- Email: [email protected]
Isolation, purification and characterization of antioxidative steroid derivative from methanolic extract of Carissa carandas (L.) leaves
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
Contents lists available at ScienceDirect
Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Isolation, purification and characterization of antioxidative steroid derivative from methanolic extract of Carissa carandas (L.) leaves Bhushan S. Bhadane, Ravindra H. Patil
MARK
⁎
Department of Microbiology and Biotechnology R. C. Patel Arts, Commerce and Science College, Shirpur 425405 Maharashtra, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Phytochemicals Bioautography DPPH FT-IR and GC-HRMS
Present study evaluates antioxidative and erythrocyte membrane stabilizing potential of methanol extract of Carissa carandas leaves Cc(L)M and its purified fraction CM 1. Phytochemically Cc(L)M revealed presence of alkaloid, steroids, saponins and tanins. The results of antioxidant assays of Cc(L)M and CM 1 found to be dose dependent. However, in TLC bioautography, CM 1 showed yellow coloured scavenged band of DPPH reagent. The results revealed that CM 1 has significant (p≤0.05) free radical scavenging potential compared to Cc(L)M. The purified fraction CM 1 was found to be most potent with lowest EC50 values for DPPH (546.4 µg/ml), OH(498.5 µg/ml) radical scavenging and 57.48 µg/ml for reducing power assay. It also erythrocytes membrane stabilization effect with lowest EC50 1.31 mg/ml as compared to Cc(L)M (2.56 mg/ml) and standard diclofenac sodium (1.00 mg/ml). The FT-IR and GC-HRMS analysis of CM 1 reveal the presence of steroid derivative 20hydroxypregnan 18-oic acid.
1. Introduction
of biological membranes and tissue damage (Lavanya et al., 2010). The drugs which commonly used to treat inflammation are non-steroidal anti-inflammatory drugs which are having adverse effects like gastric ulcers (Kumar et al., 2012). Earlier reports suggests that herbal drug containing principles had ability to stabilize biological membranes when exposed to induced lyses (Sadique et al., 1989; Gandhidasan et al., 1991; Syed et al., 1997). Hence, there is increase in demand of plant derived anti-inflammatory drugs to treat inflammatory conditions. C.carandas belongs to family Apocynaceae; native and common throughout India, Sri Lanka, Java, Malaysia, Myanmar and Pakistan (Motwani et al., 2012). Different parts of this plant are reported for pharmacological activities like spasmolytic, analgesic, anti-inflammatory, Antihyperglycemic, hepato-protective, antipyretic and cardiotonic (Kirtikar and Basu, 1987; Mishra et al., 2012; Balakrishnan et al., 2011) and purgative and snake bite antidote (Pakrashi et al., 1968). Different parts of Carissa carandas are enriched with wider group of phytochemicals. The roots comprises Triterpenes like lupeol, ursolic acid, αamyrin and oleanolic acid. Sesquiterpene, β-sitosterol and cholest-5-en3β-ol are also reported (Itankar et al., 2011). Moreover, fruits were enriched with flavonoids like rutin, epicatechin, quercetin and kaemferol. Patil et al. (2012) reported phenolics like syringic acid, vanillic acid and caffeic acid in the ripened fruits of C. carandas. Leaves of the plant were found to contain triterpene such as betulinic acid (Naim et al., 1988).
Reactive Oxygen Species (ROS) are derived from the metabolism of oxygen. Free radicals such as hydroxyl, superoxide and hydrogen peroxide are generated as byproducts of biological reactions or from exogenous factors are act as ROS (Krishnaiah et al., 2011). The free radicals mostly attack membrane lipid, protein, and DNA and are ultimately responsible for many disorders (Rackova et al., 2007) such as cancer, diabetes mellitus (Kinnula and Crapo, 2004; McCune and Johns, 2002), neurogenerative and inflammatory diseases (Matteo and Esposito, 2003; Sreejayan and Rao, 1996). The antioxidants are the substances that delays, prevent or removes oxidative damage (Gulcin, 2007). The antioxidants exert their activity by inhibiting free radical oxidation, act as chain-breaking antioxidants, singlet oxygen quenchers and reducing agents (Halliwell, 2007; Darmanyan et al., 1998; Heim et al., 2002). However, use of synthetic antioxidants such as BHA and BHT has been restricted because they may be responsible for liver damage and carcinogenesis (Wichi, 1988; Madhavi and Salunkhe, 1995). Therefore, interest in the use of natural antioxidants has been increased. Inflammation is sequential process induced by biological stimuli or tissue injury caused by noxious chemical, physical trauma or microbiological agents (Osadebe and Okoye, 2003). In inflammatory conditions ROS release from activated neutrophils and macrophages. Overproduction of these ROS attributed to tissue injury by lipid peroxidation
⁎
Corresponding author. E-mail address: [email protected] (R.H. Patil).
http://dx.doi.org/10.1016/j.bcab.2017.03.012 Received 14 February 2017; Received in revised form 14 March 2017; Accepted 18 March 2017 Available online 20 March 2017 1878-8181/ © 2017 Published by Elsevier Ltd.
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
The aim of present study was to evaluate in vitro antioxidant and erythrocyte membrane stabilizing potential of C.carandas leaf extract and its purified fraction. The attempt has also been made to purify and partially characterize bioactive compound by chromatographic and spectroscopic methods. Identification and structural confirmation of bioactive compound was done by GC-HRMS analysis.
the reaction mixture indicates higher free radical scavenging activity. The scavenging activity of DPPH radical was calculated using the following equation:
DPPH scavenging effect(%) = [(A 0 − A1/A 0) × 100] where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample. Each test was carried out in three replicates. The EC50 value of DPPH was determined by plotting values of percent scavenging against extract concentration. The EC50 indicates the concentration of antioxidant that scavenges DPPH radical about 50%.
2. Materials and methods 2.1. Collection and extraction of plant material Leaves of the healthy plant of C.carandas were collected as per standard procedure from Aner river forest region (21.26°N, 75.11°E) Dhule district, India. The plant specimen was identified and authenticated by plant taxonomist; Herbarium of the specimens (RCP/DBT-05/ 2012) was deposited at the Department of Biotechnology R. C. Patel Arts, Commerce, and Science College, Shirpur, MS, India. Leaves were washed to dry in dark at room temperature and further ground to obtain fine powder. The powdered leaves (500 g) were defatted using hexane by soxhlet method. Residue remains after defatting was subjected to extraction using methanol for 48 h. After extraction, solvent was evaporated under reduced pressure using rotary vacuum evaporator (Equitron, Mumbai, India) to afford a gummy solid residue. The resulting extract was stored in a refrigerator at 4 °C until use.
2.4.2. Reducing power assay The reducing power of test extract was determined according to the method previously described by Qyaizu (1986). Different concentrations (10–100 μg/ml) of test extract and standard ascorbic acid (1 ml) mixed with 2.5 ml, 0.2 M phosphate buffer (pH 6.6) and 2.5 ml potassium ferricyanide (1%). The mixture was incubated at 50 °C for 20 min. A portion of 2.5 ml of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The supernatant (2.5 ml) of the solution was mixed with 2.5 ml distilled water and 0.5 ml FeCl3 (1%) and the absorbance of reaction mixtures was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid was used as a standard. Phosphate buffer was used as a blank. Each test was carried out in three replicates. The EC50 value of reducing power assay was determined by plotting values of absorbance against extract concentration. The EC50 indicates the concentration of antioxidant that reduces ferric ion about 50%.
2.2. Phytochemical screening The phytochemical analysis of the plant extract for major phytoconstituents was done as per the published procedures (Kokate et al., 1995). Test extract was screened for the presence of phytochemicals like alkaloids, flavonoids glycoside, steroids and triterpenoids, saponins, tannins etc.
2.4.3. Hydroxyl radical scavenging assay Hydroxyl radical scavenging activity of the test extract was measured by the salicylic acid method as described by Smirnoff and Cumbes (1989) with slight modifications. Reaction mixture contained 1.0 ml of 1.5 mM FeSO4, 0.7 ml of 6 mM H2O2, 0.3 ml of 20 mM sodium salicylate and 1 ml of test extract. After incubation at 37 °C for 60 min, absence of the hydroxylated salicylate complex was measured at 562 nm. Scavenging activity was determined as follows.
2.3. Total phenols Total phenolic content of crude extract was determined spectrophotometrically using the method of Singleton and Rossi (1965) with little modification. 1 ml of 10% Folin-ciocalteu's reagent was added to 1 ml crude extract (1000 μg/ml) and mixed thoroughly. To the mixture, 4 ml sodium carbonate (7.5%) and 10 ml of distilled water was added and allowed to stand for 2 h. The absorbance of reaction mixture was recorded at 760 nm. A standard curve was obtained using various concentrations of gallic acid (10–100 μg/ml). Total phenols were expressed as mg of GAE gallic acid equivalent/gm of extract.
Scavenging ability(%) = [(T1 −T2 / T1) × 100] where T1 was the absorbance of the control reaction and T2 the absorbance in the presence of the crude extract. Each test was carried out in three replicates. Ascorbic acid was used as positive controls. The EC50 value of hydroxyl radical was determined by plotting the values of percent scavenging against extract concentration. The EC50 indicates the concentration of antioxidant that scavenges hydroxyl radicals about 50%.
2.4. Antioxidant activity 2.4.1. Free radical scavenging activity by DPPH method 2.4.1.1. Qualitative assay. TLC bioautography method (Takao et al., 1994; Wang et al., 2012; Olech et al., 2012) was used to determine free radical scavenging activity of test extract. Briefly, the test extracts were separated by suitable solvent system. After TLC separation, the developed plates were sprayed with 0.2% DPPH solution prepared in methanol. Plates were then kept in dark for 30 min and examined in daylight. The formation of a yellow coloured spot against violet coloured background indicated DPPH radical scavenging activity.
2.5. Erythrocyte membrane stabilization assay Erythrocyte membrane stabilization assay was carried out using human red blood cells (Oyedapo et al., 2010). Briefly, blood from a healthy human was collected in vials containing an anticoagulant (0.8% sodium citrate) and centrifuged at 3000 rpm for 10 min. The supernatant containing plasma or leucocytes were carefully removed and RBCs pelleted at the bottom of the vial were washed in isosaline (0.85%, w/v, NaCl) for four to five times or until a clear supernatant is obtained. Hematocrit (2%, v/v) were prepared and used for membrane stabilization assay (Oyedapo et al., 2004). Different concentrations (0.5–3.5 mg/ml) of test extract and standard drug diclofenac sodium (1 ml) were prepared in isosaline and mixed with 2 ml of hyposaline (0.25%, w/v, NaCl), 1 ml of 0.15 M sodium phosphate buffer (pH 7.4), 0.5 ml of 2% hematocrit. Drug control and blood control were also prepared. Drugs were omitted in the blood control, while the drug control did not contain the RBC suspension. All the reaction mixtures with a final volume of 4.5 ml were incubated at 56 °C for 30 min in
2.4.1.2. Quantitative assay. Free radical scavenging activity of test extract was determined by using 1, 1 diphenyl 2 picrylhydrazyl (DPPH) assay (Shimada et al., 1992). Briefly, extract concentrations (100–1000 μg/ml) with final volume 4 ml was mixed with 1 ml 0.2 mM methanolic solution of 1, 1 diphenyl 2 picrylhydrazyl (DPPH). The mixture was shaken vigorously and left to stand for 30 min in the dark at room temperature. After incubation, the absorbance of the reaction mixture was recorded spectrophotometrically (Shimadzu 1750) at 517 nm using methanol as blank. The decrease in the absorbance of 217
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
was mixed with dry potassium bromide (KBr) to homogenized and dried in mortar and pestle. The homogenized mixture was placed on the disc to form a KBr pellet and FT-IR spectrum was analyzed (Liu et al., 2006; Were et al., 2015).
water bath. After incubation, mixture was centrifuged at 5000 rpm for 10 min. The supernatant was taken in a separate tube and the absorbance of the supernatant was recorded at 560 nm. The percent membrane stability was estimated using following equation.
%Membranestabil 100–(Absorbanceoftestdrug –Absorbanceofdrugcontrol ) = × 100 (Absorbanceofbloodcontrol )
2.6.5. GC-HRMS analysis GC-HRMS analysis of column eluted fraction was carried out using Agilent 7890-5 GC (Santa Clara, USA) fused with HP-5 silica capillary column (30 m×0.20 mm, phase thickness 0.25 µm) coupled with a mass spectrometer (Jeol Accu TOF GCV) and FID detector. The inlet temperature was at 250 °C. The injection volume was 10 µl injected in split mode with a ratio of 20:80 and the flow rate of carrier gas was maintained at 1 ml per minute. The identification of compounds present in column eluted bioactive fraction was compared using NIST 05 library search database.
The EC50 value of this assay was determined by plotting the values of percent protection against extract concentration. The EC50 indicates the concentration of antioxidant that stabilizes erythrocytes membrane about 50%. 2.6. Purification and characterization of bioactive compound 2.6.1. Thin layer chromatography (TLC) The methanolic extract (10 µl) of C.carandas leaves was spotted on the TLC plates using Spraylin V automatic sample applicator (Aetron, India). The spots were air dried and the plate was developed in a presaturated chamber containing different mobile phases (Wagner and Bladt, 1996). Chromatographic separation was carried out at room temperature in ascending manner using mobile phases such as; toluene: ethyl acetate (7:2), toluene: ethyl acetate: diethylamine (7:2:1); Chloroform: ethyl acetate (7:3); Chloroform:Glacial acetic acid:Methanol:Water (65:32:12:8); Ethyl acetate:Formic acid: Glacial acetic acid:Water (100:11:11:26). After chromatographic separation plates were derivatized using 10% ethanolic sulfuric acid or anisaldehyde sulfuric acid reagent and heated in a hot air oven to further visualized in UV and daylight.
2.7. Statistical analysis Values are means ± SD of three consistent readings of each experiment. Data from all the experiments further subjected to Analysis of variance (ANOVA) to determine the significance variations. The means were compared according to Dunnet test (P≤0.05). The processing of data to present the results was done using XLSTAT version 16 (Addinsoft, US). 3. Results 3.1. Phytochemical screening and phenolic content Results of preliminary phytochemical screening in Cc(L)M showed dominantly presence of alkaloids, steroids, saponins and tannins and moderately presence of flavonoids (Table 1). However, total phenolic content of Cc(L)M was obtained from linear regression analysis equation (y=0.0027x+0.0141; R2=0.956) from the standard graph (Fig. 1) of gallic acid and it was found to be 102.33 mg GAE/gm of extract.
2.6.2. Column chromatography The mobile phase which gave better separation in TLC was used for elution of column. The chromatographic separation was carried out in silica 60–120 mesh size column chromatography. The silica slurry prepared in mobile phase was poured into column (30×1.8 cm) and improve column packing by tapped it from outside. After column packing, test extract prepared in dry silica was loaded on the top of column bed to get even layer (Ode et al., 2011). After loading of sample, mobile phase was poured through side wall of column and fractions were collected in 5 ml quantity. During elution, the constant flow rate of eluent was maintained for all fractions. Simultaneously, TLC of eluted fraction was carried out to check presence of single compound.
3.2. Antioxidant activity 3.2.1. DPPH radical scavenging assay TLC bioautography of Cc(L)M and purified fraction CM 1 revealed yellow coloured band as a result of DPPH scavenging (Fig. 2). The quantitative assay of Cc(L)M and CM 1 showed a dose dependent (R2=0.952) radical scavenging activity (Fig. 3). The maximum DPPH radical scavenging activity of Cc(L)M and CM 1 was obtained at 1000 µg/ml with scavenging effect of 77.08 ± 0.66% and 81.87 ± 0.27% respectively. Results of present findings indicated that CM 1 was found to be more effective DPPH radical scavenger than Cc(L)
2.6.3. HPLC analysis The fraction obtained from column chromatography was subjected to HPLC analysis in order to check purity of resulting fraction. HPLC was carried out using, LC 20 Ad liquid chromatography system (Shimadzu, Japan) equipped with SPD-M20A PDA detector and C18 120 (250×4.60) mm column with 5 µm internal diameter. The fractions were filtered using 0.45 μ filter and sonicate prior to analysis. 20 µl sample was injected and chromatogram was developed using mobile phase methanol: deionized water in different combinations (80:20, 50:50, 60:40) with the constant flow at 0.5–1 ml/min. Detection of eluting compound was monitored in the range of 280–350 nm (Dubber and Kanfer, 2004).
Table 1 Phytochemical analysis of methanolic extract of C.carandas leaves. Phytochemicals
Test
Cc(L)M
Alkaloids
Mayer's test Wagner's test Dragendorff's test Hager's test Shinoda test Zinc HCl test Keller-Killiani test Borntrager's test Salkowski Ferric chloride test Gelatin test Froth formation test
+++ ++ +++ ++ + – – – +++ +++ ++ +++
Flavonoids
2.6.4. UV and FT-IR spectroscopic analysis The diluted sample of purified fraction was subjected to UV analysis using UV–visible spectrophotometer (Shimadzu 1750, Japan). The absorption spectrum of sample was taken in the spectral range of 200–800 nm (Kasal et al., 2010). However, FT-IR analysis of column eluted purified fraction was measured in the spectral range of 4000–400 cm−1. (Shimadzu 8202 PC, Japan). Samples were prepared using KBr as a matrix for FT-IR analysis. A small quantity of test sample
Glycosides Steroids and triterpenoids Tanins Saponins
(–) Not detected; (+) present in low concentration; (++) present in moderate concentration; (+++) present in high concentration.
218
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Fig. 4. Reducing power activity of methanolic extract of Cc(L)M, purified fraction CM 1 and standard ascorbic acid. Data is shown as mean ± SD (n=3).
Fig. 1. Calibration curve of gallic acid.
3.2.2. Reducing power assay The extracts (Cc(L)M and CM 1), as well as standard ascorbic acid, carried out the reduction of the Fe3+ to ferrous form which indicated by green or blue shades in the test sample. The results of this finding revealed that a dose-dependent activity (R2=0.962). However, the reducing potential of the test extracts, Cc(L)M and CM 1 at tested concentrations (10–100 µg/ml) was found to be lower than standard drug ascorbic acid (Fig. 4).
3.2.3. Hydroxyl radical scavenging assay The results of hydroxyl radical scavenging activity for Cc(L)M and CM 1 were found to be dose-dependent (R2=0.957). Among them, the activity of CM 1 (73.04 ± 0.27%) was greater than Cc(L)M (61.08 ± 0.49%) but slightly lower than standard drug ascorbic acid at 1000 µg/ml. The comparative OH- radical scavenging potential of control and test extracts is represented in Fig. 5. Table 2 represents EC50 values of standard drug, Cc(L)M and CM 1 for DPPH radical scavenging activity, reducing power and hydroxyl radical scavenging potential. 3.2.3.1. Erythrocyte membrane stabilization activity. Test extract Cc(L)M and purified fraction CM 1 showed a dose-dependent membrane stabilization activity (R2=0.960) in comparison with standard drug diclofenac sodium (Fig. 6). The result of this finding indicated that a dose of 3.5 mg/ml of purified fraction of CM 1 has maximum membrane stabilization activity (93.21 ± 0.72%) than Cc(L)M (92.13 ± 0.12%). The activity of CM 1 was also masked activity of standard drug (91.67 ± 0.15%) at tested concentration. EC50 values of Standard drug, Cc(L)M and CM 1 found to be 1.00 ± 0.012 mg/ml, 2.56 ± 0.009 mg/ml and 1.31 ± 0.012 mg/ml respectively.
Fig. 2. TLC bioautography using DPPH reagent.
Fig. 3. DPPH radical scavenging activity of methanolic extract of Cc(L)M, purified fraction CM 1 and standard ascorbic acid. Data is shown as mean ± SD (n=3).
M. The resulting activity of CM 1 was found to be equivalent to standard drug ascorbic acid (82.55 ± 1.15%) at concentration 1000 µg/ml. Fig. 5. Hydroxyl radical scavenging activity of methanolic extract of Cc(L)M, purified fraction CM 1 and standard ascorbic acid. Data is shown as mean ± SD (n=3).
219
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Table 2 EC50 values of Cc(L)M and CM 1 comparable with standard as a measure of antioxidant activity. Test extract
DPPH scavenging effect (µg/ml)
Reducing potential (µg/ml)
OH- scavenging effect (µg/ml)
Cc(L)M CM 1 Standard (Ascorbic acid)
630.4 ± 0.009 546.4 ± 0.011 365.8 ± 0.014
62.09 ± 0.008 57.48 ± 0.009 57.73 ± 0.008
606.9 ± 0.006 498.5 ± 0.012 469.8 ± 0.013
The values correspond to means ± SD of three replicates.
Fig. 6. Erythrocyte membrane stabilizing potential of methanolic extract of Cc(L)M, purified fraction CM 1 and standard diclofenac sodium. Data is shown as mean ± SD (n=3).
Fig. 7. TLC profile of crude extract Cc(L)M and purified fraction CM 1with Rf=0.44.
presence of functional groups. Hence in order to determine volatile bioactive compounds present in CM 1, it was subjected to GC-HRMS analysis. GC-HRMS spectra of CM 1 revealed presence of a major peak at 15.11 min with additional minor peaks (Fig. 10A). Resulting peak was identified as 20-hydroxypregnan 18-oic acid (Fig. 10B). Result indicated that CM 1 was steroid derivative as major phytoconstituent as per previous report (Mohamed et al., 2013). Its identification was duly confirmed by functional groups of steroids revealed in FT-IR analysis (Kasal et al., 2010).
3.3. Purification and characterization of bioactive compound 3.3.1. TLC and column chromatography Test extract of Cc(L)M was separated by TLC using various mobile phases. Better separation was obtained in mobile phase Chloroform: Glacial acetic acid: Methanol: Water (65:32:12:08). TLC result of Cc(L) M revealed presence number of phytochemicals characterized by different colour bands. After spraying with anisaldehyde-sulfuric acid reagent (Wagner and Bladt) followed by observation under UV (at 365 nm) revealed a sharp blue coloured band of Rf 0.44 (Fig. 7). Cc(L)M was further subjected to column chromatographic separation and eluted with same mobile phase that used for TLC separation. Total 81 fractions with 5 ml quantity were collected from column and simultaneously analyzed by TLC. Fractions 36–50 showed single band with Rf 0.44 with excellent bioactivities. Therefore, fraction 36–50 were mixed together and designated as CM 1. The remaining fractions did not reveal unique separation in TLC hence discarded.
4. Discussion Under stressful condition, human body produces reactive oxygen species (ROS) which leads to cell damage and health related problems (Aruoma, 1998; Steer et al., 2002; Peuchant et al., 2004). The results of present finding indicated that Cc(L)M and CM 1 has marked antioxidant and erythrocyte membrane stabilization potential. The antioxidant potential of compound is linked with formation of a non-radical form of DPPH-H (Oktay et al., 2003) as well as ability to donate hydrogen atom to break free radical chain formation (Duh et al., 1999). The activity of CM 1 found to be greater than Cc(L)M and comparable to standard drug. In erythrocyte membrane stabilization assay, standard drug as well as Cc(L)M and CM 1 were found to inhibit hemolysis of erythrocyte. The activity attributes to altered surface charges of the membrane or repels the charges which lead to hemolysis (Oyedapo et al., 2010). Various researchers have reported number of phytochemicals for their antioxidant potential. Diterpenoids from Calatropis procera exerted in vitro antioxidant and erythrocyte membrane stabilizing activity (Patil et al., 2016). Flavonoids (Pathak et al., 1991; Middleton, 1996) and saponins from Fagonia cretica are known to exert the anti-inflammatory potential by stabilizing lysosomal membrane (El-Shabrawy et al., 1997). This study for the first time reports new steroid derivative 20hydroxypregnan 18-oic acid for their marked antioxidant and erythrocyte membrane stabilizing potential. Our results are agreement with the earlier findings of Djeridane et al. (2010) where they proposed steroid derivative (17-(4-hydroxy-1,5-dimethylhexyl)-2,3,7-(acetyloxy) gona-
3.3.2. HPLC analysis In order to check the purity of the fraction CM 1, it was further analyzed by high performance liquid chromatography (HPLC). It revealed a major peak at 310 nm with RT 4.85 min. Moreover, numbers of minor peaks were also revealed when methanol: double distilled water (80:20) used as a mobile phase (Fig. 8). Reproducibility of the analysis was also checked by multiple repetitions. 3.3.3. UV and FT-IR analysis The result of UV–visible spectrophotometric analysis revealed major peak at 350 nm along with few minor peaks in the range of 200–400 nm (Fig. 9A). The results of FT-IR spectrophotometric analysis of CM 1 revealed presence of common bonds (Fig. 9B) in functional group region; for OH stretch at 3400, CH stretch at 2927, C=O stretch at 1728 and bond for aromatic ring at 1604 cm−1. However, bond present in fingerprint region for a C-O stretch at 1082 cm−1. 3.3.4. GC-HRMS analysis TLC and HPLC analyses of CM 1 revealed its purity, FT-IR revealed 220
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Fig. 8. HPLC profile of purified fraction CM1.
dichloromethane and toluene extract of C.carandas were used for antimicrobial activity (Verma and Chaudhary, 2011). Verma et al. (2015) studied DNA damage inhibition using methanolic extract of C.carandas leaves. Besides, ethanolic extract of C. carandas fruits and hydroalcoholic extract of roots were used to study antiemetic, analgesic, anti-inflammatory as well as antipyretic activities (Bhaskar and Balakrishnan, 2009). Galipalli et al. (2015) carried out bioassay guided fractionation of methanolic extract of C.carandas roots. In this study they investigated lupeol, carrisone, stigmasterol and scopoletin as
1,3,5(10)-trien-15-ol) from Cleome arabica for its powerful antioxidant potential than six different tested standard antioxidant drugs. Steroids exert their vital role against oxidative stress (Reyes et al., 2006; Vento et al., 2009). A detailed review of Patel and Savjani (2015) reports antiinflammatory potential of steroids from Trigonella foenum graecum, Solanum xanthocarpum, Boswellia serrata, Glycyrrhiza glabra, Commiphora mukul and Withania somnifera. In earlier studies, crude extract of different parts of C.carandas were used to investigate various biological activities. Aqueous as well as
Fig. 9. (A) UV spectrum of CM 1, (B) FT-IR spectrum of CM 1.
221
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Fig. 10. (A) Gas chromatogram of CM 1, (B) Mass spectrum analyses of purified fraction CM 1. Dubber, M.J., Kanfer, I., 2004. High-performance liquid chromatographic determination of selected flavonols in Ginkgo biloba solid oral dosage forms. J. Pharm. Pharm. Sci. 7, 303–309. Duh, P.D., Tu, Y.Y., Yen, G.C., 1999. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum morifolium Ramat). LWT-Food Sci. Technol. 32 (5), 269–277. El-Shabrawy, O.A., El-Gindi, O.D., Melek, F.R., Abdel-Khalik, S.M., Haggag, M.Y., 1997. Biological properties of saponin mixtures of Fagonia cretica and Fagonia mollis. Fitoterapia 68 (3), 219–222. Galipalli, S., Patel, N.K., Prasanna, K., Bhutani, K.K., 2015. Activity-guided investigation of Carissa carandas (L.) roots for anti-inflammatory constituents. Nat. Prod. Res. 29 (17), 1670–1672. Gandhidasan, R., Thamaraichelvan, A., Baburaj, S., 1991. Anti-inflammatory action of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia 62, 81–83. Gülçin, I., 2007. Comparison of in vitro antioxidant and antiradical activities of L-tyrosine and L-Dopa. Amino Acids 32 (3), 431–438. Halliwell, B., 2007. Biochemistry of oxidative stress. Biochem. Soc. Trans. 35 (5), 1147–1150. Heim, K.E., Tagliaferro, A.R., Bobilya, D.J., 2002. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J. Nutr. Biochem. 13 (10), 572–584. Itankar, P.R., Lokhande, S.J., Verma, P.R., Arora, S.K., Sahu, R.A., Patil, A.T., 2011. Antidiabetic potential of unripe Carissa carandas Linn. fruit extract. J. Ethnopharmacol. 135 (2), 430–433. Kasal, A., Budesinsky, M., Griffiths, W.J., 2010. Spectroscopic Methods of Steroid Analysis. In Steroid Analysis. Springer, Netherlands, pp. 27–161. Kinnula, V.L., Crapo, J.D., 2004. Superoxide dismutases in malignant cells and human tumors. Free Radic. Biol. Med. 36 (6), 718–744. Kirtikar, K.R., Basu, B.D., 1987. ICS. Indian medicinal plants. Blatter, E., Caius, J.F., Mhaskar, K.S., editors, 3, pp. 707–1708. Kokate, C.K., Purohit, A.P., Gokhale, S.B., 1995. Methods of Crude Drug Evaluation, Pharmacognosy 10. Nirali Prakasan, Pune, pp. 88–99. Krishnaiah, D., Sarbatly, R., Nithyanandam, R., 2011. A review of the antioxidant potential of medicinal plant species. Food Bioprod. Process. 89 (3), 217–233. Kumar, V., Bhat, Z.A., Kumar, D., Khan, N.A., Chashoo, I.A., 2012. Evaluation of antiinflammatory potential of leaf extracts of Skimmia anquetilia. Asian Pac. J. Trop. Biomed. 2 (8), 627–630. Lavanya, R., Maheshwari, S.U., Harish, G., Raj, J.B., Kamali, S., Hemamalani, D., Varma, J.B., Reddy, C.U., 2010. Investigation of in-vitro anti-inflammatory, anti-platelet and anti-arthritic activities in the leaves of Anisomeles malabarica Linn. Res. J. Pharm. Biol. Chem. Sci. 1 (4), 745–752. Liu, H.X., Sun, S.Q., Lv, G.H., Chan, K.K., 2006. Study on Angelica and its different extracts by Fourier transform infrared spectroscopy and two-dimensional correlation IR spectroscopy. Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 64 (2), 321–326. Madhavi, D.L., Salunkhe, D.K., 1995. Toxicological aspects of food antioxidants. Food Science and Technology-New York-Marcel Dekker, pp. 267–360. Matteo, V., Esposito, E., 2003. Biochemical and therapeutic effects of antioxidants in the treatment of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Curr. Drug Targets-CNS Neurol. Disord. 2 (2), 95–107. McCune, L.M., Johns, T., 2002. Antioxidant activity in medicinal plants associated with
potent anti-inflammatory agents against proinflammatory mediators like TNF-α, IL-1b and nitrous oxide. However, the antioxidant and erythrocyte membrane stabilization activity of purified fraction from leaves extract of this plant has not been reported so far. As per our knowledge, this is first attempt to report antioxidant and erythrocyte membrane stabilizing activity of steroidal derivative from methanolic extract of C. carandas leaves. 5. Conclusion It can be concluded the purified fraction CM 1 from C.carandas has potent in vitro antioxidant and erythrocyte membrane stabilizing potential. Both activities of CM 1 may attribute to presence of steroid derivative in it. Moreover, activities reported by steroid derivative will lead to develop potential drug candidate or commercial food additives. However, further in vivo studies with the precise mechanism of this bioactive compound are needed to validate its commercial exploration. Conflict of interest Authors declare that there are no conflicts of interest. References Aruoma, O.I., 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. J. Am. Oil Chem. Soc. 75 (2), 199–212. Balakrishnan, N., Balasubaramaniam, A., Sangameswaran, B., Bhaskar, V., 2011. Hepatoprotective activity of two Indian medicinal plants from Western Ghats, Tamil Nadu. J. Nat. Pharm. 2 (2), 92–98. Bhaskar, V.H., Balakrishnan, N., 2009. Analgesic, anti-inflammatory and antipyretic activities of Pergularia daemia and Carissa carandas. DARU J. Pharm. Sci. 17 (3), 168–174. Darmanyan, A.P., Gregory, D.D., Guo, Y., Jenks, W.S., Burel, L., Eloy, D., Jardon, P., 1998. Quenching of singlet oxygen by oxygen-and sulfur-centered radicals: evidence for energy transfer to peroxyl radicals in solution. J. Am. Chem. Soc. 120 (2), 396–403. Djeridane, A., Yousfi, M., Brunel, J.M., Stocker, P., 2010. Isolation and characterization of a new steroid derivative as a powerful antioxidant from Cleome arabica in screening the in vitro antioxidant capacity of 18 Algerian medicinal plants. Food Chem. Toxicol. 48 (10), 2599–2606.
222
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
product of browning reaction prepared from glucosamine. Jpn. J. Nutr. 44, 307–315. Rackova, L., Oblozinsky, M., Kostalova, D., Kettmann, V., Bezakova, L., 2007. Free radical scavenging activity and lipoxygenase inhibition of Mahonia aquifolium extract and isoquinoline alkaloids. J. Inflamm. 4 (1), 1–7. Reyes, M.R., Sifuentes-Alvarez, A., Lazalde, B., 2006. Estrogens are potentially the only steroids with an antioxidant role in pregnancy: in vitro evidence. Acta Obstet. Et. Gynecol. Scand. 85 (9), 1090–1093. Sadique, J., Al-Rqobah, W.A., Bughaith, M.F., El-Gindy, A.R., 1989. The bio-activity of certain medicinal plants on the stabilization of RBC membrane system. Fitoterapia 60, 525–532. Shimada, K., Fujikawa, K., Yahara, K., Nakamura, T., 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40 (6), 945–948. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Vitic. 16 (3), 144–158. Smirnoff, N., Cumbes, Q.J., 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28 (4), 1057–1060. Sreejayan, N., Rao, M.N., 1996. Free radical scavenging activity of curcuminoids. Arzneim. Forsch. 46 (2), 169–171. Steer, P., Millgård, J., Sarabi, D.M., Basu, S., Vessby, B., Kahan, T., Edner, M., Lind, L., 2002. Cardiac and vascular structure and function are related to lipid peroxidation and metabolism. Lipids 37 (3), 231–236. Syed, I.T., Gopalakrishnan, S., Hazeena, B.V., 1997. Biochemical modes of action of Gmelina asiatica in inflammation. Indian J. Pharmacol. 29 (5), 306–309. Takao, T., Kitatani, F., Watanabe, N., Yagi, A., Sakata, K., 1994. A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shellfish. Biosci. Biotechnol. Biochem. 58 (10), 1780–1783. Vento, M., Aguar, M., Escobar, J., Arduini, A., Escrig, R., Brugada, M., Izquierdo, I., Asensi, M.A., Sastre, J., Saenz, P., Gimeno, A., 2009. Antenatal steroids and antioxidant enzyme activity in preterm infants: influence of gender and timing. Antioxid. Redox Signal. 11 (12), 2945–2955. Verma, K., Shrivastava, D., Kumar, G., 2015. Antioxidant activity and DNA damage inhibition in vitro by a methanolic extract of Carissa carandas (Apocynaceae) leaves. J. Taibah Univ. Sci. 9 (1), 34–40. Verma, S., Chaudhary, H.S., 2011. Effect of Carissa carandas against clinically pathogenic bacterial strains. J. Pharm. Res. 4 (10), 3769–3771. Wagner, H., Bladt, S., 1996. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer Science & Business Media, Berlin-Heidelberg. Wang, J., Yue, Y.D., Tang, F., Sun, J., 2012. TLC screening for antioxidant activity of extracts from fifteen bamboo species and identification of antioxidant flavone glycosides from leaves of Bambusa textilis McClure. Molecules 17 (10), 12297–12311. Were, P.S., Waudo, W., Ozwara, H.S., Kutima, H.L., 2015. Phytochemical analysis of Warburgia ugandensis Sprague using Fourier transform infra-red (FT-IR) spectroscopy. Int. J. Pharmacogn. Phytochem. Res. 7, 201–205. Wichi, H.P., 1988. Enhanced tumor development by butylated hydroxyanisole (BHA) from the prospective of effect on forestomach and oesophageal squamous epithelium. Food Chem. Toxicol. 26, 717–723.
the symptoms of diabetes mellitus used by the indigenous peoples of the North American boreal forest. J. Ethnopharmacol. 82 (2), 197–205. Middleton Jr., M.D., E., 1996. Biological properties of plant flavonoids: an overview. Int. J. Pharmacogn. 34 (5), 344–348. Mishra, C.K., Sasmal, D., Shrivastava, B., 2012. An In vitro evaluation of the anthelmintic activity of unripe fruits extract of Carissa carandas Linn. Int. J. Drug Dev. Res. 4 (4), 393–397. Mohamed, S.S., El-Refai, A.M.H., El-Raoof Sallam, L.A., Abo-Zied, K.M., Hashem, A.G.M., Ali, H.A., 2013. Biotransformation of progesterone to hydroxysteroid derivatives by whole cells of Mucor racemosus. Malay J. Microbiol. 9, 237–244. Motwani, S.M., Pandey, E.P., Sonchhatra, N.P., Desai, T.R., Patel, V.L., Pandya, D.J., 2012. Pharmacognostic and phytochemical study of aerial parts of Carissa carandas. Int. J. Biol. Pharm. Res. 3 (1), 75–81. Naim, Z., Khan, M.A., Nizami, S.S., 1988. Isolation of a new isomer of ursolic acid from fruits and leaves of Carissa carandas. Pak. J. Sci. Ind. Res. 31, 753–755. Ode, O.J., Asuzu, I.U., Ajayi, I., 2011. Bioassay-guided fractionation of the crude methanol extract of Cassia singueana leaves. J. Adv. Sci. Res. 2 (4), 81–86. Oktay, M., Gülçin, İ., Küfrevioğlu, Ö.İ., 2003. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. LWT-Food Sci. Technol. 36 (2), 263–271. Olech, M., Komsta, Ł., Nowak, R., Cieśla, Ł., Waksmundzka-Hajnos, M., 2012. Investigation of antiradical activity of plant material by thin-layer chromatography with image processing. Food Chem. 132 (1), 549–553. Osadebe, P.O., Okoye, F.B.C., 2003. Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves. J. Ethnopharmacol. 89 (1), 19–24. Oyedapo, O.O., Akinpelu, B.A., Orefuwa, S.O., 2004. Anti-inflammatory effect of Theobroma cacao, root extract. Trop. Med. Plants 5 (2), 161–166. Oyedapo, O.O., Akinpelu, B.A., Akinwunmi, K.F., Adeyinka, M.O., Sipeolu, F.O., 2010. Red blood cell membrane stabilizing potentials of extracts of Lantana camara and its fractions. Int. J. Plant Physiol. Biochem. 2 (4), 46–51. Pakrashi, S.C., Datta, S., Ghosh-Dastidar, P.P., 1968. Indian medicinal plants—XVII. 3 phytochemical examination of Carissa spp. Phytochemistry 7 (3), 495–496. Patel, S.S., Savjani, J.K., 2015. Systemetic review of plants steroids as potential antiinflammatory agent: current status and future perspectives. J. Phytopharm. 4, 121–125. Pathak, D., Pathak, K., Singla, A.K., 1991. Flavonoids as medicinal agents-recent advances. Fitoterapia 62, 371–389. Patil, R.P., Pai, S.R., Pawar, N.V., Shimpale, V.B., Patil, R.M., Nimbalkar, M.S., 2012. Chemical characterization, mineral analysis, and antioxidant potential of two underutilized berries (Carissa carandus and Eleagnus conferta) from the Western Ghats of India. Crit. Rev. Food Sci. Nutr. 52 (4), 312–320. Patil, S.G., Patil, M.P., Patil, R.H., 2016. In vitro anti-hypercholesterolemic activity of Calotropis procera (Aiton) using human erythrocytes. Biocatal. Agric. Biotechnol. 5, 104–110. Peuchant, E., Brun, J.L., Rigalleau, V., Dubourg, L., Thomas, M.J., Daniel, J.Y., Leng, J.J., Gin, H., 2004. Oxidative and antioxidative status in pregnant women with either gestational or type 1 diabetes. Clin. Biochem. 37 (4), 293–298. Qyaizu, M., 1986. Studies on products of browning reactions: antioxidative activities of
223
Contents lists available at ScienceDirect
Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Isolation, purification and characterization of antioxidative steroid derivative from methanolic extract of Carissa carandas (L.) leaves Bhushan S. Bhadane, Ravindra H. Patil
MARK
⁎
Department of Microbiology and Biotechnology R. C. Patel Arts, Commerce and Science College, Shirpur 425405 Maharashtra, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Phytochemicals Bioautography DPPH FT-IR and GC-HRMS
Present study evaluates antioxidative and erythrocyte membrane stabilizing potential of methanol extract of Carissa carandas leaves Cc(L)M and its purified fraction CM 1. Phytochemically Cc(L)M revealed presence of alkaloid, steroids, saponins and tanins. The results of antioxidant assays of Cc(L)M and CM 1 found to be dose dependent. However, in TLC bioautography, CM 1 showed yellow coloured scavenged band of DPPH reagent. The results revealed that CM 1 has significant (p≤0.05) free radical scavenging potential compared to Cc(L)M. The purified fraction CM 1 was found to be most potent with lowest EC50 values for DPPH (546.4 µg/ml), OH(498.5 µg/ml) radical scavenging and 57.48 µg/ml for reducing power assay. It also erythrocytes membrane stabilization effect with lowest EC50 1.31 mg/ml as compared to Cc(L)M (2.56 mg/ml) and standard diclofenac sodium (1.00 mg/ml). The FT-IR and GC-HRMS analysis of CM 1 reveal the presence of steroid derivative 20hydroxypregnan 18-oic acid.
1. Introduction
of biological membranes and tissue damage (Lavanya et al., 2010). The drugs which commonly used to treat inflammation are non-steroidal anti-inflammatory drugs which are having adverse effects like gastric ulcers (Kumar et al., 2012). Earlier reports suggests that herbal drug containing principles had ability to stabilize biological membranes when exposed to induced lyses (Sadique et al., 1989; Gandhidasan et al., 1991; Syed et al., 1997). Hence, there is increase in demand of plant derived anti-inflammatory drugs to treat inflammatory conditions. C.carandas belongs to family Apocynaceae; native and common throughout India, Sri Lanka, Java, Malaysia, Myanmar and Pakistan (Motwani et al., 2012). Different parts of this plant are reported for pharmacological activities like spasmolytic, analgesic, anti-inflammatory, Antihyperglycemic, hepato-protective, antipyretic and cardiotonic (Kirtikar and Basu, 1987; Mishra et al., 2012; Balakrishnan et al., 2011) and purgative and snake bite antidote (Pakrashi et al., 1968). Different parts of Carissa carandas are enriched with wider group of phytochemicals. The roots comprises Triterpenes like lupeol, ursolic acid, αamyrin and oleanolic acid. Sesquiterpene, β-sitosterol and cholest-5-en3β-ol are also reported (Itankar et al., 2011). Moreover, fruits were enriched with flavonoids like rutin, epicatechin, quercetin and kaemferol. Patil et al. (2012) reported phenolics like syringic acid, vanillic acid and caffeic acid in the ripened fruits of C. carandas. Leaves of the plant were found to contain triterpene such as betulinic acid (Naim et al., 1988).
Reactive Oxygen Species (ROS) are derived from the metabolism of oxygen. Free radicals such as hydroxyl, superoxide and hydrogen peroxide are generated as byproducts of biological reactions or from exogenous factors are act as ROS (Krishnaiah et al., 2011). The free radicals mostly attack membrane lipid, protein, and DNA and are ultimately responsible for many disorders (Rackova et al., 2007) such as cancer, diabetes mellitus (Kinnula and Crapo, 2004; McCune and Johns, 2002), neurogenerative and inflammatory diseases (Matteo and Esposito, 2003; Sreejayan and Rao, 1996). The antioxidants are the substances that delays, prevent or removes oxidative damage (Gulcin, 2007). The antioxidants exert their activity by inhibiting free radical oxidation, act as chain-breaking antioxidants, singlet oxygen quenchers and reducing agents (Halliwell, 2007; Darmanyan et al., 1998; Heim et al., 2002). However, use of synthetic antioxidants such as BHA and BHT has been restricted because they may be responsible for liver damage and carcinogenesis (Wichi, 1988; Madhavi and Salunkhe, 1995). Therefore, interest in the use of natural antioxidants has been increased. Inflammation is sequential process induced by biological stimuli or tissue injury caused by noxious chemical, physical trauma or microbiological agents (Osadebe and Okoye, 2003). In inflammatory conditions ROS release from activated neutrophils and macrophages. Overproduction of these ROS attributed to tissue injury by lipid peroxidation
⁎
Corresponding author. E-mail address: [email protected] (R.H. Patil).
http://dx.doi.org/10.1016/j.bcab.2017.03.012 Received 14 February 2017; Received in revised form 14 March 2017; Accepted 18 March 2017 Available online 20 March 2017 1878-8181/ © 2017 Published by Elsevier Ltd.
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
The aim of present study was to evaluate in vitro antioxidant and erythrocyte membrane stabilizing potential of C.carandas leaf extract and its purified fraction. The attempt has also been made to purify and partially characterize bioactive compound by chromatographic and spectroscopic methods. Identification and structural confirmation of bioactive compound was done by GC-HRMS analysis.
the reaction mixture indicates higher free radical scavenging activity. The scavenging activity of DPPH radical was calculated using the following equation:
DPPH scavenging effect(%) = [(A 0 − A1/A 0) × 100] where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample. Each test was carried out in three replicates. The EC50 value of DPPH was determined by plotting values of percent scavenging against extract concentration. The EC50 indicates the concentration of antioxidant that scavenges DPPH radical about 50%.
2. Materials and methods 2.1. Collection and extraction of plant material Leaves of the healthy plant of C.carandas were collected as per standard procedure from Aner river forest region (21.26°N, 75.11°E) Dhule district, India. The plant specimen was identified and authenticated by plant taxonomist; Herbarium of the specimens (RCP/DBT-05/ 2012) was deposited at the Department of Biotechnology R. C. Patel Arts, Commerce, and Science College, Shirpur, MS, India. Leaves were washed to dry in dark at room temperature and further ground to obtain fine powder. The powdered leaves (500 g) were defatted using hexane by soxhlet method. Residue remains after defatting was subjected to extraction using methanol for 48 h. After extraction, solvent was evaporated under reduced pressure using rotary vacuum evaporator (Equitron, Mumbai, India) to afford a gummy solid residue. The resulting extract was stored in a refrigerator at 4 °C until use.
2.4.2. Reducing power assay The reducing power of test extract was determined according to the method previously described by Qyaizu (1986). Different concentrations (10–100 μg/ml) of test extract and standard ascorbic acid (1 ml) mixed with 2.5 ml, 0.2 M phosphate buffer (pH 6.6) and 2.5 ml potassium ferricyanide (1%). The mixture was incubated at 50 °C for 20 min. A portion of 2.5 ml of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The supernatant (2.5 ml) of the solution was mixed with 2.5 ml distilled water and 0.5 ml FeCl3 (1%) and the absorbance of reaction mixtures was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid was used as a standard. Phosphate buffer was used as a blank. Each test was carried out in three replicates. The EC50 value of reducing power assay was determined by plotting values of absorbance against extract concentration. The EC50 indicates the concentration of antioxidant that reduces ferric ion about 50%.
2.2. Phytochemical screening The phytochemical analysis of the plant extract for major phytoconstituents was done as per the published procedures (Kokate et al., 1995). Test extract was screened for the presence of phytochemicals like alkaloids, flavonoids glycoside, steroids and triterpenoids, saponins, tannins etc.
2.4.3. Hydroxyl radical scavenging assay Hydroxyl radical scavenging activity of the test extract was measured by the salicylic acid method as described by Smirnoff and Cumbes (1989) with slight modifications. Reaction mixture contained 1.0 ml of 1.5 mM FeSO4, 0.7 ml of 6 mM H2O2, 0.3 ml of 20 mM sodium salicylate and 1 ml of test extract. After incubation at 37 °C for 60 min, absence of the hydroxylated salicylate complex was measured at 562 nm. Scavenging activity was determined as follows.
2.3. Total phenols Total phenolic content of crude extract was determined spectrophotometrically using the method of Singleton and Rossi (1965) with little modification. 1 ml of 10% Folin-ciocalteu's reagent was added to 1 ml crude extract (1000 μg/ml) and mixed thoroughly. To the mixture, 4 ml sodium carbonate (7.5%) and 10 ml of distilled water was added and allowed to stand for 2 h. The absorbance of reaction mixture was recorded at 760 nm. A standard curve was obtained using various concentrations of gallic acid (10–100 μg/ml). Total phenols were expressed as mg of GAE gallic acid equivalent/gm of extract.
Scavenging ability(%) = [(T1 −T2 / T1) × 100] where T1 was the absorbance of the control reaction and T2 the absorbance in the presence of the crude extract. Each test was carried out in three replicates. Ascorbic acid was used as positive controls. The EC50 value of hydroxyl radical was determined by plotting the values of percent scavenging against extract concentration. The EC50 indicates the concentration of antioxidant that scavenges hydroxyl radicals about 50%.
2.4. Antioxidant activity 2.4.1. Free radical scavenging activity by DPPH method 2.4.1.1. Qualitative assay. TLC bioautography method (Takao et al., 1994; Wang et al., 2012; Olech et al., 2012) was used to determine free radical scavenging activity of test extract. Briefly, the test extracts were separated by suitable solvent system. After TLC separation, the developed plates were sprayed with 0.2% DPPH solution prepared in methanol. Plates were then kept in dark for 30 min and examined in daylight. The formation of a yellow coloured spot against violet coloured background indicated DPPH radical scavenging activity.
2.5. Erythrocyte membrane stabilization assay Erythrocyte membrane stabilization assay was carried out using human red blood cells (Oyedapo et al., 2010). Briefly, blood from a healthy human was collected in vials containing an anticoagulant (0.8% sodium citrate) and centrifuged at 3000 rpm for 10 min. The supernatant containing plasma or leucocytes were carefully removed and RBCs pelleted at the bottom of the vial were washed in isosaline (0.85%, w/v, NaCl) for four to five times or until a clear supernatant is obtained. Hematocrit (2%, v/v) were prepared and used for membrane stabilization assay (Oyedapo et al., 2004). Different concentrations (0.5–3.5 mg/ml) of test extract and standard drug diclofenac sodium (1 ml) were prepared in isosaline and mixed with 2 ml of hyposaline (0.25%, w/v, NaCl), 1 ml of 0.15 M sodium phosphate buffer (pH 7.4), 0.5 ml of 2% hematocrit. Drug control and blood control were also prepared. Drugs were omitted in the blood control, while the drug control did not contain the RBC suspension. All the reaction mixtures with a final volume of 4.5 ml were incubated at 56 °C for 30 min in
2.4.1.2. Quantitative assay. Free radical scavenging activity of test extract was determined by using 1, 1 diphenyl 2 picrylhydrazyl (DPPH) assay (Shimada et al., 1992). Briefly, extract concentrations (100–1000 μg/ml) with final volume 4 ml was mixed with 1 ml 0.2 mM methanolic solution of 1, 1 diphenyl 2 picrylhydrazyl (DPPH). The mixture was shaken vigorously and left to stand for 30 min in the dark at room temperature. After incubation, the absorbance of the reaction mixture was recorded spectrophotometrically (Shimadzu 1750) at 517 nm using methanol as blank. The decrease in the absorbance of 217
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
was mixed with dry potassium bromide (KBr) to homogenized and dried in mortar and pestle. The homogenized mixture was placed on the disc to form a KBr pellet and FT-IR spectrum was analyzed (Liu et al., 2006; Were et al., 2015).
water bath. After incubation, mixture was centrifuged at 5000 rpm for 10 min. The supernatant was taken in a separate tube and the absorbance of the supernatant was recorded at 560 nm. The percent membrane stability was estimated using following equation.
%Membranestabil 100–(Absorbanceoftestdrug –Absorbanceofdrugcontrol ) = × 100 (Absorbanceofbloodcontrol )
2.6.5. GC-HRMS analysis GC-HRMS analysis of column eluted fraction was carried out using Agilent 7890-5 GC (Santa Clara, USA) fused with HP-5 silica capillary column (30 m×0.20 mm, phase thickness 0.25 µm) coupled with a mass spectrometer (Jeol Accu TOF GCV) and FID detector. The inlet temperature was at 250 °C. The injection volume was 10 µl injected in split mode with a ratio of 20:80 and the flow rate of carrier gas was maintained at 1 ml per minute. The identification of compounds present in column eluted bioactive fraction was compared using NIST 05 library search database.
The EC50 value of this assay was determined by plotting the values of percent protection against extract concentration. The EC50 indicates the concentration of antioxidant that stabilizes erythrocytes membrane about 50%. 2.6. Purification and characterization of bioactive compound 2.6.1. Thin layer chromatography (TLC) The methanolic extract (10 µl) of C.carandas leaves was spotted on the TLC plates using Spraylin V automatic sample applicator (Aetron, India). The spots were air dried and the plate was developed in a presaturated chamber containing different mobile phases (Wagner and Bladt, 1996). Chromatographic separation was carried out at room temperature in ascending manner using mobile phases such as; toluene: ethyl acetate (7:2), toluene: ethyl acetate: diethylamine (7:2:1); Chloroform: ethyl acetate (7:3); Chloroform:Glacial acetic acid:Methanol:Water (65:32:12:8); Ethyl acetate:Formic acid: Glacial acetic acid:Water (100:11:11:26). After chromatographic separation plates were derivatized using 10% ethanolic sulfuric acid or anisaldehyde sulfuric acid reagent and heated in a hot air oven to further visualized in UV and daylight.
2.7. Statistical analysis Values are means ± SD of three consistent readings of each experiment. Data from all the experiments further subjected to Analysis of variance (ANOVA) to determine the significance variations. The means were compared according to Dunnet test (P≤0.05). The processing of data to present the results was done using XLSTAT version 16 (Addinsoft, US). 3. Results 3.1. Phytochemical screening and phenolic content Results of preliminary phytochemical screening in Cc(L)M showed dominantly presence of alkaloids, steroids, saponins and tannins and moderately presence of flavonoids (Table 1). However, total phenolic content of Cc(L)M was obtained from linear regression analysis equation (y=0.0027x+0.0141; R2=0.956) from the standard graph (Fig. 1) of gallic acid and it was found to be 102.33 mg GAE/gm of extract.
2.6.2. Column chromatography The mobile phase which gave better separation in TLC was used for elution of column. The chromatographic separation was carried out in silica 60–120 mesh size column chromatography. The silica slurry prepared in mobile phase was poured into column (30×1.8 cm) and improve column packing by tapped it from outside. After column packing, test extract prepared in dry silica was loaded on the top of column bed to get even layer (Ode et al., 2011). After loading of sample, mobile phase was poured through side wall of column and fractions were collected in 5 ml quantity. During elution, the constant flow rate of eluent was maintained for all fractions. Simultaneously, TLC of eluted fraction was carried out to check presence of single compound.
3.2. Antioxidant activity 3.2.1. DPPH radical scavenging assay TLC bioautography of Cc(L)M and purified fraction CM 1 revealed yellow coloured band as a result of DPPH scavenging (Fig. 2). The quantitative assay of Cc(L)M and CM 1 showed a dose dependent (R2=0.952) radical scavenging activity (Fig. 3). The maximum DPPH radical scavenging activity of Cc(L)M and CM 1 was obtained at 1000 µg/ml with scavenging effect of 77.08 ± 0.66% and 81.87 ± 0.27% respectively. Results of present findings indicated that CM 1 was found to be more effective DPPH radical scavenger than Cc(L)
2.6.3. HPLC analysis The fraction obtained from column chromatography was subjected to HPLC analysis in order to check purity of resulting fraction. HPLC was carried out using, LC 20 Ad liquid chromatography system (Shimadzu, Japan) equipped with SPD-M20A PDA detector and C18 120 (250×4.60) mm column with 5 µm internal diameter. The fractions were filtered using 0.45 μ filter and sonicate prior to analysis. 20 µl sample was injected and chromatogram was developed using mobile phase methanol: deionized water in different combinations (80:20, 50:50, 60:40) with the constant flow at 0.5–1 ml/min. Detection of eluting compound was monitored in the range of 280–350 nm (Dubber and Kanfer, 2004).
Table 1 Phytochemical analysis of methanolic extract of C.carandas leaves. Phytochemicals
Test
Cc(L)M
Alkaloids
Mayer's test Wagner's test Dragendorff's test Hager's test Shinoda test Zinc HCl test Keller-Killiani test Borntrager's test Salkowski Ferric chloride test Gelatin test Froth formation test
+++ ++ +++ ++ + – – – +++ +++ ++ +++
Flavonoids
2.6.4. UV and FT-IR spectroscopic analysis The diluted sample of purified fraction was subjected to UV analysis using UV–visible spectrophotometer (Shimadzu 1750, Japan). The absorption spectrum of sample was taken in the spectral range of 200–800 nm (Kasal et al., 2010). However, FT-IR analysis of column eluted purified fraction was measured in the spectral range of 4000–400 cm−1. (Shimadzu 8202 PC, Japan). Samples were prepared using KBr as a matrix for FT-IR analysis. A small quantity of test sample
Glycosides Steroids and triterpenoids Tanins Saponins
(–) Not detected; (+) present in low concentration; (++) present in moderate concentration; (+++) present in high concentration.
218
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Fig. 4. Reducing power activity of methanolic extract of Cc(L)M, purified fraction CM 1 and standard ascorbic acid. Data is shown as mean ± SD (n=3).
Fig. 1. Calibration curve of gallic acid.
3.2.2. Reducing power assay The extracts (Cc(L)M and CM 1), as well as standard ascorbic acid, carried out the reduction of the Fe3+ to ferrous form which indicated by green or blue shades in the test sample. The results of this finding revealed that a dose-dependent activity (R2=0.962). However, the reducing potential of the test extracts, Cc(L)M and CM 1 at tested concentrations (10–100 µg/ml) was found to be lower than standard drug ascorbic acid (Fig. 4).
3.2.3. Hydroxyl radical scavenging assay The results of hydroxyl radical scavenging activity for Cc(L)M and CM 1 were found to be dose-dependent (R2=0.957). Among them, the activity of CM 1 (73.04 ± 0.27%) was greater than Cc(L)M (61.08 ± 0.49%) but slightly lower than standard drug ascorbic acid at 1000 µg/ml. The comparative OH- radical scavenging potential of control and test extracts is represented in Fig. 5. Table 2 represents EC50 values of standard drug, Cc(L)M and CM 1 for DPPH radical scavenging activity, reducing power and hydroxyl radical scavenging potential. 3.2.3.1. Erythrocyte membrane stabilization activity. Test extract Cc(L)M and purified fraction CM 1 showed a dose-dependent membrane stabilization activity (R2=0.960) in comparison with standard drug diclofenac sodium (Fig. 6). The result of this finding indicated that a dose of 3.5 mg/ml of purified fraction of CM 1 has maximum membrane stabilization activity (93.21 ± 0.72%) than Cc(L)M (92.13 ± 0.12%). The activity of CM 1 was also masked activity of standard drug (91.67 ± 0.15%) at tested concentration. EC50 values of Standard drug, Cc(L)M and CM 1 found to be 1.00 ± 0.012 mg/ml, 2.56 ± 0.009 mg/ml and 1.31 ± 0.012 mg/ml respectively.
Fig. 2. TLC bioautography using DPPH reagent.
Fig. 3. DPPH radical scavenging activity of methanolic extract of Cc(L)M, purified fraction CM 1 and standard ascorbic acid. Data is shown as mean ± SD (n=3).
M. The resulting activity of CM 1 was found to be equivalent to standard drug ascorbic acid (82.55 ± 1.15%) at concentration 1000 µg/ml. Fig. 5. Hydroxyl radical scavenging activity of methanolic extract of Cc(L)M, purified fraction CM 1 and standard ascorbic acid. Data is shown as mean ± SD (n=3).
219
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Table 2 EC50 values of Cc(L)M and CM 1 comparable with standard as a measure of antioxidant activity. Test extract
DPPH scavenging effect (µg/ml)
Reducing potential (µg/ml)
OH- scavenging effect (µg/ml)
Cc(L)M CM 1 Standard (Ascorbic acid)
630.4 ± 0.009 546.4 ± 0.011 365.8 ± 0.014
62.09 ± 0.008 57.48 ± 0.009 57.73 ± 0.008
606.9 ± 0.006 498.5 ± 0.012 469.8 ± 0.013
The values correspond to means ± SD of three replicates.
Fig. 6. Erythrocyte membrane stabilizing potential of methanolic extract of Cc(L)M, purified fraction CM 1 and standard diclofenac sodium. Data is shown as mean ± SD (n=3).
Fig. 7. TLC profile of crude extract Cc(L)M and purified fraction CM 1with Rf=0.44.
presence of functional groups. Hence in order to determine volatile bioactive compounds present in CM 1, it was subjected to GC-HRMS analysis. GC-HRMS spectra of CM 1 revealed presence of a major peak at 15.11 min with additional minor peaks (Fig. 10A). Resulting peak was identified as 20-hydroxypregnan 18-oic acid (Fig. 10B). Result indicated that CM 1 was steroid derivative as major phytoconstituent as per previous report (Mohamed et al., 2013). Its identification was duly confirmed by functional groups of steroids revealed in FT-IR analysis (Kasal et al., 2010).
3.3. Purification and characterization of bioactive compound 3.3.1. TLC and column chromatography Test extract of Cc(L)M was separated by TLC using various mobile phases. Better separation was obtained in mobile phase Chloroform: Glacial acetic acid: Methanol: Water (65:32:12:08). TLC result of Cc(L) M revealed presence number of phytochemicals characterized by different colour bands. After spraying with anisaldehyde-sulfuric acid reagent (Wagner and Bladt) followed by observation under UV (at 365 nm) revealed a sharp blue coloured band of Rf 0.44 (Fig. 7). Cc(L)M was further subjected to column chromatographic separation and eluted with same mobile phase that used for TLC separation. Total 81 fractions with 5 ml quantity were collected from column and simultaneously analyzed by TLC. Fractions 36–50 showed single band with Rf 0.44 with excellent bioactivities. Therefore, fraction 36–50 were mixed together and designated as CM 1. The remaining fractions did not reveal unique separation in TLC hence discarded.
4. Discussion Under stressful condition, human body produces reactive oxygen species (ROS) which leads to cell damage and health related problems (Aruoma, 1998; Steer et al., 2002; Peuchant et al., 2004). The results of present finding indicated that Cc(L)M and CM 1 has marked antioxidant and erythrocyte membrane stabilization potential. The antioxidant potential of compound is linked with formation of a non-radical form of DPPH-H (Oktay et al., 2003) as well as ability to donate hydrogen atom to break free radical chain formation (Duh et al., 1999). The activity of CM 1 found to be greater than Cc(L)M and comparable to standard drug. In erythrocyte membrane stabilization assay, standard drug as well as Cc(L)M and CM 1 were found to inhibit hemolysis of erythrocyte. The activity attributes to altered surface charges of the membrane or repels the charges which lead to hemolysis (Oyedapo et al., 2010). Various researchers have reported number of phytochemicals for their antioxidant potential. Diterpenoids from Calatropis procera exerted in vitro antioxidant and erythrocyte membrane stabilizing activity (Patil et al., 2016). Flavonoids (Pathak et al., 1991; Middleton, 1996) and saponins from Fagonia cretica are known to exert the anti-inflammatory potential by stabilizing lysosomal membrane (El-Shabrawy et al., 1997). This study for the first time reports new steroid derivative 20hydroxypregnan 18-oic acid for their marked antioxidant and erythrocyte membrane stabilizing potential. Our results are agreement with the earlier findings of Djeridane et al. (2010) where they proposed steroid derivative (17-(4-hydroxy-1,5-dimethylhexyl)-2,3,7-(acetyloxy) gona-
3.3.2. HPLC analysis In order to check the purity of the fraction CM 1, it was further analyzed by high performance liquid chromatography (HPLC). It revealed a major peak at 310 nm with RT 4.85 min. Moreover, numbers of minor peaks were also revealed when methanol: double distilled water (80:20) used as a mobile phase (Fig. 8). Reproducibility of the analysis was also checked by multiple repetitions. 3.3.3. UV and FT-IR analysis The result of UV–visible spectrophotometric analysis revealed major peak at 350 nm along with few minor peaks in the range of 200–400 nm (Fig. 9A). The results of FT-IR spectrophotometric analysis of CM 1 revealed presence of common bonds (Fig. 9B) in functional group region; for OH stretch at 3400, CH stretch at 2927, C=O stretch at 1728 and bond for aromatic ring at 1604 cm−1. However, bond present in fingerprint region for a C-O stretch at 1082 cm−1. 3.3.4. GC-HRMS analysis TLC and HPLC analyses of CM 1 revealed its purity, FT-IR revealed 220
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Fig. 8. HPLC profile of purified fraction CM1.
dichloromethane and toluene extract of C.carandas were used for antimicrobial activity (Verma and Chaudhary, 2011). Verma et al. (2015) studied DNA damage inhibition using methanolic extract of C.carandas leaves. Besides, ethanolic extract of C. carandas fruits and hydroalcoholic extract of roots were used to study antiemetic, analgesic, anti-inflammatory as well as antipyretic activities (Bhaskar and Balakrishnan, 2009). Galipalli et al. (2015) carried out bioassay guided fractionation of methanolic extract of C.carandas roots. In this study they investigated lupeol, carrisone, stigmasterol and scopoletin as
1,3,5(10)-trien-15-ol) from Cleome arabica for its powerful antioxidant potential than six different tested standard antioxidant drugs. Steroids exert their vital role against oxidative stress (Reyes et al., 2006; Vento et al., 2009). A detailed review of Patel and Savjani (2015) reports antiinflammatory potential of steroids from Trigonella foenum graecum, Solanum xanthocarpum, Boswellia serrata, Glycyrrhiza glabra, Commiphora mukul and Withania somnifera. In earlier studies, crude extract of different parts of C.carandas were used to investigate various biological activities. Aqueous as well as
Fig. 9. (A) UV spectrum of CM 1, (B) FT-IR spectrum of CM 1.
221
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
Fig. 10. (A) Gas chromatogram of CM 1, (B) Mass spectrum analyses of purified fraction CM 1. Dubber, M.J., Kanfer, I., 2004. High-performance liquid chromatographic determination of selected flavonols in Ginkgo biloba solid oral dosage forms. J. Pharm. Pharm. Sci. 7, 303–309. Duh, P.D., Tu, Y.Y., Yen, G.C., 1999. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum morifolium Ramat). LWT-Food Sci. Technol. 32 (5), 269–277. El-Shabrawy, O.A., El-Gindi, O.D., Melek, F.R., Abdel-Khalik, S.M., Haggag, M.Y., 1997. Biological properties of saponin mixtures of Fagonia cretica and Fagonia mollis. Fitoterapia 68 (3), 219–222. Galipalli, S., Patel, N.K., Prasanna, K., Bhutani, K.K., 2015. Activity-guided investigation of Carissa carandas (L.) roots for anti-inflammatory constituents. Nat. Prod. Res. 29 (17), 1670–1672. Gandhidasan, R., Thamaraichelvan, A., Baburaj, S., 1991. Anti-inflammatory action of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia 62, 81–83. Gülçin, I., 2007. Comparison of in vitro antioxidant and antiradical activities of L-tyrosine and L-Dopa. Amino Acids 32 (3), 431–438. Halliwell, B., 2007. Biochemistry of oxidative stress. Biochem. Soc. Trans. 35 (5), 1147–1150. Heim, K.E., Tagliaferro, A.R., Bobilya, D.J., 2002. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J. Nutr. Biochem. 13 (10), 572–584. Itankar, P.R., Lokhande, S.J., Verma, P.R., Arora, S.K., Sahu, R.A., Patil, A.T., 2011. Antidiabetic potential of unripe Carissa carandas Linn. fruit extract. J. Ethnopharmacol. 135 (2), 430–433. Kasal, A., Budesinsky, M., Griffiths, W.J., 2010. Spectroscopic Methods of Steroid Analysis. In Steroid Analysis. Springer, Netherlands, pp. 27–161. Kinnula, V.L., Crapo, J.D., 2004. Superoxide dismutases in malignant cells and human tumors. Free Radic. Biol. Med. 36 (6), 718–744. Kirtikar, K.R., Basu, B.D., 1987. ICS. Indian medicinal plants. Blatter, E., Caius, J.F., Mhaskar, K.S., editors, 3, pp. 707–1708. Kokate, C.K., Purohit, A.P., Gokhale, S.B., 1995. Methods of Crude Drug Evaluation, Pharmacognosy 10. Nirali Prakasan, Pune, pp. 88–99. Krishnaiah, D., Sarbatly, R., Nithyanandam, R., 2011. A review of the antioxidant potential of medicinal plant species. Food Bioprod. Process. 89 (3), 217–233. Kumar, V., Bhat, Z.A., Kumar, D., Khan, N.A., Chashoo, I.A., 2012. Evaluation of antiinflammatory potential of leaf extracts of Skimmia anquetilia. Asian Pac. J. Trop. Biomed. 2 (8), 627–630. Lavanya, R., Maheshwari, S.U., Harish, G., Raj, J.B., Kamali, S., Hemamalani, D., Varma, J.B., Reddy, C.U., 2010. Investigation of in-vitro anti-inflammatory, anti-platelet and anti-arthritic activities in the leaves of Anisomeles malabarica Linn. Res. J. Pharm. Biol. Chem. Sci. 1 (4), 745–752. Liu, H.X., Sun, S.Q., Lv, G.H., Chan, K.K., 2006. Study on Angelica and its different extracts by Fourier transform infrared spectroscopy and two-dimensional correlation IR spectroscopy. Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 64 (2), 321–326. Madhavi, D.L., Salunkhe, D.K., 1995. Toxicological aspects of food antioxidants. Food Science and Technology-New York-Marcel Dekker, pp. 267–360. Matteo, V., Esposito, E., 2003. Biochemical and therapeutic effects of antioxidants in the treatment of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Curr. Drug Targets-CNS Neurol. Disord. 2 (2), 95–107. McCune, L.M., Johns, T., 2002. Antioxidant activity in medicinal plants associated with
potent anti-inflammatory agents against proinflammatory mediators like TNF-α, IL-1b and nitrous oxide. However, the antioxidant and erythrocyte membrane stabilization activity of purified fraction from leaves extract of this plant has not been reported so far. As per our knowledge, this is first attempt to report antioxidant and erythrocyte membrane stabilizing activity of steroidal derivative from methanolic extract of C. carandas leaves. 5. Conclusion It can be concluded the purified fraction CM 1 from C.carandas has potent in vitro antioxidant and erythrocyte membrane stabilizing potential. Both activities of CM 1 may attribute to presence of steroid derivative in it. Moreover, activities reported by steroid derivative will lead to develop potential drug candidate or commercial food additives. However, further in vivo studies with the precise mechanism of this bioactive compound are needed to validate its commercial exploration. Conflict of interest Authors declare that there are no conflicts of interest. References Aruoma, O.I., 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. J. Am. Oil Chem. Soc. 75 (2), 199–212. Balakrishnan, N., Balasubaramaniam, A., Sangameswaran, B., Bhaskar, V., 2011. Hepatoprotective activity of two Indian medicinal plants from Western Ghats, Tamil Nadu. J. Nat. Pharm. 2 (2), 92–98. Bhaskar, V.H., Balakrishnan, N., 2009. Analgesic, anti-inflammatory and antipyretic activities of Pergularia daemia and Carissa carandas. DARU J. Pharm. Sci. 17 (3), 168–174. Darmanyan, A.P., Gregory, D.D., Guo, Y., Jenks, W.S., Burel, L., Eloy, D., Jardon, P., 1998. Quenching of singlet oxygen by oxygen-and sulfur-centered radicals: evidence for energy transfer to peroxyl radicals in solution. J. Am. Chem. Soc. 120 (2), 396–403. Djeridane, A., Yousfi, M., Brunel, J.M., Stocker, P., 2010. Isolation and characterization of a new steroid derivative as a powerful antioxidant from Cleome arabica in screening the in vitro antioxidant capacity of 18 Algerian medicinal plants. Food Chem. Toxicol. 48 (10), 2599–2606.
222
Biocatalysis and Agricultural Biotechnology 10 (2017) 216–223
B.S. Bhadane, R.H. Patil
product of browning reaction prepared from glucosamine. Jpn. J. Nutr. 44, 307–315. Rackova, L., Oblozinsky, M., Kostalova, D., Kettmann, V., Bezakova, L., 2007. Free radical scavenging activity and lipoxygenase inhibition of Mahonia aquifolium extract and isoquinoline alkaloids. J. Inflamm. 4 (1), 1–7. Reyes, M.R., Sifuentes-Alvarez, A., Lazalde, B., 2006. Estrogens are potentially the only steroids with an antioxidant role in pregnancy: in vitro evidence. Acta Obstet. Et. Gynecol. Scand. 85 (9), 1090–1093. Sadique, J., Al-Rqobah, W.A., Bughaith, M.F., El-Gindy, A.R., 1989. The bio-activity of certain medicinal plants on the stabilization of RBC membrane system. Fitoterapia 60, 525–532. Shimada, K., Fujikawa, K., Yahara, K., Nakamura, T., 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40 (6), 945–948. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Vitic. 16 (3), 144–158. Smirnoff, N., Cumbes, Q.J., 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28 (4), 1057–1060. Sreejayan, N., Rao, M.N., 1996. Free radical scavenging activity of curcuminoids. Arzneim. Forsch. 46 (2), 169–171. Steer, P., Millgård, J., Sarabi, D.M., Basu, S., Vessby, B., Kahan, T., Edner, M., Lind, L., 2002. Cardiac and vascular structure and function are related to lipid peroxidation and metabolism. Lipids 37 (3), 231–236. Syed, I.T., Gopalakrishnan, S., Hazeena, B.V., 1997. Biochemical modes of action of Gmelina asiatica in inflammation. Indian J. Pharmacol. 29 (5), 306–309. Takao, T., Kitatani, F., Watanabe, N., Yagi, A., Sakata, K., 1994. A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shellfish. Biosci. Biotechnol. Biochem. 58 (10), 1780–1783. Vento, M., Aguar, M., Escobar, J., Arduini, A., Escrig, R., Brugada, M., Izquierdo, I., Asensi, M.A., Sastre, J., Saenz, P., Gimeno, A., 2009. Antenatal steroids and antioxidant enzyme activity in preterm infants: influence of gender and timing. Antioxid. Redox Signal. 11 (12), 2945–2955. Verma, K., Shrivastava, D., Kumar, G., 2015. Antioxidant activity and DNA damage inhibition in vitro by a methanolic extract of Carissa carandas (Apocynaceae) leaves. J. Taibah Univ. Sci. 9 (1), 34–40. Verma, S., Chaudhary, H.S., 2011. Effect of Carissa carandas against clinically pathogenic bacterial strains. J. Pharm. Res. 4 (10), 3769–3771. Wagner, H., Bladt, S., 1996. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer Science & Business Media, Berlin-Heidelberg. Wang, J., Yue, Y.D., Tang, F., Sun, J., 2012. TLC screening for antioxidant activity of extracts from fifteen bamboo species and identification of antioxidant flavone glycosides from leaves of Bambusa textilis McClure. Molecules 17 (10), 12297–12311. Were, P.S., Waudo, W., Ozwara, H.S., Kutima, H.L., 2015. Phytochemical analysis of Warburgia ugandensis Sprague using Fourier transform infra-red (FT-IR) spectroscopy. Int. J. Pharmacogn. Phytochem. Res. 7, 201–205. Wichi, H.P., 1988. Enhanced tumor development by butylated hydroxyanisole (BHA) from the prospective of effect on forestomach and oesophageal squamous epithelium. Food Chem. Toxicol. 26, 717–723.
the symptoms of diabetes mellitus used by the indigenous peoples of the North American boreal forest. J. Ethnopharmacol. 82 (2), 197–205. Middleton Jr., M.D., E., 1996. Biological properties of plant flavonoids: an overview. Int. J. Pharmacogn. 34 (5), 344–348. Mishra, C.K., Sasmal, D., Shrivastava, B., 2012. An In vitro evaluation of the anthelmintic activity of unripe fruits extract of Carissa carandas Linn. Int. J. Drug Dev. Res. 4 (4), 393–397. Mohamed, S.S., El-Refai, A.M.H., El-Raoof Sallam, L.A., Abo-Zied, K.M., Hashem, A.G.M., Ali, H.A., 2013. Biotransformation of progesterone to hydroxysteroid derivatives by whole cells of Mucor racemosus. Malay J. Microbiol. 9, 237–244. Motwani, S.M., Pandey, E.P., Sonchhatra, N.P., Desai, T.R., Patel, V.L., Pandya, D.J., 2012. Pharmacognostic and phytochemical study of aerial parts of Carissa carandas. Int. J. Biol. Pharm. Res. 3 (1), 75–81. Naim, Z., Khan, M.A., Nizami, S.S., 1988. Isolation of a new isomer of ursolic acid from fruits and leaves of Carissa carandas. Pak. J. Sci. Ind. Res. 31, 753–755. Ode, O.J., Asuzu, I.U., Ajayi, I., 2011. Bioassay-guided fractionation of the crude methanol extract of Cassia singueana leaves. J. Adv. Sci. Res. 2 (4), 81–86. Oktay, M., Gülçin, İ., Küfrevioğlu, Ö.İ., 2003. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. LWT-Food Sci. Technol. 36 (2), 263–271. Olech, M., Komsta, Ł., Nowak, R., Cieśla, Ł., Waksmundzka-Hajnos, M., 2012. Investigation of antiradical activity of plant material by thin-layer chromatography with image processing. Food Chem. 132 (1), 549–553. Osadebe, P.O., Okoye, F.B.C., 2003. Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves. J. Ethnopharmacol. 89 (1), 19–24. Oyedapo, O.O., Akinpelu, B.A., Orefuwa, S.O., 2004. Anti-inflammatory effect of Theobroma cacao, root extract. Trop. Med. Plants 5 (2), 161–166. Oyedapo, O.O., Akinpelu, B.A., Akinwunmi, K.F., Adeyinka, M.O., Sipeolu, F.O., 2010. Red blood cell membrane stabilizing potentials of extracts of Lantana camara and its fractions. Int. J. Plant Physiol. Biochem. 2 (4), 46–51. Pakrashi, S.C., Datta, S., Ghosh-Dastidar, P.P., 1968. Indian medicinal plants—XVII. 3 phytochemical examination of Carissa spp. Phytochemistry 7 (3), 495–496. Patel, S.S., Savjani, J.K., 2015. Systemetic review of plants steroids as potential antiinflammatory agent: current status and future perspectives. J. Phytopharm. 4, 121–125. Pathak, D., Pathak, K., Singla, A.K., 1991. Flavonoids as medicinal agents-recent advances. Fitoterapia 62, 371–389. Patil, R.P., Pai, S.R., Pawar, N.V., Shimpale, V.B., Patil, R.M., Nimbalkar, M.S., 2012. Chemical characterization, mineral analysis, and antioxidant potential of two underutilized berries (Carissa carandus and Eleagnus conferta) from the Western Ghats of India. Crit. Rev. Food Sci. Nutr. 52 (4), 312–320. Patil, S.G., Patil, M.P., Patil, R.H., 2016. In vitro anti-hypercholesterolemic activity of Calotropis procera (Aiton) using human erythrocytes. Biocatal. Agric. Biotechnol. 5, 104–110. Peuchant, E., Brun, J.L., Rigalleau, V., Dubourg, L., Thomas, M.J., Daniel, J.Y., Leng, J.J., Gin, H., 2004. Oxidative and antioxidative status in pregnant women with either gestational or type 1 diabetes. Clin. Biochem. 37 (4), 293–298. Qyaizu, M., 1986. Studies on products of browning reactions: antioxidative activities of
223