Dihydrochalcone glycosides in a traditional Chinese medicine baeckea fruit and a preparation method and application thereof

Abstract

The application discloses a dihydrochalcone glycoside compound in traditional Chinese medicine bryonia and a preparation method and application thereof. The preparation method is as follows: taking cup-stem bryonia as initial raw material, taking the aboveground part, crushing, extracting at room temperature with 95% ethanol, concentrating the extract to obtain an extract; dispersing the extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, recovering the solvents to obtain a petroleum ether part, an ethyl acetate part and an n-butanol part; dissolving the ethyl acetate part extract with 10% ethanol, separating by polyamide column chromatography to obtain three parts A, B and C; and separating the B part by polyamide column chromatography with (ethyl acetate: ethanol: glacial acetic acid: water = 10:5:1:1) to obtain compounds 1, 2 and 3, i.e. dihydrochalcone glycoside compounds. The preparation process is simple, the extraction rate is high, and the development and utilization of the traditional Chinese medicine bryonia are facilitated; the compounds have antioxidant, antitumor and antibacterial activities, and can be applied to antioxidant and inhibition of proliferation of hepatoma cells.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine compound technology, and in particular to a dihydrochalcone glycoside compound from the traditional Chinese medicine *Hemiberlesia asiatica*, its preparation method, and its application. Background Technology

[0002] Dihydrochalcone is a class of compounds with a benzo[a]chromone core structure and possesses antioxidant, antitumor, antidiabetic, antibacterial, and estrogen-like activities.

[0003] The earliest record of *Balanophora subcupularis* as a medicinal herb is found in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and also in the *Compendium of Materia Medica*, where it is called "Ge Ru" and is known in Chinese folk medicine as "Wen Wang Yi Zhi Bi" (King Wen's Brush). It is widely distributed in southwestern regions such as Sichuan, Guizhou, and Yunnan, and is one of the "Four Famous Medicines" widely used in folk medicine among ethnic minorities in southern China. In Miao medicine, the entire plant of *Balanophora subcupularis* is often used. It is bitter and astringent, and has a cold nature. It is believed to have effects such as cooling the blood and stopping bleeding, clearing heat and detoxifying, promoting qi circulation and relieving pain, tonifying the kidneys and strengthening yang, stopping bleeding and promoting tissue regeneration, and relieving hangovers. It is mainly used to treat wind-heat rashes, metrorrhagia, intestinal bleeding, syphilis, boils, hemorrhoids, cough with hemoptysis, and penile swelling in children. In traditional Chinese medicine and Miao medicine, *Balanophora subcupularis* is derived from various sources, including *Balanophora subcupularis*, *Balanophora tubeosa*, and *Balanophora rubescens*. The *Balanophora subcupularis* used in this invention is *Balanophora subcupularis* Tam.

[0004] Since 1950, research on the chemical constituents of plants in the genus *Heliotropium* has been reported. To date, triterpenes, iridoids, steroids, flavonoids, phenylpropanoids, phenolic acids, and tannins have been isolated from this genus. Furthermore, novel flavonoid glycosides with significant pharmacological activities have been continuously isolated, including antitumor, anti-inflammatory, analgesic, hangover-relieving, liver-protective, anti-aging, antioxidant, and anti-fatigue effects. Studies have shown that *Heliotropium* polysaccharides and flavonoids have certain therapeutic effects on hepatitis and cirrhosis in clinical practice. Polyphenolic components can effectively inhibit the proliferation of liver cancer cells, and the entire process of liver cancer cell deformation and necrosis can be observed under a microscope.

[0005] This invention uses ethanol as the extraction solvent, and petroleum ether, ethyl acetate, and n-butanol as extraction solvents. The obtained petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction are separated by polyamide column chromatography to purify the extract of *Phloretin* stalk, yielding three new dihydrochalcone components: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Through activity screening and other studies, the antioxidant efficacy of *Phloretin* has been preliminarily understood, providing a material basis for the development of new antioxidant drugs and the quality evaluation standards of *Phloretin*, and also providing a scientific basis for the further development and utilization of *Phloretin* resources. Summary of the Invention

[0006] The purpose of this invention is to provide a dihydrochalcone glycoside compound from the traditional Chinese medicine *Smilax china*.

[0007] Another objective of this invention is to provide a method for preparing dihydrochalcone glycosides from the traditional Chinese medicine *Hemiberlesia asiatica*.

[0008] Another objective of this invention is to provide the application of dihydrochalcone glycosides from the traditional Chinese medicine *Hemiberlesia asiatica* in the preparation of antioxidant drugs.

[0009] This invention is achieved through the following technical solution:

[0010] A dihydrochalcone glycoside compound from the traditional Chinese medicine *Phloretin*, wherein the dihydrochalcone glycoside compound is: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside), with the following structural formula:

[0011]

[0012] The preparation method of the present invention includes the following steps:

[0013] (1) Take the fresh aerial parts of the cup stem snake rice, crush them with a juicer, extract them with ethanol solution at room temperature, and concentrate the extract to obtain an extract.

[0014] (2) The extract was dispersed in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvent was recovered to obtain the petroleum ether fraction, the ethyl acetate fraction and the n-butanol fraction.

[0015] (3) Dissolve part of the ethyl acetate extract in ethanol solution, and separate it by polyamide column chromatography using an eluent to obtain three parts A, B and C;

[0016] (4) The B part was separated by polyamide column chromatography using an eluent to obtain compound 1, compound 2 and compound 3.

[0017] The preparation method of the present invention specifically includes the following steps:

[0018] (1) Take 20 kg of fresh aerial parts of *Heliotropium indicum*, crush them using a juicer, and extract them three times with ethanol solution at room temperature. Combine the extracts and concentrate to obtain 900 g of extract;

[0019] (2) The extract was dispersed in 2L of water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvent was recovered to obtain 170g of petroleum ether fraction, 244g of ethyl acetate fraction and 150g of n-butanol fraction.

[0020] (3) Dissolve part of the ethyl acetate extract in ethanol solution, and separate it by polyamide column chromatography using an eluent to obtain three parts A, B and C;

[0021] (4) The B part was separated by polyamide column chromatography using an eluent to obtain compound 1 24 mg, compound 2 46 mg, and compound 3 40 mg.

[0022] The ethanol solution mentioned in step (1) of this invention is 95% ethanol.

[0023] The ethanol solution mentioned in step (3) of this invention is 10% ethanol.

[0024] The polyamide in step (3) of this invention has a mesh size of 200 to 300 mesh.

[0025] The eluent in step (3) of this invention is: ethanol / water = 30% to 70%, gradient elution.

[0026] The eluent in step (4) of this invention is: ethyl acetate: ethanol: glacial acetic acid: water = 10:5:1:1.

[0027] The application of dihydrochalcone glycoside compounds 1, 2, and 3 from the traditional Chinese medicine *Smilax china* in the preparation of antioxidant drug formulations.

[0028] The formulations described in this invention are prepared by adding pharmaceutically acceptable excipients and using conventional processes to form pharmaceutically acceptable formulations. The pharmaceutically acceptable formulations are solid or liquid formulations; the solid formulations are granules, capsules, tablets, or pills; and the liquid formulations are injectable formulations or oral liquids.

[0029] Beneficial effects of this invention:

[0030] 1. In this invention, three new compounds were isolated and identified from Phloretin: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Compounds 1, 2, and 3 have not been reported in existing literature.

[0031] 2. Antioxidant activity experiments revealed that DPPH + and ABTS + Free radical scavenging experiments showed that compounds 1, 2, and 3 had antioxidant capacity comparable to that of the positive control vitamin C. Attached Figure Description

[0032] Figure 1 UV spectrum of compound 1

[0033] Figure 2 IR spectrum of compound 1

[0034] Figure 3 HR-ESI-MS spectrum of compound 1

[0035] Figure 4 Compound 1 1 H-NMR spectrum

[0036] Figure 5 Compound 1 13 C-NMR spectrum

[0037] Figure 6 HSQC spectrum of compound 1

[0038] Figure 7 COSY spectrum of compound 1

[0039] Figure 8 HMBC spectrum of compound 1

[0040] Figure 9 ROESY spectrum of compound 1

[0041] Figure 10 UV spectrum of compound 2

[0042] Figure 11 IR spectrum of compound 2

[0043] Figure 12 HR-ESI-MS spectrum of compound 2

[0044] Figure 13 Compound 2 1 H-NMR spectrum

[0045] Figure 14 Compound 2 13 C-NMR spectrum

[0046] Figure 15 HSQC spectrum of compound 2

[0047] Figure 16 COSY spectrum of compound 2

[0048] Figure 17 HMBC spectrum of compound 2

[0049] Figure 18 ROESY spectrum of compound 2

[0050] Figure 19 Compound 3 1 H-NMR spectrum

[0051] Figure 20 Compound 3 13 C-NMR spectrum

[0052] Figure 21 HSQC spectrum of compound 3

[0053] Figure 22 COSY spectrum of compound 3

[0054] Figure 23 HMBC spectrum of compound 3

[0055] Figure 24 ROESY spectrum of compound 3

[0056] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0057] Example 1

[0058] The dihydrochalcoside compounds are: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside).

[0059]

[0060] Example 2: Preparation method of dihydrochalcone glycosides from the traditional Chinese medicine *Smilax china*

[0061] (1) Take 20 kg of fresh aerial parts of *Heliotropium indicum*, crush them using a juicer, and extract them three times at room temperature with 95% ethanol solution. Combine the extracts and concentrate to obtain 900 g of extract;

[0062] (2) The extract was dispersed in 2L of water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvent was recovered to obtain 170g of petroleum ether fraction, 244g of ethyl acetate fraction and 150g of n-butanol fraction.

[0063] (3) Dissolve the ethyl acetate extract in 10% ethanol solution, elute with a gradient of ethanol / water = 30% to 70%, separate by polyamide (200 to 300 mesh) column chromatography, and detect by polyamide thin-layer chromatography to obtain three parts A, B and C;

[0064] (4) Analysis of Part B using silica gel thin-layer chromatography, reversed-phase silica gel thin-layer chromatography, and polyamide thin-layer chromatography revealed that this part mainly contains flavonoids with extremely similar structures, making separation difficult using silica gel and reversed-phase silica gel. Polyamide thin-layer chromatography analysis showed that the ethanol:water system also failed to separate these components. Through repeated trials of eluent composition and ratio, the solvent system of ethyl acetate:ethanol:glacial acetic acid:water = 10:5:1:1 was found to have better separation performance. Therefore, Part B was separated by polyamide column chromatography using ethyl acetate:ethanol:glacial acetic acid:water = 10:5:1:1 to obtain compound 1 24 mg, compound 2 46 mg, and compound 3 40 mg.

[0065] Example 3

[0066] Take any one, two, or three of the following compounds as raw materials: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Add pharmaceutically acceptable excipient dextrin, granulate, and obtain granules.

[0067] Example 4

[0068] Take any one, two, or three of the following compounds as raw materials: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Add pharmaceutically acceptable excipient dextrin, mix well, and fill into capsules to obtain capsules.

[0069] Example 5

[0070] Take any one, two, or three of the following compounds as raw materials: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Add pharmaceutically acceptable excipient dextrin, granulate, and compress to obtain tablets.

[0071] Example 6

[0072] Take any one, two, or three of the following compounds as raw materials: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Add pharmaceutically acceptable excipient dextrin, mix well, compress into pellets, and dry to obtain pills.

[0073] Example 7

[0074] Take any one, two, or three of the following compounds as raw materials: compound 1 (Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside), compound 2 (Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside), and compound 3 (phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside). Add 10 times the volume of water for injection, mix well, filter, and sterilize to obtain the injection.

[0075] Experimental Example 1

[0076] Physicochemical data of compound 1:

[0077] Phloretin-4'-O-(4”-O-galloyl)-β-D-glucoside(1), white amorphous powder; [α]2 D 0–149.7(c 0.1,MeOH); HR-ESI-MS m / z:589.1551[M+H] + ;UV(MeOH):λ max 220.5, 281.5 nm; IR(KBr)ν max cm -1 :3349,2947,1704,1630,1598,1515,1438,1353,1176,1030,828; 1 H-NMR (400MHz, MeOD): δ7.05(2H,d,J=8.5Hz,H-2,6),6.72(2H,d,J=8.5Hz,H-3,5),2.87(2H, dd,J=8.7,6.8Hz,H-7),3.32(2H,overlapped,H-8),6.14(2H,s,H-3',5'),5.07(1H,d,J=7.2 Hz,H-1”),3.62(1H,overlapped,H-2”),3.79(1H,m,H-3”),5.07(1H,m,H-4”),3.85(1H,t,J= 9.3Hz,H-5”),3.72(1H,m,H-6”a),3.63(1H,overlapped,H-6”b),7.16(2H,s,galloylH-2,6); 13C-NMR (100MHz, MeOD): δ133.8(s,C-1),130.3(d,C-2,6),116.1(d,C-3,5),156. 2(s,C-4),31.1(t,C-7),47.4(t,C-8),207.1(s,C-9),106.9(s,C-1'),165.2(s ,C-2',6'),96.5(d,C-3',5'),164.7(s,C-4'),100.9(d,C-1”),74.8(d,C-2”), 76.3(d,C-3”),72.1(d,C-4”),75.6(d,C-5”),62.0(t,C-6”),120.9(s,galloyl C-1), 110.4 (d, galloyl C-2, 6), 146.4 (s, galloyl C-3, 5), 140.1 (s, galloyl C-4), 167.7 (s, galloyl C-7).

[0078] Experimental Example 2

[0079] Physicochemical data of compound 2:

[0080] Phloretin-4'-O-(3”-O-galloyl)-β-D-glucoside(2), white amorphous powder; [α]2 D 0–208.4(c 0.1,MeOH); HR-ESI-MS m / z:589.1560[M+H] + ;UV(MeOH):λ max 219.0, 280.5 nm; IR(KBr)ν max cm -1 :3356,2943,1629,1598,1515,1446,1384,1348,1130,1078,983,830; 1H-NMR (400MHz, MeOD): δ7.06(2H,d,J=8.4Hz,H-2,6), 6.72(2H,d,J=8.4Hz,H-3,5), 2.88(2H,dd,J=8.5,6 .7Hz,H-7),3.33(2H,overlapped,H-8),6.14(2H,s,H-3',5'),5.11(1H,d,J=7.8Hz,H-1”),3.73(1H,ove rlapped,H-2”),5.25(1H,t,J=9.5Hz,H-3”),3.62(1H,ddd,J=9.5,5.1,2.3Hz,H-4”),3.73(1H,overlapp ed,H-5”),3.95(1H,dd,J=12.2,2.2Hz,H-6”a),3.80(1H,dd,J=12.2,5.1Hz,H-6”b),7.18(2H,s,galloyl H-2,6); 13 C-NMR (100MHz, MeOD): δ133.8(s,C-1),130.3(d,C-2,6),116.1(d,C-3,5),156. 4(s,C-4),31.2(t,C-7),47.5(t,C-8),207.1(s,C-9),107.0(s,C-1'),165.3(s ,C-2',6'),96.5(d,C-3',5'),164.8(s,C-4'),101.1(d,C-1”),73.2(d,C-2”), 78.9(d,C-3”),69.4(d,C-4”),78.2(d,C-5”),62.1(t,C-6”),121.7(s,galloyl C-1), 110.4 (d, galloyl C-2, 6), 146.4 (s, galloyl C-3, 5), 139.8 (s, galloyl C-4), 168.2 (s, galloyl C-7).

[0081] Experimental Example 3

[0082] Physicochemical data of compound 3:

[0083] phloretin-4′-O-(6″-O-galloyl)-β-D-glucoside(3): white amorphous powder; 1H-NMR (400MHz, MeOD): δ7.03(2H,d,J=8.4Hz,H-2,6), 6.68(2H,d,J=8.4Hz,H-3,5), 2.85(2H,dd,J= 8.5,6.4Hz,H-7),3.30(2H,overlapped,H-8),6.08(2H,s,H-3′,5′),4.97(1H,d,J=7.1Hz,H-1″),3. 47(1H,overlapped,H-2″),3.51(1H,overlapped,H-3″),3.52(1H,overlapped,H-4″),3.74(1H,m,H -5″),4.45(1H,dd,J=12.0,4.9Hz,H-6″a),4.55(1H,dd,J=12.0,2.3Hz,H-6″b),7.08(2H,s,galloyl H-2,6); 13 C-NMR (100MHz, MeOD): δ133.9(C-1),130.3(C-2,6),116.1(C-3,5),156.4(C-4),31.2(C-7),47.5(C-8),207.1(C-9),107.0(C-1′),165.4 (C-2′,6′),96.5(C-3′,5′),164.8(C-4′),101.1(C-1″),74.6(C-2″),77.7(C-3″),71.2(C-4″),75.7(C-5″),64.3(C-6″),121.3(galloyl C-1),110.3(galloyl C-2,6),146.5(galloyl C-3,5),139.9(galloyl C-4),168.4(galloyl C-7).

[0084] Experiment Example 4 Antioxidant Activity Experiment

[0085] (1) Pretreatment of experimental materials

[0086] Preparation of DPPH solution: Accurately weigh 3.94 mg of DPPH and dissolve it in methanol. Dilute the solution to 10 mL in a brown volumetric flask. Take 1 mL of the DPPH solution and dilute it to 10 mL in a brown volumetric flask to obtain a DPPH concentration of 0.01 mmol / L. Protect from light.

[0087] Preparation of ABTS solution: Accurately weigh 38.41 mg of ABTS and dilute with distilled water to 10 mL; weigh 66.1 mg of K₂S₂O₈ and dilute with distilled water to 100 mL; then mix the ABTS solution and K₂S₂O₈ solution in equal volumes, store in the dark for 12-16 hours, and finally... + The stock solution was diluted with distilled water to achieve an absorbance of approximately 1.1 at a wavelength of 734 nm.

[0088] Sample preparation: Dissolve approximately 10 mg of each test sample in ethanol, and dilute the concentrations to 10 / 1, 50 / 1, 100 / 1, 500 / 1, and 1000 / 1, respectively.

[0089] Reference standard preparation: Take V C Approximately 1g was fully dissolved in ethanol to prepare gradient concentrations of 0.1mg / mL, 0.5mg / mL, 1mg / mL, 2.5mg / mL, and 5mg / mL.

[0090] (2) Experimental methods

[0091] The experiment was divided into three groups: (1) blank group, (2) sample group, and (3) control group. Three replicate wells were set for each sample concentration, and the average value was taken.

[0092] DPPH + Determination of scavenging ability: The sample was reacted at room temperature in the dark for 30 min, and its absorbance A0 was measured at 517 nm.

[0093] Table 1 DPPH + Experimental sample addition method

[0094]

[0095] DPPH. + Free radical scavenging rate (%) = [1-(A i -A j ) / A0]×100.

[0096] ABTS. + Determination of scavenging ability: After adding the sample, shake the 96-well plate to mix for 10 s, react at room temperature for 6 min, and measure the absorbance at 734 nm.

[0097] Table 2 ABTS. + Experimental sample addition method

[0098]

[0099] ABTS. + Free radical scavenging rate = (A0-A1) / A0 × 100%

[0100] (3) Experimental Results

[0101] Table 3. Compounds DPPH and ABTS IC 50 ±SD

[0102]

[0103] Table 3 shows that DPPH + and ABTS + Free radical scavenging experiments showed that compounds 1, 2, and 3 had antioxidant capacity comparable to that of the positive control vitamin C.

[0104] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing dihydrochalcone glycosides from the traditional Chinese medicine *Smilax china*, characterized in that, The preparation method includes the following steps: (1) Take the fresh aerial parts of the cup stem snake rice, crush them with a juicer, extract them with ethanol solution at room temperature, and concentrate the extract to obtain an extract. (2) The extract was dispersed in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvent was recovered to obtain the petroleum ether fraction, the ethyl acetate fraction and the n-butanol fraction. (3) Dissolve the ethyl acetate extract in ethanol solution, and separate it by polyamide column chromatography using an eluent to obtain three parts A, B and C; the eluent is: ethanol / water = 30%~70%, gradient elution; (4) Part B was separated by polyamide column chromatography using an eluent to obtain dihydrochalcone glycoside compounds 1 and 2, whose structural formulas are as follows: Compound 1 Compound 2.

2. The method for preparing dihydrochalcone glycosides from the traditional Chinese medicine *Smilax china* according to claim 1, characterized in that, The preparation method specifically includes the following steps: (1) Take 20 kg of fresh aerial parts of the cup stem snake grass, crush them with a juicer, extract them three times with ethanol solution at room temperature, combine the extracts, and concentrate them to obtain 900 g of extract. (2) The extract was dispersed in 2 L of water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. The solvent was recovered to obtain 170 g of petroleum ether fraction, 244 g of ethyl acetate fraction and 150 g of n-butanol fraction. (3) Dissolve the ethyl acetate extract in ethanol solution, and separate it by polyamide column chromatography using an eluent to obtain three parts A, B and C; the eluent is: ethanol / water = 30%~70%, gradient elution; (4) The B part was separated by polyamide column chromatography using an eluent to obtain compound 1 24 mg and compound 2 46 mg; the eluent was ethyl acetate: ethanol: glacial acetic acid: water = 10:5:1:

1.

3. A method for preparing dihydrochalcone glycosides from the traditional Chinese medicine *Smilax china* according to any one of claims 1 or 2, characterized in that, The ethanol solution in step (1) of the preparation method is 95% ethanol.

4. A method for preparing dihydrochalcone glycosides from the traditional Chinese medicine *Smilax china* according to any one of claims 1 or 2, characterized in that, The ethanol solution in step (3) of the preparation method is 10% ethanol.

5. A method for preparing dihydrochalcone glycosides from the traditional Chinese medicine *Smilax china* according to any one of claims 1 or 2, characterized in that, The mesh size of the polyamide in step (3) of the preparation method is 200~300 mesh.

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