Preparation and application of active part of large-leaf rhododendron

Abstract

The application relates to preparation and application of an active part of large-leaf rhododendron. The preparation method of the active part comprises the following steps: taking dry roots of rhododendron farrerae, crushing and sieving to obtain powder, taking a certain amount of the powder, adding a methanol solution with a volume fraction of 80%, ultrasonic extraction, centrifugal separation and collection of supernatant, concentration of the supernatant to 1 / 2-1 / 3 of the volume, D101 macroporous adsorption resin column chromatography, elution with distilled water, 30% ethanol solution, 50% ethanol solution and 70% ethanol solution in sequence, 2 column volumes of each eluent, collection of the fractions obtained by different eluents, vacuum concentration and drying, and obtaining F1, F2, F3 and F4 components in sequence; wherein the F2 and F3 components are the active part.

Description

Technical Field

[0001] This invention belongs to the field of extraction of active ingredients from traditional Chinese medicine, specifically involving the preparation and application of the active components of *Gynostemma pentaphyllum*. Background Technology

[0002] *Bergeniae Scopulosae Rhizoma*, also known as Qinling Rock White Vegetable or Ground White Vegetable, is the dried rhizome of *Bergenia scopulosa* TP. Wang, a plant belonging to the genus *Bergenia* in the family Saxifragaceae. It is one of the famous "Seven Medicines" of Taibai Mountain and is distributed in the Qinling and Qilian Mountains of Shaanxi Province. It has an astringent taste and neutral properties, and enters the spleen, liver, bladder, and lung meridians. It has the effects of astringing and consolidating the intestines, stopping bleeding and leukorrhea, stopping dysentery and diarrhea, and strengthening the spleen and eliminating dampness. Clinically, it is mainly used to treat diarrhea, dysentery, metrorrhagia, leukorrhea, abdominal distension and pain, impetigo, tinea capitis, and scabies.

[0003] To date, a total of 26 components have been isolated from Qinling rock cabbage, including 10 phenolic acid components, 8 flavonoid components, 2 anthraquinone components, and 6 other components. The phenolic components in Qinling rock bergamot mainly include bergenin, 4-O-gallobergenin, 11-O-gallobergenin, arbutin, 6′-O-gallobergenin, gallic acid, catechin, syringic acid, protocatechuic acid, and methyl gallate; the flavonoid components mainly include catechin, 7-O-gallocatechin, catechin-7-O-α-D-glucoside, kaempferol, quercetin, afostigmoside, rutin, and hyperoside; the anthraquinone components include emodin methyl ether and emodin-8-O-β-D-glucoside; in addition, Qinling rock bergamot also contains other types of components such as succinic acid, isobutyl anisole, phenylalanine, stigmasterol, β-sitosterol, and carotene.

[0004] The inventors previously used UHPLC-Q Exactive Focus MS / MS to resolve 66 chemical components from *Gynostemma pentaphyllum*, including 2 free amino acids, 7 bergenin derivatives, 15 flavonoids, 15 organic acids, 25 glycosides, and 2 other compounds. They also analyzed the blood-transfer components of the *Gynostemma pentaphyllum* extract, detecting 35 blood-transfer components, of which 6 were precursor components and 29 were metabolites. Among the precursor components, there were 2 phenolic acids, 1 isocoumarin, 2 C-glycosides, and 1 O-glycoside. Arbutin, bergenin, salicylic acid, and ellagic acid were identified by comparison with reference standards. The blood-transfer metabolites mainly originated from compounds containing structural units such as gallic acid, mallonanoside A, bergenin, catechin, and ellagic acid. Their metabolic pathways primarily involved glucuronidation, sulfation, and methylation, with phase II metabolism being the dominant process. The inventors previously constructed liquid phase fingerprint spectra of 10 batches of Dapanlong 7, identifying a total of 14 common peaks. By comparing with mixed reference solutions, 7 common peaks were identified, namely arbutin, gallic acid, bergenin, catechin, 4-O-galloylbergenin, 11-O-galloylbergenin, and catechin gallate.

[0005] Literature reports that extracts of *Brassica rapa* from the Qinling Mountains possess antibacterial and antioxidant effects. Ma reported that *Brassica rapa* exhibits strong antibacterial activity against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Escherichia coli*, with stronger inhibition against *Staphylococcus aureus* than against *Pseudomonas aeruginosa* and *Escherichia coli*. The extract also exhibits high antioxidant activity; its scavenging effect on three free radicals—·OH, ·O2-, and H2O2—gradually increases with increasing concentration, showing a good linear relationship. However, existing research on the pharmacological effects of *Brassica rapa* from the Qinling Mountains focuses on the extract level and does not address the study of its active components. This invention uses macroporous adsorption resin to separate different fractions from the *Brassica rapa* extract and tests the total flavonoids, total polyphenols, total tannins, antioxidant, antitumor, and α-glucosidase inhibitory effects of these fractions. Furthermore, UHPLC-QExactive Focus MS is used to analyze the chemical composition of the active fractions, aiming to discover more potential active ingredients from *Brassica rapa* from the Qinling Mountains and provide a basis for the comprehensive development and utilization of this herb. Summary of the Invention

[0006] This invention provides an antitumor active fraction of *Gynostemma pentaphyllum*, characterized in that the preparation method of the antitumor active fraction includes the following steps:

[0007] Dried rhizomes of *Brassica rapa* from the Qinling Mountains were pulverized and sieved to obtain powder. A certain amount of powder was weighed and added to an 80% methanol solution. After ultrasonic extraction, the powder was centrifuged and the supernatant was collected. The supernatant was concentrated to 1 / 2-1 / 3 of its volume and then subjected to D101 macroporous adsorption resin column chromatography. The powder was eluted sequentially with distilled water, 30% ethanol solution, 50% ethanol solution, and 70% ethanol solution, with each eluent eluting for 2 column volumes. The fractions obtained with different eluents were collected, concentrated under reduced pressure, and dried to obtain fractions F1, F2, F3, and F4. Among them, fraction F2 is the antitumor active site.

[0008] After pulverization, it is preferable to pass the powder through a 50-mesh sieve. The amount of 80% methanol solution used is preferably 8-12 mL (preferably 10 mL) of 80% methanol solution per gram of powder. The ultrasonic time is preferably 15-60 min, and the ultrasonic frequency is selected from 20-40 kHz (preferably 30 kHz). The centrifugation conditions are preferably 3000 rpm for 5 min. For macroporous adsorption resin column chromatography, it is preferable to let the sample stand for 10-12 h after loading before elution. The tumor is preferably HepG-2 liver cancer cells.

[0009] The HPLC chromatograms of the above-mentioned antitumor active fraction of *Gynostemma pentaphyllum* under the following chromatographic conditions are basically as follows: Figure 1 As shown in C;

[0010] Chromatographic conditions: Agilent 5TC-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; phase A: acetonitrile, phase B: 0.1% phosphoric acid aqueous solution; gradient elution: 0–12 min, 2% A; 12–14 min, 2–11% A; 14–40 min, 11–12% A; 40–85 min, 12–24% A; 85–90 min, 24% A; flow rate: 1.0 mL / min; detection wavelength: 275 nm; column temperature: 25 °C; injection volume: 10 μL.

[0011] The total ion chromatograms of the above-mentioned antitumor active fraction of *Gynostemma pentaphyllum* under the following chromatographic and mass spectrometric conditions, in both positive and negative ion modes, are basically as follows: Figure 2 As shown in F2;

[0012] Chromatographic conditions: Agilent InfinityLab Poroshell 120SB-C 18 The chromatographic column was 4.6 mm × 150 mm with a diameter of 2.7 μm. The column temperature was 30 °C, and the flow rate was 0.8 mL / min. The mobile phase A was methanol, and the mobile phase B was 0.1% formic acid aqueous solution. The gradient elution was as follows: 0–35 min, 2%–40% A; 35–60 min, 40%–95% A; 60–65 min, 95% A.

[0013] Mass spectrometry conditions: The ion source was a heated electrospray ionization source (HESI), with positive and negative ion monitoring modes. The spray voltages for positive and negative ion modes were 3.5 kV and 2.8 kV, respectively. The sheath gas flow rate was 70 flow units, the auxiliary gas flow rate was 20 flow units, the capillary temperature was 400 °C, the nebulization temperature was 500 °C, the S-lens RF level was 55, and the scanning mode was Full MS / dd-MS. 2 Full MS resolution 70,000, dd-MS 2 Resolution 17500, scan range m / z 80~1200; collision energy NCE 20, 40, 60eV.

[0014] The UHPLC-Q Exactive Focus MS analysis of the chemical components of the antitumor active fraction of the above-mentioned Dapanlongqi is shown in Table 4.

[0015] Another embodiment of the present invention provides the use of the above-mentioned antitumor active fraction of *Gynostemma pentaphyllum* or its pharmaceutically acceptable salt in the preparation of an antitumor drug. The tumor is preferably HepG-2 liver cancer cells.

[0016] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses the above-mentioned antitumor active fraction of *Gynostemma pentaphyllum* or a pharmaceutically acceptable salt thereof as the active ingredient. The pharmaceutical composition may also optionally contain other antitumor drugs. The pharmaceutical composition may also optionally include pharmaceutically acceptable excipients.

[0017] This invention provides a site on *Gynostemma pentaphyllum* that inhibits α-glucosidase activity, characterized in that the preparation method of the site includes the following steps:

[0018] Dried rhizomes of *Brassica rapa* from the Qinling Mountains were pulverized and sieved to obtain powder. A certain amount of powder was weighed and added to an 80% methanol solution. After ultrasonic extraction, the mixture was centrifuged and the supernatant was collected. The supernatant was concentrated to 1 / 2-1 / 3 of its volume and then subjected to D101 macroporous adsorption resin column chromatography. The mixture was eluted sequentially with distilled water, 30% ethanol solution, 50% ethanol solution, and 70% ethanol solution, with each eluent eluting for 2 column volumes. The fractions obtained with different eluents were collected, concentrated under reduced pressure, and dried to obtain fractions F1, F2, F3, and F4. Among them, fraction F3 is the site that inhibits α-glucosidase activity.

[0019] After pulverization, it is preferable to pass the powder through a 50-mesh sieve. The amount of 80% methanol solution used is preferably 8-12 mL (preferably 10 mL) of 80% methanol solution per gram of powder. The ultrasonic time is preferably 15-60 min, and the ultrasonic frequency is selected from 20-40 kHz (preferably 30 kHz). The centrifugation conditions are preferably 3000 rpm for 5 min. For macroporous adsorption resin column chromatography, it is preferable to let the sample stand for 10-12 h after loading before elution.

[0020] The HPLC chromatogram of the above-mentioned α-glucosidase inhibitory site of *Gynostemma pentaphyllum* under the following chromatographic conditions is basically as follows: Figure 1 As shown in D;

[0021] Chromatographic conditions: Agilent 5TC-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; phase A: acetonitrile, phase B: 0.1% phosphoric acid aqueous solution; gradient elution: 0–12 min, 2% A; 12–14 min, 2–11% A; 14–40 min, 11–12% A; 40–85 min, 12–24% A; 85–90 min, 24% A; flow rate: 1.0 mL / min; detection wavelength: 275 nm; column temperature: 25 °C; injection volume: 10 μL.

[0022] The above-mentioned inhibitory α-glucosidase activity site of *Gynostemma pentaphyllum* under the following chromatographic and mass spectrometric conditions, its total ion chromatograms in positive and negative ion modes are basically as follows: Figure 2 As shown in F3;

[0023] Chromatographic conditions: Agilent InfinityLab Poroshell 120SB-C 18 The chromatographic column was 4.6 mm × 150 mm with a diameter of 2.7 μm. The column temperature was 30 °C, and the flow rate was 0.8 mL / min. The mobile phase A was methanol, and the mobile phase B was 0.1% formic acid aqueous solution. The gradient elution was as follows: 0–35 min, 2%–40% A; 35–60 min, 40%–95% A; 60–65 min, 95% A.

[0024] Mass spectrometry conditions: The ion source was a heated electrospray ionization source (HESI), with positive and negative ion monitoring modes. The spray voltages for positive and negative ion modes were 3.5 kV and 2.8 kV, respectively. The sheath gas flow rate was 70 flow units, the auxiliary gas flow rate was 20 flow units, the capillary temperature was 400 °C, the nebulization temperature was 500 °C, the S-lens RF level was 55, and the scanning mode was Full MS / dd-MS. 2 Full MS resolution 70,000, dd-MS 2 Resolution 17500, scan range m / z 80~1200; collision energy NCE 20, 40, 60eV.

[0025] The chemical components of the above-mentioned Dapanlongqi inhibiting α-glucosidase activity site are analyzed by UHPLC-Q ExactiveFocus MS, as shown in Table 5.

[0026] Another embodiment of the present invention provides the use of the above-mentioned α-glucosidase-inhibiting site of *Gynostemma pentaphyllum* or a pharmaceutically acceptable salt thereof in the preparation of α-glucosidase inhibitors.

[0027] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses the above-mentioned α-glucosidase inhibitory site of *Gynostemma pentaphyllum* or a pharmaceutically acceptable salt thereof as the active ingredient. The pharmaceutical composition may also optionally contain other α-glucosidase inhibitors. The pharmaceutical composition may also optionally include pharmaceutically acceptable excipients.

[0028] Unless otherwise specified, the concentrations of the eluent, mobile phase, and extraction solvent mentioned in this invention refer to volume percentage concentrations.

[0029] Compared with the prior art, the advantages of the present invention are as follows: the present invention obtained the optimal extraction solvent, namely 80% methanol solution, through screening of extraction solvents; the present invention obtained the antitumor active part and the α-glucosidase inhibitory part, and conducted component analysis on them, providing a basis for the comprehensive development and utilization of Qinling rock cabbage. Attached Figure Description

[0030] Figure 1 These are HPLC chromatograms of crude extracts and different polar chemical components of *Gnaphalium affine* from the Qinling Mountains; A. Crude extract of *Gnaphalium affine* from the Qinling Mountains; B. Component F1; C. Component F2; D. Component F3; E. Mixed reference solution; F. Component F4; 1. Bergenin; 2. Catechin; 3. 4-O-Galloylbergenin; 4. 11-O-Galloylbergenin; 5. Catechin gallate.

[0031] Figure 2 This is the total ion chromatogram of components F2 and F3 of *Brassica qinlingensis* in positive and negative ion modes. Detailed Implementation

[0032] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0033] 1. Instruments and reagents

[0034] 1.1 Instruments

[0035] UltiMate 3000 ultra-high performance liquid chromatography system and Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Inc., USA), SQP 1 / 100,000 electronic analytical balance and 1 / 10,000 electronic analytical balance [Sartorius Scientific Instruments (Beijing) Co., Ltd.], ELISA reader (Thermo Fisher Scientific, Inc., USA), KQ-400DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), TD4 low-speed benchtop centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.).

[0036] 1.2 Test Drugs

[0037] The *Bergenia scopulosa* plants were collected from Baoji, Shaanxi Province, and identified by Associate Researcher Chen Zhiyong of the Shaanxi Academy of Traditional Chinese Medicine as *Bergenia scopulosa* TPWang, a plant belonging to the genus *Bergenia* of the family Saxifragaceae. D101 macroporous adsorption resin (batch number: C12896490), Folin-Ciocalteu (batch number: C13547993), DPPH (batch number: C14357523), ABTS (batch number: C14565258), casein (batch number: C12913946), and potassium persulfate (batch number: C14951199) were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and Trolox (batch number: 1027J021) was purchased from Beijing Solarbio Technology Co., Ltd. Hepatocellular carcinoma cells (HepG-2), DMEM medium (Gibico), fetal bovine serum (BI), trypsin (Gibico), penicillin-streptomycin solution (double antibody), PBS (Yobibio), water was ultrapure water, methanol was chromatographic grade, formic acid was mass spectrometry grade, and all other reagents were analytical grade.

[0038] 2 methods

[0039] 2.1 Selection of the optimal extraction solvent

[0040] An appropriate amount of dried rhizomes of *Brassica rapa* from the Qinling Mountains was pulverized and passed through a 50-mesh sieve to obtain powder. 1.5 g of the powder was weighed and added to 15 mL of 0%, 20%, 40%, 60%, 80%, and 100% methanol aqueous solutions (v / v) respectively in 50 mL centrifuge tubes. The tubes were sonicated for 30 min (frequency 30 kHz), centrifuged at 3000 rpm for 5 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm membrane and stored at 4℃ to obtain crude extracts. The polyphenol content and antioxidant activity of the six crude extracts were then determined sequentially to identify the optimal extraction solvent.

[0041] 2.2 Preparation of Components with Different Polarities

[0042] Take an appropriate amount of dried Qinling rock cabbage rhizomes, crush them, pass them through a 50-mesh sieve to obtain powder, weigh 50.0g of powder, add 500mL of 80% methanol solution, sonicate for 30min (frequency 30kHz), centrifuge at 3000rpm for 5min, collect the supernatant, concentrate at 45℃ to about 1 / 2 of the original volume (about 250mL), pass through a D101 macroporous adsorption resin column, after loading the sample and standing for 12h, elute sequentially with distilled water, 30% ethanol, 50% ethanol and 70% ethanol, elute with each eluent for 2 column volumes, collect the fractions obtained with different eluents, concentrate under reduced pressure and dry, to obtain fractions F1, F2, F3 and F4 sequentially.

[0043] 2.3 Determination of polyphenolic components

[0044] 2.3.1 Determination of total flavonoids

[0045] Mix 120 μL of appropriately diluted sample solution with 8 μL of 50 mg / mL sodium nitrite solution, let stand for 6 min, then add 8 μL of 100 mg / mL aluminum trichloride solution. After incubating at room temperature for 5 min, add 100 μL of 40 mg / mL sodium hydroxide solution to each well, incubate in the dark at room temperature for 30 min, and then measure at 410 nm using a microplate reader. The results are expressed as rutin equivalents per g of dry weight of the herb.

[0046] 2.3.2 Determination of total polyphenols

[0047] Mix 20 μL of appropriately diluted sample solution with 40 μL of 25% Folin-Ciocalteau reagent and incubate at room temperature for 5 min. Add 140 μL of 700 mM sodium carbonate solution to each well and incubate in the dark at 40 °C for 30 min. Measure the results at 765 nm using a microplate reader. The results are expressed as gallic acid equivalents per g of dry weight of the herb.

[0048] 2.3.3 Determination of total tannins

[0049] 25 mL of appropriately diluted sample solution was mixed with 600 mg of casein and incubated at room temperature for 3 h with constant shaking. The mixture was centrifuged at 3000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The procedure was followed according to "2.3.2 Determination of Total Polyphenols". Total tannins are equal to the difference in total phenol content before and after the reaction with casein. The results are expressed as gallic acid equivalents per g of dry weight of the medicinal material.

[0050] 2.4 Antioxidant Activity Assay

[0051] 2.4.1 DPPH free radical scavenging activity

[0052] 40 μL of freshly prepared 1 mM DPPH solution and 190 μL of methanol were added to 10 μL of sample solutions of different concentrations, respectively. The mixture was vortexed and incubated in the dark at room temperature for 30 min. Measurements were then taken at 517 nm using a microplate reader. Trolox was used as a positive control, and blanks were included in each group. The DPPH free radical scavenging rate was [1-(A S -A SB ) / (A C -A CB )]×100%, where A S It is the absorbance of the sample solution, A SB It is the absorbance of the blank sample solution, A. C It is the absorbance of the negative control solution, A. CB It is the absorbance of the negative blank solution.

[0053] 2.4.2 ABTS free radical scavenging activity

[0054] 2.5 mL of freshly prepared 7 mM ABTS and 44 μL of freshly prepared 140 mM potassium persulfate were incubated overnight (12–16 h) at room temperature in the dark to prepare the ABTS stock solution. Before use, the solution was diluted with 10 mM phosphate buffer (pH = 7.4) to a absorbance of 0.70 ± 0.02 at 734 nm to prepare the ABTS working solution. 200 μL of the ABTS working solution was added to 10 μL of sample solutions of different concentrations. The solutions were incubated at 30°C in the dark for 5 min, and the absorbance was measured at 734 nm using a microplate reader. A positive control was trolox, and blanks were included in each group. The ABTS free radical scavenging rate was [1-(A S -A SB ) / (A C -A CB )]×100%, where A S It is the absorbance of the sample solution, A SB It is the absorbance of the blank sample solution, A. C It is the absorbance of the negative control solution, A. CB It is the absorbance of the negative blank solution.

[0055] 2.5 Assay for α-glucosidase activity inhibition

[0056] 50 μL of sample solutions of different concentrations were placed in a 96-well plate. 50 μL of 0.25 U / ml α-glucosidase was added to each well, vortexed to mix, and incubated at room temperature for 10 min. Then, 50 μL of 5 mM PNPG was added to each well, vortexed to mix, and incubated at room temperature for 10 min. The α-glucosidase inhibitory activity was measured at 405 nm using a microplate reader. S -A SB ) / (A C -ACB )]×100%, where A S It is the absorbance of the sample solution, A SB It is the absorbance of the blank sample solution, A. C It is the absorbance of the negative control solution, A. CB It is the absorbance of the negative blank solution.

[0057] 2.6 Cytotoxic Activity Assay

[0058] Cell culture: HepG-2 cells are adherent cells, cultured in DMEM medium with 10% FBS and 1% antibiotics, and cultured at 37°C in a 5% CO2 incubator. Cells are passaged when they cover 90% of the bottom of the culture dish.

[0059] Viability assay: HepG-2 cells in logarithmic growth phase were used, and the cells were inoculated at a rate of 1.5 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates and incubated at 37°C in a 5% CO2 incubator. After cell attachment, 200 μL of the test sample was added to each well, and 200 μL of culture medium was added to the negative control. After 18 h of incubation, 100 μL of culture medium and 10 μL of CCK-8 reagent were added, and the cells were incubated for another 1 h. The absorbance (A) was measured at 450 nm using a microplate reader. Cell growth inhibition rate (%) = (1 - A) 样品组 / A 阴性对照组 )×100%.

[0060] 2.7 HPLC Analysis

[0061] 2.7.1 Chromatographic conditions

[0062] Agilent 5TC-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; the mobile phase was acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), with gradient elution (0–12 min, 2% A; 12–14 min, 2–11% A; 14–40 min, 11–12% A; 40–85 min, 12–24% A; 85–90 min, 24% A), the flow rate was 1.0 mL / min, the detection wavelength was 275 nm, the column temperature was 25 °C, and the injection volume was 10 μL.

[0063] 2.7.2 Preparation of the test solution

[0064] Take different component samples, weigh them accurately, add 60% methanol aqueous solution to prepare samples with a mass concentration of 1.50 mg / mL, filter them through a 0.22 μm microporous membrane to obtain the final product.

[0065] 2.7.3 Preparation of mixed reference solution

[0066] Take appropriate amounts of bergenin, catechin, 4-O-gallo-bergenin, 11-O-gallo-bergenin, and catechin gallate reference standards, accurately weigh them, and add 60% methanol aqueous solution to prepare mixed reference standard solutions with mass concentrations of 150, 100, 50, 100, and 100 μg / mL, respectively.

[0067] 2.8 Liquid chromatography-mass spectrometry (LC-MS) analysis

[0068] 2.8.1 Chromatographic conditions

[0069] Agilent InfinityLab Poroshell 120SB-C 18 The chromatographic column (4.6 mm × 150 mm, 2.7 μm), column temperature 30 °C, flow rate 0.8 mL / min, and mobile phase of methanol (A)-0.1% formic acid aqueous solution (B) gradient elution (0–35 min, 2%–40% A; 35–60 min, 40%–95% A; 60–65 min, 95% A).

[0070] 2.8.2 Mass Spectrometry Conditions

[0071] The ion source was a heated electrospray ionization source (HESI), with positive and negative ion monitoring modes. The spray voltages for positive and negative ion modes were 3.5 kV and 2.8 kV, respectively; the sheath gas flow rate was 70 flow units, the auxiliary gas flow rate was 20 flow units, the capillary temperature was 400℃, the atomization temperature was 500℃, and the S-lens RF level was 55. The scanning mode was Full MS / dd-MS. 2 Full MS resolution 70,000, dd-MS 2 Resolution 17500, scan range m / z 80~1200; collision energy (NCE) 20, 40, 60eV.

[0072] 3. Results

[0073] 3.1 Effects of different methanol concentrations on polyphenol content and antioxidant capacity

[0074] Different concentrations of methanol solutions (0, 20, 40, 60, 80, 100% methanol, v / v) were used to extract *Brassica juncea* from Qinling rock. The contents of total flavonoids, total polyphenols, and total tannins in the sample solutions were determined, as well as their scavenging activities against DPPH and ABTS. The results are shown in Table 1. The table shows that with increasing methanol concentration, the contents of total flavonoids and total polyphenols in the extracted sample solutions gradually increased, and the scavenging activities against DPPH and ABTS also gradually increased, reaching the highest levels with 80% methanol extraction, while the scavenging activities with 100% methanol extraction decreased. The total tannin results showed that the total tannin content was highest with 60% methanol extraction, and then decreased with increasing methanol concentration. However, the total tannin content in the 80% methanol extract was still relatively high. Therefore, considering all factors, 80% methanol was selected as the optimal extraction solvent for *Brassica juncea* samples from Qinling rock.

[0075] Table 1. Effects of methanol extraction at different concentrations on polyphenol content and antioxidant activity.

[0076]

[0077] 3.2 Polyphenol content and antioxidant activity of components with different polarities

[0078] The contents of total flavonoids, total polyphenols, and total tannins in the crude extract of *Brucea qinlingensis* and its different polar components were determined, and the results are shown in Table 2. The results showed that component F2 had the highest contents of total flavonoids, total polyphenols, and total tannins per unit weight of raw medicinal material, followed by F3, while components F1 and F4 had the lowest contents of total flavonoids, total polyphenols, and total tannins. The antioxidant activity of the crude extract of *Brucea qinlingensis* and its different polar components was studied by investigating their DPPH and ABTS scavenging activities, and the results are shown in Table 2. The results showed that the crude extract of *Brucea qinlingensis* had good DPPH and ABTS free radical scavenging activity. Component F2 contributed the most to the DPPH and ABTS free radical scavenging activity of the crude extract, while F3 contributed less than F2. Components F1 and F4 contributed the least to the DPPH and ABTS free radical scavenging activity of the crude extract. Overall, component F2 had the highest content of total flavonoids, total polyphenols, and total tannins, and made the greatest contribution to the in vitro scavenging activity of DPPH and ABTS free radicals of *Brassica rapa* 'Qinling Rock'.

[0079] Table 2. Polyphenol content and antioxidant activity of different polar components

[0080]

[0081]

[0082] 3.4 α-Glucosidase inhibitory activity and cytotoxic activity of different polarity components

[0083] The α-glucosidase inhibitory activity and cytotoxic activity of crude extracts and different polar components of *Brassica juncea* were determined, and the results are shown in Table 3. The results indicate that the α-glucosidase inhibitory activity, in descending order, is as follows:

[0084] The order of cytotoxic activity against HepG-2 liver cancer cells is: F3 > F2 > F4 > BS, with F3 exhibiting the strongest activity.

[0085] Table 3. α-Glucosidase inhibitory activity and cytotoxic activity of different polar components

[0086]

[0087] Note: F1, F2, F3, and F4 are the four components of the extract of *Brassica rapa* 'Qinlingyan'; BS is the crude extract of *Brassica rapa* 'Qinlingyan'; all values ​​in the table represent IC50. 50 The inhibitory activity or toxicity of the corresponding component (μg / mL) against α-glucosidase or HepG-2 is expressed as follows.

[0088] 3.5 HPLC Analysis

[0089] Analysis of crude extracts and F1-F4 of *Brassica rapa* from the Qinling Mountains ( Figure 1 Compounds in F1, F2, and F3 were all well separated with few overlapping peaks, while the peak in F4 was very indistinct and the yield was the lowest. Figure 2 It can be seen that the five components, namely bergenin, catechin, 4-O-gallo-bergenin, 11-O-gallo-bergenin, and catechin gallate, are all concentrated in the F2 fraction.

[0090] 3.6 UHPLC-Q Exactive Focus MS Analysis of Components with Different Polarities

[0091] The above studies show that component F2 in *Brassica rapa* from the Qinling Mountains contributes the most to its in vitro antioxidant activity, exhibits the strongest cytotoxic activity against HepG-2 liver cancer cells, and has the highest content of total flavonoids, total polyphenols, and total tannins. s It exhibits the strongest inhibitory activity against α-glucosidase. Therefore, we selected components F2 and F... s Further analysis of its chemical composition was performed using UHPLC-QExactive Focus MS. Solutions with a mass concentration of 1.00 mg / mL were prepared using 60% methanol, filtered through a 0.22 μm microporous membrane, and analyzed using the "2.8 LC-MS" method. The injection volume for F2 was 1 μL. s The injection volume was 2 μL. Their total ion chromatograms in positive and negative ion modes are shown below. Figure 2Based on the precise relative molecular mass information of the compound obtained from first-order mass spectrometry, and compared with the chemical components previously resolved from *Baijia qinlingensis*, the analysis was performed. [3] Comparative analysis revealed 29 chemical components in the F2 fraction of *Brassica rapa* 'Qinling Rock' (Table 4), including 6 isocoumarins, 8 flavonoids, and 15 phenolic acids and their glycosides; 24 chemical components were extracted from the F3 fraction (Table 5), including 5 isocoumarins, 15 flavonoids, and 4 phenolic acids. The total ion chromatogram showed that the F2 fraction of *Brassica rapa* 'Qinling Rock' was dominated by isocoumarins and phenolic acids. s The main components are flavonoids.

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Claims

1. The use of the antitumor active fraction of *Gynostemma pentaphyllum* or its pharmaceutically acceptable salt in the preparation of antitumor drugs, characterized in that... The preparation method of the antitumor active fraction of Dapanlongqi includes the following steps: Dried rhizomes of *Brassica rapa* from the Qinling Mountains were pulverized and sieved to obtain powder. A certain amount of powder was weighed and added to an 80% methanol solution. After ultrasonic extraction, the powder was centrifuged and the supernatant was collected. The supernatant was concentrated to 1 / 2-1 / 3 of its volume and then subjected to D101 macroporous adsorption resin column chromatography. The powder was eluted sequentially with distilled water, 30% ethanol solution, 50% ethanol solution, and 70% ethanol solution, with each eluent eluting for 2 column volumes. The fractions obtained with different eluents were collected, concentrated under reduced pressure, and dried to obtain fractions F1, F2, F3, and F4. Among them, fraction F2 is the antitumor active site.

2. The application according to claim 1, characterized in that... The HPLC chromatographic conditions for the antitumor active fraction of *Gynostemma pentaphyllum* are as follows. Chromatographic conditions: Agilent 5 TC-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; phase A: acetonitrile, phase B: 0.1% phosphoric acid aqueous solution; gradient elution: 0–12 min, 2% A; 12–14 min, 2–11% A; 14–40 min, 11–12% A; 40–85 min, 12–24% A; 85–90 min, 24% A; flow rate: 1.0 mL / min; detection wavelength: 275 nm; column temperature: 25℃; injection volume: 10 μL.

3. The application according to any one of claims 1-2, characterized in that... The total ion chromatography and mass spectrometry conditions for the antitumor active components of *Dracaena cochinchinensis* under positive and negative ion modes are as follows. Chromatographic conditions: Agilent InfinityLab Poroshell 120 SB-C 18 Chromatographic column, 4.6 mm × 150 mm, 2.7 μm, column temperature 30℃, flow rate 0.8 mL / min, mobile phase A: methanol, mobile phase B: 0.1% formic acid aqueous solution, gradient elution: 0~35 min, 2%~40% A; 35~60 min, 40%~95% A; 60~65 min, 95% A; Mass spectrometry conditions: Ion source was a heated electrospray ionization source, with positive and negative ion monitoring modes. The spray voltages for positive and negative ion modes were 3.5 kV and 2.8 kV, respectively. The sheath gas flow rate was 70 flow units, the auxiliary gas flow rate was 20 flow units, the capillary temperature was 400℃, the nebulization temperature was 500℃, the S-lens RF level was 55, and the scanning mode was Full MS / dd-MS. 2 Full MS resolution 70,000, dd-MS 2 Resolution 17500, scan range m / z 80~1200; collision energy NCE 20, 40, 60 eV.

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