Cellular and transcriptional responses of SH-SY5Y human neurocytes following in vitro exposure to Gelsemium sempervirens

Background: Gelsemium sempervirens (Gelsemium s.) is a highly toxic plant but is employed at low doses and/or high dilutions as an anxiolytic and antidepressant. Previous investigations in our laboratory [1,2] have shown a significant anxiolytic-like activity of Gelsemium s., using emotional response models in laboratory mice. Although there is some biochemical evidence of a possible role of neurosteroid metabolism [3], the cellular and molecular mechanisms involved in the effects of Gelsemium s. at the level of nervous system are largely unknown. To help determine these pathways, we used human neurocytes (SH-SY5Y cell line) treated in vitro with different dilutions of Gelsemium s. and evaluated their vitality and gene expression changes. Methods: The drugs were produced by Boiron Laboratoires, Lyon (F), starting from a whole-plant-hydroalcoholic extract of Gelsemium s. Solutions 1C, 2C, 3C, 4C, 8C and 29C (C= centesimal dilution/dynamization prepared in 30% ethanol/distilled water) were provided in 30-ml glass bottles, wrapped in aluminium foil and were stored in the dark at room temperature in a metal cupboard. Control solutions (“placebo”) were serially diluted/dynamized 30% ethanol/distilled water. Before each experiment, 0.05 ml samples of Gelsemium s. and placebo were added to 5 ml of distilled sterile and apyrogenic water in a 15 ml Falcon polystyrene plastic tube, closed and shaken in mechanical shaker DinaA for 7.5 sec (150 strokes) to obtain the final 2C, 3C, 4C, 5C, 9C and 30C succussed dilutions, with ethanol concentration of 0.3% (v/v) (final 0.03% in the assay system). Human neuroblastoma cell line SHSY5Y was grown in DMEM-F12 medium (Lonza), with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 mg/ml). To assess cell viability and metabolism, 20,000 cells per well were seeded in 96 microplate wells in 200 μl of medium. After overnight incubation, 22 μl of drug or placebo were added and the plate was incubated at 37°C with 5% CO2 in a humidified atmosphere for 72 hours. Then the viability test with reagent WST-1 (Roche) was performed for 3 hours and the absorbance was detected with multiplate reader. A total of 17 experiments, each done with six replicate microwells. To make a relative measurement of protein, the cells were lysed and a Bradford assay was done directly in the plate. The Student t-test and the sign rank test for paired data were utilized for data analysis. To obtain a profile of gene expression, cells were pre preconditioned with Gelsemium s./placebo dilutions for 24 h, then RNA was isolated and analysed by microarray and RT-PCR. SHSY5Y cells were plated onto Petri dishes (day 1) and the day after the medium was replaced with the medium with 2% FBS (day 2). After 24h, 10%v/v Gelsemium s. or placebo dilutions were added to the medium (day 3) and cells were incubated for a further 24h. On day 4, cells were then harvested and the RNA extracted using the Qiagen RNAeasy Mini Kit following the manufacturer’s instructions. Microarray analysis was performed on a custom 12 x 135 k human NimbleGen microarray containing 45033 genes with 3 probes per target gene. Four biological replicates were analysed for each condition. Analysis of differentially expressed genes was performed using linear modelling and empirical Bayes methods and p-values were adjusted for multiple testing with the Benjamini and Hochberg method. A Human Neurotransmitter Receptors and Regulators RT2 Profiler PCR array (Qiagen) was performed in profiling the expression of genes involved in modulating the biological processes of neurotransmitter biosynthesis, uptake, transport and signaling through neurotransmitter receptors. Results: In viability tests, cells treated with Gelsemium s. showed slightly higher metabolic activity (3-4 %) than those treated with placebo. Overall comparison of the data for the whole sample of placebo versus that of Gelsemium s. using the Student t-test showed a small but significant difference (p < 0.001). Furthermore, a non parametric approach comparing the two treatments at the same dilution yielded a significant difference under the sign test (p < 0.01) and Wilcoxon rank test for paired data (p < 0.05), so that the values of differences were also considered. The differences between groups having the same dilution (placebo 2C versus Gelsemium s. 2C etc.) were significant in four dilutions: 2 C, 3 C, 4C (p < 0.01), 9 C (p < 0.02), while 5 C and 30 C yielded non-significant values. No changes due to Gelsemium s. were detected using protein assay, suggesting that the viability test revealed effects on metabolic activity instead of on cell proliferation. In microarray analysis, transcripts expression was analyzed and genes differentially expressed by the Gelsemium s. dilutions were selected. A gene was considered to be differentially expressed if it showed an absolute value of log-ratio greater than or equal to 0.5, an index that translates to a fold-change of 1.4 in transcript quantity. Out of a total of 45,033 transcripts, exposure to Gelsemium s. 2C promoted the selective downexpression of 49 genes (p values adj

Homeopathic Medicines Used as Prophylaxis in Kolkata during the COVID-19 Pandemic: A Community-Based, Cluster-Randomized Trial

Introduction There is some evidence that homeopathic treatment has been used successfully in previous epidemics, and currently some countries are testing homeoprophylaxis for the coronavirus disease 2019 (COVID-19) pandemic. There is a strong tradition of homeopathic treatment in India: therefore, we decided to compare three different homeopathic medicines against placebo in prevention of COVID-19 infections. Methods In this double-blind, cluster-randomized, placebo-controlled, four parallel arms, community-based, clinical trial, a 20,000-person sample of the population residing in Ward Number 57 of the Tangra area, Kolkata, was randomized in a 1:1:1:1 ratio of clusters to receive one of three homeopathic medicines (Bryonia alba 30cH, Gelsemium sempervirens 30cH, Phosphorus 30cH) or identical-looking placebo, for 3 (children) or 6 (adults) days. All the participants, who were aged 5 to 75 years, received ascorbic acid (vitamin C) tablets of 500 mg, once per day for 6 days. In addition, instructions on healthy diet and general hygienic measures, including hand washing, social distancing and proper use of mask and gloves, were given to all the participants. Results No new confirmed COVID-19 cases were diagnosed in the target population during the follow-up timeframe of 1 month—December 20, 2020 to January 19, 2021—thus making the trial inconclusive. The Phosphorus group had the least exposure to COVID-19 compared with the other groups. In comparison with placebo, the occurrence of unconfirmed COVID-19 cases was significantly less in the Phosphorus group (week 1: odds ratio [OR], 0.1; 95% confidence interval [CI], 0.06 to 0.16; week 2: OR, 0.004; 95% CI, 0.0002 to 0.06; week 3: OR, 0.007; 95% CI, 0.0004 to 0.11; week 4: OR, 0.009; 95% CI, 0.0006 to 0.14), but not in the Bryonia or Gelsemium groups. Conclusion Overall, the trial was inconclusive. The possible effect exerted by Phosphorus necessitates further investigation. Trial registration: CTRI/2020/11/029265.

Randomized Double-Blind, Placebo-Controlled Feasibility Study, Evaluating the Efficacy of Homeopathic Medicines in the Prevention of COVID-19 in a Quarantined Population

Introduction Exploring preventive therapeutic measures has been among the biggest challenges during the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We explored the feasibility and methods of recruitment, retention, and potential signal of efficacy, of selected homeopathic medicines as preventive measure for developing COVID-19 in a multi-group study. Materials and Methods A six-group, randomized, double-blind, placebo-controlled prophylaxis study was conducted in a COVID-19 exposed population in a quarantine facility in Mumbai, India. Each group received one of the following: Arsenicum album 30c, Bryonia alba 30c, a combination (Arsenicum album 30c, Bryonia alba 30c, Gelsemium sempervirens 30c, and Influenzinum 30c), coronavirus nosode CVN01 30c, Camphora 1M, or placebo. Six pills twice a day were administered for 3 days. The primary outcome measure used was testing recruitment and retention in this quarantined setting. Secondary outcomes were numbers testing positive for COVID-19 after developing symptoms of illness, number of subjects hospitalized, and days to recovery. Results Good rates of recruitment and retention were achieved. Of 4,497 quarantined individuals, 2,343 sought enrollment, with 2,294 enrolled and 2,233 completing the trial (49.7% recruitment, 97.3% retention). Subjects who were randomized to either Bryonia alba or to the CVN01 nosode signaled (p <0.10) a lower incidence of laboratory-confirmed COVID-19 and a shorter period of illness, with evidence of fewer hospitalizations, than those taking placebo. The three other groups did not show signals of efficacy. Conclusion This pilot study supports the feasibility of a larger randomized, double-blind, placebo-controlled trial. Bryonia alba 30c and CVN01 30c should both be explored in disease prevention or shortening the course of disease symptomatology in a COVID-19-exposed population.

Impact of Bias in Data Collection of COVID-19 Cases

Background/Objective Prognostic factor research (PFR), prevalence of symptoms and likelihood ratio (LR) play an important role in identifying prescribing indications of useful homeopathic remedies. It involves meticulous unbiased collection and analysis of data collected during clinical practice. This paper is an attempt to identify causes of bias and suggests ways to mitigate them for improving the accuracy in prescribing for better clinical outcomes and execution of randomized controlled studies. Methods A prospective, open label, observational study was performed from April 2020 to December 2020 at two COVID Health Centers. A custom-made Excel spreadsheet containing 71 fields covering a spectrum of COVID-19 symptoms was shared with doctors for regular reporting. Cases suitable for PFR were selected. LR was calculated for commonly occurring symptoms. Outlier values with LR ≥5 were identified and variance of LRs was calculated. Results Out of 1,889 treated cases of confirmed COVID-19, 1,445 cases were selected for pre-specified reasons. Nine medicines, Arsenicum album, Bryonia alba, Gelsemium sempervirens, Pulsatilla nigricans, Hepar sulphuricus, Magnesia muriaticum, Phosphorus, Nux vomica and Belladonna, were most frequently prescribed. Outlier values and large variance for Hepar sulphuricus and Magnesia muriaticum were noticed as indication of bias. Confirmation bias leading to lowering of symptom threshold, keynote prescribing, and deficiency in checking of all symptoms in each case were identified as the most important sources of bias. Conclusion Careful identification of biases and remedial steps such as training of doctors, regular monitoring of data, checking of all pre-defined symptoms, and multicenter data collection are important steps to mitigate biases.

Homeopathic Remedies in COVID-19: Prognostic Factor Research

Background/Objective Coronavirus disease 2019 (COVID-19) is a new disease; its clinical profile and natural history are evolving. Each well-recorded case in homeopathic practice is important for deciding the future course of action. This study aims at identifying clinically useful homeopathic remedies and their prescribing symptoms using the prognostic factor research model. Methods This was an open-label, multi-centric, observational study performed from April 2020 to July 2020 at various public health care clinics. The data were collected prospectively from clinical practice at integrated COVID-19 care facilities in India. Good-quality cases were selected using a specific set of criteria. These cases were analyzed for elucidating prognostic factors by calculating the likelihood ratio (LR) of each frequently occurring symptom. The symptoms with high LR values (>1) were considered as prescribing indications of the specific remedy. Results Out of 327 COVID-19 cases reported, 211 met the selection criteria for analysis. The most common complaints were fatigue, sore throat, dry cough, myalgia, fever, dry mouth and throat, increased thirst, headache, decreased appetite, anxiety, and altered taste. Twenty-seven remedies were prescribed and four of them—Arsenicum album, Bryonia alba, Gelsemium sempervirens, and Pulsatilla nigricans—were the most frequently used. A high LR was obtained for certain symptoms, which enabled differentiation between the remedies for a given patient. Conclusion Homeopathic medicines were associated with improvement in symptoms of COVID-19 cases. Characteristic symptoms of four frequently indicated remedies have been identified using prognostic factor research, findings that can contribute to accurate homeopathic prescribing during future controlled research in COVID-19.

Updates from China: clinical and research experience of homeopathy in the pandemic

Background: Hong Kong is one of the regions which is most affected by the COVID-19 pandemic in China. In homeopathy, common pathognomonic symptoms are less informative, while homeopathic symptoms are always more helpful for homeopathic prescriptions. Aims: The first case series from Hong Kong was presented and the homeopathic repertorization symptoms observed in the 18 confirmed/suspected cases of COVID-19 in Hong Kong in early 2020 was reported. Methodology: A standard questionnaire on common homeopathic symptoms was filled in by all diagnosed/suspected COVID-19 patients, with or without the help of a consulting-room assistant, before the consultation. Repertorization symptoms from the case series were reported from 18 consecutive patients who used homeopathy as an adjunctive measure, in addition to the conventional or traditional chinese medicine, for symptomatic relief from COVID-19. Anonymized clinical information for research and academic purposes was used and approved by patients. The authors focused mainly on the homeopathic symptoms, i.e. the more individualised and characteristic symptoms which were available in the homeopathic repertories. In the presentation, the demographics, prevelance of homeopathic medicines, common repertorization symptoms in each of the top-ranked remedies, and differentiating symptoms for each top-ranked remedy were presented. Differentiating features were defined as homeopathic symptoms that were present in at least 50% of cases for the top-ranked remedy and in no more than 2 cases for other top-ranked remedies. The data collection project was further extended under the Clificol COVID-19 Support Project, a multinational, combined retrospective, and prospective longitudinal study. Results and discussion: Homeopathic symptoms of 18 cases were presented. All of the cases were categorized as mild. Eighteen common symptoms screened out of 79 selected repertorization symptoms constituted two sets of homeopathic symptom pictures: Bryonia alba (n = 4) and Gelsemium sempervirens (n = 12). Eight and seven differentiating features, respectively, were identified for Bryonia alba and Gelsemium sempervirens. Most updated data from the ongoing COVID-19 Support project was also presented. Conclusion: The common symptoms of 18 mild COVID-19 cases constituted two sets of homeopathic symptom pictures, indicating Bryonia alba or Gelsemium sempervirens; they were indicated in 4 and 12 cases, respectively, out of the 18 in total.

Survey COVID-19 first peak 2020

Background: In Belgium, the first peak of COVID-19 was observed between March and April 2020. This represented more than 300 deaths a day beginning April and more than 1500 new cases every day during this period. Aims: This retrospective observational survey of the practice of Homeopathic Medical Doctors in Belgium has been conducted to check Belgian Homeopathic Doctors (HMD) have treated their patients presenting mild symptoms of COVID-19. Methodology: 6 HMDs accepted to provide data from all consecutive cases treated for COVID-19 symptoms in March and April 2020. This systematic retrospective data collection was compared with information from a free exchange HMDs’ forum. 13 HMDs regularly participated in this forum. Results and discussion: this survey included 313 patients presenting mild COVID-19 symptoms. The efficiency score (healing of symptoms in less than eight days) is relevant for 83,1% of these patients. The average duration of COVID-19 symptoms is 20 days. The four most used homeopathic medicines (Bryonia alba/Arsenicum album/Phosphorus/Gelsemium sempervirens) were prescribed to 66% of them. 1,3% needed hospitalization for some time. Not any loss of chance to cure occurred for patients that used homeopathy for COVID-19 symptoms. Market data also reveals that homeopathy has been largely used to prevent and heal during the early peak of COVID-19 in Belgium. This survey allows us to conclude that during such an epidemic situation for which no effective therapy exists and no vaccination is possible, homeopathy should be considered a valuable approach.

HOMEOPATIA NO TRATAMENTO DA ANSIEDADE

A ansiedade é frequentemente relatada por pacientes que buscam tratamentos alternativos ou complementares, como a Homeopatia, por exemplo. A matéria médica compreende grande variedade de medicamentos homeopáticos e evidenciou o uso em pessoas sadias. Contudo, os estudos clínicos carecem de informações sobre os efeitos alcançados. O extrato da planta Ginkgo biloba mostrou efeitos ansiolíticos em animais roedores, assim como Gelsemium sempervirens, Ignatia amara, Achilea milefolium e outras substâncias de origem vegetal. A busca pelo medicamento individual é um diferencial do tratamento homeopático, no intuito de encontrar o Simillimum ideal para o paciente que sofre ao seu modo determinado sintoma. Este projeto se propõe a levantar evidências sobre o uso da Homeopatia no tratamento de transtornos de ansiedade por meio de casos clínicos no intuito de contribuir para o uso de medicamentos homeopáticos na melhora da saúde dos indivíduos.

Clinical Characteristics and Remedy Profiles of Patients with COVID-19: A Retrospective Cohort Study

Objective The aim of the study was to identify indicated homeopathic remedies based on the clinical characteristics of coronavirus disease 2019 (COVID-19) patients in India. Methods In this retrospective, cohort study, confirmed COVID-19 patients admitted at a COVID Health Centre in New Delhi between April 29 and June 17, 2020 were given conventional and homeopathic treatment. Patients were grouped into mild, moderate or severe categories of disease. Their symptomatologic profiles were analyzed to identify indicated homeopathic medicines. Results A total of 196 COVID-19 patients were admitted. One hundred and seventy-eight patients had mild symptoms; eighteen patients had moderate symptoms; no patients with severe symptoms were included as they were referred to tertiary care centers with ventilatory support. The mean age of patients with mild symptoms was significantly lower (38.6 years; standard deviation or SD ± 15.8) compared with patients in the moderate category (66.0 years; SD ± 9.09). The most important symptoms identified were fever (43.4%), cough (47.4%), sore throat (29.6%), headache (18.4%), myalgia (17.9%), fatigue (16.8%), chest discomfort (13.8%), chills (12.6%), shortness of breath (11.2%) and loss of taste (10.2%). Twenty-eight homeopathic medicines were prescribed, the most frequently indicated being Bryonia alba (33.3%), Arsenicum album (18.1%), Pulsatilla nigricans (13.8%), Nux vomica (8%), Rhus toxicodendron (7.2%) and Gelsemium sempervirens (5.8%), in 30C potency. Conclusion Data from the current study reveal that Arsenicum album, Bryonia alba, Pulsatilla nigricans, Nux vomica, Rhus toxicodendron and Gelsemium sempervirens are the most frequently indicated homeopathic medicines. A randomized controlled clinical trial based on this finding is the next step.