Preparation method of paniculatoside C

Abstract

The application discloses a preparation method of a pregnane glycoside Paniculatoside C. The preparation method of the pregnane glycoside Paniculatoside C comprises the following steps: after a crushed Qiqielou is extracted with ethanol for several times to obtain alcohol extracts, the alcohol extracts are suspended with water, petroleum ether, ethyl acetate and n-butanol are sequentially used for extraction for several times to obtain petroleum ether layer extracts, ethyl acetate layer extracts and n-butanol layer extracts, the petroleum ether layer extracts and the ethyl acetate layer extracts are combined and suspended with water, an acid-base extraction method is used to obtain A ① part extracts; a crude segmentation is performed by using a normal phase silica gel column gradient elution method, each segment is obtained, and then gel column chromatography, normal phase silica column chromatography, reverse phase silica gel column chromatography and preparation thin layer chromatography are comprehensively used for purification and extraction to obtain the Paniculatoside C, and the method has a high yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical drugs, in particular to a preparation method of Paniculatoside C. BACKGROUND

[0002] Tylophora floribunda is a plant of Tylophora genus in Asclepiadaceae, which is produced in Jiangsu, Zhejiang, Fujian, Jiangxi, Hunan, Guangdong, Guangxi and Guizhou provinces, and grows in shrubs or sparse forests with sufficient sunlight at an altitude of 500 meters. The roots of Tylophora floribunda are medicinal and have the effects of treating infantile convulsions, diphtheria, sprains, joint pain and snake bites. The chemical composition of Tylophora floribunda is relatively limited, and the reported chemical components mainly include antofine, β-Sitosterol and Daucosterol, hancolupenone and hancolupenol, and phenolic acid such as vanillin acid, ferulic acid and syringic acid.

[0003] Paniculatoside C, chemical name: glaucogenin A 3-O-β-L-diginopyranoside, molecular formula: C 28 H 40 O9, CAS number: 52700-50-2, is a C 21 steroid, which is a kind of pregnane glycoside, and its steroid nucleus belongs to 13:14, 14:15-double split ring type pregnane skeleton, which is composed of glaucogenin A and L-diginopyranose (pyran type) connected to the 3-OH position of the steroid nucleus in the form of β. According to the literature, Paniculatoside C has anti-tobacco mosaic virus (TMV) activity.

[0004] Paniculatoside C is a natural organic compound with a complex skeleton structure, has multiple chiral centers, and is difficult to carry out organic chemical total synthesis. At present, the compound is generally obtained by extraction and separation and purification from natural products. The existing extraction and separation and purification process of Paniculatoside C in the related art has the disadvantages of long time consumption, complex process and low extraction rate, and therefore a new extraction and separation and purification process needs to be developed to realize the efficient preparation of Paniculatoside C, so as to meet the needs of the subsequent research on the pharmacological mechanism of the anti-TMV activity of Paniculatoside C or other biological activities of Paniculatoside C. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes in a first aspect a preparation method of a Pregnane glycoside Paniculatoside C, which has a high extraction rate.

[0006] According to the preparation method of the Pregnane glycoside Paniculatoside C provided by the first aspect of the present application, the Pregnane glycoside Paniculatoside C has the following structural formula:

[0007]

[0008] The preparation method of the Pregnane glycoside Paniculatoside C comprises the following steps:

[0009] S1, after the crushed Qiqiaolou is extracted with ethanol for several times to obtain an alcohol extract, the alcohol extract is suspended in water, and then extracted with petroleum ether, ethyl acetate and n-butanol for several times to obtain a petroleum ether layer extract, an ethyl acetate layer extract and an n-butanol layer extract, respectively; the petroleum ether layer extract and the ethyl acetate layer extract are combined and suspended in water, the water phase is adjusted to be acidic, extracted with chloroform for several times to obtain A ① part, the water phase is adjusted to be alkaline, and then extracted with chloroform and n-butanol for several times to obtain A ② layer and A ③ layer, respectively; the A ② layer and the A ③ layer are discarded; the remaining A ① part is rotary evaporated under reduced pressure to remove the solvent to obtain an A ① part extract;

[0010] S2, the A ① part extract is dry mixed to obtain a sample silica gel layer, and then eluted with a petroleum ether-ethyl acetate system to obtain each fraction; the 9th to 15th fractions of petroleum ether: ethyl acetate = 1:1 are combined to obtain Fr 2 component.

[0011] S3, the Fr 2 component is dissolved in dichloromethane-methanol, and eluted with dichloromethane-methanol as an eluent to obtain G1 component by combining the 21st to 56th fractions;

[0012] S4, the Fr 2-G1 component is dissolved in methanol-water, and eluted with methanol-water solution on a reversed-phase column to obtain R8 component by combining the fractions eluted by 100% methanol;

[0013] S5, the Fr 2-G1-R8 component is dissolved in n-hexane-acetone, and eluted with n-hexane-acetone as an eluent on a normal-phase column to obtain S3 component by combining the 1st to 20th fractions eluted by n-hexane-acetone = 10:1 and the 1st to 8th fractions eluted by 5:1;

[0014] S6, the Fr 2-G1-R8-S3 component is dissolved with dichloromethane-methanol, isocratic elution is carried out by using a gel chromatographic column and using dichloromethane-methanol as an eluent, and the 44th-78th fractions are combined to obtain the G1 component;

[0015] S7, the Fr 2-G1-R8-S3-G1 component is dissolved with n-hexane-acetone, gradient elution is carried out by using an n-hexane-acetone eluent in sequence by using a normal phase chromatographic column, and the 118th-211th fractions eluted by n-hexane-acetone=12:1 are combined to obtain the S4 component;

[0016] S8, the Fr 2-G1-R8-S3-G1-S4 component is dissolved with dichloromethane-methanol, gradient elution is carried out by using a dichloromethane-methanol eluent in sequence by using a normal phase chromatographic column, and the 7th-24th fractions eluted by dichloromethane-methanol=100:1.5 are combined to obtain the S1 component;

[0017] S9, the Fr 2-G1-R8-S3-G1-S4-S1 component is dissolved with methanol-water, isocratic elution is carried out by using 80% methanol aqueous solution as an eluent by using a reversed phase chromatographic column, and the 12th-15th fractions of 80% methanol are combined to obtain the R1 component;

[0018] S10, the Fr 2-G1-R8-S3-G1-S4-S1-R1 component is dissolved with dichloromethane-methanol, thin layer chromatography is carried out by using dichloromethane-methanol as a developing agent, after the solvent is completely developed, drying is carried out and second development is carried out, after the development is completed, a color developing band distribution height is observed by using a color developing method, silica gel powder in a color developing area is obtained, compounds in the silica gel powder are eluted out by using ethyl acetate, and the compound Paniculatoside C is obtained.

[0019] The preparation method of the Pregnane glycoside Paniculatoside C according to the embodiment of the present application has at least the following beneficial effects:

[0020] The present application provides a method for preparing the Paniculatoside C from the plant of the Morinda officinalis How of the family of the Morindae in the Morindae, and the compound Paniculatoside C can be obtained through steps S1-S10, and the method has a high yield.

[0021] According to some embodiments of the present application, in step S2, the mesh number of the silica gel in the sample silica gel layer is 100-200 mesh.

[0022] According to some embodiments of the present application, in step S2, gradient elution is carried out in sequence according to the volume ratio of petroleum ether and ethyl acetate as 25:1, 20:1, 4:1, 2:1 and 1:1.

[0023] According to some embodiments of the present application, in step S3, the volume ratio of dichloromethane and methanol in the eluent is 1:1.

[0024] According to some embodiments of the present application, in step S4, 30%, 40%, 50%, 60%, 70%, and 100% methanol aqueous solution are used in sequence for elution.

[0025] According to some embodiments of the present application, in step S5, the mesh number of the silica gel filler in the normal phase column is 300-400 mesh.

[0026] According to some embodiments of the present application, in step S5, the volume ratio of n-hexane and acetone is 15:1, 10:1, 5:1, and 3:1 in sequence for gradient elution.

[0027] According to some embodiments of the present application, in step S6, the volume ratio of dichloromethane and methanol in the eluent is 1:1.

[0028] According to some embodiments of the present application, the mesh number of the silica gel filler in the normal phase column in step S7 and step S8 is 300-400 mesh.

[0029] According to some embodiments of the present application, in step S7, the volume ratio of n-hexane and acetone is 15:1, 12:1, and 10:1 in sequence for gradient elution.

[0030] According to some embodiments of the present application, the volume ratio of dichloromethane and methanol is 100:1 and 100:1.5 in sequence for gradient elution.

[0031] According to some embodiments of the present application, in step S9, the model of the reverse phase column is C 18 SMB 100-20 / 45.

[0032] According to some embodiments of the present application, in step S10, the volume ratio of dichloromethane and methanol in the developing agent is 200:(10-13).

[0033] According to some embodiments of the present application, the chromogenic agent is anisaldehyde chromogenic agent.

[0034] According to some embodiments of the present application, in step S10, the thin layer chromatography silica gel plate in the thin layer chromatography satisfies at least the following conditions:

[0035] i pH value is 6-8;

[0036] ii The particle size of the silica gel powder is 10-30 μm.

[0037] According to some embodiments of the present application, the model of the gel column is Sephadex LH-20 type gel column.

[0038] Other features and advantages of the present application will be set forth in the description that follows, and in part will be apparent from the description, or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0039] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0040] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of Paniculatoside C prepared in Example 1 of the present application;

[0041] Figure 2 is the nuclear magnetic resonance carbon spectrum of Paniculatoside C prepared in Example 2 of the present application. DETAILED DESCRIPTION

[0042] The following are specific examples of the present application, and the technical solutions of the present application are further described in conjunction with the examples, but the present application is not limited to these examples.

[0043] The reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field, unless otherwise specified.

[0044] Example 1

[0045] Example 1 provides a preparation method of the Pregnane glycoside Paniculatoside C, and the preparation method is as follows:

[0046] S1, 19.8 kg of dried Qizhenglou medicinal material was crushed by a traditional Chinese medicine crusher, and then extracted by a traditional solvent extraction method. The crushed medicinal material was soaked in 20 L of 95% ethanol at room temperature for 7 days. The extraction was repeated 3 times. The obtained extracts were combined, and the solvent was removed by rotary evaporation under reduced pressure to obtain a total extract of about 1728 g. The total extract was suspended in water, and then sequentially extracted with petroleum ether, ethyl acetate, and n-butanol 5 times by a sequential extraction method to obtain a petroleum ether layer extract of 121 g, an ethyl acetate layer extract of 731 g, and a n-butanol layer extract of 361 g. TLC analysis was performed. The petroleum ether layer extract and the ethyl acetate layer extract were combined, and the n-butanol layer extract was discarded to remove the more polar part. Then, the combined extract was suspended in water by an acid-base extraction method, and the pH value of the water phase was adjusted to 1-2 by adding concentrated hydrochloric acid. The water phase was extracted with chloroform 3 times to obtain A 1 part. Then, the pH value of the water phase was adjusted to 9-10 by adding ammonia water, and the water phase was extracted with chloroform and n-butanol 3 times, respectively, to obtain A 2 layer and A 3 layer, respectively. The A 2 layer and the A 3 layer were discarded to remove alkaloid impurities. The A 1 part mainly contains neutral, weakly basic, and acidic compounds with low polarity. The solvent of the A 1 part was removed by rotary evaporation under reduced pressure to obtain an A 1 part extract;

[0047] S2, about 319 g of the A 1 part was dry mixed, and then dissolved in dichloromethane. The solution was added to 100-200 mesh silica gel powder with a weight of 1-2 times the sample weight, and stirred uniformly. The dichloromethane was evaporated to obtain a sample silica gel layer. The fractionation and enrichment of the A 1 part were performed by column chromatography. A 200-300 mesh normal phase silica gel was used to fill the chromatographic column, and the filling volume was about 10 Bv (10 column volumes). The sample silica gel layer was added to the top of the column, and then sequentially eluted with petroleum ether-ethyl acetate 25:1, 20:1, 4:1, 2:1, and 1:1 (v / v) to obtain each fraction. After TLC and LC-MS comprehensive analysis, some fractions were discarded. The 17th to 34th fractions of petroleum ether: ethyl acetate = 2:1 and the 1st to 8th fractions of 1:1 were combined to obtain component Fr 1 of about 24.0 g. The 9th to 15th fractions of petroleum ether: ethyl acetate = 1:1 were combined to obtain component Fr 2 of about 7.9 g.

[0048] S3, Fr 2 was dissolved in an appropriate amount of dichloromethane-methanol, and then wet-loaded on a Sephadex LH-20 (Pharmacia Biotec AB) gel column soaked and packed with dichloromethane-methanol = 1:1. Each fraction was obtained by isocratic elution with dichloromethane-methanol = 1:1 as the eluent. The 21st to 56th fractions were combined to obtain G1 component with a net weight of about 1.28 g.

[0049] S4, Fr 2-G1 component was dissolved in an appropriate amount of methanol-water, and then wet-loaded on a C 18SMB 100-20 / 45 type reversed phase column eluted with 30%, 40%, 50%, 60%, 70%, 100% methanol solution in sequence, the fraction eluted with 100% methanol was combined as R8 fraction, and the net weight was about 2.7g.

[0050] S5, Fr 2-G1-R8 component was dissolved with appropriate amount of n-hexane-acetone, wet loaded on a chromatographic column packed with 300-400 mesh normal silica gel, eluted with eluent n-hexane-acetone = 15:1, 10:1, 5:1, 3:1 in sequence, the first-20 fractions eluted with n-hexane-acetone = 10:1 and the first-8 fractions eluted with 5:1 were combined as S3 component, about 1.0g.

[0051] S6, Fr 2-G1-R8-S3 component was dissolved with appropriate amount of dichloromethane-methanol, wet loaded on a Sephadex LH-20 type gel column soaked and packed with dichloromethane-methanol = 1:1, isocratic elution was carried out with dichloromethane-methanol = 1:1 as eluent to obtain fractions, the 44-78 fractions were combined as G1 component, the weight was about 936.1mg.

[0052] S7, Fr 2-G1-R8-S3-G1 component was dissolved with appropriate amount of n-hexane-acetone, wet loaded on a chromatographic column packed with 300-400 mesh normal silica gel, eluted with eluent n-hexane-acetone = 15:1, 12:1, 10:1 in sequence, the 118-211 fractions eluted with n-hexane-acetone = 12:1 were combined as S4 component, the weight was about 227.4mg.

[0053] S8, Fr 2-G1-R8-S3-G1-S4 component was dissolved with appropriate amount of dichloromethane-methanol, wet loaded on a chromatographic column packed with 300-400 mesh normal silica gel, eluted with eluent dichloromethane-methanol = 100:1, 100:1.5 in sequence, the 7-24 fractions eluted with dichloromethane-methanol = 100:1.5 were combined as S1 component, the weight was about 132.5mg.

[0054] S9, Fr 2-G1-R8-S3-G1-S4-S1 component was dissolved with appropriate amount of methanol-water, wet loaded on a C 18 SMB100-20 / 45 type reversed phase column eluted with 80% methanol solution, the 12-15 fractions eluted with 80% methanol were combined as R1 component, the weight was about 30.7mg.

[0055] S10, Fr 2-G1-R8-S3-G1-S4-S1-R1 component is dissolved with appropriate amount of dichloromethane-methanol, and is uniformly spotted on the bottom of a prepared thin layer chromatography silica gel plate. After the solvent is volatilized and dried, the plate is developed in a dichloromethane-methanol = 200:12 solvent system. After the solvent is developed to the top, it is taken out, the solvent is volatilized and dried, and then it is placed in the system for secondary development. After the development is completed, the plate is placed in a dark-line type three-purpose ultraviolet analyzer to observe the ultraviolet or fluorescence band distribution height. Then the edge of the silica gel plate is trimmed, and the color band distribution height is observed by using anisaldehyde color reagent. A relatively accurate band area range containing the purified compound is obtained. The silica gel powder in the area is scraped off, and the compound in the silica gel powder is eluted with ethyl acetate. After the solvent is dried, the compound Paniculatoside C in a colorless oil form is obtained, and the weight is about 25 mg.

[0056] Using the extraction and separation and purification process, 25 mg of the target compound Paniculatoside C is purified from about 19.8 kg of medicinal materials, and the yield is 1.26 mg / kg. Compared with the prior art, the yield is higher.

[0057] Further, the structure of the prepared and purified Paniculatoside C is identified by nuclear magnetic resonance hydrogen spectrum and carbon spectrum, and the results are as follows: Figure 1 and Figure 2 The corresponding hydrogen spectrum and carbon spectrum data are as follows:

[0058] 1 H NMR (500 MHz, Chloroform-d) δ 6.28 (s, 1H), 5.43 (d, J = 4.7 Hz, 1H), 5.31 (s, 1H), 4.73 (dd, J = 9.7, 1.5 Hz, 1H), 4.16 (dd, J = 8.4, 7.2 Hz, 1H), 3.85 (t, J = 9.3 Hz, 1H), 3.73-3.67 (m, 3H), 3.66-3.63 (m, 1H), 3.46 (s, 3H), 3.29-3.21 (m, 2H), 2.64-2.52 (m, 1H), 2.51-2.38 (m, 3H), 2.33 (d, J = 8.7 Hz, 3H), 2.30-2.22 (m, 2H), 2.17-2.02 (m, 4H), 1.64 (ddt, J = 15.5, 8.1, 4.1 Hz, 2H), 1.54 (s, 3H), 1.31 (d, J = 6.2 Hz, 3H), 1.27-1.23 (m, 3H), 1.06-0.97 (m, 2H), 0.95 (s, 3H). 13C NMR (126 MHz, Chloroform-d) δ 175.6, 143.6, 139.2, 120.7, 118.2, 113.9, 98.1, 86.8, 75.2, 72.3, 71.3, 69.8, 67.6, 57.5, 55.8, 52.7, 43.9, 40.0, 39.3, 37.6, 34.0, 29.7, 28.0, 24.7, 23.7, 19.3, 18.9, 18.1. HRMS (ESI) [M + Na] + C 28 H 40 C9Na: 543.2570, found: 543.2537.

[0059] The above has been described in detail in combination with the embodiments of the present application, but the present application is not limited to the above-described embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the gist of the present application.

Claims

1. A process for the preparation of a pregnane glycoside Paniculatoside C, characterized by, The structural formula of the pregnane glycoside Paniculatoside C is as follows: The preparation method of the pregnane glycoside Paniculatoside C includes the following steps: S1. At room temperature, the pulverized *Seven Layers of Fragrance* was extracted several times with ethanol to obtain an alcohol extract. The extract was then suspended in water and successively extracted several times with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract, respectively. The petroleum ether extract and ethyl acetate extract were combined and suspended in water. The aqueous phase was adjusted to acidity and extracted several times with chloroform to obtain fraction A①. The aqueous phase was then adjusted to alkalinity and extracted several times with chloroform and n-butanol to obtain layers A② and A③, respectively. Layers A② and A③ were discarded. The remaining fraction A① was evaporated under reduced pressure to remove the solvent, yielding fraction A① extract. S2. The extract of A① was dry-mixed to obtain a silica gel layer. The sample was then eluted sequentially using a petroleum ether-ethyl acetate system to obtain the various fractions. Fractions 9 to 15 of petroleum ether:ethyl acetate = 1:1 were combined to obtain component Fr 2. S3. Dissolve the Fr 2 fraction in dichloromethane-methanol, elute isocratically with dichloromethane-methanol as the eluent, and combine fractions 21-56 to obtain fraction G1. S4 and Fr 2-G1 components were dissolved in methanol-water and eluted sequentially with methanol-water solution on a reversed-phase column. The fractions eluted with 100% methanol were combined to form component R8. S5 and Fr 2-G1-R8 fractions were dissolved in hexane-acetone and eluted sequentially with hexane-acetone on a normal phase column. The fractions 1-20 eluted with hexane-acetone = 10:1 and fractions 1-8 eluted with hexane-acetone = 5:1 were combined to form fraction S3. S6 and Fr2-G1-R8-S3 fractions were dissolved in dichloromethane-methanol and eluted isocratically using a gel chromatography column with dichloromethane-methanol as the eluent to obtain each fraction. Fractions 44-78 were combined to obtain fraction G1. S7 and Fr2-G1-R8-S3-G1 fractions were dissolved in n-hexane-acetone and eluted sequentially with n-hexane-acetone using a normal phase column. Fractions 118-211 eluted with n-hexane-acetone = 12:1 were combined and identified as fraction S4. S8 and Fr2-G1-R8-S3-G1-S4 fractions were dissolved in dichloromethane-methanol and eluted sequentially with dichloromethane-methanol using a normal phase column. The fractions 7-24 eluted with dichloromethane-methanol = 100:1.5 were combined and identified as fraction S1. S9 and Fr2-G1-R8-S3-G1-S4-S1 fractions were dissolved in methanol-water and eluted using a reversed-phase column with 80% methanol-water solution. Fractions 12-15 of the 80% methanol solution were combined to form fraction R1. S10 and Fr2-G1-R8-S3-G1-S4-S1-R1 components were dissolved in dichloromethane-methanol. Using dichloromethane-methanol as the developing solvent, thin-layer chromatography was performed. After the solvent was fully developed, the mixture was dried and then developed a second time. After development, the height of the colored bands was observed by colorimetric method to obtain the silica powder in the colored region. The compound in the silica powder was eluted with ethyl acetate to obtain the compound Paniculatoside C.

2. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S2, the silica gel in the sample silica gel layer has a mesh size of 100-200 mesh.

3. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S3, the volume ratio of dichloromethane to methanol in the eluent is 1:

1.

4. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S5, the silica gel packing material in the normal phase chromatography column has a mesh size of 300-400 mesh.

5. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S6, the volume ratio of dichloromethane to methanol in the eluent is 1:

1.

6. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, The silica gel packing material in the normal phase chromatography column described in steps S7 and S8 has a mesh size of 300-400 mesh.

7. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S9, the model of the reversed phase chromatographic column is C 18 SMB 100-20 / 45.

8. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S10, the volume ratio of dichloromethane to methanol in the developing agent is 200:(10~13).

9. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, The colorimetric reagent used in the colorimetric method is anisaldehyde colorimetric reagent.

10. The method for preparing pregnane glycoside Paniculatoside C according to claim 1, characterized in that, In step S10, the thin-layer chromatography silica gel plate used in the thin-layer chromatography method must at least meet the following conditions: The pH value is 6~8; ii. The particle size of the silica powder is 10~30μm.

Images