|
Ethnobotanical Leaflets 13: 850-64, 2009. Antimicrobial
Activity of Carica Papaya (Pawpaw Leaf) on Some
Pathogenic Organisms of Clinical Origin from South-Western Nigeria *I.I.
Anibijuwon and A.O. Udeze Department of Microbiology, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria���������������������� �*Corresponding Author: kunledoexploit@yahoo.com Issued 01 July 2009 Abstract The bioactive compound of leaf and root extracts
of Carica papaya was extracted, using
water and organic solvents, and were investigated for antibacterial activity
against some human pathogenic bacteria using the agar diffusion method. The
aqueous extracts of the root extracts did not show significant activity, but
the organic extracts had significant activity with the methanol extracts
demonstrating the highest activity against the test bacteria. The root
extracts demonstrated higher activities against all the gram-positive
bacteria than the gram-negative bacteria tested, with the highest activity
(14 mm zone of inhibition) demonstrated against Pseudomonas aeruginosa while the aqueous leaf extract showed
pronounced inhibition demonstrating higher activities against the test
bacteria than the organic solvents. The extracts demonstrated higher
activities against all the gram-positive bacteria than the gram-negative
bacteria tested, with the highest activity (4.2 mm zone of inhibition)
demonstrated against Pseudomonas aeruginosa.
Increase in temperature enhanced the activity of the extracts, while alkaline
pH decreased the activity. The Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) of the root extracts ranged between
50-200 mg/ml. Preliminary phytochemical analyses
showed that the extracts contain alkaloids, tannins, saponins,
glycosides and phenols. Carica papaya
may be used for the treatment of gastroenteritis, uretritis,
otitis media and wound infections. Key words: Antimicrobial
activity, Carica papaya. pathogenic organisms. South-Western
Nigeria Introduction In recent years, the growing
demand for herbal products has led to a quantum jump in volume of plant
materials traded across the countries. Therefore, the use and history of
herbs dates back to the time of early man, who had the crudest tools as his
implements and use stones to start his fire. They used herbs in their raw and
cooked forms to keep fit. Since that time, the use of herbs has been known
and accepted by all nations and has been known also as the first art of treatment
available to man (Kafaru, 1994). The importance of herbs in the
management of human ailments cannot be over emphasized. It is clear that the
plant kingdom harbours an inexhaustible source of active ingredients
invaluable in the management of many intractable diseases. Furthermore, the
active components of herbal remedies have the advantage of being combined
with other substances that appear to be inactive. However, these
complementary components give the plant as a whole a safety and efficiency much
superior to that of its isolated and pure active components (Ahmad, 2001). There is no plant that does not
have medicinal value. The active components are normally extracted from all
plant structures, but the concentrations of these components vary from structure
to structure. However, parts known to contain the highest concentration of
the principles are preferred to therapeutic purposes and it can either be the
leaves, stems, barks, roots, bulks, corms, rhizomes, woods, flowers, fruits
or the seeds (Kafaru, 1994). Some of the active principles
singly or in combination inhibit greatly the life processes of microbes,
especially the disease causing ones. They do this by binding their protein
molecules, acting as chelating agents (selective binding polyvalent metal
ions so that the latter loses its biological activities), altering their
biochemical systems, preventing utilization of available interests to the microorganisms, other causes inflammation analysis of
microbial cells (Garrod et al., 1995). The bitter taste, pungent and repulsive smell in
some plants; have been found to have repressive ability over the metabolic
activities of a wide range of microorganisms (Mitscher et al., 1992).����������� Sofowora,
(1982) and Baladrin et al., (1985) defined medicinal plants as a plant in which one
or more of the organs contains substances that can be used for therapeutic
purposes or which it precursors for the manufacturing of drugs are useful for
disease therapy. The use of medicinal plants predates the introduction of antibiotics
and other modern drugs into the African continent. ������� Since medicinal plants do not merely save people from
feeling pain but also permit them to emerge unscathed, then they deserve
investigation. The active components in these medicinal attribute are
expected to be inimical to the growth of at least some microorganisms
especially the disease causing ones e.g. Escherichia
coli, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumoniae,
Pseudomonas aeruginosa etc. therefore, many
studies and researches had been done on the antimicrobial properties of many
plants but for this study, the leaf of Carica papaya will be discussed. Carica papaya
belongs to the family Caricaceae. It has the
following common names; pawpaw tree, papaya, papayer,
tinti, pepol, chich put, fan kua, wan shou kuo, kavunagaci,
kepaya etc. The parts that are usually used include
the leaves, fruit, seed, latex, and root. The plant is described as a fast
growing, erect, usually unbranched tree or shrub,
7-8m tall with copious latex, trunk of about 20cm in diameter. The plant is
also described in a documented property forms and it act as analgesic, amebicide, antibacterial, cardiotonic,
cholagogue, digestive, emenagogue,
febrifuge, hypotensive, laxative, pectoral,
stomachic and vermifuge. It is distributed
throughout Asia, Nigeria etc (Afolayan, 2003). Carica papaya contains many biochemically active compounds. Two important compounds
are chymopapain and papain,
which are supposed to aid in digestion. Papain is
used in the treatment of arthritis. The leaves of Carica papaya is used as soap substitute which are supposed to remove
stains. The papain, the proteolytic
enzyme has a wealth of industrial uses. It has milk-clotting (rennet) and
protein digesting properties. Active over a wide pH range, papain is used in medicine, combating dyspepsia and other
digestive orders. In liquid preparations, it has been used for reducing
enlarged tonsils. Nearly 80% of American beer is treated with papain, which digests the precipitable
protein fragmented and then the beer remains clear on cooling. Papain is also used for degumming
natural silk. But most of the papin imported in the
U.S is used for meat-tenderizers and chewing gums. Also used to extract the
oil from tuna liver cosmetically, it is used in some dentifrices, shampoos
and face-lifting preparations. Use to cleat silks and wools before dying and
to remove hair from hides during tanning (James, 1983). it is also used in
the manufacture of rubber from heaven (Morton, 1977). Recently, FDA has cleared
chymopapain for intradiscal
injection in patients with documented herniated lumbar inter-vertebral discs
whose signs and symptoms have not responded to conservative therapy over an
adequate period of time. The medicinal folk uses the
leaves poultice onto nervous pains and elephantoid
growths. The leaf smoked for asthma relief in various remote areas. Javanese
believe that eating papaya prevent rheumatism. Dietary papaya does reduce
urine acidity in humans while the flowers have been used for jaundice. The
young leaves and to lesser degree other parts contain carpain
an active bitter alkaloid which has a depressing action on heart. The plant
is strong amoebicide (Reed, 1976). The efficacy of treatment with C. papaya is dependent on the quantity
of the different compounds in the preparations. In Indonesia, papaya leaves
are used as feed for animals after parturition-2 leaves boiled in water fed
every 2 days for 1week. It also has been reported that papaya leaf extracts
is used as a profilaxis against malaria, though no
studies on this use could be found in the literature (Satrija
et al., 1994). In Nigeria, it is used for smooth
upper respiratory tract ailment and tumour (uterus). In Ivory Coast, it is
used for treating madness. In Trinidad, it is used for treating scorpion
bites and hypertension. In cote d�Ivoire and Sama,
it is used for toothache and tuberculosis in Mexico. In Honduras and Turkey,
it is used for liver ailments, constipation and laxative. In Philippines,
India, Malagasy and Malaya, it is used for treating arthritis and rheumatism.
In Java, Panama, Sri Lanka and Turkey, it is used for treating abortifacient. In Honduras, Japan, Panama and West
Africa, it is used for the treatment of diarrhea
and dysentery. Materials
and Methods Sterilization
of Materials All glasswares
were washed with detergent and rinsed with distilled water properly. These
were then air dried before wrapping with aluminium foil and sterilized in hot
air oven at 170�c for 2hours. Prepared media such as nutrient agar and nutrient
broth were sterilized in an autoclave at 121�c for 15minutes. Cork borer,
glass rods and forceps were sterilized by dipping in 70% ethanol which was
then flamed in Bunsen flame. The inoculating loop was also sterilized by
heating in Bunsen flame. The inoculating loop was also sterilized by heating
to redness using naked flame before and after each use. Collection
and Maintenance of Test Organisms The test organisms that were used
were all human pathogenic organisms from clinical origin. These isolates
include Escherichia coli, Pseudomonas aeruginosa, Klebseilla pneumoniae, Staphylococcus aureus,
and Proteus mirabilis. They were obtained from the Department of
Microbiology and Parasitology laboratory in
University of Ilorin Teaching Hospital (U.I.T.H).
The organisms were collected on sterile agar slants and incubated at 37�c for
48hours. They were then kept as stock cultures in the refrigerator set at
4�c. Biochemical analysis was carried out on each of the test organisms for
confirmatory purposes. Collection of Plant
Materials ����������� The plant Carica papaya was used for this project work and it was identified as
pawpaw leaf. These were then collected in a sterile polythene bag, rinsed, sundried and made into a powdery form before use. Preparation of Plant
Material ����������� The
pawpaw leaf Carica papaya was separately extracted with
cold ethanol, cold methanol and hot water. These were prepared using the
method as described by Oyagade et al., 1999. These were carried out by suspending 25grams of the
finely ground leaves in 125milliliter of distilled water or 95% ethanol or
methanol. The hot water extraction was done at 80�c in a water bath for
1/2hours. The ethanolic and methanolic
extraction was done at 28�1�c for 120hours. The extracts were then decanted
and filtered through a Whatman filter paper.� The filtered extract was then sterilized
using a membrane filter and evaporated to dryness at 45�c.� the residues obtained were reconstituted in
95% ethanol� at stock
concentration� of 0.2g/ml. the extract
solution were then stored in the refrigerator at 4�2�c until used (Omojosola and Awe, 2004). Standardization of
Test Organisms ����������� All
inoculums were standardized using the Mcfarland nephelometer method (Albert et al., 1991).� To prepare
this, 11 (eleven) large test tubes, 1% each of barium chloride and sulphuric
acid were also used. The protocol for preparing this solution is as stated in
the Table 1. ����������� Table 1 shows the various
concentrations of 1% each of barium chloride and sulphuric acid that would be
added in the various tubes. The reaction gives rise to turbid solutions but
the degree of turbidity differs in each test tubes. This are then kept on the
work bench for use. After then, liquid broth of the test organisms were made
in other test tubes and their turbidity were used to match the standard
solutions turbidity such that any one that has turbidity similar to the
standard solution is considered as having the corresponding number of
bacterial suspension per ml. However for this project, the standard that was
used is the one that corresponds to 15�108/bacteria suspension per
millilitre. To prepare this 0.5ml of already prepared nutrient broth was
pipette into a sterile test tube aseptically and the pure culture of the
particular test organism was dissolved into it until the bacterial suspension
was corresponding to the standard. Antimicrobial Assay
of Extracts The agar well method of the agar diffusion
technique was used to determine the antibacterial activity of the plant
extracts. One millilitre of� the
different standardized organisms were introduced separately and thoroughly
mixed with 30milliliters of molten nutrient agar each in a sterile Petri dish
and allowed to set then labelled. A sterile 8mm cork borer was then used to
punch holes (i.e. 5wells) in the inoculated agar and the agar was then
removed. Four wells� that were formed
were filled with different concentrations of the extract which were labelled
accordingly; 200mg/ml, 100mg/ml, 50mg/ml and 25mg/ml while the 5th
well contained the extractant i.e. the solvent used
for the extraction to serve as control. These were then left on the bench for
1hour for adequate diffusion of the extracts and incubated at 37�c for
48hours. After incubation, the diameter of the zones of inhibition around
each well were measured to the nearest millimetres along two axis i.e. 90� to
each other and the mean of the two readings were then calculated. Minimum Inhibitory
Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the
extracts ����������� The MIC of the extracts was determined
by using the broth dilution technique (Adebayo et al., 1989). The MIC helps to
measure more exactly the concentration of an antibiotic necessary to inhibit
growth of a standardized inoculums under defined conditions (Geo et al., 2001). Serial dilutions of
the extract in liquid medium were prepared. These were then challenged with
small inoculums of an overnight broth culture of the test organisms. The
culture was then challenged with small inoculums of an overnight broth
culture of the test organisms. The culture was then incubated at 37�c for
48hours. The smallest concentration that inhibits the growth was taken as the
MIC. The determination of the value of MBC follows the determination of MIC
by the broth dilution technique. The MBC is the lowest concentration of the
antibacterial agent that kills at least 99.9% of the test organism (Geo et al., 2001). To determination this
value, about ) 0.5ml of the sample was removed from the test tubes used in
the determination of MIC in which there was no desirable growth was spread
over the surface of the oven dried nutrient agar plates. The lowest
concentration of the agent that prevent the growth of less than 0.1% of the
test organism on the recovery plate was taken to be the MBC value for the
extract. Results and
Discussion Nature of Extracts ����������� The colours of the methanolic and ethanolic
extracts were both green while that of the hot water was brown (Table 2). The
presence of bioactive substances have been reported to confer resistance to
plants against bacteria, fungi and pests and therefore explains the
demonstration of antibacterial activity by the plant extracts used in this
study (Srinivasan et al., 2001). The results of this study showed that the organic
extracts were more effective than aqueous extracts and the methanol extracts
demonstrated the highest activity. This may be due to the better solubility
of the active components in organic solvents (de Boer et al., 2005). Among the Gram-positive and Gram-negative bacteria
tested against the root extract of C.
papaya, the Gram-negative bacteria were more susceptible especially P. aeruginosa
to the extracts. This result, however, is at disparity with an
earlier report indicating that plant extracts are more active against
Gram-positive bacteria than Gram-negative bacteria while that of the leaf
extract of C. papaya was next to
the most sensitivity with the Gram-negative bacteria especially Proteus mirabilis (Jigna
and Sumitra, 2006). There may be several factors
that will predispose bacteria to antibacterial agents such as previous
encounters with the agents or the nature of medium used, which may affect the
diffusability of the agent. The activity of the
extracts was comparable to those of antibiotics. The demonstration of
activity against the test bacteria provides scientific bases for the local
usage of these plants in the treatment of various ailments. The fact that the
extracts were active against both Gram-negative and Gram-positive bacteria
tested may indicate a broad spectrum of activity. This observation is very
significant because of the possibility of developing therapeutic substances
that will be active against multidrug-resistant
organisms. The
low MIC value observed for S. aureus is a
good indication of high efficacy against this bacterium. This outcome is
remarkable considering that boil, breast abscess and surgical wound infection
etc (caused by S. aureus) is on the rise and
also becoming recalcitrant to first-line antibiotics for its treatment in
developing countries, including Nigeria. High MIC may be an indication of low
efficacy or that the organisms have the potential for developing resistance
to the bioactive compounds. Temperature stability of plant extracts has been
reported earlier (Doughari, 2006). This may be an
indication that the bioactive compounds are heat stable and explains the
ethno-botanical application process of the plants where boiling at very high
temperatures for extended time periods are often practiced without the
concoctions losing their efficacy. ����������� The impotency of the order
extracts on the test organisms especially those of the methanolic
and ethanolic leaf extract of all the
concentrations used and the water extract for the root of C. papaya at concentrations 50, 100, 150 and 200mg/ml could be
as a� result of the following as stated
by Bernice (1997); 1.
Location of harvest
should never be areas treated with insecticides. 2.
Time of collection
should be when the leaf or plant sprouts most and collection should also be
during the day. 3.
Drying method. Sometimes,
active agents of leaves are destroyed by direct exposure to sunlight or by
drying in the hot air oven especially if the active agent is volatile. 4.
Once dried, herbs
should be dated, labelled and stored in area not exposed to light, moisture
or heat. 5.
It could also be due
to the extractant used and the method used to
obtain the active component (Unaeza and Abrikwa, 1989). Therefore, since this
work had revealed the ineffectiveness of methanolic
and ethanolic leaf extracts and aqueous and ethanolic (50mg/ml) root extracts, further research
should be made using the leaves dried at room temperature and not sun drying
nor oven drying to see if this had contributed to the impotency of the
extracts. ����������� It
could be however concluded that the demonstration of antimicrobial activity
against both gram-negative and gram-positive bacteria is an indication that
the plant is a potential source for production of drugs with a broad spectrum
of activity. The results of the study also supports the traditional application
of the plant and suggests that the plant extracts possess compounds with
antibacterial properties that can be used as antibacterial agents in novel
drugs for the treatment of gastroenteritis, uretritis,
otitis media, and wound infections. Further pharmacological
evaluations, toxicological studies and possible isolation of the therapeutic
antibacterial from this plant are the future challenges. References Adebayo, A. C., Oloke, J. K., Aladesanmi, A. J. 1989. Antimicrobial activities of the leaf of Eugenia uniflora. Phytotherapy Res. 3(6): 258-259.��� Afolayan,
A.J. 2003. Extracts from the shoots of Arctotis artotoides inhibit the growth of
bacteria and fungi. Pharm. Biol. 41: 22-25.���� Ahmad, I. and
Beg, A. Z. 2001. Antimicrobial and phytochemical studies
on 45 Indian medicinal plants against multi-drug resistant human pathogens. J. Ethnopharm. 74:
87- 91. Albert
B, William, J. H, Jr., Keneth,
L. H, Henry, D. I., and Jean, H. S. 1991. Manual
of Clinical Microbiology. American Society for Microbiology. Baladrin,
M. F., Clocke, J .A, Wurtele,
E. S. and Bolinge, W. H. 1985. National Plant
Chemicals: Source Industrial and Medicinal Materials Science, pp. 228,
1154-1160. Benjamin,
T.V. and Lamikanra, A. 1981. Investigation of Cassia alata, a
plant use in Nigeria in the treatment of skin diseases. Quart J. Guide Drug. 19 (2&3), 93-96. Bernice,
T. 1997. Medicinal Herbs: Current uses of some not so new medicines. PMAP
Conference 1997. Bibitha,
B., Jisha, V. K., Salitha,
C. V., Mohan, S., and Valsa, A. K. 2002.
Antibacterial activity of different plant extracts. Short Communication. Indian J. Microbiol.
42: 361-363. Chinoy,
N. J. Joshi H. and Ghosh S. (2007. Antifertility investigations of alcoholic papaya seed
extract in female rats. Journal of
Medicinal and Aromatic Plant Sciences
19 (2):�� 422-426. Chinoy,
N. Joshi, H. and Ghosh, S. 2007. Reversible effects
of aqueous extract of papaya seed on microenvironment and sperm metabolism of
cauda epididymis of rat. Journal of Medicinal and Aromatic Plant Sciences 19 (3):
717-723. de Boer, H.
J., Kool, A., Broberg,
A., Mziray, W. R., Hedberg,
I. and Levenfors, J. J. 2005. Antifungal and
antibacterial activity of some herbal remedies from Tanzania. J. Etnopharm.
96: 461 469. Doughari,� J. H. 2006. Antimicrobial activity of Tamarindus indica Linn.
Tropical J. Pharm.
Res. 5(2): 592-603. Fawole, M. O. and Oso, B. A. 2004. Laboratory
Manual of Microbiology. Spectrum Book Ltd., Ibadan,
Nigeria. Pp. 15-25. Garrod,
L. P., Lambert, H. P. and O�Gray, F. 1995. Antibiotics and Chemotherapy, 4th
Ed, Churchill, Livingstons, Edinburgh, London and
New York. pp.501-512. Geo,� F., Brooks, J. S. B. and Stephen,� A. M. 2001. Jawetz, Melnick and � Adelberg�s� Medical Microbiology,� 22nd Ed. pp. 607. Gerard,
J.T., Berdell, R. F. and Christine, L.C. 1992. An Introduction to Microbiology, 14th
Ed, The Benjamin and Cumings Publishing Company,
pp. 220-225. James,
A. D. 1983. Handbook of Energy Crops,
Unpublished, Purdue online. Jigna,
P. and� Sumitra,
C. 2006. In-vitro antimicrobial activities of extracts of Launaea
procumbns Roxb. (Labiateae), Vitis vinifera L. (Vitaceae) and Cyperus rotundus L.
(Cyperaceae). Afr. J. Biomed. Res. 9(2): 89-93. Kafaru,
E. 1994. Immense Help from Natives
Workshop, 1st Ed, Elizabeth Kafaru,
Lagos, Nigeria. pp. 11-14. Mitscher,� L. A., Ryey Ping,
L., Bathala, M.S., Wu-wu-Nan,
D. and Roger, W. 1992. Antimicrobial agents from Higher Plants :
Introduction, Rational and Methodology, Llaydia 35(2). Morton,
1977. FDA� DrugBull 12 (3): 17-18. ����������� Omojasola,
P. F. and Awe, S. 2004. The Antibacterial activity of the leaf extract of Anacardium occidentale and
Gossyptum hirsutum against
some selected microorganisms. Bioscience Research Communication 16 (1). Osborn,
E. M. 1943. The occurrence of antibacterial substances in green plants. Brit .J.Exp Pathol. pp. 24-227. Oyagade,
J.O., Awotoye, J.T, Adewumi,
A., Thorpe, H.T. 1999. Antimicrobial activity of some Nigerian medicinal
plants. Bio. Res. Comm. 11(3): 193
� 197. Reed,
L.J. and Muench , H. 1976. A simple method of
estimating fifty percent end� point. Chemotherapy 22:211-220. Satrija,
F., Nansen, P., Bjorn, H., Murtini
S and He, S. 1994. Effects of Papaya latex
Ascaris suum in
naturally infected pigs. Journal of Helminthology, 68: 343-346. Sofowora,
A. 1982. Medical Plants and Traditional
Medicine in West Africa, 1st Ed., John Wiley and Sons, London.
pp. 5-22. Sofowora,
A. 1982. Medical Plants and Traditional
Medicine in West Africa, Spectrum Books Ltd., Ibadan,
in association with John Wiley and Sons, London. pp. 204-208. Srinivasan,� D., Perumalsamy,� L., Nathan, P. Sures,
T. 2001. Antimicrobial activity of certain Indian medicinal plants used in
folkloric medicine. J. Ethnopharm. 94: 217-222. Unaeze,
M.C. and Abarikwa, P. O. 1986. Antimicrobial
activity of certain medicinal plant used in traditional medicine in Nigeria:
A preliminary study. Journal of
Microbiology 6 (1-2), 32-40. Table 1. Protocol for
test tubes for Mcfarland nephelometer
in 10ml.
Source:
Albert et al., (1991). Table 2. Nature of Carica papaya extracts.
Table 3. Minimum
Inhibitory Concentration Of Carica papaya.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||