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In vitro antioxidant activity of Retama monosperma (L.) Boiss. a
a
Zoubir Belmokhtar & Meriem Kaid Harche a
Laboratoire des Productions Valorisations Végétales et Microbiennes (LP2VM), Faculty of Natural Sciences and Life, University of Sciences and Technology, Mohamed Boudiaf (USTOMB), Oran 31000, Algeria Published online: 17 Jul 2014.
To cite this article: Zoubir Belmokhtar & Meriem Kaid Harche (2014) In vitro antioxidant activity of Retama monosperma (L.) Boiss., Natural Product Research: Formerly Natural Product Letters, 28:24, 2324-2329, DOI: 10.1080/14786419.2014.934237 To link to this article: http://dx.doi.org/10.1080/14786419.2014.934237
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 24, 2324–2329, http://dx.doi.org/10.1080/14786419.2014.934237
SHORT COMMUNICATION In vitro antioxidant activity of Retama monosperma (L.) Boiss. Zoubir Belmokhtar* and Meriem Kaid Harche Laboratoire des Productions Valorisations Ve´ge´tales et Microbiennes (LP2VM), Faculty of Natural Sciences and Life, University of Sciences and Technology, Mohamed Boudiaf (USTOMB), Oran 31000, Algeria
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(Received 21 April 2014; final version received 10 June 2014) The relationship between the antioxidant activity and the phenolic contents (total polyphenol, flavonoid and condensed tannin) of Retama monosperma (Fabaceae), used commonly in the traditional medicine of Mediterranean regions, was investigated. The antioxidant activities of the various fractions (toluene, chloroform, ethyl acetate and butanol) of the hydromethanolic extract of the seeds, stems and flowers have been evaluated using in vitro 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging activities and Phosphomolybdic acid assays and were compared to ascorbic acid. A significant high Pearson’s correlations between flavonoid content and antioxidant activities (r ¼ 0.91) with Phosphomolybdic acid assays and (r ¼ 2 0.79) with IC50 DPPH radical scavenging activities. However, there was no correlation between condensed tannin and antioxidant activities. The results obtained in the present study indicate that the ethyl acetate fraction of seeds is a potential source of natural antioxidant for R. monosperma. Keywords: Retama monosperma; polyphenol; antioxidant; relationship
1. Introduction Biological systems are continuously exposed to oxidants, either generated endogenously by metabolic reactions or exogenously, such as air pollutants or cigarette smoke. Reactive oxygen z z species, such as the superoxide anion ðO2 2 Þ and the hydroxyl radical ( OH), are highly unstable species with unpaired electrons, capable of initiating oxidation of proteins, lipids and nucleic acid leading to alterations in cell structures and mutagenesis. The damage is cumulative and may be the trigger for diseases such as artheriosclerosis, cancer and even Parkinson’s disease in man (Halliwell & Whiteman 2004; Rahman 2005). Currently, phenolic compounds are the subject of increasing scientific interest owing to their antioxidant capacity (free radical scavenging and metal chelating activities) and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease and other pathologies. Much of the literature refers to a single group of plant phenolics, the flavonoids (Bravo 1998). Retama monosperma commonly called R’tem, belongs to the family Fabaceae, endemic of the Mediterranean regions (Quezel & Santa 1962). It is a toxic plant, used in small doses as a purgative, antihelmintic, vermifuge, disinfectant and an effective abortive (Bellakhdar 1997; Benrahmoune & Dubruille 2003; Ouarghidi et al. 2013). Phytochemical studies have shown that R. monosperma is very rich of alkaloids (Touati et al. 1996; Fdil et al. 2012). However, studies on the biological activities show a potential anticancer
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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activity against cervical cancer cell lines in vitro (Merghoub et al. 2011; Benbacer et al. 2012), anti-inflammatory effects in vivo (Gonza´lez-Mauraza et al. 2013) and a significant antileukaemic activity (Belayachi et al. 2014). It is necessary to elucidate the pharmacological activities of this species. Nevertheless, their antioxidant capacity is still not known. The objective of the present study was to investigate the antioxidant activity in vitro of the crud extract of seeds, flowers and stems and their fractions of R. monosperma. Total phenolic, flavonoid and condensed tannin content of the fractions was also determined in order to evaluate a coefficient of correlation between the antioxidant activity and the three types of compounds. 2. Results and discussion 2.1. Extraction and solvent fractionation The yields of crude extracts of seeds, stems, flowers and theirs fractions (toluene, chloroform, ethyl acetate, butanol) are presented in Table 1. The maximum yield was obtained for the crude extracts of flowers (34.8%). The n-hexane fractions of three samples and the chloroform fraction of seed are negligible and undetectable. 2.2. Screening of phenolic compound and antioxidants activity by TLC Plant extracts were shown to fluorescence under the UV at a wavelength of 254 and 365 nm showing the presence of secondary metabolites. Compounds are indicated with different colour fluorescent bands. The chromatograms revealed a complex mixture of compounds which exhibited different coloured reactions with the vanillin –H2SO4 spray reagent. Antioxidant compounds appear as yellow spots against a purple background. The numbers of active compounds identified on a plate depend on the mobile phase used in the development of the plate. Antioxidant bioautography by TLC-2,2-diphenyl-1-picrylhydrazyl radical (DPPH) analyses shows that the fraction of ethyl acetate presents three spots clearly separated and strongly coloured yellow with Rf values: 0.12, 0.21 and 0.46 on the mobile phase: ACOET– MeOH – H2O (100:13.5:10) (Supplementary Figure S1). The butanol fraction of flowers demonstrated no antioxidant activity by TLC-DPPH analyses. 2.3. Estimation of total polyphenol, flavonoids and condensed tannins content Total phenolic content and total flavonoids content are determined respectively by the method of Singleton and Rossi (1965) and Dewanto et al. (2002). The highest phenolic and flavonoid contents were recorded in the ethyl acetate fraction of seeds (524.81 ^ 1.23 mg GAE/g and 300.2 ^ 0.37 mg CE/g respectively), while the lowest phenolic and flavonoid contents were in the n-butanol fraction of flowers (90.81 ^ 0.16 mg GAE/g and 17.1 ^ 0.03 mg CE/g respectively). R. monosperma has a high content of total phenols and flavonoids, if compared to Retama raetam growing in Tunisia (Edziri et al. 2008).
Table 1. The percentage yield of the crude extracts and various fractions.
Stems Flowers Seeds
Methanol 70%
n-hexane
Toluene
Chloroform
Ethyl acetate
n-butanol
23.26 34.8 18.13
– – –
– 0.075 0.05
0.6 1.1 –
0.47 1.5 1.2
4.55 11.3 2.06
– 148.05 ^ 0.20 227.13 ^ 0.42 137.72 ^ 0.33 212.05 ^ 0.19
– 60.6 ^ 0.19 93.38 ^ 0.17 39.56 ^ 0.17 19.41 ^ 0.06
Flav (mg CE/g) – 9.60 ^ 0.33 2.5 ^ 0.21 5.97 ^ 0.45 8.01 ^ 0.35
Cond.tan (mg CE/g)
Flav (mg CE/g) 46.76 ^ 0.27 41.5 ^ 0.12 31.33 ^ 0.02 17.1 ^ 0.03 17.42 ^ 0.02
Poly (mg GA/g) 146.37 ^ 0.17 182.16 ^ 0.06 229.19 ^ 0.47 90.81 ^ 0.16 200 ^ 0.4
Flowers
2.5 ^ 0.27 5.58 ^ 0.23 4.67 ^ 0.48 2.01 ^ 0.11 6.01 ^ 0.5
Cond.tan (mg CE/g)
Notes: mg GAE/g, milligram gallic acid equivalent per gram dry extract. mg CE/g, catechin equivalent per gram dry extract.
Toluene Chloroform Ethyle acetate n-butanol Methanol 70%
Poly (mg GAE/g)
Stems
263.78 ^ 0.12 – 524.81 ^ 1.23 232.01 ^ 0.28 239.85 ^ 0.04
Poly (mg GAE/g)
Table 2. Total polyphenol, flavonoid and condensed tannin contents in stems, flowers and seeds of R. monosperma extracts.
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130.85 ^ 0.43 – 300.2 ^ 0.37 97.39 ^ 0.54 47.23 ^ 0.07
Flav (mg CE/g)
Seeds
3.6 ^ 0.23 – 7.56 ^ 0.61 4.01 ^ 0.34 3.02 ^ 0.27
Cond.tan (mg CE/g)
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Table 3. Antioxidative activity of stems, flowers and seeds of cruds extracts and theirs fractions of R. monosperma expressed as IC50 values for DPPH expressed as (mg/mL) and (mg/g) of AAE for Phosphomolybdic essay. Stems
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Toluene Chloroform Ethyl acetate n-butanol Methanol 70%
Flowers
DPPH IC50 (mg/mL)
TAC (mg/g) DPPH IC50 of AAE (mg/mL)
– 3.15 ^ 0.2 1.51 ^ 0.11 2.88 ^ 0.15 3.87 ^ 0.22
– 86.29 ^ 0.52 68.22 ^ 0.48 58.01 ^ 0.29 43.14 ^ 0.31
TAC (mg/g) of AAE
Seeds DPPH IC50 (mg/mL)
TAC (mg/g) of AAE
3.13 ^ 0.21 63.26 ^ 0.38 0.31 ^ 0.13 129.8 ^ 0.39 2.56 ^ 0.19 107.87 ^ 0.72 – – 3.32 ^ 0.17 39.56 ^ 0.20 0.15 ^ 0.11 197.95 ^ 0.98 24.1 ^ 0.71 26.24 ^ 0.22 1.17 ^ 0.15 111.07 ^ 0.45 4.08 ^ 0.25 34.11 ^ 0.15 1.66 ^ 0.13 57.23 ^ 0.31
–: not determined.
The condensed tannin contents were determined by a modification of the vanillin – HCl method of Burns (1971). They are present in very lower abundance; its contents are ranged from 2.01 ^ 0.11 mg CE/g in n-butanol fraction of flowers to 9.60 ^ 0.33 in chloroform extract of stems (Table 2).
2.4. Study of antioxidant activity The total antioxidant capacity (TAA) of the extract fractions was measured by the method described by Prieto et al. (1999). The phosphomolybdenum assay showed that the ethyl acetate fraction of seeds has higher TAA, and it was 197.95 mg ascorbic acid equivalent (AAE)/g dry extract, and in contrast the lowest one was 26 mg (AAE)/g dry extract of the butanol extract of flowers (Table 3). The antioxidant activity was determined by the radical scavenging activity method using DPPH by the method described by Brand-Williams et al. (1995). The results showed a significant variations in the capacity of the test samples to scavenge the DPPH radical with IC50 ranging from 0.15 in ethyl extract of seeds to 4.08 mg/mL in methanol extract of flowers (Table 3). However, at 7 –10 mg/mL, butanol fraction of flowers showed a plateau of scavenging ability of 18 –20%. (Supplementary Figure S2).
2.5. Correlation between phenolic compounds and antioxidant capacity The coefficient of correlation between phenolic compounds and DPPH or phosphomolibdic scavenging activity was studied using linear regression analysis (Supplementary Figures S3 and S4). Results show a high positive correlation coefficient between the TAA with total phenolic content and flavonoid compounds (r ¼ 0.78 and r ¼ 0.91, respectively). However, negative correlation appeared between IC50 for DPPH scavenging with total phenolic (r ¼ 2 0.66) and flavonoid (r ¼ 2 0.79) contents. The quantitative analysis by HPLC-MS-MS showed that the major flavonoid of R. monosperma of Spain was the isoflavone genistein (Gonza´lez-Mauraza et al. 2013). Numerous studies have shown that genistein is a strong antioxidant and increases the activity of other antioxidant enzymes such as catalase, glutathione peroxidase, superoxide dismutase and glutathione reductase (Qiuyin & Huachen 1996; Trieu et al 1999). The correlation coefficient between TAA and the condensed tannin contents was found to be very small 0.26; however, a positive correlation coefficient between condensed tannin contents determined by vanillin – HCl assay and IC50 for DPPH scavenging 0.3 suggests there was no correlation.
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These surveys propose that flavonoid compounds are major contributors to antioxidant activity. 3. Conclusion Flavonoids are the contributor of the antioxidant power in ethyl acetate extracts of seeds of R. monosperma. However, further studies will be needed to identify the exact molecular structures of the antioxidant compounds. Supplementary material
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Experimental details relating to this article are available online alongside Figures S1– S4. References Belayachi L, Aceves-Luquero C, Merghoub N, Bakri Y, Ferna´ndez de Mattos S, Amzazi S, Villalonga P. 2014. Retama monosperma n-hexane extract induces cell cycle arrest and extrinsic pathway-dependent apoptosis in Jurkat cells. BMC Complement Altern Med. 14:38. Bellakhdar J. 1997. La pharmacope´e marocaine traditionnelle. Me´decine arabe ancienne et savoirs populaires [Traditional Moroccan pharmacopoeia. Ancient Arabic medicine and popular knowledge]. Paris: IBIS Press. 764 p. Benbacer L, Merghoub N, El Btaouri H, Gmouh S, Attaleb M, Morjani H, Amzazi S, El Mzibri M. 2012. Antiproliferative effect and induction of apoptosis by Inula viscosa L. and Retama monosperma L. extracts in human cervical cancer cells. In: Rajamanickam, editor. Topics on cervical cancer with an advocacy for prevention. InTech. ISBN: 978-953-51-0183-3; doi:10.5772/30025. Benrahmoune Z, Dubruille C. 2003. Invitation a` l’amour des plantes. Guide floristique illustre´ de la Reserve Biologique de Sidi Boughaba [Invitation to love plants. Floristic Illustrated Guide Biological Reserve Sidi Boughaba]. Edition Acriptura, 319 p. Brand-Williams W, Cuvelier M, Berset C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Technol. 28:25–30. Bravo L. 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev. 56:317–333. Burns RE. 1971. Method for estimation of tannin in grain sorghum. Agron J. 63:511–512. Dewanto V, Wu X, Adom KK, Liu RH. 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem. 50:3010–3014. Edziri H, Mastouri M, Ammar S, Matieu M, Patrich G, Hiar R, Mahjoub MA, Ali SM, Laurent G, Zine M, Aouni M. 2008. Antimicrobial, antioxidant, and antiviral activities of Retama raetam (Forssk.) Webb flowers growing in Tunisia. World J Microbiol Biotechnol. 24:2933–2940. Fdil R, Nawal, Hamdani N, El Kihel A, Sraidi K. 2012. Distribution of alkaloids in the aerial parts of Retama monosperma (L.) Boiss. in Morocco. Ann Toxicol Anal. 24(3):139–143. Gonza´lez-Mauraza H, Martı´n-Cordero C, Alarco´n-de-la-Lastra C, Rosillo MA, Leo´n-Gonza´lez AJ, Sa´nchez-Hidalgo M. 2013. Anti-inflammatory effects of Retama monosperma in acute ulcerative colitis in rats. J Physiol Biochem. 70:163–172. Halliwell B, Whiteman M. 2004. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol. 142:231–255. Merghoub N, Benbacer L, El Btaouri H, Ait Benhassou H, Terryn C, Attaleb M, Madoulet C, Benjouad A, El Mzibri M, Morjani H, Amzazi S. 2011. In vitro antiproliferative effect and induction of apoptosis by Retama monosperma L. extract in human cervical cancer cells. Cell Mol Biol. 57(Suppl):OL1581– OL1591. Ouarghidi A, MartinG J, Powell B, Esser G, Abbad A. 2013. Botanical identification of medicinal roots collected and traded in Morocco and comparison to the existing literature. J Ethnobiol Ethnomed. 9:1–13. Prieto P, Pineda M, Aguilar M. 1999. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 269:337–341. Qiuyin C, Huachen W. 1996. Effect of dietary genistein on antioxidant enzyme activities in SENCAR mice. Nutr Cancer. 25(1):1–7. Quezel, Santa. 1962. In: Tome I, editor. Nouvelle flore d’Alge´rie et des re´gions de´sertiques me´ridionales [New flora of Algeria and southern desert regions]. CNRS. 475 p.
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Rahman I. 2005. The role of oxidative stress in the pathogenesis of COPD: implications for therapy. Treat Respir Med. 4:175–200. Singleton VL, Rossi JA. 1965. Colorimetry of total phenolics with phosphomolybdic- phosphotungstic acid reagents. Am J Enol Vitic. 16:144–158. Touati D, Allain P, Pellecuer J, Fkih-tetouani S, Agoumi A. 1996. Alkaloids from Retama monosperma ssp. Eumonosperma. Fitoterapia. 67(1):49– 52. Trieu VN, Dong Y, Zheng Y, Uckun FM. 1999. In vivo antioxidant activity of genistein in a murine model of singlet oxygen-induced cerebral stroke. Radiat Res. 152(5):508–516.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
In vitro antioxidant activity of Retama monosperma (L.) Boiss. a
a
Zoubir Belmokhtar & Meriem Kaid Harche a
Laboratoire des Productions Valorisations Végétales et Microbiennes (LP2VM), Faculty of Natural Sciences and Life, University of Sciences and Technology, Mohamed Boudiaf (USTOMB), Oran 31000, Algeria Published online: 17 Jul 2014.
To cite this article: Zoubir Belmokhtar & Meriem Kaid Harche (2014) In vitro antioxidant activity of Retama monosperma (L.) Boiss., Natural Product Research: Formerly Natural Product Letters, 28:24, 2324-2329, DOI: 10.1080/14786419.2014.934237 To link to this article: http://dx.doi.org/10.1080/14786419.2014.934237
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 24, 2324–2329, http://dx.doi.org/10.1080/14786419.2014.934237
SHORT COMMUNICATION In vitro antioxidant activity of Retama monosperma (L.) Boiss. Zoubir Belmokhtar* and Meriem Kaid Harche Laboratoire des Productions Valorisations Ve´ge´tales et Microbiennes (LP2VM), Faculty of Natural Sciences and Life, University of Sciences and Technology, Mohamed Boudiaf (USTOMB), Oran 31000, Algeria
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(Received 21 April 2014; final version received 10 June 2014) The relationship between the antioxidant activity and the phenolic contents (total polyphenol, flavonoid and condensed tannin) of Retama monosperma (Fabaceae), used commonly in the traditional medicine of Mediterranean regions, was investigated. The antioxidant activities of the various fractions (toluene, chloroform, ethyl acetate and butanol) of the hydromethanolic extract of the seeds, stems and flowers have been evaluated using in vitro 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging activities and Phosphomolybdic acid assays and were compared to ascorbic acid. A significant high Pearson’s correlations between flavonoid content and antioxidant activities (r ¼ 0.91) with Phosphomolybdic acid assays and (r ¼ 2 0.79) with IC50 DPPH radical scavenging activities. However, there was no correlation between condensed tannin and antioxidant activities. The results obtained in the present study indicate that the ethyl acetate fraction of seeds is a potential source of natural antioxidant for R. monosperma. Keywords: Retama monosperma; polyphenol; antioxidant; relationship
1. Introduction Biological systems are continuously exposed to oxidants, either generated endogenously by metabolic reactions or exogenously, such as air pollutants or cigarette smoke. Reactive oxygen z z species, such as the superoxide anion ðO2 2 Þ and the hydroxyl radical ( OH), are highly unstable species with unpaired electrons, capable of initiating oxidation of proteins, lipids and nucleic acid leading to alterations in cell structures and mutagenesis. The damage is cumulative and may be the trigger for diseases such as artheriosclerosis, cancer and even Parkinson’s disease in man (Halliwell & Whiteman 2004; Rahman 2005). Currently, phenolic compounds are the subject of increasing scientific interest owing to their antioxidant capacity (free radical scavenging and metal chelating activities) and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease and other pathologies. Much of the literature refers to a single group of plant phenolics, the flavonoids (Bravo 1998). Retama monosperma commonly called R’tem, belongs to the family Fabaceae, endemic of the Mediterranean regions (Quezel & Santa 1962). It is a toxic plant, used in small doses as a purgative, antihelmintic, vermifuge, disinfectant and an effective abortive (Bellakhdar 1997; Benrahmoune & Dubruille 2003; Ouarghidi et al. 2013). Phytochemical studies have shown that R. monosperma is very rich of alkaloids (Touati et al. 1996; Fdil et al. 2012). However, studies on the biological activities show a potential anticancer
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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activity against cervical cancer cell lines in vitro (Merghoub et al. 2011; Benbacer et al. 2012), anti-inflammatory effects in vivo (Gonza´lez-Mauraza et al. 2013) and a significant antileukaemic activity (Belayachi et al. 2014). It is necessary to elucidate the pharmacological activities of this species. Nevertheless, their antioxidant capacity is still not known. The objective of the present study was to investigate the antioxidant activity in vitro of the crud extract of seeds, flowers and stems and their fractions of R. monosperma. Total phenolic, flavonoid and condensed tannin content of the fractions was also determined in order to evaluate a coefficient of correlation between the antioxidant activity and the three types of compounds. 2. Results and discussion 2.1. Extraction and solvent fractionation The yields of crude extracts of seeds, stems, flowers and theirs fractions (toluene, chloroform, ethyl acetate, butanol) are presented in Table 1. The maximum yield was obtained for the crude extracts of flowers (34.8%). The n-hexane fractions of three samples and the chloroform fraction of seed are negligible and undetectable. 2.2. Screening of phenolic compound and antioxidants activity by TLC Plant extracts were shown to fluorescence under the UV at a wavelength of 254 and 365 nm showing the presence of secondary metabolites. Compounds are indicated with different colour fluorescent bands. The chromatograms revealed a complex mixture of compounds which exhibited different coloured reactions with the vanillin –H2SO4 spray reagent. Antioxidant compounds appear as yellow spots against a purple background. The numbers of active compounds identified on a plate depend on the mobile phase used in the development of the plate. Antioxidant bioautography by TLC-2,2-diphenyl-1-picrylhydrazyl radical (DPPH) analyses shows that the fraction of ethyl acetate presents three spots clearly separated and strongly coloured yellow with Rf values: 0.12, 0.21 and 0.46 on the mobile phase: ACOET– MeOH – H2O (100:13.5:10) (Supplementary Figure S1). The butanol fraction of flowers demonstrated no antioxidant activity by TLC-DPPH analyses. 2.3. Estimation of total polyphenol, flavonoids and condensed tannins content Total phenolic content and total flavonoids content are determined respectively by the method of Singleton and Rossi (1965) and Dewanto et al. (2002). The highest phenolic and flavonoid contents were recorded in the ethyl acetate fraction of seeds (524.81 ^ 1.23 mg GAE/g and 300.2 ^ 0.37 mg CE/g respectively), while the lowest phenolic and flavonoid contents were in the n-butanol fraction of flowers (90.81 ^ 0.16 mg GAE/g and 17.1 ^ 0.03 mg CE/g respectively). R. monosperma has a high content of total phenols and flavonoids, if compared to Retama raetam growing in Tunisia (Edziri et al. 2008).
Table 1. The percentage yield of the crude extracts and various fractions.
Stems Flowers Seeds
Methanol 70%
n-hexane
Toluene
Chloroform
Ethyl acetate
n-butanol
23.26 34.8 18.13
– – –
– 0.075 0.05
0.6 1.1 –
0.47 1.5 1.2
4.55 11.3 2.06
– 148.05 ^ 0.20 227.13 ^ 0.42 137.72 ^ 0.33 212.05 ^ 0.19
– 60.6 ^ 0.19 93.38 ^ 0.17 39.56 ^ 0.17 19.41 ^ 0.06
Flav (mg CE/g) – 9.60 ^ 0.33 2.5 ^ 0.21 5.97 ^ 0.45 8.01 ^ 0.35
Cond.tan (mg CE/g)
Flav (mg CE/g) 46.76 ^ 0.27 41.5 ^ 0.12 31.33 ^ 0.02 17.1 ^ 0.03 17.42 ^ 0.02
Poly (mg GA/g) 146.37 ^ 0.17 182.16 ^ 0.06 229.19 ^ 0.47 90.81 ^ 0.16 200 ^ 0.4
Flowers
2.5 ^ 0.27 5.58 ^ 0.23 4.67 ^ 0.48 2.01 ^ 0.11 6.01 ^ 0.5
Cond.tan (mg CE/g)
Notes: mg GAE/g, milligram gallic acid equivalent per gram dry extract. mg CE/g, catechin equivalent per gram dry extract.
Toluene Chloroform Ethyle acetate n-butanol Methanol 70%
Poly (mg GAE/g)
Stems
263.78 ^ 0.12 – 524.81 ^ 1.23 232.01 ^ 0.28 239.85 ^ 0.04
Poly (mg GAE/g)
Table 2. Total polyphenol, flavonoid and condensed tannin contents in stems, flowers and seeds of R. monosperma extracts.
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130.85 ^ 0.43 – 300.2 ^ 0.37 97.39 ^ 0.54 47.23 ^ 0.07
Flav (mg CE/g)
Seeds
3.6 ^ 0.23 – 7.56 ^ 0.61 4.01 ^ 0.34 3.02 ^ 0.27
Cond.tan (mg CE/g)
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Table 3. Antioxidative activity of stems, flowers and seeds of cruds extracts and theirs fractions of R. monosperma expressed as IC50 values for DPPH expressed as (mg/mL) and (mg/g) of AAE for Phosphomolybdic essay. Stems
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Toluene Chloroform Ethyl acetate n-butanol Methanol 70%
Flowers
DPPH IC50 (mg/mL)
TAC (mg/g) DPPH IC50 of AAE (mg/mL)
– 3.15 ^ 0.2 1.51 ^ 0.11 2.88 ^ 0.15 3.87 ^ 0.22
– 86.29 ^ 0.52 68.22 ^ 0.48 58.01 ^ 0.29 43.14 ^ 0.31
TAC (mg/g) of AAE
Seeds DPPH IC50 (mg/mL)
TAC (mg/g) of AAE
3.13 ^ 0.21 63.26 ^ 0.38 0.31 ^ 0.13 129.8 ^ 0.39 2.56 ^ 0.19 107.87 ^ 0.72 – – 3.32 ^ 0.17 39.56 ^ 0.20 0.15 ^ 0.11 197.95 ^ 0.98 24.1 ^ 0.71 26.24 ^ 0.22 1.17 ^ 0.15 111.07 ^ 0.45 4.08 ^ 0.25 34.11 ^ 0.15 1.66 ^ 0.13 57.23 ^ 0.31
–: not determined.
The condensed tannin contents were determined by a modification of the vanillin – HCl method of Burns (1971). They are present in very lower abundance; its contents are ranged from 2.01 ^ 0.11 mg CE/g in n-butanol fraction of flowers to 9.60 ^ 0.33 in chloroform extract of stems (Table 2).
2.4. Study of antioxidant activity The total antioxidant capacity (TAA) of the extract fractions was measured by the method described by Prieto et al. (1999). The phosphomolybdenum assay showed that the ethyl acetate fraction of seeds has higher TAA, and it was 197.95 mg ascorbic acid equivalent (AAE)/g dry extract, and in contrast the lowest one was 26 mg (AAE)/g dry extract of the butanol extract of flowers (Table 3). The antioxidant activity was determined by the radical scavenging activity method using DPPH by the method described by Brand-Williams et al. (1995). The results showed a significant variations in the capacity of the test samples to scavenge the DPPH radical with IC50 ranging from 0.15 in ethyl extract of seeds to 4.08 mg/mL in methanol extract of flowers (Table 3). However, at 7 –10 mg/mL, butanol fraction of flowers showed a plateau of scavenging ability of 18 –20%. (Supplementary Figure S2).
2.5. Correlation between phenolic compounds and antioxidant capacity The coefficient of correlation between phenolic compounds and DPPH or phosphomolibdic scavenging activity was studied using linear regression analysis (Supplementary Figures S3 and S4). Results show a high positive correlation coefficient between the TAA with total phenolic content and flavonoid compounds (r ¼ 0.78 and r ¼ 0.91, respectively). However, negative correlation appeared between IC50 for DPPH scavenging with total phenolic (r ¼ 2 0.66) and flavonoid (r ¼ 2 0.79) contents. The quantitative analysis by HPLC-MS-MS showed that the major flavonoid of R. monosperma of Spain was the isoflavone genistein (Gonza´lez-Mauraza et al. 2013). Numerous studies have shown that genistein is a strong antioxidant and increases the activity of other antioxidant enzymes such as catalase, glutathione peroxidase, superoxide dismutase and glutathione reductase (Qiuyin & Huachen 1996; Trieu et al 1999). The correlation coefficient between TAA and the condensed tannin contents was found to be very small 0.26; however, a positive correlation coefficient between condensed tannin contents determined by vanillin – HCl assay and IC50 for DPPH scavenging 0.3 suggests there was no correlation.
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These surveys propose that flavonoid compounds are major contributors to antioxidant activity. 3. Conclusion Flavonoids are the contributor of the antioxidant power in ethyl acetate extracts of seeds of R. monosperma. However, further studies will be needed to identify the exact molecular structures of the antioxidant compounds. Supplementary material
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