Saponin composition from Siraitia grosvenorii (Swingle) C. Jeffrey root, and preparation method thereof

Abstract

The present disclosure relates to the technical field of extraction, separation, and purification of traditional Chinese medicine ingredients, and in particular to a saponin composition from a Siraiti

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of extraction, separation, and purification of traditional Chinese medicine (TCM) ingredients, and in particular to a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root, and a preparation method thereof. BACKGROUND

[0002] Siraitia grosvenorii (Swingle) C. Jeffrey is a medicinal and edible local plant in Guilin, Guangxi, China, and is also the second one of the ten major medicinal herbs in the future in Guangxi. The Siraitia grosvenorii (Swingle) C. Jeffrey industry has become an important pillar industry for rural revitalization in Guilin, and has an output value of 10 billion (RMB). Siraitia grosvenorii (Swingle) C. Jeffrey has a medicinal history of more than 300 years in the folk, and is clinically used to treat diseases such as diabetes, tuberculosis, and acute and chronic tracheitis. Mogroside, namely a cucurbitane triterpenoid saponin, is a major active ingredient of Siraitia grosvenorii (Swingle) C. Jeffrey, and mogroside can relieve various inflammatory diseases such as colitis, neuroinflammation, and acute pancreatitis, and can play a hypoglycemic role by improving a function of pancreatic P-cells, improving the insulin resistance, and inhibiting the absorption of glucose in an intestinal tract.

[0003] In recent years, the Siraitia grosvenorii (Swingle) C. Jeffrey industry has expanded rapidly, and a planting area of Siraitia grosvenorii (Swingle) C. Jeffrey has increased from 120,000 mu in 2016 to 250,000 mu in 2021. Currently, Siraitia grosvenorii (Swingle) C. Jeffrey is cultivated once a year, and roots of Siraitia grosvenorii (Swingle) C. Jeffrey are dug up every year. With the increase of the planting area of Siraitia grosvenorii (Swingle) C. Jeffrey, increasing Siraitia grosvenorii (Swingle) C. Jeffrey roots are produced. According to documents such as "Chinese Materia Medica", Siraitia grosvenorii (Swingle) C. Jeffrey roots have functions of "removing dampness, arresting diarrhea, relaxing muscles and tendons, and mainly treating diarrhea, enlarged tongue and meningitis sequelae, and are a medicinal material used by people in Guilin to treat hepatitis and boils. The major components of Siraitiagrosvenorii (Swingle) C. Jeffrey roots are also cucurbitane triterpenoid saponins. However, the research on Siraitia grosvenorii (Swingle) C. Jeffrey roots, especially the research on a pharmacological activity of Siraitia grosvenorii (Swingle) C. Jeffrey roots, is very limited. As a result, Siraitia grosvenorii (Swingle) C. Jeffrey roots are often directly discarded, resulting in a great waste of resources.

[0004] The current related research on Siraitia grosvenorii (Swingle) C. Jeffrey roots mainly 04 02 25 focuses on the separation and identification of chemical components. Wang Xuefen, Si Jianyong, Chen Dihua et al. have successively isolated decarbonized cucurbitane tetracyclic triterpenic acids (siraitic acid A-H), and Li Dianpeng et al. have isolated the two saponin components of Siraitic IIB and Siraitic IIC from Siraitia grosvenorii (Swingle) C. Jeffrey roots. CN101440118 A discloses two saponin compounds extracted from a Siraitia grosvenorii (Swingle) C. Jeffrey root, and a preparation method and use thereof, where the preparation method is as follows: a Siraitia grosvenorii (Swingle) C. Jeffrey root is subjected to extraction with 80% to 100% of methanol or ethanol under reflux, saponin components are enriched with a macroporous resin column, and then the saponin compounds of siraitic acid IIB and siraitic acid II C are separated by silica gel column chromatography; and siraitic acid IIB has a significant inhibitory effect on lung cancer cells A549 and liver cancer cells Hep-G2, and siraitic acid II C also has a significant inhibitory effect on liver cancer cells Hep-G2.

[0005] However, only a few types of compounds have been isolated from Siraitia grosvenorii (Swingle) C. Jeffrey roots, and chemical components in Siraitia grosvenorii (Swingle) C. Jeffrey roots and activities thereof have not been fully elucidated, which affects further development and utilization of Siraitia grosvenorii (Swingle) C. Jeffrey roots. In addition, there are very limited separation methods for saponin components from Siraitia grosvenorii (Swingle) C. Jeffrey roots, and the repeated separation by a silica gel column is time-consuming and causes a low recovery rate due to serious dead adsorption of silica gel for saponin components. SUMMARY

[0006] A technical problem to be solved by the present disclosure is to provide a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root, and a preparation method thereof.

[0007] In order to solve the above technical problem, the present disclosure provides the following technical solutions:

[0008] The present disclosure provides a method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root, including the following steps:

[0009] SI. ultrasonic extraction: providing a Siraitiagrosvenorii (Swingle) C. Jeffrey root powder, mixing the Siraitia grosvenorii (Swingle) C. Jeffrey root powder with a first methanol or ethanol solution to obtain a mixture, and subjecting the mixture to the ultrasonic extraction to obtain a Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution;

[0010] S2. macroporous resin adsorption and elution: subjecting the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution to macroporous resin adsorption and elution to obtain a macroporous resin eluate, and concentrating the macroporous resin eluate to obtain a crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root;

[0011] S3, liquid-liquid extraction (LLE): subjecting the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root to the LLE to obtain an aqueous phase, concentrating the aqueous phase to obtain a concentrate, and drying the concentrate to obtain a refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root;

[0012] S4. high-speed counter-current chromatography (HSCCC): subjecting the refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root to separation by the HSCCC, and collecting separated fractions as a first mixed fraction and a second mixed fraction according to an outflow time order; and separately subjecting the first mixed fraction and the second mixed fraction to concentration, drying, and dissolution with a second methanol aqueous solution to obtain a dissolved first mixed fraction solution and a dissolved second mixed fraction,

[0013] where the HSCCC is eluted for 240 min in total, with a fraction eluted before 160th min being the first mixed fraction, and a fraction eluted after the 160th min being the second mixed fraction;

[0014] S5. semi-preparative high-performance liquid chromatography (HPLC): subjecting the dissolved first mixed fraction solution and the dissolved second mixed fraction solution to separation by the semi-preparative HPLC to obtain two separation products, respectively,

[0015] where a mobile phase used for subjecting the dissolved first mixed fraction solution to the separation by the semi-preparative HPLC is an acetonitrile aqueous solution with a volume fraction of 30%; and a mobile phase used for subjecting the dissolved second mixed fraction solution to the separation by the semi-preparative HPLC is an acetonitrile aqueous solution with a volume fraction of 35%; and

[0016] S6. mixing the two separation products to obtain the saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root.

[0017] In some embodiments, in SI, the mixing is conducted by adding the first methanol or ethanol solution with a volume percentage of 40% to 60% to the Siraitia grosvenorii (Swingle) C. Jeffrey root powder at a liquid-to-solid ratio of 30 mL:l g to 50 mL:l g, and then mixing to be uniform; and the ultrasonic extraction is conducted at a frequency of 10 kHz to 50 kHz and a temperature of 30°C to 50°C for 30 min to 60 min.

[0018] In some embodiments, in SI, a volume fraction of methanol or ethanol in the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution is in a range of 15% to 20%.

[0019] In some embodiments, in S2, the elution is gradient elution, and conducted as follows: eluting a macroporous resin column with methanol aqueous solutions having volume fractions of 20%, 40%, and 70% sequentially, where a volume of each methanol aqueous solution used for the elution each time is 2 to 3 times a volume of the macroporous resin column; and a solution eluted by the methanol aqueous solution having a volume fraction of 70% is collected as the macroporous resin eluate.

[0020] In some embodiments, in S3, a solvent used for the LLE is a four-component solution consisting of w-hexane, ethyl acetate, methanol, and water; a volume ratio of the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root to the four-component solution is in a range of l:(30-50); and in the four-component solution, a volume ratio of the / / -hexane, the ethyl acetate, the methanol, and the water is 3:7:3:7.

[0021] In some embodiments, in S4, a separation solvent system used for the HSCCC includes chloroform, methanol, and water in a volume ratio of 2:2:1;

[0022] the HSCCC is conducted with an upper phase as a stationary phase and a lower phase as a mobile phase; and the HSCCC is conducted at a rotational speed of 860 r / min with an elutionreverse separation mode, where elution is conducted for 180 min at a flow rate of 2 mL / min and reverse separation is conducted for 60 min at a flow rate of 5 mL / min.

[0023] In some embodiments, in S4, fractions separated by the reverse separation are collected at an amount of 10 mL / fraction and numbered according to an outflow time order to obtain 66 fractions numbered 1 to 66; and fractions numbered 1 to 28 are discarded, fractions numbered 29 to 32 are mixed to obtain the first mixed fraction, and fractions numbered 47 to 60 are mixed to obtain the second mixed fraction.

[0024] In some embodiments, in S5, for subjecting the dissolved first mixed fraction solution and the dissolved second mixed fraction solution to the separation by the semi-preparative HPLC, the mobile phases both have a flow rate of 3.0 mL / min, elution procedures both are isocratic elution, and detection wavelengths both are 230 nm.

[0025] The present disclosure also provides a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root prepared by the method described above.

[0026] In some embodiments, the separation product separated from the first mixed fraction includes a monomeric compound A, a monomeric compound B, a monomeric compound C, and a monomeric compound D; the separation product separated from the second mixed fraction includes a monomeric compound E, a monomeric compound F, a monomeric compound G, and a monomeric compound H;

[0027] the monomeric compound A has a structural formula of formula (1): formula (1)

[0028] in formula (1), R is -P-D-glucosyl(l—>6)-P-D-glucoside, where 1—>6 means that position 1 of glucosyl in front is linked to position 6 of glucosyl in back;

[0029] the monomeric compounds B to E each have a general formula of formula (2): formula (2)

[0030] where for a structural formula of the monomeric compound B, in formula (2), Ri is -oxygen-P-D-glucosyl, R2 is -P-D-glucoside, and R3 is hydrogen;

[0031] for a structural formula of the monomeric compound C, in formula (2), Ri is -hydroxyl, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is hydrogen;

[0032] for a structural formula of the monomeric compound D, in formula (2), Ri is hydrogen, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is methyl; and

[0033] for a structural formula of the monomeric compound E, in formula (2), Ri is -oxygen-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is hydrogen; and

[0034] the monomeric compounds F to H each have a general formula of formula (3): formula (3)

[0035] where for a structural formula of the monomeric compound F, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, R3 is hydrogen, and R4 is methyl;

[0036] for a structural formula of the monomeric compound G, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, R3 is hydrogen, and R4 is hydroxymethylene; and

[0037] for a structural formula of the monomeric compound H, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-apiose(l—>2)P-D-glucosyl(l—>6)-P-D-ghicoside, R3 is hydrogen, and R4 is methyl.

[0038] Compared with the prior art, the present disclosure has the following beneficial effects:

[0039] (1) In the present disclosure, eight saponin compounds are isolated from the Siraitia grosvenorii (Swingle) C. Jeffrey root, and these compounds each have a novel specific structure, an excellent activity, and a high use value.

[0040] (2) In the present disclosure, ultrasonic extraction is applied for the Siraitia grosvenorii (Swingle) C. Jeffrey root, which greatly shortens an extraction time.

[0041] (3) In the preparation method of a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root provided in the present disclosure, an extract is adsorbed on a macroporous resin with a 15% to 20% alcohol solution and then subjected to gradient elution, then an eluate is refined through a combination of LLE and HSCCC, and then saponins are separated by semi-preparative HPLC under guidance of liquid chromatography-mass spectrometry (LC-MS) analysis. The preparation method involves simple steps and easy operations, and the macroporous resin, Cl8 material, LLE, and HSCCC used in the preparation method all have little or no dead adsorption for a sample. Therefore, the preparation method has advantages such as high efficiency and high recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGs. 1A-1C show HPLC spectra of a sample before HSCCC separation and a first mixed fraction and a second mixed fraction after HSCCC separation in Example 1 according to the method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root provided in the present disclosure, where FIG. 1A shows an HPLC spectrum of the sample before HSCCC separation, FIG. IB shows an HPLC spectrum of the first mixed fraction after HSCCC separation, and FIG. IC shows an HPLC spectrum of the second mixed fraction after HSCCC separation.

[0043] FIGs. 2A-2E show HPLC spectra for extracts extracted with different solvents in Comparative Example 1 according to the method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root provided in the present disclosure, where FIG. 2A shows an HPLC spectrum for the extract extracted with water, FIG. 2B shows an HPLC spectrum for the extract extracted with 50% methanol, FIG. 2C shows an HPLC spectrum for the extract extracted with 50% ethanol, FIG. 2D shows an HPLC spectrum for the extract extracted with 100% methanol, and FIG. 2E shows an HPLC spectrum for the extract extracted with 100% ethanol.

[0044] FIGs. 3 A-3B show HPLC spectra for extracts extracted by ultrasonic extraction and soaking extraction of a Siraitia grosvenorii (Swingle) C. Jeffrey root in Comparative Example 2 according to the method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root provided in the present disclosure, where FIG. 3A shows an HPLC spectrum corresponding to the extract extracted by ultrasonic extraction and FIG. 3B shows an HPLC spectrum corresponding to the extract extracted by soaking.

[0045] FIGs. 4A-4D show HPLC spectra for extraction of a crude saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root in Comparative Example 3 according the method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root provided in the present disclosure, where FIG. 4A shows an HPLC spectrum corresponding to an ethyl acetatewater (1:1, v / v) organic phase, FIG. 4B shows an HPLC spectrum corresponding to an w-hexane-ethyl acetate-methanol-water (3:7:3:7, v / v / v / v) organic phase, FIG. 4C shows an HPLC spectrum corresponding to the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root before extraction, and FIG. 4D shows an HPLC spectrum corresponding to a refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root after extraction with a hexaneethyl acetate-methanol-water organic phase. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] The principles and features of the present disclosure are described below with reference to the accompanying drawings. The listed embodiments are only used to explain the present disclosure, rather than to limit the scope of the present disclosure.

[0047] The Siraitia grosvenorii (Swingle) C. Jeffrey roots used in the embodiments of the present disclosure are harvested from a Siraitia grosvenorii (Swingle) C. Jeffrey production area, where Siraitia grosvenorii (Swingle) C. Jeffrey is planted in March and April of the year, and after Siraitia grosvenorii (Swingle) C. Jeffrey fruits are harvested in January of the following year, root samples are dug up.

[0048] The cucurbitane tetracyclic triterpenoids extracted from the Siraitia grosvenorii (Swingle) C. Jeffrey root in the present disclosure have a novel structure, and these natural products are relatively rare.

[0049] The present disclosure provides a method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root, including the following steps:

[0050] SI. ultrasonic extraction: a Siraitia grosvenorii (Swingle) C. Jeffrey root powder is provided and mixed with a first methanol or ethanol aqueous solution to obtain a mixture, the mixture is subjected to ultrasonic extraction and filtration to obtain a filtrate, and the filtrate is concentrated under a reduced pressure at a low temperature to remove a part of the alcohol solution in the filtrate to obtain a Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution.

[0051] In some embodiments, the mixing is conducted by adding the first methanol or ethanol aqueous solution with a volume percentage of 40% to 60% to the Siraitia grosvenorii (Swingle) C. Jeffrey root powder at a liquid-to-solid ratio of 30 mL:l g to 50 mL:l g, and then mixing to be uniform; and the ultrasonic extraction is conducted at a frequency of 10 kHz to 50 kHz and a temperature of 30°C to 50°C for 30 min to 60 min. The extraction is conducted with the the first methanol or ethanol aqueous solution with a volume percentage of 40% to 60%, such that target saponins can be effectively extracted by the solvent.

[0052] In some embodiments, in SI, the ultrasonic extraction is conducted for 30 min to 60 min. The ultrasonic extraction accelerates the release, diffusion, and dissolution of intracellular effective substances based on a cavitation effect, a mechanical effect, and a thermal effect of ultrasonic waves to significantly improve the extraction efficiency.

[0053] In some embodiments, in SI, the filtrate is concentrated under a reduced pressure at a low temperature until a volume fraction of methanol or ethanol in the filtrate is 15% to 20% to obtain the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution. 15% to 20% of methanol or ethanol is retained in the extraction solution such that some components with high polarity are not adsorbed, but target components with low polarity are effectively adsorbed on the resin, which can improve the adsorption selectivity and desorption efficiency.

[0054] S2. Macroporous resin adsorption and elution: the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution is subjected to macroporous resin adsorption and elution to obtain a macroporous resin eluate, and the macroporous resin eluate is concentrated under a reduced pressure to obtain a crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey roots.

[0055] In some embodiments, in S2, the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution obtained in SI is first added to a macroporous resin column and subjected to adsorption; and then gradient elution is conducted by eluting the macroporous resin column with methanol aqueous solutions having volume fractions of 20%, 40%, and 70% sequentially, and a solution eluted by a 70% methanol aqueous solution is collected as the macroporous resin eluate.

[0056] In some embodiments, during the gradient elution, a volume of each methanol aqueous solution used for elution is 2 to 3 times a volume of the macroporous resin column.

[0057] S3. LLE: the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root is subjected to the LLE to obtain an aqueous phase, and the aqueous phase is concentrated under a reduced pressure and dried to obtain a refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root.

[0058] In some embodiments, in S3, a solvent used for the LLE is an / / -hexane-ethyl acetatemethanol-water (3:7:3:7, v / v / v / v) system including an aqueous phase and an organic phase that are mutually incompatible. Compared with a two-component solvent system for LLE, the four-component solvent system for LLE has higher selectivity and can remove more non-target compounds.

[0059] S4. HSCCC: the refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root is subjected to the HSCCC, a plurality of fractions are collected, and corresponding fractions are mixed to obtain a first mixed fraction and a second mixed fraction; and the first mixed fraction and the second mixed fraction each are concentrated, dried, and then dissolved in a methanol aqueous solution to obtain a dissolved first mixed fraction solution and a dissolved second mixed fraction solution.

[0060] In some embodiments, in S4, a solvent system used for the HSCCC includes chloroform, methanol, and water in a volume ratio of 2:2:1. The HSCCC is conducted with an upper phase as a stationary phase and a lower phase as a mobile phase; and the HSCCC is conducted at a rotational speed of 860 r / min with an elution-reverse separation mode, where elution is conducted for 180 min at a flow rate of 2 mL / min and reverse separation is conducted for 60 min at a flow rate of 5 mL / min. 66 tubes of fractions are collected by an automatic fraction collector in total, with 10 mL / tube. HSCCC has characteristics such as high recovery rate, excellent reproducibility, and high separation efficiency, and exhibits unique advantages in efficient separation of main components and enrichment of trace components.

[0061] The 25th to 66th tubes of fractions with chromatographic absorption peaks each are centrifuged, dried, dissolved in methanol, and then filtered through a microporous filter membrane; and then subjected to HPLC-ESI-IT-TOF-MSn analysis. HPLC analysis is conducted under the following conditions: Inertsil ODS-3 (150 x 4.6 mm, 5 gm) analysis column, column temperature: 35°C, flow rate: 0.8 mL / min, injection volume: 10 pL, detection wavelength: 230 nm, mobile phase: water and acetonitrile, and gradient elution: 20% to 50% acetonitrile from 0 min to 40 min. Instrument conditions for MS analysis in a negative ion mode are as follows: a spray voltage, a cone voltage, and a collision energy are 3,000 V, 40 V, and 10 V, respectively; a vaporizer temperature and a capillary temperature are 350°C and 250°C, respectively; a nitrogen (N2) pressure and an auxiliary gas pressure are 40 psi and 10 psi, respectively; an atomization gas flow rate is 6.0 L / min; and a mass range is set as 150 to 1,500 (m / z) in a full scan mode.

[0062] In the present disclosure, analysis results are analyzed, and according to MS fragments, it can be determined that fractions in the 29th to 32nd tubes mainly include monomeric compounds A, B, C, and D, and fractions in the 47th to 60th tubes mainly include monomeric compounds E, F, G, and H; thus the monomeric compounds A, B, C, and D are mixed to obtain a first mixed fraction and the monomeric compounds E, F, G, and H are mixed to obtain a second mixed fraction.

[0063] S5. Separation by semi-preparative HPLC: the dissolved first mixed fraction solution and the dissolved second mixed fraction solution each are subjected to separation to obtain two separation products, respectively.

[0064] The dissolved first mixed fraction solution and the dissolved second mixed fraction solution each are subjected to separation by semi-preparative HPLC, where products at different chromatographic peaks are collected, and a solvent is recovered under reduced pressure to obtain dry powders. According to spectral data of LC-MS, 1HNMR, and 13CNMR, it is determined that monomeric compounds A, B, C, and D are obtained from the first mixed fraction and monomeric compounds E, F, G, and H are obtained from the second mixed fraction.

[0065] In some embodiments, a mobile phase for the semi-preparative HPLC is an acetonitrile aqueous solution with a volume fraction of 35%; a flow rate of the mobile phase is 3.0 mL / min; and an elution procedure for the semi-preparative HPLC is isocratic elution, and a detection wavelength is 230 nm.

[0066] The separation of the refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey roots with HSSCCC and then the combination and separate preparation of different fractions have the following advantages: target compounds can first be enriched and purified by HSCCC; HSCCC and semi-preparative HPLC can complement each other's advantages due to their different separation principles; and HSCCC can separate compounds with a low resolution on an HPLC column, such that the separate preparation by semi-preparative HPLC will not be interfered, which can effectively improve a purity of a product.

[0067] S6. The two separation products are mixed to obtain the saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root.

[0068] The monomeric compound A has a structural formula of formula (1): formula (1)

[0069] in formula (1), R is -P-D-glucosyl(l —>6)-P-D-glucoside;

[0070] the monomeric compounds B to E each have a general formula of formula (2): formula (2)

[0071] where for a structural formula of the monomeric compound B, in formula (2), Ri is -oxygen-P-D-glucosyl, R2 is -P-D-glucoside, and R3 is hydrogen;

[0072] for a structural formula of the monomeric compound C, in formula (2), Ri is -hydroxyl, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is hydrogen;

[0073] for a structural formula of the monomeric compound D, in formula (2), Ri is hydrogen, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is methyl; and

[0074] for a structural formula of the monomeric compound E, in formula (2), Ri is -oxygen-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is hydrogen; and

[0075] the monomeric compounds F to H each have a general formula of formula (3): formula (3)

[0076] where for a structural formula of the monomeric compound F, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, R3 is hydrogen, and R4 is methyl;

[0077] for a structural formula of the monomeric compound G, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, R3 is hydrogen, and R4 is hydroxymethylene; and

[0078] for a structural formula of the monomeric compound H, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-apiose(l—>2)P-D-glucosyl(l—>6)-P-D-ghicoside, R3 is hydrogen, and R4 is methyl.

[0079] Effects of the present disclosure are specifically described below with reference to specific examples:

[0080] Example 1

[0081] Monomeric compounds A, B, C, D, E, F, G, and H were prepared by the method of the present disclosure.

[0082] In this example, for the ultrasonic extraction in SI, a methanol aqueous solution with a volume percentage of 50% was added to a root powder at a liquid-to-solid of 45 mL:l g; and the ultrasonic extraction was conducted for 40 min.

[0083] The separation by semi-preparative HPLC in S5 was conducted under the following conditions: Agilent 1200 prep-HPLC chromatograph; Pack ODS-A (250 mm x 10 mm, 5 pm) column; flow rate: 3.0 mL / min; detection wavelength: 230 nm; and isocratic elution: acetonitrile (35%) and water (65%). The monomeric compounds A, B, C, and D were separated from the first mixed fraction, and the monomeric compounds E, F, G, and h were separated from the second mixed fraction. Spectra are shown in FIGs. 1A-1C.

[0084] Example 2

[0085] Inhibitory activities of the monomeric compounds for a-glucosidase were tested.

[0086] In this example, the saponin composition of the Siraitia grosvenorii (Swingle) C. Jeffrey 12 root prepared in Example 1 was subjected to activity assay by a p-nitrophenyl-a-Dglucopyranoside (PNPG) assay. 20 pL of 0.2 U / mL a-glucosidase and 50 pL of a sample were added successively to a 96-well plate, and then the plate was incubated at 37°C for 5 min; 20 pL of a 1 mM PNPG solution was added, and then the plate was incubated at 37°C for 30 min; and 50 pL of a 0.2 M Na2COs solution was added to stop a reaction, and the absorbance at 405 nm was determined. The above process was repeated with the sample replaced by acarbose to allow an experiment of a positive control group. Three replicates were set for each group.

[0087] As shown in Table 1, with acarbose as a positive control, the monomeric compounds Ato H each exhibit a specified inhibitory activity for a-glucosidase in a same mass concentration range (1 mg / mL), with an inhibitory rate of 19.73% to 58.48% and an IC50 value of 0.32 to 5.73 mg / mL, where the monomeric compound C has a higher inhibitory activity than the commercially-available acarbose.

[0088] Table 1 Inhibitory activities of the monomeric compounds A to H for a-glucosidase No. Compound names Inhibition (%) IC50 (mg / mL) 1 A 42.73 1.71 2 B 50.59 1.09 3 C 58.48 0.32 4 D 53.12 0.99 5 E 36.74 2.11 6 F 19.73 5.73 7 G 21.34 5.36 8 H 26.67 5.55 9 acarbose 55.15 0.86

[0089] Comparative Example 1

[0090] In this comparative example, extraction effects of different extraction solvents were compared.

[0091] 5 groups of a Siraitia grosvenorii (Swingle) C. Jeffrey root powder was prepared, where each group had 2.0 g of the Siraitia grosvenorii (Swingle) C. Jeffrey root powder; water, 50% methanol aqueous solution, 50% ethanol aqueous solution, 100% methanol, and 100% ethanol were added to the 5 groups, respectively; resulting mixtures each were thoroughly shaken, and subjected to ultrasonic extraction for 1 h to obtain extraction solutions; and the extraction solutions each were tested by HPLC. It can be seen from the comparative analysis of spectra (FIGs. 2A-2E) that 50% methanol aqueous solution and 50% ethanol aqueous solution have equivalent extraction effects and lead to higher peak heights than other solvents; and the extraction with 100% ethanol leads to the least peaks with the lowest peak height. Therefore, it is considered that 50% methanol aqueous solution or 50% ethanol aqueous solution has an excellent extraction effect.

[0092] Comparative Example 2

[0093] In this comparative example, extraction effects of ultrasonic extraction and soaking extraction were compared.

[0094] (1) Ultrasonic extraction: 2.0 g of a Siraitia grosvenorii (Swingle) C. Jeffrey root powder was accurately weighed, 50 mL of 50% methanol aqueous solution was added to the powder, a resulting mixture was shaken to be uniform and then subjected to an ultrasonic treatment at ambient temperature for 1 h, and a resulting supernatant was collected and subjected to HPLC analysis.

[0095] (2) Soaking extraction: 2.0 g of a Siraitia grosvenorii (Swingle) C. Jeffrey root powder was accurately weighed, 50 mL of 50% methanol aqueous solution was added to the powder, a resulting mixture was shaken to be uniform and then placed at ambient temperature overnight (about 12 h) to allow cold soaking, and a resulting supernatant was collected and subjected to HPLC analysis.

[0096] It can be seen from HPLC analysis results (FIGs. 3A-3B) that, with the same solvent, the height of each chromatographic peak of a sample obtained after ultrasonic extraction for 1 h is slightly higher than that of a sample obtained after soaking extraction for 12 h, which fully indicates that the ultrasonic extraction has higher extraction efficiency.

[0097] Comparative Example 3

[0098] In this comparative example, extraction effects of different solvent systems for LLE were compared. Specifically, an extraction effect of an / / -hexane-ethyl acetate-methanol-water (3:7:3:7, v / v / v / v) solvent system was compared with an extraction effect of an ethyl acetate-water (1:1, v / v) solvent system.

[0099] The / / -hexane-ethyl acetate-methanol-water (3:7:3:7, v / v / v / v) solvent system and the ethyl acetate-water (1:1, v / v) solvent system each were used for extraction of a crude saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root. It can be seen from HPLC analysis results that when the ethyl acetate-water (1:1, v / v) solvent system is adopted, monomeric compounds G and H enter a resulting organic phase and thus lose (FIG. 4A), but when the n-hexane-ethyl acetate-methanol-water (3:7:3:7, v / v / v / v) solvent system is adopted, the two compounds do not lose basically (FIG. 4B). It can also be seen that, chromatographic peaks in a spectrum of the refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root (FIG. 4D) are reduced compared with chromatographic peaks in a spectrum of the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root (FIG. 4C), indicating that a purity of a sample can be improved by changing the extraction solvent.

[0100] Comparative Example 4

[0101] In this comparative example, the monomeric compounds A to H were separated by a conventional method, and an extraction and separation effect of the conventional method was compared with an extraction and separation effect of the method of the present disclosure.

[0102] The conventional method were conducted as follows: 4.75 kg of a Siraitia grosvenorii (Swingle) C. Jeffrey root powder was taken and subjected to soaking extraction with 75% ethanol aqueous solution three times, where the soaking extraction was conducted for 7 days each time; resulting extraction solutions were combined, concentrated under a reduced pressure, and passed through an HPD-100 macroporous resin chromatographic column; then gradient elution was conducted with pure water, 20% methanol aqueous solution, 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, and 100% methanol successively, and eluates were collected; and eluates eluted by 80% methanol and 100% methanol each were concentrated under a reduced pressure to obtain portions Fr3 (30.88 g) and Fr4 (20.86 g). The Fr3 (30.88 g) portion was fully dissolved in a small amount of methanol, a resulting solution was passed through an MCI chromatographic column, and then gradient elution was conducted with pure water, 20% methanol aqueous solution, 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, and 100% methanol successively; and similar fractions were combined and then concentrated under a reduced pressure to obtain five portions Fr31, Fr32, Fr33, Fr34, and Fr35. The Fr32 (4.09 g) portion was further passed through an RP-C18 chromatographic column, elution was conducted with different gradients of methanol, eluates were tested by a thin layer chromatography (TLC) silica gel plate, combined, and concentrated, and a concentrate was repeatedly passed through an RP-C18 column to obtain a monomeric compound F. The Fr34 (1.55 g) portion was prepared by HSCCC (with a solvent system consisting of ethyl acetate, / / -butanol, and water in 6:3:8.5) under the following conditions: number of revolutions: 860 r.min'1, host: forward rotation, and flow rate: 2 mL.min'1, where a fraction was collected by an automatic collector every 5 min, and corresponding fractions were combined to obtain monomeric compounds E, G, and H.

[0103] The Fr4 (20.87 g) portion was fully dissolved in a small amount of methanol, a resulting solution was passed through an MCI chromatographic column, and then gradient elution was conducted with pure water, 20% methanol aqueous solution, 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution, and 100% methanol successively; and similar fractions were combined and then concentrated under a reduced pressure to obtain four portions Fr41, Fr42, Fr43, and Fr44. The Fr42 (2.58 g) was subjected to silica gel column chromatography with an ethyl acetate-methanol-water solvent system (5:1:0.1, v / v / v) and then to separation by an RP-C18 chromatographic column, and isocratic elution (60% methanol-water) was conducted to obtain monomeric compounds A and B. The Fr43 (2.90 g) was passed through an MCI chromatographic column, then elution was conducted with different concentrations of methanol aqueous solutions successively, an eluate eluted by 80% methanol aqueous solution was collected, concentrated, and introduced into a preparative liquid phase (36% acetonitrile-water system) to obtain monomeric compounds C and D.

[0104] It can be seen that the conventional extraction method is time-consuming and involves many separation steps, while the method of the present disclosure has a shorter extraction time and a higher separation efficiency.

[0105] The above are merely preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, and the like made within the spirit and principle of the present disclosure shall fall within the scope of the present disclosure.

Claims

1. A method for preparing a saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root, comprising the following steps:SI. ultrasonic extraction: providing a Siraitia grosvenorii (Swingle) C. Jeffrey root powder, mixing the Siraitia grosvenorii (Swingle) C. Jeffrey root powder with a first methanol or ethanol aqueous solution to obtain a mixture, and subjecting the mixture to the ultrasonic extraction to obtain a Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution;S2. macroporous resin adsorption and elution: subjecting the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution to the macroporous resin adsorption and elution to obtain a macroporous resin eluate, and concentrating the macroporous resin eluate to obtain a crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root;S3, liquid-liquid extraction (LLE): subjecting the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root to the LLE to obtain an aqueous phase, concentrating the aqueous phase to obtain a concentrate, and drying the concentrate to obtain a refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root;S4. high-speed counter-current chromatography (HSCCC): subjecting the refined saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root to separation by the HSCCC, and collecting separated fractions as a first mixed fraction and a second mixed fraction according to an outflow time order; and separately subjecting the first mixed fraction and the second mixed fraction to concentration, drying and dissolution with a second methanol aqueous solution to obtain a dissolved first mixed fraction solution and a dissolved second mixed fraction, wherein the HSCCC is eluted for 240 min in total, with a fraction eluted before 160th min being the first mixed fraction, and a fraction eluted after the 160th min being the second mixed fraction;S5. separation by semi-preparative high-performance liquid chromatography (HPLC): separately subjecting the dissolved first mixed fraction solution and the dissolved second mixed fraction solution to the separation by the semi-preparative HPLC to obtain two separation products, wherein a mobile phase used for subjecting the dissolved first mixed fraction solution to the separation by the semi-preparative HPLC is an acetonitrile aqueous solution with a volume fraction of 30%; and a mobile phase used for subjecting the dissolved second mixed fraction solution to the separation by the semi-preparative HPLC is an acetonitrile aqueous solution with a volume fraction of 35%; andS6. mixing the two separation products to obtain the saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root.

2. The method as claimed in claim 1, wherein in SI, the mixing is conducted by adding the first methanol or ethanol solution with a volume percentage of 40% to 60% to the Siraitia grosvenorii (Swingle) C. Jeffrey root powder at a liquid-to-solid ratio of 30 mL: 1 g to 50 mL:l g, and then mixing to be uniform; and the ultrasonic extraction is conducted at a frequency of 10 kHz to 50 kHz and a temperature of 30°C to 50°C for 30 min to 60 min.

3. The method as claimed in claim 1, wherein in SI, a volume fraction of methanol or ethanol in the Siraitia grosvenorii (Swingle) C. Jeffrey root extraction solution is in a range of 15% to 20%.

4. The method as claimed in claim 1, wherein in S2, the elution is gradient elution, and conducted as follows: eluting a macroporous resin column with methanol aqueous solutions having volume fractions of 20%, 40%, and 70% sequentially, wherein a volume of each methanol aqueous solution used for the elution is 2 to 3 times a volume of the macroporous resin column; and a solution eluted by the methanol aqueous solution having a volume fraction of 70% is collected as the macroporous resin eluate.

5. The method as claimed in claim 1, wherein in S3, a solvent used for the LLE is a four-component solution consisting of w-hexane, ethyl acetate, methanol, and water; a volume ratio of the crude saponin composition from the Siraitia grosvenorii (Swingle) C. Jeffrey root to the four-component solution is in a range of 1 :(30-50); and in the four-component solution, a volume ratio of the w-hexane, the ethyl acetate, the methanol, and the water is 3:7:3:7.

6. The method as claimed in any one of claims 1 to 5, wherein in S4, a separation solvent system used for the HSCCC comprises chloroform, methanol, and water in a volume ratio of 2:2:1;the HSCCC is conducted with an upper phase as a stationary phase and a lower phase as a mobile phase; and the HSCCC is conducted at a rotational speed of 860 r / min with an elutionreverse separation mode, wherein elution is conducted for 180 min at a flow rate of 2 mL / min and reverse separation is conducted for 60 min at a flow rate of 5 mL / min.

7. The method as claimed in claim 6, wherein in S4, fractions separated by the reverse separation are collected at an amount of 10 mL / fraction and numbered according to an outflow time order to obtain 66 fractions numbered 1 to 66; and fractions numbered 1 to 28 are discarded, fractions numbered 29 to 32 are mixed to obtain the first mixed fraction, and fractions numbered 47 to 60 are mixed to obtain the second mixed fraction.

8. The method as claimed in claim 7, wherein in S5, for subjecting the dissolved first mixed fraction solution and the dissolved second mixed fraction solution to the separation by the semipreparative HPLC, the mobile phases both have a flow rate of 3.0 mL / min, elution procedures both are isocratic elution, and detection wavelengths both are 230 nm.

9. A saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root prepared by the method as claimed in any one of claims 1 to 8.

10. The saponin composition from a Siraitia grosvenorii (Swingle) C. Jeffrey root as claimed in claim 9, whereinthe separation product separated from the first mixed fraction comprises a monomeric compound A, a monomeric compound B, a monomeric compound C, and a monomeric compound D; the separation product separated from the second mixed fraction comprises a monomeric compound E, a monomeric compound F, a monomeric compound G, and a monomeric compound H;the monomeric compound A has a structural formula of formula (1):0ch3formula (1)in formula (1), R is -P-D-glucosyl(l —>6)-P-D-glucoside;the monomeric compounds B to E each have a general formula of formula (2):□ch3formula (2)wherein for a structural formula of the monomeric compound B, in formula (2), Ri is -oxygen-P-D-glucosyl, R2 is -P-D-glucoside, and R3 is hydrogen;for a structural formula of the monomeric compound C, in formula (2), Ri is -hydroxyl, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is hydrogen;for a structural formula of the monomeric compound D, in formula (2), Ri is hydrogen, R2 is -P-D-glucosyl(l —>6)-P-D-glucoside, and R3 is methyl; andfor a structural formula of the monomeric compound E, in formula (2), Ri is -oxygen-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, and R3 is hydrogen; andthe monomeric compounds F to H each have a general formula of formula (3):wherein for a structural formula of the monomeric compound F, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, R3 is hydrogen, and R4 is methyl;for a structural formula of the monomeric compound G, in formula (3), Ri is -P-D-glucosyl(l—>6)-P-D-glucoside, R2 is -P-D-glucosyl(l—>6)-P-D-glucoside, R3 is hydrogen, and R4 is hydroxymethylene; andfor a structural formula of the monomeric compound H, in formula (3), Ri is -P-D-20glucosyl(l^6)-P-D-glucoside, R2 is -P-D-apiose(l^2)P-D-glucosyl(l^6)-P-D-glucoside, R3 is hydrogen, and R4 is methyl.

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