Pharmaceutico Analytical and Experimental Study of Amalakyadi Churna in Jwara with Special Reference to Antipyretic Activity

Ayurvedic pharmaceutics involves processing medicinal substances into dosage forms suitable for administration. Churna Kalpana is an Upakalpana in which dried raw drugs are reduced to a fine powder and mixed according to the formulation. The preparation method described in Sharangadhara Samhita provides the pharmaceutical basis for processing and combining the ingredients.¹

Jwara occupies an important position in Ayurvedic literature and is discussed both as a disease and as a manifestation associated with other conditions. Classical descriptions emphasize the involvement of Dosha, Agni and Ama in its development.² In contemporary physiology, fever or pyrexia involves an increase in body temperature associated with elevation of the hypothalamic thermoregulatory set point.³ Experimental evaluation of an Ayurvedic Jwarahara formulation can therefore assess its effect on induced pyrexia while retaining the distinction between the classical disease concept and the experimental temperature endpoint.

Amalakyadi Churna, described in Bharata Bhaishajya Ratnakara, contains Amalaki, Chitraka, Pathya or Haritaki, Pippali and Saindhava Lavana in equal proportions. The formulation is traditionally indicated for Jwara and is described as possessing Bhedana, Ruchikara, Dipana, Pachana and Kaphahara properties.⁴ Its ingredients are readily available, and the powder preparation is comparatively simple to perform.

The need for scientific evaluation of the classical indication of Amalakyadi Churna in Jwara prompted the present study. Hence, the study was conducted to evaluate its pharmaceutical preparation, analytical characteristics and experimental antipyretic activity.

MATERIALS AND METHODS

The study comprised three components: pharmaceutical preparation, analytical evaluation and experimental assessment of the antipyretic activity of Amalakyadi Churna.

1. Pharmaceutical Study

1.1 Preparation of Churnodaka and Chitraka Shodhana

Churnodaka was prepared using 12.5 g of Sudha Churna and 3 L of water. The mixture was left undisturbed for nine hours, after which the clear liquid was collected.⁵ Chitraka roots were cut into small pieces and immersed in sufficient Churnodaka overnight. The material was collected on the following day and dried. The Shodhana procedure followed Rasa Tarangini, chapter 24, verse 575.⁶

During Churnodaka preparation, the freshly mixed liquid appeared milky white and subsequently became clear, with a white surface layer. During Chitraka Shodhana, the liquid changed from light brownish pink to dark brownish pink after approximately nine hours.

1.2 Preparation of Amalakyadi Churna

Amalakyadi Churna was prepared according to Bharata Bhaishajya Ratnakara, following the general Churna preparation procedure described in Sharangadhara Samhita. Raw materials were checked for authenticity before use.¹

Table 1: Ingredients and Quantities of Amalakyadi Churna

Cleaning and drying

The herbal ingredients were cleaned, washed and dried under sunlight in a clean place. Materials were monitored for fungal infestation and contamination. Chitraka was dried after Shodhana. Each ingredient was weighed according to the prescribed proportion. Saindhava Lavana was handled separately.

Powdering sieving and mixing

Amalaki, Pippali, Chitraka and Haritaki were pounded separately using Khalwa Yantra and then powdered individually using a mixer grinder. The fibrous nature of Chitraka required thorough pounding. Saindhava Lavana was finely powdered separately. The herbal powders were individually sieved, combined and mixed thoroughly to obtain a homogeneous preparation. Saindhava Lavana was added at the end. The finished Churna was stored in an airtight container.

Precautions included thorough drying of the raw drugs, use of Shodhita Chitraka, separate processing of the ingredients and avoidance of moisture during storage. Weight changes during grinding and sieving were recorded.

2. Analytical Evaluation

The prepared Amalakyadi Churna was subjected to organoleptic and physicochemical evaluation for characterization of the formulation. Powder microscopy and HPTLC fingerprinting were also performed.

Physical appearance, colour, odour, taste and consistency were examined. The physicochemical assessment included pH, loss on drying, total ash, acid-insoluble ash, water-soluble ash, alcohol-soluble extractive value, water-soluble extractive value, bulk density, tapped density, Carr’s index and Hausner’s ratio.⁷

For loss on drying, 10 g of the sample was dried at 105 °C for five hours and further dried to constant weight. Total ash was determined by incinerating 2 g of sample at a temperature not exceeding 450 °C until carbon-free ash was obtained. Acid-insoluble ash was determined from the ash residue using dilute hydrochloric acid, filtration and ignition to constant weight. Water-soluble ash was determined by boiling the ash with water and subtracting the insoluble residue from total ash.⁷

For extractive values, 4 g of the sample was treated with 100 mL of the respective solvent, shaken intermittently for six hours and left to stand for 18 hours. A measured portion of the filtrate was evaporated, dried at 105 °C and weighed. The aqueous sample preparation for pH measurement was 1% w/v, and the instrument was calibrated using standard buffers. Bulk and tapped densities were recorded, with the latter measured after 50 tappings. Powder microscopy was performed after mounting the sample in glycerine, using a Zeiss Axio trinocular microscope with an attached camera.

One gram of Amalakyadi Churna was extracted with 10 mL of ethanol. Aliquots of 3, 6 and 9 µL were applied as 7 mm bands to precoated silica gel F??? aluminium plates using a Linomat 5 applicator. The mobile phase was Toluene:Ethyl acetate:Formic acid (5.0:4.0:1.0). Developed plates were visualized under short and long ultraviolet light and after derivatization with vanillin-sulphuric acid. Densitometric scans were recorded at 254, 366 and 620 nm, the latter following derivatization. Rf values and band colours were documented.

3. Experimental Study

The antipyretic activity of Amalakyadi Churna was evaluated using Brewer’s yeast-induced pyrexia in healthy Wistar albino rats of either sex, weighing 180–250 g. Animals were housed in standard plastic cages with stainless-steel coverings and wheat-straw bedding under controlled environmental conditions, with a balanced pelleted diet and water. Pregnant animals, diseased animals and animals involved in other experiments were excluded.

The experimental protocol was approved by the Institutional Animal Ethics Committee under approval number SDMCRA/IAEC/SDM-KAMC-MR-14.

Eighteen rats were randomly divided into three groups of six animals each. The Control group received distilled water, the Standard group received Paracetamol and the Test group received Amalakyadi Churna.

Table 2: Experimental Grouping

3.1 Induction of Pyrexia

Animals were fasted overnight before the experiment. Initial normal rectal temperatures were recorded using a digital thermometer. Pyrexia was induced by subcutaneous injection of Brewer’s yeast suspension at 1 mL/100 g body weight. Rectal temperature was recorded 18 hours after injection to confirm pyrexia.

3.2 Dose Fixation and Treatment

Following confirmation of pyrexia, the corresponding treatments were administered orally using a feeding tube. The Control group received distilled water, the Standard group received Paracetamol at 100 mg/kg body weight and the Test group received Amalakyadi Churna at the calculated rat dose.

The rat dose was calculated using the formula: human dose × 0.018 × 5 per kilogram body weight. The human dose was one Karsha, equivalent to 12 g, corresponding to a calculated rat dose of 1.08 g/kg body weight.

3.3 Assessment of Antipyretic Activity

Antipyretic activity was assessed by recording rectal temperature before and after yeast administration and at subsequent observation intervals following treatment. The stated protocol called for hourly readings for five hours and a further reading at 24 hours. Temperature responses and percentage comparisons were recorded for the three groups.

PGE? results were evaluated for the Control, Standard and Test groups as a biochemical outcome alongside rectal temperature.

3.4 Statistical Analysis

Experimental data were statistically analyzed using analysis of variance (ANOVA).

RESULTS

Pharmaceutical findings

Amalakyadi Churna was prepared as a fine brown powder with the characteristic odour of its ingredients. No fungal infestation was observed during preparation. Processing losses occurred during grinding, sieving and handling, with some fibrous material remaining after grinding. The weights recovered from the individual ingredients are shown in Table 3.

Table 3: Weight Changes During Grinding and Sieving

Table 4: Organoleptic Characteristics

Analytical findings

Table 5: Physicochemical Parameters of Amalakyadi Churna

HPTLC demonstrated multiple bands under different visualization conditions. Under short UV, bands were reported at Rf 0.26, 0.48, 0.56 and 0.78. Under long UV, bands were observed at Rf 0.56, 0.65 and 0.73. Following derivatization, bands were recorded at Rf 0.15, 0.28, 0.47, 0.80 and 0.87.

Experimental findings

Brewer’s yeast administration increased rectal temperatures in all three groups. This increase was reported as statistically significant relative to the initial normal temperature. The reported means after yeast injection were 39.63 ± 0.1961 °C in Control, 40.30 ± 0.1183 °C in Standard and 39.72 ± 0.2272 °C in Test. The rectal temperatures are presented in Table 6.

Table 6: Reported Rectal Temperature Summary

Mean ± SEM.

Amalakyadi Churna did not produce statistically significant antipyretic activity compared with Control. At the first, third and fourth hourly observations, the Test group had lower reported mean temperatures than Control. The reported percentage comparisons are shown in Table 7. Temperature reductions were reported for the Standard group at several observations.

Table 7: Reported Percentage Comparisons in Rectal Temperature

At the 24-hour observation, the Test mean was 39.27 ± 0.2305 °C and Control was 39.44 ± 0.1631 °C. The Standard mean was 39.92 ± 0.2417 °C. The temperature-based antipyretic response of the Test group remained statistically non-significant compared with Control.

Reported PGE? findings

Reported PGE? values were 250.34 ± 2.34 in Control, 55.18 ± 1.22 in Standard and 108.56 ± 3.55 in Test. Reported reductions were 77.95% and 56.63% for Standard and Test, respectively, with the results marked as p < 0.01 and p < 0.05 (Table 8). This biochemical finding is presented separately from the temperature-based antipyretic conclusion.

Table 8: Reported Effect on Pge? Levels

Mean ± SEM; *p < 0.05, **p < 0.01.

DISCUSSION

Classical rationale and pharmaceutical preparation

The selection of Amalakyadi Churna was based on its classical indication in Jwara and the properties attributed to its constituents. The classical rationale considers Amalaki in relation to its Sheeta Virya and Rasayana properties; Haritaki in relation to digestion and removal of Ama; Pippali in relation to Katu Rasa, Ushna Virya and Rasayana properties; Chitraka in relation to Dipana, Pachana and Vata-Kapha-hara actions; and Saindhava Lavana in relation to Dipana, Pachana and Ruchya properties. This classical rationale informed the experimental investigation; it does not substitute for demonstration of antipyretic efficacy.

The pharmaceutical process involved cleaning, drying, Chitraka Shodhana, separate pounding and powdering, sieving and mixing. Chitraka was processed in Churnodaka before incorporation into the formulation. Separate powdering allowed the fibrous ingredients to be handled individually, while addition of Saindhava Lavana at the final stage followed the preparation procedure.

The individual processing losses ranged from 1% for Saindhava Lavana to 8% for Chitraka. Losses during grinding, sieving and handling, together with retention of fibrous material, accounted for the reduced recovered weights. The resulting product was a fine brown powder with characteristic odour and mixed astringent, sweet, pungent and salty taste.

Interpretation of the analytical profile

Analytical assessment provided a physicochemical and chromatographic profile of the prepared formulation. The pH of 7.05 indicated a value close to neutral under the test conditions. Loss on drying documented the sample’s drying-related weight loss, while total ash, acid-insoluble ash and extractive measurements characterized the preparation. Bulk density, tapped density, Carr’s index and Hausner’s ratio documented powder packing and flow-related characteristics. The Carr’s index and Hausner’s ratio were interpreted as indicating good flow properties.

HPTLC demonstrated bands at different Rf values under short UV, long UV and after derivatization, indicating the presence of multiple detectable constituents and a characteristic chromatographic profile of Amalakyadi Churna.

The analytical parameters were reported to be within preliminary standard limits. The measured profile provides a basis for characterization of the prepared sample and comparison with subsequent preparations made using the same procedure.

Interpretation of the experimental findings

In the experimental pyrexia study, Amalakyadi Churna showed reductions in rectal temperature at selected observation intervals. At the first hour, the mean temperature in the Test group was 39.22 ± 0.2227 °C, compared with 39.60 ± 0.1592 °C in Control and 39.06 ± 0.3614 °C in the Standard group. At the third hour, the Test mean was 39.22 ± 0.1600 °C, compared with 39.40 ± 0.3502 °C in Control. At the fourth hour, the Test mean was 39.10 ± 0.1461 °C, compared with 39.58 ± 0.4505 °C in Control and 39.20 ± 0.4012 °C in the Standard group. Although these observations showed lower mean temperatures than Control, the antipyretic effect of Amalakyadi Churna remained statistically non-significant.

The observed temperature reductions may be considered in relation to the combined Dipana, Pachana, Amapachana and Jwarahara properties attributed to Amalaki, Haritaki, Pippali, Chitraka and Saindhava Lavana. In the Ayurvedic account of Jwara, these properties are considered to address Agnimandya and Ama. They provide the classical rationale for evaluating the formulation’s antipyretic activity.

At the 24-hour observation, the mean rectal temperature in the Test group was 39.27 ± 0.2305 °C, compared with 39.44 ± 0.1631 °C in Control. The difference was statistically non-significant. The lower mean temperature represents a numerical trend, while the statistical comparison did not establish a significant antipyretic effect.

Overall, the pharmaceutical and analytical findings characterized the prepared Amalakyadi Churna. The experimental findings showed temperature reductions at selected intervals, but did not establish statistically significant antipyretic activity compared with Control under the study conditions.

CONCLUSION

Amalakyadi Churna was prepared using the selected classical formulation and standard operating procedure, including Chitraka Shodhana followed by separate powdering, sieving and mixing of the ingredients. Organoleptic, physicochemical, microscopic and HPTLC assessments documented the characteristics of the prepared sample. The analytical parameters were reported within preliminary standard limits.

In Brewer’s yeast-induced pyrexia in Wistar albino rats, Amalakyadi Churna did not demonstrate statistically significant antipyretic activity compared with Control under the experimental conditions.

REFERENCES

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