JPH01228992A - Tocopherol derivative, production and use thereof - Google Patents

Abstract

NEW MATERIAL:Phenylpropanoide-substituted tocopherol shown by formula I or formula II (R is group shown by formula III). USE:A natural antioxidant having high safety. PREPARATION:First, fresh leaves of a plant (e.g., Prunus donarium Sieb.) are extracted with chloroform and the extracted solution is filtered. Then the filtrate is concentrated under reduced pressure, an oily substance comprising leaf wax is treated by silica gel chromatography to give a fraction having antioxidation activity. Further the fraction is purified by silica gel chromatography twice and the active fraction is successively treated by silica gel thin layer chromatography and liquid chromatography to isolate the aimed substance.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for producing a tocopherol compound having antioxidant activity as a physiological effect from a plant of the genus Prunus (genus Sakura), and an excellent method thereof. It is applied to the food industry, pharmaceutical industry, cosmetics industry, etc. due to its antioxidant activity.

(Prior Art and Problems) Foods are generally manufactured from agricultural products, marine products, livestock products, and the like. However, during the storage and preservation processing of food raw materials and products, food raw materials and products deteriorate due to microbial contamination and putrefaction, as well as chemical and physical effects, reducing their commercial value. For this reason, various food additives have been developed, as well as temperature treatment, deoxidation treatment, vacuum packaging, low temperature storage,
Methods such as radiation treatment have been developed and put into practical use.

Among these types of deterioration of food materials and products, the most problematic is the oxidation or peroxidation reaction of food components caused by oxygen in the air. Oxygen is important for maintaining the life of living organisms through respiration, but on the other hand, as it is a highly reactive compound, it reacts with various components in food, oxidizing or peroxidizing them, reducing their value as products. It is known that not only does it reduce the amount of carbon dioxide, but it also leads to the production of harmful substances in food. For example, nutritionally essential unsaturated fatty acids such as linoleic acid and lylunic acid contained in foods are easily peroxidized by oxygen in the air, producing peroxidized fatty acids and reactive radicals (free radicals). It has also been reported that other carcinogenic substances such as malondialdehyde are produced. It has also been reported that peroxidized lipids, which are produced when unsaturated fatty acid molecules in lipids are peroxidized, alter proteins and nucleic acids in living organisms through chemical reactions, and have carcinogenic effects on living organisms. . (“Mutagen and Toxicity”
Vol. 5, p. 243 (1982), "Food Packaging" No. 1
7, p. 106 (1986)).

In order to prevent such peroxidation of lipids, packaging techniques such as removing oxygen from packaging with an oxygen scavenger, vacuum packaging, and nitrogen gas displacement packaging are used. On the other hand, with the development of the chemical industry, synthetic antioxidants such as butylated hydroxyanisole (DIIA) and butylated hydroxytoluene ([1llT) have been commonly used. However, as the use of these synthetic antioxidants increases, food pollution increases, leading to major safety issues, and consumers' negative reaction to synthetic antioxidants becomes stronger, leading to a decline in their usage. The current situation is that

On the other hand, as mentioned above, peroxides and carcinogens that are generated in animal bodies due to the toxic effects of oxygen are thought to have a negative effect on animal cells. is thought to be related to cell aging and, ultimately, to lifespan (cree radical aging theory). Therefore, highly safe, naturally derived antioxidant substances are useful in the technical fields of food, especially health foods and nutritional foods, as well as pharmaceuticals and cosmetics, as substances that support the antioxidant defense mechanism of the living body. There are high expectations for this.

However, natural antioxidants that are expected to be used in place of synthetic antioxidants that pose food pollution problems include vitamin C produced by chemical synthesis and vitamin E (tocopherol) extracted and purified from natural products. ) has just been put into practical use.

In order to appropriately use these naturally derived antioxidants in foods, medicines, cosmetics, etc., it is important to discover natural antioxidants with different properties and utilize them under conditions that exhibit their respective characteristics.

In the present invention, we have succeeded in extracting these naturally derived and highly safe antioxidant substances from natural plants and mass producing them, and we have developed a natural antioxidant that can replace synthetic antioxidants. It provides new technology to provide

(Problem to be solved by the invention) To find a naturally derived antioxidant substance from plants in the natural world that has a strong antioxidant effect in place of vitamin C and vitamin E, which are currently used as naturally derived antioxidants. This makes it possible to apply it to foods, medicines, and cosmetics.

(Means for Solving the Problems) In the present invention, in order to solve these problems, we searched for antioxidant substances from plants in the natural world. As a result, Pru
We have discovered a new substance that has strong antioxidant effects in plants belonging to the genus Nus (genus Sakura).

The present invention was completed based on such basic research.

Specifically, leaf wax contained in the leaves of a wide variety of plants was extracted and the antioxidant activity contained therein was analyzed.
We found strong antioxidant activity in leaf wax prepared from leaves of plants belonging to the genus Sakura, and isolated antioxidant substances from this as a pure standard. When the structure was analyzed using the latest analytical equipment, it was discovered that the compound had a completely new structure that had not been reported before, and the present invention was completed.

The surface layer of plant leaves is protected by a hydrophobic substance called leaf wax, which plays an important role in maintaining normal leaf cell activity. Antioxidant effect is thought to be one of its protective effects. Eucalyptus leaf wax is widely used as a food additive due to its aroma, but eucalyptus leaf wax also contains
Recognized antioxidant properties. (Agricultu
ral and Biological Chemist
ry, agricultural and biological
Chemistry, vol. 45, p. 735, 1981).

FIG. 1 shows the structural formula of a new antioxidant substance discovered from the leaf wax of a plant belonging to Prunusa (Prunus genus) according to the present invention. These have a structure in which tocopherol and metabolic intermediates of phenolic compounds in plant cells are combined, and are thought to be produced during the metabolic process. Substances I and 2 shown in the two structural formulas shown in FIG. 1 are optical isomers of each other. These are called phenylpropanoid-substituted tocopherols.

The plants used in the present invention are plants belonging to the Prunus genus (Prunus genus), such as Prunus Grayana.
Maxill, (Uwamizuzakura L Prunus v.
oehne (kasumi cherry) in erecunda,
Prunus donariu+s 5ieb, (Yamazakura), etc. are used. Using leaf wax extracted from the leaves of these plants as a standard, we analyzed the antioxidant activity using Rodan iron, which is commonly used to measure antioxidant activity.We found that synthetic antioxidant (BIIA) and natural antioxidant Antioxidant activity was found to be comparable to that of other agents such as α-tocopherol.

The iron rhodan test, which is used for the analysis of antioxidant substances, uses linoleic acid as a reaction substrate, and is a method in which air-induced autooxidation is colored by rhodan iron salt, and is used to examine lipid overbreeding.

(Agricultural and Biological Chemistry, Vol. 45, p. 735, 1981).

Next, a method for extracting and purifying phenylpropanoid-substituted tocopherol from leaves of Prunusa (Sakura a) plant will be described.

The collected fresh leaves of the genus Prunus are extracted with chloroform, filtered, and concentrated under reduced pressure to obtain an oily substance. This was subjected to silica gel chromatography and eluted with a mixed solvent of n-hexane and ethyl acetate. Fractions with antioxidant activity are collected and subjected to silica gel chromatography again, and eluted with a mixed solvent of benzene and ethyl acetate. After the fraction with antioxidant activity is concentrated to dryness, it is extracted with n-hexane to obtain a fraction soluble in n-hexane. This was further subjected to silica gel chromatography, eluted with a mixed solvent of n-hexane and ethyl acetate, and the fractions with antioxidant activity were collected, and subjected to silica gel thin layer chromatography and then liquid chromatography to obtain single substances I and II. get.

FIG. 2 shows the state of development of the thus prepared single component having antioxidant activity in silica gel thin layer chromatography.

Structural analysis of compounds with antioxidant activity isolated as single components is performed using commonly used equipment.
We analyzed its chemical structure. First, the structural analysis of substance I will be described. Figure 3 shows an analytical diagram of substance I by electron impact mass spectrometry (EI-MS).The molecular ion peak of substance fil is m/z 562, and from this the molecular formula is C,, Hs, 0. It was decided. On the other hand, analysis by nuclear magnetic resonance (NMR) spectroscopy suggested the presence of a benzene ring in substance I. “C-N
It was analyzed by MR spectroscopy and compared with the signal of tocopherol, and it was determined that Substance I contained tocopherol molecules.

In addition to these structural analysis methods, infrared absorption spectroscopy (IR) shown in Figure 4, ultraviolet absorption spectroscopy (UV) shown in Figure 5, and "C-N" shown in Figure 6
As a result of analysis using MR spectroscopy, it was concluded that Substance I was a phenylpropanoid-substituted tocopherol, as shown in FIG.

Next, we will discuss the structural analysis of substance ■. The analysis diagram of substance (2) by electron impact mass spectrometry was exactly the same as substance I, and the molecular ion peak of substance (2) was ra/z 562, similar to substance I. Also,'
Also in H-NMR spectroscopy, IR spectroscopy, and UV spectrum 13C-NMR spectroscopy, substance (2) was the same as substance (2).

However, as shown in Figure 2, according to silica gel thin layer chromatography, there is a clear difference in the development states of substance I and substance ■, and circular digloism spectroscopy of substance I and substance ■ CD) analysis (Figure 7), it was concluded that Substance ■ and Substance ■ are mutually optical isomers, and the chemical structure of Substance H was determined to be the phenylpropanoid-substituted tocopherol shown in Figure 1. Ta.

The present invention will be explained using the following examples and test examples, but these are illustrative and do not limit the present invention.

Example I 35 kg of fresh leaves of Prunus Grayana Maxim, (Uwamizuzakura) were collected. 1 for this
Extracted with 52 chloroform at room temperature. The extract was separated from the leaves by filtration, extracted again by adding ISO chloroform, and then the extract was separated from the leaves by filtration. The extracts were combined and concentrated under reduced pressure to completely evaporate the chloroform, yielding 400 g of leaf wax.

The obtained leaf wax was divided into 130 to 140 g, each adsorbed on a silica gel adsorption column with a diameter of 10 cI11 and a length of 65 cm, and fractionated by elution with a mixed solvent of n-hexane and ethyl acetate. At this time, an elution method was performed in which the ratio of ethyl acetate was increased stepwise.The fractions eluted with a mixed solvent with a ratio of 6n-hexane and ethyl acetate of 6:4.5:5.4:5 were collected and concentrated and dried. After solidification, 49 g of a compound with antioxidant activity was obtained. This was adsorbed again on a silica gel column with a diameter of 8 cm and a length of 60 cm, and then eluted with a mixed solvent of benzene and ethyl acetate. At this time, an elution method was performed in which the proportion of ethyl acetate was increased stepwise. Antioxidant activity was observed in the fractions eluted with mixed solvents with benzene and ethyl acetate ratios of 8:2 and 7:3, so these fractions were collected and concentrated to dryness under reduced pressure. .7 g of compound material was obtained. From this, we investigated better purification conditions, and since the product contained impurities insoluble in n-hexane, only the n-hexane soluble portion was collected and concentrated to dryness to obtain 2.6 g of an intermediate purified product.

For further purification, it was adsorbed onto a silica gel column with a diameter of 4 cm and a length of 40 cm, and then eluted with a mixed solvent of n-hexane and ethyl acetate, resulting in a mixed solvent with a ratio of n-hexane and ethyl acetate of 7:3. Antioxidant activity was observed in the fraction eluted by

This fraction was concentrated to dryness to obtain 310 mg of intermediate purified product.

The intermediate purified product thus obtained was further purified to a single component by the commonly used thin layer chromatography method and liquid chromatography method, resulting in a substance! is 21B, substance■
obtained 1g of 15I.

Example 2 As a plant belonging to the genus Prunus (genus Sakura), Pr
oehne to unus Grayana Maxim, (Uramizzakura), Prunus verecunda
(Prunus donarium), Prunus donarium
5ieb.

500 g of each fresh leaf of (Yamazakura) was collected. In the same manner as in Example 1, each was extracted with IQ chloroform and concentrated to dryness under reduced pressure to obtain leaf waxes of 5.4 g of Umamizuzakura, 4.2 g of Kasumizakura, and 5.6 g of Yamazakura, respectively.

Next, the leaf wax of Uwamizuzakura was adsorbed onto a silica gel column having a diameter of 4 c+w and a length of 40 c+m, and then eluted with a mixed solvent of n-hexane and ethyl acetate in the same manner as in Example 1. The substance! Since it contained 1 and 2, these were collected and concentrated to dryness under reduced pressure. In the same manner, impurities were removed from leaf waxes obtained from Kasumizakura and Yamazakura by silica gel chromatography to obtain leaf waxes.

The leaf wax thus obtained was used as a standard to analyze the amounts of the substances if and H contained therein. As a standard substance, use the substance purified to a single component in Example 1! It was measured using liquid chromatography, which is used in ordinary separation and analysis methods, using As a result, 1000g of leaf wax was added to Uwamizuzakura.
The amount of substance (①) was 96 mg, and the amount of substance (②) was 72 mg.

Also, in Kasumizakura, the substance ■ is 87B, and the substance ■ is 7B.
It is 1mg.

In Yamazakura, substance I is 104 mg, and substance gn is 78 m.
It was g.

Test Example 1 Using the phenylpropanoid-substituted tocopherol substances I and ■, which were isolated and purified to a pure state in Example 1, as standards, the antioxidant activity was determined using rhodanic iron, which is commonly used for the analysis of antioxidant activity. was measured.

Rodan iron properties are published in Agricultural and Biological Chemistry, vol. 45, p. 735 (1'118
1), but the principle will be briefly described.

Using linoleic acid, an unsaturated fatty acid, as a substrate,
The production of hydroperoxide through the autoxidation reaction of linoleic acid will be monitored using oxygen in the air as the oxidizing agent. The generated hydroperoxide reacts with ferrous ions and ammonium thiocyanate to form red ferric thiocyanate. By measuring the red color development with absorbance at 500 nm, we can determine the autooxidation of linoleic acid. Amounts can be measured. The antioxidant activity of the specimen is determined by the degree of inhibition of autooxidation of linoleic acid in such a reaction system.

What was obtained by the present invention through such a method? The antioxidant activities of tI and ■ are shown in FIG. Synthetic antioxidant (B
HA) and tocopherols, the substances ■ and ■
has been shown to have strong antioxidant activity.

(Effects of the Invention) The present invention provides a new type of naturally-derived antioxidant substance, which can be used as an alternative to conventionally produced synthetic antioxidants.

It is safely used as a raw material for foods, medicines, cosmetics, etc.

[Brief explanation of the drawing]

FIG. 1 shows the structural formulas of substances 1 and 2 of the present invention. FIG. 2 is a thin layer chromatogram of substances I and ■. The thin layer was formed using silica gel, the developing solvent was a mixture of n-hexane and ethyl acetate at a ratio of 3:1 (by volume), and the substance was detected using 40% sulfuric acid for color development. (1) is substance 1. (2) is a substance ■, and (3) is a tocopherol. FIG. 3 is a chart of the electron impact mass spectrum (EI-MS) of substance 1. FIG. 4 shows the optical external absorption spectrum of substance 'II. FIG. 5 shows the ultraviolet absorption spectrum of Substance I. FIG. 6 is a 13C-NMR (nuclear magnetic resonance) spectrum diagram of Substance I. FIG. 7 shows the circular dichroism spectra (CD) of substance [1 and substance 2]. □: Substances - 112 Substances■ Figure 8 is a measurement diagram of antioxidant activity based on Rodan iron properties. 10-2 control group 10-: α-tocopherol group 10-: BHA group 1-: substance l -he 1: substance ■ Agent Patent attorney Door 1) Parent Male Figure 1 (1) (n) Do : (shiH2)3(twoH(nito12)3C)((
CH223C1-1cH3 Fig. 2 Fig. 5 Wave- & (nm) Fig. 7-40

Claims (1)

[Claims] 1. Substance I or II of the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) Where R is ▲ Numerical formulas, chemical formulas, tables, etc. Phenylpropanoid-substituted tocopherols are represented by ▼. 2. Substance I of the formula shown in item 1, which is extracted from a plant belonging to the genus Prunus (genus Sakura)
or a method for producing a phenylpropanoid-substituted tocopherol represented by II. 3. An antioxidant containing a phenylpropanoid-substituted tocopherol represented by substance I or II of the formula shown in item 1 as an active ingredient.