KR890003711B1 - Purifing method for shikonin from lithospermum officinale linne var - Google Patents

Abstract

Shikonins are prepd. by culturing cells of pre-multiplied lithospermum (by conventional solid or liq. cultivation) on the white liq. culture media added adsorbent i.e. silica gel, zeolite or aluminum oxide. Wt. ratio of lithospermum cells to adsorbent is 8:1- 2:1. Shikonins are useful for treating dermatopathy, eczema, hemorrhoids, etc..

Description

Method for producing sikonin pigment by culturing the cells of branch plants

The present invention relates to a method for producing naphthoquinone-based Shikonin pigments and other useful substances by aseptically cultivating the leaves, stems and roots of the larvae belonging to a dentifer. Chichi plants are perennial herbaceous plants grown in Northeast Asia such as Northeast China and Japan. Its roots are known to contain naproquinone-based sikonin pigments of general formula (I). There are about ten kinds of siconin pigments.

(Ⅰ)

As a pharmacological action of these siconin pigments, antibacterial, anti-inflammatory and granulation angiogenesis have been found, and have been used since various skin diseases, eczema, frostbite, burns, cuts, hemorrhoids and the like. However, the content of the active ingredient of extractable siconin pigments is very small in the roots of branched plants, and it is difficult to stably supply the active ingredient due to the limitation of the yearly production due to the temporal and climatic obstacles.

Therefore, research has been conducted on a method of proliferating dental plants and mass-producing siconin pigments by the cell culture method without being subjected to timely, climatic and quantitative restrictions using the cell culture method.

In recent years, the production of sikonin pigments by cell culture methods, such as copper, manganese, molybdenum, ammonium or other mediums such as organic acids, amino acids, vitamins, or other media such as auxin and cytokine It is reported as a production method of cyanine dyes by adding a growth regulator and adding pigmented cellulose fibers, liquid paraffin or chitin, pectinic acid, activated carbon, oils and fats to flow the pigment production. However, the method reported by them has the drawback that the amount of pigment production of sikonins is very small. Accordingly, the present inventors have developed a liquid medium suitable for the proliferation of chichi cells and the production of sikonin pigments by cell culture, thereby producing a large amount of sikonin pigments as well as the proliferation of chichi cells.

The present invention relates to a method for efficiently producing a large amount of siconins and other useful components by adding a predetermined amount of various adsorbents to a medium for proliferation and pigment production of branched cells. That is, the present invention relates to a cell culture method of a dental clinic plant comprising using a liquid medium containing at least one adsorbent among silica gel, zeolite and aluminum oxide, and the basic medium used is used for tissue culture of conventional plants. Any medium used may be used, but in the present invention, NH ₄ NO ₃ 1650mg, KNO ₃ 1900mg, H ₃ BO ₃ 6.2mg, KH ₂ PO ₄ 170mg, KI0.83mg, Na ₂ M ₀ O ₄ 2H ₂ O0.025mg, CoCl ₂ · 6H ₂ O0.025mg, CaCl ₂ · 2H ₂ O440mg, MgSO ₄ · 7H ₂ O370mg, MnSo ₄ · 4H ₂ O22.3mg, ZnSO ₄ · 7H ₂ O8.6mg, CuSO ₄ · 5H ₂ O0.025 mg, Na ₂ EDTA 37.3 mg, FeSO ₄ · 7H ₂ O 27.8 mg, Chiamine hydrochloride (Thiamine HCI) 0.4 mg, Sucrose 30 g, Inositol 100 mg ) Medium was used. Silica bells have various types of particle size, but among them, silica gel before and after the particle size of 100 mesh was used for 5-20 g per 1 l of the liquid medium. Zeolites had various forms depending on the adsorption capacity and shape, but the composition or powder was 5-30 g per 1 L of liquid medium. Auxins and cytokinins were added to the liquid medium at a concentration of 0.1-10 ppm, and various organic compounds such as yeast extract, peptone, and coconut milk were used as needed.

When explaining the present invention in more detail the seed of the plant aseptic or pollen cultivation in a greenhouse to obtain a plant where the roots, leaves, a portion of the stem aseptically collected 25-30 days in a white solid medium at 25-30 ℃ The cells were cultured to obtain amorphous cell masses. The amorphous cell masses were transferred to the adsorbent-containing liquid medium of the present invention and shaken at 100-120 ppm at 25-30 ° C. The grown suspension culture cells or cell masses were transferred to freshly prepared white medium containing sorbents and other existing mediums, and cultured to produce large amounts of sikonins and other useful substances. Extraction and quantification of sikonins were carried out in the same manner as the extraction from natural roots.

Example 1

In a 250ml Erlenmeyer flask, 70ml of the white medium of the composition and about 1g of each adsorbent were added and sterilized at 121 ° C for 15 minutes. The cells obtained by proliferation by solid and liquid culture were put into 2 g of sterile medium cooled to room temperature and cultured for 2 weeks at 120 rpm in a dark place at 28 ° C. After measuring the dry cell dry weight after removing the additives, the content of the siconin pigments adsorbed on the cells and the additives was measured by UV sprctrophotometer. The results are shown in Table 1 in terms of 1 l of liquid medium.

Table 1.

Comparison of Siconin Content with Addition of Adsorbents

Example 2

Using the white medium of the composition as the base medium, the base medium 70ml and various adsorbents as shown in Table 2 in a 250ml Erlenmeyer flask were sterilized for 15 minutes at 121 ℃.

The cells obtained in advance were cultured in a preparation medium cooled to room temperature as shown in Table 2 and shaken at 28 ° C. for 2 weeks at 120 rpm in the dark.

The culture medium was filtered to obtain cells, dried at 35 ° C. for 48 hours, after which the additives were removed, and the dry weight was measured.

The content of siconins adsorbed on dry cells or additives is shown in Table 2.

Table 2.

Comparison of Siconin Content by Cell and Adsorbent Ratio

Claims (2)

Cultivating cells of Lithospermum officinale Linne var. Erythrorhizon Maximowicz characterized by culturing the cells of chichi grown in a conventional solid or liquid culture method in a white liquid medium containing silica gel or zeolite or aluminum oxide. To produce a shikonin dye. The method according to claim 1, wherein a weight ratio of the cells of the branched tree to the adsorbent is 8: 1: 1: to cultivate the cells of the branched plants to produce the siconin pigment.