CN101263811B - Method for improving yield of glycyrrhizae hairy root secondary metabolism product - Google Patents
Method for improving yield of glycyrrhizae hairy root secondary metabolism product Download PDFInfo
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- CN101263811B CN101263811B CN2008100069389A CN200810006938A CN101263811B CN 101263811 B CN101263811 B CN 101263811B CN 2008100069389 A CN2008100069389 A CN 2008100069389A CN 200810006938 A CN200810006938 A CN 200810006938A CN 101263811 B CN101263811 B CN 101263811B
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Abstract
The invention discloses a method to improve the synthetic quantity of secondary metabolites in liquorice hairy roots; the method processes the liquorice hairy roots in a liquid medium by a matter capable of regulating the osmotic pressure of cells to form drought stress, and then liquorice hairy roots with a higher content of secondary metabolites is obtained. The matter capable of regulating the osmotic pressure of cells is PEG or tween. The method of the invention can improve the content of secondary metabolites in liquorice hairy roots by processing the liquorice hairy roots with the matter capable of regulating the osmotic pressure of cells to form drought stress. Experiment showed the method to improve the synthetic quantity of secondary metabolites in liquorice hairy roots has the advantages that, the content of total flavone in the liquorice hairy roots processed by the method is increased to 44% to 55%; the invention provides a new way to rapidly and efficiently obtain high yield liquorice effective secondary metabolites by utilizing liquorice hairy roots culture system.
Description
Technical field
The present invention relates to a kind of method that improves secondary metabolite synthetic amount in the Radix Glycyrrhizae hairy root.
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 drug actions of a prescription, is described as " kings of many medicines ".The main effectively secondary metabolite of Radix Glycyrrhizae comprises triterpenes glycyrrhizic acid and licoflavone, and wherein glycyrrhizic acid is very precious antipoison, can prevent and treat diseases such as virus hepatitis, high fat of blood, cancer and acquired immune deficiency syndrome (AIDS); That licoflavone has is anti-oxidant, antitumor, anti-inflammatory, antiulcer, anti-ageing, pharmacological action such as protect the liver.Radix Glycyrrhizae is not only important medicinal material, also is widely used in daily chemicals and field of food, and wherein glycyrrhizic acid is a kind of natural sweetener, and licoflavone is a class natural antibacterial agent and a preservative.Yet the endangered deep development and use that hindered Radix Glycyrrhizae of Radix Glycyrrhizae wild resource.
Along with improving constantly of biotechnology, utilizing hairy root to cultivate the living metabolite of parity in next life becomes the research focus, and its growth rapidly, branch is many, do not need exogenous hormone, and, be one of effective way of resources of medicinal plant sustainable development than the easy amplification of cell culture.By the Radix Glycyrrhizae hairy root system that agrobacterium rhizogenes infection plant forms, fast growth, genetic stability is good, is the good culture systems of producing the Radix Glycyrrhizae active component.But still there is the low problem of active component in Radix Glycyrrhizae hairy root culture systems, has influenced further developing of Radix Glycyrrhizae hairy root and has amplified and industrialization.General flavone content as Zhang Yinlin report Ural Radix Glycyrrhizae hairy root such as (1990) is 0.39% of a dry weight, well below the content 2.38% of crude drug root; The Ural Radix Glycyrrhizae hairy root flavones content of Du Min researchs such as (2001) also is about 0.39%; It is 0.78% that Wang Yiwei detections such as (2004) glycyrrhiza glabra hairy root general flavone behind growth 31d reaches high-load; Yang Shihai (2006) etc. has detected the content of various different flavones in the Radix Glycyrrhizae hairy root of Ural, and its content of total flavone is about 0.15%.Research to glycyrrhizic acid composition detection in the Radix Glycyrrhizae hairy root is less, and the someone reports and do not contain glycyrrhizic acid in the Radix Glycyrrhizae hairy root, contains glycyrrhizic acid in the Radix Glycyrrhizae hairy root as studies show that of Japan's high capital show etc.; And some researchs are reported in and do not detect glycyrrhizic acid in the Radix Glycyrrhizae hairy root, may be relevant with the source with the kind of Radix Glycyrrhizae.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves secondary metabolite synthetic amount in the Radix Glycyrrhizae hairy root.
The method of secondary metabolite synthetic amount in the raising Radix Glycyrrhizae hairy root provided by the present invention, be to cause Radix Glycyrrhizae hairy root in the mass treatment liquid nutrient medium of drought stress, obtain the Radix Glycyrrhizae hairy root that secondary metabolite content improves with adjustable ganglion cell's osmotic pressure.
It is PEG and/or tween that described adjustable ganglion cell's osmotic pressure causes the material of drought stress.The polyethylene glycol (PEG) that described PEG can be different extent of polymerizations comprises PEG6000, PEG8000, and PEG20000 is preferably PEG8000.
In the described method, the concentration of treatment of described PEG is 0.02-0.08g/mL, is preferably 0.02g/mL; The concentration of treatment of described tween is 0.01-0.04g/mL, is preferably 0.02g/mL.
In the described method, the time of the mass treatment of described adjustable ganglion cell's osmotic pressure is 6-72 hour, is preferably 6-48 hour.The time that described PEG handles separately is 24-72 hour, is preferably 48 hours; The time that described tween is handled separately is 6-36 hour, is preferably 24 hours.
In the described method, described liquid nutrient medium is the MS liquid nutrient medium.
In the described method, described Radix Glycyrrhizae hairy root is that the tip of a root position with the Radix Glycyrrhizae hairy root is inoculated in the MS liquid nutrient medium successive transfer culture 2-4 generation and obtains.The tip of a root position of described Radix Glycyrrhizae hairy root is the tip of a root from tip of a root tip 2-3cm; Described successive transfer culture changed a subculture in per 20 days.
In the described method, described secondary metabolite is a licoflavone.
The redox reaction of organism in the aerobic metabolism process generates with reactive oxygen species (ultra-oxygen anion free radical, hydroxy radical, singlet oxygen, hydrogen peroxide etc.), can cause biomembrane polyunsaturated fatty acid generation peroxidatic reaction of lipid.Damage membrane structure and function, large biological molecules such as damage sugar, protein and nucleic acid cause function and metabolic disorder.Infer that drought stress has stimulated the anti-oxidative defense mechanism of licorice cell, thereby can stimulate a large amount of synthetic of flavonoids.
Method of the present invention realizes improving the content of secondary metabolite in the hairy root by the mass treatment Radix Glycyrrhizae hairy root that causes drought stress with adjustable ganglion cell's osmotic pressure, the general flavone content that experiment showed, the Radix Glycyrrhizae hairy root that obtains after method of the present invention is handled increases 44%-55%.Method of the present invention comes the effective secondary metabolite of acquisition high yield Radix Glycyrrhizae rapidly and efficiently that novel approach is provided for utilizing Radix Glycyrrhizae hairy root cultivating system.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is that the PEG8000 of 0.02g/ml handles general flavone content histogram behind the Radix Glycyrrhizae hairy root different time for concentration.
Fig. 2 is that the PEG8000 of variable concentrations handles general flavone content histogram behind the Radix Glycyrrhizae hairy root; 1 is contrast among Fig. 2, and 2 be that the PEG8000 of concentration 2% (g/ml) handles, and 3 for concentration is that the PEG8000 of 4% (g/ml) handles, and 4 for concentration is the PEG8000 processing of 6% (g/ml), 5 for concentration be the PEG8000 processing of 8% (g/ml).
Fig. 3 is a general flavone content histogram behind same concentrations 0.02g/ml, the variety classes PEG processing Radix Glycyrrhizae hairy root 48h; Among Fig. 3,1 is contrast, and 2 is PEG6000, and 3 is PEG8000, and 4 is PEG20000.
Fig. 4 is that the Tween-80 of variable concentrations is handled general flavone content histogram behind the Radix Glycyrrhizae hairy root 24h; 1 is contrast among Fig. 4, and 2 is the Tween-80 processing of 0.01g/ml, and 3 is the Tween-80 processing of 0.02g/ml, and 4 is the Tween-80 processing of 0.03g/ml, and 5 is the Tween-80 processing of 0.04g/ml.
Fig. 5 is a general flavone content histogram behind Tween-80, the 0.02g/ml concentration processing Radix Glycyrrhizae hairy root different time.
Embodiment
One, it is synthetic that PEG induces the secondary metabolite of Ural Radix Glycyrrhizae hairy root
In the present embodiment, be material with Ural Radix Glycyrrhizae hairy root; Polyethylene glycol (PEG) with different extent of polymerizations comprises PEG6000, PEG8000, and the PEG (0.02g/ml-0.2g/ml) of PEG20000 or variable concentrations and identical type handles the Radix Glycyrrhizae hairy root.The concrete operations step is as follows:
1, will induce that eugonic hairy root white tip of a root position 2-3cm downcuts in the Ural Radix Glycyrrhizae hairy root solid culture of generation, be seeded in the 300ml blake bottle that contains 100ml MS liquid nutrient medium, the inoculum concentration of every bottle of hairy root is 1g, wherein do not contain any hormone in the MS liquid nutrient medium, in temperature is 25 ℃, rotating speed is a dark culturing about 40 days (approximately subculture is twice) on the shaking table of 100rpm/min, changes a subculture in 20 days.
2, collect the Radix Glycyrrhizae hairy root that obtains of 1 liquid culture in steps, remove the dead hairy root of brownization, all the other hairy root mean allocation are in the 300ml blake bottle, every bottle of hairy root weight in wet base is 2g, be divided into 8 groups, each adds 100ml and contains variety classes and same concentrations PEG (PEG6000, PEG8000, PEG20000, concentration is 0.02g/ml) or variable concentrations and identical type PEG (0.02g/ml, 0.04g/ml, 0.06g/ml, 0.08g/ml or 0.1g/ml) the MS medium, every then component becomes three groups, respectively under the condition identical with the front successive transfer culture promptly: temperature is 25 ℃, and rotating speed is to induce on the shaking table of 100rpm to handle 24,48 or 72 hours, the hairy root of handling the identical time with the MS culture fluid that does not contain the PEG elicitor was contrast (CK).
Two, PEG handles the detection of general flavone content in the Radix Glycyrrhizae hairy root of back.
1, Radix Glycyrrhizae hairy root and the contrast hairy root after step 1 respectively being organized PEG and being handled with aseptic water washing three times, removed residual medium respectively, suck dry moisture on filter paper is put in 60 ℃ of baking ovens and is dried to constant weight back 60 mesh sieves of crossing of pulverizing, take by weighing the 0.1g powder, add 5ml methyl alcohol, soaked overnight (about 12 hours), ultrasonic (40Hz) extracted 30 minutes, suction filtration, filter residue again repetitive operation once, merging filtrate is settled to 10ml.
2, ultraviolet spectrophotometry detects that step 1 obtains respectively organizes general flavone content (representing with the quality percentage composition) in the sample.With the aurantiin is standard sample, makes calibration curve.Calculate the concentration of institute's test sample product according to calibration curve, thereby calculate the quality percentage composition of general flavone in the 0.1g Radix Glycyrrhizae hairy root powder.
Shown in table 1-table 3 (corresponding histogram is as shown in Figure 1-Figure 3), the result shows through the ultraviolet spectrophotometry testing result:
1) after concentration was the PEG8000 processing Ural Radix Glycyrrhizae hairy root different time of 0.02g/ml, general flavone content was improved from hairy root between the 24h to 72h, especially with general flavone content in the hairy root of 48h the highest (as table 1, shown in Figure 1).
Table 1. concentration is the general flavone content of the Ural Radix Glycyrrhizae hairy root handled of the PEG8000 of 0.02g/ml
| Project | General flavone content (quality percentage composition %) | ||
| |
24h | 48h | 72h |
| Contrast (ck) | 1.02 | 1.04 | 1.03 |
| PEG8000(0.02g/ml) | 1.4 | 1.64 | 1.37 |
2) in the hairy root after identical type and variable concentrations PEG handle, general flavone content is handled from 0.02g/ml-0.08g/ml PEG and is improved, but effectively raising scope is between the 0.02g/ml to 0.06g/ml, especially be that 0.02g/ml handles after 48 hours to the highest with PEG concentration, general flavone content comparison photograph has improved 44% (as table 2, shown in Figure 2); 1 is contrast among Fig. 2, and 2 be that the PEG8000 of concentration 2% (g/ml) handles, and 3 for concentration is that the PEG8000 of 4% (g/ml) handles, and 4 for concentration is the PEG8000 processing of 6% (g/ml), 5 for concentration be the PEG8000 processing of 8% (g/ml).
Table 2.PEG8000 handles the general flavone content of the Ural Radix Glycyrrhizae hairy root of different time
| Project | General flavone content (quality percentage composition %) | |||
| PEG8000 concentration (g/ml) | 2％ | 4％ | 6％ | 8％ |
| Contrast (ck) | 1.330 | |||
| PEG8000 | 1.920 | 1.802 | 1.697 | 1.501 |
3) variety classes and same concentrations (being 0.02g/ml) PEG handle general flavone content difference in the hairy root behind the 48h, and general flavone content the highest (as table 3, the shown in Figure 3) comparison after handling with PEG8000 is according to having improved 55.20%.Among Fig. 3,1 is contrast, and 2 is PEG6000, and 3 is PEG8000, and 4 is PEG20000.
The general flavone content of the Ural Radix Glycyrrhizae hairy root that table 3. variety classes PEG handles
| PEG 1500 | Contrast (ck) | PEG6000 | PEG8000 | PEG20000 |
| Flavones content (quality percentage composition %) | 0.683 | 0.880 | 1.06 | 0.749 |
General flavone content detected after embodiment 2, Tween-80 were handled the Radix Glycyrrhizae hairy root
Method according to the step 1 in the step 1 of embodiment 1 is cultivated the Radix Glycyrrhizae hairy root that obtains liquid culture, then with the Radix Glycyrrhizae hairy root of this liquid culture through twice subculture (MS medium, temperature is 25 ℃, rotating speed is a dark culturing on the shaking table of 100rpm) after the growth, collect all hairy root, remove the dead hairy root of brownization, all the other hairy root mean allocation are in the 300ml blake bottle, every bottle of hairy root weight in wet base is 2g, each adds 100ml and contains variable concentrations Tween-80 (0.01g/ml, 0.02g/ml, 0.03g/ml MS medium or 0.04g/ml), with front successive transfer culture the same terms under induce and handle 6,12,24 and 36 hours, the hairy root of handling the identical time with the MS culture fluid that does not contain elicitor was contrast (ck).
Detect the Radix Glycyrrhizae hairy root after handling and the general flavone content of contrast hairy root according to the method for the step 2 of embodiment 1, the result is shown in table 4, table 5 (its histogram is Fig. 4, Fig. 5), and the result shows:
(1) in the hairy root that the Tween-80 of variable concentrations is handled, the general flavone content of handling from 0.01g/ml to 0.04g/ml is improved, especially the Tween-80 that with concentration is 0.02g/ml is handled the highest of 24h, comparison is according to high by 47.30% (as table 4, shown in Figure 4,1 is contrast among Fig. 4, and 2 is the Tween-80 processing of 0.01g/ml, and 3 is the Tween-80 processing of 0.02g/ml, 4 is the Tween-80 processing of 0.03g/ml, and 5 is the Tween-80 processing of 0.04g/ml):
The general flavone content of the Radix Glycyrrhizae hairy root after table 4. variable concentrations tween is handled
| Tween-80 concentration | 0.01g/ml | 0.02g/ml | 0.03g/ml | 0.04g/ml |
| Tween-80 is handled back general flavone content (%) | 0.831 | 1.09 | 0.863 | 0.952 |
| Ck general flavone content (%) | 0.740 | |||
(2) be that the Tween-80 of 0.02g/ml is handled in the hairy root of different time sections with concentration, the general flavone content of handling from 6h to 36h is improved, and the highest with 24h especially is higher than 49% (shown in Fig. 5, table 5) of contrast.
Table 5. tween concentration of treatment is the general flavone content of the Radix Glycyrrhizae hairy root behind the 0.02g/ml processing different time
| | |
12h | 24h | 36h | ||
| Ck general flavone content (%) | 0.57 | 0.583 | 0.588 | 0.589 | ||
| Tween-80 (0.02g/ml) is handled back general flavone content (%) | 0.666 | 0.827 | 0.850 | 0.717 |
Claims (9)
1. method that improves licoflavone synthetic amount in the Radix Glycyrrhizae hairy root, be with the Radix Glycyrrhizae hairy root in polyethylene glycol and/or the tween treat liquid medium, wherein the concentration of treatment of polyethylene glycol is 0.02-0.08g/mL, the concentration of treatment of tween is 0.01-0.04g/mL, obtains the Radix Glycyrrhizae hairy root that licoflavone content improves.
2. method according to claim 1 is characterized in that: the concentration of treatment of described polyethylene glycol is 0.02g/mL; The concentration of treatment of described tween is 0.02g/mL.
3. method according to claim 2 is characterized in that: the time that described polyethylene glycol and/or tween are handled is 6-72 hour.
4. method according to claim 3 is characterized in that: the time that described polyethylene glycol and/or tween are handled is 6-48 hour.
5. method according to claim 3 is characterized in that: described polyethyleneglycol stay alone the reason time be 24-72 hour; The time that described tween is handled separately is 6-36 hour.
6. method according to claim 5 is characterized in that: described polyethyleneglycol stay alone the reason time be 48 hours; The time that described tween is handled separately is 24 hours.
7. method according to claim 5 is characterized in that: described liquid nutrient medium is the MS liquid nutrient medium.
8. method according to claim 7 is characterized in that: described Radix Glycyrrhizae hairy root is that the tip of a root position with the Radix Glycyrrhizae hairy root is inoculated in the MS liquid nutrient medium successive transfer culture 2-4 generation and obtains.
9. method according to claim 8 is characterized in that: the tip of a root position of described Radix Glycyrrhizae hairy root is the tip of a root from tip of a root tip 2-3cm; Described successive transfer culture changed a subculture in per 20 days.
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| CN102021137A (en) * | 2009-09-08 | 2011-04-20 | 北京未名凯拓农业生物技术有限公司 | Method for preparing glycyrrhiza total flavonoids from glycyrrhiza hairy roots and culture solution thereof |
| CN103695458A (en) * | 2013-11-08 | 2014-04-02 | 宁夏农林科学院 | Methods for efficient induction production of Glycyrrihiza uralensisi Fisch hairy roots and production of licorice root secondary metabolites by using Glycyrrihiza uralensisi Fisch hairy roots |
| CN110622808B (en) * | 2019-09-26 | 2021-11-23 | 中国科学院昆明植物研究所 | Planting method for promoting growth of bletilla striata and accumulation of effective components |
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| CN1484942A (en) * | 2003-08-15 | 2004-03-31 | 锦 王 | Planting process of herbal plant of strip liquorice , oxalic acid content increased liquorice |
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