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Living organism requires oxygen for important biochemical reaction i.e. Oxidation, is essential for the production of energy to fuel biological processes, during this process free redicals and other reactive oxygen species (ROS) are produced. These free radicals and ROS are unstable species, reacts with bimolecules leads to cell structural damage tissue injury or gene mutation and ultimately resulting into development of various health disorders such as Alzheimer's disease, cancer, atherosclerosis, diabetes mellitus, hypertension and ageing (Hajar et al., 2010). Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress (Ozsoy et al., 2008)4.3 It has been suggested that natural antioxidants are more safe and healthy than synthetic antioxidants. ( Xiao, Zhen & Jia, 2009). Therefore there is an increasing interest in natural antioxidants, particularly fruits and vegetables become more popular among scientific communities and consumers because epidemiological studies have indicated that frequent consumption of natural antioxidants is associated with a lower risk of cardiovascular disease and cancer (Renaud et al., 1998; Temple, 2000). These effect in natural antioxidants is because of phytoconstituent e.g., polyphenols, vitamins and carotenoids (Kriengsak, 2006) (Scholer, 1990) present in fruits and vegetables, which might help prevent oxidative damage (Silva et al., 2005). Polyphenols possess ideal structural chemistry for free radical-scavenging activity, and they have been shown to be more effective antioxidants in vitro than tocopherols and ascorbate. The phenolic compounds in herbs act as antioxidants due to their redox properties, allowing them to act as reducing agents, hydrogen donors, free radical quenchers and metal chelators (Javanraedi et al., 2003). The many plant species have been investigated in the search for novel antioxidants but generally there is still a demand to find more information concerning the antioxidant potential of plant species. Recently there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury. Besides well known and traditionally used natural antioxidants are already exploited commercially either as antioxidant additives or a nutritional supplement. Also many other plant species have been investigated in the search for natural antioxidants, (Y.Chu, 80: 561-566 (2000). but generally there is still a demand to find more information concerning the antioxidant potential of plant species.
Luffa acutangula var. amara (Cucurbitaceae) is a scrambling or climbing shrub, different plant parts were used in traditional system of medicine in India for the treatment of diverse ailments. Conditions for which the plant is used include mastitis (leaf) jaundice, fever, cough, asthma, and piles. (fruits) emetic, cathartic. Small doses as expectorant and demulcent (seed). (kirtikar and basu) InTraditional system of medicine the plant is used to cures "vata", "kapha", biliousness, anaemia, liver complaints, leucoderma, piles, inflammation, bronchitis, ascites, tumours, tuberculous glands, uterine and vaginal tumours; useful in rat bite. recommended in splenic enlargements.(dravyagunvidnyan) On scientific hand the L. amara fruit contains bitter principle luffin and Colocynthin allied principle.(nandkarni) a triterpenoid amarinin was isolated. (Mukherjee, 1986). Luffa amara fruit powder extract exhibited dose-dependant CNS- depressant activity as reported by Misar et al. Indra kumar et al. and Daniel Prabu.E, et al. reported as ethyl acetate extract and ethanol extract of the leaves of Luffa acutangula var. amara showed marked hepatoprotective and free radical scavenging activity. The traditional claims of the plant may be by increasing immunity or by reducing oxidative stress. Therefore, this study will investigate the antioxidant activity of the four successive extracts (pet ether, ethyl acetate, ethanol and aqueous) from the fruit without seed of Luffa amara and compare the differences in content of total phenolic and flavonoids.
2. Materials and methods
Folin Ciocalteu reagent (Merck Co.), P-coumaric acid (Sigma Ltd., USA) and sodium carbonate (Na2CO3), (S.D-Fine Chemicals, Mumbai), quercetin (Sigma Ltd., USA), aliminium nitrate and Sodium acetate, catechin (Sigma-Aldrich Chemie, Steinheim, Germany), vanillin (Merck Co.) and sulphuric acid, 2, 2, Diphenyl-1-Picryl hydrazyl DPPH (Sigma-Aldrich Chemie, Steinheim, Germany), Dimethyl sulfoxide (DMSO), Methanol, 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) ABTS, Potassium per sulfate, Ascorbic acid, Sulphuric acid, sodium sulphate, ammonium molybdate, Nitro blue tetrazolium (NBT), riboflavin, Gallic acid (standard solution) (Loba Chemie, Mumbai), All other reagents used were of analytical grade.
2.2 Plant material:
The fruits of L. amara were at maturity were collected and dried under shade in Oct 2009 from Shirpur, Dhule District, Maharashtra, India. The plant was identified by its local name and later taxonomic identification was conducted by Prof. D. A. Patil, Dept. of botany, SSVP college, Dhule, MS, India. A voucher specimen was deposited in the herbarium of our laboratory under the number (Bot/H/3352).
2.2. Extract preparation:
The Luffa amara fruits were cleaned and dried in shed, the seeds were separated form pericarp. The separated pericarp (500gm) were powdered in a pulverizer to 60-mesh size and used for solvent extraction. For extract preparation, 500 g of dried sample was extracted successively with pet ether 60-800, ethyl acetate, ethanol by hot continuous percolation and with water twice 250C for 48 h. by maceration. The extracts were filtrated through Whatman No. 1 and followed by concentration using a rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) under reduced pressure at 400C. The dry extract obtained with each solvent was weighed. The percentage yield was expressed in terms of air dried weight of plant material.
2.4. Determination of total phenolics
The total phenolics were determined by the spectrophotometric method (Singleton and Rossi, 1965). With slight modification In brief, a 10mg of extracts were individually dissolved in 100 ml of methanol. Then 0.1 ml of these solutions was mixed with 0.2 ml of 10-fold diluted Folin-Ciocalteau reagent, and 2.0 ml of 15% sodium carbonate (Na2CO3). After 2 h incubation period at 500C for 10 min, the absorbance of the reaction mixtures were measured at 760 nm by using a spectrophotometer(Shimadzu 2405) The standard curve for total phenolics was made using gallic acid standard solution (0-100 mg/l) under the same procedure as above. The total phenolics were expressed as milligrams of gallic acid equivalents (GAE) per g of dried sample.
2.5. Determination of total flavonoids
The total flavonoid content was determined following the method described by Liu et al. (2005). 10 mg of extracts were dissolved in 100 ml of methanol and filtered through Whatman filter paper No. 42 (125 mm). In a 10 ml test tube, 0.5 ml of diluted extract, ethanol (1.5Â ml), Al(NO3)3 (0.1 ml, 10%), CH3COONa (0.1Â ml, 1Â M) and H2O (2.8Â ml) were thoroughly mixed and kept at ambient temperature for 40Â min. The absorbance of reaction mixture at 415Â nm. was measured with a spectrophotometer (Shimadzu 2405)Total flavonoid content was calculated according to a standard curve established with quercetin. The total flavonoid was expressed as Âµg quercetin equivalent.
2.5. Determination of total Proanthocyanidin
Total proanthocyanidin content of L. amara extracts was determined using method reported (Porter, 1989). The proanthocyanidins were detected in TLC test; therefore total proanthocyanidins were determined using vanillin-H2SO4 method. Briefly, 1ml of extract (100Âµg/ml) was mixed with 2 ml of freshly prepared vanillin solution (1% vanillin in 70% of H2SO4) and maintained for 15 min at 20 0C. The absorbance was measured at 500 nm. Calibration curved was drawn using aqueous solution of epicatechin (8-40 Âµg/ml) as a reference standard. .All experiment were performed by using Microplate spectrophotometer (Biotek,USA)
2.6. Antioxidant assays
For determination of antioxidant activity the different extracts were dissolved in 95% methanol at a concentration 1 mg/ml and then diluted to prepare the series concentrations for antioxidant assays. Reference chemicals were dissolved as above at concentration 100mcg/ml, used for comparison in all assays.
Radical scavenging activity of plant extracts against stable DPPH (2, 2- diphenyl-2-picrylhydrazyl hydrate, Sigma-Aldrich Chemie, Steinheim, Germany) was determined spectrophotometrically. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from deep-violet to light-yellow) were measured at 517 nm wavelength. Radical scavenging activity of extracts were measured by slightly modified method of Mohammad et al.(2010) as described below. Extract stock solutions were prepared in 1mg/ml in methanol. The different extract was not fully soluble in methanol (even after treating solutions for 5 min in an ultrasonic bath), therefore it was dissolved in dimethylsulphoxide. The assay was carried out in a 96 well microtitre plate (BIO-Tek Power wave TM XS). To 100Âµl of DPPH solution, 100Âµl of each of the test sample or the standard solution was added separately in wells of the microtitre plate. The final concentration of the test and standard solutions used are 800 to 50 Âµg/ml and 100 to 10 Âµg/ml respectively. The plates were incubated at 370C for 20 minutes and the absorbance of each well was measured at 517 nm, using ELISA reader (Biotek, USA) against the corresponding test and standard blanks and the remaining DPPH was calculated. The experiment was carried out in triplicate. Radical scavenging activity was calculated by the following formula:
% Inhibition = [(AB-AA)/AB] X 100
AB= Absorption of blank sample (t = 0 min)
AA= Absorption of tested extract solution (t = 30 min).
Commercial standard Gallic acid was also tested against DPPH and used as a reference.
ABTS radical cation scavenging activity
The ABTS radical cation scavenging activity was performed with slight modifications (Badami et al 2008), briefly, stock solution was prepared by reacting 2 mM ABTS with 17 mM potassium persulfate in proportion of 50:0.3 kept overnight in the dark to yield a dark coloured solution i. e. to generate the ABTS cation chromophore. The experiment was carried out by mixing 0.16 ml of ABTS reagent, 1 ml of distilled DMSO and 0.2 ml of various concentrations of the extract or standards, were added to make a final volume of 1.36 ml. Absorbance was measured spectrophotometrically, after 20 min at 734 nm using ELISA reader (BIO-Tek Power wave TM XS, Model-96 well micro plate). IC50 value obtained is the concentration of the sample required to inhibit 50 % ABTS radical monocation. It is calculated by formula as DPPH method.
Evaluation of Total antioxidant capacity by phosphomolybdenum method
The total antioxidant capacity of L. amara extracts, was evaluated by the method of Gudda darangavvanahally et al. and Pongtip et al., (2005). An aliquot of 0.1 ml of sample (100 Î¼g) solution was combined with 1 ml of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 950 C for 90 min. After the samples had cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against blank containing 0.1 ml of distilled water and 1 ml of reagent. in Genesys-5-UV spectrophotometer (Milton Roy, New York). The antioxidant capacity was expressed as equivalents of ascorbic acid (mmol/g of Luffa amara extracts).
Super Oxide Free Radical Scavenging Activity by Riboflavin-Light-NBT System:
The Super Oxide Free Radical Scavenging Activity was carried out as per Bafna and Misra (2005) with slight modification. The 200 ïl EDTA, 100 ïl NBT, 50 ïl riboflavin, 2.5 ml phosphate buffer pH 8.0 and 200 ïl various extracts cts dissolved in methanol were mixed in test tube. Reaction was started by illuminating the reaction mixture for 15 minutes. After illumination the absorbance was read at 590 nm in micro titer plate ELIZA reader (BIO-Tek Power wave TM XS, Model-96 well micro plate). Same procedure was followed for control by using methanol in place of samples. Ascorbic acid and BHA were used as positive control.IC50 values for each extracts and standards were calculated and expressed as ïg/ml.
Reducing Power Assay [Oyaizu et al., 1986; Mondal et al., 2006]
The reducing power assay was measured using modified method as described previously (Oyaizu et al., 1986). The reducing capability was measured by the transformation of Fe3+ - Fe2+ in the presence of different test extracts. Reducing power of different extracts of L. amara were determined on the basis of the ability of antioxidants to form colored complex with potassium ferricyanide, TCA and FeCl3. Different concentrations of various extracts (50, 100, 200, 400 and 800ïg/ml) and standard i.e. ascorbic acid (10, 20, 40, 80, and 100ïg/ml) were mixed with 2.5 ml phosphate buffer (pH 6.6) and 2.5 ml potassium ferricyanide (1%). The mixture was incubated at 50°C for 20 min. 2.5 ml TCA (10%) was added to it and centrifuged at 3000 rpm for 10 min. 2.5 ml of supernatant was mixed with 2.5 ml of water and 0.5 ml of FeCl3 (0.1%) were added to it and absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.
Extraction yields, total phenolics and flavonoid content
The percentage yield of total methanol extracts and different successive extract of luffa amara are shown in table 1. The percent extractive yield ranges form 1.2 to 11.2 % w/w, among all successive extract ethanol extract (11.2 %) shown heist yield while pet ether extract shown lowest % yield (1.2%). The extraction ability of ethanol and water is almost same while extraction yield of pet ether solvent is least.
Total phenolic content
The phenoilic content in different extracts of plant were expressed in gallic acid equivalent (GAE). It ranges between 12 mg it 112 mg of gallic acid equivalent /gm of dry weight extract. While total methanol extract shows 212 mg of gallic acid equivalent /gm of dry weight extract. (table 1.) the result are comparable with literature (abdille eta l, 2005, sakanaka 2005). The highest total phenolic content was found in ethanol extract 25.5Â± mg GAE/gm of DW, while least in pet ether extract 8.3 Â± mg GAE/DW which is in arrangement with report of Ao, Li Elzawele., Xuan and twata (2008). The phenolic compound form the plant are known to be good source of natural antioxidant (Duh et al 1999, Nagappa et al 2007) because of their scavenging ability due to presence of hydroxyl groop in their chemical structure. (hatano et al 1989), Etuo et al (2000) also suggested relationship between high phemolic content and antioxidant activity of extract.3
Total flavonoid content
the total flavonoid content were expressed in quericitin equivalants, varied form 4.1 to 18.2 Â± as quericitin equivalants 1 gm of extract. The results given in table 1. Shown 22Â± , 4.1 Â± , 18.2Â± , 15.2 Â± and 10.2 Â± mg of quericitin equivalent gm extract in totol methanol, pet ether , ethyl acetate, ethanol and water receptively, it shows correlation between total phenolics and flavonoid content. The content of flavanoid in different extracts were comparable with other plant extracts described in litreture (Ao et al 2008)2, the high flavonoid content plant shows good antioxidant activity and their effect on human nutrition and health care are considerable. The fruits and vigitables shows good antioxidant capacity because of their main phytocontituents i.e. phenoilc acid and flavonoid (Bahrmikia, ardestani and Yazdanparast 2009 ) 2. The mechanism of action of flavonoids are through scavenging, chelating process (M. Kessler, G Ubeaunel and L jug 2003) 4 and protect against lipid peroxidation.
DPPH Radical scavenging acitivity
The DPPH radical is consider to be model for lipophillic radicals, it is stable and widely used as tool for estimating free radical scavenging activity of antioxidant. It accept and electron / hydrogen at room temperature to become stable diamagnetic molecule (Sores et al 1997,4, sanchenz - Moreno 2002, 1). The reduction capability of DPPH radicals was determine by decrease in absorbance at 517 nm, which is because of free radicals scavengers, the scavenging ability of all extracts were found to be dose dependant manner in range of 50 - 800Âµg/ml and given in table 2. It is compared with ascorbic acid. The ethyl acetate extract shows least IC50 (inhibitory concentration) i.e. 87.39 Â± while aqueous extract shows highest i.e. 774.77 Â± . the scanging ability of DPPH were descending order as
Ethanol - ethyl acetate - aqueous - pet ether
The antioxidant activity in DPPH assay correlated well with phenoilc and flavonoid content of individual extract.
ABTS radical cation scavenging activity
The different strategies were implicated for generation of ABTS.+ radical cation. One of the most widely used by chemical reaction with potassium persulphate (Pellegrini 1999)5, the ABTS radical is quite stable over time and pH, is soluble in water and organic solvent enabling the determination of antioxidant capacity of both hydrophilic and lipophillic compounds (LuÄ±Â´s M. Magalha et al., 2008 5). The reduction capability of ABTS radicals was determined by decreased in absorbance at 745 nm which is induced by antioxidants. The different extracts of L. amara at range concentration (50 - 800Âµg/ml) the ABTS scavenging activity of different extract was found to be dose dependant manner. The scavenging activity of sample can be ranked as Ethanol - ethyl acetate - aqueous- pet ether extract. The ethanol and ethyl acetate extract from fruits of L. amara exhibited the highest radical scavenging activity. In contrast, pet ether extract showed least ABTS radical scavenging capacity. Similarly the efforts was produced by ascorbic acid nearly at concentration 12.11 Âµg/ml. the result was shown in table 2.
Super Oxide Free Radical Scavenging Activity by Riboflavin-Light-NBT System:
Superoxide radical is very harmful cellular component can generate more reactive oxygen species. These ROS can cause tissue or DNA damage leads to various disease, therefore it is recommended to measure comparative interceptive ability of antioxidant extract to scavenge superoxide molecule. (vani, rajani, Shankar and shishoo 1997) 2, (Halliwell and gufferidge, 1999) 1. The decrease in absorbance at 560 nm with palnt extracts and reference compound ascorbic acid indicates their ability to quench superoxide redical in reaction mixture. The super oxide redical scavenging activity of different extract of fruits of L. amara were determined over concentration range of 50- 800 Âµg/ml. the IC 50 value in superoxide scavenging activity were found in desending in order as Ethyl acetate - ethanol- aqueous - pet ether. All extracts showed dose dependant superoxide radical scavenging activity. The ethyl acetate extract shown least IC50 i.e. 78.14 while pet ether showed the highest IC 50 i.e. 398.74 â€¦ . when these finding compared with standard ascorbic acid IC50. The super oxide scavenging activity of extracts was found to be significant. It may be due to presence of flavonoid and phenolic content.
Total antioxidant capacity by phosphomolybdenum method
The phasphomolybdate method has been routinely used to evaluate the antioxidant capacity of extracts (prieto, pineda and aguliar, 1999) 2. In the presence of extract MO (VI) is reduced to Mo (V) and forms a green colored phasphomolybdenum V complex, which shows maximum absorbance at 700 nm. The total antioxidant capacity of different extracts of L. amara where determined with refrence to ascorbic acid. It can be ranked in descending order as ethyl acetate - ethanol - aqueous - pet ether. The ethyl acetate and ethanol extract showed 50.68 and 49.82 â€¦. Mg od ascorbic acid equvalant / gm of extracts. While aqueous extract shown less total antioxidant capacity i.e. 3.34 .. mg of ascorbic acid equivalent /gm if extract. The total antioxidant capacity of ethyl acetate and ethanol extracts were comparable with flavonoid and phenol content.
Reducing power assay:
Reducing power assay is commonly used for assessing the antioxidant activity. The significant reducing capacity of compounds is indicator of its antioxidant potential. The reducing capacity is due to prevention of chain initiation, decomposition of peroxide, reducing capacity and scavenging capacity. This is important mechanism of phenolic antioxidant action and can be strongly correlated with antioxidant capacity. (Dorman, Peltoketo, Hiltunen, & Tikkanen, 2003). (Yildirim A 2000) Fig. 8 shows the dose-response curves for the reducing powers of the extracts from L amara fruits. The reducing power of the ethanol and ethyl acetate extract increased from 0.245 Â± 0.006 and 0.116 Â± 0.001 at 50 Âµg/ml, respectively to 0.615 Â± 0.004 and 0.512 Â± 0.004 at 800 Âµg/ml, espectively. The reducing power of aqueousl and pet ether extracts increased from 0.101 Â± 0.001 and 0.068 Â± 0.004, respectively at 50 Âµg/ml l to 0.488 Â± 0.003 at 0.451 Â± 0.006 at 800 Âµg/ml, respectively. At a dosage of 400 to 800 Âµg/ml extracts showed high reducing values of 0.345 - 0.615, almost equal to that of gallic acid (0.472 -0.754) at a concentration of 40 - 80 Âµg/ml.
The EC50 value (the effective concentration at which the absorbance was 0.5) was very high for the pet ether extract (886.91 Â± 0.16 Âµg/ml), compared with the aqueous, ethyl acetate and ethanol extracts, of which the EC50 values were 819.67 Â± 0.045, 487.80 Â± 0.037 and 241.54 Â± 0.039 Âµg/ml, respectively. EC50 values of gallic acid (42.372 Â± 0.003 Âµg/ml). Effectiveness in reducing power inversely correlated with EC50 values and was in descending order:
Ethanol > Ethyl acetate > Aqueous > Pet ether.
The free radicals are responsible for development of several degenerative diseases such as Parkinson's disease, Alzheimer's type of dementia, Diabetes, Arthritis etc. the production of free radicals ad activity of scavenger enzyme against those radicals such as superoxide dismutase (SOD) are correlated with life expectancies. We have demonstrated that extracts of L. amara fruits contain high levels of total flavonoid and phenolic compounds and were capable of inhibiting lipid peroxidation, directly quenching free radicals to terminate the radical chain reaction, acting as reducing agents, and chelating transition metals to suppress the initiation of radical formation. It is well-known that phenolic compounds present in the plant kingdom are mainly responsible for the antioxidant potential of plants. Accordingly in this study, a significant and linear relationship was found between the antioxidant activity and phenolic and flavonoid content, indicating that flavonoid and phenolic compounds could be major contributors to antioxidant activity. To explore the suitability of different extracting solvents with different polarity, we have compared the yield, total phenolic, flavonoid content and antioxidant properties of ethanol, ethyl acetate, aqueous and pet ether extracts. Ethanol and ethyl acetate extracts showed the highest scavenging activities against DPPH, ABTS superoxide radical and reducing power assay. The data on phenolic content and antioxidant activity assessment obtained in these experiments single out ethanol and ethyl acetate as the most promising sources for the isolation of natural antioxidative compounds from the fruit of L. amara. Although the antioxidant activity found in an in vitro experiment is only indicative of the potential health benefit, these results remain important as the first step in screening antioxidant activity of L. amara fruit. It can be concluded that, powder and Juices of L. amara leaves, in the way which they are used as medicine in traditional system of medicine, can be used as an accessible source of natural antioxidants with consequent health benefits. Further scientific work in our laboratory is in progress to ensure the medicinal properties of the plant in vivo correlate with its antioxidant activity.
The authors would like to thank Prof. Dr. D. A. Patil from the Prof, Dept of Botany, SSVPS college, Dhule, MS. for the identification of the plant. The authors are also thankful of SE society for providing the facilty for research.
Totoal antioxidant capacity (AAE mg/ml)
ability on DPPH
ability on super
ability on ABTS
Reducing pawar assay
Pet ether (60-800)
474.28 Â± 1.75
319.79 Â± 0.55
56.76 Â± 0.15
866.58 Â± 0.39
13.22 Â± 0.50
232.02 Â± 0.84
75.23 Â± 0.43
46.00 Â± 0.30
245.14 Â± 0.94
30.72 Â± 0.73
84.00 Â± 0.76
77.69 Â± 0.06
43.76 Â± 0.62
487.51 Â± 1.88
28.22 Â± 0.37
414.83 Â± 2.56
109.18 Â± 1.20
49.26 Â± 0.32
821.65 Â± 1.21
20.72 Â± 0.37
41.89 Â± 0.36
20.72 Â± 0.07
12.16 Â± 0.04
42.41 Â± 0.47
Total phenolics (mg gallic acid equivalent/g)
Total flavonoids (mg catechine equivalent/g)
Extract yield (%)
Pet ether (60-800)
3.85 Â± 0.003
5.07 Â± 0.001
21.26 Â± 0.008
86.50 Â± 0.074
30.11 Â± 0.005
73.64 Â± 0.011
21.42 Â± 0.004
19.34 Â± 0.005