Evaluation of Antibacterial Activity of Sodium Copper Chlorophyll in from Mimosa pudica L.

 

Rajalakshmi. K*, N. Banu

Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies, Chennai, Tamil Nadu, India

 

ABSTRACT:

Synthetic water soluble form of sodium copper salt of chlorophyll is Chlorophyll in. Antibacterial efficiency of Cholorophyllin from Mimosa pudica was tested against Escherichia coli, Staphylococcus aureus, proteus vulgaris, Klebsiella pneomoniae, Bacillus subtilis, and pseudomonas arogenosa. Agar well diffusion method was used to determine the Antibacterial activity and minimum inhibitory concentration (MIC). Sodium copper chlorophyll in from Mimosa pudica shows significant antibacterial activity against all tested micro organisms. Similarly standard sodium copper chlorophyll in also exhibited significant activity. The minimum zone of inhibition and comparatively greater inhibitory concentration were determined in Escherichia coli, Staphylococcus aureus and showed significant activity in other tested organisms. The Spectrum of activity observed in the present study may be the leaf extracts of sodium copper chlorophyll in from Mimosa pudica could be a possible source to obtain new and effective herbal medicines to treat infectious diseases.

 

 

 

INTRODUCTION:

Mimosa pudica L. (Mimosaceae) is the herb called sensitive plant in English and lajvanti or chuimui in hindi. It possesses antibacterial, anti-inflammatory, antiasthma tic, antidepressant activities. The roots and leaves are majorly used for the treatment as bitter, constipating cooling vulnerary, astringent, alexipharmic, acrid, emetic, diuretic, and febrifuge.¹ Compared to synthetic medicine, herbal medicine has lesser side effect and hence demand has been increased from last decade. From the word “mimic” Mimosa pudica is derived. It means to allude, to sensitivity of leaves. The meaning of “Pudica” is shrinking and bashful. It is sensitivity to light, gravity, time of day; it reacts to the contact of insect. So mimosa is also known as sensitive plant, humble plant, sleeping plant, touch me not etc.²

 

Mimosa pudica leaves are used in the treatment of piles and fistula.³ It has been used for the treatment in various ailments and has been used in disease arising from corrupted blood, bile, fever, piles, jaundice, leprosy, ulcers, and small pox.⁴ Traditionally many plants species have been used; it has antimicrobial and antiviral  properties.⁵ It raises optimistic thinking of scientists about the future of phyto-antimicrobial agents.⁶ Water-soluble derivative of chlorophyll (Chl) is Chlorophyll in (CHL) in which magnesium has been replaced with copper and the phytol chains lost. It has been used as a medicine safely. Derifil is used primarily to control body odor in geriatric patients for many years⁷and is available as a dietary supplement. Chl is present in our diet in green, leafy vegetables, reaching levels of 5.7% in spinach.⁸ CHL is also known as sodium copper chlorophyll in. Sodium/ copper derivative used as a food additive and its alternative medicine.  It has anticancer property against hepatocellular carcinoma.⁹ In our present study we evaluated the antibacterial activity of Chlorophyllin from Mimosa pudica. We observed significant activity of Chlorophyllin in all the tested microorganisms were used for the study. 

 

MATERIALS AND METHODS:

Plant Material:

Mimosa pudica leaves were collected from Vels University garden, Chennai. Extraction of Chlorophyllin (Schertz, 1928)¹⁰: Ten grams of fresh Mimosa pudica leaves were taken and 1gm of sodium carbonate was added to neutralize the acidity. The plant material was ground with 50 – 100ml acetone and filtered using filter paper. This procedure is repeated until the residue becomes colorless. It was then washed with 50 – 150ml 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 a yellow color separates off which consists of flavones. The solution was poured into a bottle and 10 – 25ml of methanol saturated with potassium hydroxide pellets was added. The solution was shaken thoroughly and kept in icebox for overnight. The alkaline solution of chlorophyll in was poured into a separating funnel and 100ml diethyl ether was added and left for 30mins. Chlorophyll in separates off greenish layer which was removed. The ether layer was washed off with dilute potassium hydroxide and distilled water, to remove traces of chlorophyll in salts. The filtrate was evaporated to dryness in rotary evaporator and the extract was stored in ice box.

 

Microorganisms:

The microorganisms used for the study includes Escherichia coli, Staphylococcus aureus, proteus vulgaris, Klebsiella pneomoniae, Bacillus subtilis, and pseudomonas arogenosa.

 

Antibacterial Assay:

In vitro antibacterial was carried out by agar well diffusion method. Bacterial strains grown on nutrient agar (37^oC for 24h) and was suspended in a saline solution (0.85 NaCl) and adjusted to a turbidity of 0.5 McFarland standards. From the stock 512, 256, 128, 64, 32 µg/ml concentrations were prepared. Bacterial culture was inoculated uniformly using L-rod on labeled medium plate.  A sterile a sterile cork borer was used to make 5 mm well on the agar. 100μl of various concentrations of prepared samples were dropped into the wells and incubated at 37^oC for 24 hours. Antimicrobial activity was determined by measuring the diameter of zones of inhibition (mm) produced after incubation. O.O5% of chloramphenicol was used as positive control. Diethyl ether used as a negative control. Determination of Minimum Inhibitory Concentration (MIC) The lowest concentration able to inhibit visible bacterial growth is minimum inhibitory concentration. It was measured with various concentrations of the test samples such as Chlorophyllin from Mimosa pudica and Standard chlorophyllin assayed against the test bacteria. The minimum inhibitory concentration was defined as the lowest concentration able to inhibit any visible bacterial growth¹¹ (NCCLS, 2000).

 

RESULTS:

The antibacterial activities and minimum inhibitory activity for Sodium copper chlorophyllin extracts obtained from Mimosa pudica leaves and Standard Chlorophyllin against tested organisms are depicted  in Table1-3. The result obtained in the present study revealed that sodium copper Chlorophyllin from Mimosa pudica possesses potential Antibacterial activity against the Escherichia coli, Staphylococcus aureus, proteus vulgaris, Klebsiella pneomoniae, Bacillus subtilis, and pseudomonas arogenosa. The lowest concentration showed inhibition was 32μg/ml in Escherichia coli and 64μg/ml in other tested microorganism. Sodium copper Chlorophyllin extracted from Mimosa pudica was compared with the standard Chlorophyllin and the results was comparable and more or less similar.

 

Table 1: Antibacterial activity of Chlorophyllin from Mimosa Pudica

Values are mean of three independent replicates Standard Deviation

 

Table 2: Antibacterial activity of Standard Chlorophyllin

Name of the organisms	Zone of the Inhibition (mm)					Chloramphenicol
Concentration(μg/ml)	-	64	128	256	512	50
Escherichia coli	-	-	22±1.1	28±1.5	34±0.5	36±0.5
Staphylococcus aureus	-	-	-	26±0.5	34±1.1	34±0.5
proteus vulgaris	-	-	16±0.5	-	32±2.6	30±1.1
Klebsiella pneomoniae	-	-	-	24±1.1	26±1.5	36±1.5
Bacillus subtilis	-	-	12±1.1	21±0.5	24±0.5	38±1.1
Pseudomonas arogenosa	-	-	-	18±1.1	28±1.1	30±0.5

Values are mean of three independent replicates Standard Deviation

Table 3: Minimum inhibitory concentration (μg/ml) of Chlorophyllin from Mimosa pudica (M) and Standard Chlorophyllin (S):

 

The minimum inhibitory concentrations of the sodium copper Chlorophyllin range between 32-256μg/ml. The highest inhibitory concentration was seen in Escherichia coli (32). In Staphylococcus aureus and Bacillus subtilis inhibition observed at the concentration of 64μg/ml. In Klebsiella pneomoniae the effective inhibitory concentration observed at 128μg/ml. In standard Chlorophyllin Staphylococcus aureus MIC was observed at 128μg/ml. Hence the Chlorophyllin is active and effective against the tested microorganism

 

DISCUSSIONS:

The antimicrobial activity of ethanolic extract of Mimosa pudica was against Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumonia.¹² Antimicrobial activity of chloroform, methanol, and ethyl acetate extracts of leaf, root and pod in both gram positive and gram negative bacteria. Evaluation of antimicrobial activity against different bacterial strains E.coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Streptococcus pyogenes. In this study 17.65 -20 mm zone of inhibition was observed in the methanolic crude extract and the MIC ranges between 225-250mg/ml concentration.^{13, 14}

 

Antimicrobial activity of Mimosa pudica crude extract against Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumonia. The zone of inhibition was seen between the ranges of 1.2-2.0cm. It was more or less similar to the current study.¹⁵ The sensitivity of test srains was, in decreasing order: Escherichia coli> Staphylococcus aureus> Bacillus subtilis> pseudomonas arogenosa> Proteus vulgaris. Gram positive were more sensitive than gram negative bacteria. which may be due the composition of cell wall.³

 

CONCLUTION:

The present evaluation of antibacterial activity of sodium copper Chlorophyllin from Mimosa pudica was compared with the standard chlorophyllin and observed effective inhibitory activities. The antibacterial activity may vary with the plant species. Sodium copper Chlorophyllin beneficial for human health. It can be used for the treatment of various infectious diseases caused by the bacteria which showed significant activity. Thus the Chorophyllin from Mimosa pudica can be used as an antibacterial agent and may serve as leads for the Pharmaceutical industries.

 

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4.     Rekha Rajendran, S. Hemalatha, K. Akasakalai, C. H. Madhukrishna, Bavan Sohil, Vittal and R. Meenakshi Sundaram. Hepatoprotective activity of Mimosa pudica leaves against carbontetrachoride induced toxicity. journals of natural products. 2009; vol 2 116-122.

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7.     Young, R.W. et al. (1980) Use of chlorophyll in in the care of geriatric patients. J Am. Geriatr. Soc., 28, 46–47.

8.     Dashwood, R. (1997) Chlorophylls as anticarcinogens. Int. J. Oncol., 10, 721–727.

9.     Breinholt V, Hendricks J, Pereira C, Arbogast D, Bailey G. Dietary chlorophyllin is a potent inhibitor of aflatoxin B1 hepatocarcinogenesis in rainbow trout. Cancer Res. 1995;55(1):57-62.

10.  Schertz, F. M. 1928. The extraction and separation of chlorophyll (a + b ), carotin and xanthophyll in fresh green leaves, preliminary to their quantitative determination. Plant Physiol. 3: 211-216.

11.  NCCLS, 2000, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard M7-A5, 5th edn. NCCLS, Wayne, PA.

12.  Rajesh Singh Tomar, Vikas Shrivastava and Shuchi Kaushik.  2014, In vitro efficacy of methanolic extract of Mimosa pudica against selected micro-organisms for its broad spectrum antimicrobial activity. Int. J. Curr. Microbiol. App. Sci (2014) 3(4): 780.

13.  S. K. Gangai Abirami, K. Sudha Mani, M. Nisha Devi, P. Nirmala Devi. 2014. International Journal of Ayurveda and Pharma Research.  The Antimicrobial activity of Mimosa pudica L. 2(1): 105-108.

14.  Bhawana pandey and Nisreen Husain. 2015. Antimicrobial activity of Mimosa pudica Linn. against some microbes. Indian J. L. Sci. 5(1): 058-061.

15.  Ghani. A. medicinal plants of Bangladesh with chemical constituents and uses. Dhaka. Asiatic Society of Bangladesh. 1998; Second Ed

 

 

 

Accepted on 04.01.2017           © RJPT All right reserved