Evaluation of Anti-Oxidant and Anti-Pyretic Activity of Fruit of Garcenia Padunculata

Most of the synthetic drugs used at present as analgesic and anti-inflammatory agents cause many side effects and toxic effects. Many medicines of plant origin with analgesic and anti-inflammatory activity have been used since long time without adverse effects. The purpose of the study was to explore the anti-pyretic and anti-oxidant activity of fruits of Garcinia pedunculata. The fruit of G. pedunculatawas collected from Shillong, Meghalaya and extraction is done by using methanol as a solvent. The methanol extract shows the presence of carbohydrate, glycoside, alkaloids, phenol and flavonoids. Acute antipyretic activity for the extract was investigated in pyrexia rat. Temperature level was determined after 1hours, 4 hours, 8 hours and 12 hours after giving the extract dose of 100mg/kg and200mg/kg body weight, and it was found to produce a significant reduce in fever. DPPH radical scavenging activity (In-vitro anti-oxidant activity of G. pedunculata) of methanolic extract shows 38.31 as IC50 (μg/ml) whereas Ascorbic acid as standard in the same concentration shows34.81 IC50 (μg/ml). The obtained result justified the traditional use of G. pedunculata as anti-pyretic and antioxidant purpose.


INTRODUCTION:
ever or pyrexia is an elevated body temperature above the normal level ranges 36.5-37.5 °C (97. 7-99.5 °F) characterized by an increase in thermoregulatory set point, which results from the interaction of the central nervous and immune system 1 . Fever is body's natural defense mechanism against infectious agent which can damage the tissue. This triggers the enhanced formation of pro-inflammatory cytokines like necrosis factor TNF-α, interleukin1β, Prostaglandin PGE-2 which increases near the hypothalamus area and thereby triggers the hypothalamus to elevate the body temperature 2 . The treatment for pyrexia is most often done by using synthetic NSAIDs which are very toxic to the body, thus it has been focus on the medicinal plant which having large numbers of evidences and has increased their uses all over the world as various folk medicines 3 . Generation of free radicals or reactive oxygen species (ROS) from various metabolism or may be environmental sources has found to interact continuously in biological system and their uncontrolled formation may directly correlates with the molecular basis of various diseases along with the lipid peroxidation 4 . Plants are rich in antioxidants; so much attention has been directed towards the development of ethnomedicines because of their safety profile and reach in valueable chemical constituents such as phenols, flavonoids, alkaloids, glycosides, tannins, vitamins, terpenoids, and many more phytochemical constituents which are responsible for various pharmacological activities 5 . Recent research data has reported that ingestion of the natural antioxidants has been found to be associated with less risk of many graded diseases 6 .
Garcinia. pedunculata, popularly known asbor thekera in Assamese and soh danei in khasi, it is an evergreen tree related to the more familiar purple mangos teen (Garcinia mangostana). Garcinia is well flourish in evergreen forest mostly available in areas having low rain fall 7 . It grows wild in the Ri-Bhoi region of state Meghalaya and also in sunitpur district, Assam, having a high content of polyphenols, flavonoids, tannins, alkaloids, glycosides and ascorbic acid which are natural anti-oxidant and anti- aflatoxins that help in preventing the coronary artery disease and atherosclerosis 8 . There are claimed that it has a number of medicinal benefits right from the anti-diarrheal, anti-diabetic, anti-cancer to anti-flatulent. Besides cure and control of disease G. pedunculata protects internal organs like liver, kidneys, and pancreas 9 .

MATERIAL AND METHODS:
Collection of the plant material was done from Shillong, Meghalaya, identification and authentication of plant material G. pedunculata used in this study were obtained from The Department of Botany, Guwahati University, Assam, India.

Preparation of plant extract:
G. pedunculata fruits were dried at room temperature until a constant weight was obtained then pulverized into fine powder. 100g powder was taken for soxhlet extraction by using 1000 ml of methanol as solvent for 72 hours. The mixture was filtered and evaporated to dryness under reduced pressure in a rotary evaporator. The extract was reconstituted into doses of 150, 300 and 600 mg/kg body weight which were used for the experiment 1 .

In-vitro antioxidant activity:
DPPH radical scavenging activity: The free radical scavenging activity of six different essential oils was measured by 2-2-diphenyl-2picrylhydrazyl (DPPH). One ml of 0.1 m mol solution of DPPH in methanol was mixed with 0.3 ml of methanolic extract of G. pedunculata in various concentration (20, 40, 80, 160 mcg/ml) during 30 min at room temperature and the absorbance was recorded at 517 nm using UV visible spectrophotometer. Each experiment was performed with appropriate blank 10 . A positive control without extract was set up in parallel. Ascorbic acid at various concentrations was included as a standard. The scavenging activity of the extract was estimated base on the percentage of DPPH radical scavenged (1%) using equation- Where A sample is the absorbance of a sample solution, and A control is the absorbance of the control solution (containing all of the reagents, except the test sample) 10,11 .

In-vivo Anti-pyretic activity:
The experiments was designed by taking groups rats (n=6) were injected subcutaneously with 10/kg body weight of 10% yeast solution to induce hyperthermia. The rectal temperature of each animal was recorded prior to the 24 hours of yeast solution injection. Thereafter, the test group was treated orally with 1ml (500 mg/kg body weight) crude suspension of methanolic extract ofG. pedunculata. Similarly for positive control group 1 ml (4 mg/kg body weight) aqueous solution of indomethacin was give orally. Post treatment the rectal temperature of each animal was measured and recorded at 60, 90, 120 minutes. Each reading was calculated as the mean of three readings 12,13 .

Experimental animals:
Healthy rats (50-60gm) were used for testing the antipyretic activity. Animal were procured and kept in animal house for 15 days prior to the study. All the procedure was carried out as per the norms and regulation of CPCSEA and IAEC of Assam down town University which has approved the study (AdtU/IAEC/2017/03, dated 11/11/2017).

RESULTS AND DISCUSSION:
Qualitative Phytochemical Estimation: The methanolic extract was examined for preliminary phytochemical screening by using different standards test reagent shows the presence of alkaloids, phenolic groups, tannins, glycosides flavonoids and carbohydratein the investigation, on the basis of secondary metabolites as shown in (Table1) 14 .

In-vivo test for Anti-pyretic activity on animals (rats):
The In-vivo test for anti-pyretic activity was performed on rats by inducing hyperthermia with the help of Brewer's yeast after fasting for 1 day. After 18 hours the initial body temperature was measured in each animal of different groups.The methanolic extract of plant fruit was administered orally and the temperature was noted after 1 hour, 4 hour, 8 hour and 12 hours for both test as well as positive control group. The temperature for in-vivo antipyretic activity obtained was shown in the table below (Table-2  Discussion: The obtained result from the experiment suggested that the methanolic extract of fruits of G. pedunculata possesses anti-pyretic activity at the tested doses by inhibiting the pro inflammatory mediator's cytokines, TNF-α and prostaglandins. The anti-pyretic activity as observed can be attributed to the presence of flavonoids, glycosides 15 .

In-vitro antioxidant activity study:
In-vitro investigation of methanolic extract of G. pedunculata fruits was carried out using DPPH radical scavenging activity method with the help of a standard antioxidant ascorbic acid of different concentration as shown in the table below (Table-3) 16 . .

DISCUSSION:
DPPH (2-2-diphenyl-2-picrylhydrazyl) is the most commonly used free radicals for testing the preliminary radical scavenging activity of the plant extract. Scavenging of DPPH radical is related to the inhibition of lipid peroxidation 17 . An antioxidants either it transfer an electron or a hydrogen atom to the DPPH, result into neutralization of free radical character. In this investigation, the effect of methanolic extracton the DPPH radical scavenging was concomitantly increasing with increase in the concentration of methanolic extracts of the fruits from 10 to 50µg/ml 10 . The percentage of inhibition obtained wereranging from 32.06 at 10µg/ml to 58.29 at 50 µg/ml with 38.31 as anIC 50 (µg/ml) for the extract and for positive control (ascorbic acid) were 34.05 at 10µg/ml and 61.25 at 50 µg/ml with 34.81 as anIC 50 (µg/ml). (Table: 3). From the obtained results it can conclude that the speciesG. pedunculata, possess hydrogen donating capabilities for methanolic extract and does it undergoes scavenging of free radicals.

CONCLUSION:
This investigation confirmed the ethnomedicinal claim about the anti-pyretic and anti-oxidant use of fruits of G. pedunculata plant. In the perspective of identifying traditional herbal drugs which might be useful in preventing excessive and also because of the harmful side effects of synthetic products. The methanolic extract of G. pedunculata fruits shows a promising anti-oxidant (DPPH radical scavenging activity) and anti-pyretic activity. These studies suggest that it is having a source of active compound that can prevent the increase in temperature and its complications; the anti-oxidant activity was estimated by comparing with a standard anti-oxidant Ascorbic acid. The obtained result concluded that G. pedunculata has a good anti-pyretic and anti-oxidant activity.