Evaluation of anti-bacterial activity of Dashapushpam in the form of Ghritham

The state Kerala in India is famous for its plant resource both culturally and medicinally. There is a cluster of ten sacred medicinal plants commonly known as Dashapushpam. These herbs are of great importance in the cold rainy season. Each plant of this group possesses many medicinal values. There are many formulations using these herbs. Ancient people knew the value of using these herbs in a cluster, so they included them in their diet to improve immunity in the monsoon season. There are many Ayurvedic texts which mention the uses of these sacred herbs. Formulations that use all the members of Dashapushpam are rare. The Dasapushpagritham is one such formulation taken from the text vishavaidhya jyostnika. The present work intends to evaluate the anti-bacterial property of Dashapushpam when used in an Ayurvedic formulation known as Dasapushpa Ghritham. The bacterial strains used as the test micro-organisms for the study was Pseudomonas aeruginosa and Bacillus cereus. The method of the anti-bacterial evaluation was done through agar-gel diffusion technique. The samples including the plant extracts possessed a varying level of anti-bacterial activity against these two bacteria, and their values obtained were compared with standard antibiotic amoxicillin. Based on these results, it was concluded that the anti-bacterial property of the Ayurvedic drug Dasapushpa Ghritham has significant value when compared to the drug base and other individual plant extracts. Hence the present study proves the significant usage of the Dashapushpam plants in various therapeutics used as an anti-microbial agent.

Dasapushpa Ghritham, anti-bacterial property, Dashapushpam, Agargel diffusion technique ABSTRACT The state Kerala in India is famous for its plant resource both culturally and medicinally. There is a cluster of ten sacred medicinal plants commonly known as Dashapushpam. These herbs are of great importance in the cold rainy season. Each plant of this group possesses many medicinal values. There are many formulations using these herbs. Ancient people knew the value of using these herbs in a cluster, so they included them in their diet to improve immunity in the monsoon season. There are many Ayurvedic texts which mention the uses of these sacred herbs. Formulations that use all the members of Dashapushpam are rare. The Dasapushpagritham is one such formulation taken from the text vishavaidhya jyostnika. The present work intends to evaluate the anti-bacterial property of Dashapushpam when used in an Ayurvedic formulation known as Dasapushpa Ghritham. The bacterial strains used as the test micro-organisms for the study was Pseudomonas aeruginosa and Bacillus cereus. The method of the anti-bacterial evaluation was done through agar-gel diffusion technique. The samples including the plant extracts possessed a varying level of anti-bacterial activity against these two bacteria, and their values obtained were compared with standard antibiotic amoxicillin. Based on these results, it was concluded that the anti-bacterial property of the Ayurvedic drug Dasapushpa Ghritham has signi icant value when compared to the drug base and other individual plant extracts. Hence the present study proves the signi icant usage of the Dashapushpam plants in various therapeutics used as an anti-microbial agent.

INTRODUCTION
Pandemic diseases have always shattered the economic and healthy stability of society.
The current work is intended to analyse the antibacterial property of the individual aqueous extract of the plants and their combinational formulation as the drug Dasapushpa Ghritham when tested against Bacillus cereus and Pseudomonas aeruginosa. The Ayurvedic formulation of Dasapushpa Ghritham is from "visha vaidhya jyostnika" which is an old text in Ayurveda used in ancient times. The formulation constitutes the aqueous extract of the Dashapushpam herbs as the actual content and a drug base like ghee, turmeric, sandal etc. (Krishnapriya et al.,

MATERIALS AND METHODS
The Dashapushpam herbs were collected from different parts of Malappuram district, Kerala, India. The plants were irst washed in tap water and then dried in the shade and powdered.

Extraction preparation
The Dashapushpam plants were washed in tap water and shade dried. 50g of powdered sample was weighed and taken and added to 300ml distilled water. This solution was heated for 15 min and stirred continuously. The plant extract was then cooled for 24hrs at room temperature. After that, the solution was iltered through a Whatman ilter paper [no.1] and vacuum pump. The iltrate obtained was concentrated at 40 o c until all the solvents evaporated completely. Then each sample was dissolved in sterile distilled water separately (Parekh and Chanda, 2006).

Drug preparation
The Ayurvedic formulation of Dasapushpa Ghritham is taken from the Ayurvedic text Vishavaidhya Jyostnika. The method of preparation of the drug is given in the sixth chapter of the text, which deals with the viper venom and non-healing ulcers.
The content of the drug is mainly classi ied into two parts that are the paste of plant parts known as Kalkkam and the fresh juice of ten sacred plants known as Swarasam. The paste of Kalkam is prepared by using the various parts of plants.

Micro-organisms used
Test organisms used were Gram-negative bacteria as Pseudomonas aeroginosa, and a gram-positive bacterium was Bacillus cereus.

Anti-bacterial activity
Agar-gel diffusion technique was done to analyse the anti-bacterial property of the plant extract samples. Medium for bacterial culture was nutrient agar plates. On the 20ml solidi ied nutrient agar, 1ml of each bacterial suspension was inoculated by spread plate method. Each plate had 6mm diameter wells cut out in it. The samples, positive control (Amoxicillin 30µg/ml), negative control (sterile distilled water) was added in the irst, second and third well respectively. 20 µl of the plant extract (0.5 g/ml) was added into the irst well of one grampositive and one gram-negative plate as the sample. The Dasapushpa Ghritham (Ayurvedic drug) and the base(Ayurvedic drug without the plant extract) was added as the sample to the Pseudomonas aeroginosa and Bacillus cereus cultured nutrient agar plates with a positive and negative control. After inoculation, the incubation of the plates was done at 37 o c for 2-3 days. Inhibition zone was formed, and the diameter was measured around the sample well and the positive control.

RESULTS AND DISCUSSION
The anti-bacterial assay results obtained from the agar-gel diffusion technique was given in Table 3. The Ayurvedic drug Dasapushpa Ghritham has a diameter of 1.54cm in inhibition zone with the gram-positive bacteria Bacilluscereus and 1.74cm with the gram-negative bacteria Pseudomonasaeruginosa, as shown in Figure 1  The positive control used was the standard drug amoxicillin which has a diameter of 1.75cm in inhibition zone with Gram-negative bacteria Pseudomonasaeruginosa and 1.85cm diameter of inhibition zone with the Gram-positive bacteria Bacilluscereus. Negative control (distilled water) used did not give a zone of inhibition. Ipomea sepiaria from the Dashapushpam family was the sample which gave the highest value for the anti-bacterial assay with a zone of inhibition of 1.85cm diameter for both the test organisms. Thus this result is equivalent to that of the standard drug (positive control) amoxicillin. Evolvulus alsinoides was the sample that gave the least value with the test organisms with the diameter of 1cm and 1.24cm each. The rest of the members of the Dashapushpam family have the antibacterial assay results ranging between these values.

CONCLUSION
After performing the anti-bacterial assay, the results prove that the members of the Dashapushpam, when used in a combination as a drug, shows signi icant anti-bacterial activity than when used as an individual plant extract. The Ayurvedic drug shows the anti-bacterial activity as that of the positive control antibiotic amoxicillin in the case of Pseudomonas aeruginosa. More studies are required to understand the therapeutic potential of the drug Dasapushpa Ghritham. Another important aspect of this work is that it encourages the use of a combinational formulation of the ten sacred plants (Dashapushpam) for many other therapeutic drugs. Using the plant extracts as a medical remedy has always been of great interest. The present study also proves the ef iciency of using Ayurvedic herbs in medicinal formulations which can be used against diseasecausing micro-organisms. A natural drug for microbial infection always has an excellent acceptance in the world of modern medicine.

Funding Support
No external funding support all the works are done by myself.

Con lict of Interest
We declare that we have no con lict of interest for this study.  Ipomoea sepiaria Ro+b.