CN102295545B - Method for biosynthesizing abietane diterpenoid compounds by using cephalotaxus fortune culture cells - Google Patents
Method for biosynthesizing abietane diterpenoid compounds by using cephalotaxus fortune culture cells Download PDFInfo
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Abstract
The invention relates to a plant cell culture technology, in particular to a method for biosynthesizing abietane diterpenoid compounds by using cephalotaxus fortune culture cells. The method comprises the following steps of: placing cephalotaxus fortune cells in a common culture medium, and culturing, wherein50 to 150g of the cells are inoculated in each liter of wet weight of the culture medium; and adding an inducer at the final concentration of 10 to 2,000 mu M and 50 to 200g of adsorbent into each liter of culture medium at the initial stage of cell exponential growth, wherein the inducer is an abiological inducer or fungal elicitor. The method is a biosynthesis method and is stable and environment-friendly; and products are completely adsorbed onto the adsorbent, so that the operation process of separation and purification is greatly simplified, the cost is reduced and industrial production is easy to realize. Meanwhile, seven abietane diterpenoid compounds comprising a new compound are synthesized. The abietane diterpenoid compounds have high bioactivity, and are important lead compounds, and the technology has important commercial application prospect.
Description
Technical field
The present invention relates to plant cell culture technology, a kind of method of inducing the synthetic abietane diterpene-kind compound of Cephalotaxus fortunei fluid suspension culture cell biological specifically, product comprises a new compound simultaneously.
Background technology
The abietane diterpene-kind compound belongs to the tricyclic diterpene compounds, is the important natural compounds of a class.Why this compounds causes that people's research interest is mainly because a lot of materials in them have good activity, contains this class material in a lot of medications among the people.Such as, the hinokiol in the present invention (compound 2) is inhibited to streptococcus aureus, suis, intestinal bacteria, Pseudomonas aeruginosa etc., is good antibiotic lead compound.Yet this compounds is mainly derived from from plant and extracts at present, about the report of its chemosynthesis seldom, and also in the laboratory study stage.Because the content of this compounds in plant is very low, obtain difficulty, greatly limit its further research and application.
Utilize plant cell culture technology produce biologically active substance because of its environmental friendliness, with short production cycle, controllability is strong etc., and advantage is subject to people's attention day by day, becomes the potential source of natural active matter.And the subject matter that at present people run into is the pathways metabolism of how regulating plant and then the product that obtains expection, as controlling chemosynthesis, control biosynthesizing.
At present common regulate and control method is mainly the production that promotes the secondary metabolite that existed in cell, namely improves its productive rate.
Summary of the invention
The purpose of this invention is to provide a kind of Cephalotaxus fortunei (Cephalotaxus fortunei that utilizes, Hook, f.) fluid suspension culture cell, induce the 7 kinds of abietane diterpene-kind compounds of combined regulating technology biosynthesizing with the combination of sorbent material Adsorption Phase with inductor, comprise the method for a new abietane diterpene compound.
For achieving the above object, the technical solution used in the present invention is:
Cephalotaxus cell is placed in to its substratum commonly used to be cultivated, the cell inoculum size is 50～150g/L weight in wet base, at the cell index early growth period, add inductor that 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 of lower concentration in the crude extract of evaporate to dryness, alkali lye mass volume ratio example 1g: the 3～15ml of crude extract and lower concentration;
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, and the elutriant that simultaneously utilizes thin-layer chromatography to collect sample bottle is detected, when elutriant used is hexanaphthene-ethyl acetate (8: 1, v/v), the time, obtain respectively the crude product of compound 1 and 4; Hexanaphthene-ethyl acetate (5: 1, in the time of v/v), obtain respectively the crude product of compound 3 and 6; Hexanaphthene-ethyl acetate (2: 1, in the time of v/v), obtain 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-hydroxy-2-isopropyl-6-methyl-5-(4-methyl-3-oxopentyl) naphthalene
Compound 2:abieta-8,11,13-triene-3 β, 12-diol
Compound 3:12-hydroxyabieta-6,8,11,13-tetraene-3-one
Compound 4:abietatriene-3 beta-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 7:3 β, 11-dihydroxy-12-methoxyabieta-8,11,13-triene-7-one
The substratum commonly used of Cultured Cells of Cephalotaxus Fortunei consists of, and adding final concentration in the MS basic medium is 0.1～2.5mg/L 2,4 dichloro benzene ethoxyacetic acid, 0.1～1mg/L kinetin and 10～50g/L sucrose, and the pH value is 5.4～6.2;
The cell index early growth period is 4th～10 days after cell is inoculated in substratum.
The culture condition of 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.
Abiotic inductor is Silver Nitrate, Whitfield's ointment, jasmonic or methyl jasmonate; Sorbent material for terpenoid is had to stronger adsorptive power and to cell nontoxic macroporous adsorbent resin, comprise XAD-7HP, XAD-4, XAD-1600 or HP2MG.
While extracting for the first time, polarity used or the organic solvent of middle polarity comprise 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.
While extracting for the second time, the alkali lye of lower concentration used refers to the potassium hydroxide that ammoniacal liquor that volumetric concentration is 0.1～1%, sodium hydroxide that mass concentration is 0.05～0.5% or mass concentration are 0.05～0.5%; While extracting for the second time, non-polar solvent used refers to chloroform or methylene dichloride.
The eluent components of silicagel column is followed successively by hexanaphthene, hexanaphthene-ethyl acetate (60～10: 1, v/v), hexanaphthene-ethyl acetate is (8: 1, v/v), hexanaphthene-ethyl acetate is (5: 1, v/v), hexanaphthene-ethyl acetate is (3: 1, v/v), hexanaphthene-ethyl acetate is (2: 1, v/v), hexanaphthene-ethyl acetate (1: 1, v/v), ethyl acetate.
The filler of gel column is Sephadex
TMLH-20; Elutriant used is methyl alcohol or the volume ratio methyl alcohol-methylene dichloride of 1: 1.
The inventive method principle:
By adding inductor and sorbent material, can induce Cephalotaxus fortunei fluid suspension culture cell to synthesize the abietane diterpene-kind compound, its principle mainly contains following three aspects: 1. inductor can activate specific pathways metabolism as signaling molecule, causes building-up reactions; 2. sorbent material can adsorbed product, increases the storage site of product, eliminates feedback inhibition, can prevent the degraded of product simultaneously.3. both must combine use, may be due to product to the toxic effect of cell, only use inductor and do not add sorbent material if make, although can bring out reaction, product has just accumulated cell has been produced to restraining effect, by cell degradation, can not obtain product.
The present invention has following advantage:
1. the combined utilization inductor is induced with sorbent material and is adsorbed to induce Cephalotaxus fortunei fluid suspension culture cell to synthesize the abietane diterpene-kind compound, the artificial controlled production that has realized this compounds have not been reported, and this compounds is mainly derived from the extraction of plant at present.
2. obtained a new abietane diterpene compound in the present invention.
3. in the present invention, product is attracted in sorbent material fully, is easy to separation and purification, has reduced cost, for further suitability for industrialized production provides very large possibility.
4. the cell culture condition in the present invention can guarantee the Fast Growth of cell and guarantee good homogeneity.
5. in the present invention, utilize low-concentration alkali liquor to remove impurity, greatly improved purification efficiency.
The contained natural product of the Cephalotaxus fortunei of recording in document is mainly alkaloid, and the abietane diterpene-kind compound have not been reported.Process is carried out the analysis of HPLC to leaf, the stem of Cephalotaxus fortunei and the fluid suspension culture cell that process is not induced, and seven compounds in the present invention all do not detected.
Cephalotaxus is 9 kinds of genus, at present, only separates to such an extent that be the abietane diterpene in Japanese caephalotaxus sinensis in cephalotaxus plant, and have not been reported in other 8 kinds.
Characteristics of the present invention are, the product in the present invention do not detected in the Cephalotaxus fortunei fluid suspension culture cell before inducing, and the Cultured Cells of Cephalotaxus Fortunei after inducing have synthesized 7 kinds of abietane diterpene-kind compounds, comprises a new abietane diterpene compound.Illustrate that the present invention is a kind of effective ways that utilize the synthetic abietane diterpene-kind compound of Cephalotaxus fortunei fluid suspension culture cell biological.
Embodiment
Embodiment 1
(1) cultivation of cephalotaxus cell:
Adopt Cephalotaxus fortunei fluid suspension culture cell:
The callus suspension culture that cell strain in the present invention is induced by the young stem of Cephalotaxus fortunei (C.fortunei) at first, adopt Murashige-Skoog (MS) substratum, adding concentration is 2mg/L 2, the 4-dichlorophenoxyacetic acid, 0.1mg/L kinetin and 30g/L sucrose, Medium's PH Value is 5.8.The 7.5 gram wet cells that filtration is obtained are inoculated in the 500ml shaking flask that contains the above-mentioned substratum of 100ml, then are placed on rotary shaking table and cultivate, and rotating speed is 100rpm, and 25 ℃, the dark cultivation.
(2) combined induction of abietane diterpene is synthetic:
Within the 7th day, in substratum, add methyl jasmonate (MJA) (final concentration in substratum is 300 μ M/L) and XAD-7HP resin (100g/L) in Growth of Cells, continue to be cultured to the 14th day, difference harvested cell and XAD-7HP, after measured, product is attracted in XAD-7HP fully, product do not detected in cell.
(3) extraction of abietane diterpene-kind compound, purifying and Structural Identification:
Under room temperature, the sorbent material 300g after vacuum filtration is first used to methanol extraction 3 times, the volume that at every turn extracts methyl alcohol used is 0.9L, and each the extraction comprises mechanical shaking extraction 1 hour, and then supersound extraction is 30 minutes.Then merge the methanol extract liquid of three times, filter, the rotation evaporate to dryness.Add 0.5% (v/v) ammoniacal liquor 2L and isopyknic methylene dichloride in the methanol extract of evaporate to dryness.Extract after 12 hours, dichloromethane layer is separated and rotated evaporate to dryness, then be placed on silicagel column and separate, used silica gel is 200～300 order silica gel, and type of elution is gradient elution.The eluent components of silicagel column is followed successively by hexanaphthene (300ml), hexanaphthene-ethyl acetate (50: 1, v/v, 500ml), hexanaphthene-ethyl acetate (30: 1, v/v, 300ml), hexanaphthene-ethyl acetate (20: 1, v/v, 300ml), hexanaphthene-ethyl acetate (10: 1, v/v, 500ml), hexanaphthene-ethyl acetate (8: 1, v/v, 400ml), hexanaphthene-ethyl acetate (5: 1, v/v, 400ml), hexanaphthene-ethyl acetate (3: 1, v/v, 400ml), hexanaphthene-ethyl acetate (2: 1, v/v, 600ml), hexanaphthene-ethyl acetate (1: 1, v/v, 300ml), ethyl acetate (300ml).Collect continuously elutriant with sample bottle, each sample bottle is collected 10ml elutriant, utilizes thin-layer chromatography to be detected simultaneously, when elutriant used be hexanaphthene-ethyl acetate (8: 1, in the time of v/v), obtain respectively the crude product of compound 1 and 4; Hexanaphthene-ethyl acetate (5: 1, in the time of v/v), obtain respectively the crude product of compound 3 and 6; Hexanaphthene-ethyl acetate (2: 1, in the time of v/v), obtain respectively the crude product of compound 2,5 and 7.The crude product of these 7 compounds is placed in respectively on the gel column that filler is LH-20 and completes further purifying, elutriant used is methyl alcohol or the volume ratio methyl alcohol-methylene dichloride of 1: 1, obtain the sterling of 7 compounds, compound 1 (3.8mg) wherein, compound 2 (10.4mg), compound 3 (14.2mg), compound 4 (6.5mg), compound 5 (200mg), compound 6 (4mg), compound 7 (12.8mg), through Spectrum Analysis, compound 1 is new compound, and the structure of seven compounds is 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
The faint yellow oily solid of 3-hydroxy-2-isopropyl-6-methyl-5-(4-methyl-3-oxopentyl) naphthalene (acetone).
1H NMR (CD
3COCD
3, 500MHz) δ 1.08 (6H, d, J=6.9Hz, H-18 and H-19), δ 1.32 (6H, d, J=6.85Hz, H-16 and H-17), δ 2.42 (3H, s, H-20), δ 2.67 (1H, sept, J=6.9Hz, H-4), δ 2.73 (2H, t like, H-1a and H-1b), δ 3.16 (2H, t like, H-2a and H-2b), δ 3.41 (1H, sept, J=6.85Hz, H-15), δ 7.07 (1H, d, J=8.3Hz, H-6), δ 7.29 (1H, s, H-11), δ 7.52 (1H, d, J=8.3Hz, H-7), δ 7.61 (1H, s, H-14), δ 8.59 (1H, s, 12-OH).
13C NMR (CD
3COCD
3, 125.7MHz) δ 18.53 (CH
3, C-18), δ 18.53 (CH
3, C-19), δ 20.04 (CH
3, C-20), δ 22.95 (CH
3, C-16), δ 22.95 (CH
3, C-17), δ 23.61 (CH
2, C-2), δ 28.18 (CH, C-15), δ 40.37 (CH
2, C-1), δ 41.34 (CH, C-4), δ 105.81 (CH, C-11), δ 126.49 (CH, C-14), δ 126.69 (CH, C-7), δ 126.93 (CH, C-6), δ 129.00 (C, C-8), δ 132.76 (C, C-10), δ 132.96 (C, C-5), δ 132.96 (C, C-9), δ 136.89 (C, C-13), δ 154.93 (C, C-12), δ 213.60 (C, C-3) .EIMS 70eV, m/z (rel.int.): 298[M]
+(39), 280[M-H
2O]
+(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
20H
26O
2]
+, 298.1933.
Compound 2
abieta-8，11，13-triene-3β，12-diol
Clear crystal (acetone).
1HNMR (CD
3COCD
3, 500MHz) δ 0.87 (3H, s, H-19), δ 1.06 (3H, s, H-18), δ 1.15 (3H, s, H-20), δ 1.18 (6H, t, J=6.8Hz, H-16 and H-17), δ 1.24 (1H, dd, J=12.3, 2.2Hz, H-5), δ 1.44 (1H, td, J=12.85, 4.9Hz, H-1a), δ 1.71 (1H, m, H-6a), δ 1.74 (2H, m, H-2a and H-2b), δ 1.86 (1H, m, H-6b), δ 2.17 (1H, dt, J=13.05, 3.5Hz, H-1b), δ 2.72 (1H, m, H-7a), δ 2.82 (1H, m, H-7b), δ 3.21 (1H, sept, J=6.8Hz, H-15), δ 3.24 (1H, m, H-3), δ 3.45 (1H, d, J=5.3Hz, 3-OH), δ 6.7 (1H, s, H-11), δ 6.78 (1H, s, H-14), δ 7.67 (1H, s, 12-OH).
13CNMR (CD
3COCD
3, 125.7MHz) δ 16.06 (CH
3, C-19), δ 19.93 (CH
2, C-6), δ 22.93 (CH
3, C-16), δ 23.04 (CH
3, C-17), δ 25.24 (CH
3, C-20), δ 27.48 (CH, C-15), δ 28.72 (CH
3, C-18), δ 28.98 (CH
2, C-2), δ 30.84 (CH
2, C-7), δ 38.02 (CH
2, C-1), δ 38.08 (C, C-10), δ 39.75 (C, C-4), δ 51.09 (CH, C-5), δ 78.51 (CH, C-3), δ 111.56 (CH, C-11), δ 126.34 (C, C-8), δ 127.09 (CH, C-14), δ 132.83 (C, C-13), δ 148.51 (C, C-9), δ 153.19 (C, C-12) .EIMS 70e V, m/z (rel.int.): 302[M]
+(48), 284[M-H
2O]
+(57), 269[M-H
2O-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
20H
30O
2]
+, 302.2246.
Compound 3
12-hydroxyabieta-6，8，11，13-tetraene-3-one
Pale yellow powder shape solid (acetone).
1H?NMR(CD
3COCD
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).
13C?NMR(CD
3COCD
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
20H
26O
2]
+，298.1933.
Compound 4
abietatriene-3β-ol
Clear crystal (acetone).
1H NMR (CD
3COCD
3, 500MHz) δ 0.89 (3H, s, H-19), δ 1.07 (3H, s, H-18), δ 1.17 (3H, s, H-20), δ 1.18 (3H, s, H-16), δ 1.20 (3H, s, H-17), δ 1.27 (1H, dd, J=12.35, 2.25Hz, H-5), δ 1.45 (1H, td, J=12.9, 4.7Hz, H-1a), δ 1.74 (1H, m, H-6a), δ 1.76 (2H, m, H-2a and H-2b), δ 1.90 (1H, m, H-6b), δ 2.30 (1H, dt, J=13.05, 3.5Hz, H-1b), δ 2.80 (1H, m, H-15), δ 2.81 (1H, m, H-7a), δ 2.90 (1H, m, H-7b), δ 3.24 (1H, m, H-3), δ 3.43 (1H, d, J=5.25Hz, 3-OH), δ 6.88 (1H, s, H-14), δ 6.96 (1H, d, J=8.15Hz, H-12), δ 7.16 (1H, d, J=8.15Hz, H-11).
13C (CD
3COCD
3, 125.7MHz) δ 16.06 (CH
3, C-19), δ 19.72 (CH
2, C-6), δ 24.36 (CH
3, C-16 and C-17), δ 25.31 (CH
3, C-20), δ 28.73 (CH
3, C-18), δ 28.99 (CH
2, C-2), δ 31.52 (CH
2, C-7), δ 34.29 (CH, C-15), δ 37.97 (CH
2, C-1), δ 38.15 (C, C-10), δ 39.79 (C, C-4), δ 51.12 (CH, C-5), δ 78.50 (CH, C-3), δ 124.62 (CH, C-12), δ 125.23 (CH, C-11), δ 127.45 (CH, C-14), δ 135.49 (C, C-8), δ 146.27 (C, C-13), δ 148.03 (C, C-9) .EIMS 70eV, m/z (rel.int.): 286[M]
+(20), 271[M-CH
3]
+(52), 253[M-CH
3-H
2O]
+(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
20H
30O]+, 286.2297.
Compound 5
11，12-dihydroxy-8，11，13-abietatriene-3，7-dione
Clear crystal (acetone).
1H NMR (CD
3COCD
3, 500MHz) δ 1.16 (6H, s, H-18 and H-19), δ 1.21 (3H, d, J=6.9Hz, H-16), δ 1.23 (3H, d, J=6.9Hz, H-17), δ 1.49 (3H, s, H-20), δ 2.00 (1H, dt, J=13.9,8.4Hz, H-1a), δ 2.44 (1H, m, H-6a), δ 2.48 (1H, m, H-5), δ 2.60 (2H, m, H-2a and H-2b), δ 2.66 (1H, m, H-6b), δ 3.31 (1H, sept, J=6.9Hz, H-15), δ 3.40 (1H, m, H-1b), δ 7.55 (1H, s, H-14).
13C (CD
3COCD
3, 125.7MHz) δ 18.00 (CH
3, C-20), δ 21.07 (CH
3, C-19), δ 22.95 (CH
3, C-17), δ 23.06 (CH
3, C-16), δ 27.21 (CH
3, C-18), δ 27.40 (CH, C-15), δ 34.92 (CH
2, C-2), δ 36.39 (CH
2, C-1), δ 36.52 (CH
2, C-6), δ 39.65 (C, C-10), δ 47.61 (C, C-4), δ 50.40 (CH, C-5), δ 117.40 (CH, C-14), δ 125.58 (C, C-8), δ 134.38 (C, C-13), δ 137.71 (C, C-9), δ 143.96 (C, C-11), δ 148.52 (C, C-12), δ 196.91 (C, C-7), δ 215.33 (C, C-3) .EIMS 70eV, m/z (rel.int.): 330[M]
+(100), 315[M-CH
3]
+(40), 297[M-CH
3-H
2O]
+(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
20H
26O
4]
+, 330.1831.
Compound 6
11-hydroxy-12-methoxyabieta-8,11,13-triene-3,7-dione colourless acicular crystal (chloroform).
1H NMR (CDCl
3, 500MHz) δ 1.17 (3H, s, H-18), δ 1.18 (3H, s, H-19), δ 1.25 (3H, d, J=6.9Hz, H-16), δ 1.27 (3H, d, J=6.9Hz, H-17), δ 1.47 (3H, s, H-20), δ 2.01 (1H, dt, J=14, 8.4Hz, H-1a), δ 2.46 (1H, dd, J=14.3, 3.2Hz, H-5), δ 2.60 (1H, m, H-6a), δ 2.64 (2H, m, H-2a and H-2b), δ 2.64 (1H, m, H-6b), δ 3.22 (1H, sept, J=6.9Hz, H-15), δ 3.33 (1H, m, H-1b), δ 3.83 (3H, s, 12-OCH
3), δ 6.14 (1H, s, 11-OH), δ 7.63 (1H, s, H-14).
13C (CDCl
3, 125.7MHz) δ 17.68 (CH
3, C-20), δ 20.80 (CH
3, C-19), δ 23.46 (CH
3, C-16), δ 23.54 (CH
3, C-17), δ 26.79 (CH, C-15), δ 26.99 (CH
3, C-18), 34.43 (CH
2, C-2), δ 35.38 (CH
2, C-1), δ 36.15 (CH
2, C-6), δ 38.89 (C, C-10), δ 47.13 (C, C-4), δ 49.60 (CH, C-5), δ 61.95 (CH
3, 12-OCH
3), δ 117.42 (CH, C-14), δ 128.32 (C, C-8), δ 135.63 (C, C-9), δ 139.94 (C, C-13), δ 146.70 (C, C-11), δ 149.41 (C, C-12), δ 197.52 (C, C-7), δ 215.89 (C, C-3) .EIMS 70eV, m/z (rel.int.): 344[M]
+(100), 329[M-CH
3]
+(53), 311[M-CH
3-H
2O]
+(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
21H
28O
4]
+, 344.1988.
Compound 7
3β，11-dihydroxy-12-methoxyabieta-8，11，13-triene-7-one
Faint yellow needle-like crystal (acetone).
1H NMR (CD
3COCD
3, 500MHz) δ 0.95 (3H, s, H-19), δ 1.07 (3H, s, H-18), δ 1.21 (3H, d, J=6.9Hz, H-16), δ 1.23 (3H, d, J=6.9Hz, H-17), δ 1.42 (3H, s, H-20), δ 1.48 (1H, td, J=13.75, 3.6Hz, H-1a), δ 1.75 (2H, m, H-2a and H-2b), δ 1.78 (1H, m, H-5), δ 2.58 (2H, m, H-6a and H-6b), δ 3.26 (1H, sept, J=6.9Hz, H-15), δ 3.30 (1H, m, H-3), δ 3.40 (1H, dt, J=13.8, 3.55Hz, H-1b), δ 3.52 (1H, d, J=5.25Hz, 3-OH), δ 3.78 (3H, s, 12-OCH
3), δ 7.52 (1H, s, H-14), δ 7.86 (1H, s, 11-OH).
13C (CD
3COCD
3, 125.7MHz) δ 15.97 (CH
3, C-19), δ 17.97 (CH
3, C-20), δ 23.75 (CH
3, C-16), δ 23.88 (CH
3, C-17), δ 27.32 (CH, C-15), 28.48 (CH
3, C-18), δ 28.76 (CH
2, C-2), δ 35.63 (CH
2, C-1), δ 35.89 (CH
2, C-6), δ 39.92 (C, C-4), δ 40.91 (C, C-10), δ 50.87 (CH, C-5), δ 61.96 (CH
3, 12-OCH
3), δ 77.81 (CH, C-3), δ 116.98 (CH, C-14), δ 129.45 (C, C-8), δ 138.95 (C, C-9), δ 140.21 (C, C-13), δ 148.43 (C, C-11), δ 150.72 (C, C-12), δ 198.02 (C, C-7) .EIMS 70eV, m/z (rel.int.): 346[M]
+(53), 331[M-CH
3]
+(18), 313[M-CH
3-H
2O]
+(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
21H
30O
4]
+, 346.2144.
Comparative Examples 1
The culture condition of cephalotaxus cell is with embodiment 1, and difference from Example 1 is, does not add MJA and XAD-7HP in the combined induction building-up process, directly continues to cultivate.Analyze through HPLC, above-mentioned 7 kinds of products do not detected.Illustrate that these 7 kinds of products induce synthetic.
Comparative Examples 2
The culture condition of cephalotaxus cell is with embodiment 1, and difference from Example 1 is, only adds MJA in the combined induction building-up process or only adds XAD-7HP, adds concentration with embodiment 1.Analyze through HPLC, if only add MJA, above-mentioned 7 kinds of products do not detected in cell extract.If only add XAD-7HP, all do not detect this 7 kinds of compounds in cell extract and XAD-7HP extract.Illustrate that these 7 kinds of products only when inductor and sorbent material are used simultaneously, can synthesize.
Comparative Examples 3
The culture condition of cephalotaxus cell and inductive condition are with embodiment 1, and difference from Example 1 is, does not use ammoniacal liquor in leaching process.
Analyze and find through the HPLC to dichloromethane extract, while in extraction, using ammoniacal liquor, foreign matter content is significantly less than the foreign matter content while not using ammoniacal liquor, do not use ammoniacal liquor if extract, be difficult to obtain sterling during next step purifying, even or obtain sterling, productive rate also reduces greatly.Illustrate that ammoniacal liquor is the committed step of removing impurity.
In prior art, using ammoniacal liquor when extracting alkaloid is for alkaloid is dissociated from its salt, and the alkaloid after then making to dissociate is dissolved in organic layer.And use ammoniacal liquor in the present invention, be in order to remove the impurity such as pigment.
Comparative Examples 4
The culture condition of cephalotaxus cell and inductive condition, and extraction conditions is with embodiment 1.Difference from Example 1 is, the elutriant of silicagel column be followed successively by chloroform-methanol (5: 1, v/v), chloroform-methanol (3: 1, v/v), and chloroform-methanol (2: 1, v/v), chloroform-methanol (1: 1, v/v) time, product is eluted together, can not separate.
Illustrate, in the present invention, selected silicagel column elutriant polarity meets the needs of separation and purification of products, and after silicagel column, these 7 compounds can access good separation.
Embodiment 2
Culture condition and purification condition are with embodiment 1, difference from Example 1 is, induce when synthetic and within the 4th day, to add MJA (final concentration in substratum is 150 μ M/L) and XAD-7HP (100g/L) in Growth of Cells, continue to be cultured to 14 days, result is 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)
Embodiment 3
Culture condition and purification condition are with embodiment 1, difference from Example 1 is, induce while synthesizing and within the 7th day, add jasmonic (final concentration in substratum is 150 μ M/L) and XAD-7HP (100g/L) in Growth of Cells, continue to be cultured to 14 days, result is as follows: compound 1 (7.5mg), compound 2 (16mg), compound 3 (20mg), compound 4 (3mg), compound 5 (190mg), compound 6 (6mg), compound 7 (11.7mg)
Embodiment 4
Culture condition and purification condition are with embodiment 1, difference from Example 1 is, induce when synthetic and within the 7th day, in substratum, to add MJA (final concentration in substratum is 150 μ M/L) and HP-2MG (100g/L) in Growth of Cells, continue to be cultured to 14 days, result is 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.
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| CN106279200B (en) * | 2015-06-01 | 2021-05-14 | 中国科学院上海药物研究所 | Abietane diterpenoid compound with hypolipidemic activity, and preparation method and application thereof |
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| CN114890870A (en) * | 2022-06-07 | 2022-08-12 | 新疆维吾尔自治区中药民族药研究所 | Abietane diterpene in schizonepeta bracteata extract as well as preparation method and application thereof |
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