Asian Journal of Chemical Sciences 6(1): 1-5, 2019; Article no.AJOCS.48643 ISSN: 2456-7795 Phaeophytin and Triterpenoids from Brachystelma togoense Schltr, a Nigerian Medicinal Herb Abiche Ekalu1,2* Rachael Gbekele-Oluwa Ayo2, James D. Habila2 and Ibrahim Hamisu2 1 Nigerian Army School of Education, Ilorin, Kwara, Nigeria. 2 Ahmadu Bello University, Zaria, Kaduna, Nigeria. Authors’ contributions This work was carried out in collaboration among all authors. Author AE designed the study, performed the experimental study and wrote the protocol and the first draft of the manuscript. Authors RGOA, JDH and IH supervised the work. All authors read and approved the final manuscript. Article Information DOI: 10.9734/AJOCS/2019/v6i118990 Editor(s): (1) Dr. V. Sivamurugan, PG & Research Department of Chemistry, Pachaiyappa’s College, University of Madras, Chennai, India. Reviewers: (1) Dr. Phillip Minnaar, Agricultural Research Council, South Africa. (2) Francisco José Queiroz Monte, Universidade Federal do Ceará, Brasil. (3) Marcelo Barcellos da Rosa, Federal University of Santa Maria, Brazil. Complete Peer review History: http://www.sdiarticle3.com/review-history/48643 Original Research Article Received 31 January 2019 Accepted 27 April 2019 Published 07 May 2019 ABSTRACT The medicinal herb Brachystelma togoense schtlr (Apocynaceae) is used traditionally for treatment of ailments. The secondary metabolites, phaeophytin a, α-amyrin and lupeol were isolated from the 1 CH2Cl2 and MeOH extracts of Brachystelma togoense. The structures were elucidated using H, 13 C and 2D NMR. These phytochemicals have previously being reported to have various biological activities such as anti-inflammatory, anti-fungal and anti-cancer. The presence of phaeophytin a, αamyrin and lupeol in Brachystelma togoense justified the use of the plant for medicinal purpose in Nigeria. Keywords: Secondary metabolites; phaeophytin a; α-amyrin; lupeol; Brachystelma togoense schtlr. _____________________________________________________________________________________________________ *Corresponding author: E-mail: ekalumiracle@gmail.com; Ekalu et al.; AJOCS, 6(1): 1-5, 2019; Article no.AJOCS.48643 1. INTRODUCTION and to 100% EtOH/Ac to yield various fractions (fr. 1-100). Fr.20 was spotted on the TLC plate using 100% CH2Cl2 and appeared a pure compound 1 (51.0 mg). The same procedure was repeated for the MeOH extract yielding compounds 2 (32.0 mg) and 3 (28.0 mg) which were spotted as pure compounds using CH2Cl2 /EtOH/Ac (7:3) from fr.30. Brachystelma was first described by Robert Brown in 1822. The genus Brachystelma R. Br. (Apocynaceae: Asclepiadoideae) is represented by about 100-120 species [1]. It is an erect perennial herb, growing up to 30 cm high. The genus Brachystelma is chiefly distributed in South Africa, South-East Asia and Australasia [2]. A total of 18 species are known in India [3] and out of them, 3 species in Maharashtra. Brachystelma is found from Ghana to Nigeria, in lowlands to montane areas [4]. The raw tuber is said to be edible [4]. Many of the tuberous Brachystelma are known to be used medicinally for the treatment of headache, stomachache and colds in children [5]. Brachystelma togoense has being medicinally used for the treatment of dysentery, cough and cold, wounds, stomach ache, typhoid and erectile dysfunction. 2. MATERIALS AND METHODS 2.1 Collection The aerial parts of Brachystelma togoense was collected during April 2018 from the Ugbokolo forest in Okpokwu local government area of Benue State-Nigeria. The plant was collect and stored in a plastic container before it was airdried. The collected specimen was positively identified by Mr. Namadi Sanusi, a botanist at Ahmadu Bello University, Zaria as Brachystelma togoense. A specimen (no. 25856) had been retained at the Department of Biological Sciences, Ahmadu Bello University, Zaria-Nigeria (Fig. 1). Fig. 1. Brachystelma togoense in its natural habitat [16] 2.3 General Experimental Procedure NMR spectra were recorded in CDCl3 on a 400MHz or 500 MHz Bruker AVANCE III NMR instrument at room temperature. HREIMS were recorded on an Agilent Technologies 6550 iFunnel Q-TOF LC/MS with samples dissolved in CH2Cl2. Infrared spectra were recorded using a Perkin-Elmar (2000 FTIR) spectrometer on NaCl plates. 2.2 Extraction and Isolation The air-dried B. togoense was manually reduced to powder using mortar and pestil. Exactly 1000 g of the powdered plant material was extracted on a shaker at room temperature using 100% dichloromethane (CH2Cl2) for 72 h. The extracts were concentrated using a rotary evaporator at 40˚C resulting in a brown gum-like texture (32 g). The same procedure was used for methanol (MeOH) which yielded a brown gum-like texture (36 g). The CH2Cl2 and MeOH extracts were separated by flash chromatography (Biotage system) over silica gel using three solvents. Firstly, a hexane/ CH2Cl2, gradient starting with 100% hexane and gradually increasing the polarity to 100% CH2Cl2. Secondly, CH2Cl2/ EtOH/Ac from a 100% CH2Cl2 to 50% EtOH/Ac 3. RESULTS AND DISCUSSION The following following compounds phaophytin a (51.0 mg; 0.16%), α-amyrin (32.0 mg; 0.10%) and lupeol (28.0 mg; 0.09%) were isolated from Brachystelma togoense using flash chromatography (biotage system). These compounds (Fig. 2) were elucidated based on comparison of previous data [6–8]. Phaeophytin-a was isolated as a dark green solid from the CH2Cl2 extract of the aerial parts of B. 2 Ekalu et al.; AJOCS, 6(1): 1-5, 2019; Article no.AJOCS.48643 Fig. 2. Structures of isolated compounds 1-3 from B. togoense schtlr 1. Phaeophytin a; 2. α-Amyrin; 3. Lupeol spectra for compound 1 were assigned using HSQC and HMBC as given in Table S1. togoense that was previously described [6]. The IR spectrum showed absorbance bands for vinyl proton (3056 cm-1) and sp3 CH (2987, 2932 cm-1) -1 and carbonyl (1736 cm ) groups. A molecular ion could not be seen in the HRMS spectrometer despite repeated attempts. Amyrin (α) was isolated as a brown solid from the CH2Cl2 extract of the aerial parts of B. togoense, which had been isolated previously from the methanol extract of Sacoglottis uchi [7]. The IR spectrum showed absorbance bands for -1 3 -1 hydroxyl (3055 cm ) and sp CH (2987 cm ) in conjugation and unsymmetrical ethylenic double -1 -1 bond (1733 cm ) and olefinic carbon (1422 cm ) groups. From the 1H and 13C NMR spectra, it was evident that phaeophytin-a belonged to the phaeophytin class. This was particularly evident by the downfield shifts at δH 9.32 s, 9.48 s and 8.56 s which could be assigned as H-5, H-10 and H-20 respectively. The deshielded methyl groups proton resonances occurred at δH 3.19 (3H-2’), δH 3.3 (3H-7’) and δH 3.38 (3H-12’) and a methoxy group proton resonance occurred at δH 3.89 (3H-134). The presence of a C-20 phytol tail was evident from the presence of four methyl protons (δH 0.80 d, J = 7.3, δH 0.82 d, J = 7.3, δH 0.79 s, δH 1.61 s) and ester carbonyl resonance 3 at δC 173.8 (C-13 ). A comparison of the NMR data of phaeophytin-a against literature values for phaeophytin a showed the enabled assignment of a keto group carbon resonances at δC 189.9 to C-131 [6,9]. The 1H and 13C NMR The molecular ion was not observed in the HRMS spectrum, however 30 carbons could be 13 counted in the C NMR spectrum, indicating the compound was a triterpenoid. 1 13 The H and C NMR spectra (spectrum 2.2 and 2.3) showed the presence of one trisubstituted double bond. A hydroxyl group was placed on C3 confirmed by the C-3 (δC 79.3) resonance correlating with both the 3H-23 (δH 0.99 s), 3H24 (δH 0.78 s) and H-5 (δH 0.73 d, J = 11.5) resonances. A further singlet (δH 0.79, 0.93, 3 Ekalu et al.; AJOCS, 6(1): 1-5, 2019; Article no.AJOCS.48643 0.99, 0.78 and 1.24) and two doublet (δH 0.86 d, J= 6.2 and δH 0.95 d, J= 6.2) methyl group proton resonances were present and the typical 12-olaenene double bond (δH 5.25, δC 126.1, δC 138.2) was seen. A comparison against literature data [7] confirmed that this compound was αamyrin which has been isolated previously from the stem bark of Sacoglottis uchi (Humiriaceae) [7]. had been reported for lupeol [15]. Ref [16] gives the picture of B.togoense in its natural habitat. 4. CONCLUSION Phaeophytin a, α-amyrin and lupeol are reported here for the first time from B. togoense. This was also the first report of the phytochemical quantification in B. togoense in Nigeria. However, these secondary metabolites, i.e phaeophytin a, α-amyrin and lupeol were reported previously to show various biological activities. Therefore, the results of chemical compound analysis of B. togoense justified the ethnomedicinal uses of this plant in Nigeria. The configuration of the hydroxyl group at C-3 was confirmed as β by the coupling constant of H-3 (J = 5.1, 11.3 Hz). The configurations at the chiral centres were confirmed using the NOESY spectrum. The 1H and 13C NMR spectra for compound 2 were assigned using HSQC and HMBC as given in Table S2. ACKNOWLEDGMENTS Lupeol was isolated as a brown solid from the MeOH extract of the aerial parts of B. togoense which had been isolated previously from the hexane extract of Magnolia salicifilia [10] as well as synthesised [8]. The IR spectrum showed an -1 absorbance band for hydroxyl (3363 cm ). The molecular ion was no seen in the HRMS spectrum, however 30 carbons could be counted in the 13C NMR spectrum indicating the compound was a triterpenoid. The author wishes to thank the Natural Product Research Group, University of Surrey, UK for the opportunity to carry out my research work using their laboratory, Chemicals and Instruments. The NMR spectra of lupeol showed the presence of an iso-propenyl group typical of the lupenetype of pentacyclic triterpenoids. Coupled 2H-29 methylene protons (δH 4.69 d, J = 2.1, δH 4.57 d, J = 2.4) and 13C NMR resonances (δC 105.9, δC 151.2, δC 19.5) could be assigned to two H-29 and C-29, C-20 and C-30 respectively [11]. REFERENCES COMPETING INTERESTS Authors have interests exist. 1. 2. Compound 3 was identified as the known 3βhydroxylup-20(29)-ene, commonly referred to as lupeol. A literature search revealed that the 13C NMR chemical shifts were similar to those of lupeol. 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