CN109369769B - Method for extracting and separating hecolone monomer from stems and leaves of tribulus terrestris - Google Patents

Abstract

The invention discloses a method for extracting and separating hecodone monomers from stems and leaves of Tribulus terrestris, and belongs to the field of natural medicinal chemistry research. In the present invention, the stems and leaves of Tribulus terrestris are used as raw materials to extract, filter, concentrate, and dilute the concentrated solution with water, and then adsorb through a macroporous adsorption resin column. Elution is carried out to obtain the total spirol saponin containing the hexamethylene monomer, and the total spirol saponin is taken and separated by silica gel column chromatography to obtain the hexacodone monomer. The preparation method of the invention has high extraction efficiency, simple operation, and is suitable for industrial production.

Description

A method for extracting and separating hecodone monomer from stems and leaves of Tribulus terrestris

technical field

The invention relates to a method for extracting and separating hecodone monomers from stems and leaves of Tribulus terrestris, and belongs to the field of natural medicinal chemistry research.

Background technique

Tribulus terretris L. is an annual or perennial herb. The stem is branched from the base and spreads flat on the ground. The length of the branches can reach 30-60cm. The leaves are pinnately compound leaves, small and pointed. It is a traditional Chinese medicine, also known as Tribulus terrestris, Tribulus terrestris, Tribulus terrestris, and Tribulus terrestris. It is acrid, bitter, slightly warm in nature, has a small poison, enters the liver and lung meridians, and has the functions of calming the liver and relieving depression, promoting blood circulation, improving eyesight, and relieving itching. Pharmacological studies have shown that Tribulus terrestris has antibacterial, anticancer, and diuretic effects, especially in the prevention and treatment of cardiovascular and cerebrovascular diseases.

So far, a variety of steroidal saponins have been isolated from Tribulus terrestris, including furosteric steroidal saponins and spirosteroidal steroidal saponins, which are the main active components of Tribulus terrestris. Modern research has found that the content and types of steroidal saponins in the stems and leaves of Tribulus terrestris are similar to those in fruits, and the extracts from stems and leaves of Tribulus terrestris also have the effects of improving cardiovascular and cerebrovascular, lowering blood lipids, and antifungal. Hecogenone is a spirosteroidal steroidal saponin contained in the stems and leaves of Tribulus terrestris, which has the functions of scavenging hydroxyl radicals, inhibiting tyrosinase and antifungal.

SUMMARY OF THE INVENTION

The invention provides a method for extracting and separating hecodone monomer from stems and leaves of Tribulus terrestris.

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

Using the stems and leaves of Tribulus terrestris as raw materials, extract, filter, concentrate, and dilute the concentrated solution with water, and then adsorb through a macroporous adsorption resin column, first eluate with an aqueous solution of a low-concentration organic solvent, and then use an aqueous solution of a high-concentration organic solvent. Elution is carried out, followed by TLC monitoring to obtain total spirosterin saponins, and the total spirosterol saponins are taken for separation by silica gel column chromatography, and finally the hexamethylene monomer is obtained.

The method for extracting the stems and leaves of Tribulus terrestris is as follows: take and pulverize the stems and leaves of Tribulus terrestris, extract three times by refluxing with an aqueous organic solvent solution, combine the three extracts, and recover the solvent. The aqueous solution of the organic solvent is acetone water, methanol water or ethanol water, preferably 70%-85% acetone water, methanol water or ethanol water.

The macroporous adsorption resin is selected from one of AB-8, D103, D101, HP20 or a mixed resin of two or more of them.

The organic solvent is selected from ethanol, methanol, acetone or two or more mixed solvents thereof.

The aqueous solution of the low-concentration organic solvent is an aqueous solution with a volume percent concentration of less than or equal to 40%.

The aqueous solution of the high-concentration organic solvent is an aqueous solution with a volume percent concentration greater than 40%.

After the tribulus terrestris stem and leaf extract is adsorbed with macroporous adsorption resin, it is firstly eluted with an aqueous solution of an organic solvent whose volume percentage concentration is less than 15%, and then eluted with an aqueous solution of an organic solvent whose volume percentage concentration is less than 15% to less than 40%. Furosteroid saponins are finally eluted with an aqueous solution of an organic solvent with a concentration of more than 40% by volume to obtain total spirosteroidal saponins containing hecodone monomer.

The total spirosterol saponins column chromatography uses ethyl acetate: ethanol: water as the eluent, preferably ethyl acetate: ethanol: water with a volume ratio of 5: 3: 1 as the mobile phase to perform silica gel column chromatography separation to obtain a After the component B of the heikodone monomer; the component B is separated by silica gel column chromatography with petroleum ether: ethyl acetate: ethanol: water as the mobile phase again to obtain the heikodone monomer, and the preferred volume ratio is 6: 1:1:0.1. The preparation method of the invention has the advantages of reasonable process, high extraction efficiency, simple operation and high purity of the hecodone, and is suitable for industrial production.

The hecodone monomer obtained by the present invention can be used for preparing pharmaceutical compositions and other products.

Detailed ways

Preferred embodiments of the present invention will be described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

Example 1

Take 5Kg of dried Tribulus terrestris stems and leaves and pulverize, and extract three times with 70% ethanol aqueous solution. Solvent until there is no ethanol odor. The concentrated solution was diluted with water, the diluted solution was adsorbed by AB-8 macroporous adsorption resin, washed with deionized water until colorless, eluted with 35% ethanol solution of 8 times the column volume, and the eluate was concentrated with chloroform-methanol- Water (8:4:0.9) was used as the developing solvent to carry out thin layer chromatography test, and reagent A (50ml acetic acid was used to dissolve 0.5ml anisaldehyde, and 1ml sulfuric acid was added) and reagent E (1% p-dimethylaminobenzaldehyde ethanol solution: Concentrated hydrochloric acid (4; 1)) developed color, the result showed that the eluted component at this time was fururosteroid saponin, after the fururosteroid saponin was completely eluted, the spirosteroid saponin was eluted with 80% ethanol solution , the solvent was recovered under reduced pressure and dried to obtain total spirosterol saponins.

Take 30 g of total spirosterol saponins, and use ethyl acetate: ethanol: water = 5:3:1 (volume ratio) as the mobile phase for separation by silica gel column chromatography to obtain 10.1 g of component B containing hecodone monomer; Component B is again separated by silica gel column chromatography with petroleum ether: ethyl acetate: ethanol: water (6:1:1:0.1, volume ratio) as the mobile phase to obtain the hecodone monomer.

The structure of the obtained hecodone monomer was confirmed by NMR, and its purity was 99.5% detected by HPLC-ELSD.

The NMR data of hecodone are as follows

¹³ C NMR (C ₅ D ₅ N, 150 MHz) δc: 38.12 (C-1), 38.02 (C-2), 209.72 (C-3), 44.7 (C-4), 46.37 (C-5), 28.75 (C-6), 31.43(C-7), 34.31(C-8), 54.96(C-9), 36.37(C-10), 38.12(C-11), 212.44(C-12), 55.47( C-13), 55.77(C-14), 31.53(C-15), 79.75(C-16), 54.42(C-17), 16.19(C-18), 10.88(C-19), 42.74(C -20), 14.01(C-21), 109.41(C-22), 31.89(C-23), 29.31(C-24), 30.63(C-25), 67.04(C-26), 17.40(C-20) 27).

¹ H NMR (C ₅ D ₅ N, 600 MHz) δ _H : 4.37 (1H, m H-16), 3.48 (1 H, dd J=10.7 Hz H-26), 3.35 (1 H, t J=10.8 Hz H- 26), 2.66-1.06 (25H, m), 0.82 (3H, s H-18), 1.00 (3H, s H-19), 1.24 (3H, d J=6.1Hz H-21), 0.57 (3H, d J = 6.3 Hz H-27).

The HPLC-ELSD chromatographic conditions are as follows

Column: Agilent Eclipse XDB-C18 (ODS, 4.6×250nm, 5μm)

Mobile phase: acetonitrile-water (45:55); flow rate: 1L/min; column temperature: 30°C

Injection volume: 20 μL; ELSD detector drift tube temperature 98°C; Impactor status: off.

Carrier gas pressure: 0.45Mpa Nebulizer flow rate: 2.6L/min Magnification: 1.0 times

Example 2

Take 5Kg of dried stems and leaves of Tribulus terrestris and pulverize, and extract three times with 70% methanol aqueous solution. The amount of methanol aqueous solution is 10, 8, and 6 times (W/V), respectively. Solvent to no alcohol odor. The concentrated solution was diluted with water, the diluted solution was adsorbed by D101 macroporous adsorption resin, washed with deionized water until colorless, eluted with 30% methanol solution of 8 times the column volume, and the eluent was concentrated with chloroform-methanol-water ( 8:4:0.9) as the developing agent, carry out thin layer chromatography test, use reagent A (50ml acetic acid to dissolve 0.5ml anisaldehyde, add 1ml sulfuric acid) and reagent E (1% p-dimethylaminobenzaldehyde ethanol solution: concentrated hydrochloric acid) (4; 1)) color development, the results show that the components eluted at this time are furosteroid saponins. After the furosteroid saponins are completely eluted, the spirosteroid saponins are eluted with 80% methanol solution. The solvent was recovered under reduced pressure and dried to obtain total spirosterol saponins.

Take 30 g of total spirosterol saponins, and use ethyl acetate: ethanol: water (5:3:1, volume ratio) as the mobile phase to carry out silica gel column chromatography separation to obtain 9.2 g of component B containing hecodone monomer; Component B is again separated by silica gel column chromatography with petroleum ether: ethyl acetate: ethanol: water (6:1:1:0.1, volume ratio) as the mobile phase to obtain the hecodone monomer.

HPLC-ELSD detection, its purity is 99.2%.

Example 3

Take 5Kg of dried stems and leaves of Tribulus terrestris and pulverize, and extract three times with 85% acetone aqueous solution. Solvent recovery. The concentrated solution was diluted with water, the diluted solution was adsorbed by D4020 macroporous adsorption resin, washed with deionized water until colorless, eluted with 25% acetone solution of 8 times the column volume, and the eluent was concentrated with chloroform-methanol-water ( 8:4:0.9) as the developing agent, carry out thin layer chromatography test, use reagent A (50ml acetic acid to dissolve 0.5ml anisaldehyde, add 1ml sulfuric acid) and reagent E (1% p-dimethylaminobenzaldehyde ethanol solution: concentrated hydrochloric acid) (4; 1)) color development, the results show that the eluted component is furosteroid saponin at this time, after the furosteroid saponin is completely eluted, the spirosteroid saponin is eluted with 85% acetone solution. The solvent is recovered under pressure and dried to obtain total spirosterol saponins.

Take 30 g of total spirosterol saponins, and use ethyl acetate: ethanol: water (5:3:1, volume ratio) as the mobile phase to separate by silica gel column chromatography to obtain 9.5 g of component B containing hecodone monomer; Component B is again separated by silica gel column chromatography with petroleum ether: ethyl acetate: ethanol: water (6:1:1:0.1, volume ratio) as the mobile phase to obtain the hecodone monomer.

HPLC-ELSD detection, its purity is 98.0%.

Example 4

Take 5Kg of dry stems and leaves of Tribulus terrestris and pulverize them, extract three times with 90% ethanol aqueous solution, the amount of ethanol aqueous solution is 10, 8, and 6 times (W/V) respectively, and the reflux time is 3h, 2h, 1h, and the three extracts are combined, The solvent is recovered until there is no ethanol odor. The concentrated solution was diluted with water, the diluted solution was adsorbed by HP20 macroporous adsorption resin, washed with deionized water until colorless, eluted with 40% ethanol solution of 8 times the column volume, and the eluent was concentrated with chloroform-methanol-water ( 8:4:0.9) as the developing agent, carry out thin layer chromatography test, use reagent A (50ml acetic acid to dissolve 0.5ml anisaldehyde, add 1ml sulfuric acid) and reagent E (1% p-dimethylaminobenzaldehyde ethanol solution: concentrated hydrochloric acid) (4; 1)) color development, the result shows that the eluted component is furosteroid saponin at this time, after the furosteroid saponin is completely eluted, the spirosteroid saponin is eluted with 95% ethanol solution to reduce The solvent is recovered under pressure and dried to obtain total spirosterol saponins.

Take 30 g of total spirosterol saponins, and use ethyl acetate: ethanol: water (5:3:1, volume ratio) as the mobile phase for separation by silica gel column chromatography to obtain 8.5 g of component B containing hecodone monomer; Component B was separated again by silica gel column chromatography with petroleum ether: ethyl acetate: ethanol: water (6:1:1:0.1, volume ratio) as the mobile phase to obtain the hecodone monomer.

HPLC-ELSD detection, its purity is 97.5%.

The foregoing embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the foregoing embodiments have described the present invention in detail, for those skilled in the art, they can still understand the The recorded technical solutions are modified, or some technical features thereof are equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (4)

1. a method for extracting and separating hecodone monomer from the stems and leaves of Tribulus terrestris, it is characterized in that: get dry stems and leaves of Tribulus terrestris 5Kg and pulverize, extract three times with 70% aqueous ethanolic reflux, and the aqueous ethanolic consumption is respectively 10, 8, 6 times (W/V), the reflux time is 3h, 2h, 1h, the three extracts are combined, and the solvent is recovered until there is no ethanol smell; the concentrate is diluted with water, the diluted solution is adsorbed by AB-8 macroporous adsorption resin, and washed with deionized water to colorless, eluted with 35% ethanol solution of 8 times the column volume, concentrated the eluate, and used chloroform-methanol-water with a volume ratio of 8:4:0.9 as the developing solvent to carry out thin-layer chromatography. 50ml of acetic acid dissolves 0.5ml of anisaldehyde, add 1ml of sulfuric acid reagent A and a 4:1 volume ratio of 1% p-dimethylaminobenzaldehyde ethanol solution and concentrated hydrochloric acid reagent E to develop color, the results show that the components eluted at this time It is furosteroid steroid saponins. After the furosteroid steroid saponins are completely eluted, the spirosteroid saponins are eluted with 80% ethanol solution, the solvent is recovered under reduced pressure, and dried to obtain the total spirosteroid saponins; take the total spirosteroid saponins. 30g, ethyl acetate: ethanol: water with a volume ratio of 5:3:1 was used as the mobile phase for separation by silica gel column chromatography to obtain 10.1g of component B containing hecodone monomer; the volume ratio of component B was 6:1:1:0.1 of petroleum ether: ethyl acetate: ethanol: water was used as the mobile phase, and the silica gel column chromatography was carried out again to obtain the hecodone monomer. 2. a method for extracting and separating hecodone monomer from the stems and leaves of Tribulus terrestris, it is characterized in that: get dried stems and leaves of Tribulus terrestris 5Kg and pulverize, with 70% methanol aqueous solution reflux extraction three times, the methanol aqueous solution consumption is respectively 10, 8, 6 times (W/V), the reflux time is 3h, 2h, 1h, the three extracts are combined, and the solvent is recovered until there is no alcohol smell; the concentrate is diluted with water, the diluted solution is adsorbed by D101 macroporous adsorption resin, and washed with deionized water until there is no alcohol. color, eluted with 30% methanol solution of 8 times the column volume, concentrated the eluate, and used chloroform-methanol-water with a volume ratio of 8:4:0.9 as the developing solvent to carry out thin-layer chromatography inspection, with 50ml of acetic acid. Dissolve 0.5ml of anisaldehyde, add 1ml of sulfuric acid reagent A and 1% p-dimethylaminobenzaldehyde ethanol solution with a volume ratio of 4:1 and reagent E of concentrated hydrochloric acid to develop color. The results show that the components eluted at this time are Furosteroid steroid saponins, after the furosteroid steroid saponins are completely eluted, the spirosteroid saponins are eluted with 80% methanol solution, the solvent is recovered under reduced pressure, and dried to obtain total spirosteroid saponins; take 30 g of total spirosteroid saponins , using ethyl acetate: ethanol: water with a volume ratio of 5:3:1 as the mobile phase to carry out silica gel column chromatography separation to obtain 9.2 g of component B containing hecodone monomer; the volume ratio of component B is 6:1:1:0.1 of petroleum ether: ethyl acetate: ethanol: water is used as the mobile phase to carry out the separation by silica gel column chromatography again to obtain the hecodone monomer. 3. a method for extracting and separating hecodone monomer from Tribulus terrestris stems and leaves, it is characterized in that: get dry Tribulus terrestris stems and leaves 5Kg and pulverize, extract three times with 85% aqueous acetone backflow, and the aqueous acetone consumption is respectively 10, 8 , 6 times (W/V), the reflux time is 3h, 2h, 1h, the three extracts were combined, and the solvent was recovered; the concentrated solution was diluted with water, the diluted solution was adsorbed by D4020 macroporous adsorption resin, washed with deionized water until colorless, and then used 8 times the column volume of 25% acetone solution to elute, the eluent was concentrated and then used chloroform-methanol-water with a volume ratio of 8:4:0.9 as the developing solvent for thin-layer chromatography, and 50ml of acetic acid was used to make 0.5ml Dissolve anisaldehyde, add 1 ml of sulfuric acid reagent A, 1% p-dimethylaminobenzaldehyde ethanol solution with a volume ratio of 4:1 and reagent E of concentrated hydrochloric acid to develop color. The result shows that the eluted component is furosteroid After the furosteroid saponins are completely eluted, the spirosteroid saponins are eluted with 85% acetone solution, the solvent is recovered under reduced pressure, and dried to obtain the total spirosteroid saponins; 5:3:1 ethyl acetate:ethanol:water was used as the mobile phase for separation by silica gel column chromatography to obtain 9.5g of component B containing hecodone monomer; the volume ratio of component B was 6:1: 1: 0.1 petroleum ether: ethyl acetate: ethanol: water is used as the mobile phase and is separated by silica gel column chromatography again to obtain the hecodone monomer. 4. a method for extracting and separating hecodone monomer from Tribulus terrestris stems and leaves, it is characterized in that: get dry Tribulus terrestris stems and leaves 5Kg and pulverize, extract three times with 90% aqueous ethanolic reflux, and the aqueous ethanolic consumption is respectively 10, 8 , 6 times (W/V), the reflux time is 3h, 2h, 1h, the three extracts are combined, and the solvent is recovered until there is no ethanol smell; the concentrate is diluted with water, and the dilution is adsorbed by HP20 macroporous adsorption resin, and washed with deionized water until Colorless, eluted with 8 times the column volume of 40% ethanol solution, concentrated the eluate, and used chloroform-methanol-water with a volume ratio of 8:4:0.9 as the developing solvent to carry out thin layer chromatography test, using 50ml Acetic acid dissolves 0.5ml of anisaldehyde, adds 1ml of sulfuric acid reagent A and 1% p-dimethylaminobenzaldehyde ethanol solution with a volume ratio of 4:1 and reagent E of concentrated hydrochloric acid to develop color. The results show that the components eluted at this time are Furosteroid saponins, after the furosteroid saponins are completely eluted, the spirosteroid saponins are eluted with 95% ethanol solution, the solvent is recovered under reduced pressure, and dried to obtain total spirosteroid saponins; take 30 g of total spirosteroid saponins , using ethyl acetate: ethanol: water with a volume ratio of 5:3:1 as the mobile phase to carry out silica gel column chromatography separation to obtain 8.5 g of component B containing hecodone monomer; the volume ratio of component B is 6:1:1:0.1 of petroleum ether: ethyl acetate: ethanol: water is used as the mobile phase to carry out the separation by silica gel column chromatography again to obtain the hecodone monomer.