Ntrop4mohamadsyakir-checked.docx

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

BIOACTIVITIES OF JATROPHA CURCAS LINN LATEX Mohamad Syakir M.S.1, Ismail J.1, Zaini A.2, Nur Diyana I.1 1

Department of Plant Science and Environmental Ecology 2 Department of Chemistry Faculty of Resource Science and Technology Universiti Malaysia Sarawak (MALAYSIA) e-mails: [email protected]

Abstract Jatropha curcas L. is a perennial tree under Euphorbiaceae family and well known for alternative source of biodiesel obtained from its seed. The study examined the latex of J. curcas crude that might be toxic to human and plant pathogen fungi namely Fusarium oxyporum, F. solani and Aspergillus niger; wood decay fungi which were Trametes versicolor and Gleophylum trabeum. The specific objectives of this study were firstly to determine the toxicity of crude powdered J.curcas latex, secondly to determine the chemical compositions of crude powdered J. curcas latex and thirdly to determine the chemical constituents of crude powdered J.curcas latex. Three different concentrations of J. curcas latex (100 mg/ml, 50mg/ml and 25 mg/ml) were prepared for in vitro bioassay test using agar well diffusion method. The wells were filled with concentration prepared and the growths of microorganisms were observed after 72 hours incubation period. The crude latex of J.curcas was subjected to composition and constituent analyses using Gas Chromatography – Mass Spectroscopy (GC/MS). In toxicity test, all tested fungi showed significant inhibition to all fungus tested except A. niger. The main compounds detected were dotriacontane (24%), pentatriacontane (20%), hexatriacontane (20%), 1,2-benzenedicarboxylic acid (41%) and -sitosterol (35%). Further study should be carried out to tap the potential of J. curcas latex as active ingredient in the pharmaceutical and pesticide production. Keywords: Jatropha curcas; pure powdered latex; toxicity; chemical composition; chemical constituent.

1

INTORDUCTION

Jatropha curcas Linn. belongs to the family Euphorbiaceae, known as ‘Jarak Pagar’ in Malay and widely known as physic nut. It is a drought resistant and perennial tropical plant that can be grown in low to high rainfall areas either in the farms as a commercial crop or on the boundaries as a hedge to protect fields from grazing animals and to prevent erosion (Irvine, 1961). It grows under a wide range of rainfall regimes from 250 to over 1200 mm per annum (Katwal & Soni, 2003). The extract of its leaves has antifungal properties (Garcia & Lawas, 1990). Fruit of J. curcas is highly toxic and may lead to death if consumed. According to Goonasekera et al. (1995), the fruit may cause pregnancyterminating in rat which benefit the pest management industry. However, the toxicity can be removed and utilized to many other purposes. The water extract of J. curcas branches showed inhibition on the HIV induced cytopathic effect with low cytotoxicity (Matsuse et al., 1999).

1.1

Jatropha curcas Latex

The latex of J. curcas obtained from its branches reported to contains an alkaloid known as jatrophine, which is believed to be having anti-cancerous properties (Henning, 2003). The healing effect of curcain a proteolytic enzyme from the latex on wound has been demonstrated (Nath & Dutta, 1991). The latex combined with the powdered leaves is applied to sluggish wounds while when formulated as enema it is used for the treatment of gonorrhoea (Irvine, 1961). The latex also used in healing of wounds, refractory ulcers, and septic gums and as a styptic in cuts and bruises. A proteolytic enzyme (curcain) has been reported to have wound healing activity in mice (Nath & Dutta, 1997). The latex is used to treat fungal infections in the mouth, bee and wasp stings and digestive problems of children in Mexico (Schmook & Serralta-Peraza, 1997). The chemical composition of J. curcas latex was reported to contain curcacycline A that has anti tumour properties (Van den Berg et al., 1995) and curcain, a protease (Nath & Dutta, 1991). Previous study on the latex using IR spectrum of ethyl acetate extract reported the presence of aromatic phenolic compounds and phenolic compounds which generally behave as acids that have high antimicrobial activity (Gisvold, 1977). In phytochemical screening

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

study done by Suhaili et al. (2011) on the crude latex extract revealed the presence of saponins and tannins as had previously been reported on other parts of the plant. Saponins and tannins, in particular, have been reported to possess antimicrobial activity (Zakaria et al., 2010)

2 2.1

METHODOLOGY Latex Collection

The fresh latex of J. curcas was collected from Jatropha orchard owned by Carbon Capital Coperation in Sadong Jaya, Kota Samarahan. Latex were collected and stored into clean sterilized bottle warped with aluminum foil to prevent phytochemical reaction. Sample collected were then directly brought into the laboratory and stored in 4°C.

2.2

Drying and Powdering of Latex

Latex was spread on sterilized glass Petri dish and kept in fume cupboard in dark condition for 24 hours. Dried latex were subsequently scrapped off carefully using sterilized glass slide and mashed to obtain finer powder. The powder was then sterilized using UV light inside laminar flow for 10 minutes.

2.3

Fungi Preparation

Trametes versicolor, Aspergillus niger, Fusarium oxysporum, F. solani, and Gloeophylum trabeum cultured on Malt Extract Agar (MEA) was used for culturing all the fungi. 15ml of MEA solution was poured into sterile disposable Petri dish and left cools until it solidified. Fungi were then inoculated from stock culture onto agar plate and were re-inoculated after one week to obtain pure culture of fungus.

2.4

Toxicity test

The agar well diffusion method adapted from Suhaili et al. (2011) and Oyi et al. (2007) was employed in this study with several modifications. Three well were made using cork borer (10 mm diameter) in 25 ml agar. Three different concentrations of J.curcas latex diluted using metanol (25 mg/ml, 50 mg/ml, 25 mg/ml) filled into well, each 30µl and left for 20 minutes. Plug of test fungi (± 5mm X 5mm) from cultured stock were next placed on the centre of solidified agar. Then it was incubated at 30 ºC and the data were recorded on 5th day after incubation. A plate with single well was prepared and filled with 30µl methanol for each fungi tested as a control. Antifungal activities were calculated by measuring the zone of inhibition or clear zone. Inhibition zones above 20 mm were classified as “strong”, 19 mm to 15 mm as “moderate” and below 14mm as “weak” activities.

2.5

Fractionation and Chemical Analysis

Column chromatography was employed to fractionate the pure powdered Jatropha curcas latex using 19 solvent systems with different ratio of n-hexane, dichloromethane, ethyl acetate and metanol. The fractions were then subjected to Gas Chromatography equiped with Mass Spectrometer. Identification of chemical components was made based on comparison with GC retention times of standard references materials. It was also assisted by comparing the mass spectra obtained during analysis with those mass spectra stored in the National Institute of Standard and Technology (NIST) standard library incorporated in the GC/MS data system.

3 3.1

RESULTS AND DISCUSSION Toxicity Test

The toxicity tests of crude powdered latex of J. curcas were carried out by using 100mg/ml, 50 mg/ml and 25 mg/ml concentrations diluted in methanol. From the test, it was observed that J.curcas latex has toxicity towards T. versicolor, G. trabeum, F. oxysporum, F. solani in all concentrations of crude powdered J.curcas latex applied. Fusarium solani was recorded to have the least inhibition for all concentrations of powdered J.curcas latex. Fig. 1 shows the example for the formation of inhibition zone against F. solani with different concentrations of crude J. curcas latex observed from toxicity test.

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

However, A. niger showed no significant effect of J.curcas latex on the growth of this fungus in any concentrations of J. curcas latex (Fig. 2). 100 mg/ml concentration showed strong inhibition with inhibition diameter more than 20 mm. Gleophylum trabeum was observed to strongly inhibited with 27 mm inhibition zone. F. Oxysporum and T. versicolor recorded 25.7 mm zone of inhibition. F. solani formed inhibition zone of 20 mm. For 50 mg/ml concentration, inhibition zone of T. versicolor was 22 mm, indicated strong inhibition while the lowest inhibition diameter was F. solani with 16 mm indicated moderate activity. Both F. oxysporum and G. trabeum showed inhibition zone of 21 mm. For 25 mg/ml concentration, inhibition zone was strong on both F. oxysporum and T. versicolor with 20 mm while less inhibition was observed on F. solani with 14 mm which indicated moderate activity. G. trabeum was observed to inhibited by 19 mm zone of inhibition which indicated moderate inhibition.

Figure 1: Formation of inhibition zone against Fusarium solani.

Figure 2: No inhibition zone showed against Aspergillus niger.

Table 1 summarize zone of inhibitions of J.curcas latex and the readings were taken as mean of three replicates. Fig. 3 visualized the inhibition classification of different types of fungi and its concentration and shows different fungi has different inhibition strength for each concentration. Pure latex of J.curcas has toxicity affect on several human and plant pathogen fungi. Study done by Schmook and Serralta-Peraza (1997) proved that J. curcas latex can be used to treat fungal infection Henning (2003) analyzed the presence of alkaloid in J. curcas called jatrophine and it’s believed to have anti dermatomucosal disease. Ambuse & Bhale (2012) reported J. curcas latex showed toxicity toward the growth of Fusarium proliferatum and F. pytium. It also been reported that the crude extracts of ethyl acetate and methanol from stem bark has no significant affect towards A. niger (Gupta et al., 2010).

Table 1: Inhibition zones of fungal species in agar well diffusion plate assay of powdered J. curcas latex after 5 days incubation Conc. of latex (mg/ml) FS

Fungi / Zone of inhibition (mm) FO TV GT

AN

100

20 ± 2.3

25.7 ± 0.3

25.7 ± 0.8

27 ± 1.5

-

50

16.3 ± 1.2

21.7 ± 0.3

22 ± 1.2

21.7 ± 0.9

-

25

14 ± 1.2

20 ± 0.6

20 ± 2.3

19 ± 1.7

-

Methanol

-

-

-

-

-

FS = Fusarium solani; FO = Fusarium oxysporum; TV = Trametes versicolor; GT = Gleophylum trabeum; AN = Aspergillus niger; - = No inhibition

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

Fig3: Inhibition classification of three concentrations of J. curcas latex against tested fungi

3.2

Chemical Constituents and Compound Analysis

The GC/MS analyses revealed that the most abundantce compounds in hexane and methanol fractions were dotriacontane and 1,2-benzenedicarboxylic acid.

3.2.1

n-hexane Fraction

Major chemical constituents of hexane fraction of J.curcas latex was dotriacontane with percentage of composition 24% (Fig 4). Other significant compounds were pentatriacontane (20%), hexatriacontane (19%), tetracosane (19%), tetratricontane (10%) and nonacosane (5%) (Table 2). The chemical constituents in hexane fraction contain nonacosane was reported to responsible in the inhibition of several types of bacterial formation (Yayli et al, 2006). Nonacosane is known as a plant compound that responsible in formation many of waxy layer of long chain paraffin (Hankin & Kolattukudy, 1968).

Fig 4: Gas chromatogram traced by GC/MS for n-hexane fraction of powdered J.curcas latex Tetratriacontane was also reported in the extract of Dictyopteris membracea and showed significants for antimicrobial activities (Ozdemir et al, 2006). Dotriacontane was reported to show toxicity affect on the microbe tested. (Amin & Sleem, 2007). This compound was also found in extract of Sideritis scardica and it showed antimicrobial properties (Vanja et al., 2012). Hexatriacontane is classified as flavanoid and has very high medicinal value (Vaishali et al., 2009). Yassa et al. (2009) reported

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

Hexatriacontane can be used as active preventing agent for many diseases and also has antioxidant affects. Tetracosane was also reported in flower extract of Allium atroviolaceum and showed positive antibacterial activity (Dehpour et al., 2011). It also found in the essential oil of Geranium columbinum that has toxicity affects (Radulovic et al., 2011) Table 2: Major chemical constituents identified in n- hexane fraction of pure latex of J.curcas

3.2.2

Methanol Fraction

Methanol fraction is assumed to consist of intermediate polar compounds such as fatty acids and most abundance chemical identified was mainly classified in alcohol group. Figure 5 shows presence of 1,2benzenedicarboxylic acid (40.96%), ß-Sitosterol (35.06%), 2-Hexyl-1-decanol (9.30%) and 4,6Cholestadien-3.beta.-ol (4.72%) (Table 3). 2-hexyl-decanol was reported to has antibacterial activities (Najiah et al,. 2008) and also found in Thai honey bee which has antibiotic affect for many human diseases (Suwannapong et al., 2011). 1,2benzenedicarboxylic acid is a plasticizer compound reported to have high toxicity and antimicrobial effect against Listeria (Hsouna, 2011) and reported as the active phytochemical compound (Chen et al., 2011). Compound 4,6-Cholestadien-3.beta.-ol was found in extract of Azadiracta indica which had showed antibacterial activity (Moorthy & Boominathan, 2011).

Fig 5: Gas chromatogram traced by GC/MS for methanol fraction of powdered J.curcas latex

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

Compound ß-Sitosterol was also been found in the root and kernel meal of J.curcas in phenolics and flavonoids analyses by HPLC analysis (Oskoueian et al., 2011). The compound was also found in the leaves of J. curcas. ß-Sitosterol has no antifungal effect (Hegazy et al, 2010), but it had significant effect on bacteria (Gohari et al., 2009) Table 3: Major chemical constituents identified in methanol fraction of pure latex of J.curcas

4

CONCLUSION

The toxicity test showed J. curcas latex inhibited Trametes versicolor, Gleophyllum trabeum, Fusarium oxysporum, Fusarium solani, but negative to Aspergillus niger indicate the powdered latex has limited inhibition affect different type of organisms.The main compounds detected include dotriacontane, pentatriacontane, hexatriacontane, 1,2-benzenedicarboxylic acid and -sitosterol. Extensive research need to be done with various concentrations of Jatropha curcas latex and organisms. Extraction of pure compounds to obtain significant active ingredient and finding which inert ingredients have the potential to be amplified for chemical activation, will lead to the diversification of J. curcas utilization, and hence benefit industries related to Jatropha.

REFERENCES Ambuse, M. G., & Bhale, U. N. (2012). Evaluation of antifungal activity of plant latex extracts against resistant isolates of pathogens associated on Rumex Acetosa L. International Journal Of Ayurvedic And Herbal Medicine, (2) 2:389-393 Amin, W. M. A., & Sleem, A. A. (2007). Chemical and biological study of aerial parts of Anethum graveolens L. Egypt Journal of Biomedical Science, (23) 13: 188-190 Bruce, A. & Highley, T. L. (1991). Control of growth of wood decay Basidiomycetes by Trichoderma spp. and other potentially antagonistic fungi. Forest Product Journal, 41: 6367. Chen, H., Yang, Y., Xue, J., Wei, J., Zhang, Z., & Chen H. (2011). Comparison of compositions and antimicrobial activities of essential oils from chemically stimulated agarwood, wild agarwood and healthy Aquilaria sinensis (Lour.) Gilg trees. Molecules Journal. (16): 4884-4896 Dehpour, A. A., Babakhani, B., Khazaei, S., & Asadi, M. (2011). Chemical composition of essential oil and antibacterial activity of extracts from flower of Allium atroviolaceum. Journal of Medicinal Plants Research, (5) 16: 3667-3672 Garcia, R.P., & Lawas, P. (1990). Note: Potential plant extracts for the control of Azolla fungal pathogens. Philippine Agriculture, (73) 4: 343-348 Gisvold, O. (1977). Phenols and their derivatives. In: Textbook of organic Medicinal and Pharmaceutical Chemistry 7th Edition by Wilson, C. O., Gisvold, O & George, R. F. (1977). Philadephia.: J.B Lippincott company, pp. 182-184.

4th Regional Conference on Natural Resources in the Tropics (NTrop4), 2012

Gohari, A. R., Saeidnia, S., Shaverdia, A. R., Yassa, N., Malmir, M., Mollazade, K., & Naghinejad, A. R. (2009). Phytochemistry and antimicrobial compounds of Hymenocrater calycinus. EurAsia Journal of BioScience, (3): 64-68 Goonasekera, M. M., Gunawardana, V. K., Jayasena, K., Mohammed, S. G., & Balsubramaniam, S. (1995). Pregnancy terminating effect of Jatropha curcas in rats. Journal of Ethnopharmacology, (47) 3:117–123 Gupta, D. D., Haque, M. E., Islam, M. N., Mondal, M. S. I., & Shibib, B. A. (2010) Antimicrobial and cytotoxic activities of Jatropha curcas (Euphorbiacea). Dhaka Universities Journal of Pharmaceutical Sciences, (9) 2: 139-142 Hankin, L., & Kolattukudy, P. E. (1968). Metabolism of a Plant Wax Paraffin (n-Nonacosane) by a Soil Bacterium (Micrococcus cerificans). Microbiology, 51: 457 Hegazy, A. K., Sayed, A. M., & Kabiel, H. F. (2010). Study on combined antimicrobial activity of some biologically active constituent from wild Moringa peregrine Forssk. Journal of yeast and fungal research, (1) 1:15-24 Henning, R.K. (2003). A Guide to Jatropha Promotion in Africa. Weissensberg, Germany: bagani GbR. Hsouna, A. B., Trigui, M., Mansour, R. B., Jarraya, R. M., Damak, M., & Jaoua, S. (2011). Chemical composition, cytotoxicity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat. International Journal of Food Microbiology, (148) 1: 66-72 Irvine, F. R. (1961). Woody Plants of Ghana 1st Edition. London: Oxford University Press. Katwal, R. P. S., Soni, P. L. (2003). Biofuels: an opportunity for socioeconomic development and cleaner environment. Indian Forester, (129): 939–949. Matsuse, I. T., Lim, Y. A., Hattori, M., Correa, M., & Gupta, M.P. (1999). Search of anti-viral properties in Panamanian medicinal plants, the effects on HIV and its essential enzymes. Journal of Ethnopharmacology, (64): 15-22. Moorthy, V., & Boominathan, M. (2011). The antimicrobial activities of crude extracts and fraction of Psidium guajava and Azadirachta indica against Staphylococcus aureus in chronic disease affected patients. International Journal of Universal Pharmacy and Life Sciences, (1) 2: 160173 Najiah, M., Wei, L. S., Seng, C. T., Wee, W., & Leong L. K. (2008). Potential of edible plants as remedies of systemic bacterial disease infection. Cultured Fish Global Journal of Pharmacology 2, (2): 31-36 Nath, L. K., & Dutta, S. K., (1992). Extraction and purification of curcain, a protease from the latex of Jatropha curcas L. J. Pharm. Pharmacology, (43) 111–114. Oskoueian, E., Norhani Abdullah, Syahida Ahmad, Wan Zuhainis Saad, Abdul Rahman Omar, & Ho, Y. W. (2011). Bioactive compounds and biological activities of Jatropha curcas L. kernel meal extract. International Journal of Molecular Science, (12): 5955-5970 Oyi, A. R. Onaolapo, J.A & Adigun, J. O. (2002). Phytochemical and antimicrobial Screening of the latex of Jatropha curcas Linn (Euphorbiaceae). Journal of phytomedicine and Therapeutical (1&2), 63-74. Ozdemir, G., Horzum, Z., Atakan, S., & Ulku, K. Y. (2006) Antimicrobial activities of volatile and various component and extracts of Dictyopteris membranacea and Cystoseira barbata from the coast of Izmir, Turkey. Turkish Journal of Chemistry, (3) 44: 183-188 Radulovic, N., Deki, M., Stojanovi, Z. R., & Pali’c, R. (2011). Chemical composition and antimicrobial activity of the essential oils of Geranium columbinum L. and G. lucidum L. (Geraniaceae). Turkish Journal of Chemistry, (35): 499-512 Schmook, B., Serralta, P. L. (1997) Jatropha curcas: distribution and uses in the Yucantan Penisula. Proceeding of First International Symposium on Biofuel and Industrial Product from Jatropha curcas and other Tropical Oil Seed Plants, Managua, Nicaragua.

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Suhaili, Z., Yeo, C. C., Yasin, H. N., Badaludin, N. A., & Zakaria, Z. A. (2011). Antibacterial profile of Jatropha curcas latex extracts against selected human pathogenic bacteria. African Journal of Microbiology Research, (5) 29:5147-5154 Suwannapong, G., Benbow, M. E., & Nieh, J. C. (2011). Biology of Thai honeybees: natural history and threats. Thailand: Nova Science. pp. 1-98. Vaishali, A., Khatiwora, E., Manik, K., Tambe, A., Pawar, P. & Deshpande, N. (2009). GC-MS study of fatty acid, ester and alcohol from leaves of Ipomea carnea. International Journal of Pharmaceutical Technology Reseach, (1) 4: 1224-1226 Van den Berg, A. J., Horsten, S. F., Kettenes van den Bosch, J. J., Kroes, B. H., Beukelman, C. J., Loeflang, B. R., & Labadie, R. P., (1995). Curcacycline A: a novel cyclic octapeptide isolated from the Jatropha curcas. FEBS Letters, (358): 215–218. Vanja, T., Dragica, B., Ivana, A., Sofija, D., & Ksenija, A. (2012). Chemical and antimicrobial evaluation of supercritical and conventional Sideritis scardica L. extract. Molecules Journal, 17: 2683-2703 Yassa, N., Masoomi, F., Rohani, R. S. E., & Hadjiakhoondi, A. (2009). Chemical composition and antioxidant activity of the extract and essential oil of Rosa damascena from Iran, population of Guilan. DARU Journal of Pharmaceutical Sciences, (17) 3: 175-179 Yayli, N., Conan, G. Osman, U. C., Ahmet, Y., Ulker, S., Kamil, C., & Salih, T. (2006). Composition and Antimicrobial Activities of Volatile Components of Minuartia meyeri. Turkish Journal of Chemistry, 3: 71-76 Zakaria, Z. A., Sufian, A. S., Ramasamy, K., Ahmat, N., Sulaiman, M. R., Arifah, A. K., Zuraini, A., Somchit, M. N. (2010). In vitro antimicrobial activity of Muntingia calabura extracts and fractions. African Journal of Microbiology Research, (4) 4: 304-308.