CN111574573A - Fifteen phenylethanoid glycoside compounds and separation and purification method and application thereof - Google Patents

Abstract

The invention discloses fifteen phenylethanoid glycoside compounds, a separation and purification method thereof and application thereof in preparing anti-inflammatory functional foods and medicaments, wherein the fifteen phenylethanoid glycoside compounds have the structures shown in formulas (1) to (15). The fifteen phenylethanoid glycosides compounds provided by the invention are used as natural compounds, and have low toxicity to normal cells and small side effect; the composition also shows obvious anti-inflammatory effect below the cytotoxic concentration, and has the advantages of moderate effective dose, obvious curative effect, small toxic and side effects and the like, so the composition has wide application prospect.

Description

Fifteen phenethyl glycosides and their separation and purification methods and applications

technical field

The invention relates to the technical field of traditional Chinese medicines, in particular to fifteen phenethyl glucoside compounds and a separation and purification method and application thereof, in particular to fifteen new phenethyl glucoside compounds, a separation and purification method thereof, and the Application of glycol glycoside compound in the preparation of anti-inflammatory functional food and medicine.

Background technique

Cistanche Herba, also known as golden bamboo shoots, goblin, Cistanche, and Dayun, is the dry, scaly stem of Cistanche deserticola Y.C.Ma or C.tubulosa (Schrenk) Wight of Lidanaceae. It was first recorded in "Shen Nong's Materia Medica" and was listed as a top grade. Later, it was recorded in the classical "Ben Cao Jing Shu", "Ben Cao Hui Yan", "Yue Hua Zi Materia Medica" and so on. Li Shizhen said: "This thing is tonic but not severe, so it has a calmness." Cistanche is sweet, salty and warm in nature. It has the functions of invigorating the kidney and strengthening yang, filling essence and marrow, nourishing blood and moisturizing dryness, and prolonging life. Constipation, etc. It has been used in my country for more than 2,000 years, and it ranks first in the prescriptions of Zengli traditional Chinese medicines, and second only to ginseng in anti-aging and anti-aging prescriptions. Due to the increasing depletion of wild resources of Cistanche, it has been listed as a second-class protected species in my country, and has been included in the "International List of Wild Plant Protection".

Cistanche is mainly produced in Inner Mongolia, Gansu, Xinjiang, Qinghai and other places; it is warm in nature and sweet and salty in taste. Modern pharmacological studies have shown that Cistanche deserticola has the functions of anti-fatigue, anti-inflammatory, anti-aging, enhancing immunity and enhancing memory, in addition to tonifying the kidney and strengthening yang, moistening the intestines and laxative, and no adverse reactions have been found.

Since the 1970s, scholars at home and abroad have begun to systematically study the chemical constituents and pharmacological activities of Cistanche. So far, nearly 100 chemical components and trace elements have been isolated and identified from Cistanche deserticola, mainly including phenylethanol glycosides, iridoid glycosides, lignan glycosides, monosaccharides, disaccharides, polysaccharides, amino acids, polypeptides, proteins , triterpenes, sterols, polyols, etc. Among them, phenylethanoid glycosides have the highest content in the original medicinal materials, and are one of the main active components of Cistanche spp. They are also the index components for identification and content determination under the Chinese Pharmacopoeia "Cistanberry". They have antioxidant, anti-inflammatory, and promote substance metabolism. , Improve learning and memory, enhance sexual function and so on. At present, it is still necessary to expand the types of phenylethanoid glycosides to increase the selectivity in terms of antioxidant, anti-inflammatory, promoting substance metabolism, improving learning and memory, and enhancing sexual function.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides fifteen phenethyl glucoside compounds and their separation and purification methods and applications.

To achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention is to provide fifteen kinds of phenethyl alcohol glycoside compounds, the structural formulas are as follows (1)-(15):

The second aspect of the present invention is to provide the above-mentioned fifteen kinds of separation and purification methods of phenethyl alcohol glycoside compounds, comprising the following steps:

S1: at room temperature, multiple times of cold dipping and percolation extraction are performed on Cistanche deserticola with ethanol, and the extracts are combined, then, the solvent is recovered under reduced pressure, and concentrated to obtain an ethanol extract;

S2: above-mentioned ethanol extract is suspended in water, and then extracted with cyclohexane, ethyl acetate and n-butanol respectively to obtain cyclohexane extract, ethyl acetate extract CE, n-butanol extract and water Partial extract;

S3: Dissolve the above-mentioned ethyl acetate extract CE with chloroform and methanol, mix samples with column chromatography silica gel, then load into a silica gel column, load samples, and perform gradient elution; use thin-layer TLC analysis, and combine the same fractions Then 16 components CE-1～CE16 were obtained;

S4: Take CE-7, dissolve it with methanol and filter, and obtain 4 fractions CE-7-1～CE-7-4 through Rp-18 high performance liquid chromatography; take the component CE-7-1 and dissolve it with methanol Filtration, rough separation by Rp-18 high performance liquid chromatography, and compound 1 and compound 2 were obtained at retention times of 42-43min and 84-85min respectively; take the component CE-7-4, dissolve it with methanol and filter, pass through Rp-18 High performance liquid chromatography was used for crude separation to obtain compounds 3, 4, 5, 6 and 7 at retention times of 12.0-12.5min, 15.5-16.0min, 17.5-18.0min, 42.0-42.5min and 56.8-57.2min respectively;

S5: Take CE-9, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 6 components CE-9-1 ~CE-9-6; take the component CE-9-6, dissolve it in methanol and filter it, pass through Rp-18 high performance liquid chromatography for rough separation, and obtain compound 8 at a retention time of 23.5-24.0 min;

S6: Take CE-10, dissolve it with a small amount of dichloromethane-methanol, and perform gel column chromatography to implement isocratic elution; use thin-layer TLC analysis, and combine the same fractions to obtain 3 components CE-10- 1～CE-10-3; take the component CE-10-2, dissolve it with methanol and filter, and pass through Rp-18 high performance liquid chromatography for rough separation, the retention times are 18.0-18.5min, 18.8-19.2min and 20.0- Compounds 9, 10 and 11 were obtained in 23.5 min;

S7: Take CE-12, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 5 components CE-12-1 ~CE-12-5; take the component CE-12-5, dissolve it in methanol and filter it, pass through Rp-18 high performance liquid chromatography for rough separation, and obtain compound 12 at a retention time of 43.8-44.2min;

S8: Take CE-13, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 9 components CE-13-1 ~CE-13-9; take the component CE-13-7, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography, and obtain compound 13 and compound 14 at about 25.1 min and 26.4 min respectively at retention time. .

S9: Take CE-14, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 4 components CE-14-1 ~CE-14-4; take the component CE-14-1, dissolve it in methanol and filter, pass through Rp-18 high performance liquid chromatography for rough separation, and obtain compound 15 at a retention time of 34.0-34.5 min.

Further, the concentration of ethanol in S1 was 85%.

Further, the eluents adopted in the silica gel column gradient elution in S3 are successively dichloromethane, dichloromethane-methanol with a volume ratio of 100:1, dichloromethane-methanol with a volume ratio of 20:1, and a volume ratio of 10:1 dichloromethane-methanol, 5:1 volume ratio of dichloromethane-methanol, 2:1 volume ratio of dichloromethane-methanol, methanol.

Further, the above-mentioned column chromatography silica gel is 100-200 mesh column chromatography silica gel.

Further, the gel of the above-mentioned gel column chromatography is Sephadex LH-20.

Further, the eluent used in the above-mentioned gel column chromatography is dichloromethane-methanol, wherein the volume ratio of dichloromethane and methanol is 7:3.

Further, the flow rate of the above isocratic elution is 200ml/h, and each 100ml is concentrated as a fraction.

Further, the specifications of the chromatographic column used for the above-mentioned high-performance liquid chromatography rough separation are Cosmosil 5C ₁₈ -MS-II, 5 μm, 20×250 mm; the flow rate used is 8 mL/min, and the fluidity is methanol/water.

The second aspect of the present invention is to provide a pharmaceutical preparation, comprising any one or more of the above-mentioned fifteen phenethyl glycoside compounds.

The third aspect of the present invention is to provide the application of the above-mentioned fifteen phenethyl glycoside compounds in the preparation of anti-inflammatory functional foods and medicines.

The present invention adopts the above technical scheme, compared with the prior art, has the following technical effects:

The fifteen phenethyl glycoside compounds provided by the present invention, as a natural compound, have low toxicity to normal cells and less side effects; below the cytotoxic concentration, they also exhibit significant anti-inflammatory effects and have a moderate effective dose. , the curative effect is remarkable, the toxicity and side effects are small and so on, therefore, it has the broad application prospect.

Description of drawings

Figures 1-9 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 1 in the present invention;

Figures 10-18 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 2 in the present invention;

Figures 19-27 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 3 in the present invention;

Figures 28-36 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 4 in the present invention;

Figures 37-41 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV images of compound 5 in the present invention;

Figures 42-50 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR, and UV diagrams of compound 6 in the present invention;

Figures 51-59 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR, and UV diagrams of compound 7 in the present invention;

Figures 60-68 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 8 in the present invention;

Figures 69-77 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 9 in the present invention;

Figures 78-86 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR, and UV diagrams of compound 10 in the present invention;

Figures 87-95 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR, and UV diagrams of compound 11 in the present invention;

Figures 96-104 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV diagrams of compound 12 in the present invention;

Figures 105-113 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV images of compound 13 in the present invention;

Figures 114-118 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR, and UV diagrams of compound 14 in the present invention;

Figures 119-123 are ¹ H NMR, ¹³ C NMR, 2D-NMR, MS, IR and UV images of compound 15 in the present invention;

Figure 124 shows a graph of the results of the cytotoxicity of phenethyl glycosides of the present invention to RAW 264.7 cells;

Figure 125 shows a bar graph of the inhibitory rate of the phenethyl glycosides of the present invention on LPS-induced NO release in RAW 264.7 cells.

Detailed ways

The present invention provides fifteen kinds of phenethyl glucoside compounds, and expands the types of phenethyl glucoside compounds of Cistanche deserticola plants at present. Through a series of experiments, it is found that these compounds have good anti-inflammatory and immune-enhancing effects, and can become new anti-inflammatory functional foods and medicines on the market in the future.

The present invention will be described in detail and concretely below through specific embodiments, so as to make the present invention better understood, but the following embodiments do not limit the scope of the present invention.

Example 1

The present embodiment provides a phenethyl glycoside compound, which has the following general formulas (I)-(III):

wherein R represents a different type of substituent.

In particular, the above-mentioned phenethyl glycoside compounds include compounds with the following structural formulas (1)-(15):

According to the structural formula (1), the phenethyl alcohol glycoside compound 1 can be named 2-(4-hydroxyphenethyl)-[4-O-transcaffeoyl]-β-D-mannoside (2-(4 -hydroxyphenylethyl)-[4-O-trans-caffeoyl]-β-D-mannoside), referred to as cisstanoloside A;

According to the structural formula (2), the phenethyl glycoside compound 2 can be named 2-(4-hydroxyphenethyl)-[4-O-transcaffeoyl]-β-D-alloside (2-( 4-hydroxyphenyl)ethyl-[4-O-trans-caffeoyl]-β-D-allopyranoside), referred to as cistanoloside B;

According to the structural formula (3), the phenethyl alcohol glycoside compound 3 can be named as 2-(4-hydroxyphenethyl)-[4-O-transcoumaroyl]-β-D-mannoside (2-( 4-hydroxyphenylethyl)-[4-O-trans-coumaroyl]-β-D-mannoside), referred to as cisstanoloside C;

According to the structural formula (4), the phenethyl alcohol glycoside compound 4 can be named as 2-(4-hydroxyphenethyl)-[4-O-transcoumaroyl]-β-D-alloside (2- (4-hydroxyphenyl)ethyl-[4-O-trans-coumaroyl]-β-D-allopyranoside), referred to as cisstanoloside D;

According to the structural formula (5), the phenethyl alcohol glycoside compound 5 can be named as 2-(4-hydroxyphenethyl)-[4-O-cis-coumaroyl]-β-D-mannoside (2-( 4-hydroxyphenyl)ethyl-[4-O-cis-coumaroyl]-β-D-allopyranoside), referred to as cisstanoloside E;

According to the structural formula (6), the phenethyl glycoside compound 6 can be named 2-(4-hydroxyphenethyl)-rhamnose (1→3)-[4-O-trans-cinnamoyl]-β- D-glucoside (2-(4-hydroxyphenyl)ethyl-O-α-L-rhamnopyranosyl-(1→3)-[4-O-trans-cinaamoyl]-β-D-glucopyranoside), referred to as cisstanoloside F;

According to the structural formula (7), the phenethyl alcohol glycoside compound 7 can be named 2-phenethyl alcohol-rhamnose (1→3)-[4-O-trans-coumaroyl]-β-D-glucoside (2-phenylethyl-O-α-L-rhamnopyranosyl-(1→3)-[4-O-trans-coumaroyl]-β-D-glucop yranoside), referred to as cisstanoloside G;

According to the structural formula (8), the phenethyl glycoside compound 8 can be named 2-(3-methoxy-4-hydroxyphenethyl)-rhamnose (1→3)-[4-O-trans Coumaroyl]-β-D-glucoside (2-(3-methoxy-4-hydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1→3)-[4-O-trans-coumaroyl)]-β-D -glucopyranoside), referred to as cisstanoloside H;

According to the structural formula (9), the phenethyl glycoside compound 9 can be named (7S)-2-ethoxy-(3,4-dihydroxyphenethyl)-rhamnose (1→3)-[2 -Acetyl-4-O-transcaffeoyl]-β-D-glucoside ((7S)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-( 1→3)-[2-acetyl-4-O-trans-caffeoyl]-β-D-glucopyranoside), referred to as cisstanoloside I;

According to the structural formula (10), the phenethyl glycoside compound 10 can be named as (7R)-2-ethoxy-(3,4-dihydroxyphenethyl)-rhamnose (1→3)-[2 -Acetyl-4-O-transcaffeoyl]-β-D-glucoside ((7R)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-( 1→3)-[2-acetyl-4-O-trans-caffeoyl]-β-D-glucopyranoside), referred to as cistanoloside J;

According to the structural formula (11), the phenethyl glycoside compound 11 can be named 2-(3,4-dihydroxyphenethyl)-rhamnose(1→3)-[2-acetyl-4-O- ciscaffeoyl]-β-D-glucoside (2-(3,4-dihydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1→3)-2-acetyl-[4-O-cis-caffeoyl)]- β-D-glucopyranoside), referred to as cisstanoloside K;

According to the structural formula (12), the phenethyl glycoside compound 12 can be named 2-(4-hydroxyphenethyl)-rhamnose (1→3)-[6-O-transcoumaroyl]-β -D-glucoside (2-(4-hydroxyphenyl)ethyl-O-α-L-rhamnopyranosyl-(1→3)-[6-O-cis-coumaroyl]-β-D-glucopyranoside), abbreviated as cisstanoloside L ;

According to the structural formula (13), the phenethyl glycoside compound 13 can be named (7S)-2-ethoxy-(3,4-dihydroxyphenethyl)-rhamnose (1→3)-[4 -O-transcaffeoyl]-β-D-glucoside ((7S)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-(1→3)- [4-O-trans-caffeoyl]-β-D-glucopyranoside), referred to as cisstanoloside M;

According to the structural formula (14), the phenethyl glycoside compound 14 can be named (7R)-2-ethoxy-(3,4-dihydroxyphenethyl)-rhamnose (1→3)-[4 -O-transcaffeoyl]-β-D-glucoside ((7R)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-(1→3)- [4-O-trans-caffeoyl]-β-D-glucopyranoside), referred to as cisstanoloside N;

According to structural formula (15), the phenethyl glycoside compound 15 can be named 2-(4-hydroxyphenethyl)-[6-O-ciscaffeoyl]-β-D-glucose

(2-(4-hydroxyphenyl)-ethyl-[6-O-cis-caffeoyl)]-β-D-glucopyranoside), referred to as cisstanoloside O.

Embodiment 2

The present embodiment provides methods for the separation and purification of fifteen kinds of phenethyl alcohol glycoside compounds in the embodiment, including the following steps:

Step 1, at room temperature, take 5.5kg of air-dried Cistanche deserticola, carry out multiple cold immersion and percolation extraction with 85% ethanol, combine the extracts, then recover the solvent under reduced pressure at 55°C, and concentrate to obtain 2.2kg of ethanol extract ; Suspended the ethanol extract in water, then extracted with cyclohexane, ethyl acetate, n-butanol respectively to obtain 77.0g of cyclohexane extract, 20.0g of ethyl acetate extract (CE), normal Butanol extract 41.0g and water part extract 2000g.

Step 2, take 20.0 g of the ethyl acetate extract CE, dissolve it with chloroform and methanol, mix the sample with 30 g of silica gel for column chromatography (100-200 mesh), dissolve the silica gel with dichloromethane and pack it into a column, and use two Equilibrate the column with methyl chloride until the surface of the silica gel no longer drops, dry the sample, and then use dichloromethane, dichloromethane-methanol with a volume ratio of 100:1, dichloromethane-methanol with a volume ratio of 20:1, and Dichloromethane-methanol with a ratio of 10:1, dichloromethane-methanol with a volume ratio of 5:1, dichloromethane-methanol with a volume ratio of 2:1, and methanol were subjected to gradient elution, and the flow rate was controlled at 1000ml/h , each 500ml is concentrated as a fraction. Using thin-layer TLC analysis, 16 components CE-1 to CE16 were obtained by combining the same fractions.

Step 3, take the 7th fraction CE-7 (1.0g) (obtained by dichloromethane-methanol 10:1 elution), dissolve and filter with methanol, and crudely separate by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 254nm), mobile phase is methanol:water (60:40, v/v), 4 fractions CE-7-1～CE- 7-4. Take the component CE-7-1, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 254nm), The mobile phase was methanol:water (35:65, v/v), and compound 1 (2.6 mg) and compound 2 (21.4 mg) were obtained at retention times of about 42.9 min and about 84.9 min, respectively. Take the component CE-7-4, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 205nm), The mobile phase is methanol: water (53:47, v/v), and compounds 3 (1.1 mg) and 4 ( 4.5 mg), 5 (2.6 mg), 6 (19.3 mg) and 7 (11.6 mg).

Step 4, take the 9th fraction CE-9 (3.0g) (obtained by dichloromethane-methanol 10:1 elution), dissolve it with a small amount of dichloromethane-methanol, and carry out Sephadex LH-20 gel (23 ×595mm) column chromatography, wherein the eluent used is dichloromethane-methanol, the volume ratio is 7:3, isocratic elution is performed, the flow rate is controlled at 200ml/h, and each 100ml is concentrated as a fraction. Using thin-layer TLC analysis, 6 components CE-9-1 to CE-9-6 were obtained after combining the same fractions. Take the component CE-9-6, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 205nm), The mobile phase was methanol:water (50:50, v/v), and compound 8 (4.1 mg) was obtained at a retention time of about 23.8 min.

Step 5, take the 10th fraction CE-10 (0.6g) (obtained by dichloromethane-methanol 10:1 elution), dissolve it with a small amount of dichloromethane-methanol, and carry out Sephadex LH-20 gel (15 ×350mm) column chromatography, wherein the eluent used is dichloromethane-methanol, the volume ratio is 7:3, and isocratic elution is performed; the flow rate is controlled at 200ml/h, and each 100ml is concentrated as a fraction. Using thin-layer TLC analysis, three components CE-10-1 to CE-10-3 were obtained after combining the same fractions. Take the component CE-10-2, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 205nm), The mobile phase was methanol: water (52:48, v/v), and compounds 9 (2.9 mg), 10 (3.1 mg) and 11 (4.5 mg were obtained at retention times of about 18.2 min, 19.0 min and 20.3 min, respectively). ).

Step 6, take the 12th fraction CE-12 (1.8g) (obtained from dichloromethane-methanol 5:1), dissolve it with a small amount of dichloromethane-methanol, and carry out Sephadex LH-20 gel (23 ×595mm) column chromatography, wherein the eluent used is dichloromethane-methanol, the volume ratio is 7:3, isocratic elution is performed, the flow rate is controlled at 200ml/h, and each 100ml is concentrated as a fraction. Using thin-layer TLC analysis, five components CE-12-1 to CE-12-5 were obtained after combining the same fractions. Take the component CE-12-5, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 330nm), The mobile phase was methanol:water (47:53, v/v), and compound 12 (1.5 mg) was obtained at a retention time of about 44.0 min.

Step 7, take the 13th fraction CE-13 (3.6g) (obtained from dichloromethane-methanol 5:1), dissolve it with a small amount of dichloromethane-methanol, and carry out Sephadex LH-20 gel (23 ×595mm) column chromatography, wherein the eluent used is dichloromethane-methanol, the volume ratio is 7:3, isocratic elution is performed, the flow rate is controlled at 200ml/h, and each 100ml is concentrated as a fraction. Using thin-layer TLC analysis, 9 components CE-13-1 to CE-13-9 were obtained after combining the same fractions. Take the component CE-13-7, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 205nm), The mobile phase was methanol:water (42:58, v/v), and compound 13 (0.5 mg) and compound 14 (0.5 mg) were obtained at retention times of about 25.1 min and 26.4 min, respectively.

Step 8, take the 14th fraction CE-14 (0.2g) (obtained from dichloromethane-methanol 5:1), dissolve it with a small amount of dichloromethane-methanol, and carry out Sephadex LH-20 gel (15 ×350mm) column chromatography, wherein the eluent used is dichloromethane-methanol, the volume ratio is 7:3, and isocratic elution is performed; the flow rate is controlled at 200ml/h, and each 100ml is concentrated as a fraction. By thin-layer TLC analysis, four components CE-14-1 to CE-14-4 were obtained by combining the same fractions. Take the component CE-14-1, dissolve it in methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography (Cosmosil 5C ₁₈ -MS-II, 5μm, 20×250mm, flow rate: 8mL/min, wavelength: 205nm), The mobile phase was methanol:water (45:55, v/v), and compound 15 (1.0 mg) was obtained at a retention time of about 34.1 min.

In addition, the natural phenethyl glycoside compounds of the present invention can also be prepared by other extraction processes or chemical synthesis methods.

Embodiment 3

The present embodiment identifies the structures of fifteen kinds of phenethanol glycosides in Example 1, and the specific operations and results are as follows:

Structural identification of compound 1

Yellow amorphous powder, HR-ESI-MS (negative) gave a quasi-molecular ion peak [MH] ^- m/z 461.1877 (calcd.C ₂₃ H ₂₅ O ₁₀ , 461.1853), suggesting a molecular weight of 462; combined ¹ H and ¹³ _The molecular formula was determined to be _C23H26O10 by C NMR, and the unsaturation was calculated to be ₁₁ . The UV spectrum has a strong absorption at 213 nm, suggesting the existence of phenethyl group, while the larger absorption at 327 nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3359cm ^-1 ), α,β-unsaturated ester (1632cm ^-1 ), benzene ring skeleton (1599,1516cm ^-1 ), glycosidic bond (824cm ^-1 ) and the like.

Referring to Figures 1-9, in conjunction with Table 1 and Table 2, on ¹ H NMR, 4 hydrogen proton formations of δ _H 7.04 (2H, d, J=8.0 Hz), 6.66 (2H, d, J=8.0 Hz) _A set of _A2B2 systems. In DEPT135°, the peaks of δ _C 34.74 and 70.03 all show downward, and the HMBC spectrum shows δ _H 2.74(2H,m,H-7) and δC 129.76( _C -2,6), 115.00(C-3,5 ) is related to 34.74(C-7), and a 4-hydroxyphenethyl moiety is deduced in the structure. In addition, another ³ hydrogen protons of δ _H 7.04(1H,s),7.00(1H,d,J=8.0Hz),6.76(1H,d,J=8.0Hz) in 1H-NMR spectrum form a group of ABX System, δ _H 7.46 (1H, d, J=15.9Hz), 6.26 (1H, d, J=15.9Hz) are two protons of the trans double bond, which suggests the existence of transcaffeoyl in the compound molecule . A sugar end group signal at δ _H 44.33 (1H, d, J=8.0 Hz), which was suggested in the HMQC spectrum to be related to δ _C 102.54 (C-1'), suggested that this sugar was an oxyglucoside. Hydrogen signal on sugar δ _H 4.88(1H,t,J=9.3Hz),4.33(1H,d,J=8.0Hz),and 3.67(1H,m) and carbon signal δC 102.54( _C -1'), 76.61(C-2'), 71.46(C-3'), 77.68(C-4'), 67.97(C-5') and 60.68(C-6') suggest that this sugar is different from that commonly found in Cistanche plants. The central sugar glucose. After acid hydrolysis, the derivatization of sugar residues was analyzed by HPLC, and the standard sugar was also derivatized by comparative analysis to determine that the central sugar of the compound was D-mannose. The HMBC spectrum shows that the correlation between δ _H H-1' (δ _H 4.33) of the central sugar and δ _C 70.03 (C-8) proves that the 4-hydroxyphenethyl group is attached to the C-1' position of the central sugar mannose, The H-4' of the central sugar (δ _H 4.88) is related to C-9" (166.09), which suggests that the caffeoyl group may be attached to the C-4' position of mannose. Therefore, it is inferred that this compound 1 is a 2- (4-hydroxyphenylethyl)-[4-O-trans-caffeoyl]-β-D-mannoside, which is abbreviated as cistanoloside A.

Structural identification of compound 2

Yellow amorphous powder, HR-ESI-MS (negative) gave a quasi-molecular ion peak [MH]-m/z 461.1849 (calcd.C ₂₃ H ₂₅ O ₁₀ , 461.1853), suggesting a molecular weight of 462; combined ¹ H and ¹³ C NMR determined its molecular formula to be C ₂₃ H ₂₅ O ₁₀ , and the calculated degree of unsaturation was 11. The UV spectrum has a strong absorption at 216nm, suggesting the existence of phenethyl group, while the larger absorption at 327nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3272 cm ^-1 ), α,β-unsaturated ester (1630 cm ^-1 ), benzene ring skeleton (1560, 1516 cm ^-1 ), glycosidic bond (824 cm ^-1 ) and the like.

Referring to Figures 10-18, in conjunction with Tables 1 and 2, the ¹ H NMR and ¹³ C NMR spectra of this compound are very similar to those of compound 1, except for the configuration of the central sugar. After acid hydrolysis, the derivatization of sugar residues was analyzed by HPLC, and the standard sugar was also derivatized by comparative analysis to determine that the central sugar of the compound was D-allose. Therefore, the structure of compound 2 was deduced as

2-(4-hydroxyphenyl)ethyl-[4-O-trans-caffeoyl]-β-D-allopyranoside, abbreviated as cisstanoloside B.

Structural identification of compound 3

Yellow amorphous powder, HR-ESI-MS (negative) gave a quasi-molecular ion peak [MH] ^- m/z was 445.1947 (calcd. for C ₂₃ H ₂₅ O ₉ , 445.1941) suggesting a molecular weight of 446; combined with ¹ H and Its molecular formula was determined to be C ₂₃ H ₂₆ O ₉ by ¹³ C NMR, and the unsaturation was calculated to be 11. The UV spectrum has a strong absorption at 208 nm, suggesting the existence of phenethyl group, while the larger absorption at 309 nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3362 cm ^-1 ), α,β-unsaturated ester (1630 cm ^-1 ), benzene ring skeleton (1603, 1514 cm ^-1 ), glycosidic bond (825 cm ^-1 ) and the like.

Referring to Figures 19-27, combined with Table 1 and Table 2, the NMR spectrum of compound 3 is very similar to that of compound 1, there is no ABX signal on ¹ HNMR, and there is an additional set of A ₂ B ₂ system signals δ _H 7.54 (2H , d, J=8.0Hz), 6.79 (2H, d, J=8.0Hz), and with the trans double bond signal δ _H 7.54 (1H, d, J=15.9Hz), 6.40 (1H, d, J= 15.9 Hz), which indicates that the substituent of this compound is trans-coumaroyl. Other signals were similar to compound 1, and the presence of D-mannose was also confirmed by HPLC alignment of acid-hydrolyzed sugar residues. Therefore, the structure of this compound is 2-(4-hydroxyphenylethyl)-[4-O-trans-coumaroyl]-β-D-mannoside, abbreviated as cisstanoloside C.

Structural identification of compound 4

Yellow amorphous powder, HR-ESI-MS (negative) gave a quasi-molecular ion peak [MH] ^- m/z was 445.1934 (calcd. for C ₂₃ H ₂₅ O ₉ , 445.1941), suggesting a molecular weight of 446; bound ¹ H ^and13C NMR determined its molecular _formula to be _C23H26O9 , and the calculated unsaturation was ₁₁ . The UV spectrum has a strong absorption at 218 nm, suggesting the existence of phenethyl group, while the larger absorption at 312 nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3274cm ^-1 ), α,β-unsaturated ester (1630cm ^-1 ), benzene ring skeleton (1603,1515cm ^-1 ), glycosidic bond (832cm ^-1 ) and the like.

Referring to Figures 28-36, combined with Table 1 and Table 2, the NMR spectrum of compound 4 is very similar to that of compound 2, there is no ABX signal on ¹ HNMR, and there is an additional set of A ₂ B ₂ system signals δ _H 7.55 (2H , d, J=8.0Hz), 6.79 (2H, d, J=8.0Hz), and with the trans double bond signal δ _H 7.55 (1H, d, J=15.9Hz), 6.38 (1H, d, J= 15.9 Hz), which indicates that the substituent of this compound is trans-coumaroyl. Other signals were similar to compound 2, and the presence of D-allose was also confirmed by HPLC alignment of acid-hydrolyzed sugar residues. Therefore, the structure of this compound is 2-(4-hydroxyphenyl)ethyl-[4-O-trans-coumaroyl]-β-D-allopyranoside, abbreviated as cisstanoloside D.

Structural identification of compound 5

Yellow amorphous powder, HR-ESI-MS (positive) gave a quasi-molecular ion peak [M+Na] ⁺ m/z of 469.1469, suggesting a molecular weight of 446; combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₂₃ H ₂₆ O ₉ , the calculated unsaturation is 11. The UV spectrum has a strong absorption at 217 nm, suggesting the existence of phenethyl group, while the larger absorption at 312 nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3353 cm ^-1 ), α,β-unsaturated ester (1700 cm ^-1 ), benzene ring skeleton (1603, 1515 cm ^-1 ), glycosidic bond (831 cm ^-1 ) and the like.

Referring to Figures 37-41, in combination with Table 1 and Table 2, the NMR spectrum of compound 5 is only the difference in the double bond configuration compared with the spectrum of compound 4. The double bond coupling constant of compound 4 is 15.9HZ, while that of compound 5 The double bond coupling constant is 12.9 Hz, and the double bond hydrogen signal is shifted upfield, δ _H 6.84 (1H, d, J=12.9 Hz), 5.72 (1H, d, J=12.9 Hz), therefore, this suggests compound 5 Contains a pair of cis double bonds. Other signals were similar to compound 4, and the presence of D-allose was also confirmed by HPLC alignment of acid-hydrolyzed sugar residues. Therefore, the structure of this compound is 2-(4-hydroxyphenyl)ethyl-[4-O-cis-coumaroyl]-β-D-allopyranoside, abbreviated as cistanoloside E.

Table 11H NMR (400MHz) data of compounds ^1-5 in _DMSO -d6

Table ^213C NMR (100MHz) data of compounds 1-5 in _DMSO -d6

Structural identification of compound 6

Light yellow amorphous powder, HR-ESI-MS (positive) gives a quasi-molecular ion peak [M+Na] ⁺ m/z of 599.2097 (calcd.for C ₂₉ H ₃₆ O ₁₂ Na, 599.2099), suggesting a molecular weight of 576 ; Combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₂₉ H ₃₆ O ₁₂ , and the calculated degree of unsaturation was 12. The UV spectrum has strong absorption at 212nm, suggesting the existence of phenethyl group. The IR spectrum shows that it contains hydroxyl group (3354cm ^-1 ), α,β-unsaturated ester (1709cm ^-1 ), benzene ring skeleton (1516cm ^-1 ), glycosidic bond (834cm ^-1 ) and the like.

See Figures 42-50, in conjunction with Table 3, on ¹ H NMR, 4 hydrogen protons at δ _H 7.06 (2H, d, J=8.0 Hz), 6.68 (2H, d, J=8.0 Hz) form a set of A ₂ B ₂ system, and two methylene signals δ _H 3.91(1H,d,J=8.0Hz), 3.63(1H,d,J=8.0Hz), 2.77(2H,m), there is one inferred structure 4-Hydroxyphenethyl fragment. In addition, five aromatic hydrogen signals on the benzene ring δ _H 7.70 (1H, d, J=2.0Hz), 7.44 (4H, m) and a pair of trans double bond signals δ _H 7.65 (1H, d, J=15.9 Hz) ), 6.59 (1H, d, J=15.9Hz) indicates the existence of trans-cinnamoyl group in the molecule of this compound. The two sugar end group signals are respectively at δ _H 5.05(1H,s), 4.38(1H,s), and the HMQC spectra suggest that they are related to δ _C 101.20(C-1"'), 102.28(C-1'), It is suggested that the two sugars are both oxosides. After comparing with the literature, the two sugars are glucose and rhamnose respectively. The HMBC spectrum shows that the central sugars are δ _H H-1' (δ _H 4.38) and δ _C 70.38 (C The correlation at -8) proves that the 4-hydroxyphenethyl group is attached to the C-1' position of the central sugar glucose, and the H-4' (δ _H 4.77) of the central sugar is related to the C-9" (165.30), which It is suggested that the caffeoyl group may be connected to the C-4' position of glucose, and the end group signal H-1"' (δ _H 5.05) of rhamnose is coupled with the C-3' (δ _C 79.01) position of the central sugar, then It shows that rhamnose is attached to the 3-position of glucose. Therefore, the structure of this compound is

2-(4-hydroxyphenyl)ethyl-O-α-L-rhamnopyranosyl-(1→3)-[4-O-trans-cinaamoyl]-β-D-glucopyranoside, abbreviated as cisstanoloside F.

Structural identification of compound 7

Light yellow amorphous powder, HR-ESI-MS (positive) gives a quasi-molecular ion peak [M+Na] ⁺ m/z of 599.2091 (calcd.for C ₂₉ H ₃₆ O ₁₂ Na, 599.2099), suggesting a molecular weight of 576 ; Combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₂₉ H ₃₆ O ₁₂ , and the calculated degree of unsaturation was 12. The UV spectrum has a strong absorption at 207 nm, suggesting the existence of phenethyl group, while the larger absorption at 314 nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3373 cm ^-1 ), α,β-unsaturated ester (1704 cm -1 ), benzene ring skeleton (1603, 1515 cm ^-1 ), glycosidic bond (834 cm ^-1 ) and the like.

51-59, in conjunction with Table 3, the NMR information of compound 7 shows that it is a structural analog with jionoside C, the difference is that compound 7 is a trans-coumaroyl group, and compound jionoside C is a trans-caffeoyl group. The coupling at H-2", 6" (δ _H 7.51) and C-7" (δ _C 145.23)/C-8" (δ _C 113.48) on the HMBC spectrum further confirmed the above inference. Therefore, the structure of compound 7 is 2-phenylethyl-O-α-L-rhamnopyranosyl-(1→3)-[4-O-trans-coumaroyl]-β-D-glucopyra noside, abbreviated as cistanoloside G.

Table 3 ¹ H NMR and ¹³ C-NMR data of compounds 6, 7 and gionoside C in DMSO-d ₆

Structural identification of compound 8

Light yellow amorphous powder, HR-ESI-MS (positive) gave a quasi-molecular ion peak [M+Na] ⁺ m/z of 645.2156, suggesting a molecular weight of 622; combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₃₀ H ₃₈ O ₁₄ , calculated as 12 for unsaturation. The UV spectrum has a strong absorption at 206nm, suggesting the existence of phenethyl group, while the larger absorption at 315nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3376 cm ^-1 ), α,β-unsaturated ester (1702 cm ^-1 ), benzene ring skeleton (1603, 1516 cm ^-1 ), glycosidic bond (824 cm ^-1 ) and the like.

Referring to Figures 60-68, combined with Table 4, the NMR information of compound 8 shows that it is a structural analog with isosyringaldehyde 3'-α-L-rhamnopyranoside, the difference is that compound 8 has one more methoxy group, HMBC shows The methoxy signal δ _H 3.75 correlated with C-3 (δ _C 147.33), indicating that the methoxy group was attached to the 3-position of the phenethyl group. Therefore, the structure of compound 8 is

2-(3-methoxy-4-hydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1→3)-[4-O-trans-co umaroyl)]-β-D-glucopyranoside, abbreviated as cisstanoloside H.

Table 4 ¹ H NMR and ¹³ C-NMR data of compound 8 and isosyringaldehyde 3'-α-L-rhamnopyranoside in DMSO-d ₆

Structural identification of compound 11

Light yellow amorphous powder, HR-ESI-MS (positive) gave a quasi-molecular ion peak [M+Na] ⁺ m/z of 689.2057, suggesting a molecular weight of 666; combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₃₁ H ₃₈ O ₁₆ , calculated unsaturation 13. The UV spectrum has a strong absorption at 206nm, suggesting the existence of phenethyl group, while the larger absorption at 330nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3363 cm ^-1 ), α,β-unsaturated ester (1742 cm ^-1 ), benzene ring skeleton (1600, 1518 cm ^-1 ), glycosidic bond (823 cm ^-1 ) and the like.

See Figures 87-95, combined with Table 5, the NMR information of compound 11 shows that it is a structural analog with cis-acryloside, the difference is that compound 11 has an additional acetyl group, HMBC shows the central sugar 2-position hydrogen signal H-2' (δ _H 4.67) is related to the acetyl C-2"" (δ _C 169.10), indicating that the acetyl group is attached to the 2-position of the central sugar. Therefore, the structure of compound 11 is

2-(3,4-dihydroxyphenyl)-ethyl-O-a-L-rhamnopyranosyl-(1→3)-2-acetyl-[4-O-cis-caff eoyl)]-β-D-glucopyranoside, abbreviated as cisstanoloside K.

Table 5 ¹ H NMR and ¹³ C-NMR data of compound 11 and cis-acteoside in DMSO-d ₆

Structural identification of compound 12

Light yellow amorphous powder, HR-ESI-MS (negative) gave a quasi-molecular ion peak [MH] ^- m/z was 591.2548 (calcd. for C ₂₉ H ₃₅ O ₁₃ , 591.2539), suggesting a molecular weight of 592; combined with ¹ H and ¹³ C NMR determined the formula to be C ₂₉ H ₃₆ O ₁₃ , and the calculated degree of unsaturation was 12. The UV spectrum has a strong absorption at 208nm, suggesting the existence of phenethyl group, while the larger absorption at 310nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3378cm ^-1 ), α,β-unsaturated ester (1649cm ^-1 ), benzene ring skeleton (1603cm ^-1 ), glycosidic bond (825cm ^-1 ) and the like.

Referring to Figures 96-104, in conjunction with Table 6, the NMR information of compound 12 shows that it is a structural analog with carnoside B ₆ , the difference lies in the coupling constant of the double bond of compound 12, the double bond proton hydrogen signal δ _H 6.85 (1H, d, J=12.9Hz), 5.78 (1H, d, J=12.9Hz) suggests that compound 12 contains a pair of cis double bonds. Therefore, the structure of compound 12 is

2-(4-hydroxyphenyl)ethyl-O-α-L-rhamnopyranosyl-(1→3)-[6-O-cis-coumaroyl]-β-D-glucopyranoside, abbreviated as cisstanoloside L.

Table 6 ¹ H NMR and ¹³ C-NMR data of compound 12 and Osmanthuside B ₆ in DMSO-d ₆

Structural identification of compound 13 and compound 14

Yellow amorphous powder with quasi-molecular ion peaks [M+Na] ⁺ m/z 691.2205[M+Na] ⁺ and 691.2204[M+Na] ⁺ (calculated for C ₃₁ H ₄₀ O ₁₆ Na , 691.2209), suggesting _a molecular weight of 668; combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₃₁ H ₄₀ O ₁₆ , and the calculated degree of unsaturation was 12.

Referring to Figures 105-113 and 114-118, in conjunction with Tables 7 and 8, the NMR information of compounds 13 and 14 show structural analogs to acteoside. First, the ABX system signal [δ _H 7.02(1H,s),6.98(1H,d,J=8.0Hz),6.76(1H,d,J=6.8Hz)] and a pair of trans double bond signals [ _δH 7.46(1H,d,J=15.9Hz) and 6.19(1H,d,J=15.9Hz)] suggest the existence of transcaffeoyl. Secondly, another ABX system signal [δ _H 6.70(1H,s),6.69(1H,d,J=8.0Hz),6.58(1H,dd,J=2.0,8.0Hz)] and a methine signal δ _H 4.35 (1H, dd, J=3.0, 8.0 Hz, H-7), one methylene signal 3.64 (1H, m, H-8a), 3.22 (1H, m, H-8b), indicating phenethyl The 2-position of is substituted by an oxo group, and the NOESY correlation at H-7 (δ _H 4.35) and H-8a (δ _H 3.64) also confirmed the above inference. In addition, H-9 (δ _H 3.32) correlated with NOESY at H-7 (δ _H 4.35) and H-10 (δ _H 1.09), and H-10 (δ _H 1.09) with C-9 (δ _C 63.38 The HMBC correlation at ) suggests that the above-mentioned oxy group is an ethoxy group and is attached to the 7th position of the phenethyl group. The NMR signal of compound 14 is very similar to that of 13, with only a 0.2 ppm difference in the hydrogen at the 8-position of the phenethyl group, which is due to the difference in the configuration of the hydrogen at the 7-position. To confirm their configurations, we performed optical rotation tests and finally determined that compound 13 was 7S because its optical rotation value was [α] _D -4.8°, similar to S-suspensaside ([α] _D = -4.7°) and S-suspensaside methylether ([α] _D = -4.7°). Compound 14 is 7R, and its optical rotation is [α] _D -34.4°, which is similar to R-suspensaside [α] _D = -31.3°. Therefore, the structures of compounds 13 and 14 were confirmed as

(7S)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-(1→3)-[4-O-trans-caffeoyl]-β-D-glucopyranoside and

(7R)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-(1→3)-[4-O-trans-caffeoyl]-β-D-glucopyranoside, They are abbreviated as cistanoloside M and cisstanoloside N, respectively.

Structural identification of compound 9 and compound 10

Yellow amorphous powder, HR-ESI-MS (positive) gives quasi-molecular ion peaks [M+Na] ⁺ m/z are 733.2316[M+Na] ⁺ and 733.2318[M+Na] ⁺ , suggesting a molecular weight of 710 ; Combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₃₃ H ₄₂ O ₁₇ , and the calculated degree of unsaturation was 13.

Referring to Figures 69-77 and 78-86, combined with Table 7 and Table 8, the NMR information of compounds 9 and 10 shows that they are structural analogs with compounds 13 and 14, the difference is that compounds 9 and 10 have an additional acetyl group on the 2-position of glucose base. By comparing the hydrogen spectrum (9, δ _H 3.50, 3.60; 10, δ _H 3.50, 3.80) and optical rotation values (9, [α] _D = -4.6°; 10, [α] _D = -31.1°) of the compounds, The structures of compounds 9 and 10 were finally determined as

(7S)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-(1→3)-[2-ac etyl-4-O-trans-caffeoyl]-β -D-glucopyranoside and

(7R)-2-ethyoxyl-2-(3,4-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl-(1→3)-[2-a cetyl-4-O-trans-caffeoyl]-β -D-glucopyranoside, abbreviated as cisstanoloside I and cisstanoloside J, respectively.

Table 7 ¹ H NMR data of compounds 9, 10, 13, 14 in DMSO-d ₆

Table 813C-NMR data of compounds 9, 10, ¹³ , 14 in DMSO-d ₆

Structural identification of compound 15

Yellow amorphous powder, HR-ESI-MS (positive) gave a quasi-molecular ion peak [M+Na] ⁺ m/z of 485.1418, suggesting a molecular weight of 462; combined with ¹ H and ¹³ C NMR, the molecular formula was determined to be C ₂₃ H ₂₆ O ₁₀ , the calculated unsaturation is 11. The UV spectrum has a strong absorption at 207 nm, suggesting the existence of phenethyl group, while the larger absorption at 320 nm is the strong absorption peak of cinnamoyl. The IR spectrum shows that it contains hydroxyl group (3362 cm ^-1 ), α,β-unsaturated ester (1632 cm ^-1 ), benzene ring skeleton (1603, 1514 cm ^-1 ), glycosidic bond (825 cm ^-1 ) and the like.

Referring to Figures 119-123, in conjunction with Table 9, the NMR information of compound 15 shows that it is a structural analog to (E)-isosyringalide A, except that compound 15 contains a pair of cis double bonds. The proton hydrogen signal on the double bond H-7" (δ _H 6.86)/H-8" (δ _H 5.80) correlates with the HMBC at C-9" also confirms the above inference. Therefore, the structure of compound 15 is 2- (4-hydroxyphenyl)-ethyl-[6-O-cis-caffeoyl)]-β-D-glucopyranoside, abbreviated as cistanoloside O.

Table 9 ¹ H NMR and ¹³ C-NMR data of compound 15 and (E)-isosyringaldehyde A in DMSO-d ₆

Embodiment 4

In this example, the in vitro cytotoxicity test was performed on fifteen kinds of natural phenethyl glucoside compounds, and the adopted cell line was RAW 264.7 cells. The specific test methods and results are as follows:

MTT method: RAW 264.7 cells were seeded in a 96-well cell culture plate, 200 μL per well (containing 10×10 ⁴ tumor cells), in a 37°C, 5% CO ₂ incubator, and in DMEM containing 10% FBS In the medium, culture for 24h, add any one of the fifteen kinds of natural phenethyl glucoside compounds provided by the present invention at different concentrations (100, 50, 0μg/ml), and continue to culture for 48h; 4h before the end of the experiment, add 20μL of MTT (5 mg/mL), continue to incubate for 4 h at 37°C, 5% CO ₂ , absorb the culture medium, add 150 μL of dimethyl sulfoxide, shake until the crystals are completely dissolved, and then measure the absorbance on a microplate reader. The wavelength of 570 nm and the reference wavelength of 630 nm were used to calculate the survival rate of the compounds of the present invention on RAW 264.7 cells. The experimental results are shown in Figure 124.

It can be seen from Figure 124 that the fifteen natural phenylethanoid glycosides have no obvious cytotoxicity at the concentrations implemented in this experiment. The anti-inflammatory experimental drug concentrations in Example 5 all refer to the experimental results.

Embodiment 5

In this example, fifteen kinds of natural phenylethanoid glycosides were used to conduct in vitro anti-inflammatory experiments, and the cell line used was RAW 264.7 cells. The specific experimental methods and results are as follows:

RAW 264.7 cells were seeded in a 24-well cell culture plate, 500 μL per well, in a 37°C, 5% CO ₂ incubator, and in DMEM medium containing 10% FBS, cultured for 24 h, adding 100 ng/ml lipopolysaccharide (LPS) and any one of the fifteen natural phenylethanoid glycosides provided by the present invention, continue to cultivate under the conditions of 37° C. and 5% CO ₂ . After 24 hours, the supernatant of the medium was collected, and the operation was performed according to the instructions of the NO assay kit to determine the NO release inhibition rate.

The experimental results show that, as shown in Figure 125, most of the fifteen natural phenethyl glycosides can inhibit the release of NO in LPS-induced macrophage RAW 264.7, which proves that the phenethyl glycosides have good in vitro anti-inflammatory properties. active.

The specific embodiments of the present invention have been described above in detail, but they are only used as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

Claims (10)

1. fifteen kinds of phenethyl glucoside compounds, it is characterised in that the structural formula is as follows:

2. the separation and purification method of fifteen kinds of phenethyl alcohol glycosides compounds as described in claim 1, is characterized in that, comprises the steps: S1: at room temperature, multiple times of cold dipping and percolation extraction are performed on Cistanche deserticola with ethanol, and the extracts are combined, then, the solvent is recovered under reduced pressure, and concentrated to obtain an ethanol extract; S2: the ethanol extract is suspended in water, and then extracted with cyclohexane, ethyl acetate and n-butanol respectively to obtain cyclohexane extract, ethyl acetate extract CE, n-butanol extract and water part extract; S3: Dissolve the ethyl acetate extract CE with chloroform and methanol, mix samples with column chromatography silica gel, then load into a silica gel column, load the sample, and perform gradient elution; use thin-layer TLC to analyze, combine the same streams 16 components CE-1～CE16 were obtained after fractionation; S4: Take CE-7, dissolve it with methanol and filter, and obtain 4 fractions CE-7-1～CE-7-4 through Rp-18 high performance liquid chromatography; take the component CE-7-1 and dissolve it with methanol Filtration, rough separation by Rp-18 high performance liquid chromatography, respectively at retention time of 42-43min and 84-85min to obtain the compound 1 and compound 2; take component CE-7-4, dissolve with methanol and filter, pass through Rp -18 high performance liquid chromatography crude separation, the compounds 3, 4, 5, 6 and 7; S5: Take CE-9, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 6 components CE-9-1 ~CE-9-6; take the component CE-9-6, dissolve it with methanol and filter, and obtain the compound 8 at a retention time of 23.5-24.0min after rough separation by Rp-18 high performance liquid chromatography; S6: Take CE-10, dissolve it with a small amount of dichloromethane-methanol, and perform gel column chromatography to implement isocratic elution; use thin-layer TLC analysis, and combine the same fractions to obtain 3 components CE-10- 1～CE-10-3; take the component CE-10-2, dissolve it with methanol and filter, and pass through Rp-18 high performance liquid chromatography for rough separation, the retention times are 18.0-18.5min, 18.8-19.2min and 20.0- The compounds 9, 10 and 11 were obtained in 23.5 min; S7: Take CE-12, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 5 components CE-12-1 ~CE-12-5; take the component CE-12-5, dissolve it with methanol and filter, and obtain the compound 12 at a retention time of 43.8-44.2min after rough separation by Rp-18 high performance liquid chromatography; S8: Take CE-13, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 9 components CE-13-1 ~CE-13-9; take the component CE-13-7, dissolve it with methanol and filter it, and conduct rough separation by Rp-18 high performance liquid chromatography. compound 14; S9: Take CE-14, dissolve it with a small amount of dichloromethane-methanol, and perform isocratic elution by gel column chromatography; use thin-layer TLC analysis, and combine the same fractions to obtain 4 components CE-14-1 ~CE-14-4; take the component CE-14-1, dissolve it in methanol, filter, and carry out rough separation by Rp-18 high performance liquid chromatography, and obtain the compound 15 at a retention time of 34.0-34.5 min. 3. The separation and purification method according to claim 2, wherein the concentration of the ethanol in S1 is 85%. 4. separation and purification method according to claim 2, is characterized in that, the eluent that silica gel column gradient elution described in S3 adopts is successively methylene dichloride, volume ratio is the dichloromethane-methanol of 100:1, Dichloromethane-methanol in a volume ratio of 20:1, dichloromethane-methanol in a volume ratio of 10:1, dichloromethane-methanol in a volume ratio of 5:1, and dichloromethane-methanol in a volume ratio of 2:1 Methanol, methanol. 5 . The method for separation and purification according to claim 2 , wherein the column chromatography silica gel is 100-200 mesh column chromatography silica gel. 6 . 6. The separation and purification method according to claim 2, wherein the gel of the gel column chromatography is Sephadex LH-20. 7. separation and purification method according to claim 2, is characterized in that, the eluent that described gel column chromatography adopts is dichloromethane-methanol, wherein, the volume ratio of dichloromethane and methanol is 7:3 . 8. separation and purification method according to claim 2, is characterized in that, the specification of the chromatographic column that described high performance liquid chromatography rough separation adopts is Cosmosil 5C ₁₈ -MS-II, 5 μm, 20 × 250mm; was 8 mL/min, and the fluidity was methanol/water. 9. A pharmaceutical preparation, characterized in that it comprises any one or more of the above-mentioned fifteen kinds of phenethyl alcohol glycoside compounds. 10. The application of fifteen kinds of phenylethanoid glycoside compounds as claimed in claim 1 in the preparation of anti-inflammatory functional food and medicine.

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