What Are the Uses of Steviol Glycosides?

Steviol glycosides are a sweetener extracted from the perennial Asteraceae plant Stevia rebaudiana, which is native to Paraguay and other parts of South America [1]. Steviol glycosides are currently the most representative compound family of natural, non-nutritive, high-potency sweeteners. Both domestically and internationally, they are unanimously recognised as the world's “third sugar source”. Steviol glycosides are 200-350 times sweeter than sucrose, but only 1/300 of the calories of sucrose. At the end of 2008, the US FDA approved Rebaudioside A in stevia as GRAS (Generally Recognized as Safe).

In November 2011, the European Commission approved stevioside as a food additive. Numerous experiments by domestic and foreign scholars have shown that stevioside has no acute or subacute toxicity, genetic toxicity or carcinogenicity, and also has pharmacological activities such as preventing atherosclerosis [2], hyperglycemia [3], obesity, hypertension, heart disease, dental caries and other diseases [4], as well as certain anti-inflammatory [5] and anti-cancer [6] effects. It has also been found to be used as an immune enhancer [7]. Therefore, stevia can replace sucrose and is a natural functional sweetener[8]. This article mainly reviews the metabolic pathway of stevioside in vivo and the biological activities of stevioside, such as lowering blood pressure and blood sugar, inhibiting bacteria, improving immunity, and anti-diarrhea, in the hope of providing a reference for follow-up research.

1 The sweetening components and structure of steviol glycosides

The sweetening components of the more than 30 steviol glycosides found in the leaves of Stevia rebaudiana are all ent-kaurenoic acid/ester compounds. The sugar units in these compounds are all connected to the parent compound via a β-bond and can be divided into four different kaurene structures [9]. , of which the most abundant is stevioside {13-[(2-O-β-D -glucopyranosyl-β-D -glucopyranosyl)oxy]kaur-16-en-18-oic acid, β-D-glucopyranosyl ester, Stevioside, St}, while the best-tasting one is Rebaudioside A{13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaur-16-en-18-oic acid-β-D -glucopyranosyl ester, Rebaudioside A, RA}, the aglycone of which is steviol [(5β, 8α, 9β, 10α, 13α) -13 -Hydroxykaur - 16-en-18-oic acid, steviol]. Analyzing their stereochemistry, they are all steviol (glycoside) C-19 and C-13 positions grafted with an unequal number of glucose or rhamnose groups, forming stevioside derivatives with different sweetness and taste qualities (Table 1) [10].

2 In vivo metabolism of stevioside

The aglycone and glycosyl groups of stevioside are mainly connected by β-1,2 glycosidic bonds, which makes it impossible for the stevioside to be directly absorbed by the organism in the digestive tract. This has also been verified in in vitro experiments. Digestive enzymes (amylase, protease, and trypsin) and liver tissue cannot degrade stevioside and rebaudioside A, but they can be metabolized by microorganisms in the intestines of pigs, mice, and humans to become steviol [11-12]. Regarding the in vivo metabolism of stevioside, Geuns and Simonetti et al. [11, 13-14] found that the gastrointestinal tract absorbs very little stevioside, and that stevioside reaching the colon is metabolized by microorganisms into steviol, which is the only metabolite that can be detected in the feces. stevioside is metabolised by microorganisms to steviol, which is the only metabolite that can be detected in the faeces. This transformation is mainly carried out by Bacteroides sp. in the lower gastrointestinal tract [11]. Geuns et al. [14] showed in an analysis of key human tissues and organs that stevioside accumulates preferentially in the large and small intestines. After 3 days of stevioside consumption, no free stevioside was detected in the feces, only free steviol.

Simonetti et al. [11] did not find free steviol in the peripheral blood of volunteers, but only in bound form, which indicates that all free steviol in the peripheral blood is converted by the liver into steviol glucosides. Free steviol was not detected in the urine, and steviol was only found as a glycoside base. Steviol glucoside was the only bound compound that could be detected in the urine [13, 15]. The presumed metabolic pathway of stevioside in the body is shown in Figure 1. It is completely degraded by intestinal bacteria in the cecum to steviol, and some of the steviol is absorbed and passes through the enterohepatic circulation. It is transported via the portal vein, converted to steviol glucuronide (steviol 19-O-β-D-glucopyranosiduronic acid, SG) in the enterohepatic circulation, and then transported via the bloodstream and finally excreted in the urine via the kidneys. (steviol glucuronide, steviol 19-O-β-D-glucopyranosiduronic acid, SG), which is then transported via the bloodstream and ultimately excreted in the urine via the kidneys.

 Glucuronide is readily formed in the liver, similar to soy flavonoids, and is metabolized into a complex after ingestion, which is then degraded by β-glucuronidase and sulfatase [16].

Wheeler et al. [15] compared the pharmacokinetics of steviol and steviol glucosides after oral administration of rebaudioside A (5 mg/kg body weight) and stevioside (4.2 mg/kg body weight) to healthy men. Steviol glycosides were found in the plasma of all subjects after administration of either rebaudioside A or stevioside. The median Tmax after intake was 12 h and 8 h, respectively. For both compounds, the T1/2 values for the elimination of steviol glycosides from the blood were similar, at about 14 h. Steviol glycosides were mainly excreted in the urine collected over 72 hours, accounting for 59% and 62% of the dose of rebaudioside A and stevioside, respectively. Steviol glycosides were not detected in the feces. Pharmacokinetic analysis showed that rebaudioside A and stevioside are metabolized and excreted in the human body by the same route, and that steviol glucosiduronic acid is mainly excreted in the urine.

3 Biological activity of stevioside

3.1 Effect on blood glucose levels

Diabetes is a metabolic disease caused by a deficiency in insulin secretion or a disturbance in the action of insulin. For patients with type 2 diabetes, who have low insulin activity or insufficient insulin secretion, the intake of sugar needs to be strictly quantified. Stevioside can promote insulin secretion [17] and has the characteristics of being highly sweet and low in calories. Therefore, for diabetic patients, stevioside can be used instead of sucrose [17].

Chen [17] studied the mechanism by which stevioside can lower blood sugar and found that stevioside can stimulate insulin secretion by pancreatic β-cells and the β-cell line INS-1 cells, enhance insulin sensitivity, and reduce blood sugar, radionuclide and cholesterol levels over the long term [18]. The blood glucose concentration increased from 3.3 mmol/L to 16.7 mmol/L to stimulate insulin release, while stevioside significantly stimulated insulin secretion at 1 mmol/L. Long-term consumption of stevioside by hypertensive patients with normal glucose levels does not affect fasting blood glucose [19]. However, it is interesting to note that in a long-term study on GK rats, rebaudioside A was found to fail to promote insulin secretion [20]. For patients with type 2 diabetes, taking rebaudioside A for 16 did not affect the homeostasis of blood sugar, and had no effect on blood lipids or blood pressure [21]. This shows that Rebaudioside A does not have a pharmacological effect in the body similar to that of stevioside in regulating glucose.

3.2 Blood pressure lowering effect

Currently, blood pressure is lowered mainly by six methods: calcium channel blockers, angiotensin converting enzyme inhibitors, beta-blockers, alpha-blockers, vasodilators, and diuretics. Stevioside has a blood pressure lowering effect [8, 22]. The mechanism by which stevioside lowers blood pressure is similar to that of calcium channel blockers, mainly by reducing the influx of extracellular Ca2+ and stimulating the production of vasodilators (prostaglandins) [23]. Early animal and human experiments have shown that a liquid extract of dried stevia leaves can promote vasodilation in systemic and renal blood vessels, thereby causing hypotension, weakening the renal autoregulation function, and causing negative effects such as polyuria and urinary sodium excretion [24].

Melis et al. [25] proposed a hypothetical mechanism for the hypotensive effect of stevioside, suggesting that stevioside or its metabolites act directly on renal function. Intravenous administration of stevioside (16 mg/kg) to rats increased the excretion of water, sodium and potassium, which would produce a vasodilatory effect and result in a reduction in blood pressure. Because oral intake is too low, it is unclear whether these effects also occur after oral administration [26]. Melis further believes that stevioside's vasodilatory activity contributes to the blockade of Ca2+ channels. Due to this effect, calcium influx into smooth muscle cells is suppressed, resulting in vasodilation [27].

In a study of stevioside metabolism, volunteers with normal blood pressure (114/74 mmHg) were given oral doses of 10–15 mg/kg body weight of stevioside and no effect on blood pressure was observed [14]. In a 7-week, 11-week and 6-week experiment, the intake of three doses (3.75, 7.5 and 15 mg/kg body weight) had no effect on the systolic and diastolic blood pressures of mildly hypertensive patients (140/94 mmHg) [28]. These results show that stevioside intake of up to 15 mg/kg body weight does not affect blood pressure in the normal blood pressure population.

3.3 Antibacterial effect

The increased resistance of the human body to antibiotics and the increased incidence of new and recurring infectious diseases have prompted the urgent need to discover antibacterial substances with different structures and to study their mechanisms of action [29]. Therefore, plant extracts and phytochemicals with antibacterial properties are of great importance in treatment options [30]. Debnath et al. [31] found that the use of sucrose in people susceptible to fungal or streptococcal infections can aggravate the infection, while the use of stevioside instead can alleviate it. Therefore, stevioside is believed to inhibit the growth of certain bacteria and other infectious organisms [32]. At the same time, the antibacterial activity of extracts from different stevia solvents (water, acetone, methanol, chloroform, ethyl acetate, hexane) was studied, and it was found to have antibacterial activity against Salmonella typhimurium, Aeromonas hydrophila, Vibrio cholerae, Bacillus subtilis and Staphylococcus aureus [30-31, 33-34]; however, fermented stevia extract can only inhibit Salmonella typhimurium, Staphylococcus aureus and Bacillus subtilis [31, 33]. Tomita et al. [35] also found that a hot water extract of stevia had inhibitory activity against enterohemorrhagic Escherichia coli and other foodborne pathogenic bacteria.

3.4 Immune effects

The immune system constitutes the host defence against invading pathogens, foreign substances and cancer cells. Inflammatory processes, including the release of pro-inflammatory cytokines and the formation of reactive oxygen and nitrogen species, are an important part of the immune response. In this process, the immune system, epithelial cells and endothelial cells, among others, interactively participate in the removal of harmful stimuli and healing. The inflammatory response is an early host immune response mediated by immune cells and cytokines. Pathogenic bacteria and other infectious substances can directly activate monocytes or macrophages, initiating a cytokine cascade and an immune response in the inflammatory process. Monocytes are stimulated to release a large number of cytokines, such as the biologically active peptide/tumor necrosis factor TNF-α and interleukin IL-1β. In addition, active free radicals/nitric oxide (NO) also play a role in inflammation [36]. Therefore, interfering with the production of TNF-α, IL-1β, and nitric oxide is often used as an indicator to evaluate the anti-inflammatory effect of natural products [37].

Boonkaewwan et al. [38] reported that in human monocyte THP-1 cells stimulated with lipopolysaccharide (LPS), stevioside (1 mmol/L) significantly reduced the release of pro-inflammatory factors (TNF-α and IL-1β) from THP cells, and there was also a slight decrease in nitric oxide production. Toll-like receptor 4 (TLR4) is one of the essential receptors for LPS signaling. Stevioside may bind to TLR4 as a competitive inhibitor of LPS. The three glucose units of stevioside may play a crucial role in the interaction of stevioside with TLR4 in THP-1 cells. For monocytes at this level, stevioside is beneficial for enhancing the innate immunity of healthy people. On the other hand, in LPS-stimulated THP-1 cells, stevioside at the same concentration inhibited the release of TNF-α, IL-1β and NO by interfering with the NF-κB signaling pathway. NF-κB is a transcription factor that controls the expression of pro-inflammatory cytokines in immune cells (Figure 2).

Kochikyan [39] found that stevioside can prevent gastric ulcers because stevioside and steviol can inhibit the production of 12-O-14-alkenephorbol-13-acetate (12-O-tetrade can oylphorbol-13 - acetate (TPA) [40], which is a major factor in the formation of skin cancer and local inflammation [40]. Nakamura et al. [41] found that stevioside can inhibit the growth of TPA-induced tumours in skin cancer. Stevioside enhances cell regeneration and blood coagulation, inhibits tumour growth and strengthens blood vessels [4, 42–43]. Mizushina et al. [44] found that steviol could inhibit the growth of three human cancer cell lines by inhibiting the activity of DNA polymerase and DNA topoisomerase II, thereby exerting an anti-cancer effect.

3.5 Other biological activities

Diarrhoea is a common large intestine disease, usually caused by intestinal infection with bacteria and viruses. Enterohaemorrhagic E. coli can cause severe bloody diarrhea. Tomita et al. [35] found that stevioside has antibacterial activity against enterohaemorrhagic E. coli, and thus inferred that stevioside has the potential to treat diarrhea. Stevioside and its congeners mainly treat secretory diarrhea caused by excessive intestinal fluid secretion by affecting Cl- secretion in the intestine [45]. Stevioside has the effect of restoring memory. Sharma et al. [46] used the water maze method to evaluate the learning and memory abilities of mice. They found that stevioside can restore the memory of mice after scopolamine caused memory damage. Oral intake of stevioside can prevent dental caries and can be considered a calcium antagonist [47]. Stevioside also has a certain inhibitory effect on atherosclerosis. Holvoet's research found that after mice ingested stevioside, the deposition of oxidized low-density lipoprotein (ox-LDL) was reduced, the expression of the antioxidant superoxide dismutase (SOD) was increased, and the degree of atherosclerosis was reduced [48].

The main function of the proximal renal tubule is reabsorption. It can also remove various foreign substances and compounds through the organic anion and cation secretion systems. Therefore, inhibition or interference with this secretion and transport system can reduce the clearance of therapeutic drugs by the proximal renal tubule and enhance the therapeutic effect of the drugs themselves. Steviol glycosides have a small degree of interaction with organic anion and cation transport systems. After oral intake of steviol glycosides, they are converted to steviol in the body. The interaction with the renal transport system during this process makes steviol have potential medical value. It can help delay the removal of therapeutic drugs, so that the efficacy of the drugs themselves can be better exerted before being removed, thereby achieving the purpose of treatment [49].

4 Outlook

Steviol glycosides are a safe sweetener that has been approved for use by the China and US FDA and is being recognized by more and more countries and organizations. In addition to its sweet taste, it also has pharmacological and biological activities such as lowering blood pressure, lowering blood lipids, antibacterial and immune, which are also being developed and utilized in the pharmaceutical and health industries. In the 21st century, which advocates green and safety, stevioside, as a natural functional sweetener, has a very broad application prospect. In-depth research on the metabolic pathway and metabolites of stevioside can provide a clearer understanding of its safety in the body and also provide a reference for the application of its biological activity.

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