Evaluation of in vitro antioxidant, antiglycation and antimicrobial potential of indigenous Myanmar medicinal plants

Accepted Manuscript Original Research Article Evaluation of in vitro antioxidant, antiglycation and antimicrobial potential of indigenous Myanmar medicinal plants The Su Moe, Htet Htet Win, Thin Thin Hlaing, War War Lwin, Zaw Min Htet, Khin Mar Mya PII: DOI: Reference:

S2095-4964(18)30083-9 https://doi.org/10.1016/j.joim.2018.08.001 JOIM 51

To appear in:

Journal of Integrative Medicine

Received Date: Accepted Date:

2 February 2018 8 June 2018

Please cite this article as: T.S. Moe, H.H. Win, T.T. Hlaing, W.W. Lwin, Z.M. Htet, K.M. Mya, Evaluation of in vitro antioxidant, antiglycation and antimicrobial potential of indigenous Myanmar medicinal plants, Journal of Integrative Medicine (2018), doi: https://doi.org/10.1016/j.joim.2018.08.001

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JIM-01-2018-OA-ER-0029 Original Research Article

Evaluation of in vitro antioxidant, antiglycation and antimicrobial potential of indigenous Myanmar medicinal plants The Su Moea, Htet Htet Wina, Thin Thin Hlaingb, War War Lwina, Zaw Min Hteta, Khin Mar Myac a Pharmaceutical Research Laboratory, Biotechnology Research Department, Ministry of Education, Kyaukse 05151, Mandalay Division, Myanmar b Department of Research and Innovation (DRI), Ministry of Education, Yangon 11081, Myanmar c Cell Culture Laboratory, Biotechnology Research Department, Ministry of Education, Kyaukse 05151, Mandalay Division, Myanmar ABSTRACT OBJECTIVE: Myanmar has a long history of using medicinal plants for treatment of various diseases. To the best of our knowledge there are no previous reports on antiglycation activities of medicinal plants from Myanmar. Therefore, this study was aimed to evaluate the antioxidant, antiglycation and antimicrobial properties of 20 ethanolic extracts from 17 medicinal plants indigenous to Myanmar. METHODS: In vitro scavenging assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), superoxide (SO) radicals were used to determine the antioxidant activities. Folin-Ciocalteu’s method was performed to determine the total phenolic content. Antiglycation and antimicrobial activities were detected by bovine serum albumin-fluorescent assay and agar well diffusion method. RESULTS: Terminalia chebula Retz. (Fruit), containing the highest total phenolic content, showed high antioxidant activities with inhibition of 77.98% ± 0.92%, 88.95% ± 2.42%, 88.56% ± 1.87% and 70.74% ± 2.57% for DPPH, NO, SO assays and antiglycation activity respectively. It also showed the antimicrobial activities against Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans with inhibition zone of 19, 18, 17, 25 and 15 mm, respectively. Garcinia mangostana Linn. showed the strongest activities for SO and antiglycation assays with inhibition of 93.68% ± 2.63% and 82.37% ± 1.78%. Bark of Melia sp. was the best NO radical scavenger with inhibition rate of 89.39% ± 0.60%. CONCLUSION: The results suggest that these plants are potential sources of antioxidants with free radicals-scavenging and antiglycation activities and could be useful for decreasing the oxidative stress and glycation end-products formation in glycation-related diseases. Please cite this article as: Moe TS, Win HH, Hlaing TT, Lwin WW, Htet ZM, Mya KM. Evaluation of in vitro antioxidant, antiglycation and antimicrobial potential of indigenous Myanmar medicinal plants. J Integr Med. 2018; Epub ahead of print. Keywords: Medicinal plants; Antiglycation; Antioxidant; 2,2-Diphenyl-1-picrylhydrazyl; Nitric oxide; Superoxide; Anti-infective agents Received February 2, 2018; accepted June 8, 2018. Correspondence: The Su Moe; E-mail: [email protected]. The Su Moe and Htet Htet Win contributed equally.

1. Introduction Oxidative stress and nonenzymatic protein glycation may be associated with the very common chronic disease, diabetes mellitus. The formation of advanced glycation end-products (AGEs) is accelerated in hyperglycemic conditions. Oxidation process plays a major role in accelerating the rate of formation of AGEs. On the other hand, AGEs generate reactive oxygen species, in addition to radicals and other reactive intermediates, from autoxidation reactions. Free radical-derived oxidative stress plays a significant role in 1 / 12

causing several physiological conditions, including aging and diseases like diabetes, cancer, cardiovascular diseases and Parkinson’s disease [1–3]. Plant samples that have both antioxidant and antiglycation properties may have greater potential for treating various biological disorders. For example, diabetes can be treated more effectively using compounds that have a combination of antioxidant and antiglycation properties [4–6]. Additionally, the antiglycation activity of plant extracts has been attributed to the presence of phenolic compounds, which have also been correlated with the free radical-scavenging activity of a compound [4,7–12]. These days, several types of natural and artificial antioxidants are in regular use worldwide to treat oxidative stress; plants are known to produce natural antioxidants to control their own oxidative stress [13]. On the other hand, antimicrobial drugs have been effectively used for the control of bacterial infections. However, the emergence of antibiotic-resistant pathogenic microbes has made this conventional treatment less effective [14]. New antimicrobial compounds are needed for fighting these challenging pathogenic microbes. At this point, natural products, especially medicinal plants, are considered an important source for new drug discoveries. Pharmacologically active compounds and phytochemicals isolated from endemic and indigenous plants that have been used in traditional medicine are of great interest for drug discovery [15–17]. Myanmar has a long tradition of medicinal practices that relies on natural products for primary health care. Indigenous plants of Myanmar have been used in traditional medicine to manage diabetes, but many interesting traditional uses have not been rigorously studied. To fill this knowledge gap, the present study evaluates the in vitro antioxidant, antiglycation and antimicrobial properties of selected indigenous medicinal plants from Myanmar which have traditionally been used for the management of diabetes. 2. Materials and methods 2.1. Plant materials The plant samples were collected from Kyaukse district, Mandalay region, Myanmar. The plants were manually cleaned, air-dried and ground into fine powder with a grinding machine (10 HP, Hein Min, Myanmar). Plant tissue was extracted using a maceration method. The samples were soaked in 95% ethanol for one month, prior to filtering. The solvent was evaporated to dryness in a rotary evaporator (RE 2000B, China), and the residues were transferred to screw-capped vials and stored in the refrigerator until used. The botanical identifications of plant species were made by the authorized botanist from Mandalay University, Mandalay, Myanmar. The selected Myanmar medicinal plants used in this study are shown in Table 1. Table 1 Selected Myanmar medicinal plants. Sample code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Botanical name Costus specosus Smith. Coccinia indica W. & A. Syn. Cephalandra indica Naud. Argemene mexicana Linn. Jatropa gossypifolia Linn. (Bronze) Jatropa gossypifolia Linn. (Green) Mimosa pudica Linn. Melia sp. Melia sp. Melia azedarach. Linn.(Blue) Melia azedarach. Linn.(Blue) Jasminum sessiliflorum Vahl. Jasminum sessiliflorum Vahl. Ficus bengalensis Linn. Poinciana regia Bojer. Terminalia belerica Roxb. Rauwolfia serpentina Benth. Annona muricata Linn. Azima sarmentosa Benth. Garcinia mangostana Linn. Terminalia chebula Retz.

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Family name Zingiberaceae Cucurbitaceae Papaveraceae Euphorbiaceae Euphorbiaceae Mimosaceae Meliaceae Meliaceae Meliaceae Meliaceae Oleaceae Oleaceae Moraceae Caesalpiniaceae Combretaceae Sapotaceae Annonaceae Salvadoraceae Hypericaceae Combretaceae

Parts used Leaves Whole plant Whole plant Whole plant Whole plant Whole plant Roots Bark Leaves Bark Leaves Stems Bark Flowers Fruits Whole plant Fruits Leaves Rings Fruits

2.2. Chemicals All chemicals used for these experiments were of analytical grade. Chemicals such as 2,2-diphenyl-1picrylhydrazyl (DPPH), sodium nitroprusside, sulphanilic acid, fructose, sodium azide, N-(1-naphthayl) ethylenediamine dihydrochloride, Folin-Ciocalteu’s reagent, ethylenediamine tetra-acetic acid (EDTA), nitro blue tetrazolium (NBT), riboflavin and bovine serum albumin (BSA) were purchased from Sigma Chemicals Co. (St. Louis, USA), or Himedia (Mumbai), or TCI Development Co. Ltd (Shanghai, China). 2.3. In vitro DPPH free radical-scavenging assay The free radical-scavenging activity of the extracts was determined by using the stable DPPH free radicalscavenging assay as described by Lee et al. [18]. The reaction mixture containing 5 µL of test sample (0.5 mg/mL) in dimethyl sulfoxide (DMSO) and 95 µL of DPPH (300 µmol/L) in ethanol, was placed in individual wells of a 96-well microtiter plate. After keeping the microplate in the darkness at 37 ˚C for 30 min, the optical density (OD) of each well was read using a SPECTROstar Nano microplate reader (BMG, Labtech, Germany) at the wavelength of 515 nm. DMSO was used as the blank, and ascorbic acid was used as the standard. The inhibition rate was calculated using the following formula: inhibition (%) = (1 – OD tested/OD control) × 100. 2.4. In vitro nitric oxide radical-scavenging assay The free radical-scavenging activity of the extracts was determined with the nitric oxide (NO) radicalscavenging assay as described by Hertog et al. [19]. The reaction mixture, containing 10 µL of test sample (0.5 mg/mL) in DMSO, 20 µL of phosphate buffer saline (0.1 mol/L, pH 7.4) and 70 µL of sodium nitroprusside (10mmol/L), was incubated at 25 ˚C for 90–100 min to allow formation of nitrite ions. After incubation, 50 µL of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) was added and allowed to stand for 5 min for completion of diazotisation. Then 50 µL of N-(1-naphthyl) ethylenediamine dihydrochloride (0.1% w/v) was added, stirred and allowed to stand for 20 min. A pink-colored chromophore was formed under the diffused light. The reduction of the pink-colored chromophore was measured at 540 nm against the corresponding blank solution. Gallic acid was used as the standard. Inhibition rate of nitric oxide radical formation was calculated using the following formula: inhibition (%) = (1 – OD tested/OD control) × 100. 2.5. In vitro superoxide radical-scavenging assay The free radical-scavenging activity of the extracts was determined with a superoxide (SO) radicalscavenging assay modified from the protocol described by Patel Rajesh et al. [20]. Briefly, the reaction mixture, containing 10 µL of test sample (0.5 mg/mL) in DMSO, 160 µL of potassium phosphate buffer (0.067 mol/L, pH 7.4), 15 µL of EDTA (4.5 mmol/L), 10 µL of NBT (1 mg/mL) and 5 µL of riboflavin (0.2 mg/mL), was incubated for 5 min under fluorescence light. The OD of the solution was measured at 560 nm against the corresponding blank solution. Gallic acid was used as the standard. Inhibition rate was calculated by using the following formula: inhibition (%) = (1 – OD tested/OD control) × 100. 2.6. Total phenolic content measurements The total amount of phenolic compounds in each plant extract was measured with the method described by Spanos and Wrolstad [21] with a few modifications. Briefly, 100 μL of the extracts (1 mg/mL) were oxidized with 500 μL of freshly diluted 10% Folin-Ciocalteu’s reagent. Then, the reaction was neutralized by adding 500 μL of 7.5% (w/v) sodium carbonate, and samples were vortexed for 20 s. Next, the samples were incubated at 37 °C for 1 h and the OD was measured with a digital photo colorimeter (Apel PD 303, Japan) at a wavelength of 765 nm. For each sample, three replicate assays were performed. The total phenolic content (TPC) was calculated as gallic acid equivalent (GAE) with the following equation: T = C ×V/M. T is the TPC in mg/g of the extracts in units of GAE; C is the concentration of gallic acid, established from the calibration curve in mg/mL; V is the volume of the extract solution in mL and M is the weight of the extract in g. 2.7. In vitro antiglycation assay The in vitro antiglycation activity of the extracts was determined by measuring the ability of the extracts to inhibit the formation of AGE, as described by Choudhary et al. [22]. In a 96-well black fluorescence plate, the reaction mixture, containing 10 µL of BSA (10 mg/mL), 70 µL of sodium phosphate buffer (0.1 mol/L, pH 7.4), 100 µL of fructose (500 mmol/L) and 20 µL of test samples (0.5 mg/mL) in DMSO, was incubated at 37 ˚C for 7 days in the darkness. All the chemicals were dissolved in sodium phosphate buffer to get the 3 / 12

desired concentration. To avoid the microbial contamination, sodium azide (0.1 mmol/L) was added to the sodium phosphate buffer. After incubation, inhibition of AGE formation was measured at the fluorescence’s intensity of excitation (340 nm) and emission (440 nm) by using an Agilent Cary Eclipse Fluorescence spectrophotometer (G9800, US). Rutin (1mmol/L) was used as a standard. The reaction mixture, without fructose, was used as the negative control and reaction mixture without extracts was used as a positive control. Inhibition rate (%) was calculated with the following formula: inhibition (%) = (1 – OD tested/OD control) × 100. 2.8. Evaluation of antimicrobial activity 2.8.1. Tested microorganisms Two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), three Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis and Bacillus cereus) and one strain of fungus, Candida albicans, were used as test microorganisms for this experiment. B. cereus and C. albicans were provided by Department of Biotechnology, Mandalay Technological University, Myanmar. Other bacterial strains were provided by the Public Health Laboratory (PHL), Mandalay, Myanmar. 2.8.2. Agar well diffusion method The agar well diffusion method was used for evaluating antimicrobial activity, and followed a method modified from Schlegel et al. [23]. Muller Hinton Broth (Himedia, India) was inoculated with test microorganisms and incubated at 37 ˚C overnight. The overnight broth culture was then diluted with normal saline to obtain the OD600 nm of 0.08 to 0.1. Muller Hinton agar plates were prepared and sterilized in an autoclave at 121 ˚C for 15 min. The broth inoculums were evenly spread on the Muller Hinton agar plates with sterile cotton swabs to obtain a lawn of growth. After a plate was inoculated, 8-mm diameter wells were made on the agar medium with a sterile cork borer. The wells were then filled with 50 µL of extracts with the concentration of 25 mg per 50 µL. Ethanol (70%) was used to prepare the extracts and as a solvent control. Chloramphenicol (30 µg/well) was used as positive control. The plates were then incubated at 37 ˚C for 24 h and the diameter of the zone of growth inhibition was recorded and compared with the positive control. 2.9. Statistical analysis All data were expressed as mean ± standard error of the mean of triplicate measurements. One-way analysis of variance and Dunnett’s multiple comparison test were performed to compare the difference between the plant extracts and standard control or negative control for each of the following assays: DPPH radicals, NO radicals, SO radicals and fluorescent AGEs. Tukey’s multiple comparison test was performed to compare the difference between the tested plant extracts for TPC. In each analysis, P< 0.05 was set at the threshold for statistical significance. For the gallic acid standard curve, the OD of the gallic acid was plotted against the concentrations of 0, 1.7, 5, 15, 44, 131, 392 and 1176 mg/L. The linear regression points were used to determine the GAEs of the phenolic concentration in tested samples. The correlations between antioxidant activity, antiglycation activity and TPC of extracts were also evaluated with correlation and linear regression analysis. Statistical analyses were performed using the Microsoft Office 2010 and GraphPad Prism version 7.0. 3. Results 3.1. In vitro DPPH free radical-scavenging assay DPPH inhibition by the extracts is shown in Table 2. Of the plant extracts, extract 20 (T. chebula) had the strongest radical-scavenging activity against DPPH radicals, with an inhibition rate of 77.98% ± 0.92%, while the standard antioxidant, ascorbic acid, had an inhibition rate of 84.78% ± 0.39%. Statistical analysis tested the difference between inhibition of DPPH radicals by each extract and the data are shown in Table 2 and Fig. 1. While many extracts had significantly lower activity than the standard, extract 20 was not significantly different from the standard, which means its activity was comparable to the standard.

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Table 2 Biological activities in vitro and total phenolic content of Myanmar medicinal plant extracts. Sample code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ascorbic acid Gallic acid Rutin

Inhibition rate (%) DPPH free radicalscavenging activity 8.14 ± 2.67* 21.56 ± 1.72* 20.87 ± 0.88* 40.94 ± 1.83* 57.57 ± 0.77* 58.83 ± 1.46* 54.93 ± 2.67* 58.72 ± 1.89* 16.39 ± 0.28* 15.83 ± 1.12* 11.12 ± 1.79* – 48.85 ± 1.27* 65.59 ± 1.06* 63.76 ± 1.19* 38.76 ± 0.28* 30.5 ± 1.11* 46.79 ± 2.76* 42.09 ± 6.98* 77.98 ± 0.92 84.78 ± 0.39 – –

Nitric oxide radicalscavenging activity

Superoxide radicalscavenging activity

Antiglycation activity

70.41 ± 2.25 59.31 ± 1.23* 53.51 ± 1.25* 57.27 ± 1.55* 72.21 ± 0.48 71.85 ± 2.42 79.89 ± 0.33 89.39 ± 0.60 51.36 ± 2.08* 82.52 ± 5.87 51.49 ± 1.77* – 76.05 ± 4.42 77.07 ± 3.87 87.55 ± 4.56 64.95 ± 1.96* 48.96 ± 4.47* 61.12 ± 5.08* 67.07 ± 1.67* 88.95 ± 2.42 78.96 ± 1.71 76.59 ± 0.38 –

57.45 ± 5.31 58.83 ± 13.46 55.46 ± 9.09 59.53 ± 2.64 78.95 ± 7.53 62.69 ± 2.39 84.12 ± 5.61 68.14 ± 8.29 47.57 ± 7.96* 60.04 ± 6.93 51.29 ± 2.38* 53.93 ± 5.90* 79.08 ± 4.62 49.65 ± 5.25* 86.38 ± 8.05 40.80 ± 2.41* 43.15 ± 8.62* 67.61 ± 5.02 93.68 ± 2.63 88.56 ± 1.87 – 83.24 ± 1.73 –

49.83 ± 2.59 68.94 ± 1.14 # 33.62 ± 1.95 62.00 ± 4.30 52.40 ± 1.64 70.01 ± 1.39 72.90 ± 1.14 # 77.38 ± 0.96 66.64 ± 2.12 68.80 ± 1.56 72.75 ± 2.48 72.63 ± 2.41 # 77.59 ± 2.21 67.82 ± 3.99 # 78.96 ± 2.85 # 21.21 ± 6.07 # 41.23 ± 2.94 60.22 ± 2.64 # 82.37 ± 1.78 70.74 ± 2.57 – – 87.68 ± 1.75

#

TPC (mg GAE/g of extract) 20.69 ± 0.00k 10.96 ± 0.01n 7.56 ± 0.01o 10.76 ± 0.01n 15.29 ± 0.01m 15.96 ± 0.00l 47.94 ± 0.01f 115.63 ± 0.39d 16.16 ± 0.01l 26.60 ± 0.01i 2.09 ± 0.00r 23.23 ± 0.01j 132.83 ± 0.03c 65.14 ± 0.01e 191.18 ± 0.03b 38.34 ± 0.01g 3.36 ± 0.00q 4.69 ± 0.01p 33.00 ± 0.00h 203.23 ± 0.01a – – –

Results were shown as the average of triplicates of experiments ± standard error of mean. *P < 0.05, vs standard in each group. #P < 0.05, vs negative control. Different letters (a to r) indicated significant difference (P < 0.05) from each group. DPPH: 2,2-diphenyl-1-picrylhydrazyl; GAE: gallic acid equivalent; TPC: total phenolic content.

Fig. 1. Comparison of inhibition rate of DPPH radicals of 20 ethanolic extracts. Values are expressed as mean ± standard error of the mean of three independent experiments. *P < 0.05, vs standard. DPPH: 2,2diphenyl-1-picrylhydrazyl. 3.2. In vitro NO radical-scavenging assay Inhibition of NO radicals by the extracts is shown in Table 2; statistical comparisons between each plant extract and the standard was carried out. Samples that were significantly different (P < 0.05) from the standard are indicated in Fig. 2. According to our results, Extract 8 (the bark of Melia sp.) showed the greatest activity against NO radicals among the tested samples. The inhibition rate was 89.39% ± 0.60% which was stronger than both of the standards, ascorbic acid and gallic acid, which showed the inhibition 5 / 12

rates of 78.96% ± 1.71% and 76.59% ± 0.38%, respectively. Extract 20 (T. chebula) also had strong activity with the inhibition rate of 88.95% ± 2.42%, which was not very different from the Extract 8. Further, Extract 10 (M. azedarach) and Extract 15 (T. belerica.) also showed strong activity with inhibition rates of 82.52% ± 5.87% and 87.55% ± 4.56%, respectively. Therefore, 4 plant extracts were found to be as highly active as the standards.

Fig. 2. Comparison of inhibition rate of nitric oxide radicals of 20 ethanolic extracts. Values are mean ± standard error of the mean of three independent experiments. *P < 0.05, vs the standards. GA: gallic acid; AA: ascorbic acid. 3.3. In vitro SO radical-scavenging assay The reduction of NBT-diformazan is directly proportional to the inhibition of SO radicals by the tested samples. Rates of SO inhibition by the extracts are shown in Table 2. Among the 20 tested extracts, 4 extracts, Extract 7 (roots of Melia sp.), Extract 15 (T. belerica), Extract 19 (G. mangostana), and Extract 20 (T. chebula), showed very strong activity, with inhibition rates of 84.12% ± 5.61%, 86.38% ± 8.05%, 93.68% ± 2.63% and 88.56% ± 1.87%, respectively. Their activity can be compared to the standard gallic acid, which had an inhibition rate of 83.24% ± 1.73%. Comparison of the SO radical inhibition rates of the 20 extracts to the standard, gallic acid, is shown in Fig. 3.

Fig. 3. Comparison of the inhibition rate of superoxide radicals of 20 ethanolic extracts. Values are mean ± standard error of the mean of three independent experiments. *P < 0.05, vs the standard. 3.4. TPC measurements In this experiment, TPCs of the plant extracts were determined using Folin-Ciocalteu’s reagent. A gallic acid calibration curve was prepared at the concentrations of 0, 1.7, 5, 15, 44, 131, 392 and 1176 mg/L (Fig. 4). Comparison of TPCs of the extracts, expressed as GAEs, is shown in Fig. 5. The highest TPC, (203.23 ± 0.01) mg GAE/g, was found in Extract 20 (T. chebula; Table 2). The second highest TPC was (191.18 ± 0.03) mg GAE/g of extract and was found in Extract 15 (T. belerica). Extract 8 (the bark of Melia sp.) and Extract 13 (the bark of F .bengalensis) also had high TPC ((115.63 ± 0.39) and (132.83 ± 0.03) mg GAE/g, respectively).

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Fig. 4. Calibration curve of the gallic acid standard at concentrations of 0, 1.7, 5, 15, 44, 131, 392 and 1176 mg/L

Fig. 5. TPC of extracts determined by the Folin-Ciocalteu’s assay and calculated as GAE in mg/g extract based on dry weight. Results were the average of triplicates ± standard error of the mean. Different letters (a to r) indicated significant difference (P < 0.05) from each group. GAE: gallic acid equivalent; TPC: total phenolic content. 3.5. In vitro antiglycation assay The inhibition of AGE formation was evaluated by a nonenzymatic glycation reaction. Results are shown in Table 2 and activities were compared statistically to the negative control (reaction without extracts; Fig. 6). Among all the tested samples, Extract 19 (G. mangostana) demonstrated the highest antiglycation activity (inhibition rate: 82.37% ± 1.78%) and was comparable to the standard rutin (inhibition rate: 87.68% ± 1.75%).

Fig. 6. Comparison of inhibition rate of fluorescent AGEs of 20 ethanolic extracts. Values are mean ± standard error of the mean of three independent experiments. *P< 0.05, vs negative control. 3.6. Correlations between antioxidant activity, antiglycation activity and TPC of plant extracts The correlations between antioxidant activity, antiglycation activity and TPC of tested extracts were also investigated. We found that TPC was well correlated with DPPH radical-scavenging activity (r = 0.7), NO 7 / 12

radical-scavenging activity (r = 0.8) and SO radical-scavenging activity (r = 0.6), but poorly correlated with antiglycation (r = 0.4; Fig. 7).

Fig. 7. Correlation between TPC and percentage inhibition of DPPH, NO, SO and AGE of 20 ethanolic extracts. TPC: total phenolic content; DPPH: 2,2-diphenyl-1-picrylhydrazyl; NO: nitric oxide; SO: superoxide; AGE: glycation end-product. – 3.7. Evaluation of antimicrobial activity The antimicrobial activities of the crude extracts against two Gram-negative bacteria (E. coli and P. aeruginosa), three Gram-positive bacteria (S. aureus, E. faecalis and B. cereus) and one strain of fungus (C. albicans) are shown in Table 3. S. aureus appeared to be most strongly affected by Extract 1 (C. specosus), with a 30 mm diameter zone of inhibition, while the standard, chloramphenicol, had a diameter of 27 mm. For the antimicrobial activity against B. cereus, E. coli, E. Faecalis and C. albicans, the extracts produced inhibition zones ranging from 10 mm to 20 mm in diameter. However, the activities were weak, and the zones of inhibition were generally much smaller than made by the standard. For the antimicrobial activity against P. aeruginosa, Extract 4 (J. gossypifolia) and Extract 16 (R. serpentina) had the strong activities, with inhibition zone diameters of 30 mm. This strain was resistant to chloramphenicol. Further, 70% ethanol produced no inhibition zone against any tested bacteria.

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Table 3 Antimicrobial activity of extracts of 20 medicinal plants from Myanmar. Sample code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 70% ethanol Chloramphenicol

Staphylococcus aureus 30 12 13 15 12 11 18 14 25 – – 12 12 12 15 14 10 – 20 19 – 27

Bacillus cereus 15 – 13 13 13 13 16 14 20 12 11 12 11 12 15 – 14 11 13 18 – 24

Inhibition zone diameter (mm) Escherichia Enterococcus coli faecalis – 12 – 11 – 13 – 12 – 14 – 10 – 12 – 16 10 11 – – – 11 – 14 – 15 14 14 – – 13 14 – 14 – – – 15 17 – – – 24 24

Pseudomonas aeruginosa 16 19 20 30 23 22 20 13 20 17 17 22 13 15 20 30 22 20 17 25 – –

Candida albicans – – 13 17 15 13 12 11 13 12 15 13 12 15 14 16 14 11 13 15 – 30

4. Discussion Traditional practitioners of Myanmar have extensively used medicinal plants to cure various diseases. There is a growing trend in seeking evidence-based validation for traditional medicine. In the present work, we investigated the antioxidant, antiglycation and antimicrobial potentials of 20 ethanolic extracts from 17 plants indigenous to Myanmar, using six different bioassays. The DPPH radical-scavenging activity was measured by reducing the stable, violet DPPH radical to the yellow DPPH-H. The degree of color change depends on the free radical-scavenging activity of the tested samples through their hydrogen-donating ability. The sample with the strong antioxidant activity will have the high percentage of radical-scavenging activity [24,25]. In our results, the order of antioxidant activities was 20 > 14 > 15 > 6 > 8 > 5 > 7 > 13 > 18 > 19 > 4 > 16 > 17 > 2 > 3 > 9 > 10 > 11 > 1 > 12. Only 5% of the tested samples exhibited scavenging activities similar to the standard; 30% were moderately active and 65% had only weak activity. NO radical-scavenging activity was also measured in the present study. It is well known that NO is an important bioregulatory molecule that has a number of physiological effects, including control of blood pressure, neural signal transduction, platelet function, antimicrobial and antitumor activity. On the other hand, NO can also be toxic when it reacts with oxygen and SO radicals, which can alter the structure and function of many cellular components and cause the cellular damage [26,27]. We found that 50% of the tested samples had strong inhibition activity of > 70% of NO radicals, the remaining 50% had significantly lower activity than the standard. Another antioxidant assay was performed to evaluate the SO radical-scavenging activity of the plant extracts. SO radical, generated by numerous biological and photochemical reactions, is biologically important, as it can form singlet oxygen and hydroxyl radicals. It can be highly toxic, leading to redox imbalance and harmful physiological consequences [20,28]. Therefore, plant extract activity against SO radicals may also be very useful for preventive or therapeutic purposes. We found that 30% of the extracts had significantly lower activity than the standard. Nonetheless, 30% of the tested samples were found to be highly active, while 40% showed moderately active. 9 / 12

In plants, phenolic compounds are secondary metabolites; they are involved in a wide range of specialized physiological functions and are important for cellular metabolism of the plants [29]. These compounds are capable of inhibiting free radicals and can prevent adverse effect of excessive free radicals. There may be variations in the chemical makeup of the TPC found in different plant extracts, as species, plant maturity, geographic location and plant part used can affect secondary metabolite levels. The correlation analysis found a linear relationship between the free radical-scavenging activity and TPC of the extracts that we tested. These data support previous studies that found plants with high phenolic content to have good antioxidant activity; this is why phenolic compounds are regarded as major contributors to antioxidant activity [30,31]. AGEs and oxidative stress, caused by nonenzymatic protein glycation, lead to numerous biological disorders such as diabetes, Alzheimer’s disease, rheumatoid arthritis and atherosclerosis [32,33]. Therefore protein glycation inhibition is crucial to the prevention of these disorders. Recently, much research has sought natural or synthetic compounds that function as AGE inhibitors. Synthetic compounds like aminoguanidine, metformin and irbesartan have shown adverse health effects; natural compounds have been considered to be safer and less expensive alternatives for the inhibition of AGEs [17]. In our results, 45% the tested extracts had strong antiglycation activity, with inhibition above 70%. Moderate activity was found in 30% of the tested plants and 25% had weak activity. These data suggest that nearly half of the tested plant extracts could inhibit glycation. Among the tested samples, T. chebula was the most potent, with high radicalscavenging activity and good antiglycation activity. It could have the dual function of decreasing oxidative stress and AGE formation in glycation-related diseases. On the other hand, mankind has used various plants and plant products for the prevention and treatment of bacterial infections for millennia. Research has also shown that medicinal plants can have potent antimicrobial activity. Therapeutic compounds isolated from these plants have been helping in the fight against several infectious diseases [34]. In the present research, the antimicrobial activities of 20 plant extracts were tested against two Gram-negative bacteria (E. coli and P. aeruginosa), three Gram-positive bacteria (S. aureus, E .faecalis and B. cereus) and one strain of fungus (C. albicans), using the well diffusion assay and chloramphenicol as a reference drug. The results revealed that Extract 1 (C. specosus), Extract 4 (J. gossypifolia) and Extract 16 (R. serpentina) had strong antimicrobial activities against S. aureus and P. aeruginosa which was comparable to that of the reference drug. It is well known that ethanol is a common disinfectant and has antibacterial properties if used in high concentrations. However, 70% ethanol, which was used to prepare the extracts and used as negative control, did not form a zone of inhibition against the tested bacteria. This result was in agreement with the other research. One research group tested the effect of different ethanol concentrations against human bacterial pathogens. In their assay, 50%, 70% and even 90% ethanol was used as control and showed no inhibition zone at any concentration [35]. The reason why bacteria can withstand the 70% ethanol is still not clear. However, these findings confirm that the extract can be prepared with 70% ethanol for the antimicrobial assays without confounding the results. In the present study, crude ethanolic extracts were used for the evaluation of biological activities, because ethanol can extract most of the polar and moderately polar constituents. Fractionation and purification of these crude extracts, in order to identify individual phytochemical constituents present in the extracts with highest activity, are a logical next step. Additionally, further studies on the antiglycation activity of extracts will be necessary to confirm these findings in vivo. 5. Conclusions In conclusion, among the selected medicinal plants from Myanmar, T. chebula was found to be the most promising extract. It demonstrated free radical-scavenging activity in each assay and also had good antiglycation activity. Thus, it could be useful for decreasing the oxidative stress and AGE formation in glycation-related diseases. Additionally, the extracts from selected plants may be a source for plant-based antimicrobials, as was shown by their activities against five different clinically important microbes. Therefore, this research uses an evidence-based approach for identification of plant-derived compounds that have high antioxidant, antiglycation and antimicrobial activities, which is very important for traditional drug development industry. 10 / 12

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