Study on Medicine Plant Luo Han Guo

Siraitia grosvenorii (Swingle) C. Jef-frey ex Lu et Z. Y. Zhang is the dried fruit of a dioecious, dioecious perennial vine in the family Cucurbitaceae. It is cool in nature, sweet in taste, and enters the lung meridian. It has the effects of clearing away heat from the lungs, moistening the lungs, relieving sore throat and improving voice, and lubricating the intestines to promote bowel movement. It is used to treat cough due to lung heat, sore throat with loss of voice, and constipation due to dryness in the intestines [1]. As a precious medicinal and sweetening plant unique to China, Luo Han Guo is mainly produced in Yongfu, Lingui and Longsheng, etc., in Guangxi. The fruit of Luo Han Guo contains a variety of sweet glycosides, among which Mogroside V is one of the strongest non-sugar sweeteners in the world, about 300 times sweeter than sucrose. It is widely used in foods, health products and medicines, and is an ideal sugar substitute for diabetics, obese people and hypertensive patients [2]. This paper reviews the taxonomic status, geographical distribution, main cultivated varieties, breeding and cultivation techniques, chemical composition, pharmacological effects and molecular biology research of Luo Han Guo, with a view to providing a reference for further in-depth research, development and utilization of Luo Han Guo.

1 Taxonomic status of Siraitia grosvenori

Regarding the taxonomy of Siraitia grosvenori, Merrill first established a new genus (Siraitia) in 1934 with S. silomaradjae as the type species, but it was not recognized. In 1941, the American Swingle classified Luo Han Guo as a species of the genus Momordica in the family Cucurbitaceae after identifying it based on specimen Tan Yinghua No. 1 (collected in Yongfu County, Guangxi). In 1979, after studying this specimen, the British botanist Jeffrey concluded that Luo Han Guo did not belong in the genus Momordica but in the genus Thladiantha based on the fact that the tendrils of the plant bifurcate at the tip and and twisted up and down at the bifurcation point, he believed that Luohan fruit did not belong to the genus Momordica but to the genus Thladiantha, and named it Thladiantha grosvenorii (Swingle) C. Jeffrey. In this regard, many scholars in China are skeptical [3]. In 1997, Zhuang Weijian et al. [4] finally determined that the chromosome number of Luo Han Guo is x = 14, while that of the genus Tribulus is x = 9, and that of the genus Momordica is x = 11 or 14. In addition, the pollen morphology of Luo Han Guo is quite different from that of Tribulus terrestris, and is more similar to that of Momordica charantia and Tribulus terrestris, except for the pollen size and mesh size [5].

In 1980, during Jeffrey's visit to China to examine specimens, Lu Anmin et al. from the Institute of Botany, Chinese Academy of Sciences [6] concluded that the male anther locules of the Lohan fruit group were inflexed or arched, and the seeds were significantly larger, which was clearly different from the genus Momordica. They also concluded that it could not be placed in the genus Momordica because these plants had no bracts on the female and male flower stalks, the stamens are not three but five, and the fruit skin is smooth and free of tuberous projections. Therefore, it was proposed that Siraitia should be separated out and a new genus established. Subsequently, Jeffrey used the genus name Siraitia proposed by Merrill in his article “East Asian Cucurbitaceae” and transferred all species of Siraitia to the genus Siraitia. Currently, the botanical taxonomy generally accepts the taxonomic claim of Jeffrey and Lu Anmin et al. [6] in 1984, that the scientific name of Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang is Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang, and that it is a plant in the genus Siraitia in the family Cucurbitaceae. However, in early literature, Siraitia grosvenorii is still sometimes written as Thladi-antha or Momordica grosvenorii.

2. Distribution of Siraitia grosvenorii

Siraitia grosvenorii resources are mainly distributed in some mountainous areas of Guangxi, Guangdong, Hunan, Guizhou, Hainan and Jiangxi provinces (regions) in China. These areas have a humid tropical and subtropical climate, with an altitude of 250–1,400 m, an easting of 106.5°–115.0°, and a north latitude of 21.0°–24.5°.   However, its distribution is not even. Among them, Yongfu and Lingui counties in Guangxi are the centers of cultivation of Luo Han Guo [7]. Luo Han Guo is widely distributed in Guangxi, from Zhaoping County in Hezhou City in the east, Pubei County in Qinzhou City in the south, Lingyun County in Baise City in the west, and Lingui County in Guilin City in the north. Among them, Dayao Mountain in Jinxiu Yao Autonomous County has the most concentrated distribution [8].

3 Main cultivated varieties of Luohanguo

The main cultivated varieties of Luohanguo are Qingpiguo, Dongguanguo, Lajianguo, Changtan Guo, Hongmaoguo and Chashanguo. Due to the serious harm caused by viral diseases, root-knot nematode diseases, bacterial wilt and fruit flies, etc., and the use of tissue culture for seedling propagation can achieve the purpose of rapid propagation and purification of good varieties, so in recent years, tissue culture seedlings of varieties such as Bolin and Qingpi have been widely promoted in the Luo Han Guo producing areas. Among them, Bolin No. 3 is a new strain of Luo Han Guo that was successfully selected from Bolin No. 2 green-skinned fruit. After apical shoot disinfection and rapid propagation, directional cultivation and purification and rejuvenation, it was successfully cultivated in 2003.

It has excellent characteristics such as wide adaptability, well-developed roots, vigorous growth, drought and fertilizer tolerance, concentrated flowering, early fruiting and high yield, low pest and disease damage, few physiological fruit cracks, high rate of medium and large fruits, good quality, and stable seed characteristics [9]. Yongqing No. 1 is a female clonal variety that was produced by crossing a winter melon fruit as the male parent with a Longjiang green-skinned fruit as the female parent, followed by two years of single plant selection and tissue culture breeding. The fruits of this variety are long and oval, with a large fruit rate of up to 73.48% with an average single fruit weight of 100 g. The content of sweetener V, total fruit glycoside, total sugar, water extract and vitamin C is as high as 1.03%, 8.84%, 17.40%, 37.90% and 3.02 mg/g, respectively. It has good yield and strong stress resistance, with up to

165,000 per hectare [10].

4 Propagation and cultivation techniques for Luohanguo

The main traditional propagation techniques for Luohanguo are seed propagation, tuber propagation, layering propagation, cuttings propagation and grafting propagation. In view of the current serious situation of viral diseases, root-knot nematode diseases and fruit fly infestations in the production areas, as well as the adverse consequences of traditional planting patterns, such as deforestation of mountain forests at the expense of the environment, leading to soil erosion, habitat degradation, etc., On the one hand, traditional propagation techniques have been changed, and significant progress has been made in experimental research using tissue culture to obtain virus-free seedlings [11, 12]. Among them, the Guangxi Medicinal Botanical Garden, the Guangxi Institute of Botany, and Guilin High-tech Zone Berlin Biotechnology Co., Ltd. have been engaged in the selection, breeding, and rapid propagation of excellent strains of Luohanguo in recent years, which has improved the stress resistance of Luohanguo to a certain extent and achieved the goal of increasing production and improving quality. At present, the tissue culture seedlings of varieties such as Qingpi Fruit and Bolin have been widely promoted in production areas; on the other hand, there have been many discussions and attempts to transform the traditional hill planting method of Luohanguo to a flatland cultivation method [13].

5 Chemical composition of Luohanguo

5.1 Cucurbitane triterpenoids and their glycosides

At present, the main triterpenoid and its glycosides isolated and identified from the fruit of Luohanguo are: Simonoside I, Mogroside II E, Mogroside III, Mogroside III E, Mogroside IV, Mogroside V, 11-oxo-Mogroside V [14], Mogroside ⅣA and Mogroside ⅡA1 [15], Luohanguo digolyl benzoate [16], and Luohanguo neoglycoside [17]. The above components are the main sweet components of Luohanguo, accounting for 3.755% to 3.858% of the dried fruit content. Among them, Mogroside V is the main sweetening component, while Simonoside I is the sweetest component of the cucurbitane triterpene glycosides. When its content is 0.01%, the sweetness of the two is 256 to 344 times and 563 times that of a 5% sucrose aqueous solution, respectively. In addition, Luo Han Guo acid A and Luo Han Guo acid B [18] and Luo Han Guo acid E [19] have been isolated and identified from the root of Luo Han Guo.

5.2 Proteins and amino acids

Xu Weikun et al. [20] found that the protein content of dried Luo Han Guo fruit was 7.1% to 7.8%. In its hydrolysate, except for tryptophan, which was not detected, all 18 amino acids are present, including 8 essential amino acids for the human body. The highest content is glutamic acid and aspartic acid, which shows that Luo Han Guo has certain nutritional value.

5.3 Flavonoids

Si Jianyong et al. [21] isolated two flavonoid glycoside components from fresh Luo Han Guo aqueous extract: kaempferol-3,7-α-L-dirhamnoside and kaempferol-3-O-α-L-rhamnoside-7-O-[β-D-glucopyranosyl-(1-2)-O-L-rhamnoside]. Chen Quanbin et al. [22] isolated and identified kaempferol-3,7-O-α-L-dirhamnoside and quercetin-3-O-β-D-glucose-7-O-α-L-rhamnoside from the leaves of Luo Han Guo, with kaempferol and quercetin as the aglycones. Liao Riquan et al. [23] isolated and identified luohangol benzoate, bis[5-formylfurfuryl] ether, 5-hydroxymethyl furfuroic acid, succinic acid, magnolol, kaempferol, kaempferol-3,7-0-a-L-dirhamnoside, of which bis[5-formylfurfuryl] ether, 5-hydroxymethyl furfural and magnolol were isolated from Luo Han Guo for the first time.

5.4 Other ingredients

The fresh fruit of Luo Han Guo contains D-mannitol [24]; the ripe fruit contains 26 inorganic elements (16 of which are essential to the human body) [25] and a large amount of glucose and fructose [26]; the seed oil is rich in unsaturated fatty acids [27].

6 Pharmacological effects of Luo Han Guo

6.1 Expectorant and cough suppressant effects

Luo Han Guo Mogroside V can increase the secretion of phenol red in the trachea of mice, promote the movement of mucus in the esophagus of frogs, and inhibit ammonia-induced coughing in mice. However, it has no significant effect on guinea pig asthma induced by citric acid, indicating that Luo Han Guo sweet glycosides have expectorant and antitussive effects [28].

6.2 Antibacterial effect

Su Huanqun et al. [29] found by turbidimetry that Luo Han Guo extract can significantly limit the growth and acid production ability of Streptococcus mutans, thereby inhibiting its cariogenic effect. The inhibition rates of the ethanol extract of the leaves and stems of Luo Han Guo (50.0 mg/mL) on Pseudomonas aeruginosa were 90.9% and 76.7%, respectively. In addition, the inhibition rate of the above stem extract on Escherichia coli was also as high as 70.2% [30].

6.3 Immune function

Luo Han Guo sweet glycoside has no significant effect on the immune function of normal mice, but can significantly improve the phagocytic function of macrophages and the proliferative effect of T cells in cyclophosphamide (CTX) immunosuppressed mice [31]. Luo Han Guo water extract can enhance the humoral and cellular immune functions of normal rats and improve the non-specific immune function of mice [29].

6.4 Anticancer effect

In vitro tests on mice with Luo Han Guo extract found that the anticancer effect of Luo Han Guo sweetin V is comparable to or stronger than that of stevioside [32].

6.5 Antioxidant effect

Monk fruit extract can effectively remove free radicals, inhibit the oxidation and hemolysis of red blood cells, and the production of malondialdehyde (MDA), indicating that it has antioxidant activity, and monk fruit glycosides are the main antioxidant active ingredients [33].

7 Molecular biology research on monk fruit

Early molecular biology research on Luo Han Guo was limited to studies using molecular marker techniques such as RFLP [34], RAPD [35–37], ISSR [38], AFLP [39] and SRAP [40], which were used to study the genetic diversity, kinship, sex identification, genetic background, fingerprints and genetic maps of Luo Han Guo. Some researchers used the second-generation high-throughput Solexa sequencing technology to perform high-throughput sequencing of the transcriptome of the Luohanguo fruit and the expression profiles 3 d, 50 d and 70 d after pollination, obtaining 43,891 Unigenes, of which 739 were related to Luohanguo secondary metabolism, 60 Unigenes involved in terpene skeleton synthesis. Using transcriptome data, all the genes in the biosynthetic pathway of Luo Han Guo sweet glycoside V were discovered, involving 20 Luo Han Guo glycoside skeleton synthesis genes and 2 types of structural modification genes (cytochrome P450 genes and glycosyltransferase genes). In addition, 80 P450 genes, 72 glycosyltransferase genes and 90 glucosyltransferase genes were obtained. Combined with expression profile screening, 6 candidate UDPG genes that may be related to the synthesis of loganin V were obtained. Using RACE technology, the full-length of 16 skeleton synthesis genes and 2 genes with 5′ or 3′ fragments were cloned, as well as 6 full-length CYP450 genes and 7 full-length glucosyltransferase genes. The deciphering of the transcriptome and expression profile lays a solid foundation for the functional genome of Luo Han Guo and the research on the molecular mechanism of the biosynthesis of sweetener V.

8 Outlook

As an important medicinal and edible plant resource, Luo Han Guo is a well-known specialty and traditional export commodity in China with a long-standing reputation at home and abroad. However, viral diseases, root-knot nematode diseases and fruit flies have always restricted the healthy development of the Luo Han Guo industry. Therefore, it is necessary to implement comprehensive control methods focusing on agriculture and biological control to minimize and strictly control the use of chemical agents to avoid pesticide pollution of the planting environment, plants, pollution of fruits; at the same time, efforts should be made to breed Luohanguo varieties that are resistant to diseases and insects.

Luohanguo has strict requirements for the planting environment, a narrow planting area, and a replanting barrier. Crops in continuous cropping are extremely vulnerable to pests and diseases, and changing the planting area every year requires a lot of manpower, material resources and financial resources. In addition, the current production areas still mainly use traditional planting methods, which often involve cutting down mountain forests, leading to adverse consequences such as soil erosion and habitat deterioration and other adverse consequences. For this reason, the author believes that research on soil microorganisms in planting fields must be strengthened to overcome the obstacle of continuous cropping of Luo Han Guo as soon as possible.

Luo Han Guo is a unisexual, dioecious plant. The pollen of male flowers is heavy and sticky, and has a bitter taste. Relying solely on wind or insects to spread pollen will not result in a high rate of fertilization. Only artificial pollination can ensure production, and pollination is the process that requires the most labor. In recent years, a method that is easier and more effective than pollination with a bamboo skewer has been developed in production practice: Press the male flower petals to the fruit stalk to expose the stamens, and lightly touch the side with dense pollen to the female stigma. Investigations have found that the same species of plant, the winged Luohanguo, is highly resistant, has large fruits, and a high yield, making it a good material for breeding. Moreover, individual plants have bisexual flowers that bear fruit without human intervention, which is an atavistic phenomenon that provides insight into the evolution of angiosperms. Further research is needed on the winged Luohanguo, which has bisexual flowers, in order to solve the problem of Luohanguo's dependence on human intervention for pollination.

Due to the destruction of the planting environment, the germplasm of Luohanguo has seriously deteriorated, and it is necessary to carry out germplasm innovation of Luohanguo.

The application of polyploidy breeding technology in the research and development of new varieties of Luohanguo is an effective way to overcome the defects of the original varieties. Since the monosaccharide only exists in the flesh and peel of the fruit, and the seeds do not contain the monosaccharide but do contain a large amount of seed oil, which greatly increases the difficulty of extraction and purification and production costs, therefore, breeding a seedless Luohanguo with a higher content of monosaccharide than conventional varieties and a high utilization rate of the whole fruit is a milestone for the development of the entire Luohanguo industry.

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