Evaluation of antibacterial activities from major bioactive constituents of the whole plant of Hedyotis corymbosa

Triterpenes Hedyotis corymbosa is locally known as rumput mutiara from the Rubiaceae family, widely distributed in tropical regions of Asia. Researchers provided scientific evidences the beneficial impact of this plant for pharmacologic alactivity. This study aimed to isolate and evaluate the bioactive constituents based on their biological activities. In this study, the whole plant of H. corymbosa was extracted using methanol. Extract of H. corymbosa was sequentially partitioned using ethyl acetate and n-butanol. The ethyl acetate layer was further fractionated and isolated using chromatographic techniques to obtain the pure compounds. The bioactive compounds structure was determined using spectroscopic analysis especially the Nuclear Magnetic Resonance (NMR). The investigation of H. corymbosa resulted in the isolation of eight compounds, were identified as ursolic acid, 3βhydroxyolean-11-en-28,13β-olide, β-sitosterol, stigmastane-3,6-dione, ferulic acid, scopoletin, 2-hydroxy-1-methoxyanthraquinone, 3-hydroxy-1,2-dimethoxyanthraquinone. The antimicrobial effect of the crude extract, partitions and fractions were evaluated using agar well diffusion for antimicrobial susceptibility test and for the pure compounds were evaluated using minimum inhibitory concentration. The ethyl acetate layer and crude extract displayed the higher antimicrobial activities than nbutanol and water layer. The Minimum Inhibitory Concentration (MIC) for the pure compound was shown that most of the compounds have the ability to inhibit human pathogenic bacteria with average 100 μg/mL. The antimicrobial activities showed by the crude extracts, fractions, and pure compounds of H. corymbosa can be used as a commercial product for antimicrobial agent against S. aureus, S. enterica, E. coli and B. substilis.


Introduction
Hedyotis corymbosa (L). Lam (syn; Oldenlandia coymbosa), is a flowering plant from the Rubiaceae family and commonly called diamond flower (or rumput mutiara). Most of the species from the Rubiaceae family possess medicinal properties and is used as ingredients in Chinese Herbal Medicine for treatment of cancer, anti-inflammatory and hepatoprotective in male rat has been reported (Lin et al., 2002) Previous phytochemical studies on some of genus Hedyotis showed that the genus contained iridoids, flavonoids, anthraquinones, alkaloids, lignans, coumarins and triterpenes (Ahmad et al., 2006). Since then, glucan (Cui et al., 2006), naphthoquinones and cyclotides (Ding et al., 2014) have been obtained from the species of this genus. A comparative chemotaxonomy study of different species demonstrated that iridoids are the predominant and characteristic constituents of genus Hedyotis (Chen et al., 2005;Van Long et al., 2013).
Based on the traditional uses, researchers provided substantial scientific evidence revealing the health beneficial impact of this plant. This study aimed to isolate and evaluate the typical components based on the secondary metabolites of the whole plant of H. corymbosa.

Plant material
The whole plant of H. corymbosa were collected in around Department of Biological Science and Technology, NPUST, Pingtung county Taiwan,

ORIGINAL RESEARCH
Open Access 40 fresh sample of H. corymbosa were transferred to cutting and drying process. The total weight of the dried sample from the whole plant of H. corymbosa was 9.8 kg. The dried sample of H. corymbosa was macerated by methanol 95% for a 10 days. The methanol as a result of maceration was evaporated using rotary evaporation at 50°C.

Identification by Nuclear Magnetic Resonance (NMR)
1 H NMR and 13 C NMR spectra were measured using Varian Mercury Plus 400 MHz, NMR. Proton or carbon nuclear test was conducted by dissolved samples in a deuterated solvent of chloroform-d or dimethylsulfoxide (DMSO), kept the sample in 5 mm NMR tube, determined by NMR which yield the peaks as electromagnetic wave absorption signal. Chemical shift is expressed in parts per million (ppm) unit relative to TMS as an internal standard (Lambert and Eugene, 2004)

Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of the compound was determined using the microdilution technique with 96 well plates and resazurin as an indicator cell growth based on Sarker et al. (2007), was assessed on four pathogenic bacteria from both gram positive (S. aureus and B. substilis) and gram negative bacteria (E. coli and S. enterica).

Minimum Inhibitory Concentration (MIC)
The MIC of the compounds was determined using microdilution with resazurin as an indicator of cell growth. The data obtained through MIC revealed variability in the inhibitory concentrations for five pure compounds (S1, S2, An1, An2, T1) against pathogenic bacteria. The MIC values of the different compound against the gram negative bacteria and gram positive bacteria were found in the range of 4.69 -600 µg/mL. The MIC value of each isolated compound was shown in Table 1. According to Holetz et al. (2002), the isolated compound less than 100 µg/mL is good antimicrobial activity, from 100-500 µg/mL is moderate antimicrobial activity, whereas the MICs over than 1000 is inactive. From Table 13, results showed that almost of the isolated compounds were considered good antimicrobial activity against the test strains. Except, compound An1 and T1 showed moderated activity against S. enterica and S. aureus and compound An1 shown weak activity against S. aureus.