Advances in Pharmacological Research of Psidium Guajava L. Leaves in Resisting Diabetes and Its Complications

Weibo DAI, Weiwen PENG, Gengting DONG, Leyu LI

Zhongshan Hospital of Traditional Chinese Medicine, Affiliated to Guangzhou University of Chinese Medicine, Zhongshan 528401, China

Abstract Modern pharmacological studies have shown that Psidium Guajava L. leaves have a variety of pharmacological effects, such as anti-inflammation, anti-oxidation, anti-tumor, reducing blood lipid, and reducing blood sugar. In recent years, the effect of Psidium Guajava L. leaves on diabetes and its complications has become the focus of research. This paper reviews the pharmacological research trends of effect of Psidium Guajava L. leaves on diabetes and its complications, in order to provide reference for the application and research and development of this plant.

Key words Psidium Guajava L. leaves, Diabetes, Pharmacology, Research progress

1 Introduction

PsidiumGuajavaL., commonly known as Bazi, Jishiguo, Bale, Hongxinguo, Labaguo and so on, is a small evergreen tree or shrub of the Myrtaceae family. Native to tropical America and Southeast Asia, it is widely distributed and cultivated in tropical and subtropical regions.P.GuajavaL. leaf extract not only has the effects of anti-diarrhea, antioxidation, scavenging free radicals and anti-prostate cancer, but also has a variety of pharmacological effects such as cardioprotection, lowering blood sugar and blood pressure. It has a long history of medicinal use in Peru, Mexico, the Caribbean, Africa and Asia.

In the 1970s, the Diabetes Research Group of Guangxi Medical College conducted a clinical study on the treatment of diabetes withP.GuajavaL. leaves[2-5]. A total of 284 subjects were enrolled, which confirmed the hypoglycemic effect ofP.GuajavaL. leaves on patients with type 2 diabetes from the point of view of clinical trials. However, limited to the research level and conditions at that time, a systematic and comprehensive study on the material basis and mechanism of pharmacodynamics could not be carried out. On the basis of long-term medical practice and clinical observation,P.GuajavaL. leaf prescribed preparation "Xiaoke Jiangtang capsule" has been included in Volume 15 of thePharmaceuticalStandardTraditionalChineseMedicinePrescriptionoftheMinistryofHealth, which is used for the clinical prevention and treatment of diabetes and its complications, however, the specific active components and action mechanism have not been determined.

The complexity of chemical composition ofP.GuajavaL. leaves determines its unique advantages of multi-level anti-diabetes. One of the key issues to scientifically clarify the anti-diabetic mechanism ofP.GuajavaL. leaves is to clarify the specific composition and structure of its active components, and another key problem is to clarify the molecular mechanism and pathway of its action. This paper reviews the research progress on the improvement of blood sugar and complications ofP.GuajavaL. leaves in recent years, in order to provide some reference for the development of this medicinal plant.

2 Pharmacological mechanism in the treatment of diabetes

2.1 Improving the function of islet cells

2.1.1Improving insulin resistance and enhancing islet function. The mechanism of insulin resistance (IR) is complex, and the rapid degradation of insulin is one of the reasons. Insulin-degrading enzyme (IDE) is a kind of metalloproteinase that exists in cells, and its main function is to degrade insulin and inactivate it. After intragastric administration ofP.GuajavaL. leaf decoction to streptozotocin (STZ) diabetic rats for 8 weeks, Cui Rongjunetal.[7]found that its effect on reducing fasting blood glucose was similar to that of metformin, a positive control drug. Through the investigation of various indexes of insulin, it was found thatP.GuajavaL. leaf decoction could significantly reduce the gene expression of IDE, increase insulin sensitivity index, reduce insulin resistance, up-regulate the expression of pancreaticoduodenal homologous gene (PDX-1), stimulate the proliferation and differentiation of islet β-cells, and increase insulin secretion.

In recent years, another research hotspot related to IR is protein tyrosine phosphokinase 1B (PTP1B). PTP1B plays a negative role in the upstream signal transduction chain of insulin, which can dephosphorylate protein tyrosine, thus blocking the signal transduction of insulin binding to receptor and leading to insulin resistance. Zhangetal.[8]conducted many studies of biochemical genes and small molecules, which showed that PTP1B was an important target for the treatment of diabetes and obesity. Ohetal.[9]injected 10 mg/kg methanol extract ofP.GuajavaL. intraperitoneally into 1-month-old and 3-month-old Leprdb/Leprdb mice, which could significantly reduce blood sugar in mice. Through screening the methanol extract and its extracts with inhibitory effects on PTP1B, it was found that all of them had certain inhibitory activity, so it was speculated that the hypoglycemic mechanism was at least partly due to the inhibition of PTP1B activity.

Li Xiucunetal.[10]found that total triterpenes ofP.GuajavaL. leaves could significantly increase the viability and inhibit the differentiation of 3T3-L1 preadipocytes, significantly promote glucose consumption of insulin resistant adipocytes, and significantly inhibit the production of free fatty acids. Total triterpenes (0.3 and 3 μg/L) ofP.GuajavaL. leaves significantly increased the secretion of adiponectin and inhibited the secretion of TNF-α in IR adipocytes. Total triterpenes (3 μg/L) ofP.GuajavaL. leaves significantly inhibited the secretion of resistin, but had no significant effect on the secretion of leptin; significantly down-regulated the mRNA expression of proteintyrosine phosphatase-1B (PTP1B) in IR adipocytes; significantly up-regulated the level of p-IRS-1/IRS-1; significantly up-regulated the protein level of p-Akt/Akt. This suggested that total triterpenes ofP.GuajavaL. leaves could significantly improve the IR of 3T3-L1 adipocytes, which may be related to the down-regulation of PTP1B mRNA expression and up-regulation of p-IRS-1/IRS-1 and p-Akt/Akt protein levels in IR adipocytes.

The studies of Yan Xiaoguangetal.[11]showed that 50 μg/mL total flavonoids ofP.GuajavaL. leaves could promote the proliferation of islet β in HIT-T15 under high glucose, insulin secretion, insulin receptor mRNA expression and insulin receptor substrate 1 protein expression. The results showed that total flavonoids ofP.GuajavaL. leaves could promote insulin secretion of HIT-T15 islet β cells in high glucose environment, which may be related to the increase of insulin receptor mRNA expression and insulin receptor substrate (IRS-1) protein expression.

2.1.2Protecting islet function and improving insulin sensitivity. Wang Boetal.[12]found that compared with the diabetic model control group, the diabetic mice in theP.GuajavaL. leaf water extract group fasted for 3 h, the fasting blood glucose level decreased by 33.4%, and the glucose tolerance was significantly improved. The pathological changes such as the reduction of islet volume, irregular shape and β cell degeneration were significantly alleviated, the number of β cells increased, and the expression of insulin per unit area increased significantly. The level of glutathione in liver tissue increased significantly, which indicated that the water extract ofP.GuajavaL. leaves could protect the islets of diabetic mice and reduce the damage of the structure and function of islet β cells in mice. Lin Juannaetal.[13]found that ursolic acid, a triterpenoid fromP.GuajavaL. leaves, could significantly promote the proliferation and differentiation of 3T3-L1 preadipocytes, significantly increase glucose consumption of insulin resistant adipocytes, significantly reduce the production of free fatty acids and promote adiponectin secretion in insulin resistant adipocytes, and up-regulate the expression of PPARγ protein, but had no significant effect on PTP1B protein expression. This suggested that ursolic acid could promote the proliferation and differentiation of 3T3-L1 preadipocytes, increase glucose uptake of adipocytes, inhibit the production of free fatty acids, promote adiponectin secretion, and improve insulin resistance of adipocytes. The mechanism may be related to the up-regulation of PPARγ protein expression and insulin sensitivity. Li Leyuetal.[14]found that compound preparation ofP.GuajavaL. could significantly increase body weight, decrease fasting blood glucose (FBG) and fasting serum insulin content (FINS), and increase insulin sensitivity index (ISI) in type 2 diabetic rats with insulin resistance, indicating that compound preparation ofP.GuajavaL. could improve hyperglycemia, hyperinsulinemia and insulin sensitivity in type 2 diabetic rats with insulin resistance.

2.1.3Regulating islet β-cell dedifferentiation. Su Tongetal.[15]found thatP.GuajavaL. leaf (8 g/kg) could significantly reduce the levels of fasting blood glucose and fasting insulin in Zucker diabetic obese rats. There were 9 kinds of differential expression of microRNA in pancreas (7 up-regulated and 2 down-regulated), among which the expression of miR-361-5p was significantly up-regulated. The results indicated that the extract ofP.GuajavaL. affected the expression of microRNA in rat pancreas, and might regulate the dedifferentiation of islet β cells by regulating the expression of miR-361-5p, thus lowering blood glucose.

2.1.4Promoting islet cell regeneration. Studies by Li Jieetal.[16]showed that total flavonoids ofP.GuajavaL. leaves could significantly increase the expression of PDX-1, Ngn3, Nkx6.1 mRNA in the pancreas of streptozotocin diabetic mice and promote islet regeneration in diabetic mice. Studies by Guo Shengnanetal.[17]showed that total flavonoids ofP.GuajavaL. leaves could significantly reduce blood glucose and increase the number of islets in diabetic mice, significantly increase the expression of GK, GLUT2, IGF-1, IRS-1 and IRS-2 protein, and decrease the expression of GKRP protein. It suggested that the total flavonoids ofP.GuajavaL. leaves could significantly reduce the level of blood glucose in STZ diabetic mice, which may be related to the enhancement of liver GK, GLUT2, IGF-1, IRS-1, IRS-1 expression, the decrease of liver GKRP expression and the promotion of islet regeneration.

2.1.5Inhibiting islet cell apoptosis. Qian Luetal.[18]found thatP.GuajavaL. leaf triterpenes could improve the islet cell injury induced by STZ in diabetic rats, reduce the apoptosis rate of islet cells, increase the relative viability of islet cells, and increase the protein expression levels of AKT, ERK, Caspase-3 and Bcl-2 in islet cells, indicating thatP.GuajavaL. leaf triterpenes could reduce blood glucose in diabetic rats, which may be related to the up-regulation of PI3K/Akt pathway to regulate apoptosis and inhibit islet cell apoptosis.

2.2 Inhibiting α-amylase and α-glucosidaseInhibition of α-glucosidase and α-amylase carbohydrate hydrolase can slow down the absorption of glucose and reduce postprandial hyperglycemia, which is one of the ways to treat diabetes. The aqueous extract ofP.GuajavaL. leaves was used to test the α-glucosidase inhibition of streptozotocin-induced diabetes in Kunming mice, and the inhibition rate reached 80%. Based on the analysis of the double reciprocal kinetic curve of the inhibitory effect ofP.GuajavaL. leaf water extract on sucrase and maltase, it is inferred that the inhibitory effect ofP.GuajavaL. leaf water extract on intestinal mucosal α-glucosidase in diabetic mice is competitive and non-competitive, and the inhibitory effect is related to the content of extract and catalytic substrate, that is, ifP.GuajavaL. leaf water extract is used to control diabetic blood glucose and the dosage ofP.GuajavaL. leaf water extract is increased while reducing the intake of carbohydrates can enhance the inhibitory effect of the extract on α-glucosidase[19].

2.3 Promoting the synthesis of liver glycogenShenetal.[20]evaluated the effects of aqueous and alcohol extracts ofP.GuajavaL. leaves on hyperglycemia and blood glucose metabolism in type 2 diabetes induced by streptozotocin and nicotinamide. The levels of plasma glucose and insulin in type 2 diabetic mice were measured by oral glucose tolerance test. The results showed that the activity of hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase, which were the most important rate-limiting enzymes in liver glycogen in theP.GuajavaL. leaf water extract treatment group, was higher than that in the diabetic group. In the alcohol extract treatment group, only the activity of hexokinase and phosphofructokinase could be improved. Therefore, it was considered thatP.GuajavaL. leaf extract could increase the activity of hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase in glycolysis pathway and pentose-phosphate shunt, resulting in the decrease of blood glucose in diabetic rats. At present, related studies have shown that polyphenolic flavonoids play a hypoglycemic effect through this mechanism. Cai Danzhaoetal.[21]found that the muscle glycogen content of rats treated with total flavonoids ofP.GuajavaL. leaves increased significantly, indicating that flavonoids could promote glycogen synthesis and improve the disorder of glucose metabolism in diabetic rats. Chengetal.[22]examined the effect ofP.GuajavaL. leaf water extract on glucose uptake by cloned rat hepatocytes and looked for effective components. The hypoglycemic effects of these components were evaluated by the absorption of 2-[1-14C] deoxy-D-glucose by cloned rat hepatocytes. The results showed that phenolic compounds were the main components ofP.GuajavaL. leaf extract, and the highly polar components could increase the glucose absorption of cloned rat hepatocytes, and quercetin was the main effective constituent. It was believed that quercetin promoted glucose uptake by hepatocytes. Wang Hongetal.[23]found that the total flavonoids ofP.GuajavaL. leaves significantly improved the blood glucose, insulin index and pathological changes of type 2 diabetic mellitus (T2DM) mice, and significantly decreased the protein expression levels of ERRγ and CREBH and the mRNA expression levels of PGC1α and TORC2 in liver tissue. This showed that the total flavonoids ofP.GuajavaL. leaves had obvious hypoglycemic and protective effects on liver and pancreas in T2DM model mice, and the mechanism of hypoglycemic effect may be related to the inhibition of ERRγ/CREBH signal pathway in liver. Duan Yingetal.[24]found that after 4 weeks of treatment,P.GuajavaL. leaf extract (2 g/kg) significantly decreased insulin level, improved glucose tolerance and HOMA-IR index, down-regulated TNF-α gene expression in adipose tissue, up-regulated PPAR-γ gene expression in adipose tissue, and up-regulated liver AKT protein expression, but had no significant effect on body mass, serum TG, TC, LDL-C and FFA in obese KKay mice. It suggested thatP.GuajavaL. leaf extract could affect the expression of AKT protein in liver glucose metabolism and improve glucose tolerance and insulin resistance by regulating the expression of TNF-α and PPAR-γ genes in adipose tissue.

2.4 Regulating lipid metabolismDai Peietal.[25]found thatP.GuajavaL. leaf extract could significantly reduce the body weight, blood glucose and low density lipoprotein of diabetic db/db mice, increase the expression of PGC-1α and FNDC5 protein in myoblasts, and increase the expression of Prdm16 protein in SVF cells in adipose tissue. This showed thatP.GuajavaL. leaf extract could regulate lipid metabolism of diabetes by promoting the differentiation of white adipocytes into brown adipocytes and increasing the level of brown fat. Liang Fanetal.[26]found thatP.GuajavaL. leaf extract could significantly reduce the body weight, blood glucose and insulin levels of diabetic db/db mice, and increase the expression of AMPK-α, PGC-1α, FNDC5 protein in skeletal muscle and PGC-1α and UCP-1 protein in adipose tissue of diabetic mice. It suggested thatP.GuajavaL. leaf extract could induce browning of white adipose tissue by enhancing AMPK-PGC-1α-FNDC5 signal transduction pathway in skeletal muscle, so as to reduce blood glucose and body weight. Zhang Yuyingetal.[27]found thatP.GuajavaL. could significantly reduce fasting blood glucose, improve glucose tolerance in diabetic mice, decrease the levels of TC, TG and LDL-C, increase HDL-C and decrease liver index in hyperlipidemic mice.

3 Pharmacological mechanism in the treatment of diabetic complications

Diabetic complications are common chronic complications, such as foot disease (foot gangrene, amputation), nephropathy (renal failure), ophthalmopathy (blurring, blindness), encephalopathy (cerebrovascular disease), and heart disease. They are the most common complications of diabetes, bringing a lot of inconvenience to people’s life.

P.GuajavaL. leaf extract not only had the effect of improving diabetes, but also had a significant alleviating effect on diabetic complications.

3.1 Improving diabetic nephropathyThe studies of Ge Wenjunetal.[28]showed that total triterpenes ofP.GuajavaL. leaves could significantly reduce FBG, 24-h urinary protein, BUN and Scr, reduce the expression of mTOR, S6K1 and Nephrinm RNA in renal tissue, and decrease the average protein water level of p-mTOR/mTOR and p-S6K1/S6K1 in renal tissue of rats with diabetic nephropathy (DN). Pathologically, it could significantly improve pathological injuries such as glomerular basement membrane thickening, irregular cyst outline, uneven cell distribution and steatosis, renal tubule atrophy and collapse, irregular wall cells and subcellular structural changes. This suggested that total triterpenes ofP.GuajavaL. leaves could alleviate the pathological changes of renal tissue and enhance renal function in DN rats. The mechanism may work by inhibiting mTOR/S6K1 pathway, reducing the expression of mTOR, S6K1 and the level of pmTOR/mTOR, p-S6K1/S6K1, and up-regulating the expression of Beclin1, LC3 and Nephrin. The studies of Gao Feietal.[29]showed that total flavonoids ofP.GuajavaL. leaves (200 mg/kg) could significantly reduce blood sugar (BS), blood urea nitrogen (BUN), urinary albumin/creatinine ratio (ACR) and serum creatinine (SCr), and increase the expression of Nrf2 and HO-1 protein in diabetic nephropathy rats, indicating thatP.GuajavaL. leaf flavonoids had a certain therapeutic effect on diabetic nephropathy in rats. The mechanism may be related to the decrease of blood glucose and the activation of Nrf2/HO-1 signal pathway. Rao Jiaoyuetal.[30]found thatP.GuajavaL. leaf triterpenes had a good protective effect on renal function in rats with diabetic nephropathy, improved glucose and lipid metabolism, decreased renal index, 24-h urinary protein and pathological damage of renal tissue in rats with diabetic nephropathy, and significantly improved renal function in rats. It could down-regulate the phosphorylation level of NF-κB-p65 and IKKβ protein, down-regulate the expression of TNF-α and IL-6mRNA gene, and reduce the expression of IL-6 in renal tissue. The mechanism may be related to its inhibition of the activity of NF-κB pathway and the expression of inflammatory factors TNF-α and IL-6 mRNA. The studies of Duan Yingetal.[31]showed thatP.GuajavaL. leaf extract could significantly reduce body weight and blood glucose in db/db mice, significantly improve glomerular mesangial cell proliferation, juxtaglular organ proliferation and granular degeneration in db/db mice, and significantly down-regulate the expression of TGF-β1 protein in the kidney of db/db mice. This suggested thatP.GuajavaL. leaf extract could improve renal fibrosis in db/db mice by inhibiting the expression of TGF-β1 immunohistochemical protein in kidney tissue.

3.2 Improving retinal injury in diabetic ratsZhang Qiaoetal.[32]found that the total triterpenes ofP.GuajavaL. leaf interfered with streptozotocin-induced diabetic rats for 12 weeks, decreased the expression of glial fibrillary acidic protein (GFAP), NF-κB and TNF-α protein, and significantly increased the density of retinal ganglion cells (RGC). This suggested that total triterpenes ofP.GuajavaL. leaves had a protective effect on RGC damage in diabetic rats, and the mechanism may be related to lowering blood glucose, down-regulating the expression of GFAP in retina and inhibiting inflammation.

3.3 Improving gestational diabetesThe studies of Chen Yanetal.[33]showed thatP.GuajavaL. leaf water extract could significantly reduce fasting blood glucose, C-reactive protein (CRP) and tumor necrosis factor α (TNF-α) in gestational diabetic rats, indicating thatP.GuajavaL. leaf water extract could effectively improve the blood glucose level of gestational diabetic rats, and its mechanism may be related to the regulation of CRP and TNF-α levels.

3.4 Improving diabetic peripheral neuropathyThe studies of Ye Kaiheetal.[34]showed that total triterpenes ofP.GuajavaL. leaves (120 mg/kg) could significantly improve the neuropathy of sciatic nerve in diabetic rats, significantly inhibit the expression of mRNA of TNF-a, IL-6 and iNOS, and significantly down-regulate the expression of p-AKT/AKT, p-IKKα/IKKα and p-p65/p65. This suggested that total triterpenes of pomegranate leaves could significantly improve peripheral neuropathy in diabetic rats, and its mechanism may work through down-regulating the signal pathway mediated by NF-κB, thus reducing the production of inflammatory mediators.

3.5 Resisting protein glycosylationProtein glycosylation is a reaction between uronyl group and protein amino group, and it is a non-enzymatic glycosylation reaction which is closely related to diabetic complications and aging. The formation and accumulation of advanced glycation end products in human tissues will aggravate diabetes, so the supplement of antioxidants for protein modification inhibition is a therapeutic strategy for the treatment of diabetic complications.

Baeetal.[35]confirmed that quercetin was a good anti-glycosylation substance bothinvitroandinvivo, which could prevent β-cell damage caused by STZ and so on. Wuetal.[36]studied the inhibitory effect ofP.GuajavaL. leaves and compounds on glycosylation in albumin/glucose model systeminvitro. The results showed thatP.GuajavaL. leaf extract was a potential anti-glycosylation substance, and its inhibitory effect was better than that of aminoguanidine and green tea polyphenol 60. Phenolic compounds had a strong inhibitory effect on the end products produced by protein glycosylation, especially the inhibition rate of quercetin at 100 μg/mL was as high as 95%. Therefore,P.GuajavaL. leaf extract could effectively resist glycosylation and was of great value in preventing diabetic complications caused by glycosylation.

3.6 Inhibiting hypercoagulemiaThe hypercoagulable state of blood in patients with diabetes is related to hyperglycemia, which makes it easy to form thrombus in the blood vessels of patients with diabetes. Hsiehetal.[37-38]determined the anticoagulant activity ofP.GuajavaL. leaf extractinvitro. It was found thatP.GuajavaL. leaf extract could prolong clotting time and inhibit hypercoagulemia. Improving the hypercoagulable state of diabetic patients was helpful to controlling their blood sugar.

4 Conclusion

At present, the incidence of diabetes in China is increasing rapidly.P.GuajavaL. leaves are diverse in chemical constituents (especially flavonoids and phenols), having obvious hypoglycemic pharmacological effects. The active components of flavonoids and phenols inP.GuajavaL. leaves can be used as the leading compounds of potential antidiabetic drugs and have good application prospect and development value. In addition, the hypoglycemic activity of heteroterpenes inP.GuajavaL. leaves is also worthy of further research and development, which brings new opportunities and hopes for the treatment of diabetes. Some thorny problems still need to be further solved: (i) the mechanism of lowering blood sugar in part is not clear, or even just speculated; (ii) the action of the effective compound ofP.GuajavaL. leafinvivois complex, and the monomer and its chemical composition and structure of the active component have not been known; (iii) many experimental studies are still in the animal experimental stage, some areinvitroexperiments, and human trials are not enough, which need to be further studied.

To sum up, in the future work, we should further investigate the molecular structure and structure-activity relationship of the main effective components, study pharmacological effects and screen biological effects, find out more new chemical components, improve the research on other medicinal parts of the plant, prepare for further drug development, and strive to develop new hypoglycemic drugs and other innovative drugs fromP.GuajavaL. leaves.