CN112480196A - Hypoglycemic component in camellia bee pollen and extraction method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of natural botanical drug preparations, and particularly relates to a hypoglycemic component in camellia bee pollen, and an extraction method and application thereof. The extraction method of the hypoglycemic component in the camellia bee pollen comprises the following steps: degreasing by using n-hexane, extracting by using ethanol solution water bath ultrasound, and purifying and enriching by using SPE small columns; wherein: the feed-liquid ratio of camellia bee pollen to n-hexane is 1g (4-6) mL; the mass concentration of the ethanol solution is 70%, the material-liquid ratio of the camellia bee pollen to the ethanol solution is 1g: (6-10) mL. The extraction method can extract the functional flavonoid compounds in the camellia bee pollen to a great extent. The obtained extract can remarkably inhibit glucose absorption and transport of human intestinal epithelial cells, thereby lowering blood sugar.

Description

Hypoglycemic component in camellia bee pollen and extraction method and application thereof

Technical Field

The invention belongs to the technical field of natural botanical drug preparations, and particularly relates to a hypoglycemic component in camellia bee pollen, and an extraction method and application thereof.

Background

Hyperglycemia is one of the so-called "three highs," which refers to a patient's blood glucose level that persists beyond normal levels for a long period of time. The normal blood glucose value is not higher than 6.1mmol/L in fasting state, and not higher than 7.8mmol/L after two hours of meal. If it is higher than this range, it is called hyperglycemia.

Under normal conditions, the human body can ensure blood sugar balance through hormone regulation and nerve regulation. However, for patients with hypertension, hyperlipidemia, diabetes, obesity, and long-term improper diet, the two major regulatory functions are disordered and hyperglycemia is easily caused. Hyperglycemia easily induces a plurality of complications such as cardiovascular and cerebrovascular diseases, and the like, and seriously threatens human health. Therefore, the development of natural hypoglycemic drugs has important significance for assisting in reducing blood sugar.

Bee pollen is known as a concentrated nutrition library of all natural food, and is rich in various nutritional ingredients and active ingredients. Early researches have found that polysaccharide components, polyphenol compounds and flavonoid compounds in bee pollen have the potential of reducing blood sugar.

For example, CN104998263A discloses a method for obtaining α -glucosidase inhibitors from camellia pollen. The inhibitor inhibits the digestion of ingested polysaccharide, oligosaccharide and disaccharide into glucose, fructose and other monosaccharides by inhibiting the activity of alpha-glucosidase, namely, the aim of reducing blood sugar is achieved by preventing the generation of more monosaccharides.

However, monosaccharides such as glucose present in food can still be absorbed and transported by human intestinal epithelial cells after ingestion, and blood glucose levels in patients can be increased.

Disclosure of Invention

The first purpose of the invention is to provide a method for extracting blood sugar reducing components from camellia bee pollen. The obtained blood sugar reducing component can remarkably inhibit the absorption and transportation of human intestinal epithelial cells on glucose, thereby realizing the blood sugar reducing effect.

The extraction method of the hypoglycemic component in the camellia bee pollen comprises the following steps: degreasing by using normal hexane, and performing ultrasonic extraction by using an ethanol solution in a water bath; finally purifying and enriching by SPE small column; wherein:

the feed-liquid ratio of camellia bee pollen to n-hexane is 1g (4-6) mL;

the mass concentration of the ethanol solution is 70%, the material-liquid ratio of the camellia bee pollen to the ethanol solution is 1g: (6-10) mL.

The extract obtained by the conventional bee pollen extraction process can only be used as an alpha-glucosidase inhibitor, and the reduction of blood sugar is realized by inhibiting the conversion of monosaccharide. However, we found through studies that there are some active ingredients in camellia bee pollen, such as kaempferol-3-O- (6 ' -O-trans-coumaroyl) -glucopyranoside (active ingredient A), kaempferol-3-O- (2',6' -di-O-trans-p-coumaroyl) - β -D-glucopyranoside (active ingredient B), in addition to the known α -glucosidase inhibitors, which have a different hypoglycemic mechanism from that of the inhibitors. Unlike the hypoglycemic action mechanism of the previous inhibitor, the active ingredients can realize the hypoglycemic effect by inhibiting the absorption and transportation of glucose after ingestion by human intestinal epithelial cells.

For this reason, we expect to maximize the availability of this fraction of active ingredient by improving the existing extraction process.

In the research, more active components in camellia bee pollen are extracted by adopting a 95% ethanol routine, but the obtained extracting solution is found to have higher viscosity, and cannot be purified and enriched by adopting an SPE small column; because the extract has more interference components such as saccharides and the like, the content ratio of flavonoid substances in the extract is greatly reduced, and the hypoglycemic effect is difficult to play.

The replacement of an extraction reagent is tried, and although the viscosity of an extracting solution is reduced to a certain degree, the extraction degree of flavonoids is reduced; attempts have also been made to replace the SPE cartridge with ethyl acetate extraction, but the results are still less than ideal.

Through continuous attempts, a technological process of firstly removing grease, then extracting, and finally purifying and enriching is finally provided, and an optimal matching use combination mode is obtained by screening a grease removing reagent and an extracting reagent, so that an extracting solution with low viscosity, high flavonoid content and less interference components is obtained, the operability of purification and enrichment of a subsequent SPE small column is further realized, and the blood sugar reducing effect of the final extract is improved.

Although normal hexane and 70% ethanol are common extraction reagents in the field, the problem that the conventional extraction process cannot adopt SPE (solid phase extraction) small columns for purification and enrichment is solved by selecting normal hexane for degreasing and extracting and compounding with 70% ethanol in the scheme, which is obviously different from the conventional effects of normal hexane and 70% ethanol.

The conditions of the water bath ultrasound are as follows: the power is 60-100 KHz, and the temperature is 50-70 ℃. By controlling this condition, the extraction rate of the hypoglycemic agent can be further improved, and the hypoglycemic effect can be further improved.

The ultrasonic extraction time is 60-90 min, which is determined according to actual conditions.

Preferably, the filler of the SPE cartridge is modified styrene-divinylbenzene polymer Bond Elut-PPL; the specification is 500mg/6 mL. Research shows that compared with other fillers, the Bond Elut-PPL filler is more beneficial to adsorption of flavonoid compounds, so that the enrichment degree of the bee pollen hypoglycemic components (including the active component A and the active component B) is improved, and the bee pollen extract with higher hypoglycemic activity is obtained.

The purifying and enriching comprises the following steps: activating the SPE small column by using 4-5 mL of methanol, and washing the methanol by using 4-5 mL of pure water; before sample loading, residual water in the column is pumped out under pressure so as to play a good purifying and enriching role of the SPE small column. The concentration of active ingredients in the extract obtained after purification and enrichment is higher, and the extract has practical application value.

The sample loading refers to adding camellia bee pollen extracting solution into the SPE small column. In the sample loading process, the flow rate is not too fast, so that the camellia bee pollen extracting solution flows down naturally without pressurization; and after the sample loading is finished, washing the SPE small column by using 4-5 mL of pure water.

Preferably, the SPE cartridge washed by the pure water needs to be eluted by 4-6 times by methanol, 4-5 mL each time, until the cartridge is completely eluted; collecting eluate with small tube, and blowing the collected liquid with nitrogen until methanol is volatilized to obtain yellow extract as blood sugar lowering component of camellia bee pollen.

Preferably, the extraction method further comprises, before the purifying and enriching, subjecting the extraction liquid to rotary evaporation; the conditions of the rotary evaporation are as follows: the rotating speed of the rotary evaporator is 50-60 r/min, and the temperature is 45-50 ℃; the rotary steaming time is determined according to the volume of the residual solution, and the rotary steaming is generally stopped until 20-30% of the volume of the solution is remained, so as to obtain the camellia melissa extracting solution without ethanol.

The second purpose of the invention is to provide the hypoglycemic component in the camellia bee pollen obtained by the extraction method.

The camellia bee pollen contains the components for reducing blood sugar, and comprises the following components:

kaempferol-3-O- (6' -O-trans-coumaroyl) -glucopyranoside, the content of which is at least more than 10.0 mg/g;

kaempferol-3-O- (2',6' -di-O-trans-p-coumaroyl) -beta-D-glucopyranoside, the content of which is at least more than 8.0 mg/g.

The third purpose of the invention is to provide the application of the hypoglycemic component of camellia bee pollen in preparing a hypoglycemic medicament for inhibiting the absorption and transportation of glucose by human intestinal epithelial cells.

The invention has the following beneficial effects:

the extraction method can greatly extract the functional flavonoid compounds in the camellia bee pollen, including the active ingredient A and the active ingredient B. The obtained extract can remarkably inhibit glucose absorption and transport of human intestinal epithelial cells, thereby lowering blood sugar.

Drawings

Fig. 1 is a mass spectrum of active ingredient a and active ingredient B in a positive ion scan mode.

Wherein A1 and A2 are respectively an extracted ion flow graph and a secondary mass spectrogram of an active ingredient A, wherein the active ingredient A in a sample is on the upper graph, and the active ingredient A of a standard product is on the lower graph;

b1 and B2 are respectively an extracted ion flow diagram and a secondary mass spectrogram of the active ingredient A, wherein the active ingredient B in the sample is shown in the upper diagram, and the active ingredient B in the standard product is shown in the lower diagram.

Fig. 2 is a mass spectrum of active ingredient a and active ingredient B in a negative ion scan mode.

Wherein A1 and A2 are respectively an extracted ion flow graph and a secondary mass spectrogram of an active ingredient A, wherein the active ingredient A in a sample is on the upper graph, and the active ingredient A of a standard product is on the lower graph;

b1 and B2 are respectively an extracted ion flow diagram and a secondary mass spectrogram of the active ingredient A, wherein the active ingredient B in the sample is shown in the upper diagram, and the active ingredient B in the standard product is shown in the lower diagram.

FIG. 3 is a graph of the difference in glucose uptake by human intestinal epithelial cells between different treatment groups.

FIG. 4 shows the difference in transport of glucose from the inner compartment to the outer compartment by human intestinal epithelial cells between different treatment groups.

FIG. 5 shows the difference in the absorption of fluorescently labeled glucose by human intestinal epithelial cells between different treatment groups.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The embodiment provides a method for extracting blood sugar reducing components from camellia bee pollen, which comprises the following specific steps:

1) accurately weighing 5.0g of camellia bee pollen powder, adding 20mL of n-hexane for degreasing, filtering to remove n-hexane lipid extract, adding 40mL of 70% ethanol solution according to the material-to-liquid ratio of 1:8, and performing water bath ultrasonic extraction under the conditions of 80KHz power, 60min time and 60 ℃, filtering after extraction and collecting ethanol extract.

2) And (3) carrying out low-pressure rotary evaporation on the ethanol extracting solution to remove the ethanol solvent, setting the rotating speed of a rotary evaporator at 50 rpm and the temperature at 50 ℃, and stopping rotary evaporation when the volume of the residual solution in the rotary evaporation bottle is about 10mL to obtain the camellia bee pollen extracting solution without ethanol.

3) Selecting SPE cartridge with 500mg/6mL specification and modified styrene-divinylbenzene polymer (Bond Elut-PPL) as filler, activating with 5mL of methanol, washing off the methanol with 5mL of pure water, pressurizing and pumping out residual water in the cartridge before loading to play good purifying and enriching effects of the SPE cartridge.

4) Adding camellia bee pollen extracting solution into the SPE small column, wherein the flow rate is not too high in the sample loading process, the camellia bee pollen extracting solution is required to flow down naturally without pressurization, after the sample loading is finished, washing the SPE small column by 5mL of pure water, then eluting by 5 times by methanol, 5mL of methanol each time until the small column is completely eluted, collecting the eluent by a small tube, and blowing nitrogen to the collecting solution until the methanol is completely volatilized to obtain a yellow extract which is the blood sugar reducing component of the camellia bee pollen.

The hypoglycemic component of camellia bee pollen obtained in the embodiment is subjected to ultra performance liquid chromatography-tandem quadrupole-time-of-flight-high resolution mass spectrometry, wherein the content of an active component A in the hypoglycemic component of the camellia bee pollen is 10.0mg/g of extract, and the content of an active component B in the hypoglycemic component of the camellia bee pollen is 8.0mg/g of extract, and mass spectrograms of the active components A and B are shown in fig. 1 and fig. 2.

Test example 1:

this test example was conducted to examine the hypoglycemic effect of the hypoglycemic components of camellia bee pollen obtained in example 1.

The specific detection steps are as follows:

(1) establishing an intestinal epithelial cell transport model: human intestinal epithelial cells, i.e., Caco-2 cells, were treated at 1X10⁵One cell/well was seeded on a Transwell plate (12 cells per plate, 0.4 μm pore size polycarbonate membrane at the bottom of the inner chamber, 1.12cm membrane area²) The culture solution in the inner chamber is 0.5 mL/hole, the culture solution in the outer chamber is 1.5 mL/hole, the culture solution is updated every 1 day, the resistance value of a monolayer Caco-2 cell formed in each chamber is detected by using a Millicelle ERS-2 cell resistance meter before the culture solution is updated every time, and the resistance value exceeds the resistance value of a blank chamber (namely the chamber without inoculated cells) by 300 omega continuously until the resistance value is continued for 18-21 days, so that the establishment of an intestinal epithelial cell transport model is successful.

(2) Grouping tests: the 12 wells of the Transwell plate were set to 2 blank cells, 5 control cells, 5 sample cells.

Blank, 2, no cell-seeded chamber, and no glucose administration and sample treatment;

control group, 5, cells inoculated with Caco-2 cells, and glucose treatment was performed to give a glucose concentration of 2 mg/mL;

sample group, 5, cells inoculated with Caco-2 cells, and simultaneously subjected to glucose administration treatment and sample treatment, glucose concentration was 2mg/mL, sample concentration was 100. mu.g/mL, and the sample was the hypoglycemic component of camellia bee pollen obtained in example 1.

(3) The test steps are as follows:

1. when the intestinal epithelial cell transport model was successfully established, the culture medium in the 12 cells of the Transwell well plate was replaced with HEPES buffer solution consisting of 20mM HEPES, 137mM NaCl, 4.7mM KCl, 1.8mM CaCl₂And 1.2mM MgSO₄And (4) forming. Then, the test treatment is carried out according to the test grouping condition.

2. After incubation for 2h, the outer and inner chamber solutions were collected separately and assayed. The chamber was then washed 2 times with pre-chilled PBS buffer, 500 μ L/well of 0.1M NaOH was added to the chamber and incubated at 37 ℃ for 30min to lyse the cells on the inner chamber membrane, and the cell lysate was collected and assayed.

3. And (4) carrying out glucose content detection on the collected outer chamber solution, inner chamber solution and cell lysate by using a glucose detection kit.

4. Placing a cell slide (the inner diameter of the slide is 24mm) at the center of each well in a 6-well plate, and placing Caco-2 cells at 1x10⁵Uniformly inoculating each well with one/mL, setting 3 wells as a control group, namely adding 200 mug/mL Fluorescein Isothiocyanate (FITC) labeled glucose; 3 wells were used as a sample group, to which 200. mu.g/mL FITC-labeled glucose and 100. mu.g/mL of a sample, which is a hypoglycemic component of camellia bee pollen obtained in example 1, were added simultaneously.

5. After incubation for 2h, the cell slide was taken out and placed in a new 6-well plate, washed 3 times with pre-cooled PBS buffer solution, then fixed for 30min with pre-cooled methanol acetone (volume ratio 1:1) mixed solution, washed 3 times with pre-cooled PBS buffer solution, then stained for 5min with 4', 6-diamidino-2-phenylindole (DAPI), and finally observed and photographed with a Leica laser confocal microscope.

(4) And (3) test results:

the glucose detection results show that:

the glucose concentration of the sample group external chamber solution is obviously lower than that of the control group external chamber solution, and the average inhibition rate reaches 53.6 percent (shown in figure 3), thereby reflecting that the sample has obvious effect of inhibiting the glucose transport of human intestinal epithelial cells;

the glucose concentration of the cell lysate of the sample group is obviously lower than that of the cell lysate of the control group, and the average inhibition rate reaches 30.8 percent (as shown in figure 4), so that the sample has obvious effect of inhibiting the absorption of the glucose by the human intestinal epithelial cells;

the results of the observation pictures under the laser confocal microscope show that:

the fluorescence signal of the cells in the sample group is significantly lower than that of the cells in the control group (as shown in fig. 5), which also reflects that the sample has significant effect of inhibiting the absorption of glucose by the human intestinal epithelial cells.

Comparative example 1:

this comparative example provides a comparison of the hypoglycemic activity of the camellia bee pollen obtained in example 1 with 2 known flavonoids (i.e., phloretin and phlorizin) which lower blood glucose by inhibiting the absorption and transport of glucose by human intestinal epithelial cells.

The method comprises the following specific steps:

(1) after constructing the intestinal epithelial cell transport model, the culture medium in the 12 cells of the Transwell plate was replaced with HEPES buffer solution. Then, test treatment is carried out according to test grouping conditions:

a control group, in which 3 chambers, chambers inoculated with Caco-2 cells, and glucose-administration treatment were set to give a glucose concentration of 2 mg/mL;

a sample group in which 3 chambers were set, a chamber inoculated with Caco-2 cells, and glucose administration treatment and sample treatment were simultaneously performed, the glucose administration concentration was 2mg/mL, and the sample concentration was 100. mu.g/mL; the sample is the hypoglycemic component of camellia bee pollen obtained in example 1.

A phloretin group, which was set to 3 chambers, was inoculated to the chamber of Caco-2 cells, and was subjected to simultaneous glucose administration treatment at a glucose concentration of 2mg/mL and phloretin concentration of 1. mu.g/mL;

a phlorizin group, which was set to 3 chambers, was inoculated with the chambers of Caco-2 cells, and was subjected to simultaneous glucose administration treatment at a glucose concentration of 2mg/mL and phlorizin treatment at a phlorizin concentration of 1. mu.g/mL;

(2) the outer and inner chamber solutions were collected after 2h incubation, respectively. The inner chamber was then washed 2 times with pre-cooled PBS buffer, and then 500. mu.L/well of 0.1M NaOH was added to the chamber and incubated at 37 ℃ for 30min to lyse the cells on the inner chamber membrane, and the cell lysate was collected. And (4) carrying out glucose content detection on the collected outer chamber solution, inner chamber solution and cell lysate by using a glucose detection kit.

After comparison, the hypoglycemic component of camellia bee pollen obtained in example 1 was found to be equivalent to the inhibitory ability of phloretin and phlorizin administered at a concentration of 1 μ g/mL to glucose absorption and transport in human intestinal epithelial cells at an administration concentration of 100 μ g/mL (as shown in fig. 3 and 4).

Therefore, the hypoglycemic component of the camellia bee pollen provided by the invention can play a remarkable role in reducing blood sugar, and has a wide application prospect in the field of preventing and treating hyperglycemia.

Comparative example 2

The difference from the extraction process of example 1 is that: directly extracting camellia bee pollen without degreasing, wherein the extraction reagent is ethanol with the mass concentration of 95%. As a result, it was found that:

the camellia bee pollen extracting solution which is obtained after rotary steaming and does not contain ethanol has higher viscosity, and SPE column processing can not be carried out, so that the interference components in the extract are more, and the activity of reducing blood sugar is obviously reduced by at least more than 50%.

Comparative example 3

The difference from the extraction process of example 1 is that: the ethanol-free camellia bee pollen extract obtained by the steps 1) and 2) in the example 1 is not processed by SPE column, but extracted by ethyl acetate, and the specific operation is as follows:

adding 40mL of ethyl acetate into the camellia bee pollen extracting solution without ethanol obtained in the step 1) and the step 2) of the embodiment 1, performing ultrasonic extraction in a water bath under the condition of 80KHz power for 60min at the temperature of 60 ℃, standing for 30min, taking supernate, and drying by nitrogen to obtain a yellow extract.

The obtained yellow extract is re-dissolved in methanol, and subjected to ultra performance liquid chromatography-tandem quadrupole-time of flight-high resolution mass spectrometry.

The content of the active ingredient A and the active ingredient B in the obtained extract is obviously reduced by about 20 percent compared with the content of the active ingredient A and the active ingredient B in the extract obtained in example 1.

Meanwhile, the extract obtained in comparative example 2 was subjected to cell tests according to the procedure described in test example 1, and it was found by comparison that the hypoglycemic activity of the extract obtained in comparative example 2 was significantly reduced by about 20% as compared to that of the extract obtained in example 1.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may 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 (9)

1. An extraction method of components for reducing blood sugar in camellia bee pollen is characterized in that n-hexane is used for degreasing, then ethanol solution is used for water bath ultrasonic extraction, and finally SPE small columns are used for purification and enrichment; wherein:

the feed-liquid ratio of camellia bee pollen to n-hexane is 1g (4-6) mL;

the mass concentration of the ethanol solution is 70%, the material-liquid ratio of the camellia bee pollen to the ethanol solution is 1g: (6-10) mL.

2. The extraction method of hypoglycemic components in camellia bee pollen according to claim 1, wherein the water bath ultrasonic conditions are as follows: the power is 60-100 KHz, and the temperature is 50-70 ℃.

3. The method for extracting hypoglycemic components from camellia bee pollen as claimed in claim 2, wherein the filler of the SPE cartridge is Bond Elut-PPL.

4. The method for extracting hypoglycemic components from camellia bee pollen according to claim 3, wherein the SPE cartridge is activated by 4-5 mL of methanol, and then washed by 4-5 mL of pure water; before loading, the residual water in the column is pumped out under pressure.

5. The method for extracting hypoglycemic components in camellia bee pollen according to claim 4, wherein the sample loading is adding camellia bee pollen extracting solution into a pointed SPE column; wherein, in the sample loading process, the flow rate of the camellia bee pollen extracting solution is natural flow-down; after the sample loading is finished, washing the SPE small column by using 4-5 mL of pure water;

the SPE small column washed by pure water needs to be eluted by methanol for 4-6 times, and each time is 4-5 mL; collecting the eluate with small tube, and blowing the collected liquid with nitrogen until the methanol is volatilized.

6. The method for extracting hypoglycemic components from camellia bee pollen as claimed in claim 5, wherein the method further comprises the steps of performing rotary evaporation on the extracting solution before the purification and enrichment;

the conditions of the rotary evaporation are as follows: the rotating speed of the rotary evaporator is 50-60 revolutions per minute, and the temperature is 45-50 ℃.

7. The camellia bee pollen obtained by the extraction method of any one of claims 1 to 6 contains hypoglycemic components.

8. The camellia bee pollen of claim 7 containing hypoglycemic components, characterized by comprising:

kaempferol-3-O- (6' -O-trans-coumaroyl) -glucopyranoside, the content of which is at least more than 10.0 mg/g;

kaempferol-3-O- (2',6' -di-O-trans-p-coumaroyl) -beta-D-glucopyranoside, the content of which is at least more than 8.0 mg/g.

9. Use of the camellia bee pollen of claim 7 or 8 for reducing blood glucose in the preparation of a hypoglycemic agent for inhibiting the absorption and transport of glucose by human intestinal epithelial cells.

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