Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P.

19 25

Nigerian Journal of Pharmaceutical Sciences Vol. 7, No. 1, March, 2008, ISSN: 0189-823X All Rights Reserved

ISOLATION OF STIGMASTEROL, -SITOSTEROL AND 2HYDROXYHEXADECANOIC ACID METHYL ESTER FROM THE RHIZOMES OF STYLOCHITON LANCIFOLIUS PYER AND KOTCHY (ARACEAE)
*1

Pateh, U. U., 1Haruna A. K., 1Garba, M., 1Iliya, I., 1Sule, I. M., 2Abubakar, M. S. and 2Ambi A.A.
1

Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria, Nigeria 2 Department of Pharmacognosy and Drug Development, Ahmadu Bello University, Zaria, Nigeria

*Author for Correspondence

ABSTRACT General Phytochemical screening of the rhizome of Stylochiton lancifolius Kotschy and Peyr (Araceae) revealed the presence of steroids/triterpenes, saponins, fatty acids and tannins. Extensive Phytochemical investigation of the petroleum ether extract of the rhizome afforded white crystalline mixture of -sitosterol, stigmasterol and 2-hydroxy hexadecanoic acid methyl ester which were identified by I.R, 1HNMR, 13CNMR and FABS-MS. The compounds are reported for the first time from this plant. Key words: Phytochemical, Steroids, Fatty acid and Stylochiton lancifolius

INTRODUCTION Stylochiton lancifolius Kotschy and Peyr (Araceae) is a small herbaceous plant with short rootstock found in savanna woodland, from Senegal to Southern Nigeria and wide spread in the drier savanna of tropical Africa (Burkill, 1985).The Rhizome has been reputedly being used by the Fulanis of the Northern Nigeria as an anti-inflammatory and analgesic agent during their customary annual sharo (Jumba 2000.). In addition, the leaves are employed as analgesic and antibacterial agent in the treatment of cellulitis commonly known as whitlow (Waziri 2002.). However, so far, no ethno medical value has been documented for the plant apart from its utilization as food during famine after prolong heating with common salt or asben salt to remove the acidity in Northern Nigeria and its use in making a yellow dye in Togo for 19

dyeing cloth (Burkill, 1985). The family Araceae is vast with 2000 species and 106 genera. It contains numerous horticultural ornamentals, especially for indoors and a few are grown for food e.g. Colocasia for taro, Monstera for its fruit (Mexican breadfruit). Alkaloids are reported in some of the family members. Proanthocyanidins are sometimes very abundantly present the most common being cyanidin. Favonoids are either present or absent and when present it is mostly kaempferol or quercetin or both. Saponin/Sapogenins are present in some genera (Watson and Dallwitz, 2005). EXPERIMENTAL Collection, Identification and Preparation of plant materials The whole plant (aerial and the underground

Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 19 25

Rhizome and roots) was collected around Samaru, village, Zaria in the month of September 2001. The plant was authenticated in the herbarium unit, Department of Biological Sciences, Ahmadu Bello University, Zaria (with voucher number 1407). The aerial part (leaves) were manually separated from the rhizome and the rhizome was air dried, powdered, sieved, weighed and stored in air tight container and subsequently referred to as powdered rhizome. Extraction and Isolation Powdered rhizome of Stylochiton lancifolius (1.3kg) was defatted exhaustively with light petroleum ether (60-80oc) in a soxhlet extractor. The solvent was recovered under reduced pressure to afford a dark greenish oily mass (41gm), which was labeled as petroleum extract (RPE) and kept in the refrigerator. The resulting marc was air dried at room temperature and then exhaustively extracted with methanol using the soxhlet extractor. The methanol was recovered under reduced pressure to yield a dark brownish waxy mass (414.7gm). This was labeled as methanol extract (RME). Chromatographic Separation The petroleum ether extract of rhizomes was subjected to thin layer chromatography using silica gel as stationary phase and petroleum ether: methanol (1:1) and petroleum ether: chloroform: methanol (5:2:1) as mobile phase. The chromatograms when developed in iodine chamber yielded seven and eight spots respectively that showed zones for steroidal nucleus with Liebermann Buchard visualizing reagent. Column chromatography of RPE (2g) was conducted using silica gel (Mesh 60-120) that was packed using wet packing method. The column was run using petroleum ether, chloroform and methanol by gradient elution technique. T.L.C was used to monitor the eluates. A total of 157 eluates were collected. Similar fractions were pooled 20

together to yield fifteen fractions. Eluates A, and G were worked upon to yield As, and G1 respectively. As, and G1 all yielded a single spot when subjected to T.L.C using several solvent systems including petroleum ether: ethyl acetate (70:30), petroleum ether: methylene chloride (50:20), and Petroleum ether: Chloroform: Methanol (60:30:10) and it showed it to be homogenous compound. G1 a white crystalline powder (9mg) with a melting point (144-146oC) was subjected to TLC using various solvent systems such as petroleum ether: chloroform: methanol (5:3:1), methylene chloride: petroleum ether (50:50) indicated it to be homogenous compound. G1 was further subjected to 1HNMR, 13CNMR and HHCOSY to ascertain the chemical structure. As a white amorphous powder (8.3mg) with a melting point (59-60oC ) was also obtained and subjected to thin layer chromatography using various solvent systems such as petroleum ether: chloroform: methanol (5:3:1), methylene chloride: petroleum ether (50:50) indicated it to be homogenous compound. As was further subjected to IR 1HNMR, and 13CNMR to ascertain the chemical structure. RESULTS AND DISSCUSSION Compound G1 G1: The IR absorption spectrum showed absorption peaks at 3384.3cm-1(O-Hstr.); 3218cm-1 (HC==CH cyclic); 3025.8cm-1(CHstr.); 2868.4-1cm-1(C-Hstr.); 1665.5cm1 (C=C absorption peak); other absorption peaks includes 1462cm-1([CH2] n); 1 1382.7cm (OH def.) and 1046cm1 (cycloalkane). HNMR of G1: 1HNMR has given signals at 3.52(m), 5.358(br,s), 0.68(s), 1.01 (s), 0.92(d, J= 6.4), 0.814 (d, J=6.5), 0.833 (d, bj=6.5) and 0.845 (d, j=7.5) ppm. Other peaks are observed at 3.52(m), 5.357 (br, 5), 0.699(s), 1.02(d,j=7.5), 0.795(d,j=6.5),0.846 (d,j=6.5)
1

Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 19 25

and 0.804 (t, j=7.5) ppm. (Table 1). NMR of G1: 13CNMR has given signal at 140.8 and 1221.7ppm for C5=C6 double bond respectively, 71.8 for C3 -hydroxyl group 19.4 and 11.9 for angular methyl carbon atoms for C19 and C18 respectively (table 1). FAB-MS Spectrometry showed the molecular ion peaks at 414 and 412 that correspond to the molecular formula. C29H50O and C29H48O respectively. Ion peaks were also observed at m/z 382, 273, 255, 213, 187, 159, 133, 107, 81 and 55. Other ion peaks were further observed at m/z 367, 329, 273, 303, 273, 199, 145 and 199. The column chromatographic of separation of RPE on silica gel yielded G1, which is a white crystalline substance with a melting point of 144 - 146oC. G1gave a positive test to Liebermann Buchard reagent for steroidal nucleus. On subjection to I.R Spectroscopic analysis, the observed 1 absorption bands are 3384cm (b) that is characteristic of O-H stretching. Absorption at 3218 cm1 is due to cyclic olefinic HC= CHstr, 3025 cm1 due to =CH str and 2868 cm1 assigned to C-H str. Other absorption frequencies include 1665 cm1 as a result of C=C absorption, however, this band is weak (Pretsch et al., 2000). 1462 cm1 is a bending frequency for cyclic (CH2) n and 1382 cm1 for CH2 (CH3)2. The absorption frequency at 1332 cm1 can be attributed to OH def. while absorption at 1046cm signifies cycloalkane. These absorption frequencies resemble the absorption frequencies observed for Stigmasterol (Grasselli, 1973). The Proton NMR has revealed the existence of signals for Olefinic proton at 5.358 (br.,s.), Angular methyl proton at 0.68 (s), 0.699 (s) and 1.01 (s) corresponding to C18 and C19 proton respectively. The 13CNMR has shown recognizable signals 140.8 and 121.7ppm, which are assigned C5 and C6 double bonds respectively as in 5 spirostene (Agrawal et al., 1985). The value at 71.0ppm is due to C.3 hydroxy group (Pretsch et al., 2000). 21
13

The signals at 19.4 and 11.9ppm corresponds to angular carbon atom (C19 and C18 respectively). The value for C18 is lower due to -gauche interaction that increases the screening of the C18 hence lower chemical shift. However, the loss of H in C6 results in decrease in screening of the C19 leading to increase in 13C chemicals shift to higher frequency (Smith, 1978). This is also tenable as in chemical shift of 21.2 and 11ppm (for C19 and C18 respectively). The weak molecular ions were given at m/z 414.4 and m/z 412.8 and the characteristics peaks were given at m/ =367 that corresponds, to (M-45) or the loss of HO+=CH-CH3. The ion peak at m/z 382 is also due to the loss of HO.-CH3 (M-32). These suggest that the sample G1 contains two compounds with molecular weight of 414.4 and 412.3. Other ion peaks are m/z=273 because of the loss of C10H21 and C13H27 that corresponds to the (M-183) and (M-181) respectively (Biemann, 1962). The molecular weights and the fragmentation pattern indicate that the compounds present in G1 are sitosterol and stigmasterol respectively. The dehydration of fragment at m/z 273 would yield m/z 255, which on successive dealkylation would yield ions at m/z 213, 187, 159, 133, 107, 18 and 55 for -sitosterol. While in stigmasterol the fragment at m/z 273 will lose C4H9=+OH to yield fragment at m/z 199 which on further dealkylation would yield fragments at m/z 173, 145 and 119. Another pattern is from m/z 412 to m/z 329 (M-83) then to m/z 303. The above IR, 1HNMR, 13 CNMR, HHCOSY and FABS-MS spectal data and a comparison of the 13CNMR signal with those described in the literatures (Agrawal et al., 1985; Grasselli, 1973; Pretsch et al., 2000; Kokdil et al., 2003 and Mc. Farlane 1972) showed the structure of G1 to be the mixture of -sitosterol and stigmasterol. The only difference between the two compounds is the presence of C22=C23 double bond in Stigmasterol and C22-C23 single bond in Sitosterol hence, the lack of practical

Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 19 25

difference in their Rf despite the use of several solvent systems. Furthermore, literatures have shown that sitosterol is difficult to be obtained

in pure state. (Pollock and Stevens, 1965; Fieser and Fieser, 1962).

Table 1: 13 C and 1H NMR data of G1 (-sitosterol and Stigmasterol) S/No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 -sitosterol 13 C 37.3 31.6 71.8 42.2 140.8 121.7 31.9 31.9 51.2 36.5 21.1 39.8 42.3 56.8 24.3 28.3 56 11.9 19.4 36.2 18.8 33.9 26.1 45.9 29.2 19.8 19.3 23.1 12.2
1

3.52 m

5.358 br s

0.680 s 1.01 s 0.92 (d, 6.4)

0.814 (d, 6.5) 0.833 (d, 6.5) 0.845 (t, 7.5)

stigmasterol 13 C 37.3 31.6 71.8 42.3 140.8 121.7 31.9 31.9 51.2 36.5 21.1 39.7 42.3 56.9 24.4 28.4 56.1 11 21.2 40.5 21.2 138.3 129.3 51.2 31.9 21.2 19 25.4 12.1

3.52 m

5.357 br s

0.699 s 1.01 s 1.02 (d, 7.5)

0.795 (d, 6.5) 0.846 (d, 6.5) 0.804 (t, 7.5)

Measured in CD3Cl (400 MHz)

22

Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 19 25

29

G1
21 20 22 23

28 24 25 27

18 17 13 14 1 2 3 5 4 6 10 9 8 7

12 19 11

26 16 15

HO

-sitosterol C29H50O Mol. Wt.: 414.71

HO
Stigmasterol C29H48O Mol. Wt.: 412.69

Compound As As: The I.R absorption spectrum showed absorption peaks at 3209.8cm-1 for O-H (str.), 2849 cm -1 and 1734 cm -1 for C=O in aliphatic carbonyl compounds; while, 1462 cm -1 and 1384cm-1 are for O-H; . Other peaks are 1119cm-1 and 1020cm-1 for C-OH and CO respectively. The absorption peak at 720cm1 indicates the presence methylene protons. 1 HNMR of As: 1HNMR has given signal at 5.38, 5.34, 4.09, 4.07, 2.33, 2.30, 2.28, 1.65, 1.63, 1.58, 1.41, 1.27, 1.21, 1.04, 0.95, 0.92 and 0.8 ppm Other peaks are 0.6, 0.70, 0.71, 0.81ppm. 13CNMR of As: The 13CNMR have given the characteristic signal at 174.4 ppm, 64.8, 34.80, 32.39, 30.09, 26.34, and 25.44 and 23.09 ppm. The spectra have indicated the presence of 17-carbon atom. Infrared spectrometry of As yielded absorption peaks at 3210cm-1 that corresponds to O-H str. Hence it is broad due to H. bonding C-H (str) absorption frequency is observed at 2849cm-1 and the absorption frequency at 1734cm-1 is assigned to C=O(str) in aliphatic carbonyl compounds. The O-H (b) is indicated by the 23

absorption frequency at 1385cm-1 while the C-O-C that is characteristics of ester linkage is given at absorption frequency at 1176cm-1, COH and C-O absorption frequencies were noticed at 1119 and 1020cm-1 respectively (Pretsch et al., 2000 and Kemp, 1991). The absorption at 762, 720, 658, and 617cm-1 were assigned to methylene protons in the fatty acid chain. 1HNMR has indicated the presence of methylene proton adjacent to carbonyl group at 2.28-2.33 (multiple). The characteristics chemical shift of hydroxyl proton are noticeable at 4.05, 4.07 and 4.09 (Pretsch et al., 2000) Multiplates at 1.61, 166 caused by the second proton in the -carbon atom where also observed. 13CNMR data allowed the identification of hydroxyl bearing carbon atom (64.8), ester linked methyl group (23.09) (Hartmann et al., 1994) carbonyl function at 174 that is characteristic of carboxylic acid is also detected. All other signals as indicated for non-functionalized saturated carbons (Rie Ker Jie and Cheng, 1995). The above analysis coupled with the comparison of the melting point and spectral

Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 19 25

data (Isbell and Mund, 1998; Hartmann et al., 1994; Blaise et al., 1997 and Pretsch et al.,

2000) suggested that compound As is 2hydroxy hexadecanoic acid methyl ester [III].

CH3-O-CO-CH (OH) CH2 (CH2)12 CH3 [III] CONCLUSION From the above findings, -sitosterol, stigmasterol and 2-hydroxyhexadecanoic acid methyl ester were isolated from petroleum ether extract of the rhizomes of Stylochiton lancifolius Kotschy and Peyr (Araceae) and their chemical structures elucidated respectively.

REFERENCE Agrawal, P.K., Jain, D.C, Gupta, R.K, and Thakur, R.S. (1985) Carbon -13 NMR spectroscopy of steroidal sapgenins and steroidal saponins Phytochemistry Res., vol24, 11: 2476-2496. Blaise, P., Fairness, M., and Soulier, J. (1997). Identification of cyclopentenyl fatty acids by 1H and 13C Nuclear Magnetic resonance. J. Am.oil. Chem Soc. 74, 727-730 Burkill, H.M. (1985). The useful plant of West Tropical Africa vol.1, Royal Botanical Gardens Kew Pp 193-209. Fieser, L.F. and Fieser, M. (1962) Organic Chemistry 3rd Ed. Wiley New York pp.250,353 Gamze Kokdil, Gulacti Topac, Ahmet C. Coren and Wolfgang Voelter (2002). Steroids and Terpenoids from Ajuga relicta: Z Naturforsch. 57b 957-960 Grasselli, J.G. (1973) CRC spectral Data and physical constants for organic compound. CRC press Hartman, S., Minnikin, D.Z., Romming, H.J., Baird, M.S., Ratledge, C. and Wheeler, P.R. (1994). Synthesis of methyl 3-(2octadecylcyclopropen-1-yl)propanoate and methyl 3-(2-octadecylcyclopropen-1-yl) 24

pentanoate and cyclopropen fatty acids as possible inhibitors of mycolic biosynthesis. Chem. Phys. Lipids, 71, 99-108 Isbell, T.A. and Mund, M.S. (1998). Synthesis of secondary ethers derived from meadow foam oil J. Am oil Chem. Soc. 75,1021-1029. Jumba Adamu communication (2000). Personal

Klaus Biemann (1962). Mass spectrometryorganic chemical Applications. McGraw-Hill Book Co. Pp 46-361Annual report on NMR spectrorscopy vol. 8 Academic Press inc. London Pp 199-226 Lie Ken Jie, M.S.F. and Chen, K.L. (1995). Nuclear Magnetic resonance spectroscopic analysis of homo allylic and bis homo allylic substituted methyl fatty ester derivatives. Lipids 30, 115-120 Mc. Farlane (1972). Application of Nuclear Magnetic resonance spectroscopy in: Bentley K.W and Kirby G.W (ed.), Technique of chemistry vol. IV Elucidation of organic structures by physical and chemical methods 2nd Ed. Wiley inter science pp. 225-322 Pollock, J.R.A, Stevem R.S (ed.) (1965) Dictionary of organic compounds 4th ed., vol. 5 Eyre and spottiswoode (Publishers) Ltd.

Pateh et al., Nig. Journ. Pharm. Sci., March, 2009, Vol. 8 No. 1, P. 19 25

Pretsch E. Buhlmann and Affolter, A. (2000) structure Determination of organic compounds Table of spectral data springerverlag Berlin Heidelberg Pp 71-150. Smith, W.B (1978) Carbon-13NMR Spectroscopy of steroids in: Webb G.A (Ed.) Annual reports on NMR spectrorscopy vol. 8 Academic Press inc. London Pp 199-226

Wazirin Badaromo communication.

(2002).

Personal

Watson, L. and Dallwitz, M.J. (2005). The families of flowering plants. Description, Illustration, identification, information retrieval. Angiosperm Families-Araceae Jusshttp:// delta-intkey.com/

25