An In Vitro Study to Evaluate the Synergy of Baptisia tinctoria against Salmonella typhi

Abstract

As a consequence of gradual emergence of anti-microbial resistance, the anti-biogram profile of the microbe Salmonella typhi has undergone substantial changes. Patients frequently use homeopathy for the flu and other serious illnesses. Although the use of Baptisia tinctoria (BT) is extensively described in homeopathic literature and in phytomedicine, scientific exploration has never been done. In this study, we have made an effort to understand the anti-typhoidal effect of BT at different concentrations. S. typhi (ATCC 6539) from a freshly prepared stock culture was used in this experiment. We also procured an international strain of Escherichia coli (ATCC 25922 strain) which was taken as control. The antibiotic susceptibility patterns of the isolates were determined by the Kirby-Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines and interpretative criteria (National Committee for Clinical Laboratory Standards 2000) using antibiotic discs (Hi Media Laboratory. Pvt. Ltd., Mumbai, Maharashtra, India), namely cefotaxime (30 μg), ceftazidime (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), streptomycin (10 μg), ampicillin (10 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg) and trimethoprim (5 μg). BT in its 6CH potency showed the greatest growth inhibition zone, not just amidst the homeopathic potency gradient. It has shown the highest growth inhibition zone of 28mm, more than any conventional antibiotic. Ethanolic extract of BT can be considered to combat against array of infections caused by S. typhi species alongside that it can also be considered as adjuvant with conventional antibiotics for better result.

Keywords

Baptisia tinctoria - S. typhi - synergy - anti-microbial resistance - homeopathy

Introduction

Salmonella enterica (sub.sp. typhi) is a gram-negative rod that commonly infects through contaminated food and drinks to cause enteric fever. Globally, 14·3 million (95% uncertainty interval [UI] 12·5–16·3) cases of typhoid and paratyphoid fevers occurred in 2017, a 44·6% (42·2–47·0) decline from 25·9 million (22·0–29·9) in 1990.[1] Morbidity has also come down from 2.35 to 1.36 million (41.6%) from 1990 to 2017.[1] The Southeast Asia alone has 10.3 million cases which are 71.8% of the global burden and Sub-Saharan Africa has 1073 million cases which are 12.1% of the total burden.[1] The Global Burden of Disease Study estimated in 2017 shows that the incidence of typhoid/paratyphoid in India was 586 cases per 1,00,000 person-years. However, these figures were mostly extrapolated from regional data because there have been relatively few population-based studies in India.[1] In addition to the ongoing research gaps in the area, an additional factor that is raising concerns amongst researchers and medical professionals is the rise in the number of multi-drug-resistant (MDR) typhoid illnesses. Salmonella typhi bacteria that are MDR, or resistant to ampicillin, chloramphenicol and cotrimoxazole, have appeared more frequently during the 1990s. Treatment procedures changed to include the use of fluoroquinolones as a result of the rise in MDR S. typhi.[2] [3] [4] [5] Another systematic review that analysed the prevalence of MDR typhoid was published, and it found that the majority (276/384; 72% of the studies) were from South Asia.[6] Aside from a few scattered studies clarifying the anti-typhoid effects of medicinal plants and a study on the anti-typhoid effects of Baptisia tinctoria (BT), we were unable to find any high-quality research articles regarding the use of complementary, and alternative medicine (CAM) in treating typhoid fever.[7] [8] The World Health Organization has released a report that lays out the possibility of CAM to achieve exponential growth over the next 10 years.[9] The increased awareness regarding the drug induced complications due to consumption of modern medicine has further encouraged the use of CAM in different parts of the globe.[10] At least 80% of the world's population receives their basic medical care through the use of homeopathy and ayurveda; two of the oldest and most popular types of CAM.[11] By 2020, the market for herbal supplements and treatments is expected to reach USD115 billion due to consumer demand for natural therapies over the previous 20 years.[12] Another survey reported that the market for homeopathy products is expected to reach around USD 50,203.3 million in value by 2028, from an anticipated value of USD 17,948.5 million in 2021, with a compound annual growth rate of roughly 18.7%.[13] It is widely known that the indiscriminate use of commercially available antibiotics has led to the development of MDR strains and S. typhi is not an exception to that.[14] Moreover, misuse or over use of these drugs has different side effects that affect patients, such as stroke, heart attack, irregular heartbeat, liver toxicity, seizures, psychoses, allergic responses, immune system suppression and mortality.[15] The ever-changing scenario has led to the hunt for new anti-microbial chemicals that are therapeutically more potent with lower toxicity. To evaluate whether BT may be used as an adjuvant to increase the killing effect of the drug in a situation where the efficacy of antibiotic drugs is being questioned, we also tried to understand how various antibiotics would interact with BT extracts to combat S. typhi.

Phytochemical Analysis

The plant BT, commonly known as Wild Indigo, belongs to the Fabaceae family. This is a North American native plant that grows mostly in dry hilly woods from North Carolina to Southern Canada.[16] The active constituents of Baptisia include the poisonous alkaloid, Baptitoxin (also known as Cytisine), otherwise known as Baptisine.[17] [18] [19] [20] It also contains glycosides like Baptisin, glycoproteins and arabinogalactan-proteins (AGPs).[21] [22] [23] Glycoprotein derivatives from Baptisia root demonstrated immunomodulatory activity in vitro by stimulating lymphocyte DNA synthesis.[22] Isolated and purified AGPs from Baptisia root were demonstrated to increase production of immunoglobulin M (IgM; via polyclonal activation of B-lymphocytes), nitrate and IL6 (via activation of macrophages) in vitro.[23] Another study made by Nikishin et al showed that 10% ethanolic extract of BT showed the significant presence of quinolizidine alkaloids mainly cytisine ([Fig. 1]), N-methylcytisine ([Fig. 2]) and the presence of sparteine was also noted in the lesser amount.[24] We think the immune stimulant, antiseptic, anti-parasitic, hepato-protective actions of Baptisia extracts might be due to the effect of these aforementioned compounds.[22] [23] [24] [25] [26] [27] [28] The conventional application of cytisine has been as a partial agonist of α4β2-nicotinic receptors and has been used for antidepressant efficacy.[29] The AGPs extracted from the roots of BT are characterised by the occurrence of terminal Rha(p) and 4-linked Gal(p) residues, which were usually absent in other AGPs.[30] Hydroxyproline is a typical amino acid of AGPs, and the linkage of the carbohydrate moieties to the protein backbone through b-D-Gal (p)-hydroxyproline bonds has been proposed.[23] According to the analysis of Classen et al, it is the location of the small protein moiety (10%) in the centre of AGPs, surrounded by large AG substituents; it seems likely that mainly the carbohydrate part of AGPs is important for activities.[23] Guided by the shreds of evidence here in this experiment we have attempted to find out the synergy between common antibiotics and BT on S. typhi by comparison of growth inhibition zone (GIZ) and to also find out the antibacterial activity by measuring GIZ.

Fig. 1 Cytisine.

Fig. 2 N-Methylcytisine.

[Figs. 1]-[3] show structures of the bio-active compounds of BT.

Fig. 3 Sparteine.

Materials and Methods

Materials:

• Collection of microorganisms: Freshly cultured S. typhi (ATCC 6539) from a stock culture was used in this experiment. This international strain was used for proper interpretation in this experiment. We also procured an international strain of Escherichia coli (ATCC 25922 strain) which was taken as control.

• Collection of medicines: BT in mother tincture, 6CH and 30CH dilutions were directly purchased from a good manufacturing practice (GMP)-certified manufacturer of homeopathic drugs.

• Procurement of antibiotic: All the antibiotic discs used in this experiment were procured from Hi Media Pvt. Ltd, listed in [Table 1].

• Collection of culture media: Mueller Hinton Agar media was procured from Hi Media Pvt. Ltd.

Table 1 List of antibiotics and their concentrations used in the experiments

Name of the antibiotic	Concentration (μg)
Cefotaxime	30
Ceftazidime	30
Ciprofloxacin	5
Tetracycline	30
Chloramphenicol	30
Nalidixic acid	30
Streptomycin	10
Ampicillin	10
Gentamicin	10
Kanamycin	30
Trimethoprim	5

Methods

The antibiotic susceptibility patterns of the isolates were determined by the Kirby-Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and interpretative criteria (National Committee for Clinical Laboratory Standards 2000) using antibiotic discs (Hi Media Laboratory. Pvt. Ltd., Mumbai, India), namely cefotaxime (30 μg), ceftazidime (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), streptomycin (10 μg), ampicillin (10 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg) and trimethoprim (5 μg). Briefly, a well-isolated single colony was inoculated in peptone water and incubated for 4 to 6 hours. The growth was adjusted to match 0.5 MacFarland standards. The adjusted inoculum was applied to dried Mueller Hinton agar. Round-shaped discs of Whatman filter paper no. 41 of 6mm diameter were used after sterilisation in the autoclave at 121°C. About 0.06 mL of BT was given per disc and incubated overnight at 370 °C. A separate experiment was conducted with the anti-microbial disc and incubated overnight at 37°C. Another experiment was conducted with 0.06 mL of BT given per disc to which anti-microbial discs were applied to the inoculated plates and incubated overnight at 370 °C. Measurement of the GIZ was done and tabulated for statistical analysis. Here for the control group 0.06 mL of dispensing alcohol was used which contains concentration of 95% of ethanol. The same experiment was conducted on Escherichia coli (ATCC 25922 strain) as a control in mother tincture, 6CH and 30CH dilutions and with anti-microbial discs of ciprofloxacin (5 μg), chloramphenicol (30 μg) separately as well as in combination with one another. All the experiments were repeated three times for each medicine and potency as well as for controls.

Results

Zone of sensitivity was observed in all the plates ([Figs. 1] [2] [3] [4] [5] [6]). The results, however, varied in all the medicine and their respective potencies as tabulated in [Table 2]. However, [Table 3] shows the combined effect of antibiotics with the homeopathic drug in its potency gradient. As observed from the conducted experiment, BT in its 6CH potency showed the greatest GIZ, not just amidst the homeopathic potency gradient but also when compared against the conventional antibiotics except in a few cases ([Figs. 4] [5] [6] [7] [8] [9]). It has shown the highest GIZ of 28 mm, more than any conventional antibiotic. There is another conclusion that can be drawn from here that the GIZ found individually in antibiotics is less than when antibiotics were given in combination with BT. For example, trimethoprim showed a GIZ of 24.6666 when individually tested but gave the highest GIZ in combination with BT which was 24.666 with the mother, 26 with the 6CH potency and 27.3333 with 30CH which is the highest found in this particular study. Details of the results obtained, and the statistical interpretation are shown in [Tables 4] to [16], [Graphs 1] to [9], [Figs. 4] to [9]. The control trial conducted with Escherichia coli showed the highest GIZ in 6 CH and a poorer synergistic effect of the combination of antibiotics with BT ([Figs. 8], [9]). The results are shown in [Tables 17] to [20] and [Graph 5] to [6].

Fig. 4 C₃₀ -GIZ of chloramphenicol. GEN₁₀-GIZ of gentamicin. B_GEN ¹⁰-GIZ of Baptisia tinctoria mother tincture + gentamicin. 6_GEN ¹⁰-GIZ of Baptisia tinctoria 6CH + gentamicin. 30_GEN ¹⁰-GIZ of Baptisia tinctoria 30CH + gentamicin. B_NA ³⁰-GIZ of Baptisia tinctoria mother tincture + nalidixic acid. 6_NA ³⁰-GIZ of Baptisia tinctoria 6CH + nalidixic acid. 30_NA ³⁰-GIZ of Baptisia tinctoria 6CH + nalidixic acid. GIZ, growth inhibition zone.

Fig 5 A - GIZ of ethanol. AMP₁₀-GIZ of ampicillin. CIP₅-GIZ of ciprofloxacin. B_AMP ¹⁰-GIZ of Baptisia tinctoria mother tincture + ampicillin. 6_AMP ¹⁰-GIZ of Baptisia tinctoria 6CH + ampicillin. 30_AMP ²⁵-GIZ of GIZ of Baptisia tinctoria 30CH + ampicillin. B_CIP ¹⁰-GIZ of Baptisia tinctoria mother tincture + ciprofloxacin. 6_CIP ¹⁰-GIZ of Baptisia tinctoria 6CH + ciprofloxacin. 30_CIP ²⁵-GIZ of Baptisia tinctoria 30CH + ciprofloxacin. GIZ, growth inhibition zone.

Fig. 6 (B)-GIZ of Baptisia tinctoria mother tincture. B₆ -GIZ of Baptisia tinctoria 6CH. B₃₀ -GIZ of Baptisia tinctoria 30CH. B_C ³⁰ -GIZ of Baptisia tinctoria mother tincture + chloramphenicol. S₂₅ -GIZ of streptomycin. B_S ²⁵ -GIZ of Baptisia tinctoria mother tincture + streptomycin. 6_S ²⁵ -GIZ of Baptisia tinctoria 6CH + streptomycin. 30_S ²⁵ -GIZ of GIZ of Baptisia tinctoria 30CH + streptomycin. GIZ, growth inhibition zone.

Fig. 7 C₃₀ -GIZ of chloramphenicol. B-GIZ of Baptisia tinctoria mother tincture. B₆ -GIZ of Baptisia tinctoria 6CH. B₃₀-GIZ of Baptisia tinctoria 30CH. S₂₅-GIZ streptomycin. B_S ²⁵-GIZ of Baptisia tinctoria mother tincture + streptomycin. 6_S ²⁵-GIZ of Baptisia tinctoria 6CH + streptomycin. 30_S ²⁵-GIZ of Baptisia tinctoria 30CH + streptomycin. B_C ¹⁰-GIZ of Baptisia tinctoria mother tincture + chloramphenicol. 6_C ¹⁰ -GIZ of Baptisia tinctoria 6CH + chloramphenicol. 30_C ¹⁰ -GIZ of Baptisia tinctoria 30CH + chloramphenicol. GIZ, growth inhibition zone.

Fig. 8 (B)- GIZ Baptisia tinctoria mother tincture. B₆ -GIZ Baptisia tinctoria 6CH. B₃₀ -GIZ Baptisia tinctoria 30CH. A-GIZ of ethanol. C₃₀ -GIZ of chloramphenicol. B_C ³⁰ -GIZ of Baptisia tinctoria mother tincture + chloramphenicol. 6_C ³⁰ -GIZ of Baptisia tinctoria 6CH + chloramphenicol. 30_C ³⁰-GIZ of Baptisia tinctoria 30CH + chloramphenicol. GIZ, growth inhibition zone.

Fig. 9 B- GIZ Baptisia tinctoria mother tincture. B₆ -GIZ Baptisia tinctoria 6CH. B₃₀ -GIZ Baptisia tinctoria 30CH. A-GIZ of ethanol. CIP₅ -GIZ of ciprofloxacin. B_CIP ⁵ -GIZ of Baptisia tinctoria mother tincture + ciprofloxacin. 6_CIP ⁵ -GIZ of Baptisia tinctoria 6CH + ciprofloxacin. 30_CIP ⁵-GIZ of Baptisia tinctoria 30CH + ciprofloxacin. GIZ, growth inhibition zone.

Graph 1 GIZ of Baptisia tinctoria. GIZ, growth inhibition zone.

Graph 2 GIZ of antibiotics. GIZ, growth inhibition zone.

Graph 3 GIZ in combination of antibiotics and Baptisia tinctoria (BT). GIZ, growth inhibition zone.

Graph 4 Synergistic effect of antibiotics with Baptisia tinctoria. GIZ, growth inhibition zone.

Graph 5 SD of GIZ in individual reagent. GIZ, growth inhibition zone; SD, standard deviation.

Graph 6 SD of GIZ in combination of reagent. GIZ, growth inhibition zone; SD, standard deviation.

Graph 7 GIZ of control. GIZ, growth inhibition zone.

Graph 8 Synergistic effect of antibiotics with Baptisia tinctoria of control. GIZ, growth inhibition zone.

Graph 9 SD of control. GIZ, growth inhibition zone; SD, standard deviation.

Table 2 GIZ of antibiotics against Salmonella typh i

Sl. no.	Reagent	Trial no.	GIZ (mm)	Mean ± SD
1.	Baptisia tinctoria mother tincture	I	25	25.3333 ± 1.5275
		II	24
		III	27
2.	Baptisia tinctoria 6CH	I	28	28 ± 1
		II	27
		III	26
3.	Baptisia tinctoria 30CH	I	26	24.6666 ± 1.1547
		II	24
		III	24
4.	Cefotaxime	I	23	24 ± 1
		II	25
		III	24
5.	Ceftazidime	I	16	16.6666 ± 0.5773
		II	17
		III	17
6.	Ciprofloxacin	I	24	24 ± 0
		II	24
		III	24
7.	Tetracycline	I	22	21.6666 ± 0.5773
		II	21
		III	22
8.	Chloramphenicol	I	26	25.3333 ± 0.5773
		II	25
		III	25
9.	Streptomycin	I	18	16.666 ± 1.5275
		II	17
		III	15
10.	Ampicillin	I	20	21.3333 ± 1.5275
		II	21
		III	23
11.	Gentamicin	I	22	22.3333 ± 1.5275
		II	21
		III	24
12.	Kanamycin	I	24	25.3333 ± 1.5275
		II	25
		III	27
13.	Nalidixic acid	I	16	15 ± 1.0000
		II	15
		III	14
14.	Trimethoprim	I	24	24.6666 ± 0.5773
		II	25
		III	25
15.	Ethanol	I	25	25.3333 ± 1.5275
		II	24
		III	27

Abbreviations: GIZ, growth inhibition zone; SD, standard deviation.

Table 3 GIZ of antibiotics in combination with Baptisia tinctoria

Sl. no.	Antibiotic	Combined drug in its respective potency	Trial no.	GIZ (mm)	Mean ± SD
1.	Cefotaxime	Baptisia tinctoria mother tincture	I	20	21.3333 ± 1.2575
			II	21
			III	23
		Baptisia tinctoria 6CH	I	25	25.3333 ± 1.5275
			II	24
			III	27
		Baptisia tinctoria 30CH	I	18	19.6666 ± 2.0816
			II	19
			III	22
2.	Ceftazidime	Baptisia tinctoria mother tincture	I	18	17.3333 ± 1.1547
			II	16
			III	18
		Baptisia tinctoria 6CH	I	22	22.3333 ± 1.5275
			II	21
			III	23
		Baptisia tinctoria 30CH	I	20	19.3333 ± 1.1547
			II	18
			III	20
3.	Ciprofloxacin	Baptisia tinctoria mother tincture	I	22	21.6666 ± 0.5773
			II	21
			III	22
		Baptisia tinctoria 6CH	I	24	23 ± 1.0000
			II	22
			III	23
		Baptisia tinctoria 30CH	I	24	23.6666 ± 0.5773
			II	23
			III	24
4.	Tetracycline	Baptisia tinctoria mother tincture	I	22	22 ± 0
			II	22
			III	22
		Baptisia tinctoria 6CH	I	23	23.6666 ± 0.5773
			II	24
			III	24
		Baptisia tinctoria 30CH	I	23	23.6666 ± 1.1547
			II	23
			III	25
5.	Chloramphenicol	Baptisia tinctoria mother tincture	I	25	24.3333 ± 0.5773
			II	24
			III	24
		Baptisia tinctoria 6CH	I	28	26.3333 ± 1.5275
			II	25
			III	26
		Baptisia tinctoria 30CH	I	25	24.3333 ± 0.5773
			II	24
			III	24
6.	Streptomycin	Baptisia tinctoria mother tincture	I	19	17.3333 ± 2.0816
			II	18
			III	15
		Baptisia tinctoria 6CH	I	22	21.6666 ± 1.5275
			II	20
			III	23
		Baptisia tinctoria 30CH	I	24	23.3333 ± 2.0816
			II	21
			III	25
7.	Ampicillin	Baptisia tinctoria mother tincture	I	18	19.6666 ± 1.5275
			II	20
			III	21
		Baptisia tinctoria 6CH	I	24	23.3333 ± 1.1547
			II	23
			III	24
		Baptisia tinctoria 30CH	I	24	24.6666 ± 1.5275
			II	23
			III	24
8.	Gentamicin	Baptisia tinctoria mother tincture	I	24	22.6666 ± 2.0816
			II	21
			III	25
		Baptisia tinctoria 6CH	I	25	24 ± 1.0000
			II	24
			III	23
		Baptisia tinctoria 30CH	I	24	24 ± 0
			II	24
			III	24
9.	Kanamycin	Baptisia tinctoria mother tincture	I	24	24.3333 ± 0.5773
			II	25
			III	24
		Baptisia tinctoria 6CH	I	23	22 ± 1.0000
			II	21
			III	22
		Baptisia tinctoria 30CH	I	26	25.3333 ± 0.5773
			II	25
			III	25
10.	Nalidixic acid	Baptisia tinctoria mother tincture	I	6	6.3333 ± 1.5275
			II	8
			III	5
		Baptisia tinctoria 6CH	I	18	20.3333 ± 2.0816
			II	21
			III	22
		Baptisia tinctoria 30CH	I	22	22 ± 0
			II	22
			III	22
11.	Trimethoprim	Baptisia tinctoria mother tincture	I	25	24.6666 ± 0.5773
			II	25
			III	24
		Baptisia tinctoria 6CH	I	26	26 ± 0
			II	26
			III	26
		Baptisia tinctoria 30CH	I	28	27.3333 ± 1.5275
			II	27
			III	29

Abbreviations: GIZ, growth inhibition zone; SD, standard deviation.

Table 4 Matrix of p-values between sensitivity zones with Baptisia tinctoria with different potency

	Mother tincture	6 CH	30 CH	Ethanol
Mother tincture	_
6 CH	0.0647	–
30 CH	0.1703	0.0194	–
Ethanol	0.0020	0.0004	0.0017	–

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 5 Matrix of p-values between sensitivity zones with cefotaxime with Baptisia tinctoria in different potency

	Cefotaxime	Cefotaxime + mother tincture	Cefotaxime + 6 CH	Cefotaxime + 30 CH
Cefotaxime	_
Cefotaxime + mother tincture	0.0647	_
Cefotaxime + 6 CH	0.2748	0.0327	_
Cefotaxime + 30 CH	0.0314	0.3261	0.0191	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 6 Matrix of p-values between sensitivity zones with ceftazidime with Baptisia tinctoria in different potency

	Ceftazidime	Ceftazidime + mother tincture	Ceftazidime + 6 CH	Ceftazidime + 30 CH
Ceftazidime	_
Ceftazidime +mother tincture	0.4216	_
Ceftazidime +6 CH	0.0039	0.0106	_
Ceftazidime + 30 CH	0.0232	0.1012	0.0533	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 7 Matrix of p-values between sensitivity zones with ciprofloxacin with Baptisia tinctoria in different potency

	Ciprofloxacin	Ciprofloxacin + mother tincture	Ciprofloxacin + 6 CH	Ciprofloxacin + 30 CH
Ciprofloxacin	_
Ciprofloxacin + mother tincture	0.0022	_
Ciprofloxacin + 6 CH	0.1583	0.1161	_
Ciprofloxacin + 30 CH	0.3738	0.0132	0.3739	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 8 Matrix of p-values between sensitivity zones with tetracycline with Baptisia tinctoria in different potency

	Tetracycline	Tetracycline + mother tincture	Tetracycline + 6 CH	Tetracycline + 30 CH
Tetracycline	_
Tetracycline + mother tincture	0.3738	_
Tetracycline + 6 CH	0.0132	0.0075	_
Tetracycline + 30 CH	0.0550	0.0668	1.0000	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 9 Matrix of p-values between sensitivity zones with chloramphenicol with Baptisia tinctoria in different potency

	Chloramphenicol	Chloramphenicol + mother tincture	Chloramphenicol + 6 CH	Chloramphenicol + 30 CH
Chloramphenicol	_
Chloramphenicol + mother tincture	0.1003	_
Chloramphenicol + 6 CH	0.3483	0.1012	_
Chloramphenicol + 30 CH	0.1003	1.0000	0.1012	_

Table 10 Matrix of p-values between sensitivity zones with streptomycin with Baptisia tinctoria in different potency

	Streptomycin	Streptomycin + mother tincture	Streptomycin + 6 CH	Streptomycin + 30 CH
Streptomycin	_
Streptomycin + mother tincture	0.6778	_
Streptomycin + 6 CH	0.0327	0.0438	_
Streptomycin + 30 CH	0.0111	0.0242	0.3261	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 11 Matrix of p-values between sensitivity zones with ampicillin with Baptisia tinctoria in different potency

	Ampicillin	Ampicillin + mother tincture	Ampicillin + 6 CH	Ampicillin + 30 CH
Ampicillin	_
Ampicillin + mother tincture	0.2524	_
Ampicillin + 6 CH	0.1890	0.0295	_
Ampicillin + 30 CH	0.0686	0.0160	0.2943	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 12 Matrix of p-values between sensitivity zones with gentamicin with Baptisia tinctoria in different potency

	Gentamicin	Gentamicin + mother tincture	Gentamicin + 6 CH	Gentamicin + 30 CH
Gentamicin	_
Gentamicin + mother tincture	0.8340	_
Gentamicin + 6 CH	0.1890	0.3739	_
Gentamicin + 30 CH	0.1318	0.3294	1.0000	_

Table 13 Matrix of p-values between sensitivity zones with kanamycin with baptisia tinctoria in different potency

	Kanamycin	Kanamycin + mother tincture	Kanamycin + 6 CH	Kanamycin + 30 CH
Kanamycin	_
Kanamycin +mother tincture	0.3488	_
Kanamycin +6 CH	0.0341	0.0249	_
Kanamycin +30 CH	1.0000	0.1012	0.0075	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 14 Matrix of p-values between sensitivity zones with nalidixic acid with Baptisia tinctoria in different potency

	Nalidixic acid	Nalidixic acid + mother tincture	Nalidixic acid + 6 CH	Nalidixic acid + 30 CH
Nalidixic acid	_
Nalidixic acid +mother tincture	0.0012	_
Nalidixic acid + 6 CH	0.0161	0.0007	_
Nalidixic acid + 30 CH	0.0003	0.0001	0.2378	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 15 Matrix of p-values between sensitivity zones with trimethoprim with Baptisia tinctoria in different potency

	Trimethoprim	Trimethoprim +mother tincture	Trimethoprim +6 CH	Trimethoprim +30 CH
Trimethoprim	_
Trimethoprim + mother tincture	1.0000	_
Trimethoprim + 6 CH	0.0161	0.0161	_
Trimethoprim + 30 CH	0.0474	0.0474	0.2051	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 16 Matrix of p-values between sensitivity zones with Baptisia tinctoria with different potency in Escherichia col i

	Mother tincture	6 CH	30 CH	Ethanol
Mother tincture	_
6 CH	0.0026	–
30 CH	0.0329	0.1874	–
Ethanol	0.0600	0.0109	0.1963	–

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 17 GIZ of Escherichia coli

Sl. no.	Reagent	Trial no.	GIZ (mm)	Mean ± SD
1.	Baptisia tinctoria mother tincture	I	12	9.6666 ± 2.0816
		II	8
		III	9
2.	Baptisia tinctoria 6CH	I	25	22.3333 ± 2.5166
		II	22
		III	20
3.	Baptisia tinctoria 30CH	I	22	18 ± 4.0000
		II	14
		III	18
4.	Ciprofloxacin	I	25	24.3333 ± 0.5773
		II	24
		III	24
5.	Chloramphenicol	I	26	25.3333 ± 0.5773
		II	25
		III	25
6.	Ethanol	I	16	14 ± 2
		II	12
		III	14

Abbreviations: GIZ, growth inhibition zone; SD, standard deviation.

Table 18 GIZ of Escherichia coli in combination of antibiotics and Baptisia tinctoria

Sl. no.	Antibiotic	Combined drug in its respective potency	Trial no.	GIZ (mm)	Mean ± SD
1.	Ciprofloxacin	Baptisia tinctoria mother tincture	I	25	24.6666 ± 0.5773
			II	24
			III	25
		Baptisia tinctoria 6CH	I	26	26.3333 ± 0.5773
			II	26
			III	27
		Baptisia tinctoria 30CH	I	23	21.6666 ± 1.5275
			II	22
			III	20
2.	Chloramphenicol	Baptisia tinctoria mother tincture	I	24	22.6666 ± 2.3094
			II	24
			III	20
		Baptisia tinctoria 6CH	I	26	26 ± 0
			II	26
			III	26
		Baptisia tinctoria 30CH	I	24	21.6666 ± 2.0816

Abbreviations: GIZ, growth inhibition zone; SD, standard deviation.

Discussion

There is a flagrant disregard for hygiene standards, faecal-contamination of drinking water, poverty and unchecked population growth contributing to the escalation of typhoid cases. This in turn causes a huge burden on healthcare in South Asian nations. The number of MDR typhoid infections in India has increased to 7% as a result of this situation.[31] In India, occasional reports of strains resistant to ceftriaxone have been identified.[32] MDR organisms (resistant to ampicillin, co-trimoxazole and chloramphenicol) and extremely drug-resistant S. typhi strains have emerged as a result of decades of empiric antibiotic use (resistant to chloramphenicol, ampicillin, co-trimoxazole, fluoroquinolones and third-generation cephalosporin). According to a study published in The Lancet, people with resistant infections have a mortality risk and financial burden that are two times higher than those with non-resistant illnesses.[33] The study also reported the need for the strict implementation of programs like the antibiotic stewardship program in the country which is difficult to implement.[33] In such a scenario, we believe homeopathic medicine BT can be a potent therapeutic agent to combat this crisis. AGPs from the extract of the plant Baptisia have shown significant immunomodulatory effects and antigen-specific reactivity.[23] [34] During the infective stage, S. typhi actively interferes with the phagolysosomal fusion and it inhibits the interferon-γ (IFN-γ) induced bacterial killing.[35] This is the reason why patients with primary genetic deficiencies in IFN-γ receptor signalling suffer from repeated disseminated Salmonella infections.[36] [37] Thus, Th1 cells play an important role in mediating protective immunity in both human and murine Salmonellosis. B cells can contribute to protective immunity via antigen presentation to Salmonella-specific Th1 cells, or as an important source of inflammatory cytokines during infection.[38] [39] [40] [41] In 1992, Egert and Beuscher studied antigen specificity of immune reactive AGPs from this plant by performing a series of experiments on the cross-reactivity of antibodies that were raised against AGPs derived from BT.[34] The pivotal role of B cells in protective immunity against salmonella has been demonstrated by Nanton et al in their experiment.[42] Their experiment on mice model revealed that mice lacking B cells had much higher bacterial loads in both the spleen and liver compared with wild-type mice.[42] What is even more significant is that in 2006 Classen et al showed that AGP extracted from BT rooms can increase IgM interleukin-6 (IL-6) and NO2 production in alveolar macrophages of mice.[23] They also postulated that the lymphocyte-activating potential of IL-6 and the strong effects of AGP from BT on IgM production might be due to a direct influence on B-lymphocytes or indirectly caused by the activation of macrophages followed by the release of IL6. A recent study showed that B cell production of IL-6 is crucial for optimal Th17 responses and B cell production of IFN contributed to Th1 development, even though B lymphocytes may directly present antigen and trigger Salmonella-specific Th1 responses.[40] Additionally, a current investigation has demonstrated that B cells can influence subsequent responses to Salmonella infection through a MyD88 and IL-10-dependent pathway.[43] By presenting antigens and producing certain cytokines that promote effector lineage commitment during early responses, B cells are therefore likely to contribute to protective CD4 responses. Another patient-based study was published in homeopathy where researchers considered 66 different homeopathic medicines where BT showed a significant role against this pathogen.[8] Apart from S. typhi BT has a significant role in non-typhoidal salmonella as well. An experiment performed by Shaw et al with typhoidal Salmonella strains, S. Enteritidis, S. Orion, S. Istanbul, S. Kentucky showed a positive agglutination reaction with BT mother tinctures, which was absent when tested with other drugs.[44] Another study showed BT extract can increase the number of circulating leukocytes amongst which an increased number of neutrophils and eosinophils were significant.[45] All the aforementioned findings not only justify our search but also uphold our belief of BT is a potent therapeutic option against the pathogen. Though it is still unclear in which state molecules of alkaloids remain in such dilutions, that cannot be substantial enough to neglect these finding. The microorganisms are natural symbionts that share many structural and functional similarities with different natural compounds. The effect of BT against the genus Salmonella might be due to some unexplored structural similarities which favour the AGPS to block the antigen. Natural alkaloids have a significant role in the propagation and inhibition of these organisms. We also believe the utility of such natural compounds should be explored to combat the emergence of drug-resistant organisms.

Conclusion

Homeopathy has always been a popular choice of treatment amongst patients but the lack of acceptable scientific data in favour lags homeopathy behind. Despite the ignorance of the scientific fraternity, homeopathy effectively treats many acute and chronic conditions. The homeopathic literature also supports the use of Baptisia in typhoid. We think more high-quality trials should be performed to understand the predictors of using such compounds.

Table 19 Matrix of p-values between sensitivity zones with ciprofloxacin with Baptisia tinctoria in different potency in Escherichia col i

	Ciprofloxacin	Ciprofloxacin + mother tincture	Ciprofloxacin + 6 CH	Ciprofloxacin +30 CH
Ciprofloxacin	_
Ciprofloxacin + mother tincture	0.5185	_
Ciprofloxacin + 6 CH	0.0132	0.0241	_
Ciprofloxacin + 30 CH	0.0474	0.0335	0.0078	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Table 20 Matrix of p-values between sensitivity zones with chloramphenicol with Baptisia tinctoria in different potency in Escherichia coli

	Chloramphenicol	Chloramphenicol + mother tincture	Chloramphenicol + 6 CH	Chloramphenicol + 30 CH
Chloramphenicol	_
Chloramphenicol + mother tincture	0.1243	_
Chloramphenicol + 6 CH	0.1161	0.0668	_
Chloramphenicol + 30 CH	0.0424	0.6072	0.0226	_

Legend

	Statistically significant
	Very statistically significant
	Extremely statistically significant
	Not quite statistically significant

Conflict of Interest

None declared.

Ethical Approval

Approval was obtained from the Institutional Ethics committee.

• Reference

• 1 Stanaway JD, Reiner RC, Blacker BF. et al; GBD 2017 Typhoid and Paratyphoid Collaborators. The global burden of typhoid and paratyphoid fevers: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Infect Dis 2019; 19 (04) 369-381
• 2 Arora RK, Gupta A, Joshi NM, Kataria VK, Lall P, Anand AC. Multidrug resistant typhoid fever: study of an outbreak in Calcutta. Indian Pediatr 1992; 29 (01) 61-66
• 3 Bhattacharya SS, Das U, Choudhury BK. Occurrence &antibiogram of Salmonella Typhi & S. Paratyphi A isolated from Rourkela, Orissa. Indian J Med Res 2011; 133 (04) 431-433
• 4 Choudhary A, Gopalakrishnan R, Nambi PS, Ramasubramanian V, Ghafur KA, Thirunarayan MA. Antimicrobial susceptibility of Salmonella enterica serovars in a tertiary care hospital in southern India. Indian J Med Res 2013; 137 (04) 800-802
• 5 Das U, Bhattacharya SS. Multidrug resistant Salmonella typhi in Rourkela, Orissa. Indian J Pathol Microbiol 2000; 43 (02) 135-138
• 6 Browne AJ, Kashef Hamadani BH, Kumaran EAP. et al. Drug-resistant enteric fever worldwide, 1990 to 2018: a systematic review and meta-analysis. BMC Med 2020; 18 (01) 1
• 7 Akwa TE, Nguimbous SP. Common plants used in the treatment of typhoid fever, their active components and toxicity related issues: a review. Natural Resources Human Health. 2021; 1 (01) 36-42
• 8 Banerji P, Banerji P, Das GC, Islam A, Mishra SK, Mukhopadhyay S. Efficacy of Baptisia tinctoria in the treatment of typhoid: its possible role in inducing antibody formation. J Complement Integr Med 2012; 9: 15
• 9 World Health Organization. WHO Traditional Medicine Strategy: 2014–2023. Geneva, Switzerland: World Health Organization; 2013
• 10 Bodeker G, Ong CK. WHO Global Atlas of Traditional, Complementary and Alternative Medicine. Kobe, Japan: World Health Organization; 2005
• 11 Ekor M. The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol 2014; 4: 177
• 12 Analysts GI. Inc. The global herbal supplements and remedies market: trends, drivers and projections, February 2015.
• 13 Global Herbal Medicine Market By Form (Liquid & Gel, Tablets & Capsules, And Powder), By Application (Personal Care & Baby Products, Food & Beverages, Pharmaceutical, And Nutraceutical) - And By Region - Global And Regional Industry Overview, Market Intelligence, Comprehensive Analysis, Historical Data, And Forecasts 2022 - 2028. Accessed January 25, 2024 at: https://www.zionmarketresearch.com/news/global-homeopathy-products-market
• 14 Buhner SH. Herbal Antibiotics: Natural Alternatives for Treating Drug-Resistant Bacteria. New York: Storey Publishing; 2012
• 15 McKenna J. Antibiotics–Are They Curing Us or Killing Us?: The Catastrophic Impact of the Over-prescription of Antibiotics on Our Health. Dublin, Ireland: Gill & Macmillan Ltd; 2014
• 16 Chevallier A. Encyclopedia of Medicinal Plants. London: Dorling Kindersley; 2001
• 17 Felter HW, Lloyd JR. King's American Dispensatory. Sandy, Oregon: Eclectic Medical Publications; 1898
• 18 Felter HW. The Eclectic Materia Medica, Pharmacology and Therapeutics. Sandy, Oregon: Eclectic Medical Publications; 1922
• 19 Sayre LE. A Manual of Organic Materia Medica and Pharmacognosy. 4th edition. 1917 . Accessed January 25, 2023 at: http://www.swsbm.com/SayreMM/SayreMM.html
• 20 Ellingwood R. American Materia Medica, Therapeutics and Pharmacognosy. 1919 . Accessed January 25, 2023 at: http://www.swsbm.com/Ellingwoods/Ellingwoods.htm
• 21 Remington JP, Wood HC. Eds. The Dispensatory of the United States of America. 20th edition. 1918. . Accessed January 25, 2023 at: http://www.swsbm.com/Dispensatory/USD-1918-complete.pdf
• 22 Beuscher N, Scheit KH, Bodinet C, Kopanski L. Immunologisch aktive Glykoproteine aus Baptisia tinctoria. [Immunologically active glycoproteins of Baptisia tinctoria] Planta Med 1989; 55 (04) 358-363
• 23 Classen B, Thude S, Blaschek W, Wack M, Bodinet C. Immunomodulatory effects of arabinogalactan-proteins from Baptisia and Echinacea. Phytomedicine 2006; 13 (9-10): 688-694
• 24 Nikishin DA, Teryoshina NS, Tashlitsky VN. Investigation of the alkaloids of homeopathic indigo (Baptisia) tinctures. Pharmacy (Basel) 2006; 7
• 25 Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke PA. Handbook of Medicinal Herbs. 2nd edition. Boca Raton, FL: CRC Press; 1929
• 26 Lyle TJ. Physio-Medical Therapeutics, Materia Medica and Pharmacy. London: Medical College of Herbal Practitioners; 1897
• 27 Hoffmann D. The Complete Illustrated Holistic Herbal. Dorset: Element Books; 1996
• 28 Bone K. A Clinical Guide to Blending Liquid Herbs: Herbal Formulations for the Individual Patient. St Louis, Missouri: Churchill Livingstone; 2003
• 29 Han J, Wang DS, Liu SB, Zhao MG. Cytisine, a partial agonist of α4β2 nicotinic acetylcholine receptors, reduced unpredictable chronic mild stress-induced depression-like behaviors. Biomol Ther (Seoul) 2016; 24 (03) 291-297
• 30 Nothnagel EA. Proteoglycans and related components in plant cells. Int Rev Cytol 1997; 174: 195-291
• 31 Ochiai RL, Acosta CJ, Danovaro-Holliday MC. et al; Domi Typhoid Study Group. A study of typhoid fever in five Asian countries: disease burden and implications for controls. Bull World Health Organ 2008; 86 (04) 260-268
• 32 Sah R, Donovan S, Seth-Smith HMB. et al. A novel lineage of ceftriaxone-resistant Salmonella Typhi from India that is closely related to XDR S. typhi found in Pakistan. Clin Infect Dis 2020; 71 (05) 1327-1330
• 33 Pustake M, Giri P, Tambolkar S, Nayak S. Extensively drug-resistant typhoid fever: a call to action. Indian J Community Med 2022; 47 (01) 153-154
• 34 Egert D, Beuscher N. Studies on antigen specifity of immunoreactive arabinogalactan proteins extracted from Baptisia tinctoria and Echinacea purpurea. Planta Med 1992; 58 (02) 163-165
• 35 Mastroeni P, Grant A, Restif O, Maskell D. A dynamic view of the spread and intracellular distribution of Salmonella enterica. Nat Rev Microbiol 2009; 7 (01) 73-80
• 36 Dorman SE, Holland SM. Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev 2000; 11 (04) 321-333
• 37 MacLennan C, Fieschi C, Lammas DA. et al. Interleukin (IL)-12 and IL-23 are key cytokines for immunity against Salmonella in humans. J Infect Dis 2004; 190 (10) 1755-1757
• 38 Mastroeni P, Simmons C, Fowler R, Hormaeche CE, Dougan G. Igh-6(-/-) (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar typhimurium and show impaired Th1 T-cell responses to Salmonella antigens. Infect Immun 2000; 68 (01) 46-53
• 39 Ugrinovic S, Ménager N, Goh N, Mastroeni P. Characterization and development of T-Cell immune responses in B-cell-deficient (Igh-6(-/-)) mice with Salmonella enterica serovar Typhimurium infection. Infect Immun 2003; 71 (12) 6808-6819
• 40 Barr TA, Brown S, Mastroeni P, Gray D. TLR and B cell receptor signals to B cells differentially program primary and memory Th1 responses to Salmonella enterica. J Immunol 2010; 185 (05) 2783-2789
• 41 Barr TA, Brown S, Mastroeni P, Gray D. B cell intrinsic MyD88 signals drive IFN-gamma production from T cells and control switching to IgG2c. J Immunol 2009; 183 (02) 1005-1012
• 42 Nanton MR, Way SS, Shlomchik MJ, McSorley SJ. Cutting edge: B cells are essential for protective immunity against Salmonella independent of antibody secretion. J Immunol 2012; 189 (12) 5503-5507
• 43 Neves P, Lampropoulou V, Calderon-Gomez E. et al. Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during Salmonella typhimurium infection. Immunity 2010; 33 (05) 777-790
• 44 Shaw N, Singh S, Das S. In-vitro clumping of food poisoning strains of Salmonella by extract of Baptisia Tinctoria (Baptisia Ѳ). European J Biomedical Pharmaceutical Sci 2017; 4 (07) x
• 45 Rautenbach H. “A Study On The Effect Of A Complex Formula Consisting Of Homoeopathic Thuja Occidentalis Dl And Baptisia Tinctoria Mother Tincture On Circulating Leukocytes” PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Accessed January 25, 2023 at: https://ujdigispace.uj.ac.za

Publication History

Article published online:
03 May 2024