Stevia extract

The sweet ingredients in stevioside are all glycosides. Glycoside (glycoside or heteroside) is a compound composed of sugar or sugar derivative and another non-sugar compound, connected by the terminal carbon atom of sugar. After hydrolysis, glycoside can generate two parts, sugar and non-sugar. The non-sugar part is called aglycone or ligand, glycoside (agly-cone, genin). Foreign research has separated 8 different sweet ingredients from stevia extracts: stevioside; rebaudiside A, B, C, D, E. Dulcoside A and B, these 8 ingredients are glycosides composed of the same diterpene ligand, belonging to tetracyclic diterpene compounds, they are connected to a glucose on the α-carboxyl group at the C-4 position, followed by a disaccharide or trisaccharide at the C-13 position. Among them, stevioside (Stv) with a sophorose molecule attached to C-13 and rebaudioside A (RebA) with a glucose molecule attached to the sophorose molecule on C-13 are the main

Stevia
There are 6 confirmed sweet ingredients in stevia: stevioside; rebaudioside A, D, C, E; dulcoside A. They are all glycoside compounds with the same aglycone - steviol. The difference between them lies in the different types and composition of sugars bound to the glycoside bonds.

In Japan, highly purified stevia extracts have been approved for use in food and beverages for decades. In the past five years, the world's leading food safety and regulatory agencies have been positive about the use of purified stevia extracts in food and beverages as a safe sweetener.
These agencies include: Joint Expert Committee on Food Additives (JECFA), AN-SES (National Food, Environmental and Occupational Health Agency) of France, Food and Standards Australia New Zealand (FSANZ), US Food and Drug Administration (FDA) and most recently European Food Safety Authority (EFSA).

Stevia
Preclinical and clinical studies have shown that the use of stevia extracts is safe for the general population, including diabetics, children and pregnant women, as well as people with unexplained side effects or allergies.

2 Pharmacological effects

2.1 Hypoglycemic effect

Diabetes is a metabolic disease caused by insufficient insulin secretion or insulin resistance[32]. Studies have shown that among the hypoglycemic components of stevia, steviol has the strongest hypoglycemic effect, followed by stevioside and rebaudioside A. Ahmad et al.[33] studied the effects of stevia water extract on diabetic rats and found that stevia water extract can significantly reduce the body weight, blood sugar and glycosylated hemoglobin levels of diabetic rats, and improve the insulin and liver glycogen levels of rats. Li et al.[34] showed that stevioside can promote insulin secretion and improve glucose intolerance in mice. Its mechanism of action may be to enhance the activity of transient receptor potential subfamily M member 5 (TRPM5). Dandin et al. [35] studied the effects of stevioside on zebrafish models of obesity induced by a high-fat diet. The results showed that stevioside 5 mg/L can reduce blood sugar by inhibiting the expression of fibroblast growth factor 21 and tumor necrosis factor-α (TNF-α), and can also reduce serum nitric oxide levels and enhance the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST). In addition, stevia root polysaccharides can significantly reduce the fasting blood sugar level of type 2 diabetic mice, improve insulin resistance, and reduce oxidative stress. Its mechanism of action is related to stevia root polysaccharides improving intestinal flora and increasing the content of short-chain fatty acids. It can be used to treat type 2 diabetes [36].

2.2 Anti-tumor

Stevia is used as a sugar substitute to help reduce the level of glucose in the blood circulation and deprive tumor cells of their energy source [6]. Stevia extract has a significant inhibitory effect on the growth of various tumor cells and has no significant toxicity to normal cells. Studies have found that in human fibroblasts, stevia extract has no cytotoxicity when the concentration is less than 1,000 mg/mL [37]. Many studies have shown that stevioside can significantly inhibit the proliferation of human liver cancer HepG2 cells [38-39]. Velesiotis et al. [40] pointed out in their study on the effect of stevia on the functionality of breast cancer cells that stevioside 40 μmol/L can inhibit different breast cancer cells; at the same time, it can also significantly inhibit the proliferation of prostate cancer-related fibroblasts by increasing the expression levels of B-cell lymphoma-2 (Bcl-2)-related X protein and caspase-3, and induce cancer cell death [37]. Voloshina et al. [41] studied the cytotoxic effect of steviol on human breast cancer MCF-7 cells. The results showed that steviol 10 μmol/L showed cytotoxicity in MCF-7 cells at the same level as azithromycin, and was selective for cancer cells. Its mechanism may be related to the induction of cell apoptosis via the mitochondrial pathway, thereby inducing cell apoptosis.

2.3 Antioxidant

Oxidative stress is a pathological damage caused by the imbalance between the production of reactive oxygen and reactive nitrogen and antioxidant defense in the body [42]. Studies have shown that stevioside exerts antioxidant effects by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) pathway [43], and can also reduce malondialdehyde content and SOD activity, and can exert antioxidant activity by scavenging free radicals [44-45]. Ferreira et al. [46] found that 500 μg/mL of stevia extract can effectively scavenge 2,2′-azobis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt and 1,1-diphenyl-2-trinitrophenylhydrazine free radicals. Carrera-Lanestosa et al. [47] found that 25 mg/kg of stevia extract can reduce the level of malondialdehyde in the liver, pancreas and kidney tissues of diabetic mice, thereby improving oxidative damage.

2.4 Blood pressure reduction

Hypertension is a common cardiovascular disease and the single largest factor in global disease burden and mortality. Its incidence rate is also increasing year by year [48]. Wang et al. [49] studied the therapeutic effect of stevioside on hypertensive rats and found that stevioside 2 500 μg/mL has a strong aldehyde level, thereby improving the angiotensin converting enzyme (ACE) inhibitory activity and has the potential to reduce blood pressure. In addition, stevioside can increase vascular elasticity, reduce calcium accumulation on the vascular wall, and reduce diastolic blood pressure in a dose-dependent manner, improving hypertension [50-51]. Studies by Yesmine et al. [52] showed that consuming stevia can cause relaxation of the aorta in rats and reduce systolic blood pressure, which may be related to the inhibition of calcium channels.

2.5 Anti-inflammatory

Inflammation refers to an immune response produced after unknown substances invade the body, but it also causes certain damage to the body [53]. Studies have shown that stevia has a certain improvement effect on inflammation such as enteritis and arthritis [54]; Mostafa et al. [55] showed that pretreatment with stevia water extract 80 mg/kg can significantly reduce the degree of colon lesions in rats with ulcerative colitis, reduce crypt degeneration, maintain the integrity of the intestinal lining, and reduce the expression levels of TNF-α, interleukin-1β (IL-1β), myeloperoxidase and NF-κB, thereby alleviating ulcerative colitis. El Nashar et al. [56] found that stevia can exert anti-inflammatory effects by downregulating the expression levels of NF-κB and IL-6. Wu et al. [57] found that stevioside can alleviate osteoarthritis by inhibiting the expression of NF-κB in chondrocytes of osteoarthritis mice and activating the nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway.

2.6 Lipid regulation

Non-alcoholic fatty liver disease (NAFLD) is generally accompanied by increased levels of triglyceride (TG) and low density lipoprotein (LDL) [58-59]. Studies have shown that stevia extract can reduce serum triglyceride and LDL levels by enhancing liver-induced lipase activity and promoting fecal excretion [60]. Stevia root polysaccharides can reduce the blood lipid level of NAFLD rats by upregulating the expression of peroxisome proliferator-activated receptor α (PPARα) and adenosine phosphate activated protein kinase (AMPK) α [61], thereby improving NAFLD. At the same time, the enhanced expression of PPARα can also promote lipid autophagy and thus reduce the levels of triglycerides and total cholesterol in serum [39].

2.7 Other pharmacological effects

In addition, stevia also has antibacterial, weight control, and liver damage prevention effects. Studies have found that 12.5% ​​stevia alcohol extract can inhibit Streptococcus mutans and Lactobacillus acidophilus [62]. Khatun et al. [63] showed that the antibacterial effect of stevia may be caused by caffeoylquinic acid. Clinical research results show that replacing added sugar in the diet with stevia can significantly reduce the weight and waist circumference of overweight patients after 90 days [64]. Ramos-Tovar et al. [65] found that 100 mg/kg of stevia powder can prevent liver damage by upregulating Nrf2 and inhibiting NF-κB expression, preventing hepatocyte necrosis and cholestasis. The mechanism of stevia pharmacological action is shown in Figure 4.

3 Q-Marker prediction analysis

As a treasure of the Chinese nation, Chinese medicine has been a good medicine for curing patients for thousands of years. Its quality plays an important role in clinical efficacy and drug safety [66-67]. Quality standardization has always been the core of Chinese medicine application, which greatly affects human health [68-69]. In recent years, more and more scholars have paid attention to the quality control of Chinese medicinal materials and have made significant progress [70-71]. However, the following challenges still exist. (1) Some indicator components cannot represent the overall chemical characteristics of Chinese medicinal materials; (2) There is currently little evidence to confirm the strong relationship between the chemical markers of Chinese medicine and clinical efficacy; at the same time, there are few reasonable quantitative methods related to Q-Markers. Therefore, there is an urgent need to develop a method to explore the Q-Marker of Chinese medicine to establish a connection between chemical characteristics and efficacy [72]. Academician Liu Changxiao[73] proposed the concept of Q-Marker in 2016 to improve the quality and quality control of traditional Chinese medicine. The process of Q-Marker research and discovery can be discussed from the perspectives of plant affinity and chemical component specificity, component measurability, component effectiveness and traditional medicinal properties[74-75]. Through the systematic compilation and analysis of relevant literature, the Q-Marker of stevia is predicted and analyzed. The prediction and analysis of stevia Q-Marker is shown in Figure 5.

3.1 Plant affinity and chemical component specificity

The chemical composition of traditional Chinese medicine is relatively complex. Drugs of the same genus but different origins may contain different components, and one component may also appear in different drugs, which makes it difficult to reflect the characteristics of the drug[76]. Therefore, the affinity and chemical component specificity of stevia are analyzed to find the indicator components that can represent the quality standard of stevia. Stevia is native to South America and is a herbaceous plant of the Asteraceae family[77]. It is widely distributed throughout the world, but less so in tropical areas. There are 13 subfamilies, 1,911 genera, and 32,913 species in the world[78]. In my country, there are more than 2,000 species in 200 genera[79], distributed throughout the country, including chrysanthemum, dandelion, Artemisia annua, sunflower, and other plants. Stevia contains a variety of components. Currently, diterpenoid glycosides, flavonoids, quercetin, chlorogenic acid, polysaccharides, and other components have been isolated[80]. Among them, diterpenoids are the main components. Inulin is an important component of Asteraceae plants and also an important secondary product. However, different types of plants may grow in the same environment, and as plants of the same genus, their morphology is sometimes difficult to distinguish. Due to the complex growth environment, the content of their components may also be different, resulting in different clinical applications. Therefore, analyzing chemical components from the perspective of affinity will make stevia quality control Q-Marker more practical. Both stevioside and rebaudioside are unique components of stevia. The content of stevia from different origins varies. Studies have found that the amount of rebaudioside A in stevia from different origins varies[81]. Abdul-Qader et al.[82] analyzed the content of quercetin in different parts, and the content of quercetin in leaves was much higher than that in rhizomes. Tang Taoxia et al.[83] showed that the contents of stevioside, rebaudioside A and rebaudioside C in different varieties of stevia are different. Therefore, quercetin, rebaudioside A, rebaudioside C and stevioside can be considered as the basis for the selection of Q-Markers for stevia.

3.2 Measurability of chemical components

In the 2019 edition of the "Anhui Province Traditional Chinese Medicine Preparation Specifications", stevioside is used as the main content determination component[84]. Stevia contains a variety of rebaudioside[85], among which rebaudioside A was discovered the earliest and is also regarded as the characteristic component of stevia. HPLC is a commonly used method for qualitative and quantitative analysis of traditional Chinese medicine ingredients. HPLC was used to determine the content of stevioside and rebaudioside A in stevia, and it was found that the content of stevioside and rebaudioside A was quite different[86]. Borgo et al.[12] studied the phytochemical differences of stevia from different origins and found that the content of flavonoid glycosides in the two types of stevia was significantly different. Pacifico et al.[87] used ultra-high performance liquid chromatography and quadrupole time-of-flight mass spectrometry for chemical analysis. The results showed that stevia contains a large amount of chlorogenic acid substances, and the chlorogenic acid content is relatively high. Based on the above analysis, stevioside, rebaudioside A, chlorogenic acid and flavonoid glycosides can be used as important choices for stevia Q-Marker.

3.3 Correlation between ingredients and efficacy

The ultimate goal of controlling the quality standards of traditional Chinese medicine is to control the efficacy of traditional Chinese medicine. Therefore, effectiveness is the core of Q-Marker selection[88]. In the treatment of various diseases, various chemical components in stevia play different roles. Kamal et al. [89] separated stevioside and rebaudioside A from stevia water extract by HPLC and repeated column chromatography, and found that stevioside showed significant inhibitory effects on pancreatic lipase, α-amylase and α-glucosidase. Studies have shown that stevia can be used as a complementary medicine for the prevention and treatment of metabolic changes associated with type 2 diabetes. Sari et al. [90] also showed that stevioside has a hypoglycemic effect. Secondly, some studies have shown that the lipid-regulating effect of stevia is also related to stevioside. Park et al. [39] studied the effects of stevioside and rebaudioside on liver steatosis in mice. Both reduced body and liver mass and serum total cholesterol and triglyceride levels. Mlambo et al. [50] showed that quercetin and flavonoids have anti-inflammatory and antibacterial effects. Based on the above analysis, stevioside, flavonoids, and quercetin can all be used as important bases for the selection of Q-Markers to characterize their efficacy.

3.4 Traditional medicinal properties

Stevia has a light fragrance and extremely sweet taste. It enters the lung and stomach meridians[84]. Stevia is often used as a sweetener, such as in beverages or candies[91-92], which is related to its extremely sweet taste. The sweetness of stevia mainly comes from stevioside and rebaudioside A. The occasional bitterness of stevia is due to the presence of dulcoside[93]. With the deepening of modern research, it is found that the sweetness in stevia is the result of multiple substances such as glycosides, proteins, and lipids. Stevia contains a large amount of volatile oil substances[94], which makes stevia have a light fragrance. According to the above analysis, stevioside, rebaudioside A, lipids and volatile oil substances in stevia are the material basis of its "light fragrance and extremely sweet taste". Among them, the specific components and contents of lipids and volatile oil substances need further study. Stevioside and rebaudioside A can be used as the basis for the selection of stevia Q-Marker.

4 Conclusion and Outlook

Stevia contains a variety of chemical components, which have multiple pharmacological effects such as hypoglycemic, anti-tumor, and anti-inflammatory. Stevioside is the main pharmacologically active ingredient. It can not only be used as a sweetener or sugar substitute, but also as an auxiliary for diabetes treatment.