Stellaria media tea protects against diabetes-induced cardiac dysfunction in rats without affecting glucose tolerance

Abstract

Background and aim

Common chickweed (Stellaria media) tea has traditionally been applied for treatment of various metabolic diseases including diabetes in folk medicine; however, experimental evidence to support this practice is lacking. Therefore, we aimed to assess the effect of Stellaria media tea on glucose homeostasis and cardiac performance in a rat model of diabetes.

Experimental procedure

Hot water extract of Stellaria media herb were analyzed and used in this study, where diabetes was induced by fructose-enriched diet supplemented with a single injection of streptozotocin. Half of the animals received Stellaria media tea (100 mg/kg) by oral gavage. At the end of the 20-week experimental period, blood samples were collected and isolated working heart perfusions were performed.

Results and conclusion

Compared to the animals receiving standard chow, serum fasting glucose level was increased and glucose tolerance was diminished in diabetic rats. Stellaria media tea did not affect significantly fasting hyperglycemia and glucose intolerance; however, it attenuated diabetes-induced deterioration of cardiac output and cardiac work. Analysis of the chemical composition of Stellaria media tea suggested the presence of rutin and various apigenin glycosides which have been reported to alleviate diabetic cardiomyopathy. Moreover, Stellaria media prevented diabetes-induced increase in cardiac STAT3 phosphorylation. We demonstrated for the first time that Stellaria media tea may beneficially affect cardiac dysfunction induced by diabetes without improvement of glucose homeostasis. Rutin and/or apigenin glycosides as well as modulation of STAT3 signaling may be implicated in the protection of Stellaria media tea against diabetic cardiomyopathy.

Graphical abstract

Highlights

• •Stellaria media (SM) tea attenuates diabetic deterioration of cardiac performance.
• •SM tea contains apigenin glycosides and presumably rutin which have been reported to alleviate diabetic cardiomyopathy.
• •SM tea restores diabetes-induced cardiac STAT3 phosphorylation.
• •SM tea does not affect fasting hyperglycemia and glucose intolerance.

List of abbreviations

Akt

protein kinase B

ALP

alkaline phosphatase

ALAT

alanine aminotransferase

ASAT

aspartate aminotransferase

AUC

area under the curve

CK

creatine kinase

CK-MB

creatine kinase – myocardial band

DPPH

2,2-diphenyl-1-picryl-hydrazyl-hydrate

ELISA

enzyme-linked immunosorbent assay

Erk

extracellular signal-regulated kinase

HbA1c

haemoglobin A1c

HDL

high-density lipoprotein cholesterol

HPLC

high performance liquid chromatography

LC-MS/MS

liquid chromatography-tandem mass spectrometry

LDH

lactate dehydrogenase

LVDP

left ventricular developed pressure

LVEDP

left ventricular end-diastolic pressure

OGTT

oral glucose tolerance test

ORAC

oxygen radical absorbance capacity

QE

quercetin equivalent

Stellaria m.

Stellaria media

STAT3

signal transducer and activator of transcription 3

STZ

streptozotocin

UPLC

ultra-performance liquid chromatography

1. Introduction

Diabetes is a common metabolic disease characterized by elevated blood glucose level and impaired glucose tolerance. The prevalence of diabetes for adults was approximately 171 million patients worldwide in 2000 and according to predictions, this number is expected to be doubled by 2030.¹ Due to the continuously increasing trend in diabetes morbidity, investigation of harmful consequences of the disease and potential therapeutic interventions is a relevant current research area.¹ In the absence of proper therapy diabetes may lead to development of various complications including cardiomyopathy, cataract, kidney failure, as well as neuronal damage. Diabetic cardiomyopathy is characterized by diastolic and/or systolic dysfunction which can lead to heart failure without the presence of classic risk factors such as dyslipidaemia, hypertension, or coronary artery disease.^2,3

Nowadays, medicinal plants are gaining popularity in prevention and treatment of various diseases including diabetes. There are some herbs with a well-described and scientifically proven anti-hyperglycaemic properties, for instance aqueous leaf extract of stinging nettle (Urtica dioica)^4,5 or powdered fenugreek seeds (Trigonella foenum graecum).^6,7 In folk medicine, Stellaria media is believed to be a remedy to lose weight⁸ and it is suggested to be used for its believed beneficial effects on blood lipid profile.⁹ According to a popular Hungarian traditional healer, Stellaria media tea improves general metabolism, lowers blood glucose level, making it an adjuvant therapy for diabetic patients.¹⁰ Moreover, consumption of chickweed tea for lowering blood glucose level is recommended by some websites dealing with medicinal plants and health issues.^11,12 Although, an animal experiment proposed an anti-hyperglycaemic effect of intraperitoneally administered alcoholic Stellaria media extract,¹³ the clinical translation of this design is limited since Stellaria media is mostly consumed as tea. Nevertheless, potential effects of Stellaria media tea on diabetes-induced cardiac dysfunction has never been investigated.

Taken together, there is no firm experimental or clinical evidence supporting the anti-diabetic effect of Stellaria media tea. Therefore, the aim of the present study was to investigate the potential therapeutic efficacy of Stellaria media tea on the severity of diabetes and on harmful cardiac consequences induced by diabetes.

2. Materials and methods

2.1. Animals

The experiment conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health [National Institutes of Health publication 85–23, revised 1996] and the regulations of the Hungarian Act No. XXVIII of the year 1998 on protection and care of animals were strictly followed. The study was approved by the local animal ethics committee of the University of Szeged (XV.1181/2013).

Altogether 30 adult (9-week old) male Wistar rats were used in this study (purchased from Charles River Laboratories, Göttingen, Germany), weighing 292–420 g at the onset of the experiments. Rats were kept under standard climatic conditions (22 ± 2 °C room temperature, 12h light/dark cycles) in pairs in individually ventilated cages (Sealsafe IVC system, Buguggiate, Italy) and had ad libitum access to tap water and rat chow.

2.2. Experimental setup

After one week of acclimatization, the rats were randomized into three groups: Control, Diabetes, Diabetes + Stellaria media treatment. Rats in the control group (n = 10) received standard laboratory rat chow for 20 weeks. The other 20 rats were fed a special fructose-enriched diet, i.e. a standard laboratory rat chow (Innovo Ltd., Isaszeg, Hungary) supplemented with 60% (w/w) fructose (Floravita Ltd., Soltvadkert, Hungary) for 20 weeks¹⁴ and were treated with a low-dose streptozotocin (STZ) injection (20 mg/kg body weight, ip.) on the 17th week. Combination of fructose-enriched diet/drinking water and low-dose STZ treatment has been reported to be an alternative approach to achieve experimental type 2 diabetes^{15, 16, 17} (Fig. 1A). In order to prevent STZ-induced hypoglycaemia, drinking water containing 10% glucose was given to the animals after STZ injection. The diet of 10 animals receiving fructose-enriched chow was further supplemented with Stellaria media tea administered by oral gavage (Diabetes + Stellaria media) in order to examine the potential effects of Stellaria media tea on the glucose homeostasis and cardiac function, while the other rats received equal amount of distilled water. One animal in the Control group and two animals in the Diabetes group were excluded due to technical problems occurring during the treatments.

Fig. 1.

Experimental protocol: rats were divided into Control, Diabetes and Diabetes + Stellaria media groups receiving either a standard chow or a chow supplemented with 60% fructose, respectively, for 20 weeks (A). Fasting blood glucose measurement was performed every four weeks until week 16. Oral glucose tolerance test (OGTT) was performed on week 12, 16 and 19. At week 17, rats in the Diabetes and Diabetes + Stellaria media groups were injected with a low-dose streptozotocin (20 mg/kg body weight) intraperitoneally. Fasting blood glucose was measured on week 18 and 19 to monitor the effect of streptozotocin injection. On week 20, the animals were anaesthetised and sacrificed. Blood samples were collected from the abdominal aorta, hearts were isolated and subjected to working heart perfusion according to Neely (A). Body weight (B) and weight gain (C) during 20 weeks in the Control group (circles), Diabetes group (squares) and Diabetes + Stellaria media group (triangles). Results are means ± SEM (n = 8–10), analyzed by repeated measures two-way ANOVA or one-way ANOVA with Holm-Sidak post hoc test,∗p < 0.05 Diabetes vs. Control.

Fasting blood glucose level measurements were performed every 4 weeks, accompanied by oral glucose tolerance (OGTT) tests at weeks 12, 16 and 19 (Fig. 1A). On the 20th week, rats were anaesthetised with sodium pentobarbital (Euthasol, 50 mg/kg body weight, ip., Produlab Pharma b.v., Raamsdonksveer, The Netherlands), the abdominal cavity was opened, and blood samples were taken from the abdominal aorta. Collected blood was allowed to clot and was centrifuged (2000 g, 20 min, 4 °C), then serum was separated for analysis of various serum parameters. Then the rats were given 500 U kg⁻¹ heparin intravenously into the vena cava inferior. Isolated hearts were subjected to working heart perfusion according to Neely in order to evaluate cardiac function.

2.3. Preparation of Stellaria media tea

Stellaria media was harvested in Algyő (Hungary) by ‘Ezerjófű’ Association in 2017. Voucher specimen (No: 882) was deposited in Herbarium of the University of Szeged, Faculty of Pharmacy, Department of Pharmacognosy. The drug was dried and stored at room temperature. The dried and grounded drug was extracted with boiling water (1:10 w/v ratio) for 15 min by ultrasonication. The highly dense extract was separated from solid particles by mechanical press. The aqueous extract was dry-freezed. 1.5 g of dark-green lyophilized powder was equal to 10 g of dried drug. The lyophilized powder was dissolved in distilled water to achieve a final concentration of 100 mg/ml. The extract and the vehicle (distilled water) were then stored at 4 °C and were brought to room temperature before administration.

2.4. Stellaria media administration

Rats in the Diabetes + Stellaria media group received 100 mg/kg body weight Stellaria media tea with traditional oral gavage technique once a day since the onset of the experiment. The Control and Diabetes groups received equal amount of distilled water. The treatment was carried out at the same 2-h time range every day to minimalize the possible influence of circadian rhythm. The 100 mg/kg body weight dose of Stellaria media tea was considered as equal to the recommended human daily dose, that is 2 cups of tea, prepared with 3 g dried herb per cup, calculated according to Nair and Jacob considering the differences in body surface area.¹⁸

2.5. Working heart perfusion

Cardiac performance and function was assessed in isolated Neely working hearts, as described earlier.^19,20 Cardiac functional parameters including heart rate, coronary flow, aortic flow, cardiac output, left ventricular developed pressure (LVDP) and its first derivatives (dp/dt max and dp/dt min), and left ventricular end-diastolic pressure (LVEDP) were measured. Cardiac work was calculated by multiplying cardiac output and maximum pressure. For more information, see Supplementary Material.

2.6. Measurements of serum parameters

Several serum parameters describing pancreas, liver and kidney function, cardiac markers, lipid panel and electrolytes were analyzed by Roche Cobas 8000 Analyzer System using enzymatic colorimetric assays from Roche (Mannheim, Germany). For further details, please see Supplementary Material. Haemoglobin A1c (HbA1c) levels were analyzed by DCA Vantage Analyzer System (Siemens) provided by Diagnosticum Ltd. (Budapest, Hungary).

2.7. Measurement of serum insulin levels

Serum insulin levels were determined by enzyme-linked immunosorbent assay (ELISA) technique (Mercodia, Ultrasensitive Rat Insulin ELISA) according to the instructions of the manufacturer.²¹

2.8. Total flavonoid content and screening for flavonoids

The total flavonoid content was determined as quercetin equivalent (QE) using the aluminum chloride colorimetric method. The lyophilizate powder was dissolved in methanol to get a solution with a concentration of 1 mg/mL. For calibration curve 5, 10, 25, 50 μg/mL methanolic solutions of quercetin were prepared. As reagent, 2% aluminum chloride methanolic solution was used. Reaction mixtures were prepared by mixing 2 mL of solution and 2 mL of aluminum chloride, respectively. After 60 min of incubation at room temperature, the absorbance was measured against blank by applying UV-VIS spectrophotometer (Helios β ThermoSpectronic) at 420 nm. The total flavonoid concentration was calculated using a calibration plot (R² = 0.9999). The measurements were carried out in triplicate.

The flavonoid content of the lyophilized Stellaria media aqueous extract was screened by ultra-performance liquid chromatography (UPLC). For this experiment 1 mg/mL methanolic solution was prepared from the lyophilizate. The presence of ubiquitous flavonoids in plants, namely apigenin, apigenin-7-glucoside, kaempferol, luteolin, quercetin, and rutin was screened by UPLC. Experiments were carried out on a Shimadzu Nexera X2 UHPLC liquid chromatography system. For more information, see Supplementary Material.

2.9. Liquid chromatography-tandem mass spectrometry analysis

In order to gain more information about the chemical composition, the aqueous extract was filtered through a 0.22 μm membrane filter and diluted tenfold with 0.1% formic acid and analyzed by data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a Waters MClass nanoUPLC system online coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer. MS2 data were subjected to a spectral library search against the MzCloud database using the Compound Discovery software. For more information, see Supplementary Material.

2.10. Measurement of the antioxidant activity

The measurement of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging activity was carried out according to the method of Fukumoto et al. with some modifications.²² The Oxygen Radical Absorbance Capacity (ORAC) assay was carried out according to the method of Mielnik et al.²³ For further details, please see Supplementary Material.

2.11. Western blot analysis

Phosphorylation of key proteins of well-known cardioprotective signaling pathways, signal transducer and activator of transcription 3 (STAT3), protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) proteins and activation of apoptosis-related Bax and Bcl-XL proteins and GLUT4 glucose transporter were detected by Western blot technique as described earlier.²⁴ For more information, see Supplementary Material.

2.12. Statistical analysis

All values are presented as mean ± SEM. Repeated measures two-way ANOVA was applied for analysis of time-dependent body weight change. One-way ANOVA was used to determine the differences among the three experimental groups and p < 0.05 was accepted as statistically significant difference, using Holm-Sidak post hoc test. In the analysis of the working heart perfusion and Western blot results, those data which were out of mean ± 2SD range were excluded in order to minimalize the effect of extremities.

3. Results

3.1. Time course of body weight, fasting blood glucose and OGTT values

Body weight was measured every 3 days in order to calculate Stellaria media tea dosage and to monitor weight gain. It showed a continuous increase from 356 ± 7 g at the onset of the experiment to 482 ± 16 g at week 20 in the Control group fed with standard laboratory rat chow (Fig. 1B). Compared to the controls, fructose-enriched diet plus STZ injection reduced body weight increase in the last 6 weeks in the Diabetes group, however, this effect was not significant in the Diabetes + Stellaria media group (Fig. 1B). Weight gain during the feeding protocol was significantly lower in the Diabetes group compared to the Control group (Fig. 1C). Similarly, there was a tendency of decrease in the Diabetes + Stellaria media group compared to the controls; however, it did not reach the level of statistical significance (Fig. 1C).

Fasting blood glucose levels were measured every 4 weeks since the onset of the experiment, accompanied by OGTT at weeks 12, 16 and 19. In fasting blood glucose levels and glucose tolerance, there was no significant difference among the three groups until week 16 (Fig. 2A and B). On the 17th week, the rats in the Diabetes and Diabetes + Stellaria media groups were injected with a low dose of STZ injection (20 mg/kg body weight, ip.), while the Control group was treated with equal amount of vehicle (citrate buffer). Following the STZ injection a significant elevation in fasting blood glucose levels could be observed in the Diabetes group compared to the Control group, achieving an experimental diabetes state (Fig. 2A). Although on week 18 the blood glucose elevation in the Diabetes + Stellaria media group was significantly lower compared to the Diabetes group, this difference was faded away by week 19 (Fig. 2A). According to our OGTT results, there was a significant elevation in the area under the curve (AUC) levels in the Diabetes group compared to the Control group on week 19 (Fig. 2B) representing an impairment of glucose tolerance. Stellaria media treatment did not affect glucose intolerance (Fig. 2B).

Fig. 2.

Fasting blood glucose levels (A) and area under the curve (AUC) (B) values of oral glucose tolerance tests (OGTT) measurements. Results are means ± SEM (n = 8–10), analyzed by one-way ANOVA followed by Holm-Sidak post hoc test,∗p < 0.05 vs. Control, #p < 0.05 vs. Diabetes.

3.2. Parameters reflecting endocrine and exocrine function of the pancreas

At the end of the 20-week experiment, elevated fasting blood glucose, non-significantly increased HbA1c (3.6 ± 0.1% in Control and 4.3 ± 0.5% in Diabetes, respectively) and serum insulin levels decreased by approximately 20% indicated impaired endocrine pancreatic function (Fig. 3A–C) in the Diabetes group. Stellaria media tea failed to improve these parameters (Fig. 3A–C), and HbA1c was 4.6 ± 0.4% in this group. Pancreas weight was significantly lower in the Diabetes group compared to the Control group (Fig. 3D), showing that there might have been pancreatic damage due to the fructose-enriched diet plus STZ injection. Stellaria media did not affect this alteration. The serum activity of α-amylase was significantly elevated both in the Diabetes and Diabetes + Stellaria media groups (Fig. 3E), while there was no difference among the three groups in the activity of lipase enzyme (Fig. 3F).

Fig. 3.

Parameters representing pancreatic function at week 20: fasting blood glucose at termination (A), HbA1c levels (B), serum insulin levels (C), pancreas weight (D), enzyme activities of α-amylase (E) and lipase (F). Results are means ± SEM (n = 8–10 except for serum insulin measurement where n = 6–8), analyzed by one-way ANOVA followed by Holm-Sidak post hoc test,∗p < 0.05 vs. Control.

3.3. Serum parameters of liver and kidney function, cardiac markers, lipid panel and electrolytes

At termination of the animals, blood samples were collected from the abdominal aorta and several serum parameters were measured. Markers of liver function, such as alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP) enzyme activities, as well as albumin and total protein concentrations were measured. Diabetes caused a significant decrease in ALAT and ASAT enzyme activities and an elevation in ALP enzyme activity without affecting albumin or total protein concentration indicating lack of considerable liver damage (Table 1). Serum parameters describing kidney function were also measured. Urea levels were significantly lower in the Diabetes group compared to the Control group (Table 1). There was no difference in the creatinine levels. Stellaria media tea did not affect liver and kidney function compared to the Diabetes group (Table 1). There was no significant difference among the three groups in creatine kinase (CK), Creatine kinase – myocardial band (CK-MB) and lactate dehydrogenase (LDH) activities suggesting the lack of significant cellular injury in the heart and other tissues (Table 1). Cholesterol, high-density lipoprotein (HDL) cholesterol, as well as relevant electrolytes, such as sodium, potassium and chloride levels were not changed significantly among the groups (Table 1).

Table 1.

Serum parameters representing liver and kidney function, cardiac markers, lipid panel and electrolytes.

	Control	Diabetes	Diabetes +  Stellaria m.	p
Liver function:
ALAT (U/L)	34.7 ± 2.0	23.9 ± 2.2∗	29.1 ± 2.5	<0.05
ASAT (U/L)	65.1 ± 2.5	50.4 ± 1.8∗	55.6 ± 1.9∗	<0.05
ALP (U/L)	46.9 ± 1.9	86.5 ± 12.7∗	101.4 ± 10.4∗	<0.05
Albumin (g/L)	40.8 ± 0.8	41.8 ± 0.6	42.0 ± 0.4	ns
Total protein (g/L)	54.0 ± 1.5	55.9 ± 0.4	57.3 ± 0.7∗	<0.05
Kidney function:
Urea (mmol/L)	6.3 ± 0.3	3.8 ± 0.7∗	3.8 ± 0.3∗	<0.05
Creatinine (μmol/L)	32.1 ± 1.3	35.3 ± 1.8	35.2 ± 1.6	ns
Cardiac markers:
CK (U/L)	276.9 ± 25.7	235.1 ± 19.4	289.7 ± 29.8	ns
CK-MB (U/L)	438.6 ± 29.1	433.8 ± 37.9	524.8 ± 55.6	ns
LDH (U/L)	281.7 ± 26.0	298.4 ± 23.3	359.3 ± 42.9	ns
Lipid panel:
Cholesterol (mmol/L)	1.58 ± 0.05	1.74 ± 0.16	1.78 ± 0.11	ns
HDL-Cholesterol (mmol/L)	0.95 ± 0.04	1.03 ± 0.08	1.08 ± 0.10	ns
Electrolytes:
Sodium (mmol/L)	142.1 ± 0.9	141.5 ± 0.9	140.2 ± 1.1	ns
Potassium (mmol/L)	4.8 ± 0.1	5.1 ± 0.1	5.1 ± 0.2	ns
Chloride (mmol/L)	101.9 ± 0.5	101.3 ± 0.8	101.0 ± 0.5	ns

Results are means ± SEM (n = 8–10), analyzed by one-way ANOVA followed by Holm-Sidak post hoc test,∗p < 0.05 vs. Control. ALP alkaline phosphatase, ALAT alanine aminotransferase, ASAT aspartate aminotransferase, CK creatine kinase, CK-MB creatine kinase – myocardial band, HDL high-density lipoprotein cholesterol, LDH lactate dehydrogenase, ns non-significant.

3.4. Ex vivo working heart perfusion

Cardiac dysfunction is a frequent consequence of diabetes, therefore we assessed cardiac performance in hearts subjected to working heart perfusion. Aortic flow, cardiac output and cardiac work, reflecting systolic heart function, were significantly impaired in the Diabetes group compared to the Control group (Fig. 4A and B, Table 2), indicating diabetic adverse effects on the heart. Stellaria media treatment significantly improved cardiac output and cardiac work suggesting that Stellaria media tea may have beneficial effects on the heart in a diabetic state (Fig. 4A and B). The diastolic function of the hearts was assessed by measurements of LVEDP, which inversely correlates with the function. LVEDP showed a tendency of elevation in the Diabetes group compared to the Control group, however, Stellaria media had no prominent effect on this parameter (Fig. 4C). There were no significant alterations in the other analyzed cardiac functional parameters among the three experimental groups (Table 2).

Fig. 4.

Cardiac function in isolated hearts subjected to working perfusion according to Neely: cardiac output (A), cardiac work (B), left ventricular end diastolic pressure (LVEDP) (C). Results are means ± SEM (n = 8–10), analyzed by one-way ANOVA followed by Holm-Sidak post hoc test,∗p < 0.05 vs. Control, #p < 0.05 vs. Diabetes.

Table 2.

Parameters measured by working heart perfusion according to Neely.

	Control	Diabetes	Diabetes +  Stellaria m.	p
Aortic flow (mL)	44.4 ± 2.6	27.3 ± 2.4∗	34.4 ± 2.3∗	p < 0.05
Coronary flow (mL)	22.8 ± 0.7	21.8 ± 1.5	23.7 ± 1.0	ns
Max dp/dt (mmHg/s)	6323 ± 282	6260 ± 439	6431 ± 487	ns
Min dp/dt (mmHg/s)	– 4520 ± 188	– 4512 ± 397	– 4496 ± 374	ns
Aortic diastolic pressure (mmHg)	37.6 ± 0.5	37.9 ± 0.8	37.8 ± 1.1	ns
Aortic systolic pressure (mmHg)	114.8 ± 2.5	110.4 ± 3.3	114.9 ± 3.1	ns
LVDP (mmHg)	136.2 ± 4.6	130.0 ± 4.5	131.3 ± 5.2	ns
Heart rate (1/min)	240 ± 10	211 ± 16	231 ± 11	ns

Results are means ± SEM (n = 8–10), analyzed by one-way ANOVA with Holm-Sidak post hoc test,∗p < 0.05 vs. Control. LVDP left ventricular developed pressure, ns non-significant.

3.5. Total flavonoid content, LC-MS/MS analysis and antioxidant activity of Stellaria media tea

Flavonoids are active compounds in several medicinal herbs and are related to anti-oxidative properties, therefore we analyzed the flavonoid content of the Stellaria media tea. The total flavonoid content determined by means of UV-VIS absorbance was 9.88 ± 0.10 mg quercetin equivalent/gram (QE/g). The flavonoid screening by means of UPLC indicated rutin being a possible component, based on comparison of its retention time and UV spectrum with a reference standard.

In the LC-MS/MS analysis, several components have been detected. These comparisons revealed altogether five possible components out of the ten most intense sample components, which appeared to be various glycosylated apigenin-derivatives. MS2 spectrum of m/z 595.1660 displayed reasonable resemblance (MzCloud best match: 62.9) to MzCloud spectral library entries to two isomeric compounds, 6-arabinosyl-8-galactosylapigenin (corymboside) and 6-β-d-glucopyranosyl-8-β-D-ribopyranosylapigenin (schaftoside), while MS2 spectrum of m/z 595.1660 showed good agreement (MzCloud best match: 82.8) with apigenin-6,8-di-C-glucoside (vicenin). Two further components, m/z 757.2195 and 933.2657 showed similarity to MS2 data acquired on 2″-O-α-l-rhamnopyranosyl-isovitexin, a compound with the same apigenin base structure. The molecular mass of these two components were 178.1 and 354.1 Da higher compared to the database entry. Six further components (m/z 274.2737, 535.1448, 679.2974, 677.2817, 611.1608 and 381.0793) did not show any resemblance to MzCloud entries indicating that these components were not included with an MS2 spectrum in the spectral library.

Moreover, the lyophilized powder was evaluated for antioxidant activity using DPPH and ORAC assays. The lyophilized aqueous extract of Stellaria media exerted low direct antioxidant capacity in both assays: EC₅₀ 168.30 ± 11.06 μg/L in the DPPH assay and 0.97 ± 0.16 Trolox Equivalent mmol/g in the ORAC study.

3.6. Cardiac signaling pathways

Activation of STAT3 is proposed to play a role in diabetes-induced cardiac dysfunction. The phosphorylation of STAT3 was significantly elevated in the Diabetes group which was attenuated by Stellaria media treatment (Fig. 5A), suggesting an association with the beneficial cardiac effect of Stellaria media. There were no significant differences among the three groups in the phosphorylation of Akt and Erk proteins (Fig. 5B and C). Since increased cardiac apoptosis represents greater risk for the development of diabetic cardiomyopathy,^{25, 26, 27} we examined apoptosis related proteins in our study. Diabetes and Stellaria media treatment had no effects on the proapoptotic Bax and antiapoptotic Bcl-XL proteins (Fig. 5D and F). We also investigated GLUT4 transporter expression as GLUT4-mediated glucose uptake may play a relevant role in diabetes. Diabetes and Stellaria media treatment did not influence GLUT4 protein expression (data not shown).

Fig. 5.

Western blot analysis of phosphorylation of proteins: signal transducer and activator of transcription 3 (STAT3) (A), protein kinase B (Akt) (B), extracellular signal-regulated kinase (Erk) (C), proapoptotic Bax (D), representative bands (E), antiapoptotic Bcl-XL (F). Results are means ± SEM (n = 7), analyzed by one-way ANOVA followed by Fisher LSD post hoc test,∗p < 0.05 vs. Control, #p < 0.05 vs. Diabetes.

4. Discussion

In the present study, we tested the effects of Stellaria media tea on the severity of diabetes as well as on diabetes-induced cardiac consequences in an in vivo rat model. Based on our findings, Stellaria media tea appears to have beneficial effects on cardiac dysfunction induced by diabetes, since the treatment ameliorated the impaired cardiac output and cardiac work. However, this effect seems to be independent of the modulation of diabetes severity as the application of the tea treatment did not influence fasting hyperglycaemia or glucose intolerance.

In folk medicine Stellaria media is consumed mainly as tea. To the best of our knowledge, this is the first study investigating the hot water extract of chickweed in the settings of diabetes. In our study, Stellaria media was not effective in lowering blood glucose level or improving glucose tolerance in diabetic rats. In the literature only one other study examined specifically the effect of Stellaria media on the severity of diabetes. Ethanolic leaf extract of Stellaria media in a dose of 100–400 mg/kg/day administered by intraperitoneal injection has been shown to attenuate hyperglycaemia in a 21-day alloxan-induced diabetic rat model.¹³ In that study, Stellaria media treatment attenuated fasting blood glucose levels, decreased haemoglobin A1c levels and inhibited pancreatic α-amylase and β-glucosidase enzyme activities.¹³ There are also some studies using various models where glucose homeostasis was estimated among other metabolic parameters. For instance, ethanolic radix extract of another member of the Stellaria genus (Stellaria dichotoma) improved glucose homeostasis in high-fat-diet fed mice.²⁸ In contrast, Chidrawar et al. found that ethanolic extract of Stellaria media was ineffective to decrease hyperglycaemia in both cafeteria-diet- and progesterone-induced obesity models.^29,30 However, they also found that 200 and 400 mg/kg methanolic extract significantly attenuated serum glucose levels in these models.^29,30 These controversial results may be explained by the differences in (i) the composition of the extracts, (ii) delivery time, administration and dose of Stellaria media, (iii) the applied animal models and strains. It should be also noted that intraperitoneal application of ethanolic and methanolic extracts of Stellaria media has limited translational value in the view of the human consumption of this medicinal plant.

Diabetic cardiomyopathy is one of the major consequences of diabetes. In our study, aortic flow, cardiac output and cardiac work were significantly decreased in the Diabetes group in comparison to the Control group, showing that this experimental diabetes model has some adverse effects on the heart. Stellaria media tea treatment significantly improved cardiac output and cardiac work, suggesting that Stellaria media tea may have beneficial effects on the heart in a diabetic state. To the best of our knowledge, in the literature currently there is no other experimental data concerning the effects of Stellaria media on cardiac function or cardiomyopathy. Our research group tested the effects of Stellaria media tea lyophilizate in another chronic metabolic disease, i.e. hypercholesterolaemia, where we demonstrated that the treatment has no blood cholesterol lowering effect in diet-induced hypercholesterolaemia in rats.³¹ In the same study, we investigated some safety issues of the Stellaria media treatment on the heart. Our transthoracic echocardiographic measurements showed that Stellaria media treatment did not affect cardiac morphology and parameters related to cardiac function in diet-induced hypercholesterolaemia.³¹

Various mechanisms may play a role in the development of cardiac dysfunction in diabetes, e.g. oxidative stress, diffuse apoptosis of cardiomyocytes, dysregulation of cardiac signaling pathways, mitochondrial dysfunction, fibrosis or hypertrophy.^26,32 Interestingly, there are more than 50 medicinal herbs, such as sesame,³³ which have beneficial effects on experimental diabetic cardiomyopathy (for review please see Refs. ^32,34). These plants have been suggested to exert antioxidant properties that may attenuate oxidative stress or inflammation, and to reduce apoptosis, and cardiac remodelling.³² In the literature, there are some experimental data suggesting similar antioxidant and anti-inflammatory properties of Stellaria media, which can be associated with the improvement of certain cardiac parameters in our present study.³⁵ Therefore, we tested the in vitro antioxidant capacity of Stellaria media tea, and we found a low antioxidant activity. This phenomenon was observed by another study too, where the antioxidant activity of the aqueous and methanolic extract was weak.³⁶ Stellaria media has been reported to contain active metabolites e.g. phenolic compounds, flavonoids or steroid saponins, that may play a role in pharmacological activities.³⁵ We determined the total flavonoid content of Stellaria media tea and it was 9.88 ± 0.10 mg QE/g. The flavonoid content of Stellaria media has been discussed by several papers.^{36, 37, 38} In a phytochemical study, the flavonoid content was determined not less than 1.2% in raw plant material.³⁸ In an experiment, the total flavonoid content (determined by HPLC) of a lyophilized juice was 25.6 mg/g, and in an ethanolic extract 63.9 mg/g³⁶. Our extract was prepared with hot water (in accordance with the human use) and this might explain the lower flavonoid content. UPLC analysis of flavonoid screening indicated rutin being a possible component in the Stellaria media tea in our present study. The beneficial cardiovascular effect of rutin in diabetes has been proposed. Some studies demonstrated that rutin alleviates diabetic cardiomyopathy and improve left ventricular dysfunction in STZ-induced diabetes^{39, 40, 41, 42} and in high-carbohydrate, high-fat diet models.⁴³ Rutin was also shown to exert neuroprotective effects which may be beneficial in diabetes as well.⁴⁴ LC-MS/MS analysis of the extract we used in our study afforded identification of various glycosylated apigenin-derivatives, which finding is in accordance with literature data.³⁶ The glycosylated flavonoid derivatives can be poorly absorbed in the intestines directly, therefore during digestion there is a deglycosylation step, and after that only the aglycone part of the molecule will be absorbed,⁴⁵ therefore being responsible for the biological effects. The aglycone part of these components is apigenin, which has already been reported to alleviate STZ-induced diabetic cardiomyopathy⁴⁶ and to exert protective effects against cardiac dysfunction in myocardial infarction in diabetic rats,⁴⁷ suggesting that apigenin-derivatives may contribute to the beneficial cardiac effects of Stellaria media tea.

The limited antioxidant capacity of the water extract raised the question whether signaling pathways are involved in the cardiac effects. Based on literature data, STAT3 is proposed as a key mediator of diabetes-induced cardiac dysfunction.^{48, 49, 50} In conjunction with other studies^{51, 52, 53} (for specific review please see Ref. 54), we showed that diabetes increased cardiac STAT3 phosphorylation. Moreover, Stellaria media tea treatment prevented diabetes-induced cardiac STAT3 dysregulation, suggesting a protective role. Indeed, some studies demonstrated that attenuation of the enhanced cardiac STAT3 activation may be beneficial in diabetes. In a high-glucose, high-fat diet and STZ injection-induced diabetic rat model, losartan attenuated the cardiac STAT3 phosphorylation and improved heart function.⁵³ In STZ-induced diabetic mice hearts, inhibition of diabetes-induced activation of EGFR-STAT3 signaling was associated with restoring cardiac fibrosis and hypertrophy related factors, and prevented diabetes-induced cardiac dysfunction.⁵⁰ Interestingly, the possible presence of rutin in Stellaria media tea may be a feasible explanation for the observed effects in our study, as rutin was suggested to exert cytoprotection by inhibiting STAT3 phosphorylation.^55,56 Similarly, apigenin and its derivatives has been shown to attenuate STAT3 activation in tumor cells,^57,58 which is in agreement with our present findings observed in cardiac tissue and may be related to the beneficial effect of Stellaria media tea on diabetic cardiomyopathy.

Nevertheless, a better understanding of the molecular mechanism underlying the proposed beneficial cardiac effects of Stellaria media tea would require additional research in the future. Further investigation of STAT3 signaling pathway could identify downstream targets or direct cause-effect mechanisms. Applying other doses of the tea, using a different extraction method or combination with standard therapies may also contribute to a deeper knowledge. Testing the effects of the tea in female rats could reveal sex differences in the effectiveness of the extract. It would be worth studying the effects of Stellaria media tea not only in a rat model, but in humans as well whether we can observe functional cardiac improvement in diabetic patients who drink chickweed tea regularly.

Although we provide interesting data on the effects of Stellaria media in diabetes, as always, there may be some possible limitations of our present study. The animals received Stellaria media tea from the onset of the experiments, together with the fructose-enriched diet. It would be worthwhile testing its effects in a developed diabetic state, which may have greater clinical relevance. Moreover, elucidation of the causal relation between the tea's beneficial effect and STAT3 phosphorylation with state-of-the-art experiments or further analysis of downstream targets in diabetic cardiomyopathy would be straightforward in the future. We tested the effects in a rat model, which might differ from the human metabolism. Moreover, the lack of a group receiving only herbal treatment without fructose-enriched diet can also be considered as a possible limitation.

In conclusion, the tea made of Stellaria media (i.e. common chickweed) may protect against diabetes-induced cardiac dysfunction; however, this effect seems to be independent of the modulation of fasting hyperglycemia or glucose tolerance in rats. Stellaria media prevented diabetes induced STAT3 phosphorylation in the heart, which may play a role in the beneficial cardiac effect. Nevertheless, further studies are needed to reveal the exact molecular mechanisms underlying the proposed cardioprotective effect of Stellaria media in diabetic conditions.

Declaration of competing interest

None.

Funding

This work was supported by the Economic Development and Innovation Operative Programme [GINOP-2.3.2-15-2016-00012 and GINOP-2.3.2-15-2016-00006] and EFOP-3.6.2-16-2017-00006, and by grants from the National Research, Development and Innovation Office [K115990, K115796 and PD128271] the Ministry of Human Capacities, Hungary [20391–3/2018/FEKUSTRAT] and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. HCEMM has received funding from the EU's Horizon 2020 research and innovation program under grant agreement No. 739593. V.D., A.F. and F.D.G. were supported by the New National Excellence Program of the Ministry of Human Capacities, respectively [UNKP-19-3-SZTE-47, UNKP-20-2–SZTE-60, UNKP-20-2–SZTE-61].

Appendix A. Supplementary data

The following is the Supplementary data to this article: