Herba geranii extract, preparation method thereof and application of herba geranii extract in resisting rheumatoid arthritis

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and relates to a geranian extract, a preparation method thereof and an application of the geranian extract in resisting rheumatoid arthritis, dry geranian powder is subjected to ethanol extraction and then is subjected to liquid separation extraction with ethyl acetate to obtain a geranian ethyl acetate extract with anti-inflammatory activity, and the geranian ethyl acetate extract is subjected to silica gel column chromatography separation and purification to obtain the geranian extract with anti-inflammatory activity. The geranian extract which contains ellagic acid, geraniin and corilagin and has remarkable anti-inflammatory activity is separated from the geranian extract, and the geranian extract has a remarkable improvement effect on rheumatoid arthritis. The invention provides an efficient and low-toxicity plant extract for developing medicines for improving rheumatoid arthritis, the source of the raw material geranian is convenient, the extraction method is simple and convenient, the cost is low, the geranian extract containing ellagic acid, geraniin and corilagin has development potential in the aspect of treating inflammation, and the geranian extract containing ellagic acid, geraniin and corilagin has a wide application prospect. The compound can be applied to the development of efficient and low-toxicity plant-derived anti-inflammatory agents.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and relates to a geranium seedling extract, a preparation method thereof, and application of the extract in treating rheumatoid arthritis. Background Art

[0002] Inflammation is the body's defensive response to injury or infection, and it's also a very common pathological reaction that occurs alongside other diseases. Local manifestations include redness, swelling, heat, pain, and dysfunction. Systemic reactions may include fever and changes in white blood cell count, which can be life-threatening in severe cases. Currently, the main drugs used in clinical treatment of inflammation include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and some topical medications and antibiotics. However, these drugs have drawbacks such as limited applicability, numerous side effects, and a single target. Therefore, the search for highly effective, low-toxic anti-inflammatory drugs is of vital importance.

[0003] Plants are rich in bioactive substances, and their anti-inflammatory properties have garnered widespread attention both domestically and internationally. Geranium chinense (Germanicum chinense) belongs to the Geraniaceae family, a family of plants with medicinal properties known for their meridian-opening, rheumatism-relieving, and antidiarrheal properties. It is a hot topic of research in traditional Chinese medicine. Geranium chinense is a traditional Chinese folk herbal medicine, containing bioactive ingredients that inhibit bacteria, dispel rheumatism, promote blood circulation, and clear away heat and toxins. However, methods for extracting anti-inflammatory compounds from geranium chinense have not been reported. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing a geranium seedling extract containing ellagic acid, geranin and corilagin, thereby providing a high-efficiency, low-toxic plant extract for the development of drugs for improving rheumatoid arthritis.

[0005] To achieve the above objectives, the present invention uses geranium seedlings as raw material and obtains a geranium seedling extract containing ellagic acid, geraniol, and corilagin, which exhibits anti-inflammatory activity, through ethanol extraction, petroleum ether and ethyl acetate extraction, and silica gel column chromatography. This extract, at a concentration of 100 μg / mL, significantly reduces the levels of pro-inflammatory factors in LPS-induced RAW 264.7 inflammatory cells and exhibits a significant alleviating effect on rheumatoid arthritis in mice at a dose of 750 mg / kg.

[0006] The present invention provides a method for preparing a geranium seedling extract, which comprises the following steps:

[0007] In the first step, the raw material of the Geranium truncatum plant is crushed to obtain a Geranium truncatum dry powder; the dry powder is mixed with anhydrous ethanol at a material-liquid ratio of 1 (g): 8 (mL), ultrasonically shaken at room temperature for 24 hours, and then vacuum filtered to obtain a primary ethanol extract of the Geranium truncatum. The filtration residue is repeated twice according to the above method, and the resultant mixture is combined to obtain a primary ethanol extract of the Geranium truncatum;

[0008] The second step is to concentrate the primary ethanol extract of the Geranium chinense seedlings by vacuum distillation using a rotary evaporator to obtain the primary ethanol extract of the Geranium chinense seedlings;

[0009] The third step is to extract the obtained primary ethanol extract of the geranium seedlings twice with ethyl acetate and water at a volume ratio of 1:1, combine the ethyl acetate phases, and concentrate them by vacuum distillation to obtain the ethyl acetate extract of the geranium seedlings;

[0010] In the fourth step, the ethyl acetate extract of the above-mentioned geranium seedling is separated by silica gel column chromatography and gradient eluted with a methanol-ethyl acetate solvent system to obtain 12 components, which are Fr.1-Fr.12 in elution order, among which Fr.5 is a highly anti-inflammatory active component containing ellagic acid, geraniol and corilagin, which is the geranium seedling extract.

[0011] The chemical structural formulas of ellagic acid, geraniol and corilagin described in the present invention are:

[0012]

[0013] Ellagic acid, geranylgeranin, corilagin

[0014] The present invention also provides the use of the geranium seedling extract in the preparation of an anti-rheumatoid arthritis drug, which can significantly alleviate rheumatoid arthritis at a dose of 750 mg / kg.

[0015] Compared with the existing technology, the present invention develops a new preparation process for the geranium seedling extract, and also provides a plant extract for the development of drugs with the function of significantly improving rheumatoid arthritis. The natural plant geranium seedling is convenient to obtain, the extraction method is simple, the cost is low, and it is low-toxic and environmentally friendly. The main anti-rheumatoid arthritis components in the geranium seedling are established, and can be used to develop high-efficiency, low-toxic plant anti-rheumatoid arthritis agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the HPLC test result of the Fr.5 component of Example 1 of the present invention, wherein Figures A and B are standard solutions of corilagin, geraniol and ellagic acid, and Figure C is the Fr.5 solution; 1. Corilagin, 2. Geraniol, 3. Ellagic acid.

[0017] Figure 2This is the in vitro anti-inflammatory activity test result of the Fr.5 component involved in the present invention, wherein A is IL-6 and B is TNF-α.

[0018] Figure 3 This figure shows the effect of the Fr.5 component of the present invention on the toe thickness of rheumatoid arthritis model mice.

[0019] Figure 4 The present invention relates to H&E and Safranin O-Fast Green staining images of fresh ankle joint tissue sections of mice. DETAILED DESCRIPTION

[0020] The present invention will be further described below through specific examples.

[0021] Example 1

[0022] The present application relates to a method for preparing a geranium seedling extract containing ellagic acid, geranin and corilagin, which is extracted from the geranium seedling plant. The specific steps are as follows:

[0023] (1) The whole plant of Geranium truncatum was dried at room temperature and crushed in a grinder to obtain Geranium truncatum dry powder. 100 g of dry powder was added to 800 mL of anhydrous ethanol and extracted with ultrasonic oscillation at room temperature for 24 h. The clarified Geranium truncatum ethanol primary extract was obtained by vacuum filtration. The filter residue was repeatedly extracted twice and the Geranium truncatum ethanol primary extract was combined. The Geranium truncatum ethanol primary extract was concentrated to dryness by vacuum distillation on a rotary evaporator to obtain 8.45 g of Geranium truncatum ethanol primary extract. The ethanol primary extract was fully suspended in 800 mL of distilled water and an equal volume of ethyl acetate was added. The mixture was allowed to stand for 4 h. After separation, the lower aqueous phase was extracted again with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated by vacuum distillation on a rotary evaporator to obtain 4.05 g of Geranium truncatum ethyl acetate extract.

[0024] (2) The ethyl acetate extract of the geranium seedling was separated by silica gel (200-300 mesh) column chromatography, and gradient elution was performed with an ethyl acetate-methanol solvent system (the volume ratio of ethyl acetate to methanol was 1:2, 1:4, 1:7, 1:9, and 100% methanol, respectively). The eluted components were filtered through silica gel (GF 254 ) Thin layer chromatography was used for detection. The same fractions were combined and concentrated separately, and finally there were 12 fractions in total: Fr.1-Fr.12 in the order of elution.

[0025] Each component of Fr.1-Fr.12 was dissolved in methanol to prepare a test solution with a concentration of 1 mg / mL, and the HPLC test was carried out under the following chromatographic conditions: an Agilent 5TC-C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile (A) and 0.1% phosphoric acid (B), with a gradient elution (0-10 min, 4%-5% A; 10-15 min, 5%-12% A; 15-20 min, 12%-14% A; 20-30 min, 14%-15% A; 30-40 min, 15%-21% A; 40-50 min, 21% A; 50-60 min, 21%-45% A; 60-62 min, 45% A); the column temperature was 30°C; the detection wavelengths were 280 nm (0-32 min) and 360 nm (32-62 min); the volume flow rate was 1 mL / min; injection volume 10 μL. Test results are as follows Figure 1 As shown, Figures A and B are standard solutions of corilagin, geraniol and ellagic acid, and Figure C is Fr.5 solution.

[0026] from Figure 1 It can be seen that the Fr.5 component is composed of the geranium seedling extract containing ellagic acid, geraniol and corilagin.

[0027] Example 2:

[0028] This example relates to the in vitro anti-inflammatory activity assay of the Geraniol extract containing ellagic acid, geraniol, and corilagin in Example 1, specifically:

[0029] A 96-well plate was taken and divided into three groups: normal cell group, extract intervention group and lipopolysaccharide (LPS) cell model group. 60 μL of complete culture medium was added to each well, pipetted and mixed, and RAW264.7 cell suspension was added, with about 20,000 cells per well; after 24 hours of cell attachment and growth, LPS (1000 ng / mL) was added to the extract intervention group and LPS cell model group to induce an inflammatory model; after 24 hours, the culture medium was replaced, and the normal cell group and LPS cell model group were replaced with complete culture medium, while the extract intervention group was replaced with complete culture medium containing Fr.5 (100 μg / mL). After 24 hours of intervention, the anti-inflammatory effect was observed; the cell supernatants of the normal cell group, LPS cell model group and extract intervention group were collected, and the expression levels of two pro-inflammatory factors, interleukin 6 (IL-6) and tumor necrosis factor (TNF-α), in the cells of each treatment group were determined by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 2. Figure 2 As shown. Figure 2As can be seen, compared with the LPS cell model group, the expression levels of IL-6 and TNF-α in the extract intervention group were significantly reduced (p<0.001), indicating that the geranium extract has significant anti-inflammatory activity against LPS-induced inflammation in RAW264.7 cells.

[0030] Example 3:

[0031] This example relates to the in vivo anti-inflammatory activity determination of the Geraniol extract prepared in Example 1, specifically:

[0032] The Fr.5 fraction of the G. geraniolii extract was dissolved in a 0.5% sodium carboxymethylcellulose aqueous solution to prepare a 75 mg / mL G. geraniolii extract solution for later use. After one week of adaptive feeding, 30 SPF male DBA mice were randomly divided into a normal control group (NC), an extract intervention group (AT), and a collagen-induced arthritis model group (AC). Bovine type II collagen was used to establish the AT and AC groups, and subcutaneous collagen strips were visible after injection, thus establishing a collagen-induced rheumatoid arthritis model. Then, the extract intervention group (AT) was given a daily dose of 750 mg / kg G. geraniolii extract by gavage (i.e., 10 mL of the above-mentioned G. geraniolii extract solution per kg of mouse). The normal control group (NC) was given a daily dose of 0.5% sodium carboxymethylcellulose aqueous solution by gavage. The collagen-induced arthritis model group (AC) was given a daily dose of 0.5% sodium carboxymethylcellulose aqueous solution by gavage. Each group received an equal volume of gavage solution once daily for a total of 8 weeks. All mice were fed AIN-93M feed. The weight of the mice was measured and recorded daily. The geranium extract had no significant effect on the mice's weight. The thickness of the mice's toes was measured and photographed every other day. The images were taken on the 60th day of intervention ( Figure 3 ).

[0033] After the intervention, fresh ankle joint tissue sections of mice in each group were taken for hematoxylin and eosin (H&E) staining and safranin O-fast green staining. Figure 4 Pathological changes in the ankle joints of mice, including inflammation, bone erosion, cartilage damage, and proteoglycan loss, were observed and scored. Inflammation and bone erosion were assessed using H&E staining, while cartilage damage and proteoglycan loss were assessed using Safranin-O fast green staining.

[0034] from Figure 3 and Figure 4It can be seen that the geranium seedling extract containing ellagic acid, geraniol, and corilagin significantly reduced toe swelling, inflammatory cell infiltration, bone erosion area, proteoglycan loss, and cartilage damage in mice with rheumatoid arthritis. In summary, the geranium seedling extract has a significant ameliorative effect on rheumatoid arthritis.

Claims

1. A method for preparing a geranium seedling extract, characterized in that: The specific steps are as follows: In the first step, the raw material of the Geranium truncatum plant is crushed to obtain a Geranium truncatum dry powder; the dry powder is mixed with anhydrous ethanol at a material-liquid ratio of 1 (g): 8 (mL), ultrasonically shaken at room temperature for 24 hours, and then vacuum filtered to obtain a primary ethanol extract of the Geranium truncatum. The filtration residue is repeated twice according to the above method, and the resultant mixture is combined to obtain a primary ethanol extract of the Geranium truncatum; The second step is to concentrate the primary ethanol extract of the Geranium chinense seedlings by vacuum distillation using a rotary evaporator to obtain the primary ethanol extract of the Geranium chinense seedlings; The third step is to extract the obtained primary ethanol extract of the geranium seedlings twice with ethyl acetate and water at a volume ratio of 1:1, combine the ethyl acetate phases, and concentrate them by vacuum distillation to obtain the ethyl acetate extract of the geranium seedlings; In the fourth step, the ethyl acetate extract of the above-mentioned geranium seedling is separated by silica gel column chromatography and gradient eluted with a methanol-ethyl acetate solvent system to obtain 12 components, which are Fr.1-Fr.12 in elution order, among which Fr.5 is a highly anti-inflammatory active component containing ellagic acid, geraniol and corilagin, which is the geranium seedling extract.

2. The geranium seedling extract prepared by the preparation method according to claim 1.

3. Use of the Geranium chinense seedling extract according to claim 2 in the preparation of anti-rheumatoid arthritis drugs.

4. The use of the Geraniol extract according to claim 3 in the preparation of an anti-rheumatoid arthritis drug, characterized in that: It can significantly alleviate rheumatoid arthritis at a dose of 750 mg / kg.

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