CN102258578A - Novel lotus leaf total alkaloid extract and preparation method as well as application thereof - Google Patents
Novel lotus leaf total alkaloid extract and preparation method as well as application thereof Download PDFInfo
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Abstract
The invention relates to a novel lotus leaf total alkaloid extract and a preparation method as well as application thereof, and particularly discloses the lotus leaf total alkaloid extract, wherein the lotus leaf total alkaloid content can reach 70-98 percent (w/w); nuciferine content can reach 14-28 percent (w/w); and the weight ratio of the lotus leaf total alkaloid to the nuciferine is (3.5-5):1. The invention also discloses a preparation method of the lotus leaf total alkaloid extract, a composition containing the lotus leaf total alkaloid and application of the lotus leaf total alkaloid extract to preparing a product for treating and/or preventing diseases or symptoms related to abnormal glycolipid metabolism. The extract has high objective substance content; and the preparation method is simple and is suitable for industrialization.
Description
Technical field
The invention belongs to medical technical field, relate to Folium Nelumbinis extract, be particularly related to a kind of preparation method of total alkaloid of lotus leaves extraction and a kind of simple and reliable total alkaloid of lotus leaves extraction of novelty, and the particularly application in treating and/or preventing glycolipid metabolism abnormal diseases such as obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension of the purposes of described extract.
Background technology
Folium Nelumbinis is the dry blade of nymphaeaceae plant lotus (Nelumbo nucifera Gaertn), has multiple efficacies such as clearing away summer-heat, Weight-reducing and lipid-lowering, sending up the lucid YANG, cooling blood for hemostasis.Modern pharmacology studies show that the alkaloid in the Folium Nelumbinis has blood fat reducing, suppresses cholesterol, antibacterial and pharmacologically actives such as spasmolytic, antioxidation, is the effective constituent that mainly contains of Folium Nelumbinis.
Bibliographical information adopts the total alkaloid of lotus leaves content of resin absorption technology preparation lower to the more employing macroporous resin of the extraction purification partition method of total alkaloid of lotus leaves.(Xiao Wenjun in the document, Hu Xiangwen, Hu Yunling etc. the technical research of lotus leaf alkaloid column separating purification. Food Science [J] .2007,28 (6): be raw material 120-124) with the Folium Nelumbinis cured leaf, behind 30% ethanol extraction, studied polyamide, three kinds of macroporous resins of D101, the HP-20 process to lotus leaf alkaloid static adsorption and desorbing, in its finally prepd lotus leaf alkaloid extract total alkaloid of lotus leaves content is 32.56%.
CN101085041A (Chinese patent application numbers 2007101226448, on December 12nd, open day) a kind of total alkaloid of lotus leaves extraction and preparation method thereof disclosed, wherein adopt macroporous resin to handle, and total alkaloid content is lower than 58% in the extract that is obtained.
Therefore, provide a kind of method of easy extraction lotus leaf alkaloid, obtain to contain the alkaloidal Folium Nelumbinis extract of higher degree, be still the problem that those skilled in the art are eager to solve.
Summary of the invention
The objective of the invention is to overcome the existing deficiency that exists in the lotus leaf alkaloid technology of extracting, make extraction process simplify reliable and/or obtain to have higher degree alkaloid extraction thing.The inventor is surprisingly found out that, adopts the inventive method to prepare total alkaloid of lotus leaves extraction, and medicinal material extract is complete, and step is simple, and solvent load is few, toxicity is low, is suitable for suitability for industrialized production; Prepared extract total alkaloid of lotus leaves content after measured can reach 70～98% (w/w), and nuciferine content can reach 14～28% (w/w).The present invention is based on above-mentioned discovery and be accomplished.
Summary of the invention:
First aspect present invention provides a kind of total alkaloid of lotus leaves extraction, wherein comprises total alkaloid of lotus leaves and nuciferine, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
According to each total alkaloid of lotus leaves extraction of first aspect present invention, wherein said total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight.In one embodiment, described total alkaloid of lotus leaves accounts for 72～96%, 72～95%, 75～95%, 78～95%, 78～93%, 80～95%, 80～92% or 80～90% of this extract weight.
According to each total alkaloid of lotus leaves extraction of first aspect present invention, wherein said nuciferine accounts for 14～28% (w/w) of this extract weight.In one embodiment, described total alkaloid of lotus leaves accounts for 14～28%, 15～28%, 15～26%, 15～25%, 16～25%, 17～25%, 18～25%, 19～25% or 20～25% of this extract weight.
According to each total alkaloid of lotus leaves extraction of first aspect present invention, wherein comprise total alkaloid of lotus leaves and nuciferine, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1, and described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight, and described nuciferine accounts for 14～28% (w/w) of this extract weight.
According to each total alkaloid of lotus leaves extraction of first aspect present invention, it is to prepare by the method that may further comprise the steps: make the Folium Nelumbinis ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid, carrying out Soxhlet then extracts, the extracting solution that obtains extracts with aqueous hydrochloric acid solution, the water layer ethyl acetate extraction, organic layer is concentrated, drying, promptly.In one embodiment, the described ethanol that contains hydrochloric acid be contain 0.05～0.4%, 0.06～0.4%, 0.07～0.35%, 0.08～0.3%, 0.08～0.25%, about 0.09%, about 0.1%, about 0.12%, about 0.15%, about 0.2% or the ethanol of about 0.25% hydrochloric acid.In one embodiment, described ethanol is 50～80% ethanol (for example 55～80%, 60～80%, 65～80%, 65～75%, about ethanol of 65%, about 70% or about 75%).In one embodiment, the described ethanol that contains hydrochloric acid is 65～75% ethanol that contain 0.08～0.25% hydrochloric acid.In one embodiment, the described ethanol that contains hydrochloric acid is 68～72% ethanol that contain 0.09～0.15% hydrochloric acid.In one embodiment, the described ethanol that contains hydrochloric acid is about 70% ethanol that contains 0.1% hydrochloric acid of having an appointment.In one embodiment, described extraction is that merceration extracts.In one embodiment, described ethanol extract extracts the back and regulates pH to 8.5～10.5, for example regulates pH to 9～10, and then it is concentrated.In one embodiment, described Soxhlet extraction is to extract with ethyl acetate.In one embodiment, described Soxhlet extracting solution pH is 1～2 aqueous hydrochloric acid solution extraction.In one embodiment, described Soxhlet extracting solution is regulated pH to 11～12, the reuse ethyl acetate extraction after extracting with aqueous hydrochloric acid solution.
According to each total alkaloid of lotus leaves extraction of first aspect present invention, it is by the method preparation that may further comprise the steps:
A) make Folium Nelumbinis 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid, the extracting solution that obtains is regulated pH to 8.5～10.5, concentrates, and obtains first concentrated solution;
B) concentrated solution that step a) is won is mixed with kieselguhr, drying, reuse ethyl acetate are carried out the Soxhlet extraction in apparatus,Soxhlet's, and extracting solution is concentrated, and obtain second concentrated solution;
C) making the step b) gained second concentrated solution pH is the extraction of 1～2 aqueous hydrochloric acid solution, and water layer is regulated pH to 11～12, and the reuse ethyl acetate extracts, and the organic layer that obtains is concentrated, drying, promptly.
Second aspect present invention provides a kind of total alkaloid of lotus leaves extraction, wherein comprises total alkaloid of lotus leaves and nuciferine, and wherein said total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight.In one embodiment, described total alkaloid of lotus leaves accounts for 72～96%, 72～95%, 75～95%, 78～95%, 78～93%, 80～95%, 80～92% or 80～90% of this extract weight.
According to each total alkaloid of lotus leaves extraction of second aspect present invention, wherein said nuciferine accounts for 14～28% (w/w) of this extract weight.In one embodiment, described total alkaloid of lotus leaves accounts for 14～28%, 15～28%, 15～26%, 15～25%, 16～25%, 17～25%, 18～25%, 19～25% or 20～25% of this extract weight.
According to each total alkaloid of lotus leaves extraction of second aspect present invention, wherein comprise total alkaloid of lotus leaves and nuciferine, described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight, and described nuciferine accounts for 14～28% (w/w) of this extract weight.
According to each total alkaloid of lotus leaves extraction of second aspect present invention, wherein comprise total alkaloid of lotus leaves and nuciferine, described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight, described nuciferine accounts for 14～28% (w/w) of this extract weight, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
According to each total alkaloid of lotus leaves extraction of second aspect present invention, it is to prepare by the method that may further comprise the steps: make the Folium Nelumbinis ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid, carrying out Soxhlet then extracts, the extracting solution that obtains extracts with aqueous hydrochloric acid solution, the water layer ethyl acetate extraction, organic layer is concentrated, drying, promptly.In one embodiment, the described ethanol that contains hydrochloric acid be contain 0.05～0.4%, 0.06～0.4%, 0.07～0.35%, 0.08～0.3%, 0.08～0.25%, about 0.09%, about 0.1%, about 0.12%, about 0.15%, about 0.2% or the ethanol of about 0.25% hydrochloric acid.In one embodiment, described ethanol is 50～80% ethanol (for example 55～80%, 60～80%, 65～80%, 65～75%, about ethanol of 65%, about 70% or about 75%).In one embodiment, the described ethanol that contains hydrochloric acid is 65～75% ethanol that contain 0.08～0.25% hydrochloric acid.In one embodiment, the described ethanol that contains hydrochloric acid is 68～72% ethanol that contain 0.09～0.15% hydrochloric acid.In one embodiment, the described ethanol that contains hydrochloric acid is about 70% ethanol that contains 0.1% hydrochloric acid of having an appointment.In one embodiment, described extraction is that merceration extracts.In one embodiment, described ethanol extract extracts the back and regulates pH to 8.5～10.5, for example regulates pH to 9～10, and then it is concentrated.In one embodiment, described Soxhlet extraction is to extract with ethyl acetate.In one embodiment, described Soxhlet extracting solution pH is 1～2 aqueous hydrochloric acid solution extraction.In one embodiment, described Soxhlet extracting solution is regulated pH to 11～12, the reuse ethyl acetate extraction after extracting with aqueous hydrochloric acid solution.
According to each total alkaloid of lotus leaves extraction of second aspect present invention, it is by the method preparation that may further comprise the steps:
A) make Folium Nelumbinis 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid, the extracting solution that obtains is regulated pH to 8.5～10.5, concentrates, and obtains first concentrated solution;
B) concentrated solution that step a) is won is mixed with kieselguhr, drying, reuse ethyl acetate are carried out the Soxhlet extraction in apparatus,Soxhlet's, and extracting solution is concentrated, and obtain second concentrated solution;
C) making the step b) gained second concentrated solution pH is the extraction of 1～2 aqueous hydrochloric acid solution, and water layer is regulated pH to 11～12, and the reuse ethyl acetate extracts, and the organic layer that obtains is concentrated, drying, promptly.
Third aspect present invention provides the method for preparing first aspect present invention each or each described total alkaloid of lotus leaves extraction of second aspect, this method may further comprise the steps: make the Folium Nelumbinis ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid, carrying out Soxhlet then extracts, the extracting solution that obtains extracts with the saline acid solution, the water layer ethyl acetate extraction, organic layer is concentrated, drying, promptly.In one embodiment, the described ethanol that contains hydrochloric acid be contain 0.05～0.4%, 0.06～0.4%, 0.07～0.35%, 0.08～0.3%, 0.08～0.25%, about 0.09%, about 0.1%, about 0.12%, about 0.15%, about 0.2% or the ethanol of about 0.25% hydrochloric acid.In one embodiment, described ethanol is 50～80% ethanol (for example 55～80%, 60～80%, 65～80%, 65～75%, about ethanol of 65%, about 70% or about 75%).In one embodiment, the described ethanol that contains hydrochloric acid is 65～75% ethanol that contain 0.08～0.25% hydrochloric acid.In one embodiment, the described ethanol that contains hydrochloric acid is 68～72% ethanol that contain 0.09～0.15% hydrochloric acid.In one embodiment, the described ethanol that contains hydrochloric acid is about 70% ethanol that contains 0.1% hydrochloric acid of having an appointment.In one embodiment, described extraction is that merceration extracts.In one embodiment, described ethanol extract extracts the back and regulates pH to 8.5～10.5, for example regulates pH to 9～10, and then it is concentrated.In one embodiment, described Soxhlet extraction is to extract with ethyl acetate.In one embodiment, described Soxhlet extracting solution pH is 1～2 aqueous hydrochloric acid solution extraction.In one embodiment, described Soxhlet extracting solution is regulated pH to 11～12, the reuse ethyl acetate extraction after extracting with aqueous hydrochloric acid solution.
According to each method of third aspect present invention, it may further comprise the steps:
A) make Folium Nelumbinis 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid, the extracting solution that obtains is regulated pH to 8.5～10.5, concentrates, and obtains first concentrated solution;
B) concentrated solution that step a) is won is mixed with kieselguhr, drying, reuse ethyl acetate are carried out the Soxhlet extraction in apparatus,Soxhlet's, and extracting solution is concentrated, and obtain second concentrated solution;
C) making the step b) gained second concentrated solution pH is the extraction of 1～2 aqueous hydrochloric acid solution, and water layer is regulated pH to 11～12, and the reuse ethyl acetate extracts, and the organic layer that obtains is concentrated, drying, promptly.
Fourth aspect present invention provides a kind of compositions, and it comprises the total alkaloid of lotus leaves extraction that treats and/or prevents effective dose and optional pharmaceutically acceptable carrier.
According to each compositions of fourth aspect present invention, wherein said total alkaloid of lotus leaves extraction comprises total alkaloid of lotus leaves and nuciferine.
According to each compositions of fourth aspect present invention, wherein said total alkaloid of lotus leaves extraction comprises total alkaloid of lotus leaves and nuciferine, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～4.5): 1.
According to each compositions of fourth aspect present invention, wherein said total alkaloid of lotus leaves extraction is each a total alkaloid of lotus leaves extraction of first aspect present invention, and/or described total alkaloid of lotus leaves extraction is each a total alkaloid of lotus leaves extraction of second aspect present invention.
According to each compositions of fourth aspect present invention, it is a pharmaceutical composition.
According to each compositions of fourth aspect present invention, it is a pharmaceutical composition, and it is the form that is tablet, granule, capsule, pill, drop pill.
According to each compositions of fourth aspect present invention, it is a pharmaceutical composition, and it is to be mixed and made in tablet, granule, capsule, pill, the drop pill any with one or more pharmaceutical excipients.
Fifth aspect present invention provide total alkaloid of lotus leaves extraction preparation be used for the treatment of and/or the product of prevention and unusual diseases associated of glycolipid metabolism or disease in purposes.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, described and the unusual diseases associated of glycolipid metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Sixth aspect present invention provide total alkaloid of lotus leaves extraction preparation be used for the treatment of and/or the product of prevention and unusual diseases associated of glycolipid metabolism or disease in purposes, and/or be used for blood fat reducing, suppress cholesterol, the purposes of antibacterial, spasmolytic, antioxidative product, wherein said total alkaloid of lotus leaves extraction is as first aspect present invention as described in each, perhaps as second aspect present invention as described in each.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, wherein said and the unusual diseases associated of glycolipid metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Seventh aspect present invention is provided for treating and/or preventing the product with unusual diseases associated of glycolipid metabolism or disease, comprises the total alkaloid of lotus leaves extraction that treats and/or prevents effective dose and optional pharmaceutically acceptable carrier in this product.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, described and the unusual diseases associated of glycolipid metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Eighth aspect present invention is provided for treating and/or preventing the product with unusual diseases associated of glycolipid metabolism or disease, and/or be used for blood fat reducing, suppress cholesterol, antibacterial, spasmolytic, antioxidative product, comprise the total alkaloid of lotus leaves extraction that treats and/or prevents effective dose and optional pharmaceutically acceptable carrier in this product, described total alkaloid of lotus leaves extraction is as first aspect present invention as described in each, perhaps as second aspect present invention as described in each.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, described and the unusual diseases associated of glycolipid metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Ninth aspect present invention is provided among the experimenter who needs and treats and/or prevents and unusual diseases associated of glycolipid metabolism or disease method, and/or blood fat reducing in the experimenter who needs is arranged, suppressing cholesterol, antibacterial, spasmolytic, antioxidation method, described method comprises the total alkaloid of lotus leaves extraction of using effective dose to described experimenter.In one embodiment, described total alkaloid of lotus leaves extraction is as first aspect present invention as described in each, perhaps as second aspect present invention as described in each.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described total alkaloid of lotus leaves extraction, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, described and the unusual diseases associated of glycolipid metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
The feature that each had of either side of the present invention or this either side is equally applicable to each of other either side or this other either side, as long as they can be not conflicting, certainly at where applicable each other, necessary words can be done suitably to modify to individual features.In the present invention, when for example, mentioning " first aspect present invention each ", should " each " be meant the arbitrary sub-aspect of first aspect present invention, when others are mentioned in a similar manner, also have identical meanings.
Detailed Description Of The Invention:
Be further described with characteristics to various aspects of the present invention below.
All documents that the present invention quoted from, their full content is incorporated this paper by reference into, and if the expressed implication of these documents and the present invention when inconsistent, be as the criterion with statement of the present invention.In addition, various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art, nonetheless, the present invention still wishes at this more detailed description and interpretation to be made in these terms and phrase, term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.
As described herein, term " Folium Nelumbinis " can meet the regulation under the version Pharmacopoeia of the People's Republic of China corresponding entry in 2005.
As described herein, term " effective dose " is meant the dosage that can realize treating, prevent, alleviate and/or alleviating disease of the present invention or disease in the experimenter.
As described herein, term " compositions ", it can also be meant pharmaceutical composition, is used in to realize treating, prevent, alleviate and/or alleviating disease of the present invention or disease among the experimenter.
As described herein, term " experimenter " can refer to that patient or other accept the present composition to treat, to prevent, to alleviate and/or to alleviate the animal of disease of the present invention or disease, particularly mammal, for example people, Canis familiaris L., monkey, cattle, horse etc.
As described herein, term " disease and/or disease " is meant a kind of condition of described experimenter, this condition is relevant with disease of the present invention and/or disease.
As described herein, percent symbol " % ", it has as the definition in the version Pharmacopoeia of the People's Republic of China note on the use in 2005.Especially, for ethanol of the present invention, be meant ethanol water with prescribed concentration; For the % concentration of hydrochloric acid of the present invention, it is meant concentration of volume percent.In some cases, as do not specialize, generally be meant the percentage ratio of w/w when being solid, generally be meant the percentage ratio of weight/volume when being liquid for total material for total material.
As described herein, term " total alkaloid of lotus leaves extraction " is meant that a kind of Folium Nelumbinis that particularly meets the regulation under the version Pharmacopoeia of the People's Republic of China " Folium Nelumbinis " item in 2005 by the plant Folium Nelumbinis through a kind of extract that extraction obtains, wherein contains the alkaloid (being referred to as total alkaloids) of some kinds.
As described herein, term " total alkaloid of lotus leaves " be Folium Nelumbinis after extracting, obtain comprise some alkaloidal active component groups, wherein comprise monomeric compound nuciferine and other alkaloid, for example alkaloid such as N-demethyl nuciferine and/or the O-demethyl nuciferine etc. put down in writing of prior art.
The present invention is surprisingly found out that, by to medical material with the acid alcohol merceration extract, Soxhlet is extracted and simple three steps of solvent extraction can easily prepare total alkaloid of lotus leaves extraction, and total alkaloid of lotus leaves content can reach 70～98% in the prepared extract.When medicinal material extract, select the method for acid alcohol merceration for use, than the safer convenience of reflux extraction, energy savings, impurity content is low in the extract, is convenient to the raising of total alkaloid of lotus leaves content in the subsequent step.In addition, hydrochloric ethanol extract, when repeatedly leaching, last cooling bath can be used for having reduced solvent load when the merceration solvent first time of next group time Folium Nelumbinis merceration extraction.In the Soxhlet extraction step, kieselguhr is admixed in the concentrated solution of preceding step acquisition, can better disperse above-mentioned concentrated solution, make it more abundant with contacting of organic solvent (for example ethyl acetate) when Soxhlet is extracted backflow, extract more complete.In addition, the solvent that uses in different step (particularly organic solvent) is convenient to recycling for solvent single, low toxicity.
In one embodiment, extract of the present invention can be by the method preparation that may further comprise the steps; Perhaps in one embodiment, preparation method of extract of the present invention may further comprise the steps:
(1) the medical material merceration extracts: 50%～80% ethanol with hydrochloric 0.05%～0.5% serves as to extract solvent, and extracting solvent load is 16～18 times, and merceration extracts 3 times, merge the merceration extracting solution, regulating extracting solution pH is 9～10, and concentrating under reduced pressure reclaims solvent, the concentrated solution of winning;
(2) Soxhlet extraction separation: first concentrated solution, adding kieselguhr is mixed thoroughly, after the drying, carries out Soxhlet with ethyl acetate and extracts, and extracts the back concentrating under reduced pressure and reclaims solvent, gets second concentrated solution;
(3) solvent extraction and separation: second concentrated solution, with the aqueous hydrochloric acid solution extraction of pH=1～2 of 1～2 times of volume 2～4 times, tell and merge the sour water layer; After regulating sour water layer pH to 11～12, with the ethyl acetate extraction of 1～2 times of volume 2～4 times, collect ethyl acetate layer, concentrating under reduced pressure reclaims solvent, after the drying, and must total alkaloid of lotus leaves extraction.
In one embodiment, preparation is during extract, and the optimum solvent that Folium Nelumbinis medical material merceration extracts is that hydrochloric 0.1% concentration of volume percent is 70% ethanol.
Adopt the inventive method to prepare total alkaloid of lotus leaves extraction, medicinal material extract is complete, and step is simple, and solvent load is few, toxicity is low, is suitable for suitability for industrialized production; Prepared extract is measured total alkaloid of lotus leaves content through acid-dye colorimetry can reach (for example more than 80%) more than 70%, measures nuciferine content through HPLC and can reach (for example more than 15%, 18% or 20%) more than 14%.
Pharmaceutical composition of the present invention can be made acceptable dosage form clinically, comprises tablet, granule, capsule, pill, drop pill or the like.Its preparations shaping technology can adopt the preparation method of routine well known to those skilled in the art, and the present invention does not limit at this.The adjuvant commonly used that allows use in the oral formulations be can adopt, disintegrating agent, filler, lubricant, binding agent, correctives, fluidizer, surfactant etc. comprised.
The present invention further describes total alkaloid of lotus leaves extraction of the present invention at the beneficial effect that treats and/or prevents aspect unusual diseases associated of glycolipid metabolism or disease by the biological experiment example.
Liver and peripheral tissues are the target tissues of insulin action, it is the main position that insulin resistant produces, it is the emphasis of inquiring into the pathophysiology link of mechanism that insulin resistant forms and glucose metabolism obstacle, total alkaloid of lotus leaves can effectively suppress fatty precursor differentiation, reduce accumulating of triglyceride in the adipose cell, point out it to have fat-reducing and improve the insulin resistant effect.
Total alkaloid of lotus leaves extraction can suppress the secretion of catecholamine (CA) of the high potassium mediation of n acetyl choline receptor agonists acetylcholine (ACh) on the cell membrane, voltage dependent form sodium-ion channel opener veratridine alkali (Ver), voltage dependent form calcium channel open environment, and have dose dependent, can significantly suppress the interior stream of cell calcium of acetylcholine-induced simultaneously again.Illustrate that total alkaloid of lotus leaves extraction suppresses the catecholamine secretion by reducing the intracellular calcium concentration performance, this result of study prompting Folium Nelumbinis can be used for glycolipid metabolism abnormal diseases such as obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension except that being applied to heatstroke.
Description of drawings
Fig. 1 shows nuciferine reference substance HPLC chromatogram (1-nuciferine peak).
Fig. 2 shows the HPLC chromatogram (1-nuciferine peak) of total alkaloid of lotus leaves extraction of the present invention.
Fig. 3 shows that total alkaloid of lotus leaves extraction is to ACh, Ver, the excretory influence of the inductive catecholamine of high potassium.
Fig. 4 shows that the variable concentrations total alkaloid of lotus leaves extraction is to ACh (A), Ver (B), the excretory influence of the inductive CA of high potassium (C).
The specific embodiment
Can further describe the present invention by following example, yet scope of the present invention is not limited to following example.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.
The present invention carries out generality and/or concrete description to the material and the test method that are used in the test.Though for realizing that employed many materials of the object of the invention and operational approach are well known in the art, the present invention still does detailed as far as possible description at this.
The preparation of preparation example 1---total alkaloid of lotus leaves extraction
(1) the medical material merceration extracts: get 100g Folium Nelumbinis medical material, and 70% ethanol merceration extraction with hydrochloric 0.05% 3 times, solvent load is respectively 1800mL, 1800mL, 1600mL, each 36 hours, merge extractive liquid, was regulated extracting solution pH to 8.5, be evaporated to about 100mL, the concentrated solution of winning;
(2) Soxhlet extraction separation: with first concentrated solution, add 50g kieselguhr and mix thoroughly, put into apparatus,Soxhlet's after the drying, use 200mL ethyl acetate extraction 2 times, each 3 hours, merge extractive liquid, was evaporated to about 100mL, got second concentrated solution;
(3) solvent extraction and separation: second concentrated solution, use the aqueous hydrochloric acid solution 100mL of pH=1 to extract 3 times, merge the sour water layer, regulate pH to 11, with 300mL ethyl acetate extraction 3 time of sour water layer, the combined ethyl acetate layer, concentrate drying gets total alkaloid of lotus leaves extraction.
The preparation of preparation example 2---total alkaloid of lotus leaves extraction
(1) the medical material merceration extracts: 100g Folium Nelumbinis medical material, and 50% ethanol merceration extraction with hydrochloric 0.1% 3 times, solvent load is respectively 1600mL, 1600mL, 1600mL, each 36 hours, merge extractive liquid, was regulated extracting solution pH to 10, be evaporated to about 100mL, the concentrated solution of winning;
(2) Soxhlet extraction separation: with first concentrated solution, add 50g kieselguhr and mix thoroughly, put into apparatus,Soxhlet's after the drying, use 200mL ethyl acetate extraction 2 times, each 3 hours, merge extractive liquid, was evaporated to about 100mL, got second concentrated solution;
(3) solvent extraction and separation: second concentrated solution, use the aqueous hydrochloric acid solution 100mL of pH=2 to extract 2 times, merge the sour water layer, regulate pH to 12, with 200mL ethyl acetate extraction 2 time of sour water layer, the combined ethyl acetate layer, concentrate drying gets total alkaloid of lotus leaves extraction.
The preparation of preparation example 3---total alkaloid of lotus leaves extraction
(1) the medical material merceration extracts: 100g Folium Nelumbinis medical material, add 80% the ethanol that 1700mL contains 0.5% hydrochloric acid respectively, and merceration extracts 3 times, and each 36 hours, merge extractive liquid, was regulated extracting solution pH to 10.5, is evaporated to about 100mL, the concentrated solution of winning;
(2) Soxhlet extraction separation: with first concentrated solution, add 50g kieselguhr and mix thoroughly, put into apparatus,Soxhlet's after the drying, use 200mL ethyl acetate extraction 2 times, each 3 hours, merge extractive liquid, was evaporated to about 100mL, got second concentrated solution;
(3) solvent extraction and separation: second concentrated solution, use the aqueous hydrochloric acid solution 100mL of pH=1 to extract 4 times, merge the sour water layer, regulate pH to 11, with 400mL ethyl acetate extraction 3 time of sour water layer, the combined ethyl acetate layer, concentrate drying gets total alkaloid of lotus leaves extraction.
In different preparation examples, use different extraction conditions, and the performance parameter of product is listed in table 1.
Analytical test example 1---the content of total alkaloid of lotus leaves in the total alkaloid of lotus leaves extraction
Measure
Adopt acid-dye colorimetry to measure.
The preparation of reference substance solution: it is an amount of to get the nuciferine reference substance, and accurate the title decides, and being mixed with concentration with chloroform is 0.04mgmL
-1The nuciferine reference substance solution.
The preparation of bromocresol green buffer: precision takes by weighing bromocresol green 125mg, uses 0.2molL
-1NaOH 12.5mL dissolving, add an amount of Potassium Hydrogen Phthalate, add the low amounts of water dissolving, regulate pH value to 4.0.
The drafting of standard curve: accurate respectively absorption 0,0.5,1.0,1.5,2.0,2.5mL reference substance solution add chloroform to scale in the 5mL measuring bottle, jolting moves in the separatory funnel, accurate bromocresol green buffer and the 0.2molL of adding
-1Each 0.5mL of NaOH, shake up, leave standstill, getting clarifying chloroform layer and measure ultraviolet absorptivity under the 415nm wavelength, is vertical coordinate with nuciferine concentration, and absorbance is an abscissa, get regression equation: Y=71.5109X-0.01476, r=0.9986, the range of linearity 0.004～0.02mg.m L
-1
The mensuration of total alkaloids in the total alkaloid of lotus leaves extraction: precision takes by weighing the 8mg total alkaloid of lotus leaves extraction and places the 10ml volumetric flask, adds chloroform to scale, shakes up, the accurate 1ml that draws is in the 10ml volumetric flask, add chloroform to scale, shake up, get need testing solution.Get the 1mL need testing solution and place the separatory funnel that adds the 5mL chloroform, add bromocresol green buffer 3mL and 0.2molL again
-1NaOH solution 1mL, jolting 1min leaves standstill 30min, gets clarifying chloroform soln layer and measures absorbance in the 415nm place, the substitution regression equation calculation obtains total alkaloid contents in the total alkaloid of lotus leaves extraction.
Above the measurement result of the total alkaloid of lotus leaves extraction that obtains in the preparation example sees Table 1.
Analytical test example 2---the assay of nuciferine in the total alkaloid of lotus leaves extraction
Adopt the HPLC method to measure.
The preparation of reference substance solution: it is an amount of to get the nuciferine reference substance, and accurate the title decides, and being mixed with concentration with methanol is 0.0154mgmL
-1The nuciferine reference substance solution.
The preparation of need testing solution: it is an amount of to get total alkaloid of lotus leaves extraction, and accurate the title decides, and being mixed with concentration with methanol is 0.176mgmL
-1
HPLC chromatographic condition: chromatographic column: Phenomsil C
18(4.6mm * 250mm, 5 μ m); Mobile phase: acetonitrile-water-triethylamine-glacial acetic acid (27: 70.6: 1.6: 0.78); Detect wavelength: 270nm; Flow velocity: 1.0mLmin
-1Column temperature: 25 ℃.
The drafting of standard curve: it is an amount of to get the nuciferine reference substance, and accurate the title decides, and is made into the reference substance solution of series concentration with methanol.With peak area (Y) is vertical coordinate, and sample size (X) is the abscissa mapping, and getting the nuciferine regression equation is Y=3.22X+12.02, r=0.9998, and the range of linearity is 9.56 * 10
-3～0.478 μ g.
Sample determination: the accurate reference substance solution 5 μ l that draw, need testing solution 5 μ l inject high performance liquid chromatograph successively, measure the nuciferine peak area, press one point external standard method and calculate, and get nuciferine content.
Above the measurement result of the total alkaloid of lotus leaves extraction that obtains in the preparation example sees Table 1.The HPLC collection of illustrative plates of the extract of nuciferine reference substance and preparation example 1 is seen Fig. 1 and Fig. 2 respectively.Calculate in this extract the weight rate of total alkaloids and nuciferine simultaneously.
In table 1, total alkaloid of lotus leaves extraction sample or its compositions, nuciferine and total alkaloid contents measurement result
| Sample | Total alkaloids (%) * | Nuciferine (%) ** | Total alkaloids/nuciferine *** |
| Preparation example 1 | 70.0 | 14.0 | 5∶1 |
| Preparation example 2 | 98.0 | 28.0 | 3.5∶1 |
| Preparation example 3 | 85.0 | 19.3 | 4.4∶1 |
Annotate:
*The expression total alkaloids accounts for the percetage by weight (%) of extract;
*The expression nuciferine accounts for the percetage by weight (%) of extract;
* *The total alkaloids in the expression extract and the weight rate of nuciferine.
Formulation example 1, preparation comprise the pharmaceutical composition of total alkaloid of lotus leaves extraction
Get the total alkaloid of lotus leaves extraction 100g of preparation example 1, starch 50g, microcrystalline Cellulose 60g, carboxymethyl starch sodium 120g, polyvinylpolypyrrolidone 50g.Behind the mix homogeneously, containing 90% alcoholic solution system soft material of 1% hydroxypropyl emthylcellulose, 16 mesh sieves are granulated, 60 ℃ of dryings and granulate.Add micropowder silica gel 20g again, magnesium stearate 2g with the dried particles mix homogeneously, is pressed into the tablet of 1000 pharmaceutical compositions of the present invention, the bag film-coat.
Biological test example 1, total alkaloid of lotus leaves extraction are to fatty precursor differentiation and fat
The influence that fat is accumulated
Obesity and/or obesity are the key factors that causes diabetes insulin opposing, hyperinsulinemia, also are the independent hazard factors of metabolism syndrome and cardiovascular disease.Have that the Chinese medicine that suppresses the lipid material Absorption can be assisted and the dietetic therapy of alternative prediabetes.Some Chinese medicine is by the decomposition of inhibition lipase active, minimizing monoacylglycerol and free fatty, thus the absorption of minimizing lipid material; Some Chinese medicine is to reduce obesity and/or obesity degree by the formation that suppresses adipose cell.
Adipose cell progressively breaks up under the effect of multiple factor, grows and form by originating from mesoblastic pluripotent stem cell, its process has roughly experienced adult stem cell, mesenchymal precursor cells, preceding adipose cell, mature fat cell several stages, and each stage cell is referred to as fatty precursor before the mature fat cell.The degree that how much directly influences obesity and/or obesity of adipose cell quantity.The inventor has studied the influence of total alkaloid of lotus leaves extraction to fatty precursor differentiation, and methods and results is as follows:
The cultivation of 3T3-L1 fat precursor and induce differentiation: the 3T3-L1 cell is by 3.33 * 10
4Cell/ml inoculating cell is in 48 orifice plates, at 37 ℃ of incubator (5%CO
2, 95% air) in cultivate, cell culture medium is the high sugared complete culture solution of DMEM, every 2d changes a not good liquor, treats to begin to induce after cell merges fully; Culture fluid changes into and contains 0.5mmol/L IBMX, 1 μ mol/L dexamethasone, and the high sugared DMEM complete culture solution of 10 μ g/ml insulins is induced differentiation; After cultivating 48h, change the high sugared DMEM complete culture solution that contains 5 μ g/ml insulins and cultivate, change high sugared DMEM complete culture solution behind the 48h and keep cultivation, every 2d changes a not good liquor.Be cultured to the 12nd day, cell 90% is divided into mature fat cell.
The total alkaloid of lotus leaves extraction of preparation example 2 is dissolved in the DMSO solution, be prepared into 64mg/ml solution, reuse culture fluid dilution back adds cell culture system, final concentration is 100,30,10,3,1,0.3 μ g/ml, DMSO content is lower than 0.5%, and this concentration DMSO content cell growth does not have influence.The results are shown in Table 2.
Table 2, total alkaloid of lotus leaves extraction are to fatty precursor suppression ratio
| Total alkaloid of lotus leaves extraction concentration (μ g/ml) | 100 | 30 | 10 | 3 | 1 | 0.3 |
| Suppression ratio % | 100±1.9 | 60±5.7 | 66±1.8 | 71±4.0 | 35±4.8 | 19±7.6 |
IC
50＝1.40μg/ml
The result shows that total alkaloid of lotus leaves extraction can effectively suppress fatty precursor differentiation.
Biological test example 2, total alkaloid of lotus leaves extraction are to the bovine adrenal medullary epithelium
The excretory influence of tea phenol amine
Catecholamine (CA, catecholamine comprise norepinephrine, epinephrine, dopamine) is the endogenous material that body is kept normal activities, and participating in organism physiology stress change, too or not as good as all causing pathologic damage.Catecholamine occupies very important effect regulating on the cardiovascular system normal function, but since the catecholamine that stress cause secretion can excessive participation and increases the weight of such as heart failure, atherosclerosis, coronary heart disease and cardiovascular primary diseases such as hypertension and/or vascular hypertension.Therefore it is significant aspect prevention and the treatment disease relevant with catecholamine to study it.Catecholamine and obesity and/or obesity and hyperglycemia are in close relations, and it both can stimulate hepatic glucose to generate, and also can limit glucose utilization.And by intervening the synthetic of carbohydrate metabolism influence fat.Its major physiological machining function shows: 1. impel glycogenolysis, it is synthetic to reduce glycogen; 2. promote glyconeogenesis; 3. suppress insulin secretion and glucose uptake.The inventor has studied total alkaloid of lotus leaves extraction to the excretory influence of catecholamine, and method and result are as follows:
Adopt In vitro culture bovine adrenal medullary epithelium, detect content, the intracellular calcium content of catecholamine in the cell culture fluid respectively by HPLC-ECD, FlexstationIII fluorescence method, investigate total alkaloid of lotus leaves extraction (extract with preparation example 3 is tested) to the excretory influence of the inductive catecholamine of different stimulated agent, and under the different stimulated agent total alkaloid of lotus leaves extraction concentration change to the secretion of catecholamine and the influence of calcium current.
The result sees Fig. 3 and Fig. 4 respectively.Wherein, Fig. 3 shows that total alkaloid of lotus leaves extraction is to ACh, Ver, the excretory influence of the inductive catecholamine of high potassium; Fig. 4 shows that the variable concentrations total alkaloid of lotus leaves extraction is to ACh (A), Ver (B), the excretory influence of the inductive CA of high potassium (C).
The result shows: compare 50mgL with contrast (control)
-1Total alkaloid of lotus leaves extraction can significantly suppress acetylcholine (ACh), veratridine alkali (Ver, veratridine), high potassium (HighK
+) inductive catecholamine secretion, its suppression ratio is respectively: 42.86%, 35.33%, 37.46% (
*P＜0.01); 0.5,5,50mgL
-1The concentration total alkaloid of lotus leaves extraction is the inductive catecholamine secretion of energy dose-dependent inhibition acetylcholine, veratridine alkali and high potassium (IC all
50Value is respectively 34.85mgL
-1, 41.62mgL
-1And 27.63mgL
-1), compare with acetylcholine, veratridine alkali and high potassium with single respectively, have significant difference (
*P＜0.05,
*P＜0.01).The metabolic defect in cellular calcium ion concentration that total alkaloid of lotus leaves extraction can also significantly suppress acetylcholine-induced raises.Above result of study shows that total alkaloid of lotus leaves extraction passes through nAChR passage, voltage dependent form sodium-ion channel and the performance of voltage dependent form calcium channel and suppresses the sympathetic activity effect.
Above biological test has illustrated: adopt the total alkaloid of lotus leaves extraction of the inventive method preparation to can be used for treating the glycolipid metabolism abnormal diseases.
Claims (10)
1. a total alkaloid of lotus leaves extraction wherein comprises total alkaloid of lotus leaves and nuciferine, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
2. according to the total alkaloid of lotus leaves extraction of claim 1, it is characterized in that following (1) to (3) each or multinomial:
(1) described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight;
(2) described nuciferine accounts for 14～28% (w/w) of this extract weight;
(3) weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1, and described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight, and described nuciferine accounts for 14～28% (w/w) of this extract weight.
3. according to each total alkaloid of lotus leaves extraction of claim 1 to 2, it is to prepare by the method that may further comprise the steps: make the Folium Nelumbinis ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid, carrying out Soxhlet then extracts, the extracting solution that obtains extracts with aqueous hydrochloric acid solution, the water layer organic solvent extraction, organic layer is concentrated, drying, promptly.
4. a total alkaloid of lotus leaves extraction wherein comprises total alkaloid of lotus leaves and nuciferine, and wherein said total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight.
5. according to the total alkaloid of lotus leaves extraction of claim 4, it is characterized in that following (1) to (3) each or multinomial:
(1) described nuciferine accounts for 14～28% (w/w) of this extract weight;
(2) described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight, and described nuciferine accounts for 14～28% (w/w) of this extract weight;
(3) described total alkaloid of lotus leaves accounts for 70～98% (w/w) of this extract weight, and described nuciferine accounts for 14～28% (w/w) of this extract weight, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
6. according to each total alkaloid of lotus leaves extraction of claim 4 to 5, it is to prepare by the method that may further comprise the steps: make the Folium Nelumbinis ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid, carrying out Soxhlet then extracts, the extracting solution that obtains extracts with aqueous hydrochloric acid solution, the water layer organic solvent extraction, organic layer is concentrated, drying, promptly.
7. each the method for total alkaloid of lotus leaves extraction of preparation claim 1 to 6, this method may further comprise the steps: make the Folium Nelumbinis ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid, carrying out Soxhlet then extracts, the extracting solution that obtains extracts with aqueous hydrochloric acid solution, the water layer organic solvent extraction, organic layer is concentrated, drying, promptly.In one embodiment, said method comprising the steps of:
A) make Folium Nelumbinis 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid, the extracting solution that obtains is regulated pH to 8.5～10.5, concentrates, and obtains first concentrated solution;
B) concentrated solution that step a) is won is mixed with kieselguhr, drying, reuse ethyl acetate are carried out the Soxhlet extraction in apparatus,Soxhlet's, and extracting solution is concentrated, and obtain second concentrated solution;
C) making the step b) gained second concentrated solution pH is the extraction of 1～2 aqueous hydrochloric acid solution, and water layer is regulated pH to 11～12, and the reuse ethyl acetate extracts, and the organic layer that obtains is concentrated, drying, promptly.
8. compositions, said composition comprises the total alkaloid of lotus leaves extraction that treats and/or prevents effective dose and optional pharmaceutically acceptable carrier, wherein said total alkaloid of lotus leaves extraction comprises total alkaloid of lotus leaves and nuciferine, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1; Perhaps, said composition comprises the total alkaloid of lotus leaves extraction that treats and/or prevents effective dose and optional pharmaceutically acceptable carrier, and wherein said total alkaloid of lotus leaves extraction is as claim 1 to 6 as described in each.
Total alkaloid of lotus leaves extraction preparation be used for the treatment of and/or the product of prevention and unusual diseases associated of glycolipid metabolism or disease in purposes.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
Total alkaloid of lotus leaves extraction preparation be used for the treatment of and/or the product of prevention and unusual diseases associated of glycolipid metabolism or disease in purposes, and/or be used for blood fat reducing, suppress cholesterol, the purposes of antibacterial, spasmolytic, antioxidative product, wherein said total alkaloid of lotus leaves extraction is as claim 1 to 6 as described in each.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in the described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
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| CN105726640A (en) * | 2016-02-24 | 2016-07-06 | 中国药科大学 | Preparing method and application of high-purity lotus leaf total alkaloid extract |
| CN105911174A (en) * | 2016-04-15 | 2016-08-31 | 广西壮族自治区梧州食品药品检验所 | Determination method for nuciferine in lotus leaf |
| CN105929036A (en) * | 2016-04-15 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for extracting nuciferine from lotus leaves |
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| CN105726640A (en) * | 2016-02-24 | 2016-07-06 | 中国药科大学 | Preparing method and application of high-purity lotus leaf total alkaloid extract |
| CN105911174A (en) * | 2016-04-15 | 2016-08-31 | 广西壮族自治区梧州食品药品检验所 | Determination method for nuciferine in lotus leaf |
| CN105929036A (en) * | 2016-04-15 | 2016-09-07 | 广西壮族自治区梧州食品药品检验所 | Method for extracting nuciferine from lotus leaves |
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