The antidiarrhoeal evaluation of Psidium guajava L. against enteropathogenic Escherichia coli induced infectious diarrhoea

Journal Pre-proof The antidiarrhoeal evaluation of Psidium guajava L. against enteropathogenic Escherichia coli induced infectious diarrhoea Jayshri R. Hirudkar, Komal M. Parmar, Rupali S. Prasad, Saurabh K. Sinha, Amarsinh D. Lomte, Prakash R. Itankar, Satyendra K. Prasad PII:

S0378-8741(19)31047-5

DOI:

https://doi.org/10.1016/j.jep.2020.112561

Reference:

JEP 112561

To appear in:

Journal of Ethnopharmacology

Received Date: 15 March 2019 Revised Date:

13 October 2019

Accepted Date: 8 January 2020

Please cite this article as: Hirudkar, J.R., Parmar, K.M., Prasad, R.S., Sinha, S.K., Lomte, A.D., Itankar, P.R., Prasad, S.K., The antidiarrhoeal evaluation of Psidium guajava L. against enteropathogenic Escherichia coli induced infectious diarrhoea, Journal of Ethnopharmacology (2020), doi: https:// doi.org/10.1016/j.jep.2020.112561. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

The antidiarrhoeal evaluation of Psidium guajava L. against enteropathogenic Escherichia coli induced infectious diarrhoea Jayshri R. Hirudkara, Komal M. Parmara, Rupali S. Prasada, Saurabh K. Sinhab, Amarsinh D. Lomtea, Prakash R. Itankar,a, Satyendra K. Prasada,* a

Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University,

Nagpur, Maharashtra-440033, India b

Department of Pharmaceutical Sciences, Mohanlal Shukhadia University, Udaipur,

Rajasthan-313001, India

* For Correspondence Dr. Satyendra K. Prasad Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India Phone No- , +919604421621 Email: [email protected] 1

Abstract Ethnopharmacological relevance: The plant Psidium guajava L. (Myrtaceae), commonly used as an edible fruit is traditionally used worldwide in treatment of various gastrointestinal problems including diarrhoea. The leaves of the plant have also been evaluated for antidiarrhoeal activity in various chemical induced diarrhoea models. Objective: The main objective of the present study was to evaluate the potency of P. guajava leaves and its major biomarker quercetin against enteropathogenic Escherichia coli (EPEC) induced infectious diarrhoea using preclinical and computational model. Material and methods: P. guajava alcoholic leaf extract (PGE) was initially standardized using HPLC taking quercetin as a biomarker and was then subjected to antidiarrhoeal evaluation on rats in an EPEC induced diarrhoea rat model. The study included assessment of various behavioral parameters, initially for 6 h and then for up to 24 h of induction which was followed by estimation of stool water content, density of EPEC in stools and blood parameters evaluation. The colonic and small intestinal tissues of the treated animals were subjected to various biochemical estimations, in vivo antioxidant evaluation, estimation of ion concentration, Na+/K+–ATPase activity, assessment of pro-inflammatory cytokines and histopathological studies. Further, the major biomarker off PGE, quercetin was subjected to molecular docking studies with Na+/K+–ATPase and EPEC. Results: The results demonstrated a significant antidiarrhoeal activity of quercetin (50 mg/kg,, PGE at 200 and 400 mg/kg, p.o., where quercetin and PFGE at 200 mg/kg, p.o. were found to be more prominent, as confirmed through higher % protection, water content of stools and density of EPEC in stools. PGE and its biomarker quercetin also significantly recovered the WBC, Hb, platelets loss and also revealed a significant restoration of altered antioxidants level, pro-inflammatory cytokines (IL-1β and TNF-α) expression and had positive influence on Na+/K+–ATPase activity. The docking studies of quercetin with 2

Na+/K+–ATPase showed favourable interactions and residues Glu 327, Ser 775, Asn 776, Glu 779 and Asp 804 of Na+/K+–ATPase were adequately similar to quercetin for donating ligands for binding, while quercetin was also found to terminate the linkage between mammalian cells and EPEC thus, preventing further infection from EPEC. Conclusion: Inhibition in intestinal secretion, reduced nitric oxide production and inflammatory expression along with reactivation of Na+/K–ATPase activity could be attributed to the observed antidiarrhoeal potential of PGE against infectious diarrhoea, where quercetin was confirmed to be the main contributing factor.

Keywords: Antidiarrhoeal activity, diarrhoea score, enteropathogenic Escherichia coli, Na+/K+–ATPase, Psidium guajava, quercetin

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MANUSCRIPT The antidiarrhoeal evaluation of Psidium guajava L. against enteropathogenic Escherichia coli induced infectious diarrhoea

1. Introduction Diarrhoea is defined as a disorder linked with increased volume or fluidity of stools, increased frequency of defecation and is mostly accompanied by cramps. Most recent evidences have shown that, the disorder affects almost 3–5 billion people every year and accounts for about 5-6 million deaths among children younger than 5 years of age. Due to poor and compromised hygiene, shortage of safe drinking water and malnutrition, diarrhoea has been a major concern for developing countries like India (Thapar and Sanderson, 2004; Palla et al., 2015; Prasad et al., 2017). Pathogenic Escherichia coli, Shigella spp and Vibrio cholera, are considered to be the most common causative agent of diarrhoea in humans, which contributes to about 14–17% in developing countries. The other major causative organisms includes Salmonella spp, Campylobacter spp and Yersinia spp (Yakubu and Salimon, 2015; Thapar and Sanderson, 2004). Even though, antibiotics have been found effective in treatment of various infectious diseases including diarrhoea, however their inappropriate, irregular and continuous use may lead to development of resistant microbes resulting in decreased efficacy of regimens. Therefore, people are shifting their focus towards medicinal plants for fulfilment of their basic healthcare needs and also to combat against various ailments (Barbour et al., 2004; Parmar et al., 2019). The plant Psidium guajava L. (Myrtaceae) commonly known as guava, native to Caribbean, and Central and South America, is cultivated as food and is medicinally used throughout the tropical and subtropical region worldwide (Piña-Vázquez et al., 2017; 4

Machado et al., 2018). Traditionally, the plant has been used globally in treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, indigestion, inflammation, ulcers, as antiamoebic and as antispasmodic (Gutiérrez et al., 2008; Tetali et al., 2009; Birdi et al., 2010; Koriem et al., 2019). Pharmacological studies have reported its antitrypanosoma, antileishmania, cytotoxic, antimalarial, antidiarrhoeal and antiparasitic activities (de Souza et al., 2017; Machado et al., 2018). Phytochemical evaluations have reported the presence of gallic acid, rutin, morin, morin-3-O-lyxoside, morin-3-O arabinoside, quercetin and quercetin-3-O-arabinoside as major active constituents in the plant (Koriem et al., 2019). The leaves of the plant P. guajava have been reported for its antidiarrhoeal property in various chemical induced diarrhoea model (Ojewole et al., 2008; Lin et al., 2002) and have also been reported for its efficacy against Citrobacter rodentium (a common mouse pathogen) induced infectious diarrhoea, which is specific to mouse (Gupta and Birdi, 2015). Literature has also revealed the in vitro antibacterial potential of P. guajava against pathogen susceptible to diarrhoea (Birdi et al., 2010). Further, infectious diarrhoea has been a major concern for developing country like India, attributing to third largest cause of death among children throughout the world (Prasad et al., 2013). Therefore, keeping the above view into consideration, the present investigation was undertaken to evaluate the potency of standardized P. guajava leaf extract and its biomarker quercetin in treatment of infectious diarrhoea.

2. Materials and methods 2.1. Plant material and extraction The leaves of plant P. guajava were collected from the medicinal plant garden of Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India and were authenticated by Dr. Nitin Dongarwar, Department of 5

Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India. The voucher specimen (No.: 1031) of the plant has been deposited in our Department for future reference. The collected leaves (500 g) of the plant were dried in shade, coarsely powdered and was extracted with ethanol (2.0 L) using maceration method for a period of five days. The extract so obtained, was concentrated and evaporated under reduced pressure using rotary evaporator (BUCHI India Pvt. Ltd, Mumbai, India) and then was kept in desiccator until use (yield: 6.33% w/w).

2.2. Phytochemical standardization The ethanol extract of P. guajava was further subjected to preliminary phytochemical screening to identify the presence of various phytoconstituents following the standards procedure (Trease and Evans, 2002). Further, quantitative estimations of identified phytoconstituents were done which included determination of total alkaloid content following gravimetric analysis (Wagner and Bladt, 1996). Total carbohydrate content in the extract was determined by using anthrone reagent as described by Yemm and Willis (1954), while total phenolics and total tannin contents were estimated following the method proposed by Hagerman et al. (1998) using Folin ciocalteau reagent. Total flavonoid content in the leaf extract was determined by adopting the aluminium chloride method (Kumaran and Karunakaran, 2006), whereas total saponin content was estimated taking diosgenin as standard using sulphuric acid reagent, (Prasad et al., 2013). P. guajava extract (PGE) was than standardized using quercetin (Sigma-Aldrich, St. Louis, MO, USA, purity 95%) as a biomarker compound with the help of High Performance Liquid Chromatography (HPLC) after confirming its presence through Thin Layer Chromatography. The separation was carried out on a Cosmosil C18 column (150 mm × 4.6 mm, 5 µm particle), where the mobile phase consisted of a mixture prepared from methanol 6

and water containing 0.1% formic acid in the ratio of 80:20 (Al-Rifai et al., 2015). A stock solution of sample (PGE) at 0.5 mg/ml and standard quercetin at 0.1 mg/ml was prepared in methanol. The flow rate was kept at 1.0 mL/min, while the injection volume was kept at 10 µL. The data was collected at wavelength 350 nm while the peaks were identified by comparing its retention time with that of standard.

2.3. Experimental animals The experimental animals for this study included healthy Wistar rats of either sex weighing between 150 to 200 g and were obtained from the Central Animal House (Reg. No.: 92/1999/CPCSEA, dated: 28/04/1999) of Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India. The animals were kept under standard conditions of light and dark cycle and at an ambient temperature of 25 ±1°C with relative humidity of 45-55%. The rats were allowed to acclimatize for 7 days to the environment before the commencement of the experiment and were fed with commercially available rat feed and water ad libitum. All experimental protocols were approved from the Central Animal Ethical Committee of Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur (Letter No.: IAEC/UDPS/2017/43 dated 14/08/2017) and were conducted according to the accepted standard guidelines of National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85–23, revised 1985).

2.4. Induction of diarrhoea For induction of diarrhoea, the rats were initially fasted for 6 h, and were then divided into two groups, i.e. normal control group and diarrhoeal model group. The rats in the normal control group were administered sterile water by gavage (1 ml), while the diarrhoea model group was administered with suspension (1 ml) of enteropathogenic Escherichia coli (EPEC) 7

[MTCC-724], at a concentration of 3.29×109 CFU/ml procured from Microbial Type Culture

Collection (MTCC) Chandigarh, India, as per the procedure described previously (Parmar et al., 2019; Kamgang et al., 2006). The rats were then kept under observation for any symptoms of diarrhoea, which initiated after 40-50 min.

2.5. Grouping of animals After the induction of diarrhoea, the rats were divided into seven groups that included,: Group 1: normal control rats administered with normal saline (1 ml/kg, p.o.), Group 2: diarrhoea control group administered with normal saline, Group 3: diarrhoeal rats treated with PGE at 100 mg/kg, p.o., Group 4: diarrhoeal rats treated with PGE at 200 mg/kg, p.o., Group 5: diarrhoeal rats treated with PGE at 400 mg/kg, p.o., Group 6: diarrhoeal rats treated with quercetin at 50 mg/kg, p.o. (Gálvez et al., 1993) procured from Sigma-Aldrich, St. Louis, MO, USA and Group 7: diarrhoeal rats treated with norfloxacin at 5.7 mg/kg, p.o., which was used as a standard drug. PGE, its biomarker quercetin and standard norfloxacin were administered as suspension prepared by using 0.5% carboxymethyl cellulose and were administered per orally one hour after EPEC administration. The dose of the PGE was decided as per acute oral toxicity study (data included as supplementary document) and literature available on the leaves of the plant (Lin et al., 2002).

2.6. Behavioral evaluations After grouping of animals, they were kept separately into cages containing plastic sheets at the base of the cage and were kept under observation of various behavioral parameters for 6 h initially and then for up to 24 h as described previously (Kamgang et al., 2015; Parmar et al., 2019).

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2.7. Estimation of stool water content The water (moisture) content in stool was measured at 6th and 24th h following PGE administration. The stools were initially weighed followed by drying them at 37 °C in an incubator for about 48 h. The difference obtained between the initial wet weight and final dry weight of stools was used to calculate the % of water content in the stools (Parmar et al., 2019; Prasad et al., 2017).

2.8. Estimation of the level of EPEC in stools Stool samples, collected at 2nd, 4th, 6th, and 24th h after PGE treatment was used to determine the level of EPEC in stools. About 0.5 g of feces was homogenized in 4.5 ml of sterile saline, which was further serially diluted and from this, 500 µl of each dilution was spread over Salmonella Shigella Agar (SS agar) plate. The plates were then incubated for 24 h at 37 °C and the number of CFU was calculated (Kamgang et al., 2015).

2.9. Blood cells count After 24 h of PGE treatment, blood was withdrawn from the retro-orbital plexus of each animal. The collected blood was then used for determination of white blood cells (WBCs), red blood cells (RBCs), platelets (PCs) and haemoglobin level as per the standard procedure described previously (Kamgang et al., 2015).

2.10. Biochemical analysis and determination of ion concentration The rats were sacrificed after 24 h of PGE treatment using intraperitoneal administration of thiopental sodium (65 mg/kg) and the colonic portion of the rats were dissected out, removed and rinsed with the tyrode solution. The tissue samples was homogenized in phosphate buffer followed by centrifugation and the supernatant was used 9

for determination of different biochemical parameters such as nitric oxide [NO] (Kamgang et al., 2015), total carbohydrate (Yemm and Willis, 1954), total DNA (Burton, 1956) and total protein content (Lowry et al., (1951) following the standard procedures described previously. The Levels of antioxidants viz. superoxide dismutase [SOD], catalase [CAT] and lipid peroxidation [LPO] (Laloo et al., 2013) were also determined in vivo, in tissues samples to ascertain the antioxidant status after treatment. The study also includes estimation of liver enzymes such as alkaline phosphatase [ALP], aspartate transaminase [AST], alanine transaminase [ALT] by employing the methods previously described (Sengar et al., 2015). The supernatant from the tissue homogenate was also analysed for determination of ion concentration i.e. Cl-, Na+, K+, and Ca2+ in the treated animals using Nulyte Electrolyte Analyzer (Tech Medisystems, Chandigarh, India) as per the instructions given by manufacturer.

2.11. Measurement of IL-1β, and TNF-α The colonic tissues of rats were also subjected to determination of level of proinflammatory cytokines IL-1β and TNF-α following enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits (Komabiotech, Korea) as per manufacturers guidelines.

2.12. Determination of Na+/K+–ATPase activity After sacrificing the animals, 24 h following PGE treatment, small intestine was dissected out, rinsed, homogenized and the supernatant was collected following the method described by Gal-Garber et al. (2003). The supernatant so obtained was then subjected to Na+/K+–ATPase assay as per the method adopted by Parmar et al. (2019).

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2.13. Histopathological studies Some portion of the colons were immediately blotted, dried and was fixed in 10% formalin for histopathological examination. The tissue samples were dehydrated in acetone and were embedded in paraffin wax followed by sectioning (4 µm) using microtome. The staining of the tissue sections were done using hematoxylin-eosin (H & E) and then were subjected to examination under microscope (Leica DM-2000, Leica, Germany).

2.14. Molecular docking study To study the interactions of quercetin with Na+/K+–ATPase and EPEC, docking simulations were done on the 3-Dstructure of Na+/K+–ATPase (PDB Code: 3B8E) and crystal structure of C-terminal 282-residue fragment of EPEC intimin (Organism: E. coli O127:H6, strain E2348/69 / EPEC) (PDB Code: 1F00). Molecular docking simulations were performed using version 1.5.6rc3 of the program AutoDock along with AutoDockTools using the Lamarckian Genetic Algorithm (LGA). The preparation of proteins and quercetin input structure and the description of the binding sites were carried out under a GRID-based procedure; a rectangular grid box (70×114×68 ˚A3) was constructed with a spacing of 0.375 ˚A. The structure of Na+/K+–ATPase and EPEC was pre-processed, refined, optimized and cleaned of its water molecules. Resulting docking orientations within 1.0 ˚A in the root mean square deviation lenience of each other were clustered together and represented by the result with the most favourable binding energy (Morth et al., 2007).

2.15. Statistical analysis All the results presented in the investigation are represented as mean ± S.E.M., with six animals in each group, which was followed by One Way Analysis of variance (ANOVA). Post-hoc Newman–Keuls multiple comparison test was used for determining the statistical 11

significance between different groups. However, Two Way ANOVA followed by Bonferroni’s post-hoc test was performed for determining the water content in stools and EPEC density in stools. GraphPad Prism, software version 5 (San Diego, CA, USA) was preferred for all statistical analysis and P<0.05 were considered to be significant.

3. Results 3.1. Phytochemical standardization Preliminary phytochemical screening and quantitative estimation of PGE revealed the presence of mainly flavonoids, phenols, tannins, alkaloids, carbohydrates and steroids as a major component while saponins were reported to be in minor quantity. Total phenolic and tannin content in PGE was reported to be 186.63 ±8.25 mg/g gallic acid equivalent and 40.44 ±2.16 mg/g tannic acid equivalent respectively. Total flavonoid and saponons quantified in PGE were 335.41 ±6.23 mg/g rutin equivalent and 16.35 ±1.63 mg/g diosgenin equivalent, while total alkaloid and carbohydrates estimated in PGE was reported to be 0.41 ±0.03% w/w and. 65.05 ±3.02 mg/g d-fructose equivalent respectively, The HPLC analysis of PGE revealed a well resolved and sharp peak of quercetin at Rt of 4.3 min, which was found to be similar to standard quercetin, thus confirming its presence. From the HPLC analysis, the quantity of quercetin in PGE was reported to be 6.78% w/w (Figure 1).

3.2. Antidiarrhoeal evaluation Administration of EPEC to rats resulted in initiation of diarrhoea, 40 to 50 min after its administration, where it was found to be more severe after 3rd hour as confirmed through higher aggressiveness among rats from negative control group. From the behavioral analysis, it was observed that, treatment with quercetin, PGE at 200 and 400 mg/kg, p.o. demonstrated a highly significant recovery from diarrhoea. More specifically, the results demonstrated 12

more pronounced antidiarrhoeal potential of quercetin and PGE at 200 mg/kg, p.o., which was confirmed through significant (p< 0.05) reduction in the total number of diarrhoeal stools, weight of stools and mean defecation rate of stools taken after 6th and 24th h of treatment. Further, the obtained diarrhoea score and % protection also justified the protective nature of quercetin and PGE more at 200 mg/kg, p.o. and was quite comparable with standard treated group (Table 1). PGE and its biomarker quercetin also showed a significant decline in the water content in stools of rats after 6th and 24th h (Figure 2) and also revealed a significant decline the level of EPEC in stools after 4th h of treatment (Figure 3). From the overall observation, the rats treated with PGE at 400 mg/kg, p.o. demonstrated almost similar effect to that of PGE at 200 mg/kg, p.o. therefore, we may presume that PGE showed ceiling effect at 200 mg/kg, p.o. and hence, taking above results into consideration, further, evaluations were carried out only on the most effective dose level of PGE i.e. at 200 mg/kg, p.o. From the results, it was observed that, EPEC caused a significant decline in the blood parameters evaluated viz. WBC, Hb and platelet cells as observed in EPEC induced negative control group rats. However, on treatment with PGE at 200 mg/kg, p.o. and quercetin, there was a significant recovery in WBC, Hb and platelets loss, while no significant changes in the level of RBC was observed in treatment group (Table 2). The biochemical estimations revealed a significant increase in the nitric oxide level of EPEC induced diarrhoeal rats, which was found to decline significantly on treatment with PGE and quercetin. The cellular proliferative factors such as protein, DNA and carbohydrates evaluated in the present study were also found to significantly increase on treatment with PGE and quercetin. Further, it was observed that, EPEC caused a significant decline in the levels of in vivo antioxidant enzymes SOD and CAT, while a significant elevation in level of LPO was observed. However, on treatment with PGE and quercetin, there was a significant restoration of altered antioxidant status of treated animals (Table 3). The liver enzymes 13

evaluated in the study revealed no significant difference in their levels as compared to EPEC control group (Data not included). Figure 4 represents the effect of PGE and quercetin treatment on pro-inflammatory cytokines IL-1β and TNF-α, where EPEC caused a significant increase in the expression of these cytokines. However, on treatment with PGE, a significant (p < 0.05) decline in the expression of IL-1β and TNF-α was observed. The results from the ion concentration analysis revealed a significant decline in the levels of Cl-, Na+, and K+ in EPEC control group, whereas on treatment with PGE and quercetin, a significant recovery from the ionic loss in the colonic tissue was observed. Further, there were no significant changes in the level of Ca2+ ion among all the tested groups (Table 4). The results obtained from the Na+/K+–ATPase assay, showed a significant decline in the enzyme activity on administration of EPEC, which was confirmed after comparing it with normal control rats. Nevertheless, on treatment with PGE and quercetin, there was a significant recovery in the enzyme activity and was found to be higher, even when compared with normal control rats, while maximum effect was observed in standard norfloxacin treated group (Figure 5). In histopathological examination of colons, distinct and intact epithelia with normal glands were observed in case of normal control rat colons, while EPEC induced rat colons were found to be disturbed and distracted due to necrosis. However, on treatment with PGE and its biomarker quercetin, a very less destruction of epithelia was observed confirming the protective nature of PGE and quercetin in pathogenic diarrhoea (Figure 6).

3.3. Molecular docking study To study the binding interactions of quercetin with Na+/K+–ATPase, we have performed simulation study on two sites of enzyme, first on Rb+ binding site and for better 14

outcome later on C-terminal cavity of α- subunit of Na+/ K+–ATPase. Docking simulations of quercetin revealed that, ligand interacted with Pro 326, GLu 327, Ser 775, Asn 776, Glu 779, Asp 804, Asp 808 amino acids (Figure 7A) and the hydroxyl group of quercetin showed Hbonding with Asn 776 on Rb+ binding site (Figure 7B). In C-terminal cavity quercetin showed interactions with Met 845, Arg 933, Arg 934, Arg 938, Arg 1003, Arg 1004, Arg 1005, Glu 1014, Tyr 1016 amino acids (Figure 7C) and one hydroxyl group of quercetin showed bi-hedral H-bonding with Arg 998 and second hydroxyl group with Gln 849 (Figure 7D). Quercetin was further simulated with C-terminal 282-residue fragment of EPEC intimin, where it showed interactions with Cys 860, Cys 937, Gln 781, Ser 808, Lys 866, Lys 861, Gly 864, Ser 841 and Asn 862 (Figure 7E).

4. Discussion The most common causative agents for infectious diarrhoea mainly includes E. coli, Shigella spp and V. cholera that mostly spread through the fecal-oral route (Prasad et al., 2013). Therefore, the present investigation was undertaken to evaluate the potency of standardized extract of P. guajava and its biomarker quercetin against infectious diarrhoea mediated through EPEC. Pathogenicity of E. coli has been reported to cause disorders such as diarrhoea, hemolytic uremic syndrome, hemorrhagic colitis and thrombocytopenic purpura (Ahmed et al., 2014). Specifically, EPEC have been reported to induce diarrhoea by binding intimately to the epithelial surface of intestine, especially to the colon via adhesive bundleforming pilus (BFP), which results in formation of lesion followed by the destruction of microvilli, which contributes to malabsorption and diarrhoea (Chen and Frankel, 2005; Evans Jr and Evans, 1996). Our results also demonstrated that, administrations of EPEC suspension produced diarrhoea at an onset time of approximately 40 to 50 min. This may be attributed to a massive destruction of microvilli, which was also confirmed through the histopathological 15

examination of the EPEC control group. Nevertheless, on treatment with PGE and quercetin, a significant recovery from diarrhoea was observed which was depicted through a significant decline in the number of diarrhoeal stools, resulting in higher % protection. The results also confirmed the antidiarrhoeal potential of PGE through a marked decline in the water content of stools and EPEC density in treated rat stools. Literature have revealed that, some of the pathogenic E. coli especially enterohemorrhagic E. coli causes a severe blood loss due to hemorrhagic colitis and hemolytic-uremic syndrome (Donnenberg and Whittam, 2001). Keeping the above view into consideration, various blood parameters were evaluated in the present study. Our observation showed a significant decline in the level of WBC and platelets in EPEC control group however, on treatment with PGE and quercetin, a significant recovery in their loss was observed, which may be as a result of enhanced immune potential of treated group (Croxen and Finlay, 2010; Evans Jr and Evans, 1996). Further, PGE and quercetin also caused a significant recovery from the haemoglobin loss due to EPEC administration confirming the nutritional potential of PGE. During the entire protocol, there were no signs of haemorrhage or haemolysis, as depicted through appearance of non-bloody stools in EPEC control animals. Studies have reported that, EPEC mediated infectious diarrhoea causes a massive tissue damage due to release of inflammatory mediators like IL-1β, and TNF-α (as observed) at the site of infection, resulting in stressful condition (Kamgang et al., 2015; Chen and Frankel, 2005; Evans Jr and Evans, 1996). The above consequences results in the expression of inducible NOS (iNOS), that results in higher production of NO in colons. Likewise, our study also demonstrated a significant increase in the level of NO, however, on treatment with PGE and quercetin there was a significant decline in the level of NO that may be attributed to a significant decline in the elevated expression of inflammatory mediators. The elevated level of NO has also been reported to alter physiological conditions like blood pressure, platelet 16

function and host defences (Korhonen et al., 2005; Kamgang et al., 2015). Therefore, elevated level of NO might have also contributed in the previously observed altered levels of platelets and WBC in EPEC control group, which was significantly restored on PGE and quercetin treatment. The inflammation mediated through EPEC has also been reported to impair the antioxidant defences mechanism as a result of lipid peroxidation, free radical chain reaction and auto-oxidation. This condition contributes in release of reactive oxygen species (ROS) such as peroxide anion, hydrogen peroxide, and hypochlorous acid causing additional alleviation in inflammatory expression (Moorthy et al., 2007). Our study demonstrated that, PGE and quercetin treatment showed a significant antioxidant potential of P. guajava against EPEC mediated oxidative stress, where a significant decline in the level of LPO, and a significant increase in the level of antiperoxidative enzymes SOD and CAT was observed (Nieto et al., 2000). Pathogenic diarrhoea has also been found to hamper the process of protein and DNA synthesis, which results in mucosal atrophy that lowers cell turnover (Petri et al., 2008). Our study also showed a significant decline in the level of protein and DNA content in colonic tissues of EPEC control animals, which was found to significantly recover on treatment with PGE and quercetin. Such conditions have also attributed in a marked loss of carbohydrates (Prasad et al., 2013), resulting in instant weakness. Administration of PGE has revealed a significant recovery from carbohydrate loss that confirmed its contribution in storing and transporting energy. Studies have reported the significant role of EPEC in altering electrolyte balance across the membranes, due to inhibition in NaCl absorption and acceleration of Cl– secretion through a Type III secretion system contributing in a concomitant decline in absorption of water. The above consequence also attribute in a significant loss of K+ that occurs via enhanced solvent drag phenomenon (Croxen and Finlay, 2010; Bruins et al., 2006; Chen and 17

Frankel, 2005). The observed imbalance in transport of electrolyte may also be due to decrees in Na+/K+–ATPase activity, a basolateral protein that plays a pivotal role in efficient nutrient and ion absorption. Literature have revealed that, EPEC induces inflammation to the protein, which results in its inhibition through Na+/K+–ATPase endocytosis in a EspF‐dependent manner, (Tapia et al., 2017). Our study also showed a significant decline in the Na+/K+– ATPase activity of EPEC control group, which may have attributed in significant decrease in reabsorption of ions and water. However, treatment with PGE and quercetin, there was a significant increase in the Na+/K+–ATPase activity that contributed in significant restoration in altered concentration of ions. Further, the protective nature of PGE and its biomarker quercetin against infectious diarrhoea was also justified through the histopathological examination showing a marked recovery from EPEC mediated destruction of colonic cells including microvilli as observed in EPEC control group. The leaves of the plant P. guajava have already been reported to have a potential antidiarrhoeal activity in chemical induced diarrhoea (Ojewole et al., 2008; Lin et al., 2002) and is also reported for its potent in vitro antibacterial activity against pathogens susceptible to infectious diarrhoea (Birdi et al., 2010). P. guajava along with its major biomarker quercetin, has also been reported for its spasmolytic activity both in vitro and in vivo, where quercetin was found to be major responsible factor for the observed antidiarrhoeal activity, of P. guajava (Gutiérrez et al., 2008; Lozoya et al., 1994). Thus, from the above evidences and the observed results from our study, quercetin was confirmed to be the major biomarker of P. guajava and hence, was subjected to molecular docking studies. Molecular docking simulations studies have become an important tool in understanding the binding mode and binding interactions between protein and ligand that helps in proper characterization of the nature of molecules at the binding site. Previous docking studies conducted on Na+/K+–ATPase (Morth et al., 2007; Paulsen et al., 2013) 18

confirmed that, the bound K+ ions and Rb+ occupies the similar site. In the present study, we have removed Rb+ and docked quercetin with Na+/K+–ATPase on the same site and found favourable interactions. The residues Glu 327, Ser 775, Asn 776, Glu 779 and Asp 804 were adequately close to the quercetin to donate ligands for binding. Glu 327 was related absolutely with K+/Rb+ which controls the extracellular gate of the occlusion cavity. The residues Glu 327, Asn 776, Glu 779, Asp 804, Asp 808, and Gln 923 of the Na+/K+–ATPase provides oxygen ligands for Ca2+ binding and facilitates binding of residues in two of the three Na+ sites of the Na+/K+–ATPase (Einholm et al., 2007). The sensitivity of the Na+/K+ pump activity with respect to the membrane potential is directed by Arg 1003, Arg 1004 and Arg 1005 together with Arg 933, Arg 934 and Arg 998 which makes the area around the C terminus in the membrane edge region. In the Na+/K+–ATPase the arginine cluster is allied with the C terminus, which functions as a control point and modifies the C terminus in its binding pocket during depolarization/repolarization (Jiang et al., 2003; Bass et al., 2002). Intimin and its translocated intimin receptor (Tir) are bacterial proteins (in EPEC) that arbitrate linkage between mammalian cells and effacing pathogens. Intimin has an aminoterminal bacterial membrane anchor and C-terminal domains required for Tir binding. EPEC intimin has a pair of cysteine residues 860 and 937, the main structural description required for binding with Tir (Luo et al., 2000). Thus, we can say that, quercetin engage the same binding site of K+ ion i.e. Pro 326, GLu 327, Ser 775, Asn 776, Glu 779, Asp 804 and Asp 808 in Na+/ K+–ATPase and also controls the C-terminal cavity of Na+/K+–ATPase by binding with Arg 1003, Arg 1004, Arg 1005, Arg 933, Arg 934 and Arg 998. Further, we also articulate that, quercetin terminate the linkage between mammalian cells and EPEC by binding with cysteine residues (Cys 860 and Cys 937) of intimin and therefore, may be the major biomarker in P. guajava, that may be responsible for its potential antidiarrhoeal activity against EPEC infectious diarrhoea. 19

In conclusion, the present investigation scientifically validated the antidiarrhoeal potential of P. guajava against enteropathogenic E. coli induced infectious diarrhoea. From the overall observation, quercetin a major biomarker of P. guajava was found to terminate linkage between mammalian cells and EPEC through cysteine residues of intimin and also reactivated Na+/K+–ATPase activity. This significantly contributed in inhibition of EPEC mediated expression of cytokines, reduced nitric oxide production along with restoration of antioxidant status and intestinal electrolyte balance. Thus, quercetin could be considered as a major contributing factor for the observed activity of P. guajava. The outcome of the present study could be useful in preparation of a cost effective formulation, as the plant is easily available in treatment of infectious diarrhoea.

Acknowledgment: The authors would like to acknowledge the valuable contribution of Science and Engineering Research Board, Department of Science and Technology, Government of India for providing financial support for the present research work (DST Letter No: ECR/2015/000090, Dated 15/03/2016) under Early Career Research Grants to Dr. Satyendra K. Prasad.

Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the writing and content of the paper.

Author’s contribution SKP and PRI had designed the framework of the study, while JRH and KMP have performed the phytochemical evaluations and standardization of extract. Further, JRH, KMP and RSP were involved in the initial behavioural evaluation and pharmacological studies. RSP and 20

ADL have performed the biochemical evaluations, while SKS and SKP performed the molecular docking studies and its interpretation. JRH, KMP and ADL contributed significantly in compilation of results and interpretation of data. RSP, SKS, SKP and PRI contributed in initial drafting of the manuscript, which was finalized by PRI and SKP.

References Ahmed, A.S., McGaw, L.J., Elgorashi, E.E., Naidoo, V., Eloff, J.N., 2014. Polarity of extracts and fractions of four Combretum (Combretaceae) species used to treat infections and gastrointestinal disorders in southern African traditional medicine has a major effect on different relevant in vitro activities. J. Ethnopharmacol. 154, 339–350. Al-Rifai, A., Aqel, A., Awaad, A., ALOthman, Z.A., 2015. Analysis of Quercetin and Kaempferol in an Alcoholic Extract of Convolvulus pilosellifoli using HPLC, Commun. Soil Sci. Plant Anal. 46, 1411–1418. Barbour, E.K., Sharif, M.A., Sagherian, V.K., Habre, A.N., Talhouk, R.S., Talhouk, S.N., 2004. Screening of selected indigenous plants of Lebanon for antimicrobial activity. J. Ethnopharmacol. 93, 1–7. Bass, R.B., Strop, P., Barclay, M., Rees, D.C., 2002. Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science 298, 1582–1587. Birdi, T.1., Daswani, P., Brijesh, S., Tetali, P., Natu, A,. Antia, N., 2010. Newer insights into the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea. BMC Complement Altern Med. 10, 33. Bruins, M.J., Cermak, R., Kiers, J.L., Van der Meulen, J., Van Amelsvoort, J.M., Van Klinken, B.J., 2006. In vivo and in vitro effects of tea extracts on enterotoxigenic Escherichia coli-induced intestinal fluid loss in animal models. J. Pediatr. Gastroenterol. Nutr. 43, 459– 469. 21

Burton, K., 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315–323. Chen, H.D., Frankel G., 2005. Enteropathogenic Escherichia coli: unravelling pathogenesis. FEMS Microbiol. Rev. 29, 83–98. Croxen, M.A., Finlay, B.B., 2010. Molecular mechanisms of Escherichia coli pathogenicity. Nat. Rev. Microbiol. 8, 26–38. de Souza, C.E.S., da Silva, A.R.P., Gomez, M.C.V., Rolóm, M., Coronel, C., da Costa, J.G.M., Sousa, A.K., Rolim, L.A., de Souza, F.H.S., Coutinho, H.D.M., 2017. AntiTrypanosoma, anti-Leishmania and cytotoxic activities of natural products from Psidium brownianum Mart. ex DC. And Psidium guajava var. Pomifera analysed by LC–MS. Acta Trop. 176, 380–384. Donnenberg, M.S., Whittam, T.S., 2001. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J. Clin Invest. 107, 539–548. Einholm, A.P., Andersen, J.P., Vilsen, B., 2007. Importance of Leu99 in transmembrane segment M1 of the Na+,K+-ATPase in the binding and occlusion of K+. J. Biol. Chem. 282, 23854–23866. Evans, D.J., Evans, D.G., 1996. Escherichia coli in Diarrheal Disease. Medical Microbiology. 4th edition, Galveston (TX): University of Texas, USA. Gal-Garber, O., Mabjeesh, S.J., Sklan, D., Uni, Z., 2003. Nutrient Transport in the Small Intestine: Na+, K+-ATPase Expression and Activity in the Small Intestine of the Chicken as Influenced by Dietary Sodium. Poult. Sci. 82, 1127–1133. Gálvez, J., Crespo, M. E., Jiménez, J., Suárez, A., Zarzuelo, A., 1993. Antidiarrhoeic activity of quercitrin in mice and rats. J. Pharm. Pharmacol. 45, 157–159. Gupta, P., Birdi, T., 2015. Psidium guajava leaf extract prevents intestinal colonization of Citrobacter rodentium in the mouse model. J. Ayurveda Integr. Med. 6, 50–52. 22

Gutiérrez, R.M., Mitchell, S., Solis, R.V., 2008. Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. J. Ethnopharmacol. 117, 1-27. Hagerman, A.E., Riedl, K.M., Jones, G.A., Sovik, K.N., Ritchard, N.T., Hartzfeld, P.W., 1998. High molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric. Food Chem. 46, 1887–1892. Jiang, Y., Ruta, V., Chen, J., Lee, A., MacKinnon, R., 2003. The principle of gating charge movement in a voltage-dependent K+ channel. Nature 423, 42–48. Kamgang, R., Pouokam, K.E., Fonkoua, M.C., Penlap, N.B., Biwole, S.M., 2006. Activities of aqueous extracts of Mallotus oppositifolium on Shigella dysenteriae A1-induced diarrhoea in rats. Clin. Exp. Pharmacol. Physiol. 33, 89–94. Kamgang, R., Tagne, M.A., Kamga, H.G., Noubissi, P.A., Fonkoua, M.C., Oyono, J.L., 2015. Activity of Aqueous Ethanol Extract of Euphorbia scordifolia on Shigella dysenteriae Type 1-Induced Diarrhoea in Rats. Int. J. Pharm. Sci. Drug. Res. 7, 40–45. Korhonen, R., Lahti, A., Kankaanranta, H., Moilanen, E., 2005. Nitric oxide production and signaling in inflammation. Curr. Drug Targets Inflamm. Allergy 4, 471–479. Koriem, K.M.M., Arbid, M.S., Saleh, H.N., 2019. Antidiarrheal and protein conservative activities of Psidium guajava in diarrheal rats. J. Integr. Med. 17, 57–65. Kumaran, A., Karunakaran, J., 2006. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. Food Sci. Technol. 40, 344–352. Laloo, D., Prasad, S.K., Sairam, K., Hemalatha, S., 2013. Gastroprotective activity of ethanolic root extract of Potentilla fulgens Wall. ex Hook. J. Ethnopharmacol. 148, 505-514. Lin, J.1., Puckree, T., Mvelase, T.P.. 2002. Anti-diarrhoeal evaluation of some medicinal plants used by Zulu traditional healers. J. Ethnopharmacol. 79, 53–56. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall RT., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–276. 23

Lozoya, X., Meckes, M., Abou-Zaid, M., Tortoriello, J., Nozzolillo, C., Arnason, J.T., 1994. Quercetin glycosides in Psidium guajava L. leaves and determination of a spasmolytic principle. Arch. Med. Res. 25, 11–15. Luo, Y., Frey, E.A., Pfuetzner, R.A., Creagh, A.L., Knoechel, D.G., Haynes, C.A., Finlay, B.B., Strynadka, N.C., 2000. Crystal structure of enteropathogenic Escherichia coli intiminreceptor complex. Nature 405, 1073–1077. Machado, A.J.T., Santos, A.T.L., Martins, G.M.A.B., Cruz, R.P.1., Costa, M.D.S., Campina, F.F., Freitas, M.A., Bezerra, C.F., Leal A.L.A.B., Carneiro, J.N.P., Coronel, C., Rolón, M., Gómez, C.V., Coutinho, H.D.M., Morais-Braga, M.F.B., 2018. Antiparasitic effect of the Psidium guajava L. (guava) and Psidium brownianum MART. EX DC. (araçá-de-veado) extracts. Food Chem. Toxicol. 119, 275–280. Moorthy, G., Murali, M.R., Devaraj, S.N., 2007. Protective role of lactobacilli in Shigella dysenteriae1–induced diarrhea in rats. Nutrition 23, 424–433. Morth, J.P., Pedersen, B.P., Toustrup-Jensen, M.S., Sørensen, T.L., Petersen, J., Andersen, J.P., Vilsen, B., Nissen, P., 2007. Crystal structure of the sodium–potassium pump, Nature 450, 1043–1049. Nieto, N., Torres, M.I., Fernandez, M.I., Giro, M.D., Rios, A., Suarez, M.D., Gil, A., 2000. Experimental ulcerative colitis impairs antioxidant defense system in rat intestine. Dig. Dis. Sci. 45, 1820–1827. Ojewole, J.A.1., Awe, E.O., Chiwororo, W.D., 2008. Antidiarrhoeal activity of Psidium guajava Linn. (Myrtaceae) leaf aqueous extract in rodents. J. Smooth Muscle Res. 44, 195– 207. Palla, A.H., Khan, N.A., Bashir, S., Ur-Rehman, N., Iqbal, J., Gilani, A.H., 2015. Pharmacological basis for the medicinal use of Linum usitatissimum (Flaxseed) in infectious and non-infectious diarrhea. J. Ethnopharmacol. 160, 61–68. 24

Parmar, K.M., Bhagwat, D.S., Sinha, S.K., Katare, N.T., Prasad, S.K., 2019. The potency of eriosematin E from Eriosema chinense Vogel. against enteropathogenic Escherichia coli induced diarrhoea using preclinical and molecular docking studies. Acta Trop. 193, 84–91. Paulsen, P.A., Jurkowski, W., Apostolov, R., Lindahl, E., Nissen, P., Poulsen, H.,2013. The C-terminal cavity of the Na,K-ATPase analyzed by docking and electrophysiology. Mol. Membr. Biol. 30, 195–205. Petri, W.A., Miller, M., Binder, H.J., Levine, M.M., Dillingham, R., Guerrant, L.R., 2008. Enteric infections, diarrhea, and their impact on function and development. J. Clin. Invest. 118, 1277–1290. Piña-Vázquez, D.M., Mayoral-Peña, Z., Gómez-Sánchez, M., Salazar-Olivo, L.A., ArellanoCarbajal, F., 2017. Anthelmintic effect of Psidium guajava and Tagetes erectaon wild-type and Levamisole-resistant Caenorhabditis elegans strains. J. Ethnopharmacol. 202, 92–96. Prasad, S.K., Laloo, D., Kumar, M., Hemalatha, S., 2013. Antidiarrhoeal evaluation of root extract, its bioactive fraction and lupinifolin isolated from Eriosema chinense. Planta Med. 79, 1620–1627. Prasad, S.K., Parmar, K.M., Danta, C.C., Laloo, D., Hemalatha, S., 2017. Antidiarrhoeal activity of eriosematin E isolated from the roots of Eriosema chinense Vogel. Phytomedicine 24, 127–133. Sengar, N., Joshi, A., Prasad, S.K., Hemalatha, S., 2015. Anti-inflammatory, analgesic and anti-pyretic activities of standardized root extract of Jasminum sambac. J. Ethnopharmacol. 160, 140–148. Tapia, R., Kralicek, S.E., Hecht, G.A., 2017. EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity. Cell Microbiol. 19.

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Tetali, P., Waghchaurea, C., Daswani, P.G., Antia, N.H., Birdi, T.J., 2009. Ethnobotanical survey of antidiarrhoeal plants of Parinche valley, Pune district, Maharashtra, India. J. Ethnopharmacol. 123, 229–236. Thapar, N., Sanderson, I.R., 2004. Diarrhoea in children: an interface between developing and developed countries. Lancet 363, 641–653. Trease, G.E., Evans, W.C., 2002. Pharmacognosy. 15th Edition, W.B. Saunders Company Ltd., London. Wagner, H., Bladt, S., 1996. Plant drug analysis. 2nd Ed., Springer-Verlog Berlin Heidelberg, Germany. Yakubu, M.T., Salimon, S.S., 2015. Antidiarrhoeal activity of aqueous extract of Mangifera indica L. Leaves in female albino rats. J. Ethnopharmacol. 163, 135–141. Yemm, E.W., Willis, A.J., 1954. The estimation of carbohydrates in plant extracts by Anthrone. Biochem. J. 57, 508–514.

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TABLES Table 1. Effect of PGE and its biomarker quercetin on various behavioral parameters in EPEC induced diarrhoea rat model. Behavioural Parameters Total no of faeces Total no of wet faeces Loss in body weight (g) Total wt. of faeces (g) Mean defecation Diarrhoea score % protection

Time 6 hr 24 hr 6 hr 24 hr 6 hr 24 hr 6 hr 24 hr 6 hr 24 hr 6 hr 24 hr 6 hr 24 hr

Normal Control 3.03 ±0.75 5.26 ±0.20 0.08 ±0.01 0.23 ±0.02 0.48 ±0.03 0.30 ±0.03 0.50 ±0.12 0.21 ±0.01 100 100

EPEC Control 15.33 ±0.84a 13.68 ±0.85a 6.33 ±0.98 8.16 ±0.38 1.21 ±0.11a 0.94 ±0.06a 4.50 ±0.34a 4.11 ±0.28a 2.55 ±0.14a 0.57 ±0.03a 21.00 ±1.34 17.83 ±1.01 -

PGE 100 mg/kg 12.83 ±0.74a 8.70 ±0.46a 8.33 ±0.95 6.53 ±0.29 0.91 ±0.06a 0.49 ±0.02a 2.46 ±0.15ab 2.60 ±0.33ab 2.13 ±0.12a 0.36 ±0.01b 16.33 ±1.42b 13.33 ±0.88b 22.22 ±6.80 25.09 ±4.95

PGE 200 mg/kg 8.66 ±0.80ab 6.33 ±0.34b 4.91 ±0.55b 4.01 ±0.25b 0.71 ±0.09ab 0.26 ±0.01b 1.53 ±0.10ab 1.41 ±0.16ab 1.44 ±0.13ab 0.26 ±0.01b 7.50 ±0.84b 5.33 ±0.42b 64.28 ±4.03 70.03 ±2.36

PGE 400 mg/kg 8.83 ±0.90ab 6.63 ±0.25b 5.33 ±0.40b 4.18 ±0.32b 0.83 ±0.04ab 0.27 ±0.02b 1.76 ±0.23ab 1.50 ±0.19ab 1.47 ±0.15ab 0.27 ±0.01b 8.00 ±0.57b 5.66 ±0.61b 61.90 ±2.74 68.16 ±3.45

Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control In Table, PGE: Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

Quercetin 50 mg/kg 8.50 ±0.61ab 6.48 ±0.64b b 5.0 ±0.36 b 3.95 ±0.31 ab 0.73 ±0.09 b 0.27 ±0.04 ab 1.58 ±0.07 ab 1.34 ±0.09 ab 1.43 ±0.09 b 0.26 ±0.04 b 7.33 ±0.80 b 4.91 ±0.37 65.07 ±3.87 71.07 ±2.20

Norfloxacin 5.7 mg/kg, 7.33 ±0.27ab 5.66 ±0.20b 4.83 ±0.22b 3.45 ±0.29b 0.67 ±0.02ab 0.21 ±0.01b 1.48 ±0.04ab 1.04 ±0.10ab 1.22 ±0.04ab 0.23 ±0.008b 6.83 ±0.43b 3.83 ±0.40b 67.46 ±2.09 78.46 ±2.25

Table 2. Effect of PGE and its biomarker quercetin on various blood parameters in EPEC induced diarrhoea rat model. EPEC Control Quercetin 50 Norfloxacin 5.7 Normal PGE 200 mg/kg Blood mg/kg mg/kg, Control Parameters WBC (103/mm3) 8.39 ±0.67 7.15 ±0.38a 8.16 ±0.24b 8.03 ±0.35b 8.28 ±0.38b a b b Hb (g/dL) 13.61 ±0.74 10.01 ±0.65 13.58 ±0.65 12.33 ±0.56 12.35 ±0.63b 6 3 a RBC (x10 /mm ) 8.16 ±0.30 7.73 ±0.40 8.03 ±0.85 7.76 ±0.32 7.93 ±0.38 PC (x105/mm3) 9.80 ±0.44 7.33 ±0.35a 9.03 ±0.49b 8.76 ±0.43b 8.80 ±0.65b Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control In Table,. WBC: White Blood Cells, Hb: Hemoglobin, RBC: Red Blood Cells, PC: Platelet Cells, PGE: Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

Table 3. Effect of PGE and its biomarker quercetin on various biochemical parameters in EPEC induced diarrhoea rat model. Biochemical Parameters Normal Control EPEC Control PGE 200 mg/kg Quercetin 50 mg/kg Norfloxacin 5.7 mg/kg, NO (Units in mole/mg of protein) 0.91 ±0.11 4.40 ±0.22a 2.11 ±0.18ab 2.16 ±0.25ab 2.32 ±0.23ab Total protein (Units in mg/100 mg of tissue) 2.06 ±0.22 0.60 ±0.07a 1.51 ±0.11ab 1.49 ±0.16ab 1.25 ±0.20ab a b b Total DNA (Units in mg/100 mg of tissue) 2.48 ±0.19 1.08 ±0.18 2.03 ±0.14 2.21 ±0.14 2.11 ±0.12b a ab ab Total carbohydrates (mg/g of tissue) 2.09 ±0.19 0.73 ±0.08 1.76 ±0.19 1.88 ±0.13 1.51 ±0.10ab a ab ab TBARS (Units in mole/mg of protein) 2.78 ±0.34 13.47 ±0.90 5.43 ±0.61 5.24 ±0.34 5.17 ±0.59ab a ab ab CAT (µmol H2O2 consumed/min/mg of protein) 140.42 ±4.36 81.93 ±4.81 111.21 ±7.10 123.15 ±3.92 116.13 ±7.14ab a ab ab SOD (Units/mg of protein) 2.72 ±0.23 1.02 ±0.09 2.16 ±0.17 2.43 ±0.21 2.22 ±0.12ab Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control. In Table, NO: Nitric Oxide, TBARS: Thiobarbituric Acid Reactive Substance, CAT: Catalase, SOD: Superoxide Dismutase, PGE: Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

Table 4. Effect of PGE and its biomarker quercetin on concentrations of ions in EPEC induced diarrhoea rat model. Ion concentration Normal Control EPEC Control PGE 200 mg/kg Quercetin 50 mg/kg Norfloxacin 5.7 mg/kg, (mmol/L) C l116.30 ±4.94 81.43 ±2.62a 105.48 ±4.10b 108.78 ±4.91b 110.40 ±3.57b + a b b K 7.06 ±0.62 3.33 ±0.26 6.05 ±0.44 6.33 ±0.40 6.21 ±0.41b + a ab ab Na 155.52 ±4.91 112.14 ±4.74 135.73 ±4.49 140.80 ±3.72 140.28 ±3.48ab 2+ Ca 1.04 ±0.07 1.07 ±0.06 1.11 ±0.07 1.12 ±0.02 1.16 ±0.06 Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control. In Table, PGE: Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

FIGURES

Figure1. HPLC chromatogram of quercetin. In figure, A: HPLC chromatogram of standard peak of quercetin, B: HPLC chromatogram of quercetin in ethanolic extract of P. guajava.

Water Content (%)

50 40 30

a a

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Figure 2. Effect of PGE and its biomarker quercetin on stool water content in EPEC induced diarrhoea rat model. Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control. In figure, PGE: Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

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Hours Figure 3. Effect of PGE and its biomarker quercetin on density of EPEC (log10 transformed) in stool of EPEC induced diarrhoea rat model. Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control In figure, , PGE: Psidium guajava leaf extract, EPEC: Enteropathogenic E. coli.

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Figure 4. Effect of PGE and its biomarker quercetin on IL-1β (A) and TNF-α (B) in colonic tissue of EPEC induced diarrhoeal rat. Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control In figure, , PGE: Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

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Na+/K+ ATPase activity (µmol Pi/mg Protein/Hour)

1500

Figure 5. Effect of PGE and its biomarker quercetin on Na+/ K+–ATPase activity in small intestine of EPEC induced diarrhoea rat model. Values are mean ±S.E.M. (n = 6). Where a: P < 0.05 vs. Normal control and b: P < 0.05 vs. EPEC induced diarrhoea control In figure, Psidium guajava leaf extract and EPEC: Enteropathogenic E. coli.

Figure 6. Histopathological view of colonic section of EPEC induced diarrhoea rats on treatment with Psidium guajava leaf extract and its biomarker quercetin [10 ×, Scale Bar 100µm]. In figure, A: Normal control rat colon, B: EPEC induced diarrhoeal control rat colon, C: EPEC induced diarrhoeal rat colon treated with Psidium guajava leaf extract (200 mg/kg, p.o.), D: EPEC induced diarrhoeal rat colon treated with quercetin and E: EPEC induced diarrhoeal rat colon treated with norfloxacin (5.7 mg/kg, p.o) and EPEC: Enteropathogenic E. coli (Arrow in the figure indicates localised destruction of colonic cell including microvilli).

Figure 7. Molecular docking study of quercetin, a major biomarker from Psidium guajava on Na+ / K+–ATPase enzyme and enteropathogenic Escherichia coli. In figure, A: Binding mode of quercetin in the Na+/ K+–ATPase binding pocket, B: Hydrogen bonding interaction of quercetin in the Na+/ K+–ATPase binding pocket, C: Binding mode of quercetin in the C-terminal cavity of Na+/K+–ATPase, D: Hydrogen bonding interaction of quercetin in the C-terminal cavity of Na+/K+– ATPase and E: The binding mode of quercetin in the enteropathogenic Escherichia coli intimin-receptor complex.