CN112279811B - C 20 Diterpenoid alkaloids, their preparation and use for treating pain related diseases - Google Patents
C 20 Diterpenoid alkaloids, their preparation and use for treating pain related diseases Download PDFInfo
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Abstract
The invention discloses a 20 Diterpenoid alkaloids, their preparation and use for treating pain related disorders, in particular, the invention discloses novel C 20 The diterpene alkaloid compound can play a dose-dependent analgesic effect activity under a safe dose.
Description
Technical Field
The invention relates to C with a novel framework, which is extracted, separated and purified from traditional Chinese medicine monkshood 20 Application of diterpene alkaloid compounds, derivatives thereof and medicinal salts in preparation of medicines for preventing and treating diseases such as acute inflammation, acute or chronic pain and the like. Belongs to the technical field of medicine.
Background
Radix Aconiti lateralis Preparata is processed from lateral root of Aconitum carmichaeli Debx of Aconitum of Ranunculaceae, collected from late 6 to late 8 months, removed from mother root, fibrous root and silt to obtain what is called "mud radix Aconiti lateralis Preparata", and processed by different methods to obtain common and commonly-called medicinal products such as "salted radix Aconiti lateralis Preparata", "black radix Aconiti lateralis" and "white radix Aconiti lateralis [1] . The monkshood has the effects of restoring yang and rescuing from collapse, tonifying fire and supporting yang, and dispelling cold and relieving pain, and is used for treating symptoms such as yang exhaustion and collapse, cold limbs and small pulse, insufficient heart yang, chest obstruction and pain, deficiency cold vomiting and diarrhea, abdominal psychroalgia, deficiency and decline of kidney yang, impotence and cold womb, yin cold edema, yang deficiency and exogenous syndrome, cold-dampness arthralgia and the like in traditional Chinese medicine. Meanwhile, fu Zi belongs to the toxic "lower-grade" Chinese medicine. The definite clinical curative effect and toxicity of aconite root have been known for a long timeIs one of the objects of the study of the scholars.
Research on the chemical components and pharmacological activity of monkshood discovers that the chemical components are similar to other plants in the same genus and mainly comprise diterpene alkaloids with complex and unique structures, and the diterpene alkaloids have obvious effects on anti-inflammation, analgesia, arrhythmia resistance and the like. Thus, diterpene alkaloids are considered to be the main active ingredient of aconite [4-7] . Specifically comprises aconitane type C 19 Diterpene alkaloid, hyphenane type C 20 Diterpene alkaloid, azineone type C 20 Diterpene alkaloids, isoquinoline alkaloids, uracil and ceramide compounds. In addition, flavonoids and steroidal trisaccharides have also been reported. Among them, higenamine, also called aconite one, is clinically used as a cardiotonic active ingredient found in aconite.
The monkshood has the effect of restoring yang and rescuing the collapse, is a common traditional Chinese medicine, has strong toxicity, can cause poisoning by mistaken eating or careless administration, is carried by Shen nong herbal Jing, is pungent and warm in nature and toxic, is listed as a toxic herb in Ben Cao gang mu of Ming dynasty Li Shi Zhen, and is recorded by Wu Fu poison which is not used for critical diseases. The toxic component of radix Aconiti lateralis is liposoluble aconitine alkaloid, and the main components are aconitine and N-methyl substituted homolog mesaconine, which has strong toxicity (toxicity level mesaconine > aconitine > 3-acetyl aconitine) [39] The main toxic action of aconitine is to inhibit respiration and cause arrhythmia, and the toxic action to heart is caused by exciting center and direct action to heart, and further experimental results indicate that the direct toxic action of aconitine to heart makes myocardial cell Na + Channel opening, acceleration of Na + Causes depolarization of the cell membrane, thereby causing arrhythmia.
The research of separating and identifying the pharmacological activity of chemical components in monkshood mainly comprises the following steps: (1) analgesic and sedative effects: early researches find that the processed monkshood can reduce the times of writhing reaction caused by potassium antimony tartrate or acetic acid injected into the abdominal cavity and prolong the latent period of response of mice to heat pain. Further studies have shown that aconite mediates neuropathic pain via k-opioid receptorsRats have analgesic effects, and their active ingredient is aconitine, which has a reducing effect on the peptide transmitters contained in the ganglion and the ganglion of the spinal cord posterior fibers, presumably reducing substance P, reducing the nerve ending substances which transmit pain, and thus reducing pain. In acetic acid writhing and tail flick experiments, mesaconitine has strong analgesic activity, and the conclusion can be drawn by researching the analgesic mechanism of mesaconitine in comprehensive literature, and the mesaconitine can activate a downlink inhibitor alpha 2 Epinephrine and 5-hydroxytryptamine neurons to inhibit pain transmission. (2) anti-inflammatory action: researches find that monkshood has the effects of inhibiting mouse auricle swelling caused by xylene and rat toe swelling caused by carrageenan, inhibiting granuloma formation and adjuvant arthritis, and compound peony Gan Fuzi decoction, peony Gan Fuzi decoction, liquorice monkshood decoction and the like which are subjected to compatibility also have the obvious effect of treating arthritis, and the anti-inflammatory effect is not obvious any more after monkshood is removed in a prescription. (3) cardiotonic effect: many scholars use different preparations to prove that monkshood has positive cardiotonic effect on different animal models, and particularly has more remarkable effect on experimental animals with cardiac insufficiency. The early research on the cardiotonic component of aconite is considered to be the comprehensive action of decomposition products of aconitine and other components and non-alkaloid components thereof, but the aconitine has no obvious effect of enhancing myocardial contraction proved under the condition of dose not causing arrhythmia. Other compounds have blood pressure lowering and myocardial contractility inhibiting effects. As for the heart strengthening component and the heart strengthening mechanism in monkshood, a plurality of scholars also carry out more intensive research, and find that uracil, aconitine glycoside, higenamine, vauqueline, norsalsoline, mesaconine, hypaconine, beiwutinine and other components separated from monkshood all show obvious heart strengthening effect, wherein the mesaconine has protective effect on myocardial ischemia reperfusion injury of rats, including improvement of sexual muscle force effect and left ventricular diastolic function, but has almost no influence on heart rate. (4) antiarrhythmic effect: research proves that the aconite extract and the chemical components thereof, including hypaconitine, mesaconitine, norsalsoline and the like, have obvious effects of reducing and alleviating the range and degree of animal hypoxia and acute myocardial ischemia injury, can improve the hypoxia tolerance of mice and can also improve the myocardial ischemia of ratsAnd arrhythmia. Experiments prove that the substance causing arrhythmia in the monkshood is aconitine, but simultaneously proves that the aconitine-resistant substance exists in the monkshood. The water soluble part of radix Aconiti lateralis can specifically prevent and treat aconitine-induced arrhythmia, and the action intensity in the range of 200-400mg/kg is enhanced with the increase of dosage. (5) others: a large number of researches also find that the extract and the chemical components of the monkshood have the effects of resisting cancers, aging and tumors, enhancing immunity and the like.
The anti-inflammatory analgesic drugs are widely used in clinic, and have very large usage amount regardless of prescription drugs or non-prescription drugs. At present, the anti-inflammatory analgesic drugs with the widest clinical application range are traditional non-steroidal drugs, and the representative drugs mainly comprise aspirin, acetaminophen and ibuprofen. However, in the treatment process of the traditional non-steroidal anti-inflammatory analgesic drugs, the anti-inflammatory and analgesic effects are achieved by inhibiting the exertion of the effect of cyclooxygenase II and further inhibiting the synthesis of inflammatory prostaglandin. However, in the process of exerting the drug action, cyclooxygenase I is also inhibited, so that the synthesis of physiological prostaglandin is greatly reduced, gastrointestinal side effects such as gastrointestinal mucosal erosion, ulcer and the like are caused to the patients, and in severe cases, the patients have the symptoms of blood coagulation dysfunction, renal toxicity and the like. In addition, the blood system symptoms, the cardiovascular system symptoms and the like are also adverse reactions frequently occurring in the taking process of the anti-inflammatory analgesic, and the clinical application range of the traditional anti-inflammatory analgesic is greatly limited by the safety venereal disease seriously influencing the medication of patients. Therefore, there is an urgent need to develop new anti-inflammatory analgesic drugs with less side effects and wide clinical application range.
The search for anti-inflammatory analgesic active ingredients from natural products is becoming a research focus in recent years. Researches find that natural products such as saponin, polysaccharide, alkaloid, flavone, coumarin and the like have certain anti-inflammatory and analgesic effects, and the effects are mainly shown in that inflammatory swelling or granuloma caused by chemical and physical stimulation can be remarkably inhibited, the permeability of abdominal cavity capillaries after inflammation can be reduced, and the expression of inflammatory factors and genes thereof can be inhibited; the analgesic effect of natural products is generally manifested by an ability to extend the pain threshold of a variety of pain-causing factors such as thermal pain, tenderness, and chemical stimulation. The compound in the application is a natural product with anti-inflammatory and analgesic effects, which is obtained by separating from traditional Chinese medicine monkshood.
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Although the literature reports that various chemical components as described above are obtained from monkshood and the pharmacological activities of the chemical components in monkshood extract and part of monkshood, the present invention has been directed to C having a novel skeleton 20 Diterpenoid alkaloids and derivatives thereof are not obtained from radix Aconiti lateralis, or from other animals, plants and microorganisms by separation and purification or by chemical synthesisAnd biosynthesis; further, it has not been reported that these compounds or derivatives thereof have an analgesic effect.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel drug with analgesic effect.
In order to solve the technical problem, the invention provides the following technical scheme:
the first aspect of the technical scheme of the invention provides a novel C shown as general formulas (I) and (II) 20 Diterpene alkaloids and their derivatives.
Specifically, compounds represented by general formula (I) and pharmaceutically acceptable salts thereof are provided:
wherein R is 1 Selected from H, CH 3 、CH 2 CH 3 、CH 2 CH 3 OH；R 2 Selected from H, O, OH, OCH 3 、 OCH 2 CH 3 、SO 3 H；R 3 Selected from H, O, OH, OCH 3 、OCH 2 CH 3 、SO 3 H；R 4 Selected from H, O, OH, OCH 3 、OCH 2 CH 3 ；R 2 The attachment to C-17 may be selected from single or double bonds; the connection mode of C-15 and C-16 can be selected from single bond or double bond; the C-16 and C-17 linkage may be selected from single or double bonds.
Compounds of the general formula (II) and pharmaceutically acceptable salts thereof are provided:
wherein R is 1 Selected from H, CH 3 、CH 2 CH 3 、CH 2 CH 3 OH；R 2 Selected from H, O, OH, OCH 3 、 OCH 2 CH 3 、SO 3 H、SO 3 - ；R 3 Selected from H, O, OH, OCH 3 、OCH 2 CH 3 、SO 3 H、SO 3 - ； R 4 Selected from H, O, OH, OCH 3 、OCH 2 CH 3 R 2 The attachment to C-17 may be selected from single or double bonds; the connection mode of C-15 and C-16 can be selected from single bond or double bond; the C-16 and C-17 linkage may be selected from single or double bonds.
Further preferred compounds of the invention are selected from the group consisting of:
in a second aspect of the present invention, there is provided a process for the preparation of a compound according to the first aspect.
Drying monkshood, crushing, extracting for 2-4 times by using distilled water at 35-50 ℃ for 4-8 hours each time, combining extracting solutions, recovering a solvent under reduced pressure to obtain an extract, performing chromatographic separation by using a macroporous resin column, and sequentially using water: gradient elution with ethanol 1:0-0:1, TLC or HPLC monitoring, eluting each fraction until no sample is eluted obviously, recovering solvent under reduced pressure to obtain corresponding eluted part, wherein 50% ethanol part is separated with MCI resin, and sequentially adding water: ethanol 1:0-0:1 gradient elution, TLC or HPLC monitoring combined the same fractions. Wherein the water elution part is separated by C-18 reverse phase silica gel column chromatography, and the water: methanol 1:0-0:1 gradient elution, TLC or HPLC monitoring combined the same fractions to obtain the corresponding elution fraction (C1-1-C1-12). Subjecting C1-4 to Sephadex LH-20 gel column chromatography, separating with purified water as mobile phase, and mixing the same components by TLC or HPLC to obtain eluate (C1-4-1-C1-4-4). And (3) separating the subfraction C1-4-3 by C-18 reverse phase silica gel column chromatography, eluting with 10-15% methanol water in sequence, monitoring by TLC or HPLC, and mixing the same components to obtain C1-4-3-1-C1-4-3-10, wherein C1-4-3-8 is separated by C-18 reverse phase silica gel column chromatography, 5-10% methanol water is used as a mobile phase, and monitoring by TLC or HPLC, and mixing the same components to obtain C1-4-3-8-1-C1-4-3-8-7. And (3) performing chromatography on the C1-4-3-8-6 by using a HW-40F gel resin column, eluting by using 5-10% methanol water, and combining the same components by monitoring by TLC or HPLC to obtain the component C1-4-3-8-6-1-C1-4-3-8-21. Wherein C1-4-3-8-7 is purified by HW-40F gel resin column chromatography, methanol: dichloromethane 1:1 as mobile phase separation and combined with reverse phase HPLC preparation, compounds (I) and (II) are obtained.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising as an active ingredient a novel compound C represented by the general formulae (I) and (II) 20 Diterpene alkaloid, and carrier commonly used in pharmaceutical field.
Typically, the pharmaceutical compositions of the present invention contain 0.1 to 95% by weight of a compound of the present invention.
Pharmaceutical compositions of the compounds of the invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention can, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants and brought into a suitable administration form or dosage form for use as a human or veterinary medicine.
The compound of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be intestinal or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum, etc., preferably oral.
The route of administration of the compounds of the invention or the pharmaceutical compositions containing them may be by injection. Injections include intravenous, intramuscular, subcutaneous, intradermal, and the like.
The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.
The extract or the compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.
In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.
For example, to form the administration units into pills, various carriers well known in the art are widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc.
For example, to encapsulate the administration units, the active ingredient, extract or compound of the invention is mixed with the various carriers described above and the mixture thus obtained is placed in hard gelatin or soft gelatin capsules. The effective component of the compound can also be prepared into microcapsules, suspended in an aqueous medium to form a suspension, and also can be filled into hard capsules or prepared into injections for application.
For example, the extract or compound of the present invention may be formulated into injectable preparations such as solutions, suspensions, emulsions, lyophilized powders, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. These adjuvants are commonly used in the art.
In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.
For the purpose of administration and enhancing the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.
The dose of the compound, pharmaceutical composition of the present invention to be administered depends on many factors such as the nature and severity of the disease to be prevented or treated, sex, age, body weight, character and individual response of the patient or animal, administration route, administration frequency, therapeutic purpose, and thus the therapeutic dose of the present invention can be widely varied. Generally, the dosage of the pharmaceutical ingredients of the present invention used is well known to those skilled in the art. The prophylactic or therapeutic objectives of the present invention can be accomplished by appropriate adjustment of the actual amount of drug contained in the final formulation of the compound composition of the present invention to achieve the desired therapeutically effective amount. A suitable daily dosage range of the compound of the invention is 0.001-150mg/kg body weight, preferably 0.01-100mg/kg body weight, more preferably 0.01-60mg/kg body weight, most preferably 0.1-10mg/kg body weight. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms which is limited by the clinical experience of the administering physician and by dosage regimens which include the use of other therapeutic means.
The total dose required for each treatment can be divided into multiple doses or administered as a single dose. The compound and the composition of the invention can be taken alone or combined with other therapeutic drugs or symptomatic drugs and the dosage is adjusted.
The fourth aspect of the technical proposal of the invention provides a novel C shown as general formulas (I) and (II) 20 Diterpenoid alkaloids are used for preparing medicines for treating diseases such as somatic pain, visceral pain, neuropathic pain or cancer pain.
The invention also relates to the application of the aconite extract in preparing medicines for preventing or treating acute or chronic pain and other diseases. The various pains include pains associated with the central nervous system or the peripheral nervous system, various acute or chronic pains, nociceptive pains, somatic pains, visceral pains, neuropathic pains, or cancer pains.
The inventor finds that the compounds (I) and (II) and pharmaceutically acceptable salts have certain analgesic effects. Therefore, the compounds (I) and (II) and pharmaceutically acceptable salts thereof of the present invention relate to methods for treating and improving diseases related to analgesia. The method comprises administering to a patient in need of treatment a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or a pharmaceutical composition thereof.
The compounds (I) and (II) are shown to have good analgesic effect on the whole animal level. Compounds (I) and (II) or pharmaceutically acceptable salts have not been reported in the public.
Advantageous technical effects
In the research process of the active ingredients of the traditional Chinese medicine monkshood, the inventor of the invention separates C with a novel skeleton from monkshood by an activity tracking method 20 Diterpenoid alkaloids 1 and 2. The compounds are subjected to activity evaluation through an acetic acid writhing experiment, and the results show that the compounds 1 and 2 have good analgesic effect on the whole animal level, have dose dependence and belong to lead compounds with novel structures in the research and development process of analgesic drugs.
Drawings
FIG. 1, analgesic Effect of Compound 1
FIG. 2, analgesic Effect of Compound 2
Detailed Description
The following experimental examples further illustrate the invention but do not limit it in any way.
Example 1, compounds 1 and 2 are novel C isolated and purified from aconite 20 The diterpenoid alkaloid is separated, purified and identified as follows:
drying radix Aconiti lateralis 50Kg, pulverizing, extracting with water at 40 deg.C for 3 times, each for 6 hr, mixing extractive solutions, recovering solvent under reduced pressure to 120L, separating with macroporous resin (HPD-110, 19kg) column chromatography (20 × 200 cm), sequentially eluting with water (50L), 30% ethanol (120L), 50% ethanol (120L), and 95% ethanol (100L), recovering solvent under reduced pressure to obtain corresponding eluate (A-D), wherein the C part is separated with MCI resin (CHP 20P), and sequentially eluting with water (10L), 30% ethanol (30L), 50% ethanol (20L), and 95% ethanol (10L) to obtain corresponding eluate (C1-C4). The water eluted fraction (C1, 750 g) was separated with C-18 reverse Silica gel (Ulrapure Silica Dels), and eluted sequentially with 0-50% methanol in water (80L) and 100% methanol in alcohol (10L) to give the corresponding eluted fraction (C1-1-C1-12). C1-4 (75 g) was subjected to Sephadex LH-20 (H) 2 O) separating to obtain an elution part (C1-4-1-C1-4-4). And the subfraction C1-4-3 (26 g) is separated by C-18 reverse Silica gel (Ulrapure Silica Dels) (10% -15% methanol water) to obtain C1-4-3-1-C1-4-3-10, wherein C1-4-3-8 (20 g) is separated by C-18 reverse Silica gel (Ulrapure Silica Dels) (6% methanol water) to obtain C1-4-3-8-1-C1-4-3-8-7. Chromatography of C1-4-3-8-6 (12 g) on a HW-40F gel column, CH 3 OH-H 2 And eluting the O (6%) at a fixed degree to obtain a component C1-4-3-8-6-1-C1-4-3-8-21. Wherein C1-4-3-8-7 (2 g) is isolated by HW-40F gel column chromatography (methanol: dichloromethane = 1:1) to yield C1-4-3-8-7-1-C1-4-3-8-7-8. Wherein C1-4-3-8-7-7 (52 mg) was prepared by reverse phase HPLC (ADME column,14% CH) 3 CN in H 2 O, conjugation 0.2% TFA,2.0 mL/min) to give Compound 1 (3.4 mg R =14 min) and compound 2 (3.4mg R ＝14min)。
Compound 1 colorless Cristobalite (CH) 3 OH-H 2 O,5:1),m.p.＞300℃； 1 H NMR(D 2 O,600 MHz) data are shown in Table 1; (+) -HRESIMS m/z 422.1991[ 2] M + H] + (calcd.for C 22 H 32 NO 5 S,422.1996)；(-)-HRESIMS m/z 420.1844[M-H] - (calcd.for C 22 H 30 NO 5 S,420.1850).
Compound 2 is a white amorphous powder; 1 H NMR(D 2 o,600 MHz) data are shown in Table 1; (+) -HRESIMS m/z 424.2152, [ M ] +H] + (calcd.for C 22 NH 34 O 5 S,424.2152)；(-) -HRESIMS m/z 422.2009[M-H] - (calcd.for C 22 H 32 NO 5 S,422.2007).
TABLE 1 preparation of Compounds 1 and 2 1 H NMR data
Example 2 analgesic Effect, effect of Compounds 1 and 2 on acetic acid-induced pain in mice
The acetic acid writhing experiment is a classical model of peripheral analgesia, which is caused by mouse peritoneal inflammation caused by acetic acid stimulation and is one of methods for evaluating and screening analgesics. Acetic acid with a certain volume and concentration is injected into the abdominal cavity of a mouse to stimulate the visceral layer and parietal peritoneum to cause inflammatory pain with a large deep part and a long time, so that the mouse has behavioral reactions such as abdominal indent, trunk and hind limb stretch, hip rising and the like, which are called writhing reaction. The frequency of the reaction is high within 15min after injection, so the number of writhing times or the number of mice which react within 15min after injection is taken as the quantitative index of pain. [ Wei Wei, wu Ximei, li Yuanjian pharmacological experimental methodology, national sanitation Press, 4 th edition, p770 ]
Test animals: healthy adult female ICR mice, clean grade, weight 18-22g. The experimental animals are raised in an independent environment with 12h-12h day and night alternation, the room temperature is maintained at 24 +/-2 ℃, water is freely drunk and food is eaten, and the experiment is carried out after the experimental animals are adapted to the environment for 1 week. All treatments for animals were in compliance with the requirements of the ethical committee of the international society for pain research.
Test compounds: the test substances are compounds 1 and 2, and the positive drug is morphine
The method comprises the following steps: dividing ICR female mice into 5 groups, each group comprises 10 mice, and the groups respectively comprise a model control group (normal saline) and a positive control group (morphine, 0.3 mg/kg); a compound high dose group (1.0 mg/kg), a compound medium dose group (0.3 mg/kg), and a compound low dose group (0.1 mg/kg). Except for the model group, the other physiological saline was distilled water. In each group, 1% (0.1 ml/10 g) of chemical stimulant acetic acid solution is injected into the abdominal cavity 30 minutes after administration, the number of writhing of mice within 15 minutes after acetic acid injection is recorded, the inhibition rate of the drug on writhing reaction is calculated according to the following formula, and the analgesic effect of the drug is judged:
inhibition% = [ (average number of twists of negative control group-average number of twists of experimental group)/(average number of twists of negative control group) ] × 100%.
The experimental results are as follows: compared with the model group, the average number of writhing times of the high, medium and low dose groups of the compounds 1 and 2 is less than that of the model group, and the compounds have obvious dose-dependent effect. The analgesic effect of the positive drug is very obvious, and the analgesic inhibition rate reaches 65.47%. The compounds 1 and 2 are shown to have significant analgesic effects and dose-dependent properties. Specific results are shown in table 2, fig. 1 and fig. 2.
TABLE 2 analgesic Effect of Compounds 1 and 2
Note: data are expressed as mean ± sem, # p <0.01, and # p <0.001 indicates significance compared to model groups.
Claims (8)
1. A compound represented by the following group and pharmaceutically acceptable salts thereof:
2. the compound according to claim 1, wherein the pharmaceutically acceptable salt is selected from salts of compound 1 with organic or inorganic acids.
3. A process for the preparation of a compound according to any one of claims 1 and 2, and pharmaceutically acceptable salts thereof, wherein said compound is prepared by:
drying monkshood, crushing, extracting for 2-4 times by using distilled water at 35-50 ℃ for 4-8 hours each time, combining extracting solutions, recovering a solvent under reduced pressure to obtain an extract, performing chromatographic separation by using a macroporous resin column, and sequentially using water: gradient elution with ethanol 1:0-0:1, TLC or HPLC monitoring, eluting each fraction until no sample is eluted obviously, recovering solvent under reduced pressure to obtain corresponding eluted part, wherein 50% ethanol part is separated with MCI resin, and sequentially adding water: gradient elution of 1:0-0:1 with ethanol, TLC or HPLC monitoring to combine the same components; wherein the water elution part is separated by C-18 reverse phase silica gel column chromatography, and the water: gradient eluting with methanol 1:0-0:1, monitoring by TLC or HPLC, and mixing the same fractions to obtain corresponding eluate fraction (C1-1-C1-12); subjecting C1-4 to Sephadex LH-20 gel column chromatography with purified water as mobile phase, and monitoring by TLC or HPLC to combine the same components to obtain eluate (C1-4-1-C1-4-4); performing chromatographic separation on the subfraction C1-4-3 by using a C-18 reverse phase silica gel column, sequentially eluting by using 10-15% methanol water, monitoring by TLC or HPLC, and combining the same components to obtain C1-4-3-1-C1-4-3-10, wherein the C1-4-3-8 is subjected to chromatographic separation by using the C-18 reverse phase silica gel column, using 5-10% methanol water as a mobile phase, and monitoring by TLC or HPLC, and combining the same components to obtain C1-4-3-8-1-C1-4-3-8-7; subjecting the C1-4-3-8-6 to HW-40F gel resin column chromatography, eluting with 5-10% methanol water, monitoring by TLC or HPLC, and mixing the same components to obtain a component C1-4-3-8-6-1-C1-4-3-8-21; wherein C1-4-3-8-7 is purified by HW-40F gel resin column chromatography, methanol: dichloromethane 1:1 as a mobile phase separation and combined with reverse phase HPLC preparation, compounds 1 and 2 were obtained.
4. A pharmaceutical composition comprising a compound of any one of claims 1 and 2, and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
5. Use of a compound according to any one of claims 1 and 2, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to claim 4, for the manufacture of a medicament for the prevention or treatment of pain.
6. The use according to claim 5, wherein the pain is associated with the central or peripheral nervous system.
7. The use of claim 5, wherein the pain comprises acute pain or chronic pain.
8. Use according to claim 5, wherein the pain is somatic pain, visceral pain or neuropathic pain.
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| CN101434581A (en) * | 2008-12-24 | 2009-05-20 | 西南科技大学 | Preparation of aconite primary diterpene alkaloid component and use in disinsection |
| CN101440064A (en) * | 2008-12-24 | 2009-05-27 | 西南科技大学 | Aconite napelline type diterpene alkaloid acylate, synthesis and use thereof |
| CN102127016A (en) * | 2010-12-30 | 2011-07-20 | 成都大学 | Hetisin type diterpenoid alkaloid and preparation method and application thereof |
| CN109942491A (en) * | 2017-12-20 | 2019-06-28 | 中国医学科学院药物研究所 | With the C of anti-inflammatory and analgesic effect in monkshood20Diterpene alkaloid and application thereof |
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| CN101434581A (en) * | 2008-12-24 | 2009-05-20 | 西南科技大学 | Preparation of aconite primary diterpene alkaloid component and use in disinsection |
| CN101440064A (en) * | 2008-12-24 | 2009-05-27 | 西南科技大学 | Aconite napelline type diterpene alkaloid acylate, synthesis and use thereof |
| CN102127016A (en) * | 2010-12-30 | 2011-07-20 | 成都大学 | Hetisin type diterpenoid alkaloid and preparation method and application thereof |
| CN109942491A (en) * | 2017-12-20 | 2019-06-28 | 中国医学科学院药物研究所 | With the C of anti-inflammatory and analgesic effect in monkshood20Diterpene alkaloid and application thereof |
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