Stigmasterol and Stigmasterone from Methanol Extract of Calophyllum soulattri Burm . F . Stem Bark H )

Received: 11th November 2020 Revised: 30 March 2021 Accepted: 30 March 2021 Online: 30th April 2021 Stigmasterol and Stigmasterone from Methanol Extract of Calophyllum soulattri Burm. F. Stem Bark. Calophyllum soulattri Burm. F. has been widely used for herbal medicine. Phytochemical investigation of C. soulattri contains a secondary metabolite of the steroid class. Steroid compounds have various biological activities, such as anti-inflammatory, antioxidant, antiproliferative, antibacterial, antimalarial, and anticancer. Two secondary metabolites steroids have been isolated and identified from the stem bark extract of C. soulattri. Isolation was carried out through the extraction (maceration), fractionation, and purification stages. Maceration is carried out using methanol as a solvent. Fractionation was carried out by vacuum liquid chromatography (VLC), and purification was by flash column chromatography. Identification of combined fractions and determination of pure isolates were used through thin-layer chromatography (TLC). The solvent used in the chromatography methods was a mixture of n-hexane and ethyl acetate. The structure isolates were identified by FTIR, XH NMR, and 13C NMR and compared with literature data. Secondary metabolites steroids that have been isolated are identical compounds to stigmasterol and stigmasterone.


Introduction
The Calophyllum genus is a type of plant that is often found in the tropical forests of Indonesia. The community has used this plant as herbal medicine, including a diuretic, blood pressure, rheumatism, malaria, sexually transmitted diseases, varicose veins, hemorrhoids, infections of the skin, nephritis, and antiinflammatory drugs [1]. One species of the genus Calophyllum is Calophyllum soulattri, known as slatri.
Exploration of compounds in the stem bark of C. soulattri has not been widely carried out, especially the isolation of secondary metabolites of the steroid group. The steroid group has anti-inflammatory, antidiabetic [6], antioxidant, anti-tumor, anti-osteoarthritis, antimutagenic [ 7 ], and antibacterial activities [8].
Therefore, it is necessary to explore C. soulattri , especially in the stem bark section, contributing to adding a database of steroid group compounds. Furthermore, the database can be used as a source for potential medicinal compounds.
The C. soulattri stem bark powder ( 2.5 kg) was macerated using methanol (10 L) solvent for 3 x 24 hours. The filtrate was evaporated to give 385 g of thick blackish brown extract. The 13 g methanol extract was fractionated using VLC with a solvent mixture of nhexane: ethyl acetate (10: ; 0:10) with a grading system that produced 20 fractions. The selection of fractions to be purified based on TLC analysis. TLC results were seen with a UV lamp ( 254 ) then sprayed with spotting reagent Ce(S 04 ) 2. The fractions having the same TLC profile were combined and purified by flash column chromatography.
Based on the TLC profile, fractions 1-9 were further purified using a mixture of eluent n-hexane: ethyl acetate with a ratio of 9.5 : 0.5 (200 mL); while each ratio of 9: 1, 8:2, 7 : 3 , 6: 4 , and 5 : 5 in a volume of 100 mL, and 150 mL of 100% acetone, which gives 72 fractions. The fractions F 19 -21 and F28 were chosen to be tested for purity because they showed one spot. The fraction F 19 -21 hereinafter referred to as F 19 , and F28 were identified for their structure using FTIR, XH NMR, and 13 C NMR spectroscopy methods.

. Identification of F 19 Compound
The isolation of the stem bark extract of C. soulattri resulted in two pure isolates, namely F 19 and F28. Analysis of the IR spectrum of F 19 shows the presence of hydroxy group ( -OH) absorption at around 3400 -3500 cnr 1 (broad). The IR spectrum also shows the presence of aliphatic C-H stretching vibrations at around 2900 -2800 cm -1 (sharp) and weak absorption of alkenes (C = C) around 1600 cm -1 (sharp). The absorption at 1400 cnr 1 (sharp) is the absorption of CH2 bending, while the absorption at around 1000-1100 cnr 1 (sharp) is the absorption of cycloalkanes. Based on FTIR results, and compared with literature data, isolate F 19 is a steroid compound [ 9 ]. The FTIR absorption data are shown in Figure 1 and Table 1. Further analysis of the NMR and 13 C NMR data was carried out.    were then compared with literature data [ Figure 4 . Stigmasterol which has been isolated from the stem bark of C. soulattri, is also found in many other plants, such as Neocarya macrophylla [11], Ficus hispida [12], and Terminalia schimperiana [ 13 ]. Based on the analysis of FTIR, TI NMR, and 13 C NMR data, as well as the results of comparisons with literature data, compound F 19 is a compound identical to stigmasterol (F 19* ) with the molecular formula C 29 H 48 O, as shown in Figure 4 . Stigmasterol which has been isolated from the stem bark of C. soulattri , is also found in many other plants, such as Neocarya macrophylla [11], Ficus hispida [12], and Terminalia schimperiana [ 13 ].

. Identification of F28 compound
Analysis of the F28 compound IR spectrum shows the absorption at around 3400 -3500 cm 1 (broad ), which is characteristic of the hydroxy (-OH) group. The presence of aliphatic C-H stretching vibration and alkene (C = C) vibration was shown at around 2900 -2800 cm 1 (sharp) and around 1600 cm -1 (sharp). The presence of a carbonyl group (C = 0) is indicated by absorption at around 1700 cm ' 1 (sharp). The absorption at 1400 cnr 1 (sharp) is the CH2 bending vibration. Besides, there is cycloalkane absorption at around 1000-1100 cm 1 (sharp). The IR spectrum of F28 compared with the literature [ 9 , 14 ] are shown in Figure 5 and Table 3 .
Further analysis of the JH NMR and 13 C NMR data was carried out.
H-25 ). There is an alkene proton signal on chemical shift 5.17 (lH, dd, / = 8.55 ; 15.1 Hz, H-22); 5.04 (lH, dd, / = 8.6; 15.1 Hz, H-23 ), which is the main feature of the steroid framework. This lH NMR data analysis is supported by 13  were compared with literary data [ 9 , 10], as shown in Table 4 .   [ 9 , 11] C alkene CDCIj C carbonyl Identification of F28 was carried out in CDC 13 solvent using NMR spectroscopy, including 13   Based on the overall results of FTIR, XH NMR, and 13 C NMR data analysis, and compared with literature data, the suggested compound structure for compound F28 is similar to stigmasterone with the molecular formula C 29 H 46 O [ 15 ], the structure of stigmasterone is shown in Figure 2. Based on a literature review, stigmasterone was first discovered in the bark of C. soulattri. Two secondary metabolites of the steroid class that have been isolated from the bark of C. soulattri, namely stigmasterol and stigmasterone, contribute to increasing the database of C. soulattri, which can then be used as a source of medicinal compounds. Based on the literature review, stigmasterone was isolated from C. soulattri for the first time. Apart from C. soulattri stigmasterone, it is found in Amaranthus spinosus [16] and Virola surinamensis [ 17 ].