[ VOLUME 5 I ISSUE 4 I OCT.– DEC. 2018] E ISSN 2348 –1269, PRINT ISSN 2349-5138 “In - vitro study for anti-fungal activity of Homoeopathic Medicines against plant fungus Ashbya gossypii”

Anuj Deepakrao Chinche1, Suyash Arunrao Kathade2, Pashmin Kaur Anand2, Arun Bhargav Jadhav3, Bipinraj Nirichan Kunchiraman4 & Chetan Hanamantrao Shinde5. 1Post - Graduate Scholar, Department of Homoeopathic Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Homoeopathic Medical College & Hospital, Katraj – Dhankawadi, Pune, India. 2Department of Microbiology, Bharati Vidyapeeth (Deemed to be) University, Rajiv Gandhi Institute of Information Technology and Biotechnology, Katraj – Dhankawadi, Pune, India. 3Principal, Bharati Vidyapeeth (Deemed to be) University, Homoeopathic Medical College and Hospital, Katraj - Dhankawadi, Pune, India. 4Assistant Professor, Department of Microbiology, Bharati Vidyapeeth (Deemed to be) University, Rajiv Gandhi Institute of Information Technology and Biotechnology, Katraj – Dhankawadi, Pune, India. 5Associate Professor, Department of Homoeopathic Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Homoeopathic Medical College & Hospital, Katraj – Dhankawadi, Pune, India.

Received: July 05, 2018 Accepted: August 22, 2018

ABSTRACT Ashbya gossypii is a plant fungus which causes stigmatomycosis disease in cotton and different variety of crops around the world including India. This study aimed to screen Homoeopathic medicines and their potencies for inhibition of Ashbya gossypii. The Homoeopathic medicines Calcarea Carbonica, Lachesis Mutus, Mercurius Solubilis, Silicea and Thuja occidentalis in 6C, 12C, 30C, 200C, 1M, 10M, CM potencies were screened by agar well diffusion method and MIC assay for anti fungal activity against Ashbya gossypii, which shows inhibition for Calcarea Carbonica in (12C, 30C and 1M), Mercurius Solubilis 30C and Thuja occidentalis 1M potencies. Whereas most of the potencies of above mentioned Homoeopathic medicines showing inhibition in MIC against Ashbya gossypii (NCIM- 1193).

Keywords: Anti-fungal, In-vitro, Homoeopathic medicines, Ashbya gossypii, Calcarea Carbonica, Lachesis Mutus, Mercurius Solubilis, Silicea, Thuja occidentalis.

Introduction: - Stigmatomycosis has majorly affected cash crops like cotton, , , or citrus fruits. Causative organism, leading towards stigmatomycosis is found to be Ashbya gossypii also familiar as . [1] The organism Eremothecium gossypii was discovered in 1926 by Ashby and Nowell. [2] The fungus requires host from the Pyrrohocoridae family to cause stigmatomycosis. The filamentous ascomycete Ashbya gossypii is used as a fungal model in understanding its pathogenicity for discovering new antifungal agents, such as the exciting fertilizers and fungal agents seen to be having drug resistant. [2] This organism has been studied as multi-drug resistant transporter in Saccharomyces cerevisiae. [3] This organism has an industrial application in manufacture of Riboflavin (Vitamin B2) which is prepared by direct fermentation of this fungus. [4] Discovery of biotin (Vitamin B7) has made Ashbya gossypii an important application in industrial research. [5] This fungus is found to produce rose-scented essential oil, which is been studied under pharmacological aspects which is similar to natural rose essential oil. [6] Among the various agricultural crops, cottons are many more profound due to their requirements throughout the year for industrial use. A fungal pathogen Ashbya gossypii was affecting on cotton plant which hampers the growth of cotton in definite regions of the subtropesics, causing profound economical losses. Maturation of hair cells inside the cotton bolls affected by Ashbya gossypii and when transmitted to citrus fruits, which leads to drying up and collapse. [7] Various Complementary and alternative therapies has been used as an alternative option for drug resistance of this fungus. Homoeopathy is a classical therapeutic science, which is actively used in studying various antimicrobial in-vitro researches. Homoeopathic medicines namely Calcarea Carbonica, Lachesis Mutus, Mercurius Solubilis, Silicea and Thuja occidentalis are seen to have anti- fungal activities in humans as mentioned in Synthesis Repertory 9.0 [8] and Murphy Repertory [9] for fungicidal activities. The above mention Homoeopathic medicines in 6C, 12C, 30C, 200C, 1M, 10M, CM potencies were made available from standard manufacturer which was used in the present research work.

466 IJRAR- International Journal of Research and Analytical Reviews Research Paper [VOLUME 5 I ISSUE 4 I OCT. – DEC. 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 Material and Methods: -

Media and Chemicals - Procurement of all media and chemicals was done from Hi-Media Lab, Mumbai, India. All the chemicals and reagents used are of AR grade and procured from MERK India. Homoeopathic Medicines- Homoeopathic medicines such as Calcarea Carbonica, Lachesis Mutus, Mercurius Solubilis, Silicea and Thuja occidentalis in 6C, 12C, 30C, 200C, 1M, 10M and CM liquid potencies were bought from GMP approved standard Homoeopathic manufacturing unit. Control- Acetylsalicylic acid used as chemical control [10] and ethanol (Dispensing alcohol 90%) used as vehicle control. [11] Distilled water and PDB is used as negative control. [12] Organism- Ashbya gossypii (NCIM-1193) was procured from National Collection of Industrial Microorganisms (NCIM), Pune in the form of active slants and kept it in the refrigerator for further use. For all studies, culture was grown in potato dextrose broth with pH 6.5 and incubated at 25ºC for 24 hrs. [10]

Preparation of media- Potato dextrose agar pH 6.5 (with the help of 1 molar NaOH and 1 molar HCl) medium was prepared and adjusted for antifungal studies and autoclaved it at 15 PSI for 121ºC for 15 minutes.

Slide culture technique - Potato dextrose agar of 6.5 pH is prepared and autoclaved. Afterwards streaking was done with loop. Cover slip inserted into a streaking area of petri plate and incubated at 25ºC for 24 hrs. After 24 hrs. of incubation petri plate was removed from incubator and kept under laminar flow for further staining procedure. Cover slip was draw from petri plate and kept on glass slide. After staining of cover slip with lacto - phenol cotton blue is kept under a compound microscope for observing the morphology (spores) of Ashbya gossypii.

Anti-fungal assay - Anti-fungal activity of the selected Homoeopathic medicines determined by using agar well diffusion assay. [12, 13, 14] Accordingly, culture suspension (100 µl) was spread on potato dextrose agar plates using spreader and incubated at 25ºC for 30 min. Various potencies of selected Homoeopathic medicines (20 µl) was added in wells made by 5 mm borer. It was followed by incubation at 25ºC for 48 hrs. Acetylsalicylic Acid (0.1mg, 100/ml) was added as the chemical control. [10] Anti-fungal activity was scored by measuring the diameter of the zone of inhibition around the wells.

MIC value - MIC value of the Homoeopathic medicines was found out by using the broth dilution assay. [15] Total volume in each test tube was 1.5ml with Potato Dextrose Broth (0.5ml), varying potencies of Homoeopathic medicines (0.5ml) and Ashbya gossypii culture (0.5ml). The experiment was conducted along with the negative control (Potato dextrose broth), chemical control (Acetylsalicylic acid) and ethanol (Dispensing alcohol 90%) as a vehicle control. [11] MIC value of the fungus was calculated after 24 hours. [10] Inoculum size was standardized 0.5 OD at 600nm for all the experiments.

Results: - 1) Slide culture technique: - Morphology of Ashbya gossypii were observed using slide culture technique and it was observed under 40x and 100 x objective lens. [Figure 1]

Figure 1 - Ashbya gossypii under microscope.

2) Antifungal activity: – Nearby all selected homoeopathic medicines for study shown anti-fungal activity. Effectiveness of Homoeopathic medicines was seen by observing zone of inhibition, which ranges from 0.55 to 1.10 c.m. for different potencies. (Table.1).

Research Paper IJRAR- International Journal of Research and Analytical Reviews 467 [ VOLUME 5 I ISSUE 4 I OCT.– DEC. 2018] E ISSN 2348 –1269, PRINT ISSN 2349-5138

Figure 2 – Zone of inhibition against Ashbya gossypii with various Homoeopathic medicines.

Table 1:Antifungal Activity of Homoeopathic medicines Zone of inhibition Name of Homoeopathic Potency against Ashbya gossypii Medicines (Mean ± Standard deviation) in c.m. Calcarea Carbonica 6C 0.95± 0.07 12C 1.05±.0.07 30C 1.05±0.07 200C 0.90±0.00 1M 1.05±0.21 10M 0.80±0.14 CM 0.70±0.14 Lachesis Mutus 6C 0.80±0.00 12C 0.65±0.07 30C 0.70±0.14 200C 0.75±0.07 1M 0.70±0.00 10M 0.85±0.07 CM 0.75±0.07 Mercurius Solubilis 6C 0.70±0.00 12C 0.90±0.14 30C 1.05±0.07 200C 0.65±0.07 1M 0.65±0.07 10M 0.75±0.07 CM 0.95±0.21 Silicea 6C 0.75±0.07 12C 0.55±0.07 30C 0.85±0.07 200C 0.70±0.14 1M 0.90±0.14 10M 0.75±0.07 CM 0.75±0.07 Thuja occidentalis 6C 0.65±0.07 12C 0.70±0.00 30C 0.85±0.21 200C 0.85±0.07 1M 1.10±0.14 10M 0.95±0.07 CM 0.85±0.07 Acetylsalicylic Acid 10 µg/ml 1.10±0.28 Alcohol (Dispensing) 90% 0.65±0.07 468 IJRAR- International Journal of Research and Analytical Reviews Research Paper [VOLUME 5 I ISSUE 4 I OCT. – DEC. 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 Distilled Water -- 0.50±0.00 Blank -- 0.50±0.00

3) MIC value: - The MIC of the Homoeopathic medicine varied from 6C to CM potencies as given in the (Table 2). Table 2. MIC of the Homoeopathic medicines MIC Sr. No. Homoeopathic Medicine (Homoeopathic Potency) 1 Calcarea Carbonica CM 2 Lachesis Mutus CM 3 Mercurius Solubilis CM 4 Silicea CM 5 Thuja occidentalis CM

Discussion: - There is very few research study performed against Ashbya gossypii with Homoeopathic medicines as far as my literature survey. In present research work Homoeopathic medicines showed an inhibitory effect against Ashbya gossypii. Ashbya gossypii affect to the cotton ball by developing hair cell in it, then it can transfer to citrus fruit causes dry rot disease. In this cotton, fruit dried and collapse, [7] where as this study may effective against various plant diseases. All this process carried out with the help of hemipteran vector so here Fungicides do not control stigmatomycosis but insecticides decrease hemipteran vector populations and the incidence of stigmatomycosis. The present study shows the effectiveness of Homoeopathic medicines in controlling the Ashbya gossypii in - vitro. The purpose of this study was to determine antifungal activity of Homoeopathic medicines for inhibition of Ashbya gossypii. The outcome of these in - vitro studies are very much favorable and have shown that the majority of the Homoeopathic drugs have specific inhibitory activity against Ashbya gossypii selected on the basis of Synthesis Repertory [8] and Murphy repertory. [9] Modern fungicides being known to cause many side - effects in cotton plant such as development of brown spots over leaves with burnt foliage [16] where as Homoeopathic medicines are known for no side effects. That’s why treating with Homoeopathic medicines will be an effective choice for a healthy plant cycle. Further studies may discover a new outlook even in the resistant strain treatment as well. Homoeopathic treatment is useful, cost effective and preventive mode of treatment in the agriculture field. Homoeopathy has always been termed as placebo therapy, but the results of this experiment prove that the ultra diluted medicines are effective in in-vitro fungal testing as evidence based medicine.

Conclusion: - Now a day Agro - Homoeopathy is a new technique to treat various types of diseases found in agricultural field with Homoeopathic medicines. To evaluate the results achieved in present study support the concept of the ‘evidence based medicine’, clearly represent that Homoeopathic medicines have specific inhibitory activity against Ashbya gossypii. All the Homoeopathic medicines are found to have inhibitory activity viz. Calcarea Carbonica 12C (1.05 ± 0.07), 30C (1.05 ± 0.07) and 1M (1.05 ± 0.21), Mercurius Solubilis 30C (1.05 ± 0.07), Thuja occidentalis 1M (1.10 ± 0.14) showing the best inhibitory activity and effective against Ashbya gossypii to treat stigmatomycosis diseases in cotton plants. Further study is required field trials to check activity against Ashbya gossypii and identifying the mechanism of action of Homoeopathic medicines.

Acknowledgement: - The author wishes to thank B.V.D.U.H.M.C. and B.V.D.U.R.G.I.T.B.T. Pune - 411043 for providing Laboratory Facilities and kind support. Author also extends thanks to Dr. Mrs. Anita S. Patil, PG Coordinator and Dr. Mrs. Manisha P. Gajendragadkar Head of Department, Homoeopathic Pharmacy, B.V.D.U.H.M.C., Pune – 411043.

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