Naturally Occurring Pest Bioregulators - American Chemical Society

Chapter 28

A Search for Agrochemicals from Peruvian Plants 1

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D. Howard Miles , Jorge Meideros , Liza Chen , Vallapa Chittawong , Colin Swithenbank , Zev Lidert , Allen Matthew Payne , and Paul A. Hedin 1

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Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch028

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Department of Chemistry, University of Central Florida, Orlando, FL 32826 Rohm and Haas Company, Spring House, PA 19477 Crop Science Laboratory, U.S. Department of Agriculture, Mississippi State, M S 39762 2

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The objective of this study was the isolation and identification of the antifeedant, antifungal, and antibacterial active constituents from Peruvian plants. 5,7-Dihydroxy-6,8-dimethyl-4'-methoxyflavanone (matteucinol) was isolated from the root of Miconia cannabina. This is the first report of the spectral data of matteucinol. Matteucinol showed significant antifungal activity (Pythium ultimum 135%). Also reported is the isolation of sitosterol and medicarpin from the toluene extract of the wood of Dalbergia monetaria. Medicarpin possessed high antifungal (Rhizoctonia solani, 145% and Helminthosporium teres, 100%) and antibacterial activity (Xanthomonas campestris, 100%) along with high antifeedant activity against the cotton boll weevil (Anthonomus grandis, 98%). As long as mankind continues to cultivate and grow plants for food, feed, and fiber, crops will be threatened by various enemies and agents such as insects, fungi, and bacteria. Prevention and control of insect pests and plant diseases will remain an important objective of agrochemical research. In this study, the isolation and structure determination of several compounds showing insect antifeedant activity and antifungal activity from Peru plants are presented. The plant Miconia cannabina Markgr. (Melastomaceae) is common to Iquitos, Peru. No previous chemical study has been undertaken on this plant; however, examples of studies on related plants are known. A study of the protein and water contents of several parts of the plant M. theaezans was undertaken by Gaulin and Craker (1). In another study Marini-Bettolo (2) isolated primin and miconidin from the ethyl acetate extract of a nonidentified species of Miconia. Primin has biological activity against various Gram positive and negative microorganisms and fungi as well as antitumor activity against sarcoma of mice (Swiss). Miconidin is less active against common microorganisms, but inhibits Mucobacterium sp and Gibberella fulikuroi (2). These two compounds also showed antifeedant activity (3) against six insect species - desert locust Schistocerca gregaria, the migratory locust Locusta migratoria, the armyworms S. littoralis and S. extemota, the budworm Heliothis amigera and caterpillars of the cabbage white butterfly Pieris brassicae. Dalbergia, a wood climber, belongs to the family Leguminosae and subfamily Papilionaceae. Nearly 120 Dalbergia species are known to occur in nature (4). The chemical components of this genus of plants provides a great number of structures belonging to widely different chemical categories such as, neoflavonoids, isoflavonoids, flavonoids, furans, isoflavans, benzophenones, styrenes, sterols, and terpenoids. 0097-6156/91/0449-0399$06.00A) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

Materials and Methods Sample collection. — Roots of M. canabina (voucher number 7261) and wood of D. monetaria (voucher number 7000) were collected in Iquitos, Peru in 1982, and identified


by Dr. Sidney McDaniel, Department of Biological Sciences, Mississippi State University. The plant material was air dried and ground in a Wiley mill before extraction. Extraction. — M. Canabina (6.8 kg dry weight) was extracted in a soxhlet apparatus with methylene chloride (16 h.) and then ethanol (16 h.). The solvent was evaporated under reduced pressure to yield 6.7 g of methylene chloride extract (fraction A , Figure 1) and 22.6 g of ethanol extract (fraction B , Figure 1). Plant Material 6.8 kg Soxhlet CH Cl (16h.) 2

2

Fraction A 6.7 g I P ++ R + H

Ins. Soxhlet EtOH(16h) Fraction B 22.6 g I P ++++ R H

Ins.

Silica gel column 1

1 20% C H -C H 7

8

6

1 2

4 30% Et 0-C H 2

1 1

P R H

7

8

3 100% CHC1

I

+ ++ -

p

R H

... , 4 100% 3

CH3OH

1

1

1

1

+

+

++++ +++

P R H

1 +++ -

P R H

+ --

Purification Compound 1 P - Pythium ultimum; R - Rhizoctonia solani; H - Helminthosporium teres diameter of the clear zone of inhibition around the disk: + - 1-2 mm; ++ - 2-5 mm; +++ - 5-10; ++++ - > 10 mm Figure 1. Fractionation Scheme for Miconia cannabina D. monetaria (25.6 kg dry weight) was extracted in sequence with methylene chloride (16 h.) and ethanol (16 h.) in a Soxhlet apparatus. The solvent was removed in vacuo to yield 211.1 g of methylene chloride extract (fraction A , Figure 2) and 174.4 g of extract ethanol (fraction B , Figure 2). Fraction A was sequentially extracted with hot hexane (fraction A l , Figure 2) and toluene (fraction A 2 , Figure 2). Fraction B was extracted with ether. The ether-insoluble fraction (72.2 g) (fraction C, Figure 2) was

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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A Search for Agrochemicals from Peruvian Plants


sequentially extracted with hexane : ether (2:1), and chloroform. The ether-soluble fraction (60.9 g) was partitioned between chloroform and water (1:1). Fraction A 2 (73.8 g) was extracted with chloroform. The chloroform solution was extracted with a 5% hydrochloric acid solution. The aqueous layer was neutralized with a 5% sodium hydroxide solution and shaken several times with chloroform. The chloroform was removed in vacuo to yield 4 g of fraction A2-b (Figure 3). The original chloroform layer was partitioned between chloroform and 5% aqueous sodium hydroxide solution (1:1). After separation of the layers, the aqueous layer was neutralized with 5% hydrochloric acid and extracted repeatedly with chloroform. The combined chloroform extracts were then dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo to yield 12.0 g of residue (fraction A2-a). Plant Material 25.6 kg Soxhlet CH Cl2(16h) 2

Fraction A 211.1 g Hexane (hot) Fraction A l 54.1 g P R H

h

INSOLUB. I Soxhlet EtOH(16h)

Ins. 138.0 g

Fraction B 174.49 g I ether

Toluene Fraction C 72.2 g (Ins.) ++ |— 79 Fraction A2 73.8 g Fraction A3 Hexane: ether (Ins.) (Sol.) 79 (2:1) 64.2 g

Sol162.9 i —i Ins^ P R H Ii

1 Fraction D 4.8 g (Sol.)

P R H Ij

h

P R H I,

80 95

h

76 98

P R H Ij

h

93 98

h

Fraction E 58.5 g

^cLci3 Sol. 4.5 g P R H 89 I, h 93

Ins. ^3.61 P R H 11 1 2

-

diameter of the clear zone of inhibition around the disk: + -1-2 mm; ++ - 2-5 mm; +++ - 5-10 mm; ++++ - >10 mm Boll weevil antifeedant activity (I): Iflo = [(# holes blank - # holes sample) / (# holes blank)] x 100 n = # mg / cm paper 2

Figure 2. Fractionation Scheme for Dalbergia monetaria

Fraction A2-a was chromatographed on silica gel (60-200 mesh, 800 g) with eluents of increasing polarity from hexane through toluene to chloroform, and then to methanol. Fractions of 250 ml were collected, the solvent was removed in vacuo, and those fractions exhibiting similar T L C profiles were combined. Bioassay Antifungal and Antibacterial Bioassay. — Fractions were screened for activity against the fungi Helminthosporium teres, Pythium ultimum, and Rhizoctonia solani and the bacteria Xanthonomas campestris by the paper disc method (5). Each fraction to be tested was



402

Fraction A2 73.8 g (1) CHC1 (2) HC1 (5%) 3

~B 0

cricIT

2

(1) 5%aq.NaOH

aq. NaOH (5%) CHC1

1—

"So

3

H 0

1

2


(1) HC1 (5%)

HoO

(2) CHCI3 CHCI3 A2-b4g

(2) CHCI3 CiHCI3 A2-a 12.0 g Silica gel column

1— 3 n

6 12

T

50%

Compound 2

C7H0 ^

h

1

H R P h I9

1— 10 CHCl,

+++ +++ +++ 95.0 98.0 "T 11

12

CH3OH

++++ +++ +++ 97.5 99.0 Purification

Compound 3 diam. of the clear zone of inhibition around the disk: + -1-2 mm; ++ - 2-5 mm; +++ - 5-10 mm; ++++ - >10 mm Boll weevil antifeedant activity (I): In % = [l-(# holes sample / # holes blank)] x 100 n = # mg / cm paper 2

Figure 3. Purification of Fraction A2 of Dalbergia monetaria

dissolved in an appropriate solvent. In the preliminary tests of semi-purified plant extracts, a 100,000 ppm solution was prepared, while in the final test of pure compounds a 10,000 ppm solution was used. Filter paper discs (6 mm dia.) were soaked in the test solution and allowed to dry overnight. Petri plates containing solidified agar (DIFCO Potato-Dextrose for H. teres and R. solani, DIFCO cornmeal for P. ultimum, and DIFCO nutrient agar for X. campestris) were inoculated with fungi. The inoculation with H. teres and X. campestris was performed by mixing 100 ml of the appropriate agar with the filtrate of an aqueous suspension of their spores and then placing 5 ml of the mixture on top of the solidified agar layer. For R. solani and P. ultimun, pieces of the mycelium were cut from the culture plate and placed in the center of the bioassay plate. Three of the prepared paper disks were then placed on each Petri plate. A paper disk soaked only in the solvent used and dried by the usual method was also placed on each plate as a blank. As a control, a 10,000 ppm solution of Dithane M-45 (from Rhom & Hass Co.), a commercially available fungicide, was used. Inhibition was determined by measuring the diameter of the clear zone (if present) around the disc. Boll weevil antifeedant bioassay. — The bioassay was performed according to the procedure of Hedin et. al. (6, 7). Agar plugs were formed by boiling 3 grams of agar and



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A Search for AgrochemicalsfromPeruvian Plants

3 grams of freeze-dried cotton bolls in 100 mis of distilled water to effect a viscous sol. The sol. was poured into 18 mm diameter tubing, and gelation occurred after cooling. These gelatinous rods were cut into individual 3.6 cm plugs. The extracts of the plant samples were applied to preweighed 4.5 x 4.5 cm squares of Whatman #1 chromatography paper by dipping the paper into a solution of the test sample. After air-drying, the paper was weighed. A blank was prepared by dipping one of the papers into the solvent used on the test sample. The papers were wrapped around the agar-cotton plugs and fastened with four staples. The ends of the plugs were sealed with corks. The plugs were then placed staple-side down in the petri dishes. Twenty newly emerged boll weevils were placed in 14 x 2 cm petri dishes containing the test and control plugs. The bioassay was carried out in the dark at 25°C for 4 hours. When the bioassay was finished the paper was removed from the plugs and the number of punctures counted. Antifeedant activity (IJ was expressed as: (IJ % = [l-(# holes sample/^ holes blank] x 100 where n indicates the amount used (in grams) of the compound per surface units (cm ) of the filter paper. 2

Results and Discussion The ethanolic extract of M. cannabina revealed the highest antifungal activity when tested against P. ultimum (P), R. solani (R), and H. teres (H)» using the preliminary "paper disc" method (5). Fractionation of the ethanolic extract was performed by column chromatography (Figure 1) with silica gel as the support. The resulting chromatographic fractions were monitored by the "paper disc" bioassay. The material which was eluted with 30% (v/v) ether in toluene was further purified by repetitive crystallization from methanol to yield the compound 1. The high resolution mass spectrum suggested a molecular formula of C H 0 for compound 1. This compound developed the characteristic color reactions of flavonoids (8,9). The U V spectrum of compound 1 in methanol, consisted of two major absorption maxima, one of which occurred at 294 nm. The other band at 328 nm was less intense and was suggestive of the presence of a flavonoid lacking conjugation between the A - and B-rings (9,10,11). A doublet of doublets at 5 2.83 and 3.08 in the H N M R spectrum of compound 1 suggested the presence of a flavanone. 1 8

1 8

5

l

7

r ^

6

^ O Skeleton of flavanones

Examination of the U V spectra of compound 1 in methanol with the shift reagents NaOMe, A1C1 , A1C1 + HC1, NaOAc, and NaOAc + H B 0 indicated that compound 1 had the basic skeleton of 5,7-dihydroxyflavanone. The absence of signals between 8 5.7 6.9 in the *H N M R spectrum indicated that the C-6 and C-8 positions in the A-ring were substituted. The presence of protons, at C-3', C-5', C-2', and C-6' was indicated by two pairs of ortho coupled doublets at 5 6.98 and 7.42 (J = 10 Hz). A mass spectral fragment at m/z 180, suggested that the two methyl groups observed in the *H N M R spectrum (8 2.06 and 2.08 were at C-6 and C-8; therefore, the methoxy group at 8 3.84 ppm in the *H N M R spectrum could be located at C-4'. These spectral properties are consistent with 3

3

3

3


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the assignment of the structure of compound \ as 5,7-dihydroxy-6,8-dimethyl-4'methoxyflavanone. A compound of this structure was first isolated from Matteucia orientalis by Munesada (12) and was named (-)-matteucinol. Later the isolation of matteucinol was reported from two species of Rhododendron, by Arthur and Hui (13) and by Tanabe, Kondo and Takahashi (14); however, the structural assignments were not unequivocal since no spectral data was reported. Thus, this paper provides the first report of the spectral data of (-)-matteucinol and confirms the structure assignment as 5,7-dihydroxy-oc,8dimethyl-4'-methoxyflavanone. Matteucinol demonstrated excellent antifungal and antibacterial activity (Table I). The highest antifungal activity was observed against R. solani for which a threshold concentration of 15.1 p,g/cm was determined by regression analysis. A threshold concentration of 7 x ltf u,g/cm was determined for the activity against the bacterium X. campestris.


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Table I. Antifungal and antibacterial activity* of Matteucinol and (-)-Medicarpin

P. ultimum

MICROORGANISM R. solani H. teres

X. campestris

Matteucinol

25

135

30

69

(-)-Medicarpin

75

145

100

80

'relative activity of compound (10,000 ppm) to the standard (10,000 ppm): Relative activity (%) =

diam. of clear zone for test compound diam. of clear zone for standard

X

100

In a continuing search for compounds with antifungal and insect antifeedant activity, the active constituents from the wood of D. monetaria were also examined. The toluene soluble material (fraction A-2) from the methylene chloride extract of the wood of this plant showed both potent insect antifeedant activity against the boll weevil (A. grandis Boheman) and high antifungal activity (Figure 2). Partitioning of fraction A-2 between chloroform and 5% aqueous hydrochloric acid resulted in the concentration of the basic components in the aqueous layer. The basic fraction (fraction A-2-b), obtained as shown in Figure 3 presented no antifungal or insect antifeedant activity.

[

O

Matteucinol

Partitioning the chloroform layer between chloroform and 5% aqueous sodium hydroxide (Figure 3) resulted in concentration of the acid components in the aqueous layer. T7ie acid fraction (fraction A-2-a) showed high antifungal activity (P. ultimum - +++;/?. solani - +++; H. teres - +++) and antifeedant activity against boll weevils (feeding inhibition activity of 95.0 and 98.0% at the dose level of 1 and 2 mg/cm of the filter 2



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A Search for AgrochemicalsfromPeruvian Plants

paper). Fraction A-2-a was fractionated on a silica gel column with eluants of increasing polarity from hexane through toluene to chloroform and then to methanol as shown in Figure 3. The material was eluted with toluene-hexane (1:1) and was further purified by recrystallization from benzene to produce compound 2. A more polar material was eluted with toluene and purified by flash chromatography using hexane-acetone (2:1) as the eluant. Fractions 13-18 were combined and further purified by preparative T L C using 10% methanol in chloroform (v/v) to obtain compound 3. Compound 2, m.p. 134 - 135°C, showed a NT at m/e 414. The *H N M R , U V , and IR spectra suggested the presence of a steroid. The color reactions developed with the Liebermann-Burchard and Carr-Price reagents were also in accord with a steroid structure. Compound 2 was identified as sitosterol by comparing its m.p. and *H N M R , IR, U V , and M S spectra data with those of an authentic sample (Aldrich Chemical Company, Inc., Milwaukee, WI). Compound 3 had a molecular formula of C H 0 , as determined by high resolution mass spectrum. The *H N M R spectrum was similar to those observed for pterocarpans (13). Compound 3 was identified as (-)-Medicarpin by comparing its m.p. and *H N M R , IR, U V , and M S with those of an authentic sample (Dr. H . D . Van Etton, Cornell University). (-)-Medicarpin showed excellent antifungal and antibacterial activity (Table I). The highest antifungal activity (14.5%) was observed against R. solani. 16

14

4

O

(-)-3 -Hydroxy-9-methoxypterocaipan [(-)-medicarpin]

Conclusion This is the first report of the isolation of matteucinol from Af. cannabina and (-)medicarpin from D. monetaria. Matteucinol demonstrated excellent antifungal activity (R. solani 135%). (-)-Medicarpin showed activity against the fungi P. ultimum (75%), R. solani (145%), H. teres (100%), the bacteria X. campestris (80%), and the boll weevil (100% antifeedant at a dose level of 2 mg/ml). Thus matteucinol and (-)-medicarpin have potential as new agrochemicals. Acknowledgments This study was supported by the Rohm & Haas Co. We are indebted to Drs. Catherine Costello and Thomas Dorsey of the M I T mass spectrometry facility for their invaluable assistance in obtaining the mass spectrum of compounds. We thank Dr. Jim Dechter and Vicki Farr of the University of Alabama N M R facility for their help in obtaining H N M R data. !

Literature Cited 1. Gaulin, S.J.C.; Carker, L.E. J. Agric. Food Chem.1974,27(4), 791. 2. Marini Bettolo, G.B.; Delle Monache, F.; Goncalves Da Lima, O.; de Barros Coelho, S. Gazz. Chim. Ital.1971,101, 41. 3. Benays, E.; Lupi, A.; Marini Bettolo, R.; Mastrofrancesco, C.; Tagliatesta, P. Experientia,1984,41, 1010. 4. Chawla, H.M.; Chibber, S.S. J. Scientific and Industrial Research1981,40, 313-325.



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5. The Official Methods of Analysis of the Association of Official Analytical Chemist, Official First Action, Eleventh Ed., 1970, p. 843. 6. Hedin, P.A.; Thompson, A.C.; Minyard, J.P. J. Econ. Entomol.1966,59, 181. 7. Struck, R.F.; Frye, J.; Shealy, Y.F.; Hedin, P.A.; Thompson, A.C.; Minyard, J.P. J. Econ. Entomol.1961,61, 270. 8. Zweig, G.; Sherma, J., (Ed.s), Handbook of Chromatography, Vol. 2, The Chemical Rubber Co., Clevelent, 1972. 9. Mabry, T.J.; Markham, K.R.; Thomas, M.B. In: The Systematic Identification of Flavonoids, Springer-Verlag, New York, 1970. 10. Jurd, L., In: "The Chemistry of Flavonoid Compounds", Geissman, T.A., (Ed.), MacMillan, New York, 1962. 11. Harbone, J.B.; Mabry, T.J. In: "The Flavonoid Compounds", Academic: New York, 1975, Vol.1. 12. Munesda, J. Pharm. Soc. Japan, 1924, 505, 185. 13. Arthur, H.R.; Hui, W.K.; J. Chem.Soc.,1954,2782. 14. Yoshihisa, T.; Kondo, S.; Takahashi, K.; Kanazawa Daigaku Yakugakubu Kenkyu Nempo,1962,12, 7-14. RECEIVED May 16, 1990


Naturally Occurring Pest Bioregulators - American Chemical Society

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