CN112028958A - Method for preparing dammarane type triterpene from marine mangrove hornberry leaf - Google Patents

Abstract

The invention relates to the technical field of medicinal chemistry, and specifically discloses a method for preparing dammarane-type triterpenes from marine mangrove silique leaves. The method comprises the following steps: (1) extracting silique leaves with ethanol to obtain an ethanol extract; (2) applying the ethanol extract to a silica gel column and eluting with an elution solvent composed of chloroform:methanol to obtain fraction 1 (3) Fraction 1 is applied to silica gel column again, and the elution solvent of petroleum ether: ethyl acetate is eluted to obtain fraction 2; (4) Fraction 2 is separated with preparative HPLC to obtain dammarane type three. Terpenes. The invention provides a brand-new method for preparing dammarane-type triterpenes from silique leaves; the method can simultaneously prepare two dammarane-type triterpenes, and the obtained two dammarane-type triterpenes have purity high.

Description

A kind of method for preparing dammarane-type triterpenes from marine mangrove silique leaves

technical field

The invention relates to the technical field of medicinal chemistry, and specifically discloses a method for preparing dammarane-type triterpenes from marine mangrove silique leaves.

Background technique

Silique leaves are the leaves of mangrove silique trees, the Latin scientific name of siliques is Ceriops tagal (perr.) C.B.Rob., alias: scissors tree, sea flail, Haidianzi (Guangdong, Hainan), produced in Guangdong Xuwen, Hainan's northeast to southern beaches, Taiwan's Kaohsiung Port and other places.

The inventors obtained two dammarane-type triterpenes, Cereotagalol C and Cereotagalol D, from the silique leaves in the previous study, and the two compounds showed certain antitumor activity and had certain application value (see Xin for details). Wu,Hongbo Liao,Xiaohui Zhu,Hongyu Lu,Xiaobin Zeng,Liao Cui,Xiaohua Lv,Yanqun Li and Chaohua Zhang.Two New Dammarane Triterpenes from the Leaves of Ceriops tagal,Rec.Nat.Prod.10:5(2016)628- 632.). Although the above-mentioned document discloses the method for separating Cereotagalol C and Cereotagalol D, in the above-mentioned document, Cereotagalol C and Cereotagalol D are obtained through macroporous resin column chromatography, silica gel column chromatography, ODS column chromatography and preparative high performance liquid chromatography etc. It can only be obtained by combining various chromatographic separation techniques, with many preparation steps and difficult preparation; and Cereotagalol C and Cereotagalol D are separated in different fractions after being separated by various chromatographic columns, which need to be prepared separately and cannot be in the same fraction at one time. The preparation is obtained, which further increases the preparation steps and the preparation difficulty. Therefore, it is of great significance to develop a relatively easy method for preparing Cereotagalol C and Cereotagalol D with few preparation steps.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for preparing dammarane-type triterpenes from marine mangrove silique leaves. The method can simultaneously prepare the dammarane-type triterpenes Cereotagalol C and Cereotagalol D in the same fraction; the preparation steps of the existing preparation method are simplified.

The above-mentioned technical problems to be solved by the present invention are realized through the following technical solutions:

A method for preparing dammarane-type triterpenes from marine mangrove silique leaves, comprising the following steps:

(1) silique leaf is extracted with ethanol to obtain ethanol extract;

(2) the ethanol extract is placed on a silica gel column, and eluted with an elution solvent composed of chloroform: methanol to obtain fraction 1;

(3) with fraction 1 on silica gel column again, carry out elution with petroleum ether: the elution solvent of ethyl acetate composition obtains fraction 2;

(4) Fraction 2 is separated by preparative HPLC to obtain dammarane-type triterpenes.

The marine mangrove silique leaves in the present invention refer to the leaves of siliques grown in marine mangroves.

In the present invention, two silica gel column chromatography is carried out successively by using an elution solvent system composed of chloroform: methanol and an elution solvent system composed of petroleum ether: ethyl acetate, and through the two silica gel column chromatography, fraction 2 is enriched with A large amount of Cereotagalol C and Cereotagalol D; further separation by preparative HPLC can realize the simultaneous preparation of dammarane-type triterpenes Cereotagalol C and Cereotagalol D in the same fraction. The method only needs three separation and purification steps to simultaneously prepare the dammarane-type triterpenes Cereotagalol C and Cereotagalol D; the method simplifies the preparation steps of the triterpenes Cereotagalol C and Cereotagalol D, and greatly improves the preparation efficiency.

Preferably, the extraction with ethanol in step (1) refers to leaching with ethanol with a volume fraction of 70-95%, the leaching time is 48-72 hours, and the extract is concentrated and dried to obtain an ethanol extract.

Most preferably, the extraction with ethanol in step (1) refers to leaching with ethanol with a volume fraction of 95%, the leaching time is 60h, and the extract is concentrated and dried to obtain an ethanol extract.

Preferably, the specific conditions for obtaining fraction 1 by elution with an elution solvent composed of chloroform:methanol described in step (2) are: firstly use chloroform:methanol with a volume ratio of 95:5 to elute and remove impurities; then Then 85:15 chloroform:methanol was used for elution, and the 85:15 chloroform:methanol elution part was collected and concentrated and dried to obtain fraction 1.

Further preferably, the specific conditions for eluting fraction 1 with an elution solvent consisting of chloroform and methanol as described in step (2) are: first, use chloroform with a volume ratio of 5 to 8 times the column volume of 95:5: Methanol is used for elution and impurity removal; then chloroform:methanol in a volume ratio of 6 to 10 times the column volume is used for elution, and the 85:15 chloroform:methanol elution part is collected and concentrated and dried to obtain fraction 1.

Preferably, in step (2), the weight and dosage of silica gel in the silica gel column is 20-30 times that of the ethanol extract; the mesh number of the silica gel is 200-300 meshes.

Preferably, in step (3), use petroleum ether: the eluting solvent that ethyl acetate is formed to carry out elution to obtain the specific condition of fraction 2 as follows: first use petroleum ether with a volume ratio of 96:4: ethyl acetate to elute Then use petroleum ether:ethyl acetate with a volume ratio of 92:8 for elution; collect the 92:8 petroleum ether:ethyl acetate elution part and concentrate and dry to obtain fraction 2.

Further preferably, in step (3), use petroleum ether: the elution solvent composed of ethyl acetate to carry out elution to obtain fraction 2 The specific conditions are: first use petroleum ether with a volume ratio of 6 to 10 times the column volume to be 96:4 : ethyl acetate for elution and impurity removal, and then eluted with petroleum ether: ethyl acetate with a volume ratio of 8 to 12 times the column volume of 92: 8; collect the 92: 8 petroleum ether: ethyl acetate elution part Concentrate and dry to obtain fraction 2.

Preferably, in step (3), the weight and dosage of silica gel in the silica gel column is 40-60 times that of the ethanol extract; the mesh number of the silica gel is 300-400 meshes.

The composition of the elution solvent and the selection of the elution solvent ratio for the above two silica gel column chromatography plays a crucial role in whether Cereotagalol C and Cereotagalol D can be enriched in fraction 2. Improper composition of elution solvent or improper selection of elution solvent ratio in the two silica gel column chromatography will result in the failure to enrich Cereotagalol C and Cereotagalol D in Fraction 2 at the same time; A sort of. After a large number of experimental studies in the present invention, it is concluded that fraction 2 rich in Cereotagalol C and Cereotagalol D can be obtained under the above-mentioned two silica gel column chromatography conditions. It provided decisive conditions for the subsequent preparative HPLC to simultaneously prepare dammarane-type triterpenes CereotagalolC and CereotagalolD.

Preferably, the specific conditions for separation by preparative HPLC are: use a preparative C18 chromatographic column, an ultraviolet detector, and a detection wavelength of 230-254 nm; use acetonitrile as mobile phase A and water as mobile phase B for gradient elution; The gradient elution described is: 0～5min, the volume change of mobile phase A is 0～12%; 5～15min, the volume change of mobile phase A is 12～26%; 15～35min, the volume of mobile phase A is 26% % remained unchanged; the eluted parts corresponding to the chromatographic peaks at 24.1 min and 30.8 min were collected respectively, concentrated and dried to obtain dammarane-type triterpenes Cereotagalol C and Cereotagalol D.

Fraction 2 containing Cereotagalol C and Cereotagalol D was obtained although enrichment after two silica gel column chromatography. But how to separate Cereotagalol C and Cereotagalol D from fraction 2 is also a technical problem. For preparative HPLC separation, the choice of mobile phase and the elution conditions of the mobile phase play a decisive role in whether Cereotagalol C and Cereotagalol D can be separated. Improper selection of elution conditions for the mobile phase also failed to separate Cereotagalol C and Cereotagalol D from fraction 2. After a large number of experimental studies in the present invention, it is concluded that Cereotagalol C and Cereotagalol D can be successfully separated under the specific conditions for separation by the above-mentioned preparative HPLC.

Beneficial effects: The present invention provides a brand-new method for preparing dammarane-type triterpenes from marine mangrove silique leaves.

The method only needs three separation and purification steps to realize the simultaneous preparation of dammarane-type triterpenes Cereotagalol C and Cereotagalol D; by performing silica gel column chromatography twice under the silica gel column chromatography conditions of the present invention, Cereotagalol C and Cereotagalol D can be separated into Cereotagalol D is enriched in the same part, and the dammarane-type triterpenes Cereotagalol C and Cereotagalol D can be simultaneously prepared by one HPLC separation. Compared with the prior art, the method simplifies the preparation steps of the triterpenes Cereotagalol C and Cereotagalol D, and greatly improves the preparation efficiency.

Detailed ways

The present invention is further explained below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the scope of the embodiments.

Method for preparing dammarane-type triterpenes from marine mangrove silique leaves:

(1) leaching the air-dried marine mangrove silique leaves with 5 times the volume fraction of 95% ethanol, the leaching time is 60h, and the extract is concentrated and dried to obtain an ethanol extract after standing for 48h;

(2) The ethanol extract is placed on a silica gel column (the amount of silica gel in the silica gel column is 25 times that of the ethanol extract; the mesh number of the silica gel is 200-300 mesh), and the volume ratio of 6 times the column volume is 95:5. The chloroform:methanol was used for elution and impurity removal; then 8 times the column volume was used for elution with a volume ratio of 85:15 chloroform:methanol, and the 85:15 chloroform:methanol elution part was concentrated and dried to obtain fraction 1;

(3) Put fraction 1 on the silica gel column again (the weight amount of silica gel in the silica gel column is 50 times that of fraction 1; the mesh number of the silica gel is 300 to 400 meshes), and the volume ratio of 8 times the column volume is 96:4. Petroleum ether: ethyl acetate is eluted to remove impurities, and then eluted with petroleum ether: ethyl acetate with a volume ratio of 10 times the column volume of 92: 8; the 92: 8 petroleum ether: ethyl acetate elution part is collected Concentrate and dry to obtain fraction 2;

(4) Fraction 2 is separated by preparative HPLC to obtain dammarane-type triterpenes; the specific conditions of separation by preparative HPLC are: a preparative C18 chromatographic column is used, and an ultraviolet detector is used, and the detection wavelength is 245 nm; is mobile phase A, and water is mobile phase B for gradient elution; the gradient elution is: 0-5min, the volume change of mobile phase A is 0-12% (the volume change of mobile phase B is 100-88% ); 5～15min, the volume change of mobile phase A is 12～26% (the volume change of mobile phase B is 88～74%); 15～35min, the volume of mobile phase A is 26% and remains unchanged (mobile phase B The volume of 24.1min and 30.8min corresponding to the chromatographic peaks were collected respectively, concentrated and dried to obtain dammarane-type triterpenes Cereotagalol C and Cereotagalol D.

The compound obtained by concentrating and drying the eluted part corresponding to the 24.1min chromatographic peak was characterized by mass spectrometry and nuclear magnetic resonance. The results showed that the molecular formula of the compound was determined by high-resolution mass spectrometry as: m/z 483.3856. 1H NMR spectrum showed: δH1.69 ( 1H,m), 0.98(1H,m), 1.61(1H,m), 3.63(1H,t,8.5), 1.34(1H,m), 1.49(1H,m), 1.32(1H,m), 1.51 (1H,m), 1.25(1H,m), 0.88(1H,m), 1.52(1H,m), 1.25(1H,m), 1.52(1H,m), 1.44(1H,m), 1.62( 1H,m), 1.44(1H,m), 1.05(1H,m), 1.84(1H,m), 1.24(1H,m), 1.73(1H,m), 0.96(3H,s), 0.88(3H ,s),1.14(3H,s),1.47(2H,t,8.5),2.05(2H,m),5.11(1H,m),1.62(3H,d,1.0),1.68(3H,d,1.0 ), 3.71(1H,d,10.4), 3.42(1H,d,10.4), 0.87(3H,s), 0.88(3H,s). 13CNMR spectrum shows: δC 38.7, 26.9, 76.6, 41.9, 50.6, 18.3, 34.9, 40.3, 50.4, 36.9, 21.5, 24.7, 42.2, 50.3, 31.1, 27.5, 49.8, 15.5, 16.4, 75.3, 25.4, 40.4, 22.5 , 124.7, 131.6, 25.7, 17.7, 71.9, 11.3, 16.5.

The compound obtained after concentrating and drying the eluted part corresponding to the 30.8min chromatographic peak was characterized by mass spectrometry and nuclear magnetic resonance. The results showed that the molecular formula of the compound was determined by high-resolution mass spectrometry as: m/z 481.3692, 1H NMR spectrum showed: 1.76(1H, m),1.06(1H,m),1.62(1H,m),3.76(1H,dd,11.7,4.6),1.29(1H,m),1.49(1H,m),1.32(1H,m),1.57 (1H,m), 1.23(1H,m), 1.43(1H,m), 1.53(1H,m), 1.27(1H,m), 1.52(1H,m), 1.44(1H,m), 1.63( 1H,m), 1.44(1H,m), 1.06(1H,m), 1.84(1H,m), 1.23(1H,m), 1.74(1H,m), 0.96(3H,s), 0.89(3H ,s),1.14(3H,s),1.47(2H,t,8.5),2.05(2H,m),5.12(1H,m),1.62(3H,t,1.0),1.69(3H,t,1.0 ), 9.38(1H,s), 1.05(3H,s), 0.89(3H,s). 13C NMR spectrum shows: δC38.6, 26.4, 71.8, 55.5, 48.9, 21.0, 34.6, 40.8, 50.6, 36.1, 21.6, 24.8, 42.2, 50.3, 31.1, 27.4, 49.8, 15.5, 16.4, 75.4, 25.5, 40.4 , 22.5, 124.6, 131.7, 25.7, 17.7, 207.0, 8.6, 16.4.

The above mass spectrometry and nuclear magnetic structure characterization data show that the compound obtained after concentrating and drying the elution part corresponding to the 24.1min chromatographic peak is Cereotagalol C; the eluting part corresponding to the 30.8min chromatographic peak is concentrated and dried. The present invention successfully realizes the simultaneous preparation of dammarane-type triterpenes CereotagalolC and Cereotagalol D through the above method; and the preparation steps are simple, and the preparation efficiency of Cereotagalol C and Cereotagalol D is greatly improved.

Claims (10)

1. A method for preparing dammarane type triterpenes from leaves of ocean mangrove hornberries is characterized by comprising the following steps:

(1) extracting folium Caricae with ethanol to obtain ethanol extract;

(2) passing the ethanol extract through a silica gel column, eluting with chloroform: eluting with an eluting solvent consisting of methanol to obtain fraction 1;

(3) fraction 1 was again applied to a silica gel column and purified with petroleum ether: eluting with an elution solvent consisting of ethyl acetate to obtain fraction 2;

(4) separating fraction 2 by preparative HPLC to obtain dammarane type triterpene.

2. The method as claimed in claim 1, wherein the ethanol extraction in step (1) is performed by extracting with 70-95% ethanol by volume for 48-72 h, concentrating the extractive solution, and drying to obtain ethanol extract.

3. The method according to claim 2, wherein the ethanol extraction in step (1) is performed by extracting with 95% ethanol by volume for 60h, concentrating the extractive solution, and drying to obtain ethanol extract.

4. The method of claim 1, wherein the step (2) is performed using chloroform: the specific conditions for eluting with the elution solvent consisting of methanol to obtain fraction 1 are as follows: firstly, using a mixture with the volume ratio of 95: 5 chloroform: eluting with methanol to remove impurities; then, the following steps of 85: 15 chloroform: methanol was eluted and 85: 15 chloroform: concentrating and drying the methanol elution part to obtain fraction 1.

5. The method of claim 4, wherein the step (2) is performed using chloroform: the specific conditions for eluting with the elution solvent consisting of methanol to obtain fraction 1 are as follows: firstly, using a volume ratio of 5-8 times of column volume as 95: 5 chloroform: eluting with methanol to remove impurities; then, the volume ratio of 6-10 times of the column volume is 85: 15 chloroform: methanol was eluted and 85: 15 chloroform: concentrating and drying the methanol elution part to obtain fraction 1.

6. The method according to claim 1, wherein the amount of the silica gel in the silica gel column in the step (2) is 20 to 30 times of the weight of the ethanol extract; the mesh number of the silica gel is 200-300 meshes.

7. The method according to claim 1, wherein in step (3) the mixture is treated with petroleum ether: the specific conditions for eluting with the elution solvent consisting of ethyl acetate to obtain fraction 2 are as follows: firstly, using a mixture with the volume ratio of 96: 4 petroleum ether: eluting with ethyl acetate to remove impurities, and then carrying out elution with a volume ratio of 92: 8 petroleum ether: eluting with ethyl acetate; and (4) collecting 92: 8 petroleum ether: concentrating and drying the elution part of the ethyl acetate to obtain a fraction 2.

8. The method according to claim 7, wherein in step (3) the mixture is treated with petroleum ether: the specific conditions for eluting with the elution solvent consisting of ethyl acetate to obtain fraction 2 are as follows: firstly, using a column with the volume ratio of 6-10 times of column volume as 96: 4 petroleum ether: eluting with ethyl acetate to remove impurities, and then carrying out elution with a volume ratio of 8-12 times of column volume of 92: 8 petroleum ether: eluting with ethyl acetate; and (4) collecting 92: 8 petroleum ether: concentrating and drying the elution part of the ethyl acetate to obtain a fraction 2.

9. The method according to claim 1, wherein the amount of the silica gel in the silica gel column in the step (3) is 40 to 60 times of the weight of the ethanol extract; the mesh number of the silica gel is 300-400 meshes.

10. The method according to claim 1, wherein the separation by preparative HPLC is carried out under the following conditions: a preparative C18 chromatographic column and an ultraviolet detector are adopted, and the detection wavelength is 230-254 nm; performing gradient elution by taking acetonitrile as a mobile phase A and water as a mobile phase B; the gradient elution is as follows: the volume change of the mobile phase A is 0-12% in 0-5 min; 5-15 min, wherein the volume change of the mobile phase A is 12-26%; keeping the volume of the mobile phase A unchanged at 26 percent for 15-35 min; collecting the elution parts corresponding to chromatographic peaks of 24.1min and 30.8min respectively, concentrating and drying to obtain the dammarane type triterpene Cereotagalol C and Cereotagalol D.