CN102258578B - Lotus leaf total alkaloid extract and preparation method as well as application thereof - Google Patents
Lotus leaf total alkaloid extract and preparation method as well as application thereof Download PDFInfo
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- CN102258578B CN102258578B CN201010181495.4A CN201010181495A CN102258578B CN 102258578 B CN102258578 B CN 102258578B CN 201010181495 A CN201010181495 A CN 201010181495A CN 102258578 B CN102258578 B CN 102258578B
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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 easy reliable total alkaloid of lotus leaves extraction of novelty, and the particularly application in treating and/or preventing the Abnormality of Glycolipid Metabolism 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 the multiple efficacies such as clearing away summer-heat, Weight-reducing and lipid-lowering, sending up the lucid YANG, cooling blood for hemostasis.Modern pharmacology research shows, the pharmacologically actives such as the alkaloid in Folium Nelumbinis has blood fat reducing, suppresses cholesterol, antibacterial and spasmolytic, antioxidation, are the effective constituents that mainly contains of Folium Nelumbinis.
The extraction purification more employing macroporous resin partition method of bibliographical information to total alkaloid of lotus leaves, the total alkaloid of lotus leaves content that adopts resin absorption technology to prepare is lower.(Xiao Wenjun in document, Hu Xiangwen, Hu Yunling etc. the technical research of lotus leaf alkaloid column separating purification. Food Science [J] .2007,28 (6): 120-124) take Folium Nelumbinis cured leaf as raw material, after 30% ethanol extraction, studied polyamide, D101, the process of tri-kinds of macroporous resins of HP-20 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 No. 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 process, and in the extract obtaining total alkaloid content lower than 58%.
Therefore, provide a kind of method of easy extraction lotus leaf alkaloid, obtain and contain the alkaloidal Folium Nelumbinis extract of higher degree, be still the problem that those skilled in the art are eager solution.
Summary of the invention
The object of the invention is to overcome the deficiency existing in existing extraction lotus leaf alkaloid technology, make extraction process simplify reliably and/or obtain and there is the alkaloidal extract of higher degree.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 after measured total alkaloid of lotus leaves content 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 the total alkaloid of lotus leaves extraction of first aspect present invention any one, 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 the total alkaloid of lotus leaves extraction of first aspect present invention any one, 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 the total alkaloid of lotus leaves extraction of first aspect present invention any one, 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, described nuciferine accounts for 14～28% (w/w) of this extract weight.
According to the total alkaloid of lotus leaves extraction of first aspect present invention any one, it is prepared by method by comprising the following steps: make the ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid for Folium Nelumbinis, then carry out Soxhlet extraction, the extracting solution obtaining extracts with aqueous hydrochloric acid solution, water layer is extracted with ethyl acetate, make organic layer concentrated, dry, obtain.In one embodiment, the ethanol that contains hydrochloric acid described in be contain 0.05～0.4%, 0.06～0.4%, 0.07～0.35%, 0.08～0.3%, 0.08～0.25%, approximately 0.09%, approximately 0.1%, approximately 0.12%, approximately 0.15%, approximately 0.2% or the ethanol of approximately 0.25% hydrochloric acid.In one embodiment, described ethanol is 50～80% ethanol (for example 55～80%, 60～80%, 65～80%, 65～75%, approximately 65%, approximately 70% or approximately 75% ethanol).In one embodiment, the ethanol that contains hydrochloric acid described in is 65～75% ethanol that contain 0.08～0.25% hydrochloric acid.In one embodiment, the ethanol that contains hydrochloric acid described in is 68～72% ethanol that contain 0.09～0.15% hydrochloric acid.In one embodiment, the ethanol that contains hydrochloric acid described in is approximately 70% ethanol containing 0.1% hydrochloric acid of having an appointment.In one embodiment, described extraction is that merceration extracts.In one embodiment, after described ethanol extract extracts, regulate pH to 8.5～10.5, for example, regulate pH to 9～10, and then concentrated.In one embodiment, described Soxhlet extraction is to extract by ethyl acetate.In one embodiment, the aqueous hydrochloric acid solution extraction that described Soxhlet extracting solution is 1～2 with pH.In one embodiment, described Soxhlet extracting solution, with after aqueous hydrochloric acid solution extraction, regulates pH to 11～12, then is extracted with ethyl acetate.
According to the total alkaloid of lotus leaves extraction of first aspect present invention any one, it is prepared by method by comprising the following steps:
A) make 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid for Folium Nelumbinis, the extracting solution obtaining regulates pH to 8.5～10.5, concentrated, obtains the first concentrated solution;
B) make step a) gained the first concentrated solution mix with kieselguhr, dry, then with ethyl acetate, in apparatus,Soxhlet's, carry out Soxhlet extraction, extracting solution is concentrated, obtain the second concentrated solution;
C) make step b) the aqueous hydrochloric acid solution extraction that is 1～2 with pH of gained the second concentrated solution, water layer regulates pH to 11～12, then extracts by ethyl acetate, makes the organic layer that obtains concentrated, dry, obtains.
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 the total alkaloid of lotus leaves extraction of second aspect present invention any one, 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 the total alkaloid of lotus leaves extraction of second aspect present invention any one, 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 the total alkaloid of lotus leaves extraction of second aspect present invention any one, 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 the total alkaloid of lotus leaves extraction of second aspect present invention any one, it is prepared by method by comprising the following steps: make the ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid for Folium Nelumbinis, then carry out Soxhlet extraction, the extracting solution obtaining extracts with aqueous hydrochloric acid solution, water layer is extracted with ethyl acetate, make organic layer concentrated, dry, obtain.In one embodiment, the ethanol that contains hydrochloric acid described in be contain 0.05～0.4%, 0.06～0.4%, 0.07～0.35%, 0.08～0.3%, 0.08～0.25%, approximately 0.09%, approximately 0.1%, approximately 0.12%, approximately 0.15%, approximately 0.2% or the ethanol of approximately 0.25% hydrochloric acid.In one embodiment, described ethanol is 50～80% ethanol (for example 55～80%, 60～80%, 65～80%, 65～75%, approximately 65%, approximately 70% or approximately 75% ethanol).In one embodiment, the ethanol that contains hydrochloric acid described in is 65～75% ethanol that contain 0.08～0.25% hydrochloric acid.In one embodiment, the ethanol that contains hydrochloric acid described in is 68～72% ethanol that contain 0.09～0.15% hydrochloric acid.In one embodiment, the ethanol that contains hydrochloric acid described in is approximately 70% ethanol containing 0.1% hydrochloric acid of having an appointment.In one embodiment, described extraction is that merceration extracts.In one embodiment, after described ethanol extract extracts, regulate pH to 8.5～10.5, for example, regulate pH to 9～10, and then concentrated.In one embodiment, described Soxhlet extraction is to extract by ethyl acetate.In one embodiment, the aqueous hydrochloric acid solution extraction that described Soxhlet extracting solution is 1～2 with pH.In one embodiment, described Soxhlet extracting solution, with after aqueous hydrochloric acid solution extraction, regulates pH to 11～12, then is extracted with ethyl acetate.
According to the total alkaloid of lotus leaves extraction of second aspect present invention any one, it is prepared by method by comprising the following steps:
A) make 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid for Folium Nelumbinis, the extracting solution obtaining regulates pH to 8.5～10.5, concentrated, obtains the first concentrated solution;
B) make step a) gained the first concentrated solution mix with kieselguhr, dry, then with ethyl acetate, in apparatus,Soxhlet's, carry out Soxhlet extraction, extracting solution is concentrated, obtain the second concentrated solution;
C) make step b) the aqueous hydrochloric acid solution extraction that is 1～2 with pH of gained the second concentrated solution, water layer regulates pH to 11～12, then extracts by ethyl acetate, makes the organic layer that obtains concentrated, dry, obtains.
Third aspect present invention provides the method for preparing total alkaloid of lotus leaves extraction described in first aspect present invention any one or second aspect any one, the method comprises the following steps: make the ethanol extraction that contains 0.05～0.5% (v/v) hydrochloric acid for Folium Nelumbinis, then carry out Soxhlet extraction, saline acid solution extraction for the extracting solution obtaining, water layer is extracted with ethyl acetate, make organic layer concentrated, dry, obtain.In one embodiment, the ethanol that contains hydrochloric acid described in be contain 0.05～0.4%, 0.06～0.4%, 0.07～0.35%, 0.08～0.3%, 0.08～0.25%, approximately 0.09%, approximately 0.1%, approximately 0.12%, approximately 0.15%, approximately 0.2% or the ethanol of approximately 0.25% hydrochloric acid.In one embodiment, described ethanol is 50～80% ethanol (for example 55～80%, 60～80%, 65～80%, 65～75%, approximately 65%, approximately 70% or approximately 75% ethanol).In one embodiment, the ethanol that contains hydrochloric acid described in is 65～75% ethanol that contain 0.08～0.25% hydrochloric acid.In one embodiment, the ethanol that contains hydrochloric acid described in is 68～72% ethanol that contain 0.09～0.15% hydrochloric acid.In one embodiment, the ethanol that contains hydrochloric acid described in is approximately 70% ethanol containing 0.1% hydrochloric acid of having an appointment.In one embodiment, described extraction is that merceration extracts.In one embodiment, after described ethanol extract extracts, regulate pH to 8.5～10.5, for example, regulate pH to 9～10, and then concentrated.In one embodiment, described Soxhlet extraction is to extract by ethyl acetate.In one embodiment, the aqueous hydrochloric acid solution extraction that described Soxhlet extracting solution is 1～2 with pH.In one embodiment, described Soxhlet extracting solution, with after aqueous hydrochloric acid solution extraction, regulates pH to 11～12, then is extracted with ethyl acetate.
According to the method for third aspect present invention any one, it comprises the following steps:
A) make 50～80% ethanol extractions that contain 0.05～0.5% (v/v) hydrochloric acid for Folium Nelumbinis, the extracting solution obtaining regulates pH to 8.5～10.5, concentrated, obtains the first concentrated solution;
B) make step a) gained the first concentrated solution mix with kieselguhr, dry, then with ethyl acetate, in apparatus,Soxhlet's, carry out Soxhlet extraction, extracting solution is concentrated, obtain the second concentrated solution;
C) make step b) the aqueous hydrochloric acid solution extraction that is 1～2 with pH of gained the second concentrated solution, water layer regulates pH to 11～12, then extracts by ethyl acetate, makes the organic layer that obtains concentrated, dry, obtains.
Fourth aspect present invention provides a kind of compositions, and it comprises and treats and/or prevents the total alkaloid of lotus leaves extraction of effective dose and optional pharmaceutically acceptable carrier.
According to the compositions of fourth aspect present invention any one, wherein said total alkaloid of lotus leaves extraction comprises total alkaloid of lotus leaves and nuciferine.
According to the compositions of fourth aspect present invention any one, 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 the compositions of fourth aspect present invention any one, wherein said total alkaloid of lotus leaves extraction is the total alkaloid of lotus leaves extraction of first aspect present invention any one, and/or described total alkaloid of lotus leaves extraction is the total alkaloid of lotus leaves extraction of second aspect present invention any one.
According to the compositions of fourth aspect present invention any one, it is pharmaceutical composition.
According to the compositions of fourth aspect present invention any one, it is pharmaceutical composition, and it is the form that is tablet, granule, capsule, pill, drop pill.
According to the compositions of fourth aspect present invention any one, it is pharmaceutical composition, and it is to be mixed and made into any in tablet, granule, capsule, pill, drop pill with one or more pharmaceutical excipients.
Fifth aspect present invention provides total alkaloid of lotus leaves extraction in the purposes for the preparation for the treatment of and/or preventing in the product of the disease relevant with Abnormality of Glycolipid Metabolism or disease.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, the described disease relevant with Abnormality of Glycolipid Metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Sixth aspect present invention provides total alkaloid of lotus leaves extraction in the purposes for the preparation for the treatment of and/or preventing in the product of the disease relevant with Abnormality of Glycolipid Metabolism or disease, and/or for blood fat reducing, suppress the purposes of cholesterol, antibacterial, spasmolytic, antioxidative product, wherein said total alkaloid of lotus leaves extraction is as described in first aspect present invention any one, or as described in second aspect present invention any one.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, the wherein said disease relevant with Abnormality 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 of disease or the disease relevant with Abnormality of Glycolipid Metabolism, comprises and treat and/or prevent the total alkaloid of lotus leaves extraction of effective dose and optional pharmaceutically acceptable carrier in this product.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, the described disease relevant with Abnormality 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 of disease or the disease relevant with Abnormality of Glycolipid Metabolism, and/or for blood fat reducing, suppress cholesterol, antibacterial, spasmolytic, antioxidative product, in this product, comprise and treat and/or prevent the total alkaloid of lotus leaves extraction of effective dose and optional pharmaceutically acceptable carrier, described total alkaloid of lotus leaves extraction is as described in first aspect present invention any one, or as described in second aspect present invention any one.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in described product, the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.In one embodiment, the described disease relevant with Abnormality of Glycolipid Metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Ninth aspect present invention provides in having the experimenter who needs and treats and/or prevents the disease relevant with Abnormality of Glycolipid Metabolism or disease method, and/or blood fat reducing in having the experimenter who needs, 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 described in first aspect present invention any one, or as described in second aspect present invention any one.In one embodiment, comprise total alkaloid of lotus leaves and nuciferine in 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, the described disease relevant with Abnormality of Glycolipid Metabolism or disease are selected from obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
The feature that any one of either side of the present invention or this either side has is equally applicable to any one 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, for example, while, mentioning " first aspect present invention any one ", be somebody's turn to do the arbitrary sub-aspect that " any one " refers to first aspect present invention, when other side is mentioned in a similar manner, also there is identical meanings.
detailed Description Of The Invention:
Be further described with feature to various aspects of the present invention below.
All documents that the present invention quotes from, their full content is incorporated to herein by reference, and if when the expressed implication of these documents and the present invention are 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, these terms and phrase to be described in more detail and to be explained, the term of mentioning and phrase, if any inconsistent with known implication, are as the criterion with the implication that the present invention was explained.
As described herein, term " Folium Nelumbinis " can meet the regulation under version < < Pharmacopoeia of People's Republic of China > > corresponding entry in 2005.
As described herein, term " effective dose " refers to the dosage that can realize treatment in experimenter, prevent, alleviate and/or alleviate disease of the present invention or disease.
As described herein, term " compositions ", it can also refer to pharmaceutical composition, is used in experimenter and realizes and treat, prevent, alleviate and/or alleviate disease of the present invention or disease.
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, such as people, Canis familiaris L., monkey, cattle, horse etc.
As described herein, term " disease and/or disease " refers to a kind of condition of described experimenter, and 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 People's Republic of China > > note on the use in 2005.Especially, for ethanol of the present invention, refer to the ethanol water with prescribed concentration; For the % concentration of hydrochloric acid of the present invention, it refers to concentration of volume percent.In some cases, as do not specialize, while being solid for total material, generally refer to the percentage ratio of w/w, while being liquid for total material, generally refer to the percentage ratio of weight/volume.
As described herein, term " total alkaloid of lotus leaves extraction " refers to that a kind of Folium Nelumbinis that particularly meets the regulation under version < < Pharmacopoeia of People's Republic of China > > " Folium Nelumbinis " item in 2005 by plant Folium Nelumbinis, through extracting a kind of extract obtaining, wherein contains the alkaloid (being referred to as total alkaloids) of some kinds.
As described herein, to be Folium Nelumbinis obtain term " total alkaloid of lotus leaves " after extracting comprises some alkaloidal active component groups, wherein comprise monomeric compound nuciferine and other alkaloid, the alkaloid of having recorded such as prior art is as N-demethyl nuciferine and/or O-demethyl nuciferine etc.
The present invention is surprisingly found out that, by acid alcohol merceration extraction for medical material, Soxhlet extraction and simple three steps of solvent extraction, can easily prepare total alkaloid of lotus leaves extraction, and in prepared extract, total alkaloid of lotus leaves content can reach 70～98%.When medicinal material extract, select the method for acid alcohol merceration, than the safer convenience of reflux extraction, energy savings, in extract, impurity content is low, is convenient to the raising of total alkaloid of lotus leaves content in subsequent step.In addition, hydrochloric ethanol extract, when repeatedly leaching, last cooling bath can be used for, when the solvent of merceration for the first time of next batch Folium Nelumbinis merceration extraction, having reduced solvent load.In Soxhlet extraction step, kieselguhr is admixed in the concentrated solution of preceding step acquisition, can better disperse above-mentioned concentrated solution, make it for example, with contacting of organic solvent (ethyl acetate) more abundant when Soxhlet is extracted backflow, extract more complete.In addition, the solvent using in different step (particularly organic solvent), for solvent single, low toxicity, is convenient to recycling.
In one embodiment, extract of the present invention can be prepared by the method comprising the following steps; Or in one embodiment, the preparation method of extract of the present invention comprises the following steps:
(1) medical material merceration extracts: hydrochloric 0.05%～0.5% 50%～80% the ethanol of take is to extract solvent, and extracting solvent load is 16～18 times, and merceration extracts 3 times, merge merceration extracting solution, regulating extracting solution pH is 9～10, and concentrating under reduced pressure reclaims solvent, obtains the first concentrated solution;
(2) Soxhlet is extracted separated: the first concentrated solution, and add kieselguhr to mix thoroughly, after being dried, by ethyl acetate, carry out Soxhlet extraction, after extracting, concentrating under reduced pressure reclaims solvent, obtains the second concentrated solution;
(3) solvent extraction and separation: the second concentrated solution, with the aqueous hydrochloric acid solution extraction of pH=1～2 of 1～2 times of volume 2～4 times, separates and merge sour water layer; Regulate behind sour water layer pH to 11～12, by the ethyl acetate of 1～2 times of volume, extract 2～4 times, collect ethyl acetate layer, concentrating under reduced pressure reclaims solvent, after being dried, obtains total alkaloid of lotus leaves extraction.
In one embodiment, while preparing extract, the optimum solvent that Folium Nelumbinis medical material merceration extracts is the ethanol that hydrochloric 0.1% concentration of volume percent is 70%.
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 can reach more than 70% (for example, more than 80%) through Wan withAcid-dye Colorimetry total alkaloid of lotus leaves content, measures nuciferine content can reach more than 14% (for example, more than 15%, 18% or 20%) through HPLC.
Pharmaceutical composition of the present invention can be made acceptable dosage form clinically, comprises tablet, granule, capsule, pill, drop pill etc.Its preparations shaping technology can adopt the preparation method of routine well known to those skilled in the art, and the present invention is in this no limit.The conventional adjuvant that allows use in 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 by biological experiment example and is treating and/or preventing the disease relevant with Abnormality of Glycolipid Metabolism or the beneficial effect aspect disease.
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 mechanism of insulin resistant formation and the pathophysiology link of impaired glucose metabolism, total alkaloid of lotus leaves can effectively suppress Preadipocyte differentiation, reduce accumulating of triglyceride in adipose cell, point out it there is fat-reducing and improve Insulin Resistance.
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 cell membrane, voltage dependent form sodium-ion channel opener veratridine alkali (Ver), voltage dependent form calcium channel open environment, and there is dose dependent, can significantly suppress again the interior stream of cell calcium of acetylcholine-induced simultaneously.Illustrate that total alkaloid of lotus leaves extraction suppresses Secretion of Catecholamine by reducing intracellular calcium concentration performance, this result of study prompting Folium Nelumbinis, except being applied to heatstroke, can be used for the Abnormality of Glycolipid Metabolism diseases such as obesity and/or obesity, diabetes, hyperlipemia, hypertension and/or vascular hypertension.
Accompanying drawing explanation
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 the impact of total alkaloid of lotus leaves extraction on the Secretion of Catecholamine of ACh, Ver, the induction of high potassium.
Fig. 4 shows the impact of variable concentrations total alkaloid of lotus leaves extraction on the CA secretion of ACh (A), Ver (B), high potassium (C) induction.
The specific embodiment
Example by below can conduct further description the present invention, yet scope of the present invention is not limited to following example.One of skill in the art can understand, and is not deviating under the prerequisite of 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 use in test.Although be well known in the art for realizing many materials and the operational approach that the object of the invention used, 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) medical material merceration extracts: get 100g Folium Nelumbinis medical material, 70% the ethanol merceration with hydrochloric 0.05% extracts 3 times, solvent load is respectively 1800mL, 1800mL, 1600mL, each 36 hours, merge extractive liquid,, regulate extracting solution pH to 8.5, be evaporated to about 100mL, obtain the first concentrated solution;
(2) Soxhlet is extracted separated: by the first concentrated solution, add 50g kieselguhr to mix thoroughly, after being dried, put into apparatus,Soxhlet's, by 200mL ethyl acetate, extract 2 times, each 3 hours, merge extractive liquid,, was evaporated to about 100mL, obtains the second concentrated solution;
(3) solvent extraction and separation: the second concentrated solution, with the aqueous hydrochloric acid solution 100mL of pH=1, extract 3 times, merge sour water layer, regulate pH to 11, by 300mL ethyl acetate extraction 3 times for sour water layer, combined ethyl acetate layer, concentrate drying, obtains total alkaloid of lotus leaves extraction.
the preparation of preparation example 2---total alkaloid of lotus leaves extraction
(1) medical material merceration extracts: 100g Folium Nelumbinis medical material, and 50% the ethanol merceration extraction with hydrochloric 0.1% 3 times, solvent load is respectively 1600mL, 1600mL, 1600mL, each 36 hours, merge extractive liquid,, regulated extracting solution pH to 10, be evaporated to about 100mL, obtain the first concentrated solution;
(2) Soxhlet is extracted separated: by the first concentrated solution, add 50g kieselguhr to mix thoroughly, after being dried, put into apparatus,Soxhlet's, by 200mL ethyl acetate, extract 2 times, each 3 hours, merge extractive liquid,, was evaporated to about 100mL, obtains the second concentrated solution;
(3) solvent extraction and separation: the second concentrated solution, with the aqueous hydrochloric acid solution 100mL of pH=2, extract 2 times, merge sour water layer, regulate pH to 12, by 200mL ethyl acetate extraction 2 times for sour water layer, combined ethyl acetate layer, concentrate drying, obtains total alkaloid of lotus leaves extraction.
the preparation of preparation example 3---total alkaloid of lotus leaves extraction
(1) medical material merceration extracts: 100g Folium Nelumbinis medical material, add respectively 1700mL containing 80% ethanol of 0.5% hydrochloric acid, and merceration extracts 3 times, and each 36 hours, merge extractive liquid,, regulated extracting solution pH to 10.5, is evaporated to about 100mL, obtains the first concentrated solution;
(2) Soxhlet is extracted separated: by the first concentrated solution, add 50g kieselguhr to mix thoroughly, after being dried, put into apparatus,Soxhlet's, by 200mL ethyl acetate, extract 2 times, each 3 hours, merge extractive liquid,, was evaporated to about 100mL, obtains the second concentrated solution;
(3) solvent extraction and separation: the second concentrated solution, with the aqueous hydrochloric acid solution 100mL of pH=1, extract 4 times, merge sour water layer, regulate pH to 11, by 400mL ethyl acetate extraction 3 times for sour water layer, combined ethyl acetate layer, concentrate drying, obtains 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 total alkaloid of lotus leaves extraction
measure
Adopt acid-dye colorimetry to measure.
The preparation of reference substance solution: get nuciferine reference substance appropriate, accurately weighed, being mixed with concentration with chloroform is 0.04mgmL
-1nuciferine reference substance solution.
The preparation of bromocresol green buffer: precision takes bromocresol green 125mg, uses 0.2molL
-1naOH 12.5mL dissolve, add appropriate Potassium Hydrogen Phthalate, add a small amount of water dissolution, regulate pH value to 4.0.
The drafting of standard curve: accurate absorption 0,0.5,1.0,1.5,2.0,2.5mL reference substance solution, in 5mL measuring bottle, add chloroform to scale respectively, and jolting, moves in separatory funnel, and precision adds bromocresol green buffer and 0.2molL
-1each 0.5mL of NaOH, shake up, standing, get the chloroform layer of clarification and measure ultraviolet absorptivity under 415nm wavelength, take nuciferine concentration as vertical coordinate, absorbance is abscissa, obtain 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 total alkaloid of lotus leaves extraction: precision takes 8mg total alkaloid of lotus leaves extraction and is placed in 10ml volumetric flask, adds chloroform to scale, shakes up, the accurate 1ml that draws is in 10ml volumetric flask, add chloroform to scale, shake up, obtain need testing solution.Get 1mL need testing solution and be placed in the separatory funnel that adds 5mL chloroform, then add bromocresol green buffer 3mL and 0.2molL
-1naOH solution 1mL, jolting 1min, standing 30min, the chloroform soln layer of getting clarification is measured absorbance in 415nm place, substitution regression equation calculation obtains the content of total alkaloids in total alkaloid of lotus leaves extraction.
The measurement result of the total alkaloid of lotus leaves extraction above obtaining in preparation example is in Table 1.
analytical test example 2---the assay of nuciferine in total alkaloid of lotus leaves extraction
Adopt HPLC method to measure.
The preparation of reference substance solution: get nuciferine reference substance appropriate, accurately weighed, being mixed with concentration with methanol is 0.0154mgmL
-1nuciferine reference substance solution.
The preparation of need testing solution: get total alkaloid of lotus leaves extraction appropriate, accurately weighed, 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
-1; Column temperature: 25 ℃.
The drafting of standard curve: get nuciferine reference substance appropriate, accurately weighed, with methanol, be made into the reference substance solution of series concentration.The peak area (Y) of take is vertical coordinate, and sample size (X) is abscissa mapping, and obtaining 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 nuciferine peak area, calculate by one point external standard method, obtain nuciferine content.
The measurement result of the total alkaloid of lotus leaves extraction above obtaining in preparation example is in Table 1.The HPLC collection of illustrative plates of the extract of nuciferine reference substance and preparation example 1 is shown in respectively Fig. 1 and Fig. 2.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, the assay result of nuciferine and total alkaloids
| 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 |
Note:
*represent that total alkaloids accounts for the percetage by weight (%) of extract;
*represent that nuciferine accounts for the percetage by weight (%) of extract;
* *total alkaloids in expression extract and the weight rate of nuciferine.
the pharmaceutical composition that formulation example 1, preparation comprise 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.After mix homogeneously, with the 90% alcoholic solution soft material processed containing 1% hydroxypropyl emthylcellulose, 16 mesh sieves are granulated, 60 ℃ of dry and granulate.Add micropowder silica gel 20g, magnesium stearate 2g, mixs homogeneously with dried particles, is pressed into the tablet of 1000 pharmaceutical compositions of the present invention, film coating again.
biological test example 1, total alkaloid of lotus leaves extraction are to Preadipocyte differentiation and fat
the impact that fat is accumulated
Obesity and/or obesity are the key factors that causes diabetes insulin opposing, hyperinsulinemia, are also the independent hazard factors of metabolism syndrome and cardiovascular disease.Have that the Chinese medicine that suppresses lipid material Absorption can be assisted and the dietetic therapy of alternative prediabetes.Some Chinese medicine is by suppressing the decomposition of 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 suppressing the formation of adipose cell.
Adipose cell progressively breaks up, grows and form by originating from mesoblastic pluripotent stem cell under the effect of many factors, its process has roughly experienced adult stem cell, mesenchymal precursor cells, front adipose cell, several stages of mature fat cell, and before mature fat cell, each phase cell is referred to as Preadipocyte.How many direct degree that affect obesity and/or obesity of adipose cell quantity.Inventor has studied the impact of total alkaloid of lotus leaves extraction on Preadipocyte differentiation, and methods and results is as follows:
The cultivation of 3T3-L1 Preadipocyte and induction differentiation: 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, starts induction after cell merges completely; Culture fluid changes into and contains 0.5mmol/L IBMX, 1 μ mol/L dexamethasone, and the DMEM in high glucose complete culture solution of 10 μ g/ml insulins is induced differentiation; Cultivate after 48h, change containing the DMEM in high glucose complete culture solution of 5 μ g/ml insulins and cultivate, change DMEM in high glucose complete culture solution maintain after 48h, 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 DMSO solution, be prepared into 64mg/ml solution, again with adding cell culture system after culture fluid dilution, 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 is without impact.The results are shown in Table 2.
Table 2, total alkaloid of lotus leaves extraction are to Preadipocyte 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
Result shows that total alkaloid of lotus leaves extraction can effectively suppress Preadipocyte differentiation.
biological test example 2, total alkaloid of lotus leaves extraction are to bovine adrenal medullary epithelium
the impact of tea phenol amine secretion
Catecholamine (CA, catecholamine comprise norepinephrine, epinephrine, dopamine) is the endogenous material that body maintains 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 cardiovascular system normal function, but because the Secretion of Catecholamine that stress cause can excessive participation and increase the weight of cardiovascular primary diseases such as heart failure, atherosclerosis, coronary heart disease and hypertension and/or vascular hypertension.Therefore study it significant aspect the prevention disease relevant to catecholamine with treatment.Catecholamine and obesity and/or obesity and hyperglycemia are in close relations, and it both can stimulate Hepatic glucose production, also can limit glucose utilization.And by intervening the synthetic of carbohydrate metabolism impact fat.Its major physiological machining function shows: 1. impel glycogenolysis, reduce glycogen synthetic; 2. promote glyconeogenesis; 3. suppress insulin secretion and glucose uptake.Inventor has studied the impact of total alkaloid of lotus leaves extraction on Secretion of Catecholamine, and method and result are as follows:
Adopt In vitro culture bovine adrenal medullary epithelium, by HPLC-ECD, FlexstationIII fluorescence method, detect respectively content, the intracellular calcium content of catecholamine in cell culture fluid, investigate the impact of total alkaloid of lotus leaves extraction (testing with the extract of preparation example 3) on the Secretion of Catecholamine of different stimulated agent induction, and the impact of total alkaloid of lotus leaves extraction concentration change on the secretion of catecholamine and calcium current under different stimulated agent.
Result is shown in respectively Fig. 3 and Fig. 4.Wherein, Fig. 3 shows the impact of total alkaloid of lotus leaves extraction on the Secretion of Catecholamine of ACh, Ver, the induction of high potassium; Fig. 4 shows the impact of variable concentrations total alkaloid of lotus leaves extraction on the CA secretion of ACh (A), Ver (B), high potassium (C) induction.
Result shows: compare 50mgL with contrasting (control)
-1total alkaloid of lotus leaves extraction can significantly suppress acetylcholine (ACh), veratridine alkali (Ver, veratridine), high potassium (HighK
+) induction Secretion of Catecholamine, its suppression ratio is respectively: 42.86%, 35.33%, 37.46% (
*p < 0.01); 0.5,5,50mgL
-1concentration total alkaloid of lotus leaves extraction is the Secretion of Catecholamine (IC of energy dose-dependent inhibition acetylcholine, veratridine alkali and the induction of high potassium all
50value is respectively 34.85mgL
-1, 41.62mgL
-1and 27.63mgL
-1), compare with alone acetylcholine, veratridine alkali and high potassium 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 sympathetic activity effect.
Above biological test has illustrated: the total alkaloid of lotus leaves extraction that adopts the inventive method to prepare can be used for treating Abnormality of Glycolipid Metabolism disease.
Claims (9)
1. a total alkaloid of lotus leaves extraction, 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, described total alkaloid of lotus leaves accounts for 70～98w/w% of this extract weight, described total alkaloid of lotus leaves extraction is prepared by the method by comprising the following steps: make the ethanol extraction that contains 0.05～5v/v% hydrochloric acid for Folium Nelumbinis, then carry out Soxhlet extraction, the extracting solution obtaining extracts with aqueous hydrochloric acid solution, water layer organic solvent extraction, make organic layer concentrated, dry, obtain.
2. according to the total alkaloid of lotus leaves extraction of claim 1, it is characterized in that:
Described nuciferine accounts for 14～28w/w% of this extract weight.
3. a total alkaloid of lotus leaves extraction, wherein comprise total alkaloid of lotus leaves and nuciferine, wherein said total alkaloid of lotus leaves accounts for 70～98w/w% of this extract weight, described total alkaloid of lotus leaves extraction is prepared by the method by comprising the following steps: make the ethanol extraction that contains 0.05～5v/v% hydrochloric acid for Folium Nelumbinis, then carry out Soxhlet extraction, the extracting solution obtaining extracts with aqueous hydrochloric acid solution, water layer organic solvent extraction, make organic layer concentrated, dry, obtain.
4. according to the total alkaloid of lotus leaves extraction of claim 3, it is characterized in that following (1) is to (3) any one 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, 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.
5. prepare the method for the total alkaloid of lotus leaves extraction of claim 1 to 4 any one, the method comprises the following steps: make the ethanol extraction that contains 0.05～0.5v/v% hydrochloric acid for Folium Nelumbinis, then carry out Soxhlet extraction, the extracting solution obtaining extracts with aqueous hydrochloric acid solution, water layer organic solvent extraction, make organic layer concentrated, dry, obtain.
6. the method for claim 5, said method comprising the steps of:
A) make 50～80% ethanol extractions that contain 0.05～0.5v/v% hydrochloric acid for Folium Nelumbinis, the extracting solution obtaining regulates pH to 8.5～10.5, concentrated, obtains the first concentrated solution;
B) make step a) gained the first concentrated solution mix with kieselguhr, dry, then with ethyl acetate, in apparatus,Soxhlet's, carry out Soxhlet extraction, extracting solution is concentrated, obtain the second concentrated solution;
C) make step b) the aqueous hydrochloric acid solution extraction that is 1～2 with pH of gained the second concentrated solution, water layer regulates pH to 11～12, then extracts by ethyl acetate, makes the organic layer that obtains concentrated, dry, obtains.
7. a compositions, said composition comprises and treats and/or prevents the total alkaloid of lotus leaves extraction of effective dose and optional pharmaceutically acceptable carrier, and wherein said total alkaloid of lotus leaves extraction is as described in claim 1 to 4 any one.
8. total alkaloid of lotus leaves extraction is in the purposes for the preparation for the treatment of and/or preventing in the product of the disease relevant with Abnormality of Glycolipid Metabolism or disease, and/or for blood fat reducing, suppress the purposes of cholesterol, antibacterial, spasmolytic, antioxidative product, wherein said total alkaloid of lotus leaves extraction is as described in claim 1 to 4 any one.
9. the purposes of claim 8, comprises total alkaloid of lotus leaves and nuciferine in described product, and the weight ratio of described total alkaloid of lotus leaves and nuciferine is (3.5～5): 1.
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