In vitro anti-hypercholesterolemic activity of Calotropis procera (Aiton) using human erythrocytes

Biocatalysis and Agricultural Biotechnology 5 (2016) 104–110

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In vitro anti-hypercholesterolemic activity of Calotropis procera (Aiton) using human erythrocytes Samadhan G. Patil, Mohini P. Patil, Ravindra H. Patil n Department of Microbiology and Biotechnology, R. C. Patel Arts, Commerce and Science College, Shirpur 425405, Maharashtra, India

art ic l e i nf o

a b s t r a c t

Article history: Received 27 October 2015 Received in revised form 4 January 2016 Accepted 4 January 2016 Available online 6 January 2016

This study evaluates in vitro hypocholesterolemic and antioxidant potential of Calotropis procera root extracts and its diterpenoid rich fraction. In vitro hypocholesterolemic activity was determined by measuring cholesterol concentration in the normal and hypercholesterolemic human red blood cells (hRBCs) after incubating with the test extracts (1, 10 and 100 mg/mL) for 24 h. The bioactivity guided fractionation of crude acetone root extract yielded a fraction with EC50 63.03 7 1.10 mg/mL in relation to ascorbic acid standard (EC50 39.95 7 1.25 mg/mL). In case of isolated erythrocytes of diseased donors, the purified fraction reduced the membrane cholesterol up to 1.627 0.22 mmole CH/mL (52%), whereas it was 1.01 70.75 mmole CH/mL (35%) in case of quercetin standard. The whole blood, when incubated with purified fraction and quercetin standard at 10 mg/mL for 24 h, it showed significant decrease in the cholesterol level of hRBCs of both, healthy (P o0.001) as well as diseased (P o0.05) donors. GC-HRMS analysis of the bioactive diterpenoid rich fraction revealed cyclic diterpenoids – Phenol, 2,4 bis (1,1dimethylethyl) ester and 1,2 benzedenedicarboxylic acid, bis(2-methylpropyl) ester. In conclusion, C. procera root extract and its diterpenoid rich fraction exhibited potential hypocholesterolemic effect possibly by inhibiting membrane cholesterol synthesis and improving antioxidant levels in hypercholesterolemic hRBCs. & 2016 Elsevier Ltd. All rights reserved.

Keywords: Hypocholesterolemic activity Di-terpenoids hRBCs Hypercholesterolemic donors Membrane cholesterol

1. Introduction Coronary heart disease (CHDs) is the major cause of death and disability in both developed and developing countries. It accounts for more than one third of total deaths globally and is becoming a worldwide epidemic with increasing clinical and economic burden (Kreatsoulas and Anand, 2010). A number of factors such as hypercholesterolemia, hypertension and smoking contribute to the development of CHDs (Patil et al., 2010). Hypercholesterolemia has been the major contributing factor for the development of CHDs (Patil et al., 2011). The strategies for preventing and treating hypercholesterolemia include dietary management, bile acid sequestration and inhibition of cholesterol biosynthesis. Besides, the existing study also shows involvement of free radicals in the etiology of CHDs (Fruchart and Duriez, 1994). It has been observed that, antioxidant levels are significantly lowered in the patients with symptomatic CHDs (Shanmugasundaram, 1995). Several studies have revealed the important role of cholesterol content of red blood cells (RBCs) membranes in the development of atherosclerosis (Tabas, 2002). Since the erythrocyte membrane contains n

Corresponding author. E-mail address: [email protected] (R.H. Patil).

http://dx.doi.org/10.1016/j.bcab.2016.01.002 1878-8181/& 2016 Elsevier Ltd. All rights reserved.

the large amount of cholesterol, they are the important contributing factor for the development of atherosclerotic plaques (Zhong et al., 2012). In recent times, the need for combination of multiple therapy approaches to overcome CHDs is widely accepted by majority of health care systems (Mohammad et al., 2013). Several epidemiological studies have demonstrated that diet with fruits and vegetables rich in antioxidants, are associated with lower incidences of CHDs (Aviram et al., 2005). Hence increased intake of natural antioxidants and hypocholesterolemic substances would have beneficial effects. Many plants in the Indian system of medicine have been reported for anti-hypercholesterolemic properties and are rich sources of natural antioxidant (Devasagayam et al., 2004). Clerodendron colebrookianum (Boruah et al., 2014), Commiphora mukul (Ramesh and Saralakumari, 2012), Celatrus paniculatus (Patil et al., 2010), Terminalia arjuna (Patil et al., 2011), Mucuna pruriens and Ionidium suffruticosum (Dharmarajan and Arumugam, 2012) are known to have cardio protective activities. The antioxidants present in these plants have been shown to lower serum cholesterol levels by varying mechanisms and thus have potential to reduce the progression of atherosclerosis (Heber, 2001). Calotropis procera (Aiton) R.Br. Asclepiadaceae is an ancient tribe shrub grown uncultivated and widespread in tropical

S.G. Patil et al. / Biocatalysis and Agricultural Biotechnology 5 (2016) 104–110

regions. The plant is known for its abundant latex which is mainly recovered from the green part of the plant. Various parts of C. procera possess a number of biological activities mainly proteolytic (Kumar and Jagannadham, 2003), hypoglycemic, antioxidant (Yadav et al., 2014), anti-inflammatory (Freitas et al., 2012), analgesic (Dewan et al., 2000), anti-fertility (Kamath and Rana, 2002) and anti-arthritic (Kumar and Roy, 2007). Besides this various parts of C. procera, its latex has some medicinal activities like insecticidal, anti-fungal and wound healing (Cahiwut et al., 2010).Several bioactive molecules with different biological activities have been reported in C. procera. Phenol, 2,4 bis(1,1-dimethylethyl) ester with antioxidant and antiviral activity against spot syndrome virus have been reported (Prakash and Suneetha, 2014; Velmurugan et al., 2012). The other species of the genus Calotropis such as C. gigantea is reported for its antioxidant and hypocholesterolemic potential (Jaiswal et al., 2014). However, to the best of our knowledge, no report is available on in vitro hypocholesterolemic potential of the root extracts of C. procera. In the present study, the methanol, acetone root extracts and purified fraction of C. procera were further investigated for its in vitro hypocholesterolemic, membrane stabilizing and antioxidant activities. The attempt has also been made to characterize and identify its active ingredient by chromatographic and spectroscopic methods. The structural confirmation of the compound has been done by GC-HRMS analysis.

2. Materials and methods 2.1. Plant material and preparation of extract The roots of plant C. procera were collected from the agricultural out field near the campus of R. C. Patel Arts, Commerce and Science College, Shirpur, India. Plant was identified and authenticated by the expert taxonomist and the voucher specimen was deposited (RCP-02/2015) at Department of Botany, R. C. Patel Arts, Commerce and Science College, Shirpur, India. The roots were thoroughly washed, shade dried at 37 °C and grounded in grinder to obtain fine powder of it. The powdered roots (500 g) were subjected to successive extraction in the Soxhlet extractor for 48– 78 h at 65 °C with increasing polarity of solvents like acetone and methanol. The extracts were filtered and subjected to dryness in rotary vacuums evaporator (Equitron, India) and stored in refrigerator until used. 2.2. Phytochemical screening The total ash, acid soluble and water soluble ash values were calculated according to method described in the Indian Pharmacopoeia (2007). The preliminary phytochemicals like alkaloids, flavonoids, glycosides, tannins and triterpenoids were investigated as per previously reported methods (Aiyegoro and Okoh, 2010). Concentration of phenolic in the test extracts was calculated by linear regression analysis and the results were expressed as gallic acid equivalents (GAE). 2.3. In vitro antioxidant activity 2.3.1. DPPH radical scavenging assay The DPPH radical quenching ability of the acetone, methanol crude extracts and purified fraction was performed as per the previously reported method (Aiyegoro and Okoh, 2010). Briefly, 1 mL of methanolic DPPH (2 mM) solution was mixed with different concentrations of test extracts (10–100 mg/mL) and ascorbic acid standard, after 30 min incubation in dark, absorbance was measured at 510 nm. The ability to scavenge the DPPH radical was

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calculated using the following formula:

DPPH radical scavenging assay (%)=(A 0 –A1) / A 0 × 100 where A0 is the absorbance of control, A1 is the absorbance of sample. 2.3.2. Reducing power assay The reducing power of the acetone, methanol crude extracts and purified fraction was determined as per the method of Aiyegoro and Okoh (2010). Different concentrations of the test extracts (10–100 mg/mL) were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min, cooled at room temperature, and then 2.5 mL of 10% tricarboxylic acid was added to the mixture and centrifuged at 600g for 10 min. The supernatant solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride (FeCl3) and the absorbance was measured at 700 nm. Increase in the absorbance of the reaction mixture indicated the reducing power. Ascorbic acid and phosphate buffer were used as standard and blank, respectively. 2.4. Membrane stabilization assay Membrane stabilizing activity of acetone, methanol crude extracts and purified fraction was performed using human red blood cell membrane stabilization (hRBCs) assay performed as per the previously reported method (Oyedapo et al., 2010). Fresh human blood sample were collected into an anticoagulant (0.8% sodium citrate, 0.05% citric acid and 0.42% NaCl) solution and centrifuged at 300g for 10 min at room temperature. Supernatant was removed and packed red blood cells were washed in normal saline (0.84% NaCl). Hematocrit (2%) was prepared and used for membrane stabilization assay as reported previously (Oyedapo et al., 2010). Test extracts (0.5–2.5 mg/mL) and ibuprofen standard was mixed with 2 mL hypo-saline, 1 mL sodium phosphate buffer (pH 7.4), and 0.5 mL hematocrit (2%). The reaction mixture was incubated at 56 °C for 30 min in water bath. After incubation, reaction mixture cooled at room temperature and again centrifuged at 600g for 10 min, the supernatant content was separated and released hemoglobin was estimated spectrophotometrically at 560 nm. The percent hemolysis was calculated by using following formula.

% hemolysis = 100−( A1 −A2 )/A3 × 100 where, A1 is absorbance of test, A2 is absorbance of drug control and A3 is absorbance of blood control. 2.5. In vitro hypocholesterolemic activity In vitro hypocholesterolemic activity of all test extracts was carried out according to the method described in earlier report with slight modifications (Duchnowicz et al., 2012). Blood samples from hypercholestermic and healthy donors (sample size n ¼6) were collected from clinical pathological laboratories of Shirpur city. The study protocol was approved by the institutional research ethics committee (RCPREC 2/2014) and informed consent was obtained from each participant. Study was carried out on whole blood and isolated erythrocytes. The hRBCs were washed twice with phosphate buffer (pH 7.4) and centrifuged. The isolated erythrocytes (5% hematocrit) and whole blood were suspended separately in the incubation medium containing 140 mM NaCl, 10 mM KCL, 1.5 mM MgCl2, 10 mM glucose, 100 mg/mL streptomycin, 5 mM Tris–HCl buffer (pH 7.4). Whole blood and hematocrit were incubated with and without test extracts, purified fraction and quercetin standard and for 24 h at 37 °C at concentrations

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of 1, 10, and 100 mg/mL. After incubation, erythrocytes were washed five times with 0.9% NaCl and lipids were extracted by hexane: propanol (3:1 v/v) solvent extraction (Rodriguez-Vico et al., 1991). Cholesterol concentration was determined using standard cholesterol estimation kits (Nicholas Piramal, India) (Allain et al., 1974). 2.6. Separation of active compound using silica gel column chromatography The crude extracts were further subjected to column chromatography. The chromatographic separation was carried out using silica gel (60–120 mesh size) column chromatography. The crude extract was mixed with silica at the ratio of (1:1). The mixture was applied to column (30 cm  1.5 cm) which was preconditioned with hexane. The column was eluted with hexane (100%), hexane: ethyl acetate (80:20, 60:40, and 50:50 v/v) and finally with 100% ethyl acetate. A total of 51 fractions of 5 mL fraction size were collected. Fractions 5–30 yielded colorless semisolid compound which showed two bands of Rf 0.70 and 0.65, did not showed significant antioxidant and hypocholesterolemic activity hence discarded. Fractions 31–50 afforded a red color semisolid compound of Rf 0.42 with excellent antioxidant and hypocholesterolemic activities and therefore mixed together and subjected for further characterization. Ten microliter of each acetone and methanol crude extracts of C. procera was applied onto pre-coated silica gel TLC plate (Merck, Darmstadt, Germany) by using Spraylin V applicator (Aetron, Mumbai, India). The TLC plate was developed in a pre-saturated chamber containing the different mobile phases (Wagner and Bladt, 1996). The plate development was carried out in ascending way using various mobile phases such as chloroform: methanol (9:1, 8:2, 7:3, 6:4,1:1 v/v); hexane: ethyl acetate (9:1, 8:2, 7:3, 6:4, 1:1 v/v); n-butanol:2 M NO4OH (1:1 v/v); benzene: ethyl acetate (9.9:0.1 v/v); chloroform:methanol: 25% NH3 (8:2:0.5 v/v) and ethyl acetate: glacial acetic acid: formic acid: water (100:11:11:10 v/v). After separation, plates were air dried, sprayed with 10% sulfuric acid and heated at 105 °C in a hot air oven for 10 min to develop color of spots. 2.7. Identification of the bioactive compounds using GC-HRMS The partially purified fraction obtained from silica gel column having antioxidant and hypocholesterolemic activities was subjected for spectroscopic and chromatographic characterization. GC-HRMS analysis was carried out using Agilent 7890-GC (Santa Clara, CA, USA) fitted with HP-5 fused silica capillary column (30 m  0.20 mm, phase thickness 0.25 mm) coupled with Jeol Accu TOF GCV mass spectrometer with flame ionization detector FID detector. The inlet temperature was 250 °C. The injection volume was 10 ml and the injection was carried out in a split mode at the split ratio of 20:80. The gas flow rate was 1 mL per minute. Analysis of compounds in the fraction was done by NIST library search. 2.8. Statistical analyses Each value is the mean of three replicates. Values of different parameters were expressed as the mean 7 standard deviation (SD). The statistical significance between healthy and hypercholestermic individuals was carried out using one way ANOVA and multiple comparison was done by Tukey's test. The statistical analyses and EC50 values were calculated using Graph Pad Prism 6 software (San Diego, CA, USA).

3. Results 3.1. Phytochemical investigation The yield of the acetone and methanol extract was found to be 8.0% and 8.5%, respectively. The total ash was found to be 27.10% whereas, the water and acid soluble ash was found to be 2.95% and 1.85%, respectively. Preliminary phytochemical screening of acetone and methanol crude extract revealed the presence of alkaloids, triterpenoids, phenols, flavonoids, tannins and saponins in agreement with a previous report (Mainasara et al., 2012). Phytochemical investigation of crude extract as well as the purified fraction showed dominant presence of terpenoids. The methanol and acetone crude extracts showed the presence of polyphenols at 582.94 and 823.20 mg/GAE, respectively. 3.2. In vitro antioxidant activity 3.2.1. DPPH radical scavenging assay The DPPH radical scavenging activity of the acetone and methanolic crude extracts, purified fraction and ascorbic acid was found to be dose dependent (Fig. 1). The radical scavenging effect of acetone and methanol extract, at 100 mg/mL, was found to be 83.127 0.10% and 78.7870.10%, respectively. The purified fraction and ascorbic acid standard showed 85.78 7 10% and 79.46 70.10% radical scavenging effect at 100 mg/mL. The purified fraction, at 100 mg/mL, showed significant (P o0.05) free radical scavenging activity as compared to standard. 3.2.2. Reducing power assay The results of reducing power of the acetone and methanolic crude extracts, purified fraction and ascorbic acid standard are shown in Fig. 2. The reducing power potential of the purified fraction was found to be dose dependent (R2 ¼0.905) and was significantly (Po0.05) higher than the standard at 80 mg/mL. The results clearly demonstrate the excellent reducing power potential of purified fraction. 3.3. Membrane stabilization assay The membrane stabilization activity of crude extracts, purified fraction and ibuprofen standard was measured at the concentration range of 0.5–2.5 mg/mL and expressed in terms of the percent protection of hRBCs hemolysis (Fig. 3). The acetone extract showed 86.147 3.02% inhibition of hemolysis of hRBCs at 2.5 mg/mL in comparison with ibuprofen standard which showed 98.12 73.87% inhibition of hemolysis at the same concentration. The purified

DPPH scavenging acticity(%)

106

100 90 80 70 60 50 40 30 20 10 0

Acetone crude extract Methanol crude extract Purified fraction Ascorbic acid

10

20 30 40 50 60 70 80 90 Concentration of test extract (µg/mL)

100

Fig. 1. DPPH radical scavenging effect of acetone, methanol crude extracts, purified fraction and ascorbic acid standard. Data is shown as mean 7 SD.

S.G. Patil et al. / Biocatalysis and Agricultural Biotechnology 5 (2016) 104–110

0.8

0.6

Absorbace at 765 nm

Table 2 Concentration of cholesterol (mmole CH/mL packed cells) in erythrocytes of whole blood after 24 h incubation.

Ascorbic acid Purified Fraction Acetone Crude extract Methanol crude extract

0.7

Concentration of plant Concentration of cholesterol (lmole CH/mL) extracts/standard (lg/mL) Healthy donor Hypercholesterolemic donor (n¼ 6) (n¼ 6)

0.5 0.4 0.3 0.2 0.1 0

10

20

30 40 50 60 70 80 Concentration of test extract (µg/mL)

90

100

Fig. 2. Reducing power potential of acetone crude, methanol crude, purified fraction and ascorbic acid. Data is shown as mean7 SD.

60

40

Before incubation After incubation (Control) Acetone extract 1 10 100 Methanol extract 1 10 100 Purified fraction 1 10 100 Quercetin 1 10 100 ANOVA a

% Protection

107

b

20

3.09 7 0.51 2.95 7 0.49

4.50 7 1.27 4.31 71.14

2.60 7 0.36 2.30 7 0.29 1.99 7 0.07

3.80 7 1.24 3.447 1.33 3.19 71.25a

2.53 7 0.31 2.217 0.15 2.117 0.11

3.377 1.40 2.717 1.20 3.02 7 0.96a

2.3570.69 2.02 7 0.45a 1.69 7 0.32a

3.727 1.24 3.02 7 1.14a b 2.727 0.88a

1.45 7 0.19 1.22 7 0.21 1.077 0.16 Po 0.05

2.62 7 1.16 2.187 1.17 1.63 7 1.3a P o 0.001

Po 0.001 vs control after incubation. P o 0.05 vs concentration 1 mg/mL.

Purified Fraction Acetone extract

3.4. In vitro hypocholestrolemic activity

Methanolic Extract Ibuprofen

0 1

0.5

1.5

2

2.5

Concentration of test extracts (µg/ml) Fig. 3. Membrane stabilizing activity of acetone crude, methanol crude, purified fraction and ibuprofen. Data is shown as mean 7 SD.

fraction of the C. procera extract showed a dose dependent (R2 ¼ 0.968) inhibition of hemolysis of hRBCs with maximum (82.077 3.03%) inhibition at the concentration of 2.5 mg/mL. The test extracts showed excellent radical scavenging activity with the EC50 values ranging from 37.7071.46 to 40.71 71.36 mg/mL and reducing power potential ranging from 41.37 74.15 to 50.25 75.21 mg/mL (Table 1). The EC50 values of the crude extract are significantly less than the purified fraction indicates that the cumulative effect of other metabolites present in the crude extract. The results clearly demonstrate the membrane stabilization effect of the various crude extracts and the purified fraction of the C. procera.

Table 1 EC50 values of different test extracts. Test extract

DPPH radical scavenging effect (mg/mL)

Reducing power potential (mg/mL)

Membrane stabilization effect (mg/mL)

Acetone crude Methanol crude Purified fraction Standard

40.71 71.36

41.37 74.15

02.077 0.10

37.7071.46

50.25 75.21

01.73 7 0.35

63.03 71.10

45.49 73.25a

1.247 0.15

39.95 71.25

44.12 73.76

0.86 7 0.20

a

a

P o0.05 vs standard.

The results of in vitro hypocholesterolemic activity of the extracts, purified fraction and quercetin standard in whole blood are summarized in Table 2. The whole blood treated with purified fraction and quercetin standard, showed decrease in the cholesterol level (mmole CH/mL) in both, healthy (Po0.05) as well as diseased (P o0.001) donors. The initial level of cholesterol in healthy and diseased donors before incubation was found to be 3.09 70.51 and 4.50 71.27 mmole CH/mL, respectively (Table 2). After 24 h incubation of whole blood of hypercholesterolemic donors with 100 mg/mL of extracts (acetone and methanol) and purified fraction, the concentration of cholesterol was found to be reduced significantly (Po 0.001). The purified fraction at 10 mg/mL concentration showed 70% reduction in the whole blood cholesterol of hypercholesterolemic individuals in comparison with quercetin standard (48%). The control tubes containing the whole blood from the normal as well from the hypercholesterolemic individual do not show statistically significant (P 40.05) change in the cholesterol concentration after incubation. The cholesterol concentration in the isolated erythrocytes was also altered significantly as a function of treatment with test extracts (Table 3). The initial levels of cholesterol in isolated erythrocytes of healthy and diseased donors before incubation were found to be 2.84 70.16 and 3.107 0.09 mmole CH/mL, respectively. After 24 h incubation with 100 mg/mL, of extracts and purified fraction, the cholesterol concentration in isolated erythrocytes of healthy donor were found to decrease significantly (P o0.05). The concentration of cholesterol was found to be 1.46 70.15 mmole CH/mL when the erythrocytes were incubated with 100 mg/mL of purified fraction in comparison with standard quercetin (1.36 70.42 mmole CH/mL). In case of isolated erythrocytes of diseased donors, the purified fraction reduced the cholesterol up to 1.62 70.22 mmole CH/mL (52%), whereas, it was 1.01 70.75 mmole CH/mL (35%) in case of standard. The control tubes containing the isolated erythrocytes from the normal as well from the hypercholesterolemic individual do not show statistically significant (P 40.05) change in the cholesterol concentration after

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S.G. Patil et al. / Biocatalysis and Agricultural Biotechnology 5 (2016) 104–110

Table 3 Concentration of cholesterol (mmole CH/mL packed cells) in isolated erythrocytes after 24 h incubation. Concentration of plant Concentration of cholesterol (lmole CH/mL) extracts/standard (lg/mL) Healthy donor Hypercholesterolemic donor (n¼ 6) (n¼ 6) Before incubation After incubation (Control) Acetone extract 1 10 100 Methanol extract 1 10 100 Purified fraction 1 10 100 Quercetin 1 10 100 ANOVA a b

2.84 70.16 2.747 0.15

3.10 70.09 2.95 7 0.08

2.4 7 0.20 2.26 70.15 1.917 0.12a

2.84 7 0.30 2.417 0.29 2.277 0.26

2.39 70.26 2.247 0.27 2.06 70.19

3.01 70.14 2.777 0.13 2.34 7 0.59

2.187 0.37 1.89 7 0.40 1.46 7 0.15a

2.56 7 0.15 1.917 0.24 1.62 70.22a,b

1.95 7 0.68 1.60 7 0.59 1.36 7 0.42a P o 0.05

1.94 70.84 1.56 70.80 1.017 0.75a P o0.05

P o0.05 vs control after incubation. Po 0.001 vs concentration 10 mg/mL.

incubation. The results demonstrate the significant in vitro cholesterol lowering activity of the purified fraction of C. procera in both healthy and diseased donor. 3.5. Identification of the bioactive compounds Based on the results of bioactivity studies, the acetone extract was chosen for further characterization of bioactive metabolite (s) present in it. The preliminary phytochemical screening revealed the presence of phyto-constituents like alkaloids, flavonoids, triterpenoids in acetone and methanol crude extracts of C. procera. In TLC studies, out of various mobile phases tested, the best resolution was obtained when hexane:ethyl acetate (80:20 v/ v) was used as mobile phase. The TLC results of the crude acetone extract revealed a number bands indicating presence of several phytoconstituents. After derivatization, the crude extract as well as the purified fraction showed the identical band at Rf 0.42 (Fig. 4). The compound showed faint yellow fluorescence when observed at long wavelength (365 nm) which is the characteristic feature of the terpenes (Wagner and Bladt, 1996). The crude acetone extract was subjected to the column chromatography using non-polar to polar mobile phases. When the column was eluted with hexane: ethyl acetate (80:20 v/v), the fractions 31 to 50 afforded a red color semisolid which showed a single compound of Rf 0.42 with excellent bioactivities. These fractions were mixed together and used for GC-HRMS studies.

Fig. 4. TLC profile of crude extract (A) and purified fraction (B) of C. procera.

3.6. Structural characterization using GC-HRMS The GC-HRMS spectra and the bioactive metabolites present in the purified fraction are represented in Fig. 5 and Table 4. The spectra revealed six identified compounds in order of their elution from the column which are Phenol, 2,4-bis (1,1-dimethylethyl) ester; Disisobutyl phthalate; 1,2 benzedenedicarboxylic acid, bis (2-methylpropyl) ester; dibutyl phthalate (two peaks) and phthalic acid pentyl 2-pentyl ester. The results indicate that the purified fraction contents diterpens as major phytoconstituent in agreement with the previous report (Velmurugan et al., 2012).

Fig. 5. Gas chromatogram of the purified fraction.

4. Discussion The free radicals and the reactive oxygen species (ROS) are

S.G. Patil et al. / Biocatalysis and Agricultural Biotechnology 5 (2016) 104–110

Table 4 The GC-HRMS profile of the compounds present in the bioactive fraction of the C. procera. Retention time

Molecular weight

Molecular formula

Compound name as per NIST library

10.80

206

C14H22O

15.82 16.56

278 223

C16H22O4 C16H22O4

17.42 17.82 19.63

278 278 306

C16H22O4 C16H22O4 C18H26O4

Phenol, 2,4-bis (1,1-dimethylethyl) ester. Disisobutyl phthalate 1,2 benzedenedicarboxylic acid, bis(2-methylpropyl) ester Dibutyl phthalate Dibutyl phthalate Phthalic acid pentyl 2-pentyl ester

known to induce oxidative stress and it has been implicated in the pathology of cardiovascular diseases, inflammatory conditions, cancer and ageing (Velioglu et al., 1998). The results of the study indicated the strong antioxidant and hypocholestrolemic effects in vitro. The purified fraction of C. procera has strong antioxidant activity and it was found to be better than the, ascorbic acid standard. The reducing power of the compound(s) is commonly linked with the presence of the reductones, which exert antioxidant action by donating hydrogen atom and thus breaks the free radical chain and prevent the peroxide formation (Duh et al., 1999). It is known that oxidative stress and the elevated cholesterol level in the RBC membrane has been recognized as an important factor contributing the development of atherosclerosis and CHDs (Kanakaraj and Singh, 1989). Several studies suggested that erythrocytes entering the plaque promote plaque peroxidation (Kolodgie et al., 2003). RBC membranes consist of more than 40% of cholesterol and are significant contributors for the development of atherosclerotic plaques (Yeagle, 1985). Our study shows the significant decrease in the cholesterol concentration of RBCs incubated with the test extracts. Down regulating the cholesterol level in RBC membrane can improve the rheological properties of the blood and thus minimize the risk of atherosclerosis. Various biological activities of the different parts of C. procera are well documented. However, the reports available on bioactive terpenoids present in the root bark of C. procera are relatively few. We found that the terpenoids rich fraction of the bark extract of C. procera has potent in vitro antioxidant and hypocholestrolemic activities. Terpenoids are widely recognized for their ability to improve dyslipidemia by altering the level of serum cholesterol. Several in vivo studies have demonstrated the protective role of triterpenoids against inflammation and oxidative stress. A pentacyclic triterpene with broad range of pharmacological properties have been isolated and characterized from C. procera, the latex of the plant has been reported for potent anti-inflammatory, wound healing, anti-cancer and hepatoprotective activity in animal models (Sangraula et al., 2002; Choedon et al., 2006). Cyclic diterpenoids, phenol, 2,4 bis (1,1-dimethylethyl) ester and 1,2 benzedenedicarboxylic acid, bis(2-methylpropyl) ester are reported in C. procera with antimicrobial and antioxidant activities in earlier reports (Velmurugan et al., 2012) Our study is the first report demonstrating a decrease in cholesterol concentration in hRBC membranes in whole blood as well as of isolated erythrocytes with purified fraction of C. procera. More decrease in cholesterol concentration of isolated erythrocytes as compared to whole blood was observed in agreement with previous findings with hypolipidemic effects of phenolic compounds (Duchnowicz et al., 2012). Moreover, the compounds like Disisobutyl phthalate and phthalic acid pentyl ester are reported for the first time in our studies.

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5. Conclusion It can be concluded that the purified fraction of C. procera root extract has potent in vitro antioxidant and hypocholesterolemic activities. The hypocholesterolemic activity of the purified fraction in the isolated erythrocytes and in the whole blood may attribute to diterpenoids present in it. Also, there have been increased concerns over the use of experimental animals for in vivo studies. Therefore, development of an appropriate in vitro assay system for evaluating biological activities is the need of time. The method described in this report is not only rapid, convenient and economical but can also be used as easy alternative for primary screening of the compounds with hypocholesterolemic potential. To the best of our knowledge, this is the first report on hypocholesterolemic potential of cyclic diterpenoids from C. procera. However, a further in vivo study with the large-scale clinical investigation and precise mechanism underlying these finding is necessary.

Acknowledgment This research was supported by the University Grant Commission (UGC File No. 42-456/2013 SR) New Delhi, India.

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