Dried stem bark powder of Leptadania hastata was subjected to
maceration with methanol to afford crude methanol extract, which was
partitioned with n-hexane, ethylacetate, chloroform and n- butanol to
afford different their respective fractions. Extensive phytochemical
screening of the n-hexane fraction using column chromatography resulted
to the isolation of a white solid substance. The substance was
identified as of lupeol using IR, 1D \u2013 NMR, 2D \u2013 NMR data
and by comparison with reference spectral data

ACHIKA, JI

ATINGA, V

BILA, AH

HASSAN, HS

MAILAFIYA, MM

MUSA, TL

PATEH, UU

SULE, MI

Bioline International

 *Corresponding Author Email: mariamailafiya@gmail.com; Tel: +2348030971798 PRINT ISSN 1119-8362 Electronic ISSN 1119-8362    J. Appl. Sci. Environ. Manage.  Vol. 24 (10) 1835-1838 October 2020 Full-text Available Online at https://www.ajol.info/index.php/jasem http://ww.bioline.org.br/ja Isolation of Lupeol from the Stem Bark of Leptadenia hastata (Pers.) Decne  *1MAILAFIYA, MM; 2PATEH, UU; 2HASSAN, HS; 2SULE, MI; 2BILA, AH; 1MUSA TL, 1ATINGA, V; 3ACHIKA, JI  *1Department of Pharmaceutical and Medicinal Chemistry, Department of Pharmacognosy and Drug Development, Gombe State University, Gombe, Nigeria 2Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria, Nigeria 3Department of Chemistry, Federal University Lokoja, Kogi State, Nigeria *Corresponding Author Email: mariamailafiya@gmail.com; Tel:+2348030971798  ABSTRACT: Dried stem bark powder of  Leptadania hastata was subjected to maceration with methanol to afford crude methanol extract, which was partitioned with n-hexane, ethylacetate, chloroform and n- butanol to afford different their respective fractions. Extensive phytochemical screening of the n-hexane fraction using column chromatography resulted to the isolation of a white solid substance. The substance was identified as of lupeol using IR, 1D – NMR, 2D – NMR data and by comparison with reference spectral data.  DOI: https://dx.doi.org/10.4314/jasem.v24i10.19  Copyright: Copyright © 2020 Mailafiya et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.  Dates: Received: 15 August 2020; Revised: 22 September 2020; Accepted: 19 October 2020  Keywords: Leptadenia hastata, Isolation, Lupeol, 1D – NMR, 2D – NMR  Leptadenia hastata (Pers.) Decne. (Asclepiadaceae) is an important emergent local food of Africa with the ability to grow under harsh environmental conditions, is a widely distributed tropical African herb used as vegetable (Sena et al., 1998). L. hastata is edible non-domesticated vegetable and it is collected in wild throughout Africa. It is typically grown in tropical dry lands in sandy soil. Wild foods like L. hastata provide food security during seasonal changes and are used medicinally in many areas as anti-snake venoms, analgesics, anti-inflammatory, anti tumors, anti-hypertensive and anti-diabetic among others (Thomas, 2012). Leptadenia hastata (Pers.) Decne (L. hastata) is a perennial species of the Apocynaceae family that includes the subfamily Asclepediaceae, (Meve and Liede, 2004). Asclepiediaceae plants are widely used in traditional medicine and have been reported to be rich in steriodial glycosides, cardenolides, flavonoids, triterpenes and polyoxypregnanederivatives (Bazzaz and Haririzadeh, 2003; Atta and Mouneir, 2005; Cioffi et al., 2006). The family is mostly herbs and shrubs with white sap comprising about 250 genera and 2,000 species, many of which are lianous and some of which are cactus like succulents with reduced leaves (Thomas, 2012). Six new polyoxypregnane esters and three new glycosides together with five known esters were separated, purified and elucidated from chloroform extract of the bark of L. hastata (Aquino et al., 1996). Triterpene has been isolated from the latex of the leaves of L. hastata (Nikeima et al., 2001). β – sitosterol has been isolated from the methanol leaves extract of Leptadenia hastata and stigmasterol glycoside has been isolated from the methanol root bark extract of Leptadenia hastata (Mailafiya et al., 2017; Mailafiya et al., 2020). Seven flavonoids were isolated from the butanol fraction of the methanol extract of the aerial parts of Cynanchum acutum L. which belongs to the same family with Leptadenia hastata (Asclepiadaceae). All of which have been isolated for the first time from the genus Cynanchum. Their structures were established as quercetin 3-O- galacturonopyranoside , quercetin 7-O- α -glucopyranoside , tamarixtin 3-O-galacturonopyranoside, kaempferol 3-O-galacturonopyranoside , 8-hydroxyquercetin 3-O -galactopyranoside , tamarixtin 3-O-α-rhamnopyranoside, and tamarixtin 7-O-α-arabinopyranoside  on the basis of their chromatographic properties, chemical and spectroscopic data (Ghada et al., 2008). A pentacyclic triterpenoid compound has been reported to be isolated from the fruits of Dregea volubilis Benth Asclepiadaceae (Bikash and Haldar, 2009). Lupeol, a phytosterol and triterpene, is widely found in edible fruits and vegetables and there is a growing interest in natural triterpenoids due to their wide spectrum of biological activities. Various in vitro and pre clinical animal studies suggests that lupeol has a potential to act as an anti-inflammatory, anti-cancer, anti-microbial, anti protozoal, anti-proliferative, anti-Isolation of Lupeol from the Stem Bark…..                                                                                                    1836 MAILAFIYA, MM; PATEH, UU; HASSAN, HS; SULE, MI; BILA, AH; MUSA TL, ATINGA, V; ACHIKA, JI invasive, anti-angiogenic and cholesterol lowering agent (Siddique and Saleem, 2011).  The literature survey reveals that there is no report on isolation and elucidation of any pentacyclic triterpene from the stem bark of L. hastata. However, the present work reports herein the isolation and identification of lupeol for the first time from hexane fraction of the methanol stem bark of L. hastata.  MATERIALS AND METHODS Collection and Identification of Plant Material: The plant sample of Leptadenia hastata was collected in July 2015 at Zaria Local Government area of Kaduna State of Nigeria. It was identified at the Herbarium unit, Department of Biological Sciences, Ahmadu Bello University, Zaria Nigeria by comparing with herbarium reference voucher specimen (No. 900220). The stem bark  was  shade dried, pounded to powder, labelled and stored for use.  Preparation of extract:The powdered stem bark (4.3 kg) was extracted with 90 % methanol using maceration method. The extract was concentratedin-vacuo using rotary evaporator at 400C to yeild athe crude stem methanol extract (118 g). A portion methanol extract (92 g) was suspended in distilled water and partitioned exhasutively and successively with n-hexane, chloroform, ethylacetate and n-butanolto obtain hexane fraction, chlorofom fraction, ethylacetate fraction and n-butanol fraction respectively. The hexane fraction was subjected to colum chromatography.  Isolation by column chromatography:The hexane fraction (5 g) was weighed and subjected to extensive column chromatography; 5 ml of methanol was added to dissolve the fraction. It was followed by addition of silica gel, dried and crushed into fine powder with mortar and pestle before it was mounted on the column.The column was packed with n-Hexane and silica gel using wet slurry method. The sample was mounted on top of the silica gel, but prevented from having direct contact with the silica gel by the use of cotton. Wet method of packing was used in packing the column. The column was eluted using hexane, hexane/ethylacetate as gradient mixture solvent systems of increasing polarity. Several eluates (30 mls) collected were monitored using TLC, and those with similar TLC profile were pooled together as a fractions, evaporated at reduced pressure coded A – K.Further purification of fraction D (295 mg) was carried out by subjection to extensive column chromatography. Exactly 2 ml of methanol was added to dissolve fraction ‘D’, followed by addition of silica gel, dried and crushed into fine powder with mortar and pestle before it was mounted to the column. The column was packed using n-hexane and silica gel using wet slurry method, the sample was mounted directly on top of the silica gel but prevented from having direct contact with the eluting solvent by use of cotton.The column was eluted using 100 % n- hexane; hexane/ethylacetate. Several eluates (5 ml) were collected and monitored using TLC. Those with similar TLC profile were pooled together as sub-fractions, evaporated at reduced pressure coded D1 – D7.  Purification of isolated compound by gel filtration column chromatography: D4 - D6 were subjected to gel filtration using sephadex LH-20 and methanol as eluting solvent. Two (2) ml each of a total of 6 collections were made and combined based on their TLC profile which led to the isolation of Compound M2. M2was subjected to 1H-NMR, 13C-NMR and 2D-NMR to ascertain the chemical structure.    RESULTS AND DISCUSSION Solubility Profile, Chemical Tests and Melting point of M2: The compound gave a violet single spot on the TLC plate with Rf value of 0.36, it was soluble in methanol, and a positive Liebermann Buchard test. M2 is a white amorphous solid substance with a melting point of 2140C. Spectral Analysis: IR Vmax cm-1 of M2:The IR spectrum of M2showed broad absorption bands at 3433cm-1 for hydrogen bonded OH stretch, for C-H stretch in CH2 and CH3 at 2942cm-1 and 2357 cm -1 respectively, 1663 cm -1  for C = C  symmetric stretch, 1564 cm -1 for C=C asymmetric stretch, 1417 cm -1 for C – H deformation in CH2 and CH3, 1035 cm-1 (C – O stretch of Secondary alcohol), = C – H bonding exocyclic CH2at 889 cm-1. 1HNMR (500 MHz, Deuterated chloroform) of M2:  The proton spectrum of M2  showed signals at δ 4.69ppm, 4.58 (H – 29, d, d, 2H), 2.43 (H-19, m, 1H),1.43 (H-18, t, H), 1.10 (H-15, d. 1H), 3.16 (H-3, t, 1H), 0.93 (H-23, s. 3H), 0.75 (H-5, t, 1H), 1.68 (H-30, s, 3H) (Table 1). 13C-NMR of M2: The 13C-NMR spectrum of M2 revealed the presence of 30 carbon signals at 38.43, 26.64, 79.82, 40.22, 152.10, 110.28 e.t.c (Table 1). APT of M2: The Attached Proton Test spectrum indicated the following carbons and their multiplicity; C = 6, CH = 6, CH2 = 11, CH3 = 7, 152.10 (q), 110.28 (CH), 56.97 (CH), 52.02 (CH), 39.66 (CH2), 35.67 (CH2), 36.85 (q), 177.70 (CO-), 16.26(CH3), 15.15 (CH3), 16.72 (CH3). HSQC of M2: H-5 (0.71) # C-5 (56.97), H-18 (1.43) # C-18 (49.68), H-3 (3.16) # C-3 (79.83), H-19 (2.43) # C-19(48.20), H-29a and H-29b (4.69, 4.58) # C-29 (110.3). HMBC of M2:  H-30 (1.68) # C-20(152.10),C-29 (110.3), and C-19 (48.20), H-25(0.89) # C-5 (56.97), C-9 (52.02) and C-4 (38.43), H-29a (4.69) # C-19 (48.20), C-30 (19.60), H-Isolation of Lupeol from the Stem Bark…..                                                                                                    1837 MAILAFIYA, MM; PATEH, UU; HASSAN, HS; SULE, MI; BILA, AH; MUSA TL, ATINGA, V; ACHIKA, JI 28 (0.83) # C-18 (49.68), C-16 (35.67) and C-17 (42.20), H-23 (0.96) # C-3 (79.8), C-5 (56.97), C-4 (38.43), and C-24 (16.00), H-24 (0.79) # C-3 (79.82), C-5 (56.97), C-4 (38.43) and C-2 (26.64) . 1H-1H COSY of M2:  H-3 (3.16) # H-2 (1.65), H-29a (4.69) # H-29b (4.58), H-19 (2.43) # H-22 (1.37), H-29a (4.69) # H-30 (1.68), H-29b (4.58) # H-30 (1.68).  Table 1: 13C-NMR and 1H-NMR data of M2 and Lupeol from literature Position δ1H δ 13C δ1H δ 13C  (M2);           (Suryati et al., 2011) 1    38.9 2 1.65 26.64 1.62 27.6 3 3.16 79.82 3.18(1H, t) 79.2 4  38.43  39.0 5 0.71 56.97 0.67 (1H, t) 55.5 6 1.53 18.55 1.38 (2H, m) 18.5 7  -  34.4 8  40.22  41.0 9  52.02 1.25 (1H, t) 50.6 10  36.85  37.3 11  19.69  21.1 12  -  25.3 13  -  38.2 14  41.19  43.0 15 1.10 28.19  27.7 16  35.67  35.8 17  42.20  43.2 18 1.43 49.68 1.35 (1H, dd) 48.5 19 2.43 48.20 2.36 (1H, m) 48.2 20  152.10  151.2 21 1.05 28.76  30.0 22 1.19, 1.37 39.66  40.2 23 0.96 28.78 0.96 (3H, s) 28.2 24 0.79 16.00 0.75 (3H, s) 15.6 25 0.89 16.86 0.82 (3H, s) 16.2 26 1.13 16.72 1.02 (3H, s) 16.3 27 1.01 15.15 0.94 (3H, s) 14.7 28 0.83 16.86 0.78 (3H, s) 18.1 29 a. 4.69 b.4.58 110.28 a. 4.68 (1H, d) b. 4.56 (1H, d) 109.5 30 1.68 19.60 1.67 (3H, s) 19.5 CH3COO-  31.03   CO-  177.70    The positive Liebermann Buchard test given by compound M2 suggests that the compound is a terpernoid. M2 is a white powder with a melting point of 2140C which is the characteristic colour and melting point of pentacyclictriterpene.IR spectrum of the isolated compound showed an intensively broad band at 3433cm-1 for O – H bond vibration of the hydroxyl group. A weakly intense band was seen around 1663 for C=C vibrations. The methylenic part vibration was shown by the band at 2356. The C – H out of plane vibration of the unsaturated part was observed at 889cm -1. Corresponding C – C vibration was seen as a weak intense band at 1035 cm-1.The 1H and 13C-NMR spectra of the isolated compound showed a characteristic pattern of a triterpenoid, comparison of the spectra data of M2 and that of lupeol from literature are similar. In the proton NMR spectrum of M2, olefinic protons of H-29 (110.28)showed signals at δH 4.69ppm (1H, d) and 4.58 respectively, this supported the double bond between methylene carbon(C– 29) and quaternary carbon (C – 20).  H-3 proton appeared as triplet at 3.16ppm. Seven methyl protons also appeared at 1.68, 1.13, 1.01, 0.96, 0.89, 0.83 and 0.79 (3H each, s, CH3).The 13C-NMR spectrum of the isolated compound revealed the presence of 30 carbon atoms which were further classified into 7 methyls at positions C – 23, C – 24, C – 25, C – 26, C – 27, 28 and C - 30, 11 methylenes at C - 1, C – 2, C – 6, C – 7, C – 11, C - 12, C – 15, C – 16, C – 21, C – 22 and C – 29,  6 methines at C – 3, C – 5,  C – 9, C – 13, C – 18,  C - 19  and 6 quaternary carbon atoms at C – 4, C – 8, C – 10, C – 14, C – 17 and C - 20 by the APT spectrum of the isolated compound. The HSQC correlation shows the correlation of protons to their corresponding carbons atoms. The proton at δ 0.71 (H - 5) which is a methine proton is correlating with the carbon at δ 56.97 (C - 5) which shows that H – 5 is bounded to C – 12. Isolation of Lupeol from the Stem Bark…..                                                                                                    1838 MAILAFIYA, MM; PATEH, UU; HASSAN, HS; SULE, MI; BILA, AH; MUSA TL, ATINGA, V; ACHIKA, JI  Fig 1: Lupeol (C30H50O)  The protons at δ 4.69 and δ 4.58 which methylene protons at position H – 29a and H – 29b respectively is correlating with the carbon at δ 110. 3 (C – 29) which shows that H – 29a and H – 29b are bounded to C – 29. The proton signal at δ 3.16 (H - 3) correlated with methine carbon at δ 79.82 ppm (C - 3) reveals that it belongs to methyneoxy proton C – 3. Long range correlation 1H    13C of HMBC spectrum was used to prove bonding structure relationship of the isolated compound. Long range correlation of proton at δ 1.68 (H - 30) with quaternary carbon at δ 152.10 (C - 20), methylene carbon at δ 110.3 (C – 29) and methine carbon at δ 48.20 (C - 19) revealed that the methyl carbon atom C – 30 binds to the quaternary carbon C – 20. HMBC correlation of methyl protons at δ 0.96 (H - 23) with methine carbons at δ 79.8 (C - 3), δ 56.97 (C - 5), quaternary carbon δ 38.43 (C - 4), methyl carbon at δ 16.00 (C - 24) and the methyl protons at δ 0.79 (C - 24) correlating with δ38.43 quaternary carbon (C – 4), methine carbons at δ 56.9 (C – 5), δ 79.82 (C – 3) and methylene carbon at δ 26.64 (C – 2), supported the dimethyl position at C – 4.Methyneoxy proton H – 3 was coupled by methylene proton H – 2 at 1.65ppm (2H, m) this correlation was established by COSY analysis. The 1H-NMR,13C-NMR, DEPT, HSQC, HMBC and 1H-1H COSY spectral data and comparison with the data described in literature (Suryati et al., 2011) showed the structure of M2 to be a lupeol (Figure 1). In conclusion, lupeol was isolated from the stem bark of Leptadenia hastata. The presence of lupeol in the root extract of this plant may be responsible for the observed ethnomedicinal uses of the plant since various pharmacological activities of lupeol have been reported in literature.  REFERENCE Aquino, R; Peluso, G; De Tommasi, N; De Simone, F;  Pizza,  C (1996). New polyoxypregnane ester derivative from Leptadeniahastata. J. Nat. Prdt. (59): 555-564   Atta, AH; Mouneir, SM (2005). Evaluation of some medicinal plants extracts for antidiarrhoeal activity. Phytotheraph Research. (19): 481-485 Bazzaz, BSF; Harrizadeh, G (2003). Chemotaxonomic analysis of the genus Cryptolepsis. Biochem. SystE and Eco.(31): 155-166  Biskash, KS; Haldar, PK (2009). In vitro Anti-Leishmanial and Anti-Tumour Activities of a PentacyclicTriterpenoid Compound Isolated from the Fruits of DregeavolubilisBenthAsclepiadaceae.Trop. J. Pharm. Res., 8 (2):1-8  Cioffi, G; Sanogo, R; Vassallo, A; Dal Piaz, F; Autore, G; Marzocco S; De Tommasi N (2006). Pregnane glycosides from Leptadeniapyrotechnica. J  Nat. prdts..(69): 625:635  Ghada, AF; Hossam, MA; Mohamed, SAM; Fathy, MS; Amany, AS (2008). Ant-diabetic and Anti-oxidant Activity of Major Flavonoids of Cynanchumacutum L.  Asclepiadaceae) Growing in Egypt. Int. J Ani and Vet.Adv 4(4): 303-305  Mailafiya, MM; Pateh, UU; Iliya, I; Yusuf, AJ; Mohammed, GT; Sanusi, A (2017). Isolation of B-Sitosterol from the Methanol Leaf extract of Leptadeniahastata (Pers) Decne (Asclepiadaceae). Nig. J. of Pharm and Biomed. Res.2(2):132-138.  Mailafiya, MM; Pateh UU; Hassan, HS; Sule, MI; Yusuf, AJ; Bila AH (2020). Isolation and Characterization of Stigmasterol glycoside from the root bark of Leptadenia hastata.  FUW Trends in Sci. Technol. J. 5(2): 394-398 e-ISSN: 24085162; p-ISSN: 20485170  Meve, U and Leide, S (2004). Subtrail division of ceropegieae (Apocynaceae - Asclepiadaceae). Taxon,53: 61-72  Nikiema, JB; Vanhaelen-Fastre, R; Vanhaelen, J; Fontaine, CD; Heenen, M. (2001). Effect of anti-inflammatory triterpenes isolated from Leptadeniahastata latex on Keratinocyte Proliferation. Phytother. Res. (15): 131-134  Sena, LP; Vanderjagt, DJ; Rivera C; Tsin, AT; Muhamadu, I; Mahamadou, O; Millison, M; Pastuszyn, A; Glew, RH (1998). Analysis of nutritional components of eight famine foods of the republic of Niger. Plants food for Hum Nutri. 52: 17-30  Siddique, HR; Saleem, M. (2011). Beneficial health effects of lupeoltriterpene: a review of preclinical studies. Life Sciences. 88(7-8), 285 – 293  Suryati, H; Dachriyanus D; Md, NHL. (2011). Structure elucidation of antibacterial compound from Ficus deltoidea Lack leaves. Indonesia J. of Chem. 11 (1): 67-70.  Thomas, SD (2012). Leptadenia hastata: A Review of its Traditional uses and its Pharmacological Activity. Med chemistry. 2:148-150. 

Isolation of Lupeol from the Stem Bark of Leptadenia hastata   (Pers.)
Decne

Isolation of Lupeol from the Stem Bark of Leptadenia hastata (Pers.) Decne

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