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Triterpenoid and Steroid from the Rind of Chisocheton macrophyllus (Meliaceae)

Terpenoids form numerous and structurally diverse family of secondary metabolites derived from C5 isoprene units joined in a head-to-tail fashion. Among the terpenoids, triterpenoids emerge as a unique and large group of phytochemicals. There is secondary metabolites steroid, which is modified triterpenoids containing tetracyclic ring system but losing three methyl at C-4 and C-14. Triterpenoids and steroids can be obtained in the family Meliaceae. The Chisocheton genus which is part of the Meliaceae family is known for secondary metabolites, such as terpenoids, phenolics, and steroids. The purpose of this research is to isolate and to elucidate triterpenoids and steroid compounds from rind of Chisocheton macrophyllus. Stigmastane-type steroid Stigmast-22-en-3β-ol (1) and pentacyclic triterpenoid moronic acid (2), were isolated from the n-hexane extract of the rind of C. macrophyllus using column chromatography. The chemical structures of compounds 1-2, were identified on the basis of spectroscopic evidence and by comparison with those spectra previously.


Introduction
Terpenoids are a large and structurally diverse family of natural compounds derived from C 5 isoprene units. Isoprene was known as a fundamental building block to form terpenoids. Dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) identified as active isoprene units derived by mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway. Terpenoids structure joined in a head-to-tail fashion, classified as hemiterpenes (C 5 ), monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), sesterterpenes (C 25 ), triterpenes (C 30 ), and tetraterpenes (C 40 ) 1 . Among the terpenoids, triterpenoids emerge as a unique and large group of phytochemicals 2 . Triterpenoids were not formed by an extension of adding IPP but two molecules of farnesyl diphosphate (FPP) are joined in a tail-to-tail fashion resulting squalene skeleton 1 . Squalene can fold into two different conformation resulting two different cations such as dammarenyl cation and protosteryl cation. Protostheryl cation undergoes further cyclizations and series of proton,methyl Wagner-Meerwein 1,2-shisfts until form a lanosterol skeletal structure. Lanosterol is a precursor for cholesterol and other steroids in animals. In plants, cycloartenol take a role as a precursor for steroids 1 1,3 . All steroid classes and their metabolites play important roles in the physiology and biochemistry of living organism 3 .
During the course of our continuing search for novel pentacyclic triterpenoid and stigmastanetype steroid from the Chisocheton species, we isolated and described the pentacyclic triterpenoids, Betulonic acid from C. macrophyllus 4 and stigmastane-type steroid, 7Į-hydroxy-ȕ-sitosterol, stigmasta-4,6-diene-3-one, stigmasterol, 7ȕ-methoxy-3ȕ-sitosterol, stigmast-5-en-3ȕ-ol, and ȕsitosterol-3-O-acetate from C. tomentosus 5 , C. macrophyllus 6 , and C. cumingianus 7 In the further screening for pentacyclic triterpenoids and stigmastane-type steroids in Chisocheton species, we found that the methanolic extract of C. macrophyllus exhibited the presence the triterpenoid and steroid based on the phytochemical test. C. macrophyllus is a higher plant and mainly distributed in Sumatera, Java, and Papua Island in Indonesia 8 . Previous phytochemical studies reported the presence of limonoids, steroids, and triterpenoids from leaves, seed, and stembark of C. macrophyllus 4,6,9 . Triterpenoids and steroids from the rind of C. macrophyllus is still unknown. We report herein the isolation and structure elucidation of pentacylic triterpenoids and stigmastane-type steroids.

General Experimental Procedure
Melting points were measured on a melting point M-565 apparatus. IR spectra were recorded by Perkin Elmer Spectrum 100 FT-IR Spectrometer in KBr. 1 H-and 13 C-NMR spectra of compound 1 and 2 were obtained with a JEOL 500 MHz spectrometer, using TMS as internal standard. Mass spectra were determined with Waters Q-TOF Xevo mass spectrometer instrument. Chromatographic separations were carried out on Merck silica gel 60 (70ñ230 mesh and 230ñ400 mesh). TLC glass plates were precoated with silica gel GF254 (Merck, 0.25 mm). Spots on the plates were detected under UV light (254 and 365 nm) and visualized by spraying with 10% sulfuric acid in ethanol, followed by heating.

Plant Materials
The rind of C. macrophyllus was collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia in August 2018. The plant was identified by Center for Plant Conservation Botanic Gardens, Bogor, Indonesia.

Extraction and Isolation
Powder of dried rind of C. macrophyllus (3.2 kg) was extracted with methanol at room temperature, filtered, and concentrated. The methanol extract (335.1 g) was resuspended in a mixture of water and methanol, then extracted with n-hexane, ethyl acetate, and n-butanol successively. Each extract was evaporated to yield the n-hexane extract (156.2 g, 5.4%), ethyl acetate extract (62.8 g, 2.2%), and n-butanol extract (40.2, 1.3%). The n-hexane extract (156.2 g) was separated by vacuum liquid chromatography on silica gel 60 using a gradient n-hexane and ethyl acetate to give fourteen subfractions (A-N). Subfraction E (51.0 g) was subjected to silica gel column chromatography using gradient elution of n-hexane-ethyl acetate to afford eight subfractions (E1-E8). Subfraction E5 (11.1 g) was further separated by column chromatography on silica gel using gradient elution of n-hexanechloroform-ethyl acetate to afford six subfractions (E5a-E5f). Subfraction E5b (2.9 g) was separated by column chromatography on silica gel using gradient n-hexane-chloroform-ethyl acetate to give three subfractions (E5b1-E5b3). Subfraction E5b2 (540.7 mg) was further separated by column chromatography on ODS using gradient of water-methanol to afford compound 2 (31.2 mg). Subfraction K (11.3 g) was further separated by column chromatography on silica gel using gradient

Result and Disscussion
The n-hexane extract of the rind of C. macrophyllus was subjected to column chromatography and recrystallization to afford stigmastane-type steroid stigmast-22-en-3ȕ-ol (1) and pentacyclic triterpenoid moronic acid (2). Compounds 1 were isolated from this plant for the first time. A total twenty nine carbon resonances were observed in the 13 C-NMR spectrum. These were assigned by DEPT and HSQC experiments to six methyls, eleven sp 3 methylenes, seven sp 3 methines, two quaternary carbons, two olefinic methines at į C 129.3 and 138.2, and one oxygenated methine carbons at į C 71.3. These functionalities accounted for one out of the total five degrees of unsaturation. The remaining four degrees of unsaturation were consistent with the molecule containing four rings which characteristic for stigmastane-type steroid structure 10 .
The presence of stigmastane-type steroid structure in compound 1 was confirmed from HMBC experiment, (Figure 2). The HMBC spectra showed the correlation between proton of methyl groups Me-18, Me-19, Me-21, Me-26, Me-27, Me-29 with neighbor carbons supporting the presence of stigmastane-type steroid in 1.
The stereochemistry of compound 1 were determined based on NOESY spectrum. In the NOESY spectrum showed cross peaks from į H 1.03 (H-9) to 3.44 (H-3) were indicated that H-3 is Į orientation consequently configuration for hydroxyl at C-3 is ȕ orientation.
A comparison of the NMR data of compound 1 with those of stigmasterol isolated from Sesbania grandiflora 10 , revealed that the structures of the two compounds are similar but different at C-5 and C-6 consequently compound 1 was identified as stigmast-22-ene-3ȕ-ol.  29.0 q A total thirty carbon resonances were observed in the 13 C-NMR spectrum. These were assigned by DEPT and HSQC experiments to seven methyls, ten sp 3 methylenes, three sp 3 methines, six quaternary carbons, one querternary sp 2 carbon at į C 136.6, one olefinic carbons at į C 133.4, one carboxyl group at į C 181.2, and one ketone carbonyl at į C 218.2. These functionalities accounted for three out of the total eight degrees of unsaturation. The remaining five degrees of unsaturation were consistent with the molecule containing oleanane-type pentacyclic triterpenoid structure 11 .
A comparison of the NMR data of compound 2 with those of moronic acid isolated from Phoradendron reichenbachianum 11 , revealed that the structures of the two compounds are similar, consequently compound 2 was identified as moronic acid.