CN108478575B - Magnoflorine preparation, preparation method and application thereof - Google Patents
Magnoflorine preparation, preparation method and application thereof Download PDFInfo
- Publication number
- CN108478575B CN108478575B CN201810470007.8A CN201810470007A CN108478575B CN 108478575 B CN108478575 B CN 108478575B CN 201810470007 A CN201810470007 A CN 201810470007A CN 108478575 B CN108478575 B CN 108478575B
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- magnoflorine
- preparation
- phospholipid
- phospholipid complex
- phase
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- A61K31/47—Quinolines; Isoquinolines
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
本发明公开了木兰花碱制剂和制备方法及应用，其中一种为木兰花碱磷脂复合物，另外一种为鼻用木兰花碱磷脂复合物‑温敏型原位凝胶。本发明的制备方法具有分离效果好、制备量大、损失少、操作简便的特点。所涉及的木兰花碱磷脂复合物中木兰花碱和磷脂按质量比为1～4:2～1，所涉及的制备方法是将配方量的组分在有机溶剂中溶解混合，在适宜温度下制备。将所得木兰花碱磷脂复合物再加入温敏型原位凝胶辅料制备而成木兰花碱磷脂复合物‑温敏型原位凝胶。经动物药效学实验研究表明，木兰花碱及其两种制剂对于抑郁症、失眠疾病具有显著的改善和治疗作用。
The invention discloses a magnolia alkaloid preparation, a preparation method and an application, wherein one is a magnolia alkaloid phospholipid complex, and the other is a nasal magnoli phospholipid complex-thermosensitive in-situ gel. The preparation method of the invention has the characteristics of good separation effect, large preparation amount, less loss and simple operation. The magnolidine and phospholipids in the magnolidine phospholipid complex are in a mass ratio of 1-4:2-1, and the related preparation method is to dissolve and mix the components of the formula in an organic solvent, and at a suitable temperature preparation. The obtained magnolanine phospholipid complex is further added with thermosensitive in situ gel auxiliary materials to prepare a magnolia alkali phospholipid complex-thermosensitive in situ gel. Experiments on animal pharmacodynamics show that magnolia and its two preparations have significant improvement and therapeutic effects on depression and insomnia.
Description
Technical Field
The invention relates to a magnoflorine preparation as an effective alkaloid component, a preparation method thereof and application thereof in treating depression and insomnia, belonging to the field of medicines.
Background
The depression is a syndrome with obvious and lasting mood depression as a main clinical manifestation, serious people can have suicide and self-disabled behaviors, the incidence rate of global depression reaches 3.3 percent at present, but exceeds 6 percent in developed countries, more than 7 hundred million of global depression patients and the prevalence rate of depression in China is between 3 and 5 percent, and the data continuously and continuously rises along with the development of the society. Insomnia refers to the failure of normal sleep, and the specific symptoms are rolling and difficult sleep, poor sleep quality, reduced sleep time, and severe people may have insomnia all over the night[3]. In the modern society, more and more people suffer from insomnia, and the insomnia seriously affects the normal life of people and brings great mental pain to patients, so the people are widely concerned by the society[4]. Studies find that insomnia is closely related to depression. Most patients with depression suffer from insomnia, and the incidence rate of depression of the patients with insomnia is far higher than that of the normal population. Long-term clinical tests show that western medicines for treating depression and insomnia patients have a great deal of side effects, such as memory decline and drug dependence, and can even lead the disease condition to rebound and increase the risk degree, but the traditional Chinese medicines have good treatment effect and few side effects,is suitable for a wide range of people, so that the Chinese medicament has more and more attention to the treatment of depression and insomnia.
The researches have proved that the magnoflorine has good effects of protecting cardiovascular system, regulating immunity and resisting oxidation. Due to the poor lipid solubility of magnoflorine, permeability and bioavailability thereof are low, so that antidepressant effect of magnoflorine is seriously affected. Aiming at the problems of the magnoflorine, the magnoflorine preparation is developed to effectively improve the problems.
The rate and extent of penetration of antidepressant, insomnia drugs into the bloodstream or cerebrospinal fluid through the intestinal or meninges depends on the solubility, i.e. lipid solubility, of the drug in lipids; the lipid solubility also affects the pharmacokinetic properties of the drug molecule, particularly the absorption, diffusion and release of the drug. Phospholipid complexes are a novel drug delivery system. As an amphoteric material, phospholipids have polar groups (hydrophilic part) and long-chain fatty acid chains (lipophilic part). Therefore, the phospholipid can not only increase the hydrophilicity of the drug but also increase the lipophilicity of the drug. Many natural drugs, especially alkaloids, flavones, have significant affinity for phospholipid molecules, facilitating the binding of the drug to the phospholipid molecules. The drug-phospholipid complex, as a novel drug delivery system, exhibits superior chemical and physical properties and enhances the tissue permeability of the drug.
Modern medicine believes that the treatment of central nervous system diseases by nasal cavity into brain administration has the following advantages: 1. direct delivery of drugs into the brain bypassing the blood brain barrier; 2. the first pass effect of the liver is avoided; 3. can relieve initial shock effect of the medicine. The nasal preparations which are put into clinical application at present are mainly divided into spray, film agent, gel and the like, but the nasal cavity has a unique physiological structure, and the pharmaceutical dosage forms all face the problems of difficult use, easy interference from external environment, low bioavailability and the like, so that the novel nasal temperature-sensitive in-situ gel integrates the advantages of the traditional nasal preparation and can improve the defects of the nasal preparation to a certain extent becomes a research hotspot. The temperature sensitive in-situ gel is characterized in that the gel is free flowing liquid at room temperature, can be rapidly converted into semisolid gel at the ambient temperature condition by spraying or dropping into nasal cavities, and can be attached to the surface of nasal mucosa and enter the body through nasal mucosa capillaries and sieve pores.
Disclosure of Invention
The invention provides a magnoflorine preparation, a preparation method and application thereof, and solves the problems of low lipid solubility and low bioavailability of magnoflorine in the prior art.
The invention is realized by the following technical scheme:
a magnoflorine phospholipid complex is characterized in that the mass ratio of magnoflorine to phospholipid in the magnoflorine phospholipid complex is 1-4: 2-1, and the mass concentration of magnoflorine is 2.5-7.5 mg/mL.
The phospholipid is selected from soybean lecithin, egg yolk lecithin or synthetic phospholipid, and the synthetic phospholipid is at least one of synthetic phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine.
The magnoflorine is extracted and separated from the spina date seed or is selected from natural medicines with similar pharmacological effects through a chemical synthesis method.
The preparation method of the magnoflorine comprises the following steps:
(1) preparation of crude extract:
crushing spina date seeds, sieving with a 40-mesh sieve, degreasing with petroleum ether, adding 6-12 times by mass of an ethanol water solution with the volume percentage concentration of 60% -80%, heating and refluxing for 2-3 times, each time for 1-3 hours, filtering, combining extracting solutions, concentrating ethanol in a filtrate until no alcohol smell exists, dissolving with 2-6 times of water, extracting with n-butyl alcohol for 4-6 times, concentrating the extracting solution under reduced pressure, recovering a solvent to obtain a n-butyl alcohol crude extract, namely a sample to be separated;
(2) separating magnoflorine by high-speed counter-current chromatography:
taking ethyl acetate, n-butanol and water in a volume ratio of 1-3: 1-5: 2-7, placing the ethyl acetate, n-butanol and water in a separating funnel to prepare a two-phase solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase; filling a chromatographic column of a countercurrent chromatography with the stationary phase, rotating the main machine forward at the temperature of 25-35 ℃ at the rotating speed of 300-900 r/min, and pumping the mobile phase at the flow rate of 1.5-10 ml/min until the two-phase solvent reaches an equilibrium state in the column; and (2) dissolving the mixed crude extract prepared in the step (1) by using a mixed solvent with the volume ratio of the upper phase to the lower phase being 1:1, injecting a sample, collecting effluent of the magnoflorine under an ultraviolet detector respectively, concentrating and drying to obtain the magnoflorine respectively.
A preparation method of magnoflorine phospholipid complex comprises the following steps: dissolving magnoflorine in absolute ethyl alcohol at the concentration of 2.5-7.5mg/mL, adding phospholipid according to the mass ratio of 1-4: 2-1, stirring for 1-3 hours at the temperature of 50-60 ℃, and removing the solvent; adding appropriate amount of dichloromethane to dissolve reaction product, centrifuging to remove precipitate, evaporating filtrate to remove solvent, and vacuum drying at 30 deg.C for 24 hr to obtain magnoflorine phospholipid complex.
The application of the magnoflorine and phospholipid complex in preparing the medicine for preventing and treating insomnia and depression.
A magnoflorine phospholipid complex-temperature sensitive in-situ gel is prepared by taking magnoflorine as an active ingredient to prepare a magnoflorine phospholipid complex and adding a temperature sensitive in-situ gel auxiliary material, wherein the weight percentages of the active ingredient and the auxiliary material are as follows:
poloxamer 40716-20%, poloxamer 1880-6%, benzalkonium chloride 0.02% and magnoflorine and phospholipid complex 1-3%.
A preparation method of magnoflorine phospholipid complex-temperature sensitive in-situ gel comprises the following steps: weighing 1-3% of magnoflorine phospholipid complex, adding a proper amount of distilled water, carrying out ultrasonic dissolution, then adding 16-20% of poloxamer 407, 0-6% of poloxamer 188 and 0.02% of benzalkonium chloride, placing in an ice water bath at 4 ℃, carrying out magnetic stirring, after uniform dispersion, placing in a refrigerator at 4 ℃, and fully swelling for 24 hours to obtain the clear and transparent magnoflorine phospholipid complex-temperature-sensitive in-situ gel.
The magnoflorine phospholipid complex-temperature sensitive in-situ gel is used for preparing the medicine for preventing and treating insomnia and depression.
The invention has the beneficial effects that:
the lipid solubility of the magnoflorine provided by the invention is increased after the magnoflorine forms the phospholipid complex, and the permeability of the blood brain barrier and the intestinal tract of the magnoflorine is favorably improved, so that the oral availability is improved, and the better anti-depression and anti-insomnia effect is exerted. Compared with the traditional nasal preparation, the magnolia alkaloid phospholipid complex-temperature-sensitive in-situ gel has the characteristics of convenience in use, difficulty in quick removal, quick response, high bioavailability and the like.
Drawings
FIG. 1 is an HPLC chromatogram of a crude mixture of magnoflorine and spruce, 6' -feruloylspirocin prepared in example 1;
FIG. 2 is a high-speed countercurrent chromatography (HSCCC) chart of example 1;
FIG. 3 is an HPLC chromatogram of magnoflorine compound 1 prepared in example 1;
FIG. 4 is a mass spectrum of magnoflorine;
FIG. 5 shows magnoflorine NMR spectra;
FIG. 6 is a NMR carbon spectrum of cymbidine;
FIG. 7 DSC scan of magnoflorine phospholipid complex; wherein, the mangnoline comprises (1) magnoflorine, (2) lecithin, (3) a physical mixture of magnoflorine and lecithin, (4) magnoflorine and phospholipid complex;
FIG. 8 IR spectrum of magnoflorine phospholipid complex; wherein, the mangnoline comprises (1) magnoflorine, (2) lecithin, (3) a physical mixture of magnoflorine and lecithin, (4) magnoflorine and phospholipid complex;
FIG. 9 is a transmission electron micrograph of magnoflorine phospholipid complex;
FIG. 10 particle size distribution of magnoflorine phospholipid complexes;
FIG. 11 oil-water partition coefficients of magnoflorine at different pH; wherein, the mangnoline (1) and the magnoflorine phospholipid complex (2) are mixed;
FIG. 12 plasma concentration-time curves for magnoflorine and magnoflorine phospholipid complexes ((C))
n＝6)；
FIG. 13 is a response surface of the interaction of P407 and P188;
fig. 14 contour lines of P407 interaction with P188.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention, but are not intended to limit the embodiments of the invention in accordance with the prior art known in the art. Any modification or variation of the individual steps of the invention will fall within the scope of the invention without departing from the spirit and principles of the invention.
Example 1
Preparation method of magnoflorine
(1) Preparation of crude extract:
crushing 250g of spina date seed, sieving with a 40-mesh sieve, degreasing with petroleum ether, respectively adding 1200ml, 800ml and 600ml of ethanol water solution with the percentage concentration of 70%, heating and refluxing for 3 times, each time for 1 hour, filtering, combining extracting solutions, concentrating the filtrate with ethanol until no ethanol smell exists, dissolving with 2 times of water, extracting with n-butanol for 5 times, concentrating the extracting solution under reduced pressure, and recovering the solvent to obtain n-butanol crude extract, namely a sample to be separated, wherein the HPLC (high performance liquid chromatography) spectrum of the sample is shown in figure 1.
(2) Separating magnoflorine and stecinolone, 6' -feruloyl stecinolone by high-speed counter-current chromatography:
taking n-butanol, ethyl acetate and water in a volume ratio of 2:3:5, placing the mixture in a separating funnel to prepare a two-phase solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase; filling the stationary phase in a chromatographic column of a counter-current chromatograph, rotating the main machine forward at 25 ℃ at a rotating speed of 500r/min, and pumping the mobile phase at a flow rate of 7ml/min until the two-phase solvent reaches an equilibrium state in the column; taking the mixed crude extract prepared in the step (1), dissolving the mixed crude extract by using a mixed solvent with the volume ratio of an upper phase to a lower phase being 1:1, then injecting a sample, detecting effluent liquid by using an ultraviolet detector with the wavelength of 280nm, respectively merging and collecting effluent liquid corresponding to 72min to 84min, 86min to 100min and 176min to 202.5min according to a high-speed countercurrent chromatogram, and concentrating and drying the effluent liquid to constant weight to respectively obtain magnoflorine (fraction I), steinocin (fraction II) and 6' -feruloylscinonide (fraction III).
(3) Monomer purity and Structure determination
The obtained monomer compounds were detected by high performance liquid chromatography. ChromatographyColumn: agilent TC-C18Columns (4.6 mm. times.250 mm, 5 μm); acetonitrile-0.1% acetic acid water solution, gradient elution; the running time is 40 min; 0min 10% acetonitrile, 5min 20% acetonitrile, 5-40 min 20% -30% acetonitrile; flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction amount: 20 mu L of the solution; the detection wavelength is 280 nm. The purity of the compound magnoflorine was found to be 95.0% (fig. 3). By Mass Spectrometry (MS) (FIG. 4),1H-NMR and13and C-NMR analysis, structural analysis and comparison with literature data show that the obtained monomer compounds are 250mg of magnoflorine respectively.
Identifying the structure of magnoflorine: the crystal is yellow needle crystal, blue fluorescence is shown under an ultraviolet lamp at 254nm, and the improved bismuth potassium iodide reagent shows positive color. Subjecting it to nuclear magnetic resonance spectroscopy, which1H-NMR (400MHz DMSO) (FIG. 5) chemical shifts are as follows: 6.504(1H, s), 6.356(IH d, J ═ 8.0Hz), 6.598(IH d, J ═ 8.0 Hz); it is composed of13C-NMR(100MHz MeOD-d4) (FIG. 6) chemical shifts are as follows: 43.5, 53.8(N-Me), 24.6(2 Me), 56.0, 56.3(10,11-MeO), 150.8, 151.7(2, 10), and the compound was identified as magnoflorine (magnoflorine) with the formula C20H24NO4 +Molecular weight 342.4.
Example 2
Pharmacodynamic experiment of magnoflorine on CUMS depression insomnia mouse model
1 materials of the experiment
1.1 Experimental animals
Healthy male ICR mice (weight 20 +/-2 g) were purchased from the Experimental animals center of the institute of Radiology and medicine, academy of Chinese sciences; the animals are raised for 3 days before the experiment and are adaptive to the environment. The room temperature is 22 +/-2 ℃, and the relative humidity is 65-70%. The day is 12h, and the diet and water intake are free.
1.2 reagents and drugs:
magnoflorine, spidroin (self-made in this laboratory); venlafaxine hydrochloride sustained release capsules (Chengdu Kanghong pharmaceutical industry group member Co., Ltd.)
1.3 instruments
Stopwatches (Hoyere Orida, Switzerland);
analytical balance model AUX120 (shimadzu corporation);
ZH-YLS-1A animal autonomic activity recorder (Anhui Zhenghua biological instruments equipment Co., Ltd.);
KQ-200KDB model high power digital control ultrasonic cleaner (ultrasonic instruments Co., Ltd., Kunshan city, Jiangsu).
2 method of experiment
2.1 animal grouping and administration
The mice are randomly divided into 6 groups, each group comprises 10 mice, and the groups respectively comprise a blank group, a model group, a western medicine positive group (venlafaxine), a Chinese patent medicine positive group (depression relieving and nerve calming granules), a water decoction group and a magnoflorine group. Except for the blank group, the other groups of mice are molded according to the method of 2.2; gavage was administered 8:00-9:00 a day earlier for 21 days from the first day of molding, and the dosing schedule is shown in table 1.
TABLE 1 animal grouping and dosing regimens
2.2 method for establishing CUMS depression insomnia mouse model
10 stressors were randomized within 21d, and any two stimuli were given daily, with an average of 4 per stimulus given to avoid adaptation in mice.
(1) And (3) moist breeding: the mouse pad is moistened by adding a proper amount of water and survives for 12 hours in a humid environment.
(2) Fasting: and (5) stopping eating for 12 h.
(3) Water forbidding: and water is cut off within 12 hours.
(4) Obliquely feeding: mice were reared with 45 ° inclination for 12 h.
(5) Tail clamping: the mice were placed in a stationary cage and held 1cm from the caudal root for 1min with hemostats.
(6) Feeding without padding: mice were housed in litterless mouse cages for 12h each time.
(7) Sleep deprivation: depriving sleep for 24 h. A small platform water environment method is adopted, a platform with the diameter of 3cm and the height of 2cm is arranged at the bottom of the container, water (the water depth is 0.5cm) is filled at the periphery, and the mouse can fall into the water when sleeping.
(8) Cold water swimming: the animals were placed in a container (water depth 5cm) containing water at 10 ℃ for 5 min.
(9) Swimming with hot water: the animals were placed in a container (water depth 5cm) containing water at 45 ℃ for 5 min.
(10) The day and night are reversed: the cages of mice were placed in dark space without lighting at 7:00 a.m., and mice were placed in lighted rooms until 7:00 a.m. the next day by 19:00 a.m.
2.3 Observation index and method
(1) Body weight
The body weight change of the mice during the experiment, i.e., the body weight on day 1, day 3, day 10, day 17, and day 21 was measured, and the body weight gain of each group of mice was calculated.
(2) Tail suspension experiment (TST)
The tail end of the mouse is attached to a horizontal wooden stick, the wooden stick is lifted off the ground to enable the animal to be in a suspended and inverted hanging shape, and the two sides of the hanging shape block the sight of the mouse by using partition plates. Animals struggle to move in order to overcome abnormal body positions, but after moving for a certain time, intermittent 'immobility' appears to show a 'disappointed' state. The experiment was run for 6min and the immobility time within 4min after the experiment was recorded.
(3) Forced Swimming Test (FST)
The mice were placed individually in transparent glass jars containing water at 45 ℃ and a depth of over 10cm and the experiment was carried out for 6 min. The mouse stops struggling after struggling to run away to feel hopeless, and only the head is exposed out of the water surface and the limbs are in a fixed state, and the immobility time of the mouse within 4min after recording.
(4) Pentobarbital sodium synergistic effect experiment
The sub-threshold dose of sodium pentobarbital is injected into the abdominal cavity after the intragastric administration for 0.5h on the 8 th day of the animal experiment, and the dose is 28 mg/kg. The number of mice in each group that sleep was recorded. The suprathreshold dose of sodium pentobarbital is injected into the abdominal cavity after the intragastric administration for 0.5h on the 9 th day, and the dose is 38 mg/kg. After injection, timing was started and sleep latency and sleep time were recorded. The time when the righting reflex disappears is taken as the sleep starting time, and the righting reflex is recovered as the sleep ending time. The time from intraperitoneal injection to sleep onset is sleep latency, and the latency exceeds 15min and is recorded as 15 min.
3 results
3.1 Effect of each administration group on the immobility time of the TST experiment of CUMS mice
The model group showed a significant increase in TST immobility time compared to the blank group of mice. The TST immobility time of the magnoflorine group is lower than that of the model group, and the difference has statistical significance (P is less than 0.05); the lower percentage of time spent on TST immobility compared to the model group was comparable to the water-decocted group, and the results are shown in table 2.
TABLE 2 Effect of each administration group on the time of immobility of tail suspension of CUMS depressed insomnia mice: (
n＝10)
Comparison with model group P <0.05 x comparison with model group P <0.01
3.2 Effect of each administration group on the immobility time of the FST experiment of CUMS mice
The FST immobility time of the magnoflorine group is obviously reduced compared with that of the model group, and the statistic difference (P is less than 0.01) is obvious, and the difference between the positive medicine group and the water decoction group is of statistical significance (P is less than 0.05) compared with that of the model group. The percentage of FST immobility time of the magnoflorine group compared with the model group is equivalent to that of the blank group, the water-decocted group and the positive drug group, and the results are shown in Table 3.
TABLE 3 Effect of the groups on the immobility time of forced swimming of mice: (
n＝10)
Comparison with model group P <0.05 x comparison with model group P <0.01
3.3 Effect of each administration group on the subthreshold dose sodium pentobarbital experiment in mice
The experimental results in table 4 show that the percentage of sleeping time of each administration group is significantly increased compared to the model group, wherein the percentage of sleeping time of the magnoflorine group is equivalent to that of the water-decocted group.
Table 4 effect of each dosing group on the subthreshold dose sodium pentobarbital experiments in mice
3.4 Effect of Each administration group on the synergistic Effect of suprathreshold dose of sodium pentobarbital
The data in table 5 show that there was no statistical difference in sleep latency between the groups. The sleep time of each administration group is longer than that of the model group, wherein the sleep time of the magnoflorine group is obviously longer than that of the model group, the difference has statistical significance (P is less than 0.05), and the administration group is similar to the depression-relieving and nerve-soothing granule group and the water-decocting group.
Table 5 suprathreshold dose pentobarbital sodium experiment in mice with each dosing group
Comparison with model P <0.05 x comparison with model group P <0.01
Example 3
Preparation and detection of magnoflorine phospholipid complex
1 materials of the experiment
1.1 drugs and reagents
Magnoflorine (self-made in laboratory, purity > 98%);
soya bean lecithin (Shanghai blue season Bio Inc., batch number: 20160628);
absolute ethyl alcohol, dichloromethane, methanol, ethyl acetate, n-butanol (analytical purity, tianjin standard chemical reagents, ltd.);
1.2 instruments
RCT basic magnetic stirrer (guangzhou instrumental laboratory technologies ltd);
type 214 differential scanning calorimetry analyzer (german navy corporation);
a type 380 fourier infrared spectrometer (hitachi high new technologies, japan);
HT7700 type transmission electron microscope (hitachi high and new technologies, japan);
agilent1100 high performance liquid chromatograph (Agilent technologies, ltd);
analytical balance model AUX120 (shimadzu corporation);
2 method of experiment
2.1 preparation of magnoflorine phospholipid complexes
Dissolving magnoflorine in anhydrous ethanol at a concentration of 2.5-7.5mg/mL, adding soybean lecithin at a mass ratio of 1:2, stirring at 50-60 deg.C for 1-3h, and removing solvent; adding appropriate amount of dichloromethane to dissolve reaction product, centrifuging to remove precipitate, evaporating filtrate to remove solvent, and vacuum drying at 30 deg.C for 24 hr to obtain magnoflorine phospholipid complex.
2.2 determination of the Complex Rate of magnoflorine phospholipid Complex
Accurately weighing a certain amount of magnoflorine phospholipid complex, dissolving with sufficient dichloromethane, centrifuging, washing the precipitate with dichloromethane for multiple times to obtain uncomplexed magnoflorine or other impurities. Dissolving the precipitate with methanol, filtering, and measuring magnoflorine content by HPLC. According to the following formula, the complex rate of the magnoflorine phospholipid complex can be calculated.
The complex rate (%) of magnoflorine and lecithin is (A1-A2)/A1 × 100%
Note: a1 is the initial magnoflorine dosed mass; a2 is the magnoflorine mass in the precipitation of dichloromethane insolubles.
2.3 Differential Scanning Calorimetry (DSC)
Respectively taking magnoflorine, lecithin, physical mixture of magnoflorine and lecithin (1:2), and magnoflorine phospholipid complex, taking an empty aluminum crucible as reference, and heating at 10 deg.C/min-1Scanning at 0-300 deg.C under nitrogen flow protection, and performing differential scanning calorimetryAnd (6) analyzing.
2.4 Infrared Spectroscopy (IR)
Respectively taking appropriate amount of magnoflorine, lecithin, physical mixture of magnoflorine and lecithin (1:2) and magnoflorine phospholipid compound, performing IR analysis by taking KBr as reference, wherein the wavelength range is 400-4000 cm-1。
2.5 morphological analysis
Taking a small amount of magnoflorine phospholipid complex powder, adding a proper amount of distilled water, uniformly mixing by a vortex oscillator, dropwise adding the mixture onto a 300-mesh copper net, sucking off the redundant filtrate, taking out and placing under a transmission electron microscope for observation.
2.6 particle size analysis
Taking a small amount of magnoflorine phospholipid complex powder, adding a proper amount of distilled water, uniformly mixing by using a vortex oscillator, and placing in a Malvern laser particle size analyzer to measure the particle size distribution.
2.7 investigation of solubility Properties
Placing a proper amount of magnoflorine phospholipid complex in a 10mL volumetric flask, adding water-saturated n-octanol, and diluting to a scale to prepare a drug-containing n-octanol solution. Precisely measuring 1mL of the medicinal solution, placing in a centrifuge tube, respectively adding water saturated with n-octanol, hydrochloric acid solution with pH 1.2, phosphate buffer solution with pH 2.0, 2.5, 4.0, 5.8, 6.8, 7.4 and 8.0, placing in a constant temperature oscillator at 37 ℃, shaking for 24h for balancing, centrifuging at 3000r/min for 30min, sucking lower layer water solution, diluting to a certain concentration with methanol, introducing sample by using a liquid chromatograph, measuring the content of magnoflorine, and calculating the oil-water distribution coefficient (P) according to formula 1-1. The oil-water distribution coefficient (P) is that (W)1/VOil)/(W2/VWater (W)) (1-1)
W1: the dosage of the oil phase at the end is the dosage of the initial oil phase-the dosage of the water phase at the end; w2: the dosage of the water phase at the end
2.8 pharmacokinetic and bioavailability Studies
12 SD rats (body mass 200g + -20 g) were randomly divided into two groups of 6 rats, and fasted for 12h before administration with free water. The mangnoline is injected into the tail vein of the first group (the dosage is 12mg/kg), the mangnoline phospholipid complex is injected into the tail vein of the second group (the dosage is equivalent to 12mg/kg of the mangnoline), 0.3mL of blood is taken from orbital venous plexus for 5min, 10min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h and 24h, the mixture is placed into a heparin-containing blood collection tube, the centrifugation is carried out for 10min at 3000r/min, and the upper plasma is taken and stored in a refrigerator at minus 20 ℃.
Plasma sample analysis chromatography conditions column: agilent TC-C18Columns (4.6 mm. times.250 mm, 5 μm); acetonitrile-0.1% acetic acid water solution, gradient elution; the running time is 35 min; 10-20% acetonitrile in 0-10min, 20-23% acetonitrile in 10-35 min; flow rate: 1 mL/min; column temperature: 25 ℃; sample introduction amount: 20 mu L of the solution; the detection wavelength is 280 nm. Pharmacokinetic parameters were analyzed using the pharmacokinetic software DAS 2.0.
3 results of the experiment
3.1 complexing ratio of magnoflorine phospholipid Complex
The high performance liquid chromatography is used for determining that the complexing rate of the magnoflorine phospholipid complex is 99.1%.
3.2 differential scanning calorimetry
As shown in the results of FIG. 7, magnoflorine has an endothermic peak at 82.64 ℃; in the DSC curve of lecithin, lecithin has two endothermic peaks at 92.24 ℃ and 259.97 ℃ and one exothermic peak at 189.11 ℃; the DSC curve of the physical mixture of the magnoflorine and the lecithin according to the mass ratio of 1:2 is basically the superposition of main peaks of the magnoflorine and the lecithin, the endothermic peak rise of the magnoflorine in the curve is 103.40 ℃, and the magnoflorine and the endothermic peak at the temperature of 92.24 ℃ possibly are mixed together; in a DSC curve of the magnoflorine phospholipid complex, three endothermic peaks of the magnoflorine phospholipid complex are 101.86 ℃, 182.05 ℃ and 260.06 ℃ respectively, while a physical mixture of magnoflorine and lecithin is an exothermic peak at the temperature of 175-200 ℃, which indicates that complex chemical compounding is not performed between magnoflorine and lecithin in the magnoflorine phospholipid complex instead of simple physical mixing, and strong acting force is formed between the magnoflorine and the lecithin, and the acting force can be destroyed only by raising the temperature to a certain temperature and absorbing enough energy.
3.3 Infrared Spectroscopy
As shown in FIG. 8, in the infrared of magnoflorineIn the spectrum, the stretching vibration peak (v OH, 3362.1 cm) of hydroxyl in the magnoflorine structure can be seen-1) And a stretching vibration peak of a benzene ring skeleton (v C, 1645.9 cm)-1，1515.7cm-1) Out-of-plane bending vibration peak of carbon-hydrogen bond on benzene ring (v C-H, 833.2 cm)-1) Stretching vibration peak of carbon-oxygen single bond (v Ar-O-C, 1283.4 cm)-1，1250.5cm-1). In the infrared spectrum of lecithin, the hydrocarbon stretching vibration peak (v C-H, 2925.2 cm) of saturated aliphatic hydrocarbon chain in the lecithin structure can be seen-1，2853.8cm-1) The peak of carbonyl stretching vibration in fatty acid ester (v C ═ O, 1739.8 cm)-1) Stretching vibration peak of methyl group (v C-H, 1463.3 cm)-1，1375.6cm-1) Stretching vibration peak of phosphorus-oxygen double bond (v P ═ O, 1237.1 cm)-1) Stretching vibration peak of phosphorus-oxygen single bond (v P-O, 1061.2 cm)-1). As can be seen by comparison, the infrared spectrum of the magnoflorine-lecithin physical mixture is basically the superposition of main stretching vibration peaks of magnoflorine and lecithin, which indicates that no interaction occurs between magnoflorine and lecithin in the physical mixture, and magnoflorine still exists in a crystal form. In the infrared spectrum of the magnoflorine-phospholipid complex, the peak position of the stretching vibration peak of hydroxyl in a magnoflorine structure is obviously changed from 3362.1cm-1Becomes 3332.2cm-1(ii) a The stretching vibration peak of the benzene ring framework is changed, and v C is C1515.7 cm-1The peak of (a) disappears; the out-of-plane bending vibration peak of the carbon-hydrogen bond on the benzene ring is 833.2cm-1Moved to 829.4cm-1(ii) a Stretching vibration peak of carbon-oxygen single bond (v Ar-O-C, 1283.4 cm)-1，1250.5cm-1) Disappearance; the stretching vibration peak of the phosphorus-oxygen single bond in the lecithin structure is 1061.2cm-1Displaced to 1101.9cm-1(ii) a The peak positions of the hydrocarbon stretching vibration peak of the saturated fatty hydrocarbon chain at the lipophilic end of lecithin and the carbonyl stretching vibration peak in fatty acid ester are not changed. In conclusion, the magnoflorine phospholipid complex is supposed to form a complex through charge migration between oxygen anions of methoxyl and hydroxyl groups of magnoflorine and nitrogen positive ions of quaternary ammonium groups at the hydrophilic end of lecithin.
3.4 morphological analysis
The aqueous solution of the magnoflorine-phospholipid complex is in a galactocolloid state at high concentration and in a solution state at low concentration. Observing dilute solution of magnoflorine phospholipid complex under a transmission electron microscope, as shown in fig. 9, it can be seen that the appearance of magnoflorine phospholipid complex is substantially spheroidal particle with a particle size of about 200nm, the dispersion is uniform, and sparse coating, suspected to be hydrophobic long chain of lecithin, can be seen on the surface of the particle.
3.5 particle size analysis
As shown in FIG. 10, the average hydrated particle size of magnoflorine phospholipid complex is about 208nm, which is similar to the particle size of magnoflorine phospholipid complex observed under a transmission electron microscope.
3.6 investigation of solubility Properties
As shown in fig. 11, at different pH, the average oil-water partition coefficient (P value) of the magnoflorine monomer is 0.868, and after the magnoflorine and lecithin form a complex, the average oil-water partition coefficient (P value) of the magnoflorine is 1.113, so that the content of the drug in the aqueous phase is reduced, the solubility of the drug in n-octanol is increased, and the lipid solubility of the magnoflorine is obviously increased.
3.7 pharmacokinetic and bioavailability Studies
The plasma concentration-time curve of the magnoflorine and magnoflorine phospholipid complex after intravenous injection administration of rats is shown in figure 12, and the main pharmacokinetic parameters of the magnoflorine and magnoflorine phospholipid complex are shown in table 6.
TABLE 6 major pharmacokinetic parameters of magnoflorine and magnoflorine phospholipid complexes ((C))
n＝6)
The results in Table 6 show that the half-life t is eliminated after the magnoflorine is prepared into the magnoflorine phospholipid complex1/2Beta is prolonged, the elimination rate CL is reduced, and the area AUC under the time curve of the blood concentration(0-t)Improved by 2.42 times, the magnoflorine phospholipid complexThe relative bioavailability is 249.46%, which shows that the magnoflorine phospholipid compound can slow down the elimination rate of magnoflorine, prolong the action time of magnoflorine, and remarkably improve the bioavailability of magnoflorine.
Example 4
Preparation of magnoflorine phospholipid complex-temperature sensitive in-situ gel
1 materials of the experiment
1.1 drugs and reagents
Poloxamer 407 (Beijing coupled science and technology, Inc., lot number 20170215);
poloxamer 188 (Beijing coupling technologies, Inc., lot number 20161119);
benzalkonium chloride (Tianjin Ding national biotechnology, Limited liability company, lot number: XS0902GA 14).
1.2 instruments
AUX120 one-ten-thousandth analytical balance (Shimadzu, Japan);
RCT basic magnetic stirrer (guangzhou instrumental laboratory technologies ltd).
2 method of experiment
2.1 preparation of magnoflorine phospholipid Complex-temperature sensitive in situ gel
Weighing a proper amount of magnoflorine phospholipid complex, adding a proper amount of distilled water, carrying out ultrasonic dissolution, then adding a proper amount of P407, P188 and benzalkonium chloride, placing in an ice water bath at 4 ℃, carrying out magnetic stirring, placing in a refrigerator at 4 ℃ after uniform dispersion, and fully swelling for 24 hours to obtain the clear and transparent magnoflorine phospholipid complex-temperature-sensitive in-situ gel.
2.2 determination of temperature sensitive in situ gel gelation temperature
And (3) measuring the gelling temperature by adopting a test tube inversion method, adding 3mL of temperature-sensitive in-situ gel into a thin-wall penicillin bottle, and placing the thin-wall penicillin bottle in a water bath at the temperature of 10 ℃ to slowly raise the temperature of the water bath at the speed of about 1 ℃/min. The temperature at which the liquid does not flow for 30s when the tube is inverted is recorded and the temperature at this time is determined as the gelling temperature. Each sample was assayed in duplicate 3 times and the results averaged.
2.3 Star test design optimization prescription process
On the basis of single factor investigation, 2 factors having a large influence on the gelation temperature were determined to be the P407 concentration and the P188 concentration. A star point design effect surface method is adopted, a P407 concentration (A) and a P188 concentration (B) are used as investigation objects, a gelling temperature (Y) is used as an evaluation index, the preparation process of the magnoflorine phospholipid complex-temperature-sensitive in-situ gel is optimized according to five levels of two factors, and the experimental design scheme is shown in Table 7.
TABLE 7 factors and levels of the design of the Star test
2.4 prescription Process verification
Preparing 3 batches of samples according to the prescription process, measuring the gelation temperature of the samples, and calculating the deviation between the actual gelation temperature and the predicted value of the samples.
3 results of the experiment
3.1 prescription Process optimization results
3.1.1 Star test design results
A star point design effect surface method is adopted, a P407 concentration (A) and a P188 concentration (B) are taken as investigation objects, a gelling temperature (Y) is taken as an evaluation index, the preparation process of the magnoflorine phospholipid complex-temperature-sensitive in-situ gel is optimized, and test results are shown in Table 8.
TABLE 8 design results of the asterisk test (
n＝3)
3.1.2 mathematical model construction and significance testing
Performing binary regression fitting analysis on the test result by using Design-Expert 8.0.6 to obtain two values between the gelation temperature (Y) and each factorA polynomial equation model of degree: y is 184.62-6.21A +12.25B-0.64AB +0.40A2+0.34B2. The mathematical model is subjected to variance analysis to obtain the equation significance test result, and the result is shown in table 9.
As shown in Table 9, P<0.0001 indicates that the mathematical model has a high degree of significance; first order item A, B, interaction item AB, second order item A2、B2The P values of (A) are all less than 0.01, which shows that the P values are all significant influencing factors and have significant influence on the gelling temperature. Coefficient of determination R of quadratic polynomial equation model20.9955, the equation has good fitting degree and can accurately predict the response value; correction decision coefficient R2 AdAnd j is 0.9922, which shows that the regression model can well reflect the change of the response value, and 99.22% of the change of the response value can be explained by the model, so that the mathematical model can be applied to the analysis and prediction of the temperature-sensitive in-situ gel gelation temperature.
TABLE 9 binomial equation model analysis of variance
3.2 response surface optimization and prediction
Through the response curve and contour lines of the interaction between P407 and P188 (FIGS. 13 and 14), it can be seen that the gelation temperature of in-situ gel is concentration-dependent on poloxamer 407 and 188, the former is in negative correlation, and the latter is in positive correlation, that is, the gelation temperature gradually decreases with the increase of the concentration of P407; while the gelling temperature gradually increased with increasing concentration of P188. The optimal value is that the temperature of the nasal cavity of the human body is 33-35 ℃, so that the gelation temperature is selected to be within the range of 32-33 ℃.
3.3 prescription Process verification
As shown in table 10, the deviation between the actual gelation temperature and the predicted value of the sample is small, which indicates that the optimal prescription process obtained by the star point design-effect surface method is accurate and reliable, and the gelation temperature can be well predicted by the fitting model.
Table 10 verification of test results (
n＝3)
Example 5
Effect of magnoflorine and phospholipid complex on resisting depression and insomnia
1 materials of the experiment
1.1 animals
Healthy male ICR mice (weight 20g + -2 g) were purchased from the institute of radiology, institute of Chinese medical sciences, laboratory animal center; before the experiment, the food is adapted to a laboratory for 3 days, the room temperature is 22 +/-2 ℃, the relative humidity is 65-70 percent, the daily illumination is 12 hours, and the food can be eaten freely and can absorb water.
1.2 drugs
Sodium pentobarbital (Shandong-West Asia chemical industry Co., Ltd., batch No. 57-33-0);
amitriptyline hydrochloride tablets (Hunan Dongting pharmaceutical industry Co., Ltd., batch No. B170421);
magnoflorine phospholipid complex (self-made in laboratory).
2 method of experiment
2.1 establishment of mouse model for insomnia caused by chronic stress depression
The procedure was as described in example 2 under "2.2".
2.2 Observation indicators and methods
The same test indexes and methods as those in the section "2.3" in example 2.
3 results of the experiment
3.1 Effect of oral Magnoliine phospholipid complexes on FST immobility time in mice
Compared with the model group, the FST immobility time of mice in the blank group and each administration group is obviously reduced, and the statistical significance is achieved (P is less than 0.01), which indicates that the magnoflorine phospholipid complex has good anti-depression activity when the mice are orally administered.
TABLE 11 Effect of oral Magnoliine phospholipid complexes on FST immobility time in mice: (
n＝10)
Note: p <0.01 compared to model control group.
3.2 Effect of oral Magnoliine phospholipid complexes on mouse TST immobility time
Compared with a model group, the TST immobility time of mice in the blank group and each administration group is obviously reduced, and the statistical significance is achieved (P <0.05 or P <0.01), wherein the TST immobility time of the mice in the magnolia alkaloid phospholipid complex group is similar to that of the blank group, and the fact that the magnolia alkaloid phospholipid complex has certain antidepressant activity when the mice are orally administered is shown.
TABLE 12 Effect of oral Magnoliine phospholipid complexes on mouse TST immobility time ((S))
n＝10)
Note: p <0.05 compared to model control; p <0.01 compared to model control group.
3.3 Effect of oral Magnoliine phospholipid complexes on suprathreshold dose sodium pentobarbital experiments in mice
The mice in the model group are excited in emotion, poor in sleeping quality, not easy to enter a sleeping state, long in sleeping latency period and short in sleeping time. Compared with a model group, the sleep latency of mice in the blank group and each administration group is obviously reduced, and the statistical significance is achieved (P is less than 0.01), wherein the sleep latency of the mice in the magnoflorine phospholipid complex group is similar to that of the amitriptyline group, and the magnoflorine phospholipid complex is shown to enable the mice to be calmed and to rapidly enter a sleep state. In the aspect of sleep time, compared with a model group, the sleep time of mice in a blank group and mice in each administration group are obviously prolonged, and the statistical significance is achieved (P <0.01), so that the magnoflorine phospholipid complex has a good calming effect after oral administration.
TABLE 13 Effect of magnoflorine phospholipid complexes on suprathreshold dose sodium pentobarbital experiments in mice: (
n＝10)
Note: p <0.01 compared to model control group.
Example 6
Antidepressant effect of nasal magnoflorine phospholipid complex-temperature-sensitive in-situ gel
1 materials of the experiment
1.1 animals
Healthy male ICR mice (weight 20g + -2 g) were purchased from the institute of radiology, institute of Chinese medical sciences, laboratory animal center; before the experiment, the food is adapted to a laboratory for 3 days, the room temperature is 22 +/-2 ℃, the relative humidity is 65-70 percent, the daily illumination is 12 hours, and the food can be eaten freely and can absorb water.
1.2 drugs
Magnolia liliiflora alkali phospholipid complex-temperature sensitive in-situ gel (self-made in laboratories).
2 method of experiment
2.1 Forced Swimming Test (FST)
The procedure is as in example 2 under "2.3".
2.2 Tail suspension experiment (TST)
The procedure is as in example 2 under "2.3".
3 results of the experiment
3.1 influence of nasal magnoflorine phospholipid Complex-temperature sensitive in situ gel on mouse FST and TST immobility time
Compared with a model control group, the immobility time of the FST and the TST of the magnoflorine phospholipid complex-temperature-sensitive in-situ gel group mouse is obviously reduced, the statistical significance is achieved (sig is less than 0.01), and the magnoflorine phospholipid complex-temperature-sensitive in-situ gel group mouse has good anti-depression activity after being administrated through the nasal cavity.
TABLE 14 influence of magnoflorine phospholipid Complex-temperature sensitive in situ gels on mouse FST and TST immobility time ((S))
n＝10)
Note: sig <0.01 compared to model control group.
Claims (2)
1. The preparation method of the magnoflorine and phospholipid compound is characterized in that magnoflorine is extracted and separated from spina date seeds and is prepared by the following steps:
(1) preparation of crude extract:
crushing spina date seeds, sieving with a 40-mesh sieve, degreasing with petroleum ether, adding 6-12 times by mass of an ethanol water solution with the volume percentage concentration of 60% -80%, heating and refluxing for 2-3 times, each time for 1-3 hours, filtering, combining extracting solutions, concentrating ethanol in a filtrate until no alcohol smell exists, dissolving with 2-6 times of water, extracting with n-butyl alcohol for 4-6 times, concentrating the extracting solution under reduced pressure, recovering a solvent to obtain a n-butyl alcohol crude extract, namely a sample to be separated;
(2) separating magnoflorine by high-speed counter-current chromatography:
taking ethyl acetate, n-butanol and water in a volume ratio of 1-3: 1-5: 2-7, placing the ethyl acetate, n-butanol and water in a separating funnel to prepare a two-phase solvent system, taking an upper phase as a stationary phase and a lower phase as a mobile phase; filling a chromatographic column of a countercurrent chromatography with the stationary phase, rotating the main machine forward at the temperature of 25-35 ℃ at the rotating speed of 300-900 r/min, and pumping the mobile phase at the flow rate of 1.5-10 ml/min until the two-phase solvent reaches an equilibrium state in the column; dissolving the mixed crude extract prepared in the step (1) by using a mixed solvent with the volume ratio of the upper phase to the lower phase being 1:1, then injecting a sample, collecting effluent of the magnoflorine under an ultraviolet detector, concentrating and drying to obtain the magnoflorine;
(3) dissolving magnoflorine in absolute ethyl alcohol at the concentration of 2.5-7.5mg/mL, adding phospholipid according to the mass ratio of 1-4: 2-1, stirring for 1-3 hours at the temperature of 50-60 ℃, and removing the solvent; adding a proper amount of dichloromethane to dissolve a reaction product, centrifuging to remove precipitates, evaporating a filtrate to remove a solvent, and then carrying out vacuum drying at 30 ℃ for 24 hours to obtain a magnoflorine phospholipid compound; the phospholipid is selected from soybean lecithin.
2. The use of the phospholipid complex prepared according to the method of claim 1 in the preparation of a medicament for preventing and treating depression.
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