TWI308871B - Preparation of alkali soluble polysaccharides from panax quinquefolium, process for making and use in enhancing immunity of the same - Google Patents

Description

1308871 ..

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V IX. OBJECTS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to a Western-style signing, a six-year-old four-salt/ready polysaccharide preparation, a preparation method thereof, and an application for boosting immunity, especially Western glutinous rice (fine χ χ / / o " Mm). Root remnant - an aqueous solution of the water extract of the Western scorpion test soluble multi-vine preparation. Prior Art • A ^ {Panax ginseng CA Meyer) ^ ® ^ ^ {Panax is a genus of genus ceple), the roots of which have a long history of medicinal history in Asia, especially China, are also considered by traditional Chinese medicine and traditional medicine. It is a safe and effective noble medicine. Chinese people have always attached great importance to the treatment and replenishment effects of human beings. People are considered to have the effects of making up the vital energy, making up the deficiency, promoting metabolism, blood circulation and strengthening the body. People are suitable for the elderly and those who are weak after illness. There are also many academic papers that mention the effects of human sputum on immune response. For example, human sputum can improve the resistance of users to foreign infections. 'Reducing adverse allergic reactions, inhibiting tumor growth in vivo, and observing from the cellular level. To the regulation of basic immune factors by humans. Western oysters are generally considered to have similar effects to human mites, but they are still different plants. Chinese medicine believes that there is a considerable difference in efficacy between the two. Western oysters are mainly produced in the United States, Canada and other places. The quality of the western oysters produced in the state of Wisconsin is superior. Western is cold and cold, so it is best for drinking when it is hot. Citigroup can replenish qi, reduce phlegm and heat, 1308871 refreshing and spleen appetite, suitable for people who are busy with work and cause sleep deprivation. Traditionally, it has been considered that the active ingredient in human mites and yam is Ginsenosides. In most of the early academic research, saponins were the main research objects. Recently, some scholars have begun to pay attention to the physiological activities of various polysaccharides and components in human mites. Among them, Professor Tomada's research room at Kyoritsu College of Pharmacy has done a series of related experiments (Panax ginseng CA Meyer) ^ m % 5 Two acidic polysaccharides, Ginsenan PA and Ginsenan PB, were extracted from human I mash extracted with hot water [Tomoda et al., 1993, Biol Pharm. Bull., 16, 22-25.; Tomoda et al. , 1994, Biol Pharm. Bull., 17, 1287-1291.] » ^ According to the compositional analysis, the molecular weights of Ginsenan PA and Ginsenan PB are 160,000 and 55,000, respectively. The single-sound composition of Ginsenan PA is 21.3% arabinose, 53.4% galactose, 2.0% rhamnose, 16.0% galacturomic acid and 2.7% glucuronic acid, with a composition ratio of 11:22:1:6:1. The monosaccharide composition of Ginsenan PB is ii_〇% arabin〇se, Lu 32.2% galactose, 8.1% rhamnose, 39.9% galacturomic acid and 5.0% glucuronic acid, with a composition ratio of 3:7:2:8:1. According to the experimental results, both of these polysaccharides have an obvious effect of enhancing immune activity. The Yamada Research Team of the Toyo Medical Research Institute of the Kitasato Institute in Japan isolated a number of polysaccharides from the leaves and roots of human c. γβγα) and compared the differences in structure and activity. [Gao et al., 1989, Planta Mediea, 55, 9-12.]. The leaves and roots of the human cockroaches obtained from mainland China are first extracted with alcohol to remove the components of saponins. Then the residue is extracted with water, and the residue after water extraction is 1308871.

- The residue is extracted with 0.5M NaOH aqueous solution to separate the water-soluble polysaccharide GL-2 and the alkali-soluble polysaccharide GLA-2 from the leaves of the human cockroach, and the water-soluble polysaccharide GR-2 and the alkali-soluble polysaccharide GRA can be separated from the root of the human cockroach. -2. These four types of gambling are based on the amount of acidic components that make up the monosaccharide, and further separation and purification can produce many different polysaccharide components, several of which have special physiological activities. For example, GL-3 has the strongest Anti-Complementary-Effect and GL-BIII have obvious effects on protecting mucosal cells and reducing the incidence of gastric ulcer. However, according to the literature published by the Yaniada team [Sun et al., 1991, J. Ethnopharmacol., 31, 101-107.], the yield of the test-soluble polysaccharide extracted from human 蔘Λ/e er is very low. The yield of the alkali-soluble polysaccharide (GRA_2) was only 0.49% (% by weight;) 'After 100 g of human cockroaches, only 49 g of alkali-soluble polysaccharide was extracted. Although GRA-2 has a very significant inhibitory effect on gastric ulcer in mouse experiments, it is difficult to produce enough alkali-soluble polysaccharides for subsequent experiments without further investigation. Most of the research is biased towards the polysaccharide fraction extracted from the leaves of human cockroaches. A research team at Bethune Medical University in mainland China isolated a water-soluble polysaccharide PPq_丄 from the western carp [Ma et al., 1997, Baiqiuen Yike Daxue Xuebao, 23, 236-238.; Zhu et al., 1997, Zhongguo Yaolixue Tongbao, 13, 76-78.1, this polysaccharide can stimulate the spleen lymphocytes of mice to produce cytokines, promote the transformation and proliferation of white blood cells, and thus improve immunity. CV Technologies, a Canadian biotech company, is also working on the development of the immunological activity of the polysaccharides of the yam, which extracts the water-soluble polysaccharide component CVT-E002 [Shan et al" 2002, US patent 6,432,454] from the yam. PVT2, PQ223 and other components were isolated and purified from CVT-002 polysaccharide. These components have obvious ability to enhance immune activity. From the preclinical studies in mice, these water-soluble polysaccharides can stimulate the differentiation of spleen lymphocytes into immune B cells. B cells produce a large amount of antibodies. Increasing the amount of immunoglobulins such as IgG in serum also stimulates the production of various cytokines (Cytokine) such as IL-1, IL-6 and TNF-α by megatuber cells. The ability of these water-soluble polysaccharides to enhance their immunological activity can be used to help humans resist foreign infections or diseases associated with autoimmunity. The company has obtained US Patent No. 6,432,454 and has been the water-soluble polysaccharide CVT_E of American yam. 〇2 Developed as a healthy food buckle COLD-fX® for preventing colds, it has a good response in the European and American markets. Currently cVTE〇〇2 At the same time, human and clinical trials in humans are carried out in the country. A large part of the yam is used to extract saponins and water-soluble vinegar and other ingredients. These factories for extracting yam are produced with a lot of ginsengmarc. After extracting the saponin and the water-soluble polysaccharide by ethanol and hot water, the dried yam slices will produce about 50 kg of yam. These slags are traditionally considered as low-value by-products. So far, the inventors It is known that there is no literature or patent that mentions that Western slag has any medical application value, and Western slag is usually sold as an animal feed additive by the extraction plant S, and one kilogram of slag is sold at around RMB. ^ ^ 1308871

SUMMARY OF THE INVENTION A primary object of the present invention is to provide a dry weight of 50% from the yam. Western wilting) to extract the technology of the medicinal part. The present invention discloses a novel alkali-soluble polysaccharide preparation obtained by extracting and further purifying from the root of the yam (7 > gw called we/o/iMw) using an aqueous alkaline solution. The present invention also discloses a method of producing the cicada-soluble polysaccharide preparation, and a medical application of the cicada-soluble polysaccharide preparation. The sliced root of the yam is subjected to hot water and/or alcohol-extracted slag, and the polysaccharide preparation extracted by the alkaline aqueous solution and further purified has been experimentally proven to have a remarkable effect of improving immunity. The activity of the immune cells can be significantly improved under the preparation. Clinically, it can be used to treat or prevent diseases related to immunity, such as colds, AIDS and cancer. Preferred embodiments of the invention include, but are not limited to, the following items: 1. A method of preparing a campanol-soluble polysaccharide preparation comprising the following φ steps: A) taking an anthrax (A) in an aqueous solution "Kiss such as (four) root residue" wherein the residue of the root of the sea urchin contains a human 蔘t glycoside and a water-soluble polysaccharide extracted from the root of the yam with a polar solvent, and a residue portion obtained by solid-liquid separation of the inert solvent extraction mixture; B) a liquid-liquid separation step obtained by the solid-liquid separation step A) to obtain a liquid portion containing an alkali-soluble polysaccharide; and C) purifying the liquid portion containing the alkali-soluble polysaccharide to obtain a neutral A test soluble polysaccharide preparation. 1308871 2. The method of the method of item 1 wherein the purification of step c) comprises the steps of: neutralizing the liquid portion of step B) by adding an acid; drying the neutralized liquid obtained, Obtaining a neutral alkali-soluble polysaccharide preparation 3. The method of the method of item 1 wherein the purification of step c) comprises the steps of: adding CrC3 alcohol to the liquid portion of the step B), thereby producing more alkali solubility Precipitation; • obtaining a precipitate of the alkali-soluble polysaccharide by solid-liquid separation means; mixing the precipitate of the test soluble polysaccharide with a mixed solvent selected from the group consisting of alcohol or water, Cl_C3 alcohol to obtain a mixture in which the alkali solution polysaccharide is concentrated; Neutifying a mixture thickened with an alkali solution polysaccharide by adding an acid; removing an alkali-soluble polyterpene solid from the neutralized mixture by solid-liquid separation means; and drying the alkali-soluble polysaccharide solid to obtain a neutral alkali solubility The method of claim 3, wherein the CVC3 alcohol is ethanol. 5. The method of item 1, wherein the purifying of step C) comprises the step of: filtering the liquid portion of step B) with a molecular sieve. a portion having a molecular mass greater than that of the 3 sacral ear; neutralizing the portion of the molecular mass greater than 30 angstroms by adding an acid; and drying the neutralized molecular mass greater than 3000 A portion of the otolysin which is a neutral, alkali-soluble polysaccharide preparation. 6. The method of item 1, wherein the purification of step C) comprises the following step 10 1308871 to filter the liquid portion of step B) with a molecular sieve Molecular mass obtained More than 10,000 ear-swallowed parts; neutralize the part of the molecule with a mass greater than 10,000 ear-swallows by adding an acid; and • dry the neutralized molecular mass to more than 10,000 ear-swallowed parts to obtain a medium A test-soluble multi-enzyme preparation. 7. The method of step 1 wherein the purification of step c) comprises the following steps: • filtering the liquid portion of step 3) with a molecular sieve to obtain a molecular mass greater than 3000 amps. Part; mixing the portion of the molecular mass greater than 3000 amps with water; and filtering the mixture of the portion of the molecular mass greater than 3 amps with water by a molecular sieve to obtain a second sputum a molecular mass greater than 3 parts of the deafness; neutralizing the second filtered mass of the molecular mass greater than 3 000 deaf by adding an acid; and • drying the neutralized molecular mass A neutral test soluble polysaccharide preparation is obtained with more than 3000 ear swabs. 8. The method of item 1, wherein the purifying of step C) comprises the steps of: filtering the liquid portion of step B) with a molecular sieve to obtain a molecular mass of more than 10,000 auricular portions; the molecular mass is greater than 10,000 The part of the scorpion scorpion is mixed with water; the molecular mass of the part of the scorpion and the water is filtered by a molecular sieve to obtain a molecular mass of the second filtration greater than ι〇_ 耳Part of the cockroach; 1308871

Neutralizing the portion of the second filtered molecular mass greater than 10,000 ear-irradiated by adding -acid; and drying the neutralized molecular mass greater than 1,000,000 deafness to obtain a neutral alkali solubility Polysaccharide preparation. 9. The method of clause 5, 6, 7, or 8, wherein the drying is spray drying or freeze drying. The method of item 1, wherein the neutral alkali-soluble polysaccharide preparation has a molecular weight of between 5,0 Å and 1, 〇〇〇, 〇〇〇. 11. The method of clause 10, wherein the neutral alkali-soluble polysaccharide preparation has two major portions, the first portion having a molecular weight of about 10 000 and the second portion having a molecular weight of about 900,000. 12. The method of item 11, wherein the first portion of the cardinic assay-soluble multi-enzyme preparation is greater than the second portion. 13. The method of clause 11, which further comprises isolating the first portion and the second portion of the neutral alkali-soluble polysaccharide preparation, wherein the first portion or the second portion of the Lu is used as the neutral Alkali soluble polysaccharide preparation. 3. The method of claim 1, wherein the root residue of the sea urchin contains a residue portion obtained by extracting the root of the scorpion from the root of the sputum with water, a glycol or a mixture thereof. On the eve of the day, π η π· causes the lower margin slag to contain (10) the part of the residual edge produced by the second extraction of the root of the oriental carp. For example, the extraction of the method 'in which step Α), w 8 ° ° C. The liquid aqueous solution of the method of the method of the present invention, wherein the method of the method of item 16, wherein the step A) is 12 1308871. A pH value of not less than 12. 18. The method of item 16, wherein the alkaline aqueous solution of step a) is a 0.4-10 g/L aqueous NaOH solution and the extraction is carried out at room temperature for 〇$. A kite-soluble polysaccharide preparation prepared by the method according to any one of the preceding items. An immunostimulating composition comprising an anthraquinone-soluble polysaccharide preparation according to item 19, which is an effective ingredient for enhancing immunity. Embodiments The present invention discloses a method for preparing a neutral cold-testing preparation from the residual edge of the root of the yam, and an experiment for stimulating the proliferation of immune cells of the preparation, such as human peripheral blood mononuclear cells. (Human p-heral Bl00d MononucIear Ceu, pBMCs) to evaluate the activity of the φ agent to boost immunity. The invention also includes performing a basic molecular weight analysis of the formulation from which the different molecular weight fractions or monosaccharides are separated, and the ability of the different molecular weight lesions or monosaccharides to enhance immunological activity is assessed. The extraction and purification of saponins and water-soluble polysaccharides from western roots is a well-established technique, such as the aforementioned U.S. Patent 6,432,454. The patent content is incorporated into the case by reference. There are many companies in North America and China that produce extracts of Acacia. The water-soluble polysaccharide extracted from the common yam is usually extracted by hot water, and the extracted saponin is usually extracted and purified using a different ratio of water/13 ethanol mixed solvent. The western and western roots (four) which are suitable for use in the present invention as a starting material are subjected to the extraction of the residue of the roots of the yam, which is residual after the water-soluble polysaccharide and the saponin of the yam. Experiment The present invention cuts 40 kg of dried yam roots purchased from Canada, extracts 400 liters of pure water 90 〇c for i hours, and removes the residual curtain with 320 A liters of pure water for 9 hours. The two extractions are combined to obtain an extract rich in saponins and water-soluble polysaccharides, which are collected after two hot water extractions, and then passed through (10). c Drying, pulverizing after a 6 〇 net purpose 4 ’ can get about 20 kg of dried yam powder for storage. The extract of the field containing saponins and water-soluble polysaccharides is spray-dried to obtain a mixture of about 5 kg of saponins and water-soluble polysaccharides, which are extracted at different concentrations of alcohol, and purified according to a conventional purification process. It is a saponin SA2 rich in human saponin Rbi fine We Rbl, a saponin SA3 rich in saponins, and a water soluble polysaccharide SB4 from yam. SA1, SA2, SA3, SB4 were dissolved in an appropriate amount of methanol, and then analyzed by HPLC on an Rp_ci8 column. From the analysis results, it was found that SA1 contains 7〇% or more of total saponins of yam, and sapon of saponin contained in SA2 accounts for 80.1% of total saponins; SA3 contains saponin which is soluble in total saponins. The total saponin content of the polysaccharide SB4 is below 1%. The laboratory manufacturing process for preparing a neutral alkali-soluble polysaccharide preparation (hereinafter referred to as ST-GRAPs) from the spare dry dried yam powder is shown in Fig. j 1308871. Extracting the alkali solubility contained in it by using two different extraction conditions. The extraction conditions a. were mild extraction conditions and were extracted with a mixture of % (w/v) sodium aroxide at room temperature for 1 hour. Extraction conditions b. are more aggressive extraction conditions 'with 1% (w/v) sodium hydroxide, 6 Torr. (: stirring for 1 hour extraction. Strong test • After extraction, use a centrifuge for solid-liquid separation, centrifuge speed 3000 rpm, 1 〇 minutes. After centrifugation, take the supernatant, the lower sludge solid is discarded. The supernatant is added to the same volume. The 95% medicinal alcohol dehydrates the alkali-soluble polysaccharide to form a colloid. φ Because the mixture of alcohol and water will produce many bubbles at the same time, the bubbles adhere to the polysaccharide colloid, so that the colloid will be suspended in the upper layer of the aqueous alcohol solution. Gently pour out and discard the lower aqueous alcohol solution. The poured transparent colloid uses rapid stirring to allow the bubbles adhering to the colloid to escape, so that the polysaccharide colloid can be precipitated in the solution and centrifuged at a speed of 3 rpm. i 〇 minute. Collect the precipitated beige precipitate and discard the supernatant. Add the white polysaccharide residue to 60% alcohol solution, stir and wash, add 3NHc丨 to adjust the pH to neutral (pH=6.8~7), adjust After the pH value, it was centrifuged by a centrifuge, and the white alkali-soluble polysaccharide precipitate was collected by rotating at 3000 rpm for 10 minutes. The white polysaccharide precipitate was dried overnight in a 50 ° C oven. The yellow flakes are then powdered in a small pulverizer and sieved (1 〇〇 mesh) to obtain a semi-mouthed mouth of ST-GRAPs. The semi-finished product of ST_GRAPs is dissolved in water to confirm that the pH of the aqueous solution is neutral. The insoluble particles are removed by suction filtration using ordinary filter paper. The clarified filtrate is added to an equal volume of 95% medicinal alcohol, and the precipitate is collected and dried to obtain a finished product of neutral saponin-soluble polysaccharide preparation (ST_GRAPs). After milder extraction conditions ( After the above extraction conditions a) and the above purification step, about 6 g of st_GRAPs 15 is 1308871 can be separated per 100 g of the yam residue. - Finished product SB1 (yield. After the more vigorous extraction conditions (previous extraction condition b)) In the purification step, about 14 g of ST-GRAPs finished product SB2 (yield 14%) can be separated per gram of yam yam. The present invention is purified from the production of neutral alkali-soluble polysaccharide preparation purified from yam. The procedure is about 6 to 15%, which is significantly higher than the yield of the alkali-soluble polysaccharide (GRA-2) separated from the human cockroach (household (4) by c. 12 3〇)). Panax quinquefolium and k spring, panax ginseng c. Α, are different kinds of plants, and there are also significant differences in the efficacy of traditional Chinese medicine. The present invention also finds that the western slag contains The water-soluble polysaccharide SB4 of the yam has been purified from the hot water extract of the root of the yam, and the slag is extracted with an aqueous alkali solution to obtain the ST. -GRAPs finished SB1 and SB2 'a total of three polysaccharides. The three polysaccharide preparations of sbi, 8 phantom and SB4 were respectively subjected to colloidal filtration chromatography (Gpc) for molecular weight distribution analysis. P-lyethylene oxide (pE) 〇) as a molecular weight reference standard. The results of the analysis are shown in Figures 2a, 2b and 2c. It can be clearly seen in Figures 2a and 2b that SB1 and sB2 of ST_GRAPs have similar molecular weight distributions, and the molecular weight distribution patterns of the two have obvious retention times of 145 and 18 minutes. The peaks correspond to molecular weights of 900,000 and 10,000, respectively. The area of high molecular weight peaks (residence time = 14.5) of SB2 is smaller than the area of high molecular weight peaks (residence time = 145) of SB1, indicating that the average molecular weight of SB2 is slightly smaller. The average molecular weight of SB1 may be due to the more intense extraction of the test-soluble polysaccharide contained in A Φ artichoke residue.

(S 16 1308871 .

Conditions result in molecular chain scission, while mild extraction conditions of SB1 preserve a relatively complete polysaccharide structure. The molecular weight distribution of the water-soluble polysaccharide preparation SB4 of yam is significantly different from that of ST-GRAPs. As shown in Figure 2c, SB4 has a maximum molecular weight of less than 500,000 and a very broad molecular weight distribution with no distinct peaks. The molecular weight of SB4 is widely distributed between 1 〇〇〇 and 1 〇〇〇〇〇. From the analysis of molecular weight, the water-soluble polysaccharide preparation contained in the root of the yam is very different from the alkali-soluble polysaccharide preparation contained in the yam. The molecular weight distribution, the alkali soluble polysaccharide preparation and the water soluble polysaccharide preparation should be two types of polysaccharide preparations having different polysaccharide compositions. In order to compare the immunomodulatory activities of various preparations extracted from the yam, the present invention utilizes immune cells cultured in vitro to extract and purify six different kinds of yam extracts from the yam, SA1 (salt total saponin), SA2 (Aristotle saponin rich in ginsenoside Rbl), SA3 (Western saponin rich in ginsenoside Re), SB1 (more soluble in Western carp extracted by mild conditions), SB2 (by severe conditions) The extracted anthraquinone-soluble polysaccharide) and SB4 (the water-soluble polysaccharide of the oriental jelly) were subjected to preliminary immunomodulatory activity assay. The human immune cells cultured in vitro were treated with various concentrations of extracting swords (SA1, SA2, SA3, SB1, SB2, SB4; 0-200 Kg/mL), together with commercially available immunopotentiators and inhibitors. The immunomodulatory activity and effective concentration were tested. In this experiment, the immune cells of the blood samples of three healthy subjects were treated with different concentrations of the extracted components, and then the commercially available immunopotentiators and inhibitors were detected to detect the cell activity, cell survival rate, and The duration of action is used to assess the immunomodulatory activity of each extract. 17 Immune cells (human peripheral jk monocytes) separation of experimental steps: 1. Place the purchased 7.5 mL white blood cell thick solution in a centrifuge tube under room temperature aseptic environment 'Add 22.5 mL buffer solution (Hank's balanced salt solution; HBSS) Dilute the blood cells, mix well and slowly inject into a centrifuge tube with 15 mL Ficoll-Paque Plus (Density = 1.077 ± 0.001 g/mL) and centrifuge. 2. Collect the middle human peripheral blood mononuclear cell layer 'and add appropriate amount of HBSS buffer to dilute' to remove the Ficoll-Paque Plus solution by centrifugation. 3' After adding 9 mL of sterile water to break the red blood cells, quickly add 1 mL of OX HBSS to mix to maintain cell osmotic pressure. 4 · Centrifugal removal of the supernatant. After adding an appropriate amount of medium to uniformly suspend the cells, the cells were counted. The cell proliferation test was performed using a 96 well plate, and each well was dispensed with 2 X 1 〇 5 cells, and various extraction preparations at different concentrations were added to make the final concentration of the preparations 3-125, 6.25, 12.5, respectively. , 25, 50, 100, 200 pg/mL. The different drugs and concentrations of each cell were repeated four times, and phytoagglutinin (PHA) and cyclosporin A (Cyclo A) were used as the positive control group and the negative control group respectively. After 72 hours of culture, Using the WST_i method, it was tested whether the different concentrations of the extracts affected the cell viability of human peripheral blood mononuclear cells (PBMCs) cultured in vitro, and probed at 1308871.

Whether it can promote or inhibit the proliferation of cells. WST-l Assay - Cell viability and proliferative response assay Cell number and activity were measured using the activity of dehydrogenase in the cell line. Where the mitochondria are respiration, there are many reduction reactions catalyzed by dehydrogenase, and the more vigorous the respiration of the cells, the higher the activity of the dehydrogenase. WST-1 (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetra-zolio]-l, 3-benzene disulfonate) Hydrogenase removes B hydrogen ions to form a water-soluble formazan. As shown in Figure 3, it has a strong absorbance at a wavelength of 45 〇 nm, and the higher the cell activity or the larger the number of cells, the darker the color. Therefore, WST-l can detect the activity of cells and the number of cells. 'Because the produced formazan is water-soluble, it is suitable for both suspension cells and adherent cell lines', and it is also accurate in sensitivity and analysis. The statistical method was used to calculate the experimental results of three healthy human cells. After standardization, the leaf φ its mean and standard deviation 'to analyze whether each group has statistical significance (p value greater than 0.05 is not statistically significant, p value is less than 〇.〇5 Statistically significant, * : P < 0.05, " : p < 0.01), and the drawing is shown below. The percentage of promotion obtained by the formula: (dosing group - blank control group) / blank control group * 100% is attached to the chart (Figure 4 to Figure 9). Results and Discussion In each group of experiments, both PHA and Cyclo. A were added as a positive control group and a negative control group. In the actual data, the cellular activity of PBMCs with significantly increased PHA was observed, and the average cell activity was increased. 1308871 cell activity has inhibitory effect, the experiment can see very 71.8%' and as a negative control group Cycl〇. A obviously on the pBMCs fine 'average inhibition of cell activity up to 56.4% ^ the results of the kidneys of each group, confirmed This immune self-b force regulation experiment performed in this experiment is an effective method for clearing. As shown in Fig. 4 and Fig. 5, SA1 (salt total saponin) and sa2 (mandarin saponin rich in ginSenoside Rbl) promoted the cell viability of pBMCs, compared with the blank control group, # sai added concentration It is 25 us/mT. Bi· , ηίτ,, λα /〇, /--.,.

The activity-proliferation curves are very similar. In addition, it can be seen in Fig. 6 that when SA3 (rich • saponin containing ginsen〇sideRe) is added at a concentration lower than 50 pg/mL, there is no significant promoting effect on cell viability, and it is required to reach a very high concentration. 200 pg/mL will have a significant promoting effect, the percentage of promotion is 40.86%, only about 62 times the percentage of PHA promotion of 66 〇8%. Combining the results of these three images, it can be seen that the main two components of the saponins, the saponins Rbl and Re', are more effective in promoting immunity, which is saponin Rbl, while saponin Re has almost no immunity. effect. This part of the experiment confirmed that the extract of Dioscorea zingiberensis can stimulate the proliferation of immune cells and increase the activity of immune cells. The main effective saponin component is suffocating.

Cs 20 1308871

Rbl. The activity of immune cells increased at a low concentration of saponin (3 125~25 called/melon), and gradually increased with the increase of saponin concentration. When the concentration of saponin added exceeded 5〇Mg/mL, the cell activity decreased significantly. And Fig. 8 is an experimental result of the neutral alkali-soluble polysaccharide preparation disclosed by the present invention - ST-GRAPs stimulates proliferation of immune cells, and Fig. 7 is an addition of ST-GRAPs preparation SB1 extracted by mild conditions in three different subjects. In the cell viability assay of peripheral blood mononuclear cells (PBMCs), there are very consistent results. SB1 can significantly stimulate immune cell proliferation, and the immune cell proliferation activity is positively correlated with the amount of SB1 added. Pg/mL also did not cause cell death, and there was a significant difference in cell death when the concentration of saponin RM was more than 50 pg/mL. When the concentration of SB 1 was 25 pg/mL, the percentage of cell activity was promoted. 162.21%, higher than the promotion effect of PHA by 2. π times, and the promotion effect of saponin is equivalent. When the added concentration of SB1 reaches 2 〇〇 / 〇 1 [, the percentage of cell activity promotion can be Achieved 3.95 times PHA. Figure 8 shows the addition of ST-GRAPs preparation SB2 which was extracted by violent conditions. Basically, SB2 promoted the activity of immune cells to be the same as SB 1. Activity increased with the addition of SB2. The promotion effect of SB2 at low concentration was slightly lower than that of SB1. When the concentration of SB2 was 25 pg/mL, the percentage of cell activity was 59.63%, only 73 times that of PHA. The concentration of SB2 was 2〇〇0§/ At 1111^, the percentage of cell viability was 24〇.13<)/., which was 2.96 times higher than that of PHA. The effect of SB2 on promoting immune cell activity was significantly lower than that of SB 1 'this may be due to higher temperature and Extraction with a higher concentration of aqueous sodium hydroxide solution, resulting in the molecular structure of the saponin-soluble polysaccharide 1308871

V. This can also be confirmed by the molecular weight analysis of GPC. In the experimental data (Fig. 2), it can be seen that the molecular weight distribution of SB2 is different from that of SB1, and the molecular weight of the polysaccharide molecules is reduced by intense conditions. • The above experimental results show that ST-GRAPs of the present invention is an effective promoter of immune cell activity, and it has been confirmed in vitro that ST-GRAPS can significantly enhance the activity of human peripheral blood mononuclear cells (PBMCS). • The water-soluble polysaccharide SB4' of the additive used in the experiment of Figure 9 can purify a variety of water-soluble polysaccharides from hot water extracts of mandarin or yam, which is the direction of many research teams. CV Technologies, a biotechnology company in Canada, has extracted the water-soluble polysaccharide component CVT-E002 from yam, and successfully developed CVT_E〇〇2 as a health food for improving immunity and preventing influenza (US patent 6,432,454). Some clinical trials. The present invention utilizes the water-soluble polysaccharide SB4 of the yam, which is purified from the hot water extract of the sea bream, to perform the same in vitro stimulation of the activity of human peripheral blood mononuclear cells (PBMCs). In Figure 9, it can be seen that SB4 can significantly increase the activity of immune cells at a very low concentration. When the concentration of SB4 is 3.125 pg/mL, the percentage of cell activity is 158.86%, which is 1.95 times that of PHA. When the concentration exceeds 625 Hg/mL, the activity of immune cells begins to decrease with the increase of the concentration of SB4. This activity of immune cells exceeding a certain concentration decreases with the increase of the concentration, and the addition of human asphyxiation In the experiment of Pu Re, 'the situation observed is similar. This may be due to excessive drug concentration causing some cells to be killed by poisoning' or the cells being over-stimulated to cause 22 1308871 膂 death. Based on the above experimental results, it can be found that Sai, SA2, SBl, SB2, and SB4 have an effect of promoting human immune cell activity in vitro. In the experiments of the SARS and SA2 extracts, the effect of promoting immune cell activity reached a maximum at an addition concentration of 25 pg/mL, and then the promoting effect decreased with increasing concentration. In the SB4 experiment of water-soluble polysaccharides of the yam, the effect of promoting immune cell activity reached a maximum at a very low concentration of 3 125 pg/mL, and then decreased rapidly with increasing concentration. In the experiments of the scorpion-soluble polysaccharide preparations SB1 and SB2 disclosed in the present invention, the immune cell proliferation activity and the added concentration are positively correlated, and even if the concentration is as high as 2 〇〇 Kg/mL, the immune cell death is not caused. These three different experimental results represent a different mechanism for stimulating immune cell proliferation in the two extracts of the yam. Previous studies on the efficacy of all kinds of yam are concentrated on the traditional squid and water-soluble polysaccharides that can be dissolved in hot water boiling. The slag after hot water extraction has not seen any use in national patents and research literature. the study. The present invention prepares an alkali-soluble polysaccharide preparation from the yam residue and proves that it has a high potential to be developed as an immuno-promoting drug or a health food' which will be used for preventing or treating immune-related diseases such as the common cold, influenza, AIDS and cancer. The present invention also proposes an ST-GRAPi amplification mass production process for preparing a neutral alkali-soluble polysaccharide preparation from yam. As shown in the figure, 3 gram dry dried yam> powder, using mild room temperature extraction conditions with 1 $ liter 0. 1% (w / v) sodium hydroxide, stirring 25 〇〇卬 melon at room temperature , i hour extraction. After centrifugation and filtration to remove solid particles above μ45 μηη, first use the molecular 23 1308871 10,00 〇 ultrafiltration system (Cut-off = 1 〇, 〇〇〇 Da (Dora)) will clarify The extract was concentrated 10 times, diluted with 9 times the volume of pure water, and subjected to a second ultrafiltration to wash away sodium hydroxide, salts and other small molecules with a molecular weight of less than 10,000. The ultra-filtered concentrate was adjusted back to neutral with a small amount of hydrochloric acid (pH = 6.8~7_0). After lyophilization, the sieve was pulverized (100 mesh) to obtain 28.8 g of the finished product of the alkali-soluble polysaccharide preparation ST_GRAPs, yield 9.6. %. This mass production process avoids the use of precipitated polysaccharides to avoid alcohol recovery and saves production costs, while avoiding excessive hydrazine (NaCl) residues in the finished product of the alkali soluble polysaccharide formulation ST_GRAPs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart showing the laboratory manufacture of a neutral, soluble polysaccharide preparation prepared from dried yam powder according to the method of the present invention. Figure 2a shows the picture! The manufacturing process of the present invention uses 〇l% (w/v) sodium hydroxide and a stirring condition of stirring for 1 hour at room temperature to prepare a knee-filter chromatography (Gpc) of neutral barium lysine SB1. Molecular weight distribution map. The figure shows the manufacturing process according to the invention of Figure 1 with 1.0. /. Colloidal filtration chromatography (Gpc) molecular weight distribution map of (w/v) sodium hydroxide and neutral barcelonic soluble formulation SB2 prepared by stirring at 60 ° C for 1 hour. Fig. 2c shows the molecular weight distribution map of colloidal filtration chromatography (GPC) of the water-soluble polysaccharide SB4 of Anthraquinone purified from the hot water extract of the root of the yam. Figure 3 shows WST-1 -tetra-zolio]-1 (4-[3-(4.1odophenyl)-2-(4-nitrophenyl).2H-5 24 1308871 .

V 3-benzene disulfonate) is a reaction form in which the dehydrogenase in the granulocyte is removed to remove hydrogen ions to form a water-soluble f〇rmazan. Figure 4 shows the effect of the addition of SA1 (total saponin) on the cell viability of human PBMCs (peripheral blood mononuclear cells) cultured in vitro, • which also showed the immunostimulant PHA (phytohemagglutinin) and The inhibitor (cycl〇sporin A) was the result of the positive and negative control groups. Figure 5 shows the effect of adding SA2 (rich in human saponin RM) on the cell viability of human PBMCs (peripheral blood mononuclear cells) cultured in vitro for a short period of time, which also showed the use of the immunostimulant PHA (plant agglutination) The inhibitor and the inhibitor (cycl〇sporin A) are the results of the positive and negative control groups. Figure 6 shows the effect of the addition of SA3 (rich in human saponin Re) on the cell viability of human PBMCs (peripheral blood mononuclear cells) cultured in vitro, together with the immunostimulant PHA (phytohemagglutinin) and The inhibitor (cyCi〇sporin A) was the result of the positive and negative controls. Figure 7 is a graph showing the effect of adding the neutral barley-soluble polysaccharide preparation SB1 of the present invention of Fig. 2a on the cell survival rate of human PBMCs (peripheral blood mononuclear cells) cultured for 72 hours, which is also shown to promote immunity. The agent PHA (phytohemagglutinin) and the inhibitor (cycl〇sp〇rin A) were the results of the positive and negative control groups. Figure 8 is a view showing the effect of adding the neutral barley-soluble polysaccharide preparation SB2 of the present invention of Figure 2b to the cell viability of human pBMCs (peripheral blood mononuclear cells) cultured in vitro for 72 hours, in which an immunostimulant was simultaneously shown. PHA (plant globulin lectin) and inhibitor (cyel〇sp〇rin A) were the results of the positive and negative control groups. 25 1308871 Figure 9 shows the effect of the addition of 赆Λ2, a traditional water-soluble polysaccharide SB4 of Figure 2C, on the cell viability of human PBMCs (peripheral blood mononuclear cells) cultured in vitro for 72 hours, pot + agglutination 'Dangan at the same time The results of the positive and negative control groups of the immunostimulating agent PHA (plant blood cell ', prime) and inhibitor (eycl〇sporin A) were shown. Fig. 1 Flow chart of mass production of 〇 _ polysaccharide preparation 颂 shows the preparation of neutral alkali solubility from dried yam powder according to the method of the invention

26 4

Claims (1)

.1308871 Revised) Patent application scope: [丨ff HH 1 · A method for preparing a campanol-soluble polysaccharide preparation 1 comprises the following steps: A) an alkaline aqueous solution having a pH of not less than 12 at room temperature to 8 Torr .匸0 /〇(4) root residue, wherein the residue of the root of the yam contains a water-soluble mixture of water, methanol or ethanol or a mixture thereof, and the water-soluble mixture is extracted by solid-liquid separation. a residue portion produced; B) a solid aqueous extraction step obtained in the solid-liquid separation step a) to obtain a liquid portion containing an alkali-soluble polysaccharide; and C) purifying the liquid portion containing the test-soluble polysaccharide A neutral alkali-soluble polysaccharide preparation is obtained, wherein the purification of step C) is selected from the purification methods of the group consisting of the following (1) to (vi): The purification method (1) comprises the following steps: neutralizing step B) by adding an acid a liquid portion; drying the neutralized liquid to obtain a neutral test-soluble polysaccharide preparation; and the purification method (ii) comprises the steps of: adding CrC3 alcohol to the liquid portion of the step B), thereby producing alkali solubility Precipitation of polysaccharide; precipitation of the alkali-soluble polysaccharide by means of solid-liquid separation; precipitation of the alkaline soluble polysaccharide with a mixture selected from Ci-Cs alcohol or water and (: 1-(:3 alcohol) The solvent were mixed to obtain a mixture enriched test solution to listen to; 27 1308871-, (Amended in October 2008) a mixture in which the alkali solution is concentrated by adding an acid; the test-soluble polysaccharide enclosure is taken out from the neutralized mixture by solid-liquid separation means; and the base is dried Solving the polysaccharide solid to obtain a neutral alkali-soluble polysaccharide preparation; the purification method (iii) comprises the steps of: filtering the liquid portion of the step B) with a molecular sieve to obtain a molecular mass of more than 3,000 ear-swallowed portions; Neutralizing the portion of the molecule having a mass greater than 3000 amps by adding an acid; and drying the neutralized molecular mass greater than 3000 amp portions to obtain a neutral test soluble polysaccharide preparation; purification method (iv The method comprises the steps of: filtering the liquid portion of step B) with a molecular sieve to obtain a molecular mass of more than 10,000 ear-swallowed portions; _ neutralizing the portion of the molecular mass greater than 10,000 ear-sucking by adding an acid And drying the neutralized molecular mass of more than 10,000 ear-swallowed portions to obtain a neutral alkali-soluble polysaccharide preparation; the purification method (v) comprises the following steps: filtering the steps with a molecular sieve The liquid portion of step B) obtains a molecular mass greater than 3,000 ear-swallowed portions; the portion of the molecular mass greater than 3 amps is mixed with water; and the molecular mass is greater than 3000 by a molecular sieve. Department of Daodean 28 1308871·. ^ . „ (Amended in October 2008) = dimer to obtain the second part of the molecularly devastated portion; neutralize by adding an acid The molecular mass of the second pass is greater than 3,000 auricular portions; and the neutralized molecular mass is greater than 3 得 to obtain a neutral test soluble polysaccharide preparation; and 'purified and purified Method (vi) comprises the following steps: U - molecular step B) (iv) body (iv) greater than 10 _ ot of the ear; the obtained knives mass the mass of the 〇 道 道 耳 耳 再 道 - - - The molecular mass is greater than the mixture of water and the water to obtain the second filtered part of the ear swallowing part of the ear; the sub-mass is greater than 10000 and neutralizing the second 10000 ear swallow by adding an acid Part; and the mass of the knife is greater than the mass of the neutralization a neutral test-soluble polysaccharide preparation. Part of the ear-swallowed alcohol. 2. For the application of the full-time item, wherein the Cl.c3 alcohol is dry::: dry: the method of the first item, the drying For example, the method of claim 1, wherein the neutral alkali-soluble polysaccharide preparation has a molecular weight of 5,000 to 1,000,000. The method of clause 4, wherein the neutral test strip polysaccharide preparation has two major portions, the first portion having a molecular weight of about 1 Torr and the second portion having a molecular weight of about 900,000. 6. The method of claim 5, further comprising separating the first portion and the second portion of the neutral alkali-soluble polysaccharide preparation, wherein the first portion or the second portion is used as the middle portion Sexually soluble polysaccharide preparation. 7. The method of claim 5, wherein the aqueous test solution of step a) is 0.4_10 g/L Na〇I^ solution, and the extract of the root residue of the yam is at room temperature for 5-2 hours. A composition comprising any one of the first to seventh aspects of the invention, which is prepared by the method described above. A composition having an immunity-enhancing composition comprising a composition for increasing the amount of immunity as an active ingredient, such as a caricillin-soluble polysaccharide according to item 8 of the patent application.匕3 30 L 3 •1308871 ;'>.· (Amended in October 2008) Η Φ 鹅 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 1.E+09 1.E+08 1.E+07 l.E+06 u|H :1.E+05 2 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 N- 12 13 14 15 16 17 18 19 Stay time (minutes) 20 Figure 2a I ooooooooooo oooooooooo oooooooooo oooooooooo 0987654321 9876543210 oooooooooo .E+.E+.E+.E+.E+.E+.E+.E+.E+.E+ 1± 1X 11 11 1111 111A 20 9 11 8 11 7 6 11 5 11 4 3 11 2 11 Residence time (minutes) 2b set — ooooooooooo oooooooooo oooooooooo oooooooooo 0987654321 14 15 16 17 18 19 20 1.E+09 1.E+08 1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E +01 1.E+00 2 11 3 11 Residence time (minutes) Figure 2c