Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)The Protective role <strong>of</strong> Luffa acutangula fruit MethanolicFraction against t-BHP Induced Oxidative damage in HumanErythrocytesBoreddy Purushotham Reddy*, S. Venkata Mohan <strong>and</strong> P. N. SarmaBioengineering <strong>and</strong> Environmental Centre, Indian Institute <strong>of</strong> ChemicalTechnology Hyderabad 500 607, India*For correspondence - poory_boreddy@yahoo.com1073AbstractThe present study was designed to examinethe ability <strong>of</strong> partially purified fractions <strong>of</strong> Luffaacutangula (Cucurbitaceae) fruit, to attenuatet-BHP induced oxidative damage in humanerythrocyte as an in vitro model. Initially, weinvestigated the antioxidant property <strong>of</strong> thehexane, methanolic <strong>and</strong> aqueous extracts (FHE,FME <strong>and</strong> FAE, respectively) by DPPH freeradical method <strong>and</strong> 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 <strong>and</strong>51.84±3.76%, respectively). Hence, this extractwas further partially purified chromatographically<strong>and</strong> out <strong>of</strong> these fractions (F1, F2, F3, F4, F2-1,F2-2, F2 -3 <strong>and</strong> 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 <strong>and</strong>glutathione in t-butyl hydroperoxide (t-BHP)treated-erythrocytes. Pretreatment with fractionF2-3 significantly inhibited lipid peroxidation in adose <strong>and</strong> time dependent manner compared tocontrol (40.6±3.2, 27.9±2.4 <strong>and</strong> 75.5±5.2 nmolMDA/gHb, at 30, 90 min <strong>and</strong> control,respectively). Catalase, SOD <strong>and</strong> GSH levelswere also brought up in a dose <strong>and</strong> time dependantmanner compared to control (treated <strong>and</strong> controlwere CAT: 100.7±4.7 <strong>and</strong> 51.3±3.2 µMH 2O 2/gHb/min, SOD: 9.68±0.87 <strong>and</strong> 1.15±0.12 IU/gHb, GSH: 21.3±1.23 <strong>and</strong> 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·-) <strong>and</strong> hydroxylradical (·OH), etc. are reactive oxygen species(ROS) generated by aerobic metabolism inbiological system <strong>and</strong> also by exogenous sourcessuch as drugs, ultra violet light, ionizing radiation<strong>and</strong> pollution. Under normal conditions, generatedROS are neutralized by inbuilt <strong>and</strong> defaultantioxidant enzymes present in the body such ascatalase, superoxide dismutase, glutathioneperoxidase, etc. (1, 2). Non-enzymatic antioxidantmolecules such as ascorbic acid, glutathione <strong>and</strong>uric acid also play a key role in detoxifying thefree radicals (3). Antioxidants are a type <strong>of</strong>complex 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 <strong>of</strong> antioxidantRole <strong>of</strong> Luffa acutangula in Oxidative damage
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1073-1082 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1074mechanism leads to over production <strong>of</strong> freeradicals that may exceed system capacity toremove them. This situation ultimately culminatesinto damage <strong>of</strong> macromolecules such as proteins,lipids <strong>and</strong> 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 molecules<strong>and</strong> data supporting the proposal that healthbenefits associated with vegetables <strong>and</strong> fruits areprobably linked to the phenol antioxidants theycontain. Phenols are present in a variety <strong>of</strong> plantsutilized as important components <strong>of</strong> both human<strong>and</strong> animal diets (6, 7, 8). A general consensushas been reached during the last few years thatdiet has a major role in the development <strong>of</strong> chronicdiseases, such as cancer, coronary heart disease,obesity, diabetes type 2, hypertension <strong>and</strong> cataract(9, 10). This consensus suggests that apredominantly plant-based diet rich in fruits <strong>and</strong>vegetables, pulses <strong>and</strong> minimally processedstarchy staple foods reduces the risk fordevelopment <strong>of</strong> these diseases significantly (11,12).The recommendations are mainly based onepidemiological studies, are thus, that fruits,vegetables <strong>and</strong> less processed staple foods providethe best protection against the development <strong>of</strong>disease 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 <strong>of</strong>antioxidants from several plant parts has achievedhuge attention. Management <strong>of</strong> diseases withminimal side effects is still a complicated medicalchallenge. There is an increasing dem<strong>and</strong> 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 <strong>and</strong>yellow flowers belongs to Cucurbitaceae family.Tea <strong>of</strong> these leaves is used as a diuretic, seedshave laxative properties <strong>and</strong> juice <strong>of</strong> the fruit isused against internal hemorrhage in traditionalmedicine. The results suggest that free radicalsmight play a role in the development <strong>of</strong> brain injuryfollowing brain hemorrhage (4). The fruit <strong>of</strong> 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 <strong>of</strong> itstherapeutic potentials are scientifically evaluatedexcept robisome inactivating activity (15).Therefore we set out to determine the effect <strong>of</strong>various doses <strong>of</strong> L.acutangula fruit extract onlipid peroxidation, glutathione (GSH) <strong>and</strong>Superoxide dismutase (SOD), Catalase (CAT)activities in erythrocytes in a time <strong>and</strong> 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 <strong>of</strong> dietary foods (16). t-BHP) is a wellknown cytotoxin <strong>and</strong> oxidative stress inducer,induces oxidative damage in different organs likeliver (17) testes (18), etc. mainly by mobilization<strong>of</strong> arachidonic acid (AA) from membranephospholipids under cytotoxic conditions.Materials <strong>and</strong> methodsReagents : The medicinal plant L.acutangulawas purchased from Dr. Madhava Shetty,Department <strong>of</strong> Botany, Sri VenkateswaraUniversity,Tirupati, Andhra Pradesh, India.Thiobarbituric acid (TBA), 1,1,3,3,tetraethoxypropane (TEP), t-butylPurushotham Reddy et al.