JPH0144232B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing antioxidants from sage.

Conventionally, various measures have been taken to prevent food from deteriorating due to oxidation, one of which is the addition of synthetic antioxidants, but recently synthetic additives have been introduced from a safety standpoint. There is a growing need for a reconsideration of antioxidants, and effective natural antioxidants are therefore desired.

Traditionally, it has been known that herbal spices have the effect of preventing oxidation of oils and fats and oil-based foodstuffs, but it is very difficult to use spices themselves as antioxidants because the flavor is too strong. There is a problem that it can only be used for some purposes.

The present inventors conducted various studies with the aim of finding safer and more effective antioxidant substances, and as a result, they found that a specific fraction obtained from sage achieves the above objectives. I found out.

That is, by using sage as a raw material, extracting it with n-hexane or methylene chloride, subjecting the obtained extract to steam distillation, and collecting the weakly acidic fraction from the obtained residue according to a conventional method. , the antioxidant components in sage migrate into the steam distillation residue and further into the weakly acidic fraction, and therefore the weakly acidic fraction obtained by performing the above process exhibits an extremely high antioxidant effect. I found out. Furthermore, this fraction contains carnosol represented by the following formula (1), and 7β,11,12-trihydroxy-6,10- represented by the formula (2).
Epoxymethanoabieta-8,11,13-trien-20-one (rosmanol), a new compound 12-hydroxy-6,7-seco-11,10 represented by formula (3)
-Epoxymethano-20-Oxo-Aviator-8,
Contains 11,13-triene-6,7-dial (rosmazial), 5,4'-dihydroxy-6,7-dimethoxyflavone represented by formula (4), etc., and these components We found that this fraction is one of the factors that gives sage a high antioxidant effect, and that the fraction has almost no strong odor characteristic of sage, and that this fraction can be used very effectively as an antioxidant. , has completed the present invention.

(2) 7β,11,12-trihydroxy-6,10-
epoxymethano―abieta―8, 11, 13―trien―
20-one (rosmanol) (3) 12-hydroxy-6,7-seco-11,10-
epoxymetha-20-oxo-abieta-8, 11, 13
-trien-6,7-dial (rosmadial) (4) 5,4'-dihydroxy-6,7-
dimethoxyflavone The present invention will be explained in more detail below.

In the method for producing an antioxidant of the present invention, sage is used as a raw material. Sage can be fresh or dried. In addition, the whole plant, the above-ground part, etc. of sage can be used as a cut or powdered product. Note that the place of origin of sage is not particularly limited.

Extraction treatment is performed using n-hexane or methylene chloride as an organic solvent, and the extract is collected.
The extraction treatment can be carried out according to a conventional method, for example, a method can be adopted in which the above-mentioned solvent is added to sage and the extraction treatment is carried out at room temperature or the reflux temperature of the solvent for 0.5 to 24 hours. After extraction, the extract is separated into an extract and an extraction residue using conventional methods such as filtration, centrifugation, decantation, etc., and the extract is collected. In addition, for the extraction treatment, one of the above solvents may be used alone or a mixture of both solvents may be used, and the extraction residue may be subjected to the extraction treatment repeatedly if necessary, but in this case, the extraction solvent It is also possible to use a different solvent from the above solvents.

The extract obtained in this manner is directly proceeded to the next step with or without distilling off the solvent. Here, the product from which the solvent has been distilled off may be further subjected to an extraction treatment using another solvent, if necessary, and the resulting extract may be collected.

Next, in the present invention, after performing the extraction treatment step using the organic solvent, a step of subjecting this extraction to steam distillation treatment and collecting the residue is performed.
This makes it possible to obtain an antioxidant that is highly effective and has almost no odor. In this case, the solvent is preferably distilled off from the organic solvent extract and the resultant is subjected to steam distillation treatment.

Note that the steam distillation treatment can be performed according to a conventional method. In the present invention, steam distillation is continued until almost no essential oil components are recognized in the steam distillate, and then the steam distillation residue is collected. Since the antioxidant components in the sage remain in this steam distillation residue, this is recovered.

Next, in the present invention, a weakly acidic fraction is collected from the obtained steam distillation residue according to a conventional method. In this step, the steam distillation residue is first dissolved in a water-insoluble organic solvent, and an aqueous solution of a weak alkaline compound is added thereto for extraction treatment to collect a water-insoluble organic solvent layer.

Here, the water-insoluble organic solvents include ethyl ether, n-hexane, methylene chloride, petroleum ether, ligroin, cyclohexane, carbon tetrachloride, chloroform, 1,2-dichloroethane, toluene, benzene, ethyl acetate, n-butanol, Examples include isobutanol and gasoline, and one or more of these can be used in combination.

In addition, weak alkaline compounds include sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium phosphate, sodium hydrogen phosphate, sodium pyrophosphate, sodium acetate, sodium citrate, potassium phosphate, and potassium hydrogen phosphate. , potassium pyrophosphate, potassium acetate, potassium citrate, etc. In the present invention, one or more of these weak alkaline compounds are used, usually in an amount of 0.1% by weight to
It is dissolved in water in a saturating amount and used as an aqueous solution.

When performing an extraction treatment by adding this weak alkali compound aqueous solution to the water-insoluble organic solvent solution of the extract, the weak alkali compound aqueous solution may be added to the water-insoluble organic solvent solution of the extract and immediately treated. However, in the present invention, an acid such as dilute sulfuric acid or dilute hydrochloric acid is first added to a solution of the extract in a water-insoluble organic solvent and shaken, and after separating the acid layer, an aqueous solution of a weak alkaline compound is added to the water-insoluble organic solvent layer. It is preferable to adopt a method in which the final fraction is treated by adding .This can reduce contamination of the final fraction with impurities. The treatment with an aqueous solution of a weak alkaline compound can be carried out according to a conventional method, for example, a method of shaking for about 1 to 10 minutes at room temperature or under heating can be adopted. It is also desirable to operate in a nitrogen or inert gas stream to avoid contact with oxygen in the air during extraction.

After the treatment with the weak alkaline compound, the mixture is left to stand and separated into a water-insoluble organic solvent layer and a weak alkaline compound aqueous solution layer, and the water-insoluble organic solvent layer is collected. According to the present invention, the antioxidant components in the sage are transferred to this water-insoluble organic solvent layer by the above-mentioned operation, and are collected.

Furthermore, in order to obtain a weakly acidic fraction, an aqueous solution of a strong alkaline compound such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. is added to the thus obtained water-insoluble organic solvent layer for extraction. It is preferable to process the sage and collect the strong alkaline compound aqueous solution layer.This allows the antioxidant components in the sage to transfer to the strong alkaline compound aqueous solution layer in a more concentrated state, so this strong alkaline compound aqueous solution layer is collected. This results in a more effective antioxidant. In this case, the strong alkali compound can be dissolved in water at a concentration of 0.1 to 10% by weight, and the treatment method with the strong alkali compound should be the same as the treatment method with the weak alkali compound. Can be done. The present invention provides this strong alkaline compound aqueous solution layer,
That is, the weakly acidic fraction is collected as the final fraction, but in this case, it is preferable to neutralize this aqueous solution layer, its concentrate, or add a water-insoluble organic solvent to the aqueous solution layer after neutralization. After extraction, the water-insoluble organic solvent layer to which the antioxidant components have been transferred again is collected, and the solvent is distilled off from this layer, which is used as an antioxidant.

In addition, in the present invention, if necessary, decolorization treatment can be performed during any of the above steps using an adsorbent such as activated carbon, diatomaceous earth, or acid clay, thereby making it possible to obtain a light-colored antioxidant. can.

The antioxidants obtained by the above-mentioned method include carnosol, rosmanol, rosmadial, 5,4′-
It contains antioxidant components such as dihydroxy-6,7-dimethoxyflavone, has a high antioxidant effect, and has very little odor, so it is suitable for various uses. For example, the antioxidant of the present invention may be used in fish oil, lard, tallow, head, chicken oil, soybean oil, linseed oil, cottonseed oil, surfflower oil, rice oil, corn oil, coconut oil, palm oil, sesame oil, cacao oil, etc. Oils and fats made from animals and plants such as fat, castor oil, and fallen raw oil, butter, cheese, margarine, starch toning, mayonnaise, dressing, ham, sausage, potato chips, fried rice crackers, fried ramen, curry roux, soy sauce, and soft drinks. It can be used by being added to alcohol, fruit liquor, ketchup, jam, fish or meat paste products, other foods, cosmetics such as hair cosmetics, skin cosmetics, mouth cosmetics, and pharmaceuticals. In this case, the amount of the antioxidant 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. or ethanol, propylene glycol, glycerin,
Alternatively, it may be dissolved and dispersed in a mixture of these 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 sage, it has almost no sage aroma, and even when added to foods, etc., the flavor is hardly impaired. Since it is a waste product and can be obtained by separating it from natural sage, it is suitable for foods such as oil and fat products, cosmetics, pharmaceuticals, etc., as the safety of synthetic additives is currently a problem. It can be used.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples.

〔Example〕

An antioxidant was obtained using crushed dried leaves of Greek sage according to the process shown in FIG.

That is, 500g of crushed sage and 2400g of n-hexane.
ml was added and extracted by stirring at room temperature for 24 hours. After extraction, the mixture was filtered and separated into an extract and an extraction residue, and the extract was collected. This operation was repeated three times and the extracts were combined.

Next, the concentrate of this extract (crude ext. fraction (steam distillation residue) and the volatile fraction was concentrated (fraction V).

The non-volatile fraction (steam distillation residue) was extracted with 500 ml of ethyl ether and concentrated to give a concentrate of 19.6
g was obtained, and 500 ml of ethyl ether was added thereto to dissolve the concentrate. Next, 200 ml of 2N hydrochloric acid was added to this ethyl ether solution, and after shaking for 2 minutes, the mixture was allowed to stand to separate into a hydrochloric acid layer and an ethyl ether layer. After repeating this operation three times, the hydrochloric acid layer was neutralized with sodium hydroxide, re-extracted with ethyl ether, and the ethyl ether layer was concentrated to obtain a basic fraction. On the other hand, 200 ml of a saturated aqueous sodium bicarbonate solution was added to the ethyl ether layer separated from the hydrochloric acid layer, and the mixture was shaken for 2 minutes, left to stand, and separated into an aqueous sodium bicarbonate layer and an ethyl ether layer. The layer was neutralized with hydrochloric acid, then re-extracted with ethyl ether, and the ethyl ether layer was concentrated to obtain a strongly acidic fraction (fraction SA). Add 200ml of 1N sodium hydroxide aqueous solution to the ethyl ether layer separated from the sodium bicarbonate aqueous solution layer.
was added, shaken for 2 minutes, and allowed to stand to separate into a sodium hydroxide aqueous solution layer and an ethyl ether layer.
After repeating this operation three times, the sodium hydroxide aqueous solution layer was neutralized with hydrochloric acid, re-extracted with ethyl ether, and the ethyl ether layer was concentrated to obtain 5.70 g of a weakly acidic fraction (fraction WA). The ethyl ether layer separated from the sodium hydroxide aqueous solution layer was concentrated.
12.81 g of a neutral fraction (fraction N) was obtained.

Furthermore, 1000 ml of methylene chloride was added to the extraction residue after the n-hexane extraction, and the extract was collected. Thereafter, steam distillation treatment, acid and alkali treatment were carried out in the same manner as described above,
Extract concentrate (crude ext.M), volatile fraction (fraction V′), strongly acidic fraction (fraction SA′), weakly acidic fraction (fraction WA′) and neutral fraction (fraction minutes N′) were obtained, respectively. The yield of the weakly acidic fraction was 4.30 g, and the yield of the neutral fraction was 5.75 g.

Next, the antioxidant effect of the above fractions was measured by the Rodan iron method and the TBA method. For comparison, a control (no antioxidant added, control), a natural antioxidant substance α-tocopherol (α-
The antioxidant effect of Toc) was also measured.

The Rodan iron method and TBA method were as follows, and the above fractions and α-tocopherol were both used at a concentration of 0.01%.

[Rodin iron method]

Test products (above fractions and α-tocopherol)
2 ml of a solution made to the specified concentration with 99.5% ethanol, 2.051 ml of a 2.51% linoleic acid solution prepared by dissolving linoleic acid in 99.5% ethanol, 0.05 M phosphate buffer (PH
7.0) Add 4 ml and water to make 10 ml of the test solution.
Store in the dark at 40℃. 75% in 0.1ml of this test solution
9.7 ml of ethanol, 0.1 ml of 30% ammonium thiocyanate solution, 2 x 10 ^-2 M ferrous chloride solution (3.5%
After adding 0.1ml (in hydrochloric acid), stir for exactly 3 minutes,
Measure the absorbance at 500nm.

[TBA method]

A.Ottolenghi Arch.Biochem.Biophs, 79 ,
355 (1959).

The above results are shown in FIGS. 2 and 3. Figure 2 shows the results of the fraction extracted with n-hexane, and Figure 3 shows the results of the fraction extracted with methylene chloride. Also, 1 is Rodin Iron Law, 2 is
These are the results obtained using the TBA method.

From the results shown in Figures 2 and 3, the weakly acidic fraction (fraction
WA, WA') and neutral fractions (fractions N, N') were recognized to have a strong antioxidant effect, and the fractions surrounded by the dotted line in Figure 1, especially the weaker fractions surrounded by the double line. It has been found that the acidic fraction is suitable as an antioxidant.

In addition, from the above experiment, a strong antioxidant effect was observed in the weakly acidic fractions, so fractions WA and WA' were analyzed using silica gel chromatography.
and 14 fractions, and the antioxidant effects of each fraction were evaluated using the Rodan iron method and the TBA method.
Here, the conditions for silica gel chromatography are:
Silica gel 60 manufactured by Merck was used, and benzene-acetone (9:1 to 0:10) was used as the elution solvent, followed by methanol.

Figure 4 shows the results of fractions 1 to 17 of fraction WA (weakly acidic fraction from n-hexane extract), and Figure 5 shows the results of fraction WA' (weakly acidic fraction from methylene chloride extract). The results for fractions 1 to 14 are shown respectively. Note that 1 is the result of the Rodan iron method and 2 is the result of the TBA method, and the reference numbers in the figure indicate the fraction numbers, respectively. Also, BHT is the result when dibutylhydroxytoluene was used. In addition, in measuring the antioxidant effect, the sage fraction was
BHT was used at a concentration of 0.02% and 0.01%.

From the results shown in Figure 4, an antioxidant effect was observed from fraction WA over a wide range from fractions 1, 3 to 12, and 16, and in particular, fractions 1 and 7 had an antioxidant effect comparable to that of BHT. . Also, from the results shown in Figure 5, from fraction WA', fractions 1 to
Although the effect was observed in all fractions 14, a particularly remarkable antioxidant effect was observed in fractions 8 to 11.

Furthermore, the active fractions of the weakly acidic fraction (fraction WA) from the n-hexane extract and the weakly acidic fraction (fraction WA') from the methylene chloride extract were subjected to changes in the polarity of the elution solvent under the same conditions as above. It was finely divided and purified by repeated chromatography.

As a result, Carnosol was obtained from fractions 5 to 8 of fraction WA, fractions 4 to 5 of fraction WA', and Rosmanol (7β,11,12-trihydroxy-6 , 10-epoxymethanoabieta-8,11,13-trien-20-one) was obtained. Also, from fraction 2 of fraction WA, the above formula
(3) The new compound Rosmadial (12-hydroxy-6,7-seco-
11,10-epoxymethano-20-oxo-abieta-8,11,13-triene-6,7-dial) was obtained, and further fractions 8 to 5 of fraction WA' were obtained.
4'-dihydroxy-6,7-dimethoxyflavone (Flavon) was obtained.

In addition, the characteristic values of the new compound Rosmadiar are shown below.

Structural formula Melting point 225℃ (benzene) Color reaction FeCl ₃ positive; 2,4-
DNDpositive Specific rotation [α] ²⁴ _D -216.8 (EtOH, c0.54) Elemental analysis value (C ₂₀ H ₂₄ O ₅ ) Found: C, 69.59; H, 7.07%. Calcd: C, 69.75; H, 7.02%. High MS m/z 344, 1602 (Calcd.
forC ₂₀ H ₂₄ O ₅ 344.1622) UVλmax (EtOH) nm 203 (logε3.96), 234
(4.15), 2.90 (3.88), 356 (3.83). IRνmax (nujol) cm ^-1 3140, 2730, 1803,
1786, 1713, 1655, 1615, 1380, 1370,
1240, 1170, 1020. Furthermore, the results of measuring the antioxidant effects of the above-mentioned compounds are shown in FIG. Figure 6 1 is Rodin iron method, 2 is
This is a result of the TBA method. The antioxidant effect was measured at a concentration of 0.02% for the above compound and 0.01% for α-tocopherol and BHT.

[Brief explanation of drawings]

FIG. 1 is a process diagram illustrating an embodiment of the present invention;
Figure 2 shows the antioxidant effect of the n-hexane extracted fraction of sage, Figure 3 shows the antioxidant effect of the methylene chloride extracted fraction of sage, and Figure 4 shows the weakly acidic fraction of sage extracted with n-hexane. Fig. 5 shows the antioxidant effect of fractions obtained by fractionating sage with silica gel chromatography. Figure 6 is a graph showing the antioxidant effects of the compounds isolated in the weakly acidic fractions from n-hexane extract and methylene chloride extract, respectively. 1 in the figure is the measurement result using the Rodan iron method, and 2 is the measurement result using the TBA method.

Claims (1)

[Claims] 1 Extract sage with n-hexane or methylene chloride, collect the extract, perform steam distillation on this extract to collect the residue, and further fractionate and collect the weakly acidic fraction from this residue. A method for producing an antioxidant, characterized by: