Antimicrobial, herbicidal and antifeedant activities of mansonone E from the heartwoods of Mansonia gagei Drumm.

Journal of Integrative Agriculture 2016, 15(12): 2795–2802 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Antimicrobial, herbicidal and antifeedant activities of mansonone E from the heartwoods of Mansonia gagei Drumm. Rachsawan Mongkol1, 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 biological activities including anti-phytopathogenic fungi, antibacterial, antifeedant and herbicidal activities of the extracts from the heartwoods of Mansonia gagei Drumm. were evaluated. The dichloromethane (DCM) extract displayed antifungal activity against four plant pathogenic fungi (Alternaria porri, Colletotrichum gloeosporioides, Fusarium oxysporum and Phytophthora parasitica) higher than the methanolic (MeOH) extract. The separation of the DCM extract using bioassay guided antifungal activity against P. parasitica led to the isolation of mansorins A, B, and C, mansonones C, E, G and H. Among isolated compounds, mansonone E displayed the highest antifungal activity against P. parasitica, followed by mansonone C, mansorin B and mansonone G. This potent compound revealed the same minimum inhibitory concentrations (MIC) of 31 µg mL–1 against C. gloeosporioides and P. parasitica, and minimum fungicidal concentration (MFC) of 31 and 125 µg mL–1, respectively. Moreover, mansonone E exhibited highly significant antibacterial activity against both Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) with MIC and minimum bactericidal concentration (MBC) as 7.8 and >500 µg mL–1, respectively. This compound furthermore could inhibit the feed of Spodoptera litura with 45.9% antifeedant and significantly herbicidal activity reduced the shoot and root growth of Brassica chinensis, Oryza sativa, Mimosa pigra and Echinochlooa crus-galli. Mansonone E has potential as a new natural pesticide for agricultural plant pathogen management. Keywords: Mansonia gagei Drumm., mansonone E, plant pathogens, antifungal, antibacterial, antifeedant, herbicidal activities

1. Introduction Nowadays, the crop production has serious problems

Received 24 February, 2016 Accepted 1 June, 2016 Rachsawan Mongkol, E-mail: [email protected]; Correspondence Warinthorn Chavasiri, Tel: +66-2-2187625, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61444-2

from plant diseases caused by plant pathogens such as fungi, bacteria, insects and weeds (Agrios 2004). Even though synthetic pesticides can be utilized to control plant pathogens on management of plant diseases for several years, recently it has been realized that many fungi adopt themselves to resist pesticides (Mukalazi et al. 2001). Thus, there is a need for the development of alternative type of selective control or crop protection methods and reduced use of conventional pesticides. Plants may provide this ideal alternative to control plant pathogens since they constitute a rich source of bioactive chemicals. Therefore, the secondary metabolites from plants have evidenced to protect themselves from the damage of microbial pathogens

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or stress environment (Benner 1993). Mansonia gagei Drumm. (Sterculiaceae) was the only species of Mansonia found in Thailand (Akavipak 1992). The common names in Thai are Chan-cha-mod, Chan-hom and Chan-pha-ma. This traditional medicinal plant has been used as cardiac stimulant, onilivertigo, antiemetic, antidepressant and refreshment agent (Pongboonrod 1976). Certain reports on the chemical constituents of Mansonia genus were addressed. The heartwoods of M. altissima constituted primarily 1,2-naphthoqiunones of the mansonone type: mansonones A–H (Bettòlo et al. 1965; Tanaka et al. 1966), mansonones I and L (Galeffi et al. 1969). Three new coumarins as mansonrins A, B, and C, six new mansonones N, O, P, Q, R, and S, and a novel neolignan as mansoxetane were later addressed as constituents from the heartwoods of M. gagei (Tiew et al. 2002a, b, 2003b). The biological activities of the constituents from M. gagei have been reported. While mansonones C, E and mansorin A displayed high activities against Candida albicans and Aedes aegypti, the first compound additionally exhibited good antihistamine. Among isolated compounds, only mansonone N revealed radical scavenging activity (Tiew et al. 2003a; Tiengtham et al. 2004). Moreover, mansonone E displayed the highest anti-Alzheimer’s disease to inhibit acetyl- and butyryl-cholinesterase (Changwong et al. 2012). Mansonones F and S showed the most potent anti-estrogenic activity (El-Halawany et al. 2007, 2011, 2013). Most studies indicated that the investigation of M. gagei focused on human pathogens and bacteria. To our best knowledge, this is the first report for anti-phytopathogenic fungi, antibacterial, phytotoxicity (herbicidal) and antifeedant activities from the constituents of the heartwoods of M. gagei.

2. Material and methods 2.1. General experimental procedures Silica gel (40–63 and 63–200 µm, Merck Ltd., Germany) were used for column chromatography. Thin layer chromatography (TLC) analysis was performed on silica gel 60 F254 on an aluminium sheet and detected under UV light (254 and 365 nm) or visualized with potassium permanganate (KMnO4) followed by heating. The pure compounds were identified by nuclear magnetic resonances (NMR) and confirmed by 1H and 13C NMR spectra on a Bruker AV 400 spectrophotometer and TMS as an internal reference.

2.2. Plant materials The heartwoods of M. gagei were purchased from a local drug store in Bangkok, Thailand, in 2012. This plant was identified by taxonomist, Parinyanoot Klinratana and her-

barium number A015376 (BCU) at the herbarium of Botany Department, Chulalongkorn University, Bangkok, Thailand.

2.3. Extraction and isolation Dried heartwoods (15 kg) were milled and extracted three times with DCM at room temperature. The residue was further extracted with MeOH. The extracts were filtered and the solvent was removed by vacuum rotatory evaporator to give DCM (473 g) and MeOH (648 g) extracts. Both extracts were tested for antifungal assay, the DCM extract showed higher activity than the MeOH extract. The DCM extract (220 g) was fractionated by silica gel quick column (No. 7729, Merck). A stepwise elution was conducted by hexane and increasing polarity by ethyl acetate (EtOAc) and final with 5% MeOH in EtOAc. The fractions were combined according to TLC results to give seven fractions (MGH1–MGH7). All fractions were submitted for antifungal activity against Phytophthora parasitica. MGH3–MGH5 displayed the most potent active fractions with 60, 73 and 63% inhibition, respectively. 26 g of MGH3 were subjected to silica gel column (No. 7734, Merck) using a stepwise system of hexane-CH2Cl2, CH2Cl2-EtOAc. The fractions were collected and combined according to TLC results to 5 fractions (MGH3/1–MGH3/5). Subfractions MGH3/2 and MGH3/3 were separately purified by silica gel column (No. 9385, Merck) using 2% EtOAc-hexane as a mobile phase to give three pure compounds and verified purity of compounds by NMR and compared the spectroscopic data (NMR) with previously reported (Tiew et al. 2002b, 2003b). According to NMR spectra, three pure compounds from these fractions were identified as mansorin A (1, yellowish crystal), mansorin C (3, white crystal) and mansonone C (4, orange needle). Following the same procedure, MGH4 (35 g) was rechromatographed on silica gel column using stepwise system of hexane-CH2Cl2, CH2Cl2-EtOAc to afford five fractions (MGH4/1–MGH4/5). MGH4/1 and MGH4/2 were separately subjected on silica gel column (No. 9385, Merck), eluting with 5% EtOAc-hexane as a mobile phase to get yellow powder of mansorin B (2), orange solid of mansonone E (6) and orange needle of mansonone G (5). All pure compounds were identified by NMR and compared the spectrum data with previously reported (Tiew et al. 2002b; Wang et al. 2004). MGH5 (51 g) was separated by silica gel column (No. 9385, Merck) using 20% EtOAc-hexane. The fractions were collected and combined according to TLC behaviour in order to achieve five fractions (MGH5/1– MGH5/5). Mansonones G and H (red platelet) were isolated from this fraction. Finally, we got seven compounds: mansorin A (1, 788 mg), mansorin B (2, 70 mg), mansorin C (3, 35 mg), mansonone C (4, 85 mg), mansonone E (6, 228 mg), mansonone G (5, 10.9 g) and mansonone H (7,

Rachsawan Mongkol et al. Journal of Integrative Agriculture 2016, 15(12): 2795–2802

216 mg). Mansonone G (5) as the major compound was isolated from both MGH4 and MGH5.

2.4. Antimicrobial activity Preparation of bacterial and fungal inoculum Four fungal plant pathogens: Alternaria porri DOAC 1601 (purple blotch), Colletotrichum gloeosporioides DOAC 2047 (anthracnose), Fusarium oxysporum DOAC 1258 (vascular wilt) and Phytophthora parasitica DOAC 2052 (heart rot and root rot) and two bacteria: Xanthomonas oryzae pv. oryzae TB0006 (Xoo) (leaf blight disease) and X. oryzae pv. oryzicola TS8203 (Xoc) (leaf streak disease) were used in this research. These microorganisms were supplied by the Division of Plant Disease and Microbiology, Department of Agriculture, Ministry of Agriculture and Cooperative, Bangkok, Thailand. The bacteria were cultivated on nutrient agar (NA) and incubated at 37°C for 24 h. The preparation of the inoculum was carried out by adjusting with 0.85% sterile sodium chloride to cell density of 1.5×108 CFU mL–1. The fungus was cultured on potato dextrose agar (PDA) and incubated at 27°C for 7 days. Spore suspension was prepared using mycelial of fungi (7 days old) added sterilized water and mix. After that, filtrated and adjusted the concentration of spore suspension at 106 spores mL–1 which observed by haemacytometer under light microscope. Anti-phytopathogenic assay The bioassays against four plant pathogenic fungi were conducted using the agar incorporation method. For screening, the extracts of M. gagei or isolated compounds were dissolved in dimethylsulfoxide (DMSO) before adding to sterile PDA (sterilization at 15 psi and 121°C) to obtain 1 000 µg mL–1 as the final concentration. The effects on mycelial growth were examined by 8-mm diameter of mycelial disc, taking from the margin of stock cultures using the sterilized cork borer to remove and placed at the center of Petri dish. The agar plates amend with DMSO as negative control and metalaxyl (100 µg mL–1) (Dr. Ehrenstorfer GmbH, Germany) as positive control. The Petri dishes were incubated at 27°C for 5–7 days. Radial measurements of fungal growth were taken when fungi reached the edge of the control plate; colony diameters were measured and calculated as percentage mycelial growth inhibition according to the formula (Parveen et al. 2014). Percentage inhibition of mycelial growth=(dc–dt)/ dc×100% Where, dc=average diameter of the fungal colony of control plate and dt=average diameter of the fungal colony of treatment plate. All treatments were replicated three times. Antibacterial assay The antibacterial activity of crude extracts and isolated compounds were determined using agar well diffusion method as described by Irshad et al. (2012) with some modification. 20 mL of nutrient broth

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agar (NA) were melted and poured into sterilized Petri dish plate (9 cm diameter). After the agar was allowed to set and harden, the bacterial inoculum was swabbed on NA. The numbers of holes were cut using a sterile cork borer (6 mm diameter) and agar plugs were removed. The 30 µL of extracts or isolated compounds (1 000 µg mL–1) were added into the wells. The tested plates were incubated at 37°C for 24 h. The diameter of zones inhibition (mm) was measured and the results were reported as mean standard deviation (SD). DMSO used as a negative control and Nativo 750 WG as positive control. Minimum inhibitory concentrations (MIC) and minimum fungicidal concentration (MFC) or minimum bactericidal concentration (MBC) The MIC was conducted by microwells dilution method using 96 well-microtitre plates for the compound that showing the highest antimicrobial activities in above. The tested compounds were dissolved at two-fold serial dilutions (3.9 to 500 µg mL–1) in nutrient broth (NB). The 100 µL of bacteria suspension was added into the wells. After incubated, the lowest concentration of tested compounds that completely inhibited microbial growth was determined as MIC value. A loopful of each concentration was streaked on fresh NA and incubated. The minimal concentration without the growth of bacteria was determined as MBC. The antiphytopathogenic activities compounds were dissolved in DMSO and added two-fold serial dilutions (1 000 to 3.9 µg mL–1) to sterilize potato dextrose broth (PDB). Then 100 µL of fungal spore suspension was added and inoculated at 27°C for 48 h. DMSO (0.4% v/v) using as a control had no effect on the fungal growth. After incubation, turbidity of PDB medium was measured to determine MIC. Furthermore, 20 µL of tested suspension was dropped on fresh PDA and incubated at 27°C for 24 h, the minimal concentration that the mycelial of fungi could not grow was determined as MFC. Herbicidal assay The herbicidal assay of crude extracts and isolated compounds from the heartwood of M. gagei on crops and weeds seedling growth were investigated. The seeds of Chinese cabbage-PAI TSAI (Brassica chinensis Jusl var. parachinensis (Bailey) Tsen & Lee), giant sensitive plant (Mimosa pigra Linn.), Jasmin rice (Oryza sativa Linn.) and Barnyard grass (Echinochlooa crus-galli Linn. T. Beauv) were used for this bioassay. The seeds were discarded by floating in tap water for germination (1–3 days). The preparation of M. pigra seeds was immersed in the preheated water at 90°C and later changed into the water at room temperature for another day. The only swollen seeds were selected for this investigation (Premasthira and Zungsontiporn 1994). Each crude extract and isolated compounds were dissolved in acetone at the concentration of 1 000 µg mL–1. 2 mL or each solution was added to sterile Petri dish

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(9 cm diameter) with filter paper (Whatman #1) and allowed to evaporate to dryness in a laminar flow. 3 mL of sterile distilled water were added and 20 germinated seeds were placed on the filter paper for each treatment and control plate as acetone. Three replicates were arranged in complete randomized design. The plates were incubated in growth chamber for 3 days at 25°C under constant light illumination. The length of shoot, root and fresh weight were recorded as the indication for plant growth. Antifeedant assay The antifeedant activity of extracts and isolated compounds from the heartwoods of M. gagei was investigated using leaf disc no-choice method. The fresh leaf of Kale (Brassica albroglabra) was washed with tap water, prepared with cork borer (10 mm diameter) and then dropped with 1 000 µg mL–1 of each extract or compound in acetone. After air dried at room temperature, the leaf discs of each treatment were kept in 24 well plates. The second instar larvae of castor (Spodoptera litura) were purchased from Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology Unit, Thailand and pre-starved (6 h) larvae were allowed to feed on the treated leaf discs for 24 hours (27°C). The leaf discs dropped with acetone were used as negative control and 0.1% (w/v) azadirachtin as positive control. For each treatment, ten replicates and three times were maintained. At the end of the experiment, the uneaten area of the leaf discs was recorded in control and treated discs using graph sheet method (Jeyasankar et al. 2014). The percent antifeedant activity was calculated based on the formula (Aricoli and Tennyson 2012): Percent antifeedant activity= Leaf disc consumed by the larvae in the control– Leaf disc consumed by the larvae in the treated Leaf disc consumed by the larvae in the control+ Leaf disc consumed by the larvae in the treated

gel column furnished seven compounds (Fig. 1). Mansonone E (6) exhibited the highest activity against P. parasitica with 94% inhibition at 1 000 µg mL–1. Mansonones C (4) and G (5), and mansorin B (2) also displayed potent activity against this fungus with 84, 81 and 61% inhibition, respectively (Table 1). In addition, the MIC of mansonone E (6) against C. gloeosporioides and P. parasitica exhibited the same value of 31 µg mL–1 against A. porri and F. oxysporum as 250 and 500 µg mL–1, respectively. Whereas the MFC displayed against C. gloeosporioides, P. parasitica, A. porri and F. oxysporum as 31, 125, 250 and 1 000 µg mL–1, respectively.

3.2. Antibacterial activity For antibacterial activity, the DCM extract exhibited the inhibitory activity against Xoo and Xoc with the inhibition zone as 12.3 and 14.3 mm, whereas the MeOH extract showed the inhibition zone against these bacteria as 9.0 and 9.7 mm, respectively. Among the isolated compounds, mansonone E was the most active against both bacteria with the diameter of inhibition zone as 25.7 and 23.4 mm, respectively at the same concentration tested. The inhibition zone of the other compounds against bacteria could be observed ranging from 11.3 to 21.7 mm (Table 1). While mansorin C showed less toxic to Xoc and non-inhibited to Xoo. The MIC and MFC of mansonone E against both Xoo and Xoc were 7.5 and

CH3

CH3 O

O

O

×100% R

Statistical analysis All measurements were done in triplicate and expressed as means±standard deviation (SD). The radial growth inhibition data were analyzed by the Windows SPSS program (ver. 21). The difference of means was performed by one-way ANOVA, Duncan’s multiple range test (DMRT) (P<0.05).

For preliminary study, the DCM extract showed higher antifungal activity than the MeOH extract against all four phytopathogenic fungi tests. The former extract displayed antifungal activity against P. parasitica, F. oxysporum, A. porri and C. gloeosporioides with 73, 54, 53 and 25% inhibition, respectively. Further separation of the DCM extract by silica

O

H 3C

CH3 (1) R = OMe (2) R = OH CH3

(3)

O

CH3

O

O

3. Results 3.1. Antifungal activity against plant pathogens

CH3

CH3 H 3C

R H 3C

CH3 CH3 (4) R = H (5) R = OH

O

O

R

CH3

H 3C

O

(6) R = H (7) R = OH

Fig. 1 Structures of isolated compounds from the heartwoods of Mansonia gagei.

Rachsawan Mongkol et al. Journal of Integrative Agriculture 2016, 15(12): 2795–2802

>500 µg mL–1, respectively (Table 2).

3.3. Herbicidal activity According to Table 3, the extracts and isolated compounds were tested at 1 000 µg mL–1 on four representatives: two crops (B. chinensis and O. sativa) and two weeds (M. pigra and E. crus-galli). The DCM extract showed excellent inhibition on the root growth of Chinese cabbage PAI TSAI (B. chinensis) and completely inhibited the root growth of barnyard grass (E. crus-galli). Mansonone E displayed the highest significant herbicidal agent to all tested crops and weeds. It reduced the seedling growth of dicotyledon (B. chinensis and M. pigra) more than monocotyledon (E. crus-galli and O. sativa). Moreover, this compound retarded the growth of root more than shoot. To justify the effectiveness on the shoot and root growth of M. pigra, mansorins A and C, mansonones C, G and H inhibited the shoot growth; nonetheless, all of them stimulated the root growth, except for mansonone H. The shoot growth of monocotyledon E. crus-galli was inhibited by mansonone

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E, followed by mansorin A and mansonone C, respectively. The root length of this weed was inhibited by mansonone E, followed by mansorin A, mansonones C, G and H. For the shoot growth of jasmin rice (O. sativa), the DCM extract and mansonone E also have herbicidal activity against the growth of this crop, followed by mansorin C and mansonone G. On the other hand, the fresh weight of tested extracts and isolated compounds are presented in Fig. 2. Mansonone E revealed the highest reduction of the fresh weight of all tested plants. The current results suggested that the test compounds had herbicidal potentials and some compounds had stimulatory effects on crops and weeds.

3.4. Antifeedant activity The antifeedant activity of the extract and compounds was investigated by comparing the average of the leaf areas consumed in the treated with the control. The efficiency of tested compounds was assayed with the second instar larvae of S. litura. The average food consumption by castors in the control was 62.8 mm2 at the starting of leaf area as 86 mm2.

Table 1 Antifungal and antibacterial activities of crude extracts and isolated compounds from the heartwood of Mansonia gagei at 1 000 µg mL–1 Compounds1)

Mycelial growth inhibition (%) Phytophthora parasitica

Control DCM extract MeOH extract Mansorin A Mansorin B Mansorin C Mansonone C Mansonone E Mansonone G Mansonone H Metalaxyl Nativo 750 WG

0.0±0.0 k 73.3±1.0 e 26.7±1.0 i 38.9±0.8 g 61.1±0.4 f 17.8±1.2 j 84.2±2.6 c 94.4±2.2 b 81.2±1.8 d 33.3±1.5 h 100.0±0.0 a NT

Diameter of inhibition zone (mm) Xanthomonas X. oryzae pv. oryzae pv. oryzae (Xoo) oryzicola (Xoc) 0.0±0.0 g 0.0±0.0 g 12.3±0.6 d 14.3±1.2 d 9.0±0.0 f 9.7±0.6 f 11.3±0.6 e 11.3±2.1 e NT NT 0.0±0.0 g 9.0±0.0 f 12.7±0.6 d 13.7±1.2 d 25.7±0.6 a 24.3±1.2 a 18.3±0.6 c 17.0±0.0 c 21.7±0.6 b 18.3±1.2 b NT NT 12.50±0.07 d 13.00±0.14 d

1)

DCM, dichloromethane; metalaxyl, 100 µg mL–1; Nativo 750 WG, 1 mmol L–1. Data are means±SD. Different letters above columns indicate significant differences among the microbial species at P<0.05 (ANOVA followed by DMRT). NT, not test. The same as below.

Table 2 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)/minimum fungicidal concentration (MFC) (µg mL–1) of mansonone E against plant pathogenic bacteria and fungi Microorganisms Xoo Xoc Alternaria porri Colletotrichum gloeosporioides Fusarium oxysporum Phytophthora parasitica

MIC 7.8 7.8 250 31.2 500 31.2

MBC/MFC >500 >500 250 31.2 1 000 125

Compared with control, reduced food consumption was observed in all treated leaf discs eaten by castor except mansonone C which showed the progressive consumption leaf area more than the control (–3.8% antifeedant). Mansonone E displayed the highest significant antifeedant activity with 45.9%, followed by mansonone G, the DCM extract with 40.7 and 36.3%, respectively at 1 000 µg mL–1 concentration (Fig. 3). The other compounds expressed antifeedant activity less than 20%.

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4. Discussion The botanical natural products are a rich source of organic chemicals on earth. In nature, many plants have unpalatable substances like high content of phenols, alkaloids, flavonoids, terpenes, quinones, coumarins which play a de-

fensive role against particularly agriculture plant pathogens and insect pests (Jeyasankar et al. 2014). The antimicrobial compounds from plant are the results of coevolution with plant pathogens and their functions are to alleviate the harm of pathogens but unable to harm themselves. Therefore, the secondary compounds from plants are impossible to be

Table 3 Herbicidal activity of crude extracts and isolated compounds from the heartwood of Mansonia gagei against Brassica chinensis, Mimosa pigra, Echinochlooa crus-galli and Oryza sativa at 1 000 µg mL–1 B. chinensis Shoot Radical 7.9±0.5 cd 29.6±1.6 de 8.7±1.4 de 1.5±0.3 a 8.1±0.9 d 29.9±0.8 de 6.1±0.5 b 32.6±0.9 ef 7.1±0.2 cd 33.7±2.9 f 7.6±0.6 cd 18.5±1.2 c 1.9±2.2 a 1.0±0.03 a 8.5±0.3 de 29.0±1.4 d 9.2±0.7 e 10.1±1.8 b

1000

e

800

c

Crops

Weeds

B. chinensis

M. pigra

d

c

e b

600

e

d

400 a

200

D

C

M

C

on tro ex l M eO tra c H ex t t r M an act so ri M an n A s M an orin so C M non an e so C n M an one so E M non an e so G no ne H

0

3 500 3 000 2 500 2 000 1 500 1 000 500 0

O. sativa Shoot Root 35.6±1.3 de 50.7±0.6 e 26.2±1.7 a 21.4±0.7 a 37.0±1.1 e 35.9±0.7 d 34.8±0.8 d 55.1±1.3 g 30.7±1.2 b 53.0±0.3 f 44.1±0.7 f 49.7±1.2 e 26.3±0.6 a 23.6±1.3 b 32.7±0.8 c 36.5±0.8 d 34.7±1.0 d 34.2±0.7 c

g

f

c

d

b

bc

e

a

e

C on C M trol e M eO xtr H act ex tr M an act so r M an in A so M an ri so n C n M an one so C n M an one so E M no an ne so G no ne H

Fresh weight (mg)

Control DCM extract MeOH extract Mansorin A Mansorin C Mansonone C Mansonone E Mansonone G Mansonone H

Plants growth (mm) M. pigra E. crus-galli Shoot Radical Shoot Root 45.9±1.0 e 47.4±1.6 bc 26.4±0.4 f 32.2±2.1 f 43.3±0.9 de 56.3±2.6 e 15.2±0.6 c 0.0±0.0 a 46.2±0.9 e 50.5±2.1 cd 24.8±0.9 e 14.5±1.0 de 28.2±0.3 b 50.5±0.4 cd 7.7±0.5 b 5.7±0.5 b 35.2±2.7 c 48.9±0.9 cd NT NT 31.1±1.7 b 53.2±4.0 de 17.8±0.4 d 9.5±1.1 c 11.2±2.7 a 2.6±0.3 a 5.3±0.6 a 3.8±0.1 b 40.3±2.6 d 47.9±4.9 bc 26.8±0.9 f 13.1±0.9 d 41.2±1.7 d 43.5±2.7 b 26.3±0.8 f 15.8±1.6 e

D

Compounds

Dicotyledon O. sativa c

0

0

tro C on

C M D

C

a

c

d

a

tra ct an s M or an in so A no M an ne C so no M ne an so E no M n an e G so no ne H

50

c

M

1 000

M

b

l

100

C

cd

150

2 000

D

d

200

ct

a

d

ex

c

H

b

tra

b

ex

3 000

b

eO

b

M

e

4 000

E. crus-galli 250

on tro ex l M eO tra c H ex t t r M an act so ri M an n A so M ri an so n C M non an so e C n M an one so E M non an e so G no ne H

Fresh weight (mg)

5 000

Monocotyledon

Fig. 2 Herbicidal activity of crude extracts and isolated compounds from the heartwood of M. gagei on the fresh weight of crops (Brassica chinensis and Oryza sativa) and weed (Mimosa pigra and Echinochlooa crus-galli) at 1 000 µg mL–1. Error bars show means±SD; different lower case letters for each column indicate a significant difference at P<0.05 (DMRT). The same as below.

Rachsawan Mongkol et al. Journal of Integrative Agriculture 2016, 15(12): 2795–2802

very toxic to pathogens; otherwise the plants themselves will suffer injury (Deng et al. 2010). In this research, the potential use of the extracts and isolated compounds from the heartwoods of M. gagei was investigated. Mansonone E revealed highly significant antifungal, antibacterial, antifeedant and herbicidal activities to all plant pathogens. Biological activities against plant pathogens (anti-phytopathogenic fungi, antibacterial, antifeedant and herbicidal) of the constituents from M. gagei have not been previously investigated for anti-phytopathogenic fungi against A. porri, C. gloeosporioides, F. oxysporum and P. parasitica. Among the microbial isolates, Xoo appeared to be the susceptible to mansonone E. Boonsri et al. (2008) reported that mansonone E from the heartwoods of T. populene exhibited significant cytotoxic and antibacterial activities with MIC against Bacillus subtillis as 4.69 µg mL–1 (Boonsri et al. 2008). Our results demonstrated that the DCM extract and the isolated mansonone E significantly inhibited plant pathogens, herbicidal and antifeedant activities. Moreover, mansonone E did not show specific herbicidal activity to both crops and weeds; hence it should be applied before plantation. The same as the pervious study, Tiew and co-workers reported the antifungal activity against Cladosporium cucumerinum, mansonone E also displayed strong inhibit the growth of fungi (Tiew et al. 2003a). According to previous reports, mansonones have been addressed as phytoalexins, anticancer, anti-leukemia, antibacterial, anti-inflammatory and anti-topoisomerase I activities (Dumas et al. 1986; Duchesne et al. 1994; Banerjee et al. 2006; Lee and Rhee 2012; Sadeghi et al. 2013). Mansonone E has been reported cytotoxic to human cell line, when using as pesticide, it should be considered using a safe technique such as wearing protective clothes as recommended, and not directly touch chemicals the same as that applied for other pesticides. However the maintaining Good Agricultural Practice (GAP) is also essential in agri-

a

80 60

b

d

40

e

20

f

f

or in an A so no ne M an C so no ne M an E so no ne M an G so no ne Az H ad ira ch tin

H eO

g

c

an s

M

M

D

C

–20

M

0

M

Antifeedant (%)

100

Compounds

Fig. 3 Percent antifeedant activity from the heartwoods of M. gagei extract and isolated compounds against the second instar larvae of Spodoptera litura at 1 000 µg mL–1.

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culture cultivation (Zhang et al. 2015). This present study disclosed that mansonone E was the key biological activity constituent in M. gagei. On the view point of structure-antifungal activity relationship, mansorins displayed lower activity than 1,2-naphthoquinones (mansonones). Mansonones C (4), E (6) and G (5) revealed good activity. This implied the essence of substituent on the activity. The presence of a hydroxyl group at 6-position (4 vs. 5, 6 vs. 7), and a pyran ring (4 vs. 6, 5 vs. 7) in mansonone system deteriorated the activity. It is obvious that this compound possesses multiple bioactivities against plant pathogens and has high application and exploitation values in pesticide field in the future study.

5. Conclusion The DCM extract from the heartwoods of M. gagei displayed more pronounced biological activities than MeOH extract. Mansorins A, B, and C, mansonones C, E, G and H were isolated from the DCM extract. Among the isolated compounds according to bioactivity guided fractions, mansonone E showed high antifungal against P. parasitica, followed by mansonone C, mansorin B and mansonone G. Mansonone E was tested for antifungal activity against other plant pathogens: A. porri, C. gloeosporioides, F. oxysporum. Moreover, mansonone E exhibited the highest antibacterial against Xoo and Xoc, antifeedant against S. litura and herbicidal activities against the growth of B. chinensis, M. pigra, E. crus-galli and O. sativa. Mansonone E could be considered as an alternative natural pesticide for controlling many agricultural plant pathogens.

Acknowledgements The authors would like to thank the office of the Higher Education Commission, Thailand for supporting grant fund under the program Strategic Scholarships for Frontier Research Network for the Ph D Program Thai Doctoral degree for this research (77/2551) and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund, GCUGR11244525026D24).

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