KR100861473B1 - Process for producing Valeriana fauriei roots, Valeriana fauriei roots produced by the process, and process for producing valerenic acids and valepotriates from the roots - Google Patents

Abstract

The present invention is a method for producing Valeriana fauriei var. Dasycarpa Hara roots by culturing in a bioreactor, Valerian roots produced by the method and valerianic acid as an active ingredient from the Valerian roots. The present invention relates to a method for producing acids and valepotriates, and according to the present invention, it is possible to economically produce a large amount of Valerian root having a valerian acid and valeric acid content comparable to field cultured Valerian root. .

Valerian, Culture Root, Valeriana fauriei var. dasycarpa Hara, In-flight culture, Valerensan, Valefort

Description

Process for producing Valeriana fauriei roots, Valeriana fauriei roots produced by the process, and process for producing valerenic acids and valepotriates from the roots}

1 is a graph showing the production of valerian acid and valeric acid when juvenile Valerian roots were cultured for 7 weeks by the method according to the present invention;

2 is a graph showing the change in total valerenic acid content of Valerian cultivation root;

Figure 3 is a graph showing the change in total valerian fort content of Valerian cultivars.

The present invention relates to a method for producing Valeriana fauriei root, a method for producing valerian root produced therefrom, and a method for producing valerennic acids and valepotriates therefrom, and more specifically, A method of producing Valerian root, which can economically produce large amounts of Valerian root having valerian acid and valeric acid content comparable to field-cultivated Valerian root by culturing in a bioreactor, Valerian root produced by the same and a method for producing valeric acid and valeric acid from the same.

Valerian is a perennial herbaceous plant belonging to the Valerianaceae Valeriana, mainly in northern Europe and temperate regions of Asia. The mature herb is about 50 ㎝ and grows straight. The leaves are intact, stiff, and large. They are prickly, divided into 5 ~ 7, and the lobes are serrated. Flowers bloom in May-July with reddish black color and are in the shape of mountains. The hairs on the fruit are called Valerian fauriei var.dasycarpa Hara, and the leaves are not sawtoothed on the leaf lobes. Valerian fauriei var. Integra . In particular, Gwangneung Jute is a Korean urine, which is distributed in China and Japan.

Valerian root has been used as a medicinal herb for the treatment of neuropsychiatric diseases such as sedation, malaise, insomnia, anxiety and hysteria.The essential oils obtained from the roots of this plant are fragrances, tonics, liquors, foods, It has been used as a fragrance for cosmetics and tobacco, and has recently been shown to have anti-HIV effects. As mentioned above, valerian plants have soothing, tectonic and anti-anxiety properties, mainly because they produce valeric acid and valeric acid.

Valerian breeding methods include seed breeding and dispensing methods, but both have high viral infection rates, and seeds have a constant fruiting period and have a characteristic of easily scattering with fluffy when the wind blows or falling easily when it rains. In addition, the dispensing method is capable of dispensing only about two to five from one mother bottle, and it has been a big problem that difficulties in seeding and propagation have arisen, such as infection of pathogens caused by dispensing and labor increase.

Nevertheless, no method of mass production of Valerian roots by culturing in a bioculture has been established until now, and thus, large amounts of economical cultivation of Valerian roots containing useful physiologically active ingredients by incubating in a bioculture There has been a great demand for the development of a method that can produce.

The present inventors have carried out continuous research to develop a method for mass production of Valerian root through in vitro culture without plant growth regulators or transformation. As a result, it was confirmed that it is possible to mass-produce the Valerian root having an active ingredient content comparable to the open cultivated Valerian root by incubating the Valerian root under specific conditions in a specific medium, thereby completing the present invention.

Therefore, a first object of the present invention is to provide a culture medium in the bioreactor of Valerian root.

A second object of the present invention is to provide a method of producing Valerian root by culturing in a bioreactor.

A third object of the present invention is to provide a Valerian root produced by the method.

It is a fourth object of the present invention to provide a method for producing valeric acid and valeric acid from the produced Valerian root.

The first aspect of the present invention relates to a culture medium in a bioreactor of Valerian root, containing 7% sucrose in B ₅ medium and adjusted to pH 5.5-6.0. In the medium, it is preferable that the concentration ratio of KNO ₃ : (NH ₄ ) ₂ SO ₄ is 24 to 25: 1.0, and the concentration of NaH ₂ PO ₄ is 0.3 to 0.4 mM.

The second aspect of the present invention relates to a method of producing Valerian root, comprising culturing Valerian root in a bioreactor containing the medium. In the said method, it is preferable to incubate for 4 to 6 weeks at 15-25 degreeC by the air injection amount of 0.2-0.5 vvm.

The third aspect of the invention relates to Valerian root produced by the method. Although the Valerian root is a cultured root, it has an active ingredient content comparable to that of the cultivated Valerian root.

The fourth aspect of the present invention

(1) cultivating Valerian root in a bioreactor containing the medium;

(2) Extracting valerianic acid and valeric acid from cultured Valerian root:

It relates to a method for producing valeric acid and valeric acid, comprising the step.

Hereinafter, the present invention will be described in detail.

In the present invention, it was intended to establish a method for mass production of Valerian root through culturing in a bioreactor. Therefore, in order to identify the root growth titration medium through in-flight culture, various media, carbon sources, light conditions, pH (4.5, 5.0, 5.3, 5.5, 5.7 or 6.0), temperature (15, 20, 25, 30 or 35) Experiments were carried out. As a result, root growth was best when 7% sucrose was added as a carbon source to the B ₅ medium and the pH was adjusted to 5.5 to 6.0, especially 5.7. Also, valencene was cultured by adding KNO ₃ : (NH ₄ ) ₂ SO ₄ with a concentration ratio of 24 to 25: 1.0, especially 24.8: 1.0 as a nitrogen source, and 0.3 to 0.4 mM as a phosphoric acid source, especially 0.32 mM NaH ₂ PO ₄ . The productivity of acid and valericulite was shown to be the best. On the other hand, germination rate of Valerian seed did not show any significant difference in light condition, media type and medium treatment concentration, and low germination rate (<30%) under all conditions.

Furthermore, in the present invention, the content of valerenic acid and valeric acid in open field and incubation tissue of Valerian was analyzed by HPLC-PDA (High Performance Liquid Chromatography-PhotoDiode Array) method. The content of valeric acid and valeric acid in different tissues was similar in open field and in-plant cultures, where valeric acid and valeric acid were mainly synthesized in muscle (> 70%) and less in shoots. Sheep were synthesized. Didrovaltrate was detected in all incubation muscles, but only in samples collected in April in cultivation muscles.

As a result, the optimal conditions for the production of valerenic acid and valeric acid in bioreactor culture were 7% sucrose in B ₅ medium and the pH was adjusted to 5.5-6.0, especially 5.7. 1.0, in particular KNO ₃ : (NH ₄ ) ₂ SO ₄ of 24.8: 1.0, 0.3 ~ 0.4 mM, in particular 0.32 mM NaH ₂ PO _{4 It} was confirmed that the case of the addition of phosphoric acid. In addition, inoculate 20 g (fresh wt.) By slicing the inoculum into 1 to 2 cm and adjust the air injection amount to 0.2 to 0.5 vvm, especially 0.3 vvm, at 4 to 6 weeks at 15 to 25 ° C., especially at 20 ° C. When cultured weekly, the reproduction rate was 15.12 times, and the productivity of valerenic acid and valeric acid was 41.60 mg / l and 26.15 mg / l, respectively. That is, the content of valeric acid is 2.10 mg / g, the content of valeric acid is 1.32 mg / g, the percent content of valeric acid is 0.210%, and the percent content of valeric acid is 0.132%.

Valerian culture root having an active ingredient content as described above is provided for the first time by the present invention, Valerian root produced according to the production method of the present invention is also within the scope of the present invention.

In addition, by extracting valerian and valeric acid, which are active ingredients, according to a conventional extraction method, from Valerian root produced as described above, it is possible to produce valerenic acid and valeric acid. Production methods are also within the scope of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only intended to help the understanding of the present invention, and the scope of the present invention is not limited by them in any way.

Example 1 In Vitro Culture of Valerian Root

In this experiment, stock 1 was dissolved in 1 liter of water (2 times (× 2) concentration), but 50 ml of stock 1 was used, and stocks 2, 3, and 4 were used as they were, and B ₅ medium (see [ Gamborg et al., 1968, Exp. Cell Res. 50: 151-158]. In addition, the concentration ratio of KNO ₃ : (NH ₄ ) ₂ SO ₄ was 24.8: 1.0, and the concentration of NaH ₂ PO ₄ was 0.32 mM.

7% sucrose obtained by adding 70 g of sucrose to 1 liter of water was added to the B ₅ medium prepared as above, wherein the pH was 5.7. The medium was introduced into the bioreactor.

The inoculum was inoculated into 1 to 2 cm finely inoculated with 20 g (fresh wt.) And incubated for 7 weeks in a dark condition of 20 ℃ by adjusting the air injection amount to 0.3 vvm. The results are shown in FIG. As can be seen from Figure 1, the high reproduction rate and high valerian acid and valeric acid productivity was obtained when the Valerian root was incubated for 4-6 weeks, especially 15.12 times the reproduction rate and valerian acid when cultured for 5 weeks. Valleportate could be obtained with high contents of 2.10 mg / g (0.210%) and 1.32 mg / g (0.132%), and high productivity of 41.60 mg / L and 26.15 mg / L, respectively.

Example 2: Determination of the active ingredient content of Valerian

HPLC-PDA method was used to analyze the content of valeric acid and valeric acid in the field and incubation tissue of Valerian.

Specifically, Waters 600 controller, Waters 996 photodiode array detector, Waters 600 pump and Waters 717 plus auto-sampler were used. Data acquisition was performed using a Millennium ³² PDA program.

The elution solvent was analyzed in a linear gradient mode flowing from distilled water: acetonitrile: phosphate = 80: 20: 0.05 in mixed solution A at 40 minutes in distilled water: acetonitrile: phosphate = 20: 80: 0.05. The flow rate at this time was 1.5 ml per minute.

Extraction and analysis by HPLC of valerian acid and valeric acid as active ingredients in Valerian herb were based on the method of Bicchi et al. (2000). That is, the urine was collected, washed twice with distilled water, lyophilized, and 10 ml of methanol was added to 1 g of the ground powder sample, sonicated at room temperature for 5 minutes, and filtered. To the residue was added 5 ml of solvent again, the extraction operation was repeated two more times, and the filtrate was combined and concentrated three times. 2 ml of methanol was added, followed by filtration through a 0.20 µm membrane filter to obtain a sample for HPLC analysis. In consideration of the difference in UV maximum absorption wavelengths of valerenic acid and valeric acid, data were analyzed at a PDA detector of valence acid at 220 nm and valeric acid at 256 nm, and the injection volume was 20 μl.

Table 1 shows the contents of valerenic acid and valerian formate in different tissues of Valerian from the open field in April to September. First, germination of seeds in the cabin and incubation in solid medium to see if in-flight culture is possible Table 2 shows the contents of valerenic acid and valeric acid in the culture tissue.

HVA: hydroxyvaleric acid; AVA: acetoxyvaleric acid; VA: valerenic acid; AV: acebaltrate; IVA: isobaltrate, VAL: baltrate; DVA: Dedrovalrate, TVA: Total Valerenic Acid, TVP: Total Vallefort

*-: Not detected

In addition, the contents of total valerenic acid and total valerian formate in the cultivated roots of Valerian grass are shown in FIGS. 2 and 3, respectively. Valleportate content in culture roots was significantly higher than in cultivated fields. Recently, valetrate has been reported not only to block viruses that cause immunodeficiency, but also to prevent viruses from spreading to body organs (Nobutoshi). Murakami et al.).

As can be seen from the table and figures, the content of valerenic acid and valeric acid in dissimilar tissues was found to be similar in the field culture and in-flight cultivation plants, where valeric acid and valeric acid were mainly synthesized in muscle ( > 70%), small amounts were synthesized in the seedlings. Didrobaltrate was detected in all incubation muscles, but only in samples collected in April in cultivars.

According to the present invention, it is possible to economically produce large amounts of Valerian root having a valerian acid and valeric acid content comparable to the bare cultivated Valerian root through culture in a bioreactor.

Claims (6)

Culture medium in the bioreactor of Valerian root, containing 7% sucrose in B ₅ medium and adjusted to pH 5.5-6.0. The medium for culture in a bioreactor of Valerian root according to claim 1, wherein the concentration ratio of KNO ₃ : (NH ₄ ) ₂ SO ₄ is 24-25: 1.0, and the concentration of NaH ₂ PO ₄ is 0.3-0.4 mM. A method of producing Valerian root, comprising culturing Valerian root in a bioreactor containing a medium according to claim 1. The method of claim 3, which is incubated for 4-6 weeks at 15 ~ 25 ℃ with an air injection amount of 0.2 ~ 0.5 vvm. delete delete

Images