JPH10175989A - Para-aminobenzoic acid glycoside, its production and preparation containing the same as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new para-aminobenzoic acid glycoside excellent in water solubility and useful for ultraviolet light absorbents, medicines, cosmetics, etc., without passing through complicated synthesis processes. SOLUTION: A compound of formula I or II [R is H, glucose reside (1→6)]. For example, para-aminobenzoyl β-D-glucopyranoside. The compound of formula I or II is obtained by culturing culture cells derived from the tissue piece of a Eucalyptus plant such as Eucalyptus perriniana, adding para-aminobenzoic acid to the culture product, further continuing the culture, harvesting the cultured cells, and subsequently extracting the harvested cells with a solvent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel para-aminobenzoic acid glycoside by a plant cell culture technique and a method for producing the same. The present invention also relates to a pharmaceutical or cosmetic preparation using the para-aminobenzoic acid glycoside.

[0002]

2. Description of the Related Art Para-aminobenzoic acid is a compound having the following structure:

[0003]

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[0004] It is contained in burdock and the like in the form of a vitamin B complex, and is used in cosmetics and the like as an ultraviolet absorber.

[0005] As is well known, ultraviolet rays in the sun's rays, particularly in the region of 290 to 320 nm (so-called "UVB" light), have a direct effect on living organisms, and cause erythema and blisters on the skin due to irritation. It causes inflammation such as formation, causes pigment formation and skin aging, and also causes DNA damage and skin cancer due to chronic exposure. Therefore, development of an ultraviolet ray protective agent that absorbs ultraviolet rays in the UVB region and prevents the above-mentioned obstacles caused by ultraviolet rays has been promoted.

[0006] Para-aminobenzoic acid has been used as an ultraviolet absorber because it effectively absorbs ultraviolet rays in the UVB region. However, due to the presence of two highly polar groups in the molecule, a remarkable solvent effect occurs with a polar solvent such as water, causing a shallow color shift in which the absorption maximum wavelength moves to a shorter wavelength side than 290 nm. As a result, the ultraviolet absorbing effect is reduced.
Therefore, use of para-aminobenzoic acid as an ultraviolet absorber has been restricted in its use form. In addition, since para-aminobenzoic acid itself is poorly soluble in water, its formulation is limited, and the range of use of para-aminobenzoic acid is limited, for example, it is not possible to manufacture an aqueous substrate. is there.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide, as one means for solving the above-mentioned restriction on the use of para-aminobenzoic acid, a method using para-aminobenzoic acid as a glycoside. Accordingly, it is an object of the present invention to provide a para-aminobenzoic acid derivative which has water solubility and can exhibit an ultraviolet absorbing effect without being affected by a solvent. Another object is to provide a method for producing the same by a plant cell culture technique. The object of the present invention includes providing various preparations, such as medicines or cosmetics, containing the thus obtained para-aminobenzoic acid as an active ingredient.

[0008]

The paraaminobenzoic acid glycoside of the present invention is a monoglycoside having the structure of the following formula I and a diglycoside having a structure of the following formula II.

[0009]

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[0010]

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Wherein R is H or a glucose residue (1
→ Represents 6). The method for producing a para-aminobenzoic acid glycoside of the present invention, which gives the above-mentioned glycoside, comprises culturing cultured cells derived from a tissue piece of a Eucalyptus plant, and adding para-aminobenzoic acid thereto. The conversion is performed by continuing the culture, and the cultured cells are harvested and extracted with a solvent to obtain a glycoside as a conversion product.

Paraaminobenzoic acid added to the culture medium and given to the cells of the plant of the genus Eucalyptus is characterized in that an OH group at the 1-position of glucose is ester-linked to the carboxyl group of paraaminobenzoic acid by the action of intracellular glycosylase. It becomes a monoglycoside composed mainly of one glucose, that is, the compound of the formula (I). Subsequently, when the 1-position of glucose is bonded to the amino group of para-aminobenzoic acid, a diglycoside of the formula (II) is formed.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The conditions for cell culture and the conditions for recovering para-aminobenzoic acid glycoside by solvent extraction are as follows:
Those skilled in the art will readily be able to determine this according to techniques known in the art and with reference to the examples that follow. Since the monoglycoside of the formula (I) and the diglycoside of the formula (II) have a slight difference in the degree of water solubility, the former is, for example, water-soluble.
It can be separated and extracted with a mixed solvent of methanol and the latter with water.

[0014] as eucalyptus plant of the genus, E. perriniana
(Tsuki-nuki eucalyptus), E. globulus (eucalyptus),
E. FIG. calophylla , E. radiata , E. dives , E. citrio
dora (lemon eucalyptus) and E. It is preferable to use those selected from polybractea .

The medium to which para-aminobenzoic acid is added is optional, but may be any of MS (Murashige / Skoog) medium, LS (Rinsmeyer / Skoog) medium, and Gambog (Gambog) medium.
rg) B5 medium, niche (Nitsch & Nitsch) medium, White medium or their modified medium are suitable.

[0016]

[Example] E. Cultured cells were derived from aseptic seedlings of perriniana ( Papula persica ) in a conventional manner. The cells were further placed on an agar medium, allowed to stand in a dark place kept at 25 ° C, and subcultured every 3 to 4 weeks to obtain a cell line with good growth.

The agar medium is prepared by adding 0.9% of agar, 1 ppm of benzyladenine and 3% (w / v) of agar to an MS medium having the following composition and dissolving by heating. It was prepared by dispensing 40 ml each, autoclaving and then solidifying.

[0018] Component amount _{(mg / l) NH 4 NO} 3 1650 KNO 3 1900 KH 2 PO 4 170 H 3 BO 3 6.2 MnSO 4 · 4H 2 O 22.3 ZnSO 4 · 4H 2 O 8.6 KI 0.83 Na ₂ MoO ₄ .2H ₂ O 0.25 CoCl ₂ .6H ₂ O 0.025 CuSO ₄ .5H ₂ O 0.025 CaCl ₂ .2H ₂ O 440 MnSO ₄ .7H ₂ O 370 FeSO ₄ .7H ₂ O 27.8 Na ₂ -EDTA 37.3 Inositol 100 Thiamine hydrochloride 0.1 Nicotinic acid 0.5 Pyridoxyli hydrochloride 0.5 Glycine 2 Cell cultures for the production of glycosides contain 1 ppm of benzyladenine About 250 ml of the MS modified medium is poured into a 500-ml Erlenmeyer flask, and about 30 g of the above-mentioned cultivated Tsukinuki eucalyptus cells are added to each Erlenmeyer flask. This was performed by rotating culture in a dark place for 3 weeks.

Thereafter, 25 mg of para-aminobenzoic acid per aseptic flask was aseptically administered as an ethanol solution.

After a further week of rotation culture, the cells were harvested and separated from the medium. After the medium is first adsorbed to the medium through a column of ion exchange resin "Diaion HP-20", the column is washed with water, methanol is eluted, and the methanol solvent is distilled off under reduced pressure to obtain a methanol eluted fraction. Was.

After the cells were homogenized, they were subjected to methanol cold soaking to obtain an extract. This methanol extract was dissolved in water and extracted with diethyl ether. The aqueous layer was further extracted with ethyl acetate, and the remaining aqueous layer was passed through a column of "Diaion HP-20" to be adsorbed. After washing the column with water, the column was eluted with a water-methanol mixed solvent containing 20% methanol, and separated into a 20% methanol elution fraction and a water elution fraction. The solvent was distilled off from the diethyl ether layer and the ethyl acetate layer under reduced pressure.

The above operation is shown in FIG. 1 as an operation system diagram.

After confirming the conversion product by thin layer chromatography, silica gel chromatography and H
Purified using PLC, para-aminobenzoic acid monoglycoside
(I) and diglycoside (II) were obtained. The results are as follows: Cell weight Growth rate Dose Glycoside amount Conversion rate 310 gFW 3 times 150 mg (I) 18.9 mgDW 12.6% (II) 15.7 mgDW 10.4% Paraaminobenzoic acid monoglycoside The properties of body (I) are shown below.

(I) para-aminobenzoyl β-D-glucopyranoside UV λmax (EtOH) nm 224.0, 295.5 FAB-MS m / z 300 [M + H] ⁺ , 138
[MH-Glc] _{^{+ 1 H-NMR (CD 3}} OD) δppm J (Hz) 3.39~3.45 (2H, m, J = 7.0, H-4'a
nd 5 ') 3.45 to 3.51 (2H, m, J = 7.0, H-2'a
nd 3 ') 3.69 (1H, dd, J = 12.0, 5.0, H-6'
a) 3.85 (1H, dd, J = 12.0, 2.0, H-6 '
b) 5.65 (1H, d, J = 8.0, H-1 ') 6.63 (2H, d, J = 9.0, H-4 and 6) 7.81 (2H, d, J) = 9.0, H-3 and 7).

The properties of paraaminobenzoic acid diglycoside (II) are shown below.

(II) para (N-β-D-glucopyranosylamino) benzoyl β-D-glucopyranoside UV λmax (EtOH) nm 224.0, 295.7 FAB-MS m / z 484 [ M + Na] ⁺ , 462
[M + H] _{^{+ 1 H-NMR (CD 3}} OD) δppm J (Hz) 3.36 (1H, dd, J = 8.0,8.0, H-2 ") 3.47 (1H, dd, J = 8.0, 8.0, H-2 ') 3.35 to 3.50 (6H, m, H-4' and 5 ') 3.45 to 3.51 (2H, m, J = 7. 0, H-
3 ', 4', 5 'and 3 ", 4", 5 ") 3.67 (1H, dd, J = 12.0, 5.0, H-6'
a or 6 "a) 3.70 (1H, dd, J = 12.0, 5.0, H-6 '
a or 6 "a) 3.86 (1H, dd, J = 12.0, 2.0, H-6 '
b or 6 "b) 4.63 (1H, d, J = 8.0, H-1") 5.66 (1H, d, J = 8.0, H-1 ') 6.81 (2H, d, J = 9.0, H-4 and 6) 7.90 (2H, d, J = 9.0, H-3 and 7).

The ¹³ C NMR spectra of the two glycosides are as follows.

Compounds (I) (II) Agricone 1 167.4 167.2 2 117.6 119.5 3 133.1 132.8 4 114.3 114.0 5 155.3 153 .46 114.3 114.0 7133.1 132.8 O-glucose 1 '95.8 95.9 2' 74.1 74.1 3 '78.2 78.2 4' 71.2 71. 25 '78.8 78.8 6' 62.4 62.4 N-glucose 1 "85.6 2" 74.5 3 "78.6 4" 71.7 5 "79.2 6" 62.7

[0029]

Industrial Applicability According to the present invention, a novel substance, a glycoside of paraaminobenzoic acid, is obtained by utilizing a plant cell culture technique.
It can be obtained without having to go through complicated synthesis steps. Glycosides are water-soluble, so that various formulations containing para-aminobenzoic acid as an active ingredient, including those using an aqueous substrate, can be made into various formulations. On the other hand, the use of glycosides can prevent the ultraviolet absorption maximum wavelength from being shifted by the solvation of water into a shallow color, so that the ultraviolet absorption effect of para-aminobenzoic acid can be sufficiently utilized.

[Brief description of the drawings]

FIG. 1 is an operation system diagram showing a treatment operation of a cell culture after administration of para-aminobenzoic acid in the production of para-aminobenzoic acid glycoside according to the method of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C09K 3/00 104 C09K 3/00 104Z C12N 5/04 C12N 5/00 F (C12P 19/44 C12R 1:91) (C12N 5 / 04 C12R 1:91)

Claims (6)

[Claims] 1. A para-aminobenzoic acid glycoside represented by the following formula I or II: Embedded image [Wherein, R represents H or a glucose residue (1 → 6). ] 2. A culturing a eucalyptus (Eucalyptus) cultured cells derived from the tissue pieces of plants, this was done to transform by continuing further culture by adding para-aminobenzoic acid, and harvested cultured cells, A method for producing a para-aminobenzoic acid glycoside, comprising obtaining a glycoside as a conversion product by solvent extraction. 3. A plant belonging to the genus Eucalyptus, E. coli . perriniana
(Tsuki-nuki eucalyptus), E. globulus (eucalyptus),
E. FIG. calophylla , E. radiata , E. dives , E. citrio
dora (lemon eucalyptus) and E. 3. The method according to claim 2, wherein a substance selected from polybractea is used. 4. A medium to which para-aminobenzoic acid is added, MS (Murasige / Skoog) medium, LS (Rinsmeyer / Skoog) medium, Gamborg BS
3. The method according to claim 2, wherein the method is carried out using a medium, a niche (Nitsch & Nitsch) medium, a white medium, or an improved medium thereof. 5. A preparation containing the para-aminobenzoic acid glycoside according to claim 1 as an active ingredient. 6. The preparation according to claim 5, wherein an aqueous substrate is used.