CN113788869B

CN113788869B - Preparation method of four iridoid glycoside compounds

Info

Publication number: CN113788869B
Application number: CN202111149499.9A
Authority: CN
Inventors: 曾小斌; 葛岚岚; 江园园; 谢秋杰; 李阳芳; 缪雨阳
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2023-04-28
Anticipated expiration: 2041-09-29
Also published as: CN113788869A

Abstract

The invention discloses a preparation method of four iridoid glycoside compounds, which comprises a compound (1), a compound (2), a compound (3) and a compound (4). The preparation method of the four iridoid glycoside compounds has the advantages that the four iridoid glycoside compounds are used as a natural compound, and have low toxicity to normal cells and small side effects; the preparation method has the advantages of moderate effective dose, obvious curative effect, small toxic and side effects and the like, and has high efficiency, high stability and wide application prospect.

Description

Preparation method of four iridoid glycoside compounds

Technical Field

The invention relates to the technical field of traditional Chinese medicine chemistry, in particular to a preparation method of four iridoid glycoside compounds.

Background

Gardenia is called as yellow gardenia, gardenia jasminoides ellis and white toad, is a dry mature fruit of gardenia Gardenia jasminoides Ellis of Rubiaceae, belongs to a first medicine and food dual-purpose resource issued by the ministry of health, and has the effects of protecting liver, promoting bile flow, reducing blood pressure, calming, stopping bleeding, relieving swelling and the like. The Chinese herbal medicines are widely distributed in various places in China and are common clinical traditional Chinese medicines, and the calendar pharmacopoeia is recorded.

Gardenia jasminoides ellis is a evergreen shrub. Bitter and cold in property, nontoxic, and enters heart, liver, lung and stomach meridians. Has effects of purging pathogenic fire, relieving restlessness, clearing heat, promoting diuresis, cooling blood, and removing toxic substances; external use detumescence and pain relieving. It is used for treating vexation, jaundice due to damp-heat, stranguria, hematemesis, conjunctival congestion, swelling and pain, sore and abscess due to fire toxin. At present, the research on chemical components and biological activity of gardenia is mainly focused on iridoid components, and the active components of the gardenia have various pharmacological activities such as liver protection, cholagogue, anti-inflammatory, analgesic, antioxidant, antitumor, anti-atherosclerosis and the like, but the research on iridoid glycoside compounds is further improved.

Disclosure of Invention

The invention mainly aims to provide a preparation method of four iridoid glycoside compounds, which can effectively solve the problems in the background technology.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the preparation method of four iridoid glycoside compounds comprises a compound (1), a compound (2), a compound (3) and a compound (4), and is characterized in that the structure of the compound (1) is determined to be 6' -O- [ (E) -sinapoyl ] -6 beta-hydroxy-gardoside through detection, and the chemical structural formula is as follows

The structure of the compound (2) is determined to be 6' -O- [ (E) -sinapoyl ] shanzhiside by detection, and the chemical structural formula is

The structure of the compound (3) is determined to be 6' -O- [ (E) -sinapoyl ] ixoside through detection, and the chemical structural formula is

The structure of the compound (4) is determined to be 6' -O- [ (E) -sinapoyl ] geniposidic acid through detection, and the chemical structural formula is

The preparation method of the four iridoid glycoside compounds comprises the following steps:

s1: extracting fructus Gardeniae with ethanol at room temperature by cold soaking and percolating for several times, mixing extractive solutions, recovering solvent under reduced pressure, and concentrating to obtain ethanol extract;

s2: suspending the ethanol extract in water, and extracting with petroleum ether and ethyl acetate respectively to obtain petroleum ether extract, ethyl acetate extract and water extract;

s3: dissolving the water extract with a small amount of pure water, performing gradient elution by using a macroporous resin column, and recovering the solvent to obtain 4 elution components Fr A-Fr D;

s4: taking Fr C, dissolving and filtering with methanol, performing gradient elution by using a reverse chromatographic column, and recovering the solvent to obtain 4 elution components Fr C20, fr C30, fr C50 and Fr C80;

s5: taking Fr C30, dissolving with a small amount of dichloromethane-methanol, and performing gel column chromatography to perform isocratic elution; adopting thin layer TLC analysis, and obtaining 6 components Fr C30-1 to Fr C30-6 after combining the same flow;

s6: dissolving the component Fr C30-1 with a small amount of dichloromethane-methanol, and performing gel column chromatography to perform isocratic elution; adopting thin layer TLC analysis, and obtaining 3 components Fr C30-1-1 to Fr C30-1-3 after combining the same flow; dissolving component Fr C30-1-1 in methanol, filtering, and performing Rp-18 high performance liquid chromatography for coarse separation to obtain compound (1) with retention time of 42.0-43.0 min;

s7: dissolving the component Fr C30-3 with a small amount of dichloromethane-methanol, and performing gel column chromatography to perform isocratic elution; adopting thin layer TLC analysis, and obtaining 5 components Fr C30-3-1 to Fr C30-3-5 after combining the same flow components; dissolving component Fr C30-3-2 in methanol, filtering, and performing Rp-18 high performance liquid chromatography for coarse separation to obtain compound (2) with retention time of 42.0-44.0 min; dissolving component Fr C30-3-3 in methanol, filtering, and performing Rp-18 high performance liquid chromatography for coarse separation to obtain compound (3) and compound (4) respectively with retention time of 59.0-62.0min and 65.0-67.0 min.

Preferably, the concentration of ethanol in the step S1 is 85%.

Preferably, the macroporous resin in the step S3 is D101, and the eluent used in gradient elution sequentially comprises water and a volume ratio of 30:70 ethanol-water, volume ratio of 50:50 ethanol-water ratio of 80:20 ethanol-water.

Preferably, the reverse chromatography in the step S4 is ODS, and the eluent used in the gradient elution is in a volume ratio of 20:80, methanol-water, volume ratio of 30:70 methanol-water, volume ratio of 50:50 methanol-water and volume ratio of 80:20 methanol-water.

Preferably, the gel of the gel column chromatography in the step S5 is Sephadex LH-20, and the isocratic eluent is dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol is 50:50.

Preferably, the gel of the gel column chromatography described in the above steps S6 and S7 is Sephadex LH-20, and the isocratic eluent is dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol is 70:30.

Preferably, the specification of a chromatographic column used for the high performance liquid chromatography crude separation is Cosmosil5C18-MS-II,5 μm,20 x 250mm; the flow rate adopted by the method is 8mL/min, and the mobile phase is methanol/water.

Compared with the prior art, the invention has the following beneficial effects:

the four iridoid compounds provided by the invention are taken as a natural compound, and have low toxicity to normal cells and small side effects; the preparation method has the advantages of moderate effective dose, obvious curative effect, small toxic and side effects and the like, and the preparation method is efficient and high in stability, so that the preparation method has a wide application prospect.

Drawings

FIGS. 1 to 8 are 1H NMR, 13C NMR, 2D-NMR, MS, HPLC views of compound (1) of the present invention;

FIGS. 9 to 16 are 1H NMR, 13C NMR, 2D-NMR, MS, HPLC views of compound (2) of the present invention;

FIGS. 17 to 24 are 1H NMR, 13C NMR, 2D-NMR, MS, HPLC views of compound (3) of the present invention;

FIGS. 24 to 32 are 1H NMR, 13C NMR, and 2D-NMR, MS, HPLC views of compound (4) of the present invention;

FIG. 33 is a graph showing the result of cytotoxicity of four iridoid compounds on RAW 264.7 cells in the present invention;

fig. 34 shows bar graphs of inhibition of LPS-induced NO release from RAW 264.7 cells by four iridoid compounds according to the invention.

Detailed Description

The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.

In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be configured and operated in the specific direction, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "provided," "connected," and the like are to be construed broadly, and may be fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.

Example 1

Four iridoid glycosides compounds, including compound (1), compound (2), compound (3) and compound (4), characterized in that the structure of compound (1) is determined to be 6' -O- [ (E) -sinapoyl ] -6 beta-hydroxy-gardoside by detection, and the chemical structural formula is

Example 2

crushing 17kg of dried gardenia fruits at room temperature, performing cold leaching and diacolation extraction for a plurality of times by using 85% ethanol, combining the extracting solutions, then recovering the solvent under reduced pressure at 55 ℃, and concentrating to obtain 3.0kg of ethanol extract; suspending the ethanol extract in water, and extracting with petroleum ether and ethyl acetate respectively to obtain 2600g of petroleum ether extract, ethyl acetate extract and water extract.

Taking 2600g of the water part extract, and dissolving with a small amount of pure water for later use. Simultaneously, 5kg D101 macroporous resin is loaded with pure water, the pure water is eluted until the cylindrical surface is no longer declined, the wet method is used for loading samples, and then the pure water and the volume ratio are sequentially 30:70 ethanol-water, volume ratio of 50:50 ethanol-water ratio of 80:20, controlling the flow rate at 1000ml/h, concentrating the eluting solvent with different proportions as one fraction, and recovering the solvent to obtain 4 eluting components (Fr A,1000 g), 30% ethanol water eluting component (Fr B,300 g), 50% ethanol water eluting component (Fr C,800 g) and 80% ethanol water eluting component (Fr D,350 g).

And step three, fr C is taken, and is dissolved and filtered by a small amount of methanol for standby. ODS reverse chromatography is loaded with pure water, the pure water is eluted until the cylindrical surface is not lowered any more, wet loading is carried out, and the volume ratio is 20:80, methanol-water, volume ratio of 30:70 methanol-water, volume ratio of 50:50 methanol-water and volume ratio of 80:20 in methanol-water gradient elution, controlling the flow rate at 1000ml/h, concentrating the eluting solvent as one fraction in different proportions, and recovering the solvent to obtain 4 eluting components, namely 20% methanol-water eluting component (Fr C20), 30% methanol-water eluting component (Fr C30), 50% methanol-water eluting component (Fr C50) and 80% methanol-water eluting component (Fr C80).

Step four, a 30% methanol water elution component Fr C30 is taken, dissolved by a small amount of methylene dichloride-methanol, and subjected to Sephadex LH-20 gel (15X 250 mm) column chromatography, wherein the eluent is methylene dichloride-methanol, and the volume ratio is 50:50, performing isocratic elution; the flow rate was controlled at 200ml/h, and 100ml each was concentrated as one fraction. The same fractions were combined by thin layer TLC analysis to give 6 fractions Fr C30-1 to Fr C30-6.

Step five, taking a component Fr C30-1, dissolving with a small amount of dichloromethane-methanol, performing Sephadex LH-20 gel (15X 350 mm) column chromatography, wherein the eluent used is dichloromethane-methanol with the volume ratio of 70:30, and performing isocratic elution; controlling the flow rate at 200mL/h, concentrating 100mL each as a fraction, adopting thin-layer TLC analysis, combining the same fractions to obtain 3 components Fr C30-1-1 to Fr C30-1-3, dissolving and filtering the component Fr C30-1-1 by methanol, and performing coarse separation by Rp-18 high performance liquid chromatography (Cosmosil 5C18-MS-II,5 mu m, 20X 250mm, flow rate: 8mL/min, wavelength: 254 nm), wherein the mobile phase is methanol: water (25:75, v/v) gave compound (1) (23.7 mg) at a retention time of about 42.3 min.

Step six, taking a component Fr C30-3, dissolving with a small amount of dichloromethane-methanol, performing Sephadex LH-20 gel (15X 350 mm) column chromatography, wherein the eluent used is dichloromethane-methanol with the volume ratio of 70:30, and performing isocratic elution; the flow rate is controlled at 200ml/h, 100ml each is used as one fraction for concentration, thin layer TLC analysis is adopted, and 5 components Fr C30-3-1-Fr C30-3-5 are obtained after the same fractions are combined. Dissolving component C Fr C30-3-2 in methanol, filtering, and performing coarse separation by Rp-18 high performance liquid chromatography (Cosmosil 5C18-MS-II,5 μm, 20X1250 mm, flow rate: 8mL/min, wavelength: 254 nm), wherein the mobile phase is methanol: water (28:72, v/v) for about 42.3min to give compound (2) (33.9 mg); dissolving component C Fr C30-3-3 with methanol, filtering, and performing coarse separation by Rp-18 high performance liquid chromatography (Cosmosil 5C18-MS-II,5 μm, 20X1250 mm, flow rate: 8mL/min, wavelength: 254 nm), wherein the mobile phase is methanol: water (30:70, v/v) gave compounds (3) (45.2 mg) and 4 (52.8 mg) at retention times of about 42.3min and about 42.3min, respectively.

Example 3

The structures of the four iridoids in example 1 were identified in this example, and specific operations and results are as follows:

structural identification of Compound (1)

Yellow uncertainty powder, HR-ESI MS (negative) gave an excimer ion peak [ M-H ] -M/z595.1078, suggesting a molecular weight of 596; the molecular formula was determined to be C27H32O15 by combination of 1H and 13C NMR (Table 1).

Referring to FIGS. 1-8, the compounds are shown to contain an iridoid aglycone fragment, a beta glucose and a phenylpropanoid fragment in combination with the NMR spectra of tables 1,1H and 13C. First is the iridoid aglycone fragment, δH 7.26 (1H, s, H-3) and 5.37 (1H, s, H-1) on 1H NMR are characteristic hydrogens on the iridoid dihydrofuran ring, while δH 5.12 (2H, s, H-10) is an exocyclic double bond hydrogen signal. δC 95.5 (C-1), 150.5 (C-3), 109.5 (C-4), 36.1 (C-5) and 41.8 (C-9) in the carbon spectrum belong to the carbon on the iridoid dihydrofuran ring, δC 151.0 (C-8) and 110.5 (C-10) are exocyclic double bond carbon signals, and δC 74.4 (CH) and 72.9 (CH) are signals of C-6 and C-7 on the iridoid aglycone, respectively. The presence of the iridoid aglycone fragment can also be confirmed by analyzing 1H-1H correlation, HSQC correlation and HMBC correlation. δh4.50 (1H, d, j=8.0 Hz) is the β -glucose end group hydrogen signal. Furthermore, the presence of two aromatic ring hydrogens δH 7.02 (2H, s, H-2", 6"), a pair of double bond hydrogens 7.56 (1H, d, J=15.9 Hz, H-7 "), 6.57 (1H, d, J=15.9 Hz, H-8"), and two methoxy δH 3.80 (6H, s,3",5" -OCH3 ") in the 1H-NMR spectrum can be inferred to be the presence of trans-sinapoyl groups in the molecule of the compound. The three fragments are combined, and the compound (1) is an iridoid glycoside compound containing sinapoyl substitution. Based on the hydrocarbon signals of C-6 and C-7, it was initially inferred that both C-6 and C-7 were linked to-OH. The literature demonstrates that 6-OH is in the beta configuration when the coupling constants of H-6 and H-7 are less than 1.0Hz and the displacement difference between C-3 and C-4 is ΔδC=41. According to literature alignment, 7-OH is also the beta configuration. Combining the 1H NMR and 13C NMR spectra, compound (1) contained an iridoid glycoside very similar to the known compound zaluzioside (6β -hydroxy-gardoside methyl ester), with only one methyl ester substitution at position 11. The HMBC spectra show that H-6 '(δH 4.36,4.28)) of glucose correlates with C-9 "(δC166.5) of sinapoyl, suggesting that our sinapoyl is linked to glucose at position 6 on iridoid glycoside, which is also demonstrated by the shift of C-6' (δC63.3) of glucose to the high field. Thus, the compound (1) was deduced to be 6' -O- [ (E) -sinapoyl ] -6β -hydroxy-gardoside, which was abbreviated as gardoside A.

Structural identification of Compound (2)

The yellow uncertainty powder, HR-ESI MS (positive), gave an excimer ion peak [ M+Na ] +m/z621.1831, suggesting a molecular weight of 598; the molecular formula was determined to be C27H34O15 by combination of 1H and 13C NMR (Table 1).

Referring to FIGS. 9-16, the combination of the NMR spectra of Table 1,1H and 13C shows that the compound is also an iridoid glycoside compound containing sinapoyl substitution. Comparing the 1H NMR and 13C NMR spectra, the iridoid glycoside contained in the compound (2) is very similar to the known compound shanzhiside. Furthermore, the H-6 '(δH 4.36,4.19) of glucose was shown to be related to sinapoyl at C-9 "(δC166.6) in the HMBC spectra, suggesting that our sinapoyl is linked to glucose at position 6 on iridoid glycoside, which is also demonstrated by the shift of glucose C-6' (δC63.3) to the high field. Thus, the compound (2) was assumed to be 6' -O- [ (E) -sinapoyl ] shanzhiside, which was abbreviated as gardeoside B.

Structural identification of Compound (3)

The yellow uncertainty powder, HR-ESI MS (positive), gave an excimer ion peak [ M+Na ] +m/z617.0876, suggesting a molecular weight of 594; the molecular formula was determined to be C27H30O15 by combination of 1H and 13C NMR (Table 1).

Referring to FIGS. 17-24, the combination of the NMR spectra of Table 1,1H and 13C shows that the compound is also an iridoid glycoside compound containing sinapoyl substitution. H-7 (δH26.67) on 1H NMR is a ring double bond hydrogen signal, δH27.30 (1H, s, H-3) and 5.77 (1H, d, J=3.0 Hz, H-1) are characteristic hydrogens on the iridoid dihydrofuran ring, and the β -glucose end group hydrogen signal H-1' (1H, d, J=8.0 Hz) at δH2.46 suggests that compound (3) contains iridoid glycosides very similar to known compound ixoside. Furthermore, the H-6 '(δH 4.28,4.19) of glucose was shown to be related to sinapoyl at C-9 "(δC166.7) in the HMBC spectra, suggesting that our sinapoyl is linked to glucose at position 6 on iridoid glycoside, which is also demonstrated by the shift of glucose C-6' (δC63.5) to the high field. Thus, the compound (3) was deduced to be 6' -O- [ (E) -sinapoyl ] ixoside, which was abbreviated as gardeoside C.

Structural identification of Compound (4)

The yellow uncertainty powder, HR-ESI MS (positive), gave an excimer ion peak [ M+Na ] +m/z603.1529, suggesting a molecular weight of 580; the molecular formula was determined to be C27H32O14 by combination of 1H and 13C NMR (Table 1).

Referring to FIGS. 25 to 32, it was revealed by combining the NMR spectra of tables 1,1H and 13C that the compound (4) was an iridoid glycoside compound having sinapoyl group substitution. Comparing the 1H NMR and 13C NMR spectra, the iridoid glycoside contained in the compound (2) is very similar to the known compound geniposidic acid. Furthermore, the H-6 '(δH 4.30,4.19) of glucose was shown to be related to sinapoyl at C-9 "(δC166.7) in the HMBC spectra, suggesting that our sinapoyl is linked to glucose at position 6 on iridoid glycoside, which is also demonstrated by the shift of glucose C-6' (δC63.4) to the high field. Thus, the compound (4) was assumed to be 6' -O- [ (E) -sinapoyl ] geniposidic acid, which was abbreviated as gardeoside D.

TABLE 1H NMR (400 MHz) and 13C NMR (100 MHz) data for compounds (1) - (4) in DMSO-d6

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Example 4

In this example, in vitro cytotoxicity tests were performed on four natural iridoid compounds, wherein the cell lines used were RAW 264.7 cells, and specific test methods and results were as follows:

MTT method: RAW 264.7 cells were inoculated into 96-well cell culture plates, 200. Mu.L per well (containing 10X 104 tumor cells), cultured in a 5% CO2 incubator at 37℃and in a DMEM medium containing 10% FBS for 24 hours, and any one of the four natural iridoid compounds provided by the present invention was added at different concentrations (100, 50, 0. Mu.g/ml) and cultured for 48 hours further; after adding 20. Mu.L of MTT (5 mg/mL) for 4 hours before the experiment is finished, continuously incubating for 4 hours under the conditions of 37 ℃ and 5% CO2, absorbing the culture solution, adding 150. Mu.L of dimethyl sulfoxide, shaking until crystals are completely dissolved, detecting the absorbance of the culture solution by an enzyme-labeled instrument, detecting the absorbance at a wavelength of 570nm and a reference wavelength of 630nm, and calculating the survival rate of the compound of the invention to RAW 264.7 cells, wherein the experimental result is shown in FIG. 33.

As can be seen from fig. 33, all four natural iridoids were not significantly cytotoxic at the concentrations used in the present experiment.

Example 5

In this example, four natural iridoid compounds were used for in vitro anti-inflammatory experiments, wherein the cell lines used were RAW 264.7 cells, and the specific experimental methods and results are as follows:

RAW 264.7 cells were inoculated into 24-well cell culture plates, 500. Mu.L per well, cultured in a 5% CO2 incubator at 37℃and in a DMEM medium containing 10% FBS for 24 hours, and 100ng/ml of Lipopolysaccharide (LPS) and any one of the four natural iridoid compounds provided by the present invention were added to continue the culture at 37℃under 5% CO 2. After 24 hours, the culture supernatant was collected, and the NO release inhibition was measured by performing the procedure according to the NO measurement kit.

As shown in figure 34, the experimental results show that the four natural iridoid compounds can obviously inhibit the release of NO in macrophage RAW 264.7 induced by LPS (p is less than 0.01), and the gardenia iridoid compounds have obvious in-vitro anti-inflammatory activity.

It should be noted that: the four iridoid compounds can be applied to the preparation of anti-inflammatory functional foods and medicines.

The invention can provide a pharmaceutical preparation which comprises any one or more of four iridoid compounds with effective treatment dose and a pharmaceutically acceptable carrier thereof. It is worth noting that the pharmaceutically acceptable carrier is, for example, one or several selected from diluents, excipients, fillers, binders, wetting agents, lubricants, disintegrants, absorption promoters and surfactants.

The dosage form of the pharmaceutical preparation is selected from any one of the following: tablets, capsules, pills, granules, suspensions, oral liquids, smears, cataplasms, sprays, powder injections and water injections. For another example, when the above pharmaceutical preparation is an injection, the injection may be any of a subcutaneous injection, an intraperitoneal injection, an intramuscular injection, and an intravenous injection.

The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. The preparation method of four iridoid glycoside compounds comprises a compound (1), a compound (2), a compound (3) and a compound (4), and is characterized in that the structure of the compound (1) is determined to be 6' -O- [ (E) -sinapoyl ] -6 beta-hydroxy-gardoside through detection, and the chemical structural formula is as follows

s7: dissolving the component Fr C30-3 with a small amount of dichloromethane-methanol, and performing gel column chromatography to perform isocratic elution; adopting thin layer TLC analysis, and obtaining 5 components Fr C30-3-1 to Fr C30-3-5 after combining the same flow components; dissolving component Fr C30-3-2 in methanol, filtering, and performing Rp-18 high performance liquid chromatography for coarse separation to obtain compound (2) with retention time of 42.0-44.0 min; dissolving component Fr C30-3-3 in methanol, filtering, and performing Rp-18 high performance liquid chromatography for coarse separation to obtain compound (3) and compound (4) respectively at retention time of 59.0-62.0min and 65.0-67.0 min;

the specification of a chromatographic column adopted by the high performance liquid chromatography crude separation is Cosmosil5C18-MS-II,5 mu m,20 mm; the flow rate adopted by the method is 8mL/min, and the mobile phase is methanol/water.

2. The method for producing four iridoid glycosides according to claim 1, wherein the concentration of ethanol in step S1 is 85%.

3. The method for preparing four iridoid glycoside compounds according to claim 1, wherein the macroporous resin in the step S3 is D101, and the eluent used in gradient elution sequentially comprises water and a volume ratio of 30:70 ethanol-water, volume ratio of 50:50 ethanol-water ratio of 80:20 ethanol-water.

4. The method for preparing four iridoid glycoside compounds according to claim 1, wherein the reverse chromatography in the step S4 is ODS, and the eluent adopted in gradient elution is in turn in a volume ratio of 20:80, methanol-water, volume ratio of 30:70 methanol-water, volume ratio of 50:50 methanol-water and volume ratio of 80:20 methanol-water.

5. The method for preparing four iridoid glycoside compounds according to claim 1, wherein the gel column chromatography in the step S5 is Sephadex LH-20, and the isocratic eluent is dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol is 50:50.

6. The method for preparing four iridoid glycoside compounds according to claim 1, wherein the gel of the gel column chromatography in the steps S6 and S7 is Sephadex LH-20, the isocratic eluent is dichloromethane-methanol, and the volume ratio of dichloromethane to methanol is 70:30.