay were used. The experimental protocol was approved by 1 ml of the test compound in various concentrations the Institutional Animal Ethics Committee (50, 100,150, 200 & 250 µg/ml) was added to 1ml of (APSC/CPCSEA/-04/IAEC/2013) and animals were 0.1 mm solution of DPPH in methanol. After 30 maintained under standard conditions in the animal minutes, absorbance was measured at 517 nm, using a house approved by Committee for the Purpose of spectrophotometer (SHIMADZU,UV 1800).A 0.1 mM Control and Supervision on Experiments on Animals solution of DPPH in methanol was used as blank, (CPCSEA). whereas ascorbic acid was used as a reference standard. Collection and Authentication All tests were performed in triplicate. Percent Genus –Anaphyllum beddomei Engl. and Anaphyllum inhibition was calculated using equation, wightii Schott were reported from the high ranges of Percent inhibition = [(Control ×Test)/Control]×100 Western Ghats.The two species in this genus are © Sakun Publishing House (SPH): IJPLS 4050 Research Article [Dharsana et al., 5(12): Dec., 2014:4049-4054] CODEN (USA): IJPLCP ISSN: 0976-7126 Superoxide Scavenging Assay21,22,23 The absorbance of the extracts increased with the rise 1ml of the test compound in various
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