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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf fluid extract a
b
a
J.C. Escalona-Arranz , R. Perez-Rosés , J. Rodríguez-Amado , c
a
a
H.J. Morris-Quevedo , L.B. Mwasi , O. Cabrera-Sotomayor , d
e
e
R. Machado-García , O. Fong-Lórez , A. Alfonso-Castillo & E. Puente-Zapata
e
a
Department of Pharmacy, Oriente University, Avenida Patricio Lumumba s/n, 90500Santiago de Cuba, Cuba
Click for updates
b
Departament Farmacología i Química Terapéutica, Universitat de Barcelona, Barcelona, Spain c
Centre for Studies in Industrial Biotechnology (CEBI). Oriente University, Santiago de Cuba, Cuba d
Department of Chemistry, Oriente University, Santiago de Cuba, Cuba e
Medical Toxicology Centre (TOXIMED). Medical Sciences University, Santiago de Cuba, Cuba Published online: 10 Mar 2015.
To cite this article: J.C. Escalona-Arranz, R. Perez-Rosés, J. Rodríguez-Amado, H.J. MorrisQuevedo, L.B. Mwasi, O. Cabrera-Sotomayor, R. Machado-García, O. Fong-Lórez, A. AlfonsoCastillo & E. Puente-Zapata (2015): Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf fluid extract, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1019350 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1019350
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.
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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, 2015 http://dx.doi.org/10.1080/14786419.2015.1019350
SHORT COMMUNICATION Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf fluid extract J.C. Escalona-Arranza*, R. Perez-Rose´sb, J. Rodrı´guez-Amadoa, H.J. Morris-Quevedoc, L.B. Mwasia, O. Cabrera-Sotomayora, R. Machado-Garcı´ad, O. Fong-Lo´reze, A. Alfonso-Castilloe and E. Puente-Zapatae
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
a
Department of Pharmacy, Oriente University, Avenida Patricio Lumumba s/n, 90500 Santiago de Cuba, Cuba; bDepartament Farmacologı´a i Quı´mica Terape´utica, Universitat de Barcelona, Barcelona, Spain; c Centre for Studies in Industrial Biotechnology (CEBI). Oriente University, Santiago de Cuba, Cuba; d Department of Chemistry, Oriente University, Santiago de Cuba, Cuba; eMedical Toxicology Centre (TOXIMED). Medical Sciences University, Santiago de Cuba, Cuba (Received 19 November 2014; final version received 10 February 2015)
In the scientific community, there is a growing interest in Tamarindus indica L. leaves, both as a valuable nutrient and as a functional food. This paper focuses on exploring its safety and antioxidant properties. A tamarind leaf fluid extract (TFE) wholly characterised was evaluated for its anti-DPPH activity (IC50 ¼ 44.36 mg/mL) and its reducing power activity (IC50 ¼ 60.87 mg/mL). TFE also exhibited a high ferrous ionchelating capacity, with an estimated binding constant of 1.085 mol L21 while its influence over nitric oxide production in human leucocytes was irregular. At low concentrations, TFE stimulated NO output, but it significantly inhibited it when there was an increase in concentration. TFE was also classified as a non-toxic substance in two toxicity tests: the acute oral toxicity test and the oral mucous irritability test. Further toxicological assays are needed, although results so far suggest that TFE might become a functional dietary supplement. Keywords: Tamarindus indica L; antioxidant; toxicological evaluation; functional foods; tamarind leaves
1. Introduction Nowadays, there is a growing interest in functional foods that are able to provide additional physiological benefits for human health other than basic nutritional and energetic requirements. A remarkable activity present in some functional foods is their antioxidant capacity.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
J.C. Escalona-Arranz et al.
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
Antioxidants play an important role in preventing many diseases induced by reactive oxygen species – diseases which result in oxidative damage to DNA, proteins and other macromolecules. Those oxidative damages are associated with pathological events, such as cancer, diabetes, atherosclerosis and neurodegenerative disorders (Rajendran et al. 2014). Tamarindus indica leaves are edible and are used to make curries, salads, stews and soups in many countries. Crude protein ratios in tamarind leaves are high, leaves are also rich in fat, fibre and some vitamins such as thiamine, riboflavin, niacin, ascorbic acid and b-carotene (Bhadoriya et al. 2011). Yet, toxicological tests that ensure their safety are scarce. Since it might be very interesting to employ a tamarind leaf fluid extract (TFE) as a functional food, this paper focuses on exploring its safety and antioxidant properties. 2. Results and discussion 2.1. DPPH radical-scavenging activity and reducing power assay The calculated IC50 of quercetin and TFE in the DPPH assay was 10.88 ^ 0.81 mg/mL and 44.36 ^ 3.72 mg/mL, respectively. Even when TFE inhibitory activity was lower than that of reference compound, the antioxidant activity of this crude extract can be considered good. In the reducing power assay, activity was good as well (TFE IC50 ¼ 60.87 ^ 1.07 mg/mL while quercetin IC50 ¼ 21.94 ^ 0.89 mg/mL). These results point to tamarind leaves as a plant part with strong antioxidant activity. 2.2. Ferrous ion-chelating activity assay Results in this assay (Figure 1) showed three peaks for TFE (235, 273 and 338 nm). All peaks were modified when 0.01 mM of ferric sulphate was added. The last peak (338 nm) switched to 418 nm in a concentration-dependent mode. Quercetin behaviour was similar, its main band switched to 450 nm. The appearance of a band in the visible region (410 – 450 nm) is characteristic of polyphenol compounds (Andjelkovic´ et al. 2006). This behaviour agrees with the high levels of polyphenols present in TFE (Escalona-Arranz et al. 2011). Estimated binding constants were 1.085 mol L21 for TFE and 2.00 mol L21 for quercetin. TFE exhibited a lower ferrous ion-chelating capacity than reference substance although it was similar to that of some pure catechol compounds (Andjelkovic´ et al. 2006). The stability of formed complexes was manifest when the addition of EDTA 0.01 mM/L almost did not affect them. Nevertheless,
Figure 1. Absorbance peaks from TFE and quercetin with/without addition of ferric sulphate (0.01 mM).
Natural Product Research
3
EDTA at 0.1 mM/L had a significant effect on the TFE complex (only 10% of it remained), rather than over the quercetin complex (69% of it remained).
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
2.3. Nitric oxide production in human leucocytes Results on NO production (Figure 2) showed that at low concentrations TFE stimulated NO output while it significantly inhibited NO production when there was an increase in concentration. The calculated IC50 value (311.0 mg/mL) was high in contrast with reference substance (L -NMMA, IC50 ¼ 36.5 mg/mL). TFE was revealed as a mild although significant inhibitor of NO production in lipopolysaccharides-stimulated human leucocytes. Published papers on tamarind leaves activity had not yet explored its influence on NO production in human leucocytes. TFE inhibitory activity on NO production can be related to its high content of flavonoids and polyphenols. The proved antioxidant capacity of TFE is certainly related to the ethnopharmacological use of tamarind leaves. Rajendran et al. (2014) , in a recent review of the evidence on antioxidant nutrients’ effect on human diseases, highlighted the positive action of those antioxidant nutrients over diabetes, arthritis and atherosclerosis. Use of tamarind leaves in treating those illnesses or others closely related is documented in the literature (Havinga et al. 2010). 2.4. Acute oral toxicity evaluation Clinical observations in rats treated with TFE 2000 mg/mL did not evidence clinical signs that might be associated with systemic toxic effects. No changes in the animal’s hair or skin were observed. Mucous or eye coloration were normal, and so was their behaviour and somato-motive activity. Weight increments and food and daily water consumptions were normal for the species. The assay finished with a 100% survival. Histopathological studies did not show any significant difference between the animals that received treatment and the control group. TFE was qualified as a ‘not classified’ product (meaning not toxic) within the scale of toxic class substances (OECD/OCDE 423 2012).
Figure 2. Inhibition of nitric oxide production in human leucocytes by TFE and L -NMMA.
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
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J.C. Escalona-Arranz et al.
2.5. Oral mucous irritability Macroscopic evaluation of the oral mucous of Syrian hamsters exposed to TFE in the right malar bag evidenced a very light erythema according to the methodology employed (ISO 10993-10 2002). No significant changes were observed in the weight of the animals. Important damages to the oral mucous that could put at risk food and water consumption did not occur. However, the microscopic examination did show a trivial degeneration of the epithelium and a mild vascular congestion in the muscular tissue of four animals. TFE was then classified as a light irritant. With several organic acids (tartaric, malic and citric) as well as other substances such as polyphenols in its composition, TFE could be a very mild irritant to the skin. In an already published work, TFE toxicity was partially explored. TFE did not cause blood protein denaturalisation or haemolysis on human red blood cells. Also, erythrocyte membrane damage caused by the action of oxidative H2O2 displayed a steady reduction with increasing TFE concentrations (EscalonaArranz et al. 2014). However, the fundamental element that suggests little or no toxicity in TFE is its employment in human nutrition and its widespread use in traditional medicine by many communities (Havinga et al. 2010; Bhadoriya et al. 2011). 3. Conclusions In this study, TFE demonstrated its antioxidant capacity through various tests. TFE was also classified as a non-toxic substance in two conducted toxicity tests. Experimental data summarised here add evidential support to the traditional consumption and ethnopharmacological use of tamarind leaves by some communities. Consumption of a tamarind TFE as a functional food may play a role in the prevention of oxidative stress and the bolstering of cellular antioxidant defence systems. However, while further toxicological assays are needed, the bioguided fractionation of the TFE will surely be an important area of research in the future. Supplementary material Experimental details relating to this article are available online. References Andjelkovic M, Van Camp J, Demeulenaer B, Depaemelaere G, Socaciu C, Verloo M, Verhe R. 2006. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem. 98:23– 31. doi:10.1016/j. foodchem.2005.05.044. Bhadoriya SS, Ganeshpurkar A, Narwaria J, Rai G, Jain AP. 2011. Tamarindus indica: extent of explored potential. Pharmacogn Rev. 5:73–81. doi:10.4103/0973-7847.79102. Escalona-Arranz JCJ, Garcia-Diaz JJ, Perez-Rose´s RR, De la Vega JJ, Rodrı´guez-Amado JJ, Morris-Quevedo HJ. 2014. Effect of Tamarindus indica L. leaves’ fluid extract on human blood cells. Nat Prod Res. 28:1485–1488. doi:10. 1080/14786419.2014.911296. Escalona-Arranz JC, Rodrı´guez-Amado J, Pe´rez-Rose´s R, Can˜izares-Lay J, Sierra-Gonza´lez G, Morris-Quevedo HJ, Licea-Jimenez I. 2011. Metabolites extraction optimization in Tamarindus indica L. leaves. Bol Latinoam Caribe Plant Med Aromat. 10:359–369. Havinga RM, Hartl A, Putscher J, Prehsler S, Buchmann C, Vogl CR. 2010. Tamarindus indica L. (Fabaceae): patterns of use in traditional African medicine. J Ethnopharmacol. 127:573 –588. doi:10.1016/j.jep.2009.11.028. ISO 10993-10. 2002. International Organization for Standardization. Biological evaluation of medical devices. Part 10: Tests for irritation and delayed-type hypersensitivity. OECD/OCDE 423. 2012. OECD Guideline for Testing of Chemicals. Acute Oral Toxicity – Acute Toxic Class Method. Available from: http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD_GL423.pdf Rajendran P, Nandakumar N, Rengarajan T, Palaniswami R, Gnanadhas EN, Lakshminarasaiah U, Gopas J, Nishigaki I. 2014. Antioxidants and human diseases. Clin Chim Acta. 436:332 –347. doi:10.1016/j.cca.2014.06.004.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf fluid extract a
b
a
J.C. Escalona-Arranz , R. Perez-Rosés , J. Rodríguez-Amado , c
a
a
H.J. Morris-Quevedo , L.B. Mwasi , O. Cabrera-Sotomayor , d
e
e
R. Machado-García , O. Fong-Lórez , A. Alfonso-Castillo & E. Puente-Zapata
e
a
Department of Pharmacy, Oriente University, Avenida Patricio Lumumba s/n, 90500Santiago de Cuba, Cuba
Click for updates
b
Departament Farmacología i Química Terapéutica, Universitat de Barcelona, Barcelona, Spain c
Centre for Studies in Industrial Biotechnology (CEBI). Oriente University, Santiago de Cuba, Cuba d
Department of Chemistry, Oriente University, Santiago de Cuba, Cuba e
Medical Toxicology Centre (TOXIMED). Medical Sciences University, Santiago de Cuba, Cuba Published online: 10 Mar 2015.
To cite this article: J.C. Escalona-Arranz, R. Perez-Rosés, J. Rodríguez-Amado, H.J. MorrisQuevedo, L.B. Mwasi, O. Cabrera-Sotomayor, R. Machado-García, O. Fong-Lórez, A. AlfonsoCastillo & E. Puente-Zapata (2015): Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf fluid extract, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1019350 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1019350
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.
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
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, 2015 http://dx.doi.org/10.1080/14786419.2015.1019350
SHORT COMMUNICATION Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf fluid extract J.C. Escalona-Arranza*, R. Perez-Rose´sb, J. Rodrı´guez-Amadoa, H.J. Morris-Quevedoc, L.B. Mwasia, O. Cabrera-Sotomayora, R. Machado-Garcı´ad, O. Fong-Lo´reze, A. Alfonso-Castilloe and E. Puente-Zapatae
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
a
Department of Pharmacy, Oriente University, Avenida Patricio Lumumba s/n, 90500 Santiago de Cuba, Cuba; bDepartament Farmacologı´a i Quı´mica Terape´utica, Universitat de Barcelona, Barcelona, Spain; c Centre for Studies in Industrial Biotechnology (CEBI). Oriente University, Santiago de Cuba, Cuba; d Department of Chemistry, Oriente University, Santiago de Cuba, Cuba; eMedical Toxicology Centre (TOXIMED). Medical Sciences University, Santiago de Cuba, Cuba (Received 19 November 2014; final version received 10 February 2015)
In the scientific community, there is a growing interest in Tamarindus indica L. leaves, both as a valuable nutrient and as a functional food. This paper focuses on exploring its safety and antioxidant properties. A tamarind leaf fluid extract (TFE) wholly characterised was evaluated for its anti-DPPH activity (IC50 ¼ 44.36 mg/mL) and its reducing power activity (IC50 ¼ 60.87 mg/mL). TFE also exhibited a high ferrous ionchelating capacity, with an estimated binding constant of 1.085 mol L21 while its influence over nitric oxide production in human leucocytes was irregular. At low concentrations, TFE stimulated NO output, but it significantly inhibited it when there was an increase in concentration. TFE was also classified as a non-toxic substance in two toxicity tests: the acute oral toxicity test and the oral mucous irritability test. Further toxicological assays are needed, although results so far suggest that TFE might become a functional dietary supplement. Keywords: Tamarindus indica L; antioxidant; toxicological evaluation; functional foods; tamarind leaves
1. Introduction Nowadays, there is a growing interest in functional foods that are able to provide additional physiological benefits for human health other than basic nutritional and energetic requirements. A remarkable activity present in some functional foods is their antioxidant capacity.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
J.C. Escalona-Arranz et al.
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
Antioxidants play an important role in preventing many diseases induced by reactive oxygen species – diseases which result in oxidative damage to DNA, proteins and other macromolecules. Those oxidative damages are associated with pathological events, such as cancer, diabetes, atherosclerosis and neurodegenerative disorders (Rajendran et al. 2014). Tamarindus indica leaves are edible and are used to make curries, salads, stews and soups in many countries. Crude protein ratios in tamarind leaves are high, leaves are also rich in fat, fibre and some vitamins such as thiamine, riboflavin, niacin, ascorbic acid and b-carotene (Bhadoriya et al. 2011). Yet, toxicological tests that ensure their safety are scarce. Since it might be very interesting to employ a tamarind leaf fluid extract (TFE) as a functional food, this paper focuses on exploring its safety and antioxidant properties. 2. Results and discussion 2.1. DPPH radical-scavenging activity and reducing power assay The calculated IC50 of quercetin and TFE in the DPPH assay was 10.88 ^ 0.81 mg/mL and 44.36 ^ 3.72 mg/mL, respectively. Even when TFE inhibitory activity was lower than that of reference compound, the antioxidant activity of this crude extract can be considered good. In the reducing power assay, activity was good as well (TFE IC50 ¼ 60.87 ^ 1.07 mg/mL while quercetin IC50 ¼ 21.94 ^ 0.89 mg/mL). These results point to tamarind leaves as a plant part with strong antioxidant activity. 2.2. Ferrous ion-chelating activity assay Results in this assay (Figure 1) showed three peaks for TFE (235, 273 and 338 nm). All peaks were modified when 0.01 mM of ferric sulphate was added. The last peak (338 nm) switched to 418 nm in a concentration-dependent mode. Quercetin behaviour was similar, its main band switched to 450 nm. The appearance of a band in the visible region (410 – 450 nm) is characteristic of polyphenol compounds (Andjelkovic´ et al. 2006). This behaviour agrees with the high levels of polyphenols present in TFE (Escalona-Arranz et al. 2011). Estimated binding constants were 1.085 mol L21 for TFE and 2.00 mol L21 for quercetin. TFE exhibited a lower ferrous ion-chelating capacity than reference substance although it was similar to that of some pure catechol compounds (Andjelkovic´ et al. 2006). The stability of formed complexes was manifest when the addition of EDTA 0.01 mM/L almost did not affect them. Nevertheless,
Figure 1. Absorbance peaks from TFE and quercetin with/without addition of ferric sulphate (0.01 mM).
Natural Product Research
3
EDTA at 0.1 mM/L had a significant effect on the TFE complex (only 10% of it remained), rather than over the quercetin complex (69% of it remained).
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
2.3. Nitric oxide production in human leucocytes Results on NO production (Figure 2) showed that at low concentrations TFE stimulated NO output while it significantly inhibited NO production when there was an increase in concentration. The calculated IC50 value (311.0 mg/mL) was high in contrast with reference substance (L -NMMA, IC50 ¼ 36.5 mg/mL). TFE was revealed as a mild although significant inhibitor of NO production in lipopolysaccharides-stimulated human leucocytes. Published papers on tamarind leaves activity had not yet explored its influence on NO production in human leucocytes. TFE inhibitory activity on NO production can be related to its high content of flavonoids and polyphenols. The proved antioxidant capacity of TFE is certainly related to the ethnopharmacological use of tamarind leaves. Rajendran et al. (2014) , in a recent review of the evidence on antioxidant nutrients’ effect on human diseases, highlighted the positive action of those antioxidant nutrients over diabetes, arthritis and atherosclerosis. Use of tamarind leaves in treating those illnesses or others closely related is documented in the literature (Havinga et al. 2010). 2.4. Acute oral toxicity evaluation Clinical observations in rats treated with TFE 2000 mg/mL did not evidence clinical signs that might be associated with systemic toxic effects. No changes in the animal’s hair or skin were observed. Mucous or eye coloration were normal, and so was their behaviour and somato-motive activity. Weight increments and food and daily water consumptions were normal for the species. The assay finished with a 100% survival. Histopathological studies did not show any significant difference between the animals that received treatment and the control group. TFE was qualified as a ‘not classified’ product (meaning not toxic) within the scale of toxic class substances (OECD/OCDE 423 2012).
Figure 2. Inhibition of nitric oxide production in human leucocytes by TFE and L -NMMA.
Downloaded by [Simon Fraser University] at 10:37 14 March 2015
4
J.C. Escalona-Arranz et al.
2.5. Oral mucous irritability Macroscopic evaluation of the oral mucous of Syrian hamsters exposed to TFE in the right malar bag evidenced a very light erythema according to the methodology employed (ISO 10993-10 2002). No significant changes were observed in the weight of the animals. Important damages to the oral mucous that could put at risk food and water consumption did not occur. However, the microscopic examination did show a trivial degeneration of the epithelium and a mild vascular congestion in the muscular tissue of four animals. TFE was then classified as a light irritant. With several organic acids (tartaric, malic and citric) as well as other substances such as polyphenols in its composition, TFE could be a very mild irritant to the skin. In an already published work, TFE toxicity was partially explored. TFE did not cause blood protein denaturalisation or haemolysis on human red blood cells. Also, erythrocyte membrane damage caused by the action of oxidative H2O2 displayed a steady reduction with increasing TFE concentrations (EscalonaArranz et al. 2014). However, the fundamental element that suggests little or no toxicity in TFE is its employment in human nutrition and its widespread use in traditional medicine by many communities (Havinga et al. 2010; Bhadoriya et al. 2011). 3. Conclusions In this study, TFE demonstrated its antioxidant capacity through various tests. TFE was also classified as a non-toxic substance in two conducted toxicity tests. Experimental data summarised here add evidential support to the traditional consumption and ethnopharmacological use of tamarind leaves by some communities. Consumption of a tamarind TFE as a functional food may play a role in the prevention of oxidative stress and the bolstering of cellular antioxidant defence systems. However, while further toxicological assays are needed, the bioguided fractionation of the TFE will surely be an important area of research in the future. Supplementary material Experimental details relating to this article are available online. References Andjelkovic M, Van Camp J, Demeulenaer B, Depaemelaere G, Socaciu C, Verloo M, Verhe R. 2006. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem. 98:23– 31. doi:10.1016/j. foodchem.2005.05.044. Bhadoriya SS, Ganeshpurkar A, Narwaria J, Rai G, Jain AP. 2011. Tamarindus indica: extent of explored potential. Pharmacogn Rev. 5:73–81. doi:10.4103/0973-7847.79102. Escalona-Arranz JCJ, Garcia-Diaz JJ, Perez-Rose´s RR, De la Vega JJ, Rodrı´guez-Amado JJ, Morris-Quevedo HJ. 2014. Effect of Tamarindus indica L. leaves’ fluid extract on human blood cells. Nat Prod Res. 28:1485–1488. doi:10. 1080/14786419.2014.911296. Escalona-Arranz JC, Rodrı´guez-Amado J, Pe´rez-Rose´s R, Can˜izares-Lay J, Sierra-Gonza´lez G, Morris-Quevedo HJ, Licea-Jimenez I. 2011. Metabolites extraction optimization in Tamarindus indica L. leaves. Bol Latinoam Caribe Plant Med Aromat. 10:359–369. Havinga RM, Hartl A, Putscher J, Prehsler S, Buchmann C, Vogl CR. 2010. Tamarindus indica L. (Fabaceae): patterns of use in traditional African medicine. J Ethnopharmacol. 127:573 –588. doi:10.1016/j.jep.2009.11.028. ISO 10993-10. 2002. International Organization for Standardization. Biological evaluation of medical devices. Part 10: Tests for irritation and delayed-type hypersensitivity. OECD/OCDE 423. 2012. OECD Guideline for Testing of Chemicals. Acute Oral Toxicity – Acute Toxic Class Method. Available from: http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD_GL423.pdf Rajendran P, Nandakumar N, Rengarajan T, Palaniswami R, Gnanadhas EN, Lakshminarasaiah U, Gopas J, Nishigaki I. 2014. Antioxidants and human diseases. Clin Chim Acta. 436:332 –347. doi:10.1016/j.cca.2014.06.004.
