CN1535975A - Method for synthesizing secoiridoid glycosides compound - Google Patents
Method for synthesizing secoiridoid glycosides compound Download PDFInfo
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- CN1535975A CN1535975A CNA031261116A CN03126111A CN1535975A CN 1535975 A CN1535975 A CN 1535975A CN A031261116 A CNA031261116 A CN A031261116A CN 03126111 A CN03126111 A CN 03126111A CN 1535975 A CN1535975 A CN 1535975A
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Abstract
The present invention relates to a method for synthesizing secoiridoid glycoside compound, belonging to the field of biolgoical conversion synthesis technology of organic compound. Said method includes four steps of preparation of culture medium, induction of explant, tissue (cell) culture and extraction of target molecule. Said invention only can utilize solid culture medium to make culture and extraction, also can utilize solid culture medium to make culture firstly, then utilize liquid culture medium to make culture and extraction. Said method and process are simple, and high in yield.
Description
Technical field
The invention belongs to the bio-transformation synthesis technical field of organic compound, the method for the synthetic secoiridoid glycoside compound of particularly a kind of utilization tissue (cell) culture method.
Background technology
That the secoiridoid glycoside compound is that a class has is antibiotic, the material of anti-inflammatory, effect such as antiviral, anti-oxidant, anticancer, hypoglycemic, and toxicity is very low, can be used for medical treatment and nourishing function aspect.But this compounds content in natural phant is very low, and the chemical structure complexity, utilizes chemical process to carry out complete synthesis very difficulty, and uneconomical, has seriously limited its further development and utilization.
Summary of the invention
The object of the present invention is to provide a kind of bio-transformation synthetic method that is suitable for the synthetic secoiridoid glycoside compound of suitability for industrialized production promptly to organize (cell) cultural method.
In order to achieve the above object, the present invention adopts following technical scheme: a kind of method of synthetic secoiridoid glycoside compound, comprise the preparation of substratum, four steps of extraction of inducing, organizing (cell) cultivation and target molecule of explant: in the explant induction step, be inoculated on the solid medium after getting Oleaceae Genus Syringa, Ligustrum, sweet-scented osmanthus genus, Olea, Jasminum, fontanesia Shu He the plant leaf of Ash genus, leaf bud or rhizome sterilization, cultivated 15-108 days in 15-32 ℃; At the target molecule extraction step, place extractor to use organic solvent refluxing extraction 2-6 hour or cold soaking 8-48 hour the callus of gathering in the crops, remove organic solvent and get target molecule secoiridoid glycoside compound.
Contain sal epsom 160-689mg/l in the solid medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6-furfuryladenine, 6-benzyladenine, one of 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, agar 4500-32000mg/l, take by weighing one by one except that sucrose, the medium component that agar is outer, use water dissolution, mixing and thin up are to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
Also contain pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in the solid medium.
Organic solvent comprises alcohols, ketone and ether organic solvent.
Organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus weight.
A kind of method of synthetic secoiridoid glycoside compound, comprise the preparation of solid medium and liquid nutrient medium, four steps of extraction of inducing, organizing (cell) cultivation and target product of explant: in the explant induction step, be inoculated on the solid medium after getting Oleaceae Genus Syringa, Ligustrum, Olea, Jasminum He the plant leaf of Ash genus, leaf bud or rhizome sterilization, cultivated 14-49 days in 15-32 ℃; Taking out callus again transfers in the liquid nutrient medium in concussion incubator or shaking table or bio-reactor or reactor and cultivated 10-60 days at 18-32 ℃; At the target product extraction step, culture and substratum filtration are earlier separately placed the extractor organic solvent extraction respectively again, remove organic solvent and get target product secoiridoid glycoside compound.
Contain sal epsom 160-689mg/l in the liquid nutrient medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6One of-furfuryladenine, 6-benzyladenine, 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, take by weighing the medium component except that sucrose one by one, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get liquid nutrient medium; Solid medium is than increasing the agar composition in the liquid culture based formulas, content is 4500-19500mg/l, its method for making is: take by weighing the medium component except that sucrose, agar, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
Respectively contain pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in solid medium and the liquid nutrient medium.
Organic solvent comprises alcohols, ketone and ether organic solvent.
Organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus.
The present invention adopts the synthetic secoiridoid glycoside compound of tissue (cell) cultural method in biology and the cell engineering; not limited by natural resources; and help preserving the ecological environment; the plant cell culture medium of substratum for extensively adopting in tissue (cell) cultivation in the method; composition is easy to get; contain macroelement such as nitrogen, phosphorus, potassium in the substratum; trace elements such as boron, zinc, manganese, molybdenum, copper, cobalt, sodium, iodine, iron; VITAMIN and 2 such as thiamines, pyridoxol, nicotinic acid, inositol, 4-D, N
6-furfuryladenine, 6-benzyladenine, 6-chaff aminoadenine, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, plant growth regulating substances such as 6-trichloropyridine formic acid, can provide tissue (cell) to cultivate required various nutritive ingredients, the culture condition gentleness, easy extraction.Entire method is easy to suitability for industrialized production.In two kinds of methods of the present invention, first method only adopts solid medium to cultivate; Second method adopts the solid medium cultivation to change liquid nutrient medium then over to earlier and cultivates, and is easier to large-scale industrialization production, and shortens incubation time, and product yield improves greatly, and production cost is reduced greatly.
Description of drawings
Fig. 1 is the chemical structural formula of target product secoiridoid glycoside compound representative oleuropein among the present invention.
Embodiment
Embodiment 1, and a kind of method of synthetic secoiridoid glycoside compound is got sal epsom 370mg, potassium primary phosphate 170mg, saltpetre 1900mg, ammonium nitrate 1650mg, calcium chloride 440mg, thiamines 1.0mg, inositol 100mg, boric acid 6.2mg, manganous sulfate 15.6mg, zinc sulfate 8.6mg, Sodium orthomolybdate 0.025mg, copper sulfate 0.025mg, cobalt chloride 0.025mg, potassiumiodide 0.085mg, ferrous sulfate 27.8mg, EDTA sodium salt 37.3mg, 2,4-D 1.0mg, 6-chaff aminoadenine 2.0mg, naphthylacetic acid 3.0mg, sucrose 30000mg, agar 12000mg takes by weighing one by one except that sucrose, the medium component that agar is outer is used deionized water dissolving, mix, take by weighing formula ratio sucrose and agar then, add to dissolve in the above-mentioned solution and add deionized water and be diluted to 1000ml, transfer PH=5.8, through 121 ℃ of sterilizations after 25 minutes solid medium.
On the solid medium that is inoculated in after daphne lilac blade or young shoot or the sterilization of tender stem after the sterilization, in 25 ℃ of incubators, induce and produce callus and cultivated 49 days, take out product, place apparatus,Soxhlet's to add the dehydrated alcohol extraction 2 times of 10 times of weight at every turn, steam ethanol then, get target molecule.Its representative oleuropein structural formula is seen Fig. 1, and its NMR data are listed in the table 1.
Embodiment 2, and the glossy privet blade after will sterilizing in the present embodiment places on the solid medium after the sterilization, induce in 28 ℃ of incubators and cultivate 49 days, take out product, place apparatus,Soxhlet's to add the dehydrated alcohol extraction 2 times of 15 times of weight at every turn, steam ethanol then, target molecule.Other is with embodiment 1.
Insert on the solid medium after getting the sterilization of daphne lilac blade, cultivated 42 days for 28 ℃, the callus that obtains is changed in the liquid nutrient medium, in the shaking culture case, cultivated 21 days for 25 ℃, filter separately culture and nutrient solution.Culture adds the methyl alcohol of 10 times of weight with methanol extraction 2 times at every turn; Nutrient solution adds the methyl alcohol of 3 times of weight with methanol extraction 2 times at every turn.The united extraction thing is a target product.Its representative oleuropein structural formula is seen Fig. 1, and its NMR data are listed in the table 1.
Embodiment 4, in the present embodiment, solid medium is cultivated the gained callus change in 50 liters of reactors that fill liquid nutrient medium, cultivate 18 days for 25 ℃, filter separately culture and nutrient solution, use methanol extraction respectively.Extract is a target product.Other are with embodiment 3.
NMR data (the CD of table 1 oleuropein
3COCD
3)
H δ
H(ppm) J(Hz) C δ
C(ppm)
1 5.91s 1 94.3
3 7.47s 3 154.1
5 3.94dd 9.6，4.0 4 108.7
6a 2.39dd 14.0，9.6 5 31.1
6b 2.70dd 14.0，4.0 6 40.4
8 6.01q 6.8 7 171.6
10 1.64d 6.8 8 124.0
9 129.8
10 13.3
11 167.1
OMe 3.67s OMe 51.4
1′ 4.87d 8.0 1′ 100.1
2′，4′，5′?3.41～3.45m 2′ 74.1
3′ 3.55t 8.8 3′ 77.2
6′a 3.66dd 11.6，6.0 4′ 70.9
6′b 3.87d 11.6 5′ 77.4
6′ 62.3
1″a 4.07dt 10.8，7.2 1″ 65.9
1″b 4.18dt 10.8，7.2 2″ 34.6
2″ 2.74t 7.2 3″ 130.0
4″ 6.73d 1.6 4″ 116.5
7″ 6.74d 8.0 5″ 145.4
8″ 6.56dd 8.0，1.6 6″ 144.1
7″ 115.8
8″ 120.7
Claims (10)
1, a kind of method of synthetic secoiridoid glycoside compound, the invention is characterized in, comprise the preparation of substratum, four steps of extraction of inducing, organizing (cell) cultivation and target molecule of explant: in the explant induction step, be inoculated on the solid medium after getting Oleaceae Genus Syringa, Ligustrum, sweet-scented osmanthus genus, Olea, Jasminum, fontanesia Shu He the plant leaf of Ash genus, leaf bud or rhizome sterilization, cultivated 15-108 days in 15-32 ℃; At the target molecule extraction step, place extractor to use organic solvent refluxing extraction 2-6 hour or cold soaking 8-48 hour the callus of gathering in the crops, remove organic solvent and get target molecule secoiridoid glycoside compound.
2, the method of claim 1, it is characterized in that, contain sal epsom 160-689mg/l in the solid medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6-furfuryladenine, 6-benzyladenine, one of 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, agar 4500-32000mg/l, take by weighing one by one except that sucrose, the medium component that agar is outer, use water dissolution, mixing and thin up are to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
3, method as claimed in claim 2 is characterized in that, also contains pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in the solid medium.
4, as claim 1,2 or 3 described methods, it is characterized in that organic solvent comprises alcohols, ketone and ether organic solvent.
5, method as claimed in claim 4 is characterized in that, organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus weight.
6, a kind of method of synthetic secoiridoid glycoside compound, the invention is characterized in, comprise the preparation of solid medium and liquid nutrient medium, four steps of extraction of inducing, organizing (cell) cultivation and target product of explant: in the explant induction step, be inoculated on the solid medium after getting Oleaceae Genus Syringa, Ligustrum, Olea, Jasminum He the plant leaf of Ash genus, leaf bud or rhizome sterilization, cultivated 14-49 days in 15-32 ℃; Taking out callus again transfers in the liquid nutrient medium in concussion incubator or shaking table or bio-reactor or reactor and cultivated 10-60 days at 18-32 ℃; At the target product extraction step, culture and substratum filtration are earlier separately placed the extractor organic solvent extraction respectively again, remove organic solvent and get target product secoiridoid glycoside compound.
7, method as claimed in claim 6, it is characterized in that, contain sal epsom 160-689mg/l in the liquid nutrient medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6One of-furfuryladenine, 6-benzyladenine, 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, take by weighing the medium component except that sucrose one by one, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get liquid nutrient medium; Solid medium is than increasing the agar composition in the liquid culture based formulas, content is 4500-19500mg/l, its method for making is: take by weighing the medium component except that sucrose, agar, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
8, method as claimed in claim 7 is characterized in that, respectively contains pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in solid medium and the liquid nutrient medium.
9, as claim 6,7 or 8 described methods, it is characterized in that organic solvent comprises alcohols, ketone and ether organic solvent.
10, method as claimed in claim 9 is characterized in that, organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389571C (en) * | 2005-03-25 | 2008-05-21 | 华为技术有限公司 | Method for detecting chain circuit fault between end-to-end notes in mixed network |
| CN105820196A (en) * | 2016-04-21 | 2016-08-03 | 成都中医药大学 | 1,2-secoiridoid compound with neuroprotective effect and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0223880A (en) * | 1988-07-13 | 1990-01-26 | Kanebo Ltd | Production of bitter component of plant of family gentianaceae |
| ES2220090T3 (en) * | 1998-07-23 | 2004-12-01 | Creagri, Inc. | WATER SOLUBLE OLIVE EXTRACT. |
| JP2002128678A (en) * | 2000-10-17 | 2002-05-09 | Tama Seikagaku Kk | Method for producing extract composition containing oleuropein |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389571C (en) * | 2005-03-25 | 2008-05-21 | 华为技术有限公司 | Method for detecting chain circuit fault between end-to-end notes in mixed network |
| CN105820196A (en) * | 2016-04-21 | 2016-08-03 | 成都中医药大学 | 1,2-secoiridoid compound with neuroprotective effect and preparation method thereof |
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| CN100362012C (en) | 2008-01-16 |
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