Journal of Ethnopharmacology 113 (2007) 284–291
Antioxidant activity of Caesalpinia digyna root R. Srinivasan a , M.J.N. Chandrasekar b , M.J. Nanjan a,∗ , B. Suresh a a
b
TIFAC CORE in Herbal Drugs, JSS College of Pharmacy, Ootacamund 643001, The Nilgiris, Tamilnadu, India Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Ootacamund 643001, The Nilgiris, Tamilnadu, India Received 11 August 2006; received in revised form 6 June 2007; accepted 18 June 2007 Available online 26 June 2007
Abstract The antioxidant properties of three successive extracts of Caesalpinia digyna Rottler root and the isolated compound, bergenin, were tested using standard in vitro and in vivo models. The amount of the total phenolic compounds present was also determined. The successive methanol extract of Caesalpinia digyna root (CDM) exhibited strong scavenging effect on 2,2-diphenyl-2-picryl hydrazyl (DPPH) free radical, 2,2 -azino-bis(3ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt (ABTS) radical cation, hydrogen peroxide, nitric oxide, hydroxyl radical and inhibition of lipid peroxidation. The free radical scavenging effect of CDM was comparable with that of reference antioxidants. The CDM having the highest content of phenolic compounds and strong free radical scavenging effect when administered orally to male albino rats at 100, 200 and 400 mg/kg body weight for 7 days, prior to carbontetrachloride (CCl4 ) treatment, caused a significant increase in the levels of catalase (CAT) and superoxide dismutase (SOD) and significant decrease in the levels of lipidperoxidation (LPO) in serum, liver and kidney in a dose dependent manner, when compared to CCl4 treated control. These results clearly indicate the strong antioxidant property of Caesalpinia digyna root. The study provides a proof for the ethnomedical claims and reported biological activities. The plant has, therefore, very good therapeutic potential. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Caesalpinia digyna; Free radicals; Bergenin; In vivo; CCl4
1. Introduction Free radicals and reactive oxygen species (ROS) are well known inducers of cellular and tissue pathogenesis leading to several human diseases such as cancer, inflammatory disorders, as well as in aging processes (Halliwell, 1994; Aviram, 2000). Antioxidants provide protection to living organisms from damage caused by uncontrolled production of ROS and the concomitant lipid peroxidation, protein damage and DNA strand breaking (Ghosal et al., 1996). Several anti-inflammatory, digestive, antinecrotic, neuroprotective and hepatoprotective drugs have recently been shown to have antioxidant and/or radical scavenging mechanism as part of their activity (Lin and Huang, 2000; Repetto and Llesuy, 2002). The use of traditional medicine is widespread, and plants still present a large source of natural antioxidants that might serve as leads for the development of novel drugs (Perry et al., 1999). Several members of the species of genus Caesalpinia like
∗
Corresponding author. Tel.: +91 423 2447135; fax: +91 423 2447135. E-mail address:
[email protected] (M.J. Nanjan).
0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.06.006
Caesalpinia sappan and Caesalpinia bonducella, etc., are used traditionally for a wide variety of ethnomedical properties such as anti-inflammatory, antidiabetic, antioxidant and hepatoprotective (Badami et al., 2003), etc. Among them, Caesalpinia digyna Rottler (Family: Leguminosae) is a large, scandent, prickly shrub or climber, upto 10 m in height, growing wild in the scrub forests of the eastern Himalayas in Assam and West Bengal, the Eastern Ghats in Andhra Pradesh, Madhya Pradesh and also in Ceylon and Malay Islands. The plant is one of the ingredients of an indigenous drug preparation “Geriforte”, which has been used for curing senile prurites with excellent results. The drug also exhibits antifatigue effect in rats (Anon., 1992). The root has marked astringent properties. It is given internally in pthisis and scrofulous affections; when sores exist, it is applied externally as well. It is also used in diabetes. In some parts of the Burma the root, pounded and mixed with water, is drunk as a febrifuge. It is said to have intoxicating effect (Kiritikar and Basu, 1999). The ethanol water extract of roots inhibits the growth of Mycobacterium tuberculosis (Patel et al., 1966). Chemical investigations of the plant have shown the presence of caesalpinine A, cellallocinnine, ellagic acid, gallic acid, bergenin, bonducellin, intricatinol and tannins (Biswas,
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1944; Chaudhry et al., 1954; Evans and Bell, 1978; Mahato et al., 1983, 1985; Boonsri et al., 2005; Chantrapromma et al., 2006). In view of the several ethnobotanical uses of Caesalpinia digyna described above, it was proposed to screen its successive extracts and isolated compound(s) for the in vitro and in vivo antioxidant activity using standard procedures. 2. Materials and methods 2.1. Plant material The root of Caesalpinia digyna was purchased from Abirami Botanicals, Tuticorin, Tamilnadu, India, and authenticated by Dr. D. Suresh Baburaj, Survey of Medicinal Plants and Collection Unit, Ootacamund, India. A voucher specimen (TIFAC 01) has been deposited for further reference at J.S.S College of Pharmacy herbarium, Ootacamund, India. 2.2. Chemicals 2,2-Diphenyl-2-picryl hydrazyl (DPPH) and 2,2 -azinobis(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt (ABTS) were obtained from Sigma–Aldrich Co., St. Louis, USA. Rutin and p-nitroso dimethyl aniline (p-NDA) were obtained from Acros Organics, NJ, USA. Naphthyl ethylene diamine dihydrochloride (NEDD) was from Roch-Light Ltd., Suffolk, UK, ascorbic acid, nitro blue tetrazolium (NBT) and butylated hydroxy anisole (BHA) were from SD Fine Chemicals Ltd., Mumbai, India and 2-deoxy-d-ribose was from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. Sodium nitroprusside and Silymarin were from Ranbaxy Laboratories Ltd., Mohali, India. Sulphanilic acid used was from E-Merck (India) Ltd., Mumbai, India. All chemicals used were of analytical grade. 2.3. Animals Healthy male albino rats of wistar strain (180–220 g) were obtained from the animal house, J.S.S. College of Pharmacy, Ootacamund, India, and were maintained under standard environment conditions (22–28 ◦ C, 60–70% relative humidity, 12-h dark:12-h light cycle) and were fed with standard rat feed (M/S Hindustan Lever Ltd., Bangalore, India) and water ad libitum. The experiments were conducted as per the guidelines of CPCSEA, Chennai, India (approval no. JSSCP/IAEC/Ph.D/PH.Chemistry/01/2005–2006). 2.4. Extraction procedure The root was chopped to small pieces and dried in shade. The dried root was powdered and passed through sieve no. 20 and extracted (100 g) successively with 600 mL each of petroleum ether (60–80 ◦ C), methanol and water in a Soxhlet extractor for 18–20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature (40–50 ◦ C). The petroleum ether extract yielded a yellowish green sticky semisolid, weighing 0.3 g (0.30%). The methanol and water extracts yielded brown and dark brown semi-solid residues,
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weighing 7.0 g (7.0%) and 0.9 g (0.9%), respectively. All the extracts were preserved in a refrigerator till further use. 2.5. Isolation of bergenin The powdered root was extracted (100 g) with 600 mL of methanol in a Soxhlet extractor for 18–20 h. The extract was concentrated and dissolved in a minimum amount of methanol and kept in room temperature for 24 h. A crystalline solid settled down to the bottom of the container. The crystalline solid was separated and washed with acetone. Repeated recrystallation of combined crystalline solids with methanol yielded a colourless crystalline compound. This was characterized by comparing its melting point, IR, NMR and mass spectrum with a pure specimen of bergenin (Taneyama et al., 1983). 2.6. Preparation of test and standard solutions All the three extracts of Caesalpinia digyna Rottler root, the isolated compound and the standard antioxidants (ascorbic acid, rutin, butylated hydroxy anisole and alpha tocopherol) were dissolved in distilled dimethyl sulphoxide (DMSO) separately and used for the in vitro antioxidant assays using seven different methods except the hydrogen peroxide method. For the hydrogen peroxide method (where DMSO interferes with the method), the extracts and the standards were dissolved in distilled methanol and used. The stock solutions were serially diluted with the respective solvents to obtain lower dilutions. A suspension of CDM and standard drug silymarin were prepared in sodium CMC (0.5%, w/v) using distilled water and used for in vivo experiments. 2.7. Total phenolic compounds estimation Antioxidant compounds generally contain phenolic group(s) and hence, the amount of phenolic compounds in all the three extracts of the root was estimated by using Folin–Ciocalteu reagent (Sadasivam and Manikam, 1992). In a series of test tubes, 0.4 mL of the extract in methanol was taken, mixed with 2 mL of Folin–Ciocalteu reagent and 1.6 mL of sodium carbonate. After shaking, it was kept for 2 h and the absorbance was measured at 750 nm using a Shimadzu-UV-160 spectrophotometer. Using gallic acid monohydrate, a standard curve was prepared. The linearity obtained was in the range of 1–10 g/mL. Using the standard curve, the total phenolic compounds content was calculated and expressed as gallic acid equivalent in mg/g of extracts. 2.8. In vitro antioxidant activity The three extracts and the isolated compound were tested for their in vitro antioxidant activity using the standard methods. In all these methods, a particular concentration of the extract or standard solution was used which gave a final concentration of 1000–0.45 g/mL after all the reagents were added. Absorbance was measured against a blank solution containing the extract or standard, but without the reagents. A control
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test was performed without the extracts or standards. Percentage scavenging and IC50 values ± S.E.M. (IC50 value is the concentration of the sample required to inhibit 50% of radical) were calculated. 2.8.1. Scavenging of ABTS radical cation To 0.2 mL of various concentrations of the extracts, the compound or standard, 1.0 mL of distilled DMSO and 0.16 mL of ABTS solution were added and incubated for 20 min. Absorbance of these solutions were measured spectrophotometrically at 734 nm (Re et al., 1999). 2.8.2. DPPH radical scavenging method A 10 L aliquot of the different concentrations of extracts, the compound and standards were added to 200 L of DPPH in methanol solution (100 M) in a 96-well microtitre plate (Tarson Products (P) Ltd., Kolkota, India). After incubation at 37 ◦ C for 20 min, the absorbance of each solution was determined at 490 nm using ELISA reader (Bio-Rad Laboratories Inc., CA, USA, Model 550) (Hwang et al., 2001). 2.8.3. Scavenging of hydrogen peroxide A solution of hydrogen peroxide (20 mM) was prepared in phosphate-buffered saline (PBS at pH 7.4). Various concentrations of the extracts, the compound and standard in methanol (1 mL) were added to 2 mL of hydrogen peroxide solution in PBS. After 10 min the absorbance was measured at 230 nm (Jayaprakasha et al., 2004). 2.8.4. Lipid peroxidation inhibitory activity Lipid peroxidation inhibitory activity of three extracts, the compound and standard were carried out according to the method of Duh et al., 2001. Egg lectin (3 mg/mL, phosphate buffer, pH 7.4) was sonicated. The test sample of different concentrations was added to 1 mL of liposome mixture, control was without test sample. Lipid peroxidation was initiated by adding 10 L ferric chloride (400 mM) and 10 L l-ascorbic acid (200 mM). After incubation for 1 h at 37 ◦ C, the reaction was stopped by adding 2 mL of 0.25N HCl containing 15% trichloro acetic acid and 0.375% thiobarbituric acid and the reaction mixture was boiled for 15 min then cooled, centrifuged and the absorbance of the supernatant was measured at 532 nm. 2.8.5. Nitric oxide radical inhibition assay The reaction mixture (6 mL) containing sodium nitroprusside (10 mM, 4 mL), phosphate buffer saline (1 mL) and the extracts, the compound and standard solutions (1 mL) were incubated at 25 ◦ C for 150 min. After incubation, 0.5 mL of the reaction mixture was removed and 1 mL of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) was mixed and allowed to stand for 5 min for completion of diazotization reaction and then 1 mL of NEDD was added, mixed and allowed to stand for another 30 min in diffused light. The absorbance was measured at 540 nm against the corresponding blank solutions in a 96-well microtitre plate (Tarsons Product (P) Ltd., Kolkata, India) using ELISA
reader (Bio-Rad Laboratories Inc., Model 550) (Garrat, 1964; Marcocci et al., 1994). 2.8.6. Scavenging of hydroxyl radical by deoxyribose method Various concentrations of the extracts, the compound and standard in DMSO (0.2 mL) were added to the reaction mixture containing deoxyribose (3 mM, 0.2 mL), ferric chloride (0.1 mM, 0.2 mL), EDTA (0.1 mM, 0.2 mL), ascorbic acid (0.1 mM, 0.2 mL) and hydrogen peroxide (2 mM, 0.2 mL) in phosphate buffer (pH, 7.4, 20 mM) to give a total volume of 1.2 mL. The solutions were then incubated for 30 min at 37 ◦ C. After incubation, ice-cold trichloro acetic acid (0.2 mL, 15%, w/v) and thiobarbituric acid (0.2 mL, 1%, w/v) in 0.25N HCl were added. The reaction mixture was kept in a boiling water bath for 30 min, cooled and the absorbance was measured at 532 nm (Halliwell et al., 1987). 2.8.7. Scavenging of hydroxyl radical by p-NDA method Various concentrations of the extracts, the compound and standard in distilled DMSO (0.5 mL) were added to a solution mixture containing ferric chloride (0.1 mM, 0.5 mL), EDTA (0.1 mM, 0.5 mL), ascorbic acid (0.1 mM, 0.5 mL), hydrogen peroxide (2 mM, 0.5 mL) and p-NDA (0.01 mM, 0.5 mL) in phosphate buffer (pH 7.4, 20 mM), to produce a final volume of 3 mL. Absorbance was measured at 440 nm (Elizabeth and Rao, 1990). 2.8.8. Scavenging of super oxide radical by alkaline DMSO method To the reaction mixture containing 0.1 mL of NBT (1 mg/mL solution in DMSO) and 0.3 mL of the extracts, the compound and standard in DMSO, 1 mL of alkaline DMSO (1 mL DMSO containing, 5 mM NaOH in 0.1 mL water) was added to give a final volume of 1.4 mL and the absorbance was measured at 560 nm (Elizabeth and Rao, 1990). 2.9. In vivo antioxidant activity Animals were divided into six groups comprising of six animals in each group. Group I served as normal and received 1 mL of 0.5% sodium carboxy methyl cellulose (CMC). Group II served as CCl4 treated control and received 1 mL of 0.5% sodium CMC. Groups III received standard silymarin at 100 mg/kg body weight. Group IV, Groups V and VI received the successive methanolic extracts at 100, 200 and 400 mg/kg body weight, respectively. All these treatments were given orally for 7 days. On day 8, except for group I, all the other groups received 1 mL/kg body weight of CCl4 , intraperitoneally. On the day 9, the rats were anesthetized using diethyl ether and blood was collected from abdominal artery and kept at 37 ◦ C in the incubator for 30 min. Later, it was cold centrifuged at 2000 rpm for 15 min to get clear supernatant serum, which was used for biochemical estimations. Liver and kidneys were removed, weighed and homogenized immediately with Elvenjan homogenizer fitted with Teflon plunger, in ice-chilled 10% KCl solution (10 mg/g of tissue). The suspension was centrifuged
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3.3. In vitro antioxidant assay
3. Results
Among the three successive extracts and the isolated compound tested for in vitro antioxidant activity, CDM exhibited potent antioxidant activity in ABTS, DPPH, hydrogen peroxide, inhibition of lipid peroxidation and nitric oxide radical inhibition assays and the IC50 values were found to be 0.92 ± 0.05, 4.86 ± 0.90, 24.89 ± 0.64, 27.5 ± 0.43 and 178.16 ± 2.17 g/mL, respectively (Table 1). The values were found to be comparable to those obtained for the standards used. However, CDM was found to be moderate to low in scavenging hydroxyl radical by p-NDA, deoxy ribose and superoxide radical by alkaline DMSO methods (Table 1). The petroleum ether extract showed poor antioxidant activity in ABTS method with an IC50 value 770 ± 2.30 g/mL and was found to be inactive in all the other methods tested (Table 1). The water extract showed good scavenging activity in ABTS method with IC50 value 6.80 ± 0.72 g/mL. In DPPH, nitric oxide, hydrogen peroxide and inhibition of lipid peroxidation assays, it has shown moderate antioxidant activity with IC50 value 89.72 ± 1.05, 218.23 ± 3.23, 143.05 ± 1.76 and 195 ± 1.06 g/mL, respectively. Water extract was, however, found to be inactive in scavenging hydroxyl radical by p-NDA, deoxy ribose and superoxide radical by alkaline DMSO methods. The isolated compound, bergenin, exhibited good antioxidant activity in hydrogen peroxide, ABTS, DPPH and inhibition of lipid peroxidation assays with IC50 values 32.54 ± 1.78, 75.06 ± 0.97, 165.35 ± 1.60 and 365.12 ± 2.78, respectively. It has shown low antioxidant activity in scavenging of nitric oxide method and deoxy ribose method with IC50 values 785.63 ± 2.03 and 815.63 ± 2.95, respectively. Bergenin, however, was found to be inactive in scavenging hydroxyl radical by p-NDA and superoxide radical by alkaline DMSO methods.
3.1. Isolation of bergenin
3.4. In vivo antioxidant assay
The structure of the colourless crystalline compound (yield 0.75–0.8%) isolated was confirmed as bergenin (Fig. 1) based on its mp 238 ◦ C; UVmax (MeOH): 275 nm; IR bands (KBr): 3425, 2885, 2724, 1702, 1614, 1528, 1464, 1421, 1375, 1341, 1233, 1093 and 1046 cm−1 ; FAB-MS: m/z 329 (M + 1) C14 H16 O9 ; 13 C NMR (125 MHz, DMSO-d6 ); δppm C1 –C7 : 117.918, 115.864, 147.927, 140.556, 150.801, 109.419, 163.208. C1 –C6 : 72.064, 73.641, 79.710, 70.662, 81.665, 61.023, OCH3: 59.706. The homogeneity and purity of the isolated bergenin was confirmed by HPTLC using an ethyl acetate–methanol–glacial acetic acid (8:1.5:0.2, v/v/v) as the mobile phase. Rf value was 0.22 and a spot was detected under UV irradiation at 275 nm.
CDM has shown potent in vitro antioxidant activity compared to other extracts and the isolated compound. Hence, it was selected for the in vivo antioxidant screening. The administration of CDM at 100, 200 and 400 mg/kg bodyweight for 7 days prior to CCl4 treatment caused a significant increase in the levels of catalase and SOD and a significant decrease in the levels of LPO in serum, liver and kidney.
Fig. 1. Structure of bergenin.
at 2000 rpm at 4 ◦ C for 10 min and the clear supernatant was used for biochemical estimations. Catalase was estimated by following the breakdown of hydrogen peroxide according to the method of Beers and Sizer (1952). Superoxide dismutase (SOD) was assayed according to Misra and Fridovich (1972) based on the inhibition of epinephrine autooxidation by the enzyme. Lipid peroxidation was measured in terms of malondialdehyde (MDA) content following the thiobarbituric acid method of Ohkawa et al. (1979). 2.10. Statistical analysis Results are expressed as mean ± S.E.M. Comparisons among the groups were tested by one-way ANOVA using Graph Pad Prism, Version 4.0 (Graph Pad Software, San Diego, CA, USA). When the p-value obtained from ANOVA was significant (p < 0.05), the Tukey test was applied to test for differences among groups.
3.2. Total phenolic compounds estimation The total phenolic compounds of three successive extracts were expressed as gallic acid equivalent in mg/g of extracts. Methanol extract had the highest phenolic content 44.70 mg/g, followed by water extract 14.2 mg/g and petroleum ether extract does not contain any phenolic compounds.
3.4.1. In vivo lipid peroxidation The localization of radical formation resulting in lipid peroxidation, measured as MDA in serum, liver and kidney are shown in Table 2. MDA content in the serum, liver and kidney significantly increased in CCl4 control group compared to the normal group (p < 0.001). The pretreatment of CDM at all the three dose levels significantly inhibited the MDA level by 31.16, 59.39 and 86.91%, respectively, for serum, 39.84, 62.15 and 84.29%, respectively, for liver and 30.47, 46.33 and 74.25%, respectively, for kidney when compared to CCl4 control. At the same time, the percentage of inhibition for silymarin (100 mg/kg) on MDA levels in CCl4 was
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Table 1 In vitro antioxidant activity of Caesalpinia digyna root extracts and bergenin Extract/compound
IC50 values ± S.E.M.a (g/mL) DPPH
Hydrogen peroxide
Lipid peroxidation
Nitric oxide
Deoxy ribose
p-NDA
Super oxide
Petroleum ether Methanol (CDM) Water Bergenin
770.00 ± 2.30 0.92 ± 0.05 6.80 ± 0.72 75.06 ± 0.97
>1000.00 4.86 ± 0.90 89.72 ± 1.05 165.35 ± 1.60
>1000.00 24.89 ± 0.64 143.05 ± 1.76 32.54 ± 1.78
>1000.00 27.50 ± 0.43 195.00 ± 1.06 365.12 ± 2.78
>1000.00 178.16 ± 2.17 218.23 ± 3.23 785.63 ± 2.03
>1000.00 335.76 ± 1.78 >1000.00 815.63 ± 2.95
>1000.00 485.36 ± 1.66 >1000.00 >1000.00
>1000.00 820.10 ± 2.43 >1000.00 >1000.00
Standards Ascorbic acid Rutin BHA ␣-Tocopherol
11.25 ± 0.49 0.51 ± 0.26 – –
4.97 ± 0.67 8.91 ± 0.15 – –
187.33 ± 3.51 36.16 ± 0.25 24.75 ± 1.53 –
– – – 91.66 ± 1.67
– 88.47 ± 2.54 – –
– – 74.66 ± 1.49 –
>1000.00 203.63 ± 3.25 – –
– – – –
(–) Means not done. a Average of three determinations.
Table 2 Effect of CDM and silymarin on antioxidant enzymes and lipid peroxidation in CCl4 -induced rats Treatment
Dose (mg/kg body wt)
Catalase (IU/min/mg of tissue)
SOD (unit/min/mg of tissue)
Serum Liver Kidney Serum 0.5 mL sodium 1.523 ± 0.082 3.796 ± 0.262 1.875 ± 0.125 0.328 ± 0.009 CMC +++ +++ +++ Control (CCl4 ) 1 mL 0.702 ± 0.051 1.561 ± 0.092 0.810 ± 0.045 0.155 ± 0.014+++ Silymarin + CCl4 100 1.439 ± 0.067*** (89.71) 3.558 ± 0.182*** (89.35) 1.672 ± 0.081*** (81.10) 0.301 ± 0.008*** (84.67) CDM + CCl4 100 0.914 ± 0.049 (25.82) 2.369 ± 0.184* (36.15) 1.093 ± 0.065 (27.19) 0.200 ± 0.009* (26.18) CDM + CCl4 200 1.150 ± 0.049*** (54.56) 2.891 ± 0.202*** (59.50) 1.409 ± 0.054*** (56.61) 0.235 ± 0.007*** (46.03) CDM + CCl4 400 1.426 ± 0.088*** (88.19) 3.502 ± 0.163*** (86.85) 1.665 ± 0.127*** (80.45) 0.295 ± 0.014*** (80.55) The data in the parenthesis indicate percentage protection in individual biochemical parameters from their elevated values caused by the CCl4 . The percentage of protection is * p < 0.05, ** p < 0.01, *** p < 0.001, when compared with CCl treated control. +++ p < 0.001, when compared to normal group. 4 Normal
LPO (n mol of MDA/mg of tissue) Liver 0.318 ± 0.008
Kidney 0.301 ± 0.009
Serum 3.672 ± 0.265
0.139 ± 0.012+++ 0.162 ± 0.006+++ 6.467 ± 0.296 ± 0.014*** (87.67) 0.281 ± 0.010*** (85.10) 3.945 ± 0.196 ± 0.013 (31.90) 0.215 ± 0.009** (37.63) 5.596 ± 0.242 ± 0.025*** (57.61) 0.257 ± 0.009*** (68.35) 4.807 ± 0.294 ± 0.007*** (86.17) 0.280 ± 0.006*** (84.82) 4.038 ± calculated as 100 × (values of CCl4 control − values of sample)/(values
Liver 3.940 ± 0.165
Kidney 3.385 ± 0.163
0.611+++ 8.408 ± 0.570+++ 7.326 ± 0.297+++ 0.123*** (90.23) 4.400 ± 0.337*** (89.70) 4.272 ± 0.154*** (77.49) 0.222 (31.16) 6.628 ± 0.186** (39.84) 6.125 ± 0.164** (30.47) 0.116** (59.39) 5.631 ± 0.168*** (62.15) 5.500 ± 0.172*** (46.33) 0.128*** (86.91) 4.642 ± 0.161*** (84.29) 4.402 ± 0.153*** (74.25) of CCl4 control − values of vehicle control). Results are mean ± S.E.M. (n = 6).
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ABTS
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90.23, 89.70 and 77.49%, respectively, for serum, liver and kidney. 3.4.2. In vivo antioxidant enzymes The protective effect of CDM on SOD activity in serum, liver and kidney are shown in Table 2. SOD activity of the serum, liver and kidney in CCl4 control group was found to be significantly lower than in normal group (p < 0.001). The protective effect of CDM at 100, 200 and 400 mg/kg groups is indicated by significant increase in the SOD levels by 26.18, 46.03 and 80.55%, respectively, for serum. In liver and kidney, the protective effect of CDM at 100, 200 and 400 mg/kg showed an increase in SOD levels by 31.90, 57.61, 86.17, 37.63, 68.35 and 84.82%, respectively. The protective effect of silymarin (100 mg/kg) on SOD levels in CCl4 increased by 84.67, 87.67 and 85.10%, respectively, for serum, liver and kidney. Catalase activities in the serum, liver and kidney are shown in Table 2. Catalase activity of CCl4 control group was seen to be strikingly lower than the normal group (p < 0.001). Serum catalase protective activities increased by 25.82, 54.56 and 88.19%, respectively, for all the three dose levels. In addition, liver and kidney catalase protective activities for all the three dose levels increased significantly by 36.15, 59.50, 86.85, 27.19, 56.61 and 80.45%, respectively. The protective effect of silymarin (100 mg/kg) on catalase levels in CCl4 increased by 89.71, 89.35 and 81.10%, respectively, for serum, liver and kidney. 4. Discussion In recent years, attention has been focused on the role of biotransformation of chemicals into highly reactive metabolites that initiate cellular toxicity. Many compounds, including clinically useful drugs, can cause cellular damage through metabolic activation of the chemical to highly reactive species such as free radicals, carbenes and nitrenes. CCl4 has probably been studied more extensively both biochemically and pathologically than any other hepatotoxin. CCl4 hepatotoxicity depends on the reductive dehalogenation of CCl4 catalysed by Cyt 450 in the liver cell endoplasmic reticulum leading to the generation of an unstable complex CCl3 • radical. This trichloromethyl radical has been shown to be a highly reactive species, capable of attacking microsomal lipids leading to its peroxidation. This also covalently binds to microsomal lipids and proteins initiating secondary biochemical processes which is the ultimate cause for the unfolding of the panorama of pathological consequences of CCl4 metabolism (Wei, 1998). Further, oxidative stress, the consequence of an imbalance of prooxidants and antioxidants in the organism, is also gaining recognition as a key phenomenon in chronic illnesses like inflammation and heart diseases, hypertension and some forms of cancer (Oh et al., 2001). ROS produced through mechanism of signaling leads to deleterious effects. Hydrogen peroxide (ROS) has reported as an important mediator of signaling oriented to the activation of transcription factors which are sensible to redox cycle and activators of responsible genes of cancerigenic cells growth and of some inflammatory processes. Oxidative stress results in toxicity when the rate at which the ROS are generated
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exceeds the cell capacity for their removal. Lipid peroxidation is an autocatalytic process, which is a common consequence of cell death. This process may cause peroxidative tissue damage in inflammation, cancer and toxicity of xenobiotics and aging. MDA is one of the end products in the lipid peroxidation process (Kurata et al., 1993). The data obtained in our present study (Table 2) clearly shows an increase in the MDA level in serum, liver and kidney of rats treated with CCl4 suggesting enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with CDM, however, is seen to significantly reverse these changes in a dose dependent manner. CDM at 400 mg/kg significantly (p < 0.001) inhibited the formation of MDA levels in CCl4 treated group and this is seen to be comparable with the standard drug silymarin. Biological systems protect themselves against the damaging effects of activated species by several means. These include free radical scavengers and chain reaction terminators; enzymes such as SOD and CAT system (Proctor and McGinness, 1986). The SOD converts superoxide radicals (O2 − ) into H2 O2 plus O2 , thus participating with other antioxidant enzymes, in the enzymatic defense against oxygen toxicity. The present study (Table 2) reveals that there is an increase of SOD activity in a dose dependent manner suggesting that the CDM has an efficient protective effect in response to ROS. CDM at 400 mg/kg and silymarin at 100 mg/kg significantly (p < 0.001) restores the SOD activity in CCl4 treated groups. CAT is a key component of the antioxidant defense system. Inhibition of this protective mechanism results in enhanced sensitivity to free radical induced cellular damage. The reduction in the activity of CAT may, therefore, result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide (Sampathkumar et al., 2005). In our study (Table 2), administration of CDM increases the CAT level in CCl4 induced liver damage to rats thus preventing the accumulation of excessive free radicals and protects the liver from CCl4 intoxication. CDM at 400 mg/kg and silymarin at 100 mg/kg almost significantly (p < 0.001) restored the enzyme activity to the near normal levels. During hepatic injury, superoxide radicals generate at the site of damage and modulate SOD and CAT, resulting in the loss of activity and accumulation of superoxide radical, which damages liver. Decreased CAT activity is linked up to exhaustion of the enzyme as a result of oxidative stress caused by CCl4 . The reduced levels of parameters of SOD and CAT, in CCl4 treated rats were significantly increased by treatment with plant extracts evidently shows the antioxidant property of the extract against oxygen free radicals (Badami et al., 2005; Rai et al., 2006). The phytochemical studies carried out on CDM reveal the presence of steroid, terpenoid, tannins, flavonoid, coumarin and carbohydrates, etc. An analysis of the data given in Table 1 reveals that the observed in vitro antioxidant activity of three successive extracts of Caesalpinia digyna correlates with its phenolic content. A number of scientific reports indicate certain terpenoids, steroids and phenolic compounds such as tannins, coumarins and flavonoids have protective effects due to its antioxidant properties (Bors and Michel, 2002; DeFeudis et al.,
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2003; Takeoka and Dao, 2003; Chandrasekar et al., 2006). In the present study CDM shows the presence of bergenin as a major compound which has good to moderate antioxidant activity in different in vitro models as seen in Table 1 and it could be the relevant contributor for the synergistic activity of antioxidant metabolites of CDM extract. Caesalpinia digyna is traditionally used for the treatment of diabetes and inflammatory diseases. ROS and free radicals are known to play a causative role in these diseases, so free radical scavenging and antioxidant nature of CDM proved in this study may be, at least in part, can contribute for these activities. The study also provides proof for the ethnomedical use and its relation with the ethnopharmacology of the species. In conclusion, the present study clearly reveals that the CDM has potent in vitro free radicals scavenging effect in different in vitro models, and exhibits a dose dependent antioxidant activity by inhibiting lipid peroxidation and enhancing antioxidant enzymes such as SOD and CAT level, in CCl4 intoxicated rat model. CDM is, therefore, a potential therapeutic, thus making it an excellent candidate for more detailed investigations. Further work is under progress, to identify and isolate the active principle responsible for its antioxidant activity. References Anon., 1992. The Wealth of India, vol. 3. CSIR, New Delhi, India, pp. 6–16. Aviram, M., 2000. Review of human studies on oxidative damage and antioxidant protection related to cardiovascular diseases. Free Radical Research 33, S85–S97. Badami, S., Moorkoth, S., Rai, S.R.M.S., Kannan, E., Bhojraj, S., 2003. Antioxidant activity of Caesalpinia sappan heartwood. Biological and Pharmaceutical Bulletin 26, 1534–1537. Badami, S., Rai, S.R., Suresh, B., 2005. Antioxidant activity of Aporosa lindleyana root. Journal of Ethnopharmacology 101, 180–184. Beers Jr., R.F., Sizer, T.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of Biological Chemistry 195, 133–140. Biswas, H.G., 1944. The constitution of the tannin from Indian teripods. Journal of Indian Chemical Society 21, 32. Boonsri, S., Chantrapromma, S., Fun, H.K., Karalai, C., Kanjana-opas, A., Anjum, S., 2005. 2,3-Dihydro-7-hydroxy-3-[(4-methoxyphenyl)methylene]-4H-1-benzopyran-4-one. Acta Crystallographica Section E 61, 3930–3932. Bors, W., Michel, C., 2002. Chemistry of the antioxidant effects of polyphenols. Annals of the New York Academy of Science 957, 57–69. Chandrasekar, M.J.N., Praveen, B., Nanjan, M.J., Suresh, B., 2006. Chemoprotective effect of Phyllanthus maderaspatensis in modulating cisplatin-induced nephrotoxicity and genotoxicity. Pharmaceutical Biology 2, 100–106. Chantrapromma, S., Boonsri, S., Fun, H.K., Anjum, S., Kanjana-Opas, A., 2006. 2,3-Dihydro-7,8-dihydroxy-3-[(4-methoxyphenyl)methylene]-4H-1benzopyran-4-one. Acta Crystallographica Section E 62, 1254–1256. Chaudhry, G.R., Sharma, V.N., Dhar, M.L., 1954. Chemical examination of the roots of Caesalpinia digyna. Journal of Scientific and Industrial Research. Part B 13, 147–148. DeFeudis, F.V., Papadopoulos, V., Drieu, K., 2003. Ginkgo biloba extracts and cancer: a research area in its infancy. Fundamental and Clinical Pharmacology 17, 405–417. Duh, P.D., Yen, G.C., Yen, W.J., Chang, L.W., 2001. Antioxidant effects of water extracts from barley (Hordeum vulgare L.) prepared under different roasting temperatures. Journal of Agricultural and Food Chemistry 49, 1455–1463. Elizabeth, K., Rao, M.N.A., 1990. Oxygen radical scavenging activity of curcumin. International Journal of Pharmaceutics 58, 237–240.
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