Comparative Evaluation of Antipyretic Activity of an Ayurvedic Herbo-mineral Formulation Dhatryadi Churna and Its Modified Dosage form in Albino Wistar Rats

Introduction: Experimental studies are essential and expected part of a new drug development. The studies are conducted to know about the toxic effects (if any), produced by the drug & to access safety of the drug. Studies also help to ascertain the efficacy of the drug & measures to be taken so as to curtail the toxic effects. Aim & Objective: Study was aimed to do comparative evaluation of antipyretic activity of an Ayurvedic herbo-mineral formulation Dhatryadi Churna and its modified dosage form in experimental albino rats. Materials and Methods: Present study was carried out at Animal house attached with the Institute. Original Research Article Rathi et al.; JPRI, 33(46A): 289-300, 2021; Article no.JPRI.73771 290 24 healthy Albino wistar rats of either sex weighing 180-200 gm were randomly selected and divided into 4 groups. Pyrexia was induced in rat by subcutaneous injection of 10 mL/kg b.w. of a 20% aqueous suspension of brewer's yeast. The trial drug Dhatryadi Churna and Dhatryadi Vati administered orally in the form of suspension to the albino rats. Observation and Results: The present result shows that the Dhatryadi Churna (DC) and Dhatryadi Vati (DV) have a significant antipyretic effect in yeast induced pyrexia in rats. Hematological parameters were found to be within the normal biological and laboratory limits on comparison with the values of the control group. Conclusion: Dhatryadi churna and Dhatryadi Vati does not show significant adverse effects on the blood, blood cells, and target organs at the doses used and can be safely used in human being.


INTRODUCTION
In olden days all the leading authorities of Ayurveda have stressed the importance of four main Pramanas (modes of examining and acquiring the knowledge) to obtain the real knowledge of any Padartha (subject of knowledge). In other words, these four Pramanas served as different evidences or different methods to prove truthfulness of a matter. They are Prathyaksha (direct evidence), Anumana (Inference), Yukti (rational) and Aptopadesha (authoritative person) [1-2]. The present era is of sciences. Scientific fraternity believes only in proved facts or they require rationality behind facts. The entire suggestion has to be proved by the available, affordable parameters or experiments to establish the facts. Then only that will be accepted by the modern people. Ayurveda has also accepted the importance of visible facts by saying that so it is essential to prove all the Ayurveda theories and drug efficacy scientifically. In the process of new drug development, experimental study is the first step to know the efficacy and safety of the drug. Experimental studies are essential and expected part of a new drug development. The studies are conducted to know about the toxic effects (if any), produced by the drug & to access safety of the drug. Studies also help to ascertain the efficacy of the drug & measures to be taken so as to curtail the toxic effects [3].According to ethics it has been stated that before application of the new drug to the society it should be tested on animals having similar behavior pattern and physiological function as that of humans. Pharmacological studies, conducted on experimental models helps in ascertaining the mode of action of drug, along with its pharmaco-kinetic and pharmacodynamic properties [4]. So it is essential to prove all the Ayurvedic principles and drug efficacy scientifically by modern parameters, through which Ayurveda can excel in current era as "Evidence based, well documented system of medicine. The study was aimed to do comparative evaluation of antipyretic activity of an Ayurvedic herbo-mineral formulation Dhatryadi Churna and its modified dosage form in experimental albino rats. Herbal powder is one of the most often used traditional dose forms in Ayurvedic medicine, and it is used to treat a variety of ailments. Churna is the fine powder of a completely dry medication sifted through a clean cloth in general [5].

MATERIALS AND METHODS
Collection of the raw drugs: All the ingredients were procured from the Ayurveda pharmacy of the institute. All the raw drugs were authenticated from Department of Dravyaguna of the same institute. The formulation selected for the present study Dhatryadi churna was prepared in the P.G Lab of Rasa Shastra and Bhaishajya Kalpana department under expert supervision. Its modified form Dhatryadi vati was prepared by giving the three bhavana of decoction of the same ingredients to the Dhatryadi churna and then punched through tableting machine. Each tablet weighed 250 mg [6].

Materials: Digital Clinical thermometer:
Obtained from animal house. Thermometer has thermo-sensitive and digital display screen to display temperature in Fahrenheit scale which beeps on recording accurate temperature. Glycerine applied thermo sensitive tip is inserted into the rectum of the rat and should be kept for one minute for obtaining the accurate temperature. Brewer's yeast [Baker's yeast] -500g of dried Brewer's yeast was purchased online from Amazon. Calpol [Paracetamol] suspension (5ml containing 120 mg of Paracetamol) -Manufactured by Dr. Reddy Pharmaceuticals Limited was purchased from local medical store as standard drug. Selection of animals: 24 healthy Albino wistar rats of either sex weighing 180-200 gm were randomly selected and grouped into four (Group I to Group IV), so that each group consisted of 6 rats. They were marked with sketch pens for their individual identification.
Rat Maintenance: Animals were well maintained under identical condition of place, light, temperature, humidity, food and other conditions at the animal house. Before the commencement of the experiment all 8 cages required for the experiment were cleaned once in 3 days and there after regularly till the end of experiment. Cages were washed with detergent followed by disinfectant phenol solution to maintain the hygiene. Bedding material was prepared using paddy husk, which was changed once in three days till the end of experiment.
Feeding Schedule: The quantity of food for rats weighing180-200 gm was about 18-20 gm / day. Readymade rat feed prepared by Lipton India Ltd was procured & used. Water was provided as required with the feeding bottles.

METHODOLOGY
Brewer's Yeast Induced Pyrexia Method: Pyrexia was induced in rat by subcutaneous injection of 10 mL/kg b.w. of a 20% aqueous suspension of brewer's yeast in the back below the nape of the rat [7-8]. It induces pyrexia in 1 hr. The trial drug Dhatryadi Churna and Dhatryadi Vati administered orally in the form of suspension to the albino rats in calculated dose as shown in the Table 2.
Procedure: Animals were kept on fasting overnight, but were provided with drinking water. Next morning, the initial rectal temperatures of all rats were recorded. After two hours of induction of fever, the respective DC and DV group drugs were administered through stainless steel oral feeding needles. Calculated quantity of drug was taken in the syringe and pushed directly in a single dose. Rectal temperatures recorded at a regular interval of 1 hr for 14 hours.
Hematological Findings: Twenty-four hours after the last dose, blood samples were collected for estimation of hematological parameters CBC-Hb%, RBC count, Platelet count, MCH, MCHC, MCV and HCT were assessed using an automated machine (Automated CBC Analyzer: Sysmex KX-21).

Evaluation:
The difference between actual values and initial values were registered for each time interval. The maximum reduction in rectal temperature in comparison to the standard positive was calculated and results were compared with the effect of standard drug, Paracetamol.

DISCUSSION
Pyrexia inducing action of yeast Brewer's yeast is a fungi containing lipo-polysaccharide, which is a cell wall component of gram negative bacteria [9]. It binds with macrophages, releasing cytokines, interleukin -1 etc into the blood circulation, leading to antigen-antibody reaction. Then it reduces blood brain barrier and releases Arachidonic acid mediated by the enzymes phospholipase, prostaglandin E2 synthase, and cyclooxygenase [10]. Finally synthesis and release of PGE2 into anterior hypothalamus result in pyrexia [11][12]. Experimental evaluation of antipyretic effect has been carried out in albino wistar rats. Trial drugs were subjected to evaluation in a set of standard experimental albino rats. They were divided into 4 groups as NC group, SC group, DC group and DV group consisting of six rats in each group [13]. 20% yeast solution was prepared in normal saline and given orally with 1ml / 100gm body weight dose. Group NC was treated with distilled water to serve as Control.

Group
Before t/t After t/t elevated body temperature, graft rejection or other disease state. Regulation of body temperature requires a delicate balance between production and loss of heat and the hypothalamus regulates the set point at which body temperature is maintained. Most of the antipyretic drugs inhibit COX-2 expression thus inhibiting PGE2 biosynthesis to reduce elevated body temperature [10]. The present result shows that the Dhatryadi Churna (DC) and Dhatryadi Vati (DV) have a significant antipyretic effect in yeast induced pyrexia in rats.

Graph 9. Comparison of MCHC in four groups before and after t/t Graph 10. Comparison of MCV in four groups before and after t/t
Graph 11. Comparison of HCT in four groups before and after t/t

Before t/t After t/t
There is an extensive evidence to implicate free radicals in the development of disease. Free radicals have been implicated in the causation of ailments such as fever, diabetes, liver cirrhosis etc [14]. The antioxidant activity of Ascorbic acid, Riboflavin, Tannin, Ellagic acid, Lupeol, Tannic acid, Sesamin and plumbagin may helpful in either inhibiting or scavenging radicals. Reactive oxygen damages an important cellular components causing tissue injury through covalent binding and lipid per oxidation. In a previous study, the increase in the body temperature intensified the lipid peroxidation process, which indicates that pyrexia is associated with increased oxidative stress [15]. The antioxidant supplementation decreased the lipid peroxidation process. The flavonoids reported to have antioxidant activity. Hence, antioxidant activity may be one of the possible mechanisms by which it reduces the elevated body temperature. Yeast-induced pyrexia is called pathogenic fever and its etiology involves production of prostaglandins.
Most of the marketed preparations, drug compounds and toxic substances which are foreign to our body target the blood system ie. hematopoietic system, an important index of bodies physiology involved in the creation of the cells of blood and determining the pathological status in rodent and human being [16]. Hematological analysis shows the picture of the values of the different parameters examined like complete blood count (CBC), hemoglobin Hb%, Red blood cell (RBC) count, Platelet count, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), Mean corpuscular volume (MCV) and HCT were found to be within the normal biological and laboratory limits on comparision with the values of the control group. No significant changes were observed in all the treated groups as compared to the normal control groups. This indicates that the Dhatryadi churna and Dhatryadi Vati does not show significant adverse effects on the blood, blood cells, and target organs at the doses used. In the present study, from result it is cleared that, both the herbo mineral formulations DC and DV also significantly increases the platelet count as compare to normal control thus played an important role in increasing immunity and due to presence of many active substances like flavonoids may be involve in balancing the redox parameter thus protect from oxidative stress.
Dhatryadi Churna (DC) and Dhatryadi Vati (DV) showed significant antipyretic activity and the effects are comparable to standard drug Paracetamol (PCM) that may be due to inhibition of prostaglandin synthesis. Again, many researchers reported that the formulations containing alkaloids, tannins, carbohydrates and flavonoids have been reported antipyretic potential in various studies. In many earlier studies flavonoids compounds have been reported to exhibit antipyretic effect, as some flavonoids are predominant inhibitors of cyclo oxygenase or lipo oxygenase [17]. So Dhatryadi Vati (DV) form is therapeutically more effective than Dhatryadi Churna (DC). Paracetamol is an analgesic but is also an effective febrifuge. It is a poor inhibitor of cyclo oxygenase in the presence of peroxides that are found in inflammatory lesions. In contrast, its antipyretic effect may be explained by its ability to inhibit cyclo oxygenase in the brain, where peroxide tone is low. Further, it does not inhibit neutrophil activation. In suprapharmacologic doses it inhibits NF-kB stimulation of inducible nitric oxide synthase [18]. In the present study, in the standard control group the rise in temperature was consistent and significant in comparison to the initial values. In the Negative control group also the rise in temperature was significant; however, the magnitude was slightly less in comparison to that in the standard control group in the initial stages. Both the Dhatryadi Churna and Dhatryadi Vati samples produced very good antipyretic effect in a dose-dependent manner and the observed effect were almost similar to that in the paracetamol-treated group. Churna Kalpana being the most commonly preferred dosage form holds certain disadvantages like higher chances of oxidation and hydrolysis, non-palatability, low shelf life to overcome these issues DC modified into DV, holding the advantages as higher shelf life, easy for administration and fixed dosage.

CONCLUSION
The comparison of Anti Pyretic activity in experimental study clearly indicates that DV possess significantly higher efficacy than DC and PCM (p value -0.005). Thus, Dhatryadi Churna can be replaced by modified dosage from Dhatryadi Vati and the present formulation should be studied in clinical study in human trials to assess the antipyretic action as it may be a replaced by standard paracetamol of alternate natural safe formulation.

RESEARCH SIGNIFICANCE
The study highlights the efficacy of " an Ayurvedic formulation " which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.