ANTIPYRETIC ACTIVITY OF SEED EXTRACTS OF Gynocardia Odorata roxb. IN EXPERIMENTAL ANIMALS

About Authors:
Urmila moyal*, Ram garg
NIMS Institute of Pharmacy, NIMS University,
Shobha Nagar, Jaipur (Rajasthan)
*urmilamoyal@gmail.com

Abstract:
The study was designed to evaluate antidiabetic activity of ethanolic and aqueous seed extracts of Gynocardia  Odorata  in

• Brewer’s yeast induced pyrexia in rats.
• TAB vaccine-induced pyrexia in rabbits.

The seeds were collected, authenticated and shade dried. Shade dried seeds were then grinded into coarse powder and processed for further studies. Ethanolic and aqueous extracts were prepared and the phytochemical screening was performed. Ethanolic and aqueous both extracts were evaluated for the antipyretic activity by Brewer’s yeast induced pyrexia in rats and TAB vaccine-induced pyrexia in rabbits. Both the extracts showed the significant antipyretic activity but the maximum antipyretic activity by aqueous extract of Gynocardia odorata roxb.seeds. From study we concluded that both ethanolic and aqueous, seed extracts of Gynocardia odorata  have antipyretic activity, but the aqueous extract of Gynocardia odorata showed more significance then ethanolic extract.

REFERENCE ID: PHARMATUTOR-ART-1671

Introduction:
Pyrexia
It is defined as a controlled elevation of body temperature above the normal range of 36.7 to 37.0°C. Pyrexia is a manifestation of many disease states other than infection. Many drugs have been identified as causes of pyrexia. Drug-induced pyrexia is defined as persistent pyrexia in the absence of infection or other underlying condition. The pyrexia must coincide temporally with the administration of the offending agent and disappear promptly upon its withdrawal, after which it remains normal.¹

Pyrexia is caused as a secondary impact of infection, malignancy or other diseased states. It is the body’s natural function to create an environment where infectious agents or damaged tissues cannot survive. Normally the infected or damaged tissue initiates the enhanced formation of pro inflammatory mediators (cytokines, such as interleukin 1β, α, β, and TNF- α), which increase the synthesis of prostaglandin E2 (PgE2) near hypothalamic area and thereby trigger the hypothalamus to elevate the body temperature. When body temperature becomes high, the temperature regulatory system, which is governed by a nervous feedback mechanism, dilates the blood vessels and increases sweating to reduce the temperature. When the body temperature becomes low, hypothalamus protects the internal temperature by vasoconstriction. High pyrexia often increases faster disease progression by increasing tissue catabolism, dehydration, and existing complaints, as found in HIV.²As Gynocardia Odorata Roxb.seeds are an old traditional medicament used in fever  Hence, the present study was designed to determine the antipyretic effect of petroleum ether and ethyl acetate soluble fractions ofethanol extract

Gynocardia Odorata is a very large East Indian tree. The leaves are glossy, entire, and alternate. The flowers are yellow and sweet-scented. The fruit is round, ash-colored, and when mature, averages in weight from 10 to 20 pounds. The numerous seeds are imbedded in its pulp, and contain oil, which, according to Roxburgh, is mixed with fresh butter, and used by the natives as a remedy for cutaneous diseases. It is known as Chaulmoogra (or Chaulmugra), and are said, when powdered, to have been used with advantage in scrofula, skin diseases, and rheumatism, the dose being about 6 grains. The seeds are grayish, irregularly ovoid, compressed, and angular and smooth, a little over an inch long, and have an oily taste and a peculiar, nauseous odor.It is a medicinal plant growing wildly throughout India and tropical countries of the world.^3,4,5

Materials and Methods:

Plant Material:
The driedseeds of Plant Gynocardia Odorata were procured from Crude Drug Supplier in Delhi and authenticatedby Dr. ZeaUiHasan Botanist, Saifia College of Science, Bhopal.The seeds were then shade dried and grinded and made a coarse powder and the coarse powder were used for further studies.

Preparation of Extract:⁶
Extraction was done according to standard procedures using analytical grade solvents. For ethanolic extract 250 gm. powdered seed was taken in a pouch of filter paper and kept inside the soxhlet thistle then it was extracted with petroleum ether for 48-72 hours for defatting after that it was extracted with ethanol (99.9%) for 48-72 hours. Aqueous extract was separately prepared by maceration process. The extracts were then concentrated until dryness under reduced pressure and controlled temperature (40-50ºC). .The % yield of ethanolic and aqueous extracts was found to be 7.2% and 10.3%. Then Preliminary Phytochemical screening was performed. Furthermore, the dried ethanolic and aqueous extracts were used for evaluation of Pyrexia activities.

Preliminary Phytochemical Screening:
The extracts were subjected to preliminary phytochemical qualitative screening to evaluate the presence of various primary or secondary metabolites following standard procedures. In Previous phytochemical studies the presence of phytoconstituents such as Flavonoids, Tannins, Proteins, Alkaloids, Saponins and Triterpenoids were already reported.⁷

Experimental Animals:

Evaluation of antipyretic activity was done by usingBrewer’s yeast induced pyrexia in rats and TAB vaccine-induced pyrexia in rabbits were used and the animals were maintained under standard laboratory conditions with access to standard diet and water ad libitum. The experiment was carried out according to the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.⁸ Before starting experimentation on test animals, permission of Institutional Animal Ethics Committee (IAEC) was obtained.

Acute toxicity study:
Acute oral toxicity studies were performed according to OECD no. 423 guidelines. Three rat and mice of either sex were selected for the study. The animals were fasted over night for food with free access for water prior to test extract. Gynocardia odorata roxb.aqueous and ethanolic extracts were administration orally up to dose 2000 mg/kg. Individual animal was observed after dosing at least once during first 30 min., periodically during 24 hrs. with a special attention given during the first 4 hrs. and daily thereafter, for a 7 days.⁹

Activities

Anti-Pyretic Activity
Anti-Pyretic Activity were carried out by using two different models -
(a)  Brewer’s yeast induced pyrexia in rats.
(b)  TAB - Vaccine induced pyrexia in rabbits.
In this activity Both Ethanolic and Aqueous extract dose were taken 100 mg/kg.

Procedure
(a)  Brewer’s yeast induced pyrexia in rats:¹⁰
All animal were divided in to five groups each group having six healthy rats weighing (160-200 gm.). Group I was normal and received vehicle only while Group II- V were made hyper-thermic by a subcutaneous injection of 20% yeast suspension in 0.9% saline at a dose of 1mL/100 g body weight. Group II was pyretic and received vehicle only. Group III was received Ethanolic extract of Gynocardia odorata roxb.(100mg/kg, p.o.). Group IV was received aqueous extract of Gynocardia 0dorata roxb.(100mg/kg, p.o.)and Group V was received standard drug Paracetamol (150 mg/kg p.o.). The rectal temperatures of different groups of animals were recorded by using an electric telethermometer at 1 hour interval for six hours.

Statistical analysis:
The significance of difference among the control group and various treated groups were analyzed by means of one-way ANOVA followed by Dunnett’s multiple comparison tests. The experimental results are represented as ± SEM (standard error mean).

(b)  TAB - Vaccine induced pyrexia in rabbits:¹¹
The study was performed in rabbits. The animals were maintained in the laboratory for 24 hours prior to the experiment. In this method the rabbits were divided into five groups, each group consisting six animals.

Group I served as Normal control and received vehicle only while Group II-V were made hyperthermic by single intravenous injection of TAB Vaccine into the marginal ear vein of rabbits at a dose of 0.5 ml/rabbit. Group II was pyretic and received vehicle only; Group III was received Ethanolic extract of Gynocardia odorata roxb.(100 mg/kg p.o.), Group IV was received Aqueous extract of Gynocardia odorata roxb. (100 mg/kg p.o.), and Group V were received standard drug Paracetamol (150 mg/kg).The rectal temperature of a group of rabbits was recorded by an electric telethermometer. Gynocardia odorata roxb.extracts were administered orally after 60 min of TAB vaccine when there was significant pyrexia. The rectal temperature of groups of rabbits was recorded for a period of 6 hours by using Telethermometer.

Statistical analysis:
The significance of difference among the control group and various treated groups were analyzed by means of one-way ANOVA followed by Dunnett’s multiple comparison tests. The experimental results are represented as ± SEM (standard error mean)

Result:

Results of acute toxicity study:
There was no change in normal behavioral pattern of animals and no sign and symptoms of toxicity were observed during the observations which was done continuously for the first two hours and then observed up to twenty four hours for mortality. The extracts were safe up to a maximum dose of 2000 mg/ kg body weight.

6.2. Preliminary Phytochemical study:-
The % yield of ehanolic and aqueous extracts was found to be 7.2% and 10.3%.Thisstudy showed the presence of phytoconsituents such as proteins, carbohydrates,alkaloids, fatty acids, amino acids, flavonoids, triterpenoids and cynogenic glycosides were observed.

Anti-Pyretic activity of seed extracts of Gynocardia odorata roxb.onBrewer’s yeast induced pyrexia in rats:
After 18 hours of brewer’s yeast administration there was an increases in the rectal temperature of groups of animals when compared with the normal temperature of same groups, which is (≥1.2⁰C), these animal were considered as pyretic.

In the normal group there was no change in rectal temperature and remain similar throughout the experiment.In all other groups the temperature increased about 1.2⁰C after 18 hours of brewer’s yeast administration,in pyretic untreated group from (37.0±0.22 to 38.6±0.42),in Gynocardia odorata roxb.ethanolic extract at dose of 100mg/kg from (35.9+0.19 to37.5± 0.12), in Gynocardia odorata roxb.aqueous extract at dose of 100mg/kg from (36.6+0.28 to 38.6±0.17) and in standard group from (36.6± 0.14 to 38.9± 0.13).When 0 hours (37.5± 0.12) temperature of Gynocardia odorata roxb. ethanolic extract group was compared with the temperature of 1^sthour (37.1±0.20) and 2^nd hour (36.7±0.23) of the same group, there was no significant difference observed. When 0 hours (37.5± 0.12)the temperature ofGynocardia odorata roxb.ethanolic extract group was compared with the temperature of 3^rdhour (36.4±0.30)there was a less significant difference (P≤ 0.05) observed. When 0 hours (37.5± 0.12)the temperature of Gynocardia odorata roxb.ethanolic extract group was compared with the temperatureof 4^thhour (36.3±0.26), 5^thhour (36.3±0.28), 6^thhour (36.3± 0.25)there was a less significant difference ( P≤ 0.01) observed.

When 0 hours (38.6±0.17) temperature of Gynocardia odarataroxb.aqueous extract group was compared with the temperature of 1^st hour (38.2± 0.19) and 2^nd hour (37.9± 0.15) of the same group there was no significant difference observed.When 0 hours (38.2± 0.19) the temperature of Gynocardia odarataroxb.aqueousextract group was compared with the temperature.and the aqueous extract group was compared with the temperature of 3^rd hour (37.6± 0.17)and 4^th hour (37.6± 0.15)of the same group there was a moderate significant difference (P≤ 0.01) observed.When 0 hours (38.2± 0.19) the temperature of Gynocardia odorata roxb.aqueous extract group was compared with the temperature.and the aqueous extract group was compared with the temperature of 5^th hour (37.4± 0.19),6^th hour (37.2± 0.22) there was a highly significant difference (P≤ 0.001) observed.When 0 hours (38.9± 0.13)temperature of standard drug Paracetamol group was compared with the temperature of 1^st hour (38.2± 0.29)of the same group there was no significant difference observed.When 0 hours (38.9± 0.13) the temperature of standard drug Paracetamol group was compared with the temperature and 2^nd hour (37.7± 0.23) of the same group there was a moderate significant difference (P≤ 0.01) observed.When 0 hours (38.9± 0.13) the temperature of standard drug Paracetamol group was compared with the temperature of 3^rd hour (37.4± 0.18), 4^th hour (37.2± 0.22), 5^th hour (37.0± 0.20) and 6^th hour (36.8± 0.16) there was a highly significant difference (P≤ 0.001) observed.

Anti-Pyretic activity of seed extracts of Gynocardia odorata roxb. onTAB-Vaccine induced pyrexia in rabbits
After administration of TAB-Vaccinethere was an increase in the rectal temperature of groups of animals when compared with the normal temperature of same groups, which is (≥1.2⁰C), these animals were considered as pyretic.

In the normal group there was no change in rectal temperature and remain similar throughout the experiment. In all other groups the temperature increased about 1.2⁰C after 1 hours of TAB-Vaccineadministration, in pyretic untreated group from (38.2 ± 0.04) and (39.4±0.01), in Gynocardia odorata roxb., the ehanolic extract at dose of 100mg/kg from (38.36±0.03) to (39.38±0.07), in Gynocardia odarataroxb., the aqueous extract at dose of 100mg/kg from (38.36±0.02 to 39.40±0.0) and in standard group from (38.1±0.00 to 39.5±0.07).When 0 hours (39.38±0.07) the temperature of Gynocardia odarataroxb.ethanolic extract group was compared with the temperature of 1^st hour (39.05±0.01), 2^nd hour (38.91±0.02), 3^rd hour (38.83±0.02), 4^th hour (38.78±0.02), 5^th hour (38.77±0.03) and 6^th hour (38.77±0.03) there was a highly significant difference (P≤ 0.001) observed.When 0 hours (39.40±0.07) the temperature of Gynocardia odarataroxb.aqueous extract group was compared with the temperature of 1^st hour 39.05±0.00 2^nd hour (38.91±0.01),3^rd hour (38.80±0.01), 4^th hour (38.75±0.01), 5^th hour (38.69±0.01)and 6^th hour (38.64±0.01) there was a highly significant difference (P≤ 0.001) observed.When 0 hours (39.5±0.07) the temperature of standard drug Paracetamol group was compared with the temperature of 1^st hour (39.2±0.03) 2^nd hour (39.0±0.03),3^rd hour (38.8±0.02), 4^th hour (38.6±0.00), 5^th hour (38.4±0.01) and 6^th hour (38.2±0.00) therewas a highly significant difference P≤ 0.001 observed.

Discussion:

Brewer’s yeast induced pyrexia in rats:
The extract of Gynocardia odorata roxb.exhibited antipyretic activity in Brewer’s yeast induced pyrexia in rats. It is currently accepted that prostaglandin E2 (PGE2) is the final fever mediator in the brain, specifically in the pre -optic area of the anterior hypothalamus ¹², thus it may be plausible to conclude that the extract inhibits the synthesis of prostaglandins. However, it must be noted that several biochemical events occur leading ultimately to the synthesis of PGE2. Fever is believed to result from a finely tuned, complex event that involves both the peripheral immune system and the brain, through which a series of inflammatory and metabolic processes are regulated. It is established that there are two pathways leading to the transcription and induction of cyclooxygenase (COX)-2, the rate limiting enzyme for prostaglandin (PGE2) synthesis. Both pathways are activated by cytokines e.g. IL-1, IL-6 and tumor necrosis factor (TNF) which trigger central mechanisms that act via the transcription factors such nuclear factor (NF) B and signal transducer and activator of transcription (STAT-3) . It may therefore be worthwhile to investigate the exact point in the biochemical events where the extract exerts it antipyretic effect.¹³

Pyrexia is thought to be produced by several endogenous substances including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), macrophage protein-1 (MIP-1) and prostaglandins. A. Brewer’s yeast induces both TNF-α and prostaglandin synthesis.¹⁴

For better characterization of the antipyretic activity of the EFR, two models of pyresis including Tab vaccine induced and brewer’s yeast tinduced fever were employed in this study. Antipyretics such as ASA and other nonsteroidal anti-inflammatory drugs (NSAIDs) reduce fever by depressing inflammatory messages at both peripheral sites of tissue inflammation and within central nervous system thermoregulatory sites.These agents suppress peripheral production of pyrogenic cytokines such as TNF-α and interleukin-1β, while lowering the thermoregulatory set point by blocking central cyclooxygenase production of prostaglandin E2 (PGE2)¹⁵.

In this study brewer’s yeast induced increases in rectal temperature in rats After 18 hours of brewer’s yeast administration there was an increases in the rectal temperature of groups of animals when compared with the normal temperature of same groups,which is ≥1.20C,these animal were considered as pyretic. In the normal group there was no change in rectal temperature and remain similar throughout the experiment. From the observation it was observed that standard drug Paracetamol and aqueous showed maximum effect of antipyretic activity and ethanolic extract of Gynocardia odorata roxb. showed moderate effect against Brewer’s yeast induced pyrexia.

Tab vaccine induced Pyrexia in Rabbits:
TAB vaccine is a sterile suspension, 1ml containing 1 × 109 S. typhiand 7.5 × 108 each of S. paratyphiA and B organisms in 5 and 10ml vials. It is probable that the TAB induced hyperpyrexia indirectly by causing release of endogenous pyrogen. It was of interest to note that the extracts of Gynocardia odorata roxb. produced a rather modest decrease in the body temperature in hyperthermic rats. Regulation of body temperature requires a delicate balance between the production and loss of heat, and the hypothalamus regulates the set point at which body temperature is maintained. The cause of this decrease may be central and/or peripheral in origin. Clinically available antipyretic drugs, such as paracetamol and the non-steroidal anti-inflammatory drugs are able to lower the body temperature only in feverish patients. In general, non-steroidal anti-inflammatory drugs produce their antipyretic action through inhibition of prostaglandin synthetase within the hypothalamus. ^16,17

The preliminary phytochemical analysis reveals that the major chemical Constituents of the alcoholic extract are terpenoids and flavonoids. Thus phytochemicals found in alcoholic extract, which might be responsible for the antipyretic activity. Thus, the results of the present study provide support to the traditional usage of Gynocardia odorata roxb. in fever, even if further studies are needed to better evaluate these activities and the potential of the plant.

Table no. 2:Effects of Gynocardia odorata roxb. Seed extracts on TAB-Vaccine induced pyrexia in rabbits.

Values are expressed as mean ± S.E.M (n = 6); ***P≤0.001, **P≤0.01,*P≤0.05. Compared in the same group with 1hr. of Tab Vaccine. G.O.E.E-Gynocardia odorata  roxb.ethanolic extract, G.O.A.E-Gynocardia odorata roxb.aqueous extract

Figure Legends

Figure no. 1-Effect Gynocardia odorata roxb. seeds extracts on Brewer’s yeast induced pyrexia in rats

Figure no. 2- Effects of Gynocardia odorata roxb.Seeds extract on TAB-Vaccine induced pyrexia in rabbits

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