Physicochemical and Phytochemical Profiling with Antimicrobial Activity of Selected Kushtaghna Mahakashaya Drugs

INTRODUCTION

The skin, the body's outermost layer, serves as a protective barrier for underlying tissues and organs. Skin disorders account for approximately 1.79% of all diseases worldwide (Deshmukh and Kshirsagar, 2024). Multiple factors contribute to the development of these conditions, often categorized under the term "Kushta" in Ayurvedic literature, which may result from environmental changes and lifestyle factors, such as altered work schedules that increase stress. Traditionally, a group of ten medicinal plants, known as Kushthaghna Mahakashaya, has been used to treat skin ailments. These herbs are believed to possess anti-inflammatory, immunomodulatory, and antioxidant properties that can help manage skin disorders (Saini et al., 2022). From this group, five plants, Khadira (Acacia catechu Willed), Haritaki (Terminalia chebula Retz), Amalaki (Emblica officinalis Gaertn.), Haridra (Curcuma longa Linn.) and Vidanga (Embelia ribes)-were selected based on their high therapeutic utility. These plants were evaluated for their physical and phytochemical properties, as well as the antimicrobial activity of their combined preparation, Kwatha Churna (powder).

MATERIALS AND METHODS

Raw drugs of Khadira (Acacia catechu Willed), Haritaki (Terminalia chebula Retz) Fruit, Amalaki (E. officinalis Gaertn.) Fruit, Haridra (C. longa Linn.) Rhizome, Vidanga (Embelia ribes) seed were collected from KLE GMP Certified Pharmacy, Belagavi, Karnataka. The authentication and identification of raw drugs were done at the AYUSH-Approved ASU Drug Testing Laboratory, Belagavi. Dry drugs were used to study the physical, phytochemical parameters, and antimicrobial activity (Patel et al., 2019).

Ethical Statement

This in vitro study did not involve human or animal subjects; thus, ethical clearance was not required.

Statistical Analysis

The obtained data were analyzed descriptively based on physicochemical, phytochemical, High-Performance Thin-Layer Chromatography (HPTLC), and antimicrobial observations. Results were presented in tabular and graphical form wherever applicable. No advanced statistical analysis was performed, as the study primarily focused on qualitative and observational evaluation of the selected Kushtaghna Mahakashaya drugs.

Reagents and Instruments

Every chemical reagent and solvent utilized in the investigation was of analytic quality. watch glass, microscopic slide, Simple microscope, compound microscope, flask shaker, sonicator, hot-water bath, muffle furnace and other common glassware were used in this experiment. Chemical reagents like Dragendorff ’s, Millon’s, and Benedict’s were used for physicochemical studies

Macroscopy

Organoleptic characters like color, texture, form, and odor were assessed by observing the dried raw drugs.

Microbial Load

The microbial load of Khadira (Acacia catechu Willed), Haritaki (Terminalia chebula Retz) fruit, Amalaki (E. officinalis Gaertn.), Haridra (C. longa Linn.), and Vidanga (Embelia ribes) was evaluated using Nutrient Agar, MacConkey Agar, Sabouraud dextrose Agar, Soybean Casein Digest Agar, Mannitol Salt Agar, cetrimide Agar, Xylose Lysine Deoxycholate Agar (XLD), buffer, and Soybean Casein Digest Medium broth at the Microbiology Laboratory of the AYUSH-Approved ASU Drug Testing Laboratory, Belagavi.

Physicochemical Testing

Physicochemical characteristics, including foreign organic matter, total ash, acid-insoluble ash, alcohol-and water-soluble extractives, and loss on drying, were measured using established techniques. All of these quantitative experiments employed air-dried Fine powder (40 #) samples. Using ethanol and water, extractive values were calculated.

Phytochemical Assessment

Standard protocols were used for phytochemical screening. Alkaloids, glycosides, phenols, flavonoids, and other phytoconstituents were detected using chemical assays on the alcohol-soluble floral extract, whereas proteins, carbohydrates, and saponins were detected using an aqueous extract (Khandelwal, 2013).

HPTLC Analysis

Chemical and reagents

The HPTLC plate used measured 4×10 cm and was precoated with silica gel 60 F254 TLC (0.2-mm thickness) on an aluminum sheet support (E. Merck). A sample application was performed using a CAMAG Linomat V Automatic Sample Spotter (Camag, Muttenz, Switzerland) with a 100-μL Hamilton syringe. The plate was developed in a CAMAG glass twin-trough chamber (10×10 cm), and densitometric analysis was carried out using a CAMAG TLC Scanner 3 connected to WINCATS software. The mobile phase consisted of chloroform: ethyl acetate: formic acid (5:4:1).

Preparation of Test Sample

1 g of the sample was weighed. For the aqueous extract, the extract was initially prepared with 5 mL of water, and the solution was evaporated to dryness. The residue was then further extracted with 5 mL of methanol. For the alcoholic extract, the sample was extracted with 5 mL of ethanol, evaporated to dryness, and the residue was subsequently extracted with 5 mL of methanol. The resulting methanol solution was used for HPTLC. 5 μL of the extract was applied to the HPTLC plate and developed to a distance of 8 cm using a solvent system of chloroform: ethyl acetate: formic acid (5:4:1). After development, the plate was air-dried and observed under UV light at 254 and 366 nm.

Method

The plate was developed in a twin-trough chamber that had been presaturated with the solvent system for 30 min. The syringe was rinsed twice with methanol before use. After development, the plate was dried and placed in the scanner. A new file was opened, and all scanning, integration, and spectral parameters were set. For the absorption-reflection mode, the plate was scanned at 425 nm using a tungsten lamp. Each track was scanned individually, and the UV spectrum of every track was recorded. The fingerprint profile of each track was obtained, and the UV spectral spots were compared using the spectrum display.

Antimicrobial Study

Kashaya (decoction) of compound drugs -comprising Haridra, Amalaki, Vidanga, Haritaki, and Khadira was prepared from coarse powder (40#). 2g of each coarse powder was added to 160 ml of purified water and allowed to soak for 30 mins. The mixture was then boiled on medium flame until the part, that is, 20 mL. After the reduction, the decoction was allowed to cool and filtered. volume reduced to 1/8 th of its initial volume (20ml), after which it was filtered.

Microorganisms and Media

The sensitivity test was carried out against Gram-negative and fungal species, namely Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, respectively. In 10 different concentrations.

Serial dilutions of the test compounds were prepared in different rows of sterile 96-well microplates and were exposed to fixed concentrations of standardized suspension of the test organism overnight. If the test compounds were able to inhibit the bacterial growth, then the solution in the test wells would remain clear in the respective dilutions. If there were no bacterial growth inhibition, then the growth medium in the test wells would be turbid. The last well in each row that shows no growth (remains clear) is considered the Minimum Inhibitory Concentration (MIC) value of the test product against the targeted organism. This could be confirmed by the addition of an indicator dye such as resazurin sodium.

RESULTS

Macroscopic Description

The form was kwatha churna, the color was brownish yellow, and the odor was aromatic.

Physicochemical Studies

Moisture content at 110ºC was found to be 9.8 and 0.52%, respectively. The total ash and acid-insoluble ash values were found to be 4.3 and 0.61%, respectively. The water-soluble extractives and alcohol soluble extractives values were found to be 17.9 and 20.6%, respectively. Physicochemical studies results are shown in Table 1.

Phytochemical Evaluations

It has been demonstrated that the alcoholic extract contains reducing sugar, monosaccharides, proteins, flavonoids, and tannins, whereas the aqueous extract contains carbohydrates, monosaccharides, reducing sugars, and proteins. Table 2 displays preliminary phytochemical results.

HPTLC Analysis

HPTLC profiling of the aqueous and alcoholic extracts revealed the presence of multiple phytoconstituents with distinct banding patterns at different R_f values. The chromatographic patterns observed under UV 254 and 366 nm demonstrated characteristic fingerprints for the extracts. The HPTLC chromatograms recorded at 254 and 366 nm are presented in Figures 3 and 4, respectively. The corresponding R_f values and peak details obtained from HPTLC analysis are summarized in Tables 3 and 4 for the aqueous and alcoholic extracts, respectively. Densitometric analysis further showed multiple peaks corresponding to different chemical constituents, confirming the phytochemical complexity of the extracts. The densitograms of the aqueous and alcoholic extracts are shown in Figures 1 2 3 4, respectively.

Antimicrobial Activity (MIC Determination)

The minimum inhibitory concentrations of the Kashaya (Aqueous extract) were evaluated against Gram-positive, Gram-negative and fungus. The extract exhibited greater potency, showing inhibition of S. aureus at 1.60% (vol/vol), P. aeruginosa at 3.13% (vol/vol) and C. albicans at 1.60% (vol/vol). P. aeruginosa (Gram-negative) remains relatively less susceptible. Results are shown in Table 5.

DISCUSSION

The physicochemical evaluation of the churna reveals parameters that collectively affirm its quality, purity, and suitability for medicinal use. The moisture content of 9.8% is within acceptable limits for powdered herbal formulations, minimizing the risk of microbial growth and ensuring stability during storage. Excessive moisture can promote microbial contamination and degradation of active constituents; thus, the observed value suggests good handling and drying practices during preparation. The foreign matter content was found to be 0.52%, indicating minimal contamination with extraneous materials such as soil, sand, or plant debris. This low value reflects adherence to proper collection, cleaning, and processing protocols, aligning with pharmacopeial standards for crude drug materials. The total ash content was determined to be 4.3%, reflecting the total mineral residue remaining after incineration. This value encompasses both physiological ash (natural plant mineral content) and any extraneous inorganic matter. The relatively moderate ash level suggests acceptable purity and minimal adulteration. The acid-insoluble ash value of 0.6% further confirms the low presence of siliceous matter such as sand or silica, which is particularly significant in assessing sample cleanliness and proper processing. High acid-insoluble ash would indicate contamination with insoluble minerals, but the current low value signifies good quality control (Government of India, 1990 India, -2008)). The phytochemical screening of the compound kwatha churna revealed the presence of flavonoids, tannins, reducing sugars, monosaccharides, and proteins, many of which are pharmacologically relevant to skin health. Flavonoids and tannins, present in both aqueous and alcoholic extracts, contribute antioxidant, anti-inflammatory, and antimicrobial effects that support their use in managing skin conditions (Kumar and Pandey, 2013; Trease and Evans, 2009). The presence of simple sugars and proteins may further enhance hydration and skin barrier function. These findings support the therapeutic potential of the formulation in dermatologic applications.

The formulation was analyzed through both aqueous and alcoholic extracts, revealing distinct chemical signatures. The alcoholic extract exhibited seven peaks, including a prominent peak at R_f 0.93 (18.26% area) (Ansari et al., 2005), which is suggestive of curcumin, the principal bioactive ofC. longa. Curcumin has been shown to possess potent anti-inflammatory, antioxidant, and antimicrobial effects, supporting its widespread use in acne, eczema, psoriasis, and wound healing (Vijayakaran et al., 2025).

A shared peak near Rf 0.51 in aqueous extracts indicates the presence of ellagic acid, commonly found in E. officinalis and T. chebula. These compounds exhibit antioxidant, photoprotective, and melanogenesis-inhibiting activities, making them beneficial for skin brightening and antiaging effects (Aggarwal et al., 2007; Naik et al., 2003). The strong antioxidant capacity of Amalaki, particularly due to ascorbic acid and polyphenols, enhances collagen biosynthesis, a crucial factor in maintaining skin elasticity and preventing premature aging (Sharma et al., 2009).

The peak at Rf 0.62 ± 0.02 (1.09% area) in aqueous extract and a may represent embelin, the key active of Embelia ribes. (KPR Vijayan et al,., 2021). Embelin possesses broad-spectrum antimicrobial and antifungal activity, contributing to the formulation's role as a krimighna (antimicrobial) agent (Scartezzini and Speroni, 2000). in Ayurvedic dermatology. (Sharma V et al,., 2022) Acacia catechu, represented likely by peaks around Rf 0.49 and 0.54, contains quercetin and catechin (Srivatsav et al,. 2024), which demonstrate anti-inflammatory and antioxidant effects. These flavonoids also enhance microvascular protection and inhibit matrix metalloproteinases, both critical in the prevention of dermal degradation in inflammatory and aging skin conditions (Poojari et al., 2013).

Click to enlarge

Click to enlarge

The Kashaya showed concentration-dependent antimicrobial activity against S. aureus, P. aeruginosa, and C. albicans. Higher concentrations (50-3.13%) exhibited significant antimicrobial effects, whereas lower concentrations showed resistance, indicating reduced activity due to lower phytochemical concentrations (Cowan, 1999). The kashaya also demonstrated antifungal activity against C. albicans, suggesting the presence of bioactive compounds capable of disrupting fungal cell membranes and metabolic pathways (Silva et al., 2012). Overall, the results suggest that the Kashaya possesses broad-spectrum antimicrobial activity, likely due to phytochemicals such as flavonoids, tannins, phenols, and alkaloids present in the formulation.

CONCLUSION

The prepared kwatha churna exhibited satisfactory physicochemical characteristics. Was found to possess a diverse range of bioactive phytoconstituents, particularly flavonoids and tannins known for their therapeutic relevance in skin disorders. HPTLC analysis established the presence of important phytochemical markers, confirming the authenticity and chemical consistency of the formulation. Furthermore, the aqueous extract demonstrated significant antimicrobial efficacy. The overall findings scientifically support the traditional application of the formulation and indicate its promising potential as a natural dermatologic and antimicrobial agent.

Click to enlarge

SUMMARY

This paper focused on the physicochemical, phytochemical, fluorescence, High-Performance Thin-Layer Chromatography (HPTLC), and antimicrobial evaluation of selected Kushtaghna Mahakashaya drugs. Standard analytic methods were used to profile the raw drugs and aqueous decoction. The study also assessed antimicrobial activity against selected bacterial and fungal strains to explore the therapeutic relevance of these formulations.

ACKNOWLEDGEMENT

The author expresses their sincere gratitude to the guide for valuable guidance, constant encouragement, and support throughout the completion of this research work. The authors also acknowledge the support provided by the department and laboratory staff for facilitating the analytic and antimicrobial studies carried out during the study.

ABBREVIATIONS

PhD

Doctor of Philosophy

AYUSH

Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homeopathy

ASU

Ayurveda, Siddha and Unani

GMP

Good Manufacturing Practice

SDA

Sabouraud Dextrose Agar

SCDA

Soybean Casein Digest Agar

MSA

Mannitol Salt Agar

XLD

Xylose Lysine Deoxycholate Agar

SCDM

Soybean Casein Digest Medium

HPTLC

High-Performance Thin-Layer Chromatography

MIC

Minimum Inhibitory Concentration

UV

Ultraviolet

Rf

Retention Factor

HAVAK

Haridra, Abhaya, Vidanga, Amalaki, and Khadira.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

AUTHOR CONTRIBUTIONS

1st Author: literature review, experimental work, data collection, analysis, interpretation of results, and manuscript preparation. 2nd Author: Supervised the research work, provided scientific guidance, reviewed the manuscript, and approved the final version for submission. Competing Interests.