INTRODUCTION:

Garcinia is the most important genus of Guttiferae family, comprises about 40 genera and 1200 species¹which is widely distributed in tropical Africa, Asia, New Caledonia and Polynesia.

This plant has been listed in IUCN Red List of Threatened Species in 1998 and has become one of the endangered species in Malaysia. Medicinal plants such as Garcinia has been playing an important role in the survival of the ethnic communities, who live in remote villages and forests². It is well known to be rich in a variety of oxygenated and prenylated phenol derivatives^3,4. Some of these plants exhibited a wide range of biological activities such as antioxidant, anti-fungal, anti-inflammatory and anti-HIV activities^5-8. Some Garcinia species are well known in Asia as medicinal plants and are widely used in traditional medicines. One of their species is Garcinia prainiana that native to Malaysia and Thailand. The fruit is cultivated in Southeast Asia, by a few backyard growers in South Florida, and at the Whitman Rare Fruit Pavilion of Florida's Fairchild Tropical Botanic Garden⁹. In Malaysia, this plant is locally known as cerapu, cerpu, mencupu and kecup. It is a small to medium-sized tree up to 18 m high with oppositely arranged of dark green leaves and produces white latex. The fruit is orange coloured with sweet-sour flavoured pulp. This plant requires adequate cold protection, hand pollination, shelter from strong winds and regular watering¹⁰. To date, there is no report on the bioactivities including antioxidant and antimicrobial activity from leaves, fruit and twig of Garcinia prainiana. Hence, in this paper we report its bioactivities (antioxidant and antimicrobial) including total phenolic and total flavonoid content from different parts of the plant.

MATERIALS AND METHODS:

Plant material:

Various parts (leaves, fruit and twig) ofGarcinia prainiana were collected from a Fruit Farm located in Jeli, Kelantan, Malaysia. A sample of the plant is deposited to Herbarium of Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin.

Extraction procedure:

The samples were washed, chopped into small pieces and dried in the oven at 60˚Cfor a few days until the weight was constant. After that, the dried samples were grounded to powder and extracted using 1L ethyl acetate until super saturation for 72 hours at room temperature¹¹. The samples extracts were kept away from sunlight and were stirred several times with a sterile glass rod. The supernatant was filtered through Whatman No. 1 filter paper. The collected extracts were then unloaded to the rotary evaporator at 40˚C for the ethyl acetate to evaporate. Finally, the extracts were re-suspended in 15 % dimethyl sulfoxide (DMSO).

Antioxidant assays:

Free radical diphenylpicrylhydrazyl (DPPH) assay:

Using the method by Wang et al. (2007), free radical scavenging activity was measured using DPPH free radical¹². About 0.1 M DPPH stock solution was prepared by dissolving 1.5 mg of DPPH in the 50 ml methanol solution. Next,plantextracts at different concentrations (0.5, 0.4, 0.3, 0.2 and 0.1 mg/mL)were mixed with 200 µL of 0.1 M of DPPH solution in methanol. Then, the mixture was shaken and incubated in dark condition for 30 minutes at room temperature. After that, the absorbance was measured at 517 nm with a microplate reader. Trolox was used as a standard. The inhibition percentage of the radical scavenging is calculated using the equation below.

Inhibition (%) = 100 – 100 (A_s ÷ A_o)

Where (A_o) is absorbance of the control and (A_s) is absorbance of standard and samples. IC₅₀value (sample concentration that is required to scavenge 50 % of free radical) was estimated from the percentage inhibition graph and compared with the standard. Three replicates from each samples were tested and the results were expressed in mg/mL.

Antimicrobial assay:

Preparation of bacterial strain:

Bacterial strains used in this study included gram positive- Bacillus cereus ATCC 14579; Staphylococcus aureus ATCC 33591; Enterococcus faecalis ATCC 29212 and gram negative bacteria- Escherichia coli ATCC 35218; Pseudomonas aeruginosa ATCC 27853; Acinetobacter baumannii ATCC 19606 and Salmonella typhimurium ATCC 14028. These bacteria were cultured from the glycerol stock available in Microbiology Laboratory, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Terengganu, Malaysia. Reviving process began by thawing the bacteria at the room temperature, followed by transferring into Mueller Hinton broth (MHB) and the cultures were left overnight at 37 °C for growth. After an overnight incubation, the bacteria broth was streaked on Nutrient Agar (NA) for single colony formation. Single colony without contamination was used in the antibacterial activity test. The plate was kept at 4 °C. For long term storage, 20 %glycerol stocks were prepared for each strain of bacteria and was stored at -80 °C.

Disc diffusion method:

Antibacterial activity of Garcinia prainiana extracts were done using the standard procedure of disc diffusion method by Nostro et al. (2000)¹³. A concentration of 25 mg/ml, 50 mg/ml and 100 mg/ml were prepared from the stock solution. Cultures of the bacteria tested were inoculated separately on the surface of Mueller Hinton agar plates by surface spreading using sterile cotton swab and was allowed to stand for a few minutes to enable the pre-diffusion of the inoculated organisms. Each bacteria strain was evenly spread over the entire surface of agar to obtain a uniform inoculum. Disc (6 mm diameter) was sterilized by autoclaving. The blank sterile disc was dropped with 10 μL of respected plant extract before placing aseptically on the inoculated Mueller Hinton Agar (MHA) surface by using a sterile forceps and was gently pressed to enable even contact. Gentamycin was used as control at a concentration of 50 μg/ml. The plates were incubated at 37 °C for 24 hours. All tests were performed in triplicate. After incubation, the zone of clearance was measured using metric ruler by taking measurement from the edge of the zone of inhibition to the other edge. The diameter of zone of inhibition represents antibacterial activity of the extracts tested^14,15.

Estimation of Total Phenol Content (TPC):

Total phenolic content was determined using Folin-Ciocalteu reagent following method described by Riemann et al. (2010)¹⁶. First of all, gallic acid¹⁷standard solution in different concentration of 200-60 μg/ml was used to construct the calibration curve. The samples were prepared at 1 mg/ml final concentration. A volume of 60 μl of each sample was combined with 40 μl of methanol and 200 μl of Folin-Ciocalteu reagent and vortex the mixture. Then, 800 μl of 7 % Na₂CO₃ solution was added and the mixture allowed to incubate for 2 hour at room temperature in dark. Absorbance was measured at 725 nm with microplate reader. Finally, the results were expressed as mg of gallic acid equivalents (GAE) per g (mg GAE/g).

Estimatimation of Total Flavonoid Content (TFC):

Total flavonoid content was determined by aluminium chloride method, as described by Quettier-Deleu et al. (2000)¹⁸. The samples were prepared at 1 mg/ml of final concentration. About 140 μl of sample extract was mixed with 150 μl of 10 % aluminium chloride (AlCl₃)solution. Then, 150 μl of potassium acetate (KC₂H₃O₂)solution and mixed by votex. Next, the mixture was topped-up with 260 μl distilled water and vortex again. Then, the mixture was allowed to incubate at room temperature for 30 minutes and the absorbance of the mixture was measured at 430 nm with microplate reader. The flavonoid content was expressed in mg per g of quercetin equivalents per gram of dry plant extract (mg QE/g).

RESULTS AND DISCUSSION:

Different plant parts (leaves, fruit and twig) of Garcinia prainiana were dried and macerated using ethyl acetate due to its ability to dissolve polar and non polar constituents. Thus, it is possible to extract a diverse range of metabolites¹⁹. Antioxidant capacity of the extracts were determined based on the reaction of DPPH absorbance by calculating percentage of antioxidant activity²⁰. In this experiment, all samples (leaves, twig and fruit) of Garcinia prainiana were used to evaluate its potential antioxidant activity through free radical diphenylpicrylhydrazyl (DPPH) assay. Molecule that neutralizing free radicals intermediate by removing the chain reaction is an antioxidant molecule²¹. A protonated DPPH radicals can be observed by its absorbance decrement at 517 nm and it is visually notiecable from discoloration from purple to yellow²².

DPPH method is a preferred method because it is fast, easy and reliable and does not require a special reaction and device. Result for antioxidant activity is presented in Table 1. The highest inhibition percentage show by leaves (77.41 %) at 0.4 mg/ml, twig (39.78 %) at 0.5 mg/ml and fruit (36.02 %) at 0.4 mg/ml respectively. Higher inhibition profile indicate higher level of antioxidant activities in the plant sample due to high activity of free radical scavenging^23,24,25. However, IC₅₀ was only recorded from leaves extract of Garcinia prainiana at 0.19 mg/ml compared to Trolox at 0.023 mg/ml (Fig. 1). Whereas, IC₅₀ value for twig and fruit extract cannot be determined due to lower antioxidant activity profile showed. Trolox was used since it is a natural antioxidant and widely used as a standard drug for many assays²⁶. From this study, it is showed that different parts of Garcinia prainiana exhibit different antioxidant profiles. Results showed that ethyl acetate extract of the leaves (77.41%) has better potential as antioxidant agent compared to others. Based on Kruawan and Kangsadalampai (2006), he mentioned that if the inhibition percentage was between 60% - 90%, the plant has moderate potential of antioxidant activity, whereas, if the value is below 60% inhibition, the sample has low potential antioxidant activity²⁷.

Fig. 1: Antioxidant activity of ethyl acetate extract of leaves, twig and fruit of Garcinia prainiana (A) and Trolox as standard (B).

Table 2: Antimicrobial activity of Garcinia prainiana ethyl acetate extracts against pathogenic bacteria strains.

Concentration of extracts (mg/ml)		Microorganisms/Zone of inhibition (mm)Mean ± SD
		Gram-Positive strain			Gram-Negative strain
		BC	EF	SA	AB	EC	PA	ST
25	Leaves	-	-	-	-	-	-	-
	Twig	-	-	-	-	-	-	-
	Fruit	-	-	-	-	-	-	-
50	Leaves	4±1.13	-	9±3.06	-	10±2.25	-	-
	Twig	-	-	8±2.52	-	8±1.87	-	-
	Fruit	-	-	-	-	-	-	-
100	Leaves	10±2.08	-	10±3.21	6±3.51	12±1.43	-	-
	Twig	8±2.52	-	9±1.32	-	11±1.82	-	-
	Fruit	7±2.64	-	-	-	-	-	-
Gentamycin (50μg/ml)		16±1.5	14±3.2	15±3.1	16±2.5	19±2.5	16±1.8	20±2.3

Abbreviations: BC= B.cereus ATCC 14579, EF= E. faecalis ATCC 29212 , SA= S. aureus ATCC 33591, AB= A.baumanni ATCC19606, EC= E.coli ATCC 35218, PA= P. aeruginosa ATCC 27853, ST= S. thyphimurium ATCC 14028, - = No zone of inhibition. Each value is expressed as a mean ± SD of triplicates.

The activity was determined qualitatively using disc diffusion method in order to evaluate potential of Garcinia prainiana extracts as antimicrobial agent. From the result (Table 2), the highest zone of inhibition was shown by leaves extract at 12mm followed by twig extract at 11mm against Escherichia coli ATCC 35218. Additionally, 100 mg/ml of leaves extract managed to inhibit Bacillus cereus ATCC 14579 at 10 mm zone of inhibition followed by twig extract (8mm) and fruit extract (7mm) as compared to Gentamycin as control (16mm). Meanwhile, for 50 mg/ml of leaves extract, the highest inhibition was at 10 mm followed by twig extract at 8 mm against Escherichia coli ATCC 35218. Beside that, inhibition against Staphylococcus aureus ATCC33591 was also recorded at 50 mg/ml of leaves and twig extracts with 9 mm and 8 mm zone of inhibition, respectively. The same leaves extract at the concentration of 100mg/ml also managed to inhibit Acinetobacter baumannii ATCC 19606 with 6 mm zone of inhibition. For the rest of the bacteria strains tested, no inhibition profile was recorded against all extracts. Previous study by Nargis et al. (2013) on Garcinia hombroniana showed thatethyl acetate extract was able to inhibit the growth of S. aureus and E. coli strains³⁴. This activity may be due to the presence of biflavonoids and related metabolites that have been implicated for their antimicrobial activities^35,36. Other researchers also suggested that plant antimicrobial components such as terpenoid, alkaloid and phenolic compounds would interact with enzymes and proteins associated with microbial cell membrane causing its disruption to disperse a ﬂux of protons towards cell exterior which induces cell death or may inhibit enzymes necessary for aminoacids biosynthesis^37,38.

Phenolic compounds are excellent oxygen radical scavengers because it has the ability to destroy radicals due to hyroxyl groups presence^39,40. These phenolic plant components give up hydrogen atoms from their hydroxyl groups to radicals and form stable phenoxyl radicals^41,42. Therefore, it is important to determine the quantity of phenolic compounds presence in order to determine the antioxidant capacity of the plant extracts. The content of total phenolic was determined by extrapolation from the calibration curve prepared from gallic acid concentrations and expressed in milligram of gallic acid. The result indicated that phenolic content was higher in leaves extract (86.047 ± 3.039 mg/g) followed by fruit extract (21.811 ± 5.022 mg/g) and twig extract (2.512 ± 3.446 mg/g) (Fig. 2).Different levels of phenolic content were observed in this study may be attributed to the diferent part of plant used. Higher amount of phenolic compound was found in leaves extract compared to twig and fruit extract of Garcinia prainiana ethyl acetate extractfollowing the order: leaves > fruit> twig. High amount of phenolic compounds in leaves extract was probably because the leave samples were collected at fully matured stages⁴³compared to twig and fruit.

Fig. 2: Comparison between total phenolic content (mg/g) and inhibition percentage of different parts of Garcinia prainiana ethyl acetate extract. Each value is expressed as a mean ± SD of triplicates.

This finding was consistent with antioxidant assay which exhibited higher antioxidant activity in leaves extract compared to other parts of the plant (Fig. 2). Previous study by Aksoy et al. (2013) stated that the antioxidant activity observed may be due to the presence of significant amount of phenolic compounds such as phenols and phenolic acids⁴¹. Healso stated that phenolic compounds and polyphenols are the most abundant components in plants which contribute to the antioxidant profile. Furthermore, the usage of Folin-Ciocalteu reagent isonly measured based on colour measurement which is non-specific to phenol. Possibly there are other components that can react with the reagent such as ascorbic acid and many more⁴⁴. Besides, different compounds of phenolic have different response to this assay. In the present study, the responses of the plant extracts in this assay may arise from the variety and quantity of phenolics found in the ethyl acetate extracts of Garcinia prainiana. Several studies have also reported strong relationship between total phenolic content and antioxidant activity in selected grains, herbs and vegetables^45,46,47. Some authors found a correlation between the phenolic content and the antioxidant activity, while others found no such correlation⁴⁷. Fig. 2 shows that leaves extract of Garcinia prainiana was found to be the highest in antioxidant activity and also the highest in phenolic content. This finding shows correlation between antioxidant ability with total phenolic content.

Flavonoids are small molecule of secondary metabolites which synthesized by plants for various biological activities including plants’ interaction with organisms (animals, microorganisms and other plants) and their interactions to environmental stresses⁴⁸.The content of total flavonoid was determined by extrapolation from the calibration curve prepared from quercetin concentration and expressed in miligram of quercetin.Based on Fig. 3, total flavonoid content in fruit extract (73.188± 1.056 mg/g) was significantly higher compared to twig extract (49.932 ± 3.057 mg/g) and leaves extract (26.676 ± 2.062 mg/g) of Garcinia prainiana ethyl acetate extract. The flavonoid content in leaves extract was found to be significantly lower compared to other parts of the plant. In fact, fruit of Garcinia prainiana contain the highest in flavonoid content (Fig. 3). From this study, total flavonoid content is significantly different among all plant parts following the order: fruit > twig > leaves. Result from the total flavonoid content also revealed that there werenegative correlation between total flavonoid content and antioxidant activity with the highest flavonoid content was observed in fruit,but contain the lowest antioxidant activity (Fig. 3).Flavonoids can also suppress reactive oxygen formation, chelate trace elements involved in free-radical production, scavenge reactive species and up-regulate and protect antioxidant defenses⁴⁰. But, in this study, itis showed that flavonoid compounds in Garcinia prainiana leaves extract had no influence in antioxidant activity by DPPH method. Other than that, high flavonoid content may be attributed to the antimicrobial activity of the extract which has been reported involved in inhibition of nucleic acid biosynthesis and other metabolic processes⁴².

Flavonoids are synthesized in all parts of the plant andimportantfor providing color, fragrance and taste to the fruits, flowers and seeds. This makes plant attractants for insects, birds or mammals, which aid in pollen or seed transmission⁴⁸. Flavonoids are also cytotoxic which contribute to their function in plant defense mechanism against plant feeding insects and herbivores^49,50. Their presence can alter the palatability of the plants and reduce their nutritive value, decrease digestibility or even act as toxins⁵⁰.Besides that, antifungal and antimicrobial activity of phenolic and flavonoid compound has also been reported previously in Garcinia cambogia^12,45. Flavonoids such as catechins are the most common group of polyphenolic compound in the human diet and are found ubiquitously in plant^51,52.

Fig.3:Comparison between total flavonoid content (mg/g) and inhibition percentage of different parts of Garcinia prainiana ethyl acetate extract. Each value is expressed as a mean ± SD of triplicates.

CONCLUSION:

In this study, free radical scavenging activity, antimicrobial activity, total phenolic and total flavonoid content of ethyl acetate extract of Garcinia prainiana, an endangered plant were determined. All plant parts tested (leaves, twig and fruit) of Garcinia prainiana showed different level of antioxidant and antimicrobial properties. The leaves extract shows moderate potential of antioxidant activity with the IC₅₀ value at 0.19 mg/ml. Antimicrobial activity againts leaves and twig extract showed zone of inhibition against Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 33591, Acinetobacter baumannii ATCC 19606 and Bacillus cereus ATCC 14579. Fruit extract managed to show a weak inhibition profile against Bacillus cereus ATCC 14579. From this study, phenolic compounds in Garcinia prainiana leaves extracts were themajor contributor in its antioxidant activities by DPPH method. The presence of phenolics and flavonoids compounds in this plant are considered to be responsible for their antioxidant and antimicrobial effects, but more in-depth study is required to identify the active compounds.As a result, Garcinia prainianacan be potentially developed as pharmaceutical products for a source of natural antioxidants. Therefore, we think studies on how to ensure the existence of this species, which is in danger of extinction, should be emphasised.

ACKNOWLEDGEMENTS:

We acknowledge Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin for providing necessary chemicals and equipments. This study was supported and funded by UniSZA Research Grant (UniSZA/2017/DPU/08).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.06.2018 Modified on 25.07.2018

Research J. Pharm. and Tech 2018; 11(9): 3752-3758.

DOI: 10.5958/0974-360X.2018.00687.X

Trolox (Standard)		*Garcinia prainiana* ethyl acetate extract
Concentration (mg/ml)	% Inhibition (Mean ± SD)	Concentration (mg/ml)	% Inhibition (Mean ± SD)
Concentration (mg/ml)	% Inhibition (Mean ± SD)	Concentration (mg/ml)	Leaves	Twig	Fruit
0.01	23.02± 0.33	0.1	19.99 ± 0.36	14.51 ± 0.40	6.11 ± 0.42
0.02	43.52 ± 0.24	0.2	47.85 ± 0.23	20.00 ± 0.37	10.22 ± 0.40
0.03	64.26 ± 0.15	0.3	64.56 ± 0.16	27.80 ± 0.33	15.14 ± 0.38
0.04	75.57 ± 0.10	0.4	77.41 ± 0.01	35.31 ± 0.30	36.02 ± 0.29
		0.5	76.15± 0.11	39.78 ± 0.28	10.89 ± 0.40