Anti fungal activity of metallochlorophyllins 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.

*Corresponding Author E-mail:



Metallochlorophyllin belongs to a group of compound, porphyrins containing  a chelated metal ion in the center of the molecule. The central metal atom consists of metals like copper, magnesium, zinc etc with loss of phytol chain. The present study was to extract the metallochlorophyllin from Leucas aspera L. and to check its antifungal activity. The result shows that the metallochlorophyllins (Sodium copper chlorophyllin, Sodium magnesium chlorophyllin, Sodium zinc chlorophyllin) of Leucas aspera L. has significant anti fungal activity against Aspergillus niger and Rhizopus microsporus with Amphotericin-B (Std) 20 µg /ml as positive control. The active zone of inhibition of Sodium copper chlorophyllin was around 7-15 mm, Sodium magnesium chlorophyllin 15-22 mm, Sodium zinc chlorophyllin  6-10 mm against the pathogenic fungi. This result confirms that the medicinal value of metallochlorophyllins of Leucas aspera L. as an anti fungal sources.


KEYWORDS: Metallochlorophyllins, antifungal, Medicinal plants.




Medicinal plants produce many chemical compounds for biological functions, including defence against fungi, insects and mammals. Ethnobotany, the study of traditional human uses of plants, is recognized as  an effective way to discover future medicines1,2. Researchers found around 120 compounds isolated from  the higher plants that were used in modern medicine, from traditional plant sources and more than 80% show a  positive correlation between modern therapeutic use and the traditional use of the plants derived3. The World  health organization (WHO) estimates the population of some Asian and African countries presently use herbal  medicine for some aspect of primary health care. By comparing modern medicines, herbal medicines are  grown from seed and gathered from nature for little or no cost.


Chlorophyllin (CHL), a water soluble sodium salt containing sodium and copper as the central metal ion. Metallochlorophyllin is a derivative of chlorophyll in which the chelated metal magnesium is replaced by other metals such as copper, zinc etc. Sodium copper chlorophyllin (SCC), copper as the chelated metal was found to be a mixture of copper chelate of chlorophyll derivatives4. It is used as a food additive 5 and medicine for gastrointestinal disorders6. Sodium zinc chlorophyllin (SZC) also known as chlorophyll zinc salt, is a natural green raw material  obtained from chlorophyll, made of magnesium with zinc substitution, dark green powder or crystal, soluble in  water with green transparent solution without precipitation, has good water solubility and stability, but also has a suitable zinc ion dissolution. Sodium zinc chlorophyllin used as active ingredient in oral care products, has the  presence of poor stability and toxicity problems . In an effort to expand the spectrum of antifungal agents from natural resources, Leucas aspera L. belonging to  the family Lamiaceae has been selected. In the Indian literature, the plant have been described to be useful against skin diseases, psoriasis, scabies and chronic rheumatism and it is used for the treatment of scorpion bites, sinusitis and  intestinal worms diseases7,8. In the present investigation ,a screening of metallochlorophyllin of Leucas aspera L. leaves against pathogenic fungi is done in order to detect  new sources  of antifungal agents.



Collection of Plant Materials:

The healthy leaves of the plant  Leucas aspera L. were collected between April and June, 2015 from various  areas of vellore, Tamil Nadu. The plant parts were selected on the basis of  knowledge and their use in different  medicinal system of health care.


Solvent Extraction:

Sodium Copper Chlorophyllin (SCC):

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. This procedure is repeated until the residue becomes  colorless. Wash it with 50–150 ml of diethyl ether to remove acetone. The mixture was poured into a  separating funnel. This was repeated until yellow color separates off  which consists of flavones. The solution  was poured into a bottle to that 10 ml of methanol saturated with potassium hydroxide pellets was added. The solution was shaken thoroughly and kept in the icebox for overnight. To the separating funnel, add 100 ml diethyl ether 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 chlorophyll in salts. The filtrate was evaporated to dryness in a rotary evaporator, and the extract was stored in ice box9.


Sodium Magnesium Chlorophyllin (SMC):

Leaf sample was weighed and boiled with water and caustic soda (NaOH) for 30 mins. The solution was filtered and cooled. Acidified with HCl and agitated to obtain a green precipitate. 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 10 mins. 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 storage.


Sodium Zinc Chlorophyllin (SZC):

Fresh and healthy leaves were weighed and 1 gm of sodium carbonate was added to neutralize the acidity. This was ground with acetone and filtered, procedure was repeated until the residue is colorless. It was washed with diethyl ether to wash of acetone. The extract was then poured into a separating funnel and acetone was washed off using distilled water. Zinc Sulphate was added to saturated methanol and 2 gms of pellets of potassium hydroxide was added and shaken thoroughly and incubated in ice box overnight. The alkaline solution of Sodium zinc chlorophyllin salts was poured into a separating funnel. The bottle was washed several times with distilled water and the ether to remove traces of pigments. Diethyl ether was added to the funnel and left for 30 minutes. The Sodium zinc chlorophyllin separates as a light greenish layer below. The filtrate was evaporated to dryness in a rotary evaporator. The extracted Sodium zinc chlorophyllin was stored in ice box.


Culture Media:

Stock cultures were maintained at 4°C on Sabouraud Dextrose agar slant. Active cultures for experiments were prepared by transferring the stock cultures into the test tubes containing sabouraud dextrose broth that were incubated at 48 hrs at room temperature. The assay was performed by agar disc diffusion method.


Antifungal Activity:

Antifungal activity of metallochlorophyllins  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: 1000 µg /ml, 750 µg /ml and 500 µg /ml) 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.


Statistical Analysis:

All experiments were performed in triplicates and the values were calculated statistically by standard deviation




Medicinal plants are responsible for several biological activities. In recent periods, ethno medicine and traditional pharmacology acheives great appreciation in modern medicines, though search for new medicinal plants often  based on ethanomedicinal origin10. In the present work, the metallochlorophyllins obtained from  Leucas aspera L. shows strong activity against the tested pathogenic fungal strains. The results were compared with standard antibiotic drugs. The antifungal activity of metallochlorophyllins of Leucas aspera L. leaves was evaluated using agar disc diffusion method. The metallochlorophyllins exhibits high significant activity in Leucas aspera L. against all the tested pathogenic fungi compared with the standard Amphotericin-B (20 µl/disc). Metallochlorophyllin shows good activity against the fungal isolates with the active zone of inhibition of Sodium copper chlorophyllin (SCC)was around 7-15 mm, Sodium magnesium chlorophyllin (SMC) 15-22 mm, Sodium zinc chlorophyllin(SZC) 6-10 mm against the tested pathogenic fungi. By concluding  the results, the metallochlorophyllins of Leucas aspera L. has a broad spectrum agent which can be used against all the tested pathogenic fungi. Our results strongly report that the medicinal use of this plant in traditional medicine that can be  used as antimicrobial agents in the search for new drugs. With the emergence of multiple strains of antibiotic resistance microorganism, great interest has been generated in search for potential compounds from plants for  therapeutic, medicinal and aromatic uses.



Table 1:Antifungal activity of metallochlorophyllins against pathogenic fungi tested by disc diffusion assay



Zone of inhibition (mm) Concentration (µg /ml).

Amphotericin-B (µg /ml)

Aspergillus niger




















Rhizopus microsporus
















The values are expressed statistically by standard deviations



Many medicinal plants remain under explored. Scientists from divergent fields investigating new plants to their antimicrobial usefulness. The results justifies that the metallochlorophyllins of Leucas aspera L. plant is a good  source of antifungal activity. The result of present investigation supports strongly the efficiency of crude  solvent plant extracts contain medicinally important bioactive compounds due to the strongly presence of plants  which can be used to fight against resistant fungi for the treatment of different diseases in traditional medicine.



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



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Received on 24.11.2016             Modified on 05.12.2016

Accepted on 22.12.2016           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(2): 513-515.

DOI: 10.5958/0974-360X.2017.00102.0