A neutral polysaccharide from American ginseng and its application in the preparation of uric acid-lowering drugs

Abstract

This invention provides a neutral polysaccharide from American ginseng and its application in the preparation of uric acid-lowering drugs, belonging to the field of polysaccharide preparation technology. The molecular weight of the American ginseng neutral polysaccharide PQ-7R is 54.5 kDa. This invention isolates and purifies a new neutral polysaccharide PQ-7R from American ginseng processed through a seven-steaming and seven-drying process. The molecular weight of the neutral polysaccharide is 54.5 kDa, and the linkage types mainly include →4)-Glcp-(1→, →6)-Galp-(1→, Glcp-(1→, →4,6)-Glcp-(1→). The American ginseng neutral polysaccharide PQ-7R provided by this invention has good uric acid-lowering activity and low toxicity, showing good development prospects.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation technology, and in particular to a neutral polysaccharide from American ginseng and its application in the preparation of uric acid-lowering drugs. Background Technology

[0002] American ginseng ( Panax quinquefolius Ginseng (L.) belongs to the Araliaceae family and is a perennial herb native to eastern Canada, the eastern and central United States. Also known as American ginseng, it is a globally recognized precious medicinal plant and tonic with a medicinal history dating back hundreds of years. It occupies an important position in both Eastern and Western medicine systems.

[0003] American ginseng contains a variety of chemical components, including ginsenosides, polysaccharides, amino acids, fatty acids, flavonoids, volatile oils, and trace elements. Among these, ginsenosides and polysaccharides are the key material basis for its pharmacological effects. However, due to its cold nature, long-term use by those with weak spleen and stomach may easily lead to adverse reactions such as abdominal pain and cold extremities.

[0004] American ginseng undergoes multiple steaming and drying processes, similar to the processing of fresh ginseng into black ginseng, hence the name "black American ginseng." The composition and content of its saponins and polysaccharides change accordingly, affecting its pharmacological activity and mitigating its cooling properties. To fully explore the medicinal effects of polysaccharides in steamed American ginseng, this invention studies the polysaccharide components of "seven-steamed and seven-dried" American ginseng and isolates a new neutral polysaccharide. This polysaccharide exhibits strong uric acid-lowering activity. Summary of the Invention

[0005] The purpose of this invention is to provide a neutral polysaccharide from American ginseng and its application in the preparation of uric acid-lowering drugs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a neutral polysaccharide PQ-7R from American ginseng, wherein the molecular weight of PQ-7R is 54.5 kDa and the structural formula is as follows: .

[0007] This invention provides a method for preparing the American ginseng neutral polysaccharide PQ-7R, comprising the following steps: (1) Steam fresh American ginseng at 105~115℃ for 1.5~2.5h and then dry it at 60~70℃ for 12~16h. Repeat the steaming and drying process for a total of seven times to obtain "seven-steamed and seven-dried" American ginseng. (2) After crushing the "seven-steamed and seven-dried" American ginseng obtained in step (1), it was extracted by heating water under reflux. The extraction temperature was 95~105℃, the mass-to-volume ratio of "seven-steamed and seven-dried" American ginseng to water was 1g:2~4mL, the extraction time was 1.5~2.5h, and it was extracted twice. The extract was collected and concentrated to obtain the "seven-steamed and seven-dried" American ginseng water extract. (3) Mix the "seven-steamed and seven-dried" American ginseng water extract obtained in step (2) with ethanol, adjust the ethanol content in the mixture to 60%, and collect the solid part after standing for 20-28 hours to obtain "seven-steamed and seven-dried" American ginseng crude polysaccharide. (4) Dissolve the crude polysaccharide of American ginseng obtained in step (3) in water, first extract it with petroleum ether 5 times, each time for 3-5 hours; then extract it with Sevage reagent 5 times, each time for 5-7 hours; concentrate the aqueous solution containing polysaccharide obtained after extraction to obtain polysaccharide extract after removing esters and proteins. (5) Dissolve the polysaccharide extract obtained in step (4) after removing esters and proteins in water, add it to the top of the DEAE-52 chromatographic column, elute with ultrapure water, collect the eluent, concentrate it and freeze dry to obtain the American ginseng neutral polysaccharide PQ-7R.

[0008] Preferably, the Sevage reagent in step (4) is composed of chloroform and n-butanol in a volume ratio of 4:1.

[0009] This invention provides the application of the American ginseng neutral polysaccharide PQ-7R or the American ginseng neutral polysaccharide PQ-7R obtained by the preparation method in the preparation of uric acid-lowering drugs.

[0010] This invention provides a uric acid-lowering drug, wherein the drug contains the American ginseng neutral polysaccharide PQ-7R or the American ginseng neutral polysaccharide PQ-7R obtained by the preparation method.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention isolates and purifies a novel neutral polysaccharide, PQ-7R, from American ginseng processed through a seven-steaming and seven-drying process. The molecular weight of this neutral polysaccharide is 54.5 kDa, and its linkage types primarily include →4)-Glcp-(1→, →6)-Galp-(1→, Glcp-(1→, →4,6)-Glcp-(1→). The American ginseng neutral polysaccharide PQ-7R provided by this invention exhibits good uric acid-lowering activity and low toxicity, showing promising development potential. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 American ginseng neutral polysaccharide PQ-7R 1 H NMR spectrum; Figure 2 American ginseng neutral polysaccharide PQ-7R 13 C NMR spectrum; Figure 3 American ginseng neutral polysaccharide PQ-7R 1 H- 1 H COSY spectrum; Figure 4 HMQC spectrum of PQ-7R, a neutral polysaccharide from American ginseng; Figure 5 HMBC spectrum of PQ-7R, a neutral polysaccharide from American ginseng; Figure 6 Infrared spectrum of PQ-7R, a neutral polysaccharide from American ginseng; Figure 7 The structure of the neutral polysaccharide PQ-7R from American ginseng. Detailed Implementation

[0014] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0015] Example 1

[0016] The preparation method of American ginseng neutral polysaccharide PQ-7R is as follows: (1) Fresh American ginseng (purchased from Wendeng Chuanfu Ginseng Industry Co., Ltd., Weihai City) was washed and steamed at 110℃ for 2 hours. After steaming, it was dried at 65℃ for 14 hours. The steaming and drying process was repeated for a total of seven times to obtain "seven-steamed and seven-dried" American ginseng.

[0017] (2) The obtained "seven-steamed and seven-dried" American ginseng was pulverized and passed through a 200-mesh sieve. The sieve residue was collected and subjected to water heating and reflux extraction. The extraction was carried out in a 100℃ water bath with a solid-liquid ratio of 1g:3mL for 2 hours. The extraction was repeated twice. The extracts were combined and filtered. The filtrate was concentrated to 1 / 6 of its original volume to obtain the "seven-steamed and seven-dried" American ginseng water extract.

[0018] (3) The obtained “seven-steamed and seven-dried” American ginseng water extract was mixed with an ethanol-water solution, the ethanol content was adjusted to 60%, and it was left for 24 hours to allow the polysaccharides in the water extract to precipitate completely. After filtration, the crude polysaccharide of “seven-steamed and seven-dried” American ginseng was obtained.

[0019] (4) 30g of the obtained "seven-steamed and seven-dried" American ginseng crude polysaccharide was dissolved in water at a mass-volume ratio of 1g:8mL and extracted five times with petroleum ether. Each extraction used 800mL of petroleum ether for 4 hours to remove fat-soluble components. The polysaccharide aqueous solution after deesterification was subjected to rotary evaporation under reduced pressure (pressure 200 mbar, temperature 50℃) to remove residual petroleum ether. Subsequently, the polysaccharide aqueous solution after deesterification was extracted five times with Sevage reagent (chloroform: n-butanol = 4:1, v / v). Each extraction used 800mL of Sevage reagent for 6 hours to remove proteins mixed in the polysaccharide. The polysaccharide aqueous solution after protein removal was concentrated under reduced pressure (pressure 100 mbar, temperature 50℃) to remove residual organic solvents, yielding a polysaccharide extract after deesterification and protein removal.

[0020] (5) Take 20g of the obtained polysaccharide extract after removing esters and proteins, and purify it by DEAE-52 ion exchange column chromatography. Dissolve the polysaccharide extract after removing esters and proteins in water, add it to the top of the DEAE-52 column, and elute with ultrapure water. Collect one tube for every 50mL, for a total of 40 tubes. Detect the polysaccharide content in each tube using the sulfuric acid-phenol method. Combine the eluents rich in polysaccharides and concentrate them under reduced pressure to 100mL. Freeze-dry the concentrated eluent (cold trap temperature -40℃, freeze-drying time 24h) to obtain 1.5g of "seven-steamed and seven-dried" neutral polysaccharide from American ginseng, named American ginseng neutral polysaccharide PQ-7R. The yield of American ginseng neutral polysaccharide PQ-7R (PQ-7R / total sugars loaded) was 7.5%.

[0021] Example 2: Structural Analysis of American Ginseng Neutral Polysaccharide PQ-7R

[0022] The neutral polysaccharide PQ-7R from American ginseng isolated in Example 1 was analyzed using Nicolet IS50, HPGPC high-performance liquid chromatography-gel chromatography, and Burker AVIII HD 600 MHz nuclear magnetic resonance spectrometer. Its infrared (IR), molecular weight, and nuclear magnetic (NMR) spectra were determined. The monosaccharide composition and linkage sites were also analyzed. The results are as follows: Figures 1-6 As shown in Table 1, a novel neutral polysaccharide structure was identified.

[0023] The molecular weight of the neutral polysaccharide PQ-7R from American ginseng was determined to be 54.5 kDa using multi-angle laser light scattering. The monosaccharide composition of PQ-7R, determined by acid hydrolysis, was Gal (15.0%), Glc (81.6%), and Ara (3.4%), including three types of monosaccharides (Ara content was low, and its signal was masked by other high-content sugars in NMR, therefore it was not detected in subsequent analysis). Methylation analysis showed that the main monosaccharide linkage types of PQ-7R were →4)-Glcp-(1→, →6)-Galp-(1→, Glcp-(1→, →4,6)-Glcp-(1→).

[0024] 3373 cm⁻¹ in the IR spectrum -1 and 2938 cm -1 The absorption peaks are due to the stretching vibrations of OH and CH. (PQ-7R) 1 The 1H NMR spectrum showed four hydrogen signal peaks at the glycosidic terminals, at δH 5.34, 5.28, 4.90, and 4.57, respectively. These hydrogen signals were determined by correlation analysis of the HSQC spectrum. 13 The relevant carbon signals in the C NMR spectrum are δ C Based on the hydrogen signals, the sugar residue fragments were numbered A, B, C, and D (Table 1) at concentrations of 99.63, 99.93, 98.59, and 104.50. Through... 1 H NMR, 13 C NMR, HSQC, HMBC, 1 H- 1 H COSY spectroscopy was used to determine the H and C signals of each sugar residue fragment in segments A, B, C, and D. Figures 1-5 ). 1 δ in H NMR spectrum H The signals of 4.20-3.30 ppm were attributed to the signals of each sugar residue fragment C-2~C-6. 1 The δH signal of 5.34 ppm in the 1H NMR spectrum is the telomere hydrogen signal of the sugar in fragment A, and the associated carbon signal is δH. C 99.63, the remaining signals pass through 1 H- 1 The H COSY, HSQC, and HMBC spectra were assigned. The signals in segment A were δ 71.55 (3.58), 73.27 (3.89), 76.76 (3.59), 71.02 (3.77), and 60.23 (3.76), respectively. Segment A was inferred to be →4)-α-D-Glcp-(1→;δ H The 5.28 ppm signal represents the hydrogen signal at the sugar terminus of fragment B, and the associated carbon signal is δ. C 99.93, passed 1 H- 1The H COSY, HSQC, and HMBC spectra were assigned. The signals in segment B are δ 71.72 (3.56), 72.92 (3.64), 77.12 (3.79), 71.55 (3.60), and 69.29 (3.36), respectively. Segment B is inferred to be →4,6)-α-D-Glcp-(1→;δ H The 4.90 ppm signal represents the hydrogen signal at the sugar terminus of fragment C, and the associated carbon signal is δ. C 98.59, the remaining signals pass through. 1 H- 1 The H COSY, HSQC, and HMBC spectra were assigned. The signals in segment C were δ 71.55 (3.54), 72.75 (3.63), 71.72 (3.37), 73.10 (3.89), and 60.58 (3.79), respectively. Therefore, segment C was inferred to be α-D-Glcp-(1→;δ H The 4.57 ppm signal represents the telomere hydrogen signal of the sugar in fragment D, and the associated carbon signal is δ. C 104.50, the remaining signals pass through 1 H- 1 The H COSY, HSQC, and HMBC spectra were assigned. The signals in segment D were δ 71.37 (3.61), 74.51 (3.78), 70.31 (3.91), 73.63 (3.59), and 70.31 (3.97), respectively. Segment D was inferred to be →6)-β-D-Galp-(1→).

[0025] The base linkage order of the structural fragments of PQ-7R was further determined by the correlation signals of the HMBC spectrum. In the HMBC spectrum, the correlation signals of H-A1 and C-A4 confirm the presence of the structure →4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→) in the PQ-7R structure; the correlation signals of H-B1 and C-A4 confirm the presence of the structure →4,6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→) in the PQ-7R structure; the correlation signals of H-C1 and C-D6 confirm the presence of the structure α-D-Glcp-(1→6)-β-D-Galp-(1→) in the PQ-7R structure; and the correlation signals of H-D1 and C-B4 confirm the presence of the structure →6)-β-D-Galp-(1→4)-α-D-Glcp-(1,6→) in the PQ-7R structure.

[0026] Based on the methylation determination results, NMR analysis results and PQ-7R, Figure 6Based on the infrared spectrum results shown, the structure of PQ-7R is inferred. Based on the monosaccharide composition and methylation data, it is speculated that PQ-7R has a main sugar chain structure of (→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→). Figure 7 ).

[0027] Table 1 PQ-7R 1 H NMR (600 MHz, D2O) and 13 C10 NMR data (150 MHz, D2O)

[0028] Example 3: Study on the uric acid-lowering activity of American ginseng neutral polysaccharide PQ-7R

[0029] 1. Construction and administration of a mouse model of hyperuricemia

[0030] SPF-grade male Kunming mice (purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd.) were selected and, after 7 days of acclimatization, were randomly divided into 6 groups of 6 mice each according to their body weight. The specific groups were: normal control group (CON), model group (MOD), positive control group (allopurinol), low-dose PQ-7R group (PQ-7R-L), medium-dose PQ-7R group (PQ-7R-M), and high-dose PQ-7R group (PQ-7R-H).

[0031] Except for the normal control group, all other groups were equipped with a hyperuricemia (HUA) mouse model by intraperitoneal injection of potassium oxonate (300 mg / kg), gavage administration of hypoxanthine (300 mg / kg), and feeding with a 20% high-yeast diet (purchased from Jinan Haicheng Biotechnology Co., Ltd.). Modeling was initiated daily at 8:00 AM for 21 consecutive days. One hour after the end of each day's modeling, the corresponding drugs were administered by gavage according to the experimental groups. PQ-7R-L, PQ-7R-M, and PQ-7R-H were administered 50 mg / kg, 100 mg / kg, and 200 mg / kg of American ginseng neutral polysaccharide PQ-7R, respectively, by gavage. The positive control group was administered 5 mg / kg of allopurinol by gavage, while the normal control group and the model group were administered an equal volume of physiological saline by gavage.

[0032] 2. Determination of serum uric acid levels

[0033] After the last administration, all mice were fasted for 12 hours but allowed free access to water. The final fasting weight of the mice was accurately weighed and recorded. Blood was collected by enucleation. Whole blood was collected in sterile centrifuge tubes and allowed to clot naturally at room temperature for 2 hours. The tubes were then centrifuged at 3000 r / min for 30 min at 4°C. The supernatant pale yellow serum was carefully aspirated, aliquoted, and immediately frozen at -80°C. Uric acid (UA) levels were measured using a uric acid assay kit and an ELISA reader. The results are shown in Table 2.

[0034] Table 2. Uric acid-lowering effect of American ginseng neutral polysaccharide PQ-7R

[0035] Serum uric acid (UA) level is the most crucial and intuitive indicator for evaluating the successful establishment of a hyperuricemia model and the efficacy of uric acid-lowering drugs. Compared with the normal control group, the serum UA level of mice in the model group was significantly increased, indicating the successful establishment of the HUA mouse model. After intervention with the positive control drug, UA levels were significantly suppressed. In the PQ-7R administration group, PQ-7R at all doses significantly reduced the abnormally elevated serum uric acid levels in HUA mice. Among them, PQ-7R showed a highly significant uric acid-lowering effect at the high dose (200 mg / kg), and the UA reduction at the high dose exceeded that of the positive control group.

[0036] 3. Determination of xanthine oxidase (XOD) activity in the liver

[0037] To elucidate the intervention mechanism of ginseng neutral polysaccharide PQ-7R at the source of uric acid production, the activity of xanthine oxidase (XOD) in liver tissue was detected. After blood collection, mice were immediately euthanized by cervical dislocation, and the abdominal cavity was quickly opened and dissected on ice. The liver was completely dissected. Approximately 50 mg of liver tissue was added to a sterile centrifuge tube and quickly washed twice with pre-cooled PBS to remove residual blood. Pre-cooled PBS was added at a weight (g):volume (mL) ratio of 1:9, with two grinding beads added to each tube. The mixture was homogenized at 50 Hz for 3 min, followed by centrifugation at 4000 rpm for 15 min. 200 μL of the supernatant was carefully transferred to a new tube using a pipette tip. XOD enzyme activity was measured using an XOD assay kit. The results are shown in Table 3.

[0038] Table 3. Effects of American ginseng neutral polysaccharide PQ-7R on XOD activity in the liver.

[0039] Table 3 shows that the liver XOD activity in the model group was significantly higher than that in the normal control group, indicating that uric acid synthesis and metabolism in the liver are abnormally hyperactive under HUA conditions. In the treatment groups, PQ-7R significantly reduced XOD levels in HUA mice at all doses. Among them, the PQ-7R group showed extremely significant XOD inhibitory activity at high dose (200 mg / kg), and the inhibitory activity exceeded that of the positive control group, indicating that the neutral polysaccharide PQ-7R of American ginseng exerts its uric acid-lowering effect by inhibiting XOD activity.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A neutral polysaccharide PQ-7R from American ginseng, characterized in that, The molecular weight of the American ginseng neutral polysaccharide PQ-7R is 54.5 kDa, and its structural formula is as follows: 。 2. A method for preparing the American ginseng neutral polysaccharide PQ-7R according to claim 1, characterized in that, Includes the following steps: (1) Steam fresh American ginseng at 105~115℃ for 1.5~2.5h and then dry it at 60~70℃ for 12~16h. Repeat the steaming and drying process for a total of seven times to obtain "seven-steamed and seven-dried" American ginseng. (2) After pulverizing the "seven-steamed and seven-dried" American ginseng obtained in step (1), water was heated and refluxed for extraction. The extraction temperature was 95~105℃, the mass-to-volume ratio of "seven-steamed and seven-dried" American ginseng to water was 1g:2~4mL, the extraction time was 1.5~2.5h, and the extraction was performed twice. The extract was collected and concentrated to obtain the "seven-steamed and seven-dried" American ginseng water extract. (3) Mix the "seven-steamed and seven-dried" American ginseng water extract obtained in step (2) with ethanol, adjust the ethanol content in the mixture to 60%, and collect the solid part after standing for 20-28 hours to obtain "seven-steamed and seven-dried" American ginseng crude polysaccharide. (4) Dissolve the crude polysaccharide of American ginseng obtained in step (3) in water, first extract it with petroleum ether 5 times, each time for 3-5 hours; then extract it with Sevage reagent 5 times, each time for 5-7 hours; concentrate the aqueous solution containing polysaccharide obtained after extraction to obtain polysaccharide extract after removing esters and proteins. (5) Dissolve the polysaccharide extract obtained in step (4) after removing esters and proteins in water, add it to the top of the DEAE-52 chromatographic column, elute with ultrapure water, collect the eluent, concentrate it and freeze dry to obtain the American ginseng neutral polysaccharide PQ-7R.

3. The preparation method according to claim 2, characterized in that, The Sevage reagent mentioned in step (4) is composed of chloroform and n-butanol in a volume ratio of 4:

1.

4. The application of the American ginseng neutral polysaccharide PQ-7R according to claim 1 or the American ginseng neutral polysaccharide PQ-7R obtained by the preparation method according to claim 2 or 3 in the preparation of uric acid-lowering drugs.

5. A uric acid-lowering drug, characterized in that, The drug contains the American ginseng neutral polysaccharide PQ-7R as described in claim 1 or the American ginseng neutral polysaccharide PQ-7R obtained by the preparation method described in claim 2 or 3.

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