|Year : 2010 | Volume
| Issue : 3 | Page : 367-370
Antipyretic activity of Guduchi Ghrita formulations in albino rats
BK Ashok1, B Ravishankar1, PK Prajapati2, Savitha D Bhat3
1 Pharmacology laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Gujarat, India
2 Department of Rasashastra and Bhaishajya Kalpana, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Gujarat, India
3 Department of Dravyaguna, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Gujarat, India
|Date of Web Publication||26-Feb-2011|
B K Ashok
Research Assistant, Pharmacology Laboratory, IPGT and RA, Dhanvantari Mandir, Gujarat Ayurved University, Jamnagar
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The present pharmacological investigation was undertaken to study the anti-pyretic activity of Guduchi ghrita formulations in albino rats against yeast induced pyrexia. Seven groups of six animals were used for the experiment. The yeast induced pyrexia method was standardized first by injecting 12.5% yeast suspension (s.c) followed by recording the rectal temperature at regular intervals. Then the evaluation of anti-pyretic activity of Guduchi ghrita formulations was carried out by using this standard procedure. Both the Guduchi ghrita samples including vehicle significantly attenuated the raise in temperature after three hours of yeast injection. After 6 and 9 hours of yeast injection also both the Guduchi ghrita samples attenuated the raise in temperature in a highly significant manner in comparison to both yeast control and vehicle control groups. The data generated during study shows that both the Guduchi ghrita formulations having significant anti-pyretic activity.
Keywords: Guduchi Ghrita , pyrexia, Brewer′s yeast, paracetamol, Tinospora cordifolia (Willd.) Miers. medicated ghee.
|How to cite this article:|
Ashok B K, Ravishankar B, Prajapati P K, Bhat SD. Antipyretic activity of Guduchi Ghrita formulations in albino rats. AYU 2010;31:367-70
| Introduction|| |
Guduchi [Tinospora cordifolia (Willd.) Miers.] has been used in the indigenous system of medicine since the Vedic period. It is a very common drug and is quite frequently mentioned in most of the Samhitas, Nighantus, and Granthas for its effectiveness in various diseases. It is considered the best drug in terms of availability, economy, ease of administration, etc. and further, at the dose levels employed clinically it is well tolerated. This plant is used in Ayurveda as single drug in the form of Swarasa, Kalka, Kwatha, Hima, Churna, and Ghrita and also as one of the important ingredients in many other formulations used for treating various ailments. The accepted botanical source of Guduchi throughout India is Tinospora cordifolia (Willd.) Miers., of the family Menispermaceae. This plant is dioecious, with male and female flowers borne on separate plants. However, during collection of the plant material for medicinal purposes this factor is not given due consideration. It is well established that environmental, agricultural, genetic, storage, and other factors affect the quality of the crude drug. Thus the plant's gender may also impact the therapeutic efficacy of the extract. Many previous studies have shown that in many dioecious species, phyto-constituents are more in the female plant than in the male; thus the source will obviously affect the biological activity.
Ghritas are medicated ghee preparations containing the fat-soluble components of the ingredients. Preparation involves protracted boiling of ghee with prescribed decoctions and a fine paste of the drug to dehydration or near dehydration, whereby the fat-soluble principles are transferred to the Ghrita from the drug ingredients or decoctions or expressed juice as the case may be. The Ghrita formulation has several advantages over other pharmaceutical preparations in terms of absorption, shelf-life, route of administration, etc.
Guduchi is renowned in Ayurvedic therapeutics for its usefulness in the treatment of Jwara (fever).  Guduchi Swarasa and Guduchi Kalka (expressed juice and paste, respectively) prepared from the stem of T cordifolia (Willd.) Miers. was used for processing the Ghrita (ghee) in this formulation, which is indicated for the treatment of fever. , Thus prepared formulation of Guduchi from male and female plants was subjected to screening for anti-pyretic activity to assess the impact of gender and formulation on expression of pharmacological activity of Guduchi.
| Materials and Methods|| |
Wistar strain albino rats of either sex weighing between 140-160 g were used. The animals were obtained from the animal house attached to the pharmacology laboratory of Institute for Post Graduate Teaching and Research in Ayurveda. The rats were exposed to natural day and night cycles under ideal ambient laboratory conditions (temperature 22±2°C and humidity 50%-60%). They were fed with Amrut® rat pellet feed (Pranav Agro Industries) and tap water was supplied ad libitum.
The experiments were carried out after obtaining permission from the institutional animal ethics committee (approval number - IAEC/3/07-08/01).
The dose of the test formulations were calculated by extrapolating the human dose (900 mg/kg) to animals based on the body surface area ratio) by referring to the standard table of Paget and Barnes, (1964).  The study was carried out using two dose levels, i.e., the therapeutically equivalent dose (TED) of 900 mg/kg and TED × 2 (i.e., 1800 mg/kg).
Animal grouping : Wistar albino rats of body weight ranging from 140 g to 160 g were used as experimental animals. They were divided into seven groups as follows:
Standardization of yeast-induced antipyretic model for the present study
- Group I: Yeast control (YC)
- Group II: Vehicle control (VC) (given 900 mg/kg plus yeast injection)
- Group III: Guduchi Ghrita prepared from male plant (900 mg/kg plus yeast injection)
- Group IV: Guduchi Ghrita prepared from male plant (1800 mg/kg plus yeast injection)
- Group V: Guduchi Ghrita prepared from female plant (900 mg/kg plus yeast injection)
- Group VI: Guduchi Ghrita prepared from female plant (1800 mg/kg plus yeast injection)
- Group VII: Paracetamol (reference standard) (100 mg/kg plus yeast injection)
Fever can be induced in experimental animals by intravenous or subcutaneous injection of pyrogens. To evaluate the antipyretic activity of test drugs, the most commonly employed method to induce fever involves injection of lipopolysaccharides (LPS) or brewer's yeast in rabbits or rats. 
However, the fact that small animals do not present reliable pyrogen-induced fever makes antipyretics screening difficult and expensive, since the amount of drug necessary for these tests increases proportionally with the size of the animal. The use of different pyrogens in the literature certainly provide an additional source of variation for antipyretic screening results, since it has been reported that lipopolysaccharide-induced fever is dependent on the serotype of its source.  In addition, there are several reports of hypothermia, instead of fever, after LPS and yeast administration to rats and mice. 
Presently available methods of yeast-induced pyrexia in rats include development of fever usually 9-18 h after yeast injection.  It is essential to develop a model in which fever ensues within a few hours (4-5 h) of yeast injection to test the classical antipyretic drug. It is also necessary to have long-lasting fever episodes (up to 24 h) as the onset of therapeutic action and duration of therapeutic action of classical drugs is believed to be more.
Against this background, the yeast-induced pyrexia model was standardized prior to the experiment proper. Since the Charles Foster rats available in the animal house of our institute did not develop pyrexia on injection of Brewer's yeast, this study was carried out on albino rats of the Wistar strain. Young rats (three males and three females) were selected. It is reported that in yeast-induced pyrexia, adult rats develop fever within 9-18 h [ 7] and that age can influence the response to the pyrogen, which may account for different immune responses. ,
The animals were fasted for 18 h before the commencement of the experiment, but drinking water was provided ad libitum. Rectal temperature (T R ) was measured by inserting a lubricated thermostat probe (external diameter 6 mm) 3 cm into the rectum of the animal. The probe was linked to a digital device, which displayed the temperature at the tip of the probe with 0.1°C precision. The values displayed were manually recorded.
Immediately after measuring the initial basal rectal temperature, the animals were injected with brewer's yeast in normal saline (12.5%; 1 ml/100 g body weight, subcutaneously). The rectal temperature changes were recorded every hour up to 12 h, and expressed as the difference from the basal value. Since it has been previously reported that stress related to handling and temperature measurment can alter rectal temperature,  these animals were handled carefully so as to minimize the posible stress. The increase in T R occurred almost at the end of 2 h after yeast injection. This increase was around 2.25 ± 0.08°C (mean ± SEM where n=6) at 3 h, 2.50 ± 0.07°C at 6 h, and 2.73 ± 0.08° C at 9 h. After this time interval, the T R did not increase steadily up to 12 h; instead, a decrease in T R was observed in some rats. This standardized method was used for the present study.
The test drugs and reference standard were administered to the respective groups. One hour after drug administration, the yeast injection was given s.c. and the rectal temperature was recorded at the end of the 3 rd , 6 th , and 9 th hours. The rectal temperature of the control groups (yeast control) was compared with rectal temperature of the rats administered the test drugs.
The results are presented as mean ± SEM. The difference between the groups was statistically analyzed using the unpaired Student's t test and analysis of variance (ANOVA) followed by Dunnett's t test for all the treated groups (except reference standard group, where only the unpaired Student's t test was applied). P<.05 was considered statistically significant. The level of significance was noted and interpreted accordingly.
| Results|| |
[Table 1] shows data related to the effect of Guduchi Ghrita on yeast-induced pyrexia at different time intervals. Yeast injection in experimental animals caused significant rise in body temperature at the various time intervals as recorded rectally with the help of a tele-thermometer. Paracetamol, a well-established antipyretic drug attenuated the rise in temperature to a significant extent at all time intervals. Both the Guduchi Ghrita samples including vehicle significantly attenuated the rise in temperature 3 h after yeast injection. After 6 and 9 h of yeast injection also both the Guduchi Ghrita samples attenuated the raise in temperature in a highly significant manner in comparison to both yeast control and vehicle control groups. The vehicle itself had a moderate antipyretic effect in the initial stages; at the later stages, however, the observed antipyretic activity was only marginal.
|Table 1: Effect of Guduchi Ghrita prepared from male and female plants of T cordifolia on yeast-induced pyrexia at various time intervals in albino rats|
Click here to view
| Discussion|| |
Fever is a surrogate marker for disease activity in many infectious and inflammatory disorders. According to the classical view, the genesis of fever is induced by inflammatory mediators (i.e., cytokines, namely interleukin-1, interleukin-6, tumor necrosis factor, and others) that are predominantly released by activated peripheral mononuclear phagocytes and other immune cells. , Due to the fact that direct access of the large hydrophilic cytokine proteins to the temperature-controlling brain structures within the pre-optic/anterior hypothalamic areas is prevented by the blood-brain barrier, the mechanisms described below have been suggested for producing pyrexia.
Cytokines which are transported by the bloodstream could act at sites lacking a tight blood-brain barrier, the so-called circumventricular organs.  Alternatively, circulating cytokines could interact with their specific receptors on brain endothelial cells  or perivascular cells  and thereby stimulate these cells to release pyrogenic mediators into the abluminal brain tissue. It has been proposed that fever-promoting cytokines are transported from the blood into the brain via specific carriers.  An assumed manifestation of a febrile response produced by these mechanisms is termed as the humoral hypothesis of fever induction. Within the brain, prostaglandin E 2 (PGE 2 ), produced by cyclooxygenase (COX)-2, is regarded as the principle downstream mediator of fever  acting on thermosensitive or thermointegrative hypothalamic neurons.
Fever is tightly regulated by the immune response. Inflammatory stimuli triggering the generation of pro-pyretic messages provoke the release of endogenous antipyretic substances.  PGE 2 is synthesized from arachidonic acid, which is released from cell membrane lipid by phospholipase. Arachidonic acid is metabolized by two isoforms of the COX enzyme, COX-1 and COX-2. COX-1 usually is expressed constitutively and generates prostanoids important for housekeeping functions supporting homeostasis.  COX-2, on the other hand, is inducible by inflammatory signals such as the pyrogenic cytokines, IL-1b, TNF, and IL-6, as well as bacterial lipopolysaccharide. Many cells, including synoviocytes, macrophages, endothelial cells, and chondrocytes, have the capacity to rapidly up-regulate the expression of the COX-2 during inflammation.  The most likely cell type in the central nervous system responsible for producing PGE 2 is the microvascular endothelial cell, which expresses COX-2 exuberantly after stress. An effective febrifuge might interrupt pyrexogenesis at any step that connects peripheral inflammation with the central production of PGE 2 . Stated differently, an antipyretic might blunt peripheral inflammation or depress central pyrogenic signals, or it may affect both. Inhibiting central production of PGE 2 is a well-known mechanism of antipyretic agents, but activated leukocytes and endothelial cells in peripheral areas of inflammation also represent potential drug targets.
Paracetamol is an analgesic but is also an effective febrifuge. It is a poor inhibitor of cyclooxygenase in the presence of peroxides that are found in inflammatory lesions. In contrast, its antipyretic effect may be explained by its ability to inhibit cyclooxygenase in the brain, where peroxide tone is low. Further, it does not inhibit neutrophil activation. In supra-pharmacologic doses it inhibits NF-kB stimulation of inducible nitric oxide synthase. 
In the present study, in the yeast control group the rise in temperature was consistent and significant in comparison to the initial values. In the vehicle control group also the rise in temperature was significant; however, the magnitude was slightly less in comparison to that in the yeast control group in the initial stages. Both the Guduchi Ghrita samples produced very good antipyretic effect in a dose-dependant manner and the observed effect was almost similar to that in the paracetamol-treated group.
Previous studies by Ikram et al.  and Leghari et al. [22 ] have reported the antipyretic effect of Tinospora cordifolia in Himalayan rabbits. Vedavathy and Rao,  showed water-soluble fractions of a 95% ethanolic extract of T cordifolia plant to possess significant antipyretic activity (when given orally) against yeast-induced pyrexia. Many authors have ascribed the antipyretic properties of T cordifolia to the presence of berberine or other bitter substances. 
| Conclusion|| |
From the analysis of the data generated in this study it becomes clear that the dioecious nature of the plant does not influence the expression of the antipyretic activity since no statistically significant difference could be found between male and female plant-derived Ghritas. The second point to be noted is that though the vehicle itself exhibits a short-lived mild to moderate antipyretic activity, the test Ghritas have significantly higher activity in comparison to the vehicle control. This clearly shows that the active principles present in the plant per se possess significant antipyretic activity, which might be supplemented by the vehicle. Further, in an earlier study (Savrikar, 2006)  it was observed that the antipyretic activity of the expressed juice per se is short lived but when administered in the form of Ghrita it is long-lasting. In the present study also a long-lasting antipyretic activity was observed. Thus, the study provides unequivocal evidence of the presence of antipyretic activity in the test Ghritas.
In this study no attempt was made to ascertain the mechanism of the observed antipyretic activity. However, it can be suggested that it may be acting through either the peripheral or central mechanism enumerated above. It is also possible that both the mechanisms may be involved. Considering the lipoid nature of the vehicle used it seems that lipid-soluble constituents may be responsible for the observed effect.
| References|| |
|1.||Sushruta Samhita with 'Nibandha Sangraha'. Dalhanacarya. Yadavaji T. Krishnadaas Academy. Varanasi: Oriental Publishers and Distributors; 1998. p. 203. |
|2.||Ashtanga Hridaya. commentaries; Arunadatta's Sarvanga Sundara and Hemadri's Ayurveda Rasayana. Paradakar HS. 7 th ed. Chikitsa Sthana. Lane Varanasi: Choukhamba Orientalia; 1982. p. 94. |
|3.||Ashtanga Sangraha. Indu, Aathavale AD, Aathavale MA, editors. Pune: Shrimad Atreya Prakashan; 1980. p. 22. |
|4.||Paget GE , Barnes JM. Evaluation of drug activities. In: Pharmacometrics Laurence DR, Bacharach AL, editors. New York: Academic press; 1964. p. 161. |
|5.||Gerhard VH, WolfgangVH. Chapter H-4 Antipyretic Activity. In: Drug Discovery and Evaluation, Springer: Pharmacological Assays; 2002. p. 418-20. |
|6.||Dogan MD, Ataoglu H, Akarsu ES. Effects of different serotypes of Escherichia coli lipopolysaccharides on body temperature in rats. Life Sci 2000;67:2319-29. |
|7.||Dogan MD, Ataoglu H, Akarsu ES. Characterization of the hypothermic component of LPS-induced dual thermoregulatory response in rats. Pharmacol Biochem Behav 2002;72:143-50. |
|8.||Santos FA, Rao VS. A study of the antipyretic effect of quinine, an alkaloid effective against cerebral malaria, on fever induced by bacterial endotoxin and yeast in rats. J Pharm Pharmacol 1998;50:225-9. |
|9.||Balmagiya T, Rozovski SJ. Age-related changes in thermoregulation in male albino rats. Exp Gerontol 1983;18:199-210. |
|10.||Ferguson AV, Turner SL, Cooper KE, Veale WL. Neurotransmitter effects on body temperature are modified with increasing age. Physiol Behav 1985;34:977-81. |
|11.||Thompson CI, Brannon AJ, Heck AL. Emotional fever after habituation to the temperature-recording procedure. Physiol Behav 2003;80:103-8. In: Jorgete Tomazetti. Baker yeast-induced fever in young rats - Characterization and validation of an animal model for antipyretics screening. J Neurosci Meth 2005;147:29-35. |
|12.||Zeisberger E. From humoral fever to neuroimmunological control of fever. J Therm Biol 1999;24:287-326. |
|13.||Roth J. Endogenous antipyretics. Clinica Chimica Acta 2006;371:13-24. |
|14.||Roth J, Harre EM, Rummel C, Gerstberger R, Hubschle T. Signaling the brain in systemic inflammation: role of sensory circumventricular organs. Front Biosci 2004;9:290-300. |
|15.||Matsumura K, Kobayashi S, Signaling the brain in systemic inflammation: the role of endothelial cells. Front Biosci 2004;9:2819-26. |
|16.||Schiltz JC, Sawchenko PE. Signaling the brain in systemic inflammation: the role of perivascular cells. Front Biosci 2003;8:1321-9. |
|17.||Banks WA, Plotkin SR, Kastin AJ. Permeability of the blood-brain barrier to soluble cytokine receptors. Neuroimmunomodulation 1995;2:161-5. |
|18.||Kluger MJ. In: David M, Aronoff MD, Neilson EG, Antipyretics: Mechanisms of action and clinical use in fever suppression. American J Medicine 1998;111:304-15. |
|19.||Simon LS. In: David M, Aronoff MD, Neilson EG. Antipyretics: Mechanisms of action and clinical use in fever suppression. American J Medicine 1999;111:304-15. |
|20.||Goodman and Gilman's, The Pharmacological Basis of Therapeutics. Joel GH, Lee EL. 10 th ed. New York: McGraw-Hill Medical Publishing Division; 2001. p. 703-04. |
|21.||Ikram M, Khattak SG, Gilani SN. Antipyretic studies on some indigenous Pakistani medicinal plants: II. J Ethnopharmacology 1987;19:185-92. |
|22.||Leghari MY, Muzaffar NA, Haq IU. Pharmacological testing of antipyretic activity of Tinospora cordifolia. J Pharmacy University 1984;3:31-41. |
|23.||Vedavathy S, Rao KN. Short communication: Antipyretic activity of six indigenous medicinal plants of Tirumala Hills, Andhra Pradesh, India. J Ethnopharmacology 1991;33:193-6. |
|24.||Rao EV. Chemistry and Pharmacological studies on Tinospora species - A Review. Indian Drugs 1999;36:78-86. |
|25.||Savrikar SS. Comparative Study of Physico-chemical characteristics of 'Accha Sneha' and 'Siddha Sneha' with reference to 'Guduchi Ghrita', the butter fat, medicated with Tinospora cordifolia (Willd.) Miers . PhD thesis submitted to Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India. 2006. |
|This article has been cited by|
||Synergistic analgesic, anti-pyretic and anti-inflammatory effects of extra virgin olive oil and ibuprofen in different experimental models in albino mice
| ||Wallaća A. Osman,Dina A. Labib,Mona O. Abdelhalim,Elsayed M. Elrokh |
| ||International Journal of Rheumatic Diseases. 2017; |
|[Pubmed] | [DOI]|
||Estimation of the novel antipyretic, anti-inflammatory, antinociceptive and antihyperlipidemic effects of silymarin in Albino rats and mice
| ||Mohamed Mahmoud Amin,Mahmoud Soliman Arbid |
| ||Asian Pacific Journal of Tropical Biomedicine. 2015; 5(8): 619 |
|[Pubmed] | [DOI]|
||Remarks on "Tinospora cordifolia: One plant, many roles"
| ||Rohit Sharma,Rohit Galib,PK Prajapati |
| ||Ancient Science of Life. 2014; 33(3): 192 |
|[Pubmed] | [DOI]|
||Evaluation of the Antihyperlipidemic, Anti-inflammatory, Analgesic, and Antipyretic Activities of Ethanolic Extract of Ammi majus Seeds in Albino Rats and Mice
| ||Khaled M. M. Koriem,Gihan F. Asaad,Hoda A. Megahed,Hanan Zahran,Mahmoud S. Arbid |
| ||International Journal of Toxicology. 2012; 31(3): 294 |
|[Pubmed] | [DOI]|