Saussurea involucrata acetate extract, preparation method and application thereof

Abstract

The application discloses a preparation method of a Saussurea involucrata extract, in particular, an ethyl acetate extract. The method can efficiently separate different polarity extracts from Saussurea involucrata, and has the advantages of simple operation, solvent recyclability, and suitability for large-scale preparation. The ethyl acetate extract of Saussurea involucrata obtained by the method has excellent anti-rheumatoid arthritis and anti-aging effects, and thus can be used as an active ingredient of an anti-rheumatoid arthritis and anti-aging drug.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, specifically to a method for preparing snow lotus extract and the application of the prepared snow lotus extract in anti-rheumatoid arthritis and anti-aging. Background Technology

[0002] Xinjiang snow lotus (Saussurea involucrata) is a perennial herbaceous plant belonging to the genus Saussurea in the family Asteraceae. It is an endangered species endemic to China and is classified as a Class II protected wild plant in China. This plant mainly grows near the snow line at altitudes of 2600-4000 meters in the Tianshan and Kunlun Mountains of Xinjiang Uygur Autonomous Region, commonly found in harsh environments such as rock crevices or sandy riverbanks. The snow lotus typically has a growth cycle of 5-8 years and flowers only once in its lifetime, thus being considered a rare resource among high-altitude medicinal plants. In terms of chemical composition research, more than 70 active ingredients have been isolated and identified from Tianshan snow lotus, including phenylpropanoids, flavonoid glycosides, coumarin derivatives, lignans, sesquiterpene lactones, steroidal compounds, ceramides, and polysaccharides. Among them, rutin and chlorogenic acid, due to their significant pharmacological activity and high content, have been listed as key indicator components for quality evaluation in the current Pharmacopoeia of the People's Republic of China. Studies have shown that extracts of Tian Shan snow lotus have a wide range of biological activities, including anti-inflammatory (used to treat inflammatory diseases such as RA), regulating lipid metabolism, anti-tumor, improving gynecological and reproductive system diseases, promoting blood circulation, and relieving cold symptoms.

[0003] Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease caused by abnormal activation of the body's immune system, leading to inflammation of the synovium and subsequent destruction of joint structure and loss of function. Its main pathological features are symmetrical polyarthritis, chronic synovitis, bone destruction, and loss of joint function, with a global incidence of approximately 1%-2%. Typical symptoms of RA include symmetrical inflammation of the small joints of the hands and feet, often accompanied by extra-articular multi-organ damage and abnormal production of autoantibodies. Current treatments suffer from limitations such as difficulty in effectively halting progressive joint destruction, significant long-term toxicity, high treatment costs, a significantly increased risk of secondary infections due to immunosuppression, and unclear long-term efficacy for some drugs.

[0004] Therefore, there is an urgent clinical need to develop safer, more effective, and lower-cost anti-RA drugs. Xinjiang snow lotus, as a traditional anti-RA medicinal material, has been widely used in clinical practice in compound preparations, but there is a lack of research on its active components and the dual regulatory mechanism of "aging-inflammation". Summary of the Invention

[0005] The purpose of this invention is to provide a method for efficiently separating extracts of different polarities from snow lotus of the Tianshan Mountains.

[0006] Another objective of this invention is to provide a snow lotus extract that has good anti-inflammatory and anti-aging effects both in vivo and in vitro.

[0007] Another objective of this invention is to provide an anti-inflammatory and anti-aging product. In a first aspect, the present invention provides a method for preparing snow lotus extract, the method comprising the following steps: (1) Extraction of snow lotus using organic solvents; (2) After extraction, filter the solution, collect the filtrate, and retain the residue; (3) Extract the filter residue using organic solvents; (4) After extraction, filter and collect the filtrate; (5) Combine all filtrates, concentrate under reduced pressure to remove solvent, and obtain total extract; (6) Disperse the total extract in distilled water to form a homogeneous suspension; (7) Liquid-liquid partition extraction was performed using an organic solvent. The extract phases were combined and concentrated under reduced pressure to constant weight to obtain snow lotus extract.

[0008] In a preferred embodiment, the snow lotus is the flower of the Tian Shan snow lotus.

[0009] In a preferred embodiment, the snow lotus flower in step (1) is first dried, pulverized, and then sieved to obtain snow lotus flower powder.

[0010] In a preferred embodiment, the sieving in step (1) is sieved through a 30-60 mesh sieve; preferably a 40 mesh sieve.

[0011] In a preferred embodiment, the extraction described in steps (1) and (3) is ultrasound-assisted extraction.

[0012] In a preferred embodiment, the conditions for ultrasound-assisted extraction are: ultrasound frequency 30–60 kHz, power 400–800 W, extraction time 20–60 min per extraction, and repeated 3–5 times.

[0013] In a preferred embodiment, the conditions for ultrasound-assisted extraction are: ultrasound frequency 40 kHz, power 500 W, room temperature, repeated 3 times, each extraction lasting 30 min.

[0014] In specific implementations, the organic solvents in steps (1) and (3) may be the same or different; preferably the same organic solvent.

[0015] In a specific implementation, the organic solvent in steps (1) and (3) is anhydrous ethanol.

[0016] In a preferred embodiment, in step (3), the filter residue is extracted with an organic solvent of 60% to 80% by volume; preferably 70%.

[0017] In a preferred embodiment, the material-to-liquid ratio of the ethanol extract with a volume fraction of 60% to 80% is 1:5 to 1:10 (g / mL); preferably 1:7 (g / mL).

[0018] In a preferred embodiment, in step (6), the mass ratio of the total extract to distilled water is 1:0.5 to 1:2; preferably 1:1.

[0019] In a specific embodiment, the organic solvent used in step (7) is selected from one or more of the following: petroleum ether, ethyl acetate, and n-butanol.

[0020] In a preferred embodiment, the n-butanol is water-saturated n-butanol.

[0021] In a preferred embodiment, in step (7), the liquid-liquid partitioning extraction can be performed 2-4 times.

[0022] In a preferred embodiment, in step (7), the volume ratio of solvent to suspension is 1:0.5 to 1:2 for each solvent extraction; preferably 1:1.

[0023] In a preferred embodiment, in step (7), the petroleum ether extract is concentrated under reduced pressure at 30°C, the ethyl acetate extract is concentrated under reduced pressure at 45°C, and the saturated n-butanol extract is concentrated under reduced pressure at 50°C.

[0024] In a specific embodiment, the organic solvent used in step (7) is selected from petroleum ether or ethyl acetate.

[0025] In a specific implementation, the organic solvent used in step (7) is ethyl acetate.

[0026] In a preferred embodiment, the chemical composition of the snow lotus extract obtained using ethyl acetate mainly consists of sesquiterpene lactones, flavonoids, lignins, and phenolic acids.

[0027] In a preferred embodiment, the chemical composition of the ethyl acetate extract of snow lotus is as follows: (1) Sesquiterpene lactones: dehydrocostus lactone, dihydrodehydrocostus lactone, Saussurea macrocarpa lactone-O-β-D-glucoside, 11βH-11,13-dihydrodehydrocostus lactone-8α-O-(6'-acetyl)-β-D-glucopyranoside, 3α-hydroxy-11β-11,13-dihydrodehydrocostus lactone-8-O-β-D-glucopyranoside, etc.

[0028] (2) Flavonoids: isoquercitrin, hyperoside, astragaloside, quercetin, rutin, calciferol, luteolin-O-glucoside, senna-O-glucoside, zeylan flavonoids, methyl quercetin, senna-O-glucoside, kaempferol-3-O-rutin glycoside, etc.

[0029] (3) Lignans: compounds such as arctigenin.

[0030] (4) Phenolic acids: 5-O-caffeoylquinic acid, chlorogenic acid, 3-O-caffeoylquinic acid, neochlorogenic acid, 1-O-caffeoylquinic acid, 1,3-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, p-coumarylquinic acid, 4-coumarylquinic acid, 5-coumarylquinic acid, feruloylquinic acid, protocatechuic acid-O-glucoside, dihydroxybenzoic acid, protocatechuic acid, gentianic acid, p-coumaric acid-O-glucoside, caffeoylquinic acid-O-glucoside, galloyl diglucoside, digaloyl glucose, etc.

[0031] (5) Other types: citric acid, yazoline, eucommia alcohol and other compounds.

[0032] In a second aspect, the present invention provides a snow lotus extract, which is obtained by the preparation method described in the first aspect.

[0033] In a preferred embodiment, the snow lotus extract is an ethyl acetate extract.

[0034] In a preferred embodiment, the chemical composition of the ethyl acetate extract of snow lotus mainly consists of sesquiterpene lactones, flavonoids, lignins, and phenolic acids.

[0035] In a third aspect, the present invention provides the use of the snow lotus extract described in the second aspect, or the snow lotus extract obtained by the preparation method described in the first aspect, in the preparation of a medicament with anti-inflammatory and / or anti-aging activity.

[0036] In a specific implementation, the inflammation is rheumatoid arthritis.

[0037] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the snow lotus extract as described in the second aspect, or the snow lotus extract obtained by the preparation method described in the first aspect, and a pharmaceutically acceptable excipient.

[0038] In a fifth aspect, the present invention provides a method for treating rheumatoid arthritis and / or anti-aging, comprising the step of administering the snow lotus extract described in the second aspect, or the snow lotus extract obtained by the preparation method described in the first aspect, or the pharmaceutical composition described in the fourth aspect, to a subject in need of such treatment.

[0039] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0040] Figure 1 This is the UHPLC-Q-MS base peak ion chromatogram of the ethyl acetate extract of *Saussurea involucrata* in this embodiment of the invention. In the figure, (A) is the total ion chromatogram of positive ions, and (B) is the total ion chromatogram of negative ions; Figure 2 The following are nematode lifespan curves of different solvent extracts of snow lotus in this invention embodiment; Figure 3 The effect of snow lotus extract on the expression levels of LPS-induced macrophage inflammatory factors IL-6 (A) and TNF-α (B) in this embodiment of the invention; Figure 4 This is a diagram showing the mouse CIA modeling and the drug administration cycle of snow lotus extract in this embodiment of the invention; Figure 5 This invention illustrates the effect of snow lotus extract on the degree of joint swelling in CIA mice. Figure 6 This invention illustrates the effect of snow lotus extract on the expression levels of plasma inflammatory factors IL-1β (A) and TNF-α (B) in CIA mice. Figure 7 This invention illustrates the effect of snow lotus extract on the synovial tissue of the knee joint in CIA mice. Figure 8 This invention illustrates the effect of snow lotus extract on knee cartilage in CIA mice. Figure 9 This figure illustrates the effect of snow lotus extract on knee joint bone destruction in CIA mice. In the figure, (A) shows the effect of snow lotus extract on knee joint bone density in CIA mice; (B) shows the effect of snow lotus extract on the number and intertrabecular spacing of trabeculae in the knee joint of CIA mice. Figure 10 This invention illustrates the effect of snow lotus extract on immunofluorescence markers of knee joint aging in CIA mice. In the figure, (A) shows the effect of snow lotus extract on the percentage of p16INK4a positive cells in the knee joint of CIA mice; (B) shows the effect of snow lotus extract on the percentage of p21 positive cells in the knee joint of CIA mice; and (C) shows the effect of snow lotus extract on the percentage of γH2AX positive cells in the knee joint of CIA mice. Figure 11 This invention illustrates the effect of snow lotus extract on the spleen coefficient of CIA mice. Detailed Implementation

[0041] Through extensive and in-depth research, the inventors unexpectedly discovered that after obtaining the total extract of snow lotus using traditional methods, a further liquid-liquid fractionation extraction of the total extract yielded significantly higher anti-inflammatory and anti-aging effects both in vivo and in vitro compared to the total extract. This makes it well-suited for anti-inflammatory and anti-aging applications, particularly in the treatment of rheumatoid arthritis. This invention was completed based on this discovery.

[0042] Terminology Definition The scientific and technical terms used herein are the same as those conventionally understood by those skilled in the art. For the purpose of clear understanding of the invention, some terms are defined below.

[0043] Snow Lotus In this article, "Xinjiang Snow Lotus ( Saussurea involucrata "Snow Lotus" and "Naturally Born Snow Lotus" have the same meaning, both referring to perennial herbaceous plants of the genus Saussurea in the family Asteraceae. Xinjiang Snow Lotus is an endangered species endemic to China and a rare resource among plateau medicinal plants. In this invention, the flowers of the Tian Shan Snow Lotus are particularly suitable.

[0044] The method of the present invention and the snow lotus extract obtained therefrom In this invention, the total extract of snow lotus is creatively subjected to liquid-liquid partition extraction again, thereby efficiently separating extracts of different polarities from Tian Shan snow lotus. The method of this invention is simple to operate, the solvent is recyclable, and it is suitable for large-scale preparation.

[0045] In a specific implementation, dried Tian Shan snow lotus flowers are first taken, pulverized, and sieved to obtain snow lotus flower powder. Then, a fractional extraction strategy is employed, using anhydrous ethanol as the solvent, with an ultrasonic-assisted extraction at a material-to-liquid ratio of 1:5 to 1:10 (g / mL). After extraction, the mixture is filtered, the filtrate is collected, and the residue is retained. The residue is then subjected to ultrasonic-assisted extraction using 60%–80% ethanol (volume fraction) as the solvent, at the same material-to-liquid ratio. After extraction, the mixture is filtered, and the filtrate is collected. All extracts are combined and concentrated under reduced pressure until no solvent flows out, yielding a total extract. The obtained total extract is redispersed in distilled water to form a homogeneous suspension. Liquid-liquid partition extraction is then performed sequentially using organic solvents such as petroleum ether, ethyl acetate, and saturated n-butanol. The extract phase is concentrated under reduced pressure to constant weight to obtain the snow lotus flower extract.

[0046] The petroleum ether or ethyl acetate extracts of snow lotus obtained in this invention exhibit good anti-inflammatory and anti-aging effects both in vitro and in vivo. In particular, the ethyl acetate extract of snow lotus shows the best efficacy in treating collagen-induced arthritis (CIA) in mice with rheumatoid arthritis and in anti-aging applications.

[0047] Extraction and extraction In this document, "extraction" and "extraction" have the meanings conventionally understood by those skilled in the art. Specifically, extraction is the process of separating a desired substance from a mixture, with the aim of obtaining the target substance. Extraction involves the comprehensive application of various separation methods, encompassing a variety of separation techniques, which may include physical methods such as filtration and evaporation, as well as chemical methods. Extraction, on the other hand, is a specific physical method of extraction. Extraction utilizes the difference in solubility of a solute in two immiscible solvents to transfer the solute from the solvent with lower solubility to the solvent with higher solubility.

[0048] Therefore, in this invention, the total extract obtained by ultrasonic-assisted extraction of the flowers of Tian Shan snow lotus using anhydrous ethanol as solvent, followed by filtration, collection of the filtrate, and concentration under reduced pressure is called the extract; while the product obtained by liquid-liquid partition extraction of the total extract is called the extract.

[0049] Pharmaceutical Composition Based on the compounds of the present invention, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of the extract of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0050] The pharmaceutical compositions of the present invention can be formulated into dosage forms suitable for various routes of administration. A dosage is an amount of medicine that effectively improves or eliminates one or more symptoms. For the treatment of a specific disease, an effective amount is an amount sufficient to improve or alleviate, in some way, the symptoms associated with the disease. Such a dosage may be administered as a single dose or may be administered according to an effective treatment regimen. The dosage may cure the disease, but administration is usually intended to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. The dosage of the medicine will be determined based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0051] The pharmaceutical compositions of the present invention can be administered to any mammal, provided they achieve the desired therapeutic effect. Humans are the most important of these mammals.

[0052] The pharmaceutical compositions of the present invention can be manufactured by known methods. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolving, or freeze-drying processes. When manufacturing oral formulations, solid excipients and active compounds can be combined and the mixture can be selectively ground. If necessary or required, an appropriate amount of excipients can be added, and the granular mixture can be processed to obtain tablets or tablet cores.

[0053] Suitable excipients, especially fillers, include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or dicalcium phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added. Adjuvants, especially flow conditioners and lubricants, include silica, talc, stearates such as calcium magnesium stearate, stearic acid, or polyethylene glycol. If necessary, a suitable coating that resists gastric juices can be provided to the tablet core. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, varnish solutions, and suitable organic solvents or solvent mixtures. To prepare a coating resistant to gastric juice, a suitable cellulose solution, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments can be added to the coating of the tablet or lozenge core, for example, to identify or characterize combinations of active ingredient dosages.

[0054] Treatment for rheumatoid arthritis and / or anti-aging Based on the above-described pharmaceutical composition, the present invention further provides a method for treating rheumatoid arthritis and / or anti-aging, comprising the step of administering the snow lotus extract or pharmaceutical composition of the present invention to a subject in need of such treatment.

[0055] Administration methods include, but are not limited to, various administration methods known in the art.

[0056] Advantages of this invention: 1. The method of the present invention can efficiently separate extracts of different polarities from Tian Shan snow lotus; 2. The method of the present invention is simple to operate, the solvent can be recovered, and it is suitable for large-scale preparation; 3. The petroleum ether and ethyl acetate extracts of snow lotus obtained by the method of this invention have good anti-inflammatory and anti-aging effects both in vivo and in vitro; 4. The ethyl acetate extract of snow lotus of the present invention has the best effect in treating rheumatoid arthritis in mice with collagen-induced arthritis (CIA) and anti-aging.

[0057] The technical solution of the present invention is further described below with reference to specific implementation examples. However, the following implementation examples do not constitute a limitation on the present invention. All application methods based on the principles and technical means of the present invention are within the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0058] Example Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0060] Example 1: Preparation of Ethanol Extract from Snow Lotus 3.1 kg of dried Tian Shan snow lotus flowers were crushed and passed through a 40-mesh sieve. A staged extraction strategy was adopted: First, anhydrous ethanol was used as the solvent, and ultrasonic-assisted extraction was performed at a material-to-liquid ratio of 1:7 (g / mL) (frequency 40 kHz, power 500 W, room temperature, 30 min each time, repeated 3 times). After each extraction, the mixture was filtered (using filter paper), and the filtrates from the three anhydrous ethanol extractions were combined, retaining the residue. The residue was then extracted three more times (30 min each time) with 70% (v / v) ethanol as the solvent, at the same material-to-liquid ratio and ultrasonic conditions. The filtrates from the three 70% ethanol extractions were combined. All extracts (i.e., the filtrates from the anhydrous ethanol and 70% ethanol extractions) were combined and concentrated under reduced pressure at 45℃ until no solvent flowed out, yielding a total extract of 810 g.

[0061] Example 2: Extraction of Ethanol Extract from Snow Lotus The extract obtained in Example 1 was redispersed in distilled water at a mass ratio of 1:1 to form a homogeneous suspension. Liquid-liquid partition extraction was performed sequentially using petroleum ether, ethyl acetate, and saturated n-butanol, with each solvent extracted three times, and the solvent volume to suspension volume ratio being 1:1 each time. The extracts with the same solvent were combined and concentrated under reduced pressure to constant weight (the difference between two weighings ≤ 0.1 g). The petroleum ether extract was concentrated at 30°C, the n-butanol extract at 50°C, and the ethyl acetate extract at 45°C, also concentrated under reduced pressure to constant weight. The final yield was 95 g of the petroleum ether extract, 23 g of the ethyl acetate extract, and 53 g of the saturated n-butanol extract.

[0062] Example 3: Chemical composition analysis of ethyl acetate extract of snow lotus The ethyl acetate extract of Saussurea involucrata obtained in Case 2 was analyzed by UHPLC-Q-MS: 1. Equipment and materials Ultimate 3000 UHPLC-Q Exactive liquid chromatography-mass spectrometry system (Thermo Scientific, US); formic acid, acetonitrile (chromatographic grade, Fisher); ultrapure water.

[0063] 2. Sample processing The sample was filtered through a 0.22 μm microporous membrane and then directly injected for LC-MS testing.

[0064] 3. Chromatographic conditions Chromatograph: Dinonex Ultimate 3000 UHPLC Chromatographic column: ACQUITY UPLC BEH C18 100mm × 2.1mm, 1.7μm Injection volume: 3.0 μL Column temperature: 30℃ Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile Gradient elution conditions: 4. Mass spectrometry conditions Mass spectrometer: Thermo Scientific Q Exactive Ion source: HESI Air-lift rate: 40 arb Auxiliary gas rate: 10 arb Spray voltage: 3.8kV for positive ions, 3.0kV for negative ions. Capillary temperature: 320℃ Needle temperature: 250℃ S-lens: 50% Scan mode: Fullms / dd-ms2 top10 Scan range: Level 1 scan: resolution 70000, 67-1000 m / z; Level 2 scan: resolution 17500, starting ion 50 m / z; Shattering Energy: HCD 15, 30, 45 5. Test Results The UHPLC-Q-MS base peak ion chromatogram of the ethyl acetate extract of Saussurea involucrata is shown below. Figure 1 The main chemical components are as follows: (see Table 1 for details) (1) Sesquiterpene lactones: dehydrocostus lactone, dihydrodehydrocostus lactone, Saussurea macrocarpa lactone-O-β-D-glucoside, 11βH-11,13-dihydrodehydrocostus lactone-8α-O-(6'-acetyl)-β-D-glucopyranoside, 3α-hydroxy-11β-11,13-dihydrodehydrocostus lactone-8-O-β-D-glucopyranoside, etc.

[0065] (2) Flavonoids: isoquercitrin, hyperoside, astragaloside, quercetin, rutin, calciferol, luteolin-O-glucoside, senna-O-glucoside, zeylan flavonoids, methyl quercetin, senna-O-glucoside, kaempferol-3-O-rutin glycoside, etc.

[0066] (3) Lignans: compounds such as arctigenin.

[0067] (4) Phenolic acids: 5-O-caffeoylquinic acid, chlorogenic acid, 3-O-caffeoylquinic acid, neochlorogenic acid, 1-O-caffeoylquinic acid, 1,3-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, p-coumarylquinic acid, 4-coumarylquinic acid, 5-coumarylquinic acid, feruloylquinic acid, protocatechuic acid-O-glucoside, dihydroxybenzoic acid, protocatechuic acid, gentianic acid, p-coumaric acid-O-glucoside, caffeoylquinic acid-O-glucoside, galloyl diglucoside, digaloyl glucose, etc.

[0068] (5) Other types: citric acid, yazoline, eucommia alcohol and other compounds.

[0069] Table 1 Chemical composition of ethyl acetate extract of snow lotus Example 4: Effect of Saussurea involucrata extract on the lifespan of Caenorhabditis elegans The anti-aging activity of different solvent extracts of Saussurea involucrata was evaluated using Caenorhabditis elegans as a model organism. This nematode has the following significant advantages: First, its life cycle is relatively short, with an average lifespan of 14-21 days under culture conditions at 20℃, which facilitates lifespan experiments; second, its aging regulatory pathways are highly conserved with those of mammals.

[0070] Lifespan experiments were conducted on *C. elegans* nematodes using different solvent extracts of *Saussurea involucrata* obtained in Example 2. The results showed that both petroleum ether and ethyl acetate extracts significantly prolonged the lifespan of the nematodes. The results are shown in Table 2 and... Figure 2 Specific experimental steps: First, nematodes were synchronized until they reached the L4 stage (identified by ventral crescent morphological characteristics). They were then randomly divided into a control group (DMSO) and treatment groups using various snow lotus extracts (200 μg / mL). The nematodes were transferred to NGM medium containing 50 μM 2-deoxy-5-fluorouridine to inhibit progeny reproduction. The medium was changed every 48 hours, and treatment continued until day 10. Afterward, all nematodes were transferred to normal NGM medium for further culture. The entire experiment was conducted at a constant temperature of 20℃. The number of dead nematodes was observed and recorded daily until all nematodes had died.

[0071] Table 2. Experimental data on nematode lifespan using different extracts from snow lotus. Example 5: Efficacy of snow lotus extract on lipopolysaccharide (LPS)-induced inflammation model in RAW264.7 cells The anti-inflammatory activity of two Saussurea involucrata extracts (petroleum ether extract SU20H05 and ethyl acetate extract SU20H11) exhibiting nematode anti-aging effects from Example 3 was evaluated in an LPS-induced RAW264.7 cell inflammation model. The results are as follows: Figure 2 As shown, compared with the control group, the levels of inflammatory factors IL-6 and TNF-α were significantly increased in the LPS-induced model group after 24 h of induction, while the positive control dexamethasone group significantly reduced the levels of these two inflammatory factors, proving the successful construction of the cellular inflammation model. Compared with the model group, both snow lotus extracts significantly reduced the levels of IL-6 and TNF-α inflammatory factors (…). Figure 3 ).

[0072] Specific experimental steps: SU20H05 (50, 100 μg / mL) and SU20H11 (50, 100 μg / mL) were co-cultured with 0.5 μg / mL LPS for 24 h. The levels of inflammatory factors IL-6 and TNF-α in the cell culture supernatant were detected using ELISA kits, and the expression levels of IL-6 and TNF-α were detected using enzyme-linked immunosorbent assay (ELISA). Commercially available kits (Mouse IL-6 ELISA Kit, EK206) and (Mouse TNF-α ELISA Kit, EK282) were used, and the procedures were strictly followed according to the instructions. The specific steps are as follows: First, the cell culture supernatant was collected, centrifuged at 1200 rpm for 10 minutes at 4℃ to remove cell debris, and the supernatant was aliquoted and stored at -80℃ for later use. Before detection, an IL-6 standard curve of 0-700 pg / mL was prepared using standards (R0 required). 2(≥0.99). In the formal experiment, standards and test samples were added to 96-well plates and incubated at room temperature for 2 hours; then washed 6 times with washing buffer; HRP-labeled secondary antibody was added, and the plate was reacted at 37°C for 45 minutes; after washing 6 more times, TMB chromogenic solution was added, and the plate was incubated at room temperature in the dark for 15 minutes; finally, stop solution was added to terminate the reaction, and the absorbance at 450 nm was immediately measured using a microplate reader. To ensure experimental reliability, three biological replicates were set up for each sample group.

[0073] Example 6: Efficacy of snow lotus extract in a mouse model of collagen-induced arthritis (CIA) The anti-inflammatory and anti-aging activities of the two Saussurea involucrata extracts (petroleum ether extract SU20H05 and ethyl acetate extract SU20H11) exhibiting in vitro anti-inflammatory effects in Example 4 were evaluated in a CIA mouse model, specifically including the following: 1. Mouse CIA modeling Male DBA / 1 mice, aged 7-8 weeks and weighing 22±2 g, were used in the experiment. After receiving the animals, they were acclimatized in the animal room for 3-7 days before modeling was performed. First, the syringe tip was sealed with a three-way valve and placed on ice. Complete Freund's adjuvant (or incomplete Freund's adjuvant for enhanced injection) and bovine type II collagen were added to the syringe in a 1:1 ratio and stirred with a high-speed mixer to prepare an emulsion. The mice were then fixed in place, and 100 μL of the emulsion was injected into each mouse 1-2 cm from the base of the tail (first immunization). On day 21 post-immunization, following the same emulsification method, 2 mg / mL bovine type II collagen and incomplete Freund's adjuvant were mixed 1:1 and emulsified. 100 μL of the emulsion was injected into each mouse 0.5 cm from the base of the tail (second immunization). Joint swelling was assessed in the limbs of the mice every two days. Mice with a combined limb score of 4 or higher were considered successfully modeled. The paw swelling scores of mice were continuously monitored until day 38. It was found that most mice had mild paw swelling. Therefore, on day 38, mice received a third booster immunization with the emulsion, and swelling scores were continued to be monitored. On day 55, except for the normal control group, mice in each group were grouped according to the principle of having similar swelling scores. Five groups were set up: normal control group (0.9% saline), model control group (0.9% saline), positive control group (methotrexate 1 mg / kg intraperitoneal injection), and experimental group (snow lotus petroleum ether SU20H05 and ethyl acetate SU20H11 extract 50 mg / kg orally by gavage). Except for methotrexate administered every two days, all other groups received daily administration at fixed times for 16 consecutive days. Figure 4 During this period, the mouse's weight change was recorded every two days.

[0074] 2. Effects of Saussurea involucrata extract on joint swelling scores in CIA mice Starting from the first day of administration, researchers scored the arthritis index of the mice's limb joints every two days according to the scoring criteria in Table 3, and the scores of each limb were accumulated and recorded as the total score.

[0075] Table 3 Joint swelling score The results showed that, compared with the normal group, the paws of mice in the model group were significantly swollen, and the joint swelling in the positive control drug MTX group was significantly reduced compared with the model group, indicating that the arthritis model was successfully established. Administration of petroleum ether (SU20H05) and ethyl acetate (SU20H11) extracts of Saussurea involucrata significantly improved the degree of joint swelling in the model group mice. Figure 5 ).

[0076] 3. Effects of Saussurea involucrata extract on the expression levels of plasma inflammatory factors in CIA mice. After drug administration, the levels of inflammatory factors IL-1β and TNF-α in mouse plasma were detected using an ELISA kit. The results showed that, compared with the control group, the plasma levels of IL-1β and TNF-α in the model group mice were significantly increased; compared with the model, the levels in the positive control group (MTX) were significantly decreased, and the petroleum ether extract (SU20H05) and ethyl acetate extract (SU20H11) of *Saussurea involucrata* showed significant inhibitory effects. Figure 6 A and Figure 6 B).

[0077] 4. Effects of snow lotus extract on synovial tissue of the knee joint in CIA mice Synovial tissue, an important structure of the joint, is located around the joint cavity. Significant synovial inflammation is frequently observed in CIA model mice, manifesting as joint swelling and other clinical symptoms. Histological examination reveals typical pathological features of synovial inflammation, including inflammatory cell infiltration and synovial tissue hyperplasia. Hematoxylin-eosin staining was used to analyze the knee joint tissue of mice. Basic hematoxylin staining turned the cell nuclei purple-blue, while acidic eosin staining the cytoplasm and extracellular matrix red. Knee joint staining scores were assigned to each group of mice, and the specific scoring criteria are shown in Table 4. The results showed that, compared with the normal control group, RA mice exhibited significant inflammatory cell infiltration and synovial hyperplasia in the knee joint after modeling, characterized by increased hematoxylin staining in the synovial region and a significant reduction in normal reticular connective tissue (the dashed box in the figure indicates the synovial region). Administration of petroleum ether extract (SU20H05) and ethyl acetate extract (SU20H11) of Saussurea involucrata significantly improved the pathological condition of the knee joint, showing reduced inflammatory cell infiltration and synovial hyperplasia. Among them, the ethyl acetate extract of snow lotus showed the most significant improvement effect. Figure 7 ).

[0078] Table 4. Hematoxylin-eosin staining scoring criteria for the knee joint 5. Effects of snow lotus extract on knee cartilage tissue in CIA mice Cartilage, another key structure of the joint, is mainly composed of chondrocytes and their secreted extracellular matrix, covering the joint surface. In CIA model mice, abnormally proliferating synovial cells invade cartilage tissue and degrade the cartilage matrix by releasing matrix metalloproteinases, ultimately leading to cartilage destruction and loss. We used the Safranin O-Fix Green staining method to analyze the cartilage of the mouse knee joint. The basic dye Safranin O binds to the basophilic cartilage matrix, turning it red, while the acidic dye Fast Green binds to the eosinophilic bone, turning it green, thus distinguishing cartilage and bone tissue. The specific scoring criteria are shown in Table 5. The experimental results showed that compared with the control group, the RA model group mice showed significant cartilage destruction and loss in the knee joint, as evidenced by a significant reduction in the Safranin O staining area. After treatment with petroleum ether extract (SU20H05) and ethyl acetate extract (SU20H11) of Saussurea involucrata, the cartilage damage in both groups of mice was significantly improved. The improvement effect was most significant in the group treated with ethyl acetate extract of Saussurea involucrata, as evidenced by a significant increase in the Safranin O staining area. Figure 8 ).

[0079] Table 5. Scoring criteria for safranin O-fast green staining of the knee joint 6. Effects of Ethyl Saussurea involucrata Extract on Knee Bone Tissue in CIA Mice Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and bone destruction. Its pathological process involves systemic inflammatory responses and abnormal osteoclast activation, which in turn affects osteoblast function. This study used Micro-CT technology to scan and analyze the knee joint and distal femur of CIA mice, focusing on bone mineral density (BMD), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Trabeculae, also known as cancellous bone, are a three-dimensional network structure composed of interconnected bone plates and rods, playing a crucial role in maintaining skeletal mechanical properties. The results showed that, compared with the control group, the CIA model group mice had significantly lower bone mineral density (BMD). Figure 9 A) The number of trabeculae is significantly reduced, and the intertrabecular gaps are significantly increased. Figure 9 B). Treatment with MTX and ethyl acetate extract of Saussurea involucrata (SU20H11) significantly increased bone mineral density, improved the number of trabeculae, and increased the intertrabecular spacing. These data indicate that ethyl acetate extract of Saussurea involucrata can improve bone destruction in CIA mice.

[0080] 7. Effects of Ethyl Saussurea involucrata Extract on Immunofluorescence of Knee Joint Aging Indicators in CIA Mice Rabies (RA) is an age-related disease. Our research aims to treat RA by targeting aging. p16 and p21 are two cyclin-dependent kinase inhibitors present in cells, crucial for maintaining the proliferative arrest of senescent cells. γH2AX is a DNA damage-related protein. Using immunofluorescence staining, specific antibodies were used to identify and quantify these aging-related biomarkers in mouse knee joint tissue. Experimental results showed that, compared with the saline control group, p16 (…) was significantly higher in the joint tissue of RA model mice. Figure 10 A), p21 ( Figure 10 B) and γH2AX ( Figure 10 C) The percentage of positive cells increased significantly. Treatment with MTX and ethyl acetate extract of Saussurea involucrata (SU20H11) significantly reduced the number of positive cells, indicating aging. These results suggest that RA progression is accompanied by significant cellular senescence, and that ethyl acetate extract of Saussurea involucrata can effectively alleviate this pathological process, suggesting that it may exert a therapeutic effect on RA by regulating aging pathways.

[0081] 8. Effect of Ethyl Saussurea involucrata Extract on Spleen Coefficient in CIA Mice Rabies (RA), a typical systemic autoimmune disease, is closely related to immune regulatory dysfunction in its pathogenesis. Numerous studies have shown that RA patients exhibit extensive infiltration of abnormally functioning inflammatory cells, including T cells, B cells, and macrophages, in the synovium of their joints. Notably, early RA onset is associated with immune system self-tolerance defects and immune cell remodeling, with T cell senescence being a key characteristic of RA. The spleen, as an important peripheral immune organ, is not only a major distribution site for T and B lymphocytes but also a crucial organ for regulating specific immune responses. Therefore, this study first systematically evaluated the effect of ethyl acetate extract of Saussurea involucrata (SU20H11) on the spleen coefficient (spleen mass / body weight) by measuring spleen mass in experimental and control mice. The results showed that compared to the normal control group, the spleen coefficient of mice in the CIA model group was significantly increased, indicating activation of splenic immune cells after modeling. Treatment with SU20H11 significantly reduced the spleen coefficient, indicating that ethyl acetate extract of Saussurea involucrata can inhibit excessive activation of splenic immune cells. Figure 11 ).

[0082] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing snow lotus extract, the method comprising the following steps: (1) Extraction of snow lotus using organic solvents; (2) After extraction, filter the solution, collect the filtrate, and retain the residue; (3) Extract the filter residue using organic solvents; (4) After extraction, filter and collect the filtrate; (5) Combine all filtrates, concentrate under reduced pressure to remove solvent, and obtain total extract; (6) Disperse the total extract in distilled water to form a homogeneous suspension; (7) Liquid-liquid partition extraction was performed using an organic solvent. The extract phases were combined and concentrated under reduced pressure to constant weight to obtain snow lotus extract.

2. The preparation method according to claim 1, characterized in that, The organic solvents used in steps (1) and (3) may be the same or different; preferably the same organic solvent.

3. The preparation method according to claim 1, characterized in that, The organic solvent in steps (1) and (3) is anhydrous ethanol.

4. The preparation method according to claim 1, characterized in that, The organic solvent used in step (7) is selected from one or more of the following: petroleum ether, ethyl acetate, n-butanol.

5. The preparation method according to claim 4, characterized in that, The organic solvent used in step (7) is selected from petroleum ether or ethyl acetate.

6. The preparation method according to claim 5, characterized in that, The organic solvent used in step (7) is ethyl acetate.

7. A snow lotus extract, said snow lotus extract being obtained by the preparation method according to any one of claims 1-6.

8. Use of the snow lotus extract as described in claim 7, or the snow lotus extract obtained by any one of claims 1-6, in the preparation of a medicament with anti-inflammatory and / or anti-aging activity.

9. The use as described in claim 8, characterized in that, The inflammation in question is rheumatoid arthritis.

10. A pharmaceutical composition comprising the snow lotus extract as described in claim 7, or the snow lotus extract obtained by any one of claims 1-6, and a pharmaceutically acceptable excipient.

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