Differential analysis of secondary metabolites between medicinal and edible Polygonatum sibiricum and study on active ingredients and mechanisms of action for improving insulin resistance
Insulin resistance (IR) generally refers to a decrease in the sensitivity of insulin target cells to quantitative concentrations of insulin at normal concentrations, resulting in impaired insulin promotion of glucose uptake and utilization, compensatory increase in insulin secretion, and decreased insulin sensitivity and impaired glucose utilization in tissues such as skeletal muscles, liver, and adipose tissue. Among them, IR is one of the important pathogenesis of type 2 diabetes, which runs through the whole process of type 2 diabetes. At present, the main drugs used to treat IR are insulin sensitizers, but there are side effects such as fractures, edema, and liver injury.

Polygonatum sibiricum Red. is a plant belonging to the Liliaceae family and the Polygonatum genus. It is one of the three types of Polygonatum included in the 2020 edition of the Chinese Pharmacopoeia. It is used as medicine with dried tubers and is a common medicinal and edible product. Due to its nodular and curved cylindrical shape resembling a chicken head, it is commonly known as chicken head Polygonatum sibiricum. It is widely distributed in Sichuan, Hunan, Jiangxi and other regions. It has a sweet and mild taste, is non-toxic, and has the effects of nourishing qi and yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is used to treat spleen and stomach qi deficiency, fatigue and weakness, internal heat and thirst. It mainly contains polysaccharides, saponins, flavonoids, alkaloids, anthraquinone and other ingredients. In clinical practice, Huangjing compound is widely used in the treatment of type 2 diabetes. For type 2 diabetes with deficiency of both qi and yin, it can alleviate the patients’ TCM syndromes, reduce blood sugar and lipids, and improve the function of pancreatic islets. According to literature reports, the mechanism by which Huangjing compound improves pancreatic function suggests that its active ingredients act on targets such as PI3K and STAT3, further affecting protein serine/threonine activity, PI3K-AKT signaling pathways, and exerting their effects; The mechanism of Huangjing monotherapy in improving pancreatic islet function suggests that flavonoids act on the postsynaptic membrane, altering biological processes such as cell sensitivity to drugs, neurotransmitter receptor activity, and regulating signaling pathways such as calcium ion and hormone resistance to improve insulin resistance.

At present, HPLC, LC-MS/MS, UPLC-Q-TOF-MS and other techniques are commonly used for qualitative and quantitative research on the chemical components of Polygonatum sibiricum. It has been reported that Polygonatum sibiricum contains flavonoids, alkaloids and other chemical components, but it is generally difficult to reflect all metabolic products contained in Polygonatum sibiricum due to the small number of detected components. In recent years, with the development of metabolomics technology, the widely targeted metabolomics technology based on UPLC-ESI-MS/MS has been widely used for the study of traditional Chinese medicine metabolites. Compared with traditional common chromatographic techniques, it has the advantages of high sensitivity, low cost, and high throughput. Due to the lack of reports on the differential analysis of secondary metabolites between medicinal and edible Polygonatum sibiricum, this study focuses on medicinal Polygonatum sibiricum Red and edible Polygonatum alternatirrhosum Hand. – Mzt. (commonly known as fruit Polygonatum) as research objects. For the first time, UHPLC-ESI-MS/MS widely targeted metabolomics technology was used to comprehensively detect their secondary metabolites, and principal component analysis (PCA), HCA clustering analysis, and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for comparative analysis of metabolites to screen for differential metabolites; Secondly, network pharmacology analysis was conducted on the top ten compounds with upregulated relative changes in metabolites between medicinal and edible Polygonatum sibiricum to improve insulin resistance. Molecular docking was performed on the active ingredients and key targets, and HepG2 cells were selected for cell experiment validation. On the one hand, we aim to comprehensively understand the differential metabolites between medicinal and edible Polygonatum sibiricum, laying the foundation for its quality evaluation. On the other hand, we plan to screen potential active ingredients for treating IR, providing a basis for elucidating the mechanism of action of Polygonatum sibiricum in treating IR.

 

This article analyzes the secondary metabolites of medicinal and edible Polygonatum sibiricum using metabolomics based on UHPLC-ESI-MS/MS technology. A total of 319 secondary metabolites were identified, including 86 phenolic acids, 96 flavonoids, 5 anthraquinones, 25 lignans and coumarins, 3 tannins, 51 alkaloids, 11 terpenes, 21 steroids, and 21 other metabolites. There is a significant difference in secondary metabolites between medicinal and edible Polygonatum sibiricum, with PCA and HCA showing significant differences. A total of 106 differential metabolites were screened in the comparison group between medicinal and edible Polygonatum sibiricum, including 85 up-regulated compounds and 21 down regulated compounds. Medicinal Polygonatum sibiricum has significant advantages in the quantity and relative content of secondary metabolites. To further compare the changes in secondary metabolites between medicinal and edible Polygonatum sibiricum, the differential metabolites were annotated by KEGG database. The KEGG classification and enrichment results showed that medicinal Polygonatum sibiricum was significantly enriched in the metabolic pathways of flavonoids, flavonols, isoflavones, and alkaloids, which may be related to the formation of medicinal Polygonatum sibiricum quality. According to literature reports, plants produce a wide variety of secondary metabolites during their growth process due to environmental stress or adversity. These metabolites often have specific biological functions such as drought resistance, disease resistance, and pest control. At the same time, the accumulation of these secondary metabolites is also beneficial for improving the medicinal quality of plant parts. For example, the accumulation of flavonoids is beneficial for improving antioxidant, anti-inflammatory, and anti-tumor effects; The accumulation of alkaloids exhibits high anti liver cancer effects and inhibition of alpha glucosidase activity; Steroid saponins have diverse bioactive components, such as anti-inflammatory and anti-tumor effects. Therefore, in-depth analysis of these differentially accumulated flavonoids, alkaloids, and steroidal compounds is beneficial for screening quality markers related to the quality of medicinal Polygonatum sibiricum.

At present, there is no content limit for secondary metabolites such as flavonoids and alkaloids under the Huangjing section of the 2020 edition of the Chinese Pharmacopoeia. This study compared the top ten compounds in terms of relative content changes of metabolites between medicinal and edible Polygonatum sibiricum, and found that 50% of the metabolites belonged to flavonoids, which is consistent with the KEGG classification and enrichment results of differential metabolites. Among them, metabolites with high pharmacological activity, such as isorhamnetin, have good anti-tumor, anti myocardial hypoxia, ischemia, relieving angina pectoris, anti arrhythmia, treating coronary heart disease and hypertension, clearing oxygen free radicals, reducing serum cholesterol, protecting the cardiovascular system, promoting blood circulation and other biological effects, and are widely used in clinical practice; Thin yam saponins also have certain physiological activities in anti-tumor, antibacterial, parasitic killing, hemolysis, lipid-lowering, anti osteoporosis, anti mutation and other aspects. The differential upregulation of resveratrol and dioscin was only observed in Polygonatum sibiricum Red, a species derived from Polygonatum sibiricum. Based on this, the next step is to use Polygonatum sibiricum (fruit Polygonatum sibiricum) as a control to explore Polygonatum sibiricum from the pharmacopoeia et Hemsl.、 Polygonatum cyrtonema Hua and locally medicinal Polygonatum sibiricum varieties (such as the widely planted Polygonatum sibiricum in Nanchong, Sichuan, Xiushan, Chongqing, Xinhua, Hunan, etc.) were used as experimental groups to analyze the changes in secondary metabolites in each comparative group based on extensive targeted metabolomics. Combined with the biological activity of specific categories of metabolites, metabolites related to the quality of traditional Chinese medicine Polygonatum sibiricum were screened, providing a basis for the quality evaluation and quality control of authentic Polygonatum sibiricum.

In order to further validate the activity of differentially upregulated compounds screened by metabolomics, this study combined the traditional efficacy of Huangjing in strengthening the spleen and treating internal heat and thirst syndrome. Using IR as the disease model, network pharmacology analysis was conducted. The results of KEGG pathway analysis showed that three differentially upregulated compounds, namely isorhamnetin, naringin, and isonaringin, were involved in the PI3K Akt signaling pathway, mTOR signaling pathway, VEGF signaling pathway, and estrogen signaling pathway The enrichment of AMPK and other signaling pathways is highly significant. Through reviewing and analyzing previous literature, it can be found that the PI3K/AKT signaling pathway is involved in cell cycle, apoptosis, proliferation, and glucose metabolism, while insulin mainly acts on muscle, liver, and adipose tissue. It activates the PI3K/AKT signaling pathway by binding to insulin receptors, thereby regulating glucose and fat metabolism processes; The mTOR signaling pathway participates in regulating skeletal muscle glucose intake and insulin resistance. Activation of the pathway can increase insulin sensitivity, but excessive activation can inhibit insulin sensitivity; VEGFA, as an important target protein of the VEGF signaling pathway, improves insulin resistance by increasing insulin sensitivity in fat and muscle, thereby controlling blood glucose; Adenosine activated protein kinase (AMPK) is an important substance that regulates energy metabolism in the body. Studies have shown that various natural compounds can activate AMPK, enhance insulin sensitivity, and are potential targets for improving insulin resistance. In addition, the target of Huangjing in improving IR may also act on related pathways such as the estrogen signaling pathway, regulate the pituitary adrenal axis and other tissues and organs, and directly or indirectly improve IR. The above research results are consistent with the reported results of the monomer isorhamnetin in the treatment of diabetes. Furthermore, molecular docking technology was used to further reveal the mechanism of action of Polygonatum sibiricum in improving IR. The results showed that key targets of PI3KAkt signaling pathway, mTOR signaling pathway, VEGF signaling pathway, estrogen signaling pathway, AMPK signaling pathway, etc. had good docking effects with isorhamnetin, kaempferol glycoside, and isokaempferol glycoside, with binding energies all ≤ -20.9 kJ/mol. Therefore, it is speculated that the binding of active ingredients of Polygonatum sibiricum to key targets may be the key to the therapeutic effect of Polygonatum sibiricum on IR. Further cell experiments confirmed that isorhamnetin, a differential metabolite, can effectively improve the IR status of HepG2 cells, mainly by affecting the expression of PI3K, AKT1, VEGF, and mTOR signaling pathway related proteins. The results showed that isorhamnetin can increase the expression levels of PI3K and AKT1 proteins and decrease the expression levels of VEGF and mTOR proteins in IR HepG2 cells.

In summary, this article accurately and comprehensively identified the secondary metabolites in medicinal and edible Polygonatum sibiricum through extensive targeted metabolomics technology. Compared with edible Polygonatum sibiricum, the upregulated compounds such as isorhamnetin in medicinal Polygonatum sibiricum act on the PI3K, AKT1, VEGFA, and mTOR signaling pathways through core targets such as PI3K, AKT1, VEGF, and mTOR to improve IR, fully reflecting the pharmacological characteristics of traditional Chinese medicine with multiple components, targets, and pathways. The research results lay the foundation for the screening and quality evaluation of active ingredients in Polygonatum sibiricum, and provide a basis for elucidating the mechanism of action of Polygonatum sibiricum in treating IR.