Vol. 18(13), pp. 275-279, 27 March, 2019
Article Number: 190043D60561
Author(s) retain the copyright of this article
African Journal of Biotechnology
Ultra high dilutions: A review on in vitro studies
Aditya Dilipkumar Patil, Anuj Deepakrao Chinche, Atul Kumar Singh, Sana Parvej Peerzada,
Snehal Ashok Barkund, Jay Nilesh Shah and Arun Bhargav Jadhav*
Department of Homoeopathic Pharmacy, Homoeopathic Medical College and Hospital, Bharati Vidyapeeth (Deemed to
be University), Katraj, Pune – 411043, Maharashtra, India.
Received 20 November 2018; Accepted 13 March, 2019
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.
Key words: Homeopathy, in vitro, manuscript information score, review.
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 × 1023) which
is implausible in interpreting the concept with dose-
dependent model of modern pharmacology (Teixeira and
In vitro studies are aimed to create evidence,
*Corresponding author. E-mail: email@example.com. Tel: (+91) 7387191547.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
276 Afr. J. Biotechnol.
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 (Table no.1 and Table no. 2). This can
lead to a mean development in the field of homeopathy in
form of a new preclinical Homeopathic Materia Medica on
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).
In the study of Chakraborty et al. (2015), Aconite
napellus (6C, 30C, 200C) showed bactericidal activity
against Staphylococcus aureus and Escherichia coli and
also identifed the nano-sized particle through Field
Emission Scanning Electron Microscope of Aconite
(Chakraborty et al., 2015).
Damin et al., (2015) studied homeopathic medicines
like Arsenicum album, Calcarea carbonica, Kali iodatum,
Phosphorus, Silicea, Staphysagria, Spodoptera
frugiperda, Sulphur, and Th. occidentalis against
Metarhizium anisopliae (strain UNIOESTE 22) which
showed all the treatments as compatible in their dilutions
(24CH, 30CH, 100CH; 3CH, 30CH; 6CH, 30CH, 100CH;
30CH, 100CH, 200CH; 200CH, respectively)( Damin et
In Gupta et al. (2015) study, homeopathic medicine
Acid benzoicum, Apis mellifica, K. iodatum, Mezereum,
Petroleum, Sulphur, Tellurium, Sulphur iodatum,
Graphites, Sepia, Silicea and T. occidentalis in 30C and
200C acted as an evidenced based medicine that
conformed both in vitro and in vivo on oral candidiasis.
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
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,
In the study of Prajapati et al. (2017), homeopathic
mother tincture S. jambolanum, F. religiosa, O. sanctum,
A. cepa, T. occidentalis and H. antidysenterica showed
inhibitory action against human pathogenic fungi Candida
albicans (Prajapati et al., 2017).
Passeti et al., (2017) experiment proved that
Belladonna and bacterial nosode in 6CH and 30CH,
Silicea and Hepar sulphur in 6CH, 12CH and 30CH, and
oxacillin showed a significant reduction (p< 0.001) on
Methicillin-resistant S. aureus (Passeti et al., 2017).
Shinde et al. (2018) had conducted two in vitro studies
on both Pityrosporum ovale and C. albicans (NCIM-
3557), regarding fungal culture homeopathic medicine
Selenium, Cinchona officinalis, Azadiracta indica,
Phosphorus, Acidum benzoicum, Zingiber, Sulphur,
Acidum sulphuricum, Iodium, and Zincum metallicum in
6CH, 12CH, 30CH, 200CH, 1M respectively that showed
inhibitory effects in both fungal culture (Shinde et al.,
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
Patil et al. 277
Table 1. Summary of in vitro studies evaluated with Manuscript Information Score (MIS) ≥ 5.
Methods and assay
Asha et al.
Fusarium, Aspergillus flavus,
Cello tape flag method,
Sterile water, rectified spirit,
Q, 30 C, 200 C, 1 M,
10 M and 50 M
against the fungi
Chakraborty et al.
spread plate technique,
Control plates of both the
6C, 30C, 200C
Activity in bacteria in
Silvana Damin et.al.
(strain UNIOESTE 22)
Insecticidal activity, CFU,
Calcarea carbonica, Kali
24C; 30C; 100CH
3CH; 30CH; 6CH,
Compatible in fungus
Girish Gupta et al.
Disc diffusion method.
spirit, distilled water.
Acid benzoicum, Apismellifica,
Kali iodatum, Mezereum,
Petroleum, Sulphur, Tellurium,
Graphites, Sepia, Silicea and
Toledo et al.
sporulation and conidial
Distilled water and
solution with dynamizations
Sulphur, Silicea terra,
sulphuricumand Kali iodatum
6CH and handled to
12, 30 and
Activity in control of
black spot disease in
AsmaHanif et al.
spp. and Macrophomina
Growth inhibition percent
Paper Disc Diffusion
Sterilized distilled water,
Thuja occidentalis and
Activity against non-
Suneel Prajapati et
C.albicans (MTCC No. 3017)
Agar disc diffusion
Ketoconazole, 90% alcohol
sanctum, Alliumcepa, Thuja
Mother tincture (Q)
Silicea, Hepar sulph,
Belladonna and bacterial
6 CH, 12 CH and 30
CH in sterile 30%
Activity different in
different potency of live
Chetan H.shinde et
Anti-dandruff assay, MIC,
Dispensing alcohol, Zinc
Selenium, Cinchona officinalis,
against the fungus
278 Afr. J. Biotechnol.
Table 1 Contd.
Chetan H.Shinde et al.
Candida albicans (NCIM-
Agar diffusion assay,
Germ Tube Inhibition
Vehicle Control (Dispensing
Acidum Sulphuricum, Acidum
indica, Cinchona officinalis,
Selenium, Sulphur, Zincum
Metallicum, Zingiber officinale
6C, 12C, 30 C, 200 C,
Inhibitory effect against
MIC= Minimum Inhibitory Concentration; C or CH= Centesimal Scale (1:99); M=1000CH; NCIM= National collection of Industrial Micro-organism; CFU= Colony Forming Unit; FESEM= Field Emission
Scanning Electron Microscopic; MFC= Minimum Fungicidal Concentration; MTCC= Microbial Type Culture Collection; UNIOESTE= Universidade do Oeste do Parana; MIS= Manuscript Information
Table 2. Manuscript Information Score of the included studies.
Description in the study
Asha et al.
Fusarium, Aspergillus flavus,Bipolaris, Exserohilum, Curvularia
Monalisa Chakraborty et al.
Staphylococcus aureus, Escherichia coli
Silvana Damin et al.
Metarhiziumanisopliae(strain UNIOESTE 22)
Girish Gupta et al.
Márcia Vargas Toledo et al.
AsmaHanif et al.
Rhizoctoniasolani, Fusarium spp. And Macrophominaphaseolina.
Suneel Prajapati et al.
C.albicans(MTCC No. 3017)
TâniaAguiarPasseti et al.
Multi-Resistant Staphylococcus Aures (MRSA)
Chetan H. Shinde et al.
Chetan H. Shinde et al.
Candida albicans (NCIM- 3557)
and in vivo studies in homeopathy.
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.
CONFLICT OF INTERESTS
The authors have not declared any conflict of
The authors appreciate Dr. Manikrao Salunkhe,
Vice Chancellor, Bharati Vidyapeeth (Deemed to
be University), Pune, India; staff of Bharati
Vidyapeeth (Deemed to be University), Pune,
India; staff of Homoeopathic Medical College and
Hospital, Department of Post Graduate and Research
Centre, Pune, India: Dr. Anita S. Patil (P.G. Coordinator),
Dr. Manisha P. Gajendragadkar and Dr. Chetan H.
Shinde, for the support, assistance, guidance and
relentless supervision throughout the course of the
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