WO2018151334A1 - Xanthine oxidase inhibitor and method for producing same - Google Patents

Abstract

Provided are a xanthine oxidase inhibitor which is safe and has an excellent effect, and a method for producing the xanthine oxidase inhibitor. A novel xanthine oxidase inhibitor can be produced by extraction from the shoots of camomile (Matricaria recutita). By using camomile, a familiar plant that is known to be safe and the flowers of which are used an herb, it is possible to produce a xanthine oxidase inhibitor which is safe and has an excellent effect. The method for producing a xanthine oxidase inhibitor includes subjecting the shoots of camomile to extraction using water, a hydrophilic organic solvent or a mixed solvent thereof to produce an extract, and separating an aqueous layer from the extracted extract. The separated aqueous layer is eluted with a solvent mixture composed of water and the hydrophilic organic solvent. The main component in an elution fraction is concentrated and crystallized to produce the xanthine oxidase inhibitor.

Description

Xanthine oxidase inhibitor and method for producing the same

The present invention relates to a novel xanthine oxidase inhibitor, which can efficiently extract its active ingredients from familiar plants that are well known to be safe, The present invention relates to a production method, and further relates to a utilization method for pharmaceuticals, foods and the like using the production method.

Metabolism of purine, which is a nucleobase in vivo, is xanthine oxidase (xanthine oxidase and xanthine dehydrogenase are enzymes that reversibly convert each other, and they are collectively referred to as xanthine oxidoreductase. According to conventional practice, it is known that the enzyme is finally oxidized to uric acid by the action of xanthine oxidase as an oxidation function of uric acid metabolism of the enzyme. Uric acid itself functions as an antioxidant when it is present in the blood, and effectively acts to remove active oxygen. However, when it is taken into cells, it generates active oxygen inside the cells. It is known that it causes an inflammatory reaction. Therefore, the uric acid concentration in the living body needs to be maintained within a preferable range.

However, if the uric acid level in the blood continuously exceeds 7 mg / dL due to abnormal metabolic pathway or abnormal excretion of uric acid from the kidney, it is diagnosed as hyperuricemia and manifestation of various pathological conditions It becomes the cause that leads to. The solubility of uric acid greatly depends on pH.For example, the solubility decreases in a weakly acidic state, and in the process of gout development, urate crystals are precipitated in joint fluid, and this is phagocytosed by macrophages and other innate immune mechanisms. It is known to provoke.
In macrophages, the NLRP3 inflammasome is activated to produce inflammatory cytokines such as IL-1β, which stimulates peripheral synovial cells to produce chemokines and migrate to them. It is known that neutrophils develop gout pathology by damaging joint tissue.
Furthermore, excessive production of uric acid and a decrease in excretion are closely correlated with the causes of renal failure, and there is also a correlation between heart failure and hyperuricemia. It is clear that high uric acid levels in blood are closely associated with the development of metabolic syndrome with various symptoms, and maintaining blood uric acid levels in a moderate range is extremely important for maintaining health Has been pointed out.

The blood uric acid level is controlled by the xanthine oxidase production of uric acid and the reuptake of uric acid via a transporter such as URAT1 within a concentration range preferable for healthy bodies. Therefore, for hyperuricemia, both the use of a xanthine oxidase inhibitor that suppresses the production of uric acid and the promotion of uric acid excretion by a URAT1 inhibitor are basically available. At present, xanthine oxidase inhibitors such as allopurinol and febuxostat are mainly used for the treatment of hyperuricemia, and benzbromarone and the like are known as URAT1 inhibitors, but the latter increases uric acid production. May cause uric acid stones and should be used with caution.
Xanthine oxidase is remarkably expressed in the liver and small intestine in the body, but is also known to be highly expressed in adipose tissue, blood vessels and the like. Uric acid has an antioxidant effect that deactivates active oxygen, and has a function to prevent cell and tissue damage due to active oxygen generated in various parts of the body, but when the activity of xanthine oxidase is enhanced, for example, Various functions in the body can be maintained normally by moderately inhibiting the action of xanthine oxidase, such as attenuating the action of nitric oxide that relaxes vascular smooth muscle and inducing hypertension.

A variety of xanthine oxidase inhibitors are known to inhibit xanthine oxidase activity and maintain blood uric acid levels at normal levels. Allopurinol is the first choice especially for the treatment of hyperuricemia Although it dominates the market as a drug, it interferes with the action of enzymes related to nucleic acid metabolism other than xanthine oxidase, causing various side effects, and the burden on renal function is often a problem. Is a problem. Febuxostat is mentioned as a xanthine oxidase inhibitor with another skeleton, and it is expected to be widely applied by reducing the burden on renal function. There is a need for xanthine oxidase inhibitors that can be used with greater peace of mind and safety.

As mentioned above, xanthine oxidase generates reactive oxygen species in the process of oxidizing hypoxanthine to xanthine in the body and further oxidizing xanthine to uric acid. Therefore, when xanthine oxidase activity is enhanced, the concentration of uric acid only increases in the body. In addition, it becomes a factor causing various obstacles due to the influence of the generated active oxygen. Therefore, various antioxidant substances including polyphenols such as catechins are preferably used in the health supplement field such as supplements for the purpose of deactivating active oxygen. However, as such effects, problems such as low bioavailability and difficulty in introducing them into the systemic circulatory system to an effective concentration to show pharmacological action have been pointed out.
In this regard, since the generation of active oxygen can be reduced by inhibiting the activity of xanthine oxidase, which is greatly involved in the production of active oxygen in the body, the use of xanthine oxidase inhibitor not only reduces the uric acid level but also various Since it also has the effect of reducing the generation of active oxygen, which also causes organ damage, it is also expected to be very effective in preventing the development of metabolic syndrome.

It has been known for a long time to contain components exhibiting favorable pharmacological activity among herbs and various medicinal plants, and has various xanthine oxidase inhibitory effects and is useful for preventing gout. Various plant resources have been reported (see, for example, Non-Patent Documents 1 and 2). Non-Patent Document 1 reports that xanthine oxidase inhibitory activity is observed in the phenol component contained in licorice. Non-Patent Document 2 reports that three types of rosemary, clove, and sage contain a phenolic component that exhibits xanthine oxidase inhibitory activity.
However, in any case, the ratio of the inhibitory component contained in the plant is small, and the inhibitory activity is insufficient unless the active ingredient is extracted and concentrated using a large amount of raw materials, and it is extremely difficult to use this practically. Met.

In addition to its use as a pharmaceutical as a xanthine oxidase inhibitor, it can be used to prevent daily hyperuricemia and prevent metabolic syndrome related to it by using daily supplements, health foods, health drinks, etc. Be expected. In order to use for this purpose, in addition to the effect as a xanthine oxidase inhibitor, there is little burden on the accumulation and metabolism in the body and the liver function, kidney function, etc. due to daily intake, There is a need for materials that are free of various safety concerns, such as lack of drug interactions. Herbs and various medicinal plants contain various physiologically active ingredients in addition to active ingredients, so it is important to be able to extract highly safe ingredients including these effects efficiently and with high purity. In addition, even if other components are mixed, it is preferable that the component has no problem with respect to safety to the living body.

Patent Document 1 discloses a xanthine oxidase inhibitor containing a plant or a propolis such as pearl millet, cinnamon, cedron, and grape. Patent Document 2 discloses a xanthine oxidase inhibitor extracted from wolfberry, broom, lemon balm, rosemary, spearmint, peppermint, and the like. Patent Document 3 discloses a xanthine oxidase inhibitor, which is an extract from kahakuzansho, koryang, cumin, and rose. Patent Document 4 further discloses a xanthine oxidase inhibitor extracted from pimenta, marjoram, guava, and the like. It is shown. As a method for extracting an active ingredient that acts as a target xanthine oxidase inhibitor from these various plants, extraction is performed at a temperature around room temperature using an aqueous solution containing an organic solvent such as hot water or alcohols. At that time, there is a problem that the target product or other components contained in the process are extracted during the extraction process to be denatured, the xanthine oxidase inhibitory activity is impaired, or the compound is denatured into an undesirable side effect. Therefore, a production method that does not affect inhibitory activity and can be easily extracted under simple conditions without denaturation, and a xanthine oxidase inhibitor obtained thereby, which satisfies both the effect and safety, and the production thereof There is a need for a method.

As described above, various xanthine oxidase inhibitors extracted from a variety of conventionally known plant resources are actually obtained as a mixture of many kinds of compounds. It is often the case that knowledge such as effectiveness and safety is not obtained.
For example, Patent Document 5 shows the results of high-performance liquid chromatography (HPLC) analysis of various active ingredients that act as xanthine oxidase inhibitors extracted from black turmeric. Therefore, it is not easy to verify the effectiveness, side effects, and safety of each of these components, and there is a problem in actually using them.

An example is also shown in which an active ingredient that acts as a xanthine oxidase inhibitor is isolated from a plant resource and the effect thereof is shown. For example, Patent Document 6 shows that quercetin glycosides and Patent Document 7 show that isoquercetin glycosides have high activities comparable to allopurinol as xanthine oxidase inhibitors, respectively. Such quercetin glycosides are a kind of flavonoids abundantly contained in vegetables and fruits, and it has been reported that the average intake of quercetin by Japanese is about 10 to 35 mg per day regardless of gender age. Therefore, since this level of quercetin glycoside has already been ingested on a daily basis, it is not expected that the effect can be exerted even if a similar amount of the same compound is ingested again. Furthermore, overdose is dangerous because quercetin has been reported to be lethal to mice at 160 mg. In addition, it needs to be used with care because it interacts with certain antibiotics and diminishes its action.
Therefore, as a xanthine oxidase inhibitor, in addition to a method of using as a pharmaceutical, a method of using that can be enjoyed through health foods, health drinks and supplements that can be taken on a daily basis is preferable. For that purpose, a xanthine oxidase inhibitor that can be easily and inexpensively extracted from familiar plants well known to be safe is preferable.

From the conventional technologies and their problems as described above, it is possible to efficiently extract active ingredients from familiar plants that are well known to be safe at present. There is a demand for an oxidase inhibitor and a method for producing the same, and there is also a need for a method of using the same for pharmaceuticals, foods, and the like.

JP 2002-121145 A JP 2003-252776 A JP 2010-37334 A JP 2010-37335 A JP 2011-236133 A JP 2002-145875 A JP 2003-171283 A

An object of the present invention is to provide a safe and effective xanthine oxidase inhibitor and a method for producing the same.

In order to solve the above problems, the xanthine oxidase inhibitor of the present invention is produced, for example, by extracting from the above-ground part of chamomile (Matricaria recutita). A safe and highly effective xanthine oxidase inhibitor can be obtained by using chamomile of a familiar plant that is known to be safe and uses flower parts and whole above-ground plants including them as herbs. Conventionally, chamomile flower parts have been widely used as herbs, but the current situation is that stems and leaves are hardly used. The present inventors have obtained knowledge that, in addition to the flower part, an active ingredient that acts effectively as a xanthine oxidase inhibitor is contained in the stem part and the leaf part, and further, the active ingredient in the future They have found a production method that efficiently extracts the.

In the chamomile line, an active ingredient that effectively acts as a xanthine oxidase inhibitor can be efficiently extracted from German chamomile (Matricaria chamomilla L.). Moreover, the active ingredient which has the xanthine oxidase inhibitory activity extracted from the above-ground part of a Roman chamomile (Chamaemelum nobile) may be contained in the xanthine oxidase inhibitor derived from a German chamomile. An active ingredient derived from German chamomile and an active ingredient derived from Roman chamomile have common medicinal effects and can be used as xanthine oxidase inhibitors.
Here, it is preferable to extract from the stem part and the leaf part excluding the flower part of chamomile. Traditionally, chamomile flower parts are widely used as herbs, but chamomile stems and leaves are rarely used, but chamomile stems and leaves act effectively as xanthine oxidase inhibitors. The compound to be included is contained in a high concentration.

The xanthine oxidase inhibitor of the present invention comprises (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid ((E) -2-β-D-glucopyranosyloxy) represented by the following chemical formula (1): -3-methoxycinnamic acid) as an active ingredient. Here, the compound of the chemical formula (1) may be artificially synthesized, but it is preferably derived from the above-mentioned extract of the above-mentioned chamomile (Matricaria recutita) from the viewpoint of ensuring safety.

The active ingredient of the above chemical formula (1) represents an E-form in which both are in the trans position with respect to the relative arrangement of the carboxyl group and the phenyl group bonded to the double bond of the cinnamic acid skeleton, and both are in the cis position. Differentiated from the body. The effect of the present invention as a xanthine oxidase inhibitor is remarkably exhibited in the E form, whereas the effect of the Z form is much inferior to that of the E form. The xanthine oxidase inhibitor of the present invention is a case where at least the E-form represented by the chemical formula (1) is contained, and may further contain a Z-form together with the E-form.

The pharmaceutical and food of the present invention contain the above xanthine oxidase inhibitor of the present invention.

Next, the manufacturing method of the xanthine oxidase inhibitor of this invention is demonstrated.
The method for producing a xanthine oxidase inhibitor of the present invention comprises a step of extracting from the above-ground part of chamomile (Matricaria recutita).
Specifically, the method for producing a xanthine oxidase inhibitor of the present invention comprises the following steps 1) to 3).
1) A step of extracting the above-ground portion of chamomile (Matricaria recutita) with water, a hydrophilic organic solvent or a mixed solvent thereof.
2) A step of separating an aqueous layer from the extracted extract and eluting with a mixed solvent of water and a hydrophilic organic solvent.
3) A step of concentrating and crystallizing the eluted fraction.

In the extraction step of 1) above, it is preferable to extract from the above-ground part of chamomile after removing the essential oil component contained in chamomile, and it is also preferable to use German chamomilla (Matricaria chamomilla L.) as the chamomile. You may mix the active ingredient which extracted the above-ground part of Roman chamomile (Chamaemelum nobile) with the mixed solvent of water and a hydrophilic organic solvent with the xanthine oxidase inhibitor derived from a German chamomile.
As the hydrophilic organic solvent, lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol and isobutanol are preferable, and methanol or ethanol and a mixed solvent with water containing these are particularly preferable.

In addition, the separated aqueous layer of 2) is subjected to gel separation, and after elution with water, it is eluted with a mixed solvent of water-alcohol-organic solvent. The obtained eluate may be separated and purified by a gel filtration carrier.
The above extraction is performed under mild conditions of 10 to 40 ° C. This is because when extraction is performed at a temperature below 10 ° C., it takes a very long time to extract the components, and a sufficient yield cannot be obtained. On the other hand, when extraction is performed at a temperature exceeding 40 ° C., This is because xanthine oxidase inhibitory activity is lost due to heat denaturation.
In addition, in the method for producing a xanthine oxidase inhibitor of the present invention, (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid ((E) -2- (2)) represented by the above chemical formula (1) is used. A xanthine oxidase inhibitor containing β-D-glucopyranosyloxy-3-methoxycinnamic acid) as an active ingredient is obtained.

According to the present invention, a safe and effective xanthine oxidase inhibitor can be obtained by extracting the above-ground parts of familiar chamomile that are well known to be safe, and to pharmaceuticals and foods using the same. There is an effect that can be used.

Flow scheme for extracting target components from the above-ground part of chamomile Production flowchart of xanthine oxidase inhibitor Positive ion mode mass spectrometry chart of sodium salt of compound represented by chemical formula (1) Negative ion mode mass spectrometry chart of the compound represented by chemical formula (1) ¹ H-NMR chart of the compound represented by the chemical formula (1) ¹³ C-NMR chart of the compound represented by the chemical formula (1) FT-IR chart of compound represented by chemical formula (1) ¹ H-NMR spectrum chart of extracted component CPPG HPLC analysis chart of samples collected from mice before oral administration of CPPG HPLC analysis chart of sample collected from mice after oral administration of CPPG The graph which shows the time change of the blood concentration of CA which moved into the blood after oral administration

The present invention provides a familiar plant with chamomile (Matricaria recutita), in addition to the flower part normally used as a herb, the stem part and the leaf part, which have been rarely used so far, are newly added. Was found to contain a high concentration of compounds that act effectively as xanthine oxidase inhibitors. The chamomile that can be used in the present invention is so-called German chamomile (Matricaria chamomilla L .; Japanese name chamomile), but similar components are also found in the above-ground parts of roman chamomile (Chamaemelum nobile) of different strains. It is known to be included, and since its medicinal properties are common, it can also be used in combination with German chamomile.

Chamomile is an annual herb belonging to the family Asteraceae native to Southern and Eastern Europe. Its distribution is spread all over the world and is widely distributed in Europe, Asia, North Africa, South America, North America, Australia and New Zealand. Hungary is the main production area, and flower parts are exported to Germany, where essential oils and other components are extracted and distributed. The optimum temperature for growth is preferably around 10 to 20 ° C., and it is known that growth is possible even at lower temperatures. As soil for growth, it is possible to grow even in soil with relatively little nutrient, and it is possible to grow well even in alkaline soil, so that their cultivation is relatively easy. Chamomile has long been known to contain compounds that exhibit various pharmacological effects on the flower parts of the above-ground parts. Typical components include (1) essential oil components, (2) phenolic components, and (3) coumarins.

The essential oil component of (1) includes azulenes such as α-bisabolol, cis-spiroether, chamazulene, their oxides, and various volatile components, which can be used as essential oils. Representative. The scent is known to exhibit mental depression and sedation, and is used in aromatherapy and cosmetics. Furthermore, it is used as an anti-inflammatory agent, disinfectant, disinfectant, digestive system diseases and inflammation, or It is used for anti-inflammatory action against various allergic symptoms. Chamomile tea, in which a dried product of the chamomile flower part is immersed in hot water to extract the essential oil component, and an oil component obtained by concentrating the essential oil component by a method such as steam distillation have a sedative effect and an anti-inflammatory effect. The effect which relieves the symptom of the pain originating in the etc. has been utilized. Such essential oil components are mainly contained in the flower part, and are produced from the whole above-ground part or only the flower part using a method such as steam distillation. When the xanthine oxidase inhibitor of the present invention is obtained by extraction from chamomile, when such an essential oil component is contained, the compound of the above formula (1), which is an active ingredient as a xanthine oxidase inhibitor, is extracted from the extract. When obtained, since it may be difficult to separate the extract from the extract and the yield may be reduced, the xanthine oxidase inhibitor of the present invention preferably contains no essential oil component.

(2) Examples of phenolic components include apigenin, quercetin, patuletin, luteolin, and their glycosides, and their pharmacological effects include relief of flatulence, anxiety disorder, insomnia, etc. The effect is mentioned. Among these phenolic components, particularly quercetin is known to have a xanthine oxidase inhibitory action, but the xanthine oxidase inhibitor of the present invention does not contain these phenolic components.

(3) As coumarins, herniarin and umbelliferone are known as typical components. These coumarin compounds are converted to cinnamic acid by the action of the enzyme PAL (phenylalanine ammonia lyase) in the metabolic pathways of plants, and further oxidized to p-coumaric acid by the action of oxidase, and then as an intermediate. It is known that it is cyclized via 2-glucopyranosyloxy-p-coumaric acid and converted to umbelliferone which is a coumarin compound. It is also known that hernialin is biosynthesized from p-coumaric acid via 2-glucopyranosyloxy-4-methoxycinnamic acid (GMCA). On the other hand, the xanthine oxidase inhibitor of the present invention does not contain these coumarin compounds.
In particular, it has been reported that GMCA is produced in the flower part and leaf part of chamomile, and hernialin is produced from GMCA under the influence of various stresses (Repcak M et al, Journal of Plant Physiology, Volume 158, Issue 8). , 2001, Pages 1085-1087). Although these coumarins have been conventionally used as a herbal component of chamomile, they do not have an action as a xanthine oxidase inhibitor as in the present invention. Therefore, it is preferable to suppress the formation of coumarins in chamomile and increase the content of the active ingredient of chemical formula (1).

Essential oil extracted from the flower part of chamomile is the most used ingredient at present, but research on various ingredients contained in these parts has also been reported for the above-ground part and root other than the flower part. It is known that essential oil components also exist in the above-ground parts and roots other than the flower parts, but unlike the ingredients contained in the flower parts, these have been extracted and used as ingredients. It was never discarded, and was partly disposed of except for being used as a bath salt. Moreover, it has been analytically known that coumarins and precursors thereof, such as GMCA, exist in the leaf part in addition to the flower part, but there exists an active ingredient having xanthine oxidase inhibitory action in these. Was not known at all.

The inventors of the present invention have conducted research on the useful components contained in a wide variety of resource plants and their actions for many years, and the chamomile above-ground part that has not been studied in detail in the course of the research, In particular, in addition to the flower part, the ground stem part and leaf part were found to contain extremely high concentrations of xanthine oxidase inhibitory activity, and as a method for producing xanthine oxidase inhibitor, the extraction and purification method of the ingredient was examined. Furthermore, as a result of detailed examination of its xanthine oxidase inhibitory activity, the present invention has been achieved.

In addition to the conventionally used chamomile flower part, using the above-ground stem part and leaf part that have not been used conventionally, the ingredient extracted from this is the ingredient having the xanthine oxidase inhibitory effect of the present invention. They found a manufacturing method and its usage for effective use. As shown in the examples to be described later, in addition to the chamomile flower part, the stem part and the leaf part, which are the above-ground part, are used as raw materials, and an extraction medium is added to this to extract under mild conditions with a high yield. It was found that the obtained extract component exhibits a remarkable xanthine oxidase inhibitory activity. Further, 2-glucopyranosyloxy-4-methoxycinnamic acid ((E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid) having the structure represented by the chemical formula (1) is highly concentrated in the extracted component. It was found that this has a remarkable effect as a xanthine oxidase inhibitor.

As described above, the compound represented by the chemical formula (1) represents an E-form in which both the carboxyl group and phenyl group bonded to the double bond of the cinnamic acid skeleton are in the trans position, and both are in the cis position. It is distinguished from a certain Z body. The effect of the present invention as a xanthine oxidase inhibitor is remarkably exhibited in the E form, whereas the effect of the Z form is much inferior to that of the E form. The xanthine oxidase inhibitor of the present invention is a case where at least the E-form represented by the chemical formula (1) is contained, and may further contain a Z-form together with the E-form.

Cinnamic acid and its derivatives are known to cause isomerization between E-form and Z-form by the action of light irradiation or catalyst. Thermodynamically, E-form is more stable than Z-form, but in various plants such as chamomile, it is isomerized from E-form to Z-form by exposure to sunlight, which is further cyclized to hernia as a coumarin derivative It is known to produce phosphorus (7-methoxy coumarin) (“The variability of (Z)-and (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acids and apigenin glucosides in diploid and tetraploid Chamomilla recutita ”, Food Chemistry, Volume 111, Issue 3, 1 December 2008, Pages 755-757). Along with the compound of the above chemical formula (1), a Z isomer which is a geometric isomer thereof may be contained. As a preferred embodiment, the E-form which is the compound of the chemical formula (1) is contained at a ratio of at least 1% by mass, and even if the Z-form and other types of components are contained, It is preferable that the ratio of the E isomer in the sum of is at least 1% by mass or more. Further, it is known that the compound of the chemical formula (1) is changed to a Z form by light irradiation, and when the compound of the chemical formula (1) is used or stored in a state exposed to light, the Z form is gradually increased. Is included in this. As shown in the examples described later, the Z form has no effect as a xanthine oxidase inhibitor or is extremely weak, but there is no adverse effect when used together with the compound of the chemical formula (1), but both are included. Therefore, an equilibrium state is established between the E body and the Z body, and the ratio between the two is maintained in a stable state, which can be used very preferably.

The raw material that can be used for the purpose of producing the xanthine oxidase inhibitor of the present invention includes German chamomile (Matricaria chamomilla L .; Japanese name chamomile) as a chamomile, excluding the flower part, or together with the flower part Leaves and stems that are above-ground parts can be used as raw materials. By allowing the following extraction medium to act on these, an extract that effectively acts as a xanthine oxidase inhibitor can be efficiently obtained.

For the ingredients contained in flower parts, see the literature (“Quantitative determination of phenolic compounds by UHPLC-UV – MS and use of partial least-square discriminant analysis to differentiate chemo-types of Chamomile / Chrysanthemum flower heads”, Journal of Pharmaceutical and Biomedical Analysis, Volume 88, 25 January 2014, Pages 278-288) As well as the compound of chemical formula (1) related to the present invention, it includes Z-forms and apigenin derivatives as phenolic components, etc. It is known that
On the other hand, it has been clarified in the present invention that the compound of the chemical formula (1) is contained at a very high concentration in the leaves and stems as the above-ground parts other than the flower parts. Can be used commercially, and a remarkable action as a xanthine oxidase inhibitor has been found.
Here, when roman chamomile (Chamaemelum nobile) is used as chamomile, the flower part of this contains almost no compound of chemical formula (1), and it is obtained by extraction and purification from the ground part other than the flower part. Since the yield of the compound represented by the chemical formula (1) is relatively small, it is preferable to mainly use German chamomile for the purpose of the present invention.

In the present invention, the flower part is used together with the leaf and stem as the above-mentioned part of the above chamomile, and the extract extracted therefrom can be used as the xanthine oxidase inhibitor. In this case, the essential oil component mainly contained in the flower part together with the compound represented by the chemical formula (1) and the apigenin derivative is contained together with the compound of the chemical formula (1) in the extract, so that separation of them becomes extremely difficult. Addition of effects (aroma and taste) other than the xanthine oxidase inhibitory activity targeted by the present invention may cause problems when applied to foods and pharmaceuticals as usage forms. Therefore, in the xanthine oxidase inhibitor of the present invention, it is preferable to use an extract obtained from these by using chamomile leaves and stems as raw materials, without using flower parts, and after removing essential oil components Extracting from the leaves and stems of chamomile is preferred. From a commercial point of view, it is possible to extract and use the compound of the above chemical formula (1) from the residue left after the extraction of essential oil components or the steam distillation process that has been used industrially. Since it can do, it leads to the effective utilization of the conventional waste, and is very preferable.

The extraction medium that can be used in the present invention is water or a hydrophilic organic solvent, or a mixed solvent thereof. Examples of the hydrophilic organic solvent include lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. In particular, a mixed solvent of methanol or ethanol and water containing these can be most preferably used. The conditions for extracting chamomile from above-ground stems and leaves using these extraction solvents are preferably mild, and the temperature is preferably in the range of 10 to 40 ° C. When extraction is performed at the following temperatures, it takes a very long time to extract the components of the present invention, and in the target present invention, the compound of the chemical formula (1) cannot be obtained in a sufficient yield. There is a case. Or when extracting at the temperature exceeding 40 degreeC, the effect may be lost because an extract denatures with a heat | fever.

The above-ground portion of chamomile that can be used in the present invention can be used for extraction as it is after being collected without being dried. Regarding nitrogen fertilizer in soil used for the cultivation of chamomile, the proportion of the compound of chemical formula (1) contained in the above-ground part of chamomile increases when the nitrogen content is deficient or relatively small. Regarding the time of collection, the content of the compound of the chemical formula (1) is relatively high when collected in the state of young leaves. In the compound of the chemical formula (1), the ratio of the Z form produced by isomerization may change during the drying process, and the extract obtained from the above-ground part in a fresh state without drying does not contain the E form. Is relatively high. Alternatively, the above-ground portion of chamomile can be used after being collected, but it is not preferable to dry at a temperature exceeding 90 ° C. when drying, when drying at a temperature of 90 ° C. or lower. Can be preferably used as in the case of undried. Furthermore, the above-described extraction medium may be added to the above-ground part, and the extraction may be performed in a standing state, or the time required for extraction may be obtained by finely cutting or finely pulverizing and extracting with a mixer or a homogenizer. It is also preferable to shorten the length. As described above, it is known that the compound of the chemical formula (1) isomerizes to the Z form upon exposure to light. Furthermore, it is known that in the cultivation of chamomile, the compound of the chemical formula (1) isomerizes into a Z form due to the influence of sunlight during the daytime, and the rate at which this is further cyclized to hernialin increases. Therefore, when cultivating and harvesting chamomile, it is preferable to avoid sunlight during the day, and after cutting, store it in a cool and dark place away from direct sunlight, and handle it so that it is not exposed to light as much as possible even when extracting from there. Is preferred.

In the above extraction step, the extract obtained by the extraction solvent is further added with a solvent in which three kinds of solvents of chloroform / methanol / water are mixed in a preferable ratio, so that an upper hydrophilic layer and a lower hydrophobic layer are added. The compound of the chemical formula (1) can be effectively separated from other components other than the active component by being selectively contained in the upper hydrophilic layer. As shown in the examples described later, as a preferred ratio of the above three solvents of chloroform / methanol / water, it is most preferable that each ratio is included in this order at 4: 2: 3. When used in a range of 1: 1: 1 to 1: 2: 1 apart, it becomes difficult to separate the hydrophilic layer and the hydrophobic layer, and the purity and yield of the extract obtained therefrom are lowered. Furthermore, the time required for extraction may increase.

The fraction separated as a hydrophilic layer using the above mixed solvent of chloroform / methanol / water is further purified by adsorbing and separating the compound of the formula (1) on the surface using an adsorption carrier. Can be increased. At this time, activated carbon or various silica gels can be used as the adsorption carrier, but the adsorption carrier having the best adsorption ability for organic substances having fine pores composed of crosslinked polymer beads as the synthetic adsorbent is the most. It can be preferably used. Examples of synthetic adsorbents that can be used for such purposes include Diaion HP20 or HP21 (manufactured by Mitsubishi Chemical), which is a styrene-divinylbenzene synthetic adsorbent having large pores. The compound of the chemical formula (1) can be preferably used for adsorption purification.

The compound of the chemical formula (1) adsorbed and supported on the adsorption carrier is further subjected to a washing operation with water and alcohol to separate and purify extract components other than the chemical formula (1), which are components, It can be separated and eluted from the adsorption carrier with an alcohol mixed solvent. As the water / alcohol mixed solvent that can be used in this case, a mixed solvent in which water and methanol are mixed in equal amounts can be preferably used.

The solution containing the compound of the chemical formula (1) eluted from the adsorption carrier with the above mixed solvent can be further purified by gel filtration chromatography. In this case, as a carrier for gel filtration chromatography that can be preferably used, Sephadex (made by GE Healthcare Japan) can be mentioned.

The fraction purified by the above gel filtration chromatography can be obtained as high-purity colorless crystals by evaporating the alcohol to precipitate the compound of the formula (1) as crystals. The compound of the chemical formula (1), which is a high-purity component thus purified, was previously shown by structural analysis using a mass spectrometer, NMR, FT-IR, etc., as shown in the examples described later. It was revealed that it was (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid having the structure of chemical formula (1).
The final yield is between 0.02 and 0.04% by weight with respect to the above-ground portion of the undried chamomile, which is the raw material used first, from about 1 kilogram of undried raw material to about The compound of formula (1) was obtained in high purity with a yield of 0.2 to 0.4 grams. However, for applications such as foods containing the xanthine oxidase inhibitor of the present invention, the chemical formula (1) obtained by concentrating from the fraction obtained in the intermediate step without necessarily passing through all the above steps. It is also possible to use it in the state of a mixture containing the compound.

It was confirmed in Examples described later that the compound of the chemical formula (1) in the xanthine oxidase inhibitor of the present invention is extremely stable against heat. That is, it was confirmed that even when an aqueous solution in which the compound of the chemical formula (1) was dissolved was heated at a temperature of 90 ° C., for example, the properties of the components did not change and the composition was extremely stable against heat. However, when long-term storage is required, it is preferable to store the compound of the chemical formula (1) alone, foods and raw materials containing the compound alone in a cool and dark place.

The xanthine oxidase inhibitory action of the compound of the chemical formula (1) and the extract containing the compound obtained as described above can be evaluated as follows. That is, a sample solution having a constant concentration was added to a phosphate buffer solution in which xanthine was dissolved, preincubated for 15 minutes at 37 ° C., and a predetermined amount thereof was taken and added to a phosphate buffer solution in which xanthine oxidase was dissolved. By incubating for a minute and reacting, the inhibitory effect can be quantitatively estimated from the ratio of the change in xanthine concentration due to the addition of the sample solution containing the xanthine oxidase inhibitor to the change in xanthine concentration when the sample solution is not added. Is possible. The inhibitory effect of the compound of the chemical formula (1) thus determined was confirmed to increase in action in a dose-dependent manner, and showed an action equivalent to or higher than that of the comparative allopurinol in the state of the crude extract. became. Details will be described in an embodiment described later.

Furthermore, as shown in the examples described later, the compound of the chemical formula (1) is characterized in that it is absorbed into the body very efficiently in the process of being orally administered and reaches the digestive tract, and exhibits high bioavailability in the flow of the systemic circulation. is there. The compound of formula (1) may undergo chemical changes due to the action of various enzymes during the absorption process, but after oral administration, approximately 70 to 80% or more of the whole is not subject to chemical changes. It rides in the systemic circulation as it is, acts on xanthine oxidase existing on the surface of endothelial cells of blood vessels and various organs in the body, and effectively inhibits this to lower the excessive uric acid concentration in the body. Moreover, the effect is moderately moderated.For example, when compared with the action of allopurinol used as a therapeutic agent for hyperuricemia, when allopurinol is administered, the uric acid concentration in the blood is significantly lower than the normal range. However, when a xanthine oxidase inhibitor containing the compound of formula (1) is administered, the effect is not excessively expressed and the blood uric acid concentration is avoided to fall below the normal range. it can. It is known that uric acid exhibits a remarkable reducing action in the body and prevents various damages caused by active oxygen in the body by maintaining the blood concentration within an appropriate concentration range. For this reason, it is strictly forbidden to use a drug that exhibits a strong uric acid level-lowering action such as allopurinol, particularly when the blood uric acid level is in the normal range. When the xanthine oxidase inhibitor of the present invention is used, since its pharmacological action is limited, it does not cause blood uric acid levels below normal values in normal use, so it can be used safely. it can.

It has not been conventionally known that the compound of the chemical formula (1) can be efficiently extracted from the above-ground part of chamomile using a relatively simple method as described above. It has been clarified by the present inventors for the first time that it exhibits an oxidase inhibitory action. As an object of the present invention, it is possible to efficiently extract an active ingredient from familiar plants well known to be safe, and to find a xanthine oxidase inhibitor excellent in effect and a method for producing the same. From this point of view, chamomile is a very familiar plant and can be easily cultivated. Therefore, the novel xanthine oxidase represented by the structure of the compound of the chemical formula (1) clarified in the present invention It becomes clear that an inhibitor can be efficiently extracted from the above-ground portion of chamomile by a simple method, and it can be said that the utility value of the present invention is high in that resources that have not been used can be effectively used.

When the above-described extract of chamomile obtained from the present invention is used as a xanthine oxidase inhibitor for humans, it is preferably used by oral administration, and the dose is generally one. It is preferably used in an amount of 0.01 to 50 mg / kg of body weight per day, preferably once or several times a day. When an amount below this range is used, the target xanthine oxidase inhibitory effect of the present invention may not be observed. In addition, when an amount exceeding the above range is used, the bioavailability may decrease due to an increase in the ratio of excretion without being absorbed into the body.

The extract using the above-ground portion of chamomile obtained in the present invention can be used as a medicine containing this as a xanthine oxidase inhibitor. In this case, the dosage form can be used as a solid agent such as a tablet, granule, powder or capsule, or as a liquid agent such as a solution, suspension, emulsion, syrup or spray. Examples of pharmaceutical carriers that can be used in the preparation include glucose, sucrose, lactose, starch, mannitol, dextrin, gelatin, collagen, hyaluronic acid, albumin, polyethylene glycol, amino acid, water, and physiological saline. I can do it. Furthermore, you may use together a pH adjuster, a stabilizer, a wetting agent, an emulsifier, a lubricant, an isotonic agent etc. as needed.

The extract using the above-ground portion of chamomile obtained in the present invention can be used as a food containing this as a xanthine oxidase inhibitor. In that case, the food form can be used as an additive in various foods usually eaten. For example, when used as a food in the form of a beverage, it can be used by adding in the range of 0.01 mg to 100 mg per liter to various beverages that are normally distributed. In drinking, it is possible to heat and drink, but preferably it is stored at a temperature of room temperature or lower and supplied for drinking. As a preferable example, it can be added to milk, carbonated drinks, semi-solid foods such as yogurt, and the like, and can be provided as a beverage having a preventive effect on hyperuricemia. As other foods, for example, it may be added to processed meat foods such as ham and sausage, fishery processed foods such as chikuwa and kamaboko, bread and confectionery. Furthermore, as supplements and health supplements, it is provided in the form of tablets, granules, powders, capsules and other solids containing the above extract, or liquids such as solutions, suspensions, emulsions, syrups and sprays. Is possible.

The food containing the xanthine oxidase inhibitor obtained in the present invention is particularly preferably used as a beverage. By taking the xanthine oxidase inhibitor in the body in a preferable amount range, it is possible to suppress the production of uric acid due to the metabolism of purine to an appropriate range and prevent the development of hyperuricemia. Hyperuricemia is characteristically observed in adult males, and in addition, the uric acid level is often high in the layer that prefers alcoholic beverages, more than 20% of adult men, especially more than 30% of men in their 30s and 40s Has been reported to show hyperuricemia. If hyperuricemia is left unattended, there is a high possibility of a gout attack, and the number of gout patients exceeds 1 million per year as of 2016. Furthermore, it is said that there are 10 million hyperuricemia patients nationwide as a gout reserve army, and as guidelines for life as an improvement measure against this, restrictions on purine intake and alcohol intake are guidelines. Are listed. However, at present, there are limits to the effects of these intake restrictions, and there is no effective method for lowering uric acid levels other than medical intervention, so it is difficult to respond positively until a doctor diagnoses hyperuricemia. There is a problem. As an example of a beverage containing a xanthine oxidase inhibitor obtained by the present invention, a beverage obtained by adding the compound of the formula (1) according to the present invention to an alcoholic beverage has an effect of lowering the uric acid level in the blood. It is preferably used as a means for preventing the disease. Furthermore, as mentioned above, by inhibiting the action of xanthine oxidase, it suppresses not only uric acid but also the generation of active oxygen, thereby suppressing and preventing various disorders due to the effect of active oxygen in various organs in the body. It is possible.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. In addition, the percentage in an Example is a mass reference | standard unless there is a notice.

(Extraction method using the above-ground part of chamomile and structural analysis of xanthine oxidase inhibitor obtained thereby)
Fresh ground parts of chamomile (Matricaria chamomilla L.) cultivated in Setun University's Medicinal Botanical Garden were harvested, and 1520 grams were put into a 5 liter glass container in a fresh state (undried) of the ground stem part and leaves. Methanol-soluble components were extracted by charging 3.9 liters of methanol and allowing to stand at room temperature for 2 days. Methanol was distilled off under reduced pressure to obtain 56.2 grams (3.7% of the raw material) of a crude extract. Using 28.0 grams of the crude extract, this was further mixed with an organic layer (chloroform-methanol layer) and a hydrophilic layer (chloroform-methanol layer) using a mixed solvent of chloroform-methanol-water (4: 2: 3) (volume ratio). (Methanol-water layer) was separated, and the latter hydrophilic layer (about 25.0 grams) was recovered. With respect to what was collected from the hydrophilic layer, it was further distributed using a mixed solvent of butanol-water (1: 1) (volume ratio) to obtain 22.0 g as a water-soluble crude extract (MR). As components contained in the crude extract (MR), the compound of the chemical formula (1) was contained in 2.3% by mass with respect to the whole, and the Z-form was contained in 9.5% by mass. In MR, saccharides and amino acid compounds are mainly included as components other than these, and compounds such as essential oil components and phenols are substantially not included. As described later, this crude extract (MR) was measured for xanthine oxidase inhibitory activity, and was found to exhibit extremely excellent inhibitory activity.

The crude extract was further subjected to a gel separation operation using a polystyrene gel for adsorption (DIAION (registered trademark) HP-20 manufactured by Mitsubishi Chemical Corporation) (φ45 mm × 270 mm), and the eluent was first eluted with water. Subsequently, elution was performed with a mixed solvent of water-methanol (1: 1) (volume ratio), and finally elution was performed with methanol alone. Among these, it was confirmed that the component of the chemical formula (1) was selectively contained in the fraction eluted with a water-methanol (1: 1) mixed solvent. A portion of the fraction thus separated is further used for separation and purification using a carrier for gel filtration (SEPHADEX (registered trademark) 分離 LH-20) (φ30 mm × 360 mm). As a fraction, 1.1 grams of a light brown powder was obtained. As a water-methanol solution, methanol was evaporated and concentrated to give 0.2 g of colorless crystals showing a single spot on TLC (Thin-Layer Chromatography). The above separation process is shown in FIG. FIG. 1 represents a flow scheme for extracting the component of the chemical formula (1) from the above-ground stem portion and leaf portion of chamomile.

FIG. 2 shows a production flow chart of a xanthine oxidase inhibitor. As shown in FIG. 2, the xanthine oxidase inhibitor of this invention performs the extraction process by the methanol with respect to the above-ground part of chamomile first (step S01). Next, a separation and purification process is performed on the extract extracted in step 01 with a mixed solvent of chloroform, methanol, and water (step S02). Next, using a mixed solvent of butanol and water, a separation and purification process is performed on the hydrophilic layer separated into the organic layer and the hydrophilic layer (methanol-water layer) in step S02 (step S03). Then, after performing the gel separation and purification process for the aqueous layer (step S04), the elution process such as elution with water first, elution with water and methanol, and elution with methanol again is performed (step S05). Then, a separation and purification step is performed on the fraction eluted with water and methanol by the gel filtration carrier (step S06), and a concentration step for concentrating and crystallizing the eluted fraction is performed (step S07). Crystallization proceeds by transpiration, and the compound of the chemical formula (1) is produced as a crystalline product of the active compound having the intended effect of inhibiting xanthine oxidase (step S08).

The crystals obtained above were further analyzed by mass spectrometry, and the mass of the sodium salt of the compound represented by the chemical formula (1) of m / z: 379 [M + Na] ⁺ was analyzed by pos ion FAB-MS shown in FIG. An analysis chart was given. FIG. 3 represents a positive ion mode mass spectrometry chart of the sodium salt of the compound represented by the chemical formula (1).

Similarly, the analysis result in neg mode in mass spectrometry gave a peak of m / z: 355 [M−H] ⁻ as shown in FIG. 4 and was consistent with the structure of chemical formula (1). . FIG. 4 shows a negative ion mode mass spectrometry chart of the compound represented by the chemical formula (1).

The crystal obtained above was further analyzed using ¹ H-NMR, and it was confirmed from the assignment of each peak that it had a structure represented by chemical formula (1). FIG. 5 shows a ¹ H-NMR chart of the compound represented by the chemical formula (1).

Similarly, the above crystal was analyzed using ¹³ C-NMR, and it was confirmed from the assignment of each peak that it had a structure represented by chemical formula (1). FIG. 6 shows a ¹³ C-NMR chart of the compound represented by the chemical formula (1).

Similarly, the above crystal was analyzed using FT-IR, and it was confirmed from the assignment of each peak that it had a structure represented by chemical formula (1). FIG. 7 shows an FT-IR chart of the compound represented by the chemical formula (1). From the above results, in this example, the compound having the structure represented by the chemical formula (1) can be obtained in high yield as a high-purity compound in the form of crystals from the ground stem part and leaf part excluding the flower part of chamomile. done. It is clear that the compound is mainly composed of (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid, which contains a slight amount of (Z) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid It became.

(Evaluation of xanthine oxidase inhibitory activity of crude extract)
In the extraction step described above, xanthine oxidase was obtained by using the following method using a part of 22.0 grams of a water-soluble crude extract (MR) separated by a mixed solvent of butanol-water (1: 1) (volume ratio). Inhibitory activity was evaluated. The crude extract used contained 2.3% by mass of the component of the chemical formula (1), further contained 9.5% by mass of Z-form, and other components contained saccharides and amino acids. It was.

A commercially available xanthine oxidase was dissolved in a 0.1 molar phosphate buffer (pH 7.8) to prepare a xanthine oxidase buffer (3.2 kg units / mL). As a substrate, xanthine phosphate buffer (65.7 μM) in which xanthine was similarly dissolved in phosphate buffer was prepared. For comparison, allopurinol which is a commercially available xanthine oxidase inhibitor was dissolved in a phosphate buffer to prepare an allopurinol buffer (11.9 μM). As a sample to be evaluated in this example, the above-mentioned water-soluble crude extract separated with butanol-water (1: 1) (volume ratio) was dissolved in a phosphate buffer to a concentration of 1.12 mg / mL. Prepared and used as sample solution. The concentration of the compound of chemical formula (1) contained in the sample solution was 51.3 μM.

Using the sample solution prepared above and allopurinol buffer, each was added to xanthine phosphate buffer and preincubated at 37 ° C. for 15 minutes. Xanthine oxidase buffer was further added to these, and incubation was performed at 37 ° C. for 40 minutes. The obtained reaction solution was filtered through a membrane filter, and the absorbance of the sample was measured at a wavelength of 280 nm using an ultraviolet-visible spectrophotometer. As the xanthine oxidase inhibition rate, the absorbance of each blank was subtracted from the absorbance of each sample, and the ratio to the absorbance when no inhibitor was added was determined as the inhibition rate. As a result, it was found that the inhibition rate of allopurinol was 72%, but the inhibition rate of the sample solution of the present invention was 99%, showing an extremely high inhibition rate. From the above, according to this example, it is clear that the crude extract obtained in high yield from the above-ground stem part and leaf part of chamomile shows a high xanthine oxidase inhibitory activity exceeding allopurinol which is a commercially available drug. became.

(Evaluation of xanthine oxidase inhibitory action using chemical formula (1))
Using the compound of the chemical formula (1) finally purified in Example 1 and obtained in a crystalline state, the effect as a xanthine oxidase inhibitor in a pure state was examined. That is, the compound of the formula (1) was dissolved in a phosphate buffer solution at a concentration of 3.25 μM, and a phosphate buffer solution in which allopurinol was dissolved at a concentration of 3.51 μM, which is its IC50, was used for comparison. In the same manner as in 1, xanthine oxidase inhibitory activity was examined. As a result, the compound of the chemical formula (1) showed a result of 15% as an inhibition rate, and allopurinol was 55%. From this result, it was revealed that the compound of the chemical formula (1) obtained by the method for producing a xanthine oxidase inhibitor of the present invention exhibits extremely remarkable xanthine oxidase inhibitory activity.

(Evaluation of heat stability of chemical formula (1))
Using the compound of the chemical formula (1) finally purified in Example 1 and obtained in the crystalline state, the stability to heat was examined. That is, the compound of the formula (1) was dissolved in distilled water and heated at 90 ° C. for 1 hour, but no change was observed in the ultraviolet-visible absorption spectrum of the compound, and it was extremely stable against heat. Was confirmed.

(Pharmaceuticals containing the xanthine oxidase inhibitor of the present invention)
Using the compound of the chemical formula (1) which is the crystal obtained in Example 1, for example, a tablet was produced as follows. That is, 23% by mass of the compound of the chemical formula (1), lactose 60%, corn starch 15%, guar gum 1% and magnesium stearate 1% were uniformly mixed to produce tablets according to a conventional method.

(Food containing the xanthine oxidase inhibitor of the present invention)
Using the crude extract (MR) obtained in Example 1, for example, juice was prepared as follows. That is, banana ripening component (1%), oligosaccharide (10%), fresh lemon juice (1%), fresh grape juice (1%), natural water (86%) with respect to 1% by mass of crude extract (MR) %) Was added to prepare a total volume of 200 L. This solution (200 L) is put into a stainless steel container (inner diameter 567 mm, outer height 890 mm), and subjected to stirring treatment (sterilization treatment according to the Food Sanitation Law) at 90 ° C. for 30 minutes with a stirrer to prepare as a functional beverage did.

(Beverage containing the xanthine oxidase inhibitor of the present invention)
Alcohol beverage containing xanthine oxidase inhibitor according to the present invention by adding 10 mg of the compound of chemical formula (1) obtained in Example 1 to 100 mL of commercially available sake (Laurel Wreath (registered trademark) “Sugar Zero”) Was made. No influence on the taste, fragrance, flavor, etc. due to the addition of the compound of the chemical formula (1) was observed, and no adverse effect on the quality of sake was found.

In Example 1, the components extracted from fresh above-ground portions of chamomile (Matricaria chamomilla L.) using methanol were mixed with an organic layer (chloroform using a mixed solvent of chloroform-methanol-water (4: 2: 3) (volume ratio). -Methanol layer) and a hydrophilic layer, and the hydrophilic layer is further purified with a mixed solvent of butanol-water (1: 1) to obtain a polystyrene gel for adsorption (DIAION (registered trademark) HP-20 manufactured by Mitsubishi Chemical). Gel separation operation using (φ45 mm × 270 mm) was performed to obtain 2.7 g of component CPPG eluted with a water-methanol (1: 1) (volume ratio) mixed solvent. The following tests were performed using this.

As a result of analyzing the obtained extracted component CPPG by ¹ H-NMR, the spectrum of FIG. 8 was obtained. FIG. 8 shows a ¹ H-NMR spectrum chart of the extracted component CPPG used in this example. When the ratio of the other components contained together with the compound of the chemical formula (1) is analyzed from the peak area ratio of the signal appearing on the spectrum of FIG. 8, the compound of the chemical formula (1) has a concentration of about 16% by mass in CPPG. In addition, it was found that about 74% by mass of the Z-form was contained, and about 10% as a hydrocarbon compound not containing an aromatic component was contained as a component other than these.

(About oral administration test results using mice)
Using the extracted component CPPG, an animal experiment on oral administration to mice and blood transferability was performed as follows. As a mouse, a 7-week-old ICR mouse (male) obtained from Japan SLC was used.
A feed was prepared by dissolving 30 mg or 60 mg of the CPPG in 300 microliters of a 0.5% strength carboxymethylcellulose solution, and these were orally administered to mice as doses per animal (performed in each group n = 3). Comparison was made using the average value in each group).
Before oral administration and when 15 minutes, 60 minutes, and 180 minutes have passed after oral administration, 60 microliters of blood was collected from the tail vein of the mouse, and this was deproteinized by centrifugation, and CPPG contained in the plasma supernatant and The metabolite concentration of this was analyzed by HPLC (quantitative).

For the HPLC measurement, a pump (JASCO PU-2080 Plus), a column (Gaschrom Industries ODS-3 4.6 × 100 mm), and a column oven (30 ℃, JASCO CO-2067 Plus) are used. Absorbance at a wavelength of 315 nm was measured using JASCO UV-4075). As the mobile phase, acetonitrile / water / phosphoric acid = 150/850/1 was used. FIG. 9 shows the results of HPLC analysis of samples collected from mice before oral administration under the above conditions. FIG. 9 shows a chart when a sample collected from a mouse before CPPG is orally administered is analyzed by HPLC.

Next, a chart obtained by HPLC analysis from a sample collected after oral administration is shown in FIG. FIG. 10 shows a chart when a sample collected from mice after oral administration (60 mg administration group, 60 minutes after administration) is analyzed by HPLC. From this chart, CPPG-derived components contained in mouse blood after oral administration of CPPG mainly have three peaks that appear at elution times of 3.4 minutes, 4.7 minutes, and 5.9 minutes. The component of was not recognized. Among them, the peak component with an elution time of 4.7 minutes is contained in a ratio of 70 to 80% with respect to all the three components, and this is not metabolized and is transferred into the blood with the compound of the formula (1) Identification of the retention time given by the crystal obtained in Example 1 and the FAB-MS in both pos- and neg- モ ー ド modes are both Z-forms (hereinafter, the component including both is referred to as CA). (Molecular weight 356), but the other two peak components were small compared to the CA component in blood, and structural analysis was difficult.

FIG. 11 shows the results of quantifying the concentration of components derived from CPPG contained in blood from the HPLC chart obtained as described above, and plotting the blood concentration of CA after administration against time. FIG. 11 shows the time change of the blood concentration of CA transferred to the blood after oral administration. As a result, in this experiment, the blood concentration reached the maximum value in the vicinity of 60 minutes after the administration at any oral dose, and the result decreased thereafter. In this case, the maximum concentration of the compound of the chemical formula (1) in the blood showed an extremely high value of 6.3 μM. With respect to this numerical value, it is a numerical value comparable to 3.25 μM which is the concentration of the compound of the chemical formula (1) used in Example 2, and from this, the concentration of the compound of the chemical formula (1) transferred into the blood is It was clarified that the efficacy is comparable to that of allopurinol as a comparison. That is, from this example, when the extract from the above-ground chamomile obtained by the present invention was orally administered to mice, it was transferred to the blood in the form of the compound of the chemical formula (1) at a high concentration. It was revealed that the concentration was comparable to the effective concentration of allopurinol to be compared.

(Comparative evaluation on the xanthine oxidase inhibitory action of Z form on the compound of chemical formula (1))
In Example 1, a separation and purification process was performed on the fraction eluted with water and methanol using the carrier for gel filtration (step S06), and a concentration process for concentrating and crystallizing the eluted fraction was performed (step S07). Crystallization progressed to obtain a compound of the chemical formula (1). In this final step, a solution (comparative example) containing mainly Z form from the supernatant excluding crystals and substantially free of the compound of chemical formula (1) was obtained. Produced. As a result of evaluating the inhibition rate of xanthine oxidase in the same manner as in Example 1 for the produced comparative example, it was 0%, and no inhibitory effect was observed.

The xanthine oxidase inhibitor of the present invention can be used as pharmaceuticals, foods (including various beverages such as carbonated drinks and alcoholic drinks), supplements and the like for the purpose of preventing or treating hyperuricemia. Furthermore, it can be used as a health food for preventing excessive active oxygen generation in various organs in the body and preventing symptoms such as hypertension, diabetes and obesity.

Claims (16)

1. (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid ((E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid) represented by the following chemical formula (1) is an active ingredient Xanthine oxidase inhibitor contained as
   

   
2. The active ingredient further contains (Z) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid ((Z) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid). 1 xanthine oxidase inhibitor.
3. The xanthine oxidase inhibitor according to claim 1 or 2, wherein the active ingredient is derived from an extract of a chamomile (Matricaria recutita) above-ground part.
4. The xanthine oxidase inhibitor according to claim 1 or 2, wherein the active ingredient is derived from an extract of a stem part and a leaf part excluding a flower part of chamomile (Matricaria recutita).
5. The xanthine oxidase inhibitor according to claim 3 or 4, wherein the chamomile is German chamomile (Matricaria chamomilla L.).
6. The xanthine oxidase inhibitor according to claim 5, further comprising a compound derived from an extract of the above-ground part of Roman chamomile (Chamaemelum nobile).
7. A pharmaceutical comprising the xanthine oxidase inhibitor according to any one of claims 1 to 6.
8. A food comprising the xanthine oxidase inhibitor according to any one of claims 1 to 6.
9. The manufacturing method of the xanthine oxidase inhibitor provided with the step extracted from the above-ground part of a chamomile (Matricaria recutita).
10. 10. The xanthine oxidase inhibitor according to claim 9, wherein the extracting step is performed by extracting from a stem part and a leaf part excluding a chamomile flower part.
11. Extracting the above-ground portion of chamomile with water or a hydrophilic organic solvent or a mixed solvent thereof;
    Separating the aqueous layer from the extract and eluting with a mixed solvent of water and a hydrophilic organic solvent;
    Concentrating and crystallizing the main components of the eluted fraction,
    A method for producing a xanthine oxidase inhibitor according to claim 9 or 10.
12. The method for producing a xanthine oxidase inhibitor according to any one of claims 9 to 11, wherein in the extracting step, the essential oil component contained in chamomile is removed and then extracted from the above-ground portion of chamomile.
13. The method for producing a xanthine oxidase inhibitor according to any one of claims 9 to 12, wherein the extracting step uses German chamomile (Matricaria chamomilla L) as chamomile.
14. The method for producing a xanthine oxidase inhibitor according to claim 13, further comprising a step of mixing a compound obtained by extracting the above-ground portion of Roman chamomile (Chamaemelum nobile) with a mixed solvent of water and a hydrophilic organic solvent.
15. The method for producing a xanthine oxidase inhibitor according to any one of claims 9 to 14, wherein the extraction is performed at 10 to 40 ° C.
16. (E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid ((E) -2-β-D-glucopyranosyloxy-4-methoxycinnamic acid) represented by the following chemical formula (1) is an active ingredient The method for producing a xanthine oxidase inhibitor according to any one of claims 9 to 15, wherein
    

    

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