677 | P a g e International Standard Serial Number (ISSN): 2319-8141

International Journal of Universal Pharmacy and Bio Sciences 2(6): November-December 2013

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES
IMPACT FACTOR 1.89***
ICV 5.13***
Pharmaceutical Sciences RESEARCH ARTICLE!!!

INVITRO XANTHINE OXIDASE INHIBITORY ACTIVITY OF VARIOUS PART OF

CARICA PAPAYA.

STALIN.C*, VIVIEKANANDAN.K, SYAMALA S, SUSANA B, REDDY VAHIDA S.

Department of Pharmacology, Raos college of pharmacy, Nellore.

KEYWORDS:
Carica papaya, Xanthine
oxidase, Allopurinol.
For Correspondence:
STALIN.C*
ABSTRACT
Aim of the Study: To evaluate the in-vitro xanthine oxidase
inhibitory activity of the ethanolic extract of various parts
(leaves, petioles, seeds, unripe fruit peels and flowers) of Carica
papaya. Materials and Methods: xanthine oxidase inhibitory

Address: activity was assayed spectrophotometrically under aerobic

Department of conditions and the degree of enzyme inhibition was determined

pharmacology, Raos by measuring the increase in absorbance at 295nm associated

Pharmacy College. with uric acid formation. Results and Discussion: The plant

Nellore , Andhra was selected based on their frequent medicine usages. Among

Pradesh,India. the six extracts tested the leaf extract of Carrica papaya

Tel.: +91-9160348634. exhibited highest potency of xanthine oxidase inhibition

E- Mail address: (42.890.64g/ml) this was followed by unripe fruit extract

stalinmpharm@gmail.co (63.760.39g /ml), unripe fruit peel extract (72.290.42g

m. /ml), petioles extract (89.230.32g /ml), flower extract

(92.660.42g /ml).Compared with Ic50 value of the standard
allopurinol of 6.6 g/ml.

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INTRODUCTION:
Enzymatic degradation of hypoxanthine and xanthine leads to the production of uric acid. Gout and
hyperuricemia are some of the common metabolic disorders in humans associated with an reduced uric acid
level in the blood and it leads to the deposition of urate crystals in the joints and kidneys leads to Gouty
arthritis and uric acid nephrolithiasis 1. Xanthine oxidase and xanthine dehydrogenase are two interconvertable
forms of same gene product known as Xanthine oxidoreductase. It catalyses hypoxanthine to xanthine and
then to uric acid by oxidation process and final reaction in the metabolism of purine bases with generation of
2
reactive oxygen species, Superoxide anion radical (or) hydrogen .The deposition of needle shaped
monosodium urate crystals in synovial fluid of the major joints produces an severe painful arthritis with
repeated attacks of Gout. The increased risk of hyperuricemia has been also joined with the development of
hypertension (blood pressure) hyperlipidaemia, cancer, diabetes and obesity. Xanthine oxidase may also
involve in pathogenesis of Neutrophil mediated Lung injury 3, Congestive heart failure 4 and may contribute to
cerebral ischemia inflammation 5 and Neurogenerative disorders. 6
The treatment of Gout is increasing excretion of uric acid or reducing the uric acid production. Xanthine
oxidase inhibitors are very useful because they possess lesser side effects compared to Uricosuric and anti-
inflammatory agents4.
Allopurinol is the only clinically used Xanthine oxidase inhibitor also having many side effects such as
7,8
Hypersensitivity syndrome, Stevens Johnson syndrome and renal toxicity . It is necessary to develop
compounds with Xanthine oxidase inhibitory activities which are devoid of the undesirable side effects of
Allopurinol. A potential source of such compounds can be obtained from medicinal plants8. Flavonoids and
polyphenolic crude extracts have been reported to possess Xanthine oxidase inhibitory activity 9,10.
Many Indian medicinal plants have been used for the prevention and treatment of Gout, Hyperuricemia, and
related inflammatory disorders 11 but they lack scientific evidence.
Carica papaya belonging to the family Caricaceae. It is used as a Medicine for Dyspepsia, Hyperacidity,
Dysentery and Constipation12. It is reported that it helps in the prevention of Diabetic Heart diseases, Papaya
lowers high cholesterol level as it is a good source of fibre. Carrica papaya is used for the treatment of
numerous diseases like Warts, Corns, Sinuses, Eczema, Cutaneous tubercles Glandular tumours, Blood
pressure; Amenorrhoea.
Carica papaya contain a milky juice like enzyme called papain which is present in the bark, leaves, peel
extract and fruit extracts are dried and used as a chewing gum for digestive problems, tooth paste and meat

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 679 | P a g e International Standard Serial Number (ISSN): 2319-8141

tenderizer. It also contain many biological active compounds include chymo papain and papain which is
the ingredient that aids in digestive system and Arthritis.
Collection and authentication of plant
The whole plant of carica papaya (Carricaceae) was collected from the local regions of Nellore, identified
and authentified by Dr.Madhava chetty Department of Botony, SV.University Tirupathi chittoor(dist).
Andrapradesh.
Plant collection: the plant material consists of dried powered leaves seeds; peels; unripe fruit of Carica
papaya belonging to the family Caricacae.
Preparation of the extract: Seed and unripe fruits of the Carica papaya plant were collected and dried in
shade under room temperature; powdered and sieved through No.20mesh sieve. The finely powdered leaves
are kept in an air tight container until the time of use. About 120g of the dried powdered was soaked with
1200ml of ethanol: water (6:4) for 12h and then macerated at room temperature using a mechanical shaker for
4hr .the extract was filtered off and the marc was again filtered .the filtrates were then combined; concentrated
under reduced pressure and evaporated at 40degee centigrade. About 50g of peel powdered was taken in
Soxhlet apparatus; and the extracted with 450ml of ethanol: water after extractions; filtered and the filtrate was
concentrated and evaporated at 40 degrees centigrade. the fresh juice was collected from the fruit and dried at
40 degrees .
DRUGS AND CHEMICALS: Xanthine oxidase , xanthine and allopurinol were purchased from Himedia
Labs , Pvt Ltd, Mumbai , India. All other drugs and chemicals used in the study were obtained commercially
and were of analytical grade.
20
In vitro xanthine oxidase inhibitory (XOI) activity: The xanthine oxidase inhibitory activity was
assayed spectrophotometrically under aerobic conditions using xanthine as the substrate. The extract and
the standard drug allopurinol (1 mg/ml) were prepared by dissolving in dimethyl sulphoxide (not
exceeding more than 5% of total volume) initially and then made up to the required volume with potassium
dihydrogen phosphate buffer (pH 7.5). The assay mixture consisted of 1 ml of extract at different
concentrations (5-100 g/ml), 2.9 ml of potassium dihydrogen phosphate buffer (pH 7.5) and 0.1 ml of
xanthine oxidase enzyme solution (0.1U/ml in potassium dihydrogen phosphate buffer, pH 7.5, prepared
immediately before use). After preincubation at 25C for 15 min, the reaction was initiated by the addition
of 2 ml of substrate solution (150 M xanthine in phosphate buffer, pH 7.5). The assay mixture was
incubated at 25C for 30 minutes the reaction was then stopped by the addition of 1N HCl. The absorbance
was measured at 290 nm against blank. Allopurinol (5 100g/ml), a known inhibitor of XO, was used as
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the positive control. One unit of XO is defined as the amount of enzyme required to produce 1mmol of uric
acid/min at 25C 12,13. XOI activity was expressed as the percentage inhibition of XO in the above assay
system calculated as
% Inhibition = {(A-B) (C-D) / (A-B)} X 100
Where A is the activity of the enzyme without the fraction, B is the control of A without the fraction and
enzyme; C and D are the activities of the fraction with and without XO, respectively. The assay was done
in triplicate and IC50 values were calculated from the percentage inhibition.
RESULTS AND DISCUSSION
Recent findings show that the occurrence of gout is increasing worldwide, possibly due to the changes in
dietary habits like intake of food rich in nucleic acids, such as meat, sea foods, etc. 9. Hypouricemic agents
are commonly employed for the treatment of chronic gouty arthritis, which includes xanthine oxidase
inhibitors and uricosuric agents 14.
Xanthine oxidase inhibitors such as allopurinol interfere with the conversion of hypoxanthine to xanthine
and then to uric acid. In general, allopurinol is the drug of choice; however it has been observed that
allopurinol induces side effects such as hepatitis, nephropathy and allergic reaction. Thus, new alternatives
with an increased therapeutic activity and less side effects are desired. An alternative to allopurinol is the
use of medicinal plants, which possess phytochemical constituents like flavonoids are a group of
polyphenolic compounds, which have been reported to possess xanthine oxidase inhibitory activity. We
thus began our program to look for Xanthine oxidase inhibitors of phytochemical of dried powered leaves,
petioles ,seeds, unripe fruit peels and flowers, of Carica papaya belonging to the family Caricacae which
are traditionally used by the local people and tribals in India to treat various diseases.
All the extract elicited a dose- dependent inhibition of xanthine oxidase resulted in a decreased production
of uric acid which was measured spectrophotometrically .Of the 6 extracts assayed 5 extracs (whole plant
of the Carica papaya) demonstrated XOI activity at a concentration of 100/ml; showing an inhibition
greater than 50%. Carica papaya leaves extract was found to have the highest activity (68.520.64) at a
concentration of 50g/ml. All the 5 extracts showed IC50 values below 100g/ml concentration. Seeds
extracts did not show prominent xanthine oxidase inhibitory activity. In general, the leaf extracts were
found to be more active than the unripe fruit peels and flower extracts. The IC50 values of leaves extract
were found to be 42.890.64g/ml respectively. These results were compared with that of the standard
drug allopurinol, (Graph-1) which showed 91.021.28 % inhibition at 100 g/ml concentration with an
IC50 value of 6.60.20g/ml (Table-1)
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Conclusion
Recent findings showed that the occurrence of gout is increasing worldwide, possibly due to the changes in
dietary habits like intake of food rich in meat, sea foods etc. Xanthine oxidase inhibition such as
allopurinol in the use of medicinal plants, we thus began our work to both for xanthine oxidase inhibition
of phytochemical origin.
Finally it was concluded that the ethanolic extract of Carica papaya leaves have shown promising
Xanthine oxidase inhibitory activity as compared to the seeds, petioles, flowers, unripe fruits and unripe
fruit peels.
Table-1 In vitro xanthine oxidase inhibitory activity of the extract leaves, petioles, seeds, unripe fruit
peels and flowers of Carica papaya
Extracts Percentage Xanthine Oxidase Inhibition
IC50(g/ml)
5g/ml 10g/ml 25g/ml 50g/ml 100g/ml

leaves 23.540.96 34.710.29 41.250.53 54.440.89 68.520.64 42.890.64

Petioles 12.620.25 19.820.33 28.650.94 41.280.99 51.350.48 89.230.32

seeds 7.720.46 16.610.37 23.720.84 36.240.88 48.430.32 -

Ethanolic Unripe fruits 21.810.53 27.710.42 39.810.74 47.180.78 69.540.81 63.760.39
extract of
carica Unripe fruit
15.180.68 22.560.43 29.980.62 44.720.67 56.610.74 72.290.42
papaya peels

flowers 19.660.78 32.380.55 39.480.56 42.580.57 51.740.68 92.660.42

Allopurinol (Std)
44.870.32 65.380.55 75.630.84 84.610.55 91.021.28 6.660.20

Values are mean SEM of three parallel measurements.

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Graph-1 Comparison of xanthine oxidase inhibitory activity of various extracts

100

90

80
leaves
70
%Xanthine Petioles
60
oxidase seeds
50
inhibition Unripe fruits2
40 Unripe fruit peels
30 flowers
20 Allopurinol

10

0
5g/ml 10g/ml 25g/ml 50g/ml 100g/ml
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