March 24, 2021 Longchang Chemical

1. Introduction to Rutin and Isoquercitrin

Rutin, also known as vitamin P, has a molecular formula of C27H30O16. The chemical structure of rutin is shown in Figure 1. It is a natural flavonoid glycoside with anti-inflammatory, anti-oxidant, anti-allergic and anti-viral effects. The melting point of rutin is about 178 ℃, and it is a yellow crystal at room temperature. It becomes darker in color when exposed to light and has a bitter taste. It has low solubility in water, easily soluble in methanol, ethanol, and insoluble in organic reagents with low polarity such as petroleum ether. Rutin is widely found in the roots, stems, leaves and other parts of plants, and its content is higher in plants such as Sophora japonicus, Rutinous herb, Eclipta prostrata, and buckwheat. At present, rutin is mainly extracted from Sophora japonica in industrial production, and its content can reach more than 23.0%. In terms of geographical distribution, the rutin content in Sophora japonica is higher in Henan, Shandong, and Hebei provinces. In addition, some studies have shown that buckwheat, red dates, mulberry leaves and other plants also contain a certain amount of rutin.
Figure 1 The chemical structure of rutin
Isoquercitrin, also known as apocynum A, is a derivative of rutin derived from derhamnosyl. Its molecular formula is C21H20O12. The chemical structure of isoquercitrin is shown in Figure 2. Isoquercitrin has a melting point of about 226 °C and is a yellow crystal at room temperature. Its solubility in water is low, only 25.9 mg/L at room temperature. It becomes darker after being dissolved in alkaline water. Isoquercitrin is widely distributed in plants, including Saururus chinensis, Houttuynia cordata, Rhododendron gold leaf, rhododendron, Ginkgo biloba, Morus mulberry, and Elaeagnus seabuckthorn, etc. However, the natural content of isoquercitrin in plants is low, only a few ten thousandths on average, so it is mostly prepared by synthetic methods. Modern pharmacological studies have shown that the pharmacological activity of isoquercitrin is significantly higher than that of rutin, and its medicinal value is higher.
Figure 2 The chemical structure of isoquercitrin

2. Preparation of isoquercitrin

Isoquercitrin has a low natural content in plants, and it is often prepared by acid hydrolysis, high pressure hydrolysis and other methods in the industry. Studies have used column chromatography to separate isoquercitrin monomer from plant extracts. For example, Shi Xin et al. used semi-preparative high performance liquid chromatography to separate isoquercitrin with a purity of > 98.0% from Yidianhong medicinal materials; Yin Li used macroporous resin column chromatography combined with semi-preparative high performance liquid chromatography to obtain isoquercitrin from 580 mg Huangdingju 9.3 mg isoquercitrin was isolated from the alcohol extract, and its purity was 95.8%. However, due to the low natural content of isoquercitrin in plants, this method not only has a low yield, but also has a large workload and a lot of reagent consumption, which largely limits its application in industrial practice. Yu Ting et al. combined high-pressure hydrolysis with SG64 resin chromatographic separation technology to establish a rapid preparation method for isoquercitrin. However, the hydrolysis conditions are not easy to control, and the yield of isoquercitrin is low. The product contains a large amount of unhydrolyzed rutin and quercetin obtained by further hydrolysis, which increases the difficulty of subsequent separation.
Biotechnology such as microbial transformation and enzyme-catalyzed transformation is essentially a metabolic reaction that uses free enzymes or complex enzymes to modify the structure of foreign compounds. It has the advantages of mild conditions, strong selectivity, few by-products, clean and environmentally friendly, and low cost; natural; Glycoside compounds usually contain more glycosyl groups and are very polar, so they are not the best structure to exert their pharmacological activity. Converting them into low glycosides, aglycones or other products will help to better exert their efficacy. Wang Yuanyuan et al. used Streptomyces griseus to biotransform rutin, and separated 6 conversion products including isoquercitrin by silica gel column chromatography. Further studies found that the reactions involved in this process are more complicated, including methylation and Glycoside hydrolysis, etc., indicate that Streptomyces griseus has poor specificity for rutin transformation.
The enzymatic hydrolysis method has the advantages of mild reaction conditions, strong specificity, and easy control of the reaction, and can overcome the shortcomings of the above-mentioned method for preparing isoquercitrin. For example, Wu Di et al. used α-L-rhamnosidase produced by microorganisms to transform rutin; the results showed that the yield of isoquercitrin was 49.4%, and its purity could reach 98.3% after purification by silica gel column chromatography. Sun Guoxia et al. used hesperidinase to hydrolyze rutin to prepare isoquercitrin, and used ionic liquids to increase the yield of isoquercitrin. The conversion rate of the final product reached 99.27 ± 0.55%.

3. Introduction to the pharmacological activities of rutin and isoquercitrin

Rutin is an important component of traditional Chinese medicine safflower and other medicines for promoting blood circulation and removing blood stasis. It has certain effects on cardiovascular and cerebrovascular diseases such as cerebral thrombosis and angina pectoris. Jin Ming et al. found that a certain concentration of rutin can antagonize the specific binding of platelet activating factor and rabbit platelet receptors, thereby inhibiting the platelet adhesion mediated by the activating factor and the increase of free Ca2+ concentration in platelets.
Guardia et al. studied three flavonoids of hesperidin, quercetin and rutin, and the anti-inflammatory action effects of these three flavonoids on rats. First, a rat model of acute and chronic inflammation was constructed. After intraperitoneal administration at a dose of 80 mg/kg·d, the three flavonoids can inhibit the acute and chronic phases of the experimental inflammation model; among them, rutin has the strongest effect on chronic inflammation. Yoo et al. studied the anti-inflammatory effects of rutin on the pro-inflammatory response of human umbilical vein endothelial cells (HUVECs) induced by high mobility group protein 1 (HMGB1) and related signal pathways. The results show that rutin can inhibit the release of HMGB1 and reduce the migration of mouse leukocytes. Further studies have found that rutin can also inhibit the production of tumor necrosis factor alpha and interleukin 6 induced by HMGB1, which proves that rutin can treat various severe vasculitis diseases by inhibiting the HMGB1 signaling pathway.
Yang et al. measured the antioxidant activity of rutin and compared it with the standard antioxidant butyl hydroxytoluene (BHT) and ascorbic acid (Vc). The results show that rutin has a strong ability to scavenge DPPH free radicals. When the concentration is 0.05 mg/mL, the inhibition rates of Vc, BHT and rutin can reach 92.8%, 58.8% and 90.4% respectively; in addition, rutin has a strong effect on lipids. Qualitative peroxidation also has a significant inhibitory effect.
Alonso-Castro et al. used the MTT method to detect the cytotoxic effect of rutin on human cancer cells and non-tumorigenic cell lines. Different doses of rutin were intraperitoneally injected into nu/nu mice with SW480 colon carcinoma for 32 days; the serum vascular endothelial growth factor (VEGF) levels, survival time, and toxicological effects on body weight and the weight of the organs were analyzed. The results show that rutin has the highest cytotoxic effect on SW480 cells (IC50 = 125 μM), and has no toxic effect on other organs of mice; compared with untreated mice, the average survival time is prolonged by 50 days, and the serum VEGF level A decrease of 55%. Saleh et al. compared the anticancer effects of rutin and orlistat on two breast cancer models (in vivo EAC and in vitro MCF7) and pancreatic cancer cell lines (PANC-1). Tumor volume, CEA (carcinoembryonic antigen) level, cholesterol content, FAS antigen, antioxidant effect, and histopathological examination showed that both rutin and orlistat had anti-cancer activity in the body. In addition, both are cytotoxic to MCF-7 and PANC-1 cell lines by promoting apoptosis.
Modern pharmacological studies have shown that the pharmacological activities of isoquercitrin in anti-oxidation, anti-tumor and other aspects are significantly higher than that of rutin. Jung et al. isolated seven compounds such as isoquercitrin from Platycladus orientalis and tested their antioxidant activity. The results showed that isoquercitrin has an effect on the retinal ganglion cell line RGC-5 induced by hydrogen peroxide (H2O2). The inhibitory effect of cell death is the strongest. At the same time, isoquercitrin is tolerated orally, so it can be used to treat diseases such as glaucoma. Rogerio et al. studied the anti-inflammatory effects of quercetin and isoquercitrin on mouse models of asthma; the results showed that these two flavonoids are effective eosinophilic inflammation inhibitors and have certain potential for the treatment of allergic diseases.
Huang et al. studied the mechanism of the effect of isoquercitrin on liver cancer. In in vitro experiments, it was found that isoquercitrin can inhibit the proliferation of cancer cells while promoting their apoptosis, and at the same time, reduce the expression level of PKC in human liver cancer cells; in in vivo experiments, isoquercitrin can also cause transplanted tumors in nude mice. The growth rate of the cells is significantly reduced. It is confirmed that isoquercitrin can significantly inhibit the occurrence and development of liver cancer, and its molecular mechanism may be related to the PKC and MAPK signal pathways.
Ji Lili compared the in vitro hypoglycemic activity of isoquercitrin and total flavonoids of Moringa oleifera leaves. The results show that both can significantly increase the consumption of glucose by HepG2 cells, and the hypoglycemic effect of isoquercitrin is significantly stronger than that of total flavonoids; further studies have found that its hypoglycemic mechanism is mainly increased by inhibiting the activity of DPP-4 and the secretion of insulin also up-regulates the expression of InsR, PKA and PKCα, thereby enhancing the effect of insulin and promoting the proliferation of liver and pancreatic islet cells.
Yun et al. discussed the antifungal activity of isoquercitrin and its mechanism of action; the results showed that isoquercitrin has a strong effect in the susceptibility test of pathogenic fungi, and no hemolysis was found. In addition, candida albicans was also tested for the release of malonyl iodide and potassium, confirming that isoquercitrin can interfere with the cell membrane and increase its permeability to promote membrane damage, thereby exerting antibacterial activity. Kim et al. found that isoquercitrin can inhibit the replication of influenza A and B viruses, and when used in conjunction with amantadine and oseltamivir, it can effectively inhibit the emergence of resistant viruses, indicating that isoquercitrin can effectively inhibit the emergence of resistant viruses. It has certain application potential for the treatment of viral influenza.
In addition to the above effects, isoquercitrin also has physiological activities such as anti-osteoporosis, lowering blood pressure, blood lipids, neuroprotection and anti-depression. The application of α-L-rhamnosidase in the biotransformation of rutin will be further introduced later.

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