CN102295545A - 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 specifically method of inducing the synthetic abietane diterpene-kind compound of Cephalotaxus fortunei fluid suspension culture cell biological comprises a new compound in the product 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 mainly is 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 among the present invention (compound 2) is inhibited to streptococcus aureus, suis, intestinal bacteria, Pseudomonas aeruginosa etc., is the excellent 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 is in the laboratory study stage.Because the content of this compounds in plant is very low, obtain difficulty, limit greatly it is further studied and uses.
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 present people run into is the product of how regulating and control the pathways metabolism of plant and then obtaining expection, controls biosynthesizing as the control chemosynthesis.
At present common regulate and control method mainly is the production that promotes the secondary metabolite that existed in the cell, just 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 that combine with adsorbents adsorb 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:
Place its substratum commonly used to cultivate cephalotaxus cell, the cell inoculation amount is 50～150g/L weight in wet base, at the cell index early growth period, adding concentration in every liter of substratum simultaneously is the inductor of 10～2000 μ M and the sorbent material of 50～200g, and described inductor is abiotic inductor or fungal elicitor; Continue to cultivate after 7～14 days, respectively harvested cell and sorbent material;
Extract for the first time: under the room temperature, with the sorbent material behind the vacuum filtration earlier with polarity or middle polarity organic solvent extraction 2～5 times; United extraction liquid filters, the rotation evaporate to dryness;
Extract for the second time: in the crude extract of evaporate to dryness, add the alkali lye and the extraction of isopyknic non-polar organic solvent of lower concentration, alkali lye mass volume ratio example 1g: the 3～15ml of crude extract and lower concentration;
Separation and purification: extract after 8～16 hours, organic layer is separated, organic layer is rotated evaporate to dryness, place on the silicagel column then and separate, used silica gel is 200～300 order silica gel, type of elution is a gradient elution, the volume of the used elutriant of each gradient is 200-800ml, collect elutriant continuously with sample bottle, each sample bottle is collected the 5-15ml elutriant, and the elutriant that utilizes thin-layer chromatography that sample bottle is collected simultaneously detects, when used elutriant is hexanaphthene-ethyl acetate (8: 1, v/v) time, obtain the crude product of compound 1 and 4 respectively; Hexanaphthene-ethyl acetate (5: 1, in the time of v/v), obtain the crude product of compound 3 and 6 respectively; Hexanaphthene-ethyl acetate (2: 1, in the time of v/v), obtain the crude product of compound 2,5 and 7 respectively.The crude product of these 7 compounds placed respectively finish further purifying on the gel column, obtain the pure product 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 Cephalotaxus fortunei culturing cell 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 cell inoculation the 4th～10 day behind 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, goes down to posterity once every 14～21 days.
Abiotic inductor is Silver Nitrate, Whitfield's ointment, jasmonic or methyl jasmonate; Sorbent material comprises XAD-7HP for terpenoid is had stronger adsorptive power and the nontoxic macroporous adsorbent resin of pair cell, XAD-4, XAD-1600 or HP2MG.
The used polarity or the organic solvent of middle polarity comprise methyl alcohol, ethanol, acetone or ethyl acetate when extracting for the first time; In each leaching process, sorbent material and organic solvent mass volume ratio example 1g: 3-15ml, the each extraction adopts vibration to extract earlier 30 minutes～3 hours, and supersound extraction is 10～60 minutes then.
The alkali lye of used lower concentration is meant that volumetric concentration is that 0.1～1% ammoniacal liquor, mass concentration are that 0.05～0.5% sodium hydroxide or mass concentration are 0.05～0.5% potassium hydroxide when extracting for the second time; Used non-polar solvent is meant chloroform or methylene dichloride when extracting for the second time.
The elutriant composition 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; Used elutriant is methyl alcohol or methyl alcohol-methylene dichloride of 1: 1 of volume ratio.
The inventive method principle:
Can induce Cephalotaxus fortunei fluid suspension culture cell to synthesize the abietane diterpene-kind compound by adding inductor and sorbent material, 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 unite use, may be because the toxic effect of product pair cell, if make and only use inductor and do not add sorbent material, though can bring out reaction, it is that pair cell produces restraining effect that product has just accumulated, and by cell degradation, can not obtain product.
The present invention has following advantage:
1. the combined utilization inductor is induced with adsorbents adsorb and is induced Cephalotaxus fortunei fluid suspension culture cell to synthesize the abietane diterpene-kind compound, the artificial controlled production that has realized this compounds yet there are no report, and this compounds is mainly derived from the extraction of plant at present.
2. obtained a new abietane diterpene compound among the present invention.
3. product is attracted in the sorbent material fully among the present invention, is easy to separation and purification, has reduced cost, for further suitability for industrialized production provides very big possibility.
4. the cell culture condition among the present invention can guarantee the quick growth of cell and guarantee favorable uniformity.
5. utilize low-concentration alkali liquor to remove impurity among the present invention, improved purification efficiency greatly.
The contained natural product of the Cephalotaxus fortunei of writing down in the document mainly is an alkaloid, and the abietane diterpene-kind compound yet there are no report.Pass through leaf, stem and do not carry out the analysis of HPLC, all do not detect seven compounds among the present invention through inductive fluid suspension culture cell to Cephalotaxus fortunei.
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 yet there are no report in other 8 kinds.
Characteristics of the present invention are, do not detect the product among the present invention in the Cephalotaxus fortunei fluid suspension culture cell before inducing, and the Cephalotaxus fortunei culturing cell 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:
Cell strain among the present invention is at first by the young stem inductive of Cephalotaxus fortunei (C.fortunei) callus suspension culture, adopt Murashige-Skoog (MS) substratum, adding concentration is 2mg/L 2, the 4-dichlorophenoxyacetic acid, 0.1mg/L kinetin and 30g/L sucrose, medium pH value are 5.8.The 7.5 gram wet cells that filtration is obtained are inoculated into the 500ml that contains the above-mentioned substratum of 100ml and shake in the bottle, place then on the rotary shaking table and cultivate, and rotating speed is 100rpm, and 25 ℃, the dark cultivation.
(2) combined induction of abietane diterpene is synthetic:
In substratum, add methyl jasmonate (MJA) (final concentration in the substratum is 300 μ M/L) and XAD-7HP resin (100g/L) in the 7th day of cell growth, continue to be cultured to the 14th day, difference harvested cell and XAD-7HP, after measured, product is attracted among the XAD-7HP fully, does not detect product in the cell.
(3) extraction of abietane diterpene-kind compound, purifying and structure are identified:
Under the room temperature sorbent material 300g behind the vacuum filtration is used methanol extraction 3 times earlier, the volume that at every turn extracts used methyl alcohol is 0.9L, and each extraction comprising vibrating extracted 1 hour, and supersound extraction is 30 minutes then.Merge three times methanol extract liquid then, filter, the rotation evaporate to dryness.In the methanol extract of evaporate to dryness, add 0.5% (v/v) ammoniacal liquor 2L and isopyknic methylene dichloride.Extract after 12 hours, dichloromethane layer is separated and rotated evaporate to dryness, place on the silicagel column then and separate, used silica gel is 200～300 order silica gel, and type of elution is a gradient elution.The elutriant composition 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 elutriant continuously with sample bottle, each sample bottle is collected 10ml elutriant, utilizes thin-layer chromatography to detect simultaneously, when used elutriant be hexanaphthene-ethyl acetate (8: 1, in the time of v/v), obtain the crude product of compound 1 and 4 respectively; Hexanaphthene-ethyl acetate (5: 1, in the time of v/v), obtain the crude product of compound 3 and 6 respectively; Hexanaphthene-ethyl acetate (2: 1, in the time of v/v), obtain the crude product of compound 2,5 and 7 respectively.It is to finish further purifying on the gel column of LH-20 that the crude product of these 7 compounds is placed filler respectively, used elutriant is methyl alcohol or methyl alcohol-methylene dichloride of 1: 1 of volume ratio, obtain the pure product 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) is 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), and δ 28.18 (CH, C-15), δ 40.37 (CH
2, C-1), and δ 41.34 (CH, C-4), δ 105.81 (CH, C-11), and 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), and δ 132.96 (C, C-5), δ 132.96 (C, C-9), and 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), and 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), and 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), and 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), and 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), and 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), and 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), and δ 1.17 (3H, s, H-20), δ 1.18 (3H, s, H-16), and 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), and 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), and 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), and δ 34.29 (CH, C-15), δ 37.97 (CH
2, C-1), and 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), and δ 1.23 (3H, d, J=6.9Hz, H-17), δ 1.49 (3H, s, H-20), and 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), and 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), and 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), and δ 39.65 (C, C-10), δ 47.61 (C, C-4), and δ 50.40 (CH, C-5), δ 117.40 (CH, C-14), and δ 125.58 (C, C-8), δ 134.38 (C, C-13), and δ 137.71 (C, C-9), δ 143.96 (C, C-11), and 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), and 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), and 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), and 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), and 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), and 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), and 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), and 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), and 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), and 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, do not detect above-mentioned 7 kinds of products.Illustrate that these 7 kinds of products are to 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,, in cell extract, do not detect above-mentioned 7 kinds of products if only add MJA.If only add XAD-7HP, in cell extract and XAD-7HP extract, all do not detect this 7 kinds of compounds.Illustrate that these 7 kinds of products only when inductor and sorbent material use 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 the leaching process.
Analyze discovery through the HPLC to dichloromethane extract, when using ammoniacal liquor in the extraction, foreign matter content is significantly less than the foreign matter content when not using ammoniacal liquor, do not use ammoniacal liquor if extract, be difficult to obtain pure product during next step purifying, even or obtain pure product, productive rate also reduces greatly.Illustrate that ammoniacal liquor is the committed step of removing impurity.
In the prior art, using ammoniacal liquor when extracting alkaloid is for alkaloid being dissociated from its salt, the alkaloid after dissociating being dissolved in the organic layer.And use ammoniacal liquor among the present invention is in order to remove 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 that the elutriant of silicagel column is followed successively by chloroform-methanol, and (5: 1, v/v), (3: 1, v/v), (2: 1, v/v), (1: 1, in the time of v/v), product was eluted chloroform-methanol chloroform-methanol chloroform-methanol together, can not separate.
Illustrate that selected silicagel column elutriant polarity meets the needs of separation and purification of products among the present invention, can access good separation through these 7 compounds behind the silicagel column.
Embodiment 2
Culture condition and separation and purification condition are with embodiment 1, difference from Example 1 is, induced when synthetic in the 4th day of the cell growth and add MJA (final concentration in the substratum is 150 μ M/L) and XAD-7HP (100g/L), continue to be cultured to 14 days, the 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 separation and purification condition are with embodiment 1, difference from Example 1 is, induce when synthesizing and added jasmonic (final concentration in the substratum is 150 μ M/L) and XAD-7HP (100g/L) in 7 days in the cell growth regulation, continue to be cultured to 14 days, the 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 separation and purification condition are with embodiment 1, difference from Example 1 is, induced when synthetic in the 7th day of the cell growth and add MJA (final concentration in the substratum is 150 μ M/L) and HP-2MG (100g/L) in substratum, continue to be cultured to 14 days, the 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 (8)
1. utilize the method for Cephalotaxus fortunei culturing cell biosynthesizing abietane diterpene-kind compound, it is characterized in that: place its substratum commonly used to cultivate cephalotaxus cell, the cell inoculation amount is 50～150g/L weight in wet base, at the cell index early growth period, adding final concentration in every liter of substratum simultaneously is the inductor of 10～2000 μ M and the sorbent material of 50～200g, and described inductor is abiotic inductor or fungal elicitor; Continue to cultivate after 7～14 days, respectively harvested cell and sorbent material;
Extract for the first time: under the room temperature, with the sorbent material behind the vacuum filtration earlier with polarity or middle polarity organic solvent extraction 2～5 times; United extraction liquid filters, the rotation evaporate to dryness;
Extract for the second time: in the crude extract of evaporate to dryness, add the alkali lye and the extraction of isopyknic non-polar organic solvent of lower concentration, alkali lye mass volume ratio example 1g: the 3～15ml of crude extract and lower concentration;
Separation and purification: extract after 8～16 hours, organic layer is separated, organic layer is rotated evaporate to dryness, place on the silicagel column then and separate, used silica gel is 200～300 order silica gel, type of elution is a gradient elution, the volume of the used elutriant of each gradient is 200-800ml, collect elutriant continuously with sample bottle, each sample bottle is collected the 5-15ml elutriant, and the elutriant that utilizes thin-layer chromatography that sample bottle is collected simultaneously detects, when used elutriant is hexanaphthene-ethyl acetate (8: 1, v/v) time, obtain the crude product of compound 1 and 4 respectively; Hexanaphthene-ethyl acetate (5: 1, in the time of v/v), obtain the crude product of compound 3 and 6 respectively; Hexanaphthene-ethyl acetate (2: 1, in the time of v/v), obtain the crude product of compound 2,5 and 7 respectively; The crude product of these 7 compounds placed respectively finish further purifying on the gel column, obtain the pure product 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
2. according to the described method of claim 1, it is characterized in that: the substratum commonly used of described Cephalotaxus fortunei culturing cell consists of, adding final concentration in the MS basic medium is 0.1～2.5mg/L 2, the 4-dichlorophenoxyacetic acid, 0.1～1mg/L kinetin and 10～50g/L sucrose, pH value are 5.4～6.2;
Described cell index early growth period is cell inoculation the 4th～10 day behind 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, goes down to posterity once every 14～21 days.
4. according to the described method of claim 1, it is characterized in that: described abiotic inductor is Silver Nitrate, Whitfield's ointment, jasmonic or methyl jasmonate;
Described sorbent material comprises XAD-7HP for terpenoid is had stronger adsorptive power and the nontoxic macroporous adsorbent resin of pair cell, XAD-4, XAD-1600 or HP2MG.
5. according to the described method of claim 1, it is characterized in that: the described first time, used polarity or the organic solvent of middle polarity comprised methyl alcohol, ethanol, acetone or ethyl acetate when extracting;
In each leaching process, sorbent material and organic solvent mass volume ratio example 1g: 3-15ml, the each extraction adopts vibration to extract earlier 30 minutes～3 hours, and supersound extraction is 10～60 minutes then.
6. according to the described method of claim 1, it is characterized in that: the described second time, the alkali lye of used lower concentration when extracting was meant that volumetric concentration is that 0.1～1% ammoniacal liquor, mass concentration are that 0.05～0.5% sodium hydroxide or mass concentration are 0.05～0.5% potassium hydroxide;
The described second time, used non-polar solvent was meant chloroform or methylene dichloride when extracting.
7. according to the described method of claim 1, it is characterized in that: the elutriant gradient composition of described 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 (2: 1, v/v), hexanaphthene-ethyl acetate (1: 1, v/v), ethyl acetate.
8. according to the described method of claim 1, it is characterized in that: the filler of described gel column is Sephadex
TMLH-20; Used elutriant is methyl alcohol or methyl alcohol-methylene dichloride of 1: 1 of volume ratio.
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| WO2016192631A1 (en) * | 2015-06-01 | 2016-12-08 | 中国科学院上海药物研究所 | Abietane-type diterpene compound having lipid-lowering activity, method for preparing same, and use of same |
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| CN114044734A (en) * | 2021-09-08 | 2022-02-15 | 新疆维吾尔自治区中药民族药研究所 | Abietane diterpene and preparation method and application thereof |
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| CN106279200A (en) * | 2015-06-01 | 2017-01-04 | 中国科学院上海药物研究所 | There is the Diterpene compounds of hypolipidemic activity, Preparation Method And The Use |
| 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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