Seven-day Oral Intake of Orthosiphon stamineus Leaves Infusion Exerts Antiadhesive Ex Vivo Activity Against Uropathogenic E. coli in Urine Samples^{[ # ]}

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimentation procedures, solvents, reagents
• Biomedical study design
• Urine samples preparation and analysis
• Cell Culture and Microbiology
• T24 cells cell line and growth conditions
• Uropathogenic E. coli (UPEC) strains and growth conditions
• Monitoring of bacterial growth in urine
• Adhesion assay with urine samples by quantitative flow cytometry
• Invasion assay with urine samples 38
• Motility assay
• Quantitative real-time PCR (qPCR)
• Tamm-Horsfall protein assay
• Statistical analysis
• Funding information
• Contributorsʼ Statement
• References

Abstract

Orthosiphon stamineus leaves (Java tea) extract is traditionally used for the treatment of urinary tract infections. According to recent in vitro data, animal infection studies, and transcriptomic investigations, polymethoxylated flavones from Java tea exert antiadhesive activity against uropathogenic Escherichia coli (UPEC). This antiadhesive activity has been shown to reduce bladder and kidney lesion in a mice infection model. As no data on the antivirulent activity of Java tea intake on humans are available, a biomedical study was performed on 20 healthy volunteers who self-administered Orthosiphon infusion (4 × 3 g per day, orally) for 7 days. The herbal material used for the study conformed to the specification of the European Pharmacopoeia, and ultra high-performance liquid chromatography (UHPLC) of the infusion showed rosmarinic acid, caffeic acid, and cichoric acid to be the main compounds aside from polymethoxylated flavones. Rosmarinic acid was quantified in the tea preparations with 243 ± 22 µg/mL, indicating sufficient reproducibility of the preparation of the infusion. Urine samples were obtained during the biomedical study on day 1 (control urine, prior to Java tea intake), 3, 6 and 8. Antiadhesive activity of the urine samples was quantified by flowcytometric assay using pre-treated UPEC NU14 and human T24 bladder cells. Pooled urine samples indicated significant inhibition of bacterial adhesion on day 3, 6 and 8. The urine samples had no influence on the invasion of UPEC into host cells. Bacterial proliferation was slightly reduced after 24 h incubation with the urine samples. Gene expression analysis (qPCR) revealed strong induction of fitness and motility gene fliC and downregulation of hemin uptake system chuT. These data correlate with previously reported datasets from in vitro transcriptomic analysis. Increased bacterial motility was monitored using a motility assay in soft agar with UPEC UTI89. The intake of Java tea had no effect on the concentration of Tamm-Horsfall Protein in the urine samples. The present study explains the antiadhesive and anti-infective effect of the plant extract by triggering UPEC from a sessile lifestyle into a motile bacterial form, with reduced adhesive capacity.

Introduction

Uncomplicated urinary tract infections (UTI) are one of the most common infectious conditions, with an estimated global incidence of more than 150 million cases per year [1]. UTIs are caused, in about 80% of all cases, by uropathogenic Escherichia coli (UPEC) but Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, group B Streptococcus, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp. can also be involved in the pathogenesis of UTIs [2]. One of the first and most relevant steps in the pathogenesis of the infection is the specific recognition of the host cells of the lower urinary tract, followed by bacterial adhesion and subsequent invasion into the cell [3]. Therefore, the development of specific inhibitors of bacterial adhesion provides a new molecular target for combating UTIs. Antiadhesive entities act mainly at a very early stage of the infection and can also be involved in reducing potential reinfections. Subsequently, inhibition of host cell recognition and adhesion to the host cell membrane reduces the invasion of UPEC into the epithelial cells, the destruction of the epithelial cells and the formation of intracellular bacterial communities (IBC) by UPEC. IBC are, in many cases, responsible for the high degree of infection recurrence [4], [5], [6], [7], [8]. Furthermore, inhibitors of UPEC invasion can reduce the intensity of infection [9]. The main adhesins of UPEC are mannose-sensitive type 1-pili (FimH, the most relevant adhesin in clinical isolates), and digalactoside-specific Pap-pili. FimH interacts with highly mannosylated uroplakins on umbrella cells of the host bladder and kidney cells. Additionally, many other pili contribute to bacterial adhesion. Afimbrial Afa/Dr adhesins bind to type IV collagen [10]. Interaction of UPEC with DAF proteins (human decay-accelerating factor) is fundamental for the internalization of the bacteria into intracellular vacuoles [11]. Additionally, amyloid-like curli interact with proteins of the extracellular matrix, leading to enhanced adhesion of UPEC to the host cells [12].

A variety of traditional herbal remedies have been investigated against UTI focusing on the elucidation of the underlying mode of action. Besides antibacterial compounds (e.g., arbutin, isothiocyanates), anti-inflammatory extracts (e.g., Ononis spinosa L. roots [13], Matricaria recutita L. flowers, Achillea millefolium L. flowers, inhibitors of human hyaluronidase-1 (e.g., clitorienolacton B from O. spinosa roots [13], [14], inductors of Tamm-Horsfall protein as part of the innate immune defence of the renal system (e.g., Vaccinium macrocarpon Aiton fruits [15]), as well, and anti-adhesive and anti-invasive extracts and compounds have been identified (e.g., phthalides from Apium graveolens L. fruits [16], flavones and flavonols from V. macrocarpon fruits [17], flavan-4-ol derivatives from Zea mays L. stigmata [5], and polymethoxylated flavones from Orthosiphon stamineus leaves [18]).

In rational phytotherapy, extracts from the leaves of O. stamineus BENTH. (syn. O. aristatus MIQ., Java tea) from the plant family of Lamiaceae are widely used for UTI. The plant origins from the tropical Asia and is mainly cultivated in Indonesia. Tea preparation from O. stamineus is widely used in the tropical areas and Java tea is popularly known also as “Kumis Kucing” in Indonesia and “Miasai Kucing” in Malaysia. The herbal material used in the Western countries is mainly imported from Indonesia and freshly brewed Java tea is widely used as a refreshing tea in the food sector but is also known as an herbal remedy for medical use and for treatment and prophylaxis of UTI. This medical use is supported by monographs of competent authorities such as the Herbal Medicinal Product Committee (HMPC) of the European Medicines Agency [19], or other scientific bodies such as the European Scientific Cooperative on Phytotherapy [20]. The traditional use is described in the recommendations of HMPC, where the use of aqueous (mainly) and hydroalcoholic extracts (20 to 60% EtOH content) are both accepted for medical registration in the EU.

The phytochemical composition of O. stamineus leaves has been investigated extensively (for review, see [21]).

Within a systematically investigation of O. stamineus for the treatment of UTI, an aqueous extract with the designation OWE (Orthosiphon water extract) has been prepared by hot water (yield 20% w/w, related to the dried starting material; herbal material/extract ratio = 1 : 5) and analytical quantification of the extract by ICH2 guideline-validated HPLC revealed a content of the three analytical marker compounds of OWE as follows: caffeic acid 7.1 ± 0.3 mg/g, cichoric acid 7.9 ± 0.4 mg/g and rosmarinic acid 10.9 ± 0.4 mg/g [8]. A detailed description of the manufacture of OWE and the analytical protocols, including UPLC chromatograms is displayed in [8].

In vitro testing of the OWE indicated concentration-dependent and significant antiadhesive effects against UPEC [8]. OWE showed no direct cytotoxicity in UPEC, as bacterial proliferation was not influenced by OWE. In addition, cellular vitality and mitochondrial activity of human bladder and kidney cells was not negatively influenced by OWE [8]. Detailed investigation and bioassay-guided fractionation of OWE identified polymethoxylated flavones as compounds which are responsible for the observed antiadhesive effect [18]. This is interesting, as it is known from literature that polymethoxylated flavones such as sinensitin, nobiletin, eupatorin, or tangeretin are bioavailable in rats after oral application, and metabolites of these flavones as the respective glucuronides, partially demethylated flavones and sulfates can be detected in urine samples [22], [23], [24]. OWE demonstrated strong influence on bacterial quorum sensing and significantly reduced the gene expression of fimH under in vitro conditions [8]. OWE increased the expression of the motility/fitness gene fliC, which promoted a change of phenotypes towards an increased bacterial motility. These effects have additionally been investigated in detail by transcriptomic analysis of OWE-treated UPEC, which indicated that the reduced bacterial adhesion is due to a decreased formation of the bacterial fimbriae due to disturbance of the chaperone-usher system [21]. Anti-adhesion can cause a relevant anti-infective effect, as has been shown in a mice infection model with animals orally treated with OWE, leading to significant reduced infection of bladder and kidneys [8]. In vivo animal studies (mouse infection model) showed that OWE has significant anti-infective effects (750 mg OWE/kg, p. o. treatment of the animals for 3 and 5 days) and lowered the bacterial colonization in the kidneys and bladder after transurethral infection with UPEC strain CFT073 [8]. Furthermore, 4- and 7-day pre-treatment of the mice with OWE prior to infection with UPEC NU14 reduced the colonization of the bladder [8]. From the published data described above, OWE seems to be a potent antiadhesive agent. Conversely, it is important to recognize that results obtained from in vitro or other preclinical experiments cannot be directly translated into clinical results [19]. The use of biomedical studies with volunteers, evaluating kinetic aspects of the orally administered remedy, could help to close the gap between preclinical studies and clinical investigations. Additionally, monitoring potential functionality by ex vivo investigations could help in clarifying the bioavailability of active compounds after oral ingestion.

The present study describes the results of a biomedical study during which Orthosiphon herbal tea was used, according to the recommendation of the HMPC. Urine samples obtained from volunteers were to be investigated within the present study on potential antiadhesive effects against UPEC to clarify the occurrence of antivirulent effects.

Results and Discussion

The influence of a 7-day oral intake of aqueous infusions prepared from O. stamineus leaves was investigated by a biomedical study. The herbal material used for tea preparation conforms to the specification of European Pharmacopoeia for O. stamineus leaves [25]. Preparation of the O. stamineus leave infusion was performed using 3.0 g herbal material in a standard cellulose tea bag, the addition of 200 mL of boiling water, and 10 min extraction time. Analytical investigation of these Java tea infusions revealed a dry yield of 0.91 g, corresponding to 30.3% (w/w), related to the herbal material (herbal substance : extract ratio = 3 : 1) after lyophilization. A typical U(H)PLC chromatogram obtained from the lyophilized extract, obtained from an exemplary herbal infusion, is displayed in the Supplementary Data (Figure 1S), indicating a qualitatively very similar profile compared to the hot water extract OWE, described in previous investigations in the literature [8], [18], [21]. Main compounds were – as expected – rosmarinic acid, cichoric acid, caffeic acid and minor amounts of polymethoxylated flavones. To investigate the reproducibility of the tea preparation prior to the biomedical study, 9 randomly selected subjects not involved in the study were asked to prepare a java tea. They were instructed to do so according to the same protocol as the study participants (extraction of a tea bag containing 3.0 g of Java tea with 200 mL of boiling water in a teacup for 10 min, moving the tea bag every 2 to 3 min and squeezing it after 10 min).

The tea samples prepared in this way were analyzed by U(H)PLC [8] for content of rosmarinic acid against the respective reference standard after calibration. Average content of rosmarinic acid was calculated with 243.3 ± 21.5 µg/mL. From this, a relative standard deviation of ± 9% was calculated, indicating sufficient reproducibility and validity of the study product.

From the previously quantified content of rosmarinic acid in Java tea (1.8%, c. f. Material and Methods), an amount of 54 mg per tea bag (3 g) can be calculated. The quantified content per serving (ca. 50 mg in 200 mL infusion) fits this very well.

For the biomedical study, Java tea infusions from 3 g of the herbal material were self-administered four times a day. This dosage is based on the recommendation of the HMPC for Orthosiphon tea [19]. The primary goal of the study was to investigate the potential antiadhesive capacity of the urine samples against UPEC as well as the influence of the urine samples on UPEC invasion into T24 bladder cells, UPEC proliferation, gene expression, and potential changes in the UPEC phenotype. After acceptance of the study by the ethics commission, twenty-two volunteers in the U. K. (average age 26.7 years, 12 males, 10 females) were enrolled in the study. After collection of urine samples at days 1, 3, 6 and 8, the urine of two participants was excluded due to a non-continuous intake of the tea preparation. Within the scope of the 7-day consumption of Orthosiphon tea, no intolerances were observed.

The monitoring of morning urine samples obtained from the volunteers before (day 1) and during the consumption (day 3, 6, 8) of Java tea showed no abnormalities related to standard urine parameters, except that the potassium concentration tended to decrease over the study time. Thirty-three percent (day 6) of the urine samples had a relatively high osmolarity at the upper limit (> 700 mosmol/L) of the reference standard range (50 to 1200 mosmol/L). This seems to be due to the nutritional habits of the volunteers, with either a low water intake or a high daily salt consumption. The high osmolarity affected the outcome of the subsequent functional testing: during the 30 min incubation of host cells together with the urine samples (> 700 mosmol/L) drastic morphological changes of the T24 bladder were observed, possibly due to osmotic stress. Consequently, investigation of the urine samples for potential antiadhesive activity could not be performed by co-incubation of the host cells together with UPEC and urine samples. Instead, a 2 h pre-incubation of the bacteria with the urine samples was performed. Subsequently, the urine was removed by a washing step after the preincubation of UPEC. The pre-treated bacteria were added to the T24 bladder cells, and the mixture was incubated for one hour. Evaluation of the adhesion of the fluorescent-labelled bacteria was performed by flow cytometry and the relative adhesion was calculated for every individual against the day 1 control urine.

The analysis of the individual urine samples was performed by three independent assays with n ≥ 2 technical replicates. The individual relative adhesion values showed a homogenous distribution ([Fig. 1 a]) except for two data points, which were statistically investigated by Grubbsʼ test for outliers. After elimination of the two data points, on day 3 and 6, a significant reduction of the relative adhesion on day 3 (mean adhesion 80%) and day 8 (mean 83%) was observed ([Fig. 1 b]); adhesion values on day 6 were reduced (mean 92%) as well, but not significantly when compared to the respective control values from day 1. Investigation of the pooled urine samples indicated significant inhibition of bacterial adhesion to T24 bladder cells on day 3, day 6 and day 8 ([Fig. 1 c]).

Evaluation of the data concerning a potential influence of ethnicity on the outcome of the adhesion assay indicated no significant differences between Caucasian and Asian participants (data not shown).

Subgroup analysis for investigation on the potential influence of the volunteersʼ gender indicated moderate benefits of Orthosiphon application for male individuals, with significantly reduced bacterial adhesion on day 3 and day 8 ([Fig. 1 d]). This effect was more pronounced than that obtained for female participants. Nevertheless, these correlations need to be interpreted carefully, as the evaluated data originated from a limited number of samples.

The results of this preliminary study show that a 7-day oral administration of Orthosiphon tea evokes a significant antiadhesive effect of urine and limits the interaction of UPEC with bladder cells. Based on these results, it can further be inferred that the compounds contained in the OWE are systemically bioavailable after oral intake and that active metabolites are eliminated through the urinary system. Note that antiadhesion can cause a relevant anti-infective effect, as has been shown in a mice infection model with animals orally treated with Orthosiphon aqueous extract, leading to significant reduced infection of bladder and kidneys [8].

To exclude that the observed antiadhesive effect was due to antiproliferative or direct cytotoxicity of the urine samples against UPEC, the pooled urine samples from day 1, 3, 6 and 8 were tested over 24 h for any direct effect on in vitro proliferation of UPEC NU14 ([Fig. 2]).

UPEC grown in day 1 urine samples showed a slightly higher proliferation rate compared to the samples grown in day 3, 6 and 8 urine, but interestingly, the differences after 24 h of incubation were negligible. To assure that the results obtained within the adhesion assay were not hampered by such slight antiproliferative effects, a short time study over 2 h incubation of the fluorescent-labelled bacteria in day 1, 3, 6, 8 urine samples was performed. No differences were found in all test groups (data not shown). This data suggest that urine samples had no relevant antiproliferative effects which would have influenced the adhesion data.

While bacterial adhesion to host cells is the initial step of the pathogen-host interaction, successful infection requires that the attached UPEC be internalized by membrane fusion into the cell. As some reports on natural products interfering with bacterial invasion into the host cell [9] are available, a specific invasion assay was performed with UPEC NU14, which had been pre-incubated for 2 h with the pooled urine samples (day 1, 3, 6, 8). After removal of the urine, the pre-treated bacteria were incubated together with T24 cell. Non-invaded bacteria were eliminated by gentamycin. T24 cells were lyzed, and aliquots of the cell lysate were plated onto agar. Colony-forming units were quantified after 24 h. A slight, non-significant reduction in intracellular bacteria count was recorded, unfortunately with a high standard deviation, due to the complexity of the assay ([Fig. 3]). Comparison of these results with the data obtained from the adhesion assay did not reveal relevant differences ([Fig. 3]).

From these data, it can be concluded that the reduction in the number of intracellular bacteria is not due to an anti-invasive effect, but caused primarily by inhibition of the bacterial adhesion to the host cell.

In vitro studies with aqueous Orthosiphon extract on the gene expression of UPEC indicated significant downregulation of bacterial adhesins (curli, type 1-, F1C-, and P-fimbriae and of the chaperone-mediated protein folding/unfolding and pilus assembly) [21]. In contrast, flagellar and motility-related genes had been shown to get upregulated [21]. As gene expression, measured under in vitro conditions by use of a crude extract in contact with the bacteria may be different compared to the in vivo situation, similar qPCR analysis experiments were performed ex vivo by using the pooled day 8 urine samples, which were obtained from the Orthosiphon-treated volunteers. After 2 h incubation time of UPEC NU14 with the respective urine samples the following genes were monitored by qPCR: fimH, fimC and fimD (type 1-fimbriae), csgA (thin aggregative fimbriae-curli), papGIII, papC, papD, prsGIII (P-fimbriae), sfaG (S-fimbriae), focG (F1C-fimbriae), fyuA (Yersiniabactin siderophore), chuT (hemin uptake system), fliC (flagellum H-antigene, motility and fitness). Significant upregulation was observed for the motility and fitness gene fliC ([Fig. 4]), in accordance to recent data described for Orthosiphon’s in vitro studies [21].

Day 8 urine pre-treatment resulted in about 40% downregulation of the chuT transcript, responsible for the formation of proteins responsible for iron uptake. This downregulation is in accordance with data reported recently for the extract under in vitro conditions [21]. No other genes showed relevant or significant changes. Interestingly, no changes in the expression of genes responsible for attachment was observed. This is in contrast to the reported in vitro data, which showed strong downregulation of type 1 fimbriae-associated genes as a consequence of contact between Orthosiphon extract and UPEC [8], [21].

It could well be that under in vitro conditions, significantly high concentrations of the extract (2 mg/mL) will influence the chaperone-usher mediated formation of pili, while the effect does not occur after incubation with the ex vivo urine samples, because of much lower concentrations of the respective metabolites.

To investigate whether this effect of the urine samples on gene expression level is further reflected by changes in bacterial phenotype, a motility assay was performed, which is strongly related to the activity of the flagellin FliC [8], [26]. Bacteria were pre-incubated for 2 h in pooled urine samples (day 1, 3, 6, and 8) and stabbed on semi-solid agar. It is known that bacteria in semi-solid agar solutions will not only disperse on top (as they do on solid agar 1%), but also insert and move inside and through the soft agar, causing the matrix to appear turbid; increased fitness and motility of the bacteria will thereby result in different spreading behaviour in the agar [8], [27]. As an increased motility of UPEC is a disadvantage for an adequate adhesion, the increased expression of fliC, leading to a high motility phenotype appears as a cause for reduced bacterial adhesion [28], [29]. Day 3, 6, and 8 urine-treated bacteria showed higher motility compared to the untreated bacteria ([Fig. 5]). The respective data cannot be considered significant due to the high standard deviation, but a clear tendency for higher motility can be observed in the individual experiments. Thus, data obtained from the gene expression analysis, indicating increased fliC expression, can be correlated to the changed phenotypic behaviour.

Finally, the influence of the Orthosiphon application on the concentration of Tamm-Horsfall Protein (THP, syn. uromodulin) in the urine samples was investigated. THP is known to be part of the innate immune defence and is produced exclusively by the renal tubular cells in the Henle loop of the kidney [30], [31].

It is known that THP secretion in the urine can be stimulated by exogenous noxes, and extract of cranberry fruit (Vaccinium macrocarpon) has been especially shown to be a strong inductor of THP formation, which subsequently leads to significantly elevated anti-adhesive activity of urine against UPEC [15]. Therefore, all urine samples were investigated using a specific ELISA for THP concentration ([Fig. 6]). No significant changes between day 1, 3, 6, and 8 urine were recorded. As THP formation in the kidney is also gender-specific, subgroup analysis for male and female participants was performed, but no relevant difference was found. From this point of view, Orthosiphon infusion has no influence on THP formation in the kidney.

In summary, the present study clearly proves strong and significant antiadhesive and anti-virulent effects of Java tea in urine samples after oral administration. Direct cytotoxic effects against UPEC can be excluded, but a strong influence in the pathogen-host interaction is obvious. The clinical data obtained within the present study are in good accordance with previously reported data obtained by in vitro experiments and animal infection studies. It seems promising that under in vivo conditions, antiadhesive and antivirulent effects are observed in the urine samples, indicating that the compounds from O. stamineus leaves are responsible for the observed effects are bioavailable after oral ingestion. Additionally, the respective metabolites are obviously excreted in the urine. Based on the recent in vitro studies, polymethoxylated flavones have been claimed to be the main antiadhesive compounds in Java tea extracts [18]. In particular, flavones with high lipophilicity due to multiple methoxylations in the A- and B-rings (e.g., sinensetin, ladanein, 5,6,7,4′-tetra-methoxyflavone, 5-hydroxy-6,7,3′,4′-tetra-methoxyflavone) exert high anti-adhesive activity. Polymethoxylated flavones (e.g., sinensitin, eupatorin) have been shown to be bioavailable after oral administration to rats and partially demethoxylated metabolites have been identified in rat urine samples [23], [24]. From this point of view the fraction of polymethoxylated flavones from Java tea infusion is assessed to be responsible for the observed antiadhesive effects found for the urine samples in the above-described biomedical study. Unfortunately, despite the use of sensitive and specific LC-MS protocols (data not shown), we had been not able to quantify the amount of these compounds in the urine samples. This is due to the high variability of different polymethoxylated flavones in Java tea (> 6 different aglycones have been described for O. stamineus leaves by [18]) and subsequent metabolic demethylation will additionally lead to the formation of even more products, excreted also in the urine. This complex mixture has not been accessible to our LC-MS analytical protocols. However, there is no doubt of the antiadhesive functionality of the investigated urine samples against UPEC. The underlying mechanism still seems to be not clear. A stimulation of host-innate immune defence via THP-induction, similar to the mode of action described for cranberry extracts for UTI [15] can be excluded. Also, direct inhibition of the bacterial adhesins (e.g., FimH) by Java tea metabolites is unlikely, based on the findings that the expression of relevant fimbrial genes is not influenced by the urine samples, and that FimH-mediated yeast agglutination is not reduced by the test samples. It is interesting, however, that the motility gene fliC is significantly upregulated, which is also reflected by increased motility in the respective UPEC phenotypic. From this perspective, it can be assumed that Orthosiphon triggers the bacterial cell into a high motile form, which again leads to a strongly reduced sessile form and bacterial adhesion to the host cells. Motility and adhesion are, in general, two different living forms contrary to one other: reduced adhesion during high motility phase or increase attachment of UPEC to host cells in cases where the flagella activity is downregulated. As these Orthosiphon effects have been shown in the present study by the urine samples as well as in a recent report by an in vitro transcriptomic study [21], the assumption that Orthosiphon triggers the UPEC lifestyle from a more sessile into a motile form has been confirmed. Which molecular factors are responsible for this switch remains unclear at the moment and detailed molecular studies on the influence of polymethoxylated flavones on the flagella regulation are required to pinpoint the exact molecular mechanism.

Materials and Methods

General experimentation procedures, solvents, reagents

If not stated otherwise, solvents, reagents, and consumables were obtained from VWR International (Darmstadt, Germany). All solvents and reagents were of analytical quality. Water was produced by a Millipore Simplicity 185 system (Schwalbach, Germany). Dried leaf material from O. stamineus (Orthosiphonis folium), batch no 201 808 131 005, was obtained by Martin Bauer Group. The material was identified by AH and complied with the specifications of the European Pharmacopoeia [25] (identity TLC: conforms, loss on drying: conforms, foreign matter: conforms, total ash: conforms, content rosmarinic acid HPLC 1.8% w/w): conforms, pesticides: complies to EU396/2005, Ph. Eur. 2.8.13). Additionally, identity testing for polymethoxylated flavones, caffeic acid, and cichoric acid was performed by U(H)PLC as described recently [8]. A voucher specimen of the material is retained in the archives of Institute of Pharmaceutical Biology and Phytochemistry, Münster, Germany under the designation IPBP 495.

Manufacture of the study teas for the biomedical study was performed in a community pharmacy store under controlled conditions complying with GMP. 3.0 g of Orthosiphon leaves were filled into tea filter bags (Caesar & Loretz). Each tea bag was labelled with the relevant information for every volunteer.

Quantitation of rosmarinic acid in Java tea infusions was performed by use of rosmarinic acid reference compound (Sigma-Aldrich, content 97.0% HPLC) in the range of 50 to 530 µg/mL.

Biomedical study design

The study protocol “An intervention with Java tea (Orthosiphon stamineus) – Metabolite changes in urine” was approved (March 20, 2019) by the UCL Research Ethics Committee, London, U. K. (project: 5101/001).

Healthy men and women with an age of 18 to 40 years were included. Exclusion criteria is listed in the supplementary material.

Twelve male and ten female participants gave written informed consent to participate in the study. The mean age was 26.7 years, median 25.5 years, ranging from 21 to 35 years. Ethnicity: 50% were Caucasian (11/22), 41% Asian (9/22), 2 participants (9%) mixed ethnicity. Preferred diet of the volunteers was documented by the questionnaires. Normal balanced diet was recorded for 82%, vegetarian and pescatarian diets were stated for one participant each, and 14% reported an unbalanced, carbohydrate-loaded diet. Concerning the potential intake of drugs during the biomedical study, 9% of the volunteers reported on the intake of hormone-based contraceptives at the time of the study, while the rest of the volunteers reported no use of concomitant medications. All participants volunteered to take Orthosiphon tea (4× daily, 3 g per dosing) over a 7-day period (one cup in the morning, one at noon, one in the afternoon and one in the evening), and to collect a sample of their first morning urine of the day on day 1 (prior to Orthosiphon tea application = control urine), 3, 6, and 8 for ex vivo studies.

Before starting the trial, volunteers were instructed to abstain from consumption of any other products containing cranberry or phytochemically or botanically similar fruits (especially from the plant family Ericaceae) two weeks before and during the study. Each participant was asked to drink a cup of freshly prepared Orthosiphon tea, made from prepacked tea sachets, each containing 3.0 g of the herbal material, four times a day, reasonably distributed through the morning, afternoon, and evening, regardless of food. The daily intake was equivalent to 12.0 g herbal material per day for 7 days, based on the recommendation of the Committee on Herbal Medicinal Products [19]. Preparation of the Java tea infusion was performed by infusing one filtered tea bag for 10 min in 200 mL of boiling water. The addition of sugar, honey or lemon juice after preparation was allowed. The tea could be taken independently of meals, but the volunteers were instructed to take it every day at approximately the same time (± 2 hours) and to check the box of the corresponding scheduled intake on the detailed calendar provided.

Generally, the first midstream urine of the day was collected and used for functional and analytical investigations. A control urine sample (day 1) was collected prior to the consumption of the tea.

Volunteers were instructed to collect the samples and store them in a refrigerator (+ 2 to 8 °C), or, if possible, in a freezer (< − 5 °C), until handing it over to the research facility the very same morning of collection. The urine was finally stored in a − 20 °C freezer until analysis.

By the end of the study, two drop-outs (one male, one female) were identified and removed from the data collection, due to non-continuous intake of infusions; one participant did not collect day 8 urine. Samples of day 1, 3 and 6 of the participants were included. Therefore, 79 urine samples from 20 volunteers were included in the subsequent ex vivo evaluation.

Urine samples preparation and analysis

The urine samples were stored at − 20 °C until use. Two mL of each urine sample from all volunteers were pooled and named day 1 PU (pooled urine), day 3 PU, day 6 PU, day 8 PU.

All urine samples obtained were tested for their pH, density, and content of creatinine, leukocytes, erythrocytes, sodium, potassium, chloride, bilirubin, urobilinogen, glucose, nitrite, protein, ketones (Supplementary Data, Table 1S and Table 2S). Urine osmolality was determined after dilution with highly purified water (Millipore quality) using a Semi-Micro Osmometer K-7400 (Knauer) (Supplementary Data, Table 3S)

Cell Culture and Microbiology

T24 cells cell line and growth conditions

T24 cells (ATCC HTB-4) represent a human epithelial bladder cell line, derived from the bladder carcinoma of an 82-year-old Swedish female [32]. These cells have been demonstrated suitable for adhesion and invasion in in vitro assays with UPEC [33] and were kindly provided by Prof. Straube (University of Jena, Germany). Cultivation was performed as described by [18].

The cultivation of the cells was performed in Dulbeccoʼs Modified Eagle Medium with high glucose and L-glutamine (DMEM) (Biochrom), supplemented with 10% FCS (Biochrom) and 0.5% penicillin/streptomycin (Biochrom) at 37 °C and 5% CO₂.

Uropathogenic E. coli (UPEC) strains and growth conditions

Bacterial strains: UPEC strain NU14 (NCBI txid569 579), a fully sequenced clinical isolate, obtained from a patient with an acute cystitis, was provided by Prof. Dr. U. Dobrindt, University of Münster, Germany [34]. The clinical cystitis isolate UPEC UTI89, (NCBI: txid364106) was provided by Prof. Dr. U. Dobrindt, University of Münster, Germany [35].

Optical density (OD) was determined at λ = 640 nm and 1 × 10⁹ colony-forming units were equivalent to an OD₆₄₀ of 5.0.

Bacteria from frozen stocks were cultivated for 48 h on UPEC agar (Agar-Agar 15 g, Bacto Tryptone 10 g, NaCl 8 g, glucose 1 g, yeast extract 1 g, CaCl₂ 2 g, purified water 1 L) were used. CaCl₂ supplementation is supposed to increase the type 1 fimbria expression [36].

Urine culture: 1 CFU of overnight agar-grown bacteria was transferred to 10 mL in 50 mL tubes and incubated at 37 °C overnight (~ 15 h) in a steady culture at 37 °C/5% CO₂.

Monitoring of bacterial growth in liquid culture and adhesion assay by quantitative flow cytometry was performed as described by [5], [15], [17].

Monitoring of bacterial growth in urine

Pooled urine samples from day 1 (control urine), 3, 6, and 8 were transferred in aliquots of 180 µL each into a 96-well plate. Additionally, pooled urine supplemented with gentamycin (100 µg/mL) was used as positive control. Overnight agar-grown bacteria (UPEC NU14) were harvested and suspended in UPEC liquid medium. The OD_640 nm adjusted to 0.5. 20 µL of the suspension was added to the urine samples and gently mixed. The plate was incubated at 37 °C and bacterial growth was monitored by measuring the optical density every 60 min over a 6- h period and after 24 h at λ = 640 nm. Moreover, the OD of the urine samples without any additional bacteria was determined to ascertain that there are no differences in the OD due to different coloured urine.

Adhesion assay with urine samples by quantitative flow cytometry

In general, FITC-labelling of UPEC and flow cytometric adhesion assay was performed as described by [4], [37]. Agar-grown UPEC NU14 were labelled under light protection with fluorescein isothiocyanate (FITC) as follows: UPEC were re-suspended in 1 mL sterile saline solution (NaCl 150 mM, Na₂CO₃ 100 mM, pH 8.0). 14 × 10⁸ bacteria were re-suspended in 900 µL of saline solution. One hundred µL of a FITC solution (10% in DMSO) were added and incubated for 60 min at 37 °C in a thermomixer (Eppendorf) at 300 rpm. The labelling process was terminated by pelleting the bacteria (10.000 × g, 5 min). Labelled bacteria were washed 3 × with 1 mL PBS to remove excess FITC, re-suspended in 1 mL PBS and adjusted to OD_640 nm of 4 (determined from a 1 : 20 dilution). All further steps with FITC-labelled E. coli were carried out under direct light protection. For sample preparation, 900 µL of individual or pooled urine samples were mixed with 100 µL of FITC-labelled bacteria suspension (OD_640 nm 0.4) and incubated for 2 h at 37 °C/5% CO₂. Subsequently the suspensions were centrifuged (10.000 × g, 5 min) and pelleted bacteria washed twice with 1 mL PBS each. Washed bacteria were re-suspended in 1 mL DMEM and added to the prior prepared T24 cells. T24 cells (1.25 × 105 cells/well) were seeded into 6-well plates and incubated at 37 °C/5% CO₂ until 90% confluence was reached (corresponding to 800.000 cells, after approximately 48 h of incubation). The medium was removed, and cells were washed twice with PBS (1 mL) and once with DMEM (1 mL). For adhesion experiments, the bacteria-to-cell ratio (BCR) of 100 : 1 was used. UPEC and T24 cells were incubated for 1 h at 37 °C. Subsequently, unattached UPEC were removed by gently washing the cells 3 × with 1 mL PBS/well. Cells were detached by addition of 1 mL trypsin/EDTA for 4 min at 37 °C. Trypsinization was stopped by addition of 2.5 mL DMEM. The content of each well was transferred to tubes and centrifuged for 5 min at 450 × g. The supernatant was discarded, and the cells re-suspended in 700 µL of DMEM. Fluorescence of the cell suspension was measured by flow cytometry (FACS Calibur, Software: BD CellQuest Pro V 5.2, BD Biosciences). For data evaluation, 10.000 counts per sample were used. Day 1 urine samples were used as untreated control urine, while day 1 urine added with antiadhesive hydroalcoholic Zea mays extract [5] (1 mg/mL) served as positive control.

Invasion assay with urine samples [38]

Cells (1.25 × 10⁵ cells/well) were seeded into 6-well plates and incubated at 37 °C/5% CO₂ until 90% confluence was reached (corresponding to 800.000 cells, after approximately 48 h of incubation). After this incubation, T24 cell culture medium was removed, and cells were washed twice with PBS and once with DMEM. Agar-grown UPEC NU14 were harvested and suspended in 1 mL DMEM. The OD_640 nm was adjusted to 4. 100 µL of the suspension were added to 900 µL of each urine sample and incubated for 2 h at 37 °C/5% CO₂. Subsequently, the suspensions were centrifuged (10.000 × g, 5 min) and pelleted bacteria washed twice with 1 mL PBS each. Washed bacteria were re-suspended in 1 mL DMEM and added to the prior prepared T24 cells. Bacteria and T24 cells were incubated for 1 h at 37 °C/5% CO₂. Bacteria which did not interact during the incubation with T24 cells were removed by 3 × washing of the T24 cells with 1 mL PBS/well. Subsequently, DMEM containing 100 µg/mL of the membrane-impermeable antibiotic gentamycin was added for 1 h at 37 °C to the samples to eliminate selectively extracellular bacteria. The antibiotic was removed by rinsing three times with PBS. Finally, cells were lyzed/15 min, room temperature) by the addition of 0.1% Triton X-100; the lysate was plated in 1 : 250 dilution onto UPEC agar and incubated for 24 h at 37 °C. Lysis released bacteria which had already been invasive before the addition of gentamycin and gave them the possibility to multiply on the agar plates. Anti-invasive activity was evaluated by counting CFU after incubation time. Bacteria incubated in control urine (day 1) served as the untreated control.

Motility assay

Motility was evaluated using soft agar plates (0.25% agar), which were prepared the day prior to use and left at room temperature overnight. UPEC strain UTI89 (one colony from 24 h agar-grown bacteria) were cultivated and suspended in 1 mL UPEC liquid medium. OD_640 nm was adjusted to 20. Fifty µL of the suspension was mixed with 950 µL of the pooled urine samples and incubated for 2 h at 37 °C/5% CO₂. One µL of the suspension was transferred into a soft agar plate, which was incubated for 48 h at 37 °C/5% CO₂. Motility was determined by viewing wet mounts of bacterial cultures and measuring the diameter of motility.

Quantitative real-time PCR (qPCR)

Preparation of E. coli strain UTI89: One colony of 24 h agar-grown bacteria strain UTI89 was inoculated in 10 mL of pooled human urine (urine received independently of the study) and incubated overnight (~ 15 h) in a steady culture at 37 °C/5% CO₂. Bacteria were pelleted by centrifugation (5000 × g, 5 min), re-suspended in 500 µL of fresh urine and the OD_640 nm adjusted to 1. One hundred µL of the suspension was transferred to 9.9 µL day 1 and day 8 urine samples and incubated at 37 °C/5% CO₂ for 2 h to reach the mid-logarithmic phase. The suspensions were centrifuged (5000 × g, 5 min), the supernatant removed, and the pellets re-suspended in 500 µL PBS. Immediately, 1 mL of RNAprotect Bacteria reagent was added to the pellet.

Bacterial RNA isolation: Total RNA was extracted using of RNeasy mini kit according to the manufacturerʼs instruction. Briefly, the tubes, which contained the bacteria and RNAprotect suspension were centrifuged (8.000 × g, 10 min), the supernatant was decanted, and 100 µL of TE buffer, containing 1 mg/mL lysozyme, were added to each tube. After 8 min of incubation at RT an appropriate volume of RLT buffer and ethanol were added. 700 µL of the mixture were transferred to an RNeasy spin column. After removal of impurities, 35 µL of RNase-free water were directly added to the column and the RNA was eluted by centrifugation.

DNA digestion: DNA was digested using a TURBO DNA-free Kit following the manufacturerʼs instruction. ≤ 200 µg/mL isolated RNA was transferred to a 0.2 mL tube and 5 µL of 10× Turbo DNAse buffer as well as 1 µL Turbo DNAse was added, then mixed and incubated for 30 min at 37 °C in a Thermocycler. After adding 5 µL inactivation reagent and incubation for 5 min, the mixture was centrifuged (10.000 × g, 1.5 min) and the supernatant including RNA transferred to a fresh tube.

cDNA synthesis: RNA was transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit following the manufacturerʼs instruction. To 1 µg RNA (13 µL), 2 µL random hexamer primer was added. After 10 min incubation at 65 °C, 4 µL reverse transcriptase buffer, 0.5 µL protector RNase inhibitor, 2 µL deoxynucleotide mix and 0.5 µL reverse transcriptase were added and the mixture incubated in the thermocycler (10 min 25 °C, 60 min 50 °C, 5 min 85 °C.

q-PCR was performed with an equivalent of 15 ng of total RNA using the iTaq Universal SYBR Green supermix (BioRad,) according to the protocol recommended by the manufacturer, using a CFX96 Real-Time SystemC1000 Touch (BioRad). qPCR parameters were as follows: polymerase activation and initial denaturation for 15 s at 95 °C followed by 39 cycles of 5 s at 95 °C and 30 s at 60 °C. Afterward, an additional melt curve analysis was performed (65 – 95 °C, ramp 0.5 °C pro cycle, 1 cycle = 5 s, 60 cycles in total). Data were evaluated with the BioRad CFX Manager 3.0 software based on the comparative CT method (2−ΔΔCT method) and normalized to the endogenous reference gene coding for the 16S rRNA. Primers for the qPCR were designed with the Universal Probe Library Assay Design Center (Roche, Switzerland). Oligonucleotides were obtained from Eurofins MWG Operon, Luxembourg. Primer sequences used for the differential gene expression analysis are listed in [Table 1].

Tamm-Horsfall protein assay

Concentration of THP in urine was quantified by an in-house sandwich-like ELISA, modified accordingly [39]. 96-Well Nunc Maxisorp (Thermo Fisher) were coated with 100 µL of a solution (concentration 10 µg/mL) of wheat germ agglutinin from Triticum vulgaris (Sigma-Aldrich), diluted in coating buffer (\pH 9.6, Na₂CO₃ 50 mM, NaHCO₃ 349 mM, NaN₃ 0.02% (w/v) in H₂O) for 2 hours at room temperature while gently shaking. After rinsing with washing buffer (Tween 20, 0.05% in PBS), non-specific binding sites were blocked with 200 µL of blocking buffer (2% (m/v) BSA in washing buffer) for 2 h while gently shaking. After blocking, plates were washed 3× with washing buffer. Residual buffer was removed by air drying.

Urine samples were diluted 1 : 10 (or 1 : 20 in cases where absorption at λ = 450 nm was too high) with blocking buffer. 100 µL of the sample were added to the pre-coated wells and incubated for 2 h at room temperature while gently shaking. Samples and standards were run in duplicate. Blocking buffer served as blank. After incubation, plates were washed 3 × with washing buffer. Plates were then placed top-down on absorbent paper to remove residual buffer. Gentle tapping is recommended. 100 µL of Sheep Anti-Human Tamm-Horsfall Glycoprotein (BioRad) (1 : 1000 in blocking buffer) was dispensed into each well and incubated for 2 h at room temperature while gently shaking. Plates were washed as described above. One hundred µL of Rabbit Anti-Sheep IgG (H+L)-HRP Conjugate (BioRad) (1 : 1000 in blocking buffer) was added and incubated for 1 h at room temperature. The well plate was washed again as previously described. For photometric detection, 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB Liquid Substrate System for ELISA) (Sigma-Aldrich) was dispensed on each well and incubated for up to 20 min in the absence of light. Measurements were performed immediately after addition of 100 µL sulfuric acid 4 mol/L at λ = 450 nm with λ = 550 nm as reference wavelength. The intensity of this signal is directly proportional to the concentration of THP present in the original specimen. Concentration of THP was calculated by plotting a four-parameter logistic curve fit for standard concentrations and then interpolation of sample absorbances.

Statistical analysis

Statistical results were obtained using GraphPad Prism statistics (version 3) (GraphPad Software). Results are expressed as the mean value (MV) ± standard deviation (± SD). Data (n ≥ 3) were processed by analysis of variance (one-way ANOVA). Subsequent post hoc testing was conducted using Tukeyʼs test to determine the statistical significance of differences between mean values of two with each other compared groups. p < 0.05 was determined as statistically significant (*), p < 0.01 as high significant (**) and p < 0.001 as very high significant.

Funding information

This project received no direct external funding.

Contributorsʼ Statement

MD and FS performed the experiments on UPEC and host cells, BM analysed THP, MH mentored the biomedical study, AH designed the study and mentored the lab work.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

Many thanks to Dr. Matthias Lechtenberg for help within the quantification of rosmarinic acid in the Java tea preparations! Also, many thanks to Mrs. Cathleen Possemeyer and Mrs. Ursula Liefländer-Wulf for organisation of the “tea study” and HPLC analysis of the infusion samples! We are grateful to all participants of the biomedical study, mostly students at the School of Pharmacy, UCL, London, U. K., and volunteers from the University of Münster, Germany, for preparation of Java tea infusions for analytical rosmarinic acid quantification.

^# Dedicated to Prof. Gerhard Franz, University of Regensburg, Germany, on the occasion of his 85th birthday.

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Correspondence

Publication History

Received: 12 May 2021

Accepted after revision: 30 July 2021

Article published online:
14 September 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: Epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015; 13: 269-284
  Search in Google Scholar
• 2 Ronald A. The etiology of urinary tract infection: Traditional and emerging pathogens. Dis Mon 2003; 49: 71-82
  CrossrefPubMedSearch in Google Scholar
• 3 Martinez JJ, Mulvey MA, Schilling JD, Pinkner JS, Hultgren SJ. Type 1 pilus-mediated bacterial invasion of bladder epithelial cells. EMBO J 2000; 19: 2803-2812
  CrossrefPubMedSearch in Google Scholar
• 4 Rafsanjany N, Senker J, Brandt S, Dobrindt U, Hensel A. In vivo consumption of Cranberry exerts ex vivo antiadhesive activity against FimH-dominated uropathogenic Escherichia coli: A combined in vivo, ex vivo, and in vitro study of an extract from Vaccinium macrocarpon . J Agric Food Chem 2015; 63: 8804-8818
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• 5 Rafsanjany N, Sendker J, Lechtenberg M, Petereit F, Scharf B, Hensel A. Traditionally used medicinal plants against uncomplicated urinary tract infections: Are unusual, flavan-4-ol- and derhamnosylmaysin derivatives responsible for the antiadhesive activity of extracts obtained from stigmata of Zea mays L. against uropathogenic E. coli and Benzethonium chloride as frequent contaminant faking potential antibacterial activities?. Fitoterapia 2015; 105: 246-253
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• 6 Messing J, Thöle C, Niehues M, Shevtsova A, Glocker E, Borén T, Hensel A. Antiadhesive properties of Abelmoschus esculentus (Okra) immature fruit extract against Helicobacter pylori adhesion. PLoS One 2014; 9: e84836
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• 7 Sarshar S, Sendker J, Qin X, Goycoolea FM, Asadi Karam MR, Habibi M, Bouzari S, Dobrindt U, Hensel A. Antiadhesive hydroalcoholic extract from Apium graveolens fruits prevents bladder and kidney infection against uropathogenic E. coli . Fitoterapia 2018; 127: 237-244
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  CrossrefPubMedSearch in Google Scholar
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• 14 Addotey JN, Lengers I, Jose J, Gampe N, Béni S, Petereit F, Hensel A. Isoflavonoids with inhibiting effects on human hyaluronidase-1 and norneolignan clitorienolactone B from Ononis spinosa L. root extract. Fitoterapia 2018; 130: 169-174
  CrossrefPubMedSearch in Google Scholar
• 15 Scharf B, Sendker J, Dobrindt U, Hensel A. Influence of Cranberry extract on Tamm-Horsfall Protein in human urine and its antiadhesive activity against uropathogenic Escherichia coli . Planta Med 2019; 85: 126-138
  Thieme ConnectPubMedSearch in Google Scholar
• 16 Grube K, Spiegler V, Hensel A. Antiadhesive phthalides from Apium graveolens fruits against uropathogenic E. coli . J Ethnopharmacol 2019; 237: 300-306
  CrossrefPubMedSearch in Google Scholar
• 17 Scharf B, Schmidt TJ, Rabbani S, Stork C, Dobrindt U, Sendker J, Ernst B, Hensel A. Antiadhesive natural products against uropathogenic E. coli: What can we learn from cranberry extract?. J Ethnopharmacol 2020; 257: 112889
  CrossrefPubMedSearch in Google Scholar
• 18 Deipenbrock M, Hensel A. Polymethoxylated flavones from Orthosiphon stamineus leaves as antiadhesive compounds against uropathogenic E. coli . Fitoterapia 2019; 139: 104387
  CrossrefPubMedSearch in Google Scholar
• 19 European Medicines Agency. Herbal medicines for human use: The official European Union Herbal Monographs of the Committee on Herbal Medicinal Products (HMPC). Orthosiphonis folium . Accessed August 20, 2018 at: http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_Community_herbal_monograph/2011/01/WC500100376.pdf
  PubMed
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