Exploring Homoeopathic Potential Against Typhoid: Baptisia tinctoria, Rhus toxicodendron, and Eupatorium perfoliatum in growth Inhibition of Salmonella typhi – In-vitro study

doi:10.21203/rs.3.rs-4432701/v1

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Exploring Homoeopathic Potential Against Typhoid: Baptisia tinctoria, Rhus toxicodendron, and Eupatorium perfoliatum in growth Inhibition of Salmonella typhi – In-vitro study

https://doi.org/10.21203/rs.3.rs-4432701/v1

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Typhoid fever, caused by Salmonella typhi, is a global health issue with high morbidity and mortality rates, especially in low-income regions. The rise of antibiotic-resistant strains necessitates new treatment strategies. This study explores the potential of homoeopathic medicines, known for their safety and non-induction of resistance, as an alternative treatment.

Three homoeopathic medicines, Rhus toxicodendron, Eupatorium perfoliatum, and Baptisia tinctoria, were tested for their inhibitory effects on S. typhi using the agar disc diffusion method. The medicines, along with a control antibiotic, ceftriaxone, were applied to agar plates with S. typhi cultures.

All tested homoeopathic medicines showed inhibitory effects against S. typhi. Rhus toxicodendron Q and Baptisia tinctoria 200 CH showed significant inhibition zones of 16 mm and 14 mm, respectively. Baptisia tinctoria 30 CH and Eupatorium perfoliatum 30 CH also showed inhibitory effects, while ceftriaxone exhibited the largest inhibition zone.

This study highlights the need for innovative treatments against antibiotic-resistant infections. It confirms the antimicrobial potential of homoeopathic medicines against S. typhi, suggesting a possible solution for this infectious disease. Further research is needed to assess their clinical effectiveness in managing typhoid fever.

Salmonella typhi

antimicrobial activity

agar disc diffusion method

Baptisia tinctoria

Rhus toxicodendron

Eupatorium perfoliatum

Typhoid fever

As per the World Health Organization (WHO), typhoid fever continues to impact between 11 to 20 million individuals annually and is accountable for 128,000 to 161,000 deaths each year. In urban locations of India, the rate of culture-confirmed typhoid fever was between 576 to 1173 cases per 100,000 child-years, whereas in rural areas, it was 35 cases^[2]. When analyzing data from hospitals, the estimated occurrence ranged from 12 to 1622 cases for individuals aged 6 months to 14 years, and from 108 to 970 cases for those aged 15 years and older. The incidence rate for paratyphoid fever was 68 cases per 100,000 child-years.

Typhoid fever, caused by the bacterium Salmonella typhi, is transmitted through person-to-person contact via contaminated food and water ^[8]. This transmission transpires through the fecal-oral route, implying that contaminated feces (and sometimes urine) may infiltrate water or food supplies, subsequently consumed by others causing infection. While infrequent in industrialized nations, it poses a significant threat in certain low-income countries ^{[1, 16]}. The symptoms of typhoid fever range from mild to severe, generally appearing one to three weeks after exposure to the bacteria. These symptoms encompass fever, headache, nausea, constipation or diarrhea, loss of appetite, and the presence of a rose-colored rash on the body. Symptoms of typhoid fever bear resemblance to other prevalent gastrointestinal illnesses. The only definitive method to ascertain a person's affliction with typhoid is by testing their blood or feces for Salmonella typhi. Salmonella typhi exclusively thrives in humans, with no animal reservoir.^[8]

Complications arising from typhoid fever encompass intestinal hemorrhage, intestinal perforation, encephalitis, metastatic abscess, and others. Paratyphoid and Typhoid conjugate vaccines are authorized in India. For the past 60-70 years, antibiotics have remained the sole mode of treatment since the discovery of Salmonella. Consequently, drug-resistant organisms have emerged, homoeopathic medicines do not cause drug resistance even when administered for long time. Hence our proposal aims to assess the In-vitro Efficacy of Homoeopathic Medicine against Salmonella typhi. The homeopathic remedies employed in this study encompass Baptisia tinctoria, Eupatorium perfoliatum, and Rhus toxicodendron in mother tincture, as well as potencies of 30 CH and 200 CH.

OBJECTIVE:

To determine the inhibitory effect of the various homoeopathic drugs against Salmonella typhi confirming the antimicrobial activity of potentized drugs in higher dilutions.

Medicines:^[4]

As per Homoeopathic pharmacopoeia of India, standardised homoeopathic Medicines Baptisia tinctoria, Rhus toxicodendron and Eupatorium perfoliatum in different potencies like Q, 30 CH and 200 CH along with Ethanol of 90% v/v were procured from GMP certified Drug Manufacturing unit of Dr.Willmar Schwabe, India.

Microorganism:

The bacterial culture of S.Typhi (MTCC No: 3231) procured as lyophilized freeze-dried culture strain from the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, was used to evaluate the antimicrobial activity of homoeopathic drugs.

Preparation of Agar medium:^[8]

The agar medium was prepared by dissolving 4.95 g of MacConkey agar in 100 ml of distilled water and dissolved completely with the help of a hot plate. Then the media was sterilised by autoclaving at 15 lb pressure (121⁰C) for 15 minutes. The sterilised agar was transferred into the petri plates (100 mm × 15 mm) and was allowed to solidify. These procedures were completed in laminar air flow under aseptic conditions.

Preparation of bacterial culture:^[8]

The bacterial strain of Salmonella typhi (MTCC NO. 3231), lyophilized freeze-dried culture was aseptically opened in a laminar air flow and the suspension was made as per protocol. 0.5 ml of buffered peptone water was taken in the microfuge tube and freeze-dried culture was transferred into it and mixed well. The mixture was allowed for 20 minutes before it was transferred to solid media. Petri plates containing MacConkey agar medium (HiMedia, Mumbai, India, Catalogue No. M081) was incubated for 24 hours at 37⁰C to give white round colonies against a mild reddish pink background. Incubation period of Salmonella typhi is 24 hours. This culture was kept as Broth culture/stock culture. Bacterial cultures were repeatedly sub cultured using streaking methods and maintained to obtain pure isolation on the MacConkey medium for further drug sensitivity assay.

Identification of Salmonella typhi:^[8]

Catalase Test:^[8]

2 ml of hydrogen peroxide solution was taken into the test tube and by using a glass rod, samples of several colonies from the Broth Culture were immersed into the hydrogen peroxide solution. Formation of an immediate bubble confirmed the presence of Salmonella typhi.

Preparation of disc for antimicrobial assay:

Agar disc diffusion method was adopted for determining the antimicrobial activity of different homoeopathic medicines in various potencies. Whatman filter paper No.1 of size approximately 6 mm in diameter was used to prepare the standard discs and autoclaved at 121⁰C at 15 lbs for 15 minutes. These discs were soaked in Baptisia tinctoria, Rhus toxicodendron, Eupatorium perfoliatum under different potencies like Q, 30CH and 200CH, Ethanol of 90% v/v and Ceftriaxone for 30 to 45 minutes. after which they were taken out and allowed to dry under aseptic conditions.

Determination of Antimicrobial Activity:^{[7, 8, 19]}

The antimicrobial activity of the selected homoeopathic medicines under different potencies were evaluated using the agar disc diffusion method by measuring the diameter of growth inhibition zones. Ceftriazone was used as a positive control and Ethanol of 90% v/v was used as a negative control. The discs were inverted and placed in the petri well plates in an incubator set to 37⁰C. After 24-hour growth, an inhibition zone around the discs was measured. The diameter of the growth inhibition zone around the discs was measured using a scale. Each petri well plate was divided into five compartments. Three compartments were allocated for different homoeopathic potencies and one for vehicle Ethanol (V) and one for Antibiotic (AB).

All the three homoeopathic medicine in various potencies exhibited the inhibitory action against Salmonella typhi (Table 1 and Figure 2). Among these, Rhus toxicodendron Q and Baptisia tinctoria 200 CH exhibited maximum inhibition zone of diameter of 16 mm and 14 mm respectively followed by Baptisia tinctoria 30 CH and Eupatorium perfoliatum 30 CH showing the least zone of inhibition of 11 mm and 10.9 mm respectively. However the antibiotic positive control ceftriaxone showed the maximum zone of inhibition.

Table 1: Zone of Inhibition of Homoeopathic Medicines in different potencies

Drugs	Mother Tincture	30 CH	200 CH	Control (Ceftriaxone)	Vehicle (Ethanol 90% v/v)
Baptisia tinctoria	12 mm	11 mm	14 mm	12 mm	7 mm
Rhus toxicodendron	16 mm	12 mm	13 mm	19 mm	7.1 mm
Eupatorium perfoliatum	13 mm	10.9 mm	12 mm	18 mm	6.9 mm

From the sources of various homoeopathic Materia Medica, Baptisia tinctoria in low dilutions produces a form of antibodies to bac.typhosus, viz, the agglutinins thus it raises the bodily natural resistances to the invasion of the bacillary intoxication, which produces the typhoid syndrome^[4]. Rhus toxicodendron indicated therapeutically for typhoid fever^[4]. From the sources of drug proving and clinical verification by pioneers the antibacterial activity has been recorded even before the antimicrobial era. This study reiterates the fact and scientifically confirms the antimicrobial activity of Baptisia tinctoria and Rhus toxicodendron. Eupatorium perfoliatum is a well-known antiviral remedy^[4], however it exhibits antimicrobial activities.

In recent days, repeated use of antibiotics in frequent intervals has caused drug resistance in patients, which poses a serious threat to the community. This resistance has pushed them to discover new generations of antibiotics year after year. Homoeopathic medicines do not cause any resistance and do not produce undesirable side-effects as it is being dispensed from low attenuation to ultra-high dilutions since the Hahnemannian era onwards. This evidence based in-vitro study contributes to the scientific and literature fact that homoeopathic medicine has definite antimicrobial activity against Salmonella typhi.

CONFLICT OF INTEREST:

None

Author Contribution

Dr.K.Narayanasamy - Conceptualization, Material method, Manuscript preparation Manuscript editing, software ,Manuscript review.Dr.P. Bhuvanesh of intellectual Concepts, investigation Design, content, Manuscript reviewG. Sridevi-content ,writing, material and methods, Supervision Senthilkumaran.G - Manuscript review, Result, Software JP.Jerin- discussion,Manuscript review, Manuscript editing

Acknowledgement

We thank our colleagues at The Tamilnadu Dr. MGR Medical University, Tamilnadu, India, for their support, and the reviewers of BMC Microbiology for their valuable feedback.

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Exploring Homoeopathic Potential Against Typhoid: Baptisia tinctoria, Rhus toxicodendron, and Eupatorium perfoliatum in growth Inhibition of Salmonella typhi – In-vitro study

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INTRODUCTION

MATERIALS AND METHODOLOGY

RESULTS

DISCUSSION

CONCLUSION

Declarations

CONFLICT OF INTEREST:

Author Contribution

Acknowledgement

References

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