ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF RED OCHRE TRITURATED WITH PUNICA GRANATUM (DADIM BHAVIT GAIRIKVATI)

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF RED OCHRE TRITURATED WITH PUNICA GRANATUM (DADIM BHAVIT GAIRIKVATI)

Avinash Ashok Shete *, Jayashree Patil, Ashwin Ashok Shete and Arati Prabhakar Dubewar

Department of Streerogaevam Prasutitantra, Dr. D. Y. Patil College of Ayurveda and Research Centre, Pimpri, Pune, Maharashtra, India.

ABSTRACT: Antibiotic or antifungal drug resistance is a major challenge in this era. Because the discovery and development of new drugs takes a long time. So, ancient medicine formulations are being checked for antimicrobial properties. Red ochre triturated with Punica granatum (Dadim bhavit gairikvati) is herbo-mineral formulation prepared by red ochre (Gairika/Ferric Oxide fe₂O₃) triturated with Punica granatum (Dadima-pomengrate juice). The process is done according to the classical text Sharangadhara Samhita. This formulation is useful in naushea, vomiting, hyperacidity, anemia, piles, anorexia, diarrhea, burning micturation, etc. Analytical testing shows normal results within the standard range. The formulation is tested with reference to standard drugs for antibacterial study as two types of gram positive bacteria, viz. Staphylococcus aureus and Bacillus subtilis, and two types of gram negative bacteria, viz. E. coli and Proteus mirabilis. Two fungi, named Candida albicans and Aspergillus niger are also tested again with the same formulation. Five batches of samples for each organism are tested. A statistical tool such as the mean (a measure of central tendacy) is used for analysis. Staphylococcus aures (14.6mm), Bacillus subtilis (16mm), Escheria coli (15mm), Proteus mirabilis (16.4mm), Candida albicans (17.2mm) and Aspergillus niger (19.2mm) were found to have a zone of inhbition in the formulation. The positive result shows about 50% antibacterial and antifungal activity as compared with standard drugs (Streptomycin and fluconazole).

Keywords: Red ochre, Gairik, Antifungal, Antimicrobial, Zone of inhibition, Punica granatum

INTRODUCTION: Since their discovery, antibiotics have saved countless lives ¹. Many antibiotics found effective previously are not potent in the present era ². Antimicrobial drugs that are available on the market are being challenged by microbes. Antibiotics show resistance or fewer effects. Antibiotics are misused worldwide ³. Microbial resistance to present antimicobial drugs causes death or health calamities ⁴.

New drug discoveries are being carried out, but it may need more time in modern medicine. To overcome this challenge, alternative but traditional systems of medicine can show rays of light. The Herbo-minerals formulations are being tested in this study ⁵. Herbs show antibacterial activity with the presence of phytochemicals and alkaloids ⁶. Herbo-mineral preparations were also found to be effective as antimicrobial drugs ⁷_.

Red ochre triturated with Punica granatum (Dadimbhavitgairikvati) is herbo-mineral formulation prepared by red ochre (Garika / Ferric Oxide fe₂O₃) triturated with Punica granatum (Dadima-pomengrate juice). Then it was dried in shadow and subjected to tableting of 250mg. The process is done according to the classical text Sharangadhara Samhita ⁸. This formulation is useful in naushea, vomiting, hyperacidity, anemia, piles, anorexia, diarrhea, burning micturation, etc. The formulation has not been assessed as an antibiotic or antifungal. Hence, the study was done to find out the antimicrobial activity of the formulation. The formulation was tested for antibacterial study as two types of gram positive bacteria viz. Staphylococcus aureus ⁹ and Bacillus subtilis ¹⁰ and two types of gram negative bacteria viz. E. coli ¹¹ and Proteus mirabilis ¹². Two fungi named Candida albicans ¹³ and Aspergillus niger ¹⁴ are also tested again with the same formulation.

The standard antibiotic range is used for reference. Staphylococcus aureus causes pneumonia, endocarditis, Bacillus subtilis causes bacteremia, endocarditis, pneumonia, septicemia etc. E. coli is the cause of watery or bloody diarrhea, fever, abdominal cramps, nausea and vomiting. P. mirabilis is capable of causing urinary tract infections, including cystitis and pyelonephritis ¹⁵. Candida albicans causes candidiasis and Aspergillus niger causes aspergillosis.

MATERIAL AND METHOD:

Materials:

Punica granatum (Dadima-Pomengranate): This is an ancient fruit described in classical texts ¹⁶. Its antioxidant activity is already known ¹⁷. Classical texts explained it for digestion and nutrition along with a therapuetic effect ¹⁸. It paliates all tridosha (three vital elements of the body) and provides vigour with vitality ¹⁹. Red ochre (Gairika-Ferric Oxide fe₂O₃) is indicated for hiccups, vomiting, naushea and hyperacidity ²⁰. Red ochre (Gairika-Ferric Oxide fe₂O₃) is purified by frying with cow ghee as per the classical text Rasa tarangini ²¹. In addition, cow ghee is also mentioned with medicinal properties in classical texts ²².

Method: Raw material authentification is done at Agharkar Research Institute, Pune (an autonomous body under the department of science and technology, Govt. of India) with authentification voucher no. AUTH 22-49 dated 10/05/2022. Raw Red ochre (Gairika-Ferric Oxide fe₂O₃) is shallow fried with 1/4^th cow ghee as a process of purification. This is done with reference to Rasa Tarangini. Red ochre (Gairika-Ferric Oxide fe₂O₃) in shudhha or purified form is trichurated with dadim (pomegranate) juice. This mixture is subjected to tableting of 250mg. Its physico-chemical analysis is done Table 1. Macroscopic studies or organoleptic properties are assessed as color, odour and taste. Analytical studies such as pH, loss on drying, hardness, disintegration, average weight, friability, etc. are tested with standard methods and instruments Table 1.

Antimicrobial Study:

Antimicrobial Activity Test (Kirby-Bauer Method): An antimicrobial activity test was performed by the commonly used agar diffusion method, which is designed to determine the smallest amount of the antibiotic needed to inhibit the growth of microorganisms ²³.

Requirements;

• 18 hr nutrient broth culture of the test organism
• Standard Chloramphenicol
• Mueller-Hinton agar plates
• Cork borer
• Sterile standard bioassay filter paper disc²⁴.
• Sterile cotton swabs
• Alcohol
• Ruler
• Laminar flow chamber
• Test samples of which the activity has to be

Procedure ²⁵: A sterile cotton swab was taken and dipped into a culture of test organism suspension Inoculated the entire agar surface of each plate first in a horizontal and then in vertical direction to ensure the even distribution of the organism over the agar surface using the swab. The agar surface was allowed to dry for 5 minutes. A cork borer was sterilised by autoclaving. Mueller-Hinton agar plates were obtained and holes (4 mm) in the agar were aseptically punched with a cork borer. Using a wax pencil, marks were made on the underside of the Petri to label the wells. With the help of a micropipette, the test solution was added to the well. Repeat the procedure for all wells. In an incubator, all plates were incubated at 370°F for 24-48 hours. Observations are recorded and a statistical study is done.

RESULTS: A pharmaceutical study of the formulation results in a brownish-red, odourless, astringent sweet tablet of 250mg being formed. Table 1. The pH is 7.04, with 30% LOD, hardness of 1.7kg/cm, disintegration time of 2.1 minutes, and zero percent friability Table 1.

TABLE 1: PHYSICOCHEMICAL ANALYSIS OF DADIMBHAVITGAIRIKVATI

Examined all the plates for the clear zone of inhibition surrounding the disc. The diameter of zone of inhibition is measured in musing a ruler on the underside of the plate Fig. 1, 2, 3.

FIG. 1: ANTIBACTERIAL ACTIVITY OF DADIMGIRIJVATI WITH STAPHYLOCOCCUS AUREUS AND BACILLUS SUBTILIS

FIG. 2: ANTIBACTERIAL ACTIVITY OF DADIMGIRIJVATI WITH E. COLI, PROTEUS MIRABILIS

FIG. 3: ANTIFUNGAL ACTIVITY OF DADIMGIRIJVATI WITH CANDIDA ALBICANS AND ASPERGILLUS NIGER

The zone size recorded and prepared a table comparing the zone obtained with the known concentration verses the zone of inhibition of standard antibiotic Table 2.

TABLE 2: ANTIMICROBIAL STUDY ON SAMPLE OF DADIMBHAVIT GAIRIKVATI

DISCUSSION: Red ochre triturated with Punica granatum (Daim BhavitGairikVati) formulation is prepared as per the standards of the classical text Sharangadharsamhita. Raw material authentic-fication was done at Agharkar Research Institute, Pune with authentification voucher no AUTH 22-49 dated on 10^th May 2022. Oraganoleptic characteristics are found as reddish brown in colour without odor. An astringent sweet taste is observed Table 2.

The colour of aqueous extract (10%) is reddish while 30% is lost on drying. So the formulation is much more stable. The tablet's hardness is 1.7kg/cm, making it easy to absorb in the body Table 2.

The disintegration time of a tablet is found to be 2.1 min. So, it disintegrates easily in gastric juices. The average weight is 500mg as it was made on a tabletting machine. No weight variation is seen. Friability testing is a laboratory technique used to test the durability of tablets during transit. The formulation was found to be extremely durable, with no friability Table 2.

In an antimicrobial study, five batches of samples for each organism are made. Streptomycin is used as an antibiotic for two gram-positive and two gram-negative bacteria. The zone of inhabitation of the formulation for Staphylococcus aureus is 14.6mm, whereas streptomycin shows 21.6mm. Bacillus subtilis responded by 16 mm to the formulation as streptomycin has a 26 mm zone of inhibition. Escheria coli has a 15mm and 31.2mm zone of inhibition for the formulation and standard. Proteus mirabilis has a 16.4 mm and a 25 mm zone of inhibition for the formulation and standard.

An antifungal study is done with reference to the standard drug fluconazole. Candida albicans responded with 17.2 mm and 28 mm of zone of inhibition for the experimental formulation and the standard, respectively. Aspergillus niger has a 19.2 mm zone of inhibition for the formula and a standard of 30 mm for the standard.

Red ochre triturated with Punica granatum (Dadimbhavitgairikvati) shows antibiotic as well as antifungal activity, approximately half of the standard. Although this formulation is not meant for antimicrobial activity, the activity is still found. This activity can be enhanced by additives or synergism with other herbal drugs.

CONCLUSION: According to the classical text Sharangadharsamhita, red ochre triturated with Punica granatum (Dadimbhavitgairikvati) is herbo-mineral formulation.

Red ochre triturated with Punica granatum (Dadimbhavitgairikvati) is standardised with analytical study parameters at both the raw phase and finished product phase. That shows the drug is standardised and manufactured with Standard Operating Procedures. The results found are satisfactory as there are five batches of the drug in each plate for a single organism. The zone of inhibition in five samples was observed and the average was calculated and compared with the present antibiotic and/or antifungal. Even though the formulation is not claimed to have antimicrobial property, the formulation shows positive results as antimicrobial and antifungal. So, with therapuetic properties in the said diseases, antimicrobial action can be expected in a limited range. As a further scope of the study, potensifying the drug with additives or synergism can enhance the zone of inhibition to function as an antimicrobial drug.

ACKNOWLEDGEMENT: Thankful to Dept of Rasashastra Evam Bhaishajya kalpana, Dr. D. Y. Patil college of Ayurved and research centre, Pimpri, Pune 411018 for pharmaceutical study and analytical study. Special gratitude to Agharkar research Institute, Pune for raw drug standardisation. Antimicrobial study is done by Late Prin B. V. Bhide foundation research institute Pune. Special thanks to this institute and staff.

CONFLICT OF INTEREST: The author(s) declared no potential conflict of interest with respect to the research, authorship and/or publication of this article. No funding agency or institutional support for the research work. As present study is in-vitro study, no animals or human subjects are involved in the study.

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    How to cite this article:

    Shete AA, Patil J, Shete AA and Dubewar AP: Antibacterial and antifungal activity of red ochre triturated with Punica granatum (dadim bhavit gairikvati). Int J Pharm Sci & Res 2023; 14(7): 3482-86. doi: 10.13040/IJPSR.0975-8232.14(7).3482-86.

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