CN105175238B - New component and its extraction separation method in a kind of Reishi sporule - Google Patents

Abstract

The invention discloses a new component in Ganoderma lucidum spore oil and its extraction and separation method, wherein the chemical name of the new component is 3β,5α,6β,11-tetrahydroxy-(22E,24R)-9,11-schisergoster -7,22-diene-9-ketone, the extraction and separation method includes extraction-degreasing-gradient elution-reversed-phase column-gradient elution-isocratic elution-recrystallization and other steps. The new component in the ganoderma spore oil provided by the invention and the extraction and separation method thereof successfully extract and separate a new component from the ganoderma spore, which provides a material basis for the further pharmacological effects of the ganoderma spore oil.

Description

A new component in Ganoderma lucidum spore and its extraction and separation method

technical field

The invention relates to the technical field of extraction and separation, in particular to a new component in ganoderma lucidum spores and an extraction and separation method thereof.

Background technique

Ganoderma lucidum is also known as Lin Zhongling (scientific name: Ganoderma Lucidum Karst), and the Ganoderma lucidum grown in the forest is the best. The shape is umbrella-shaped, and the cap is kidney-shaped, semi-circular or nearly round. It is the fruiting body of the polyporaceae fungus Ganoderma lucidum. It has the effects of invigorating qi and calming the nerves, relieving cough and relieving asthma, and is used for dizziness, insomnia, palpitation, shortness of breath, cough and asthma due to fatigue.

Ganoderma spore powder is the extremely tiny oval reproductive cells ejected from the gills of Ganoderma lucidum, that is, the seeds of Ganoderma lucidum during the growth and maturity stage. Each spore of Ganoderma lucidum is only 4-6 microns. It is a living organism with a double-walled structure and is surrounded by hard chitin cellulose, which is difficult for the human body to fully absorb. After breaking the wall, it is more suitable for direct absorption by the human stomach. It condenses the essence of Ganoderma lucidum, and has all the genetic material and health care functions of Ganoderma lucidum.

The main components of Ganoderma lucidum spore oil are Ganoderma acid, unsaturated fatty acids, and Ganoderma polysaccharides. Induce or promote the phagocytosis of macrophages to kill tumor cells directly and indirectly. This product can improve the body's immunity and reduce the occurrence of poor spirits, loss of appetite, vomiting, and hair loss caused by radiotherapy and chemotherapy.

It is generally believed that the clinical effects of Ganoderma lucidum spore oil are closely related to its rich components, but all components of Ganoderma lucidum spore oil have not yet been identified.

Contents of the invention

The invention provides a new component in ganoderma lucidum spores and an extraction and separation method thereof.

A new component in Ganoderma lucidum spores, the chemical name of the new component is 3β,5α,6β,11-tetrahydroxy-(22E,24R)-9,11-schisergosta-7,22-diene-9 - Ketone, the chemical structure is

A method for extracting and separating new components in the ganoderma spores, comprising the following steps:

(1) Take the Ganoderma lucidum spore powder, put it into a supercritical extraction kettle, extract it through the extraction kettle and pass through the separation kettle I and the separation kettle II for secondary separation, and collect the Ganoderma lucidum spore oil in the separation kettle II;

(2) Ganoderma lucidum spore oil is extracted three times with ethanol with a volume fraction of 75-85%, and the ethanol extract is extracted twice with petroleum ether for further degreasing, and the dilute alcohol solution is concentrated under reduced pressure to obtain the ethanol extract;

(3) Put the ethanol extract on a silica gel column, and use petroleum ether-acetone for gradient elution with a mass ratio of 100:0, 98:2, 95:5, 9:1, 8:2, 7:3, and 0:100;

(4) Combine petroleum ether-acetone mass ratio 9:1～7:3 eluted component samples, put the component samples on the reverse phase column, use methanol-formic acid with mass ratio 7:3, 8:2, 9 :1, 100:0 gradient elution;

(5) The methanol-formic acid mass ratio is 100:0 to obtain two components A and B from the eluted part;

(6) The two components were separately sent to HPLC, eluted isocratically with 65-75% volume fraction of acetonitrile, and the peak at t _R =41-44min was collected, recrystallized with methanol, and a new compound was obtained from one of the components.

Preferably, in the step (1), the pressure of the extraction tank is 25-35 MPa and the temperature is 55-65 °C; the pressure of the separation tank I is 11-15 MPa and the temperature is 50-60 °C; the pressure of the separation tank II is 5-7 MPa , the temperature is 40-50° C.; the entrainer is 95% ethanol; the extraction time is 60-120 minutes.

Preferably, in the step (2), the Ganoderma lucidum spore oil is extracted with 80% ethanol by volume fraction.

Preferably, in the step (3), the silica gel in the silica gel column is 100-200 mesh.

Preferably, in the step (4), the filler of the reversed-phase column is ODS.

Preferably, in the step (5), the volume fraction of formic acid is 1%.

Preferably, in the step (6), the volume fraction of acetonitrile is 70%.

The new component in the ganoderma spores and the extraction and separation method provided by the invention successfully extract and separate a new component from the ganoderma spores, which provides a material basis for the further pharmacological effects of the ganoderma spore oil.

Description of drawings

Fig. 1 is the molecular structural formula of the new component extracted and separated by the present invention.

detailed description

The following examples are further detailed descriptions of the present invention.

Example 1

A method for extracting and separating new components in Ganoderma lucidum spores, comprising the following steps:

(1) Take the Ganoderma lucidum spore powder, put it into a supercritical extraction kettle, extract it through the extraction kettle and pass through the separation kettle I and the separation kettle II for secondary separation, and collect the Ganoderma lucidum spore oil in the separation kettle II; wherein the pressure of the extraction kettle is 25MPa , temperature is 55 DEG C; separation tank I pressure is 11MPa, temperature is 50 DEG C; separation tank II pressure is 5MPa, temperature is 40 DEG C; entrainer is 95% ethanol; extraction time is 60 minutes;

(2) Ganoderma lucidum spore oil is extracted three times with ethanol with a volume fraction of 75%, and the ethanol extract is extracted twice with petroleum ether to further degrease, and the dilute alcohol solution is concentrated under reduced pressure to obtain the ethanol extract;

(3) Put the ethanol extract on a silica gel (100g, 100 mesh) column, use petroleum ether-acetone at a mass ratio of 100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 0: 100 gradient elution;

(4) Combine petroleum ether-acetone mass ratio 9:1～7:3 eluted component samples, put the component samples on the reverse phase column (ODS, 40-75μm), use methanol-formic acid with a mass ratio of 7: 3. 8:2, 9:1, 100:0 gradient elution;

(5) Methanol-1% formic acid mass ratio 100:0 elution part obtains two components A, B;

(6) The two components were separately subjected to HPLC, eluted isocratically with 65% volume fraction of acetonitrile, and the peak at t _R =41-44min was collected, recrystallized with methanol, and a new compound was obtained from one of the components.

Example 2

A method for extracting and separating new components in Ganoderma lucidum spores, comprising the following steps:

(1) Take Ganoderma lucidum spore powder, put it into a supercritical extraction kettle, extract it through the extraction kettle and separate it through the separation kettle I and the separation kettle II, and collect the Ganoderma lucidum spore oil in the separation kettle II; wherein the pressure of the extraction kettle is 35MPa , temperature is 65 DEG C; separation tank I pressure is 15MPa, temperature is 60 DEG C; separation tank II pressure is 7MPa, temperature is 50 DEG C; entrainer is 95% ethanol; extraction time is 120 minutes;

(2) Ganoderma lucidum spore oil is extracted three times with ethanol with a volume fraction of 85%, and the ethanol extract is extracted twice with petroleum ether to further degrease, and the dilute alcohol solution is concentrated under reduced pressure to obtain the ethanol extract;

(3) Put the ethanol extract on a silica gel (100g, 150 mesh) column, use petroleum ether-acetone at a mass ratio of 100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 0: 100 gradient elution;

(4) Combine petroleum ether-acetone mass ratio 9:1～7:3 eluted component samples, put the component samples on the reverse phase column (ODS, 40-75μm), use methanol-formic acid with a mass ratio of 7: 3. 8:2, 9:1, 100:0 gradient elution;

(5) Methanol-1% formic acid mass ratio 100:0 elution part obtains two components A, B;

(6) The two components were separately subjected to HPLC, and eluted isocratically with 75% volume fraction of acetonitrile, and the peak at t _R =41-44min was collected, recrystallized with methanol, and a new compound was obtained from one of the components.

Example 3

A method for extracting and separating new components in Ganoderma lucidum spores, comprising the following steps:

(1) Take the Ganoderma lucidum spore powder, put it into a supercritical extraction kettle, extract it through the extraction kettle and pass through the separation kettle I and the separation kettle II for secondary separation, and collect the Ganoderma lucidum spore oil in the separation kettle II; wherein the pressure of the extraction kettle is 30MPa , temperature is 60 DEG C; separation tank I pressure is 13MPa, temperature is 55 DEG C; separation tank II pressure is 6MPa, temperature is 45 DEG C; entrainer is 95% ethanol; extraction time is 60 minutes;

(2) Ganoderma lucidum spore oil is extracted three times with 80% ethanol by volume fraction, and the ethanol extract is extracted twice with petroleum ether for further degreasing, and the dilute alcohol liquid is concentrated under reduced pressure to obtain the ethanol extract;

(3) Put the ethanol extract on a silica gel (100g, 200 mesh) column, use petroleum ether-acetone at a mass ratio of 100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 0: 100 gradient elution;

(4) Combine petroleum ether-acetone mass ratio 9:1～7:3 eluted component samples, put the component samples on the reverse phase column (ODS, 40-75μm), use methanol-formic acid with a mass ratio of 7: 3. 8:2, 9:1, 100:0 gradient elution;

(5) Methanol-1% formic acid mass ratio 100:0 elution part obtains two components A, B;

(6) The two components were separately subjected to HPLC, and eluted isocratically with 70% acetonitrile by volume fraction, and the peak at t _R =41-44min was collected, recrystallized with methanol, and a new compound was obtained from one of the components.

The new ingredients were identified as follows:

3β,5α,6β,11-tetrahydroxy-(22E,24R)-9,11-schistoster-7,22-dien-9-one [3β,5α,6β,11-tetrahydroxy-(22E,24R )-9,11-secoergosta-7,22-dien-6-one,BZY-16]: white solid; [α] _D ²⁵ –5.5 (c 0.18, MeOH); UV(MeOH)λ _max nm(logε) : 203 (3.98), 230 (sh.); 1H NMR (600MHz, C ₅ D ₅ N) δ: 6.90 (1H, br s, H-7), 5.29 (1H, dd, J＝15.3, 8.2Hz, H-22),5.20(1H,dd,J＝15.3,7.8Hz,H-23),4.76(1H,m,H-3),4.71(1H,br s,H-6),4.33(1H, dd,J=14.0,9.0Hz,H-11a),4.13(1H,dd,J=14.0,9.0Hz,H-11b),3.78(1H,dd,J=9.0,6.0Hz,H-14), 2.93(1H,br t,J=12.2Hz,H-4b),2.77(1H,br t,J=13.6,H-1a),2.46(1H,br d,J=12.2,H-4a),2.31 (1H,m,H-2),2.28(1H,m,H-20),1.95-2.03(3H,overlapped,H-1b,H-2b,H-12a),1.83-1.90(3H,overlapped, H-12b, H-17, H-24), 1.82 (1H, s, H-19), 1.74 (1H, m, H-16b), 1.60 (1H, m, H-15a), 1.58 (1H, m,H-16a),1.47(1H,m,H-15b),1.41(1H,m,H-25),1.10(1H,d,J=6.8,H-21),1.00(1H,s, H-18),0.92(1H,s,J=6.8,H-28),0.83(1H,s,J=6.8,H-27),0.81(1H,s,J=6.8,H-26). ¹³ C NMR (150MHz, C ₅ D ₅ N) δ: 29(C-1), 32.2(C-2), 67(C-3), 41.4(C-4), 77(C-5), 72.9 (C-6), 141.5(C-7), 136.6(C-8), 205.4(C-9), 49.1(C-10), 58.5(C-11), 42.4(C-12), 46.8( C-13), 43.3 (C-14), 26.1 (C -15), 27.6(C-16), 50.5(C-17), 18.1(C-18), 22.2(C-19), 49.1(C-20), 21.9(C-21), 135.9(C- 22), 132.7(C-23), 43.2(C-24), 33.5(C-25), 29.9(C-26), 20.3(C-27), 17.8(C-28); ESIMS m/z: 947[2M+Na] ⁺ ,485[M+Na] ⁺ ,959[2M+Cl] ^– ,497[M+Cl] ^– ,461[M–H] ^– .; ESI(+)MS m/z: 947[2M+Na] ⁺ , 485[M+Na] ⁺ ; ESI(-)MS m/z: 959[2M+Cl] ^- , 497[M+Cl] ^- , 461[M-H] ^- .

Table 1. ¹ H(600MHz) and ¹³ C(150MHz) NMR data

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (8)

1. the compound in a kind of Reishi sporule, it is characterised in that：The chemical name of the compound is 3 β, 5 α, 6 β, 11- tetra- Hydroxyl-(22E, 24R) -9,11- splits ergot steroid -7,22- diene -9- ketone, and chemical structural formula is

。

2. a kind of extraction separation method of the compound in Reishi sporule as claimed in claim 1, it is characterised in that including with Lower step：

（1）Lucidum spore powder is taken, is loaded into supercritical extraction reactor, is extracted through extraction kettle and passes through separating still I, separating still II The second-order separation, collects ganoderma lucidum spore oil in separation reactor I I；

（2）Ganoderma lucidum spore oil with the ethanol of volume fraction 75 ~ 85% extract three times, alcohol extraction liquid petroleum ether extraction it is secondary enter one Degreasing is walked, the dilute alcohol that is concentrated under reduced pressure liquid obtains ethanol extract；

（3）Silicagel column on ethanol extract, with petroleum ether-acetone with mass ratio 100:0、98:2、95:5、9:1、8:2、7:3、0: 100 gradient elutions；

（4）Merge petroleum ether-acetone quality and compare 9:1~7:3 elution fractions obtain component sample, reversed-phase column on the component sample, use first Alcohol-formic acid is with mass ratio 7:3、8:2、9:1、100:0 gradient elution；

（5）Methyl alcohol-formic acid mass ratio 100:0 elution fraction obtains two components A, B；

（6）Two components enter HPLC respectively, with the acetonitrile isocratic elution of volume fraction 65 ~ 75%, collect t_R=41-44 min peak, With recrystallizing methanol, one of component obtains compound.

3. the compound extraction separation method in a kind of Reishi sporule as claimed in claim 2, it is characterised in that：The step （1）In, extraction kettle pressure is that 25 ~ 35MPa, temperature are 55 ~ 65 DEG C；Separation reactor I pressure is that 11 ~ 15MPa, temperature are 50 ~ 60 DEG C； Separation reactor I I pressure is that 5 ~ 7MPa, temperature are 40 ~ 50 DEG C；Entrainer is 95% ethanol；Extraction time is 60 ~ 120 minutes.

4. the compound extraction separation method in a kind of Reishi sporule as claimed in claim 2, it is characterised in that：The step （2）In, ganoderma lucidum spore oil is extracted with the ethanol of volume fraction 80%.

5. the compound extraction separation method in a kind of Reishi sporule as claimed in claim 2, it is characterised in that：The step （3）In, the silica gel in silicagel column is 100 ~ 200 mesh.

6. the compound extraction separation method in a kind of Reishi sporule as claimed in claim 2, it is characterised in that：The step （4）In, the filler of reversed-phase column is ODS.

7. the compound extraction separation method in a kind of Reishi sporule as claimed in claim 2, it is characterised in that：The step （5）In, formic acid volume fraction is 1%.

8. the compound extraction separation method in a kind of Reishi sporule as claimed in claim 2, it is characterised in that：The step （6）In, acetonitrile volume fraction is 70%.