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Revista Cubana de Química ISSN: 0258-5995 [email protected] Universidad de Oriente Cuba

Escalona-Arranz, Julio César; Pérez-Rosés, Renato; Licea Jiménez, Irina; Rodríguez- Amado, Jesús; Argota-Coello, Humberto; Cañizares-Lay, Javier; Morris-Quevedo, Humberto J.; Sierra-González, Gustavo CHEMICAL CONSTITUENTS OF Tamarindus indica L. Revista Cubana de Química, vol. XXII, núm. 3, 2010, pp. 65-71 Universidad de Oriente Santiago de Cuba, Cuba

Available in: http://www.redalyc.org/articulo.oa?id=443543720011

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative CHEMICAL CONSTITUENTS OF Tamarindus indica L. LEAVES Julio César Escalona-Arranz *; Renato Pérez-Rosés **; Irina Licea Jiménez *; Jesús Rodríguez-Amado *;" Humberto Argota-Coello ***, Javier Cañizares-Lay *, Humberto J. Morris-Quevedo ***, Gustavo Sierra-González *** * Pharmacy Department, Oriente University, ** Departament Farmacología i Química Terapéutica, Universitat de Barcelona, Spain *** Laboratory. Empresa Geominera-Oriente (EGMO), Santiago de Cuba, **** Centre for Studies in Industrial Biotechnology, Oriente University, Finlay Institute. Ciudad de la Habana. Cuba

z Resumen El (Tamarindus indica L.) es un árbol tropical con múltiples usos, empleado principalmente por las propiedades nutritivas de sus hojas y frutos. Ambas partes presentan también propiedades medicinales reportadas, pero para el caso de las hojas, la información disponible sobre su composición química es limitada. En este trabajo, se determina la composición química de dos extractos de las hojas empleando para ellos técnicas de cromatografía gaseosa/espectrometría de masas (GC/MS), cromatografía en capa delgada de alta resolución/espectroscopía UV (HTLC-UV) y espectrometría por plasma inductivamente acoplado (ICP-AES). Los resultados confirman la presencia de aceites esenciales, ácidos grasos libres y esterificados, flavonoides y compuestos relacionados, pero también describen la presencia de ocho nuevos compuestos e importantes niveles de selenio y otros micro-elementos nunca antes reportados para la planta. En su conjunto, muchos de los compuestos determinados pueden ser relacionados con las propiedades atribuidas por la población: la antibacteriana y la antioxidante, en especial la relacionada con desórdenes hepáticos. Palabras clave: tamarindus indica, ácidos grasos, aceites esenciales, flavonoides, microelements. z Abstract

Tamarind (Tamarindus indica L.) is a multipurpose tropical used primarily because its and leaves have eatable properties. Both parts of the have also medicinal uses, but in the specific case of the leaves, little information about their chemical composition is available. In this paper, we explore the tamarind leaves´ composition of two extracts employing Gas Chromatography/Mass Spectrometry (GC/MS), High Performance Thin Layer Chromatography-Ultraviolet spectroscopy (HTLC-UV) and Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) techniques. Results confirm the production of essential oils, free and conjugated fatty acids, flavonoids, and other compounds, but also describe the presence of eight new compounds for this part of the plant and important levels of Selenium and other micro-elements not previously reported. As a whole, many of the detected compounds can be related to the traditional folk use: Antibacterial and antioxidant, particularly in liver diseases. Keywords: tamarindus indica, fatty acids, , flavonoids, micro-elements.

z Introduction eaten fresh or processed, used as a or ; but also as medicine beverages. In fact, their Tamarindus indica L. or tamarind, as it is fruits are the most studied and valuable medicinal part commonly known, is a medium-sized tree belonging to of the plant, which has often been reported as curative the Caesalpinaceae family. It’s a multipurpose in several pharmacopoeias. This millenary tree has tropical tree used primarily for its fruits, which are been taken into consideration from ancient times;

Vol. XXII, Nº 3, 2010 65 documented evidences about its cultivation appear in activity is suggested; giving rise to this investigation Egypt around 400BC, and it is mentioned in the Indian in which an exploration of the phytochemical Brahmasamhita Scriptures between 1200-200 BC. constituents is proposed. Also about 370-287 BC Theophrastus wrote on and two descriptions refer to tamarind, even though z Material and methods not named as such /1/. Plant material Thai traditional medicine recognizes Tamarindus indica as digestive, carminative, Tamarind leaves were collected in November , expectorant and blood tonic /2/. Many 2008 from a tamarind population in Santiago de Cuba, other properties have been also reported like eastern part of Cuba. A voucher specimen registered Hypolipemic and antioxidant /3/, anti-inflammatory as 052216 was deposited at the herbarium of the /4/, antimicrobial /5/, cytotoxic /6/, against biology department, University of Oriente, Cuba. gastrointestinal spasms /7/, and modifying the complement system /8/. Plant extraction

These extraordinary properties of tamarind fruits The collected leaves were sun dried and then along with the interest on the polysaccharide and powdered with the help of a blender. Two kinds of antioxidant seed compounds, keep the attention of the extracts were prepared. scientific community on tamarind tree, leaving relegated Extract 1: Twenty-five grams of the powder to a secondary level the other parts of the plant. was placed in the thimble and extracted with 150 mL On the other hand, tamarind leaves retain a of Chloroform, using a Soxhlet apparatus for 12 h. The more empiric use. As the fruits, tamarind leaves extract was concentrated using rotary flash evaporator are also edible and are used to make , under reduced pressure at temperatures below 35 °C. salads, and soups in many countries, but Extract 2: Fifty grams of the powered leaves especially in times of scarcity, even when their were prepared as fluid extract by percolation method ratios (4,0-5,8%) /9/ are not too far from with four-day extractions, using ethanol 70 % as those reported for fruits (2,0-7,1%) /10/. In fact, solvent. most of the chemical studies of tamarind leaves are focused in their eatable properties, with flavonoid Fractions from extract 2: Fluid extract was compounds as the only exception, which have been fractioned by a successive liquid-liquid separation reported with certain frequency /11,12/. Other method described for flavonoid and other related classes of compounds reported are the triterpenoids compounds /16/. Three extractions with 50 mL of n- lupanone and lupeol /13/, and some essential oil hexane were used to generate the first fraction in this with benzyl benzoate and limonene as major liquid-liquid separation. Successive extractions with compounds /14/. chloroform, ethyl acetate and n-butanol were employed to obtain the other three fractions. All four fractions During the latest years, the descriptions of the were dried by reduced pressure at temperatures pharmacological potential of tamarind leaves are below 50 °C and re-dissolved in absolute ethanol. coming out. The hepatoprotective and the antimicrobial activity look like the most prominent Chemical analysis activities, both associated to flavonoid and/or phenol presence. Our research group was investigating Gas Chromatography-Mass spectrometry the real influence of these compounds on the (GC-MS) analysis antimicrobial activity, but no quantitative relationship was possible to establish /15/. Due to the relative The extract 1 and the two first fractions of the poor knowledge about the chemical composition of extract 2 were analyzed by a Gas Chromatography- tamarind leaves, no specific association compound/ Mass spectrometry (GC-MS) analysis on a FISONS

66 Vol. XXII, Nº 3, 2010 Trio 1000 system. The experimental conditions each element. The calibration curves and the were an SPB-1 fused silica column of 30 m ⋅ data were statistically analyzed by using fitting 0,32 mm, 0,25 µm film thickness, with a of straight line by the least square method. As a temperature program from 30 0C (3min), 4 0C/ way to evaluate the total inorganic substance, min till 250 0C (10 min.). Carrier gas (helium) Total Ash determination was developed as it is flow rate was 1 ml/min and mass spectra were described in literature /23/. measured by electron ionization (EI) at 70 eV. Identification was made comparing mass spectra z Results and discussion and GC retention indices (RI) with those of our IDENT data bank included in the system. Some Gas Chromatography-Mass spectrometry mass spectra are also compared with literature data /17/. (GC-MS) analysis

The Gas Chromatography-Mass spectrometry High Performance Thin Layer (GC-MS) analysis on the first extract reveals 18 Chromatography-Ultraviolet spectroscopy peaks, yielding an acceptable 90,83 % (table 1). (HTLC-UV) Taking into consideration the chemical nature of the compounds extracted; great varieties of The last two fractions of the second extract chemical compounds (essential oils, fatty acids, were analyzed by High Performance Thin Layer polyphenols and others) yield the extract 1. Chromatography (HPTLC LINOMAT IV) Previous papers signed the essential oils coupled with a UV-visible densitometer production in tamarind leaves /15,16/. Limonene, (SCANNER II) both CAMAG from Switzerland. linalool anthranilate, and p-cymene are some of A bi-dimensional method employing butanol- acetic acid-water (65:15:25 v/v/v) and acetic those before mentioned. However, the acid 5 % as mobile phases was used, meanwhile compounds, Diphenyl-ether, Longifolene, Caryophyllene and 6,10,14-trimethylpentadeca- Silica gel 60F254 plates 10x10 with internal fluorescent indicator (Merck EMD, Germany), 5,9,13-trien-2-one are also essential oils but no were employed as static phase /18/. For all previous reports appear in literature. The amino compounds detected, three UV-spectrums were substituent of the volatile linalool anthranilate determined controlled by the Peak Purity option compound can be the cause of why tamarind included in CATS software (Planar leaves give an intermittent and weak evidence Chromatography Manager) version 3.20. of alkaloid /24/. Compound assignation was developed comparing the data obtained with literature /19-22/ and The Diphenyl-ether is a compound with a considering the previous tamarind leaves limited distribution on plants. Nevertheless some flavonoids reports. reports in green tea, and others species as Isatis tinctoria, Capparis spinosa L., Rosa Inductively Coupled Plasma Atomic damascena Mill, y Mangifera indica L. has Emission Spectrometry (ICP-AES) been found /25/. Compounds 6 and 7 have a phenolic nature. Compound 6 (2,6-di-tert-butyl- Elemental assay: A Nitric Acid - Perchloric 4-methylphenol) is a well recognized natural Acid digestion of one gram of dried leaves, and antioxidant widely distributed on plants, and both extracts were investigated for elemental compound 7 is a chemical derivate from them, analysis by ICP-AES, in a SPECTRO ARCOS but not commonly reported in the plant kingdom. spectrometer from Germany. Appropriate These two compounds have not been previously working standard solution was prepared for reported for tamarind plant before.

Vol. XXII, Nº 3, 2010 67 TABLE 1. IDENTIFIED COMPOUNDS IN TAMARINDUS INDICA L. LEAVES EXTRACT 1 BY GC-MS

MF= Molecular Formula, MW= Molecular weight, RI= Retention index, % = Relative percentage Polyacetylenes are plant metabolites in tamarind growing at the American derivates from the latest steps of acetate continent. As well as these previous studies, pathway, being expressed in the final stages of C16:0, and C18:n, appear as the main fatty acids this metabolic via and common in high- in tamarind leaves, but also the long chain fatty development plants. The compound 3-eicosine acid Methyl 15-tricosenoate is detected. belongs to this class of metabolites that has been cited as potent antimicrobial and citotoxic This diversity of compounds provides new substance. Compound Cryptopinone have a elements to justify the two more reported carbonyl function, and has been referred in pharmacological properties for tamarind leaves: several species on plant Kingdom, but neither 3- Antioxidant and antimicrobial activity. This study eicosine nor Cryptopinone were previously also affords eight compounds not previously reported for Tamarindus indica L. specie. reported for Tamarindus indica leaves.

Free and conjugated fatty acids and β- In the GC-MS analysis of extract 2, n-hexane Sitosterol are also detected in the extract 1. and chloroform fractions results only in six and These kinds of compounds with a high value not seven identified peaks as is shown in table 2. only as nutrient but also as antioxidant and The relative high solvent polarity employed in antimicrobial /26,27/ were reported in previous this second extract (ethanol 70%) should be the papers in India and Pakistan /28,29/, but never cause of these few peaks found.

68 Vol. XXII, Nº 3, 2010 TABLE 2. IDENTIFIED COMPOUNDS IN TAMARINDUS INDICA L. LEAVES EXTRACT 2 BY GC-MS (N-HEXANE AND CHLOROFORM FRACTIONS)

MF= Molecular Formula, MW= Molecular weight, RI= Retention index, % = Relative percentage In n-hexane fraction, all extracted compounds absorption in UV region and this fact has been used result already identified in the extract 1. Even when in their chemical detection, quantification and chloroform and n-hexane belong to the same category characterization. Our studies reveal a total of 8 and 9 of non-polar solvents, their diverse polarities allow spots in ethyl-acetate and n-butanol fractions chloroform fraction to extract different kinds of respectively, but only 3 and 4 spots were detected in compounds, mainly those with acidic characteristic. enough concentration to be considered as main appears in higher concentration. This compounds and with conditions to be analyzed by the compound is reported as the responsible of the most technique employed. These results are showed in outstanding characteristic of tamarind: its sweet acidic table 3. . Many other acidic compounds´-types are also extracted in this fraction. Hexadecanoic and 10- From table 3 it is inferred that spots from 1 to 6 are Octadecenoic acids identification occurs in both flavonoid derivates, and more specific flavone type, fractions, confirming that the most common fatty acid because the two characteristic band of absorption in η η chains in tamarind leaves are C and C . the range 260-270 m (band II) and 330-365 m 16 18 (band I), while spot 7 should be a polyphenol, kind of High Performance Thin Layer compound usually extracted together with flavonoids. Chromatography-Ultraviolet Spectroscopy Another important information from the flavonoid UV (HTLC-UV) spectrum is the arithmetic difference between the The latest ethyl acetate and n-butanol fractions both absorbance peaks to define the pattern of from extract 2 were analyzed by High Performance substitution in B ring. Values under 83 mean a single Thin Layer Chromatography-Ultraviolet spectroscopy OH substitution (Spots 3 and 6), whereas over 83 (HTLC-UV). mean double OH substitution (spots 1, 2, 4 and 5). Because of the methodology employed, these Spot 3, gives a perfect chromatography and fractions should be rich in flavonoids and related spectroscopic match with apigenin, while spots 1 and compounds. Those compounds have a strong 2 with almost identical spectroscopy features but

Vol. XXII, Nº 3, 2010 69 different chromatography behavior, were experimental data with the data bank and matched with luteolin 7-o-glucoside and luteolin literature. All the identified compounds were respectively. The same analysis was developed previously reported for tamarind leaves by for the n-butanol fraction, matching the et al. in 2006 /12/. TABLE 3. IDENTIFIED COMPOUNDS IN THE EXTRACT 2 BY HPTLC-UV (ETHYL ACETATE AND N-BUTANOL FRACTIONS)

Inductively coupled plasma atomic common as cadmium, chromium, cobalt, manganese, emission spectrometry (ICP-AES) nickel, strontium, molybdenum and vanadium. The results of these cation determinations in dried Dried leaves and both extract were investigated leaves and both prepared extracts are showed in table for elemental analysis. Traditionally, this kind of study 4. Metals in plants normally appear forming or determines common elements as Sodium, Phosphorus, complexes, but usually retain their hydrophilic nature, Magnesium, , Potassium and others. In fact, for this reason the low results obtained for extract 1 the few reports found refer these elements /1, 29/. But are not a surprise, due to the non-polar nature of none of these elements is associated to the antioxidant chloroform solvent. Even when ethanol 70 % is much or antimicrobial activity, for this reason we focused polar than chloroform, it cannot extract high quantities our study on multi-valence cations in which an electron of the total metals present in leaves, meaning a transfer may happen and indeed be involved in the maximum of 30 % for Manganese and minimum of antioxidative process, or elements which form parts 1,13 % for Nickel. The values of total ashes determined of antioxidant metallo-proteins. By this way, common in leaves and both extracts confirms these elements as aluminium, iron, copper, lead, selenium observations. These differences between one metal and were considered, as well as others less to other must depend on the substrate nature. TABLE 4. ELEMENTS CONCENTRATIONS AND TOTAL ASHES DETECTED IN TAMARINDUS INDICA L. LEAVES BY ICP-AES

70 Vol. XXII, Nº 3, 2010 Copper and Iron appears in upper concentrations 8. Librandi AP, Chrysóstomo TN, Azzolini AE, Vargas-Recchia than previous studies /1, 29/, also Lead concentrations CG, Uyemura SA, Assis-Pandochi AI. Food Chem Toxicol. 45: 1487–1495 (2007). are superior, but not in toxic levels. On the other hand, 9. Duke, JA. Handbook of of World Economic cadmium, zinc and specially manganese are detected Importance. Plenum Press, New York, USA, pp 228-230, in lower concentrations. (1981) . 10. Ishola MM, Agbaji EB, Agbaji AS. J. Sci. Food Agric., 51: In spite of all, tamarind leaves have good quantities 141-143, (1990). of the most prominent pro-oxidant/antioxidant cations: 11. Chitra Arya. Res J Chem Envirom. 3: 305-17, (1999). iron, cupper and selenium, therefore this property 12. Dehesa M, Jauregui O, Cañigueral S. Revista de Fitoterapia, should be expressed in polar tamarind formulations. 6: 79-82, (2006). 13. Imam S, Azhar I, Mohtasheemul MH, Ali MS, Waseemuddin  Conclusion A. Pak. J. Pharm. Sci. 20(2): 125-127, (2007). 14. Pino JA, Escalona JC, Licea I, Pérez R, Agüero J. J Essent Tamarind leaves are worldwide reported for Oil Res; 14:187-8, (2002). their antioxidant and antimicrobial activity, but it 15. Escalona-Arranz JC, Péres-Rosés R, Urdaneta-Lafitta I, hasn´t been possible to establish a relationship Camacho-Pozo MI, Rodríguez-Amado J, Licea-Jimenez I. Phcog Mag. 6(23), 242-247, (2010). with the chemical composition due to the scanty 16. Petersen F. and Amstutz R. (Eds.). Progress in Drug Research, information availed. In this study, we detected Vol. 65 Birkhäuser Verlag, Basel, Switzerland, pp 232, eight components not previously reported, and (2008) confirmed the high fatty acid and polyphenol 17. Adams RP. 1995. Identification of essential oil by Gas production in Tamarindus indica L leaves. In Chromatography/Mass Spectroscopy. Carol Stream, Illinois: addition, high concentration of the most prominent Allured publisher corp. USA. pro-oxidant/antioxidant cations is described. All 18. Andersen ØM, Markham KR. FLAVONOIDS, Chemistry, Biochemistry and Applications. CRC Press Taylor & Francis this information give light to the pretended intention Group, Boca Raton, FL, USA. pp 10-16, (2006). to find chemical proof that support the 19. Mabry TJ, Markham KR, Thomas MB. The Systematic pharmacological activities previously described Identification of Flavonoids, Springer-Verlag, New York. for tamarind leaves. USA. (1970) 20. George S, Maute A. Chromatographia, 15: 419-452, (1982). Bibliofraphy 21. Harborne JB, Mabry TJ. (eds). The Flavonoids: Advances in Research since 1980, Chapman & Hall, London, United 1. El-Siddig K, Gunasena HPM, Prasad BA, Pushpakumara Kingdom: pp 99–124, (1988). DK, Ramana KVR, Vijayanand P, Williams JT. Tamarind, 22. Merken HM, Beecher GR. J. Agric. Food Chem. 48: 577, Tamarindus indica. Southampton Centre for Underutilised (2000). Crops, Southampton, United Kingdom: pp 13-27, (2006). 23. Ahmad I, Aqil F, Owais M. (eds). Modern Phytomedicine. 2. Komutarin T, Azadi S, Butterworth L, Keil D, Chitsomboon WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, B, Suttajit M, Meade BJ. Food Chem. Toxicol. 42: 649–658, Germany pp: 35, (2006). (2004). 24. Doughari JH. Trop J Pharm Res. 5 (2): 597-603, (2006). 3. Martinello F, Soares SM, Franco JJ, Santos AC, Sugohara A, Garcia SB, Curti C, Uyemura SA. Food Chem Toxicol. 44(6): 25. Pino JA, Mesa J, Muñoz Y, Martí MP, Marbot R. J. Agric. 810–818, (2006). Food Chem. 2005; 53: 2213-2223, (2005). 4. Paula FS, Kabeya LM, Kanashiro A, Figueiredo A, Azzolini 26. Krishnaiah D, Sarbatly R, Bono A. Biotech. Mol. Biol. Rev. AE, Uyemura SA, Lucisano-Valim YM. Food Chem Toxicol. Vol. 1 (4): 97-104, (2007). 47: 163–170, (2009). 27. Desbois AP, Smith VJ. Appl. Microbiol. Biotechnol. 85:1629– 5. Norhana MN, Azman MN, Poole SE, Deeth HC, Dykes GA. 1642, (2010). Int. J. Food Microbiol. 136: 88–94, (2009). 28. Srihar R, Lakshminarayana G. J. Agric. Food Chem. 41(1): 6. Kobayashi A, Adenan ML, Kajiyama SI, Kanzaki H, Kawazu 61-63, (1993) K. J. Biosciences, 51(3-4): 233-242, (1996). 29. Khanzada SK, Shaikh W, Shahzadi S, Kazi TG, Usmanghani 7. Coutino-Rodriguez R, Cruz-Hernandez P, Gills-Rios H. K, Kabir A, Sheerazi TH. Pak. J. Bot. 40(6): 2553-2559, Arch. Med. Res. 32(4): 251-259, (2001). (2008).

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