KR20060092308A - Method for producing guinarine by tissue culture of poppy family - Google Patents

Abstract

The present invention relates to a method for producing raw guinarin, and more particularly, in the production of fresh guinealine by tissue culture of a poppy family containing a high content of raw guinealine, a medium for plant growth and production of guinealine To increase the production of guinarine by varying the composition of the chlorine and to increase the amount of guinealine, and to dry the plant cell culture medium containing the guinarine obtained from this, to make a vinegar extract using organic acids and organic solvents, add salt The present invention relates to a method for producing raw guirin by tissue culture of poppy plants, which can improve both productivity and recovery rate of fresh guinealine by efficiently separating the raw guinealine from the plant cell culture solution.

According to the present invention, the productivity and recovery rate of raw guinealine can be significantly improved compared to the conventional method, and the safety can be increased by using an extraction method that is harmless to the human body. By enabling mass production, there is an effect of reducing the production cost of cosmetics, food additives and medical preparations using the same.

Raw curiosity, element, safety, recovery

Description

Method for preparing guinarine by tissue culture of poppy family {Method for Preparing Sanguinarine Using Tissue Culture of Papaveraceae Plant}

Figure 1 (a) is used when Botrytis cinerea ( Elysit ) as an eliminator, (b) when the yeast extract (yeast extract) is used as an eliminator, (c) is used to purify the yeast extract In the case of the production of fresh guinarin and dihydro guinarine is a graph showing a comparison with the control (control) without the addition of the eliminator.

Figure 2 is a graph showing the extraction efficiency when the extraction of raw guinarin by adding acetic acid, lactic acid, citric acid.

The present invention relates to a method for producing raw guinarin, and more particularly, in the production of fresh guinealine by tissue culture of a poppy family containing a high content of raw guinealine, a medium for plant growth and production of guinealine To increase the production of guinarine by varying the composition of the chlorine and to increase the amount of guinealine, and to dry the plant cell culture medium containing the guinarine obtained from this, to make a vinegar extract using organic acids and organic solvents, add salt The present invention relates to a method for producing raw guirin by tissue culture of poppy plants, which can improve both productivity and recovery rate of fresh guinealine by efficiently separating the raw guinealine from the plant cell culture solution.

Sanguinarine is a folk remedy used by indigenous people living in the Americas, and is rooted in the Sanguinaria Canadensis Linne plant of the Papaveraceae in North America and Canada. It is a kind of benzophenanthridine alkaloids-based compound mainly contained in perennial plants such as Park, Rak-hee, Baekchulchae and Celandine, and plants such as Eschscholtzia california .

Raw Guinarin is known to exhibit a wide range of antibiotic and anticancer effects, such as bacteria, fungi, yeast, protozoa, etc., and is particularly widely used as a dental antibiotic. Due to the broad antibiotic effect of Saint-Ginarin, the application of not only medicine, but also cosmetics and food additives has been expanded, and US Patent No. 4,335,110, US Patent No. 4,145,412, US Patent No. 4,689,216, etc. have.

On the other hand, methods for producing raw guinarin include synthetic methods, plant cell culture methods, and direct cultivation methods, which are not suitable for industrial mass production due to its complicated steps and low yield. . In addition, the direct cultivation method is to grow a poppy family known to contain the highest content of raw guinea to date to obtain raw guinea from this, although the relatively simple method, the content of the raw guinarin is environmentally and geographically affected. There is a drawback that the production is not much because it receives a lot. In the case of Alban Muller International, France, which produces commercially fresh raw guinarin, the final yield of raw guinealine from the roots of the poppy family is around 1% of the root dry weight.

For these reasons, in recent years, attempts have been made to produce raw guinealine using plant cell cultures. Plant cell culture is a method used when production by other methods is not possible or economical, as well as easy to control the production, there is an advantage that the harvesting process is relatively simple because it uses the cells grown in the incubator. In particular, in the countries where the cultivation of wild creeper plants containing raw guinea plants in relation to psychotropic drugs is prohibited by law, plant cell culture is the most appropriate method for producing raw guinea pigs.

As a related art related to the production of raw guinealine through the cell culture of the poppy family, Korean Patent Application No. 10-1995-12880 discloses the elimination of suspended cell culture solution of Park, Rak-hee and Mccleya microkappa plants containing high contents of guinealine. Disclosed is a method of improving the productivity of guinarine and its derivatives by adding a seed. The patent has a meaning in that the plant of the genus McCleya, which has not been studied in the past, has been used, but it is possible to use hydrochloric acid and methanol, which are harmful to the human body, in the free state of guinarine and its derivatives in the culture medium. There is a problem in safety by extraction according to the known method used, there is a disadvantage that it does not improve the amount of recoverable raw guinarin and its derivatives and did not increase the final production of raw guinarine.

In addition, Korean Patent Application No. 10-1994-26350 discloses a method for increasing the productivity of secondary metabolites by culturing plant cells, such as anthraquinone, menthol and taxol, including guinealine, by adding cyclodextrin to plant cell culture medium. Is disclosed. In addition, there is a problem of safety and the failure to improve the extraction process of secondary metabolites by the demanding plant cell culture, there is a problem that does not improve the recovery rate of the final guinarin.

Therefore, the present inventors have made intensive efforts to satisfy both the productivity and the recovery rate of fresh guinea pigs. As a result, in the culture of plant cells for the production of fresh guinea pigs, the composition of the medium for plant growth and the production of fresh guinarin and The productivity of raw guinealine can be improved by adding a citrate, and the guinealine contained in the plant cell culture medium is prepared by adding organic acid and ethanol which are harmless to the human body to make the raw guinea extract, and extracting with added salt. It was confirmed that the can be effectively separated and recovered, to complete the present invention.

After all, it is an object of the present invention to provide a method for producing fresh guinarin by tissue culture of poppy plants, which can improve both safety, productivity and recovery rate of fresh guinarine.

In order to achieve the above object, the present invention is a suspension culture of the callus of the poppy family to produce a high concentration of raw guirin, and recovering the raw guirin through the following steps:

(a) drying the plant cell culture solution, and then preparing a fresh extract using a first organic solvent and at least one acid selected from the group consisting of citric acid, acetic acid or lactic acid; (b) extracting the liquid / liquid and separating the aqueous layer by adding a second organic solvent to the fresh extract; (c) adjusting the pH of the separated aqueous layer to 10 or more, followed by adding a third organic solvent to extract liquid / liquid and separating the aqueous layer; And (d) distilled water containing at least one acid selected from the group consisting of citric acid, acetic acid, or lactic acid to the separated aqueous layer, extracts liquid / liquid, and then adds a salt to precipitate the precipitate. It provides a method for producing guinarine characterized in that it comprises the step of producing.

In the present invention, the poppy family may be characterized in that the Park Rak Hee, Sanguinaria Canadensis Linne ( Sanguinaria Canadensis Linne), baekryechae, celandine or Eschscholtzia california , preferably a gold film You can do

In the present invention, in the suspension culture, at least one elite selected from the group consisting of Botrytis cinerea, yeast extract, phytophthora megasperma, chitin, chitosan and glucan can be added. And, it may be characterized in that the Botrytis cinerea, yeast extract.

In the present invention, the first organic solvent may be characterized in that the ethanol, the salt may be characterized in that the magnesium sulfate, potassium nitrate, calcium chloride, sodium chloride, copper sulfate or aluminum chloride, preferably sodium chloride It may be characterized in that, the salt may be characterized in that it is added in 10 ~ 15g with respect to 100ml of fresh guinea extract.

In the present invention, the second organic solvent may be hexane, the third organic solvent may be characterized in that the methylene chloride (methylene chloride), the liquid / liquid extraction is performed repeatedly 3 to 5 times It may be characterized by.

In the present invention, the step of recovering the raw fish may further comprise the step of centrifuging the precipitate produced in step (d), followed by washing with methanol and drying.

Hereinafter, the present invention will be described in detail.

In general, alkaloid-based compounds, such as guinarine, produced during the cell culture of poppy plants exhibit effective physiological or pharmacological activity, but not only have a very low concentration of intracellular or extracellular discharge, but also degrade or be converted into other metabolites. Very low recovery. Accordingly, the present invention provides a method for producing fresh guinarin, which can significantly increase productivity and improve safety and recovery rate of poppy guinealine in cell culture of poppy plants.

Among the poppy family, the stem of the gold film was selected and sectioned about 0.5 ~ 2cm, and 2,4-D (2,4-dichlorophenoxy acetic acid) and NAA (napthaleme acetic acid) were each 0.1 ~ 10.0mg / ℓ. Induced callus by inoculating MS medium added in an amount of preferably 0.1-5.0 mg / L, and then inducing the callus in an amount of 0.1-5.0 mg / L NAA, preferably 2.0 mg. Proliferation is induced in MS medium added in an amount of / l. At this time, the pH of the medium can be propagated to the maximum callus under the conditions of 5.5 to 6.0, the culture temperature is 18 to 24 ℃. In addition, when the growth regulator affects callus proliferation, each of NAA, IBA, and 2,4-D is added at 0.1 to 2.0 mg / l.

In addition to MS (murashige-skoog) medium, in addition to MS (murashige-skoog) medium, SH (schenk-hildebrandt) medium, B5 (gamborg) medium, white medium, and the like can be used. Although the effects are almost the same, the most desirable results can be obtained in MS medium.

After the suspension culture of the proliferated callus tissue, by changing the composition of the medium used for the growth of the plant and the production of raw guinarin culture can be improved productivity of the raw guinarin, preferably NAA as a plant growth regulator In the SH medium added in the amount of 4.0 to 6.0 mg / L can greatly improve the productivity of raw guinarin. In addition, in order to further improve the productivity of raw guinarin, an elicitor may be added that can stimulate the biosynthetic pathway of raw guinarin. In the present invention, by adding an elite element such as Botrytis cinerea , yeast extract, yeast extract, phytophthora megasperma, chitin, chitosan or glucan, the production amount of guinea pigs can be drastically increased. have.

In the plant cell culture obtained as above, alkaloid derivatives such as chelerythrine, sanguilutine, sanguirubine, and chelilutine are present in addition to chlorinthrine. In the present invention, the cell culture solution is extracted with an organic acid of citric acid, acetic acid or lactic acid and a first organic solvent, and then separated from the aqueous layer by a second organic solvent and a third organic solvent, distilled water containing citric acid After re-extracting, the salts can be treated to effectively separate and recover only raw Guinarin from a large number of alkaloid compounds.

In the present invention, by using organic acids and ethanol, such as citric acid, lactic acid, acetic acid, which is safer for the human body, instead of hydrochloric acid (HCl) and methanol, which are harmful to the human body, which is conventionally used in the extraction of conventional raw fish, can increase safety.

In addition, after the salt is treated in the present invention, a precipitate can be obtained even by leaving the culture solution alone, but it is preferable to centrifuge for easy recovery, and the type of salt used is not particularly limited, but preferably magnesium sulfate , Potassium nitrate, calcium chloride, sodium chloride, copper sulfate, aluminum chloride, more preferably sodium chloride.

In the present invention, the cell culture solution includes a cell culture solution containing cells or a culture filtrate from which cells are removed.

In addition, in the present invention, by extracting the raw guinarin with distilled water containing acid, which is harmless to the human body, such as citric acid, there is no problem of toxicity and side effects.

Although the present invention has been described using only the gold film of the poppy family as an example, the present invention can be applied to all plant species containing raw guirin, such as Park, Lak-hee, Sanguinaria Canadensis Linne, white oyster and celandine. It is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 : Selection of Optimum Condition for Raw Fish Production

1-1. Callus Induction and Proliferation

After sterilizing and disinfecting the stem of gold film, a kind of poppy family, it is cut into 0.5 ~ 2cm sections, and then 2,4-D (2,4-dichlorophenoxy acetic acid) 0.1mg / ℓ, NAA 2.0mg / L was added to murashige-skoog (MS) medium and transferred to a culture chamber maintained at 23 ° C. in the dark to induce callus.

In order to select the conditions for the maximum growth in the callus induced as described above in the shortest period, the experiment was as follows. That is, 30 ml of MS medium to which NAA 2.0 mg / l was added was dispensed into a disposable Petri dish, and then the callus of the gold film obtained in Example 1-1 was sectioned and inoculated. The pH of the medium was divided into 4.0, 5.0, 5.5, 6.0, 6.5, and 7.0, and the callus growth was observed by dividing the culture temperature into 15 ° C, 17 ° C, 19 ° C, 21 ° C, 23 ° C, and 25 ° C.

As a result, it was confirmed that the range of the medium pH 5.5 to 6.0 and the culture temperature of 18 to 24 ° C was the optimal condition for callus growth.

1-2. Effect of Growth Regulators on Callus Growth

The callus of the gold film obtained in Example 1-1 was fixed at pH 6.0, and each of NAA, IBA, 2,4-D was 0.1 mg / L, 1.0 mg / L, 2.0 mg / L, 3.0 as a growth regulator. The effect of growth regulators on callus proliferation was examined by placing the MG / L and 5.0 mg / L into MS medium. After dental treatment, the cells were transferred to fresh medium of the same composition every two weeks, and after 10 weeks, the extent and condition of callus were observed.

As a result, as shown in Table 1, the growth in the growth-treated medium was superior to the medium without the growth regulator, and also in the medium added IBA and NAA showed a more than four-fold increase in live weight , The titration concentration was confirmed to be 1.0 ~ 2.0mg / ℓ. On the other hand, the addition of 2,4-D significantly increased the body weight, it can be seen that the optimum concentration is 0.1 ~ 1.0mg / ℓ.

1-3. Effect of Medium Composition on Unprecedented Productivity

Callus grown at the optimum condition of 1-2 was transferred to a liquid medium to which 2,4-D 1.0 mg / L was added to induce liquid suspension culture. A 300 ml Erlenmeyer flask containing 50 ml of liquid medium was induced and about 1.0 g of fine callus was shaken at a speed of 120 rpm in a rotary shaker controlled at 23 ° C. in a dark state, followed by culturing of liquid at 7 days intervals. Passed.

In order to determine the productivity of fresh guinarin according to the change of the composition of the medium, incubated for 3 weeks in the liquid suspension culture medium, and then collected only well-grown cells step by step NAA at a concentration of 0 ~ 12mg / ℓ as a plant growth regulator Incubate for 2 more weeks in the added SH medium to compare the productivity of raw guinarin.

To quantify the vitreous rinse, the plant cell suspension culture was filtered with Whatman filter paper, weighed and dried in an oven to measure the amount of drying, and the cells were sonicated for 20 minutes at room temperature for measurement of intracellular content. Extracted by breaking with. In addition, extraction was performed using hexane to measure the content in the filtrate. HPLC was performed to quantify the content of virinin in the cells and extracts. At this time, HPLC was carried out according to a known analytical method using a mixed solution of acetonitrile and water (35:65) as a supelcosil-18-DB column (diameter 4.6 mm × 15 cm). .

As a result, as shown in Table 2, when the composition of the medium was changed after suspension culture, the production of raw guinarin increased, and when the NAA was added at a concentration of 4.0 to 6.0 mg / L, It was confirmed that the increase significantly.

1-4. Effect of Eliator on Unprecedented Productivity

In order to improve the productivity of alkaloids, a method of stimulating a biosynthetic pathway is used. In the present invention, after treating various eliminators, the effect of the treatment of the eliminator on the productivity of the raw guinarin was examined. In general, the stimulation of the biosynthetic pathway can be used for non-living materials such as heavy metals and biological materials such as fungal cell wall components.

After the addition of the eliminator of the botrytis cinerea and yeast extract into the medium of Examples 1-3, the productivity improvement of the guinearin by the addition of the elite was measured.

1-4-1 When Botrytis Cinerea is used as an eliminator

Botrytis Cinerea was used as the elementator. Figure 1 (a) is a graph showing the increase in production of raw guinarin and dihydro raw guinarin when the Botrytis cinerea is used as an eliminator compared to the control without the addition of the eliminator. Looking at the graph, it can be seen that the production of fresh guinarin and dihydro fresh guinarin increased more than three times with the addition of Botrytis cinerea.

1-4-2 Using Yeast Extract as Eliminator

Yeast extract was used as an eliminator. Figure 1 (b) is an increase in the production of raw guinealine and dihydro guinealine when the yeast extract is used as an eliminator and (c) is an increase in the production of raw guinealine and dihydro guinealine when the yeast extract is purified It is compared with a control without adding a sheeter. Looking at the graph, it can be seen that when yeast extract is added, the production of raw guinarin and dihydro virgin guinarin is increased by 6 to 10 times.

In addition, after the addition of various kinds of elite in the medium of Example 1-3, the productivity improvement of the raw guinarin according to the addition of the elite was measured. Cell wall extract of phytophthora megasperma was used by extracting about 80 mg of cell wall components from 1.5 L liquid cultured cells for 1 week according to a known method. Chitin, chitosan, glucan and benzoic acid were used. , Cellulase, methyl jasmone (methyl jasmone), cis jasmone (cis jasmone), ethylene (ethylene) was purchased from Sigma used.

The results are shown in Table 3 below. In the case of Botrytis cinerea and yeast extract, the production of raw guinarin was significantly increased, and the production of fresh guinarin increased about 1.5 to 2.5 times with the addition of phytophthora megasperma, chitin, chitosan, and glucan. It was. Conversely, the addition of cellulase and cis jasmon decreased the production of guinarin rather.

Example 2:  Selection of Optimum Condition for Raw Fish Collection

2-1. Raw Extracts Using Organic Acids and Ethanol

In addition to raw guirin, alkaloid derivatives such as fresh guirin, fresh guirubin, celeryrin, and cellirutin are present in the cell culture medium after plant cell culture. Therefore, in order to select extraction conditions for effectively separating and recovering only raw guirin, Experimented together.

After drying the cells incubated for 2 weeks using the method of Example 1, 2 g of the extract was added with 10 ml of an extraction solvent of pH 2 and 3 and ethanol to which acetic acid, lactic acid, citric acid were added, and ethanol. Sonication was performed at 50 ° C. for 60 minutes, vortexing was performed for 20 minutes, and then centrifuged to separate only the supernatant. After passing through a 0.45μm membrane filter (membrane filter) was analyzed by HPLC.

As a result of the analysis, when extracted with lactic acid (pH 2) as shown in Fig. 2, the extraction efficiency was high as 1598 ppm of guinealine, 7179 ppm of celeryrin, and 4638 ppm of dihydro guinarine.

2-2. Recovery of Raw Guina

The cell suspension culture obtained in Example 2-1 was filtered with a filter paper, and extracted with the same amount of hexane. After repeating this process three times, 1N NaOH aqueous solution was added to the obtained aqueous layer to adjust the pH to 10, and then re-extracted using the same amount of methylene chloride, followed by the same amount of distilled water containing citric acid (pH 2.30, citric acid 3 ~). 5 g / 100 ml) was added and extracted again.

2-3. Recovery Effect of Raw Guinea with Addition of Salt

10 to 15 g of sodium chloride (NaCl) salt was added per 100 ml of the extract obtained in the same manner, followed by stirring, followed by centrifugation. After centrifugation, the supernatant and the precipitate were separated, and distilled water was added thereto, respectively, and HPLC was performed under the conditions described in Examples 1-3, to compare the recovery of guinea saline.

As a result, as shown in Table 4 below, when sodium chloride salt was added to the extraction step, it was confirmed that the recovery rate of raw Guinarin was significantly improved as compared with the case without addition.

2-4. Recovery Effect of Fresh Guinarin by Addition Salts

By varying the type of salt added, the extraction process was performed in the same manner as in Example 2-2, to determine the recovery effect of raw guinarin according to the type of added salt. That is, magnesium sulfate, potassium nitrate, calcium chloride, sodium chloride, copper sulfate, and aluminum chloride at a concentration of 1 M are added and mixed well, followed by centrifugation, and a supernatant and a precipitate are separated, and distilled water is added thereto, respectively. HPLC was carried out under the conditions described in to determine the recovery of guinarine.

As a result, it was found that the best recovery was obtained when sodium chloride salt was added. Accordingly, when the salt was added to the plant cell culture solution, and especially the sodium chloride salt was added, it was confirmed that the guinarine was more effectively separated and recovered.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described in detail above, according to the present invention, the productivity and recovery rate of raw guinarin can be significantly improved compared to the conventional method, and the safety can be increased by using an extraction method that is harmless to the human body, and it is used in a wide range of fields as an antimicrobial material. By enabling the mass production of fresh guinarin, there is an effect of reducing the production cost of cosmetics, food additives and medical preparations using the same.

Claims (13)

Method of producing a raw guinarin, characterized in that to produce a high concentration of raw guinealine by suspending cultured callus of the poppy family, and recovering the raw guinealine by the following steps: (a) drying the plant cell culture solution, and then preparing a fresh extract using a first organic solvent and at least one acid selected from the group consisting of citric acid, acetic acid or lactic acid; (b) extracting the liquid / liquid and separating the aqueous layer by adding a second organic solvent to the fresh extract; (c) adjusting the pH of the separated aqueous layer to 10 or more, then adding a third organic solvent to extract liquid / liquid and separating the aqueous layer; And (d) distilled water containing at least one acid selected from the group consisting of citric acid, acetic acid, or lactic acid to the separated aqueous layer, extracts liquid / liquid, and then adds a salt to precipitate the precipitate. Generating. The method of claim 1, wherein the poppy family is Rak Hee, Sanguinaria Canadensis Linne, white buckwheat, celandine or Eschscholtzia california . The method of claim 2, wherein the poppy family is Eschscholtzia california . The method of claim 1, wherein in the suspension culture, at least one element selected from the group consisting of Botrytis cinerea, yeast extract, phytophthora megasperma, chitin, chitosan and glucan is added. How to. The method of claim 4, wherein the eliminator is Botrytis cinerea or yeast extract. The method of claim 1, wherein the first organic solvent is ethanol. The method of claim 1 wherein the salt is magnesium sulfate, potassium nitrate, calcium chloride, sodium chloride, copper sulfate or aluminum chloride. 8. The method of claim 7, wherein said salt is sodium chloride. The method of claim 1, wherein the second organic solvent is hexane. The method of claim 1, wherein the third organic solvent is methylene chloride. The method according to claim 1, wherein the salt is added in an amount of 10 to 15 g based on 100 ml of fresh quinine extract. The method of claim 1, wherein the liquid / liquid extraction is performed 3 to 5 times repeatedly. The method of claim 1, wherein the step of recovering the raw fish further comprises the step of centrifuging the precipitate produced in step (d), followed by washing with methanol and drying.

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