Chemistry and Pharmacology of Siraitia Grosvenorii: a Review LI Chun1, LIN Li-Mei2, SUI Feng1*, WANG Zhi-Min1, HUO Hai-Ru1, DAI Li1, JIANG Ting-Liang1

Chinese Journal of Natural Chinese Journal of Natural Medicines 2014, 12(2): 0089−0102 Medicines

doi: 10.3724/SP.J.1009.2014.00089

Chemistry and pharmacology of Siraitia grosvenorii: A review LI Chun1, LIN Li-Mei2, SUI Feng1*, WANG Zhi-Min1, HUO Hai-Ru1, DAI Li1, JIANG Ting-Liang1

1 Institute of Chinese Material Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China; 2 Hunan University of Traditional Chinese Medicine, Changsha 410208, China Available online 20 Feb. 2014

[ABSTRACT] Siraitia grosvenorii is a perennial herb endemic to Guangxi province of China. Its fruit, commonly known as Luo hanguo, and has been used for hundreds of years as a natural sweetener and as a traditional medicine for the treatment of pharyngitis, pharyn- geal pain, as well as an anti-tussive remedy in China. Based on ninety-three literary sources, this review summarized the advances in chemistry, biological effects, and toxicity research of S. grosvenorii during the past 30 years. Several different classes of compounds have been isolated or detected from various parts of S. grosvenorii, mainly triterpenoids, flavonoids, polysaccharides, amino acids, and essential oils. Various types of extracts or individual compounds derived from this species exhibited a wide array of biological effects e.g. anti-tussive, phlegm-relieving, anti-oxidant, immunomodulatory, liver-protecting, glucose-lowering, and anti-microbial. The existing research has shown that extracts and individual compounds from S. grosvenorii are basically non-toxic. Finally, some suggestions for further research on specific chemical and pharmacological properties of S. grosvenorii are proposed in this review.

[KEY WORDS] Siraitiagrosvenorii; Cucurbitaceae; Chemical constituents; Pharmacological effects [CLC Number]R284; R285 [Document code] A [Article ID]2095-6975(2014)02-0089-14

[2] . In 1987, S. grosvenorii fruit was listed as a medicinal Introduction and edible species by the China Ministry of Health [3]. To Siraitia grosvenorii is a perennial vine of the Cucur- date, S. grosvenorii fruit has been shown to have the fol- bitaceae family, and its fruit is commonly known as Luo lowing effects: antitussive, anti-asthmatic, anti-oxidation, hanguo (LHG). A total of seven species belong to the ge- liver-protection, glucose-lowering, immunoregulation, and [4] nusSiraitia, and these plants are distributed mostly in south anti-cancer . S. grosvenorii contains triterpenoids, flavon- [5] China, the Indo-China Peninsula, and Indonesia. There are oids, vitamins, proteins, saccharides, and a volatile oil . four species in China, among which Siraitia grosvenorii Mogrosides, a group of triterpenoid glycosides isolated (Swingle) C. Jeffreyex A. M. Lu and ZhiY. Zhang and from S. grosvenorii fruit, are regarded as the main active Siraitiasiamensis(Craib) C. Jeffrey ex S. Q. Zhong & D. ingredients for the sweet taste, and responsible for the Fang are usually used as medicinal plants. S. grosvenoriis main biological effects of S. grosvenorii. LHG products endemic to China, and principally grows in Guangxi prov- have been approved as dietary supplements in Japan, the ince, where it has been cultivated for more than 200 years [1] United States, New Zealand and Australia. Currently, the (Fig. 1). S. grosvenorii fruit has been used for centuries in extracts or some compounds from LHG are used mainly for China as a natural sweetener and as a traditional medicine their anti-tussive, expectorant, anti-diabetic, or sweet prop- for the treatment of lung congestion, colds, and sore throat erties in various Chinese herbal compound prescriptions or dietary supplements. In this review, the research progress of S. grosvenorii during the last 30 years is summarized.

[Received on] 15-Dec.-2012 Chemical Composition [Research funding] This project was supported by the Beijing Joint Project Specific Funds, National Natural Science Foundation of Several different classes of compounds were previously China (Nos. 30873393, 81274112, 81373986) and the Beijing Mu- isolated from various parts of S. grosvenorii, with the main nicipal Natural Science Foundation (Nos. 7112098, 7132152). groups being triterpenoids, particularly the cucurbitane-type [*Corresponding author] SUI Feng: Prof., Tel.: 86-10-64041008, triterpenoid glycosides, flavonoids, polysaccharides, proteins Fax: 86-10-64041008, E-mail: [email protected] These authors have no conflict of interest to declare. and essential oils.

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of one part in ten thousand are 425 and 563 times as sweet as that of 5% sucrose, respectively [17]. In addition, a series of cucurbitane tetracyclic triterpenoid acids, including siratic acids A-F were isolated from the root of S. grosvenorii [18-21]. So far, a total of forty-seven triterpenoids (Table 1) have been isolated, and/or detected in the fruit, roots, or leaf materials of S. grosvenorii. As shown in studies of the structure-taste relationships for the glycosides of 3β-hydroxy-cucurbit-5-ene derivatives, the number of glucose units, the oxygen function at the 11-position of the aglycone moiety, the location of the gly- cosyl units, and the hydroxylation of the side chain, are ap- parently responsible for the perception of taste [17, 30-31]. The Fig. 1 Line drawing of S. grosvenorii: 1. stem; 2. leaf; 3. presence of at least three sugar units in the molecule is essen- inflorescence; 4. fruit tial for the occurrence of taste. For example, compounds 6, 9

Cucurbitane glycosides are the main components, and also and 13 are tasteless due to their failure to meet the the active ingredients of S. grosvenorii fruit. Ever since above-mentioned basic structural requirements. Glycosides of Takemoto et al. isolated mogrosides IV, V, and VI from S. the 11α-hydroxy compounds taste sweet, such as compounds grosvenorii fruit in 1983 [6-8], more than thirty similar 10, 11, 14 and 15, while glycosides in the 11β-hydroxy series compounds have been obtained from the fruit [9-13]. are tasteless, and the 11-oxo compounds, as well as the dehy- These compounds share the mogrolaglycone structure, dro derivatives, taste bitter. The relationship between the [10α-cucurbit-5-ene-3β, 11α, 24(R), 25-tetraol], with two to allocation of glucosyl units and sweetness is also noteworthy. six glucose units attached (see Fig. 2). Most of them taste SiamenosideI (16), which has four glucosyl units, is the sweet, so they are collectively called mogrosides, and are the sweetest compound among the glycosides of this type so far main active components of S. grosvenorii fruit. Mogrosides isolated, and showed a similar sweetness to mogrosideV (14) [14] are present at 1.19% in the fresh fruit , and 3.82% in the which has five glucosyl units, while mogroside IVA (10) [15] dried fruit of S. grosvenorii . Mogroside V is the main and IVE (11), with the same number of glucosyl units as 16, is component, with a content of 0.5%−1.4% in the dried fruit of less sweet than 16. Additionally, hydroxylation of the side S. grosvenorii. Siamenoside I is the sweetest among the cu- chain also affects the taste. For example, the bitter 11-oxo curbitane glycosides so far isolated [16-17]. The sweetness glycoside became sweet on hydroxylation of the side chain values of mogroside V and siamenoside I at the concentration double bond with osmium tetroxide.

R1 R2 R3 R4 R5 R1 R2 R3 R4 R5

1 H H H α-OH H2 16 glc H α-OH H2

2 H glc H α-OH H2 H H 17 α-OH 2

3 glc H H α-OH H2 18 H α-OH H2

4 H H α-OH H2 19 glc H α-OH H2

5 H H α-OH H2 20 H H H =O H2

6 glc glc H α-OH H2 21 H glc H =O H2

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7 H H α-OH H2 22 glc H H =O H2

8 glc H α-OH H2 23 H H =O H2

9 glc H α-OH H2 24 glc glc H =O H2

10 H α-OH H2 25 glc H =O H2

11 H α-OH H2 26 H =O H2

12 glc H glc α-OH H2 27 H =O H2

13 glc H α-OH H2 28 glc glc H α-OH =O

14 H α-OH H2 29 H α-OH =O

15 H H 30 glc H H H α-OH 2 2 2

Fig. 2 Structures of thetriterpenoid compounds isolated from S. grosvenorii

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Table 1 Triterpenoid compounds isolated from S. grosvenorii No. Compound name Plant parts References 1 Mogrol Fruit [6-9]

2 Mogroside IA (MogrosideIA1) Fruit [8-9]

3 Mogroside IE1 Fruit [8-9]

4 Mogroside IIA1 Fruit [8, 10]

5 Mogroside IIA2 Hydrolysis product [8] 6 Mogroside IIE Fruit [8-9, 11, 17-19]

7 Mogroside IIIA1 Fruit [8, 12]

8 Mogroside IIIA2 Fruit [8, 10] 9 Mogroside IIIE Fruit [8, 17]

10 Mogroside IVA Fruit [8-9, 19, 12-13]

11 Mogroside IVE Fruit [8-9, 12, 18] 12 Mogroside IIB Fruit [10] 13 Mogroside III Fruit [9, 17-19] 14 Mogroside V Fruit [8-9, 12-13, 17-18] 15 Mogroside VI Fruit [8] 16 Siamenoside I Fruit [9, 12, 17] 17 Neomogroside Fresh Fruit [18] 18 Isomogroside V Fruit [13] 19 Grosmomoside I Fruit [20] 20 11-Oxomogrol Hydrolysis product [9]

21 11-Oxomogroside IA1 Fruit [9, 19]

22 11-Oxomogroside IE1 Fruit [9]

23 11-Oxomogroside IIA1 Fruit [10] 24 11-Oxomogroside IIE Unripe fruit [19] 25 11-Oxomogroside III Unripe fruit [21]

26 11-Oxomogroside IVA Fruit [10, 21] 27 11-Oxomogroside V Fruit [9, 17] 28 7-Oxomogroside IIE Fruit [10] 29 7-Oxomogroside V Fruit [10] 30 11-DeoxymogrosideIII Fruit [10, 21]

31 20-Hydroxy-11-oxomogroside IA1 Unripe fruit [19]

32 5α, 6α-Epoxymogroside IE1 Fruit [9] 33 5-Dehydro-karounidiol dibenzoate Fruit [9] 34 Karounidiol dibenzoate Fruit [9] 35 Karounidiol 3-benzoate Fruit [9] 36 Isomultiflorenol Fruit [9] 37 β-Amyrin Fruit [9, 22] 38 10α-Cucurbitadienol Fruit [9] 39 Siratic acid A Root [23-24] 40 Siratic acid B Root [23-24] 41 Siratic acid C Root [23-24] 42 Siratic acid D Root [25] 43 Siratic acid E Root [18] 44 Siraitic acid F Root [26] 45 Mogroester Fruit [27-28] 46 Siraitic acid IIA Root [29] 47 Siraitic acid IIB Root [29]

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Li et al studied the variation of mogrol glycosides in S. the pulp (7.55%) and lowest in the seeds (3.12%) [46]. In 2003, grosvenorii fruit during different growth periods. The results two polysaccharides, SGPS1 and SGPS2, were first isolated showed that the mogrol glycosides emerged after 5 days of fromLHG and their relative molecular weights were deter- pollination, with mogroside IIE (6) as the main component in mined by HPLC to be 430 000 and 650 000, respectively [47]. the young fruits in the first 30 days. Mogroside III (13) started After that, Li et al reported on the monosaccharide composi- to appear after 30 days and reached the highest content in the tion and connection mode of SGPS1 and SGPS2 with the

55th day, then mogroside IVA (10) and mogroside IVE (11) improvement of the extraction and purification technology formed, and their contents reached the peak in the 70th day, [48-51]. Finally, SGPSl was determined as an and mogroside V(14) was generated after 70 days, and devel- acidicheteropolysaccharide, which was composed of Rha, oped to be the main sweet component after 85 days of pollina- Ara, Xyl, Gal, Glc, and GlcA in a molar ratio of 1.00 : 2.30 : [32] tion, then the fruit began to be ripe . Consequently, the op- 1.40 : 9.07 : 39.53 : 2.46. Its basic structure was made up of timum harvest time of S. grosvenorii fruit should be in the (1→4) linked glucose and (1→3) linked galactose residues in 90th day after pollination. Other studies have come to similar the main chain, and (1→3) linkedglucose, (1→6) linked glu- conclusions [33-34]. cose, (1→4) linked galactose, and (1→2) linked rhamnose Regarding the phenolic compounds of S. grosvenorii, units in the side chains. Sidechains attached the main chain to several authors reported the presence of various flavonoids in 2-O, 3-O, and 6-O of glucose, and to 6-O of galactose. On the fruit, leaves, stems, and flowers of the plant [35-38]. These average, among the twenty main chain residues, there were compounds possessed the aglycone of kaempferol or five branches. By means of acid hydrolysis and TLC analy- quercetin. So far, a total of seven flavonoid compounds were isolated and/or detected from S. grosvenorii. With quercetin sis, SGPS2 was determined to contain Rha and GlcA. 13 and kaempferol as reference substances, the content of the However, the IR and C NMR analysis results showed that total flavones in the fresh fruits, and the mogrosides in the SGPS2 was comprised of Rha, GlcA, and amino sugars. leaves of S. grosvenorii were determined by HPLC. The re- Yan et al studied the composition of the polysaccharides from sults showed the content of total flavones was about 5−10 mg S. grosvenoriiroot,and investigated its effect on subcutaneous [52] in a fresh fruit, with 1.42% as the mogrosides, and 1.62% in H22-implanted mice . Asa result, the polysaccharides were the leaf of S. grosvenrii, respectively [39-40]. Furthermore, the shown to be composed of glucose, arabinose, and xylose, and total flavone contents in the various parts of S. grosvenorii compared with the model group, the tumor growth was not were different, and the total flavone contents from high to significantly suppressed (inhibition rate < 50%), but the in- low were leaf (3.718%) > stem (1.688%) > root (0.129%) [41]. dex of thymus increased (P < 0.01), and the index of spleen For the dried fruits of different sizes, the content of decreased (P < 0.01) in the polysaccharide-treated groups．As kaempferol was between 1.63% to 3.49%, and the contents early as the 1980s, Xu et al measured the contents of various from high to low were peel (4.71%) > pulp (0.855%) > seed types of nutritious ingredients in S. grosvenorii fruit [53]. The (0.197%) [42]. However, if kaempferitrin was used as refer- content of crude protein was between 8.67% to 13.35% in the ence substance, the content of total flavones determined by dried fruit of the wild type and three cultivars of S. grosve- UV in the leaf and stem of S. grosvenrii were 5.85% and norii. Furthermore, the hydrolysis products of S. grosvenorii 0.29%, and the content of kaempferitrin was 1.76% and fruit contained eighteen amino acids, including eight of the 0.167%, respectively [43-44]. Chen et al studied the variation of essential amino acids. Among the eighteen amino acids, the mogroside V and the flavonol glycosides in S. grosvenorii content of aspartic acid was the highest (939−1 125 mg/100 g fruit at different growth stages, and the results showed since dried fruits), and the content of γ-aminobutyric acid was the the date of fruiting, the content of mogroside V increased lowest (15.9−35.6 mg/100 g dried fruits) [54]. Additionally, rapidly after 50 days, and became stable after 80 days, while there were a lot of sugars in the dried fruit of S. grosvenorii the content of the flavonol glycosides increased rapidly from mainly fructose, glucose, and non-reducing sugars [53]. The the 40th to 50th days, and reached the highest about 50 days content of total sugar in S. grosvenorii fruit ranged from later, and started to decrease after 60 days, and became stable 25.17% to 38.31%, and that of the reducing sugars in it [33] when it dropped to the level of the 20th day . was between 16.11% and 32.74%. Besides the flavonoids, three phenolic acids, two an- S. grosvenorii fruit is rich in vitamin C. The content of thraquinones, three alkaloids, three sterols, three aliphatic vitamin C in the fresh fruit of S. grosvenorii reached 339−461 acids, and three other compounds have been isolated from the mg/100 g, but it decreased to 24.6−38.7 mg/100 g in the dried [22, 28, 45] fruit or leaf of S. grosvenorii . The structures of fla- fruit [53]. With the differences in varieties, forms, producing vonoids and other related compounds of S. grosvenorii are area, growing period, and ripeness, the content of vitamin C shown in Table 2 and Fig. 3. in S. Grosvenorii fruit was remarkably different, but was Polysaccharides are also important ingredients of S. several times higher than that in citrus, apples, pears, grapes, grosvenrii. The content of polysaccharide was between and persimmons. Within a certain range, the content of vi- 2.88% to 5.65% in LHG of different sizes, with the highest in tamin C was positively correlated with altitude, and 30 days

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Table 2 Flavonoids and related compounds isolated from S. grosvenorii No. Compound name Plant parts References 48 Kaempferol Fruit, Leaf, Flower [35, 37] 49 Kaempferol 7-O-α-L-rhamnopyranoside Fruit, Flower [35, 37] Kaempferol 3-O-α-L-rhamnopyranoside-7-O- 50 Fruit, Flower [35-37] [β-D-glucopyranosyl(1→2)]-α-L-rhamnopyranoside Kaempferol 3, 7-di-O-α-L-rhamnopyranoside 51 Leaf, Fresh fruit [35-36, 38] (kaempferitrin) Quercetin-3-O-β-D-glucopyranoside-7-O-α-L- 52 Leaf [38] rhamnopyranoside 53 7-Methoxy-kaempferol 3-O-α-L-rhamnopyranoside Flower [37] 54 7-Methoxy-kaempferol 3-O-β-D-glucopyranoside Flower [37] 55 Magnolol Fruit [28] 56 Vanillic acid Fruit [45] 57 p-Hydroxybenzylic acid Leaf [22] 58 1-Acetyl-β-carboline Fruit [45] 59 Cyclo-(leu-pro) Fruit [45] 60 Cyclo-(ala-pro) Fruit [45] 61 Aloe emodin Leaf [22] 62 Aloe-emodin acetate Leaf [22] 63 5, 8-Epidioxy-24(R)-methylcholesta-6, 22-dien-3β-ol Leaf [22] 64 β-Sitosterol Fruit [45] 65 Daucosterol Leaf [22] 66 Succinic acid Fruit [28] 67 n-Hexadecanoic acid Leaf [22] 68 12-Methyltetradecanoic acid Leaf [22] 69 5, 5'-Oxydimethylene-bis-(2-furfural) Fruit [28] 70 5-(Hydroxymethyl)-furoic acid Fruit [28] 71 5-Hydroxymaltol Fruit [45] after flowering, the content of vitamin C increased gradu- from dried and fresh fruit were significantly different. ally in the fruit of young plants [55]. n-Hexadecanoic acid (45.609%) and 9, 12-octadecadienoic S. grosvenorii seed oil contained a number and large acid (36.151%) were the most abundant in the essential oil quantity of fatty aldehydes, such as fagni aldehyde, valeral- from dried fruit [59], while 2-butenoic acid butyl ester dehyde, hexanal, and nonanal, and the content of fagni alde- (20.80%) and 2-heptanol (13.86%) were the main compo- [56] hyde in the oil reached 52.14% . The oil content of S. gros- nents of the fresh fruit. venorii seed kernel was 48.5%, among which the first three Both the ripe fruits and roots of S. grosvenorii contained components were squalene (51.52%), [Z, Z]-9, 12- octade- sixteen essential trace elements and a wide variety of inor- cadienoic acids (23.89%) and 3-hydroxy-1, 6, 10, 14, 18, 22- ganic elements. Its fruit has higher contents of potassium, tetracosahexaene [57]. HPLC analysis showed the content of calcium and magnesium than its root, with a ratio of 1.229%, squalene was 12.5% in the seed kernel oil of S. grosvenorii 0.667% and 0.55%, respectively [61]. [58]. It was reported that squalene had body-building and anti-fatigue functions, and that it can be used to treat liver Additionally, the content of selenium in S. grosvenorii −1 diseases. fruit reached 0.186 4 mg·kg , which was 2−4 times high than Although the essential oil is sometimes mentioned that in grains. Mo et al. measured the contents of Al, Cd, Cu, among the chemical components of S. grosvenorii, there are Fe, Mg, Mn, P, Pb and Zn in S. grosvenorii fruit by only a few reports dealing with its detailed analysis. The ICP-AESN, and the results showed that the content of these volatile oil content in the dried fruit of S. grosvenorii was nine trace elements was between 0.1−2 440 ppm, and that the about 0.2%−0.3%, but it was only 0.03% in the fresh fruit concentrations in order were P highest, then Mg, Fe, Zn, Mn, [62] [59-60]. Moreover, the main components of the volatile oil Al, Cu, Pd and Cd .

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Fig. 3 Structures of flavonoids and related compounds from S. grosvenorii

Anti-tussive, phlegm-expelling, and dyspnea-relieving functions Biological Activities The anti-tussive, phlegm-expelling, and dyspnea reliev- Traditional Chinese medicine (TCM) believes that S. ing activities of S. grosvenorii fruit have been reported for a grosvenorii fruits sweet, sour, and cool, and it has many long time. Oral consumption of the water extract of S. gros- −1 physiological functions, such as clearing away the lung-heat, venorii fruit at doses of 25 or 50 g·kg markedly reduced the eliminating phlegm, and relaxing bowels, and it can be used mouse cough induced by ammonia water or sulfur dioxide in [63] to treat cough, sore throat, and constipation. TCM also holds a dose-dependent manner . Mogrosides (purity > 98%), the that S. grosvenorii root can be used for the treatment of car- main active constituents of LHG, also displayed a strong buncle, furuncle, and swollen boils, and that the fruit hair inhibitory effect on the mouse cough evoked by inhalation of can be used to heal cuts. As summarized below, the pharma- ammonia water dose-dependently, with the minimum inhibi- −1 [64] cological and clinical investigations carried out during the tory concentration of 80 mg·kg . In another in vivo last 30 years have shown that extracts and individual com- mouse study, oral administration of doses of 75, 150 or 300 −1 pounds (especially the mogrosides) isolated from different mg·kg of mogroside V (purity > 94%) derived from LHG parts of the plant have specific biological effects, including could significantly reduce the number of coughing times relieving cough, eliminating phlegm, preventing asthma, induced by ammonia water, and the latency to the time that immunostimulation, eliminating free radicals to prevent oxi- the mice start to cough could also be prolonged for the high −1 [65] dizing pathology, regulating blood sugar and doses beyond 75 mg·kg . It was also reported that hista- ing blood-fat, anti-bacterial, anticarcinogen, and anti-fatigue mine-induced mouse trachea spasm could be distinctly an- functions. tagonized by oral administration of mogroside V at doses of

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2.5 or 5.0 g·L−1 [65]. of red blood cells, and lipid peroxidation induced by Fe2+ or

Regarding the effect of S. grosvenorii fruit on cough, the H2O2 in vitro indicating that mogrosides has antioxidant ef- water extract showed a significant phlegm-releasing action fects, and mogroside V may be the main antioxidant compo- on mice by increasing the excretion of phenol red from the nent of the mogrosides [69]. mouse trachea, as well as the excretion of phlegm from the In a more recent in vitro study with different extracts rat trachea [63]. It was reported in a mouse model study that from the stem of S. grosvenorii, the water, ethanol, ethyl mogrosides at doses of 50, 100 or 200 mg·kg−1 could signifi- acetate, and chloroform extracts all exhibited excellent anti- cantly increase the excreted amount of phlegm oxidant activity, superior to the control group butylatedhy- dose-dependently. In addition, mogrosides could promote droxytoluene (BHT), but inferior to the control with rutin significantly the motility of the ciliated cells of the frog res- applied [70]. Similarly, the anti-oxidant capacities of the total piratory tract when locally applied [63]. flavones from the leaves of S. grosvenorii were evaluated in a Immunostimulatory actions recent in vitro study, and it was found that the radical scav- Ever since the 1990s, the immunomodulatory effect of enging abilities of the tested compounds were much higher LHG has been reported in a number of in vivo experiments, than that of BHT, a synthetic anti-oxidant, suggesting that it performed mainly with rats and mice. According to the re- might be pursued as a potential natural food anti-oxidant [71]. sults of an earlier experiment [66], oral administration of the The anti-oxidant capacity of five flavonol glycosides isolated aqueous extract of S. grosvenorii fruit to rats at doses of 25 or from the flowers of S. grosvenorii was also conducted re- 50 g·kg−1 showed an evident increase in the percentage of cently by the FRAP, TEAC, and ORAC assays. At the same acid α-naphthyl acetate acid enzyme-positive lymphocytes in time, the structure-activity relationships of these compounds the peripheral blood, as well as in the ratio of rosette-forming were investigated. The results showed that two out of the five cells. No marked influence was found on the neutrophil flavonoids had significant antioxidant activity. Based on the phagocytosis rate in the peripheral blood. These data support correlation between the structure and activity, 7-hydroxyl and the fact that LHG could significantly improve both cellular 3-hydroxyl groups on the aglycone were found to be posi- and humoral immunity processes, with no effect on the tively related to the activity, and that methylation of non-specific immunity of the rats. In an another study with the 7-hydroxy group, and/or glycosylation of the 3-hydroxy mice, oral administration of S. grosvenorii fruit wa- group decreases activity [37]. ter-extraction, alcohol-precipitation extract displayed a Liver protection and transaminase reduction activities marked inhibitory effect on the induced decrease of mononu- Administration of S. grosvenorii fruit water-extraction clear phagocytic function by hydrocortisone, suggesting S. alcohol-precipitation extract at a dose of 50 g·kg−1 by gavage grosvenorii fruit possesses the potential to enhance the re- could reduce the biological activities of transaminase in the duced immune function of the body [63]. serum of the tested mice with liver injury induced by carbon In addition, S. grosvenorii fruit-derived mogrosides also tetrachloride or thioacetamide [63]. Similarly, protection showed a marked positive effect on the inhibited mouse against mouse liver injury was also shown in another in vivo macrophage phagocytic function and T cell proliferation by study performed recently by Yao et al. They demonstrated cyclophosphamide (CTX) in comparison with the control that oral administration of a 75% ethanol extract of S. gros- group. No significant influence on the immune cells of nor- venorii fruit could promote both SOD and GSH activities in mal mice was found, indicating that mogrosides could im- the liver tissue of the experimental mice [72]. Mogrosides prove the immune functions markedly [67]. Recently, it was were also shown to be effective in the reduction of carbon reported that in a study in mice, S. grosvenorii polysaccharide, tetrachloride-induced acute liver injury, tuberculosis vaccine over a seven-day administration period, could significantly plus lipopolysaccharide-induced immunological liver injury, elevate the mass of immune organs, such as thymus and and carbon tetrachloride-induced chronic liver injury. An spleen. In addition, the level of serum hemolysin, along anti-lipid peroxidation-associated effect of mogrosides on with boosted peritoneal macrophage phagocytic percentage, improving pathological conditions of liver tissue was re- phagocytic index of chicken red blood cells, and lymphocyte ported by Wang et al [73-74]. transformation rate, were strongly suggestive of its immune Anti-diabetic effects system-strengthening actions [68]. The effect of S. grosvenorii fruit extract, and individual Anti-oxidative effects compounds derived from S. grosvenorii fruit, on diabetes The anti-oxidant effects of LHG extracts and individual has been extensively studied, mainly in in vivo experiments. compounds from various parts of S. grosvenorii, have been For example, 30-day administration of 0.5, 1.0 or 3.0 g·kg−1 S. extensively studied. In an in vivo study performed with grosvenorii fruit powder or extract was conducted by Qi et al. rats by the test methods of D-deoxyribose, superoxide anion, in mice. The results showed that all of the doses of S. gros- and spectrophotometry, it was found that both mogrosides venorii fruit extract could significantly lower the fasting and (total mogroside > 98%) and mogroside V displayed signifi- after-meal blood glucose of diabetic mice induced by alloxan cant inhibitory effects on the oxygen free radical, hemolysis (P < 0.01). Furthermore, the effect for the S. grosvenorii fruit

– 96 – LI Chun, et al. / Chin J Nat Med, 2014, 12(2): 89−102 powder was dose-dependent, with the high dose showing the S. grosvenorii fruitextract supplemented group, which the best effect. On the other hand, for the S. grosvenorii fruit may explain the greater capacity to secrete insulin during the extract, an inverse relationship between activities and doses OGTT. Thiobarbituric acid-reactive substances in both the was displayed, with the low dose having the best effect [75]. liver and the plasma were lower in the S. grosvenorii fruitex- To study the effect of mogroside extract on splenic lympho- tract supplemented group than in the control, suggesting that cytes subsets and expression levels of the cytokines of type 1 an absorbable component in S. grosvenorii fruit extract has diabetes mellitus (TIDM) mice, an experiment with Balb/c an anti-oxidative effect on lipid peroxidation, thereby coun- mice was designed and conducted by Chen et al [76]. After teracting the oxidative stress caused by a diabetic state. Ex- 30-day gavage administration of mogroside extract at a dose creted urine volume and urinary albumin level for 24 h of 150 or 300 mg·kg−1 body weight, the expression levels of were both reduced in the S. grosvenorii fruit extract supple- IFN-γ and TNF-α at both mRNA and protein were markedly mented group, suggesting the attenuation of kidney damage down-regulated, as well as lowering the blood glucose levels caused by diabetes. These data indicated that S. grosvenorii of the tested mice. Also, both the abnormal values for the fruit extract supplementation may be suitable for the type 2 [78] CD4/CD8 ratio and the number of CD4-positive splenic lym- diabetes, along with its sweet characteristics . phocytes in diabetic mice returned to normal levels at the end Anticancer effects of the experiment, and the expression level of IL-4 was pro- To search for cancer chemopreventive agents from natu- moted [77]. To evaluate the action of a mogroside extract (MG) ral resources, many phytochemicals and food additives from S. grosvenorii fruit on reducing oxidative stress, hyper- have been screened. An experimental in vivo study perglycemia, and hyperlipidemia in alloxan-induced diabetic formed by Takasaki et al showed that the two natural sweet- mice, as well as on the oxygen free radical scavenging activ- eners mogroside V and 11-oxo-mogroside V, isolated from ity in vitro, experimental studies by Qi et al [78] were carried LHG, have a strong inhibitory effect on the primary screening out. As a result, a significant increase in the levels of serum test indicated by the induction of Epstein-Barr virus early glucose, total cholesterol (TC), triacylglycerol (TG), and antigen (EBV-EA) by a tumor promoter, 12-O-tetradecanoyl- hepatic malondialdehyde (MDA), as well as a reduction in phorbol-13-acetate (TPA). These sweet glycosides with a the level of hepatic high-density lipoprotein cholesterol cucurbitane triterpenoida glycone exhibited signiﬁcant in- (HDL-C) associated with diminution of the corresponding hibitory effects in the two-stage carcinogenesis test of mouse skin tumors induced by peroxynitrite (ONOO ) as an initiator antioxidant enzymes, such as glutathione peroxidase 2 and TPA as a promoter. Furthermore, 11-oxo-mogroside V (GSH-Px) and superoxide dismutase, were observed in all also exhibited a remarkable inhibitory effect in a two-stage diabetic mice. Moreover, treatment of the diabetic mice with carcinogenesis test of mouse skin tumor induced by 7, MG (100, 300 and 500 mg·kg−1 body weight) for four weeks 12-dimethylbenz[a]-anthracene (DMBA) as an initiator and significantly decreased serum glucose, TC, TG, and hepatic TPA as a promoter [79]. MDA levels (P < 0.05), whereas it increased serum HDL-C To clarify the cancer chemopreventive action of the S. level and reactivated the hepatic antioxidant enzymes (P < grosvenorii fruit waterextract, which has anti-oxidative prop- 0.05) in alloxan-induced diabetic mice (P < 0.05). The hypo- erties, a two-stage liver carcinogenesis model in partially glycemic, hypolipidemic, and anti-oxidative activities of MG hepatectomized male ICR mice was employed by Matsumoto −1 (100 mg·kg body weight) were all higher compared with all et al [81]. The mice were maintained on a diet containing di- of the other diabetic groups. Furthermore, the antioxidant cyclanil at a concentration of 1 500 ppm for nine weeks after capacity evaluated in vitro showed that MG and mogroside V, a single intraperitoneal injection of diethylnitrosamine (DEN) which was the main component of MG, possessed strong, at a dose of 30 mg·kg−1 to experimentally induce the patho- oxygen free radical scavenging activities. These results dem- logical model, and were then given water containing 2 500 onstrate that the extract may have the capacity to inhibit hy- ppm of LHG extract for eleven weeks after two week’s ad- perglycemia induced by diabetes, and the data suggest that ministration on dicyclanil. The LHG extract inhibited the administration of the extract may be helpful in the prevention induction of γ-glutamyl transpeptidase-positive hepatocytes, of diabetic complications associated with oxidative stress and lipid peroxidation, and gene expression of Cyp1a1, all of hyperlipidemia [77]. To investigate the anti-diabetic effect of which were caused by dicyclanil. To examine whether the LHG, an experimental study of type 2 diabetic Goto-Kakizaki LHG extract indirectly inhibited Cyp1a1 expression in- (GK) rats was designed and conducted. After 13-week ad- duced by inhibition of aryl hydrocarbon receptor ministration of a diet supplemented with 0.4% of the S. gros- (Ahr)-mediated signal transduction caused by dicyclanil, venorii fruit extract, the anti-diabetic effects were evaluated. mice with high (C57BL/6J mice) and low affinities (DBA/2J The LHG extract had no effect on food intake or body weight. mice) to Ahr were given dicyclanil-containing diet and/or S. In oral glucose tolerance tests (OGTT), LHG extract supple- grosvenoriiextract-containing tap water for two weeks. mentation improved the insulin response at 15 min (P < 0.05), Cyp1a1 gene expression was significantly lower in C57BL/6J and reduced the plasma glucose level at 120 min after the mice administered dicyclanil + LHG extract than in glucose administration (P < 0.05). The total amount of insulin C57BL/6J mice administered dicyclanil alone. There was no in the whole pancreas taken from fasting rats was higher in significant difference in the Cyp1a1 expression between

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DBA/2J mice administered dicyclanil + LHG extract and from LHG was further researched by Pan et al using in vitro dicyclanil alone. These results suggest that the S. grosvenorii murine RAW264.7 cells by the methods of Western blotting extract suppressed the induction of Cyp1a1, leading to inhibi- and reverse transcriptase polymerase chain reaction (RT-PCR) tion of reactive oxygen species (ROS) generation, and con- analyses [83]. The results demonstrated that the mogrosides sequently inhibited hepatocarcinogenesis, probably due to significantly blocked protein and mRNA expression of iNOS suppression of Ahr activity. and COX-2 in LPS-induced macrophages. Treatment with Inhibitory effect on bacteria mogrosides resulted in the reduction of LPS-induced nuclear The anti-bacterial activity of the ethanol extracts from S. translocation of nuclear factor-κB (NF-κB) subunit and the grosvenoriileaf and stem on Staphylococcus aureus, Es- dependent transcriptional activity of NF-κB, by blocking cherichia coli, Pseudomonas aeruginosa, Micrococcus luteus phosphorylation of inhibitor jB(IjB)a and p65, and the sub- and Candida albicans were performed by Ye et al [82]. The sequent degradation of IjBa. Transient transfection experi- samples for test were extracted using 50% ethanol, and ments using NF-κB reporter constructs indicated that the the bacteriostatic rate was measured. As a result, the higher mogrosides inhibited the transcriptional activity of NF-κB in the concentration of the ethanol extracts of S. grosvenorii leaf LPS-stimulated mouse macrophages. Mogrosides also inhib- and stem was, the higher the bacteriostatic rate. The bacterio- ited LPS-induced activation of PI3K/Akt, extracellular sig- static rates of the ethanol extracts of leaf and stem on Pseu- nal-regulated kinase1/2, and p38MAPK. Taken together, domonas aeruginosa reached 90.9% and 76.7%, respectively these results show that the mogrosides could down-regulate when the concentration was 50 mg·mL−1. It was also found inflammatory iNOS and COX-2 gene expression in macro- that bacteriostatic rates of the ethanol extracts of leaf and phages by inhibiting the activation of NF-κB, through inter- stem on Staphylococcus aureus, Micrococcus luteus, and fering with the activation of PI3K/Akt/IKK and MAPK. Candida albicans were all below 50%. These results have important implications for using the To explore the antibacterial activity of S. grosvenorii, mogrosides towards the development of effective [83] various extracts were prepared from S. grosvenorii fruit, anti-inflammatory agents . leaves, vine, and roots, and the bacterial activity of these Improving physiological function extracts were tested using a colorimetric detection method Both the ethanol extract of S. grosvenorii fruit and the and agar plating. The results showed that different extracts total flavonoids of S. grosvenorii leaf had effects on improv- from all parts of S. grosvenorii exhibited strong antibacterial ing physiological function of experimental animals. Based on activity against the oral bacteria Streptococcus mutans. To the incremental load swimming training experimental model, further identify the bioactive fractions, the extracts were pu- Yao et al investigated the effect of the ethanol extract of S. rified with Amberlite chromatography, and the purified fac- grosvenorii fruit on the capacity of hypoxia tolerance, [85] tions were further tested for antibacterial activity. The anti- heat-resistance, and exercise of swimming mice . The re- bacterial activities of the positive fractions were further sults showed that the improvement of physiological function tested by the blood agar plating method. The experimental of mice was proportional to the dosage of the ethanol extract data demonstrated that various extracts of S. grosvenorii within a certain range, but as the dosage continues to increase, −1 −1 leaves, vines, fruits, and roots exhibited strong antibacterial the inhibition effect decreased, and 15 g·kg ·d was the activities, suggesting these parts of S. grosvenorii may have optimum dosage to exert an effect. Further study found that the potential to be used for pharmaceutical preparations and the exhaustion swimming time of mice was significantly dietary supplements to treats infection [84]. The capacity of prolonged after administering S. grosvenorii fruit ethanol nine compounds isolated from the leaves of S. grosvenorii extract. Furthermore, immediately after exhaustive exercise, were evaluated in vitro against the growth of oral bacterial and 24 h after recovery, hemoglobin and the activity of su- species S. mutans, Actinobacillus actinomycetemcomitans, peroxide dismutase and glutathione peroxidase in the liver Fusobacterium nucleatum, and the yeast C. albicans, and were higher in the treated group than that in the control group, their minimum inhibition concentrations were determined. while blood lactate, serum lactate dehydrogenase, alanine The result showed aloe emodin had the strongest activities aminotransferase, and the content of malonaldehyde (MAD) [73] against all the tested bacteria and yeast, with minimum in- in liver were lower . MAD is the metabolic product of hibitory concentration values ranging from 1.22 to 12.20 lipid peroxide, and it can indirectly reflect the level of free µg·mL−1 [22]. radicals in the body. These results established that the ethanol Anti-inflammatory action extract of S. grosvenorii fruit could significantly inhibit the A tablet made from an extract of LHG significantly in- increase of MAD content, timely eliminate excess free radi- hibited mouse swelling induced by cotton, ear swelling in- cals, prevent or inhibit the body lipid peroxidation, and has a duced by dimethylbenzene, and paw swelling induced by protective effect on the damage of liver tissue or its mem- carrageenan, respectively. Additionally, the considerable brane structure caused by movement. Chen et al found the pain-relieving effect of this preparation was also demon- duration of endurance training for rats swimming to exhaus- strated in an acetic acid writhing test [63]. The molecular basis tion was prolonged after applying the total flavonoid prepara- of the anti-inflammatory effects of the mogrosides derived tion of S. grosvenorii leaf [86]. Furthermore, the total flavon-

– 98 – LI Chun, et al. / Chin J Nat Med, 2014, 12(2): 89−102 oid fraction of S. grosvenorii leaf showed anti-oxidative ef- heart, liver, kidney, lung and spleen. Therefore, the fects and protection against free-radical damage in the rat mogrosides were actually non-toxic. experiment. PureLo, a powdered concentrated of S. grosvenorii fruit, Other effects has been introduced for use in the US as a tabletop sweetener In in vitro experiments, the water extract of S. grosve- of foods. The product is produced by Guilin JiFuSi Biotech- norii fruit antagonized the spasm of isolated ileum of rabbit nology Limited Corporation. The overall sweetness of the caused by acetylcholine or adrenaline, and also exhibited the PureLo concentrate has been estimated to be up to 200 times same effect on the spasm of isolated ileum of mice caused by that of sucrose. The active components responsible for the acetylcholine [63]. Hossen et al found that the water extract sweetness are mogrosides, members of the family of triter- and glycoside fraction (a complex of sweet components) of S. pene glycosides, which are shown by high-performance liq- grosvenorii fruit significantly inhibited histamine-induced uid chromatography to constitute 62% of the PureLo prepara- nasal rubbing, and compound 48/80-induced skin scratch- tion. Other components include protein and melanoidins. ing behavior in ICR mice after consecutive treatment for four Mogroside V comprises approximately 39% of PureLo. A weeks, but they were inactive when administered in a single 28-day dietary study was conducted in Sprague Dawleyrats to dose, even at a dose of 1 000 mg·kg−1 [87]. Furthermore, both evaluate the safety of PureLo, and the results showed PureLo the extract and glycoside fraction inhibited the histamine was well tolerated and no significant adverse effects were [92] release induced by compound 48/80 at concentrations of produced . A combined 28-day and 90-day oral study was 300 or 1 000 μg·mL−1. Therefore, it was assumed that the conducted in male and female dogs to investigate the safety inhibition of nasal rubbing and skin scratching behavior in- of PureLo. Three dogs of each sex were administered 10 −1 −1 duced by S. grosvenorii fruit occurred through a mast mL·kg ·d of either an aqueous solution or distilled water −1 −1 cell-dependent mechanism. In an in vitro experiment, 0.05 providing 3 000 mg·kg ·d of PureLo for either 28 days or −0.5 g·mL−1 of water extract of S. grosvenorii fruit exhibited 90 days. Measurements regarding clinical observations, body a promotional effect on the growth and conservation vitality weight, food consumption, hematology, blood chemistry, of Lactobacillus plantarum, Leuconostoc mesenteroides, and urinalysis, gross necropsy, organ weight, and histopathology Streptococcus thermophilus[88]. Recently, Wang et al. found were performed, and no significant adverse effects were [93] that the total flavones from S. grosvenorii leaf provided pro- found . tection against the injury of endothelial cells induced by Conclusions phenanthroline copper [89]. A great number of pharmacological and phytochemical Toxicology studies carried out during last 30 years have demonstrated the Both S. grosvenorii fruit and the mogrosides are safe vast medicinal potential of S. grosvenorii, especially its foods. In 1999, Wang first investigated the acute toxicity of marked anti-tussive, phlegm-relieving, and anti-asthmatic the aqueous extract of S. grosvenorii fruit in mice [63]. The effects. Various types of preparations, extracts, and individual results showed that the maximum tolerated dose was more compounds derived from this species have been found to than 100 g·kg−1 after administering the S. grosvenorii fruit possess a number of pharmacological effects on organs such extract by gavage. Jin et al carried out a 13-week repeated as the liver, blood, and respiratory and gastrointestinal sys- dose toxicity study in Wistar Hannover (GALAS) rats for tems, as well as some other biological actions, e.g. antioxi- assessing the safety of the extract of S. grosvenorii fruit [90]. dant and immunomodulatory activities, relief of cough and The animals were given a diet containing 0%, 0.04%, 0.2%, asthma, and relaxing bowel properties. Moreover, some 1% and 5% of S. grosvenorii extract for thirteen weeks. As a other biological activities, including anticarcinogenic, anti- result, no deaths were observed in any groups, and there were bacterial and antifatigue effects have been reported for ex- no remarkable changes in general appearance, body weight, tracts or individual compounds of S. grosvenorii fruit . food and water consumption, hematological and serum bio- Among several classes of biologically active compounds chemical parameters, organ weight, and histopathological identified in S. grosvenorii, the mogrosides are assumed to be aspects between the control and treated groups. On the basis the main active principles, responsible for the majority of the of these data, the maximum non-toxic concentration of S. pharmacological actions. However, other components de- grosvenoriiextract for Wistar Hannover rats was considered scribed in this review, such as flavonoids, sterols, coumarins, to be beyond 5% (2 520 mg·kg−1·d−1 for males and 3 200 amino acids, vitamins, polysaccharides or the essential oil mg·kg−1·d−1 for females). may, to some degree, augment the pharmacological effects of The LD50 of mogrosides by gavage to mice was more the plant. than 10 000 mg·kg−1, and the result for the Ames test was The data summarized above, together with the low toxic- negative [91]. Dogs were given (i.g.) mogrosides (3 g·kg−1) for ity potential of the plant, strongly support the view that S. four weeks, and the results showed there were no change in grosvenorii has beneficial therapeutic properties indicating its the dog’s hematologic parameters, liver and kidney func- potential as an effective anti-tussive and expectorant tions, blood and urine sugar levels, and the morphology of plant- based therapy. However, further studies are needed to

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Cite this article as: LI Chun, LIN Li-Mei, SUI Feng, WANG Zhi-Min, HUO Hai-Ru, DAI Li, JIANG Ting-Liang. Chemistry and pharmacology of Siraitiagrosvenorii: A review [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 89-102

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