Establishment and application of fingerprint of Taohong Siwu decoction

Abstract

This invention discloses a fingerprint spectrum of Taohong Siwu Decoction and its determination method. The method employs high-performance liquid chromatography (HPLC), and the chromatographic conditions include: using an octadecylsilane-bonded silica column, with phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, and performing gradient elution, which is suitable for detecting the fingerprint spectrum of Taohong Siwu Decoction.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to the establishment and application of a fingerprint spectrum for Taohong Siwu Decoction. Background Technology

[0002] Taohong Siwu Decoction is a classic formula recorded in ancient books, with the functions of nourishing blood, promoting blood circulation, and removing blood stasis. It is mainly used to treat blood deficiency, manifested as irregular menstruation in women, heavy menstrual bleeding with clots, dark purple and sticky blood, abdominal pain and distension, etc. The reference sample of Taohong Siwu Decoction is the substance obtained after decoction, in its concentrated and dried form. The reference sample loses the original morphological fingerprint of the medicinal slices; qualitative identification based solely on indicator components is insufficient to reflect its quality. Therefore, the quality control of the reference sample relies primarily on overall quality control using fingerprint spectroscopy.

[0003] The main active ingredients of Taohong Siwu Decoction are flavonoids, phenylethanol glycosides, phenolic acids, monoterpenes and their glycosides, volatile oils, cyanogenic glycosides, and polysaccharides. Among them, caffeic acid has the functions of constricting and strengthening microvessels, increasing coagulation factors, and raising white blood cells and platelets. It stimulates the maturation of megakaryocytes, thereby promoting the release of platelets from megakaryocytes and reducing the destruction of platelets by drugs, and is used to prevent bleeding or stop bleeding. It is one of the main pharmacologically active components of Taohong Siwu Decoction. However, caffeic acid is unstable, and the detection of caffeic acid is not disclosed in the currently published chromatograms and quality control methods of Taohong Siwu Decoction. Chinese Patent Publication No. CN107478749A, entitled "Detection Method of Taohong Siwu Decoction for Pharmaceutical Preparation", discloses a fingerprint spectrum of Taohong Siwu Decoction. It selects peak 7 (hydroxysaffron yellow A) as the internal reference peak in the fingerprint spectrum, confirms the common characteristic peaks peak 7 (hydroxysaffron yellow A), peak 8 (paeoniflorin), and peak 9 (ferulic acid), and determines the relative retention times of 11 common characteristic peaks of Taohong Siwu Decoction granules, but does not include the characteristic peak of caffeic acid.

[0004] Based on the important role of caffeic acid in Taohong Siwu Decoction, the inventors believe that identifying caffeic acid in the fingerprint spectrum of Taohong Siwu Decoction is more important and can more accurately and comprehensively evaluate the quality of Taohong Siwu Decoction. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a fingerprint spectrum and determination method for Taohong Siwu Decoction that can detect multiple substances such as caffeic acid, which is suitable for accurate and comprehensive evaluation of the quality of Taohong Siwu Decoction.

[0006] This invention provides a method for obtaining fingerprint spectroscopy results for Taohong Siwu Decoction, comprising the following steps:

[0007] A fingerprint chromatogram of Taohong Siwu Decoction was established using high performance liquid chromatography-visible ultraviolet detection. The chromatographic conditions included: an octadecylsilane-bonded silica column, phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution.

[0008] According to the present invention, the gradient elution involves the following steps: within 0 to 20 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 15%B to 25%B at a uniform rate; within 20 to 40 minutes, the volume ratio of 25%B gradually changes from 40%B to 40%B at a uniform rate; and within 40 to 80 minutes, the volume ratio of 40%B gradually changes from 40%B to 70%B at a uniform rate.

[0009] According to the present invention, the test solution is prepared by accurately weighing 1g of Taohong Siwu Decoction standard, placing it in a stoppered conical flask, accurately adding 25ml of water, sonicating for 30min, extracting three times with ethyl acetate, 25ml each time, combining the ethyl acetate, evaporating to dryness, dissolving the residue in methanol, transferring to a 10ml volumetric flask, diluting with methanol to the mark, shaking well, filtering, and discarding the filtrate to obtain the test solution.

[0010] According to the present invention, the determination method is as follows: the test solution is pipetted into a liquid chromatograph, measured, and the chromatogram is recorded to obtain the solution.

[0011] According to the present invention, the chromatographic column has a length of 250 mm and a diameter of 4.6 mm.

[0012] According to the present invention, the detection wavelength is 230–250 nm.

[0013] According to the present invention, the column temperature is 30°C.

[0014] In this invention, the injection volume is 10 μl.

[0015] According to the present invention, the specific steps for determining the fingerprint spectrum are as follows: preparing a test solution, injecting the sample into a liquid chromatograph, and recording the chromatogram.

[0016] This invention also provides the application of fingerprint spectrum of Taohong Siwu Decoction, which can be used to identify each herb in Taohong Siwu Decoction and to evaluate the quality of Taohong Siwu Decoction.

[0017] Effect 1. The method established in this invention is applicable to the material reference and preparation of Taohong Siwu Decoction, and controls the key quality attributes of the material reference and preparation.

[0018] 2. Through gradient elution experiments, this invention has found that using phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B can effectively separate the characteristic chromatographic peaks of Taohong Siwu Decoction.

[0019] 3. This invention utilizes high performance liquid chromatography-visible ultraviolet detection to establish a fingerprint spectrum of Taohong Siwu Decoction by comprehensively considering the combined effects of various parameters on separation and detection. Detailed Implementation

[0020] To make the advantages of the present invention clearer, the technical solution of the present invention will be described in more detail below.

[0021] 1. Instruments

[0022] Shimadzu LC-2030 High Performance Liquid Chromatograph

[0023] Column C 18 (4.6×250mm)

[0024] Electronic balance (1 / 100,000) Mettler Toledo Instruments (Shanghai) Co., Ltd. Electronic balance (1 / 10,000) Mettler Toledo Instruments (Shanghai) Co., Ltd.

[0025] Ultrasonic Cleaner Kunshan Ultrasonic Instrument Co., Ltd.

[0026] 2. Selection of chromatographic column conditions

[0027] Procedure for high-performance liquid chromatography determination of material standards:

[0028] Preparation of the test solution: Weigh 1g of the material standard powder accurately, place it in an Erlenmeyer flask, add 25ml of water, sonicate for 30min, centrifuge, take the supernatant, extract three times with 25ml of ethyl acetate, combine the extracts, evaporate to dryness, dissolve in methanol, transfer to a 10ml volumetric flask, dilute to the mark with methanol, filter, and take the filtrate to obtain the test solution.

[0029] Column: octadecylsilane-bonded silica gel as the packing material

[0030] Mobile phase: 0.1% phosphoric acid (A) - acetonitrile (B)

[0031] Flow rate: 1 ml / min

[0032] Column temperature: 30℃

[0033] Gradient elution program: From 0 to 20 min, the volume ratio of mobile phase B is gradually changed from 15% to 25% at a constant rate; from 20 to 40 min, the volume ratio of 25% B is gradually changed from 40% B at a constant rate; from 40 to 80 min, the volume ratio of 40% B is gradually changed from 70% B at a constant rate.

Claims

1. A method for constructing a fingerprint spectrum of Taohong Siwu Decoction, characterized in that... The method includes the preparation and determination of the test solution; The determination method described herein employs high-performance liquid chromatography-ultraviolet detection. The chromatographic conditions include: an octadecylsilane-bonded silica column; phosphoric acid solution as mobile phase A; acetonitrile as mobile phase B; and gradient elution. The elution gradient is as follows: from 0 to 20 min, the volume ratio of mobile phase A to mobile phase B gradually changes from 15%B to 25%B; from 20 to 40 min, it gradually changes from 25%B to 40%B; and from 40 to 80 min, it gradually changes from 40%B to 70%B.

2. The preparation of the test sample according to claim 1 is as follows: Take 1g of Taohong Siwu Decoction standard, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, sonicate for 30min, extract with ethyl acetate three times, 25ml each time, combine the ethyl acetate, evaporate to dryness, dissolve the residue in methanol, transfer to a 10ml volumetric flask, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain the test sample.

3. The method according to claim 1, characterized in that, The mobile phase consists of 0.1% phosphoric acid solution A to acetonitrile B, with gradient elution.

4. The method according to potential requirement 1, characterized in that, The chromatographic column is 250 mm long and 4.6 mm in diameter; the detection wavelength is 240 nm.

5. The method according to potential requirement 1, characterized in that, The flow rate was 0.8–1.2 ml per minute, and the column temperature was 28–35 °C.

6. The method according to claim 1, characterized in that, The injection volume was 10 μL.

7. The fingerprint spectrum of Taohong Siwu Decoction constructed by any one of the methods described in claims 1 to 6.

8. The application of the fingerprint spectrum of Taohong Siwu Decoction as described in claim 7, characterized in that: ① Used to identify whether Taohong Siwu Decoction contains wine-processed Angelica sinensis, Ligusticum chuanxiong, wine-processed Paeonia lactiflora, or wine-processed Carthamus tinctorius; ② Used to evaluate the quality of Taohong Siwu Decoction.