Antiinflammatory Activity of Ethanolic Leaf Extract from Carica papaya in Rats Orogastrically Dosed with Salmonella typhi and Staphylococcus aureus
The anti-inflammatory activity of ethanolic extract of Carica papaya was investigated in Swiss Albino rats with induced multiple infections from Staphylococcus aureus and Salmonella typhi. The first group was given the standard inoculums of Staphylococcus aureus only while the second group was given the standard inoculum=s of S. aureus and treated with ethanol extract of Carica papaya. The rats in the third group were given standard inoculums of Salmonella typhi only and those in the fourth category were given standard inoculums of S. typhi and treated with the extract. The Albino rat in the last group was placed on basal diet and water only to serve as the control. The anti-inflammatory activity of the extract as haematological indices was determined by monitoring the amount, occurrence and distributions of total and differential White Blood Count (WBC), haemoglobin level and Pack Cell Volume count (PCV) before and after infection with the pathogenic bacteria. The rats infected with Staphylococcus aureus without extract treatment gave a PCV value of 33%, a WBC of 3650 mm3, an Hb value of 2.2H1012 LG1, a neutrophil count of 60%, lymphocyte 40% and monocytes of 1%. The rats infected with Staphylococcus aureus and treated with C. papaya showed an increase in PCV count of 36%, the WBC increase to 4050 mm3, an Hb count 2.4H1012 LG1 and increase in neutrophil to 74% and reduction in lymphocyte to 24% while the monocytes count was 2%. The rats infected with S. typhi only gave a PCV count of 24%, WBC 2050 mm3, a RBC 1.0H1012 LG1, a neutrophil value of 60%, lymphocyte 38% and monocytes count of 2%. The rats infected with S. typhi and treated showed an increase in PCV value to 38%, reduction in WBC to 4275 mm3, an Hb count of 1.9H1012 LG1, neutrophil 67%, a lymphocyte count of 30% and Eosiniphils 1%. The control group gave values that were within the acceptable limit. The urinalysis showed that the rats infected with Staphylococcus aureus only had a pH of 6, negative to glucose and nitrite, positive to ascorbic acid, ketone, bilirubin, blood (Ca250) and normal urobilinogen with 100 mg mLG1 of protein. The group infected with Staphylococcus aureus and treated with the extract showed an increase in pH to 7, negative to glucose, ascorbic acid, ketoses and nitrite, 30 mg mLG1 of protein, bilirubin, blood (Ca50) and urobilinogen were in the normal range. The animals infected with Salmonella typhi only showed a pH of 6, negative to glucose, bilirubin and blood (Ca50) and permissible level of urobilinogen. The rats infected with Salmonella typhi and treated with the extract showed reduction in pH to 5, negative to glucose, ketone, nitrite and blood, positive to ascorbic acid, protein, bilirubin and normal urobilinogen. The values of all the urinary compositions were normal except blood (Ca250) for the control. The results obtained justify the scientific bases for the use of the plant in ethnomedicine.
There is an ever growing interest in investigating difference species of plants
to identify their potential therapeutic applications. This is due to a tremendous
historical legacy in folk medicine use of plants as remedy for treating diseases
(Steenkanp, 2003). The use of traditional medicine remain
widespread in developing countries while the use of complementary medicine is
increasing rapidly in developed countries (Oladunmoye, 2006).
Their uses have a comparative advantage over the conventional chemotherapeutic
agents for their easy availability, cost effectiveness, accessibility and presumed
safely. In the past, scientific studies on plants used in ethnomedicine have
led to the discovery of many valuable drugs such as pilocarpine and vincrinstine
and others (Akinyemi et al., 2003).
Researches on the chemical and biological activities of plants during the past
two centuries have yielded compounds for the development of modern drugs (Arivazhagan
et al., 2000).
Despite little information on the composition and biological activity of many
plants substances, there has been little effort developed to the development
of chemotherapeutic and prophylactic agents from these plants (Boom,
1989). The use of medicinal plants all over the world predates the introduction
of antibiotics and other modern drugs into Africa continent (Akinyemi
et al., 2003; Oladunmoye, 2006).
Vertebrates are continually exposed to micro organisms and their metabolic
products that can cause disease. The immune system is composed of widely distributed
cells, tissues and organs that recognize foreign substances and microorganisms
and act to destroy them. Immunity refers to the specific defensive response
of a host to an invasion by foreign organisms (Weir and Stewart,
The plant Carica papaya belongs to the family Caricaceae. C. papaya
biologically active compounds are chymopapain and papain which helps to
aid digestion. Vitamins and traces of an alkaloid called carpain have also been
found in the latex. Apart from natural oils, the seeds of the fruit also contain
carbohydrates, carpasemine benzyl senevol and a glycoside (Arivazhagan
et al., 2000). Other compounds found in the parts of Carica papaya
are the Nicotine, tannins and Flavones (Atlas, 2004).
Carica papaya can be used as a diuretic, antihelmintic and to treat
various disorders. Parts of the plant are also used to combat dyspepsia and
other digestive disorders (Akah et al., 1997).
It also has the ability to inhibit Candida albcans. The extracts have
a bacteriostatic property against Staphylococcus aureus, Salmonella
typhi, Bacillus subtilis and other bacteria. Furthermore, Alpha-D
mannosidase and N-acetyl beta D glucosaminidase isolated from the latex acted
synergistically to inhibit yeast growth. Its antiinfertility properly is well
documented (Chinoy and Padman, 1996; Chinoy
et al., 1997).
In vitro assessment of the antimicrobial activity of C. papaya
have been reported (Akah et al., 1997); which
was linked to the presence of certain biomocules like saponin, tannin, flavonoids
and some essential oils (Arivazhagan et al., 2000).
However, investigations regarding the anti-inflammatory activity of the plant
were neglected or if available at all are largely unknown. The lack of scientific
investigations in this area prompted the present study which was aimed at assessing
the anti-inflammatory potentials of ethanolic leaf extract from C. papaya
in animals with induce infections with S. aureus and S. typhi
by monitoring certain haematological indices involved in inflammatory responses
in animals and thus establish the in vivo antimicrobial activity of this
MATERIALS AND METHODS
Collection, Extraction and Fractionation
The Carica papaya plant was collected from the vicinity of Federal
University of Technology, Akure in June 2004. It was identified by Dr. M.A.
Awodun of Crop, Soil and Pest Management Department, Federal University of Technology,
Akure, Nigeria. The leaves of Carica papaya were sun dried for six days
and then blended with blender into powdery form. A 95% ethanol was the solvent
for the extraction. The extract obtained was concentrated in rotary evaporator.
The extract was dissolved in 0.1 M Tris-HCL buffer (pH 7.0, 5 mL) and applied
to a column (5x85 cm) of Sephacryl S-300 HR, pre-equilibrated and developed
with the same buffer. Fractions corresponding to the peak were pooled together
concentrated and freeze dried. The powder was dissolved in water and applied
to a Sephadex G-25, column (1.5x50 cm), then eluted with water and fractions
were collected. The eluate obtained was concentrated and lyophilized.
Source of Microorganisms Used and Preparation of Standard Inoculums
The pure isolates of Staphylococcus aureus and Salmonella typhi
used in this study were obtained from Microbiology Department, Obafemi Awolowo
University, Ile-Ife, Nigeria. The isolates were maintained in pure culture on
agar slants and store in refrigerator prior to use. Standard inoculums were
prepared from the broth cultures of the organisms and adjusted to contain approximately
1.0x106 cfu mL-1 using Mac- Ferland turbidometry standard.
Source of Laboratory Animals Used
Swiss albino rats were obtained from Pharmacy Department, University of
Ibadan, Oyo state. Nigeria. The rats have average body weights between 110 and
200 g. The rats were given animal feeds (Bendel Feeds) and water ad libitum.
They were housed in standard environmental conditions of temperature and
humidity and a 12 h light and 12 h dark cycle. All international ethics and
guidelines with respect to animal care in research were carefully followed and
The rats were divided into five groups of eight per treatment. Two groups
were given the standard inoculums of Staphylococcus aureus and Salmonella
typhi each. Two other groups were each treated with the ethanolic extract
of Carica papaya after inoculation with the two pathogenic bacteria.
The last set of animals was feed with basal diet and water only and serves as
the control. Haematological test and urinalysis of the various treatments were
carried out before and after infection.
Total and differential WBC, PCV and haemoglobin concentration were determined
using standard methods with slight modifications. Total White blood count was
estimated using the haemocytometer method. Packed cell volume measurements was
carried out using the microhaematocrit technique by the use of a microhaematocrit
centrifuge and spinning for 5 min at 1000 rev min-1 before reading
with the haematocrit reader. Hemoglobin levels were measured colorimetrically
by the oxyhaemoglobins methods using Reicherts haemoglobinometer while
the differential is done by the use of Leishmans stain before viewing
under the microscope (Baker et al., 2001).
Urinalysis was carried out by the using standard method to determine blood,
urobilinogen, bilirubin, protein, nitrite, ketone, ascorbic acid, glucose and
pH in urine (Monica, 2002).
RESULTS AND DISCUSSION
The total and differential White Blood Counts (WBC), Packed Cell Volume (PCV)
as well as the Hemoglobin concentration (Hb) were found to differ in animals
that were administered with C. papaya extract after inoculation with
the pathogenic bacteria and those that were not treated (Table
1). The ability of the extract to alter the distribution and occurrence
of lymphocyte, neutrophil, eosiniphils, basophil and monocytes suggest the potentiality
of the extract acting as an immunostimulant. The rats infected with S. aureus
and not treated with extract (A) showed a lower WBC than the treated (B) and
the control group (E). This is contrary to the expectation as more white cells
were suppose to be produced during infection in readiness for phagocytosis.
The unexpected result might be explain in terms of the physiology of S. aureus
that are known to produce coagulase enzyme (Jawetz et al.,
2004). The enzyme helps in blood clotting by converting soluble fibrin in
the blood to insoluble fibrinogen. This might have form a protective coat around
the cell of the bacterium and thus protects it from phagocytosis. However neutrophil
count was higher and lymphocyte reduced in the animals that were infected but
treated with extracts than the infected but without orally treated with the
plant extract. The reduction may result from possible migration into tissue
in response to the infection from the pathogenic organism. Neutrophil is the
most abundant circulating granulocyte and their granules contain numerous microbicidal
molecules and when a chemotactic factor is produced as a result of infection
or injury, in an extracellular site, these cells enter the tissues Weir
and Stewart, 1999). However the trend was reversed with lymphocytes that
remain higher in untreated animals (A and C) than treated after infection with
the pathogens (B and D). This can be explain in terms of lymphocyte remaining
the only freely circulating granulocyte after migration of neutrophil into tissue
during inflammation. The lower values in the treated animals are probably due
to the in vivo antibacterial activity of the extract against the pathogen.
This antagonistic property can be linked to the presence of certain biomolecules
of pharmacological importance in the plant. Phenolic derivatives had been known
to be potent antimicrobial agents (Oladunmoye, 2006).
The data relating the amount of the various types of granulocytes obtained from
rats with salmonella infection followed similar pattern with those of S.
aureus. The same reason adduced to the former observations may also be responsible
for these findings.
The haemoglobin level and PCV were higher in infected but treated groups of
rats (Band D) than those infected without oral administration of extract from
Carica papaya (A, C and E). Acute inflammation from most pathogenic microorganisms
results in haemolysis which is manifested in lower haemoglobin level and PCV
(Kumarnsit et al., 2006). The higher values of
these haematological indices in rats treated with the extract after infection
from the pathogens can be due to their inability to cause haemolysis resulting
from the anti-inflammatory potentials inherent in the C. papaya
The values showed that the protein level was abnormally high during infection
with S. aureus but became normal after the infectivity stage (Table
2). The amount of blood was equally high. This might be that during infection
with these organisms, there was inflammation of the excretory organs and this
became manifested in the urinary composition.
The values however became normal after treatment of the infected rats with
the extract suggesting a possible killing of the pathogens and thus their inability
to cause inflammation in the excretory organs. This may also be as a result
of presence of phytoreactants in the plant (Trease and Evans,
RECOMMENDATIONS AND CONCLUSIONS
This research clearly show that extract of C. papaya has anti-inflammatory activity in rats orogastrically dosed with S. aureus and S. typhi as revealed by the distribution, occurrence and patterns of certain haematological indices produce during inflammatory process. This activity was also confirmed by the variation in composition of rats urine during infection and after the infectios was treated with the plant extract. These findings thus provide scientific bases for the use of C. papaya in ethnomedicine.
Further research is necessary to establish the actual constituent(s) responsible for the anti-inflammatory activity. There is also the need for toxicological studies of the extract for possible histopathological damages to the vital organs using enzyme markers.
The technical assistance of Mr. B.A. Erinle of the University Health Center, Federal University of Technology, Akure, Nigeria for the haematological analysis is appreciated.
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