Assessment of phytochemicals, antioxidant, anti-lipid peroxidation and anti-hemolytic activity of extract and various fractions of Maytenus royleanus leaves

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Assessment of phytochemicals, antioxidant, anti-lipid peroxidation and anti-hemolytic activity of extract and various fractions of Maytenus royleanus leaves
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  RESEARCH ARTICLE Open Access Assessment of phytochemicals, antioxidant,anti-lipid peroxidation and anti-hemolyticactivity of extract and various fractions of  Maytenus royleanus  leaves Maria Shabbir † , Muhammad Rashid Khan * and Naima Saeed † Abstract Background:  Maytenus royleanus  is traditionally used in gastro-intestinal disorders. The aim of this study was toevaluate the methanol extract of leaves and its derived fractions for various antioxidant assays and for its potentialagainst lipid peroxidation and hemolytic activity. Methods:  Various parameters including scavenging of free-radicals (DPPH, ABTS, hydroxyl and superoxide radical),hydrogen peroxide scavenging, Fe 3+ to Fe 2+ reducing capacity, total antioxidant capacity, anti-lipid peroxidationand anti-hemolytic activity were investigated. Methanol extract and its derived fractions were also subjected forchemical constituents. LC-MS was also performed on the methanol extract. Results:  Qualitative analysis of methanol extract exhibited the presence of alkaloids, anthraquinones, cardiacglycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids. LC-MS chromatogram indicatedthe composition of diverse compounds including flavonoids, phenolics and phytoestrogens. Methanol extract, itsethyl acetate and  n -butanol fractions constituted the highest amount of total phenolic and flavonoid contents andshowed a strong correlation coefficient with the IC 50  values for the scavenging of DPPH, hydrogen peroxideradicals, superoxide radicals, anti-lipid peroxidation and anti-hemolytic efficacy. Moreover,  n -butanol fractionshowed the highest scavenging activity for ABTS radicals and for reduction of Fe 3+ to Fe 2+ . Conclusions:  Present results suggested the therapeutic potential of   Maytenus royleanus  leaves, in particular,methanol extract, ethyl acetate and  n -butanol fraction as therapeutic agent against free-radical associated damages. The protective potential of the extract and or fraction may be attributed due to the high concentration of phenolic,flavonoid, tannins and terpenoids. Keywords:  Maytenus Royleanus , Antioxidant Activities, Phenolic Content, Solvent Extraction Background Nowadays, plants provide raw materials for new sourcesof drugs and pharmaceutical products. A wide variety of naturally occurring constituents such as polyphenolics,terpenoids and pro-vitamins have received much atten-tion as alternative therapeutic agents to fight against various oxidative stress induced diseases [1-3]. Numer- ous natural antioxidants from medicinal plants havebeen isolated and characterized. In particular, phenoliccompounds are suggested to exert therapeutic activitiesbecause of their anti-oxidative and anti-inflammatory properties. The polyphenolic compounds impart mul-tiple antioxidant properties such as scavenging of freeradicals, reducing abilities or act as metal chelators. Fur-thermore, anti-microbial, anti-thrombotic and vasodila-tor properties of phytochemicals have increased theinterest as alternative and complementary medicines [4].The ability of cells to detoxify the reactive intermediatesthat are produced as a result of excessive metabolism iscompromised, which disturbs the equilibrium between * Correspondence: mrkhanqau@yahoo.com † Equal contributorsDepartment of Biochemistry, Faculty of Biological Sciences, Quaid-i-AzamUniversity Islamabad, Islamabad 45320, Pakistan © 2013 Shabbir et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the srcinal work is properly cited. Shabbir  et al. BMC Complementary and Alternative Medicine  2013,  13 :143http://www.biomedcentral.com/1472-6882/13/143  antioxidant and reactive oxygen species (superoxide rad-ical, hydroxyl radical, peroxide radical, and nitric oxide).In addition to the threats posed by oxidative stress tobiological system, it is also responsible for inducingchronic diseases (cancer, cardiovascular, aging, diabetes,cataract) [5]. Various studies have suggested an associ-ation between the dietary intake of these phytochemicalsand the prevention of various stress induced anomalies[6-11]. It is need of the day that medicinal plants should be characterized for their pharmacological properties asmajority of the population in developing countries usetraditional medicines [11-13]. Antioxidants (polyphenols, vitamin C, vitamin E, selen-ium,  β -carotene, lycopene, lutein and other carotenoids)present in large amounts in many medicinal plants andherbs, eliminate free radicals by acting as antioxidant, by neutralizing, quenching, reducing or through decomposingperoxides [14]. Compounds carrying antioxidant potentialcan be isolated and used as a remedy against oxidative stressand related diseases [15]. Recent studies are focusing on re-placement of synthetic antioxidants with naturally occurringantioxidants to avoid the potential toxicity of synthetic ones[16-18].  Maytenus royleanus  belongs to the family Celastraceae,Its flowering season is March-April and is distributedthroughout lower Kaghan on dry sunny slopes. It is ashrub having stiff branches, usually with straightthorns, white flowers in short axillary clusters, fruit is3 angled capsule [19]. Paste of bark is applied withmustard oil to destroy pediculi and it is also used lo-cally against gastro-intestinal diseases [20]. Smoke of the seeds is believed to be valuable against toothache.Leaves are often used as fodder. Branches are used forrepair of houses and fuel. In spite of the popular use,there is no report about phytochemical compositionand antioxidant properties of   M. royleanus  leaves. In- volvement of oxidative stress as a key role in variousdiseases, we aimed to evaluate the phytochemical andantioxidant potential of this plant. Methods Chemicals All the chemicals used in these assays were of high qual-ity. Na 2 CO 3,  ascorbic acid, gallic acid, rutin, Folin-Ciocalteu ’ s phenol reagent, NaNO 2 , AlCl 3 .6H 2 O, rutin,2, 2-Diphenyl-1-Picrylhydrazyl (DPPH), 2,2- azino-bis(3-ethylbanzthiazoline-6- sulphonic acid (ABTS), potas-sium oxidopersulphate, H 2 SO 4,  ammonium molybdate,riboflavin, phenazine methosulphate (PMS), nitrobluetetrazolium (NBT), H 2 O 2,  2- deoxyribose, ferric chlo-ride, potassium chloride, trichloroacetic acid (TCA),thiobarbituric acid (TBA), potassium ferricynide, Mayer ’ sreagent, NaOH, FeCl 3  were obtained from SigmaChemicals Co. (St. Louis, MO, USA). Solvents and otherreagents were of analytical grade. Collection of plant Collection of the plant material was made in March2011 from village Lehtrar, Tehsil Kotli Sattian of Pakistan. After identification a voucher specimen(# 032564) was deposited at the Herbarium of Pakistan Museum of Natural History, Islamabad. Preparation of extract Powder of dried leaves (500 g) was extracted with 95%methanol twice (2000 ml for each) for 48 h with occa-sional shaking and filtered. Extract was dried at 40°Cunder reduced pressure giving a yield of 12.2% to that of the powder (Panchun Scientific Co., Kaohsiung, Taiwan).Extract was suspended in 50 ml of distilled water andfractions were made by adding solvents (200 ml twice)successively with increasing polarity i.e.,  n -hexane,chloroform, ethyl acetate,  n -butanol and shake vigor-ously. The layers were separated accordingly and the sol-uble remainder was used as residual aqueous fraction.Fractions were dried with the yields  n -hexane (7.5%),chloroform (3.4%), ethyl acetate (8.9%),  n - butanol(7.8%) and residual aqueous fraction (10.7%) to that of the methanol extract [21]. Phytochemical screening The methanol extract of   M. royleanus  was analyzed by LC-MS to get the fingerprint of compounds it carriesand was found to carry number of compounds (reten-tion time: 0.5-45 min, m/z  “ mass ”  restriction: 100-3200Da, peak height >500 counts, relative height: > 2.5%,limit to largest: 300 compounds). Dried sample of methanol extract (~15 mg), was dissolved 1 ml of methanol (15 mg/ml). It was spun down to remove par-ticles and 10  μ l was injected under C18 or HILIC chro-matography and positive (C18) or negative (C18/HILIC)ionization (targeting flavonoids and phenolic acids). Qualitative determination of the chemical constituents Presence of alkaloids, anthraquinones, cardiac glyco-sides, coumarins, flavonoids, saponins, phlobatannins,tannins and terpenoids in the extract and various frac-tions was confirmed individually by following standardprocedures. Test for alkaloids Mixture of methanol extract of   M. royleanus  leaves andits various derived fractions (0.4 g) in 8 ml of 1% HClwas warmed on water bath. After filtration 2 ml filtratefrom the extract and each fraction was allowed to reactwith few drops of potassium mercuric iodide and withpotassium bismuth, separately. Turbidity or precipitation Shabbir  et al. BMC Complementary and Alternative Medicine  2013,  13 :143 Page 2 of 13http://www.biomedcentral.com/1472-6882/13/143  formation was considered as a confirmation for presenceof alkaloids [22]. Test for saponins The criterion of oil emulsion formation of saponins wasused for the screening of saponins [22]. Briefly, extractand various fractions (20 mg) suspended in 20 ml of dis-tilled water and boiled for 5 min. In 10 ml of the abovefiltrate 5 ml of distilled water was added and mixed wellto develop the froth. Development of emulsion aftermixing the froth with olive oil confirmed the existenceof saponins. Test for terpenoids Briefly, 2 ml of chloroform was mixed with 5 ml (1 mg/ml) of each sample in a test tube then 3 ml of concen-trated H 2 SO 4  was added to develop the color. Exhibitionof reddish brown coloration at the interface confirmedthe presence of terpenoids [22]. Test for anthraquinones To a volume of 6 ml of 1% HCl, 200 mg of each samplewas added separately and boiled. Benzene (5 ml) wasmixed with the filtrate and after separation of benzenelayer 2 ml of 10% ammonia solution was lowered. Devel-opment of pink, violet or red color in the ammonicalphase indicated the existence of anthraquinones [22]. Cardiac glycosides determination An aliquot of 5 ml of methanol extract of   M. royleanus leaves and its various fractions (10 mg/ml in methanol)were added in the sequence of glacial acetic acid (2 ml)and FeCl 3  solution (one drop). Concentrated H 2 SO 4 (1 ml) was added and the formation of brown ring at theinterface confirmed the presence of cardiac glycosides[23]. Test for coumarins In a vial having 300 mg/ml of the extract and each frac-tion was plugged with filter paper dipped in 1 N NaOHand boiled in a boiling water bath for few minutes. Yel-low fluorescence of filter paper under UV light con-firmed the presence of coumarins [23]. Test for phlobatannins An amount of 80 mg of the extract and various fractionswas boiled in 1% HCl. Development of red precipitateindicated the existence of phlobatannins [23]. Test for flavonoids Mixture of methanol extract and various fractions of   M.royleanus  leaves were prepared by adding 50 mg of eachsample to 100 ml of distilled water and filtered. An ali-quot of 5 ml of dilute ammonia solution was mixed with10 ml of the filtrate. Appearance of yellow coloration by addition of few drops of concentrated sulfuric acid indi-cated the presence of flavonoid [24]. Test for tannins A mixture was prepared by mixing 50 mg of methanolextract and each fraction in 20 ml of distilled water andboiled. Appearance of brownish green or blue-black col-oration after mixing few drops of 0.1% FeCl 3  confirmedthe existence of tannins [24]. Quantitative determination of the chemical constituents Quantification of alkaloids Quantification of alkaloids was performed by followingthe method reported previously (22). Each fraction(50 mg) was mixed with 200 ml of acetic acid (10%) inethanol; the beaker was covered and incubated for 4 h.The mixture was concentrated up to one third of itstotal volume. Ammonium hydroxide was added dropwise in the mixture until it formed precipitate. The pre-cipitate was washed with ammonium hydroxide andthen filtered. The filtrate (alkaloids) was calculated aspercentage of the dried fraction. Quantification of tannins For quantification of tannins each fraction (50 mg) wassuspended in 100 ml of distilled water, it was shaken for1 h in a mechanical shaker and then filtered. Each sam-ple (5 ml) was added with ferric chloride (2 ml) inhydrochloric acid (0.1 N) and potassium ferricyanide(0.008 M). Absorbance was taken at 120 nm within10 min and tannins contents were calculated as percent-age of the dried fraction (24). Quantification of saponins Methanol extract and each fraction (50 mg) were mixedin 100 ml of ethanol (20%). It was kept on heating for4 h with continuous stirring at 55°C, than diluted withdiethyl ether (20 ml) and washed with 5% sodium chlor-ide. Saponins were estimated as percentage of the driedfraction (22). Estimation of leaf protein For the determination of leaf protein content, monobasicsodium phosphate (16 ml) and dibasic sodium phos-phate (84 ml) was combined to get the desire pH (7.5) of phosphate buffer.Reagent 1: Sodium carbonate (2 g), sodium chloride(0.4 g) 0.1 N and Na-K tartarate 1 g was dissolved in dis-tilled water (100 ml).Reagent 2: Copper sulphate 0.5 g was mixed in dis-tilled water (100 ml).Reagent 3: Solution A (50 ml) and solution B (1 ml)both were mixed in a flask. Shabbir  et al. BMC Complementary and Alternative Medicine  2013,  13 :143 Page 3 of 13http://www.biomedcentral.com/1472-6882/13/143  Reagent 4: Folin phenol reagent was added to distilledwater (1:1 ratio).Fresh leaves (0.1 g) were homogenized in 1 ml of phosphate buffer (pH 7.5). The homogenate wascentrifuged for 10 min at 3000 rpm. The 0.1 ml of supernatant was taken in a test tube and finally volumewas raised by adding distilled water up to 1 ml. ReagentD (0.1 ml) was mixed after shaking for 10 min. After30 min of incubation, absorbance of each sample wastaken at 650 nm and protein content was measured withhelp of standard (BSA) (23). Sugar estimation Fresh plant material was used to estimate the sugar con-tent; plant material was homogenized and then treatedwith concentrated sulphuric acid. The sample was incu-bated for 4 h at 25°C. Optical density of each samplewas observed at 420 nm (24). Total phenolic content  Briefly, a mixture was prepared by adding 9 ml of dis-tilled water and 1 ml of each sample. To the mixture1 ml of Folin-Ciocalteu ’ s phenol reagent was addedfollowed by the addition of 10 ml of Na 2 CO 3  solutionand total volume was made to 25 ml by adding distilledwater. The optical density was determined after 90 minat 750 nm at 23°C. Quantity of total phenolics was de-termined as mg of gallic acid equivalent (GAE) per g of dried sample. For blank distilled water was used [25]. Total flavonoid content  According to this method [26], 0.3 ml of the filtrate theextract and each fraction (50 mg) in 10 ml of 80%methanol was mixed with a reagent; 3.4 ml of 30%methanol, 0.15 ml of NaNO 2  (0.5 M) and 0.15 ml of AlCl 3 .6H 2 O (0.3 M). Then NaOH was added properly mixed. Absorbance was recorded at 506 nm after 5 minof mixing. Total flavonoid content was calculated as mgof rutin equivalent per g of dried extract or fraction. In vitro antioxidant assays A stock solution (1 mg/ml) of the methanol extract andeach fraction was prepared in 95% methanol and dilutedaccordingly for various antioxidant and reducing assays.Antioxidant power of each assay was compared with theefficacy of standard chemicals. DPPH radical scavenging activity  Antioxidant potential of the extract and each fractionwas assessed by using 1,1-diphenyl 1-2-picryl-hydrazyl(DPPH) assay [27]. DPPH (2.4 mg) was dissolved in100 ml of methanol and diluted with methanol to obtainan absorbance of about 0.98 (± 0.02) at 517 nm. An ali-quot of 0.01 ml of the extract and each fraction atdifferent concentrations of 25-250  μ g/ml was added in3 ml of the DPPH solution. After incubation for 15 minin dark; absorbance of the mixture was determined at517 nm. Following formula was applied to determine theDPPH radical scavenging activity; Percentage inhibition  ¼ ð control absorbance − sample absorbance ½ Þ =  control absorbance ð Þ   100 : IC 50  is the concentration value which scavenged 50%of the DPPH radicals. Ascorbic acid and rutin were usedas reference compounds [28,29]. Superoxide radical scavenging Procedure of Beauchamp and Fridovich [30] wasfollowed to determine the scavenging potential of the ex-tract and fractions with respect to superoxide radicals.Briefly, 0.5 ml of 50 mM phosphate buffer (pH 7.6),0.3 ml of 50 mM riboflavin, 0.25 ml of 20 mM phena-zine methosulphate (PMS) and 0.1 ml of 0.5 mM nitroblue tetrazolium (NBT) were mixed, before the additionof 100  μ l of the methanol extract and each fraction at varying concentrations of 25-250  μ g/ml. Mixture was il-luminated in the fluorescent light for 20 min and560 nm wave length was used to record the absorbanceof the mixture. The percent inhibition was calculated by using the following formula: Percentage inhibition  ¼ ð 1 − absorbance of sample = absorbance of control Þ   100 : Reference compound used was ascorbic acid in thisassay. Total antioxidant capacity  Phosphomolybdate assay system was used to determinethe total antioxidant activity of the methanol extract and various fractions [31]. To a reagent solution; sulphuricacid (0.6 M), sodium phosphate (28 mM) and ammo-nium molybdate (4 mM); 100  μ l of each sample wasadded and incubated at 95°C in a water bath for 90 min.After cooling to room temperature; absorbance wasrecorded at 765 nm against reagent blank. Total antioxi-dant capacity of the ascorbic acid was also estimated forreference. The total antioxidant capacity was determinedby using following formula: Total Antioxidant capacity   % ð Þ¼ ð control absorbance − sample absorbance ½ Þ =  control absorbance ð Þ   100 : Reducing assays Hydroxyl radical scavenging A method described earlier was adapted to assess the hy-droxyl radical scavenging ability of various samples [32]. Shabbir  et al. BMC Complementary and Alternative Medicine  2013,  13 :143 Page 4 of 13http://www.biomedcentral.com/1472-6882/13/143  Briefly, reagent solution was prepared by sequential additionof ferric chloride (10 mM), 0.25 ml of 2-deoxyribose(2.8 mM) in 50 mM phosphate buffer (pH 7.4), 0.1 ml of 1 mM (1:1; v/v) EDTA solution and 0.1 ml of 10 mM H 2 O 2 .A volume of 0.1 ml of the extract and various fractions wasindividually added to 0.01 ml of reagent solution. Then0.1 ml of ascorbate (1 mM) was added and incubated at37°C for 1 h. In the mixture thiobarbituric acid (TBA) 0.5%;w/v in 1 ml of 50 mM NaOH and 1 ml of 10% w/v trichloroacetic acid (TCA) was added and cooled to roomtemperature after incubation in a boiling water bath for15 min. Intensity of chromogen was read at 532 nm. Thehydroxyl radical scavenging activity was estimated as;Hydroxyl radical scavenging activity   % ð Þ¼  1 − Abs : of sample = Abs : of control ð Þ   100 : Hydrogen peroxide scavenging Methods described previously  [33-35] were followed to de- termine the ability of extract and various fractions to scav-enge H 2 O 2 . Hydrogen peroxide (2 mM) working solutionwas made by mixing with 50 mM phosphate buffer(pH 7.4). Reaction mixture was prepared by the addition of 0.1 ml of extract and each fraction with 0.4 ml of 50 mMphosphate buffer (pH 7.4) followed by the addition of 0.6 mlof 50 mM H 2 O 2  and allowed to stand for 10 min. At230 nm absorbance of the mixture was recorded. Followingequation was used to determine the capacity to scavengeH 2 O 2 ;Hydrogen peroxide scavenging activity   % ð Þ¼  1 − absorbance of sample = absorbance of control ð Þ  100  ABTS radical scavenging Method of Re et al. [36] was used for the ABTS radicalscavenging activity of the extract and each fraction. Forthe development of ABTS radicals potassium persulfate(2.45 mM) solution was mixed with ABTS (7 nM) andincubated overnight in the dark to get a dark colored so-lution. The standard solution of ABTS was diluted by the addition of 60% methanol to have an absorbance of 0.70 (± 0.02) at 745 nm at 30°C. An aliquot of 0.3 ml of extract or fraction was mixed with 1 ml of ABTS andabsorbance was recorded after one minute. Reducingcapacity was determined according to the formula: %  inhibition  ¼ ð control absorbance − sample absorbance ½ Þ =  control absorbance ð Þ   100 : Reduction of Fe 3+ to Fe  2+ Reducing capability of the extract and each fraction wasestimated by following the method of Oyaizu [37]. A volume of 2 ml of 0.2 M phosphate buffer (pH 6.6) and2 ml of potassium ferricyanide was mixed with 2 ml of the extract and each fraction (10 mg/ml) and incubatedfor at 50°C for 20 min. From the reaction mixture 2 mlwas taken after the addition of 2 ml of 10% TCA andwas mixed with 0.4 ml of 0.1% ferric chloride and 2 mlof distilled water. After 10 min of incubation opticaldensity of the chromogen formed was read at 700 nm.High reducing power ability was associated with highabsorbance values. Reducing power of ascorbic acid wasconsidered as reference. In vitro anti-lipid peroxidation assay  Standard method for estimation of TBARS was used toassay the degree of lipid peroxidation [38]. The study procedure for the animal care and experimentation waspermitted by Ethical Committee of Quaid-i-Azam Uni- versity Islamabad. From freshly excised liver of rat 10×homogenate was made in cold phosphate buffer saline(pH 7.4). Extract and each fraction were added to 100  μ lof (15 mM) ferrous sulphate followed by addition of 3 ml of homogenate. After incubation for 30 min; 0.1 mlof this reaction mixture was mixed with 1.5 ml of 10%TCA. After 10 min of incubation it was filtered andsupernatant was added in a tube having 1.5 ml of 0.67%TBA (in 50% acetic acid) and placed in a boiling waterbath for 30 min. Concentration of chromogen formedwas measured at 535 nm. Anti-lipid peroxidation wasassessed by using the following formula: %  Inhibition  ¼  control − test ð Þ = control    100 Anti-hemolytic activity Anti-hemolytic activity was assessed by following thespectrophotometric method [39]. From a normal healthy individual 5 ml of blood was taken and centrifuged at1500 rpm for 3 min. Pellet of blood was washed threetimes in sterile phosphate buffer saline solution (pH 7.2).The pellet was re-suspended in normal 0.5% saline solu-tion. A volume of 0.5 ml of the extract and various frac-tions (10, 50, 100, 200, 250  μ g/ml in saline) were addedin 0.5 ml of cell suspension. After incubation the mix-ture at 37°C for 30 min it was centrifuged at 1500 rpmfor 10 min. Anti-hemolytic activity was assessed by measuring the absorbance at 540 nm. For positive andnegative control distilled water and phosphate buffer sa-line were used respectively. The study protocol was incompliance with Helsinki Declaration. Statistical analysis  In vitro  and other parametric assays were performed intriplicate and results are shown as mean± SD. Antioxi-dant potential of different assays was determined as IC 50 Shabbir  et al. BMC Complementary and Alternative Medicine  2013,  13 :143 Page 5 of 13http://www.biomedcentral.com/1472-6882/13/143
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