Quality evaluation method of rhizoma cyperi

Abstract

The invention belongs to the technical field of medicine detection and quality evaluation, and provides a quality evaluation method of rhizoma cyperi. According to the method, the biological surfactant is used for extracting the rhizoma cyperi, so that active ingredients in the rhizoma cyperi can be efficiently extracted in an environment-friendly manner; detecting the rhizoma cyperi extract in combination with high performance liquid chromatography-tandem triple quadrupole mass spectrometry to obtain chromatographic information of the rhizoma cyperi extract; and obtaining the content of each active substance in the rhizoma cyperi extract based on a preset standard curve. According to the quality evaluation method provided by the invention, the biological surfactant is used for extracting the rhizoma cyperi, so that the extraction efficiency is improved, and the method is more environment-friendly; meanwhile, due to the setting of high performance liquid chromatography parameters and triple quadrupole mass spectrometry parameters, more components can be detected, and the overall mass of the rhizoma cyperi can be reflected more accurately.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection and quality evaluation, and in particular to a quality evaluation method for cyperus rotundus. Background Art

[0002] Cyperus rotundus is a perennial herbaceous plant of the Cyperaceae family. Cyperus rotundus (CyperiRhizoma) is the dried rhizome of Cyperus rotundus, which has rich nutritional and medicinal value and has a long history as an edible and medicinal plant in China, India, Japan and other countries. Cyperus rotundus was first recorded as a traditional Chinese medicine in "Ming Yi Bie Lu". According to traditional Chinese medicine theory, Cyperus rotundus has the effects of soothing the liver and relieving depression, regulating qi and relieving pain. It has been widely used clinically for diseases such as infertility, dermatitis, breast cancer and depression. The chemical components of Cyperus rotundus are mainly sesquiterpenes, flavonoids and phenolic acids, which have significant antioxidant, anti-inflammatory and anti-tumor effects. Therefore, these active ingredients can be used as alternative markers for the quality evaluation of Cyperus rotundus.

[0003] To date, a variety of analytical methods and strategies have been used for the detection of Cyperus rotundus, such as liquid chromatography tandem ultraviolet spectroscopy (LC-UV), liquid chromatography tandem mass spectrometry (LC-MS) and gas chromatography tandem mass spectrometry (GC-MS). However, the extraction process of the quality evaluation methods currently reported is mostly heating reflux or selecting organic reagents as extraction solvents, which has problems such as low efficiency and environmental pollution. In addition, during the detection stage, the focus component is relatively single, which is not enough to reflect the overall quality of Cyperus rotundus. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a quality evaluation method for Cyperus rotundus. The quality evaluation method provided by the present invention uses a surfactant to extract Cyperus rotundus, which improves the extraction efficiency and is more environmentally friendly; at the same time, the collection method constructed based on high performance liquid chromatography tandem triple quadrupole mass spectrometry technology can detect more active ingredients and more accurately reflect the overall quality of Cyperus rotundus.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for evaluating the quality of Cyperus rotundus, comprising the following steps:

[0007] The cyperus rotundus is extracted by using a biosurfactant to obtain a cyperus rotundus extract;

[0008] The cyperus rotundus extract is detected by high performance liquid chromatography tandem triple quadrupole mass spectrometry to obtain chromatographic information of the cyperus rotundus extract;

[0009] Based on a preset standard curve, the content of each active substance in the Cyperus rotundus extract is obtained;

[0010] The high performance liquid chromatography tandem triple quadrupole mass spectrometer comprises high performance liquid chromatography and triple quadrupole mass spectrometer;

[0011] The parameters of the high performance liquid chromatography include: the chromatographic column is a Waters Xbrige C18 chromatographic column, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, the mobile phase B is acetonitrile, and the gradient elution program is: 0-5min, the volume fraction of the mobile phase B changes from 12% to 40% at a uniform rate; 5-6min: the volume fraction of the mobile phase B is maintained at 40%; 6-11min, the volume fraction of the mobile phase B changes from 40% to 42% at a uniform rate; 11-15min: the volume fraction of the mobile phase B changes from 42% to 50% at a uniform rate; 15-20min, the volume fraction of the mobile phase B changes from 50% to 82% at a uniform rate;

[0012] The parameters of the triple quadrupole mass spectrometer include: the ion source is an electrospray ionization source, and the ion source parameters include: the drying gas temperature is 300°C, the drying gas flow rate is 11L / min, the nebulizing gas pressure is 15psi, and the capillary voltage is 4kV; the acquisition mode is multiple reaction monitoring, and the parameters of the multiple reaction monitoring are shown in Table 1:

[0013] Table 1 Multiple reaction monitoring parameters of eight active ingredients in Cyperus rotundus

[0014]

[0015] Preferably, the parameters of the HPLC also include: a flow rate of 0.4 mL / min, an injection volume of 5 μL, and a column temperature of 30°C.

[0016] Preferably, the Cyperus rotundus extract contains syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, vecinin-2, nootkatone, α-cyperone and cyperenone.

[0017] Preferably, the method of extracting Cyperus rotundus by using a biosurfactant comprises the following steps:

[0018] The cyperus rotundus powder and the sophorolipid aqueous solution are mixed and extracted in a water bath and under ultrasonic conditions to obtain the cyperus rotundus extract.

[0019] Preferably, the mass concentration of the sophorolipid aqueous solution is 0.1-1%.

[0020] Preferably, the solid-liquid ratio of the Cyperus rotundus powder to the sophorolipid aqueous solution is 1:10 to 1:250 g / mL.

[0021] Preferably, the power of the ultrasound is 100-500W.

[0022] Preferably, the temperature of the water bath is 30-60°C.

[0023] Preferably, the extraction time is 10 to 50 minutes.

[0024] Preferably, the mass concentration of the sophorolipid aqueous solution is 0.2%, the solid-liquid ratio of the Cyperus rotundus powder and the sophorolipid aqueous solution is 1:75 g / mL, the power of the ultrasound is 250 W, the temperature of the water bath is 30° C., and the extraction time is 10 min.

[0025] The invention provides a quality evaluation method for Cyperus rotundus.

[0026] The quality evaluation method provided by the present invention utilizes a biosurfactant to extract the Cyperus rotundus, thereby improving the extraction efficiency and being more environmentally friendly; meanwhile, the setting of high performance liquid chromatography parameters and triple quadrupole mass spectrometry parameters can detect more components and more accurately reflect the overall quality of the Cyperus rotundus.

[0027] Furthermore, sophorolipids are used as biosurfactants to extract the Cyperus rotundus powder, thereby avoiding the use of organic solvents. When the solvent is water, efficient extraction of active ingredients in Cyperus rotundus can be achieved. Compared with the prior art, the extraction method of the present invention is non-toxic and environmentally friendly.

[0028] Furthermore, the present invention limits the mass concentration of the sophorolipid aqueous solution, the solid-liquid ratio of the Cyperus rotundus powder and the sophorolipid aqueous solution, the power of ultrasound, the temperature of the water bath and the extraction time, thereby further improving the extraction efficiency of the eight active ingredients in Cyperus rotundus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the effect of different concentrations of sophorolipid aqueous solutions on the extraction of eight active ingredients;

[0030] Figure 2 The effect diagram of different material-liquid ratios on the extraction of eight active ingredients;

[0031] Figure 3 This is a graph showing the effect of different extraction times on the extraction of eight active ingredients;

[0032] Figure 4 The figure shows the effect of different ultrasonic powers on the extraction of eight active ingredients;

[0033] Figure 5 This is a graph showing the effect of different water bath temperatures on the extraction of eight active ingredients. DETAILED DESCRIPTION

[0034] The present invention provides a method for evaluating the quality of Cyperus rotundus, comprising the following steps:

[0035] The cyperus rotundus is extracted by using a biosurfactant to obtain a cyperus rotundus extract;

[0036] The cyperus rotundus extract is detected by high performance liquid chromatography tandem triple quadrupole mass spectrometry to obtain chromatographic information of the cyperus rotundus extract;

[0037] Based on a preset standard curve, the content of each active substance in the Cyperus rotundus extract is obtained;

[0038] The high performance liquid chromatography tandem triple quadrupole mass spectrometer comprises high performance liquid chromatography and triple quadrupole mass spectrometer;

[0039] The parameters of the high performance liquid chromatography include: the chromatographic column is a Waters Xbrige C18 chromatographic column, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, the mobile phase B is acetonitrile, and the gradient elution program is: 0-5min, the volume fraction of the mobile phase B changes from 12% to 40% at a uniform rate; 5-6min: the volume fraction of the mobile phase B is maintained at 40%; 6-11min, the volume fraction of the mobile phase B changes from 40% to 42% at a uniform rate; 11-15min: the volume fraction of the mobile phase B changes from 42% to 50% at a uniform rate; 15-20min, the volume fraction of the mobile phase B changes from 50% to 82% at a uniform rate;

[0040] The parameters of the triple quadrupole mass spectrometer include: the ion source is an electrospray ionization source, and the ion source parameters include: the drying gas temperature is 300°C, the drying gas flow rate is 11L / min, the nebulizing gas pressure is 15psi, and the capillary voltage is 4kV; the acquisition mode is multiple reaction monitoring, and the parameters of the multiple reaction monitoring are shown in Table 1:

[0041] Table 1 Multiple reaction monitoring parameters of eight active ingredients in Cyperus rotundus

[0042]

[0043]

[0044] Unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0045] The invention utilizes biological surfactant to extract the cyperus rotundus to obtain the cyperus rotundus extract.

[0046] In the present invention, the extraction of Cyperus rotundus by using a biosurfactant preferably comprises the following steps:

[0047] The cyperus rotundus powder and the sophorolipid aqueous solution are mixed and extracted in a water bath and under ultrasonic conditions to obtain the cyperus rotundus extract.

[0048] In the present invention, the particle size of the Cyperus rotundus powder is preferably 50 to 70 meshes, specifically preferably 60 meshes. The preparation method of the Cyperus rotundus powder preferably comprises the following steps: crushing the Cyperus rotundus decoction pieces and then sieving them to obtain the Cyperus rotundus powder; the present invention does not specifically limit the crushing and sieving operations, as long as the Cyperus rotundus powder with a particle size of 50 to 70 meshes can be obtained.

[0049] In the present invention, the mass concentration of the sophorolipid aqueous solution is preferably 0.1-1%, specifically preferably 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0050] In the present invention, the solid-liquid ratio of the Cyperus rotundus powder and the sophorolipid aqueous solution is preferably 1:10 to 1:250 g / mL, preferably 1:10 to 1:100 g / mL, specifically preferably 1:10 g / mL, 1:20 g / mL, 1:30 g / mL, 1:40 g / mL, 1:50 g / mL, 1:60 g / mL, 1:70 g / mL, 1:75 g / mL, 1:80 g / mL, 1:90 g / mL, 1:100 g / mL, 1:150 g / mL, 1:200 g / mL or 1:250 g / mL.

[0051] In the present invention, the power of the ultrasound is preferably 100-500 W, more preferably 200-300 W, specifically preferably 100 W, 150 W, 200 W, 250 W, 300 W, 350 W, 400 W, 450 W or 500 W.

[0052] In the present invention, the temperature of the water bath is preferably 30-60°C, and specifically preferably 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C.

[0053] In the present invention, the extraction time is preferably 10 to 50 min, specifically preferably 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min or 50 min.

[0054] After the extraction, the present invention preferably further comprises: filtering the obtained extract, and the obtained filtrate is the Cyperus rotundus extract. In the present invention, the pore size of the filter membrane for the filtration is preferably 0.22 μm.

[0055] In the present invention, the Cyperus rotundus extract preferably contains syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, vecinin-2, nootkatone, α-cyperone and cyperenone.

[0056] The present invention uses sophorolipids as additives to extract the cyperus rotundus powder, avoiding the use of organic solvents. When the solvent is water, the extraction of active ingredients in the cyperus rotundus can be achieved. Compared with the prior art, the extraction method of the present invention is non-toxic and environmentally friendly. Furthermore, the present invention limits the mass concentration of the sophorolipid aqueous solution, the solid-liquid ratio of the cyperus rotundus powder and the sophorolipid aqueous solution, the power of ultrasound, the temperature of the water bath and the extraction time, further improving the extraction efficiency of the eight active ingredients in the cyperus rotundus.

[0057] After obtaining the Cyperus rotundus extract, the present invention uses high performance liquid chromatography tandem triple quadrupole mass spectrometry to detect the Cyperus rotundus extract to obtain chromatographic information of the Cyperus rotundus extract.

[0058] In the present invention, the high performance liquid chromatography tandem triple quadrupole mass spectrometry includes high performance liquid chromatography and triple quadrupole mass spectrometry; the high performance liquid chromatography is preferably performed on an Agilent 1200HPLC, and the triple quadrupole mass spectrometry is preferably performed on an Agilent 6430 mass spectrometer.

[0059] In the present invention, the parameters of the high performance liquid chromatography include: the chromatographic column is a Waters Xbrige C18 chromatographic column, the size of the Waters Xbrige C18 chromatographic column is preferably 2.1×100 mm, 3.5 μm; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, the mobile phase B is acetonitrile, and the gradient elution program is: 0 to 5 minutes, the volume fraction of the mobile phase B changes from 12% to 40% at a uniform rate; 5 to 6 minutes: the volume fraction of the mobile phase B is maintained at 40%; 6 to 11 minutes, the volume fraction of the mobile phase B changes from 40% to 42% at a uniform rate; 11 to 15 minutes: the volume fraction of the mobile phase B changes from 42% to 50% at a uniform rate; 15 to 20 minutes, the volume fraction of the mobile phase B changes from 50% to 82% at a uniform rate.

[0060] In the present invention, the parameters of the HPLC also include: the flow rate is preferably 0.4 mL / min, the injection volume is preferably 5 μL, and the column temperature is preferably 30°C.

[0061] In the present invention, the parameters of the triple quadrupole mass spectrometer include: the ion source is an electrospray ionization source, and the ion source parameters include: the drying gas temperature is 300°C, the drying gas flow rate is 11L / min, the nebulizing gas pressure is 15psi, and the capillary voltage is 4kV; the acquisition mode is multiple reaction monitoring, and the parameters of the multiple reaction monitoring are shown in Table 1.

[0062] After obtaining the chromatographic information of the Cyperus rotundus extract, the present invention obtains the content of each active substance in the Cyperus rotundus extract based on a preset standard curve.

[0063] In the present invention, the preset standard curve includes a syringic acid standard curve, a chlorogenic acid standard curve, a luteolin standard curve, a luteolin-7-O-glucuronide standard curve, a vesinian-2 standard curve, a nootkatone standard curve, an α-cyperone standard curve and a cyperenone standard curve.

[0064] The present invention does not specifically limit the method for establishing the preset standard curve, and those skilled in the art can establish it according to conventional means.

[0065] The quality evaluation method of the present invention can obtain the content of active ingredients in Cyperus rotundus to achieve quality evaluation of Cyperus rotundus; at the same time, the present invention uses biological surfactants to extract Cyperus rotundus, and then combines high performance liquid chromatography tandem triple quadrupole mass spectrometry to achieve detection of the active ingredients in Cyperus rotundus.

[0066] The quality evaluation method of Cyperus rotundus provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Reagents and medicines

[0068] Example 1 Investigation of detection methods

[0069] 1.1 Chemical substances and reagents

[0070] Syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, nootkatone, vecinin-2, α-cyperone and cyperenone were provided by Chengdu Desit Biotechnology Co., Ltd. The purity of the above reference substances was higher than 98%. Sophorolipids were purchased from Shandong Yousuo Chemical Technology Co., Ltd. Chromatographic grade methanol and acetonitrile were purchased from Fisher Chemical. Chromatographic grade formic acid was purchased from Anaqua Chemicals Supply. Ultrapure water was produced from Millipore ultrapure water machine.

[0071] 1.2 Instruments and analytical conditions

[0072] High performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS / MS) detection method, specifically: Anjie Agilent 1200HPLC tandem with Agilent 6430 mass spectrometer: Waters Xbrige C18 column(2.1×100mm, 3.5μm) is used for chromatographic separation; the mobile phase includes mobile phase A (0.1% formic acid water) and mobile phase B (acetonitrile), and the elution gradient program is: 0-5min, the volume fraction of mobile phase B changes from 12% to 40% at a uniform rate; 5-6min: the volume fraction of the mobile phase B is maintained at 40%; 6-11min, the volume fraction of the mobile phase B changes from 40% to 42% at a uniform rate; 11-15min: the volume fraction of the mobile phase B changes from 42% to 50% at a uniform rate; 15-20min, the volume fraction of the mobile phase B changes from 50% to 82% at a uniform rate; the flow rate is 0.4mL / min, the injection volume is 5μL and the column temperature is 30°C.

[0073] Agilent 6430 mass spectrometer was equipped with an electrospray ionization source (ESI), and the ion source parameters included: drying gas temperature of 300°C; drying gas flow rate of 11L / min; nebulizer gas pressure of 15psi; capillary voltage of 4kV. Multiple reaction monitoring (MRM, parameters see Table 1) acquisition mode was used for the detection of Cyperus rotundus extract in order to obtain a highly specific and sensitive quantitative method.

[0074] 1.3 Preparation of reference solution

[0075] Accurately weigh an appropriate amount of reference substance and dissolve it in methanol to prepare a 1 mg / mL reference substance stock solution. Take an appropriate amount of each component reference substance stock solution to prepare a mixed reference substance solution with a concentration of 1600 ng / mL (syringic acid, chlorogenic acid, quercetin, quercetin-7-O-glucuronide, vecinin-2 and α-cyperone), 1200 ng / mL (nokatone) and 2000 ng / mL (cyperone), and use methanol to dilute stepwise to the target concentration. The above reference substance solutions are all stored at 4°C.

[0076] 1.4 Methodological considerations

[0077] The standard curve was constructed using the concentration (X) of the analyte and the peak area (Y) of the chromatographic peak. The linearity of the standard curve and the sensitivity of the detection method were evaluated using the correlation coefficient (r), the limit of detection (LOD, S / N=3) and the limit of quantification (LOQ, S / N=10). The results are shown in Table 2.

[0078] Table 2 Standard curves, LOD and LOQ of eight chemical components in Cyperus rotundus extract

[0079]

[0080]

[0081] It can be seen from Table 2 that the HPLC-MS / MS established in the present invention can detect eight active ingredients in a wide concentration range (r>0.9995).

[0082] The samples were prepared in parallel for 6 times, and the contents of each component were determined by the established HPLC-MS / MS method to evaluate the repeatability of the method. The RSDs of the repeatability of syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, vecinin-2, nootkatone, α-cyperone and cyperenone were 1.9%, 1.3%, 3.4%, 1.8%, 4.7%, 3.4%, 3.9% and 4.2%, respectively, indicating that the method proposed in the present invention has good repeatability.

[0083] Take an appropriate amount of each reference solution, mix well, and prepare QC samples at three concentration levels (low, medium, and high) for specificity, precision, stability, and dilution integrity. The specificity of the detection method was investigated by comparing the chromatograms of the mixed reference solution, sample solution, and blank matrix. At the same retention time, there was no significant interference peak at the peak position of the target compound in the blank matrix sample, indicating that the method has strong specificity and can be used for target component detection. The intra-day and inter-day precision were evaluated by examining the relative standard deviation (RSD) and relative error (RE) of the peak area of ​​the target component in the QC sample for six consecutive injections or three consecutive days on the same day. The results are shown in Table 3. The QC samples were stored in the autosampler for 0h, 2h, 4h, 8h, 12h, and 24h, and the stability of the target component was evaluated by RSD and RE. The results are shown in Table 3.

[0084] Table 3 Intra-day and inter-day precision and stability of eight compounds in Cyperus rotundus extract

[0085]

[0086]

[0087] As can be seen from Table 3: the RSD of intra-day precision and inter-day precision of each active ingredient is 0.12% to 4.57%, and the RE is -4.47% to 4.60%, indicating that the instrument has good precision and can be used for the detection of target ingredients. The RSD of the stability of each active ingredient is 0.53% to 4.27%, and the RE is -4.83% to 4.75%, indicating that the target analyte is stable in the blank matrix within 24 hours.

[0088] High-concentration QC samples were prepared in parallel and diluted 10 times to evaluate the dilution integrity. The results are shown in Table 4.

[0089] Table 4 Dilution integrity of eight analytes in Cyperus rotundus extract

[0090]

[0091]

[0092] It can be seen from Table 4 that the RSD of each active ingredient is no higher than 2.8%, and the RE is no more than ±2.9%, indicating that the dilution process does not affect the concentration detection of the target ingredient.

[0093] Weigh 6 portions of 20 mg of Cyperus rotundus decoction powder in parallel, add equal amounts of target analytes, and prepare Cyperus rotundus extract in parallel according to the preparation method of the test solution, inject and measure, and calculate the recovery rate. The results show that the recovery rates of syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, vecinin-2, nokatone, α-cyperone and cyperenone are 97.0%, 103.0%, 103.5%, 104.6%, 95.3%, 98.1%, 97.2% and 100.6%, respectively, and the corresponding RSDs are all less than 5.0%. It shows that the content determination method established in the present invention can accurately and quantitatively analyze the content of the target components in the Cyperus rotundus extract.

[0094] Example 2

[0095] Take a batch of Cyperus rotundus decoction pieces purchased on the market, crush them and sieve them through a 60-mesh sieve to obtain Cyperus rotundus powder. Take 40 mg of Cyperus rotundus powder, accurately weigh and transfer to 2 mL of sophorolipid aqueous solution with mass concentrations of 0%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0%, respectively, and extract for 30 minutes at 500W ultrasonic power in a 30°C water bath; after the extraction, the obtained extract is filtered using a 0.22μm filter membrane, and the filtrate is diluted 25 times in an appropriate amount and then sampled for detection. The detection parameters are the same as in Example 1, and the detection results are shown in Figure 1 .

[0096] Figure 1 The effect of different concentrations of sophorolipid aqueous solution on the extraction of eight active ingredients is shown in Figure 2. Figure 1 It can be seen that the concentration of the sophorolipid aqueous solution plays a vital role in the extraction. The micelle critical concentration of sophorolipids is 0.0043% (43 mg / L). Therefore, a single factor investigation of six levels of sophorolipid aqueous solution concentration was carried out, including 0%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0%. The results showed that the content of active ingredients increased with the increase of the concentration of sophorolipid aqueous solution. This may be due to the increase in sophorolipid micelles encapsulating more active ingredients. The T test results showed that there was a significant difference only between the ultrapure water and the sophorolipid aqueous solution with a concentration of 0.2%. As the concentration of the sophorolipid aqueous solution increased, the total content of active ingredients no longer increased significantly. Therefore, a sophorolipid aqueous solution with a concentration of 0.2% was selected as the extraction agent for subsequent optimization.

[0097] Example 3

[0098] Take 40 mg of Cyperus rotundus powder (same as in Example 2), accurately weigh and transfer to different volumes of a 0.2% mass concentration of sophorolipid aqueous solution, so that the solid-liquid ratio is 1:10 g / mL, 1:50 g / mL, 1:100 g / mL, 1:150 g / mL, 1:200 g / mL and 1:250 g / mL, respectively, and extract for 30 min at 500 W ultrasonic power in a 30 ° C water bath; after the extraction, the obtained extract is filtered using a 0.22 μm filter membrane, and the filtrate is diluted 25 times in an appropriate amount and then sampled for detection. The detection parameters are the same as in Example 1, and the detection results are shown in Figure 2 .

[0099] Figure 2 The influence diagram of different material-liquid ratios on the extraction of eight active ingredients is shown in Figure 2. Figure 2 It can be seen that the solid-liquid ratio can directly affect the viscosity of the extract and the dissolution of the active ingredients. Therefore, the solid-liquid ratio is a key parameter affecting the extraction efficiency. The T test results show that there is a significant difference between the solid-liquid ratio of 1:50g / mL and 1:10g / mL, and there is no significance and difference between the other adjacent levels (p<0.05). This phenomenon may be due to the fact that when the solid-liquid ratio is 1:50g / mL, the emulsification capacity of the sophorolipid micelles is basically saturated and more target components cannot be solubilized. Therefore, 1:50g / mL was selected as the solid-liquid ratio for subsequent optimization.

[0100] Example 4

[0101] Take 40 mg of Cyperus rotundus powder (same as in Example 2), accurately weigh and transfer to 2 mL of a 0.2% mass concentration of sophorolipid aqueous solution to make the solid-liquid ratio 1:50 g / mL, and extract for 10 min, 20 min, 30 min, 40 min and 50 min respectively in a 30 ° C water bath at 500 W ultrasonic power; after the extraction, the obtained extract is filtered using a 0.22 μm filter membrane, and the filtrate is diluted 25 times in an appropriate amount and then sampled for detection. The detection parameters are the same as in Example 1, and the detection results are shown in Figure 3 .

[0102] Figure 3 The effect of different extraction time on the extraction of eight active ingredients is shown in Figure 2. Figure 3 It can be seen that the appropriate extraction time is conducive to sufficient interaction between ultrasound, extraction solvent and Cyperus rotundus powder. The results show that the content of the target component does not increase significantly with the extension of extraction time. Therefore, the extraction time is set to 10 minutes to improve the extraction efficiency and reduce energy consumption.

[0103] Example 5

[0104] Take 40 mg of Cyperus rotundus powder (same as in Example 2), accurately weigh and transfer to 2 mL of a 0.2% mass concentration.The extract was filtered using a 0.22 μm filter membrane, and the filtrate was diluted 25 times and then injected for testing. The test parameters were the same as those in Example 1, and the test results are shown in Table 1. Figure 4 .

[0105] Figure 4 The effect of different ultrasonic powers on the extraction of eight active ingredients is shown in Figure 2. Figure 4 It can be seen that ultrasonic power is a key parameter for extraction. The content of active ingredients in the Cyperus rotundus extract gradually increased with the increase of ultrasonic power, and no longer increased significantly after 200W. This may be because as the ultrasonic power increases, the cavitation effect increases, thereby increasing the damage to the cell wall. However, the use of higher ultrasonic power means higher energy consumption. Therefore, 200W was selected as the ultrasonic power for extraction for subsequent research.

[0106] Example 6

[0107] Take 40 mg of Cyperus rotundus powder (same as in Example 2), accurately weigh and transfer to 2 mL of a 0.2% mass concentration of sophorolipid aqueous solution, so that the solid-liquid ratio is 1:50 g / mL, and extract for 10 min at 200 W ultrasonic power in a water bath at 30 ° C, 40 ° C, 50 ° C and 60 ° C; after the extraction, the obtained extract is filtered using a 0.22 μm filter membrane, and the filtrate is diluted 25 times in an appropriate amount and then sampled for detection. The detection parameters are the same as in Example 1, and the detection results are shown in Figure 5 .

[0108] Figure 5 The effect of different water bath temperatures on the extraction of eight active ingredients is shown in Figure 2. Figure 5 It can be seen that appropriate water bath temperature can improve the dissolution effect of active ingredients. The contents of syringic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucuronide increased with the increase of water bath temperature. In contrast, nootkatone, α-cyperone, and cyperenone decreased with the increase of water bath temperature. The component loss phenomenon occurred in the three volatile components. Considering the whole, the total content of active ingredients did not increase with the increase of water bath temperature. Finally, the water bath temperature was set to 30°C.

[0109] Example 7

[0110] Orthogonal experimental design can be used to evaluate the interaction between different factors and provide the optimal extraction process. Therefore, an orthogonal experimental table with 7 factors and three levels was established using the Orthogonal Experiment Assistant software (version 3.1), as shown in Table 5. The 7 factors are A (concentration of the aqueous solution of sophorolipids), B (solid-liquid ratio), AB (interaction between factor A and factor B), C (ultrasonic power), AC (interaction between factor A and factor C), BC (interaction between factor B and factor C) and D (error).

[0111] Table 5 Orthogonal experimental design optimization results

[0112]

[0113]

[0114] Note: Ki represents the mean value of each level; R represents the range of each factor; A represents the concentration of sophorolipid aqueous solution; B represents the solid-liquid ratio; C represents the ultrasonic power; D represents the error column.

[0115] It can be seen from Table 5 that the range values ​​of each factor are B>C>A>BC>AC>AB from large to small, indicating that there is no interaction between the three factors. The results of the single-factor variance show that the F value of the solid-liquid ratio is 20.17 (higher than the F critical value of 19.0 and p<0.05), and the content of the target components obtained by the three-level extraction is 1:75>1:50>1:25 from high to low. Therefore, 1:75 was selected as the optimal solid-liquid ratio. In order to obtain higher extraction efficiency, the concentration of the sophorolipid aqueous solution and the ultrasonic power of the extraction were set to 0.2% and 250W, respectively, following the optimal extraction process proposed by the orthogonal design experiment.

[0116] Example 8

[0117] 21 batches of Cyperus rotundus decoction pieces purchased on the market were crushed and sieved to obtain Cyperus rotundus powder with a particle size of 60 mesh. The 21 Cyperus rotundus powders were respectively extracted under the following conditions:

[0118] 40 mg of Cyperus rotundus powder was accurately weighed and transferred to 3 mL of a 0.2% sophorolipid aqueous solution, and extracted for 10 min in a 30°C water bath, 250 W ultrasonic power and 40 kHz ultrasonic frequency. The obtained extract was filtered using a 0.22 μm filter membrane, and the filtrate was diluted 25 times in an appropriate amount and then sampled for detection. The detection parameters were the same as in Example 1, and the results are shown in Tables 6 and 7.

[0119] Table 6 Contents of four active ingredients in 21 batches of Cyperus rotundus (μg / g)

[0120]

[0121]

[0122] Table 7 Contents of four active ingredients in 21 batches of Cyperus rotundus (μg / g)

[0123]

[0124]

[0125] It can be seen from Tables 6 and 7 that the total amount of eight active ingredients in Cyperus rotundus is 472.50 μg / g to 1248.66 μg / g. In addition, there are significant differences in the content of the same active ingredient between different batches. Taking luteolin-7-O-glucuronide as an example, the content of this active ingredient in sample No.12 can reach 92.73 μg / g, while the content in sample No.11 is only 0.80 μg / g. The RSD of the content of luteolin-7-O-glucuronide in each batch of samples is 165.0%, indicating that the consistency of the content of active ingredients in Cyperus rotundus is poor between batches. In addition, the content of cyperus rotundus enone in Cyperus rotundus is generally high (the content is not less than 333.17 μg / g), while the content of vecinin-2 is generally low (the content is not higher than 6.64 μg / g).

[0126] The present invention uses ultrasound-assisted micellar extraction to improve the yield of active ingredients in Cyperus rotundus, and based on orthogonal experimental design, investigates the interaction of different extraction factors, and at the same time, obtains the optimal extraction process: the concentration of the sophorolipid aqueous solution is 0.2%, the solid-liquid ratio is 1:75g / mL, and the ultrasonic power is 250W; the HPLC-MS / MS method for simultaneous quantitative analysis of eight active ingredients in Cyperus rotundus is successfully established and verified, and applied to the content determination study of 21 batches of Cyperus rotundus slices. The present invention not only confirms the ability of biosurfactants (sophorolipids) to enrich chemical components, but also provides a reference for the improvement of the efficient, convenient, and environmentally friendly quality evaluation system of traditional Chinese medicine.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for evaluating the quality of Cyperus rotundus, characterized in that: The following steps are involved: The cyperus rotundus is extracted by using a biosurfactant to obtain a cyperus rotundus extract; The cyperus rotundus extract is detected by high performance liquid chromatography tandem triple quadrupole mass spectrometry to obtain chromatographic information of the cyperus rotundus extract; Based on a preset standard curve, the content of each active substance in the Cyperus rotundus extract is obtained; The high performance liquid chromatography tandem triple quadrupole mass spectrometer comprises high performance liquid chromatography and triple quadrupole mass spectrometer; The parameters of the high performance liquid chromatography include: the chromatographic column is a Waters Xbrige C18 chromatographic column, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, the mobile phase B is acetonitrile, and the gradient elution program is: 0-5min, the volume fraction of the mobile phase B changes from 12% to 40% at a uniform rate; 5-6min: the volume fraction of the mobile phase B is maintained at 40%; 6-11min, the volume fraction of the mobile phase B changes from 40% to 42% at a uniform rate; 11-15min: the volume fraction of the mobile phase B changes from 42% to 50% at a uniform rate; 15-20min, the volume fraction of the mobile phase B changes from 50% to 82% at a uniform rate; The parameters of the triple quadrupole mass spectrometer include: the ion source is an electrospray ionization source, and the ion source parameters include: the drying gas temperature is 300°C, the drying gas flow rate is 11L / min, the nebulizing gas pressure is 15psi, and the capillary voltage is 4kV; the acquisition mode is multiple reaction monitoring, and the parameters of the multiple reaction monitoring are shown in the following table:

2. The quality evaluation method according to claim 1, characterized in that: The parameters of the HPLC also include: a flow rate of 0.4 mL / min, an injection volume of 5 μL, and a column temperature of 30°C.

3. The quality evaluation method according to claim 1, characterized in that: The cyperus rotundus extract contains syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, vecinin-2, nootkatone, α-cyperus rotundus ketone and cyperus rotundus ketone.

4. The quality evaluation method according to claim 1 or 3, characterized in that: The method of extracting Cyperus rotundus by utilizing a biosurfactant comprises the following steps: The cyperus rotundus powder and the sophorolipid aqueous solution are mixed and extracted in a water bath and under ultrasonic conditions to obtain the cyperus rotundus extract.

5. The quality evaluation method according to claim 4, characterized in that: The mass concentration of the sophorolipid aqueous solution is 0.1-1%.

6. The quality evaluation method according to claim 5, characterized in that: The solid-liquid ratio of the Cyperus rotundus powder and the sophorolipid aqueous solution is 1:10 to 1:250 g / mL.

7. The quality evaluation method according to claim 4, characterized in that: The power of the ultrasound is 100-500W.

8. The quality evaluation method according to claim 4, characterized in that: The temperature of the water bath is 30-60°C.

9. The quality evaluation method according to claim 4, characterized in that: The extraction time is 10 to 50 minutes.

10. The quality evaluation method according to claim 4, characterized in that: The mass concentration of the sophorolipid aqueous solution is 0.2%, the solid-liquid ratio of the Cyperus rotundus powder and the sophorolipid aqueous solution is 1:75 g / mL, the power of the ultrasound is 250 W, the temperature of the water bath is 30° C., and the extraction time is 10 min.

Images