JP3131627B2 - Phenylpropanoid glycosides and uses thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a phenylpropanoid glycoside and its use, and more particularly, to a novel phenylpropanoid glycoside and a lipoxygenase activity inhibitor containing the glycoside as an active ingredient.

[0002]

2. Description of the Related Art In recent years, the number of patients suffering from asthma and allergic diseases has rapidly increased in Japan due to changes in living environment and eating habits, which has become a problem. In addition, the number of adult diseases related to the circulatory system such as thrombus and arteriosclerosis is increasing rapidly, and it is expected that the situation will become even more serious in an aging society in the future. It is known that metabolites of arachidonic acid are involved in these various diseases. Arachidonic acid is present in vivo as a fatty acid component of phospholipids of cell membranes in a living body, and is hydrolyzed by phospholipase and released from phospholipids when tissues or cells are subjected to some kind of stimulation. The metabolic pathway of arachidonic acid is called an arachidonic acid cascade. Representative examples thereof include a cyclooxygenase pathway that synthesizes a prostaglandin series and a thromboxane series, and a lipoxygenase pathway that synthesizes a leukotoluene series. Further, the lipoxygenase pathway is mainly divided into a 5-lipoxygenase pathway and a 12-lipoxygenase pathway depending on the type of lipoxygenase.

Cyclooxygenase acts on arachidonic acid released from phospholipids to produce prostaglandin G _2.
Is an enzyme that converts into prostaglandin H ₂ through
When this prostaglandin H ₂ is further metabolized, thromboxanes such as thromboxane A ₂ and other prostaglandins such as prostaglandin E ₂ and prostacyclin, which play a major role in the inflammatory reaction, are synthesized. Thromboxane A ₂ causes thrombosis by causing platelet aggregation and vasoconstriction, and prostaglandins are involved in fever and inflammation. Therefore, if there is a compound that can effectively inhibit the activity of cyclooxygenase, the effect of preventing and treating thrombus caused by thromboxane A ₂ can be expected, and the effect of preventing and treating fever and inflammation caused by prostaglandins can be improved. Can be expected.

[0004] 5-lipoxygenase converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (hereinafter referred to as 5-lipoxygenase).
It may be abbreviated as -HPETE. ). Various leukotrienes are biosynthesized using this compound as an intermediate. Among these leukotrienes, the leukotriene B ₄ is a mediator of inflammation that activates leukocytes, leukotrienes C ₄ and D ₄ are mediators of asthma causing sustained contraction the bronchi,
It is also known to be a slow-reacting substance of type I allergy anaphylaxis. Therefore, if there is a compound capable of effectively inhibiting the activity of 5-lipoxygenase, a preventive and therapeutic effect on allergic diseases, asthma, edema and various inflammations caused by overproduction of leukotrienes can be expected.

[0005] In addition, 12-lipoxygenase converts arachidonic acid to 12-hydroperoxyeicosatetraenoic acid (hereinafter sometimes abbreviated as 12-HPETE). This compound is rapidly reduced to form 12-hydroxyeicosatetraenoic acid (hereinafter referred to as 12-HETE).
May be abbreviated. ). It has been pointed out that the physiological action of metabolites of 12-lipoxygenase promotes platelet aggregation, adhesion and migration in vascular smooth muscle, and thus is involved in arteriosclerosis. In addition, activation of 12-lipoxygenase, leukocyte migration and agglutination are associated with allergic reactions, and furthermore, 12-HETE promotes adhesion and metastasis of certain cancer cells such as Lewis lung cancer to vascular endothelial cells. It is clear. Therefore, compounds that can effectively inhibit 12-lipoxygenase activity include thrombosis,
Prevention and treatment of arteriosclerosis, allergic diseases, etc., and prevention of cancer cell metastasis can be expected.

[0006]

Therefore, if there is a compound capable of effectively inhibiting the activities of cyclooxygenase and lipoxygenase, various compounds derived from metabolites such as prostaglandins, thromboxanes, and leukotrienes are available. In addition to being effective for the prevention and treatment of arachidonic acid metabolic disorders that cause diseases and the like, it is also expected to have the effect of suppressing metastasis of certain types of cancer cells. Therefore, development of a drug that has an action of sufficiently inhibiting the activities of cyclooxygenase and lipoxygenase and that has reduced side effects is desired.

[0007] Citrus fruits, which are consumed in large quantities in Japan, are not only eaten as they are, but also processed into cans, jams, fruit juices and the like. Squeezed residue such as pericarp and carrots generated during juice processing is 50% of the fresh weight of the fruit.
Has also reached. Although some of these squeezed slags are used as feed for livestock and fertilizers for crops, most of them are currently incinerated. Therefore, there is a demand for the development of a usage method that gives added value to them.

[0008] The present inventors have been studying to add a new added value to the juice of citrus as well as the juice of citrus fruits, and the inhibitory action against cyclooxygenase and 12-lipoxygenase is mainly contained in the components extracted from the peel. It was found that this was the case (Japanese Patent Application Laid-Open No. 8-245412). That is, the cyclooxygenase and lipoxygenase inhibitory effects of the pericarp extract were tested on 45 representative varieties of fruits in Japan, and the effective varieties, differences in the inhibitory activities among the varieties, etc. were clarified, and the substances having the inhibitory activities were identified. The manufacturing method was established. And it is strong 12-
It was determined that a component having a lipoxygenase inhibitory activity was contained, and that the active component was present particularly in albedo, flavedo, carrot, and fruit juice, but it was not possible to identify a compound having the activity. Thus, the present inventors have isolated and purified the compound having the above-mentioned activity, and have conducted repeated studies to determine its structure.
The has been completed.

[0009]

Means for Solving the Problems The present invention according to claim 1 is a phenylpropanoid glycoside represented by the following formula 1.

[0010]

Embedded image (Where R ₁ is

[0011]

Embedded image And R ₂ represents COCH ₃ or H. )

The present invention according to claim 2 comprises the phenylpropanoid glycoside according to claim 1 as an active ingredient.
It is a lipoxygenase activity inhibitor .

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The phenylpropanoid glycoside of the present invention represented by the above-mentioned formula 1 has two kinds of compounds I and II depending on the substituent R ₂ in the formula. For example, it is contained in all of the juice, squeezed residue and pericarp of Ponkan fruit. The method for isolating and purifying the present compound from Ponkan fruit is described below. In order to isolate and purify from the squeezed residue, water or a mixed solution of alcohols such as methanol and ethanol and dimethyl sulfoxide or acetone and dimethylformamide can be used as an extraction solvent. Since the present invention intends to use the above compound mainly as a food material or the like,
A solvent suitable for this purpose should be selected. for that reason,
Water and alcohols such as ethanol and methanol are preferred. The raw squeezed residue may be used as it is, but if it is degreased with hexane in advance and further unnecessary components are removed with chloroform, the subsequent purification operation is facilitated. When a dried product or a powdered product is used, extraction can be performed efficiently. The extraction can be carried out by adding a solvent to the squeezed residue and stirring the mixture overnight, or by using a Soxhlet extractor. It is preferable that the extract is freeze-dried or concentrated using an evaporator because the subsequent separation operation becomes easy.

Next, the concentrated extract was diluted 10 times or more with water, and added to an Amberlite XAD-2 column equilibrated with water, and the mixture was diluted with water, 20% methanol, 40% methanol, 60% methanol, and 80% methanol. , 100% methanol and 100% acetone. The two compounds I and II elute in the 60% methanol and 80% methanol fractions. These are collected and concentrated by an evaporator or the like, and then added to Sephadex LH-20 resin equilibrated with 100% methanol. The addition amount is preferably 1/20 or less of the resin volume. Then elute with 100% methanol. Compounds I and II elute between 1.1 and 1.5 times the resin volume. This is collected and concentrated by an evaporator or the like. HPLC uses an ODS resin column and a guard column as separation columns. The samples are separated by a two-part gradient method. As the eluent at this time, acetonitrile and water, acetonitrile and water each containing 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA), methanol and water each containing 0.05% TFA are used. be able to. Among them, 0.05% of T
Acetonitrile and water containing FA, and methanol and water containing 0.05% TFA provide good separation. However, considering the possibility of hydrolysis of the compound by TFA, it is better to use acetonitrile and water as eluents. preferable. The separated compounds are tested for the inhibition of 12-lipoxygenase in each fraction and, if necessary, purified again by HPLC.

When the compound of the present invention is isolated and purified from juice, the juice is centrifuged at 10,000 g for 30 minutes, and the supernatant is added to an Amberlite XAD-2 column.
Thereafter, it can be isolated and purified in the same manner as in the case of the above-mentioned juice extract. Further, the operation of isolating and purifying the above-mentioned compound, which is the phenylpropanoid glycoside of the present invention, from the peel may be basically performed in the same manner as in the case of extracting from squeezed scum, and the details thereof are described in Test Examples below. It was shown to.

Next, an assay system for measuring the arachidonic acid metabolizing enzyme inhibitory activity of the phenylpropanoid glycoside obtained as described above will be described. In the case of 5-lipoxygenase, a male Wistar-king rat (body weight 250-300 g) was intraperitoneally injected with a 5% glycogen solution at a volume of 20 ml / kg, and after 4 hours, polymorphonuclear from the abdominal cavity. Leukocytes are collected while suspended in 0.9% sodium chloride. A 9-fold volume of a 0.83% ammonium chloride solution is added to the collected cell suspension, followed by shaking at 37 ° C. for 10 minutes to lyse erythrocytes and the like. The cell suspension is centrifuged at 1200 rpm for 10 minutes to precipitate leukocytes, and the precipitate containing leukocytes is collected. In order to wash the precipitate, about 10 ml of 0.9% sodium chloride solution is added to suspend, and the suspension is centrifuged again at 1200 rpm for 10 minutes, and the supernatant is discarded. After repeating this washing operation two more times, 50 mM potassium phosphate (pH 7.
4) was added to a concentration of 7 × 10 ⁸ cells / ml, the cells were sonicated to crush the cells, and then centrifuged at 100,000 × g for 1 hour. The centrifuged supernatant thus obtained is used as an enzyme solution of 5-lipoxygenase. The separately prepared sample was added to the container together with the enzyme solution, and the composition of the reaction solution was 50 mM potassium phosphate (pH 7.4), 3 mM CaCl ₂ , 3 mM ATP.
(PH 7.4) and pre-incubated at 37 ° C. for 1 to 10 minutes, preferably 5 minutes.
^[14 C] Arachidonic acid (0.05 μCi, final concentration 4 μM) is added and incubated at 37 ° C. for 5 to 10 minutes, preferably 5 minutes.

Then, the reaction is stopped by adding 0.2 ml of 0.5N formic acid, and the metabolite of arachidonic acid is extracted with 3 ml of ethyl acetate. The extract is dried in a stream of nitrogen and dried in a small amount (4
(0 μl) in ethyl acetate, quantitatively spotted on a silica gel plastic sheet, and developed with diethyl ether-petroleum ether-acetic acid (50: 50: 1 v / v) at 4 ° C. The obtained radioactive metabolite is detected by an autoradiography or a radioisotope imaging scanner, and the enzyme inhibitory activity of 5-lipoxygenase is quantified from the radioactivity. According to this method,
Mainly two spots appear. That is, 5-hydroxy-5,8,10,14-eicosatetraenoic acid (5-H) produced by being metabolized by 5-lipoxygenase
Arachidonic acid remaining unmetabolized as a result of ETE) and enzyme inhibition. The enzyme inhibitory activity of 5-lipoxygenase is determined from the intensity of these radioactivity compared to the 5-HETE control.

Next, in the case of 12-lipoxygenase,
Blood was collected from normal Wistar-king rats (body weight 150 to 400 g), and ethylenediaminetetraacetate (EDTA) was added thereto as an anticoagulant (0.5 mM).
The blood is centrifuged at 1200 rpm for 10 minutes. The obtained supernatant (platelet-rich plasma) is further centrifuged at 3000 rpm for 10 minutes. Precipitated platelets were washed with 1 mM ED
25 mM Tris-HCl containing TA, 130 mM
After washing twice with a NaCl solution (pH 7.4), the washed platelets are suspended in the same solution containing 1 mM EDTA and subjected to ultrasonic treatment. This ultrasonically treated platelet is used as an enzyme solution for arachidonic acid metabolism. A separately prepared sample was added to the container along with the platelet homogenate (1-2 mg protein / ml), and preliminarily incubated at 37 ° C. for 1-10 minutes, preferably 5 minutes, to give [1- ¹⁴ C] arachidonic acid (0%). .05
Add μCi) and incubate at 37 ° C. for 5-10 minutes, preferably 5 minutes.

Then, the reaction is stopped by adding 0.2 ml of 0.5N formic acid, and the metabolite of arachidonic acid is extracted with 3 ml of ethyl acetate. The extract is dried in a stream of nitrogen and dried in a small amount (4
0 μl) of ethyl acetate, quantitatively spotted on a silica gel plastic sheet, and chloroform-methyl alcohol-acetic acid-water (90: 8: 1: 0.8 v / v).
To the developing solvent. The obtained radioactive metabolite is detected by an autoradiography or radioisotope imaging scanner, and the arachidonic acid metabolite inhibitory activity is quantified from the radioactivity. According to this method, mainly four spots appear. That is, thromboxane B ₂ produced by being metabolized by cyclooxygenase
(TXB ₂ ), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 12-hydroxy-5,8,10-heptadeca-triene produced by metabolism by 12-lipoxygenase Acid (12-
HHT) and arachidonic acid remaining unmetabolized as a result of enzyme inhibition. The enzyme inhibitory activities of cyclooxygenase and 12-lipoxygenase were TXB ₂ , 12-
Measurements are taken from the intensity of these radioactivity compared to the HETE control. However, 12-HHT is not quantified in this experiment due to lack of biological activity.

[0020] The phenylpropanoid glycosides of the present invention Li
A place to use as an active ingredient of oxygenase activity inhibitor
In this case , the amount of the glycoside is 0.01 μg / ml or more, preferably 0.1
By adding ２０20 μg / ml, 5-lipoxygenase and 12-lipoxygenase can be effectively inhibited. 0.01- for 5-lipoxygenase
1 μg / ml is 0.1 μg for 12-lipoxygenase.
1 to 10 μg / ml is appropriate.

[0021]

EXAMPLES The present invention will be described below in more detail with reference to examples and the like, but the present invention is not limited to these examples. Test Example 1 An extraction solvent used for extracting the phenylpropanoid glycoside of the present invention was examined. (1) Extraction of Phenylpropanoid Glycoside 3.0 g of powder obtained by pulverizing freeze-dried Ponkan peel was used as a sample, and 25 ml of water, ethanol, methanol, chloroform or hexane was added thereto as a solvent. This was placed in a 50 ml glass centrifuge tube and shaken at 25 ° C. (room temperature) for 12 hours. After that, 1 at 2000 rpm
After centrifugation for 0 minutes, the supernatant was collected. Further, 25 ml of the above solvent was added to the precipitate and centrifuged again to collect the supernatant. This operation was further repeated twice, and the obtained supernatants were combined to obtain 100 ml of an extract. Then, the extract was diluted 10-fold with distilled water, and adsorbed on 10 g of resin (trade name: Seppak Bag _C18 , manufactured by Waters) for reverse phase chromatography (ODS).
After washing this with a 10% aqueous solution of methyl alcohol,
Eluted with 1% methyl alcohol. The obtained alcohol fraction was dried under reduced pressure at 40 ° C. using a rotary evaporator to prepare extracts with the respective solvents. The inhibitory effect of these extracts on 12-lipoxygenase was tested by the following method.

(2) Test for 12-lipoxygenase inhibitory effect Normal Wistar-king rats (weight: 150-400 g)
Blood was collected from 0 animals, and 0.5 mM EDTA was added thereto as an anticoagulant, followed by centrifugation at 1200 rpm for 10 minutes. The obtained supernatant (platelet-rich plasma) was further added for 30 minutes.
Centrifuged at 00 rpm for 10 minutes. The precipitated platelets were washed with 25 mM Tris-HCl containing 1 mM EDTA,
After washing twice with a 130 mM NaCl solution (pH 7.4), the washed platelets were suspended in the same solution containing 1 mM EDTA and subjected to sonication, and the sonicated platelets were used as an enzyme solution for arachidonic acid metabolism. The sample prepared in the above (1) is added to a container together with the platelet homogenate (1-2 mg protein / ml), preliminarily incubated at 37 ° C. for 5 minutes, and [1- ¹⁴ C] arachidonic acid (0.05 μC
i) was added and incubated at 37 ° C. for 5 minutes.

Then, the reaction was stopped by adding 0.2 ml of 0.5N formic acid, and the metabolite of arachidonic acid was extracted with 3 ml of ethyl acetate. The extract is dried in a stream of nitrogen and 40 μl
Of ethyl acetate, spotted quantitatively on a silica gel plastic sheet, and developed with a developing solvent of chloroform-methyl alcohol-acetic acid-water (90: 8: 1: 0.8 v / v). The obtained radioactive metabolite was detected by an autoradiography or radioisotope imaging scanner, and the arachidonic acid metabolite inhibitory activity was quantified from the radioactivity. According to this method, there are mainly four spots, thromboxane B ₂ (TXB ₂ ), which is metabolized and formed by cyclooxygenase, 12-
12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 12-hydroxy-5,8,10 produced by metabolism by lipoxygenase
-Heptadeca-trienoic acid (12-HHT) and arachidonic acid which remained unmetabolized as a result of enzyme inhibition appeared. The enzyme inhibitory activities of cyclooxygenase and 12-lipoxygenase were compared with the control values of TXB ₂ and 12-HETE, respectively, and were measured from their radioactivity intensities. However, 12-HHT is not quantified because it has no biological activity. The results are shown in Table 1.

As is clear from the table, no inhibitory activity against 12-lipoxygenase was observed in the fractions extracted with chloroform and hexane. On the other hand, when water, ethanol and methanol were used as the extraction solvent, an inhibitory activity against the enzyme was observed, but among these solvents, ethanol and methanol were found to be particularly suitable.

[0025]

[Table 1] Table 1 重量 Solvent extract weight ¹⁾ Inhibitory activity ²⁾ Total activity ³⁾ (mg) (% / 10 μg / ml) (× 10 ⁴ ) {Water 33.2 45.99 1527 ethanol 46.4 47.84 2220 methanol 80.2 37.82 3033 chloroform 12.00 hexane 7.0 00} ─────────────────────────────── ¹⁾ Extracted from 3.0 g of dried peel ²⁾ Inhibitory activity ³ per 10 μg / ml ⁾ Inhibitory activity x weight (% mg)

Production Example 1 Extraction of Phenylpropanoid Glycoside from Ponkan Peel 300 g of Ponkan peel crushed after freeze-drying was used as a sample, and a fraction containing the glycoside was extracted using a Soxhlet extractor. First, after extraction with 1,000 ml of hexane overnight, the residue was air-dried. The residue after air-drying was extracted again with 1,000 ml of chloroform overnight, and the residue was air-dried. The residue was extracted overnight with 1,000 ml of ethanol, and the obtained extract was recovered and then extracted again with 1,000 ml of ethanol overnight. The extracts were combined. Next, the residue was air-dried, and extracted overnight with 1,000 ml of methanol.
The mixture was extracted again with 1,000 ml of methanol overnight, and the obtained extracts were combined. Each extract thus obtained was dried with a rotary evaporator, and the extract was weighed and tested for its inhibitory effect on 12-lipoxygenase in the same manner as in Test Example 1 (2).
The results are shown in Table 2.

[0027]

[Table 2] Table 2 ──────────────────────────────────── Total solvent weight inhibitory activity Activity ¹⁾ (g) (% / 10 μg / ml) (× 10 ⁴ ) ──────────────────────────────── {Hexane 1.4300 Chloroform 3.002 00 Ethanol 61.32 50.73 31.11 Methanol 47.81 36.71 17.55} ────────────────────── ¹⁾ Inhibitory activity × weight (% × g).

As a result, no inhibitory activity against 12-lipoxygenase was observed in the hexane and chloroform fractions. In addition, although the ethanol and methanol fractions exhibited inhibitory activity against the enzyme, a comparison of the two shows that the ethanol fraction had a higher extract weight and a higher inhibitory activity per 10 μg / ml. It became clear. Therefore, the following purification operation was proceeded using this ethanol fraction.

Production Example 2 Purification of Compound by Amberlite XAD-2 The ethanol fraction obtained in Production Example 1 was used as a sample, and further purified. Amberlite XAD equilibrated with water
-2 Ethanol extract 2 of Production Example 1 in 500 ml of resin
8 g was added, and elution was performed using 1 L each of water, 20% methanol, 40% methanol, 60% methanol, 80% methanol, 100% methanol, and 100% acetone in order. For each eluted fraction, 12
-The inhibitory effect on lipoxygenase was tested in the same manner as in Test Example 1 (2). The results are shown in Table 3.

[0030]

[Table 3] Table 3 溶 Solvent weight inhibitory activity (g) ( % / 10 μg / ml) ───────────────────────────────── water 23.8 0 20% methanol 0.381 0 40% methanol 0.451 18.84 60% methanol 0.568 62.94 80% methanol 0.247 56.11 100% methanol 0.699 15.68 100% acetone 0.087 0 ───────────────────────────

As is clear from the table, the inhibitory activity against 12-lipoxygenase was 10% from 20% methanol.
Although it was distributed in 0% methanol, particularly, 60% methanol and 80% methanol had high activity, and these were combined and concentrated by an evaporator. The yield was 815 mg.

Production Example 3 Purification of Compound by Sephadex LH-20 815 mg of the highly active fraction obtained in the above Production Example 2 was dissolved in 10 ml of methanol, and Sephadex LH-20 (Pharmacia) was equilibrated with 100% methanol. Made)
Added to 400 ml. The eluent used was 100% methanol. The eluate was fractionated into test tubes in 20 ml portions, concentrated and dried using a centrifugal evaporator, and then tested for 12-lipoxygenase inhibitory activity in the same manner as in Test Example 1 (2). The inhibitory activity of each fraction fractionated by Sephadex LH-20 is shown in FIG. As shown,
Inhibitory activity was observed between fractions 18 to 40, and particularly high activity was observed in fractions 22 to 30. Therefore, these fractions were combined, concentrated and dried to obtain a sample.

Production Example 4 Purification of Compound by HPLC 256 mg of highly active sample obtained in Production Example 3 above
Was purified by HPLC. HPLC was performed on an ODS column (20 m) with a guard column (20 mm × 50 mm).
mx 250 mm (manufactured by YMC) was used as a separation column, and the eluate was 0.05% TFA aqueous solution as solution A,
Using a 0.05% TFA-methanol solution as solution B,
A linear gradient was set such that the liquid became 40% to 80% in 60 minutes, and the flow rate was 5 ml / min. The sample was dissolved in a small amount of 50% methanol and injected in 100 μl portions. An ultraviolet light spectrophotometer was used as a detector, and the detection wavelength was 280 nm. Fractionation was performed for each elution peak, and each peak was concentrated with a centrifugal evaporator and dried.
The inhibitory activity of 12-lipoxygenase was tested on Test Example 1 (2).
It measured similarly to. Further, in the first HPLC, Compounds I and II eluted at the same time and had the same peak. Therefore, when HPLC was performed again, two kinds of glycosides could be separated. The first eluting compound is Compound I,
The next eluting compound was designated as compound II. The elution conditions at this time were isocratics in which one eluate was fixed at 50%, and the other conditions were the same as in the first HPLC. By this operation, two types of phenylpropanoid glycosides could be isolated. FIG. 2 shows the elution chromatogram by the first HPLC, and FIG. 3 shows the elution chromatogram by the second HPLC. Table 4 shows the weight of the fraction having an inhibitory activity and the inhibitory activity per 10 μg / ml in each treatment step. As is clear from the table, both Compound I and Compound II have inhibitory activity against 12-lipoxygenase.

[0034]

[Table 4]

Production Example 5 Determination of Structure of Phenylpropanoid Glycoside The structure analysis of Compound I and Compound II isolated by the above Production Example by nuclear magnetic resonance (NMR) was attempted. Methanol and hydrochloric acid were added to 1 mg of each of Compound I and Compound II to prepare a sample solution. On this occasion,
The 1st-position caffeic acid ester and the 2nd-position ferulic acid ester in the structure are hydrolyzed with a hydrochloric acid-methanol solution having a hydrochloric acid concentration of 9%, and only the 2-position ferulic acid is hydrolyzed with 3% LiOH or NaOH. did. Further, arabinose moiety identified by GC-MS, caffeic acid, ferulic acid, ¹ H each for arabinose partial hydrolyzate caffeic acid is ester bonded ^{NMR, 13 C, H-Hcosy} , HMQ
Each spectrum data of C and HBMC was measured. Table 5 shows the ¹ H, ¹³ C NMR spectrum data of Compound I. As is clear from the table, the compound I was a compound in which caffeic acid was ester-bonded to the 1-position of arabinose and ferulic acid was bonded to the 2-position, and the 4-position was acetylated. Compound II had an ester bond of caffeic acid at the 1-position and ferulic acid at the 2-position of arabinose.

[0036]

[Table 5]

Example 1 The inhibitory activity against lipoxygenase of compound I and compound II whose structures were clarified by Production Example 5 was measured. The inhibitory effects on 5-lipoxygenase and 12-lipoxygenase when the concentrations of compound I and compound II were changed are shown in FIGS. 4 and 5, respectively. In the figure, ○ indicates compound I, and ● indicates compound II. Also,
The concentration that inhibits the activity of 5-lipoxygenase and 12-lipoxygenase by 50% was also measured. The results are shown in Table 6.

The test for the inhibitory effect on 5-lipoxygenase was carried out by the following method. 5 male Wistar-king rats (weight: 250-300 g) intraperitoneally
% Glycogen solution was injected at a volume of 20 ml / kg body weight, and after 4 hours, 0.9% of polymorphonuclear leukocytes were injected from the peritoneal cavity.
The sample was collected while suspended in% sodium chloride. After adding 18 ml of a 9-fold volume 0.83% ammonium chloride solution to 2 ml of the collected cell suspension, the mixture was shaken at 37 ° C. for 10 minutes,
Red blood cells and the like were lysed. This cell suspension was 1200 rpm
and centrifuged at 10 m for 10 minutes to precipitate leukocytes. About 10% of 0.9% sodium chloride solution was added to wash the collected precipitate.
After adding and suspending ml, the mixture was centrifuged again at 1200 rpm for 10 minutes, and the supernatant was discarded. After repeating this washing operation two more times, 50 mM potassium phosphate (pH 7.4) was added to the precipitated cells at a concentration of 7 × 10 ⁸ cells / ml.
After crushing the cells by sonication, 100000 × g
For 1 hour. The obtained supernatant was used as an enzyme solution of 5-lipoxygenase. 20 μl of the sample obtained in Production Example 5 was added to a container together with this enzyme solution, and the composition of the reaction solution was 50 mM.
Potassium phosphate (pH 7.4), 3 mM CaCl ₂ , 2
mM ATP (pH 7.4).
After pre-incubation for 5 minutes at 37 ° C., [1- ¹⁴ C] arachidonic acid (0.05 μCi, final concentration 4 μM) was added to the mixture.
Incubated at 5 ° C for 5 minutes.

Then, the reaction was stopped by adding 0.2 ml of 0.5N formic acid, and the metabolite of arachidonic acid was extracted with 3 ml of ethyl acetate. The extract is dried in a stream of nitrogen and 40 μl
Of ethyl acetate, spotted quantitatively on a silica gel plastic sheet, and developed at 4 ° C. with a developing solvent of diethyl ether-petroleum ether-acetic acid (50: 50: 1 v / v). The obtained radioactive metabolite was detected by an autoradiography or a radioisotope imaging scanner, and the enzyme inhibitory activity of 5-lipoxygenase was quantified from the radioactivity. By this method, 2
Two spots appeared. That is, 5-hydroxy-5,8,5 produced by being metabolized by 5-lipoxygenase
It is 10,14-eicosatetraenoic acid (5-HETE) and arachidonic acid remaining without being metabolized as a result of enzyme inhibition. The enzyme inhibitory activity of 5-lipoxygenase is 5-HE
It was determined from the intensity of these radioactivity compared to the TE control. The test of the inhibitory effect on 12-lipoxygenase was performed in the same manner as in Test Example 1 (2).

As a result, Compound I inhibited 72.75% of 5-lipoxygenase at a concentration of 0.1 μg / ml,
It was found to be completely inhibited at a concentration of 1.0 μg / ml. The 50% inhibitory concentration of compound I on 5-lipoxygenase was 0.024 μg / ml (0.0452 μg / ml).
M). On the other hand, it inhibits 12-lipoxygenase at a concentration of 10 μg / ml at 72.85% and at 50%
The inhibitory concentration was 0.7 μg / ml (1.32 μM). Compound II was 0.1 μg to 5-lipoxygenase.
/ Ml concentration and 19.48% inhibition, and the 50% inhibitory concentration of compound II against 5-lipoxygenase is 0.52 μg / m 2.
1 (1.1 μM). On the other hand, 12-lipoxygenase was inhibited by 66.44% at a concentration of 10 µg / ml, and the 50% inhibitory concentration was 2.0 µg / ml (4.1 µM).

[0041]

Table 6 Table 6 Compound I (μM) Compound II (ΜM) ５− 5-lipoxygenase 0.0452 1.32 12- Lipoxygenase 1.1 4.1────────────────────────────────────

[0042]

According to the present invention, a novel phenylpropanoid glycoside having an effective inhibitory action on 5- and 12-lipoxygenase can be obtained from citrus fruits such as Ponkan fruit. Furthermore, this compound does not show harmful side effects on the human body, and is expected to be used as a preventive or therapeutic agent for arachidonic acid metabolic disorder . In addition, the novel compound is obtained from waste such as squeezed residue of citrus fruit, which has hardly been used so far, so that the present invention is highly practical from the viewpoint of effective use of resources.

[Brief description of the drawings]

FIG. 1 shows the fractionation of phenylpropanoid glycoside-containing components by Sephadex LH-20 column and the lipoxygenase inhibitory activity of the fractions.

FIG. 2 shows a first HPLC elution chromatogram of a fraction having an inhibitory activity.

FIG. 3 shows a second HPLC elution chromatogram of a fraction having an inhibitory activity.

FIG. 4 shows the inhibitory effects of compounds I and II on 5-lipoxygenase.

FIG. 5 shows the inhibitory effects of compounds I and II on 12-lipoxygenase.

Continuation of the front page (56) References JP-A-8-245412 (JP, A) JP-A-4-1344092 (JP, A) Agric. Food Chem. , Feb. 1997, Vol. 45, No. 2, p. 373-377 of Chromatography a, 1995, Vol. 718, No. 1, p. 89-97 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07H 1/00-23/00 A23L 1/05-1/09 A61K 31/33-33/44 BIOSIS (DIALOG) CA (STN ) REGISTRY (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A phenylpropanoid glycoside represented by the following formula 1. Embedded image (Wherein R ₁ is And R ₂ represents COCH ₃ or H. ) 2. A lipoxygenase activity inhibitor comprising the phenylpropanoid glycoside according to claim 1 as an active ingredient.