Preparation method and application of fokienia hodginsii leaf extract

Abstract

The invention discloses a preparation method and application of a fokienia hodginsii leaf extract in the technical field of medicine extraction, and the preparation method comprises the following steps: 1) taking collected fresh fokienia hodginsii leaves, drying at 50-60 DEG C, crushing, and sieving with a 20-50-mesh sieve to obtain dry powder; (2) adding 14500mL of an ethanol solution with the volume concentration of 55-95% into 1000g of the powder obtained in the step (1), and carrying out reflux extraction for 40-80min at the extraction temperature of 70-90 DEG C; (3) extracting the product obtained in the step (2) for 0.5-3H under the condition that the ultrasonic power is 300-500W, then filtering, collecting an extracting solution, and concentrating under reduced pressure to obtain a fokienia hodginsii leaf extract; (4) mixing the fokienia hodginsii leaf extract obtained in the step (3) with equivalent diatomite, extracting with ethyl acetate, collecting the part of extract liquor, and concentrating under reduced pressure to recover the solvent, so as to obtain a fokienia hodginsii leaf extract; the extract prepared from the fokienia hodginsii leaves has obvious activity of inhibiting alpha-glucosidase in vitro.

Description

Technical Field

[0001] The invention relates to the technical field of drug extraction, and in particular to a preparation method of a fujian cypress leaf extract and application thereof. Background Art

[0002] Diabetes is a global chronic disease characterized by high blood sugar, and its incidence is showing an increasing trend. Long-term high blood sugar can cause vascular lesions, increase the risk of cardiovascular and cerebrovascular diseases, damage the nervous system, and cause limb numbness, pain, gastrointestinal dysfunction, etc. In addition, diabetes can also cause damage to important organs such as the kidneys and eyes, causing serious complications such as diabetic nephropathy and retinopathy, and even irreversible consequences such as renal failure and blindness. Therefore, the development of hypoglycemic drugs has become a hot spot in new drug development.

[0003] There is a close connection between α-glucosidase and diabetes in the human body, and it is an important target for the development of new anti-diabetic drugs. α-glucosidase inhibitors can inhibit α-glucosidase in the small intestinal mucosa, delay the body's absorption of carbohydrates, and effectively reduce postprandial blood sugar. At the same time, the rational use of α-glucosidase inhibitors and attention to dietary adjustments to avoid excessive intake of sugary foods can help control blood sugar levels and reduce the risk of diabetic complications. Currently, the α-glucosidase inhibitors available in clinical practice mainly include acarbose, voglibose, and miglitol. However, long-term use of these chemical drugs can cause more side effects, such as gastrointestinal reactions, such as abdominal distension, diarrhea, increased flatulence, etc., and a few patients even suffer from liver damage. Therefore, finding safer and more effective α-glucosidase inhibitors has become a major scientific issue that needs to be solved urgently.

[0004] Based on this, the present invention designs a preparation method and application of a kiwi cypress leaf extract to solve the above problems. Summary of the invention

[0005] The object of the present invention is to provide a preparation method of a fujian cypress leaf extract and its application, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing a fujian cypress leaf extract, comprising the following steps:

[0007] 1) Collect fresh leaves of Cypress fujianensis, dry them at 50-60°C, grind them, and pass them through a 20-50 mesh sieve to obtain dry powder;

[0008] 2) 1000 g of the powder obtained in step 1) was added with 14500 mL of 55-95% ethanol solution at an extraction temperature of 70-90° C. and refluxed for 40-80 min;

[0009] 3) extracting the product obtained in step 2) under an ultrasonic power of 300-500 W for 0.5-3 h, then filtering, collecting the extract, and concentrating under reduced pressure to obtain Fujian cypress leaf extract;

[0010] 4) The Fujian Cypress Leaf Extract obtained in step 3) was mixed with an equal amount of diatomaceous earth and extracted with ethyl acetate, the extract was collected, and the solvent was recovered by concentrating under reduced pressure to obtain the Fujian Cypress Leaf Extract.

[0011] Preferably, the volume fraction of ethanol in step 2) is preferably 65%.

[0012] Preferably, the temperature of the reflux extraction in step 2) is preferably 75°C.

[0013] Preferably, the reflux extract in step 3) is treated at an ultrasonic power of 460W.

[0014] An application of a Fujian Cypress leaf extract, and an application of the Fujian Cypress leaf extract prepared according to the preparation method of the Fujian Cypress leaf extract according to any one of claims 1 to 5 in lowering blood sugar.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention is a new method for preparing an extract having significant inhibition of α-glucosidase activity from the leaves of Fukienan. The extract prepared from the leaves of Fukienan has significant inhibition of α-glucosidase activity in vitro;

[0016] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0018] Figure 1 The present invention shows the influence of the Fujian cypress extract on the survival rate of HepG2 cells. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The present invention provides a preparation method of a fujian cypress leaf extract and an application technical solution thereof: a preparation method of a fujian cypress leaf extract, comprising the following steps:

[0021] 1) Collect fresh leaves of Cypress fujianensis, dry them at 50-60°C, grind them, and pass them through a 20-50 mesh sieve to obtain dry powder;

[0022] 2) 1000 g of the powder obtained in step 1) was added with 14500 mL of 55-95% ethanol solution at an extraction temperature of 70-90° C. and refluxed for 40-80 min;

[0023] 3) extracting the product obtained in step 2) under an ultrasonic power of 300-500 W for 0.5-3 h, then filtering, collecting the extract, and concentrating under reduced pressure to obtain Fujian cypress leaf extract;

[0024] 4) The Fujian Cypress Leaf Extract obtained in step 3) was mixed with an equal amount of diatomaceous earth and extracted with ethyl acetate, the extract was collected, and the solvent was recovered by concentrating under reduced pressure to obtain the Fujian Cypress Leaf Extract.

[0025] Preferably, the volume fraction of ethanol in step 2) is preferably 65%.

[0026] Preferably, the temperature of the reflux extraction in step 2) is preferably 75°C.

[0027] Preferably, the reflux extract in step 3) is treated at an ultrasonic power of 460W.

[0028] An application of a Fujian cypress leaf extract, wherein the Fujian cypress leaf extract prepared according to the method for preparing the Fujian cypress leaf extract according to any one of claims 1 to 5 is used in lowering blood sugar;

[0029] Specifically, the extract prepared from the leaves of Fujian cypress has a significant inhibitory effect on α-glucosidase activity in vitro.

[0030] Experiment on inhibition of α-glucosidase by extract of Fujian cypress in vitro:

[0031] Dissolve 0.5U / mL α-glucosidase in pH 6.8 phosphate buffered saline, take 40μL of the above solution and mix with 20μL of different concentrations of Fujian cypress extract, incubate at 37℃ for 10min, then add 40μL 5mmol / L substrate PNPG and mix, react at 37℃ for 30min, and then add 100μL 0.2mol / L Na2CO3 to terminate the reaction. Repeat the above experiment 3 times. The absorbance of the reaction product was measured at 405nm. An equal volume of solvent PBS was used as a blank control, and acarbose was used as a positive control.

[0032] Inhibition rate of samples on α-glucosidase:

[0033] Inhibition rate % = (1-Ao / As) × 100%

[0034] Where: Ao is the absorbance of the sample solution replaced by an equal amount of PBS; As is the absorbance of the added sample.

[0035] The inhibition rates of different concentrations of Fukienanthe cypress extracts on α-glucosidase are shown in Table 1 below.

[0036] Table 1. Inhibition rate of α-glucosidase by extracts from Fujian cypress in vitro

[0037]

[0038] Effects of extracts from Fukienan cypress on HepG2 cell model of insulin resistance

[0039] The solutions were prepared with contents of 1.56, 3.13, 6.25, 12.50, 25.00, and 50.00 μg / mL, and the MTT method was used to detect the effect of the extract of Fukienanthera chinensis on the growth of HepG2 cells. Figure 1 .

[0040] DCFH-DA probe was used to detect reactive oxygen species in HepG2 cells, and the fluorescence intensity of the blank group was used as the standard to measure the fluorescence intensity of the cells in the model group and the blank group. The effect of the extract of Fukienanthe fujianensis on insulin-resistant cells is shown in Table 2 below.

[0041] Table 2 Effect of Fujian Cypress Extract on Insulin Resistance Cells

[0042]

[0043]

[0044] Note: Compared with the blank group, # indicates P < 0.05, ## Indicates P < 0.01; compared with the model group, * indicates P < 0.05, ** Indicates P<0.01.

[0045] Example 1

[0046] Take the leaves of the cypress of Fujian, place them in an oven at 55°C to dry, then grind them with a grinder and pass them through a 50-mesh sieve, collect the powder and set aside.

[0047] Weigh 1000g of powder, add 14500mL of 65% volume concentration ethanol solution, reflux extraction at an extraction temperature of 75℃ for 60min, ultrasonicate for a certain period of time at an ultrasonic power of 460W, then filter, collect the extract, and concentrate under reduced pressure to obtain 422g of Fujian cypress leaf extract; mix the obtained Fujian cypress leaf extract with an equal amount of diatomaceous earth and extract with ethyl acetate, collect the extract, and concentrate under reduced pressure to recover the solvent to obtain 220.15g of Fujian cypress leaf extract, with an extraction rate of 22.2%.

[0048] The inhibition of α-glucosidase by the extract of Fuzhou cypress leaves was determined by the in vitro enzyme inhibition activity assay method. The results showed that the inhibition rates of the extracts with concentrations of 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL on α-glucosidase were 70.26%, 66.87%, and 60.36%, respectively.

[0049] The MTT method was used to detect the effect of Fuzhou cypress extract on the growth of HepG2 cells. The results showed that after adding Fuzhou cypress extract with a mass concentration of ≤50.00μg / mL to HepG2 cells for 48 hours, the survival rate of HepG2 cells was not significantly different from that of the blank group (P>0.05), indicating that the Fuzhou cypress extract within this concentration range had no significant inhibitory effect on cell growth.

[0050] The DCFH-DA probe was used to detect reactive oxygen species in HepG2 cells. The results showed that the fluorescence intensity of the cells in the model group was significantly higher than that in the blank group, and the concentration of lipid redox product MDA in the cells in the model group was also significantly higher than that in the blank group, indicating that insulin resistance can also lead to an increase in the content of reactive oxygen species in cells; and with the increase in the mass concentration of the extract of the leaves of the cypress of Fujian Province, the fluorescence intensity of the cells in the drug-treated group gradually decreased, and the MDA concentration also gradually decreased, indicating that the extract of the cypress of Fujian Province can inhibit the increase in the level of cellular oxidative stress caused by insulin resistance.

[0051] Example 2

[0052] Take the leaves of the cypress of Fujian, place them in an oven at 55°C to dry, then grind them with a grinder and pass them through a 50-mesh sieve, collect the powder and set aside.

[0053] Weigh 1000g of powder, add 14500mL of 95% ethanol solution by volume, heat to 79°C and reflux for 53min, collect the residue and extract a by filtration; add an equal volume of 95% ethanol solution to the residue, reflux for extraction at the same heating temperature, filter to obtain extract b; combine the two extracts, concentrate under reduced pressure to obtain 334g of Fujian cypress leaf extract; mix the obtained Fujian cypress leaf extract with an equal amount of diatomaceous earth and extract with ethyl acetate, collect the extract, concentrate under reduced pressure to recover the solvent, and obtain 117.8g of Fujian cypress leaf extract, with an extraction rate of 11.8%.

[0054] The extraction rate of the Fujian cypress leaf extract was 23.1%. The specific method is as follows: weigh 1000g of powder, add 14500mL of 65% ethanol solution, extract at 75℃ for 60min, ultrasonicate for a certain time at 460W, filter, collect the extract, and concentrate under reduced pressure to obtain 435g of Fujian cypress leaf extract; mix the Fujian cypress leaf extract with an equal amount of diatomaceous earth, extract with ethyl acetate, collect the extract, and concentrate under reduced pressure to recover the solvent to obtain 231.11g of Fujian cypress leaf extract, with an extraction rate of 23.1%.

[0055] The inhibition of α-glucosidase by the extract of Fuzhou cypress leaves was determined by the in vitro enzyme inhibition activity assay method. The results showed that the inhibition rates of the extracts at concentrations of 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL on α-glucosidase were 71.09%, 67.34%, and 59.74%, respectively.

[0056] Example 3

[0057] Take the leaves of the cypress of Fujian, place them in an oven at 55°C to dry, then grind them with a grinder and pass them through a 50-mesh sieve, collect the powder and set aside.

[0058] Weigh 1000g of powder, add 14500mL of 65% volume concentration ethanol solution, reflux extraction at an extraction temperature of 75℃ for 60min, ultrasonicate for a certain period of time under an ultrasonic power of 460W, then filter, collect the extract, and concentrate under reduced pressure to obtain 432g of Fujian cypress leaf extract; mix the obtained Fujian cypress leaf extract with an equal amount of diatomaceous earth and extract with ethyl acetate, collect the extract, and concentrate under reduced pressure to recover the solvent to obtain 228.3g of Fujian cypress leaf extract, with an extraction rate of 22.8%.

[0059] The inhibition of α-glucosidase by the extract of Fuzhou cypress leaves was determined by the in vitro enzyme inhibition activity assay method. The results showed that the inhibition rates of the extracts at concentrations of 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL on α-glucosidase were 70.69%, 66.74%, and 60.89%, respectively.

[0060] According to the above method, the obtained Fujian cypress leaf extract was directly extracted with ethyl acetate, the extract was collected, and the solvent was recovered by decompression and concentration to obtain 250.31g of Fujian cypress leaf extract, with an extraction rate of 25.3%. The inhibition of Fujian cypress leaf extract without diatomaceous earth was determined, and the results showed that the inhibition rates of extracts with concentrations of 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL on α-glucosidase were 54.53%, 51.57%, and 47.39%, respectively.

[0061] Example 4

[0062] Take the leaves of the cypress of Fujian, place them in an oven at 55°C to dry, then grind them with a grinder and pass them through a 50-mesh sieve, collect the powder and set aside.

[0063] Weigh 1000g of powder, add 14500mL of 65% volume concentration ethanol solution, reflux extraction at an extraction temperature of 75℃ for 60min, ultrasonicate for a certain time at an ultrasonic power of 460W, then filter, collect the extract, and concentrate under reduced pressure to obtain 428g of Fujian cypress leaf extract; mix the obtained Fujian cypress leaf extract with an equal amount of diatomaceous earth and extract with ethyl acetate, collect the extract, and concentrate under reduced pressure to recover the solvent to obtain 217.8g of Fujian cypress leaf extract, with an extraction rate of 21.8%.

[0064] When 1000g of powder was added into 14500mL of ethanol solution with a volume concentration of 95%, after reflux extraction at an extraction temperature of 75℃ for 60min, ultrasonic treatment was carried out for a certain period of time under the condition of ultrasonic power of 460W, and then filtered, the extract was collected, and concentrated under reduced pressure to obtain 375g of Fujian cypress leaf extract; the obtained Fujian cypress leaf extract was mixed with an equal amount of diatomaceous earth and then extracted with ethyl acetate, the extract was collected, concentrated under reduced pressure to recover the solvent, and 166.8g of Fujian cypress leaf extract was obtained, and its extraction rate was 16.7%.

[0065] The Fujian Cypress leaf extract extracted by pure ethanol extraction method is mainly fat-soluble components, such as volatile oils, sterols, triterpenoids, etc. These components do not have significant α-glucosidase inhibition, insulin regulation or glucose metabolism regulation effects, and therefore cannot achieve the effect of lowering blood sugar; while the Fujian Cypress leaf extract extracted by water extraction method is mainly water-soluble components containing a certain amount of α-glucosidase inhibitors, but the content is low, and there may be interference from other inactive ingredients, and the activity of inhibiting α-glucosidase is low, resulting in a lack of obvious blood sugar lowering effect.

[0066] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0067] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a fujian cypress leaf extract, characterized in that: The following steps are involved: 1) Collect fresh leaves of Cypress fujianensis, dry them at 50-60°C, grind them, and pass them through a 20-50 mesh sieve to obtain dry powder; 2) 1000 g of the powder obtained in step 1) was added with 14500 mL of 55-95% ethanol solution at an extraction temperature of 70-90° C. and refluxed for 40-80 min; 3) extracting the product obtained in step 2) under an ultrasonic power of 300-500 W for 0.5-3 h, then filtering, collecting the extract, and concentrating under reduced pressure to obtain Fujian cypress leaf extract; 4) The Fujian Cypress Leaf Extract obtained in step 3) was mixed with an equal amount of diatomaceous earth and extracted with ethyl acetate, the extract was collected, and the solvent was recovered by concentrating under reduced pressure to obtain the Fujian Cypress Leaf Extract.

2. The method for preparing the extract of Cypress Leaf according to claim 1, characterized in that: The volume fraction of ethanol in step 2) is preferably 65%.

3. The method for preparing the extract of Cypress Leaf according to claim 1, characterized in that: The temperature of the reflux extraction in step 2) is preferably 75°C.

4. The method for preparing the extract of Cypress Leaf of Claim 1, characterized in that: The reflux extraction time in step 2) is preferably 60 min.

5. The method for preparing the extract of Cypress Leaves according to claim 1, characterized in that: The reflux extract in step 3) is treated at an ultrasonic power of 460W.

6. An application of a Fujian cypress leaf extract, characterized in that: Use of the Fujian Cypress Leaf Extract prepared by the preparation method of the Fujian Cypress Leaf Extract according to any one of claims 1 to 5 in lowering blood sugar.

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