CN1173043C - Process for synthesizing beta-dihydroagarofuran sesquiterpene polyolester compounds - Google Patents
Process for synthesizing beta-dihydroagarofuran sesquiterpene polyolester compounds Download PDFInfo
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Abstract
The present invention relates to a method for synthesizing beta-dihydroagarofuran sesquiterpene polyolester compounds, which comprises three steps: preparing a culture medium, cultivating tissues and extracting a goal product. The goal product can be cultivated and extracted only through a solid culture medium, and can be cultivated through the solid culture medium firstly and then a liquid culture medium and extracted. The method has the advantages of simple process, easy operation, high yield of products, low production cost and easy industrialized application.
Description
The invention belongs to the biosynthesis technology field of hydroxyl carbonate, particularly a kind of method with the synthetic beta-dihydroagarofuran sesquiterpene polyolester compound of tissue culture method.
The beta-dihydroagarofuran sesquiterpene polyolester compound is the material that a class has insecticidal activity and insect antifeedant, and this compounds has good disinsection effect, and is little to natural enemy lethality, to characteristics such as people and animals' low toxicities, can be used as the high-efficiency low-toxicity environmental-pollution insecticide.But this compounds content in natural phant (existing only in the celastrus angulatus plant) is very low, and the chemical structure complexity, and chemosynthesis is difficulty very, and is very uneconomical, seriously limited its further development and utilization.
It is tissue culture method that the object of the invention is to provide a kind of biosynthetic means that is suitable for the synthetic beta-dihydroagarofuran sesquiterpene polyolester compound of suitability for industrialized production.
For reaching above-mentioned purpose, the present invention adopts following technical scheme: a kind of method of synthetic beta-dihydroagarofuran sesquiterpene polyolester compound comprises solid medium preparation, tissue culture and three steps of target product extraction; In the tissue culture step, be inoculated on the solid medium after getting the sterilization of celastrus angulatus blade or rhizome, cultivated 14-90 days in 16-32 ℃; In the target product extraction step, the callus that forms is used organic solvent reflux extraction 4-6 hour in extractor, boil off organic solvent and get target product beta-dihydroagarofuran sesquiterpene polyolester compound.
Contain sal epsom 185-740mg/l in the solid medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 75-340mg/l, saltpetre 950-3800mg/l, ammonium nitrate 825-3300mg/l, calcium chloride 220-880mg/l, sucrose 15000-60000mg/l, agar 6000-24000mg/l; Thiamines 0.25-1.0mg/l, pyridoxol 0.25-1.0mg/l, nicotinic acid 0.025-0.10mg/l, inositol 50-200mg/l; Boric acid 3.2-12.4mg/l, manganous sulfate 7.8-31.2mg/l; Zinc sulfate 4.3-17.2mg/l, Sodium orthomolybdate 0.125-0.50mg/l, copper sulfate 0.0125-0.050mg/l, cobalt chloride 0.0125-0.05mg/l, potassiumiodide 0.415-1.66mg/l, ferrous sulfate 13.9-55.6mg/l, EDTA sodium salt 18.6-74.6mg/l or EDTA molysite 21.5-86mg/l, 2,4-D 0.1-10mg/l, naphthylacetic acid, naphthalene butyric acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine carboxylic acid, two, three, four kind of 0.1-40mg/l, N
6One of-furfuryladenine, 6-benzyladenine, 6-chaff amido VITAMIN B4, two, three kind of 0.1-30mg/l; Take by weighing above-mentioned substratum composition except that sucrose, agar one by one, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose and agar then and add in the above-mentioned solution and dissolve, transfer PH=5.5-6.5, through sterilize solid medium.
A kind of method of synthetic beta-dihydroagarofuran sesquiterpene polyolester compound comprises that solid medium and liquid nutrient medium preparation, tissue culture and target product extract three steps; In the tissue culture step, be inoculated on the solid medium after getting the sterilization of celastrus angulatus blade or rhizome earlier, cultivated 14-50 days in 16-32 ℃, take out callus again and move in the liquid nutrient medium and in shaking culture case or shaking table, cultivated 14-50 days in 16-32 ℃; In the target product extraction step, culture and nutrient solution filtration are earlier separately used organic solvent extraction more respectively, get the beta-dihydroagarofuran sesquiterpene polyolester compound.
Contain in the liquid nutrient medium: sal epsom 185-740mg/l, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 75-340mg/l, saltpetre 950-3800mg/l, ammonium nitrate 825-3300mg/l, calcium chloride 220-880mg/l, sucrose 15000-60000mg/l; Thiamines 0.25-1.0mg/l, pyridoxol 0.25-1.0mg/l, nicotinic acid 0.025-0.10mg/l, inositol 50-200mg/l; Boric acid 3.2-12.4mg/l, manganous sulfate 7.8-31.2mg/l, zinc sulfate 4.3-17.2mg/l, Sodium orthomolybdate 0.125-0.50mg/l, copper sulfate 0.0125-0.05mg/l, cobalt chloride 0.0125-0.05mg/l, potassiumiodide 0.415-1.66mg/l, ferrous sulfate 13.9-55.6mg/l, EDTA sodium salt 18.6-74.6mg/l or EDTA molysite 21.5-86mg/l; 2,4-D 0.1-10mg/l, naphthylacetic acid, naphthalene butyric acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine carboxylic acid, two, three, four kind of 0.1-30mg/l, N
6One of-furfuryladenine, 6-benzyladenine, 6-chaff amido VITAMIN B4, two, three kind of 0.1-30mg/l; Take by weighing one by one above-mentioned except that sucrose the substratum composition, water-soluble, mix and thin up to desired concn, add the formula ratio sucrose dissolved then, transfer PH=5.5-6.5, through sterilize liquid nutrient medium.Solid medium is than increasing the agar composition in the liquid culture based formulas, content is 6000-24000mg/l, its method for making is: take by weighing the substratum composition except that sucrose, agar, with water dissolution, mix and thin up to desired concn, adding sucrose and agar dissolves, transfer PH=5.5-6.5, through sterilize solid medium.
Fig. 1 is target product representative structural representation among the present invention.
The present invention adopts biological method for tissue culture to synthesize the beta-dihydroagarofuran sesquiterpene polyolester compound, not limited by natural resources, the culture medium of culture medium for extensively adopting in the tissue cultivation in the method, composition is easy to get, contain nitrogen in the culture medium, phosphorus, the a great number of elements such as potassium, boron, zinc, manganese, molybdenum, ketone, cobalt, iodine, iron, the trace elements such as sodium, thiamines, pyridoxol, nicotinic acid, the vitamins and 2 such as inositol, 4-D, methyl α-naphthyl acetate, the naphthalene butyric acid, indole-3-acetic acid, 4 amino-3,5,6-trichloropyridine carboxylic acid, N6The plant growths such as-furfuryladenine, 6-benzyladenine, 6-chaff amido adenine are transferred The joint material can provide tissue to cultivate required various nutritions. The conditions of tissue culture gentleness, extraction Method is simple. Whole method is easy to industrial applications. In two kinds of methods of the present invention, first method Only adopt solid medium to cultivate; Second method then successively adopts solid medium and Liquid Culture Base is cultivated, and Liquid Culture is easier to large-scale industrialization production, can be cultivated by a small amount of callus Obtain a large amount of target products, product yield improves greatly, and production cost is able to decrease.
Further specify the present invention below in conjunction with embodiment.
Embodiment 1, and a kind of method of synthetic beta-dihydroagarofuran sesquiterpene polyolester compound is got sal epsom 370mg, potassium primary phosphate 170mg; Saltpetre 1900mg, ammonium nitrate 1650mg, calcium chloride 440mg, 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 0.83mg, ferrous sulfate 27.8mg, EDTA sodium salt 37.3mg, thiamines 0.5mg, pyridoxol 0.5mg, nicotinic acid 0.05mg, inositol 120mg, 2,4-D 2.5mg, 6-chaff amido VITAMIN B4 1.2mg, naphthylacetic acid 5mg uses water dissolution, mix, thin up is to 1000ml, take by weighing sucrose 36000mg again, agar 14000mg adds to heat in the above-mentioned solution to be made it to dissolve, transfer PH=5.8 with the KOH of 0.1mol/l, through 121 ℃ of sterilizations 20 minutes, cool off solid medium.
With being inoculated on the solid medium after the sterilization of celastrus angulatus blade, in 25 ℃ of incubators, cultivated 42 days, take out the callus that forms, in apparatus,Soxhlet's, add petroleum ether extraction, steam sherwood oil, get target product.
Embodiment 2, and solid medium is implanted in the celastrus angulatus stem segment sterilization back after will sterilizing in the present embodiment, cultivates 28 days in 28 ℃ of incubators, takes out callus, and other is with embodiment 1.
Embodiment 3, and the solid culture based formulas is sal epsom 444mg/l in the present embodiment, potassium primary phosphate 204mg/l, saltpetre 2280mg/l, ammonium nitrate 1980mg/l, calcium chloride 528mg/l, boric acid 7.4mg/l, manganous sulfate 18.8mg/l, zinc sulfate 10.3mg/l, Sodium orthomolybdate 0.30mg/l, copper sulfate 0.03mg/l, cobalt chloride 0.03mg/l, potassiumiodide 0.99mg/l, ferrous sulfate 33.2mg/l, EDTA sodium salt 44.5mg/l, thiamines 0.6mg/l, pyridoxol 0.6mg/l, nicotinic acid 0.06mg/l, inositol 120mg/l, 2,4-D 2mg/l, 6-benzyladenine 1mg/l, indole-3-acetic acid 5mg/l, sucrose 30000mg/l, agar 12000mg/l.Other is with embodiment 1.
Embodiment 4, a kind of method of synthetic beta-dihydroagarofuran sesquiterpene polyolester compound, and in the present embodiment, solid medium and method for making are with embodiment 1.The liquid culture based formulas is: sal epsom 296mg/l, potassium primary phosphate 136mg/l, saltpetre 1520mg/l, ammonium nitrate 1320mg/l, calcium chloride 325mg/l, boric acid 5.0mg/l, manganous sulfate 12.4mg/l, zinc sulfate 6.9mg/l, Sodium orthomolybdate 0.20mg/l, copper sulfate 0.02mg/l, cobalt chloride 0.02mg/l, potassiumiodide 0.66mg/l, ferrous sulfate 22.3mg/l, EDTA sodium salt 29.4mg/l, thiamines 0.4mg/l, pyridoxol 0.4mg/l, nicotinic acid 0.04mg/l, inositol 80mg/1,2,4-D2mg/l, 4-amino-3,5,6-trichloropyridine carboxylic acid 6mg/l, N
6-furfuryladenine 2mg/ll, sucrose 30000mg/l.Method for making is: get above-mentioned except that sucrose composition water-soluble, mix and thin up to desired concn, add sucrose dissolved, transfer PH=6.0, sterilize liquid nutrient medium.Get celastrus angulatus blade sterilization back and implant on the interior solid medium of 25 ℃ of incubators, cultivated 42 days, get callus.The callus immigration is not contained in the liquid nutrient medium of agar, in the shaking culture case, cultivated 14 days for 25 ℃.Filter separately culture and nutrient solution, use methanol extraction respectively, get target product.
Target product representative Celangulin A among the embodiment 1,2,3,4
1H and
13CNMR spectrum data see Table
1, representative Celangulin A structural formula is seen Fig. 1.
Table 1 Celangulin A
1H and
13C NMR spectral data
C δC H δH
1 75.0 1 5.49d
2 67.3 2 5.39dd
3 41.1 3 2.02dd?2.12d
4 72.1 4
5 91.5 5
6 76.8 6 5.22s
7 53.5 7 2.58d
8 73.8 8 5.62dd
9 75.3 9 6.06d
10 50.5 10
11 84.5 11
12 61.7 12 4.87d?4.65d
13 24.2 13 1.78s
14 26.3 14 1.61s
15 30.0 15 1.72s
Ethanoyl
a 169.5?169.6
b 20.5 21.1 1.55s?2.11s
Isobutyryl
a 175.8?176.7
b 34.1 34.3 2.38sept?2.85sept
c 18.4 18.6?19.0?19.1 0.92d?0.95d?1.353d?1.348d
Benzoyl
a 165.7
b 129.4
c 129.2 7.85m
d 128.6 7.42m
e 133.4 7.55m
Claims (2)
1, the synthetic method of a kind of Celangulin A is characterized in that, comprises solid medium preparation, tissue culture and three steps of target product extraction; In the tissue culture step, be inoculated on the solid medium after getting the sterilization of celastrus angulatus blade or rhizome, cultivated 14-90 days in 16-32 ℃; In the target product extraction step, the callus that forms is used organic solvent reflux extraction 4-6 hour in extractor, boil off organic solvent and get target product Celangulin A; Contain sal epsom 185-740mg/l in the solid medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 75-340mg/l, saltpetre 950-3800mg/l, ammonium nitrate 825-3300mg/l, calcium chloride 220-880mg/l, sucrose 15000-60000mg/l, agar 6000-24000mg/l; Thiamines 0.25-1.0mg/l, pyridoxol 0.25-1.0mg/l, nicotinic acid 0.025-0.10mg/l, inositol 50-200mg/l; Boric acid 3.2-12.4mg/l, manganous sulfate 7.8-31.2mg/l; Zinc sulfate 4.3-17.2mg/l, Sodium orthomolybdate 0.125-0.50mg/l, copper sulfate 0.0125-0.050mg/l, cobalt chloride 0.0125-0.05mg/l, potassiumiodide 0.415-1.66mg/l, ferrous sulfate 13.9-55.6mg/l, EDTA sodium salt 18.6-74.6mg/l or EDTA molysite 21.5-86mg/l; 2,4-D0.1-10mg/l, naphthylacetic acid, naphthalene butyric acid, indole-3-acetic acid, 4-amino-3,5, a kind of, two kinds, three kinds or four kinds of 0.1-40mg/l of 6-trichloropyridine carboxylic acid, N
6A kind of, two kinds or three kinds of 0.1-30mg/l of-furfuryladenine, 6-benzyladenine, 6-chaff amido VITAMIN B4; Take by weighing above-mentioned medium component except that sucrose, agar one by one, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose and agar then and add in the above-mentioned solution and dissolve, transfer PH=5.5-6.5, through sterilize solid medium.
2, the synthetic method of a kind of Celangulin A is characterized in that, comprises solid medium and liquid nutrient medium preparation, tissue culture and three steps of target product extraction; In the tissue culture step, be inoculated on the solid medium after getting the sterilization of celastrus angulatus blade or rhizome earlier, cultivated 14-50 days in 16-32 ℃, take out callus again and move in the liquid nutrient medium and in shaking culture case or shaking table, cultivated 14-50 days in 16-32 ℃; In the target product extraction step, culture and nutrient solution filtration are earlier separately used organic solvent extraction more respectively, get target product Celangulin A; Contain sal epsom 185-740mg/l, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 75-340mg/l, saltpetre 950-3800mg/l, ammonium nitrate 825-3300mg/l, calcium chloride 220-880mg/l, sucrose 15000-60000mg/l in the liquid nutrient medium; Thiamines 0.25-1.0mg/l, pyridoxol 0.25-1.0mg/l, nicotinic acid 0.025-0.10mg/l, inositol 50-200mg/l; Boric acid 3.2-12.4mg/l, manganous sulfate 7.8-31.2mg/l; Zinc sulfate 4.3-17.2mg/l, Sodium orthomolybdate 0.125-0.50mg/l, copper sulfate 0.0125-0.05mg/l, cobalt chloride 0.0125-0.05mg/l, potassiumiodide 0.415-1.66mg/l, ferrous sulfate 13.9-55.6mg/l, EDTA sodium salt 18.6-74.6mg/l or EDTA molysite 21.5-86mg/l; 2,4-D0.1-10mg/l, naphthylacetic acid, naphthalene butyric acid, indole-3-acetic acid, 4-amino-3,5, a kind of, two kinds, three kinds or four kinds of 0.1-30mg/l of 6-trichloropyridine carboxylic acid, N
6A kind of, two kinds or three kinds of 0.1-30mg/l of-furfuryladenine, 6-benzyladenine, 6-chaff amido VITAMIN B4; Take by weighing one by one above-mentioned except that sucrose medium component, water-soluble, mix and thin up to desired concn, add the formula ratio sucrose dissolved then, transfer PH=5.5-6.5, through sterilize liquid nutrient medium; Solid medium is than increasing the agar composition in the liquid culture based formulas, content is 6000-24000mg/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, adding sucrose and agar dissolves, transfer PH=5.5-6.5, through sterilize solid medium.
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| CN103073529B (en) * | 2013-01-03 | 2015-09-30 | 西北农林科技大学 | 2-ketone type-β-dihydroagarofuran sesquiterpenoids its preparation method and application in Siberian cocklebur seven |
| CA2939219C (en) * | 2014-02-11 | 2023-02-28 | Mitokinin Llc | Compositions and methods using the same for treatment of neurodegenerative and mitochondrial disease |
| EP3641545A4 (en) | 2017-06-21 | 2021-02-24 | Mitokinin, Inc. | Compositions and methods using the same for treatment of neurodegenerative and mitochondrial disease |
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