Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves , Barks , Stems and Fruits of Alphitonia philippinensis ( Rhamnaceae ) From Brunei Darussalam

Cite this article: Ahmed J, Salim KA, Lim LBL, Jama AM. Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves, Barks, Stems and Fruits of Alphitonia philippinensis (Rhamnaceae) From Brunei Darussalam. Pharmacog J. 2019;11(5):951-61. Phcogj.com Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves, Barks, Stems and Fruits of Alphitonia philippinensis (Rhamnaceae) From Brunei Darussalam


INTRODUCTION
Throughout history, humans have relied on nature to manage their basic needs for food, shelter, garments, transportation, fertilizers, medicines and essence.Numerous medicines have been derived from natural sources for the treatment of various types of diseases in humans and animals from the very beginning of human civilization and most of them come from plants.These plantbased treatments play a vital role in many health care systems.The World Health Organization (WHO) reported that approximately 80% of the world's inhabitants rely mainly on traditional medicines for their primary health care. 1,2[7][8][9] Free radicals, oxidants, and various reactive oxygen species (ROS) are produced by common essential metabolic processes or from external sources such as cigarette smoking, air pollutants, ozone, industrial chemicals, and exposure to X-rays.Free radicals are liable to generate a large number of diseases including cardiovascular disease, neural disorders (Alzheimer's disease, Parkinson's disease, muscular dystrophy), cancer, hypertension, inflammatory diseases (e.g., arthritis, vasculitis, glomerulonephritis), aging and degenerative diseases.1][12] Synthetic antioxidants such as butylatedhydroxytoluene (BHT), propyl gallate (PG), butylated hydroxyanisole (BHA), and tertbutylhydroquinone are firmly regulated to use in food and medicinal products due to suspicions of their safety and side effects like carcinogenesis and liver damage. 13Thus, increased attention is now turned to natural antioxidants such as those of phytochemicals which are relatively safer, more abundant and have the stronger efficiency to scavenge free radicals compared to synthetic antioxidants. 14phitonia, a genus of the Rhamnaceae family with, approximately 20 species, are available in the tropical regions of Southeast Asia, Polynesia and Oceania. 15,16[18][19][20] Alphitonia philippinensis Braid is vernacularly known by the Malays in Brunei Darussalam as "balik angin".Locally it is used in traditional medicine where its leaves are used for the treatment of stomachaches and herbal bath for women to regain strength after delivery.Freshly cut and separated bark and stem of the plant produce a pleasant aromatic smell.Isolated triterpenoids (lupeol, betulinic acid, ceanothic acid) and flavonoid glycosides (derivatives of quercetin and isorhamnetin) from the stems of A. philippinensis exhibited cytotoxicity against human PC-3 cells and hepatoma HA22T cells.Replication of herpes simplex virus type-1 was inhibited in trial by some of these compounds. 20is current study was designed to screen methanolic extracts of leaves, stems, barks and fruits of this plant for phytochemicals, and to determine their total phenolic, flavonoid, flavonol contents and antioxidant activity.To date and to the best of our knowledge, no report has been published on the phytochemical screening and antioxidant activity of A. philippinensis.This study will, therefore, provide further insight into the potential of A. philippinensis as a medicinal plant.

Plant materials
The leaves, stems, barks and fruits of A. philippinensis were collected from a slope of a vegetated roadside area in Kampung Katok, situated in the Brunei-Muara District, Brunei Darussalam in May 2015.The species was identified by Dr. Kamariah Abu Salim (Universiti Brunei Darussalam) and authenticated by comparing it with a reference specimen at the National Herbarium of Brunei Darussalam (BRUN).A voucher specimen (No.JA-1) was prepared and deposited in the Universiti Brunei Darussalam Herbarium (UBDH).

Preparation of methanolic extracts
Different parts of the plant (leaves, stems, barks and fruits) were separated, rinsed, and cut into small pieces.The plant materials were dried in open air at 25°C under the shade and then freeze dried under vacuum for two days.All the four parts of the plants were ground into fine powder using a laboratory mill (Model: MF 10 B, IKA).The powder (35 g) was placed in a thimble and extracted with 250 mL of absolute methanol using a soxhlet apparatus for 6-8 h.The solvent was then evaporated under reduced pressure at 50°C using a rotary evaporator (BuchiRotavapor, Model: R-114).The crude extracts were dried in a freeze dryer until a constant mass was obtained and stored at 4°C in the dark.All the measurements for total phenolic content (TPC), total flavonoid content (TFC) and total flavonol content (TFlC) and others of experiments were expressed in gram of freeze dried powder sample which is converted from the gram freeze-dried crude extract.

Phytochemical screening
All the plant extracts were subjected to a variety of phytochemical tests following the methods as described by Evans 5 and Sofowora 8 to detect the presence of tannins, saponins, steroids, alkaloids, cardiac glycosides, anthraquinones, and terpenoids.The presence or absence of a particular phytochemical was confirmed by color change or precipitate formation by visual observation.

Determination of TPC
The total phenolic content was determined using the standard Folin-Ciocalteu method by Chlopicka et al., with minor modification. 21The plant extract (0.3 mL of 1 mg/mL solution) was mixed with the Folin-Ciocalteu phenol reagent (2.25 mL) and 6% sodium carbonate (2.25 mL) was added after 5 min.The mixture was left to stand for 90 min at 25°C and then the absorbance was measured at 725 nm using a UVvisible spectrophotometer (UV-1800, Shimadzu).Following the same procedure, a standard calibration curve of gallic acid was prepared in the concentration range of 0-200 µg/mL.The results were expressed as mg gallic acid equivalents per gram dry weight of powdered plant parts (mg GAE/g DW).

Determination of TFC
Aluminum chloride colorimetric method by Lin and Tang, 22 with some modifications, was used to determine the total flavonoid content (TFC) of each of the plant extracts.Quercetin was used as the standard and a calibration curve was prepared in the concentration range 0-200 µg/ mL.Both standard and extract (0.5 mL) were placed in different test tubes and to each 10% aluminum chloride (0.1 mL), 1 M potassium acetate (0.1 mL), 80% methanol (1.5 mL) and distilled water (2.8 mL) were added and mixed.Likewise, a blank was prepared in the same manner but distilled water (0.5 mL) was used instead of the sample or standard and aluminum chloride was also replaced by distilled water in the same amount.All the tubes were incubated at 25°C for 30 min, and the absorbance was measured at 415 nm using a UV-visible spectrophotometer.The concentration of flavonoid was expressed as mg quercetin equivalents per gram dry weight of powdered plant parts (mg QE/g DW).

Determination of TFlC
Total flavonol content (TFlC) was determined using the aluminum chloride colorimetric method as described by Kumaran and Karunakaran, 23 with minor modification.A standard calibration curve was prepared using quercetin in the concentration range of 0-35 µg/mL.The plant extract (1 mL) and standard solution (1 mL) were placed in different test tubes and then 2% aluminum chloride (1 mL), 5% sodium acetate (3 mL) were added and mixed well.The reaction mixture was then centrifuged at 3000 rpm for 20 min at 25°C and the absorbance of standard and sample were measured at 440 nm using a UV-visible spectrophotometer.The concentration of flavonol was expressed as mg quercetin equivalents per gram dry weight of powdered plant parts (mg QE/g DW).

DPPH (1,1-Diphenyl-2-picrylhydrazyl) free radical-scavenging assay:
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging capacity of the methanolic extracts (leaves, barks, stems and fruits) was determined using the assay method as described by Abdul-Wahab et al., 24 with minor modifications.A methanol solution of 0.1 mM DPPH was prepared as well as all the extracts in different concentrations ranging from 1-200 µg/mL.Trolox and ascorbic acid were used as the standards and a series of these standards were prepared in the concentration range of 1-12 µg/mL.The free radical scavenging activity was expressed as EC 50 (effective concentration in µg/mL of sample or standard which reduces the absorbance of DPPH by 50% as calculated from the standard graph).
The extracts (leaves, barks, stems and fruits; 1.5 mL each) and standard solutions (1.5 mL) were placed in different test tubes.The DPPH solution (1.5 mL) was added to each of the test tubes.The reaction was allowed to react in the dark for 30 min and its absorbance was measured at 517 nm by using a UV-visible spectrophotometer.The percentage (%) inhibition of both samples and standards were calculated for each concentration and plotted in graphs of percentage inhibition against concentration.The EC 50 values of standards and samples were calculated from these graphs .The percentage inhibition was calculated according to the equation ( 2 ABTS radical scavenging assay 2,2´-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging assay was carried out according to the method as described by Lobo et al., 25 with minor modifications.Methanolic extracts (leaves, barks, stems and fruits) were prepared at different concentrations ranging from 5 to 200 µg/mL.Trolox and ascorbic acid were used as the standards, and a series of standards were prepared in the concentration range from 1 to 12 µg/mL.The ABTS stock solution was prepared by mixing 7 mM ABTS solution and 2.4 mM potassium persulfate solution in the ratio of 1:1 and followed by incubation at 25°C in the dark for 12 h.The working solution was prepared by diluting the resulting solution (1 mL) with methanol (60 mL) until an absorbance of 0.70 ± 0.01 arbitrary units at λ = 734 nm using a UVvisible spectrophotometer.All the extracts and standards were then allowed to react with the ABTS working solution at a ratio of 1:1 for 30 min at 25°C in the dark and the absorbance was measured at λ = 734 nm.The free radical scavenging activity was expressed as EC 50 (effective concentration in µg/mL of sample extract or standard that reduces the absorbance of ABTS by 50% calculated from the standard graph).The percentage (%) inhibition of samples and standards were calculated for each concentration using equation (II) above.Graphs of percentage inhibition against concentration were plotted and the EC 50 values of the test sample and two standards were calculated from these graphs.

FRAP assay
The assay was conducted according to the methods as described by Thiapong et al., 26 with minor modifications.The FRAP reagent was freshly prepared by mixing 300 mM sodium acetate buffer (100 mL), 10 mM TPTZ solution (10 mL) and 20 mM FeCl 3 .6H 2 O (10 mL) solution and kept warmed at 37°C until used.Trolox and ascorbic acid were used as the standards and the calibration curve for each was prepared in the range 1-250 µg/mL.The extracts (leaves, barks, stems and fruits; 150 µL each) and the standard solution (150 µL) were allowed to react with the FRAP solution (2850 µL) in different test tubes for 30 min in the dark at 25°C.The increase in absorbance due to the reduction of Fe 3+ -TPTZ to Fe 2+ -TPTZ (ferrous-tripyridyltriazine complex) was measured at 593 nm by using a UV-visible spectrophotometer.The results were expressed as mg of trolox equivalent and mg of ascorbic acid equivalent per gram dry weight of powdered plant parts (mg TE/g DW and mg AAE/g DW), which were estimated from standard graphs.

ABTS assay
The ABTS assay methods as described by Thiapong et al., 26 was used to determine the antioxidant activity of the extracts in terms of weight of trolox and ascorbic acid equivalents.The ABTS stock solution was prepared by mixing an equal amount of 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution followed by incubation at 25°C in the dark for 12 h.The working solution was prepared by diluting the resulting solution (1 mL) with methanol (20 mL) until reach at an absorbance of 1.1 ± 0.05 arbitrary units at λ = 734 nm using a UVvisible spectrophotometer.A series of standards were prepared in the concentration range of 1-125 µg/mL.Standard solution (150 µL) and sample extract (150 µL) were placed in different test tubes and then the ABTS working solution (2850 µL) was added to each oe.All the mixtures were allowed to react in the dark for 30 min, and their absorbance was measured at 734 nm by using a UV -visible spectrophotometer.The percentage (%) inhibition of the two standards and extracts were calculated for each concentration and plotted in graphs of percentage inhibition against concentration.The results were expressed as mg of trolox or ascorbic acid equivalent per gram dry weight of powdered plant parts (mg TE/g DW and mg AAE/g DW), which were estimated from standard graphs.

Statistical analyses
All the experiments were carried out in triplicates and the results were expressed as means ± standard deviations (unless otherwise stated).The results of antioxidants contents and antioxidant activity were analyzed using correlation and regression of Microsoft Excel 2007.Statistical analyses were done using SPSS software version 20.0 (IBM Corp., Armonk, NY, USA).Analysis of variance (ANOVA), Kruskall-Wallis, Tukey's HSD, and independent sample t-tests were applied for comparisons of means and medians, where significant differences were considered at P < 0.05.The Pearson's correlation coefficient test was done using SPSS to verify inter-relationships between TPC, TFC, TFlC, and antioxidant activity.

Yield of crude extracts
The extraction yields of crude methanolic extracts of leaves, barks, stems, and fruits were 34.60, 12.70, 3.18, 18.81 % per g of the freezedried weight of the sample, respectively, and they differed significantly (P < 0.05) from each other (Figure 1).Thus, ranked from highest to lowest, the % yield is in the order of leaves > fruits > barks > stems.

Phytochemical screening
The phytochemical screening of crude methanolic extracts of the leaves, barks, stems and fruits of A. philippinensis showed the presence of some secondary metabolites such as steroids, tannins, saponins and cardiac glycosides (Table 1).Cardiac glycosides and steroids were detected in all plant parts of the A. philippinensis.Saponins were detected in leaves and barks whereas tannins were only found in leaves.Alkaloids and anthraquinone glycosides were absent from all plant parts.

Total phenolic, flavonoid, and flavonol contents
The total phenolic, flavonoid and flavonol contents of the extracts of A. philippinensis varied significantly (P < 0.05) with different plant parts (Table 2).Among the four plant parts (leaves, stems, barks and fruits), leaves exhibited the highest amount of total phenolic, flavonoid and flavonol contents, followed by fruits, barks and stems.The TPC was also the highest of all four plant parts, followed by the TFC and TFlC.

Evaluation of antioxidant activity
The antioxidant activities of extracts of A. philippinensis were determined using three different assays i.e.DPPH, ABTS and FRAP, as well as two different standards, trolox and ascorbic acid (Tables 3 and 4).Different assays and standards were used to evaluate the antioxidant activities of samples since a single method can detect only a very specific antioxidant from its large and many varieties and cannot cover the whole antioxidant activity measurement of a sample.The delocalization of the spare electron of DPPH makes it a stable free radical.When DPPH accepts an electron from the antioxidant, its deep purple color changes to yellow.The ABTS is also a stable bluegreen free radical and losses its color by the addition of an antioxidant.
The FRAP method measures the ability of antioxidants based on the reduction of the complex of ferric ion and TPTZ to the ferrous form at low pH.All these three methods are simple, inexpensive and reliable to measure antioxidant activity.

Correlation between antioxidant contents and antioxidant activity
According to the Pearson's correlation analysis, correlation is positively high if 0.61≤ r ≤0.97 and negatively high if -0.61≤ r ≤ -0.97. 26,27Negative value signifies that the relations between two parameters are inversely correlated.In this study we determined the correlations between antioxidants contents (TPC, TFC and TFlC) of leaves, barks, stems and fruits extracts of A. philippinensis and their activities detected by different in vitro antioxidant assays (ABTS, FRAP and DPPH).The results are presented in Tables 5A to 5D.

DISCUSSION
Extraction yields of the different plant parts of this study were compared with some publish data and previous report showed that extraction of A. philippinesis stem in ethanol using maceration technique gave 1.50%, 20  Values are means ± SD (n = 3), a TE trolox equivalent, b AAE ascorbic acid equivalent.Mean values were significantly different between (P < 0.05) columns except that with the asterisk.also used to facilitate the extraction of phytochemicals that are soluble in water and/or organic solvent. 29Similarity of polarity and molecular weight of extraction solvent and extracted chemicals from a given sample have also a good relation.It can be explained with 'like dissolve like' concept. 30Therefore, it is quite impossible to extract all types of phenolic compounds from a sample in a single solvent.
A research study used five different solvents as hexane, petroleum ether, chloroform, ethyl acetate and methanol for extraction and results revealed that methanol extract contain highest number of Phytochemicals compared to other solvent extracts. 31Another two published reports also claimed to have found highest amount of total phenolic content from methanolic extract when compared with some other solvent extracts in different species. 32,33In this study, methanol a highly polar solvent was chosen as the extraction solvent to extract more polar compounds as well as phenolic compounds.Extracting all the four plant parts (leaves, barks, stems, and fruits) using the same solvent and extraction method allows easier and consistent comparison of results.
In terms of phytochemical screening study similar secondary metabolites have also been reported in other species of this genus and other genus of this family.For example, fruits of A. neocaledonic, leaves of Zizyphus spina-christi, leaves and stem barks of Z. mauritiana, were all reported to have similar Phytochemicals. 14,34,35 annins are considered as a potent anticancer agent with antioxidant properties. 39Saponins are natural detergents known to have antiinflammatory, expectorant, immune stimulating, and antineoplastic effects.They reduce blood cholesterol level by binding with cholesterol and bile salts.Once bound to saponins, bile salts are unable to form small micelles with cholesterol and thus prevent its absorption in the intestinal tract. 40Steroids found in both plants and animals have many therapeutic uses.Steroids from plants are important source of medicine having cardiotonic, antibacterial and insecticidal properties. 41nce there were no reports on the TPC, TFC and TFlC of Alphitonia species, the results obtained for A. philippinensis in this study were compared to other known medicinal plants as shown in Table 3.The TPC of A. philippinensis leaves in the present study was higher than those of Colubrina asiatica, 42 Ginkgo biloba, 43 Etlingera coccinea, 4 Zizyphus oxyphylla, 44 and Morus alba, 43 but lower than Zizyphus spinachristi, 45 Zizyphus xylopyrus, 46 and Goniothalamus velutinus. 2The value of TPC for the barks was lower than those of Z. spina-christi 45 and G. velutinus, 2 whereas higher value was obtained for the fruits than those of Z. jujube, 47 Z. mistol, 48 and Z. spina-christi, 45  than Z. lotus, 49 Z. oxyphylla, 44 and Z. xylopyrus. 46The TPC result for stems in this study was higher than that of C. asiatica, 42 but lower than E. coccinea, 4 and Z. oxyphylla. 44In terms of the total flavonoid content, leaves of A. philippinensis was also higher than those of Etlingera belalongensis (3.77 mg catechin equivalent (CE) /g), E. volutina (7.63 mg CE/g) and Zingiber pseudopungens (2.87 mg CE/g). 50The leaves also had the highest flavonol content (4.60 mg QE/g DW) compared to barks (0.12 mg QE/g DW), stems (0.001 mg QE/g DW) and fruits (1.82 mg QE/g DW).
Phenolic compounds from plants had been reported to show a variety of biological functions such as antioxidant, antibacterial, antiinflammatory, anticarcinogenic, antiatherosclerotic and antiviral activities. 17,18,20They also have a vital role in preventing osteoporosis, neurodegenerative disease, diabetes and major cardiovascular ailments like hypertension. 36,38ong polyphenols, flavonoids are a large group of compounds having potent antioxidant activities.The special chemical structures of flavonoids, compared with other polyphenols, such as the position of hydroxyl group, carbonyl group and double bond are responsible for their antioxidant activities. 51They have a variety of biological activities such as antibacterial, anti-inflammatory, antiallergic, antithrombotic, and vasodilatory activities.They also hamper the diffusion of free radicals by stabilizing membrane fluidity and thus prevent the peroxidation reaction. 36Flavonols such as quercetin, kaempferol, myricetin, rutin, and azaleatin, have also been reported to show antioxidant properties. 52enolic compounds are the constitutive products of plants and accumulate during normal growth and development.Some phenolic compounds like flavonols and flavones are formed in plant in presence of sunlight and mainly concentrated in the outer tissues. 534][55] These reports supports the results of this study as A.phillipinensis has the highest phenolic contents in leaves followed by fruits, barks and stem which is in agreement with other published reports. 4,56e antioxidant capacity of different plant parts of A. phlippinensis varied significantly (P < 0.05) amongst them (Table 4).For each assay (FRAP and ABTS), the rank of antioxidant activity, from highest to lowest was, leaves > fruits > barks > stems.In comparison to some other known medicinal plants, leaves of A. philippinensis showed higher antioxidant activity than those of Z. jujuba (Rhamnaceae), Paeonia lactiflora (Paeoniaceae), Cimicifuga foetida (Ranunculaceae), Lobelia chinensis (Campanulaceae) and M. alba (Moraceae) with antioxidant capacity values of 546, 85, 349, 26 and 23 µmol TE/g, respectively.These plants have been reported to exhibit antispasmodic, anti-inflammatory, analgesic and anticancer effect.Their uses in the treatment of fever, stomach ache, swelling, sores and improved sleep by soothing nerves have scientifically been proven. 55,57The antioxidant activity of fruits of A. philippinensis was also higher than those of M. alba (1.32 µmol TE/g), Ligustrum lucidum of the Oleaceae (2.34 µmol TE/g) and Gardenia jasminoides of the Rubiacea (1.32 µmol TE/g).However, its barks exhibited lower antioxidant activity than those of Cinnamomum cassia of the Lauraceae (28 mg GAE/g DW) and Magnolia officinalis of the Magnoliaceae (32 mg GAE/g DW).Similarly, the activity of stems was lower than those of Spatholobus suberectus (Leguminosae) and Dendrobium nobile (Orchidaceae) with antioxidant capacity values of 90 mg GAE/g DW and 22 mg GA, respectively. 54e antioxidant activities (expressed as EC 50 values) of methanolic extracts of A. philippinensis in comparison to trolox and ascorbic acids as standard references, differed significantly amongst the four plant parts for DPPH (Figure 2) and ABTS (Figure 3), except for leaves and fruits.The rank from best to lowest scavengers for each assay was Trolox > ascorbic acid > leaves > fruits > barks > stems.This study found a good correlation evidence between antioxidant content with antioxidant property and to the best of our knowledge this is the first complete reported "antioxidant profile" for A. philippinensis.TPC of leaves extract (Table 5A) showed good significant correlation with TFC (r = 0.99, p<0.05) but not TFlC.Both TPC and TFC showed linear correlation with ABTS and FRAP, when either trolox or ascorbic acid was used as the standard, however the correlation was significant only for FRAP.Strong negative correlations were observed for TPC, TFC with DPPH EC 50 and ABTS EC 50 , indicating that increasing TPC and TFC in leaves would increase the antioxidant activity.TFlC of the leaves extract only showed correlation with DPPH EC 50 (r = 0.61).
Unlike the leaves extract which showed no correlation between TFlC with TPC and TFC, the bark extract (Table 5B), however, showed good linear correlations between the three with r value 0.98.Strong linear correlations were also observed for FRAP assays with TPC, TFC and TFLC.Of these, significant correlations was found between FRAP and TFC (r = 1.00, p < 0.05), while TPC and TFlC also showed significant negative correlations with DPPH EC 50 (r = -0.99,p < 0.05 and r = -1.00,p < 0.01, respectively).
As for the stem extract, except for FRAP assay using ascorbic acid as the standard, generally there were correlations between TPC, TFC, TFlC and all the antioxidant assays.However, none were significant.For the DPPH and ABTS EC 50 determination, TFC showed the highest negative correlation (r value -0.98 and -0.85), followed by TPC (r value -0.97 and -0.80) and TFlC (r value -0.69 and -0.36, respectively).
Of the four extracts studied, the fruit extract (Table 5D) showed maximum correlation not only between TPC, TFC and TFlC but also with FRAP and ABTS, all giving significant correlation of r = 1.00 and p < 0.01 or p < 0.05.This is in line with literature reports in which fruits and fruit juices have been known to show high correlation between TPC and antioxidant activities. 60,61However, no correlation was observed for EC 50 of DPPH and ABTS.
Among the four plant parts leaves and fruits showed highest correlation between the antioxidant contents and assays followed by barks and fruits.These variations of correlation with antioxidant contents and assays may happen due to the variation of mechanism of action, delocalization of electron, solubility and redox potential. 27,62RAP can measure the activity of hydrophilic antioxidants, ABTS can measure both hydrophilic and lipophilic antioxidants, and DPPH can react only with those antioxidants that are soluble in organic solvents. 2,4,6The evaluation of antioxidant capacity of these extracts with different assay methods has made the results more reliable since it has been shown that a single method of evaluating antioxidant activity may not be sufficient as no one method will be able to fully evaluate the antioxidant property of a given sample. 63Our results are consistent with other reports 4,26,47 and indicate that phenolic compounds could have a major contribution to the antioxidant property of A. philippinensis.This is in line with reports where high antioxidant activity is associated with high TPC and such observation is not just limited to medicinal plants, but also in wild and cultivated plants 64 , flour, 65 seaweed, 66 fruits, 67 etc.However, it must be noted that a report 68 showed in their study that other plant constituents such as reducing carbohydrate, terpenes, ascorbates etc could also be responsible for the antioxidant activity of a given plant.

CONCLUSIONS
The results of current study showed that methanolic extracts of different plant parts (leaves, barks, stems and fruits) of A. philippinensis revealed the presence of cardiac glycosides, steroids, terpenoids, saponins and tannins.These phytochemicals have vital roles in modern medicines and plants containing these constituents are considered as important natural sources.Based on the antioxidant contents (TPC, TFC, TFlC) and in vitro antioxidant assays (DPPH, ABTS and FRAP), leaves of A. philippinensis showed the highest antioxidant activity followed by fruits, barks and stems.Comparative studies with different published data of some medicinal plants conclude that the leaves of A. philippinensis are strong source of natural antioxidants and can be used as a substitute for synthetic antioxidants in food and pharmaceutical industries to extent the shelf life of their products.A. philippinensis, known for its use in traditional medicine in Brunei Darussalam, has not been sufficiently explored.This is the first attempt and reports to evaluate its medicinal properties.Further studies on the pharmacological evaluation and the isolation of bioactive compounds of the plant are currently being carried out.

Ahmed,Figure 1 :
Figure 1: Percentage yield (mean, N=3) of methanolic extracts of leaves, barks, stems and fruits of A. philippinensis.The bars represent standard errors of means.

Figure 2 :
Figure 2: Scavenging capacity (EC 50 ) of methanolic extracts of different plant parts of A. philippinensis in comparison to standards determined by DPPH assay.Values are means ± SD (n = 3) and the bars represent standard errors of means.

Figure 3 :
Figure 3: Scavenging capacity (EC 50 ) of methanolic extracts of different plant parts of A. philippinensis in comparison to standards determined by ABTS assay.Values are means ± SD (n = 3) and the bars represent standard errors of means.
Ahmed, et al.: Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves, Barks, Stems and Fruits of Alphitonia philippinensis (Rhamnaceae) From Brunei Darussalam

Table 1 : Phytochemicals detected in methanolic extracts of leaves, barks, stems and fruits of A. philippinensis.
+ Present, -absent

Table 2 : Total phenolic, flavonoid and flavonol contents of methanolic extracts of leaves, barks, stems and fruits of A. philippinensis.
Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves, Barks, Stems and Fruits of Alphitonia philippinensis (Rhamnaceae) From Brunei Darussalam higher by approximately two fold with methanol extract but close to water extract.Percentage yields of different plant species may vary due to the difference in genetic makeup of the plants, sampling location, harvesting time, extraction method and solvent used in the extraction.Different solvents have different polarity and hence have the ability to extract different compounds, giving different percentage yields of extracts.Sometimes the aqueous solution of organic solventsAhmed, et al.:

Table 4 : Antioxidant activities of extracts of different plant parts of A. philippinensis determined using ABTS and FRAP assays.
Evaluation of Antioxidant Activity and Phytochemical Screening of Leaves, Barks, Stems and Fruits of Alphitonia philippinensis (Rhamnaceae) From Brunei Darussalam [36][37][38]hemicals are known to have important traditional and medicinal uses.Crude extracts of plants having cardiac glycosides had been used in ancient time for arrow coatings, homicidal or suicidal aids, rat poison, heart tonics, diuretics and emetics.Modern medicine has also developed drugs to treat congestive heart failure and cardiac arrhythmia from purified plant extracts or synthetic analogues of some of these cardiac glycosides.[36][37][38]et al.: