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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 13  |  Issue : 3  |  Page : 150-158

Antimicrobial activity of different homoeopathic drugs and their potencies against 'Aspergillus niger' In vitro


1 Dr. D.P. Rastogi Central Research Institute for Homoeopathy, Noida, Uttar Pradesh, India
2 Central Council For Research in Homoeopathy, Janakpuri, New Delhi, India

Date of Submission23-Aug-2018
Date of Acceptance23-Oct-2019
Date of Web Publication4-Oct-2019

Correspondence Address:
Mr. Suneel Prajapati
Dr. D.P. Rastogi-Central Research Institute for Homoeopathy, Noida - 201 301, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijrh.ijrh_46_18

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  Abstract 


Background: Homoeopathic remedies are widely used all over the world for different disease conditions. Approximately 70% are derived from the plant; however, their preclinical evaluation is still a major concern. Objective: This study was undertaken with an aim to explore the antimicrobial effect of different homoeopathic drugs and its potencies against the Aspergillus niger. Materials and Methods: Fifteen homoeopathic mother tinctures (Θ) and their potencies (3x, 6x, 12x) were tested for their biological activity against the human pathogenic fungi A. niger using disc diffusion method according to clinical and laboratory standard (CLSIM44-A) with slight modifications. The diameter of zone of inhibition was measured and compared with vehicle control (Alcohol 90%). The experiment was performed twice to check the reproducibility. Results: The marked antifungal activity was observed with Θ of Zingiber officinale; the growth of A. niger was inhibited and showed maximum zone of inhibition up to 15.4 ± 2.88 mm followed by Holarrhena antidysenterica (13.2 ± 1.09) and Terminalia chebula (10.6 ± 1.14). Different potencies (3x, 6x and 12x) were also exhibited significant zone of inhibition, especially Allium cepa 6x (10.4 ± 0.89), Caesalpinia bonducella 6x and 12x (12.8 ± 0.54 and 10.4 ± 1.14, respectively), Eucalyptus globulus 12x (11.3 ± 1.94), Ruta graveolens 12x (15.0 ± 2.23), Thuja occidentalis 6x (10.8 ± 0.83), and Zingiber officinale 3x and 6x (13.0 ± 2.73 and 11.4 ± 2.30, respectively) as compared to control. Conclusion: The findings of study concluded that Θ and potencies can effectively inhibit the growth of A. niger in vitro. This study paves the way for development of homoeopathic antifungal treatments. However, further investigations are required to get more information about the mechanistic approach, their mode of action and in vivo evaluation.

Keywords: Antifungal, Aspergillus niger, Disc diffusion, Homoeopathic drugs, In vitro


How to cite this article:
Prajapati S, Sharma M, Kumar A, Gupta P, Dwivedi B, Arya BS, Arya R, Nayak D. Antimicrobial activity of different homoeopathic drugs and their potencies against 'Aspergillus niger' In vitro. Indian J Res Homoeopathy 2019;13:150-8

How to cite this URL:
Prajapati S, Sharma M, Kumar A, Gupta P, Dwivedi B, Arya BS, Arya R, Nayak D. Antimicrobial activity of different homoeopathic drugs and their potencies against 'Aspergillus niger' In vitro. Indian J Res Homoeopathy [serial online] 2019 [cited 2019 Nov 22];13:150-8. Available from: http://www.ijrh.org/text.asp?2019/13/3/150/268518




  Introduction Top


Aspergillus is airborne fungus and can cause allergy as well as invasive infection worldwide. Moulds with highest toxicity come from the Aspergillus genus and are considered as highly pathogenic for humans. Although since ancient time, it was thought that Invasive Aspergillosis (IA) is caused by Aspergillus fumigates,[1] later on it was reported that the emergence of IA can be caused by non-fumigatus species such as: A. flavus, A. ochraceus, A. niger, A. versicolor or A. terreus.[2],[3] Aspergillus species also are saprophytic, thermo tolerant fungi which are ubiquitous in the air and environment. There are 185 species of genus Aspergillus reported, and out of these, 20 can cause human infections. Aspergillus fumigatus is the most common species found in human infections all over the world.[4],[5] The incidence of A. niger complex IA has been found to be 0.048% in organ transplantation patients and 0.16% in stem cell transplant recipients.[6],[7] Although humans inhale Aspergillus spores at the rate of hundreds per day, they rarely experience complications. However, under special circumstances, Aspergillus species has ability to produce spectrum of diseases involving lungs and later on other organs and tissues.[5]

Aspergillosis infections can also strike as sinus disease in immunocompromised hosts. If left untreated, IA can have mortality approaching 100%. In cases of suspected IA, an extensive diagnostic workup is necessary, but treatment should be initiated early to reduce morbidity and mortality.[8],[9],[10] Over the past two decades, emergence of such saprophytic fungi are increasing day by day, and one of the major challenges for immunocompromised patients, such as those with hematologic malignancies, bone marrow transplant and HIV infection is reported to cause cutaneous infection, paranasal aspergilloma and osteitis of the middle ear in such patients. A. niger, also known as black mould, are filamentous fungi having ability of fast growth with high pH tolerance ability and consist of smooth and colourless conidiophores and spores.[11] A. niger is also reported to cause endocarditis after heart surgery and infection of exenterated orbit even in immunocompetent patients.[12] A. niger was also found to cause allergic bronchopulmonary infection, IA or may be a coloniser of natural or preformed cavities of the human body. Resistance of Aspergilli to some clinically used antifungal agents brings a worrying clinical prognosis in people attacked by aspergillosis. For over 50 years, antibiotics have been applied for treating or inhibiting infections. The wide use and sometimes misuse of chemoantimicrobials in both human and animal medicine have been responsible for drug resistance.[13],[14]

However, the management of Aspergillus infections faces a number of problems including limited resources and high cost of effective antifungal agents, and their adverse effects. Besides these, the indiscriminate and prolonged use of antifungal drugs has led to therapeutic failures associated with an emergence of multidrugs' resistance to pathogenic organisms. This has necessitated a search for new antifungal alternative therapeutics, which are safe and cost-effective in terms of Aspergillus management. Since the 20th century, the researchers have focused on the homoeopathic system due to its efficacy, minimal side effects and cost-effectiveness. Therefore, we proposed to evaluate the in vitro efficacy of homoeopathic medicines against the A. niger an opportunistic pathogen.

Although scientific reports revealed that substantial work has been done on microbial activities of many plants in other systems of medicine. Besides their traditional uses as antimicrobial agents, their antifungal activity as homoeopathic medicine has not been reported so far. The present study aims to evaluate the efficacy of antifungal activities of the selected homoeopathic medicines; (selection based on their traditional use in other systems of medicine and scientific literature survey). Thus, the particular plant materials Allium cepa, Allium sativum, Caesalpinia bonducella, Calotropis gigantean, Cassia angustifolia, Eucalyptus globulus, Ficus religiosa, Holarrhena antidysenterica, Kalmia latifolia, Ocimum sanctum, Ruta graveolens, Syzygium jambolanum, Thuja occidentalis, Terminalia chebula and Zingiber were processed for homoeopathic preparation, and tested for the antifungal property against the human pathogenic fungi A. niger.


  Materials and Methods Top


Plant material

Fifteen Plants were selected for this study based on their medicinal use, reported literature and clinical indications. Fresh plant parts were collected from the tribal villages. Plants materials of Allium cepa (Bulb), Allium sativum (Bulb), Caesalpinia bonducella (fruit), Eucalyptus globulus(leaves), Ficus religiosa (leaves), Holarrhena antidysenterica (bark), Ocimum sanctum (whole plant), Syzygium jambolanum (Seed), Thuja occidentalis (leaves) and Zingiber officinale (Rhizome) were collected from Nilgiris District of Tamil Nadu State and taxonomically identified/authenticated by the Centre of Medicinal Plants Research in Homeopathy (CMPRH), Nilgiri District, Udagamandalam, Tamil Nadu. However, the mother tincture of C. gigantean, C. angustifolia, K. latifolia, R. graveolens and T. chebula were procured from a Homoeopathic pharmaceutical company (SBL, Haridwar, Uttarakhand, India).

Processing of the plant materials

The raw plant materials were collected, sorted, washed and chopped into small pieces, wherever necessary, before drying. The materials were dried outdoors; however, leaves were dried in the shade. The dried plant materials were ground to various degrees of fineness depending on their botanical structures and further used for the preparation of alcohol-based homoeopathic mother tincture.

Preparation of mother tinctures, potencies and standard

Mother Tinctures (MT) of Homoeopathic drugs (Θ) of Allium cepa, Allium sativum, Caesalpinia bonducella, Eucalyptus globulus, Ficus religiosa, Holarrhena antidysenterica, Ocimum sanctum, Syzygium jambolanum, Thuja occidentalis and Zingiber officinale were prepared and processed for different potencies (3x, 6x, 12x) according to the procedures mentioned in Homoeopathic Pharmacopoeia of India. For the remaining 5 homoeopathic mother tinctures potencies were prepared in house as per the standard procedure mentioned in Homoeopathic Pharmacopoeia of India. Ketoconazole (10 μg/ml) (Sigma-Aldrich, GmbH Germany) was used as standard antifungal drug and 90% alcohol (unsuccussed) used as vehicle control whereas, double-distilled water was also used as one of the negative control to highlight the effect of alcohol.

Microorganism

For this study, the fungal culture of A. niger (MTCC No. 282) procured as lyophilized freeze-dried culture strain was obtained from the MTCC, Institute of Microbial Technology, Chandigarh for evaluating antifungal activity of homoeopathic drugs.

Preparation of fungal culture

The fungal strain of A. niger (MTCC No. 282), freeze-dried culture, was aseptically opened in a biosafety cabinet and suspension was made in CMPRH0.4 ml sterilised water. It was then taken in a micro centrifuge tube and freeze-dried culture was transferred in water, mixed well and was left to stand for 20 minutes before transferring it on solid media. Petri plates containing Sabouraud dextrose agar (SDA; Hi Media, Mumbai, India, Catalogue No. M063) medium and Czapek Yeast Extract Agar medium (CYEA; Hi Media, Mumbai, India, Catalogue No. M1335) was incubated for 24–48 h at 30°C to give white round colonies against a yellowish/light yellowish background. Approximately, 1 mm colonies were picked up and suspended in 5 ml of sterile Sabouraud dextrose broth and kept as broth culture/stock culture. Microorganisms were repeatedly subcultured using spreading method and maintained to obtain pure isolation on the CYEA for further drug sensitivity assay.

Morphological Identification of Aspergillus niger (direct microscopy by KOH stain)

Morphological features of A. niger species were identified according to the method previously described.[15] In brief, one drop of KOH stain was placed on centre of clean grease free glass microscope slide and transferred a loop of culture growth from CYEA media containing A. niger and mixed gently with the stain and covered with a cover slip. The preparation was examined using the low power (10x, 20x) objective of Inverted phase contrast microscope (RTC-7, Radical scientific equipment's Pvt., Ltd., Ambala, India). High power (45x) objective was then used to confirm observations [Figure 1].
Figure 1: Identification and characterization of fungal strain Aspergillus niger at (a) 10X, (b) 20X and (c) 45X magnification

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Preparation of disc for antifungal assay

For determining antifungal activity of different homoeopathic mother tinctures and potencies, agar disc diffusion method was used. Filter paper (Whatman no. 1) was used to prepare discs approximately 6 mm in diameter, which were placed in a  Petri dish More Details and sterilised in a hot air oven. Sterilised discs of filter paper were soaked in homoeopathic mother tincture, as well as different potencies of same drugs and allowed to stand for 30 min. Commercially available antifungal drug Ketoconazole was used as positive control and 90% alcohol was used as vehicle control. Drug-impregnated discs were used for further drug sensitivity assay.

Preparation of growth media (Sabouraud dextrose agar/Czapek Yeast Extract Agar)

Media with pH 5.6 ± 0.2 containing relatively high concentration of glucose (40%) were prepared by mixing SDA and distilled water, and CYEA medium containing relatively high concentration of sucrose (30%) was prepared by mixing with double-distilled water at 121°C for 15 min. Twenty ml of molten (45°C) SDA medium was aseptically transferred into each sterile petri plates (100 mm × 15 mm) and allowed to solidify in a biological safety cabinet (Model No. AC2-4S8-NS, ESCO Micro Pvt. Ltd., Singapore).

Preparation of Muller Hinton Agar, 2% Glucose with Methylene Blue for Drug sensitivity assay

Media containing relatively high concentration of Glucose (20%) were prepared by mixing Muller Hinton Agar, 2% Glucose with Methylene Blue (MHAGMB) (HIMEDIA, Mumbai, India, Catalogue No: M1825) in distilled water and autoclaved at 121°C for 15 min. Twenty ml of molten (45°C) MHAGMB medium was aseptically transferred into each sterile petri plates (100 mm × 15 mm) and allowed to solidify in a biological safety cabinet.

Inoculum suspension

Spore suspensions of A. niger were prepared in sterile saline from fresh colonies grown on CYEA media at 35°C. Cell concentration was adjusted to final concentrations of 0.5 McFarland (1–5) ×106 CFU/ml. These suspensions were used directly for the inoculation purpose. The fungal strains were allowed to grow in respective agar medium, and the fungal mycelia before the spore formation was used for antifungal assay to avoid the spreading of spores throughout the plate which will interrupt in the radial growth measurement.

Disc diffusion assay

In vitro antifungal activities were examined as per CLSI document M 44-A (CLSI/NCCLS 2004), with minor modifications. Antifungal activities of homoeopathic drug against the pathogenic Aspergillus niger were investigated by the agar disk diffusion method.[16] Ketoconazole (10μg/ml) were used as standard drug. Antifungal activity of homoeopathic medicines and their potencies were determined by measuring the diameter of zone of inhibition. To screen the antifungal activity, a sterile cotton swab was dipped into the adjusted suspension and swabbed over MHAGMB media with sterile cotton bud on the entire dried surface of a MHAGMB plate. Then, filter paper discs containing the 15 homoeopathic mother tinctures, as well as their different potencies were placed on the agar surface. 90% alcohol was used as vehicle control for the antifungal activity against the A. niger.

The plates were inverted and placed in an incubator set at 35°C ± 2°C for 15 min after the discs were applied. Zone diameters (mm) were determined after 24 hours of incubation at 35°C and measured the point at which total growth inhibition zone was noted. Each assay was performed in duplicate on two different days, and the mean diameters were reported.

Determination of percentage of zone of inhibition

Following the observation for fungal inhibition by homoeopathic medicines, the diameter of zone inhibition values were determined according to the equation as below:



Statistical analysis

Experiment was performed twice to check the reproducibility of the results. Data were expressed as mean diameter of zone inhibition (mm) and analysed using one-way analysis of variance followed by Dunnett's post hoc test to monitor significance among groups using the Graph Pad prism version 7.0. The statistical comparison having standard deviation P < 0.05 was considered as significant. Ethical approval was not required for this study.


  Results Top


In the present study, mother tinctures and their different potencies (3x, 6x and 12x) of homoeopathic drugs Allium cepa, Allium sativum, Caesalpinia bonducella, Calotropis gigantean, Cassia angustifolia, Eucalyptus globulus, Ficus religiosa, Holarrhena antidysenterica, Kalmia latifolia, Ocimum sanctum, Ruta graveolens, Syzygium jambolanum, Thuja occidentalis, Terminalia chebula and Zingiber officinale have shown varying results against the growth of A. niger.

Microscopy recorded under the different objectives [Figure 1] reveals fungal hyphae with conidial head biseriate, radiate, conidia in chains or detached and dispersed. Single or paired conidia (at an angle of approximately 45°) confirmed the characteristical identification of A. niger.

Antimicrobial properties of medicinal plants are being reported from different parts of the world. World Health Organization estimates that plant extract or their active constituent's preparation are used as folk medicine in traditional therapies of 80% of the world's population. In the present work, homoeopathic drugs were prepared from different plant material, and some of them show strong activity against fungal strains. In this screening work, the antifungal activity was accessed by disc diffusion assay; this method is most suited to Homoeopathy system and most acceptable as well. Broth microdilution assay is not feasible to perform with homoeopathic drugs because in homoeopathic drug dilution, the concentration of the active constituents is unknown; hence, determination of concentration of the compound is difficult. Therefore, the in vitro antifungal activity of homoeopathic mother tinctures and their potencies were tested using the disc diffusion method according to clinical and laboratory standard (CLSIM44-A) with slight modifications against the A. niger. The diameter (mm) of zone of inhibition was measured and compared with standard (Ketoconazole) [Table 1] and [Figure 2]. The per cent zone of inhibition for each medicine was also calculated and depicted in [Figure 3]. Mother tincture of Z. officinale was found to be the most potent against A. niger and showed maximum zone of inhibition up to 15.4 ± 2.88 mm followed by H. antidysenterica (13.2 ± 1.09) and T. chebula (10.6 ± 1.14). Different potencies (3x, 6x and 12x) exhibited significant zone of inhibition, especially A. cepa with 6x (10.4 ± 0.89), C. bonducella with 6x and 12x (12.8 ± 0.54 and 10.4 ± 1.14, respectively), E. globulus with 12x (11.4 ± 1.94), R. graveolens with 12x (15.0 ± 2.23), T. occidentalis with 6x (10.8 ± 0.83) and Z. officinale with3x and 6x (13.0 ± 2.73, 11.4 ± 2.30) as compared to vehicle control. The antifungal activity of reference drug Ketoconazole showed zone of inhibition (11.9 ± 0.22) compared to control group.
Table 1: Zone of inhibition of the homoeopathic mother tincture (ø) and their potencies (3x, 6x and 12x) against fungal strain Aspergillus niger

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Figure 2: Effect of homoeopathic medicines against fungal strain Aspergillus niger

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Figure 3: Per cent zone of inhibition of homoeopathic medicine against the Aspergillus niger

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The maximum percentage of zone inhibition (92.5%) was found in mother tincture of Zingiber officinali s compared to other medicines used in the study. In case of different potencies (3x, 6x and 12x), the maximum percentage of zone of inhibition was recorded in R. graveolens with 12x (87.50%), E. globulus with 12x (42.50%), T. occidentalis with 6x (35%) and Z. officinale with 3x and 6x (62.50% and 42.50%, respectively).

Published research data have suggested that antimicrobial components of the plant extracts (terpenoid, alkaloid and phenolic compounds) interact with enzymes and proteins of the microbial cell membrane and cause cell wall disruption to disperse a flux of protons toward cell exterior which induces cell death or may inhibit enzymes necessary for amino acids biosynthesis.[17],[18] Other research studies attributed the inhibitory effect of these plant extracts to hydrophobicity characters may enable them to react with protein of microbial cell membrane and mitochondria disturbing their structures and changing their permeability.[19],[20] The result of present study showed variable antifungal activity of tested homoeopathic mother tincture and their potencies against the growth of A. niger. However, justification about the observed activity could not be explained as no uniform pattern (decreasing or increasing order) of zone of inhibition was seen in any of the ultrahigh dilutions potencies of homeopathic drugs. Hence, correlation between the effect of mother tincture and different potencies could not be established. Some previous studies about homoeopathic medicine also revealed that these homoeopathic preparations have capability to inhibit the growth of pathogenic microbes;[21],[22],[23] however, their mechanism of action is unknown till date. This study suggests that homoeopathic drugs would be helpful for treating diseases of human beings caused by A. niger.


  Conclusion Top


The results of the present study demonstrated that homoeopathic drugs, namely Z. officinale, H. antidysenterica, T. chebula, A. cepa, C. bonducella, E. globulus, R. graveolens and T. occidentalis have significant antifungal activity against human pathogenic fungi A. niger. In conclusion, the findings of this experiment confirmed that homoeopathic drugs can be used as natural fungi toxicant to control the growth of pathogenic fungi (A. niger) and reduce the dependence on the synthetic fungicides. Further, in vivo experiments are required to confirm their fungicidal activity, mechanism of action to recognise them as better alternative for antifungal treatment in the current scenario.

Acknowledgement

The authors are thankful to CCRH for financial assistance, Dr. Raj K. Manchanda former Director General and Dr. Anil Khurana, Director General I/c for providing necessary facilities and encouragement.

Financial support and sponsorship

This study was financially supported by Central Council for Research in Homoeopathy.

Conflicts of interest

None declared.



 
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