Anti-pathogenic potential of a classical ayurvedic Triphala formulation

Test formulation

Triphala formulation (TF) (Emami Ltd; batch no. EM0029; Proportion of 3 constituent plant species: 1:1:1 i.e. P. emblica, T. bellerica, and T. chebula) was purchased from a local market in Ahmedabad, India). For assay purpose, 150 mg of this formulation was suspended in 5 ml of DMSO (Merck, Mumbai), followed by vortexing for 15 min. Then it was centrifuged at 8,000 rpm for 30 min at ambient temperature, and resulting supernatant was collected in a sterile 15 ml glass vial (Borosil) and stored under refrigeration till further use. Remaining pellet was subjected to drying in an oven at 70-80°C until the solvent was completely evaporated, followed by weighing of the dried plant material. Subtracting the latter from the initial weight of 150 mg, the concentration of test formulation in supernatant was calculated to be 22.94 mg/ml. This way the whole formulation was found to contain 70% DMSO soluble fraction, which was used for our experiments.

Bacterial strains

In vivo assays

In vivo efficacy of TF against bacterial infections was tested in the nematode host Caenorhabditis elegans N2-Bristol (maintained at the Institute of Science, Nirma University). Maintenance and synchronization of the worm population was done as previously described in Joshi (2019). Worms were maintained on NGM [Nematode Growing Medium; 2.5 g/L peptone (HiMedia, RM001-500G), 3 g/L NaCl (HiMedia, MB023-500G), 1 M CaCl₂ (HiMedia, GRM534-500G), 1 M MgSO₄ (Merck, 1.93645.0521), 5 mg/mL cholesterol (HiMedia, TC101-5G), 1 M phosphate buffer of pH 6, 17 g/L agar-agar (HiMedia, GRM666-500G)] agar plate with E. coli OP50 (LabTIE B.V. OP50 V.2; batch # 002, JR Rosmalen, the Netherlands) as food. For synchronization of the worm population, adult worms from a 4–5 days old NGM plate were first washed with distilled water, and then treated with 1 mL of bleaching solution [1N NaOH (HiMedia MB095-100G) + 4% NaOCl (Merck 61842010001730) + water in 1:1:3 proportion], followed by centrifugation (at 1500 rpm at 22°C) for 1 min. Eggs in the resultant pellet were washed with sterile distilled water, and then transferred onto a new NGM plate seeded with E. coli OP50. L3-L4 stage worms appearing on this plate after 2–3 days of incubation at 22°C were used for further experiments.

Three different types of in vivo assays were done as under, employing the methodology described in reference cited in parenthesis following the assay name:

Catechin (Sigma Aldrich; C1251-5G) and standard antibiotics (HiMedia; Ampicillin CMS645-1G; Gentamicin TC026-1G; Chloramphenicol CMS218-5G; Vancomycin CMS217-500MG) were used as positive controls. Catechin was employed at 100 µg/ml, whereas different sub-MIC concentrations (0.1–5 µg/ml) of the antibiotics were used against different organisms as per their susceptibility.

Videos of some of the in vivo assays were captured on the fifth day of the experiment, using an inverted microscope (Nikon Eclipse Ti) under 4X objective lens, wherein 100 µl of the liquid content from 24-well assay plate was transferred onto a large cover slip for observation and video capturing [see extended data (Patel et al., 2019c)].

In vitro assays

After confirming the in vivo anti-pathogenic efficacy of the TF, we performed following in vitro assays to gain insight into interaction of this formulation with the pathogenic bacteria, as per the methodology described in respective references mentioned in the parenthesis:

Effect of TF on probiotic strains

Bifidobacterium bifidum (NCDC 255), Enterococcus faecium (NCIM 5366), and Lactobacillus plantarum (MTCC 2621) were grown in Lactobacillus MRS broth (HiMedia GM369-500G) containing TF, in screw capped tubes at 37°C for 22-24 h. For B. bifidum, this medium was supplemented with 0.05% cysteine (HiMedia PCT0305-25G). Effect of TF (10–100 µg/ml) on these bacteria was interpreted by comparing their cell density [OD₆₅₅ measured with microplate reader (Biorad 680)] in TF-supplemented media to that in TF-free media.

Statistical analysis

All the experiments were performed in triplicate, and measurements are reported as mean ± standard deviation (SD) of 3 independent experiments. Statistical significance of the data was evaluated by applying t-test using Microsoft Excel^® 2013. p values ≤0.05 were considered to be statistically significant. Trial version of GraphPad Prism 7 was used to make Kaplan-Meier survival curve for worms.

In vivo experiments

Anti-infective assay. When all the five pathogens were pre-treated with 0.5-100 µg/ml of TF before being allowed to attack C. elegans, Triphala formulation (TF) was able to attenuate virulence of all test pathogens except S. pyogenes at ≤20 µg/ml [Figure 1; underlying data (Patel et al., 2019c)]. Worms challenged with TF-treated pathogens demonstrated 18–45.50% better survival than those challenged with TF-unexposed pathogens. Effect of catechin and standard antibiotics used as positive controls on bacterial virulence is shown in Figure 2 [underlying data (Patel et al., 2019c)].

Figure 1. Anti-infective activity of Triphala formulation against test pathogens.

TF-treatment attenuated virulence of four of the test pathogens towards C. elegans. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of any of the bacterium towards C. elegans; DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. (A) TF at 0.5 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml allowed 32%±5.10, 29%***±9.60, 41.9%***±2.40, 38.4%***±7.10, 39.9%***±9.40, and 41.4%***±7.10 better survival of the worm population respectively, when challenged with S. marcescens. Also see videos a-b submitted as part of extended data. (B) TF at 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml allowed 18%***±5.01, 40%***±7.01, 45.5%***±0, and 45%***±5.00 better survival of the worm population respectively, when challenged with P. aeruginosa. (C) TF at 20 µg/ml, 50 µg/ml and 100 µg/ml allowed 31.5%±***2.35, 41.5%***±2.35and 44.5%***±14.14 better survival of the worm population respectively, when challenged with S. aureus. (D) TF-treatment did not attenuate virulence of S. pyogenes towards the worms. (E) TF at 0.5µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml allowed 31%***±5.77, 29.5%***±8.81, 29%***±1.92, 29%***±1.92, 32.3%***±1.92, and 32.3%**±5.09 better survival of the worm population respectively, when challenged with C. violaceum. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation.

Figure 2. Anti-infective activity of positive controls (catechin and antibiotics) against test pathogens.

Catechin was able to reduce virulence of different test bacteria towards C. elegans by 50–100% (p≤0.05). Various standard antibiotics at sub-MIC level could reduce bacterial virulence by 55–100% (p≤0.05).

After confirming the anti-infective activity of TF, we investigated (as described in Patel et al., 2019a) whether this formulation exerts any post-extract effect (PEE - https://doi.org/10.32388/359873) on the test pathogens i.e. whether the virulence-attenuating effect suffered by the parent bacterial culture is retained even in their daughter population never receiving any direct exposure to TF. When the TF-treated bacteria were subsequently subcultured on TF-free media, their daughter populations were unable to exert virulence at par with that of control (DMSO-treated parent culture having no TF-exposure). In case of P. aeruginosa and S. aureus, this PEE lasted up to the second subculturing, whereas in the case of S. marcescens PEE lasted until first subculturing [Figure 3; underlying data (Patel et al., 2019c)].

Figure 3. Post extract effect of Triphala on test pathogens.

TF-treatment reduces the virulence of all the test pathogens towards C. elegans even after subculturing of cells in TF-free media. DMSO present in the ‘vehicle control’ at 0.5% v/v did not affect virulence of the bacteria towards C. elegans; DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. (A) S. marcescens obtained after first subculturing on TF-free media were able to kill 38%***±4.71 lesser worms than control; (B) P. aeruginosa obtained after first and second subculturing on TF-free media were able to kill 18.5%**±2.35 and 15.5%*±2.35 lesser worms respectively, than control; (C) S. aureus obtained after first and second subculturing on TF-free media were able to kill 31.5%***±2.35 and 23%***±0 lesser worms respectively, than control. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation.

TF as a post-infection therapy. To test the therapeutic efficacy of TF in already-infected worm population, we first allowed different pathogenic bacteria, not previously exposed to TF, to infect C. elegans either for 6 h or 24 h, and then exposed the infected worms to two different concentrations of TF. TF failed to exert any therapeutic effect on worms infected with P. aeruginosa. However, it could exert significant (p≤0.05) therapeutic effect on worms infected with C. violaceum or S. marcescens. Against S. aureus, TF was effective only if the worms were given TF-treatment early (i.e. 6 hour-post infection) [Figure 4; underlying data (Patel et al., 2019c)]. TF could also not rescue the worms if they already had a mixed infection (S. aureus and P. aeruginosa).

Figure 4. Assessing efficacy of Triphala formulation as a post-infection therapy in pre-infected worms.

DMSO (0.5%v/v) did not affect survival of pre-infected worms. DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. A1–D1: TF employed 6 h post-infection A2–D2: TF employed 24 h post-infection (A1) TF at 10 µg/ml, and 50 µg/ml used as therapy for already S. marcescens infected C. elegans after 6 h incubation conferred 31.5%***±2.35 and 28.5%***±2.35 survival benefit, respectively; (A2) TF at 10 µg/ml, and 50 µg/ml used as therapy for already S. marcescens infected C. elegans after 24 h incubation conferred 11.5%***±2.35 and 13%*** ±0.51 survival benefit, respectively; (B1–B2):TF could not rescue P. aeruginosa-infected worms when tested as post-infection remedy. (C1) TF at 20 µg/ml, and 50 µg/ml used as therapy for already S. aureus-infected C. elegans after 6 h incubation conferred 11.5%***±2.35 and 21.5%***±2.35 survival benefit, respectively; (C2) TF could not rescue S. aureus infected worms when tested as post-infection remedy. (D1) TF at 10 µg/ml, and 50 µg/ml used as therapy for already C. violaceum infected C. elegans after 6 h incubation conferred 18.5%***±2.35 and 28.5%***±2.35 survival benefit, respectively; (D2) TF at 10 µg/ml, 50 µg/ml used as therapy for already C. violaceum infected C. elegans after 24 h incubation conferred 22%***±2.35 and 28.5%***±2.35 survival benefit, respectively. (E) TF could not rescue the worms in face of mix-culture infection by S. aureus and P. aeruginosa, when tested as post-infection remedy. Survival benefit refers to the difference between number of worms surviving in experimental and control wells. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation.

Prophylactic potential of TF. To investigate whether previous feeding with TF can make the worm population tolerate subsequent challenge with pathogenic bacteria, not treated with TF, better; worms were first maintained in a TF-containing M9 buffer for 96 h, and then challenged with different bacterial pathogens. Such TF-fed worms scored 14.50–41.50% better survival in face of pathogen challenge [Figure 5; underlying data (Patel et al., 2019c)]. However, TF did not confer any prophylactic benefit on the worm population against mix-culture challenge of P. aeruginosa and S. aureus. Since prophylactic activity of any formulation can be said to stem mainly from its effect on the host, we also compared whether TF imparts any extension of longevity on the worm. Worms fed with TF (10–20 µg/ml) registered marginally better survival up to 11 days (Figure 6).

Figure 5. TF-pre-exposed C. elegans exhibit better resistance to subsequent bacterial challenge.

Pre-treatment of worms with DMSO (0.5%v/v) did not alter their susceptibility to subsequent challenge with pathogenic bacteria. DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. Among positive controls, catechin (100 μg/ml) pre-treatment conferred 23%±0 protection on worm population against subsequent S. marcescens, P. aeruginosa, S. aureus, and C. violaceum challenge; (A) Ampicillin (5 μg/ml) pre-treatment conferred 26%±0 protection on worm population against subsequent S. marcescens challenge; TF (100 µg/ml) pre-treatment conferred 18%***±2.35 protection on fifth day worm population against subsequent S. marcescens challenge; (B) Gentamicin (0.5 μg/ml) pre-treatment conferred 26% protection on worm population against subsequent P. aeruginosa challenge; TF pretreatment at 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml, conferred 18%***±0, 33%***±2.35, 38%***±2.35, 41.5%**±0 and 34.5%***±2.35 protection on worm population against subsequent P. aeruginosa challenge (C) Vancomycin (0.1 μg/ml) pre-treatment conferred 26% protection on worm population, TF pre-treatment at 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml, conferred 14.5%***±2.35, 13%***±4.71, 11.5%***±2.35, 21.5%***±2.35, and 33%***±0 protection on worm population against subsequent S. aureus challenge (D) Chloramphenicol (0.5 μg/ml) pre-treatment conferred 26% protection on worm population against C. violaceum; TF pre-treatment at 0.5 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml, conferred 14.5%***±0, 19.5%***±4.71, 14.5%***±2.35, 28%***±7.07, 34.5%***± 2.35 and 35.5%***±0 protection on worm population against subsequent C. violaceum challenge. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation.

Figure 6. Triphala formulation imparts marginal longevity extension on C. elegans.

Worms fed with TF (10 µg/ml or 20 µg/ml) scored 11.5%***±1.20, and 16%***±0.96 better survival on 11^th day. All worms (not fed with TF) in control were dead by the 11^th day. DMSO (0.5%v/v) at tested concentration had no effect on worm longevity. ***p≤0.001; TF: Triphala Formulation.

Repeated exposure of test pathogens to TF. Since one of the major challenges with even the most potent antimicrobial agents/formulations is development of resistance against them by the pathogen populations, we tested whether repeated exposure of the test pathogens to TF can induce any resistance in them. For this, we subcultured two of the gram-negative test pathogens (P. aeruginosa and S. marcescens) in TF (50 µg/ml)-containing broth for 10 subsequent times, and then the 'TF-habituated' cultures thus obtained were tested for their virulence towards the nematode host. Since antibiotic-resistant strains of P. aeruginosa and Entrerobacteriaceae (to which S. marcences belongs) are recognized as serious threats (https://www.cdc.gov/drugresistance/biggest-threats.html; https://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf), these two bacteria were used in this assay. Since higher concentration of antimicrobial formulations can be expected to exert higher selection pressure on the susceptible bacterial population, we chose the highest TF concentration (50 µg/ml) beyond which TF does not exert any statistically superior anti-infective effect.

Repeated TF-exposure was found to induce resistance in S. marcescens [Figure 7A; underlying data (Patel et al., 2019c)]. Though TF-habituated P. aeruginosa could kill more worms than its counterpart receiving single TF-exposure, it still could not kill as many worms as TF-unexposed P. aeruginosa [Figure 7B; underlying data (Patel et al., 2019c)]. These results indicate that it may be difficult for the pathogenic bacteria to develop complete resistance against polyherbal formulations like Triphala, but not impossible. These results indicate that it may be difficult for the pathogenic bacteria to develop complete resistance against polyherbal formulations like Triphala, but not impossible. Though our previous results on other polyherbal formulations (Joshi et al., 2019; Patel et al., 2019a) and multicomponent crude plant extracts (Joshi, 2019) have indicated that the probability of pathogens developing resistance against multi-component anti-virulence preparations is low, such a probability can certainly not be ruled out (Kalia et al., 2014; Singh, 2014)

Figure 7. Effect of repeated exposure to Triphala formulation on test pathogens.

DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worms. (A) C. elegans challenged with TF (50 μg/ml) treated- S. marcescens registered 35%***±2.35 better survival. No survival benefit was available to C. elegans, when challenged with S. marcescens subcultured 5 times or 10 times on TF (50 μg/ml) containing media. (B) P. aeruginosa obtained after 5 and 10 subculturings in TF (50 μg/ml)-containing media were able to kill 21.5%***±2.35 and 21%***±7.07 lesser worms respectively, as compared to control (DMSO-treated) bacterial population. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation.

In vitro experiments

Since TF showed significant in vivo anti-pathogenic potential in the C. elegans model, we performed various in vitro experiments to gain insight into its interaction with the target pathogens. TF was able to modulate production of quorum sensing (QS)-regulated pigments in all the three gram-negative bacteria [Figure 8; underlying data (Patel et al., 2019c)]. It did so with S. marcescens without affecting its growth, which is characteristic of an ideal anti-virulence agent i.e. attenuation of virulence without exerting any selection pressure on the susceptible pathogen. However, TF did exert a growth-inhibitory effect on P. aeruginosa, wherein its IC₅₀ was observed to be near 50 µg/ml. The quorum modulatory effect of TF on pigment production in P. aeruginosa was observed not to be amenable to be described by a linear dose-response curve. It seems to fall within the realm of hormesis (Calabrese, 2004). For example, production of both pigments was not affected maximally at the highest test concentration. Pyocyanin production was inhibited more at 0.5 µg/ml than at 20 µg/ml. Effect of TF on pyoverdine production followed a linear threshold model within concentration range of 0.5–50 µg/ml, but it took an inverted-U shape over 20-100 µg/ml. Though the exact mechanism responsible for a non-linear dose-response relationship is not known, it may be the varying magnitude of bacterial adaptive response at different concentrations of the test agent, which generates such non-linear response curves (Lushchak, 2014).

Figure 8. Effect of Triphala on growth and QS-regulated pigment production in test pathogens.

Bacterial cell density was quantified as OD_750. DMSO (0.5%v/v) in the vehicle control did not affect growth and pigment production in any of the test pathogens. Pigment Units were calculated to nullify the effect of any change in cell density on pigment production (A) S. marcescens: OD of prodigiosin was measured at 490 nm, and Prodigiosin Unit was calculated as the ratio OD₄₉₀/OD₇₅₀ (an indication of prodigiosin production per unit of growth). Catechin (100 µg/ml) inhibited prodigiosin production by 13.05%*±0.10 without affecting bacterial growth; Ampicillin (5 µg/ml) inhibited growth and prodigiosin production by 8.48%**±0.02 and 40.60%*±0.23, respectively. (B) P. aeruginosa: OD of pyoverdine and pyocyanin was measured at 405 nm and at 490 nm. Pyoverdine Unit and Pyocyanin Unit was calculated as the ratio OD₄₀₅/OD₇₅₀ and OD₄₉₀/OD₇₅₀ (an indication of pyoverdine and pyocyanin production per unit of growth). Catechin (100 µg/ml) inhibited, pyoverdine and pyocyanin production by 3.85%*± 0.38 and 12.74%*± 2.60 respectively without affecting bacterial growth; Gentamicin (0.5 µg/ml) inhibited, pyoverdine and pyocyanin production by 10.53%*±2.07 and 57.93%***±6.47 without affecting bacterial growth; (C) C. violaceum: OD of violacein was measured at 570 nm, and Violacein Unit was calculated as the ratio OD₅₇₀/OD₇₅₀ (an indication of violacein production per unit of growth). Catechin (100 µg/ml) did not affect growth as well as violacein production; Chloramphenicol (0.5 µg/ml) inhibited growth by 40.31**%±0.44 without affecting violacein production. (D) S. aureus: OD of staphyloxanthin was measured at 450 nm, and Staphyloxanthin Unit was calculated as the ratio OD₄₅₀/OD₇₅₀ (an indication of staphyloxanthin production per unit of growth). Catechin (100 µg/ml) and vancomycin (0.1 µg/ml) did not affect growth as well as staphyloxanthin pigment production. (E) S. pyogenes: TF or catechin (100 µg/ml) did not affect the growth when measured as OD₆₅₅; Chloramphenicol (0.5 µg/ml) inhibited growth by 7.56%**±3.46. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation; QS: Quorum Sensing.

We also tested the effect of TF on two important virulence traits of the bacterial pathogens i.e. haemolytic activity, and biofilm. Though TF could not curb haemolytic activity of any of the gram-negative bacteria, this activity of S. aureus was heavily inhibited under the influence of TF [Figure 9; underlying data (Patel et al., 2019c)]. While P. aeruginosa biofilm was not affected by TF, TF was able to reduce biofilm formation by S. marcescens, and S. aureus. When TF was applied on pre-formed bacterial biofilms, it seemed to enhance synthesis of the biofilm matrix material (quantified thorough crystal violet assay), and also the metabolic activity (measured in terms of organism's ability to reduce MTT) of the bacterial biofilm [Figure 10; underlying data (Patel et al., 2019c)]. It may be speculated that TF-treatment induces stress in the bacterial population residing in biofilm form, and this causes the bacteria to mount stress-response. Slow metabolism is a general characteristic of bacterial biofilms (Singh et al., 2017), but TF seems to have forced the biofilms of two of our test bacteria to enhance the rate of their metabolic activity, as well as synthesis/secretion of biofilm matrix components (e.g. polysaccharides, proteins, and extracellular DNA). Enhanced production of exopolysaccharide and e-DNA is believed to occur in stressed bacterial populations (Chang et al., 2007; Zatorska et al., 2018). Sub-inhibitory concentrations of beta-lactam antibiotics have been reported to induce extracellular DNA release and biofilm formation in some S. aureus strains (Kaplan et al., 2012).

Figure 9. Effect of Triphala on haemolytic activity of test pathogens.

TF had no effect on hemolytic activity of S. marcescens (A), P. aeruginosa (B), and C. violaceum (C). However, it curbed haemolytic potential of S. aureus notably. Hemoglobin released as a result of haemolysis was quantified as OD₄₉₀; 1% triton (OD₄₉₀ = 1.2), and PBS (pH 7.4) were used as positive and negative control respectively; *p<0.05, ***p<0.001; TF: Triphala Formulation; PBS: Phosphate Buffer Saline.

Figure 10. Effect of Triphala formulation on biofilm of test pathogens.

Crystal violet assay was performed to quantify biofilm formation or eradication, wherein amount of this dye retained by the biofilm was read at 570 nm after extracting it in ethanol. MTT assay was performed to quantify biofilm viability (metabolic activity), wherein change in colour of the MTT dye owing to bacterial metabolism was read at 570 nm. Catechin (100 µg/ml) for all test bacteria, ampicillin (5 µg/ml) for S. marcescens, gentamicin (0.5 µg/ml) for P. aeruginosa, and vancomycin(0.1 µg/ml) for S. aureus were used as positive controls.

(A)Effect of TF on S. marcescens biofilm. Catechin inhibited S. marcescens biofilm formation by (29.80±9.38)%***, eradicated pre-formed biofilm by (49.60 ±12.00)%*, and reduced metabolic activity of biofilm by (86.20 ±0.88)% ***. Ampicillin inhibited all three by 29.25*** ±7.30, 54.23*** ±3.67, 86.11*** ±0.21 for S. marcescens. (B) Effect of TF on P. aeruginosa biofilm. Catechin inhibited P. aeruginosa biofilm formation by (22.18±1.16)%***, eradicated pre-formed biofilm by (30.54 ±3.50)%**, and enhanced metabolic activity of biofilm by (32.27 ± 4.74)%***. Gentamicin did not affect biofilm formation, eradicated pre-formed biofilm by (23.27±4.91)%***, reduced metabolic activity of biofilm by (123.99 ±26.81)% ***. (C) Effect of TF on S. aureus biofilm. Catechin inhibited S. aureus biofilm formation by (26.24±7.35)%***, enhanced pre-formed biofilm by (22.54 ±10.90)%**, and enhanced metabolic activity of biofilm by (177.71 ± 16.49)% ***. Vancomycin inhibited biofilm formation by (41.53±5.49)% ***, eradicate pre-formed biofilm by (42.37±11.21)%***, enhanced metabolic activity of biofilm by (70.90 ±5.10)% ***. *p<0.05, **p<0.01, ***p<0.001; TF: Triphala Formulation.

During the host-pathogen interaction, host defense mechanisms play a determinant role in deciding the outcome of this interaction. Since lysozyme is an important component of the innate defense machinery of human immune system against invading microbes (Herbert et al., 2007), we also studied whether TF can have any effect on susceptibility of the test pathogens to lysozyme. TF-treated cells of S. marcescens and S. aureus were found to suffer marginal (albeit statistically significant; p≤0.05) increase in their susceptibility to lysis by lysozyme. P. aeruginosa's lysozyme-susceptibility was found to increase heavily (by ~25-43%) upon TF-pretreatment [Figure 11; underlying data (Patel et al., 2019c)].

Figure 11. Triphala formulation increases susceptibility of test pathogens to lysozyme.

(A) S. aureus; (B) S. marcescens; (C) P. aeruginosa *p≤0.05 ***p≤0.001.

Most conventional antibiotics suffer from an inherent limitation of not being selectively inhibitory to pathogenic bacteria, and they simultaneously inhibit resident bacterial members of the human microbiome; which may lead to gut dysbiosis (Wipperman et al., 2017). Thus an ideal anti-pathogenic formulation should exert anti-pathogenic effects without inhibiting indigenous members of human microbiome. We tested TF's effect on three such bacteria (Enterococcus faecium, Bifidobacterium bifidum, and Lactobacillus plantarum) which are part of human microbiome, and also used as probiotic strains. Though TF did not exert any prebiotic potential by promoting growth of the probiotic bacteria, it also had no negative effect on them [Extended data: Figure S1 (Patel et al., 2019c)].

Underlying data

Figshare: Anti-pathogenic potential of a classical ayurvedic formulation- Triphala. https://doi.org/10.6084/m9.figshare.8052143.v2 (Patel et al., 2019c)

Extended data

Figshare: Anti-pathogenic potential of a classical ayurvedic formulation- Triphala. https://doi.org/10.6084/m9.figshare.8052143.v2 (Patel et al., 2019c)

This project contains the following extended data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).