Plantago major L. Extracts Reduce Blood Glucose in Streptozotocin-Induced Diabetic Mice

Plantago major L. (P. major L.) is a perennial plant belonging to the family Plantaginaceae. It has been used as a folk remedy for diabetes in Europe andAsia. However, the biologically active constituents responsible for the antidiabetic effects have not been reported. e objectives of this study aimed at determining the chemical components of Plantago major L. and evaluating the antidiabetic activity of the extracts using streptozotocin(STZ-) induced diabetic mice. In this study, the Swiss mice were fed a high-fat diet to gain weight before STZ injections to induce diabetic conditions. e STZ-induced diabetic mice were orally treated with P. major L. extracts. e blood glucose test results from the treated diabeticmice and nontreated diabeticmice were compared.We found that a 15-day treatment with EP6 extract from P.major L. at a dose of 400mg/kg could reduce the blood glucose level to the same level as a 15-day treatment with glucophage at a dose of 70mg/kg. e major chemical components and structural characterization of EP6 extract were also reported. AST (aspartate transferase) andALT (alanine aminotransferase) indicators of liver damage weremeasured in the treated and nontreated diabetic mice to give an overall view of the antidiabetic effect of P. major L. extracts.


Introduction
Diabetes mellitus, commonly known as diabetes, is a metabolic disorder causing high blood glucose and can lead to serious complications such as cardiovascular disease, stroke, chronic kidney disease, and nerve and vision damage [1].
ere are two types of diabetes: type 1 diabetes results from the pancreas's disability to make enough insulin due to the loss of beta cells. Patients who suffer from type 1 diabetes are subjected to life-long insulin supplement therapy and are required to monitor blood insulin level regularly; type 2 diabetes often happens in overweight individuals, starting with cells that stop responding to insulin properly and possibly progressing to insulin deficiency. Among the two conditions, type 2 diabetes is considered to be more manageable and reversible by treatments with insulin sensitizers of biguanides, such as metformin, phenformin, and buformin, or thiazolidinediones, such as rosiglitazone, pioglitazone, and troglitazone, or Lyn kinase activators, such as tolimidone. However, these drugs have been linked to increased risks of lactic acidosis and liver damage [2][3][4][5].
Nature has been a generous source of bioactives for medicinal chemistry. P. major L. is a perennial plant populated in Europe and Asia. It has been used by the locals for the treatment of diabetes and other illnesses. Studies of chemical compositions of P. major L. reveal that it contains flavonoids, terpenoids, and phenolic [6]. ose are phytochemicals that are produced by the plant through secondary metabolism and can possibly possess biological activities that serve as the plants' defense against competitors, predators, or pathogens. To our best knowledge, studies of the antidiabetic effect of P. major L. in type 2 diabetes treatment are limited. A few studies have shown that the extracts from P. major L. were effective in improving blood glucose in type 2 diabetic mice [7][8][9]. ese studies show that the antidiabetic activity test using STZ-induced diabetic mice at a dose of 1000 mg alcoholic extract/kg rat body weight of P. major L. reduced the blood glucose to a comparable level of the control group at the 5 th day until the 14 th day of the study; oral administration of P. major L. extract at the doses of 600 mg aqueous extract/kg rat b.w. for alloxan monohydrate-induced diabetic rat significantly decreases the blood glucose level and 500 mg methanol extract/kg body weight promotes glucose uptake in mice with efficient insulin-secreting pancreas. However, thorough isolation and chemical characterization of the extract components of P. major L. and the determination of bioactives responsible for antidiabetic effects have not been reported. us, we carried out a thorough extraction and then column chromatography isolation and structural determination of P. major L. extracts by NMR (nuclear magnetic resonance) spectroscopy. To test the glycemic control activity of P. major L. extracts, an in vivo experiment on mice was performed starting with preparing obese mice, inducing diabetes with STZ, followed by the treatment with P. major L. extracts. e antihyperglycemic effect was determined via a blood glucose test. Importantly, to identify any negative effects of the extracts on liver function and biosafety of the extract, liver lipid and enzymes profile, including cholesterol, TG (triglyceride), AST, and ALT were measured. Furthermore, liver enzymes are associated with the diabetic nonalcoholic liver fatty disease. Our studies show that P. major L. extracts were capable of reducing elevated liver enzymes in diabetic mice compared to the control group. Hence, this paper provides important information to fill the knowledge gaps in P. major L. antihyperglycemic activity research: chemical structural determination of the antihyperglycemic components of P. major L. and in vivo assessment of the safety of P. major L. in treatment of the hyperglycemic condition in mice.

Plant Source.
e plant sample was grown and harvested in September 2018 in Hoai Duc province, Vietnam. e plant was identified as P. major L. by Dr. Trieu Anh Trung, Faculty of Biology, Hanoi National University of Education. All parts of P. major L. plant were washed with water to remove soil and dried in the shade until drained. Fresh plant samples were dried at 40 degrees until complete dryness. e dry sample is ground into a powder using a homemade blender.

Extract Preparation and Compounds Isolation.
P. major L. (5 kg) was immersed in ethyl acetate at room temperature. After 7 days, the extract was collected, and the residue was repeatedly extracted two times. e combined extract was concentrated under vacuum to yield a solid EP (306 g). e extract was subjected to silica gel column chromatography eluting with a gradient system of n-hexane/ ethyl acetate (20 : 1 ⟶ 0 : 1) to provide 6 fractions (EP1-EP6). Fraction EP3 was further purified by silica gel column chromatography eluting with a gradient system of n-hexane/ acetone (20 : 1) to provide 4 fractions (EP3.1-EP3.4).

Animals and Induction of Diabetes in Obese Mice.
Swiss mice weighing 18-22 g were provided from the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, and were humanely treated in accordance with the Ministry of Health Guidelines for the Care and Use of Laboratory Animals. e protocols were approved by the Ethics Committee of VNU Pharmaceutical Chemistry Laboratory (permit number: A 2019-0033). Animals were kept 10 mice per group of a total of 8 groups and divided into two experiments: the high-fat diet-fed (HFD) group with 70 animals (groups 1-7) and the normal control with 10 animals (group NC). e animals were housed in clean metabolic cages placed in a well-ventilated house with an optimum condition (temperature: 25 ± 2°C; photoperiod: 12 h natural light and 12 h dark; and humidity: 45-50%). e cleaning of the cages was done on a daily basis. e HFD group were provided with a high-fat diet consisting of carbohydrate (40%), fat (30%), protein (20%), and other vitamins and minerals (5%). e NC mice were given a standardized diet consisting of protein carbohydrate (53%), protein (20%), and fat (6%). Body weight was recorded weekly. After 8 weeks, HFD mice show a significant increase in body weight (158%-204%) compared with NC mice (78%), as shown in Table 1.
Mice whose body weights were over 50 g in the HFD groups (cages 1-6) were injected with STZ (120 mg/kg bw).
e high-fat-fed NCF mice (average body weight 33.52 g) were added with physiological saline only. e blood glucose level in both STZ-induced diabetic group and control group was measured by the One Touch Ultra device before the STZ injection and at 48 h, 72 h, 5 days, 7 days, and 10 days after the injection [15,16]. e results are tabulated in Table 2.
After 10 days of STZ injection, mice with blood glucose levels >18 mmol/L were considered type 2 diabetic [17]. e diabetic animals were subjected to further treatment (Table 3). Diabetic mice in Cage 1, the normal control diabetic group, are only treated with saline solution. Diabetic mice in Cage 2: the positive control group, are treated with Glucophage (70 mg/kg). e diabetic mice in Cages 3, 4, 5, and 6 received 400 mg/kg of extractions EP, EP3, EP4, and EP6, accordingly.
Mice in those cages were given the treatments at exactly 8 a.m. daily. e blood glucose level in each cage was measured before and at 2 h, 4 h, 8 h, 3 days, and 15 days after the oral intake. e results are shown in Figure 3 and Table 4.

Determination of Blood Glucose Level and Biochemical
Parameters.
e venous blood of mice was collected from the tail for measuring the blood glucose levels using a glucose monitoring system (One Touch brand). Four groups from obese mice diabetic group treated with EP6 (Cage 6), obese mice diabetic with no treatment (Cage 1), high-fat-fed mice (NCf ), and normal mice (NC) were chosen, and their blood was collected and centrifuged at 3,000 rpm for 5 minutes. e separation serum was used for the biochemistry assay. e serum parameters: cholesterol level and triglycerides level, alanine aminotransferase level (ALT), and aspartate transaminase level (AST) were estimated using QuickDetect Total cholesterol/Triglyceride/AST/ALT (Rat) ELISA Kit (Ray Biotech). Consequently, these blood samples were centrifuged at a velocity of 3000 rpm for 15 minutes. Sera were then analyzed with commercially available kits (Sandwich method) according to the manufacturer's protocol by ELISA apparatus (Photometer 5010 V5 + , Robert Riele, Germany). Changes in color were checked at a wavelength of 450 nm. According to manufacture information, both the intra-assay and interassay coefficients of variation were below 12%.

Statistics.
Data are presented as mean ± SD. Statistical significance was assessed by group comparison with the use of one-way ANOVA followed by the Tukey-Kramer test. Significance was accepted at p < 0.05. All data were analyzed with the SPSS version 26 (64-bit edition) for Windows (SPSS Inc., Chicago, IL).    Table 1: Body weight of mice in the high-fat diet-fed group (HFD) and the normal control group (NC) group before and after 8-week diet.

Group
Week 0 (g)     Figure 3 and Table 4, the blood glucose levels of obese mice in the nontreated group Cage 1 (NCd) showed no decrease throughout the 15-day treatment but significantly decreased in the mice that were treated with Glucophage (Cage 2), up to 55.7% only after 3 days of treatment in comparison with the controls at the same time (p < 0.05). In the groups that were treated with P. major L. extracts EP (Cage 3), EP3 fraction (Cage 4), EP4 fraction (Cage 5), and EP 6 fraction (Cage 6) with a dose of 400 mg/kg, in general, we also observed a significant decrease in blood glucose level after 2 h, 4 h, 8 h, 3 days, and 15 days (p < 0.05). A substantial decrease was recorded in groups treated with extract EP4 (Cage 5) and EP 6 (Cage 6). With EP4 treatment (Cage 5), the blood glucose dropped quickly after the first few hours of oral intake and showed a 39.1% and 51.51% decrease compared to the controls (p < 0.01) after 3 days and 5 days, respectively. After 3 days of EP6 treatment (Cage 6), the blood glucose levels in this group dropped as low as 65.72% (p < 0.01) and stabilized during the rest of the experiment.

Determination of the Blood Glucose Concentration Using P. major L. Extracts in In Vivo Study. From
Obese mice have high levels of cholesterol and triglycerides. Based on Table 5, mice that received the treatment with EP6 extract (Cage 6) have a significantly higher level of cholesterol and triglycerides (2.00 mmol/L, 1.25 mmol/L) compared with those of the diabetic obese mice with no treatment (Cage 1-NCd) (1.22 mmol/L, 0.83 mmol/L) but lower than those of the obese mice (NCf ) (2.91 mmol/L, 1.99 mmol/L). High AST and ALT levels are reasonably sensitive indicators of liver damage. In the normal mice (NC) group, AST and ALT levels were 21.9 (U/L) and 11.85 (U/L), respectively. In the treated group (cage 6), those levels were 23.1 (U/L) and 26.5 (U/L) (AST, ALT). However, those levels were significantly lower than those of the diabetic group without treatment (Cage 1), 100.02 (U/L), and 99.59 (U/L) (AST, ALT).

Discussion
e STZ administration to damage the pancreas in obese mice led to an increase in their blood sugar levels. e condition accompanied by high consumption of food and water indicates signs of diabetes in mice [18,19]. In this study, the use of selective fractions and crude extract from P. major L. to control blood sugar levels of obese mice with type 2 diabetes showed positive results. Fraction EP6 showed the ability to reduce blood glucose in type 2 diabetes obese mice by 67.73% due to the presence of two flavonoid compounds: 7-O-methylapigenin (EPL7) and 7, 4′-Odimethylapigenin (EPL8).
is result was supported by previous research where the glycemic control effect of P. major L. extracts was contributed due to the presence of flavonoids compounds. e characteristic structures of these compounds contain aromatic rings with hydroxyl groups and are similar to standard flavonoid compounds used for antioxidants or inhibiting α-glucosidase activity, such as quercetin and resveratrol [20][21][22][23][24]. e efficacy of EP6 fraction is comparable to the result obtained from the treatment with Glucophage (a standard antihyperglycemic drug currently being used to treat type 2 diabetes patients) in the positive control group after 15 days. is demonstrates that P. major L. extracts can reduce the blood glucose level in type 2 diabetic mice. In an oral glucose tolerance test aiming at measuring the efficacy to eliminate excess glucose after an oral glycemic load [9], fasting mice were given a 500 mg/kg dose of P. major L. extract before the administration of glucose 1.25 g/kg orally. It was observed that there is a 27% reduction in blood glucose in diabetic mice given the extract at 120 minutes compared to the nontreated group. Unfortunately, the blood glucose levels were only measured at 30, 60, 90, and 120 minutes, so there is not enough information to conclude about the long-term antiglycemic effect of the P. major L. extract. e research, however, provided valuable results when a glucose test was carried out on normal mice. Even within the normal nondiabetic group, those given P. major L. extract have lowered blood glucose to normal ones without treatment (a 23% reduction) and this level is comparable to normal ones given glibenclamide, a commonly used prescription in diabetes mellitus type 2 (a 26% reduction compared to normal ones without treatment). is supports our result that extract from P. major L. could contain chemicals for reducing blood glucose in diabetes mellitus. In a thorough 14-day experiment on the antiglycemic effect of P. major L. extracts in mice [7], a dose of 1000 mg/kg was able to reduce blood glucose in diabetic mice by 47% compared to the nontreated group after 5-day treatment. is result was followed by a 43% reduction on the 5 th day and a 54% reduction on the 14 th day. In comparison, our P. major L. extract, used at a dose of 400 mg/kg, showed a 65% reduction in blood glucose of the diabetic mice compared to the nontreated group after 3 days of treatment and an almost 70% reduction ensuing 15-day treatment. Clearly, our extract shows improvement in the dosage used and time to take effect. We hypothesize that the oral intake of the extracts can enhance the capacity of β-cell to upregulate insulin synthesis or trigger the renewal of β-cell or preservation of pancreatic β-cell mass as indicated in other research [25,26]. is leads to improved glycemic control and cellular glucose uptake. Still, more quantitative investigation on the morphology of pancreatic islets needs to be done to check if islets were restored after using P. major L. extracts and led to the restoration of plasma glucose and insulin level in diabetic mice. As a consequence, serum insulin levels also need to be checked to confirm this effect. However, these experiments are beyond the scope of this paper. Meanwhile, we did assess biochemical parameters on diabetic mice after 15 days of treatment with EP6 fraction and compared with other groups of mice. ere was no improvement in cholesterol and triglycerides within the group given EP6 extract, so it is possible that EP6 extract had no effect on fat metabolism [27,28]. However, differences in AST and ALT levels among those groups are observed. Abnormal changes in AST and ALT in the blood are indicators of liver damage due to intoxication or pathology, which was reported in studies using STZ on mouse models leading to the damage of the pancreas and liver. [29,30]. e AST and ALT levels of the EP6-treated group were significantly lower than the nontreated diabetic mice (Cage 1), slightly less than the nontreated and nondiabetic obese mice (NCf ) and comparable to normal mice. us, it is possible that EP6 extract improved liver function, reducing elevated liver enzymes in diabetic mice to a similar level of AST in the control group (NC).

Conclusion
e present research indicates the potential of P. major L. to control the blood sugar of type 2 diabetes in obese mice at a lowered dose and shorter time to take effect compared to previous research. We have provided thorough isolation and bioactive structural determination of P. major L. extracts. e in vivo study also pointed out the efficacy of fraction EP6 in reducing blood glucose to a level comparable to that obtained from Glucophage treatment. Two components of EP6 fraction were also identified, 7-O-methylapigenin (EPL7) and 7, 4′-O-dimethylapigenin (EPL8). Fortunately, EP6 does not show any damage to liver functions as usually observed in other diabetic treatments. Further studies on the glycemic regulating mechanism of P. major L. extract via the release of pancreatic insulin should be considered by tracking the insulin level in the bloodstream and urine excretion.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest. Journal of Chemistry 7