Evaluation of Antipyretic and Anti-Inflammatory Activity of Aqueous Extract of Leptadenia Reticulata in Animal Models

Objective: To study the antipyretic and anti-inflammatory effects of the Aqueous Extract of Leptadenia Reticulata (AELR) in different animal models. Materials and Methods: Acute toxicity studies were performed and extract was found to be safe upto 2000 mg/kg. Two doses were selected for the “evaluation of antipyretic and anti-inflammatory activity of aqueous extract of Leptadenia reticulata in animal models” i.e 200 mg/kg and 400 mg/kg b. w. Antipyretic activity was evaluated using baker’s yeast induced pyrexia in albino rats and cow milk induced pyrexia in albino rabbits. Antiinflammatory activity was evaluated using carrageenan induced paw edema and turpentine oil induced paw edema in albino rats. Results: In all the animal models AELR at the dose of 200 mg/kg b.w and 400 mg/kg b.w showed significant (P<0.01) antipyretic and anti-inflammatory activity. Conclusion: These finding could justify the inclusion of aqueous extract of Leptadenia reticulata in the management of pyrexia and inflammation. Evaluation of Antipyretic and Anti-Inflammatory Activity of Aqueous Extract of Leptadenia Reticulata in Animal Models Bherji Sneha*, M. Ganga Raju and Namile Divya Department of Pharmacology, Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad – 500090, Andhra Pradesh, India *Author for correspondence Email: snehabherji@gmail.com JOURNAL OF NATURAL REMEDIES


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Pyrexia or fever may be the result of infection, tissue damage, inflammation or other disease states. A common feature of these conditions is the enhanced formation of cytokines such as interleukin-1, interleukin-6, interferon-α and β and tumor necrosis factor. The cytokines increase the synthesis of PGE2 by activating arachidonic acid pathway. PGE2 triggers the hypothalamus to elevate the body temperature by promoting increase in heat generation and decrease in heat loss 1 . Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate healing process 2 . Many synthetic drugs are used to treat fever and inflammation, but these drugs have several side effects. Therefore, many medicinal plants are used to cure diseases with lesser side effects.
Leptadenia reticulate (Retz.) Wight & Arn. is a much branched twining shrub of family Asclepiadaceae. It is commonly called as 'jiwanti". This plant is distributed in the Southern parts of India. Traditionally this plant has stimulant and restorative properties. The leaves and roots are used in skin infections such as ring worm, wounds, nose and ear disorders, asthama and fever. The main chemical constituents reported are: α-amyrin, β-amyrin, ferulic acid, luteolin, diosmetin, rutin, β-sitosterol, stigmasterol, hentriacontanol, a triterpene alcohol simiarenol, apigenin, pregnane glycosides 3 . Aerial parts of Leptadenia reticulata is reported to contain tocopherol and possess several pharmacological activities such as galactogogue, antimicrobial and anti-inflammatory

Preliminary Phytochemical Studies
The preliminary phytochemical screening of the aqueous extract of Leptadenia reticulata revealed the presence of sterols, triterpenoids, flavonoids, proteins and carbohydrates 5 .

Experimental Animals
Wistar albino rats (150-200 g) and albino rabbits (1-2 kg) were maintained on a standard pellet, cabbage and carrot diet respectively and water ad libitum. They were housed in polypropylene cages and maintained under standard conditions (12 h light-dark cycle; 23-25 °C; 35-60 % relative humidity). All the experimental protocols for animal care procedures were approved by the ethical committee of Gokaraju Rangaraju College of Pharmacy. Principles of laboratory animal care guidelines were followed and prior permission was sought from the Institute Animal Ethics Committee (IAEC) for conducting the experiments. Present study was carried out in CPCSEA approved animal house (Reg. no. 1175/ac/08/CPCSEA) of Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad, India.

Acute Toxicity Study
Acute toxicity studies were performed as per OECD guidelines 425. According to these guidelines a limit test was carried out using 5 female mice. A test dose of 2000 mg/kg of aqueous extract of Leptadenia reticulata was administered orally to the first mice and it was observed for 48 hrs. When there was no mortality seen, other four mice were given the same dose and were observed for 14 days. The oral administration of aqueous extract of Leptadenia reticulata did not exhibit any signs of toxicity and mortality at 2000 mg/kg bd. wt. All animals were safe even after 14 days of observation.

Selection of Dose
From the above toxicity studies, 2000 mg/kg bd. wt was identified and the working dose was considered as 1/10 th i.e., 200 mg. To perform this study two doses were selected i.e. 200 mg/kg bd. wt and 400 mg/kg bd. wt 6 .

Baker's Yeast Induced Pyrexia in Albino Rats
Antipyretic activity was assessed using baker's yeast (containing Saccharomyces cerevisiae) induced pyrexia in Wistar albino rats. The rectal body temperature was measured by introducing a digital thermometer coated with glycerine, inserted into the rectum to a depth of 3 cm. After measuring the basal rectal temperature, animals were given a subcutaneous injection of 20% baker's yeast suspended in 0.9 % saline (10 mL/kg bd. wt). Rats were then returned to respective cages.

Cow Milk Induced Pyrexia in Rabbits
Antipyretic activity was assessed using cow milk induced pyrexia in rabbits, as per the method described by M. Thirumal et al 9 . Group-I served as disease control, received cow milk and saline, group-II received cow milk and AELR (200 mg/kg bd. wt), group-III cow milk and AELR (400 mg/kg bd. wt) and group-IV received cow milk and paracetamol (10 mg/kg bd. wt) p.o. The temperature was measured with a thermometer hourly upto 4 hours after cow milk injection.

Carrageenan Induced Paw Edema in Rats
Anti-inflammatory activity was carried out using carrageenan induced paw edema in rats, as per the method described by Kalabharathi

Turpentine Oil Induced Paw Edema in Rats
Anti-inflammatory activity was carried out using turpentine oil induced paw edema in rats, as per the method described by Surender Singh et al 10 . Group-I served as disease control, received turpentine oil and saline, group-II received turpentine oil and AELR (200 mg/kg bd. wt), group-III turpentine oil and AELR (400 mg/kg bd. wt) and group-IV received turpentine oil and indomethacin (10 mg/kg bd. wt) p.o. Paw edema was measured by mercury plethysmometer hourly upto 4 hours. The difference between the '0' and '3 rd ' h reading gives the actual edema.

Statistical Analysis
All the results are expressed as mean ± SEM of six animals in each group. The data was evaluated using ANOVA followed by Dunnet's 't' test. A significant value of p<0.01 was considered statistically significant.

Results
In baker's yeast induced pyrexia in rats, AELR (at the doses 200 mg/kg bd. wt and 400 mg/kg bd. wt) significantly (P<0.01) reduced the body temperature at 19 h, 20 h, 21 h and 22 h of yeast administration when compared to the control group.
In cow milk induced pyrexia in rabbits AELR (at the doses 200 mg/kg bd. wt and 400 mg/kg bd. wt) significantly (P<0.01) reduced the body temperature in  In carrageenan and turpentine oil induce inflammation in rats AELR (at the doses 200 mg/kg bd. wt and 400 mg/kg bd. wt) significantly (P<0.01) reduced paw edema in rats when compared to the control group.

Discussion
Pyrexia or fever may be the result of infection, tissue damage, inflammation or other disease states. A common feature of these conditions is the enhanced formation of cytokines such as interleukin-1, interleukin-6, interferon-α and β and tumor necrosis factor. The cytokines increase the synthesis of PGE2 by activating arachidonic acid pathway. PGE2 triggers the hypothalamus to elevate the body temperature by promoting increase in heat generation and decrease in heat loss. Antipyretic drugs act by inhibiting the enzyme cyclooxygenase and reduce the levels of PGE2 within the hypothalamus 1 . In baker's yeast induced pyrexia in rats and cow milk induced pyrexia in rabbits, AELR at the doses of 200 mg/kg bd. wt and 400 mg/kg bd. wt significantly reduced the body temperature.
Carrageenan induced paw edema is a commonly used primary test for the screening of new anti-inflammatory agents and it is believed to be biphasic. The first phase (1-2 hr) is due to the release of histamine or serotonin and the second phase of edema is due to the release of prostaglandin 2 .
Turpentine oil induced paw edema is characterized by a triphasic release of inflammatory mediators. The initial phase is mediated by histamine and serotonin, intermediate phase by kinin like substance and the late phase by cycloxygenase and lipoxygenase products 7 .
In carrageenan and turpentine oil induced paw edema in rats, AELR at the doses of 200 mg/kg bd. wt and 400 mg/kg bd. wt significantly inhibits the edema induced by carrageenan and turpentine oil in rats.
The earlier reports of Krishnamoorthi Mahalakshmi et al. 11 , supports the role of flavonoids, triterpenoids and  Values are expressed as mean ± SEM; n=6. * P<0.01 compared with the control steroids in antipyretic and anti-inflammatory activity. Since these chemical constituents are also present in AELR, may be responsible for antipyretic and antiinflammatory activity.

Conclusion
Antipyretic activity was performed using two models i.e. baker's yeast induced pyrexia in albino rats and cow milk induced pyrexia albino rabbits. AELR at 200 mg/kg bd. wt and 400 mg/kg bd. wt significantly decreased the elevated body temperature in both the animal models. Anti-inflammatory activity was performed using two models i.e. carrageenan induced paw edema in rats and turpentine oil induced paw edema in rats. AELR at 200 mg/kg bd. wt and 400 mg/kg bd. wt significantly reduced the paw edema in both the animal models.
These finding could justify the inclusion of aqueous extract of Leptadenia reticulata in the management of pyrexia and inflammation.
The present study therefore supports the claims of traditional use of the plant for antipyretic and antiinflammatory activities. To know the exact mechanism of action of AELR, further study with purified fractions of the bioactive compound is needed.