Aromatase promoting activity of composition containing rehmannia and quality evaluation method of composition based on aromatase promoting activity

Abstract

The invention discloses an aromatase activity determination method for a composition containing rehmannia, which comprises the following steps: (1) constructing an aromatase activity reaction system and a control system to respectively obtain an aromatase activity reaction solution and a control solution; (2) preparing a blank solution; (3) taking a proper amount of the aromatase active reaction solution, the contrast solution and the blank solution, and carrying out high performance liquid detection to obtain a detection result; and (4) obtaining aromatase activity information of the composition containing the rehmannia according to a detection result, the composition containing the rehmannia comprises prepared rehmannia root, wine-processed fructus corni, moutan bark, Chinese yam, poria cocos and rhizoma alismatis. The determination method disclosed by the invention is simple, convenient, rapid, accurate and reliable, can be used for biological evaluation of the quality of the composition containing the rehmannia glutinosa, and is expected to be applied to quality control work of the composition containing the rehmannia glutinosa.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a method for evaluating the aromatase promoting activity of a composition containing Rehmannia glutinosa and the quality of the composition based on the activity. BACKGROUND

[0002] Aromatase is expressed on the endoplasmic reticulum membrane of cells in various tissues, and the human aromatase gene CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1) is located on chromosome 19q21.1 and contains 10 exons: exon 1 is related to gene tissue-specific expression but does not participate in protein coding; exons 2-10 encode an aromatase protein composed of 503 amino acids. The specific promoter determines the tissue-specific expression of the CYP19A1 gene; due to the same coding region, the aromatase synthesized by various tissues is only one kind and has the same function.

[0003] There is no method for evaluating the aromatase promoting activity of a composition containing Rehmannia glutinosa and the quality of the composition based on the activity in the prior art. SUMMARY

[0004] Therefore, the present application provides a method for determining the aromatase activity of a composition containing Rehmannia glutinosa, which comprises the following steps:

[0005] (1) constructing an aromatase activity reaction system and a control system to obtain an aromatase activity reaction solution and a control solution, respectively;

[0006] (2) preparing a blank solution;

[0007] (3) taking an appropriate amount of the aromatase activity reaction solution, the control solution and the blank solution for high-performance liquid detection to obtain a detection result; and

[0008] (4) obtaining the aromatase activity information of the composition containing Rehmannia glutinosa according to the detection result;

[0009] The composition containing Rehmannia glutinosa comprises Rehmannia glutinosa, wine dried meat, peony root bark, Chinese yam, poria cocos and alisma.

[0010] The chromatographic conditions of the high-performance liquid detection are as follows: using a Diamonsil C 18 chromatographic column, methanol as mobile phase A, water as mobile phase B, isocratic elution, a flow rate of 0.5-1.5 mL / min, a column temperature of 20-30℃, a detection wavelength of 220-260 nm, and a sample injection amount of 15-25 μL.

[0011] Further, the method for constructing the aromatase activity reaction system comprises the following steps: sequentially adding the Tris-HCL buffer solution, the testosterone solution, the water extract of the composition containing Rehmannia glutinosa, the aromatase solution and the NADPH solution into a centrifuge tube, mixing and reacting, the reaction temperature is 30-45℃, the reaction time is 10-90 min, then 400-600 μL of methanol is added to terminate the reaction, and filtration is performed to obtain the aromatase activity reaction solution.

[0012] Further, the method for constructing the control system comprises the following steps: sequentially adding the Tris-HCL buffer solution, the testosterone solution, the aromatase solution and the NADPH solution into a centrifuge tube, mixing and reacting, the reaction temperature is 30-45℃, the reaction time is 10-90 min, then 400-600 μL of methanol is added to terminate the reaction, and filtration is performed to obtain the control solution.

[0013] Further, the method for preparing the blank solution comprises the following steps: sequentially adding the Tris-HCL buffer solution, the testosterone solution and the NADPH solution into a centrifuge tube, adding 400-600 μL of methanol, and performing filtration to obtain the blank solution.

[0014] Further, in the method for constructing the aromatase activity reaction system, the volume ratio among the Tris-HCL buffer solution, the testosterone solution, the water extract of the composition containing Rehmannia glutinosa, the aromatase solution and the NADPH solution is about 200: about 50: about 50: about 100: about 100.

[0015] Further, in the method for constructing the control system, the volume ratio among the Tris-HCL buffer solution, the testosterone solution, the aromatase solution and the NADPH solution is about 250: about 50: about 100: about 100.

[0016] Further, in the method for preparing the blank solution, the volume ratio among the Tris-HCL buffer solution, the testosterone solution and the NADPH solution is about 350: about 50: about 100.

[0017] Further, the pH value of the Tris-HCL buffer solution is about 7.4.

[0018] Further, the volume of the methanol is about 500 μL.

[0019] Further, the reaction temperature is about 37℃.

[0020] Further, the reaction time is about 20 min or about 30 min.

[0021] Further, the filtration is membrane filtration.

[0022] Furthermore, the pore size of the filter membrane is 0.45 μm.

[0023] Furthermore, the preparation method of the testosterone solution comprises the following steps: weighing an appropriate amount of a testosterone standard and placing it in a volumetric flask, adding methanol, and fixing the volume to obtain the testosterone solution.

[0024] Furthermore, the mass of the testosterone standard is 5-7 mg, for example, about 5.77 mg.

[0025] Furthermore, the concentration of the testosterone solution is 0.5 to 2 mmol / L, for example, about 1 mmol / L.

[0026] Furthermore, the aromatase solution is diluted with Tris-HCl buffer.

[0027] Furthermore, the concentration of the aromatase solution is 0.25-2 μg / mL, for example, about 1 μg / mL.

[0028] Furthermore, the preparation method of the NADPH solution includes the following steps: weighing an appropriate amount of NADPH powder, adding Tris-HCl buffer to dissolve it, and constant volume to obtain the NADPH solution.

[0029] Furthermore, the mass of the NADPH powder is 1-2 mg, for example, about 1.67 mg.

[0030] Furthermore, the concentration of the NADPH solution is 0.2-2 mmol / L, for example, about 1 mmol / L.

[0031] Furthermore, when the testosterone in the aromatase activity reaction solution is within the range of 3.125 to 200 μmol / L, the correlation coefficient r of the linear relationship of the determination method is ≥0.9995.

[0032] Furthermore, the preparation method of the aqueous extract of the composition containing Rehmannia glutinosa comprises the following steps: weighing an appropriate amount of the composition containing Rehmannia glutinosa, grinding to obtain a composition powder, adding water to dissolve the composition powder, ultrasonically extracting, filtering to obtain a filtrate; evaporating the filtrate to dryness in a water bath, adding water to redissolve it, and obtaining the aqueous extract of the composition containing Rehmannia glutinosa.

[0033] Furthermore, the mass of the composition powder is 0.1-1 g, for example, about 0.5 g.

[0034] Furthermore, the water is purified water.

[0035] Furthermore, the mass / volume (g / mL) ratio of the composition powder to the water in the water dissolution is 0.01 to 0.05, for example, about 0.02.

[0036] Furthermore, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0037] Furthermore, the concentration of the aqueous extract of the composition containing Rehmannia glutinosa is 1 to 20 mg / mL, for example, about 5 mg / mL or about 10 mg / mL.

[0038] Furthermore, the preparation method of the composition containing Rehmannia root comprises: weighing appropriate amounts of Rehmannia root, wine-soaked Cornus fruit, peony bark, yam, Poria cocos and Alisma orientalis, grinding them into fine powder, sieving, mixing and obtaining a mixture; and making pills from the mixture to obtain the composition containing Rehmannia root.

[0039] Furthermore, the pill-making method is selected from the following: (a) alternately adding the mixture and ethanol to a mold to form pills, and drying to obtain the product; (b) adding refined honey and water to the mixture to form pills, and drying to obtain the product; or (c) adding refined honey to the mixture to form pills, and drying to obtain the product.

[0040] Furthermore, in method (b), the mass ratio of the refined honey to the mixture is 0.3 to 0.6, such as 0.35 to 0.5.

[0041] Furthermore, in method (c), the mass ratio of the refined honey to the mixture is 0.7 to 1.2, such as 0.8 to 1.1.

[0042] Furthermore, the mass ratio of the Radix Rehmanniae Preparata, the Fructus Corni with Wine Wine, the Cortex Moutan, the Dioscorea Oppositae, the Poria and the Rhizoma Alismatis is (10-20):(4-12):(2-10):(4-12):(2-10):(2-10).

[0043] Furthermore, the mass ratios of the Radix Rehmanniae Preparata, the Fructus Corni with Wine Wine, the Cortex Moutan, the Dioscorea Oppositae, the Poria and the Rhizoma Alismatis are (14-18):(6-10):(4-8):(6-10):(4-8):(4-8).

[0044] Furthermore, the mass ratio of the Rehmannia root, the wine-soaked Cornus fruit, the Paeonia suffruticosa moutan bark, the Dioscorea opposita, the Poria cocos and the Alisma orientalis is about 16: about 8: about 6: about 8: about 6: about 6.

[0045] Furthermore, the mass of the Rehmannia root is 140-180 g, for example, about 160 g.

[0046] Furthermore, the weight of the wine-cooked cornus fruit is 60-100 g, for example, about 80 g.

[0047] Furthermore, the mass of the peony bark is 40-80 g, for example, about 60 g.

[0048] Furthermore, the mass of the yam is 60-100 g, for example, about 80 g.

[0049] Furthermore, the mass of the Poria cocos is 40-80 g, for example, about 60 g.

[0050] Furthermore, the mass of the Rhizoma Alismatis is 40-80 g, for example, about 60 g.

[0051] Furthermore, the detection result is the peak area of ​​the testosterone chromatographic peak in the chromatogram obtained by high performance liquid chromatography detection of the aromatase activity reaction solution, the control solution and the blank solution.

[0052] Furthermore, the aromatase activity information is calculated based on the recorded peak area of ​​the testosterone chromatographic peak in the aromatase activity reaction solution, the peak area of ​​the testosterone chromatographic peak in the control solution, and the peak area of ​​the testosterone chromatographic peak in the blank solution, and the aromatase activity promotion rate is calculated based on the testosterone conversion amount in the aromatase activity reaction solution and the testosterone conversion amount in the control solution.

[0053] Furthermore, the calculation formula for the testosterone conversion amount in the aromatase activity reaction solution is: testosterone conversion amount in the aromatase activity reaction solution = peak area of ​​the testosterone chromatographic peak in the blank solution - peak area of ​​the testosterone chromatographic peak in the aromatase activity reaction solution.

[0054] Furthermore, the calculation formula for the testosterone conversion amount of the control solution is: testosterone conversion amount of the control solution = peak area of ​​the testosterone chromatographic peak in the blank solution - peak area of ​​the testosterone chromatographic peak in the control solution.

[0055] Furthermore, the calculation formula of the aromatase activity promotion rate is: (testosterone conversion amount in the aromatase activity reaction solution-testosterone conversion amount in the control solution) / testosterone conversion amount in the control solution×100%.

[0056] Furthermore, the volume ratio of the mobile phase A to the mobile phase B in the HPLC detection is about 7:about 3.

[0057] Furthermore, the flow rate is about 1.0 mL / min.

[0058] Furthermore, the column temperature is room temperature, for example, about 25°C.

[0059] Furthermore, the detection wavelength is 243 nm.

[0060] Furthermore, the injection volume is about 20 μL.

[0061] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0062] Further, the aromatase is an in vitro aromatase.

[0063] According to another aspect of the present application, there is provided a quality evaluation method of a composition comprising Rehmannia glutinosa, the quality evaluation method comprising the steps of:

[0064] (a) optionally, obtaining a reference sample of the composition according to the aromatase activity determination method described above to obtain an aromatase activity promotion rate of the reference sample of the composition;

[0065] (b) obtaining a test sample of the composition according to the aromatase activity determination method described above to obtain an aromatase activity promotion rate of the test sample of the composition; and

[0066] (c) comparing the aromatase activity promotion rate of the test sample of the composition obtained in step (b) with the aromatase activity promotion rate of the reference sample of the composition obtained in step (a), if the difference between the average values of the two is ≤10%, the composition is a qualified product, if the difference between the average values of the two is >10%, the composition is an unqualified product;

[0067] (c') alternatively, if the aromatase activity promotion rate of the test sample of the composition is ≥65%, the composition is a qualified product, if the aromatase activity promotion rate of the test sample of the composition is <65%, the composition is an unqualified product.

[0068] According to another aspect of the present application, there is provided a use of the determination method or the quality evaluation method described above in the quality detection and / or quality evaluation and / or quality control of a composition comprising Rehmannia glutinosa.

[0069] According to another aspect of the present application, there is provided a use of a composition comprising Rehmannia glutinosa in the preparation of a medicament for promoting aromatase activity;

[0070] wherein the composition comprising Rehmannia glutinosa comprises Rehmannia glutinosa, Euonymus alatus, Paeonia suffruticosa, Dioscorea opposita, Poria cocos and Alisma orientale; and the mass ratio between the Rehmannia glutinosa, Euonymus alatus, Paeonia suffruticosa, Dioscorea opposita, Poria cocos and Alisma orientale is about 16:about 8:about 6:about 8:about 6:about 6.

[0071] Further, the aromatase is an in vitro aromatase.

[0072] Further, the aromatase is an in vitro aromatase.

[0073] Advantages of the present application:

[0074] The present invention uses aromatase activity calibrated by T conversion rate and HPLC to determine T content, establishing a rapid and effective in vitro aromatase activity evaluation system, thereby providing a basis for quality control of compositions containing Rehmannia glutinosa. The assay method of the present invention is simple, rapid, accurate, and reliable, and can be used for biological quality evaluation of compositions containing Rehmannia glutinosa, with the goal of applying it to quality control of compositions containing Rehmannia glutinosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0076] Figure 1 The HPLC chromatograms of compositions containing Rehmannia glutinosa extracted with different solvents are shown.

[0077] Figure 2 Schematic diagram of the effect of different substrate concentrations on enzyme activity.

[0078] Figure 3 Schematic diagram of the effect of different NADPH concentrations on enzyme activity.

[0079] Figure 4 Schematic diagram of the effect of different aromatase concentrations on enzyme activity.

[0080] Figure 5 Schematic diagram of the effect of different reaction times on enzyme activity.

[0081] Figure 6 Schematic diagram of the effect of different reaction temperatures on enzyme activity.

[0082] Figure 7 Schematic diagram of the specificity test results. A: HPLC chromatogram of testosterone solution; B: HPLC chromatogram of testosterone blank solution; C: HPLC chromatogram of test solution; 1: testosterone.

[0083] Figure 8 Schematic diagram of the testosterone standard curve. DETAILED DESCRIPTION

[0084] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0085] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those skilled in the art in the field of the present invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0086] The present invention is intended to encompass all alternatives, modifications, and equivalents that may come within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0087] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0088] In the present invention, the term "comprising" is synonymous with "including." As used herein, the terms "comprises," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0089] As described in the background section, the aromatase-promoting activity of a composition containing Rehmannia glutinosa, as claimed in the present invention, and a method for evaluating the quality of the composition based on this activity, are not known in the prior art. To address the above issues, the present invention provides a method for determining the aromatase activity of a composition containing Rehmannia glutinosa, comprising the following steps:

[0090] (1) constructing an aromatase activity reaction system and a control system to obtain an aromatase activity reaction solution and a control solution, respectively;

[0091] (2) preparing a blank solution;

[0092] (3) taking appropriate amounts of the aromatase activity reaction solution, the control solution, and the blank solution for high performance liquid chromatography (HPLC) detection to obtain the detection results; and

[0093] (4) obtaining aromatase activity information of the composition comprising Rehmannia glutinosa based on the test results;

[0094] The composition containing Rehmannia root includes Rehmannia root prepared from Radix Rehmanniae, Fructus Corni with wine, Cortex Moutan, Rhizoma Dioscoreae, Poria cocos and Rhizoma Alismatis;

[0095] The chromatographic conditions for the HPLC detection are as follows: using Diamonsil C 18 Chromatographic column, mobile phase A is methanol, mobile phase B is water, isocratic elution, flow rate is 0.5-1.5 mL / min, column temperature is 20-30°C, detection wavelength is 220-260 nm, and injection volume is 15-25 μL.

[0096] In the present invention, when flow rate, column temperature, wavelength, injection amount, mass ratio, temperature, time, volume, mass, concentration, ratio, or other value or parameter is expressed as a range, preferred range, or a range limited by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed alone. For example, when the range "20-30" is disclosed, the described range should be interpreted as including the range "20-30", "20-28", "20-26", "20-24", "20-22", "22-30", "22-28", "22-26", "22-24", "24-30", "24-28", "24-26", "26-30", "26-28", "28-30", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0097] In a preferred embodiment, the method for constructing the aromatase activity reaction system comprises the following steps: adding Tris-HCl buffer, testosterone solution, an aqueous extract of a composition containing Rehmannia root, an aromatase solution, and an NADPH solution to a centrifuge tube in sequence, mixing and reacting, the reaction temperature is 30-45°C, the reaction time is 10-90 min, then adding 400-600 μL of methanol to terminate the reaction, filtering, and obtaining the aromatase activity reaction solution.

[0098] In a preferred embodiment, the method for constructing the control system comprises the following steps: adding Tris-HCl buffer, testosterone solution, aromatase solution and NADPH solution to a centrifuge tube in sequence, mixing and reacting, the reaction temperature is 30-45°C, the reaction time is 10-90 min, then adding 400-600 μL of methanol to terminate the reaction, filtering, and obtaining the control solution.

[0099] In a preferred embodiment, the method for preparing the blank solution comprises the following steps: sequentially adding Tris-HCl buffer, testosterone solution and NADPH solution to a centrifuge tube, adding 400-600 μL of methanol, and filtering to obtain the blank solution.

[0100] In a preferred embodiment, in the method for constructing the aromatase activity reaction system, the volume ratio of the Tris-HCl buffer, the testosterone solution, the aqueous extract of the composition containing Rehmannia glutinosa, the aromatase solution, and the NADPH solution is about 200: about 50: about 50: about 100: about 100.

[0101] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 200" includes ±5% of 200, or from 190 to 210; "about 50" includes ±5% of 50, or from 47.5 to 52.5; and "about 100" includes ±5% of 100, or from 95 to 105.

[0102] In a preferred embodiment, in the method for constructing the control system, the volume ratio of the Tris-HCL buffer, the testosterone solution, the aromatase solution and the NADPH solution is about 250:about 50:about 100:about 100.

[0103] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5; "about 50" includes ±5% of 50, or from 47.5 to 52.5; and "about 100" includes ±5% of 100, or from 95 to 105.

[0104] In a preferred embodiment, in the method for preparing the blank solution, the volume ratio of the Tris-HCL buffer, the testosterone solution and the NADPH solution is about 350:about 50:about 100.

[0105] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 350" includes ±5% of 350, or from 332.5 to 367.5; "about 50" includes ±5% of 50, or from 47.5 to 52.5; and "about 100" includes ±5% of 100, or from 95 to 105.

[0106] In a preferred embodiment, the pH of the Tris-HCl buffer is about 7.4.

[0107] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7.4" includes ±5% of 7.4, or from 7.03 to 7.77.

[0108] In a preferred embodiment, the volume of methanol is about 500 μL.

[0109] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0110] In a preferred embodiment, the reaction temperature is about 37°C.

[0111] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 37" includes ±5% of 37, or from 35.15 to 38.85.

[0112] In a preferred embodiment, the reaction time is about 20 min or about 30 min.

[0113] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21; "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0114] In a preferred embodiment, the filtration is membrane filtration.

[0115] In a preferred embodiment, the pore size of the filter membrane is 0.45 μm.

[0116] In a preferred embodiment, the method for preparing the testosterone solution comprises the following steps: weighing an appropriate amount of a testosterone standard and placing it in a volumetric flask, adding methanol, and fixing the volume to obtain the testosterone solution.

[0117] In a preferred embodiment, the mass of the testosterone standard is 5-7 mg, for example, about 5.77 mg.

[0118] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5.77" includes ±5% of 5.77, or from 5.4815 to 6.0585.

[0119] In a preferred embodiment, the concentration of the testosterone solution is 0.5-2 mmol / L, such as about 1 mmol / L.

[0120] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0121] In a preferred embodiment, the aromatase solution is diluted with Tris-HCl buffer.

[0122] In a preferred embodiment, the concentration of the aromatase solution is 0.25-2 μg / mL, such as about 1 μg / mL.

[0123] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0124] In a preferred embodiment, the method for preparing the NADPH solution comprises the following steps: weighing an appropriate amount of NADPH powder, adding Tris-HCl buffer to dissolve it, and constant volume to obtain the NADPH solution.

[0125] In a preferred embodiment, the mass of the NADPH powder is 1-2 mg, for example, about 1.67 mg.

[0126] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.67" includes ±5% of 1.67, or from 1.5865 to 1.7535.

[0127] In a preferred embodiment, the concentration of the NADPH solution is 0.2-2 mmol / L, for example, about 1 mmol / L.

[0128] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0129] In a preferred embodiment, when the testosterone in the aromatase activity reaction solution is in the range of 3.125 to 200 μmol / L, the correlation coefficient r of the linear relationship of the determination method is ≥ 0.9995.

[0130] In a preferred embodiment, the preparation method of the aqueous extract of the composition containing Rehmannia glutinosa comprises the following steps: weighing an appropriate amount of the composition containing Rehmannia glutinosa, grinding to obtain a composition powder, adding water to the composition powder to dissolve it, ultrasonically extracting it, filtering to obtain a filtrate; evaporating the filtrate to dryness in a water bath, adding water to redissolve it, and obtaining the aqueous extract of the composition containing Rehmannia glutinosa.

[0131] In a preferred embodiment, the mass of the composition powder is 0.1-1 g, for example, about 0.5 g.

[0132] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0133] In a preferred embodiment, the water is purified water.

[0134] In a preferred embodiment, the mass / volume (g / mL) ratio of the composition powder to the water in the water dissolution is 0.01 to 0.05, such as about 0.02.

[0135] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.02" includes ±5% of 0.02, or from 0.019 to 0.021.

[0136] In a preferred embodiment, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0137] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0138] In a preferred embodiment, the concentration of the aqueous extract of the composition comprising Rehmannia glutinosa is 1 to 20 mg / mL, such as about 5 mg / mL or about 10 mg / mL.

[0139] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0140] In a preferred embodiment, the preparation method of the composition containing Rehmannia root comprises: weighing appropriate amounts of Rehmannia root, wine-soaked Cornus fruit, peony bark, yam, Poria cocos and Alisma orientalis, grinding them into fine powder, sieving, mixing and obtaining a mixture; and making pills from the mixture to obtain the composition containing Rehmannia root.

[0141] In a preferred embodiment, the pill-making method is selected from the following: (a) alternately adding the mixture and ethanol to a mold to form pills, and drying to obtain the product; (b) adding refined honey and water to the mixture to form pills, and drying to obtain the product; or (c) adding refined honey to the mixture to form pills, and drying to obtain the product.

[0142] In a preferred embodiment, in method (b), the mass ratio of the refined honey to the mixture is 0.3 to 0.6, such as 0.35 to 0.5.

[0143] In a preferred embodiment, in method (c), the mass ratio of the refined honey to the mixture is 0.7 to 1.2, such as 0.8 to 1.1.

[0144] In a preferred embodiment, the mass ratio of the Rehmannia root, the wine-processed Cornus fruit, the Paeonia suffruticosa bark, the Dioscorea opposita, the Poria cocos and the Alisma orientalis is (10-20):(4-12):(2-10):(4-12):(2-10):(2-10).

[0145] In a preferred embodiment, the mass ratio of the Rehmannia root, the wine-processed Cornus fruit, the Paeonia suffruticosa bark, the Dioscorea opposita, the Poria cocos and the Alisma orientalis is (14-18):(6-10):(4-8):(6-10):(4-8):(4-8).

[0146] In a preferred embodiment, the mass ratio between the Rehmannia glutinosa, the Fructus Corni, the Cortex Moutan, the Rhizoma Dioscoreae, the Poria and the Alisma is about 16: about 8: about 6: about 8: about 6: about 6.

[0147] In the present application, "about" means a value that is ±5% of a particular value. For example, "about 16" includes ±5% of 16, or from 15.2 to 16.8; "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0148] In a preferred embodiment, the mass of the Rehmannia glutinosa is 140-180 g, for example about 160 g.

[0149] In the present application, "about" means a value that is ±5% of a particular value. For example, "about 160" includes ±5% of 160, or from 152 to 168.

[0150] In a preferred embodiment, the mass of the Fructus Corni is 60-100 g, for example about 80 g.

[0151] In the present application, "about" means a value that is ±5% of a particular value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0152] In a preferred embodiment, the mass of the Cortex Moutan is 40-80 g, for example about 60 g.

[0153] In the present application, "about" means a value that is ±5% of a particular value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0154] In a preferred embodiment, the mass of the Rhizoma Dioscoreae is 60-100 g, for example about 80 g.

[0155] In the present application, "about" means a value that is ±5% of a particular value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0156] In a preferred embodiment, the mass of the Poria is 40-80 g, for example about 60 g.

[0157] In the present application, "about" means a value that is ±5% of a particular value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0158] In a preferred embodiment, the mass of the Alisma is 40-80 g, for example about 60 g.

[0159] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0160] In a preferred embodiment, the detection result is the peak area of ​​the testosterone chromatographic peak in the chromatogram obtained by high performance liquid chromatography detection of the aromatase activity reaction solution, the control solution and the blank solution.

[0161] In a preferred embodiment, the aromatase activity information is calculated based on the recorded peak area of ​​the testosterone chromatographic peak in the aromatase activity reaction solution, the peak area of ​​the testosterone chromatographic peak in the control solution, and the peak area of ​​the testosterone chromatographic peak in the blank solution, and the aromatase activity promotion rate is calculated based on the testosterone conversion amount in the aromatase activity reaction solution and the testosterone conversion amount in the control solution.

[0162] In a preferred embodiment, the calculation formula for the testosterone conversion amount in the aromatase activity reaction solution is: testosterone conversion amount in the aromatase activity reaction solution = peak area of ​​the testosterone chromatographic peak in the blank solution - peak area of ​​the testosterone chromatographic peak in the aromatase activity reaction solution.

[0163] In a preferred embodiment, the calculation formula for the testosterone conversion amount of the control solution is: testosterone conversion amount of the control solution = peak area of ​​the testosterone chromatographic peak in the blank solution - peak area of ​​the testosterone chromatographic peak in the control solution.

[0164] In a preferred embodiment, the calculation formula of the aromatase activity promotion rate is: (testosterone conversion amount in aromatase activity reaction solution-testosterone conversion amount in control solution) / testosterone conversion amount in control solution×100%.

[0165] In a preferred embodiment, the volume ratio of the mobile phase A to the mobile phase B in the HPLC detection is about 7:about 3.

[0166] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7" includes ±5% of 7, or from 6.65 to 7.35; "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0167] In a preferred embodiment, the flow rate is about 1.0 mL / min.

[0168] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0169] In a preferred embodiment, the column temperature is room temperature, such as about 25°C.

[0170] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0171] In a preferred embodiment, the detection wavelength is 243 nm.

[0172] In a preferred embodiment, the injection volume is about 20 μL.

[0173] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0174] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0175] In a preferred embodiment, the aromatase is an in vitro aromatase.

[0176] According to another aspect of the present invention, a method for evaluating the quality of a composition containing Rehmannia glutinosa is provided, the method comprising the following steps:

[0177] (a) Optionally, obtaining the aromatase activity promotion rate of a reference sample of the composition according to the aromatase activity determination method described above;

[0178] (b) taking the aqueous extract of the composition and testing it according to the above aromatase activity assay method to obtain the aromatase activity promotion rate of the test sample of the composition; and

[0179] (c) comparing the aromatase activity promotion rate of the test sample of the composition obtained in step (b) with the aromatase activity promotion rate of the reference sample of the composition obtained in step (a); if the difference between the average values ​​of the two is ≤10%, the composition is a qualified product that meets the requirements; if the difference between the average values ​​of the two is greater than 10%, the composition is an unqualified product that does not meet the requirements;

[0180] (c') Alternatively, if the aromatase activity promotion rate of the composition in the sample to be tested is ≥65%, the composition is a qualified product that meets the requirements; if the aromatase activity promotion rate of the composition in the sample to be tested is <65%, the composition is an unqualified product that does not meet the requirements.

[0181] According to another aspect of the present invention, there is provided a use of the above-mentioned determination method or the above-mentioned quality evaluation method in quality detection and / or quality evaluation and / or quality control of a composition containing Rehmannia glutinosa.

[0182] According to another aspect of the present application, there is provided use of a composition comprising Rehmannia in the preparation of a medicament for promoting aromatase activity.

[0183] wherein the composition comprising Rehmannia includes Rehmannia glutinosa, Fructus Corni, Cortex Moutan, Dioscorea, Poria and Alisma; the mass ratio between the Rehmannia glutinosa, the Fructus Corni, the Cortex Moutan, the Dioscorea, the Poria and the Alisma is about 16: about 8: about 6: about 8: about 6: about 6.

[0184] In the present application, "about" means a value within ±5% of a specified value. For example, "about 16" includes ±5% of 16, or from 15.2 to 16.8; "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0185] In a preferred embodiment, the promoting aromatase activity is increasing testosterone conversion rate.

[0186] In a preferred embodiment, the aromatase is in vitro aromatase.

[0187] The present application is further described in conjunction with the following examples. It should be understood that these examples are merely for illustrative purposes and do not limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally performed according to conventional conditions or the conditions recommended by the manufacturers.

[0188] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are for illustrative purposes only and are not intended to limit the scope of the application.

[0189] The above features mentioned in the present application, or the features mentioned in the examples, can be combined in any manner. All features disclosed in the present patent specification can be used in any combination, and each feature disclosed in the present specification can be replaced by any alternative feature that provides the same, equivalent or similar results. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0190] Examples

[0191] 1. Experimental materials

[0192] 1.1. Medicinal materials and reagents

[0193] Rehmannia root, wine-soaked cornus fruit, moutan bark, yam, poria, and oriental dextrose were purchased from commercial suppliers and were authenticated as authentic. Testosterone (batch number: AFCF1351) was purchased from Chengdu Aifa Biotechnology Co., Ltd.; aromatase (batch number; CSB-EP006394HUa0) was purchased from Huamei Biotechnology; NADPH (batch number: 481973) was purchased from Merck, Germany; and Tris-HCl buffer, pH 7.4 (batch number: AC17151) was purchased from Shanghai Jizhi Biochemical.

[0194] 1.2 Instruments

[0195] Waters e2695 high-performance liquid chromatograph (Waters, USA); XSE 105DU electronic analytical balance (Mettler, Switzerland); KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Axio Scope A1 biological microscope, Zeiss Technology Suzhou Co., Ltd.; Multiskan FC microplate reader, Thermo Fisher Scientific Inc., USA; Revolution ECHO upright and inverted integrated electric intelligent microscopy imaging system, Echo Corporation, USA; electric blast drying oven, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0196] 2. Experimental methods

[0197] 2.1. Study on different solvent extraction methods of compositions containing Rehmannia root

[0198] 2.1.1. Preparation method of concentrated pills containing Rehmannia root

[0199] 160g of Rehmannia root, 80g of wine-soaked cornus fruit, 60g of peony bark, 80g of Chinese yam, 60g of Poria cocos, and 60g of Alisma orientalis are weighed, crushed into fine powder, sieved, and mixed to obtain a mixture; the mixture is made into pills to obtain the composition containing Rehmannia root. The pill-making method is selected from the following: (a) alternately adding the mixture and ethanol to a mold to form pills, and drying to obtain the composition; (b) adding 35-50g of refined honey and an appropriate amount of water per 100g of the mixture to form pills, and drying to obtain the composition; or (c) adding 80-110g of refined honey to the mixture to form pills, and drying to obtain the composition.

[0200] 2.1.2 Preparation of test solution

[0201] Take an appropriate amount of concentrated pills of the composition containing Rehmannia glutinosa, grind them, take about 0.5 g of the powder respectively, accurately weigh it, put it in a conical flask, add 25 mL of purified water, 80% ethanol, pure methanol, 80% methanol, 70% methanol, and 50% methanol respectively, ultrasonically extract for 30 minutes, shake well, filter with a 0.45 μm filter membrane, and take the filtrate to obtain the product.

[0202] 2.1.3 Chromatographic conditions

[0203] Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B; the chromatographic column was Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm); the gradient elution was as shown in Table 1; the column temperature was 25°C; the volume flow rate was 1.0 mL / min; and the injection volume was 10 μL.

[0204] Table 1 Gradient elution program

[0205] Time (min) Acetonitrile (%) Water (0.1% phosphoric acid)% 0 3 97 6 3 97 28 22 78 42 60 40 53 75 25 60 90 10 62 3 97 78 3 97

[0206] 2.1.4. Calculation of the yield rate

[0207] Take an appropriate amount of concentrated pills of the composition containing Rehmannia glutinosa, grind them, take about 0.5 g of the powder respectively, weigh it accurately, put it in a conical flask, add 25 mL of pure water, 80% ethanol, pure methanol, 80% methanol, and 50% methanol respectively, and ultrasonically extract for 30 minutes. Filter the filtrate, evaporate it to dryness in a water bath to obtain an extract, and calculate the extract rate.

[0208] 2.2. In vitro aromatase activity assay

[0209] 2.2.1 Solution preparation

[0210] Preparation of the aqueous extract of the composition containing Rehmannia: Grind an appropriate amount of concentrated pills of the composition containing Rehmannia into fine powder, weigh 0.5 g of the powder of the composition containing Rehmannia, add 25 ml of water and ultrasonically extract for 30 minutes, filter, take the filtrate, evaporate to dryness in a water bath, reconstitute with water to a concentration of 10 mg / mL, and store in a refrigerator at -20°C.

[0211] Testosterone solution: Accurately weigh 5.77 mg of testosterone standard into a 10 mL dry volumetric flask and dilute to 2 mmol / L testosterone solution with methanol.

[0212] Aromatase (CYP 19 A1) Solution preparation: Take out 0.5 mg / mL aromatase solution from a -20°C freezer and dilute it to the desired concentration with Tris-HCl buffer.

[0213] NADPH solution: Use an analytical balance to accurately weigh 1.67 mg of NADPH powder, dissolve it in Tris-HCl buffer and adjust the volume to 2 mmol / L NADPH solution.

[0214] Testosterone blank solution: 250 μL Tris-HCl buffer, 50 μL methanol, 100 μL 1 μg / mL CYP 19A1 solution and 100 μL of 1 mmol / L NADPH solution were added to the centrifuge tube in sequence, and 500 μL of methanol was added to terminate the reaction (to remove protein) to obtain a testosterone blank solution.

[0215] Preparation of test solution: 250 μL Tris-HCl buffer, 50 μL 1 mmol / LT solution, 100 μL 1 μg / mL CYP 19 A1 solution and 100 μL of 1 mmol / L NADPH solution were added to the centrifuge tube in sequence, and 500 μL of methanol was added to terminate the reaction to obtain the test solution.

[0216] 2.2.2 Reaction system composition

[0217] After repeated exploration, a total of 500 μL reaction system was established, 200 μL Tris-HCl buffer (PH = 7.4), 50 μL T solution, 50 μL LWDHW solution (experimental group) / 50 μL Tris-HCl buffer (control group), 100 μL CYP 19 A1 and 100 μL NADPH solution were added to the centrifuge tube in sequence, with a total reaction volume of 500 μL, mixed evenly, and reacted at 37°C for 30 minutes. After the reaction was completed, 500 μL methanol was added to terminate the reaction, and the mixed solutions were taken as the experimental group and the control group respectively. The blank group was added with 350 μL Tris-HCL buffer (PH=7.4), 50 μL T solution, and 100 μL NADPH solution, and 500 μL methanol was added after 0 minutes of reaction (methanol was added immediately after the blank group reactants were added to terminate the reaction) (as shown in Table 2). Each group was operated in triplicate. The solutions of each group were filtered through a 0.45 μm microporous filter membrane, and samples were prepared and the T peak area was detected by HPLC. Aromatase activity was expressed as T μmol·L -1 T conversion of the experimental group = T peak area of ​​the blank group - T peak area of ​​the experimental group; T conversion of the control group = T peak area of ​​the blank group - T peak area of ​​the control group; aromatase activity promotion rate (%) = (T conversion of the experimental group at 30 min - T conversion of the control group at 30 min) / T conversion of the control group at 30 min * 100%.

[0218] Table 2 Composition of in vitro aromatase activity assay system

[0219]

[0220]

[0221] 2.2.3 Chromatographic conditions for testosterone content determination

[0222] Chromatographic column: Beijing Dima Diamonsil C 18(5μm, 250mm×4.6mm); mobile phase: methanol-water (volume ratio 70:30); column temperature: room temperature; detection wavelength: 243nm; flow rate: 1.0mL / min; injection volume: 20μL.

[0223] 2.2.4. Optimization of reaction conditions

[0224] (1) Effect of T concentration on enzyme activity

[0225] First, determine the optimal concentration of substrate T. Keeping aromatase at a fixed concentration of 1 μg / mL and NADPH at 1 mM, gradually increase the T concentration to 50, 100, 500, 1000, 1500, and 2000 μM. Incubate at 37°C for 30 minutes. Analyze by HPLC. Calculate the T concentration based on the T peak area.

[0226] (2) Effect of NADPH concentration on enzyme activity

[0227] In order to determine the optimal concentration of NADPH in the reaction system established in this study, the range of 200-2000 μmol / L NADPH was selected.

[0228] (3) Effect of enzyme reaction concentration on enzyme activity

[0229] After fixing the concentration of substrate T and NADPH, the enzyme concentration required for the reaction system was investigated. The aromatase stock solution was diluted with Tris-HCl buffer to a series of concentrations: 0.25, 0.5, 1, 2, and 4 μg / ml, respectively. The reaction was carried out at 37°C for 30 minutes.

[0230] (4) Effect of reaction time on enzyme activity

[0231] The enzyme concentration, substrate T concentration, NADPH concentration and reaction temperature were fixed, and the effects of different reaction times on enzyme activity were determined. The different reaction times selected were: 10 min, 20 min, 30 min, 60 min, and 90 min.

[0232] (5) Effect of reaction temperature on enzyme activity

[0233] The enzyme concentration, substrate T concentration, and NADPH concentration were fixed, the reaction time was set to 20 min, and different temperature values ​​were set to investigate the effects on enzyme activity. The temperature values ​​were: 25°C, 37°C, 45°C, 50°C, and 60°C.

[0234] 2.2.5 Effect of aqueous extracts of compositions containing Rehmannia root on aromatase activity

[0235] 200 μL Tris-HCl buffer (PH=7.4), 50 μL 1 mmol / LT solution, 50 μL 7.5 mg / mL LWDHW solution (experimental group) / 50 μL Tris-HCl buffer (control group), 100 μL 1 μg / mL CYP 19 A1 and 100 μL of 1 mmol / L NADPH solution were added to a centrifuge tube in sequence, resulting in a total reaction volume of 500 μL. The mixture was mixed thoroughly and allowed to react at 37°C for 20 min. After completion, 500 μL of methanol was added to terminate the reaction. The mixed solution was used as the experimental and control groups. For the blank group, 350 μL of Tris-HCl buffer (pH 7.4), 50 μL of 1 mmol / L NADPH solution, and 100 μL of 1 mmol / L NADPH solution were added after 0 min of reaction. 500 μL of methanol was added after 0 min of reaction. The reaction groups are shown in Table 3. Each group was run in triplicate.

[0236] Calculate the rate (%) of aromatase activity promotion by the composition containing Rehmannia glutinosa. Experimental group T conversion = blank group T peak area at 0 min - experimental group T peak area at 20 min; control group T conversion = blank group T peak area at 0 min - control group T peak area at 20 min; aromatase activity promotion rate (%) = (experimental group T conversion at 20 min - control group T conversion at 20 min) / control group T conversion at 20 min × 100%.

[0237] Table 3 Composition and concentration of each reaction group

[0238] category Experimental group (μL) Control group (μL) Blank group (μL) Tris-HCl (PH=7.4) 200 250 350 T (1mmol / L) 50 50 50 LWDHW (7.5 mg / mL) 50 0 0 <![CDATA[CYP 19 A1(1μg / mL)]]> 100 100 0 NADPH (1mmol / L) 100 100 100

[0239] 2.2.6 Methodological Investigation

[0240] (1) Specificity inspection

[0241] Accurately pipette testosterone solution, testosterone blank solution, and test sample solution, take 20 μL of each and inject it into the liquid chromatograph to examine the testosterone peak shape and its separation from other components under the above chromatographic conditions.

[0242] (2) Linear relationship investigation

[0243] Testosterone solution was precisely pipetted into a series of concentration gradients (200, 100, 50, 25, 12.5, 6.25, and 3.125 μmol / L) for liquid chromatography-mass spectrometry. A standard curve was plotted with testosterone concentration as the abscissa (X) and peak area as the ordinate (Y), yielding the regression equation and regression coefficient.

[0244] (3) Repeatability experiment

[0245] Accurately measure 6 portions of the test solution, take 20 μL of each and inject it into the liquid chromatograph to determine the T peak area and relative standard deviation (RSD).

[0246] (4) Precision test

[0247] Accurately pipette 20 μL of the test solution and inject it 6 times continuously to determine the T peak area and relative standard deviation (RSD).

[0248] (5) Stability test

[0249] At room temperature, accurately pipette 20 μL of the test solution at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h, inject it into the liquid chromatograph, and measure the T peak area and relative standard deviation (RSD).

[0250] (6) Sample recovery experiment

[0251] Accurately measure 6 portions of 250 μL T solution (200 μmol / L), add 5 portions of 250 μL T blank solution to each, add 500 μL of methanol to 1 mL, filter through a 0.45 μm filter membrane, take 20 μL for injection, and calculate the recovery and relative standard deviation.

[0252] 3. Experimental results

[0253] 3.1. Results of different solvent extraction methods for compositions containing Rehmannia root

[0254] The chromatograms of the composition containing Rehmannia root extracted with different solvents are as follows: Figure 1 The chromatograms of the composition containing Rehmannia glutinosa extracted with different solvents are shown in Table 4. The yield of the paste is as follows: water > 50 methanol > 80 methanol > 80 ethanol > pure methanol. Comprehensive analysis of the different solvent extraction methods and the yield of the paste of the composition containing Rehmannia glutinosa showed that when water was used as the extraction solvent, the HPLC chromatogram had more peaks and the highest yield of the paste. Therefore, water was selected as the extraction solvent for the composition containing Rehmannia glutinosa.

[0255] Table 4: The yield of the composition containing Rehmannia root extracted by different solvents

[0256] solvent Powder weight (g) Cream weight (g) Cream yield% water 0.5027 0.2433 48.40 50methanol 0.5016 0.1782 35.53 80 Methanol 0.5026 0.1727 34.36 Pure methanol 0.5014 0.1377 27.46 80 ethanol 0.5084 0.1558 30.65

[0257] 3.2. In vitro aromatase activity assay results

[0258] 3.2.1 Effect of T concentration on enzyme activity

[0259] Table 5 and Figure 2 The results showed that when the substrate concentration ranged from 50 to 1000 μmol / L, the enzyme activity increased with increasing substrate concentration, showing a good linear relationship. Further increases in substrate concentration had little effect on enzyme activity. Therefore, the optimal substrate concentration for this study was 1000 μmol / L.

[0260] Table 5 Effects of different substrate concentrations on enzyme activity

[0261]

[0262] Wherein, T0: the measured concentration of substrate at the start time of enzyme reaction; T 30 : Measured concentration of substrate after 30 min of enzyme reaction;

[0263] 3.2.2 Effect of NADPH concentration on enzyme activity

[0264] Table 6 and Figure 3 The results showed that enzyme activity increased with increasing NADPH concentrations within the range of 200-1000 μmol / L, while enzyme activity decreased at 2000 μmol / L. Therefore, 1000 μmol / L NADPH was selected as the optimal concentration for this study.

[0265] Table 6 Effects of different NADPH concentrations on enzyme activity

[0266]

[0267] 3.2.3 Effect of enzyme reaction concentration on enzyme activity

[0268] Table 7 and Figure 4 The results showed that when the aromatase concentration was in the range of 0.25-1 μg / ml, the enzyme activity first increased with the increase of enzyme concentration, reaching the maximum at 1 μg / ml, and then decreased as the enzyme concentration continued to increase. Therefore, the optimal enzyme concentration was selected as 1 μg / ml in this study.

[0269] Table 7 Effects of different aromatase concentrations on enzyme activity

[0270]

[0271] 3.2.4 Effect of reaction time on enzyme activity

[0272] Table 8 and Figure 5 The results showed that the enzyme activity reached its maximum value when the reaction time was 20 minutes. As the reaction time was prolonged, the enzyme activity decreased. Therefore, the optimal reaction time was selected as 20 minutes.

[0273] Table 8 Effects of different reaction times on enzyme activity

[0274]

[0275] 3.2.5 Effect of reaction temperature on enzyme activity

[0276] Table 9 and Figure 6The results showed that when the enzyme reaction temperature was 37℃, the enzyme activity reached its maximum value, and the enzyme activity would decrease when the temperature was lowered or increased. Therefore, the optimal reaction temperature was determined to be 37℃.

[0277] Table 9 Effects of different reaction temperatures on enzyme activity

[0278]

[0279] 3.2.6 Effect of Compositions Containing Rehmannia glutinosa on Aromatase Activity

[0280] The testosterone peak area measurement results of each group are shown in Table 10. The composition containing Rehmannia root can promote aromatase activity and increase T conversion. The T conversion amount of the experimental group is 84088, and the T conversion amount of the control group is 50103. The promotion rate of aromatase activity of the composition containing Rehmannia root is 67.83%.

[0281] Table 10 T peak area measurement results of each group

[0282] Experimental group control group Blank group 1 969792 996427 1068011 2 967228 1007579 1042775 3 993366 1028333 1071864 average value 976795 1010780 1060883

[0283] 3.2.7. Specificity Inspection

[0284] Figure 7 The results showed that under the above chromatographic conditions, the chromatographic peak of testosterone standard was symmetrical and well separated from other components.

[0285] 3.2.8. Linear Relationship Investigation

[0286] Table 11 and Figure 8 The results showed that testosterone showed a good linear relationship in the range of 3.125 to 200 μmol / L, and the regression equation was Y = 18946X + 13089, R 2 =0.9997.

[0287] Table 11 Experimental results of investigation on linear relationship of T

[0288]

[0289] 3.2.9. Repeatability Experimental Results

[0290] The results in Table 12 show that the RSD value of the measured values ​​of the 6 T test solutions is 1.86%, indicating that the repeatability of the operation method is good.

[0291] Table 12 Repeatability test results

[0292]

[0293] 3.2.10 Precision test results

[0294] The results in Table 13 show that the RSD of the six determinations of testosterone is 0.18%, indicating that the instrument has good precision.

[0295] Table 13 Precision test results

[0296]

[0297] 3.2.11 Stability test results

[0298] The results in Table 14 show that the RSD of the six measurements within 6 hours is 0.23%, indicating that the solution is stable within 6 hours. To ensure accurate and reliable results, the experiment should be completed within 6 hours or the test solution should be prepared and used immediately.

[0299] Table 14 Stability test results

[0300]

[0301]

[0302] 3.2.12. Sample recovery test results

[0303] The results in Table 15 show that the average recovery of the six samples was 100.73% and the RSD was 0.56%, indicating that the determination method was accurate and feasible.

[0304] Table 15 Sample recovery experimental results

[0305]

[0306] 4. Summary

[0307] The optimal conditions for the in vitro aromatase activity assay were: testosterone 1 mmol / L, aromatase 1 μg / mL, NADPH 1 mmol / L, reaction temperature 37°C, and reaction time 20 min. The assay of a composition containing Rehmannia root using this bioactivity assay showed that the composition promoted aromatase activity by 67.83%. Methodological studies demonstrated that other components in the reaction solution did not interfere with the testosterone assay. The testosterone concentration showed good linearity within the range of 3.125 to 200 μmol / L, with a correlation coefficient of r = 0.9997. The RSD for repeatability was 1.86%, the RSD for precision was 0.18%, the RSD for stability within 6 hours was 0.23%, and the recoveries ranged from 99.98% to 101.50%.

[0308] The above has carried out the detailed introduction to the embodiment of the application, the principle and implementation mode of the application have been described in this paper by applying specific examples, the above embodiment description is only used for helping understanding the method of the application and its core idea. Meanwhile, the changes or deformations made by the person skilled in the art according to the idea of the application, based on the specific implementation mode and application range of the application, all belong to the protection range of the application. In summary, the content of the specification should not be understood as the limitation of the application.

Claims

1. A method for determining aromatase activity of a composition comprising Rehmannia glutinosa, characterized in that: The determination method comprises the following steps: (1) constructing an aromatase activity reaction system and a control system to obtain an aromatase activity reaction solution and a control solution, respectively; (2) preparing a blank solution; (3) taking appropriate amounts of the aromatase activity reaction solution, the control solution, and the blank solution for high performance liquid chromatography (HPLC) detection to obtain the detection results; as well as (4) obtaining aromatase activity information of the composition comprising Rehmannia glutinosa based on the detection results; The composition comprising Rehmannia root comprises Rehmannia root prepared, Cornus officinalis, Paeonia suffruticosa root, Dioscorea opposita, Poria cocos and Alisma orientalis; The chromatographic conditions for the HPLC detection are as follows: using Diamonsil C 18 Chromatographic column, mobile phase A is methanol, mobile phase B is water, isocratic elution, flow rate is 0.5-1.5 mL / min, column temperature is 20-30°C, detection wavelength is 220-260 nm, and injection volume is 15-25 μL.

2. The measuring method according to claim 1, wherein The method for constructing the aromatase activity reaction system comprises the following steps: sequentially adding a Tris-HCl buffer, a testosterone solution, an aqueous extract of a composition containing Rehmannia root, an aromatase solution, and an NADPH solution into a centrifuge tube, mixing them evenly and reacting them at a temperature of 30 to 45° C. for 10 to 90 minutes, then adding 400 to 600 μL of methanol to terminate the reaction, and filtering to obtain the aromatase activity reaction solution; Preferably, the method for constructing the control system comprises the following steps: sequentially adding Tris-HCl buffer, testosterone solution, aromatase solution and NADPH solution to a centrifuge tube, mixing and reacting at a reaction temperature of 30-45° C. for 10-90 min, then adding 400-600 μL of methanol to terminate the reaction, filtering, and obtaining the control solution; Preferably, the method for preparing the blank solution comprises the following steps: sequentially adding Tris-HCl buffer, testosterone solution and NADPH solution to a centrifuge tube, adding 400-600 μL of methanol, and filtering to obtain the blank solution; More preferably, in the method for constructing the aromatase activity reaction system, the volume ratio of the Tris-HCL buffer, the testosterone solution, the aqueous extract of the composition containing Rehmannia root, the aromatase solution, and the NADPH solution is about 200: about 50: about 50: about 100: about 100; More preferably, in the method for constructing the control system, the volume ratio of the Tris-HCL buffer, the testosterone solution, the aromatase solution, and the NADPH solution is about 250: about 50: about 100: about 100; More preferably, in the method for preparing the blank solution, the volume ratio of the Tris-HCL buffer, the testosterone solution and the NADPH solution is about 350:about 50:about 100.

3. The measuring method according to claim 2, wherein The pH value of the Tris-HCl buffer is about 7.4; Preferably, the volume of methanol is about 500 μL; Preferably, the reaction temperature is about 37°C; Preferably, the reaction time is about 20 min or about 30 min; More preferably, the filtration is membrane filtration; More preferably, the pore size of the filter membrane is 0.45 μm.

4. The measuring method according to claim 2, wherein The preparation method of the testosterone solution comprises the following steps: weighing an appropriate amount of a testosterone standard and placing it in a volumetric flask, adding methanol, and adjusting the volume to obtain the testosterone solution; Preferably, the mass of the testosterone standard is 5 to 7 mg, for example, about 5.77 mg; Preferably, the concentration of the testosterone solution is 0.5 to 2 mmol / L, for example, about 1 mmol / L; More preferably, the aromatase solution is diluted with Tris-HCl buffer; More preferably, the concentration of the aromatase solution is 0.25 to 2 μg / mL, for example, about 1 μg / mL; Preferably, the method for preparing the NADPH solution comprises the following steps: weighing an appropriate amount of NADPH powder, adding Tris-HCl buffer to dissolve it, and constant volume to obtain the NADPH solution; Still more preferably, the mass of the NADPH powder is 1 to 2 mg, for example, about 1.67 mg; Still more preferably, the concentration of the NADPH solution is 0.2 to 2 mmol / L, for example, about 1 mmol / L; Even more preferably, when the testosterone in the aromatase activity reaction solution is in the range of 3.125 to 200 μmol / L, the correlation coefficient r of the linear relationship of the determination method is ≥ 0.9995.

5. The measuring method according to claim 2, wherein The preparation method of the aqueous extract of the composition containing Rehmannia root comprises the following steps: weighing an appropriate amount of the composition containing Rehmannia root, grinding to obtain a composition powder, adding water to dissolve the composition powder, ultrasonically extracting, filtering to obtain a filtrate; evaporating the filtrate to dryness in a water bath, and redissolving it in water to obtain the aqueous extract of the composition containing Rehmannia root; Preferably, the mass of the composition powder is 0.1 to 1 g, for example, about 0.5 g; Preferably, the water is purified water; Preferably, the mass / volume (g / mL) ratio of the composition powder to the water in the water dissolution is 0.01 to 0.05, for example, about 0.02; Preferably, the ultrasonic extraction time is 20 to 40 minutes, for example, about 30 minutes; Preferably, the concentration of the aqueous extract of the composition comprising Rehmannia glutinosa is 1 to 20 mg / mL, for example, about 5 mg / mL or about 10 mg / mL; More preferably, the preparation method of the composition comprising Rehmannia root comprises: weighing appropriate amounts of Rehmannia root, wine-soaked Cornus fruit, peony bark, Chinese yam, Poria cocos and Alisma orientalis, grinding them into fine powder, sieving, and mixing to obtain a mixture; and making pills from the mixture to obtain the composition comprising Rehmannia root; Preferably, the pill-making method is selected from the following: (a) alternately adding the mixture and ethanol to a mold to form pills, and drying to obtain the product; (b) adding refined honey and water to the mixture to form pills, and drying to obtain the product; or (c) adding refined honey to the mixture to form pills, and drying to obtain the product; More preferably, in method (b), the mass ratio of the refined honey to the mixture is 0.3 to 0.6, for example 0.35 to 0.5; Even more preferably, in method (c), the mass ratio of the refined honey to the mixture is 0.7 to 1.2, such as 0.8 to 1.

1.

6. The measuring method according to claim 1, wherein The mass ratio of the Radix Rehmanniae Preparata, the Fructus Corni with Wine Wine, the Cortex Moutan, the Rhizoma Dioscoreae, the Poria and the Rhizoma Alismatis is (10-20):(4-12):(2-10):(4-12):(2-10):(2-10); Preferably, the mass ratio of the Radix Rehmanniae Preparata, the Fructus Corni Wine Extract, the Cortex Moutan, the Rhizoma Dioscoreae, the Poria and the Rhizoma Alismatis is (14-18):(6-10):(4-8):(6-10):(4-8):(4-8); More preferably, the mass ratio of the Radix Rehmanniae Preparata, the Fructus Corni of Wine Wine, the Cortex Moutan, the Dioscorea Oppositae, the Poria and the Rhizoma Alismatis is about 16: about 8: about 6: about 8: about 6: about 6; Preferably, the mass of the Rehmannia glutinosa is 140-180 g, for example, about 160 g; Preferably, the weight of the wine-boiled cornus fruit is 60-100 g, for example, about 80 g; Preferably, the mass of the peony bark is 40 to 80 g, for example, about 60 g; Preferably, the mass of the yam is 60-100 g, for example, about 80 g; Preferably, the mass of the Poria cocos is 40 to 80 g, for example, about 60 g; More preferably, the mass of the Rhizoma Alismatis is 40-80 g, for example, about 60 g.

7. The measuring method according to claim 1, wherein The test result is the peak area of ​​the testosterone chromatographic peak in the chromatogram obtained by high performance liquid chromatography detection of the aromatase activity reaction solution, the control solution, and the blank solution; Preferably, the aromatase activity information is calculated based on the recorded peak area of ​​the testosterone chromatographic peak in the aromatase activity reaction solution, the peak area of ​​the testosterone chromatographic peak in the control solution, and the peak area of ​​the testosterone chromatographic peak in the blank solution, and the aromatase activity promotion rate is calculated based on the testosterone conversion amount in the aromatase activity reaction solution and the testosterone conversion amount in the control solution; More preferably, the calculation formula for the testosterone conversion amount in the aromatase activity reaction solution is: testosterone conversion amount in the aromatase activity reaction solution = peak area of ​​the testosterone chromatographic peak in the blank solution - peak area of ​​the testosterone chromatographic peak in the aromatase activity reaction solution; More preferably, the calculation formula for the testosterone conversion amount of the control solution is: testosterone conversion amount of the control solution = peak area of ​​the testosterone chromatographic peak in the blank solution - peak area of ​​the testosterone chromatographic peak in the control solution; Preferably, the calculation formula of the aromatase activity promotion rate is: (testosterone conversion amount in aromatase activity reaction solution - testosterone conversion amount in control solution) / testosterone conversion amount in control solution × 100%; Furthermore, preferably, the volume ratio of the mobile phase A to the mobile phase B in the high performance liquid chromatography (HPLC) assay is about 7: about 3; Also preferably, the flow rate is about 1.0 mL / min; Also preferably, the column temperature is room temperature, for example, about 25°C; Furthermore, preferably, the detection wavelength is 243 nm; Also preferably, the injection volume is about 20 μL; More preferably, the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; Even more preferably, the aromatase is an in vitro aromatase.

8. A method for evaluating the quality of a composition comprising Rehmannia root, characterized in that: The quality evaluation method comprises the following steps: (a) optionally, obtaining an aromatase activity promotion rate of a composition reference sample according to the aromatase activity assay method described in any one of claims 1 to 7; (b) taking a water extract of the composition and performing an aromatase activity assay according to any one of claims 1 to 7 to obtain an aromatase activity promotion rate of the composition sample; and (c) comparing the aromatase activity promotion rate of the test sample of the composition obtained in step (b) with the aromatase activity promotion rate of the reference sample of the composition obtained in step (a); if the difference between the average values ​​of the two is ≤10%, the composition is a qualified product that meets the requirements; if the difference between the average values ​​of the two is greater than 10%, the composition is an unqualified product that does not meet the requirements; (c') Alternatively, if the aromatase activity promotion rate of the composition in the sample to be tested is ≥65%, the composition is a qualified product that meets the requirements; if the aromatase activity promotion rate of the composition in the sample to be tested is <65%, the composition is an unqualified product that does not meet the requirements.

9. Use of the assay method according to any one of claims 1 to 7 or the quality evaluation method according to claim 8 in quality detection and / or quality evaluation and / or quality control of a composition comprising Rehmannia root.

10. Use of a composition comprising Rehmannia root in the preparation of a medicament for promoting aromatase activity; in, The composition comprising Rehmannia root comprises Rehmannia root prepared, cornus officinalis, moutan bark, Chinese yam, Poria cocos and Alisma orientalis; the mass ratio of the Rehmannia root prepared, cornus officinalis, moutan bark, Chinese yam, Poria cocos and Alisma orientalis is about 16: about 8: about 6: about 8: about 6: about 6; Preferably, the promoting aromatase activity is to increase the testosterone conversion rate; Preferably, the aromatase is an in vitro aromatase.

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