KR20130010987A - The way of refining panaxan which has efficacies of boosting hematopoiesis and immunity against cancer. and an analysis of defining its attributes and a composition for the efficacies - Google Patents

Abstract

PURPOSE: A method for purifying panaxan polysaccharide from ginseng is provided to enhance anticancer immunity and to directly and indirectly treat immune diseases. CONSTITUTION: A method for purifying panaxan from ginseng comprises: a step of washing raw ginseng or dried ginseng and dipping 70-100% high purity ethanol to wash the raw ginseng or dried ginseng; a step of crushing the raw ginseng or dried ginseng; a step of extracting the raw ginseng or dried ginseng using water at 80-95 deg.C.; a step of separating into a ginseng extract and ginseng cake using a centrifugal dehydrator or by filter press; a step of passing through ultracentrifuge and removing fine particles, macromolecular carbohydrate, and proteins; a step of passing through activated charcoal and a terra alba filter and adsorbing heavy metal ions and agrochemical ingredients; a step of removing a material with high molecular weight from inert hydrocarbon using a filter system; a step of disposing a solution containing low molecular weight material and metallic ion ingredients and preparing purified ginseng polysaccharide panaxan liquid; and a step of concentrating and drying the prepared ginseng extract. [Reference numerals] (AA) Raw ginseng, dried ginseng + ethanol; (BB) Raw ginseng, dried ginseng + water; (CC) Removing ginseng cake; (DD) Removing protein; (EE) Reflux extraction; (FF) Scrapping a solvent; (GG) Crushing; (HH) Extraction process; (II) Centrifugal dehydration, filter press; (JJ) First refining process(ultracentrifugation); (KK) (second refining process) activated charcoal-based filtration; (LL) Removing organic components; (MM) Panaxan powder; (NN) Panaxan liquid; (OO) Removing heavy metal and inorganic salt; (PP) Removing low molecular materials; (QQ) Removing microorganisms and macropolymers; (RR) Extremely low temperature-based freezing and drying process; (SS) Decompression thickening process; (TT) (fifth refining process) N/F system; (UU) (fourth refining process) U/F system; (VV) (third refining process) M/F system

Description

Method for Purifying Panaxane Polysaccharides Having Anticancer Immunity Promoting and Hematopoietic Promoting Effects from Ginseng, Assays for Characterizing Purified Panaxane Polysaccharides and Anticancer Immunity Enhancing and Hematopoietic Promoting Including Ginseng Panaxane Polysaccharides The way of refining Panaxan which has efficacies of boosting hematopoiesis and immunity against cancer. And an analysis of defining its attributes and a composition for the efficacies}

The present invention provides a method for purifying panaxane polysaccharide from ginseng and an analytical method for defining physical and chemical properties of the purified panaxan polysaccharide and a composition for anticancer immunity enhancement and hematopoiesis including the analyzed ginseng panaxane polysaccharide. It is about.

Background art of the invention is a patent (Application No. 10-1993-23385) filed for separating acidic polysaccharides from ginseng or alcohol extract ginseng.

Hereinafter will be described the background of the invention of the present invention.

Ginseng has long been used as a tonic or a first aid for prolonging the life of dying patients in oriental medicine, and has been rarely used as a medicine to help rejuvenation or immortality.

In the twentieth century, due to the development of science and technology, the chemical pure separation of ginseng, which has been accumulated in the mystery for thousands of years, has been promoted in earnest, and the chemical structure, efficacy, and effect of the saponin ingredient uniquely contained in the plant of ginseng Intensive research has led to the development of medical and health foods that highlight saponin. Table 1 shows the chemical components of Korean ginseng.

Organic water



Saponin
(3-6%) * Protoparnaxadiol-based saponins (22 types)
* Proto-Panax Triol Saponins (11 types)
* Oleananiline Saponin (1 type) Nitrogen-containing compound
(12-16%) * Protein * Amino Acid * Peptide
* Nucleic acid * alkaloids Fat-soluble ingredients
(1 to 2%) * Lipids * Fatty acids * Essential oils * Phytosterols * Organic acids * Phenolic compounds
* Polyacetylene * Terpenoids vitamin
(0.05%) * Water Soluble Vitamin carbohydrate
(60-70%) * Polysaccharides * trisaccharides * disaccharides * monosaccharides
* Crude Fiber * Pectin Inorganic Ash
(4-6%) Mineral moisture 9-11%

Recently, as it is gradually revealed that there are various pharmacological activities in non-saponin fractions other than the saponin component, pharmacological activity search studies centering on these active fractions have been developed in various ways. Of course, some of the non-saponin substances found in ginseng are bioactive components found in other herbals and natural products, but the components that exhibit pharmacological activity as ingredients unique to ginseng have recently been revealed. It contributes greatly to understanding.

The water-soluble extract of ginseng contains various forms of sugars. Monosaccharides, including glucose and fructose, and disaccharides, trisaccharides, tetrasaccharides, pentasaccharides and many monosaccharides including maltose and sucrose, are also found in ginseng (Ovodov et al. 1966).

Ginseng pectin, a polysaccharide component isolated from ginseng, was hydrolyzed by acid hydrolysis, revealing that its major constituents were galacturonic acid, glucose, galactose, arabinose, and the minor constituents were xylose, ranose and galacturonan (Solov). 'eva et al. 1967 1969). However, in the early studies until the 1960s, little chemical research was carried out on the ginseng polysaccharide, which is a polymer, and little research was conducted on the physiological function of ginseng saccharides. In recent years, with the development of life sciences, carbohydrates have been recognized as an important role not only as a simple biostructure material and energy source, but also as a signaling material for biological function. In particular, polysaccharides and derivatives thereof from plants and microorganisms have been developed as immunomodulators, and oligosaccharides have been actively developed as medical preparations and functional food materials such as anti-inflammatory and antibacterial agents. Studies on the polysaccharide components of ginseng and their effectiveness have been carried out much later than the saponin studies due to the structural complexity of these components. In 1984, Hikino and Tomoda of Japan first reported the separation of polysaccharides A, B, C, D and E from Korean Korean ginseng (Tomoda et al. 1984, 1985, Konno et al. 1984). . Subsequently, continued research was carried out by the researchers, resulting in the separation of a total of 21 panaxans from panaxan F to U (Konno et al. 1985, 1987, Hikino et al. 1985, Oshima et al). 1985).

However, these polysaccharide components are combined into the same or different monosaccharides, and the major constituent sugars differ depending on the polysaccharide type, but are glucose, arabinose, galactose, xylose, and rhamnose. Among them, Panaxane A is composed of glucose (92.1%) and a small amount of peptide (1.7%), molecular weight of about 14,000, Panaxan B contains 95.9% glucose, 0.7% peptide and molecular weight of about 1,800,000 Doglycans reveal chemical partial structures (Tomoda et al. 1984, 1985).

The physiological activity of these polysaccharide components has been shown in animal experiments to have a hypoglycemic effect that significantly inhibits hyperglycemia induced by experimental diabetes-producing alloxan treatment (Konno et al. 1984). , Ng et al. 1985). Yang et al. Also reported that administration of polysaccharide components from ginseng to rats (50-200 mg / kg, intraperitoneal, subcutaneous injection), hypoglycemic activity, hepatic glycogen content, and insulin secretion effect. (Yang et al. 1990).

In addition, there are reports that polysaccharide components isolated from ginseng enhance immune function. The antitumor activity of most polysaccharide components is known to be due to immunomodulatory activity, not direct toxicity to cancer cells. At present, the polysaccharide fraction of ginseng is used as an anticancer agent in clinical trials. Therefore, the pharmacological activity of the ginseng root and leaves is isolated from the polysaccharide component. Wang et al. Orally orally intraperitoneally administered ginseng polysaccharide components to normal and cancer-causing animals (mouse), which enhances the foreign body's ability to detect foreign bodies of the reticuloendothelial system that is responsible for immune function and promotes antibody production. The clinical application of these components to cancer patients has also been reported to improve (Wang et al. 1985).

The water-soluble and alkaline (0.5 M NaOH) -soluble polysaccharide fractions isolated from the roots and leaves of ginseng have anti-complementary activity, especially the acidic polysaccharide fractions showed the strongest activity (Gao et al. al. 1989). The complement system plays an important role in immunity, inflammation and allergic reactions. Polysaccharides with antitumor effects have been reported to have anticomplement activity (Okuda et al. 1972). Crude acid polysaccharide content is higher in the roots than in the leaves, and their chemical properties are different and the roots contain mainly pectin and glucan, whereas the leaves contain pectin and heperoglycans (Gao et al. 1989). However, the chemical structure of these acidic polysaccharides is not known, and further studies have recently revealed that two new acidic polysaccharide components, ginsenan PA and ginsenan PB, are derived from water-soluble extracts of Korean ginseng. They have been isolated, which have the effect of enhancing anti-complementary activity and the activity of the immune system in the immune system by phagocytosing foreign substances coming from outside (Tomoda et al. 1993).

The molecular weight of these components is about 16,000 and 5,500, respectively, and the chemical structural forms have structural units of the α-arabino-β-3,6-galactan type and rhamnogalacturonan type. Ginsenan PA consists of L-arabinose, D-galactose, L-rhamnose, D-galacturonic acid (11: 22: 1: 1: 6: 1, molar ratio) and 1.3% O-acetyl groups. Investigation of phagocytosis and anticomplement activity on the degradation products of scavenger showed that aramiose units had a significant effect on immunological activity (Tomoda et al. 1994).

In Korea, research on the efficacy of ginseng polysaccharide components in relation to cancer immunity is being carried out with interest. The ginseng polysaccharide fraction administered to mice against the anti-cancer cyclophosphamide (CY) immunotoxicity had the effect of suppressing the immunotoxicity (reduction of hemolytic plaque forming cell count, white blood cell count, spleen weight, etc.) caused by CY treatment ( Kim et al, 1990). In addition, administration of ginseng polysaccharide component suppressed cancer in cancer cells (Sarcoma-180) transplanted mice, increased hemolytic plaque forming cells (PFC) related to antibody production, and enhanced phagocytic activity of the intra-net system. Strong activity was observed in the acidic polysaccharide fraction of (Kim et al. 1990, 1991).

On the other hand, Okuda, Japan, has been separating acidic polysaccharides from water extracts of Korean red ginseng through research on bioactive substances other than saponins contained in Korean red ginseng over the past decade. The chemical structure of this component was found to have an α-1,4-polygalturonan backbone similar to pectin with several acetoxy groups (Lee et al. 1990, Okuda 1990).

In general, cancer causes weight loss, and the weight loss of these cancer patients is known to be due to body fat breakdown and appetite-inducing effects of toxohormone-L secreted from cancer cells. It has pharmacological activity that inhibits the action of (Okuda et al. 1984, Okuda 1989). In addition, these acidic polysaccharides have been found to have activity in inhibiting lipase (lipase) activity, cholesterol estase inhibitory activity, and serum triglycerides in the pancreas. This action is expected to be effective in the prevention of so-called obesity or hyperlipidemia, because it can inhibit or delay the absorption of sugar or fat in the intestine (Okuda et al. 1993). However, the content distribution of acidic polysaccharides with these activities is higher in red ginseng than in white ginseng (Okuda et al. 1990, 1992), and in roots (body parts) than coarse roots, i.e. .

In addition, saponins are mainly distributed in the outer layer and hardly in the central part of the endothelial region, whereas acidic polysaccharides are contained in the inner inner region, which is in contrast to the distribution of saponins (Okuda et al. 1992).

Recently, Han et al. (1992) established an analysis method of ginseng polysaccharides using alcian blue dye, and compared the contents of fresh ginseng, white ginseng, and red ginseng. The highest contents were reported in the root and brain head. Since ancient times, ginseng was not used as a medicinal part with high saponin content, and in the case of rice ginseng, the root was mainly used as a medicinal part in folk medicine or herbal medicine, although saponin content was more than twice that of main root. This suggests that the medicinal value standard of ginseng has other quality factors besides the absolute content of saponin. Considering this point, there are some scholars' opinions that the pharmacological activity of the acidic polysaccharide component contained in the roots is well matched with the existing medicinal value of ginseng (Okuda et al. 1990), and the polysaccharide component is the main active ingredient of ginseng. In the future, more research on the chemical and physiological activities of polysaccharide components having physiological functions is expected. The pharmacological activity of the ginseng polysaccharide component is summarized in Table 2 below.

Polysaccharide Ingredients Pharmacologically active castle Related Statements Panaxane A, B, C... U
(21 types separated) Antidiabetic action
* Hyperglycemia Konno, Hikino,
Tomoda et al.
(1984, 1985, 1987) Ginseng Crude Polysaccharide Ingredients
(Undefined) Antidiabetic action
* Lowering blood sugar, glycogen lowering in the liver
Promotes Dragon and Insulin Secretion
Immune enhancement
Activation of Intranet Function Antibody Production
Increase * Mouse cancer occurrence suppression * Cancer disease
Improvement of clinical symptoms Yang et al. (1990)
Ng et al. (1985)
Wang et al. (1985)
Kjm et al. (1990, 1991) Ginseng Polysaccharide Ingredients
(Pectin and glucan bonds,
Ginse PA, PB,
S-IA, S-IIA) Immune enhancement
Anticomplement activation, phagocytosis of the intranet system
Dragon activation Gao et al. (1989)
Tomoda et al.
(1993, 1994) Acid of Ginseng Roots and Leaves
Polysaccharide fraction Anti-ulcer effect
* Suppression of gastric lesion formation, gastric acid secretion and peptide
Repression Sun et al. (1991, 1992) Water Solubility of Red Ginseng
Acid Polysaccharide Ingredients Amirase, activity of pancreatic lipase and
Inhibition of Cholesterol Estrase
* Prevention of obesity and hyperlipidemia
Lipolysis and Appetite of Toxohormone-L
Suppression of decay Okuda et al. 1990)
Lee et al. (1980)

Saponins, ginsenosides, which are indicator components of ginseng or red ginseng, exist within a narrow range of 88 to 1400 in the range of monosaccharides and disaccharides, so they are not only simple to analyze items and methods that can characterize substances. As it has an isomeric structure with similar physical and chemical properties, compatible manufacturing techniques are also very simple.

However, since ginseng polysaccharides have a wide range of molecular weights ranging from 1,000 to 2,000,000 with a low binding range of monosaccharides, disaccharides, and trisaccharides, the physical and chemical properties are very different depending on the degree of polymer bonds. The analysis items and methods that can be defined have not been established until now, and the development of commercialized manufacturing technology that can selectively purify high purity by selectively separating inactive carbohydrates and active ginseng polysaccharides from different groups of ginseng polysaccharides with different physical and chemical properties It was a very difficult situation. Nevertheless, many researchers at home and abroad have shown that the pharmacological effect of polysaccharides in ginseng is as excellent as the indicator substance saponin on the immune system in vivo. It is becoming a key substance in biological immune response modifier (BRM), which is an important field of preventive medicine.

Among the numerous studies, briefly describe the efficacy of ginseng polysaccharides published by the Atomic Energy Research Institute. Jinsan increases the ability of macrophages to produce IL-1 and IL-12 in bioimmune mechanisms, and by the action of these factors, As well as the ability to produce cytokines such as IL-2 and IFN- [gamma], the reactivity of various cells to these cytokines increases, thereby increasing the activity of cytotoxic T cells, LAK cells, NK cells, macrophages and the like.

In addition, proliferation of bone marrow cells reduces side effects caused by various anticancer drugs or radiation causing bone marrow failure. More specifically, the anti-cancer immunity effect was first examined in the case of LAK cell production ability, the spleen cells of mouse were cultured in vitro in FCS-RPMI1640 medium supplemented with 0.5 µg / ml of acid, and the killing activity of tumor cells YAC-1 was measured. The result was 10 times stronger than the control group. In addition, the efficacy of ginseng polysaccharides published through many studies in Korea and abroad has been shown to be effective in preventing and treating various immune diseases such as asthma, atopy, AIDS, sepsis, and bacterial hepatitis. As ginseng products can be developed, it is expected to create a new market using ginseng in the functional food and supplemental medicine fields of the alternative medicine market. Most of the ginseng industry consists of red ginseng-oriented products and saponin-based products, and the Southeast Asian market accounts for more than 95% of the global export market. In the EU, the situation is completely ignored.

In particular, the present inventors have identified ginseng polysaccharide panaxan, which is recognized as an important field in alternative medicine among non-saponin-based materials of ginseng, and has proven excellent efficacy in the field of biological immune response modulator, which is rapidly growing in the world market. The chemical structure of 2-3 polysaccharide fractions characteristic of various fractions of ginseng polysaccharides obtained by extracting, dehydrating and purifying under specific conditions during the ongoing study to purify them from high ginseng was identified and these polysaccharide fractions were contained. Completed the present invention by confirming that the ginseng polysaccharide is a panaxan based on the main component, and found that the panaxan has an anticancer immune boosting and hematopoietic effect, and has direct and indirect healing effects against cancer as well as immune diseases. It was.

An object of the present invention is to provide a method for purifying panaxic acid, which has anticancer immunity and hematopoietic promoting effect from ginseng.

Another object of the present invention is to provide an analytical method for defining the chemical and physical properties of ginseng polysaccharide panaxic acid.

Still another object of the present invention is to provide a composition for anticancer immunity enhancement and hematopoietic promotion comprising the polysaccharide panaxonic acid purified from the ginseng.

In order to achieve the above object, the present invention provides a method for purifying panaxic acid from ginseng, comprising the following steps:

(1) washing the fresh ginseng or dried ginseng root with water and then immersing it in 70% to 100% high purity ethanol to rinse the alcohol while circulating the ethanol, and then roughly crushing it;

(2) extracting at 80 to 95 ° C. using 3 to 24 times water by weight of ginseng;

(3) separating the ginseng extract and ginseng foil by centrifugal dehydrator or filter press;

(4) removing the fine particles, the large carbohydrate and the protein suspended in the ginseng extract by passing through the ultra-fast centrifugal separator;

(5) adsorbing heavy metal ions and pesticide components remaining in the ginseng extract by passing through the activated carbon and clay filter;

(6) removing the macromolecular weight material of the inert carbohydrates using a filtration system equipped with a 0.45 μm micromembrane filter or ceramic filter;

(7) Using a filtration system equipped with 1K-Dalton and 0.5K-Dalton nano-membrane in order to faithfully discard the aqueous solution containing the low molecular weight material and the metallic ionic component to obtain a purified Ginseng Polysaccharide Panaxate. step; And

(8) The obtained ginseng extract was concentrated using a reduced pressure concentrator so that the measured sugar content was 20 Brix, and then powdered by using a cryogenic freeze dryer or spray dryer and a fluidized bed dryer to obtain ginseng polysaccharide panaxuric acid powder. Or or concentrating the ginseng extract obtained in the above to obtain a ginseng polysaccharide panaxuric acid concentrate by using a reduced pressure concentrator so that the measured sugar content is 65 Brix or more.

In order to achieve the above another object, the present invention provides a method for analyzing the ginseng polysaccharide panaxuric acid purified according to the above method by measuring the irradiated content of irradiated with a saturated butanol method.

In addition, in order to achieve the above object, the present invention provides a method for analyzing the ginseng polysaccharide paroxane purified according to the method by measuring the total sugar content by the D.N.S method using a D.N.S (Dinitro salicylic acid) reagent.

In addition, in order to achieve the above object, the present invention provides a method for analyzing the ginseng polysaccharide panaxuric acid purified according to the above method by measuring the acidic polysaccharide content using the carbazole-sulfuric acid method.

In addition, in order to achieve the above object, the present invention provides a method for analyzing the ginseng polysaccharide panaxuric acid purified according to the above method by measuring the molecular weight distribution of the ginseng polysaccharide paroxane using GPC (Gel Permeation Chromatography). do.

In order to achieve the above another object, the present invention comprises a ginseng polysaccharide panaxuric composition having anticancer immunity enhancing and hematopoietic effect, including ginseng polysaccharide panaxic acid having a specific structural properties purified according to the above method, such a composition Examples include pharmaceuticals or processed foods.

According to the present invention, various physical, chemical and structural properties of ginseng polysaccharides purified according to the method for purifying ginseng polysaccharide panaxane are identified as characteristics and structures that become a common denominator, and an analytical method is secured with precision, accuracy and reproducibility. By using this production method, we have developed a new ginseng product based on ginseng polysaccharides with excellent efficacy against various immune diseases by breaking the scope of ginseng industry focused on red ginseng and saponin products. By recognizing ginseng as the biological immune response regulator, which is the most important material in the huge market of alternative medicine and supplementary medicines, it is expected to provide an opportunity to expand the market of ginseng industry to the world beyond Southeast Asia.

1 shows a method for purifying polysaccharide panaxane from ginseng.
Figure 2 shows the dilution ratio used in the assay of ginseng polysaccharide panaxuric acid extract.
Figure 3 is a graph showing the absorbance (OD) value according to the concentration of the acidic polysaccharide.
4 is a graph showing absorbance (OD) values according to glucose concentrations.
Figure 5 shows the calibration curve of the polysaccharide.
6 is a graph of analysis of acidic polysaccharides.
7 is a graph showing the total equivalent weight analysis of polysaccharides.
8 is a graph of the polysaccharide irradiated with a checkponin.
9 shows the results of GPC analysis for each manufacturing process of panaxane.
10 shows the results of GPC analysis of panaxane.

The technical terms and scientific terms used in the present invention can be construed as meaning ordinary meanings understood by those of ordinary skill in the art without departing from the scope of the present invention.

The method for purifying panaxic acid from the ginseng of the present invention

(1) washing the fresh ginseng or dried ginseng root with water and then immersing it in 70% to 100% high purity ethanol to rinse the alcohol while circulating the ethanol, and then roughly crushing it;

(2) extracting at 80 to 95 ° C. using 3 to 24 times water by weight of ginseng;

(3) separating the ginseng extract and ginseng foil by centrifugal dehydrator or filter press;

(4) removing the fine particles, the large carbohydrate and the protein suspended in the ginseng extract by passing through the ultra-fast centrifugal separator;

(5) adsorbing heavy metal ions and pesticide components remaining in the ginseng extract by passing through the activated carbon and clay filter;

(6) removing the macromolecular weight material of the inert carbohydrates using a filtration system equipped with a 0.45 μm micromembrane filter or ceramic filter;

(7) Using a filtration system equipped with 1K-Dalton and 0.5K-Dalton nano-membrane in order to faithfully discard the aqueous solution containing the low molecular weight material and the metallic ionic component to obtain a purified Ginseng Polysaccharide Panaxate. step; And

(8) The obtained ginseng extract was concentrated using a reduced pressure concentrator so that the measured sugar content was 20 Brix, and then powdered by using a cryogenic freeze dryer or spray dryer and a fluidized bed dryer to obtain ginseng polysaccharide panaxuric acid powder. Alternatively, the ginseng extract obtained above is concentrated using a reduced pressure concentrator so that the measured sugar content is 65 Brix or more, thereby obtaining a ginseng polysaccharide panaxuric acid concentrate.

In step 1 of the method for purifying ginseng panaxuric acid of the present invention, ethanol washing is performed under the conditions of 50 ° C. or less, and the ginseng raw material is washed for 2 hours or more by reflux.

Extraction in step 2 of the method for purifying ginseng panaxuric acid of the present invention is carried out by extracting the roughly crushed ginseng raw material into the extraction tank for water extraction at a temperature in the range of 80 ~ 95 ℃ for more than 6 hours to precipitate a large carbohydrate and protein The temperature of the ginseng extract can be quenched below 50 ℃ by adding cooling water into the double jacket of the extraction tank.

In step 2 of the method for purifying ginseng panaxuric acid of the present invention, in the case of fresh ginseng, 3-6 times the weight of water is added, and in the case of dried ginseng, 12-24 times the weight of water is extracted. .

In step 4 of the purification method of ginseng panaxuric acid of the present invention, the ultra-high speed centrifugal separator is characterized in that it has a rotation speed of 15,000rpm or more.

In step 6 of the method for purifying ginseng panaxuric acid of the present invention, bacterial microorganisms and giant carbohydrates remaining in the ginseng extract are removed by 99% or more, and in step 7, low molecular weight ginseng components other than the ginseng polysaccharides remaining in the ginseng extract And 90% or more of the metallic and nonmetallic inorganic ionic components.

In step 8 of the method for purifying ginseng panaxuric acid of the present invention, the ginseng polysaccharide panaxuric acid extract is concentrated under reduced pressure until the sugar content is about 20 Brx at a heat source of 70 ° C. or less.

In the present invention, in order to produce a high concentration of the ginseng polysaccharide panaxuric acid extract, in step 8, the sugar content measured in the high concentration of 65Brix or more can be finally produced a high concentration of the ginseng polysaccharide panaxuric acid.

In the present invention, the step 4 and step 5 may be performed simultaneously to purify the ginseng polysaccharide panaxuric acid.

In the present invention, the ginseng polysaccharide panaxuric acid purified according to the above method was used to measure the irradiated butanin content using the saturated butanol method, and the total sugar content was measured by the DNS method using a DNS (Dinitro salicylic acid) reagent, and the carbazole-sulfuric acid method. Ginseng polysaccharides can be analyzed by measuring the acidic polysaccharide content using or by measuring the molecular weight distribution of ginseng polysaccharides panaxic acid using GPC (Gel Permeation Chromatography). The phosphate polysaccharide panacyl analyzed according to the present invention has a criterion as shown in Table 3 below.

Analysis item Analysis method Criteria Joe Saponin Saturated Butanol Method ★ crude saponin content less than 3% Per total D.N.S Act ★ 50% or more total sugar content Acidic polysaccharides Carbazole
-sulfuric acid method ★ acid polysaccharide content 5% or more Molecular weight distribution G P C Act ★ Higher than 2,000,000 at molecular weight 3,500 or more
Molecular distribution area is over 30% of total peak area
Should be
★ Higher than 2,000,000 at molecular weight 3,500 or more
Retention, the time zone of the molecular distribution
1 time in 20-23 minutes, 23-26 minutes, and 26-30 minutes
The presence of more than one polymer peak should be confirmed.

In addition, in the present invention, according to the method of the GPC analysis of ginseng polysaccharides panaxuric acid composition having anti-cancer immunity enhancement and hematopoietic effect, including purified ginseng polysaccharides panaxuric acid appearing at a specific time period, for example, pharmaceuticals or processed foods can do.

According to the present invention, various physical, chemical and structural properties of ginseng polysaccharides purified according to the method for purifying ginseng polysaccharide panaxane are identified as characteristics and structures that become a common denominator, and an analytical method is secured with precision, accuracy and reproducibility. By using this production method, we have developed a new ginseng product based on ginseng polysaccharides with excellent efficacy against various immune diseases by breaking the scope of ginseng industry focused on red ginseng and saponin products. By recognizing ginseng as the biological immune response regulator, which is the most important material in the huge market of alternative medicine and supplementary medicine, it is expected to provide an opportunity to expand the market of ginseng industry worldwide from Southeast Asia.

Hereinafter, the present invention will be described in more detail based on the following Examples and Test Examples. However, the following Examples and Test Examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  One

(Step 1) Raw Material Preparation and Washing Process

Fresh ginseng or dried ginseng is mainly purchased and washed in running water through the washer. The washed ginseng or dried ginseng is subdivided into 2 ~ 3kg each in a sack and put into a shower reflux washing device.

Into the shower reflux washing apparatus is injected into the alcohol alcohol solution of purity of 70% to 100% to the upper layer of the sackcloth ginseng to wash the reflux for about 2 hours. At this time, the reflux washing is performed at a temperature ranging from room temperature to 50 ° C.

After the reflux washing is completed, the alcohol alcohol solution is removed and the ginseng is put into the crusher, and the particles are crushed into coarse particles having a size of about 2-5 mm and put into a stirred water extraction tank.

(Step 2) Extraction Process

When the crushed ginseng is added to the stirred water extraction tank, 3 to 6 times the weight of fresh ginseng, and 12 to 24 times the weight of dry ginseng. When ginseng and water are added, stirring is started while raising the extraction temperature to 80 ~ 95 ℃.

Stirring extraction is performed with an extraction time of 6 hours from the time of temperature increase to 80-95 degreeC which is extraction temperature.

When the extraction is completed, the cooling water is circulated inside the double jacket of the extraction tank to cool the temperature of the ginseng extract below 50 ° C.

(Step 3) Dewatering Process

The discharge port at the bottom of the extraction tank is connected to the centrifugal dehydrator or filter press to slowly discharge the extraction solution and ginseng foil while operating the stirrer of the extraction tank.

Pass the ginseng gourd and extract through a centrifugal dehydrator or filter press to separate the extract and ginseng gourd. The separated ginseng meal is discarded and the extract is collected in a primary purification tank for feeding into an ultrafast centrifuge.

(Step 4) First purification process

In order to remove fine particles such as protein and carbohydrate suspended in the ginseng extract from the ginseng extract, connect the discharge port of the primary purification tank and the ultra-high speed centrifuge of the cylinder type. When the high speed centrifuge is operated and the rotation speed reaches more than 15,000 r.p.m, the discharge port of the primary purification tank is opened to allow the ginseng extract to pass through the high speed centrifuge. The clear and light brown primary ginseng purified liquid, which has passed through a high speed centrifuge, is collected in a secondary purification tank.

If the flow rate of the primary ginseng tablet solution decreases rapidly, stop the operation of the ultra-high speed centrifuge, completely remove the solids present in the state of cake in the rotating cylinder, and not pass through the ultra-high speed centrifuge, and repeat the procedure as above. Operate.

(Step 5) Secondary purification process

Pallet-type activated carbon filtration and white soil filtration are passed through to remove organic compounds and heavy metals in the primary ginseng purification liquid. In order to prevent the contamination of activated carbon particles in the secondary ginseng tablet liquid that has passed through the activated carbon filtering device, the membrane is passed through a 1 μm filter membrane and collected in a third purification tank.

(Step 6) 3rd purification process

The third refining tank is a double jacket type, and the second ginseng refining liquid must be manufactured in a structure that can circulate the cooling water to prevent the temperature from rising during the refining process and to maintain the temperature below 30 ° C.

To remove microorganisms and macromolecules with molecular weight of 2,000,000 or more in the secondary ginseng tablet liquid contained in the tertiary purification tank, a micro filter system or ceramic filtration device equipped with a 0.45 μm membrane for disinfection is passed.

The primary panaxuric acid purified liquid portion of the absorbent liquid passing through the micro filter system is collected in the fourth purification tank. Secondary ginseng purified liquid from the concentrate portion that cannot pass through the micro filter system is repeatedly passed through the micro filter system to increase the amount of primary panic acid purified liquid absorbed and the concentration of the concentrated liquid increases to put a load on the micro filtration system. An appropriate amount of pure water prepared in the pure water production apparatus is added to the tertiary purification tank to lower the concentration of the concentrate to operate the micro filter system.

If the amount of absorbent liquid that passes through the microfilter system is drastically reduced, stop the microfilter system and discard any concentrate that has not passed, and wash it with an aqueous solution of caustic soda to remove contaminants in the attached membrane or ceramic filter.

(Step 7) 4th purification process

The fourth refining tank is a double jacket type, which should be made of a structure that can circulate the cooling water to prevent the temperature of the first panic acid refining liquid from rising during the refining process and to maintain the temperature below 30 ℃.

An ultrafilter system equipped with a 1k-dalton-sized membrane is passed through to remove low molecular weight substances with a molecular weight of 1,000 or less present in the primary panic acid purification liquid contained in the fourth purification tank. Secondary panaxuric acid tablets from the concentrate portion that do not pass through the ultra filter system are collected in a fifth purification tank.

Discard some of the absorbent liquid that passes through the ultrafilter system and repeat the remaining portion through the ultrafilter system to increase the amount of secondary panic acid purified liquid that is concentrated and to increase the concentration of the concentrated liquid, which puts a load on the ultrafilter system. The ultrafiltration system is operated by adding an appropriate amount of pure water produced in the pure water production apparatus to the fourth purification tank to lower the concentration of the concentrate.

If the amount of absorbent liquid that passes through the ultrafilter system is drastically reduced, the ultrafilter system is shut down and washed with an aqueous solution of caustic soda to remove contaminants from the membrane.

(Step 8) 5th purification process

The fifth refining tank is a double-jacket type, which must be made of a structure that can circulate the cooling water in order to prevent the temperature of the second panic acid refining liquid from rising during the refining process and to maintain the temperature below 30 ° C. Pass a low pressure reverse-osmosis system equipped with a 500-dalton-sized desalting membrane to remove heavy metal ions and inorganic salts in the secondary panaxuric refining liquid in the fifth refining tank. .

The final panic acid purified liquid from the concentrate portion that does not pass through the nano filtration system is collected in a reduced pressure concentrator.

Part of the filtrate passing through the nano filtration system is discarded, and if the concentration of the concentrate increases and the load is placed on the nano filtration system, an appropriate amount of pure water prepared in the pure water production apparatus is added to the fifth purification tank to lower the concentration of the concentrate. Start up.

If the amount of absorbent liquid that passes through the nanofiltration system decreases drastically, the nanofiltration system is shut down and washed with an aqueous solution of caustic soda to remove contaminants from the membrane.

(Step 9) vacuum concentration

The final panaxuric acid purified liquid in the decompression concentrator usually has a sugar concentration of about 8 to 10 Brix, and in order to condense it to 15 to 20 Brix, the decompression concentrator is concentrated under a pressure not exceeding 70 ° C.

At this time, the heat source input to the jacket of the concentration tank to raise the temperature of the decompression concentrator should not use steam above 100 ℃ and hot water below 90 ℃.

In order to process the final 15 ~ 20Brix final panic acid purified liquid which has been completed under reduced pressure concentration into a drink type final product, it is stored in a freezer at -5 ℃ and concentrated in the freezer for 6 months or longer. The sugar concentration of the final panaxuric acid purified solution is concentrated to 65 Brix and stored in the freezer.

(Step 10) Lyophilization

In order to cryogenically freeze 15 ~ 20Brix final panic acid purified liquid into -70 ℃ freezer, it is subdivided into shelf which meets freezer standard and has large surface area and puts into freezer.

When the final panaxuric acid purification liquid is subdivided into the shelf and finished in the freezer, it is frozen at very low temperature to -70 ℃. When the freezing of the final panaxuric acid purification liquid is completed, the shelf is placed in a freeze dryer, and dried under vacuum while gradually raising the temperature from -60 ° C to 35 ° C for about 48 hours.

When the drying is completed, the solidified panaxuric acid is pulverized through a mill while being careful not to contaminate, thereby preparing a high purity panaxuric tablet powder capable of preparing a final product in a capsule or tablet form.

Panaxane refined powder is stored in a place equipped with UV sterilization, ozone generating sterilizer, dehumidifier, and air conditioner for safe storage until it is formed into final product.

Example  2: acidic polysaccharide colorimetry ( Acidic Polysaccharides Ginseng polysaccharide Panaconic  analysis

The ginseng polysaccharide panaxate purified according to the purification method of Example 1 was analyzed by acidic polysaccharide colorimetric method (Acidic Polysaccharides).

Weigh out 2 to 5 g of the extracted powder and place it in a Erlenmeyer flask to dissolve by adding 50 ml of distilled water (extracted solvent dilution multiple value).

Extract the Erlenmeyer flask by heating it in a 80 ° C water bath using a reflux condenser for 1 hour. (In this case, the upper inlet of the reflux condenser with the Erlenmeyer flask should be sealed.)

When the extraction is completed, cover the inlet of the Erlenmeyer flask with foil and allow it to cool.

The sample in the Erlenmeyer flask was placed in an ultrafast centrifuge tube and centrifuged by setting the centrifugation conditions at a rotational speed of 10.000 rpm, a rotation time of 20 min, and a cooling temperature of 4 ° C.

Take 2 ml of the supernatant of the centrifuged sample, place it in an ultra-centrifuge tube, and add 8 ml of ethanol to form a precipitate.

When the precipitate is formed, the centrifugation is performed once again by setting the centrifugation conditions to 10.000 rpm, 10 min rotation time, and 4 ° C. cooling temperature. After completion of centrifugation, remove all supernatant and dissolve by adding 2 ml of distilled water to the precipitate.

Dilute the sample solution in which the precipitate is completely dissolved in the test tube as shown in FIG. 2 (at this time, using a vortex mixer to mix the distilled water and the sample solution well).

In another test tube, 0.5 ml of the diluted sample solution was added to each test tube, and 0.25 ml of carbazole reagent (0.1 wt% ethanol) was added thereto, followed by mixing using a vortex mixer to prepare a sample.

Another test tube is prepared by adding 0.5 ml of the diluted sample solution according to each dilution factor and adding 0.25 ml of ethanol and mixing using a vortex mixer.

3 ml of H ₂ SO ₄ is added to the test sample and the blank prepared according to the dilution factor (X2, X4, X8, X16, X32, X64, X128).

The test tube containing the measurement sample and the blank test sample of H ₂ SO ₄ treated dilution factor (X2, X4, X8, X16, X32, X64, X128) was heated in a water bath heated to 85 ° C for 5 minutes, and then heated to about 15 minutes. Preparation of the absorbance measurement sample is completed by cooling to room temperature for a minute.

The spectrophotometer is started and stabilized 30 minutes before measurement, and the measurement wavelength is fixed at 525 nm. The quartz cell containing distilled water is added thereto to set the reference value (O.D value = 0).

The absorbance is measured with a stabilized spectrophotometer containing the measured sample and blank test sample diluted in each dilution factor in a quartz cell. In order to convert the measured O.D value into the sample concentration value, repeat the above operation using the acidic polysaccharide standard to determine the absorbance value for each acid polysaccharide concentration to determine the calculation formula for obtaining the acid polysaccharide concentration value.

Figure 3 is a graph showing the absorbance (O.D) value of each concentration of the acidic polysaccharide.

Using the O.D values (Y-axis) measured for each acidic polysaccharide content (X-axis), the first equation is obtained: Y = 0.0062X + 0.0044.

After subtracting the OD value of the blank test sample from the OD value of the measured sample, substitute the equation above to obtain the sample concentration and obtain the acidic polysaccharide concentration value by substituting Equation 1 below. OD value minus OD value, multiplied by 160 may be used as a sample concentration value.)

Example  3: total sugar Total Saccharides Ginseng Polysaccharide by D.N.S Analysis Panaconic  analysis

Weigh 1 ~ 2g extract powder into 250ml saccharification flask and add 50ml of distilled water to dissolve it. 4 ml of 36% HCl stock solution is added to the Erlenmeyer flask with the extracted powder.

Acid disintegration of the Erlenmeyer flask using a reflux condenser in an 80 ° C water bath for 3 hours in a hot water bath.

After acid decomposition is completed, the inlet of the Erlenmeyer flask is covered with foil and allowed to cool.

In a Erlenmeyer flask, add 4% of 36% NaOH (solution dissolved in a small amount of NaOH in a small amount of distilled water and finally adjusted to 100 ml), and immerse the electrode sense of the calibration pH meter in the sample solution. Neutralize the sample solution to the pH value of 7.0 by adding a small amount of the solution dissolved in the solution up to 1,000 ml. (If it is too alkaline, add 1N-HCl to make it neutral again.)

Fill the sample solution of the Erlenmeyer flask with a 100 ml volumetric flask and fill it with distilled water from the volume line to the 100 ml mark. (The reason for filling the distilled water up to the 100 ml mark is to set the dilution factor of the extraction solvent to 100 in the calculation. For sake.)

Insoluble matters are removed by filtering the sample liquid of the volumetric flask using a vacuum filter and a filter paper.

Dilute the sample liquid filtered in the test tube in the same ratio as in FIG. 2 (at this time, using a vortex mixer to mix the distilled water and the sample liquid well).

In another test tube, add 1 ml of the diluted sample solution according to each dilution factor, dissolve 10 g of DNS reagent (10 g of dinitrosalicylic acid reagent in 1 ml of 1N-NaOH), add 300 g of Rochelle salt, and thoroughly Melt and distilled water to make up to 1,000ml mark) 1ml is added and mixed using a vortex mixer.

The test tube containing the diluted sample and DNS reagent diluted according to the dilution factor (X2, X4, X8, X16, X32, X64, X128) is heated in boiling water for 5 minutes in a boiling water, and then allowed to cool and 8 ml of distilled water in each test tube. Preparation of the measurement sample is completed by adding each and mixing with a vortex mixer.

Start the spectrophotometer 30 minutes before measurement, stabilize it, fix the measurement wavelength at 540nm, and add quartz cell with distilled water to set the default value (O.D value = 0).

The absorbance is measured with a stabilized spectrophotometer containing a sample diluted in each dilution factor in a quartz cell. In order to convert the measured O.D value into the sample concentration value, the above operation is repeated using glucose to measure the absorbance value for each glucose concentration to determine a calculation formula for obtaining a glucose reference sample concentration value. Figure 4 is a graph showing the absorbance (O.D) value for each glucose concentration.

Using the O.D value (Y-axis) measured for each glucose content (X-axis), the first equation is obtained and Y = 0.0007X 0.0141.

The sample concentration is obtained by substituting the OD value of the measured sample into the above equation and substituting the total equivalent equation of Equation 2 below to obtain a total equivalent value.

Example  4: Crude saponin  analysis( Saturated Butanol Method Ginseng Polysaccharide Using Panaconic  analysis

Take 1-2g sample, dissolve in 60ml distilled water, transfer to separatory funnel and extract fat with 60ml ether. 60 ml of saturated butanol was added to a separatory funnel and shaken to separate: recovery of upper layer (3 times).

Combine the recovered butanol layers and wash once with 50 ml distilled water:

The collected solution was placed in a weighed flask and concentrated under reduced pressure at 80 ° C. After concentration, it is dried for 15 minutes in a 105 ℃ drying oven. Cool completely in the dryer and weigh it. The content of the irradiated phononine is calculated using the following equation.

Example  5: molecular weight distribution analysis ( Come Permeation Chromatography Ginseng using Polyche Panaconic  analysis

50.000mg of the collected sample is placed in a test tube, 5ml of distilled water is added, and completely dissolved using a vortex mixer. The dissolved sample is placed in a syringe and passed through a 0.45 μm nylon syringe filter to complete the preparation of the sample for GPC analysis.

Configuration of the analyzer

1.Pump: Shimadzu LC-20AD

2. Inserter: Shimadzu SIL-20A

3. Column Oven: Shimadzu CTO-20A

4. Column: PSS Suprema 10000Å + 3000Å + 30Å

5. Explorer: ELSD

Analysis condition

1.Flow rate: 1.0ml / min

2. Insertion volume: 50µl

3. Oven temperature: 40 ℃

4. Analysis time: 40-50min

Calibration curve ( Calibration Curve ) And functions ( Function ) Get

Using the PSS dextran 1,400,000 333,000 164,000 35,600 8,100 908 as a standard sample is prepared in the same manner as the preparation of the analytical sample. The calibration curve is shown in FIG. 5.

Function f (x) = 7.655291e-004 * X ^ 3-3.181748e-002 * X ^ 2 + 0.1386024 * X + 6.373336 (X = x-T.LIMIT)

     R ^ 2 = 0.9999212 Dispersion = 0.0093897 T.LIMIT = 15min. Calibration results are shown in Table 4 below.

# Time (minutes) Molecular Weight error(%) activation One 21.452 1400000 -0.6085 Available 2 24.375 333000 3.4946 Available 3 25.431 164000 -2.5961 Available 4 27.732 35600 -2.0459 Available 5 29.925 8100 2.0257 Available 6 33.246 908 -0.4096 Available

Example  6: ginseng polysaccharide ( Panaksan Analytical evaluation of)

A. Evaluation criteria for each analysis item of ginseng polysaccharide (panaxane) are as shown in Table 5 below.

Analysis item Analysis method Criteria Joe Saponin Saturated Butanol Method ★ crude saponin content less than 3% Per total D.N.S Act ★ 50% or more total sugar content Acidic polysaccharides Carbazole
-sulfuric acid method ★ acid polysaccharide content 5% or more Molecular weight distribution G P C Act ★ Higher than 2,000,000 at molecular weight 3,500 or more
Molecular distribution area is over 30% of total peak area
Should be
★ Molecular weight 3,500 and above 2,000,000
Retention, the time frame of the distribution of polymers
time 20 to 23 minutes, 23 to 26 minutes, 26 to 30 minutes each
More than one polymer peak must be identified
do.

B. Acid Polysaccharide Analysis Evaluation

The sample amount (g) was 2.042 once, 2.006 twice, and 2.013 three times. The collected sample is placed in a Erlenmeyer flask and 50 ml of distilled water is added to dissolve completely. Insert the Erlenmeyer flask into the reflux condenser (top inlet seal) and extract for 1 hour in an 80 ° C water bath. After extraction, allow to cool and centrifuge the sample liquid by setting the operating conditions of the ultra-high speed centrifuge to 10,000rpm, 20min, 4 ℃. After centrifugation is completed, 2 ml of the supernatant is collected, and 8 ml of ethanol is added and mixed with a vortex mixer to form a precipitate. When the precipitate is generated, centrifugation is performed by setting the operating conditions of the ultra-high speed centrifuge at 10,000 rpm, 10 min, and 4 ° C.

After centrifugation is completed, the supernatant is removed and 2 ml of distilled water is added to the precipitate to completely dissolve the precipitate. Dilute 2 ml of the sample solution in each test tube according to the specified dilution factor (X2, X4, X8, X16, X32, X64). 0.5 ml of each diluted sample solution was added to another test tube and 0.25 ml of carbazole solution was added to prepare a sample sample solution for absorbance measurement. 0.5 ml of each diluted sample solution is added to another test tube, and 0.25 ml of ethanol (99% ↑) is added to prepare a blank test sample solution for absorbance measurement.

Concentrated H ₂ SO ₄ in the sample solution and blank sample solution diluted according to each dilution factor. Add 3 ml each. The test tube to which the concentrated H ₂ SO ₄ was added was heated in a water bath in a water bath at 85 ° C. for 5 minutes, cooled to room temperature, and mixed well with a vortex mixer.

Measure the wavelength of the spectrophotometer with 525nm and insert the quartz cell with distilled water, adjust it to the default setting (O.D = 0), and put the sample in the quartz cell to measure the absorbance. O.D values according to the dilution factor of the sample sample and the blank test sample are shown in Table 6 below.

(1 time) Dilution X2 X4 X8 X16 X32 X64 Sample O.D value Max. 1.871 1.482 0.994 0.683 0.397 O.D value of blank 0.435 0.264 0.130 0.066 0.059 0.047 Calculated O.D Value - 1.607 1.352 0.928 0.624 0.350 (Episode 2) Dilution X2 X4 X8 X16 X32 X64 Sample O.D value Max. 1.968 1.513 1.005 0.752 0.424 O.D value of blank 0.428 0.307 0.174 0.073 0.062 0.049 Calculated O.D Value - 1.661 1.339 0.932 0.690 0.375 (3rd time) Dilution X2 X4 X8 X16 X32 X64 Sample O.D value Max. 1.959 1.520 0.973 0.728 0.399 O.D value of blank 0.447 0.334 0.162 0.068 0.055 0.045 Calculated O.D Value - 1.625 1.358 0.905 0.673 0.354

OD value according to the dilution factor of the sample is calculated according to Equation 4 below the sample concentration value based on the acidic polysaccharide is shown in Table 7.

(1 time) Dilution X2 X4 X8 X16 X32 X64 Sample concentration value - 258.5 217.4 149.0 99.9 55.7 (Episode 2) Dilution X2 X4 X8 X16 X32 X64 Sample concentration value - 267.2 215.3 149.6 110.6 59.8 (3rd time) Dilution X2 X4 X8 X16 X32 X64 Sample concentration value - 261.4 218.3 145.3 107.8 56.4

The sample concentration value, the dilution factor and the extraction solvent dilution factor were obtained from the acidic polysaccharide content of the analytical sample according to Equation 5 shown in Table 8 below.

※ When calculating the concentration of sample (㎍ / ㎖), you can multiply the OD value by 160 as an approximation.

Dilution Acidic polysaccharide (%) once Acidic polysaccharide (%) twice Acidic polysaccharide (%) 3 times 2 - - - 4 2.53 2.67 2.60 8 4.26 4.29 4.34 16 5.84 5.97 5.77 32 7.83 8.82 8.57 64 8.74 9.54 8.96 Average value (times) 5.97 6.36 6.23 Total average value 6.19

※ The lowest and highest acidic polysaccharide values in each experiment were excluded from the average calculation.

+ 5% of total mean: 6.50%

-5% of total mean: 5.88%

6 shows an analysis graph of acidic polysaccharides.

C. Total Equivalence Analysis Assessment

The sample amount g shall be 2.061 once, 2.000 twice, and 2.057 three times. The collected sample is placed in a Erlenmeyer flask and 50 ml of distilled water is added to dissolve completely. Add 4 ml of concentrated HCl (36%) to the fully dissolved sample solution. Insert the Erlenmeyer flask into the reflux condenser (top inlet seal) for acid decomposition and heat the bath for 3 hours in an 80 ° C water bath.

After the acid decomposition is completed, the mixture is cooled with the inlet of the Erlenmeyer flask closed and neutralized to pH 7 by adding 4 ml of 36% NaOH and adding a small amount of 1N-NaOH. Put sample liquid into 100 ml volumetric flask and fill distilled water to 100 ml mark. Solvent dilution drainage 100ml

Fold the filter paper into the tripod and filter the sample to remove insoluble matters. Dilute the filtered sample solution according to the dilution factor (X2, X4, X8, X16, X32, X64, X128) specified in the test tube.

In another test tube, add 1 ml of each diluted sample solution and add 1 ml of D.N.S reagent.

The test tube to which the DNS reagent was added was heated in boiling water for 5 minutes, allowed to cool, and added to each test tube by 8 ml of distilled water, and mixed well with a vortex mixer. Measure the wavelength of the spectrophotometer with 540nm, insert the quartz cell with distilled water, adjust it to the set reference value (OD = 0), and put the sample in the quartz cell to measure the absorbance. OD values according to the dilution factor of the sample are shown in Table 9 below.

 (1 time) Dilution factor X2 X4 X8 X16 X32 X64 X128 O.D value 2.126 1.356 0.899 0.495 0.312 0.162 0.088 (Episode 2) Dilution factor X2 X4 X8 X16 X32 X64 X128 O.D value 2.119 1.337 0.909 0.518 0.308 0.153 0.079 (3rd time) Dilution factor X2 X4 X8 X16 X32 X64 X128 O.D value 2.130 1.345

The sample concentration value based on glucose based on the OD value according to the dilution factor of the sample is calculated according to Equation 6 below.

(1 time) Dilution factor X2  X4 X8 X16 X32 X64 X128 Sample concentration value 3057.3 1957.3 1304.4 727.3 465.9 251.6 145.9 (Episode 2) Dilution factor X2 X4 X8 X16 X32 X64 X128 Sample concentration value 3047.3 1930.1 1318.7 760.1 460.1 238.7 133.0 (3rd time) Dilution factor X2 X4 X8 X16 X32 X64 X128 Sample concentration value 3063.0 1941.6 1284.4 748.7 454.4 237.3 141.6

The sample concentration value, the dilution factor, and the extraction solvent dilution factor were calculated by using Equation 7 below.

O.D value 0.4 is correct in the calculation, so average the remaining values except X2, X64 and X128.

Dilution Total equivalent amount (%) Total equivalency (%) twice Total equivalency (%) 3 times 2 29.67 30.47 29.78 4 37.99 38.60 37.76 8 50.63 52.75 49.95 16 56.46 60.81 58.24 32 72.33 73.62 70.69 64 78.12 76.39 73.83 128 90.59 85.12 88.10 Average value (times) 54.35 56.45 54.16 Total average value 54.99

+ 5% of total average: 57.74%

-5% of total average: 52.24%

Figure 7 shows the total equivalent analysis graph.

D. Evaluation of Crude Saponin Assay

3.015 g of the collected sample is placed in a Erlenmeyer flask, and 180 ml of distilled water is added to dissolve completely. 250 ml Erlenmeyer flasks are prepared and weighed.

Dissolve 60 ml of the sample solution completely dissolved in 180 ml into three 250 ml separatory funnels. Add 60 ml of ether to the Erlenmeyer flask containing 60 ml of sample solution, shake several times, extract the fat and separate the layers. After separation of the ether and sample liquid layers in the separatory funnel, remove the ether layer and leave only the sample liquid layer in the separatory funnel.

To the Erlenmeyer flask containing 60 ml of sample solution, 60 ml of saturated butanol was added, shaken several times to extract saponin, and the layers were separated. After the separation of the saturated butanol layer and the sample liquid layer in the separatory funnel is completed, the saturated butanol layer (upper layer) is recovered and placed in a weighed 250 ml Erlenmeyer flask, and the sample solution is added to the separatory funnel again. The process was repeated three times in total to collect 180 ml of a saturated butanol layer in a 250 ml Erlenmeyer flask.

The saponin extraction is completed, discard the sample solution, and put the 180ml saturated butanol layer to the separatory funnel again, 50ml distilled water is added to wash the saturated butanol layer. 180 ml of the washed saturated saturated butanol layer was placed in a weighted Erlenmeyer flask, and an Erlenmeyer flask was mounted on the evaporator, and the water temperature of the bath was concentrated at 80 캜. Put the Erlenmeyer flask with reduced pressure concentration into the drying oven set at 105 ℃, dry it for about 15 minutes, cool in the dryer, and weigh the Erlenmeyer flask again. Weigh a 250 ml Erlenmeyer flask containing irradiated phononine.

When the weight measurement of the Erlenmeyer flask is completed, the content of the irradiated saponin was calculated using Equations 8 and 9, and the results are shown in Table 12 below.

Amount of sample by conical flask (g): 1.005 division Irradiated Phonine Content Ratio (%) Irradiation amount (mg / g) Error range (±%) Erlenmeyer flask 1 2.59 25.87 -4% Erlenmeyer flask 2 2.69 26.87  0% Erlenmeyer flask 3 2.79 27.86 + 4% Average value 2.69 26.87

+ 5% from average: 28.21 g

-5% from average: 25.52 g

8 is a graph of the investigationalponin analysis.

E. Evaluation of Molecular Weight Distribution Analysis

Sample volume (mg): 50.000 X 3 times

Molecular weight distribution analysis result table (once) Peak # Ret. Time Weight Average Molecular Weight Area (%) One 19.62 2,567,504 1.95 2 21.54 1,357,533 0.40 3 25.04 214,761 3.81 4 26.99 60,650 17.58 5 29.04 14,641 16.41 6 30.88 4,148 4.07 7 32.38 1,555 18.55 8 35.13 328 36.46 9 37.71 120 Area (%) Total 100.00 Peak number (Area ratio between Mw 2,000,000 and 3,500)
5 Area (%) 42.27

Molecular weight distribution analysis result table (twice) Peak # Ret. Time Weight Average Molecular Weight Area (%) One 18.21 3,279,365 8.66 2 20.63 1,916,448 3.35 3 23.63 493,216 9.81 4 25.46 165,379 11.73 5 27.04 58,265 13.07 6 29.54 10,344 6.34 7 31.88 2,140 19.56 8 34.96 356 27.02 9 37.96 112 0.45 Area (%) Total 100.00 Peak number (Area ratio between Mw 2,000,000 and 3,500)
5 Area (%) 44.31

Molecular weight distribution analysis result table (three times) Peak # Ret. Time Weight Average Molecular Weight Area (%) One 19.04 2,912,867 6.20 2 20.79 1,809,377 2.84 3 23.88 428,990 11.18 4 27.71 36,927 26.03 5 30.54 5,199 5.24 6 32.88 1,140 15.14 7 36.88 156 32.96 8 37.96 112 0.43 Area (%) Total 100.00 Peak number (Area ratio between Mw 2,000,000 and 3,500)
4 Area (%) 45.29

Experiment repeat count (Area ratio between Mw 2,000,000 and 3,500) Experiment repeat count Peak number Area (%) One 5 42.27 2 5 44.31

9 shows the results of GPC analysis for each manufacturing process of panaxane, and FIG. 10 shows the results of GPC analysis of panaxan.

As a result of GPC analysis for measuring the molecular weight distribution of Panaxan, the molecular weight distribution pattern of Panaxan was very similar even after three times of experiments. It is judged to be a very suitable analysis method in analyzing the molecular weight distribution of.

According to the present invention, various physical, chemical and structural properties of ginseng polysaccharides purified according to the method for purifying ginseng polysaccharide panaxane are identified as characteristics and structures that become a common denominator, and an analytical method is secured with precision, accuracy and reproducibility. By using this production method, we have developed a new ginseng product based on ginseng polysaccharides with excellent efficacy against various immune diseases by breaking the scope of ginseng industry focused on red ginseng and saponin products. By recognizing ginseng as the biological immune response regulator, which is the most important material in the huge market of alternative medicine and supplementary medicine, it is expected to provide an opportunity to expand the market of ginseng industry worldwide from Southeast Asia.

Claims (21)

A method for purifying panaxane from ginseng, comprising the following steps:
(1) washing the fresh ginseng or dried ginseng root with water and then immersing it in 70% to 100% high purity ethanol to rinse the alcohol while circulating the ethanol and then roughly crushing it;
(2) extracting at 80 to 95 ° C. using 3 to 24 times water by weight of ginseng;
(3) separating the ginseng extract and ginseng foil by centrifugal dehydrator or filter press;
(4) removing the fine particles, the large carbohydrate and the protein suspended in the ginseng extract by passing through the ultra-fast centrifugal separator;
(5) adsorbing heavy metal ions and pesticide components remaining in the ginseng extract by passing through the activated carbon and clay filter;
(6) removing the macromolecular weight material of the inert carbohydrates using a filtration system equipped with a 0.45 μm micromembrane filter or ceramic filter;
(7) Using a filtration system equipped with 1K-Dalton and 0.5K-Dalton nano-membrane in order to faithfully discard the aqueous solution containing the low molecular weight material and the metallic ionic component to obtain a purified Ginseng Polysaccharide Panaxate. step; And
(8) The obtained ginseng extract was concentrated using a reduced pressure concentrator so that the measured sugar content was 20 Brix, and then powdered by using a cryogenic freeze dryer or spray dryer and a fluidized bed dryer to obtain ginseng polysaccharide panaxuric acid powder. Or or concentrating the ginseng extract obtained in the above to obtain a ginseng polysaccharide panaxuric acid concentrate by using a reduced pressure concentrator so that the measured sugar content is 65 Brix or more. The method of claim 1,
The ethanol wash in step 1 is carried out under the conditions of 50 ℃ or less, characterized in that the ginseng raw material is washed for 2 hours or more by reflux method. The method of claim 1,
Extract the roughly crushed ginseng raw material of step 2 into the extraction tank to extract water for 6 hours at a temperature in the range of 80 ~ 95 ℃ and then cool the water into a double jacket of the extraction tank to precipitate the large carbohydrate and protein. Purification method characterized by quenching the temperature of the ginseng extract to 50 ℃ or less. The method of claim 1,
In step 2, in the case of ginseng, 3 to 6 times the weight of water is added to the ginseng, and in the case of dry ginseng, 12 to 24 times the weight of water to purify. The method of claim 1,
Purification method characterized in that the ultra-high speed centrifugal separator in step 4 has a rotation speed of 15,000rpm or more. The method of claim 1,
Purification method characterized in that to remove more than 99% bacterial microorganisms and giant carbohydrates remaining in the ginseng extract in step 6. The method of claim 1,
In step 6, the purified water is mixed with the ginseng polysaccharide extract several times so that the filtration load is not increased in order to increase the yield of ginseng polysaccharide panaxuric acid having activity such as anticancer immunity enhancement and hematopoietic promotion. Purification method. The method of claim 1,
Purification method characterized in that to remove the low-molecular ginseng components and metallic, non-metallic inorganic ionic components other than the ginseng polysaccharide remaining in the ginseng extract in step 7 above 90%. The method of claim 1,
In step 7, purified water was mixed with the ginseng polysaccharide extract several times to avoid the filtration load in order to increase the yield of ginseng polysaccharide paroxane having activity such as anticancer immunity enhancement and hematopoietic promotion. Purifying method characterized in obtaining the ginseng polysaccharide panaxuric acid solution. The method of claim 1,
In step 8, the ginseng polysaccharide panaxuric acid extract is concentrated under reduced pressure in a heat source at 70 ° C. or lower until it reaches about 20 Brix so that thermal degeneration does not occur at a high temperature, such as a freeze-drying device, a spray drying device, or a fluidized bed drying device. Purification method, characterized in that to obtain a ginseng polysaccharide as the paroxane powder. The method of claim 1,
Purifying the ginseng polysaccharide panaxuric acid extract in step 9 under reduced pressure to a sugar content of 65Brix or higher in a heat source at 70 ℃ or less so as not to cause thermal denaturation at a high temperature to obtain a ginseng polysaccharide panaxic acid high purity concentrate. The method of claim 1,
Purification method characterized in that the steps 4 and 5 are performed at the same time. The method of claim 1,
Purification method characterized in that the steps 6 and 7 are performed at the same time. A method of analyzing the ginseng polysaccharide panaxuric acid according to any one of claims 1 to 13 by measuring the irradiated butanin content using a saturated butanol method. A method for analyzing ginseng polysaccharide panaxuric acid according to any one of claims 1 to 13 by measuring the total sugar content by the D.N.S method using a D.N.S (Dinitro salicylic acid) reagent. A method for analyzing the ginseng polysaccharide panaxuric acid according to any one of claims 1 to 13 by measuring the acid polysaccharide content using the carbazole-sulfuric acid method. A method for analyzing the ginseng polysaccharide paroxane purified according to any one of claims 1 to 13 by measuring the molecular weight distribution of the ginseng polysaccharide (panaxic acid) using GPC (Gel Permeation Chromatography). Ginseng polysaccharide composition for anticancer immunity enhancement and hematopoietic promotion comprising the ginseng polysaccharide panaxic acid purified according to any one of claims 1 to 13. The method of claim 18,
The ginseng polysaccharide panaxuric acid has an analytical value as shown in Table 3 below, ginseng polysaccharide composition for anti-cancer immunity promotion and hematopoietic promotion.

The method of claim 18,
Ginseng polysaccharide panaxuric composition, characterized in that the composition is a pharmaceutical. The method of claim 18,
Ginseng polysaccharide panaxuric composition, characterized in that the composition is processed food.

Images