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Antimutagenic and antioxidant activity of Ficus benghalensis stem bark and Moringa oleifera root extract
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 4 5 e4 8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijcas
Original Article
Antimutagenic and antioxidant activity of Ficus benghalensis stem bark and Moringa oleifera root extract A. Satish a, R. Punith Kumar a, D. Rakshith b, S. Satish b, Faiyaz Ahmed c,* a
Nutra Bio Innovations, Mysore 570017, Karnataka, India Department of Studies in Microbiology, University of Mysore, Mysore, India c Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Government of India, Thanjavur, India b
article info
abstract
Article history:
Objective: To investigate antimutagenic and antioxidant potency of the aqueous heat
Received 19 February 2013
treated Ficus benghalensis stem bark (FBH) extract and Moringa oleifera root (MRH) extract
Accepted 23 March 2013
against sodium azide in TA100 tester strains of Salmonella typhimurium and their inhibition
Available online 10 June 2013
of microsomal lipid peroxidation (LPO). Methods: Mutagenicity was assayed by the standard Ames test (standard plate incorpora-
Keywords:
tion assay) and antioxidant potency was investigated by employing ex vivo inhibition of
Lipid peroxidation
lipid peroxidation in liver Microsomes.
Microsomes
Results: Both FBH and MRH showed strong antimutagenic effect on S. typhimurium TA100
Antimutagenic
strains against sodium azide (NaN3). IC50 values of aqueous extract of FBH and MRH ex-
Antioxidant
tracts were 70.24 mg/ml and 99.20 mg/ml respectively. FBH extract showed maximum in-
Salmonella typhimurium
hibition of microsomal lipid peroxidation responses than MRH. IC50 values of aqueous extract of FBH and MRH extracts were 80.24 mg/ml and 92 mg/ml respectively. FBH and MRH exhibited a dose dependent antioxidant activity. Conclusion: The aqueous heat-treated FBH and MRH have antimutagenic as well as antioxidant activity. Further studies are in progress to evaluate the effect of both extracts by other antioxidant and antimutagenic assays and to identify the factors responsible for these activities. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Mutagens are not only involved in genotoxicity and carcinogenesis but also in the inception and pathogenesis of
several chronic degenerative diseases including hepatic disorders, cardiovascular disorders, diabetes, arthritis, chronic inflammation and in the process of aging. One of the best way to minimize the detrimental effects of mutagens is by
* Corresponding author. Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Government of India, Pudukkottai Road, Thanjavur, India. Tel.: þ91 7373068427. E-mail address: [email protected] (F. Ahmed). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.03.008
46
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 4 5 e4 8
the use of natural antimutagens. Naturally occurring antimutagenic principles present in plants, human diet and other sources have protective effects against mutagens, these include flavonoids, phenolics, coumarins, carotenoids, antraquinones, tannins, saponins and many more. Natural antimutagens from edible and medicinal plants are of particular importance because they may be useful for human cancer prevention.1 Numerous studies from four decades have been out in order to identify compounds, which might protect humans against DNA-damage and its consequences.2 The rich diversity of Indian medicinal plants have not yet systematically screened for antimutagenic activity. Many plant species are known to elicit antimutagenesis and thus have a full range of prospective applications in human healthcare. Even for populations using herbs traditionally, encouraging the use of species with chemopreventive actions could be helpful as part of life expectancy improvement strategies: costs are significantly low, herbs have usually little or no toxicity during long-term oral administration and are relatively available at large scale. It has been suggested that regularly consuming anticarcinogens and antimutagens in the diet may be the most effective way of preventing human cancer and search for novel antimutagens acting in chemoprevention is a promising field in phytotherapy.3 Ficus benghalensis a genus of the family Moraceae is an important medicinal and widely distributed tree found in different regions of India. The species of this family have significant antidiabetic, antiinflammatory, antitumor activity, anticancer, cytoprotective and antiulcer activity, antinociceptive, antioxidant, hypolipidemic, antihyperglycemic, and antipyretic. The phytochemical constituents of the F. benghalensis are rutin, friedelin, taraxosterol, lupeol, b-amyrin along with psoralen, bergapten and b-sisterol, quercetin-3galactoside.4 Moringa oleifera Lam (Moringaceae) is a highly valued plant, distributed in many countries of the tropics and subtropics. It has an impressive range of medicinal uses with high nutritional value. Different parts of this plant contain a profile of important minerals, and good source of protein, vitamins, b-carotene, amino acids and various phenolics. The Moringa plant provides a rich and rare combination of zeatin, quercetin, b-sitosterol, caffeoylquinic acid and kaempferol. Various parts of this plant such as the leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, possess antitumor, antipyretic, antiepileptic, antiinflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal activities, and are being employed for the treatment of different ailments in the indigenous system of medicine, particularly in South Asia.5 Pharmacological effects of the medicinal plants are related to their free-radical scavenging properties which include inhibition of lipid peroxidation, maintaining integrity and permeability of cell walls and the protection of neurons against oxidative stress. Free radical induced lipid peroxidation has been associated with many neurodegenerative diseases.6 Hence, the present study was planned to explore the antioxidant and antimutagenic activity of F. benghalensis stem bark and M. oleifera root extract.
2.
Materials and method
2.1.
Materials
Salmonella typhimurium strain TA100 was purchased from microbial type culture collection (MTCC), India. Sodium azide was purchased from Sisco Research Lab (India), All other chemicals and reagents used in the study were of analytical grade.
2.2.
Collection of plant materials
F. benghalensis stem bark and M. oleifera root were collected from mature trees in the campus of University of Mysore, India and was identified by Dr. Niranjan, Department of Botany, University of Mysore, India. The bark and root was cut into small pieces, dried at 50 C overnight, powdered and passed through 60 mesh sieve and stored in an air tight container at 4 C till further use.
2.3.
Heat treatment
The bark and root powder was subjected to heat treatment in a vacuum oven at 100 C for 60 min, cooled in a desiccators and used for the preparation of the heat-treated extract F. benghalensis (FBH) and M. oleifera (MRH).
2.4.
Preparation of extracts
Aqueous extracts were prepared by extracting heat-treated bark and root powders with distilled water (1:8 w/v) on a mechanical shaker, for 24 h, at room temperature.
2.5.
Antimutagenicity assay
Toxicity and mutagenicity were assayed by the standard Ames test (standard plate incorporation assay).7 In the antimutagenicity test, the inhibitions of mutagenic activity of the sodium azide by the test samples were determined. The sample extract (25, 50, 100, 150, 200 and 250 mg/plate) were assayed by plating with molten soft agar (2 ml) containing 10 h old culture (0.1 ml) of strain of S. typhimurium TA100. Positive and negative controls were also included in each assay. Sodium azide was used as a diagnostic mutagen (1.5 mg per plate) in positive control and plates without sodium azide and without test samples were considered as negative controls. His þ revertants were counted after incubation of the plates at 37 C for 48 h. Each sample was assayed using duplicate plates and the data presented here are mean of triplicates. þSD of three independent assays. The mutagenicity of sodium azide in the absence of test samples was defined as 100% or 0% inhibition. The calculation of % inhibition was done according to the formula given by.8 % inhibition ¼ [1T/M] 100 where T is number of revertants per plate in presence of mutagen and test sample and M is number of revertants per plate in positive control. The number of spontaneous revertants was subtracted from numerator and denominator. Antimutagenic inhibitory effect (%): Strong e 40%, Moderate e 25e40%, Weak or negative e 25%.9
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 4 5 e4 8
2.6.
Inhibition of microsomal lipid peroxidation (LPO)
Liver excised from adult male Wistar rats was homogenized (20% w/v) in 0.02 M tris buffer pH (7.4). Microsomes were isolated according to calcium aggregation method.10 One hundred microliters (0.5 mg of protein) of liver microsomal suspension was mixed with FeSO4 (1 Mm) and ascorbic acid with or without the isolated compound in a total volume of 1 ml of 0.1 M phosphate buffer (pH 7.4) and incubated at 37 C for 60 min. This was followed by addition of 1 ml each of TCA (10%) and TBA (0.67%) and boiled in a water bath for 15 min. The absorbance of the supernatant was read at 535 nm, and the thiobarbituric acid reactive substances (TBARS) value of the supernatant was calculated using tetraethoxypropane as the standard. The TBARS value was taken as a measure of lipid peroxide generation.11
2.7.
47
Fig. 2 e Inhibition of microsomal lipid peroxidation (LPO) of FBH and MRH.
Statistical analysis
The data was analyzed by ANOVA followed by Tukey’s multiple comparisons test for significant differences, using SPSS 14.0 computer software. The values were considered significant when p 0.01. IC50 values were calculated by Boltzmann’s dose response analysis using Origin 6.1 software.
3.
Results
3.1.
Antimutagenicity assay
The antimutagenic potency of the aqueous heat-treated F. benghalensis stem bark (FBH) extract and M. oleifera root (MRH) extract are presented in (Fig. 1) was studied using TA100 tester strains of S. typhimurium against induced mutagenicity of sodium azide (NaN3). Both extracts exhibited nontoxicity in S. typhimurium strain and shows significant antimutagenic activity against sodium azide (NaN3) in a dose dependent manner. IC50 value of aqueous extract of FBH and MRH extracts were 70.24 mg/ml and 99.20 mg/ml respectively. The antimutagenic activity of FBH was significantly higher ( p 0.01) when compared with that of MRH, and as a result the IC50 value of FBH was lower than that of MRH.
Fig. 1 e Antimutagenicity assay e the inhibitions of mutagenic activity of the sodium azide by the test samples of FBH and MRH.
3.2.
Inhibition of microsomal lipid peroxidation (LPO)
The inhibition of microsomal lipid peroxidation (LPO) of the two extracts FBH and MRH at different concentration (100e500 mg) is given in (Fig. 2). Both extracts showed significantly ( p 0.01) in a dose dependent manner. Maximum inhibition of microsomal lipid peroxidation responses were observed in FBH extract when compared with MRH extract. IC50 value of aqueous extract of FBH and MRH extracts were 80.24 mg/ml and 92 mg/ml respectively.
4.
Discussion
Free radicals can damage DNA and cause mutagenicity and cytotoxicity and thus play a key role in carcinogenesis, it is believed that reactive oxygen species (ROS) can induced mutations and inhibit DNA repair process, that result in the inactivation of certain tumor suppressor genes, leading to cancer.1 The present study evaluated the antioxidant and antimutagenic activity of F. benghalensis stem bark (FBH) and M. oleifera root extract (MRH). In the present investigation, both extract effectively exhibited nontoxicity in S. typhimurium strain against induced mutagenicity of sodium azide (NaN3). A strong antimutagenic effect was observed in FBH when compared with MRH extract. The variations in the antimutagenic activity in both plant extracts might be due to the differences in the bioactive constituents in the extract. Several mechanisms have been proposed for antimutagenic activity due to presence of phytochemical constituents such as tannins, saponin, flavonoids, steroids, terpenoids and glycoside. However, enhanced antimutagenic activity of FBH may be due to its oligomeric nature. Oligomeric hydrolyzable tannins have more antitumor promoting activities than monomeric hydrolyzable tannins. Several tannins have been found to reduce the mutagenic activity of a number of mutagens.12 The acetone extract of Terminalia arjuna showed very promising antimutagenic activity against base pair substitution mutagen sodium azide.11 Vitamin A, C and E have shown to be antimutagenic against doxorubicin induced
48
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 4 5 e4 8
chromosomal aberrations and Methyl Azoxy Methanol (MAM) induced mutagenesis in S. typhimurium strain TA100.13,14 Flavonoids are a class of phytochemicals constituents that posses antimutagenic properties. A number of known flavonoids including glycosides and isoflavones were reported to posses significant antimutagenic activity such as glaberene, quercetin, myricentin kaemferol and hesperiden.15 Phenolics compounds namely curcumin and eugenol respectively were found to inhibit the mutagenicity produced by direct acting mutagens such as N-Methyl-N-nitro-N-nitrosoguanidine using S. typhimurium strains TA100. However, the significant antimutagenic activity was observed in both plant extracts against direct acting mutagens (sodium azide) suggests that these extracts may directly protect DNA damage from mutagen,16 and has been related to their antioxidative property, which is important in protecting cellular oxidative damage including lipid peroxidation. However, the inhibition of mutagenesis is often complex, acting through multiple mechanisms. The antioxidants, with particularly emphasis on naturally derived antioxidants, which may inhibit ROS production and may display protective effects. Maximum inhibition of microsomal lipid peroxidation responses were observed in FBH extract when compared with MRH extract suggesting that extracts may have a mixture of biomolecules with hydroxyl groups that prevent the abstraction of hydrogen atom from the double bond of lipid bilayer thereby avoiding the damage to lipid membrane.17 Several plant extracts have been shown to inhibit lipid peroxidation by acting as chain-breaking peroxyl-radical scavengers, and can protect LDL from oxidation as measured by the levels of TBARS.18 Phenolic compounds have been reported to have a capacity to scavenge free radicals. The antioxidant activity of phenolic is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition, they have a metal chelation potential.19 The results of our study indicate that FBH and MRH extracts are rich source in phytochemical compounds which possess potent antioxidant properties by inhibition of microsomal lipid peroxidation (LPO) activities and protects the S. typhimurium strains TA100 against sodium azide (NaN3) induced mutagenicity.
5.
Conclusion
The results of the present study support antimutagenic activity and inhibition of microsomal lipid peroxidation potential has been related to their antioxidant property, which is important in protecting cellular oxidative damage including lipid peroxidation. However, the inhibition of mutagenesis is often complex, acting through multiple mechanisms. Antimutagen present in the extracts may interact with the specific enzyme systems, which are necessary for activation of mutagens. Further studies are underway to confirm these results by isolating and characterizing bioactive compounds responsible for the antimutagenic and antioxidant activities to achieve lead molecules in the search of novel herbal drugs.
Conflicts of interest All authors have none to declare.
references
1. Sanjib B. Natural antimutagens: a review. Res J Med Plant. 2011;5:116e126. 2. De Flora S, Ramel C. Mechanisms of inhibitors of mutagenesis and carcinogenesis. Classification and overview. Mutat Res. 1988;202:285e306. 3. Gowri S, Chinnaswamy P. Evaluation of in-vitro antimutagenic activity of Caralluma. J Nat Prod Plant Resour. 2011;1:27e34. 4. Baby Joseph, Justin Raj S. Phytopharmacological and phytochemical properties of three. Intl J Phar Bio Sci. 2010;1:246e253. 5. Farooq A, Sajid L, Muhammad A, Anwarul Hassan G. Moringa oleifera: a food plant with multiple medicinal uses. Phytother Res. 2007;21:17e25. 6. Ravindra PS, Shashwat S, Suman K. Free radicals and oxidative stress in neurodegenerative diseases. JIACM. 2004;3:218e225. 7. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983;113:173e215. 8. Ong T, Wong WZ, Stewart JD, Brockman HE. Chlorophyllin: a potent antimutagen against environmental and dietary complex mixture. Mutat Res. 1986;173:111e115. 9. Ikken Y, Morales P, Martinez A, Marin ML, Haza AI, Cambero MI. Antimutagenic effect of fruit and vegetable ethanolic extracts against N-nitrosamines evaluated by the Ames test. J Agric Food Chem. 1999;47:3257e3264. 10. Kamath SA, Rubin E. Interaction of calcium with microsomes: a modified method for the rapid isolation of rat liver microsomes. Biochem Biophys Res Commun. 1972;49:52e59. 11. Satwinderjeet K, Subodh K, Prabhjit K, Madhu C. Study of antimutagenic potential of Phytoconstituents isolated from Terminalia arjuna in the Salmonella/Microsome assay. Am J Biomed Sci. 2010;2:164e177. 12. Chung KT, Wong TY, Wei C, Huang YW, Lin Y. Tannins and human health: a review. Crit Rev Food Sci Nutr. 1998;38:421e464. 13. Tavan E, Maziere S, Narbonne JF, Cassend P. Effects of vitamins A and E on methylazoxymethanol induced mutagenesis in S. typhimunum strains TA100. Mutat Res. 1997;377:231e237. 14. Kuroda Y. Antimutagenic activity of vitamins in cultured mammalian cells. Basic Life Sci. 1990;52:233e256. 15. Miyazawa M, Hisama M. Antimutagenic activity of flavonoids from Chrysanthemum morifolium. Biosci Biotechnol Biochem. 2003;67:2091e2099. 16. Soudamini KK, Unnikrishnan MC, Sukumaran K, Kuttan R. Mutagenicaity and antimutagenicity of some selected spices. Indian J Physiol Pharmacol. 1995;39:347e353. 17. Suaib L, Suchita S, Ritesh K, Anil K, Debabrata C. Experimental assessment of Moringa oleifera leaf and fruit for its antistress, antioxidant, and scavenging potential using in vitro and in vivo assays. Evid Based Complement Alternat Med. 2012;2012:1e12. 18. O’Byme DJ, Devaraj S, Grundy SM, Jialal I. Comparison of antioxidant effects of Concord grape juice flavonoids and atocopherol on markers. Am J Clin Nutr. 2002;76:1367e1374. 19. Pietta PG. Flavonoids as antioxidants. J Nat Prod. 2000;63:1035e1042.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijcas
Original Article
Antimutagenic and antioxidant activity of Ficus benghalensis stem bark and Moringa oleifera root extract A. Satish a, R. Punith Kumar a, D. Rakshith b, S. Satish b, Faiyaz Ahmed c,* a
Nutra Bio Innovations, Mysore 570017, Karnataka, India Department of Studies in Microbiology, University of Mysore, Mysore, India c Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Government of India, Thanjavur, India b
article info
abstract
Article history:
Objective: To investigate antimutagenic and antioxidant potency of the aqueous heat
Received 19 February 2013
treated Ficus benghalensis stem bark (FBH) extract and Moringa oleifera root (MRH) extract
Accepted 23 March 2013
against sodium azide in TA100 tester strains of Salmonella typhimurium and their inhibition
Available online 10 June 2013
of microsomal lipid peroxidation (LPO). Methods: Mutagenicity was assayed by the standard Ames test (standard plate incorpora-
Keywords:
tion assay) and antioxidant potency was investigated by employing ex vivo inhibition of
Lipid peroxidation
lipid peroxidation in liver Microsomes.
Microsomes
Results: Both FBH and MRH showed strong antimutagenic effect on S. typhimurium TA100
Antimutagenic
strains against sodium azide (NaN3). IC50 values of aqueous extract of FBH and MRH ex-
Antioxidant
tracts were 70.24 mg/ml and 99.20 mg/ml respectively. FBH extract showed maximum in-
Salmonella typhimurium
hibition of microsomal lipid peroxidation responses than MRH. IC50 values of aqueous extract of FBH and MRH extracts were 80.24 mg/ml and 92 mg/ml respectively. FBH and MRH exhibited a dose dependent antioxidant activity. Conclusion: The aqueous heat-treated FBH and MRH have antimutagenic as well as antioxidant activity. Further studies are in progress to evaluate the effect of both extracts by other antioxidant and antimutagenic assays and to identify the factors responsible for these activities. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Mutagens are not only involved in genotoxicity and carcinogenesis but also in the inception and pathogenesis of
several chronic degenerative diseases including hepatic disorders, cardiovascular disorders, diabetes, arthritis, chronic inflammation and in the process of aging. One of the best way to minimize the detrimental effects of mutagens is by
* Corresponding author. Indian Institute of Crop Processing Technology, Ministry of Food Processing Industries, Government of India, Pudukkottai Road, Thanjavur, India. Tel.: þ91 7373068427. E-mail address: [email protected] (F. Ahmed). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.03.008
46
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 4 5 e4 8
the use of natural antimutagens. Naturally occurring antimutagenic principles present in plants, human diet and other sources have protective effects against mutagens, these include flavonoids, phenolics, coumarins, carotenoids, antraquinones, tannins, saponins and many more. Natural antimutagens from edible and medicinal plants are of particular importance because they may be useful for human cancer prevention.1 Numerous studies from four decades have been out in order to identify compounds, which might protect humans against DNA-damage and its consequences.2 The rich diversity of Indian medicinal plants have not yet systematically screened for antimutagenic activity. Many plant species are known to elicit antimutagenesis and thus have a full range of prospective applications in human healthcare. Even for populations using herbs traditionally, encouraging the use of species with chemopreventive actions could be helpful as part of life expectancy improvement strategies: costs are significantly low, herbs have usually little or no toxicity during long-term oral administration and are relatively available at large scale. It has been suggested that regularly consuming anticarcinogens and antimutagens in the diet may be the most effective way of preventing human cancer and search for novel antimutagens acting in chemoprevention is a promising field in phytotherapy.3 Ficus benghalensis a genus of the family Moraceae is an important medicinal and widely distributed tree found in different regions of India. The species of this family have significant antidiabetic, antiinflammatory, antitumor activity, anticancer, cytoprotective and antiulcer activity, antinociceptive, antioxidant, hypolipidemic, antihyperglycemic, and antipyretic. The phytochemical constituents of the F. benghalensis are rutin, friedelin, taraxosterol, lupeol, b-amyrin along with psoralen, bergapten and b-sisterol, quercetin-3galactoside.4 Moringa oleifera Lam (Moringaceae) is a highly valued plant, distributed in many countries of the tropics and subtropics. It has an impressive range of medicinal uses with high nutritional value. Different parts of this plant contain a profile of important minerals, and good source of protein, vitamins, b-carotene, amino acids and various phenolics. The Moringa plant provides a rich and rare combination of zeatin, quercetin, b-sitosterol, caffeoylquinic acid and kaempferol. Various parts of this plant such as the leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, possess antitumor, antipyretic, antiepileptic, antiinflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal activities, and are being employed for the treatment of different ailments in the indigenous system of medicine, particularly in South Asia.5 Pharmacological effects of the medicinal plants are related to their free-radical scavenging properties which include inhibition of lipid peroxidation, maintaining integrity and permeability of cell walls and the protection of neurons against oxidative stress. Free radical induced lipid peroxidation has been associated with many neurodegenerative diseases.6 Hence, the present study was planned to explore the antioxidant and antimutagenic activity of F. benghalensis stem bark and M. oleifera root extract.
2.
Materials and method
2.1.
Materials
Salmonella typhimurium strain TA100 was purchased from microbial type culture collection (MTCC), India. Sodium azide was purchased from Sisco Research Lab (India), All other chemicals and reagents used in the study were of analytical grade.
2.2.
Collection of plant materials
F. benghalensis stem bark and M. oleifera root were collected from mature trees in the campus of University of Mysore, India and was identified by Dr. Niranjan, Department of Botany, University of Mysore, India. The bark and root was cut into small pieces, dried at 50 C overnight, powdered and passed through 60 mesh sieve and stored in an air tight container at 4 C till further use.
2.3.
Heat treatment
The bark and root powder was subjected to heat treatment in a vacuum oven at 100 C for 60 min, cooled in a desiccators and used for the preparation of the heat-treated extract F. benghalensis (FBH) and M. oleifera (MRH).
2.4.
Preparation of extracts
Aqueous extracts were prepared by extracting heat-treated bark and root powders with distilled water (1:8 w/v) on a mechanical shaker, for 24 h, at room temperature.
2.5.
Antimutagenicity assay
Toxicity and mutagenicity were assayed by the standard Ames test (standard plate incorporation assay).7 In the antimutagenicity test, the inhibitions of mutagenic activity of the sodium azide by the test samples were determined. The sample extract (25, 50, 100, 150, 200 and 250 mg/plate) were assayed by plating with molten soft agar (2 ml) containing 10 h old culture (0.1 ml) of strain of S. typhimurium TA100. Positive and negative controls were also included in each assay. Sodium azide was used as a diagnostic mutagen (1.5 mg per plate) in positive control and plates without sodium azide and without test samples were considered as negative controls. His þ revertants were counted after incubation of the plates at 37 C for 48 h. Each sample was assayed using duplicate plates and the data presented here are mean of triplicates. þSD of three independent assays. The mutagenicity of sodium azide in the absence of test samples was defined as 100% or 0% inhibition. The calculation of % inhibition was done according to the formula given by.8 % inhibition ¼ [1T/M] 100 where T is number of revertants per plate in presence of mutagen and test sample and M is number of revertants per plate in positive control. The number of spontaneous revertants was subtracted from numerator and denominator. Antimutagenic inhibitory effect (%): Strong e 40%, Moderate e 25e40%, Weak or negative e 25%.9
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 4 5 e4 8
2.6.
Inhibition of microsomal lipid peroxidation (LPO)
Liver excised from adult male Wistar rats was homogenized (20% w/v) in 0.02 M tris buffer pH (7.4). Microsomes were isolated according to calcium aggregation method.10 One hundred microliters (0.5 mg of protein) of liver microsomal suspension was mixed with FeSO4 (1 Mm) and ascorbic acid with or without the isolated compound in a total volume of 1 ml of 0.1 M phosphate buffer (pH 7.4) and incubated at 37 C for 60 min. This was followed by addition of 1 ml each of TCA (10%) and TBA (0.67%) and boiled in a water bath for 15 min. The absorbance of the supernatant was read at 535 nm, and the thiobarbituric acid reactive substances (TBARS) value of the supernatant was calculated using tetraethoxypropane as the standard. The TBARS value was taken as a measure of lipid peroxide generation.11
2.7.
47
Fig. 2 e Inhibition of microsomal lipid peroxidation (LPO) of FBH and MRH.
Statistical analysis
The data was analyzed by ANOVA followed by Tukey’s multiple comparisons test for significant differences, using SPSS 14.0 computer software. The values were considered significant when p 0.01. IC50 values were calculated by Boltzmann’s dose response analysis using Origin 6.1 software.
3.
Results
3.1.
Antimutagenicity assay
The antimutagenic potency of the aqueous heat-treated F. benghalensis stem bark (FBH) extract and M. oleifera root (MRH) extract are presented in (Fig. 1) was studied using TA100 tester strains of S. typhimurium against induced mutagenicity of sodium azide (NaN3). Both extracts exhibited nontoxicity in S. typhimurium strain and shows significant antimutagenic activity against sodium azide (NaN3) in a dose dependent manner. IC50 value of aqueous extract of FBH and MRH extracts were 70.24 mg/ml and 99.20 mg/ml respectively. The antimutagenic activity of FBH was significantly higher ( p 0.01) when compared with that of MRH, and as a result the IC50 value of FBH was lower than that of MRH.
Fig. 1 e Antimutagenicity assay e the inhibitions of mutagenic activity of the sodium azide by the test samples of FBH and MRH.
3.2.
Inhibition of microsomal lipid peroxidation (LPO)
The inhibition of microsomal lipid peroxidation (LPO) of the two extracts FBH and MRH at different concentration (100e500 mg) is given in (Fig. 2). Both extracts showed significantly ( p 0.01) in a dose dependent manner. Maximum inhibition of microsomal lipid peroxidation responses were observed in FBH extract when compared with MRH extract. IC50 value of aqueous extract of FBH and MRH extracts were 80.24 mg/ml and 92 mg/ml respectively.
4.
Discussion
Free radicals can damage DNA and cause mutagenicity and cytotoxicity and thus play a key role in carcinogenesis, it is believed that reactive oxygen species (ROS) can induced mutations and inhibit DNA repair process, that result in the inactivation of certain tumor suppressor genes, leading to cancer.1 The present study evaluated the antioxidant and antimutagenic activity of F. benghalensis stem bark (FBH) and M. oleifera root extract (MRH). In the present investigation, both extract effectively exhibited nontoxicity in S. typhimurium strain against induced mutagenicity of sodium azide (NaN3). A strong antimutagenic effect was observed in FBH when compared with MRH extract. The variations in the antimutagenic activity in both plant extracts might be due to the differences in the bioactive constituents in the extract. Several mechanisms have been proposed for antimutagenic activity due to presence of phytochemical constituents such as tannins, saponin, flavonoids, steroids, terpenoids and glycoside. However, enhanced antimutagenic activity of FBH may be due to its oligomeric nature. Oligomeric hydrolyzable tannins have more antitumor promoting activities than monomeric hydrolyzable tannins. Several tannins have been found to reduce the mutagenic activity of a number of mutagens.12 The acetone extract of Terminalia arjuna showed very promising antimutagenic activity against base pair substitution mutagen sodium azide.11 Vitamin A, C and E have shown to be antimutagenic against doxorubicin induced
48
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chromosomal aberrations and Methyl Azoxy Methanol (MAM) induced mutagenesis in S. typhimurium strain TA100.13,14 Flavonoids are a class of phytochemicals constituents that posses antimutagenic properties. A number of known flavonoids including glycosides and isoflavones were reported to posses significant antimutagenic activity such as glaberene, quercetin, myricentin kaemferol and hesperiden.15 Phenolics compounds namely curcumin and eugenol respectively were found to inhibit the mutagenicity produced by direct acting mutagens such as N-Methyl-N-nitro-N-nitrosoguanidine using S. typhimurium strains TA100. However, the significant antimutagenic activity was observed in both plant extracts against direct acting mutagens (sodium azide) suggests that these extracts may directly protect DNA damage from mutagen,16 and has been related to their antioxidative property, which is important in protecting cellular oxidative damage including lipid peroxidation. However, the inhibition of mutagenesis is often complex, acting through multiple mechanisms. The antioxidants, with particularly emphasis on naturally derived antioxidants, which may inhibit ROS production and may display protective effects. Maximum inhibition of microsomal lipid peroxidation responses were observed in FBH extract when compared with MRH extract suggesting that extracts may have a mixture of biomolecules with hydroxyl groups that prevent the abstraction of hydrogen atom from the double bond of lipid bilayer thereby avoiding the damage to lipid membrane.17 Several plant extracts have been shown to inhibit lipid peroxidation by acting as chain-breaking peroxyl-radical scavengers, and can protect LDL from oxidation as measured by the levels of TBARS.18 Phenolic compounds have been reported to have a capacity to scavenge free radicals. The antioxidant activity of phenolic is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition, they have a metal chelation potential.19 The results of our study indicate that FBH and MRH extracts are rich source in phytochemical compounds which possess potent antioxidant properties by inhibition of microsomal lipid peroxidation (LPO) activities and protects the S. typhimurium strains TA100 against sodium azide (NaN3) induced mutagenicity.
5.
Conclusion
The results of the present study support antimutagenic activity and inhibition of microsomal lipid peroxidation potential has been related to their antioxidant property, which is important in protecting cellular oxidative damage including lipid peroxidation. However, the inhibition of mutagenesis is often complex, acting through multiple mechanisms. Antimutagen present in the extracts may interact with the specific enzyme systems, which are necessary for activation of mutagens. Further studies are underway to confirm these results by isolating and characterizing bioactive compounds responsible for the antimutagenic and antioxidant activities to achieve lead molecules in the search of novel herbal drugs.
Conflicts of interest All authors have none to declare.
references
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