CN1058290C - Arnebia euchroma (Royle) Johnst. cell cultivation and prodn. process by solid two step method - Google Patents

Abstract

In the present invention, different organs of Arnebia euchroma seedlings are used as explants for callus induction and the obtainment of a cell line; a two-step production technological process for cell solid culture is established, wherein an AG-7 growth culture medium and an AP-5 production culture medium are used in the process. Tests prove that the content of shikonin derivatives in cell culture substances obtained by the method is 5 to 9 times as high as that of the original plants; the content of acetylshikonin with antibiotic activity and anti-tumor activity is 7 to 11 times as high as that of the original plants, and the content of wax accounts for only 1/16 of the content of natural extracts.

Description

Two steps of lithospermum euchromum Royle cell solid are cultivated production technique

The invention belongs to field of plant cell engineering technology, specifically it is a kind of method of lithospermum euchromum Royle cell solid culture.

Lithospermum euchromum Royle (Arnebia euchroma) is China's tradition herbal medicine, and its effective constituent shikonin and derivative thereof are naphthoquinone compound.Be mainly used in treatment skin burn, frostbite, scald, ulcer obstinate and gynecological inflammation etc.Clinically also be used for various skin disease diseases, shallow as eczema, papule, acne, cuticle disease, women's color spot and head property dittany etc.Especially the acetylshikonin in the shikonin derivative has anti-tumor activity.Shikonin and derivative thereof still are fabulous natural pigment in addition, look valency height, and physicochemical property is stable.Can be used for makeup, food and dyestuff etc.But because lithospermum euchromum Royle is grown in the alpine belt of about 3000 meters of height above sea level, so the method that wild alkanna extracts shikonin and derivative thereof is still excavated at present in introducing culture not success as yet so far.The shortcoming of this technology is:

1. lithospermum euchromum Royle is a perennial plant, and shikonin and derivative thereof only are present in the root of natural plant, so gather root " sacrificing future gains to satisfy present needs " beyond doubt, wild resource is had serious destructive.For this reason, lithospermum euchromum Royle is classified as national protective plant by Ministry of Forestry already.

2. the wild plant strain growth of Asian puccoon is in different growth areas, the content instability of effective ingredient in its root, and also content is lower, is generally the 1-4% of dry weight.

3. the wax that contains 15-30% in shikonin that from natural plant root, extracts and the derivative thereof.

Except that from wild lithospermum euchromum Royle root, extracting shikonin and the derivative thereof, China the Northeast, not generic Liaoning Asian puccoon (Lithospermum erythrorhizon) introducing culture success in recent years.Also there is following shortcoming in the approach that this kind produced shikonin and derivative thereof:

1. China's clinical practice proves, the clinical drug effect of lithospermum euchromum Royle far surpasses Liaoning Asian puccoon.

2. in the lithospermum euchromum Royle root content of shikonin and derivative thereof also than higher in the Radix Arnebiae (Radix Lithospermi) of Liaoning.

3. artificial culture will occupy a large amount of farmlands, can plough under the situation that area day by day dwindles, and this behave should be considered as very unwise move.

4. artificial culture can not be broken away from the influence of physical environment, and as bad climatic condition, disease and pest etc. are so the content of effective ingredient and quality are with instability.

The objective of the invention is at the problems referred to above and have defective, propose a kind of two steps of lithospermum euchromum Royle cell solid to cultivate production method.This method adopts a large amount of culture techniques of vegetable cell, has realized suitability for industrialized production shikonin and derivative thereof.

Below be main contents of the present invention:

1. producing of lithospermum euchromum Royle clone:

The Different Organs (root, plumular axis, cotyledon and true leaf etc.) of A, employing lithospermum euchromum Royle seed seedling is made the explant induction callus.Concrete operations are as follows: will pick up from the lithospermum euchromum Royle seed of Xinjiang region, the clorox through 8% soaks 12 minutes to carry out surface sterilization, be sowed at through autoclaving to be soaked with on the filter paper of sterilized water, and be 25 ± 1 ℃ of dark cultivations down in temperature, begin after 5 days to germinate.Treat that the seedling true leaf forms, root and plumular axis grow into 0.8--1.2cm, and seedling is divided into root, plumular axis, cotyledon and true leaf.This four parts explant is seeded in additional KT 0.5mg/l and 2 respectively, on the LS substratum of 4-D1mg/l, is positioned over 25 ± 1 ℃ of darkrooms and cultivates.Cultivate through 6 weeks, can form callus at the cutting part of explant.

B. callus is gone up succeeding transfer culture (25 ± 1 ℃ of dark cultivations) at growth medium (AG-7), changes generation once in per 25 days, can obtain A-1 clone through the succeeding transfer culture in 10-30 generation.

2. substratum is formed in two step of the lithospermum euchromum Royle cell solid culture method:

A. growth medium B, production culture medium A G-7 growth medium moiety AP-5 produce the substratum moiety

(the grass position: mg/L) (unit: mg/L) KNO3 1,9O0.0 KNO3 30.0 (NH4) 2,SO4 463.0 Ca (NO3) 4H2O 694.0KH2PO4 170.0 MgSO47H2O 750.0MgSO47H2O 370.0 NaH2PO42H2O 19.0CaCl22H2O 440.0 KCl 65.0H3BO3 6.2 Na2SO4 1480.0KI 0.83MnSO44H2O 22.3 FeSO47H2O 27.8ZnSO47H2O 8.6 Na2EDTA 37.3Na2M4oO42H2O 0.25 ZnSO47H2O 3.0CuSO45H2O 0.025 H3BO3 4.5CoC16H2O 0.025 CuSO45H2O 0.025FeSO47H2O 27.8 KT 1.0Na2EDTA 37.3 Sucrosse 20,000.0-50,000.0Inositol 100.0 Agar 7,000.0-10,000.0VjtamineB1-HCl 0.1 * pH before autoclave sterilization transfers to 4.8-6.2IAA 0.2KT 1.0Sucrosse 20,000.0-50,000.0Agar 7,000.0-10 000.0* pH before autoclave sterilization transfers to 4.8-6.2

3. two steps of lithospermum euchromum Royle cell solid are cultivated the technological process of production

(A) form the preparation growth medium by the prescription of AG-7 growth medium of the present invention.Measure the pH value with pH meter, and regulate the medium pH value in the 4.8-6.2 scope with the HCl of 1N and the KOH of 1N.Add 0.7-1% agar, boil the back branch and be filled in the square vase in No. 5 (Beijing glass 5 factories product) every bottle of 20-60 milliliter.With sealing after film seals, carry out autoclave sterilization, pressure is 1.0-1.2kg/cm2, the time is 15-25 minute.

(B) the A-1 clone that will obtain through succeeding transfer culture is inoculated in Bechtop in the above-mentioned growth medium, inoculum size be 1.5-5.0% (w/v, g.fw/ML).Postvaccinal culturing bottle is put into dark culturing room cultivated 18-28 days, temperature is 15-30 ℃.

(C) by (A) described program, to produce the prescription of substratum by present patent application AP-5 and form, substratum is produced in preparation.With (B) 18-28 days callus of growth on growth medium, by to producing in the substratum, be 3.0-7.0 gram fresh weight/bottle in the Bechtop transfer by kind of amount, culturing room's condition is (B) item together.Cultivate after 20-28 days, culture is all taken out, put into the enamel dish, send in 50 ℃ the baking oven and dry, carry out the separation and Extraction of following the 4th shikonin derivative with the stainless steel spoon.

Fig. 1 cultivates technological process of production synoptic diagram in two steps of lithospermum euchromum Royle cell solid.

4. the separation-extraction technology of Shikonin Derivative Formation:

Get the Plant cell culture thing after 60 ℃ of oven dry of air circulation drier, be ground into pulverizer 20 purpose meal are loaded in the combined type continuous extractor, do leaching with the benzinum of 30-60 boiling range Solvent carries out circulating leaching. The fluid coefficient of leachate is controlled at below 5. Leachate after filtration After use again rotary evaporator recovered under reduced pressure benzinum, leachate is condensed into paste, again in 60 ℃ Dry Shikonin Derivative Formation (AC-90) product that gets.

Evidence is used culture medium provided by the invention under solid culture, its cell growth rate Can reach 10.0 gram dry weights/liter/month, the content of Shikonin Derivative Formation reaches as high as the culture dry weight 7.0%; Liquid suspension is cultivated, and cell growth rate can reach 22.0 gram dry weights/liter/month, The content of Shikonin Derivative Formation can reach 17.0%; Applying biological bioreactor culture (10-30 liter) Cell growth rate can reach 18.0 dry weights/liter/month, and the content of Shikonin Derivative Formation reaches as high as training Support 12.0% of thing dry weight.

Fig. 2 is Shikonin Derivative Formation extraction process flow chart.

The present invention compares than prior art has following advantage and good effect:

(1). lithospermum euchromum Royle is the state guarantee plant, utilizes plant mass cell culture to produce Asian puccoon Peaceful derivative is walked the approach of suitability for industrialized production, can effectively protect wild resource, and protection is ecological Environment.

(2). application cell is cultivated the biosynthesis Shikonin Derivative Formation in a large number, can obtain stable quality And output, be not subjected to the restriction of natural conditions and affect.

(3). do not need to take the farmland, only need to take limited factory building and culturing room, highly intensive.

(4). by manual adjustment and the growth of control cell and synthesizing of secondary metabolite, can be greatly Improve output and the content (seeing Table 1) of Shikonin Derivative Formation, especially improve in the Shikonin Derivative Formation The content ratio of the Acetylshikonin (Acetylshikonin) of tool antibiotic (seeing Table 2), antitumor activity Example (seeing Table 3). Than the high 4-8 of Shikonin Derivative Formation content in the former plant root doubly, Acetylshikonin The high 6-10 of content times, wax content only is 1/16 of natural extract.

(5). this technology, do not use bioreactor to carry out suitability for industrialized production, purchase so can remove from The expensive investments of bioreactor. The one-time investment amount is little, output and stable content, pollution rate Extremely low.

Below narrate embodiments of the invention: embodiment 1

Prescription by AG-7 growth medium of the present invention is formed, the preparation growth medium, and sucrose concentration is 30 grams per liters, and adjust pH is 5.8, and agar concentration is 0.7%.With square vase in No. 5,40 milliliters of every bottled nutrient solutions.Autoclave sterilization, pressure are 1.0Kg/CM2,20 minutes.Insert A-1 clone, inoculum size is 3.0%, puts into dark culturing room and cultivates, and temperature is 24 ° ± 1 ℃.Cultivate after 23 days every bottle of fresh weight and can reach 15.0 grams.It is forwarded to AP-5 produces in the substratum (the same growth medium of preparation procedure), inoculum size is 5.0 gram fresh weight/bottles, the same growth phase of culturing room's condition.Cultivate results after 23 days, every bottle can be obtained culture 0.68 gram dry weight.Cell culture is used high pressure liquid chromatographic analysis after dividing high extraction, and with natural plant in the shikonin derivative that extracts to carry out results of comparison as follows: the comparison of table 1 lithospermum euchromum Royle cell culture and natural plant shikonin derivative content and characteristic

Total percent of ash wax content pure compound content ratio (%) * look valency

(％) (％) E10％1CM

Natural plant 0.12 15.6 1.8 11.6 6.0 5.2 0.2 〉=138 cell culture of A B C D E (520nm) 0.23 0.62 7.3 21.6 20.6 15.0 4.2 〉=167*.A. deoxyshikonin, B. β, beta-dimethyl-acryloyl Shikonin C. acetylshikonin, D.2,3-dimethyl pentene acyl Shikonin E. beta-hydroxyisovalerylshiderivative

Embodiment 2

Press embodiment 1 and surpass the shikonin derivative that natural plant is extracted by its anti-microbial activity of shikonin derivative that the culture extraction separation obtains.Test-results such as following table.BA-I represents β in the table, beta-dimethyl-acryloyl Shikonin, and BA-II represents acetylshikonin, total shikonin compound that the BA-P representative is extracted from plant, total shikonin compound that the BA-C representative is extracted from cell culture.

The anti-microbial effect of table 2 pair 15 kinds of bacteriums

MIC (μ g/MI) tests bacterium

100＞50＞100＞100 Shigella flexneris 50 50 of the BA-I BA-II BA-P BA-C gold bacterium 209p of Portugal 0.39 0.19 0.78 0.19 gold medal Portugal bacterium, 15 0.39 0.39 3.125 0.78 form staph 26,069 50＞50＞100＞100 sarcines, 1.56 0.78 6.25 0.78 hay bacillus 6,633 50 25 25 12.5 Escherichia coli, 22＞100＞50＞100＞100 Pseudomonas aeruginosa 11 100＞50＞100＞100 Pseudomonas aeruginosas, 17 100＞50＞100＞100 proteus 9 100 50＞100＞100 pneumobacilluses, 14 100＞50＞100＞100 shigella flexneris＞100 50 typhoid bacilluses, 100＞50＞100＞100 salmonella typhimuriums, 100＞50＞100＞100 serratia marcescens 100＞50＞100＞100

Embodiment 3

Press embodiment 1 and surpass the shikonin derivative that natural plant is extracted by its anti-tumor activity of shikonin derivative that the culture extraction separation obtains.Test-results such as following table.Total shikonin compound that the BA-P representative is extracted from plant in the table, total shikonin compound that the BA-C representative is extracted from cell culture, BA-II represents acetylshikonin.

The specific activity of the anti-ehrlich carcinoma of table 3 shikonin derivative (EC) is than sample dose mouse increase in life span %

(Mg/Kg/ day) BA-P 5 22BA-C 5 107BA-II 5 69

Claims (3)

1. a paniculatum cell is, it is characterized in that: A. is inoculated in additional kinetin KT 0.5mg/l and synthetic hormone 2 with the root of lithospermum euchromum Royle seedling or plumular axis or cotyledon or true leaf explant, on the LS substratum of 4-D 1mg/l, being positioned over 25 ± 1 ℃ of darkrooms cultivates, after 6 weeks, can obtain callus;

B. callus succeeding transfer culture on following culture medium A G-7 changeed generation once in per 25 days, can obtain paniculatum cell system through the succeeding transfer culture in 10-30 generation;

AG-7 growth medium moiety (unit: mg/L) wherein

KNO ₃ 1,900.0

(NH ₄) ₂S0 ₄ 463.0

KH ₂PO ₄ 170.0

MgSO ₄·7H ₂O 370.0

CaCl ₂·2H ₂O 440.0

H ₃BO ₃ 6.2

KI 0.83

MnSO ₄·4H ₂O 22.3

ZnSO ₄·7H ₂O 8.6

Na ₂MoO ₄·2H ₂O?0.25

CuSO ₄·5H ₂O 0.025

CoCl·6H ₂O 0.025

FeSO ₄·7H ₂O 27.8

Na ₂EDTA 37.3

Inositol 100.0

Vitamins B ₁-HCl 0.1

IAA 0.2

Kinetin KT 1.0

Sucrose 20,000.0-50,000.0

Agar 7,000.0-10,000.0

2. the production method of paniculatum cell system, it is characterized in that: A. is seeded in additional kinetin KT 0.5mg/l and 2 respectively with the root of lithospermum euchromum Royle seedling or plumular axis or cotyledon or true leaf explant, on the LS substratum of 4-D 1mg/l, being positioned over 25 ± 1 ℃ of darkrooms cultivates, after 6 weeks, can obtain callus;

B. callus succeeding transfer culture on following culture medium A G-7 changeed generation once in per 25 days, can obtain paniculatum cell system through the succeeding transfer culture in 10-30 generation;

AG-7 growth medium moiety (unit: mg/L)

KNO ₃ 1,900.0

(NH ₄) ₂SO ₄ 463.0

KH ₂PO ₄ 170.0

MgSO ₄·7H ₂O 370.0

CaCl ₂·2H ₂O 440.0

H ₃BO ₃ 6.2

KI 0.83

MnSO ₄·4H ₂O 22.3

ZnSO ₄·7H ₂O 8.6

Na ₂MoO ₄·2H ₂O 0.25

CuSO ₄·5H ₂O 0.025

CoCl·6H ₂O 0.025

FeSO ₄·7H ₂O 27.8

Na ₂EDTA 37.3

Inositol 100.0

Vitamins B ₁-HCl 0.1

IAA 0.2

Kinetin KT 1.0

Sucrose 20,000.0-50,000.0

Agar 7,000.0-10,000.0

3. the paniculatum cell of a claim 1 ties up to the application of producing in shikonin and the derivative thereof.

Images