A quality evaluation method for Cyperus rotundus

Abstract

This invention belongs to the field of drug detection and quality evaluation technology, and provides a method for quality evaluation of Cyperus rotundus. This invention utilizes biosurfactants to extract Cyperus rotundus, achieving efficient and environmentally friendly extraction of its active ingredients. The extract is then analyzed using high-performance liquid chromatography (HPLC) tandem triple quadrupole mass spectrometry (TMS) to obtain chromatographic information. Based on a preset standard curve, the content of each active substance in the extract is determined. The quality evaluation method provided by this invention utilizes biosurfactants for extraction, improving extraction efficiency and being more environmentally friendly. Furthermore, the optimized settings of HPLC and TMS parameters allow for the detection of more components, more accurately reflecting the overall quality of Cyperus rotundus.

Description

Technical Field

[0001] This invention relates to the field of drug detection and quality evaluation technology, and in particular to a method for quality evaluation of Cyperus rotundus. Background Technology

[0002] Cyperus rotundus is a perennial herbaceous plant belonging to the Cyperaceae family. (Cyperus rotundus (…)) Cyperi Rhizoma Cyperus rotundus (Xiangfu) is the dried rhizome of Cyperus rotundus, possessing rich nutritional and medicinal value. It has a long history of use as an edible and medicinal plant in China, India, Japan, and other countries. Cyperus rotundus was first recorded as a traditional Chinese medicine in the *Mingyi Bielu* (Records of Famous Physicians). Traditional Chinese medicine theory holds that Cyperus rotundus has the effects of soothing the liver and relieving depression, regulating qi and relieving pain. Clinically, it has been widely used for diseases such as infertility, dermatitis, breast cancer, and depression. The main chemical components of Cyperus rotundus are sesquiterpenes, flavonoids, and phenolic acids, which have significant antioxidant, anti-inflammatory, and anti-tumor effects. Therefore, these active ingredients can serve as candidate markers for the quality evaluation of Cyperus rotundus.

[0003] To date, various analytical methods and strategies have been used for the detection of Cyperus rotundus, such as liquid chromatography-tandem ultraviolet spectrometry (LC-UV), liquid chromatography-tandem mass spectrometry (LC-MS), and gas chromatography-tandem mass spectrometry (GC-MS). However, the quality assessment methods currently reported mostly involve extraction processes that involve heating and reflux or using organic reagents as extraction solvents, which are inefficient and environmentally unfriendly. Furthermore, in the detection stage, the focused components are relatively singular and insufficient to reflect the overall quality of Cyperus rotundus. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a quality evaluation method for Cyperus rotundus. The quality evaluation method provided by this invention uses surfactants to extract Cyperus rotundus, which improves extraction efficiency and is more environmentally friendly. Simultaneously, the acquisition method based on high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS) can detect more active ingredients and more accurately reflect the overall quality of Cyperus rotundus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

[0007] Cyperus rotundus extract was obtained by using biosurfactants;

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

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

[0010] The high performance liquid chromatography-tandem triple quadrupole mass spectrometry includes high performance liquid chromatography and triple quadrupole mass spectrometry;

[0011] The parameters of the high-performance liquid chromatography (HPLC) include: a Waters Xbrige C18 column; mobile phases A and B, wherein mobile phase A is a 0.1% (v / v) aqueous solution of formic acid; mobile phase B is acetonitrile; and the gradient elution program is as follows: 0–5 min, the volume fraction of mobile phase B is uniformly reduced from 12% to 40%; 5–6 min, the volume fraction of mobile phase B is maintained at 40%; 6–11 min, the volume fraction of mobile phase B is uniformly reduced from 40% to 42%; 11–15 min, the volume fraction of mobile phase B is uniformly reduced from 42% to 50%; and 15–20 min, the volume fraction of mobile phase B is uniformly reduced from 50% to 82%.

[0012] The parameters of the triple quadrupole mass spectrometer include: the ion source is an electrospray ionization source, and the ion source parameters include: drying gas temperature of 300℃, drying gas flow rate of 11 L / min, nebulizing gas pressure of 15 psi, and capillary voltage of 4 kV; 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 components in Cyperus rotundus.

[0014]

[0015] Preferably, the parameters of the high performance liquid chromatography further 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, and luteolin-7- O - Glucuronide, Vizenin-2, Nocaketone, α-Cyperone and Cyperone.

[0017] Preferably, the extraction of Cyperus rotundus using biosurfactants includes the following steps:

[0018] Cyperus rotundus powder and sophorolipid aqueous solution were mixed and extracted under water bath and 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 ratio of the cyperus 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℃.

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

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

[0025] This invention provides a method for evaluating the quality of Cyperus rotundus.

[0026] The quality evaluation method provided by this invention utilizes biosurfactants to extract Cyperus rotundus, which improves extraction efficiency and is more environmentally friendly. At the same time, the setting of high performance liquid chromatography parameters and triple quadrupole mass spectrometry parameters allows for the detection of more components and more accurately reflects the overall quality of Cyperus rotundus.

[0027] Furthermore, using sophorolipids as a biosurfactant to extract Cyperus rotundus powder avoids the use of organic solvents. With water as the solvent, the active ingredients in Cyperus rotundus can be extracted efficiently. 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 material-liquid ratio of Cyperus rotundus powder and the sophorolipid aqueous solution, the ultrasonic power, the water bath temperature, and the extraction time, thereby further improving the extraction efficiency of eight active ingredients in Cyperus rotundus. Attached Figure Description

[0029] Figure 1 The effect of different concentrations of sophorolipid aqueous solution on the extraction of eight active ingredients is shown in the figure.

[0030] Figure 2 The effect of different solid-liquid ratios on the extraction of eight active ingredients is shown in the figure.

[0031] Figure 3 The effect of different extraction times on the extraction of eight active ingredients is shown in the figure.

[0032] Figure 4 The effect of different ultrasonic powers on the extraction of eight active ingredients is shown in the figure.

[0033] Figure 5 The graph shows the effect of different water bath temperatures on the extraction of eight active ingredients. Detailed Implementation

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

[0035] Cyperus rotundus extract was obtained by using biosurfactants;

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

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

[0038] The high performance liquid chromatography-tandem triple quadrupole mass spectrometry includes high performance liquid chromatography and triple quadrupole mass spectrometry;

[0039] The parameters of the high-performance liquid chromatography (HPLC) include: a Waters Xbrige C18 column; mobile phases A and B, wherein mobile phase A is a 0.1% (v / v) aqueous solution of formic acid; mobile phase B is acetonitrile; and the gradient elution program is as follows: 0–5 min, the volume fraction of mobile phase B is uniformly reduced from 12% to 40%; 5–6 min, the volume fraction of mobile phase B is maintained at 40%; 6–11 min, the volume fraction of mobile phase B is uniformly reduced from 40% to 42%; 11–15 min, the volume fraction of mobile phase B is uniformly reduced from 42% to 50%; and 15–20 min, the volume fraction of mobile phase B is uniformly reduced from 50% to 82%.

[0040] The parameters of the triple quadrupole mass spectrometer include: the ion source is an electrospray ionization source, and the ion source parameters include: drying gas temperature of 300℃, drying gas flow rate of 11 L / min, nebulizing gas pressure of 15 psi, and capillary voltage of 4 kV; 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 components in Cyperus rotundus.

[0042]

[0043] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0044] This invention utilizes biosurfactants to extract Cyperus rotundus, obtaining Cyperus rotundus extract.

[0045] In this invention, the extraction of Cyperus rotundus using biosurfactants preferably includes the following steps:

[0046] Cyperus rotundus powder and sophorolipid aqueous solution were mixed and extracted under water bath and ultrasonic conditions to obtain the Cyperus rotundus extract.

[0047] In this invention, the particle size of the Cyperus rotundus powder is preferably 50-70 mesh, and more preferably 60 mesh. The preparation method of the Cyperus rotundus powder preferably includes the following steps: crushing Cyperus rotundus slices and then sieving them to obtain the Cyperus rotundus powder. This invention does not specifically limit the crushing and sieving operations, as long as Cyperus rotundus powder with a particle size of 50-70 mesh can be obtained.

[0048] In this 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%.

[0049] In this invention, the preferred material-to-liquid ratio of the Cyperus rotundus powder and the sophorolipid aqueous solution is 1:10 to 1:250 g / mL, more preferably 1:10 to 1:100 g / mL, and 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.

[0050] In this invention, the power of the ultrasound is preferably 100~500W, more preferably 200~300W, and specifically preferably 100W, 150W, 200W, 250W, 300W, 350W, 400W, 450W or 500W.

[0051] In this invention, the temperature of the water bath is preferably 30~60℃, and more preferably 30℃, 35℃, 40℃, 45℃, 50℃, 55℃ or 60℃.

[0052] In this invention, the extraction time is preferably 10 to 50 minutes, and more preferably 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes.

[0053] Following the extraction, the present invention preferably further includes filtering the obtained extract, wherein the resulting filtrate is the Cyperus rotundus extract. In this invention, the pore size of the filter membrane is preferably 0.22 μm.

[0054] In this invention, the Cyperus rotundus extract preferably contains syringic acid, chlorogenic acid, luteolin, and luteolin-7- O - Glucuronide, Vizenin-2, Nocaketone, α-Cyperone and Cyperone.

[0055] This invention uses sophorolipid as an additive to extract Cyperus rotundus powder, avoiding the use of organic solvents. The active ingredients in Cyperus rotundus can be extracted using only water as the solvent. Compared to existing technologies, the extraction method of this invention is non-toxic and environmentally friendly. Furthermore, this invention limits the mass concentration of the sophorolipid aqueous solution, the material-to-liquid ratio of Cyperus rotundus powder and the sophorolipid aqueous solution, the ultrasonic power, the water bath temperature, and the extraction time, further improving the extraction efficiency of eight active ingredients in Cyperus rotundus.

[0056] After obtaining Cyperus rotundus extract, the present invention uses high performance liquid chromatography-tandem triple quadrupole mass spectrometry to detect Cyperus rotundus extract and obtain chromatographic information of Cyperus rotundus extract.

[0057] In this 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 1200 HPLC, and the triple quadrupole mass spectrometry is preferably performed on an Agilent 6430 mass spectrometer.

[0058] In this invention, the parameters of the high-performance liquid chromatography (HPLC) include: a Waters Xbrige C18 column, preferably with dimensions of 2.1 × 100 mm and 3.5 μm; mobile phases including mobile phase A and mobile phase B, wherein mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile; the gradient elution program is as follows: 0–5 min, the volume fraction of mobile phase B changes from 12% to 40% at a constant rate; 5–6 min, the volume fraction of mobile phase B remains at 40%; 6–11 min, the volume fraction of mobile phase B changes from 40% to 42% at a constant rate; 11–15 min, the volume fraction of mobile phase B changes from 42% to 50% at a constant rate; 15–20 min, the volume fraction of mobile phase B changes from 50% to 82% at a constant rate.

[0059] In this invention, the parameters of the high performance liquid chromatography 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.

[0060] In this 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℃, 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.

[0061] 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.

[0062] In this invention, the preset standard curves include syringic acid standard curve, chlorogenic acid standard curve, luteolin standard curve, and luteolin-7- O -Standard curves for glucuronide, vezin-2, nocaconide, α-cyperone, and cyperone.

[0063] The present invention does not specifically limit the method for establishing the preset standard curve; those skilled in the art can establish it using conventional methods.

[0064] The quality evaluation method of this invention can obtain the content of active ingredients in Cyperus rotundus, so as to realize the quality evaluation of Cyperus rotundus; at the same time, this invention uses biosurfactants to extract Cyperus rotundus, and then combines high performance liquid chromatography-tandem triple quadrupole mass spectrometry to realize the detection of active ingredients in Cyperus rotundus.

[0065] The following detailed description of the quality evaluation method for Cyperus rotundus provided by the present invention, in conjunction with specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0066] reagents and medicines

[0067] Example 1: Examination of the detection method

[0068] 1.1 Chemical substances and reagents

[0069] Syringic acid, chlorogenic acid, luteolin, luteolin-7- O - Glucuronide, Nocaketone, Viczerine-2 α Cyperone and cyperene were provided by Chengdu Desite Biotechnology Co., Ltd. The purity of all the above reference standards was higher than 98%. Sophorolipids were purchased from Shandong Yousuo Chemical Technology Co., Ltd. Chromatographically pure methanol and acetonitrile were purchased from Fisher Chemical. Chromatographically pure formic acid was purchased from Anaqua Chemicals Supply. Ultrapure water was produced using a Millipore ultrapure water system.

[0070] 1.2 Instruments and Analytical Conditions

[0071] A high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS) method was employed, specifically as follows: The equipment used was an Agilent 1200 HPLC system tandem with an Agilent 6430 mass spectrometer; a Waters Xbrige C18 column (2.1 × 100 mm, 3.5 μm) was used for chromatographic separation; the mobile phase consisted of mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile). The elution gradient program was as follows: 0–5 min, the volume fraction of mobile phase B was uniformly increased from 12% to 40%; 5–6 min, the volume fraction of mobile phase B was maintained at 40%; 6–11 min, the volume fraction of mobile phase B was uniformly increased from 40% to 42%; 11–15 min, the volume fraction of mobile phase B was uniformly increased from 42% to 50%; 15–20 min, the volume fraction of mobile phase B was uniformly increased from 50% to 82%; the flow rate was 0.4 mL / min, the injection volume was 5 μL, and the column temperature was 30 °C.

[0072] The Agilent 6430 mass spectrometer is equipped with an electrospray ionization (ESI) source. Ion source parameters include: drying gas temperature of 300°C; drying gas flow rate of 11 L / min; nebulizing gas pressure of 15 psi; and capillary voltage of 4 kV. Multiple reaction monitoring (MRM, parameters shown in Table 1) acquisition mode was used for the detection of Cyperus rotundus extract to obtain a highly specific and sensitive quantitative method.

[0073] 1.3 Preparation of reference solution

[0074] Accurately weigh an appropriate amount of reference standard and dissolve it in methanol to prepare a 1 mg / mL reference standard stock solution. Take appropriate amounts of each component reference standard stock solution to prepare a solution with a concentration of 1600 ng / mL (syringic acid, chlorogenic acid, quercetin, quercetin-7- O - Glucuronium, Viclin-2 and α A mixed reference solution of 1200 ng / mL (cyperone) and 2000 ng / mL (norcacon) was prepared and serially diluted with methanol to the target concentration. All reference solutions were stored at 4°C.

[0075] 1.4 Methodological Examination

[0076] A standard curve was constructed using the concentration of the analyte (X) and the peak area of ​​the chromatographic peak (Y). 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 quantitation (LOQ, S / N=10). The results are shown in Table 2.

[0077] Table 2 Standard curves, LOD, and LOQ of eight chemical components in Cyperus rotundus extract.

[0078]

[0079] As can be seen from Table 2, the HPLC-MS / MS established in this invention can detect eight active ingredients over a wide concentration range (r>0.9995).

[0080] The reproducibility of the method was evaluated by preparing six parallel samples and determining the content of each component using the established HPLC-MS / MS method. Syringic acid, chlorogenic acid, luteolin, and luteolin-7- O - Glucuronide, Vicine-2, Norcadone α The RSDs for the repeatability of cyperone and cyperene 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 this invention has good repeatability.

[0081] Take appropriate amounts 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 assessment. The specificity of the detection method was assessed by comparing the chromatograms of the mixed reference solution, sample solution, and blank matrix. At the same retention time, the peak position of the target compound did not show significant interference peaks in the blank matrix sample, indicating that the method has strong specificity and can be used for target component detection. 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 samples for six consecutive injections or three consecutive days within the same day; the results are shown in Table 3. The stability of the target component was evaluated by storing the QC samples in an autosampler for 0h, 2h, 4h, 8h, 12h, and 24h using RSD and RE; the results are shown in Table 3.

[0082] Table 3. Intra-day and inter-day precision and stability of eight compounds in Cyperus rotundus extract.

[0083]

[0084] Table 3 shows that the RSDs for intra-day and inter-day precision of each active ingredient are 0.12%–4.57%, and the REs are -4.47%–4.60%, indicating good instrument precision suitable for the detection of target components. The RSDs for the stability of each active ingredient are 0.53%–4.27%, and the REs are -4.83%–4.75%, indicating that the target analytes are stable in the blank matrix within 24 hours.

[0085] High-concentration QC samples were prepared and diluted 10-fold in parallel to evaluate the integrity of the dilution. The results are shown in Table 4.

[0086] Table 4. Dilution integrity of eight analytes in Cyperus rotundus extract.

[0087]

[0088] As can be seen from Table 4, 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 detection of the target ingredient concentration.

[0089] Six 20mg portions of Cyperus rotundus powder were weighed in parallel, and equal amounts of the target analyte were added to each. Cyperus rotundus extract was prepared in parallel according to the method for preparing the test solution. The extracts were then injected for analysis, and the recovery rates were calculated. Results showed that syringic acid, chlorogenic acid, luteolin, and luteolin-7-... O - Glucuronide, Vicine-2, Norcadone α The recoveries of cyperone and cyperene were 97.0%, 103.0%, 103.5%, 104.6%, 95.3%, 98.1%, 97.2%, and 100.6%, respectively, with corresponding RSDs all less than 5.0%. This indicates that the content determination method established in this invention can accurately and quantitatively analyze the content of the target components in Cyperus rotundus extract.

[0090] Example 2

[0091] A batch of Cyperus rotundus slices purchased from the market was pulverized and sieved through a 60-mesh sieve to obtain Cyperus rotundus powder. 40 mg of Cyperus rotundus powder was accurately weighed and transferred to 2 mL of sophorolipid aqueous solutions with mass concentrations of 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%, respectively. Extraction was performed in a 30°C water bath at 500 W ultrasonic power for 30 min. After extraction, the resulting extract was filtered through a 0.22 μm filter membrane. The filtrate was diluted 25 times and then injected for analysis. The analysis parameters were the same as in Example 1, and the results are shown in [Figure 1]. Figure 1 .

[0092] Figure 1 The graph shows the effect of different concentrations of sophorolipid aqueous solution on the extraction of eight active ingredients. Figure 1 It can be seen that the concentration of the sophorolipid aqueous solution plays a crucial role in the extraction process. The critical micelle concentration of sophorolipid is 0.0043% (43 mg / L). Therefore, a single-factor study was conducted at six levels of sophorolipid aqueous solution concentration, including 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%. The results showed that the content of active ingredients increased with increasing sophorolipid aqueous solution concentration. This may be due to the increased number of sophorolipid micelles encapsulating more active ingredients. The T-test results showed that there was a significant difference only between ultrapure water and a 0.2% sophorolipid aqueous solution level; as the concentration of the sophorolipid aqueous solution increased, the total content of active ingredients no longer increased significantly. Therefore, a 0.2% sophorolipid aqueous solution was selected as the extraction agent for subsequent optimization.

[0093] Example 3

[0094] 40 mg of Cyperus rotundus powder (same as in Example 2) was accurately weighed and transferred to different volumes of 0.2% sophorolipid aqueous solution to achieve material-to-liquid ratios of 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. Extraction was performed in a 30°C water bath at 500 W ultrasonic power for 30 min. After extraction, the extract was filtered through a 0.22 μm filter membrane. The filtrate was diluted 25 times and then injected for analysis. The parameters were the same as in Example 1, and the results are shown in [Figure 1]. Figure 2 .

[0095] Figure 2 The graph shows the effect of different feed-to-liquid ratios on the extraction of eight active ingredients. Figure 2 It can be seen that the solid-liquid ratio directly affects the viscosity of the extract and the dissolution of active ingredients; therefore, the solid-liquid ratio is a key parameter affecting extraction efficiency. The t-test results showed a significant difference between solid-liquid ratios of 1:50 g / mL and 1:10 g / mL, while no significant differences were found between other adjacent levels (p<0.05). This phenomenon may be because the emulsifying capacity of sophorolipid micelles is essentially saturated at a solid-liquid ratio of 1:50 g / mL, making it impossible to solubilize more target components. Therefore, 1:50 g / mL was chosen as the solid-liquid ratio for subsequent optimization.

[0096] Example 4

[0097] 40 mg of Cyperus rotundus powder (same as in Example 2) was accurately weighed and transferred to 2 mL of 0.2% sophorolipid aqueous solution to make a material-to-liquid ratio of 1:50 g / mL. Extraction was performed in a 30°C water bath at 500 W ultrasonic power for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. After extraction, the extract was filtered through a 0.22 μm filter membrane. The filtrate was diluted 25 times and then injected for analysis. The detection parameters were the same as in Example 1, and the results are shown in [Figure 1]. Figure 3 .

[0098] Figure 3 The graph shows the effect of different extraction times on the extraction of eight active ingredients. Figure 3 It can be seen that an appropriate extraction time facilitates sufficient interaction between ultrasound, extraction solvent, and Cyperus rotundus powder. The results show that the content of the target component did not significantly increase with prolonged extraction time. Therefore, an extraction time of 10 min was set to improve extraction efficiency and reduce energy consumption.

[0099] Example 5

[0100] 40 mg of Cyperus rotundus powder (same as in Example 2) was accurately weighed and transferred to 2 mL of 0.2% sophorolipid aqueous solution to make a material-to-liquid ratio of 1:50 g / mL. Extraction was performed in a 30°C water bath at ultrasonic powers of 100 W, 200 W, 300 W, 400 W, and 500 W for 10 min, respectively. After extraction, the extract was filtered through a 0.22 μm filter membrane. The filtrate was diluted 25 times and then injected for analysis. The analysis parameters were the same as in Example 1, and the results are shown in [Figure 1]. Figure 4 .

[0101] Figure 4 The graph shows the effect of different ultrasonic powers on the extraction of eight active ingredients. Figure 4 It can be seen that ultrasonic power is a key parameter for extraction. The content of active ingredients in Cyperus rotundus extract gradually increases with increasing ultrasonic power, but no longer increases significantly after 200W. This may be because as ultrasonic power increases, the cavitation effect increases, thereby enhancing the destructive effect on the cell wall. However, the use of higher ultrasonic power means higher energy consumption. Therefore, 200W was selected as the ultrasonic power for extraction in subsequent studies.

[0102] Example 6

[0103] 40 mg of Cyperus rotundus powder (same as in Example 2) was accurately weighed and transferred to 2 mL of 0.2% sophorolipid aqueous solution to make a material-to-liquid ratio of 1:50 g / mL. Extraction was performed for 10 min at 200 W ultrasonic power in water baths at 30℃, 40℃, 50℃, and 60℃ respectively. After extraction, the extract was filtered through a 0.22 μm filter membrane. The filtrate was diluted 25 times and then injected for analysis. The detection parameters were the same as in Example 1, and the results are shown in [Figure 1]. Figure 5 .

[0104] Figure 5 The graph shows the effect of different water bath temperatures on the extraction of eight active ingredients. Figure 5 It can be seen that an appropriate water bath temperature can improve the dissolution effect of active ingredients. Syringic acid, chlorogenic acid, luteolin, luteolin-7- O The content of glucuronide increased with increasing water bath temperature. Conversely, nocarbone, α-cyperone, and cyperone decreased with increasing water bath temperature. This component loss phenomenon occurred in the three volatile components. Overall, the total content of active ingredients did not increase with increasing water bath temperature. Ultimately, the water bath temperature was set to 30°C.

[0105] Example 7

[0106] Orthogonal experimental design can be used to evaluate the interactions between different factors and provide the optimal extraction process. Therefore, an orthogonal experimental table with 7 factors and 3 levels was established using the Orthogonal Experiment Assistant software (version 3.1), as shown in Table 5. The 7 factors are A (concentration of sophorolipid aqueous solution), 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).

[0107] Table 5. Optimization results of orthogonal experimental design

[0108]

[0109] Note: Ki represents the average value of each level; R represents the range of each factor; A represents the concentration of sophorolipid aqueous solution; B represents the material-to-liquid ratio; C represents the ultrasonic power; D represents the error column.

[0110] Table 5 shows that the range values ​​of each factor, from largest to smallest, are B>C>A>BC>AC>AB, indicating that there is no interaction among the three factors. The one-way variance results show that the F-value for the feed-liquid ratio is 20.17 (higher than the critical F-value of 19.0 and...). p The content of the target component obtained from the three levels of extraction (<0.05) decreased from 1:75 to 1:50 to 1:25. Therefore, 1:75 was selected as the optimal material-liquid ratio. To obtain higher extraction efficiency, following the optimal extraction process proposed by orthogonal design experiments, the concentration of the sophorolipid aqueous solution and the ultrasonic power for extraction were set to 0.2% and 250W, respectively.

[0111] Example 8

[0112] Twenty-one batches of Cyperus rotundus slices purchased from the market were pulverized and sieved to obtain Cyperus rotundus powder with a particle size of 60 mesh. The 21 Cyperus rotundus powders were then extracted under the following conditions:

[0113] 40 mg of Cyperus rotundus powder was accurately weighed and transferred to 3 mL of 0.2% sophorolipid aqueous solution. The solution was extracted for 10 min at 30 °C water bath, 250 W ultrasonic power and 40 kHz ultrasonic frequency. The extract was filtered through a 0.22 μm filter membrane. The filtrate was diluted 25 times and then injected for analysis. The analysis parameters were the same as in Example 1. The results are shown in Tables 6 and 7.

[0114] Table 6. Content (μg / g) of four active ingredients in 21 batches of Cyperus rotundus

[0115]

[0116] Table 7. Content (μg / g) of four active ingredients in 21 batches of Cyperus rotundus

[0117]

[0118] Tables 6 and 7 show that the total amount of the eight active ingredients in Cyperus rotundus ranged from 472.50 μg / g to 1248.66 μg / g. Furthermore, the content of the same active ingredient varied significantly between different batches. For example, luteolin-7- O Taking glucuronide as an example, the content of this active ingredient reached 92.73 μg / g in sample No. 12, while the content was only 0.80 μg / g in sample No. 11. In each batch of samples, luteolin-7- O The RSD of glucuronide content was 165.0%, indicating poor batch-to-batch consistency in the content of active ingredients in Cyperus rotundus. Furthermore, the content of cyperone in Cyperus rotundus was generally high (not less than 333.17 μg / g), while the content of veselene-2 ​​was generally low (not more than 6.64 μg / g).

[0119] This invention utilizes ultrasound-assisted micelle extraction to improve the yield of active ingredients in Cyperus rotundus. Based on orthogonal experimental design, the interactions of different extraction factors were investigated, and the optimal extraction process was determined: a sophorolipid aqueous solution concentration of 0.2%, a solid-liquid ratio of 1:75 g / mL, and an ultrasonic power of 250 W. An HPLC-MS / MS method for the simultaneous quantitative analysis of eight active ingredients in Cyperus rotundus was successfully established and validated, and applied to the content determination of 21 batches of Cyperus rotundus decoction pieces. This invention not only demonstrates the ability of biosurfactants (sophoracil) to enrich chemical components but also provides a reference for improving the quality evaluation system of traditional Chinese medicine in a more efficient, convenient, and environmentally friendly manner.

[0120] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for evaluating the quality of Cyperus rotundus, characterized in that, Includes the following steps: Cyperus rotundus extract was obtained by extracting Cyperus rotundus using a biosurfactant; the Cyperus rotundus extract contains syringic acid, chlorogenic acid, luteolin, luteolin-7-O-glucuronide, virzalin-2, nocaconone, α-cyperone and cyperone-co ... The extraction of Cyperus rotundus using biosurfactants includes the following steps: Cyperus rotundus powder and sophorolipid aqueous solution were mixed and extracted under water bath and ultrasonic conditions to obtain Cyperus rotundus extract; the mass concentration of sophorolipid aqueous solution was 0.2%, the material-to-liquid ratio of Cyperus rotundus powder to sophorolipid aqueous solution was 1:75 g / mL, the ultrasonic power was 250 W, the water bath temperature was 30 °C, and the extraction time was 10 min; The extract of Cyperus rotundus was detected by high performance liquid chromatography-tandem triple quadrupole mass spectrometry to obtain the chromatographic information of the extract; Based on a preset standard curve, the content of each active substance in the Cyperus rotundus extract was obtained; The high performance liquid chromatography-tandem triple quadrupole mass spectrometry includes high performance liquid chromatography and triple quadrupole mass spectrometry; The parameters of the high-performance liquid chromatography (HPLC) include: a Waters Xbrige C18 column; mobile phases A and B, wherein mobile phase A is a 0.1% (v / v) aqueous solution of formic acid; mobile phase B is acetonitrile; and the gradient elution program is as follows: 0–5 min, the volume fraction of mobile phase B is uniformly reduced from 12% to 40%; 5–6 min, the volume fraction of mobile phase B is maintained at 40%; 6–11 min, the volume fraction of mobile phase B is uniformly reduced from 40% to 42%; 11–15 min, the volume fraction of mobile phase B is uniformly reduced from 42% to 50%; and 15–20 min, the volume fraction of mobile phase B is uniformly reduced from 50% to 82%. The parameters of the triple quadrupole mass spectrometer include: an electrospray ionization source with the following parameters: drying gas temperature of 300℃, drying gas flow rate of 11 L / min, nebulizing gas pressure of 15 psi, and capillary voltage of 4 kV; the acquisition mode is multiple reaction monitoring (MRM), and the parameters of MRM are shown in the table below: 。 2. The quality evaluation method according to claim 1, characterized in that, The parameters of the high-performance liquid chromatography also include: flow rate of 0.4 mL / min, injection volume of 5 μL, and column temperature of 30 °C.

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