In vitro Evaluation of Crude Extracts and Isolated Compounds from Goniothalamus rongklanus and Goniothalamus latestigma for Bioactive Properties

The genus Goniothalamus belongs to the Annonaceae family. The pure compounds from Goniothalamus rongklanus and Goniothalamus latestigma were isolated by repeated column chromatography. The structures were recognized by NMR spectral methods. The chemical compounds of ethyl acetate extract (EtOAc) from the stems of G. rongklanus were identified as goniotriol (1) and stigmasterol glucoside (2). The EtOAc leaves extract of G. latestigma yielded a compound, which was identified as pinocembrin (3). The ethyl acetate (EtOAc) and methanol (MeOH) extract were tested for anti-HIV-1 RT and cytotoxic activities against P-388, KB, HT 29, MCF-7, A 549, ASK and CL cell lines. The EtOAc and MeOH extract of G. rongklanus showed evidence of anti-HIV-1 RT inhibition at 76.44 and 88.48 %, respectively. The EtOAc extract of G. rongklanus and G. latestigma showed cytotoxic activities on KB and HT 29 with an ED50 at <4 μg/mL. In addition, antibacterial study on extracts and isolated compounds was also performed. Antibacterial study was evaluated using nine strains (Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli 0157: H7, Escherichia coli (ETEC), Escherichia coli (EPEC), Proteus mirabilis, Salmonella typhimuriam, Shigella flexneri and Vibrio cholera) by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) method. Goniotriol was the most effective antibacterial with a MIC in the range <0.16 0.6 mg/mL and MBC in the range 0.3 >5 mg/mL. The antibacterial activity of goniotriol has been reported for the first time.


Introduction
The genus Goniothalamus (Annonaceae) include about 185 species, of which 25 species are found in Thailand 1 . This genus is widely distributed in the tropical forests of Southeast Asia and are used in traditional medicine. Root barks of G. cheliensis and seeds of G. amuyon are used in the treatment of edema and arthritis 2,3 . The stem bark of G. laoticus has been applied as roborant 4 .

Plant Materials
The stems of G. rongklanus (BKF. 152884) were collected from Phuhin Rongkhla National Park, Amphoe Nakhonthai, Phitsanulok Province, Northern Thailand in 2017. The leaves of G. latestigma (BKF. 163333) were collected from Surat Thani provinces, Thailand in 2017. The plant materials were authenticated by Narong Nantasean, The Forest Herbarium, Ministry of Natural Resources and Environment, Bangkok.

General Procedures
1 H (500 MHz), 13 C (125 MHz) and 2D NMR spectra were noted on a BrÜker AV-500 spectrometer in deuterated chloroform (CDCl 3 ) and deuterated methanol (CD 3 OD) solutions. The tetramethylsilane (TMS) was internal standard of chemical shifts (δ) in ppm. The mass spectra were recorded on a Thermo Finnigan Polaris Q mass spectrometer at 70 eV (probe) and EIMS were measured by a BrÜker Esquire apparatus. Infrared spectra (IR) were recorded at potassium bromide (KBr) solid with a Shimadzu 8900 FT-IR spectrophotometer and major bands (λ max ) were noted in wave number (cm -1

Anti-HIV-1 RT (Reverse Transcriptase) Assay
The extracts of G. rongklanus and G. latestigma were tested for anti-HIV-1 RT assay and cytotoxicity at the Service Centre of Department of Microbiology, Mahidol University, Thailand. The anti-HIV activities were determined by testing RT inhibition 15

Cytotoxicity Assay
The extracts of G. rongklanus and G. latestigma were also studied for cytotoxicity, using the standard Sulforhodamine B (SRB) assay. Ellipticine was used as the positive control 15,17 . The concentration of the samples was 20 -0.16 µg/mL in 0.5 % DMSO. The cancer cell lines used were Murine lymphocytic leukemia (P-388), Human oral cavity carcinoma (KB), Human colon adenocarcinoma (HT 29), Human breast adenocarcinoma (MCF-7), Human lung, adenocarcinoma (A 549), Rat glioma cell (ASK) and Chang liver (CL). MEM (minimum essential medium with Earles salt and L-glutamine) in 10% FBS were used for culturing of cell lines. The cell lines were maintained t 37 °C for 72 hours (48 hours for P-388) in 5% CO 2 and 100% relative humidity, followed by stabilizing with 20% trichloroacetic acid at 4 °C for 60 min and then stained for 30 min by 0.4 % SRB in 1% acetic acid at room temperature. The unbound dye was washed by 1% acetic acid, already dried stain was mixed with 10 mM Tris base with pH = 10. The absorbance was read at 510 nm on a microplate reader and 50% effective dose (ED 50 ) was calculated.

Bacterial Strains
In vitro antibacterial studies were carried out against nine strains (

Minimum Bactericidal Concentration (MBC)
The MBC assay was determined for samples which did not showed any visible growth and was subsequently sub-cultured on to nutrient agar plate. These plates were incubated at 37 °C for 24 hours. MBC was recorded only for the lowest concentration of the bacteria that did not retrieve or a single colony. 18

Biological Activities
The bioactivities screening of EtOAc and MeOH extract of G. rongklanus and G. latestigma comprised of anti-HIV-1 RT and cytotoxicity assay on P-388, KB, HT 29, MCF-7, A 549, ASK and CL cell lines. The results are shown in Table 1.
The extract and isolated compounds were studied for antibacterial activity against nine bacterial by micro well dilution assay. The tested concentrations of the extracts ranged between 6.25 -200 mg/mL and 0.16 -5 mg/mL for isolated compounds. The MIC and MBC of samples are shown in Table 2.

Discussion
Compound (1) (a styryllactone) and (2) (a steroid glucoside) isolated from EtOAc stem extract of G. rongklanus were identified as goniotriol 20 and stigmasterol glucoside 19 respectively. Compound (3) (a flavanone) isolated from EtOAc leaves extract of G. latestigma was identified as pinocembrin 21 based on Mass, NMR spectra and analogy with literature data.

Conclusion
Crude extracts of G. rongklanus and G. latestigma yielded three compounds which were identified as goniotriol (1), stigmasterol glucoside (2) and pinocembrin (3). Extracts of G. rongklanus showed effective activity in anti-HIV-1 RT assay. Extracts of G. rongklanus and G. latestigma were cytotoxic against KB and HT 29 cell lines. Goniotriol showed potent antibacterial activity against all tested micro-organisms.

Acknowledgement
Financial support from the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation is gratefully acknowledged.