FINAL REPORT

APPLICATION OF PLANT-DERIVED PRESERVATIVES TO IMPROVE DURABILITY OF RATTAN AND RATTAN-BASED PRODUCTS

Project number: RPI-2

2008

August 31, 2008

Duration: December 1, 2006 – May 31, 2008

Total Budget: US$ 5,500

“Application of Plant-derived Preservatives to Improve Durability of Rattan and Rattan-Based Products is funded under the Rattan Research Grant Program of the

ITTO-Philippines-ASEAN Rattan Project (PD 334/05 Rev. 2 (I)”

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Preface

In order to improve the quality of rattan and rattan-based products, preservation treatment plays very improtant role. This report describe the results of the exploration of plant extract and their active substances to be developed as rattan preservatives. The purposes of this research is to inventory potent plant extract having biological activity, to isolate the active substances and to test their efficacy to be used as rattan preservative. This work was funded under the Rattan Research Grant Program of the ITTO-Philippines-ASEAN Rattan Project (Grant contract PD 334/05 Rev 2 (I) in collaboration with Mulawarman University. The authors wish to acknowledge the funding provided for this work by ITTO, Government of Philippines and ASEAN Rattan project to Rattan Research Grant program.

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Project Participants

Dr. Irawa

Akhmad

Supriyad

Supriyan

n W Kusuma Research Scientist

Wijaya, M.Agr Research Scientist

i Technician

to Technician

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TABLE OF CONTENTS Cover page 1 Preface 2 Project Participants 3 List of Acronyms Used 5 Executive Summary …………………………………………………………………. 6 Chapter 1 Project Background …………………………………………………… 7 Chapter 2 Project Objectives …………………………………………………….. 10 Chapter 3 Methodologies ………………………………………………….......... 11 Chapter 4 Results and Discussion………………………………………........... 16 Chapter 5 Conclusion .......………………………………………………………. 27 Chapter 6 Recommendations ……………………………………………………. 28 Chapter 7 Publication and Presentations ………………………………………. 29 Chapter 8 Report on Expenditures.......... ………………………………………. 30

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LIST OF ACRONYMS USED

C. sappan - Caesalpinia sappan CCA - Chromated Copper Arsenate EI - Electron impact Ionization MIC - Minimum Inhibitory Concentration NMR - Nuclear Magnetic Resonance P. indicus - Pterocarpus indicus PDA - Potato Dextrose Agar

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EXECUTIVE SUMMARY

As a continuation effort to discover high efficacy and environmentally-benign preservative agents to improve durability and resistance of rattan and rattan-based products against biological deterioration agents, research into exploration of natural preservatives from tropical plants is conducted. Thirty-two plant species including herbs and trees were collected from two villages in East Kalimantan, Indonesia. The plant species were collected on the basis of the availability of ethnobotanical information and historical uses by local community. The plants collected from field were identified and air dried prior to delivery to laboratory in Samarinda, Indonesia. The plants collected were converted into meals and extracted with several organic solvents. The extracts obtained were applied to laboratory assays using several decay and mold fungi commonly known as rattan-degrading organisms. The plant extracts showing inhibition against the test degrading organisms are assumed to contain such phytochemicals possible to be used as natural preservatives. Two plant species, i.e.: Pterocarpus indicus and Caesalpinia sappan, were selected to be further investigated based on their biological activity to inhibit the growth of test fungi. Bioassay-guided fractionation was conducted to isolate the active substances from both target plants. Two active substances from P. indicus and an active compound from C. sappan were isolated by combination of various chromatographic methods. Identification of the active compounds from both tree species was conducted based on the instrumental analysis and the physical properties. Bioactive substances from two tropical plants, Pterocarpus indicus and Caesalpinia sappan displayed potent activity against rattan-degrading fungi in vitro. However, only bioactive compound from P. indicus showed activity against post-powder beetles. To determine the potential of the natural products as preservatives in the field, several tests were conducted. In field test of the natural products in preserving the rattan and rattan products, the results show that the rattan samples treated with the isolated compounds have better resistance against microorganisms attack than those of without any treatment. Result of the present research displayed high potential of plant-derived preservatives, in term of P. indicus and C. sappan, to be applied as rattan preservative. Budget Headings Annual budget in US$ Total Expenditure

(in US$) Annual Expenditure

as % of Budget Year 1 Year 2 Year n

0 0 0 0 0 100 800 300 100 400 800 100 200 100 0 100 200 100 800 400 400 0 800 100 600 200 400 0 600 100 400 100 100 200 400 100

2000 800 550 650 2000 100 700 100 200 400 700 100

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CHAPTER 1

PROJECT BACKGROUND

Globally, there are about 600 species of rattans under 13 genera, most of them indigenous to South East Asia. Indonesia has 80% share in the international market, with a value of US $ 1.147 billion. All the rattan species are used by the local people, but 20 species are regularly used for plantation and commercial purposes (Anonimous, 2004). Several important rattan-based products are furniture, shopping and laundry baskets and serving trays as the major products from urban areas, and carrier and storage baskets as the main products at the rural level. The quality of finished products is variable. Rattan has relatively high starch content, which make it particularly liable to be infested by fungi and insects. As a result of infections by staining fungi and beetle attack, severe losses will be resulted. Fungi cause discoloration and decay of the canes, while beetles cause pinholes or worm holes. Blue stain fungi are the most common causal agent of staining, which penetrate with their hyphae deep inside the stem, utilizing starch and sugar. Stained canes are often coloured to hide the defect. Through intensive marketing, furniture in various colours has become fashionable. However, heavily stained material cannot be used for furniture since its bending strength is reduced. At moisture levels of more than 20 percent, decay fungi can also attack the stem. Such infections are often noticed only at a later stage, when the fruit bodies appear, and they can cause serious structural degradation of rattan in service. Rattan with a moisture content of 50 to 100 percent is liable to insect attack, mostly by the powder-post beetle. Jasni and Suprapti (1992) reported that several species of mold and decay fungi such as Penicillium sp, Aspergillus sp, Schyzophyllum commune and Polyporus sp as well as several insect species such as Dinoderus minutus Farb., Heterobostrychus aequalis Wat., Lyctus buneus Steph., and Mynthea sp. have been found to be associated with biological deterioration of rattan.

Fig 1. Rattan and rattan-based products For protection against insects and fungi, insecticide and fungicide have to be applied very early. Several chemicals, so called fungicides and insecticides, have been used to prevent and kill fungi and insects in various environments. A major barrier in protecting rattan with preservatives from the assortment of degradative agents found environmentally is the time period that the rattan must be protected without supplementary treatment. Furthermore, formulations of preservative developed must be

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environmentally benign and still economically competitive. Although several synthetic preservatives have been used with good results, the disadvantages of using them are their potential negative effects on the environment, for instance toxicity, accumulation in soil and water and build-up of resistance in fungal and insect populations, limited their applications. In relation with that, the availability and legal acceptance of suitable preservatives differ from country to country. Another solution is to treat the rattan with a wood preservative, such as the oilborne, organic pentachlorophenol and creosote systems and the water-borne, inorganic ‘‘chromated copper arsenate’’ (CCA-type C) preservative. Most of the treated wood products, about 80% by wood volume, are treated with CCA (Micklewright, 1998). CCA is highly effective in protecting wood against a wide variety of wood-destroying organisms, besides being inexpensive, water-soluble, and leach-resistant. However, the perceived environmental hazards of the disposal of wood which contain such metals may limit the future use of CCA in the US (Preston, 2000). Indeed, the availability of CCA-treated lumber has been greatly reduced, and CCA use has been restricted in many European countries and Japan. Again the consequences of pollution must be taken into consideration.

Fig 2. Rattan processing in medium-scale industry. On the other hand, various different natural substances from plant extracts have been investigated on fungal and insect activities, e.g.: activity of essential oil from Argentinean aromatic plants against Alternaria solani, Sclerotium cepivorum, and Colletotrichum coccodes (Zygadlo and Grosso 1995), examination of 11 plant extract against Rhizoctonia solani (Singh et al. 1998), activity of Ocimum gratissimum, Zingiber cassumunar, Cymbopogon citratus and Caesulia axilliaris against Aspergillius flavus (Dubey et al. 2000), and activity of origanum commercial oil (Manohar et al. 2001). The plants can be act as the potent sources of biologically active substances which can be applied as preservative to protect rattan and its products from biological deterioration.

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Due to the main disadvantages of the present preservative agents, the public demand has grown for more environmental friendly methods. Therefore, it is important that more effective and less toxic naturally-occurring preservative agents with novel mechanisms of action be discovered and developed. The proposed research activity will be directed to discover a new, effective and environmentally-acceptable plant-derived preservative and the suitable preservative treatment method.

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CHAPTER 2

PROJECT OBJECTIVES

In continuation of our investigation to discover and develop plant-derived preservative agents and appropriate application method, the research activity aims:

1. to discover the potent plant extracts and the active substances to be applied as preservative for rattan and rattan-based products due to their susceptible for biological deterioration;

2. to develop economical preservative treatment methods for rattan and rattan-based products;

3. to standardize the plant-derived preservatives including the product quality and the safety aspect for a wide-ranging application.

The expected outputs of this study are:

1. Novel, high efficacy, environmentally-benign and standardized natural preservatives for rattan and rattan-based products;

2. Economical treatment method for the discovered natural preservatives.

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CHAPTER 3

METHODOLOGIES

1. Collection of plant and rattan samples Plant species were selected based on our preliminary investigation on screening of Indonesian plants having antifungal activity. The plant collection was conducted in March 2007. Thirty-two plant samples, trees and herbs, were collected from the Laburan and Selo batu villages at Paser regency in East Kalimantan (Fig 3.), representing the availability of ethnobotanical information (Table 1). The Laburan and Selo batu villages were two areas where the local people (Paser tribe) of Paser regency are exist. Known uses and historical application of the plants as preservative agent by the Paser were taken as the main consideration in collecting the plant samples. In the field the plants collected, including parts of leaf and stem were labelled, preserved with ethanol, pressed in plastic bags and delivered to our laboratory. In the laboratory, the samples were converted into meals to give 100-200 g plant meals kept in sample bags. The plant meals were,then preserved in constant room and their moisture content were measured. In the same time, voucher specimens of the plant samples were deposited in laboratory.

Location 2

Location 1

Fig 3. Location of plant sample collection

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2. Plant extraction Plant extraction was conducted by cold extraction using methanol (Merck, reagent grade) as the solvent. Plants meals were weighed in erlenmeyer flasks (10 g each) and poured with methanol (100-150 ml). The mixtures were placed on a shaker (Fig 4.) and the extraction was conducted for 24 hours each. After 24 hours the methanol solution in the erlenmeyer was taken and the sample was poured again with new methanol with the same method as described previously.

Fig 4. Cold methanol extraction of plants collected from field. The methanolic solution obtained twice extraction was filtrated using a filter paper in a buchner filtration apparatus, and then evaporated using a rotary evaporator. The syrup-like extract obtained from rotary evaporator was dried in a vaccum oven dryer to dryness� 3. Laboratory assay All plant extracts were subjected to bioassays using several rattan-degrading fungi, i.e.: Schyzophyllum commune, Pleurotus pulmonarius, Aspergillus niger and Fusarium oxysporum. The former two fungi known decaying fungi of wood and other lignoselulosic materials, including rattan, while the latter belongs to molds and known as the causal agents of staining and colorization of rattan. The agar dilution and agar diffusion method (Quiroga et al. 2001) were referred to conduct bioassay. Each plant extract (2 mg/Petri dish) was dissolved in acetone and mixed with 20 ml of sterile PDA in a Petri dish. After inoculated by test fungi, the Petri dish was incubated for 5-7 days in the dark to let the fungi grow. The fungal growth in the treated Petri dish was compared with that of control and the antifungal activity was determined as growth inhibition. 4. Isolation and identification of the active substances Boactivity assays against several rattan-degrading agents were conducted. Two plant extracts, Pterocarpus indicus and Caesalpinia sappan, showed the most potent activity. Both species were subjected to plant extraction and solvent-solvent fractionation to

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obtain active soluble. Each soluble fraction from P. indicus and C. sappan extract was subjected to laboratory assay to determine the bioactivity of plant extracts against several biological deterioration agents of rattan. In this step, activities against decay and colorization or moulds of rattan were evaluated. Bioassays against the test organisms showed that diethyl ether soluble of P. indicus and C. sappan possess the most activity. Isolation of the active compounds were conducted by a bioassay-guided fractionation with combination of silica gel column chromatography and thin-layer chromatography methods and bioassays (antifungal and antifeedant assays). Only the most active fractions were applied to separation process. Active compounds were purified either by preparative thin-layer chromatography or recrystallization. Identification of the active compounds was conducted on the basis of spectral data, physical data and comparison to those of reference compounds . Instrumental analyses including ultraviolet, mass spectrometry and nuclear magnetic resonance spectroscopy were done.

Fig 5. Silica gel column chromatography of the plant extract. 5. Field test of rattan natural preservative The active fractions were subjected to a small-scale efficacy studies for uses as new plant-derived preservatives. Rattan samples were prepared in sticks of 2 cm length. The samples were sprayed with acetone solution containing several concentrations of the active subtances. Controls were sprayed with acetone only. After left for 24 hours, the

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samples were inoculated by test fungus by spraying with spore suspension of the Aspergillus, Fusarium and Penicillium spore. The inoculated samples were incubated for 2 weeks at room temperature in the dark. As a positive control, cypermethrin (a commercial wood preservative) was used at 2% concentration. Clear zone of the treated sample sticks and controls was observed and rated daily on the scale of 0-5 (0=no growth appearance; 1 = <30% growth appearance; 2 = 30-50% growth appearance; 3=>50-70% growth appearance and 4 = >70% growth appearance). 6. Development of treatment method and standardization of rattan preservatives Cold soaking treatment for rattan preservation was tested. This is based on the economical and easy application for rattan farmer and industry to apply the preservative agents. Testing samples were collected from several species of rattan. Rattan stem were cut in 2 cm length for preservative treatment moisture content measurement. Five consentration levels of compound A and compound B in water and a few acetone (0.1%, 0.25%, 0.5%, 1% and 2%,) were applied in triplicates for each experiment. Three rattan species with 20% average moisture content were used as samples. The air dried samples in 2 cm length were soaked in preservative solution (compound A and compound B). Preservative agents (compounds A and B), positive control (cypermethrin) and negative control (water only) were prepared in several preservative chambers. Rattan samples already weighed were put into the chamber containing preservatives and stacked in several rows. The samples were left for 3 x 24 hours to let the preservative penetrate the samples. After soaking period, the rattan samples were air dried in constant room the let the samples completely dried. The rattan samples (treated samples and control) were tested for resistance againts post-powder bettles and staining fungi. The rattan samples (treated samples and control) were put into chamber containing 20 post-powder beetles and left for 15 days. Observation was conducted daily. Insect mortality was determined. 7. Data Analysis Data obtained from laboratory and field test were tabulated, classified and analyzed statistically using appropriate computer software. Every experimental value were calculated from representative replication of experiments. Timetable of the research implementation is presented below.

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YEAR 1 YEAR 2 ACTIVITIES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Preparatory phase Rattan and plant sample collection Extraction of plant in laboratory Laboratory assays Isolation of the active substances Field test of rattan preservatives Trial of preservative treatment methods Standardization of the preservatives Data analysis Monitoring and evaluation Report writing and publication

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CHAPTER 4

RESULTS AND DISCUSSION 1. Plant extracts Plant species were selected based on our preliminary investigation on screening of Indonesian plants having antifungal activity. The plant collection was conducted in March 2007. Thirty-two plant samples, trees and herbs, were collected from the Laburan and Selo batu villages at Paser regency in East Kalimantan (Fig 3.), representing the availability of ethnobotanical information (Table 1).

Fig 6. Some of plant samples collected from field. Table 1. Plant samples collected for screening of antifungal activity

No Plants Family Part used Methanolic

extract (%)

1. Alpinia galanga Zingiberaceae Rhizome 6.42% 2. Annona muricata Annonaceae Leaf 12,49 3. Aporusa elmeri Euphorbiaceae Stem 8.57 4. Caesalpinia sappan Caesalpiniaceae Stem 10.31 5. Cassia alata Leguminosae Leaf 12.26 6. Dialium platysepalum Caesalpiniaceae Stem 7.78 7. Diospyros ebena Ebenaceae Stem 9.25 8. Dryobalanops aromatica Dipterocarpaceae Stem 10.40 9. Erythrina fusca Leguminosae Leaf 8.87 10. Ficus benjamina Moraceae Leaf 4.56 11. Gluta velutina Anacardiaceae Stem 6.98

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Table 1. Continued

No Plants Family Part used Methanolic extract (%)

12. Intsia palembanica Fabaceae Stem 8.60 13. Lansium domesticum Meliaceae Leaf 9.41 14. Lepisanthes arnoena Sapindaceae Leaf 5.99 15. Melaleuca cajuputi Myrtaceae Stem 3.64 16. Mimusop elengi Sapotaceae Leaf 15.80 17. Palaquium gutta Sapotaceae Stem 9.39 18. Pallava grantha Granthaceae Stem 8.47 19. Peronema canescens Verbenaceae Leaf 5.41 20. Pluchea indica Compositae Leaf 10.3 21. Pterocarpus indicus Leguminosae Stem 8.56 22. Schima walichii Theaceae Stem 6.93 23. Shorea leprosula Dipterocarpaceae Stem 10.31 24. Sindora velutina Caesalpiniaceae Stem 8.90 25. Swietenia mahagoni Meliaceae Stem 3.51 26. Tamarindus indica Linn. Leguminoceae Leaf 15.20 27. Tinospora crispa Menispermaceae Leaf 11.31 28. Tristania maingayi Myrtaceae Stem 7.41 29. Uncaria cordata Rubiaceae Leaf 8.72 30. Vitex pubescens Verbenaceae Stem 8.11 31. Xylocarpus granatum Meliaceae Stem 8.27 32. Zingiber purpureum Zingiberaceae Rhizome 2.78

All plant extracts were subjected to bioassays using several rattan-degrading fungi, i.e.: Schyzophyllum commune, Pleurotus pulmonarius, Aspergillus niger and Fusarium oxysporum. The former two fungi known decaying fungi of wood and other lignoselulosic materials, including rattan, while the latter belongs to molds and known as the causal agents of staining and colorization of rattan. Of the 32 plant samples tested, extracts of Pterocarpus indicus and Caesalpinia sappan displayed most potential activity against several rattan-degrading organisms (Table 2). This result informed that these two plant species can be expected as sources of natural preservatives to be applied in rattan preservation. Table 2. Antifungal activity of Indonesian Plant Methanolic Extracts

Antifungal activity (%)*No Plants Family Part used

AN FO SC PP 1. Annona muricata Annonaceae 2. Aporusa elmeri Euphorbiaceae Leaf + ++ + - 3. Averhoa belimbi Oxalidaceae Stem - + ++ - 4. Caesalpinia sappan Caesalpiniaceae Leaf +++ ++ +++ + 5. Cassia alata Leguminosae Stem ++ +++ + +

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Table 2. Continued

Antifungal activity (%)*No Plants Family Part used

AN FO SC PP 6. Dialium platysepalum Caesalpiniaceae Leaf - ++ + ++ 7. Diospyros ebena Ebenaceae Stem + ++ + - 8. Dryobalanops aromatica Dipterocarpaceae Stem - - + - 9. Erythrina fusca Leguminosae Leaf ++ + + ++ 10. Ficus benjamina Moraceae Stem - - ++ - 11. Gluta velutina Anacardiaceae Stem ++ ++ + + 12. Intsia palembanica Fabaceae Stem + + ++ + 13. Kompassia excelsa Leguminosae Leaf + +++ + - 14. Lepisanthes arnoena Sapindaceae Leaf - - - - 15. Melaleuca cajuputi Myrtaceae Leaf - - - + 16. Mimusop elengi Sapotaceae Stem ++ + ++ - 17. Palaquium gutta Sapotaceae Stem - + - + 18. Pallava grantha Granthaceae Leaf - ++ ++ - 19. Peronema canescens Verbenaceae Leaf - - - - 20. Pluchea indica Compositae Stem + + ++ - 21. Pterocarpus indicus Leguminosae Stem + +++ +++ ++ 22. Schima walichii Theaceae Stem + + + - 23. Shorea leprosula Dipterocarpaceae Stem - - + - 24. Sindora velutina Caesalpiniaceae Stem + - - + Stachytarpheta mutabilis Acanthaceae Leaf + + - + 25. Swietenia mahagoni Meliaceae Stem - - - - 26. Tamarindus indica Linn. Leguminoceae Leaf + ++ - + 27. Tinospora crispa Menispermaceae Leaf + + ++ - 28. Tristania maingayi Myrtaceae Stem - - + + 29. Vitex pubescens Verbenaceae Stem - ++ ++ + 30. Xylocarpus granatum Meliaceae Stem + ++ + ++ 31. Zingiber purpureum Zingiberaceae Rhizome ++ ++ + ++

*The extract concentration (dry matter) in the culture medium was 5 mg ml-1 in all cases. The inhibition was reported as (-) <10% growth inhibition, (+) between 10 and 30%, (++) between 30 and 60%, (+++) > 60%; AN=Aspergillus niger; FO=Fusarium oxysporum; SC=Schyzophyllum commune; PP=Pleurotus pulmonarius.

Fig 7. Antifungal activity of the P. indicus extract against a Fusarium oxysporum, a rattan ning fungus (left = control; right = plant extract). stai

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Fig 8. Antifungal activity of the plant extract against a rattan-degrading fungus, Pleurotus

monarius. pul

Fig 9. Antifungal activity of the plant extract against a rattan-degrading fungus, zophyllum commune. Schy

Based on the bioactivity assays, the most active plant extracs, Pterocarpus. indicus and Caesalpinia sappan were selected to be further investigated.

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Figure 10. Caesalpinia sappan, a promising plant as a source of rattan natural preservative.

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Figure 11. Pterocarpus indicus, a promising plant as a source of rattan natural

preservative. 2. Isolation and indentification of the active substances Each soluble fraction from P. indicus and C. sappan extract was subjected to laboratory assay to determine the bioactivity of plant extracts against several biological deterioration agents of rattan. In this step, activities against decay and colorization or moulds of rattan were evaluated. Bioassays against the test organisms showed that diethyl ether soluble of P. indicus and C. sappan possess the most activity. Woodmeal of P. indicus was extracted with methanol and the methanol solution obtained was evaporated with a rotary evaporator to give methanolic extracts. The methanolic extract was subjected to a bioassay-guided fractionation using n-hexane, diethyl ether, ethyl acetate and n-butanol, respectively, to give soluble fraction of the respective solvents. Antifungal assays against the test fungi informed that n-hexane and diethyl ether solubles possess higher activity than others Diethyl ether soluble fraction of P. indicus was applied to a silica gel column chromatography with gradient of chloroform-methanol as eluent to give six fractions. Two of them, fractions no 2 and no 3, showed most activity. Fraction 3 was further separated by silica gel column chromatography with elution of chloroform-methanol to give another 8 fractions. Among others, fraction 3.4 and 3.6 were more active in inhibiting the growth of test fungi than others. Further silica gel column chromatography of fraction 3.4 gave six more fractions, which then subjected to an antifungal assay. Fraction 3.4.6 showed more activity than others in inhibiting the fungal growth. Fraction 3.4.6 showed 2 major spots on thin-layer chromatography chromatogram. This fraction was then subjected to

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silica gel column chromatography with elution of dichloromethane-methanol to give an active compounds A, which was purified by recrystallization from aqueous ethanol. Compound A, C15H12O4 (M+=256), mp. 202-205oC, was made up of yellowish crystals. Based on this feature and the comparison with mass spectra of several class of chemicals, compound A was suggested to be a flavanone-related chalcone. Characteristic A1

+• (m/z=137) and BB1+• (m/z=120) ions were also observed in the

spectrum and the compound was suggested to be 2, 4, 4’-trihydroxychalcone, isoliquiritigenin. Acetylation with pyridine and acetic anhydride gave a crystalline triacetate, mp. 120-121 C. EI mass spectrum of the acetate showed a molecular ion peak at m/z 382, indicated the presence of three hydroxyl moieties in structure of A. In the H NMR spectrum of the acetate, signals for a para-disubstituted ring A, a 1, 2, 4-trisubstituted ring B, and an α, β-unsaturated double bond at 7.58 and 7.12 ppm were observed. Furthermore, signals for two acetoxy groups at 2.32 ppm and one acetoxy group at 2.24 ppm attributed to the substituents at A-4’, B-4 and B-2, respectively, were observed in the spectrum. C NMR spectrum was very informative in identifying the compound. These data were in good agreement with those for authentic isoliquiritigenin synthesized from resacetophenone and p-hydroxybenzaldehyde (Nadkarni and Wheeler 1938). The mixed-melting point of compound A and authentic isoliquiritigenin was undepressed. Therefore, compound A was identified as isoliquiritigenin with chemical structure as described in Fig 1.

o

1

13

OH

O

OH

HO

B

A

1

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4

5

6

2'3'

4'

5'6'β

α

Fig 12. Chemical Structure of Compound A from P. indicus. C. sappan wood was ground and thrice extracted with cold methanol. The methanolic extracts were evaporated and suspended in water, and then subsequently partitioned with n-hexane, diethyl ether and ethyl acetate respectively, to give the corresponding solubles. The soluble fractions were subjected to agar dilution antifungal assays against the test fungi to guide the fractionation and isolation of the active compound. Repeated column chromatography was conducted to give fractions that will subjected to bioassay. Diethyl ether soluble fraction of C. sappan showed the most activity in antifungal assays and, therefore, subjected to column chromatography separation. Column chromatography over silica gel with eluent chloroform-methanol dan benzene-acetone yielded several further fractions. After carefully repeated silica gel column chromatography guided by antifungal assays, an active compound, compound B was purified as a pale red crystal after recrystallized from chloroform-methanol. Based on the NMR spectroscopy data with method as previosuly described, compound B was identified as brazilein. The chemical structure of brazilein is presented in Fig 13.

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OHO

OH

OHO

Fig 13. Chemical structure of Brazilein (Compound B) from Caesalpinia sappan. Each compound was tested its antifungal activity against several rattan-degrading fungi, i.e.: Pleurotus pulmonarius, Schyzophyllum commune, Aspergillus niger and Fusarium oxysporum. Active compounds were prepared in several level concentrations (0.03-2,5 mg/disk) to determine the minimum inhibitory concentrations (MIC). The results showed that both active compounds (compounds A and B) possessed potent antifungal compound with relatively low MIC values. Tabel 3. MIC value of compound A (from P. indicus) and compound B (from C. sappan).

MIC (mg/disk) Samples P. pulmonarius S. commune A. niger F. oxysporum Compound A 0.07 0.07 0.60 0.15 Compound B 0.15 0.07 0.60 0.30

*Experiments were done in triplicates. Concentration levels were: 0.03, 0.07, 0.15, 0.30, 0,60, 1,3 and 2,5 mg/disk.

Fig 13. Antifungal activity of the the active compound from C. sappan against a rattan-coloring fungus, Aspergillus niger.

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Fig 14. Antifungal activity of the the active compound from C. sappan against a rattan-

staining, Fusarium oxysporum, at concentration 0.03-2.5 mg/dish.

3. Field test of rattan natural preservative The active fractions were subjected to a small-scale efficacy studies for uses as new plant-derived preservatives. Rattan samples were prepared in sticks of 2 cm length. The samples were sprayed with acetone solution containing several concentrations of the active subtances. Controls were sprayed with acetone only. After left for 24 hours, the samples were inoculated by test fungus by spraying with spore suspension of the Aspergillus, Fusarium and Penicillium spore. The inoculated samples were incubated for 2 weeks at room temperature in the dark. As a positive control, cypermethrin ( a commercial wood preservative) was used at 2% concentration. Clear zone of the treated sample sticks and controls was observed and rated daily on the scale of 0-5 (0=no growth appearance; 1 = <30% growth appearance; 2 = 30-50% growth appearance; 3=>50-70% growth appearance and 4 = >70% growth appearance). The result showed that addition of the active compounds (compound A from P. indicus and compound B from C. sappan) improved resistance of the treated sample against the test fungi comparing to control until the 1st week incubation with almost the same activity relative to positive control. However, for a longer time, the resistances were lower than

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the positive control since the growth of the test fungi was appeared after 1st week as can be seen in the Fig. 11. Compound 1 from P. indicus showed more activity than compound 2, however, less activity relative to positive control. Tabel 4. Antifungal Activity of Compound A (from P. indicus) and Compound B

(from C. sappan).

Antifungal activity (%) Compound A. niger F. oxysporum Penicillium sp A 1 0 1 B 2 1 1 Cypermethrin 1 0 0

Remarks: 0=no growth appearance; 1 = <30% growth appearance; 2 = 30-50% growth appearance; 3=>50-70% growth appearance and 4 = >70% growth appearance.

a b c d

Fig 15. Field test of natural products from P. indicus and C. sappan as a rattan preservative.

Remarks: a=control; b=sample treated with 2% cypermethrin; c=sample treated with 2% of compound 1 from P. indicus; d=sample treated with 2% of compound 2 from C. sappan.

4. Development of treatment method and standardization of rattan preservatives Cold soaking treatment for rattan preservation was tested. This is based on the economical and easy application for rattan farmer and industry to apply the preservative agents. The results showed that mortality of D. minutus as an important rattan-damaging agent was considerably high after the treatment of preservatives A (compound A), while the preservative B did not cause mortality of the beetles. This indicated that treatment of preservative B to rattan in several concentrations improved the resistance of the rattan against the post-powder beetles. Mortality of D. minutus is an indicator of attacking ability of the insect against rattan. The stronger of insect resistance, the higher attacking ability of the insect. In this research, three species of rattan samples tested showed relatively same resistance against post-powder beetles after preservative treatment. Soaking of rattan in preservative A in several concentration for 24 hours improved the

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resistance of rattan, significantly. The result showed that preservative B preserve the rattan by concentration-dependent activity. This means the higher concentration of the preservative, the higher resistance of the rattan. In soaking treatment for 24 hours, the lowest concentration of preservative A (0.1 %) caused 43% mortality effect, while the highest concentration of the preservative A (2%) caused 91% mortality effect. This data indicated high potential of preservative A to be applied as a rattan preservative agents to increase rattan resistance against post-powder beetles. Generally, preservative causing more that 70% insect mortality has good potential to be applied.

Fig 16. Insect deterrent assay of rattan treated by preservatives (a=control; b=treated by

compound B, c=treated by compound A; d=treated by cypermethrin, concentration=2%)

High mortality of D. minutus informed that compound A possessed high toxicity against this insect while compoud B was not toxic. Based on the concentration level applied, 2% of the natural preservatives can be expected to be safe for human and environment. Therefore, the most effective treatment of rattan preservative is by soaking the rattan samples in 2% preservative B (contains active compound B) for 24 hours. However, 1% of the preservative solution caused 82% mortality of the insect and can be expected will have more toxicity if the soaking time is prolonged. Tabel 5. Mortality of post-powder beetles on the treatment of preservative.

Mortality (%) Compound 0,1% 0,25% 0,5% 1% 2% A 43 51 65 82 91 B 20 33 40 47 53 Cypermethrin 68 82 100 100 100

Remarks: soaking time was 24 hours. Experimental procedures see chapter 3

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CHAPTER 5

CONCLUSION

Based on the extraction of the target plants and isolation of the active substances, high efficacy and environmentally-benign preservative agents, in vitro, were obtained. In the field experiment, the active compound displayed potent activity against rattan-degrading fungi and post-powder beetles. The research result open possibility to apply extract of Pterocarpus indicus and Caesalpinia sappan and their active compounds as natural rattan preservatives. Based on the characteristic of plant uses and purposes, the extract and active compounds offer more more safety application for human and animals, and more environmentally friendly than chemical synthetic preservatives because they are easily degradable in nature during disposal.

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CHAPTER 6

RECOMMENDATIONS Consideration for application of rattan preservative is not only on the basis of the efficacy of preservatives, but also the safety aspect of environmental concern. To improve potential application of the natural preservatives from P. indicus and C. sappan, more field test with wide–ranging degrading organisms should be done. To confirm the safety aspect, long-term application and effect of long-term exposure to preservative agent with active ingredients compound A or B should be done to confirm chronic and sub-chronic toxicity against human and animal.

29

CHAPTER 7

PUBLICATIONS AND PRESENTATIONS

Draft of manuscript to be submitted to Bioresource Technology Journal (Elsevier ltd., www.elsevier.com/locate/biortech) entitled: “Search for potential tropical plant extracts as natural preservatives” is in preparation.

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CHAPTER 8

REPORT ON EXPENDITURES

Budget headings

Total approved project budget

(in US$)

Total budget release (in US$)

Actual expenditure (in

US$)

Total Unexpended

Balance (US$) Project

personnel 0 0 0 0 Other labor 800 800 800 0

Administration consultant 200 200 200 0

DSA 800 800 800 0 Transportation 600 600 600 0 Capital items 400 400 400 0 Consumables 2000 2000 2000 0 Miscellaneous 700 700 700 0