Evaluation of antibacterial activity potential of extracts of Ricinus communis, Zingiber officinalis and Punica granatum in a Polyherbal Extract

 

Ranjith Anishetty^1,2*, Satya Swapna¹, B. Aishwarya¹, K. Chaitanya Sravanthi¹

¹Vignan Institute of Pharmaceutical Sciences, Deshmukhi Village, Nalgonda-508 824,Andhra Pradesh, India.

²Department of Quality Assurance, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara - 144402, Punjab, India

 

ABSTRACT:

Pharmaceutical industries have produced a number of new antibiotics in the last decades, resistance to these drugs by micro organisms has also increased. For a long period of time, plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies.  The use of plant extracts and phytochemicals both with known antimicrobial properties can be of great significance in therapeutic treatments. The main objective is to study the anti microbial activity of the combined plant extracts which was evaluated with antibiotic susceptible and resistant microorganisms. The possible synergistic effects were studied.  Extracts from the following plants were utilized: Ricinus communis (castor), Zingiber officinalis (ginger) and Punica granatum (pomegranate). The highest antimicrobial potential were observed for the extracts of pomegranate and castor and comparative studies were made in order to know the activity of combined extracts of Ginger and pomegranate, pomegranate and castor,  Ginger and castor and also pomegranate, ginger and castor and the results showed synergistic antibacterial activity against antibiotic resistant bacteria.

 

 

INTRODUCTION:

Even though pharmacological industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents.  Such a fact is cause for concern, because of the number of patients in hospitals who have suppressed immunity, and due to   new bacterial strains, which are multi-resistant. Consequently, new infections can occur in hospitals resulting in high mortality¹. The problem of microbial resistance is growing and the outlook for the use of antimicrobial drugs in the future is still uncertain.

 

Therefore, actions must be taken to reduce this problem, for example, to control the use of antibiotic, develop research to better understand the genetic mechanisms of resistance, and to continue studies to develop new drugs, either synthetic or natural.

 

The ultimate goal is to offer appropriate and efficient antimicrobial drugs to the patient.  For a long period of time, plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies². The use of plant compounds for pharmaceutical purposes has gradually increased. Medicinal plants would be best source to obtain a variety of drugs³. Many plants have been used because of their antibacterial activity which is due to compounds synthesized in the secondary metabolism of plants like Phenolic compounds which are part of essential oils and tannins. From ancient history of Ayurveda many plant extracts are combined in order to get maximum activity^{4, 5}. Therefore the present proposal is being undertaken to evaluate anti bacterial potential of various combined extracts of Zinziber officinalis, Ricinus communis, Punica granatum.

 

TEST ORGANISMS:

The test organisms include the following Gram positive bacteria: Staphylococcus aureus (MTCC740), Bacillus subtilis (MTCC441) and Gram negative bacteria: Pseudomonas aeruginosa (MTCC424), Escherichia coli (MTCC41), Proteus vulgaris (MTCC426). All the strains were obtained from Institute of Microbial Technology, Chandigarh, India.

MATERIALS AND METHODS:

The leaves of Ricinus communis were collected during the month of December from Vignan Hills of Deshmukhi. The authentification was done by Mr. / Dr. K. Chaitanya sravanthi, Asstt. Prof., Department of Pharmacognosy, Vignan Institute of Pharmaceutical Sciences. Required amount of rhizomes of Zingiber officinale and fruits of punica granatum were collected from market.

 

Ricinus communis: The leaves of Ricinus communis were shade dried at room temperature and undergone size reduction using pulverizer. It is sieved several times to ensure uniformity of the powdered plant material. The powdered leaves (100 gms) were undergone ethanolic extraction using soxhlet apparatus. After extraction the solvent is removed. Trace amounts of the solvent may be present in the semi solid extract obtained. This solvent remnant was removed by subjecting it to desiccation. A desiccator with activated silica was used for this purpose. The extract obtained was 7mg/ml.

 

Zingiber officinalis: 100 gms of ginger was collected and washed with water. Fresh ginger was cut into small pieces and grinded to obtain fine paste. The liquid extract from this paste is obtained by decanting with muslin cloth. The liquid extract is refrigerated.  The percentage yield of Ginger was 3.3%.

 

Punica granatum: 100 gms of pomegranate is collected and washed with water. Fresh pomegranate is cut into pieces and grinded to obtain fine paste. The paste is taken in muslin cloth and pressed to get the liquid extract (seed + rind). The percentage yield of pomegranate was 3.3%

 

Preparation of pure cultures:

A small amount of culture is placed on the tip of an inoculation loop/needle and is inserted into flask containing nutrient broth. This is carried out in laminar air flow. The flask is plugged with cotton. These flasks were incubated to allow the growth of organisms for 18 to 24 hrs which is used as pure cultures for activity.

 

Spread Plate Method: 

20ml of nutrient agar media was transferred into each petri plate. The petri plates were left undisturbed for 1-2hrs. 100μl of each pure culture was transferred into petri plates using micro pipette. The pure cultures were evenly spread with the help of sterile bent glass rod. They were kept for incubation for 24hrs.

 

PROCEDURE FOR ANTIBACTERIAL ACTIVITY TESTING:

Drug substances that either suppress or influence the growth of microorganisms are generally analyzed by microbial method. The procedure employed for this testing is Cup plate method or Agar well diffusion method.

 

Cup plate method:

The antibacterial activity of the extracts was determined by using the agar well diffusion technique. Mueller- Hinton agar plates (Himedia, Mumbai) were seeded with 0.1 ml of overnight culture, allowed to incubate for 24hrs. Cups were made in Petri plates using sterile cork borer (0.85 cm) and 50 μl of each extract was added into each well. Then bacterial plates were incubated at 37˚ C 24 hrs⁶. Each test compound has got six bores for which zone of inhibition diameter and mean values were determined. Antibacterial activity was determined by measurement of zone of inhibition around each well in plate using zone reader⁷. Measured inhibition zones were recorded as mean diameter in mm⁸. Gentamycin antibiotic was used as control.

 

Castor, Ginger and Pomegranate fruit extract were taken in equal ratio with three different combinations and are mixed in a beaker and 50 µl are taken with micro pipette and introduced into the bores made in the petri dishes.

 

RESULTS:

The observation of combined antibacterial activity of Ginger and Pomegranate extracts was found to be as shown in Fig.no:1.  The diameters of zone of inhibition of ginger and pomegranate were recorded in Table-1.

 

 

 

Table 1: Combined activity of Ginger and Pomegranate

 

Fig.no:1 Graphical representation of combined activity of Ginger and Pomegranate.

 

 

The observation of combined antibacterial activity of ginger and castor extracts was found to be as shown in Fig. no: 2. The diameters of zone of inhibition of ginger and castor were recorded in Table- 2

 

Table 2: Combined activity of Ginger and Castor

S.no	Bacillus subtilis	Escherichia coli	Proteus vulgaris	Staphylococcus  aureus	Pseudomonas aeroginosa
1.	11mm	9mm	25mm	21mm	13mm
2.	12mm	14mm	10mm	16mm	11mm
3.	7mm	10mm	10mm	7mm	14mm
4.	10mm	11mm	11mm	8mm	15mm
5.	11mm	9mm	9mm	11mm`	10mm
6.	9mm	10mm	8mm	10mm	11mm

 

Fig.no: 2 Graphical representation of combined activity of Ginger and Castor

 

The observation of combined antibacterial activity of pomegranate and castor extracts was found to be as shown in Fig.no:3. The diameters of zone of inhibition of pomegranate and castor were recorded in Table -3

 

Table 3: Combined activity of Pomegranate and Castor

 

Fig.no:3 Graphical representation of combined activity of Pomegranate and Castor

 

Table 4: Zone of inhibition of extracts and standard, Gentamycin against test organisms

Plant Extracts	Bacillus Subtilis	Pseudomonas aeruginosa	Escherichia coli	Proteus vulgaris	Staphylococcus Aureus
Ginger and pomegranate	17.66±3.50238	12.83±2.2286	10.33±4.36654	8.5±1.3784	13.6±2.16025
Ginger and castor	10±1.72225	10.5±1.8708	12.16±6.3692	12.16±5.344	12.33±1.9663
Castor and pomegranate	10.16±1.47196	9.5±1.04881	12.33±1.63299	8.16±1.1695	11±1.41421
Castor, ginger and pomegranate	18.5± 1.5	20.6± 1.1	17.8± 1.86	19.3± 2.1	16.8± 2.26
Standard(GENTAMYCIN)	16.8± 2.26	10±0.67	12.8± 1.06	10±0.86	11.6± 2.2

 

DISCUSSION:

The results of antibacterial activity of Table 1 shows that the combination of the Ginger and Pomegranate compared to standard drug 14.1± 1.4 have good activity on B. subtilis with zone of inhibition and standard deviation as 22mm and   17.66±3.50238 respectively. Therefore from these results we can access that the combination of Ginger and Pomegranate is highly active towards Gram positive bacteria.

 

The combination of Ginger and Castor extracts has antibacterial activity and results are shown in Table 2 with zone of inhibition of 25mm diameter and standard deviation 12.16±5.344 on Proteus vulgaris. It is higher than the activity on staphylococcus aureus and Escherichia coli.  Therefore from these values we can assess that the combination of Ginger and Castor may have a high antibacterial activity on gram negative bacteria when compared to standard drug 10±0.86. Pomegranate and Castor combination shows a good antibacterial activity when compared to standard drug and the results are depicted in Table 3. The zone of inhibition was in a moderate range from 8 to 16mm in diameter and this shows broad spectrum of activity when compared with standard.

 

The combination of three extracts Ginger, Castor and Pomegranate in equal ratio shows a high antibacterial activity on all bacterial strains when compared to standard. Thus, the three herbs (Zingiber, Ricinus and Pomegranate) which were used in combinations showed potent Antibacterial activity towards gram negative and gram positive when compared to that of standard drug gentamycin 12.16±5.344. 

This particular Study has a wide scope in future for development of herbal drugs with   less or no side effects and there are some void in these three drugs which should be filled by research in near future.

 

ACKNOWLEDGEMENT:

The authors are thankful to K. Chaitanya Sravanthi, Assistant Professor, Department of Pharmacognosy, for her constant support and motivation throughout the project.

 

REFERENCES:

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4.       Jansen, A.M.; Cheffer, J.J.C.; Svendsen, A.B. Antimicrobial activity of essential oils: a 1976-1986 literature review. Aspects of test methods. Planta Med. 40, 395-398, 1987. 

5.       Saxena, G.; McCutcheon, A.R.; Farmer, S.; Towers, G.H.N.; Hancock, R.E.W. Antimicrobial constituents of Rhus glabra. J. Ethnopharmacol. 42, 95-99, 1994. 

6.       Agarwal V.S. Drug plants of India, Kalyani Publishers New Delhi, Vol 1, 52.

7.       Ramesh Londonkar and Ranirukmini R.K. Antimicrobial activity of Butea frondosa Roxb, Journal of Pharmacognosy, Vol. 1, Issue 1. 2010.

8.       Bibi Sedigheh Fazly Bazza, Mehrangiz Khajehkaramandin and Hamid Reza Shokooheizadeh. In vitro antibacterial activity of Rheum ribes extract obtained from various plant parts against Clinica isolates of Gram-negative pathogens. Iranian Journal of Pharmaceutical Research. 2: 87-91. 2005.

 

 

Received on 26.09.2012       Modified on 14.10.2012

Accepted on 19.10.2012      © RJPT All right reserved