Anti-Diabetic Effects of Edgeworthia chrysantha and Edgeworthia gardneri Flower Buds – An Ethnic Herbal Tea in China

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analgesic activities [2]. In western Hunan Province, people of Tujia ethnic group pick the EC flower buds, dry them and then soak them in hot water to make herbal tea for daily consumption to tranquilize the mind, remove obstruction and improve digestion [3].
(EG) distributes in areas at high altitude in eastern Tibet and northwest Yunnan Province. Its dried flower buds, named "Lu luohua" in Chinese, are marketed as precious Tibetan floral tea which is renowned for its broad pharmacological activities in treating diabetes mellitus, cardiovascular diseases and hyperlipidemia. As reported by Geng and colleagues, the water extract of EG flowers could inhibit-D-glucosidase in vitro and significantly lower blood glucose level of alloxan-induced diabetic mice [4]. Recently, it was further elucidated that the water extract of EG flowers considerably decreased blood glucose level and ameliorated insulin resistance in diabetic mice through the regulation on gut bacterial phylotypes [5]. Owing to its promising anti-diabetic effect, EG has been used as the major component of a patented auxiliary blood sugardecreasing composite in China since 2014 [6].
Many compounds have been isolated from EC and EG flowers [7][8][9][10]. They can be mainly grouped as coumarins, flavonoids, flavone glycosides and triterpenes. Among these compounds, some isolated from EG have been proven to possess anti-diabetic effects. Edgeworin and daphnoretin were evaluated for their effects on two critical enzymes for carbohydrates digestion and metabolism. Edgeworin displayed strong inhibitory activity against α-glucosidase, while daphnoretin exhibited potent inhibitory effect against both α-glucosidase and α-amylase [11]. Another compound, tiliroside,was identified as the major constituent of EG flowers. It showed a significant α-glucosidase inhibitory activity and acted as a competitive inhibitor. Moreover, oral administration of tiliroside at 300 mg/kg could significantly reduce the fasting and postprandial blood glucose levels of streptozotocin (STZ)-induced diabetic mice [12]. Besides tiliroside, quercetin was also found to be a promising anti-diabetic agent. Quercetin induced insulin secretion in MIN-6 cell line in a glucose-dependent manner. Treatment of diabetic mice with quercetin resulted in remarkable decrease in blood glucose level, increase in insulin secretion and protection of pancreatic islets [13].
As shown in Figure 1, the appearance of flowers and dried flower buds of EC and EG are very similar in shape, color and size. Therefore, people of Tujia ethnic group very often collect EC flower buds as a substitute for EG and they believe that EC can help in managing diabetes mellitus as well. Although there are several scientific papers reporting the anti-diabetic effect of EG both in vitro and in vivo [4,5,14,15], the effect of EC on basal or postprandial plasma glucose level is still unknown and needs to be investigated. Hence, in the present study, the hypoglycemic and antihyperglycemic effects of water extract of EC flower buds, in parallel with that of EG, were evaluated and compared in diabetic mice for the first time. Moreover, the chemical profiles of the two extracts were analyzed and the contents of those active anti-diabetic compounds (edgeworin, daphnoretin, tiliroside and quercetin) were quantified for comparison.

Materials and Methods
Dried flower buds of EC and EG were collected in Shaoyang were performed as previously reported with minor modifications [16]. Metformin (200 mg/kg) was used as a positive control.
In general, one person consumes 5 g of dried flower buds a day.
As the aforementioned extraction yield is about 20 %, one person intakes 1 g of extract powder daily. Human Equivalent Dose (HED) is 1 g/ 60 kg, i.e. 16.7 mg/kg. After normalization of body surface area [17], mouse dose is 16.7 x 37/3,i.e. 206 mg/kg. In the animal studies, 200 mg/kg was therefore used as the testing dose for both EC and EG aqueous extracts.

Results
The UPLC profiles of EC and EG aqueous extracts are shown in  Table 1. Effects of 14-day treatment with EC or EG aqueous extract on body weights and basal plasma glucose levels of overnight-fasted diabetic mice are shown in Figure 3. It was found that the body weights of all groups of mice increased gradually during the course of experiment ( Figure 3A). However, weight gain of mice in EG group was the least. On average, their weight increased by 7.7 % on Day 15 when compared with that on Day 1 whilst other groups, including water, EC and metformin groups, had around 17 % increase in body weight. As shown in Figure 3B, the mice of EC and EG groups had lower plasma glucose levels than the water (control) group on Day 8. However, the effect was not statistically significant and the trend of decrease disappeared on Day 15. It implied that EC or EG aqueous extract at 200 mg/kg did not exert hypoglycemic effect in our current experiment setting.   Data are mean ± SEM (n = 6-13). No significant difference was observed among groups on the same experiment day.    Figure 4(A). Data are mean ± SEM (n = 9-14). *p<0.05, **p<0.01 and ***p<0.001 when compared with water (control) group.

Discussion
As a substitute of EG, flower buds of EC are always used by people of Tujia ethnic group for the management of diabetes mellitus. In the present study, the chemical composition and anti-diabetic effect of EC aqueous extract were investigated and compared with that of EG.
In the hypoglycemic animal experiment, the weight gain of mice in EG group was the least ( Figure 3A). Moreover, 4 out of 10 mice in EG group died before the end of experiment (data not shown). Taking these pieces of information together, the dosage of EG at 200 mg/ kg might be too high for diabetic (+db/+db) mice. However, Geng and colleagues reported that single intragastric administration of EG aqueous extract at 1000, 2500 and 5000 mg/kg did not cause any acute toxicity to normal mice [4]. The inconsistency between our findings and the reported ones might be due to the variations in animal strains, animal conditions and dosing intervention. On the other hand, continuous treatment with EC at 200 mg/kg for 14 days seemed to be safe to the animals.
It was reported that intragastric application of aqueous extract of EG at 1000, 2000 and 3000 mg/kg for 4 weeks could lower fasting blood glucose of high-fat diet-and STZ-induced diabetic mice [18]. However, the hypoglycemic effect of EG at lower dosage (200 mg/kg) for shorter treatment duration (14 days) could not be demonstrated in our study using genetically diabetic mice. Similar to EG, EC did not exhibit any promising effect in lowering basal blood glucose level either ( Figure 3B).
According to traditional practice, floral tea is usually prepared by soaking flower buds in hot water. Under this extraction condition, edgeworin and quercetin are not available for consumption as reflected by the fact that they were not detected in EC or EG aqueous extract (Table 1). Even though they have been reported to possess anti-diabetic effects [11,13], edgeworin and quercetin were unlikely to contribute to the activity of EC or EG aqueous extract.
Tiliroside was identified as the major constituent of EG flowers [12]. As determined using UPLC, EC aquoues extract contained 1.311 % of tiliroside, which was double the amount in EG aqueous extract (0.660 %) ( Table 1). It has been reported that tiliroside at a dose of 300 mg/kg could significantly reduce the fasting and postprandial blood glucose levels of STZ-induced diabetic mice [12]. However, tiliroside might not have contributed a lot to the antihyperglycemic effect of EC or EG (Figure 4) because its actual amount in 200 mg/kg of EC and EG aqueous extracts were only 2.6 mg/kg and 1.3 mg/kg, respectively, which were much lower than the reported active dose (300 mg/kg). Therefore, the in vivo antidiabetic effect of EC aqueous extract was not significantly different from that of EG aqueous extract, despite containing higher tiliroside content.

Conclusion
In conclusion, the results of the present study showed for the first time the mild antihyperglycemic effect of EC, supporting its traditional usage by ethnic people in China. At tested dosage (200 mg/kg, according to traditional human use in making tea), EC and EG exhibited similar potency. Although EG is more common and commercially available as products, our findings suggested that EC can also be considered as an alternative of EG in terms of diabetes management.