Antibacterial activity of Sodium Copper Chlorophyllin (SCC) from Leucas aspera L.

 

Geetha Rani. R¹*, Banu. N²

¹Research Scholar, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Vels University, Chennai, Tamil Nadu, India.

²Associate Professor, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies(VISTAS), Vels University, Chennai, Tamil Nadu, India.

 

ABSTRACT:

Sodium copper chlorophyllin (SCC) belongs to a group of compound, porphyrins containing a chelated metal ion in the center of the molecule. It is a sodium salt containing copper as the central metal atom with loss of phytol chain. The present study was to extract the sodium copper chlorophyllin from Leucas aspera L. and to check its antibacterial activity. The results show that sodium copper chlorophyllin of Leucas aspera L. has significant anti- bacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi and Pseudomonas aeroginosa with Ampicillin (Std) 20 µg /ml as positive control. The active zone of inhibition was around 6-11 mm against the tested human  pathogens .The Minimal Inhibitory Concentration (MIC) was determined and found to be around 31.2-125 µg /ml. This result confirms that the medicinal value of Leucas aspera L. of sodium copper chlorophyllin as an anti bacterial sources .

 

 

 

INTRODUCTION:

The drugs derived from natural sources plays a vital role in the prevention and treatment of human diseases. In many developed and developing countries, traditional medicine is one of the primary health care system.^1,2 Herbs are widely established in the traditional medicine  and their potentials are well documented.³ About 61% of new drugs was developed during  20^th  century was based on natural products and they are very successful in the areas of  cancer and infectious disease.⁴ Natural products of higher plants produce new source of  antibacterial agents with possible novel mechanisms of action.^5,6 The effects of plant  extracts on bacteria have been studied by a very large number of researchers in different parts  of the world.⁷ Much work has been done on ethno medicinal plants in India.⁸

 

The modern medicines are produced indirectly from medicinal plants. Leucas aspera L. (Lamiaceae) represent a rich source of antibacterial activity.⁹ Leucas aspera L. possess to have antimicrobial, antifungal, antioxidant, antinociceptive and cytotoxic activities.¹⁰ Leucas aspera L. is well-known for its medicinal properties particularly for the treatment of scorpion bites. It is also an antipyretic.¹¹ It has been traditionally used in the treatment of various ailments including sinusitis, as well as headaches and also be used to treat intestinal worms in children. The plant Leucas aspera L. is used as an insecticide and traditional medicine for chronic skin eruption, coughs, colds and painful swelling.¹²The leaves of Leucas aspera L. were used in psoriasis, scabies and chronic rheumatism.¹³ Compounds isolated from Leucas aspera L. include long aliphatic chain, triterpenes, sterols and phenolic compounds.¹⁴ The present study was done to determine the antibacterial activity of Sodium copper chlorophyllin from Leucas aspera L. The Sodium copper chlorophyllin is semi-synthetic water soluble sodium salt containing copper as the central metal atom. During the synthesis of chlorophyllin the magnesium atom at the center of the ring is replaced with copper and the phytol tail is lost. Unlike natural chlorophyll, chlorophyllin is water-soluble.¹⁵ The antibacterial activity of SCC of Leucas aspera L. was proved by disc diffusion method on Muller Hinton agar (MHA) medium.

 

MATERIALS AND METHODS:

Plant Material:

The fresh and healthy leaves of the plant Leucas aspera L. were collected from Chennai, Tamil Nadu. Plant material was labelled, numbered, and noted with the date of collection, locality, and their medicinal uses were recorded.

 

Solvent Extraction (Sodium Copper Chlorophyllin):

Ten grams of fresh leaves were taken and 1 gm of sodium carbonate was added to neutralize the acidity. The plant material was ground with 50–100 ml acetone and filtered using filter paper. This procedure is repeated until the residue becomes colorless. It was then washed with 50–150 ml of diethyl ether to wash off acetone. The mixture was poured into a separating funnel and acetone was washed off using distilled water. This was repeated until yellow colour separates off which consists of flavones. The solution was poured into a bottle and 10–25 ml of methanol saturated with potassium hydroxide pellets was added. The solution was shaken thoroughly and kept in the icebox for overnight. The alkaline solution of chlorophyllin was poured into a separating funnel, and 100 ml diethyl ether was added and left for 30 min. Chlorophyllin separates off greenish layer which was removed. The ether layer was washed off with dilute potassium hydroxide and distilled water, to remove traces of chlorophyllin salts. The filtrate was evaporated to dryness in a rotary evaporator, and the extract was stored in ice box ¹⁶.

 

Purification:

The extracted Sodium copper chlorophyllin of Leucas aspera L. was purified by column chromatography using silica gel by the elution with Di ethyl ether and acetone in the ratio of 8:2.

 

Source and Maintenance of Organism:

Bacterial cultures of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi and Pseudomonas aeroginosa were collected from King’s Institute of Preventive Medicine, Guindy, Chennai.

 

Culture Media:

Stock cultures were maintained at 4°C on Nutrient agar Slant. Active cultures for experiments were prepared by transferring a loop full of culture from the stock cultures into the test tubes containing nutrient broth, and were incubated for 24 hrs at 37ºC. The Assay was performed by agar disc diffusion method.

 

Antibacterial Activity:

Antibacterial activity of Sodium copper chlorophyllin against five pathogenic bacteria were investigated by the agar disc diffusion method. Muller Hinton Agar (MHA) medium was poured on to the petriplates. Once the medium was solidified, the inoculums were spread on the solid plates with sterile swab moistened with the bacterial suspension. The disc were impregnated in MHA plates and 20 µl of sample were loaded in the concentration of (1000µg, 750 µg and 500 µg). The zones of growth inhibition around the disc were measured in triplicates after 18 to 24 hours of incubation at 37°C. The sensitivity of the microorganism were determined by measuring the sizes of inhibitory zones including the diameter of disc on the agar surface.

 

Minimum Inhibitory Concentration (MIC):

Sample Preparation:

The 1mg of Sodium copper chlorophyllin was taken and mixed it with 1ml of DMSO obtaining the concentration of 1mg/ml. The Minimum Inhibitory Concentration of Sodium copper chlorophyllin of Leucas aspera L. was done through the method of successive dilution. 1ml of sterile LB broth was added in each tube and autoclaved under constant pressure at the temperature of 121°C. Add 1 ml of diluted sample in tube1 and perform serial dilution until tube 8. 100μl of bacterial cultures were added to all the tubes. The tubes were vortexed well and incubated at 37°C for 24 hrs. The resulting turbidity was observed after 24 hrs. MIC was determined to be where growth was no longer visible by assessment of turbidity by optical density readings at 600 nm.

 

Statistical Analysis:

All experiments were performed in triplicates and the values were calculated statistically by standard deviation (SD).

 

RESULTS AND DISCUSSION:

The result obtained in the present study of Sodium copper chlorophyllin of Leucas aspera L. possesses potential antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi and Pseudomonas aeroginosa. The test material was effectively inhibited all test pathogens at all the tested concentrations. The concentrations (1000μg/ml, 750 μg/ml and 500 μg/ml) shows a zone of inhibition against all the tested pathogens. The Sodium copper chlorophyllin of Leucas aspera L .showed almost similar antibacterial activity against Staphylococcus aureus (6.5 mm-11 mm) , Bacillus subtilis (7 mm-9 mm), Escherichia coli (6 mm-9 mm), Salmonella typhi (6 mm-9.5 mm) and Pseudomonas aeroginosa (6 mm-10 mm) at the concentration of 1000 μg/ml, 750 μg/ml and 500 μg/ml. Sodium copper chlorophyllin standard shows almost similar or higher zone of inhibition in the entire five test organism. Antibacterial activity in standard chlorophyllin and Sodium copper chlorophyllin of Leucas aspera L .were almost similar. Zone of inhibition was also almost similar or lower when compared with the Ampicillin. Standard Chlorophyllin recorded better antibacterial activity than Sodium copper chlorophyllin of Leucas aspera L. Sodium copper chlorophyllin of Leucas aspera L .recorded potential antibacterial activity against the tested microorganism with the range of 6 mm-11 mm at 1000-500μg/ml. The Minimum Inhibitory Concentration (MIC) was determined and found to be around 31.2-125 µg /ml against the test bacteria. The Minimum Inhibitory Concentration was defined as the lowest concentration that able to inhibit any visible bacterial growth.^17,18

 

Table1: Zone of the Inhibition of Leucas aspera L. Sodium copper chlorophyllin for antibacterial activity

The values are expressed statistically by standard deviations

 

CONCLUSION:

The present study justified the uses of plant parts in the traditional medicine to treat various infectious disease caused by the microbes. However, further studies need to evaluate the potential effectiveness of the crude extracts as the antibacterial agents. This results in the form of basis for selection of plants for further investigation in the potential discovery of new natural bioactive compounds. Future studies which aim the isolation and structure elucidation of antibacterial active constituents from the plant have been initiated.

 

ACKNOWLEDGMENT:

The authors are very much grateful to the Chancellor Ishari K. Ganesh, Vels University for providing all the facilities to carry out my research.

 

REFERENCES:

1.     Farnsworth NR. Ethno pharmacology and future drug development: The North American experience. J. Ethnopharmacol. 1993; 38:145–52.

2.     Houghton PJ. The role of plants in traditional medicine and current therapy. J Alter Complement Med.1995; 1:131–43.

3.     Dubey NK, Kumar R, Tripathi P. Global promotion of herbal medicines: India's opportunity. Curr Sci.2004; 86:37–41.

4.     Cragg GM, Newman DJ. Biodiversity: A continuing source of novel drug leads. Pure Appl Chem.2005; 77:7–24.

5.     Runyoro D, Matee M, Olipa N, Joseph C, Mbwambo H. Screening of Tanzanian medicinal plants for anti-Candida activity. BMC Complement Altern Med. 2006; 6:11.

6.     Shahidi BH. Evaluation of antimicrobial properties of Iranian medicinal plants against Micrococcus luteus, Serratia marcescens, Klebsiella pneumonia and Bordetella bronchoseptica. Asian J Plant Sci. 2004; 3:82–6.

7.     Reddy PS, Jamil K, Madhusudhan P. Antibacterial activity of isolates from Piper longum and Taxus baccata. Pharmaceutical Biol. 2001; 39:236–8.

8.     Maheshwari JK, Singh KK, Saha S. Ethno botany of tribal's of Mirzapur District, Uttar Pradesh, Economic Botany Information Service, NBRI, Lucknow.1986.

9.     Balandrin MF, Klocke JA, Wurtele ES, Bollinger WH. Natural plant chemicals: Sources of Industrial and Medicinal materials. Science.1985; 228: 1154-60.

10.  Prajapati MS, Patel JB, Modi K, Shah MB. Leucas aspera: A review.2012 Apr; 28 4:857.

11.  Srinivasan R, Ravali B, Suvarchala P, Honey A, Tejaswini A, Neeraja P.Leucas aspera-Medicinal plant: A Review. International Journal of Pharma and Bio Sciences 2011; 2 (1): 153–59.

12.  Chopra RN, Nayar SL, Chopra IC. Glossary of Indian medicinal plant. National institute of science communication and information resources. New Delhi: Council of Scientific and Industrial Research (CSIR) 2002. p. 153.

13.  Rahman MS, Sadhu SK, Hasan CM. Preliminary antinociceptive, antioxidant and cytotoxic activities of Leucas aspera root. Fitoterapia. 2007; 78:552–555.

14.  Mangathayaru K, Thirumuragan D, Patel PS, Pratap DVV, David DJ, Karthikeyan J. Isolation and identification of nicotine from Leucas aspera. Indian J Pharm Sci. 2006; 68:88–90.

15.  Egner PA, Munoz, Kensler TW. Chemoprevention with chlorophyllin in individuals exposed to dietary aflatoxin. Mutation Res 2003; 524:209-16.

16.  Schertz FM, The extraction and separation of chlorophyll (α+β) carotin and xanthophyll in fresh green leaves, preliminary to their quantitative determination. Plant physiology, 1928; 3:211-16.

17.  Prescott ML, Harley J, Donald P, Klein A. In 'Antimicrobial chemotherapy'. Microbiology 2^nd edition published by C. Brown publishers, U S A, 1999; Pp 325.

18.  Shahidi Bonjar GH. Evaluation of Antibacterial properties of Iranian Medicinal plants against Micrococcus aureus, Serratia marcescens, Klebsiella pneumoniae and Bordella bronchoseptica. Asian journal of sciences.2004; 3(1):82-86.

 

 

Accepted on 16.02.2017           © RJPT All right reserved