JPH10265492A - New indole glycoside - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an indole glycoside having bloodstream accelerating effect and skin whitening effect by isolation from Calanthe discolor Lindl. SOLUTION: This new indole glycoside is 3β-Dglucopyranosyl(1→6)β- Dglucopyranosylindole (hereafter referred to as calanthoside A). This glycoside is obtained by the following process: rootstocks of Calanthe discolor Lindl. are sliced, and subjected to hot extraction with methanol followed by partition extraction with ethyl acetate and water, and the resultant aqueous phase is put to reversed-phase silica gel column chromatography followed by successive elution with water and methanol to afford a methanol elute, which, in turn, is subjected to two-stage fractionation by normal-phase silica gel column chromatography followed by repeated separation using reversed-phase high- performance liquid chromatography. Calanthoside A significantly raises rat skin's blood circulation promoting effect, having melanogenetic enzyme tyrosinase activity-suppressive effect, thus being usable in hair growers and skin whitening agents.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
lanthe discol Lindl. )).

[0002]

2. Description of the Related Art Ebinelan alcoholic solution extract
It is known that it shows a remarkable effect as a hair growth and hair growth agent (JP-A-5-294813). However, the components containing the ebinelan extract have not yet been sufficiently elucidated, and at present the active ingredients have not been identified.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have examined the fractionated components of the alcohol extract from Ebinelan and have succeeded in isolating a novel indole glycoside having a physiologically useful activity. This has led to the present invention.

[0004] That is, an object of the present invention is to provide a novel indole glycoside having a blood flow promoting action and a whitening action isolated from Ebinelan.

[0005]

The novel indole glycoside of the present invention, which has achieved the above object, is a 3-o-β-formula represented by the following chemical formula (1).
D-glucopyranosyl (1 → 6) -β-D-glucopyranosylindole (3-o-β-D-glucopyr
anosyl (1 → 6) -β-D-glucopyra
nocyl indol) (hereinafter "currant side A /
calanthoside A ").

[0006]

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[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The currant side A of the present invention can be manufactured as follows. After shredding the rhizome of Ebinelan, it is hot-extracted with, for example, methanol to obtain a methanol-extracted extract. The obtained methanol extract is partitioned and extracted with ethyl acetate and water (1: 1). The water transfer section was then subjected to reversed phase silica gel column chromatography,
Elution is carried out sequentially with methanol to obtain a methanol elution part. The methanol eluted portion is fractionated by normal-phase silica gel column chromatography, further fractionated by normal-phase silica gel column gas chromatography, and repeatedly separated using reversed-phase high-performance liquid chromatography, whereby the currant side A is isolated. .

[0008] As is clear from Test Example 1 described later, it is clear that currant side A significantly increases the skin blood flow promoting action in rats, and it can be expected to be used as a hair restorer and the like. In addition, the skin whitening effect is an indicator of preventing melamine production in the skin in order to prevent the skin from becoming black. Since melanin is produced from tyrosine or dopa by an enzyme called tyrosinase, suppressing this enzyme activity leads to whitening. As shown in Test Example 2 described below, as a result of measuring the anti-tyrosinase activity that suppresses this enzyme activity, Currantside A shows a relatively strong anti-tyrosinase effect, and it can be used as a cosmetic such as a whitening milky lotion or cream. Can be expected.

[0009]

[Example] (Isolation) After shredding fresh rhizome (7.5 kg) of shrimp from Miyazaki,
Hot extraction with MeOH (18 liters) was performed a total of three times. The three MeOH extracts were combined and the solvent was distilled off under reduced pressure to obtain a MeOH extract (330 g, 4.4%). Ebine MeOH extraction kiss (300g) with AcOE
Partitioned with t-H ₂ O (1: 1), the AcOEt layer transition (75 g, 1.0%), the H ₂ O layer transition (225 g,
3.0%). The obtained extract of the H ₂ O layer transition portion was subjected to reverse phase silica gel column chromatography [1.0 kg,
H ₂ O → MeOH] was used to remove the sugar to obtain a MeOH effluent extract (13 g, 1.7%). MeOH outflow extract 1
3 g of normal phase silica gel column chromatography {1.
0 kg, [CHCl ₃ : MeOH = 10: 1 → 3: 1 →
1: 1] → [CHCl ₃ : MeOH: H ₂ O = 65: 3
5:10 (lower layer) → 6: 4: 1] → MeOH｝. 1 (170 mg, 0.0023%), Fr.
2 (41 mg, 0.0005%), Fr. 3 (48m
g, 0.0006%), Fr. 4 (234 mg, 0.0
031%), Fr. 5 (4100 mg, 0.055
%), Fr. 6 (2700 mg, 0.036%), F
r. 7 (2000 mg, 0.027%), Fr. 8 (4
000 mg, 0.053%). Fr. 7 (430
0 mg, 0.057%) using normal phase silica gel column chromatography [40 g, CHCl ₃ : MeOH: H ₂ O].
= 7: 3: 1 (lower layer) → 65:35:10 (lower layer) →
6: 4: 1], and then reversed-phase high-performance liquid chromatography (HPLC) [column: YMC-Pack R]
& D ODS-5 (250 × 20 mmid), solvent; MeCN-H ₂ O (30:70, v / v), flow rate;
9.0 ml / min], reversed-phase high performance liquid chromatography (HPLC) [column; YMC-Pack R & D
ODS-5 (250 × 20 mm id), solvent; M
_{eOH-H 2 O (25:} 75, v / v), flow rate; 9.0
[ml / min] to obtain currant side A (81.9 mg, 0.0011%).

[0010]

[Analysis Example 1] (Identification) Currantside A is a pale yellow powder having a positive optical rotation, and the analysis results are as follows. Currant side A: amorphous powder Optical rotation: [α] _D ²⁵ + 164.0 ° (c = 0.01,
MeOH) High Resolution FAB-MS (m / z) equation Calculated ^{_{C 20 H 28 NO 11 (M}} + H) +: 458.1638 ^{Found: 458.1650 IR (KBr, cm -1} ): 3490,1618,15
54, 1458, 1028 UV (c = 0.0006, MeOH, nm, log
ε): 282 (4.7), 224 (5.4) ¹ H-NMR (CD ₃ OD, 500 MHz, δ): 4.40 (1 H, d, J = 7.6 Hz, 1 ″ -H),
4.74 (1H, d, J = 7.3 Hz, 1'-H) 6.97 (1H, ddd, J = 1.3, 6.9, 7.9)
Hz, 5-H), 7.07 (1H, ddd, J = 1.
3,6.9, 8.3 Hz, 6-H) 7.15 (1H, s, 2-H), 7.27 (1H, d
d, J = 1.3, 8.3 Hz, 7-H), 7.67 (1
H, dd, J = 1.3, 7.9 Hz, 4-H) ¹³ C-NMR (CD ₃ OD, 125 MHz, δc): C-1, C-2 112.2, C-3 138.8, C-4 118.6, C-5 119.5, C-6 122.8, C-7 112.4, C-8 135.4, C-9 121.3, C-10, C-1 ' 105.4 C-2 '75.0, C-3' 77.9 C-4 71.5, C-5 '77.3 C-6' 69.9, C-1 "104.7 C-2 "75.1, C-3" 77.9 C-4 "71.5, C-5" 77.9 C-6 "62.7, Positive-mode FAB-MS (m / z): 480 (M + Na) ) ⁺ Negative-mode FAB-MS (m / z): 456 (M-H) ^-

[0011]

[Analysis Example 2] (Identification of sugar) 5% H ₂ SO ₄ -dioxane (1: 1, v / v, 1.0 m) of currant side A (3 mg)
l) The solution was heated to reflux under a nitrogen stream and stirred for 2 hours.
The reaction solution was treated with an anion exchange resin IRA-400 (OH ^- fo
rm), the resin was separated by filtration, and the solvent was distilled off under reduced pressure to obtain a crude product (2.8 mg). The crude product was subjected to reverse phase silica gel column chromatography (0.2 g, H ₂ O → Me
OH), and the solvent was distilled off under reduced pressure to obtain a crude product (1.0 mg). L-Cysteine methyl ester hydrochloride (0.3 mg) was added to a pyridine solution of the obtained crude product, and the mixture was stirred at 60 ° C. for 1 hour, followed by N, O-bis (trimethylsilyl) trifluoroacetamide (0.3 ml). ) Was added and the mixture was stirred at 60 ° C for 1 hour, filtered, and subjected to GC analysis under the following conditions as a GC sample. GC conditions column: Supeluco STB ^™ -1
(0.25mm × 30m) Injector temp: 230 ℃ Detector temp: 230 ℃ Column temp: 230 ℃ He flow rate: 15ml / min retention time _{(t r) D-Glucose:} 24.15 L-Glucose: 25.46

From the above analysis examples 1 and 2, it was revealed that the currant side A had a molecular formula of C ₂₀ H ₂₇ NO ₁₁ by FAB-MS and high-resolution FAB-MS. In the IR spectrum, the hydroxyl group (3
490 cm ^-1 ) and a pyrrole ring (1554 cm ^-1 )
The analysis of ¹ H-NMR and ¹³ C-NMR spectra revealed that two β-linked glucoses [δ 4.40 (d, J = 7.6), δc 104.7],
The existence of [δ4.74 (d, J = 7.3), δc105.4] was confirmed. Currant side A is H ₂ SO ₄ −
When acid hydrolysis is carried out with giogisan, d-glucose (d-
glucose) is obtained, the currant side A ¹ H-
NMR and ¹³ C-NMR spectra are described in the literature.
Compared to indian shown in
Glucosylation shift of the signal at the 6-position carbon of glucose to a low field and the signal of the 5-position carbon to glucosylation shifted to a high field. It is suggested that this is a compound to which another molecule of glucose is bound.

[0013] Also of currant side A ¹ H- ^{1 H, 13}
C- ¹ HCOSY and ^{1 H- 1 H, 13 C- 1} HOHA
As a result of analysis of the HA spectrum, the signal of the sugar moiety was completely assigned, and the sugar binding mode was determined by the coupling constant of anomeric hydrogen and the chemical shift of anomeric carbon [δ4.4].
0 (d, J = 7.6), δc 104.7], [δ4.7
4 (d, J = 7.3), δc105.4],
It became clear that both glucoses were β-linked. Further, in the HMBC spectrum, the anomeric hydrogen of glucose (δ 4.74, 1H, d, J
= 7.3) and the 3-position carbon of the indole ring (δc138.
8), the anomeric hydrogen of the other glucose (δ
4.40, 1H, d, J = 7.6) and a correlation between glucose 6′-carbon (δc69.9), indicating that currant side A was located at the 6′-position of glucose in indica. It was determined to be 3-o-β-D-glucopyranosyl (1 → 6) -β-D-glucopyranosylindole (Formula 1) to which glucose was bound.

[0014]

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[0015]

Reference Example (Isolation) Fresh leaf (2.9 kg) of shrimp from Miyazaki was subjected to hot extraction with MeOH (8 liters) three times in total. 3 of MeOH extract
The batches were combined, and the solvent was removed under reduced pressure to obtain a MeOH extract (150 g, 5.2%). The Ebine MeOH extract (150 g) was partitioned with AcOEt-H ₂ O (1: 1) to give an AcOEt transition (62 g, 2.1%). The obtained AcOEt transition extract was subjected to normal phase silica gel column chromatography {1.0 kg, CHCl ₃ →
[CHCl ₃ : MeOH = 50: 1 → 10: 1 → 5:
1] → MeOH}. 1 (10.5g,
0.36%), Fr. 2 (5.4 g, 0.19%), F
r. 3 (14.2 g, 0.49%), Fr. 4 (8.0
g, 0.28%), Fr. 5 (10.7 g, 0.37
%), Fr. 6 (10.3 g, 0.36%) was obtained. F
r. 3 (14.2 g) was further subjected to normal phase silica gel column chromatography [15 g, CHCl ₃ : MeOH = 2
0: 1 → 10: 1 → 5: 1 → MeOH], normal phase silica gel column chromatography [15 g, CHCl ₃ : M
MeOH = 20: 1 → 10: 1 → 5: 1 → MeOH], and indirubin (chemical formula 3)
(128.0 mg, 0.0044%), tryptanthrin 4) (6.8 mg,
0.0002%). Fr. 5 (10.7 g) was subjected to reverse phase silica gel column chromatography [130 g, M
eOH: H ₂ O (40: 60 → 50: 50 → 80: 2
0, v / v), reversed-phase high-performance liquid chromatography [column: YMC-pack R & D ODS-5 (250 ×
20 mm i. d. ), Solvent; MeOH (45:55,
v / v) at a flow rate of 9.0 ml / min] and vomifoliol (3) (3).
8.6 mg, 0.0013%). The obtained compound was identified by comparing ¹ H, ¹³ C-NMR data and TLC, and as a result, it became clear that all of the compounds were the above-mentioned known compounds.

[0016]

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[0017]

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[0018]

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[0019]

[Test Example 1] (Radiation promoting action on rat skin blood) SLc: Wistar male rats (body weight: 180-200 g) were used as experimental animals. Skin blood flow was measured using a laser Doppler flowmeter (Laser Doppler F).
lowmeter PF2B, Perimed). The back of the rat was shaved, and the skin blood flow of the rat was measured the next day under urethane (1 g / kg) anesthesia.
Previous value. Immediately after the pre-value measurement, 25 μl of the test solution (50% ethanol solution) was applied to the pre-value measurement site,
After 40 minutes and 60 minutes, the skin blood flow at the application site was measured again, and the rate of increase relative to the previous value was calculated. The control group was coated with a 50% ethanol solution. The test solution contains the ebine MeOH extract (CDM-ext) of Example.
(0.5%, 2.0%), currant side A of the present invention
(CDH2) (0.2%) and a known substance indirubin (0.2%) of Reference Example were used. The results are shown in Table 1 and FIG.

[0020]

[Table 1]

As is apparent from Table 1, Ebine MeOH
The extracted extract significantly increased rat skin blood flow at both 0.5% and 2.0%. As shown in FIG. 1, the currant side A of the present invention significantly increased the skin blood flow in 20 minutes. Indirubin had a similar promoting effect, but was not significant.

[0022]

Test Example 2 (Whitening Action) The following experiment was conducted according to the method of Mason and Peterson. That is, 0.5 ml of L-dopa solution (0.03% in pH 6.8 PBS) is added to 0.5 ml of the test solution.
And incubated at 25 ° C. for 10 minutes, and then a tyrosinase solution derived from mushrooms (pH 6.8 PB)
(S, 45 U / ml) 0.5 ml was added and incubated at 25 ° C. for 5 minutes. After the reaction, the absorbance (D1) at 475 nm was measured. Separately, the same operation was performed without adding the L-dopa solution, the absorbance (D2) was measured, and the absorbance (D3) as the amount of dopachrome produced without addition of the test drug was measured. Using these D1, D2 and D3,
The dopachrome production inhibition rate was determined by the following formula, and used as an index of tyrosinase activity. The test liquids contained the ebine MeOH extraction extract (CDM-ext) of the example, the ethyl acetate layer transition part (AcOEtphase), the aqueous layer transition part (H ₂ Ophase) of the example, and the currant side A (C) of the present invention.
DH2), and the positive control drug kojic acid (k
ojic acid) was used. Table 2 shows the results.

[0023]

(Equation 1)

[0024]

[Table 2]

As is clear from Table 2, Ebine MeOH
The extracted extract, the ethyl acetate phase transition part and the aqueous phase transition part showed strong anti-tyrosinase activity in a dose-dependent manner. Kojic acid in the positive control group also showed strong anti-tyrosinase activity.
Considering the yield, the active ingredient of anti-tyrosinase activity is considered to be mainly located in the transition part of the aqueous layer. However, as shown in Table 3, the currant side A of the present invention also showed relatively strong anti-tyrosinase activity. .

[0026]

According to the present invention, a novel currant side A is provided.
Has a skin blood flow promoting action and can be expected to be useful for hair growth agents and the like. In addition, a skin whitening effect is recognized, and it can be used for cosmetics and the like.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the effects of currant side A and indirubin isolated from Ebinelan on promoting skin blood flow in rats.

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(Equation 1)

[Table 1]

[Table 2]

[Table 3]

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/70 ADA A61K 31/70 ADA // A61K 35/78 35/78 Y (72) Inventor Toshiyuki Murakami Yamashina, Kyoto, Kyoto 32, Shinomiya-Kakinouchi-cho, Shinnomiya-ku 418 (72) Inventor Hiromi Shimada 2-12-61 Uenozaka, Toyonaka-shi, Osaka (72) Inventor Tetsuo Sakura 1-4-4 Nishiwaki, Hirano-ku, Osaka, Osaka No. 3-101 (72) Inventor Manabu Nomura 2763 Imaizumi 7

Claims (1)

[Claims] (1) 3-o-D-glucopyranosyl (1 →
6) A novel indole glycoside comprising one β-D-glucopyranosyl indole.