A compound of a hundred li hydroquinone glycoside and its preparation method and application

Abstract

This invention discloses a thymol glycoside compound, its preparation method, and its application. The thymol glycoside compound is prepared from the root of *Hydrangea benjamina*, a plant belonging to the genus *Hydrangea* of the family Scrophulariaceae, through pretreatment, extract extraction, extraction, and separation. It is named as follows: ( S )-Thyroquinone O -(6- O (-Oleoyl)-β-D-glucopyranoside, its English name is: S )-thymoquinol O -(6- O -oleuropeoyl)- β -d-glucopyranoside, having the following structure: The thymol glycosides of the present invention can be naturally occurring organic compounds or synthetic organic compounds, possessing strong analgesic activity, and can be used as active ingredients in combination with pharmaceutically acceptable carriers or excipients to prepare analgesic drug compositions.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural product chemistry, and particularly relates to a bai-li hydrogen quinone glycoside compound and a preparation method and application thereof. BACKGROUND

[0002] Pain is an unpleasant subjective feeling and emotional experience caused by a noxious or potentially noxious stimulus from the outside or inside of the body, and has been listed as the fifth vital sign after respiration, pulse, blood pressure and body temperature in today's medicine. In clinical medical practice, pain is the most common complaint of patients seeking medical treatment, and more than 80% of patients seek medical treatment due to pain. Pain brings great harm and negative impact to individuals, and is the most common and direct factor for reducing labor capacity and working days of people, so how to relieve pain has become one of the biggest goals of clinical medicine.

[0003] Among the numerous drugs for treating pain, non-steroidal anti-inflammatory drugs (NSAIDs) and narcotic analgesics are the first choice for treating or relieving various pains, such as anti-inflammatory drugs such as aspirin, indomethacin, ibuprofen, diclofenac sodium, and central analgesics such as morphine, tramadol, and pentazocine. Compared with narcotic analgesics, non-steroidal anti-inflammatory drugs are more common and widely used. Since the advent of aspirin in the last century, more than a hundred varieties of non-steroidal anti-inflammatory drugs have been developed, including acetylsalicylic acid, pyrazolone, acetic acid, arylalkanoic acid, oxicam, and acetanilide, which are widely used to treat diseases with pain as the main symptom, such as neuralgia, headache, toothache, bone and joint pain, pain caused by sprains and injuries, stomach pain, and biliary colic, and have significant efficacy, and are one of the most frequently used drugs in the world. However, while these drugs exert anti-inflammatory and analgesic effects, they also have serious adverse reactions, which can harm the body when used repeatedly or for a long time. The main adverse reactions include gastrointestinal damage, upper abdominal pain, nausea, indigestion, esophagitis, and colitis, blood system damage, and liver and kidney damage. Opioid drugs are the strongest analgesic drugs currently discovered, and can be used for various moderate and severe pain, but these drugs also have serious adverse reactions, such as respiratory depression, cough suppression, dose dependence, addiction, nausea, vomiting, miosis, and constipation. Therefore, it is very meaningful to explore and find new analgesic drugs with good efficacy and fewer adverse reactions from natural drugs.

[0004] At present, domestic and foreign scholars have studied the analgesic active ingredients of plant medicines such as cannabis, radix aconiti, semen strychni, caulis periplocae, sinigrin and rhizoma corydalis, and obtained a batch of compounds with excellent activity, including lignans, alkaloids, diterpenes, triterpenes, steroids, flavones and other types of compounds. Although a large number of compounds with analgesic effect have been found, there are not many compounds with strong activity, and most of them have the same analgesic effect as antipyretic analgesics. At present, there are also analgesic drugs or pharmaceutical compositions made of a plurality of medicinal plants as raw materials, but the analgesic effect is not ideal. SUMMARY

[0005] The first object of the present application is to provide a thymoquinol glycoside compound; the second object is to provide a preparation method of the thymoquinol glycoside compound; and the third object is to provide an application of the thymoquinol glycoside compound.

[0006] The first object of the present application is achieved by using the roots of the plant Patrinia villosa Juss. of the Patrinia family as raw materials, and preparing by pretreatment, extractive extraction, extraction and separation, and is named as: S )-thymoquinol O -(6- O -oleuropeoyl)- S )-thymoquinol O -(6- O -oleuropeoyl)- β -d-glucopyranoside, which has the following structure:

[0007] .

[0008] The second object of the present application is achieved by using the roots of the plant Patrinia villosa Juss. of the Patrinia family as raw materials, and preparing by pretreatment, extractive extraction, extraction and separation, and specifically comprising the following steps:

[0009] A, pretreatment: crushing or cutting the roots of the plant Patrinia villosa Juss. of the Patrinia family to obtain material a;

[0010] B, extractive extraction: adding 6-10 times the mass of the organic extraction solvent to the material a, soaking and extracting at room temperature for 2-4 times, and each time for 1-2 hours, combining the extract and filtering to obtain sample extract b;

[0011] C, extraction:

[0012] 1) reducing pressure to concentrate the sample extract b to obtain extract c;

[0013] 2) The extract c is suspended in water, and sequentially extracted with petroleum ether, ethyl acetate, and n-butanol. After recovering the organic solvents by distillation under reduced pressure, the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction are obtained.

[0014] D, separation: the ethyl acetate fraction is sequentially separated by silica gel column chromatography, MCI reverse phase column chromatography, Sephadex gel column chromatography, and semi-preparative high performance liquid chromatography to obtain the target compound, baeckea frutescens hydroquinone glycoside.

[0015] The specific operation is as follows:

[0016] (1) After the Baeckea frutescens is dried, crushed, and extracted with a solvent, the extract is combined;

[0017] (2) The extract obtained in step (1) is concentrated under reduced pressure to obtain an extract;

[0018] (3) The extract obtained in step (2) is suspended in water, and sequentially extracted with petroleum ether, ethyl acetate, and n-butanol. After recovering the organic solvents by distillation under reduced pressure, the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction are obtained;

[0019] (4) The ethyl acetate fraction obtained in step (3) is separated by silica gel column chromatography, and gradient elution is performed with petroleum ether-ethyl acetate in a volume ratio of 100:1, 80:1, 50:1, 20:1, 10:1, and 1:1 to obtain 6 fractions (Fr.1~Fr.6). Fr.6 is separated by MCI reverse phase column chromatography, and gradient elution is performed with methanol-water in a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0 to obtain 8 fractions (Fr.A~Fr.H). Fr.E is separated by Sephadex LH-20 column chromatography, and elution is performed with dichloromethane-methanol in a volume ratio of 1:1 to obtain 7 fractions (Fr.E1~Fr.E7). Fr.E6 is purified by Sephadex LH-20 column chromatography (100% methanol), and then eluted by semi-preparative high performance liquid chromatography with methanol-water in a volume ratio of 47:53 at a flow rate of 3 ml / min to obtain (R)-baeckea frutescens hydroquinone (1.2 mg, tR= 29.1 min). S O (6- O -oliveacyl)-β-D-glucopyranoside (52.1 mg, t R = 29.1 min).

[0020] In step (1), the solvent can be 50%~95% ethanol / water, or 50%~95% methanol / water, or 50%~90% acetone / water, and the solvent is used in an amount of 6~10 times the weight of the Baeckea frutescens. The reflux extraction time is 2 h each time.​

[0021] The structure of the thymoquinol glycoside compound prepared in the above step can be identified by the following method:

[0022] High resolution mass spectrum shows that the molecular ion peak is m / z 517.2405 [M+Na] + (calculated value 517.2408), indicating that the molecular formula of the compound is C 26 H 38 O9, with 8 degrees of unsaturation. From 1 H and 13 C-NMR spectrum analysis and literature comparison, it can be seen that there is one carbon signal ( δ C 167.4), one double bond ( δ C 140.2, 129.7), three methylene groups ( δ C 27.2, 24.9, 23.1) and two methyl groups ( δ H 1.19, s, 3H×2; δ C 25.7, 25.1), indicating that there is one olivetolyl fragment in the compound (Tian LW, Zhang YJ, Wang YF, Lai CC, Yang CR. Eucalmaidins A-E, (+)-oleuropeic acid derivatives from the fresh leaves of δ . J Nat Prod. 2009, 72: 1608-1611). In addition, one methyl group ( Eucalyptus maideni C 14.9), six aryl signals ( δ C 150.6, 147.5, 137.4, 121.7, 119.3, 111.7) and one isopropyl functional group[ δ H 3.44 (1H, m), 1.15 (3H×2, t, J =6.4 Hz)], indicating that there is also one thymoquinol structure in the compound (Yahara S, Sakamoto C, Nohara T, Niiho Y, Nakajima Y, Ito H. Thymoquinol glucosides from δ.Shoyakugaku Zasshi. 1993,47:420-422). Remaining NMR data ( Schisandrae fructus C (103.1, 76.7, 73.8, 74.0, 70.6, 63.7) represent signals on sugars. Through H-5' ( δ H 3.60) and C-1' ( δ C 103.1), C-3' ( δ C 76.7) and H-1' ( δ H 4.72) and C-3' δ C According to the HMBC correlation of 76.7), this is a pyran-type glucose unit. Therefore, the compound consists of one oleoyl group, one thymol quinone, and one sugar unit.

[0023] From the HMBC spectrum, H-1' ( δ H 4.72) and C-5 of the thymol unit ( δ C 147) Related notes: The C-1' position of glucose is linked to the thymol unit. Furthermore, H2-6' ( δ H 4.52, 4.22) and the C-7'' of the oleoyl group ( δ C The HMBC correlation of 167.4) indicates the linking position of glucose to the oleoyl group. The coupling constant of the protons at the sugar end [ δ H 4.72(d, J = 7.4 Hz)] and GC-MS analysis of the derivatized sugars after hydrolysis determined the sugar configuration to be β -D-glucose. Methyl oleolate was obtained by hydrolysis of the compound with sodium methoxide, with an optical rotation value of […]. α ]24.5 D49.4 ( c 0.16, methanol) and the literature reported ( S )-Methyl oleuropeate is essentially the same (Manns D, Hartmann R. Monoterpene glucosides from δ Planta Med. 1994, 60:467-469). Ultimately, the compound was identified as (…). S )-Thyroquinone O -(6- O (-Olouryl)-β-D-glucopyranoside.

[0024] Hydrolysis product of the compound: Compound (25 mg) was dissolved in dry methanol, 1 M sodium methoxide-methanol solution 0.3 ml was added and stirred at room temperature for 12 h. After neutralization with formic acid, the crude product was extracted with ethyl acetate to get the crude product which was purified by silica gel column chromatography eluting with dichloromethane-methanol in the ratio of 100:0, 99:1, 90:10 to get the hydrolysis product of the compound (9.0 mg). S )-oleuropeic acid methyl ester] was colorless oil, S α ]24.5D49.4 ( c 0.16, methanol), 1 H NMR (deuterated methanol, 400 MHz) Cunila spicata : 7.00 (1H, t, J =2.8 Hz, H-2), 2.33, 2.00 (m, H-3a), 1.55 (m, H-4), 2.03, 1.23 (m, H-5), 2.54,2.17 (m, H-6), 1.18 (3H, s, H-9),1.17 (3H, s, H-10), 3.71 (3H, s, OMe); 13 C NMR (deuterated methanol, 100 MHz) δ : 131.1 (C-1), 141.2 (C-2), 27.0 (C-3), 45.5 (C-4), 24.5(C-5), 26.3 (C-6), 169.4 (C-7), 72.8 (C-8), 28.5 (C-9), 26.4(C-10), 52.0(OMe).

[0025] Physicochemical data of the compound: Compound (25 mg) was dissolved in dry methanol, 1 M sodium methoxide-methanol solution 0.3 ml was added and stirred at room temperature for 12 h. After neutralization with formic acid, the crude product was extracted with ethyl acetate to get the crude product which was purified by silica gel column chromatography eluting with dichloromethane-methanol in the ratio of 100:0, 99:1, 90:10 to get the hydrolysis product of the compound (9.0 mg). S )-thymohydroquinone O -(6- O -oliveacyl)-β-D-glucopyranoside was brownish oil, HR-ESI-MS δ : 517.2405 [M+Na] + , molecular formula C 26 H 38 O9. IR (KBr) v max : 3412, 2963, 2928, 1696, 1605, 1366, 1070, 829, and 618 cm -1 ​. UV (MeOH) λ max (log m / z ): 206 (4.06), 219 (3_99), and 286 (3_30) nm。 1 H NMR (methanol-d, 400 MHz) ε H : 6.63 (1H, s, H-3), 6.85 (1H, s, H-6), 2.13 (3H, s, H-7), 3.44 (1H, q, J = 6.6Hz, H-8), 1.15 (6H, t, J = 6.6 Hz, H-9 and H-10), 4.72 (1H, d, J = 7.4 Hz, H-1'), 3_47 (1H, m, H-2'), 3_48 (1H, m, H-3'), 3_38 (1H, m,_H-4'), 3_60 (;1H, td, J = 8.5, 1.5 Hz, H-5'), 4.52 (;1H, dd, J = 11.7, 1.5 Hz, H-6'a), 4.22 (1H, dd, J = 11.8, 7.3 Hz, H-6'b), 6.98 (1H, m, H-2''), 2.32 (1H, td, J = 19.1, 4.9 Hz, H-3''a), 2.01 (1H, m, H-3''b), 1.53 (1H, tdd, J = 11.8, 5.0, 2.1 Hz, H-4''), 1.98 (1H, m, H-5''a), 1.21 (1H, m, H-5''b), 2.46 (1H, dt, J = 17.6, 1.9 Hz, H-6''a), 2.12 (1H, m, H-6''b), 1.19(;6H, s, H-9''and H-10''); 13 C NMR (methanol-d, 100 MHz) δ C It should be noted that in the original text, there are some underscores in the translated content which are used to replace the commas in the numbers in the original text according to the format requirements to avoid confusion with the commas in the English text. You may adjust them according to the actual situation.: 121.7 (C-1), 150.6 (C-2), 111.7 (C-3), 137.4 (C-4), 147.5 (C-5), 119.3(C-6), 14.9 (C-7), 25.6 (C-8), 22.4 (C-9), 22.4 (C-10), 103.1 (C-1'), 73.8 (C-2'), 76.7 (C-3'), 70.6 (C-4'), 74.0 (C-5'), 63.7 (C-6'), 129.7 (C-1''), 140.2(C-2''), 27.2 (C-3''), 44.1 (C-4''), 23.1 (C-5''), 24.9 (C-6''), 167.4 (C-7''), 71.5 (C-8''), 25.1 (C-9''), 25.7 (C-10'').

[0026] The third object of the present application is achieved by the use of the thymohydroquinone glycoside compound in the preparation of an analgesic drug.

[0027] The use of the thymohydroquinone glycoside compound in the preparation of an analgesic drug is to prepare an analgesic drug or a pharmaceutical composition by taking the thymohydroquinone glycoside compound as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0028] The analgesic drug or pharmaceutical composition of the present application can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, nasal, oral mucosal, dermal, transdermal, subcutaneous, intradermal, peritoneal, rectal, intravenous, intramuscular, epidural, intraocular, intracranial, vaginal administration, hot spring bath administration, etc.

[0029] The administration route of the analgesic drug or pharmaceutical composition of the present application can be injection administration. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, intrathecal injection and peritoneal injection, etc.

[0030] The administration dosage form can be a liquid dosage form or a solid dosage form. For example, the solution properties of the liquid dosage form can be true solution, colloid, microparticle, emulsion, suspension. The liquid dosage form can be syrup, injection solution, non-aqueous solution, suspension or emulsion; the solid dosage form can be tablets, lozenges, capsules, dripping pills, pills, granules, powders, creams, solutions, suppositories, dispersible powders such as lyophilized powder injections, aerosols, etc.

[0031] The analgesic drug or pharmaceutical composition of the present application can be prepared into ordinary preparations, or sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0032] The pharmaceutically acceptable carriers or excipients described in the present application include oral preparation excipients, parenteral route administration or external administration excipients. The excipients used include excipients such as lactose, calcium carbonate, calcium phosphate, sodium phosphate; diluents and absorbents such as starch, cyclodextrin, lactose, sucrose, mannitol, microcrystalline cellulose sodium, calcium sulfate, etc.; wetting agents and binders such as water, ethanol, propanol, glycerol, propylene glycol, isopropyl alcohol, sugar syrup, honey, glucose, gelatin syrup, sodium carboxymethyl cellulose, potassium phosphate, etc.; disintegrants such as dried starch, agar powder, calcium carbonate, sodium bicarbonate, sodium dodecyl sulfate, methyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol triestearate, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salt, sodium dodecyl sulfate, etc.; lubricants such as talc, triethylamine magnesium stearate, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, etc. The tablets can be further prepared into coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets to delay their disintegration and absorption in the gastrointestinal tract, and thus provide sustained action over a longer period of time.

[0033] Buddleja lindleyana δ Buddleja lindleyana Wall. is a plant of the genus Buddleja of the Scrophulariaceae family, which is a single-species plant unique to the southwest region of China, mainly distributed in Yunnan, Shaanxi, Gansu, Taiwan, Hubei, Sichuan, and Guizhou of China. It has the effects of clearing heat and resolving toxins, dispelling wind and dampness, and promoting blood circulation to stop pain. It is mainly used for treating swelling and pain of the gums, rheumatism and arthralgia, amenorrhea, abdominal pain, sores, dampness, and contusions and injuries. It is used by the Yi people as a traditional medicine for treating abdominal distension and toothache. The chemical components of Buddleja lindleyana mainly include phenylpropanoid glycosides, iridoid glycosides, flavonoid glycosides, menthanoid monoterpenoid glycosides, and phenolic structures (Ma WG, Li XC, Liu YQ, et al. Phenylpropanoid and iridoid glycosides from Hemiphragmaheterophyllum . Acta Bot Yunnanica. 1995, 17:96-102; Luo Ya, Dai Jiameng, Li Yanhong, et al. Study on flavones and flavonoid glycosides from Buddleja lindleyana. Chinese Ethnic and Folk Medicine. 2017, 26(18):21-24; Luo Ya, Pu Xiaoyun, Tian Kai, et al. Study on phenolic compounds from Buddleja lindleyana. Journal of Yunnan University of Nationalities (Natural Science Edition). 2019, 28(01):16-19; Jin H, Mori A, Tanaka T, et al. A new phenylethanoid glycoside from Hemiphragma heterophyllumJpn SocFood Chem. 2006, 13:83-86). Pharmacological studies have shown that the glycosides from this plant possess multiple activities, including anti-inflammatory, antibacterial, antioxidant, antitumor, and anti-α-glucosidase activities (Ravn H, Nishibe S, Sasahara M, et al. Phenolic compounds from Hemiphragma heterophyllum . Phytochemistry. 1990,29:3627-3631; LiYH, Dai JM, Yang C, et al. Phenylpropanoid and iridoid glucosides from the whole plant of Plantago asiatica and their alpha-glucosidase inhibitory activities. Planta Med. 2020,86:205-211; Pu Xiaoyun, Gao Libin, Wang Wei, et al. Study on chemical constituents and their alpha-glucosidase inhibitory activities of Hydrangea spp. Journal of Yunnan University for Nationalities. 2019,28(05):423-427). Currently, the most studied compounds are phenylpropanoid glycosides and iridoid glycosides in this genus and their pharmacological effects, while studies on their analgesic active components have not been reported, and the mechanism of action is unclear. This invention attempts to conduct in-depth research on compounds with analgesic effects in Hydrangea spp. in order to discover analgesic active natural products and provide a basis for screening highly effective and low-toxicity analgesic drugs.

[0034] This invention discovers that solvent (e.g., ethanol solution) extracts of *Hydrangea spp.*, a plant in the *Hydrangea* genus, possess good analgesic activity. Guided by bioactivity testing, the chemical composition of the extract was studied, yielding an active ingredient: a thyme-hydroxyquinone glycoside compound that effectively inhibits writhing responses in mice. The application of this thyme-hydroxyquinone glycoside compound in the preparation of analgesic drugs or pharmaceutical compositions is for the treatment of neuralgia, bone pain, muscle pain, pain caused by falls and injuries, and cancer pain.

[0035] This invention employs the internationally recognized mouse acetic acid writhing test to assess the analgesic activity of the thymol glycosides described herein, and calculates the half-maximal effective dose (MCD) of the thymol glycosides in inhibiting mouse writhing responses. The calculation results indicate that the thymol glycosides (( S )-Thyroquinone O -(6- O (-Oleoyl)-β-D-glucopyranoside) inhibits the half-maximal effective dose (IC50) of mouse writhing response. 50) was 26.15 ± 4.28 mg / kg. The above results show that the thymoquinol glycosides (TQG) have good analgesic effect, and the analgesic effect is 2.7 times of that of the positive control aspirin (IC S )-thymoquinol O -(6- O -oliveacyl) -β-D-glucopyranoside) shows good analgesic effect, and the analgesic effect is 2.7 times of that of the positive control aspirin (IC 50 70.44 ± 3.71 mg / kg). BRIEF DESCRIPTION OF DRAWINGS

[0036] Hemiphragma heterophyllum is a schematic diagram of the activity tracking separation process of the thymoquinol glycosides described in the present application;

[0037] Figure 1 is a schematic diagram of the structural formula of the thymoquinol glycosides described in the present application;

[0038] Figure 2 is the ultraviolet absorption spectrum of the thymoquinol glycosides described in the present application;

[0039] Figure 3 is the infrared spectrum of the thymoquinol glycosides described in the present application;

[0040] Figure 4 is the high resolution mass spectrum (HRESI-MS) of the thymoquinol glycosides described in the present application;

[0041] Figure 5 is the nuclear magnetic resonance hydrogen spectrum (H NMR) of the thymoquinol glycosides described in the present application; 1

[0042] Figure 6 is the nuclear magnetic resonance carbon spectrum (C NMR) and DEPT spectrum of the thymoquinol glycosides described in the present application; 13

[0043] Figure 7 is the HSQC correlation spectrum of the thymoquinol glycosides described in the present application;

[0044] Figure 8 is the HMBC correlation spectrum of the thymoquinol glycosides described in the present application;

[0045] Figure 9 is the HSQC correlation spectrum of the thymoquinol glycosides described in the present application;

[0046] Figure 10 is the nuclear magnetic resonance hydrogen spectrum (H NMR) of the hydrolysis product of the thymoquinol glycosides described in the present application; 1

[0047] ​​​Figure 11 is the hydrolysis product of the thymoquinol glycosides compound according to the present application, and its carbon nuclear magnetic resonance spectrum is as follows: 13 C NMR);

[0048] Figure 12 is a schematic diagram of the mouse writhing reaction inhibition effect of the thymoquinol glycosides compound according to the present application. DETAILED DESCRIPTION

[0049] The present application is further described below in conjunction with the examples and drawings, but is not limited in any way by the present application, and any transformation or replacement based on the teaching of the present application belongs to the protection scope of the present application.

[0050] The thymoquinol glycosides compound according to the present application is prepared from the roots of the Scyphiphora root of the Scyphiphora plant of the Scrophulariaceae family through pretreatment, extract extraction, extraction and separation, and is named as: S thymoquinol O -(6- O -oleuropeoyl)-β-D-glucopyranoside, and its English name is: S thymoquinol O -(6- O -oleuropeoyl)- β -d-glucopyranoside, which has the following structure:

[0051] .

[0052] The preparation method of the thymoquinol glycosides compound according to the present application is prepared from the roots of the Scyphiphora plant of the Scrophulariaceae family through pretreatment, extract extraction, extraction and separation, and specifically includes the following steps:

[0053] A, pretreatment: crushing or cutting the roots of the Scyphiphora plant of the Scrophulariaceae family to obtain material a;

[0054] B, extract extraction: adding 6-10 times of organic extraction solvent to the material a, soaking and extracting at room temperature for 2-4 times, and each time for 1-2 hours, combining the extract and filtering to obtain sample extract b;

[0055] C, extraction:

[0056] 1) reducing pressure concentration of the sample extract b to obtain extract c;

[0057] 2) suspending the extract c in water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and after recovering the organic solvent by reducing pressure distillation, obtaining the petroleum ether part extract, the ethyl acetate part extract and the n-butanol part extract;

[0058] D. Separation: The ethyl acetate extract was sequentially separated by silica gel column chromatography, MCI reversed-phase column chromatography, dextran gel column chromatography, and semi-preparative high-performance liquid chromatography to obtain the target thyme glycosides.

[0059] The organic extraction solvent mentioned in step B is an aqueous solution of ethanol with a mass concentration of 50%~95%, an aqueous solution of methanol with a mass concentration of 50%~95%, or an aqueous solution of acetone with a mass concentration of 50%~90%.

[0060] The application of the thymol glycosides described in this invention is their use in the preparation of analgesic drugs.

[0061] The analgesia described is for the treatment of neuralgia, bone pain, muscle pain, cancer pain, or pain caused by falls or injuries.

[0062] The thymol glycosides described in this invention can be naturally occurring compounds or artificially synthesized compounds.

[0063] The medicinal plant *Hydrangea rubra* used in this invention is not limited by region or variety and can be used to realize this invention. Specific implementation examples are provided below to further illustrate this invention:

[0064] Example 1

[0065] Inhibitory activity of Hydrangea rotundifolia extract on acetic acid-induced writhing response in mice

[0066] Source of material: *Hydrangea villosa*, a plant belonging to the genus *Hydrangea*, was collected in Kunming, Yunnan Province, and identified by Professor Yang Qingsong of the School of Ethnic Medicine, Yunnan Minzu University. Figure 13 The specimen is preserved in the Herbarium of the School of Ethnic Medicine, Yunnan Minzu University.

[0067] Preparation of Hydrangea flavomarginata extract: The dried roots of Hydrangea flavomarginata were crushed to obtain root fragments; the root fragments were then extracted with 95% ethanol / water by reflux four times, 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.

[0068] The writhing reaction of mice was tested by using the experimental model of acetic acid writhing reaction inhibition (see technical literature: Hayashi G., Takemori A.E. The type of analgesic-receptor interaction involved in certain analgesic assays. Eur. J. Pharmacol. 1971, 16:63-66.) to test the ability of the extract of Adenocaulon bicolor to inhibit the writhing reaction of mice. 50 healthy female mice, weighing 18-22 g, were randomly divided into 5 groups, 10 mice in each group. The groups were: negative control group (0.9% sodium chloride solution), positive control group (aspirin, 100 mg / kg), high, medium and low dose groups of Adenocaulon bicolor (800 mg / kg, 400 mg / kg, 200 mg / kg). 30 min after administration, each mouse was injected with 0.6% acetic acid solution 0.2 ml. The number of writhing times within 15 min after injection of acetic acid solution was observed and recorded. The difference in the number of writhing times of mice in different groups was compared, and the inhibition rate of writhing reaction of the drug was calculated.

[0069] Inhibition rate = (average number of writhing times in control group - average number of writhing times in administration group) / average number of writhing times in control group x 100%.

[0070] The results showed that the extract of Adenocaulon bicolor had a significant ability to inhibit the writhing reaction of mice at the above concentrations. The results are shown in Table 1.

[0071] Table 1 Inhibition of acetic acid writhing reaction of mice by extract of Adenocaulon bicolor

[0072]

[0073] Note: n =3, compared with the blank group, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001.

[0074] Example 2

[0075] Further test was made with Buddleja asiatica, using 95% methanol / water, 70% ethanol / water and 70% acetone / water as extraction solvents, respectively, to repeat Example 1. The experimental results showed that the 95% methanol extract of Buddleja asiatica, the 70% ethanol extract of Buddleja asiatica and the 70% acetone / water extract of Buddleja asiatica obtained by using 95% methanol / water, 70% ethanol / water and 70% acetone / water as extraction solvents, respectively, also had significant inhibitory activity on the acetic acid writhing reaction of mice, and therefore the components in Buddleja asiatica having inhibitory effect on the acetic acid writhing reaction of mice can also be obtained by using different concentrations of ethanol / water, methanol / water or acetone / water as extraction solvents. The results are shown in Table 2.

[0076] Table 2 Inhibitory effect of different solvent extracts of Buddleja asiatica on the acetic acid writhing reaction of mice

[0077]

[0078] Note: n =3, compared with the blank group, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001.

[0079] Example 3

[0080] Isolation and identification of analgesic active compounds from Buddleja asiatica

[0081] (1) 10.0 kg of dried Buddleja asiatica whole plants were ground into particles with a particle size of 0.1 cm, to obtain Buddleja asiatica powder. The Buddleja asiatica powder was extracted by refluxing with 60 kg of 95% ethanol at a temperature of 70-74°C for 4 times, each time for 2 h. The ethanol extracts were combined and prepared for use;

[0082] (2) The ethanol extract prepared in step (1) was filtered through 80-120 micron filter paper and concentrated under reduced pressure at a temperature of 50°C using a rotary evaporator. When the specific gravity reached 1.2, 1.1 kg of extract was obtained and prepared for use;

[0083] (3) 1.1 kg of the extract in (2) was suspended in 4500 ml of water, and sequentially extracted with 4500 ml of petroleum ether, ethyl acetate and n-butanol. After recovering the organic solvents by distillation under reduced pressure, the petroleum ether fraction (158.6 g), the ethyl acetate fraction (210.0 g) and the n-butanol fraction (425.0 g) were obtained. The ethyl acetate extract (210.0 g) was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate (100:1, 80:1, 50:1, 20:1, 10:1 and 1:1) by volume. The concentrated solutions collected were detected by TLC, and similar components were combined to obtain 6 fractions with increasing polarity, i.e. Fr. 1 (13.3 g), Fr. 2 (29.5 g), Fr. 3 (12.5 g), Fr. 4 (6.3 g), Fr. 5 (58.3 g) and Fr. 6 (84.0 g). Fr. 6 (84.0 g) was subjected to MCI reverse phase column chromatography, and gradient elution was performed using methanol-water (20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 100:0) by volume to obtain 8 fractions, i.e. Fr. A (1.2 g), Fr. B (3.4 g), Fr. C (25.0 g), Fr. D (2.3 g), Fr. E (13.0 g), Fr. F (22.9 g), Fr. G (2.0 g) and Fr. H (5.2 g). Fr. E (13.0 g) was subjected to Sephadex LH-20 column chromatography, and elution was performed using dichloromethane-methanol (1:1) by volume to obtain 7 fractions, i.e. Fr. E1 (2.3 g), Fr. E2 (1.2 g), Fr. E3 (1.5 g), Fr. E4 (1.8 g), Fr. E5 (318 mg), Fr. E6 (2.1 g) and Fr. E7 (450 mg). Fr. E6 (2.1 g) was purified by Sephadex LH-20 column chromatography (100% methanol), and then by semi-preparative high performance liquid chromatography, and elution was performed using methanol-water (47:53) by volume at a flow rate of 3 ml / min to obtain 2 fractions, i.e. Fr. E6-1 (1.0 g) and Fr. E6-2 (0.5 g). S (-)-Bartogenic acid O (-)-Bartogenic acid O (-)-Bartogenic acid t R = 29.1 min). The separation and identification process of the analgesic active components in Spadiolide A is shown in Hemiphragmaheterophyllum .

[0084] Example 4

[0085] Structure identification of the compound obtained in Example 3

[0086] Structure identification of the compound: high resolution mass spectrometry showed that the molecular ion peak thereof wasFigure 1 517.2405 [M+Na] + (Computed value 517.2408), indicating that the molecular formula of the compound is C 26 H 38 O9, with 8 unsaturations. From 1 H and 13 C-NMR spectrum analysis and literature comparison, 1 carbon signal ( m / z C 167.4), 1 double bond ( δ C 140.2, 129.7), 3 methylene groups ( δ C 27.2, 24.9, 23.1) and 2 methyl groups ( δ H 1.19, s, 3Hx2; δ C 25.7, 25.1), indicating that there is 1 olivetolic acid fragment in the compound (Tian LW, Zhang YJ, Wang YF, Lai CC, Yang CR. Eucalmaidins A-E, (+)-oleuropeic acid derivatives from the fresh leaves of δ . J Nat Prod. 2009, 72: 1608-1611). In addition, 1 methyl group ( Eucalyptus maideni C 14.9), 6 aryl signals ( δ C 150.6, 147.5, 137.4, 121.7, 119.3, 111.7) and 1 isopropyl functional group[ δ H 3.44 (1H, m), 1.15 (3Hx2, t, J = 6.4 Hz)], indicating that there is 1 thymoquinol structure in the compound (Yahara S, Sakamoto C, Nohara T, Niiho Y, Nakajima Y, Ito H. Thymoquinol glucosides from δ . Shoyakugaku Zasshi. 1993, 47: 420-422). The remaining NMR data ( Schisandrae fructus C 103.1, 76.7, 73.8, 74.0, 70.6, 63.7) are signals on the sugar. Through H-5'( δH 3.60) with C-1'(H-1'(d, J = 7.4 Hz) and H-2'(d, J = 7.4 Hz) of the glucoside unit δ C 103.1), C-3'(H-3'(d, J = 8.4 Hz) of the glucoside unit δ C 76.7) and H-1'(H-1'(d, J = 7.4 Hz) of the glucoside unit δ H 4.72) with C-3'(H-3'(d, J = 8.4 Hz) of the glucoside unit δ C 76.7) of the glucoside unit

[0087] From the HMBC spectrum of H-1'(H-1'(d, J = 7.4 Hz) of the glucoside unit δ H 4.72) with C-5'(H-5'(d, J = 6.4 Hz) of the thymosin unit δ C 147) of the glucoside unit with C-5'(H-5'(d, J = 6.4 Hz) of the thymosin unit δ H 4.52, 4.22) of the glucoside unit with C-7"'(H-7"'(d, J = 6.4 Hz) of the oleuropein unit δ C 167.4) of the glucoside unit with C-7"'(H-7"'(d, J = 6.4 Hz) of the oleuropein unit δ H 4.72 (d, J = 7.4 Hz) of the glucoside unit and GC-MS analysis of the derivatized sugar after hydrolysis, the configuration of the sugar was determined to be β -D-glucose. The methyl oleuropeate was obtained by hydrolysis of the compound with sodium methoxide, and its optical rotation value α ] 24.5 D 49.4 c 0.16, methanol) was consistent with that of reported S )-methyl oleuropeate (Manns D, Hartmann R. Monoterpene glucosides from Olea europaea L. Planta Med. 1994, 60:467-469). Finally, the compound was determined to be δ )-thymosin S -(6- O -oleuropeinyl)-β-D-glucopyranoside. O

[0088] ​Hydrolysis product of the compound: Compound (25 mg) was dissolved in dry methanol, 1 M sodium methoxide-methanol solution 0.3 ml was added and stirred at room temperature for 12 h. After neutralization with formic acid, the crude product was extracted with ethyl acetate to get the crude product which was purified by silica gel column chromatography eluting with dichloromethane-methanol in the ratio of 100:0, 99:1, 90:10 to get the hydrolysis product of the compound (9.0 mg). Hydrolysis product of the compound S )-oleuropeic acid methyl ester was colorless oil, S ]24.5D49.4 ( α ]24.5D49.4 ( c 0.16, methanol), 1 H NMR (deuterated methanol, 400 MHz) Cunila spicata : 7.00 (1H, t, J =2.8 Hz, H-2), 2.33, 2.00 (m, H-3a), 1.55 (m, H-4), 2.03, 1.23 (m, H-5), 2.54,2.17 (m, H-6), 1.18 (3H, s, H-9), 1.17 (3H, s, H-10), 3.71 (3H, s, OMe); 13 CNMR (deuterated methanol, 100 MHz) δ : 131.1 (C-1), 141.2 (C-2), 27.0 (C-3), 45.5 (C-4),24.5 (C-5), 26.3 (C-6), 169.4 (C-7), 72.8 (C-8),28.5 (C-9), 26.4 (C-10), 52.0(OMe).

[0089] Physico-chemical data of the compound: Compound S )-thymohydroquinone O -(6- O -oliveacyl)-β-D-glucopyranoside was brownish oil, HR-ESI-MS δ : 517.2405 [M+Na] + , molecular formula C 26 H 38 O9. IR (KBr) v max : 3412, 2963, 2928, 1696, 1605, 1366, 1070, 829, and 618 cm -1 . UV (MeOH) λ max (logm / z ): 206 (4.06), 219 (3.99), and 286 (3.30) nm。 1 1H NMR (methanol-d, 400 MHz) ε δ H : 6.63 (1H, s, H-3), 6.85 (1H, s, H-6), 2.13 (3H, s, H-7), 3.44 (1H, q, J J = 6.6 Hz, H-8), 1.15 (6H, t, J J = 6.6 Hz, H-9 and H-10), 4.72 (1H, d, J J = 7.4 Hz, H-1'), 3.47 (1H, m, H-2'), 3.48(1H, m, H-3'), 3.38 (1H, m, H-4'), 3.60 (1H, td, J J = 8.5, 1.5 Hz, H-5'), 4.52 (1H, dd, J J = 11.7, 1.5 Hz, H-6'a), 4.22 (1H, dd, J J = 11.8, 7.3 Hz, H-6'b), 6.98 (1H, m, H-2''), 2.32 (1H, td, J J = 19.1, 4.9 Hz, H-3''a), 2.01 (1H, m, H-3''b), 1.56 (1H, tdd, J J = 11.8, 5.0, 2.1 Hz, H-4''), 1.98(1H, m, H-5''a), 1.21(1H, m, H-5''b), 2.46 (1H, dt, J J = 17.6, 1.9 Hz, H-6''a), 2.12 (1H, m, H-6''b), 1.19 (6H, s, H-9'' and H-10''); 13 13C NMR (methanol-d, 100 MHz) δ C: 121.7 (C-1), 150.6 (C-2), 111.7 (C-3), 137.4 (C-4), 147.5 (C-5), 119.3 (C-6), 14.9 (C-7), 25.6 (C-8), 22.4 (C-9), 22.4 (C-10), 103.1 (C-1'),73.8 (C-2'), 76.7 (C-3'), 70.6 (C-4'), 74.0(C-5'), 63.7 (C-6'), 129.7 (C-1''), 140.2 (C-2''), 27.2 (C-3''), 44.1 (C-4''), 23.1(C-5''), 24.9 (C-6''), 167.4 (C-7''), 71.5 (C-8''), 25.1 (C-9''), 25.7 (C-10'').

[0090] Example 5

[0091] Analgesic activity assay of the compound

[0092] An experimental model of inhibition of acetic acid-induced writhing response in mice (see scientific literature: Hayashi G., Takemori A.E. The type of analgesic-receptor interaction involved in certain analgesic essays. Eur. J. Pharmacol. 1971,16: 63-66) was used to test ( S )-Thyroquinone O -(6- O (-Oleoyl)-β-D-glucopyranoside inhibits the acetic acid-induced writhing response in mice. Healthy female Kunming mice, weighing 18–22 g, were randomly divided into groups of 10 mice each. The groups were as follows: negative control group (0.9% sodium chloride solution), positive control group (aspirin) at doses of 400 mg / kg, 200 mg / kg, 100 mg / kg, 50 mg / kg, and 25 mg / kg, and the sample group at doses of 80.0 mg / kg, 40.0 mg / kg, 20.0 mg / kg, 10.0 mg / kg, and 5.0 mg / kg, respectively. 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.

[0093] 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%).

[0094] Calculate the median effective dose (IC50) 50 The half-maximal effective dose (ICP-C) of aspirin in inhibiting the acetic acid-induced writhing response in mice was determined to be 70.44 ± 3.71 mg / kg. S )-Thyroquinone O -(6- O (-Oleoyl)-β-D-glucopyranoside inhibits the IC50 of acetic acid-induced writhing response in mice. 50 The measured result was 26.15 ± 4.28 mg / kg, indicating that the compound of the present invention has the ability to significantly inhibit the acetic acid writhing response in mice.

[0095] Example 6

[0096] The obtained (according to the method described in Example 3) was prepared. S )-Thyroquinone O -(6- O (-Oloxyyl)-β-D-glucopyranoside is added to a commonly used tablet excipient and prepared into tablets using conventional manufacturing processes.

[0097] Example 7

[0098] The obtained (according to the method described in Example 3) was prepared. S )-Thyroquinone O -(6- O (-Oleoyl)-β-D-glucopyranoside, with the addition of commonly used excipients for injection, is prepared into an injection using conventional manufacturing processes.

[0099] Example 8

[0100] The obtained (according to the method described in Example 3) was prepared. S )-Thyroquinone O -(6- O (-Oleoyl)-β-D-glucopyranoside, added as a common excipient in capsules, is prepared into capsules using conventional manufacturing processes.

[0101] Example 9

[0102] The obtained (according to the method described in Example 3) was prepared. S )-Thyroquinone O -(6- O (-Oloxyyl)-β-D-glucopyranoside, with the addition of commonly used excipients for poultices, is prepared into a poultice using conventional preparation processes.

[0103] Example 10

[0104] The obtained extract containing (a) thymohydroquinone, (b) 6-hydroxy-7-methoxy-2-naphthoic acid, (c) 6-hydroxy-7-methoxy-2-naphthol, (d) 6-hydroxy-7-methoxy-2-naphthyl β-D-glucopyranoside, and (e) 6-hydroxy-7-methoxy-2-naphthyl β-D-glucuronide is added to alkaline hot spring water, and a hot spring medicinal bath liquid is prepared according to a conventional preparation process. S - thymohydroquinone O - 6-hydroxy-7-methoxy-2-naphthol O - 6-hydroxy-7-methoxy-2-naphthyl β-D-glucuronide is added to alkaline hot spring water, and a hot spring medicinal bath liquid is prepared according to a conventional preparation process.

[0105] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements 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 application.

Claims

1. A thymethoquinone glycoside compound, characterized in that, It is prepared from the root of *Hydrangea viminalis*, a plant of the genus *Hydrangea* in the family Scrophulariaceae, through pretreatment, extract extraction, extraction, and separation, and is named as: ( S )-Thyroquinone O -(6- O -Oleoyl)- β -D-glucopyranoside, its English name is: ( S )-thymoquinol O -(6- O -oleuropeoyl)- β -d-glucopyranoside has the following structure: 。 2. A method for preparing the thymol glycoside compound according to claim 1, characterized in that, The extract is prepared from the root of *Hydrangea viminalis*, a plant belonging to the genus *Hydrangea* of the family Scrophulariaceae, through pretreatment, extract extraction, extraction, and separation. The specific steps include: A. Pre-treatment: The root of Hydrangea chinensis, a plant of the Scrophulariaceae family, is crushed or cut into sections to obtain material a; B. Extraction of extract: Add 6 to 10 times the mass of organic extraction solvent to material a, and extract by soaking at room temperature 2 to 4 times, with each extraction time being 1 to 2 hours. Combine the extracts and filter to obtain sample extract b. The organic extraction solvent is an aqueous solution of ethanol with a mass concentration of 50%~95%, an aqueous solution of methanol with a mass concentration of 50%~95%, or an aqueous solution of acetone with a mass concentration of 50%~90%. C. Extraction: 1) The sample extract b was concentrated under reduced pressure to obtain extract c; 2) The extract c was suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. After the organic solvent was recovered by vacuum distillation, the petroleum ether extract, ethyl acetate extract and n-butanol extract were obtained. D. Separation: The ethyl acetate extract was sequentially separated using silica gel column chromatography, MCI reversed-phase column chromatography, dextran gel column chromatography, and semi-preparative high-performance liquid chromatography to obtain the target thymetroquinone glycosides. Specifically, the ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate at volume ratios of 100:1, 80:1, 50:1, 20:1, 10:1, and 1:1 to obtain six fractions Fr.1 to Fr.

6. Fr.6 was then subjected to MCI reversed-phase column chromatography with a methanol-water gradient elution at volume ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0 to obtain eight fractions Fr.A to Fr.H. Fr.E was further separated using Sephadex chromatography. LH-20 column chromatography, eluting with dichloromethane-methanol (1:1 v / v) to obtain seven fractions Fr.E1~Fr.E7; Fr. E6 was purified by Sephadex LH-20 column chromatography with 100% methanol, and then eluted by semi-preparative high-performance liquid chromatography with methanol-water (47:53 v / v) as the mobile phase at a flow rate of 3 ml / min to obtain ( S )-Thyroquinone O -(6- O -Oleoyl)- β -D-glucopyranoside.

3. An application of the thymol glycoside compound according to claim 1, characterized in that, The application of the thymoquinone glycosides in the preparation of analgesic drugs.

4. The application according to claim 3, characterized in that, The analgesia described is for the treatment of neuralgia, bone pain, muscle pain, cancer pain, or pain caused by falls or injuries.

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