Protocol Standardization of Homeopathic Medicines' Preparation and Dosing for Experimental Model Systems In Vitro

Applied research in homeopathy has garnered the attention of the research fraternity, and there is a growing interest in investigating the mechanism of action of homeopathic medicines. The in vitro model systems being used rely mainly on isolated cells and cell lines. However, what has been seen from reviews of the published literature is the vast variation in the findings.[1] [2] [3] Treatment of cells reveals a clear role of homeopathic medicines in a majority of the studies, whereas some experiments yield no effects. This has led to controversies on the efficacy of homeopathic medicines at the cellular and molecular levels.[4]

When our group started working using cell-line models, we identified an urgent need for the standardization of the protocol for dosing cells in vitro to get uniform results across all studies. There were various parameters and different principles being followed by each group working in the various laboratories across the world. Some groups serially diluted the ethanolic extract of the medicines and gave 20 succussions during preparation.[5] Other groups gave 10 succussions for their experiments after dilution.[6] In another instance, the experiments were conducted by succussing the medicines just prior to addition to the cells. Therefore, the number and time of succussions varied in all the studies, which might contribute to the variations in the final results. There have been experiments done using 20 µL/mL of medicines[1] and some with 100 µL/mL in 96-well plates.[2] Some researchers have preferred to select the dose after performing the widely used MTT assay, which has been utilized to investigate effects on cell viability and cytotoxicity.[7]

The preparation of medicines and dose selection needs to become standardized so that scientists who wish to perform cell culture studies can follow a uniform protocol in aiming for replication of studies. Hence, the suggested protocol derived from numerous experiments in our own laboratory[8] [9] is outlined as follows.

Firstly, it is important to procure homeopathic medicines from established and reputable pharmaceutical companies rather than preparing them in a laboratory where the conditions will vary: for example, ethanol and water quality; potentization methods (force/manual potentization). This will ensure a uniform method of medicine preparation, at least for a given pharmaceutical company's products. The percentage of the alcohol content in the medicines should be calculated using the principle that less than 0.45% alcohol (v/v) is non-toxic to cells in culture. Furthermore, we need to negate the effect of alcohol in cell toxicity assays: for this, a solvent control should be used in all experiments. To decipher the experimental results, one then needs to confirm that there is no significant difference in the effect between control (media only) and solvent control (succussed ethanol plus media) treated cells. After ruling out that there is no difference between these two groups using cell viability assays, one can carry out the rest of the experiments using solvent control only. A crucial element to consider is the timing of succussion when preparing the solvent control. The homeopathic potencies used in our experiments are manufactured in a pharmaceutical company several days prior to their procurement for the research. Consequently, it is imperative to note that the succussed alcohol or solvent control should also be prepared at least 5 days before an experiment.

Homeopathy requires to undergo robust testing with assays that are used in contemporary science to ensure its existence and its acceptance in the scientific world. But the essence of the philosophy of homeopathic science must also not be compromised. Homeopathy often deals with dilutions beyond Avogadro's constant. Therefore, the preparation of dose as per concentration is not homeopathically sound. Standardization of both preparation and dosing protocol will help in better reproducibility of studies.

Publication History

Received: 01 March 2024

Accepted: 15 March 2024

Article published online:
13 May 2024

• References

• 1 Saha S, Hossain DMS, Mukherjee S. et al. Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit. BMC Complement Altern Med 2013; 13: 230
• 2 Thangapazham RL, Gaddipati JP, Rajeshkumar NV. et al. Homeopathic medicines do not alter growth and gene expression in prostate and breast cancer cells in vitro . Integr Cancer Ther 2006; 5: 356-361
• 3 Tournier A, Klein SD, Würtenberger S, Wolf U, Baumgartner S. Physicochemical investigations of homeopathic preparations: a systematic review and bibliometric analysis—Part 2. J Altern Complement Med 2019; 25: 890-901
• 4 Witt CM, Bluth M, Albrecht H, Weisshuhn TER, Baumgartner S, Willich SN. The in vitro evidence for an effect of high homeopathic potencies—a systematic review of the literature. Complement Ther Med 2007; 15: 128-138
• 5 Olioso D, Marzotto M, Bonafini C, Brizzi M, Bellavite P. Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative real-time PCR. Homeopathy 2016; 105: 131-147
• 6 Valle ACV, Brunel HDSS, Dallago BSL. et al. In-vitro growth kinetics of mesenchymal stem cells in cytotoxicity tests using low-diluted Viscum album . Homeopathy 2023; 112: 40-49
• 7 Mondal J, Panigrahi AK, Khuda-Bukhsh AR. Anticancer potential of Conium maculatum extract against cancer cells in vitro: drug-DNA interaction and its ability to induce apoptosis through ROS generation. Pharmacogn Mag 2014; 10 (Suppl 3) S524-S533
• 8 Arora S, Aggarwal A, Singla P, Jyoti S, Tandon S. Anti-proliferative effects of homeopathic medicines on human kidney, colon and breast cancer cells. Homeopathy 2013; 102: 274-282
• 9 Basu N, Garg M, Tandon C, Das BC, Tandon S. Arsenicum album induces cell cycle arrest and apoptosis, and inhibits epithelial-mesenchymal transition in hormone-dependent MCF7 breast cancer cells. Homeopathy 2023; 112: 160-169