Abstract: Five Shorea species which are endemic plants of Malaysia were used in this study. The methanol extract of Shorea acuminata, Shorea macroptera, Shorea leprosula, Shorea bracteolata and Shorea resinosa were investigated for total phenolic contents (TPC), total flavonoid contents, radical scavenging properties (DPPH), in vitro antioxidant and antimicrobial activities. For the antimicrobial activity, the extracts were tested against four bacteria; Gram negative bacteria Escherichia coli, Gram positive bacteria Staphylococcus pyogenes, Staphylococcus aureus and Bacillus subtilis and two fungi, Candida albicans and Aspergillus niger. The result of TPC analysis showed that S. acuminata displayed highest total phenolic content, 2731±0.09 mg/100 g, while in total flavonoid content, S. resinosa displayed the highest amount of flavonoid, 956.73±0.01 mg/100 g. However, in DPPH analysis, the samples of S. resinosa, S. macroptera and S. acuminate displayed better scavenging activity as compared to butylatedhydroxytoluene (BHT). The in vitro antioxidant activity indicated that S. macroptera showed the highest percent inhibition both in ferric thiocyanate method (FTC) (98.68%) and thiobarbituric acid method (TBA) analysis. In antimicrobial activity, S. resinosa displayed moderate inhibition against Gram negative bacteria Escherichia coli, Gram positive bacteria Streptococcus pyogenes, Staphylococcus aureus and Bacillus subtilis. Other plants were found to display selective inhibition against microbes.
INTRODUCTION
Plants contain phytochemicals with various bioactivities, including antioxidant, anti-inflammatory and anticancer activities. Currently, about 25% of the active component was identified from plants that are used as prescribed medicines (Gill et al., 2011). Several plants have been screened for their antioxidant potential (Achuthan et al., 2003; Aniya et al., 2002; Jose and Kuttan, 1995; Lin et al., 1995; Nazri et al., 2011; Shylesh and Padikkala, 1999) because the most practical way to fight degenerative diseases is to increase antioxidant activity in our body and that could be achieved by consumption of vegetables, fruits or edible plants (Oboh and Akindahunsi, 2004; Oboh, 2005). Degenerative disease is caused by free radicals in our body that are able to damage living tissues and cause cell death (Yang et al., 2002). Several plant extracts and chemical constituents have been found to show quite prominent antioxidant activity (Tripathi et al., 1996; Vani et al., 1997; Oseni and Akindahunsi, 2011). According to Chu et al. (2002), the majority of the antioxidant activity may come from active substances such as flavonoid, isoflavone, flavone, anthocyanin, catechin and iso-catechin rather than from vitamin C, E and β-carotene.
Most lowland rainforests in Malaysia are dominated by the Dipterocarpaceae family. About 67% of the total growing stock in Sabah and Sarawak is estimated to consist of Shorea, a common dipterocarp genus. Shorea is locally known as Meranti in Malaysia and for this study, five Shorea species namely S. acuminata (Meranti rambai daun), S. bracteolata (Meranti Pa’ang), S. leprosula (Meranti Tembaga), S. resinosa (Meranti Belang), S. macroptera (Meranti Melantai) were investigated for their potential as antioxidant and antimicrobial agent.
Our previous study on the chemical constituents of Shorea revealed the presence of oligostilbenoids as the main constituents (Norizan et al., 2011; Nazri et al., 2011; Zawawi and Ahmat, 2011). Oligostilbenoids found in Shorea species has been reported to possess important bioactivities such as antibacterial (Sotheeswaran et al., 1983) and 5α-reductase reactions (Hirano et al., 2001) while oligostilbenoids from other family also showed significant bioactivities such as antitumor (Ito et al., 2003), acetylchloinesterase (Sung et al., 2002), ecdysteroid antagonist (Sarker et al., 1999), anti HIV agent (Dai et al., 1998), anti-inflammation (Kitanaka et al., 1990), cytotoxic (Dai et al., 1998) and antioxidant activities (Tanaka et al., 2000). This paper describes the evaluations of total phenolic content (TPC), total flavonoid content, radical scavenging properties (DPPH), in-vitro antioxidant capacity using ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods as well as in-vitro antimicrobial activity of five Shorea species which are S. acuminata, S. macroptera, S. leprosula, S. bracteolata and S. resinosa.
MATERIALS AND METHODS
The plant samples were collected from Jengka Reserve Forest, Selangor, Malaysia in 2009 and voucher specimens were submitted to the Herbarium of Universiti Teknologi MARA, Pahang (UiTM), S. bracteolata (DO8/06/09), S. macroptera (DO7/06/09), S. acuminate (D11/06/09), S. resinosa (D10/06/09) and S. leprosula (DO9/06/09). The samples were cut into smaller pieces and ground into fine powder. The plants were macerated with methanol five times at room temperature, filtered, evaporated using rotary evaporator and the weight of the crude extracts recorded.
Antioxidant activity assay
Total phenolic content (TPC): The TPC was carried out according to Velioglu
et al. (1998) method. The 0.01 g of crude extracts was appropriately
diluted with 5 mL of methanol and 0.1 mL of the diluted sample was added with
Folin-Ciocalteu phenol reagent and placed about 20 min in a dark place. 1.5
mL of 20% Na2CO3 solution were added to the sample and
shaken vigorously. The solution was immediately marked up with distilled water
and mixed thoroughly. The absorbance was measured at 760 nm using Perkin Elmer
Lambda 35 UV-visible Spectrophotometer after incubation for 2 h in dark and
at room temperature. A calibration curve was prepared, using the regression
equation of the calibration curve of gallic acid (y = 0.013x, r2
= 0.998) and contents were expressed as mg gallic acid equivalent (GAE)/100
g of sample.
Total flavonoid content (TFC): Two milliliter of 2% AlCl3 in ethanol was added to 2 mL of the test sample and left in the dark at room temperature for 1 h. The UV absorption was measured at 420 nm. Concentration of 0.1 mg mL-1 sample solutions were used while routine concentrations of 0.02-0.12 mg mL-1 were used to obtain a calibration curve of quercetin (y = 2.519x+0.002, r2 = 0.999). Determinations were performed in triplicate.
Radical scavenging assay (DPPH): The DPPH radical-scavenging activity was determined by using the method proposed by Nazri et al. (2011) with some modification. The positive control was prepared by adding 4 mL of quercetin (0.05 mg mL-1) to 1 mL of DPPH (0.4 mg mL-1), whereas the negative control was prepared by each solvents used and 1 mL of DPPH solution. The radicals’ stock solution was prepared fresh daily. The DPPH solution (1 mL) was added to 3 mL of polyphenol extracts with different concentrations (200, 400, 600, 800, 1000 and 1200 ppm). The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 10 min. The decrease in absorbance of the resulting solution was monitored at 517 nm for 10 min. Lower absorbance of the reaction mixture indicates higher free radical-scavenging activity. Radical scavenging activity is expressed as the inhibition percentage (IP) and was calculated as follows:
The decolouration was plotted against the sample extract concentration and a logarithmic regression curve was established in order to calculate the IC50 (inhibitory concentration at 50%) which is the amount of sample extract (concentration) necessary to decrease by 50% the absorbance of DPPH.
Ferric thiocyanate method (FTC): Four milligrams of the dried solids from each fraction were dissolved in 4.0 mL of ethanol absolute and added with 4.1 mL of a 2.51% linoleic acid solution in EtOH and 8 mL of a 0.05 M phosphate buffer (pH 7.0). The mixture was incubated at 40°C in a dark screw-cap vial. During the incubation, 1.0 mL aliquot was taken from the mixture and added with 9.7 mL of 75% ethanol, followed by the addition of 0.1 mL of 30% ammonium thiocyanate. Precisely 3 min after the addition of 0.1 mL of 0.02 M ferrous chloride in 3.5% hydrochloric acid, the absorbance for the red color was measured at 500 nm .
Thiobarbituric acid method (TBA): Two milliliters of 0.67% thiobarbituric acid and 1.0 mL of 20% trichloroacetic acid solutions were added to 2.0 mL of the mixture solution containing linoleic acid which was prepared according to the FTC procedure. The mixture was then placed in a boiling water bath for 10 min. After cooling, the mixture was centrifuged at 3000 rpm for 20 min and the absorbance was measured at 532 nm.
Antimicrobial activity test
Microorganisms: The extracts were tested against four bacteria that are
Gram negative bacteria, Escherichia coli and Gram positive bacteria,
Streptococcus pyogenes, Staphylococcus aureus and Bacillus subtilis
and yeast, Candida albicans and fungus, Aspergillus niger.
Preparation of antimicrobial disc: The micropipette was used to place 20 μL extract onto a 6 mm diameters of sterile antimicrobial disc. The disc was aseptically dried and placed on media inoculated with the tested microorganisms by using sterile forceps. A maximum of only four discs were placed on the media. The plates were incubated at 37°C for 24 h. The corresponding concentrations are expressed in terms of mg of extract per mL of solvent.
Disc diffusion method: Antimicrobial tests were carried out by disc diffusion method using the suspension containing 1x108 CFU mL-1 bacteria or 1x106 CFU mL-1 yeast on Nutrient Agar (NA) or Potato Dextrose Agar (PDA), respectively. The discs were impregnated with 20 μL of extracts and placed on the inoculated agar. The inoculated plates were incubated for 24 h at 37°C for bacteria strains, 48 h for yeast. A sterile cotton swab was dipped into 18-24 h broth culture of the test organism and carefully streaked over the surface of the sterile agar plate. The plate was then turned at right angles and the swab was streaked again over the entire surface to ensure a uniform film of the test microbes. 0.02 mg mL-1. Streptomycin was used as a positive control for the bacteria whereas 100 mg mL-1 cycloheximide was used as positive control for the yeast. Antimicrobial activity was evaluated by measuring the inhibition zone against the test organisms. The assay in this experiment was repeated three times.
Statistical analysis: The experiments were done in triplicate. The result was given as the mean±SD. Analysis of Variance (ANOVA) was used for the analysis of data. Significance was accepted at p = 0.05.
RESULTS AND DISCUSSION
Five Shorea species, S. acuminata, S. macroptera, S. leprosula, S. bracteolata and S. resinosa which are endemic plants of Malaysia were investigated for their total phenolic content, total flavonoid content, in vitro antioxidant and antimicrobial activities.
The results showed that all Shorea species tested with TPC displayed high amount of phenolic with range of 2461-2731 mg/100 g GAE. Among the five Shorea samples, S. acuminata extracts displayed the highest phenolic content 2731.00±0.09 mg/100 g followed by S. leprosula extracts 2615.38±0.01 mg/100 g, S. resinosa extracts 2461.54±0.01 mg/100 g and S. macroptera extracts 2461.54±0.01 mg/100 g and the lowest total phenolic content is given by S. bracteolata extract with 2423.08±0.02 mg/100 g which is considered still very high. Most of the crude methanol extracts of these Shorea species gave total phenolic contents in the range of 2461.54±0.01 mg of GAEs to 2731.00±0.09 mg of GAEs and are presented in Table 1. Plant phenols are a major group of compounds acting as primary antioxidants or free radical scavengers (Kahkonen et al., 1999) due to their hydroxyl groups (Diplock, 1997) which contribute directly to the antioxidative action. Phenolic compounds are effective hydrogen donors, making them good antioxidants (Rice-Evans et al., 1997).
In the TFC analysis, all Shorea extracts tested displayed moderate flavonoid content with range of 535.93-956.73 mg g-1. S. resinosa displayed highest amount of flavonoid content 956.73±0.01 mg g-1 followed by S. leprosula (873.36± 0.002 ), S. acuminate ( 851.48±0.001) and S. bracteolata (766.18± 0.001) while the lowest given by S. macroptera extracts 535.93±0.02 mg g-1.
| Table 1: | Extraction yields, phenolic and flavonoid contents of Malaysian Shorea sp. |
| Values are means of triplicate, Different superscript in column indicate significant different at p<0.05, na: Not available | |
| Fig. 1: | Scavenging effect of methanolic extract on DPPH radicals, compared with BHT |
| Fig. 2: | Percent inhibitions of Shorea species by FTC method (day 6, 1 ppm) |
The total flavonoid contents findings for these Shorea extracts were summarized in Table 1. The analysis of TFC indicates that Shorea contain considerable amount of flavonoids.
In DPPH radical scavenging activity, S. resinosa, S. macroptera and S. acuminata displayed better scavenging activity as compared to the standard BHT with % scavenging of 92.50, 88.23 and 87.23, respectively (Fig. 1). Percent scavenger for BHT is 79.7%. S. bracteolata and S. leprosula showed lower % of scavenging activity (46.55 and 34.55%, respectively) when compared to BHT. The highest scavenger is shown by S. resinosa, while the lowest scavenger is S. leprosula. S. resinosa. S. macroptera and S. acuminata demonstrated significant free radical scavenging ability by giving IC50 values of 0.125, 0.140 and 0.125 μg mL-1, respectively as shown in Table 1 and are considered as excellent scavenger and even better than the standard BHT. The methanol extracts of S. bracteolata and S. leprosula did not give any values of IC50. The IC50 value is defined as the concentration that causes a decrease in the initial amount of DPPH radicals by 50% (Huang et al., 2005). It is the concentration where the active crude extract will exhibit 50% of antioxidant activity (Chiang et al., 2003) and crude extracts exhibit 50% of inhibition at concentration less than 20 μg mL-1. These concentrations are considered positive for antioxidant activity (Geran et al., 1972).
In FTC analysis, three of the Shorea species namely S. resinosa, S. leprosula and S. macroptera, exhibited strong antioxidant potential with percent inhibition between 71.11-98.68% while Quercetin and vitamin E showed percent inhibition of 99.68% and 93.45%, respectively (Fig. 2). The extract of S. Macroptera (98.68%) showed the highest percent inhibition and as good as the standard as its % inhibition is better than vitamin E. The other two of the Shorea extracts demonstrated moderate antioxidant activity at 56.75% for S. bracteolata and 57.47% for S. acuminata, respectively.
| Table 2: | Percent inhibition of Shorea sp. between FTC and TBA method (day 6,1 ppm) |
| Values are means of triplicate, Different superscript in column indicate significant different at p<0.05, when compared five with standard, FTC: Ferric thiocyanate, TBA: Thiobarbituric acid | |
The total antioxidant property of the methanol extracts of Shorea species is given as follows in descending order: S. macroptera>S. leprosula>S. resinosa> S. acuminate> S. bracteolata at 98.68, 78.42, 71.11, 57.47 and 56.75%, respectively.
The findings of this study showed that FTC results of the five Shorea species do not correlate with TPC and TFC results. The extracts of S. macroptera displayed highest antioxidant activity, 98.68% among the five Shorea species but its TPC values was the lowest amongst the five Shorea species 2461.54±0.01 mg g-1 and its TFC values was also the lowest (535.93±0.02). On the other hand, the extracts of S. acuminata displayed lowest antioxidant activity 54.79% but recorded the highest of TPC values, 2731.00±0.09 mg g-1 and moderate amount of flavonoid content (851.48±0.001) as shown in Table 1.
The plant extracts tested showed low absorbance values, indicating a high level of antioxidant activity. None of the plant extracts showed absorbance values greater than the negative controls (without plant extracts) at the end point of both methods, indicating the presence of antioxidant activity. However, the plant extracts of S. macroptera exhibited strong antioxidant activity as determined by both the FTC and TBA methods, surpassing the activity of the standard antioxidant, vitamin E. The extract of S. macroptera indicated the highest antioxidant as its absorbance was the lowest and located between both standards as shown in Table 2.
FTC test is related to the peroxide formation in the initial stage of lipid oxidation, while TBA test measures the amount of malondialdehyde (MDA) at later stage. The results in FTC showed higher inhibition than in TBA. This may indicate that the amount of peroxide in the initial stage of lipid peroxidation is much greater than the amount of peroxide in the secondary stage. This is due to the MDA, produced on the final day of the incubation period (1 day after the control reached maximum) is a very unstable compound and turn into alcohol and acid which cannot be detected by spectrophotometer (Ottolenghi, 1959) which might cause mutagenic and cytotoxic events (Zin et al., 2002). At a low pH and high temperature (100°C), MDA binds TBA to form a red complex that can be measured at 532 nm after incubation for 24 h. By determining the amount of malondialdehyde (MDA), the correlation between both FTC and TBA methods can be verified. These results correlated well with those obtained previously, using the FTC method. A comparison between FTC and TBA measured is shown in Fig. 3.
For the analysis of antimicrobial activity, the methanol extracts of Shorea species were tested against four microbes: Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis and yeast including Candida albicans and Aspergillus niger. S. acuminata extract (25-100 mg mL-1) inhibited the growth of S. pyogenes (7.0-10.0 mm) and S. aureus (6.0-12.0 mm) moderately at higher concentration, 100 mg mL-1 and weakly at a lower concentration, 25 mg mL-1.
| Fig. 3: | Lipid oxidation profile of methanol extract of Shorea sp. in linoleic acid buffer model system |
The inhibition zone was increased by 1 mm with the increment of 25 mg mL-1 dose of S. acuminata methanol extract. B. subtilis, E. coli, C. albicans and A. niger were found resistant against this methanol extract.
S. macroptera, S. leprosula, S. bracteolata and S. resinosa were able to inhibit S. aureus and B. subtilis in a dose dependant manner with the highest inhibition was shown by S. Leprosula extract (25-100 mg mL-1) against B. Subtilis (11-15 mm) and S. resinosa extract (25-100 mg mL-1) against S. pyogenes (9-15 mm) both at 100 mg mL-1 while the lowest inhibition were displayed by S. acuminata against S. aureus (6 mm) at 25 mg mL-1. All species were found able to inhibit S. aureus in a dose dependant manner. It is interesting to note that only S. resinosa was able to inhibit the growth of E. coli, a microbe commonly found in the lower intestine of warm-blooded organisms. The best result was given by S. resinosa which was able to inhibit the growth of four gram negative and gram positive microbes that are S. pyogenes, S. aureus, B. subtilis and E. coli. S. macroptera and S. bracteolata were able to inhibit the growth of three microbes: S. pyogenes, S. aureus and B. subtilis while S. acuminate and S. Leprosula only inhibited two microbes which are S. pyogenes and S. aureus and S. aureus and B. subtilis, respectively also in dose dependant manner. It is clearly shown from the results tabulated in Table 3 that none of the Shorea species was able to inhibit the growth of the two tested yeast C. albicans and A. niger.
The result indicated that most of the Shorea extracts was able to inhibit gram positive bacteria (S. pyogenes, S. aureus, B. subtilis) as compared to gram negative bacteria (E. coli). This is further confirmed by the previous studies by Kelmanson et al. (2000), Masika and Afolayane (2002), Masoodi et al. (2008), Karaman et al. (2003), Khanahmadi et al. (2010) and Zongo et al. (2010) that describe the high sensitivity of gram positive bacteria towards plant extracts and their component.
According to Zakaria (1991), E. coli was the bacteria strains not susceptible to most plant extracts which could be observed from the result of this study. These could be due to several possible reasons, in which the distinctive feature of gram-negative bacteria is the presence of a double membrane surrounding each bacterial cell. Although, all bacteria have an inner cell membrane, gram-negative bacteria have a unique outer membrane.
| Table 3: | Zone of inhibition of the methanol extracts of Shorea species |
| Values are means of triplicate, different superscript in a same row indicate significant different at p<0.05 | |
This outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram-negative bacteria are generally more resistant to antibiotics than other gram-positive bacteria. This could be the beginning for further research on the screening approach by taking into consideration the extracts preparation and the mechanism of action. E. coli in this study also exhibited the same pattern in which it was resistant to almost all extracts. However, S. resinosa methanol extract is capable of moderately inhibiting E. coli growth. Although, S. resinosa methanol extract could inhibit bacteria, it was still unable to inhibit the two fungi C. albicans and A. niger.
S. resinosa showed good antimicrobial activity in which its methanol extract inhibited the growth of all Gram-positive and Gram-negative bacteria but not the fungi. It also gave good IC50 values 0.125 μg mL-1, that may be regarded as good antioxidant agent. There were differences in cell wall structure between Gram-positive and Gram-negative bacteria, with the gram-negative outer membrane acting as a barrier to many environmental antimicrobial substances, including antibiotic. The results from the antimicrobial activity of five Shorea species may come from resveratrol oligomers which constitute as the major compound.
CONCLUSIONS
As a conclusion, S. resinosa methanol extract exhibited good antimicrobial activity against S. pyogenes, S. aureus, B. subtilis and E. coli while S. macroptera showed the highest inhibition in both FTC and TBA assays. The results of this study have revealed the potential of Malaysian Shorea as antioxidant and antimicrobial agents.
ACKNOWLEDGMENTS
The authors would like to thank Faculty of Applied Sciences, Universiti Teknologi MARA, Malaysia for financing this research project. Two of the authors were granted scholarships by UiTM fellowship and National Science Fellowship.