CN117982560A - Lotus leaf extract and preparation method and application thereof - Google Patents
Lotus leaf extract and preparation method and application thereof Download PDFInfo
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- CN117982560A CN117982560A CN202211358953.6A CN202211358953A CN117982560A CN 117982560 A CN117982560 A CN 117982560A CN 202211358953 A CN202211358953 A CN 202211358953A CN 117982560 A CN117982560 A CN 117982560A
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- lotus leaf
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a lotus leaf extract process, a quality control method and application thereof, wherein the preparation method of the lotus leaf extract comprises the following steps: taking lotus leaves, removing impurities, cutting, adding water, and decocting for 1-3 times, wherein each time is 0.5-2 hours, and each time the feed-liquid ratio is 1:5-20, filtering, mixing filtrates, concentrating into thick paste, and drying to obtain folium Nelumbinis extract. The preparation method only adopts water extraction, the preparation method is simple and safe, and the obtained extract is healthier and has rich ingredients, and can be conveniently used as raw materials for preparing products such as medicines, health-care products, foods and the like. The quality control method adopts various detection means and methods such as total alkaloid content detection, nuciferine content detection, UPLC-Q-TOF-MS analysis and the like, and can carry out comprehensive quality control on the components of the lotus leaf extract.
Description
Technical Field
The invention relates to a lotus leaf extract, a preparation method and application thereof.
Background
Lotus leaves are leaves of lotus (NelumbonuficeraGaertn) belonging to the Nymphaeaceae family, also called lotus leaves and lotus root leaves, and are planted in the north and south of China. The lotus leaf has bitter and cold property, and is effective in calming liver, spleen, stomach and heart meridian, clearing summer-heat and promoting diuresis, promoting hair growth and clearing yang, clearing heart and removing heat, stopping bleeding and promoting diuresis. The lotus leaf mainly contains alkaloid, flavonoid, polysaccharide and other components, and modern pharmacological researches show that the lotus leaf has the effects of reducing fat and losing weight, resisting oxidation and aging, inhibiting fatty liver, inhibiting bacteria, inhibiting HIV proliferation, resisting viruses, resisting inflammation, resisting allergy and the like. The lotus leaf has more clinical application in reducing weight and fat, improving liver metabolism, delaying aging and the like in a compound mode.
The lotus leaf extract is mainly prepared by taking lotus leaves as raw materials, extracting the lotus leaves by means of water, acid water or an organic solvent, and the like, and separating and purifying the lotus leaf extract by means of resin, countercurrent chromatography, and the like. The existing preparation process has the problems of complex operation steps, high cost and poor safety. Therefore, a preparation method of lotus leaf extract with simple operation, low cost and good safety is needed.
The quality control method of the lotus leaf extract is less studied, and CN202210147246.6 discloses a fingerprint construction method of an antioxidant lotus leaf extract, which aims at the liquid phase fingerprint established by the lotus leaf extract after polysaccharide is separated by macroporous resin after alkaline aqueous solution is extracted, and only 5 common peaks are marked, so that the components of the extract cannot be comprehensively represented, and the product quality cannot be comprehensively controlled.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method and application of a lotus leaf extract, wherein the preparation method of the lotus leaf extract only adopts water extraction, the preparation method is simple and safe, and the obtained lotus leaf extract is more beneficial to human health and has rich components, and can be conveniently used as raw materials for preparing products such as medicines, health products, foods and the like; the preparation method according to a further embodiment of the present invention also enables overall quality control of the obtained lotus leaf extract.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
According to a first aspect of the present invention, there is provided a method of preparing a lotus leaf extract, comprising: taking lotus leaves, removing impurities, cutting, adding water, and decocting for 1-3 times, wherein each time is 0.5-2 hours, and each time the feed-liquid ratio is 1:5-20, filtering, mixing filtrates, concentrating into thick paste, and drying to obtain folium Nelumbinis extract.
Preferably, the decoction times in the preparation method are 3 times, each time is 1 hour, and the feed-liquid ratio is 1: 15. 1:10. 1:10.
The lotus leaf extract is characterized by comprising nuciferine, nornuciferine, lotus plumule quaternary ammonium base, quercetin, kaempferol, isorhamnetin, saccharides, amino acids and procyanidins.
According to a second aspect of the present invention, the preparation method of the present invention comprises the above-mentioned quality control method of lotus leaf extract, which comprises at least one of the following methods:
(1) Total alkaloid content detection
(2) Nuciferine content detection
(3) Detection of total flavone content
(4) UPLC-Q-TOF-MS detection
(5) Moisture content detection
(6) Total sugar content detection
(7) Glucose, fructose and sucrose content detection
(8) Content detection of quercetin, kaempferol and isorhamnetin
(9) Ash content detection
(10) Amino acid content detection
(11) Procyanidine content detection
The method for detecting the total alkaloid content comprises the following steps:
And (3) preparation of a reagent: preparing bromocresol green solution with pH of 5.0;
preparing a reference substance solution: taking nuciferine reference substance, and adding chloroform to prepare reference substance solution;
preparation of test solution: extracting folium Nelumbinis extract with solvent, separating solid from liquid, and collecting liquid to obtain sample solution;
drawing a standard curve: taking 4 parts of nuciferine reference substance solutions with different amounts, respectively adding reagents to prepare solutions, measuring absorbance, and drawing a standard curve;
determination of sample content: and (5) taking the test solution to measure absorbance, and taking the absorbance into a standard curve to calculate the content.
Preferably, the solvent is 75% ethanol in the preparation step of the test sample solution, and the solid-liquid separation method is ultrasonic extraction at 50 ℃.
The method for detecting the nuciferine content in the quality control method (2) comprises the following steps:
preparing a reference substance solution: taking nuciferine reference substance, adding solvent to prepare reference substance solution;
Sample solution preparation: extracting folium Nelumbinis extract with solvent, and separating solid from liquid to obtain test solution;
Chromatographic conditions: according to high performance liquid chromatography, a chromatographic column using octadecylsilane chemically bonded silica as filler, and acetonitrile: water: triethylamine: the glacial acetic acid is used as a mobile phase for isocratic elution;
And (3) measuring: taking reference substance solution and test sample solution, respectively injecting into chromatograph, and measuring content according to external standard method.
Preferably, the specific process for preparing the reference substance solution is as follows: taking a proper amount of nuciferine reference substance, precisely weighing, and adding methanol to prepare a solution containing 15 mug per 1 mL;
The preparation method of the sample solution comprises the following specific steps: taking 0.5g of lotus leaf extract dry powder, precisely weighing, placing into a conical flask with a plug, precisely adding 50mL of methanol, weighing, performing ultrasonic treatment for 30min, cooling to room temperature, supplementing the reduced weight with methanol, shaking uniformly, filtering, precisely measuring 5mL of filtrate, placing into a volumetric flask with 25mL of filtrate, adding water to a scale mark, and shaking uniformly;
Chromatographic conditions: the detection wavelength is 270nm, the flow rate is 1.0mL/min, and the mobile phase is acetonitrile: water: triethylamine: glacial acetic acid=27:70.6:1.6:0.78.
The quality control method (4) for the lotus leaf extract comprises the following steps:
Sample solution preparation: weighing a certain amount of lotus leaf extract, dissolving with 50% methanol water, diluting to a proper concentration, filtering, and collecting filtrate;
liquid chromatography conditions: gradient elution is carried out by using a Waters BEH C18 chromatographic column with a column temperature of 30 ℃ and a flow rate of 0.2ml/min, using 0.1% formic acid water as a mobile phase A and acetonitrile as a mobile phase B according to the following table;
| Time (min) | Flow rate (mL/min) | A(％) | B(％) |
| 0.00 | 0.200 | 98.0 | 2.0 |
| 5.00 | 0.200 | 98.0 | 2.0 |
| 15.00 | 0.200 | 60.0 | 40.0 |
| 20.00 | 0.200 | 0.0 | 100.0 |
| 21.00 | 0.200 | 0.0 | 100.0 |
| 22.00 | 0.200 | 98.0 | 2.0 |
| 25.00 | 0.200 | 98.0 | 2.0 |
And (3) detection: and (5) taking a sample solution, and injecting the sample solution into a UPLC-Q-TOF-MS instrument for measurement.
Preferably, the mass spectrometry conditions are: ESI ion source, respectively collecting data in negative and positive ion modes, scanning molecular weight range: 50-1500Da, collision gas: argon gas, collision energy: capillary voltage 20-40V: (-) 2.5kV, (+) 3.0kV, taper hole voltage: 40V, source temperature: 120 ℃, atomizing gas: nitrogen, desolventizing gas: nitrogen, desolventizing gas temperature: 400 ℃, taper hole air flow: 50L/h, desolventizing gas flow: 700L/min.
In a third aspect, the present invention provides an application of the lotus leaf extract in the first aspect, wherein the lotus leaf extract is applied to making food, health care products and medicines.
The invention has the following beneficial effects:
(1) The lotus leaf extract provided by the invention is extracted by water only, and the preparation method is simple, safe and environment-friendly; the obtained extract is healthier, more comprehensively preserves the water-soluble components in the lotus leaves, and keeps the consistency with the components of the traditional lotus leaf standard decoction;
(2) The lotus leaf extract provided by the invention has stable property, is consistent with the efficacy of the traditional lotus leaf standard decoction, and can be conveniently used as raw materials for preparing products such as medicines, health-care products, foods and the like;
(3) The invention also provides a quality control method of the lotus leaf extract, and the components of the obtained lotus leaf extract can be comprehensively detected and the quality can be comprehensively controlled by adopting various detection means and methods.
Drawings
Fig. 1 is a mass spectrum of a lotus leaf extract according to one embodiment of the present invention.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments.
The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications of the relevant product. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1: preparation method of lotus leaf extract
Taking lotus leaves, removing impurities, cutting up, adding water, and decocting for three times, wherein each time is 1 hour, and the feed-liquid ratio is respectively 1: 15. 1: 10. 1:10, combining the filtrates, concentrating into thick paste, and then drying under reduced pressure to obtain the lotus leaf extract.
Example 2: preparation method of lotus leaf solid beverage
Pulverizing the lotus leaf extract prepared in example 1, sieving, adding anhydrous citric acid, crystalline fructose, maltodextrin, sodium carboxymethylcellulose and aspartame, mixing, sieving with ultrasonic vibration sieve, mixing, packaging, and packaging.
Example 3: method for measuring total alkaloid content in lotus leaf extract
1. Reagent preparation
Bromocresol green solution at ph 5.0: weighing 5g of potassium hydrogen phthalate, adding 450mL of water, adjusting the pH to 5.0 by using 0. mol.L -1 sodium hydroxide, adding 0.12g of bromocresol green, fully dissolving, and adding a proper amount of chloroform for presaturation for later use.
2. Preparation of control solution
4.5Mg of nuciferine reference substance (purchased from Chinese food and drug verification institute: 111566-201304, content of 99.9%) is precisely weighed in a 10mL volumetric flask, dissolved by adding a proper amount of chloroform, and scaled up, and shaken uniformly to prepare reference substance stock solution. Precisely measuring 1mL of the stock solution in a 10mL volumetric flask, adding a proper amount of chloroform for dilution, fixing the volume to a scale, and shaking uniformly to prepare a reference working solution.
3. Preparation of sample solutions
Precisely weighing two parts of the lotus leaf extract dry powder of the example 1, placing the two parts into 250mL conical flasks with plugs, respectively and tightly adding 50mL of 75% ethanol, carrying out ultrasonic extraction for 30min at 50 ℃, cooling, filtering, and obtaining subsequent filtrate.
4. Drawing of a Standard Curve
Precisely measuring nuciferine reference substances 0,0.5,1,2mL in a colorimetric tube, evaporating in water bath at 60deg.C, adding 4mL of bromocresol green solution with pH of 5.0, chloroform 6mL, sealing, shaking vigorously for 2min, transferring to a separating funnel, standing for 1 hr, separating chloroform layer, taking the first solution as blank, and measuring absorbance at 413 nm. And (3) carrying out regression analysis by taking the concentration as an abscissa and the absorbance as an ordinate, and drawing a standard curve.
5. Determination of sample content
1ML of the sample solution is precisely measured in a colorimetric tube, the absorbance is measured at 413nm in the same way as other operations of 1.4, and the content is calculated by taking into a standard curve.
Example 4: method for detecting nuciferine content in lotus leaf extract
1. Liquid phase conditions
The detection wavelength was 270nm, the flow rate was 1.0mL/min, and the mobile phase was acetonitrile using a C18 column: water: triethylamine: glacial acetic acid=27:70.6:1.6:0.78.
2. Preparation of control solution
Taking a proper amount of nuciferine reference substance, precisely weighing, and adding methanol to prepare a solution containing 15 mug per 1 mL.
3. Sample (solution) preparation
Taking 0.5g of the lotus leaf extract dry powder in the embodiment 1, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, weighing, ultrasonically oscillating for 30min, cooling to room temperature, supplementing the reduced weight with methanol, shaking uniformly, filtering, precisely measuring 5mL of filtrate, placing in a volumetric flask with 25mL of volume, adding water to a scale mark, and shaking uniformly to obtain the lotus leaf extract.
4. Determination of samples
Precisely sucking 20 μl of each of the control solution and the sample solution, and measuring the content by external standard method.
Example 5: method for analyzing components in lotus leaf extract by UPLC-Q-TOF-MS
1. Liquid phase conditions
Chromatographic column: waters BEH C18 (2.1 x 150mm,1.7 μm)
Column temperature: 30 DEG C
Mobile phase: a-0.1% formic acid water; b-acetonitrile
Gradient elution conditions:
2. mass spectrometry conditions
Ion source: ESI (electronic service provider interface)
Mode: negative/positive ions, sensitivity
Scanning molecular weight range: 50-1500Da
Collision gas: argon gas
Collision energy: 20-40V
Capillary voltage: (-) 2.5kV, (+) 3.0kV
Taper hole voltage: 40V
Source temperature: 120 DEG C
Atomizing gas: nitrogen gas
Desolventizing gas: nitrogen gas
Desolventizing gas temperature: 400 DEG C
Taper hole air flow: 50L/h
Desolventizing gas flow: 700L/min.
3. Sample pretreatment
A certain amount of sample is weighed, dissolved and diluted to a proper concentration by 50% methanol water, then the solution is filtered by a 0.22 mu m filter membrane to a liquid phase vial, and the solution is injected into a UPLC-Q-TOF-MS instrument for measurement.
4. Detection result
The detection results are shown in FIG. 1. The components of lotus leaf extract which show peaks in mass spectrum are analyzed, and the list is as follows:
Table 1 list of lotus leaf extract ingredients
Example 6: method for detecting other components in lotus leaf extract
1. Detection of total flavone content
After fully mixing the samples, weighing a certain amount of samples, dissolving the samples in 50% methanol, weighing 20mL, placing the samples in a 50mL volumetric flask, adding water to a scale, shaking the samples uniformly, transferring a certain volume of the sample solution into a 25mL volumetric flask, adding water to 6mL, adding 1mL of 5% sodium nitrite solution, shaking the samples uniformly, placing the samples for 6min, adding 1mL of 10% aluminum nitrate solution, shaking the samples uniformly, placing the samples for 6min, adding 10mL of 4.3% NaOH solution, adding water to the scale, shaking the samples uniformly, placing the samples for 15min, and measuring the absorbance A at 500 nm. (rutin is a reference substance)
2. Total sugar content detection (phenol sulfuric acid method)
After fully and uniformly mixing the samples, weighing a certain amount of samples, dissolving the samples in pure water, diluting the solution to a proper concentration, taking 1mL of to-be-measured solution into a 25mL test tube with a plug, adding 1mL of 5% phenol solution, gently shaking the solution uniformly, sucking 5mL of concentrated sulfuric acid by a pipette, quickly blowing in and shaking the solution uniformly by using an ear-washing ball, boiling the solution in a boiling water bath for 5min, quickly placing the solution in a cold water bath after taking out the solution, cooling the solution to room temperature, and measuring the absorbance value A at 485 nm. (glucose as a control)
3. Glucose, fructose and sucrose content detection
And detecting by a UPLC-ELSD method.
4. Content detection of quercetin, kaempferol and isorhamnetin
HPLC-UV detection.
5. Amino acid content detection
HPLC-UV detection.
6. Procyanidine content detection
And detecting by an ultraviolet spectrophotometry.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (7)
1. A method for preparing lotus leaf extract, which is characterized by comprising the following steps:
Taking lotus leaves, removing impurities, cutting, adding water, and decocting for 1-3 times, wherein each time is 0.5-2 hours, and each time the feed-liquid ratio is 1:5-20, filtering, mixing filtrates, concentrating into thick paste, and drying to obtain folium Nelumbinis extract.
2. A method for preparing lotus leaf extract according to claim 1, characterized in that:
the times of water adding and decoction are 3 times, each time is 1 hour, and the feed liquid ratio of each time is 1: 15. 1:10. 1:10.
3. A method for preparing lotus leaf extract according to claim 1, characterized in that:
The components of the lotus leaf extract comprise nuciferine, nornuciferine, lotus plumule quaternary ammonium base, quercetin, kaempferol, isorhamnetin, saccharides, amino acids and procyanidins.
4. A method of preparing a lotus leaf extract according to any one of claims 1 to 3, characterized by further comprising the following detection of the lotus leaf extract:
(1) Detecting the total alkaloid content;
(2) Detecting the nuciferine content;
(3) Detecting the total flavone content;
(4) UPLC-Q-TOF-MS detection,
Wherein, the total alkaloid content detection comprises the following steps:
And (3) preparation of a reagent: preparing bromocresol green solution with pH of 5.0;
preparing a reference substance solution: taking nuciferine reference substance, and adding chloroform to prepare reference substance solution;
preparation of test solution: extracting folium Nelumbinis extract with solvent, separating solid from liquid, and collecting liquid to obtain sample solution;
drawing a standard curve: taking 4 parts of nuciferine reference substance solutions with different amounts, respectively adding reagents to prepare solutions, measuring absorbance, and drawing a standard curve;
Determination of sample content: measuring absorbance of the sample solution, taking the sample solution into a standard curve to calculate content,
The total alkaloid content detection comprises the following steps:
preparation of test solution: the solvent is 75% ethanol, the solid-liquid separation method is ultrasonic extraction at 50 ℃,
The nuciferine content detection method comprises the following steps:
preparing a reference substance solution: taking nuciferine reference substance, adding solvent to prepare reference substance solution;
Sample solution preparation: extracting folium Nelumbinis extract with solvent, and separating solid from liquid to obtain test solution;
Chromatographic conditions: according to high performance liquid chromatography, a chromatographic column using octadecylsilane chemically bonded silica as filler, and acetonitrile: water: triethylamine: the glacial acetic acid is used as a mobile phase for isocratic elution;
and (3) measuring: taking reference substance solution and sample solution, respectively injecting into chromatograph, measuring content according to external standard method,
The nuciferine content detection comprises the following steps:
The specific preparation process of the reference substance solution comprises the following steps: taking a proper amount of nuciferine reference substance, precisely weighing, and adding methanol to prepare a solution containing 15 mug per 1 mL;
The preparation method of the sample solution comprises the following specific steps: taking 0.5g of lotus leaf extract dry powder, precisely weighing, placing into a conical flask with a plug, precisely adding 50mL of methanol, weighing, performing ultrasonic treatment for 30min, cooling to room temperature, supplementing the reduced weight with methanol, shaking uniformly, filtering, precisely measuring 5mL of filtrate, placing into a volumetric flask with 25mL of filtrate, adding water to a scale mark, and shaking uniformly;
Chromatographic conditions, comprising: the detection wavelength is 270nm, the flow rate is 1.0mL/min, and the mobile phase is acetonitrile: water: triethylamine: glacial acetic acid=27:70.6:1.6:0.78,
The UPLC-Q-TOF-MS detection comprises the following steps:
sample solution preparation, comprising: weighing a certain amount of lotus leaf extract, dissolving with 50% methanol water, diluting to a proper concentration, filtering, and collecting filtrate;
Setting liquid chromatography conditions, including: gradient elution was performed using a Waters BEH C18 column at 30 ℃, flow rate 0.2ml/min, 0.1% formic acid as mobile phase a, acetonitrile as mobile phase B, according to the following table:
Detecting, including: taking a sample solution, injecting the sample solution into a UPLC-Q-TOF-MS instrument for measurement,
Wherein the mass spectrometry conditions are set as follows: ESI ion source, respectively collecting data in negative and positive ion modes, scanning molecular weight range: 50-1500Da, collision gas is argon, collision energy is 20-40V, capillary voltage is (-) 2.5kV and (+) 3.0kV, taper hole voltage is 40V, source temperature is 120 ℃, atomization gas is nitrogen, desolvation gas temperature is 400 ℃, taper hole gas flow is 50L/h, and desolvation gas flow is 700L/min.
5. A method of preparing a lotus leaf extract as defined in claim 4, further comprising performing at least one of the following tests on the lotus leaf extract:
(5) Detecting the moisture content;
(6) Detecting the total sugar content;
(7) Detecting the content of glucose, fructose and sucrose;
(8) Detecting the content of quercetin, kaempferol and isorhamnetin;
(9) Detecting ash content;
(10) Detecting the amino acid content;
(11) And detecting the content of procyanidine.
6. A lotus leaf extract according to any one of claims 1 to 5.
7. Use of a lotus leaf extract according to claim 6 in the manufacture of a product which is one of a food product, a health product and a pharmaceutical product.
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