Structure Of Stigmasterols in Bran of Red Rice from Minahasa Regency, North Sulawesi, Indonesia

Diterima 13 Maret 2020 Disetujui 26 April 2020 Rice bran contained important bioactive phytochemical components such as tocopherol, tocotrienol and γ-oryzanol, and one of the component of γoryzanol is stigmasterol. The aims of the present study was to isolate and identify stigmasterol in native red rice bran extract. The isolation of sterol from red rice bran extract was performed by using Thin Layer Chromatography (TLC) and followed by identification by Fourier Transformed Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). The TLC single spot originated from crude red rice hexane extract was observed at 365 nm UV and the FTIR spectra analysis result showed that OH group was indicated by 3436,9 cm-1 absorption. While absorption regions of 2931,6 to 2852,5 cm-1 indicates the presence of single bond CH. The data of 1H-, 13Cand 2D-NMR of rice bran sterol indicated that the single spot obtained in TLC analysis was stigmasterol. It can be concluded that native red rice variety of North Sulawesi contained stigmasterol and potential as native antioxidant source. Key word: red rice bran Stigmasterol North Sulawesi *e-mail: emmamoko@unima.ac.id *Telp: +62 82231939278 Introduction Rice is the staple of Asia including Indonesia, where consumption's have assumed to 22.28 million tons per year [1]. In Indonesia there are several varieties of rice, among them are Cigeulis, Ciherang, IR64 and several varieties of sticky rice that are pigmented like the red rice. Rice bran is a by product of rice milling process that contains protein, dietary fiber, and natural antioxidant [2]. Pigmented rice contains procyanidine, anthocyanine and other phenolic compounds with antioxidative activities such as tocopherols, tocotrienols, oryzanols and phytosterols [3] that has many health benefits like ehancing immunity, reducing cancer risks and hepatoprotective benefits. While many previous studies have exhibited the bioactivities of rice bran from pigmented rice in regards to inhibiting activities of tyrosinase, also antialergic, anti-inflamatory, anti-mutagenic and anti-glycation activities [2]. βsitosterol is the major sterol in rice oil much as in common vegetable oils, and more than 75% of rice bran-oil sterols are esterified and collectively known as oryzanol. γ-oryzanol, which contains sterols and ferulic acids called cycloartanyl ferulate or triterpene alcohol ferulate, has anti-cancer properties and is unique to the rice plant [4]. Further research reported cycloartenol trans-ferulate (m/z 601), 24methylenecycloartanol trans-ferulate (m/z 615), campesterol trans-ferulate (m/z 575), and sitosterol trans-ferulate (m/z 589) were the major components, representing up to 90 % in the control brown rice and averaged of 75 % in the wild rice samples. Indeed, the amounts of 24-methylenecycloartanol trans-ferulate (249 μg/g), cycloartenol trans-ferulate (171 μg/g), and total γ-oryzanol (688 μg/g) obtained in the present study for long grain regular brown rice are in agreement with those reported by [5]. While [6] noted that from four different rice varieties (Super Kernel, 386, 385 and Basmati) of Pakistan contained campesterol, stigmasterol, b-sitosterol, and ∆-5-avenasterol Moko, E. M., Rahardiyan, D., 2020 17 as the major fractions of the rice bran oil sterol. Three different rice varieties of Iran (Khazar, Alikazemi, and Hashemi), all contained βsitosterol, campesterol, ∆-5-avenasterol, ∆-7avenasterol, holesterol dan brassicasterol [7]. Furthermore [8] observed that sitosterol, campesterol, ∆-5-avenasterol and stigmasterol were the main contributor of sterol components of rice bran oil of selected rice varieties in Pakistan. A study on Thai Black non glutinouse rice bran Riceberry results that Thai Black contains four phytosterols namely 24-methyleneergosta-5-en-3β-ol, 24-methylene-ergosta-7-en3β-ol, fucosterol and gramisterols. Also found in Thai Black are triterpenoids consisting of cycloeucalenol, lupenone and lupeol that are found in 2 fractions that displayed strong activities in anti leuchemia poliferations at an IC50 = 2.80 and 32.89 μg/mL [3]. Previous researches by [9] was conducted on the antioxidant characteristics of several varieties of North Sulawesi rice, indicated that crude extract of the rice bran of the red rice variety had better antioxidant characteristics compared to other varieties (Cigeulis and Superwin) which has shown by the DPPH scavenging radical activity of 88.29 ± 5.62% with the lowest IC50 value (26.26 ± 0.95 μg/ml) and highest total antocyanine content (68.61 ± 1.98 mg/g). By understanding the bioactive component profiles especially the stigmasterol content of native rice bran from Minahasa, North Sulawesi, Indonesia, therefore the importance of this research as the foundation for further applied development researches in conjunction with health and functional food of rice bran bioactives. Therefore, the objective of this research was to isolate and elucidate stigmasterol phytosterols from native red variety rice bran from the Minahasa Regency of North Sulawesi, Indonesia. Methodology Rice Bran Sterol Isolation Rice bran extraction was carried out according to the method of [10] that was slightly modified. Red rice variety of rice bran flour samples at one kg each were macerated and extracted with 3000 ml of 70% ethanol three times and kept overnight at room temperature. The ethanol extracts were then fractionated with organic solvents (hexane, ethyl acetate and n-butanol) in accordance with their polarity level. Each extract was prefiltered with Whatman paper No. 42 and then evaporated by rotary evaporator (Buchi rotavapor) under vacuum to obtain the hexane, ethyl acetate and n-butanol crude extract of rice bran. Sterol isolation from 20 mg rice bran hexane crude extract was conducted by chromatography using silica gel 60 Merck as the stationary phase and hexane-ethyl acetate for the eluent at a ratio of 9:1 with a 5% stepwise. Sterol Infrared Spectrum Infrared spectrum was taken with Fourier Transform Infrared Spectrophotometer (FTIR)Shimadzu 8400. The liquid sample is placed between two KBr pellets with the help of capillary tube. Each pellet is made of 0.2 mm thickness and it is placed in the path of the sample beam. The spectra are recorded from 4000 to 450cm-1, the number of scans being 256 at a resolution of 4cm-1, scan speed is 0.20 cm/s. Sterol Structure Elucidation 1H NMR, 13C NMR Sterol structure elucidation was performed by Nuclear Magnetic Resonance (NMR) JEOL JNM-ECA 500 spectrometer (500Mhz), JEOL Tokyo, Japan, with Chloroform-D (CDCl3) as a solvent and tetramethylsilane (TMS=0 ppm) as an internal standard. Chemical shifts are given on a δ scale (ppm). Result and Discussion Sterol Isolation Chromatograph results of hexane rice bran sterols extract was indicated by a single spot visible under 365nm UV. Thin layer chromatograph results can be viewed in figure 1 below. Moko, E. M., Rahardiyan, D., 2020


Introduction
Rice is the staple of Asia including Indonesia, where consumption's have assumed to 22.28 million tons per year [1]. In Indonesia there are several varieties of rice, among them are Cigeulis, Ciherang, IR64 and several varieties of sticky rice that are pigmented like the red rice. Rice bran is a by product of rice milling process that contains protein, dietary fiber, and natural antioxidant [2]. Pigmented rice contains procyanidine, anthocyanine and other phenolic compounds with antioxidative activities such as tocopherols, tocotrienols, oryzanols and phytosterols [3] that has many health benefits like ehancing immunity, reducing cancer risks and hepatoprotective benefits. While many previous studies have exhibited the bioactivities of rice bran from pigmented rice in regards to inhibiting activities of tyrosinase, also antialergic, anti-inflamatory, anti-mutagenic and anti-glycation activities [2].
β-sitosterol is the major sterol in rice oil much as in common vegetable oils, and more than 75% of rice bran-oil sterols are esterified and collectively known as oryzanol. γ-oryzanol, which contains sterols and ferulic acids called cycloartanyl ferulate or triterpene alcohol ferulate, has anti-cancer properties and is unique to the rice plant [4].
Previous researches by [9] was conducted on the antioxidant characteristics of several varieties of North Sulawesi rice, indicated that crude extract of the rice bran of the red rice variety had better antioxidant characteristics compared to other varieties (Cigeulis and Superwin) which has shown by the DPPH scavenging radical activity of 88.29 ± 5.62% with the lowest IC50 value (26.26 ± 0.95 µg/ml) and highest total antocyanine content (68.61 ± 1.98 mg/g).
By understanding the bioactive component profiles especially the stigmasterol content of native rice bran from Minahasa, North Sulawesi, Indonesia, therefore the importance of this research as the foundation for further applied development researches in conjunction with health and functional food of rice bran bioactives. Therefore, the objective of this research was to isolate and elucidate stigmasterol phytosterols from native red variety rice bran from the Minahasa Regency of North Sulawesi, Indonesia.

Methodology Rice Bran Sterol Isolation
Rice bran extraction was carried out according to the method of [10] that was slightly modified. Red rice variety of rice bran flour samples at one kg each were macerated and extracted with 3000 ml of 70% ethanol three times and kept overnight at room temperature.
The ethanol extracts were then fractionated with organic solvents (hexane, ethyl acetate and n-butanol) in accordance with their polarity level. Each extract was prefiltered with Whatman paper No. 42 and then evaporated by rotary evaporator (Buchi rotavapor) under vacuum to obtain the hexane, ethyl acetate and n-butanol crude extract of rice bran. Sterol isolation from 20 mg rice bran hexane crude extract was conducted by chromatography using silica gel 60 Merck as the stationary phase and hexane-ethyl acetate for the eluent at a ratio of 9:1 with a 5% stepwise.

Sterol Infrared Spectrum
Infrared spectrum was taken with Fourier Transform Infrared Spectrophotometer (FTIR)-Shimadzu 8400. The liquid sample is placed between two KBr pellets with the help of capillary tube. Each pellet is made of 0.2 mm thickness and it is placed in the path of the sample beam. The spectra are recorded from 4000 to 450cm-1, the number of scans being 256 at a resolution of 4cm-1, scan speed is 0.20 cm/s.

Result and Discussion Sterol Isolation
Chromatograph results of hexane rice bran sterols extract was indicated by a single spot visible under 365nm UV. Thin layer chromatograph results can be viewed in figure  1 below.      Table 2.  13 12,14,17 By deducing the infrared spectrum results and the spectrums of the nuclear magnetic resonance (NMR), it is suggested that rice bran sterol lipid structure would appear as in Figure 5.

Figure 5. HMBC of isolated rice brand sterol lipid
According to the chemical shift data of 1 H-and 13 C-NMR on Table 3, rice bran sterol is in comparison to stigmasterol, structure-wise results of rice brand sterol compares to other sterol.
Based on the values of the chemical shifts in Table 3 it can be concluded that rice bran sterol chemical shifts are similar to those of stigmasterol. The variance that occurred in shifts of these two compounds are most like due to the differences in solvents used during identification process via NMR and the differences in the NMR facilities that was utilized, therefore it is assumable that sterols isolated from Minahasan red rice bran from North Sulawesi is indeed the IIUPAC stigmasterol with a structure as in Figure 6.   Table 1. The absorption of OH group of 4,4-dimethylsterol from rice bran sampqle was determined at 3440 cm -1 , and [11] also observed a similar absorption at 3465 cm -1 for most probable OH group in unheated commercial rice bran oil and decrease to 3432 cm -1 which believe due to the lack of hydroperoxide formation during heat treatment.
The 1 H-NMR of rice bran sterol in figure  3 indicated the presence of 3 hydrogen signals with double bonds that appeared in shift area of δH 5,34; 5,14 and 4,98 ppm. Hydrogen signal at δH 5,34(H-6) ppm is assumed to be ethylenic carbon methyn that is attached to a double bonded quartenary carbon as illustrated on the fragment with the hydrogen indicated in red, while two hydrogen signals with the same multiplicity in δH5,14(H-22) and 4,98(H-21) ppm, therefore it is assumed to be vicinal hydrogen atoms, adjacent hydrogen atoms at a distance of 3 bonds (vicinal hydrogenhydrogen). The single hydrogen signal on δH3,52 ppm indicated the presences of a hydrogen signal bonded to an oxygenated carbon.
β-cytosterols are known for their biological activities as in decreasing blood cholesterols, anti-inflamation, and anti diabetic, while stigmasterols found in rice bran oil are known for their effects as anti-inflamation and their cytotoxic activity towards cancer cells [14,15,16]. Understanding the phytochemical composition and structures of rice bran oil, especially the compounds contributing to biological activities, will place these results as the foundation for the aplication of phytosterols of rice bran in its future developments in functional food.

Conclusion
The present study showed that a single spot was found in TLC analysis and it is believed as sterol of red rice bran extract, and further analysis using FTIR indicated that the absorption of OH group was observed at 3436.9 and the CH single bond were in the range of 2931.6 to 2852.5 cm -1 . The spectra of 1 H-NMR, 13 CNMR and 2D-NMR confirmed that the substance from native red rice bran variety extract is stigmasterol.