CN102295545B - Method for biosynthesizing abietane diterpenoid compounds by using cephalotaxus fortune culture cells - Google Patents

Abstract

The invention relates to a plant cell culture technology, in particular to a method for biosynthesizing abietane diterpenoids by using liquid suspension culture cells of cloverleaf. Cultivate the cloverleaf cells in their common culture medium, the cell inoculation amount is 50-150g/L wet weight, in the early stage of cell exponential growth, add the inducer and 50-200g of adsorbent, the inducer is an abiotic inducer or a fungal inducer. The invention is a biosynthesis method, which is stable and environment-friendly; the product is completely adsorbed on the adsorbent, greatly simplifies the operation process of separation and purification, not only reduces the cost, but also facilitates the realization of industrialized production. At the same time, the present invention has synthesized 7 abietane diterpenoids including a new compound. Abietane diterpenoids have good biological activity and are an important class of lead compounds. This technology has important commercial application prospects.

Description

Method for biosynthesizing abietane diterpenoids by culturing cells from cloverleaf

technical field

The invention relates to a plant cell culture technique, in particular to a method for inducing a liquid suspension culture cell of the cloverleaf to biosynthesize an abietane diterpenoid compound, and a new compound is included in the product at the same time.

Background technique

Abietin diterpenoids belong to tricyclic diterpenoids and are an important class of natural compounds. The main reason why this kind of compounds attract people's research interest is that many substances in them have good activity, and many folk medicines contain such substances. For example, hinokiol (compound 2) in the present invention has inhibitory effects on Staphylococcus aureus, Streptococcus, Escherichia coli, Pseudomonas aeruginosa, etc., and is a good antibacterial lead compound. However, these compounds are mainly extracted from plants at present, and there are few reports on their chemical synthesis, and they are still in the stage of laboratory research. Because the content of these compounds in plants is very low, it is difficult to obtain them, which greatly limits their further research and application.

The use of plant cell culture technology to produce biologically active substances has attracted increasing attention due to its environmental friendliness, short production cycle, and strong controllability, and has become a potential source of natural active substances. At present, the main problem that people encounter is how to regulate the metabolic pathways of plants to obtain the expected products, and to control biosynthesis like chemical synthesis.

The current common regulation method is mainly to promote the production of secondary metabolites that already exist in the cell, that is, to increase its yield.

Contents of the invention

The object of the present invention is to provide a kind of utilization Cephalotaxus fortunei (Cephalotaxus fortunei, Hook, f.) liquid suspension culture cell, with the combined control technology biosynthesis of 7 kinds of abietane diterpenoids combined with inducer induction and adsorbent adsorption , including a new method for abietane diterpenoids.

To achieve the above object, the technical solution adopted in the present invention is:

Cultivate the cloverleaf cells in its common medium, the cell inoculation amount is 50-150g/L wet weight, in the early stage of cell exponential growth, add the inducer with a concentration of 10-2000μM and 50 ～200g of adsorbent, the inducer is non-biological inducer or fungal inducer; after continuing to cultivate for 7-14 days, harvest the cells and adsorbent respectively;

First extraction: at room temperature, extract the adsorbent after vacuum filtration with polar or medium polar organic solvent for 2 to 5 times; combine the extracts, filter, and spin evaporate to dryness;

The second extraction: adding low-concentration lye and equal volume of non-polar organic solvent to the evaporated crude extract, the mass-volume ratio of crude extract to low-concentration lye is 1g: 3-15ml;

Separation and purification: After extraction for 8-16 hours, the organic layer is separated, the organic layer is rotatably evaporated to dryness, and then placed on a silica gel column for separation. The silica gel used is 200-300 mesh silica gel, and the elution method is gradient elution. The volume of the eluent used in the gradient is 200-800ml, and the eluent is collected continuously with a sample bottle, and each sample bottle collects 5-15ml of the eluent, and the eluent collected in the sample bottle is detected by thin-layer chromatography. When the eluent used was cyclohexane-ethyl acetate (8:1, v/v), the crude products of compounds 1 and 4 were obtained respectively; when cyclohexane-ethyl acetate (5:1, v/v) , the crude products of compounds 3 and 6 were obtained respectively; when cyclohexane-ethyl acetate (2:1, v/v) was used, the crude products of compounds 2, 5 and 7 were obtained respectively. The crude products of these 7 compounds were respectively placed on gel columns to complete further purification, and the pure products of 7 compounds were obtained. The seven compounds are:

Compound 1: 3-hydroxy-2-isopropyl-6-methyl-5-(4-methyl-3-oxopentyl)naphthalene

Compound 2: abieta-8, 11, 13-triene-3β, 12-diol

Compound 3: 12-hydroxyabieta-6, 8, 11, 13-tetraene-3-one

Compound 4: abietatriene-3β-ol

Compound 5: 11,12-dihydroxy-8,11,13-abietatriene-3,7-dione

Compound 6: 11-hydroxy-12-methoxyabieta-8, 11, 13-triene-3, 7-dione

Compound 7: 3β, 11-dihydroxy-12-methoxyabieta-8, 11, 13-triene-7-one

The commonly used medium composition for cultivating cells of A. cloverleaf is as follows: 0.1-2.5 mg/L 2,4-dichlorophenoxyacetic acid, 0.1-1 mg/L kinetin and 10-50 g /L sucrose, the pH value is 5.4～6.2;

The initial stage of cell exponential growth is the 4th to 10th day after the cells are inoculated in the culture medium.

The culturing conditions of the cloverleaf cells are as follows: rotary shaker, rotating speed 80-150 rpm, culturing temperature 20-30° C., culture in dark; culture period is 14-25 days, subcultured once every 14-21 days.

The non-biological inducer is silver nitrate, salicylic acid, jasmonic acid or methyl jasmonate; the adsorbent is a macroporous adsorption resin with strong adsorption capacity for terpenoids and non-toxic to cells, including XAD-7HP, XAD -4, XAD-1600 or HP2MG.

The polar or medium polar organic solvents used for the first extraction include methanol, ethanol, acetone or ethyl acetate; during each extraction process, the mass volume ratio of the adsorbent to the organic solvent is 1g: 3-15ml, each extraction First use vibration extraction for 30 minutes to 3 hours, and then ultrasonic extraction for 10 to 60 minutes.

Used low-concentration lye when extracting for the second time refers to the potassium hydroxide that volume concentration is 0.1～1% ammoniacal liquor, mass concentration is 0.05～0.5% sodium hydroxide or mass concentration is 0.05～0.5%; The non-polar solvent used during the first extraction refers to chloroform or dichloromethane.

The composition of the eluent of the silica gel column is cyclohexane, cyclohexane-ethyl acetate (60～10:1, v/v), cyclohexane-ethyl acetate (8:1, v/v), cyclohexane Hexane-ethyl acetate (5:1, v/v), cyclohexane-ethyl acetate (3:1, v/v), cyclohexane-ethyl acetate (2:1, v/v), Cyclohexane-ethyl acetate (1:1, v/v), ethyl acetate.

The filler of the gel column is Sephadex ^™ LH-20; the eluent used is methanol or methanol-dichloromethane with a volume ratio of 1:1.

Method principle of the present invention:

The addition of inducers and adsorbents can induce the liquid suspension culture cells of T. chinensis to synthesize abietane diterpenoids. The principle mainly has the following three aspects: 1. The inducer can act as a signal molecule to activate a specific metabolic pathway and trigger the synthesis reaction 2. The adsorbent can adsorb the product, increase the storage sites of the product, eliminate feedback inhibition, and prevent the degradation of the product at the same time. 3. The two must be used in combination, probably because the product has a toxic effect on the cells, so that if only the inducer is used without adding the adsorbent, although the reaction can be induced, the product will inhibit the cells as soon as it accumulates and is degraded by the cells. Can't get product.

The present invention has the following advantages:

1. Combined application of inducer induction and adsorbent adsorption to induce the synthesis of abietane diterpenoids in the liquid suspension culture cells of Cinnamon lanceolata, and the artificial controllable production of such compounds has not been reported. At present, these compounds mainly come from Extraction of plants.

2. A new abietane diterpene compound has been obtained in the present invention.

3. The product in the present invention is completely absorbed in the adsorbent, which is easy to separate and purify, reduces the cost, and provides a great possibility for further industrial production.

4. The cell culture conditions in the present invention can ensure the rapid growth of cells and ensure good uniformity.

5. In the present invention, low-concentration lye is used to remove impurities, which greatly improves the purification efficiency.

The natural products contained in Cedarwood recorded in the literature are mainly alkaloids, but abietane diterpenoids have not been reported yet. The seven compounds of the present invention were not detected by HPLC analysis on the leaves, stems and uninduced liquid suspension culture cells of C. chinensis.

There are 9 species in the genus Tricus, at present, only the perabietic diterpenoids have been isolated from Torreya japonica, but no report has been found in the other 8 species.

The present invention is characterized in that the product of the present invention is not detected in the liquid suspension culture cells of C. chinensis before induction, and 7 kinds of abietane diterpenoids are synthesized in the cultured cells of C. cloverleaf after induction, including a new abietane diterpenoids. It shows that the present invention is an effective method for biosynthesizing abietane diterpenoids by utilizing the liquid suspension culture cells of the cloverleaf.

Detailed ways

Example 1

(1) Cultivation of the cloverleaf cells:

Cells were cultured in suspension in liquid suspension:

The cell strain in the present invention is initially obtained from the suspension culture of callus induced by young stems of C.fortunei (C. fortunei), using Murashige-Skoog (MS) medium, and the added concentration is 2mg/L 2,4-dichloro Phenoxyacetic acid, 0.1mg/L kinetin and 30g/L sucrose, the pH value of the medium is 5.8. 7.5 grams of wet cells obtained by filtration were inoculated into a 500 ml shake flask containing 100 ml of the above medium, and then cultured on a rotary shaker at 100 rpm at 25° C. in the dark.

(2) Combined induced synthesis of abietane diterpenes:

On the 7th day of cell growth, methyl jasmonate (MJA) (final concentration in the medium was 300 μM/L) and XAD-7HP resin (100 g/L) were added to the medium, and the culture was continued until the 14th day, respectively. The cells and XAD-7HP were harvested. It was determined that the product was completely absorbed in XAD-7HP, and no product was detected in the cells.

(3) Extraction, purification and structural identification of abietane diterpenoids:

At room temperature, 300 g of the vacuum-filtered adsorbent was first extracted with methanol three times. The volume of methanol used for each extraction was 0.9 L. Each extraction included vibration extraction for 1 hour, and then ultrasonic extraction for 30 minutes. The three methanol extracts were then combined, filtered, and spun to dryness. Add 2 L of 0.5% (v/v) ammonia water and an equal volume of dichloromethane to the evaporated methanol extract. After 12 hours of extraction, the dichloromethane layer was separated and evaporated to dryness, and then placed on a silica gel column for separation. The silica gel used was 200-300 mesh silica gel, and the elution method was gradient elution. The composition of the eluent of the silica gel column is cyclohexane (300ml), cyclohexane-ethyl acetate (50:1, v/v, 500ml), cyclohexane-ethyl acetate (30:1, v/v , 300ml), cyclohexane-ethyl acetate (20:1, v/v, 300ml), cyclohexane-ethyl acetate (10:1, v/v, 500ml), cyclohexane-ethyl acetate ( 8:1, v/v, 400ml), cyclohexane-ethyl acetate (5:1, v/v, 400ml), cyclohexane-ethyl acetate (3:1, v/v, 400ml), cyclohexane Hexane-ethyl acetate (2:1, v/v, 600ml), cyclohexane-ethyl acetate (1:1, v/v, 300ml), ethyl acetate (300ml). Collect the eluent continuously with a sample bottle, each sample bottle collects 10ml of the eluent, and utilize thin layer chromatography to detect simultaneously, when the eluent used is cyclohexane-ethyl acetate (8:1, v/v) , the crude products of compounds 1 and 4 were obtained respectively; when cyclohexane-ethyl acetate (5:1, v/v), the crude products of compounds 3 and 6 were obtained respectively; cyclohexane-ethyl acetate (2:1, v/v), the crude products of compounds 2, 5 and 7 were obtained respectively. The crude products of these 7 compounds were respectively placed on gel columns filled with LH-20 to complete further purification, and the eluent used was methanol or methanol-dichloromethane with a volume ratio of 1:1 to obtain the pure products, wherein compound 1 (3.8mg), compound 2 (10.4mg), compound 3 (14.2mg), compound 4 (6.5mg), compound 5 (200mg), compound 6 (4mg), compound 7 (12.8mg), After spectral analysis, compound 1 is a new compound, and the structures of the seven compounds are determined as follows:

Compound 1

3-hydroxy-2-isopropyl-6-methyl-5-(4-methyl-3-oxopentyl)naphthalene

Compound 2

abieta-8, 11, 13-triene-3β, 12-diol

Compound 3

12-hydroxyabieta-6, 8, 11, 13-tetraene-3-one

Compound 4

abietatriene-3β-ol

Compound 5

11, 12-dihydroxy-8, 11, 13-abietatriene-3, 7-dione

Compound 6

11-hydroxy-12-methoxyabieta-8, 11, 13-triene-3, 7-dione

Compound 7

3β, 11-dihydroxy-12-methoxyabieta-8, 11, 13-triene-7-one

Compound 1

3-hydroxy-2-isopropyl-6-methyl-5-(4-methyl-3-oxopentyl)naphthalene Pale yellow oily solid (acetone). ¹ H NMR (CD ₃ COCD ₃ , 500MHz) δ1.08 (6H, d, J=6.9Hz, H-18 and H-19), δ1.32 (6H, d, J=6.85Hz, H-16 and H-17), δ2.42 (3H, s, H-20), δ2.67 (1H, sept, J=6.9Hz, H-4), δ2.73 (2H, t like, H-1a and H -1b), δ3.16 (2H, t like, H-2a and H-2b), δ3.41 (1H, sept, J=6.85Hz, H-15), δ7.07 (1H, d, J= 8.3Hz, H-6), δ7.29(1H, s, H-11), δ7.52(1H, d, J=8.3Hz, H-7), δ7.61(1H, s, H-14 ), δ8.59(1H, s, 12-OH). ¹³ C NMR (CD ₃ COCD ₃ , 125.7MHz) δ18.53(CH ₃ , C-18), δ18.53(CH ₃ , C-19) , δ20.04 (CH ₃ , C-20), δ22.95 (CH ₃ , C-16), δ22.95 (CH ₃ , C-17), δ23.61 (CH ₂ , C-2), δ28 .18 (CH, C-15), δ40.37 (CH ₂ , C-1), δ41.34 (CH, C-4), δ105.81 (CH, C-11), δ126.49 (CH, C-14), δ126.69 (CH, C-7), δ126.93 (CH, C-6), δ129.00 (C, C-8), δ132.76 (C, C-10), δ132 .96 (C, C-5), δ132.96 (C, C-9), δ136.89 (C, C-13), δ154.93 (C, C-12), δ213.60 (C, C -3).EIMS 70eV, m/z(rel.int.): 298[M] ⁺ (39), 280[MH ₂ O] ⁺ (69), 261(14), 243(5), 237(100 ), 213(93), 197(18), 195(15), 185(12), 179(10), 165(7), 152(4). HR-EIMS m/z 298.1939[M] ⁺ , calc .for[C ₂₀ H ₂₆ O ₂ ] ⁺ , 298.1933.

Compound 2

abieta-8, 11, 13-triene-3β, 12-diol

Colorless crystals (acetone). ¹ HNMR (CD ₃ COCD ₃ , 500MHz) δ0.87 (3H, s, H-19), δ1.06 (3H, s, H-18), δ1.15 (3H, s, H-20), δ1 .18 (6H, t, J=6.8Hz, H-16 and H-17), δ1.24 (1H, dd, J=12.3, 2.2Hz, H-5), δ1.44 (1H, td, J =12.85, 4.9Hz, H-1a), δ1.71 (1H, m, H-6a), δ1.74 (2H, m, H-2a and H-2b), δ1.86 (1H, m, H -6b), δ2.17(1H, dt, J=13.05, 3.5Hz, H-1b), δ2.72(1H, m, H-7a), δ2.82(1H, m, H-7b), δ3.21(1H, sept, J=6.8Hz, H-15), δ3.24(1H, m, H-3), δ3.45(1H, d, J=5.3Hz, 3-OH), δ6 .7(1H, s, H-11), δ6.78(1H, s, H-14), δ7.67(1H, s, 12-OH). ¹³ CNMR(CD ₃ COCD ₃ , 125.7MHz) δ16 .06 (CH ₃ , C-19), δ19.93 (CH ₂ , C-6), δ22.93 (CH ₃ , C-16), δ23.04 (CH ₃ , C-17), δ25.24 (CH ₃ , C-20), δ27.48 (CH, C-15), δ28.72 (CH ₃ , C-18), δ28.98 (CH ₂ , C-2), δ30.84 (CH ₂ , C-7), δ38.02 (CH ₂ , C-1), δ38.08 (C, C-10), δ39.75 (C, C-4), δ51.09 (CH, C-5) , δ78.51 (CH, C-3), δ111.56 (CH, C-11), δ126.34 (C, C-8), δ127.09 (CH, C-14), δ132.83 (C , C-13), δ148.51 (C, C-9), δ153.19 (C, C-12).EIMS 70e V, m/z (rel.int.): 302[M] ⁺ (48) , 284[MH ₂ O] ⁺ (57), 269[MH ₂ O-CH ₃ ] ⁺ (100), 227(30), 215(13), 199(19), 187(13), 175(11) , 157(8), 145(6), 128(3). HR-EIMS m/z 302.2248[M] ⁺ , calc.for[C ₂₀ H ₃₀ O ₂ ] ⁺ , 302.2246.

Compound 3

12-hydroxyabieta-6, 8, 11, 13-tetraene-3-one

Pale yellow powdery solid (acetone). ¹ H NMR (CD ₃ COCD ₃ , 500MHz) δ1.11 (3H, s, H-18), δ1.19 (3H, d, J=6.95, H-16), δ1.21 (3H, s, H -20), δ1.22(3H, d, J=6.95, H-17), δ1.25(3H, s, H-19), δ2.01(1H, td, J=13.95, 5.3Hz, H -1a), δ2.38(1H, m, H-2a), δ2.42(1H, m, H-1b), δ2.49(1H, t, J=2.9Hz, H-5), δ2. 90 (1H, m, H-2b), δ3.26 (1H, sept, J=6.95Hz, H-15), δ5.81 (1H, dd, J=9.6, 2.65Hz, H-6), δ6 .59 (1H, dd, J=9.6, 3.1Hz, H-7), δ6.74 (1H, s, H-11), δ6.94 (1H, s, H-14), δ8.11 (1H , s, 12-OH). ¹³ C NMR (CD ₃ COCD ₃ , 125.7MHz), δ20.30 (CH ₃ , C-20), δ22.76 (CH ₃ , C-17), δ22.84 (CH ₃ , C-16), δ23.01 (CH ₃ , C-19), δ25.39 (CH ₃ , C-18), δ27.38 (CH, C-15), δ35.06 (CH ₂ , C -2), δ35.88 (CH ₂ , C-1), δ38.10 (C, C-10), δ47.64 (C, C-4), δ52.54 (CH, C-5), δ110 .27 (CH, C-11), δ125.30 (CH, C-6), δ125.30 (C, C-8), δ125.75 (CH, C-14), δ129.76 (CH, C -7), δ132.94(C, C-13), δ145.86(C, C-9), δ155.25(C, C-12), δ214.31(C, C-3).EIMS 70eV , m/z(rel.int.): 298[M] ⁺ (28), 283[M-CH ₃ ] ⁺ (4), 243(10), 227(5), 213(100), 199(11 ), 185(69), 157(5), 141(2), 128(2). HR-EIMS m/z 298.1943[M] ⁺ , calc.for[C ₂₀ H ₂₆ O ₂ ] ⁺ , 298.1933.

Compound 4

abietatriene-3β-ol

Colorless crystals (acetone). ¹ H NMR (CD ₃ COCD ₃ , 500MHz) δ0.89 (3H, s, H-19), δ1.07 (3H, s, H-18), δ1.17 (3H, s, H-20), δ1.18 (3H, s, H-16), δ1.20 (3H, s, H-17), δ1.27 (1H, dd, J=12.35, 2.25Hz, H-5), δ1.45 ( 1H, td, J=12.9, 4.7Hz, H-1a), δ1.74 (1H, m, H-6a), δ1.76 (2H, m, H-2a and H-2b), δ1.90 ( 1H, m, H-6b), δ2.30 (1H, dt, J=13.05, 3.5Hz, H-1b), δ2.80 (1H, m, H-15), δ2.81 (1H, m, H-7a), δ2.90 (1H, m, H-7b), δ3.24 (1H, m, H-3), δ3.43 (1H, d, J=5.25Hz, 3-OH), δ6 .88(1H, s, H-14), δ6.96(1H, d, J=8.15Hz, H-12), δ7.16(1H, d, J=8.15Hz, H-11). ¹³ C (CD ₃ COCD ₃ , 125.7MHz) δ16.06 (CH ₃ , C-19), δ19.72 (CH ₂ , C-6), δ24.36 (CH ₃ , C-16 and C-17), δ25 .31 (CH ₃ , C-20), δ28.73 (CH ₃ , C-18), δ28.99 (CH ₂ , C-2), δ31.52 (CH ₂ , C-7), δ34.29 (CH, C-15), δ37.97 (CH ₂ , C-1), δ38.15 (C, C-10), δ39.79 (C, C-4), δ51.12 (CH, C- 5), δ78.50 (CH, C-3), δ124.62 (CH, C-12), δ125.23 (CH, C-11), δ127.45 (CH, C-14), δ135.49 (C, C-8), δ146.27 (C, C-13), δ148.03 (C, C-9). EIMS 70eV, m/z (rel.int.): 286[M] ⁺ (20 ), 271[M-CH ₃ ] ⁺ (52), 253[M-CH ₃ -H ₂ O] ⁺ (100), 227(11), 211(21), 199(12), 185(20), 173(16), 159(19), 143(11), 129(9), 117(5), 91(2). HR-EIMS m/z 286.2307[M] ⁺ , calc.for[C ₂₀ H ₃₀ O]+, 286.2297.

Compound 5

11, 12-dihydroxy-8, 11, 13-abietatriene-3, 7-dione

Colorless crystals (acetone). ¹ H NMR (CD ₃ COCD ₃ , 500MHz) δ1.16 (6H, s, H-18 and H-19), δ1.21 (3H, d, J=6.9Hz, H-16), δ1.23 ( 3H, d, J=6.9Hz, H-17), δ1.49 (3H, s, H-20), δ2.00 (1H, dt, J=13.9, 8.4Hz, H-1a), δ2.44 (1H, m, H-6a), δ2.48 (1H, m, H-5), δ2.60 (2H, m, H-2a and H-2b), δ2.66 (1H, m, H- 6b), δ3.31(1H, sept, J=6.9Hz, H-15), δ3.40(1H, m, H-1b), δ7.55(1H, s, H-14). ¹³ C( CD ₃ COCD ₃ , 125.7MHz) δ18.00 (CH ₃ , C-20), δ21.07 (CH ₃ , C-19), δ22.95 (CH ₃ , C-17), δ23.06 (CH ₃ , C-16), δ27.21 (CH ₃ , C-18), δ27.40 (CH, C-15), δ34.92 (CH ₂ , C-2), δ36.39 (CH ₂ , C- 1), δ36.52 (CH ₂ , C-6), δ39.65 (C, C-10), δ47.61 (C, C-4), δ50.40 (CH, C-5), δ117. 40 (CH, C-14), δ125.58 (C, C-8), δ134.38 (C, C-13), δ137.71 (C, C-9), δ143.96 (C, C- 11), δ148.52 (C, C-12), δ196.91 (C, C-7), δ215.33 (C, C-3). EIMS 70eV, m/z (rel.int.): 330 [M] ⁺ (100), 315[M- _CH3 ] ⁺ (40), 297[M- _CH3 - _H2O ] ⁺ (12), 287(16), 273(36), 259(10) , 245(16), 231(26), 219(15), 205(12), 191(6), 177(6), 161(4), 145(3), 125(6), 115(3) .HR-EIMS m/z 330.1836[M] ⁺ , calc.for[C ₂₀ H ₂₆ O ₄ ] ⁺ , 330.1831.

Compound 6

11-hydroxy-12-methoxyabieta-8,11,13-triene-3,7-dione Colorless needle-like crystals (chloroform). ¹ H NMR (CDCl ₃ , 500MHz) δ1.17 (3H, s, H-18), δ1.18 (3H, s, H-19), δ1.25 (3H, d, J=6.9Hz, H- 16), δ1.27(3H, d, J=6.9Hz, H-17), δ1.47(3H, s, H-20), δ2.01(1H, dt, J=14, 8.4Hz, H -1a), δ2.46 (1H, dd, J=14.3, 3.2Hz, H-5), δ2.60 (1H, m, H-6a), δ2.64 (2H, m, H-2a and H -2b), δ2.64(1H, m, H-6b), δ3.22(1H, sept, J=6.9Hz, H-15), δ3.33(1H, m, H-1b), δ3. 83 (3H, s, 12-OCH ₃ ), δ6.14 (1H, s, 11-OH), δ7.63 (1H, s, H-14). ¹³ C (CDCl ₃ , 125.7MHz) δ17.68 (CH ₃ , C-20), δ20.80 (CH ₃ , C-19), δ23.46 (CH ₃ , C-16), δ23.54 (CH ₃ , C-17), δ26.79 (CH , C-15), δ26.99 (CH ₃ , C-18), 34.43 (CH ₂ , C-2), δ35.38 (CH ₂ , C-1), δ36.15 (CH ₂ , C-6 ), δ38.89 (C, C-10), δ47.13 (C, C-4), δ49.60 (CH, C-5), δ61.95 (CH ₃ , 12-OCH ₃ ), δ117. 42 (CH, C-14), δ128.32 (C, C-8), δ135.63 (C, C-9), δ139.94 (C, C-13), δ146.70 (C, C- 11), δ149.41 (C, C-12), δ197.52 (C, C-7), δ215.89 (C, C-3). EIMS 70eV, m/z (rel.int.): 344 [M] ⁺ (100), 329[M- _CH3 ] ⁺ (53), 311[M- _CH3 - _H2O ] ⁺ (14), 301(16), 287(46), 273(11) , 259(16), 245(34), 233(20), 215(8), 203(8), 189(5), 173(5), 149(8), 125(7), 97(3) , 83(2).HR-EIMS m/z 344.1988[M] ⁺ , calc.for[C ₂₁ H ₂₈ O ₄ ] ⁺ , 344.1988.

Compound 7

3β, 11-dihydroxy-12-methoxyabieta-8, 11, 13-triene-7-one

Pale yellow needle-like crystals (acetone). ¹ H NMR (CD ₃ COCD ₃ , 500MHz) δ0.95 (3H, s, H-19), δ1.07 (3H, s, H-18), δ1.21 (3H, d, J=6.9Hz, H-16), δ1.23 (3H, d, J=6.9Hz, H-17), δ1.42 (3H, s, H-20), δ1.48 (1H, td, J=13.75, 3.6Hz , H-1a), δ1.75 (2H, m, H-2a and H-2b), δ1.78 (1H, m, H-5), δ2.58 (2H, m, H-6a and H- 6b), δ3.26(1H, sept, J=6.9Hz, H-15), δ3.30(1H, m, H-3), δ3.40(1H, dt, J=13.8, 3.55Hz, H -1b), δ3.52 (1H, d, J=5.25Hz, 3-OH), δ3.78 (3H, s, 12-OCH ₃ ), δ7.52 (1H, s, H-14), δ7 .86 (1H, s, 11-OH). ¹³ C (CD ₃ COCD ₃ , 125.7MHz) δ15.97 (CH ₃ , C-19), δ17.97 (CH ₃ , C-20), δ23.75 (CH ₃ , C-16), δ23.88 (CH ₃ , C-17), δ27.32 (CH, C-15), 28.48 (CH ₃ , C-18), δ28.76 (CH ₂ , C -2), δ35.63 (CH ₂ , C-1), δ35.89 (CH ₂ , C-6), δ39.92 (C, C-4), δ40.91 (C, C-10), δ50.87 (CH, C-5), δ61.96 (CH ₃ , 12-OCH ₃ ), δ77.81 (CH, C-3), δ116.98 (CH, C-14), δ129.45 ( C, C-8), δ138.95 (C, C-9), δ140.21 (C, C-13), δ148.43 (C, C-11), δ150.72 (C, C-12) , δ198.02 (C, C-7). EIMS 70eV, m/z (rel.int.): 346[M] ⁺ (53), 331[M-CH ₃ ] ⁺ (18), 313[M- CH ₃ -H ₂ O] ⁺ (100), 287(11), 271(17), 259(10), 245(64), 231(11), 219(7), 203(7), 193(5 ), 157(3), 129(3), 115(2).HR-EIMS m/z346.2153[M] ⁺ , calc.for[C ₂₁ H ₃₀ O ₄ ] ⁺ , 346.2144.

Comparative example 1

The culture conditions of the Herringbone cells were the same as those in Example 1, except that MJA and XAD-7HP were not added during the combined induction synthesis process, and the culture was continued directly. After HPLC analysis, the above 7 products were not detected. It shows that these 7 kinds of products are synthesized by induction.

Comparative example 2

The culture condition of the Herringbone cell is the same as that of Example 1, and the difference from Example 1 is that only MJA or XAD-7HP is added during the combined induction synthesis process, and the concentration is the same as that of Example 1. After HPLC analysis, if only MJA was added, the above 7 products were not detected in the cell extract. If only XAD-7HP was added, these 7 compounds were not detected in both cell extracts and XAD-7HP extracts. It shows that these 7 kinds of products can only be synthesized when the inducer and adsorbent are used at the same time.

Comparative example 3

The culture condition and induction condition of the cloverleaf cells are the same as those in Example 1, and the difference from Example 1 is that ammonia water is not used in the extraction process.

Through the HPLC analysis of dichloromethane extract, it is found that when ammonia water is used in the extraction, the impurity content is much lower than that when ammonia water is not used. Pure product, productive rate also reduces greatly. It shows that ammonia water is a key step in removing impurities.

In the prior art, the purpose of using ammonia water in the extraction of alkaloids is to dissociate the alkaloids from their salts, and then dissolve the dissociated alkaloids in the organic layer. And use ammoniacal liquor among the present invention is in order to remove impurity such as pigment.

Comparative example 4

The culture conditions, induction conditions and extraction conditions of the cloverleaf cells are the same as those in Example 1. The difference from Example 1 is that the eluent of the silica gel column is successively chloroform-methanol (5:1, v/v), chloroform-methanol (3:1, v/v), chloroform-methanol (2:1 , v/v), chloroform-methanol (1:1, v/v), the products were eluted together and could not be separated.

It shows that the polarity of the silica gel column eluent selected in the present invention meets the needs of product separation and purification, and these 7 compounds can be well separated after passing through the silica gel column.

Example 2

The culture conditions and separation and purification conditions are the same as in Example 1, except that the difference from Example 1 is that MJA (final concentration in the medium is 150 μM/L) and XAD-7HP (100 g /L), continue to cultivate to 14 days, the results are as follows: compound 1 (5.6mg), compound 2 (15.5mg), compound 3 (17.4mg), compound 4 (5.5mg), compound 5 (220mg), compound 6 ( 5mg), Compound 7 (13mg)

Example 3

The culture conditions and separation and purification conditions are the same as in Example 1, except that the difference from Example 1 is that jasmonic acid (final concentration in the medium is 150 μM/L) and XAD-7HP (100 g /L), continue to cultivate to 14 days, the results are as follows: compound 1 (7.5mg), compound 2 (16mg), compound 3 (20mg), compound 4 (3mg), compound 5 (190mg), compound 6 (6mg), Compound 7 (11.7 mg)

Example 4

The culture conditions and separation and purification conditions are the same as in Example 1, except that the difference from Example 1 is that MJA (final concentration in the medium is 150 μM/L) and HP are added to the medium on the 7th day of cell growth when the synthesis is induced -2MG (100g/L), continue to culture for 14 days, the results are as follows: Compound 1 (10mg), Compound 2 (18mg), Compound 3 (17mg), Compound 4 (8.3mg), Compound 5 (260mg), Compound 6 (7mg), Compound 7 (15.5mg)

Claims (7)

1. utilize the method for Cultured Cells of Cephalotaxus Fortunei biosynthesizing abietane diterpene-kind compound, it is characterized in that: cephalotaxus cell is placed in to its substratum commonly used and cultivates, the cell inoculum size is 50～150g/L weight in wet base, at the cell index early growth period, add inductor that final concentration is 10～2000 μ M and the sorbent material of 50～200g in every liter of substratum, described inductor is abiotic inductor or fungal elicitor simultaneously; Continue to cultivate after 7～14 days, respectively harvested cell and sorbent material;

Extract for the first time: under room temperature, by the sorbent material after vacuum filtration first with polarity or middle polarity organic solvent extraction 2～5 times; United extraction liquid, filter, the rotation evaporate to dryness;

Extract for the second time: add alkali lye and the extraction of isopyknic non-polar organic solvent in the crude extract of evaporate to dryness, crude extract and alkali lye mass volume ratio example 1g: 3～15ml;

Separation and purification: after extracting 8～16 hours, organic layer is separated, organic layer is rotated to evaporate to dryness, then be placed on silicagel column and separate, used silica gel is 200～300 order silica gel, type of elution is gradient elution, the volume of each gradient elutriant used is 200-800ml, collect continuously elutriant with sample bottle, each sample bottle is collected the 5-15ml elutriant, the elutriant that simultaneously utilizes thin-layer chromatography to collect sample bottle is detected, and when elutriant used is hexanaphthene-ethyl acetate v/v while being 8: 1, obtains respectively the crude product of compound 1 and 4; Hexanaphthene-ethyl acetate v/v is 5: 1 o'clock, obtains respectively the crude product of compound 3 and 6; Hexanaphthene-ethyl acetate v/v is 2: 1 o'clock, obtains respectively the crude product of compound 2,5 and 7; The crude product of these 7 compounds is placed in respectively on gel column and completes further purifying, obtain the sterling of 7 compounds; These 7 compounds are respectively:

Compound 1:3-hydroxyl-2-sec.-propyl-6-methyl-5-(4-methyl-3-oxygen amyl group) naphthalene

Compound 2: rosin-8,11,13-triolefin-3 β, 12-glycol

Compound 3:12-rosin hydroxyl-6,8,11,13-tetraene-3-ketone

Compound 4: rosin trialkenyl-3 β-ol

Compound 5:11,12-dihydroxyl-8,11,13-rosin triolefin-3,7-diketone

Compound 6:11-hydroxyl-12-rosin methoxyl group-8,11,13-triolefin-3,7-diketone

Compound 7:3 β, 11-dihydroxyl-12-rosin methoxyl group-8,11,13-triolefin-7-ketone

Described abiotic inductor is Silver Nitrate, Whitfield's ointment, jasmonic or methyl jasmonate;

Described sorbent material for terpenoid is had to stronger adsorptive power and to cell nontoxic macroporous adsorbent resin, macroporous adsorbent resin is XAD-7HP, XAD-4, XAD-1600 or HP2MG;

The substratum commonly used of described Cultured Cells of Cephalotaxus Fortunei consists of, and adding final concentration in the MS basic medium is 0.1～2.5mg/L2, the 4-dichlorophenoxyacetic acid, and 0.1～1mg/L kinetin and 10～50g/L sucrose, the pH value is 5.4～6.2;

Described while extracting for the second time alkali lye used refer to that volumetric concentration is 0.The potassium hydroxide that the sodium hydroxide that 1～1% ammoniacal liquor, mass concentration are 0.05～0.5% or mass concentration are 0.05～0.5%.

2. according to the described method of claim 1, it is characterized in that:

Described cell index early growth period is 4th～10 days after cell is inoculated in substratum.

3. according to the described method of claim 1, it is characterized in that: the culture condition of described cephalotaxus cell is: rotary shaking table, and rotating speed 80～150rpm, culture temperature is 20～30 ℃, the dark cultivation; Culture cycle is 14～25 days, every 14～21 days, goes down to posterity once.

4. according to the described method of claim 1, it is characterized in that: described polarity used while extracting for the first time or the organic solvent of middle polarity are methyl alcohol, ethanol, acetone or ethyl acetate;

In each leaching process, sorbent material and organic solvent mass volume ratio example 1g: 3-15ml, each extraction first adopts mechanical shaking extraction 30 minutes～3 hours, and then supersound extraction is 10～60 minutes.

5. according to the described method of claim 1, it is characterized in that:

Described while extracting for the second time non-polar solvent used refer to chloroform or methylene dichloride.

6. according to the described method of claim 1, it is characterized in that: the elutriant gradient of described silicagel column form be followed successively by hexanaphthene, hexanaphthene-ethyl acetate v/v is 60～10: 1, hexanaphthene-ethyl acetate v/v is that 8: 1, hexanaphthene-ethyl acetate v/v are that 5: 1, hexanaphthene-ethyl acetate v/v are that 3: 1, hexanaphthene-ethyl acetate v/v are that 2: 1, hexanaphthene-ethyl acetate v/v are 1: 1, ethyl acetate.

7. according to the described method of claim 1, it is characterized in that: the filler of described gel column is Sephadex ^TMLH-20; Elutriant used is methyl alcohol or the volume ratio methyl alcohol-methylene dichloride of 1: 1.