A glucuronated triterpene saponin and application thereof

Abstract

The present application relates to a glucuronic acid triterpene saponin and its use in the preparation of analgesic drugs. The glucuronic acid triterpene saponin of the present application can be a naturally occurring organic compound or a synthetic organic compound, has strong analgesic activity, and is used as an active ingredient to prepare an analgesic drug composition with a pharmaceutically acceptable carrier or excipient.

Description

Technical Field

[0001] This invention relates to a glucuronic acid triterpenoid saponin, and more particularly to its use in the preparation of analgesic drugs. Background Technology

[0002] Pain is an unpleasant subjective feeling and emotional experience caused by noxious or potentially noxious stimuli, either external or internal. It has been recognized by modern medicine as the fifth vital sign after respiration, pulse, blood pressure, and body temperature. In clinical practice, pain is the most common chief complaint among patients, accounting for over 80% of medical visits. Pain causes significant harm and negative impacts on individuals and is a common and direct factor in reducing work capacity and working days. Therefore, pain relief has become one of the most important goals of clinical medicine.

[0003] Currently, among the many medications used to treat pain, nonsteroidal anti-inflammatory drugs (NSAIDs) and narcotic analgesics are the first-line drugs for treating or relieving various types of pain. These include anti-inflammatory drugs such as aspirin, indomethacin, ibuprofen, and diclofenac, as well as central analgesics such as morphine, tramadol, and pethidine. Compared to narcotic analgesics, NSAIDs are more widely used. Since the advent of aspirin, over a hundred NSAIDs have been developed in the following century, including acetylsalicylic acid derivatives, pyrazolone derivatives, acetic acid derivatives, diclofenac, oxacin derivatives, and acetanilide derivatives. They are widely used to treat diseases with pain as the main symptom, including neuralgia, headache, toothache, bone and joint pain, pain caused by injuries, stomachache, and biliary colic, with significant efficacy. They are among the most frequently used drugs globally. However, while exerting their anti-inflammatory and analgesic effects, these drugs also have serious adverse reactions, and repeated or prolonged use can harm the body. The main adverse reactions include gastrointestinal damage, upper abdominal pain, nausea, indigestion, esophagitis and colitis, hematologic damage, and liver and kidney damage. Opioids are currently the most potent analgesics discovered and can be used for various moderate to severe pain conditions. However, these drugs also have serious adverse reactions, such as respiratory depression, cough suppression, dose dependence, addiction, nausea, vomiting, miosis, and constipation. Therefore, exploring and finding novel analgesics with good efficacy and fewer adverse reactions from natural medicines is of great significance.

[0004] Currently, scholars both domestically and internationally have studied the analgesic active components of various medicinal plants, including cannabis, aconite, strychnos nux-vomica, sinomenium, woodlice, and corydalis. A number of compounds with excellent activity have been obtained, including lignans, alkaloids, diterpenes, triterpenes, steroids, and flavonoids. Although many compounds with analgesic effects have been discovered, few possess strong activity; most are comparable in potency to antipyretic analgesics. Analgesic drugs or drug combinations made from various medicinal plants are also available, but their analgesic effects are not ideal. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] The technical problem to be solved by the present invention is to address the issue that compounds in the prior art have poor analgesic effects.

[0007] (II) Technical Solution

[0008] In order to solve the above-mentioned problems in the prior art, the present invention provides a glucuronic acid triterpenoid saponin, a method for preparing glucuronic acid triterpenoid saponins, and their use in analgesia.

[0009] The purpose of this invention is to provide a glucuronic acid triterpenoid saponin having the following structural formula (I):

[0010]

[0011] The compound of formula (I) is named: 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethyl glucuronopyranosyl quillaic acid.

[0012] The present invention also provides a method for preparing a compound of formula (I), comprising the following steps:

[0013] (1) After drying and pulverizing the plant, extract it with a solvent and combine the extracts;

[0014] (2) The extract obtained from step (1) by vacuum concentration is used to obtain an extract;

[0015] (3) The extract obtained in step (2) is suspended in water and extracted with petroleum ether to remove the small polar fraction and retain the water fraction;

[0016] (4) The water fraction obtained in step (3) was sequentially separated by D-101 macroporous resin column, normal silica gel column, dextran gel column (Sephedex LH-20) and semi-preparative high performance liquid chromatography (HPLC) to obtain compound with structural formula (I).

[0017] According to the preparation method of the compound of formula (I) above, the characteristic is that in step (1), the plant of the genus *Gnaphalium* is extracted with 6-10 times its weight of solvent for 1-3 hours, and the extraction is repeated 1-3 times. The filtrates are combined to obtain the extract solution of *Gnaphalium* medicinal material.

[0018] According to the preparation method of the compound of formula (I) described above, the characteristic is that the plant of the genus *Gynostemma* in step (1) is *Gynostemma*.

[0019] According to the preparation method of the compound of formula (I) described above, the solvent in step (1) is 50-95% by volume ethanol / water, 50-95% by volume methanol / water, or 50-90% by volume acetone / water.

[0020] Use of the compound of formula (I) in the preparation of analgesic drugs, wherein the compound of formula (I) is prepared as an active ingredient with a pharmaceutically acceptable carrier or excipient to form an analgesic drug composition.

[0021] According to the uses described in the compound of formula (I) above, the analgesia is for the treatment of neuralgia, bone pain, muscle pain, pain caused by sprains and bruises, headache, stomachache, intestinal colic, biliary colic, renal colic and cancer pain.

[0022] According to the use described in the compound of formula (I) above, the pharmaceutical composition is selected from tablets, capsules, pills, injections, and hot spring bath preparations, and the pharmaceutical composition is selected from sustained-release preparations or controlled-release preparations.

[0023] According to the uses described for the compounds of formula (I) above, the pharmaceutically acceptable carriers or excipients include excipients for oral formulations, parenteral administration, or topical administration. The routes of administration can be oral, injection, topical application, or hot spring bath administration, etc.; the dosage form can be a liquid or solid dosage form. Liquid dosage forms can be syrups, injection solutions, non-aqueous solutions, suspensions, or emulsions, while solid dosage forms can be tablets, lozenges, capsules, pellets, pills, granules, powders, creams, solutions, suppositories, or dispersible powders such as lyophilized powder for injection. Agents, aerosols, etc.; excipients used include: lactose, calcium carbonate, calcium phosphate, sodium phosphate, starch, cyclodextrin, sucrose, mannitol, sodium microcrystalline cellulose, calcium sulfate, water, ethanol, propanol, glycerol, propylene glycol, isopropanol, syrup, honey, glucose, gelatin paste, sodium carboxymethyl cellulose, potassium phosphate, dried starch, agar powder, calcium carbonate, sodium bicarbonate, sodium dodecyl sulfonate, methylcellulose, glyceryl tristearate, cocoa butter, hydrogenated oil, quaternary ammonium salts, talc, magnesium triethylamine stearate, silicon dioxide, corn starch, stearate, boric acid, and liquid paraffin.

[0024] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0025] (III) Beneficial Effects

[0026] The above-described technical solution of the present invention has the following advantages:

[0027] (1) The compound of formula (I) of the present invention showed a good inhibitory effect on the acetic acid writhing response in mice, suggesting that it has good analgesic activity and can be used as an analgesic active ingredient or lead compound, with good application prospects.

[0028] (2) The plants of the genus *Gnaphalium* that can be used in the preparation method of the compound of formula (I) of the present invention, especially *Gnaphalium*, are already cultivated in China, with abundant resources and simple raw material sources; moreover, the compound of formula I in the present invention has a high content in plants of the genus *Gnaphalium*, especially *Gnaphalium*, and is easy to obtain.

[0029] (3) The preparation method of the compound of formula (I) of the present invention can be carried out by conventional column chromatography. The compound preparation process is simple, the obtained compound has high purity, and subsequent industrial production is easy to achieve. Attached Figure Description

[0030] Figure 1 This is a flowchart of the activity tracking and separation process for the compound of formula (I) described in this invention;

[0031] Figure 2 The structural formula of compound (I) described in this invention;

[0032] Figure 3 This invention relates to high-resolution mass spectrometry (HRESI-MS) of the compound of formula (I);

[0033] Figure 4 The proton nuclear magnetic resonance spectrum of the compound of formula (I) of this invention ( 1 H NMR);

[0034] Figure 5 The carbon NMR spectrum of the compound of formula (I) of this invention ( 13 C NMR and DEPT spectra;

[0035] Figure 6 This is the COSY correlation spectrum of the compound of formula (I) of this invention;

[0036] Figure 7 This is the HSQC correlation spectrum of the compound of formula (I) of this invention;

[0037] Figure 8 This is the HMBC correlation spectrum of the compound of formula (I) of this invention;

[0038] Figure 9 This is a diagram showing the inhibitory effect of the compound of formula (I) of this invention on the writhing response in mice. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The compound of formula (I) of this invention can be a naturally occurring compound or a synthetically produced compound.

[0041] The glucuronic acid triterpenoid saponin described in this invention is obtained from the root of *Syzygium spp.*, a plant belonging to the genus *Syzygium* in the Caryophyllaceae family, through extraction by extracting the extract, organic solvent extraction, D-101 macroporous resin column chromatography, silica gel column chromatography, Sepheedx LH-20 column chromatography, and HPLC separation. The molecular formula of this compound is C1. 49 H 76 O 20 Named 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylglucuronopyranosyl quillaic acid, it has the following structural formula:

[0042]

[0043] *Psammosilene tunicoides*, a plant belonging to the genus *Psammosilene* in the family Caryophyllaceae, is a monotypic species endemic to southwestern my country, mainly distributed in Yunnan, Guizhou, Sichuan, and other southwestern regions. It possesses properties of dispersing blood stasis, relieving pain, stopping bleeding, and eliminating carbuncles and pus. It is primarily used to treat injuries from falls, rheumatic pain, stomachache, carbuncles, boils, and traumatic bleeding. *Psammosilene tunicoides* has a long history as a medicinal plant, first recorded in the *Diannan Bencao* (Herbal Classic of Yunnan). According to this text, "*Psammosilene tunicoides* is pungent and spicy, very warm in nature, and slightly toxic; ingestion causes vomiting. It is specifically used to treat facial pain due to cold, stomach and heart pain, and to treat carbuncles and pus." This herb is one of the important ingredients in the traditional classic Yunnan Baiyao and is included in the 2010 edition of the *Chinese Pharmacopoeia*. The chemical components of *Psammosilene tunicoides* mainly include triterpenoids, triterpenoid saponins, cyclic peptides, carboline alkaloids, maltol glycosides, lignans, etc. (Tan JM, Shen YH, Yang XW. Antifungal cyclic peptides from *Psammosilene tunicoides*[J]. J. Nat. Prod. 2010, 73(12): 1987-1992; Mao Zeling, Shen Yunheng, Zhou Ligang. Research progress on chemical components and bioactivity of *Psammosilene tunicoides*. Chinese Journal of Traditional Chinese Medicine. 2016, 34(12): 2883-2886; Pu Xiangyu, Zhou Jun. Research on saponins of *Psammosilene tunicoides*[J]. Yunnan Botanical Research. 1989, 11(2): 198-202.). Pharmacological studies have shown that this plant has multiple activities such as enhancing immune function, antioxidation, antibacterial, anti-inflammatory and analgesic effects. The most studied compounds in this genus are cyclic peptides and triterpenoid saponins and their pharmacological effects. Although triterpenoid saponins from *Gnaphalium affine* have been reported to have analgesic effects, no reports have been found on the analgesic activity of individual components, and the mechanism of action remains unclear. This invention aims to conduct in-depth research on compounds with analgesic effects in *Gnaphalium affine* plants, in order to discover analgesic natural products and provide a basis for screening highly effective and low-toxicity analgesics.

[0044] This invention discovers that solvent (e.g., ethanol solution) extracts of *Lysimachia christinae*, a plant of the *Lysimachia* genus, possess good analgesic activity. Under the guidance of bioactivity testing, the chemical composition of the extract was studied, yielding an active ingredient with the structure of formula (I). The compound of formula (I) exhibits good inhibitory effects on the writhing response in mice. The application of the compound of formula (I) in the preparation of analgesic drugs is characterized in that the analgesic drug or pharmaceutical composition is for treating neuralgia, bone pain, muscle pain, pain caused by falls and injuries, headaches, stomachaches, intestinal colic, biliary colic, renal colic, and cancer pain.

[0045] This invention employs the internationally recognized mouse acetic acid writhing test to assess the analgesic activity of the compound of formula (I) described herein, and calculates the half-maximal effective dose (IC50) of compound (I) in inhibiting mouse writhing responses. The calculation results show that the half-maximal effective dose (IC50) of 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin acid in inhibiting mouse writhing responses is... 50 The measured result was 11.60 mg / kg. These results indicate that 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin acid exhibits good analgesic activity, which is higher than that of the positive control aspirin (IC50). 50 It is 6.4 times that of 74.33 mg / kg.

[0046] The present invention also provides a method for preparing the compound of formula (I) above, the method comprising preparing the compound from plants of the genus *Crassula* (such as *Crassula*). Preferably, 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin is prepared from the roots, stems, leaves, and fruits of *Crassula*. Preferably, the method comprises the following steps:

[0047] (1) Dry and crush the gold iron lock, then extract it with solvent and combine the extracts;

[0048] (2) The extract obtained from step (1) by vacuum concentration is used to obtain an extract;

[0049] (3) The extract obtained in step (2) is suspended in water and extracted with petroleum ether to remove the small polar fraction and retain the water fraction;

[0050] (4) The water fraction obtained in step (3) was separated by a D-101 macroporous resin column and eluted with a methanol-water gradient (volume ratio 0:1, 8:2, 1:0) to obtain three components (Fr.1-Fr.3).

[0051] (5) The Fr.2 obtained in step (4) was subjected to silica gel column chromatography and eluted with dichloromethane-methanol (v / v ratio 1:0 to 0:1) to obtain 6 fractions (Fr.A to Fr.F). The Fr.D was subjected to silica gel column chromatography and eluted with dichloromethane-methanol (v / v ratio 35:1 to 5:1) to obtain 5 fractions (Fr.D1 to Fr.D5). The Fr.D4 was subjected to silica gel column chromatography and eluted with dichloromethane-methanol (v / v ratio 15:1 to 0:1) to obtain 5 fractions (Fr.D4a to Fr.D4e). Fr.D4e was purified by Sephadex LH-20 column chromatography (100% methanol), and then eluted by semi-preparative high performance liquid chromatography with methanol-water (70:30 v / v) as the mobile phase at a flow rate of 3 mL / min to obtain 3-O-β-D-pyranogalactosyl-(1→2)-[α-L-pyranoarabinosyl-(1→3)]-β-D-6-O-ethylpyranoglucuronic acid saponin.

[0052] In step (1), the solvent can be 50% to 95% ethanol / water, 50% to 95% methanol / water, or 50% to 90% acetone / water by volume. The amount of solvent used is 6 to 10 times the weight of *Gynostemma pentaphyllum*. The reflux extraction time is 2 hours each time, and the reflux extraction is repeated 3 times. The filtrates are combined to obtain the extract solution of *Gynostemma pentaphyllum* medicinal materials.

[0053] The present invention also provides the use of the compound of formula (I) in the preparation of analgesic drugs, wherein the compound of formula (I) is prepared as an active ingredient with a pharmaceutically acceptable carrier or excipient to form analgesic drugs or pharmaceutical compositions.

[0054] The analgesic drugs or drug compositions described in this invention can be administered in unit dose form, and the route of administration can be enteric or non-enteric, such as oral, intramuscular, nasal, oral mucosa, skin, transdermal, subcutaneous, intradermal, peritoneal, rectal, intravenous, intramuscular, epidural, intraocular, intracranial, vaginal, hot spring bath administration, etc.

[0055] The analgesic drug or pharmaceutical composition described in this invention can be administered via injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, intrathecal injection, and peritoneal injection.

[0056] Dosage forms can be liquid or solid. Liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Liquid dosage forms can include syrups, injectable solutions, non-aqueous solutions, suspensions, or emulsions. Solid dosage forms include tablets, lozenges, capsules, pellets, pills, granules, powders, creams, solutions, suppositories, and dispersible powders such as lyophilized powder for injection and aerosols.

[0057] The analgesic drugs or drug compositions described in this invention can be formulated into ordinary preparations, or into sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0058] The pharmaceutically acceptable carriers or excipients described in this invention include excipients for oral formulations, parenteral administration, or topical administration. The excipients used include: excipients such as lactose, calcium carbonate, calcium phosphate, and sodium phosphate; diluents and absorbents such as starch, cyclodextrin, lactose, sucrose, mannitol, sodium microcrystalline cellulose, and calcium sulfate; humectants and binders such as water, ethanol, propanol, glycerin, propylene glycol, isopropanol, syrup, honey, glucose, gelatin paste, sodium carboxymethyl cellulose, and potassium phosphate; disintegrants such as dried starch, agar powder, calcium carbonate, sodium bicarbonate, sodium dodecyl sulfate, and methylcellulose; disintegration inhibitors such as sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers such as quaternary ammonium salts and sodium dodecyl sulfate; and lubricants such as talc, magnesium triethylamine stearate, silica, corn starch, stearates, boric acid, and liquid paraffin. Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets, to delay their disintegration and absorption in the gastrointestinal tract and thereby provide sustained action over a longer period of time. Specific Implementation

[0060] To better understand the present invention, the following specific embodiments are provided to further explain or illustrate the content of the present invention, but these examples should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Inhibitory activity of *Gynostemma pentaphyllum* extract on acetic acid-induced writhing response in mice

[0063] Source of materials: The specimen of *Psammosilene tunicoides*, belonging to the genus *Psammosilene*, was collected in Kunming, Yunnan Province, and identified by Professor Yang Qingsong of the School of Ethnic Medicine, Yunnan Minzu University as *Psammosilene tunicoides* WCWu & C.Y.Wu. The specimen is preserved in the Herbarium of the School of Ethnic Medicine, Yunnan Minzu University.

[0064] Preparation of extract of *Crassula ovata*: The dried roots of *Crassula ovata* were crushed to obtain root fragments; the root fragments were then extracted three times by reflux with 95% ethanol / water for 2 hours each time to obtain the extract; the extracts were filtered and concentrated under reduced pressure using a rotary evaporator to obtain a paste for later use.

[0065] An experimental model of inhibition of acetic acid-induced writhing response in mice was used (see scientific literature: Hayashi G., Takemori A.E. The type of analgesic-receptor interaction involved in certain analgesicassays. Eur. J. Pharmacol. 1971, 16: 63-66.). The ability of *Gynostemma pentaphyllum* extract to inhibit acetic acid-induced writhing response in mice was tested. Fifty healthy female mice, weighing 18–22 g, were randomly divided into 5 groups of 10 mice each. The groups were as follows: negative control group (0.9% sodium chloride solution), positive control group (aspirin, 200 mg / kg), and high, medium, and low dose groups of *Gynostemma pentaphyllum* (800 mg / kg, 400 mg / kg, and 200 mg / kg). Thirty minutes after administration, each mouse was injected with 0.2 ml of 0.6% acetic acid solution. The number of writhing movements within 15 minutes after injection of acetic acid solution was observed and recorded. The differences in the number of writhing episodes among mice in different groups were compared, and the inhibition rate of the drug on the writhing response was calculated.

[0066] Inhibition rate = (average number of writhing movements in the control group - average number of writhing movements in the treatment group) / average number of writhing movements in the control group × 100%).

[0067] The results showed that at the above concentrations, the extract of *Gynostemma pentaphyllum* significantly inhibited the acetic acid-induced writhing response in mice. The results are shown in Table 1.

[0068] Table 1. Inhibitory effect of *Lysimachia christinae* extract on acetic acid-induced writhing response in mice.

[0069]

[0070] n=3

[0071] Example 2

[0072] Further experiments were conducted using *Gynostemma pentaphyllum*, with extraction solvents of 95% methanol / water, 70% ethanol / water, and 70% acetone / water, respectively, repeating Example 1. The experimental results showed that the 95% methanol extract, 70% ethanol extract, and 70% acetone / water extract of *Gynostemma pentaphyllum* obtained using 95% methanol / water, 70% ethanol / water, and 70% acetone / water as extraction solvents, respectively, all exhibited significant inhibitory activity against acetic acid-induced writhing in mice. Therefore, the components in *Gynostemma pentaphyllum* that inhibit acetic acid-induced writhing in mice can also be obtained using different concentrations of ethanol / water, methanol / water, or acetone / water as extraction solvents. The results are shown in Table 2.

[0073] Table 2. Inhibitory effects of different solvent extracts of *Lysimachia christinae* on acetic acid-induced writhing response in mice.

[0074]

[0075] n=3

[0076] Example 3

[0077] Analgesic compounds were isolated and identified from the root of *Symplocos rubra*.

[0078] (1) Dry 5kg of *Gynostemma pentaphyllum* root and crush it into particles with a particle size of 0.1cm to obtain *Gynostemma pentaphyllum* powder. Extract the *Gynostemma pentaphyllum* powder by reflux with 60kg of 95% ethanol at a temperature of 70-74℃ for 4 times, 2 hours each time. Combine the ethanol extracts for later use.

[0079] (2) The ethanol extract obtained in step (1) was filtered through 80-120 micrometer filter paper and concentrated under reduced pressure using a rotary evaporator at 50°C until the specific gravity was 1.2, yielding 204.8g of extract for later use.

[0080] (3) The 204g extract obtained in step (2) was suspended in 4500ml of water and extracted with 4500ml of petroleum ether to remove the weakly polar part. The remaining water part was subjected to D-101 macroporous resin column chromatography and eluted with methanol-water at volume ratios of 0:1, 8:2, and 1:0 to obtain three components: Fr.1-Fr.3. Fr.1 was 43.3g, Fr.2 was 79.5g, and Fr.3 was 72.5g. Fr.2 (60g) was subjected to silica gel column chromatography and eluted with a dichloromethane-methanol gradient at volume ratios of 1:0, 8:1, 7:2, 2:1, and 0:1 to obtain six fractions, Fr.A to Fr.F, with Fr.A being 2.7g, Fr.B being 5.3g, Fr.C being 9.3g, Fr.D being 19.8g, Fr.E being 5.8g, and Fr.F being 10.5g. Fr.D (19.8 g) was subjected to silica gel column chromatography and eluted with a dichloromethane-methanol gradient of 35:1, 25:1, 15:1, 10:1, and 5:1 (v / v) to obtain five fractions: Fr.D1 (1.3 g), Fr.D2 (1.2 g), Fr.D3 (3.5 g), Fr.D4 (8.5 g), and Fr.D5 (2.4 g). Fr.D4 (8.5 g) was subjected to silica gel column chromatography and eluted with a dichloromethane-methanol gradient of 15:1, 10:1, 5:1, 2:1, and 0:1 (v / v) to obtain five fractions: Fr.D4a to Fr.D4e. The fractions were: Fr.D4a (0.7 g), Fr.D4b (1.4 g), Fr.D4c (1.1 g), Fr.D4d (2.0 g), and Fr.D4e (2.5 g). Fr.D4e (2.5 g) was purified by Sephadex LH-20 column chromatography (100% methanol), and then eluted by semi-preparative high-performance liquid chromatography with a mobile phase of methanol-water (70:30 v / v) at a flow rate of 3 mL / min to obtain 3-O-β-D-pyranogalactosyl-(1→2)-[α-L-pyranoarabinosyl-(1→3)]-β-D-6-O-ethylpyranoglucuronic acid saponin (11.5 mg) (1). The isolation and identification procedure of the analgesic active ingredient in the root of *Gynostemma pentaphyllum* is described below. Figure 1 .

[0081] Example 4

[0082] Structural identification of compound 1 obtained in Example 3

[0083] Compound 1 is a white amorphous powder (in methanol), and high-resolution mass spectrometry shows its molecular ion peak at m / z 1002.5270 [M+NH4]. + (Calculated value 1002.5268), indicating that the molecular formula of this compound is C. 49 H 76 O 20 .from 1A single-peak proton signal (δ) of 6 methyl groups was read in the H-NMR spectrum. H 1.15, 1.00, 0.80, 1.39, 0.88, 0.97), hydrogen signal of 1 ethyl group [δ] H 4.22 (2H, q, J = 7.1 Hz) and 1.28 (3H, t, J = 7.1 Hz)], terminal proton signals of the three sugar groups [δ H [4.49 (d, J = 7.4 Hz), 4.81 (d, J = 7.4 Hz), 4.55 (d, J = 7.5 Hz)] and a single-peak proton (δ) at the low field. H 9.44). In 13 The corresponding six methyl signals (δ) were read from the C-NMR and DEPT spectra. C 10.8, 16.3, 17.8, 27.3, 33.5, 25.1), 1 ethoxy signal (δ C 62.69 and δ C 14.4), terminal carbon signal of 3 sugar groups (δ) C 104.6, 103.8, 104.9), and one aldehyde signal (δ). C 210.7), and one esterified carbonyl group (δ C 170.3) and 1 double bond (δ) C 145.3, 123.0). The above data indicate that the compound is a triterpenoid glycoside containing three sugar groups. Interestingly, the NMR spectrum only shows 48 carbon signals, while the high-resolution mass spectrometry data shows that the compound has 49 carbons, indicating that one carbon signal is not shown in the NMR spectrum. According to literature reports, the C-28 carbonyl signal of some triterpenoid saponins is relatively weak in NMR and may not be visible (Guo S., Kenne L., Lundgren LN, B., Sundquist B.G. Triterpenoid saponins from Quillaja saponaria. Phytochemistry, 1998, 48, 175-180). Therefore, it is further speculated that the missing carbon signal is the carbonyl signal at position 28. H-5′ (δ) was obtained from the HMBC spectrum of the glycosyl group. H 3.86) and C-1′(δ C 104.6), C-3′(δ C The correlation of 86.2), H-4′(δ) H 3.58) and C-6′(δ C The correlation of 170.3), H-3′(δ) H 3.71) and C-2′(δ CThe correlation of 78.1), H-7′(δ) H 4.22) and C-6′(δ C The correlation of 170.3), H-5″(δ) H 3.50) and C-1″(δ C 103.8), C-3″(δ C 74.6), C-6″(δ) C The correlation of 62.3), H-4″(δ H 3.79) and C-5″(δ C The correlation of 76.8) and H-1′(δ) in the COSY spectrum H 4.49) and H-2′(δ H The correlation of 3.66), H-1″(δ) H 4.81) and H-2″(δ H 3.43) related, H-7′(δ H 4.22) and H-8′(δ C The correlation with 1.28 indicates that the compound contains one ethyl pyranose glucuronide and one pyranose galactosyl group. This is very similar to the NMR data of the known compound (3-O-β-D-pyranose galactosyl-(1→2)-[β-D-pyranoxylosyl-(1→3)]-β-D-6-O-ethylpyranose glucuronide saponin) reported in the literature (Zhong Huimin, Hua Yan, Ni Wei, et al. Two new triterpenoid saponins of *Isodon japonicus* [J]. Yunnan Botanical Research, 2003(03):361-365.). The only difference is the residual sugar signal (δ) of compound 1. C The values ​​(104.9, 72.9, 75.4, 70.0, 67.8) are compared with the carbon signals (δ) of the xylose group in the known compounds mentioned above. C The values ​​(104.9, 75.3, 78.6, 70.9, 67.3) differ significantly from the carbon signal (δ) of the arabinose group of another known compound. C The values ​​(106.2, 72.9, 75.5, 70.2, 67.7) are similar to those in Tava A., Biazzi E., Ronga D., Mella M., Doria F., Accoglic R., Argentierid MP, Avato P. Triterpenic saponins from Medicago marina L. Phytochemistry, 2020, 174, 112333), indicating that compound 1 contains one pyranoarabinose molecule in addition to one ethyl pyranourelate molecule and one pyranogalactosyl molecule. From the H-5″′ (δ) of the HMBC spectrum... H 3.89) and C-1″′(δC 104.9), C-4″′(δ C The correlation of 70.0), H-4″′(δ H 3.81) and C-2″′(δ C 72.9), C-3″′(δ C The correlation of 75.4), and H-1″′ (δ) in the COSY spectrum. H 4.55) and H-2″′(δ H The correlation with 3.59) also indicates the presence of arabinose. This is further supported by the coupling constant [δ] of the terminal protons. H GC-MS analysis of the hydrolyzed derivatized sugars and [4.49 (d, J = 7.4 Hz), 4.81 (d, J = 7.4 Hz), 4.55 (d, J = 7.5 Hz)] identified three sugar units as methyl β-D-glucuronide, β-D-galactose, and α-L-arabinose. The H-1″ (δ H 4.81) and C-2′(δ C 78.1), H-1″′(δ H 4.55) and C-3′(δ C 86.2), H-1′(δ H 4.49) and C-3(δ) C The HMBC-related determination of 86.4 indicates that the sugar linkage order is as follows: galactosyl is linked to the C-2′ position of the glucuronic acid group, arabinose is linked to the C-3′ position of the glucuronic acid group, and the entire sugar side chain is linked to the C-3 position of the aglycone. Finally, compound 1 was determined to be 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylglucuronopyranosyl quillaic acid (see Formula I).

[0084] Physicochemical data of compound 1: Compound (3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin) is a white amorphous powder (methanol), HR-ESI-MS m / z: 1002.5270 [M+NH4] + Molecular formula C 49 H 76 O 20 . 1 H NMR (400MHz, CD3OD)δH :1.70(1H,m,H-1a),1.11(1H,m,H-1b),1.76(2H,m,H-2),3.87(1H,m,H-3),1.33(1H,m,H-5),1.52(1H,m,H-6a),0.91(1H,m,H-6b),1.53(1H,m,H-7a),1.24(1H,m,H-7b),1.75(1H,m,H-9),1.91(1H,m,H-11),5.30(1H,brs,H-12),1.32(1H,m,H-15a),1.84(1H,m,H-15b),4.45(1H,brs,H-16),3.03(1H,dd,J=14.0,3.7Hz,H-18),2.27(1H,t,J=13.6Hz,H-19a),1.02(1H,m,H-19b),1.91(1H,m,H-21a),1.14(1H,m,H-21b),1.87(1H,m,H-22a),1.75(1H,m,H-22b),9.44(1H,s,H-23),1.15(3H,s,H-24),1.00(3H,s,H-25),0.80(3H,s,H-26),1.39(3H,s,H-27),0.88(3H,s,H-29),0.97(3H,s,H-30),4.49(1H,d,J=7.4Hz,H-1′),3.66(1H,d,J=7.4Hz,H-2′),3.71(1H,m,H-3′),3.58(1H,m,H-4′),3.86(1H,m,H-5′),4.22(1H,q,J=7.1Hz,H-7′),1.28(1H,t,J=7.1Hz,H-8′),4.81(1H,d,J=7.4Hz,H-1″),3.43(1H,m,H-2″),3.51(1H,m,H-3″),3.79(1H,m,H-4″),3.50(1H,m,H-5″),3.77(1H,m,H-6″a),3.72(1H,m,H-6″b),4.55(1H,d,J=7.5Hz,H-1″′),3.59(1H,m,H-2″′),3.43(1H,m,H-3″′),3.81(1H,m,H-4″′),3.89(1H,m,H-5″′a),3.61(1H,m,H-5″′b); 13 C NMR(100MHz,CD3OD)δ C:39.2(C-1),25.7(C-2),86.4(C-3),56.2(C-4),49.1(C-5),21.2(C-6),33.5( C-7),40.9(C-8),48.0(C-9),37.1(C-10),24.4(C-11),123.0(C-12),145.3(C- 13),42.7(C-14),36.1(C-15),75.4(C-16),49.8(C-17),42.2(C-18),47.8(C- 19),31.4(C-20),36.6(C-21),32.5(C-22),210.7(C-23),10.8(C-24),16.3(C- 25),17.9(C-26),27.3(C-27),33.5(C-29),25.1(C-30),104.6(C-1′),78.1(C -2′),86.2(C-3′),71.4(C-4′),76.4(C-5′),170.3(C-6′),62.6(C-7′),14.4(C -8′),103.8(C-1″),73.5(C-2″),74.6(C-3″),70.8(C-4″),76.8(C-5″),62.3( C-6″), 104.9(C-1″′), 72.9(C-2″′), 75.4(C-3″′), 70.0(C-4″′), 67.8(C-5″′).

[0085] Example 5

[0086] Analgesic activity detection of the compound of formula (I)

[0087] The ability of 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglucuronic acid saponin to inhibit the acetic acid-induced writhing response in mice was tested using a mouse acetic acid-induced writhing response inhibition model (see scientific literature: Hayashi G., Takemori A.E. The type of analgesic-receptor interaction involved in certain analgesics. Eur. J. Pharmacol. 1971, 16:63-66). Healthy female Kunming mice, weighing 18–22 g, were randomly divided into groups of 10 mice each, according to the experimental requirements. The groups were as follows: negative control group (0.9% sodium chloride solution), positive control group (aspirin doses of 400 mg / kg, 200 mg / kg, 100 mg / kg, 50 mg / kg, and 25 mg / kg), and sample group (dose of 20.00 mg / kg, 10.00 mg / kg, 5.00 mg / kg, 2.50 mg / kg, and 1.25 mg / kg). Thirty minutes after administration, each mouse was injected with 0.2 ml of 0.6% acetic acid solution. The number of writhing movements within 15 minutes after acetic acid injection was observed and recorded. The differences in the number of writhing movements among the different groups were compared, and the inhibition rate of the writhing response by the drug was calculated.

[0088] Inhibition rate = (average number of writhing movements in the control group - average number of writhing movements in the treatment group) / average number of writhing movements in the control group × 100%).

[0089] Calculate the median effective dose (IC50) 50 The half-maximal effective dose (IC50) of aspirin for inhibiting acetic acid-induced writhing in mice was 74.33 mg / kg. The IC50 of 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin acid for inhibiting acetic acid-induced writhing in mice was... 50 The measured result was 11.60 mg / kg, indicating that the compound of the present invention has the ability to significantly inhibit the acetic acid writhing response in mice.

[0090] Example 6

[0091] The 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin obtained by the method described in Example 3 was added to commonly used tablet excipients and prepared into tablets according to conventional preparation processes.

[0092] Example 7

[0093] The 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin obtained by the method described in Example 3 was added to commonly used excipients for injections and prepared into an injection according to conventional preparation processes.

[0094] Example 8

[0095] The 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin obtained by the method described in Example 3 was added to common excipients for capsules and prepared into capsules according to conventional preparation processes.

[0096] Example 9

[0097] The 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin obtained by the method described in Example 3 was added to commonly used excipients for poultices and prepared into a poultice according to conventional preparation processes.

[0098] Example 10

[0099] The extract containing 3-O-β-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-6-O-ethylpyranoglobulin saponin was prepared according to the method described in Example 1. The extract was then added to alkaline hot spring water and prepared into a hot spring medicinal bath solution using conventional preparation processes.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a compound of structural formula (I), characterized in that, The structural formula (I) is as follows: Includes the following steps: (1) After drying and pulverizing the plant, extract it with a solvent and combine the extracts. The solvent is 50-95% ethanol / water, 50-95% methanol / water, or 50-90% acetone / water. (2) The extract obtained in step (1) by vacuum concentration is used to obtain an extract; (3) The extract obtained in step (2) is suspended in water and extracted with petroleum ether to remove the small polar fraction and retain the water fraction; (4) The water fraction obtained in step (3) was separated by a D101 macroporous resin column and eluted with a methanol-water volume ratio of 0:1, 8:2, and 1:0 to obtain three components Fr.1-Fr.3; (5) The Fr.2 obtained in step (4) was subjected to silica gel column chromatography and eluted with dichloromethane-methanol at a volume ratio of 1:0 to 0:1 to obtain 6 components Fr.A to Fr.F. The Fr.D was subjected to silica gel column chromatography and eluted with dichloromethane-methanol at a volume ratio of 35:1 to 5:1 to obtain 5 components Fr.D1 to Fr.D5. The Fr.D4 was subjected to silica gel column chromatography and eluted with dichloromethane-methanol at a volume ratio of 15:1 to 0:1 to obtain 5 components Fr.D4a to Fr.D4e. The Fr.D4e was purified by Sephadex LH-20 column chromatography with 100% methanol and then eluted by semi-preparative high performance liquid chromatography with methanol-water at a volume ratio of 70:30 and a flow rate of 3 mL / min to obtain the compound of structural formula (I).

2. The method for preparing the compound of structural formula (I) according to claim 1, characterized in that, Step (1) Take 5-8 times the weight of the plant and extract with solvent for 1-2 hours, extract 1-3 times, and combine the filtrates to obtain the extract solution of the plant.

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