IN-VITRO ANTIDIABETIC ACTIVITY OF A BISTORTA OFFICINALIS DELARBRE ROOT EXTRACT CAN NOT BE CONFIRMED IN THE IN-VIVO MODELS HEN’S EGG TEST AND DROSOPHILA MELANOGASTER

Bistorta officinalis Delarbre (syn. Polygonum bistorta (L.), Persicaria bistorta (L.) Samp.) is a perennial herbaceous plant commonly known by the names bistort, snakeroot or snakeweed (1-4). This plant with a height from 20 to 100 cm belongs to the Polygonaceae family and is characterized by a thick snake-like curved rhizome and a single spike inflorescence in the shades of pink, from pale to strong (1, 4), (Fig. 1). It is a decorative plant, but interestingly it is also used for food preparation (3, 4) and can therefore be considered safe for human consumption use. B. officinalis is widespread across the northern hemisphere, primarily located in Europe and Asia but also found in North America (1-4). In these regions, B. officinalis is also employed as a folk medicine against various medical conditions such as skin complaints, gastrointestinal disorders or haemorrhoids (1, 2, 4, 5). Moreover, the plant is used against snakebites in traditional medicine in China (6) or Poland (2). The anti-inflammatory and astringent properties of the plant coincide with the presence of several bioactive compounds (1). While flavones are mainly concentrated in aerial parts, the rhizome of B. officinalis is rich in tannins, phenolcarbonic acids and their derivatives and catechins (1, 4).

Fig. 1. Bistorta officinalis Delarbre.

Although there are some scientific publications on the effectiveness and mode of action of B. officinalis in relation to the prevention and treatment of diseases such as cancer (5, 7, 8), studies on the therapeutic benefit of this plant against the common metabolic disease type 2 diabetes mellitus (DM) (9) are lacking. DM is characterized by (fasting) hyperglycaemia caused by insufficient insulin action due to defects in insulin secretion (10) and/or altered insulin receptor signalling (11). Controlling postprandial hyperglycaemia is essential in diabetes management. To this end, different regulatory key points are of interest including intestinal carbohydrate-hydrolysing enzymes (α-amylase and α-glucosidase) (12), the incretin system (13, 14) or glucose transporters such as the intestinal sodium-dependent glucose transporter 1 (SGLT1) (15). Oral glucose-lowering drugs are used as part of conventional therapy. Moreover, the development of prediabetes (an intermediate stage between normal glucose regulation and diabetes) into diabetes can often be prevented or delayed by taking blood sugar-lowering drugs (16).

Accordingly, plant bioactives that have been used in folk medicine for a long time are now becoming a valuable alternative to conventional strategies for DM management in terms of financial benefits coupled with reduced adverse effects caused by conventional drugs (17).

In this context, we initially performed a screen for α-amylase and α-glucosidase inhibition to reveal root extracts with putative antidiabetic properties from our local plant extract collection Kiel in Schleswig-Holstein (PECKISH) (18). This screening approach led to the identification of an aqueous extract from B. officinalis root material. Of note, putative antidiabetic effects in humans have been mentioned for B. officinalis as part of traditional formulations (19). Following a small-scale lab production of the B. officinalis Delarbre extract (BODE), it was tested for its antidiabetic potential in enzyme- and cell culture-based assays that cover various key points of glucose regulation. Moreover, its main constituents were identified by HPLC-MS analyses. To verify the data obtained from these experiments, we employed two diabetes in-vivo models that have the additional advantage of including several pharmacological aspects that are not addressed by in-vitro and cell culture assays. A modified assay variation of the hen’s egg test-chorioallantoic membrane (HET-CAM) assay, named Gluc-HET is an innovative method for studying insulin mimetic compounds (20-23). In addition, the fruit fly Drosophila melanogaster has been established as a versatile model organism in nutritional research (24). In particular, it has been proven to be a promising model for evaluating antidiabetic potential of plant extracts by feeding a starch-based diet that induces triglyceride accumulation and, hence, an obese phenotype in the fruit fly (20). Our study revealed that, although exhibiting promising effects in in-vitro and cell culture assays, BODE did not show antidiabetic activity in our in-vivo models.

MATERIAL AND METHODS

Enzymes and chemicals

The α-amylase, α-glucosidase, p-nitrophenyl-α-D-glucopyranoside, acarbose, 3,5-dinitrosalicylic acid, sodium potassium tartrate, NaOH, gallic acid and mannitol were obtained from Sigma-Aldrich (Taufkirchen, Germany). Glucose, agarose, propionic acid and Triton X100 were purchased from Carl Roth (Karlsruhe, Germany) and Folin-Ciocalteu reagent from Merck (Darmstadt, Germany). Na2CO3 and starch were obtained from VWR (Darmstadt, Germany). Moreover, dextrose, sucrose, cornmeal, inactive dry yeast and agar Type II were purchased from Kisker (Steinfurt, Germany). Tegosept and nipagin were obtained from Genesee Scientific (San Diego, CA, USA).

Plant extract preparation

B. officinalis roots were purchased from Natur-Krauter (Freyung, Germany). The aqueous BODE was generated from dried root material according to the protocol previously described (20). In brief, 3 g of grinded plant material were stirred in 30 mL of boiling double distilled water for 1 min, followed by sonication for 1 min (Sonoplus UW 2070, Bandelin electronic, Berlin, Germany). Tubes were centrifuged for 2 min at 2000 × g. The supernatant was filtered by a folded filter (MN 615 ¼, 185 mm; Macherey-Nagel, Duren, Germany) and the aqueous BODE was stored at –20ºC.

Enzyme inhibition assays

The α-amylase, α-glucosidase and DPP4 inhibition assays were performed as previously reported (20). The α-amylase inhibitory activity of BODE was determined according to the method described by Apostolidis et al. (25) with slight modifications. Therefore, 50 µL of BODE at different concentrations (10 µg/mL to 20,000 µg/mL) were mixed with 50 µL of 0.5 mg/mL porcine pancreatic α-amylase in 20 mM sodium phosphate buffer, pH 6.9. Following 10 min pre-incubation at 25ºC, 50 µL 1% starch solution that had been cooked for 15 min in the same buffer were added. The mixture was incubated for 10 min at 25ºC. Thereafter, 100 µL of a colour reagent (1% 3,5-dinitrosalicylic acid and 30% sodium potassium tartrate in 0.4 M NaOH) were added. The mixture was incubated for an additional 5 min at 100ºC and cooled to room temperature, before the absorbance was measured at 540 nm by a microplate reader.

The inhibition of α-glucosidase by BODE was assayed following the method described by Awosika and Aluko (26) with some modifications. Therefore, 15 µL of BODE at different concentrations (10 µg/mL to 1000 µg/mL) were mixed in 96-well microtest plates with 105 µL of 0.1 M phosphate buffer, pH 6.8 and 15 µL of 0.5 U/mL α-glucosidase from Saccharomyces cerevisiae. Following 5 min pre-incubation at 37ºC, 15 µL 10 mM p-nitrophenyl-α-D-glucopyranoside in the same buffer were added as a substrate to initiate the reaction. The mixture was incubated for 20 min at 37ºC, before 50 µL 2 M Na2CO3 were added to stop the reaction. The absorbance was measured at 405 nm. Acarbose was used as a positive control in both, the α-amylase and the α-glucosidase assay. The percentage inhibition values were determined to calculate the IC50 value by nonlinear regression using GraphPad Prism.

The DPP4 assay was performed using a commercial inhibitor screening kit (MAK203, Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. In brief, 50 µL of inhibition reaction mix, containing 49 µL assay buffer and 1 µL DPP4 enzyme, were mixed in black 96-well microtiter plates with 25 µL BODE (1000 µg/mL final concentration) or the reference inhibitor sitagliptin. Following 10 min pre-incubation at 37ºC, 25 µL of an enzymatic reaction mix containing 23 µL assay buffer and 2 µL substrate was added to each well. The fluorescence signal (Ex/Em 360/465 nm) was measured at 37ºC over 30 min in 1 min intervals.

Sodium-dependent glucose transporter 1 activity in Caco-2 monolayers

1. Cell culture and transepithelial electrical resistance (TEER) measurement

The Caco-2/PD7 clone was a kind gift from Edith Brot-Laroche (Unite de Recherches sur la Differenciation Cellulaire Intestinale, Villejuif Cedex, France) (27). The cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech, Aidenbach, Germany) supplemented with 20% (v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Darmstadt, Germany). Cells were grown at 37ºC in a humidified atmosphere with 5% CO2.

For Ussing chamber experiments Caco-2/PD7 cells were seeded into 6-well Corning® Costar® Snapwell cell culture inserts (0.4 µm pore size, 1.12 cm2 surface area) (Merck, Dormstadt, Germany) at a density of 1 × 106 cells/well. 0.5 mL of the cell-containing medium were given into the upper compartment (apical side) and 2.5 mL of cell-free medium into the lower compartment (basolateral side). Cells were cultured in plates for 21 days and medium was replaced every other day. After 7 days the apical medium was modified, now lacking FBS (28).

TEER of the Caco-2/PD7 monolayer was measured against a blank well containing cell culture medium only, using a Millicell ERS-2 Volt-Ohm Meter equipped with a STX01 planar electrode (Merck, Dormstadt, Germany). Only monolayers with TEER values exceeding 400 Ω cm2 were used in further transport studies (29).

2. Ussing chamber experiments

SGLT1 inhibitory activity of BODE was examined by employing Ussing chambers (EasyMount Diffusion Chamber System, Physiologic Instruments, San Diego, CA, USA) following the protocol recently described (20). Prior to the experiments, half-chambers were filled with 5 mL of Hank’s balanced salt solution (HBSS). The HBSS in the chambers was heated to 37ºC and oxygenated by influx of carbogen-gas. Caco-2/PD7 monolayers were washed from both sides with 37ºC warm HBSS before mounting the Snapwell inserts in Ussing chamber slides (P2302). Both half-chambers were refilled with 5 mL HBSS solution containing 10 mmol/L mannitol apically and 10 mmol/L glucose basolaterally, maintained at 37ºC and continuously carbogen bubbled. The transepithelial potential difference was continuously monitored under open circuit conditions using a DVC 1000 amplifier (WPI) and recorded through Ag-AgCl electrodes and HBSS agarose bridges. Subsequently, the short-circuit current (ISC; µA cm–2) was measured via an automatic voltage clamp (VCC MC8 MultiChannel Voltage-Current Clamp; Physiologic Instruments). Recordings were collected and stored using the Acquire & Analyze Data acquisition software (Physiologic Instruments, San Diego, CA, USA).

Transepithelial resistance and ISC were allowed to stabilize for approximately 10–20 min. After that, 10 mmol/L glucose was added apically to stimulate Na+-coupled glucose transport and, for osmotic reasons, 10 mmol/L mannitol was given simultaneously to the basolateral side. When the glucose-stimulated ISC reached a stable plateau, BODE at a final concentration of 1 mg/mL or phlorizin at a final concentration of 0.1 mM as positive control was added to the apical and basolateral side of the chambers. ISC values were further recorded until they reached a stable level. The average ISC of 2 min intervals within stable plateaus were used to calculate differences in SGLT1 transport activity. The ΔISC value that indicated intestinal SGLT1-dependent glucose transport was calculated by the difference between the values after and before apical addition of glucose. The ΔISC values indicating SGLT1 inhibition were calculated by the difference between the values after the addition of the inhibitor and prior to apical addition of glucose. Finally, the percentage inhibition of SGLT1 transport activity was calculated.

Determination of total phenolic content

Determination of total phenolic content (TPC) of BODE was determined by the Folin-Ciocalteu method according to (20, 30). 100 µL BODE (1.25 mg/mL) were mixed with 100 µL of 95% ethanol, 500 µL distilled water and 50 µL Folin-Ciocalteu reagent (1:2, v/v). The mixture was incubated for 5 min before 100 µL of 5% Na2CO3 were added. The reaction mixture was incubated in the dark for 45 min and the absorbance was determined at 725 nm. TPC of BODE was expressed in mg of gallic acid equivalents (GAE) per L using a calibration curve of gallic acid.

High performance liquid chromatography-mass spectrometry analysis

BODE was analyzed for phenolic composition by using high performance liquid chromatography (HPLC) and HPLC-MS as previously described (20). High-resolution mass spectra were obtained using a Thermo Scientific LTQ Orbitrap Velios with an electrospray as well as an APCI source operated in positive and negative ionization mode. Separations were performed using a Thermo Scientific Surveyor HPLC equipped with an Accucore C18 column (150 mm × 3.0º mm, 2.6 µm particle size; Thermo Scientific, Waltham, MA, USA). The column temperature was set to 40ºC and the injection volume was 1 µL. Preconnected to MS analyses, absorbance was monitored at 260 nm. The analytes were separated by gradient elution with mobile phase A containing 0.1% formic acid (FA) in water and mobile phase B containing 0.1% FA in acetonitrile at a flow rate of 0.5 mL/min. The elution gradient starting conditions were 95% A and 80% B at 17 min for 3 min. B was reduced to 5% at 20 min until 25 min. The resolution was set to 30,000 and diisooctylphthalate (m/z=391.2843) of the MS was used as internal standard for mass calibration. Spectra were collected from 100–1000ºm/z and MS2 spectra were automatically recorded from the most intense peaks. Identification is based on high-resolution MS data and comparison to literature (2).

Hen’s egg test-chorioallantoic membrane

The HET-CAM test was used as previously reported (20–22). In brief, eggs were incubated at 38ºC for 11 days. The eggs were automatically and constantly turned, checked for fertilization via candling, and the air bladder area was marked. The eggshell was lightly pecked with a pointed pair of tweezers in this area and 300 µl of a solution containing the putative blood glucose-lowering substance were applied with a syringe into the air compartment of the egg. BODE was given at a final concentration of 600 µg/mL. 3 U/mL NovoRapid (Novo Nordisk, Bagsvaerd, Denmark) was used as positive and ddH2O as negative control. The eggs were placed back in the incubator for 60 min. After incubation, the eggshell above the air bladder was carefully removed and the eggshell membrane was equilibrated with PBS. In the next step, the eggshell membrane was removed and the chorioallantoic membrane was carefully cut with a micro-scissor. A suitable blood vessel was cut, and leaking blood was collected. The blood glucose levels were determined via a blood glucose meter (Accu-Chek Performa, Roche Diabetes Care GmbH, Mannheim, Germany).

Drosophila melanogaster experiments

The experiments were performed as previously reported (20). The D. melanogaster wild type strain w1118 (Bloomington Drosophila Stock Center #5905, Indiana University, Bloomington, USA) was used in feeding studies. Fruit flies were maintained on Caltech medium consisting of 5.5% dextrose, 3.0% sucrose, 6.0% cornmeal, 2.5% inactive dry yeast, 1.0% agar Type II with 0.15% Tegosept and 0.3% propionic acid serving as preservatives. The animals were cultured in climate cabinets (HPP750 or HPP110, Memmert, Schwabach, Germany) under standard conditions at 25ºC of ambient temperature, 60% humidity, and a 12/12 h light/dark cycle. Synchronized eggs were given onto a starch-based control diet consisting of 10% soluble starch, 4% yeast, 1% agar, 0.18% nipagin according to Abrat et al. (31) or experimental diets that were additionally supplemented with 2.5% BODE or 1.8 µg/mL acarbose. After larval development, pupation and eclosion, flies were synchronized and mated for 2 days. On day 3 after eclosion, mated female flies were sorted and further maintained by transferring the flies to the respective fresh experimental media every other day, before they were harvested on day 10. After determining their wet weights, 10 flies were homogenized in PBS containing 0.05% Triton X100 for 10 min at 4ºC and 25 Hz using a tissue lyser (Qiagen TissueLyser II, Hilden, Germany). The triglyceride and protein content of the fly lysates were determined by employing Infinity triglycerides reagent (Thermo Fisher Scientific, Waltham, USA) and the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, USA), respectively.

Statistical analysis

Ussing chamber experiments were analyzed with a two-sided paired Students t-test. DPP4 inhibition and in-ovo experiments were analyzed with an unpaired t-test. D. melanogaster experiments were analyzed by using one-way ANOVA followed by Dunnett’s multiple comparisons test. All analyses were performed in GraphPad Prism. Statistical significance was accepted at p<0.05. Asterisks indicate the following significance levels: *p<0.05; **p<0.01; ***p<0.001.

RESULTS

Bistorta officinalis Delarbre extract exhibits antidiabetic activity in-vitro

The inhibition of three major enzymes involved in blood glucose regulation by BODE was tested in-vitro. The efficacy of BODE against the two carbohydrate-hydrolysing enzymes α-amylase and α-glucosidase is shown in dose-response curves (Fig. 2). As indicated here, BODE inhibited the in-vitro enzyme activities in a dose-dependent manner. The inhibitory activity of BODE towards α-amylase was calculated with an IC50 of 81.5 µg/mL (range 67.0–98.3 µg/mL), which is approximately 5 times higher than the IC50 value obtained for our positive control acarbose with 15.7 µg/mL (range 12.7–18.6 µg/mL, data not shown, refer to (20)). Moreover, a potent α-glucosidase inhibition by BODE reflected in an IC50 value of 8.4 µg/mL (range 5.6–13.1 µg/mL) was demonstrated. Interestingly, an approximately 60 times higher IC50 value of 493 µg/mL (range 348–697 µg/mL, data not shown, refer to (20)) was obtained when the positive control acarbose was applied.

Fig. 2. Dose-response curves for the in-vitro inhibition of α-amylase and α-glucosidase by Bistorta officinalis Delarbre extract (BODE). Data represent means of triplicates. IC50 values were determined by nonlinear regression using GraphPad Prism.

Moreover, the influence of BODE on the in-vitro DPP4 activity was examined. When tested at a final concentration of 1 mg/mL, a significant (p<0.01) reduction of DPP4 enzyme activity of approximately 20% by BODE was observed (Fig. 3). However, a comparison with the positive control sitagliptin (data not shown, refer to (20)), whose IC50 value is reported at 18 nM (equivalent to 7.33 ng/mL) indicates only moderate DPP4 inhibition by BODE.

Fig. 3. Influence of Bistorta officinalis Delarbre extract (BODE) on the in-vitro activity of dipeptidyl peptidase-4 (DPP4) enzyme activity. The assays were carried out in the presence of the indicated substances (control: assay buffer; BODE 1 mg/mL). The percentage value of remaining DPP4 activity in comparison to the control is shown. Result is the mean value of n=2 duplicate. Error bars indicate the standard deviation. ** p<0.01, with significant change to control.

In order to investigate the impact of BODE on the SGLT1-mediated glucose transport, experiments mounting Caco-2/PD7 cell monolayers in Ussing chambers were conducted. It was shown that the glucose uptake via SGLT1 was significantly (p<0.05) blocked in response to BODE treatment. As demonstrated in Fig. 4 (representative run) the application of glucose to the apical side of the Ussing chamber system resulted in a rapid increase of the ISC value. When added at a final concentration of 1 mg/mL, BODE reduced the glucose-stimulated ISC from 25.48±2.00 to 2.29±2.58, corresponding to approximately 90% SGLT1 inhibition (Fig. 4). The positive control phlorizin (0.1 mM) completely blocked the glucose-induced ISC (data not shown, refer to (20)).

Fig. 4. Influence of Bistorta officinalis Delarbre extract (BODE) on sodium-dependent glucose transporter 1 (SGLT1) glucose transport in Caco-2/PD7 cell monolayers. (A): Caco-2/PD7 monolayers were mounted in Ussing chambers and the short-circuit current (ISC) was monitored over time. An exemplary run is depicted. The addition of glucose (10 mM) to the apical side led to a fast increase of the ISC. After the ISC has reached a stable plateau (approximately 10 min after glucose addition), 1 mg/mL BODE was added. (B): The corresponding calculated ISC values are shown. Error bars indicate the standard error of the mean (n=4). Statistical analysis was carried out by using a two-sided paired Students t-test (* p<0.05).

Main constituents of Bistorta officinalis Delarbre extract

In order to identify antidiabetic compounds in BODE, the polyphenolic composition was analyzed. First, the TPC of the extract was determined to be 5606±64 mg GAE/L (data not shown) indicating that the extract is rich in phenolic compounds.

Nine compounds were identified as main constituents by HPLC analysis with DAD detection and HPLC-MS.

A representative chromatogram is shown in Fig. 5 and Table 1 summarizes the related constituents. In detail, the identified compounds are gallotannins (1-O-galloyl glucose, 6-O-galloyl glucose, 1,6-O,O-digalloyl-glucose), catechins (catechin, procyanidin B1, procyanidin B7, epigallocatechin-(4,8)-epicatechin-3-O-gallate), the phenol carbonic acid chlorogenic acid and galloyl salidroside. The respective chemical structures of the identified compounds are given in Fig. 6.

Fig. 5. Representative high performance liquid chromatography analyses of Bistorta officinalis Delarbre extract. Chromatogram recorded at 260 nm. For peak identification, refer to Table 1.

Table 1. Identification of major secondary plant compounds in Bistorta officinalis Delarbre extract using liquid chromatography-mass spectrometry analysis.

Fig. 6. Chemical structures of the main polyphenolic constituents of Bistorta officinalis Delarbre extract.

Experiments in chicken embryos and D. melanogaster lacked to confirm the antidiabetic effect of Bistorta officinalis Delarbre extract in-vivo

To evaluate the antidiabetic properties of BODE in-vivo, experiments in two different model organisms were conducted. In order to test the ability of BODE to reduce blood glucose levels in chicken embryos, the extract was applied in a modified hen’s egg test (Gluc-HET). However, BODE did not significantly change blood glucose levels when hen’s eggs were incubated with 600 µg/mL extract compared to ddH2O treated eggs (Fig. 7). In contrast, application of the positive control NovoRapid led to highly significant (p<0.001) reduced blood glucose levels (refer to (20)).

Fig. 7. Influence of Bistorta officinalis Delarbre extract (BODE) on blood glucose levels in-ovo. The air compartment of 11 day old hen’s eggs were treated with ddH2O (control) or the indicated substances (NovoRapid: 3 U/mL; BODE: 600 µg/mL) dissolved in ddH2O (300 µL volume) for up to 60 min. After that, a suitable blood vessel of the chicken embryo was dissected for blood collection. Blood glucose levels were determined with a blood glucose meter. Shown are the mean values of tested eggs from two measurement days. Control (n=14); NovoRapid (n=16); BODE (n=14). Error bars indicate the standard error of the mean. ***p<0.001, with a significant decrease with respect to control.

A similar result was obtained when BODE was tested in our starch-induced D. melanogaster obesity model which is characterized by elevated triglyceride levels. It was found that compared to controls dietary supplementation of the starch-based diet with BODE (2.5%) did not significantly alter the triglyceride level of 10-day-old female fruit flies (Fig. 8). In contrast, a significant decrease (p<0.001) in triglyceride level can be observed when the positive control acarbose (1.8 µg/mL) is supplemented.

Fig. 8. The impact of Bistorta officinalis Delarbre extract (BODE) on lipid storage in Drosophila melanogaster. D. melanogaster were raised on a control diet of 10% starch, 4% yeast extract or on experimental diets that were additionally supplemented with 1.8 µg/mL acarbose and 2.5% BODE, respectively. The triglyceride to protein ratios were determined in adult females on day 10 after eclosion. Bars represent the means ±standard deviation of three independent experiments performed in triplicate (n=90 animals per group). Statistical analysis was carried out by using one-way ANOVA followed by Dunnett’s multiple comparisons test (***p<0.001).

DISCUSSION

In the present study we demonstrate that an aqueous extract of Bistorta officinalis exhibits promising antidiabetic activity in-vitro. The extract was found to be an excellent inhibitor of the carbohydrate-hydrolysing enzyme α-glucosidase, reflected in a calculated IC50 value of 8.4 µg/mL (Fig. 2). Its potency significantly exceeded that of our positive control acarbose over 50 times and is in the same range as the values previously reported for aqueous extracts from avens root and roseroot (20). For α-amylase, the inhibition value of BODE (81.5 µg/mL) was approximately 5 times higher than that of acarbose. Our data on BODE are consistent with the report of Zhao et al. (32) where similar inhibitory effects of a B. officinalis extract towards α-glucosidase and α-amylase was determined. Inhibition of the carbohydrate-hydrolysing enzymes α-amylase and α-glucosidase is a common strategy to delay glucose absorption and to reduce postprandial glucose elevation (12) and plant extracts are considered as a promising source of candidate compounds (33).

The inhibition of the enzyme DPP4 by BODE was evaluated in-vitro (Fig. 3). Inhibitors of DPP4 represent another class of oral antidiabetic agent. As a serin protease, DPP4 is responsible for the inactivation of the incretin hormones glucagon-like peptide 1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP), which stimulate insulin secretion in response to nutrient intake and thus regulate glucose homeostasis (34). In comparison to the positive control sitagliptin with an IC50 value of 18 nM, only moderate DPP4 inhibition by BODE can be assumed based on the result of 20% inhibition at a final concentration of 1 mg/mL. However, comparable results with aqueous medicinal plant extracts are reported in other studies (35).

In the present study, we found that glucose uptake via SGLT1 was significantly blocked in response to BODE treatment, when tested in the Caco-2/PD7 (a Caco-2 clone with high SGLT1 expression (27)) monolayer model mounted in Ussing chambers (20) (Fig. 4). The 90% reduction of the glucose-induced ISC by 1 mg/mL BODE was almost as effective as 0.1 mM of the positive control phlorizin, which completely blocked the glucose-induced ISC (20). Intestinal glucose absorption is mainly mediated by the glucose transporter SGLT1, whose expression is upregulated by monosaccharides in the lumen and/or stimulation of sweet taste (36). Inhibiting SGLT1 activity not only results in improved blood glucose levels due to lower glucose absorption, but also leads to an increase of incretin release (15). To the best of our knowledge, SGLT1 inhibition by B. officinalis was tested in the current study for the first time, which is in good accordance with the fact that little is known about medicinal plant extracts as SGLT1 inhibitors (37).

In particular, there are strong indications for polyphenol-rich natural extracts to have beneficial effects against inflammatory disorders, including diabetes due to their impact on pro-inflammatory mediators (38). Our analysis of polyphenolic composition revealed that phenolic compounds are abundant in the BODE. Compared to our results, a considerably lower TPC in B. officinalis roots (2336 to 3887 µg/g dry weight was determined by Demiray et al. (39). However, there are variations in phenol content depending on parameters such as the location, plant part used and the phase of its ontogenetic development (40). The identification of main constituents of BODE such as gallotannins, catechins, phenolcarbonic acids and galloyl salidroside by using HPLC with DAD detection and HPLC-MS (Table 1, Fig. 5) is in good accordance to previous reports (1, 4). Among the phenols detected there are well-studied putative antidiabetic compounds. Chlorogenic acid has long been known for its hypoglycaemic effect (41-43) e.g. by inhibiting α-amylase and α-glucosidase (44). Moreover, there is strong evidence that catechins have antidiabetic properties in-vitro and in-vivo by targeting multiple regulatory key points of glucose homeostasis (45). They have been reported to inhibit the enzymes α-amylase and α-glucosidase (46) as well as DPP4 (47). Moreover, Kobayashi et al. (48) could show that these polyphenols potently inhibited the SGLT1 transport activity. Interestingly, a rather unknown compound galloyl salidroside has also been detected in BODE, which has shown potent inhibitory activity on RNase H (49) but has not yet been tested against enzymes of glucose homeostasis. In summary, BODE contained a number of compounds that may be responsible for the observed in-vitro antidiabetic properties.

Confirmation of in-vitro data by testing in living organisms is of pivotal importance. For this purpose, BODE was evaluated in two in-vivo models. In chicken embryos, application of 600 µg/mL BODE did not lead to significant alterations in blood glucose levels (Fig. 7). This finding is of great importance as a significant reduction in blood glucose levels in the hen’s egg test has already been demonstrated for several plant extracts (20, 23, 50). Moreover, this result is consistent with a previous report of Stadlbauer et al. (50). There was no change in blood glucose level due to a B. officinalis extract in this model after 60 minutes. It should be noted, however, that although the two BODE derive from the same plant, there may be differences in phenolic composition and biological effectiveness due to the origin of the B. officinalis plants. Nevertheless, it can be summarized that there is strong evidence that B. officinalis has no effect in this model.

We have recently demonstrated that inhibitors of intestinal polysaccharide digestion, namely acarbose and an aqueous extract of Geum urbanum, cause a significant reduction of the lipid storage in a starch-induced D. melanogaster obesity model (20). However, a comparable effect could not be achieved by the supplementation of the Drosophila diet with BODE as indicated by unaltered triglyceride levels in the fruit fly compared with the controls (Fig. 8). Similarly, in a recent study, an aqueous roseroot extract exhibited excellent in-vitro data in terms of antidiabetic targets that could not be confirmed when evaluated in the in-vivo Drosophila diabetes model (20). Although it is published relatively rarely, it is not unusual that in-vitro data cannot be transferred to in-vivo situations. This is related to the fact that the concentrations of test compounds as used in in-vitro experiments are often higher than those achievable under in-vivo situations and also partly due to the low bioavailability of plant bioactive constituents (51, 52). Of note, these molecules undergo substantial metabolism such as glucuronidation and sulfation reactions in the body, associated with a loss of free hydroxyl groups which in turn may substantially decrease the antidiabetic properties of the compounds (53). Therefore, it could be suspected that despite the fact that in Drosophila experiments a high concentration of the extract (2.5%) was supplemented, a dose that is too low and/or low bioavailability of the bioactives could account for the discrepancy between our in-vitro and in-vivo outcomes (54). Overall, as frequently described for natural compounds, the inactivation of the bioactive substances in the extract via biotransformation, the influence of the intestinal microbiota or autoxidative degradation should be considered (55). In this context, the presence of an acidic region (~pH 2) in the D. melanogaster midgut might be of relevance (56).

As far as we know, the antidiabetic potential of a plant extract with a similar polyphenol pattern as BODE and chlorogenic acid in particular has not yet been examined in D. melanogaster. Investigations conducted exclusively with male fruit flies (we used females in the present study) revealed that green tea catechin (EGCG) treatment decreased the glucose concentrations, which were accompanied by an inhibition of α-amylase and α-glucosidase activity. Furthermore, the transcripts of insulin-like peptide 5 and the Drosophila homolog of leptin, unpaired 2 were downregulated (57). In this context, the results of two independent studies are of interest, which showed that supplementation with green tea catechins led to a prolongation of life span in male, but not in female fruit flies (57, 58).

Chlorogenic acid and catechin are the main polyphenols of coffee and green tea, respectively. In a recently published randomized, double-blinded, placebo-controlled crossover study in humans, a daily administration of 629 mg green tea catechin and 373 mg coffee chlorogenic acid for three weeks resulted in an improved postprandial glycaemic response in healthy volunteers (59). Furthermore, a hot-water extract of Nerium indicum (commonly known as oleander) leaves that also contained chlorogenic acid and catechin exhibited antidiabetic activity in rats by reducing postprandial blood hyperglycaemia. Again, chlorogenic acid was identified as an efficient inhibitor of α-glucosidase, while catechin had a strong inhibitory activity against α-glucosidase. In line with that, leaves of N. indicum are used as a folk remedy for DM in some regions of Pakistan (60).

By using two different models, an invertebrate and a vertebrate organism, we expect to increase the transferability to other organisms such as humans. However, the evaluation of antidiabetic activity of plant extracts in a rodent model of DM or even in humans represents the current gold standard. Mice including ob/ob (61) and db/db mice (54, 62) as well as streptozotocin and alloxan treated mice (63) are wildly used both in a nutritional and pharmacological context. Furthermore, Zucker rats are another important rodent model in diabetes research which exhibit specific characteristics of DM such as hyperinsulinemia, hyperglycemia and dyslipidemia (64). All these models have their merits and limitations and constantly undergo refining to better mimic diabetes disease in humans. The D. melanogaster has emerged as a relatively new model system in diabetes research and provides several advantages. Fruit flies can be used to screen numerous plant bioactives simultaneously which is not possible to the same extend in laboratory rodents. Furthermore, flies can be relatively easily reproduced and maintained along with financial benefits. In terms of macro and micronutrient composition well defined laboratory diets are provided to fruit flies (65). Thus, diet induced pathophysiological mechanisms related to impaired glucose and insulin homeostasis can be evaluated in D. melanogaster under well-defined experimental conditions. In this context, it should be noted that energy metabolism is evolutionary highly conserved across species. Moreover, the substantial homology of the Drosophila and the human genomes should be emphasized. Overall, the fruit fly has emerged as a significant experimental model in nutrition and diabetes research (24, 66). However, we are aware that the Drosophila model has its limitations, in part due to substantial differences in physiology compared to mammals, which restricts the transferability of the results per se. Thus, we cannot rule out that BODE may exhibit antidiabetic properties in another in-vivo model. For example, an extract of the traditional medicinal plant C. tougourensis, which is rich in secondary metabolites such as tannins, flavonoids, triterpenes or alkaloids, was shown to significantly improve hyperglycaemia induced by streptozotocin, comparable to the effect of the positive control, in mice (67). In this regard, additional data from BODE in organisms such as mouse or rat, would be of great interest to further substantiate our findings. At this point, however, it should be underlined again that, compared to the two in-vivo models used in the present study, the determination of antidiabetic activity in rodents and humans is significantly more complex in terms of approval, time and ethical issues.

In addition, the importance of further chemical analysis of BODE should be pointed out in order to more precisely identify the active compounds that may be responsible for the antidiabetic effect of BODE in-vitro. In this regard, the initial focus should be on bioassay-guided purification and fractionation of the extract (68).

Taken together, despite promising in-vitro data, our in-vivo results suggest that there are more suitable antidiabetic plant extracts and underline the importance of verifying cell culture data using appropriate in-vivo models.

Acknowledgments: This research was partly funded by the Christian Doppler Forschungsgesellschaft (Josef Ressel Center for Phytogenic Drug Research).

Conflict of interests: None declared.

REFERENCES

1. Cecotti R, Carpana E, Falchero L, Paoletti R, Tava A. Determination of the volatile fraction of Polygonum bistorta L. at different growing stages and evaluation of its antimicrobial activity against two major honeybee (Apis mellifera) pathogens. Chem Biodivers 2012; 9: 359-369.
2. Pawlowska KA, Halasa R, Dudek MK, Majdan M, Jankowska K, Granica S. Antibacterial and anti-inflammatory activity of bistort (Bistorta officinalis) aqueous extract and its major components. Justification of the usage of the medicinal plant material as a traditional topical agent. J Ethnopharmacol 2020; 260: 113077. doi: 10.1016/j.jep.2020.113077
3. Klimczak U, Wozniak M, Tomczyk M, Granica S. Chemical composition of edible aerial parts of meadow bistort (Persicaria bistorta (L.) Samp.). Food Chem 2017; 230: 281-290.
4. Voronkova MS, Vysochina GI. Bistorta Scop. Genus (Polygonaceae): Chemical Composition and Biological Activity. Chem Sustain Dev 2014; 22: 207-212.
5. Intisar A, Zhang L, Luo H, Kiazolu JB, Zhang R, Zhang W. Anticancer constituents and cytotoxic activity of methanol-water extract of Polygonum bistorta L. Afr J Tradit Complement Altern Med 2012; 10: 53-59.
6. Liu Y, Staerk D, Nielsen MN, Nyberg N, Jaeger AK. High-resolution hyaluronidase inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of anti-necrosis constituents in Chinese plants used to treat snakebite. Phytochemistry 2015; 119: 62-69.
7. Liu Y-H, Weng Y-P, Lin H-Y, et al. Aqueous extract of Polygonum bistorta modulates proteostasis by ROS-induced ER stress in human hepatoma cells. Sci Rep 2017; 7: 41437. doi: 10.1038/srep41437
8. Manoharan KP, Benny TKH, Yang D. Cycloartane type triterpenoids from the rhizomes of Polygonum bistorta. Phytochemistry 2005; 66: 2304-2308.
9. Longo M, Bellastella G, Maiorino MI, Meier JJ, Esposito K, Giugliano D. Diabetes and aging: from treatment goals to pharmacologic therapy. Front Endocrinol 2019; 10: 45. doi: 10.3389/fendo.2019.00045
10. Petersen MC, Shulman GI. Mechanisms of insulin action and insulin resistance. Physiol Rev 2018; 98: 2133-2223.
11. Batista TM, Haider N, Kahn CR. Defining the underlying defect in insulin action in type 2 diabetes. Diabetologia 2021; 64: 994-1006.
12. Tundis R, Loizzo MR, Menichini F. Natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycaemic potential in the treatment of diabetes: an update. Mini Rev Med Chem 2010; 10: 315-331.
13. Drucker DJ. Incretin action in the pancreas: potential promise, possible perils, and pathological pitfalls. Diabetes 2013; 62: 3316-3323.
14. Holst JJ, Vilsboll T, Deacon CF. The incretin system and its role in type 2 diabetes mellitus. Mol Cell Endocrinol 2009; 297: 127-136.
15. Song P, Onishi A, Koepsell H, Vallon V. Sodium glucose cotransporter SGLT1 as a therapeutic target in diabetes mellitus. Expert Opin Ther Targets 2016; 20: 1109-1125.
16. Echouffo-Tcheugui JB, Selvin E. Prediabetes and what it means: the epidemiological evidence. Annu Rev Public Health 2021; 42: 59-77.
17. Padhi S, Nayak AK, Behera A. Type II diabetes mellitus: a review on recent drug based therapeutics. Biomed Pharmacother 2020; 131: 110708. doi: 10.1016/j.biopha. 2020.110708
18. Onur S, Stockmann H, Zenthoefer M, Piker L, Doring F. The plant extract collection Kiel in Schleswig-Holstein (PECKISH) is an open access screening library. J Food Res 2013; 2: 101-106.
19. Sharafetdinov KK, Kiseleva T, Kochetkova AA, Mazo VK. Promising plant sources of anti-diabetic micronutrients. J Diabetes Metab 2017; 8: 1000778. doi: 10.4172/2155-6156.1000778
20. Gunther I, Rimbach G, Nevermann S, et al. Avens root (Geum Urbanum L.) extract discovered by target-based screening exhibits antidiabetic activity in the hen’s egg test model and Drosophila melanogaster. Front Pharmacol 2021; 12: 794404. doi: 10.3389/fphar.2021.794404
21. Stadlbauer V, Haselgrubler R, Lanzerstorfer P, et al. Biomolecular characterization of putative antidiabetic herbal extracts. PLoS One 2016; 11: e0148109. doi: 10.1371/ journal.pone.0148109
22. Haselgrubler R, Stubl F, Essl K, Iken M, Schroder K, Weghuber J. Gluc-HET, a complementary chick embryo model for the characterization of antidiabetic compounds. PLoS One 2017; 12: e0182788. doi: 10.1371/journal.pone.0182788
23. Haselgrubler R, Stadlbauer V, Stubl F, et al. Insulin mimetic properties of extracts prepared from Bellis perennis. Molecules 2018; 23: 2605. doi: 10.3390/molecules23102605
24. Staats S, Luersen K, Wagner A, Rimbach G. Drosophila melanogaster as a versatile model organism in food and nutrition research. J Agric Food Chem 2018; 66: 3737-3753.
25. Apostolidis E, Kwon Y-I, Shetty K. Potential of cranberry-based herbal synergies for diabetes and hypertension management. Asia Pac J Clin Nutr 2006; 15: 433-441.
26. Awosika TO, Aluko RE. Inhibition of the in vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates. Int J Food Sci Technol 2019; 54: 2021-2034.
27. Mahraoui L, Rodolosse A, Barbat A, et al. Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption. Biochem J 1994; 298: 629-633.
28. Ferruzza S, Rossi C, Scarino ML, Sambuy Y. A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium. Toxicol In Vitro 2012; 26: 1252-1255.
29. Stockdale TP, Challinor VL, Lehmann RP, De Voss JJ, Blanchfield JT. Caco-2 monolayer permeability and stability of Chamaelirium luteum (false unicorn) open-chain steroidal saponins. ACS Omega 2019; 4: 7658-7666.
30. Shetty K, Curtis OF, Levin RE, Witkowsky R, Ang W. prevention of vitrification associated with in vitro shoot culture of oregano. (Origanum vulgare) by Pseudomonas spp. J Plant Physiol 1995; 147: 447-451.
31. Abrat OB, Storey JM, Storey KB, Lushchak VI. High amylose starch consumption induces obesity in Drosophila melanogaster and metformin partially prevents accumulation of storage lipids and shortens lifespan of the insects. Comp Biochem Physiol, Part A Mol Integr Physiol 2018; 215: 55-62.
32. Zhao Y, Kongstad KT, Liu Y, He C, Staerk D. Unraveling the complexity of complex mixtures by combining high-resolution pharmacological, analytical and spectroscopic techniques: antidiabetic constituents in Chinese medicinal plants. Faraday Discuss 2019; 218: 202-218.
33. Alam F, Shafique Z, Amjad ST, Bin Asad MH. Enzymes inhibitors from natural sources with antidiabetic activity: a review. Phytother Res 2019; 33: 41-54.
34. Janardhan S, Sastry GN. Dipeptidyl peptidase IV inhibitors: a new paradigm in type 2 diabetes treatment. Curr Drug Targets 2014; 15: 600-621.
35. Ansari P, Hannon-Fletcher MP, Flatt PR, Abdel-Wahab YH. Effects of 22 traditional anti-diabetic medicinal plants on DPP-IV enzyme activity and glucose homeostasis in high-fat fed obese diabetic rats. Biosci Rep 2021; 41: BSR20203824. doi: 10.1042/BSR20203824
36. Margolskee RF, Dyer J, Kokrashvili Z, et al. T1R3 and gustducin in gut sense sugars to regulate expression of Na+-glucose cotransporter 1. Proc Natl Acad Sci USA 2007; 104: 15075-15080.
37. Oranje P, Gouka R, Burggraaff L, et al. Novel natural and synthetic inhibitors of solute carriers SGLT1 and SGLT2. Pharmacol Res Perspect 2019; 7: e00504. doi: 10.1002/prp2.504
38. Chojnacka K, Lewandowska U. The influence of polyphenol-rich extracts on the production of pro-inflammatory mediators in macrophages. J Physiol Pharmacol 2021; 72: 167-176.
39. Demiray S, Pintado ME, Castro PM. Evaluation of phenolic profiles and antioxidant activities of Turkish medicinal plants: Tilia argentea, Crataegi folium leaves and Polygonum bistorta roots. Int J Pharmacol 2009; 3: 74-79.
40. Feduraev P, Chupakhina G, Maslennikov P, Tacenko N, Skrypnik L. Variation in phenolic compounds content and antioxidant activity of different plant organs from Rumex crispus L. and Rumex obtusifolius L. at different growth stages. Antioxidants 2019; 8: 237. doi: 10.3390/antiox8070237
41. Ong KW, Hsu A, Tan BK. Anti-diabetic and anti-lipidemic effects of chlorogenic acid are mediated by ampk activation. Biochem Pharmacol 2013; 85: 1341-1351.
42. Meng S, Cao J, Feng Q, Peng J, Hu Y. Roles of chlorogenic acid on regulating glucose and lipids metabolism: a review. Evid Based Complement Alternat Med 2013; 2013: 801457. doi: 10.1155/2013/801457
43. Yan Y, Zhou X, Guo K, Zhou F, Yang H. Use of chlorogenic acid against diabetes mellitus and its complications. J Immunol Res 2020; 2020: 9680508. doi: 10.1155/2020/9680508
44. Oboh G, Ogunsuyi OB, Adegbola DO, Ademiluyi AO, Oladun FL. Influence of gallic and tannic acid on therapeutic properties of acarbose in vitro and in vivo in Drosophila melanogaster. Biomed J 2019; 42: 317-327.
45. Marquez Campos E, Jakobs L, Simon M-C. antidiabetic effects of flavan-3-ols and their microbial metabolites. Nutrients 2020; 12: E1592. doi: 10.3390/nu12061592
46. Yilmazer-Musa M, Griffith AM, Michels AJ, Schneider E, Frei B. Inhibition of α-amylase and α-glucosidase activity by tea and grape seed extracts and their constituent catechins. J Agric Food Chem 2012; 60: 8924-8929.
47. Fujimura Y, Watanabe M, Morikawa-Ichinose T, et al. Metabolic profiling for evaluating the dipeptidyl peptidase-IV inhibitory potency of diverse green tea cultivars and determining bioactivity-related ingredients and combinations. J Agric Food Chem 2022; 70: 6455-6466.
48. Kobayashi Y, Suzuki M, Satsu H, et al. Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism. J Agric Food Chem 2000; 48: 5618-5623.
49. Kim JA, Yang SY, Wamiru A, et al. New monoterpene glycosides and phenolic compounds from Distylium racemosum and their inhibitory activity against ribonuclease H. Bioorg Med Chem Lett 2011; 21: 2840-2844.
50. Stadlbauer V, Neuhauser C, Aumiller T, Stallinger A, Iken M, Weghuber J. Identification of insulin-mimetic plant extracts: from an in vitro high-content screen to blood glucose reduction in live animals. Molecules 2021; 26: 4346. doi: 10.3390/molecules26144346
51. Yang CS, Sang S, Lambert JD, Lee M-J. Bioavailability issues in studying the health effects of plant polyphenolic compounds. Mol Nutr Food Res 2008; 52: 139-151.
52. Cosme P, Rodriguez AB, Espino J, Garrido M. Plant phenolics: bioavailability as a key determinant of their potential health-promoting applications. Antioxidants 2020; 9: 1263. doi: 10.3390/antiox9121263
53. Wu B, Kulkarni K, Basu S, Zhang S, Hu M. First-pass metabolism via UDP-glucuronosyltransferase: a barrier to oral bioavailability of phenolics. J Pharm Sci 2011; 100: 3655-3681.
54. Schrader E, Wein S, Kristiansen K, Christensen LP, Rimbach G, Wolffram S. Plant extracts of winter savory, purple coneflower, buckwheat and black elder activate PPAR-γ in COS-1 cells but do not lower blood glucose in Db/db mice in vivo. Plant Foods Hum Nutr 2012; 67: 377-383.
55. Dei Cas M, Ghidoni R. Dietary curcumin: correlation between bioavailability and health potential. Nutrients 2019; 11: 2147. doi: 10.3390/nu11092147
56. Overend G, Luo Y, Henderson L, Douglas AE, Davies SA, Dow JA. Molecular mechanism and functional significance of acid generation in the Drosophila midgut. Sci Rep 2016; 6: 27242. doi: 10.1038/srep27242
57. Wagner AE, Piegholdt S, Rabe D, et al. Epigallocatechin gallate affects glucose metabolism and increases fitness and lifespan in Drosophila melanogaster. Oncotarget 2015; 6: 30568-30578.
58. Lopez T, Schriner SE, Okoro M, et al. Green tea polyphenols extend the lifespan of male Drosophila melanogaster while impairing reproductive fitness. J Med Food 2014; 17: 1314-1321.
59. Yanagimoto A, Matsui Y, Yamaguchi T, Hibi M, Kobayashi S, Osaki N. Effects of ingesting both catechins and chlorogenic acids on glucose, incretin, and insulin sensitivity in healthy men: a randomized, double-blinded, placebo-controlled crossover trial. Nutrients 2022; 14: 5063. doi: 10.3390/nu14235063
60. Ishikawa A, Yamashita H, Hiemori M, et al. Characterization of inhibitors of postprandial hyperglycemia from the leaves of Nerium indicum. J Nutr Sci Vitaminol 2007; 53: 166-173.
61. Tschop M, Heiman ML. Rodent obesity models: an overview. Exp Clin Endocrinol Diabetes 2001; 109: 307-319.
62. Wein S, Schrader E, Rimbach G, Wolffram S. Oral green tea catechins transiently lower plasma glucose concentrations in female db/db mice. J Med Food 2013; 16: 312-317.
63. Rees DA, Alcolado JC. Animal models of diabetes mellitus. Diabet Med 2005; 22: 359-370.
64. Pandey S, Dvorakova MC. Future perspective of diabetic animal models. Endocr Metab Immune Disord Drug Targets 2020; 20: 25-38.
65. Luersen K, Roeder T, Rimbach G. Drosophila melanogaster in nutrition research - the importance of standardizing experimental diets. Genes Nutr 2019; 14: 3. doi: 10.1186/s12263-019-0627-9
66. Pandey UB, Nichols CD. Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacol Rev 2011; 63: 411-436.
67. Bensaad MS, Dassamiour S, Hambaba L, et al. In vivo investigation of antidiabetic, hepatoprotective, anti-inflammatory and antipyretic activities of Centaurea tougourensis Boiss. & Reut. J Physiol Pharmacol 2021; 72: 439-449.
68. Mani J, Johnson J, Hosking H, et al. Bioassay guided fractionation protocol for determining novel active compounds in selected Australian flora. Plants 2022; 11: 2886. doi: 10.3390/plants11212886