Potential Antibacterial Activity of Bioactive-sitosterol from Root Bark of Rhizophora apiculata from Lampung Coastal H )

Received: 2nd February 2021 Revised: 26th March 2021 Accepted: 27th March 2021 Online: 30th April 2021 -sitosterol is an essential bioactive phytosterol naturally present in plant cell membranes. It has a coincident structure with animal cholesterol. This investigation reported isolation, structure analysis, and an antimicrobial assay of -sitosterol from the root bark of Bakau Minyak ( Rhizophora apiculata ) from Lampung coastal. The isolation of -sitosterol was carried out through maceration using methanol, separation by vacuum liquid chromatography (VLC), and purification by column chromatography (CC) using ethyl acetate/nhexane (2:8) as eluent. The structure of -sitosterol was determined using spectroscopic analysis (UV-Vis, FT-IR, 13C-NMR, TI-NMR, DEPT, and GC-MS). The pure -sitosterol has 107.4 mg of white needle crystalline compound, the compound melting point about 140.7-141.2°C, the molecular mass confirmed by m/z 414, and UV absorption detected at antimicrobial bioactivity assay has shown potential activity to be developed as a lead compound against E.coll

Mangrove forests found in Indonesia 's coastal areas exhibit habitats with high biodiversity of animals, plants, and microorganisms [11]. Mangroves have been reported to generate steroid, alkaloids, and terpenoids [16], which have biological benefits such as antidiabetic [ 17 ], antibacterial [18], anticancer [ 19 ], and antioxidant abilities [20,21]. These bioactivity properties of mangroves have impressed pharmaceutical industry interest [22]. This plant has been used as traditional medicines to treat diarrhea and asthma [ 23 ], scabies treatment, and rheumatism [ 24 ]. R. apiculate or known as Bakau Minyak in Lampung (Figure 1), is common mangrove growing on the coastal zones of Lampung. This plant is hardy, tannin-rich, and has a high density, mainly used for construction and charcoal making [ 23 ]. Bakau Minyak has a high salt tolerance. These natural properties provide several bioactive compounds produced in response to environmental stresses. The root bark of Bakau Minyak, which part has direct interaction with seawater, is mainly reported containing tannins and steroids [ 25 ], indicating it has a potential source of -sitosterol. evaporator HEI-VAP (Heidolph). The UV spectrum was obtained using UV-Vis (Shimadzu 1200). The melting point was measured using Melting Point Apparatus (Toledo HL 3 A). The -sitosterol crystals were placed in a capillary glass tube, which was then inserted on the heating side. The crystals were heated until they melted, and the temperature was recorded. The IR spectra were obtained by FTIR spectroscopy (Scimitar 2100) using KBr pellets. The mass spectrum were acquired by GC-MS model QP2010 (Shimadzu ) equipped with a VF 5 column mass spectrometer (flow rate 1.8 mL / min) with diameter of 0.25 mm, length of 30 m, thickness of 0.25 pm and under 200°C ion source / -70 eV at 40 -650°C with a flow rate of 4°C/ min (carrier gas was He). 13 C and NMR spectra were obtained by JEOL variant 500 MHz spectroscopy in acetone-d6 and TMS as the zero-shift standards.

Extraction and Isolation
All extraction and isolation steps of -sitosterol are shown in Figure 2. The dried powder of R. apiculata root bark ( 532 g) was extracted with methanol ( 4 x 2 L) under low pressure to obtain a final residue ( 58.7 g) of crude methanol extract (CME). The extraction occurred in about 72 hours with further removal of the solvent at a rotary evaporator at 50°C/ 120 rpm. CME was partitioned in a solvent to present hexane (HE) ( 17.2 g) and ethanol ( 21.4 g) extracts (EE). This study focused on the HE fractions because the EE fraction has high tannin content, making it more difficult to eliminate the tannin tint in the separation process [ 25 ]. 15.5 g HE was chromatographed using the vacuum liquid chromatography method, eluted using a gradient solvent by increasing the polarity of the eluent (hexane / ethyl acetate) to produce 33 fractions. The fractions 3 , 4 , and 5 were re-chromatographed using silica gel G-60 and eluted with hexane: ethyl acetate ( 4 : 1, v / v). Among the 16 fractions obtained, fractions 3 -9 were purified using a silica gel column to give a compound mixture ( 403.7 mg). The fraction of 403.7 mg was chromatographed on the silica gel column to produce 12 fractions. Fractions 4 -9 yielded compound 1 ( 115.8 mg). After recrystallization in hexane, this fraction was identified as -sitosterol ( 107.4 mg). Although R. apiculata plays an essential role in traditional medicine in many tropical countries [11,23 ], including Indonesia, only limited research has been scientifically reported to explore the bioactive and antimicrobial compound activity of this plant. Thus, the present study aims to investigate the antimicrobial activity of the bioactive -sitosterol from the root bark of R. apiculata to increase the potential of mangrove forests on the Lampung coast, -sitosterol was isolated using several chromatography techniques. Structural elucidation was carried out by spectroscopy analysis. The antimicrobial assay was determined by the agar diffusion method against Escherichia coli, Bacillus subtilis, and Aspergillus niger.

Methodology
The root bark of Bakau Minyak was obtained from the Research Center of Marine Development Cultivation (

. Antimicrobial Assay
The fungal and bacterial strains were acquired from the microbiology laboratory of The Indonesian Institute of Sciences LIPI-Cibinong and Biochemistry laboratory of the Chemistry department, University of Lampung collections. The antimicrobial assay was determined against E. coli, B. subtilis,and A. niger by the agar diffusion method. Both fungi and bacterial were cultured in nutrient broth (NB) medium with a conical flask serial, incubated at 37°C/24 h. Pre-warmed NB agar plates were seeded with io 7 -io 8 CFU suspension of bacteria and fungi. The paper discs ( 5 mm Whatman assay disc) were drilled into the wells plate, then the compound and the extract were slowly added until discs were filled. After all filled discs were placed in an upright position and then incubated at 37°C for 24 h, the inhibition zones were measured in millimeters ( mm). The antimicrobial assay protocol was carried out under aseptic conditions, based on a standard procedure adapted from Balouiri etal [26] with some modification.

. Results and Discussion
properties presents a proton connected to the carbon, which is correlated with the OH groups. The overlapping triplets at 5.38 ppm are assigned to the olefinic protons.        Figure 7 . The peak at m / z 301 corresponds to [M-113 ] or the loss of ( -CsHiy), the peak at m / z 273 resembles to [M-141 ] or loss of aliphatic chain ( -C10H21), and the peak at m / z 396 parallels to [M-18] or loss of hydroxy group. Based on spectroscopy analysis and comparison data from the previous report, the structure of the pure isolated compound is determined as -sitosterol. The crude extract exhibits more inhibition zones than pure isolated of -sitosterol because the crude extract contains many other bioactive compounds that have not been identified as shown in Figure 9 . Based on the zone of inhibition (ZOI) classification, according to Davis and Stout [28], the four strength that correspond to ZOI diameters: > 20 mm are classified as very strong inhibition, 10-20 mm as strong inhibition, 5 -10 mm as moderate inhibition and < 5 mm is classified as no inhibition. The -sitosterol had the potential to fight E. coli with moderate activity compared to the two positive controls as summarized in Table 3 .  Table 2 show that the -sitosterol data NMR spectrum is similar to the reported by Patra et al [ 27 ]. All the NMR data ofsitosterol are summarized in Table 2 and modelled in Figure 8.  Thesitosterol has been registered as a non-toxic and safe supplement, -sitosterol has been registered as a nontoxic and safe supplement. It has potential bioactivities in several applications, including antibacterial activity.

. Conclusion
This study reported the isolation and structure elucidation of -sitosterol from the root bark of Bakau Minyak from Lempasing coastal, South Lampung. This study supported the use of Bakau Minyak as a traditional medicine to treat several diseases such as infections by E. coli. The antibacterial activity of -sitosterol from the root bark of R. apiculata exhibited potential bioactivity to be developed as a lead compound for bacterial infections. The high potency of -sitosterol and its analogs in [