In Vitro and In Vivo Evaluation of the Antidiabetic Activity of Solidago virgaurea Extracts

Abstract

Background:

Solidago virgaurea (Asteraceae) has been used for more than 700 years for treating cystitis, chronic nephritis, urolithiasis, rheumatism, and inflammatory diseases. However, the antidiabetic activity of Solidago virgaurea has been rarely studied.

Methods:

Three extracts of Solidago virgaurea were prepared, and their antidiabetic potentials were evaluated by various cell-free, cell-based, and in vivo studies.

Results:

We found that the Solidago virgaurea contained multiple bioactive phytochemicals based on the GC-MS analysis. The Solidago virgaurea extracts effectively inhibited the functions of the carbohydrate digestive enzyme (α-glucosidase) and protein tyrosine phosphatase 1B (PTP1B), as well as decreased the amount of advanced glycation end products (AGEs). In the L6 myotubes, the Solidago virgaurea methanolic extract remarkably enhanced the glucose uptake via the upregulation of glucose transporter type 4 (GLUT4). The extract also significantly downregulated the expression of PTP1B. In the streptozotocin-nicotinamide induced diabetic mice, the daily intraperitoneal injection of 100 mg/kg Solidago virgaurea methanolic extract for 24 days, substantially lowered the postprandial blood glucose level with no obvious toxicity. The extract’s anti-hyperglycemic effect was comparable to that of the glibenclamide treatment.

Conclusion:

Our findings suggested that the Solidago virgaurea extract might have great potential in the prevention and treatment of diabetes.

1. INTRODUCTION

Diabetes mellitus is an incurable and multifactorial metabolic disorder characterized by chronic hyperglycemia, which is associated with organ dysfunction and damage, especially in the eye, kidney, cardiovascular system, and nervous system. Type 1 diabetes is caused by β-cell destruction and usually leads to absolute insulin deficiency, accounting for 5–10 % of patients with diabetes, while type 2 diabetes is highly associated with insulin resistance, accounting for 90–95 % of patients with diabetes [1, 2]. So far, many sugar-lowering drugs have been discovered to treat type 2 diabetes and metabolic syndrome [3]. Continuous hyperglycemia can also speed up the process of non-enzymatic glycation of proteins in diabetic patients in contrast to healthy individuals with normal glucose levels. The increased protein glycation results in the gradual accumulation of advanced glycation end products (AGEs) in body tissues [4]. In addition to diabetes, AGEs are involved in the development of many other degenerative diseases, such as cardiovascular diseases, rheumatoid arthritis, chronic renal failure, and neurological disorders [5]. Various AGE inhibitors have been developed and mainly used as chelators to inhibit metal-catalyzed oxidation reactions that catalyze AGE formation in diabetes [6]. Unfortunately, many AGE inhibitors either show insufficient antidiabetic effects or cause undesirable side effects [7].

Protein tyrosine phosphatase is a large family of proteins involved in the regulation of many physiological and pathological events. As an important member of this family, the protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathway by direct dephosphorylation of insulin receptors and insulin receptor substrate-1 [8]. The PTP1B overexpression causes insulin resistance in obesity models, while its downregulation or deficiency leads to increased insulin sensitization and tolerance to glucose [9–11]. Therefore, PTP1B is an important therapeutic target [7, 8], and various PTP1B inhibitors have been developed for the treatment of type 2 diabetes and obesity [8, 12].

Dietary plants are believed to lower the intake of carbohydrate-rich foods by adding more fats or fibers for weight control and hence for the management of type 2 diabetes [13]. However, a high fiber and fat-containing diet can only exert mild effects on diabetes and may cause gastrointestinal problems and obesity. To deal with these issues, a strategy that can decrease the absorption of carbohydrates through inhibition of digestive enzymes, e.g., amylase and glucosidase, has been used [14]. In addition to synthetic compounds, many phytochemicals have shown the inhibitory activities against α-amylase and α-glucosidase in the management of obesity and type 2 diabetes [15]. Among them, flavonoids, alkaloids, terpenoids, and iridoids, have also been extensively investigated for their anti-glycation activities [16, 17].

The genus Solidago has about 120 species, mostly grown wild or being purposefully cultivated as ornamental plants [18]. Solidago virgaurea (Asteraceae), commonly called goldenrod, has been used for more than 700 years for treating cystitis, chronic nephritis, urolithiasis, rheumatism, and as an anti-inflammatory drug [19, 20]. However, to the best of our knowledge, there is little research being conducted on hypoglycemic and antidiabetic activities of Solidago virgaurea. In this study, the Solidago virgaurea extracts were prepared, and their phytocomponents were analyzed by gas chromatography-mass spectrometry (GC-MS). The extracts’ inhibitory activities against α-glucosidase, AGE formation, and PTP1B were determined by cell-free enzyme assays. The extracts were also examined the L6 myoblasts in terms of their cytotoxicity, glucose uptake inhibition, and the underlying antidiabetic mechanisms. Finally, the in vivo antidiabetic activity and safety of the Solidago virgaurea extracts were investigated in the streptozotocin (STZ) -nicotinamide (NA) induced diabetic mice.

2. MATERIALS AND METHODS

2.1. Materials

α-Glucosidase, p-nitrophenol- α-D glucopyranoside, and the primers of PTP1B, GLUT4, and β-actin were purchased from Sigma-Aldrich (St. Louis, USA). PTP1B (human recombinant) was purchased from Biomol International LP (PA, USA). TRIzol Reagent and M-PER^™ Mammalian Protein Extraction Reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies, rabbitanti-GLUT4 and goat-anti-PTP1B, were purchased from Sino Biological (China). All other chemicals and reagents used were of analytical grade and purchased from VWR (Radnor, PA, USA).

Rat L6 myoblasts purchased from ATCC (Manassas, VA) were cultured in complete growth media (DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 % FBS) at 37 °C in 5 % CO₂. L6 myoblasts were differentiated by the method described previously [21, 22] with slight modification. Briefly, the medium was replaced with the DMEM supplemented with 2% FBS to induce differentiation when the cells reached 80–90% confluence. After 48 hours, the differentiated myotubes were serum-starved in 0.2 % BSA for 18 hours prior to the cell-based assays, except cytotoxicity assay.

2.2. Preparation of Plant Extracts

The Solidago virgaurea plant was collected in the spring 2018 from Elum mountain, Swat, Pakistan with the coordinates 34.6182° N, 72.3327° E, and identified by Dr. Ghulam Mujtaba Shah, Department of Botany, Hazara University, Mansehra, Pakistan. The plant samples were preserved in Herbarium at Hazara University. The aerial parts (stem and leaves) of plants were thoroughly washed and dried, and then ground to a fine powder. Since the solvent polarity influences the compounds extracted and extraction yield [23, 24], and subsequently the therapeutic effects, the powdered samples were extracted sequentially in three solvents with the polarity from low to high, i.e., ethanol, methanol, and water. Briefly, the samples were suspended in ethanol (1:10, v/v) and shaken for 24 hours, followed by centrifugation at 10,000 rpm for 15 min. The supernatant was decanted into a new container (named as ethanolic extract). The precipitate was extracted in methanol (named methanolic extract), and then the residue was extracted in water (named aqueous extract). Finally, the extracts were dried and stored in the refrigerator for further use. For biological evaluations, the dried extracts were reconstituted with the corresponding aqueous buffer.

2.3. α-Glucosidase Inhibition Assay

The α-glucosidase inhibitory activities of methanolic, ethanolic and aqueous extracts were evaluated according to the previous study [25]. Briefly, the extracts were dissolved in phosphate-buffered saline (PBS) (50 mM, pH 6.8) at various concentrations and incubated with 50 μL of α-glucosidase at 37 °C for 5 min. Then, the substrate, 1mM p-nitrophenol- α-D glucopyranoside, was added to the mixture and incubated at 37 °C for an additional 20 min. The reaction was stopped by the addition of 50 μL of Na₂CO₃ (100 μM), and the absorbance was measured at 405 nm on a Tecan Infinite M1000 Pro microplate reader (Tecan Group Ltd.). The PBS without extracts was used as the negative control and processed by the same protocol. The following equation was used for calculation:

$$\text{Inhibition}\left(\%\right)=\left(1-\text{As}/\text{Ac}\right)\times 100\%$$

Where Ac: absorbance of control, As: absorbance of sample.

2.4. Antiglycation Assay

The advanced glycation end products inhibitory assay was performed according to the protocol with little modification [26]. The reaction mixture contained 400 μL each of bovine serum albumin (BSA) (10 mg/mL), glucose anhydrous (50 mg/mL), and the sample. The glycated control contained 400 μL each of BSA, glucose, and PBS, while the blank control contained 400 μL BSA and 800 μL PBS.

The reaction mixture was incubated in the 96-well plate at 37 °C for 7 days. After incubation, 120 μL of trichloroacetic acid (TCA) was added, and the mixture was centrifuged (15,000 rpm) at 4 °C for 4 min. The pellets were washed with 120 μL of TCA, and the supernatant was removed. Then, the pellets were dissolved in 120 μL of PBS. The fluorescence was measured at the λ_ex 370 nm and λ_em 440 nm on the Tecan microplate reader. The results are calculated as follows:

$$\text{Inhibition}\left(\%\right)=\left(1-{\text{Fluorescence intensity}}_{\text{extract}}/{\text{Fluorescence intensity}}_{\text{control}}\right)\times 100\%$$

2.5. PTP1B Inhibition Assay

The inhibitory activities of the extracts were evaluated using p-nitrophenyl phosphate (p-NPP) as the substrate [27, 28]. Briefly, the extract was mixed with 2 mM p-NPP and PTP1B (0.05–0.1 μg) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol in the 96-well plates (200 μL per well) and incubated at 37 °C for 30 min. The reaction was terminated with 10 N NaOH. The absorbance of the produced p-nitrophenol was measured at 405 nm on the Tecan microplate reader. The mixture in the absence of PTP1B was measured as the non-enzymatic hydrolysis of p-NPP. The inhibition was calculated by the equation:

$$\text{Inhibition}\left(\%\right)=\left[1-\left({\text{A}}_{\text{Sample}}-{\text{A}}_{\text{Blank}}\right)/\left({\text{A}}_{\text{Control}}-{\text{A}}_{\text{Blank}}\right)\right]\times 100\%$$

2.6. Cytotoxicity Assay

The rat L6 myoblasts were seeded in the 96-well plate at a density of 1 × 10⁵ cells per well one day before the experiment. The cells were incubated with the methanolic extract at various concentrations for 48 hours. Then, the cell viability was determined by the MTT assay [29]. Briefly, the treated cells were incubated in the complete growth medium containing 0.5 mg/mL MTT at 37 °C for 3 hours. After that, the medium was aspirated and 200 μL of DMSO was added. The absorbance was measured at 540 nm on the Tecan microplate reader after gently shaking the plate for 10 min.

2.7. Glucose Uptake Assay

The differentiated L6 myotubes were treated with the methanolic extract at various concentrations for 1, 6, and 12 hours. Then, the glucose concentration in the culture medium was measured by the O-Toluidine method [30]. The cells without treatments were used as the control. The culture medium was used as the blank. The glucose uptake was calculated by the following equation:

$$\text{Glucose uptake}={\text{Glucose concentration}}_{\text{blank}}–{\text{Glucose concentration}}_{\text{treated}}$$

2.8. Real-time Quantitative PCR (qRT-PCR) of PTP1B and GLUT4

The differentiated L6 myotubes were incubated with 1mg/mL of the methanolic extract for 12 hours. Then, the RNA was extracted from the cells using TRIzol Reagent according to the manufacturer’s instructions. The cDNA was synthesized from 5 μg of total RNA samples. The primers for PTP1B, GLUT4, and β-actin genes were: PTP1B-forward (5′-TGTGATCGAGGGTGCAAAG-3′), PTP1B- reverse (5′-CTCCAGGTCTTCATGGGAAAG-3′); GLUT4-forward (5′-CAGGCCGGGACACTATACCC-3′), GLUT4-reverse(5′-GTTCCCCATCTTCAGAGCCGA-3′), β-actin-forward (5′-CCCATCTATGAGGGTTACGC-3′), and β-actin-reverse (5′-TTTAATGTCACGCACGATTTC-3′). The PCR conditions were as follows: 95 °C for 10 min, 45 cycles of 94 °C for 15 sec, and annealing/ extension at 65 °C for 1 min on a CFX Opus Real-Time PCR Systems (Bio-Rad). The relative mRNA expression was quantitated using the comparative ΔΔCT method [31]. Experiments were performed in triplicates to ensure accuracy.

2.9. Western Blot Analysis of PTP1B and GLUT4

The differentiated L6 myotubes were incubated with 1mg/mL of the methanolic extract for 12 hours. The protein expression was analyzed by western blot [21, 22]. Briefly, the treated cells were collected and washed twice by PBS, and then lysed by the M-PER^™ Mammalian Protein Extraction Reagent on ice for 1 hour. The cell lysate was centrifuged at 13,000 rpm at 4 °C for 15 min. Electrophoresis resolved the extracted proteins on a 4–15 % gradient polyacrylamide gel and subsequently transferred them to a nitrocellulose membrane. The blots were blocked with 5 % non-fat milk in TBST at room temperature for 1 hour and incubated overnight with the primary antibodies (anti-GLUT4 and anti-PTP1B) at 4 °C. After washing with TBST, the blots were incubated with the peroxidase-conjugated secondary antibodies for 1 hour. Immunoreactive bands were visualized using the chemiluminescence reagent. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was used as an internal control. Densitometric analysis was carried out by ImageJ software [32].

2.10. In Vivo Diabetic Mouse Model

The 2-month male Albino BALB/c mice weighing 25 to 30 g were purchased from Animal Facility, Department of Pharmacy, University of Peshawar, Pakistan. The mice were housed in groups of five per cage at 25 °C with a 12 hours light - dark cycle and allowed free access to food and water. All animal procedures were performed according to the animal care protocol (F.No.73/HU/ORIC/2020/677) approved by Hazara University Animal Care and Use Committees.

Diabetes was induced in mice by intraperitoneal (i.p.) injection of streptozotocin (STZ) accompanied by nicotinamide (NA) [33, 34]. Briefly, after a 12-hours fasting, the mice were injected intraperitoneally with 230 mg/kg of NA in 0.9 % saline, followed by injection of STZ (65 mg/kg) in 0.9 % saline. The postprandial blood glucose level was checked by a OneTouch^® glucometer (LifeScan, Inc.). The mice with blood glucose levels higher than 250 mg/dL were selected as the diabetic mice for further studies [35].

2.11. In Vivo Safety Study

Before the antidiabetic effect study, the in vivo safety of the plant extract injection was evaluated. Briefly, the dried methanolic extract was reconstituted in PBS containing 3% DMSO, which is much lower than the tolerated DMSO dose in rodents [36]. The doses of the extract ranging from 100 to 1000 mg/kg were injected i.p. in the induced diabetic mice. The mice were observed for 24 hours for any kinds of behavioral abnormalities and mortality.

2.12. In Vivo Antidiabetic Activity Study

The mice were randomly divided to 4 groups with 5 mice each group. Group I, non-diabetic/healthy mice; Group II, untreated diabetic mice; Group III, diabetic mice, treated with the antidiabetic drug glibenclamide; and Group IV, diabetic mice, treated with the methanolic extract. The blood glucose level of healthy mice was about 100 mg/dL, and that of diabetic mice was above 250 mg/dL, before the treatments.

Mice were given the plant extract or glibenclamide (5 mg/kg) through the i.p. injection daily for 24 days. The blood glucose level was monitored every 4^th day. The mouse body weight was measured both at the beginning and the end of the experiment. On the 24^th day, the animals were sacrificed by cervical dislocation, and the vital organs were collected and preserved in 10 % formalin. The tissues were sectioned and stained with hematoxylin and eosin (H&E) for histopathological analysis.

2.13. GC-MS Analysis of Solidago virgaurea Extracts

The Solidago virgaurea methanolic extract was analyzed by an Agilent USB-393752 gas chromatograph equipped with an HHP-5MS (5 % phenyl)-methylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm film thickness) connecting to a flame ionization detector (FID). The oven temperature program was: 70 °C for 1 min, 70 °C to 180 °C at the rate of 6 °C/min in 5 min, 180 °C to 280 °C at the rate of 5 °C/min in 20 min. Temperatures of the injector and detector were maintained at 220 °C and 290 °C, respectively. Helium gas was the carrier gas at the flow rate of 1 mL/min. The sample (1 μL) was injected manually in the split-less mode. The molecular weights of the separated constituents were determined by the connected Agilent HP-5973 mass selective detector in the electron impact mode (ionization energy: 70 eV).

2.14. Statistical Analysis

All the in vitro assays were repeated at least 3 times with independent samples. The data were expressed as mean ± standard deviation (S.D.) and analyzed by Student’s t-test using GraphPad Prism7 software. Groups were considered significantly different from the control group if * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

3. RESULTS

3.1. Inhibition of α-glucosidase by Solidago virgaurea Extracts

All the extracts showed significant α-glucosidase inhibitory activities (Fig. 1A). Among these extracts, the methanolic extract was the most active one to inhibit the α-glucosidase with an IC₅₀ value of 128.8 μg/mL, followed by the aqueous and ethanolic extracts with the IC₅₀ values of 140.4 μg/mL and 195.5 μg/mL, respectively. Acarbose, an antidiabetic drug against α-glucosidase [3], was used as a positive control and showed an IC₅₀ value of 85 μg/mL.

Fig. 1.

Antidiabetic activities of the Solidago virgaurea extracts determined by the cell-free assays. (A) α-glucosidase inhibitory activities of the Solidago virgaurea extracts. Acarbose was used as a positive control. (B) Inhibition of AGEs by the Solidago virgaurea extracts. Rutin was used as a positive control. (C) Inhibition of PTP1B by the Solidago virgaurea extracts. (D) Dose response (PTP1B inhibition) curve of the methanolic extract.

3.2. Inhibition of Glycation by Solidago virgaurea Extracts

The methanolic extract was found to be superior in inhibiting AGEs (Fig. 1B) as well with the IC₅₀ value of 24.5 μg/mL compared to 55.7 μg/mL and 69.9 μg/mL of the aqueous and ethanolic extracts, respectively. The antiglycation activity shown by the methanolic extract was comparable to the positive control, Rutin [37], which had an IC₅₀ of 22.2 μg/mL.

3.3. Inhibition of PTP1B by Solidago virgaurea Extracts

The methanolic extract also had the highest PTP1B inhibitory activity (96 %) among the three extracts (ethanolic: 55 % and aqueous: 71 %) (Fig. 1C). Furthermore, the doseresponse curve (0–1000 μg/mL) of the methanolic extract on PTP1B inhibition gave the IC₅₀ value of around 59.7 μg/mL (Fig. 1D).

3.4. Cytotoxicity

Using L6 myoblasts as the in vitro model, the cell viability was examined by the MTT assay. In the concentration range of 62.5–1000 μg/mL, the extract did not show significant cytotoxicity, suggesting that methanolic extract was safe (Fig. 2A).

Fig. 2.

Cytotoxicity and glucose uptake study of the Solidago virgaurea methanolic extract in L6 myoblasts. (A) Cell viability in the presence of the Solidago virgaurea methanolic extract determined by the MTT assay. Cell incubation time: 48 hours. (B) Effect of the Solidago virgaurea methanolic extract on glucose uptake. Data were presented as mean ± S.D. (n=3). ** P <0.01, *** P <0.001, **** P <0.0001.

3.5. Enhancement of Glucose Uptake by the Solidago virgaurea methanolic Extract

To study the therapeutic effects, the L6 myoblasts were differentiated and serum-starved, referring to L6 myotubes. Glucose uptake in L6 myotubes significantly increased in the extract-treated groups compared to the untreated control, and the effects were dose and time-dependent (Fig. 2B).

3.6. Modulation of PTP1B and GLUT4 Gene Expression by the methanolic Extract

In the presence of 1mg/mL methanolic extracts, the GLUT4 mRNA was upregulated to around 8 folds (p<0.0001) compared to that of the untreated cells (Fig. 3A), while the same treatment downregulated the PTP1B mRNA level to around 60 % (p<0.0001) (Fig. 3B). The GLUT4 (p<0.05) and PTP1B (p<0.001) mRNA data were well consistent with their protein levels, as quantitated by western blotting (Figs. 4A and B).

Fig. 3.

Relative mRNA levels of (A) GLUT4 and (B) PTP1B. Cell incubation time: 12 hours. Data were presented as mean ± S.D. (n=3). **** P <0.0001.

Fig. 4.

Western blot of (A) GLUT4 and (B) PTP1B proteins. Cell incubation time: 12 hours. Densitometric analysis was carried out by ImageJ software. The data was expressed as the mean ± S.D. (n= 3). * P <0.05, *** P <0.001.

3.7. Antidiabetic Activity in Diabetic Mice

About 80 % of the STZ-NA treated mice showed hyperglycemia (≥250 mg/dL) [35] and were considered diabetic mice for studying the in vivo safety and antidiabetic activity of the extracts. For the pilot in vivo study, a small number (5–10) of animals is recommended [33, 38] to make the study reproducible and statistically significant. In this study, five healthy or diabetic animals per group were used. The i.p. injections of the extract (100–1000 mg/kg) demonstrated no obvious abnormality and mortality in the diabetic mice, indicating the extracts were safe. The low dose, 100 mg/kg, was chosen for the antidiabetic study. In the study, the healthy mice maintained their blood glucose level at around 100 mg/dL, while the untreated diabetic mice exhibited stable hyperglycemia with a glucose level of more than 250 mg/dL. The Solidago virgaurea methanolic extract showed a strong anti-hyperglycemic effect, which was equivalent to that of the commercial drug, glibenclamide [39]. On day 24, the blood glucose levels in the extract-treated mice went down to around 120 mg/dL (p<0.0001) (Fig. 5A). During the study, only the untreated diabetic mice lost their body weights (p<0.0001), while no significant decrease in the mouse body weight was observed among the healthy, glibenclamide-treated, and extract-treated mice, further corroborating the extract’s safety (Fig. 5B).

Fig. 5.

Antidiabetic activity of the Solidago virgaurea methanolic extract in the STZ-NA induced diabetic mice. (A) Mouse blood glucose levels. The postprandial blood glucose was measured by the OneTouch^® glucometer. (B) Mouse body weights at the beginning and end of the experiment. Glibenclamide was used as a positive control. Data were presented as Means ± S.D. (n=5). **** p<0.0001.

3.8. Histopathological Analysis

Considering Solidago virgaurea extracts’ influence on glucose uptake and metabolism, the liver, kidney, and pancreas of the treated mice were excised and sectioned for histopathological examination. The normal structural features of liver, kidney and pancreas tissues were observed in healthy mice. However, in the untreated diabetic mice, some of these features were absent and the apoptotic/necrotic areas were observed, particularly in the liver. (The damaged/dead tissues were indicated by red arrows). The damaged tissues were seemingly “repaired” to some extent by the glibenclamide or methanolic extract treatment, as evidenced by the re-appearance of some of the histopathological characteristics (Fig. 6).

Fig. 6.

H&E staining. At the end of the in vivo experiment, liver, kidney, and pancreas were collected and sectioned, followed by H&E staining (scale bar: 100 μm). The arrows refer to the necrotic/apoptotic tissues (cells).

3.9. GC-MS Analysis

The GC-MS data showed that many phytochemical constituents were present in the Solidago virgaurea methanolic extract (Fig. 7). Nineteen of them were significant based on their peak heights representing their abundance in the chromatogram. These phytochemical constituents were identified by comparing the GC-MS data with the mass spectral library of National Institute of Standard and Technology (NIST). The phytochemicals include alcohols, saturated fatty acids, unsaturated fatty acids, carboxylic acids, alkanes, organic cyclic compounds, and esters (summarized in Table 1).

Fig. 7.

GC chromatogram of the Solidago virgaurea methanolic extract. The major phytochemicals were identified with the help of the mass spectral library of NIST (Table 1).

Table 1.

Major phytocomponents identified in the Solidago virgaurea methanolic extract.

S. No.	Retention Time (min)	Compound Name	Molecular Weight (amu)
1	3.22	N-Methyl glycine	89.048
2	5.08	Isopropyl acetate	102.068
3	5.25	Methyl stearate	298.287
4	5.92	di-tert-Butyl dicarbonate	218.115
5	6.18	n-Propyl acetate	102.068
6	7.38	Tridecanoic acid, methyl ester	228.209
7	7.88	Acetoin	88.052
8	9.07	Rhamnose	164.068
9	9.37	Pyrimidine,4-butyl-3,4-dihydro-5methyl	152.131
10	10.29	Butanoic acid	88.052
11	20.31	5-Furandione, dihydro-3-methylene	112.016
12	22.16	Hexanoic acid	116.084
13	24.59	di-tert-Butyl dicarbonate	218.115
14	25.24	Heptanoic acid	130.099
15	27.43	Hexadecanoic acid, methyl ester	270.256
16	28.47	4-Oxopentyl formate	130.063
17	29.28	15-Crown-5	220.131
18	30.34	9-Octadecenoic acid, methyl ester	296.272
19	31.27	5-Hydroxymethylfurfural	126.032

4. DISCUSSION

Due to the complexity of diabetes, the drugs with the antidiabetic potentials against multiple therapeutic targets are desirable [3]. Based on previous reports on the therapeutic effects of the phytochemicals, the present study aimed to explore the antidiabetic potentials of Solidago virgaurea. In this study, the methanolic, ethanolic, and aqueous extracts of Solidago virgaurea were prepared and evaluated for their inhibitory activities against the α-glucosidase, glycation, and PTP1B, which are highly associated with type 2 diabetes.

The digestion of carbohydrates is mainly mediated by pancreatic α-amylase and α-glucosidase, resulting in the postprandial increase of the blood glucose level. The agents with the inhibitory activity against these digestive enzymes can reduce the postprandial glucose level and may be used for treating diabetes [3]. Miglitol and acarbose are the major inhibitors against these digestive enzymes [3, 40]. But they may cause adverse effects such as gastric flatus and swelling [3, 40, 41]. In this study, we found that all three Solidago virgaurea extracts could inhibit the α-glucosidase activity, among which the methanolic extract was the most potent.

The formation and accumulation of AGEs is one major consequence of diabetic complications [42]. In diabetic patients, the elevated AGE level may cause microvascular damage, including neuropathy, nephropathy, cataracts, and retinopathy [43]. Compounds capable of breaking down AGEs or inhibiting the AGEs’ accumulation are potential drug candidates for treating diabetes. In line with the results of the α-glucosidase inhibition assay, all Solidago virgaurea extracts showed anti-glycation activities, among which the methanolic extract had the highest potency.

GLUT4 is a key component in glucose uptake and homeostasis [44]. Some plant extracts have shown the capability of upregulating the GLUT4 expression and enhancing glucose uptake [45]. To understand the mechanisms of the antidiabetic activity of the Solidago virgaurea extracts, L6 myoblasts were used as the in vitro model. We found that the methanolic extract of Solidago virgaurea did not have obvious cytotoxicity in the L6 myoblasts. In the presence of the methanolic extract, the cellular uptake of glucose was dramatically increased, and the effect was dose dependent. To confirm the results, the expressions of GLUT4 and PTP1B were analyzed. As evidenced by the real-time PCR and western blotting data, the methanolic extract significantly upregulated GLUT4 but downregulated PTP1B. It should be noted that the extract-induced significant transcriptional and translational changes in GLUT4 and PTP1B were observed after a relatively short incubation time (12 hours), which was most likely due to the pre-serum-starvation of L6 myotubes [46–48].

STZ can irreversibly damage pancreatic beta cells and is mostly used for the establishment of diabetic animal models [33, 34]. To maintain diabetic syndrome but keep animals alive, NA is concurrently administered [33, 34]. After the i.p. injection of STZ and NA, the diabetic syndrome was developed in the BALB/c mice, as evidenced by the moderate and persistent hyperglycemia with a postprandial blood glucose level of higher than 250 mg/dL.

Our results showed that the i.p. injection of the methanolic extract of Solidago virgaurea successfully controlled the postprandial blood glucose level and maintained the body weight. The antidiabetic activity of the methanolic extract was similar to that of the commercial anti-diabetic drug, glibenclamide. The methanolic extract did not show obvious systemic toxicity in the tested animals. Histopathological analysis indicated that methanolic extract treatment could repair the damaged tissues of liver, kidney, and pancreas to some extent, which was also close to the effects of glibenclamide.

Some of these phytochemicals in the methanolic extract confirmed by GC-MS analysis are known bioactive compounds and the plants containing these phytochemicals have been reported to have medicinal value in treating various diseases. For example, the octadecanoic acids showed antibacterial activity and the plants containing octadecanoic acids, hexadecenoic acids and their esters have been often used for treating hepatic cancer, liver cirrhosis, hepatitis, breast cancer, and inflammatory disease [49–51].

In fact, Solidago virgaurea has also showed some effectiveness against type-1 diabetes in a very recent study [52]. The new findings in the current study were well in line with the antidiabetic potential of Solidago virgaurea. Though we have identified that the Solidago virgaurea extract could downregulate PTP1B but upregulate GLUT4, other mechanisms, such as the free radical scavenging and anti-oxidant properties of the extracts, might also be involved [53]. In the future, isolation and biological evaluation of the active compounds of Solidago virgaurea and investigation of the underlying mechanisms will be imperative.

CONCLUSION

In conclusion, this preliminary study demonstrated that the Solidago virgaurea extracts contain many bioactive phytochemicals and show hypoglycemic and anti-diabetic effects through various possible mechanisms. Considering multiple functions of these phytochemicals, it was not surprising that the Solidago virgaurea could exert the actions against multiple therapeutic targets of diabetes. Our findings suggested that the methanolic extract of Solidago virgaurea might have great potential as a hypoglycemic agent for treating diabetes.

LIST OF ABBREVIATIONS

AGEs

Advanced Glycation end Products

BSA

Bovine Serum Albumin

GLUT4

Glucose Transporter Type 4

PTP1B

Protein Tyrosine Phosphatase 1B