CN113413632B - A lipid extract of Stellaria dichotoma and its preparation method - Google Patents
A lipid extract of Stellaria dichotoma and its preparation method Download PDFInfo
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Abstract
The invention provides a bupleurum root lipid extract and a preparation method thereof. The bupleurum falcatum lipid extract comprises the following components in percentage by weight: 0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid; the preparation method of the bupleurum falcatum lipid extract comprises the following steps: (1) pulverizing Stellaria dichotoma into 40-60 mesh; adding pulverized radix Stellariae into an extraction kettle, setting the extraction temperature of the extraction kettle at 40-50 deg.C, the extraction pressure at 25-35Mpa, the pressure of a separation kettle I at 50-60 deg.C, the pressure of a separation kettle II at 30-40 deg.C and the extraction time at 6-10 MPa, and obtaining radix Stellariae lipid extract. The invention analyzes the structure of the lipid component in Stellaria dichotoma, and discovers a series of lipid components comprising 7 lipid substances including 8 glycerophospholipids, 6 sphingolipids, 3 glycerides, 4 sterols, 1 pregnenolone lipid, 3 fatty acyl and 4 glycolipids, and 29 lipid components.
Description
Technical Field
The invention belongs to the technical field of extraction of chemical components of Chinese herbal medicines, and particularly relates to a starwort root lipid extract and a preparation method thereof.
Background
Ceramide, also called sphingolipid, is a lipid existing in the skin, plays an important role in the formation of the epidermal stratum corneum, is a common structural unit of (neuro) sphingolipids, fatty acids have an acid amide bond structure on the amino group of sphingosine, and when a sugar is bound thereto, they become sphingoglycolipids, and when phosphorylcholine is bound thereto, they become (neuro) sphingomyelin, which is an intermediate metabolite of sphingolipids, and particularly occupies an important position in biosynthesis, and exists only in a small amount in addition to platelets, and a large amount of ceramide is accumulated in cerebellum and kidney of a patient with Fabry disease, which is one of hereditary lipid accumulation disorders (lipidosis), and studies have shown that, in recent years, when the skin has a phenomenon of dryness, desquamation, or dehiscence, and its barrier function is significantly reduced, the skin can rapidly recover the barrier and moisturize functions by supplementing ceramide, and in view of environmental protection, the ceramide added in the skin care product is extracted from original animal brain tissues and is changed into plant extracts, such as rice, corn and other plants.
Disclosure of Invention
In order to solve the technical problems, the invention provides a bupleurum falcatum lipid extract and a preparation method thereof.
The bupleurum falcatum lipid extract comprises the following components in percentage by weight:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid;
the preparation method of the bupleurum falcatum lipid extract comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) adding pulverized radix Stellariae into an extraction kettle, setting the extraction temperature of the extraction kettle at 40-50 deg.C, the extraction pressure at 25-35Mpa, the pressure of a separation kettle I at 50-60 deg.C, the pressure of a separation kettle II at 30-40 deg.C and the extraction time at 6-10 MPa, and obtaining radix Stellariae lipid extract.
The A refers to lipid component in the extract of Stellaria dichotoma prepared by the above extraction method, the Bupleurum falcatum is a Ningxia genuine medicinal material, is a dry root of Stellaria dichotoma L.var.lanceolara Bge of Stellaria of Caryophyllaceae, has cold nature and sweet taste, and has the effects of clearing deficiency heat, removing infantile malnutrition heat and the like; currently discovered bupleurum falcatum active ingredients mainly comprise sterols, cyclic peptides, alkaloids, flavonoids, phenolic acids, volatile substances and the like, the pharmacological activity of the bupleurum falcatum active ingredients is mainly focused on the aspects of antipyretic and anti-inflammatory, antiallergic, anticancer, vasodilatation promotion and the like, but no report is found on the research on lipid substances, the current research on the extraction method of the bupleurum falcatum active ingredients mainly comprises a steam distillation method or a solvent extraction method, the two extraction methods are simple, but the extraction efficiency is low, the energy consumption is high, the safety is low, the heat stable ingredients are easily destroyed, for example, 95% ethanol is used for extraction, chloroform, ethyl acetate and n-butanol are sequentially used for extraction, and the chromatography method is used for separation, so that 10 compounds can be obtained, namely 3-hydroxy-beta-carbaryl, taraxane A, 1,2,3, 4-tetrahydro-1, 2,3, 4-trihydroxy-beta-carbaryl, 1-acetyl-beta-carbaryl, arenarine A, arenarine B, diisobutyl phthalate, dibutyl phthalate, methyl linolenate and tricin, wherein fat-soluble components extracted by different methods are different, and other fat-soluble active components in the starwort root cannot be obtained by the existing extraction method.
The research takes starwort root as a raw material and adopts supercritical CO2The extraction technology is used for extracting the active ingredients of Stellaria dichotoma, the influence of the extraction pressure, the extraction temperature and the extraction time on the yield of the Stellaria dichotoma extract is researched, the extraction technology of Stellaria dichotoma is optimized by a response surface method, and the obtained supercritical extract ingredients are analyzed by adopting the UHPLC-MS/MS technology, so that a theoretical basis is provided for further development and utilization of Stellaria dichotoma.
Further, the specific components of the lipid extract of Stellaria dichotoma provided by the invention are listed in Table 1.
TABLE 1 lipid extract distribution of Stellaria dichotoma
On the other hand, the invention also provides a method for extracting the bupleurum falcatum lipid extract from the bupleurum falcatum, wherein the bupleurum falcatum lipid extract contains the following components in percentage by weight:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid;
the preparation method of the bupleurum falcatum lipid extract comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) adding pulverized radix Stellariae into an extraction kettle, setting the extraction temperature of the extraction kettle at 40-50 deg.C, the extraction pressure at 25-35Mpa, the pressure of a separation kettle I at 50-60 deg.C, the pressure of a separation kettle II at 30-40 deg.C and the extraction time at 6-10 MPa, and obtaining radix Stellariae lipid extract.
In another aspect, the present invention also provides a method for extracting ceramide from starwort root, which comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) putting the crushed starwort roots into an extraction kettle, setting the extraction temperature of the extraction kettle to be 40-50 ℃, the extraction pressure to be 25-35Mpa, the pressure of a separation kettle I to be 6MPa-10MPa, the temperature to be 50-60 ℃, the pressure of a separation kettle II of the extraction kettle to be 6MPa-10MPa, the temperature to be 30-40 ℃, and the extraction time to be 1-3h, thus obtaining the starwort lipid extract, wherein the starwort lipid extract contains ceramide.
Further, in the step (2), the extraction temperature of the extraction kettle is set to 47 ℃, the extraction pressure is set to 35MPa, and the extraction time is set to 3 h.
Further, the lipid extract of Stellaria dichotoma prepared by using the above parameters contains the following components:
sterol 88.111%, glyceride 10.868%, fatty acyl 0.362%, pregnenolone lipid 0.013%, glycerophospholipid 0.532%, glycolipid 0.085%, sphingolipid 0.029%.
Further, in the step (2), the extraction temperature of the extraction kettle is set to be 50 ℃, the extraction pressure is set to be 30MPa, and the extraction time is set to be 3 hours.
Further, in the step (2), the extraction temperature of the extraction kettle is set to be 45 ℃, the extraction pressure is set to be 30MPa, and the extraction time is set to be 2 h.
The invention also provides an application of the bupleurum falcatum lipid extract in preparing a medicament for treatment verification, wherein the bupleurum falcatum lipid extract comprises the following components in percentage by weight:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid.
The invention also provides a preparation method of the starwort root extract, which comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) adding pulverized radix Stellariae into an extraction kettle, setting the extraction temperature of the extraction kettle at 40-50 deg.C, the extraction pressure at 25-35Mpa, the pressure of a separation kettle I at 50-60 deg.C, the pressure of a separation kettle II at 30-40 deg.C and the extraction time at 6-10 MPa, and obtaining radix Stellariae lipid extract.
The bupleurum falcatum lipid extract is obtained by extracting bupleurum falcatum, a series of lipid components including 7 lipid substances including 8 glycerophospholipids, 6 sphingolipids, 3 glycerides, 4 sterols, 1 pregnenolone lipid, 3 fatty acyl and 4 glycolipids are found in the bupleurum falcatum lipid extract, and 29 lipid components are provided, so that the bupleurum falcatum can comprehensively understand the lipid composition and excavate functional active components, provide reference for developing related products of bupleurum falcatum, and provide powerful technical support for analyzing and identifying complex and various lipid components in bupleurum falcatum.
Drawings
FIG. 1 is a line graph showing the yield of lipid extract of Bupleurum scorzonerifolium under different extraction pressures;
FIG. 2 is a line graph showing the yield of lipid extract of Bupleurum scorzonerifolium at different extraction temperatures;
FIG. 3 is a line graph showing the yield of lipid extract of Bupleurum scorzonerifolium at different extraction times;
FIG. 4 is a graph of response surface analysis of the interaction of extraction pressure and extraction temperature;
FIG. 5 is a graph of response surface analysis of the interaction of extraction pressure with extraction time;
FIG. 6 is a graph of response surface analysis of the interaction of extraction temperature and extraction time;
FIG. 7 is a chart of the ratio of the contents of the components in the lipid extract of Stellaria dichotoma;
figure 8. distribution of lipid components in Stellaria dichotoma extract a. lipid amount; b. mass percent (%) of different types of lipids; c. mass percent (%) of LPG, PC, PE, PG, PS, PI, CL, PA in glycerophospholipids; d. mass percent (%) of Cer, CerP, CerG2/3GNAc1, SM, ST, GM3 in sphingolipid; e. mass percent (%) of DG, MG, TG in glycerides; f. mass percent (%) of ChE, SiE, StE, ZyE in steroids; g. mass percent (%) of aca, OAHFA, WE in fatty acyl; h. the mass percentages (%) of MGDG, DGDG, MGMG, SQMG in glycolipids;
FIG. 9 shows the distribution of lipid components in Stellaria dichotoma extract in positive ion mode, the amount of lipid a, and the mass percentage of different types of lipid b (μ g/g);
FIG. 10 shows the distribution of ingredients in the lipids of Stellaria dichotoma extract in negative ion mode a lipid amount b mass percent (μ g/g) of different types of lipids.
Detailed Description
1 materials
1.1 Instrument
HA120-50-05 supercritical extraction device, Jiangsu Nantong Huaan supercritical extraction Co., Ltd; a high-speed universal pulverizer; AL204 electronic balance, mettler-toledo instruments (shanghai) ltd; q-exact Plus mass spectrometer (Thermo Scientific); UHPLC Nexera LC-30A ultra-high performance liquid chromatograph; (SHIMADZU) Low temperature high speed centrifuge.
1.2 herbs and reagents
Dried Stellaria dichotoma is purchased from Ningxia concentric Preview town, and identified as dried root of Stellaria dichotoma L.var.lanceolara Bge of Stellaria of Caryophyllaceae by professor Peng. of Ningxia university.
Carbon dioxide (99.9% CO2, food grade); acetonitrile (Thermo Fisher); isopropanol (Thermo Fisher); methanol (Thermo Fisher).
2 method
2.1 supercritical CO2 extraction of Stellaria dichotoma active ingredient
Weighing 1500g of bupleurum scorzonerifolium medicinal material powder which is pre-crushed (crushed to 50 meshes), putting the powder into a 5L extraction kettle, setting the extraction temperature, the extraction pressure and the extraction time of the extraction kettle according to table 3, setting the pressure of a separation kettle I to be 6MPa, the temperature to be 60 ℃, the pressure of a separation kettle II to be 6MPa and the temperature to be 60 ℃, opening a carbon dioxide gas cylinder when the temperature reaches the experimental requirements, pressurizing the extraction kettle and the separation kettle through a high-pressure pump, extracting when the pressure reaches the numerical value required by the experiment, recording the time, discharging from the separation kettle I and the separation kettle II every 30min to obtain the bupleurum scorzonerifolium lipid extract.
Wherein, the yield (%) is (supercritical extract (g)/starwort powder (g)) x 100%.
2.2 supercritical CO2 extraction Process optimization
2.2.1 Single factor experiment
The single-factor experiment is to fix other experiment factors and study the influence of the extraction temperature, the extraction pressure and the extraction time on the yield of the bupleurum falcatum lipid extract, and the levels of all factors are as follows: the extraction pressure is 20Mpa, 25Mpa, 30Mpa, 35Mpa, 40 Mpa; the extraction temperature is 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃; the extraction time is 1h, 2h, 3h, 4h and 5 h.
2.2.2 response surface optimization of supercritical CO2 extraction conditions
Through a single-factor test in the early stage, 3 main influence factors including extraction pressure, extraction temperature and extraction time are selected as research objects according to a Box-Behnken test design principle, and the extraction pressure (A), the extraction temperature (B) and the extraction time (C) are designed into 3 levels, wherein the factor levels are shown in a table 1.
TABLE 2 horizontal table of experimental design factors
2.3 LC-MS/MS analysis of lipid components of supercritical extract
2.3.1 sample Pre-treatment method
Taking a proper amount of sample (prepared bupleurum falcatum lipid extract), adding 200 mu L of water and 20 mu L of linked lipid standard mixture, MP vortex, adding 800 mu L of MTBE, vortex mixing, adding 240 mu L of precooled methanol, vortex mixing, carrying out ultrasonic treatment in a low-temperature water bath for 20min, standing at room temperature for 30min, 14000g, centrifuging at 10 ℃ for 15min, taking an upper organic phase, drying by nitrogen, adding 200 mu L of 90% isopropanol/acetonitrile solution for redissolving during mass spectrometry, fully vortex, taking 90 mu L of the redissolution, centrifuging at 14000g, at 10 ℃ for 15min, taking supernatant, injecting and analyzing.
2.3.2 chromatographic conditions
Separating the sample by adopting a UHPLC Nexera LC-30A ultra-high performance liquid chromatography system, and carrying out C18 chromatographic column; the column temperature is 45 ℃; flow rate 300 μ L/min, mobile phase composition a: aqueous acetonitrile (acetonitrile: water ═ 6:4, v/v), B: acetonitrile isopropanol solution (acetonitrile: isopropanol ═ 1:9, v/v), gradient elution procedure was as follows: 0-2min, maintaining B at 30% and A at 70%; 2-25min, B changes from 30% to 100% linearly, A maintains at 0%; and (3) for 25-35min, maintaining B at 30% and A at 70%, placing the sample in an automatic sample injector at 10 ℃ in the whole analysis process, and performing continuous analysis on the sample by adopting a random sequence in order to avoid the influence caused by the fluctuation of the detection signal of the instrument.
2.3.3 Mass Spectrometry conditions:
respectively adopting an electrospray ionization (ESI) positive ion mode and an ESI negative ion mode for detection, carrying out mass spectrometry on a sample after UHPLC separation by adopting a Q Exactive series mass spectrometer (Thermo scientific), wherein ESI source conditions are as follows: temperature 300 deg.C, sheath gas flow rate 45arb, auxiliaryGas flow rate of 15arb, purge gas flow rate of 1arb, spray voltage of 3.0KV, capillary temperature of 350 ℃, S-Lens RF Level of 50%, MS1Scanning range of (2): 200-1800, the mass-to-charge ratio of the lipid molecules and the lipid fragments, was collected as follows: 10 fragment patterns (MS) were acquired after each full scan (fullscan)2scan，HCD)，MS1Resolution is 70000 at M/Z200, MS2The resolution at M/Z200 was 17500.
2.4 data processing
The method comprises the following steps of utilizing Lipid Searcch 4.2 software (Thermo scientific TM) to realize integrated analysis of original data processing, peak extraction, Lipid identification, peak alignment, quantification and the like; realizing qualitative analysis of lipid by using an identification algorithm of the daughter ion, the parent ion and the neutral loss scan; and (3) calculating the absolute content of the object to be measured by adopting an isotope internal standard method and utilizing the response abundance ratio (peak area ratio) of the object to be measured and the internal standard and the concentration of the internal standard.
3 results
3.1 Single-factor experiment of supercritical CO2 extraction of Bupleurum falcatum Linne chemical Components
3.1.1 Effect of extraction pressure on the yield of Stellaria dichotoma lipid extract
Under the conditions of extraction temperature of 50 ℃ and extraction time of 2h, the extraction pressure of 20MPa, 25MPa, 30MPa, 35MPa and 40MPa is respectively measured, as can be seen from figure 1, the supercritical extract yield is increased along with the increase of the extraction pressure from 20MPa to 30MPa, when the extraction pressure is higher than 30MPa, the yield is reduced, and under the same conditions of temperature and time, the pressure is increased, which causes CO2The flow density is increased, the mass transfer distance between the solvent and the solute is reduced, the mass transfer efficiency is increased, the yield is increased, and the CO is reduced when the pressure is increased to a certain degree2The diffusivity causes the yield to be reduced, simultaneously, the overhigh pressure has large loss to the machine, and certain potential safety hazard exists when the equipment is used, so that the optimal extraction pressure is selected to be 30MPa in comprehensive consideration.
3.1.2 Effect of extraction temperature on the yield of Stellaria dichotoma lipid extract
Under the conditions of extraction pressure of 30MPa and extraction time of 2hThen, the extraction temperature is measured at 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C respectively, as can be seen from figure 2, when the temperature is increased from 35 deg.C to 45 deg.C, the supercritical extract yield is increased, when the extraction temperature is higher than 45 deg.C, the yield is reduced, and the influence of the extraction temperature on the extract yield is mainly two-fold, on one hand, the temperature is increased to make CO increase2Density reduction, CO2The dissolving capacity of the extract is reduced, the yield is reduced, but on the other hand, the molecular heat movement is accelerated due to the increase of the temperature, the extract yield is promoted to be increased, and CO is generated in a supercritical state2The density is very sensitive to the change of temperature and pressure, and because the pressure is higher at 30MPa, the CO2 density is reduced and slowed down due to the increase of the extraction temperature, the molecular thermal motion is accelerated, and the extract yield is gradually increased; however, after the temperature is raised to a certain degree, the density of CO2 is reduced to become a leading factor, so that the extraction yield is continuously reduced, and the optimal extraction temperature is selected to be 45 ℃ in comprehensive consideration.
3.1.3 Effect of extraction time on the yield of Stellaria delavayi lipid extract
Under the conditions of extraction pressure of 30MPa and extraction temperature of 50 ℃, the extraction time of 1h, 2h, 3h, 4h and 5h are respectively measured, as can be seen from figure 3, the extract yield increases along with the time when the time is between 1h and 2h, when the time exceeds 2h, the increase range of the extract yield is reduced, and the optimal extraction time is selected to be 2h in consideration of comprehensive experimental results and economic benefits.
3.2 response surface test results
3.2.1 response surface test design
On the basis of a single-factor experiment, Design expert12.0 software is used for designing a response surface experiment scheme, a response surface experiment of 3-factor 3 level is adopted in the experiment, and the response surface experiment is carried out according to the table 1. The protocol and results are shown in Table 3.
TABLE 3 Experimental design and results
3.2.2 response surface analysis of variance results
In order to test the reliability of the regression equation and determine the degree of influence of each factor on the yield of the lipid extract of Stellaria dichotoma, analysis of variance was performed on the regression equation, and the results are shown in Table 4. The regression equation:
Y＝0.33+0.015A+0.013B+(0.078C+7.500E-003)AB-(5.000E-003)AC+(3.750E-003)BC-0.040A2-0.029B2-0.051C2。
TABLE 4 results of ANOVA
Note: denotes P <0.01, the difference is very significant; denotes P <0.05, difference is significant;
TABLE 5 statistical analysis of regression equation errors
From Table 4, it is clear that C and A2，B2，C2The term has a very significant influence on the response value (P)<0.01), A, B, AB have a significant effect on the response value (P)<0.05), the multivariate correlation coefficient R in Table 52The larger the correlation, the better; adj R-Square and Pred R-Square (RAdj)2－RPred2< 0.2) the two values are high and close, the regression model can fully explain the process; if not, the process explanation is not sufficient, whether other significant influence factors exist needs to be considered, CV is less than 10%, and the reliability and the accuracy of the experiment are high; precision (Adeq Precision) is the effective signal and noiseThe sound ratio of more than 4 is considered reasonable, and as can be seen from table 5, the fitted regression equation conforms to the above test principle, and the adaptability is good, so that the regression equation can be used for predicting and analyzing the real experimental result.
3.2.3 analysis of response surface interactions
From the analysis result of variance, the influence degree of each factor on the yield of the starwort root extract is different and has interaction, the influence degree is not a pure linear relation, the 3D graph and the contour graph of the three obtained by optimizing the response surface method are shown in the graphs 4-6, the shape of the contour line is an ellipse, the interaction of the factors is obvious, the interaction is not obvious if the contour line is a circle, the interaction of A and B is obvious, the interaction of the other two factors is not obvious, the result is the same as the analysis result of the table 3, and the 3D graph shows that the slope of the extraction time is steeper than that of the extraction time and the extraction pressure, which indicates that the influence of the extraction time on the yield is larger than that of the extraction pressure and the extraction temperature; the radian of the slope surface of the extraction pressure is larger than that of the slope surface of the extraction time, which indicates that the influence of the extraction pressure on the yield is higher than the extraction temperature, and the influence of each factor on the yield of the bupleurum falcatum extract obtained from the response surface result is the extraction time > the extraction pressure > the extraction temperature in turn.
3.2.4 optimized Process validation test
Solving a regression equation through Design-Expert10.0.7 software to obtain the optimal extraction process conditions of the bupleurum falcatum lipid extract, wherein the optimal extraction process conditions are 47.007 ℃, the pressure is 35MPa, the extraction time is 2.737h, the theoretical yield is 0.337%, the process conditions are changed into the extraction pressure of 35MPa, the extraction temperature is 47 ℃ and the extraction time is 3h according to the actual operability of extraction equipment, verification tests are carried out according to the test conditions, 3 times of repeated verification are carried out, the yield of the bupleurum falcatum lipid extract is 0.341%, the error is 0.85%, the actual value is basically consistent with the theoretical value, and the process parameters obtained by optimizing the response surface are better, so that the application value is realized.
3.3 distribution of the lipid extract of Stellaria dichotoma extracted with supercritical CO2
As shown in Table 1, in Stellaria dichotoma lipid extract under the optimal extraction process conditions (i.e. extraction pressure of 35MPa, extraction temperature of 47 ℃, extraction time of 3h), 7 lipid substances including 8 glycerophospholipids, 6 sphingolipids, 3 glycerides, 4 sterols, 1 pregnenolone lipid, 3 fatty acyl and 4 glycolipids were detected, and 29 lipid components were detected, as shown in FIG. 7, the highest content components were sterol and glyceride components, 88.111% and 10.868%, respectively.
Since the data for determining the content of each lipid component is excessive, let Cer (ceramide) be taken as an example, as shown in table 6, the content of an active ingredient of bupleurum falcatum lipid extract, which is ceramide in a subclass (detected in a positive ion mode), is shown.
TABLE 6 content of active ingredients in the lipid extract of Stellaria dichotoma, a subclass of which is ceramide.
The lipid distribution in the lipid extract of Stellaria dichotoma is as follows:
the lipid components of Stellaria dichotoma are analyzed by UHPLC-Q Exact MS, and detected in ES I positive ion and negative ion modes, as shown in Table 7, 7 lipid substances including glycerophospholipids, sphingolipids, glycerides, sterols, pregnenolone lipids, fatty acyl and glycolipid are detected in Stellaria dichotoma, wherein the glycerophospholipids comprise LPG, PC, PE, PG, PS, PI, CL and PA 8 lipids, the sphingolipids comprise Cer, CerP, CerG2/3GNAc1, SM, ST and GM 36 lipids, the glycerides comprise DG, MG and TG 3 lipids, the sterols comprise ChE, SiE, StE and ZyE 4 lipids, the pregnenolone lipids comprise Co1 lipids, and the fatty acyl comprises AcCa, OAHFA and WE3 lipids. Glycolipids include 4 lipids MGDG, DGDG, MGMG, SQMG, for a total of 29 lipid components. As shown in FIG. 8a, 2542 lipid components were detected in the supercritical extract of Stellaria dichotoma, wherein the lipid components of glycerophospholipid, sphingolipid, glyceride, sterol, pregnenolone lipid, fatty acyl, glycolipid were 246, 196, 1630, 212, 3, 120, 138 (FIG. 8a), 0.53%, 0.03%, 10.69%, 88.29%, 0.38%, 0.09% (FIG. 8 b). The highest content of LPG, PC, PE, PG, PS, PI, CL and PA in glycerophospholipids is 0.36 percent of phosphatidylinositol (figure 8 c); lipid components including Cer, CerP, CerG2/3GNAc1, SM, ST, and GM3 in sphingolipid are 168, 11, 2, 1, 13, and 1, respectively, wherein the highest content is 0.02% of Cer by mass (FIG. 8 d); the lipid components of DG, MG and TG in glyceride were 395, 38 and 1197, respectively, and the highest content was 9.39% by mass of DG (FIG. 8 e); the sterol lipid components of ChE, SiE, StE and ZyE were 16, 48, 86 and 62 respectively, and the highest content was 61.66% by mass of zymosterol (fig. 8 f). The pregnenolone lipid Co has a lipid component of 3 wt% 0.02%, the lipid components of AcCa, OAHFA and WE in fatty acyl are respectively 1, 37 and 79, and the maximum content is WE wt% 0.28% (FIG. 8 g). The lipid component types of MGDG, DGDGDG, MGMG and SQMG in the glycolipid are 125, 7, 5 and 1 respectively, wherein the maximum content is MGDG with the mass percent of 0.08% (figure 8 h).
TABLE 7 lipid component constitution in Stellaria dichotoma extract
Wherein, the lipid of the bupleurum chinense super extract in the positive ion mode comprises the following components:
in the bupleurum falcatum extract, 7 types (glycerophospholipid, sphingolipid, glyceride, sterol, pregnenolone lipid, fatty acyl and glycolipid) of (LPG, PC, PE, PG, PS, PI, Cer, CerP, SM, ST, DG, MG, TG, ChE, SiE, StE, ZyE, Co, AcCa, WE and MGDG) and (2188 lipid components are detected in the positive ion mode, wherein the quantities of the glycerophospholipid, sphingolipid, glyceride, sterol, pregnenolone lipid and fatty acyl lipid components are respectively 184, 75, 1630, 212, 3 and 80, the mass percentages are respectively 0.51%, 0.02%, 10.71%, 88.45%, 0.01% and 0.28%, and the quantity of the lipid component in figure 9b is 4, and the content is trace. Wherein the components detected only in the positive ion mode are glycerides, sterols, pregnenolones, fatty acyl and glycolipids. The glycerophospholipids detected 6 species (LPG, PC, PE, PG, PS, PI) in positive ion mode, and the lipid component amounts were 1, 23, 90, 35, 5, and 30, respectively. Wherein the highest content is 0.36 percent of PI by mass; sphingolipids were detected in positive ion mode in 4 types (Cer, CerP, SM, ST), with lipid component types of 50, 11, 1, and 13, respectively, and the highest content was 0.02% by mass of Cer.
The lipid composition of the bupleurum chinense super-extract in the negative ion mode is as follows:
only 2 types (glycerophospholipid and sphingolipid), 7 lipid components (PG, PI, CL, PA, Cer, CerG2/3GNAc1 and GM3) and 183 lipid components were detected in the negative ion mode, as shown in FIG. 10a, wherein the amounts of the glycerophospholipid and sphingolipid components were 62 and 121, respectively, as shown in FIG. 10b, and the mass percentages were 73.79% and 26.21%. The lipid components of 4 (PG, PI, CL and PA) glycerophospholipids are respectively 38, 9, 10 and 5 under the negative ion mode, wherein the highest content of the glycerophospholipids is 30.87 percent by mass of the CL; sphingolipid is detected in negative ion mode to obtain 3 kinds (Cer, CerG2/3GNAc1, GM3) with lipid component amounts of 118, 2, and 1, wherein the highest content is Cer, and the mass percentage is 24.40%.
The content of phytosterol (sterol) in substances extracted by the supercritical extraction method accounts for 88.29% of the total lipid content; the different lipid components have different activities, so that the different lipid components have different contributions to the pharmacological functions of Stellaria dichotoma, wherein the components with the mass percentage of more than 0.1 percent are mainly glycerophospholipid, glyceride, sterol, pregnenolone lipid and fatty acyl. Glycerophospholipids are considered to be functional components against colon cancer, gastrointestinal pathogens, alzheimer's disease, depression and stress. Glycerides are the main components of vegetable oils, have the main functions of supplying and storing energy, fixing and protecting internal organs, participate in energy supply in various links such as the growth and development of pregnant mothers and ventral fetuses, and play a key role in lipid metabolism. The sterol component contained in the plant is a biologically active component widely existing in the plant, and can only be taken in by external source, but can not synthesize substances in human body. Phytosterol has good biological functions of reducing blood fat, resisting inflammation, resisting oxidation, regulating immunocompetence, resisting tumor, delaying senescence and the like, and researches show that the phytosterol has an important effect on the treatment of Alzheimer's Disease (AD) cardiovascular diseases in the future. Pregnenolone, is a naturally occurring hormone and steroid. Pregnenolone is an important intermediate for synthesizing progesterone, finasteride and the like, and is used for synthesizing an indwelling drug intermediate and a steroid drug. Pregnenolone ester can enhance brain function and memory. Currently, there is less research on the application of pregnenolone. The most abundant fatty acyl is wax, and vegetable wax is mainly present in horny layer of Bupleurum root to prevent water loss. The sphingolipid and glycolipid components have low content in Stellaria dichotoma, the two components have low content in plants, but have important biological activity, and the glycolipid has biological activity in the aspects of resisting tumor, HIV, inflammation and bacteria, enhancing body immunity and the like; sphingolipids have biological properties of anticancer, antibacterial and low cholesterol. Therefore, the research can be used for comprehensively, qualitatively and quantitatively analyzing the lipid components of Stellaria dichotoma and is of great help for the medicinal function excavation, development and utilization of Stellaria dichotoma.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (9)
1. The lipid extract of Stellaria dichotoma is characterized in that the lipid extract of Stellaria dichotoma contains the following components by weight percent:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid;
the preparation method of the bupleurum falcatum lipid extract comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) adding pulverized radix Stellariae into supercritical CO2In the extraction kettle, supercritical CO is set2The extraction temperature of the extraction kettle is 40-50 ℃, the extraction pressure is 25-35Mpa, the pressure of the separation kettle I is 6MPa-10MPa, the temperature is 50-60 ℃, the pressure of the separation kettle II of the extraction kettle is 6MPa-10MPa, the temperature is 30-40 ℃, and the extraction time is 1-3h, thus obtaining the bupleurum falcatum lipid extract.
2. The lipid extract of bupleurum falcatum of claim 1, wherein the glycerophospholipid comprises lysophosphatidylglycerol, lecithin, cephalin, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidic acid, the sphingolipid comprises ceramide, phosphoceramide, glycosphingolipid, sphingomyelin, sulfatide, ganglioside, glyceride comprises diglyceride, monoglyceride, triglyceride, sterol comprises cholesterol ester, sitosterol ester, stigmasterol ester, zymosterol comprises coenzyme, fatty acyl comprises acylcarnitine, phosphatidylethanolamine, wherein the phosphatidylethanolamine, the composition, and the composition, and the composition of the composition, and the composition of the,
(o-acyl) -1-hydroxy fatty acids and waxes, glycolipids including galactodiacylglycerol, digalactosyldiacylglycerol, galactomonoacylglycerol and sulfated monoglycerides.
3. A method for extracting Stellaria dichotoma lipid extract from Stellaria dichotoma is characterized in that the Stellaria dichotoma lipid extract comprises the following components in percentage by weight:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid; (ii) a
The preparation method of the bupleurum falcatum lipid extract comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) adding pulverized radix Stellariae into supercritical CO2In the extraction kettle, supercritical CO is set2The extraction temperature of the extraction kettle is 40-50 ℃, the extraction pressure is 25-35Mpa, the pressure of the separation kettle I is 6MPa-10MPa, the temperature is 50-60 ℃, the pressure of the separation kettle II of the extraction kettle is 6MPa-10MPa, the temperature is 30-40 ℃, and the extraction time is 1-3h, thus obtaining the bupleurum falcatum lipid extract.
4. The lipid extract of Stellaria dichotoma as claimed in claim 1, wherein the supercritical CO is set in step (2)2The extraction temperature of the extraction kettle is 47 ℃, the extraction pressure is 35MPa, and the extraction time is 3 h.
5. The lipid extract of Stellaria dichotoma as claimed in claim 1, wherein the supercritical CO is set in step (2)2The extraction temperature of the extraction kettle is 50 ℃, the extraction pressure is 30MPa, and the extraction time is 3 h.
6. The lipid extract of Stellaria dichotoma as claimed in claim 1, wherein the supercritical CO is set in step (2)2The extraction temperature of the extraction kettle is 45 ℃, the extraction pressure is 30MPa, and the extraction time is 2 h.
7. The lipid extract of bupleurum falcatum as claimed in claim 5, wherein the lipid extract of bupleurum falcatum comprises the following components by weight percentage:
sterol 88.111%, glyceride 10.868%, fatty acyl 0.362%, pregnenolone lipid 0.013%, glycerophospholipid 0.532%, glycolipid 0.085%, sphingolipid 0.029%.
8. The application of the bupleurum falcatum lipid extract in preparing the medicine for treating inflammation is characterized in that the bupleurum falcatum lipid extract contains the following components by weight percent:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid.
9. A method for extracting radix Stellariae lipid extract from radix Stellariae is characterized in that the radix Stellariae lipid extract contains the following components by weight percent:
0.5-0.6% of glycerophospholipid, 0.02-0.03% of sphingolipid, 9-11% of glyceride, 85-90.5% of sterols, 0.01-0.02% of pregnenolone lipid, 0.3-0.4% of fatty acyl and 0.07-0.09% of glycolipid;
the preparation method comprises the following steps:
(1) pulverizing Stellaria dichotoma into 40-60 mesh;
(2) adding pulverized radix Stellariae into supercritical CO2In the extraction kettle, supercritical CO is set2The extraction temperature of the extraction kettle is 47 ℃, the extraction pressure is 35MPa, the pressure of the separation kettle I is 6MPa-10MPa, the temperature is 50 ℃ -60 ℃, and supercritical CO is adopted2The pressure of a separation kettle II of the extraction kettle is 6MPa-10MPa, the temperature is 30-40 ℃, and the extraction time is 2h, thus obtaining the bupleurum falcatum lipid extract.
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