CN101139571B - Method for producing saussurea involucrate secondary metabolites - Google Patents
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Abstract
The invention discloses a method for producing secondary metabolities of saussurea involucrata, which comprises such procedures as: 1) culturing in a dedifferentiation way the outer colonizing body of saussurea involucrata to get wound-recovering tissue of saussurea involucrata, taming the wound-recovering tissue of saussurea involucrata to get a loose and rapidly-growing cell system; 2) inducing and culturing in a basic culture media with addition of colchicin the cell system of wound-recovering tissue of saussurea involucrata got in 1) to get a polyploid cell system, then culturing in an expansive way to get a lot of polyploid cells of saussurea involucrata; 3) inoculating the polyploid cells of saussurea involucrate got from 2) in a synthesizing culture media to get cells rich with secondary metabolities of saussurea involucrate. The method in the invention is for culturing and producing secondary metabolities of saussurea involucrata by using saussurea involucrata cells, can steadily achieve the industrialized production of such metabolities of saussurea involucrata as saussurea involucrata flavone. The invention has a low production cost, short culture period, is not influenced by natural environment and climate, and can achieve yearly production.
Description
Technical field
The present invention relates to a kind of method of producing saussurea involucrate secondary metabolites.
Background technology
Saussurea involucrata belongs to composite family (Compositae) cynara scolymus family (Trib.CynareaeLess.) phoenix hair Chrysanthemum (SaussureaDC.) plant, is extremely frigid zones rare Chinese herbal medicine commonly used among the people.The record of saussurea involucrata is just arranged as far back as 8th century Tibetan's Drugdocs in ancient times " month king's medicine treasure ", all on the books in " western doctor's allusion quotation ", " territory, northwest note ", " blue coloured glaze ", " brilliant pearl book on Chinese herbal medicine " and Qing Dynasty's supplementary Amplifications of the Compendium of Materia Medica afterwards, be the ethnic drug commonly used of highlandss such as Tibet, Xinjiang, Qinghai, Gansu, Yunnan, Sichuan." Xinjiang herbal medicine " claims: saussurea involucrata hardship warm in nature, little, function expelling wind and dampness have special efficacy to curing rheumatism.Prove through the clinical pharmacy research practice, the saussurea involucrata all herbal medicine, have restore menstrual flow and invigorate blood circulation, silt is loose in warm palace, dispelling cold and removing dampness, strong muscle is supporing yang, the function of hemostasis, detumescence, clinical pain, lung cold cough, palace that treatment acute and chronic rheumatic arthritis the causes illnesss such as stomachache, amenorrhoea, retention of placenta, impotence and measles without adequate eruption of trembling with fear that are mainly used in.In recent years, saussurea involucrata is receiving much concern aspect relieving inflammation and relaxing pain, antiearly pregnancy, anti-ageing the waiting for a long time as ethnic drug.Along with deepening continuously of fundamental research, show that saussurea involucrata has to remove free radical, antifatigue, strengthening immunity, antibiotic, spasmolysis, calmness, step-down, anti-gastric-ulcer and antitumor action.
Isolated nearly hundred kinds of compounds at present from saussurea involucrata, wherein main active ingredient has costunolide, dehydro-, lignanoid, arctinin, saussurea involucrata flavones, apigenin, parahydroxyacet-ophenone, Umbelliferone, various secondary metabolite, jaceosidin, hispidulin and saussurea involucrata polysaccharide etc. to be the effective constituent with various pharmaceutical actives.At present, the saussurea involucrata product has also obtained widespread use in makeup and health products trade except being used for medicine.
China is distributed with multiple saussurea involucrata, as Saussurea laniceps (Saussurea laniceps Hand.-Mazz.), Saussurea medusa (S.medusa Maxim.), Herba Saussureae Involueratae (S.involucrata Kar.Et Kir.) and Tibet saussurea involucrata (S.tridactyla Sch-Bip.).Wherein the snowy again rabbit of Tibet saussurea involucrata (S.gossypiphoraD.Don.), three finger snow hare (S.tridactyla Sch-Bip.ex Hook.f), starlike snow hare (S.stella.Maxim.), Herba Saussureae Involueratae (S.aalpina L) and clump strain snow are exempted from son (S.tridactyyla Sch-Bip var.maidugonla S.W.Liou.) and are waited several, are grown in the high mountain flowstone beach or crack of stone of height above sea level 4200-5200m scope.Tibet saussurea involucrata snow hare is the Snow Lotus Herb of some existence in the high stone riprap beach of height above sea level 4200-5200 rice, and its plant height is 20-40cm, and cauline leaf is red-purple, and epidermis is all over draping over one's shoulders white-colored hairs, and plant type is perfectly round attractive in appearance, and shape is like snow hare.
Because the habitat of saussurea involucrata all is among the snow mountain valley, boundless and indistinct no signs of human habitation ground, up to the present also can't carry out the big area artificial culture, still excavates based on the field.And the distribution range of saussurea involucrata is very narrow and small, disperses vegetatively, and wild resource is very limited, and the ultimate production of adding up in the main place of production such as Xinjiang, Tibet, Gansu and Yunnan also can not surpass 4 tons, and excavates in a large number the natural resources of saussurea involucrata is seriously damaged.In the face of pharmaceuticals, makeup and the such huge market of protective foods, starting material are obviously not enough.
Summary of the invention
The purpose of this invention is to provide a kind of method of producing saussurea involucrate secondary metabolites.
The method of production saussurea involucrate secondary metabolites provided by the present invention comprises the steps:
1) saussurea involucrata outer grown body and carried out dedifferentiation and cultivate and obtain the saussurea involucrata callus, with the saussurea involucrata callus through subculture domestication cultivate and obtain the loose clone of growth fast;
2) saussurea involucrata callus inducing culture in the minimum medium that adds colchicine that step 1) obtained is obtained polyploid cell system, carry out amplification cultivation then and obtain a large amount of snow lotus polyploid cells;
3) with step 2) the snow lotus polyploid cell inoculation that obtains cultivates the cell that obtains being rich in saussurea involucrate secondary metabolites in synthetic medium;
Described synthetic medium is for having added 20-60g/L glucose on the basis of minimum medium, 0.1-5mg/L α-naphthylacetic acid or 2,4-D or indolylacetic acid, 0.1-5mg/L6-Bian aminopurine or kinetin, 50-100mg/L phenylalanine, 50-100mg/L tyrosine, 50-150ml/L Sucus Mali pumilae, 1-20mg/L styracin, 1-40mg/L cinnamophenone, 10-50 μ l/L diacetyl oxide, 5-50mg/L2-ketoisocaproic, 1-4mg/L vitamins C, 2-10mg/L ethrel and 1-6mg/L dormin, pH value are the substratum of 5.5-7.0.
Described saussurea involucrata explant is selected from the young stem of saussurea involucrata, blade, stem-tip tissue or marrow tissue; Described dedifferentiation culture condition is for being 18-25 ℃ in temperature, and intensity of illumination is 1000-3000Lux, and cultivated under the irradiation condition 1-2 month at 12-16 hour every day.
Described dedifferentiation substratum adds 0.1-5mg/L α-naphthylacetic acid (NAA) or 2 on the basis of minimum medium, 4-D or indolylacetic acid (IAA), 0.1-5mg/ rise 6-Bian aminopurine (6-BA) or kinetin (KT), the substratum that 20-60g/L sucrose or glucose obtain.
In the described step 1), it is to add 20-50g/L sucrose, 0.1-5mg/L α-naphthylacetic acid (NAA) or 2 on the basis of minimum medium that described subculture domestication is cultivated with substratum, 4-D or indolylacetic acid (IAA), 0.1-5mg/L6-Bian aminopurine (6-BA) or the substratum that obtains of kinetin (KT) and 1-4mg/L vitamins C.
In the described method, described subculture domestication culture condition all can be 20-25 ℃, cultivates under the lucifuge condition.
Described step 2) in, the concentration of described colchicine is 10-40ml/L.
Described step 2) in, the inducing culture condition of described saussurea involucrata callus is at 20-30 ℃, under the dark condition, cultivates 5～10 days.
Described step 2) in, described amplification cultivation substratum is to be to add 20-40g/L sucrose on the basis of minimum medium, 0.1-5mg/L α-naphthylacetic acid (NAA) or 2,4-D or indolylacetic acid (IAA), 0.1-5mg/L6-Bian aminopurine (6-BA) or kinetin (KT) and 1-4mg/L vitamins C and the substratum that obtains.
Above-mentioned minimum medium is B5 medium.
Described amplification cultivation is at 20-25 ℃, cultivates 3-4 week under the lucifuge condition.
Described step 2) in, also be included in and described polyploid cell carried out the subculture domestication before the amplification cultivation and cultivate, described subculture domestication is cultivated to described polyploid cell is carried out succeeding transfer culture more than 3 generations in described subculture domestication substratum.
In the described step 3), the inoculum size of described snow lotus polyploid cell is 10～30% (weight percentages);
In the described step 3), described synthetic culture condition is at 20-30 ℃, lucifuge, and 80～120rpm/ divided on the shaking table of rotating speed shaking culture 3～7 days;
In the described method, also comprise the described cell that is rich in saussurea involucrate secondary metabolites is obtained crude extract with solvent extraction, obtain saussurea involucrate secondary metabolites through purifying again; Described solvent is ethanolic soln or the methanol solution of 50-70% for the quality percentage composition.
Method of the present invention utilizes viable cell as microreactor, takes the metabolic regulation technology to promote the synthetic of saussurea involucrate secondary metabolites fast, can be in 3～7 days with saussurea involucrata in the content of secondary metabolite saussurea involucrata flavones bring up to more than 5% of dry cell weight.Method of the present invention is produced saussurea involucrate secondary metabolites with the snow lotus polyploid cell cultures, the suitability for industrialized production of realization saussurea involucrate secondary metabolites that can be stable (as the saussurea involucrata flavones), and its production cost is lower, culture cycle is short, be not subjected to physical environment and climatic influences, can produce in the realization anniversary.
Embodiment
Embodiment 1, utilize saussurea involucrata isolated cells large scale culturing to produce saussurea involucrate secondary metabolites
1, the acquisition of the callus of Tibet saussurea involucrata
The young stem that Tibet saussurea involucrata three is referred to snow hare as explant through 70% ethanol surface disinfection 50 seconds, and then put into 5% chlorine bleach liquor's sterilization 20 minutes, behind aseptic water washing three times, under aseptic condition, the tissue behind the surface disinfection is cut into the square square of 0.5mm, be inoculated in the above-mentioned inclined-plane dedifferentiation substratum, transfer to 25 ℃, 2000Lux, carry out inducing culture under the irradiation condition at 12 hours every day, forms callus after 1 month;
Dedifferentiation culture medium preparation: in B5 medium, add 6-Bian aminopurine (6-BA) of 1mg/L α-naphthylacetic acid (NAA), 2mg/L, 20g/L sucrose, with acid or alkali the pH value is adjusted to 6.0 then, add 1.9g/LGellan Gum again, at 115 ℃, after 0.1MPa pressure was sterilized 15 minutes down, through cooling bevel dedifferentiation substratum.
The prescription of B5 medium (Gamborg, 1966): CaCl
2H
2O 150 mg/litre, KNO
31000 mg/litre, KCl 300 mg/litre, (NH
4)
2SO
4200 mg/litre, MgSO
47H
2O 250 mg/litre, NaH
2PO
4H
2The O90 mg/litre, Na
2HPO
430 mg/litre, FeSO
47H
2O 13.9 mg/litre, Na
2-EDTA18.6 mg/litre, ZnSO
47H
2O 3.0 mg/litre, CuSO
45H
2O 0.25 mg/litre, H
3BO
33.0 mg/litre, KI0.75 mg/litre, CoCl
26H
2O 0.25 mg/litre, Na
2MoO
42H
2O 0.25 mg/litre, MnSO
46H
2O 10.0 mg/litre, inositol 100 mg/litre, nicotinic acid 0.5 mg/litre, vitamin (B1) 0.5 mg/litre, pyridoxine hydrochloride (B6) 0.5 mg/litre, calcium pantothenate 0.4 mg/litre, vitamin H 0.00025 mg/litre, vitamins B
20.015 mg/litre, folic acid 0.015 mg/litre, choline chloride 60 0.2 mg/litre, vitamins C 0.4 mg/litre, M-nitro benzoic acid 0.2 mg/litre.
2, the domestication of the subculture of Tibet saussurea involucrata callus is cultivated
The described callus of formation of inducing of step 1 is seeded in the subculture domestication cultivation of carrying out in the following subculture domestication substratum more than 3 times, and culture condition is 20 ℃ of room temperatures, and lucifuge condition, culture cycle were 3 weeks.In subculture domestication culturing process, filter out the short texture and speed of growth callus cell system faster;
Subculture domestication culture medium preparation: the α-naphthylacetic acid (NAA) that on the basis of B5 medium, adds 0.5mg/L, 6-Bian aminopurine (6-BA) of 2mg/L, the 1mg/L vitamins C, 20g/L sucrose, 1.9g/LGellan Gum, through 115 ℃, 0.1MPa pressure sterilization 15 minutes is down tamed substratum after dull and stereotyped subculture is made in cooling.
3, Tibet snow lotus polyploid cell induces
In above-mentioned B5 medium, add the 10mg/L colchicine, make the liquid inducing culture, the short texture speed of growth that step 2 is obtained callus cell system faster is seeded in this inducing culture, at 22 ℃, lucifuge, 100rpm/ divided on the shaking table of rotating speed shaking culture 10 days.
Inoculate behind the collecting cell in the subculture domestication substratum of no colchicine, the subculture domestication is cultivated more than three times repeatedly, screens the polyploid cell system of the speed of growth faster (speed of growth surpasses 5 times of inoculating cell weight/culture cycle) by the biological accumulation flow measurement; After cell aceto-camine or silver dyeing, show that by microscopic inspection this cell is a poikiloploid clone.
System is seeded on the following amplification culture medium with above-mentioned polyploid cell, at 25 ℃, under the dark condition, cultivated for 4 weeks after, cell concentration more than 5 times of former inoculating cell amount that can increase.
Amplification culture medium: on the basis of B5 medium, add sucrose 30g/L, α-naphthylacetic acid (NAA) 1mg/L, 6-Bian aminopurine (6-BA) 1mg/L, the 1mg/L vitamins C, with acid or alkali the pH value is adjusted to 6 then, at 115 ℃, 0.1MPa pressure was sterilized 15 minutes down after amplification culture medium is made in cooling.
4, saussurea involucrate secondary metabolites is synthetic
The snow lotus polyploid cell inoculation of the described amplification cultivation of step 3 is arrived in the following synthetic medium, at 25 ℃, the lucifuge condition, suspension culture is 5 days in 100 rev/mins shaking table or the macro-organism reactor, filter harvested cell, but nutrient solution Returning reactor after adjusting continues to recycle.
Synthetic medium: on the basis of B5 medium, add glucose 40g/L, α-naphthylacetic acid (NAA) 0.5mg/L, 6-Bian aminopurine (6-BA) 0.5mg/L, add the combination precursor again and (add phenylalanine 50mg/L, tyrosinase 15 0mg/L, fresh cider 50ml/L, styracin 5mg/L, cinnamophenone 5mg/L) and combination metabolic regulation material (diacetyl oxide 10 μ l/L, 2-oxoglutaric acid 10mg/L, vitamins C 4mg/L, ethrel 2mg/L and dormin 1mg/L), with acid or alkali the pH value is adjusted to 6, at 115 ℃, 0.1MPa pressure sterilization was down made synthetic medium after 15 minutes.
After synthetic cultivation finishes, detect the synthetic content of cultivating the saussurea involucrate secondary metabolites in the saussurea involucrata cell that obtains with HPLC, the result shows that the saussurea involucrata flavones content accounts for more than 5% of dry cell weight.
Embodiment 2, utilize saussurea involucrata isolated cells large scale culturing to produce saussurea involucrate secondary metabolites
1, the acquisition of the callus of Tibet saussurea involucrata
With the stem apex of Tibet saussurea involucrata snow hare as explant through 70% ethanol surface disinfection 50 seconds, and then put into 8% chlorine bleach liquor's sterilization 15 minutes, behind aseptic water washing three times, under aseptic condition, the tissue behind the surface disinfection is cut into the square square of 0.5mm, be inoculated in the above-mentioned inclined-plane dedifferentiation substratum, transfer to 25 ℃, 2000Lux, carry out inducing culture under the irradiation condition at 14 hours every day, forms callus after 1 month;
Dedifferentiation culture medium preparation: in B5 medium, add the indolylacetic acid (IAA) of 2mg/L, the kinetin (KT) of 5mg/L, 25g/L sucrose, with acid or alkali the pH value is adjusted to 5.5-7.0 then, add 1.9g/LGellan Gum again, at 115 ℃, after 0.1MPa pressure was sterilized 15 minutes down, through cooling bevel dedifferentiation substratum.
2, the domestication of the subculture of Tibet saussurea involucrata callus is cultivated
The described callus of inducing formation of step 1 is seeded in carries out more than 3 times the subculture domestication in the following subculture domestication substratum and cultivate, culture condition is 22 ℃ of room temperatures, and lucifuge condition, culture cycle were 4 weeks.In subculture domestication culturing process, the screening short texture and the speed of growth be callus cell faster;
Subculture domestication culture medium preparation: the α-naphthylacetic acid (NAA) that adds 2mg/L on the basis of B5 medium, 3mg/L kinetin (KT), the 2mg/L vitamins C, 25g/L sucrose, 1.9g/L Gellan Gum, through 115 ℃, 0.1MPa pressure sterilization 15 minutes is down tamed substratum after subculture is made in cooling.
3, Tibet snow lotus polyploid cell induces
In B5 medium, add the 20mg/L colchicine respectively, make the liquid inducing culture, the short texture speed of growth that step 2 obtains callus faster is seeded in the inducing culture, at 25 ℃, lucifuge, 110rpm/ divided on the shaking table of rotating speed the vibration inducing culture 9 days.
Inoculate behind the cell behind the collection vibration inducing culture in the subculture domestication substratum of no colchicine, the subculture domestication is cultivated more than three times repeatedly, screens the speed of growth by the biological accumulation flow measurement and reaches 5 times of polyploid cell systems more than inoculating cell weight/culture cycle; After cell aceto-camine or silver dyeing, show that by microscopic inspection this cell is a poikiloploid clone.
System is seeded on the following amplification culture medium with above-mentioned polyploid cell, at 25 ℃, under the dark condition, cultivated for 3 weeks after, obtain the stable polyploid cell of raised growth.
Amplification culture medium: on the basis of B5 medium, add sucrose 30g/L, indolylacetic acid (IAA) 1mg/L, kinetin (KT) 1mg/L, the 2mg/L vitamins C, with acid or alkali the pH value is adjusted to 5.5-7.0 then, at 115 ℃, 0.1MPa pressure was sterilized 15 minutes down after amplification culture medium is made in cooling.
4, saussurea involucrate secondary metabolites is synthetic
The snow lotus polyploid cell inoculation of the described amplification cultivation of step 3 is arrived in the following synthetic medium, at 28 ℃, the lucifuge condition, suspension culture is 6 days in 90 rev/mins shaking table or the macro-organism reactor, filter harvested cell, but nutrient solution Returning reactor after adjusting continues to recycle.
Synthetic medium: on the basis of B5 medium, add glucose 50g/L, α-naphthylacetic acid (NAA) 0.5mg/L, kinetin (KT) 1mg/L, add the combination precursor again and (add phenylalanine 100mg/L, tyrosine 100mg/L, fresh cider 100ml/L, styracin 20mg/L, cinnamophenone 40mg/L) and combination metabolic regulation material (diacetyl oxide 50 μ l/L, 2-oxoglutaric acid 50mg/L, vitamins C 4mg/L, ethrel 10mg/L and dormin 6mg/L), with acid or alkali the pH value is adjusted to 5.5-7.0, at 115 ℃, 0.1MPa pressure sterilization was down made synthetic medium after 15 minutes;
After synthetic cultivation finishes, detect the synthetic content of cultivating the saussurea involucrate secondary metabolites in the saussurea involucrata cell that obtains with HPLC, the result shows that the saussurea involucrata flavones content accounts for more than 5% of dry cell weight.
Embodiment 3, utilize saussurea involucrata isolated cells large scale culturing to produce saussurea involucrate secondary metabolites
1, the acquisition of the callus of Tibet saussurea involucrata
The stem apex of Tibet saussurea involucrata clump strain snow being exempted from son as explant through 70% ethanol surface disinfection 30 seconds, and then put into 10% chlorine bleach liquor's sterilization 15 minutes, behind aseptic water washing three times, under aseptic condition, the tissue behind the surface disinfection is cut into the square square of 0.5mm, be inoculated in the above-mentioned inclined-plane dedifferentiation substratum, transfer to 25 ℃, 2000Lux, carry out inducing culture under the irradiation condition at 16 hours every day, forms callus after 1 month;
Dedifferentiation culture medium preparation: in B5 medium, add 2 of 0.1mg/L, 6-Bian aminopurine (6-BA) of 4-D, 0.1mg/L, 30g/L sucrose, with acid or alkali the pH value is adjusted to 5.5-7.0 then, add 6-8g/L agar again, at 115 ℃, after 0.1MPa pressure was sterilized 15 minutes down, through cooling bevel dedifferentiation substratum.
2, the domestication of the subculture of Tibet saussurea involucrata callus is cultivated
The described callus of inducing formation of step 1 is seeded in carries out more than 3 times the subculture domestication in the following subculture domestication substratum and cultivate, culture condition is 22 ℃ of room temperatures, and lucifuge condition, culture cycle were 4 weeks.In the succeeding transfer culture process, the screening short texture and the speed of growth be callus cell system faster;
Subculture domestication culture medium preparation: add 2 of 0.1mg/L on the basis of B5 medium, 4-D, 0.1mg/L 6-Bian aminopurine (6-BA), the 3mg/L vitamins C, 30g/L sucrose, 6g/L agar, through 120 ℃, 0.15MPa pressure sterilization 15 minutes is down tamed substratum after subculture is made in cooling.
3, Tibet snow lotus polyploid cell induces
In above-mentioned B5 medium, add the 40mg/L colchicine respectively, make the liquid inducing culture, the short texture speed of growth that step 2 obtains callus faster is seeded in this inducing culture, at 25 ℃, lucifuge, 100rpm/ divided on the shaking table of rotating speed shaking culture 7 days.Inoculate behind the collecting cell in the subculture domestication substratum of no colchicine, the subculture domestication is cultivated more than three times repeatedly, screens the speed of growth by the biological accumulation flow measurement and reaches 5 times of polyploid cell systems more than inoculating cell weight/culture cycle; After cell aceto-camine or silver dyeing, show that by microscopic inspection this cell is a poikiloploid clone.
System is seeded on the following amplification culture medium with above-mentioned polyploid cell, at 22 ℃, under the dark condition, cultivated for 4 weeks after, obtain the polyploid cell system of growing stable.
Amplification culture medium: on the basis of B5 medium, add sucrose 30g/L, 2,4-D0.5mg/L, (6-BA) 0.8mg/L, 3mg/L vitamins C are adjusted to 5.5-7.0 with acid or alkali with the pH value to 6-Bian aminopurine then, at 120 ℃, 0.15MPa pressure was sterilized 15 minutes down after amplification culture medium is made in cooling.
4, saussurea involucrate secondary metabolites is synthetic
The snow lotus polyploid callus cell of the described amplification cultivation of step 3 is inoculated in the following synthetic medium, at 25 ℃, the lucifuge condition, suspension culture is 7 days in 110 rev/mins shaking table or the macro-organism reactor, filter harvested cell, but nutrient solution Returning reactor after adjusting continues to recycle.
Synthetic medium: on the basis of B5 medium, add glucose 60g/L, 2,4-D1mg/L, 6-Bian aminopurine (6-BA) 1mg/L, add the combination precursor again and (add phenylalanine 70mg/L, tyrosine 70mg/L, fresh cider 80ml/L, styracin 15mg/L, cinnamophenone 20mg/L) and combination metabolic regulation material (add diacetyl oxide 30 μ l/L, 2-oxoglutaric acid 30mg/L, Catergen mg/L, ethrel 7mg/L and dormin 4mg/L), with acid or alkali the pH value is adjusted to 5.5-7.0, at 115 ℃, 0.1MPa pressure sterilization was down made synthetic medium after 15 minutes;
After synthetic cultivation finishes, detect the synthetic content of cultivating the saussurea involucrate secondary metabolites in the saussurea involucrata cell that obtains with HPLC, the result shows that the saussurea involucrata flavones content accounts for more than 5% of dry cell weight.
Embodiment 4, utilize saussurea involucrata isolated cells large scale culturing to produce saussurea involucrate secondary metabolites
1, the acquisition of the callus of Tibet saussurea involucrata
With young stem, stem apex, blade or the marrow tissue of Tibet saussurea involucrata Herba Saussureae Involueratae as explant through 70% ethanol surface disinfection 50 seconds, and then put into 5% chlorine bleach liquor's sterilization 20 minutes, behind aseptic water washing three times, under aseptic condition, the tissue behind the surface disinfection is cut into the square square of 0.5mm, be inoculated in the above-mentioned inclined-plane dedifferentiation substratum, transfer to 25 ℃, 2000Lux, carry out inducing culture under the irradiation condition at 12 hours every day, forms callus after 1 month;
Dedifferentiation culture medium preparation: in B5 medium, add 6-Bian aminopurine (6-BA) of 0.1mg/L α-naphthylacetic acid (NAA), 0.1mg/L, 20g/L sucrose, with acid or alkali the pH value is adjusted to 6.0 then, add 1.9g/L Gellan Gum again, at 115 ℃, after 0.1MPa pressure was sterilized 15 minutes down, through cooling bevel dedifferentiation substratum.
2, the domestication of the subculture of Tibet saussurea involucrata callus is cultivated
The described callus of formation of inducing of step 1 is seeded in the subculture domestication cultivation of carrying out in the following subculture domestication substratum more than 3 times, and culture condition is 20 ℃ of room temperatures, and lucifuge condition, culture cycle were 3 weeks.In the succeeding transfer culture process, filter out the short texture and the speed of growth faster callus carry out subculture domestication and cultivate;
Subculture domestication culture medium preparation: the α-naphthylacetic acid (NAA) that adds 0.1mg/L on the basis of B5 medium, 0.5mg/L 6-Bian aminopurine (6-BA), the 1mg/L vitamins C, 20g/L sucrose, 1.9g/L GellanGum, through 115 ℃, 0.1MPa pressure sterilization 15 minutes is down tamed substratum after dull and stereotyped subculture is made in cooling.
3, Tibet snow lotus polyploid cell induces
Add the 10mg/L colchicine in B5 medium, make the liquid inducing culture, the short texture speed of growth that step 2 obtains callus faster is seeded in this inducing culture, at 22 ℃, and lucifuge, 100rpm/ divided on the shaking table of rotating speed shaking culture 10 days.Inoculate behind the collecting cell in the subculture domestication substratum of no colchicine, the subculture domestication is cultivated more than three times repeatedly, screens the polyploid cell system of the speed of growth faster (speed of growth surpasses 5 times of inoculating cell weight/culture cycle) by the biological accumulation flow measurement; After cell aceto-camine or silver dyeing, show that by microscopic inspection this cell is a poikiloploid clone.
System is seeded on the following amplification culture medium with above-mentioned polyploid cell, at 25 ℃, under the dark condition, cultivated for 4 weeks after, cell concentration can increase more than 5 times.
Amplification culture medium: on the basis of B5 medium, add sucrose 30g/L, α-naphthylacetic acid (NAA) 0.1mg/L, 6-Bian aminopurine (6-BA) 0.1mg/L, the 4mg/L vitamins C,, with acid or alkali the pH value is adjusted to 6 then, at 115 ℃, 0.1MPa pressure was sterilized 15 minutes down after amplification culture medium is made in cooling.
4, saussurea involucrate secondary metabolites is synthetic
The snow lotus polyploid cell inoculation of the described amplification cultivation of step 3 is arrived in the following synthetic medium, at 25 ℃, the lucifuge condition, suspension culture is 5 days in 100 rev/mins shaking table or the macro-organism reactor, filter harvested cell, but nutrient solution Returning reactor after adjusting continues to recycle.
Synthetic medium: on the basis of B5 medium, add glucose 40g/L, indolylacetic acid (IAA) 0.5mg/L, 6-Bian aminopurine (6-BA) 0.5mg/L, add the combination precursor again and (add phenylalanine 50mg/L, tyrosinase 15 0mg/L, fresh cider 50ml/L, styracin 5mg/L, cinnamophenone 5mg/L) and combination metabolic regulation material (add diacetyl oxide 10 μ l/L, 2-oxoglutaric acid 10mg/L, vitamins C 1mg/L, ethrel 2mg/L and dormin 1mg/L), with acid or alkali the pH value is adjusted to 6, at 115 ℃, 0.1MPa pressure sterilization was down made synthetic medium after 15 minutes.
After synthetic cultivation finishes, detect the synthetic content of cultivating the saussurea involucrate secondary metabolites in the saussurea involucrata cell that obtains with HPLC, the result shows that the saussurea involucrata flavones content accounts for more than 5% of dry cell weight.
Embodiment 5, utilize saussurea involucrata isolated cells large scale culturing to produce saussurea involucrate secondary metabolites
1, the acquisition of the callus of Tibet saussurea involucrata
With young stem, stem apex, blade or the marrow tissue of starlike snow hare of Tibet saussurea involucrata as explant through 70% ethanol surface disinfection 50 seconds, and then put into 5% chlorine bleach liquor's sterilization 20 minutes, behind aseptic water washing three times, under aseptic condition, the tissue behind the surface disinfection is cut into the square square of 0.5mm, be inoculated in the above-mentioned inclined-plane dedifferentiation substratum, transfer to 25 ℃, 2000Lux, carry out inducing culture under the irradiation condition at 12 hours every day, forms callus after 1 month;
Dedifferentiation culture medium preparation: in B5 medium, add 6-Bian aminopurine (6-BA) of 1mg/L α-naphthylacetic acid (NAA), 0.8mg/L, 20g/L sucrose, with acid or alkali the pH value is adjusted to 6.0 then, add 1.9g/L Gellan Gum again, at 115 ℃, after 0.1MPa pressure was sterilized 15 minutes down, through cooling bevel dedifferentiation substratum.
2, the domestication of the subculture of Tibet saussurea involucrata callus is cultivated
The described callus of formation of inducing of step 1 is seeded in the succeeding transfer culture that carries out in the following subculture domestication substratum more than 3 times, and culture condition is 20 ℃ of room temperatures, and lucifuge condition, culture cycle were 3 weeks.In subculture domestication culturing process, filter out the short texture and speed of growth callus cell system faster;
Subculture domestication culture medium preparation: the α-naphthylacetic acid (NAA) that adds 0.8mg/L on the basis of B5 medium, 0.5mg/L 6-Bian aminopurine (6-BA), the 4mg/L vitamins C, 20g/L sucrose, 1.9g/L GellanGum, through 115 ℃, 0.1MPa pressure sterilization 15 minutes is down tamed substratum after dull and stereotyped subculture is made in cooling.
3, Tibet snow lotus polyploid cell induces
In above-mentioned B5 medium, add the 10mg/L colchicine respectively, make the liquid inducing culture, the short texture speed of growth that step 2 obtains callus cell system faster is seeded in this inducing culture, at 22 ℃, lucifuge, 100rpm/ divided on the shaking table of rotating speed shaking culture 10 days.Inoculate behind the collecting cell in the subculture domestication substratum of no colchicine, the subculture domestication is cultivated more than three times repeatedly, screens the polyploid cell system of the speed of growth faster (speed of growth surpasses 5 times/culture cycle) by the biological accumulation flow measurement; After cell aceto-camine or silver dyeing, show that by microscopic inspection this cell is a poikiloploid clone.
System is seeded on the following amplification culture medium with above-mentioned polyploid cell, at 25 ℃, under the dark condition, cultivated for 4 weeks after, cell concentration can increase more than 5 times.
Amplification culture medium: on the basis of B5 medium, add sucrose 30g/L, α-naphthylacetic acid (NAA) 0.5mg/L, 6-Bian aminopurine (6-BA) 0.5mg/L, the 4mg/L vitamins C, with acid or alkali the pH value is adjusted to 6 then, at 115 ℃, 0.1MPa pressure was sterilized 15 minutes down after amplification culture medium is made in cooling.
4, saussurea involucrate secondary metabolites is synthetic
The snow lotus polyploid cell inoculation of the described amplification cultivation of step 3 is arrived in the following synthetic medium, at 20 ℃, the lucifuge condition, suspension culture is 5 days in 100 rev/mins shaking table or the macro-organism reactor, filter harvested cell, but nutrient solution Returning reactor after adjusting continues to recycle.
Synthetic medium: on the basis of B5 medium, add glucose 40g/L, 2,4-D1mg/L, 6-Bian aminopurine (6-BA) 0.1mg/L, add the combination precursor again and (add phenylalanine 50mg/L, tyrosinase 15 0mg/L, fresh cider 50ml/L, styracin 5mg/L, cinnamophenone 5mg/L) and combination metabolic regulation material (add diacetyl oxide 10 μ l/L, 2-oxoglutaric acid 10mg/L, vitamins C 4mg/L, ethrel 2mg/L and dormin 1mg/L), with acid or alkali the pH value is adjusted to 6, at 115 ℃, 0.1MPa pressure sterilization was down made synthetic medium after 15 minutes.
After synthetic cultivation finishes, detect the synthetic content of cultivating the saussurea involucrate secondary metabolites in the saussurea involucrata cell that obtains with HPLC, the result shows that the saussurea involucrata flavones content accounts for more than 5% of dry cell weight.
Claims (9)
1. a method of producing saussurea involucrate secondary metabolites comprises the steps:
1) explant of saussurea involucrata is carried out dedifferentiation and cultivates and obtain the saussurea involucrata callus, with the saussurea involucrata callus through subculture domestication cultivate and obtain the loose clone of growth fast;
2) the saussurea involucrata callus cell that step 1) obtained is tied up to inducing culture obtains polyploid cell system in the B5 minimum medium that adds colchicine, carry out amplification cultivation then and obtain a large amount of snow lotus polyploid cells;
3) with step 2) the snow lotus polyploid cell inoculation that obtains synthesizes in synthetic medium and cultivates the cell that obtains being rich in saussurea involucrate secondary metabolites;
Described synthetic medium is for having added 20-60g/L glucose on the basis of B5 minimum medium, 0.1-5mg/L α-Nai Yisuan or 2,4-D or indolylacetic acid, 0.1-5mg/L 6-Bian aminopurine or kinetin, 50-100mg/L phenylalanine, 50-100mg/L tyrosine, 50-150ml/L Sucus Mali pumilae, 1-20mg/L styracin, 1-40mg/L cinnamophenone, 10-50 μ l/L diacetyl oxide, 5-50mg/L 2-oxoglutaric acid, 1-4mg/L vitamins C, 2-10mg/L ethrel and 1-6mg/L dormin, pH value are the substratum of 5.5-7.0.
2. method according to claim 1 is characterized in that: described step 2), the concentration of described colchicine is 10-40ml/L.
3. method according to claim 2, it is characterized in that: in the described step 1), it is to add 0.1-5mg/L α-Nai Yisuan or 2 on the basis of B5 minimum medium that used substratum is cultivated in described dedifferentiation, 4-D or indolylacetic acid, 0.1-5mg/L 6-Bian aminopurine or kinetin, the substratum that 20-60g/L sucrose or glucose obtain; It is to add 20-50g/L sucrose, 0.1-5mg/L α-Nai Yisuan or 2 on the basis of B5 minimum medium that described subculture domestication is cultivated with substratum, 4-D or indolylacetic acid, the substratum that 0.1-5mg/L 6-Bian aminopurine or kinetin and 1-4mg/L vitamins C obtain.
4. method according to claim 3 is characterized in that: in the described step 1), described saussurea involucrata explant is the young stem of saussurea involucrata, blade, stem-tip tissue or marrow tissue; Described dedifferentiation culture condition is for being 18-25 ℃ in temperature, and intensity of illumination is 1000-3000Lux, and cultivated under the irradiation condition 1-2 month at 12-16 hour every day; Described step 2) in, described amplification cultivation substratum is to be to add 20-40g/L sucrose on the basis of B5 minimum medium, 0.1-5mg/L α-Nai Yisuan or 2,4-D or indolylacetic acid, the substratum that 0.1-5mg/L 6-Bian aminopurine or kinetin and 1-4mg/L vitamins C obtain.
5. according to any described method among the claim 1-4, it is characterized in that: described step 2), also be included in and described polyploid cell carried out the subculture domestication before the amplification cultivation and cultivate.
6. method according to claim 5 is characterized in that: described subculture domestication culture condition is 20-25 ℃, cultivates under the lucifuge condition.
7. method according to claim 6 is characterized in that: described step 2), described inducing culture is at 20-30 ℃, and lucifuge was cultivated 5～10 days; Described amplification cultivation is at 20-25 ℃, cultivates 3-4 week under the lucifuge condition.
8. method according to claim 7, it is characterized in that: in the described step 3), the cell fresh weight weight percent of the inoculation of described synthetic cultivation snow lotus polyploid cell is 10～30%, described synthetic culture condition is at 20-30 ℃, under the dark condition, the shaking table of 80-120rpm or macro-organism reactor were cultivated 3-7 days.
9. method according to claim 8 is characterized in that: described saussurea involucrata is the Tibet saussurea involucrata; Described saussurea involucrate secondary metabolites is the saussurea involucrata flavones.
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| CN103305454A (en) * | 2012-03-14 | 2013-09-18 | 中国医学科学院药物研究所 | Method for producing Syringin, chlorogenic acid and 1, 5-dicaffeoylquinic acid by cell culture of herba saussureae involucratae |
| CN103484422B (en) * | 2013-09-22 | 2016-01-20 | 广州市恩道生物科技有限公司 | A kind of method of producing the Saussurea involucrata culture of Fu Han Yellow ketone and polysaccharide |
| CN104115747B (en) * | 2014-07-03 | 2015-12-02 | 山东农业大学 | Tissue culture method is utilized to produce apple class flavones |
| CN106701657A (en) * | 2016-11-15 | 2017-05-24 | 天津市博爱生物药业有限公司 | Medium for saussurea involucrata in vitro cells |
| CN106577279B (en) * | 2016-12-09 | 2018-08-24 | 北京林业大学 | A method of induction Tibet Saussurea laniceps callus generates anthocyanidin |
| CN106754633A (en) * | 2017-02-14 | 2017-05-31 | 天津艾赛博生物技术有限公司 | A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones |
| CN109468264B (en) * | 2018-12-20 | 2021-12-03 | 长沙学院 | Production method of geniposide |
| CN109644869B (en) * | 2018-12-20 | 2021-08-10 | 长沙学院 | Method for obtaining geniposide by tissue culture |
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| CN110923190A (en) * | 2019-11-28 | 2020-03-27 | 大连普瑞康生物技术有限公司 | Method for improving flavone phenylpropanoid compounds of saussurea involucrate cell culture |
| CN112063663A (en) * | 2020-08-24 | 2020-12-11 | 浙江省农业科学院 | Fermentation method for high-yield phellinus igniarius extracellular flavonoids |
| CN116918701A (en) * | 2020-09-30 | 2023-10-24 | 伽蓝（集团）股份有限公司 | Callus culture medium and extract of rosa tenuifolia, preparation method and application |
| CN112273227A (en) * | 2020-10-15 | 2021-01-29 | 永州职业技术学院 | Culture medium for improving survival rate of explants and preparation method and application thereof |
| CN113862212A (en) * | 2021-11-15 | 2021-12-31 | 安赛搏(重庆)生物技术有限公司 | Process for large-scale subculture and large-scale culture of saussurea involucrate cells |
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