CN111138360B

CN111138360B - Application of SCnA in extraction of nuciferine

Info

Publication number: CN111138360B
Application number: CN202010023084.6A
Authority: CN
Inventors: 任晓亮; 王萌; 孙立丽; 李承浩; 刘虹; 刘亚男; 王丽芝; 姜苗苗
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2020-01-09
Filing date: 2020-01-09
Publication date: 2021-07-09
Anticipated expiration: 2040-01-09
Also published as: CN111138360A

Abstract

The invention relates to application of SCnA in nuciferine extraction. SCnA has the general formula IIn the structure shown, n is between 4 and 8. The invention firstly utilizes the selective bonding effect of macrocyclic molecule sulfonated calixarene (especially SC6A) on nuciferine to carry out extraction and separation. The invention further researches the preferable scheme of the water-soluble macrocyclic molecules for extracting, separating and enriching nuciferine in lotus leaf medicinal materials, the method is simple, fast and environment-friendly, the extraction and purification efficiency is higher than that of the existing known method, and the method can provide technical support for clinical application development of nuciferine and development and production of related medical and health-care medicines and foods.

Description

Application of SCnA in extraction of nuciferine

Technical Field

The invention relates to the technical field of traditional Chinese medicine extraction, in particular to application of SCnA in extraction of nuciferine.

Background

The folium Nelumbinis is leaf of Nelumbo nucifera Gaertn of Nelumbo of Nymphaeaceae. The lotus leaves have been planted in China for more than 3000 years, are bitter and astringent in taste and are mild in nature. Lotus leaves have been regarded as a plant widely used in the edible and medicinal fields and have been valued by people since ancient times. In the aspect of eating, people can prepare lotus leaves and lotus flowers into tea as a nourishing beverage. In the aspect of medicine, the record of 'being taken by lotus leaves and being thin and inferior' is recorded as early as the compendium of the herbal medicine, and the lotus leaves have the functions of clearing heat and promoting diuresis, raising the hair and clearing yang, reducing fat and losing weight, stopping bleeding and dissipating blood stasis and the like. The main effective component of the lotus leaf is nuciferine, which is an aporphine alkaloid, and pharmacological research proves that the nuciferine is the main lipid-lowering component in the lotus leaf. In recent years, researches prove that nuciferine also has the effects of reducing blood sugar, resisting tumors, resisting viruses and resisting inflammation. However, nuciferine itself is less hydrophilic and therefore less soluble in water, and is less extractable by conventional water extraction methods and less efficient. The existing nuciferine extraction method mainly comprises the following steps: the traditional methods of reflux extraction, solvent extraction, acid water extraction, enzyme extraction, Soxhlet extraction, ultrasonic extraction and the like have unsatisfactory extraction effect.

SCnA is a macrocyclic molecule sulfonated calixarene, and water-soluble sulfonated calixarene often adopts a cup-like configuration in aqueous solution. The development of the application field for SCnA is under study.

The structural formula of SCnA is shown in formula I:

disclosure of Invention

Based on the defects, the invention firstly provides the application of the SCnA for extracting the nuciferine so as to expand the application field of the SCnA, wherein the SCnA has a structure shown in a general formula I.

The use according to the invention, preferably, in formula I, n is between 4 and 8, more preferably 6.

The nuciferine is proved to have extremely strong solubility in an aqueous solution of SCnA (particularly n is between 4 and 8), particularly SC 6A. It can be seen that SCnA has a solubilizing effect on nuciferine, SC6A has the strongest solubilizing effect on nuciferine, and the solubility in an aqueous solution can be more than 100 times.

The invention also provides a method for extracting nuciferine from lotus leaves, which takes SCnA as an extraction solvent.

Through verification, the method selects the SCnA as the extraction solvent to be beneficial to the extraction and separation of the nuciferine.

In the method of the present invention, preferably, the SCnA, n is between 4 and 8, more preferably 6.

The method of the present invention preferably comprises the following steps:

extraction: mixing the lotus leaves with the extraction solvent, and extracting by one of a cold soaking method and a reflux extraction method to obtain an extracting solution;

more preferably a cold dip process.

The cold soaking method has the advantages of low cost and simple and convenient operation. Through verification, the nuciferine can be well extracted by methods such as reflux extraction and the like, but the cold leaching method has outstanding advantages due to multiple factors such as comprehensive cost, extraction efficiency and the like.

Preferably, the lotus leaves and the extraction solvent (SCnA) are mixed and then are soaked for 0.2 to 2 hours; more preferably 1-2 hours.

Through verification, the better extraction efficiency can be achieved after the cold soaking is carried out for 1-2 hours.

In the method, the weight ratio of the SCnA to the lotus leaf is preferably (0.025-3): 1; more preferably (0.4-2): 1, most preferably 1.5: 1.

The extraction rate of nuciferine can be improved better by adopting the proportion. When the mass ratio of SC6A to lotus leaf is 0.4:1, the extraction rate of nuciferine reaches about 80 percent, so that the general production requirement can be met; and after the mass ratio of SC6A to lotus leaf is further improved, the extraction rate of nuciferine is continuously improved, and when the mass ratio of nuciferine to lotus leaf reaches 1.5:1, the extraction rate approaches 100%.

The method of the present invention, preferably, the SCnA is provided as an aqueous solution; in the SCnA aqueous solution, the mass-to-volume ratio of the SCnA to the water is (0.5-2) g: (10-40) mL;

more preferably (0.75-2) g: (15-30) mL (most preferably 1.5 g: (15-20) mL).

The proper feed-liquid ratio is helpful for dissolving out nuciferine during extraction.

For explanation and explanation, the above "weight ratio of SCnA to lotus leaf" means the ratio of the weight of SCnA to the weight of lotus leaf, not the ratio of the weight of an aqueous solution of SCnA to the weight of lotus leaf.

In the method, preferably, the mass-volume ratio of the lotus leaves to the water is 1 g: (10-30) mL; more preferably 1 g: 20 mL. The proper feed-liquid ratio of the lotus leaves to the water is favorable for the extraction rate of the nuciferine.

The method of the present invention, preferably, further comprises, after the step of extracting, a step of dissociating:

dissociation: adjusting the pH value of the extracting solution to be not less than 10 to obtain dissociation solution;

more preferably, the pH is adjusted to 10-12; most preferably 12;

or the like, or, alternatively,

dissociation: and adding dichloromethane into the extracting solution, and extracting the organic layer to obtain a dissociation solution.

The method can realize 100% extraction and dissociation of nuciferine.

The method according to the invention, preferably after the step of extracting or after the step of dissociating, further comprises a step of drying:

and (3) drying: and (3) performing one drying mode selected from freezing, decompression and spray drying on the extracting solution or the dissociation solution to obtain the solid extract.

More preferably, the drying is freeze drying.

The method of the present invention, preferably, further comprises a step of secondary dissociation after the step of drying:

secondary dissociation: adding methanol into the obtained solid, performing ultrasonic treatment, filtering, and spin-drying the filtrate.

By combining the technical scheme, the nuciferine can be rapidly dissociated by separating from the liquid and drying, so that the high-purity nuciferine is obtained.

As will be appreciated by those skilled in the art, centrifugation and filtration may be performed as desired. Preferably, the extraction step or dissociation step is followed by centrifugation and filtration, wherein the centrifugation conditions are 6000-8000rpm for 10-15min, and the filtration membrane specification of the filtration is 0.1-0.22 μm.

As a preferred technical scheme of the invention, the method for extracting nuciferine from lotus leaves comprises the following steps:

(1) an aqueous SC6A solution was prepared with a weight to volume ratio of SC6A to water of 1.5 g: (15-20) mL;

(2) weighing folium Nelumbinis fine powder, sieving with No. 5 sieve, adding the SC6A water solution with the weight ratio of SC6A and folium Nelumbinis of (1.5-2):1, cold soaking for 1h, filtering the extractive solution, centrifuging (8000rpm,10min), filtering with 0.22 μm filter membrane, and collecting the filtrate;

more preferably, it comprises:

(2) taking lotus leaf fine powder, sieving with a No. 5 sieve, adding the SC6A aqueous solution, wherein the weight ratio of SC6A to lotus leaf is (1.5-2):1, cold soaking for 1h to obtain an extracting solution, and performing dissociation on the extracting solution; filtering to obtain dissociation solution, centrifuging (8000rpm,10min), filtering with 0.22 μm filter membrane, and collecting filtrate.

Still further, comprising:

(2) taking lotus leaf fine powder, sieving with a No. 5 sieve, adding the SC6A aqueous solution, wherein the weight ratio of SC6A to lotus leaf is (1.5-2):1, cold soaking for 1h to obtain an extracting solution, and performing dissociation on the extracting solution; filtering the obtained dissociation solution, centrifuging (8000rpm,10min), filtering with 0.22 μm filter membrane, collecting the filtrate, and drying.

Still more preferably, after the drying step of step (2), a secondary dissociation step may be further included.

The invention firstly utilizes the selective bonding effect of macrocyclic molecule sulfonated calixarene (especially SC6A) on nuciferine to carry out extraction and separation. The method is simple, fast and environment-friendly, has higher extraction and purification efficiency than the existing method for extracting and enriching the nuciferine from the known lotus leaf medicinal materials, and can provide technical support for clinical application development of the nuciferine and development and production of related medical and health-care medicines and foods.

Of course, not all of the advantages described above need to be achieved at the same time in the practice of any one product or method of the invention.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing a comparison of peak areas of nuciferine in water, an aqueous solution of beta-CD, SBE-beta-CD, SC6A (concentration: 10 mM);

FIG. 2 is a liquid chromatogram of dissociation of nuciferine from SC 6A;

FIG. 3 is a work curve of nuciferine liquid chromatography assay;

FIG. 4 is a graph showing the comparison of extraction rates of different extraction solvents for the nuciferine extraction process from lotus leaves;

FIG. 5 is a graph comparing the extraction rate of nuciferine from lotus leaves using SC6A as the extraction solvent in different extraction modes;

FIG. 6 is a graph comparing the extraction rates of nuciferine extraction process from lotus leaves using water as the extraction solvent and different extraction methods;

FIG. 7 is a comparison graph of the mass volume ratio of lotus leaves to water for the extraction rate of nuciferine extraction process in medicinal materials;

FIG. 8 is a graph showing the comparison of extraction rates of nuciferine from herbs by different sieves;

FIG. 9 is a graph showing the comparison of extraction rates of nuciferine from herbs at different extraction times in a cold leaching process;

FIG. 10 is a graph comparing the extraction rate of nuciferine from the herbs according to different ratios of lotus leaves to SC 6A.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental instrumentation and reagent sources referred to in the following examples are as follows:

lotus leaf (origin: hunan, lot No. 1807001), nuciferine (vicker scientific and biological limited, tetrachio, lot No. 141113), methanol (sigma, usa), acetonitrile (sigma, usa), dian water, triethylamine, sulfonated calixarene (tokyo chemical co., ltd.), sulfobutyl ether β -cyclodextrin (shandong zhiyuan scientific and biological limited, lot No. 170304), β -cyclodextrin (shandong zhiyuan scientific and biological limited, lot No. 171011), phosphoric acid.

Shimadzu high performance liquid chromatograph, Cary Eclipse fluorescence spectrometer, ultrasound, magnetic stirrer, pH meter.

Chromatographic conditions for solubilization experiments of various water-soluble macrocyclic high-molecular polymers on nuciferine: a chromatographic column:

c18(4.6 × 250mm, 5 μm), mobile phase: 0.1% aqueous triethylamine solution: acetonitrile 50:50, flow rate; 1 mL/min; detection wavelength: 270 nm; the amount of the sample was 10. mu.L.

Other experimental methods and other experimental apparatuses, which are shown in the following examples, may be used as those known in the art unless otherwise specified.

Example 1

This example provides a method for extracting nuciferine from lotus leaves, using SC6A as the extraction solvent.

The specific method comprises the following steps:

preparing an SC6A aqueous solution: adding 200mg of SC6A into 80mL of water to obtain an SC6A aqueous solution;

taking 4.0g of lotus leaf fine powder, sieving with a No. 5 sieve, adding into 80mL of SC6A aqueous solution obtained above, cold soaking for 1h, taking the extract, filtering, measuring the volume, centrifuging (8000rpm,10min), filtering with a 0.22 μm filter membrane, and taking the subsequent filtrate.

Examples 2 to 12

Examples 2-12 each provide a method for extracting nuciferine from lotus leaves, differing only in comparison to example 1 in that:

the cold soaking extraction is replaced by ultrasonic extraction, and the ultrasonic extraction time is 1, 5, 10, 15, 20, 30, 40, 60, 80, 100 and 120min respectively.

The specific differences are shown in table 1:

TABLE 1

Examples 13 to 23

Examples 13-23 each provide a method for extracting nuciferine from lotus leaves, differing only in comparison to example 1 in that:

the cold soaking extraction is replaced by reflux extraction, and the reflux extraction time is 1, 5, 10, 15, 20, 30, 40, 60, 80, 100 and 120min respectively.

The specific differences are shown in table 2:

TABLE 2

Examples 24 to 25

Examples 24-25 each provide a method for extracting nuciferine from lotus leaves, differing only in comparison to example 1 in that:

the volumes of the adopted water are different, and the volumes of the water are respectively 40mL and 120 mL; and the dosages of the lotus leaf fine powder and the SC6A are kept unchanged.

The specific differences are shown in table 3:

TABLE 3

Examples 26 to 27

Examples 26-27 each provide a method for extracting nuciferine from lotus leaf, differing only in comparison to example 1 in that:

and (2) respectively sieving by a No. 2 sieve and a No. 4 sieve.

The specific differences are shown in table 4:

TABLE 4

Examples 28 to 30

Examples 28-30 each provide a method for extracting nuciferine from lotus leaves, differing only in comparison to example 1 in that:

cold soaking and extracting for 0.5h, 1.5h and 2h respectively.

The specific differences are shown in table 5:

TABLE 5

Examples 31 to 36

Examples 31-36 each provide a method for extracting nuciferine from lotus leaves, differing only in comparison to example 1:

the mass ratio of the SC6A to the lotus leaves is 0.025:1, 0.1:1, 0.2:1, 0.4:1, 0.75:1 and 1.5:1 respectively.

The specific differences are shown in table 6:

TABLE 6

Example 37

This example provides a solubilization experiment for nuciferine, as follows:

weighing 4 parts of nuciferine 2mg, respectively adding into 2mL of aqueous solution containing 10mM of SBE-beta-CD, beta-CD and SC6A and pure water solution, carrying out ultrasonic treatment for 30min in a dark place, repeating the experiment for three times, taking out the supernatant by centrifugation, filtering the supernatant by a 0.22 mu m filter membrane, and carrying out HPLC.

The results are shown in FIG. 1.

The test result shows that the nuciferine is dissolved in 15.2 mu g/mL in water, beta-CD, SBE-beta-CD and SC6A aqueous solution respectively^-1，35.8μg·mL^-1，216.1μg·mL^-1，1723.2μg·mL^-1。

As can be seen from the test results, the solubilization of beta-CD, SBE-beta-CD and SC6A on nuciferine is strongest, and can reach over 100 times of the solubility in the aqueous solution.

The beta-CD is a polysaccharide oligomer, has a hydrophobic internal and hydrophilic external macrocyclic structure, can be combined with a plurality of objects through hydrophobic acting force and van der Waals force, modifies sulfobutyl on polysaccharide hydroxyl to be SBE-beta-CD, increases a sulfonate group as an electrostatic binding site compared with the beta-CD, and has stronger binding capacity with cationic drugs. As the solubilization SBE-beta-CD is stronger than beta-CD, the bonding mode with the nuciferine is mainly electrostatic bonding with the sulfonate. Since SC6A has a large number of sulfonic acid groups and a proper size of the cavity, the sulfonic acid groups have stronger binding capacity with nuciferine and the best solubilizing effect.

Examples 38 to 44

Examples 38-44 provide dissociation experiments for nuciferine, respectively, as follows:

weighing nuciferine 3mg and SC6A15mg, adding 3mL water, ultrasonic treating for 30min, and filtering with 0.45 μm filter membrane to obtain extractive solution.

Adjusting pH of the extractive solution to 2.5, 4, 6, 8, 9, 10, and 12 with NaOH solution, respectively sampling, and filtering with 0.22 μm filter membrane. Specific extraction conditions are shown in table 7.

TABLE 7

Examples	pH value of the extract	Numbering in FIG. 2
			Example 38	2.5	1
Example 39	4	2
			Example 40	6	3
EXAMPLE 41	8	4
			Example 42	9	5
Example 43	10	6
			Example 44	12	7

The results are shown in FIG. 2 (in FIG. 2, (a) shows SC6A, (b) shows nuciferine), and tables 8-1 and 8-2.

TABLE 8-1 amount of dissociation of SC6A under different pH conditions

TABLE 8-2 nuciferine content in solution at different pH

The test results show that as the pH is gradually increased, the dissociation of nuciferine is gradually increased, the content of SC6A is not reduced, the concentration of nuciferine in the solution is reduced, and obvious nuciferine monomer precipitation is visible at the pH9, which indicates that the pH of the alkalinized solution is increased to cause the dissociation of SC6A and nuciferine. In particular, when the pH value was increased from 9 to 10, nuciferine having a low solubility in the alkaline water began to precipitate in a large amount, and it was found that pH10 was a breakthrough point at which SC6A and nuciferine were dissociated. This is because nuciferine is easily protonated under acidic conditions and thus binds to SC6A, and as pH is increased, protonation disappears and deprotonation proceeds, resulting in a decrease in binding. At pH12, the content of free nuciferine in the solution was only 3.1 μ g · mL-1, which was only 1.55% of the initial amount of nuciferine. From this, it was found that almost complete extraction and dissociation of nuciferine could be achieved by one-step extraction with SC6A bonding and elution with alkaline water at pH12, and therefore, optimal dissociation conditions were established at pH12 in an alkaline environment.

Example 45

This example provides a dissociation experiment for nuciferine, the specific method is as follows:

weighing nuciferine 3mg and SC6A15mg, adding water 3mL, ultrasonic treating for 30min, and filtering with 0.45 μm filter membrane to obtain extractive solution. Adding 3mL of dichloromethane solution into the extracting solution, performing ultrasonic treatment for 30min, extracting by using a separating funnel, filtering by using a dichloromethane solution filter membrane with the diameter of 0.22 mu m, and performing HPLC.

863 mu g/mL nuciferine in the dichloromethane solution is measured^-1。

The experimental result shows that when another polar solvent with different solubility from the nuciferine SC6A is added after SC6A is bonded with the nuciferine in the aqueous solution, the nuciferine can be dissociated from SC6A and dissolved in dichloromethane (polar solvent with different solubility), and SC6A is left in water.

Example 46

weighing nuciferine 3mg and SC6A15mg, adding 3mL water, ultrasonic treating for 30min, and filtering with 0.45 μm filter membrane to obtain extractive solution. Freeze drying the extractive solution to obtain solid, adding 3mL methanol, ultrasonic treating for 30min, filtering with 0.22 μm filter membrane, and performing HPLC. The content of nuciferine in the methanol is measured to be 1003.75 mu g/mL^-1。

The test results show that SC6A was freeze-dried to a solid after bonding with nuciferine in aqueous solution. When another polar solvent methanol with different solubility of nuciferine and SC6A is added, nuciferine can be dissociated from SC6A because nuciferine is easily soluble in methanol and SC6A is not soluble in methanol.

Example 47

The present example provides a test for extracting and separating a lotus leaf alkali component group extract from lotus leaves by using SC6A, and the specific method is as follows:

weighing 6 parts of 0.2g lotus leaf medicinal material, adding 4mL of SC6A (mass multiple of 0.15 times) solution into the first 3 parts, adding 4mL of water into the second three parts, performing ultrasonic treatment for 1h, centrifuging at 8000r/min for 10min, filtering the supernatant with 0.45 μm filter membrane into a centrifuge tube, adding NaOH solution into each extracting solution, adjusting pH to 12, centrifuging at 8000r/min for 10min, discarding the supernatant,drying overnight, weighing bottle weight m₁(total material dry weight after SC6A extraction). Redissolving with 5mL methanol, ultrasonic treating for 10min, centrifuging, collecting supernatant, filtering with 0.22 μm filter membrane, drying overnight, and weighing bottle weight m₂(weight of other extracted from SC6A), HPLC was used for sample injection, and analyzed.

The test results are shown in tables 9-1 to 9-3.

TABLE 9-1 Desorption Experimental data for nuciferine component group in folium Nelumbinis extracted by SC6A

TABLE 9-2 HPLC data of desorption experiments for nuciferine fraction from folium Nelumbinis extracted with SC6A

Tables 9 to 3

Purity%_{Extracted nuciferine}/m₁-m₂*100％

The test result shows that the SC6A can be used for extracting the lotus leaf alkali component from the lotus leaf medicinal material, the lotus leaf alkali component accounts for about 0.7-1.0 percent of the dry weight of the medicinal material, and the method is simple and is suitable for the initial separation and extraction of the nuciferine and the further processing of the lotus leaf total alkali product.

Test example 1

The experimental example provides the effect verification of different extraction solvents on the nuciferine extraction process in lotus leaves.

The specific method comprises the following steps:

taking 7.99mg of a lotus leaf standard substance, precisely weighing, placing in a 10mL brown measuring flask, adding methanol to dilute to a scale to obtain a stock solution with the mass concentration of 799 mu g/mL, precisely measuring the stock solution, diluting step by step to obtain a gradient concentration reference substance solution, respectively injecting 10 mu L from low to high concentration, and recording the peak area.

And taking the nuciferine concentration X as an abscissa and the peak area Y as an ordinate to obtain a linear regression equation Y of 29987X-10511 (r of 0.9992). The nuciferine mass concentration is in a good linear relation in the range of 0.7802-399.5 mu g/mL. As shown in fig. 3.

Weighing 2g of lotus leaves (crushed and sieved by a No. 5 sieve), adding water for dissolving, and adding 60mL of water-soluble macrocyclic high molecular polymer beta-CD, SBE-beta-CD and SC6A solution according to 0.05 times (mass times) respectively for reflux extraction. Sampling 1mL at fixed time for 1, 5, 10, 15, 20, 30, 40, 60, 80, 100, 120min, supplementing the same solvent after sampling, centrifuging the extract at 8000rpm, filtering with 0.22 μm filter membrane after 10min, and collecting the filtrate to obtain the sample solution.

The extraction yield/% -, c, was calculated_s×v_s/c×v

Wherein, c_sThe concentration (mug/mL) of nuciferine in the extracting solution is shown; v. of_sThe volume (mL) of each water-soluble macrocyclic high-molecular polymer extracting solution; c is the concentration of nuciferine extracted with methanol (μ g/mL); v is the volume of methanol extract (mL).

The test results are shown in figure 4, and the time for different macrocyclic molecule extractions to reach equilibrium is not particularly different, and the macrocyclic molecules basically reach equilibrium in about 30 min. The extraction rates of the beta-CD, the SBE-beta-CD, the SC6A and water in 30min are respectively 15.83%, 16.75%, 18.42% and 51.93%; and the extraction amount did not change significantly with the passage of time. According to the test results, under the condition of adding equal amount of auxiliary materials, the extraction amount of the SC6A in different water-soluble macrocyclic high molecular polymers to the nuciferine in the lotus leaf medicinal material is the highest.

Test example 2

The experimental example provides a research experiment of different extraction methods on the extraction effect of nuciferine in medicinal materials.

The experimental method is as follows:

3 parts of 2.000g of lotus leaf fine powder (sieved by a No. 5 sieve) are weighed, and 2.000g of lotus leaf fine powder is respectively added into 20mL of SC6A aqueous solution, wherein the mass ratio of the lotus leaf fine powder to SC6A is 1: 0.05.

A single-factor investigation method is adopted, and cold soaking, ultrasonic extraction and reflux extraction are respectively adopted to investigate the extraction efficiency of SC6A applied to nuciferine extraction in lotus leaf medicinal materials.

Sampling 1mL at fixed time for 1, 5, 10, 15, 20, 30, 40, 60, 80, 100 and 120min respectively, supplementing the same solvent after sampling, centrifuging the extract at 8000rpm for 10min, filtering with 0.22 μm filter membrane, collecting the filtrate to obtain the test solution, and inspecting the content of nuciferine in each sample by high performance liquid chromatography. The extraction efficiency of the SC6A for extracting nuciferine under each method is compared.

To better study the extraction efficiency of nuciferine from lotus leaves, the inventors supplemented the experiment of replacing the extraction solvent with water.

The specific results are shown in tables 10-1 and 10-2 and FIGS. 5 and 6.

TABLE 10-1 concentration of nuciferine in folium Nelumbinis extracted with traditional water by different extraction methods

TABLE 10-2 concentration of nuciferine fraction from folium Nelumbinis extracted by different extraction methods SC6A

The obvious observation of extracting nuciferine in different extraction modes shows that the reflux extraction effect is the highest in the traditional water extraction method. However, it was clearly observed after addition of SC6A that the different extraction modes had no significant effect on the amount of nuciferine extracted. From this, it is found that the influence of the SC6A selective binding action is stronger than the influence of the extraction method on the extraction efficiency. The cold leaching method has many advantages such as obvious energy saving and safety, so the method of cold leaching extraction is the best method.

Test example 3

The experimental example provides a research experiment of the effect of different material-liquid ratios on nuciferine extraction in medicinal materials.

The experimental method is as follows:

3 parts of 2.0g lotus leaf fine powder (sieved by a No. 5 sieve) are weighed respectively, and 3 parts of SC6A aqueous solution containing 100mg of SC6A is added respectively to obtain an extracting solution, wherein the difference of the 3 parts of SC6A aqueous solution is as follows: the volumes were 20mL, 40mL, and 60mL, respectively.

Cold soaking the above extractive solutions for 1h, filtering, measuring volume, centrifuging (8000rpm,10min), filtering with 0.22 μm filter membrane, collecting filtrate to obtain sample solution, and analyzing content of each sample by HPLC.

The results are shown in FIG. 7.

TABLE 11 amount of nuciferine component extracted from folium Nelumbinis with different feed-to-liquid ratios SC6A

Test example 4

The experimental example provides a research experiment of the effect of different crushing forms (sieving by different sieves) of raw materials on the extraction of nuciferine in medicinal materials.

The experimental method is as follows: weighing 2.0g of folium Nelumbinis fine powder with different pulverization degrees (respectively sieving with No. 2 sieve, No. 4 sieve and No. 5 sieve), and adding 40mL of SC6A water solution containing 100mg of SC6A to obtain extractive solution; cold soaking for 1h, measuring volume, centrifuging (8000rpm,10min), filtering with 0.22 μm filter membrane, collecting filtrate to obtain sample solution, and analyzing content of each sample by HPLC.

The results are shown in Table 12 and FIG. 8.

TABLE 12 concentration of nuciferine fraction extracted from folium Nelumbinis by different pulverization forms of SC6A

Test example 5

The experimental example provides a research experiment for determining the effect of the difference of extraction time on the extraction of nuciferine in medicinal materials after cold soaking extraction.

The experimental method is as follows:

3 parts of 2.0g lotus leaf fine powder (sieved through a No. 5 sieve) were weighed out, and 40mL of an aqueous SC6A solution containing 100mg of SC6A was added to obtain extracts.

Cold soaking for 0.5h, 1h, 1.5h and 2h respectively, extracting, measuring volume, centrifuging (8000rpm,10min), filtering with 0.22 μm filter membrane, collecting filtrate to obtain sample solution, and analyzing content of each sample by HPLC.

The results are shown in Table 13 and FIG. 9.

TABLE 13 concentration of nuciferine fraction from leaves of Nelumbo nucifera with different extraction times SC6A

According to the experimental result, when the cold soaking time reaches 1 hour, the extraction rate of the nuciferine is basically stable and cannot be continuously increased, so that the experimental cost and effect are integrated, and the cold soaking time of 1 hour is selected as the optimal scheme.

Test example 6

The experimental example provides a research experiment of the effect of different proportions of lotus leaves and SC6A on nuciferine extraction in medicinal materials.

The experimental method is as follows:

weighing 5 parts of 0.2g of lotus leaf fine powder (sieved by a No. 5 sieve), respectively, extracting with 4mL of SC6A aqueous solution containing 5.00mg, 10.00mg, 20.00mg, 40.00mg, 80.00mg, 150.00mg and 300.00mg (the mass ratio of lotus leaf to SC6A is 0.025:1, 0.05:1, 0.1:1, 0.2:1, 0.4:1, 0.75:1 and 1.5:1 respectively), cold soaking for 1h at the material-liquid ratio of SC6A aqueous solution (the mass-volume ratio of SC6A to water) for extraction, centrifuging (8000rpm and 10min), filtering with a 0.22 μm filter membrane, collecting the subsequent filtrate to obtain a sample solution, and analyzing the content of each sample by HPLC.

The results are shown in Table 14 and FIG. 10.

TABLE 14 concentration of nuciferine extracted from folium Nelumbinis at different ratios of SC6A to folium Nelumbinis

From the experimental results, the mass ratio of SC6A to lotus leaf reached 0.4:1, the extraction rate of nuciferine reaches about 80 percent, so that the general production requirement can be met; and after the mass ratio of SC6A to lotus leaf is further improved, the extraction rate of nuciferine is continuously improved, and when the mass ratio of nuciferine to lotus leaf reaches 1.5:1, the extraction rate approaches 100%.

All examples and test examples are combined and summarized as follows: the inventor finds that SC6A has strong capacity for selective extraction, enrichment and resolution of nuciferine through research. Through the investigation of nuciferine monomer components, the study finds that the solubilization of SC6A on nuciferine can reach more than 100 times of the solubility of nuciferine in an aqueous solution, and further, the experiment shows that nuciferine extracted by SC6A selective bonding is extracted and dissociated by using an alkalized aqueous solution, a dosage form organic solvent with large solubility difference such as dichloromethane and methanol. When the pH value is 12, the content of free nuciferine in the solution is only 1.55% of the initial nuciferine amount, the dissociation rate is 98.45%, and almost complete extraction and dissociation of nuciferine can be realized.

On the basis, the best extraction condition for extracting nuciferine from the lotus leaf medicinal material by taking SC6A as an auxiliary material is further considered, and researches show that the influence of the selective bonding effect of the SC6A material is stronger than the influence of an extraction method on the extraction efficiency, so that a cold soaking method can be adopted as the extraction method, and has the advantages of energy conservation, safety and the like. Through a large amount of researches, the optimal condition for extracting the nuciferine by SC6A cold leaching is that the medicinal materials are crushed and screened by a No. 5 sieve, the cold leaching method is adopted, the extraction time is 1 hour, and the ratio of the lotus leaf medicinal materials to water is 1: 20, the ratio of SC6A to the medicinal materials is 1.5: 1.

In addition, the method can be used for extracting and enriching nuciferine component groups in lotus leaf medicinal materials, the lotus leaf alkali components extracted from the lotus leaf medicinal materials by adopting SC6A account for about 0.7% -1.0% of the dry weight of the medicinal materials, the purity of the nuciferine contained in the lotus leaf alkali components is about 10%, compared with the traditional water extraction method, the extraction efficiency after one-step extraction and the total nuciferine purity in the extract are higher than those of the former, and the method is simple and easy to implement.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

The application of the SCnA aqueous solution as an extraction solvent to extract nuciferine is characterized in that SCnA has a structure shown in a general formula I:

wherein n is between 4 and 8;

in the SCnA aqueous solution, the mass-to-volume ratio of the SCnA to the water is (0.5-2) g: (10-40) mL.
2. The use according to claim 1, wherein SCnA has the structure of formula I:

wherein n is 6.
3. A method for extracting nuciferine from lotus leaves is characterized in that an SCnA aqueous solution is used as an extraction solvent;

the SCnA has a structure represented by formula I:

wherein n is between 4 and 8;

in the SCnA aqueous solution, the mass-to-volume ratio of the SCnA to the water is (0.5-2) g: (10-40) mL.
4. The method of claim 3, wherein the SCnA has a structure according to formula I:

wherein n is 6.
5. A method according to claim 3, characterized by the steps of:

extraction: mixing the lotus leaves with the extraction solvent, and extracting by one of a cold soaking method and a reflux extraction method to obtain an extracting solution.
6. The method as claimed in claim 5, wherein the lotus leaves and the extraction solvent are mixed and then cold-soaked for 0.2-2 hours.
7. The method as claimed in claim 6, wherein the lotus leaves and the extraction solvent are mixed and then cold-dipped for 1-2 hours.
8. The method according to claim 3, wherein the weight ratio of SCnA to lotus leaf is (0.025-3): 1.
9. the method according to claim 8, wherein the weight ratio of SCnA to lotus leaf is (0.4-2): 1.
10. the method of claim 8, wherein the weight ratio of SCnA to lotus leaf is (1.5-2): 1.
11. The method according to claim 3, wherein the mass to volume ratio of SCnA to water in the aqueous solution of SCnA is (0.75-2) g: (15-30) mL.
12. The method according to claim 3, wherein the mass to volume ratio of SCnA to water in the aqueous solution of SCnA is 1.5 g: (15-20) mL.
13. The method of claim 5, further comprising, after the step of extracting, the step of dissociating:

dissociation: adjusting the pH value of the extracting solution to be not less than 10 to obtain dissociation solution;

or the like, or, alternatively,

dissociation: and adding dichloromethane into the extracting solution, and extracting the organic layer to obtain a dissociation solution.
14. The method according to claim 13, wherein the pH of the extract is adjusted to 10 to 12 to obtain a dissociation solution.
15. The method according to claim 13, further comprising, after the step of extracting or after the step of dissociating, the step of drying:

and (3) drying: and (3) performing one drying mode selected from freezing, decompression and spray drying on the extracting solution or the dissociation solution to obtain the solid extract.
16. The method of claim 15, further comprising, after the step of drying, the step of secondary dissociation:

secondary dissociation: adding methanol into the solid extract, performing ultrasonic treatment, and filtering.
17. The method of claim 5, comprising the steps of:

(1) an aqueous SC6A solution was prepared with a weight to volume ratio of SC6A to water of 1.5 g: (15-20) mL;

(2) sieving folium Nelumbinis fine powder with No. 5 sieve, adding the SC6A water solution at weight ratio of SC6A and folium Nelumbinis of (1.5-2):1, cold soaking for 1 hr, filtering the extractive solution, centrifuging, filtering with 0.22 μm filter membrane, and collecting the filtrate.
18. The method of claim 17, wherein step (2) is:

taking lotus leaf fine powder, sieving with a No. 5 sieve, adding the SC6A aqueous solution, wherein the weight ratio of SC6A to lotus leaf is (1.5-2):1, cold soaking for 1h to obtain an extracting solution, and dissociating the extracting solution; filtering the obtained dissociation solution, centrifuging, filtering with 0.22 μm filter membrane, and collecting the filtrate.
19. The method of claim 17, wherein step (2) is:

taking lotus leaf fine powder, sieving with a No. 5 sieve, adding the SC6A aqueous solution, wherein the weight ratio of SC6A to lotus leaf is (1.5-2):1, cold soaking for 1h to obtain an extracting solution, and dissociating the extracting solution; filtering the obtained dissociation solution, centrifuging, filtering with 0.22 μm filter membrane, collecting the filtrate, and drying.