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Antioxidant activity of the ethanolic extract of Striga orobanchioides
Journal of Ethnopharmacology 85 (2003) 227–230
Antioxidant activity of the ethanolic extract of Striga orobanchioides Shrishailappa Badami∗ , Mahesh Kumar Gupta, B. Suresh Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Rocklands, Ootacamund 643 001, Tamilnadu, India Received 18 August 2002; received in revised form 16 December 2002; accepted 17 December 2002
Abstract Plants containing flavonoids have been reported to possess strong antioxidant properties. The ethanolic extract of Striga orobanchioides was screened for in vitro and in vivo antioxidant properties using standard procedures. The ethanol extract exhibited IC50 values of 18.65 ± 1.46 and 11.20 ± 0.52 g/ml, respectively in DPPH and nitric oxide radical inhibition assays. These values were less than those obtained for ascorbic acid and rutin, used as standards. In the in vivo experiments the extract treatment at 100 mg/kg body weight dose caused a significant increase in the level of the catalase in the liver and the kidneys. A significant increase in the level of SOD in the liver was observed. The treatment also caused a significant decrease in the TBA-RS and increase in the ascorbic acid levels. These results suggest strong antioxidant potentials of the ethanolic extract of S. orobanchioides. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Striga orobanchioides; Antioxidant; DPPH; Nitric oxide; Peroxidation; Free radical scavenging
1. Introduction Striga Orobanchioides Benth (Family—Scrophulariaceae), is a parasitic plant, lives on the roots of various plants mainly on Lepidagathis, Euphoria antiguorum and Dysophylla and is distributed up to 6000 ft in the hills, usually on red and gravelly soils (Gamble, 1935). In Ayurvedic medicine, the plant is described as antidiabetic (Chopra et al., 1956). Earlier studies have shown significant anti-implantation and estrogenic activity of ethanolic and distilled water extracts of the whole plant S. orobanchioides (Hiremath et al., 1994). The ethanolic extract has also shown significant antiandrogenic (Hiremath et al., 1997), antibacterial (Hiremath et al., 1996a), antihistaminic and mast cell stabilising activities (Harish et al., 2001). From the ethanolic extract, two known flavonoids, apigenin and luteolin which have also shown antifertility properties have been isolated (Hiremath et al., 1996b; Hiremath et al., 2001). Except for these studies, so far, no other chemical and biological investigations have been carried out on this plant. Lipid peroxidation has gained more importance now a days because of its involvement in pathogenesis of many diseases like atherosclerosis, cancer, diabetes mellitus, my∗ Corresponding
author. E-mail address: [email protected] (S. Badami).
ocardial infarction, and also in ageing. Free radicals or reactive oxygen species (ROS) are produced in vivo from various biochemical reactions and also from the respiratory chain as a result of occasional leakage. These free radicals are the main culprits in lipid peroxidation (Cheeseman and Scater, 1993). Plants containing flavonoids have been reported to possess strong antioxidant properties (Raj and Shalini, 1999). Hence, in the present study, the ethanolic extract of S. orobanchioides was screened for in vitro and in vivo antioxidant properties using standard procedures.
2. Material and methods 2.1. Plant material The whole plant was collected from the fields in and around Gulbarga (Karnataka, India) during September– November 2001 and authenticated in the Herbarium, Department of Botany, Gulbarga University, Gulbarga, where voucher specimens are deposited (Voucher No. GU 232). 2.2. Preparation of the extracts and standards The ethanolic extract of the shade-dried powdered whole plant of S. orobanchioides was obtained as described by (Hiremath et al., 1994). The suspension of this extract was
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00021-7
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S. Badami et al. / Journal of Ethnopharmacology 85 (2003) 227–230
prepared in sodium carboxy methyl cellulose (CMC, 0.3%) using distilled water in the case of in vivo experiments, whereas, for in vitro experiments, a weighed quantity of the extract was dissolved in distilled dimethyl sulphoxide (DMSO) and used. Solutions of ascorbic acid and rutin used as standards for in vitro studies were prepared in distilled DMSO. 2.3. Chemicals 1,1,3,3-tetramethoxypropane and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma Chemicals Co., St. Louis, MO. Sodium nitroprusside, methanol and dimethyl sulphoxide were obtained from Ranbaxy Fine Chemicals Ltd., Mohali, India. Sulphanilic acid was obtained from Himedia Laboratories Ltd., Mumbai, India. Naphthylethylenediamine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), thiobarbituric acid, trichloroacetic acid, sodium CMC were obtained from Loba Chemie, Mumbai, India. Ascorbic acid and rutin were obtained from S.D. Fine Chem., Biosar, India. Hydrogen peroxide solution (30%) was obtained from Qualigen Fine Chemicals, Mumbai, India. 2.4. Test animals Male Wistar strain rats (180–200 g) were obtained from the animal house, J.S.S. College of Pharmacy, Ootacamund, India and were maintained under standard environmental conditions and fed with Amrut rat feed, NAV Maharasthra Chakan Oil Mills Ltd., Pune, India and water ad libitum. 2.5. In vitro assays 2.5.1. DPPH method The antioxidant activity of the plant extract and the standards were assessed on the basis of the radical scavenging effect of the stable DPPH free radical (Bang et al., 2001). A total of 10 l of the ethanolic extract (from 21 mg/ml to 40 g/ml in DMSO solution) or standard was added to 200 l of DPPH in methanol solution (100 M) in a 96-well microtitre plate. After incubation at 37 ◦ C for 30 min, the absorbance of each solution was determined at 490 nm using Elisa microtitre plate reader (Bio Rad Laboratories Inc., CA; model 550). The corresponding blank readings were also taken and the remaining DPPH was calculated. IC50 value is the concentration of sample required to scavenge 50% DPPH free radical.
instead of 1-naphthylamine (5%). Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Marcocci et al., 1994). The reaction mixture (3 ml) containing sodium nitroprussude (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and extract or standard solution (0.5 ml) was incubated at 25 ◦ C for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed with 1 ml of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthylethylenediamine dihydrochloride (1%) was added, mixed and allowed standing for 30 min. A pink coloured chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank solutions in microtitre plate using Elisa reader. The IC50 value is the concentration of sample required to inhibit 50% of nitric oxide radical. 2.6. In vivo antioxidant activity Animals were divided into three groups comprising of six rats in each group. Group 1 served as control and was given the vehicle alone (sodium CMC, 0.3%). The second and third groups received ethanolic extract of S. orobanchioides orally at 50 and 100 mg/kg body weight, respectively. The treatments were given for 7 days and on the 8th day of the experiment, all the animals were sacrificed by decapitation. The liver and kidneys were removed, weighed and homogenised immediately with Elvenjan homoginizer fitted with teflon plunger, in ice chilled 10% KCl solution (10 mg/g of tissue). The suspension was centrifuged at 2000 rpm at 4 ◦ C for 10 min and the clear supernatant was used for the following estimations. Catalase was estimated by following the breakdown of hydrogen peroxide according to the method of Beer and Seizer (1952). Superoxide dismutase (SOD) was assayed according to Misra and Fridevich (1972) based on the inhibition of epinephrine auto-oxidation by the enzyme. Lipid peroxidation was measured in terms of MDA content following the thiobarbituric acid (TBA) method of Buege and Aust (1978). Ascorbic acid was measured by the method of Natelson (1963). Statistical analysis was carried out using the Student’s t-test and the results were judged significant, if P < 0.05.
3. Results 3.1. In vitro assay
2.5.2. Nitric oxide radical inhibition assay Sodium nitroprusside in aqueous solution at physiological pH, spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvoy reaction (Garrat, 1964). In the present investigation, Griess Illosvoy reagent is modified by using naphthylethylenediamine dihydrochloride (0.1% w/v)
The ethanolic extract of S. orobanchioides exhibited strong antioxidant activity in the DPPH and the nitric oxide radical inhibition assay as evidenced by the low IC50 values (Table 1). The IC50 values obtained are 18.65 ± 1.46 and 11.20 ± 0.52 g/ml, respectively in the DPPH and nitric oxide radical inhibition assays. These values were found
S. Badami et al. / Journal of Ethnopharmacology 85 (2003) 227–230 Table 1 Effect of the ethanolic extract of S. orobanchioides on free radical generation in vitro S. No.
1 2 3
Tested material
Ethanolic extract Ascorbic acid Rutin a
IC50 (g/ml) ± S.E.a DPPH method
Nitric oxide radical inhibition assay
18.65 ± 1.46 54.63 ± 3.92 43.60 ± 1.79
11.20 ± 0.52 – 37.31 ± 4.17
Average of 10 determinations.
to be less than those obtained for the reference standards, ascorbic acid and rutin. 3.2. In vivo assays The administration of ethanolic extract of S. orobanchioides to normal rats for 7 days induced a dose dependent increase in the level of catalase in the liver and kidneys. The results are significant at 100 mg/kg body weight dose of the treatment (P < 0.001 and 0.05, respectively for liver and kidneys when compared with control; Tables 2 and 3). The treatment caused a significant increase in the level of SOD in the liver (P < 0.01, when compared with control). However, the level of SOD in the kidneys of the treated rats was not dose related and found to be significantly decreased (P < 0.001 and 0.05). The treatment with ethanol extract also caused a significant and dose related decrease in the level of malondialdehyde (MDA, P < 0.05–0.01, when compared with control) formed in peroxidising system and a significant increase in the level of ascorbic acid (P < 0.05–0.001, when compared with control) in the liver and kidneys.
229
4. Discussion Free radical oxidative stress has been implicated in the pathogenesis of a wide variety of clinical disorders, resulting usually from deficient natural antioxidant defences (Hellwell and Gutteridge, 1989). Potential antioxidant therapy should, therefore, include either natural free radical scavenging enzyme or agents, which are capable of augmenting the activity of these enzymes, which include SOD and catalase (Cheeseman and Scater, 1993). The administration of the ethanol extract of S. orobanchioides at 100 mg/kg body weight significantly increased the level of catalase in liver (P < 0.001) and kidneys (P < 0.05) and the level of SOD (P < 0.01) in the liver. This shows the antioxidant nature of the extract. However, a decrease in the level of SOD was observed in the treated groups in the kidneys. Generally, results for the kidneys have shown fewer changes in antioxidant activity compared to the liver (Jadwiga et al., 2000), which could explain the present observations. The present study also showed the depletion in the lipid peroxidation as observed by the significant decrease in the MDA content of the liver and kidneys in the treated groups. Vitamin C is regarded as the first line natural antioxidant defense in plasma and powerful inhibitor of lipid peroxidation. It also regenerates the major antioxidant tocopherol in lipoproteins and cell membranes. Intracellular mechanisms exist which can regenerate ascorbate from its dehydroascorbate by reduced glutathione (Bhattacharya et al., 1999). The treatment with the ethanolic extract also caused a significant increase in the ascorbic acid level of the treated rats in the liver and kidneys. Frie (1991) has shown the ability of Vitamin C to preserve the level of other antioxidants in human plasma. Depletion of the ascorbic acid level in a biological system has been found to correlate to a loss in antioxidant capacity (Aartensson and Neister, 1991). It can be concluded that
Table 2 Effect of the ethanolic extract of S. orobanchioides on rat liver Treatment
Dose (mg/kg body weight)
Catalase (IU/mg tissue/min)
SOD (IU/mg tissue/min)
TBA-RS (nM/mg tissue)
Ascorbic acid (g/mg tissue)
Control Ethanolic extract
– 50 100
0.644 ± 0.024 0.941 ± 0.098∗ 1.352 ± 0.027∗∗∗
0.086 ± 0.008 0.091 ± 0.002 0.117 ± 0.002∗∗
0.287 ± 0.018 0.264 ± 0.012∗ 0.204 ± 0.009∗∗
4.610 ± 0.38 5.830 ± 0.39 7.440 ± 0.56∗∗∗
Results are mean ± S.E. (n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, when compared with control (Student’s t-test).
Table 3 Effect of ethanolic extract of S. orobanchioides in rat kidney Treatment
Dose (mg/kg body weight)
Catalase (IU/mg tissue/min)
SOD (IU/mg tissue/min)
TBA-RS (nM/mg tissue)
Ascorbic acid (g/mg tissue)
Control Ethanolic extract
– 50 100
1.556 ± 0.069 1.650 ± 0.045 1.921 ± 0.107∗
0.096 ± 0.008 0.056 ± 0.005∗∗∗ 0.082 ± 0.007∗
0.304 ± 0.024 0.275 ± 0.018∗∗ 0.262 ± 0.025∗∗
4.060 ± 0.230 5.190 ± 0.144 7.016 ± 0.181∗∗∗
Results are mean ± S.E. (n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, when compared with control (Student’s t-test).
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the ethanolic extract of S. orobanchioides possess strong antioxidant properties as evidenced by the significant increase in the level of catalase, SOD and ascorbic acid and decrease in the levels of TBA-RS. The in vitro studies confirm the same. The ethanolic extract is found to contain two flavonoids, apigenin and luteolin (Hiremath et al., 1996b). A large number of flavonoids including these is known to possess strong antioxidant properties (Raj and Shalini, 1999). Hence, the antioxidant activity of S. orobanchioides is probably due the presence of these flavonoids in the ethanolic extract. Acknowledgements The authors wish to place on record their heart felt thanks to Jagadguru Sri Sri Shivarathreeshwar Deshikendra Mahaswamigalavaru of Suttur Mutt for providing the facilities. References Aartensson, J., Neister, A., 1991. Glutathione deficiency disease tissue ascorbate levels in newborn rats, ascorbate spares glutathione and protects. Proceedings of the National Academy of Sciences 88, A-656– A-660. Bang, Y.H., Hang, S.K., Jeong, H.L., Young, S.H., Jai, S.R., Kyong, S.L., Jung, J.L., 2001. Antioxidant benzoylated flavan-3-ol glycoside from Celastrus orbiculatus. Journal of Natural Products 64, 82–84. Beer, R.F., Seizer, T.W., 1952. A spectrophotometric method for measuring breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 115, 130–140. Bhattacharya, A., Chatterjee, A., Ghosal, S., Bhattacharya, S.K., 1999. Antioxidant activity of tannoid principles of Emblica officinalis (Amla). Indian Journal of Experimental Biology 37, 676–680. Buege, J.A., Aust, S.D., 1978. The thiobarbituric acid assay. Methods in Enzymology 52, 306–307. Cheeseman, K.H., Scater, T.F., 1993. Free radical in medicine. British Medical Bulletin, vol. 49. Churchill Livingstone, London, pp. 479–724.
Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, p. 235. Frie, B., 1991. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage. American Journal of Clinical Nutrition 54, 113S. Gamble, J.S., 1935. Flora of the Presidency of Madras, vol. II. Adlard and Son, London, p. 967. Garrat, D.C., 1964. The Quantitative Analysis of Drugs, vol. 3. Chapman and Hall, Japan, pp. 456–458. Harish, M.S., Nagur, M., Badami, S., 2001. Antihistaminic and mast cell stabilizing activity of Striga orobanchioides. Journal of Ethnopharmacology 76, 197–200. Hellwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine, 2nd ed. Clarendon Press, Oxford. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., 2001. Antifertility and hormonal properties of flavones of Striga orobanchioides. European Journal of Pharmacology 391, 193–197. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1994. Antifertility activity of Striga orobanchioides. Biological and Pharmaceutical Bulletin 17, 1029–1031. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1997. Antiandrogenic activity of Striga orobanchioides. Journal of Ethnopharmacology 56, 55–60. Hiremath, S.P., Swamy, H.K.S., Badami, S., Meena, S., 1996a. Antibacterial and antifungal activity of Striga orobanchioides. Indian Journal of Pharmaceutical Sciences 58, 174–178. Hiremath, S.P., Badami, S., Swamy, H.K.S., 1996b. Flavones from Striga orobanchioides. Indian Drugs 33, 232. Jadwiga, J.L., Marek, M., Elzbeita, B., 2000. Effect of Sesquiterpene lactones on antioxidant enzymes and some drug metabolizing enzymes in rat liver and kidney. Planta Medica 66, 199–205. Marcocci, P.L., Sckaki, A., Albert, G.M., 1994. Antioxidant action of Ginkgo biloba extracts EGP761. Methods in Enzymology 234, 462– 475. Misra, H.P., Fridevich, I., 1972. The role of superoxide dismutase anion in the autooxidation of epinephrine and sample assay in superoxide dismutase. Journal of Biological Chemistry 241, 3170–3175. Natelson, S., 1963. Routine determinations: ascorbic acid (with dinitrophenyl hydrazine). In: Natelson, S. (Ed.), Microtechnique of Clinical Chemistry. Springfield, Illinois, p. 121. Raj, K.J., Shalini, K., 1999. Flavonoids—a review of biological activities. Indian Drugs 36, 668–676.
Antioxidant activity of the ethanolic extract of Striga orobanchioides Shrishailappa Badami∗ , Mahesh Kumar Gupta, B. Suresh Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Rocklands, Ootacamund 643 001, Tamilnadu, India Received 18 August 2002; received in revised form 16 December 2002; accepted 17 December 2002
Abstract Plants containing flavonoids have been reported to possess strong antioxidant properties. The ethanolic extract of Striga orobanchioides was screened for in vitro and in vivo antioxidant properties using standard procedures. The ethanol extract exhibited IC50 values of 18.65 ± 1.46 and 11.20 ± 0.52 g/ml, respectively in DPPH and nitric oxide radical inhibition assays. These values were less than those obtained for ascorbic acid and rutin, used as standards. In the in vivo experiments the extract treatment at 100 mg/kg body weight dose caused a significant increase in the level of the catalase in the liver and the kidneys. A significant increase in the level of SOD in the liver was observed. The treatment also caused a significant decrease in the TBA-RS and increase in the ascorbic acid levels. These results suggest strong antioxidant potentials of the ethanolic extract of S. orobanchioides. © 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Striga orobanchioides; Antioxidant; DPPH; Nitric oxide; Peroxidation; Free radical scavenging
1. Introduction Striga Orobanchioides Benth (Family—Scrophulariaceae), is a parasitic plant, lives on the roots of various plants mainly on Lepidagathis, Euphoria antiguorum and Dysophylla and is distributed up to 6000 ft in the hills, usually on red and gravelly soils (Gamble, 1935). In Ayurvedic medicine, the plant is described as antidiabetic (Chopra et al., 1956). Earlier studies have shown significant anti-implantation and estrogenic activity of ethanolic and distilled water extracts of the whole plant S. orobanchioides (Hiremath et al., 1994). The ethanolic extract has also shown significant antiandrogenic (Hiremath et al., 1997), antibacterial (Hiremath et al., 1996a), antihistaminic and mast cell stabilising activities (Harish et al., 2001). From the ethanolic extract, two known flavonoids, apigenin and luteolin which have also shown antifertility properties have been isolated (Hiremath et al., 1996b; Hiremath et al., 2001). Except for these studies, so far, no other chemical and biological investigations have been carried out on this plant. Lipid peroxidation has gained more importance now a days because of its involvement in pathogenesis of many diseases like atherosclerosis, cancer, diabetes mellitus, my∗ Corresponding
author. E-mail address: [email protected] (S. Badami).
ocardial infarction, and also in ageing. Free radicals or reactive oxygen species (ROS) are produced in vivo from various biochemical reactions and also from the respiratory chain as a result of occasional leakage. These free radicals are the main culprits in lipid peroxidation (Cheeseman and Scater, 1993). Plants containing flavonoids have been reported to possess strong antioxidant properties (Raj and Shalini, 1999). Hence, in the present study, the ethanolic extract of S. orobanchioides was screened for in vitro and in vivo antioxidant properties using standard procedures.
2. Material and methods 2.1. Plant material The whole plant was collected from the fields in and around Gulbarga (Karnataka, India) during September– November 2001 and authenticated in the Herbarium, Department of Botany, Gulbarga University, Gulbarga, where voucher specimens are deposited (Voucher No. GU 232). 2.2. Preparation of the extracts and standards The ethanolic extract of the shade-dried powdered whole plant of S. orobanchioides was obtained as described by (Hiremath et al., 1994). The suspension of this extract was
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00021-7
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S. Badami et al. / Journal of Ethnopharmacology 85 (2003) 227–230
prepared in sodium carboxy methyl cellulose (CMC, 0.3%) using distilled water in the case of in vivo experiments, whereas, for in vitro experiments, a weighed quantity of the extract was dissolved in distilled dimethyl sulphoxide (DMSO) and used. Solutions of ascorbic acid and rutin used as standards for in vitro studies were prepared in distilled DMSO. 2.3. Chemicals 1,1,3,3-tetramethoxypropane and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma Chemicals Co., St. Louis, MO. Sodium nitroprusside, methanol and dimethyl sulphoxide were obtained from Ranbaxy Fine Chemicals Ltd., Mohali, India. Sulphanilic acid was obtained from Himedia Laboratories Ltd., Mumbai, India. Naphthylethylenediamine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), thiobarbituric acid, trichloroacetic acid, sodium CMC were obtained from Loba Chemie, Mumbai, India. Ascorbic acid and rutin were obtained from S.D. Fine Chem., Biosar, India. Hydrogen peroxide solution (30%) was obtained from Qualigen Fine Chemicals, Mumbai, India. 2.4. Test animals Male Wistar strain rats (180–200 g) were obtained from the animal house, J.S.S. College of Pharmacy, Ootacamund, India and were maintained under standard environmental conditions and fed with Amrut rat feed, NAV Maharasthra Chakan Oil Mills Ltd., Pune, India and water ad libitum. 2.5. In vitro assays 2.5.1. DPPH method The antioxidant activity of the plant extract and the standards were assessed on the basis of the radical scavenging effect of the stable DPPH free radical (Bang et al., 2001). A total of 10 l of the ethanolic extract (from 21 mg/ml to 40 g/ml in DMSO solution) or standard was added to 200 l of DPPH in methanol solution (100 M) in a 96-well microtitre plate. After incubation at 37 ◦ C for 30 min, the absorbance of each solution was determined at 490 nm using Elisa microtitre plate reader (Bio Rad Laboratories Inc., CA; model 550). The corresponding blank readings were also taken and the remaining DPPH was calculated. IC50 value is the concentration of sample required to scavenge 50% DPPH free radical.
instead of 1-naphthylamine (5%). Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Marcocci et al., 1994). The reaction mixture (3 ml) containing sodium nitroprussude (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and extract or standard solution (0.5 ml) was incubated at 25 ◦ C for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed with 1 ml of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthylethylenediamine dihydrochloride (1%) was added, mixed and allowed standing for 30 min. A pink coloured chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank solutions in microtitre plate using Elisa reader. The IC50 value is the concentration of sample required to inhibit 50% of nitric oxide radical. 2.6. In vivo antioxidant activity Animals were divided into three groups comprising of six rats in each group. Group 1 served as control and was given the vehicle alone (sodium CMC, 0.3%). The second and third groups received ethanolic extract of S. orobanchioides orally at 50 and 100 mg/kg body weight, respectively. The treatments were given for 7 days and on the 8th day of the experiment, all the animals were sacrificed by decapitation. The liver and kidneys were removed, weighed and homogenised immediately with Elvenjan homoginizer fitted with teflon plunger, in ice chilled 10% KCl solution (10 mg/g of tissue). The suspension was centrifuged at 2000 rpm at 4 ◦ C for 10 min and the clear supernatant was used for the following estimations. Catalase was estimated by following the breakdown of hydrogen peroxide according to the method of Beer and Seizer (1952). Superoxide dismutase (SOD) was assayed according to Misra and Fridevich (1972) based on the inhibition of epinephrine auto-oxidation by the enzyme. Lipid peroxidation was measured in terms of MDA content following the thiobarbituric acid (TBA) method of Buege and Aust (1978). Ascorbic acid was measured by the method of Natelson (1963). Statistical analysis was carried out using the Student’s t-test and the results were judged significant, if P < 0.05.
3. Results 3.1. In vitro assay
2.5.2. Nitric oxide radical inhibition assay Sodium nitroprusside in aqueous solution at physiological pH, spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvoy reaction (Garrat, 1964). In the present investigation, Griess Illosvoy reagent is modified by using naphthylethylenediamine dihydrochloride (0.1% w/v)
The ethanolic extract of S. orobanchioides exhibited strong antioxidant activity in the DPPH and the nitric oxide radical inhibition assay as evidenced by the low IC50 values (Table 1). The IC50 values obtained are 18.65 ± 1.46 and 11.20 ± 0.52 g/ml, respectively in the DPPH and nitric oxide radical inhibition assays. These values were found
S. Badami et al. / Journal of Ethnopharmacology 85 (2003) 227–230 Table 1 Effect of the ethanolic extract of S. orobanchioides on free radical generation in vitro S. No.
1 2 3
Tested material
Ethanolic extract Ascorbic acid Rutin a
IC50 (g/ml) ± S.E.a DPPH method
Nitric oxide radical inhibition assay
18.65 ± 1.46 54.63 ± 3.92 43.60 ± 1.79
11.20 ± 0.52 – 37.31 ± 4.17
Average of 10 determinations.
to be less than those obtained for the reference standards, ascorbic acid and rutin. 3.2. In vivo assays The administration of ethanolic extract of S. orobanchioides to normal rats for 7 days induced a dose dependent increase in the level of catalase in the liver and kidneys. The results are significant at 100 mg/kg body weight dose of the treatment (P < 0.001 and 0.05, respectively for liver and kidneys when compared with control; Tables 2 and 3). The treatment caused a significant increase in the level of SOD in the liver (P < 0.01, when compared with control). However, the level of SOD in the kidneys of the treated rats was not dose related and found to be significantly decreased (P < 0.001 and 0.05). The treatment with ethanol extract also caused a significant and dose related decrease in the level of malondialdehyde (MDA, P < 0.05–0.01, when compared with control) formed in peroxidising system and a significant increase in the level of ascorbic acid (P < 0.05–0.001, when compared with control) in the liver and kidneys.
229
4. Discussion Free radical oxidative stress has been implicated in the pathogenesis of a wide variety of clinical disorders, resulting usually from deficient natural antioxidant defences (Hellwell and Gutteridge, 1989). Potential antioxidant therapy should, therefore, include either natural free radical scavenging enzyme or agents, which are capable of augmenting the activity of these enzymes, which include SOD and catalase (Cheeseman and Scater, 1993). The administration of the ethanol extract of S. orobanchioides at 100 mg/kg body weight significantly increased the level of catalase in liver (P < 0.001) and kidneys (P < 0.05) and the level of SOD (P < 0.01) in the liver. This shows the antioxidant nature of the extract. However, a decrease in the level of SOD was observed in the treated groups in the kidneys. Generally, results for the kidneys have shown fewer changes in antioxidant activity compared to the liver (Jadwiga et al., 2000), which could explain the present observations. The present study also showed the depletion in the lipid peroxidation as observed by the significant decrease in the MDA content of the liver and kidneys in the treated groups. Vitamin C is regarded as the first line natural antioxidant defense in plasma and powerful inhibitor of lipid peroxidation. It also regenerates the major antioxidant tocopherol in lipoproteins and cell membranes. Intracellular mechanisms exist which can regenerate ascorbate from its dehydroascorbate by reduced glutathione (Bhattacharya et al., 1999). The treatment with the ethanolic extract also caused a significant increase in the ascorbic acid level of the treated rats in the liver and kidneys. Frie (1991) has shown the ability of Vitamin C to preserve the level of other antioxidants in human plasma. Depletion of the ascorbic acid level in a biological system has been found to correlate to a loss in antioxidant capacity (Aartensson and Neister, 1991). It can be concluded that
Table 2 Effect of the ethanolic extract of S. orobanchioides on rat liver Treatment
Dose (mg/kg body weight)
Catalase (IU/mg tissue/min)
SOD (IU/mg tissue/min)
TBA-RS (nM/mg tissue)
Ascorbic acid (g/mg tissue)
Control Ethanolic extract
– 50 100
0.644 ± 0.024 0.941 ± 0.098∗ 1.352 ± 0.027∗∗∗
0.086 ± 0.008 0.091 ± 0.002 0.117 ± 0.002∗∗
0.287 ± 0.018 0.264 ± 0.012∗ 0.204 ± 0.009∗∗
4.610 ± 0.38 5.830 ± 0.39 7.440 ± 0.56∗∗∗
Results are mean ± S.E. (n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, when compared with control (Student’s t-test).
Table 3 Effect of ethanolic extract of S. orobanchioides in rat kidney Treatment
Dose (mg/kg body weight)
Catalase (IU/mg tissue/min)
SOD (IU/mg tissue/min)
TBA-RS (nM/mg tissue)
Ascorbic acid (g/mg tissue)
Control Ethanolic extract
– 50 100
1.556 ± 0.069 1.650 ± 0.045 1.921 ± 0.107∗
0.096 ± 0.008 0.056 ± 0.005∗∗∗ 0.082 ± 0.007∗
0.304 ± 0.024 0.275 ± 0.018∗∗ 0.262 ± 0.025∗∗
4.060 ± 0.230 5.190 ± 0.144 7.016 ± 0.181∗∗∗
Results are mean ± S.E. (n = 6). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, when compared with control (Student’s t-test).
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the ethanolic extract of S. orobanchioides possess strong antioxidant properties as evidenced by the significant increase in the level of catalase, SOD and ascorbic acid and decrease in the levels of TBA-RS. The in vitro studies confirm the same. The ethanolic extract is found to contain two flavonoids, apigenin and luteolin (Hiremath et al., 1996b). A large number of flavonoids including these is known to possess strong antioxidant properties (Raj and Shalini, 1999). Hence, the antioxidant activity of S. orobanchioides is probably due the presence of these flavonoids in the ethanolic extract. Acknowledgements The authors wish to place on record their heart felt thanks to Jagadguru Sri Sri Shivarathreeshwar Deshikendra Mahaswamigalavaru of Suttur Mutt for providing the facilities. References Aartensson, J., Neister, A., 1991. Glutathione deficiency disease tissue ascorbate levels in newborn rats, ascorbate spares glutathione and protects. Proceedings of the National Academy of Sciences 88, A-656– A-660. Bang, Y.H., Hang, S.K., Jeong, H.L., Young, S.H., Jai, S.R., Kyong, S.L., Jung, J.L., 2001. Antioxidant benzoylated flavan-3-ol glycoside from Celastrus orbiculatus. Journal of Natural Products 64, 82–84. Beer, R.F., Seizer, T.W., 1952. A spectrophotometric method for measuring breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 115, 130–140. Bhattacharya, A., Chatterjee, A., Ghosal, S., Bhattacharya, S.K., 1999. Antioxidant activity of tannoid principles of Emblica officinalis (Amla). Indian Journal of Experimental Biology 37, 676–680. Buege, J.A., Aust, S.D., 1978. The thiobarbituric acid assay. Methods in Enzymology 52, 306–307. Cheeseman, K.H., Scater, T.F., 1993. Free radical in medicine. British Medical Bulletin, vol. 49. Churchill Livingstone, London, pp. 479–724.
Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi, p. 235. Frie, B., 1991. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage. American Journal of Clinical Nutrition 54, 113S. Gamble, J.S., 1935. Flora of the Presidency of Madras, vol. II. Adlard and Son, London, p. 967. Garrat, D.C., 1964. The Quantitative Analysis of Drugs, vol. 3. Chapman and Hall, Japan, pp. 456–458. Harish, M.S., Nagur, M., Badami, S., 2001. Antihistaminic and mast cell stabilizing activity of Striga orobanchioides. Journal of Ethnopharmacology 76, 197–200. Hellwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine, 2nd ed. Clarendon Press, Oxford. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., 2001. Antifertility and hormonal properties of flavones of Striga orobanchioides. European Journal of Pharmacology 391, 193–197. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1994. Antifertility activity of Striga orobanchioides. Biological and Pharmaceutical Bulletin 17, 1029–1031. Hiremath, S.P., Badami, S., Swamy, H.K.S., Patil, S.B., Londonkar, R.L., 1997. Antiandrogenic activity of Striga orobanchioides. Journal of Ethnopharmacology 56, 55–60. Hiremath, S.P., Swamy, H.K.S., Badami, S., Meena, S., 1996a. Antibacterial and antifungal activity of Striga orobanchioides. Indian Journal of Pharmaceutical Sciences 58, 174–178. Hiremath, S.P., Badami, S., Swamy, H.K.S., 1996b. Flavones from Striga orobanchioides. Indian Drugs 33, 232. Jadwiga, J.L., Marek, M., Elzbeita, B., 2000. Effect of Sesquiterpene lactones on antioxidant enzymes and some drug metabolizing enzymes in rat liver and kidney. Planta Medica 66, 199–205. Marcocci, P.L., Sckaki, A., Albert, G.M., 1994. Antioxidant action of Ginkgo biloba extracts EGP761. Methods in Enzymology 234, 462– 475. Misra, H.P., Fridevich, I., 1972. The role of superoxide dismutase anion in the autooxidation of epinephrine and sample assay in superoxide dismutase. Journal of Biological Chemistry 241, 3170–3175. Natelson, S., 1963. Routine determinations: ascorbic acid (with dinitrophenyl hydrazine). In: Natelson, S. (Ed.), Microtechnique of Clinical Chemistry. Springfield, Illinois, p. 121. Raj, K.J., Shalini, K., 1999. Flavonoids—a review of biological activities. Indian Drugs 36, 668–676.