Steroid alkaloid as well as preparation method and application thereof in analgesic drugs

Abstract

The invention discloses a steroidal alkaloid, a preparation method thereof and application of the steroidal alkaloid in analgesic drugs. The invention provides a steroid alkaloid with a structural formula as shown in a formula I, and also provides a preparation method of the steroid alkaloid. The invention further provides application of the steroidal alkaloid in preparation of analgesic drugs, and finally provides an analgesic drug composition which comprises the steroidal alkaloid and pharmaceutically acceptable pharmaceutic adjuvants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a steroidal alkaloid extracted from Veratrum, a preparation method thereof and application thereof in analgesic drugs. BACKGROUND

[0002] Chronic pain, especially neuropathic pain, can cause serious physical harm and is a common refractory disease in clinical practice, which can deeply harm the body and mind of patients. Chronic pain is a global problem that has reached the level of an epidemic. According to the statistics of the International Pain Research Society, 1 out of 5 adults in the world is suffering from pain, and 10% of people are diagnosed with chronic pain every year, of which 17% of patients commit suicide due to unbearable pain. The analgesics currently used in clinical practice mainly include opioid drugs represented by morphine and cocaine, and non-steroidal anti-inflammatory drugs represented by aspirin and ibuprofen. Opioid drugs have strong analgesic effect, but their addiction and dependence limit their use. Non-steroidal anti-inflammatory drugs have less side effects but have less analgesic effect on neuropathic pain. Finding new analgesics with less dependence and fewer side effects has always been a research hotspot, and natural products are an important source for the development of new analgesics.

[0003] Veratrum V. mengtzeanum Loes. f. of Liliaceae is one of the original plants of the folk medicine Pima grass, which is included in the 1996 edition of Yunnan Provincial Pharmaceutical Standards, the 2005 edition of Yunnan Provincial Chinese Medicine Standards and the 2006 edition of Fujian Provincial Chinese Medicine Quality Standards. V. mengtzeanum Loes. f. is bitter and cold in nature and has large toxicity, and has the effects of removing blood stasis, swelling, analgesia, hemostasis, expectorating and opening orifices, and is mainly used for treating contusion and injury, fracture, paraplegia, epilepsy, rheumatic pain, trauma and hemorrhage, and is widely used in Yi, Bai and Dai ethnic groups. The present application aims to separate and identify the chemical components in V. mengtzeanum Loes. f., especially the steroidal alkaloids, and explore the analgesic effect, which provides a new idea for developing more effective analgesic treatment and is expected to make an important breakthrough in the field of analgesic drugs. SUMMARY

[0004] In view of the problems in the prior art, the first object of the present application is to provide a steroidal alkaloid compound, the second object of the present application is to provide a preparation method of the compound, and the third object of the present application is to provide application of the compound in the preparation of analgesic drugs.

[0005] To achieve the objects of the present application, the following technical solutions are adopted:

[0006] In a first aspect, the present application provides a steroidal alkaloid as shown in formula (I)

[0007] In a first aspect, the present application provides a steroidal alkaloid as shown in formula (I)

[0008] The compound is isolated from Veratrum V. mengtzeanum Loes. f. of Liliaceae, named Neogermitrine-N-oxide, with a molecular weight of 693, a molecular formula of C 36 H 55 NO 12 , and a chemical structural formula as shown in Formula I.

[0009] In a second aspect, the present application provides a preparation method of the steroid alkaloid, comprising the following steps:

[0010] (1) taking Veratrum medicinal materials, extracting with an alcohol solvent, and reducing pressure to concentrate the extract to obtain a total extract infusion;

[0011] (2) adding water to the total extract infusion to prepare a suspension, standing, filtering, adding chloroform to the filtrate for extraction, and concentrating the chloroform layer to obtain total alkaloids;

[0012] (3) separating the total alkaloids by silica gel column chromatography to obtain a first column chromatography sample;

[0013] (4) separating the first column chromatography sample by silica gel column chromatography to obtain a second column chromatography sample;

[0014] (5) separating the second column chromatography sample by C-18 reverse column chromatography to obtain a third column chromatography sample;

[0015] (6) separating the third column chromatography sample by preparative high performance liquid chromatography to obtain the steroid alkaloid.

[0016] Preferably, the Veratrum in step 1 is the root and rhizome of V. mengtzeanum Loes. f.

[0017] Preferably, before the extraction in step 1, the pre-treatment includes drying and crushing, and the crushing is to 20-50 mesh.

[0018] Preferably, in the extraction in step 1, the mass-volume ratio of the Veratrum medicinal materials to the solvent is 1:3-10, g / ml, and the mass-volume ratio of the raw materials to the solvent refers to the ratio of the mass of the raw materials to the volume of the solvent, for example, 3-8 ml of solvent is used for extraction per gram of raw materials.

[0019] Preferably, in step 1, the extraction method is leaching, and the extraction is performed 2-4 times, and each extraction is performed for 12-24 h.

[0020] Preferably, in step 1, the reducing pressure concentration is to the point that there is no odor of organic solvent.

[0021] Preferably, in step 2, the mass-volume ratio of the total extract infusion concentrate to water is 1:2-3.

[0022] Preferably, in step 2, the ratio of the filtrate to chloroform in the chloroform extraction of step 2 is 1:1.5-2.5, and the extraction is performed 2-4 times.

[0023] Preferably, in the silica gel column chromatography of step 3, the filler is 200-300 mesh silica gel, the mass ratio of the total alkaloid to the silica gel filler is 1:6-8, the height-diameter ratio of the silica gel column is 8-9:1, and the gradient elution is performed with petroleum ether-acetone as the mobile phase, the elution is performed with petroleum ether: acetone in the volume ratio of 25:1, 15:1, 10:1 and 5:1 in sequence, 10-20 column volumes are eluted for each gradient elution, detection is performed with a silica gel thin layer plate TLC, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide is combined, and vacuum concentration is performed to obtain a one-time column chromatography sample.

[0024] Preferably, in the silica gel column chromatography of step 4, the filler is 200-300 mesh silica gel, the mass ratio of the one-time column chromatography sample to the silica gel filler is 1:6-8, the height-diameter ratio of the silica gel column is 8-9:1, and the gradient elution is performed with petroleum ether-acetone as the mobile phase, the elution is performed with petroleum ether: acetone in the volume ratio of 15:1, 10:1, 5:1 and 1:1 in sequence, 10-20 column volumes are eluted for each gradient elution, detection is performed with a silica gel thin layer plate TLC, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide is combined, and vacuum concentration is performed to obtain a two-time column chromatography sample.

[0025] Preferably, in the C-18 reverse column chromatography of step 5, the mobile phase is methanol-water, the elution is performed with 40% methanol, 65% methanol and 80% methanol in sequence, 4-8 column volumes are eluted for each gradient elution, detection is performed with a silica gel thin layer plate TLC, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide is combined, and vacuum concentration is performed to obtain a three-time column chromatography sample.

[0026] Preferably, in the preparative high performance liquid chromatography of step 6, the chromatographic column is ZORBAX SB-C18 (9.4x250mm, 5μm), the flow rate is 10mL / min, the detection wavelength is 254nm, the mobile phase is acetonitrile-0.1% formic acid water, and the elution gradient is 10%-60% acetonitrile in the volume percentage in 0-30min; the eluate of the characteristic peak of Neogermitrine-N-oxide is collected, and vacuum concentration is performed to obtain the steroidal alkaloid.

[0027] In a third aspect, the present application provides use of the steroidal alkaloid in the preparation of an analgesic drug.

[0028] The steroidal alkaloid shows significant analgesic activity by evaluating the hot-plate test and acetic acid writhing test of mice. In the acetic acid writhing test of mice, Neogermitrine-N-oxide significantly inhibits the acetic acid writhing times of mice, and the inhibition rate reaches 86.79%, only lower than that of morphine hydrochloride. In the hot-plate test of mice, Neogermitrine-N-oxide significantly prolongs the paw withdrawal time of mice, and the paw withdrawal time after 30 minutes of administration is longer than that of morphine, and the analgesic effect is equivalent to that of morphine.

[0029] Preferably, the pharmaceutical dosage form is any one of a tablet, a capsule, an injection, a microcapsule, a spray, an ointment or a film.

[0030] In a fourth aspect, the present application further provides an analgesic pharmaceutical composition comprising the steroidal alkaloid and a pharmaceutically acceptable pharmaceutical adjuvant.

[0031] The adjuvant comprises any one of a diluent, a flavoring agent, a binder or a filler, or a combination of at least two, such as a combination of a binder and a diluent, a combination of a binder and a flavoring agent, a combination of a binder and a filler, etc., and any other combination mode can be selected, which will not be described herein.

[0032] The present application has the following beneficial effects:

[0033] 1. The steroidal alkaloid (Neogermitrine-N-oxide) provided by the present application is a new compound first isolated from Kandahar Eustoma, and has significant analgesic effect. Compared with the prior art, the discovery of the compound enriches the natural product library, and provides a candidate compound for developing a new analgesic drug.

[0034] 2. It is proved by the acetic acid writhing test of mice that Neogermitrine-N-oxide significantly inhibits the acetic acid writhing times of mice, and the inhibition rate reaches 86.79%, only lower than that of morphine hydrochloride, indicating that it has good analgesic effect.

[0035] 3. In the hot-plate test of mice, Neogermitrine-N-oxide significantly prolongs the paw withdrawal time of mice, and the paw withdrawal time after 30 minutes of administration is longer than that of morphine, and the analgesic effect is equivalent to that of morphine, proving that it has strong analgesic activity.

[0036] 4. The experimental data show that the pain threshold values of the Neogermitrine-N-oxide group after 15 minutes and 30 minutes of administration have significant differences compared with those of the physiological saline group (P<0.01), further proving that the compound has significant analgesic effect.

[0037] 5. The present invention also provides a method for extracting, separating and purifying Neogermitrine-N-oxide from Veratrum mengziensis. The method is simple to operate and has good reproducibility, providing technical support for the large-scale preparation of the compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the chemical structural formula of the steroidal alkaloid Neogermitrine-N-oxide;

[0039] Figure 2 This is the high-resolution mass spectrum of the steroidal alkaloid Neogermitrine-N-oxide;

[0040] Figure 3 Neogermitrine-N-oxide, a steroidal alkaloid 1 H-NMR spectrum;

[0041] Figure 4 Neogermitrine-N-oxide, a steroidal alkaloid 13 C-NMR spectrum;

[0042] Figure 5 The COSY spectrum of the steroidal alkaloid Neogermitrine-N-oxide;

[0043] Figure 6 This is the HMBC spectrum of the steroidal alkaloid Neogermitrine-N-oxide;

[0044] Figure 7 This is the HSQC spectrum of the steroidal alkaloid Neogermitrine-N-oxide;

[0045] Figure 8 This is the ROESY spectrum of the steroidal alkaloid Neogermitrine-N-oxide;

[0046] Figure 9 The infrared spectrum of the steroidal alkaloid Neogermitrine-N-oxide;

[0047] Figure 10 Schematic diagram of the number of writhing times in the acetic acid writhing analgesia experiment in Example 4 (Note: compared with the normal saline control group, "*" indicates P < 0.1, and "**" indicates P < 0.01);

[0048] Figure 11 This is a bar graph of the pain thresholds at 15, 30, and 60 minutes in the hot plate analgesia experiment of Example 5 (Note: compared with the normal saline control group, "*" indicates P < 0.1, and "**" indicates P < 0.01);

[0049] Figure 12 The pain threshold fold line graph of hot plate method analgesia experiment of Example 5 at 15, 30, 60 min (Note: Compared with the physiological saline control group, "*" indicates P<0.1, "**" indicates P<0.01). DETAILED DESCRIPTION

[0050] The application will be further described in detail below with reference to the accompanying drawings and examples, but in no way limits the application, any transformation or improvement based on the teaching of the application falls into the protection scope of the application.

[0051] The process, conditions, reagents, experimental methods, etc. of the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitations. The experimental methods in each example without specific conditions are usually according to the conventional conditions or according to the conditions suggested by the manufacturers.

[0052] Unless otherwise specified, the meanings of all professional terms and scientific terms used in the specification are the same as the meanings generally understood by the person skilled in the art in the technical field to which the application belongs. However, if there is a conflict, the specification containing the definition shall prevail.

[0053] V. mengtzeanum collected from Xingjing Village and Xingou Village of Caobashi, Mengzi, Yunnan Province, was identified as the root and rhizome of V. mengtzeanum, dried and ground into coarse powder;

[0054] Kunming mice (body weight in the range of 18-20 g) were purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0055] Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Co., Ltd. of Northeast Pharmaceutical Group.

[0056] Example 1 Preparation method of steroidal alkaloid Neogermitrine-N-oxide

[0057] (1) 85 kg of Kandahar aconite medicinal materials were ground to 40 mesh, 500 L of methanol was added for extraction, the extraction time was 24 h, and the extraction was performed for 3 times. The extraction liquid was combined and concentrated under reduced pressure to obtain total extract infusion;

[0058] (2) The total extract infusion was added with 2 times of distilled water to prepare a suspension, which was placed at room temperature for 12 h, filtered, and the filtrate was extracted with chloroform. The ratio of the filtrate to chloroform was 1:2 (v / v), and the extraction was performed for 3 times. The chloroform layers were combined and concentrated under reduced pressure to obtain total alkaloids;

[0059] (3) The total alkaloids were separated and purified by silica gel column chromatography, the packing material was 200-300 mesh silica gel, the mass ratio of the total alkaloids to the silica gel packing material was 1:7, and the height-diameter ratio of the silica gel column was 8:1. Petroleum ether-acetone was used as the mobile phase for gradient elution, the elution mode was that petroleum ether: acetone was used as the elution solvent in the volume ratio of 25:1, 15:1, 10:1 and 5:1 in sequence, 10-20 column volumes were used for each gradient elution, silica gel thin layer plate TLC was used for detection, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide was combined, and vacuum concentration was performed to obtain a first column chromatography sample;

[0060] (4) The first column chromatography sample was separated and purified by silica gel column chromatography, the packing material was 200-300 mesh silica gel, the mass ratio of the first column chromatography sample to the silica gel packing material was 1:8, and the height-diameter ratio of the silica gel column was 9:1. Petroleum ether-acetone was used as the mobile phase for gradient elution, the elution mode was that petroleum ether: acetone was used as the elution solvent in the volume ratio of 15:1, 10:1, 5:1 and 1:1 in sequence, 10-20 column volumes were used for each gradient elution, silica gel thin layer plate TLC was used for detection, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide was combined, and vacuum concentration was performed to obtain a second column chromatography sample;

[0061] (5) The second column chromatography sample was separated and purified by C-18 reverse column chromatography, the mobile phase was methanol-water, 40% methanol, 65% methanol and 80% methanol were used for elution in sequence, 4-8 column volumes were used for each gradient elution, silica gel thin layer plate TLC was used for detection, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide was combined, and vacuum concentration was performed to obtain a third column chromatography sample;

[0062] (6) The third column chromatography sample was separated and purified by preparative high performance liquid chromatography, the chromatographic column was ZORBAX SB-C18 (9.4×

[0063] 250 mm, 5 μm), the flow rate was 10 mL / min, the detection wavelength was 254 nm, and the mobile phase was acetonitrile-0.1% formic acid water, the elution gradient was that the acetonitrile was 0%-60% in volume at 0-30 min, the eluate containing the characteristic peak of Neogermitrine-N-oxide (15 min) was collected, and vacuum concentration was performed to obtain the steroidal alkaloid.

[0064] The compound was a white powder, and the modified bismuth potassium iodide reagent showed a positive reaction, indicating that the compound might be an alkaloid:

[0065] After 1 H-NMR (500 MHz, MeOD), 13The structural formula of the compound was confirmed by C-NMR, COSY, HMBC, HSQC, ROESY, mass spectrometry, and infrared. Figure 1 , molecular formula is C 36 H 55 NO 12 , named

[0066] (Neogermitrine-N-oxide(Ⅰ). Structural identification diagram see Figures 2-9 .

[0067] The structural analysis is as follows: ESI-MS of this compound gave m / z 694[M+H] + , combined with 1 H-NMR (500 MHz, CD3OD) and 13 C-NMR (125 MHz, MeOD) data indicate that the molecular formula of the compound is C 36 H 55 NO 12 , further analyzed by high-resolution mass spectrometry HR-ESI-MS (Measured 716.3614, Predicted 716.3616C 36 H 55 NO 12 Na) confirmed that its molecular formula is C 36 H 55 NO 12 According to the seven methyl signals in the hydrogen spectrum, δ2.66 (3H, s, H-2”), 2.05 (3H, s, H-2”’), 1.40 (3H, d, J=7.0 Hz, H-27), 1.22 (3H, s, H-21), 1.12 (3H, d, J=7.0 Hz, H-5′), 1.03 (3H, s, H-19), 0.89 (3H, t, J=7.5 Hz, H-4′), the four hydroxyl groups in the low field region The proton signals δ5.52 (H, m, H-7), 5.15 (H, d, J = 3.6 Hz, H-15), 4.86 (H, s, H-3), 4.26 (H, m, H-16), combined with the three carbonyl carbon signals δ177.0 (C-1′), 172.2 (Cl″) and 172.0 (C-1″′) in the carbon spectrum, it is speculated that there are two acetyl groups and one isovaleryl group in the compound, and there are three methyl groups and multiple hydroxyl groups on the parent nucleus. 13C-NMR shows 36 carbon signals, 7 methyl, 9 methylene, 12 methine, 8 quaternary carbon, among these carbon signals, there is a signal of hemiketal C atom δ105.2(C-4) and a signal of oxygen bridge head C δ94.1(C-9), according to the above characteristics, it is speculated that the compound is a sibiricine type alkaloid, similar to compound Germitrine. Careful comparison of compound 1 and compound Germitrine 13 C-NMR data found that, in addition to C-3, the chemical shifts of C-18 and C-26 two methylene were greatly shifted to low field, increased by more than 10ppm, reaching δ72.8(C-18) and δ72.5(C-26), according to the reference, this feature is because there is N-oxide between C-18 and C-26. In summary, the compound is speculated to be Neogermitrine-N-oxide, a new compound. Further through HMQC and HMBC, it is found that H-15 is remotely related to C-1', indicating that the isovaleryl group is substituted at C-15; H-3 is remotely related to C-1", indicating that one of the acetyl groups is connected to C-3, 5.52(H-7) is shifted to low field compared with the non-substituted analogue, indicating that the other acetyl group is connected to C-7, the structure of the compound is determined.

[0068] The method realizes efficient separation and purification of steroidal alkaloids in Kandahar by multi-step chromatographic separation technology, and the obtained product has high purity and stable yield.

[0069] Example 2 Preparation method of steroidal alkaloid Neogermitrine-N-oxide

[0070] (1) 85 kg of Kandahar medicinal materials was crushed to 50 mesh, and 850 L of methanol was added for extraction, the extraction time was 24 h, and the extraction was carried out twice, the extract was combined, and the extract was concentrated under reduced pressure to remove organic solvents to obtain total extract extract;

[0071] (2) The total extract extract was added with 3 times of distilled water to prepare a suspension, which was placed at room temperature for 12 h, filtered, and the filtrate was extracted with chloroform, the ratio of the filtrate to chloroform was 1:1.5(v / v), the extraction was carried out 4 times, the chloroform layer was combined, and concentrated under reduced pressure to obtain total alkaloids;

[0072] (3) The total alkaloids were separated and purified by silica gel column chromatography, the packing material was 200-300 mesh silica gel, the mass ratio of total alkaloids to silica gel packing material was 1:8, and the height-diameter ratio of the silica gel column was 9:1. Petroleum ether-acetone was used as the mobile phase for gradient elution, and the elution mode was elution with petroleum ether: acetone in a volume ratio of 25:1, 15:1, 10:1 and 5:1 in turn, 10-20 column volumes for each gradient elution, and detection was performed by silica gel thin layer plate TLC. The eluate containing the steroidal alkaloid Neogermitrine-N-oxide was combined, and vacuum concentration was performed to obtain a first column chromatography sample;

[0073] (4) The first column chromatography sample was separated and purified by silica gel column chromatography, the packing material was 200-300 mesh silica gel, the mass ratio of the first column chromatography sample to silica gel packing material was 1:6, and the height-diameter ratio of the silica gel column was 8:1. Petroleum ether-acetone was used as the mobile phase for gradient elution, and the elution mode was elution with petroleum ether: acetone in a volume ratio of 15:1, 10:1, 5:1 and 1:1 in turn, 10-20 column volumes for each gradient elution, and detection was performed by silica gel thin layer plate TLC. The eluate containing the steroidal alkaloid Neogermitrine-N-oxide was combined, and vacuum concentration was performed to obtain a second column chromatography sample;

[0074] (5) The second column chromatography sample was separated and purified by C-18 reverse column chromatography, the mobile phase was methanol-water, and elution was performed with 40% methanol, 65% methanol and 80% methanol in turn, 4-8 column volumes for each gradient elution, and detection was performed by silica gel thin layer plate TLC. The eluate containing the steroidal alkaloid Neogermitrine-N-oxide was combined, and vacuum concentration was performed to obtain a third column chromatography sample;

[0075] (6) The third column chromatography sample was separated and purified by preparative high performance liquid chromatography, the chromatographic column was ZORBAX SB-C18 (9.4x250mm, 5μm), the flow rate was 10 mL / min, the detection wavelength was 254 nm, and the mobile phase was acetonitrile-0.1% formic acid water, and the elution gradient was 0-30 min: 0%-60% acetonitrile in terms of volume percentage. The eluate of the characteristic peak (16 min) of Neogermitrine-N-oxide was collected, and vacuum concentration was performed to obtain the steroidal alkaloid.

[0076] It was detected that the structure of the steroidal alkaloid was the same as that of Example 1.

[0077] Preparation method of the steroidal alkaloid Neogermitrine-N-oxide

[0078] (1) Khuskhba 85 kg, crushed to 40 mesh, add methanol 255 L for extraction, extraction time is 12 h, extract 4 times, combine the extract, reduce pressure concentration to no organic solvent to get total extract infusion;

[0079] (2) The total extract infusion is added to 1.5 distilled water to make a suspension, stand at room temperature for 12 h, filter, add chloroform to the filtrate for extraction, the ratio of the filtrate to chloroform is 1:2.5 (v / v), extract 2 times, combine the chloroform layer, reduce pressure concentration to get total alkaloids;

[0080] (3) The total alkaloids are separated and purified by silica gel column chromatography, the filler is 200-300 mesh silica gel, the mass ratio of total alkaloids to silica gel filler is 1:6, and the height-diameter ratio of the silica gel column is 8:1. Petroleum ether-acetone is used as the mobile phase for gradient elution, and the elution mode is to sequentially use elution solvents with a petroleum ether:acetone volume ratio of 25:1, 15:1, 10:1 and 5:1, and each gradient elution is 10-20 column volumes, and TLC is used for detection, and the eluent containing the steroidal alkaloid Neogermitrine-N-oxide is combined and reduced pressure concentrated to obtain a one-time column chromatography sample;

[0081] (4) The one-time column chromatography sample is separated and purified by silica gel column chromatography, the filler is 200-300 mesh silica gel, the mass ratio of the one-time column chromatography sample to silica gel filler is 1:8, and the height-diameter ratio of the silica gel column is 9:1. Petroleum ether-acetone is used as the mobile phase for gradient elution, and the elution mode is to sequentially use elution solvents with a petroleum ether:acetone volume ratio of 15:1, 10:1, 5:1 and 1:1, and each gradient elution is 10-20 column volumes, and TLC is used for detection, and the eluent containing the steroidal alkaloid Neogermitrine-N-oxide is combined and reduced pressure concentrated to obtain a two-time column chromatography sample;

[0082] (5) The two-time column chromatography sample is separated and purified by C-18 reverse phase column chromatography, the mobile phase is methanol-water, and sequentially uses 40% methanol, 65% methanol and 80% methanol for elution, each gradient elution is 4-8 column volumes, and TLC is used for detection, and the eluent containing the steroidal alkaloid Neogermitrine-N-oxide is combined and reduced pressure concentrated to obtain a three-time column chromatography sample;

[0083] (6) The sample after three times of column chromatography was separated and purified by preparative high performance liquid chromatography. The chromatographic column was ZORBAX SB-C18 (9.4 x 250 mm, 5 μm), the flow rate was 10 mL / min, the detection wavelength was 254 nm, the mobile phase was acetonitrile-0.1% formic acid water, and the elution gradient was 0-30 min: 0%-60% acetonitrile by volume percentage. The eluent of Neogermitrine-N-oxide characteristic peak (15 min) was collected and concentrated under reduced pressure to obtain the steroidal alkaloid.

[0084] The steroidal alkaloid was detected to have the structure of Example 1.

[0085] Example 4, analgesic effect of Neogermitrine-N-oxide of the steroidal alkaloid on acetic acid writhing in mice

[0086] (1) Animal grouping: 24 SPF Kunming mice, half male and half female, weighing 18-20 g, were adaptively fed for 3-5 days and randomly divided into 3 groups, a normal saline control group (Normal saline), a morphine hydrochloride group (Morphine hydrochloride), and a Neogermitrine-N-oxide group, 8 mice in each group.

[0087] (2) Model construction: The drugs were administered by intraperitoneal injection. The Neogermitrine-N-oxide group and the morphine hydrochloride group were administered at a dose of 1 mg / kg with a volume of 0.2 ml / 10 g, and the normal saline group was administered at a volume of 0.2 ml / 10 g. Thirty minutes after administration, 0.7% acetic acid solution was injected intraperitoneally at a volume of 0.1 ml / 10 g, and the writhing reaction of the mice within 15 min was observed and recorded. The writhing standard was recorded: abdominal concave, stretching of the trunk and hind limbs, and high buttocks and other behavioral reactions. The pain inhibition rate was calculated according to the following formula: pain inhibition rate (%) = (model group writhing times - administration writhing times) / model group writhing times x 100%.

[0088] (3) Experimental results: The experimental results are shown in Table 1. Figure 10, the results showed that the mice in the normal saline group showed obvious writhing reaction after injection of acetic acid solution, and the number of writhing times of the mice in the normal saline group was 26.50±11.74; while the number of writhing behavior of the mice in the morphine hydrochloride group and the Neogermitrine-N-oxide group was significantly lower than that in the normal saline group (P<0.01), the number of writhing times of the mice in the morphine hydrochloride group was 0.75±1.75, and the inhibition rate of writhing was 97.17%; the number of writhing times of the mice in the Neogermitrine-N-oxide group was 3.50±3.12, and the inhibition rate of writhing was 86.79%. It is proved that Neogermitrine-N-oxide shows significant analgesic effect in the acetic acid writhing of mice.

[0089] Example 5, the analgesic effect of Neogermitrine-N-oxide, a steroid alkaloid, on the hot plate model of mice

[0090] (1) Animal grouping: SPF Kunming mice, female, fed for 3-5 days and screened. The mice were placed on the hot plate instrument at 55±0.5℃, and the pain threshold of each mouse (i.e. the time used by the mouse to contact the hot plate to the foot) was measured. The mice with pain threshold of 5-30s were screened. The qualified mice were randomly divided into 3 groups, normal saline control group (normal saline), morphine hydrochloride positive group (morphine hydrochloride), Neogermitrine-N-oxide group, 8 mice in each group;

[0091] (2) Model construction: intraperitoneal injection, the Neogermitrine-N-oxide group and the morphine hydrochloride group were given 0.2ml / 10g of drug, the dose was 1mg / kg, and the normal saline group was given 0.2ml / 10g of drug. 15min, 30min and 60min after administration, the pain response was measured, i.e. the mice were placed on the hot plate instrument at 55±0.5℃, and the time used by the mouse to contact the hot plate to the foot was measured, and the experimental results were recorded;

[0092] (3) Experimental results: the experimental results are shown in Figures 11-12 , the results showed that there was no statistical difference in the pain threshold of the normal saline group before and after administration at each time point; while the pain threshold of the morphine hydrochloride group and the Neogermitrine-N-oxide group was significantly increased, the pain threshold of the Neogermitrine-N-oxide group at 15min and 30min after administration was significantly different from that of the normal saline group (P<0.01), which proved that Neogermitrine-N-oxide had significant analgesic effect.

[0093] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A steroidal alkaloid having a chemical structure as shown in formula (I): ###0001### 2. A method for preparing the steroidal alkaloid as claimed in claim 1, comprising the following steps: (1) taking Kandurmaro medicinal materials, extracting with an alcohol solvent, and concentrating the extract to obtain a total extract; (2) adding water to the total extract to form a suspension, standing, filtering, adding chloroform to the filtrate to extract, and concentrating the chloroform layer to obtain total alkaloids; (3) separating the total alkaloids by silica gel column chromatography to obtain a first column chromatography sample; (4) separating the first column chromatography sample by silica gel column chromatography to obtain a second column chromatography sample; (5) separating the second column chromatography sample by C-18 reverse column chromatography to obtain a third column chromatography sample; and (6) separating the third column chromatography sample by preparative high performance liquid chromatography to obtain the steroidal alkaloid. In step 1, the alcohol solvent is methanol; the extraction method is leaching, the extraction time is 12-24 hours, and the extraction times is 2-4 times. In step 2, in the chloroform extraction, the volume ratio of the filtrate to chloroform is 1:1.5-2.5, and the extraction is performed 2-4 times. In step 3, in the silica gel column chromatography, the filler is 200-300 mesh silica gel, the elution is performed by using petroleum ether: acetone with a volume ratio of 25:1, 15:1, 10:1 and 5:1 in sequence as elution solvents, 10-20 column volumes are eluted for each gradient, detection is performed by using silica gel thin layer plate TLC, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide is combined, and the first column chromatography sample is obtained by concentration under reduced pressure. In step 4, in the silica gel column chromatography, the filler is 200-300 mesh silica gel, the elution is performed by using petroleum ether: acetone as mobile phase, the elution is performed by using petroleum ether: acetone with a volume ratio of 15:1, 10:1, 5:1 and 1:1 in sequence as elution solvents, 10-20 column volumes are eluted for each gradient, detection is performed by using silica gel thin layer plate TLC, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide is combined, and the second column chromatography sample is obtained by concentration under reduced pressure. In step 5, in the C-18 reverse column chromatography, the mobile phase is methanol-water, the elution is performed by using 40% methanol, 65% methanol and 80% methanol in sequence, 4-8 column volumes are eluted for each gradient, detection is performed by using silica gel thin layer plate TLC, the eluate containing the steroidal alkaloid Neogermitrine-N-oxide is combined, and the third column chromatography sample is obtained by concentration under reduced pressure. In step 6, in the preparative high performance liquid chromatography, the chromatographic column is ZORBAX SB-C18, the mobile phase is acetonitrile-0.1% formic acid water, the elution gradient is 10%-60% acetonitrile in 0-30 min in terms of volume percentage, the eluate of the characteristic peak of Neogermitrine-N-oxide is collected, and the steroidal alkaloid is obtained by concentration under reduced pressure.

3. The preparation method according to claim 2, characterized in that:

8. Use of the steroidal alkaloid as claimed in claim 1 or the steroidal alkaloid prepared by the method as claimed in claims 2-7 in the preparation of an analgesic drug.

4. The preparation method according to claim 2, characterized in that The drug dosage form is any one of a tablet, a capsule, an injection, a microcapsule, a spray, an ointment or a film.

5. The preparation method according to claim 2, characterized in that: ​ ​ 6. The preparation method according to claim 2, characterized in that: ​ 7. The preparation method according to claim 2, characterized in that: ​ ​ 9. The use according to claim 8, characterized in that ​ 10. An analgesic pharmaceutical composition comprising the steroidal alkaloid of claim 1 or the steroidal alkaloid prepared by the process of claims 2-7 and a pharmaceutically acceptable pharmaceutical excipient.

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