Hypoglycaemic and hypolipidemic activities of total flavonoids from Nymphaea candida flowers on diabetic mice

Abstract This study aimed to investigate the hypoglycaemic and hypolipidemic activities of total flavonoids from Nymphaea candida (NCTF). The result showed that NCTF could significantly ameliorate various indicators such as FBG, OGTT, TC, TG, LDL-C, and HDL-C in ALX-induced diabetic mice compared with the model group. Meanwhile, in the therapeutical effect study of NCTF on high-fat and high-sugar diets combined with STZ-induced T2DM mice, all parameters including RBG, INS, and INSR related to diabetes were significantly improved by NCTF as well as the serum ALT, AST, ALP, CR, MDA, and IL-6 activities. NCTF could significantly increase the expression levels of SOD and PPAR-γ in T2DM. Pathological observation showed that NCTF could improve the damage to pancreatic and liver tissues in T2DM mice. In conclusion, NCTF has better hypoglycaemic and hypolipidemic effects, and its mechanism may be related to its antioxidant, PPAR-γ regulation, and inhibiting inflammatory cytokine expression. Graphical Abstract


Introduction
Waterlilies (plants in Nymphaea) with more than 70 species are widely distributed all over the world, and all show significant ornamental and medicinal value, of which some waterlilies can be used to prevent diabetes and its complications (Wiersema 2001;ishrat et al. 2021).For example, N. stellata is used in South asia to treat diabetes, and its extract exhibited hypoglycaemic and hypolipidemic effects on alloxan (aLX)-induced diabetic rats as well as α-glycosidase inhibiting activity (dhanabal et al. 2007;Huang et al. 2010); N. rubra extract could reverse insulin resistance by inhibiting c-Jun nH2-terminal kinase and nF-κB activities (Gautam et al. 2014); N. nouchali extract could activate the ppaR-γ signalling pathway, increase the expression of GLut4, and then enhance insulin sensitivity in tissues, promoting fat metabolism and glucose consumption and utilisation (parimala et al. 2015).these studies indicated that there are constituents with significant hypoglycaemic effects in the waterlily's extracts.
as a member of the genus Nymphaea, N. candida (Snow-white waterlily) is mainly distributed in the Central asia region, and its flower buds have multiple efficacies such as reducing heat, nourishing the liver, relieving inflammation, moistening throat and thirst (Zhao et al. 2011).previous studies showed that N. candida has a variety of biological activities including anti-oxidation, anti-hepatitis, and neuroprotection (Wang et al. 2021;Zhao et al. 2021).Flavonoids (nicotiflorin, and astragalin, etc) are mainly the characteristic components of this plant (Zhao et al. 2008).However, systematic studies on the hypoglycaemic and hypolipidemic effect of flavonoids from N. candida and its mechanism are rarely reported.therefore, this study aimed to investigate the prevention and therapeutic effect of total flavonoids from N. candida (nCtF) on the diabetes mice and its associated diseases to provide data reference for the development and utilisation of this plant.

Result and discussion
as shown in Figure S1, the characteristic compounds of nCtF were isostrictiniin, nicotiflorin, and astragalin, their contents in nCtF were 2.21%, 11.63% and 4.68% respectively.two diabetic animal models (aLX-induced, high-sugar high-fat combined streptozotocin (StZ)-induced) were used to evaluate the hypoglycaemic and hypolipidemic activities of nCtF.
aLX is a β-cytotoxin that can destroy β cells in the pancreas, and resulting in a reduction in endogenous insulin release and a rise in the body's blood sugar concentration, so this method is commonly used to evaluate the hypoglycaemic activities of natural products.type ii diabetes (t2dm) is a disease caused by various factors such as genetics, diet, age, and pregnancy.this disease can not only damage islet cells but also produce insulin resistance, glucose and lipid metabolism disorder.High-sugar and high-fat diets can induce insulin resistance in mice.StZ can selectively destroy islet β cells in the pancreatic tissue of animals, leading to insufficient insulin secretion in the body and causing hyperglycaemia.therefore, the mice are injected by StZ after being fed high-sugar and high-fat feed for a period of time and can produce a t2dm model with steady insulin resistance.

Effects of NCTF on ALX-induced diabetic mice
the diabetic model in mice was established by aLX with intraperitoneal injection according to the previous report (tang et al. 2020).the experimental results showed that the fasting blood glucose (FBG) levels in aLX-induced diabetic mice were ≥11.1 mmol/L, and various symptoms as follows: the food intake, water intake, and urine output of mice significantly added; body weight in mice appeared significant loss, spleen and kidney index significantly increased (table S2-1).these results indicated that the model of diabetic mice was successfully established.nCtF (100, 200 mg/kg) could significantly reduce FBG, improve glucose tolerance, and effectively alleviate the "three more" symptoms in diabetic mice (p < 0.05, table S2-2, S2-3).as a metabolic disease, diabetic patients are often accompanied by varying degrees of elevated blood lipids or lipid metabolism disorders (Yan et al. 2019). in this study, the serum tG, tC and LdL-C levels were significantly increased and the HdL-C levels were significantly decreased in diabetic mice in model group (p < 0.05), while the levels of tC, tG and LdL-C were significantly decreased and the levels of HdL-C were increased in nCtF (100, 200 mg/kg) intervention group (p < 0.05, Figure S2-1).moreover, nCtF group (50,100, 200 mg/kg) could also markedly ameliorated CR level in aLX-induced diabetic mice (p < 0.05, Figure S2-1).Studies have shown that nCtF has better hypoglycaemic, hypolipidemic and renoprotective effects on aLX-induced diabetic mice.

Effects of NCTF on high-sugar and high-fat combined STZ induced T2DM mice
in this study, the mice were intraperitoneally injected with StZ (60 mg/kg, twice, every three days between) after being fed high-sugar and high-fat feed for 40 days (tang et al. 2020).Seven days after treatment with StZ, the random blood glucose (RBG) in mice was more than 16.7 mmol/L, and indicating that the t2dm model was successfully established.Subsequently, nCtF (50, 100, 200 mg/kg) and metformin (mEt, 260 mg/kg) were administered by gavage at 28 days to treatment of t2dm in mice.the experimental results showed that t2dm mice had less activity, less reaction, sparse hair, and "three more" phenomenon (polydipsia, polyuria, poly food) compared with normal mice.after treatment with nCtF and mEt, the mental status of t2dm mice were obviously improved.nCtF (200 mg/kg) also significantly improved the spleen index and kidney index (p < 0.05, Figure S3-1).However, there was no significant difference in body weight' change between various groups (table S3-1). it was found that nCtF could significantly reduce the blood glucose and insulin (FinS) levels of t2dm mice (p < 0.05), improve insulin resistance (iRi) (p < 0.05,) and effectively alleviate the symptoms of "three more" (table S3-2, Figure S3-2).Histopathological observation showed that nCtF could significantly improve pancreatic tissue injury in t2dm mice compared with the model group, such as the pancreatic islet atrophy reduced, vacuolisation decreased, the structure clearer and the number of islets increased (Figure S3-3).these results indicate that nCtF not only has the effect of lowering blood glucose but also has a certain alleviating effect on pancreatic pathological lesions in t2dm mice.
Long-term insulin resistance can cause adipocytes to produce a large amount of free fatty acids (FFa), and lead to dyslipidemia.Continuous dyslipidemia may further induce non-alcoholic fatty liver and other complications. in the experiment, the serum tG, tC, and LdL-C levels in t2dm mice were significantly increased (p < 0.05), indicating the disorder of glucose and lipid metabolism.these indicators in t2dm mice were significantly decreased by nCtF administration through improving insulin resistance (p < 0.05, Figure S3-4).ppaR-γ is a key transcription factor involved in lipid metabolism, which plays a crucial role in regulating insulin resistance, glucose and lipid metabolism.Compared with the model group, nCtF (200 mg/kg) could significantly increase the ppaR-γ protein expression in the liver of t2dm mice (p < 0.05, Figure S3-5), and indicate that nCtF had a protective effect on liver injury in t2dm mice, which may be related to the up-regulation of the expression of ppaR-γ in the liver.
in addition, disorders of glucose and lipid metabolism can induce non-alcoholic fatty liver disease, which can subsequently lead to liver damage.in this study, compared with the normal group, liver homogenate aLt, aSt, and aLp levels in t2dm mice were significantly increased (Figure S3-6), pathological change of hepatocytes in t2dm mice was also observed through a microscope, and various injuries were found such as glycogen degeneration and loose cytoplasm, lipid vacuolar degeneration, basophilic change, punctate necrosis foci, and the lobular hepatocytes hypertrophy.the results indicated that the liver of t2dm mice was damaged.after treatment with nCtF, liver homogenate aLt, aSt, and aLp levels int2dm mice were remarkably decreased (p < 0.05, Figure S3-6), and pathological changes of liver tissue were significantly improved (p < 0.05, Figure S3-7).moreover, nCtF (100, 200 mg/kg) could remarkably decrease the elevator mda levels, and improve Sod activities compared to the model group (p < 0.05, Figure S3-8).interleukin-6 (iL-6) plays an important role in the development of liver injury in t2dm mice.nCtF (50, 100, 200 mg/kg) could remarkedly decrease the elevatory iL-6 levels by t2dm (p < 0.05, Figure S3-9).t2dm leads to renal disease, and renal injury can reduce the excretion of CR and promote its increment in blood concentration.the experimental results showed that nCtF (200 mg/kg) could reduce the CR value and renal index of t2dm mice (p < 0.05, Figure S3-10), indicating that nCtF had the ability to protect the kidney function of t2dm mice.

Conclusion
through intervention in two diabetic animal models, nCtF showed a significant effect of lowering blood glucose, decreasing serum lipids, improving insulin resistance as well as alleviating liver injury and kidney injury.therefore, nCtF is expected to be a potential therapeutic drug for diabetes and its complications, but its mechanism needs to be further studied.

Disclosure statement
no potential conflict of interest was reported by the author(s).