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Research Article
 

In vitro Biological Activities of Carica papaya



Olawale H. Oladimeji , Rene Nia , Kalu Ndukwe and Emmanuel Attih
 
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ABSTRACT

The present study was designed with the aim of confirming or otherwise the ethno-medicinal claims of C. papaya in the treatment and or management of ailments such as ringworm, digestive disorders, fevers and tumors. Hence, the anti-microbial, larvicidal and brine-shrimp lethality studies on leaves, stem and roots extracts were carried out. The extracts and fractions of roots elicited good anti-microbial activity against B. subtilis, S. aureus but gave minimal activity against E. coli, S. typhi and K. pneumoniae and none against fungal isolates (A. niger and C. albicans). Both the leaves and stem, however afforded lesser activities. The larvicidal activity determined in terms of percentage mortality showed that the roots gave moderate larvicidal activity (LA%) of 40 and 55% (at 5%w/v) while the activity was potent at 70 and 80% (at 10% w/v) both at 12 and 24 h incubation respectively. However, the activity displayed by both the leaves and stem was insignificant. Interestingly, the brine-shrimp lethality assay; analyzed using the Finney probit method, showed the leaves displayed a significant LD50 value at 2.7 ppm, while the stem and roots gave moderate LD50 values at 384 and 272 ppm respectively compared with literature values below the 200 ppm which are generally considered significant. These findings indicate a potential of the plant to serve as panacea for infectious diseases and also lend scientific justification to some of the folkloric uses.

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Olawale H. Oladimeji , Rene Nia , Kalu Ndukwe and Emmanuel Attih , 2007. In vitro Biological Activities of Carica papaya. Research Journal of Medicinal Plants, 1: 92-99.

DOI: 10.3923/rjmp.2007.92.99

URL: https://scialert.net/abstract/?doi=rjmp.2007.92.99

INTRODUCTION

Carica is one the four genera in the family; Caricaceae. It comprises of 45 species of which Carica papaya is an important member. C. papaya popularly known as paw-paw was initially native to tropical America but is now cultivated in many tropical areas of the world (Hutchinson and Dalziel, 1954). It is employed in a variety of ethno-therapeutic uses. The latex is used for treating fever, psoriasis, ringworm and uterine tumors and causes severe gastritis, conjunctivitis, vesication, yellowing of palms and soles and poisoning. It can also induce asthma, rhinitis, irritation and dermatogenitis (Morton, 1977; Flath and Forrey, 1977). The leaves and seeds are used as diuretic, anti-coagulant and anti-helmintic agents. They can also be used to treat wounds, burns, ulcers, digestive disorders and combat dyspepsia (Tyler, 1987). The plant has huge economic values. It serves as a renewable energy resource in the production of alcohol (Sharma and Ogbeide, 1982), cosmetics such as creams, lotions, cleansers and shampoos (Duke, 1984) and rubber (Morton, 1977). Papain, a complex proteolytic enzyme in the plant has been found to be commercially useful in tenderizing meat, making chewing gum, treating beer and manufacturing textile (Murphy and Natarajam, 1982). Other compounds such as carpain, carpasemine and chymopapain have also been reportedly isolated from the plant (Duke, 1984). However, despite these uses, its application in malaria control at the larval stage and scientific justification for anti-tumor activity are yet to be revealed. Hence this study was designed with the aim of investigating into larvicidal activity and to confirm or otherwise the anti-tumor activity and as well as the potency of extracts and fractions to selected microbes.

MATERIALS AND METHODS

Collection of Plant
The fresh leaves, stem and roots of C. papaya were collected in the month of November, 2004 at Ikono Local Government Area of Akwa Ibom State. These plant parts were identified and authenticated by A. Williams, the taxonomist at the Department of Pharmacognosy and Traditional Medicine, University of Uyo, Uyo, Nigeria where voucher specimens NoH 50, NoH 51 and NoH 52 were deposited. Sea- water (collected from Eleko Beach, Lagos), plastic soap case, brine-shrimp eggs (Artemia salina, Leach) obtained from San Franscisco Bay Brad Inc; Newark, CA 94560,USA and Anopheles gambiae larvae bred in plastic buckets.

Chemicals, Micro-Organisms and Media
The Chemical Reagents
Acetone, Butanol, Ethanol, Ethyl-acetate, Hexane Methanol and Toluene (all of AnalaR grade; British Drug House Limited, England) were purchased in Uyo. Silica gel (254GF), Streptomycin and Nystatin (Fidson Healthcare Ltd., Lagos, Nigeria). The micro-organisms (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Aspergillus niger and Candida albicans) clinically isolated from human specimens; urine, faeces, wounds and vaginal swabs were obtained from the Medical Laboratory, University of Uyo Health Center. They were collected in sterile bottles, identified and authenticated by convectional biochemical tests (Gibson and Khoury, 1986; Murray et al., 1995) and then refrigerated at 0-5°C at the Pharmaceutical Microbiology and Parasitilogy Unit, Faculty of Pharmacy, University of Uyo, Akwa Ibom State prior to use. Also, Mueller Hinton II Agar (Biotec Laboratory Limited, Ipswich, England), Sabouraud Dextrose Agar (International Diagnostic Group PLC, Lancaster, England) and Nutient Broth (Oxiod Limited, Basingstroke, England) were used.

Extraction and Processing
The plant parts (leaves, stem and roots) were air-dried and powdered separately in an electric mill. The resultant ground powders were then extracted with cold 50% aqueous ethanol at room temperature (27±2°C) for 72 h. The filtrates were evaporated to dryness using a rotary evaporator (Buchi CH-920, Laboratorium Technic, Flawk/SG, Switzerland. The dried crude ethanolic extracts were then investigated for plant metabolites (alkaloids, saponins, tannins, cardiac glycosides, terpenes, anthraquinones and flavonoids according to the laid down phytochemical methods (Harbone, 1984; Sofowora, 1993; Trease and Evans, 1996). Also, the dried crude ethanolic extracts were separately chromatographed on silica gel (254GF) column and gradient elution carried out using hexane: Ethylacetate: Butanol (1:1:1) mixture. Eluates which showed similar TLC profiles under UV (λ 366 nm) were pooled and bulked separately to obtain the hexane, ethyl-acetate and butanol fractions which were evaporated to dryness and in addition to the parent extracts then subjected to the biological tests.

Antimicrobial Test
Determination of Zone of Inhibition
The media were prepared according to the Manufacturers’ instructions, poured into sterile petri-dishes (diameter, 13.5 cm) and then allowed to set. The bore-hole diffusion method was used for the antimicrobial screening tests. The bacteria were cultured in nutrient agar while the fungi were cultured in the sabouraud dextrose agar. The inoculum of each organism was introduced into each petri-dish. Cylindrical plugs were removed from the agar plates by means of a cork borer to produce wells of approximately 6.0 mm.The wells were equidistant from each other and the edge of the plate (Washington, 1995). Concentrations of 15 and 30 mg mL-1 of the crude ethanolic extracts and the fractions at (5 mg mL-1) dissolved in methanol/de-ionized water (1:1 v/v) were separately introduced into wells. Also, concentrations of 10 μg mL-1 of ampicillin (a standard antibiotic), 1 mg mL-1 of nystatin (a standard anti-fungal drug) and methanol :De-ionized water (1:1v/v) were introduced into other wells as positive and negative controls, respectively. The experiments were carried in triplicates. The plates were at room temperature (27±2°C) for 2 h to allow for diffusion. The plates were then incubated at 37±2°C for 24 h.

Larvicidal Assay
The Breeding of Larvae of Anopheles gambiae
The larvae were bred by keeping outdoor basins of water under growing shrubs near houses for about two weeks. After this period, at least three groups of mosquitoes larvae were dentified accurately in a container using classical methods (Sievers et al., 1949). Anopheles gambiae, Aedes agypti and Culex piper-fatigans responsible for the transmission of malaria, yellow fever and filariasis respectively were so identified. The fourth instar larvae of Anopheles gambiae were later selected, separated and the species authenticated at the Department of Entomology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria before further study.

The method employed for the determination of larvicidal activity was adopted from that described by several authors (Ojewole et al., 2000) and WHO directives on such assay with modifications (WHO, 1970). Thirty Anopheles gambiae larvae in their fourth stage were put in recovery cups (250 mL plastic jars) containing 10 mL de-ionized water (pH 7.0) at room temperature (27±2°C). Three milliliter volume each of the graded concentrations of the extracts (5 and 10% w/v) were added to 90 mL de-ionized water, mixed thoroughly and then poured into exposure cups (250 mL plastic jars containing larvae food). Each aqueous solution of the extract was set up in triplicates. Negative control (containing 90 mL de-ionized water and larvae food) as well as positive control (containing 3 mL absolute alcohol, 90 mL de-ionized water and larvae) were also set up in triplicates. Both the test controls set up were maintained in room temperature (27±2°C). The Anopheles larvae in each recovery cup were scooped and transferred by means of small nets into the test exposure cups containing the sample solutions and or control larvae food and de-ionized water. The larvae in the test and controls set up were incubated for a period of 12 and 24 h at room temperature (27±2°C). Therefore, the larvae were gently scooped into small nets, washed with de-ionized water, transferred into recovery cups containing 100 mL of de-ionized water, maintained at pH 7.0 and allowed to settle. Prior to mortality determinations, the larvae in recovery cups were gently disturbed and made to go below the water surface by agitating the water with a sterile pipette. The dead and dying larvae, which started to float on the water surface, were pushed down the recovery cups. The living larvae which were able to swim to the water surface were allowed to do so within 5 min following agitation. The larvae remaining and or staying at the bottom of the recovery cups unable to swim to the water surface were regarded as dead.

Brine-Shrimp Lethality Assay
Some sea water was placed in a small plastic tank with perforated dividing dam which was fabricated from plastic soap case. Some shrimp eggs were added to one side of the divided tank. This side was darkened by covering it with a plastic lid while the other compartment was exposed. The set-up was left for 48 h for the shrimp to hatch and mature as nauplii. Mature nauplii usually swim to the exposed compartment.

A stock solution of the sample was prepared by dissolving 20 mg of the material in 2 mL of methanol/de-ionized water (1:1 v/v). To obtain the desired final concentrations such as 1000, 100 and 10 mL-1; 0.5, 0.05 and 0.005 mL-1 of the stock solution were transferred into the three vials respectively. The solvent was then evaporated by leaving the vials in a vacuum desiccator for 24 h. Tenshrimp nauplii were counted into each vial (i.e., 30 nauplii per dilution). The total volume of solution in each vial was adjusted to 5 mL-1 by adding the sea- water (5 mL-1/vial). The control (methanol/de-ionized water 1:1 v/v) was prepared in the same way except that the sample of the extract was omitted. The vials were maintained in the laboratory with normal fluorescent illumination and the set-up left for 24 h. The number of survivors, usually swimming was counted with the aid of a magnifying lens for each f the vials at the end of 24 h. Thus the number of the dead was computed; hence the LD50 in ppm. (parts per million) was determined using the Finney probit analysis software (McLaughlin, 1988).

RESULTS AND DISCUSSION

Processing of Plant Material
The plant materials used in this present study were identified, authenticated and collected, observing basic guidelines of plant collection. The solvents and reagents used were of analytical grade. The phytochemical screening revealed the presence of alkaloids, saponins, tannins, cardiac glycosides and flavonoids (generally in trace amounts) especially in the extracts of leaves and stem while they were moderate in the roots. This confirms previous studies as contained in (Duke, 1984) and it is instructive that secondary metabolites such as alkaloids, saponins, tannins, flavonoids and cardiac glycosides present in the plant which are the basis for the curative and or management of many ailments such as wounds, ringworm, digestive disorders, fevers, tumors etc. claimed in its ethno-medicine. However, anthraquinones and terpenes were absent in the extracts (Table 1).

Anti-Microbial Sensitivity Results
The extracts and the fractions (obtained from the chromatographic purification of the extracts) were screened for anti-bacterial and anti- fungal activities using B. subtilis and S. aureus, E. coli, S. typhi and K. pneumoniae and A. niger and C. albicans to represent a desirable spectrum of microbes. The extracts were tested at 15 and 30 mg mL-1 and the while fractions were screened at 5 mg mL-1. The results presented in Table 2 show that the activities elicited were concentration-dependent. Generally, the extracts of leaves, stem and most especially those of roots were very active against B.subtilis and S. aureus but were comparably less active against E. coli, S. typhi and K. pneumoniae. This is not surprising because gram- negative bacteria posses sophisticated cell-wall which does not allow the permeation of external agents (Brown, 1975). Also, the anti-fungal activity was somewhat insignificant probably because the cell walls of fungi resemble those of higher plants and hence limits the permeation of substances into them. The antibacterial and anti-fungal activities of fractions were slightly higher than those of the crude extract because of level of purity associated with them.

Table 1: Phytochemical screening of extracts of leaves, stem and roots of C. papaya
L = Leaves, S = Stem, R = Roots, + = Trace (insignificant quantities), ++ = Moderate, - = Absent

Table 2: Antimicrobial sensitivity of extracts and fractions of C. papaya at different concentrations in methanol/de-ionized water (1:1 v/v)
Refer to Table 1 Lh, Le, Lb (hexane, ethylacetate and butanol fractions of leaves respectively); Sh, Se, Sb (hexane, ethylacetate and butanol fractions of stem, respectively); Rh, Re, Rb (hexane, ethylacetate and butanol fractions of root, respectively); A = Ampicillin (standard antibiotic or anti-bacterial drug); N = Nystatin (standard anti-fungal drug; C = Methanol/de-ionized water (1:1v/v); Nt = Not tested; Zone of inhibition recorded is diameter zone of inhibition and bore-hole cup size (6 mm)

These results are not surprising because the phytochemical screening carried out on the extracts revealed the presence of tannins and flavonoids which have been reported in previous studies (Lamikanra et al., 1990; Burapadaja and Bunchoo, 1995; Adesina et al., 2000) to be anti-microbial. It is very possible that these bioactive compounds might have been responsible for the activities observed.

Larvicidal Activity
Preliminary larvicidal assay was carried out on the crude extracts of the leaves, stem and roots at 5 w/v and 10% w/v and at 12 and 24 h incubation. The larvicidal activity (LA%) was calculated in terms of percentage mortality. The lethality furnished after 12 and 24 h incubation was concentration and time -dependent (Table 3 and 4). At 5% w/v (12 and 24 h) and 10% w/v (12 and 24 h), the roots demonstrated the highest larvicidal activity at 40 and 55%; 70 and 80% respectively compared with those given by the leaves (10 and 20%; 25 and 35%) and stem (20 and 30%; 40 and 50%) (Table 3 and 4). These results have revealed that the greatest potential for larvicidal activity resides in the roots of this plant. Interestingly, the crude extracts of the leaves, stem and roots tested positive to both alkaloids and saponins, which have shown in separate studies (Bentley et al., 1984; Ojewole et al., 2000; Oladimeji et al., 2006a; Oladimeji et al., 2006b; Nia et al., 2006) to be lethal to the fourth instar larvae of Anopheles gambiae thereby preventing the emergence of adult mosquitoes responsible for the transmission of malaria which still ravage huge populations of people around the world.

Brine-Shrimp Lethality
The brine-shrimp assay determines the lethalities of materials toward brine-shrimp larvae (nauplii) and in doing so predicts the ability to kill cancer cell cultures, kill various pests and exert a wide range of pharmacologic effects. The shrimp nauplii have been used for a number of a bioassay systems in which natural product extracts, fractions or pure isolates are tested at concentrations of 1000, 100 and 10 μg mL-1 in vials containing 5 mL of brine and ten nauplii in each of the three replicates (Meyer et al., 1982). The LD50 values in ppm are estimated with 95% confidence using the appropriate mathematical estimates; the Finney probit analysis program being the model routinely employed. The LD50 values of the extracts of leaves, stem and roots obtained in this study with those of other plants in literature (Kupahan et al., 1969; Ma et al., 1989; Obuotor et al., 1998; Oladimeji et al., 2005; Oladimeji et al., 2006b; Oladimeji et al., 2006c) are presented in Table 5. Interestingly, the leaves displayed a significant LD50 value at 2.7 ppm while the stem and roots gave marginal LD50 values at 384 and 272 ppm respectively when compared with those of Raphia hookeri (2.3 ppm), Pycnanthus angolensis (2.5 ppm), Persia major (2.6 ppm), Pogonopus speciosus (50 ppm) and Myrsine africana (114ppm) whose values below the 200 ppm are considered significant (Kupahan et al., 1969; Ma et al., 1989; Obuotor et al., 1998; Oladimeji et al., 2005; Oladimeji et al., 2006b, c).

Table 3: Larvicidal activity (LA%) of extracts of leaves, stem and roots of C. papaya at 5% w/v after 12 and 24 h incubation
Refer to Table 1

Table 4: Larvicidal activity (LA%) of extracts of leaves, stem and roots of C. papaya at 10% w/v after 12 and 24 h incubation
Refer to Table 1: PC = Positive control; NC = Negative control

Table 5: Brine- shrimp lethality assay of extracts of leaves, stem and roots of C. papaya
Raphia hookeri (mesocarp) 2.3 Key: Refer to Table 1

These results show that the leaves are more potent against tumors than the stem or the roots both of which have been found in this study to be strongly anti-microbial against clinical strains of bacteria and fungi and remarkably larvicidal against Anopheles gambiae larvae. In conclusion, the results of anti-microbial, larvicidal and brine-shrimp lethality assays lend scientific justification to the uses of the plant in the treatment and or management of bacterial infections especially those of gram- positive origin, in the fights against malaria and tumors in man.

ACKNOWLEDGMENT

The kind assitance of E. J.Akpan in the biological studies is gratefully appreciated.

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