JPH0315397A - Production of glucosyl glycyrrhetinate and anti-inflammatory agent - Google Patents

Abstract

PURPOSE:To enable advantageous production of beta-D-glucosyl glycyrrhetinate and utilization thereof by culturing a cell of a plant belonging to the genus Eucalyptus in a culture medium containing glycyrrhetinic acid or a salt thereof. CONSTITUTION:A section of young tissues of a plant belonging to the genus Eucalyptus, such as Eucalyptus perrioina, is taken from a part, such as stem or leaf, sterilized, then cut into a suitable size and planted to a culture medium prepared by adding a carbon source, such as sucrose, and a phytohormone, such as auxin, to a Murashige-Skoog culture medium, etc., to induce calli. The resultant calli are subsequently inoculated into a liquid culture medium and subjected to shaking culture. After the number of the cells attains the maximum, a solution of glycyrrhetinic acid or salt thereof in ethanol is added to subsequently continue the culture. The obtained culture solution is then separated and purified to isolate beta-D-glucosyl glycyrrhetinate expected to be extensively utilized as an anti-inflammatory agent in the form of a skin liniment or oral administration agent.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing glycyrrhetinic acid β-D-glucosyl ester and to the use of glycyrrhetinic acid β-toglucosyl ester as a pharmaceutical.

[Conventional technology]

Glycyrrhizin extracted from licorice is widely used as an anti-inflammatory agent, anti-ulcer agent, and anti-allergy agent, but it has been revealed that its pharmacological action is based on glycyrrhetinic acid, an aglycone of glycyrrhizin. It's coming.

Although there have been several attempts to obtain derivatives from glycyrrhetinic acid that can be used as medicines without side effects, there have been few attempts to obtain derivatives useful as medicines by modifying glycyrrhetinic acid to form glycosides again. One example is the synthesis of a glycoside in which β1D-glucopyranose is ester-bonded to the 30-position of glycyrrhetinic acid, and the sodium salt of glycyrrhetinic acid is dissolved in dimethylformamide.
Stir with acetobromo glucose to react and deacetylate to obtain glycyrrhetinic acid β-D-gnorecosinole ester! Monode J>6CCke Peng. Pkar Otori. Bw1l. H(7), Hth~103(1!74)).

However, this synthesis method requires complicated operations such as multi-stage chemical reactions and separation and purification steps. Incidentally, the pharmacological action of this compound has hardly been studied.

[Problem to be solved by the invention]

An object of the present invention is to provide a more advantageous method for producing and utilizing glycyrrhetinic acid β-D-glucosyl ester.

(Means for Solving the Problems) The method for producing glycyrrhetinic acid β-D-glucosyl ester of the present invention, which has succeeded in achieving the above object, includes glycyrrhetinic acid or glycyrrhetinate (hereinafter referred to as glycyrrhetin). The method is characterized in that cells of a plant of the genus Eucalyptus are cultured in a medium, and glycyrrhetinic acid β-D-glucosyl ester is collected from the culture solution.

This manufacturing method will be described below.

The medium used for culturing eucalyptus cells is a medium normally used for plant tissue culture, that is, it contains inorganic bracts and carbon sources as essential ingredients, as well as plant hormones, vitamins, amino acids, etc. An appropriately blended one can be used. As the inorganic component, nitrogen, phosphorus, potassium, iron, zinc, molybdenum, boron, cobalt, iodine, calcium, magnesium, manganese, chlorine, sodium, etc. are usually used. In addition to sucrose and other carbohydrates, organic acids and the like are used as carbon sources. Plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), 2,4-dichloro7enoxyacetic acid (!, 4-D), kinetin (K),
Cytokinins such as penzyladenine (BA) are used. Specifically, Murashige-Skoog (M++r*si (e-Sk@●g
) culture medium, Linsmeyer-Skoog (Lins-mx
ier & SkoB) medium, White
), Gamborg's B5 medium, etc., the above-mentioned carbon sources and plant hormones are added, and if necessary, the above-mentioned vitamins, amino acids, coconut milk, yeast extract, and other natural products are added. It is possible to use a medium prepared by adding the following. For culturing eucalyptus cells in the production method of the present invention, a medium prepared using Murashige Skoog's medium is particularly preferable.
An example of its composition is shown below.

KNO. N.H. No. K.H. P.O. H3BO. KI N*MeO, C ac I, -6 H . OC *C lta2 H! O MtS O a・7H, O MISO4”48!O ZIISOs”7H10 CISO4●5H, O Ns,・EDTA FeSO. -78.0 Inositol thiamine hydrochloride Nicotinic acid pyridoxine hydrochloride Base dinoleadenine trisaccharide 1900 at/al 650 170 6.2 0.8 30. 2 5 0.025 4 4 0 370 2 2.3 10.6 0.025 3 7.3 2 7 . 8 1 0 0 0.1 0.5 0.5 1.0 3 0 t/L pH 5.7 (hereinafter, this medium is referred to as MSBAI medium) In addition, the Eucalyptus plants used in the production method of the present invention are particularly Suitable examples include, but are not limited to, Eucsl71ms
psrriiia+a, EmcalyLis glo
blus, Emcal71ms cilriodor
*, Eucalytws calophylls,
bcs-121as dives, Ewcslyta
s polybr*ctes, E++ely't+
+srsdi*ts etc. can be mentioned.

Culture of Eucalyptus plant cells using the above-mentioned medium is as follows:
This is what is called callus culture. Specifically, a section of a young tissue of a Eucalyptus plant is taken from a stem, leaf, or seed, sterilized with, for example, 70% alcohol, and the surface wax removed, washed with sterile water, and then washed twice with 10% salami powder. Wash, then cut to appropriate size and attach to MSBI
Plant on agar medium and induce callus. The obtained callus is inoculated into a liquid medium and cultured with shaking under aerobic conditions in the dark at a temperature of 15 to 40°C, preferably 20 to 30°C, for 2 to 4 weeks.

Glycyrrhetin is made into an ethanol solution, and after the number of cells in liquid culture as described above preferably reaches the maximum, it is added at a ratio of 0.001 to 0.1 part by weight of glycyrrhetin to 1 part by weight of the transplanted eucalyptus cells. Added. At the same time, 0.1-1 parts by weight of glucose may be added (however, since the glucose necessary for glucosylation of glycyrrhetin is also obtained from other carbon sources in the medium,
Glucose is not an essential component of the medium used in the production method of the present invention. ). When cultured with shaking for 3 to more than 10 days, the added glycyrrhetin is absorbed into Eucalyptus plant cells and converted into glycosides in the cells to produce glycyrrhetinic acid β-D-gnorecosinole ester. During culture, eucalyptus cells do not differentiate into organs such as stems, leaves, or roots, and remain as callus.

To collect glycyrrhetinic acid β-D-glucosyl ester from the culture solution, cells are separated from the culture medium by centrifugation, the resulting cells are crushed by treatment with a homogenizer, and after filtering off the solid matter, the filtrate is The target product is isolated by any separation and purification means such as extraction or chromatography.

The present invention also provides an anti-inflammatory agent comprising glycyrrhetinic acid β-D-glucosyl ester, which can be easily produced as described above.

The anti-inflammatory effect of glycyrrhetinic acid β-D-glucosyl ester has not been known so far. It was confirmed that this compound is superior to glycyrrhizin in its anti-inflammatory effect and is almost equivalent to glycyrrhetinic acid.

Anti-inflammatory agents consisting of glycyrrhetinic acid β-D-glucosyl ester are usually available in the form of a skin application or an oral preparation. When used as a skin liniment, glycyrrhetinic acid β-D-glucosyl ester is added to the total amount of the liniment. OS is contained in an amount of about 5% by weight, preferably about 0.1 to 2.0% by weight. When formulating the formulation, moisturizers, adhesives, preservatives, surfactants,
A sequestering agent for metal ions or the like may be added. On the other hand, in the case of oral administration, sufficient effects can generally be expected by using the drug in the range of about 10 to 201 mg per day, although it depends on the patient's age and symptoms. In this case as well, if necessary, it can be made into granules, tablets, or capsules using excipients.

〔Effect of the invention〕

As mentioned above, the production method of the present invention utilizes the metabolic reaction within Eucalyptus plant cells, so glycyrrhetinic acid β-D-glucosyl ester can be obtained without complex chemical combinations or reactions, and it is economical and Excellent in terms of safety.

Second, the anti-inflammatory agent of the present invention, which uses glycyrrhetinic acid β-D-gnorecosinoleester as an effective ingredient, is expected to be widely used as an anti-inflammatory agent like glycyrrhetinic acid and glycyrrhizin. .

〔Example〕

Example l Tsukinuki eucalyptus (Eaealyli+s perri)
BAl strain [Phytocl+ssist
ry 26,715 (1917)] were inoculated into 150 500 µl Erlenmeyer 7 flasks containing 250 ml of MSBAI liquid medium with approximately 15 g of fresh Eucalyptus cells per flask, and incubated in the dark at 25°C for 70 mL. Shaking culture was performed for 10 days while shaking at rotation times/lil. after that,
To each flask, 75 mg of glycyrrhetinic acid was dissolved in 2 ml of ethanol and added in 3 portions every other day (the total amount added to all 7 couscous was 34 g).
.

Subsequently, shaking culture was performed, and 7 days after the third addition of glycyrrhetinic acid, the culture solution was centrifuged to harvest about 10 kg of cultured cells.

10M methanol was added to the obtained cultured cells, and the cells were disrupted by homogenizer treatment (10,000×5−) twice. The obtained cell lysate was filtered and concentrated under reduced pressure.
000ml. 2 liters of distilled water was added to the obtained concentrate, and the mixture was extracted twice with 4 t of ethyl acetate. The extracts were combined and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The remaining solid was dissolved in chloroform and subjected to silica gel column chromatography. ) and the solvent was distilled off under reduced pressure.

The remaining solid was recrystallized with methanol to obtain colorless needle crystals of glycyrrhetinic acid β-D-glucosyl ester 6.
92g was obtained.

In addition, after separating the cultured cells, the medium is prepared using porous resin HP-
20 (Mitsubishi Kago) 1000 Houou column to adsorb the metabolites of glycyrrhetinic acid, and then eluted with methanol. After concentrating the eluate under reduced pressure, the dried product was dissolved in a developing solvent, applied to a silica gel ram, eluted with chloroform/methanol/distilled water (5:1:0.t), and the solvent was distilled off under reduced pressure and dried. Glycyrrhetinic acid β-
D-'/Norekoshinoreesthenore 32.6 meal 1 is WIb
l'Lfko.

The physicochemical properties of glycyrrhetinic acid β-D-glucosyl ester obtained as described above were as follows, and were in good agreement with those of the synthesized specimen.

■ Color and shape: Needleless crystals (from methanol) ■
Melting point = 210-235°C ■ Optical rotation [a TB. : l 4 1' (c-
fall. MeOfl) ■ Infrared absorption W ahaa
*c@-'CKBr tablet method''): 3 4 0 0 (0
11) 2800~3000 (CI!), 1
7 5 5 (Coo-Glc) 1 6 6 0 (C
=C-C=0) ■ FAB-Mass Spectrum/! : 6 5 5
([M+Nil”)■ NMR: C-C.Hs
C311. s) δ0. H (6H,s)δ●11.1.
+5,l. H :C H (II, s) a 5.11
:CH (1B,b4)δ3.07 MiCH(
os) δ131 (III, dd) δL0 (lft,
d) δLH (III, m) δ3.13 -C H
! -OH (eickllll, dd) δ4.2lδ4
．． 37 = CH-OH (III, Id) δ3.11
Example 2 Glycyrrhetinic acid β-toglucosyl ester was produced in the same manner as in Example 1 using various Eucalyptus plants,
The glycyrrhetinic acid conversion ability was investigated.

The amount of glycyrrhetinic acid β-D-glucosyl ester produced was determined using high performance liquid chromatography 7E for the cell lysate and the culture medium. The Eucalyptus plants used were
Ewctlytms HIeb+lms, Emcxl
7tws citriedors, E*calytas
cal●pkylla, Emcalytms di
ves, Eicalylmspolybraclsa
, and Encalyt@s r*di turtles.

The results were as follows.

Table 1 Example 3: Inhibitory effect of rat lagenin on paw edema Weight 1
One group consisted of 10 male SD rats weighing 80 to 200 g.
Glycyrrhetinic acid β-D-glucosyl ester was administered to Group A as a drug, and glycyrrhetinic acid (comparative example) was administered lightly to Group B at 0.2 mM/kl suspended in 5% TveeslO. 5% Tweea8 for control group
Only 0 was administered. After 1 hour, 0.1 amount of 1% carrageenan 1/f●●t'l was applied subcutaneously to the hind leg of the rat as an inflammatory agent.
lld was administered.

Three hours after administration of carrageenan, the resulting edema was measured using a foot edema measurement device (Muromachi Kikai Co., Ltd.), and the edema rate and inhibition rate were calculated. The results are shown in Table 2.

Table 2 Visible Edema rate (%) Suppression rate (%) Control 67.2 Group A 41.3 38.5 Group B 44.9 33.2 Example 4: Suppressive effect of raft carrageenan foot edema Body weight 1
One group consists of 10 male SD rats weighing 80 to 200 g.
Group A was treated with parent wood ointment containing 1% glycyrrhetinic acid β-D-glucosyl ester (Kumiboki), and group B was treated with parent wood ointment containing 1% glycyrrhetinic acid (comparative example). 1 g was applied subcutaneously to the hind leg of the rat. To the control group, an equal amount of parent tree ointment containing no drug was applied. One hour later, 0.1 weight 1/fool psd of 1% carrageenan was injected into the application site as an inflammatory agent.

Three hours after the application of carrageenan, the resulting edema was measured using a foot edema measurement device (Muromachi Kikai Co., Ltd.), and the edema rate and inhibition rate were calculated. The results are shown in Table 3.

Table 3 Treatment Edema rate (%) Suppression rate (%) Control 64.1 Group A 49.9 22.3 Group B 47.2 26.4 (Note) Parent tree ointment set FR = White petrolatum 250 parts by weight Stearyl alcohol 200 Propylene glycol 120 Polyethylene hydrogenated castor oil 40 Glyceryl monostearate lO Methyl paraoxybenzoate l Probil paraoxybenzoate Procedure Amendment Purified water is added to bring the total amount to 1000 parts by weight.

Agent Patent Attorney Hiraboshi Gengo August 22nd Patent Office Commissioner Fumiyoshi Yoshida l. Case Description 1999 Patent Application No. 177993 2. Title of the invention: Process for producing glycyrrhetinic acid gulfucyl ester and anti-symptomatic agent 3. Relationship with the case of the person making the amendment Patent applicant Maruzen Kasho Co., Ltd. 4. Agent Address: Kyodo Pill 7th floor, 3-6-18 Kita-Aoyama, Minato-ku, Tokyo 01 (Telephone: 40●10!!) 5. Date of amendment order: Jiko ~ 16. Column 7 for detailed description of the invention in the specification to be amended. Contents of correction

Claims (3)

[Claims] (1) Glycyrrhetinic acid β-D-glucosyl ester is produced by culturing Eucalyptus plant cells in a medium containing glycyrrhetinic acid or glycyrrhetinic acid salt, and collecting glycyrrhetinic acid β-D-glucosyl ester from the culture solution. Manufacturing method. (2) Eucalytus pe as a plant of the genus Eucalyptus
Eucalytus globulus
us, Eucalytus citriedora,
Euca-lytus calophylla, Euc
alytus dives, Eucalytuspol
ybractea, Eucalytus radiat
The manufacturing method according to claim 1, using any one of a. (3) An anti-inflammatory agent containing glycyrrhetinic acid β-D-glucosyl ester as an active ingredient.