Phytochemical constituents and biological activities of pseuderanthemum palatiferum (nees) radlk leaf extracts

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2014, Vol. 59, No. 9, pp. 103-113 This paper is available online at PHYTOCHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF Pseuderanthemum palatiferum (Nees) Radlk. LEAF EXTRACTS Le Thi Phuong Hoa and Pham Thi Diu Faculty of Biology, Hanoi National University of Education Abstract. Total methanol extract and n-hexane, ethyl acetate and water fractions of P. palatiferum (Nees) Radlk. leaves were investigated for their antioxidant, antimicrobial and tyrosinase inhibitory activities in correlation with their phytochemical constituents. Of the extracts, the ethyl acetate fraction had the highest level of phenolics (327.00  56.30 mg gallic acid equivalents/g of extract), more than half of which were flavonoids (182.77  14.37 mg quercetin equivalents/g of extract), confirming the result of thin layer chromatography analysis. Ethyl acetate and water fractions had antioxidant activity by dose-dependent 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging action (IC50 = 90.01  4.12 and 82.36  2.38 g/mL, respectively), which closely correlated with the total phenolic content (R2 = 0.936). P. palatiferum leaf extracts showed stronger antibacterial activity against Escherichia coli and Bacillus subtilis than Staphylococcus aureus. Only the n-hexane fraction showed inhibitory activity on Pseudomonas sp. and Candida sp. The methanol extract of P. palatiferum (Nees) Radlk. leaves exhibited a moderate inhibitory effect on the L-3,4-dihydroxyphenylalanine (L-DOPA) oxidase activity of tyrosinase in the melanin biosynthesis pathway. Further study may lead to new bioactive compounds from ethyl acetate and n-hexane fractions. Keywords: Pseuderanthemum palatiferum, antioxidant, antimicrobial, tyrosinase inhibitory activity. 1. Introduction Pseuderanthemum palatiferum (Nees) Radlk., or Hoan ngoc in Vietnamese, is a medicinal plant of the Acanthaceae family. It was first found in the Cuc Phuong forest in northern Vietnam in the latter half of the 1990s. It is a shrub with a height of 1 to 2 m, multi-branched and rapid growing [6]. Since its discovery, it has been cultivated throughout Vietnam and has been used for both the prevention and treatment Received November 21, 2014. Accepted December 14, 2014. Contact Le Thi Phuong Hoa, e-mail address: lephhoa@yahoo.com 103 Le Thi Phuong Hoa and Pham Thi Diu of human diseases such as hypertension, diarrhea, arthritis, pharyngitis, gastritis tumor, colitis, bleeding, wounds and stomachache [6]. It is also suggested to be applied in husbandry as in raising piglets for increasing growth rate, decreasing mortality and lower the incidence and duration of diarrhea [7]. A phytochemical analysis showed that P. palatiferum leaves are composed of -sitosterol, stigmasterol, terpenoids, phenolics and flavonoids as well as the essential amino acids lysine, methionine and threonine [5, 6, 10]. Extracts of P. palatiferum leaves have been shown to be capable of biological activities. Ethyl acetate and n-butanol extracts of P. palatiferum leaves had antioxidant activities on blood peroxidase along with antibacterial and antifungal activity [8]. P. palatiferum leaf aqueous extract had acetylcholinesterase inhibitory activity in albino rats [3], antidiabetic activity in decreasing blood glucose in diabetic rats and in vitro antioxidant activity with radical scavenging activity, reducing power, lipid peroxidation inhibition and protective effects against 2-amidinopropane hydrochloride-induced hemolysis [4]. Methanol, ethanol and acetone extracts of P. palatiferum leaves also exhibited antioxidant activity with DPPH scavenging capacity, reducing power and metal chelating activity [10] as well as antibacterial and antifungal activity but at a moderate level [11]. Ethanol extract of P. palatiferum leaves showed significant anti-inflammatory activities against both acute and chronic inflammation using the ethyl phenyl propionate induced ear edema test and the cotton pellet induced granuloma model in albino rats [9]. Ethanol and water extracts of P. palatiferum leaves had antioxidant properties, in decreasing tert-butyl hydroperoxide-induced oxidative stress, and anti-inflammatory properties suppressing LPS plus IFN- -induced nitric oxide and the expression of inducible nitric oxide synthase and cyclooxygenase-2 protein levels in RAW264.7 macrophage cells [14]. However, the phytochemical constituents of P. palatiferum leaves in different extraction approaches for bioactive compound purification and other biological activities such as tyrosinase inhibitory activity in skin depigmentation for more beneficial application need characterization. This study evaluates the antioxidant, antimicrobial and tyrosinase inhibitory activities of methanol extract and its fractionation of P. palatiferum leaves and the correlation to their phytochemical constituents. 2. Content 2.1. Material and method * Materials Pseuderanthemum palatiferum (Nees) Radlk. leaves were collected in Hanoi. Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas sp. and Candida sp. were obtained from the National Institute of Hygiene and Epidemiology. Ascorbic acid, DPPH, quercetin, L-DOPA, mushroom tyrosinase and kojic acid was purchased from Sigma Chemicals (MO, USA). Gallic acid and Folin-Ciocalteu reagent were obtained from Merck Chemicals (Darmstadt, Germany). 104 Phytochemical constituents and biological activities of Pseuderanthemum palatiferum... * Sample preparation Fresh leaves were washed with distilled water to remove adhering debris and dust, and then freeze dried to constant weights. The dried tissues were ground to a powder and then extracted with methanol in an ultrasonic bath for 30 min at room temperature. The extraction was performed in three replications. The extracts were mixed and concentrated in a rotary evaporator at 40 C, and then freeze dried. The crude extract was further fractionated in distilled water, n-hexane and ethyl acetate. The three fractions were concentrated by vacuum evaporation and freeze dried. All of the extracts were stored in a refrigerator for later biochemical analysis and bioassays. * Thin layer chromatography The extracts were prepared at the concentration of 10 mg/mL in absolute ethanol. Each extract was applied as a single spot in a row along one side of the precoated silica gel aluminum plate 60F254, about 2 cm from the edge, using capillary tubes. A solvent consisting of toluene, ethyl acetate and formic acid in a ratio of 5:4:1 was used as the mobile phase. The plate was sprayed with 10% sulfuric acid, heat dried and observed under visible light. A qualitative evaluation of the plate was done by determining the migrating behavior of the separated substances compared with that of the solvent given in the form of Rf value. * Determination of total phenolic content The total phenolic content was estimated employing the method of A.L.Waterhouse [15], using Folin-Ciocalteu reagent with gallic acid as the standard. Sample solutions were prepared in ethanol at a concentration of 1 mg/mL and standard solutions were from 0 - 0.5 mg/mL. Sample or standard solution (10 L) was mixed with Folin-Ciocalteu reagent (50 L) and water (790 L). After 5 min, 150 L of 10% sodium carbonate was added. The mixture was kept at room temperature for 90 mins. The absorbance was then measured at 765 nm. The amounts of total phenolics were calculated using a gallic acid calibration curve. The results were expressed as mg gallic acid equivalents (GAE) per gram dry weight of each extract. * Determination of total flavonoid content The total flavonoid content of each extract was determined making use of the method described by Sapkota et al. [13] using quercetin as the standard. Extracts were diluted with 80% aqueous ethanol to arrive at a concentration of 1 mg/ml. Quercetin solutions were prepared in the same manner in the range of 0 - 0.3 mg/mL. Different quercetin and extract solutions (100 L) were mixed with 20 L 10% Al(NO3)3, 20 L 1MK-acetate and 860 L 80% ethanol. After standing for 40 min at room temperature, the absorbance of the mixture was determined spectrophotometrically at 415 nm. The results were expressed in mg quercetin equivalents (QE) per gram dry weight using a quercetin standard curve. 105 Le Thi Phuong Hoa and Pham Thi Diu * Antioxidant activity Antioxidant activity was evaluated through free radical scavenging capacity using DPPH according to Blois [2]. The reaction mixture contained 20 L of extract solutions at various concentrations ranging from 5 - 500 g/mL in ethanol and 180 L of 0.3 mM DPPH solution. The samples were allowed to stand in a dark place at room temperature for 20 min. The control was prepared with ethanol instead of extracts. Ascorbic acid was used for comparison with extracts. The reduction of DPPH free radicals was measured by reading at 517 nm. DPPH scavenging activity was calculated using the following formula: DPPH scavenging activity (%) = [(Acontrol – Asample)/(Acontrol)]  100 where Acontrol represents the absorbance of the control and Asample is the absorbance of the test sample. Half-maximal inhibitory concentration (IC50) of ascorbic acid and extracts were calculated based on the logarithm curve of DPPH scavenging activity vs. concentration. * Antimicrobial activity Antimicrobial activity was tested against B. subtilis, S. aureus, Pseudomonas sp., E. coli and Candida sp. using the agar well diffusion method. The 24 hour culture broth of the test microorganisms (approximately 1108 CFU/mL) was spread onto petri plates containing MPA (meat-peptone-agar) for bacteria and Hansen medium for fungi. Wells 10 mm in diameter were made aseptically in the inoculated plates. Each extract was dissolved in methanol to a final concentration of 10 mg/mL. Methanol was used as the negative control and 0.4% chloramphenicol was used as the positive control. Aliquots of 100 L of the extracts and controls were added into the respectively labeled wells. The plates were incubated at 30 C for 24 h for bacteria and 36 h for fungi in an upright position. The antimicrobial activity was determined by measuring the diameter of the inhibition zone formed around the well. * Tyrosinase inhibitory activity A Tyrosinase inhibition assay was carried out according to the procedure described by Sapkota et al. [13] using L-DOPA as the substrate. Kojic acid was used for comparison in a concentration range of 0.2 - 125 g/mL in a 0.175 M phosphate buffer at pH 6.8. Total extract concentration ranged from 50 - 1000 g/mL. One hundred L of each test sample was mixed with 20 L of phosphate buffer pH 6.8 and 40 L of 5 mM L-DOPA before added with 40 L of 110 UI/mL mushroom tyrosinase. The reaction mixture was incubated at 30 C for 2 min. The amount of DOPAchrome was determined at 475 nm. The percentage inhibition of tyrosinase activity was calculated as follows: Tyrosinase inhibition capacity (%) = [(A - B)/A]100 where A is the absorbance at 475 nm without the test sample and B is the absorbance at 106 Phytochemical constituents and biological activities of Pseuderanthemum palatiferum... 475 nm with the test sample. IC50 values were calculated based on the logarithm curve of tyrosinase inhibitory activity vs. concentration. * Statistical analysis For statistical analysis, data were analyzed using Microsoft Excel software. Results were given as means  standard deviation of three replicated determinations. 2.2. Results and discussion * Thin layer chromatography Phytochemical constituents of the crude methanol extract and fractions of Pseuderanthemum palatiferum (Nees) Radlk. leaves were screened using thin layer chromatography with a toluene:ethyl acetate:formic acid solution (5:4:1) as a solvent system. The chromatograms were visualized by spraying with 10% H2SO4 and observed under visible light at 365 nm (Figure 1). Figure 1. TLC chromatogram of P. palatiferum leaf extracts in a toluene: ethyl acetate:formic acid (5:4:1) solvent system under visible light (A), at 365 nm (B) Me: methanol extract, He: n-hexane fraction, Et: ethyl acetate fraction and W: water fraction The TLC chromatogram of P. palatiferum leaf extracts revealed various compounds. The ethyl acetate fraction and the n-hexane fraction had the highest number of bands under visible light (8 bands) with different Rf values (data not shown). The crude extract and the water fraction showed fewer bands. The dominant compounds in P. palatiferum leaf extracts are terpenoids, revealed by pink and purple bands, flavonoids (yellow and orange) and chlorophyll (green). The ethyl acetate fraction had several yellow and orange bands, suggesting the presence of a high content of flavonoids, as compared to the n-hexane and the water fraction, which requires further characterization. Blue fluorescence bands were also observed in this fraction and the n-Hexane fraction under 365 nm, represented by phenol carboxylic compounds (Figure 1B). K. Chayarop et al. also found the presence of flavonoids, terpenoids and phenol carboxylics in the ethanol extract 107 Le Thi Phuong Hoa and Pham Thi Diu of P. palatiferum leaves using a solvent system of ethyl acetate:formic acid:water solution (90:2:2) and spraying with NP/PEG or anisaldehyde sulfuric acid. However, flavonoids were not detected using the solvent system of toluene:ethyl acetate:formic acid solution (5:4.5:0.5) [5]. * Total phenolic and flavonoid content Table 1. Total phenolic and flavonoid contents of P. palatiferum leaf extracts Sample Phenolic content Flavonoid content (mg GAE/g DW) (mg QE/g DW) Methanol extract 354.50  36.06 34.79  1.69 n-Hexane fraction 80.00  16.00 71.38  0.99 Ethyl acetate fraction 327.00  56.30 182.77  14.37 Water fraction 267.07  18.00 Nd. GAE: gallic acid equivalents, QE: quercetin equivalents, DW: dry weight, Nd. Not determined The crude methanol extract of P. palatiferum leaves showed a high concentration of phenolics but a low concentration of flavonoids. The total phenolic content of the ethyl acetate fraction were the higher than the other fractions. More than half of these compounds are flavonoids. The total content of phenolics and flavonoids in the ethyl acetate fraction was approximately four times and 2.5 times, respectively, more than those in the n-hexane fraction. The result goes with the chromatogram analysis on biochemical constituents of the two fractions. The water fraction had a lower but remarkable amount of phenolics compared to ethyl the acetate fraction, which may indicate the accumulation of soluble polyphenols from P. palatiferum leaves. The P. palatiferum leaf methanol extract in this study was found to have a much higher total phenolic content but a lower total flavonoid content compared to a previous report [10]. However, the results indicate that the phenolic and flavonoid compounds of P. palatiferum leaf methanol extract are concentrated in the ethyl acetate fraction. According to Chayarop et al., the content of flavonoids equivalent to kaempferol in P. palatiferum leaf aqueous extract was also at a low level (2.2781  0.0170 mg/g extract) [4]. The water fraction collected in this study possessed a higher phenolic content than did the total water extract of P. palatiferum leaves collected by Sittisart and Chitsomboon (212.47  0.52 mg GAE/g). Accounting for approximately half of the extract were the flavonoids [14]. Phenolic compounds including flavonoids have been widely investigated in many medicinal plants and food plant as they are responsible for multiple biological effects in antiallergic, antibacterial, antidiabetic, anticancer and anti-inflammatory activities [4,8-10,14]. Previous research showed that antioxidant activity influenced blood peroxidase activity and antibacterial activity of the ethyl acetate fraction from P. palatiferum leaves and proposed the involvement of flavonoids [8]. 108 Phytochemical constituents and biological activities of Pseuderanthemum palatiferum... * Antioxidant activity The antioxidant activity of P. palatiferum leaf extracts was estimated using a DPPH free radical scavenging assay. In the presence of an antioxidant, a DPPH radical obtains one more electron, decolorizes and as a result the absorbance decreases [12]. The DPPH scavenging activity of P. palatiferum leaf extracts in different concentrations and their IC50 values were presented as follows. Table 2. DPPH scavenging activities of P. palatiferum leaf extracts Sample DPPH scavenging activity (%) IC50 (g/mL) 5 10 50 100 500 (g/mL) MeOH 11.662.36 18.962.21 44.32.67 64.4 1.84 90.7 0.08 49.702.36 n-Hex 1.990.31 4.250.43 7.180.62 22.295.59 62.736.72 337.088.31 EtoAc 2.670.22 6.471.93 28.953.19 51.914.68 83.78 6.71 90.014.12 Water 6.591.24 9.792.49 31.34.99 53.733.85 88.62 1.21 82.362.38 Ascorbic acid 8.122.47 18.361.94 72.924.61 94.311.19 96.190.73 29.050.50 MeOH: methanol extract, EtoAc: ethyl acetate fraction, n-Hex: n-hexane fraction P. palatiferum leave extracts showed a dose-dependent DPPH scavenging capacity. The ethyl acetate and water fractions showed much higher activity, IC50 value 90.01  4.12 and 82.36 2.38 g/mL, respectively, than the n-hexane fraction but lower than that of ascorbic acid (IC50 = 29.05  0.50 g/mL). These fractions also had high amounts of phenolics and flavonoids compared to the n-hexane fraction, suggesting the contribution of flavonoids and other phenolics to their antioxidant ability. As shown in Figure 2, the antioxidant activity of P. palatiferum leaf extracts closely correlated with the total phenolic content (R2 = 0.936). Figure 2. Relationship between DPPH scavenging capacity and total phenolic contents of P. palatiferum leaf extracts 109 Le Thi Phuong Hoa and Pham Thi Diu Phenolic compounds have been reported to possess redox properties such as adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, and decomposing peroxides [12]. Many previous studied showed that flavonoids and phenolic compounds were major antioxidant constituents in medicinal herbs, vegetables, fruits and spices and there is highly positive correlation between total phenolic content and the antioxidant activity of plant extracts [4, 10, 12, 13]. K. Chayarop et al. proved that P. palatiferum leaf aqueous extract had the ability to scavenge DPPH (IC50 = 221.14 g/mL), reduce Fe3+, and inhibit linoleic acid peroxidation and suggested that this is due to the contribution of flavonoids in the free radical scavenging activity and to the termination of the chain reaction due to their hydrogen donating ability [4]. In recent research, Sittisart and Chitsomboon also figured out the P. palatiferum possessed strong antioxidant properties. A water extract of P. palatiferum had significantly greater ability (IC50 = 21.55  0.06 g/mL) than the ethanol extract (IC50 = 23.45  0.12 g/mL) but less than the positive antioxidant controls such as vitamin C (IC50 = 3.94  0.01 g/mL) [14]. These fractions contained high level of phenolics and flavonoids. * Antimicrobial activity The screening of antimicrobial activity of P. palatiferum leaf extracts on two Gram-positive bacterial strains (B. subtilis and S. aureus), two Gram-negative bacterial strains (E. coli and Pseudomonas sp.) and a fungal strain (Candida sp.) was carried out using the agar well diffusion method. The activity was evaluated based on the absence or the presence of and the diameter of the zones of microbial growth inhibition around the wells. Table 3. Antimicrobial activity of P. palatiferum leaf extracts Zone of inhibition (mm) Sample B. subtilis S. aureus E. coli Pseudomonas sp. Candida sp. Control (+) 37.0  1.7 40.2  1.0 25.0  0.9 14.0  0.4 37.0  0.8 Control (-) - - - - - MeOH 3.0  0.3 8.0  0.5 13.0  0.3 - - n-Hex 3.0  0.05 8.0  1.0 8.0  0.7 5.0  0.3 3.0  0.2 EtoAc 8.0  0.5 4.0  0.8 9.0  1.1 - - Water 3.0  0.02 9.0  1.2 9.0  0.9 - - (-): no inhibition, MeOH: methanol extract, EtoAc: ethyl acetate fraction, n-Hex: n-hexane fraction The results show the antibacterial activity of P. palatiferum leaf extracts against both Gram-positive bacteria (B. subtilis, S. aureus) and Gram-negative bacteria (E. coli) with stronger activity against E. coli and S.aureus. The ethyl acetate fraction had stronger activity towards B. subtilis but weaker activity to S. aureus compared with other extracts. This antibacterial capacity of P. palatiferum leaf extracts may be associated with the 110 Phytochemical constituents and biological activities of Pseuderanthemum palatiferum... total phenolic and flavonoid content. Phenolic compounds might interact with membrane proteins or change bacterial cell permeability and obstruct membrane functions including electron transport, nutrient uptake, protein and nucleic acid synthesis and enzyme activity [1]. The observation in this study agrees with a previous report on the strong antibacterial activity of an ethyl acetate extract of P. palatiferum leaves, especially against Salmonella typhi 158, Shigella flexneri and E. coli with the probable involvement of flavonoids in this fraction [8]. According to research done by Nguyen et al., a methanol extract of P. palatiferum leaves showed antibacterial and antifungal activity with stronger activity against B. subtilis and S. aureus than E. coli and P. aeruginosa, and the antimicrobial compounds were hydrophilic [11]. Interestingly, only the n-hexane fraction inhibited the growth of Pseudomonas sp. and Candia sp. This suggests that antimicrobial compounds against these strains may be more hydrophobic. Further chemical characterization may reveal new compounds with antibacterial and antifungal activities. * Tyrosinase inhibitory activity Tyrosinase has a key role in both mammalian melanogenesis and fruit or fungi enzymatic browning. It catalyzes the rate-limiting step, the oxidation of tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA to DOPAquinone in melanin synthesis [13]. There has been a great interest in tyrosinase inhibitors for use in preventing the browning of foods and for skin whitening. P. palatiferum leaf extracts have antioxidant capacity and show antimicrobial activity. In order to further characterize its effects, P. palatiferum leaf extract was subjected to tyrosinase inhibition assay using L-DOPA as the substrate. Table 4. Tyrosinase inhibitory activity of P. palatiferum leaf extracts Sample Tyrosinase inhibition activity (%) (g/mL) 2 10 50 200 1000 MeOH 43.011.39 51.165.53 59.746.01 67.394.78 71.821.34 0.2 1 5 25 125 Kojic acid 11.230.43 18.581.79 48.481.04 85.750.73 95.430.15 MeOH: methanol extract As shown in Table 4, methanol extract of P. palatiferum leaves had an inhibitory effect on the DOPA oxidase activity of mushroom tyrosinase. The activity was in a concentration-dependent manner as the inhibition increased with the increase in the concentration of the extract though at a rate slower than that of kojic acid. The IC50 value was approximately 8.78 /mL, suggesting strong inhibitory activity at low concentration. The effect of P. palatiferum leaves on melanin synthesis should be investigated for further applications. 111 Le Thi Phuong Hoa and Pham Thi Diu 3. 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