FUNGITOXIC PRINCIPLES OF EXTRACTS OF CARICA PAPAYA ROOTS AND SEEDS AND PIPER GUINEENSE SEEDS.

D.N. Enyiukwu, A.N. Awurum *

* Department of Plant Health Management, Michael Okpara University of Agriculture, Umudike P.M.B 7267, Umuahia, Abia State, Nigeria

KEYWORDS Phytochemicals, fungitoxic activity, Carica papaya, Plant Diseases.

ABSTRACT Phytochemical screening of Carica papaya roots and seeds and Piper gunieense seeds extract revealed the presence of alkaloids, flavonoids, tannins, saponins, phenols as well as glycosides, terpenoids and steroids in the plant materials. Quantitative tests showed that alkaloids ranged between (0.16 1.63%), saponins (0.68 2.64%), flavonoids (0.34 1.11%), tannins (0.22 0.83%) and phenols (0.5 0.66%). Glycosides were absent in Piper gunieense seeds while seeds extract of Carica papaya were absent in both terpenoids and steroids. The significance of these bioactive compounds were evaluated and discussed with respect to the fungitoxic roles these plant extracts play in fungal disease control of agricultural crops.

INTRODUCTION Though chemical control of plant diseases contributes to yield increases in agricultural production, it has been reported to have several drawbacks. Exposure of target pests to repeated application of synthetic fungicides lead to the development of resistance in the organisms. For instance FAO reported that 150 pathogens of crop plants are resistant to synthetic fungicides (Par and Rajul, 1994) while Colletotrichum spp. have been reported resistant or tolerant to benomyl, carbendazim and thiophanate-methyl (Tu, 1981; Emechebe and Florini, 1997). Synthetic chemicals can cause death through poisoning, accumulate in the organism or concentrate in the food chain (Taiga and Olufolaji, 2008). There is need therefore for more selective, pathogen specific, eco-compliant and plant-safe alternatives. One such alternative is plant extracts. In Nigeria, many plant extracts have been used successfully to control plant diseases in recent times. For example Cymbopogon citratus and Uvaria chamae checked the development and spread of anthracnose of cowpea induced by Colletotrichum destructivum (Amadioha, 2001; Awurum et al., 2005). Numerous Plant Constituents exhibit pesticidal activity and have potentials for use in the development of natural fungicides (Okwu et al., 2007). To this end therefore efforts have been directed at screening plants for phytochemicals with the intent to replacing the existing multi-site synthetic ones associated with such drawbacks as mammalian toxicity, pest resistance, biological magnifications in the food chain and cost ineffectiveness among others (Awurum et al., 2005; Okwu et al., 2007). Carica papaya (Family Caricaceae) is commonly known as pawpaw or melon tree. It is a short-lived, fast growing woody large herb, native to Southern Mexico and Central America, though now, it is tropical and subtropical in distribution. The flowers five-petalled, fleshy and waxy develop into pear shaped or cylindrical fruits containing the seeds. The seeds when fresh have pungent peppery taste due to aromatic compounds

(Morton, 1987; Motherherbs, 2010). Carica papaya is used as fungicides. Antifungal effects of the seeds extract of Carica papaya on mycelial reduction of the fungal pathogen Phytophtora palmivora causing black pod diseases of cocoa has been investigated. The aqueous extract at 30 40% w/v was found superior to ridomil (metalaxyl) (Wokocha and Nwaogu, 2008). Carica papaya seed is very effective against many human pathogens such as Bacillus subtilis, Escherichia coli , Salmonella typhii, Staphylococcus aureus, Enterobacter clocae and Proteus vulgaris (Nwinyi and Abikoye, 2010). In tropical folk-medicine, not only is the plant used for antimicrobial purposes, it also serves as anthemintics, febrifuge, aphrodisiac and in treatment of jaundice and tumours (Motherherbs, 2010; Njoku and Obi, 2009). Piper guineense (Black pepper, Ashanti pepper, Uziza) a member of the Family Piperaceae is a climbing vine that grows up to 20m in length. The plant is native to West and Central Africa (Wikipedia, 2008). The flowers are unicellular or hermaphrodite with the resulting sessile drupe fruits 4-6mm in diameter. The mesocarps are pulpy and the pericarps red when ripe turn black on drying. The seeds 3-4mm in diameter weigh between 30 80mg (Purseglove, 1976). The seeds contain piperine, myristicine, elemecine, safrole and dillapiol as bioactive secondary compounds. These make P. gunieese useable in folk-medicine as well as spice in African cuisines (Adjaye Gbewonyo, 2010; Wikipedia, 2010) and for drink and perfume making. Many workers have reported the insecticidal properties of P gunieese on Sitophylus zeamais of maize, Maruca and Clavigralla Spp. of cowpea (Asawalam and Emosairue, 2006; Okparaeke, 2007); however its fungicidal attritutes against seed-borne fungi of cowpea has been demonstrated (Enyiukwu, 2002). In spite of the various uses of C. papaya and P. guineeense seeds in folk-medicine in Nigeria and as spices in native cuisines and aromatic drinks, the phytochemical constituents of these plants have not been fully documeted. This present study was undertaken to determine and quantify the phytoconstituents conferring fungitoxic attributes to these plants.

MATERIALS AND METHODS Plant Materials The experiments were conducted in the department of chemistry laboratory of Michael Okpara University of Agriculture, Umudike, Nigeria in July, 2010. Fresh roots and seeds of Carica papaya as well as seeds of Piper gunineense were collected from the University neigbourhood, Umudike in Ikwuano Local Government Area of Abia State, Nigeria.

Preparation of plant Material The plant materials were washed thoroughly in tap water, rinsed in three changes of sterile distilled water and then air-dried on the laboratory bench for 10 days. They were milled into powder using a hand milling machine (Model: Corona Lavesh 250). The powdered materials (200g for each sample) were stored until required for chemical analysis in air-tight bottles.

Phytochemical Analyses Chemical screenings were carried out on the aqueous extracts of the plant materials to identify the phytochemical constituents. For alkaloids (Mayers reagent test), saponins (Frothing test), flavonoids (Ammonia-sulphuric acid tests), glycosides (Keller-killiani test), tannins and phenols (Ferric salt tests),

steroids Chloroform-suphuric acid test) and terpenoids (salkowski tests) using standard procedures as described by Sofowora (1993), Trease and Evans (1989) and Harborne (1973). The determination of tamins were done according to the Folin-Denis spectrophotometric method described by Pearson (1976), alkaloids and flavonoids by gravimetric methods described by Harborne (1973), phenols by spectrophotometric method of A.O.A.C. (2000) and saponins according to the method of Obani and Ochuko (2001)

Statistical Analysis All data are means of triplicate determinations and were analysed using analysis of variance (ANOVA) as contained in the general linear model procedure in SAS System (2008) version. Means were separated and compared using Fishers LSD at 0.05 level of probability.

RESULTS The phytochemical profile of the assayed plant materials are shown in Table 1. The results indicate the presence of alkaloids, flavonoids, tannins, saponins, phenols as well as glycosides, terpenoids and steroids. While glycosides are absent in P. gunieense extracts, steroids and terpenoids were absent in C. papaya seed extracts.

Table 1: Phytochemical constituents of extracts of selected plant materials Extracts of Carica papaya Seeds Carica papa ++ ++ + ++ + + ++ +++ + + ++ ++ ++ ++

Phytoconstituents Alkaloids Flaronoids Tannins Saponins Phenols Terpenoids Glycosides Steroids

+ ++ +++ = = = =

Piper guineense Seeds +++ +++ +++ +++ ++ +++ +++

Constituents absent Constituent Slightly present Constituents moderately present Constituents highly present

The quantitative phytochemical content of the plant materials are shown in Table 2. The alkaloids content of Piper guineense seeds was high (1.64%), followed by C. papaya root (0.75%) while the seeds of C papaya contained 0.62% of alkaloids. The flavonoids content of P. guineense seeds was 1.20% and this was followed by 0.57% contained in C. papaya root and the seeds of C papaya were 0.34% in content of flavonoids. The trend was similar for all the other phytochemicals namely tannins, saponins and phenols.

Table 2: comparison of the yield of the same phytochemical b y the different plant materials. Plant material Alkaloids 0.623 0.753 1.637 0.083 Phytochemicals (%) Flavonoids 0.343 0.573 1.203 0.257 Tannins 0.223 0.340 0.833 0.137 Saponins 0.687 1.407 2.640 0.257 Phenols 0.083 0.053 0.660 0.122

Carica papaya seed Carica papaya root Piper guineense seed LSD

* Values are means of tripicate determination on dry matter basis

DISCUSSION The phytochemical profile in general of the plant materials revealed that the phytoconstitutents were strongly present in P. gunieense seeds, moderately present in C. papaya roots and slightly present in C. papaya seeds. The alkaloid content of P. guineense seeds was high (1.64%) and significantly (P 0.05) differed from the alkaloids content of C. papaya roots and seeds (0.75% and 0.62%) respectively and same goes for flavonoids, tannins, saponins and phenols. The low alkaloids content of P. guineense seeds against 5 8% reported by Purseglove (1976) may have been occasioned by factors such as method of extraction, extracting solvent or time of harvesting of the plant materials (Amadioha, 2001). The presence of these phytoconstituents indicate that the plants have pesticidals effects (Okwu and Ekeke, 2003). The antimicrobial activities of natural products and their effectiveness on the suppression of plant diseases have been reported (Wokocha, 1998). Leaf extracts of Piper nigrum and P. guineense strongly inhibited the phyto-fungal pathogens Rhizoctonia solani and Colletotrichum lindemuthianum in vitro and in the field while seed extracts of C. papaya were superior to ridomil (metalaxyl) in inhibition effects on phytophtora palmivora (Amadiola, 2001, 2003; Wokocha and Nwaogu, 2008). The antifungal effects P. guineense seed extract as seed dressing chemicals against Colletotrichum spp. Fusarium, Curvularia, Mucor and Aspergillus spp. have been demonstrated (Enyiukwu, 2002). Alkaloids posses antifungal bacterial properties (Okwu And Njoku, 2009). They function by blocking ion channels, interfere with neurotransmission causing loss of coordination in affected organism as well as inactivate enzymes (Anonymous, 2007; karlovsky, 2008). Piperine and piperidine are major alkaloids in P. gunieense seeds while carpaine and pseudocarpaine are contained in C. papaya. These alkaloids may be responsible for the fingicidal effects noted by the earlier investigators. Flavonoids are commonly antioxidants and play roles in disease resistance (Okwu and Ekeke, 2003). Isoflavonoids have high pesticidal activity brought about by electron transport blockage in the mitochondria and inhibiting oxidation linked to NADH2 (Friedli, 2008). The fungitoxic effects of these plant materials against phyto-pathogenic fungi may be due to flavonoids. Tannins comprise of hydrolysable or condensed tannins and exhibit toxic activity on plant pathogen (Okwu and Iroabuchi, 2000). Their mechanism of action lie in their affinity for precipitating proteins and complexing with all kinds of biochemicals (Peru, 2001; Dharmananda, 2007). This may inform their pesticidal action by interfering with enzyme activity during germination of pathogen spores and mycelial elongation resulting in the inhibitions reported by several researchers.

Saponins have soap-like nature and are used as adjuvants and surfactants for vaccines to enhance macromolecule penetration (Wikipedia, 2008). They play a role in cytolysis by complexing with cell membrane bilayers. Hence, saponins can ward off microbes including fungal and viral pathogens (Okwu and Njoku, 2009). Saponins may be responsible for the inhibition of the plant pathogenic fungi reported by various workers due to these plant materials. Phenols are aromatic alcohols (De Ruiter, 2005). They are used in the manufacture of antiseptics, bactericides, fungicides and additives to inhibit microbial growth in various range of pesticides (Greener Industry, 2009) Karlovsky (2008) noted that phenolics act by slowing growth, blocking cell division and enzyme activity. They may even impair enzymes involved in energy production of the pathogens (Okwu, and Njoku, 2009) causing swelling of hyphae tips, plasma seeping and leaking around hyphae or fusion of hyphae tips (Okwu et al., 2007). The inhibition of spores and mycelial elongation of the pathogen C. lindemuthianum, R. solani and P. palmivora and in vitro disease expressions noted by previous workers from these plant materials may be due to phenols. Terpenes are responsible for the biological activity attributed some plant extracts. Pinene, limonene and phellandruene are present in P. guineense seeds. The mode of activity of monoterpernoids are genotoxic, to interfere with GABA receptors, interact with cytoplasmic membranes, disrupt the structure of polysaccharides, fatty acids and phospholipids, resulting in leakage of radicals, calcium ions and proteins (Eschverrigaray et al., 2010). The mycelial elongation and fungitoxic activity of the extracts demonstrated by the previous researchers may be due to terpenes in the essential oil of P. guineense seeds and C. papaya roots. Phytosterols acts as structural components in cell membranes. Sterols provide protection to plants during drought stress and chilling (Wikipedia, 2008) and may be responsible for the antifungal activity of the plant materials. Glycosides act by interfering with respiration of the organism and have been noted to be strong antifungals. Benzylisothiocyanate (BITC), a glycoside isolated from the seeds of C. papaya has been shown to be fungicidal against Alternaria alternata pathogenic to tomato, at 0.5mg/ml (Reulas et al, 2003). Hence the superior fungicidal action of seeds extract of C. papaya against P. pelmivora reported by Wokocha and Nwaogu (2008) may be due to this phytochemical. The main and highly effective defence found in plants is the use of marvelous assortment of chemicals providing protection against microbial attack (Wessells and Hopson, 1988; Harborne, 2008). They are effect-specific on pathogens, causing alteration in digestion (Wessells and hopson, 1988, harborne, 2008). Extracts of C Papaya roots and seeds and P. guineense seeds have been shown to have fungicidal effects on a wide range of crop pathogen fungi including Rhizotonia solani, Collectotrichum lindemuthianum, P. palmivora, Curvularia spp., Mucor sp and Collecttrichum spp (Amadioha, 2001; 2003; Enyiukwu, 2002; Wokocha and nwaogu, 2008). This study affirms that extracts of these plant materials contain bioactive constituents which have antimicrobial activity and lend strong possibility for development of alternatives fungicides from them. The prospects of using these plant materials (C. papaya roots and seeds and P. guineense seeds) for developing natural pesticides are inviting, because these plantrs are readily available in the locality, crop and environment-safe, non mammalotoxic, less hazardous to non target species and cost friendly (Opara and Wokocha, 2008; Opara and Obana, 2010). In the light of the foregoing therefore, C. papaya roots and seeds extract and P guineense seeds extracts are possible alternatives to multi-site synthetic pesticides for control of fungal disease.

REFERENCES 1. A.O.A.C (Association of Official Analytical Chemists) (2000) Official methods of analysis international (17th Ed). Washington DC USA. Adjaye Gbewonyo, D Quaye E.C and Wubah D.A (2010). The Effects of extract of Piper guineense seeds on insect pests damage to cowpea plants. Journal of Young Investigators 20(1): 1150 1156 Amadioha, A.C. (2001). Fungicidal activity of some plant extract against Rhizoctonia solani in cowpea. Acta Phytopathologica pflanz 33: 509- 577. Amadioha, A.C (2003). Evaluation of some plant extracts against Colletotrichum lindemuthiamum in cowpea. Acta Phytopathologica et Entomologica Hungarica 38 (3-4): 259 265 Awurum, A.N., Okwu D.E and Onyeokoro, P.A (2005). In vitro and in vivo control of Colletotrichum destructivum, in cowpea using some plant extracts. Journal of Sci., Tech and Envt (1 & 2): 52 57 Anonymous (2007). Plant metabolites: Sources and effects Retrieved August 8, 2007 accessmylibrary. Asawal;am, E.F and Emosairue, S.O (2006). Comparative efficacy of Piper guineense and pirimophis methyl as poison against Sitophylus zeamais. Electronic Journal of Env. Agric and Food Chemistry online 1536 1545 Dharmananda, S (2007). Gallnuts and the uses of tannins in Chinese medicine. Retrieved April 14, 2007,. Http://www.itnonline.org/arts/gallunts.htm De Ruiter, J (2005). Alcohol structure and chemistry. In: principles of drug action 1 Spring, 2005

2.

3.

4.

5.

6.

7.

8.

9.

10. Echeverrigaray, S., Zacaria J. and Beltrao, R. (2010). Nematicidal activity of monoterpenoid against the root knot nematode Meloidogyne incognita American phytopathology 100 (2): 199 203 11. Emechebe, A.M. and Florini, D.A. (1997). Fungal and Bacterial Diseases of Cowpea. In: Adavances in Cowpea Research. Singh R.S, Morgen Raji, Daswell, K.E, and Jackai, LEN, (Eds) IITA. Pp. 176 183 12. Enyiukwu, D.N (2002). Effects of some plant extracts on germination of cowpea (Vigna unguiculata (L.) Walp ) seeds and incidence of the seed-borne fungi. An unpublished Thesis submitted to the Department of Plant Health Mgt., Michael Okpara University of Agriculture, Umudike, Nigeria.

13. Friedli (2008), Flavonoids. Retrieved http://www.friedli.com/herb.phytochem/flavanoids.html

21st

November,

2008

14. Gneener Industry (2009). Phenol Uses. Retrieved September http://www.gneenerindustry.org/pages/phenols/2phenoluses.htm

4,

2009

from

15. Harborne, J.B (1973). Phytochemical methods. Chapman and Hall, London Pp. 49 188. 16. Karlovsky, P (2008). Secondary metabolites in soil ecology. soil biology, Springer-Verlag Berlin. 17. Morton, J (1987). Papaya. In: fruits of Warm Climates. Manure Florida. Pp 336 346 18. Mortherherbs (2010). Carica Papaya http://www.motehrherbs.com.carica-papaya.html 19. Njoku, V.O and Obi C (2009). phytoChemical constituents of soma selected medicinal plants. African Journal of Pure and Applied Chemistry 3(11) :228 233. 20. Nwinyi, O.C and Abikoye, B.A (2010). Antifungal effects of pawpaw seed extracts and papain on postharvest Carica papaya L. African Journal of Agric Res. 5(12): 1531 1535. 21. Obadoni B.O. and Ochuko, P.O (2001). Phytochemical studies and comparative efficiency of the crude extracts of some Homeostatic plants in Edo and Delta States of Nigeria. Global Journal of Pure and Applied Sci 8:203-208 22. Okwu, D.E and Okeke, O (2003) Phytochemical screening and antimicrobial composition of chewing sticks in South Eastern Nigeria, Global Journal of pure and Applied Sci 9(7): 235 238 23. Okwu, D.E and Iroabuchi, F (2004). Phytochemical analysis and antimicrobial activity screening of aqueous and ethanolic root extracts of Uvaria chamae BEAV and C Ferriginea D.C Journal of Chem Soc. Nig. 29(2): 112 114 24. Okwu, D.E and Njoku, E.E (2009). Chemical composition and In vitro antifungal activity screening of seed and leave extracts from Afromomum melenguata and Monodora myristica against Sclerotium rolfsii of cowpea plant. Pest Technology 3(1): 58 67. 25. Okwu, D.E. Awurum, A,N and Okoronkwo, I. J (2007) Phytochemical composition and In virto antifungal activity screening of extracts from Citrus plant against Fusarium Oxysporium of Okra plant. Pest Technology 1:145 148 26. Opera, E.U and Obani , F.T (2010). Performance of some plant extracts and pesticides in the control of Bacterial spot disease of Solanum. Medwel Agric journal 5(2):45 49 27. Opara, E.U and Wokocha, R.C (2008) Efficiency of some plant extracts on the in vitro and in vivo control of Xanthomonas campestris pv vesicatoria, Mediwell Agric journal 3(3):163 170

28. Olufolaji, D.B. (1999). Control of wet rot of Amaranthus sp. Caused by Choanephora cucurbitarium with extracts of Azadirachta indica. Journal of Sust. Agric and Envt. 1(2). 183 190. 29. Par, Pinstrup-Anderson and Rajul, panda-lorch (1994): Alleviating poverty, intensifying agriculture and effectively managing natural resources A Food, Agriculture and Environment discussion paper Washington DC 20:13 14. 30. Pearson. D (1976). The Chemical Analysis of Foods Churchhill Livingstone, Edinburgh. Pp 17 21 31. Peru, S. C (2001). The use of tannic acid in the local treatment of burn Wounds: intriguing old and new perspectives. Wounds 13 (14) 144 158 32. Purseglove, J.W. (1976). Trophical crops (Dicolyledons). Longman, London. Pp 719 724. 33. Reules, C. Garcia, H.S and Tiznado Hermandez, M.E (2005). Effect of benzylisothiocyanate on tomato fruit infection development by Alternaria alternata www.interscience.wiley.com/journal 34. Sofowora, A (1993). Medicinal plants and traditional medicine in Africa. Ibadan, Spectrum Ltd. Pp 191 - 289 35. Taiga, A and Olufolaji, D.B (2008). In vitro screening of selected plant extracts for fungicidal properties against dry rot fungi of yam tubers (Dioscoria rotundata) in Kogi State, Nigeria Proceeding of International Society of Biotechnology, Ganglok Sikkim, India. Pp 309 311 36. Trease, G.E and Evans, W.C (1989). Pharmacognosy (11th Ed). Bailliere Tindal, London, Pp 45 50 37. Wessells, W.K and Hopson, J.L (1988). Biology. New York, Random House Press. Pp. 505 521 38. Wikipedia (2008). http://wikipedia.org/wiki.steroids 39. Wikipedia (2010). http://en.wikipedia.org/wiki/westafricanpepper 40. Wokocha, R.C (1998). Effect of crop residue on Damping off of tomato induced by Sclerotium rolfsii in the Nigeria Savana. Nigeria Journal of Plant protection 17:18 24 41. Wokocha, R.C and Nwaoju G.A (2008). Comparative toxicity of extracts of three Medicinal plants and Ridomil (Metaxyl) on phytophtora pamlivora and varietal resistance of Cocoa to the Black pod disease in Ikwunanwo L.G.A, Abia State. Nigeria Journal of Botany 21(2): 428 436