JPH0235094A - Long-term continuous production of tropane alkaloid by filariform root culture - Google Patents

Abstract

PURPOSE:To continuously produce in high efficiency for a long period the title compound for e.g., drugs by making a culture, in an ammonium ion-free culture medium containing nitrate ion at specified amount, of filariform roots induced by infecting a Solanaceae plant with Agrobacterium rhizogenes. CONSTITUTION:Filariform roots induced by infecting a Solanaceae plant (e.g., Duboisia leichhardtii) with Agrobacterium rhizogenes are put to culture in a culture medium free from ammonium ion but containing 25-100mM of nitrate ion to deliver tropane alkaloid into the culture medium in high efficiency and the resultant culture medium containing the tropane alkaloid is allowed to flow into an adsorptive resin fitted out of said culture medium to make said alkaloid absorb on the resin followed by recovering said alkaloid, thereby continuously producing the objective tropane alkaloid for a long period.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for culturing hairy roots induced from plants of the Solanaceae family to efficiently and continuously produce tropane alkaloids, which are important as pharmaceuticals, over a long period of time.

[Prior art and problems]

Tropane alkaloids such as scopolamine and l-hyocyamine contained in Solanaceous plants are used as analgesics, antispasmodics, anti-Parkinson's drugs, and the like.

These compounds are manufactured by extracting them from natural plants, but there are many challenges to production, such as the long-term cultivation of the plants containing them, the dependence on the weather, and the limited cultivation area. There is a problem with this. Therefore, attempts are being made to industrially produce useful plant components at low cost by applying plant tissue culture techniques. There have been many attempts to produce tropane alkaloids by culturing calli derived from plant explants, but in most cases, they either do not produce tropane alkaloids at all or only produce a very small amount of tropane alkaloids. Ta. Therefore, the present inventors have already selected highly proliferative and highly alkaloid-producing hairy roots from hairy roots induced by infecting plants with the soil bacterium Agrobacterium rhizogenes. developed a method for producing alkaloids by culturing them (Japanese Patent Application Laid-Open No. 61-25419). This method involves culturing hairy roots for a certain period of time, harvesting them, crushing the tissue, and accumulating it in the tissue. It requires a complicated manufacturing process to extract the alkaloids.

On the other hand, it is known that when hairy roots are cultured, alkaloids leak into the medium. However, the amount is usually small, and the existence of alkaloids in the culture medium has only been confirmed, and no effective recovery method has been known (Plant Cell Reports, 5.11).
1 (1986), JP-A-62-20579, Biot
technology Letters, 8.415
(1986)).

[Means of problem solving]

As a result of extensive research, the present inventors discovered a culture method that efficiently leaks tropane alkaloids, particularly scopolamine, into the medium from the hairy roots of Solanaceous plants, and furthermore, they have succeeded in fixing the tropane alkaloids leaked into the medium outside the medium. We have discovered a method to efficiently adsorb and recover tropane alkaloids using an adsorption resin, and to continuously produce tropane alkaloids over a long period of time by replacing the resin. That is, the present invention involves culturing the induced hairy roots of Solanaceae plants by infecting them with Agrobacterium rhizogenes in a medium that does not contain ammonium ions and contains a high nitrate ion concentration (25 to 100 mM), thereby producing tropane alkaloids. , especially a method for efficiently leaking scopolamine, and adsorption of tropane alkaloids leaked into the medium by the hairy roots infected and induced with Agrobacterium rhizogenes using a giant reticular resin attached outside the medium. Provided is a method for producing tropane alkaloids, which is characterized in that tropane alkaloids are efficiently recovered after being adsorbed by flowing a medium through a resin.

According to this method, tropane alkaloids produced by hairy roots are leaked into the medium and continuously adsorbed and collected, thereby increasing tropane alkaloid productivity of hairy roots. Continuous production became possible. Moreover, since extraction and purification becomes easy, it becomes possible to advantageously produce tropane alkaloids, especially scopolamine. Next, the present invention will be explained in detail.

In the present invention, any Solanaceae plant that produces tropane alkaloids can be used.
ia Ieichhardtii), Zboisia
Myoboroides (Duboisia va o oro)
It is preferable to use Zboisia popu-dei (厘匝凰) 中凱剋dan [) and the like.

Agrobacterium rhizogenes used to induce hairy roots is not particularly limited, and for example, Agrobacterium rhizogenes is used for inducing hairy roots.
Known strains include the 4.15834.1855.8196.2659 strain (Mo1. Gen, Gen
et, 190°204 (1983)).

The method of infecting Solanaceae plants with Agrobacterium rhizogenes to induce hairy roots is as follows: 1. Direct inoculation method on part of the plant solid; 2. Method of inoculating cultured tissue, 3. Co-culture of plant protoplasts and Agrobacterium rhizogenes, 4. Agrobacterium rhizogenes R
Methods include introducing the DNA of a plasmid or a portion thereof by electroboration, microinjection, or the like.

The hairy roots induced by the above method are sterilized by culturing them in a medium containing antibiotics such as calhenidiline and tetracycline, or by cutting out the root tip tissue and replanting it at a rapid cycle.

The sterilized hairy roots are subcultured on a medium without plant hormones. The medium is basically a known inorganic salt synthetic medium to which a carbon source is added, to which vitamins, amino acids, organic substances, etc. are added.

For example, IIF medium (Agric, Biol, Ch
eIll, 50.2715 (1986)), Heller Danchi, White Medium, Linsmeyer and Skoog (L
S) medium, Gamborg's 85 medium, etc. are used.

<Culture method of strain with high tropane alkaloid leakage> In the present invention, the hairy roots obtained by the above method are cultured in a liquid medium without addition of plant hormones.

Regarding the inorganic components of the medium excluding inorganic nitrogen,
It is based on the ingredients of known inorganic salt synthetic media, and contains vitamins, amino acids, organic substances, etc. Regarding inorganic nitrogen, it is characterized by using a medium that does not contain ammonium ions and has a nitrate ion concentration of 25 to 100 mM. Medium containing ammonium ions, medium with nitrate ion concentration lower than 25mM, and 100mM
A medium with a higher concentration than M is not preferred because the leakage amount of tropane alkaloid decreases. The compound is not specified as long as it is an inorganic salt that contains nitrate ions but does not contain ammonium ions, but examples include potassium nitrate and sodium nitrate. Furthermore, improved medium compositions of these can also be used.

In basic media that do not contain ammonium ions and have a nitrate ion concentration of less than 20 mM, such as HF medium, Heller's medium, Niche medium, Niche and Niche medium, and Kutubu medium, compounds containing nitrate ions, such as potassium nitrate,
Adjust the nitrate ion concentration to 25-100a+ with sodium nitrate.
Change to M. In basic media that contain ammonium ions and have a nitrate ion concentration lower than 25-M, such as Schenk and Hildeplant medium and Kolenbach and Schmidt medium, components containing ammonium ions are removed.
Modify nitrate 7i11 degree to 25-100mM. Contains ammonium ions and has a nitrate ion concentration of 25 to 11
00II basic medium, such as Linsmeyer and Skoog medium, Gamborg's 85 medium, etc., exclude ammonium-containing components.

As the carbon source, sigose, glucose, fructose, etc. can be used, but sucrose is particularly preferred. Sugar concentration is 1
-10χ-/V, preferably 3-5χW/V. The pH value of the medium ranges from weakly acidic to neutral, i.e. pH 4.0~
7.0 is appropriate, and the culture temperature is usually 20 to 30°C, preferably 24 to 27°C.The culture tank is preferably one that does not damage the hairy roots, such as an air lift type culture tank, a rotary culture tank, Alternatively, a rotating culture tank is suitable. In the case of rotational culture, the rotation speed is preferably about 50 to 150 rpm+. When cultured in the dark for about 1 to 6 weeks, high concentrations of scopolamine and hyocyamine leak from the hairy roots into the medium as the hairy roots proliferate.

By this method, 1-2 mg 150 ml of scopolamine
/4wk can be selected for hairy root stocks that leak into the medium.

<Method for recovering tropane alkaloids leaked into the culture medium> (1) Infusion of the hairy roots of the tropane alkaloid-rich Solanaceae plant obtained as described above into a liquid containing no plant hormones. Culture in medium. The medium is basically a known inorganic salt synthetic medium to which a carbon source is added, and to which vitamins, amino acids, organic substances, etc. are added. For example, HF medium, Heller medium, White medium, Linsmeyer medium. Andskoog medium, Gamborg's 85 medium, etc. are used. It is preferable to culture in the medium shown in the above-mentioned "Culture method for strain with high tropane alkaloid leakage" because the amount of tropane alkaloid leaked is high.

The culture device consists of a culture tank, a column containing adsorption resin, and a pump. Although a rotating culture tank, a rotating culture tank, etc. can be used as the culture tank, it is preferable to use an air lift type culture tank. In this case, aeration is performed with oxygen or air. Shown in 1.

The column is filled with adsorption resin. Ion exchange resins, silica gel, activated carbon, etc. can be used as adsorption resins, but it is preferable to use giant network structure resins, which have a high adsorption rate for tropane alkaloids such as scopolamine and hyocyamine, and a low adsorption rate for nicotine and medium components. desirable. As a giant network structure resin, Amberlite" XAD-2,
XAD-4,
Examples include P2MG (Mitsubishi Kasei).

The pump is not specified as long as it has the ability to send the culture medium from the culture tank to the column, and the flow rate is also not specified, but preferably the flow rate is 5V = 2 to 15 (processing (1) / (resin amount (1)
X time (5))).

Operate the pump and flow some of the medium into the column.

The medium poured into the column may be discarded in its entirety, partially discarded and fresh medium added, or returned in its entirety to the culture tank. Although the timing of starting the pump is not specified, it is preferable to start the pump at the same time as the start of culture, since this increases the amount of alkaloid recovered. By replacing the adsorption resin with a new one every 5 to 10 days during the culture period, production can be continued continuously for a long period of time.

(2) Tropane alkaloids adsorbed onto the resin in the above method can be eluted with an organic solvent, preferably methanol, ethanol or a mixture of methanol, ethanol and ammonia.

Purification of scopolamine and hyocyamine from a solution containing the eluted tropane alkaloid can be performed by a conventional method. For example, a purified product of scopolamine is obtained by evaporating a solution containing the tropane alkaloid to dryness, and then converting the residue to 0.5N
After dissolving in sulfuric acid and filtering, add 25% ammonia water and p
The crude alkaloid fraction is obtained by adjusting the H9 to 1O, extracting with chloroform, and drying under reduced pressure. Obtained by neutralizing with IN hydrobromic acid, concentrating, and crystallizing as scopolamine hydrobromide. .

Next, the present invention will be explained in more detail with reference to Examples. The following examples illustrate the present invention, and the present invention is not limited only to the examples listed below.

Example 1 Hairy roots of Zboisia reichhardii were subcultured in HF medium. A medium was prepared by adding potassium nitrate 301 to 50 ml of HF (containing 7.0 mM sodium nitrate) liquid medium, and was designated as IIF-3 medium. Hairy roots 100mg
Wet weight was planted in 50m1 of IIF-3 liquid platform, 85r
The cells were cultured in the dark at 25°C for 4 weeks in a rotating culture at pm.

As a result, the amount of scopolamine contained in the medium and roots is
1.4-1.9 mg, (1.5-2.4
It was n+g.

Examples 2 to 4, Comparative Examples 1 to 4 Table 1 shows the results obtained in the same manner as in Example 1 except that the inorganic nitrogen concentration was changed as shown in Table 1.

Example 5 A total of 29 plants of Zboisia reichhardei with hairy roots (
100n+g wet weight) was planted in 50ml of HF-3 medium and cultured in the dark at 25"C for 4 weeks with rotational culture at 85rp-.Among these, the amount of scopolamine leaked was the highest, 2.0
The DL47-1 strain that yielded 1.0 mg was selected.

Example 6 Scopolamine 200
reduced g/l, hyocyamine 200mg/1, nicotine 2
A solution was prepared at a concentration of mg/l. Amberlight XAD-225m1 diameter 20III, length 9
A 5 m+* cylindrical column was packed and the virtual medium was processed using a pump at a flow rate of SV = 15. Quantitative analysis of each component of both components that flowed out was performed using high performance liquid chromatography to determine the leakage rate. The adsorption amount of each alkaloid and thiamin hydrochloride, which is a medium component, was determined from the amount of virtual culture medium treated when the leakage rate was 5%.

As a result, the amount of adsorption on the resin was 0.6 mg of nicotine and 0.02 mg of thiamine hydrochloride, whereas 28 g of scopolamine was 48 mg of hyocyamine.

Examples 7 to 10 Table 2 shows the results obtained in the same manner as in Example 5, except that the adsorption resin and flow rate were changed as shown in Table 2.

Example 11 25 ml of XAD- adsorbed with 28 mg of scopolamine
When 50 ml of methanol/ammonia (19:1) solution was discharged from a column packed with 2 at a flow rate of 5V=5, 95.7% of scopolamine was recovered.

Examples 12 to 14 Table 3 shows the results obtained in the same manner as in Example 10, except that the adsorption resin, amount of scopolamine, and flow rate were changed as shown in Table 3.
Shown below.

Example 15 Zboisia reichhardei hairy@DL471 strain (planted amount, 15 g wet weight) was cultured in 21 HF-3 medium using an air lift culture tank.

Oxygen was vented from the bottom at a venting rate of IO, 51/min. From the culture tank, using a low pressure pump, at a flow rate of 5 V = 15
The medium was poured into a column packed with 25 ml, and the eluate was returned to the culture tank. The pump is operated continuously during the culture period,
The column was removed every week, 200 ml of methanol/ammonia (19:1) was passed through the column, and the eluted methanol solution was collected.

After methanol elution, the column was washed with 500 ml of water, sterilized, and then attached to the culture device again. After 4 weeks of culture, the eluted fraction from the column, the hairy roots, and the alkaloid content of the medium were measured.

As a result, the total production of scopolamine was 511B (256m
B/1), and the breakdown is 405 mg (203 m
g/1), hairy root 86IIIg (43B/1)
, the medium was 20 mg (10 mg/l). In addition, 5 mg (2.5 mg/l) of hyocyamine was adsorbed onto the column and recovered.

Of the methanol elution fraction from the column, an amount containing 395 mg of scopolamine was measured and dried under reduced pressure. 0.5
After dissolving in 401 N sulfuric acid, insoluble matter was removed by filtration, and 5 ml of ammonia was added to make p)l 10. The mixture was transferred to a branch funnel, extracted twice with 40 ml of chloroform, and the chloroform layer was taken and dried under reduced pressure to obtain a syrupy base.

After neutralizing with 1.2 ml of IN hydrobromide and removing insoluble oil by centrifugation, the supernatant was concentrated to obtain 423 mg of crystals of scopolamine hydrobromide (yield, 84.6χ). Ta.

Example 16 Culture was carried out in the same manner as in Example 14, except that the amount of hairy roots planted was changed to 30 g wet weight. As a result, after 6 weeks of culture, the amount of alkaloids in the elution fraction from the column was 475 mg (238 mg/l) of scopolamine, 1
It was 3 mg (6.5 mg/l).

[Effects of the Invention] According to the present invention, tropane alkaloids such as scopolamine, which are useful components of Solanaceae plants, can be efficiently and continuously produced.

According to the present invention, the advantages of tropane alkaloid production by hairy root culture known so far (Japanese Patent Application Laid-Open No. 61-254
In addition to i9): 1) Tropane alkaloids such as scopolamine can leak into the medium at high concentrations.

2) Since the tropane alkaloid is leaked into the medium, the complicated operations of harvesting and crushing the roots are not necessary.

3) Adsorption resins, especially giant reticular resins, selectively adsorb scopolamine and hyocyamine, but do not adsorb nicotine, culture medium components, etc., making purification operations easier.

4) Long-term continuous production is possible by replacing the adsorption resin.

5) By continuously adsorbing and removing tropane alkaloids leaked into the medium, the amount of alkaloids leaked into the medium by the hairy roots increases.

There is an advantage.

According to the present invention, 161 scopolamine was produced in 4 weeks of culture.
~203mg/1, Hyocyamine 2.5~5mg
/l, and by culturing between 6a and 23cm
It became possible to produce about 7+g/1 and 6.5+ag/1 of hyocyamine.

Claims (5)

[Claims] (1) Agrobacterium rhizogenes (￥
A. rhizogenes￥) and induced hairy roots were treated with 25mM to 100mM without ammonium ions.
A production method in which tropane alkaloids are efficiently leaked into the medium by culturing in a medium containing nitrate ions. (2) The method according to item 1, wherein the Solanaceae plant is a Zboisia plant. (3) The tropane alkaloid leaked into the medium from the hairy roots is produced by flowing the medium through an adsorption resin attached outside the medium so that the tropane alkaloid is adsorbed to the resin and then recovered and produced. Method for producing alkaloids. (4) The method according to item 3, wherein the Solanaceae plant is a Zboisia plant. (5) The method according to item 3, wherein the adsorption resin is a giant network structure resin.