2.1. Plant Parts for Preparing the Fermented Polyherbal Formulation

Fermented polyherbal formulation (AP-01) was prepared in the laboratory by boiling dried and cleaned stem barks of Holarrhena antidysenterica and Gmelina arborea mixed with large-sized dry resins of Vitis vinifera in the presence of dried flowers of Madhuca indica in potable drinking water. Dried flowers of Woodfordia fruticosa were added after the water volume lowered to one-quarter of the initial volume for initiation of fermentation while using jaggery as a source of sugar. After fermentation was completed in about 45 days, the formulation was duly filtered and kept in sterile dark glass bottles after passing through bacterial filters of 0.2-micron pore size for further use. The preparation method was modified from the Ayurvedic Pharmacopoeia of India following Sharangadhara Samhita and Vaisajyaratnavali references for different “Arishtha” formulations. All plant parts were procured from the local market and identified by qualified Ayurveda practitioners with formal academic degrees for practicing Ayurveda [19–21].

Loperamide hydrochloride (Imodium brand of Johnson and Johnson Consumer Inc.) was used as a standard drug against diarrhea induced by the effects of castor oil.

Castor oil (Erand Tail brand of Dabur) was used to induce diarrhea in animals.

Ultrapure kaolin from HiMedia Laboratories was used as a marker for determining the residual effect of AP-01 on the gastric motility of animals.

2.2. Experimental Animals

36 Sprague Dawley rats of either sex, aged around six weeks, weighing 80 to 120 grams, were obtained from the animal house of the Department of Zoology, University of North Bengal. Animals were acclimatized for seven days under standard environmental conditions, and a pellet diet of chow and water was provided in copious amounts. Experiments were conducted after the animals were deprived of both food and water for 18 to 20 hours. The study followed the National Research Council’s Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Ethics Committee of the University of North Bengal, Siliguri District, Darjeeling, West Bengal, India (Ref. No. IAEC/NBU/2018/04 dated 12.09.2018) [22].

2.3. Microorganisms with Pathogenesis and Nonpathogenic Properties

Different human pathogenic bacterial clinical isolates were thankfully received from the Gastrointestinal Tract Pathogens Repository (GTPR) of the National Institute of Cholera and Enteric Diseases (NICED), sponsored by the Indian Council of Medical Research (ICMR), the apex body to conduct clinical and preclinical research in India. Based on the availability at the repository and varying degrees of resistance against multiple antibiotics, four different strains were chosen for performing in vitro tests with AP-01. All four strains were received in sealed vials with extensive care and caution, and further experiments were conducted aseptically in the biosafety level II laboratory (Omics Laboratory) as per the guidance of ICMR. Proper disposal by autoclaving all media containing GTPR strains and thereafter incinerating the loads of the autoclave were followed. Escherichia coli 01241 (identification number assigned to each clinical isolate by GTPR) resistant to ceftazidime (CAZ), aztreonam (AT), ceftriaxone (CTR), trimethoprim/sulphamethoxazole (SXT), nalidixic acid (NA), ampicillin (AMP), and cefotaxime (CTX); Salmonella Typhi 01396 enterica serovar resistant to streptomycin (S), amoxiclav (AMC), ciprofloxacin (CIP), norfloxacin (NOR), doxycycline (DO), and erythromycin (E); Salmonella Typhi 01263 enterica serovar resistant to ofloxacin (OFX), ciprofloxacin (CIP), nalidixic acid (NA), and norfloxacin (NOR); and Shigella boydii 01399 strain resistant to nalidixic acid (NA), ciprofloxacin (CIP), cefotaxime (CTX), and kanamycin (K) were received from GTPR [23]. All four GTPR strains, along with a laboratory isolate of a nonpathogenic nature, Escherichia coli K12, were treated with the polyherbal formulation AP-01 to conduct further research after determining the susceptibility/resistance pattern of the microorganisms.

Antibiotic resistance/susceptibility profiles of the above-mentioned bacterial strains were also determined at the lab as per the guidelines of CLSI (Clinical Laboratory Standards Institute) (CLSI, 2022) using the Kirby–Bauer disk diffusion method. The laboratory findings mostly corroborated with the resistance details available with all GTPR strains. The zone of inhibition was measured and compared with breakpoints given by EUCAST/CLSI to determine the resistance/susceptibility profile of each strain against antibiotics belonging to different classes [24, 25].

2.4. Phytochemical Screening of the Formulation

Primary phytochemical studies were performed following the standard biochemical qualitative and analytical assay procedures and literature surveys for individual plant material and the polyherbal formulation to confirm the presence of phytoconstituents [26–28]. Finally, the polyherbal formulation was characterized by different spectroscopic techniques such as FTIR spectroscopy (Nicolet 6700 FTIR spectrometer, Thermo Fisher Scientific, USA), ¹H NMR spectroscopy (600 MHz, Bruker), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS, Waters, USA, HPLC-MS system equipped with model 2525 pump, ZQ detector) with acetonitrile and water (95 : 5) using conessine as the biomarker. Briefly, the alkaloid fraction was subjected to HPLC-MS using the solvent system of acetonitrile:water (both containing 0.1% acetic acid) at a flow rate of 1 ml/min [28].

2.5. Antimicrobial Assay of Polyherbal AP-01 against GTPR (GTPR Signifies Gastrointestinal Tract Pathogens Repository of Indian Council of Medical Research, Govt. of India) Pathogenic Strains

The minimum inhibitory concentration (MIC) of polyherbal preparation AP-01 against the four GTPR strains and Escherichia coli K12 was determined by the serial broth dilution method by adhering to the Clinical Laboratory Standards Institute (CLSI, 2022) reference method [24]. Polyherbal preparation AP-01 was added to sterile 5 ml Mueller–Hinton broth in different concentrations from 10 µl/ml to 200 µl/ml with a gradual increment of 5 µl/ml, and then, 1% aliquot of the mid-log-phase culture of 0.5 McFarland standard of each bacterium was inoculated in every tube equally and incubated for 24 h at 37°C. After incubation, growth was measured in a spectrophotometer (SPECTROstar Nano—BMG LABTECH) at 600 nm to determine the minimum inhibitory concentration (MIC). All the experiments were performed in triplicate and repeated twice. The overnight-grown mid-log-phase bacterial culture was inoculated both in the presence of polyherbal AP-01 at the MIC dose and without the presence of polyherbal AP-01 at 37°C and incubated for six hours to compare the optical density-based growth under both conditions using the spectrophotometer. At the same time, serially diluted aliquots from cultures grown under both conditions (with and without adding AP-01) were grown over Luria agar plates through the spread plate technique for reassurance of growth/inhibition of growth by counting the colony-forming units (cfu) of the strains. The highest dilution spread over the Luria agar plates, which showed no growth, was the minimum bactericidal concentration (MBC). In the laboratory, Escherichia coli K12 was isolated and grown in Mueller–Hinton Broth (HiMedia) with and without AP-01. This experiment was conducted to study the inhibitory pattern and determine the MIC and MBC values of AP-01 against the strains.

Furthermore, the modified Mueller–Hinton agar well diffusion method was also performed to determine the zone of inhibition produced by different volumes of undiluted AP-01 against each bacterial strain compared with the negative control (DMSO (DMSO is dimethyl sulphoxide)) and positive control (meropenem 10 μg). 100 μl aliquot of mid-log-phase culture of 0.5 McFarland standard containing 1 × 10⁹ cfu/ml of each bacterium was aseptically spread over 20 ml Mueller–Hinton agar plates, and sterile 6-mm well borers were used to dig wells. Serially diluted volumes of 150 µl, 125 µl, 100 µl, 75 µl, 50 µl, and 25 µl AP-01 in DMSO (volume adjusted to 150 μl) and 150 μl DMSO were poured into individual wells, and meropenem disks were placed in separate plates for each strain and incubated at 37°C for 24 hours to determine the zones of inhibition. Tests were performed in triplicate.

2.6. Analysis of Swarming Motility of the GTPR Pathogen Salmonella Typhi Serovar 01263 Enterica in the Presence and Absence of AP-01

A swarm agar [0.7% (W/V) Bacto-agar, 0.5% (W/V) glucose] plate was used to observe Salmonella swarming motility [29, 30]. Gentle spotting of 2 µl of fresh Salmonella culture (OD₆₀₀ value of 0.4) was performed on the surface of the swarm agar and in the center. After 8 h of incubation at 30 °C, the observed growth of the migrated cells around the inoculation site was photographed. A sub-MIC dosage of AP-01 was added to the swarm agar medium to demonstrate the inhibition of swarming motility of the same Salmonella strain following the same protocol.

2.7. Primary Acute Oral Tolerance Tests

After acclimatization, animals were distributed into several groups to check tolerance imparted by AP-01 when fed through the oral route. The standard dichromate oxidation method was applied to determine the level of ethanol in AP-01 formulation, and it was determined to be present at a quantity less than 3% v/v at the time of study (data not given). A dosage of up to 50 ml/kg for the body weight of rats was found to be well tolerated and not producing any symptoms of acute toxicity or lethality. During the study of evaluating oral acute toxicity testing, no mortality, changes in the general behavior or agility, alterations in the food intake or water intake, or abnormal changes in body weight were observed. Further studies were carried out using 5 ml/kg and 10 ml/kg dosing of AP-01 as rationalized in some earlier studies using plant extracts [31, 32] and according to the Organization of Economic Corporation Development Guidelines [33] following the principles and criteria summarized in the Humane Endpoints Guidance Document. The rationale for such dosing was based on the body surface area ratio for extrapolating human dose to animals.

2.8. Cytotoxicity Study

The human intestinal epithelial cell line IEC 18 and rat intestinal epithelial cell line RIE-1 were, respectively, procured from the National Centre for Cell Science, Pune, and the Centre for Cell and Molecular Biology, Hyderabad, India, for performing the MTT assay with different strengths of AP-01 and by diluting AP-01 with DMSO. Dulbecco’s modified Eagle medium, fortified with 0.25% sodium bicarbonate solution and 8% fetal bovine serum, was used during the maintenance of the cell culture. The cell lines were stored at 37°C in a 5% CO₂ atmosphere in a CO₂ incubator (Thermo Fischer Scientific) with 90% humidity. An equal number of cells were placed at 96-well plates with 100 μl culture media. After 24 hours of incubation, the media were replaced by 25 μl of 18.75 μl/ml, 37.5 μl/ml, 75 μl/ml, 150 μl/ml, and 750 μl/ml AP-01 after adjusting the volume to 100 μl by adding DMSO and another well with 100 μl DMSO as a control. After incubation under a similar storage condition for 24 hours, the entire contents of each well were replaced by 10 μl of 5 mg/ml MTT solution freshly prepared in phosphate buffer saline and incubated for another 6 hours. Succinate dehydrogenase enzyme present in cell lysates of viable cells would therefore be reacting with yellow MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) to form a violet-blue crystalline formazan molecule that could be dissolved in propan-3-ol or isopropyl alcohol for visible spectroscopy for quantification. As metabolically active cells could only produce formazan crystals, their quantification was used to determine the number of living cells. The maximum amount of formazan should be formed in the well containing DMSO as a control. The plates were horizontally shaken after adding 50 μl of isopropyl alcohol for 2–3 mins. Absorbance was taken at λ_max of 600 nm, concerning the violet-blue color of formazan at 490 nm, by a 96-well plate reader (CLARIOstar Plus, BMG Labtech). The percentage of cytotoxicity was calculated by comparing the OD value of AP-01-treated cells with the control, (OD_Control–OD_Treatment)/OD_Control × 100.

AP-01 was added to the cell lines as 0.25–MIC, 0.5–MIC, MIC, 2X MIC, and 10X MIC dosage towards lab isolate bacterial strain E. coli K12 [34, 35].

Human small intestine-derived cell line HIEC 6 from ATCC was further used to determine the broad-spectrum of noncytotoxicity produced by AP-01.

2.9. Experimental Design for the Action of AP-01 on the Symptom of Diarrhea Induced by Ricinoleic Acid from Castor Oil

The animals were divided into six groups, each containing six randomly selected but an equal number of male and female Sprague Dawley rats for a parallel experimental design to study the effect of AP-01 on castor oil-induced diarrhea. Pregnancy, prior illness, and underweight (<100gm) were considered the exclusion criteria of the animals. The animals were randomized and included in each group using standard randomization prompts using MS Excel software. The normal group received only distilled water (10 ml/kg p.o.), and the negative control group received only castor oil at a dose of 10 ml/kg. The positive control group was fed a per oral dose of 2 mg/kg loperamide hydrochloride suspended in distilled water half an hour before receiving castor oil (10 ml/kg per oral). The first two test groups consecutively received 5 ml/kg and 10 ml/kg AP-01 half an hour before receiving castor oil (10 ml/kg) through the oral route. However, the last test group received AP-01 at a dose of 10 ml/kg half an hour after feeding castor oil at a dose mentioned previously [31, 32]. Each animal was observed under a glass funnel lined with blotting paper for twenty-four hours after feeding castor oil and water in the case of the standard group. Observations were made to test the parameters such as the onset of symptoms of diarrhea employing excreting wet (watery/diarrheagenic) feces, cessations of diarrhea using the end time for the expulsion of wet feces, the persistence of symptoms of diarrhea, the total weight of feces, weight of wet feces, total number of stool pellets, and the total number of wet stool pellets. The percentage of inhibition of symptoms of diarrhea was determined by comparing the decrease in the persistence of diarrhea symptoms, mass of wet fecal material, and number of wet stools expelled during the observation period by animals of the positive control group and test groups to the negative control group animals. As the study did not involve any invasive procedures, the same animals were given 5–7 days of resting period before the next gastric motility-related experiments. All the test animals were rehabilitated and rehoused for further use.

2.10. The Residual Effect of AP on Body Weight, Food and Water Intake, and Gastric Motility (Using Kaolin as a Marker) before and after Intervention

The animals were again divided into six groups, each group containing six randomly selected but comprising an equal number of male and female Sprague Dawley rats for a parallel experimental design to study the effect of AP-01 on body weight, food and water intake, and gastric motility rates using kaolin as a marker. Each animal was weighed one day before experimenting and again weighed after 18–20 hours from the conclusion of the observation time of the investigation. Food and water intake was calculated one day before intervention by providing a fixed amount of pellet diet and a fixed volume of water and calculating the leftover mass of food and volume of water after twenty-four hours. Dietary intake and water intake were again measured after twenty-four hours from the interim time of twelve hours of observation of the experiment as all animals were fed with a fixed amount of diet and water after twelve hours of observation.

Before feeding the animals after the observations, each animal was administered with 2 ml/kg per oral aqueous solution of kaolin. As identified from previous studies, a dose of 40% aqueous solution of kaolin could be used as a marker of gastric motility rate as it could alter the color of stool pellets without affecting the drug action administered previously. Therefore, the measured time gap between the feeding of kaolin and expulsion of fecal matter marked with kaolin for each animal could serve as a determinant of gastric motility because it could be assessed as the time taken by the kaolin to get a passage through the gastrointestinal tract of the rats. Longer latency before the expulsion of kaolin-marked fecal matter could suggest decreasing gastric motility rates [32, 36].

2.11. Statistical Analysis

The observational and all measured values were reported as mean ± standard error of mean (S.E.M.) or mean ± standard deviation (SD). Percentage changes compared to normal values and control group data were carried out as and when required. Paired and unpaired “Student’s t-test” was employed for statistical analysis for the validation of inference at a level of significance of P < 0.001, which was considered statistically significant.

3.1. Presence of Probable Phytochemicals

The presence of different functional groups, viz., hydroxyl (alcoholic and phenolic-OH), carbonyl (C=O), alkoxy(C-O), epoxy (C-O-C), C-H, C=C, and C-H bonds was confirmed by the Fourier transform infrared (FTIR) spectra of our polyherbal formulation performed in liquid form (Figure 1(a)) [37]. The high intense peak at 3380 cm⁻¹ confirms the predominant presence of hydroxyl groups (alcoholic/phenolic) in the present formulation. This also indicates the considerable existence of alcohols and polyphenols in our formulation, which could be responsible for their significant pharmacological activity. Besides, the ¹H-NMR spectra of our current polyherbal formulation further indicate the presence of primarily aliphatic protons (δ; 1.25–4.9 ppm) and alcoholic/phenolic/benzylic protons (chemical shift values, δ 4.26, 4.57, and 4.9 ppm (Figure 1(b)).

On account of the FTIR and 1H-NMR spectra along with the LC-MS/MS data (Figure 1(c)), the presence of diverse phytochemicals, viz., quercetin(MW 302.23), β-sitosterol (MW 414.7), n-octacosanol (MW-395.72), kurchine (MW-342.6), conessine (MW 356.58), antidysentericine (MW-356.5), conimine (MW-328.53), gmelinol (MW-402.4), luteolin (MW-286.24), gmelanone (368.33), stigmasterol (MW-412.69), betulinic acid (MW-456.700), myricetin (MW-318.235), catechin (290.2681), kaempferol (MW-286.236), beta-Amyrin (MW-426.717), luteolin (MW-286.23), n-hexacosanol (MW-382.70), oleanic acid (MW-456.7), linolenic acid (MW-278.42), mannitol (MW-182.17), myricetin (MW-318.23), and their various glycosidic derivatives, is primarily inferred as bioactive compounds. The chemical composition was also compared with the literature on individual plant extracts. Hence, the major classes of phytochemicals present in our polyherbal formulation are alkaloids, flavonoids, and polyphenols.

3.2. Cytotoxicity Analysis

AP-01 was not found to be producing any cytotoxic effect in either concentration as 0.25—MIC, 0.5—MIC, MIC, 2X MIC, and 10X MIC dosages to either human or rat intestinal epithelial cell lines as at the highest concentration; cell viability was not found to be reduced for more than 10 percent as represented in Table 1.

Data are presented as mean ± SD from three independent experiments, each run in triplicate. The percentage inhibition was calculated as the percent difference between growth in DMSO and growth in the presence of AP-01 after 24 h of incubation.

3.3. Effect of AP-01 on Animals with Diarrhea Induced by Castor Oil

All fecal excrements were collected, weighed, and analyzed for the study (Figure 2). During the period of observation for twenty-four hours after dosing with castor oil, all animals of the negative control group showed symptoms of diarrhea with an early onset of the release of a wet fecal pellet within an hour (an average of 52 minutes) and the symptoms persisted as long as nineteen hours. Animals belonging to the first and second test groups showed dose-dependent delayed onset of symptoms; however, the occurrence ceased at a significantly earlier time (three hours) in the case of rats pretreated with 10 ml/kg AP-01 formulation. The third test group, which consisted of animals that received AP-01 after half an hour of castor oil feeding, also showed a delayed onset of symptoms compared to the negative control and first test groups. Treatment with loperamide as a standard drug produced a significant reduction of symptoms at a rate of 91.77, 83.1, and 89.82 in terms of the persistence of diarrhea, number of wet stools, and mass of wet stools, respectively, while the test groups having 5 ml/kg and 10 ml/kg AP-01 dosages as pretreatment and 10 ml/kg AP-01 as posttreatment with castor oil showed a consecutively reduced persistence of diarrhea at a percentage inhibition of 55.89%, 87.65%, and 73.21%, as shown in Figure 3. The test group that received 10 ml AP-01/kg body weight also showed a percentage of inhibition at 79.71% and 89.66% in terms of the number and mass of the wet fecal material considering the values of the negative control group as 100%. The duration of diarrhea was found to be significantly decreased in the case of the positive control group and second test group (that received 10 ml/kg AP-01 pretreatment) and measured consecutively as 94 minutes and 140 minutes, respectively. The normal animal group did not show any symptoms of diarrhea throughout the experiment and observational period.

3.4. The Residual Effect of AP-01 on Body Weight, Food and Water Intake, and Gastric Motility

Expulsions of stool pellets marked with kaolin were visually altered in color (Figure 4). Kaolin-marked stool pellets took a significantly longer time to pass through the gastrointestinal tract of animals in the case of both the groups treated with 10 ml/kg AP-01. However, a considerably higher latency period was shown when the dose of AP-01 was given at a later period (in the third test group). Body weight and food intake were reduced significantly in the case of the negative control group receiving only castor oil; however, there were no significant changes in the body weight or food intake for the animals belonging to any test groups and the positive control group. There was a substantial increase in the food intake for the animals of the normal group. Another significant change is shown in Figure 5, depicting the increase in the water intake only for the animals in the negative control group.

3.5. The Effect of AP-01 on Gastrointestinal Pathogens Collected from GastroIntestinal Tract Pathogens Repository

E. coli 01241, S. Typhi 01263, S. Typhi 01396, and Shigella boydii 1399 strains of pathogenic bacteria were collected from NICED to conduct in vitro antimicrobial tests on human pathogenic bacteria with AP-01. Primary tests confirmed the inhibitory effect. AP-01 was found efficacious against NICED-GTPR multiple antibiotic-resistant human pathogenic strain E. coli 01241 (resistant to CAZ, AT, CTR, SXT, NA, AMP, and CTX); S. Typhi 01396 (resistant to S, AMC, CIP, NOR, DO, and E); S. Typhi 01263 (resistant to OFX, CIP, NA, and NOR); and Shigella boydii 1399 (resistant to NA, CIP, CTX, and K) (Table 2). The minimum inhibitory concentrations of AP-01 against each GTPR strain, determined through the broth dilution method and well diffusion method, were 80 μl/ml v/v for E. coli 01241 and 100 μl/ml v/v for the other three strains. The MIC dose of AP-01 for the lab isolate E. coli K12 was determined to be 75 μl/ml v/v (Tables 3 and 4).

3.6. Analysis of Swarming Motility of the GTPR Pathogen Salmonella Typhi Serovar 01263 Enterica in the Presence and Absence of AP-01

The presence of a sub-MIC dose of AP-01 (50 μl/ml v/v) in semisolid media significantly arrested the swarming motility of the GTPR strain S. Typhi serovar 01263, the only strain to show swarming motility. In Figure 6, the first left side figure shows the swarming motility on the swarm agar plate observed without the presence of AP-01 in the media, and the adjoining figure shows the inhibition of swarming motility in the presence of the sub-MIC dose of AP-01 in media.

3.7. Statistical Analysis

Standard deviation and standard error of mean were determined using Microsoft Excel 2013 software. We used the t-test calculator (graphpad.com/quickcalcs/ttest1.cfm? Format = SD) for conducting both paired and unpaired Student’s “t-tests.” A two-sample t-test is used to compare two datasets to see if their means are statistically different. In the case of onset and cessation of diarrhea, the results of the positive control group and three test groups were found to be significantly different compared to the negative control group, whereas there were no significant differences found between the results of the positive control group fed with loperamide as a standard drug and the second test group that received 10 ml AP-01/kg body weight half an hour prior to ingestion of castor oil in paired t-test (P value <0.001). An unpaired “t-test” was performed for the statistical analysis of the mass of normal and wet fecal material and body weights of the animals of several groups. Both the positive control group and the second test group receiving 10 ml AP-01/kg body weight half an hour early showed an extremely significant difference compared to the negative control group; however, the other two test groups receiving 5 ml AP-01/kg and 10 ml AP-01/kg fed after ingestion of castor oil did not show any significant differences (P value <0.001). The gastric motility rate test using kaolin as a marker also showed a significant difference between the test groups and the negative control group. Here, the third test group receiving 10 ml AP-01/kg fed after half an hour of kaolin treatment showed a marked difference compared to the positive control group. In all three different measures of the percentage of inhibition, the positive control group and the second test group receiving 10 ml AP-01/kg body weight before receiving treatment showed a significant difference compared to the other two test groups (P value <0.01). Therefore, the statistical analysis of a multitude of factors revealed no significant difference among the positive control or the standard drug-receiving group and the second test group receiving 10 ml AP-01/kg body weight half an hour prior to receiving either castor oil or kaolin, meaning that both can produce equal and desired results in the decrease of diarrhea. However, the other two test groups did not show any extremely significant difference from the negative control group in all parameters. Hence, the comparable therapeutic efficacy could be achieved using a dose of 10 ml AP-01/kg body weight when given prior.