CN1184309C - Method for producing tripterygium alkaloids by plant suspension cultivation cell - Google Patents
Method for producing tripterygium alkaloids by plant suspension cultivation cell Download PDFInfo
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Abstract
The present invention relates to a method for producing chief tripterygium alkaloids by plant suspension cultivation cells, which comprises the steps of explant selection, surface sterilization, callus induction, subculture, cell strain selection, solid subculture, suspension culture, bulk callus separation, bulk callus preservation, the separation of granular cell lines with good dispersity, cell line suspension culture, mixing co-culture of suspension culture cell lines and bulk callus, chief alkaloids extraction, etc. The content of the chief tripterygium wilfordii alkaloids of the product obtained with the method is 2.9 times of a plant body. Besides, the present invention has the advantages of the strong stability of cell lines, short production period, high content of medicine components, no environment pollution in production process, etc. Furthermore, the present invention helps to keep the sustainable growth of the nature tripterygium wilfordii source.
Description
Technical field
The application relates to by suspension method cultivation trypterygine vegetable cell and produces the alkaloidal method of tripterygium total.
Background technology
Chinese medicine trypterygine (Tripterygium wilfordii HOOK.f.) is taken from the root of Celastraceae plant trypterygine, is treatment rheumatic and the arthritic conventional medicament of rheumatic class.Studies show that in a large number in recent years has important multiple cancer therapy drug composition, and main component is alkaloid and melampyrin, Tripterin etc.At nature, from a trypterygine seed germination to growing up to medicinal material, about 12-15.The tradition application method is used as medicine with the root skin zone of trypterygine, can utilize part few, adds disposable excavating, and is unfavorable for Sustainable utilization of resources.
People such as Yin Zuohong induce callus from stem, the leaf of trypterygine, and have carried out solid tissue's cultivation, therefrom separate obtaining diterpenic lactones such as triptolide, Triptodiolide.Xia Zhilin etc. isolate 25 compounds from trypterygine histocyte suspension culture, wherein the Triptodiolide that produces through tissue culture is higher 36 times than former plant content.
Cultivate the trypterygine vegetable cell and produce the alkaloidal method of tripterygium total and do not appear in the newspapers with suspension method.The suspension method cell cultures is compared with plate method and is more suitable for large-scale industrial production, but need overcome how technical difficulty, and not all cell can both carry out the suspension method tissue culture.
Present patent application person took to Plant Biotechnology research from 1972.Utilize higher plant cell suspension culture technology, can produce the whole medicinal effective ingredient of thallophyta.This is because after India scientist in 1964 in thorn apple flower pesticide is cultivated, obtains the first strain monoploid whole plant, experimental results show that the totipotency of vegetable cell.Up to now, lot of research and we studies have shown that, the vegetable cell isolated culture has the characteristic of the medicinal effective ingredient of all-round synthetic primary plant.This is the experiment basis and the theoretical basis of this patent.
Summary of the invention
Natural resources for the protection trypterygine; set up the production sequence of Sustainable Development and Utilization resource; adopt the suspension method tissue culture of trypterygine vegetable cell; suitability for industrialized production tripterygium total alkaloid; utilize modern technologies that the pharmaceutical compositions of trypterygine is obtained from the thallophyta of primary nature growth; being converted into from the trypterygine high yielding cell sarain of suitability for industrialized production and obtaining, is the purpose of this patent.
One, primary plant is originated:
The medicinal thallophyta that this patent relates to provides by excavating from Qi native place, Shexian County, Anhui Province town fishscale bamboo hole medicinal herb grower Ke Shangyong for defending Mao Ke plant trypterygine Tripterygium wilfordii HOOK.f..
Two, produce the cultivation of cell strain: referring to Figure of description 1
1, gets thallophyta tender leaf, stem or ripe, the full then seed of positive season growth, as the explant of evoked callus.After getting explant, note fresh-keeping and cleaning.
2, surface sterilization: explant is placed in the triangular flask of 150ml, puts 2-3 gram washing powder or 2-3 and drips Tween-60.Be placed on the water tap weep flushing 2-3 hour.On super clean bench, clip in another (120 ℃, 20 minutes steam sterilizings) 150ml triangular flask of sterilizing with the tweezers of sterilizing carefully then, pour 75% alcohol into, place 60 seconds (dry seeds 90 seconds), then to falling alcohol.Pour 0.1%HgCl sterilization into and kept 12-15 minute, pour out HgCl liquid after, with sterilized water (120 ℃, 20 minutes steam sterilizings) flushing 3 times, in sterilized water, stopped 3-4 minute for the last time.Clip on the thieving paper with the tweezers of sterilizing then and blot.The section or the segment of the explant of taking from stem or leaf being cut into 0.5 centimeter size or length with the sterilization blade.With every seed as an independent explant.
3, evoked callus: the MS minimum medium adds inositol 100mg/L, VB
10.5-1.0mg/L, VB
60.5-1.0mg/L, NA 0.5-1.5mg/L, glycine 2-10mg/L, sucrose 30-45g/L, 2,4-D 1-2mg/L, NAA 0.2-1.0mg/L, agar 6g/L, PH5.8.Through 120 ℃, 20 minutes steam sterilizings, the above-mentioned explant of taking from stem or leaf that cuts is inoculated on the substratum then.Substratum is divided in the 150ml triangular flask, every bottled 40ml, and placing temperature is evoked callus under 27 ℃ ± 2 ℃, dark culture condition.Seed germinates under the light culture condition, then, is seeded on the above-mentioned substratum evoked callus under dark culture condition respectively by upper and lower plumular axis, cotyledon.After 20 days, grow callus.
4, succeeding transfer culture: the callus of Huo Deing under these conditions, transfer on the above-mentioned identical substratum, move into 5-7 piece initial callus in every triangular flask, under the light culture condition (1500LUX fluorescent lamp), every 35-45 days subculture is once.
5, select cell strain: the callus behind 3-4 succeeding transfer culture, pick out vigorous, the granular callus of growth carefully with the sterilization injection needles.
6, the solid subculture is preserved: step 5 is selected vigorous, the granular callus of the growth that obtains, and half enters step 6, and subculture is preserved under above-mentioned identical solid medium and light culture condition.When needing, can save step 1-5, directly from the callus of this step succeeding transfer culture, pick out vigorous, the granular callus of growth, enter into step 7.
7, suspension culture: second half callus that step 5 is picked out enters step 7.Remove the liquid nutrient medium that the agar composition forms with above-mentioned solid medium, dress 75ml liquid medium in every 150ml triangular flask, the callus of every bottle graft kind 1-1.5g fresh weight is put on reciprocating type 80-90 rev/min the shaking table and cultivates, 1500LUX illumination, 27 ℃ ± 2 ℃.Subculture is for the first time made in inoculation back the tenth day, and later per 7 days subcultures once.Outwell 3/4-4/5 nutrient solution and cell at every turn, add fresh same medium then, carry out subculture next time to former liquid level.
8, return solid medium: through the suspension cell line of 5-7 subculture, under aseptic condition, filter with 150 order stainless steel mesh screens, isolate the bulk callus of diameter greater than 5mm, filtrate is filtered with 400 order stainless (steel) wires again, the leaching suspension cell, carefully with inoculating needle or shovel, the suspension cell about each 150ml triangular flask inoculation 0.5g is to above-mentioned solid medium, place under the dark condition, 27 ℃ ± 2 ℃, cultivated for three weeks, enter step 11.
9, separate the bulk callus: separate the diameter picked out bulk callus from step 8 with 150 eye mesh screens, be put on the above-mentioned identical solid medium, enter the subculture preservation of step 10 greater than 5mm.
10, the preservation of bulk callus: the bulk that step 9 is picked out, dispersive callus not, subculture is preserved standby on above-mentioned identical solid medium.
11, isolate good dispersity, particulate state clone: the suspension cell of step 8 after dark cultivation of solid through three weeks, obtains cell mass with the separation of 100 purpose screen clothes, the cell mass that 3-8 cell quick for growing, that particle is tiny formed.Enter into the clone suspension culture of step 12 then.
12, clone suspension culture: the good dispersity that the selecting of step 11 obtains, particulate state clone are by the condition of suspension culture of above-mentioned steps 7, and succeeding transfer culture expands sample, forms the cell suspension system of higher plant.
13, the suspended culture cell of step 12 system is after expanding sample, (per minute 120 changes, the long 8cm of magnetic force rotor at 20L-40L magnetic agitation reactor, diameter 0.8-1.2cm) subculture in, added the bulk callus of step 10 on the 7th day, the bulk callus that adds the 4-5g fresh weight in every bottle of suspension culture, co-cultivation 72 hours.
14, extract total alkaloids: the suspension cell of step 13 is filtered, cell is 45 ℃ ± 1 ℃ oven dry, filtered liquid is concentrated into about 10% of former liquid measure vacuumizing under the freezing condition, press tripterygium total alkaloid extracting method, as the method for " China Dispensary " 1995,6 (4): 12-13 record, extract total alkaloids, as a result, total alkaloid content accounts for 51% in the liquid, and total alkaloids accounts for 49% in the cell.
15, total alkaloids and thallophyta medicinal material compare, qualitative, quantitative assay: adopting the method for document record, compare with primary crude drug content, with the tripterygium total alkaloid that suspended culture cell is produced, is 2.9 times of primary crude drug.
Three, select the genetic stability that cell strain keeps producing cell strain:
The genetic stability of higher plant suspension cell line is to hinder the major obstacle that plant cell engineering is produced the medicinal effective ingredient of thallophyta.Selecting cell strain, to keep its stability be key problem in technology.According to higher plant cell " totipotency " theory, utilize its cells,primordial characteristic, can relatively keep the genetic stability of cell strain.Its technological process is as follows:
1, produces cell strain: the selection method and the suspension culture process of the culture process of the above-mentioned production cell strain of process, the suspension system that obtains from step 13, in case after entering logarithmic growth speed, pick out the sub-fraction suspension cell in time, move on on the following steps 2 described solid mediums.
2, the suspension cell that will select from 1 has been transferred to MS+2,4-D 0.5mg/L+BA or KT 0.1-0.2mg/L, inositol 100mg/L, VB
10.5-1.0mg/L, VB
60.5-1.0mg/L, on the 150ml triangular flask solid medium of NA 0.5-1.5mg/L, CH500-1000mg/L, sucrose 30-45g/L, agar 6g/L, every 35-45 days (preferred 45 days) subculture once, 1500LUX illumination 12 hours, growth was preserved under 27 ℃ ± 2 ℃ conditions.
3,45 days callus of step 2 growth is changeed sub-fraction to the division culture medium of other identical composition of MS+BA 0.5mg/L+KT 0.05mg/L+ step 2, measure the differentiation frequency of its regeneration plant.
4, the high yielding cell sarain of screening total alkaloids output: repeat 1,2,3 steps, constantly filter out high yielding cell sarain.
Higher plant cell has " totipotency " of regeneration plant " totipotency " and synthetic drug usefulness effective ingredient; And previous " totipotency " has only the cell of " tool embryo " just this ability can be arranged.Therefore, measure its embryo sexuality and just can guarantee its genetic stability.Stability in the heredity has been arranged, back one " totipotency " just can have been arranged.Experiment showed, " tool embryo " suspension cell line that we filter out in step 3, can keep the differentiation capability again more than 3 years more than continuous 150 generations (inferior).
The substratum that this patent relates to is that " MS " adds described supplementary component.
This patent has following characteristics:
(1) working condition is not subjected to the influence of physical environments such as region, season, weather, water quality.
(2) with short production cycle, only need tens days.
(3) pharmaceutical compositions content height is the several times of primary plant.
(4) production process does not produce a large amount of slag and effluents, has prevented environmental pollution.
(5) have very high economic benefit and social benefit.
Embodiment
1, gets the seed of mature and plump as explant.
2, seed is placed the triangular flask of 150ml, put 3 gram washing powder, on water tap, weep flushing 2-3 hour.On super clean bench, clip in another (120 ℃, 20 minutes steam sterilizings) 150ml triangular flask of sterilizing with the tweezers of sterilizing carefully then, pour 75% alcohol into, placed 90 seconds, then to falling alcohol.Pour 0.1%HgCl sterilization into and kept 15 minutes, pour out HgCl liquid after, with aseptic water washing 3 times, stopped 3-4 minute in the last sterilized water.Then with the tweezers of sterilizing clip to blot on the thieving paper after, inoculation 1-3 grain seed in each triangular flask.
3, evoked callus: the MS minimum medium adds inositol 100mg/L, VB
10.5mg/L, VB
60.5mg/L, NA 0.5mg/L, glycine 2mg/L, sucrose 30g/L, 2,4-D1mg/L, NAA0.2mg/L, agar 6g/L, PH5.8.Through 120 ℃, 20 minutes steam sterilizings, substratum was divided in the 150ml triangular flask then, and every bottled 40ml is placed under 27 ℃ ± 2 ℃ the dark culture condition.Seed germinates under light is cultivated, and then, is seeded in respectively on the above-mentioned substratum by upper and lower plumular axis, cotyledon, secretly cultivates evoked callus.After 20 days, grow callus.
4, succeeding transfer culture: the callus of Huo Deing under these conditions, transfer on the above-mentioned identical substratum, move into 5-7 piece initial callus in every triangular flask, under the light culture condition (1500LUX fluorescent lamp), every 35-45 days subculture is once.
5, select cell strain: the callus behind 3-4 succeeding transfer culture, pick out vigorous, the granular callus of growth carefully with the sterilization injection needles.
6, the solid subculture is preserved: step 5 is selected vigorous, the granular callus of the growth that obtains, and half enters step 6, and subculture is preserved under above-mentioned identical solid medium and light culture condition.When needing, can save step 1-5, directly from the callus of this step succeeding transfer culture, pick out vigorous, the granular callus of growth, enter into step 7.
7, suspension culture: with quick, granular, the vivid callus of growth that step 5 is chosen, second half enters step 7.Remove the liquid nutrient medium that the agar composition forms with above-mentioned solid medium, dress 75ml liquid medium in every 150ml triangular flask, the callus of every bottle graft kind 1.5g fresh weight is put on reciprocating type 80-90 rev/min the shaking table and cultivates, 1500LUX illumination, 27 ℃ ± 2 ℃.Subculture is for the first time made in inoculation back the tenth day, and later per 7 days subcultures once.Outwell 3/4-4/5 nutrient solution and cell at every turn, add fresh same medium then, carry out subculture next time to former liquid level.
8, return solid medium: step 7 is through suspension cultured cells system, under aseptic condition, filter with 150 order stainless steel mesh screens, isolate the bulk callus of diameter greater than 5mm, filtrate is filtered with 400 order stainless (steel) wires again, the leaching suspension cell, carefully with inoculating needle or shovel, the suspension cell about each 150ml triangular flask inoculation 0.5g is to above-mentioned solid medium, place under the dark condition, 27 ℃ ± 2 ℃, cultivated for three weeks, enter step 11.
9, separate the bulk callus: separate the diameter picked out bulk callus from step 8 with 150 eye mesh screens, be put on the above-mentioned identical solid medium, enter the subculture preservation of step 10 greater than 5mm.
10, the preservation of bulk callus: the bulk that step 9 is picked out, dispersive callus not, subculture is preserved standby on above-mentioned identical solid medium.
11, isolate good dispersity, particulate state clone: the suspension cell of step 8 after dark cultivation of solid through three weeks, obtains cell mass with the separation of 100 purpose screen clothes, the cell mass that 3-8 cell quick for growing, that particle is tiny formed.Enter into the clone suspension culture of step 12 then.
12, clone suspension culture: the good dispersity that the selecting of step 11 obtains, particulate state clone are by the condition of suspension culture of above-mentioned steps 7, and succeeding transfer culture expands sample, forms the cell suspension system of higher plant.
13, the suspended culture cell of step 12 system is after expanding sample, (per minute 120 changes, the long 8cm of magnetic force rotor at 20L-40L magnetic agitation reactor, diameter 0.8-1.2cm) subculture in, added the bulk callus of step 10 on the 7th day, the bulk callus that adds the 4-5g fresh weight in every bottle of suspension culture, co-cultivation 72 hours promptly obtains to be used for industrialization and extracts the alkaloidal tissue culture product of tripterygium total.
Claims (7)
1. one kind is passed through the alkaloidal method of suspension culture vegetable cell production tripterygium total, it is characterized in that may further comprise the steps:
(1), gets thallophyta tender leaf, stem or ripe, the full then seed of positive season growth, as the explant of evoked callus;
(2), surface sterilization: with ordinary method sterilization, afterwards stem or leaf are cut into the section or the section of 0.5 centimeter size or length, dry seeds is independent explant by every;
(3), evoked callus: the MS minimum medium adds inositol 100mg/L, VB
10.5-1.0mg/L, VB
60.5-1.0mg/L, NA 0.5-1.5mg/L, glycine 2-10mg/L, sucrose 30-45g/L, 2,4-D 1-2mg/L, NAA 0.2-1.0mg/L, agar 6g/L, PH 5.8, after the sterilization, the above-mentioned explant that cuts is inoculated on the substratum, placing temperature is evoked callus under 27 ℃ ± 2 ℃, dark culture condition, seed germinates under the light culture condition, then, be seeded on the above-mentioned substratum evoked callus under dark culture condition respectively by upper and lower plumular axis, cotyledon;
(4), succeeding transfer culture: the callus of Huo Deing under these conditions, transfer on the above-mentioned identical substratum, under the light culture condition, every 35-45 days subculture is once;
(5), select cell strain: from the callus behind 3-4 succeeding transfer culture of process, pick out vigorous, the granular callus of growth;
(6), the solid subculture is preserved: step (5) is selected vigorous, the granular callus of the growth that obtains, part subculture under above-mentioned identical solid medium and light culture condition is preserved, when needing, can save step (1)-(5), directly from the callus of this step succeeding transfer culture, pick out vigorous, the granular callus of growth, enter into step (7);
(7), suspension culture: the selected callus that goes out of step (5), another part enters this step, be seeded in above-mentioned solid medium and remove in the liquid nutrient medium of agar composition formation, cultivate under 1500LUX illumination, 27 ℃ ± 2 ℃ the condition, subculture is for the first time made in inoculation back the tenth day, per 7 days subcultures once later on, outwell 3/4-4/5 nutrient solution and cell at every turn, add fresh same medium then, carry out subculture next time to former liquid level;
(8), return solid medium: through the suspension cell line of 5-7 subculture, under aseptic condition, filter with 150 order stainless steel mesh screens, remove the callus of bulk, filtrate is filtered with 400 eye mesh screens, leaves and takes suspension cell, suspension cell is inoculated on the above-mentioned solid medium, place under the dark condition, 27 ℃ ± 2 ℃, cultivated for three weeks;
(9), separate the bulk callus: separate the bulk picked out, dispersive callus not from step (8) with 150 eye mesh screens, be put on the above-mentioned identical solid medium, enter the subculture preservation of step (10);
(10), the preservation of bulk callus: the bulk that step (9) is selected, dispersive callus not, subculture is preserved standby on above-mentioned identical solid medium;
(11), isolate good dispersity, particulate state clone: the suspension cell of step 8 after dark cultivation of solid through three weeks, obtains cell mass with the separation of 100 purpose screen clothes, the cell mass that 3-8 cell quick for growing, that particle is tiny formed.Enter into the clone suspension culture of step 12 then;
(12), clone suspension culture: the good dispersity that the selecting of step (11) obtains, particulate state clone form the cell suspension system of higher plant by the condition of suspension culture succeeding transfer culture of above-mentioned steps (7);
(13), the suspended culture cell of step (12) system after expanding sample, in the magnetic agitation reactor, behind the subculture, added the bulk callus on the 7th day, co-cultivation 72 hours promptly contains the tripterygium total alkaloid in resulting suspension cell and the suspension.
2. method according to claim 1, wherein the composition of the substratum of step (3) evoked callus is: the MS minimum medium adds inositol 100mg/L, VB
10.5mg/L, VB
60.5, NA0.5mg/L, glycine 2mg/L, sucrose 30g/L, 2,4-D 1mg/L, NAA 0.2mg/L, agar 6g/L, PH 5.8.
3. method according to claim 1, wherein the sterilising method of step (2) is: explant is placed in the triangular flask of 150ml, put 2-3 gram washing powder or 2-3 and drip Tween-60, be placed on the water tap, weep flushing 2-3 hour, move into then in another triangular flask of sterilizing, pour 75% alcohol into, placed 60 seconds, placed 90 seconds for dry seeds, then to falling alcohol, pour 0.1%HgCl sterilization into and kept 12-15 minute, pour out HgCl liquid after, with aseptic water washing 3 times, in sterilized water, stopped 3-4 minute for the last time, blot then.
4. method according to claim 1, wherein the operating condition of the described magnetic stirring apparatus of step (13) is that per minute 120 changes the long 8cm of magnetic force rotor, diameter 0.8-1.2cm.
5. method according to claim 1 is characterized in that the liquid culture condition of step (7) is: dress 75ml liquid medium in every 150ml triangular flask, the callus of every bottle graft kind 1-1.5g fresh weight is put on reciprocating type 80-90 rev/min the shaking table and cultivates.
6. according to the described method of arbitrary claim among the claim 1-5, also further comprise the step of selecting high yielding cell sarain:
(1), produce cell strain: the suspension system that Accessory Right requires the step (13) of the arbitrary claim among the 1-5 to obtain, enter logarithmic growth speed after, pick out the part suspension cell in time, move on on the described solid medium of following steps (2);
(2), the suspension cell that will select from (1) is transferred to and is contained MS+2,4-D 0.5mg/L+BA or KT 0.1-0.2mg/L, inositol 100mg/L, VB
10.5-1.0mg/L, VB
60.5-1.0mg/L, on the solid medium of NA 0.5-1.5mg/L, CH 500-1000mg/L, sucrose 30-45g/L, agar 6g/L, every 35-45 days subculture once, 1500LUX illumination 12 hours, growth was preserved under 27 ℃ ± 2 ℃ conditions;
(3), step (2) subculture callus is once changeed a part to the division culture medium of other identical composition of MS+BA 0.5mg/L+KT 0.05mg/L+ step (2), measure the differentiation frequency of its regeneration plant;
(4), screening high yielding cell sarain: repeat (1), (2), (3) step, constantly filter out high yielding cell sarain.
7. method according to claim 6 is characterized in that the once used time of step (2) subculture is 45 days.
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| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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| CN101638669B (en) * | 2009-09-09 | 2011-11-02 | 成都大学 | Method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture |
| FR2952072B1 (en) * | 2009-11-05 | 2013-09-27 | Pf Medicament | PROCESS FOR PRODUCING TRIPTOLIDE |
| CN103004590B (en) * | 2012-12-06 | 2013-12-18 | 福建农林大学 | Chinese narcissus callus preservation and tissue culture method |
| CN104020227B (en) * | 2014-05-21 | 2015-08-19 | 浙江理工大学 | A kind of tissue culture method produces the alkali content assay method of steroid alkaloid |
| CN104450787A (en) * | 2014-10-13 | 2015-03-25 | 皖西学院 | Method for producing alkaloid of anoectochilus roxburghii by suspension cultivation of cells |
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| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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