KR100760990B1 - Isolation method of a fraction containing high percentile panaxadiol saponin from korean red ginseng by ultrafiltration - Google Patents

Abstract

A method for isolating a fraction including panaxadiol saponin of Red ginseng is provided to obtain the panaxadiol saponin fraction with excellent anti-inflammatory activity, particularly a fraction containing highly percentage of Rb1 saponin. A method for isolating a fraction including panaxadiol saponin of Red ginseng comprises the steps of: (a) adding ethanol to an ethanol or a water extract of Red ginseng to remove generated precipitated materials of non-saponin polymer materials therefrom through filtration or centrifugation; (b) removing ethanol from supernatant obtained after removing the precipitated materials to concentrate it; and (c) after adding distilled water to the concentrate, filtering it through an ultrafiltration device to obtain the fraction including panaxadiol saponin not passing through the ultrafiltration membrane, wherein the obtained fraction including panaxadiol saponin is soluble in water, sparingly soluble in ethanol and methanol, and insoluble in ether and chloroform, has a pH of 4.3-4.8(in 0.1% aqueous solution) and the UV absorption of lambdamax=270-280 nanometers.

Description

Isolation method of a fraction containing high percentile panaxadiol saponin from Korean Red Ginseng by ultrafiltration}

1 is a schematic view showing a method for producing a high ratio of panaxadiol-based saponin-containing fraction (RGSF-A) using ultrafiltration in the present invention,

Figure 2 shows the TLC separation pattern of each ginsenoside in the high ratio panaxadiol-based saponin-containing fraction (RGSF-A) isolated in the present invention,

Figure 3 shows the HPLC profile of each ginsenoside in the high proportion panaxadiol family saponin-containing fraction (RGSF-A) in the present invention.

The present invention relates to a method for separating high proportion panaxadiol-based saponin-containing fractions (hereinafter referred to as "RGSF-A"), in particular high ratio Rb1 saponin-containing fractions, from red ginseng by ultrafiltration.

Ginseng is known to have about 30 ginsenosides (saponins), diols such as ginsenosides Rb1, Rb2, Rc and Rd (panaxadiol, hereinafter referred to as "PD"), and ginsenoside Re, Triols (panaxatriol, hereinafter referred to as "PT") such as Rg1 and Rf.

In particular, red ginseng is steamed ginseng with steam and dried under certain temperature conditions to gelatinize the starch of ginseng, but has a stronger effect than ginseng or pharmacological action different from ginseng. Therefore, red ginseng is treated as a separate herbal or substance from ginseng, and it is very necessary to study the diol-based (PD) ginsenoside and triol-based (PT) ginsenoside fractions from red ginseng.

Diol-based (PD) ginsenosides and triol-based (PT) ginsenosides have been reported to have opposite pharmacological actions (Food and Chemical Toxicology 41 (2003) 1381-1390, Alexa T. Smolinski, James J, Pestka) In particular, diol-based (PD) ginsenosides are known to have excellent anti-inflammatory action.

However, the pharmacological action of diol-based (PD) ginsenosides and triol-based (PT) ginsenosides is not well known, and many attempts have been made to isolate ginsenoside fractions.

Known methods for producing irradiated ginseng of ginseng are known butanol extraction and resin adsorption.

To date, the preparation of irradiated ginseng with ginseng has relied on butanol extraction. However, irradiated with butanol extract contains a large amount of non-saponin components, such as free sugar and organic acids, in addition to irradiated phosphonine, which is useful for the study of diol (PD) ginsenosides and triol (PT) ginsenoside fractions from red ginseng. Inappropriate, in particular, the irradiated phonophonine produced by the butanol extraction method cannot be used for edible purposes because of the butanol used as the extraction solvent.

In order to overcome the above problems, the present inventors have developed a method for preparing ginseng irradiated ponins by using Dion Echipi-20, an artificial synthetic adsorption resin, as known in Korean Patent Registration No. 44394. The prior art can produce irradiated saponin can be used for edible or medicinal, it is possible to develop a ginseng product with a high saponin content, but diol-based ginsenosides and triol-based ginsenosides are mixed in a large amount, red ginseng gin It is inappropriate to study the pharmacological action of senoside diol series and triol series.

Meanwhile, ultrafiltration is a method of separating and concentrating high-molecular substances and low-molecular substances by using a semipermeable membrane.In the food, pharmaceutical and medical fields, the concentration and purification of liquid seasonings and food ingredients, the recovery of active substances from various food wastewater, It is used for the removal of Pyrogen from injection water and raw materials and the manufacture of sterile water for operating room. It is used in various fields such as water and wastewater treatment, electronic and surface treatment, fisheries and aquaculture.

The ultrafiltration method is capable of separating, purifying and concentrating colloidal materials that are difficult to separate by general filtration, and also separating up to a molecular weight of 500 to 300,000.

Water or alcohol extract of red ginseng has various component distributions ranging from amino acids of about 100 molecular weight to free sugars of about 200 molecular weight, saponins of molecular weight 500 to 2,000, and polymer polysaccharides of molecular weight of 100,000 or more. The physical and chemical properties of each ginsenoside It is mixed in various ways.

Thus, the present inventors attempted to separate the diol ginsenoside and triol ginsenoside fractions from red ginseng using the ultrafiltration method.

Surprisingly, the present inventors separated by ultrafiltration a panaxadiol (PD) series saponin-containing fraction (RGSF-A), in particular a high proportion of Rb1 saponin-containing fractions, having a molecular weight of 2,000 or less, using an ultrafiltration membrane having a molecular weight of 5,000 to 100,000. The fraction has been confirmed to have a very good anti-inflammatory action as having a physicochemical properties not known so far, and completed the present invention.

It is an object of the present invention to provide a method for separating panaxadiol (PD) family saponin fraction (RGSF-A), particularly a high proportion of Rb1 saponin-containing fractions, which is separated from red ginseng by ultrafiltration and has excellent anti-inflammatory action.

In another aspect, the present invention is to provide a material of health functional food having a more enhanced function in the fraction separated from the red ginseng.

In order to achieve the above object, the present invention provides a method for separating PD-based saponin-containing fraction (RGSF-A) using ultrafiltration.

The present invention relates to a method for preparing a PD series saponin fraction (RGSF-A), in particular a high proportion containing fraction of Rb1 saponin, isolated from red ginseng using ultrafiltration.

According to the present invention, red ginseng is extracted and filtered by adding 5-10 times alcohol, ethanol or water to the sample by the weight of 5-10 times, and then concentrates the alcohol extract and the water extract. Alcohol is added to the extract to 60% alcohol concentration to induce precipitation, and then the supernatant is collected and filtered or centrifuged. The final supernatant was concentrated to remove alcohol, and then dried using vacuum filtration and reduced pressure to obtain a high proportion of PD series saponin-containing fractions (RGSF-A).

The present invention is divided into two stages: red ginseng saponin extraction, polymer material removal, and ultrafiltration.

Red ginseng saponin extraction and polymer material removal process according to the present invention is to remove the red ginseng powder by extracting the red ginseng powder with 80% alcohol or water to precipitate a polymer material such as polysaccharide present in a large amount by ethanol and filtered or centrifuged. It is a process.

In addition, the manufacturing process of the high ratio PD series saponin-containing fractions using the ultrafiltration apparatus according to the present invention is diluted by adding water to the water or alcohol extract of red ginseng from which the precipitate by alcohol is removed as described above. Millipore, USA) to remove low molecular weight sugar.

Hereinafter, the present invention will be described in detail.

In the present invention, red ginseng saponin extraction and polymer material removal process, first, 80% alcohol or water is added 5-10 times per sample weight to red ginseng powder, and extracted three times at 85 ° C. three times, followed by filtration. In the case of alcohol extracts, concentrates are removed to concentrate the final 20% solids. The water extract is also concentrated to give a final 20% solids.

Precipitation is induced by adding alcohol to each extract to 60% alcohol concentration. Supernatant is filtered or centrifuged. After adding alcohol to red ginseng water or alcohol extract to precipitate and remove polymer materials such as polysaccharides present in red ginseng, the supernatant contains a large amount of organic substances (eg, low molecular sugars) that do not form precipitates in addition to saponin.

The supernatant is concentrated to remove alcohol and then diluted with 5% solids by adding water to the resulting concentrate. The supernatant contains about 60% of alcohol, which is immediately passed through the ultrafiltration device, the supernatant contains a high concentration (about 60%) of alcohol, so the PD-based saponin passes through the ultrafiltration as it is impossible to separate Alternatively, some structures of the filtration apparatus may be denatured in the alcohol during the passage of the ultrafiltration device due to the high concentration of alcohol, so that it is preferable to concentrate the supernatant to remove and dilute the alcohol to pass through the ultrafiltration device.

The concentrate of the supernatant is diluted with water to about 5% solids. The diluent is passed through an ultrafiltration device. The dilution is passed through an ultrafiltration device (Millipore, USA) equipped with a molecular weight of 5,000 to 100,000 membrane filter cassettes and concentrated at a concentration of 1/50 to 1/2. By drying the concentrate under reduced pressure, a high proportion of PD series saponin-containing fractions (RGSF-A) using ultrafiltration can be obtained.

As described above, the passage of the ultrafiltration apparatus of the diluent liquid refers to the concentration of a substance (saponin) that is not passed through. In the present invention, as the concentration of the ultrafiltration device increases, the saponin purity increases, but the yield decreases. For example, in the present invention, the yield is 30% when the saponin purity is 17%, and the yield is 20% when the purity is 26%.

According to the present invention, the saponin fraction may be separated at a high yield even if the saponin purity is low depending on the concentration of the ultrafiltration. In the present invention, saponin fraction may be used in functional foods using saponin fraction, even if the saponin purity is low, and fractions having high purity may be used for raw materials such as pharmaceuticals.

In addition, in the present invention, the PD-based saponin has a molecular weight of 2,000 or less, but the molecular weight does not pass through 5,000 to 100,000 membranes because the molecules exist in the polymer form by forming micelles in aqueous solution regardless of the molecular weight of PD-based saponins. I guess.

The present invention was tested according to TLC method for qualitative analysis of ginseng saponin and is shown in FIG. Fractions prepared according to the present invention include a developing solvent; Chloroform: Methanol: Water = 65: 35: 10, then air-dried, sprayed with chromophore (30% sulfuric acid) and warmed at 110 ° C for about 10 minutes to reveal 10 or more red spots between Rf 0.03-0.65. When comparing the standard and the sample, red spots corresponding to Rb ₁ at Rf 0.17, Rb ₂ + Rc at Rf 0.22, Rd at Rf 0.31, Re at Rf 0.34, Rf at Rf 0.41, and Rg ₁ at Rf 0.44.

Fractions prepared by the ultrafiltration method of the present invention can be confirmed that the material having a new physical properties significantly different PD / PT and Rb1 / Rg1 ratio compared to the fractions prepared in butanol extraction method and H-20.

In another aspect, the present invention provides a panaxanadiol (PD) -based saponin fraction (RGSF-A) as a material of the health functional food having a stronger function in the fraction separated from the red ginseng.

Ginsenoside Rb ₁ (Diol saponin) has been reported to have anti-inflammatory action by inhibiting the production of inflammatory cytokine (AT Smolinski and JJ Pestka: Food and Chemical Toxocology 41 (2003): 1381-1390), the present invention Panaxadiol (PD) -based saponin fraction (RGSF-A) separated by the anti-inflammatory effect can be usefully used as a material for health functional foods with enhanced function of inflammation treatment and prevention.

In the present invention, when a panaxanadiol (PD) -based saponin fraction (RGSF-A) is added as a material of a health functional food with enhanced anti-inflammatory function, the health functional food may be added at 0.01 to 80% by weight of the total food weight. The health beverage composition may be added at a ratio of 0.02 to 30 g, preferably 0.3 to 5 g, based on 50 ml.

In addition, the present invention may further include additives such as conventional excipients, flavors, sweeteners, deodorants, etc. within the range not more than 2% by weight of the whole health functional food.

The present invention may be formulated in the form of capsules, tablets or pills according to a conventional method after powdering for convenient taking, or may be formulated in the form of a beverage or the like as a liquid form.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

Example 1 Preparation of High Ratio PD Saponin-containing Fractions (RGSF-A) Using Ultrafiltration

50 kg of 80% alcohol was added to 10 kg of dried red ginseng powder, and extracted three times for 8 hours. The extracts were combined, filtered through a filter cloth, and concentrated under reduced pressure to prepare about 3.6 kg of red ginseng alcohol extract extract.

Distilled water was added to 2kg (36% water) of red ginseng alcohol extract and dissolved to make 4 L. Then, 6 L of alcohol was added again, and left to stand overnight, followed by centrifugation. The obtained supernatant was concentrated to remove alcohol, and then distilled water was added to 20 L. This solution was concentrated (passed) until 2 L remained in the ultrafiltration unit equipped with a molecular weight 10,000 membrane (polyethersulfone) cassette. 2 L that did not pass through the membrane was dried under reduced pressure (about 10% of water) to obtain about 250 g of a high proportion of PD series saponin-containing fraction (RGSF-A).

The HPLC profile of ginsenoside in the obtained high ratio PD series saponin-containing fraction (RGSF-A) is shown in FIG. 3, and HPLC quantitative analysis showed that the ginsenosides in the high ratio PD series saponin-containing fraction (RGSF-A). Side content and composition ratio are shown in Table 1.

Table 1 Ginsenoside content in high proportion of PD series saponin-containing fractions (RGSF-A) prepared from red ginseng by ultrafiltration

                                                       [Unit: dry extract (%)]

Rb1 Rb2 Rc Rd Re Rf Rg1 Rg2 Rg3 total Diol Triol PD / PT Rb1 / Rg1 9.94 4.22 4.29 1.72 1.66 1.27 1.52 0.695 0.925 26.24 21.10 5.15 4.1 6.54

Solubility: Soluble in water, poorly soluble in ethanol and methanol, insoluble in ether and chloroform

pH: 4.56 (as measured in 0.1% aqueous solution)

Water content: 10 to 15%, total saponin: 20 to 30%

-Total lipid: 0.3-0.5%, Total protein: 3-5%, Total sugar: 50-60% (colorimetric method)

Ultraviolet absorption: λ max = 277 nm (0.1% aqueous solution)

Comparative Example 1 Preparation of Saponin-containing Fractions Using Butanol Method

50 kg of 80% alcohol was added to 10 kg of dried red ginseng, and the mixture was refluxed three times for 8 hours. The extracts were combined, filtered through a filter cloth, and concentrated under reduced pressure. About 3.6 kg of red ginseng alcohol extract with water content of about 2.3 kg (2.3 kg by alcohol extract) ) Was prepared. Water is added to the alcohol extract and dissolved to make 50 L, and then extracted twice with 50 L of ethyl ether in the fractional filter to remove lipids. The water layer (50 L) was repeatedly extracted three times with 50 L of water-saturated butanol, and then the butanol layers were combined and concentrated under reduced pressure to obtain 460 g of red ginseng saponin fraction (irradiated ponin) by extraction with butanol (20% yield compared to alcohol extract dryness). )

pH: 3.87 (as measured in 0.1% aqueous solution)

Ultraviolet absorption: λ max = 266 nm (0.1% aqueous solution)

<Comparative Example 2> Preparation of Saponin-containing Fractions from H. Ginseng Extract by HCP-20

A ginseng saponin-containing extract was prepared according to the H-20 method described in Korean Patent No. 444394.

pH: 3.48 (as measured in 0.1% aqueous solution)

Ultraviolet absorption: λ max = 283,272 nm (0.1% aqueous solution)

The ratios of PD / PT and Rb1 / Rg1 of the fractions separated in Example 1 and Comparative Examples 1 and 2 are shown in Table 2.

[Table 2] Comparison of PD / PT and Rb1 / Rg1 ratios of red ginseng saponin fractions isolated by ultrafiltration, butanol, and H-20

Way PD / PT Rb1 / Rg1 Example 1 4.10 6.54 Comparative Example 1 2.11 1.57 Comparative Example 2 1.74 1.88

The fractions obtained by the ultrafiltration method of Example 1 may be confirmed that the fractions prepared in Comparative Examples 1 and 2 and the physical properties according to pH and UV absorption have significantly different new physical properties.

In addition, the high ratio PD-based saponin-containing fraction (RGSF-A) by the ultrafiltration method of Example 1, compared to the fractions prepared in Comparative Examples 1 and 2, the PD / PT ratio is contained more than two times higher ratio, in particular Fractions containing high ratio PD-based saponins contained Rb1 / Rg1 ratio more than four times compared to Comparative Examples 1 and 2, and ginsenoside Rb ₁ was reported to have anti-inflammatory activity by inhibiting the production of inflammatory cytokine. According to the present invention, the fractions separated by the present invention have an anti-inflammatory effect and can be usefully used for the treatment and prevention of inflammation.

Preparation Example 1 Preparation of Beverage

5 g of the high ratio PD series saponin-containing fraction (RGSF-A) herbal composition prepared in Example 1, 0.4 g of champinion extract powder, 0.03 g of sucralose and 0.2 g of fruit mix flavor were added to the purified water. A total volume of 300 ml was prepared. According to the conventional food beverage manufacturing method, the beverage was prepared by stirring and heating at 85 ° C. for about 1 hour.

Preparation Example 2 Preparation of Tablet

1 g of high ratio PD-based saponin-containing fraction (RGSF-A) prepared in Example 1, lactose 20 g, starch 150 g, crystalline cellulose 10 g, inositol 6 g, silicon aluminate 3 g and food coloring 2.5 g were mixed with a tablet press Prepared.

The present invention is a novel paranaxadiol (PD) -based saponin fraction (RGSF-A), which is separated from red ginseng by ultrafiltration, having different properties from the saponin-containing fractions according to the known butanol and HI-20 methods. Provided is the Naxadiol family saponin fraction and separation method.

In particular, the present invention, panaxadiol-based saponin fraction (RGSF-A), which is isolated from red ginseng by ultrafiltration, is a high proportion of Rb1 saponin-containing fractions having excellent anti-inflammatory activity, and is used as an agent for preventing and improving inflammation, as a raw material for drinks, foods, and the like. Can be used.

Claims (14)

delete delete delete Separation method of the fraction containing the saponin of Panaxadiol (PD) series of red ginseng having the following characteristics and comprising the following steps: (a) removing the non-saponin-based polymer material, which is a precipitate produced by adding alcohol to alcohol or water extract of red ginseng; (b) a concentration step of removing the precipitate and removing alcohol from the obtained supernatant; And (c) adding distilled water to the concentrate and passing through an ultrafiltration apparatus to obtain a panaxadiol (PD) series saponin-containing fraction that did not pass through the ultrafiltration membrane. Solubility: Soluble in water, poorly soluble in ethanol and methanol, insoluble in ether and chloroform -pH: 4.3 ~ 4.7 (0.1% aqueous solution) -UV absorption: λmax = 270 ~ 280nm (0.1% aqueous solution) The method of claim 4, wherein the removing of the precipitate of step (a) is characterized in that the precipitate is removed by filtration or centrifugation. 5. The method of separating fractions according to claim 4, wherein the ultrafiltration device of step (c) is an ultrafiltration device equipped with a molecular weight of 5,000 to 100,000 membrane filter cassettes. 7. The method of claim 6, wherein the ultrafiltration device is an ultrafiltration device equipped with a molecular weight 10,000 membrane filter cassette. After adding alcohol to alcohol or water extract of red ginseng, remove the resultant non-saponin-based polymer material and concentrate the obtained supernatant and add distilled water to pass through the ultrafiltration device to pass through the ultrafiltration membrane. It contains a fraction containing the panaxanadiol (PD) series saponin, and a health functional food having an anti-inflammatory effect.       Solubility: Soluble in water, poorly soluble in ethanol and methanol, insoluble in ether and chloroform        -pH: 4.3 ~ 4.7 (0.1% aqueous solution) -UV absorption: λmax = 270 ~ 280nm (0.1% aqueous solution) delete delete delete delete A health functional food additive containing a fraction containing red ginseng panaxanadiol (PD) series saponins having the following properties. Solubility: Soluble in water, poorly soluble in ethanol and methanol, insoluble in ether and chloroform -pH: 4.3 ~ 4.7 (0.1% aqueous solution) -UV absorption: λmax = 270 ~ 280nm (0.1% aqueous solution) delete

Images