JPH0366287B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a hypotensive component obtained from wheat and a method for collecting the same.

The present inventors have conducted extensive research with the aim of isolating antihypertensive components from natural plants, and after discovering that an aqueous extract of barley leaves contains antihypertensive components, As a result of our research, we succeeded in isolating and identifying a substance that has a significant antihypertensive effect.
The present invention was completed based on this knowledge.

To collect antihypertensive components by the method of the present invention,
Wheat, especially Hordeum vulgare L.var.
Leaves (particularly young leaves) of barley plants, such as A. nudum Hook), are extracted with water and subjected to a fractionation method to collect a fraction having a hypotensive effect.

To explain this collection method in more detail, for example, young leaves of the naked barley are crushed and then heated with hot water (approximately 90%
℃), and the extract is preferably concentrated as appropriate.
Insoluble matter is removed by adding alcohol such as ethanol to obtain a supernatant. These operations are repeated as appropriate, and the resulting supernatants are combined, concentrated, lyophilized, and stored. Although this lyophilized product has a certain degree of hypotensive effect, the following operations are performed to further separate and purify the active ingredient.

That is, the above-mentioned lyophilized product is fractionated through a column of Sephadex, and a fraction having a relatively strong antihypertensive effect is collected. To test for the presence or absence of a hypotensive effect, for example, it is sufficient to administer the drug intravenously to spontaneously hypertensive rats (SHR) or sudden spontaneous hypertensive rats (SHRSP) and observe whether the blood pressure decreases or not. Collect the fraction that showed a remarkable hypotensive effect, and if necessary, use an appropriate fractionation method to extract compounds with a remarkable hypotensive effect (2)
and (3) are obtained.

The yields of compounds (2) and (3) depend on the type of raw materials, but when powdered young leaves of naked cabbage are used as described above, the yields of compounds (2) and (3) are approximately 6.0% of each weight.
×10 ^-3 % and around 1.6 × 10 ^-2 %.

Next, the identification of the chemical structures of the above compounds (2) and (3) will be described.

Both of these compounds give purple spots under UV lamp irradiation in the filter paper spot test, but when UV irradiated under an ammonia atmosphere, the compounds
(2) gives a yellow-green color, while compound (3) gives a yellow color.
In view of these facts, both C-4' and C-
Since it has a free hydroxyl group at the 5-position, it is presumed to be a flavone or a C-3-OH substituted flavonol.

In general, the UV absorption spectrum of flavones and C-3-OH substituted flavonols is between 300 and 380 nm.
It has main absorption bands 1 and 2 in the 24-280 nm region,
These are related to the excitation of π-electrons in the B and A rings, and each absorption band is optionally divided into two or three subbands. Compound
The spectra measured for the methanol solutions of (2) and (3) each have a band 1 near 340 nm.
has two subbands, band 2, around 260 and 270 nm. Band 1 of both spectra showed a bathochromic shift of about 55 nm without an increase in intensity upon addition of sodium methoxide to their methanol solutions. These shifts are due to the C-
This indicates the presence of a free hydroxyl group at the 4' position. For both compounds, the two subbands of Band 2 merged into one peak upon addition of sodium methoxide. When the weaker base anhydrous sodium acetate was added to the methanol solution of compound (2), the effect on the shift of band 1 was incomplete, leaving a shoulder at 239 nm and producing a new band at 392 nm. No fusion of subbands of Band 2 was observed for compound (2). The ortho-dihydroxyl group in the B ring is known to form a complex with boric acid in the presence of sodium acetate, resulting in a bathochromic shift of band 1. Compound (2) did not exhibit such a shift, but in the spectrum of a methanol solution of compound (3), the band underwent a bathochromic shift, giving two peaks at 373 and 432 nm. From these results, it can be understood that the presence and absence of an ortho-dihydroxyl group in the B rings of compounds (2) and (3), respectively. From the above UV spectrum data, compound (2) has one hydroxyl group (C-4' position),
It can be seen that compound (3) has two hydroxyl groups (at C-3' and C-4' positions).

Further information about the structure of these flavonoids was obtained by adding aluminum chloride. That is, the aluminum chloride addition spectrum of compound (2) gave two subbands for each of bands 1 and 2. These subbands were more or less shifted toward longer wavelengths compared to the methanol spectrum. This shift is C-4
This is believed to be due to the formation of an aluminum chelate between the keto group at the C-5 position and the hydroxyl group at the C-5 position. Since this type of chelate is acid-stable, the aluminum chloride spectrum of compound (2) was not changed by the addition of hydrochloric acid. Compound (3) gave a similar spectrum to aluminum chloride, but the subband at 420 nm of band 1 underwent a hypsochromic shift with the addition of hydrochloric acid. In this case, aluminum is thought to have coordinated to the C-4keto-C-5 hydroxyl system and the C-3'hydroxyl-C-4' hydroxyl system, since chelates with the latter system are unstable to acids. It is thought that it was disassembled. In any case, it is clear that a hydroxyl group is present at the C-5 position of both compounds.

In addition, compound (2) has a 200MHz
In the ¹ H-NMR spectrum shown in the attached drawing (see Figure 1), a pair of 2-proton doublets and 1-proton singlets derived from protons in the flavone skeleton are given in the region below 6.5 ppm. This pair of 2-proton doublets indicates that the disubstituted symmetric benzene ring, that is, ring B, is substituted only at the C-4' position. Also, 7.22ppm
A tablet and a doublet were observed at 8.17 ppm, but these are thought to be due to H-3', H-5' and H-2' protons, respectively.
The singlet shift at 6.59ppm is H-3
It is thought to be a proton. The spectrum in the intermediate region from 3.5 to 6.5 ppm originates from the protons in the two hexose groups. More detailed decoupling revealed that the signal at 4.5 to 6.5 ppm was C-1 in the sugar ring,
It is clear that this is due to the protons at the C-2 and C-3 positions. Chemical shifts and coupling constants indicate that both sugar rings are directly linked to the flavone skeleton, which is also clear from the fact that no hexoses are liberated by acid hydrolysis. . However, the hexose group was degraded to arabinose by oxidative cleavage with ferric chloride. C-6, C-7
Since no proton is observed at the and C-8 positions, these two sugar rings should be bonded to any two of the three carbons of the A ring. C-7
Since the C-glycosides attached to these positions are not known, it is believed that the linkages occur at the C-6 and C-8 positions. The two hexoses are considered to be β-D-glycopyranoses of C-1 structure from the coupling constant. Many singlets around 2 ppm are protons of acetoxyl groups. There are three free signals in the 2.3-2.6 ppm region, which are the protons of the acetoxyl group attached to the flavone skeleton, and two signals around 1.8 ppm are the acetoxyl protons at the C-2 position of the sugar ring. be. The other six singlets are acetoxy protons at the C-3, C-4 and C-6 positions of the sugar ring.

From the above confirmed facts, compound (2) is identified as apigenin-6,8-di-C-β-D-glucoside represented by the following formula: [In the formula, Gle represents a glucose residue. ] This flavone C-glucoside has been reported to exist in various plants (Wagnet
etal.; Z.Naturforsh., B27, 954-958 (1972)),
This is the first time that its existence in wheat has been confirmed, and it was completely unknown that this substance had a hypotensive effect.

Compound (3) also has ¹ H− of its acetate.
In the NMR spectrum (see Figure 2),
Two 1-proton singlets, a 1-proton doublet, and a 2-proton multiplet are provided in the region below 6.5 ppm. 6.55ppm
The singlets at 6.93 ppm are due to H-3 and H-8 protons, respectively. Doublet
The multiplet system consists of C-3' and C- of the B ring.
This is characteristic of flavonoids that have a substituent at the 4' position.

Considering these analyzes together with the results in the UV spectrum, compound (3) is a derivative of luteolin (i.e., 3',4',5,7-tetrahydroxyl flavone).

Hydrolysis of compound (3) gives one molecule of glucose, but the ¹ H-NMR spectrum of its acetate shows the presence of two hexose groups, giving 14 ring protons between 3.7 and 5.7 ppm. This means that compound (3) has one C- and one O-hexosyl group. The C-hexyl group gave arabinose upon cleavage with ferric chloride. Although it was not possible to completely determine the protons of sugars,
The free triplet at at least 5.64 ppm can be attributed to the proton at the C-2 position of the C-hexosyl group. Therefore, 5.05 ppm of the free doublet is due to the anomeric proton of the same hexose group. Large coupling constant between C-1 and C-2 protons (10.3Hz)
and a similar coupling constant between C-2 and C-3 protons when this hexosyl group is β
It is an anomer, and the coordination of protons at C-2 and C-3 are both axial. Although the coordination of the C-4 and C-5 protons is uncertain from ^{the 1H} -NMR data, this hexosyl group is glucose in comparison to the structure of other flavone glycosides isolated from barley leaves. it is conceivable that. The bonding position of O-hexosyl (obviously glucosyl from hydrolysis) is the bonding position of the other three hydroxyl groups (C-
3', C-4' and C-5) is known to be free by UV spectroscopy, so it is the C-7 position. The C-hexosyl group, which is not liberated by acid hydrolysis, is bound to the flavone skeleton and must be at the C-6 position since all other positions are already occupied. The three singlets in the 2.3-2.5 ppm region are due to the acetoxyl groups at C-3', C-4' and C-5 positions of the flavone skeleton, and the free signal at 1.74 ppm is due to the C-hexosyl group. This is due to the acetoxyl group at the -2 position. The other seven singlets around 2ppm are C-2, C-3, and C-4 of the O-hexosyl group.
and an acetoxyl group at the C-6 position and a C-
This is due to the acetoxyl groups located at the C-3, C-4 and C-6 positions of the hexosyl group.

Combining these results, compound (3) was identified as luteolin 6-C-,7-O-diglucoside (also known as isoorientin 7-O-glucoside) shown by the following formula: [In the formula, Glc represents a glucose residue. ] The properties of the above-identified compound are based on a substance previously isolated from barley and named sodium (Seikel et al.; Arch.Bio-chem.Biophys.,
99, 451-457 (1962)), but the latter is reported to be 8-C-glucoside, which is different from the compound (3) identified above, that is, 6-C-glucoside. It seems so. In any case, the presence or absence of antihypertensive effects of the above-mentioned known substances has not been investigated at all.

The above compounds (2) and (3), especially the former, exhibit excellent antihypertensive activity and are therefore useful as antihypertensive agents. In order to accurately control the dosage, it is desirable to isolate these compounds and then formulate them appropriately. However, these compounds are highly safe as understood from their origins (acute toxicity to rats when administered orally is 10 g/Kg (body weight) or more, making them virtually non-toxic). ,
Dosage accuracy is not inherently so demanding. Therefore, it must be concentrated to the extent that it does not pose a problem for practical use.
It does not necessarily have to be isolated as long as it is purified. As is clear from the above extraction operation, these compounds are relatively easily soluble in water;
It is conveniently administered dissolved in water or a mixture of water and a non-toxic, water-miscible organic solvent such as ethanol. Administration may be either oral or parenteral, although parenteral administration is generally preferred. In the case of intravenous injection, the dosage is usually in the range of 1 to 100 mg/day per adult, but of course it may deviate from this range depending on the symptoms.

The present invention will be specifically explained below with reference to Examples and Reference Examples.

Example 1 (A) Extraction and separation of antihypertensive components 50g of powdered young leaves of Hadakamori were heated to 90°C and heated to 2000 ml of hot water.
Extract by ml. After filtering the extract, concentrate to 200ml. Add 600 ml of ethanol, leave at 4°C overnight, and remove the precipitate by centrifugation.

The above operation was repeated 19 times, and the supernatant was collected, concentrated, and lyophilized. Dissolve 300 g of the freeze-dried product in a small amount of water and apply it to a Sephadex G-25 column (inner diameter 5.5 cm, length 130 cm). 1ml/
Elute with water at a flow rate of min, 0-1250 ml, 1250
~1600ml, 1600~1850ml, 1850~2150ml, 2150~
The fractions of 2,500 ml and 2,500 to 3,000 ml were designated as fractions A, B, C, D, E, and F, respectively.

The concentrate of Fraction D was dissolved in a small amount of hot water and allowed to cool. The precipitated crystals were collected and recrystallized from aqueous methanol to obtain compound (1) as pale yellow needle-like crystals.
Obtained 570mg. Melting point 226-230℃.

Analysis value (as C ₂₇ H ₃₀ O ₁₅・2H ₂ O): C,
51.43; H, 4.80. Actual value: C, 51.43; H, 5.44.

UVλ _nax (MeOH) nm: 269, 335. UVλ _nax
(MeOH + MeONa) nm: 250sh, 268, 388.
UVλ _nax (MeOH + AlCl ₃ ) nm: 277. 229, 350,
382. UVλ _nax (MeOH + AlCl ₃ + HCl) nm: 278,
298, 343, 378. UVλ _nax (MeOH+AcONa)
nm: 268, 350sh, 394. UVλ _nax (MeOH+
AcONa+ _H3BO3 ) nm: 269, ₃₃₆ .

IRν ^KCl _nax cm ^-1 : 3400 (OH), 1650 (C=O), 1614
,
1568 (C=C).

^1H -NMR (acetate) ( _CDCl3 ) δ: 1.72
(3H, s, C-acetoxyl at the 2-position of the glucosyl group), 2.0-2.2 (7 x 3H, s, other alkoxyls in the sugar ring), 2.33 (3H, s, C-4' acetoxyl), 2.48 (3H , s, C-5 acetoxyl)
5.04 (1H, d, J = 10.0Hz, H-1 of 0-glucosyl group), 5.65 (1H, dd, J = 10.0 and 9.5Hz,
0-glucosyl group H-2), 3.8 to 5.5 (12H, m,
other sugar ring protons), 6.57 (1H, s, H-3),
6.93 (1H, s, H-8), 7.24 (2H, d, J=9.3
Hz, H-3', 5'), 7.84 (2H, d, J=9.3Hz, H-
2′, 6′).

Acid hydrolysis of the present compound (1) gives only glucose as a sugar component, whereas oxidative decomposition gives approximately equimolar amounts of glucose and arabinose.

The above physicochemical properties and numerical values correspond to those of saponarin described in the literature. The chemical structure of this product was confirmed to be apigenin 6-C,7-O-diglucoside. ) The mother liquor D' after collecting compound (1) was fractionated by PPC using a solvent system of n-butanol:acetic acid:water (6:1:2) as a developing solution. Cut each paper into five bands, collect the bands with the same Rf value, and extract with 70% aqueous methanol. The fractions obtained here are D'-1, D'-2, D'-3, D'-4, in order from the larger Rf value to the smaller one.
Let it be D'-5.

Fraction D'-4, which has an Rf value of 0.19, is further purified by PPC as described above. The finally obtained fraction was freeze-dried to obtain 57 mg of compound (2) as a yellow amorphous powder.

UVλ _nax (MeOH) nm: 258, 271, 335.
UVλ _nax (MeOH+MeONa) nm: 268, 282sh,
333, 388. UVλ _nax (MeOH + AlCl ₃ ) nm: 277,
303, 345, 388. UVλ _nax (MeOH + AlCl ₃ + HCl)
nm: 278, 304, 343, 382. UVλ _nax (MeOH+
AcONa) nm: 267, 282, 298sh, 339, 392.
UVλ _nax (MeOH + AcONa + H ₃ BO ₃ ) nm: 268,
327sh, 343, 410sh.

IRν ^KCl _nax cm ^-1 : 3400 (OH), 1655 (C=O), 1617
,
1566 (C=C).

^1H -NMR (acetate) ( _CDCl3 ) δ: 1.75
(3H, s, acetoxyl at the C-2 position of the sugar ring), 1.83 (3H, s, acetoxyl at the C-2 position of the sugar ring), 2.0-2.2 (6 x 3H, s, other acetoxyls in the sugar ring) , 2.33, 2.47, 2.53 (3×
3H, s, acetoxyl in flavone skeleton)
4.84 (1H, d, J = 10.5Hz, H-1 of sugar ring), 5.18
(1H, d, J = 9.8Hz, H-1 of sugar ring), 5.15 (1H,
t, J = 9.8 Hz, sugar ring H-3), 5.29 (1H, t,
J = 9.5Hz, H-3 of sugar ring), 5.69 (1H, t, J =
10.3Hz, sugar ring H-2), 6.03 (1H, t, J = 10.0
Hz, sugar ring H-2), 6.59 (1H, s, H-3),
7.22 (2H, d, J = 9.5Hz, H-3', 5'), 8.17
(2H, d, J = 9.5Hz, H-2', 6').

Oxidative decomposition of the present compound (2) gave arabinose along with a small amount of glucose, but acid hydrolysis did not give any aldoses.

Fraction E was treated in the same manner as in the purification of compound (2) to obtain fractions E''-1, E''-2,
E''-3, E''-4 and E''-5 were obtained. From the fraction E''-4 having an Rf value of 0.25, 152 mg of compound (3) was obtained as a bright yellow crystalline solid. Recrystallization from aqueous ethanol results in needle-like crystals. Melting point 229-230
℃.

UVλ _nax (MeOH) nm: 287, 269, 348.
UVλ _nax (MeOH + MeONa) nm: 264, 404.
UVλ _nax (MeOH + AlCl ₃ ) nm: 27, 297, 332,
420. UVλ _nax (MeOH + AlCl ₃ + HCl) nm: 272,
292, 360, 393. UVλ _nax (MeOH+AcONa)
nm: 262, 409. UVλ _nax (MeOH+AcONa+
_H3BO3 ) nm: 263 _, 373, 432.

IRν ^KCl _nax cm ^-1 : 3400 (OH), 1650 (C=O), 1620
,
1570 (C=C).

^1H -NMR (acetate) ( _CDCl3 ) δ: 1.74
(3H, s, acetoxyl at the 2-position of the C-glucosyl group), 2.0-2.25 (7 x 3H, s, other acetoxyls in the sugar ring), 2.34, 2.35 (2 x 3H, s,
C-3' and C-4' acetoxyl), 2.50 (3H,
s, C-5 acetoxyl), 5.05 (1H, d, J=
10.3Hz, O-glucosyl group H-1), 5.64 (1H,
t, J = 10.3Hz, sugar ring H-2), 3.76-5.48
(12H, m, other sugar ring protons), 6.55 (1H,
s, H-3), 6.93 (1H, s, H-8), 7.36 (1H,
d, J = 8.5Hz, H-5'), 7.70 (2H, m, H-2',
6′).

Acid hydrolysis of the present compound (3) yields only glucose, whereas oxidative decomposition yields almost equimolar amounts of glucose and arabinose.

(B) Acetylation of isolated substance 5 mg of compound (2) or (3) was dissolved in 1 ml of anhydrous pyridine, and 0.5 ml of acetic anhydride was added. Leave overnight and then heat to 80℃ for 1 hour. After cooling to room temperature, it was poured into 10 ml of ice water and stirred for 1 hour. Extraction was performed three times with 5 ml of chloroform. The extracts were combined, washed three times with 5 ml of water and evaporated to dryness. This was subjected to TLC in benzene-ethyl acetate (3:7) to strip off the main deep purple zone and extracted with 20 ml of chloroform-methanol (10:1). Evaporation of the extract to dryness yields the acetate of the compound as a crystalline solid.

Reference Example 1 The mother liquor D′ (containing compound (2)) after separating compound (1) obtained in Example 1 above was freeze-dried,
The distilled water was prepared. The above solution was intravenously injected into 5-week-old SHR rats and normal rats (Wistar Kiyoto strain), and the subsequent changes in blood pressure over time were measured using the tail-pulse pick-up method. The results are shown in Figures 3A (SHR rats) and B (normal rats). In these figures,
The numbers in the box indicate the number of animals used, and the graph is created based on their average values. The dosage is indicated by the weight of the lyophilized product.
As is clear from the figure, the blood pressure drop reaches its maximum 30 minutes to 1 hour after administration. On average, the dosage
In intravenous injection of 200μg/100g (body weight), after administration
The blood pressure drop in 30 minutes was 53 mmHg. Note that the blood pressure reduction exhibited by 1 mg of the above-mentioned freeze-dried product is approximately equal to the blood pressure decrease exhibited by 32 μg of the purified product of compound (2).

When the purified product of compound (3) was tested for its hypotensive effect in the same manner as above, it was found that a dose of 1.0
When administered intravenously at mg/100g (body weight), the blood pressure drop 30 minutes after administration was 29 mmHg.

Note that the purified product of compound (1) did not substantially exhibit any blood pressure lowering effect.

[Brief explanation of drawings]

In the accompanying drawings, Figures 1 and 2 respectively show the ¹ H-NMR spectrum of acetate of compound (2) (apigenin 6,8-di-C-β--D-glucoside) obtained in the present invention and the compound.
(3) The ¹ H-NMR spectrum of acetate of (luteolin 6-C,7-O-diglucoside) is shown. FIG. 3 is a graph showing the relationship between time course and blood pressure drop after administration of a fraction containing compound (2) obtained in the present invention, and (A) is a graph showing the relationship between blood pressure reduction and the time course after administration of a fraction containing compound (2) obtained in the present invention. B) is the case when ordinary rats (Wistar/Kyoto strain) were used as experimental animals.

Claims (1)

[Claims] 1. An antihypertensive agent whose active ingredient is an aqueous ethanol-soluble component extracted from wheat leaves with water. 2 formula: [In the formula, Glc represents a glucose residue. ] An antihypertensive agent containing a compound represented by the following or a precursor that produces the compound in vivo as an active ingredient. 3 formula; [In the formula, Glc represents a glucose residue. ] An antihypertensive agent containing a compound represented by the following or a precursor that produces the compound in vivo as an active ingredient. 4. A method for collecting antihypertensive components, which comprises subjecting a water extract of barley leaves to a fractionation method to collect a fraction that has a hypotensive effect and contains aqueous ethanol-soluble components. 5 The antihypertensive component is apigenin 6,8-di-C-β-
The method for collecting D-glucoside according to item 4. 6 The antihypertensive component is luteolin 6-C-,7-O-
4. The method for collecting diglucoside according to item 4.