Journal of Dietary Supplements, 11(2):175–183, 2014 C 2014 by Informa Healthcare USA, Inc. Available online at www.informahealthcare.com/jds DOI: 10.3109/19390211.2013.859218
ARTICLE
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Determination of Antioxidant Capacity, α-Amylase and Lipase Inhibitory Activity of Crotalaria Juncea Linn In Vitro Inhibitory Activity of Crotalaria Juncea Linn Sathis Kumar Dinakaran1,2 , David Banji3 , Harani Avasarala1 , & Otilia Banji3 1
Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh, India, 2 Jawaharlal Nehru Technical University, Hyderabad, Andhra Pradesh, India, 3 Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh, India
ABSTRACT. The present study involves the determination of antioxidant capacity and in vitro α-amylase and lipase inhibitory activity of the Crotalaria juncea Linn extract. The content of polyphenols, flavonoids, and tannins in the extracts was estimated by spectrophotometry. Antioxidant activity on goat liver lipid peroxidation and linoleic acid emulsion were determined and α-amylase and lipase inhibitory activity was also evaluated. All the extracts had shown antioxidant property, α-amylase, and lipase inhibitory properties. Aqueous extract was found to show maximum antioxidant activity on goat liver. Antilipid peroxidation and antioxidant activity were determined to be 66.94 ± 0.616 (p < .01) and 59.54 ± 0.2 (p < .01), respectively. Maximum α-amylase and lipase inhibitory activities of 71.42 ± 1.37 (p < .01) and 57.14 ± 2.74% (p < .01), respectively, were exhibited by macerated methanol extract. The results had shown that all the extracts exhibited low inhibition and antioxidant activity as compared to standard. KEYWORDS. amylase, antioxidant, Crotalaria juncea Linn, flavonoids, lipase, phenolic compounds, tannins
INTRODUCTION Crotalaria juncea Linn also called as Sun hemp or Indian hemp belongs to Fabaceae family and is distributed in the tropical and subtropical regions of India, Nepal, Sri Lanka, and Southern Africa. It is an annually renewable, multi-purpose fiber crop. Due to its great potency, it is used as a medicine by many tribal communities. Generally, in the folk and Ayurvedic medicines, it is used as blood purifier, abortificient, astringent, demulcent, emetic, purgative, and used in the treatment of anemia, impetigo, menorrhagia, and psoriasis (Sathis Kumar, David, PrashanAddress correspondence to: D. Sathis Kumar, Associate Professor, Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh 533437, India (E-mail: [email protected]). (Received 1 May 2013; revised 5 August 2013; accepted 10 September 2013)
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thi, & Harani, 2011). The Alpha amylase is the one of the main enzymes in human that catalyzes the hydrolysis of 1,4-glucosidic linkage of complex carbohydrates like starch into maltose and oligosaccharides in the small intestine. Inhibition of this enzyme reduces the rate of digestion of starch and results in the decrease of postprandial blood glucose levels in diabetic patients (Karthic, Kirthiram, Sadasivam, & Thayumanavan, 2008). Reducing postprandial hyperglycemia prevents glucose uptake into adipose tissue for inhibiting the synthesis and accumulation of triacyl glycerol. On the other hand, it is well known that dietary lipid is not directly absorbed from the intestine unless it has been subjected to the action of pancreatic lipase. Lipases are enzymes that digest fats, including triacylglycerol and phospholipids. It removes fatty acids from the α and α’ position of dietary triglycerides (TGs) and yields β-monoglycerides and long chain saturated and polyunsaturated fatty acids as the lipolytic products (Shi & Burn, 2004). Lipase is responsible for the hydrolysis of 50%–70% of total dietary fats. Lingual lipase secreted by serous gland, digests approximately one third of ingested fat. Gastric lipase secreted in response to mechanical stimulation, ingestion of food or sympathetic activation accounts for the hydrolysis of 10%–40% of dietary fat (Tilbeurgh, 1992). These two enzymes, thus, potentially limit the nutritional impact of the inhibition of lipid absorption that could result from the reduction in the activity of Pancreatic Lipase alone. Pancreatic Lipase inhibition is one of the most widely studied mechanisms for the determination of the potential efficacy of natural products as antiobesity agents. One of the most important strategies in the treatment of obesity includes development of inhibitors of nutrient digestion and absorption, in an attempt to reduce energy intake through gastrointestinal mechanisms, without altering any central mechanisms. Since dietary lipids represent the major source of unwanted calories, specifically inhibiting TG digestion forms a new approach for the reduction of fat absorption. The major initial reaction products of lipid peroxidation are lipid peroxides and their quantitation serves as a direct and valuable index of the oxidative status of polyunsaturated fatty acid-containing tissues (membranes) or biosystems. Oxidatively modified human serum low-density lipoprotein (LDL) has recently gained increasing interest in atherosclerosis research. It might represent a form of LDL by which monocyte∼macrophages can be transformed to lipid-laden foam cells, a cell type that is often found in early atherosclerotic lesions (Jialal & Devaraj, 1996). Several methods currently exist for estimating the oxidation of lipids and some of them have been used to assess lipid oxidation in oxidized lipoproteins. Based on the reaction of malondialdehyde, a break down product of lipid peroxides, with thiobarbituric acid (TBA), the measurement of so-called thiobarbituric acid reactive substances has been used commonly to check lipoproteins for products of lipid peroxidation. In this present study we evaluated the antioxidant capacity, α-amylase, and lipase inhibitory activity of the Crotalaria juncea Linn extracts.
MATERIALS AND METHODS Collection and Authentification Plant material of Crotalaria juncea Linn was collected from local areas of Nalgonda, Andhra Pradesh and plant was authentified by Mr. A. Lakshma Reddy, Retired
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Professor, Dept. of Botany, Nagarjuna Govt. College (Autonomous) Nalgonda, Andhra Pradesh. The Plant was dried in shade and ground into uniform powder using milling machine.
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Chemicals Petroleum ether, chloroform, and ethanol were purchased from SD Fine chemicals Ltd., (India). Chemicals used for determination of the content of total phenols, tannins, and flavonoids were obtained from Sigma, Merck and SD Fine chemicals Ltd. All the reagents used were of laboratory and analytical grade. Diagnostic kits were purchased from the Reckon diagnostics, India. All the parameters were estimated using an automatic analyzer (Robonik Touch, version 2.622A) and Elico SL196 double beam spectrophotometer. Preparation of Extracts The extracts of Crotalaria juncea Linn were prepared by successive soxhlation with various solvents. The shade dried whole plant powder was packed in thimble kept in the soxhlet apparatus and extraction was allowed to run successively using the solvents like petroleum ether (60 ± 80◦ C), chloroform and ethanol. Finally, the marc was dried and macerated with chloroform-water for 24 hr to obtain the aqueous extract. Petroleum ether and chloroform were used to defat the final extract. Only ethanol and aqueous extracts were concentrated by evaporating the solvent on the Water-bath and the obtained extracts were weighed. The extract of Crotalaria juncea Linn was prepared using maceration technique by taking 50 g of shade dried whole plant powder suspending it and extracting with 10 volumes of methanol by shaking at room temperature for 15 hr. The extracts were filtered through filter paper, and the supernatants were pooled. The residue was re-extracted under the same conditions. Pooled extracts were condensed (and methanol was removed) with a rotary evaporator at 50◦ C. Polyphenol, Flavonoid, and Tannin Estimation Total phenolic content (Singleton, Orthofer, & Lamuela Raventos, 1999), flavonoid content (Kafkas Ebru, Ercisli, Kemal, Baydar, & Yilmaz, 2009) and condensed tannin content (Shih-chaun Liu, Jau-Tien, Chin-Kun, Hsin-Yi, & DengJye, 2009; www.scribd.com) were determined by analyzing spectrophotometrically. For phenolic content estimation, Gallic acid was used to prepare a standard curve (0.2–10 μg/mL; y = 0.06218x+0.131; r2 = 0.9850; y is the absorbance; x is the solution concentration) and the results were expressed as milligrams of Gallic acid equivalents (GAEs) per gram of powdered crude drug. For flavonoid content estimation, Rutin was used to prepare a standard curve (0.2–10 μg/mL; y = 1.4297x−0.3178; r2 = 0.9854; y is the absorbance; x is the solution concentration) and the results were expressed as milligrams of rutin equivalents (REs) per gram of powdered crude drug. For condensed tannin content, Tannic acid was used to prepare a standard curve (0.2–10 μg/mL; y = 0.0271x+0.157; r2 = 0.9951; y is the absorbance; x is the solution concentration) and the results were expressed as milligrams of tannic acid equivalents (TAEs) per gram of powdered crude drug.
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Amylase Inhibition Activity This activity was measured by test kit obtained from Reckon diagnostics Pvt. Ltd. Aliquots (50 μL) of amylase standard and samples were added and mixed gently and incubated for 5 min at 37◦ C to which the working reagent (1,000 μL) was added after initial incubation and mixed by gentle inversion, and the samples were incubated again for 1 min at 37◦ C. The rate of decrease in absorbance at 405 nm from 1 to 3 min with the regular interval of 30 s was recorded (using water as blank). Atorvastatin was used as standard. The alpha amylase activity in the samples was determined by the formula, Alpha amylase activity (IU/L) = A/min x F, where, F is a factor and is equal to (1/12.9) × (Total volume/Sample volume) × 1,000, where 12.9 is milimolar absorbance of 2-Chloro-4-Nitrophenol and 1,000 is for the conversion of activity per milliliter to per liter. Lipase Inhibition Activity Working reagent and lipase calibrator (450 U/L) were obtained from Reckon diagnostics Pvt. Ltd. Aliquots (50 μL) of lipase standard and samples were added and mixed gently and incubated for 5 min at 37◦ C. Working reagent (1,000 μL) was added and mixed by gentle inversion, and the samples were reincubated for 4 min at 37◦ C. The rate of decrease in absorbance at 340 nm from 4 to 8 min with the regular interval of 60 s was recorded (using water as blank). Atorvastatin was used as standard. The pancreatic lipase activity in the samples prepared was determined using the formula; Lipase activity (U/L) = (A/min of sample/A/min of calibrator) × calibrator value. Antilipid Peroxidation Activity in Goat Liver (Sathis Kumar, Raju, Harani, & David, 2011) Goat liver was collected from a local market in Kakinada. Goat liver perfused with normal saline through hepatic portal vein was harvested and its lobes were briefly dried between filter papers to remove excess blood and cut in to thin and small pieces with a heavy-duty blade. The small pieces were transferred in a sterile vessel containing phosphate buffer (0.1 M, pH 7.4) solution. After draining the buffer solution as completely as possible, the liver was immediately grinded to make a tissue homogenate (1 g/mL) using freshly prepared phosphate buffer (0.1 M, pH 7.4). The homogenate was centrifuged at (1,000 rpm, 15 min) to remove debris. The supernatant was used for assay to determine the lipid peroxidation activity. Extracts were used in various concentrations of 500 and 250 μg/mL separately. One hundred micrometer of 15 mM ferric chloride solution was added to 3 mL of liver homogenate and was shaken for 30 min. From the mixture, 100 μL was taken (separately into different test tubes) to which 1 mL of different concentrations of both plant extracts were added individually into different test tubes. The same procedure was followed for control and Standard (ascorbic acid). All the test tubes were incubated for 4 hr at 37◦ C. After incubation, 1.1 mL of 30% trichloroacetic acid and 1.1 mL of 0.65% TBA were added to all tubes containing the mixture. The tubes were kept in a shaking water bath for 30 min at 80◦ C. After 30 min of incubation the tubes were taken out and kept in ice-cold water for 10 min. These were then centrifuged at 800 g for 15 min. The amount of malondialdehyde was assessed by measuring the absorbance
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of supernatant at 530 nm at room temperature against an appropriate blank. The percentage inhibition of lipid peroxidation was calculated using the formula, percentage of anti lipid peroxidation = ((Control – Sample)/(Control)) × 100.
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Antioxidant Activity on Linoleic Acid (Effat Souri, Gholamreza, Hassan, Hassan, & Saba, 2008) Two dilutions of each extract (250 and 500 μg/mL) were prepared. For a typical assay an aliquot of 20 μL of each dilution was mixed with 20 μL of 2 mg/mL linoleic acid in ethanol and incubated at 80◦ C for 60 min. Incubated samples were cooled in an ice bath, followed by the addition of 200 μL of 20 mM butylated hydroxytoluene, 200 μL of 8% sodium dodecyl sulfate and 400 μL of distilled water. After mixing, 3.2 mL of 12.5 mM 1,3-diethyl-2-TBA in sodium phosphate buffer (0.125 M, pH 3.0) was added. After mixing, the tubes were heated at 95◦ C for 15 min, and cooled in an ice bath. Then 4 mL of ethyl acetate was added to each tube, vortexed to extract the pink adduct from the aqueous phase, and centrifuged at 700 g for 10 min. A control containing linoleic acid and other additives without antioxidants, representing 100% lipid peroxidation was also prepared. The absorbance of ethyl acetate layer of sample and control was measured at an excitation wavelength of 515 nm. The antioxidant activity was calculated as the percent of peroxidation inhibition using the following equation: Percent of peroxidation inhibition = ((Control − Sample)/(Control)) × 100. RESULTS AND DISCUSSION Crotalaria juncea Linn extracts were investigated for their total phenolic compounds, flavonoids, and tannins using spectrophotometry. The results of phenolic content, flavonoids, and tannins were expressed as mean ± standard deviation. In the phenolic content determination, the milligrams of GAE per gram of extracts were found to be 1.394 ± 0.092, 0.693 ± 0.025, and 0.734 ± 0.112 for ethanolic, aqueous, and macerated methanol extracts, respectively. The ethanolic extract contained maximum total phenolic content (1.394 mg GAE/g) than aqueous and macerated methanol extract. In flavonoid content estimation, the milligrams of RE per gram of extracts was found to be 0.845 ± 0.034, 0.119 ± 0.004, and 0.496 ± 0.003 for ethanolic, aqueous, and macerated methanol extracts, respectively. The ethanolic extract contained maximum total flavonoid content (0.845 mg RE/g) than aqueous and macerated methanol extract. In tannin content estimation, the milligrams of TAE per gram of extracts was found to be 0.281 ± 0.11, 0.209 ± 0.001, and 0.224 ± 0.002 for ethanolic, aqueous, and macerated methanol extracts, respectively. The ethanolic extract had maximum total condensed tannin content (0.281 mg TAE/g) than aqueous and macerated methanol extract. Figure 1 expresses the results of phenolic, flavonoid, and tannin contents in Crotalaria juncea Linn extracts. In our experiments, all the extracts with two different concentrations (250 and 500 mcg/mL) had shown in vitro inhibitory activity on amylase and lipase and antioxidant activity in a dose-dependent manner significantly (∗∗ p < .01) and also exerted a moderate inhibitory action on amylase and lipase and antioxidant activities (Table 1) when compared with standards. Standard (250 mcg/mL) had shown 90.47%
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FIGURE 1. Phenolic, flavonoid, and tannin estimation on Crotalaria juncea Linn extracts.
inhibition on amylase activity; 82.53% inhibition on lipase activity (∗∗ p < .01); 53.18% for antilipid peroxidation (∗∗ p < .01) and 57.28% for antioxidant on linoleic acid emulsion (∗∗ p < .01). Among the three plant extracts, though Macerated methanol extract (500 mg/kg) did show a greater inhibitory activity on amylase and lipase enzymes, i.e., 71.42% inhibition on amylase activity (∗∗ p < .01) and 57.14% inhibition on lipase activity (∗∗ p < .01); it had less antioxidant activity than ethanolic and aqueous extracts. Among the three extracts, aqueous extract (500 mg/kg) had high (66.94%, ∗∗ p < .01) antilipid peroxidation and 59.54% of antioxidant on linoleic acid emulsion (∗∗ p < .01) but in case of amylase and lipase inhibition, it was moderate. In all inhibitory activities, ethanolic extract had less affect than other extracts. The correlation between the active constituents and the activity of macerated methanol extract can be explained by the inhibition activity on amylase and lipase, antilipid peroxidation, and antioxidant activity on linoleic acid emulsion, which is due to high concentration of phenolic compounds, flavonoids, and tannins. Alpha amylase uses a chromogenic substrate Gal-G3-CNP, which by the reaction of alpha-Amylase, breaks down to release 2-Chloro-4-Nitrophenol. The release of 2-Chloro-4-Nitrophenol is measured at 405nm and is proportional to alpha amylase activity. Lipases catalyze the hydrolysis of triolein in the presence of colipase to form glycerides and fatty acids. The rate of decrease in turbidity measured at 340 nm is proportional to the lipase activity. As lipid peroxides are increasingly thought to have a pathogenic role in many disorders a specific and reliable test for their concentrations in plasma in greatly needed. Oxidation stress has been implicated in the pathology of many diseases and conditions including cardiovascular disease, diabetes, inflammatory conditions, cancer, and ageing. Antioxidants may offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid per-oxidation, and other mechanisms. Bioactivity of Crotalaria juncea Linn extracts may be due to the potentiating action of several compounds within the active fractions of the extracts.
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Amylase inhibitory 90.47 ± 1.37 52.38 ± 2.38∗∗ 71.42 ± 1.37∗∗ 25.39 ± 2.86∗∗ 54.76 ± 2.74∗∗ 23.80 ± 1.375∗∗ 46.82 ± 0.79∗∗
Amylase activity (IU/L)
1.62 ± 0.235 8.14 ± 0.407∗∗ 4.88 ± 0.235∗∗ 12.75 ± 0.489∗∗ 7.73 ± 0.47∗∗ 13.02 ± 0.235∗∗ 9.08 ± 0.135∗∗
78.57 ± 7.14 264.28 ± 7.14∗∗ 192.85 ± 12.37∗∗ 307.14 ± 7.14∗∗ 257.14 ± 12.37∗∗ 371.42 ± 7.14∗∗ 314.28 ± 14.28∗∗
Lipase activity (U/L)
% antilipid peroxidation 53.18 ± 0.4107∗∗ 4.31 ± 4.107 18.06 ± 0.2053 22.99 ± 1.437∗ 35.11 ± 9.446∗∗ 56.67 ± 0.616∗∗ 66.94 ± 0.616∗∗
Lipase inhibitory 82.53 ± 1.58∗∗ 41.26 ± 1.58∗∗ 57.14 ± 2.74∗∗ 31.74 ± 1.58∗∗ 42.85 ± 2.74∗∗ 17.46 ± 1.58∗∗ 30.15 ± 3.17∗∗
57.28 ± 0.41∗∗ 6.57 ± 6.36 20.53 ± 1.84∗ 20.32 ± 2.05∗ 38.19 ± 8.41∗∗ 42.29 ± 1.43∗∗ 59.54 ± 0.2∗∗
% antioxidant activity
Results represents means ± SEM, n = 6. ∗∗ p < .01; ∗ p < .05; CJI = macerated methanol extract; CJE = ethanol extract; CJW = water extract; Standard for amylase and lipase is atorvastatin whereas ascorbic acid is for antioxidant activity.
Standard (250) CJI (250) CJI (500) CJE (250) CJE (500) CJW (250) CJW (500)
Samples (mcg/mL)
TABLE 1. In Vitro Activity of Crotalaria juncea Linn Extracts
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FIGURE 2. Amylase, lipase, and antioxidant activities on Crotalaria juncea Linn extracts.
CONCLUSION By this investigation, we concluded that maceration by methanol could extract the highest concentration of polyphenols, flavonoids, and tannins from the Crotalaria juncea Linn plant which are responsible for the above-mentioned in vitro activity. So further studies are required to confirm its pharmacological potency through in vivo studies, by which we can assure its potential for exploitation to promote human and animal health. ACKNOWLEDGMENT The authors are grateful to the Management, Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh for their valuable support. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. ABOUT THE AUTHORS D. Sathis Kumar is working as an associate professor, Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh, India. His biography is being considered for the forthcoming Who’s Who in the World 2014 (31st edition). He has 80 publications in international and national journals. David Banji is a principal of Nalanda College of Pharmacy, Nalgonda, India. He has more than 200 publications and 20 years’ experience in academic and research. Harani A. is working as an assistant professor, Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh, India. She has 35 publications in international and national journals. Otilia Banji is HOD of Department of Pharmacology, Nalanda College of Pharmacy, Nalgonda, India. She has more than a good number of publications and 10 years’ experience in academic and research.
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REFERENCES Available at http://www.scribd.com/doc/6506867/Tannin-Assay, Retrieved on 29th March 2011. Effat S, Gholamreza A, Hassan F, Hassan J, Saba B. Screening of thirteen medicinal plant extracts for antioxidant activity. Iranian J Pharm Res. 2008;7(2):149–154. Jialal I, Devaraj S. Low density lipoprotein oxidation, antioxidants and atheroscelerosis: a clinical biochemistry perspective. Clin Chem. 1996;42:498–506. Kafkas E, Ercisli S, Kemal KN, Baydar K, Yilmaz H. Chemical composition of blood orange varities from Turkey: a comparative study. Pharmacognosy Mag. 2009;5(20):331. Karthic K, Kirthiram KS, Sadasivam S, Thayumanavan B. Identification of alpha amylase inhibitors from Syzygium cumini Linn seeds. Indian J Exp Biol. 2008;46:677–680. Sathis Kumar D, David B, Prashanthi G, Harani A. Pharmacognostical evaluation study on Crotalaria juncea Linn. American-Eurasian J Sci Res. 2011;6(3):139–145. Sathis Kumar D, Raju SN, Harani A, David Banji. Antilipid peroxidation activity of piper trioicum and physalis minima extract is pakistan. J Pharm Sci. 2011;24(3):411–413. Shi Y, Burn P. Lipid metabolic enzymes: Emerging drug targets for the treatment of obesity. Nat Rev Drug Discov. 2004;3:695–710. Shih-chaun L, Jau-Tien L, Chin-Kun W, Hsin-Yi C, Deng-Jye Y. Antioxidant properties of various solvent extracts from lychee (Litchi chinenesis Sonn.) flowers. Food Chem. 2009;114:578. Singleton VL, Orthofer R, Lamuela Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 1999;299:152–178. Tilbeurgh HV. Structure of the pancreatic lipase-procolipase complex. Nature. 1992;359:159–162.
ARTICLE
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Determination of Antioxidant Capacity, α-Amylase and Lipase Inhibitory Activity of Crotalaria Juncea Linn In Vitro Inhibitory Activity of Crotalaria Juncea Linn Sathis Kumar Dinakaran1,2 , David Banji3 , Harani Avasarala1 , & Otilia Banji3 1
Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh, India, 2 Jawaharlal Nehru Technical University, Hyderabad, Andhra Pradesh, India, 3 Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh, India
ABSTRACT. The present study involves the determination of antioxidant capacity and in vitro α-amylase and lipase inhibitory activity of the Crotalaria juncea Linn extract. The content of polyphenols, flavonoids, and tannins in the extracts was estimated by spectrophotometry. Antioxidant activity on goat liver lipid peroxidation and linoleic acid emulsion were determined and α-amylase and lipase inhibitory activity was also evaluated. All the extracts had shown antioxidant property, α-amylase, and lipase inhibitory properties. Aqueous extract was found to show maximum antioxidant activity on goat liver. Antilipid peroxidation and antioxidant activity were determined to be 66.94 ± 0.616 (p < .01) and 59.54 ± 0.2 (p < .01), respectively. Maximum α-amylase and lipase inhibitory activities of 71.42 ± 1.37 (p < .01) and 57.14 ± 2.74% (p < .01), respectively, were exhibited by macerated methanol extract. The results had shown that all the extracts exhibited low inhibition and antioxidant activity as compared to standard. KEYWORDS. amylase, antioxidant, Crotalaria juncea Linn, flavonoids, lipase, phenolic compounds, tannins
INTRODUCTION Crotalaria juncea Linn also called as Sun hemp or Indian hemp belongs to Fabaceae family and is distributed in the tropical and subtropical regions of India, Nepal, Sri Lanka, and Southern Africa. It is an annually renewable, multi-purpose fiber crop. Due to its great potency, it is used as a medicine by many tribal communities. Generally, in the folk and Ayurvedic medicines, it is used as blood purifier, abortificient, astringent, demulcent, emetic, purgative, and used in the treatment of anemia, impetigo, menorrhagia, and psoriasis (Sathis Kumar, David, PrashanAddress correspondence to: D. Sathis Kumar, Associate Professor, Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh 533437, India (E-mail: [email protected]). (Received 1 May 2013; revised 5 August 2013; accepted 10 September 2013)
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thi, & Harani, 2011). The Alpha amylase is the one of the main enzymes in human that catalyzes the hydrolysis of 1,4-glucosidic linkage of complex carbohydrates like starch into maltose and oligosaccharides in the small intestine. Inhibition of this enzyme reduces the rate of digestion of starch and results in the decrease of postprandial blood glucose levels in diabetic patients (Karthic, Kirthiram, Sadasivam, & Thayumanavan, 2008). Reducing postprandial hyperglycemia prevents glucose uptake into adipose tissue for inhibiting the synthesis and accumulation of triacyl glycerol. On the other hand, it is well known that dietary lipid is not directly absorbed from the intestine unless it has been subjected to the action of pancreatic lipase. Lipases are enzymes that digest fats, including triacylglycerol and phospholipids. It removes fatty acids from the α and α’ position of dietary triglycerides (TGs) and yields β-monoglycerides and long chain saturated and polyunsaturated fatty acids as the lipolytic products (Shi & Burn, 2004). Lipase is responsible for the hydrolysis of 50%–70% of total dietary fats. Lingual lipase secreted by serous gland, digests approximately one third of ingested fat. Gastric lipase secreted in response to mechanical stimulation, ingestion of food or sympathetic activation accounts for the hydrolysis of 10%–40% of dietary fat (Tilbeurgh, 1992). These two enzymes, thus, potentially limit the nutritional impact of the inhibition of lipid absorption that could result from the reduction in the activity of Pancreatic Lipase alone. Pancreatic Lipase inhibition is one of the most widely studied mechanisms for the determination of the potential efficacy of natural products as antiobesity agents. One of the most important strategies in the treatment of obesity includes development of inhibitors of nutrient digestion and absorption, in an attempt to reduce energy intake through gastrointestinal mechanisms, without altering any central mechanisms. Since dietary lipids represent the major source of unwanted calories, specifically inhibiting TG digestion forms a new approach for the reduction of fat absorption. The major initial reaction products of lipid peroxidation are lipid peroxides and their quantitation serves as a direct and valuable index of the oxidative status of polyunsaturated fatty acid-containing tissues (membranes) or biosystems. Oxidatively modified human serum low-density lipoprotein (LDL) has recently gained increasing interest in atherosclerosis research. It might represent a form of LDL by which monocyte∼macrophages can be transformed to lipid-laden foam cells, a cell type that is often found in early atherosclerotic lesions (Jialal & Devaraj, 1996). Several methods currently exist for estimating the oxidation of lipids and some of them have been used to assess lipid oxidation in oxidized lipoproteins. Based on the reaction of malondialdehyde, a break down product of lipid peroxides, with thiobarbituric acid (TBA), the measurement of so-called thiobarbituric acid reactive substances has been used commonly to check lipoproteins for products of lipid peroxidation. In this present study we evaluated the antioxidant capacity, α-amylase, and lipase inhibitory activity of the Crotalaria juncea Linn extracts.
MATERIALS AND METHODS Collection and Authentification Plant material of Crotalaria juncea Linn was collected from local areas of Nalgonda, Andhra Pradesh and plant was authentified by Mr. A. Lakshma Reddy, Retired
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Professor, Dept. of Botany, Nagarjuna Govt. College (Autonomous) Nalgonda, Andhra Pradesh. The Plant was dried in shade and ground into uniform powder using milling machine.
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Chemicals Petroleum ether, chloroform, and ethanol were purchased from SD Fine chemicals Ltd., (India). Chemicals used for determination of the content of total phenols, tannins, and flavonoids were obtained from Sigma, Merck and SD Fine chemicals Ltd. All the reagents used were of laboratory and analytical grade. Diagnostic kits were purchased from the Reckon diagnostics, India. All the parameters were estimated using an automatic analyzer (Robonik Touch, version 2.622A) and Elico SL196 double beam spectrophotometer. Preparation of Extracts The extracts of Crotalaria juncea Linn were prepared by successive soxhlation with various solvents. The shade dried whole plant powder was packed in thimble kept in the soxhlet apparatus and extraction was allowed to run successively using the solvents like petroleum ether (60 ± 80◦ C), chloroform and ethanol. Finally, the marc was dried and macerated with chloroform-water for 24 hr to obtain the aqueous extract. Petroleum ether and chloroform were used to defat the final extract. Only ethanol and aqueous extracts were concentrated by evaporating the solvent on the Water-bath and the obtained extracts were weighed. The extract of Crotalaria juncea Linn was prepared using maceration technique by taking 50 g of shade dried whole plant powder suspending it and extracting with 10 volumes of methanol by shaking at room temperature for 15 hr. The extracts were filtered through filter paper, and the supernatants were pooled. The residue was re-extracted under the same conditions. Pooled extracts were condensed (and methanol was removed) with a rotary evaporator at 50◦ C. Polyphenol, Flavonoid, and Tannin Estimation Total phenolic content (Singleton, Orthofer, & Lamuela Raventos, 1999), flavonoid content (Kafkas Ebru, Ercisli, Kemal, Baydar, & Yilmaz, 2009) and condensed tannin content (Shih-chaun Liu, Jau-Tien, Chin-Kun, Hsin-Yi, & DengJye, 2009; www.scribd.com) were determined by analyzing spectrophotometrically. For phenolic content estimation, Gallic acid was used to prepare a standard curve (0.2–10 μg/mL; y = 0.06218x+0.131; r2 = 0.9850; y is the absorbance; x is the solution concentration) and the results were expressed as milligrams of Gallic acid equivalents (GAEs) per gram of powdered crude drug. For flavonoid content estimation, Rutin was used to prepare a standard curve (0.2–10 μg/mL; y = 1.4297x−0.3178; r2 = 0.9854; y is the absorbance; x is the solution concentration) and the results were expressed as milligrams of rutin equivalents (REs) per gram of powdered crude drug. For condensed tannin content, Tannic acid was used to prepare a standard curve (0.2–10 μg/mL; y = 0.0271x+0.157; r2 = 0.9951; y is the absorbance; x is the solution concentration) and the results were expressed as milligrams of tannic acid equivalents (TAEs) per gram of powdered crude drug.
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Amylase Inhibition Activity This activity was measured by test kit obtained from Reckon diagnostics Pvt. Ltd. Aliquots (50 μL) of amylase standard and samples were added and mixed gently and incubated for 5 min at 37◦ C to which the working reagent (1,000 μL) was added after initial incubation and mixed by gentle inversion, and the samples were incubated again for 1 min at 37◦ C. The rate of decrease in absorbance at 405 nm from 1 to 3 min with the regular interval of 30 s was recorded (using water as blank). Atorvastatin was used as standard. The alpha amylase activity in the samples was determined by the formula, Alpha amylase activity (IU/L) = A/min x F, where, F is a factor and is equal to (1/12.9) × (Total volume/Sample volume) × 1,000, where 12.9 is milimolar absorbance of 2-Chloro-4-Nitrophenol and 1,000 is for the conversion of activity per milliliter to per liter. Lipase Inhibition Activity Working reagent and lipase calibrator (450 U/L) were obtained from Reckon diagnostics Pvt. Ltd. Aliquots (50 μL) of lipase standard and samples were added and mixed gently and incubated for 5 min at 37◦ C. Working reagent (1,000 μL) was added and mixed by gentle inversion, and the samples were reincubated for 4 min at 37◦ C. The rate of decrease in absorbance at 340 nm from 4 to 8 min with the regular interval of 60 s was recorded (using water as blank). Atorvastatin was used as standard. The pancreatic lipase activity in the samples prepared was determined using the formula; Lipase activity (U/L) = (A/min of sample/A/min of calibrator) × calibrator value. Antilipid Peroxidation Activity in Goat Liver (Sathis Kumar, Raju, Harani, & David, 2011) Goat liver was collected from a local market in Kakinada. Goat liver perfused with normal saline through hepatic portal vein was harvested and its lobes were briefly dried between filter papers to remove excess blood and cut in to thin and small pieces with a heavy-duty blade. The small pieces were transferred in a sterile vessel containing phosphate buffer (0.1 M, pH 7.4) solution. After draining the buffer solution as completely as possible, the liver was immediately grinded to make a tissue homogenate (1 g/mL) using freshly prepared phosphate buffer (0.1 M, pH 7.4). The homogenate was centrifuged at (1,000 rpm, 15 min) to remove debris. The supernatant was used for assay to determine the lipid peroxidation activity. Extracts were used in various concentrations of 500 and 250 μg/mL separately. One hundred micrometer of 15 mM ferric chloride solution was added to 3 mL of liver homogenate and was shaken for 30 min. From the mixture, 100 μL was taken (separately into different test tubes) to which 1 mL of different concentrations of both plant extracts were added individually into different test tubes. The same procedure was followed for control and Standard (ascorbic acid). All the test tubes were incubated for 4 hr at 37◦ C. After incubation, 1.1 mL of 30% trichloroacetic acid and 1.1 mL of 0.65% TBA were added to all tubes containing the mixture. The tubes were kept in a shaking water bath for 30 min at 80◦ C. After 30 min of incubation the tubes were taken out and kept in ice-cold water for 10 min. These were then centrifuged at 800 g for 15 min. The amount of malondialdehyde was assessed by measuring the absorbance
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of supernatant at 530 nm at room temperature against an appropriate blank. The percentage inhibition of lipid peroxidation was calculated using the formula, percentage of anti lipid peroxidation = ((Control – Sample)/(Control)) × 100.
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Antioxidant Activity on Linoleic Acid (Effat Souri, Gholamreza, Hassan, Hassan, & Saba, 2008) Two dilutions of each extract (250 and 500 μg/mL) were prepared. For a typical assay an aliquot of 20 μL of each dilution was mixed with 20 μL of 2 mg/mL linoleic acid in ethanol and incubated at 80◦ C for 60 min. Incubated samples were cooled in an ice bath, followed by the addition of 200 μL of 20 mM butylated hydroxytoluene, 200 μL of 8% sodium dodecyl sulfate and 400 μL of distilled water. After mixing, 3.2 mL of 12.5 mM 1,3-diethyl-2-TBA in sodium phosphate buffer (0.125 M, pH 3.0) was added. After mixing, the tubes were heated at 95◦ C for 15 min, and cooled in an ice bath. Then 4 mL of ethyl acetate was added to each tube, vortexed to extract the pink adduct from the aqueous phase, and centrifuged at 700 g for 10 min. A control containing linoleic acid and other additives without antioxidants, representing 100% lipid peroxidation was also prepared. The absorbance of ethyl acetate layer of sample and control was measured at an excitation wavelength of 515 nm. The antioxidant activity was calculated as the percent of peroxidation inhibition using the following equation: Percent of peroxidation inhibition = ((Control − Sample)/(Control)) × 100. RESULTS AND DISCUSSION Crotalaria juncea Linn extracts were investigated for their total phenolic compounds, flavonoids, and tannins using spectrophotometry. The results of phenolic content, flavonoids, and tannins were expressed as mean ± standard deviation. In the phenolic content determination, the milligrams of GAE per gram of extracts were found to be 1.394 ± 0.092, 0.693 ± 0.025, and 0.734 ± 0.112 for ethanolic, aqueous, and macerated methanol extracts, respectively. The ethanolic extract contained maximum total phenolic content (1.394 mg GAE/g) than aqueous and macerated methanol extract. In flavonoid content estimation, the milligrams of RE per gram of extracts was found to be 0.845 ± 0.034, 0.119 ± 0.004, and 0.496 ± 0.003 for ethanolic, aqueous, and macerated methanol extracts, respectively. The ethanolic extract contained maximum total flavonoid content (0.845 mg RE/g) than aqueous and macerated methanol extract. In tannin content estimation, the milligrams of TAE per gram of extracts was found to be 0.281 ± 0.11, 0.209 ± 0.001, and 0.224 ± 0.002 for ethanolic, aqueous, and macerated methanol extracts, respectively. The ethanolic extract had maximum total condensed tannin content (0.281 mg TAE/g) than aqueous and macerated methanol extract. Figure 1 expresses the results of phenolic, flavonoid, and tannin contents in Crotalaria juncea Linn extracts. In our experiments, all the extracts with two different concentrations (250 and 500 mcg/mL) had shown in vitro inhibitory activity on amylase and lipase and antioxidant activity in a dose-dependent manner significantly (∗∗ p < .01) and also exerted a moderate inhibitory action on amylase and lipase and antioxidant activities (Table 1) when compared with standards. Standard (250 mcg/mL) had shown 90.47%
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FIGURE 1. Phenolic, flavonoid, and tannin estimation on Crotalaria juncea Linn extracts.
inhibition on amylase activity; 82.53% inhibition on lipase activity (∗∗ p < .01); 53.18% for antilipid peroxidation (∗∗ p < .01) and 57.28% for antioxidant on linoleic acid emulsion (∗∗ p < .01). Among the three plant extracts, though Macerated methanol extract (500 mg/kg) did show a greater inhibitory activity on amylase and lipase enzymes, i.e., 71.42% inhibition on amylase activity (∗∗ p < .01) and 57.14% inhibition on lipase activity (∗∗ p < .01); it had less antioxidant activity than ethanolic and aqueous extracts. Among the three extracts, aqueous extract (500 mg/kg) had high (66.94%, ∗∗ p < .01) antilipid peroxidation and 59.54% of antioxidant on linoleic acid emulsion (∗∗ p < .01) but in case of amylase and lipase inhibition, it was moderate. In all inhibitory activities, ethanolic extract had less affect than other extracts. The correlation between the active constituents and the activity of macerated methanol extract can be explained by the inhibition activity on amylase and lipase, antilipid peroxidation, and antioxidant activity on linoleic acid emulsion, which is due to high concentration of phenolic compounds, flavonoids, and tannins. Alpha amylase uses a chromogenic substrate Gal-G3-CNP, which by the reaction of alpha-Amylase, breaks down to release 2-Chloro-4-Nitrophenol. The release of 2-Chloro-4-Nitrophenol is measured at 405nm and is proportional to alpha amylase activity. Lipases catalyze the hydrolysis of triolein in the presence of colipase to form glycerides and fatty acids. The rate of decrease in turbidity measured at 340 nm is proportional to the lipase activity. As lipid peroxides are increasingly thought to have a pathogenic role in many disorders a specific and reliable test for their concentrations in plasma in greatly needed. Oxidation stress has been implicated in the pathology of many diseases and conditions including cardiovascular disease, diabetes, inflammatory conditions, cancer, and ageing. Antioxidants may offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid per-oxidation, and other mechanisms. Bioactivity of Crotalaria juncea Linn extracts may be due to the potentiating action of several compounds within the active fractions of the extracts.
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Amylase inhibitory 90.47 ± 1.37 52.38 ± 2.38∗∗ 71.42 ± 1.37∗∗ 25.39 ± 2.86∗∗ 54.76 ± 2.74∗∗ 23.80 ± 1.375∗∗ 46.82 ± 0.79∗∗
Amylase activity (IU/L)
1.62 ± 0.235 8.14 ± 0.407∗∗ 4.88 ± 0.235∗∗ 12.75 ± 0.489∗∗ 7.73 ± 0.47∗∗ 13.02 ± 0.235∗∗ 9.08 ± 0.135∗∗
78.57 ± 7.14 264.28 ± 7.14∗∗ 192.85 ± 12.37∗∗ 307.14 ± 7.14∗∗ 257.14 ± 12.37∗∗ 371.42 ± 7.14∗∗ 314.28 ± 14.28∗∗
Lipase activity (U/L)
% antilipid peroxidation 53.18 ± 0.4107∗∗ 4.31 ± 4.107 18.06 ± 0.2053 22.99 ± 1.437∗ 35.11 ± 9.446∗∗ 56.67 ± 0.616∗∗ 66.94 ± 0.616∗∗
Lipase inhibitory 82.53 ± 1.58∗∗ 41.26 ± 1.58∗∗ 57.14 ± 2.74∗∗ 31.74 ± 1.58∗∗ 42.85 ± 2.74∗∗ 17.46 ± 1.58∗∗ 30.15 ± 3.17∗∗
57.28 ± 0.41∗∗ 6.57 ± 6.36 20.53 ± 1.84∗ 20.32 ± 2.05∗ 38.19 ± 8.41∗∗ 42.29 ± 1.43∗∗ 59.54 ± 0.2∗∗
% antioxidant activity
Results represents means ± SEM, n = 6. ∗∗ p < .01; ∗ p < .05; CJI = macerated methanol extract; CJE = ethanol extract; CJW = water extract; Standard for amylase and lipase is atorvastatin whereas ascorbic acid is for antioxidant activity.
Standard (250) CJI (250) CJI (500) CJE (250) CJE (500) CJW (250) CJW (500)
Samples (mcg/mL)
TABLE 1. In Vitro Activity of Crotalaria juncea Linn Extracts
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FIGURE 2. Amylase, lipase, and antioxidant activities on Crotalaria juncea Linn extracts.
CONCLUSION By this investigation, we concluded that maceration by methanol could extract the highest concentration of polyphenols, flavonoids, and tannins from the Crotalaria juncea Linn plant which are responsible for the above-mentioned in vitro activity. So further studies are required to confirm its pharmacological potency through in vivo studies, by which we can assure its potential for exploitation to promote human and animal health. ACKNOWLEDGMENT The authors are grateful to the Management, Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh for their valuable support. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. ABOUT THE AUTHORS D. Sathis Kumar is working as an associate professor, Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh, India. His biography is being considered for the forthcoming Who’s Who in the World 2014 (31st edition). He has 80 publications in international and national journals. David Banji is a principal of Nalanda College of Pharmacy, Nalgonda, India. He has more than 200 publications and 20 years’ experience in academic and research. Harani A. is working as an assistant professor, Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Andhra Pradesh, India. She has 35 publications in international and national journals. Otilia Banji is HOD of Department of Pharmacology, Nalanda College of Pharmacy, Nalgonda, India. She has more than a good number of publications and 10 years’ experience in academic and research.
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