Screening of Antimicrobial Activity of tin Chlorophyllin from Morinda citrifolia L.

 

Pavithra. S1*, N. Banu 2

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.

*Corresponding Author E-mail: pavithrasgopan@gmail.com

 

ABSTRACT:

Metallochlorophyllin, a water soluble analogue of the ubiquitous green pigment chlorophyll belongs to a group of compounds, porphyrins that contain a cheated metal ion in the center of the molecule. Tin chlorophyll in is the metallochlorophyllin with tin as its central metal ion and the phytol chains lost. The aim of the present study was to explore the antimicrobial activity of tin chlorophyll in from the leaves of Morinda citrifolia L. The leaves were collected and the tin chlorophyll in was extracted. The extracted tin chlorophyll in was subjected for antimicrobial study by disc diffusion method. The test organisms include Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi, Pseudomonas aeroginosa, Aspergillus niger, Rhizopus microsporus. The inhibition zones were detected and were calculated for the various organisms. The Minimal Inhibitory Concentration (MIC) was also determined and found to be 125µg/ml (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeroginosa) and 250µg/ml (Escherichia coli, Salmonella typhi). The results of the present study conclude that tin chlorophyll in from the leaves of Morinda citrifolia L. showed significant antimicrobial activity.

 

KEYWORDS: Metallochlorophyllin, Tin chlorophyll in, Antibacterial, Antifungal, MIC.

 

 


INTRODUCTION:

Medicinal plants constitute an important natural wealth of a country and they play a significant role in providing primary health care services to rural people as well as substantial amount of foreign exchange1. Plants can provide biologically active molecules and lead structures for the development of modified derivatives with enhanced activity and reduced toxicity2. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions3, and to defend against attack from predators such as insects, fungi and herbivorous mammals4.

 

The use of medicinal plants is increasing worldwide, related to the persistence and sometimes expansion of traditional medicine and a growing interest in herbal treatments5. Medicinal plants have played an essential role in the development of human culture for medicinal purposes around before the time of man6. The recent attention has been paid to biologically active extracts and compounds from plant species used in herbal medicines7. Morinda citrifolia L. commonly known as Indian mulberry is an edible and medicinal tropic plant. The whole plant was utilized in medicinal remedies and as dye for clothes. The leaves, flowers, roots, bark, stem and fruits are also involved in various combinations in almost 40 known and recorded herbal remedies8. It has been reported to have a broad range of health benefits for cancer, infections, asthma, arthritis, hypertension and diabetes9. Within the last few years interest in chlorophyll and its derivative metallochlorophyllin has increased markedly. Metallochlorophyllin, a water soluble analogue of the ubiquitous green pigment chlorophyll, belongs to a group of compounds, porphyrins that contain a chelated metal ion in the center of the molecule. The sodium and copper salt of chlorophyll in which magnesium has been replaced tin and the phytol chains lost is the tin chlorophyllin10. The loss of the phytol tail makes the resulting chlorophyll in salt soluble in water, a property that unaltered chlorophyll does not share. This improved water-solubility is the reason for many of the health benefits of the metallochlorophyllins, as it allows for far greater absorption and bioavailability within the human organism11.

 

MATERIALS AND METHODS:

Collection of Leaves:

The leaves of Morinda citrifolia L. was obtained from the fields of villages in and around Chengalpet District, Tamilnadu.

 

Extraction of Tin Chlorophyll in:

Ten grams of leaf sample was weighed and boiled with water and caustic soda (NaOH) for 30min. 2g of stannous chloride (SnCl2) was added and again boiled for 5min. The solution was filtered and cooled. It was then acidified with HCl and agitated to obtain a green precipitate. The clear liquid was separated and the remaining was filtered. Cold acetone was then added to the liquid and filtered. It was then heated with 50ml of acetone for 10min. The entire acetone was evaporated and 200ml of toluene was added. 3g of sodium bicarbonate and 100ml of acetone was added to obtain a dark precipitate. The extract was then filtered and washed with acetone and dried for storage12.

 

Purification of Tin Chlorophyll in:

Column Chromatography:

The column was mounted at the top and bottom to a ring stand using finger clamps. A small amount of glass wool was soaked in toluene and pushed to the bottom of the column using a large glass stirring rod. 10ml of toluene: acetone (8:2) was added to the column (using a funnel) to reach a height of about 15cm. 20g of silica gel was mixed well with small amount of toluene: acetone (8:2) mixture and poured into the column. The pinch clamp was opened and the excess solvent was drained into a dish until the level of the solvent is just above the top layer of the silica. 5ml of the tin chlorophyll in was pipetted onto the top of the column. 5ml of acetone was carefully added to the top of the column to produce a green band for in the column. The purified tin chlorophyll in was collected and stored at room temperature.

 

Antimicrobial Activity:

Antibacterial Activity:

Antibacterial activity of tin chlorophyll in was determined by disc diffusion method on Muller Hinton agar (MHA) medium13. The bacterial cultures such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa were inoculated in plates in Muller Hinton Agar (MHA) medium and spread on the solid plates with sterile swab. Sterile discs were placed on MHA plates and 20 µl of sample (Concentration: 1000µg, 750µg and 500 µg) were placed on the disc. The plates were then incubated at 37şC for 24 hrs. Then the antimicrobial activity was determined in triplicates by measuring the diameter of zone of inhibition.

 

Antifungal Activity:

Antifungal activity of tin chlorophyll in was determined by disc diffusion method on Sabouraud Dextrose agar (SDA) medium. The fungal cultures Aspergillus niger, Rhizopus microsporus were spread on the solid plates with sterile swab. 20 µl of sample (Concentration: 1000µg, 750µg and 500 µg) were added onto sterile discs and placed on SDA plates. Amphotericin-B was taken as positive control. The plates were incubated at 37şC for 24 hrs. Then antifungal activity was determined in triplicates by measuring the diameter of zone of inhibition.

 

Minimum Inhibitory Concentration (MIC):

Sample preparation: 1mg of sample was taken and mixed with 1ml of DMSO obtaining the concentration of 1mg/ml. A series of 8 tubes were filled with 1ml of sterile LB broth was distributed in every tube and was submitted to autoclave 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 370C for 24 hrs. After incubation, the turbidity was observed and MIC was determined to be where growth was no longer visible by assessment of turbidity by optical density readings at 600nm.

 

STATISTICAL ANALYSIS:

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

 

RESULT AND DISCUSSION:

Antimicrobial properties of several plant extracts have been attributed due to the bioactive pigments14. Pharmaceutical and scientific communities have the attention of the medicinal plants to validate the claims of their biological activity15. The extracted tin chlorophyllin from the leaves of Morinda citrifolia L. were screened against Gram positive and Gram negative bacteria (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi, Pseudomonas aeroginosa). The zone of inhibition was seen against all cultures and the maximum inhibition was shown in 1000 µg/ml of tin chlorophyllin than others and was compared with Ampicilin as positive control (Table 1). The tin chlorophyllin at a concentration of 1000 µg/ml exhibited significant antifungal activity against Aspergillus niger and Rhizopus microsporus with Amphotericin-B as positive control (Table 1).


 

Table 1 Antimicrobial activity of tin chlorophyllin

ORGANISMS

ZONE OF INHIBITION (mm)

ANTIBIOTIC (1mg/ml)

Concentration(µg/ml)

Bacteria

1000

750

500

Ampicillin

Staphylococcus aureus

0.57

0.28

0.57

21

Bacillus subtilis

0.28

0.57

0.57

15

Escherichia coli

0.57

0

1

21

Salmonella typhi

0

0.57

0

12

Pseudomonas aeroginosa

0.28

0.57

1.15

24

Fungi

 

 

 

Amphotericin-B

Aspergillus niger

0.57

1.15

0.57

25

Rhizopus microspores

0.57

1

0

25

Values are expressed as standard deviation of mean

 


The MIC values were evaluated and showed no growth at 125µg/ml for Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeroginosa and 250µg/ml for Escherichia coli, Salmonella typhi (Table 2).

 

Table 2 Minimum Inhibitory Concentration of tin chlorophyllin

Name of the organism

Minimum Inhibitory oncentration (μg/ml)

Staphylococcus aureus

125

Bacillus subtilis

125

Escherichia coli

250

Salmonella typhi

250

Pseudomonas aeroginosa

125

 

The results showed that the tin chlorophyllin of Morinda citrifolia L. is a broad spectrum agent which can be used against gram positive and gram negative bacteria as well as fungi.

 

CONCLUSION:

The bioactive compound tin chlorophyllin present in Morinda citrifolia L. can certainly find a place in treatment of various microbial infections for its high antimicrobial activity. The results from the present study are very encouraging and indicate this phytopigment should be studied more extensively to explore its potential in the treatment of infectious diseases as well.

 

CONFLICT OF INTEREST:

Conflict of interest declared none.

 

ACKNOWLEDGEMENT:

The authors thank the Chancellor, Dr. Ishari, K. Ganesh, Vels University, Chennai, Tamil Nadu, and India for the support in providing all the facilities to complete the research work.

 

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Received on 05.12.2016             Modified on 15.12.2016

Accepted on 26.12.2016           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(3): 693-695.

DOI: 10.5958/0974-360X.2017.00129.9