Antimicrobial activity from Piper sarmentosum Roxb. against rice pathogenic bacteria and fungi

Journal of Integrative Agriculture 2017, 16(11): 2513–2524 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Antimicrobial activity from Piper sarmentosum Roxb. against rice pathogenic bacteria and fungi Pragatsawat Chanprapai1, 2, Warinthorn Chavasiri2 1 2

Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

Abstract In vitro antimicrobial activity of the dichloromethane and methanol extracts of Piper sarmentosum leaves, fruits, stems, and the essential oil obtained from the fresh leaves together with their constituents were investigated against two rice pathogenic fungi: Rhizoctonia solani (sheath blight causal agent) and Bipolaris oryzae (brown spot causal agent), and two rice pathogenic bacteria: Xanthomonas oryzae pv. oryzae (Xoo) (bacterial leaf blight causal agent) and pv. oryzicola (Xoc) (bacterial leaf streak causal agent). Among them, the dichloromethane extracts of the leaves and fruits, and the essential oil showed significantly high potential anti-rice microbial activity. Based on bioassay-guided fractionation of the dichloromethane leave and fruit extracts, myristicin, sarmentine, brachystamide B, brachyamide B, and piperonal were isolated. Moreover, the major constituent of its oil was also myristicin. Myristicin and brachyamide B revealed the highest potent inhibition against R. solani and B. oryzae (half maximal inhibitory concentration (IC50) of 0.69 and 0.12 mmol L–1), respectively. Moreover, brachyamide B and piperonal displayed most antibacterial activity against Xoo (MIC/MBC 7.62/1.90 mmol L–1) and Xoc (MIC/MBC 2.59/20.75 mmol L–1), respectively. Additionally, the essential oil also exhibited the antimicrobial activity against all tested rice pathogenic bacteria and fungi. These compounds and the oil were first evaluated for anti-rice pathogenic microbial activity. Keywords: Piper sarmentosum Roxb, bioassay guided fractionation, antimicrobial activity

1. Introduction Rice microbial diseases are the most important factors to

Received 13 September, 2016 Accepted 24 April, 2017 Pragatsawat Chanprapai, E-mail: [email protected]; Correspondence Warinthorn Chavasiri, Tel: +66-2-2187625, E-mail: [email protected] © 2017 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(17)61693-9

decrease rice productivity and quality making lose values in the world. The major diseases including sheath blight (Rhizoctonia solani DOAC1406), brown spot (Bipolaris oryzae DOAC1760), bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo TB0006) and bacterial leaf streak (Xanthomonas oryzae pv. oryzicola, Xoc TS8203), have been reported from tropical areas (Seneviratne and Jeyanandarajah 2004). Many agriculturists have used chemical pesticides for suppressing these microbial diseases. Meanwhile, these synthetic chemicals will be accumulated and affected on human, animals and ecological food chains. Hence, alternative substances were searched to take place toxic chemicals; particularly those from Thai medicinal plants which have long been used traditionally to

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treat various diseases. Piper sarmentosum Roxb. (Piperaceae) is widely distributed in many countries of Asia (Mathew et al. 2004). It is locally known as Cha-Plu in Thailand. The leaves have traditionally been used in South Asian cuisines (Raman et al. 2012). This plant extract has been studied for its biological activities such as antioxidant activity (Hussain et al. 2009; Hafizah et al. 2010), anti-inflammatory and antipyretic activity, neuromuscular blocking activity (Radtitid et al. 1998, 2007; Rukachaisirikul et al. 2004; Radtitid et al. 2007; Sireeratawon et al. 2010), larvacidal activity (Chaithong et al. 2006; Qin et al. 2010; Kraikrathok et al. 2013), α-glucosidase inhibitory activities (Damsud Adisakwattana and Phuwaprisirisan 2013), natural killer cell activity and lymphocyte proliferation (Panthong and Itharat 2014), hypoglycemic effect (Peungvicha et al. 1998), allelopathic activity (Pukclai and KatoNoguchi 2010, 2011; Pukclai et al. 2012), and hypoxia inducible factor-2 (HIF-2) transcription activity and HIF-2 inhibitory activity (Bokesch et al. 2011). In addition, the plant extracts have reported antibacterial activity against Escherichia coli, Bacillus subtilis, multi-resistant Staphylococcus aureus, S. aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, and Burkholderia pseudomallei (Masuda et al. 1991; Atiax et al. 2001; Rukachaisirikul et al. 2004; Zaidan et al. 2005; Hussian et al. 2012; Panomket et al. 2012; Pscheidt and Wao 2013), and antifungal activity against Aspergillus niger and Candida albicans (Hussian et al. 2012). The reported compounds isolated from the plant included 1-allyl-2,6-dimethoxy3,4-methylenedioxybenzene, pellitorine, sarmentine, 1-piperettyl pyrrolidine, guineensine, brachyamide B, 1-(3,4-methylenedioxyphenyl)-1E-tetradecene, brachytamide B, sesamin, (+) asarinin, chaplupyrrolidones A and B, deacetylsarmentamide B, α-asarone, myristicin, α-cadinene, sarmentosine, sarmentosomols A to F, N-2´methylbutyl-2E,4E-decadieneamide, 1-nitrosoimino-2,4,5trimethoxybenzene and sarmentamide D (Stohr et al. 1999; Ee et al. 2009; Hussian et al. 2012; Damsud et al. 2013; Yang et al. 2013; Shi et al. 2016). However, the anti-rice microbial activity of the dichloromethane extract of this plant has not been reported. Thus, the present study aims to perform the extraction and fractionation of the extracts of P. sarmentosum, and to subject the fractions to in vitro anti-rice microbial activity for identifying bioactive compounds.

2. Materials and methods

plant market, Yaowarat Road, Bangkok in 2013. For leaves, there were purchased from Pak Klong Talad Market, Bangkok in 2012. This plant was labeled and deposited at the herbarium of the Kasin Suvathabandhu Herbarium (BCU-Herbarium): BCU 013525 at Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.

2.2. Rice pathogenic strains The antimicrobial susceptibility tests were performed on rice pathogenic fungi (Rhizoctonia solani, DOAC1406 and Bipolaris oryzae, DOAC1760) and bacteria Xanthomonas oryzae pv. oryzae and pv. oryzicola (Xoo TB0006 and Xoc TS8203) supplied and purchased from the plant pathology research group, plant protection research and development office, Bangkok, Thailand.

2.3. Extraction All parts of the leaves, fruits and stems were air dried, milled and extracted with CH2Cl2 and then CH3OH for three times. The extracts were filtered, evaporated and kept for antimicrobial activities (Table 1). The fresh leaves (200 g) were hydro-distilled using a modified Dean-Stark apparatus. After extraction with Et2O, the extract was evaporated under reduced pressure in rotatory evaporator. The oil yield and all test data are the average of triplicate analyses (Ho et al. 2012).

2.4. Bioassay-guided fractionation, isolation and identification of the fraction A total of 10 kg of fresh leaves were dried by sun light (yield=1 694 g) and 5 000 g of dried fruits, milled and extracted by maceration at room temperature for 72 h with CH2Cl2 and its residue was continued extracting with CH3OH. These extracts were filtered and concentrated under rotatory vacuum evaporator to obtain the CH2Cl2 and CH3OH extracts of each plant part. The yields of CH2Cl2 and CH3OH extracts were determined as 124 and 659 g for leaves, and 600 and 2 080 g for fruits, respectively (Fig. 1). The CH2Cl2 extracts Table 1 Percentage yield of the extraction of Piper sarmentosum for preliminary study Parts

2.1. Plant materials

Dried leaves Fresh leaves Dried stems Dried fruits

Piper sarmentosum fruits were purchased from medicinal

–, no extraction.

CH2Cl2 7.30 – 3.37 12.00

Percentage yield (w/w) CH3OH Essential oil 38.91 – – 0.16 6.05 – 40.00 –

Pragatsawat Chanprapai et al. Journal of Integrative Agriculture 2017, 16(11): 2513–2524

NMR comparing with literature.

Dried leaves and fruits of Piper sarmentosum (1 694 g and 5 000 g) CH2Cl2

2.5. Poisoned food techniques

Filtration CH2Cl2 extract (124 g and 600 g)

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Residue CH3OH CH3OH extract (659 g and 2 080 g)

Residue Discard

Fig. 1 The scheme of the leaves and fruits extraction of Piper sarmentosum. The dried leaves and fruits were milled and extracted with CH2Cl2 and then CH3OH for three times. The extracts were filtered, evaporated and kept for antimicrobial activities.

of dried leaves and fruits (active fractions) were selected for fractionation, isolation and identification of the active antimicrobial components. About 70 g of the bottle-green CH2Cl2 fraction from dried leaves, and effective fraction, was chromatographed over a silica gel column (100 cm×5 cm). The column was eluted with a gradient using C6H14 followed by C6H14-C4H8O2 (95:5, 90:10, 80:20, 60:40, 40:60 and 20:80), C4H8O2 and C4H8O2-CH3OH (95:5 and 90:10). Fractions of 500 mL were collected, concentrated under vacuum, and combined by thin-layer chromatography similarity to obtain eight main fractions I–VIII. By bioassay guided, fractions II (4 g) and III (14 g) were separated by silica gel column (5 cm×7 cm and 5 cm×24 cm, respectively) eluting with C6H14 followed by C6H14-C4H8O2 (95:5 and 90:10). The column fractions resulted in the isolation of Compound 1 as a major product of fractions II and III was confirmed 1H NMR and GC/MS analysis. The brownish oil CH2Cl2 fraction (200 g) of the dried fruits was chromatographed over a silica gel column (100 cm×10 cm) eluting with C6H14 followed by C6H14-C8H16O4 (95:5, 90:10, 80:20, 60:40, 40:60 and 20:80), C8H16O4 and C8H16O4-CH3OH (95:5 and 90:10). Fractions were collected for 1 000 mL, combined by thin-layer chromatography similarity and concentrated under vacuum in seven main fractions I–VII. From bioassay-guided, fractions V (20 g), VI (15 g) and VII (10 g) were selected for further separation by silica gel column (5 cm×35 cm, 5 cm×26 cm and 5 cm×17 cm, respectively) eluting with C6H14 followed by C6H14-C8H16O4 (95:5 to 20:80) and C8H16O4. Fraction V yielded compounds 2 and 3, while compounds 4 and 5 were obtained from fractions VI and VII, respectively. The structures of isolated compounds were elucidated by 1H

Stock solutions of the extracts and isolated compounds were dissolved in dimethylsulfoxide (DMSO) at 105 mg L–1 and filtered. 0.1 mL of different extracts and pure compounds with the final concentration of 1 000 mg L–1 was mixed with melted potato dextrose agar (PDA) and poured in the Petri plates. PDA mixed with only DMSO was used for the control treatment. After media setting, mycelia dish (5 mm dia.) from the margins of two-day R. solani and seven-day B. oryzae old cultures were cut, transferred and placed in the center of the Petri plates. All plates were incubated at 25–26°C and the antifungal activity was estimated using measuring the relative growth of fungus in treatment vs. control (Gupta and Datta 2012). The percentage of growth inhibition was calculated by the formula, I=(C–T)/C×100. Where, I is % inhibition, C is dia. colony in control (cm) and T is dia. colony in treatment (cm) (Kumar and Tyagi 2013). The half maximal inhibitory concentration (IC 50 ) determination was carried out by various concentrations of each active compound and calculated from the linear equation formula of percentage mycelia growth inhibition of each concentration plotted against concentration to give 50% inhibition of mycelia growth.

2.6. Agar well diffusion method The agar diffusion method modified from Barry (1999) was used for the antibacterial activity of the extracts and all isolated compounds. Briefly, selected 4–5 single colonies of each tested bacteria were cultivated into nutrient broth (NB) and incubated at 37°C for 5 h. The turbidity was adjusted to cell density to McFarland No. 0.5 by 0.85% sterile NaCl. Each melted NA (19 mL) was mixed with bacterial suspension (1 mL), poured into Petri plates, and allowed to solidify. Then, tested culture plates created the wells of 6-mm dia. were bored with cork border and the addition of 40 µL of crude extracts and DMSO (control) in each well. The plates were incubated at 37°C for 24 h. The zone of inhibition in triplication was recorded as mean±standard deviation.

2.7. Susceptibility tests The minimal inhibitory concentration (MIC) was measured by the macro-dilution broth susceptibility assay recommended by NCCL (McClatchey 2002). Ten-fold serial dilutions of isolated compounds (100 to 0.08 mg mL–1) were added with 1 mL of bacterial cultures and incubated at 37°C for 24 h. The MIC values were

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evaluated as the lowest concentration of isolated active compound that were able to inhibit the visible growth of bacteria. To find out the MBC values, one loop of the MIC condition was streaked on NA plate and incubated at 37°C for 24 h. The lowest concentration of active compound that had no bacterial colonial growth was determined as MBC mg mL–1 (Singh et al. 2011).

20.0 software. Comparison of means was performed using Duncan’s multiple range test (DMRT) and significance was accepted at the P<0.05 level. The experiment was designed as a general linear model within a completely randomized design with triplications.

2.8. Structure determination (1H NMR)

3.1. Preliminary study on the in vitro antimicrobial activity of Piper samentosum extracts

Each compound was dissolved by CDCl3. The data was collected on mercury 400-mercury 400 and pulse sequence s2pu1. The method was used in ambient temperature, relaxation delay 1.000 s, pulse 45.0 degrees Acq. time 1.985 s and width 6 389.8 Hz for eight replications. It was observed with H1, 399.863 MHz.

2.9. GC-MS analysis The isolated active compounds were analyzed by GC-MS on a Agilent 6890 GC coupled to a HP5973 MS/MS. GC was fitted with a HP-5MS column (30 m×0.25 mm×0.25 µm fused silica capillary column, film thickness) operating with the conditions: injector temperature, 250°C; transfer line temperature, 280°C; EM mode, 1 576.5 EM voltage; m/z range, 50–550; carrier gas, He; injection volume, and 1 µL min–1 (splitless). The MS ionization energy was set up to 70 eV. The chemical components of the oil was identified by comparison of their relative retention times and mass spectra with the Willey 7N electronic libraries (McLafferty and Staufer 1989; Adams 2007; Deng et al. 2009; Lu et al. 2011).

2.10. Statistical analysis All data were analyzed using the SPSS for windows version

3. Results

The results of the in vitro antimicrobial activity from the different parts and extracts of P. sarmentosum were determined and represented in Table 2. Among these extracts, the essential oil, the CH2Cl2 extracts of leaves and fruits displayed significantly high antifungal activity against Rhizoctonia solani and Bipolaris oryzae. In case of antibacterial activity, the CH2Cl2 extract of fruits significantly showed the highest activity against Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc). Thus, the oil and the CH2Cl2 extracts of leaves and fruits were selected for further studies and fractionation, respectively.

3.2. Antimicrobial activity assay of fractionated fractions of leave and fruit extracts For leave extracts, the percentage of mycelia growth inhibition is represented in Fig. 2-A. The PSLs-2 and -3 displayed significantly complete antifungal activity against R. solani. PSLs-2 and -3 showed significantly high mycelia activity than the other fractions against B. oryzae. On the other hand, PSL-8 displayed the lowest mycelia inhibition. Therefore, PSLs-2 and -3 were further separated, tested isolated and identified. An anti-fungal activity of CH2Cl2 fruit fractions against the

Table 2 Preliminary study of antimicrobial activity of different parts and extracts of Piper sarmentosum Plant part Dried Leaves Fruits Stems Fresh leaves 1)

Extracts CH2Cl2 CH3OH CH2Cl2 CH3OH CH2Cl2 CH3OH Essential oil

Fungal growth assay Mean of % mycelia growth inhibition1) R. solani B. oryzae 51.77±1.19 c 41.45±1.33 c 0g 11.26±1.78 e 71.11±0.32 b 60.74±0.74 b 32.59±2.56 e 35.56±1.29 d 39.07±1.29 d 36.11±0.55 d 27.96±1.93 f 40.78±0.96 c 100 a 100 a

Agar well diffusion assay Mean of clear zones (mm)2) Xoo Xoc 6.8±0.09 b 0d 0d 0d 13.25±1.43 a 16.25±0.61 a 3.00±0.81 c 8.50±0.41 b 2.25±0.20 c 3.50±1.22 c 3.00±0.81 c 2.50±0.41 c 5.5±0.03 b 4.00±0.06 c

R. solani, Rhizoctonia solani; B. oryzae, Bipolaris oryzae. Values were determined by: (Control–Treatment)/Control×100%, and represent the means of three replicates (±SD) at 1 000 mg L–1 of each fungi. 2) Xoo, Xanthomonas oryzae pv. oryzae; Xoc, Xanthomonas oryzae pv. oryzicola. Values mean average clear zones (mm)±SD of two replicates at 10 000 mg L–1 of each bacteria. Mean values within a column followed by a different letter are significantly different (P<0.05; DMRT).

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rice pathogenic fungi is presented in Fig. 2-B. PSFs-2, -3 and -5 displayed completely significant antifungal activity following by PSFs-6 and -7 against R. solani. For B. oryzae, PSFs-2, -6, -7, -5, and -3 appeared high potential activity, respectively. For antibacterial activity, PSFs-5, -6 and -7 displayed highly significant activity against Xoo and Xoc. The results are presented in Fig. 3. Therefore, PSFs-5, -6 and -7 were selected for isolation and identification because these fractions revealed potent antimicrobial activities.

3.3. Chemical constituents of the essential oil The oil was analyzed by GC/MS analysis and the constituents were identified based on their retention times (Rt) values as well as by comparing their mass spectra with R. solani A

120.00

d

Mycelia inhibition (%)

100.00

a d

a

a

80.00 60.00

B. oryzae

b

d

b

c

c

40.00

e

e

e

d

e

20.00

d

8 L-

7

PS

L-

6

PS

L-

5

PS

L-

4 L-

PS

3

PS

L-

2

PS

L-

PS

PS

–20.00

L-

1

0.00

Wiley 7N library (Fig. 4). GC/MS analysis of the oil displayed 63 constituents accounting for 99.99% of the oil (Table 3). Among 63 compounds, myristicin was the major component (27.27%) followed by trans-caryophyllene (18.30%), α-copaene (10.40%), germacrene-D (5.41%), β-cubebene (4.57%) and bis-(2-ethylhexyl) phthalate (3.76%).

3.4. Chemical structure of the active compounds though bioassay guided fractionation of CH2Cl2 leave and fruit extracts Five compounds isolated from the CH 2Cl 2 extracts of leaves (1) and fruits (2–5) were identified as myristicin (1), sarmentine (2), brachystamide B (3), brachyamide B (4), and piperonal (5) (Table 4), known compounds of P. sarmentosum (Thitima et al. 2004; Tuntiwuttikul et al. 2006; de Morais et al. 2007; Hussian et al. 2009, 2012). GC analysis of PSLs-2.2 and 2.3 was performed. PSL2.2 displayed only one peak at Rt 13.67 min. PSL-2.3 showed three peaks at Rt 13.73, 15.08 and 17.40 min. It could be observed that the peak at Rt 13.6 min was common in all three subfractions. PSL-2.3 was thus subjected to GC/ MS analysis, whereas PSL-2.2 was analyzed by 1H NMR. In addition, the only peak of PSL-2.2 and the first peak of PSL-2.3 were similar. GC/MS chromatogram of three peaks of PSL-2.3 was identified as myristicin (Rt: 16.9 min, 43.9%), spathulenol (Rt: 17.8 min, 4.1%) and N-(3-phenylpropanoyl)pyrrole (Rt: 19.1 min, 52.0%). All constituents were compared with the Wiley 7N library. So, the only peak of PSL-2.2 was myristicin. Myristicin (192.21 g mol–1) (1) was obtained as yellow

Dichloromethane leave fractions a

a

a

b

d

a c

b

b

c

d

e

Average of clear zones

c

14

X. oryzae pv. oryzae X. oryzae pv. oryzicola

12

a

-7 PS F

-6 PS F

5 PS F-

-4 PS F

PS F3

Dichloromethane fruit fractions

Fig. 2 Percentage of mycelia growth inhibition of rice pathogenic fungi of dichloromethane fractions of leaves (PSLs, A) and fruits (PSFs, B) at 1 000 mg L–1. Error bars show means± SD; different lower case letters for each column indicate a significant difference at P<0.05 (DMRT). R. solani, Rhizoctonia solani; B. oryzae, Bipolaris oryzae.

a

10 8 4 2

b

a

6

0

e

PS F2

100 90 80 70 60 50 40 30 20 10 0

PS F1

Mycelia inhibition (%)

B

ab abc c

abc

bc c

c c

abc

c

PSF-1 PSF-2 PSF-3 PSF-4 PSF-5 PSF-6 PSF-7 Dichloromethane fruit fractions

Fig. 3 The average of clear zone inhibition (mm) of rice pathogenic bacteria in agar well diffusion method of dichloromethane fruitfractions (PSFs) at 10 000 mg L–1. Values mean averagess±SD of triplicates of the mean inhibition zones (without diameter of agar well as 5 mm) of the extracts at 10 000 mg L–1 of each bacteria. Different lower case letters for each column indicate the significant difference at P<0.05 (DMRT). X. oryzae, Xanthomonas oryzae.

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Fig. 4 The GC/MS chromatogram of the essential oil of Piper sarmentosum fresh leaves. The GC/MS chromatogram on an HP-5MS column of the essential oil of P. sarmentosum fresh leaves with splitless injector operating with the conditions: injector temperature, 250°C; transfer line temperature, 280°C; EM mode, 1 576.5 EM voltage; m/z range, 50–550; carrier gas, He; and injection volume, 1 µL min–1 (splitless). The MS ionization energy was set up to 70 eV.

liquid and the structural formula was C11H12 O3 identifying by GC/MS at m/z 192.21. This compound was confirmed by 1H NMR by comparative assignments as follows: H-6 (δH 6.36, d, J=1.4 Hz, 1H), H-2 (δH 6.39, d, J=1.4 Hz, 2H), H-7 (δH 3.25, d, J=6.7 Hz, 2H), H-8 (δH 5.90, m, 1H, H-8), H-9a (δH 5.09, dd, J=1.7 and 17.1, 1H, H-9a), H-9b (δH 5.01, dd, J=1.7 and 8.0 Hz, 1H), -OCH2O (δH 5.92, s, 2H) and -OCH3 (δH 3.81, s, 3H) (Wan 2007). Sarmentine (221.34 g mol–1) (2) was obtained as dark brown liquid and a long unsaturated fatty acid chain cooperating with pyrrolidine. The molecular formula (C14H23NO) was identified by GC/MS at m/z 221. The 1H NMR spectrum revealed the chemical shift for H-2,5 (δH 6.04, dd, J=14.3, 7.7 Hz, 2H), H-3 (δH 7.21, dd, J=14.7, 10.7 Hz, 1H), H-4 (δH 6.13, dd, J=15.1, 10.7 Hz, 1H), H-6 (δH 2.09, quint, J=7.1 Hz, 2H), H-7 (δH 1.37, quint., J=7.2 Hz, 2H), H-8,9 (δH 1.30–1.18, m, 4H), H-10 (δH 0.83, t, J=6.9 Hz, 3H), H-1´,4´ (δH 3.52–3.42, m, 4H), H-2´ (δH 1.91, quint, J=6.7 Hz, 2H), and H-3´ (δH 1.80, quint, J=6.8 Hz, 2H). This compound was found in P. sarmentosum (Likhitwitayawuid et al. 1987). Brachystamide B (411.58 g mol–1) (3) was obtained as white powder. The structural formula was conducted as C26H37NO3. The 1H NMR resonances were identified as follows: H-2, OCH2O (δH 6.01–5.87, m, 3H), H-3 (δH 7.10, dd,

J=15.2, 10.8 Hz, 1H), H-4,5,14 (δH 6.23–6.01, m, 3H), H-6,13 (δH 2.23–2.09, m, 3H), H-7,8,9,10,11,12 (δH 1.54–1.22, m, 12H), H-15 (δH 6.33, d, J=15.8 Hz, 1H), H-2´ (δH 6.97, d, J=1.60 Hz, 1H), H-5´,6´ (δH 6.76, d, J=7.9 Hz, 1H; 6.81, dd, J=8.1, 1.6 Hz, 1H), H-7´ (δH 3.07, t, J=6.4 Hz, 2H), H-8´ (δH 1.85–1.68, m, 1H), and H-9´,9´´ (δH 0.88, d, J=6.7 Hz, 6H). It has been initially found in P. sarmentosum by Phansa (2005) and usually contained in P. brachystachyum (Parmar et al. 1997). PSF-6 was separated into four fractions. The forth fraction displayed more activity than other fractions. Brachyamide B (C20H25NO3, 327.42 g mol–1) (4) was obtained as canary brown solid. The 1H NMR spectrum revealed H-1 (δH 6.32, d, J=15.8 Hz, 1H), H-2,5´ (δH 6.79– 6.70, m, 2H), H-2´ (δH 6.87, s, 1H), H-6´ (δH 6.99–6.89, m, 1H), H-8 (δH 6.13, d, J = 15.1 Hz, 1H), H-3,6 (δH 2.41–2.25, m, 4H), H-4,5 (δH 2.20–1.79, m, 4H), H-7 (δH 6.07–5.96, m, 1H), H-9 (δH 5.93, s, 2H), H-1´´,4´´ (δH 3.51, dt, J=10.2, 6.9 Hz, 4H), H-2´´ (δH 1.95, quint, J=6.7 Hz, 2H), and H-3´´ (δH 1.85, quint, J=6.8 Hz, 2H). This compound was similar structure to Koul (1988) report. PSF-7 was obtained as brown liquid and further separated into six fractions. The third fraction displayed the highest potential activity against four rice pathogenic

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Table 3 Chemical composition of the essential oil of Piper sarmentosum (splitless) No. of peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

Rt (min)1) 6.59 7.69 8.29 9.09 9.33 9.64 11.09 11.49 11.54 11.91 13.30 13.67 16.38 16.48 16.57 17.43 17.70 18.02 18.47 18.50 18.56 18.82 18.98 19.12 19.61 20.04 20.15 20.51 20.65 20.76 20.83 21.45 21.63 24.56 24.81 24.98 25.34 25.41 25.58 25.64 25.83 26.10 26.27 26.34 26.44 28.11 29.94 33.07 34.33 34.60 35.98 36.11 37.72 37.83 38.75 39.49 41.08

Compounds2) α-Pinene β-Pinene 2,2-Dideutero heptadecanal Limonene α-Pyronene trans-β-Ocimene Linalool 2-Ethyl-2,5-dimethylcyclopent-2-enone Unknown 1,3-Cyclohexadiene,1,5,5,6-tetramethylTerpinen-4-ol α-Terpinen Safrole 2-Undecanone α-Chloronaphthalene 1,5,5-Trimethyl-6-methylene-cyclohexane (γ-pyronene) (+)-Lepidozene α-Cubebene γ-Selinene Bicycloelemene α-Cadinene α-Copaene β-Bourbonene β-Cubebene (–)-Alloaromadendrene trans-Caryophyllene β-Cubebene Zingiberene δ-Cadinene α-Humulene Myristicine Germacrene D Germacrene B Myristicin Veridiflorol δ-Cadinol Valencene γ-Eudesmol α-Cadinol Calarene (+)-β-Guaiene 7,7-Dimethyl-bicyclo[3.1.1]hept-3-ene-2-spiro-4´-(1´,3´-dioxane) Apiol cis-Isomyristicin 9,10-Dehydro-isolongifolene Spiro[2.9]dodeca-4,8-diene Butyl octyl phthalate 9,19-Cyclolanost-24-en-3-ol, acetate Isobutyl phthalate Phytol isomer bis-(2-Ethylhexyl)succinate Docosane Ional Tricosane Elemicin Diisooctyladipate Tetracosane

Molecular formula C10H16 C10H16 C17H34O C10H16 C10H16 C10H16 C10H16O6 C10H16 C9H14O C10H16 C10H18O C10H16 C10H10O2 C10H22O C10H7Cl C10H16 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C11H12O3 C15H24 C15H24 C11H12O3 C15H26O C15H26O C15H24 C15H26O C15H26O C15H24O C15H24 C12H18O2 C12H14O4 C11H12O3 C15H22 C12H18 C20H30O4 C32H52O2 C16H22O4 C20H40O C20H38O4 C22H46 C13H20O2 C23H48 C12H16O3 C22H42O4 C24H50

Composition (%)3) 0.28 1.82 0.10 0.61 0.09 1.86 0.70 0.09 0.14 0.06 0.28 0.09 0.85 0.51 0.40 0.10 1.84 1.01 0.09 0.04 0.40 10.40 0.71 4.57 0.36 18.30 1.87 0.16 0.21 1.47 1.47 5.41 2.40 27.27 0.52 0.22 0.40 0.37 0.44 0.81 1.04 0.36 0.21 0.08 0.20 0.22 0.07 0.13 0.11 0.75 0.35 0.13 0.53 0.25 0.16 0.81 0.62

(Continued on next page)

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Table 3 (Continued from preceding page) No. of peak 58 59 60 61 62 63 1) 2)

Rt (min)1) 42.06 42.61 44.17 46.00 48.26 54.07

Compounds2) bis-(2-Ethylhexyl)phthalate Hexacosane Heptacosane α-Cadinol n-Heptadecane 11,15-Dimethylheptatriacontane Total identified

Molecular formula C24H38O4 C26H54 C27H56 C24H38O4 C17H36 C39H80

Composition (%)3) 3.76 0.41 0.43 0.23 0.23 0.19 99.99

Rt, retention times. Identification of compounds based on GC/MS Wiley 7N library.

Table 4 Active compounds isolated from the CH2Cl2 extracts of Piper sarmentosum leaves and fruits Compound1) 1. Myristicin

Structure

Yield (%)2) 6.08

CH2

O O O

CH3

O

2. Sarmentine

1.33

N

3. Brachystamide B

CH3

O

H N

O

CH3

0.23

CH3

O

4. Brachyamide B

0.26

O O

N

O

5. Piperonal

O

O

0.53

O 1) 2)

Compound 1 was isolated from CH2Cl2 dried leaves and compounds 2–5 were isolated from CH2Cl2 dried fruits. Compounds are presented as a percentage with respect to the dichloromethane extract. The active fractions based on bioassay guided fractionation were further fractionation, isolation, purification, and identified the active compounds by nuclear magnetic resonance.

microorganisms. Therefore, this active fraction might be composed of antimicrobial agent which attached and disturbed cell mechanisms (Rafill et al. 2008). Piperonal (150.13 g mol–1) (5) was obtained as white crystals and the structural formula was C8H6O3 identifying by GC/MS at m/z 149. The compound was confirmed by 1H NMR as follows: H-1 (δH 9.73, s, 1H), H-2 (δH 6.01, s, 2H), H-2´ (δH 7.24, d, J=1.6 Hz, 1H), H-5´ (δH 6.86, d, J=8.0 Hz, 1H), and H-6´ (δH 7.34, dd, J=7.9 Hz, 1.6 Hz, 1H) (Wan (2007).

3.5. In vitro antimicrobial activity of the isolated active compounds and the essential oil The antimicrobial activity of isolated compounds represents in Table 5. Myristicin (1) and brachyamide B (4) exhibited greater antifungal activity against R. solani and B. oryzae with

IC50 of 0.69 and 0.12 mmol L–1, respectively. Brachyamide B (4) and piperonal (5) revealed the most antibacterial effects against Xoo and Xoc with minimum inhibitory concentration/minimum bactericidal concentration (MIC/ MBC) of 7.62/1.90 and 2.59/20.75 mmol L–1, respectively. Interestingly, brachyamide B (4) showed both high potential antifungal and antibacterial activities at low concentration. The results of antimicrobial activity displayed that the oil controlled a broad spectrum of antimicrobial activity against all antimicrobial assays with %mycelia inhibition and IC50 for anti-fungal activity, and with clear inhibition zone and MIC/MBC for antibacterial activity. The antifungal activity of the oil showed the complete inhibition for R. solani and B. oryzae (100%) with IC50 of 0.048 and 0.015 mg mL–1, respectively. In bacterial assay, the oil displayed also high activity for Xoo (11.5 mm) and Xoc (10 mm) with MIC/MBC of 3.12/6.25 and 6.25/12.50 mg mL–1.

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Table 5 The antimicrobial activity of anti-rice pathogenic active compounds from Piper sarmentosum Compounds Myristicin (1) Sarmentine (2) Brachystamide B (3) Brachyamide B (4) Piperonal (5) Nativo 750 WG (control rice microcide)

Concentrations (mmol L–1) Rice pathogenic bacteria (MIC/MBC)2) Rice pathogenic fungi (IC50)1) R. solani B. oryzae Xoo Xoc 0.69 0.42 16.23/65.03 65.03/65.03 1.02 0.92 14.76/59.15 7.38/29.57 2.87 1.81 15.19/15.19 15.19/30.37 0.97 0.12 7.62/1.90 30.52/1.90 1.11 0.26 41.57/41.57 2.59/20.75 <0.50 <0.50 <1.00/<1.00 <1.00/<1.00

1)

R. solani, Rhizoctonia solani; B. oryzae, Bipolaris oryzae. Values were calculated by linear equation formula (mg L–1), and represent the converted values (mmol L–1) by molecular weight of each compound (n=3, 4 concentrations). 2) Xoo, Xanthomonas oryzae pv. oryzae; Xoc, Xanthomonas oryzae pv. oryzicola. Values mean minimum inhibitory concentration/ minimum bactericidal concentration (mg mL–1), of converted values by molecular weight of each compound (mmol L–1).

4. Discussion This study explored the antimicrobial activity from the essential oil of P. sarmentosum and the isolated active compounds from the CH2Cl2 extracts of the plant controlling four rice pathogenic diseases. The results were differed from the previous study that the CH3OH extracts of the plant exhibited antibacterial activity against methicillinresistant Staphylococcus aureus (MRSA), S. aureus, K. pneumonia, P. aeruginosa, E. coli, B. subtilis (Masuda et al. 1991; Cheeptham and Towers 2002; Zaidan et al. 2005). The CH2Cl2 extract of the dried fruits and the essential oil of the fresh leaves of P. sarmentosum displayed higher anti-R. solani and -B. oryzae than the water extract from other plants such as Artemisia nilarigica, Artocarpous integefolia, Citrus maxima, Coix lacrymajobi, Hedychium coronarium, Lantana camera, Michelia champaka, Passiflora foetida, Punica granatum, Strobilanthes flaccidifolius, Polystichum squarrosum, Adiantum venustum, Parthenium hysterophorus, Urtica dioeca, Cannabis sativa and Piper betel. On the other hand, some plant extracts including the CH3OH extracts of P. betel, Lawsonia inermis, Polyathia longiflora, Salvadora persica, Zingiber officinale and Thymus vulgaris were similar to P. sarmentosum extracts (Seema et al. 2011; Tapwal et al. 2011; Mangang and Chhetry 2012; Al-Rahmah et al. 2013; Knaak et al. 2013). Moreover, the CH3OH extracts from the previous study revealed that antifungal activity against B. oryzae (Manimegalai et al. 2011). For antibacterial activity against Xoo and Xoc, the results were closed to the Curcuma longa extract with hot water and all extracts of Kappaphycus alvarezil (Jabeenet al. 2011; Venkatesh et al. 2011). Myristicin (1) was beforehand reported that it possessed influential antibacterial activity against S. aureus, B. subtilis, M. luteus, P. aeruginosa and E. coli with MIC ranged 0.600– 1.250 µg mL–1 (Narasimhan and Dhake 2006). This study additionally showed that it could exhibit against both rice pathogenic bacteria and fungi. Pauli and Kubeczka (2010)

proposed that the aromatic ring of phenylpropanoids would influent antimicrobial activity, besides other experimental parameters including growth medium, temperature and conditions. However, myristicin (1) has not been tested for anti-mycelia fungal activity. Thus, this study was the first report for anti-mycelia activity against rice pathogenic fungi. Two amides: sarmentine (2) and brachyamide B (4) were appraised for antibacterial activity (Rukachaisirikul et al. 2004; Hussian et al. 2012). Mueller and Bradley (2008) reported that the amide structure could also control the inhibition of RNA synthesis affecting towards mycelia growth, spore formation and structural cell wall. Moreover, the study of Almeida et al. (2009) supported that the amides could also inhibit fungal respiration, destroyed with microtubules, mitosis and cell division, disturbed on the microbial plasma membrane, and inserted in the lipophilic membrane depending on the physicochemical characteristics. Fo Maris (1995) reported that piperonal (5) has acted and denatured on proteins, and alkylated on nucleic acid within bacterial cell. Additionally, Steinhauer (2010) and Pelczar et al. (1993) explained that the action of aldehyde was only affected as a good efficacy on wide both Gram-positive and -negative bacteria embracing with mycobacteria and bacterial spores. Different active compounds from both parts of our investigations confirmed antimicrobial activity of P. sarmentosum (Lee et al. 2014). It is interesting to point out that the minor compound, brachyamide B (4) displayed higher potential anti-rice microbial activity than the main constituent as myristicin (1). In addition, the former compound revealed the broad spectrum of antirice pathogens. Therefore, brachyamide B (4) may be considered as anti-rice pathogen prototypes. The antifungal activity of the oil showed more activities than that from the essential oil of Illicium verum, Mentha piperitai, Bunium persicum, Tanacetum vulgare, Malva sp., Mentha sp., Artemisia absinthium, Ruta graveolens, Z. officinale, and Thymus vulgaris (Huang et al. 2010; Knaak et al. 2013; Khaledi et al. 2014) and closed to that from

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Calocedrus macrolepis var. formosana leaves, Cymbopogon citratus, Eucalyptus camalduensis, Mentha piperitaand clover oil (Yen et al. 2008; Al-Askar and Rashad 2010; Wonni et al. 2016). For antibacterial activity, the oil revealed potential activity against Xoo and Xoc with diameters of the zone of inhibitions and MIC/MBC. The antimicrobial activity of the essential oil of P. sarmentosum was previously investigated on oral microorganisms, Aspergilus niger, A. oryzae, Penicillium sp., Microsporum canis, A. flavus, C. albicans, Trichophyton rubrum and T. mentagrophyte (Cheeptham and Towers 2002; Taweechaisupapong et al. 2010; Nazmul et al. 2011). Based on GC/MS results of the oil, myristicin was the main constituent accounting for 27.27% of the total compound content. This major compound was previously reported (Qin et al. 2010; Hieu et al. 2014) and differed from another study that spathulenol revealed more abundant than myristicin (Cheng et al. 2008). Similarly, myristicin and β-caryophyllene were addressed as major constituents in Vietnamese P. sarmentosum leave oil (Hieu et al. 2014). Interestingly, GC/MS analysis of the oil from this study could analyze more constituents than the other reports (Cheng et al. 2008; Qin et al. 2010; Hieu et al. 2014).

5. Conclusion The present investigation concluded that antimicrobial activity of tropical medicinal plant appeared better activity to suppress rice pathogenic bacteria and fungi. Among active compounds explored, brachyamide B (4) and the oil were mainly performed on both anti-rice fungal and bacterial activities. Finally, the compound and its oil possessed the potent activity against rice pathogenic microorganisms and could be used for the new generation of rice pathogenic microcides.

Acknowledgements The research was supported by a grant from the 90th Anniversary of Chulalongkorn University Fund, Thailand (Ratchadaphiseksomphot Endowment Fund, F31GSES13). The authors would also like to thank the Natural Products Research Unit, Faculty of Science, Chulalongkorn University for facility support.

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