CN113150052A - Method for purifying quercetin glycoside in semen lepidii - Google Patents
Method for purifying quercetin glycoside in semen lepidii Download PDFInfo
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- C07H1/08—Separation; Purification from natural products
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Abstract
The invention discloses a method for purifying quercetin glycoside in pepperweed seed. The method comprises the following steps: (1) primary water extraction of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen lepidii; (2) precipitating semen Lepidii water extract with ethanol to remove impurities; (3) eluting and separating by using LX-68G type macroporous resin; (4) eluting and purifying by a CM-agarose gel FF column; (5) purifying the prepared liquid phase to obtain the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside monomeric compound. The experimental results show that: the CM-sepharose FF has good purification effect on quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, the purity of a sample purified by preparative liquid phase separation can reach more than 99.0 percent, the technical requirement that the national traditional Chinese medicine reference substance is more than 98.5 percent is met, and the CM-sepharose FF can be used as a traditional Chinese medicine reference substance.
Description
Technical Field
The invention relates to a purification method of quercetin glycoside in semen lepidii, and relates to a purification process of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen lepidii.
Background
Semen Lepidii is dry mature seed of Descurainia sophia or Lepidium Merrill of Brassicaceae, the former is known as "Nan Ting Li Zi", and the latter is known as "North Ting Li Zi". The pepperweed seed is generally not used as a monarch drug in the prescription in the traditional Chinese medicine, is generally used as an assistant drug or a single drug, and is often used for treating lung heat cough and asthma caused by various reasons. In modern clinical application, the traditional Chinese medicine composition is mainly used for being matched with other medicines in a traditional Chinese medicine compound prescription for use, and is used for treating cardiovascular diseases and respiratory diseases.
Quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is a characteristic component in the semen lepidii and is used for identifying and calibrating the semen lepidii in 2020 edition Chinese pharmacopoeia. According to literature query, after separation and purification processes of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen lepidii generally adopt modes of macroporous resin, polyamide and the like, and silica gel column chromatography or SephadexLH-20 is carried out, the separation of the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is incomplete, the purity is low, and the technical requirement that the purity of a national traditional Chinese medicine reference substance is 98.5% is difficult to achieve.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a method for purifying quercetin glycoside in pepperweed seed. The CM-sepharose FF column is selected for separation and purification, so that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the pepperweed seed is improved, and a good foundation is laid for preparing a reference substance of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for extracting quercetin glycoside from semen Lepidii comprises separating and extracting quercetin glycoside from semen Lepidii by CM-sepharose FF column, wherein the quercetin glycoside in semen Lepidii is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, and combining the prepared liquid phase to obtain quercetin glycoside monomer compound.
Preferably, the method for extracting the quercetin glycoside from the pepperweed seed comprises the following steps:
s1, mixing the dried seeds of the pepperweed seeds with deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1-2 h, repeatedly extracting for three times, combining the filtrates, concentrating the filtrate, and spray-drying to obtain a dried sample;
s2, ultrasonically dissolving the dried sample for 15-45 min by using 60% ethanol, standing for 20-30 h, filtering, concentrating the filtrate, and freeze-drying to obtain a freeze-dried sample;
s3, dissolving a freeze-dried sample by using acid water with pH4.5 for 5min in an ultrasonic mode, loading the sample on activated LX-68G type macroporous resin, standing for 1-2 h after loading, eluting 1-2 column volumes by using deionized water, then sequentially carrying out gradient elution by using 10% ethanol and 20% ethanol, wherein the elution flow rate is 1-2 mL/min, carrying out HPLC real-time detection in the elution process, carrying out gradient elution when an HPLC chromatogram shows that the peak value of impurities is reduced to 1/10 of the maximum value of the peak height, collecting 20% ethanol eluate, concentrating and freeze-drying to obtain an extract;
s4, preparing a solution with the concentration of 0.06-0.08 mg/mL by using 30% methanol, dissolving the solution by using ultrasound for 3-5 min, sucking the solution by using a dropper, slowly and uniformly spreading the solution on the surface of a regenerated CM-sepharose FF column, eluting the solution by using 30% methanol at the flow rate of 15-25 mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the eluent in real time by using HPLC (high performance liquid chromatography), collecting the eluent when the HPLC (high performance liquid chromatography) shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is contained, concentrating and freeze-drying to obtain a crude extract;
s5, taking the crude extract, ultrasonically dissolving with 30% methanol, selecting LC3000 type preparation liquid phase, eluting with 30% methanol at 254nm until no peak exists, starting sample elution, collecting eluent when the preparation liquid phase spectrum shows that there is an absorption peak, concentrating and freeze-drying the part of eluent when the HPLC detection spectrum shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, obtaining a refined extract, and determining the refined extract to be a quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside monomer compound by HPLC.
Further preferably, the ultrasonic power in the step S2 is 140-160 Hz.
Further preferably, the gauze selected in the step S2 is 200 meshes.
Further preferably, the liquid phase conditions in step S3 are: c18Reverse phase chromatography column (250 x 4.6mm,5 μm); the sample injection amount is 20 mu L; the mobile phase is as follows: acetonitrile: 0.1% glacial acetic acid = 10: 90, respectively; detection time: 20 min; flow rate: 1.0 mL/min; column temperature: 35 ℃; detection wavelength: 254 nm.
More preferably, the preparation of the acid water having a pH of 4.5 in the step S3 is adjusted by dilute hydrochloric acid.
More preferably, when the eluate is collected in step S4, the eluate is collected in units of 50mL and its components are measured by HPLC.
Has the advantages that: compared with the prior art, the invention provides a new purification technical route of quercetin glycoside in pepperweed seed, in the purification process, a CM-sepharose FF column is selected to separate and purify the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the semen lepidii, so as to improve the content of the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, and the purity of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside obtained by subsequent preparative liquid phase purification can reach more than 99.0 percent, can be used as a traditional Chinese medicine reference substance, and is obviously higher than the technical requirement of 98.5 percent of the national traditional Chinese medicine reference substance.
The invention adopts a new separation and purification method for quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the pepperweed seed, has the advantages of simple extraction and separation method, easy operation, high purity of the obtained product and the like, and has better popularization and application values. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The present invention will now be described in more detail, wherein preferred embodiments of the invention are shown, it being understood that one skilled in the art could modify the invention herein described while still achieving the beneficial results of the present invention. Thus, the following description should be understood as being broadly known to those skilled in the art, and not as limiting the present invention, and the reagents used below are all volume concentrations.
Example one
A practical separation and purification process of quercetin glycoside in semen lepidii comprises the following steps:
s1, taking 200kg of dried seeds of the pepperweed seeds, sieving the seeds with a No. four sieve, adding 4000L of deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1.5h, repeatedly extracting for three times, combining filtrates, concentrating, and spray drying to obtain dried samples.
S2, taking 500g of the dried sample, ultrasonically dissolving the sample for 15min by using 3.4L of 60% ethanol, standing the sample for 21h in the dark, filtering the solution, concentrating the filtrate, and freeze-drying the filtrate to obtain a freeze-dried sample.
S3, taking 200G of freeze-dried sample, dissolving the freeze-dried sample in 1L of acid water with the pH value of 4.5 by ultrasonic for 15min, taking acid water with the pH value of 4.5 to elute the activated LX-68G type macroporous resin, then sampling, standing for 1h after sampling, eluting 1.2 column volumes by deionized water, sequentially eluting by 10% ethanol and 20% ethanol in a gradient way, wherein the elution flow rate is 1.5mL/min, detecting the eluent by HPLC in real time, collecting 20% ethanol eluent when the HPLC chromatogram shows that the peak value of the impurity is reduced to 1/10 of the maximum value of the peak height, concentrating and freeze-drying to obtain the extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the extract is 16.40%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 1:
TABLE 1
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 1.0670 | 7396.7 | 16.40% |
S4, taking 0.4125g of extract, dissolving the extract by 5mL of 30% methanol for 3min by ultrasonic wave, slowly and uniformly spreading the extract on the surface of a regenerated CM-sepharose FF column by a dropper, eluting the surface by 30% methanol at the flow rate of 15mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the eluent by HPLC (high performance liquid chromatography) in real time, collecting the eluent when the HPLC shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, concentrating and freeze-drying to obtain the crude extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the crude extract is 52.73 percent through HPLC determination.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 2:
TABLE 2
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.2552 | 4706.8 | 52.73% |
Comparison with S3 shows: after the purification of the CM-sepharose FF column of S4, the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is greatly improved, the number of impurity peaks is obviously reduced, the separation efficiency is greatly improved, and a good foundation is laid for the subsequent separation and purification of a preparation type liquid phase.
S5, taking 41.34mg of crude extract, ultrasonically dissolving the crude extract by using 2mL of 30% methanol, selecting LC3000 type prepared liquid phase, eluting the liquid phase by using 30% methanol at the flow rate of 4mL/min under 254nm until the chromatogram shows no peak, loading and eluting the liquid phase, collecting eluent when the chromatogram of the prepared liquid phase shows an absorption peak, and concentrating and freeze-drying the part of eluent when the chromatogram shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside through HPLC detection to obtain the refined extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside measured by HPLC is 99.92%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 3:
TABLE 3
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.220 | 9585.1 | 99.92% |
Example two
A practical separation and purification process of quercetin glycoside in semen lepidii comprises the following steps:
s1, taking 200kg of dried seeds of the pepperweed seeds, sieving the seeds with a No. four sieve, adding 4000L of deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1.5h, repeatedly extracting for three times, combining filtrates, concentrating, and spray drying to obtain dried samples.
S2, taking 550g of the dried sample, ultrasonically dissolving the sample for 20min by using 3.8L of 60% ethanol, standing the sample for 26h in the dark, filtering the solution, concentrating the filtrate, and freeze-drying the filtrate to obtain a freeze-dried sample.
S3, taking 145G of freeze-dried sample, carrying out ultrasonic dissolution on 2.8L of acid water with pH of 4.5 for 30min, taking acid water with pH of 4.5 to elute the activated LX-68G type macroporous resin, then loading the sample, standing for 1.5h after loading the sample, eluting 1.5 column volumes by deionized water, then sequentially carrying out gradient elution by 10% ethanol and 20% ethanol, wherein the elution flow rate is 1.8mL/min, carrying out real-time HPLC detection on the eluent, collecting 20% ethanol eluent when the peak value of impurities is reduced to 1/10 of the maximum peak height value of the eluent when an HPLC map shows, concentrating and freeze-drying to obtain the extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the extract is determined to be 13.45% by HPLC.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 4:
TABLE 4
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 1.0480 | 5961 | 13.45% |
S4, dissolving 0.3216g of extract with 5mL of 30% methanol for 5min by ultrasound, slowly and uniformly spreading the extract on the surface of a regenerated CM-sepharose FF column by a dropper, eluting with 30% methanol at a flow rate of 20mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the eluent by HPLC (high performance liquid chromatography) in real time, and when the HPLC shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside exists, collecting the eluent, concentrating and freeze-drying to obtain crude extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the crude extract is 49.69 percent through HPLC determination.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 5:
TABLE 5
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.2820 | 5249 | 49.69% |
Comparison with S3 shows: through the purification of the CM-sepharose FF column of S4, the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is greatly improved, the number of impurity peaks is obviously reduced, the separation efficiency is improved, the result is consistent with the result shown in the first embodiment, the purification of the CM-sepharose FF column is obviously superior to the prior art, and the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is obviously improved.
S5, taking 56.34mg of crude extract, ultrasonically dissolving the crude extract by using 2mL of 30% methanol, selecting an LC3000 type preparation liquid phase, eluting the liquid phase by using 30% methanol at the flow rate of 4mL/min under 254nm until the spectrum shows no peak, loading and eluting the liquid phase, collecting eluent when the spectrum of the preparation liquid phase shows an absorption peak, and concentrating and freeze-drying the part of eluent when the spectrum shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside through HPLC detection to obtain a refined extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the refined extract is 99.36%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 6:
TABLE 6
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.210 | 9141.7 | 99.36% |
EXAMPLE III
A practical separation and purification process of quercetin glycoside in semen lepidii comprises the following steps:
s1, taking 200kg of dried seeds of the pepperweed seeds, sieving the seeds with a No. four sieve, adding 4000L of deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1.5h, repeatedly extracting for three times, combining filtrates, concentrating, and spray drying to obtain dried samples.
S2, taking 400g of the dried sample, ultrasonically dissolving the sample for 25min by using 3L of 60% ethanol, standing the sample for 20h in the dark, filtering the solution, concentrating the filtrate, and drying the concentrated filtrate under reduced pressure to obtain the dried sample.
S3, dissolving 139G of a dry sample with 3.6L of acid water with pH4.5 for 25min by ultrasonic treatment, eluting the activated LX-68G type macroporous resin with acid water with pH4.5, then sampling, standing for 1.5h after sampling, eluting 1 column volume with deionized water, then sequentially eluting with 10% ethanol and 20% ethanol in a gradient manner, wherein the elution flow rate is 1.8mL/min, detecting the eluent in real time by HPLC, collecting 20% ethanol eluent when the HPLC chromatogram shows that the impurity peak value is reduced to 1/10 of the maximum peak height value, concentrating, and drying under reduced pressure to obtain the extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the extract is 13.39% by HPLC.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 7:
TABLE 7
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 1.0560 | 5976.3 | 13.39% |
S4, dissolving 0.3876g of extract with 5mL of 30% methanol for 5min by ultrasound, slowly and uniformly spreading the extract on the surface of a regenerated CM-sepharose FF column by a dropper, eluting with 30% methanol at a flow rate of 15mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the eluent by HPLC (high performance liquid chromatography) in real time, collecting the eluent when the HPLC chromatogram shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is contained, concentrating and freeze-drying to obtain a crude extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the crude extract is 50.31%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following table 8:
TABLE 8
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.1496 | 2528.3 | 50.31% |
Compared with S3, through the purification of the CM-sepharose FF column of S4, the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is greatly improved, similar to the extraction results of the first embodiment and the second embodiment, and the purification effect is obviously better than that of the prior art.
S5, taking 52.76mg of crude extract, ultrasonically dissolving the crude extract by using 2mL of 30% methanol, selecting LC3000 type prepared liquid phase, eluting the liquid phase by using 30% methanol at the flow rate of 4mL/min under 254nm until the chromatogram shows no peak, loading and eluting the liquid phase, collecting eluent when the chromatogram of the prepared liquid phase shows an absorption peak, and concentrating and freeze-drying the part of eluent when the chromatogram shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside through HPLC detection to obtain the refined extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the refined extract is 99.44%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 9:
TABLE 9
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.222 | 9669.1 | 99.44% |
Example four
A practical separation and purification process of quercetin glycoside in semen lepidii comprises the following steps:
s1, taking 100kg of dried seeds of the pepperweed seeds, sieving the seeds with a No. four sieve, adding 2000L of deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1.5h, repeatedly extracting for three times, combining filtrates, concentrating, and spray drying to obtain dried samples.
S2, taking 325g of the dried sample, ultrasonically dissolving the sample for 20min by using 3.5L of 60% ethanol, standing the sample for 28h in a dark place, filtering the solution, concentrating the filtrate, and drying the concentrated filtrate under reduced pressure to obtain the dried sample.
S3, taking 100G of a dry sample, dissolving the dry sample with 2.5L of acid water with pH4.5 by ultrasonic sound for 20min, taking acid water with pH4.5 to elute the activated LX-68G type macroporous resin, then sampling, standing for 1.5h after sampling, eluting 2 column volumes with deionized water, then sequentially eluting with 10% ethanol and 20% ethanol in a gradient manner, wherein the elution flow rate is 1.8mL/min, detecting the eluent in real time by using HPLC, collecting 20% ethanol eluent when the HPLC chromatogram shows that the impurity peak value is reduced to 1/10 of the maximum peak height value, concentrating and freeze-drying to obtain the extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the extract is determined to be 15.89% by HPLC.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 10:
watch 10
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 1.0370 | 6965.1 | 15.89% |
S4, dissolving 0.4298g of extract with 5mL of 30% methanol for 5min by ultrasound, slowly and uniformly spreading the extract on the surface of a regenerated CM-sepharose FF column by a dropper, eluting with 30% methanol at a flow rate of 20mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the eluent by HPLC (high performance liquid chromatography) in real time, and when the HPLC shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside exists, collecting the eluent, concentrating and freeze-drying to obtain crude extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the crude extract is 52.47 percent through HPLC determination.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 11:
TABLE 11
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.2540 | 4991.6 | 52.47% |
Compared with S3, the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is greatly improved by purifying the CM-sepharose FF column of S4.
S5, taking 60.69mg of crude extract, ultrasonically dissolving the crude extract by using 2mL of 30% methanol, selecting an LC3000 type preparation liquid phase, eluting the liquid phase by using 30% methanol at the flow rate of 4mL/min under 254nm until the spectrum shows no peak, loading and eluting the liquid phase, collecting eluent when the spectrum of the preparation liquid phase shows an absorption peak, and concentrating and freeze-drying the part of eluent when the spectrum shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside through HPLC detection to obtain a refined extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the refined extract is 99.65%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 12:
TABLE 12
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.237 | 10298.2 | 99.65% |
EXAMPLE five
A practical separation and purification process of quercetin glycoside in semen lepidii comprises the following steps:
s1, taking 50kg of dried seeds of the pepperweed seeds, sieving the seeds with a No. four sieve, adding 1000L of deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1.5h, repeatedly extracting for three times, combining filtrates, concentrating, and spray drying to obtain dried samples.
S2, taking 520g of the dried sample, ultrasonically dissolving the 520L of the dried sample for 25min by using 60% ethanol 4L, standing the dissolved sample for 32h in the dark, filtering the solution, concentrating and freeze-drying the filtrate to obtain a freeze-dried sample.
S3, dissolving a freeze-dried sample 170G in 4L of acid water with pH4.5 by ultrasonic wave for 35min, eluting the activated LX-68G type macroporous resin with acid water with pH4.5, then loading the sample, standing for 2h after loading the sample, eluting 2 column volumes with deionized water, then sequentially eluting with 10% ethanol and 20% ethanol in a gradient manner, wherein the elution flow rate is 2.5mL/min, detecting the eluent in real time by HPLC, collecting 20% ethanol eluent when the HPLC chromatogram shows that the peak value of the impurity is reduced to 1/10 of the maximum peak height value, concentrating and freeze-drying to obtain the extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the extract is 11.49 percent by HPLC.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 13:
watch 13
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 1.0210 | 4959.7 | 11.49% |
S4, taking 0.3937g of extract, dissolving the extract by using 5mL of 30% methanol for 3min by ultrasound, slowly and uniformly spreading the extract on the surface of a regenerated CM-sepharose FF column by using a dropper, eluting the regenerated CM-sepharose FF column by using 30% methanol at the flow rate of 25mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by using HPLC (high performance liquid chromatography) in real time, collecting eluent when the HPLC chromatogram shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, concentrating and freeze-drying the eluent to obtain a crude extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the crude extract is 48.65%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 14:
TABLE 14
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.4912 | 8767.9 | 48.65% |
Compared with S3, the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is greatly improved by purifying the CM-sepharose FF column of S4.
S5, taking 58.43mg of crude extract, ultrasonically dissolving the crude extract by using 2mL of 30% methanol, selecting LC3000 type prepared liquid phase, eluting the liquid phase by using 30% methanol at the flow rate of 4mL/min under 254nm until the chromatogram shows no peak, loading and eluting the liquid phase, collecting eluent when the chromatogram of the prepared liquid phase shows an absorption peak, and concentrating and freeze-drying the part of eluent when the chromatogram shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside through HPLC detection to obtain the refined extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the refined extract is 99.54%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 15:
watch 15
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.240 | 10417.4 | 99.54% |
EXAMPLE six
A practical separation and purification process of quercetin glycoside in semen lepidii comprises the following steps:
s1, taking 200kg of dried seeds of the pepperweed seeds, sieving the seeds with a No. four sieve, adding 4000L of deionized water, heating and extracting in a water bath at the extraction temperature of 80 ℃ for 1.5h, repeatedly extracting for three times, combining filtrates, concentrating, and spray drying to obtain dried samples.
S2, taking 470g of dried sample, ultrasonically dissolving the 470g of dried sample for 25min by using 4.5L of 60% ethanol, standing the dissolved sample for 24h in the dark, filtering the solution, concentrating the filtrate and freeze-drying the filtrate to obtain a freeze-dried sample.
S3, taking 150G of freeze-dried sample, dissolving with 4L of acid water with pH of 4.5 by ultrasonic wave for 25min, taking acid water with pH of 4.5 to elute the activated LX-68G type macroporous resin, then sampling, standing for 1h, eluting with deionized water for 1.5 column volumes, then sequentially eluting with 10% ethanol and 20% ethanol in a gradient manner, wherein the elution flow rate is 2mL/min, detecting the eluent by HPLC in real time, collecting 20% ethanol eluent when the HPLC chromatogram shows that the peak value of the impurity is reduced to 1/10 of the maximum peak height value, concentrating and freeze-drying to obtain the extract. The content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the extract is 11.93 percent by HPLC (high performance liquid chromatography).
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 16:
TABLE 16
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 1.0080 | 5085.1 | 11.93% |
S4, dissolving 0.3742g of extract with 5mL of 30% methanol for 3min by ultrasound, slowly and uniformly spreading the extract on the surface of a regenerated CM-sepharose FF column by a dropper, eluting with 30% methanol at a flow rate of 25mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the eluent by HPLC (high performance liquid chromatography) in real time, and when the HPLC shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside exists, collecting the eluent, concentrating and freeze-drying to obtain crude extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the crude extract is 46.78%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 17:
TABLE 17
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.4248 | 7276.2 | 46.78% |
Compared with S3, the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is greatly improved by purifying the CM-sepharose FF column of S4.
S5, taking 63.29mg of crude extract, ultrasonically dissolving with 2mL of 30% methanol, selecting LC3000 type preparation liquid phase, eluting with 30% methanol at 254nm at 4mL/min until the spectrum shows no peak, loading and eluting, when the spectrum of the preparation liquid phase shows an absorption peak, collecting the eluent, and when the spectrum of HPLC detection shows that the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, concentrating and freeze-drying the part of the eluent to obtain the refined extract. HPLC determination shows that the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in the refined extract is 99.41%.
The results of measuring the content of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by HPLC are shown in the following Table 18:
watch 18
| Name (R) | Concentration (mg/mL) | Peak area | Content (wt.) |
| Sample (I) | 0.238 | 10316.4 | 99.41% |
The existing separation and purification process of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen lepidii generally adopts the separation of macroporous resin, polyamide and other modes, and then the separation of the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside is incomplete through silica gel column chromatography or SephadexLH-20, so that the purity is low, and the technical requirement of 98.5 percent of the national traditional Chinese medicine reference substance is difficult to achieve. However, the above specific examples of the present invention partially show that the effect of extracting and purifying quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by using CM-sepharose FF is obvious. CM-sepharose FF known by the technical personnel in the field is a chromatographic separation medium of a weak cation exchange medium, is mostly used for extracting and separating macromolecules such as protein, polypeptide, DNA and the like, and has no literature report that the CM-sepharose FF can be used for separating and purifying traditional Chinese medicine compounds. The key point of the invention is that a CM-sepharose FF column is selected for separating and purifying quercetin glycoside in semen lepidii, and the use of the CM-sepharose FF column can obviously improve the purification efficiency of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen lepidii, namely the CM-sepharose FF column shows obvious selective adsorption on quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in semen lepidii extract, and has poor adsorption effect on other substances (such as polysaccharide, cardiac glycoside, isothiocyanic acid and the like) in the semen lepidii extract, so that the CM-sepharose FF column has poor adsorption effect on quercetin-3-O-beta-D-glucoside in the semen lepidii extract The extraction and purification of the sugar-7-O-beta-D-gentiobioside have prominent substantive characteristics.
The separation and extraction method of the quercetin glycoside in the pepperweed seed has obvious purification technical effect, the extracted glycoside content is up to more than 99.0 percent, and the technical requirement of 98.5 percent of the national traditional Chinese medicine reference substance is met.
Although the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the details of the embodiments, and various equivalent modifications can be made within the technical spirit of the present invention, and the scope of the present invention is also within the scope of the present invention. It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (7)
1. A method for purifying quercetin glycoside in semen Lepidii is characterized in that a CM-sepharose FF column is adopted to separate and extract the quercetin glycoside in the semen Lepidii, and the quercetin glycoside in the semen Lepidii is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside.
2. A method for purifying quercetin glycoside in semen lepidii comprises the following steps:
s1, mixing semen lepidii seeds with deionized water, heating and extracting in a water bath at the temperature of 80 ℃ for 1-2 h, repeatedly extracting for three times, combining filtrates, concentrating the filtrate, and spray-drying to obtain a dried sample;
s2, ultrasonically dissolving the dried sample for 15-45 min by using 60% ethanol, standing for 20-30 h, filtering, concentrating the filtrate, and freeze-drying to obtain a freeze-dried sample;
s3, dissolving a freeze-dried sample by using acid water with pH4.5 for 5min in an ultrasonic mode, then loading the sample on activated LX-68G type macroporous resin, standing for 1-2 h after loading, eluting 1-2 column volumes by using deionized water, then sequentially performing gradient elution by using 10% ethanol and 20% ethanol, wherein the elution flow rate is 1-2 mL/min, performing HPLC real-time detection in the elution process, performing gradient elution when an HPLC chromatogram shows that the peak value of impurities is reduced to 1/10 of the maximum peak height value, collecting 20% ethanol eluent, concentrating and freeze-drying to obtain an extract;
s4, preparing a solution with the concentration of 0.06-0.08 mg/mL by using 30% methanol, dissolving the solution for 3-5 min by using an ultrasonic wave, slowly and uniformly spreading the solution on the surface of a regenerated CM-sepharose FF column after absorbing the solution by using a dropper, eluting the solution by using 30% methanol at the flow rate of 15-25 mL/min, detecting the content change of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside by using HPLC (high performance liquid chromatography), collecting eluent when the HPLC spectrum shows that the quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, concentrating and freeze-drying to obtain a crude extract;
s5, taking the crude extract to dissolve with 30% methanol by ultrasonic, selecting LC3000 preparation liquid phase to elute with 30% methanol at 254nm by 4mL/min until the spectrum shows no peak, loading and eluting, collecting the eluent when the spectrum shows an absorption peak, detecting by HPLC when the component in the eluent is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, concentrating and freeze-drying the part of eluent to obtain the refined extract.
3. The extraction method according to claim 2, wherein in S2, the ultrasonic power is 140-160 Hz.
4. The method of claim 2, wherein in step S2, 200 mesh gauze is selected for filtration.
5. The extraction method according to claim 2, wherein in S3, the liquid phase conditions are: c18Reverse phase chromatography column 250 x 4.6mm,5 μm; the sample injection amount is 20 mu L; the mobile phase is as follows: acetonitrile: 0.1% glacial acetic acid = 10: 90, respectively; detection time: 20 min; flow rate: 1.0 mL/min; column temperature: 35 ℃; detection wavelength: 254 nm.
6. The method according to claim 2, wherein the preparation of the acid water having a pH of 4.5 in S3 is adjusted by dilute hydrochloric acid.
7. The extraction method according to claim 2, wherein in S4, when the eluate is collected, the eluate is collected every 50mL and its components are measured.
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