JP5478392B2 - Method for producing lettuce pudding A extracted from red propolis bulk and method for evaluating red propolis extract using lettuce pudding A - Google Patents

Description

  The present invention relates to a method for producing lettuce pudding A obtained from, for example, a red propolis raw block made from a specific plant as a source plant, and a method for evaluating a red propolis extract using lettuce pudding A.

  Propolis is a glue-like or wax-like substance made by mixing beeswax into plant exudates, shoots and resins collected by bees for the purpose of defense and reinforcement of the nest. This propolis contains various components because bees are produced by collecting various plants around the nest box as raw materials.

  Propolis has long been known to have antibacterial and anti-inflammatory effects. Antioxidant action and immunostimulatory action are known as main physiological activities of propolis. Therefore, propolis has long been used as a raw material for pharmaceuticals or health foods. As active ingredients contained in propolis, very various kinds of active ingredients such as organic acids with high polarity, flavonoids, polyphenols, and terpenoids with low polarity have been confirmed.

  By the way, propolis production areas include, for example, South American countries such as China, Brazil, Argentina, and Uruguay, Europe such as Hungary and Bulgaria, North America such as Canada, and Oceania such as Australia and New Zealand. In recent years, as disclosed in Non-Patent Document 1, many researchers have been researching the differences in the content of propolis by production area. It is thought that the difference in the composition in each production area differs depending on the kind of the origin plant. For example, as disclosed in Patent Document 1, among Brazilian propolis, for example, Strytophendron. It is known that the propolis produced by using adstringens or the like as a main plant is red. This red propolis has been confirmed to be superior in, for example, an antioxidant action and an antibacterial action as compared with a yellow propolis produced in Brazil. These excellent physiological actions in red propolis are believed to be related to the red pigment component.

International Publication No. 2006/038690

Tazawa Shige et al .: Biopharmaceutical magazine, 54 (6), 306-313, 2000

  The present invention is known to have been contained in the propolis mass as a red pigment component from the propolis mass, which is made from the genus Crane beetle (Dalbergia) among the red propolis from Brazil. This is based on the fact that lettuce pudding A was obtained.

  An object of the present invention is to provide a method for producing lettuce pudding A and a method for evaluating a propolis extract that can be applied to the fields of foods and drinks and cosmetics, for example.

  In order to achieve the above object, the method for producing lettuce pudding A according to the first aspect of the present invention includes a step of performing an extraction treatment using an extraction solvent from a propolis bulk that is produced from a genus Crassica as a source plant. Prepare.

  The invention according to claim 2 is the method for producing lettuce pudding A according to claim 1, wherein the lettuce pudding A is at least one selected from the following general formulas (1) to (5).

Invention of Claim 3 is a manufacturing method of the lettuce pudding A of Claim 1 or Claim 2, Comprising: The process of carrying out the separation process using chromatography is further provided after the process of the said extraction process.

  The invention according to claim 4 uses lettuce purpurin A as an index in a method for evaluating a propolis extract obtained by using an extraction solvent from a propolis ingot produced from a plant of the genus Crassica as a raw material. It is characterized by.

  Invention of Claim 5 contains lettuce purpurin A in the evaluation method of the propolis extract of Claim 4 using the extraction solvent from the propolis original lump made from the plant of the genus Crane beetle as a raw material The process comprises obtaining a propolis extract, then separating the extracted components in the extract using one or more chromatography, and then detecting lettuce purine A.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the lettuce pudding A which can be applied to field | areas, such as food-drinks and cosmetics, for example, and the evaluation method of a propolis extract can be provided.

Data showing the results of mass spectrometry using electrospray ionization mass spectrometry (ESI-MS) for the red pigment component in the red propolis bulk. (A) In case of Positive mode, (b) In case of Negative mode. The data which shows the measurement result of an ultraviolet and visible light spectrum (UV / vis) about the red pigment | dye component in a red propolis ingot. The data which shows the measurement result of an infrared absorption spectrum (IR) about the red pigment | dye component in a red propolis original block. The data which shows the measurement result of a < ¹ > H-NMR spectrum about the red pigment | dye component in a red propolis original block. The data which shows the measurement result of a < ¹³ > C-NMR spectrum about the red pigment | dye component in a red propolis original block.

(First embodiment)
Hereinafter, a first embodiment in which the method for producing lettuce pudding A of the present invention is embodied will be described.
The method for producing lettuce pudding A according to the present embodiment includes a step of performing an extraction treatment using an extraction solvent from a propolis bulk that is produced using a genus Crassica as a raw material. Furthermore, preferably, after the step of performing the extraction treatment, a step of further performing a separation treatment using chromatography is provided. Lettuce pudding A is a compound having at least one configuration selected from the following general formulas (1) to (5).

Letappurpurin A represented by the general formula (1) is 6- (3,4-dihydro-7-methoxy-2H-1-benzopyran-3-yl) -4- (4-hydroxyphenyl) -2- ( 4-hydroxy-2-methoxyphenyl) -7H-1-benzopyran-7-one (C ₃₂ H ₂₆ O ₇ , molecular weight 522.54).

Letappurpurin A represented by the general formula (2) is 6- (3,4-dihydro-7-methoxy-2H-1-benzopyran-3-yl) -4- (4-hydroxyphenyl) -2- ( 4-hydroxy-2-methoxyphenyl) -7-hydroxy-1-benzopyrylium (C ₃₂ H ₂₇ O ₇ ⁺ , molecular weight 523.55). The lettuce pudding A represented by the general formula (2) has a structure obtained when the lettuce pudding A represented by the general formula (1) is subjected to acidic conditions (in an acidic aqueous solution). Regarding the lettuce purpurin A represented by the general formula (1), it is presumed that the structural formula also changes to the compounds represented by the following general formulas (3) to (5) depending on various conditions such as pH.

The lettuce pudding A is obtained from a red propolis block that is produced mainly from leguminous genus Dalbergia (Dalbergia) that inhabits in Brazil. More specifically, Dulbergia. Ecastophilum (Dalbergia. Ekastophyllum: local name: Jabo de Buzio). This lettuce pudding A is a red polyphenol compound and constitutes the red pigment component of the red propolis bulk. For example, Stryphnodendron. It has been confirmed that red propolis produced using adstringens as a primary plant is superior in anti-oxidation and antibacterial effects, for example, compared to other Brazilian yellow propolis. These excellent physiological actions in red propolis are considered to be related to lettuce purpurin A which is a red pigment component. Since lettuce purpurin A, which is the production target of this embodiment, has a polyphenol skeleton, it is expected to exhibit an excellent antioxidant effect. Therefore, lettuce pudding A obtained by the production method of the present embodiment is expected to be effectively used, for example, in the fields of foods and drinks and cosmetics.

  In addition, lettuce purple pudding A as a red pigment cannot be obtained directly from a branch, bark, etc. The red pigment is a honeybee that devours as a sap of a biological defense substance (phytoalexin) produced mainly by the perforated worms (larvae) preying on and contacting the heartwood of the plant of the genus Crassica, It exists in red propolis made by taking it back to the nest box. Although lettuce pudding A is known to be obtained from a callus of a plant of the genus Vulgaris, it is difficult to produce and obtain a callus.

  Since lettuce purple pudding A exists in the propolis bulk that is produced from the genus Cramicus, the propolis bulk that contains the plant of the genus Crab is used as an extraction raw material, and is subjected to separation and purification treatment. It can be obtained. In order to include the genus Crane beetle in the origin plant in the propolis mass, it can be achieved, for example, by placing a bee hive designed for propolis collection in a place close to a mangrove forest and a wild colony . The range for collecting the honeybee, nectar and pollen and propolis raw materials is several kilometers (within a radius of about 2 to 5 kilometers). It is preferable that the propolis bulk mass produced from the genus Crane beetle has a red color visually. This is because lettuce purpurin A as the red polyphenol compound constitutes a red pigment component in the propolis bulk.

  Letsupurpurin A as a red polyphenol compound is a step of obtaining a propolis extract as a solvent extract from an extraction raw material of a propolis bulk using an extraction solvent, preferably one or two or more of the solvent extracts. A purified product can be obtained through a step of separation using chromatography.

  As the extraction solvent, an organic solvent or a mixed solution of water / organic solvent is used. Since lettuce purpurin A as a red polyphenol compound has a relatively low polarity, it is preferable to apply an organic solvent having a low polarity as an extraction solvent. As the organic solvent used in the present embodiment, in addition to lower alcohols such as ethanol, methanol and isopropanol having a property of dissolving in water, acetone, ethers and chloroform can be appropriately selected and used. These organic solvents may be used alone or in combination of two or more. Of these, ethanol or methanol, chloroform, and a mixture thereof that can extract lettuce pudding A more efficiently are preferably applied.

  When extracting using the said solvent, it is preferable to remove impurities, such as garbage mixed at the time of extraction, and coarsely pulverize before the extraction process. When an organic solvent is applied as the extraction solvent, the use volume of the organic solvent is preferably 1 to 20 times, more preferably 3 to 10 times the mass of the extraction raw material. When the use volume of the organic solvent is less than 1 time, the extraction rate of the target component is poor, which is not preferable. On the contrary, when the use volume of the organic solvent exceeds 20 times, not only an unnecessarily large apparatus is required, but also a process such as concentration takes time, and workability is remarkably lowered. Although extraction temperature is suitably set according to the kind of extraction solvent, it is preferable that it is 5-40 degreeC. When the extraction temperature is less than 5 ° C., the extraction rate of the active ingredient is poor, which is not preferable. Conversely, when the extraction temperature exceeds 40 ° C., wax components are extracted, and the filterability after extraction may deteriorate. In addition, an extraction solvent having high volatility is not preferable because the solvent evaporates. In addition, what is necessary is just to perform extraction operation for predetermined time, for example, 4 hours or more, stirring at the said extraction temperature. And after fully extracting an active ingredient on said extraction conditions, an organic-solvent extract can be obtained by performing filtration processes, such as filter paper filtration and diatomaceous earth filtration.

  Next, lettuce purine A as a red polyphenol compound is separated and purified from the solvent extract by using one or more chromatography. As the chromatography, known chromatography such as gas chromatography, liquid chromatography, supercritical fluid chromatography, and thin layer chromatography can be used. As liquid chromatography, column chromatography can be used, for example, and more specifically, high performance liquid chromatography (HPLC) and open column chromatography can be mentioned. Examples of the chromatography carrier include ion exchange chromatography, partition chromatography (normal phase / reverse phase chromatography), adsorption chromatography, and molecular exclusion chromatography. More specifically, it is preferable to use a silica gel carrier or an ODS carrier as partition chromatography from the viewpoint of excellent separation efficiency of the extraction components. By combining them appropriately, the target component can be purified by a known method of use.

  Letapurupurin A as a red polyphenol compound obtained as described above is a novel red pigment component that has not been confirmed so far to be contained in propolis, for example, various fields such as foods and beverages and cosmetics. It can be preferably applied to.

According to the method for producing lettuce pudding A as the red polyphenol compound of the first embodiment, the following effects can be obtained.
(1) The method for producing lettuce pudding A of this embodiment is obtained from a propolis bulk that has not been known to contain lettuce pudding A. Letsapurpurin A is a red pigment component of the red propolis block that is produced from the genus Crassica, and is presumed to have an excellent physiological activity. Therefore, the lettuce pudding A obtained from the propolis bulk can be suitably used in various fields such as foods and beverages and cosmetics.

  (2) Moreover, the manufacturing method of the lettuce pudding A of this embodiment can be extracted from a red propolis bulk. Therefore, the raw material can be obtained easily and stably. The obtained lettuce pudding A can be safely applied to various uses.

  (3) Preferably, lettuce purpurin A as a red polyphenol compound is obtained by using, as an extraction raw material, a propolis bulk that is produced from a genus Crassica, using an extraction solvent, and further using chromatography. It can be obtained by separation and purification. Therefore, a purified product can be obtained at a low cost.

(4) Preferably, an organic solvent is used as the extraction solvent. Therefore, lettuce purpurin A as a red polyphenol compound can be efficiently extracted.
(5) Letapurupurin A as a red polyphenol compound is a red pigment component of propolis. Therefore, for example, it can be applied as a colorant / pigment derived from a natural product.

In addition, you may change the said embodiment as follows.
-In the above-mentioned embodiment, you may use the propolis ingot with which origin plants other than the genus Crane beetle were mixed as an extraction raw material. If the propolis raw mass contains at least the genus Crassica as a source plant, it can be used as an extraction raw material. It is considered that the extract obtained when such an extraction raw material is used also contains lettuce pudding A.

-An extraction solvent may be added to the extraction residue after obtaining the propolis extract to perform an extraction treatment. By reusing the extraction residue, the recovery rate can be increased.
-Letsapurpurin A as a red polyphenol compound obtained by the method of the above embodiment can be applied not only to foods and drinks or cosmetics consumed by humans, but also to feeds and drugs for domestic animals such as livestock You may apply to. Moreover, you may apply not only to food / beverage products or cosmetics but to miscellaneous goods and daily necessities.

(Second Embodiment)
Hereinafter, a second embodiment that embodies the propolis extract evaluation method of the present invention will be described.
The method for evaluating a propolis extract according to the present embodiment is a method for evaluating a propolis extract obtained by using an extraction solvent from a propolis bulk that is produced from a genus Cramicula as a raw material. It is characterized by doing. Using lettuce pudding A as an index means, for example, confirming the quality of propolis extract by determining the presence or absence of lettuce pudding A in the propolis extract, the content thereof, the content ratio with respect to a specific component, and the like. .

  More specifically, first, a step of obtaining a propolis extract using an extraction solvent from a propolis bulk that is produced using a genus Crane beetle as a raw material as a source plant is performed. Next, the process of separating the extracted components in the extract using one or more chromatography is performed, and finally, the process of detecting lettuce pudding A is performed. For the step of obtaining a propolis extract from the original propolis mass using an extraction solvent and the step of separating the extracted components in the extract using chromatography, the method described in the first embodiment may be appropriately adopted. it can.

  The step of detecting lettuce pudding A shown in the first embodiment is performed by qualifying the components separated by each chromatography using a known method. For example, the separated components can be qualitatively determined by determining the structure using a nuclear magnetic resonance apparatus or the like. Further, it can be qualitatively and quantitatively compared with a purified product whose components have been identified in advance. In addition, lettuce pudding A shown in the first embodiment has maximum absorption at 289 nm, 380 nm, and 511 nm in the ultraviolet / visible light spectrum, so that the target component can be detected by using a spectrophotometer. Can do. In addition, when thin-layer chromatography is used as a means for separating the extracted components, the component pattern is visualized by using, for example, sulfuric acid, dragendorf, triterpene coloring reagent, ferric chloride, bromocresol green, etc. as the coloring reagent. , Qualitatively from the Rf value.

According to the propolis extract evaluation method of the second embodiment, the following effects can be obtained.
(1) The method for evaluating a propolis extract according to the present embodiment is based on a propolis bulk that is produced from a genus Crane genus as an extraction raw material, and in an operation for confirming an extract obtained from the extraction raw material using an extraction solvent, Let's use Leturspurin A as an index.

  By the way, since propolis bulk is very difficult to ingest as it is, conventionally, for example, by an extraction method using a hydrophilic organic solvent, an extraction method using water, and a supercritical extraction method, Useful components contained in the propolis bulk are extracted, and the extract is applied to various uses. Conventionally, for example, it has been difficult to separate, extract, and qualify p-coumaric acid, which is the main extraction component, in the confirmation of an extract extracted using, for example, a hydrophilic organic solvent. Confirmation has been made by detecting the compound using quercetin, cinnamic acid derivative, or the like as an index. In addition, an extract extracted with water has been confirmed by detecting the compound using chlorogenic acids or the like, which are main extraction components, as an index.

  In the present invention, lettuce purpurin A as a red pigment component, which is a characteristic component, was purified and identified from a red propolis bulk that is produced from plants of the genus Crassica. Therefore, it is possible to confirm the solvent extract obtained from the red propolis ingot that is produced from the genus Crassica using such lettuce purpurin A as an indicator component.

  (2) The method for evaluating the propolis extract according to the present embodiment is based on the fact that when selling the red propolis ingot that is produced from the genus Crassica or the lettuce pudding A extracted therefrom, It can be applied when confirming the presence or absence or content. In addition, in the product of red propolis ingot that is made from the genus Crassica or the product of lettuce pudding A extracted from it, if the content or prescribed content of lettuce pudding A is specified / guaranteed, the product It can be applied in the standard / warranty confirmation process.

  (3) As a method for evaluating the propolis extract according to the present embodiment, the red propolis is composed of the genus Crassica by confirming that the propolis extract contains lettuce purpurin A. Can prove. Further, by measuring the content of suppurin A in the propolis extract, the color tone of red in the propolis raw material and the purity (ratio) of the constituent plant can be estimated. Moreover, it is generally known that the genus Crassica is a legume family and contains a large amount of isoflavones (isoflavans) which are active ingredients of red propolis made in Brazil. By determining the content of lettuce purpurin A in the propolis extract, the presence or absence of isoflavones (isoflavans) can be confirmed, or the approximate content of isoflavones (isoflavans) can be estimated.

In addition, you may change the said embodiment as follows.
-In the said embodiment, lettuce purpurin A isolate | separated by chromatography may be applied to the confirmation operation | work of quantitative analysis and extraction efficiency as a main component of an extract, for example.

Examples and Comparative Examples are given below to describe the above embodiments more specifically, but the present invention is not limited to these.
(Test Example 1: Separation and purification of red pigment component from red propolis bulk from Brazil)
As the propolis mass, the bee hive devised for collecting propolis is Dulbersia as a surrounding plant within a radius of several kilometers. A Brazilian red propolis block obtained by installing in mangrove forest or wild (group) containing ecastophilum for several days was used. To 117.18 g of the pulverized propolis mass, 1500 mL of chloroform / methanol = 2: 1 as an extraction solvent was added, and the mixture was extracted by stirring at room temperature (about 25 ° C.) for 3 days. Then, the stirred extract of the propolis pulverized product was suction filtered with a filter paper (No. 2 manufactured by Advantech Toyo Co., Ltd.) to remove the residue, and concentrated under reduced pressure to obtain 66.19 g of propolis extract (as solid content; 100% by mass).

  Next, 66.19 g of the propolis extract was subjected to open column chromatography (inner diameter 10 cm × length 60 cm). Silica gel (silica gel 60, manufactured by Merck) was used as a chromatography carrier. Using chloroform: methanol = 9: 1 as the eluting solvent, 33 fractions were obtained.

In order to confirm the target pigment component, these fractions were developed by thin layer chromatography (TLC; TLC silica gel 60 F ₂₅₄ , manufactured by Merck & Co., Inc.). As a developing solvent, chloroform: methanol: acetic acid = 8: 1: 1 was used. As a coloring reagent, sulfuric acid: ethanol = 1: 1 was used. After spraying the coloring reagent, it was heated and colored on a hot plate at 100 ° C. to visualize the component pattern, and those showing similar component patterns were combined to obtain 5 fractions (Fraction Nos. 1 to 5). As a result, fraction no. 5 (1400 mL, solid content 3.88 g), the spot of the pigment component was noticeable.

  Next, the above fraction No. 5 (450 mg) was further subjected to separation treatment under the following conditions using preparative reverse-phase HPLC to obtain 5 fractions (fractions 5-1 to 5-5). As a result, it was expected that fraction 5-4 (solid content 53.9 mg) had the highest absorbance and contained the most pigment component. Therefore, the fraction 5-4 was again purified by HPLC under the same conditions. Finally, 32.95 mg was obtained as a dye component.

Column: SHISEIDO CAPCELL PAK C18 UG120Å (inner diameter 20 mm × length 250 mm, manufactured by Shiseido)
Mobile phase: water: acetonitrile = 1: 1 (0.1% TFA)
Flow rate: 6.0 mL / min
Detection wavelength: 500 nm
(Test Example 2: Structural analysis of red pigment component)
(A) Measurement of molecular weight by mass spectrometry The molecular weight of the purified red pigment component was measured. The molecular weight was determined by employing electrospray ionization mass spectrometry (ESI-MS) and using LCQ (manufactured by Thermo Fisher Science). FIG. 1A shows the result of the positive mode, and FIG. 1B shows the result of the negative mode.

As a result, the molecular weight 522 was confirmed by showing a peak at (M + H) ⁺ at m / z 523.27 in the positive mode and at (M−H) ⁻ at m / z 521.13 in the negative mode.

(B) Measurement of ultraviolet / visible light spectrum (UV / vis) The purified red pigment component was measured for ultraviolet / visible light spectrum (UV / vis). The ultraviolet / visible light spectrum was measured using an ultraviolet-visible near-infrared spectrophotometer (JASCO V-560, manufactured by JASCO Corporation), after dissolving the solvent in ethanol, the absorption spectrum was measured. The results are shown in FIG. The vertical axis represents absorbance [Abs], and the horizontal axis represents wavelength (wavelength [nm]).

As shown in FIG. 2, the red pigment component purified was confirmed to have maximum absorption at 289 nm, 380 nm, and 511 nm.
(C) Measurement of infrared absorption spectrum (IR) The infrared absorption spectrum (IR) of the purified red pigment component was measured. For the infrared absorption spectrum, an infrared spectrophotometer (FT / IR-550, manufactured by JASCO Corporation) was used. The results are shown in FIG. The vertical axis represents the relative value, and the horizontal axis represents the wave number (wavenumber [cm ⁻¹ ]). As a result, the presence of carbonyl was confirmed.

(D) Structure determination using nuclear magnetic resonance apparatus (NMR) The purified red pigment component was subjected to structural analysis using a nuclear magnetic resonance apparatus (JEOL α-400, manufactured by JEOL Ltd.). Using deuterated dimethyl sulfoxide _{(DMSO-d 6)} as solvent. Data showing the measurement result of the ¹ H-NMR spectrum is shown in FIG. 4 and Table 1, and data showing the measurement result of the ¹³ C-NMR spectrum is shown in FIG. 5 and Table 1, respectively.

From the above results, it was confirmed that the purified red pigment component was lettuce pudding A having a molecular formula of C ₃₂ H ₂₆ O ₇ having a polyphenol skeleton and a molecular weight of 522.54.

Next, the technical idea that can be grasped from the above embodiment and other examples will be described below.
(A) A colorant or pigment comprising lettuce pudding A.

Claims (5)

In the method for producing lettuce pudding A,
A method for producing lettuce pudding A, comprising a step of performing extraction treatment using an extraction solvent from a propolis bulk that is produced from a genus Crassica as a source plant. The method for producing a lettuce pudding A according to claim 1, wherein the lettuce pudding A is at least one selected from the following general formulas (1) to (5).
  The method for producing the lettuce pudding A according to claim 1 or 2, further comprising a step of performing a separation treatment using chromatography after the step of performing the extraction treatment.   A method for evaluating a propolis extract characterized by using lettuce purpurin A as an index in a method for evaluating a propolis extract obtained by using an extraction solvent from a propolis bulk that is produced from a genus Crassica as a raw material .   A step of obtaining a propolis extract containing lettuce pudding A from an original propolis mass produced from a plant of the genus Cramicus as a raw material by using an extraction solvent, and then one or more of the extract components in the extract The method for evaluating a propolis extract according to claim 4, comprising a step of separating using chromatography, and then a step of detecting lettuce purine A.