JPS628474B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an antioxidant that is added to foods to prevent their oxidation, and more specifically to a novel compound represented by the following formula []. The present invention relates to a method for producing an antioxidant containing 7β·11·12-trihydroxy-6·10-(epoxymethano)avieta-8·11·13-trien-20-one. Traditionally, synthetic additives such as BHA (butylated hydroxyanisole) and BHT (dibutylated hydroxytoluene) have been used as food antioxidants, but in recent years natural antioxidants have been used due to safety and palatability concerns. There has been a demand for the use of natural antioxidants, and for this reason, research has been conducted on various natural antioxidants.In particular, the usefulness of natural spices has been reconsidered, and research on them has become widespread. Research on the search for antioxidant components present in spices began with sage (Salvia carnosa Dougl).
announced that the bitter component carnosol was isolated _from the seed (AIWhite et _al ., _{J.Am.Pharm.Assoc.Sci} . .Ed.,
p. 31, 33, 37 (1942)), and furthermore, carnosol was used in Salvia officinalis L. species and Salvia
A report was published showing that picrosalvin is the same substance as the bitter component piclosalvin from sage and rosemary (Rosmarinus officinalis L.) species triloba, and has the structure of the following formula (CHBrieskorn et al. , J.Org.Chem.
29, 2293 (1964)). Furthermore, it was revealed that carnosic acid, shown in the following formula (), exists as a major terpene component of rosemary, and its structure was determined (E.
Wenkert et al., J.Org.Chem., 30, 2931 (1965)). Recently, in addition to carnosic acid, carnosol, and phenyldinol, which have antioxidant properties, we have added flavorless and odorless antioxidant substances by using solvent extracts of rosemary and sage, washing them with water, decolorizing them, and separating their components using high-performance liquid chromatography. (SSChang et al., J.Food.Sci., 42, 1102
(1977)). There are many research reports on the search for antioxidant components in spices, but it is difficult to use spices directly as antioxidants because they have a strong, unique flavor that can affect the flavor of food. Therefore, it cannot be applied to foods that dislike strong flavors, such as Japanese food. For this reason, various proposals have been made to extract antioxidant components from spices and use them as antioxidants. For example, in Japanese Patent Publication No. 53-9595, spices (rosemary) were directly extracted with pH
It is proposed to extract the fraction containing carnosic acid by extraction with an alkaline solution of ~10 and use it as an antioxidant. As a result of various studies on the antioxidant fraction obtained from rosemary, the present inventors extracted rosemary with a non-polar solvent and washed it with steam distillation. and a weak alkaline aqueous solution with a pH lower than 10.5 to transfer the strongly acidic impurities in the residue or extract into the weak alkaline aqueous solution, and then remove this weak alkaline aqueous solution to form a non-polar aqueous solution. It was discovered that a fraction with a high antioxidant effect can be obtained by collecting the solvent layer, then adding an alkaline aqueous solution with a pH of 10.5 or higher to this non-polar solvent layer, and extracting with this alkaline aqueous solution with a pH of 10.5 or higher. did. That is, according to the above-mentioned Japanese Patent Publication No. 53-9595, it is stated that what is extracted at a pH of 10 or higher is essentially an oxidation promoter, and that it is not preferable to extract at a pH of 10 or higher. According to the results of their study, the alkali extraction fraction with a pH of 10.5 or higher has a strong antioxidant effect, and further study of this extracted fraction revealed that there are new compounds in this fraction. There are 7
β・11・12-trihydroxy-6・10-(epoxymethano)abieta-8・11・13-triene-20
In addition to containing about 1 to 10% by weight of -one, this new compound also has a strong antioxidant effect, and the antioxidant properties of the alkali extract fraction with a pH of 10.5 or higher are mainly due to this new compound. The present invention was made based on this knowledge. That is, the present invention provides a residue obtained by steam distilling an extract obtained by extracting rosemary with a non-polar solvent, or a residue obtained by steam-distilling an extract obtained by extracting rosemary with a non-polar solvent. The extracted extract is extracted with a mixture of a nonpolar solvent and a weak alkaline aqueous solution with a pH lower than 10.5, and the strongly acidic impurities in the residue or extract are transferred to the weak alkaline aqueous solution. The weak alkaline aqueous solution is removed to collect a non-polar solvent layer, then an alkali aqueous solution with a pH of 10.5 or higher is added to this non-polar solvent layer for extraction treatment, and this alkaline aqueous solution with a PH of 10.5 or higher is collected. 7β・11・12-trihydroxy-6・10
A method for producing an antioxidant containing -(epoxymethano)abieta-8,11,13-trien-20-one is provided. The present invention will be explained in detail below. In the method for producing an antioxidant according to the present invention, as the raw material rosemary, powder thereof is usually used. In the present invention, this rosemary is used as a raw material and is first subjected to an extraction treatment using a non-polar solvent and a washing and deodorizing treatment using steam distillation. In this case, either the extraction treatment or the washing and deodorizing treatment may be performed first. That is, rosemary is first extracted with a non-polar solvent, the extract is collected, and after the solvent is distilled off, the resulting extract is poured into water, steam distilled, and the residue is collected. Good too,
Alternatively, rosemary is first treated with steam distillation,
The resulting steam distillation residue may be extracted with a nonpolar solvent to obtain an extract, and if necessary, the solvent may be distilled off to collect the extract. However, in terms of operability, equipment scale, etc., a method in which extraction is first performed and then steam distillation is performed is recommended. Examples of the non-polar solvent used in the extraction process include petroleum ether, ligroin, n-hexane, cyclohexane, carbon tetrachloride, chloroform, dichloromethane, ethylene chloride (dichloroethane,
Monochloroethane, etc.), toluene, benzene, etc., and n-hexane and ethylene chloride are preferably used. in this case,
The amount used for extraction is not particularly limited, but it is preferably 3 to 10 parts by weight per 1 part by weight of the raw material. As the extraction method, a conventional extraction method such as a batch method or a reflux method may be employed. Although the extraction may be carried out at room temperature or under heating, it is more efficient to carry out the extraction under reflux. Further, the longer the extraction time is, the more preferable it is, but it is usually sufficient for 2 to 24 hours, and it is preferable to repeat the same extraction operation one to several times on the extraction residue. After the extraction operation, the extract is separated into an extract and an extraction residue using a conventional method such as filtration, centrifugation, or decantation. From the extract obtained by this extraction process, the solvent is distilled off under normal pressure or reduced pressure to collect an extract, which is sent to the next step. In addition, a normal method can be adopted as the steam distillation treatment, for example, the solid material (raw material rosemary or the extract obtained by the extraction treatment with the non-polar solvent) is preferably distilled into water at least 10 times its weight. A method may be adopted in which the spices are poured into water, stirred, dispersed in water, heated under normal pressure or reduced pressure, and boiled. Through this steam distillation process, the aromatic components in the raw spices are evaporated with water vapor, and the solids are dispersed. removed from the liquid. In this case, steam can be blown into the dispersion in the steam distillation process to promote volatilization of the aroma components. After continuing steam distillation until almost no aroma components are recognized in the steam distillate (usually 0.5 to 10 hours), the steam distillation residue is filtered, centrifuged, decanted, etc. while heated or cooled. The method separates the aqueous layer and the solid content (steam distillation residue), and the solid content is collected. The present invention provides a fraction obtained by the above-mentioned treatment,
That is, the residue obtained by steam distilling the extract obtained by extracting rosemary with a non-polar solvent, or the extract obtained by extracting the residue obtained by steam-distilling rosemary with a non-polar solvent. Preferably, the fraction obtained by the extraction treatment with the non-polar solvent and the steam distillation treatment is dissolved in a non-polar solvent such as ethyl ether in an amount of 1 to 10 parts by weight per 1 part by weight of this fraction, and the pH thereof is lower than 10.5. A low alkaline aqueous solution is added and the fraction is extracted with a mixture of a polar solvent and an alkaline aqueous solution with a pH lower than 10.5. By treating with the weak alkali aqueous solution in this manner, strongly acidic impurities in the fraction obtained by extraction with a nonpolar solvent and steam distillation are transferred to the weak alkali aqueous solution. In addition, PH is 10.5
Examples of lower alkaline aqueous solutions include saturated aqueous sodium bicarbonate solutions. Next, the alkaline aqueous solution with a pH lower than 10.5, into which the strongly acidic impurities have migrated, is separated and removed to collect the non-polar solvent layer. An aqueous alkaline solution with a pH of 10.5 or higher is added to this non-polar solvent layer to perform an extraction process. As a result, the antioxidant component containing the new compound represented by the formula [] moves to the alkaline aqueous solution side, and therefore, after the extraction process, the alkaline aqueous solution layer is separated from the non-polar solvent layer and the alkaline aqueous solution layer is collected. Fraction with definitely high antioxidant effect (antioxidant of the present invention)
is obtained. In this case, the alkaline aqueous solution is preferably used in a ratio of 0.1 to 2 parts per 1 part of the nonpolar solvent layer. It is also desirable to operate in a nitrogen or inert gas stream to avoid contact with oxygen in the air during extraction. As the alkaline aqueous solution used in this extraction process, a solution prepared by dissolving sodium hydroxide, potassium hydroxide metal hydroxide, alkaline earth metal hydroxide, etc. in water can be used, but in any case, the pH is 10.5. As mentioned above, it is necessary to use an aqueous alkali solution preferably adjusted to a pH range of 10.5 to 11.0, and by performing an extraction process using an alkaline aqueous solution in this pH range, the antioxidant components including the new compound [] can be reliably extracted. Extracted.
On the other hand, even if the extraction treatment is performed using an alkaline aqueous solution with a pH lower than 10.5, the antioxidant components including the new compound [] cannot be extracted well, and the object of the present invention cannot be achieved. After this extraction process, an alkaline aqueous solution with a pH of 10.5 or higher (into which antioxidant components including the new compound [] have migrated and is contained) is collected and used as an antioxidant. The antioxidant fraction used is obtained by neutralizing this alkaline aqueous solution with an acid, or by extracting the antioxidant component with a nonpolar solvent after neutralizing or making it acidic with an acid. It is preferable to use a residue obtained by distilling off the solvent from the extract. In the antioxidant fraction thus obtained (antioxidant of the present invention), rosemary is extracted with a non-polar solvent and steam distilled, and then mixed with a non-polar solvent with a pH lower than 10.5. After performing an extraction treatment with a mixed solution with a weak alkali aqueous solution and transferring the strong acidic impurities in the residue or extract to the weak alkali aqueous solution, the weak alkali aqueous solution is removed to collect a non-polar solvent layer, Next, add an alkaline aqueous solution with a pH of 10.5 or higher to this non-polar solvent layer to adjust the pH to 10.5.
By performing the extraction treatment with the above aqueous alkaline solution, the antioxidant components in rosemary, especially the new compound 7β, 11, 12 shown by the above formula []
-Trihydroxy-6・10- (epoxymethano)
Avieta-8,11,13-trien-20-one is transferred reliably, and this new compound is contained at about 1 to 10% by weight, and it has strong antioxidant power mainly due to this new compound. It is. In addition, in order to obtain a new compound of the formula [] from this antioxidant fraction, this antioxidant fraction (in this case, an extraction obtained by making an alkaline aqueous solution neutral or acidic and then extracting with a non-polar solvent) (using objects)
is subjected to silica gel-packed column chromatography, and fractionated into 110 subdivisions using a 90:10 (volume ratio, same below) mixed solvent of benzene and acetone as a developing solution, and the 60th fraction is extracted from the 16th fraction. . From these categories 16 to 38, carnosol, which is known as an antioxidant component, can be obtained, and in close proximity to this, the new compound described []
can be obtained. That is, carnosol is obtained by recrystallizing the solids of sections 16 to 28 obtained by removing the developing solution with benzene, and the residue obtained by concentrating the benzene solution from which carnosol has been removed and the solids of sections 29 to 28 are obtained by recrystallizing them with benzene. The solids in 38 sections were combined and subjected to silica gel packed column chromatography, developed with a 90:10 mixed solvent of benzene and acetone, and fractionated into 15 sections.Carnosol was obtained from the 7th and 8th sections. The developing solution is removed from the 10th to 11th sections, and the new compound shown in [] is obtained by recrystallizing with acetone. In addition, by recrystallizing the solid in the 39th to 60th divisions in the first chromatography with benzene and recrystallizing this crystal with acetone, a new compound [] can be obtained and benzene can be removed from this benzene-soluble part. The solid obtained was subjected to silica gel packed column chromatography using a mixture of benzene and acetone in a ratio of 90:10.
This new compound [] can also be obtained by developing it into six sections with a mixed solvent of , taking out the fourth section, removing the developing solution, and recrystallizing it with acetone. The characteristics and antioxidant effect of this new compound are as shown in the reference examples described below, and it has an antioxidant power approximately twice that of the known carnosol. The antioxidant of the present invention containing the new compound []
Fish oil, lard, tallow, head, chicken oil,
Animal and vegetable oils such as soybean oil, linseed oil, cottonseed oil, surfflower oil, rice oil, corn oil, coconut oil, palm oil, sesame oil, cacao butter, castor oil, and peanut oil, butter, cheese, Margarine, seasoning, mayonnaise, dressing, ham, sausage, potato chips, fried rice crackers, fried ramen, curry roux, soy sauce, soft drinks, sake,
It can be used by being added to fruit wine, ketchup, jam, fish meat or meat paste products, other foods, cosmetics such as hair cosmetics, skin cosmetics, mouth cosmetics, and pharmaceuticals. In this case, the amount of the compound of the present invention added is preferably 0.0005 to 1% by weight, particularly 0.001 to 0.1% by weight of the total dry product. The antioxidant according to the present invention may be used as it is, but if necessary, it may be made into a powder or granule form by adding excipients such as starch or gelatin. Alternatively, it may be dissolved and dispersed in ethanol, propylene glycol, glycerin, or a mixture thereof to obtain a liquid form. Further, the antioxidant of the present invention may contain a synergist component such as citric acid as necessary. As mentioned above, the antioxidant according to the present invention has high antioxidant power, and although it is obtained from rosemary, it has almost no rosemary aroma, so even if it is added to foods, etc., the flavor will not be impaired. Moreover, since it is obtained by separating it from natural rosemary, the safety of synthetic additives is now a problem, and it is used in foods such as oil and fat products, cosmetics, pharmaceuticals, etc. It can be suitably used for. Next, Examples and Comparative Examples will be shown to further specifically explain the present invention. Examples and Comparative Examples 500 g of dried rosemary leaves were crushed, 1.2 g of n-hexane was added, and extraction was performed with stirring overnight at room temperature. After separating the liquid by suction filtration, an equivalent amount of n-hexane was added to the residue and treated in the same manner. This operation was performed three times, and the three extracts were collected, and the solvent was distilled off under reduced pressure and concentrated to obtain 19.71 g of n-hexane extract. Next, 19.71 g of this extract was added to 200 ml of water, and after steam distillation was performed to remove 2.75 g of volatile fraction, the steam distillation residue was filtered and separated into liquid and residue. This residue was collected and dissolved in 400 ml of ethyl ether, and then the ether layer was extracted twice with 150 ml of 2N aqueous hydrochloric acid solution, and the hydrochloric acid layer was separated from the ether layer. This ether layer was washed twice with water, and then extracted three times with 150 ml of a saturated aqueous sodium bicarbonate solution in a nitrogen stream. After separating the saturated sodium bicarbonate aqueous solution layer, the ether layer was washed twice with water, and then 1N-sodium hydroxide aqueous solution 150 ml was added in the same manner in a nitrogen stream.
ml four times, and the aqueous sodium hydroxide layer was separated from the ether layer. This ether layer was washed twice with water, dried over anhydrous magnesium sulfate, and concentrated to obtain 10.2 g of a neutral fraction. On the other hand, the hydrochloric acid layer was made alkaline with a 4N aqueous sodium hydroxide solution, extracted with ethyl ether, the ether layer was separated and collected, and the solvent was distilled off to obtain a basic fraction. , the amount was only a trace. The saturated aqueous sodium bicarbonate layer was made acidic with 4N hydrochloric acid solution, extracted with ethyl ether, and the ether layer was washed with water, dried, and concentrated to obtain 0.049 g of a strongly acidic fraction. Furthermore, the sodium hydroxide aqueous solution layer is also 4N
- Acidified with hydrochloric acid solution, extracted with ethyl ether, and the ether layer was washed with water, dried, and concentrated to obtain 1.9 g of a weakly acidic fraction (antioxidant of the present invention). The results of analysis of this weakly acidic fraction showed that the new compound []
It contained 2.5%. For comparison, a neutral fraction and a weakly acidic fraction were obtained from the volatile fraction of the n-hexane extract by steam distillation in the same manner as above. Next, 0.01% by weight of each component obtained by the above method was added to lard, and the AOM method (oil chemistry,
Peroxide value (POV) according to Volume 19, No. 5, P62)
was measured. The results are shown in Table 1 and Figure 1.

【table】

[Table] In Figure 1, reference symbols A, B, C,
D, E, and F are as shown in Table 1, respectively,
A' is the peroxide value when 0.005% by weight of the weakly acidic fraction obtained from the n-hexane extract (the antioxidant of the present invention) is added to lard. As is clear from the results in Table 1 and FIG. 1, it was found that only the weakly acidic fraction, ie, the fraction obtained according to the method of the present invention, had high antioxidant power. Next, according to the reference example, the new compound 7β・11・12−
A method for producing trihydroxy-6,10-(epoxymethano)abieta-8,11,13-trien-20-one and its antioxidant effect will be explained. Reference Example Take 1.42 g of the weakly acidic fraction obtained in the example and subject it to silica gel packed column chromatography (3.0 cmφ x 24 cm, 80 g of silica gel, 0.5 ml/min)
Using a developing solution with a ratio of benzene and acetone of 90:10, divide into 110 sections (100 ml per section), take out sections 16 to 60, remove the developing solution, and obtain each solid. Ta. 348 mg of the solid from the 16th to 28th fractions was dissolved in 3 ml of benzene and recrystallized from this to obtain 180 mg of colorless needle-like crystals with a melting point of 233°C (which was confirmed to be carnosol by instrumental analysis). The residue obtained by concentrating the benzene solution from which carnosol was removed was combined with 108 mg of the solids from sections 29 to 38, and 274 mg of this solid was subjected to silica gel packed column chromatography (2.0 cmφ x 17 cm, 20 g of silica gel, 0.5
ml/min), perform a second fractionation operation, develop with a 90:10 mixture of benzene and acetone, and
The solvent was removed from the 7th to 8th sections and recrystallized with benzene to obtain 120 mg of carnosol, and the solvent was removed from the 10th to 11th sections and recrystallized with acetone. 26 mg of the indicated new compound was obtained. In addition, solids obtained from fractions 39 to 60 of the first fraction
107 mg was dissolved in 1 ml of benzene, recrystallized from this, separated into crystals and benzene soluble fraction, and the crystals were recrystallized from acetone to obtain 10 mg of the desired new compound. On the other hand, 80 mg of the solid obtained by removing the benzene from the benzene solution was subjected to silica gel packed column chromatography (1.5 cmφ x 11 cm, 8 g of silica gel, 0.35
ml/min) of benzene and acetone at a ratio of 90:10.
A third fractionation was carried out using the mixture of 6 fractions (1
The solid obtained from the fourth portion was recrystallized from acetone to obtain 10 mg of the desired new compound. This new compound 7β・11・12-trihydroxy-6・10-(epoxymethano)abieta-8・
11・13-trien-20-one exhibits the following properties and instrumental analysis values.

【table】

[Table] Next, the new compounds obtained by the above method, carnosol, the antioxidant α-tocopherol obtained from natural products, and the synthetic antioxidants BHA and BHT.
using the AOM method (Oil Chemistry, Vol. 19, No. 5,
A comparative test of the antioxidant effect was conducted using p62). As a blank, lard containing no antioxidants was taken, and each sample was added to it.
Its peroxide value (POV) was measured. The results are shown in Table 2 and Figure 5.

【table】 [Brief explanation of the drawing]

Figure 1 is a graph showing the antioxidant power of the antioxidant obtained by the method of the present invention and other fractions from rosemary, and Figure 2 is the infrared absorption spectrum of the new compound contained in the oxidant of the present invention. , Figure 3 shows the nuclear magnetic resonance spectrum of the same compound, Figure 4 shows the mass spectrum of the same compound, and Figure 5 shows the anti-oxidant spectrum of the new compound contained in the antioxidant obtained by the method of the present invention and the known antioxidant. It is a graph showing oxidizing power.

Claims (1)

[Claims] 1 For the residue obtained by steam distilling the extract obtained by extracting rosemary with a non-polar solvent, or the extract obtained by extracting the residue obtained by steam-distilling rosemary with a non-polar solvent, non-polar solvent and
Extraction treatment is performed with a mixture of a weak alkaline aqueous solution with a pH lower than 10.5 to transfer the strongly acidic impurities in the residue or extract into the weak alkaline aqueous solution, and then the weak alkaline aqueous solution is removed to form a non-polar solvent. The layer is collected, and then an aqueous alkali solution with a pH of 10.5 or higher is added to this non-polar solvent layer for extraction treatment, and this PH
Collect an alkaline aqueous solution of 10.5 or higher and
A method for producing an antioxidant, which comprises obtaining an antioxidant containing 11,12-trihydroxy-6,10-(epoxymethano)abieta-8,11,13-trien-20-one.