PRELIMINARY PHYTOCHEMICAL SCREENING, GC-MS PROFILING AND IN VITRO EVALUATION OF BIOLOGICAL ACTIVITIES OF GARCINIA ATROVIRIDIS ROOT EXTRACTS

Nur Salsabila Ahmad Roslan, Seema Zareen, Normaiza Zamri and Muhammad Nadeem Akhtar Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, 26300 Gambang, Malaysia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 01 November 2018 Final Accepted: 03 December 2018 Published: January 2019


…………………………………………………………………………………………………….... Introduction:-
Garcinia atroviridis is commonly known as 'asam gelugur' in Malaysia, India, Myanmar, and Indo-China. This plant species specifically belongs to the Guttiferae. This plant is endemic species in Peninsular Malaysia. It grows wildly in lowland and hill forest up to 600-meter altitude. It was also planted by the locals for its economic and medicinal purpose (MacKeen et al., 2000). The dried G. atroviridis fruits known as 'asam keping' are sold commercially as seasoning. It is sour and is used to season curry, dressing fish and others. G. atroviridis have medicinal values as traditionally, it has been used to treat a cough, the decoction of its leaf with roots can be used to treat an earache, and the juice of the leaf is given to female after delivery (Tisdale et al., 2003;Burkill, 1966). In the previous study (MacKeen et al., 2000), it was reported that the crude methanolic extract of the different parts of the G. atroviridis such as fruit, leaf, stem, and trunk barks showed it possessed antibacterial, antifungal, antioxidant, and antitumorpromoting properties. This study analyses the chemical compositions of the different extracts of G. atroviridis roots via preliminary phytochemical screening and GC-MS. The antibacterial activities of all extracts were determined by the disc diffusion method while antioxidant activities were evaluated based on the DPPH radical scavenging activities.

Solvent-solvent extraction
The dried roots were ground into a coarse powder using a pulverizer. Fine ground pulverized material was dissolved in different solvents as described in Figure 1. Powdered plant material was soaked in methanol for three days at room temperature and the solvent was filtered by using a sieve. This was repeated 3-4 times until the extract gave no coloration. The extract was distilled and concentrated under reduced pressure in the Buchi rotavapor yielding a gumlike residue (45.45 g). The same was repeated with organic solvents and distilled water of increasing polarity (starting with lipophilic solvent n-Hexane, ending with the more hydrophilic n-Butanol). The solvent from each extract was filtered and concentrated under reduced pressure in Buchi rotavapour. Finally, the extracts of n-hexane, dichloromethane, ethyl acetate, and n-butanol were collected, weighed (8.72 g, 4.45 g, 9.79 g and 1.08 g, respectively) and stored in the refrigerator at 4 o C for further phytochemical analysis.

Phytochemical Screening
The extract was subjected to preliminary phytochemical tests to determine the group of secondary metabolites present in the sample. The screening of the phytochemicals such as alkaloids, carbohydrates, anthraquinones, glycosides, saponin, protein and amino acids, phytosteroids, oils and fats, and phenols and flavonoids was carried out by following the standard procedure as mentioned by Sofowora (1993), Trease and Evans (1989) and Harborne (1984).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was carried out by Agilent 7980A series GC instrument. The DB-1MS column with the dimensions of 30 m Χ 0.25 mm capillary column was used for the analysis. The initial temperature was kept at 50 °C for 3 minutes and the maximum temperature was up to 250 °C. Helium was used as a carrier gas at a flow rate of 1.0 mL/min and the total run time was 60 minutes. Interpretation of mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology MS library (NIST-MS library).

Antibacterial Test Test microorganisms
The procedure was according to MacKeen et al., 2000. Four bacterial strains, i.e. Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 10536) and Proteus vulgaris (ATCC 33420) was used in this study. The nutrient broth was prepared, and the microorganism was cultured and incubated in the incubator shaker at 30°C for 24 hours. The concentration of the cultures was adjusted turbidometrically at a wavelength of 600 nm to 10 8 colony forming units (CFU) per mL.

Disc Diffusion Method
The bacterial cultures (10 8 CFU/mL) were inoculated on the prepared nutrient agar. 15 μl of samples with a concentration of 20 mg/mL was loaded onto blank filter paper discs. The loaded disc was placed on the previously inoculated agar. The plates were inverted and incubated in the incubator for 24 hours at 30 °C. The presence of clear inhibition zones around the discs showed the antibacterial activity of the extracts (MacKeen et al., 2000). The disc diffusion tests were presented in triplicate and the antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the extracts. Commercially available antibiotics discs were used as a positive control whilst the solvent used to dissolve the extracts acted as a negative control.

Antioxidant Assay α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging method
The scavenging effects of samples for DPPH radical was determined according to the method by Hui et al. (2017), with slight modification. Briefly, the initial concentration of the sample was prepared at 1 mg/mL as well as concentration for ascorbic acid which acted as standard in this assay. 0.2mM DPPH was prepared freshly and must be protected from light throughout the experiment. 100 μL of DPPH was added to 100 μL of sample with different concentrations. After incubated for 30 minutes in the dark at room temperature, the absorbance of the solution was read at 517 nm. The solution without extract; only methanol and DPPH were used as a control. All test was performed in triplicates. The data were expressed as mean ± standard deviation (SD). The calculation of the percentage of radical scavenging activity was as followed: Abs C, Absorbance of control at t= 30 minutes Abs S, Absorbance of sample t= 30 minutes

Results and Discussion:-Preliminary Phytochemical Screening
The results of the presence of important metabolites in hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), butanol (BuOH) and methanol (MeOH) extracts of Garcinia atroviridis roots are tabulated in Table 1.

GC-MS Analysis
The GC-Mass Spectrometry analysis of all extracts was given in Table 2         0.00 0.00 0.00 0.00 Tests were carried out in triplicate. Data was expressed as mean ± standard deviation (SD) ND: not determine; the particular antibiotic disc was not tested on the microorganisms +ve: positive control 59 -ve: negative control Antioxidant test DPPH assay DPPH radical scavenging method was used to evaluate the antioxidant activity of the root extracts of G. atroviridis. DPPH assay is a fast and efficient method to determine the free radical scavenging activity. The color changed from purple to yellow signifies the decline in absorbance of the DPPH radical (Jadid et al., 2017). In this study, all extracts showed a similar increasing trend in antioxidant activity with an increase in their concentrations (Table 9).
IC means inhibition concentration and IC 50 is the concentration of the sample or antioxidant that is required to inhibit 50% of DPPH radicals (Jadid et al., 2017). Hence, the lower the IC 50 , the better the antioxidant activity. Phongpaichit et al., 2007, considered the IC 50 range from 10 to 50 mg/mL to display strong antioxidant activity, while 50 to 100 mg/mL is considered intermediate and weak antioxidant activity for IC 50 value that more than 100 mg/mL. According to the IC 50 of the extracts (Table 10), butanol (50.59 μg/mL) showed better antioxidant activity followed by ethyl acetate (51.7 μg/mL) and dichloromethane (53.17 μg/mL). Meanwhile, methanol (124.7 μg/mL) and hexane (131.8 μg/mL) extracts displayed weak activity. However, none of the extracts were comparable to the standard, ascorbic acid (AA) with IC 50 of 13.21 μg/mL. Previous study (MacKeen et al., 2000), methanolic roots extracts of G. atroviridis showed significant antioxidant activity.

Conclusion:-
This paper emphasizes the phytochemical profiling of G. atroviridis roots extracts using preliminary phytochemical screening and GC-MS analysis. In this study, it was shown that the G. atroviridis roots extract contained many biologically active constituents, hence correlates with previous studies. The extracts of the sample were then tested for its antibacterial and antioxidant activity where promising results can be observed, though, further study such as bioprospecting is important to support its biological properties.