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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)The Protective role of Luffa acutangula fruit MethanolicFraction against t-BHP Induced Oxidative damage in HumanErythrocytesBoreddy Purushotham Reddy*, S. Venkata Mohan and P. N. SarmaBioengineering and Environmental Centre, Indian Institute of ChemicalTechnology Hyderabad 500 607, India*For correspondence - poory_boreddy@yahoo.com1073AbstractThe present study was designed to examinethe ability of partially purified fractions of Luffaacutangula (Cucurbitaceae) fruit, to attenuatet-BHP induced oxidative damage in humanerythrocyte as an in vitro model. Initially, weinvestigated the antioxidant property of thehexane, methanolic and aqueous extracts (FHE,FME and FAE, respectively) by DPPH freeradical method and found that fruit methanolicextract was showing higher antioxidant activity(71.4±4.46% at 1 mg/ml) compared to otherextracts (FHE&FAE were 13.93±1.3 and51.84±3.76%, respectively). Hence, this extractwas further partially purified chromatographicallyand out of these fractions (F1, F2, F3, F4, F2-1,F2-2, F2 -3 and F2-4) F2-3 showed significantantioxidant activity (73.96±6.4% at 25 µg/ml). Thisfraction was further tested for its effect on lipidperoxidation, superoxide dismutase, catalase andglutathione in t-butyl hydroperoxide (t-BHP)treated-erythrocytes. Pretreatment with fractionF2-3 significantly inhibited lipid peroxidation in adose and time dependent manner compared tocontrol (40.6±3.2, 27.9±2.4 and 75.5±5.2 nmolMDA/gHb, at 30, 90 min and control,respectively). Catalase, SOD and GSH levelswere also brought up in a dose and time dependantmanner compared to control (treated and controlwere CAT: 100.7±4.7 and 51.3±3.2 µMH 2O 2/gHb/min, SOD: 9.68±0.87 and 1.15±0.12 IU/gHb, GSH: 21.3±1.23 and 6.0±0.91 µM/g Hb,respectively). These results establish that L.acutangula fruit aqueous fraction F2-3 possessesbeneficial role in mitigating t-BHP inducedoxidative stress in erythrocyte.Key words: Fruit methanolic extract; Reactiveoxygen species , Superoxide anion, Hydroxylradical; Malondialdehyde; Superoxide dismutaseIntroductionHydrogen peroxide (H 2O 2), organoperoxide, superoxide anion (O 2·-) and hydroxylradical (·OH), etc. are reactive oxygen species(ROS) generated by aerobic metabolism inbiological system and also by exogenous sourcessuch as drugs, ultra violet light, ionizing radiationand pollution. Under normal conditions, generatedROS are neutralized by inbuilt and defaultantioxidant enzymes present in the body such ascatalase, superoxide dismutase, glutathioneperoxidase, etc. (1, 2). Non-enzymatic antioxidantmolecules such as ascorbic acid, glutathione anduric acid also play a key role in detoxifying thefree radicals (3). Antioxidants are a type ofcomplex compounds found in our diet that act asa protective shield for our body against severalacute diseases (4). Increased oxygen fluxconditions (i.e. exercise) or failure of antioxidantRole of Luffa acutangula in Oxidative damage
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1074mechanism leads to over production of freeradicals that may exceed system capacity toremove them. This situation ultimately culminatesinto damage of macromolecules such as proteins,lipids and nucleic acids followed by oxidative stress(5).Several compounds such as phenolic,flavonoids etc. obtained from different plants havestrong antioxidant capacity (4). Dietary phenolsare present in plant foods as bioactive moleculesand data supporting the proposal that healthbenefits associated with vegetables and fruits areprobably linked to the phenol antioxidants theycontain. Phenols are present in a variety of plantsutilized as important components of both humanand animal diets (6, 7, 8). A general consensushas been reached during the last few years thatdiet has a major role in the development of chronicdiseases, such as cancer, coronary heart disease,obesity, diabetes type 2, hypertension and cataract(9, 10). This consensus suggests that apredominantly plant-based diet rich in fruits andvegetables, pulses and minimally processedstarchy staple foods reduces the risk fordevelopment of these diseases significantly (11,12).The recommendations are mainly based onepidemiological studies, are thus, that fruits,vegetables and less processed staple foods providethe best protection against the development ofdisease with little or no merit in recommendingvitamin or other micronutrient supplements fordisease prevention. This is a safe principle thatpromises to provide for improved public health.In recent years, search for novel type ofantioxidants from several plant parts has achievedhuge attention. Management of diseases withminimal side effects is still a complicated medicalchallenge. There is an increasing demand to usethe natural products to prevent the free radicalinduced diseases (13, 14). In this regard, weselected L.acutangula to evaluate its antioxidantproperty because it is the popular in Indiantraditional medicine. Luffa acutangula is atropical running vine with rounded leaves andyellow flowers belongs to Cucurbitaceae family.Tea of these leaves is used as a diuretic, seedshave laxative properties and juice of the fruit isused against internal hemorrhage in traditionalmedicine. The results suggest that free radicalsmight play a role in the development of brain injuryfollowing brain hemorrhage (4). The fruit of theLuffa acutangula is rich in phenolic contents.Presently we made a hypothesis that Luffaacutangla may have potential antioxidant activitydue to which it is being used against internalhemorrhage in traditional medicine. None of itstherapeutic potentials are scientifically evaluatedexcept robisome inactivating activity (15).Therefore we set out to determine the effect ofvarious doses of L.acutangula fruit extract onlipid peroxidation, glutathione (GSH) andSuperoxide dismutase (SOD), Catalase (CAT)activities in erythrocytes in a time and dosedependent manner using Tertiary butylhydroperoxide t-BHP induction. It has beenshown that human RBCs may compose up apotent system that can be used as an in vitroexperimental model to investigate the antioxidantpotential of dietary foods (16). t-BHP) is a wellknown cytotoxin and oxidative stress inducer,induces oxidative damage in different organs likeliver (17) testes (18), etc. mainly by mobilizationof arachidonic acid (AA) from membranephospholipids under cytotoxic conditions.Materials and methodsReagents : The medicinal plant L.acutangulawas purchased from Dr. Madhava Shetty,Department of Botany, Sri VenkateswaraUniversity,Tirupati, Andhra Pradesh, India.Thiobarbituric acid (TBA), 1,1,3,3,tetraethoxypropane (TEP), t-butylPurushotham Reddy et al.
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