Abstract
This is a visual representation of the abstract.
Introduction
Diarrhea can be caused by a variety of microorganisms such as bacteria, protozoa, and viruses. However, diarrhea caused by Escherichia coli, particularly enterotoxigenic, is common in developing countries. 1 As a conditional pathogen in the intestines of humans and animals, diarrhea caused by E. coli is harmful to human health and animal husbandry production. Meanwhile, issues such as excessive residues caused by the irregular use of antibiotics, bacterial resistance, and the quality and safety of livestock and poultry products have caused serious hidden risks to food quality and safety, public health safety, and ecological safety.2,3 These issues have prompted scientists to look for alternatives from natural sources, especially medicine food homology plants.
The extracts from medicinal plants, particularly food-derived extracts, are widely concerned to be used instead of traditional antibiotics because of their high safety with antimicrobial activity.4,5 However, sometimes, the antibacterial activity of plant extracts when used alone is not as effective as compared with that being combined. 6 Increasing evidence has suggested that plant extracts, as a whole or multiple compound formulations, have better efficacy than single active ingredients, highlighting the importance of synergistic effects in therapy. 7 The combined effect of multiple plant extracts has a multi-target antibacterial effect. Furthermore, the synergistic compounds found in plants mitigate the reduction in efficacy associated with prolonged use of a single drug. 8
Houttuynia cordata Thunb., a perennial herb belonging to the Saururaceae family, is widely used in Chinese herbal medicine and as a food ingredient. This plant has several medicinal functions, including relieving fever, resolving toxins, reducing swelling, draining pus, and promoting urination. During the Severe Acute Respiratory Syndrome (SARS) outbreak, it was included in prevention formulas recognized by the Health Ministry of China. Recent studies have provided scientific evidence supporting its anti-SARS, 9 anti-inflammatory,10,11 anti-allergic,12,13 virucidal,14,15 antileukemic, 16 anti-oxidative,17,18 and anti-cancer properties. 19 Houttuynia cordata Thunb. contains various chemical components, including flavonoids, essential oils, and alkaloids. 20 Portulaca oleracea L., commonly known as purslane, is an annual herbaceous plant characterized by its reddish stems and alternate leaves. It belongs to the Portulacaceae family. Traditionally, Portulaca oleracea L. has been used to treat a wide range of ailments, including gastrointestinal disorders, respiratory issues, liver inflammation, urinary tract ulcers, fevers, insomnia, severe inflammations, and headaches.21,22 Phytochemical studies have revealed that this plant contains various beneficial compounds, such as flavonoids, alkaloids, terpenoids, organic acids, fatty acids, minerals, and vitamins.23,24 Modern pharmacological research has shown that Portulaca oleracea L. exhibits numerous biological activities, including antioxidant, 25 antimicrobial, 26 bronchodilator, 27 renoprotective, 28 neuroprotective, 29 muscle relaxant, 30 hepatoprotective, 31 antiulcerogenic, 32 and antifertility effects. 33
Houttuynia cordata Thunb. and Portulaca oleracea L. are both used as food material and herb material for clearing heat and detoxification. Nevertheless, few studies have focused on the antibacterial and antidiarrheal mechanism of the combination (CB-HHP) of H. cordata ethanol extract (HEE) and essential oil (HEO) and P. oleracea ethanol extract (PEE) against enterotoxigenic E. coli.34,35 However, in previous reports, the essential oil of H. cordata and the ethanol extract of H. cordata have good antibacterial effects, 36 while the ethanol extract of P. oleracea has good antioxidant and anti-inflammatory effects. 37 Through the analysis of these studies, we believe that CB-HHP has a synergistic effect, which can realize the inhibition of toxigenic Escherichia coli and solve the problem of diarrhea in mice. This investigation aimed to consummate the antimicrobial mechanisms of CB-HHP with the research object of enterotoxigenic E. coli on cell wall, cell membrane, cell microscopic morphology, and cell bio-film in vitro, and investigate the body weight, diarrhea rate, duodenal morphology, and immune function of mice with diarrhea induced by enterotoxigenic E. coli to prevent possible diseases.
Materials and Methods
Bacterial Strain, Plant Materials, and Chemicals
The bacterial strain enterotoxigenic E. coli (CMCC 10667) was bought from the National Center for Medical Culture Collections (CMCC, China). The dried H. cordata and P. oleracea were collected from Sichuan Chengdu Traditional Chinese Medicinal Market, China. H. cordata from Guanghan, Sichuan, and P. oleracea from Dazhou, Sichuan. The dried above-ground parts of H. cordata and P. oleracea were used in this study, and the characters were identified by reference to Pharmacopeia. 38 It was identified by Professor Zhihong Xu as H. cordata and P. oleracea, and we left plenty of medicinal specimens (HC-01, HC-02, HC-03 and PO-01, PO-02, PO-03). Propidium iodide (PI), dimethyl sulfoxide (DMSO), and Giemsa were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Other chemicals used in the study were of analytical grade.
Preparation of the Extractions
First, 100 g of H. cordata and 100 g of P. oleracea powder (sieved by 40 mesh sieve) were extracted with 500 mL of 75% ethanol for 8 h at room temperature, respectively. After filtration, the filtrate was concentrated under reduced pressure to remove ethanol, and the residue was freeze-dried. Finally, the ethanol extract of H. cordata (HEE) and ethanol extract of P. oleracea (PEE) were stored at 4 °C after freeze-drying. The essential oil was extracted from H. cordata (HEO) by steam distillation for 4 h (400 g material with 4000 mL deionized water), then dried over anhydrous Na2SO4 before storing at 4 °C.
Analysis of the Constituents in Extracts
A certain mass of HEE was weighed and dissolved in methanol to a constant volume. The solution was filtered through a 0.22 μm membrane filter. The contents of hyperoside, isoquercitrin, and quercetin in HEE were detected by a high-performance liquid chromatography (HPLC) apparatus (1260 Infinity II, Agilent Technologies Inc., Santa Clara, California, USA). The mobile phase consisted of water (A) and acetonitrile (B) using the following linear gradient program: first, from 15% (B) to 30% (B) during 0–30 min, then keeping 30% (B) for 5 min. Other conditions were maintained as follows: column temperature, 30 °C; flowrate, 0.8 mL/min; and UV detector wavelength, 275 nm. The sample injection volume was 10 μL. Meanwhile, the detection of the total flavonoid content in PEE was based on the method of Zhang et al (2019). 39 Gas chromatography-mass spectrometry (GC–MS) was used to detect the chemical constitutions of HEO based on the published method. 40
Antibacterial Activity Assay
Minimum Inhibitory Concentration (MIC) Detection
The MIC values of HEE, HEO, and PEE were determined by double broth dilution based on the method of Sopirala et al (2010). 41 The absorbance at 600 nm (A600) was measured using a microplate spectrophotometer (Multiskan™ FC; Thermo Fisher, USA). When a difference of A600 is less than 0.05 between the treatment (after deducting the blank) and the control group, the concentration of the treatment group was determined as the MIC value.
Determination of the Fractional Inhibitory Concentration index (FICI)
The determination of the combined antimicrobial activities of HEE, HEO, and PEE was performed by the checkerboard method, which was often used to assess interactive inhibition in vitro according to the method of Dong et al (2015). 42 FICI of >1.0, = 1.0, and <1.0 was performed to define synergy, addition, and antagonism, respectively.
Diameter of the Inhibitory Zone (DIZ) Assay
The DIZ of CB-HHP on E. coli was detected using the Oxford cup method according to the protocol of Kang et al (2019), 43 which could express the inhibitory activity of HEE, HEO, PEE, and CB-HHP against E. coli. In brief, 100 μL of bacterial suspension (approximately 1 × 107 CFU/mL) was evenly spread onto the NB agar plates. Then, the sterilized dry Oxford cups were put on a test plate. A 200 μL aliquot of HEE, HEO, PEE, and CB-HHP at 2 × MIC concentration was added to the Oxford cup and finally put into an incubator for incubation for 24 h at 37 °C. After incubation, the DIZ was detected by the vernier caliper.
Antibacterial Mechanism of CB-HHP
Analysis of Alkaline Phosphatase (AKP) Activity
The AKP assessment was used to assess the inhibitory effect of CB-HHP on the bacterial cell wall according to the protocol of He et al (2018). 44 CB-HHP at MIC and 2 × MIC was mixed with bacterial suspension (1 × 108 CFU/mL) and cultured in an incubator at 37 °C with time sampling (0, 2, 4, 6, 8, and 10 h). After incubation and centrifugation, the supernatant was collected to measure the content of AKP using the AKP kit (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).
Determination of Potassium ion Leakage
The protocol of Zhang et al (2017) was used to detect the potassium ion leakage. 45 CB-HHP at MIC and 2 × MIC were mixed with bacterial suspension (1 × 107 CFU/mL) and incubated at 37 °C for 10 h. After incubation and centrifugation, the supernatant was collected to measure the extracellular potassium concentration using the potassium kit (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).
Determination of Extracellular Protein Concentration
The protocol of He et al was used to detect the concentration of extracellular protein. 44 In brief, CB-HHP at MIC and 2 × MIC were mixed with bacterial suspension (1 × 107 CFU/mL) and cultured in an incubator at 37 °C with time sampling (0, 2, 4, 6, 8, 10 and 12 h). After incubation and centrifugation, the supernatant was collected to measure the content of extracellular protein by using the Coomassie Brilliant Blue method. The absorbance was measured at 595 nm using a Multiskan™ FC Spectrophotometer (Thermo Fisher, USA).
Scanning Electron Microscopy (SEM) Analysis
SEM was performed based on the previous method with some modifications to determine the efficacy of CB-HHP on the morphology of tested microorganisms according to the protocol of Hu et al (2019). 46 Finally, the cells were dried and sputter coated with gold in an ion coater for 2 min under vacuum, followed by microscopic examinations under a scanning electron microscope (Apreo S, Thermo Fisher Scientific, USA).
Anti-Biofilm Formation Assay
Crystal Violet Quantitative Assay
The anti-biofilm ability of CB-HHP on E. coli was first tested according to the protocol of Muthusamy and Shanmugam (2020) with minor modifications.
47
In brief, 100 μL of the previously activated (1 × 107 CFU/mL) E. coli strain was placed in a 96-well microplate, and then different concentrations of CB-HHP were added in the well, with the final level of MIC, 0.5 × MIC, and 0.25 × MIC. The well without CB-HHP (substituted with NB medium) was used as the negative control; the well without bacterial suspensions (substituted with NB medium) was used as the treatment control, and the well without bacterial suspensions and CB-HHP (substituted with NB medium) served as a blank. A Multiskan™ FC Spectrophotometer (Thermo Fisher, USA) was used to measure the absorbance, and the inhibition percentage of the biofilm was calculated by the following equation:
Microscopic Visualization of Biofilm
Microscopic technique was taken to detect the inhibition of biofilm formation according to the protocol of Bazargani and Rohloff (2016). 48 The formation of biofilms was observed by light microscopy at 400 × magnification.
Antidiarrheal Activity Test
Animals
Mice were obtained from Chengdu Da Shuo Co. Ltd (Sichuan, China). A total of 75 male ICR mice (body weight [BW] was 18-22 g) aged between 5 and 6 weeks were used. They were maintained in a temperature-regulated environment with a 12-h light and dark cycle. Animals had free access to commercial solid food (SCF Co. Ltd, China) and water ad libitum, and they were acclimatized for at least 1 week before the experiments.
E. coli-Induced Diarrhea Model and Sampling
As shown in Figure 1, mice were randomly divided into five groups, including the Control group (gavage 0.5% sodium carboxymethyl cellulose [CMC-Na]), Model group (gavage 0.5% CMC-Na), and CB-HHP + Model groups (gavage 20, 40, and 80 mg/mL of CB-HHP [combined with HEE, HEO, and PEE at the ratio of 1:0.56:0.5 using 0.5% CMC-Na as solvent], respectively), and several 15 mice was set in each group. After 14 days of gavage at a dose of 0.1 mL/10 g BW once a day, the body weight of mice in different groups was weighed in the morning of the 15th day, and then mice in the Model and CB-HHP + Model groups were intraperitoneally injected with E. coli (7.5 × 107 CFU/mL, 0.2 mL/10 g BW) once a day for 3 days to establish a diarrhea model. 49 The symptoms of disease or abnormal behavior were recorded as described by Leódido et al 50 The Diarrhea rate was calculated based on the following equation: Diarrhea rate = (Number of diarrhea mice/Total number of mice) × 100%. During the 3 days of E. coli injection, mice were treated by gavage as previously described. Then, the body weight of mice was weighed again on the morning of the 18th day after 3 days of injection. Mice were euthanized at day 19, and then blood samples were collected from each treatment through the eyeball. After centrifugation, the serum was harvested and stored at −20 °C for ELISA analysis. After the collection of blood, the duodenal tissue was removed, washed with normal saline, and immediately stored in 15% paraformaldehyde for subsequent morphological examination.
Design of trial on mice.
Histopathological Study
The method of Xie et al (2003) was used in this part. 51 In brief, mice duodenum tissues were isolated and sectioned (100 μm thickness) using a rotary microtome and stained with hematoxylin and eosin. Sections were observed for an alteration in architecture under a phase contrast microscope.
Immune Function Capacity
The concentrations of immunoglobulin (Ig) G, IgM, and IgA in serum were measured by their corresponding detection kits (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China).
Statistical Analysis
All data were collected in triplicate, averaged, and presented, followed by the standard deviation. The obtained results were statistically analyzed by SPSS software (version 22.0; IBM Corp, Armonk, NY). The data were analyzed via analysis of variance and independent sample T-test at p = 0.05.
Results
The Constituents in Extracts
HPLC chromatograms of standards and HEE were shown in Figure 2A and Figure 2B. The calculated results indicated the contents of hyperoside, isoquercitrin, and quercetin in HEE were 0.28%, 0.08% and 0.73%, respectively. The content of total flavonoids in PEE was 6.64% by spectrophotometry. The GC-MS total ion chromatogram and volatile constituents of HEO identified by GC-MS are shown in Figure 2C and Table 1, respectively. Essential oil extracted from H. cordata using the steam distillation method is often used as an injection in China. The results have shown that GC-MS analysis revealed the presence of 33 constituents that can be identified, representing 85.044% of the total essential oil. The major constituents of essential oil were terpinen-4-ol (15.650%), methyl-n-nonylketone (13.382%), decanal dimethyl acetal (11.688%), decanal (4.454%),1,1-dimethoxydodecane (4.217%), bornyl acetate (3.015%), spathulenol (2.838%), caryophyllene (2.818%), caryophyllene oxide (2.188%), 2-tridecanone (2.057%), methyl decanoate (2.013%) and α-terpineol (1.062%).
The HPLC chromatographic profiles of mixed standard solutions (A) and HEE (B) for the detection of hyperoside, isoquercitrin and quercetin, and the GC-MS total ion chromatogram of HEO (C).
Chemical components of essential oil extracted from H. cordata analyzed by GC-MS.
Retention Time.
Retention indices relative to C11–C21 n-alkanes on HP-5 MS capillary column.
Peak area relative to the total oil peak area.
Antibacterial Activity of HEE, HEO, PEE, and CB-HHP Against E. coli
As shown in Table 2, the MIC values of HEE, HEO, and PEE against E. coli are 16, 4.5, and 4 mg/mL, respectively. Moreover, when combining the HEE, HEO, and PEE, the MIC value of CB-HHP (MICCB−HHP) was combined with the final concentrations of 1 mg/mL of HEE, 0.56 mg/mL of HEO, and 0.5 mg/mL of PEE, and the FICI value was 0.311, which was less than 1, indicating the synergistic effect of the combination of HEE, HEO, and PEE. Meanwhile, when 2 × MIC concentration of HEE, HEO, PEE, and CB-HHP was affected by E. coli, the inhibition zone diameter detected by the Oxford cup method was 7.59 ± 0.31, 9.30 ± 0.95, 10.12 ± 0.18, and 15.57 ± 0.13 mm, respectively (Table 3). The results showed that CB-HHP had a good antibacterial effect on E. coli.
The minimum inhibitory concentration of HEE, HEO, PEE and CB-HHP.
Note: HEE represents ethanol extract from H. cordata, HEO represents essential oil from H. cordata, PEE represents ethanol extract from P. oleracea.
Diameter of the inhibition zone (mm) of HEE, HEO, PEE and CB-HHP at 2 × MIC concentration against E. coli.
Note: HEE represents ethanol extract from H. cordata, HEO represents essential oil from H. cordata, PEE represents ethanol extract from P. oleracea. Different lowercase letters represent the level of difference at P<0.05.
The above-mentioned results indicated that the antibacterial activity of the combination (CB-HHP) of HEE, HEO, and PEE was stronger than when used alone. This result might be attributed to the different extracts playing multi-target antibacterial effects to achieve a stronger antibacterial effect.
Antibacterial Mechanism of CB-HHP
Effect of CB-HHP on the Cell Wall
When the bacterial cell wall is destroyed, the AKP in the periplasmic space of the cell will leak. 52 Thus, the concentration of the extracellular AKP was detected to evaluate the effect of CB-HHP on the cell wall. The result shows that the contents of extracellular AKP increase rapidly and reach the highest level in 2 h compared with those of the control group when CB-HHP at MIC and 2 × MIC was affected by E. coli (Figure 3A). The positive correlation between the concentration of CB-HHP and AKP leakage suggested that CB-HHP could destroy the cell wall of E. coli and lead to the increased AKP leakage for a short period. In addition, concentration dependence is found when CB-HHP causes damage to the cell wall.
Effect of CB-HHP on the leakage of AKP (A) and potassium ion (B) and extracellular protein concentration (C) in E. coli, and Scanning electron micrographs of E. coli untreated with CB-HHP, treated with CB-HHP at MIC, and treated with CB-HHP at 2 × MIC (D). Different capital letters indicate that the difference among the different groups at the same incubation time is significant at the 0.05 level, and different lowercase letters indicate that the difference is significant at the 0.05 level among different incubation times in the same group.
Effect of CB-HHP on the Cell Membrane
The leakage of potassium ions and soluble protein can be used as an indicator to evaluate whether the permeability and integrity permeability of the bacterial cell plasma membrane are changed. 53 As shown in Figure 3B, when affected by CB-HHP with MIC and 2 × MIC concentration for 10 h, the leakage of potassium ion increased significantly (P < 0.05) compared with that in the control group, which indicated that the disruption of the permeability barrier might lead to the leakage of electrolytes and cause cell death. As shown in Figure 3C, the concentration of extracellular protein in the CB-HHP-treated groups increased and reached the highest level in 8 h as compared with those of the control group. The results indicated that CB-HHP might cause damage to the cell membrane of E. coli and lead to the exclusion of soluble proteins. Similarly, concentration dependence was found when CB-HHP caused damage to the cell membrane. The detection of both potassium ions and soluble protein in the extracellular environment of cells indicated that CB-HHP could damage the cell membrane, which might cause cell death. Therefore, it's inferred that the increase in cell membrane permeabilization may be related to the hydrophobicity of CB-HHP.
Effect of CB-HHP on Cell Morphology
The differences in the morphology of E. coli between the control and CB-HHP-treated groups are shown in Figure 3D. The SEM images displayed that the cells without CB-HHP treatment were regular, rod-shaped, and intact, and such cells exhibited a smooth surface in the control group. Subsequently, the surface of the bacteria was shrunk and rough, and the leakage of the intracellular substances was detected in cells after being treated with CB-HHP at MIC concentration. Furthermore, the distortion of E. coli cells was more serious after treatment with CB-HHP at 2 × MIC concentration, showing serious shrinkage of cell morphology and more leakage of intracellular substances. Therefore, the microscopic evaluation showed that CB-HHP exerts bactericidal activity by destroying the permeability or integrity of bacterial cells, which was consistent with our data on cell wall and membrane damage.
Effect of CB-HHP on the Formation of Biofilm
In the present study, the effect of CB-HHP on the formation of E. coli bacterial biofilm is shown in Figure 4A. It indicated that the addition of CB-HHP at the MIC concentration showed strong activity in the inhibition of biofilm formation with an inhibition value of 91.2%. In addition, the effect of CB-HHP on the formation of biofilm had concentration dependence, and the inhibition values were 71.3% and 20.7% at the concentrations of 1/2 MIC and 1/4 MIC, respectively. What's more, we could observe the inhibition effect of CB-HHP at the concentrations of MIC and 2 × MIC on the formation of E. coli biofilm under the optical microscope after Giemsa staining compared with the control group (Figures 4B, C, and D).
Effect of CB-HHP against the biofilm formed by crystal violet assay (A. Different capital letters indicate that the difference is significant at the 0.05 level) and light microscopy assay (B. Blank control without treatment with CB-HHP; C. Treated with CB-HHP at MIC; D. Treated with CB-HHP at 2 × MIC).
Antidiarrheal Activity of CB-HHP in Mice
Effect on the Body Weight and Diarrhea Rate
The present research has studied the effect of CB-HHP on mice treated with CB-HHP before and during E. coli infection to detect if CB-HHP can prevent possible diseases. The results indicated the body weight of mice in the Control group had no significant change (P > 0.05) from the 15th day to the 18th day; however, the weight of the mice decreased significantly (P < 0.05) in the Model group because of the injection of enterotoxigenic E. coli (Figure 5A). And the weight of the mice had no significant change (P > 0.05) in the CB-HHP3 (800 mg/kg BW) + Model group and CB-HHP2 (400 mg/kg BW) + Model group from the 15th day to 18th day during the injection of E. coli. Meanwhile, the diarrhea rate in the Model group was up to 86.5%; however, the diarrhea rate values decreased to 13% and 22% in the CB-HHP3 (800 mg/kg BW) + Model group and CB-HHP2 (400 mg/kg BW) + Model group, respectively (Figure 5B).
Body weight (A), the diarrhea rate (B) and microscopic observation of duodenal villi (C) of mice in control group treated with 0.5% sodium carboxymethylcellulose (CMC-Na), Model group treated with 0.5% CMC-Na with E. coli injection, CB-HHP1 + model group treated with 200 mg/kg BW CB-HHP with E. coli injection, CB-HHP2 + Model group treated with 400 mg/kg BW CB-HHP with E. coli injection, and CB-HHP3 + Model group treated with 800 mg/kg BW CB-HHP with E. coli injection. The ns indicates that the difference among the groups was not significant, * indicates that the difference among the groups was significant at the 0.05 level, and ** indicates that the difference among the groups was significant at the 0.01 level.
Effect on the Structure of Duodenal Villi
The tissue morphology of the duodenum was shown in Figure 5C, and we could observe that compared with the Control group, the duodenal villi of mice in the diarrhea Model group were severely broken, shedding, and irregularly arranged. Notably, the rupture and shedding of duodenal villi of mice in the CB-HHP + Model treatment groups were improved, and the villus height to crypt depth ratio was increased compared with the Model group and was concentration dependent. In the CB-HHP3 (800 mg/kg BW) + Model group, the duodenal villi structure of mice is intact, without breakage or shedding, but a slight atrophy is observed compared with the Control group.
Effect on the Concentrations of IgA, IgG, and IgM in serum
As shown in Figure 6, the concentrations of IgG (Figure 6A) and IgM (Figure 6B) in serum significantly decreased in the Model group compared with the Control group (P < 0.05), which indicated that the immune function of mice decreased after the injection of E. coli to diarrhea. In addition, no significant difference was found in the concentration of IgA between the Model group and the Control group (P > 0.05). However, treatment with 800 mg/kg BW CB-HHP could significantly increase the concentrations of IgG and IgM in serum of E. coli-injected mice, as well as the concentration of IgA (P < 0.05), compared with the Model group (Figure 6C), and the concentrations of IgG and IgM was up to the level of Control group, which indicated that CB-HHP could enhance the humoral immunity of mice injected with enterotoxigenic E. coli.
Concentration of immunoglobulin (Ig) in serum: IgG (A), IgM (B), and IgA (C). Different capital letters indicate that the difference is significant at the 0.05 level.
Discussion
Most traditional medicines are obtained from the combination of varieties of traditional herb/plant extracts. It can be concluded that the anti-infection activities of herbal medicines result from the combined effects of various herbal plant extracts. 54 For the past ten years, some studies have found that the combined use of herbal plant extracts and antibacterial drugs can significantly improve the antibacterial effect and reduce bacterial resistance, 55 however, no recent study has evaluated the antibacterial and antidiarrheal activities of combined Chinese herbal plant extracts. 56 Based on this environment, we try to find a combination prescription of Chinese medicine that can be antibacterial and antidiarrheal. To provide an excellent treatment for diarrhea caused by toxigenic E. coli.
In this study, we first conducted a principal component analysis of CB-HHP by HPLC and GC-MS to dig out the potential active ingredients, among which terpinen-4-ol, α-terpineol, bornyl acetate, and methyl-n-nonylketone are generally considered to be the quality control index components of H. cordata.57–60 First of all, we confirmed that the two medicinal materials were in line with the provisions of pharmacopoeia, 38 which ensured the credibility of this study. Secondly, we explored some potential active substances, which may be the key to play a role in the efficacy and treatment of diseases. Although these substances were not analyzed separately in this paper, it laid the foundation for the subsequent research on active substances.
From this study, it was observed that the antibacterial activity of the three extracts when they used combined was stronger than that when they used alone. This may be attributed to different extracts playing multi-target antibacterial effect together, so as to achieve a stronger antibacterial effect. Many studies have demonstrated that herbal plant extracts as a whole and/or multiple herbs in complex formulations offer better efficacies than equivalent doses of single active ingredients and/or herbs when used alone, highlighting the importance of synergistic effect in herbal therapy. 61 Therefore it was not surprising that C-HHP showed synergistic interaction (FICI < 0.5) against tested bacteria. The zone of inhibition also confirmed this conclusion.
The physiological functions of bacteria are especially concerned with subcellular structures or components including the cell membrane, the cytoplasm and the nucleic acids. Thus, the mode of bacterial inactivation caused by antibacterial agents may be confirmed by changing effects on these subcellular structures of bacteria. 62 Regarding bacteria, the proteins of the cell wall and cytoplasm are essential for maintaining their physiological functions. The loss of intracellular proteins can lead to cell death. Besides the cell membrane and proteins, DNA damage would also give rise to bacterial inactivation. 63 It was reported that H. cordata Thunb. contains many constituents such as essential oil and flavonoids, which can affect the bacterial cell wall resulting in growth inhibition. 64 Previous studies have also shown that fat-soluble substances such as houttuyfonate can directly bind to bacterial cell wall membrane proteins through hydrophobic interactions, resulting in strong antibacterial activity and enhancing the destruction of other components on bacterial cell walls. 65
In this study the AKP and protein leakage from cells of test bacteria were also observed. When the bacterial cell wall is destroyed, the AKP in the periplasmic space of the cell will leak, and when the cell membrane is destroyed, it will cause the leakage of protein. 66 According to the synergistic mechanism proposed above, it may be possible that C-HHP acted together against different cell targets to contribute to their antibacterial activity. It is believed that HCEO may penetrate the lipid layer of the bacterial cell membrane, causing loss of structural integrity or functionality. 67 Furthermore, the presence of HCE and POE are essential for inhibiting microbial growth, which can bind to and inhibit thiol enzymes within cytoplasm and cell wall. The simultaneous action of these two mechanisms could be responsible for the observed synergistic antibacterial effect. 68 SEM also confirmed that C-HHP takes antimicrobial action by changing S. aureus and E. coli cell surface and decreasing the cell density.
The gut is the major organ of nutrient digestion and absorption as well as the largest immune organ. 69 The development and integrity of the intestinal morphology does not only determine the absorption of nutrients but also affect the intestinal mucosal immune response. 70 Intraperitoneal injection of E. coli has been a classic way to induce intestinal infection. 71 The intestinal tract is the main target organ of E. coli infection. 72 Relevant studies have shown that oral gavage or intraperitoneal injection of E. coli can cause intestinal flora disorder, resulting in intestinal mucosal damages, such as intestinal villi broken off, intestinal epithelial cells necrosis and shedding, which further leads to intestinal absorption and secretion dysfunction, thereby inducing diarrhea and causing death in mice. 73 In this experiment, the infected mice showed severe diarrhea, piloerection, lethargy and weight loss. However, oral administration of C-HHP reduced the total amount of mouse diarrheal, and also prevented the loss of weight in mice. Moreover, the results showed that oral administration of 400 mg/kg of C-HHP could promote the repair of intestinal villi injury caused by E. coli infection. Intestinal villi are the basis of intestinal absorption of nutrients. 74 Therefore, C-HHP could repair duodenal villi, so as to increase intestinal absorption of nutrients as well as reduce the weight loss caused by E. coli infection.
Conclusions
This study demonstrates that the combination of HEE, HEO, and PEE produces a synergistic effect against enterotoxigenic E. coli. In vivo, the administration of CB-HHP (HEE: HEO: PEE = 1:0.56:0.5, 800 mg/kg BW) effectively preserves the structural integrity of duodenal villi, diminishes the incidence of diarrhea in mice, and sustains normal body weight in E. coli-injected mice. Consequently, the combination of CB-HHP can be utilized as a green and safe food-derived additive for antibacterial prophylaxis against diarrhea, making it suitable for use as a feed additive.
Footnotes
Abbreviations
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval
All mouse experiments in this study were approved by the Sichuan Agricultural University Animal Ethics Committee by the guidelines of the Chinese Council on Animal Care (permission no. 20210093).
Funding
We gratefully acknowledge funding by the National modern agricultural industry technology system Sichuan innovation team (SCCXTD-2024-19), and the Featured Medicinal Plants Sharing and Service Platform of Sichuan Province.
Statement of Human and Animal Rights
All experimental procedures involving animals were conducted according to the Sichuan Agricultural University guidelines for laboratory animals, approved by the Institutional Sichuan Agricultural University Animal Ethics Committee, and complied with the governmental guidelines (Science and Technology Department of Sichuan Province).
Statement of Informed Consent
There are no human subjects in this article and informed consent is not applicable.