24-Methylenedammarenol" A New Triterpene Alcohol from Shea Butter T. ITOH, T. TAMURA, and T. MATSUMOTO, College of Science end Technology,
Nihon University, 8, Kanda Surugadai, 1-chome, Chiyoda-ku, Tokyo, 101 Japan ABSTRACT
57 /~A, ion source temp 160-180C, sample temp 100-120 C, and accelerating high voltage A new triterpene alcohol was isolated 4.6 kv. The samples were introduced directly from shea butter and its structure was into the ion source through a vacuum lock. shown to be 24-methylenedammarenol Preparative argentation thin layer chroma(2 4- m e t h y l e n e - 5 a-dammar-20 [ 21 ]-entography (TLC) for the fractionation of triter3~-o I ) . Dammaradienol (5a-dammarapene acetates was carried out on the 20 x 20 20121],24-dien-3~-o1) also was isolated cm plates coated with 0.5 mm of silica gel from shea butter. (Wakogel B-10, Wako Pure Chemical Industries Ltd., Osaka, Japan) impregnated with 10% or INTRODUCTION 20% silver nitrate, using a Toyo continuous Previous work has indicated the presence of f l o w development preparative TLC (Toyo a-amyrin (1), ~-amyrin (1,2), butyrospermol Roshi Kaisha Ltd., Tokyo, Japan). The plate (1,3,4), parkeol (1,5), and 24-methylenedihy- was developed for 1 hr using hexane:benzene droparkeol (6) in the triterpene alcohol fraction (7:3) as solvent. Separated zones were observed of shea butter. Shea butter, obtained from the under ultraviolet light (3600 A) after spraykernels of Butyrospermum parkii, a sapotace- ing of a rhodamine-6G solution in ethanol ous tree of tropical Africa, is a substance used on the developed plate, and were cut off and as food by the natives and for the manufacture quantitatively extracted with ether. The argenof soap and other products. This paper describes tation silicic acid column was prepared as dethe isolation and identification of a new dam- scribed previously (1) in a similar manner as m a r a n e series triterpene alcohol, 24-meth- proposed by Vroman and Cohen (12). Gas y l e n e dammarenol (24-methylene-5a-dammar- liquid chromatography (GLC) was performed 20121]-en-3t3-ol) (Scheme I,II), and damma- under the same operating conditions as described previously (1), using a Shimadzu GC-5A r a d i e n o l (5a-dammara-20[21 ],24-dien-3r (Scheme I,I) from shea butter. Dammaradienol gas chromatograph (Shimadzu Seisakusho Ltd., (Scheme I,I) has only been known as a compo- Kyoto, Japan) equipped with a flame ionization n e n t of d a m m a r a n e series triterpenes in detector and a 2 m x 3 mm inside diameter dammar resin originating from trees of the glass column packed with 3% OV-17 on Gas Chrom-Z, 80-100 mesh. Relative retention time Dipterocarpaceae family (7-11). (RRT) was expressed by the ratio of the retention time for the substance under examination EXPERIMENTAL PROCEDURES to the retention time for sitosterol. Other proMelting points were determined with a Micro cedures such as saponification of fat, and mp apparatus (Yanagimoto Seisakusho Ltd., hydrogenation (platinum oxide catalyst, ether Kyoto, Japan) and uncorrected. All recrystaUi- solution), acetylation, and hydrolysis of triterzations were performed in acetone:methanol. penes were carried out in a similar manner as Infrared (IR) spectra (KBr) were obtained with described previously (1). a Type IRA-2, IR spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan). Optical rotaRESULTS AND DISCUSSION tions w e r e measured in CHC13 using a Carl Zeiss Polarimeter 0.01 ~ (Carl Zeiss, Ober- Isolation of Dammaradienol and kochen, Germany). Concentrations used were 24-Methylenedammarenol from Shea Butter indicated in parentheses as g/100 ml. Nuclear Unsaponifiable material (156 g) separated magnetic resonance (NMR) spectra in CDC13 from shea butter (3 kg) was acetylated in the were recorded on a JNM-C-60-HL instrument same manner as described previously (1). The (60 MHz, Japan Electron Optics Laboratory acetate (150 g) was then chromatographed on Co., Tokyo, Japan) and calibrated against argentation silicic acid (430 g) using hexane as internal tetramethylsilane as 0 ppm. Mass eluent at first. The eluate was monitored perispectra were taken with a Hitachi RMU-7M odically by GLC to remove the eluate fractions mass s p e c t r o m e t e r (Hitachi Ltd., Tokyo, (130.2 g in total, eluted with 5 liters hexane), Japan), electron energy 70 eV, target current consisting almost exclusively of the acetates of 808
24-METHYLENEDAMMARENOL IN SHEA BUTTER
809
SIDE C H A I N ( R )
at m/e 393.3537 (M- [CH 3 + H 2 0 ] , 393.3519, 2%), 315.2659 (M - [side chain + 2H], 315.2686, 5%), and 297.2570 ( M - [side chain , (i) (II) + 2H + H20] , 297.2581,3%), and base peak at m/e 109.0997. Because the sterols with unsaturated side chain are known to give a characteristic ion resulting from the loss of the entire C-17 ( III ) ( iv ) substituent together with the rearrangement of two hydrogen atoms (13), this alcohol, giving SCHEME I. Diagram of the skeleton and side chains the ion at m/e 315.2659, is expected to have a of dammarane series of triterpene alcohols. C8-side chain with two double bonds. These a-amyrin, lupeol, and butyrospermol. The col- double bonds were indicated by the IR specumn was then eluted with benzene (13 liters in trum of the I acetate (Scheme I) to exist as total) to give an eluate fraction (17.5 g) which trisubstituted double bond (831 and 820 cm-1) c o n t a i n e d considerable amounts of several and terminal methylene group (3080, 1642 and other triterpene acetates besides the above men- 885 cm-1). The I acetate (Scheme I) showed tioned three triterpene acetates. This fraction methyl signals at 0.88 (strong), 0.99, 1.64, was further fractionated on argentation silicic 1.71, and 2.05 (3fl-OCOCH3) ppm in the NMR acid column (packing t78 g) to give a fraction spectrum. The other signals also were observed (4.3 g) eluted with benzene: ether (4:1, 1 liter), at 4.51 (3a-H, multiplet [m]), 4.76 (ca. 2H, preceded by a fraction (6.5 g) ehited with ben- broad singlet [b], terminal methylene), and zene (4 liter). The fraction (4.3 g) was then 5.14 (1H, triplet [t], J 4.2 Hz) ppm. Because a fractionated by preparative argentation TLC to pair of the signals at 1.64 and 1.71 ppm is chargive a fraction (800 mg) which indicated a acteristic to the 26,27-dimethyl protons of the Rf-value almost identical with that of the au- A24-sterols (14), the trisubstituted double thentic specimen of dammaradienyl acetate iso- bond shown by IR spectrum is attributable to lated from dammar resin and was found by A24-bond. The triplet at 5.14 ppm is, hence, GLC to contain the acetates of I and II assignable to 24-H. In the spectrum of the free (Scheme I) in nearly equal proportions. Because alcohol I (Scheme I), the signals at 0.88 and the repetition of preparative argentation TLC 0.98 ppm, the part of which might be attriof this fraction gave a single broad zone instead butable to the angular methyls, and, further, of two separate zones, the upper and lower por- the signals at 1.64 and 1.72 (26,27-dimethyl), tions of the zone were cut off separately and 4.74 (b, terminal methylene), and 5.16 (t, J 4.2 the material from each portion was repeatedly Hz, 24-H) ppm, were still observed with worked up by preparative TLC in a similar scarcely any changes in their chemical shifts manner to give eventually two fractions; the from those observed for the I acetate. On the fraction from the upper portion of the zone other hand, the signals appeared also at 0.79 gave the I acetate (Scheme I) (68 rag, GLC (ca. 3H), 2.05 (b, ca. 2H), and 3.24 (3a-H) ppm purity 95%) and the other fraction from the in the spectrum of the free alcohol I. The triterlower portion of the zone afforded the II ace- pene I was found identical with the authentic specimen of dammaradienol isolated from damtate (Scheme I) (80 mg, GLC purity 93%). mar resin described later in its IR, NMR, and Both the hydrolyzed triterpene alcohols I mass spectra, RRT in GLC and mp, as well as and II (Scheme 1) showed a mobility identical TLC behavior. The alcohol (Scheme I,I) isowith that of the authentic dammaradienol in lated from shea butter is, therefore, identified TLC using silica gel plate (eluent, hexane:ether as d a m m a r a d i e n o l (5a-dammara-20[21 ],244:t). dien-3fl-o 1). Hydrogenation of the I acetate for 3 hr at Dammaradienol from Shea Butter room temperature gave the tetrahydro comand Its Tetrahydro Derivative pound, dammaranyl (Scheme I,III) acetate ( a The I acetate (Scheme I) (RRT 1.35) thus mixture of the C-20 epimers), which on recrysisolated from shea butter crystallized as plates, tallization showed mp 139-140 (plates)(lit [8], mp 151-153 C, [a]~)1+59 ~ (c 0.81) (lit [7] a mixture of the C-20 epimers of dammaranyl dammaradienyl acetate [a]D +60~ 9 Hydro- acetate, mp 139-141 C). The IR spectrum lyzed I (Scheme I) (RRT 1.11) showed mp showed no absorptions correlated to any of the 136-138 C (fine needles). Mass spectrum of the t e r m i n a l methylene and the trisubstituted a l c o h o l ( S c h e m e I) showed M+ at m/e double bond. Mass spectrum of the alcohol 426.3836 (C30H5oO , requires 426.3859, rela- ( S c h e m e I , I I I ) showed M+ at m/e 430 tive intensity 16%) with the other principal ions ( C 3 0 H S 4 0 , 30%) with the other ions at m/e LIPIDS, VOL. 10, NO. 12
810
T. ITOH, T. TAMURA, AND T. MATSUMOTO
415 (M - CH3, 10%), 412 (M - H20, 9%), 397 (M - [CH3 + H 2 0 ] , 14%), 317 (M -side chain, 73%), and 299 (M - [side chain + H 2 0 ] , 98%), and base peak at m/e 95. The presence of the strong ion (M - side chain), which is characteristic for the sterols with a saturated side chain (13), indicates that the side chain of the alcohol (Scheme I,III) is fully saturated. The free alcohol III (Scheme I) showed a predominant peak, related to one of the C-20 epimers, at RRT 0.91 with a shoulder in the front side in GLC. In the NMR spectrum of the III acetate, the signals attributable to the skeletal methyls appeared, with almost identical chemical shifts with those observed for the I acetate, at 0.87 (strong), 0.97, and 2.06 (3~-OCOCH3) ppm. A triplet assignable to 3a-H was observed at 4.50 (J 7.2 Hz) ppm. On the other hand, the 26,27-geminal dimethyl protons gave a doublet at 0.89 (J 5.4 Hz) ppm, indicating that the triterpene III (Scheme I) has a C-24 saturated side chain (14). The disappearance of the signals related to the C-20 terminal methylene and 24-H shows further that the side chain of the III is fully saturated. The free alcohol III (Scheme I) gave the methyl signals at 0.80, 0.87, 0.89 (d, J 6.0 Hz), 0.97, and 1.00 ppm, and a triplet at 3.22 (J 7.2 Hz, 3a-H) ppm. 24-Methylenedammarenol from Shea Butter and Its Tetrahydro Derivative
The new triterpene II acetate (Scheme I) (RRT 1.47) isolated from shea butter showed mp 145-147 C (plates), [a]~1+62 ~ (c 0.81). Hydrolysis of the II acetate gave free alcohol II (Scheme I) (RRT 1.21), mp 131-133 C (fine needles). In the mass spectrum, the free alcohol II showed M+ at m/e 440.4017 (C31H520, 440.4015, 20%), with the other principal ions at m/e 315.2652 (M - [side chain + 2H], 20%) and 297.2577 (M - [side chain + 2H + H 2 0 ] , 70%), and base peak at m/e 109.1028. The presence of a relatively strong ion M - (side chain + 2H) at m/e 315.2652, as is the case with the alcohol I, reveals that this alcohol (Scheme I,II) has a saturated ring system and a C9-side chain with two double bonds. The two double bonds in the side chain were recognized to exist as the terminal methylene groups because the absorptions at 3080, t 640, and 886 cm-1 were observed without any of other olefinic bands in the IR spectrum of the alcohol. The chemical shifts of the skeletal methyl signals in the NMR spectra of the triterpene alcohol II (Scheme I) and its acetate were found almost identical with those of the respective derivatives of the triterpene I (Scheme I) (dammaradienol) the acetate II (Scheme I) indicated those at 0.88 (strong), 0.99, and 2.06 LIPIDS, VOL. 10, NO. 12
(3~-OCOCH3) ppm; and the free alcohol II gave those at 0.80, 0.88, and 0.99 ppm. This fact shows unequivocally that the ring system structure of the new triterpene II (Scheme I) is identical with that of the triterpene I, i.e., 5a-dammarane type ring system. On the other hand, the triterpene II (Scheme I) gave a signal of the 26,27-geminal dimethyl protons as a doublet at 1.06 (J 6.6 Hz) ppm. This is characteristic for the sterols possessing a C-24 termin a l m e t h y l e n e group such as 24-methylenecycloartanol (24-methylene-9/3,19-cyclo-5alanostan-3t3-ol) (15). Hence, the new triterpene II is considered to possess a C-24 terminal methylene group. In the NMR spectra of the triterpene alcohol II (Scheme I) and its acetate, a broad singlet related to terminal methylene group was observed also at 4.76 ppm with an intensity (ca. 4H) stronger than that observed for the triterpene I (Scheme I) (dammaradienol) possessing one terminal methylene group at C-20. This indicates that the triterpene II (Scheme I) carries two terminal methylene groups in the side chain consistent with the observations on the IR and mass spectra mentioned above. As indicated above, because one of the terminal methylene groups was shown to be located at C-24, the other must be located at C-20 just as in the case of dammaradienol (Scheme I,I). Accordingly, the structure of the new triterpene alcohol (Scheme I,II) may be d e t e r m i n e d as 2 4 - m e t h y l e n e d a m m a r e n o l ( 2 4- m e t h y 1e n e-5~-dammar-20 [ 21 ] 7en-3/3-o1). Moreover, two broad singlets were observed in the NMR spectra of the triterpene alcohol II and its acetate, and these signals may be attributable to the methylenic protons on C-22 and C-23, both being located at a-positions relative to the olefinic carbons (16). Hydrogenation of the II acetate in the same way as described above afforded the tetrahydro c o m p o u n d (Scheme I,IV), 24-methyldammaranol (a mixture of the C-20 epimers), which on recrystallization showed mp 151-154 C (plates). The IR spectrum showed no peaks correlating with the terminal methylene group observed in the spectrum of the II. RecrystaUization followed by hydrolysis of the acetate gave free alcohol as needles, mp 121-122 C. Mass spectrum of the alcohol (Scheme I,IV) showed M+ at m/e 444 (C31Hs60, 21%) with the other principal ions at m/e 429 (M - CH3, 6%), 426 (M - H20, 8%), 411 (M - [CH 3 + H 2 0 ] , 5%), 317 (M - side chain), and 299 (M - [side chain + H 2 0 ] , 60%), and base peak at m/e 95. The presence of the relatively strong ion at m/e 317 (M - side chain) accompanied with the stronger ion involving loss of water, as is the case with the alcohol IlI, reveals that the side chain of the
24-METHYLENEDAMMARENOL IN SHEA BUTTER
alcohol (Scheme I,IV) is fully saturated. The IV acetate showed the signals related to the skeletal methyls at 0.87 (strong), 0.97, and 2.06 (3J3-OCOCH3) ppm, and the free alcohol IV indicated those at 0.79, 0.87, 0.97, and 0.99 ppm, respectively. The chemical shifts of these signals are almost identical with those observed for the triterpene II. On the other hand, a doublet related to the 26,27-geminal dimethyl protons was observed at 0.88 ppm, while the broad singlets appeared at 2.16, 2.18, and 4.76 ppm in the spectrum of the triterpene II were disappeared in the spectra of the IV for both the acetate and free alcohol. These facts suppose that during hydrogenation the side chain of the II was fully saturated and produced the IV (Scheme I). Gas chromatographic evidence affords further support to the structure IV for the hydrogenated product of the new triterpene II. The free alcohol IV indicated a major peak, one of the C-20 epimers, at RRT 1.19 with a shoulder peak in the front side in GLC as was observed also in the case of dammaranol (Scheme I,III) (major peak RRT 0.91) mentioned above. From these retention data, the separation factor IV/III was calculated as 1.31 the value was found identical with the corresponding separation factor of 24-methyl/24-unsubstituted determined on 5a-lanostane series compounds under the same GLC condition (17). Thus, the triterpene (Scheme I,IV) is regarded as a 24-methyl homologue of dammaranol (Scheme I,III), i.e., 24-methyldammaranol. Dammaradienot from Dammar Resin
An authentic specimen of dammaradienol used for the identification of the triterpene I was isolated from dammar resin as follows. Dewaxed dammar (70 g) was prepared from coarsely ground resin in a similar manner as described by Mills and Werner (7). It was suspended in hexane:benzene (4:1, 500 nil), poured onto the column of alumina (650 g), and eluted with the same solvent system. The first fraction (22.8 g) eluted with 7 liters of the eluent was left aside. The second fraction (8.4 g) eluted with 6 liters of the eluent was found to consist of nearly equal proportions of dammaradienol (RRT 1.11) and an unidentified keto alcohol (RRT 2.11). A portion (2.2 g) of the second fraction was subjected to preparative TLC (0.5 mm thick silica gel plates; eluent, hexane:ether 4:1). The zone closer to the solvent front gave dammaradienol (0.5 g), mp 136-138 C (fine needles), after recrystallization (lit [7], mp 136-138 c). This on acetylation afforded the acetate (~'99% pure by GLC), mp
811
150-152 C (plates) (lit [7], mp 151-153 C). In the literature (7), Mills and Werner reported that free dammaradienol showed IR absorptions at 3620, 3070, 1640, and 895 cm-1 in CS 2. The IR spectrum of dammaradienyl acetate isolated in this study showed the presence of terminal methylene group (3070, 1641, and 898 cm "1) and trisubstituted double bond (828 and 818 cm-l). Mass spectrum of free dammaradienol gave M+ at m/e 426 (C30H500, 30%), with the other principal ions at m/e 411 (M - CH3, 3%), 408 (M - H 2 0 , 2%), 393 (M [CHa + H 2 0 ] , 4%), 315 (M - [side chain + 2HI, 17%), and 297 (M - [side chain + 2H + H 2 0 ] , 11%), and base peak at m/e 109. In the NMR spectrum, dammaradienyl acetate indicated methyl signals at 0.87 (strong), 0.98, 1.63, 1.70, and 2.05 (3j3-OCOCH 3) ppm. Taking advantage of the observations by Lehn (18) on dammarane series triterpenes, the strong overlapping singlets at 0.87 ppm are attributed to 103-, 14a, 4a-, and 43-methyl protons, and the singlet at 0.98 p p m to 8/3methyl protons. A pair of the two signals at 1.63 and 1.70 p p m is related to the 26,27dimethyl protons deshielded by A24-bond (14), and the broad singlet observed at 4.76 (ca. 2H) ppm is assignable to C-20 terminal methylene protons. Further, the signals appearing at 4.51 (1H, m) and 5.14 (1H, t, J 4.2 Hz) are attributable to 3a-H and 24-H, respectively. Free dammaradienol gave the signals of the angular methyls, 26,27-dimethyl, C-20 terminal methylene, and 24-H, with scarcely any changes in their chemical shifts from those observed for the acetate mentioned above:83-methyl (0.99 ppm), 1013-, and ]4a-methyls (0.88 ppm), 26,27-dimethyl (1.63 and 1.70 ppm), C-20 terminal methylene (4.74 ppm), and 24-H (5.16 ppm). The 4a- and 4/3-methyl signals were, in this case, observed as two separate singlets, one of which was at 0.78 p p m and the other was overlapped with the signal at 0.99 ppm. Taking into consideration the observation of AS-lanostane series compounds by Barton, et al. ( t 9 ) , the signals at 0.78 and 0.99 ppm were attributed to 4~-methyl and 4o~-methyl protons, respectively. Moreover, a broad singlet observed at 2.05 (ca. 2H) ppm in the spectrum is presumably related to the C-22 methylenic protons which are located at a-position relative to the olefinic carbon (16). In the spectrum of the acetate mentioned above, the presence of this signal remained indistinct because of the overlapping 3~-acetoxyl signal just at 2.05 ppm. Consequently, the triterpene I isolated from shea butter is reasonably identified as dammaradienol, and the new triterpene II isolated along with the triterpene I from shea butter LIPIDS, VOL. 10 NO. 12
812
T. I T O H , T. T A M U R A , A N D T. M A T S U M O T O
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24-METHYLENEDAMMARENOL IN SHEA BUTTER
must be a 24-methylene homologue of the I, i.e., 24-methylenedammarenol. Table I summarizes the NMR signals of the dammarane series triterpenes measured in this study. ACKNOWLEDGMENTS Fuji Oil Manufacturing Co. and Cashew Co. provided shea butter and dammar resin, respectively. T. Wainai and K. Mashimo provided the facilities of NMR measurements, T. Miyao helped us in optical rotation measurements, and S. Ogawa and H. Tsukamoto gave technical assistance. Y. Toyama contributed comment and advice. REFERENCES 1. Itoh, T., T. Tamura, and T. Matsumoto, Lipids 9:173 (1974). 2. Heilbron, LM., G.L. Moffet, and F.S. Spring, J. Chem. Soc. 1934:1583. 3. Heilbron, I.M., E.R.H. Jones, and P.A. Robins, Ibid. 1949:444. 4. Lawrie, W., W. Hamilton, F.S. Spring, and H.S. Watson, Ibid. 1956:3272. 5. Lawrie, W., F.S. Spring, and H.S. Watson, Chem. Ind. 1956:1458.
6. Itoh, T., T. Tamura, and T. Matsumoto, Lipids 10:454 (19'75). 7. Mills, J.S., and A.E.A. Werner, J. Chem. Soc. 1955:3132. 8. Mills, J.S., Ibid. 1956:2196. 9. Mills, J.S., Chem. Ind. 1956:189. 10. Godson, D.H., F.E. King, and T.J. King, Ibid. 1956:190. 11. Cosserat, L., G. Ourisson, and T. Takahashi, Ibid. 1956:190. 12. Vroman, M.E., and C.F. Cohen, J. Lipid Res. 8:150 (1967). 13, Wyllie, S.G., and C.Djerassi, J. Org. Chem. 33:305 (1968). 14. Scallen, T.J., and W. Krueger, J. Lipid Res. 9:120 (1968). 15. Tamura, T., K. Takeshima, and T. Matsumoto, Yukagaku 11:212 (1962). 16, Jackman, L.M., and S. Sternhell, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry," Second edition, Pergamon Press, Oxford, England 1969, p. 161. 17. Itoh, T., T. Tamura, T. Iida, and T. Matsumoto, Steroids 26:93 (1975). 18. Lehn, J.M., Bull. Soc. Chim. Fr. 1963:1832. 19. Barton, D.H.R., D.M. Harrison, G.P. Moss, and D.A. Widdowson, J. Chem. Soc. (C) 1970:775.
[Received July 3, 1975]
A Guide for Author= it Located in Lipid= 10(January):60 (1975) LIPIDS, VOL. 10, NO. 12
Nihon University, 8, Kanda Surugadai, 1-chome, Chiyoda-ku, Tokyo, 101 Japan ABSTRACT
57 /~A, ion source temp 160-180C, sample temp 100-120 C, and accelerating high voltage A new triterpene alcohol was isolated 4.6 kv. The samples were introduced directly from shea butter and its structure was into the ion source through a vacuum lock. shown to be 24-methylenedammarenol Preparative argentation thin layer chroma(2 4- m e t h y l e n e - 5 a-dammar-20 [ 21 ]-entography (TLC) for the fractionation of triter3~-o I ) . Dammaradienol (5a-dammarapene acetates was carried out on the 20 x 20 20121],24-dien-3~-o1) also was isolated cm plates coated with 0.5 mm of silica gel from shea butter. (Wakogel B-10, Wako Pure Chemical Industries Ltd., Osaka, Japan) impregnated with 10% or INTRODUCTION 20% silver nitrate, using a Toyo continuous Previous work has indicated the presence of f l o w development preparative TLC (Toyo a-amyrin (1), ~-amyrin (1,2), butyrospermol Roshi Kaisha Ltd., Tokyo, Japan). The plate (1,3,4), parkeol (1,5), and 24-methylenedihy- was developed for 1 hr using hexane:benzene droparkeol (6) in the triterpene alcohol fraction (7:3) as solvent. Separated zones were observed of shea butter. Shea butter, obtained from the under ultraviolet light (3600 A) after spraykernels of Butyrospermum parkii, a sapotace- ing of a rhodamine-6G solution in ethanol ous tree of tropical Africa, is a substance used on the developed plate, and were cut off and as food by the natives and for the manufacture quantitatively extracted with ether. The argenof soap and other products. This paper describes tation silicic acid column was prepared as dethe isolation and identification of a new dam- scribed previously (1) in a similar manner as m a r a n e series triterpene alcohol, 24-meth- proposed by Vroman and Cohen (12). Gas y l e n e dammarenol (24-methylene-5a-dammar- liquid chromatography (GLC) was performed 20121]-en-3t3-ol) (Scheme I,II), and damma- under the same operating conditions as described previously (1), using a Shimadzu GC-5A r a d i e n o l (5a-dammara-20[21 ],24-dien-3r (Scheme I,I) from shea butter. Dammaradienol gas chromatograph (Shimadzu Seisakusho Ltd., (Scheme I,I) has only been known as a compo- Kyoto, Japan) equipped with a flame ionization n e n t of d a m m a r a n e series triterpenes in detector and a 2 m x 3 mm inside diameter dammar resin originating from trees of the glass column packed with 3% OV-17 on Gas Chrom-Z, 80-100 mesh. Relative retention time Dipterocarpaceae family (7-11). (RRT) was expressed by the ratio of the retention time for the substance under examination EXPERIMENTAL PROCEDURES to the retention time for sitosterol. Other proMelting points were determined with a Micro cedures such as saponification of fat, and mp apparatus (Yanagimoto Seisakusho Ltd., hydrogenation (platinum oxide catalyst, ether Kyoto, Japan) and uncorrected. All recrystaUi- solution), acetylation, and hydrolysis of triterzations were performed in acetone:methanol. penes were carried out in a similar manner as Infrared (IR) spectra (KBr) were obtained with described previously (1). a Type IRA-2, IR spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan). Optical rotaRESULTS AND DISCUSSION tions w e r e measured in CHC13 using a Carl Zeiss Polarimeter 0.01 ~ (Carl Zeiss, Ober- Isolation of Dammaradienol and kochen, Germany). Concentrations used were 24-Methylenedammarenol from Shea Butter indicated in parentheses as g/100 ml. Nuclear Unsaponifiable material (156 g) separated magnetic resonance (NMR) spectra in CDC13 from shea butter (3 kg) was acetylated in the were recorded on a JNM-C-60-HL instrument same manner as described previously (1). The (60 MHz, Japan Electron Optics Laboratory acetate (150 g) was then chromatographed on Co., Tokyo, Japan) and calibrated against argentation silicic acid (430 g) using hexane as internal tetramethylsilane as 0 ppm. Mass eluent at first. The eluate was monitored perispectra were taken with a Hitachi RMU-7M odically by GLC to remove the eluate fractions mass s p e c t r o m e t e r (Hitachi Ltd., Tokyo, (130.2 g in total, eluted with 5 liters hexane), Japan), electron energy 70 eV, target current consisting almost exclusively of the acetates of 808
24-METHYLENEDAMMARENOL IN SHEA BUTTER
809
SIDE C H A I N ( R )
at m/e 393.3537 (M- [CH 3 + H 2 0 ] , 393.3519, 2%), 315.2659 (M - [side chain + 2H], 315.2686, 5%), and 297.2570 ( M - [side chain , (i) (II) + 2H + H20] , 297.2581,3%), and base peak at m/e 109.0997. Because the sterols with unsaturated side chain are known to give a characteristic ion resulting from the loss of the entire C-17 ( III ) ( iv ) substituent together with the rearrangement of two hydrogen atoms (13), this alcohol, giving SCHEME I. Diagram of the skeleton and side chains the ion at m/e 315.2659, is expected to have a of dammarane series of triterpene alcohols. C8-side chain with two double bonds. These a-amyrin, lupeol, and butyrospermol. The col- double bonds were indicated by the IR specumn was then eluted with benzene (13 liters in trum of the I acetate (Scheme I) to exist as total) to give an eluate fraction (17.5 g) which trisubstituted double bond (831 and 820 cm-1) c o n t a i n e d considerable amounts of several and terminal methylene group (3080, 1642 and other triterpene acetates besides the above men- 885 cm-1). The I acetate (Scheme I) showed tioned three triterpene acetates. This fraction methyl signals at 0.88 (strong), 0.99, 1.64, was further fractionated on argentation silicic 1.71, and 2.05 (3fl-OCOCH3) ppm in the NMR acid column (packing t78 g) to give a fraction spectrum. The other signals also were observed (4.3 g) eluted with benzene: ether (4:1, 1 liter), at 4.51 (3a-H, multiplet [m]), 4.76 (ca. 2H, preceded by a fraction (6.5 g) ehited with ben- broad singlet [b], terminal methylene), and zene (4 liter). The fraction (4.3 g) was then 5.14 (1H, triplet [t], J 4.2 Hz) ppm. Because a fractionated by preparative argentation TLC to pair of the signals at 1.64 and 1.71 ppm is chargive a fraction (800 mg) which indicated a acteristic to the 26,27-dimethyl protons of the Rf-value almost identical with that of the au- A24-sterols (14), the trisubstituted double thentic specimen of dammaradienyl acetate iso- bond shown by IR spectrum is attributable to lated from dammar resin and was found by A24-bond. The triplet at 5.14 ppm is, hence, GLC to contain the acetates of I and II assignable to 24-H. In the spectrum of the free (Scheme I) in nearly equal proportions. Because alcohol I (Scheme I), the signals at 0.88 and the repetition of preparative argentation TLC 0.98 ppm, the part of which might be attriof this fraction gave a single broad zone instead butable to the angular methyls, and, further, of two separate zones, the upper and lower por- the signals at 1.64 and 1.72 (26,27-dimethyl), tions of the zone were cut off separately and 4.74 (b, terminal methylene), and 5.16 (t, J 4.2 the material from each portion was repeatedly Hz, 24-H) ppm, were still observed with worked up by preparative TLC in a similar scarcely any changes in their chemical shifts manner to give eventually two fractions; the from those observed for the I acetate. On the fraction from the upper portion of the zone other hand, the signals appeared also at 0.79 gave the I acetate (Scheme I) (68 rag, GLC (ca. 3H), 2.05 (b, ca. 2H), and 3.24 (3a-H) ppm purity 95%) and the other fraction from the in the spectrum of the free alcohol I. The triterlower portion of the zone afforded the II ace- pene I was found identical with the authentic specimen of dammaradienol isolated from damtate (Scheme I) (80 mg, GLC purity 93%). mar resin described later in its IR, NMR, and Both the hydrolyzed triterpene alcohols I mass spectra, RRT in GLC and mp, as well as and II (Scheme 1) showed a mobility identical TLC behavior. The alcohol (Scheme I,I) isowith that of the authentic dammaradienol in lated from shea butter is, therefore, identified TLC using silica gel plate (eluent, hexane:ether as d a m m a r a d i e n o l (5a-dammara-20[21 ],244:t). dien-3fl-o 1). Hydrogenation of the I acetate for 3 hr at Dammaradienol from Shea Butter room temperature gave the tetrahydro comand Its Tetrahydro Derivative pound, dammaranyl (Scheme I,III) acetate ( a The I acetate (Scheme I) (RRT 1.35) thus mixture of the C-20 epimers), which on recrysisolated from shea butter crystallized as plates, tallization showed mp 139-140 (plates)(lit [8], mp 151-153 C, [a]~)1+59 ~ (c 0.81) (lit [7] a mixture of the C-20 epimers of dammaranyl dammaradienyl acetate [a]D +60~ 9 Hydro- acetate, mp 139-141 C). The IR spectrum lyzed I (Scheme I) (RRT 1.11) showed mp showed no absorptions correlated to any of the 136-138 C (fine needles). Mass spectrum of the t e r m i n a l methylene and the trisubstituted a l c o h o l ( S c h e m e I) showed M+ at m/e double bond. Mass spectrum of the alcohol 426.3836 (C30H5oO , requires 426.3859, rela- ( S c h e m e I , I I I ) showed M+ at m/e 430 tive intensity 16%) with the other principal ions ( C 3 0 H S 4 0 , 30%) with the other ions at m/e LIPIDS, VOL. 10, NO. 12
810
T. ITOH, T. TAMURA, AND T. MATSUMOTO
415 (M - CH3, 10%), 412 (M - H20, 9%), 397 (M - [CH3 + H 2 0 ] , 14%), 317 (M -side chain, 73%), and 299 (M - [side chain + H 2 0 ] , 98%), and base peak at m/e 95. The presence of the strong ion (M - side chain), which is characteristic for the sterols with a saturated side chain (13), indicates that the side chain of the alcohol (Scheme I,III) is fully saturated. The free alcohol III (Scheme I) showed a predominant peak, related to one of the C-20 epimers, at RRT 0.91 with a shoulder in the front side in GLC. In the NMR spectrum of the III acetate, the signals attributable to the skeletal methyls appeared, with almost identical chemical shifts with those observed for the I acetate, at 0.87 (strong), 0.97, and 2.06 (3~-OCOCH3) ppm. A triplet assignable to 3a-H was observed at 4.50 (J 7.2 Hz) ppm. On the other hand, the 26,27-geminal dimethyl protons gave a doublet at 0.89 (J 5.4 Hz) ppm, indicating that the triterpene III (Scheme I) has a C-24 saturated side chain (14). The disappearance of the signals related to the C-20 terminal methylene and 24-H shows further that the side chain of the III is fully saturated. The free alcohol III (Scheme I) gave the methyl signals at 0.80, 0.87, 0.89 (d, J 6.0 Hz), 0.97, and 1.00 ppm, and a triplet at 3.22 (J 7.2 Hz, 3a-H) ppm. 24-Methylenedammarenol from Shea Butter and Its Tetrahydro Derivative
The new triterpene II acetate (Scheme I) (RRT 1.47) isolated from shea butter showed mp 145-147 C (plates), [a]~1+62 ~ (c 0.81). Hydrolysis of the II acetate gave free alcohol II (Scheme I) (RRT 1.21), mp 131-133 C (fine needles). In the mass spectrum, the free alcohol II showed M+ at m/e 440.4017 (C31H520, 440.4015, 20%), with the other principal ions at m/e 315.2652 (M - [side chain + 2H], 20%) and 297.2577 (M - [side chain + 2H + H 2 0 ] , 70%), and base peak at m/e 109.1028. The presence of a relatively strong ion M - (side chain + 2H) at m/e 315.2652, as is the case with the alcohol I, reveals that this alcohol (Scheme I,II) has a saturated ring system and a C9-side chain with two double bonds. The two double bonds in the side chain were recognized to exist as the terminal methylene groups because the absorptions at 3080, t 640, and 886 cm-1 were observed without any of other olefinic bands in the IR spectrum of the alcohol. The chemical shifts of the skeletal methyl signals in the NMR spectra of the triterpene alcohol II (Scheme I) and its acetate were found almost identical with those of the respective derivatives of the triterpene I (Scheme I) (dammaradienol) the acetate II (Scheme I) indicated those at 0.88 (strong), 0.99, and 2.06 LIPIDS, VOL. 10, NO. 12
(3~-OCOCH3) ppm; and the free alcohol II gave those at 0.80, 0.88, and 0.99 ppm. This fact shows unequivocally that the ring system structure of the new triterpene II (Scheme I) is identical with that of the triterpene I, i.e., 5a-dammarane type ring system. On the other hand, the triterpene II (Scheme I) gave a signal of the 26,27-geminal dimethyl protons as a doublet at 1.06 (J 6.6 Hz) ppm. This is characteristic for the sterols possessing a C-24 termin a l m e t h y l e n e group such as 24-methylenecycloartanol (24-methylene-9/3,19-cyclo-5alanostan-3t3-ol) (15). Hence, the new triterpene II is considered to possess a C-24 terminal methylene group. In the NMR spectra of the triterpene alcohol II (Scheme I) and its acetate, a broad singlet related to terminal methylene group was observed also at 4.76 ppm with an intensity (ca. 4H) stronger than that observed for the triterpene I (Scheme I) (dammaradienol) possessing one terminal methylene group at C-20. This indicates that the triterpene II (Scheme I) carries two terminal methylene groups in the side chain consistent with the observations on the IR and mass spectra mentioned above. As indicated above, because one of the terminal methylene groups was shown to be located at C-24, the other must be located at C-20 just as in the case of dammaradienol (Scheme I,I). Accordingly, the structure of the new triterpene alcohol (Scheme I,II) may be d e t e r m i n e d as 2 4 - m e t h y l e n e d a m m a r e n o l ( 2 4- m e t h y 1e n e-5~-dammar-20 [ 21 ] 7en-3/3-o1). Moreover, two broad singlets were observed in the NMR spectra of the triterpene alcohol II and its acetate, and these signals may be attributable to the methylenic protons on C-22 and C-23, both being located at a-positions relative to the olefinic carbons (16). Hydrogenation of the II acetate in the same way as described above afforded the tetrahydro c o m p o u n d (Scheme I,IV), 24-methyldammaranol (a mixture of the C-20 epimers), which on recrystallization showed mp 151-154 C (plates). The IR spectrum showed no peaks correlating with the terminal methylene group observed in the spectrum of the II. RecrystaUization followed by hydrolysis of the acetate gave free alcohol as needles, mp 121-122 C. Mass spectrum of the alcohol (Scheme I,IV) showed M+ at m/e 444 (C31Hs60, 21%) with the other principal ions at m/e 429 (M - CH3, 6%), 426 (M - H20, 8%), 411 (M - [CH 3 + H 2 0 ] , 5%), 317 (M - side chain), and 299 (M - [side chain + H 2 0 ] , 60%), and base peak at m/e 95. The presence of the relatively strong ion at m/e 317 (M - side chain) accompanied with the stronger ion involving loss of water, as is the case with the alcohol IlI, reveals that the side chain of the
24-METHYLENEDAMMARENOL IN SHEA BUTTER
alcohol (Scheme I,IV) is fully saturated. The IV acetate showed the signals related to the skeletal methyls at 0.87 (strong), 0.97, and 2.06 (3J3-OCOCH3) ppm, and the free alcohol IV indicated those at 0.79, 0.87, 0.97, and 0.99 ppm, respectively. The chemical shifts of these signals are almost identical with those observed for the triterpene II. On the other hand, a doublet related to the 26,27-geminal dimethyl protons was observed at 0.88 ppm, while the broad singlets appeared at 2.16, 2.18, and 4.76 ppm in the spectrum of the triterpene II were disappeared in the spectra of the IV for both the acetate and free alcohol. These facts suppose that during hydrogenation the side chain of the II was fully saturated and produced the IV (Scheme I). Gas chromatographic evidence affords further support to the structure IV for the hydrogenated product of the new triterpene II. The free alcohol IV indicated a major peak, one of the C-20 epimers, at RRT 1.19 with a shoulder peak in the front side in GLC as was observed also in the case of dammaranol (Scheme I,III) (major peak RRT 0.91) mentioned above. From these retention data, the separation factor IV/III was calculated as 1.31 the value was found identical with the corresponding separation factor of 24-methyl/24-unsubstituted determined on 5a-lanostane series compounds under the same GLC condition (17). Thus, the triterpene (Scheme I,IV) is regarded as a 24-methyl homologue of dammaranol (Scheme I,III), i.e., 24-methyldammaranol. Dammaradienot from Dammar Resin
An authentic specimen of dammaradienol used for the identification of the triterpene I was isolated from dammar resin as follows. Dewaxed dammar (70 g) was prepared from coarsely ground resin in a similar manner as described by Mills and Werner (7). It was suspended in hexane:benzene (4:1, 500 nil), poured onto the column of alumina (650 g), and eluted with the same solvent system. The first fraction (22.8 g) eluted with 7 liters of the eluent was left aside. The second fraction (8.4 g) eluted with 6 liters of the eluent was found to consist of nearly equal proportions of dammaradienol (RRT 1.11) and an unidentified keto alcohol (RRT 2.11). A portion (2.2 g) of the second fraction was subjected to preparative TLC (0.5 mm thick silica gel plates; eluent, hexane:ether 4:1). The zone closer to the solvent front gave dammaradienol (0.5 g), mp 136-138 C (fine needles), after recrystallization (lit [7], mp 136-138 c). This on acetylation afforded the acetate (~'99% pure by GLC), mp
811
150-152 C (plates) (lit [7], mp 151-153 C). In the literature (7), Mills and Werner reported that free dammaradienol showed IR absorptions at 3620, 3070, 1640, and 895 cm-1 in CS 2. The IR spectrum of dammaradienyl acetate isolated in this study showed the presence of terminal methylene group (3070, 1641, and 898 cm "1) and trisubstituted double bond (828 and 818 cm-l). Mass spectrum of free dammaradienol gave M+ at m/e 426 (C30H500, 30%), with the other principal ions at m/e 411 (M - CH3, 3%), 408 (M - H 2 0 , 2%), 393 (M [CHa + H 2 0 ] , 4%), 315 (M - [side chain + 2HI, 17%), and 297 (M - [side chain + 2H + H 2 0 ] , 11%), and base peak at m/e 109. In the NMR spectrum, dammaradienyl acetate indicated methyl signals at 0.87 (strong), 0.98, 1.63, 1.70, and 2.05 (3j3-OCOCH 3) ppm. Taking advantage of the observations by Lehn (18) on dammarane series triterpenes, the strong overlapping singlets at 0.87 ppm are attributed to 103-, 14a, 4a-, and 43-methyl protons, and the singlet at 0.98 p p m to 8/3methyl protons. A pair of the two signals at 1.63 and 1.70 p p m is related to the 26,27dimethyl protons deshielded by A24-bond (14), and the broad singlet observed at 4.76 (ca. 2H) ppm is assignable to C-20 terminal methylene protons. Further, the signals appearing at 4.51 (1H, m) and 5.14 (1H, t, J 4.2 Hz) are attributable to 3a-H and 24-H, respectively. Free dammaradienol gave the signals of the angular methyls, 26,27-dimethyl, C-20 terminal methylene, and 24-H, with scarcely any changes in their chemical shifts from those observed for the acetate mentioned above:83-methyl (0.99 ppm), 1013-, and ]4a-methyls (0.88 ppm), 26,27-dimethyl (1.63 and 1.70 ppm), C-20 terminal methylene (4.74 ppm), and 24-H (5.16 ppm). The 4a- and 4/3-methyl signals were, in this case, observed as two separate singlets, one of which was at 0.78 p p m and the other was overlapped with the signal at 0.99 ppm. Taking into consideration the observation of AS-lanostane series compounds by Barton, et al. ( t 9 ) , the signals at 0.78 and 0.99 ppm were attributed to 4~-methyl and 4o~-methyl protons, respectively. Moreover, a broad singlet observed at 2.05 (ca. 2H) ppm in the spectrum is presumably related to the C-22 methylenic protons which are located at a-position relative to the olefinic carbon (16). In the spectrum of the acetate mentioned above, the presence of this signal remained indistinct because of the overlapping 3~-acetoxyl signal just at 2.05 ppm. Consequently, the triterpene I isolated from shea butter is reasonably identified as dammaradienol, and the new triterpene II isolated along with the triterpene I from shea butter LIPIDS, VOL. 10 NO. 12
812
T. I T O H , T. T A M U R A , A N D T. M A T S U M O T O
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24-METHYLENEDAMMARENOL IN SHEA BUTTER
must be a 24-methylene homologue of the I, i.e., 24-methylenedammarenol. Table I summarizes the NMR signals of the dammarane series triterpenes measured in this study. ACKNOWLEDGMENTS Fuji Oil Manufacturing Co. and Cashew Co. provided shea butter and dammar resin, respectively. T. Wainai and K. Mashimo provided the facilities of NMR measurements, T. Miyao helped us in optical rotation measurements, and S. Ogawa and H. Tsukamoto gave technical assistance. Y. Toyama contributed comment and advice. REFERENCES 1. Itoh, T., T. Tamura, and T. Matsumoto, Lipids 9:173 (1974). 2. Heilbron, LM., G.L. Moffet, and F.S. Spring, J. Chem. Soc. 1934:1583. 3. Heilbron, I.M., E.R.H. Jones, and P.A. Robins, Ibid. 1949:444. 4. Lawrie, W., W. Hamilton, F.S. Spring, and H.S. Watson, Ibid. 1956:3272. 5. Lawrie, W., F.S. Spring, and H.S. Watson, Chem. Ind. 1956:1458.
6. Itoh, T., T. Tamura, and T. Matsumoto, Lipids 10:454 (19'75). 7. Mills, J.S., and A.E.A. Werner, J. Chem. Soc. 1955:3132. 8. Mills, J.S., Ibid. 1956:2196. 9. Mills, J.S., Chem. Ind. 1956:189. 10. Godson, D.H., F.E. King, and T.J. King, Ibid. 1956:190. 11. Cosserat, L., G. Ourisson, and T. Takahashi, Ibid. 1956:190. 12. Vroman, M.E., and C.F. Cohen, J. Lipid Res. 8:150 (1967). 13, Wyllie, S.G., and C.Djerassi, J. Org. Chem. 33:305 (1968). 14. Scallen, T.J., and W. Krueger, J. Lipid Res. 9:120 (1968). 15. Tamura, T., K. Takeshima, and T. Matsumoto, Yukagaku 11:212 (1962). 16, Jackman, L.M., and S. Sternhell, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry," Second edition, Pergamon Press, Oxford, England 1969, p. 161. 17. Itoh, T., T. Tamura, T. Iida, and T. Matsumoto, Steroids 26:93 (1975). 18. Lehn, J.M., Bull. Soc. Chim. Fr. 1963:1832. 19. Barton, D.H.R., D.M. Harrison, G.P. Moss, and D.A. Widdowson, J. Chem. Soc. (C) 1970:775.
[Received July 3, 1975]
A Guide for Author= it Located in Lipid= 10(January):60 (1975) LIPIDS, VOL. 10, NO. 12
