What Is the Use of Stevia?

Stevia, native to the high mountain grasslands along the border of Paraguay and Brazil, is used by the locals as a sweet tea or sweetener. Stevia was introduced to China in 1976 and successfully cultivated on a trial basis [1]. Due to the suitable climate and soil conditions in China, it is now cultivated in large quantities in Fujian, Yunnan and other places in China. Stevia leaves are rich in flavonoids and steviol glycosides, in addition to organic acids and inorganic impurities [2-3]. Flavonoids have various pharmacological activities such as anti-tumor and anti-oxidation; steviol glycosides are a type of sweetener with multiple uses.

1 Stevia Overview

Steviol glycosides are a mixture of structurally similar diterpenoid compounds. The basic structure and main components are shown in Table 1, of which stevioside has the highest content (about 10%), and rebaudioside A has the highest sweetness and best taste, with a content of about 1%. Stevia is a white crystal or powder that is easily soluble in water and methanol. It is stable and not easily affected by temperature, pH value or microbial fermentation. It is known as the world's third sugar source after sucrose and beet sugar. It is highly sweet (about 150-300 times that of sucrose), low in calories (about 1/250 of sucrose), and has a certain adjuvant therapeutic effect on hypertension, diabetes, obesity, dental caries, etc., and has a certain adjuvant therapeutic effect. So far, no toxicity or side effects have been found. In 2011, the European Commission allowed stevia to be used as a food additive, indicating that stevia has been widely recognized. China is the world's leading producer and exporter of stevioside. According to customs statistics, China's annual export volume of stevioside accounts for more than 80% of the global market.

2 Stevioside in the food and beverage industry

Stevia is widely used in the food and beverage industries because of its high sweetness, low calorie content, fresh taste and lack of side effects. In June 1985, the Chinese Ministry of Health approved the use of stevia as a food additive. In 1990, the Ministry of Health expanded its scope of application and approved its use as a pharmaceutical sweetener excipient; in 1999, the stevioside standard (GB 8270-1999) was formulated. To date, stevia has been widely used in beverages, candied fruits, preserved fruits, pastries, dairy products, functional foods such as those that lower blood pressure or aid weight loss, and the cigarette industry.

Although stevia is highly sweet, it has a bitter and licorice aftertaste. This may be due to the presence of bitter impurities during the extraction process or the influence of the basic structure and sugar moieties of stevia [4]. However, when stevia is mixed with citric acid, malic acid, lactic acid and amino acids, the aftertaste of stevia can be eliminated, which is beneficial to improving the taste of stevia [5].

In addition, stevia can be combined with other sweeteners to make a compound sweetener. For example, the natural combination of erythritol and stevia not only enhances its health benefits, but also reduces the cost of erythritol and masks the unpleasant taste of stevia. Stevia can replace 15% to 35% of the sucrose in the production of beverages or alcohol, without affecting the taste. Moreover, due to the bacteriostatic effect of stevia, it can extend the shelf life of beverages and improve the quality of alcohol.

The use of stevia in the production of preserved fruits and pastries not only greatly reduces costs, but also reduces calories, meeting the needs of people who want to reduce their sugar intake. The use of stevia in the production of dairy products not only improves the taste of dairy products, but also acts as a bifidobacterium growth promoter, which not only promotes the growth of bifidobacteria and lactobacilli in the human body, but also inhibits the growth of Escherichia coli and other bacteria[6]. Replacing 30% to 50% of the sucrose in processed aquatic products with stevia can prevent protein spoilage and browning caused by rancidity in aquatic products[6]. Adding stevia to condiments such as soy sauce can not only prevent browning, but also reduce the saltiness.

3 Stevioside in the pharmaceutical industry

3.1 Pharmaceutical applications

Currently, sucrose is generally used as a flavouring agent in pharmaceuticals, but there are some disadvantages in clinical applications. For example, large amounts of sucrose will limit its use by diabetics, and the presence of sucrose also causes the medicine to turn yellow, affecting its appearance.

Ruan Wenyou [7] used stevia to replace sucrose in inosine oral solution and found that the newly formulated inosine oral solution has the advantages of low viscosity, fast filtration, good transparency and color of the finished product, low cost, no effect on efficacy, and a good taste. In addition, sucrose has the disadvantages of possibly inducing cancer when consumed in large quantities, causing tooth decay; being unstable in acidic Chinese medicine solutions, which reduces its sweetness; reducing sweetness in heat clearing and detoxifying medicines and astringent medicines; and the presence of sucrose is not conducive to microbial control, which affects the quality of the medicine[8]. Stevia has the characteristics of high sweetness, low calories, no side effects, prevention of tooth decay, stability in the pH range of 3 to 10, and non-fermentability. In the pharmaceutical process, it has become a sweetener that replaces sucrose as a flavoring agent for syrups, powders, pills, and other medicines.

3.2 Pharmacological effects of stevia

Hundreds of years ago, people who used stevia as a sweetener already realized its blood pressure-lowering and blood sugar-lowering functions. Later experimental studies found that its blood pressure-lowering effect is mainly achieved by three ways: reducing the influx of extracellular Ca2+, reducing Na+ reabsorption and stimulating the production of the vasodilator prostaglandin [9]. Its blood sugar-lowering effect is achieved by stimulating the secretion of insulin and the sensitivity of peripheral tissues to insulin to promote the metabolism of glucose in the blood[10], and inhibiting the absorption of glucose in the intestines and the production of glucose in the liver[11]. In addition, stevia does not lower blood pressure in people with normal blood pressure, and only exerts its hypoglycemic effect when blood sugar is high, so it can be consumed in large quantities by normal people[12].

Stevioside and steviol have an anti-inflammatory effect by influencing the expression of cytokines and inhibiting the NF-κB signaling pathway, thereby reducing the production of pro-inflammatory factors induced by polysaccharides [13]. They also effectively inhibit the production of TPA, which causes local inflammation and skin cancer [14]. Stevioside and matrine extract have a synergistic effect on inhibiting rotavirus, which causes diarrhea in infants and young children, but the anti-diarrhea effect is reduced when either of them is used alone [15]. In rats with memory impairment induced by scopolamine, the increase in brain AChE activity and brain oxidative stress levels after taking stevioside was inhibited [16], indicating that stevia has an anti-amnesic effect.

Stevia extracts in different solvents are all mixtures with extremely complex chemical compositions, including not only stevioside compounds, but also flavonoids, nicotinic acid, riboflavin, alkaloids, tannins, etc. Stevia extract with water as the solvent does not exhibit antibacterial activity [17-18], but Stevia extract with acetone as the solvent has strong antibacterial activity against gram-positive bacteria than gram-negative bacteria. Stevia extract with acetone and ethanol solution as solvent, the antibacterial activity of stevia extract is stronger than that of the extract using only acetone as solvent, and the stevia extract using ethyl acetate as solvent shows high antibacterial activity against Trichophyton mentagrophytes and Candida albicans [17-19].

3.3 Pharmacological effects of stevia derivatives

Stevia is not only directly used in the food and pharmaceutical industries, but its derivatives can also be used in the biomedical field because they are all glycosides with the same di-terpene skeleton as the aglycone. Modifying their structure can further expand their use in the biomedical field [20-21]. Steviol can be obtained by enzymolysis or treatment with sodium periodate and a large amount of strong base. Isosteviol can be obtained by acidolysis of stevioside. Steviobioside can be obtained by heating and refluxing in a 10% potassium hydroxide solution for 1 h, as shown in Scheme 1. Experimental studies have shown that derivatives such as steviol, isosteviol and stevioside all have certain biological activities.

Isosteviol also has hypoglycemic [22] and hypotensive [23] effects, as well as a certain protective effect on ischemic hearts [24], and can be used to treat inflammation and cancer by inhibiting DNA polymerase and DNA topoisomerase II [25]. Stevioside plays an important role in the renal transport and clearance of drugs [26], and not only affects renal function, but also has a therapeutic effect on certain kidney diseases [27]. Stevioside, steviol and steviol glycosides all have a certain inhibitory effect on the growth of Mycobacterium tuberculosis H37RV, with steviol having the weakest activity and steviol glycosides having the strongest.

In the past decade, there have been continuous structural modifications to the sugar moieties of steviol glycosides, with the aim of increasing their sweetness and improving their aftertaste. Structural modifications have also been made to the aglycones of steviol and isosteviol, with the aim of enhancing their antibacterial, antitumor, antihypertensive and plant growth-regulating activities [28].

Wonganan O et al. [29] and Zou M et al. [30-31] both carried out simple structural modifications to the isosteviol skeleton and investigated its relaxing effect on the rat aorta and its inhibitory effect on different human tumor cell lines (such as HepG2, MGC-803, MDA-MB-231, etc.). The experimental results showed that some of these derivatives exhibited better antihypertensive and antitumor effects. Lin L, et al. [32] used stevioside as a raw material, Kataev V E, et al. [33-36] and  Khaybullin R N, etc. [37] used isosteviol as the raw material to synthesize a series of molecular clamp-type compounds or macrocyclic compounds containing the isosteviol skeleton, and examined their antibacterial activity and activity against Mycobacterium tuberculosis H37RV, respectively.

The results showed that some of the synthesized clamping compounds or macrocyclic compounds exhibited very superior antibacterial activity and anti-tuberculosis activity. For example, compounds 1–4 (scheme 2) showed the most superior activity against Mycobacterium tuberculosis H37RV. Their MICs against the H37Rv strains had MICs of 3.1 μg/mL, 1.7 μg/mL, 5.0 μg/mL, and 0.7 μg/mL, respectively (the MIC of the anti-tuberculosis drug pyrazinamide was 12.5 μg/mL). In addition, compound 4 showed good activity against the three strains of M. Avium, M. Terrae, and MLU, with MIC values of 0.7 μg/mL, 0.35 μg/mL, and 0.7 μg/mL, respectively.

4. Steviol glycosides in organic chemistry

Steviol glycosides can be hydrolyzed under acidic conditions to obtain isosteviol, a tetracyclic diterpene compound with a labdane skeleton. Due to its rigid molecular structure, unique groove structure, stable chemical structure, and superior chiral environment, isosteviol has been developed and applied in organic catalysis, molecular recognition, self-assembly, and other fields in recent years.

4.1 Application in organic catalysis

Since 2010, Tao Jingchao's research group has used isosteviol as a raw material to synthesize a series of difunctional thiourea catalysts for catalyzing a series of asymmetric reactions. These catalysts exhibit good asymmetric catalytic effects.

An J Y et al. [38-40] introduced 4-hydroxy-L-proline, L-threonine, and L-serine at the 19 position of isosteviol to synthesize a series of amino-thiourea compounds 5 ~10 (Scheme 3), and investigated the catalytic activity and stereoselectivity of this series of amphiphilic compounds in the direct catalytic asymmetric Aldol reaction, asymmetric α-amine oxidation reaction, asymmetric Mannich reaction and asymmetric Biginelli reaction in the organic phase and aqueous phase. The experimental results showed that the catalytic activity of compound 5 was superior to that of compound 6.

In the direct catalytic asymmetric Aldol reaction in the aqueous phase, the catalytic amount was 1%, and the ee values of the asymmetric Aldol reactions of cyclohexanone, cyclopentanone and acetone with aromatic aldehydes were 99%, 98% and 90%, respectively. Compound 6 has better catalytic activity than compound 5 for the asymmetric α-amination oxidation of aldehydes, ketones and substituted nitrobenzenes in the aqueous phase. The catalytic reaction can be completed in 3–5 min at room temperature, and the ee values are all greater than 90%. Compounds 6 and 10 have excellent selectivity in the direct asymmetric Mannich reaction of cyclohexanone, nitrobenzaldehyde and aniline substituted with a non-strong electron-donating group. Only 5% of compound 6 can be used as a catalyst to obtain an adduct with a syn configuration, while compound 10 can be used to obtain an adduct with an anti configuration, and their ee values are both as high as 99%.

In addition, 16-cyclohexanediamine and proline were introduced into isosteviol, respectively, to synthesize a series of amino-thiourea compounds 11–16 (Scheme 4), and their catalytic activities and stereoselectivities for asymmetric Michael addition and α-substitution of phenyl cyanide with N-maleimide were investigated [41–43]. The experimental results show that catalysts 11 and 12 have high catalytic activity and stereoselectivity for the asymmetric Michael addition of isobutyraldehyde and β-nitrostyrene, compounds 13 and 14 for the asymmetric Michael addition of acetylacetone and β-nitrostyrene, and compounds 13–16 for the reaction of α-substituted phenyl cyanides with N -maleimide. Compounds 11 and 13 were used as catalysts to obtain mainly R-configured adducts, while compounds 12 and 14 were used as catalysts to obtain mainly S-configured adducts. The yields were higher than 95%, and the ee values were higher than 97%. Compound 13 can catalyze the bulk reaction of α-substituted phenyl cyanide with N-maleimide without reducing its yield and ee value, and has the potential for industrial production.

4.2 Application in molecular recognition

Kataev V E et al. [44-45] used a water-chloroform layer to simulate a biofilm and investigated the ability of two isosteviol-containing clamp compounds 17–20 (Scheme 5) to transfer the chiral recognition of amino acids such as D/L-tryptophan. Unfortunately, the clamping compound 17 has the strongest ability to recognize and transfer D/L-tryptophan, but it has poor enantioselectivity. Clamping compounds 19 and 20 do not show any recognition performance for D/L-phenylalanine methyl ester.

4.3 Other

Zhang T et al. [46] synthesized a series of alkali metal salts 21–26 with an isosteviol skeleton (Scheme 6) and used the heating–cooling method to investigate their selective gelling abilities, phase transition temperatures and minimum gelling concentrations in different organic solvents. Among them, compound 24 exhibits good gelation ability in halogenated solvents. Its phase transition temperature in iodobenzene reaches 77 °C, and the minimum gelling concentration in dichloromethane and chloroform is 0.1% g/mL. It can also gel organic solvents from a large amount of water at room temperature.

Lohoelter C et al. [47] synthesized a series of benzophenone and tricyclic derivatives containing isosteviol and used quartz crystal microbalance (QCM) to test their ability to track unstable aromatic compounds as affinity materials. Among them, compound 27 (Scheme 7) can show a particularly strong signal at very low concentrations of aromatic compounds, indicating that compound 27 has high affinity for aromatic compounds. Proton screening also found that compound 27 can be used as a sensor with great potential as an affinity material for tracking very dilute aromatic compounds in the air.

Mamedova V L et al. [48] used isosteviol to first react with sodium methoxide, and then carry out exchange reactions with calcium gluconate, ferrous gluconate, cuprous chloride and nickel chloride, respectively, to generate molecular clamping compounds 28–31 (Scheme 8) with metal ions bridged at the 19-position of the isosteviol skeleton. Meanwhile, using isosteviol derivative 32 and triethylamine to react, and then separately with calcium gluconate, ferrous gluconate, cupric chloride and nickel chloride to exchange reaction to generate in isosteviol skeleton 16 with metal ion bridge molecular clamp compounds 33~36 (scheme 6), and it is believed that it can be used in pharmacology, metal catalysis or new magnetic materials.

5 Summary and outlook

Stevia is abundant in source and inexpensive. Its high sweetness and low calorie content make it widely used in the food and beverage industries. Stevia itself has biological activity, making it a highly promising lead compound in new drug research and development. Modifying the structure of stevia and its derivatives can give them higher biological activity, which is of great use in the field of new drug development. The unique rigidity, groove structure and chiral environment of the tetracyclic diterpenoid skeleton of stevioside make it also important for applications in organic chemistry, supramolecular chemistry, etc. Therefore, stevioside is a natural product resource with broad application prospects that is waiting to be further developed and utilized.

References:

[1] Tang Zhifa. The rise and development strategy of stevia [J]. China Food Industry, 1999, 6(2): 52.

[2] Zhao Yuzang, Zhang Yunsn. Research on the chemical composition, development and utilization of stevia [J]. Journal of Anyang Normal University, 2000, (3): 40.

[3] Chaturvedula V S, Clos J F, Rhea J, et al. Minor diter- penoids glycosides from the leaves of stevia rebaudiana morita [J]. Phytohem let, 2011, 4(3): 209.

[4] Wang Deji. On the sweetness, sweetness and bitter aftertaste of stevioside [J]. China Food Additives, 2007, (3): 46.

[5] Yang Yuanzhi, Li Fafa, Ju Zhengyan, et al. Current status and development prospects of stevia applications [J]. Fermentation Science and Technology Newsletter, 2011, 40(1): 40.

[6] Guo Xuexia, Zhao Renbang. The health-promoting function of stevioside and its application in foods [J]. Chinese Food and Nutrition, 2012, 18(1): 32.

[7] Ruan Wenyou. Application of stevioside as a flavoring agent in oral myoinositol solution [J]. Chinese Journal of Pharmacy, 1994, 29(12): 716.

[8] Lu Jianfeng, Zhao Xilan. Exploring the application of stevia in pharmaceuticals [J]. Chinese and Foreign Women's Health Monthly, 2014, (4X): 19.

[9]Tirapelli C R，Ambrosio S R，de Oliveira A M，et al. Hy- potensive action of naturally occurring diterpenes: a thera- peutic  promise  for  the  treatment  of  hypertension   [J] .  Fi toterapia，2010，81（7）:  690.

[10] Jeppesen P B ，Gregersen S，Poulsen C R ，et al. Stevio- side acts directly on pancreatic  β  cells to  secrete  insulin: actions  independent  of  cyclic  adenosine  monophosphate and adenosine triphosphate-sensitivie K+-channel activity[J]. Metabolism-Clinical and Experimental，2000，49（2）: 208.

[11]  Chatsudthipong  V ，Muanprasat  C.  Stevioside  and  related compounds :   therapeutic   benefits   beyond   sweetness [ J ] . Pharmacol Therapeut，2009，121（1）: 41.

[12] Maki k c，Curry L L，Carakostas M C，et al. The hemody- namic effects of rebaudioside A in healthy adults with nor- mal and low-normal blood pressure  [J]. Food Chem Toxi- col ， 2008，46（7）: 40.

[13] Wang T ，Guo M，song X，et al. Stevioside plays an anti- inflammatory  role  by  regulating  the  NF -κB  and  MAPK pathways in S. aureus-infected mouse mammary glands[J]. Inflammation ， 2014，37（5）: 1837.

[14] Yasukawa K ，Kitanaka S，Seo S. Inhibitory effect of ste- vioside on tumor promotion by  12-O-tetradecanoyl-phor-  bol-13-acetate in two-stage carcinogenesis in mouse skin [J]. Biol. Pharm. Bull.，2002，25（11）: 1488.

[15] Alfajaro M M，Rho M C ，Kim H J，et al. Anti-rotavirus effects  by  combination  therapy  of  stevioside and Sophora favescens extract[J].  Res. Vet. Sci.，2014，96（3）: 567.

[16] Sharma D，Puri M，Tiwary A K，et al. Antiamnesic effect of stevioside in scopolamine-treated rats[J]. Indian J. Phar- macol，2010，42（3）: 164.

[17] Alonso Paz E，Cerdeiras M，Fernandez J，et al. Screening of uruguayan medicinal plants for antimicrobial activity[J]. J. Ethnopharmacol，1995，45（1）: 67.

[18] Tadhani M B，Subhash R. In vitro antimicrobial activity of Stevia  rebaudiana  Bertoni  leaves[J] .  Trop  J  Pharm  Res ， 2007，5（1）: 557.

[19]  Jayaraman  S ， Manoharan  M  S ，Illanchezian  S.  In vitro antimicrobial and antitumor activities of Stevia rebaudiana （Asteraceae）leaf extracts  [J]. Trop J Pharm Res，2008，7（4）: 1143.

[20] Ogawa T，Nozaki M，Matsui M. Total synthesis of  stevio- side[J]. Tetrahedron，1980，36（18），2641-2648.

[21] Mosetting E，Nes W R. Stevioside  Ⅱ The  structure of the aglycon[J]. J. Org. Chem.，1955，20: 884.

[22] Xu D Y，Xu M，Lin L ，et al. The effect of isosteviol on hyperglycemia and dyslipidemia induced by lipotoxicity in rats fed with high-fat emulsion[J]. Life Sci，2012，90（1）: 30.

[23]  Wong  K  L ，Yang  H  Y ，Chan P ，et al. Isosteviol as a potassium  channel  opener  to  lower  intracellular  calcium concentrations  in  cultured  aortic  smooth muscle  cells [J]. Planta Med，2004，70（2）: 108.

[24] Xu D Y ，Li Y F ，Wang J P ，et al. The cardioprotective effect of isosteviol on rats with heart ischemia-reperfusion injury[J]. Life Sci，2007，80（4）: 269.

[25] Mizushina Y，Akihisa T，Ukiya M，et al. Structural analy- sis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors  [J].   Life Sci，2005，77

（17）: 2127.

[26] Wei Y，Xi L，Yao X，et al. Quantitative structure-activity relationship analysis of a series of human renal organic an- ion transporter inhibitors[J]. Arch Pharm，2012，345（10）: 759.

[27] Yuajit C，Muanprasat C，Gallagher A R，et al. Steviol re- tards renal cyst growth through reduction of CFTR expres- sion  and  inhibition  of  epithelial  cell  proliferation  in  a mouse  model  of  polycystic  kidney  disease [ J ] .  Biochem Pharmacol，2014，88（3）: 412.

[28] Mao Jinlong. Research progress on the chemical structural modification of the natural active ingredient stevioside [J]. Journal of Beijing Union University, 2011, 25(1): 70.

[29] Wonganan O，Tocharus C，Puedsing C，et al. Potent va-  sorelaxant  analogs  from  chemical  modification  and  bio-  transformation  of isosteviol  [J].  Eur  J  Med  Chem ，2013， 62C（7）: 771.

[30] Zou M，Yu S，Wang Ke，et al. Glycosylation of ent-kau-  rene derivatives and an evaluation of their cytotoxic activi- ties [J]. Chin J Nat Med，2013，11（3）: 0289.

[31] Li  J，Zhang D，Wu X. Synthesis and biological evaluation  of novel  exo  -methylene  cyclopentanone  tetracyclic  diter-  penoids as antitumor agents[J]. Bioorg. Med. Chem. Lett.， 2011，42（21）:130.

[32] Lin L ， Lee L ， Sheu S ， et al.   Study  on  the  Stevioside analogues  of  Steviolbioside ， Steviol ， and  isosteviol   19 - alkyl amide dimers: synthesis and cytotoxic and antibacte- rial activity[J]. Chem. Pharm. Bull.，2004，52（9）: 1117.

[33] Kataev V E ， Militsina O I ， Strobykina I Y，et al. Syn-  thesis  and  anti -tuberculous  activity  of  diesters based on  isosteviol  and  dicarboxylic  acids [J] .  Pharm .  Chem.  J. ， 2006，40（9）: 473.

[34] Garifullin B F ， Strobykina I Y ，Mordovskoi G G ，et al.  Synthesis   and    antituberculosis    activity  of      derivatives  of   the    diterpenoid    isosteviol   with    azine ，  hydrazide， and  hydrazone    moieties  [J]. Chem. Nat. Compd.s，2011， 47（1）: 55.

[35] Khaybullin R N ， Strobykina I Y ， Dobrynin A B ， et al. Synthesis  and  antituberculosis  activity  of novel  unfolded and   macrocyclic   derivatives   of   ent -kauranesteviol [ J ] . Bioorg. Med. Chem. Lett.，2012，22（22）: 6909.

[36] Andreeva O V ， Sharipova R R ， Strobykina I Y ，et al. Development of synthetic  approaches to  macrocyclic   gly-  coterpenoids  on  the  basis  of glucuronic  acid    and  diter- penoid isosteviol[J]. Russ. J. Org. Chem.，2015，51（9）: 1324.

[37] Khaybullin R  N ，Strobykina I Y ，Gubskaya V P ，et  al.  New  maloate  macrocycle  bearing  two  isosteviol  moieties  and its adduct with fullerene C60[J]. Mendeleev Commun， 2011，21（3）: 134.

[38] An J Y ，Zhang Y X ，Wu  Y ，et  al.  Simple  amphiphilic isosteviol - proline conjugates as chiral catalysts for the di- rect asymmetric aldol reaction in the presence of water[J]. Tetrahedron: Asymmetry，2010，21（6）: 688.

[39] An J Y，Wang C C，Xu Y Z，et al. Highly enantioselec-   tive α-aminoxylation reactions catalyzed by isosteviol-pro-  line conjugates in buffered aqueous media  [J]. Catal Lett， 2011，141（8）: 1123.

[40] An J Y，Wang C C，Liu Z P，et al. Isosteviol proline con- jugates  as  highly  efficient  amphiphilic  organocatalysts  for asymmetric three-component mannich reactionsinthe pres- ence of water[J]. Helv Chim Acta，2012，95（1）: 43.

[41] Ma Z W，Liu Y X，Zhang W J，et al. highly enantioselective   Michael    additions of isobutyraldehyde    to   nitroalkenes  promoted  by  amphiphilic  bifunctional  primary amine-thioureas in organic or aqueous medium [J]. Eur. J. Org. Chem. 2011 ，（33）: 6747.

[42]  Ma  Z  W ，Liu  Y  X ，Huo  L  J ，et  al.  Doubly  stereocon- trolled asymmetric Michael addition of acetylacetone to ni- troolefins  promoted  by  an  isosteviol -derived bifunctional thiourea [J].  Tetrahedron:  Asymmetry,2012,23（6 -7）: 443.

[43] Ma Z W，Wu Y，Sun B，et al. Thiourea-catalyzed asym- metric conjugate addition of  a-substituted cyanoacetates to maleimides[J]. Tetrahedron: Asymmetry，2013，24（1）: 7.

[44] Kataev V E，Strobykina I Y，Militsina O I，et al. Isostevi-  ol and  some  of its  derivatives  as  receptors  and  carriers  of amino acid picrates[J]. Tetrahedron Lett .，2006，47（13）: 2137.

[45] Kataev V E，Militsina O I，Strobykina I Y，et al. Synthe-  sis and anti-tubercular activity of diesters on the basis of isosteviol  and  dicarboxylic  acids [J ] .  J.  Pharm.   Chem ， 2006，40（9）: 473.

[46] Zhang T，Wu Y，Gao L，et al. A novel Na+ coordination mediated   supramolecular   organogel  based   on   isosteviol: water-assistedself-assembly ， insitu forming and selective gelation abilities[J]. Soft Matter，2013，9（3）: 638.

[47]   Lohoelter   C ， Brutschy   M ， Lubczyk   D ， et   al.  Novel  supramolecular affinity materials based on （-）-isosteviol  as molecular templates[J]. Beilstein J. Org. Chem.，2013， 9: 2821.

[48] Mamedova V L ， Sharafutdinova D R ， Nikitina K A ，et al. Metal derivatives of diterpenoid  isosteviol[J] .  Russ. J. Gen. Chem.，2014，84（4）: 700.