Ultra high dilutions : A review on in vitro studies against pathogens

Multiple in vitro studies using homeopathic medicines are conducted for their effectiveness against various microbes. The reporting of results and the methodology in many studies are a query. The present review reveals the evidence based medicinal effects of homeopathic medicines on various plant and human pathogens in vitro with the help of quality studies. The studies showed positive outcome for homeopathic medicines. Thus, homeopathy is an effective agent in in vitro studies. However, substantial evidence on these serially diluted medicines must be replicated with the help of a standardized methodology for more precise evidences and conclusion.


INTRODUCTION
Homeopathy is a bicentennial system of medicine founded by Samuel Hahnemann (1755-1843), based on principle of "Similia Similibus Curentur", which was revealed after repeated human experimentation and is currently used by approximately 500 million consumers (Manchanda, 2018).Hahnemann put forth the theory of "vital force" which believed that the succussed medicine shows medicinal effects, even beyond the Avogadro's constant unit, which turns homeopathy as a science of quantum mechanics following the principle of quantum field theory (Khuda-Bukhsh, 2003).Homeopathy has been always challenged for its high dilution properties, clinical methodologies and its mechanism of action (Manchanda, 2018).The similia principle and the dynamization phenomena of the homeopathic medicines, in these recent years, are implemented in preclinical studies (in vitro and in vivo), in testing the mechanism of these highly diluted medicines in various models of biological system (Bellavite et al., 2006;Clausen et al., 2011).The concentrations of these serial diluted medicines are found to be less than 1 g molecule surpassing the Avogadro's Constant (6.024 × 10

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) which is implausible in interpreting the concept with dosedependent model of modern pharmacology (Teixeira and Carneiro, 2017).
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License understand the mechanism and validate the role of certain medicine against a particular condition.Of late, there have been various in vitro experiments performed on plant, animal and human cellular models in plausibly explaining the action of these ultra-high diluted medicines (Manchanda, 2018).Despite the multiple studies, the reporting of results and the methodology are a query.Homeopathic Materia Medica lacks in understanding the principle of "Similia Similibus Curentur" in specific species of pathogens in their preclinical experiments (Teixeira and Carneiro, 2017).The present review reveals the evidence based medicinal effects of homeopathic medicines on various pathogens in vitro with the help of quality studies (Tables 1 and 2).This can lead to a mean development in the field of homeopathy in form of a new preclinicalHomeopathic Materia Medica on pathogens.

IN VITRO EXPERIMENTS AND HOMEOPATHY
According to Asha et al. (2014), Thuja occidentalis (Q, 30 C, 1 M, 10 M and 50 M) had a significant inhibition among all the 5 fungal genera species of Fusarium, Aspergillus flavus, Bipolaris, Exserohilum and Curvularia; however, exact mechanism of action of Thuja is unknown and can be future perspective of research in this fungal cultures (Asha et al., 2014).
The Toledo et al. ( 2016) study revealed the fungi toxicity action of homeopathic medicine against black rot disease of tomato crops.Sulphur and Staphysagria 100CH showed suppressive activity as compared to both controls in mycelium growth, even when successed distilled water at 60CH and 100CH inhibited mycelium growth.Propolis 6CH, 30CH and 60CH and Ferrum sulphuricum 6CH and 30CH caused inhibition and differed from both controls in sporulations.Also, spores germination of the pathogen was found to be reduced by Isotherapic of A. solani in 6CH, Isotherapic of ash in 6CH and Ferrum sulphuricumin 30CH medicines (Marcia et al., 2016).
According to the study of Hanif and Dawar (2016), both in vitro and in vivo experiment showed fungicidal potentials of homeopathic medicines of T. occidentalis and Arnica montana in globules 30CH against root rot disease in non-leguminous plants (Hanif and Dawar, 2016).

EXPERIMENTATION PERSPECTIVE
The above mentioned studies elaborate on scope of homeopathic medicine in the era of drug resistance to various fungal and bacterial cultures.Homoeopathic treatment can be used as an alternative therapy, as cost effective, with no adverse event observed.The experimental methodologies used by the included studies were quite different from each other; needs to be standardized and must be modified by the European Committee on Anti-microbial Susceptibility Testing (EUCAST) and Clinical Laboratory Standards (CLS) guidelines (Hombach et al., 2011) in performing experiments, specially designed with ultra-high dilution, in order to get more precise and accurate results.Also, it must be replicated in in vivo studies.In recent times, these serial diluted medicines have been proved to show the presence of nanoparticles in size of quantum dots and should be seriously taken into consideration about their nano-pharmacological aspects (Chikramane et al., 2010).Various hypothetical models have been put forth in understanding the mechanism of action of these nano medicines and attempts have been made with various molecular studies in identifying the mechanism of action of these medicines (Khuda-Bukhsh , 2003); however, a standard protocol still remains, which is a question of development for the methodologies performed in in vitro

CONCLUSION
This systematic review demonstrates homeopathy as an effective agent, in in vitro studies and can lead to a new development with the help of a new preclinical Homeopathic Materia Medica on pathogens.However, substantial evidence on these serial diluted medicines results must be replicated with a standardized methodology to provide conclusive evidence.

Table 1 .
Summary of in vitro studies evaluated with Manuscript Information Score (MIS) ≥ 5.

Table 2 .
Manuscript Information Score of the included studies.