CN102021137A - Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof - Google Patents
Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof Download PDFInfo
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Abstract
The invention provides a culture method of glycyrrhiza hairy roots and a method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof. The method comprises the following steps: culturing glycyrrhiza hairy roots in a fluid nutrient medium which does not contain Tween to the growth peak period; culturing the obtained glycyrrhiza hairy roots in the growth peak period in a fluid nutrient medium which contains Tween for more than 5 days; and eluting by ethanol with different concentration through a polyamide chromatography column, and separating the total flavonoids from the culture solution, wherein the transfer rate of the glycyrrhiza total flavonoids is 97.23%, the transfer rate of glycyrrhiza chalcone A is 98.14%, and the transfer rate of Glabridin is 97.54%. The invention also provides the glycyrrhiza total flavonoids prepared by the method and medical applications of the glycyrrhiza total flavonoids.
Description
Technical field
The invention belongs to biological technical field, particularly, the present invention relates to the cultural method of Radix Glycyrrhizae hairly root and utilize the Radix Glycyrrhizae hairly root and the method for inoculum preparation Radix Glycyrrhizae total flavones.In addition, the invention still further relates to a kind of Radix Glycyrrhizae total flavones and medical applications thereof that obtains by above-mentioned preparation method.
Background technology
Radix Glycyrrhizae is the important traditional Chinese medicine of China, has clearing heat and detoxicating, effects such as moistening lung is eliminated the phlegm, relieving spasm to stop pain, tonifying Qi benefit taste, coordinating the actions of various ingredients in a prescription, is described as " kings of hundred grass ".It is found that in recent years the various flavones that contain in the Radix Glycyrrhizae are the another kind of active substances except Potenlini, Radix Glycyrrhizae total flavones has anti-oxidant, antitumor, anti-AIDS, anti-inflammatory, antiulcer agent, the anti-ageing pharmacological action of waiting for a long time, sought-after, be applied to every field such as medicine, food, healthcare products and makeup at present.
Along with the utilization of continually developing of Radix Glycyrrhizae, wild resource is excavated excessively, has caused huge destruction for physical environment and ecological resources.September calendar year 2001, China was in order to protect day by day deficient Radix Glycyrrhizae resource, limited the excavating of wild Radix Glycyrrhizae, and banned use of wild Radix Glycyrrhizae and products thereof as the protective foods composition, and the Radix Glycyrrhizae imbalance between supply and demand is more outstanding.Being constrained to limiting the bottleneck that Radix Glycyrrhizae is used of the deficient and application of Radix Glycyrrhizae resource presses for new source, the new way of exploitation Radix Glycyrrhizae activeconstituents.
Utilizing the Radix Glycyrrhizae hairly root to produce Radix Glycyrrhizae total flavones is an effective way with good prospect, and the characteristics of many adaptation suitability for industrialized production are arranged, low as cost, and growth is fast, stable production capacity etc.But the flavones content in the Radix Glycyrrhizae hairly root is far below the content of crude drug root, having influenced further developing of Radix Glycyrrhizae hairly root amplifies and industrialization, general flavone content as report Glycyrrhiza uralensis Fisch. hairly root such as Zhang Yinlin is 0.39% of a dry weight, far below the flavones content in the crude drug root.At present more to the factor research that influences flavones content in the hairly root, reported the influence of methyl jasmonate and Whitfield's ointment as Yang Rui etc. to flavonoid content in the Saussurea medusa hairly root, adding concentration at hairly root inoculation back 10d is the methyl jasmonate of 0.02mmol/L, and the flavonoid compound comparison is according to improving 34.1%; When hairly root inoculation back 15d interpolation concentration was the Whitfield's ointment of 0.03mmol/L, the flavonoid compound comparison was according to improving 52.9%.But content, the influence factor for flavones in the hairly root nutrient solution reaches how to utilize the less of research at present, although Yu Shuhong etc. have reported the influence of tween-80 to isoflavones (isoflavone) content in the elegant jessamine hairly root, but do not relate to research, more do not relate to flavones (flavone) Study on content and enlightenment in Radix Glycyrrhizae hairly root and the nutrient solution thereof to Radix Glycyrrhizae.In addition, tween is as medicinal adjuvant commonly used, in compound Chinese medicinal preparation as auxiliary material can with Radix Glycyrrhizae or its extract isoreactivity composition formation pharmaceutical composition formulated together, as Chinese patent 95108283,02100222 and 200610035831 etc., but tween only exists as medicinal adjuvant in these medicines.
For this reason, the inventor is through studying for a long period of time, found unexpectedly, the liquid nutrient medium that utilization contains tween is cultivated long-time more than 5 days, can significantly improve content of flavonoids in hairly root that Radix Glycyrrhizae hairly root tissue culture obtains and the nutrient solution thereof, especially can impel total flavones to be discharged into the nutrient solution, improve the content of activeconstituents in the nutrient solution greatly from the hairly root of Radix Glycyrrhizae; In addition, the inventor also finds to utilize simple ethanol gradient elution polymeric amide chromatography post, can prepare highly purified Radix Glycyrrhizae total flavones.From hairly root and nutrient solution thereof, obtain Radix Glycyrrhizae total flavones like this, can effectively avoid the waste of resource, more unexpectedly be, Radix Glycyrrhizae total flavones of the present invention such as effect such as external tumor-killing in addition be better than the Radix Glycyrrhizae total flavones of existing bibliographical information.
Summary of the invention
The objective of the invention is to, for solving the contradiction of Radix Glycyrrhizae shortage of resources and Radix Glycyrrhizae demand, produce licoflavone to reach industrialization, substitute the target of Radix Glycyrrhizae resource, the Radix Glycyrrhizae hairly root that utilizes tissue culture and the method for inoculum preparation Radix Glycyrrhizae total flavones thereof are provided, improved the wherein content of Radix Glycyrrhizae total flavones, and the method for preparation or these Radix Glycyrrhizae total flavoness of purifying further is provided.Therefore, the invention provides the Radix Glycyrrhizae hairly root cultural method, Radix Glycyrrhizae hairly root and/or the application that improves the Radix Glycyrrhizae hairly root nutrient solution of Radix Glycyrrhizae total flavones content that preparation improves Radix Glycyrrhizae total flavones content, prepare the method for Radix Glycyrrhizae total flavones and by its Radix Glycyrrhizae total flavones product for preparing.
Particularly, in first aspect, the invention provides the method that cultivation can improve the Radix Glycyrrhizae hairly root and/or the Radix Glycyrrhizae hairly root nutrient solution of Radix Glycyrrhizae total flavones content, it comprises:
(1) the Radix Glycyrrhizae hairly root is cultured to vigorous period in the liquid nutrient medium that does not contain tween;
(2) the Radix Glycyrrhizae hairly root of the vigorous period that step (1) is obtained is cultivated more than 5 days in containing the liquid nutrient medium of tween.
Radix Glycyrrhizae total flavones (total flavonoids of Glycyrrhiza) is to have extracted from the enrichment of Radix Glycyrrhizae in the Radix Glycyrrhizae flavonoid compound (promptly, flavones ingredient) extract, it has anti-oxidant, antitumor, anti-AIDS, anti-inflammatory, antiulcer agent and the anti-ageing pharmacological action of waiting for a long time, one of Chang Zuowei active constituents of medicine or activeconstituents and use (referring to Xing Guoxiu etc. flavonoid The Chemical Constituents progress in the Radix Glycyrrhizae. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2003,28 (7): 593).Radix Glycyrrhizae total flavones can extract the various piece from Radix Glycyrrhizae, extracts the root from Radix Glycyrrhizae usually; When existing Radix Glycyrrhizae tissue culture, because the Radix Glycyrrhizae total flavones content in the nutrient solution is low, not too be worth reclaiming in industrialization, therefore do not appear in the newspapers and from its nutrient solution, reclaim total flavones.But the inventor finds that after cultivating, the general flavone content in Radix Glycyrrhizae hairly root and its nutrient solution has all obtained significant lifting, can be used on industry in containing the liquid nutrient medium of tween.Therefore, preferred first aspect present invention also provides cultivates the Radix Glycyrrhizae hairly root to improve the method for Radix Glycyrrhizae total flavones content in the Radix Glycyrrhizae hairly root nutrient solution, and it comprises:
(1) the Radix Glycyrrhizae hairly root is cultured to vigorous period in the liquid nutrient medium that does not contain tween;
(2) the Radix Glycyrrhizae hairly root of the vigorous period that step (1) is obtained is cultivated more than 5 days in containing the liquid nutrient medium of tween.
Composition is a lot of in the Radix Glycyrrhizae total flavones, though wherein the kind of flavonoid compound also have many, but can characterize by special component wherein.In the present invention, the content of flavonoid compound, licochalcone A and/or the glabridin that this mixture of Radix Glycyrrhizae total flavones of obtaining of the present invention can be by wherein characterizes.Therefore, the method for first aspect present invention can improve the content of flavonoid compound, licochalcone A and/or glabridin.As indicated above, the present invention especially can improve the content of the Radix Glycyrrhizae total flavones in the Radix Glycyrrhizae hairly root nutrient solution, the content of flavonoid compound, licochalcone A and/or the glabridin that improves not only is embodied in the Radix Glycyrrhizae hairly root, be also embodied in its nutrient solution, the method of therefore preferred first aspect present invention can improve the content of flavonoid compound, licochalcone A and/or glabridin in the Radix Glycyrrhizae hairly root nutrient solution more than 5 times, preferably can improve more than 10 times, doubly as 10-60.
In the method for first aspect present invention, can further include the step of collecting product, comprise and collect Radix Glycyrrhizae hairly root or its nutrient solution that step (2) obtains that more preferably the method for first aspect present invention also further comprises:
(3) collect Radix Glycyrrhizae hairly root and its nutrient solution that step (2) obtains.Like this, reclaim Radix Glycyrrhizae hairly root and its nutrient solution, can extract total flavones wherein respectively, can more effectively utilize and cultivate the total flavones that obtains, avoid waste; Simultaneously, because the general flavone content in the nutrient solution is higher, make that the cost that extracts total flavones in the nutrient solution is lower, especially can utilize the method for fourth aspect present invention to carry out, just can purify from nutrient solution by the alcoholic acid gradient elution obtains Radix Glycyrrhizae total flavones.
In the method for first aspect present invention, Radix Glycyrrhizae can be cultivated on solid medium earlier, and then is cultured to vigorous period in the liquid nutrient medium of going into not contain tween of transferring.Usually the Radix Glycyrrhizae hairly root of health can be inoculated in 0.5%～2% inoculum size and cultivate 5～10 days in the substratum, thereby reach vigorous period.In a specific embodiment of the present invention, be that the hairly root of the dead part of no brownization of 1g is inoculated in the 100mL MS liquid nutrient medium and cultivated 7 days in 25 ℃ of lucifuge shaking tables with weight in wet base, reach vigorous period.
Preferably in the method for first aspect present invention, tween can be to confirm that through test as described in the embodiment of the present application the energy evoking liquorice improves the tween kind of general flavone content, and in the specific embodiment of the present invention, this kind is a tween-80.In addition, the mass percent concentration of tween is 1～5% (w/w) in the preferred liquid substratum, 2% (w/w) more preferably, and particularly, the mass percent concentration of tween-80 is 1～5% (w/w) in the preferred liquid substratum, more preferably 2% (w/w).
Preferably in the method for first aspect present invention, the preferred liquid substratum is the MS liquid nutrient medium.The Radix Glycyrrhizae hairly root can be cultivated with conditions suitable in the liquid medium within, preferably wherein culture condition is that 20～30 ℃ of lucifuge shaking tables are cultivated more than 5 days, as 7～21 days, preferably as 10～20 days, more preferably wherein culture condition be 25 ℃, dark, on the shaking table of 100rpm rotating speed, cultivated 15 days.
In second aspect, the invention provides the application of method in the method for preparing Radix Glycyrrhizae hairly root that improves Radix Glycyrrhizae total flavones content and/or the Radix Glycyrrhizae hairly root nutrient solution that improves Radix Glycyrrhizae total flavones content of first aspect present invention; In addition, the present invention also provides preparation to improve the Radix Glycyrrhizae hairly root of Radix Glycyrrhizae total flavones content and/or has improved the method for the Radix Glycyrrhizae hairly root nutrient solution of Radix Glycyrrhizae total flavones content, comprising the step of the method for first aspect present invention.
A second aspect of the present invention provides the application of method in the method for the Radix Glycyrrhizae hairly root of preparation raising Radix Glycyrrhizae total flavones content of first aspect present invention, wherein can not collect the Radix Glycyrrhizae hairly root nutrient solution that step (2) obtains in the method for first aspect present invention, and only collect the Radix Glycyrrhizae hairly root that step (2) obtains in the method for first aspect present invention.
A second aspect of the present invention also provides the application of method in the method for the Radix Glycyrrhizae hairly root nutrient solution of preparation raising Radix Glycyrrhizae total flavones content of first aspect present invention, wherein can not collect the Radix Glycyrrhizae hairly root that step (2) obtains in the method for first aspect present invention, and only collect the Radix Glycyrrhizae hairly root nutrient solution that step (2) obtains in the method for first aspect present invention.
Each preferred feature in the method for second aspect present invention is each corresponding preferred feature in the method for first aspect present invention preferably.
In the third aspect, the invention provides the method for preparing Radix Glycyrrhizae total flavones, it comprises:
(1) carries out the method for first aspect present invention, obtain the Radix Glycyrrhizae hairly root;
(2) the Radix Glycyrrhizae hairly root that obtains of drying step (1) extracted 2-8 hour in 60 ℃-100 ℃ with its weight 10-20 water doubly, the solid retained part and with solid part in baking oven with 30 ℃ of-60 ℃ of dryings;
(3) solid part that obtains of drying step (2), and extract 1-2 time in 60 ℃-100 ℃ with 50-95% (v/v) ethanol, the residue that washing is extracted, united extraction liquid and washings, the retaining liquid part is also concentrated and/or dry.Like this, can extract Radix Glycyrrhizae total flavones in the Radix Glycyrrhizae hairly root.
Preferably in the method for third aspect present invention, step (1) thus the Radix Glycyrrhizae hairly root that obtains can be removed water-soluble impurity with water extraction before dry, for example, can be with Radix Glycyrrhizae hairly root weight 10-20 times water extraction 2-8 hour.In the method for third aspect present invention, drying can be dry under 30 ℃-60 ℃ temperature, and is preferably dry under 50 ℃-60 ℃ temperature.
Equally, for the Radix Glycyrrhizae total flavones that extracts in the Radix Glycyrrhizae hairly root nutrient solution is provided, in fourth aspect, the invention provides the method for preparing Radix Glycyrrhizae total flavones, it comprises:
(1) carries out the method for first aspect present invention, obtain Radix Glycyrrhizae hairly root nutrient solution;
(2) will choose the nutrient solution that obtains through spissated step (1) wantonly and be splined on polymeric amide chromatography post, carry out wash-out with at least two kind of 0～85% (v/v) aqueous ethanolic solution successively by alcohol concn order from low to high, collection is also concentrated and/or dry with the elutriant that concentration is not less than 60% (v/v) ethanol elution.
In the method for fourth aspect present invention, concentrate can be under 50 ℃-80 ℃ temperature concentrating under reduced pressure, can be concentrated into the 1/10-1/2 of original volume; Drying can be dry under 30 ℃-60 ℃ temperature, and is preferably dry under 40 ℃-60 ℃ temperature.Preferred in addition in the method for fourth aspect present invention, polymeric amide will be with polyamide resin with 95% (v/v) alcohol reflux 2～5 hours (as 3 hours) before the chromatography column of packing into, filter through filter cloth then, reflux in 95% (v/v) ethanol 2～5 hours (as 3 hours), filter cloth filters the back and collects resin again; Wherein, polyamide resin preferably is ground into 30-200 purpose polyamide resin, as the 30-60 order, and 60-100 order, 80-120 order or 100-200 purpose polyamide resin.In addition, in the method for fourth aspect present invention, be splined on polymeric amide chromatography post nutrient solution, be splined on polymeric amide chromatography post as the nutrient solution that obtains through spissated step (1) with the 1/2-1/5 column volume preferably through spissated.
We find, on polymeric amide chromatography post, by can purify Radix Glycyrrhizae total flavones in the nutrient solution of ethanol gradient elution.Therefore, preferably in the method for fourth aspect present invention, in step (2), water, 10～50% (v/v) ethanol and 60～95% (v/v) ethanol elution polymeric amide chromatography post successively.In addition, in the method for fourth aspect present invention, control effluent liquid flow velocity is 1～3 column volume per hour during the preferred alcohol wash-out.
We find, most Radix Glycyrrhizae total flavoness have been kept in the elutriant of 70% (v/v) ethanol elution, and before water, 30% (v/v) ethanol and 50% (v/v) ethanol successively wash-out just can remove the magazine of the overwhelming majority such as tween etc., thereby realized the purification of Radix Glycyrrhizae total flavones; And further use greater concn (as, 90% (v/v)) ethanol elution, can not significantly promote refining effect.Therefore, more preferably in the method for fourth aspect present invention, in step (2), respectively with water, 30% (v/v) ethanol, 50% (v/v) ethanol and 70% (v/v) ethanol elution polymeric amide chromatography post of 2-7 times of column volume, collect elutriant successively with 70% (v/v) ethanol elution.
The method of the present invention third and fourth aspect can be combined into the preferred method for preparing Radix Glycyrrhizae total flavones, and it comprises:
(1) carries out the method for first aspect present invention, obtain Radix Glycyrrhizae hairly root and nutrient solution thereof;
(2) the Radix Glycyrrhizae hairly root that obtains of drying step (1) extracted 2-8 hour in 60 ℃-100 ℃ with its weight 10-20 water doubly, the solid retained part and with solid part in baking oven with 30 ℃ of-60 ℃ of dryings;
(3) solid part that obtains of drying step (2), and extract 1-2 time in 60 ℃-100 ℃ with 50-95% (v/v) ethanol, the residue that washing is extracted, united extraction liquid and washings, the retaining liquid part is also concentrated and/or dry.Like this, can extract Radix Glycyrrhizae total flavones in the Radix Glycyrrhizae hairly root;
(4) will choose the nutrient solution that obtains through spissated step (1) wantonly and be splined on polymeric amide chromatography post, carry out wash-out with at least two kind of 0～85% (v/v) aqueous ethanolic solution successively by alcohol concn order from low to high, collection is also concentrated and/or dry with the elutriant that concentration is not less than 60% (v/v) ethanol elution.Wherein, preferred described each corresponding preferred feature of the method for the present invention third and fourth aspect.
Aspect the 5th, the invention provides the Radix Glycyrrhizae total flavones of the method preparation of the present invention the 3rd and/or fourth aspect, wherein flavonoid content is greater than 24%, and more preferably wherein flavonoid content more preferably wherein also is rich in licochalcone A and glabridin greater than 60%.For example, the HPLC collection of illustrative plates of described Radix Glycyrrhizae total flavones can be shown in the collection of illustrative plates of Fig. 1,2 or 3 70% ethanol eluate, and wherein the condition of HPLC is as described in the embodiment of the present application.
We find that by the method preparation back of fourth aspect present invention wherein the active ingredient loss amount is considerably less.Therefore, preferably in the Radix Glycyrrhizae total flavones of fifth aspect present invention, the licochalcone A in the described Radix Glycyrrhizae total flavones and the amount of glabridin are compared the licochalcone A in the nutrient solution that step (1) obtains in the method for fourth aspect present invention and the amount of glabridin, basic not minimizing, preferably the former amount is more than 90% of the latter, as 90%-99%, more preferably more than 95%, as 95%-99% or 97%-99%.
Because the improvement of the raising of purity and the aspects such as effective constituent proportioning that may exist, the Radix Glycyrrhizae total flavones of fifth aspect present invention is killing tumor cells more effectively, therefore, the present invention also provides pharmaceutical composition, it comprises the Radix Glycyrrhizae total flavones and the pharmaceutically acceptable carrier of fifth aspect present invention, preferred described pharmaceutical composition is used for antitumor, is particularly useful for anti-adenocarcinoma of stomach; The present invention also provides the application of the method for the Radix Glycyrrhizae total flavones of fifth aspect present invention is used for antitumor (being particularly useful for anti-adenocarcinoma of stomach) in preparation medicine; In addition, the present invention also provides the method that is used for antitumor (being particularly useful for anti-adenocarcinoma of stomach), and it comprises the Radix Glycyrrhizae total flavones of the fifth aspect present invention that gives the effective therapeutic dose of patient.
Pharmaceutically acceptable carrier is meant the pharmaceutical carrier of pharmaceutical field routine, for example: thinner, vehicle (as water etc.), weighting agent (as starch, sucrose etc.), tackiness agent (as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone), wetting agent (as glycerine), disintegrating agent (as agar, lime carbonate and sodium bicarbonate), absorption enhancer (as quaternary ammonium compound), absorption carrier (as kaolin and soap clay), lubricant (as talcum powder, calcium stearate/magnesium, polyoxyethylene glycol etc.).Other assistant agent can also be added in addition in pharmaceutical composition, as flavouring agent, sweeting agent etc.
Pharmaceutical composition can be by oral, and modes such as snuffing is gone into, rectum or administered parenterally are applied to the patient who needs this treatment.Be used for when oral, can be made into conventional solid preparation such as tablet, pulvis, granula, capsule etc., make liquid preparation such as water or oil-suspending agent or other liquid preparation such as syrup, elixir etc.; When being used for administered parenterally, can be made into solution, water or the oiliness suspension agent etc. of injection.Dosage can by the doctor according to patient's situation (as, the state of an illness, body weight, age, sex) determine, usually between 1 μ g/kg-100mg/kg weight in patients.
The present invention compares with traditional Radix Glycyrrhizae total flavones preparation method has following advantage: the licoflavone that the Radix Glycyrrhizae hairly root that obtains with biotechnology produces is the source, and the characteristics of many adaptation suitability for industrialized production such as cost is low, growth is fast, stable production capacity are arranged; With tween-80 the Radix Glycyrrhizae hairly root is induced processing, make the flavonoid content in the hairly root improve 44%-55%, make the flavonoid content in its nutrient solution improve 10-50 doubly simultaneously; Not only utilized the flavonoid compound in the hairly root, also utilized the flavonoid compound in its nutrient solution, made the flavonoid compound total amount of comprehensive acquisition improve 10-50 doubly; Wherein the licochalcone A total amount improves 10-60 doubly, and the glabridin total amount improves 10-60 doubly.The licoflavone steady sources that present method obtains is not subjected to the restriction of Application Areas, has filled up the blank in the restricted field of licorice medicinal materials, has protected limited Radix Glycyrrhizae wild resource and ecotope thereof; Present method technology is simple simultaneously, and economic worth is considerable, is the new way of licoflavone industrialization.
For the ease of understanding, below will describe in detail the present invention by concrete accompanying drawing, embodiment.It needs to be noted that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change all are conspicuous concerning one of ordinary skill in the art.In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is the same excessively in this article for their full text.
Description of drawings
Fig. 1 has shown among the embodiment 4 the high performance liquid chromatography detected result of flavones in each step elutriant.
Fig. 2 has shown among the embodiment 4 the high performance liquid chromatography detected result of licochalcone A in each step elutriant.
Fig. 3 has shown among the embodiment 4 the high performance liquid chromatography detected result of glabridin in each step elutriant.
Embodiment
The cultivation of embodiment 1, Radix Glycyrrhizae hairly root
With fresh Glycyrrhiza uralensis Fisch. seed (can available from company of Bozhou City medicinal material main office) with 70% (v/v) alcohol immersion 10 seconds, with aseptic water washing 3 times, use 2% (w/w) chlorine bleach liquor to soak then 10 minutes, with aseptic water washing 3 times, then seed is inoculated in the MS solid medium (promptly, add 1% (w/w) agar in the MS liquid nutrient medium), in 25 ℃, dark culturing 4 days, the long segment of 0.5cm is cut at its root middle part.
With segment be transferred to be added with 2,4 dichlorophenoxyacetic acid (2,4-D) in the MS solid medium of (final concentration is 2mg/L) and kinetin (KT) (final concentration is 0.2mg/L),, obtain villous adventive root (that is hairly root) in 25 ℃, dark culturing 40 days.
Then, hairly root is transferred in the MS liquid nutrient medium, in 25 ℃, dark, on the shaking table of 100rpm rotating speed, cultivated 20 days.Afterwards, remove the dead hairly root of brownization, all the other hairly root are inoculated in the 300mL culturing bottle that contains 100mL MS liquid nutrient medium, and every bottle of hairly root weight in wet base is 1g, in 25 ℃, dark, on the shaking table of 100rpm rotating speed, cultivated 7 days.Then, the hairly root of cultivating in every bottle is transferred in the MS liquid nutrient medium that 100mL contains 2% (w/w) tween-80 again, in 25 ℃, dark, on the shaking table of 100rpm rotating speed, cultivated 15 days, centrifugal then, gather in the crops Radix Glycyrrhizae hairly root and nutrient solution thereof respectively; Correspondingly, control group is to cultivate in MS liquid nutrient medium (not containing 2% (w/w) tween-80).
Wherein, the prescription of MS liquid nutrient medium is: saltpetre 1900mg/L, ammonium nitrate 1650mg/L, potassium primary phosphate 170mg/L, sal epsom 370mg/L, calcium chloride 440mg/L, potassiumiodide 0.83mg/L, boric acid 6.2mg/L, manganous sulfate 22.3mg/L, zinc sulfate 8.6mg/L, Sodium orthomolybdate 0.25mg/L, copper sulfate 0.025mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, vitamin (VB1) 0.1mg/L, pyridoxine hydrochloride (VB6) 0.5mg/L, vitamin B5 0.5mg/L, sucrose 30g/L, surplus is a water.
The preparation of total flavones in embodiment 2, the Radix Glycyrrhizae hairly root
Get Radix Glycyrrhizae hairly root 150g, place baking oven according to the tween inducing culture of embodiment 1 preparation, 60 ℃ of dryings 12 hours, exsiccant hairly root 10g.Hairly root is placed the round-bottomed flask of 500mL, add entry 200mL, soaked 7 hours in 85 ℃, be cooled to room temperature, filter paper filtering discards filtrate, again with 60 ℃ of warm water washings 3 times, each 200mL, the hairly root residue of then filtration being held back places baking oven, 60 ℃ of dryings 12 hours.Exsiccant Radix Glycyrrhizae hairly root residue is placed the round-bottomed flask of 500mL, add the ethanol 200mL of 95% (v/v), 85 ℃ of refluxing extraction 2 hours, room temperature is put in cooling, and filter paper filtering keeps filtrate.Filter residue keeps washings with the washing with alcohol 2 times of 10mL95% (v/v), and residue adds the ethanol 200mL of 95% (v/v) again, 85 ℃ of refluxing extraction 2 hours, and room temperature is put in cooling, and filter paper filtering keeps filtrate.Merge twice filtrate and washings, 60 ℃ are evaporated to 20mL, and concentrated solution is placed 60 ℃ of dryings of baking oven, flavonoid content be the Radix Glycyrrhizae total flavones 0.5g of 24.22% (w/w measures according to HPLC).
Correspondingly, repeat above process with the Radix Glycyrrhizae hairly root of control group, obtaining flavonoid content is the Radix Glycyrrhizae total flavones 0.51g of 20.56% (w/w measures according to HPLC).
This shows that cultural method of the present invention can be turned out the hairly root of high flavonoid content and is suitable for extracting preparation.
The preparation of total flavones in embodiment 3, the Radix Glycyrrhizae hairly root nutrient solution
(1) concentrating of Radix Glycyrrhizae hairly root nutrient solution: get Radix Glycyrrhizae hairly root nutrient solution 60mL, 60 ℃ of 1/2 (being 30mL) that are concentrated into original volume according to the tween-80 inducing culture of embodiment 1 preparation.
(2) pre-treatment of polyamide resin: the polyamide resin that column chromatography is used (can available from chemical reagents corporation of traditional Chinese medicines group) 100g refluxed 3 hours in 85 ℃ with 95% (v/v) ethanol 500ml, filter cloth filters, adding 95% (v/v) ethanol 500mL again refluxes 1 time in 85 ℃, filter cloth filters, to remove alcohol dissolubility impurity residual in the polyamide resin.With the polyamide resin dress post after handling, making column volume is 60mL, and each does not have the ethanol flavor with deionized water rinsing chromatography column to effluent liquid again with 300mL70% (v/v) ethanol, 50% (v/v) ethanol and 30% (v/v) alcohol flushing chromatography column successively.
(3) go up sample: will concentrate good hairly root nutrient solution 30mL through step (1) is the chromatography column capital of 60mL to column volume upward, and it is 60mL/h that controlling flow goes out flow velocity, goes up sample fully to extracting solution to enter resin bed, and static 0.5-1h allows it fully adsorb.
(4) wash-out: with 120mL deionized water rinsing resin bed, it is 60mL/h that controlling flow goes out flow velocity.Each washes chromatography column with 120mL 30% (v/v) ethanol and 50% (v/v ethanol) successively again, and it is 60mL/h that controlling flow goes out flow velocity.Use 180mL70% (v/v) ethanol elution chromatography column again, it is 60mL/h that controlling flow goes out flow velocity, collect this part elutriant, 60 ℃ are evaporated to 10mL, then concentrated solution is placed 60 ℃ of dryings of baking oven, obtaining flavonoid content is the Radix Glycyrrhizae total flavones 94.0mg of 61.49% (w/w measures according to HPLC).
Chromatography column can be used 120mL 95% (v/v) alcohol flushing at last again, and it is 60mL/h that controlling flow goes out flow velocity, usefulness for deliberation.
Correspondingly, according to aforesaid method, repeat above process with the Radix Glycyrrhizae hairly root of control group, obtaining flavonoid content only is the Radix Glycyrrhizae total flavones of 18.52% (w/w measures according to HPLC).
This shows that cultural method of the present invention can be turned out the Radix Glycyrrhizae hairly root nutrient solution of high flavonoid content and is suitable for extracting preparation.
Embodiment 4, prepare in the method for total flavones in the Radix Glycyrrhizae hairly root nutrient solution each step impurity and effectively product detect
Because some impurity will have influence on the quality of Radix Glycyrrhizae total flavones, and undue strict purifying will increase the loss of product greatly, and the therefore following product that each step wash-out (flushing) among the embodiment 3 is gone out carries out impurity and the effectively detection of product.
(1) impurity of each step elutriant detects
Because having adopted polyamide resin is sorbent material, know that according to its absorption principle the most of impurity in the substratum can be by wash-out when water is the eluting solvent removal of impurities, but tween-80 but can not be removed fully by water elution, so each step elutriant mainly detects having or not of tween-80.Particularly, get the elutriant of each step of 120mL respectively, be evaporated to 5mL in 60 ℃, with concentrated solution with wash-out (flushing) dissolution with solvents of this step and be settled to 10mL.The solution 1.0mL that gets constant volume places 60 ℃ of oven dry of baking oven, adds entry 1ml then, ultrasonic dissolution, centrifugal, get supernatant liquor and add 0.1M sodium hydroxide solution 1mL, boiled 3 minutes, be cooled to room temperature,, then contain tween-80 as producing white opacity with the acidifying of 10% (w/w) dilute hydrochloric acid.Concrete outcome sees Table 1, uses 50～70% (v/v) ethanol elution in the step of visible embodiment 3, just can effectively remove impurity such as tween-80.
Tween-80 detected result in table 1. elutriant
| The sample title | Adularescent muddiness whether | Whether tween is arranged |
| Induce the back liquid nutrient medium | Have | Have |
| Water elution liquid | Have | Have |
| 30% ethanol eluate | Have | Have |
| 50% ethanol eluate | Have | Have |
| 70% ethanol eluate | Do not have | Do not have |
| 95% ethanol eluate | Do not have | Do not have |
(2) high performance liquid chromatography detects the flavones in each step elutriant.
Get each the step elutriant that is settled to 10ml after concentrating in the above-mentioned steps (1), behind 0.45 μ m membrane filtration, carry out high performance liquid chromatography.Wherein, the high performance liquid chromatography condition is: Kromasil C18 chromatographic column (250 * 4.6mm, 5 μ m);
Detect wavelength: 365nm; Flow velocity: 1.0ml/min; Column temperature: 25 ℃; Moving phase (gradient elution) is as follows:
0-30min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 18: 82
30-50min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 18: 82 → 40: 60
50-90min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 40: 60
90-115min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 40: 60 → 55: 45
115-135min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 55: 45 → 65: 35
135-150min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 65: 35
The result as shown in Figure 1, the Radix Glycyrrhizae total flavones that contains in the Radix Glycyrrhizae hairly root nutrient solution after tween-80 is handled, behind polymeric amide absorption, Different concentrations of alcohol wash-out, in 70% ethanol eluate, obtained enrichment effectively and concentrated, the Radix Glycyrrhizae total flavones amount of other elution step losses is seldom.
(3) high performance liquid chromatography detects the licochalcone A in each step elutriant.
Get each the step elutriant that is settled to 10ml after concentrating in the above-mentioned steps (1), behind 0.45 μ m membrane filtration, carry out high performance liquid chromatography.Wherein, high-efficient liquid phase chromatogram condition is: Kromasil C18 chromatographic column (250 * 4.6mm, 5 μ m); Detect wavelength: 377nm; Flow velocity: 1.0ml/min; Column temperature: 25 ℃; Moving phase is: methyl alcohol-tetrahydrofuran (THF)-1% Glacial acetic acid=50: 10: 40.
The result as shown in Figure 2, peak with reference to the licochalcone A standard model, the licochalcone A that contains in the Radix Glycyrrhizae hairly root nutrient solution after tween-80 is handled, behind polymeric amide absorption, Different concentrations of alcohol wash-out, obtained enrichment and concentrated in 70% ethanol eluate, the licochalcone A of other elution step losses does not almost have.
(4) high performance liquid chromatography detects the glabridin content in each step elutriant.
Get each the step elutriant that is settled to 10ml after concentrating in the above-mentioned steps (1), behind 0.45 μ m membrane filtration, carry out high performance liquid chromatography.Wherein, the high performance liquid chromatography condition is: Kromasil C18 chromatographic column (250 * 4.6mm, 5 μ m); Detect wavelength: 280nm; Flow velocity: 1.0ml/min; Column temperature: 25 ℃; Moving phase (gradient elution) is as follows:
0-30min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 18: 82
30-50min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 18: 82 → 40: 60
50-90min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 40: 60
90-115min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 40: 60 → 55: 45
115-135min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 55: 45 → 65: 35
135-150min acetonitrile: water (containing 0.05% phosphoric acid) volume ratio 65: 35
The result as shown in Figure 3, the peak of reference light licoricidin standard model, the glabridin that contains in the Radix Glycyrrhizae hairly root nutrient solution after tween-80 is handled, behind polymeric amide absorption, Different concentrations of alcohol wash-out, obtained enrichment and concentrated in 70% ethanol eluate, the glabridin of other elution step losses does not almost have.
Radix Glycyrrhizae hairly root nutrient solution (before extracting) after relatively tween-80 is handled according to the HPLC method and each composition after the extraction, find, the rate of transform of Radix Glycyrrhizae total flavones is 97.23%, and the rate of transform of licochalcone A is 98.14%, and the rate of transform of glabridin is 97.54%.
The external tumor-killing experiment of embodiment 5, licorice flavone extraction of the present invention
With reference to the inscription on pottery Yihe River, Nie Xiaohua, Yin Guangyao, Deng document (effective liquorice anti tumor activity in vitro and immunocompetent research (J). Chinese medicinal materials, 2003,26 (7): 507) reported method, the external anti-tumor activity of the Radix Glycyrrhizae total flavones of the mensuration embodiment of the invention 3.In brief, handle adenocarcinoma of stomach SGC-7901 cell with 0.25% trysinization after, in 1000rpm centrifugal 5 minutes, it was resuspended with 1640 substratum that add 10%FBS to abandon supernatant, got 100 μ l and added in the 900 μ lPBS mixing and take out 10 μ l and carry out the microscopy counting.The microscopy result is as follows: getting concentration is 2 * 10
4Enchylema.Get 100 μ l enchylema and add on 96 orifice plates, in 37 ℃, 5% CO2gas incubator, cultivated 24 hours.Dissolving the flavones sample respectively with DMSO and be diluted to 1mg/ml with 1640+FBS, is 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 60 μ g/ml, 80 μ g/ml, 100 μ g/ml making the medicine final concentration with the 1%DMSO dilution.Stay 4 holes to compare 100 μ l/ holes.Cultivated 72 hours in 37 ℃ of 5% CO2gas incubator.Clean plate hole, abandon supernatant, clean plate hole with PBS; Add MTS: the MTS that adds the 1640 20 μ l of 100 μ l cultivates 2h.The 490nm place surveys the OD value, calculates tumour inhibiting rate by following formula:
Tumour inhibiting rate=(medicine blank-drug test)/medicine blank-cell blank * 100%
By the IC50 value of computed in software medicine, Radix Glycyrrhizae total flavones of the present invention is 19.66 μ g/ml to the IC50 value of adenocarcinoma of stomach cell strain SGC-7901; And the inscription on pottery Yihe River, Nie Xiaohua, Yin Guangyao, though the Radix Glycyrrhizae total flavones of reporting in the document that waits has the obvious suppression effect to adenocarcinoma of stomach cell strain SGC-7901, IC50 is up to 290.6 μ g/ml.This shows that the external tumor-killing effect of the Radix Glycyrrhizae total flavones that the present invention obtains is better than the Radix Glycyrrhizae total flavones of bibliographical information.
Claims (13)
1. cultivate the method for the Radix Glycyrrhizae hairly root and/or the Radix Glycyrrhizae hairly root nutrient solution that can improve Radix Glycyrrhizae total flavones content, it comprises:
(1) the Radix Glycyrrhizae hairly root is cultured to vigorous period in the liquid nutrient medium that does not contain tween;
(2) the Radix Glycyrrhizae hairly root of the vigorous period that step (1) is obtained is cultivated more than 5 days in containing the liquid nutrient medium of tween.
2. the described method of claim 1, it further comprises:
(3) collect Radix Glycyrrhizae hairly root and its nutrient solution that step (2) obtains.
3. claim 1 or 2 described methods, wherein tween is a tween-80, the mass percent concentration of tween-80 is 1～5% (w/w) in the preferred liquid substratum, more preferably 2% (w/w).
4. claim 1 or 2 described methods, wherein liquid nutrient medium is the MS liquid nutrient medium, preferably wherein culture condition is that 20～30 ℃ of lucifuge shaking tables were cultivated 10～20 days, more preferably 25 ℃, dark, on the shaking table of 100rpm rotating speed, cultivated 15 days..
5. claim 1 or 2 described methods, it can improve the content of flavones, licochalcone A and/or glabridin, the content that especially can improve flavones, licochalcone A and/or glabridin in the Radix Glycyrrhizae hairly root nutrient solution preferably can improve more than 10 times more than 5 times.
6. the application of the arbitrary described method of claim 1 to 5 in the method for preparing Radix Glycyrrhizae hairly root that improves Radix Glycyrrhizae total flavones content and/or the Radix Glycyrrhizae hairly root nutrient solution that improves Radix Glycyrrhizae total flavones content.
7. prepare the method for Radix Glycyrrhizae total flavones, it comprises:
(1) carries out the arbitrary described method of claim 1 to 5, obtain the Radix Glycyrrhizae hairly root;
(2) the Radix Glycyrrhizae hairly root that obtains of drying step (1) extracted 2-8 hour in 60 ℃-100 ℃ with its weight 10-20 water doubly, the solid retained part and with solid part in baking oven with 30 ℃ of-60 ℃ of dryings;
(3) solid part that obtains of drying step (2), and extract 1-2 time in 60 ℃-100 ℃ with 50-95% (v/v) ethanol, the residue that washing is extracted, united extraction liquid and washings, the retaining liquid part is also concentrated and/or dry.
8. prepare the method for Radix Glycyrrhizae total flavones, it comprises:
(1) carries out the arbitrary described method of claim 1 to 4, obtain Radix Glycyrrhizae hairly root nutrient solution;
(2) will choose the nutrient solution that obtains through spissated step (1) wantonly and be splined on polymeric amide chromatography post, carry out wash-out with at least two kind of 0～85% (v/v) aqueous ethanolic solution successively by alcohol concn order from low to high, collection is also concentrated and/or dry with the elutriant that concentration is not less than 60% (v/v) ethanol elution.
9. the described method of claim 8 is characterized in that, in step (2), and water, 10～50% (v/v) ethanol and 60～95% (v/v) ethanol elution polymeric amide chromatography post successively.
10. claim 9 or 8 described methods, it is characterized in that, in step (2), respectively with water, 30% (v/v) ethanol, 50% (v/v) ethanol and 70% (v/v) ethanol elution polymeric amide chromatography post of 2-7 times of column volume, collect elutriant successively with 70% (v/v) ethanol elution.
11. the Radix Glycyrrhizae total flavones of the arbitrary described method preparation of claim 7 to 10, wherein flavones content is greater than 24%, more preferably wherein flavones content greater than 60%, also preferably wherein also comprise licochalcone A and glabridin, for example, the HPLC collection of illustrative plates of described Radix Glycyrrhizae total flavones can be shown in the collection of illustrative plates of Fig. 1,2 or 3 70% ethanol eluate.
12. the described Radix Glycyrrhizae total flavones of claim 11, wherein the amount of licochalcone A and glabridin is compared the licochalcone A in the nutrient solution that step (1) obtains in the arbitrary described method of claim 7 to 9 and the amount of glabridin, basic not minimizing, preferably the former amount is more than 90% of the latter, more preferably more than 95%.
13. according to claim 1-12, the present invention comprise that a kind of plant hairly root is cultivated and the inductor co-culture system through containing tween synthetic and be discharged into the production method of the plant flavone class material in the liquid nutrient medium to improve plant flavone class target compound.Described method is including but not limited to Radix Glycyrrhizae.
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| CN104620987A (en) * | 2015-02-12 | 2015-05-20 | 天津大学 | Tissue-culturing method for improving content of flavone compounds in adventitious roots of glycyrrhiza uralensis |
| CN108882743A (en) * | 2016-03-29 | 2018-11-23 | 捷通国际有限公司 | For activating the nutritional supplement and correlation technique of the antioxidant system of subject |
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