phytochemical characterization and comparative

Int. J. Drug Res. Tech. 2012, Vol. 2 (5), 399-406

ISSN 2277 - 1506

International Journal of Drug Research and Technology Available online at http://www.ijdrt.com Original Research Paper PHYTOCHEMICAL CHARACTERIZATION AND COMPARATIVE EFFICACIES OF CRUDE EXTRACTS OF CARICA PAPAYA Augustine Ocloo1*, Nonye Constance Nwokolo1 and Nicholas T. K. D. Dayie2 1 Department of Biochemistry, University of Ghana, Legon P. O. Box LG 54, Legon, Accra, Ghana 2 Department of Microbiology, University of Ghana Medical School, College of Health Sciences, University of Ghana, Legon ABSTRACT Carica papaya, besides the fruits being edible, has a long history and proof of being a very effective medicinal plant. The root is said to cure piles and yaws and root infusions are used for syphilis. Studies conducted in some countries have shown that there is a significant antibacterial activity in organic extracts of different parts of C. papaya. In the present study, aqueous and organic extracts of dried seed and epicarp of one of the local varieties of pawpaw were characterised for the presence of alkaloids, flavonoids, reducing sugars, phenols, saponins, tannins and terpenoids and also tested for antibacterial activity against Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-positive) and Shigella flexneri (Gram-negative) using the disc diffusion method. The crude organic (acetone and methanol) extracts and extract fractions of the crude extracts containing phenolics and terpenoids of the seed inhibited the growth of all three organisms. No inhibition was observed in the aqueous extracts of the seed and in all the extracts of the epicarp. Therefore, the seeds of the local variety contain antibacterial agent(s) with broad spectrum of activity.

Keywords: Phytoconstituents, Antibacterial activity, Carica papaya. INTRODUCTION Infectious diseases are among the major diseases of public health concern and account for almost 50 000 deaths every day. This situation has further been complicated with the rapid development of multidrug resistance by existing microorganisms to known antimicrobial agents and emergence of new pathogenic microorganisms.1 Therefore, the search for newer sources of antibiotics has become a global challenge to research institutions, pharmaceutical companies and academia.2 Plants have the major advantage of still being the most effective and cheaper alternative sources of drugs.3 Herbal medicine, also known as botanical medicine or phytomedicine refers to the use of any part of plant for medicinal purposes.4 Long practised, alongside conventional medicine, herbal medicine is becoming more important as up-to-date analysis and research show their value in treatment and prevention of many diseases.5 The local use of natural plants as primary health remedies, due to their pharmacological properties, is quite common in Asia, Latin America and Africa.6 Drawing from the great diversity that flora offers, traditional healers have managed to find cure for certain disease conditions that have baffled western medicine for centuries such as antimicrobial drug resistance.7 About 25% of these medicinal plants are known to be useful for the cure of gastrointestinal disorders ranging from peptic ulcers and abdominal cramps to diarrhoea and dysentery.8 Some of the plants have also been found to be effective antibacterial, antiviral, antioxidant or http://www.ijdrt.com

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Augustine Ocloo et al. / International Journal of Drug Research and Technology 2012, Vol. 2 (5), 399-406

antiparasitic agents.9 Carica papaya Linnaeus (Family Caricaceae), commonly called pawpaw (English), Adiba (Ewe–Ghana) or Bofre (Twi–Ghana) is a monosexual plant of Central American origin. Besides the fruits being edible, it has a long history and proof of being a very effective medicinal plant. Pawpaw’s main medicinal use is as a digestive agent.10 The leaves, fruits and roots have been used locally with the unripe fruit being especially effective.11 The latex, used locally as an antiseptic is applied externally to speed up the healing of wounds, ulcers, warts, corns, cancers and indurations of the skin. The seeds are considered to be alexeritic, abortifacient, counter-irritant, emmenagogue and anthelmintic.12 Ethanol extract of the dried seed caused significant reduction in sperm cell count, mobility and sperm head abnormality compared to control.13 The flowers may be taken as infusions to induce menstruation and can be used for the treatment of jaundice. The root is said to cure piles and yaws and root infusions are used for syphilis. A decoction of the ripe fruit is helpful for treating persistent dysentery and diarrhoea in children.14 The ripe fruit is mildly laxative and the leaves are used to dress wounds. The efficacy of treatments with Carica papaya L. is dependent on the quantity of the different bioactive compounds in the herbal preparation.4 Studies conducted in some countries; Nigeria2,15 and Jamaica16 have demonstrated significant antibacterial activity in various extracts from different parts of the pawpaw tree. The present study investigated the antibacterial activity of extracts from one of the local breeds of pawpaw. Aqueous and organic extracts of dried seed and epicarp were tested for antibacterial activity against Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-positive) and Shigella flexneri (Gram-negative) using the disc diffusion method. The organic extracts of the seed inhibited the growth of all three organisms. No inhibition was observed in the aqueous extracts of the seed and in all the extracts of the epicarp. It was concluded therefore that, the seed of the local variety contain antibacterial agent(s) with broad spectrum of activity.

MATERIALS AND METHODS Processing of plant samples Unripe Carica papaya fruits were obtained from a farm near Dodowa in Accra, Ghana and were identified and authenticated as Carica papaya Linnaeus at the Department of Botany, University of Ghana, Legon. The unripe pawpaw fruits were washed in tap water and then rinsed in sterile distilled H2O. The epicarp and seeds were removed and sun-dried separately for about a week. The dried epicarp and seeds were then ground into fine powder with an electric blender, weighed and stored in containers at room temperature until required for analyses. Extraction of plant material Crude extract preparation Cold and hot extraction with H2O, and soxhlet extractions with methanol (99%) and acetone, as described by Junaid and co-workers,17 was carried out. Briefly, 5 g of each sample was weighed into 20 ml of the solvent (water, methanol and acetone). For cold extraction the samples and solvent were stirred every 30 min for 3 h and allowed to stand for 24 hours, while for hot extraction the samples and solvent were heated for 30 minutes and stirred every 30 minutes for 3 hours and allowed to stand for 24 hours. The extracts were first filtered with clean muslin cloth and then suction filtered. The filtrates were centrifuged at 5000 g for 15 min. The supernatants were Rotary evaporated to ½ their original volume. The extract concentrates were sterilised filtered using 0.22 m Millipore, collected in sterile falcon tubes and stored at room temperature. Fractionation of crude extract The scheme shown in Figure 1 was used for the fractionation of the acetone and methanol extracts of the dried seed of as described previously.18 http://www.ijdrt.com

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Figure 1: A general procedure for fractionating extracts into different classes according to polarity Test organisms Cultures of Escherichia coli, Staphylococcus aureus and Shigella flexneri were obtained from the Department of Biochemistry, University of Ghana, Legon. Purity plates of each of the bacterial isolates were obtained by culturing on their respective selective media. Biochemical tests were performed to reidentify and confirm the identity of the isolates. Fresh plates of the test bacteria were made from the isolate cultures obtained on agar slants. Discrete colonies of fresh cultures of the different bacterial isolates were then picked and suspended in 5 ml Nutrient broth (NB, Oxoid), in well-labelled sterile Bijou bottles, and incubated for 24 h at 37 OC prior to antimicrobial susceptibility testing. Determination of antibacterial activity The crude extracts and the fractions were tested for antibacterial activity using Escherichia coli, Shigella flexneri and Staphylococcus aureus as test organisms by the Kirby-Bauer disc diffusion method. To 90 ml of distilled water was added 1.3 g nutrient agar and dissolved. It was then autoclaved at 121 OC for 15 min for complete sterilization. The agar solution was allowed to cool and 15 ml was poured into 6 sterile glass petri dishes. The plates were allowed to set and then incubated overnight at 37 OC to test for sterility. Nutrient agar plates were inoculated with the organisms and incubated at 37 OC overnight. Colonies were picked from the plates and used to inoculate nutrient broth contained in test tubes. The tubes were incubated at 37 OC overnight. Disc of Whatman No. 3 filter paper were sterilized by heating in an oven for 30 minutes at 80 OC. Nutrient agar plates were inoculated with 100 μl of each organism and after 5 min, 5 filter paper discs, impregnated with 20 μl of the concentrated extracts, fractions or ampicillin (8 g/ml) were transferred onto the agar surface of each plate using sterile forceps. The plates were then incubated at 37 OC overnight. The effectiveness of the extract as antibiotic against the test organism was determined by measuring the diameter of zone of inhibition. Phytochemical analysis This was done on the extracts particularly to ascertain the presence of different bioactive components present in seed as compared to the epicarp. The presence of alkaloids, saponins, glycoside, tannins, http://www.ijdrt.com

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flavonoids, cardiac glycoside, steroidal terpenes, anthraquinones and carbohydrates were determined, as described previously. [4] Briefly, Fehling’s test was used for the presence of reducing sugars and ferric chloride test for tannins. The presence of flavonoids were tested by reacting 3 ml of the extracts with I ml dilute NaOH, followed by addition of 1 ml 0.1 M HCl. A colourless solution confirms the presence of flavonoids. Terpenoids were determined by mixing 5 ml of extract in chloroform and concentrated sulphuric acid carefully added. A reddish brown colouration of the interface is an indication of the presence of terpenoids. For alkaloids, 4 ml of acetic acid in ethanol was added to 1 ml of extract. This was covered and allowed to stand for 30 min. The mixture was filtered and then concentrated to one-quarter of its original volume. Concentrated ammonium sulphate was added drop wise to the filtrate. The presence of precipitate indicates the presence of alkaloids. The presence of saponins was tested by shaking 2 ml of the extract vigorously after boiling for 5 min and then allowed to stand for 10 min. A thick persistent froth indicates presence of saponins.

RESULTS The effects of the crude (aqueous and organic) extracts from the dried seed and epicarp and the positive control (ampicillin) on the growth of the test organisms are as shown in Table 1. All the organic extracts of the seed exhibited antibacterial activity against all the three organisms but the aqueous extracts did not. A similar result was reported in the root of pawpaw previously.2 The crude acetone extract produced a slightly higher inhibition against S. aureus. The inhibition against Shigella flexneri was the lowest in both the crude acetone and methanol extracts. The standard antibiotics (ampicillin) inhibited the growth of all three test organisms indicating that the organisms are not resistant to ampicillin. None of the extracts showed any activity, which again is consistent with the results of Dawkins co-workers.16 Table 2 shows the effect of the fractions from the acetone and the methanol extracts on the test organisms. For the fractions from the acetone extract, apart from the basic alkaloids and saponins contained in methanol (see method) and quaternary alkaloids in chloroform (see method) which did not inhibit the growth of S. flexneri, all extract fractions altered the growth of the test organisms used. Apart from the basic alkaloids and saponins that are contained in methanol (see method), and did not inhibit the growth of S. flexneri, all extract fractions from the methanol extract of the plant altered the growth of the test organisms used. The fractions contained in chloroform produced much higher inhibition of S. aureus. This was found to be due to the fact the solvent (chloroform) had significant inhibition effect on S. aureus producing a zone of inhibition of about 10.00 mm (result not shown). Table3 shows the data from the qualitative analysis of the phytochemical constituents of the crude extracts. The phytochemical composition of the organic extracts of the seed and epicarp were significantly different. The analysis gave positive results for Alkaloids, Phenols, Saponins, Tannins, and Terpenoids in the crude acetone extracts and Alkaloids, Glycosides, Phenols and Tannins in the crude methanol extracts of the dried seed. On the other hand, the analysis of the aqueous extracts of the seed and all extracts from the epicarp gave positive results for very few constituents. This observation indicates that the difference in activity could be due to the differences in the phytochemical composition of the extracts.

DISCUSSION Carica papaya, pawpaw is one of the tropical plants that is claimed to have several medicinal properties. In the present study, seeds and epicarp from unripe pawpaw fruit were sundried, ground into fine powder, extracted with various solvents and tested for antibacterial activity against Escherichia coli, Staphylococcus aureus and Shigella flexneri. The organic extracts of the dried seed gave activity against all three organisms producing zones of inhibition between 10-13 mm in agreement with the observations made by other researchers.15,16 However, the aqueous extracts of the seed did not produce any level of http://www.ijdrt.com

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activity against any of the test organisms. Doughari and co-workers reported similar observation in the root of C. papaya.2 Furthermore, none of the extracts of the epicarp exhibited any degree of inhibition against any of the test organisms which again is in agreement with previous report.16 In general, the bioactive compound did not show species specificity. Gram-positive and Gram-negative species were inhibited to the same extent by the extracts. This is also consistent with the observation by 2,16 and suggests that the bioactive compound have a broad spectrum of activity as explained by others.19 This will be of immense advantage in fighting the menace of antibiotic refractive pathogens that are so prevalent in recent times. S. flexneri, a Gram-negative bacterium responded slightly different to the extracts obtained by fractionation of the crude samples. Furthermore, both fractions containing basic alkaloids, saponins and quaternary alkaloids from the crude acetone extract did not inhibit the growth of S. flexneri, the fractions containing the quaternary alkaloids from the crude methanol extract inhibited its growth. The data from the qualitative phytochemical analysis showed that the extracts that did not inhibit the growth of the organisms lacked a common constituent (phenols) that are present in the extracts that showed activity, suggesting that the bioactive compound is possibly a phenolic compound. The phytoconstituents of the organic extracts of the seed in this study echoes those in the ethanolic extract of the leaves.20 However, the inability of the extracts of the epicarp to inhibit the growth of the test organisms could also be due to the presence of oils, wax, resins, fatty acids or pigment, which had been reported to be capable of blocking the active ingredients in the plant extract, thus, preventing the plant extract from accessing the bacterial cell wall.21 The difference in the composition of the crude extracts is likely to be due to the varying degrees of solubility of the active constituents in the four solvents used. Different solvents have been reported to have the capacity to extract different phytoconstituents depending on their solubility or polarity in the solvent.22 Other studies reported that most active constituents are mainly aromatic or saturated organic compounds which have better solubility in organic solvents.22 The results also suggest that water was not an effective solvent for extracting the active component. Though water was reported by traditional healers and herbalists to be the most commonly used solvent to extract biologically active compounds due to its availability,23 the observation in this study contradicts this assertion.

CONCLUSIONS The study demonstrated the presence of antibacterial compound in the seed of one of the local breeds of pawpaw. The study also showed that the anti-bacterial agent in pawpaw is active against Gram-positive and Gram-negative bacteria. Demonstration of antibacterial activity of pawpaw seeds against the test organisms has provided a scientific basis for its local usage as a medicinal plant.

ACKNOWLEDGEMENT We gratefully acknowledge the support of the technical staff of the Department of Biochemistry.

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Table 1: Effects of crude extracts of the dried seed and epicarp of unripe Carica papaya and the standard antibiotic (Ampicillin) on the test organisms Zone of inhibition in millimetres Seed Staphylococcus aureus

Extracts/Samples Shigella flexneri

Escherichia coli

Shigella flexneri

Epicarp Staphylococcus aureus

Escherichia coli

Acetone

11.67 + 0.38

13.33 + 0.51

12.67 + 0.51

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

Methanol

10.67 + 0.19

12.67 + 0.38

12.67 + 0.17

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

Cold water

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

Hot water

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

Ampicilin

17.25 + 0.08

19.50 + 0.17

15.33 + 0.38

17.25 + 0.08

19.50 + 0.17

15.33 + 0.38

Values are mean + S. E. M. of three independent experiments.

Table 2: Effects of the solvent fractions from the crude acetone and methanol extracts of the dried seed of unripe Carica papaya on the test organisms Zone of inhibition millimetres From crude acetone

Extracts fractions Shigella flexneri Terpenoids and phenolics in chloroform Basic alkaloids and saponins in methanol Quaternary alkaloids in chloroform

Staphylococcus aureus

From crude methanol Escherichia coli

Shigella flexneri

Staphylococcus aureus

Escherichia coli

15.33 + 0.51

27.97 + 0.38

15.67 + 0.25

11.67 + 0.51

26.67 + 0.38

15.00 + 1.33

0.00 + 0.00

16.00 + 0.88

14.43 + 0.28

0.00 + 0.00

18.33+ 0.19

12.00 + 0.67

0.00 + 0.00

24.63 + 1.33

10.90 + 0.19

10.67 + 1.17

25.33 + 1.59

13.67 + 0.19

Values are mean + S. E. M. of three independent experiments.


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Table 3: Phytochemical analysis data of crude extracts of the dried seed and epicarp of unripe Carica papaya. EXTRACTS Seed

Phytocompound

Epicarp

Acetone Hot water Cold water Methanol Acetone Hot water Cold water Methanol Alkaloids

+

_

_

+

_

_

_

_

Saponins

+

+

+

_

_

+

+

_

Tannins

+

+

+

+

_

_

_

_

Phenols

+

_

_

+

_

_

_

_

Flavonoids

_

_

_

_

+

+

_

_

Terpenoids

+

_

_

_

+

_

_

_

Reducing sugars

_

+

+

+

_

_

_

_

+ indicates present of the particular phytocompound; - indicates absence of the particular phytocompound.

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