phytochemical assay was conducted to establish the chemical profile of “Saye”, a mixture of leaf of Cassia alata, root of Cochlospermum planchonii and whole plant of Phyllantus amarus, used as antimarial remedy. Water and organic extracts were prepared. Characterization of phytoconstituents using specific chemical reagents was performed in tubes, by thin layer chromatography and by high performance liquid chromatography. Steroids and/or triterpenes, cathechic tannins were identified in the decocted and the macerated water extracts of “Saye”. An anthraquinone with a retention time Rt corresponding to 3.34 min was identified by the HPLC analysis.© 2015 International Formulae Group. All rights reserved.Keywords: Chemical profile, anthraquinones, steroids, triterpenes, tannins

Coulibaly, M Traoré

Da, O

Dakuyo, ZP

Kini, F

Koama, B

Matsabisa, MG

Nikiema, JB

Ouédraogo, GA

Ouédraogo, JB

Ouédraogo, JCW

Yaro, B

Yerbanga, RS

English

AJOL - African Journals Online

 Available online at http://www.ifg-dg.org  Int. J. Biol. Chem. Sci. 9(6): 2940-2946, December 2015  ISSN 1997-342X (Online),  ISSN 1991-8631 (Print)    © 2015 International Formulae Group. All rights reserved.                                                             2563-IJBCS DOI : http://dx.doi.org/10.4314/ijbcs.v9i6.33 Original Paper      http://ajol.info/index.php/ijbcs          http://indexmedicus.afro.who.int  Phytochemical screening of Saye, a traditional herbal remedy for malaria  O. DA1,6*, M. TRAORÉ COULIBALY1, J.C.W. OUEDRAOGO2, B. YARO2,  R.S. YERBANGA1, F. KINI2, B. KOAMA1, Z.P. DAKUYO4, J.B NIKIEMA3,  G.A. OUÉDRAOGO6, M.G. MATSABISA5 and J.B. OUÉDRAOGO1  1Direction Régionale de l’Institut de Recherche en Sciences de la Santé (IRSS-DRO/CNRST),  BP 545 Bobo-Dioulasso, Burkina Faso. 2Institut de Recherche en Sciences de la Santé (IRSS), 03 BP 7192 Ouagadougou, Burkina Faso. 3 Direction Générale de la Pharmacie, du Médicament et des Laboratoires (DGPML),  03 BP 7009 Ouagadougou 03, Burkina Faso. 4Pharmacie de la Comoé, Phytofla, BP 293 Banfora, Burkina Faso. 5Indigenous Knowledge System Unit of the Medical Research Council (IKS/MRC), Cape Town, South Africa, PO Box / Posbus 339, Bloemfontein 9300, Republic of South Africa / Republiek van Suid-Afrika 6Université Polytechnique de Bobo-Dioulasso, Laborati e de Recherche en Science de la Santé et Biotechnologie Animale (LARESBA), BP 1091 Bobo-Dioulasso, Burkina Faso. *Corresponding author; E-mail: olobouff@yahoo.fr ;Tel.: +226 70742977  ACKNOWLEDGMENTS  We acknowledge the financial support from UMOEA PACER-2 programme (Programme d'appui et de développement des Centres d'excellence régionaux).     ABSTRACT    A phytochemical assay was conducted to establish the chemical profile of  “Saye”, a mixture of leaf of Cassia alata, root of Cochlospermum planchonii and whole plant of Phyllantus amarus, used as antimarial remedy. Water and organic extracts were prepared. Characterization of phytoconstituents using specific chemical reagents was performed in tubes, by thin layer chromatography and by high performance liquid chromatography. Steroids and/or triterpenes, cathechic tannins were identified in the decocted and themacerated water extracts of “Saye”. An anthraquinone with a retention time Rt corresponding to 3.34 min was identified by the HPLC analysis.  © 2015 International Formulae Group. All rights reserved.  Keywords: Chemical profile, anthraquinones, steroids, triterpenes, tannins.   INTRODUCTION   The African Region continues to shoulder the heaviest malaria burden with 214 million new cases of malaria and 438 000 deaths (World malaria report, 2015). With the resistance of Anopheles mosquito to O. DA et al. / Int. J. Biol. Chem. Sci. 9(6): 2940-2946, 2015   2941insecticides and Plasmodium species to most of the drugs currently in use (White, 2004), there is a call for a return to natural products as antimalarial drugs or as sources of inspiration for the development of novel drugs (Ginsburg et al., 2011). In Burkina Faso, a traditional preparation named “Saye”, manufactured by mixing dried rhizomes of Cochospermum planchonii, leaves of Cassia alata and whole plant of Phyllanthus amarus is used for the treatment of uncomplicated malaria (Dakuyo et al., 2015). Extracts from each individual plant contained in Saye or their 2 x 2 mixture were found to inhibit malaria parasites (Da et al., 2014). In the framework of a clinical study, there was a need of chemical standardisation of “Saye”. The objective of the study was to establish the chemical profile of “Saye” and its content plants.  MATERIALS AND METHODS Plant materials The leaf powder of Cassia alata, the root powder of Cochlospermum planchonii, the whole plant powder of Phyllantus amarus and “Saye”, were all supplied by Phytofla laboratory, with market authorization  number: 0497920050N110000/2011.   Extracts preparation  Crude extracts from individual plant powder (15 g) and “Saye” (15 g) were prepared by maceration during 24 h successively with chloroform (150 mL), methanol (150 mL) and distilled water (150 mL). Organic extracts obtained were dried with an evaporator Buchi R110 type MKE 6540/2. Water extracts from individual plant powder (50 g) and “Saye” (50 g) was also prepared by maceration (24 h) or decoction (10 min) with water (1 L). After filtration, all water extracts were frozen at -20 °C and freeze-dried using a FTS Systems Dura-Dry MPII®, Floor model freeze dryer. Aliquots (1 g) of these extracts were re-dissolved in 30 mL of distilled water, acid hydrolyzed under reflux for 30 min and then extracted (liquid-liquid extraction) with chloroform.  Phytochemical analysis of the extracts by qualitative methods The detection of phytoconstituents such as alkaloids, flavonoids, steroids, tannins and saponins in the extracts was performed in tubes according to Ciulei (1982) method.  Phytochemical analysis of the fractions by thin layer chromatography (TLC)  TLC was carried out on a 60 F254 silica gel plate in glass, Merck (20 cm x 10 cm). Hydrolyzed and non-hydrolysed extracts were spotted on the plate and developed in 2 different eluent systems: (a) chloroform/acetone (20:10), (b) ethyl acetate/formic acid/acetic acid/water (50:5.5:5.5:13). The various spots were visualized by exposure of the plates to Neu, Keddee, Anisaldehyde sulfuric acid, ferric chloride (FeCl3), Liebermann-Burchard, Dragendor’ff and ammonia aqueous solution 25% reagents. A TLC monitoring of the chloroform extract by liquid-liquid extraction was done and the main compounds purified by high performance liquid chromatography (HPLC).   RESULTS  Phytochemical profile of the extracts by the qualitative method  Over all, “Saye” extracts were found to contain esters, glycosides, steroids and /or triterpens, flavonoids, carbohydrates, gallic tannins, carotenoids, saponins, flavonic aglycones, anthocyanosides, cardenolides, anthraquinones and reducing compounds. Emodols were more available in Cassia alata leave extracts. Flavone aglycones were present in Cochlospermum planchonii. Glycosides and anthocyanosides were present in the hydrolyzed phase of the methanolic and aqueous fractions of each plant. Flavonosides as well as cardenolides were present in Cochlospermum planchonii. Anthracenosides were more available in Cassia alata leaves. O. DA et al. / Int. J. Biol. Chem. Sci. 9(6): 2940-2946, 2015   2942Gallic tannins, saponins, reducing compounds and carbohydrates were present in the non-hydrolyzed methanolic fraction and in each plant aqueous extract (Table 1).  Phytochemical profile of the extracts by TLC analysis Steroids and/or triterpenes, cathechic tannins and anthraquinones were identified in the decocted and the macerated water extracts of “Saye”. The DCM/MeOH extract of “Saye” has also shown the presence of steroids and/or triterpenes. Steroids and/or triterpenes were found in the decocted and macerated extracts of each plant whereas anthraquinones and cathechic tannins were only found in Cassia alata leaves and the whole plant of Phyllanthus amarus (Table 2).  Identification of the chemical markers from TLC profiling of the fractions  Three main spots with different Rfs were identified from TLC analysis, one from each plant: Rf = 0.59, (Cochlospermum Planchonii), Rf = 0.75 (Cassia alata) and Rf = 0.90 (Phyllanthus amarus). The compound from Cassia alata purified by HPLC was identified as an anthraquinone with a retention time tR = 3.34 min (Figure 1). The spots (Rf = 0.59 and Rf = 0.90) have the characteristics of steroids and triterpenic compounds.     Figure 1: HPLC chromatogram of chloroform extract. Mobile phase in isocratic mode: acetonitrile-0.1% (v/v) formic acid in water (3:2); Flow rate: 1 mL/min; detection wavelength: λ = 250 nm. O. DA et al. / Int. J. Biol. Chem. Sci. 9(6): 2940-2946, 2015   2943Table 1: Compounds identified in the extracts by qualitative phytochemical screening.   Compounds Tisane Saye  (Decocted extract) Phyllantus amarus  Cassia alata Cochlospermum planchonii Chloroformic extract  Steroids and/or triterpenoids  ++ ++ ++ ++ Flavone Aglycones ND ND ND + Emodols (Anthracenoside and Anthracenoside Aglycone) ++ ND ++ ND Alkaloids ND ND ND  Coumarines and /or coumarin derivatives  ND ND ND ND Carotenoids + + + ++ Hydrolyzed aqueous and methanolic Fraction Glycosides ++ ++ ++ ++ Flavonosides ND ND ND + Anthracenosides ++ ND ++ ND Coumarines derivatives ND ND ND ND Cardenolides ND ND ND + Anthocyanosides + ++ ++ + Non-hydrolyzed aqueous and methanolic fraction Tannins ++ ++ + ++ Saponins ++ + + ++ Alkaloids ND ND ND ND Reducing compound ++ ++ ++ + Carbohydrates (Oses)  + + + ++ Carbohydrate (polyoses) ++ ND ND ++ ND=Not detected; + = present; ++ = more presence. O. DA et al. / Int. J. Biol. Chem. Sci. 9(6): 2940-2946, 2015   2944Table 2:  Chemical profile of Saye established by thin layer chromatography analysis.  Compounds Cassia alata  Phyllanthus Amarus  Cochlospermum planchonii  Tisane saye  DCM/MeOH M d  DCM/MeOH M D  DCM/MeOH m d  DCM/MeOH m d Alkaloid nd Nd nd  nd Nd Nd  nd nd nd  nd nd nd Flavonoid nd Nd nd  nd Nd Nd  nd nd nd  nd nd nd Steroid and/or triterpene ++ + +  ++ + +  + + +  ++ + + Tanin + Nd +  + ++ ++  + nd nd  + + + Anthraquinone + ++ +  nd Nd Nd  nd nd nd  nd ++ ++ Cardenolide nd Nd nd  nd Nd Nd  nd nd nd  nd nd nd nd= not detected; DCM /MeOH = Dichloromethane/Methanol extract; + = present; ++ = more present    O. DA et al. / Int. J. Biol. Chem. Sci. 9(6): 2940-2946, 2015   2945DISCUSSION  The main chemical groups found in “Saye” are tannins, anthraquinones, steroids and/or triterpenes. These chemical compounds, which were also found in the extracts of the individual plant, may be supportive of the antiplasmodial activity of the extracts (Da et al., 2014). In most studies, flavonoids were reported in the three plants: Phyllanthus amarus (Hemant et al., 2013); Cochlospermum planchonii (Nafiu et al., 2011); Cassia alata (Matthew et al., 2013). Previous works have also revealed the presence of tannins in the whole plant of Phyllanthus amarus (Obianime et al., 2009), Cochlospermum planchonii (Ezeja et al., 2010) and in the leaves of Cassia alata (Awomukwu et al., 2015). As in previous studies (Matthew et al., 2013) anthraquinones were found in abundance in the leave extracts of Cassia alata and in “Saye”. Triterpenes were found in Phyllanthus amarus (Hemant et al., 2013), Cochospermum planchonii (Nafiu et al., 2011) and Cassia alata (Kayembe et al., 2012). Steroids were present in Phyllanthus amarus (Hemant et al., 2013), Cochospermum planchonii (Nafiu et al., 2011) and Cassia alata (Matthew et al., 2013). Cochloxanthine and dihydrocochloxanthine were previously identified in Cochlospermum species and found to have an antimalarial activity with an IC50 estimated to 1 µg/ml (Benoit-Vical et al., 2001).  Conclusion This study showed that the main chemical components in “Saye” are tannins, anthraquinones, steroids and/or triterpenes. The results allowed establishing in part the chemical profile of “Saye” that will be used for to its quality control during the clinical study. Taking into account all the results from prior works, Cochloxanthine and dihydrocochloxanthine anthraquinones, flavonoids and kaempferol might be the target chemical compounds for the choice of the chemical marker of “Saye”.    COMPETING INTERESTS  The other authors declare that they have no competing interests.  AUTHORS’ CONTRIBUTIONS The study was conceived by MTC, JBO, JBN, JCWO, FK and MGM.  It was run by IRSS with contributions from OD, RSY, BY, JCWO, FK and BK. IRSS Bobo and IRSS Ouaga: chemical analysis. UPB: academic training of OD under supervision of MTC and GAO. Phytofla laboratory: provide plant materials. The paper was drafted by OD and supervised by JCWO, MTC and GAO. OD, RSY, JBO, MTC and JCWO participated in the overall running of the project. “Saye” and plants samples were provided by ZPD. All the authors have read and approved the final manuscript.  REFERENCES  Awomukwu DA, Nyananyo BL, Ikpeama AI, Adieze CU. 2015. Comparative chemical constituents of some Cassia species and their pharmacognistic importance in South Eastern Nigeria. Science Journal of Chemistry, 3(3): 40-49. Doi: 10.11648/j.sjc.20150303.12 ISSN: 2330-0981. Benoit-Vical F, Valentin A, Mallie M, Bessiere JM. 2001. Antiplasmodial activity of Cochlospermum planchonii and C. tinctorium tubercle essential oils. Journal of Essential Oil Research, 13(1): 65-67. DOI: 10.1080/10412905.2001. 9699609 Ciulei I. 1982. Methodology of Analysis of Vegetal Drug. Pratical Manual on Industrial Utilization of Medicinal and Aromatic Plants. Bucharest (Faculty of pharmacy), 67 p. Da O, Traore/Coulibaly M, Yerbanga RS, Koama B, Ouedraogo N, Tamboura S,. Dakuyo ZP, Sekhoacha MP, Nikiema JB, Ouedraogo GA, Matsabisa MG, Ouedraogo JB. 2014. Antiplasmodial and Antioxidant Activities of Saye: A Traditional Herbal Remedy for Malaria. American Journal of Biochemistry and O. DA et al. / Int. J. Biol. Chem. Sci. 9(6): 2940-2946, 2015   2946Molecular Biology, 4: 155-166. DOI: 10.3923/ajbmb.2014.155.166 Dakuyo Z, Meda AL, Da O, Kiendrebeogo M, Traore-Coulibaly M, Novak J, Benoit-Vical F, Weisbord E, Willcox M. 2015.: SAYE: The Story of an Antimalarial Phytomedicine from Burkina Faso. JAltern Complement Med., 21:187-195. DOI: 10.1089/acm.2014.0147 Ezeja MI,  Anaga AO. 2010. Anti-diarrhoeal Activities of the Methanolic Root Bark extract of Cochlospermum planchonii (Hook f). 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Phytochemical screening of Saye, a traditional herbal remedy for malaria

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Phytochemical screening of Saye, a traditional herbal remedy for malaria

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