CN1915994A - Indole group alkaloid of sector Gouyahua, and application for preparing drug-breaking medicine - Google Patents

Abstract

The invention relates to the technical field of natural medicinal chemistry, and discloses a new Ervatamia flabelliformis Tsiang indole alkaloid extracted and separated from Ervatamia flabelliformis Tsiang, namely flabelliformidine (EF- 14) structure and its extraction and separation method; and the indole alkaloids and their salts prepared from Bergamot scallopedus including the new alkaloid EF-14 are used in the preparation of drugs or food for the prevention and treatment of opioid dependence. Use of analgesics.

Description

Indole alkaloids of scalloped bermudagrass and its use in the preparation of detoxification drugs

technical field

The present invention relates to the field of natural medicinal chemistry and medical technology, and relates to extracting and separating new Ervatamiaf labelliformis Tsiang indole alkaloids from Ervatamiaf labelliformis Tsiang, and including the new alkaloids. The application of the indole alkaloids in the preparation of drugs or food for the prevention and treatment of opioid dependence.

Background technique

Fan-shaped dogtooth flower (Ervatamia flabelliformis Tsiang) is a common plant in tropical and subtropical Asia. It is wild and cultivated in various provinces and regions in southern my country. All parts of the plant can be used medicinally. Treatment of hypertension, sore throat, rheumatic arthralgia and other diseases [Editorial Committee of Flora of China, Chinese Academy of Sciences: Flora of China, 1977, Vol. 63, pp. 106-107]. We studied the chemical constituents and pharmacological activity of the plant, extracted and separated the alkaloid components and obtained 14 alkaloid monomers from the plant, and found no reports on the chemical constituents of Bermudagrass fanii through literature search. There is no report on its total alkaloids or monomeric alkaloids for the prevention and treatment of opioid mental and physical dependence and analgesic effects.

Contents of the invention

The purpose of the present invention is to find safe and effective natural products from natural products for the preparation of drugs or food for the prevention and treatment of opioid dependence. After continuous efforts, a new fan-shaped dog's tooth flower was finally extracted and separated from fan-shaped dog's tooth flower Indole alkaloids, EF-14: flabelliformidine; at the same time, the inventors extracted sufficient amounts of other known flabelliformidine indole alkaloids, and found flabelliformidine The total alkaloids of the indole alkaloid Bermudagrass scalloped flower and its monomer salts can be used to prepare medicines or food for preventing and treating opioid dependence or to prepare analgesic medicines. The present invention has thus been accomplished.

The present invention is accomplished through the following technical scheme: extracting the root and stem of Bermudagrass with 95% ethanol, concentrating the extract under reduced pressure to obtain the extract, dissolving it with the extract in 1% hydrochloric acid aqueous solution, and separating the acid solution, adding ammonia water to the acid solution to adjust the pH to 9-10, precipitated, filtered and dried to obtain the indole alkaloid components (alkaloid total alkaloids, represented by EFA), the total alkaloids were further chromatographically separated into the 13 known indole alkaloid monomers: EF-1: ervatamine [Yu Yang, Gao Jinming, Liu Jikai. Chemical constituents of Bermudagrass yunnanensis. Yunnan Plant Research, 1999, 21(3): 399-405], EF-2: 20-epiervatamine (20-epiervatamine) [Yu Yang, Gao Jinming, Liu Jikai. Chemical constituents of Bermudagrass yunnanensis. Yunnan Plant Research, 1999, 21(3): 399-405; Pascale Clivio, Bernard Richard, Monique Zeches, et al. Alkaloids from the leaves and stem bark of Ervatamia Malaccensis. Phytochemistry, 1990, 29(8), 2693-2696] , EF-3: 20-epi-dregamine (20-epi-dregamine) [Alain Ahond, Anne-Marie Bui, Pierre Potier.Carbon-13 nuclear magnetic resonance analysis of vobasine-likeindole alkaloids.J.Org.Chem., 1976, 41(10), 1878-1879; Yu Yang, Liu Jikai. Chemical constituents of Bermudagrass. Yunnan Plant Research, 1999, 21(2): 260-264], EF-4: dregamine [Alain Ahond, Anne-Marie Bui, Pierre Potier.Carbon-13nuclear magnetic resonance analysis of vobasine-like indole alkaloids .J.Org.Chem., 1976, 41(10), 1878-1879;], EF-5: dihydropeivine (dihydropeivine)[Total synthesis of dregamine and epidregamine.Ageneral route to 2-acylindole alkaloids. Journal of the American Chemical Society, 1978, 100(3), 938-43], EF-6: Vobasine [XiaoZhang Feng, Christiane Kan, Pierre Potier, Siew-Kwong Kan and MauriLounasmaa. Monomeric indole alkaloids from Ervatamia hainanensis.PlantaMed.1982,44:212-214], EF-7: coronaridine [Emi Okuyama, Li-Hon Gao and Mikio Yamazaki. Analgesic components from Borneanmedicinal plants, Tabernaemontana apon pauciflora and Taacquiflora and Bluem and Blume .Chem.Pharm.Bull.1992, 40(8):2075-2079], EF-8: 10-methoxycrown cinnamon (voacangine) [Kingston DG.Plant anticancer agents VI: Isolation of voacangine, voacamine , and epivoacorine from Tabernaemontana arborea sap.J Pharm Sci.1978 Feb; 67(2):271-2], EF-9: 10-methoxy-3-hydroxy-crown bermudarine (3-hydroxyvoacangine)[Albrto Madinaveitia , Matias Reina, Gabriel de la Fuente, et al.Obovamine, a New IndoleAlkaloid from Stemmadenia obovata.J.Nat, Prod., 1996, 59(2), 185-9], EF-10: 10-methoxycrown Voacanginehydroxyindolenine [Albert Madinaveitia, Gabriel de la Fuente, AntonioGonzalez. The Absolute configuration at C(7) of VoacangineHydroxyindolenine. Helvetica Chinica Acta, 1998(81), 1645-1653], EF-1 : coronaridine hydroxyindolenine (coronaridine hydroxyindolenine) [AlbrtoMadinaveitia, Matias Reina, Gabriel de la Fuente, et al.Obovamine, a New Indole Alkaloid from Stemmadenia obovata. J.Nat.Prod., 1996, 59 (2) , 185-9], EF-12: voaphylline [Pascale Clivio, Bernard Richard, Monique Zeches, et al. Alkaloids from the leaves and stem bark of Evatamia Malaccensis. Phytochemistry, 1990, 29 (8), 2693- 2696], EF-13: (-) Melanide [(-)-mehranine] [Janos Eles, Gyogy Kalaus, Istvan Greiner, et al.Synthesis of Vinca Alkaloids and Related Compounds.100.Stereoselective Oxidation Reactions of Compounds with the Aspidospermane and Quebrachamine Ring System. First Synthesis of Some Alkaloids Containing the Epoxy Ring. J. Org. Chem, 2002, 67, 7255-7260].

We also obtained a new alkaloid EF-14: flabelliformidine, whose spectral identification data [high-resolution electrospray mass spectrometry (HRESI-MS) showed quasi-molecular ion peaks: [M+H] ⁺ m/ z353.1867 (calculated value 353.1865), giving the molecular formula as C ₂₁ H ₂₄ N ₂ O ₃ ; Proton Nuclear Magnetic Resonance ( ¹ HNMR) (CDCl ₃ , δ): 2.53, (1H, dt, J=10.2, 2.4Hz , 3-Ha), 3.20 (1H, dt, J=10.2, 4.5Hz, 3-Hb), 4.97 (1H, d, J=12.0Hz, 5-Ha), 5.13 (1H, d, J=12.0Hz , 5-Hb), 8.199 (1H, dd, J=6.6, 1.2Hz, 11-H), 1.90 (1, m, 14-H), 1.13 (1H, m, 15-Ha), 1.92 (1H, m, 15-Hb), 1.81 (1H, m, 17-Ha), 3.06 (1H, dq, J=13.8, 1.2Hz, 17-Hb), 0.91 (3H, t, J=7.5Hz, 18-H ), 1.55 (2H, m, 19-H), 1.69 (1H, m, 20-H), 3.14 (1H, s, 21-H), 3.71 (3H, s, OCH ₃ ), 10.06 (1H, s , CHO); carbon nuclear magnetic resonance spectrum ( ¹³ CNMR) (CDCl ₃ , δ): 149.6 (C-2), 52.5 (C-3), 66.8 (C-5), 111.4 (C-7), 126.6 (C -8), 123.2(C-9), 120.6(C-10), 123.3(C-11), 109.2(C-12), 134.3(C-13), 25.7(C-14), 30.7(C- 15), 46.5(C-16), 35.8(C-17), 11.5(C-18), (C-), 27.0(C-19), 34.5(C-20), 56.7(C-21), 173.0 (C-22), 53.0 ( _OCH3 ), 183.4 (CHO)].

The chemical structures of 14 indole alkaloids are as follows:

EF-1 R ₁ =H R ₂ =CH ₂ CH ₃ EF-2 R ₁ =CH ₂ CH ₃ R ₂ =H

EF-3 R ₃ =HR ₄ =CH ₂ CH ₃ R ₅ =CH ₃

EF-4 R ₃ =CH ₂ CH ₃ R ₄ =H R ₅ =CH ₃

EF-5 R ₃ =H R ₄ =CH ₂ CH ₃ R ₅ =H

We have carried out related research on medical use, using the mouse model of morphine hydrochloride conditioned place preference mental dependence; morphine hydrochloride and naloxone hydrochloride dose-increasing physical dependence model tests, and found that the biological Alkali component (Ervatamiaflabelliformis alkaloids is called for short EFA) has opioid substance mental dependence and physical dependence and analgesic action, therefore, can use alkaloid component (EFA) in the fan-shaped dog's tooth flower to prepare and prevent and treat opioid substance dependence The purposes of sex medicine or food, the total alkaloids and monomer salts of the dog tooth flower of the present invention can be used for the preparation of medicines or food for the prevention and treatment of opioid dependence or the preparation of analgesic medicine.

Detailed ways

The present invention will be further illustrated by the pharmacological activity test and results of total alkaloids (total Ervatamiaflabelliformis alkaloids abbreviated as tEFA) in Bermudagrass fantocia below by embodiment.

Example 1 Alcohol extraction method to prepare total alkaloids

14 kg of dried rhizomes of Bermudagrass fanii, crushed and extracted three times under reflux with 8 times the amount (W/W) of 95% ethanol, concentrated the extract under reduced pressure until ethanol-free to obtain alcohol extract. After the extract was dissolved with 2000ml of 2% hydrochloric acid, the acid-insoluble matter was filtered off. The acidic water solution was adjusted to pH 8-10 with concentrated ammonia water, and a precipitate was formed, and 500ml of chloroform was added to extract three times. After combining the extracts, the chloroform was removed under reduced pressure, and the purified total Ervatamia flabelliformis alkaloids (tEFA for short) was obtained. .

Example 2 Silica gel column chromatography to prepare the alkaloid monomer of the fan-shaped dog's tooth flower

15g of total alkaloids of Bermudagrass fanii, separated by silica gel column chromatography (φ6*80cm, 450g), gradient washing with chloroform-methanol (100:1, 50:1, 25:1, 10:1, 8:1, 5:1) Take off, collect every 1000ml, and divide into 1-24 parts. After recovering the solvent under reduced pressure, the crystallization of compound EF-02, 03, 05, 06 and 08 can be obtained from the 5th, 7th, 10th, 13th and 14th parts. The 9th part (2.2 g) was separated by silica gel column chromatography (φ4 *40cm, 80g), petroleum ether-ethyl acetate (20:1, 10:1, 5:1) gradient elution could give compounds EHA-01, 04, 07 and 09. The 16th part (1.8g) was separated by silica gel column chromatography (φ2*40cm, 35g), and the compound EF-10 could be obtained after gradient elution of petroleum ether-ethyl acetate (5:1, 3:1, 1:1). 11 and 12. The eighteenth fraction (1.4g) was separated by silica gel column chromatography (φ2*40cm, 32g) again, and compound EF-13 and 14 could be obtained after gradient elution with chloroform-methanol (5:1, 3:1).

Example 3 The effect on the mental dependence of morphine hydrochloride and its own mental dependence experiment

3.1 Materials and methods

3.1.1 Animals: Male SD rats: 160-200 g, purchased from Shanghai Xipuer-Bikay Experimental Animal Co., Ltd. Male Kunming strain mice (clean grade): 18-22 g, provided by the Department of Experimental Animal Science, Fudan University. The animals were kept in an animal room with natural light, and they had free access to food and water.

3.1.2 Drugs and reagents

Morphine Hydrochloride (Morphine for short, code-named Mor in the table): Use physiological saline with a pH of 3.5 to 4 to make an application solution.

Total Ervatamia flabelliformis alkaloids (total Ervatamia flabelliformis alkaloids referred to as tEFA): Use physiological saline with a pH of 3.5-4 to make an application solution.

Hydrocortisone: use physiological saline to prepare the application solution.

3.2. Administration method

3.2.1 Administration method of morphine hydrochloride

In the morning, subcutaneously inject 4.0 mg·kg ^-1 of morphine hydrochloride into the white box (with medicine box), and in the afternoon, subcutaneously inject 10 ml·kg ^-1 of normal saline into the black box (without medicine box). Stay in the box for 50 minutes each time for 5 consecutive days.

3.2.2 Administration method

The mice were randomly divided into normal saline group, morphine hydrochloride 4.0 mg·kg ^-1 group, morphine hydrochloride + tEFA 10 mg·kg ^-1 group. Immediately after each subcutaneous injection of morphine hydrochloride, tEFA was injected intraperitoneally for 5 consecutive days.

3.3. Experimental method

3.3.1 Morphine mental dependence model, using mouse conditioned place preference test [Wan Xingwang, Huang Mao, Li Wanhai, Tan Yexiong, Tao Xuebin. Effect of N-nitro-L-arginine on morphine and two Inhibitory effect of hydroetorphine on mental dependence. Journal of Second Military Medical University 1999; 20(2): 105-107]

Conditional position preference box The conditional position preference shuttle box used in this experiment adopts a "straight line" two-box design, and the material is black plexiglass with a wall thickness of 1 cm. There are two left and right boxes, one black and one white. The inner diameter of the black and white boxes is 15cm×15cm×35cm. There is a shuttle door in the middle of the box, which is a twitchable baffle. The observation box has the effect of light insulation and sound insulation, and the sound intensity attenuation is 20-25dB. On the top of each box, white lighting and infrared LEDs are installed, and there are ventilation channels and small fans; both black and white boxes are equipped with a micro camera, and there are connectors for DC 12V power lines and A/V video lines ; There is a backing plate at the bottom of the box, which can effectively prevent the reflection of light and make the image clear.

Selection of experimental animals Male mice were selected. Open the gates of the two chambers, put the mice under the middle gate, and perform video acquisition at the same time, with a sampling speed of 15 frames per second. And allowed to explore the black and white box for a total of 2 minutes, and then record the time the mouse stayed in the black box and the white box within 15 minutes. After 3 days of continuous experimentation, mice with a clear preference for the black box were selected as experimental mice.

Training animals The experimental mice were randomly divided into groups according to the time of staying in the black box (without medicine box). After subcutaneous injection of morphine hydrochloride 4 mg·kg ^-1 in the morning, put them into the white box (with medicine box), and in the afternoon, after subcutaneous injection of normal saline, put them into the black box (without medicine box). Stay in the box for 50 minutes each time, for 7 consecutive days. Mice trained with morphine hydrochloride will change their natural preference, from a preference for the black box to a preference for the white box.

Observation of animals The mice that completed the training were not given any drugs on the 8th day, and were placed in the shuttle box alone, the doors of the two rooms were opened, and the black and white boxes were allowed to explore for 2 minutes, and then the mice were observed to stay in the preferred side (black box) within 15 minutes time.

3.3.2 Morphine relapse model in mice

Using the above-mentioned conditioned place preference box and method, the mice were trained for 7 days by subcutaneous injection of 4 mg·kg ^-1 morphine hydrochloride every day. On the 8th day, the administration of morphine hydrochloride was stopped, and the residence time of the mice on the preferred side (black box) was observed by the above method. It can be seen that the mice obviously preferred the white box (drug mixing box). After that, observations were made every other day until the position preference effect was observed to recede on the 7th day. After another 4 days, on the 11th day after stopping the administration of morphine hydrochloride, the mice were given foot shock (foot shock) or subcutaneous injection of hydrocortisone 20 mg·kg ^-1 , and immediately placed in a conditioned place preference box for observation .

The method of electric shock: use a box with a cover size of L 35cm×H 28cm×W 23cm, place the mouse on the stainless steel grid (0.5cm interval) at the bottom of the box, and shock the foot by transmitting a direct current. Squeaking or bouncing. The DC current was output by a CH-2 high-voltage constant-current electric stimulator with a constant voltage of 36V, an initial DC current of 0.1mA, and an increase of 0.1mA every 10 minutes for a total of 30 minutes of electric shock.

3.3.3 Mouse relapse model and the blocking effect of tEFA on the reproduction of place preference in morphine mice

3.4 Statistical processing of data

The Student t-test was used to compare between the administration group and the normal saline control group or between different administration groups. All data are presented as mean ± standard deviation (x ± s).

3.5. Experimental results

3.5.1 The effect of tEFA on morphine hydrochloride conditioned place preference and its mental dependence

3.5.1.1 Effects of tEFA administration on morphine hydrochloride conditioned place preference in mice and the mental dependence of tEFA itself

There was no difference in the residence time of the mice in the white box before and after administration in the tEFA 10 mg·kg ^-1 group. There was no difference in the residence time of mice in tEFA 10mg·kg ^-1 , EF-6 5mg·kg ^-1 , EF-7 5mg·kg ^-1 , EF-8 5mg·kg ^-1 groups before and after administration in the white box. The results are shown in Table 1.

Table 1. Effects of continuous administration of tEFA and monomeric EF-6~EF-8 on morphine hydrochloride conditioned place preference in mice and the mental dependence of tEFA itself. x±s. **P<0.01, compared with before administration; ##P<0.01, compared with normal saline group.

* Drugs Dose (mg·kg ^-1 ) Time spent in the white box (x±s, seconds) Mor(sc) drug(ip) Before administration After administration Saline MortEFAMor+EFAMor+EF-6Mor+EF-7Mor+EF-8 --44444 ----1010555 377±93410±75386±89398±83413±67394±83401±71 398±103526±116**##406±116532±158538±178472±184462±153

*Mor is morphine hydrochloride; the code in brackets is the route of administration, sc is subcutaneous injection, and ip is intraperitoneal injection.

3.5.2 Blocking effect of EFA on morphine "relapse"

3.5.2.1 Disappearance of conditioned place preference effect in morphine-dependent mice 1 to 3 days after the end of the 7-day morphine training period, the mice maintained the same intensity of conditioned place preference effect; on the 5th day, the conditioned place preference effect began to weaken , basically subsided on the 7th day (results are shown in Table 2). Therefore, selection of mice on the 11th day after the end of training induced the reappearance of the conditioned place preference effect.

Table 2. Place preference responses of mice in the same group at different times after the morphine training period.

Number of mice in each group=9, x±s. Compared with before giving morphine, ^a P>0.05, ^b P<0.05, ^c P<0.01;

Compared with normal saline group, ^d P>0.05, ^e P<0.05, ^f P<0.01. Medication (subcutaneous injection) Dose (mg·kg ^-1 ) Before giving morphine Staying time on the preferred side (x±s, seconds) Different times after cessation of morphine Day 1 3rd day day 5 day 7 Day 9 Saline Morphine Hydrochloride --4 601±46558±42 ^d 554±44 ^a 348±133 ^{c, f} 546±66 ^a 366±120 ^c,f 563±65 ^a 369±85 ^c,f 544±65 ^a 425±147 ^{b, d} 548±63 ^a 501±96 ^{a, d}

3.5.2.2 Blocking effect of EFA on reappearance of place preference in mice induced by plantar shock

On the 11th day after the end of morphine training, for mice whose place preference had disappeared, plantar electric shock could reproduce the place preference effect induced by 4 mg·kg ^-1 morphine that had disappeared, while normal saline did not change their place preference. A single administration of tEFA 40mg·kg ^-1 could block this place preference reappearance. The results are shown in Table 3.

Table 3. Blocking effect of a single tEFA on reappearance of place preference in mouse plantar shock-evoked extinction. Number of mice in each group = 9, x±s. Compared with before plantar electric shock, ^a P>0.05, ^b P<0.05, ^c P<0.01; compared with after plantar electric shock, ^d P>0.05, ^e P<0.05, ^f P<0.01; ^g P>0.05, ^h P<0.05, ⁱ P<0.01 compared with normal saline feet. drug Dose (mg·kg ^-1 ) Time to stay on the preferred side (x±s, seconds) Before plantar shock After plantar shock *tEFA40 Saline Morphine Hydrochloride -4 542±66501± ^96g 546±61 ^a 386±37 ^{c, i} 553±66 ^a 703±256 ^f

*EFA 40 is a single intraperitoneal injection of total alkaloid hydrochloride 40mg·kg ^-1 of Bermudagrass fanii.

3.5.2.3 Blocking effect of single administration of tEFA on reappearance of place preference in hydrocortisone-induced extinction in mice

On the 11th day after the end of morphine training, subcutaneous injection of hydrocortisone 20 mg·kg ^-1 to the mice whose place preference had disappeared could reproduce the place preference effect caused by 4 mg·kg ^-1 morphine which had disappeared, while physiological Saltwater has no change to its position selection. A single administration of tEFA 40mg·kg ^-1 could block this place preference reappearance. The results are shown in Table 4.

Table 4. Blocking effect of single administration of tEFA on reappearance of place preference induced by hydrocortisone (subcutaneous injection, 20 mg·kg ^-1 ) in extinction in mice. Number of mice in each group = 8, x±s. Compared with hydrocortisone before challenge, ^a P>0.05, ^b P<0.05, ^c P<0.01; compared with hydrocortisone after challenge, ^d P>0.05 , ^e P<0.05, ^f P<0.01; compared with normal saline group, ^g P>0.05, ^h P<0.05, ⁱ P<0.01. Medication (subcutaneous injection) Dose (mg·kg ^-1 ) Time to stay on the preferred side (x±s, seconds) Before hydrocortisone challenge After hydrocortisone challenge *EFA40 normal saline -- 531±73 588± ^87a 561±78 ^a

Morphine Hydrochloride 4 586± ^96g 331±148c ^,i 761± ^151f

*EFA40 is a single intraperitoneal injection of 40mg·kg ^-1 total alkaloid hydrochloride of Bermudagrass fanii.

Example 4 The analgesic effect of EFA and its influence on the analgesic effect of morphine hydrochloride

4.1 Materials and methods

4.1.1 Experimental animals

Female Kunming strain mice (clean grade): 18-22 g, provided by the Department of Experimental Animal Science, Fudan University, Shanghai, kept in animal rooms with natural light; room temperature: 20-25°C, daily temperature difference less than 3°C; free to eat and drink.

4.1.2 Reagents and their preparation

Morphine Hydrochloride (Morphine for short, code name Mor in the table): product of Qinghai Pharmaceutical Factory, made into application solution with physiological saline with pH 3.5-4.

Naloxone Hydrochloride (Naloxone Hydrochloride, referred to as Naloxone, code-named NLX in the table): Beijing Sihuan Pharmaceutical Factory, using physiological saline with a pH of 3.5-4 to make an application solution.

The total Ervatamia flabelliformis alkaloids (total Ervatamia flabelliformis alkaloids, referred to as the hydrochloride of tEFA) is prepared as an application solution with physiological saline with a pH of 3.5-4.

Hydrocortisone: Shanghai Xinyi Pharmaceutical Factory, made application solution with normal saline.

4.2 Administration method

4.2.1 Morphine

4.2.2 Dosing method for single administration of EFA

Mice were divided into random groups, i.e. normal saline group, morphine hydrochloride alone group, morphine hydrochloride+tEFA 20 mg·kg ^-1 group (denoted as Mor+tEFA ₂₀ ), morphine hydrochloride+tEFA 40 mg·kg ^-1 group (denoted as Mor+tEFA ₄₀ ) and the morphine hydrochloride+tEFA 80mg·kg ^-1 group (denoted as morphine hydrochloride+tEFA ₈₀ ).

4.3 Experimental method

Analgesic test - mouse hot plate (hot-plate) method, edited according to Xu Shuyun, Bian Rulian, Chen Xiu. Pharmacological Experimental Methodology. Second Edition. Beijing: People's Health Publishing House, 1991: 695

The hot plate instrument consists of a hot plate (copper skin material) placed in an electric heating constant temperature water bath. The temperature of the water bath is accurate to 0.1°C, and the temperature of the hot plate is maintained at 55°C±0.5°C. A stopwatch (Genque Brand Quartz Stopwatch, Shanghai Stopwatch Factory) records the time (unit s) from when the sole of the mouse touches the hot plate to when the rear paw is licked. . Mice with hot plate response latency (basic pain threshold) > 15 seconds but < 30 seconds were selected for the experiment. The basic pain threshold of each mouse is the average value of the pain response time of the mouse for 3 times, and the interval between the 4 times of measuring the pain response time is greater than 15 minutes; after administration, the mice with a latency > 60 seconds are withdrawn from the hot plate and treated with 60 seconds. seconds count. The room temperature was maintained at 23°C to 25°C during the experiment.

4.4 Statistical processing of data

T-test (Student t-test) was used for comparison between the administration group and the normal saline control group or between different administration groups. All data are presented as mean ± standard deviation (x ± s).

.5. Experimental results

4.5.1 Analgesic effect of EFA

tEFA has an analgesic effect, which increases with the increase of the dose, and is slow and persistent compared with morphine hydrochloride. Compared with morphine hydrochloride 8 mg·kg ^-1 , intragastric administration of tEFA 20 mg·kg ^-1 , tEFA 40 mg·kg ^-1 , tEFA 80 mg·kg ^-1 , and intraperitoneal injection of tEFA 40 mg·kg ^-1 all have significant The analgesic effect after intragastric administration is slower than that after intraperitoneal injection, but the intensity of the effect is equivalent to that after intraperitoneal injection, indicating that the absorption after intragastric administration is relatively complete. The results are shown in Table 5.

Table 5. Analgesic effect of tEFA on the hot plate method in mice. Number of mice in each group=9, x±s. Compared with normal saline group, ^a P>0.05, ^b P<0.05, ^c P<0.01; compared with morphine hydrochloride group, ^d P>0.05, ^e P<0.05, ^fP <0.01.

^* drugs Dose (mg·kg ^-1 ) Withdrawal latency (pain threshold) (x±s, seconds) before administration After administration Mor(sc) EFA 30 minutes 60 minutes 120 minutes 240 minutes Saline Morphine Hydrochloride EFA ₄₀ EFA ₂₀ EFA ₄₀ EFA ₈₀ --8-------- ----40(ip)20(ig)40(ig)80(ig) 24.1±2.8d ^24.2 ± ^1.8a 24.8± ^1.7a,d 25.3±1.6a ^,d 24.6±2.5a ^,d 24.0±3.8a ^,d 25.7±2.7 ^f 59.4±1.3 ^c 41.1±12.1 ^{c, f} 34.9±7.4 ^{c, f} 35.3±5.2 ^{c, f} 37.9±7.2 ^{c, f} 25.9±3.2 ^f 36.6±5.9 ^c 35.9±9.7 ^{c, d} 34.9±1.4 ^{c, d} 44.3±11.1 ^{c, d} 49.3±9.3 ^{c, f} 25.7±1.7 ^d 28.6±7.4 ^a 30.2±7.6 ^{a, d} 34.8±3.2 ^{c, e} 35.9±8.8 ^{c, d} 36.8±8.5 ^{c, e} 25.0±2.3 ^d 28.9±5.6 ^a 29.6±5.8 ^{b, d} 30.6±3.3 ^{c, d} 30.6±6.1 ^{b, d} 31.7±6.9 ^{b, d}

*Mor is morphine hydrochloride, tEFA ₂₀ , tEFA40, and tEFA ₈₀ are 20mg·kg ^-1 , 40mg·kg ^-1 or 80mg·kg ^-1 of total alkaloid hydrochloride of Bermudagrass fanii respectively; the codes in brackets indicate the route of administration , sc for subcutaneous injection, ip for intraperitoneal injection, ig for gavage (oral).

4.5.2 The effect of tEFA on the analgesic effect of morphine hydrochloride

Compared with morphine hydrochloride 4mg·kg ^-1 , tEFA 40mg·kg ^-1 has more obvious analgesic effect than morphine hydrochloride. At the same time, the analgesic effect of morphine hydrochloride could be canceled by giving the opioid receptor antagonist naloxone hydrochloride 1 mg·kg ^-1 . The analgesic effect of tEFA 40 mg·kg ^-1 can be partially blocked by intraperitoneal injection of 4 mg·kg ^-1 naloxone hydrochloride; when morphine hydrochloride 4 mg·kg ^-1 and tEFA 10 mg·kg ^-1 The analgesic effect of 4mg·kg ^-1 and tEFA 20mg·kg ^-1 appeared synergistic. The results suggest that the analgesic effect of tEFA may be achieved partly through opioid receptors. The results are shown in Table 6.

Table 6. The effect of tEFA on the analgesic effect of morphine on the mouse hot plate method. Number of mice in each group=9, x±s. Compared with normal saline group, ^a P>0.05, ^b P<0.05, ^c P<0.01; compared with morphine hydrochloride group, ^d P>0.05, ^e P<0.05, ^fP <0.01. ^* drugs ^* Dosage (mg·kg ^-1 ) Latency of hindlimb withdrawal (pain threshold, x±s, seconds) Mor(sc) NLX(ip) tEFA(ip) Before administration after administration 30 minutes 60 minutes 120 minutes 240 minutes

Normal Saline Morphine Hydrochloride Morphine Hydrochloride+NLXtEFA ₄₀ tEFA ₄₀ +NLX Morphine Hydrochloride+tEFA ₁₀ Morphine Hydrochloride+tEFA ₂₀ --4444 ----1--4---- ------40401020 21.8±3.2 ^d 22.1±1.9 ^a 22.8±2.6 ^{a, d} 21.8±2.4 ^{a, d} 22.9±3.7 ^{a, d} 21.2±2.4 ^{a, d} 22.2±2.2 ^{a, d} 22.5±3.0 ^f 39.4±8.7 ^c 22.8±3.5 ^{a, f} 55.4±8.6 ^{c, f} 54.4±9.4 ^{c, f} 40.1±10.3 ^{c, d} 41.5±12.0 ^{c, d} 21.4±3.8 ^f 33.0±4.0 ^c 22.1±3.2 ^{a, f} 56.4±7.3 ^{c, f} 34.2±7.9 ^{c, d} 31.8±7.1 ^{c, d} 40.0±9.1 ^{c, d} 21.2±2.7 ^f 28.0±5.3 ^c 22.6±5.0 ^a,d 37.1±10.1 ^c,e 32.4±10.2 ^b,d 28.0±8.8 ^a,d 35.1±11.9 ^c,d 20.5±2.3 ^d 22.8±4.5 ^a 21.8±4.5 ^{a, d} 21.0±3.7 ^{a, d} 23.4±3.6 ^{a, d} 22.4±8.4 ^{a, d} 30.9±7.5 ^{c, f}

*Mor is morphine, NLX naloxone hydrochloride, tEFA ₁₀ , tEFA ₂₀ , and tEFA ₄₀ are the total alkaloids of Bermudagrass hydrangea hydrochloride boiled 10mg·kg ^-1 , 20mg·kg ^-1 or 40mg·kg ^-1 respectively; in brackets The code name is the route of administration, sc is subcutaneous injection, and ip is intraperitoneal injection.

Opioid addiction and its treatment are closely related to its analgesic mechanism in many aspects. Muscle and joint pain all over the body is one of the main protracted symptoms after detoxification of heroin addiction, and it is also one of the important reasons for drug addicts to relapse. Therefore, non-addictive analgesics are of considerable importance in the treatment of reducing relapse. Dog tooth flower is used for rheumatic bone pain, bruises or snake bites in our country.

The results of the above analgesic experiments show that tEFA has an obvious analgesic effect in a dose-dependent manner, and has a synergistic effect with morphine hydrochloride; this effect of tEFA can be partially blocked by the opioid receptor antagonist naloxone, which shows that The analgesic effect of tEFA may be achieved partly through the opioid system. The analgesic effect of tEFA will have a positive adjuvant therapeutic effect on alleviating the protracted symptoms of drug addicts after detoxification.

The test results of the above Examples 3 and 4 show that: within the dose range tested, tEFA can significantly inhibit the conditioned place preference induced by morphine while it does not have physical dependence and mental dependence, and can block it in a dose-dependent manner. Morphine "relapse" behavior in mice induced by stress stimulation and administration of exogenous glucocorticoids. tEFA has obvious analgesic effect. The efficacy tests of tEFA given by gavage and intraperitoneal injection suggest that tEFA is absorbed completely and rapidly in the gastrointestinal tract and is suitable for oral administration. Its anti-opioid addiction mechanism partly involves the opioid peptide system and so on. Judging from the research data accumulated so far, tEFA has preliminarily met the basic conditions to be used as a drug to effectively prevent and treat heroin addiction and reduce relapse after detoxification treatment. Therefore, the total alkaloids and monomeric salts of the dog's tooth flower of the present invention can be used for the preparation of drugs or food for the prevention and treatment of opioid dependence or for the preparation of analgesic drugs.

Claims (2)

1. novel fan-shaped GOUYAHUA indoles alkaloid, flabelliformidine (flabelliformidine EF-14), is characterized in that this alkaloid has the chemical structure shown in the chemical structural formula (EF-14):

(EF-14)。

2. one kind comprises novel fan-shaped GOUYAHUA indoles alkaloid described in the claim 1, flabelliformidine (flabelliformidine, EF-14), and also comprise Ervatamine (ervatamine, EF-1), 20-table Ervatamine (20-epiervatamine, EF-2), 20-table-dregamine (20-epi-dregamine, EF-3), dregamine (dregamine, EF-4), dihydropeivine (dihydropeivine, EF-5), vobasine (vobasine, EF-6), coronaridine (coronaridine, EF-7), 10-methoxyl group coronaridine (voacangine, EF-8), 10-methoxyl group-3 hydroxyls-coronaridine (3-hydroxyvoacangine, EF-9), 10-methoxyl group jollyanine (EF-10), jollyanine (jollyanine, EF-11), voaphylline (voaphylline, EF-12) and (-) Mei Lani alkali ((-)-mehranine, EF-13) at interior fan-shaped GOUYAHUA indoles alkaloid and its esters in preparation control opioid dependent drug or anodyne or Application in Food.