CN107550954B - Application of crocodile flower in preparation of medicine for treating or preventing nerve injury and medicine - Google Patents
Application of crocodile flower in preparation of medicine for treating or preventing nerve injury and medicine Download PDFInfo
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Abstract
The invention relates to the field of medicines, and particularly relates to application of crocodile flower in preparation of a medicine for treating or preventing nerve injury and a medicine. Application of crocodile flower in preparing medicine for treating or preventing nerve injury caused by Parkinson's disease, Alzheimer's disease, cerebral apoplexy and tauopathy. A medicament for the preparation of a neuroprotective agent comprising alligator flower. The crocodile mouth flower has therapeutic effect on gradual degeneration and apoptosis of central neuron. The extract or drug of crocodile flower has effects of protecting neuron injury, thereby preventing or delaying nerve cell death; can delay the onset time or delay the development of the disease. The medicine prepared from crocodile flower can keep cells in healthy shape and proliferation and inhibit apoptosis; the medicine has remarkable protective effect on nerve cells.
Description
Technical Field
The invention relates to the field of medicines, and particularly relates to application of crocodile flower in preparation of a medicine for treating or preventing nerve injury and a medicine.
Background
The crocodile flower is dry aerial part of Lindau of crocodile flower Clinacanthus nutans (Burm.f.) of Acacia of Acanthaceae, and can be harvested all the year round. Has the main functions of clearing away heat and toxic material, promoting diuresis, removing blood stasis and detumescence, promoting blood circulation and regulating menstruation and relieving pain; can be used for treating jaundice, hepatitis ascites, menoxenia, traumatic injury, swelling and pain, fracture, and rheumatalgia.
The emphasis on treating disease varies from region to region. In Yunnan province, the crocodile flower is a Dai nationality medicine, and the Dai medicine is used for reuniting bones, treating traumatic injury and strengthening and reinforcing bones; in Hainan province, crocodile flower is used as a wild vegetable, and can be used as both medicine and food, and is commonly used for treating hepatitis, hepatitis ascites, rheumatalgia, irregular menstruation, traumatic injury, bone fracture and leptospirosis. Crocodile flowers are also used in a wide variety of other countries for the treatment of diseases, with malaysia being used to treat various cancers, including lymphoma, liver, lung, kidney, breast, nasopharynx, uterus, ovary, leukemia, brain, prostate, and the like; indonesia is used for treating various nephritis, renal atrophy, renal failure and renal calculus; thai is used for treating herpes, eczema, rash, insect and snake bites and is classified as a basic health-care medicinal plant in Thai.
Disclosure of Invention
The invention aims to provide application of crocodile flower in preparing a medicament for treating or preventing nerve injury and a medicament, and aims to provide a medicament for preparing a medicament for treating central neurodegenerative diseases.
The invention provides a technical scheme that:
application of crocodile flower in preparing medicine for treating or preventing nerve injury caused by Parkinson's disease, Alzheimer's disease, cerebral apoplexy and tauopathy.
Further, the use of the extract of crocodile flower as a neuroprotective agent. The extract of crocodile flower is mainly prepared by the following steps: extracting crocodile flower with a solvent, and separating to obtain an extracting solution; wherein the weight ratio of the crocodile flower to the solvent is 1: 5-20.
Further, the solvent is one or more selected from water, methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1, 2-propanediol, 1, 3-butanediol, acetone, and ethyl acetate.
The invention also provides a technical scheme that:
a medicament for the preparation of a neuroprotective agent comprising alligator flower.
Further, the medicine is mainly prepared by the following steps: extracting crocodile flower with a solvent, and separating to obtain an extracting solution; wherein the weight ratio of the crocodile flower to the solvent is 1: 5-20.
Further, the solvent is one or more selected from water, methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1, 2-propanediol, 1, 3-butanediol, acetone, and ethyl acetate.
Further, the solvent is 30-95% ethanol water solution by mass fraction.
Further, extracting crocodile flower with ultrasound at 30-90 deg.C for 15-60 min.
Further, the above separation to obtain the extractive solution also comprises concentrating and drying the extractive solution.
Further, the above-mentioned extract is subjected to evaporation concentration, concentration under reduced pressure or membrane concentration. Drying the concentrated extractive solution under normal pressure, reduced pressure, vacuum freeze drying or spray drying.
The application of the crocodile flower in preparing the medicine for treating or preventing the nerve injury and the medicine provided by the embodiment of the invention have the beneficial effects that:
the crocodile mouth flower has therapeutic effect on gradual degeneration and apoptosis of central neuron. The extract or drug of crocodile flower has effects of protecting neuron injury, thereby preventing or delaying nerve cell death; can delay the onset time or delay the development of the disease. The medicine prepared from crocodile flower can keep cells in healthy shape and proliferation and inhibit apoptosis; the medicine has remarkable protective effect on nerve cells.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the protective effect of the drug provided in example 1 on L-glutamate-induced HT-22 cells as measured by MTT assay;
FIG. 2 shows the protective effect of the drug provided in example 1 on L-glutamate-induced HT-22 cells observed under a microscope.
Detailed Description
The central nervous degenerative diseases are a group of neurodegenerative diseases or neurological disorders caused by gradual degeneration and apoptosis of central nervous system neurons. The etiology is mostly unknown, the pathogenesis and pathological manifestations are complex, and the clinical manifestations are different.
The central nervous degeneration diseases include Parkinson's disease combined dementia, Alzheimer's disease of Pacific Guam, Parkinson's syndrome, etc. At present, due to the lack of specific biochemical, imaging and pathophysiological characteristics, diagnosis is often made primarily on the basis of the experience of the clinician. There is currently no specific treatment for most diseases, except that a few diseases (such as Parkinson's disease) may temporarily improve clinical symptoms by means of drugs or surgery.
Neuroprotective therapy is the prevention or delay of neuronal cell death by protecting ischemia-induced hemizonal neuronal damage at an initial stage by blocking one or more of the mechanisms involved in the ischemic cascade. The rapid backflow of blood into ischemic tissue can be achieved by drugs, thereby inhibiting damage on the biochemical, metabolic and cellular levels caused by ischemia.
Therapeutic strategies for ischemic stroke, for example, include thrombolytic therapy and neuroprotective therapy. The thrombolytic drug alteplase (rt-PA) is the first and so far the only thrombolytic drug approved by the FDA for the treatment of stroke. Although rt-PA has been shown to be effective in treating acute ischemic stroke, it has a narrow therapeutic window, an undefined mechanism of action and a risk of bleeding, so that only a few patients can receive effective treatment of rt-PA in a timely manner. The compound levodopa is the first choice medicine for treating PD, but serious complications such as switching phenomenon and dose end deterioration can occur when patients take the compound levodopa for a long time. Thus, development of neuroprotective therapy may delay the time to onset or delay the progression of the condition.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The application of the crocodile flower in the preparation of the medicine for treating or preventing nerve injury and the medicine of the crocodile flower in the embodiment of the invention are specifically described below.
Application of crocodile flower in preparing medicine for treating or preventing nerve injury caused by Parkinson's disease, Alzheimer's disease, cerebral apoplexy and tauopathy.
Further, the use of the extract of crocodile flower as a neuroprotective agent. The extract of crocodile flower is mainly prepared by the following steps: extracting crocodile flower with a solvent, and separating to obtain an extracting solution; wherein the weight ratio of the crocodile flower to the solvent is 1: 5-20.
Further, the solvent is one or more selected from water, methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1, 2-propanediol, 1, 3-butanediol, acetone, and ethyl acetate.
Crocodile flower is dry aerial part of Lindau of crocodile of Acacia of Acanthaceae of dicotyledonous plant Clinacanthus (Burm.f.) and can be harvested all the year round. The crocodile mouth flower has the main functions of clearing heat and removing toxicity, promoting diuresis, removing blood stasis and reducing swelling, promoting blood circulation and regulating menstruation and relieving pain; can be used for treating jaundice, hepatitis ascites, menoxenia, traumatic injury, swelling and pain, fracture, and rheumatalgia.
Through research and discussion of the inventor, the crocodile flower has a good curative effect in preparing a medicament for treating, assisting in treating or preventing neurodegenerative diseases. The crocodile mouth flower has therapeutic effect on gradual degeneration and apoptosis of central neuron.
In the present embodiment, the "neuroprotective agent" refers to a therapeutic agent or an active substance that helps to protect nerve cells from cell damage and/or to protect against neurodegenerative processes and neuronal function impairment.
Furthermore, the crocodile flower is applied to preparing a medicament for treating or preventing neurodegenerative diseases. Neurodegenerative diseases including parkinson's disease, alzheimer's disease, huntington's disease, tauopathies (e.g., frontotemporal dementia, corticobasal degeneration, pick's disease, progressive supranuclear palsy), wilson's disease, prion diseases and other dementias including vascular dementia, Amyotrophic Lateral Sclerosis (ALS), acute stroke (nerve damage from brain stroke) and other traumatic injuries; cerebrovascular accidents (e.g., age-related macular degeneration); brain and spinal cord trauma, peripheral neuropathy, retinopathy, and glaucoma.
The invention also provides a medicament for preparing a neuroprotective agent, which comprises crocodile flower.
Further, the drug of this example was prepared mainly by the following steps: extracting crocodile flower with a solvent, and separating to obtain an extracting solution; wherein the weight ratio of the crocodile flower to the solvent is 1: 5-20.
Further, the solvent is one or more selected from water, methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1, 2-propanediol, 1, 3-butanediol, acetone, and ethyl acetate.
Specifically, the solvent is an ethanol water solution with the mass fraction of 30-95%. In this example, the ethanol solution is a 95% ethanol aqueous solution by mass fraction.
Specifically, ultrasonic extraction is adopted, and specifically, crocodile flower is subjected to ultrasonic extraction at 30-90 ℃ for 15-60 min.
Further, the above separation to obtain the extractive solution also comprises concentrating and drying the extractive solution.
Further, the above-mentioned extract is subjected to evaporation concentration, concentration under reduced pressure or membrane concentration. Specifically, concentration under reduced pressure was employed. In the preferred embodiment of the invention, concentration is performed by evaporation using a rotary evaporator.
Drying the concentrated extractive solution under normal pressure, reduced pressure, vacuum freeze drying or spray drying. Specifically, reduced pressure drying is employed in the present invention.
The crocodile mouth flower has therapeutic effect on gradual degeneration and apoptosis of central neuron. The extract or drug of crocodile flower has effects of protecting neuron injury, thereby preventing or delaying nerve cell death; can delay the onset time or delay the development of the disease. The medicine prepared from crocodile flower can keep cells in healthy shape and proliferation and inhibit apoptosis; the medicine has remarkable protective effect on nerve cells.
In addition, in other embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient.
For example: diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.
In addition, various carriers well known in the art can be widely used in order to form the administration unit into a pill. Such as: glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. For making the administration unit into a suppository, various carriers well known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like.
In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.
Further, the medicaments of the embodiments of the present invention may be administered in unit dosage form, either enterally or parenterally, such as orally, intramuscularly, subcutaneously, nasally, oromucosally, dermally, peritoneally or rectally, etc. The administration dosage forms include tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, lyophilized powder for injection, etc. Can be common preparation, sustained release preparation, controlled release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used.
In addition, the dosage of the drugs of the embodiments of the present invention to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight and individual response of the patient or animal, the specific compound used, the route of administration and the number of administrations, and the like. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms.
As mentioned above, the actual dosage level of each active ingredient in the pharmaceutical composition can be varied during the course of administration so that the resulting amount of active compound is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. Dosage levels will be selected with regard to the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is common practice in the art to start doses of the compounds at levels below those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a medicament, which is prepared by the following steps:
weighing 30.11g of crocodile flower, placing in a triangular flask, adding 300ml of 95% ethanol water solution into the triangular flask, performing ultrasonic extraction at 60 ℃ for 30min, filtering, concentrating into paste by using a rotary evaporator, placing in an evaporation dish, evaporating to dryness, and drying in a constant-temperature oven (50 ℃) to constant weight.
Example 2
This example provides a medicament, which is prepared by the following steps:
weighing 10g of crocodile flower, placing in a triangular flask, adding 50ml of 30% ethanol aqueous solution into the triangular flask, performing ultrasonic extraction at 30 ℃ for 15min, filtering, concentrating under reduced pressure to obtain extract, placing in an evaporating dish, evaporating to dryness, and drying in a constant-temperature oven (50 ℃) to constant weight.
Example 3
This example provides a medicament, which is prepared by the following steps:
weighing 10g of crocodile flower, placing in a triangular flask, adding 200ml of 60% 1, 2-propylene glycol aqueous solution into the triangular flask, ultrasonically extracting at 90 ℃ for 60min, filtering, concentrating under reduced pressure to obtain extract, and vacuum freeze-drying to constant weight.
Example 4
This example provides a medicament, which is prepared by the following steps:
weighing 10g of crocodile flower, placing in a triangular flask, adding 100ml of ethyl acetate into the triangular flask, performing ultrasonic extraction at 60 ℃ for 45min, filtering, performing membrane concentration, and drying under reduced pressure to constant weight.
Example 5
This example provides a medicament, which is prepared by the following steps:
weighing 10g of crocodile flower, placing in a triangular flask, adding 150ml of water into the triangular flask, performing ultrasonic extraction at 45 ℃ for 60min, filtering, concentrating into extract by using a rotary evaporator, placing in an evaporating dish, evaporating, and drying in a constant-temperature oven (50 ℃) to constant weight.
Test examples
This test example performed a neuroprotective test on the drug obtained in example 1.
Culture of HT-22 neural cells
Digesting (three batches) HT-22 cells from a petri dish with pancreatin (0.25%, 1mL) within 1min, blowing off to form a single cell suspension, adding a small amount of fetal bovine serum FBS (100 μ L), centrifuging at 1000rpm for 5min, removing supernatant, dispersing cell pellets in 3mL of complete medium, adding 1mL of cell suspension to 24mL of complete medium, reversing back and forth 10 times, mixing, taking 0.1mL for counting, diluting to an appropriate cell concentration (8 × 10)4cell/mL), 50. mu.L of medium was added to the 48-well plate to distribute the medium completely around the edge of the plate, and the plates were plated in 48-well plates (three plates from three lots of cells, 100. mu.L, 8 × 10)4cell/mL). Rotating the centrifugal tube while seeding, blowing and beating the cells in a half of the holes to make the cells uniform, simultaneously enabling the cell suspension to be paved at the bottom of the hole plate, observing whether the cells are uniformly dispersed under a microscope after all the cells are seeded, and shaking the cells uniformly in a cross way to uniformly disperse the cells at the bottom of the hole plate. The cells were left for 24 hours (cell fusion degree about 30-40%) for the experiment.
MTT method test
Mu.g of the drug obtained in example 1 was dispersed in 2mL of complete medium and used for 3X 48 well plate changes for MTT experiments. Well-seeded 48-well plates were divided into Control (Control), Glutamate (Glutamate), NAC (N-acetylcysteine), Trolox (water-soluble vitamin E) and sample (drug group provided in example 1). Changing culture medium for each group of three multiple wells, wherein each well is 200 μ L, and before changing culture medium, blowing the culture medium to disperse the medicine uniformly. After incubation for 1h at 37 ℃ after the exchange, glutamic acid was added to reach a final concentration of 3mM (6. mu.L). After shaking up, the cells were incubated at 37 ℃ for 24 hours. The cell morphology was observed and the cell viability was determined (MTT method), followed by incubation with 20. mu.L MTT (5mg/mL) in the dark at 37 ℃ for 2h, gentle pipetting, addition of 200. mu.L DMSO per well and shaking for 5min on a shaker. Then, the absorbance at 490nm was calculated by a multifunctional microplate reader (the data in the following figures are all measured at 490nm, and the cell survival rate of the injured group is about 20% of that of the Control group). OD values from the wells were averaged, subtracted from OD values from the blank DMSO, and then divided by the average value from the control group for normalization. The mean of the OD values of MTT of the three batches of cells was statistically different using the software SigmaPlot12.5.
The statistical results are shown in FIG. 1, in particular, FIG. 1 shows the protective effect of extract of crocodile flowers on L-glutamate-induced HT-22 cells as measured by MTT method. In the figure, "+" indicates that the corresponding test group has the corresponding component, and "-" indicates that the corresponding test group does not have the corresponding component. Wherein P <0.01, P <0.001 was compared to the glutamic acid group (one-way anova, Dunnett method). Trolox (10. mu.M) was used as a positive control.
The results in fig. 1 show that the drug provided in example 1 has a significant protective effect on HT-22 nerve cells.
3. Protective effect of crocodile flower extract observed under microscope
20 μ g of the drug provided in example 1 was dispersed in 2mL of complete medium for 3X 48 well plate changes. And (3) taking 48-well plates with planted cells, namely a Control group, a Glutamate group, a Glu (Glutamate) + NAC (N-acetyl cysteine) group, a Glu + Trolox and Glu + sample group (a medicament group provided by the embodiment 1), changing the culture medium for 200 mu L per hole, and blowing the culture medium before changing the culture medium for each time to uniformly disperse the medicaments. After incubation for 1h at 37 ℃ after the exchange, glutamic acid was added to reach a final concentration of 3mM (6. mu.L). After shaking up, the cells were incubated at 37 ℃ for 24 hours. Finally, the cell morphology was observed under a microscope and photographed, and the results are shown in FIG. 2. FIG. 2 shows the protective effect of extract of crocodile flower on L-glutamate-induced HT-22 cells observed under a microscope. As can be seen from FIG. 2, the drug provided in example 1 can maintain healthy morphology and proliferation of cells, and inhibit apoptosis caused by L-glutamate (L-glutamate).
As can be seen from this experimental example, the drug provided in example 1 is capable of maintaining healthy morphology and proliferation of cells and inhibiting apoptosis; the medicine has obvious protective effect on HT-22 nerve cells.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. Use of an extract of alligator flower for the manufacture of a medicament for the treatment or prevention of alzheimer's disease; it is characterized in that the preparation method is characterized in that,
the extract of the crocodile flower is mainly prepared by the following steps: extracting crocodile flower with a solvent, and separating to obtain an extracting solution; wherein the weight ratio of the crocodile flower to the solvent is 1: 5-20;
the solvent is 95% ethanol water solution.
2. Use according to claim 1, wherein the crocodile flower is subjected to ultrasonic extraction at 30-90 ℃ for 15-60 min.
3. The use of claim 1, wherein separating the extract further comprises concentrating and drying the extract.
4. Use according to claim 1, wherein the extraction solution is subjected to evaporative or membrane concentration; and drying the concentrated extracting solution under normal pressure, reduced pressure, vacuum freeze drying or spray drying.
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| CN103877133A (en) * | 2014-03-19 | 2014-06-25 | 江苏大学 | Extracting method of clinacanthus nutans anti-cancer active part and application thereof |
| CN107158054A (en) * | 2017-06-08 | 2017-09-15 | 广州中医药大学 | Small-leaf bonesetting extract as neuroprotective agent application |
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| CN107362185A (en) * | 2017-07-27 | 2017-11-21 | 广州中医药大学 | Application and medicine of the little Hua Asystasia gangetica (L) Anders.(A. Coromandelihas Nees)s in the medicine for preparing prevention and/or treatment nerve degenerative diseases |
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| CN103877133A (en) * | 2014-03-19 | 2014-06-25 | 江苏大学 | Extracting method of clinacanthus nutans anti-cancer active part and application thereof |
| CN107158054A (en) * | 2017-06-08 | 2017-09-15 | 广州中医药大学 | Small-leaf bonesetting extract as neuroprotective agent application |
| CN107233371A (en) * | 2017-07-27 | 2017-10-10 | 广州中医药大学 | The big application for refuting bone in the medicine for preparing prevention and/or treatment nerve degenerative diseases and medicine |
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