CN105203670B - Establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge - Google Patents

Abstract

The invention discloses a method for establishing a high-performance liquid chromatography fingerprint of loofah, relating to a method for establishing a fingerprint of loofah, a standard fingerprint and application thereof. The method of the present invention is: one, prepare need testing solution; Two, make fingerprint spectrum, need testing solution injects high performance liquid chromatography; The fingerprint spectrum of loofah of the present invention has 7 common characteristic peaks, and these common characteristic peaks constitute The fingerprint characteristics of loofah are obtained, which can be used as the standard fingerprint of loofah. The invention has the characteristics of simple and convenient method, high stability, high precision, good reproducibility and easy mastery. The use of the loofah standard fingerprint of the present invention means that the loofah fingerprint is used as a standard fingerprint for identification and quality control of the authenticity, place of origin and quality of the loofah medicinal material. The invention is applied in the field of medicine.

Description

A kind of establishment method of high performance liquid chromatography fingerprint of loofah

technical field

The invention relates to a method for establishing a loofah fingerprint and its standard fingerprint and application.

Background technique

Loofah, that is, the vascular bundle of the dry ripe fruit of the Cucurbitaceae plant loofah Luffa cylindrica (L.) Roem. It mainly contains sterols, saponins, flavonoids, phenols, proteins, amino acids, and organic acids. It has the effects of expelling wind, dredging collaterals, promoting blood circulation, and breast milk. Symptoms such as swelling and pain. Modern studies have shown that loofah has the effect of protecting myocardial ischemia, and has also achieved good results in reducing blood fat, inhibiting weight gain, anti-inflammatory, analgesic and sedative, and anti-allergic. In terms of quality control, the 2010 edition of "The Pharmacopoeia of the People's Republic of China" contains loofah medicinal materials, but only powder identification is carried out. Due to the complex origin, source, and harvest season of traditional Chinese medicine, it is necessary to adopt more scientific and reasonable methods to control its quality, which is very necessary to ensure the safety and effectiveness of clinical medication. As a technique for quality control of traditional Chinese medicine as a whole, fingerprinting has been widely recognized, and its advantages are more obvious than single-component or even multi-component qualitative and quantitative methods. At present, there is no research report on the fingerprint of loofah in the literature. Therefore, in order to scientifically and effectively control its internal quality, it is necessary to establish the fingerprint of loofah.

Contents of the invention

The purpose of the present invention is to provide a method for establishing the high-performance liquid chromatography fingerprint of loofah and its standard fingerprint and application.

The establishment method of the high performance liquid chromatography fingerprint of loofah of the present invention is to carry out according to the following steps:

One, the preparation of need testing solution: a, get loofah medicinal material, add the mass percent concentration of 10 times of quality and be 75% ethanol, soak 12h, ultrasonic 30min, filter, collect solid phase thing A and liquid phase thing A respectively, Add solid phase A to 75% ethanol at a mass percentage concentration of 10 times the mass, soak for 8 hours, ultrasonicate for 30 minutes, filter, collect liquid phase B, then mix liquid phase A and B to obtain an extract, and extract The extract was concentrated under reduced pressure to obtain the extract;

B, dissolving the extract with a small amount of distilled water and then putting it on the polyamide chromatographic column, washing 4 column volumes with distilled water earlier, then eluting 4 column volumes with 30% ethanol with a mass concentration, collecting the eluent, and then reducing The eluate was concentrated under pressure to dryness to obtain dry solid C;

c, the dry solid C obtained in step b is dissolved into a solution with a concentration of 2mg/mL with methanol, and filtered with a microporous membrane, and the filtrate is the test solution;

Two, the making of fingerprint collection: the need testing solution that step 1 obtains is injected high-performance liquid chromatograph, is that the mixed solution of methanol and acetonitrile with volume ratio 1:1 is phase A, and mass percent concentration is the acetic acid of 0.5% The aqueous solution is phase B for gradient elution; among them, the high performance liquid chromatography conditions are: chromatographic column filler Thermo C18, the specification is 250×4.6mm, 5μm; the column temperature is 35°C; the flow rate is 0.8mL/min, and the detection wavelength is 280nm. The sample volume is 20μL; analyze the solution of the test product under this condition to obtain the fingerprint of the loofah.

3. Determination of the standard fingerprint spectrum: 7 common characteristic peaks have been determined; with No. 7 peak as the contrast peak, the relative retention times of the common characteristic peaks are respectively: the relative retention time of the common characteristic peaks of No. 1 is 0.368±0.003%, and The relative retention time of peak No. 3 is 0.433±0.01%, the relative retention time of No. 3 peak is 0.462±0.025%, the relative retention time of No. 4 peak is 0.613±0.016%, the relative retention time of No. The retention time is 0.881 ± 0.028%, and the No. 7 peak has a relative retention time of 1.000. The above-mentioned common characteristic peaks constitute the fingerprint characteristics of the loofah, which can be used as the standard fingerprint of the loofah.

The use of the loofah standard fingerprint of the present invention means that the loofah fingerprint is used as a standard fingerprint in the quality control of the loofah.

Beneficial effects of the present invention:

The loofah standard fingerprint of the present invention has 7 common characteristic peaks, and these common characteristic peaks constitute the fingerprint feature of the loofah, which can be used as the standard fingerprint of the loofah. The loofah standard fingerprint of the present invention can be used in the quality control of loofah as a standard fingerprint after being verified by many batches of samples, and is used for evaluating and controlling the quality of loofah medicinal materials as a whole, which avoids the problem of only measuring one or two The one-sidedness of judging the overall quality of loofah based on chemical components also reduces the possibility of artificially adding control components to meet quality standards. The method for establishing the HPLC fingerprint of the loofah of the invention has the advantages of strong repeatability, high precision, stability and reliability, and easy mastery, and is suitable for identification and quality control of authenticity, origin and quality of loofah medicinal materials.

Description of drawings

Fig. 1 is the loofah standard fingerprint spectrum that test 1 provides; Wherein 1 to 7 are 7 common characteristic peaks of loofah;

Fig. 2 is that S1-S10 are 10 batches of luffa medicinal material fingerprints.

detailed description

The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

The specific embodiment one: the establishment method of the high-performance liquid chromatography fingerprint of the loofah of the present embodiment is to carry out according to the following steps:

One, the preparation of need testing solution: a, get loofah medicinal material, add the mass percent concentration of 10 times of quality and be 75% ethanol, soak 12h, ultrasonic 30min, filter, collect solid phase thing A and liquid phase thing A respectively, Add solid phase A to 75% ethanol at a mass percentage concentration of 10 times the mass, soak for 8 hours, ultrasonicate for 30 minutes, filter, collect liquid phase B, then mix liquid phase A and B to obtain an extract, and extract The extract was concentrated under reduced pressure to obtain the extract;

B, dissolving the extract with a small amount of distilled water and then putting it on the polyamide chromatographic column, washing 4 column volumes with distilled water earlier, then eluting 4 column volumes with 30% ethanol with a mass concentration, collecting the eluent, and then reducing The eluate was concentrated under pressure to dryness to obtain dry solid C;

c, the dry solid C obtained in step b is dissolved into a solution with a concentration of 2mg/mL with methanol, and filtered with a microporous membrane, and the filtrate is the test solution;

Two, the making of fingerprint collection: the need testing solution that step 1 obtains is injected high-performance liquid chromatograph, is that the mixed solution of methanol and acetonitrile with volume ratio 1:1 is phase A, and mass percent concentration is the acetic acid of 0.5% The aqueous solution is phase B for gradient elution; among them, the high performance liquid chromatography conditions are: chromatographic column filler Thermo C18, the specification is 250×4.6mm, 5μm; the column temperature is 35°C; the flow rate is 0.8mL/min, and the detection wavelength is 280nm. The sample volume is 20μL; analyze the solution of the test product under this condition to obtain the fingerprint of the loofah.

Embodiment 2: This embodiment differs from Embodiment 1 in that: the microporous membrane filtration in step 1 is performed by using a 0.45 μm microporous membrane. Others are the same as in the first embodiment.

Specific embodiment three: the difference between this embodiment and specific embodiment one or two is: the gradient elution sequence in step two is: 0min → 20min → 50min → 80min, and the volume ratio is 1:1 methanol and acetonitrile mixed solution 5% → 16% → 25% → 40%. Others are the same as in the first or second embodiment.

Embodiment 4: This embodiment adopts the standard fingerprint of loofah obtained by a method for establishing the high performance liquid chromatography fingerprint of loofah.

The standard fingerprint of the loofah in this embodiment has 7 common characteristic peaks, and these common characteristic peaks constitute the fingerprint characteristics of the loofah, which can be used as the standard fingerprint of the loofah.

Specific embodiment five: this embodiment is different from specific embodiment four: the standard fingerprint of loofah has 7 common characteristic peaks; With No. 7 peaks as contrast peaks, the relative retention times of common characteristic peaks are respectively: No. 1 The relative retention time of the common characteristic peaks is 0.368±0.003%, the relative retention time of peak No. 2 is 0.433±0.01%, the relative retention time of peak No. 3 is 0.462±0.025%, the relative retention time of peak No. The peak relative retention time is 0.667 ± 0.02%, the No. 6 peak relative retention time is 0.881 ± 0.028%, and the No. 7 peak relative retention time is 1.000. These 7 common characteristic peaks constitute the fingerprint feature of loofah, which can be used as the fingerprint of loofah. Standard Fingerprint. Others are the same as in Embodiment 4.

Embodiment 6: The use of the standard fingerprint of the loofah in this embodiment refers to that the fingerprint of the loofah is used as a standard fingerprint for the quality control of the loofah.

The loofah standard fingerprint of this embodiment can be used in the quality control of loofah after being verified by many batches of samples as a standard fingerprint, and is used to evaluate and control the quality of loofah medicinal materials as a whole, which avoids the problem of only measuring one or two The one-sidedness of judging the overall quality of loofah by using only one chemical component also reduces the possibility of artificially adding control components to meet quality standards.

Prove the beneficial effect of the present invention by following test:

Test 1:

1, test method: the establishment method of the HPLC fingerprint of this test loofah, is to carry out according to the following steps:

One, the preparation of need testing solution: a, get loofah medicinal material, add the mass percent concentration of 10 times of quality and be 75% ethanol, soak 12h, ultrasonic 30min, filter, collect solid phase thing A and liquid phase thing A respectively, Add solid phase A to 75% ethanol at a mass percentage concentration of 10 times the mass, soak for 8 hours, ultrasonicate for 30 minutes, filter, collect liquid phase B, then mix liquid phase A and B to obtain an extract, and extract The extract was concentrated under reduced pressure to obtain the extract;

B, dissolving the extract with a small amount of distilled water and then putting it on the polyamide chromatographic column, washing 4 column volumes with distilled water earlier, then eluting 4 column volumes with 30% ethanol with a mass concentration, collecting the eluent, and then reducing The eluate was concentrated under pressure to dryness to obtain dry solid C;

c, the dry solid C obtained in step b is dissolved into a solution with a concentration of 2mg/mL with methanol, and filtered with a microporous membrane, and the filtrate is the test solution;

Two, the making of fingerprint collection: the need testing solution that step 1 obtains is injected high-performance liquid chromatograph, is that the mixed solution of methanol and acetonitrile with volume ratio 1:1 is phase A, and mass percent concentration is the acetic acid of 0.5% The aqueous solution is phase B for gradient elution; among them, the conditions of high performance liquid chromatography are: 1. The column filler Thermo C18, the specification is 250×4.6mm, 5μm; the column temperature is 35°C; the gradient elution method is 0min→20min→ 50min→80min, methanol and acetonitrile mixed solution with a volume ratio of 1:1 5%→16%→25%→40%; flow rate 0.8mL/min, detection wavelength 280nm, injection volume 20μL;

2. Test equipment: HITACHI L-2000 high performance liquid chromatography; HITACHI L-2400 ultraviolet detector; AT-130 chromatographic column thermostat; HITACHI D-2000 chromatographic workstation.

3. Test materials: 10 batches of loofah medicinal materials come from Hunan, Shandong, Anhui, Zhejiang, Guangxi, Shanxi, Jiangsu and other places.

According to the establishment method of the high-performance liquid chromatography fingerprint of the loofah of this test, 10 batches of loofah medicinal materials have established the high-performance liquid chromatography fingerprint, and the spectrum obtained is imported into the Chinese medicine chromatographic fingerprint similarity calculation software (pharmacopoeia of the People's Republic of China Committee, 2004 version), determined 7 common characteristic peaks, with No. 7 peak as contrast peak, the relative retention time of common characteristic peak is respectively: No. 1 common characteristic peak relative retention time is 0.368 ± 0.003%, No. 2 peak relative retention time The retention time is 0.433±0.01%, the relative retention time of No. 3 peak is 0.462±0.025%, the relative retention time of No. 4 peak is 0.613±0.016%, the relative retention time of No. 5 peak is 0.667±0.02%, and the relative retention time of No. 6 peak 0.881 ± 0.028%, No. 7 peak relative retention time is 1.000, above-mentioned common characteristic peak constitutes the fingerprint characteristic of loofah, can be used as the standard fingerprint of loofah.

The standard fingerprint spectrum of the loofah that this test obtains is shown in Figure 1. As can be seen from Figure 1, the standard fingerprint of the loofah with 7 common characteristic peaks can be clearly obtained by this method, which provides a good basis for the overall evaluation of the quality of the loofah.

4. Methodological investigation

4.1 Method reproducibility test

Get 6 parts of same batch of loofah medicinal materials, prepare need testing solution according to the preparation method of loofah need testing solution in the test method, carry out fingerprint spectrum measurement, the relative retention time and relative peak area of common peak are counted respectively, the result is as follows As shown in Table 1, the RSD% is not more than 3%, showing that the method of the present invention has good reproducibility.

4.2 Fingerprint precision test

Get a part of the need testing solution that method reproducibility test is used, continuous sample injection 5 times, the relative retention time of common peak and relative peak area are counted respectively, and RSD% is all no more than 3% (as table 2), the result It shows that the method of the present invention has good precision.

4.3 Fingerprint Stability Test

Get a part of need testing solution that method reproducibility test is used, carry out fingerprint chromatogram measurement, investigate the stability of need testing solution in 48 hours, carry out statistics to the relative retention time of common peak and relative peak area respectively, RSD% average No more than 3% (as Table 3), the results showed that the test solution was stable within 48 hours.

The above test results show that the determination method is stable and reliable, and the fingerprint is relatively stable.

Table 1 Reproducibility test results (n=6)

Table 2 Precision test results (n=5)

Table 3 Stability test results (n=5)

Claims (2)

1. a method for establishing the high performance liquid chromatography fingerprint of loofah, is characterized in that the method is carried out in the following steps: One, the preparation of need testing solution: a, get loofah medicinal material, add 10 times of mass percentage concentration and be the ethanol of 75%, soak 12h, ultrasonic 30min, filter, collect solid phase thing A and liquid phase thing A respectively , adding 10 times the mass of solid phase A to 75% ethanol, soaking for 8 hours, ultrasonicating for 30 minutes, filtering, collecting liquid phase B, and then mixing liquid phase A and B to obtain an extract, Concentrating the extract under reduced pressure to obtain extract; b. Put the extract on the polyamide chromatographic column after dissolving it with distilled water, wash 4 column volumes with distilled water first, then elute 4 column volumes with 30% ethanol with a mass concentration, collect the eluent, and then decompress Concentration of the eluate to dryness afforded C as a dry solid; c, the dry solid C obtained in step b is dissolved into a solution with a concentration of 2mg/mL with methanol, and filtered with a microporous membrane, and the filtrate is the test solution; Two, the making of fingerprint collection: the need testing solution that step 1 obtains is injected high-performance liquid chromatograph, is that the mixed solution of methanol and acetonitrile with volume ratio 1:1 is phase A, and mass percent concentration is the acetic acid of 0.5% The aqueous solution is phase B for gradient elution; among them, the high performance liquid chromatography conditions are: chromatographic column filler Thermo C18, the specification is 250×4.6mm, 5μm; the column temperature is 35°C; the flow rate is 0.8mL/min, and the detection wavelength is 280nm. Sample volume 20 μ L; Analyze need testing solution under this condition, obtain the fingerprint collection of loofah; Wherein the gradient elution sequence in step 2 is: 0min→20min→50min→80min, and the mixed solution of methanol and acetonitrile with a volume ratio of 1:1 is 5%→16%→25%→40%. 2. the method for establishing the HPLC fingerprint of a kind of loofah according to claim 1, is characterized in that the microporous membrane filtration in step 1 adopts 0.45 μm microporous membrane to filter.