CN1428334A - Alkaloid compound, pain-stopping medicine and its preparation method - Google Patents
Alkaloid compound, pain-stopping medicine and its preparation method Download PDFInfo
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Abstract
The present invention relates to compounds with antinociceptive action, its medicine and preparation method of said medicine. Said invention provides two compounds, they has good antinociceptive effect, at the same time they do not produce dependency. Said invention also provides the preparation method of said two compounds, and the preparation method of the antalgesics.
Description
The technical field is as follows:
the invention relates to an alkaloid compound, in particular to a small molecular weight alkaloid and a preparation method thereof. The invention also relates to an analgesic drug, in particular to an analgesic drug containing the alkaloid provided by the invention. The invention also relates to a preparation method of the analgesic drug, which takes the marasmius androsaceus as a raw material.
Background
Humans have been afflicted with diseases. In many cases, the disease causes physical distress in addition to loss of body function and even life, such as: advanced cancer, trauma, etc. In order to alleviate the pain of patients, a number of analgesic drugs have been developed. The medicine with good analgesic effect comprises morphine and dolantin. This is currently the most effective analgesic in clinical applications. However, the greatest disadvantage of this drug is that it cannot be used for a long period of time, otherwise, it may develop dependence or addiction. Besides the analgesic drugs, Marasmius androsaceus mycelium is used for treating traumatic injuries, knife wounds and fracture pain in our country. Shanghai Chinese medicine pharmaceutical factory prepares the dried mycelium of wild Marasmius pauciflorus and the crude extract of the matrix into 'Anluojiedan tablet'. These are crude preparations, low technical content and complex ingredients. The drug standard issued by the Ministry of health of China loads four preparations of the marasmius androsaceus: anluotong tablet, Anluotong capsule, Anluotong wine and Anluotong tincture all adopt ethanol extraction process. Later, the effective components in Marasmius androsaceus are reported in the literature, for example, the analgesic effective component extracted by Fangshending et al is p-hydroxycinnamic acid (Chinese herbal medicine, 20 th 10 th volume in 1989); the analgesic effective components extracted from ZHENSHANGXIONG et al are triacontanoic acid and ergosterol (Chinese herbal medicine, 19 th volume, 8 th phase, 1988).
The extraction method of the Fangshengdong and the like comprises the following steps: soaking and washing dry mycelium and culture medium in cold water, oven drying, grinding, reflux extracting with ethanol, concentrating the alcoholic solution under reduced pressure, and filtering to obtain precipitate. The solution was concentrated under reduced pressure to give a syrup, which was subjected to silica gel column chromatography and eluted with increasing amounts of methanol in methylene chloride. Obtaining the medicanin from dichloromethane-methanol 96: 4, and obtaining the p-hydroxycinnamic acid from dichloromethane-methanol 95: 5.
The extraction method of Zhushaoxing and the like comprises the steps of extracting an marantal extract with ethyl acetate, filtering, appropriately concentrating, washing twice with a 5% sodium carbonate solution, washing with water to neutrality, drying, recovering a solvent to obtain an extract, performing G column chromatography, and performing gradient elution with petroleum ether-ethyl acetate to obtain 2, 3, 5, 6-tetrachloro-1, 4-dimethoxybenzene (petroleum ether recrystallization), triacontanoic acid (anhydrous crystal), β -sitosterol (silica gel G rotary thin-layer separation, n-hexane-ethyl acetate elution and concentration to obtain crystal), and 5, 8-peroxyergosterol (ethyl acetate recrystallization).
Disclosure of Invention
Although there are many reports in the prior art that the effective analgesic component is extracted from marasmius androsaceus, the applicant further finds a new effective analgesic component therein, and the effective analgesic component has a good analgesic effect and does not generate dependence. This is also the technical problem to be solved by the present invention.
The present invention provides compounds that are:
the molecular formula is: c7H11NO2。
Another compound provided by the present invention is:
molecular formula C6H12N2O2。
Both compounds can be synthesized by chemical methods. The invention provides an extraction preparation method using Marasmius androsaceus as a raw material, and two compounds are directly obtained. The specific method comprises the following steps:
(1) taking Marasmius androsaceus as a raw material, and extracting with ethanol;
(2) recovering ethanol to obtain fluid extract, diluting with water, adjusting pH to 8-9, defatting, and extracting with chloroform;
(3) recovering chloroform to obtain soft extract, loading on silica gel H column, and eluting with acetone;
(4) recovering acetone to obtain soft extract, washing with ethyl acetate, and vacuum filtering to obtain crystal;
(5) and (3) loading the obtained crystals on a silica gel H column, eluting with acetone to obtain a 1 st peak as alkaloid I and a 2 nd peak as alkaloid II, recrystallizing with ethyl acetate-acetone (2: 1), and further purifying and separating to obtain the alkaloid I, II.
The applicant analyzed the chemical structures of the compounds (I), (II) through experiments. The structure of the compound was confirmed to bethe structure shown above. Detailed analysis and experimental data are given in the structural analysis section attached later.
Meanwhile, the applicant also proves that the two compounds have good analgesic effect through experiments. Therefore, the compound can be used for preparing analgesic drugs.
Therefore, the invention provides an analgesic drug, wherein the effective component is the compound (I).
Preferably, the content of compound (I) is more than 25%.
The invention also provides an analgesic drug, wherein the effective component is the compound (II).
Preferably, the content of alkaloid (II) is more than 25%.
The analgesic drug provided by the invention is prepared by taking marasmius androsaceus as a raw material.
The preferred preparation method is as follows:
(1) taking Marasmius androsaceus as a raw material, and extracting with ethanol;
(2) recovering ethanol to obtain fluid extract, diluting with water, adjusting pH to 8-9, defatting, and extracting with chloroform;
(3) recovering chloroform to obtain soft extract, loading on silica gel H column, and eluting with acetone;
(4) recovering acetone to obtain soft extract, washing with ethyl acetate, and vacuum filtering to obtain crystal.
The crystals obtained by the above method contain triterpenoids in addition to the compound I, II specified in the present invention. However, the total amount of the compound I, II exceeds 51%, and the single main effective component accounts for more than 25% of the total alkaloid amount.
Mixing the above total alkaloid crystals with starch and dextrin, and sieving to obtain the final product. The content of total alkaloids is required to be more than 51%.
Experiments prove that the analgesic drug obtained by the method has good analgesic effect. It may be an analgesic drug with both peripheral and central analgesia, while dolantin is an obvious central analgesic drug.
Pharmacodynamic tests prove that the effective analgesic dose of the total alkaloid effective part is 640 mg/day/person, which is equivalent to 160g of crude drug (wheat bran and chaff solid fermentation culture) taken per day or about 53g of dry mycelium taken per day.
More importantly: the analgesic drug provided by the invention does not generate dependence.
Drawings
FIG. 1 is a graph of the ultraviolet absorption spectrum of Compound I;
FIG. 2 is a graph of the ultraviolet absorption spectrum of Compound II;
FIG. 3 is a chart of the infrared absorption spectrum of Compound I;
FIG. 4 is a chart of the infrared absorption spectrum of Compound II;
FIG. 5 Mass Spectroscopy of Compound I;
FIG. 6 Mass Spectroscopy of Compound II;
compound I nmr spectra of figures 7, 8, 9;
FIG. 10 and 11 shows NMR spectra of Compound II.
Example 1
Process for the preparation of compound I and compound II
Pulverizing Marasmius androsaceus (L.) Gaertn into coarse powder 40kg, extracting with 8 times of ethanol under reflux for 3 times each for 2 hr, mixing filtrates, recovering ethanol under reduced pressure to obtain 6kg (relative density of 1.20-1.25, measured at 60 deg.C), diluting with 10 times of water, adjusting pH with concentrated ammonia water to 8-9, adding petroleum ether 5 times, recovering petroleum ether, adding chloroform 5 times, recovering chloroform to obtain thick paste 1kg (relative density of 1.35-1.38, measured at 60 deg.C), adding 5kg of silica gel H: diatomaceous earth (2: 1), stirring, drying, loading onto silica gel H column [ silica gel H: diatomaceous earth (2: 1), loading onto acetone wet column], eluting with acetone, collecting acetone eluate to light color (less extract), recovering acetone to obtain thick paste (0.5 kg, relative density of 1.35-1.38, measured at 60 deg.C), washing with ethyl acetate, recrystallizing insoluble substance with ethyl acetate 2-3 times, filtering, and drying at below 70 deg.C to obtain total alkaloid crystal dry product of about 160 g.
And then, loading the obtained crystals on a silica gel H column, eluting with acetone, respectively collecting eluent with a 1 st peak and an eluent with a 2 nd peak, recrystallizing with an ethyl acetate-acetone (2: 1) mixed solution, and purifying and separating to obtain about 30g of alkaloid I and about 18g of compound II. The purity can reach more than 98 percent.
Structure confirmation of Compounds (I) and (II)
After further purification, the obtained compounds (I) and (II) are identified by four spectra of the university of Hunan and the university of Hunan, and are low molecular alkaloid compounds, the molecules of the compounds have obvious symmetrical structures, and the determination results are as follows: (Compounds I and II are also referred to herein as alkaloid I and alkaloid II.)
(1) Solubility:
the compound I is easily soluble in chloroform,acetone, methanol and ethanol, and slightly soluble in ethyl acetate.
The compound II is easily soluble in chloroform, ethanol and methanol, soluble in acetone and slightly soluble in ethyl acetate.
(2) Melting point:
a compound I: and (2) a compound II at 68-69 ℃: 82-84 DEG C
(3) UV spectrum:
a compound I: the instrument comprises the following steps: and scanning and measuring by using a Nippon Shimadzu PE-lambda 16 type ultraviolet-visible spectrophotometer. Lambda [ alpha]mux MeOH201.2 nm. The measurement conditions and results are shown in FIG. 1.
Compound II: the instrument comprises the following steps: and scanning and measuring by using a Japan Shimadzu PE-lambda 16 type ultraviolet-visible spectrophotometer. Lambda [ alpha]max MeOH203.3 nm. The measurement conditions and results are shown in FIG. 2.
(4) IR spectrum
A compound I: the instrument comprises the following steps: type 510P fourier transform IR spectrometer, nigh force usa.
IRνmax KBr(cm-1): 3534(NH), 2997-2465 (fatty CH, strong), 1726, 1705(c＝0)，1298，1221). The measurement conditions and results are shown in FIG. 3.
Compound II: the instrument comprises the following steps:
IRνmax KBr＝(cm-1): 3295(NH), 3090-2860 (fat CH), 1653, 1561(c ═ 0), 1445, 1366, 1289, 1236 (CH)2). The measurement conditions and results are shown in FIG. 4.
(5) Mass Spectrum (MS):
a compound I: instrument GC-17A, QP-5000 gas chromatograph-mass spectrometer, EI source bombardment voltage of 70ev, quadrupole detection, molecular weight range of 40-500, multiplier voltage of 1.1kv, sample injection rod temperature-rising program: vacuum
MS m/z：140(m+)，98(M+-42)83(m+-57)，58(m+82), 42 (100%). The measurement conditions and results are shown in FIG. 5.
Compound II: the instrument comprises the following steps: GC-17A, QP-5000 gas chromatograph-mass spectrometer, EI source bombardment voltage of 70ev, quadrupole detection, molecular weight range of 40-500, multiplier voltage of 1.1kv, sample injection rod temperature rise program: true
MS m/z：143(m+)，85(m-58)，73(m+-70)，60(m+-83)，43(m+-1O0), 30 (100%). The measurement conditions and results are shown in FIG. 6
(6) Nuclear magnetic resonance spectroscopy:
a compound I: the instrument comprises the following steps: AC-80MHz NMR spectrometer, Bruker, Switzerland, H-NMR (CDCl)3)，δPPm：1.72(CH3-)，2.13(-CH2-)，2.77(-CH2-)，9.80(＞NH)。13C-NMRδPPm 28.14(CH3-)，50.84(-CH2-), 60.5 (>C ═ O), 88 (-C-). The measurement conditions and results are shown in FIGS. 7, 8 and 9.
Compound II: the instrument comprises the following steps: AC-80MHz NMR spectrometer, Bruker, Switzerland, H-NMR (CDCl)3)δPPm：1.78(CH3-)，3.06(-CH2-)，7.77(NH)。13C-NMRδPPm：22.59(CH3-)，38.60(-CH2-, 169.40 (-C-). The measurement conditions and results are shown in the figure10、11
(7) Elemental analysis:
a compound I: the instrument comprises the following steps: model PE-2400, C, H, N element Analyzer, PE Corp, USA
And (3) measuring results: c: 53.64% H: 13.90% N: 6.56 percent
C：53.80％ H：12.88％ N：6.39％
Compound II: the instrument comprises the following steps: model PE-2400, C, H, N element Analyzer, PE Corp, USA
And (3) measuring results: c: 49.45% H: 10.59% N: 20.20 percent
C：49.65％ H：10.68％ N：20.11％
Experiment on analgesic Effect of Compound I, II
First, experimental material
1. Medicaments and agents
The extraction components of the mycelium of Marasmius androsaceus are as follows: A. alkaloid I, alkaloid B and alkaloid 2. In the experiment, 1, 2 and 4 times of the clinical dose to be used as low, medium and high dose of each product.
One of the positive control drugs, dolantin, shenyang pharmaceutical-factory, lot number: 980908.
the second positive control drug, aspirin, produced by the pharmaceutical factory in Hunan, lot number: 990704-7.
2. Animal(s) production
BALB/C line, ICR clean grade mice, weight 18-20g, available from Shanghai Sphall-Bikay laboratory animals Co.
3. Instrument for measuring the position of a moving object
BJ-084J Hot plate analgesic apparatus, produced by Jiangsu Baishi medical instruments factory.
Second, method and structure
1. Influence on twisting reaction caused by acetic acid
(1) Method of producing a composite material
90 BALB/C mice, half of each of the male and female, were randomly divided into 9 groups, except dolantin group, the other groups were administered by intragastric administration for 1 hour according to the dose of Table 1, and after dolantin group was subcutaneously injected for 20 minutes, each mouse was intraperitoneally injected with 0.7% glacial acetic acid 0.1m/10g, and the number of writhing in 5-15 minutes was recorded.
TABLE 1 Effect of alkaloid I, II on mouse writhing response (X + -SD)
Number of dose writhing of group animals
(only) (g/kg)
Blank control group 10 NS 22.6 + -7.4
Douding 100.020.8 +/-0.8**
Aspirin group 100.2017.2 + -4.2*
Low dose group 100.115.3 ± 3.6
Dose group 100.212.7 ± 3.8 in A**
High dose group 100.49.6 ± 2.7**
Low dose group 100.214.6 ± 3.1*
Dose group 100.411.5 ± 2.7 in B**
High dose group 100.810.4 ± 2.4**
Compared with the blank control group, the composition of the composition,*P＜0.05，**P＜0.01
(2) results
Table 1 shows that, compared with the blank control group, A, B mice in the low, medium and high groups have significant differences in writhing frequency, and the high, medium and high doses form a significant dose-effect relationship, which can significantly inhibit the writhing reaction of mice caused by acetic acid, indicating that alkaloid I and alkaloid II crystals both have a good analgesic effect, and dolantin has a very significant analgesic effect.
2. Influence on Hot plate pain threshold of mice
(1) Method of producing a composite material
90 BALB/C female mice meeting the requirement (the average pain threshold value is 30s) are taken, the mice are randomly divided into 9 groups, except the dolantin group, the rest groups are subjected to intragastric administration for 1 hour according to the dosage shown in the table 2, the dolantin group is subcutaneously injected with dolantin for 20 minutes, the mice are placed into a hot plate pain detector, the temperature of the hot plate is 55 +/-0.5 ℃, the time from the time when the mice are put into the hot plate to the time when the mice lick feet is taken as the pain threshold value of the mice, the test is carried out once after 30 minutes, the total time is 3 times, and the average pain threshold value is calculated for statistics.
TABLE 2 Effect of alkaloid I, II on Hot Spot pain threshold in mice (X + -SD)
Number of animals in group average pain threshold
(only) (g/kg)(s)
Blank control group 10 NS 28.0 + -5.8
Douding 100.0258.4 + -3.6**
Aspirin group 100.2038.4 + -5.2**
Low dose group 100.136.9 ± 2.8*
Dose group 100.242.2 ± 3.7 in A**
High dose group 100.444.6 + -5.4**
Low dose group 100.244.2 ± 3.1**
Dose group 100.446.5 ± 4.1 in B**
High dose group 100.849.1 ± 4.8**
Compared with the blank control group, the composition of the composition,*P＜0.05，**P＜0.01
(2) results
Compared with a blank control group, A, B has significant difference in average pain threshold values of low, medium and high dose groups, and shows that alkaloid I and alkaloid II crystals can obviously improve the hot pain resistance of mice and have better analgesic effect. The dolantin has very obvious analgesic effect.
Example 2
Preparation method of analgesic
Pulverizing Marasmius androsaceus (L.) Gaertn into coarse powder 40kg, extracting with 8 times of ethanol under reflux for 3 times each for 2 hr, mixing filtrates, recovering ethanol under reduced pressure to obtain 6kg (relative density of 1.20-1.25, measured at 60 deg.C), diluting with 10 times of water, adjusting pH with concentrated ammonia water to 8-9, adding petroleum ether for 5 times, recovering petroleum ether, adding chloroform for 5 times, recovering chloroform to obtain 1kg (relative density of 1.35-1.38, measured at 60 deg.C), adding 5kg of silica gel H and diatomaceous earth (2: 1), stirring, drying, loading onto silica gel H column (silica gel H: diatomaceous earth (2: 1), loading onto acetone by wet method), collecting acetone eluate to light color (less extract), recovering acetone to obtain soft extract (0.5 kg, relative density of 1.35-1.38, measured at 60 deg.C), washing with ethyl acetate, adding insoluble substance, recrystallizing with ethyl acetate for 2-3 times, filtering, drying at below 70 deg.C to obtain total alkaloid crystal dried product about 160g, adding starch about 50g and dextrin about 100g, mixing, pulverizing into fine powder, sieving with 100 mesh sieve, mixing, adding 50% ethanol, making soft material, sieving with 16 mesh sieve, granulating, drying at 60-80 deg.C, sieving with 16 mesh sieve, inspecting, encapsulating, sterilizing, and packaging. About 1000 granules were prepared.
Experiment on analgesic effect of analgesic drug provided by the invention
First, test materials
1. Medicaments and agents
The capsule raw material provided in example 1 of the present invention was in the specification of 0.16g (crystals)/granule.
One of the positive control drugs, dolantin, produced by pharmaceutical factory in Hubei Jingmen city, lot number: 990508
The second positive control drug, aspirin tablet, produced by Guangzhou Guangji Tang pharmaceutical factory, lot number: 990614.
2. animal(s) production
BALB/C, ICR-series clean mouse, weight 18-20g, SD-series clean rat, available from Shanghai Sphall-Bikay laboratory animals Co.
3. Instrument for measuring the position of a moving object
BJ-084J Hot plate pain measuring instrument, produced by Jiangsu Baishi medical instrument factory.
Second, method and results
The study was conducted on a mouse analgesic test using 5 doses, which were 0.025, 0.05, 0.10, 0.20, 0.40/kg (calculated as mice). Dolantin was used as a positive control.
1. Influence on twisting reaction caused by acetic acid
(1) Method of producing a composite material
Taking 70 BALBC/mouse, each half of male and female, randomly dividing into 7 groups, intragastrically administering the rest groups for 1 hr according to the dosage of table 3 except dolantin group, subcutaneously injecting dolantin into dolantin group for 20 min,
0.7% glacial acetic acid (0.1 ml/10 g) is injected into the abdominal cavity of each mouse, and the number of writhing times in 5-15 minutes is recorded.
TABLE 3 Effect of different dosages of the capsules provided by the present invention on writhing response in mice
Inhibition rate of number dose writhing frequency of group animals
(only) (g/kg) (%)
Blank control group 10 NS 36.3. + -. 13.2-
Douding 100.022.8 + -2.2**92.3
The invention provides a capsule I100.02534.5 +/-14.05.0
II 10 0.05 31.7±11.2 12.8
III 10 0.10 24.4±14.6*32.8
IV 10 0.20 18.9±12.2**52.0
V 10 0.40 13.7±8.6**62.3
Comparison with blank control group*P＜0.05，**P＜0.01。
(2) Results
Compared with a blank control group, the times of mice in the capsule III, IV and V dose groups provided by the invention have obvious difference and are in an obvious dose-effect relationship, and the capsule can obviously inhibit the mouse writhing reaction caused by acetic acid, so that the capsule provided by the invention has a good analgesic effect, and the effective analgesic dose of the mice is about 0.1 g/kg. Corresponding to 1/5 from dolantin (by dose comparison).
2. Influence on Hot plate pain threshold of mice
(1) Method of producing a composite material
70 BALB/C female mice meeting the requirements (the average pain threshold value is within 30s) are taken, the mice are randomly divided into 7 groups, except the dolantin group, the rest groups are subjected to intragastric administration for 1 hour according to the dosage of table 4, the dolantin group is subcutaneously injected with dolantin for 20 minutes, the mice are placed into a hot plate pain measuring instrument, the temperature of a hot plate is 55 +/-0.5 ℃, the time from the time when the mice are put into the hot plate to the time when the mice lick feet appears is the pain threshold value of the mice, the test is carried out once after 30 minutes, the total time is 3 times, and the average pain threshold value is obtained for statistics.
TABLE 4 Effect of different dosages of the capsules on Hot plate pain threshold in mice (X + -SD)
Number of animals in group average pain threshold
(only) (g/kg)
Blank control group 10 NS 34.7 + -10.5
Douding 100.0258.8 + -2.4**
The invention provides a capsule I100.02537.6 +/-6.9
II 10 0.05 39.8±11.0
III 10 0.10 43.1±12.3*
IV 10 0.20 48.4±14.5**
V 10 0.40 52.4±15.2**
Compared with the blank control group, the composition of the composition,*P＜0.05，**P＜0.01。
(2) results
Compared with a blank control group, the capsules III, IV and V provided by the invention have obvious difference in average pain threshold values in dose groups, and have obvious dose-effect relationship, and can obviously improve the hot pain resistance of mice, so that the capsules provided by the invention have good analgesic effect, and the effective analgesic dose of the mice is about 0.1 g/kg. Corresponding to 1/5 from dolantin (by dose comparison).
3. Study of analgesic Effect time
The analgesic effect of the mice was measured at 0.5, 1, 2, 3, 4, 5, 6 hours after administration using an effective dose of 0.10/kg in mice.
(1) Influence on twisting reaction caused by acetic acid
Taking 90 BALB/C mice, each half of male and female, randomly dividing into 9 groups, respectively comprising a blank control group, a dolantin group, a 0.5 hour group, a 1 hour group, a 2 hour group, a 3 hour group, a 4 hour group, a 5 hour group, a 6 hour group and the rest groups except the dolantin group, performing intragastric administration according to the dosage of 5 doses in the table, subcutaneously injecting dolantin into the dolantin group, wherein the former 3 groups are 30 groups after administration, the latter 5 groups are 1, 2, 3, 4, 5 and 6 hours after administration, and each mouse is injected with 0.1ml/10g of 0.7% glacial acetic acid in the abdominal cavity, and recording the times of twisting in 5-15 groups.
TABLE 5 Effect of the invention on the writhing response of mice at different times (X + -SD)
Number of dose writhing of group animals
(only) (g/kg)
Blank control group 10 NS 27.6 + -7.8
Dolantin 100.021.8 + -1.7**
0.5 hr group 100.1018.5. + -. 9.4**
1 hour group 100.1015.6. + -. 11.2**
2 hours group 100.1018.4. + -. 9.0**
3 hours group 100.1020.7. + -. 6.3**
4 hours group 100.1022.8. + -. 5.4*
5 hours group 100.1024.6. + -. 6.1
6 hours group 100.1027.0. + -. 9.5
Compared with the blank control group, the composition of the composition,*P＜0.05，**P＜0.01。
results
Compared with a blank control group, the capsule provided by the invention has significant differences in mouse writhing times of 0.5 hour, 1 hour, 2 hours, 3 hours and 4 hours, has a relatively obvious aging relationship, can obviously inhibit mouse writhing reaction caused by acetic acid, and shows that the capsule provided by the invention has a relatively good analgesic effect within 0.5-4 hours after administration.
(2) Influence on Hot plate pain threshold of mice
90 BALB/C female mice meeting the requirement (with the average pain threshold within 30s) are taken and randomly divided into 9 groups, namely a blank control group, a dolantin group, a 0.5 hour group, a 1 hour group, a 2 hour group, a 3 hour group, a 4 hour group, a 5 hour group and a 6 hour group. Except the dolantin group, other groups are administrated by intragastric administration according to the dosage of table 6, the dolantin group is injected subcutaneously, the first 3 groups are 30 minutes after administration, the last 5 groups are respectively 1, 2, 3, 4, 5 and 6 hours after administration, each group of mice are placed into a hot plate pain measuring instrument, the temperature of the hot plate is 55 +/-0.5 ℃, the time from the time when the mice are put into the hot plate to the time when the mice lick feet appears is the pain threshold value ofthe mice, the test is carried out once after 30 minutes, the total time is 3 times, and the average pain threshold value is calculated for statistics.
TABLE 6 influence of different time of the capsules on the hot-plate pain threshold of mice (X + -SD) provided by the invention
Number of dose writhing of group animals
(only) (g/kg)
Blank control group 10 NS 20.8 + -5.4
Douding 100.0255.9 + -2.4**
0.5 hr group 100.1031.5. + -. 7.2**
1 hour group 100.1034.9. + -. 2.8**
2 hours group 100.1035.2. + -. 5.0**
3 hours group 100.1029.0. + -. 4.8**
4 hours group 100.1025.6. + -. 4.4*
5 hours group 100.1023.2. + -. 5.1
6 hours group 100.1022.2. + -. 5.9
Comparing P to the blank control group to be more than 0.05,***p<0.01 compared to the blank control.
Results
Compared with a blank control group, the capsule provided by the invention has obvious difference in average pain threshold values of mice in groups of 0.5 hour, 1 hour, 2 hours, 3 hours and 4 hours, has obvious aging relationship, can obviously improve the hot pain resistance of the mice, and shows that the capsule provided by the invention has better analgesic effect within 0.5-4 hours after administration.
4. Study of analgesic site
Method of producing a composite material
50 SD clean rats are selected and male, the rats are randomly dividedinto 5 groups, 0.1ml of 20% beer yeast suspension is injected into the right hind foot sole of each rat to cause 1 hour after sterile inflammatory edema, the other groups except the dolantin group are subjected to gastric administration for 1 hour according to the dosage of table 7, after the dolantin group is injected with dolantin for 20 minutes subcutaneously, the pain threshold value of the double hind foot soles of each rat is measured by a tail pressure pain device, and the pain threshold values of the left hind foot sole and the right hind foot sole are compared.
TABLE 7 Effect of the capsules of the invention on analgesic sites in rats (X + -SD)
Group animal dosage right foot plantar pain valve left foot plantar pain valve
(only) (g/kg) (g)
Blank control group 10 NS 4.3 + -1.925.2 + -6.9
Douding 100.0221.6 + -2.5**121.0±21.5**
The invention provides a capsule low dose group 100.107.5 + -4.3*30.5±11.3
The present invention provides a dosage group 100.208 in a capsule.2±3.7**33.8±12.0*
The invention provides a capsule high-dose group 100.4010.6 + -5.8**35.3±17.8*
Compared with the control group to white,*P＜0.05，**P＜0.01 。
compared with a blank control group, the pain threshold value of the rat plantar of the low, medium and high groups of the capsule cancer (plantar in inflammation) is remarkably improved, and the increase amplitude is more than 70%; the pain threshold value of the left and right metatarsus (non-inflammatory plantar metatarsus) is obviously increased only by medium and high doses, and the increase amplitude is only 30%, which indicates that the capsule provided by the invention can be an analgesic drug with both peripheral analgesia and central analgesia, and the dolantin is an obvious central analgesia.
The analgesic drug dependence test provided by the invention
1. Test materials
1.1 drugs and reagents
The pain-relieving capsule provided by the invention is prepared into 13.3mg/ml and 26.7mg/ml suspension when in use
Meffeekang (morphine hydrochloride controlled release tablet) manufactured by southwestern pharmaceutical industry Co., Ltd, lot number 980801, was prepared to 2.9mg/ml and 6.67mg/ml when used.
1.2 animals
BALB/C clean-grade mice, half male and female, with a weight of 20-24 g, were provided by Shanghai Sepal-Bikay, Inc.
SD rats, male and female halves, body weight 200-.
2. Test methods and results
2.1 Natural withdrawal test
2.1. Natural withdrawal test for mouse
2.1.1.1 methods
The mice were divided into 4 groups of 40 mice each having 2 male and female halves, and were administered with distilled water, morphine hydrochloride, and high and low doses of the drugs, respectively. The medicine is averagely administrated four times on days 1-3 according to daily dose, the stomach is perfused once in a fixed time in the morning and afternoon of day 4-7, the medicine is continuously administrated for 7 days, the body weight is measured once every 4 hours in 72 hours from the day 7, the change condition of the body weight is recorded, the mental, activity, diet and other conditions of the animal are observed, and the change of the animalafter the medicine is stopped is recorded. See table 8.
TABLE 8 Effect on mice Natural withdrawal symptoms (X + -SD)
Group dose (mg/kg. days) animals change (only) body weight
Blank 100.01 + -0.45
200101.12 + -0.6 g morphine hydrochloride***
Medicine 400100.23 + -0.49*
Medicine 800100.19 + -0.59*
*Compared with a blank control group, P is more than 0.05,***p<0.01 compared to the blank control.
2.1.1.2 test results
After the animals are dosed for the first three days, the animals have reduced food intake and slow movement, and return to normal after three days, and within 48 hours after the dosing is stopped, the animals in each group have no abnormal movement and do not have excitation state. As can be seen from Table 8, the animal weights of the animals in the blank group did not change abnormally, the animal weights of the animals in the drug market and the animals in the two doses changed slightly within the 72-hour observation period, and the animal weights of the animals in the drug market and the animals in the two doses did not have significant difference compared with the animals in the blank control group, while the animals in the morphine hydrochloride group had obviously reduced weights. There was a very significant difference compared to the blank control group. The weight of morphine hydrochloride animals is reduced after stopping taking the medicine, and the weight is most obviously reduced within 12-24 th after stopping taking the medicine. Subsequently, body weight gradually recovered. This did not occur in the administration group.
2.1.2 rat Natural withdrawal test
Animals were randomly divided into groups, and separately gavage was administered with distilled water, morphine hydrochloride, and two high and low dose drug groups. The administration is divided into four times on the 1 st to 3 rd days according to the daily dose, the administration is divided into two times on the 4 th to 7 th days, and the administration time is 8: 00. 11: 00. 14: 00. 17: 00 and 8: 00. 4: 00. the administration was carried out for 7 days. The body weight was measured every 4 hours for 72 hours from day 7, and the changes in body weight were recorded, and the animals were observed for mental, activity, diet, etc., and the changes after withdrawal were recorded in detail. See table 9.
TABLE 9 Effect on rat Natural withdrawal symptoms (X + -SD)
Weight change (g) of animals (only) at group dose (mg/kg. day)
Blank 10-0.86 +/-0.98
Morphine hydrochloride 20010-8.54 +/-0.63***
Medicine 40010-1.24 +/-0.83*
Medicine 80010-1.62 +/-0.88*
*Compared with a blank control group, P is more than 0.05,***p<0.01 compared to blank.
2.1.2.2 test results
Animals had reduced food intake and slowed activity at the initial administration (first three days or so) and gradually returned to normal after three days. After the animal returns to normal and within 48 hours of stopping the drug, the activity of each group of animals has no abnormal condition and does not show an excited state. It can be seen from Table 9 that the blank animals showed a slight daily increase in body weight, a maximum of 0.86g of body weight loss, and very little change in body weight over the 72-hour observation period. The maximum weight lossof animals in the two drug high and low dose groups is also very small, and compared with a blank control group, the maximum weight loss is not obviously different. The weight of the morphine hydrochloride animals in the middle section of 48 hours after stopping the drug is continuously reduced, the weight is reduced maximally in 12-18 hours after stopping the drug, and then the weight is slightly increased again until the observation period is finished, the weight is still not recovered to be normal, but the weight change of the morphine hydrochloride animals in the administration group does not occur.
2.2 test of urge to withdraw
2.2.1 mice urge withdrawal test
2.2.1.1 test methods
Taking 40 mice, each half of the mice is divided into 4 groups of blank, morphine, small and large dose drug groups according to the body weight, the latter three groups are administrated by intragastric administration according to 15ml/kg twice a day, and the dosages of the first 4 morphine group and the drug groups are respectively 60mg/kg, 180mg/kg, 332mg/kg and 665 mg/kg; the blank control group was given the same volume of physiological saline for 13 consecutive days, and on day 14, 9: 00 to 12: 30mg/kg of naloxone is injected in the abdominal cavity between 00 hours, the mice are immediately placed in a large glass container to measure the jumping number in 20 minutes, the behaviors of the mice are observed for 2 hours, and the weight and the defecation are measured.
2.2.1.2 test results
As can be seen from table 10, after naloxone injection, morphine animals were significantly excited by jumps, wet dog-like tremor, diarrhea, forepaw tremor, lacrimation, etc. over the 2 hour observation period in the morphine animals. Compared with a blank control group, the morphine group mice have obvious differences in jumping number,diarrhea number and weight loss, while the analgesic II capsule has no obvious difference in large and small dose groups. See table 10.
TABLE 10 Effect of analgesic No. II capsules on the promotion of withdrawal in mice
Group mean dose N-skip weight change diarrhea
(mg/kg. day) (times) (g) (only)
Blank control group-100.6 + -1.20.06 + -0.180
Morphine group 1571022.3 + -14.8***-0.17±0.32***10***
Low dose group 332100.8 + -0.9*-0.12±0.14*0
High dose group 66510 0.3±0.5*0.02±1.3*0
*Compared with the blank control group, P is more than 0.5,***p<0.01 compared to the blank control.
2.2.2 rat urge to withdraw
2.2.2.1 test methods
Taking 40 rats, each half of male and female, randomly dividing into 4 groups, administrating morphine group and drug group by intragastric administration twice a day for 14 days continuously, and administrating physiological saline with the same volume to a blank control group; 14 at 8: after 40 minutes from the last administration of naloxone, 4mg/kg was subcutaneously injected 40 minutes, and then the withdrawal symptoms of rats within 1 hour were immediately observed and recorded. And weighing at 30-60 points.
2.2.2.2 test results
As can be seen from Table 11, morphine animals were significantly more excited than other groups of animals during the observation period after naloxone injection, and showed jumping, dog-like tremor, diarrhea, lacrimation, tooth biting, anterior paw tremor, and the like. Compared with a blank control group, the rat jumping number, diarrhea number and weight loss of morphine have obvious differences, but the indications of large and small dose groups of the analgesic capsule provided by the invention have no obvious difference.
TABLE 11 Effect of the capsules provided by the present invention on the promotion of withdrawal in rats
Group mean dose N-skipped body weight change (g) diarrhea
(mg/kg. days) (times) 30min 60min
Blank control group-100.2 + -0.450.48 + -0.240.31 + -0.180
Morphine group 1501019.4 ± 11.2***-4.62±1.64***-4.89±1.76***10***
Low dose group 300100.0 + -0.0*0.06±0.86*0.12±0.20*0
Large dose group 600100.3 + -0.5*-0.13±0.37*0.02±0.65*0
Comparison with blank control group***P＜0.01，*P＞0.05。
Discussion of 3
Through natural withdrawal and urge withdrawal tests of rats and mice, the analgesic capsule provided by the invention has no significant difference in withdrawal indexes of small dose groups and small dose groups compared with a blank control group, while morphine groups have obvious difference and show obvious dependence. The analgesic capsule provided by the invention has no physical dependence.
Claims (10)
1. A compound having the structure of formula (I).
2. A compound having the structure of formula (II).
3. A process for preparing a compound according to claim 1 or 2,
(1) taking Marasmius androsaceus as a raw material, and extracting with ethanol;
(2) recovering ethanol to obtain fluid extract, diluting with water, adjusting pH to 8-9, defatting, and extracting with chloroform;
(3) recovering chloroform to obtain soft extract, loading on silica gel H column, and eluting with acetone;
(4) recovering acetone to obtain soft extract, washing with ethyl acetate, and vacuum filtering to obtain crystal;
(5) and (3) loading the obtained crystals on a silica gel H column, eluting with acetone to obtain a 1 st peak as alkaloid I and a 2 nd peak as alkaloid II, recrystallizing with ethyl acetate-acetone (2: 1), and further purifying and separating to obtain the alkaloid I, II.
4. An analgesic drug characterized in that the active ingredient thereof is the compound I according to claim 1.
5. The analgesic drug of claim 4, wherein the content of the active ingredient is more than 25%.
6. An analgesic drug characterized in that the active ingredient thereof is the compound II according to claim 2.
7. The analgesic agent according to claim 4 or 5, which further comprises compound II.
8. The analgesic drug of claim 7, characterized in that the content of compoundII is greater than 25%
9. A process for the preparation of an analgesic drug as claimed in claim 7 or 8, characterized in that it is extracted from Marasmius androsaceus.
10. The method of claim 9, wherein the analgesic is administered in a single dose
(1) Taking Marasmius androsaceus as a raw material, and extracting with ethanol;
(2) recovering ethanol to obtain fluid extract, diluting with water, adjusting pH to 8-9, defatting, and extracting with chloroform;
(3) recovering chloroform to obtain soft extract, loading on silica gel H column, and eluting with acetone;
(4) recovering acetone to obtain soft extract, washing with ethyl acetate, and vacuum filtering to obtain crystal.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01131642A CN1428334A (en) | 2001-12-27 | 2001-12-27 | Alkaloid compound, pain-stopping medicine and its preparation method |
| PCT/CN2002/000916 WO2003082818A1 (en) | 2001-12-27 | 2002-12-26 | Alkaloids compounds, analgesic pharmaceuticals and their preparation |
| AU2002354075A AU2002354075A1 (en) | 2001-12-27 | 2002-12-26 | Alkaloids compounds, analgesic pharmaceuticals and their preparation |
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| Application Number | Priority Date | Filing Date | Title |
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| CN01131642A CN1428334A (en) | 2001-12-27 | 2001-12-27 | Alkaloid compound, pain-stopping medicine and its preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102579509A (en) * | 2012-01-18 | 2012-07-18 | 山西康欣药业有限公司 | Selenium-enriched marasmius androsaceus toadstool extract preparation and preparation method thereof |
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| CN1059896C (en) * | 1996-04-17 | 2000-12-27 | 中国科学院兰州化学物理研究所 | Cancer eliminating composition and preparation thereof |
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2001
- 2001-12-27 CN CN01131642A patent/CN1428334A/en active Pending
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2002
- 2002-12-26 AU AU2002354075A patent/AU2002354075A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102579509A (en) * | 2012-01-18 | 2012-07-18 | 山西康欣药业有限公司 | Selenium-enriched marasmius androsaceus toadstool extract preparation and preparation method thereof |
| CN102579509B (en) * | 2012-01-18 | 2014-05-14 | 山西康欣药业有限公司 | Selenium-enriched marasmius androsaceus toadstool extract preparation and preparation method thereof |
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| AU2002354075A1 (en) | 2003-10-13 |
| WO2003082818A1 (en) | 2003-10-09 |
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