Pellionia latifolia extract, preparation method thereof, related product and application of Pellionia latifolia extract

Abstract

The invention provides a Chinese ladiestresses root extract as well as a preparation method, a related product and application thereof, and belongs to the technical field of traditional Chinese medicine extraction. The preparation method comprises the following steps: mixing pedicularis chinensis and ketone, and performing reflux leaching and separation to obtain a leaching solution and medicine residues; recovering the solvent from the leaching solution to obtain a Chinese ladiestresses root extract; mixing the medicine residue with water, decocting, centrifuging the decoction, reserving the supernatant, and carrying out alcohol precipitation to obtain crude polysaccharide; reacting the crude polysaccharide with sodium periodate to obtain oxidized paniculate helminthostachys rhizome polysaccharide; and mixing 5-15 parts by weight of the paniculate helichrysum herb extract, 10-30 parts by weight of the oxidized paniculate helichrysum herb polysaccharide and 100 parts by weight of water, and carrying out ultrasonic dispersion and drying to obtain the paniculate helichrysum herb extract. The spiranthes sinensis extract obtained by the method can form a submicron-level particle structure, and can better reduce the foot swelling score of rheumatoid arthritis mice, improve the blood marker index of model mice and reduce the occurrence of hemolysis side effects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, and relates to a Panlongqi extract, its preparation method, related products and applications. Background Technology

[0002] Panlongqi is one of the seven medicinal herbs produced in Shangluo. According to existing technology CN101085093A, Panlongqi is the dried rhizome of Polygonum hydropiper, also known as Dahongfen. It is harvested in autumn, the above-ground parts, soil, and fibrous roots are removed, and it is sun-dried. It has an astringent and slightly bitter taste, and is neutral in nature. Functions and indications: It disperses blood stasis, relieves pain, stops bleeding, and reduces swelling. It is used for traumatic injuries and external bleeding (Compilation of Traditional Chinese Medicine Materials for Wartime Preparedness in Shangluo Area, page 227).

[0003] The article "Preliminary Chemical Constituent Analysis and GC-MS Analysis of Petroleum Ether Extract of Polygonum hydropiper" (Zhang Weimin et al., Progress in Animal Medicine, 2007, 28(12): 39-42) points out that Polygonum hydropiper contains sugars, polysaccharides and their glycosides (saponins, cardiac glycosides), phenols and tannins, flavonoids, coumarins and terpene lactones and anthraquinones.

[0004] Chinese invention patent CN118557665A discloses a production process for Panlong Qipian (a traditional Chinese medicine formula). The production process includes the following steps: taking Aconitum carmichaelii (Sichuan aconite), Aconitum kusnezoffii (Glycine max), and Aconitum carmichaelii (Iron Hammer), crushing and sieving them, then adding 12% water. 14 times, soak for 25 minutes 35 minutes, simmer for 2 minutes 3 hours, add water twice 8 10 times, boiled for 1 2 hours, add water three times 4 5 times, boiled for 1 After 2 hours, the extract was filtered and concentrated to obtain an extract. The remaining medicinal materials were pulverized and sieved to obtain a coarse material. The coarse material was activated and dried. A compound enzyme was added, the pH was adjusted, and the extract was extracted by ultrasound and concentrated under vacuum to obtain an extract. The extract was purified by macroporous adsorption resin column chromatography. Mannitol, isomaltitol, tert-butylhydroquinone, and magnesium stearate were added and mixed, spray-dried, compressed into tablets, and sugar-coated to obtain Panlongqi tablets. The Panlongqi tablets obtained by this method have good effects of promoting blood circulation, removing blood stasis, dispelling wind and dampness, reducing swelling and relieving pain, and have high stability, making them suitable for industrial promotion. However, the Panlongqi tablets prepared by this method involve 29 kinds of Chinese medicinal materials, resulting in a relatively large formula and more complex components. When side effects occur, it is often difficult to determine which component caused the side effects, hindering its application.

[0005] Currently, there is almost no research on the role of extracts of Panlongqi (Taibai Liao) as a single Chinese herbal medicine in autoimmune diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Panlongqi extract and related technologies to solve the technical problems such as the unclear efficacy and preparation method of Panlongqi (Taibailiao) single herb extract in the treatment of autoimmune diseases (such as rheumatoid arthritis), or a combination thereof.

[0007] To achieve the above-mentioned objectives, in a first aspect, the present invention provides a method for preparing Panlongqi extract, comprising the following steps: S1. Mix Panlongqi with ketone, reflux and extract, separate to obtain extract and residue; S2. Recover the solvent from the extract obtained in step S1 to obtain Panlongqi extract; S3. Mix the dregs obtained in step S1 with water, decoct, centrifuge the decoction and retain the supernatant, precipitate with alcohol to obtain crude polysaccharide. S4. React the crude polysaccharide obtained in step S3 with sodium periodate to obtain oxidized Panlongqi polysaccharide. S5. Take 5-15 parts by weight of the Panlongqi extract obtained in step S2, 10-30 parts by weight of the oxidized Panlongqi polysaccharide obtained in step S4, and 100 parts by weight of water, mix them, disperse them by ultrasonication, and dry them to obtain Panlongqi extract. In step S1, the ketone is acetone or 2-butanone.

[0008] In one specific embodiment of the present invention, the ketone is acetone.

[0009] In one specific embodiment of the present invention, the ketone is 2-butanone.

[0010] In step S5, the weight of the Panlongqi extract is any value in the range of 5-15 parts, specifically selected from the following values ​​or any range between two of them: 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, and 15 parts.

[0011] In step S5, the weight of the oxidized Panlongqi polysaccharide is any value in the range of 10-30 parts, specifically selected from the following values ​​or any range between two: 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, and 30 parts.

[0012] Specifically, in step S1, the Panlongqi is the pre-processed Panlongqi.

[0013] Specifically, in step S1, the weight ratio of Panlongqi to the volume ratio of the ketone is 1:4-6, in g:mL; the reflux leaching temperature is 70-75℃, the reflux leaching time is 1-3h, the reflux leaching is performed 2-4 times, and the separation is achieved by filtration.

[0014] The ratio of the weight of Panlongqi to the volume of the ketone is selected from any ratio within the range of 1:4-6, specifically selected from the following ratios or any range between the two (unit: g:mL): 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.

[0015] The reflux leaching temperature is any value within the range of 70-75℃, specifically selected from the following values ​​or any range between two of them: 70℃, 71℃, 72℃, 73℃, 74℃, and 75℃.

[0016] The reflux leaching time is any value within the range of 1-3 hours, specifically selected from the following values ​​or any range between two of them: 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h.

[0017] The number of reflux immersions is any value within the range of 2 to 4 times, specifically selected from the following values ​​or any range between two: 2 times, 3 times, and 4 times.

[0018] Specifically, in step S2, the recovered solvent is a solvent recovered under reduced pressure.

[0019] Specifically, in step S3, the weight ratio of the dregs to the volume of the water is 1:8-10, in g:mL; the decoction time is 1-3 hours, the number of decoctions is 2-4 times, the centrifugation speed is 4000-5500 rpm, and the centrifugation time is 5-20 minutes; the supernatant is the supernatant after vacuum concentration.

[0020] The ratio of the weight of the medicinal residue to the volume of the water is any ratio within the range of 1:8-10, specifically selected from the following ratios or any range between the two (unit: g:mL): 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10.

[0021] The simmering time is any value within the range of 1-3 hours, specifically selected from the following values ​​or any range between two of them: 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h.

[0022] The centrifugation speed is any value within the range of 4000-5500 rpm, specifically selected from the following values ​​or any range between two of them: 4000 rpm, 4100 rpm, 4200 rpm, 4300 rpm, 4400 rpm, 4500 rpm, 4600 rpm, 4700 rpm, 4800 rpm, 4900 rpm, 5000 rpm, 5100 rpm, 5200 rpm, 5300 rpm, 5400 rpm, and 5500 rpm.

[0023] The centrifugation time is any value within the range of 5-20 min, specifically selected from the following values ​​or any range between two of them: 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min.

[0024] More specifically, in step S3, the alcohol precipitation includes the following steps: adding ethanol to the supernatant to make the ethanol concentration in the mixture 75%vol-85%vol, letting it stand, and filtering.

[0025] The ethanol concentration in the mixture is any value within the range of 75%vol-85%vol, specifically selected from the following values ​​or any range between two of them: 75%vol, 76%vol, 77%vol, 78%vol, 79%vol, 80%vol, 81%vol, 82%vol, 83%vol, 84%vol, 85%vol.

[0026] Specifically, in step S4, the weight ratio of the crude polysaccharide to the amount of sodium periodate is 2-3:1, in g:mmol.

[0027] The ratio of the weight of the crude polysaccharide to the amount of sodium periodate is any ratio within the range of 2-3:1, specifically selected from the following ratios or any range between the two (unit: g: mmol): 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1.

[0028] Specifically, in step S4, the reaction includes the following steps: The crude polysaccharide was mixed with water to form a crude polysaccharide aqueous solution of 0.01-1 g / mL. A sodium periodate aqueous solution of 0.01-1 mol / L was added dropwise while stirring. The mixture was reacted at 10-40℃ for 10-60 min and then freeze-dried to obtain oxidized Panlongqi polysaccharide.

[0029] The concentration of the crude polysaccharide aqueous solution is any value in the range of 0.01-1 g / mL, specifically selected from the following values ​​or any range between two of them: 0.01 g / mL, 0.05 g / mL, 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 0.6 g / mL, 0.7 g / mL, 0.8 g / mL, 0.9 g / mL, and 1 g / mL.

[0030] The concentration of the sodium periodate aqueous solution is any value in the range of 0.01-1 mol / L, specifically selected from the following values ​​or any range between two of them: 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, and 1 mol / L.

[0031] The temperature of the reaction is any value in the range of 10-40℃, specifically selected from the following values ​​or any range between two of them: 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃.

[0032] The reaction time is any value within the range of 10-60 min, specifically selected from the following values ​​or any range between two of them: 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min.

[0033] Specifically, in step S5, the weight parts of the Panlongqi extract are 10 parts, and the weight parts of the oxidized Panlongqi polysaccharide are 20 parts; the ultrasonic dispersion time is 10-120 min, and the ultrasonic dispersion frequency is 20-60 kHz; the drying is freeze drying.

[0034] The ultrasonic dispersion time is any value within the range of 10-120 min, specifically selected from the following values ​​or any range between two of them: 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min.

[0035] The frequency of the ultrasonic dispersion is any value within the range of 20-60kHz, specifically selected from the following values ​​or any range between two of them: 20kHz, 21kHz, 22kHz, 23kHz, 24kHz, 25kHz, 26kHz, 27kHz, 28kHz, 29kHz, 30kHz, 31kHz, 32kHz, 33kHz, 34kHz, 35kHz, 36kHz, 37kHz, 38kHz, 39kHz, 41kHz, 42kHz, 43kHz, 44kHz, 45kHz, 46kHz, 47kHz, 48kHz, 49kHz, 51kHz, 52kHz, 53kHz, 54kHz, 55kHz, 56kHz, 57kHz, 58kHz, 59kHz, and 60kHz.

[0036] In this invention, the plant source of Panlongqi is... Polygonum taipaishanense .

[0037] As an alternative, in this invention, sodium periodate in step S4 is replaced or partially replaced with one or more of potassium periodate, metaperiodic acid, ammonium periodate, and ammonium periodate. Typical ammonium periodate includes tetramethylperiodamine (CH3)4NIO4, etc. Potassium periodate, metaperiodic acid, ammonium periodate, and ammonium periodate have very similar chemical properties to sodium periodate and can also be used to prepare *Panax notoginseng* polysaccharide and obtain the corresponding target product, *Panax notoginseng* polysaccharide.

[0038] Secondly, the present invention provides a Panlongqi extract prepared by the above-described preparation method.

[0039] Thirdly, the present invention provides a drug, the effective component of which includes the Panlongqi extract prepared by the above-described preparation method.

[0040] Specifically, the dosage form of the drug is selected from: injection, tablet, pill, powder, granule, capsule, solution, lozenge or suppository.

[0041] Specifically, this also includes pharmaceutically acceptable excipients.

[0042] Specifically, the drug is a rheumatoid arthritis drug.

[0043] Fourthly, the present invention provides the use of the Panlongqi extract or the above-mentioned drug prepared by the above-described preparation method in the preparation of a drug for treating autoimmune diseases.

[0044] Specifically, the autoimmune disease is rheumatoid arthritis. Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention provides a novel method for preparing Panlongqi extract. By coating the organic solvent extract of Panlongqi with oxidized Panlongqi polysaccharide, nano-sized Panlongqi extract particles are formed, utilizing different parts of the traditional Chinese medicine Panlongqi. Hemolysis experiments show that the nano-sized Panlongqi extract particles provided by this invention can improve the blood compatibility of Panlongqi extract.

[0045] (2) Animal experiments show that the Panlongqi extract provided by the present invention can effectively reduce the level of type II collagen antibody in the serum of mice with rheumatoid arthritis, and effectively reduce the levels of IL-6, IL-17A and TNF in the serum of mice.

[0046] (3) Animal experiments show that the Panlongqi extract provided by the present invention can effectively alleviate the symptoms of rheumatoid arthritis in mice with rheumatoid arthritis model, including symptoms such as joint redness and swelling. Detailed Implementation

[0047] Terminology and Declarations of this Invention: 1. Articles “a,” “a kind,” and “the”: These include plural objects unless otherwise explicitly specified as a single (kind) object.

[0048] 2. Numerical Range: Unless otherwise expressly stated, all ranges or ratios disclosed herein shall be construed as including any and all subranges or subratios contained herein. For example, a stated range or ratio of 1 to 30 shall be considered to be included between the minimum value of 1 and the maximum value of 30, and includes any subranges or subratios, integers, decimals, or subranges or subratios consisting of integers or decimals, including endpoints.

[0049] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0050] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0051] In the following specific embodiments, the Chinese medicinal material Panlongqi, after identification by Chinese medicinal material experts, is Polygonum hydropiper (Polygonum tinctorium). Polygonum taipaishanense The dried rhizome of ).

[0052] Example 1 A method for preparing Panlongqi extract, the steps of which are as follows.

[0053] S1. Pre-treat Panlongqi (wash to remove dirt, dry, pulverize, and pass through a 10-mesh sieve). Mix the pre-treated Panlongqi with 5 mL of 2-butanone at a ratio of 1 g Panlongqi: 5 mL 2-butanone, heat to 75°C, stir and reflux for 2 hours, and filter to separate. Repeat the above reflux process 3 times. Combine the filtrates and record them as extract, and the filter residue as medicinal residue.

[0054] S2. The solvent 2-butanone is recovered from the extract obtained in step S1 under reduced pressure to obtain Panlongqi extract.

[0055] S3. Mix the dregs obtained in step S1 with water at a weight-to-volume ratio of 1:9 g / mL, decoct for 2 hours, and repeat the decoction 3 times. Combine the decoctions, centrifuge at 4800 rpm for 13 minutes, and obtain the supernatant. Alcohol precipitation: Concentrate the supernatant under reduced pressure to 10% of its original volume, add anhydrous ethanol to make the ethanol concentration in the mixture 85% vol, let stand for 12 hours, filter to collect the precipitate, freeze-dry, and obtain crude polysaccharide.

[0056] S4. Mix the crude polysaccharide obtained in step S3 with water to obtain a 0.1 g / mL crude polysaccharide aqueous solution. Dissolve sodium periodate in water to obtain a 0.1 mol / L sodium periodate aqueous solution. Add the sodium periodate aqueous solution dropwise to the crude polysaccharide aqueous solution while stirring. The volume ratio of the crude polysaccharide aqueous solution to the sodium periodate aqueous solution is 25:10. The actual ratio of crude polysaccharide to sodium periodate added is 2.5:1 (g / mmol). React at room temperature (25℃) for 30 min. Freeze-dry to obtain oxidized Panlongqi polysaccharide.

[0057] Panlongqi polysaccharide oxidized with sodium periodate has two main effects. First, some hydroxyl functional groups are oxidized to aldehyde groups, making it possible for compounds containing functional groups such as amino, thiol, and double bonds to react. Second, some oxidation-intolerant chemical bonds in Panlongqi polysaccharide can be cleaved, improving the processability of the molecule.

[0058] S5. Take 10 parts by weight of the Panlongqi extract obtained in step S2, 20 parts by weight of the oxidized Panlongqi polysaccharide obtained in step S4, and mix with 100 parts by weight of water. Disperse by ultrasonication for 60 min at 40 kHz. Freeze-dry to obtain powdered Panlongqi extract.

[0059] Example 2 Compared with Example 1, the only difference is that 2-butanone is replaced with acetone, and everything else is the same.

[0060] Example 3 Compared with Example 1, the only difference is that in step S5, the weight of Panlongqi extract is changed to 5 parts and the weight of oxidized Panlongqi polysaccharide is changed to 30 parts, while the rest are the same.

[0061] Example 4 Compared with Example 1, the only difference is that in step S5, the weight of Panlongqi extract is changed to 15 parts and the weight of oxidized Panlongqi polysaccharide is changed to 10 parts, while the rest are the same.

[0062] Example 5 Compared with Example 1, the only difference is that in step S4, the actual ratio of crude polysaccharide to sodium periodate is 3:1 (g / mmol).

[0063] Example 6 Compared with Example 1, the only difference is that in step S4, the actual ratio of crude polysaccharide to sodium periodate is 2:1 (g / mmol).

[0064] Comparative Example 1 Compared with Example 1, the only difference is that 2-butanone is replaced with cyclohexanone, and everything else is the same.

[0065] Comparative Example 2 Compared with Example 1, the only difference is that in step S5, the weight of Panlongqi extract is changed to 20 parts and the weight of oxidized Panlongqi polysaccharide is changed to 5 parts, while the rest are the same.

[0066] Comparative Example 3 Compared with Example 1, the only difference is that step S4, which involves the oxidation of the crude polysaccharide, is omitted.

[0067] The specific preparation steps are as follows: S1. Pre-treat Panlongqi (wash to remove dirt, dry, pulverize, and pass through a 10-mesh sieve). Mix the pre-treated Panlongqi with 5 mL of 2-butanone at a ratio of 1 g Panlongqi: 5 mL 2-butanone, heat to 75°C, stir and reflux for 2 hours, and filter to separate. Repeat the above reflux process 3 times. Combine the filtrates and record them as extract, and the filter residue as medicinal residue.

[0068] S2. The solvent 2-butanone is recovered from the extract obtained in step S1 under reduced pressure to obtain Panlongqi extract.

[0069] S3. Mix the dregs obtained in step S1 with water at a weight-to-volume ratio of 1:9 g / mL, decoct for 2 hours, and repeat the decoction 3 times. Combine the decoctions, centrifuge at 4800 rpm for 13 minutes, and obtain the supernatant. Alcohol precipitation: Concentrate the supernatant under reduced pressure to 10% of its original volume, add anhydrous ethanol to make the ethanol concentration in the mixture 85% vol, let stand for 12 hours, filter to collect the precipitate, freeze-dry, and obtain crude polysaccharide.

[0070] S4. Take 10 parts by weight of the Panlongqi extract obtained in step S2, 20 parts by weight of the crude polysaccharide obtained in step S2, and 100 parts by weight of water, mix them, and sonicate for 60 min at 40 kHz. Freeze-dry to obtain Panlongqi powder extract.

[0071] Comparative Example 4 Compared with Example 1, the only difference is that the ultrasonic dispersion step in step S5 is omitted, and only stirring is performed.

[0072] The specific preparation steps are as follows: S1. Pre-treat Panlongqi (wash to remove dirt, dry, pulverize, and pass through a 10-mesh sieve). Mix the pre-treated Panlongqi with 5 mL of 2-butanone at a ratio of 1 g Panlongqi: 5 mL 2-butanone, heat to 75°C, stir and reflux for 2 hours, and filter to separate. Repeat the above reflux process 3 times. Combine the filtrates and record them as extract, and the filter residue as medicinal residue.

[0073] S2. The solvent 2-butanone is recovered from the extract obtained in step S1 under reduced pressure to obtain Panlongqi extract.

[0074] S3. Mix the dregs obtained in step S1 with water at a weight-to-volume ratio of 1:9 g / mL, decoct for 2 hours, and repeat the decoction 3 times. Combine the decoctions, centrifuge at 4800 rpm for 13 minutes, and obtain the supernatant. Alcohol precipitation: Concentrate the supernatant under reduced pressure to 10% of its original volume, add anhydrous ethanol to make the ethanol concentration in the mixture 85% vol, let stand for 12 hours, filter to collect the precipitate, freeze-dry, and obtain crude polysaccharide.

[0075] S4. Mix the crude polysaccharide obtained in step S3 with water to obtain a 0.1 g / mL crude polysaccharide aqueous solution. Dissolve sodium periodate in water to obtain a 0.1 mol / L sodium periodate aqueous solution. Add the sodium periodate aqueous solution dropwise to the crude polysaccharide aqueous solution while stirring. The volume ratio of the crude polysaccharide aqueous solution to the sodium periodate aqueous solution is 25:10. The actual ratio of crude polysaccharide to sodium periodate added is 2.5:1 (g / mmol). React at room temperature (25℃) for 30 min. Freeze-dry to obtain oxidized Panlongqi polysaccharide.

[0076] Panlongqi polysaccharide oxidized with sodium periodate has two main effects. First, some hydroxyl functional groups are oxidized to aldehyde groups, making it possible for compounds containing functional groups such as amino, thiol, and double bonds to react. Second, some oxidation-intolerant chemical bonds in Panlongqi polysaccharide can be cleaved, improving the processability of the molecule.

[0077] S5. Take 10 parts by weight of the Panlongqi extract obtained in step S2, 20 parts by weight of the oxidized Panlongqi polysaccharide obtained in step S4, and mix with 100 parts by weight of water. Stir at 4000 rpm for 60 min. Freeze-dry to obtain Panlongqi powder extract.

[0078] Comparative Example 5 The specific steps for preparing the aqueous extract of Panlongqi are as follows: Mix Panlongqi with 10 times its weight of water, decoct for 2 hours, and repeat 3 times. Combine the filtrates, concentrate under reduced pressure, and dry under vacuum to obtain Panlongqi water extract (Panlongqi extract).

[0079] Effect evaluation 1. Particle size detection of Panlongqi extract.

[0080] The Panlongqi extract powders prepared in each example and comparative example were mixed with PBS aqueous solution at pH 7.4 at a ratio of 20 mg / mL and carefully dispersed evenly by pipetting. The particle size distribution of the Panlongqi extract in the PBS aqueous solution was detected by dynamic light scattering (DLS). The detection results are shown in Table 1 below (signal intensity average particle size Z-average): Table 1

[0081] It is noteworthy that no signal peaks were detected after the water extract obtained in Comparative Example 5 was mixed with water. This indicates that the Panlongqi extract obtained in Comparative Example 5 did not form a nanostructure.

[0082] 2. Blood compatibility test of Panlongqi extract Fresh rat red blood cells were obtained from 8-week-old SD rats. Blood was collected from the orbital vein using anticoagulated blood collection tubes, and the rats were euthanized. The blood collection tubes were centrifuged at 3500 rpm for 30 min in a low-temperature centrifuge, and the pellet was collected. The red blood cells were carefully washed three times with chilled PBS, centrifuged under the same conditions each time, to obtain fresh red blood cells.

[0083] Red blood cells were diluted with PBS at a volume ratio of 1:10. 200 μL of the diluted blood was then added sequentially to 1 mL of purified water (positive control group), 1 mL of PBS (negative control group), and 1 mL of a gradient concentration of Panlongqi extract powder in PBS (experimental group). (The concentration gradient was set as follows: 1 ppm, 2 ppm, 4 ppm, 8 ppm, 16 ppm, 32 ppm, 64 ppm, 128 ppm, 250 ppm, 500 ppm, 750 ppm, 1000 ppm, 1300 ppm, 1600 ppm, 2000 ppm, 3000 ppm, 4000 ppm). The mixture was incubated at 37°C for 5 h and centrifuged at 3000 rpm. 100 μL of the supernatant from each group was added to four wells in a 96-well plate. The absorbance (OD value) was measured at 540 nm using a microplate reader. The hemolysis rate was calculated using the following formula: Hemolysis rate = (OD of experimental group - OD of negative control group) ÷ (OD of positive control group - OD of negative control group) × 100%.

[0084] The highest concentration (ppm) of Panlongqi extract obtained in each embodiment and comparative example that resulted in a hemolysis rate of no more than 2%, and the test results at that concentration are shown in Table 2 below.

[0085] Table 2

[0086] Experimental results showed that the hemolysis rates of all example groups and Comparative Example 1 were less than 2% when the material concentration was ≤1600ppm, indicating good blood compatibility. In Comparative Example 2, the amount of oxidized Panlongqi polysaccharide added was small, resulting in poor encapsulation; the hemolysis rate was >2% at a concentration of 1600ppm. In Comparative Example 3, no oxidation of Panlongqi polysaccharide was performed, resulting in poor encapsulation; the hemolysis rate was >2% at a concentration of 1300ppm. In Comparative Example 4, stirring was performed without ultrasonic dispersion, resulting in poor encapsulation of the Panlongqi extract; the hemolysis rate was >2% at a concentration of 1600ppm. Comparative Example 5 was an aqueous extract of Panlongqi, with a hemolysis rate >2% at a concentration of 1300ppm.

[0087] 3. Evaluation of the therapeutic effect of Panlongqi extract on mice with rheumatoid arthritis The experimental animal model was modeled with reference to the article "Efficacy and Preliminary Mechanism Study of In Vitro Photochemotherapy on Rheumatoid Arthritis Model Mice" (Master's Thesis, Anhui Medical University, Lin Yuwei, 2024.5.1), and the therapeutic effect of Panlongqi extract on rheumatoid arthritis (CIA) mice was evaluated.

[0088] The following is information regarding the medication used: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Beyotime (50 mL). Bovine type II collagen solution was purchased from AmyJet Scientific (Bovine Type II Collagen-Solution, catalog number: 1220-02S, protein content: 0.5 mg). Glacial acetic acid was from Shanghai Testing, GR, ≥99.8%.

[0089] Bovine type II collagen emulsion is prepared from bovine type II collagen solution, glacial acetic acid, and Freund's adjuvant. The preparation method is as follows: glacial acetic acid is mixed with deionized water to obtain a 0.05M acetic acid solution. The bovine type II collagen solution is then mixed with the 0.05M acetic acid solution to prepare a 2 mg / mL bovine type II collagen solution. Under ice bath conditions, Freund's complete adjuvant or Freund's incomplete adjuvant is repeatedly mixed with the 2 mg / mL bovine type II collagen solution at a 1:1 volume ratio until homogeneous, removing air bubbles to obtain the bovine type II collagen emulsion. This emulsion should be injected into mice within 1 hour of preparation. The emulsion obtained using Freund's complete adjuvant is designated as Emulsion 1; the emulsion obtained using Freund's incomplete adjuvant is designated as Emulsion 2.

[0090] 3.1 Animal modeling and drug administration Experimental animals: 7-8 week old male DBA / 1J mice, acclimatized for one week.

[0091] Animal modeling methods: (1) First injection on day 0: Emulsion 1 (100 μg of bovine type II collagen per mouse) was injected subcutaneously into the tail of the mouse.

[0092] (2) Enhanced injection on day 21: Emulsion 2 was injected subcutaneously into the tail of the mice (the dosage of bovine type II collagen per mouse was 100 μg, and the injection site was different from the first injection).

[0093] (3) From day 29 to 37, the mice showed symptoms of redness and swelling of the skin of the paw joints and swelling of the toes, indicating that the mouse CIA model was successfully established. Due to the reduced activity of the mice, sterile water and food were placed in the cage for easy feeding.

[0094] On day 38, animals were grouped as follows: Mice that successfully modeled the virus were randomly divided into groups of 7. Seven mice from the same batch that did not model the virus were also grouped separately. The specific groupings are as follows: Blank control group: DBA / 1J mice that have not been modeled by CIA and have normal diet and water intake.

[0095] Model control group: DBA / 1J mice modeled by CIA were administered 100 μL of sterile saline solution by gavage daily.

[0096] Positive control group: DBA / 1J mice modeled by CIA were injected intraperitoneally every 2 days with 0.5 mg / L methotrexate saline solution (containing as little as possible of the solubilizer Tween 80), with an injection volume of 3 mL / kg mouse body weight.

[0097] Each example group and comparative example group: The dried Panlongqi extract prepared in each example group and comparative example group was mixed with an appropriate amount of water and administered by gavage once a day (the dosage was 9g / kg mouse body weight per day, calculated based on the dry weight of Panlongqi extract).

[0098] The above-mentioned model control group, positive control group, each example group, and comparative group were administered the drug for a total of 16 days. During the drug administration period, the body weight of the mice was monitored, and the severity of rheumatoid arthritis symptoms was scored. After the drug administration was completed, venous blood was collected from the fundus venous plexus of the mice, and the levels of antibodies and biomarkers in the serum were detected.

[0099] 3.2 Effect Evaluation 3.2.1 Arthritis scoring test.

[0100] Starting from day 21, the degree of swelling in the mice's paws and joints was assessed every two days. The scoring criteria are shown in Table 3 below: Table 3

[0101] Each mouse's four paws were scored individually, and the scores were summed. Therefore, each mouse's score ranged from 0 to 16 points per assessment, and each group consisted of 7 mice. The scores for paw and joint swelling on days 21, 41, and 51 are shown in Table 4 below (n=7, mean ± standard deviation, rounded to one decimal place, unit: points): Table 4

[0102] As can be seen, on day 51, the embodiments provided by the present invention can significantly reduce the symptom scores of rheumatoid arthritis, especially the group of embodiments 1, which has the best effect.

[0103] 3.2.2 Detection of mouse serum anti-type II collagen antibody levels.

[0104] The level of anti-type II collagen antibody in mouse serum was detected according to the detection method provided in the kit instructions (Mouse Anti-type II Collagen Antibody Kit, Fein Biotech, EM0837-96T). The results are shown in Table 5 below (mean ± standard deviation, n=7, unit μg / mL, with the mean of the blank control group as background subtraction): Table 5

[0105] Wherein, # indicates that there are significant differences between the other embodiments, comparative examples and embodiment 1, and p < 0.001.

[0106] It is evident that the Panlongqi extract obtained in the various embodiments provided by the present invention has a better effect on reducing the level of anti-type II collagen antibodies compared with each comparative group.

[0107] 3.2.3 Detection of serum IL-6, IL-17A and TNFα levels in mice.

[0108] The levels of IL-6, IL-17A, and TNFα in mouse serum were detected according to the methods provided in the respective kit instructions (Mouse IL-6 ELISA kit, Adamas Life, P8038-1; Mouse IL-17A ELISA kit, Adamas Life, P8040-1; Tumor necrosis factor α (TNFα) ELISA kit, ELISA kit, ml022566-96T).

[0109] The test results are summarized in Table 6 below (mean ± standard deviation, n=7): Table 6

[0110] Wherein, # indicates that there are significant differences between the other embodiments, comparative examples and embodiment 1, and p < 0.001.

[0111] It is evident that the Panlongqi extracts obtained in the various embodiments provided by the present invention have a better effect on reducing the levels of IL-6, IL-17A and TNFα in mouse serum compared with the comparative groups.

[0112] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing Panlongqi extract, characterized in that, Includes the following steps: S1. Mix Panlongqi with ketone, reflux and extract, separate to obtain extract and residue; S2. Recover the solvent from the extract obtained in step S1 to obtain Panlongqi extract; S3. Mix the dregs obtained in step S1 with water, decoct, centrifuge the decoction and retain the supernatant, precipitate with alcohol to obtain crude polysaccharide. S4. React the crude polysaccharide obtained in step S3 with sodium periodate to obtain oxidized Panlongqi polysaccharide. S5. Take 5-15 parts by weight of the Panlongqi extract obtained in step S2, 10-30 parts by weight of the oxidized Panlongqi polysaccharide obtained in step S4, and 100 parts by weight of water, mix them, disperse them by ultrasonication, and dry them to obtain Panlongqi extract. In step S1, the ketone is acetone or 2-butanone.

2. The preparation method according to claim 1, characterized in that, In step S1, the Panlongqi is the pre-processed Panlongqi.

3. The preparation method according to claim 1, characterized in that, In step S1, the weight ratio of Panlongqi to the volume ratio of the ketone is 1:4-6, in g:mL; the reflux leaching temperature is 70-75℃, the reflux leaching time is 1-3h, the reflux leaching is performed 2-4 times, and the separation is achieved by filtration.

4. The preparation method according to claim 1, characterized in that, In step S2, the recovered solvent is a reduced-pressure recovered solvent.

5. The preparation method according to claim 1, characterized in that, In step S3, the weight ratio of the dregs to the volume of the water is 1:8-10, in g:mL; the decoction time is 1-3 hours, the number of decoctions is 2-4 times, the centrifugation speed is 4000-5500 rpm, and the centrifugation time is 5-20 minutes; the supernatant is the supernatant after vacuum concentration.

6. The preparation method according to claim 5, characterized in that, In step S3, the alcohol precipitation includes the following steps: adding ethanol to the supernatant to make the ethanol concentration in the mixture 75%vol-85%vol, letting it stand, and filtering.

7. The preparation method according to claim 1, characterized in that, In step S4, the weight ratio of the crude polysaccharide to the amount of sodium periodate is 2-3:1, in g:mmol. In step S4, the reaction includes the following steps: The crude polysaccharide was mixed with water to form a crude polysaccharide aqueous solution of 0.01-1 g / mL. A sodium periodate aqueous solution of 0.01-1 mol / L was added dropwise while stirring. The mixture was reacted at 10-40℃ for 10-60 min and then freeze-dried to obtain oxidized Panlongqi polysaccharide.

8. The preparation method according to claim 1, characterized in that, In step S5, the weight parts of the Panlongqi extract are 10 parts, and the weight parts of the oxidized Panlongqi polysaccharide are 20 parts; the ultrasonic dispersion time is 10-120 min, and the ultrasonic dispersion frequency is 20-60 kHz; the drying is freeze drying.

9. The preparation method according to any one of claims 1-8, characterized in that, The plant source of Panlongqi is Polygonum taipaishanense .

10. The preparation method according to any one of claims 1-8, characterized in that, The sodium periodate in step S4 is replaced or partially replaced with one or more of potassium periodate, metaperiodic acid, ammonium periodate, and ammonium periodate.

11. The Panlongqi extract prepared by the preparation method according to any one of claims 1-10.

12. A drug, characterized in that, The active ingredient of the drug includes Panlongqi extract prepared by any one of the preparation methods described in claims 1-10.

13. The medicament according to claim 12, characterized in that, The dosage form of the drug is selected from: tablets, pills, powders, granules, capsules, solutions, lozenges, or suppositories.

14. The medicament according to claim 12, characterized in that, It also includes pharmaceutically acceptable excipients.

15. The medicament according to claim 12, characterized in that, The drug in question is for the treatment of rheumatoid arthritis.

16. The use of the Panlongqi extract prepared by any one of claims 1-10 or the drug according to any one of claims 12-15 in the preparation of a drug for treating autoimmune diseases.

17. The application according to claim 16, characterized in that, The autoimmune disease mentioned is rheumatoid arthritis.

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