Chemical composition, antibacterial and antioxidant activities of acetone extract from the branches and leaves of Jasminum annamense subsp. annamense (Oleaceae)

Jasminum annamense subsp. annamense is a rare subspecies of Jasminum annamense belonging to the Oleaceae family. The aims of this study were to address the chemical profiles, antibacterial and antioxidant activities of acetone extract isolated from branches and leaves of Jasminum annamense subsp. annamense for the first time. The chemical constituents of acetone extracts of studied samples were investigated by gas chromatography-mass spectrometry. There were a total of 24 components identified from the leaf extract, including lup-20(29)-en-3-one (27.93%), levodopa (19.68%), transcinnamic acid (7.58%), linolenic acid (6.35%) as the major compounds. Meanwhile, 26 components were reported from the branch extracts which are sorbitol (25.74%), lupeol (13.3%), cis-vaccenic acid (6.97%), glycerin (6.35%) and n-hexadecanoic acid (5.86%) were the main components. The two acetone extracts of J. annamense subsp. annamense exhibited antibacterial effect against Bacillus cereus, Escherichia coli, Salmonella enteritidis and Staphylococcus aureus based on disk diffusion assay. In addition, leaf and branch extracts of the studied species also display notable antioxidant activity in the ABTS assay with IC50 values of 311.75±3.39 and 664.46±3.732 μg/ml, respectively. This is the first report on the chemical and biological properties of J. annamense subsp. annamense and provides a promising perspective for developing good sources of antioxidant and antimicrobial compounds against both Gram positive and negative bacteria.


INTRODUCTION
Vung Tau Provinces (Dam et al., 2020;Bui, 2016). To date, only one of our previous works reported the chemical components and biological properties of this species. Accordingly, Dam et al. showed 7 compounds from ethanol extracts isolated from J. annamense subsp. annamense leaves and stems using liquid chromatography/mass spectrometry analysis. Furthermore, these extracts also possessed antibacterial effects against Bacillus cereus and Salmonella typhimurium (Dam et al., 2020). Consequently, the information on the chemical composition and biological activities of acetone extracts of J. annamense subsp. annamense are limited. The present study, therefore, firstly investigated the chemical components, antibacterial and antioxidant properties of acetone extracts obtained from leaves and branches of J. annamense subsp. annamense.

Plant Material
Specimens of J. annamense subsp. annamense were collected from Binh Chau-Phuoc Buu Nature Reserve, Ba Ria-Vung Tau Province, Vietnam ( Figure 1). The location of collection is about 10 o 32'47"N; 107 o 28'57"E and the area was around 43 m above the mean sea level.

Bacterial Strains
The antibacterial activity of the acetone extracts from the leaves and the branches of J. annamense subsp. annamense was tested against four bacterial strains, including two Gram-negative bacteria (Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13976) and two Gram-positive bacteria (Bacillus cereus (ATCC 11774), Staphylococcus aureus (ATCC 25923). The strains were preserved in 20% glycerol solution at 20 o C and activated by cultivation in Luria-Bertani broth at 37 o C for 24 h prior to the antibacterial activity assay.

Extraction Procedures
The J. annamense subsp. annamense leaves and the branches were moderately dried at 50 o C until their weights were unchanged. The dried specimens were ground into powder using the electric grinder. Subsequently, 50g of the dried powder were macerated in 250 ml of 99% acetone solution at room temperature for 72 hours. The Whatman paper was used to filter the studied extracts. The process was repeated twice. The filtrate was concentrated under reduced pressure at 60 o C to obtain the brown extract, subsequently subjected to sublimation drying to completely remove the remaining acetone (Bobinaitė et al., 2013).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The chemical composition of the obtained acetone extracts of studies species was analyzed on Gas Chromatography-Mass Spectrometry (GC-MS) system equipped with an Agilent 7890A GC coupled with a 5975C VL MSD Triple-Axis selective Detector equipped with a ZB-5MS capillary non-polar column (30.0m length x 0.25mm i.d. x 0.25µm film thickness). Helium was the carrier gas used at a constant pressure of 13.209 psi. The injection temperature was 250°C; the injection volume was 0.1 µl; the split ratio was 10:1. The oven temperature was programmed to proceed from 60°C to 240°C at the rate of 3°C/min. The constituents were identified on the basis of a comparison between their mass spectra with the internal library (NIST 2017 library and the Wiley 8 th edition libraries). The equation proposed by van den Dool and Kratz was utilized to calculate the retention indices (arithmetic indices) of the oil components relative to the homologous series of C9-C17 n-alkanes.

Antibacterial Activity Assay
The method described by the CLSI (Clinical and Laboratory Standards Institute) was used to study the antibacterial activity (Clinical and Laboratory Standards Institute, 2010). The tested bacterial strains were inoculated into LB Broth for growing until the turbidity of 0.5 McFarland standards. A bacterial culture of 100 µL of bacterial culture was spread on a sterile Mueller Hinton plate. The sterile 6 mm diameter discs were placed on the surface of the inoculated Mueller Hinton plate. Each disc was then added with 10 µl of the essential oil. The plate was incubated at 37 o C for 24h and the antibacterial activity of the sample was determined via the measurement of the inhibition zone diameter of the tested bacteria. Sterilized distilled water was used as a negative control whereas Gentamycin antibiotic discs (supplied by Nam Khoa BioTek, Vietnam) were used as a positive control.

Determination of Antioxidant Activity of Extract
The ABTS radical scavenging properties of the acetone extracts from J. annamense subsp. annamense leaves and the branches were identified using Maeng et al. (2017) work. Firstly, 7 mM ABTS was added to 2.45 mM K 2 SO 4 of the sample. The mixture was slightly shaken and placed in the dark for 18 hours at 37 o C (solution A). 0.1 mL studied extract was mixed in 3 mL solution A. This mixture was diluted to 5 ml of acetone and slightly shaken and placed in the dark for 15 minutes. The UV-vis spectrophotometer (UVS 2800, Labome, USA) and UVWin6 Software v6.0.0 were used to record the absorbance of the solution at 734 nm. The reference standard was ascorbic acid. Ascorbic acid  Van et al. standard curve (0-15 ppm) was constructed with the equation y = -0.0278x + 0.421, R2 = 0.9990, where y is the absorbance at 734 nm and x is the sample concentration (µg/mL). The sample concentration was calculated from the standard curve equation and the results were expressed as µg/mL ascorbic acid.

Data Analysis
The antibacterial assay was conducted in triplicate. The oneway analysis of variance (ANOVA) and Fisher's least significant difference (LSD) procedure (Statgraphics software (Centurion XV)) were used to analyze the experimental results as well as significant differences among the means from triplicate analyses at (p<0.05). The results were presented as mean ± standard deviation (SD).

Antioxidant Activities of Acetone Extracts from J. annamense subsp. annamense
The antioxidant effects of two studied extracts depends on the extract concentrations ( Figure 3). Accordingly, the leaf and branch extracts of the studied species display notable antioxidant activity in the ABTS assay with IC 50 values of 311.75±3,39 and 664.46±3,732 µg/ml, respectively.
Several previous studies have been reported the antioxidant activities of different Jasminum plants. For instance, the antioxidant activity of the leaf ethanol extract of J. abyssinicum was determined using DPPH assay (IC 50 = 26.3 µg/mL) while 023.7 µg TE/mg extract was shown by the value of ORAC assay towards the same extract (Tauchen et al., 2015). At dose 25-400 µg/mL, the three extracts of J. arborescens leaves such as chloroform, petroleum and ethanol showed the antioxidant activities with DPPH inhibition ranging from 40-90% (Bhagath   al., 2010). In another study, the leaf ethanol extract of J. auriculatum possessed DPPH scavenging effect with an IC 50 value of 33.39 µg/mL and total phenolic content of 8.47 mg GAE/g (Arun et al., 2016). In addition, the antioxidant properties of the hydromethanolic and boiling water extracts of J. grandiflorum flower buds using different scavenging assays have been reported. As a result, the antioxidant activities of hydromethanolic extract evaluated by hydroxyl peroxide, nitric oxide, superoxide and DPPH had IC 50 values of 403.31, 225.51, 1354.30 and 189.93 µg/mL, respectively whereas 397.09, 38.27, 327.89 and 150.57 were as shown by the value of four assays towards the same extracts (Arun et al., 2016).

CONCLUSION
In this study, the chemical compositions, antibacterial and antioxidant properties of acetone extract from branches and leaves Jasminum annamense subsp. annamense were investigated for the first time. As a result, 24 compounds were reported from the leaf extract such as lup-20(29)-en-3-one (27.93%), levodopa (19.68%), trans-cinnamic acid (7.58%), linolenic acid (6.35%) as the major components. There were a total of 26 components identified from the branch extract which was characterized by the prominence of sorbitol (25.74%), lupeol (13.3%), cisvaccenic acid (6.97%), glycerin (6.35%) and n-hexadecanoic acid (5.86%). The two studied extracts were found to be effective against Bacillus cereus, Escherichia coli, Salmonella enteritidis and Staphylococcus aureus based on disk diffusion assay. In addition, leaf and branch extracts of the studied species also display notable antioxidant activity in the ABTS assay with IC 50 values of 311.75±3.39 and 664.46±3.732 µg/ml, respectively.