Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( 3 ), 31 - 35 , March (201 3 ) Res.J. Recent Sci. International Science C ongress Association 31 Physical characteristics and Chemical compositions of Leaves extracts of Sorindeia grandifolia Engl. (Anacardiaceae) harvested at Kato , Benin Noudogbessi J.P. 1 , Alitonou G.A. 1 , Avlessi F. 1 , Figueredo G. 2 , Chalard P. 3 , Chalchat J.C. 4 , Sohounhloué D.C.K. 1  1 Unité de Recherche sur les Extraits Végétaux, Laboratoire d’Etude et de Recherche en Chimie appliquée (LERCA), Ecole Polytech nique d’Abomey - Calavi, Université d’Abomey - Calavi, 01 BP 2009 Cotonou, Rép. du BENIN 2 Laboratoire d’Analyse des Extraits Végétaux et des Arômes (LEXVA Analytique), 460 Rue du Montant, 63110 Beaumont, FRANCE 3 Ecole Nationale Supérieure de Chimie, Institut de Chimie de Clermont - Ferrand, Université Blaise - Pascal, F - 63000, Clermont , FERRAND 4 Laboratoire de Chimie des Huiles Essentielles , Université Blaise - Pascal, Clermont - Ferrand II, Campus des Cézeaux, Aubière cedex, FRANCE Available online at: www.isca.in Received 7 th September 2012 , revised 14 th October 2012 , accepted 18 th November 2012 Abstract The results brought back at the end of this work concern various chemical constituents of S. grandifolia leaves collected in Benin. The major compounds (  4%) of essential oils obtained after hydrodistillation and analysis by coupling gas chromatography wi th spectrometry mass are constituted of : limonene (20.2 %), ( E) - b - ocimene (7.6 - 17.8 %), ( Z) - b - ocimene (11.4 %), g - cadinene (7 . 0 %), s elin - 11 - en - 4 - a - ol (4.5 - 5.7% ), palmitic acid (4.3 - 6.2% ), b - pinene (5.5 %), α - selinene (5.5%), a - phellandrene (5.2% ), β - selinene (4.9%), β - elemene (4.5%) .The lipidic fractions realized from petroleum ether extracts are marked by importants rates of arachidic acid (22.5 %), palmitic acid (20.3 %) and of linoleic acid (15.9 %). The phytochemical analysis showed relatively a large content of coumarins, gallic tannins , flavones , leucoanthocyans and saponins in opposition to the other secondary metabolites . Keywords : limonene , arachidic acid , metabolites , coumarins Introduction Sin ce ancient times , plant extracts , in particular aromatic , are sought for their bioactive properties . These plants are frequently used in the traditional pharmacopoeia in Benin for the therapeutic purposes in particular to relieve certain cutaneous affectio ns, respiratory disorders, gastrointestinal diseases and cardiovascular etc. It is also attributed to the aromatic plants extracts likely biological properties of potential applications in modern medicine, food industry , perfumery and cosmetics : antiradica l and anti - inflammatory powers 1 . Sorindeia grandifolia is an aromatic species of anacardiaceae family met in wooded savannas, dry forests, dense forests and humid regions of Western and Central Africa, situated in the North of the equator. This shrub , lia nescent , bears leaves and supporting leaflets oblong and lanceolate, a pale green calyx, petal pink, white or pink flowers and fruits of ellipsoidal shape at maturity 2 . In Benin , the decoction of S. grandifolia leaves, consumed in the fresh state, treats t he cough while its juice possesses antipyretic properties 3 . To date , the literature has not reported at least one bioactive chemical compounds in this plant whose therapeutic performance in traditional medicine has always proven . This work has for objectiv e the valorization of this species ( Sorindeia grandifolia Engl., Anacardiaceae) from Benin by the extraction , the identification and the characterization of bioactive compounds it contains. The purpose of this study is to determine , through appropriate ana lytical methods , the physicochemical characteristics of this plant leaf extracts . Material and Methods Plant material and distillation of the volatile constituents : Sorindeia grandifolia leaves were collected in June and July 2007 in the classified forest of Lama ( Benin). They were authenticated at the National Herbarium of the University of Abomey - Calavi . In the laboratory , these leaves are kept between 18 and 20°C in the shade of sunlight throughout the study period . Essential oil is extracted from leave s (450g) by the technique of hydrodistillation during five hours on Clevenger, according to the method used in british pharmacopoeia 4 . They was dried over anhydrous sodium sulfate and analyzed by GC/FID and GC /MS. For the determination of non - volatile comp ounds , powders were obtained from the leaves , dried in the dark for one month , by grinding with a knives machine Ika Werke MF 10 basic type . Vegetable powders collected are then sieved in the size grading 0.425 . Physical properties : Physical parameters of the essential oil of S. grandifolia leaves were determined using the methods described by AFNOR 5,6 . Density at 20°C : The density measure was carried out using a micro - pycnometer and a precision balance . Refractive index at 20°C : The refractive index wa s determined by means of the refractometer CARL ZEISS JENA 234678. Rotatory power at 20°C : The measurement was made by Carl Zeiss polarimeter 128291 . Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( 3 ), 31 - 35 , March (201 3 ) Res. J. Recent Sci. International Science Congress Association 32 Phytochemical analysis : Analysis of the volatile constituents : GC/FID: The extracts were analysed on a He wlett - Packard gas chromatograph Model 6890, equiped with a DB5 MS column (30 m x 0.25 mm, 0.25 µm), programming from 50°C (5 min) to 300°C at 5°C/min, 5 min hold. Hydrogen as carrier gas (1.0 mL/min); injection in split mode (1:60); injector and detector t emperature: 280 and 300°C respectively. Each extract is diluted in hexane: 1/30. GC/MS : The extracts compositions were analysed on a Hewlett - Packard gas chromatograph Model 5890, coupled to a Hewlett - Packad MS model 5871, equipped with a DB5 MS column (3 0 m x 0.25 mm, 0.25 µm), programming from 50°C (5 min) to 300°C at 5°C/min, 5 min hold. Helium as carrier gas (1.0 mL/min); injection in split mode (1:30); injector and detector temperature, 250 and 280°C respectively. The MS working in electron impact mod e at 70 eV; electronmultiplier: 2500 eV; ion source temperature: 180°C; mass spectra data were acquired in the scan mode in m/z range 33 - 450. The compounds assayed by GC in the different essential oils were identified by comparing their retention indices with those of reference compounds in the literature and confirmed by GC - MS by comparison of their mass spectra with those of reference substances 7,8,9 . Analysis of non - volatile constituents of leaves of S. Grandifolia : The phytochemical screening was made according to the standard techniques described by Paris and Moyse 10 , Bouquet 11 and Debray et al . 12 . Coumarins : Infused at a 10 % made from the plant powder was examined under the UV light (365 nm ). The appearance of a bluish fluorescence indicated a pos itive reaction . Tannins gallic : An aqueous infusion was prepared from 5 g of plant powder and 100 mL of boiling distilled water . After 15 min , the mixture was filtered . The residue was rinsed with hot water to bring the volume of the filtrate to 100 mL. 2 0 mL of the filtrate are saturated with sprayed sodium sulfate and then, it was added dropwise 1 mL of ferric chloride (1%).The development of a blue - black tint corresponded to the presence of gallic tannins , not precipitated by Stiasny 's reagent. Flavone s : It was introduced in a test tube, 5 mL of infused. In this content, 5 mL of hydrochloric alcohol constituted by equal volumes of ethanol at 95° , distilled water , concentrated hydrochloric acid (37% ) and 1 mL of isoamylic alcohol were added. In the prese nce of shavings magnesium, it emerged at the supernatant layer ( layer isoamyl alcohol ) a pink - orangy color indicating the presence of genins of flavonoids . Leucoanthocyans : They were identified by introducing into a test tube 5 mL of infused (5 %) and 5 mL of hydrochloric ( ethanol 95 ° + distilled water + hydrochloric acid 37% of equal volumes). The mixture was compete with 1 mL of isoamylic alcohol and then, heated to 90° through a water bath . After fifteen minutes, it had developed a red - cerise tint (or p urple) indicating the presence of leucoanthocyans. Saponins : A decoction was prepared during 30 min from two grams of plant powder and 100 mL of distilled water. After filtered the obtained mixture, the filtrate was divided into 10 different volumes (1 mL , 2 mL, 3 mL, 10 mL) in 10 calibrated tubes (internal diameter :1.3 cm). The content of each tube was adjusted to 10 mL with distilled water . After shaking each tube in a horizontal position for 15 seconds , followed by a rest of 15 min in an upright positi on , the height of the foam supernatant was measured in cm . When this height is close to 1 cm in the X th tube , the foam index ( I) is calculated by the following formula : I = Foam height ( in cm) in the X th tube × 5/0 . 0X . The presence of saponins in the pla nt is confirmed when the value of the foam index is greater than 100 . Fatty acids (FA) and unsaponifiable (Un) : Extraction of lipids : 15.0 g of the vegetable material powder were twice extracted successively by 100 mL of petroleum ether (40 - 60°C) with mag netic stirring at room temperature. After filtration and evaporation of the solvent under reduced pressure , the extracts were dried and weighed. The yields were established in calculating the average of three extractions . Saponification and fatty acids ob tention : The saponification was conducted by refluxing , during thirty minutes past one o'clock , 0.5 g of plant extract and 25 mL of an ethanolic and potassium hydroxide solution (2N). After cooling , there was added 50 mL of water and the unsaponifiable mat ter are extracted by 3 × 50 mL of cyclohexane . The soap solution produced was then acidified to precipitate the F A (5 ï‚£ pH ï‚£ 6) . The F A released were yet extracted by 3 × 50 mL of diethyl ether 13,14 . Methylation of FA : Fatty acids were converted to their me thylic esters by addition a methanolic solution ( 10%) of boron trifluoride ( BF 3 ) and the methylic esters we re extracted with cyclohexane. The analysis of FA and Un collected was made through GC/ FID and GC/ MS 15 . Results and Discussion The measurement of d ensity, refractive index and rotatory power of the essential oils samples of S. grandifolia leaves collected at different dates to Kato ( the Lama botanical forest reserve of Benin ) gave the values included in the table 1 . Table - 1 Physical characteristics of the essential oils of Sorindeia grandifolia Density (20°C) Refractive index (20°C) rotatory power (20°C) L 1 0.917 1.4935 - 7.996 L 2 0.921 1.4932 5.000 L 1 = Leaves (07 - 06 - 07) ; L 2 = Leaves (07 - 07 - 07) Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( 3 ), 31 - 35 , March (201 3 ) Res. J. Recent Sci. International Science Congress Association 33 The values determined for the density and the ro tatory power conversely varies to t hose of the refractive index after a month of harvest interval of L 1 and L 2 . According to these values , there is a slight difference between the densities of essentials oils extracted from S. grandifolia leaves . Between t he values of refractive indexes, this difference is more weak. By cons , the difference between the values of the rotatory power is very pronounced . These differences noted could be bound to several factors, in particular, edaphic, climatic as well as the cultural practices 16,17, 18 . I t is important to appreciate in the future such homogeneity over several mon ths . S. grandifolia is a little fragrant species. The yields in essential oil of the leaves of this plant are low (1.98x10 - 2 % to 2. 17x10 - 2 %). The values of these yields are seventeen for eighteen times lower than the value of the yield in essential oil of Diplolophium africanum stem - foliage reported by Koudoro et al . in 2011 19 . On the other hand, the S. grandifolia leaves seem richer in volatile extracts contrary to those of Anona senegalensis (1.4x10 - 2 %) investigated by Noudogbessi et al. in 2011 20 . The volatile compounds identified in the essential oils collected by hydrodistillation of leaves are presented in t able 2 . GC - FID and GC - MS analysis of S. grandifolia essential oil revealed 40 compounds in the sample L 1 and 46 in that of L 2 representing respectively 96.9 and 94.1% of the total weight of the essential oil ( t able 2). The main families volatile of compounds which marking L 1 and L 2 are the hydrogenated monoterpenes (31.3 to 57.9 %), hydrogenated sesquiterpenes (15.3 % to 41.5 %) and oxygenated sesquiterpenes (11.7 to 13.6 %). The proportions by oxygenated monoterpenes no neighborhood that 3.5 % in L 1 and 2.3 % in L 2 . The main constituents (  4%) susceptible to generate a bioactive character in L 1 are ( Z) - b - ocimene (11.4 %), (E) - b - ocimene (7.6 %), g - c adinene (7.0 %), s elin - 11 - en - 4 - a - ol (5.7% ), palmitic acid (6.2% ), a - selinene (5.5 %), a - phellandrene (5.2% ), b - selinene (4.9% ) and b - elemene (4.5% ). The major compounds (  4 %) of L 2 volatile extract were constitued of limonene (20.2 %), (E) - b - ocimene (17.8 %) , b - pinene (5.5 %), selin - 11 - en - 4 - a - ol (4.5 %) and palmitic acid (4.3% ). Further phytochemical investigations on various extracts realized with appropriate solvents allowed indicate the presence in S. grandifolia leaves of other metabolites, proven pharmacol ogical properties 21 . This is coumarin, gallic tannins , flavones, leucoanthocyans and saponins ( t able 3) . The presence of saponins , assessed by calculating the foam index ( 83.33), would explain the plant high aphrogene power . It was also noticed traces of carotenoids , catechic tannins and free quinone . The lipid content (specifically FA ) obtained by quantitation after extraction with petroleum ether was estimated at 0.7 % compared to the mass of plant material used . FA identified by gas chromatography cou pled with spectrometry mass are predominantly saturated (44.2%) . As for the unsaturated FA , they represent only 15.9 %. The major FA identified in S. grandifolia leaves are arachidic acid (22.5 %), palmitic acid (20.3 %), linoleic acid (15.9 %), followed by tr aces of myristic acid (1.4%) . The investigations led on the unsaponifiables did not reveal an interesting chemical composition. Indeed the compounds constituting the footsteps of Un collected could not be identified by GC/ MS. Conclusion The chromatographi c studies of S. grandifolia leaves extracts showed the presence of high proportions of monoterpenes and sesquiterpenes hydrogenated in essential oils and FA often found in the vegetable kingdom . The phytochemical screening , performed on the basis of specif ic tests allowed to characterize in S. grandifolia leaves: coumarins, gallic tannins, flavones and leucoanthocyans. The phytochemical heritage of the S. grandifolia plant, omen , because it is referenced by traditional medicine in Benin , a useful contribut ion in the development of the vegetable biotechnology . These preliminary results will be used to support a rigorous exploration of pharmacological properties of the endemic species S. grandifolia of Benin . References 1. Alitonou G.A., Huiles essentielles ext raites de plantes aromatiques acclimatées au Bénin : étude chimique, évaluation biologique et applications potentielles, Thèse de Doctorat en cotutelle, UAC et Univ. Montpellier II, (2006) 2. Akoegninou A., Van Der Burg W.J., Van Der Maesen L.J.G. Flore Analytique du Bénin , (2006) 3. Adjanohoun E.J., Médecine traditionnelle et Pharmacopée, Contribution aux études ethnobotaniques et floristiques en République Populaire du Bénin, ACCT, Paris (1989) 4. British Pharmacopoeia, 11. P. A. HMSO: London, (1980) 5. AF NOR, NF T 75 - 006, Les huiles essentielles - vocabulaire - 1ere liste, (1998) 6. AFNOR, Recueil de normes : les huiles essentielles, Tome 2, Monographies relatives aux huiles essentielles, Paris, 661 - 663 (2000) 7. Rösch P., Popp J., Kiefer W., Raman and SERS Inve stigations on Lamiaceae, J. Mol. Struct. , 121 , 480 - 481 (1999) 8. Adams R.P., Identification of essential oils by ion trap mass spectrometry, Academy Press , Inc, New - York, (1989) 9. Swigar A.A., Silverstein R.M., Monoterpènes, Infrared, Mass, NMR Spectra and Kov ats Indices, Aldrich Chem. Co. Milwaukee , WI, USA, (1981) 10. Paris R., Moyse H., Précis de matière médicinale, Masson, Paris, (1969) 11. Bouquet M., Travaux et documents de l’Orstom, Paris, 13, (1971) Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( 3 ), 31 - 35 , March (201 3 ) Res. J. Recent Sci. International Science Congress Association 34 Table - 2 Chemical compositions of essential oils from the leav es of S. grandifolia Compounds KI exp KI th (%) L 1 L 2 trans - hex - 2 - enal 848 846 0.4 1.1 trans - hex - 2 - en - 1 - ol 858 854 - 0.6 hexan - 1 - ol 867 863 - 0.3 tricyclene 925 921 2.5 - a - pinene 936 932 - 2.5 b - pinene 978 974 3.3 5.5 myrcene 991 988 0.5 1.2 met a - mentha - 1(7),8 - diene 1002 1000 - 0.3 a - phellandrene 1004 1002 5.2 2.7 p - cymene 1022 1020 - 3.2 o - cymene 1026 1022 - 0.5 limonene 1029 1024 0.4 20.2 (Z) - b - ocimene 1037 1032 11.4 0.4 (E) - b - ocimene 1046 1044 7.6 17.8 terpinolene 1090 1086 - 3.1 p - cym enene 1092 1089 - 0.5 linalool 1098 1095 1.7 0.4 p - cymen - 8 - ol 1184 1179 - 0.8 a - terpineol 1191 1186 1.4 1.1 geraniol 1254 1249 0.4 - trans - verbenyl acetate 1295 1291 - 0.3 a - longipinene 1354 1350 1.3 - a - ylangene 1377 1373 1.3 - a - copaene 1379 1974 1.1 0.5 b - elemene 1394 1389 4.5 2.2 b - longipinene 1405 1400 1.8 0.2 b - caryophyllene 1421 1417 3.2 1.7 cis - thujopsene 1432 1429 1.3 0.3 b - copaene 1433 1430 - 0.2 selina - 4(15),6 - diene 1447 - 1.6 - a - humulene 1454 1452 1.4 t selina - 4,11 - diene 1471 - 2.0 1.1 g - muurolene 1480 1478 0.7 0.5 b - selinene 1492 1489 4.9 3.6 a - selinene 1501 1498 5.5 2.7 a - bulnesene 1512 1509 1.2 0.5 g - cadinene 1515 1513 7.0 1.1 d - cadinene 1524 1522 1.5 0.7 trans - calamenene 1530 1528 1.2 - spathulenol 1579 1577 0.5 0.5 caryophyllene oxide 1584 1582 1.2 1.1 globulol 1593 1590 - 0.3 humulene epoxide II 1611 1608 0.7 0.6 1,10 - di - epi - cubenol 1622 1618 0.5 0.2 citronellyl pentanoate 1628 1624 - 0.3 1 - epi - cubenol 1632 1627 - 0.2 epi - a - cubenol 1640 - - 1.1 epi - a - muurolol 1645 1640 1.1 - Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( 3 ), 31 - 35 , March (201 3 ) Res. J. Recent Sci. International Science Congress Association 35 a - muurolol 1649 1644 0.5 - cubenol 1651 1645 - 0.4 a - cadinol 1658 1652 1.9 1.7 selin - 11 - en - 4 - a - ol 1664 1658 5.7 4.5 b - sinensal 1704 1699 - 0.6 (2Z, 6E) - farnesol 1718 1722 1.0 0.5 iso - longifolol 1723 1728 0.5 - palmitic acid 1967 19 59 6.2 4.3 oleic acid 2148 2141 0.8 - Monoterpene hydrocarbons 31.3 57.9 Oxygenated monoterpenes 3.5 2.3 Sesquiterpene hydrocarbons 41.5 15.3 Oxygenated sesquiterpenes 13.6 11.7 Total 96.9 94.1 L 1 = Leaves (07 - 06 - 07) ; L 2 = Leaves (07 - 07 - 07) T able - 3 Chemical families identified in the leaves of Sorindeia grandifolia Coumarin Gallic tannins Flavones Leucoant hocyanes Saponins (foam index) +++ +++ +++ +++ ++ (83.33) +++ : Abundant ; ++ : Average 12. Debray M., Jacquemin H., Razafindrambo R., Tr avaux et documents de l’Orstom, Paris, 8, (1971) 13. Joulain D., Konig W., the Atlas of Spectral Data of sesquiterpene hydrocarbons , E.B. – Verlag, Hamburg, Germany, (1998 ) 14. Wolff J.P., Manuel d'Analyse des Corps Gras , Azoulay, Paris, (1968) 15. Metcalfe L.D., Schm itz A.A., Rapid preparation of fatty acid esters for gas chromatography analysis , Anal. Chem ., 33, 363 - 364 (1961) 16. Demarne F . E. , Le géranium rosat , Parfums, Cosmétiques et Arômes, n°62 (1985) 17. Gilly G. , Les plantes à parfum et huiles essentielles à Grasse , L‘Harmattan, Paris 11 - 19 (1997) 18. Lis - Balchin M. , Geranium and pelargonium: the genera Geranium and Pelargonium , CRC Press, Taylor & Francis, London, 116 - 131, 147 - 165, 184 - 217, (2002) 19. Koudoro Y.A., Alitonou G.A., Sossou D.J., Yehouenou B., Avlessi F., Menut C., Sohounhloué D., Chemical Composition and biological Activities of Essential Oil from Benin Diplolophium africanum Turcz stem leaves, J. Soc. Ouest - Afr. Chim ., 032; 1 – 8 (2011) 20. Noudogbessi J.P., Natta A.K., Avlessi F., Sohounhloué D.C.K., Figueredo G. , Chalchat J.C., Chemical Composition of the Essential Oils Extracted from Two Annonaceae Required in Beninese Pharmacopeia, Australian Journal of Basic and Applied Sciences, 5(2) , 34 - 40 (2011) 21. Bruneton J., Pharmacognosie, Phytochimie, Plantes médicinal es, éd. 3, Techniques et Documentation, Lavoisier, Paris, (1999)