AAMJ / Vol. 3 / Issue 4 / July – August 2017

A A M J Anveshana Ayurveda Medical Journal

www.aamj.in ISSN: 2395-4159

Research Article

An Anti-microbial study of Rasapuṣpādi malahara

Ritesh Ramnani 1 Amit Sharma 2 Jagriti Sharma3 V. Nageswara Rao4

A b s t r a c t

Infectious diseases are great problem in present era. Gradually increasing microbial resistance

has made it more complicated. Therefore, times has come to find out new antimicrobial agent

from natural source. In this prospect Rasapuṣpādi malahara is a unique Ayurvedic formulation

described in Rasa tarangini in the management of skin disease which may be indicating anti-

microbial and antifungal activity. In vitro evaluation of its bactericidal and antifungal activity of

Rasapuṣpādi malahara against the strains of some bacteria and fungi those are responsible for

skin disease was carried out in microbiological laboratory. Agar well diffusion method was

followed to assess the anti-microbial activity like Staphylococcus aureus, Streptococcus epider-

midis, Klebsiell, E.coli, and fungus like Candida albicans, Aspergillus niger. All the bacteria

were found Susceptible (S) against two samples of Rasapuṣpādi malahara (RPM & RPM2) com-

pared to standard Ciprofloxcin. Antifungal activity of two samples of Rasapuṣpādi malahara

showed good result compared to standard fluconazole. Anti-bacterial susceptibility shows

against all bacteria and fungus in both samples in each concentration. RPM2 shows better results

against aspergillus niger than RPM1.

Keywords: Rasapuṣpādi malahara, anti-microbial activity, antifungal activity.

1&2 PG Scholar, 3 PhD Scholar, 4 Associate Professor, Department of PG Studies in Rasashastra &

Bhaishajya Kalpana, NIA, Jaipur (Rajasthan).

CORRESPONDING AUTHOR

Dr. Ritesh Ramnani

PG Scholar,

Department of Rasashastra & Bhaishajya Kalpana,

NIA, Jaipur,

Rajasthan, (India).

Email: riteshramnani@gmail.com http://aamj.in/wp-

content/uploads/Volume3/Issue4/AAMJ_1445_1448.pdf

Ritesh et.al,. : An Anti-microbial study of Rasapuṣpādi malahara

AAMJ / Vol. 3 / Issue 4 / July – August 2017 1446

INTRODUCTION

ncient Ayurveda knew the existence of microbes,

agreed that did not “tick-off” microbes as the pri-

mary casual factor in the onset for disease, unlike

conventional physician. The study of microbes has, of

course been a sizzling area of research for just over 250

years in the past.

Microbiology is one such branch, which has improved

by leaps and bounds in the past few decades thus allow-

ing for pin point diagnosis and treatment of many infec-

tive disorders. Now a day infectious disease makes a

trouble for human being. In order to avoid different infec-

tions production and use of antibiotics to be increase, this

derived from the microbial sources in synthetic manner.

However, all synthetic antimicrobial agents are local irri-

tants and are responsible for hypersensitivity reactions.

Second important thing is that repeated use of antibiotics

from microbial sources has become ineffective.

The development of bacterial resistance to presently

available antibiotics has necessitated the search for new

antibacterial agents. Now, it has become possible to in-

vestigate the action of drugs on isolated micro- organ-

isms. Thus the idea of less intrusive alternative is alluring

so due to problem like adverse effect, limited life span &

the mixture of traditional antibiotics effect is currently un-

derway to look for natural origin.

Purpose:

To lay down the procedure to perform Antimicrobial ac-

tivity to be performed in our formulation with reference

of using standard culture.

Procedure:

Precaution taken during antimicrobial activity.

Glassware to be used shall be sterilized.

Media to be used shall be pre incubated

Microbial area should be sterilized before testing.

Materials:

Micro-organisms :

Selection of microorganisms: As mentioned earlier the

Rasapuṣpādi malahara preparation is mainly used for

skin diseases. Hence the organisms responsible for the

skin infections commonly found in local community like

Gram positive, Gram negative and Fungi were selected

for the study.

Table 1: Showing bacterial strain with their MTCC No.

Microbes Species MTCC

No. Diseases

Gram

positive

Staphylo-

coccus au-

reus

737

Pyogenic granu-

loma, superficial and

deep infections etc.

Gram

positive

Staphylo-

coccus epi-

dermidis

35983 Acnevulgeris etc.

Gram

negative Klebsiella 3384

Pyogenic infections,

ulcer formations, soft

tissue infection etc.

Gram

negative E. coli 443

Pyogenic infections,

ulcer formation, septi-

caemia etc.

Fungi Candida al-

bicans 227

Affects mucous

membrane, skin and

nails.

Fungi Aspergillus

niger 872

Superficial infections

in paranasal sinuses

of ear and eye.

The viable microorganisms used in the test must not be

more than five passages removed from the original

MTCC (microbial type culture collection) culture or any

other equivalent cultures

Chemicals and Solvents- All chemicals used for the prep-

aration of nutrient media and for present study were of

analytical grade.

B.H.I.Broth

B.H.I.Agar

Saboraud Agar

Distilled water

Ethyl alcohol

Surgical spirit

Glass wares and Polywares:

All the glass wares were of sterilizable type and poly-

wares were of disposable type. Table 2:

Equipment Glassware

1) Water bath

2) Loops with holder

3) Borer

4) Hot air oven

5) Inoculation hood

6) Auto clave

7) Incubator

8) Spirit lamp

9) Weighing machine

10) Micro pipettes

1) Petri dishes

2) Conical flask

3) Test tube

4) Beakers

5) Funnels

6) Stirrer

Others:

Cotton

Whatman filter paper

A

Ritesh et.al,. : An Anti-microbial study of Rasapuṣpādi malahara

AAMJ / Vol. 3 / Issue 4 / July – August 2017 1447

Methods:

Preparation of inoculums

Prepare the inoculums.

To harvest the bacterial and fungal cultures, use ster-

ile peptone saline, wash the surface growth, collect-

ing it in suitable glassware, and adding sufficient

sterile peptone saline to obtain a microbial count of

about 1 × 108 colony-forming units (CFU) per ml.

Determine the number of CFU per ml in each suspen-

sion, using the conditions of media and microbial re-

covery incubation times 72 hours to confirm the ini-

tial CFU per ml. This value serves to calibrate the size

of inoculums used in the test. The bacterial and yeast

suspensions are to be used within 24 hours of har-

vest, but the fungal preparation may be stored under

refrigeration for up to 7 days.

Preparation of media

For weighing media use Calibrated Balance, the

glass wares and utensil are depyrogenated in oven

at 2500C for 60 min, weigh media carefully and dis-

solve in distill water, shake well and heat on hot plate

for complete dissolve.

Check the water level of autoclave if necessary ad-

just level with DMW.

Load all prepare Media, carefully close the lid of au-

toclave, check power supply & run the autoclave for

sterilization. After reach the temp. of 1210C hold 15

min on this temp then off supply and release steam

slowly.

Before testing switch on the U.V light of BIOSAFETY

CABINET, Pass Box & BIOSAFETY CABINET room

for 30 mins.

Open the lid of autoclave takes all the media on SS

trays and sent to pass Box.

Enter in air lock and then secondary change room &

change the dress and wear sterilize full dress, enter

on BIOSAFETY CABINET Room off the UV Light of

BIOSAFETY CABINET and switch on white light with

airflow.

Sterilize hands & work bench with IPA 70%.

Test Procedure

In vitro antibacterial activity of formulations was car-

ried out by using the Agar well diffusion method.

This classic method yields a zone of inhibition in mm

result for the amount of antibacterial that is needed

to inhibit growth of specific microorganisms.

Sample prepared as each purified formulation (100

& 200 mg/ml) were dissolved in DMSO.

For the determination of zone of inhibition (ZOI),

bacterial strain was taken and as a standard antibi-

otic and control DMSO for comparison of the results.

The dilution (100 & 200 mg/ml) of formulation in

DMSO and Ciprofloxacin (5 μg/ml) as antibacterial

as positive reference standards /antibiotics were

prepared in double distilled water.

Muller Hinton agar plates for bacteria were seeded

with liquid culture of bacterial strains and allowed to

stay at 37°C for 24 hours.

The zones of growth inhibition around the wells were

measured after 18 to 24 hours of incubation at 37°C

for bacterial.

The sensitivity of the microorganism species to formu-

lation were determined by measuring the sizes of in-

hibitory zones (including the diameter of well) on the

agar surface with comparison to the standard antibi-

otic zones.

Diameter of Well - 6 mm

Volume applied in each well - 100 µl

Control as DMSO and Positive control or Standard as

Ciprofloxacin & fluconazole 5 ppm.

RESULTS

Table 3 : Showing antimicrobial activity of RPM1

Micro-organism

Zone of inhibition (mm).

Standard

Test (in DMSO)

100

mg/ml

200

mg/ml

Staphylococcus au-

reus 14 7 10

Escherichia coli 18 6 8

Klebshiella 16 6 6

S.epidermidies 14 8 9

Candida albicans 14 7 9

Aspergillus niger 15 8 10

Table 4: Showing antimicrobial activity of RPM2

Micro-organism

Zone of inhibition (mm).

Stand-

ard

Test (in DMSO)

100

mg/ml

200

mg/ml

Staphylococcus au-

reus 14 7 10

Escherichia coli 18 6 8

Klebshiella 16 6 6

S.epidermidies 14 8 9

Candida albicans 14 7 9

Aspergillus niger 15 8 9

Note: Diameter of the zone of inhibition is given.

Ritesh et.al,. : An Anti-microbial study of Rasapuṣpādi malahara

AAMJ / Vol. 3 / Issue 4 / July – August 2017 1448

According to these results:

At the given concentration sample was found bioac-

tive against all micro-organisms.

All the bacteria were found Susceptible (S) against

Rasapuṣpādi malahara both samples 1 and 2 com-

pared to standard.

DMSO (negative control) did not show any activity

against test organism.

DISCUSSION

Positive (standard) control as well as negative con-

trols was used for the anti-microbial study. The dilu-

tion (100 & 200 mg/ml) of formulation in DMSO

and Ciprofloxacin (5 μg/ml) as antibacterial as pos-

itive reference standards /antibiotics were used.

Zone of inhibition was measured as diameter of

zone, including discs.

The results were positive against all the microbes as

all microbes found to be susceptible against both the

samples of Rasapuṣpādi Malahara.

Moderate to significant zone of inhibition was ob-

served against Staphylococcus aureus (MTCC 737)

and Aspergillus niger (MTCC 872) in 200mg/ml

concentration. Low zone of inhibition was there

against Klebsiella (MTCC 3384) and E-coli (MTCC

443). Comparatively, almost same zone of inhibition

as compare to standard drug in both the sample of

malahara signifies rasa puṣpa prepared by two dif-

ferent methods are found to be moderately sensitive

and quiet effective anti-microbial activity.

CONCLUSION

Both samples show Anti-bacterial susceptibility against

all bacteria and fungus in each concentration. However,

RPM1 shows better result against aspergillus niger than

RPM2. As it has mentioned in the Ayurveda classic and

particularly in Rasa shastra, alpamatraupyogitwhat….

for raas bhasmas, hence it is clear from study that ra-

saushadi shows better result than the herbal medicines as

it possesses much greater potential and hence also re-

quire in the small quantity.

ΛΛΛΛ

REFERENCES

i. Ayurvedic Formulary of India, Part I, Published by Govt.

of India, Ministry of Health and Family welfare.

ii. Textbook of Microbiology by R.Ananthanarayana and

C.K. Jayanam Paniker, 6thedition (Reprint 2002), Pub-

lished by Orient Longman Pvt. Ltd.

iii. Goodman & Gilman’s The Pharmacological Basis of Ther-

apeutics, 11th edition.

iv. Practical Microbiology ( C.P. Baveja ) page No. 128

Source of Support: Nil.

Conflict of Interest: None declared

ΛΛΛΛ

Ritesh et.al,. : An Anti-microbial study of Rasapuṣpādi

malahara. AAMJ 2017; 4:1445 – 1448.