Method for extracting kaempferol-3-o-rutinoside from tetrastigma hemsleyanum and application of kaempferol-3-o-rutinoside

Abstract

The application relates to the field of extraction of Radix Ternatae, and discloses a method for extracting kaempferol-3-O-rutinoside from Radix Ternatae, which comprises the following steps: (1) mixing the aboveground part and / or the underground part of Radix Ternatae and an ethanol-water solution to carry out reflux purification, and collecting the reflux extract; (2) sequentially extracting the reflux extract with n-hexane, ethyl acetate, chloroform and n-butanol, and obtaining n-hexane extract, ethyl acetate extract, chloroform extract and n-butanol extract after removing the solvents; and (3) separating the n-butanol extract through silica gel column chromatography and high-performance preparative liquid chromatography to obtain kaempferol-3-O-rutinoside. The method can effectively improve the extraction efficiency of kaempferol-3-O-rutinoside, and the obtained kaempferol-3-O-rutinoside has high purity.

Description

TECHNICAL FIELD

[0001] The present application relates to the extraction of Tetrastigma hemsleyanum, and in particular to a method for extracting kaempferol-3-O-rutinoside from Tetrastigma hemsleyanum. BACKGROUND

[0002] Fever is the most common symptom in clinic, and is an important clinical manifestation in the process of disease progression, which can be seen in a variety of infectious diseases and non-infectious diseases. When the body is under the action of heat source or the dysfunction of body temperature center, the heat production process increases, while the heat dissipation cannot be increased accordingly or the heat dissipation decreases, thereby causing abnormal increase of body temperature, which is called fever. For the human body, the body temperature greater than 37.3℃ is fever. After fever, adults can actively cooperate with doctors to treat and thus reduce the fever symptoms relatively quickly; for young children, since the young children have poor resistance and most cases cannot accurately inform the doctors of their symptoms to obtain accurate treatment, the fever course may be prolonged. Every year, a certain number of young children cannot obtain timely and effective treatment due to fever, thereby leading to aggravation of the disease, prolongation of the course of disease, and even causing more serious sequelae. Long-term fever can cause problems in the immune ability of the child's body, thereby increasing the probability of suffering from meningitis, otitis media, conjunctivitis and other inflammations, and also increasing the risk of viral infection. Moreover, the child may also have convulsions and dehydration, and severe cases may have brain edema and heart failure. In recent years, due to the development of medical technology, a variety of drugs for fever reduction have been developed, most of which are chemical drugs. For young children, many chemical drugs are prohibited or restricted for use, and many drugs approved for fever reduction in young children also have certain side effects, such as antibiotic drugs that can cause drug resistance, acetaminophen that can cause liver damage, ibuprofen that can cause reversible kidney damage, and severe cases that can cause central nervous system inhibition and epilepsy. Therefore, there is an urgent need to develop new drugs for fever symptoms, with small side effects, and capable of efficiently and quickly reducing the body temperature of fever patients, thereby providing help for the treatment of fever patients.

[0003] *Tetrastigma hemsleyanum* is a valuable understory medicinal herb, a perennial, evergreen herbaceous vine belonging to the genus *Tetrastigma* of the Vitaceae family. It is mainly distributed in Zhejiang, Hunan, Jiangxi, Fujian, Hubei, Guangdong, Guangxi, and Sichuan provinces. Due to different production areas, its shape and medicinal properties may vary slightly. In terms of medicinal properties, the tuberous form of *Tetrastigma hemsleyanum* from Zhejiang is considered superior among various types of *Tetrastigma hemsleyanum*. The new list of "Eight Zhejiang Herbs" includes eight medicinal herbs: *Dendrobium officinale*, *Citrus aurantium*, *Lindera strychnifolia*, *Tetrastigma hemsleyanum*, *Rubus idaeus*, *Peucedanum praeruptorum*, *Ganoderma lucidum*, and saffron. The tubers of *Tetrastigma hemsleyanum* from Zhejiang are mostly oval, relatively small, generally 1.5-3 cm long and 0.7-1.5 cm in diameter; the tubers of *Tetrastigma hemsleyanum* from Guangxi are round, oval, or irregular in shape, larger, generally 2.0-4.5 cm long and 1.0-2.5 cm in diameter. Trifoliate orange (San Ye Qing) is widely used in folk medicine for its effects of clearing heat and detoxifying, promoting blood circulation and resolving stagnation, reducing inflammation and relieving pain, dispelling wind and resolving phlegm, and regulating qi and strengthening the spleen. Modern pharmacology shows that extracts of Trifoliate orange can play a role in anti-tumor, anti-inflammatory, antipyretic, analgesic, hepatoprotective, antiviral, and in diabetes and obstructive pulmonary diseases. In folk remedies, Trifoliate orange is often used as a highly effective antipyretic for children due to its excellent antipyretic effect and lack of obvious toxic side effects, which provides new clues for the development of new, safe, and efficient heat-clearing drugs.

[0004] In terms of industrialization, patent number 2005100506495 discloses a traditional Chinese medicine preparation using *Tripterygium wilfordii* as the main raw material, which has been mass-produced. Other products include "Tripterygium wilfordii capsules," "Tripterygium wilfordii granules," and "Tripterygium wilfordii powder." Existing technologies also disclose "Jin Si Di Jia capsules," a drug developed using *Tripterygium wilfordii* tuberous roots as raw material for anti-mutation and treatment of immune diseases. It has a significant inhibitory effect on the growth of Lewis lung tumor cells, SMMC-7721 liver tumor cells, SCG-7901 gastric tumor cells, HCT-116 colon tumor cells, and HL-60 human lymphoblastic leukemia cells. In clinical use, *Tripterygium wilfordii* has been included in the list of traditional Chinese medicines eligible for public reimbursement in Zhejiang Province. Products such as "Jie Shi Kang capsules," "Hua Lun Feng Tong Bao capsules," and "Pai Shi Li Dan capsules" are all developed and manufactured using the tuberous roots or vines of *Tripterygium wilfordii* as raw materials.

[0005] As a traditional Chinese herbal medicine, the active ingredients and their corresponding biological functions of *Tripterygium wilfordii* remain unclear to this day. Traditional methods of administration, such as decoction, yield a decoction containing the water-soluble components of *Tripterygium wilfordii*. While this decoction exhibits significant heat-clearing activity, it has several drawbacks, including the inability to standardize production, the potential for allergic reactions due to unclear components, and the possibility of natural drug interactions between certain active substances, which could weaken the therapeutic effect. Furthermore, current extraction methods for *Tripterygium wilfordii* have low extraction efficiency for kaempferol-3-O-rutin, resulting in the waste of some of this glycoside. Summary of the Invention

[0006] The purpose of this invention is to overcome the problem of low extraction efficiency of kaempferol-3-O-rutin from *Trifolium repens* in the prior art, and to provide a method for extracting kaempferol-3-O-rutin from *Trifolium repens*. This method can effectively improve the extraction efficiency of kaempferol-3-O-rutin, and the obtained kaempferol-3-O-rutin has high purity.

[0007] To achieve the above objectives, a first aspect of the present invention provides a method for extracting kaempferol-3-O-rutin from *Trifolium repens*, comprising the following steps:

[0008] (1) Mix the above-ground and / or underground parts of Trifolium repens with an ethanol-water solution and reflux for purification, and collect the reflux extract;

[0009] (2) The reflux extract was extracted sequentially with n-hexane, ethyl acetate, chloroform and n-butanol, and after removing the solvent, n-hexane extract, ethyl acetate extract, chloroform extract and n-butanol extract were obtained.

[0010] (3) The n-butanol extract was separated by silica gel column chromatography and high performance preparative liquid chromatography to obtain kaempferol-3-O-rutin.

[0011] Preferably, in step (1), the amount of the above-ground and / or underground parts of *Trifolium repens* on a dry weight basis, equivalent to 1 g, is 2-6 mL of the ethanol-water solution used at one time.

[0012] Preferably, the ethanol content in the ethanol-water solution is 50-90% (by volume).

[0013] Preferably, in step (1), the reflux purification conditions include a temperature of 80-100℃.

[0014] Preferably, in step (3), the silica gel column separation includes: loading the n-butanol extract onto a silica gel column, eluting it with an ethanol-water mixture gradient, and removing the solvent to obtain fraction I and fraction II.

[0015] Preferably, the high-efficiency preparative liquid chromatography separation includes: passing fraction II through a reverse C-phase flowmeter. 18 High-efficiency preparative liquid chromatography separation, the reverse C 18 The eluent for efficient preparative liquid chromatography separation is an acetonitrile-water mixture.

[0016] More preferably, the reverse C 18 High-efficiency preparative liquid chromatography separation of reverse C 18 The particle size is 5-15 μm.

[0017] Preferably, the acetonitrile-water mixture comprises a first acetonitrile-water eluent, a second acetonitrile-water eluent, and a third acetonitrile-water eluent with gradually increasing acetonitrile content. The acetonitrile content in the first acetonitrile-water eluent is 5-15% (volume), the acetonitrile content in the second acetonitrile-water eluent is 15-85% (volume), and the acetonitrile content in the third acetonitrile-water eluent is 85-95% (volume).

[0018] More preferably, the elution time of the first acetonitrile-water eluent is 5-10 min, the elution time of the second acetonitrile-water eluent is 10-40 min, and the elution time of the third acetonitrile-water eluent is 40-50 min.

[0019] Preferably, in step (3), the silica gel column chromatography separation uses 200-300 mesh silica gel.

[0020] More preferably, the mass ratio of the silica gel to the n-butanol extract is 30-100:1, more preferably 50-80:1.

[0021] Preferably, the silica gel column used in the silica gel column chromatography separation has a height-to-diameter ratio of 10-16:1.

[0022] The second aspect of this invention provides the application of kaempferol-3-O-rutin obtained by the above-described method in the preparation of heat-clearing drugs.

[0023] Through the above technical solution, the method of the present invention purifies the above-ground and / or underground parts of *Trifolium repens* by reflux with an ethanol-water solution, followed by sequential extraction with n-hexane, ethyl acetate, chloroform, and n-butanol. The n-butanol extract is then separated and purified by silica gel column chromatography, which can improve the yield of kaempferol-3-O-rutinoside. Moreover, the above method is simple and convenient, and the kaempferol-3-O-rutinoside obtained by this method has high purity, reducing the possibility of allergic reactions during use. Attached Figure Description

[0024] Figure 1 This is the FTIR spectrum of component II-6 extracted in Example 1;

[0025] Figure 2 It is component II-6 extracted in Example 1. 1 H-NMR spectrum;

[0026] Figure 3 This is the NMR shift map of H atoms in component II-6 extracted in Example 1;

[0027] Figure 4 It is component II-6 extracted in Example 1. 13 C-NMR spectrum;

[0028] Figure 5 This is the NMR shift map of component II-6 extracted in Example 1;

[0029] Figure 6 The effects of 2,4-dinitrophenol and bacterial lipopolysaccharide (LPS) on body temperature in mice;

[0030] Figure 7 It is the heat-clearing effect of the reflux extract of *Trifolium repens* on a mouse fever model;

[0031] Figure 8 The product obtained by extracting the reflux extract of Tripterygium wilfordii with different solvents has a heat-clearing effect on a mouse fever model. Among them, the control group is the LPS model group, G-EA is the ethyl acetate extract of Tripterygium wilfordii root, G-BU is the n-butanol extract of Tripterygium wilfordii root, and G-HX is the n-hexane extract of Tripterygium wilfordii root.

[0032] Figure 9 The effects of different concentrations of kaempferol-3-O-rutin on the heat-clearing effect in a mouse fever model;

[0033] Figure 10 This is a comparison of the heat-clearing activities of kaempferol-3-O-rutin with ibuprofen and acetaminophen. Detailed Implementation

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] As mentioned above, the first aspect of the present invention provides a method for extracting kaempferol-3-O-rutin from *Trifolium repens*, comprising the following steps:

[0036] (1) Mix the above-ground and / or underground parts of Trifolium repens with an ethanol-water solution and reflux for purification, and collect the reflux extract;

[0037] (2) The reflux extract was extracted sequentially with n-hexane, ethyl acetate, chloroform and n-butanol, and after removing the solvent, n-hexane extract, ethyl acetate extract, chloroform extract and n-butanol extract were obtained;

[0038] (3) The n-butanol extract was separated by silica gel column chromatography and high performance preparative liquid chromatography to obtain kaempferol-3-O-rutin.

[0039] During the research process, the inventors discovered that the method of the present invention, which involves reflux purification of the above-ground and / or underground parts of *Trifolium repens* using an ethanol-water solution, followed by sequential extraction with n-hexane, ethyl acetate, chloroform, and n-butanol, and separation of the n-butanol extract by silica gel column chromatography and high-performance preparative liquid chromatography, can improve the yield of kaempferol-3-O-rutinoside. Moreover, the kaempferol-3-O-rutinoside obtained by the above method has a high purity.

[0040] According to the present invention, the above-ground and / or underground parts of *Tripterygium wilfordii* can be dried above-ground and / or underground parts, or they can be undried above-ground and / or underground parts. In this invention, the above-ground and / or underground parts can be directly immersed in an ethanol-water solution for reflux purification, or they can be cut into pieces and then immersed in an ethanol-water solution for reflux purification, or they can be crushed into powder and then immersed in an ethanol-water solution for reflux purification. Preferably, the above-ground and / or underground parts are first crushed into powder and then immersed in an ethanol-water solution for reflux purification.

[0041] According to the present invention, in step (2), the solvent removal method can be a solvent removal method commonly used in the prior art, and the conditions can be determined by the test personnel according to the actual situation.

[0042] The amount of the ethanol-water solution can be determined by those skilled in the art based on the actual situation, as long as the *Trifolium repens* is completely immersed in the ethanol-water solution. In order to further improve the yield of kaempferol-3-O-rutin, preferably, in step (1), the single amount of the ethanol-water solution used is 2-6 mL, which is equivalent to 1 g of the above-ground and / or underground parts of *Trifolium repens* on a dry weight basis.

[0043] To further improve the yield of kaempferol-3-O-rutin, preferably, the ethanol content in the ethanol-water solution is 50-90% (by volume).

[0044] Preferably, in step (1), the reflux purification conditions include a temperature of 80-100°C. The inventors discovered during their research that reflux purification under the above conditions can further improve the yield of kaempferol-3-O-rutin.

[0045] Preferably, in step (2), the amount of n-hexane used is 0.5-1.5 mL, the amount of ethyl acetate used is 0.5-1.5 mL, the amount of chloroform used is 0.5-1.5 mL, and the amount of n-butanol used is 0.5-1.5 mL, which is equivalent to 1 mL of reflux extract.

[0046] In order to further improve the yield of kaempferol-3-O-rutin, preferably, in step (3), the silica gel column chromatography separation includes: loading the n-butanol extract onto a silica gel column, eluting with an ethanol-water mixture gradient, and removing the solvent to obtain fraction I and fraction II.

[0047] In the gradient elution, the volume ratio of ethanol to water in the ethanol-water mixture is 7:3-9:1.

[0048] Preferably, the volume of each fraction is 1 / 20 to 1 / 40 of the volume retained by the chromatography column, and more preferably 1 / 30.

[0049] Preferably, the high-efficiency preparative liquid chromatography separation includes: passing fraction II through a reverse C2... 18 High-efficiency preparative liquid chromatography separation, the reverse C 18 The eluent for efficient preparative liquid chromatography separation is an acetonitrile-water mixture, which can effectively improve the purity of the extracted kaempferol-3-O-rutin.

[0050] The reverse C 18 High-efficiency preparative liquid chromatography separation of reverse C 18 The particle size can be determined according to the actual situation. To further improve the yield of kaempferol-3-O-rutin, preferably, the reverse C... 18 High-efficiency preparative liquid chromatography separation of reverse C 18 The particle size is 5-15 μm.

[0051] Preferably, the acetonitrile-water mixture comprises a first acetonitrile-water eluent, a second acetonitrile-water eluent, and a third acetonitrile-water eluent with gradually increasing acetonitrile content. The acetonitrile content in the first acetonitrile-water eluent is 5-15% (volume), the acetonitrile content in the second acetonitrile-water eluent is 15-85% (volume), and the acetonitrile content in the third acetonitrile-water eluent is 85-95% (volume). Using the acetonitrile-water mixture with the above-mentioned acetonitrile content for sequential elution can further improve the yield and purity of kaempferol-3-O-rutin.

[0052] In order to further improve the yield and purity of kaempferol-3-O-rutin, preferably, the elution time of the first acetonitrile-water eluent is 5-10 min, the elution time of the second acetonitrile-water eluent is 10-40 min, and the elution time of the third acetonitrile-water eluent is 40-50 min.

[0053] To further improve the separation effect of kaempferol-3-O-rutin during the extraction process, thereby increasing its yield and purity, preferably, in step (3), the silica gel column chromatography separation uses 200-300 mesh silica gel. Considering further improving the separation effect of kaempferol-3-O-rutin during the extraction process, thereby increasing its yield and purity, it is further preferred that the mass ratio of silica gel in the silica gel column to the n-butanol extract is 30-100:1, specifically 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or any value between the aforementioned two values. More preferably, the mass ratio of silica gel in the silica gel column to the n-butanol extract is 50-80:1, and more preferably 60-70:1.

[0054] Preferably, the silica gel column used in the silica gel column chromatography separation has a height-to-diameter ratio of 10-16:1. Here, the height-to-diameter ratio is the ratio of the height to the diameter of the silica gel column. Under the above-mentioned height-to-diameter ratio conditions, kaempferol-3-O-rutin in the extracted product exhibits a higher separation efficiency, thereby further improving the yield of kaempferol-3-O-rutin.

[0055] Preferably, the solvent removal method is rotary evaporation, and the conditions for rotary evaporation include: a temperature of 20-40°C and a pressure of -0.08 to -0.1 MPa.

[0056] The second invention provides the application of kaempferol-3-O-rutin obtained by the above method in the preparation of heat-clearing drugs.

[0057] The kaempferol-3-O-rutin extracted by this method has high purity and high heat-clearing activity, reducing the possibility of allergic reactions during use.

[0058] According to a particularly preferred embodiment of the present invention, a method for extracting kaempferol-3-O-rutin from *Trifolium repens* is provided, comprising the following steps:

[0059] (1) Crush *Trifolium repens* into powder, then mix the *Trifolium repens* powder with an ethanol-water solution containing 90-98% (volume) % ethanol, and extract at 95-105℃ for 0.5-2 hours, repeating 2-4 times. Then extract with an ethanol-water solution containing 40-60% (volume) % ethanol at 95-105℃ for 0.5-2 hours, repeating 2-3 times. Combine the extracts, remove the solvent, and obtain the extract concentrate; equivalent to 1g of *Trifolium repens* on a dry weight basis, the single dosage of the ethanol-water solution containing 90-98% (volume) % ethanol is 2-6mL, and the single dosage of the ethanol-water solution containing 40-60% (volume) % ethanol is 2-6mL.

[0060] (2) The reflux extract was extracted sequentially with n-hexane, ethyl acetate, chloroform and n-butanol, and after removing the solvent, n-hexane extract, ethyl acetate extract, chloroform extract and n-butanol extract were obtained;

[0061] (3) The n-butanol extract is loaded onto a silica gel column (200-300 mesh silica gel, mass ratio of silica gel to n-butanol extract phase is 60-70:1, height-to-diameter ratio of silica gel column is 10-16:1), and eluted with a gradient of ethanol-water mixture (volume ratio of ethanol to water is 2-9:1). The solvent is then removed by rotary evaporation at a temperature of 20-40℃ and a pressure of -0.08 to -0.1 MPa to obtain fraction I and fraction II. Fraction II is then subjected to reverse C... 18 High-performance preparative liquid chromatography (HPLC) was used for separation, and the mixture was eluted with acetonitrile-water mixture I (5-15% by volume) for 5-10 min, acetonitrile-water mixture II (15-85% by volume) for 10-40 min, and acetonitrile-water mixture III (85-95% by volume) for 40-50 min to obtain fractions II-1 to II-10, wherein fraction II-6 is kaempferol-3-O-rutin.

[0062] The above extraction method can effectively improve the yield of kaempferol-3-O-rutin, and the extracted kaempferol-3-O-rutin has high purity.

[0063] The present invention will be described in detail below through embodiments.

[0064] In the following examples, the chemical shift parameter of kaempferol-3-O-rutinoside was determined by NMR (Bruker AVANCE III HD 400); the group infrared absorption information parameter of kaempferol-3-O-rutinoside was determined by infrared absorption spectroscopy (Thermoelectric Nicolet 5700). The *Tripterygium wilfordii* used in this invention was obtained from Taizhou, dried at 60°C, powdered, and stored at room temperature in the dark. All other reagents were commercially available.

[0065] Example 1

[0066] (1) Crush the three leaves into powder, then mix 50g of three leaves powder with 200mL of ethanol-water solution with a content of 95% (volume) and extract for 1h at 100℃. Repeat 3 times. Then use 200mL of ethanol-water solution with a content of 50% (volume) and extract for 1h at 100℃. Repeat 3 times. Combine the extracts, remove the solvent and obtain 200mL of extract concentrate.

[0067] (2) The extract was extracted sequentially with 200 mL of n-hexane, 200 mL of ethyl acetate, 200 mL of chloroform and 200 mL of n-butanol to obtain 200 mL of n-hexane extract, 200 mL of ethyl acetate extract, 200 mL of chloroform extract and 200 mL of n-butanol extract.

[0068] (3) After desolventizing the n-butanol extract, it was reconstituted with ethanol and added to a silica gel column (200-300 mesh silica gel, mass ratio of silica gel to n-butanol extract 65:1, height-to-diameter ratio of silica gel column 12:1) for column chromatography separation. After gradient elution with an ethanol-water mixture (volume ratio of ethanol to water 7:3-9:1), it was rotary evaporated for 1.5 h at 30℃ and -0.09 MPa to obtain 0.52 g of light yellow paste I and 0.81 g of light yellow paste II. Light yellow paste II was then subjected to reverse C2 chromatography. 18 (Particle size 10 μm) High-performance preparative liquid chromatography separation was performed by elution with acetonitrile-water mixture I (10% by volume) for 10 min, acetonitrile-water mixture II (50% by volume) for 30 min, and acetonitrile-water mixture III (90% by volume) for 45 min to obtain components II-1 to II-10, wherein component II-6 is kaempferol-3-O-rutin.

[0069] Component II-6 was detected by infrared spectroscopy, proton NMR, and carbon NMR, and the resulting spectra are shown below. Figures 1-5 As shown, the main functional group present is the =CH stretching vibration (3012 cm⁻¹). -1 Deformational vibrations of —CH2 and —CH3 (2963, 2932 and 2874 cm⁻¹) -1 C=O stretching vibration (1708cm) -1 ), —C=C—stretching vibration (1655cm) -1 ) and benzene ring structure (1608-1452cm) -1 Based on these data analysis, II-6 may be a polyphenol or polyol with an aromatic structure. Further analysis... 1 H-NMR (DMSO-d6) and 13 According to C-NMR results, component II-6 is presumed to be kaempferol-3-O-rutin.

[0070] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0071] Example 2

[0072] (1) Gynostemma pentaphyllum was crushed into powder, and then 25g of Gynostemma pentaphyllum powder was mixed with 200mL of ethanol-water solution with a content of 95% (volume) and extracted for 1h at 100℃. This was repeated 3 times. Then, 200mL of ethanol-water solution with a content of 50% (volume) was used to extract for 1h at 100℃. This was repeated 3 times. The extracts were combined and desolventized to obtain 150mL of extract concentrate.

[0073] (2) The extract was extracted sequentially with 100 mL of n-hexane, 100 mL of ethyl acetate, 100 mL of chloroform and 100 mL of n-butanol to obtain 100 mL of n-hexane extract, 100 mL of ethyl acetate extract, 100 mL of chloroform extract and 100 mL of n-butanol extract.

[0074] (3) After desolventizing the n-butanol extract, it was reconstituted with ethanol and added to a silica gel column (200-300 mesh silica gel, mass ratio of silica gel to n-butanol extract 60:1, height-to-diameter ratio of silica gel column 10:1) for column chromatography separation. After gradient elution with an ethanol-water mixture (volume ratio of ethanol to water 7:3-9:1), it was rotary evaporated for 1.5 h at 25℃ and -0.1 MPa to obtain 0.30 g of light yellow paste I and 0.42 g of light yellow paste II. Light yellow paste II was then subjected to reverse C2 chromatography. 18 (Particle size 15 μm) was separated by high efficiency preparative liquid chromatography, and eluted with acetonitrile-water mixture I (15% by volume) for 5 min, acetonitrile-water mixture II (85% by volume) for 30 min, and acetonitrile-water mixture III (95% by volume) for 40 min to obtain components II-1 to II-10, wherein component II-6 is kaempferol-3-O-rutin.

[0075] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0076] Example 3

[0077] (1) Gynostemma pentaphyllum was crushed into powder, and then 50g of Gynostemma pentaphyllum powder was mixed with 200mL of ethanol-water solution with a content of 95% (volume) and extracted for 1h at 100℃. This was repeated 3 times. Then, 200mL of ethanol-water solution with a content of 50% (volume) was used to extract for 1h at 100℃. This was repeated 3 times. The extracts were combined and desolventized to obtain 190mL of extract concentrate.

[0078] (2) The extract was extracted sequentially with 200 mL of n-hexane, 200 mL of ethyl acetate, 200 mL of chloroform and 200 mL of n-butanol to obtain 200 mL of n-hexane extract, 200 mL of ethyl acetate extract, 200 mL of chloroform extract and 200 mL of n-butanol extract.

[0079] (3) After desolventizing the n-butanol extract phase, it was reconstituted with ethanol and added to a silica gel column (200-300 mesh silica gel, mass ratio of silica gel to n-butanol extract phase of 70:1, height-to-diameter ratio of silica gel column of 16:1) for column chromatography separation. After gradient elution with ethanol-water mixture (volume ratio of ethanol to water of 7:3-9:1), it was rotary evaporated at 20℃ and -0.1 MPa for 1.5 h to obtain 0.45 g of light yellow paste I and 0.75 g of light yellow paste II. The light yellow paste II was subjected to reverse C-phase chromatography. 18 (Particle size 5 μm) was separated by high efficiency preparative liquid chromatography, and then eluted with acetonitrile-water mixture I (5% by volume) for 15 min, acetonitrile-water mixture II (15% by volume) for 40 min, and acetonitrile-water mixture III (85% by volume) for 50 min to obtain components II-1 to II-10, wherein component II-6 is kaempferol-3-O-rutin.

[0080] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0081] Example 4

[0082] Kaempferol-3-O-rutin was extracted according to the method of Example 2, except that the mass ratio of silica gel in the silica gel column to the n-butanol extract was 50:1.

[0083] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0084] Example 5

[0085] Kaempferol-3-O-rutin was extracted according to the method of Example 3, except that the mass ratio of silica gel in the silica gel column to the n-butanol extract was 70:1.

[0086] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0087] Example 6

[0088] Kaempferol-3-O-rutin was extracted according to the method of Example 2, except that the mass ratio of silica gel in the silica gel column to the n-butanol extract was 30:1.

[0089] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0090] Example 7

[0091] Kaempferol-3-O-rutin was extracted according to the method of Example 3, except that the mass ratio of silica gel in the silica gel column to the n-butanol extract was 100:1.

[0092] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0093] Comparative Example 1

[0094] Kaempferol-3-O-rutin was extracted according to the method of Example 1, except that step (1) included: crushing Tripterygium wilfordii into powder, then mixing 50g of Tripterygium wilfordii powder with 200mL of ethanol and soaking for 10h to obtain 190mL of extract.

[0095] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0096] Comparative Example 2

[0097] Kaempferol-3-O-rutin was extracted according to the method of Example 1, except that step (2) included: extracting the extract sequentially with 200 mL of n-butanol, 200 mL of chloroform, 200 mL of n-hexane and 200 mL of ethyl acetate to obtain 200 mL of n-hexane extract phase, 200 mL of ethyl acetate extract phase, 200 mL of chloroform extract phase and 200 mL of n-butanol extract phase.

[0098] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0099] Comparative Example 3

[0100] Kaempferol-3-O-rutin was extracted according to the method of Example 1, except that step (2) included: extracting the extract sequentially with 200 mL petroleum ether, 200 mL ethyl acetate, 200 mL dichloromethane and 200 mL isopropanol to obtain 190 mL isopropanol extract phase.

[0101] The yield of kaempferol-3-O-rutin was calculated and is shown in Table 1. The purity of the obtained kaempferol-3-O-rutin was detected by HPLC and the results are shown in Table 1.

[0102] Test Example 1

[0103] Mice with an initial rectal temperature of 37±0.3℃ were selected for this study. After modeling with 30 mg / kg 2,4-dinitrophenol and 20 μg / kg LPS, respectively, the mice's body temperature was measured every 15 minutes until it dropped to approximately 37.0℃. A curve showing the change in body temperature was plotted. The results are as follows: Figure 6 As shown. From Figure 6 It can be seen that the fever model constructed with 20 μg / kg LPS has a long duration, while the fever model constructed with 2,4-dinitrophenol has a short duration. Therefore, 20 μg / kg LPS was used as the modeling drug in subsequent experiments.

[0104] Meanwhile, mice meeting the above conditions were selected and divided into two groups. One group served as the experimental group (administered via gavage the extract from step (1) of Example 1, at a dosage of 200 mg / kg, with physiological saline as the solvent), and the other group served as the control group (administered only physiological saline, with the same volume of physiological saline as the experimental group). The body temperature of the mice was measured every 15 minutes until the body temperature dropped to about 37.0℃. The change curve of the mouse body temperature was plotted, and the results are as follows. Figure 7 As shown. By Figure 7 It was found that, compared with the control group, the mice treated with 200 mg / kg of the extract by gavage had a faster rate of temperature reduction, indicating that the extract of *Trifolium repens* has a certain heat-clearing effect on fever in mice.

[0105] Test Example 2

[0106] The n-butanol extract, ethyl acetate extract, chloroform extract, and n-butanol extract obtained in step (2) of Example 1 were rotary evaporated at 30°C and -0.09 MPa for 1.5 h to obtain n-butanol extract, ethyl acetate extract, chloroform extract, and n-butanol extract. The four different extracts were administered to a mouse fever model via gavage at a dose of 0.2 g / kg, and the resulting temperature change curves are shown below. Figure 8 As shown. By Figure 8It was found that the three different polarity extracts obtained from the root of *Trifolium repens* all showed different degrees of heat-clearing activity when applied to a mouse fever model. However, the n-butanol extract showed the best heat-clearing activity, while the chloroform extract showed less activity and was not tested in animal experiments.

[0107] Test Example 3

[0108] The kaempferol-3-O-rutin extracted in Example 1 was administered to febrile mice by gavage at concentrations of 1, 2, 4, and 6 μmol / L (mouse selection, fever modeling, and control group procedures were consistent with those in Test Examples 1 and 2). The results are as follows: Figure 9 As shown, the rate at which the body temperature of mice decreased increased with increasing concentration of kaempferol-3-O-rutin.

[0109] Test Example 4

[0110] A mouse fever model was treated with 4 μmol / L kaempferol-3-O-rutin, ibuprofen, and acetaminophen, respectively, and the results were as follows: Figure 10 As shown, the results indicate that kaempferol-3-O-rutin can rapidly reduce the body temperature of mice to normal levels, while ibuprofen and acetaminophen require a longer time to exert their heat-clearing activity.

[0111] Table 1

[0112]

[0113]

[0114] As can be seen from the results in Table 1, the yield of kaempferol-3-O-rutin using Examples 1-7 of the present invention is significantly higher than that of the comparative example, indicating that the method provided by the present invention can effectively improve the yield of kaempferol-3-O-rutin.

[0115] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for extracting kaempferol-3-O-rutin from *Trifolium repens*, characterized in that, Includes the following steps: (1) Mix the above-ground and / or underground parts of *Trifolium repens* with an ethanol-water solution and reflux for purification, and collect the reflux extract; the reflux purification conditions include: a temperature of 80-100℃; (2) The reflux extract was extracted sequentially with n-hexane, ethyl acetate, chloroform and n-butanol, and after removing the solvent, n-hexane extract, ethyl acetate extract, chloroform extract and n-butanol extract were obtained; (3) The n-butanol extract was loaded onto a silica gel column, eluted with an ethanol-water mixture gradient, and the solvent was removed to obtain fraction I and fraction II. Fraction II was then subjected to reverse C-type chromatography. 18 High-efficiency preparative liquid chromatography (HPLC) separation yielded kaempferol-3-O-rutin, the reverse C... 18 The elution phase for efficient preparative liquid chromatography separation is an acetonitrile-water mixture.

2. The method according to claim 1, characterized in that, In step (1), the amount of the above-ground and / or underground parts of *Trifolium repens* on a dry weight basis is equivalent to 1 g, and the single dose of the ethanol-water solution is 2-6 mL.

3. The method according to claim 1, characterized in that, In step (1), the ethanol content in the ethanol-water solution is 50-90%, and the content is by volume.

4. The method according to any one of claims 1 to 3, characterized in that, The reverse C 18 High-efficiency preparative liquid chromatography separation of reverse C 18 The particle size is 5-15 μm.

5. The method according to any one of claims 1 to 3, characterized in that, The acetonitrile-water mixture includes a first acetonitrile-water eluent, a second acetonitrile-water eluent, and a third acetonitrile-water eluent with gradually increasing acetonitrile content. The acetonitrile content in the first acetonitrile-water eluent is 5-15%, the acetonitrile content in the second acetonitrile-water eluent is 15-85%, and the acetonitrile content in the third acetonitrile-water eluent is 85-95%. The content is by volume.

6. The method according to claim 5, characterized in that, The elution time of the first acetonitrile-water eluent is 5-10 min, the elution time of the second acetonitrile-water eluent is 10-40 min, and the elution time of the third acetonitrile-water eluent is 40-50 min.

7. The method according to any one of claims 1 to 3, characterized in that, In step (3), the silica gel column chromatography separation uses 200-300 mesh silica gel.

8. The method according to claim 7, characterized in that, The mass ratio of the silica gel to the n-butanol extract is 30-100:

1.

9. The method according to claim 8, characterized in that, The mass ratio of the silica gel to the n-butanol extract is 50-80:

1.

10. The method according to claim 7, characterized in that, The silica gel column used in the silica gel column chromatography separation has a height-to-diameter ratio of 10-16:1.

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