Studies on medical applications of natural antifungals - metal hexacyanoferrate(ii) complexes
Brij Bhushan Tewari* and Shivanand Singh
Department of Chemistry, Faculty of Natural Sciences, University of Guyana, P. O. Box 101110, Georgetown, Guyana, Corresponding author: Tel: +592-222-6004; Fax: +592-222-3596
Copper, mercury, molybdenum and zinc ferrocyanides were synthesized and characterized by elemental and spectral studies. The natural antifungal plants extract were collected from Cassia alata (Canicro bush), Azadirachta indica (Neem), Ocimum sanctum (Tulsi) and Cassia obtusifolia (Money bush). Antifungal activity of natural antifungals, metal ferrocyanides and natural antifungal-metal ferrocyanide complexes were tested by well known cultured fungus (Aspergillus niger). The natural antifungals with metal ferrocyanides complexes were found to be have more antifungal property in comparison to metal ferrocyanides and natural antifungals alone. The mercury ferrocyanide with Neem and copper ferrocyanide with Canicro bush complexes were found to have highest and lowest antifungal properties, respectively.
Key words: natural antifungal; metal hexacyanferrate(II)complex; medical applications, Aspergillus niger; skin infections.
Insoluble metal ferrocyanides could have produced on the primitive earth due to interaction of cyanides with readily available metal ions in the environment. Abundant cyanide ions easily formed under prebiotic conditions 1. Cyanide is a strong ligand due to presence of triple bond, small size and unidentate character 2. It has ability to form complexes with transition metal ions 3. It is believed that during the course of chemical evolution, cyanide ion form stable complexes of general formula, M2[Fe(CN)6]. xH2O, where M and x are metal cations (Cu++, Hg++, Zn2+ etc.) and number of water molecules, respectively. Insoluble metal ferrocyanides are an important class of compounds and have been widely used as adsorbents 4-5, ion- exchangers 6-7 and photosensitizer 8.
The secondary metabolites or natural antifungal extracts obtained from various plants viz: Cassia alata (Canicro bush), Cassia obtusifolia (Money bush), Ocimum sanctum (Tulsi) and Azadirachta indicia (Neem). Studies on these indigenous plants showed that they posses antifungal compounds in parts; mainly the leaves, bark and fruits. The activities of natural antifungal extracts depends on method of extraction. Such methods include wet, dry and steam distillation. Extracts are used to treat feet rashes, lotta scabies, ringworms and skin infections. In addition to the antifungal extract also have various other medicinal uses such as antibacterial, laxative etc. 9-10
To analyze the nature of the metal ferrocyanides and the natural antifungal extracts, the best suited fungal spore is Aspergillus niger. This fungus is described to be omnivorous, that is, it can survive in both plants and animals. Aspergillus niger is one of the most common and easily identifiable species of the genus Aspergillus, with its yellow mat later bearing black conidia. This is the third most common species associated with invasive pulmonary aspergillosis. It is also often acausative agent of aspergilloma and is the most frequently encountered agent of otomycosis. Aspergillus niger may also be a common laboratory contaminant 11.
Literature survey shows that no reports are available on interaction of metal ferrocyanides with natural antifungals and medicinal significance of their complexes. The studies on medical applications of natural antifungal with metal ferrocyanide complexes are seem to be very interesting. In view of this attempt were made to do such studies. However since metal ferrocyanides are believed to be excellent absorbents and ion exchangers, it should interact with these secondary metabolites because they contains organic substances which has the ability to interact with other chemical species. The complex formed should be more reactive thus showing greater effects on fungal growth.
Potassium ferrocyanide, copper(II) chloride, zinc(II) chloride, mercury(II) chloride, sodium molybdate and methylene blue (basic blue 9) were obtained from British Drug House, Poole, England. All chemicals were of analytical-reagent grade and used as such without any further purification. Solutions were prepared in doubly distilled water.
Synthesis and characterization of metal ferrocyanides
Copper, zinc and mercury ferrocyanides were prepared by Kourim's method 12 by adding potassium ferrocyanide (167 ml; 0.1 M) solution slowly to copper(II) chloride, zinc(II) chloride and mercury(II) chloride (500 ml; 0.1 M) solution with constant stirring. Reaction mixture was heated on water both at 100 oC for 2-3 h and then cured for 24 h. The precipitate was washed with distilled water and dried at 60 oC.
Molybdenum ferrocyanide complex was isolated 6, 7 by adding ethyl alcohol to a mixture containing 14 ml of 0.1M molybdic acid and 20 ml of 0.1 M hexacyanoferrate solution. Precipitate was filtered and washed with alcoholic water and dried for 48 h. All the compounds were ground and sieved to (100-150) BSS mesh size.
The compounds were characterized on the basis of elemental and spectral studies. The percentage composition of metals were determined by IL-751 atomic absorption spectrophotometer 13. Carbon, hydrogen and nitrogen analysis were carried out by CEST 118, CHN analyzer. The values are given in Table 1.
Infra-red spectra of the compounds were recorded in KBr disc on Beckman IR- 20 spectrophotometer. All three metal ferrocyanides show a broad peak at 3600 cm- 1 characteristics of water molecule and OH group. Another peak at around 1600 cm-1 is due to H-O-H bending. Two sharp band at 2000 cm-1 and 600 cm-1 are characteristics of cyanide and Fe-C stretching respectively 14. Another sharp band at around 490 cm-1 probable shows the presence of metal-nitrogen band due to polymerization 15. The infra-red spectral data are given in Table 2.
Copper, mercury, molybdenum and zinc ferrocyanides were found to have reddish, blue, green and white colours, respectively. All three metal ferrocyanides are amorphous solids and give no X-ray pattern. Metal ferrocyanides were found to be stable in acids (HCl, HNO3, H2SO4), bases (NaOH,KOH, NH4OH) and various salt (NaCl, KCl, LiCl, NH4Cl, RbCl, CsCl, BaCl2 and CaCl2 ) solutions in concentration range 1.0-2.0 M.
Preparation of natural antifungal extract
The extraction from Cassia obtusifolia will be done by wet method. The leaves of the plant will be picked and soaked by covered with 95% ethanol solvent for 24 hours. The ethanol-extract will then be filtered using glass wool. The filtrate will then be vaporized using a rotavapourizer at 45 ºC until all the ethanol is removed. This final extract will be considered the stock solution from which further dilutions would be made for the analysis. For Cassia alata, Ocimum sanctum and Azadirachta indica extraction will be done by the dry method. The leaves will be dried for three days at 45 ºC then grind using the electric mill. The powdered leaves will then be soaked by covering with 95% ethanol solvent for 24 hours. The soaking will be repeated three times. The ethanol-extract will then be filtered by gravitational filtration using Whatman filter papers. The ethanol will then be removed using a rotavapourizer until the extract solidifies.
The extracts from the plants will then be absorbed on the metal ferrocyanides and then their activity will be tested on a known cultured fungus Aspergillus niger.
Test on antifungal activity
Testing the antifungal activity of extract only
An amount of 10 mg of extract was placed by mean of washing with 20 ml ethanol in a sterilized petri dish. The fungal spores were then sprayed on the entire bottom of the dish using an electric aspirator. This procedure was repeated using different extracts.
Testing the antifungal activity of metal ferrocyanides only
An amount of 10 mg of metal ferrocyanide was placed in a sterilized petri dish. The fungal spores were then sprayed on the entire bottom of the dish using an electric aspirator. This procedure was repeated using different metal ferrocyanides.
Testing the antifungal activity of metal ferrocyanides and extract complex
An amount of 10 mg of metal ferrocyanides and 10 mg extract were placed in a sterilized petri dish with. The fungal spores were then sprayed on the entire bottom of the dish using an electric aspirator. This procedure was repeated using different extracts and ferrocyanides complexes.
Testing the antifungal activity of control (ethanol only)
A volume of 20 ml of ethanol was placed in a sterilized petri dish. The fungal spores were then sprayed on the entire bottom of the dish using an electric aspirator. The assay was to stand for 7 days in a sealed container with a dampen paper towel.
RESULT AND DISCUSSION
Antifungal activity of extract only
The result obtained from this testing is shown in Table 3. It was observed that Neem extract showed the greatest antifungal activity followed by Tulsi then Money bush and the least being Canicro bush.
Bio assay : 10 mg plant extract in petri dish.
Antifungal activity of metal ferrocyanides only
The result obtained from this testing is shown in Table 4. The antifungal activities of mercury, molybdenum, copper and zinc were studied. Mercury ferrocyanide showed the greatest antifungal activity by molybdenum then copper and finally the least active was zinc ferrocyanides.
Bioassay : 10 mg metal ferrocyanide.
Antifungal activity of metal ferrocyanides and natural antifungal extract complexes
The results obtained are shown in Table 5. It was found that the order of antifungal activity of metal ferrocyanide-natural antifungal extract complexes were:
Bioassay : 10 mg metal ferrocyanides with 10 mg plants extract in petri dish;
The most effective was found to be mercury ferrocyanides in complex with Neem extract and the least being zinc ferrocyanide with canicro bush.
Antifungal activity of control (ethanol only)
It was found that fungal spores were able to grow in the control. Testing of antifungal activities of natural antifungal extract, metal ferrocyanides and natural antifungal metal ferrocyanide complexes after spray of cultured fungus Aspergillus niger are shown in Figure 1
It can be concluded that from present studies that the metal ferrocyanides interacted with secondary metabolites thus enhancing the antifungal activity of these extracts. Neem was found to have more antifungal activity in comparison to other antifungal plants studied. Among metal ferrocyanides studied, mercury ferrocyanide was found to have maximum antifungal property. Mercury ferrocyanides-Neem extract complex and zinc ferrocyanide-Canicro bush complex were found to have maximum and minimum antifungal property, respectively. It can also be concluded from the present studies that mercury ferrocyanide - Neem extract complexes may be used as an antifungal medicine for skin infection
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