Method for separating compound from persistent calyx of winter cherry

Abstract

The invention discloses a method for separating compounds from persistent calyx of physalis alkekengi. Belongs to the field of physalis alkekengi persistent According to the invention, physalin compounds 1, 2 and 3 separated from petroleum ether and ethyl acetate parts of persistent calyx of physalis alkekengi are screened by adopting an MTT (Methyl Thiazolyl Tetrazolium) method. The physalin compound 1 and the physalin compound 2 provided by the invention have the growth inhibition rates of 19.61% and 15.20% on tumor cells MDA-MB-231 respectively, and have a relatively weak growth inhibition rate on the tumor cells; the growth inhibition rate of the compound 3 on tumor cells SW480 is 13.18%, and the compound 3 has weak growth inhibition on the cells.

Description

Technical Field

[0001] This invention belongs to the field of separating compounds from the calyx of Physalis alkekengi. Specifically, it relates to a method for separating compounds from the calyx of Physalis alkekengi. Background Technology

[0002] Physalis, a perennial herbaceous plant belonging to the Solanaceae family. To date, 216 compounds have been isolated from Physalis, including steroids, flavonoids, terpenes, phenylpropanoids, alkaloids, and other compounds. Steroids and flavonoids are the main chemical components of the Physalis calyx. When treating cancer with drugs, chemotherapy, targeted therapy, radiation, and surgery, patients experience significant suffering or side effects. Compared to chemotherapy drugs, the active ingredients of traditional Chinese medicine (TCM) have unique anti-tumor advantages. Active ingredients of TCM refer to monomeric compounds with significant pharmacological activity and certain medicinal value in clinical practice. Summary of the Invention

[0003] This invention involves the separation and purification of the petroleum ether and ethyl acetate fractions of Physalis alkekengi. The calyx of Physalis alkekengi was extracted with 95% ethanol. The crude extract was then fractionally extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the respective fractions. The chemical components of each fraction were separated and purified using normal-phase silica gel column chromatography, Seohadex LH-20, reverse-phase silica gel column chromatography, and semi-preparative high-performance liquid chromatography. The structures of the isolated monomeric compounds were identified using modern spectroscopic techniques such as IR, UV, NMR, MS, and ECD, combined with literature data comparison. The MTT assay was used to screen the physalin compounds isolated from the petroleum ether and ethyl acetate fractions of Physalis alkekengi to study their in vitro proliferation inhibitory activity against human promyelocytic leukemia (HL-60), human non-small cell lung cancer (A549), human liver cancer (SMMC-7721), human breast cancer (MDA-MB-231), and human colon cancer (SW480) cell lines.

[0004] The present invention aims to provide a method for separating compounds from the calyx of Phyllanthus urinaria, comprising the following steps:

[0005] Step 1: Place the calyx in a dark and ventilated place to dry naturally. After it is completely dry, crush it into a fine powder.

[0006] Step 2: After soaking in 95% ethanol, heat and extract three times at 60 ℃. Combine the extracts and concentrate them at 35 ℃ and 0.3 MPa until they just flow out of the concentration tank. Then further concentrate under reduced pressure to obtain crude extract.

[0007] Step 3: Thoroughly mix the extract with warm water at a 1:1 mass ratio to obtain a suspension. Add petroleum ether, dichloromethane, ethyl acetate, and n-butanol sequentially to the suspension for fractional extraction (V...水 :V 有机相 =1:1), let stand to allow separation, extract and concentrate, and slowly add dichloromethane (CH2Cl2) (V) to the suspension after removing PE. 水相 :V 有机相 =1:1), after sealing, shake well, let stand to separate layers, extract the CH2Cl2 layer for concentration, repeat the extraction multiple times until the CH2Cl2 layer becomes lighter in color and transparent, to obtain petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract and n-butanol fraction extract.

[0008] Step 4: Separate the ethyl acetate fraction of *Phyllostachys edulis*. Use a 100-200 mesh silica gel column for chromatography, mixing the sample with silica gel, and perform separation on a normal-phase, atmospheric-pressure column. Elute with a mobile phase of dichloromethane:methanol at a ratio of 50:1 → 30:1 → 20:1 → 15:1 → 10:1 → 5:1. Spot the obtained fractions onto a thin-layer silica gel plate and observe under 254 nm UV light. Combine the colorimetric results with 10% sulfuric acid ethanol. Combine identical fractions to obtain five fractions (Fr-1 to Fr-5).

[0009] Step 5: Fraction Fr-4 was further separated using a 200-300 mesh normal-phase silica gel column with dichloromethane:methanol as the mobile phase at a ratio of 15:1 → 10:1 → 5:1 → 3:1 → 2:1, yielding four fractions (Fr-4-1 ~ Fr-4-4). Fr-4-4 was then separated by normal-phase silica gel column chromatography, further purified by LH-20 gel column chromatography (MeOH), and then further purified by semi-preparative HPLC under the mobile phase conditions of (MeOH-H2O, 65:35, 2 ml / min, v / v) to obtain compound 1. The t-value of compound 1... R = 25.0 min;

[0010] Step 6: Fraction Fr-5 was further separated using a 200-300 mesh normal-phase silica gel column with dichloromethane-methanol as the mobile phase, eluting at a ratio of 10:1 → 5:1 → 3:1 → 2:1 → 1:1 to obtain three fractions (Fr-5-1 ~ Fr-5-3). Fr-5-3 was further purified by normal-phase silica gel column separation, followed by LH-20 gel column (MeOH) purification, and then further purified by semi-preparative HPLC with a mobile phase of MeOH-H2O (50:50, 2 ml / min, v / v) to obtain compound 2. The t of compound 2... R = 30.0 min;

[0011] Step 7: Separate the petroleum ether fraction of the calyx of *Phyllostachys edulis*. Take 373.46 g of the petroleum ether fraction extract and separate it by silica gel column chromatography using a 100-200 mesh screen. Elute with petroleum ether-ethyl acetate as the mobile phase at a ratio of 8:1 → 5:1 → 3:1 → 2:1 → 1:1. Combine the eluents to obtain 10 fractions (Fr-1 to Fr-10).

[0012] Fr-10 was further separated by silica gel column chromatography (200-300 mesh), using dichloromethane-methanol as the mobile phase with a ratio of 80:1→50:1→30:1→20:1→15:1→10:1. The fractions were then combined to obtain five fractions (Fr-10-1~Fr-10-5).

[0013] Fr-10-4 was subjected to LH-20 dextran gel column chromatography and eluted with methanol-dichloromethane (1:1) to obtain three components (Fr-10-4-1~Fr-10-4-3);

[0014] Fr-10-4-3 was further separated by 200-300 mesh silica gel column chromatography with gradient elution of (100:1, 80:1, 50:1) to obtain two fractions (Fr-10-4-3-1 and Fr-10-4-3-2); Fr-10-4-3-2 was separated by high performance liquid chromatography (MeCN-H2O, 34:66, v / v, 2 ml / min) to obtain compounds 9 and 10 and Fr-10-4-3-2-1; Fr-10-4-3-2-1 was purified by semi-preparative high performance liquid chromatography (MeCN-H2O, 32:68, v / v, 2 ml / min) to obtain compound 3 (t R = 20.0min).

[0015] The structural formula of compound 1 is

[0016] .

[0017] The structural formula of compound 2 is

[0018] .

[0019] The structural formula of compound 3 is

[0020] .

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Twenty compounds were isolated from the petroleum ether and ethyl acetate fractions of Physalis alkekengi. Among them were three new Physalis glycosides and seventeen known compounds. The three new Physalis glycosides were 3α-hydroxy-2,3,25,27-tetrahydro-4,7-didehydro-7-deoxyneophysalin A (1), Physalin VIII (2) and Physalin IX (3), and the seven known Physalis glycosides were Physalis glycoside L (4), Physalis glycoside D (5), alkekengilin A (6), isophysalis glycoside A (7), Physalis glycoside V (8), Physalis glycoside A (9) and Physalis glycoside N (10). Three steroidal compounds: Withaphysalin C (11), Withaminilide B (12), and stigmasterol (13); one flavonoid compound: Rhamnazin (14); five sesquiterpenoid compounds: (+)-Anhydro-β-rotunol (15), Cyperusol C (16), (3S, 5R, 8R)-3,5-dihydroxymegastigma-6,7-dien-9-one (17), Capsidiol (18), and 4,5-dihydroxybruhmol (19); and one fatty acid compound: (9Z,11E)-13-oxo-9,11-ocatadecadienoic acid (20). Compounds 11, 12, 16, and 18 are known to have been isolated from physalis for the first time. Among them, compounds 1 and 2 of physalin showed growth inhibition rates of 19.61% and 15.20% against MDA-MB-231 tumor cells, respectively, indicating a weak growth inhibition rate against this tumor cell type; compound 3 showed a growth inhibition rate of 13.18% against SW480 tumor cells, indicating a weak growth inhibition rate against this cell type; compounds 4 and 6 showed growth inhibition rates of 30.17% and 29.71% against SMMC-7221 tumor cells, respectively, indicating a weak growth inhibition rate; compound 5 showed a growth inhibition rate of more than 50% against the five types of tumor cells, and this compound was used for gradient rescreening at five concentrations.

[0023] The chemical components of the petroleum ether and ethyl acetate fractions of Physalis alkekengi were separated, purified, and identified using traditional separation techniques. Twenty compounds were isolated, including three new Physalis rubigin compounds, enriching the variety of Physalis rubigin compounds. The Physalis rubigin compounds were used for preliminary screening of in vitro antitumor activity, providing a strong basis for further in-depth research on the bioactivity of chemical components.

[0024] For a deeper understanding of the features and technical content of this invention, please refer to the accompanying detailed description and drawings. It should be noted that the drawings are provided for illustrative purposes only and are not intended to limit the scope of the invention. Attached Figure Description

[0025] Figure 1 This is a flowchart of the ethyl acetate fraction separation process;

[0026] Figure 2 This is a flow chart of the petroleum ether separation process;

[0027] Figure 3 It is compound 1 1 H- 1 Related diagrams for H COSY, HMBC, and NOESY;

[0028] Figure 4 It is compound 2. 1 H- 1 Related diagrams for H COSY, HMBC, and NOESY;

[0029] Figure 5 It is compound 3. 1 H- 1 Related diagrams for H COSY, HMBC, and NOESY. Detailed Implementation

[0030] The present invention will be described in detail below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but should not be considered as limiting the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0031] Preparation of crude extract of Physalis alkekengi calyx

[0032] Crushing of Physalis alkekengi calyx

[0033] Separate the fruit of the lycine from its calyx and place it in a dark and ventilated place to dry naturally. After it is completely dry, weigh it (16.0 kg) and grind it into fine powder using a grinder.

[0034] Preparation of Physalis alkekengi extract

[0035] The airtightness of each reaction vessel in the pilot plant was tested to ensure good airtightness. The pulverized sample was poured into an extraction vessel, soaked in 95% ethanol (solid-liquid ratio 1:10), and then heated (60 ℃) for extraction three times every 8 hours. The extracts were combined. The extract was concentrated (35 ℃, 0.3 MPa) until it could just flow out of the concentration vessel, and then further concentrated under reduced pressure to obtain a crude extract.

[0036] Extract preparation

[0037] To prevent leakage, check the airtightness of the extraction flask. Place the extract in the extraction flask and mix it thoroughly with warm water (extract:water = 1:1) to obtain a suspension. Then, add petroleum ether (PE), dichloromethane (CHCl2), ethyl acetate (EA), and n-butanol (BuOH) sequentially to the suspension for fractional extraction (V). 水 :V 有机相 =1:1), let stand to allow separation, then concentrate. Combine the obtained extracts and weigh. Slowly add dichloromethane (CH2Cl2) (V) to the suspension from which PE has been removed. 水相 :V 有机相 (The ratio of extracts to water is 1:1). After capping the bottle, shake well, allow to stand for separation, and extract the CH2Cl2 layer for concentration. Repeat the extraction multiple times until the CH2Cl2 layer becomes lighter in color and transparent. Combine the extracts and weigh them. 373.46 g of petroleum ether extract, 164.32 g of dichloromethane extract, 88.87 g of ethyl acetate extract, and 578.41 g of n-butanol extract were obtained.

[0038] Isolation and purification of chemical components of Physalis alkekengi calyx

[0039] This experiment mainly focuses on the extraction and separation of chemical components from the calyx of Physalis alkekengi. Based on the completion of the separation of the dichloromethane fraction from the calyx of Physalis alkekengi, the ethyl acetate fraction and the petroleum ether fraction are separated to search for the target compound Physalis picric acid.

[0040] The ethyl acetate fraction of *Phyllostachys edulis* was separated using a normal-phase, atmospheric-pressure column chromatography with 100-200 mesh silica gel and diluent. Gradient elution was performed using dichloromethane-methanol (50:1, 30:1, 20:1, 15:1, 10:1, 5:1, v / v). The resulting fractions were spotted onto a thin-layer silica gel plate and observed under 254 nm UV light. The colorimetric results, combined with 10% sulfuric acid-ethanol analysis, led to the merging of identical fractions, yielding five fractions (Fr-1 to Fr-5). Fraction Fr-4 was further separated using a 200-300 mesh normal-phase silica gel column with gradient elution using dichloromethane-methanol (15:1, 10:1, 5:1, 3:1, 2:1, v / v), yielding four fractions (Fr-4-1 to Fr-4-4). Fr-4-4 was separated by normal-phase silica gel column chromatography, and further purified by LH-20 gel column chromatography (MeOH) to obtain fraction Fr-4-4-2. Fr-4-4-2 was further purified by semi-preparative HPLC under the mobile phase conditions of (MeOH-H2O, 65:35, 2 ml / min, v / v) to obtain compound 1 (1.8 mg, t). R= 25.0 min). Fraction Fr-5 was further separated using a 200–300 mesh normal-phase silica gel column, with gradient elution using dichloromethane-methanol (10:1, 5:1, 3:1, 2:1, 1:1, v / v) to obtain three fractions (Fr-5-1 to Fr-5-3). Fr-5-3 was further purified by normal-phase silica gel column separation and LH-20 gel column (MeOH) to obtain fraction Fr-5-3-2. Fr-5-3-2 was further purified by semi-preparative HPLC with a mobile phase of MeOH-H2O (50:50, 2 ml / min, v / v) to obtain compound 2 (1.9 mg, t). R = 30.0 min).

[0041] The petroleum ether fraction of the calyx of *Phyllostachys edulis* was separated. 373.46 g of the petroleum ether fraction extract was taken and separated by silica gel column chromatography with a 100-200 mesh. The elution was carried out with a gradient of petroleum ether-ethyl acetate (8:1, 5:1, 3:1, 2:1, 1:1, v / v). The fractions were combined to obtain 10 fractions (Fr-1 to Fr-10).

[0042] Fr-2 (11.2 g) was purified by normal-phase silica gel column chromatography with petroleum ether-ethyl acetate mobile phase (50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, v / v) to produce six fractions (Fr-2-1 to Fr-2-6). Fr-2-4 (300.8 mg) was purified by normal-phase silica gel column chromatography to obtain Fr-2-4-1. Fr-2-4-1 was purified by normal-phase silica gel column chromatography and LH-20 gel column chromatography to obtain Fr-2-4-1-1. Fr-2-4-1-1 (31.1 mg) was further purified by semi-preparative high-performance liquid chromatography (MeCN-H2O, 65:35, v / v, 2 ml / min) to obtain compound 20 (2.3 mg, t...). R = 32.5 min).

[0043] Fr-9 was separated by silica gel column chromatography (200-300 mesh) with a gradient elution of dichloromethane-methanol (100:1, 80:1, 50:1, 30:1, 20:1), and the fractions were combined to obtain two fractions (Fr-9-1 to Fr-9-2). Fr-9-2 was further purified by LH-20 gel chromatography with elution of dichloromethane-methanol (1:1) to obtain fraction Fr-9-2-1. Fr-9-2-1 was separated by semi-preparative high-performance liquid chromatography (MeOH:H2O, 52:48, v / v, 2 ml / min) to obtain compound 14 (4.8 mg, t). R= 28.0 min). Fr-9-1 was further separated by reversed-phase silica gel column chromatography (ODS) with a methanol-water gradient (45%, 60%, 70%, 80%, 90%), and the fractions were combined to obtain four fractions (Fr-9-1-1 to Fr-9-1-4). Fr-9-1-2 was further purified by LH-20 gel chromatography with dichloromethane-methanol (1:1) to obtain fraction Fr-9-1-2-1. This fraction was further separated and purified by 200-300 mesh silica gel column chromatography with a dichloromethane-methanol gradient (100:1, 80:1) to obtain two fractions Fr-9-1-2-1-1 and Fr-9-1-2-1-2. Fr-9-1-2-1-2 was separated by high performance liquid chromatography (MeCN:H2O, 35:65, v / v, 2 ml / min) to obtain compound 4 (16.8 mg). Fr-9-1-3 (500.6 mg) was further purified by LH-20 gel column and normal-phase silica gel column chromatography to obtain Fr-9-1-3-1 and Fr-9-1-3-2; compound 16 (4.1 mg, t) was isolated from Fr-9-1-3-1 (18.1 mg) by high-performance liquid chromatography (MeCN-H2O, 32: 68, v / v, 2 ml / min). R = 45.0 min) and 17 (3.1 mg, t R = 46.2 min). Compound 18 (18.1 mg, t) was isolated from Fr-9-1-3-2 by high performance liquid chromatography (MeCN-H2O, 32: 68, v / v, 2 ml / min). R = 66.5 min) and compound 19 (3.3 mg, t R = 70.1 min).

[0044] Fr-10 was further separated by silica gel column chromatography with a 200-300 mesh, eluted with a dichloromethane-methanol gradient (80:1, 50:1, 30:1, 20:1, 15:1, 10:1), and the fractions were combined to obtain 5 fractions (Fr-10-1 to Fr-10-5); Fr-10-2 was separated by LH-20 gel column chromatography, eluted with methanol-dichloromethane (1:1), to obtain 2 fractions (Fr-10-2-1 and Fr-10-2-2). Fr-10-2-1 was further separated by silica gel column chromatography (200-300 mesh) using a gradient elution of dichloromethane-methanol (100:1, 80:1, 50:1), and the fractions were combined to obtain two fractions (Fr-10-2-1-1 and Fr-10-2-1-2). Fr-10-2-1-1 was further purified by silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate (3:1, 2:1, 1:1, 1:2) to obtain two fractions (Fr-10-2-1-1-1 and Fr-10-2-1-1-2). Fr-10-2-1-1-2 was separated by high performance liquid chromatography (MeOH:H2O, 0.2% acid, 58:42, v / v, 2 ml / min) to obtain compound 11 (24.5). Compound 12 (4.0 mg) was purified by high performance liquid chromatography (MeCN-H2O, 45:55, v / v, 2 ml / min) to obtain compound 15 (2.4 mg) and Fr-10-2-1-2-1. Fr-10-2-1-1-2-1 was purified by high performance liquid chromatography (MeCN-H2O, 52:48, v / v, 2 ml / min) to obtain compound 7 and 8 (4.1 mg). Fr-10-2-2 was further separated by silica gel column chromatography (200-300 mesh) using a gradient elution with petroleum ether-acetone (15:1, 10:1, 8:1, 5:1, 3:1, 2:1) to obtain two fractions (Fr-10-2-2-1 and Fr-10-2-2-2). Fr-10-2-2-1 was purified by high performance liquid chromatography (MeOH-H2O, 52:48, v / v, 2 ml / min) to obtain compound 6 (3.3 mg); Fr-10-2-2-2 was purified by high performance liquid chromatography (MeOH-H2O, 52:48, v / v, 2 ml / min) to obtain compound 5 (11.1 mg). Fr-10-4 was subjected to LH-20 dextran gel column chromatography and eluted with methanol-dichloromethane (1:1) to obtain three components (Fr-10-4-1 to Fr-10-4-3).Fr-10-4-1 was crystallized to give compound 13 (8.6 mg); Fr-10-4-3 was further separated by silica gel column chromatography of 200-300 mesh with gradient elution of (100:1, 80:1, 50:1) to give two fractions (Fr-10-4-3-1 and Fr-10-4-3-2); Fr-10-4-3-2 was separated by high performance liquid chromatography (MeCN-H2O, 34:66, v / v, 2 ml / min) to give compounds 9 and 10 (3.5 mg) and Fr-10-4-3-2-1; Fr-10-4-3-2-1 was purified by semi-preparative high performance liquid chromatography (MeCN-H2O, 32:68, v / v, 2 ml / min) to give compound 3 (1.8 mg, t). R = 20.0min).

[0045] Physalin compounds were extracted and separated from the petroleum ether fraction of Physalis alkekengilin. A total of 20 compounds were isolated and identified from the petroleum ether and ethyl acetate fractions of Physalis alkekengilin. Among them, 3 new physalin compounds and 17 known compounds were identified. The 3 new physalin compounds are: 3α-hydroxy-2,3,25,27-tetrahydro-4,7-didehydro-7-deoxyneophysalin A (1), Physalin Ⅷ (2) and Physalin IX (3). The 7 known physalin compounds are: Physalin L (4), Physalin D (5), alkekengilin A (6), isophysalin A (7), Physalin V (8), Physalin A (9) and Physalin N (10). Three steroidal compounds: Withaphysalin C (11), Withaminilide B (12), and stigmasterol (13); one flavonoid compound: Rhamnazin (14); five sesquiterpenoid compounds: (+)-Anhydro-β-rotunol (15), Cyberusol C (16), (3S,5R,8R)-3,5-dihydroxymegastigma-6,7-dien-9-one (17), Capsidiol (18), and 4,5-dihydroxybruhmol (19); and one fatty acid compound: (9Z,11E)-13-oxo-9,11-ocatadecadienoic acid (20). Among the known compounds, 18, 16, 11, and 12 were isolated from Physalis for the first time, further enriching the variety of Physalis bitter compounds. The stability of the type A physalin compound, physalin L, was investigated. Experiments showed that physalin L is unstable at a constant temperature of 80℃. This provides a reference for subsequent separation of physalin compounds. Two pairs of tautomers were found in the separated compounds during the separation process.The six physalin compounds were tested: 3α-hydroxy-2,3,25,27-tetrahydro-4,7-didehydro-7-deoxyneophysalin A (1), Physalin Ⅷ (2) and Physalin IX (3), Physalin L (4), Physalin D (5), and alkekengilin. A(6) conducted in vitro antitumor cell line activity studies. The results showed that, compared with positive control drugs, compounds 1 and 2 of physalin inhibited the growth of MDA-MB-231 tumor cells by 19.61% and 15.20%, respectively, exhibiting weak growth inhibition rates; compound 3 inhibited the growth of SW480 tumor cells by 13.18%, also showing weak growth inhibition; compounds 4 and 6 inhibited the growth of SMMC-7221 tumor cells by 30.17% and 29.71%, respectively, also showing weak growth inhibition rates; compound 5 inhibited the growth of all five tumor cell types by more than 50%; physalin compound 5 demonstrated a significant antitumor cell activity. This provides strong evidence for further in-depth research on the bioactivity of chemical components.

[0046] Compound 1

[0047]

[0048] 3α-hydroxy-2,3,25,27-tetrahydro-4,7-didehydro-7-deoxyneophysalin A(1) is a colorless needle-like crystal, readily soluble in methanol and chloroform. It appears dark black under 254 nm UV-Vis light and pale lemon yellow under 10% sulfuric acid in ethanol. High-resolution mass spectrometry (HR-ESIMS) shows a quasi-molecular ion peak of compound 1 at m / z 551.1891 [M + Na]. + (Calculated value 551.1893), the molecular formula of compound 1 is inferred to be C. 28 H 32 O 10 Its unsaturation degree was calculated to be 13. The IR spectrum at 3357 cm⁻¹ -1 1778cm -1 The absorption peaks at 1651 and 1651 indicate the presence of hydroxyl and carbonyl groups in compound 1. 1The 1H NMR spectrum shows that the compound has four methyl groups: 1.18 (3H, s, H-19), 1.80 (3H, s, H-21), 1.32 (3H, d, J = 7.5 Hz, H-27) and 1.37 (3H, s, H-28); it can also be inferred that the compound contains two olefinic double bonds: 6.19 (1H, dd, J = 10.41, 4.38 Hz, H-7), 6.45 (1H, d, J = 10.41 Hz, H-6) and 5.70 (1H, s, H-4), and proton signals on the two oxygen-bonded carbons: 4.52 (1H, dd, J = 1.61 Hz, H-6) and 4.47 (1H, t, J = 6.12 Hz, H-7); through 13 C NMR (CDCl 3, The 150MHz spectrum showed 28 carbon signals, including one ketone carbonyl carbon signal (δ). C 213.0 (C-1); 3 ester carbonyl signals: δ C 177.6 (C-15), 176.9 (C-26) and 171.8 (C-18), two groups of olefin double bond carbon signals: δ C 141.9 (C-5), 127.5 (C-4), 131.0 (C-6) and 129.6 (C-7); carbon signals δ of the three hydroxyl groups. C 68.5 (C-3), 80.5 (C-13), and 82.4 (C-14); δ signals of the four methyl carbons. C Based on the carbon-hydrogen correlations in the DEPT and HSQC spectra, the following segments were initially identified as steroidal: -CH2(2)-CH(3)-CH(4)-, CH(6)-CH(7)-CH(8)-CH(9)-CH2(11)-CH2(12)-, and -CH(22)-CH2(23)-. This preliminarily confirmed the aliphatic hydrocarbon structural segments of the compound. 13 C NMR data yielded four oxygen-related quaternary carbon signals: δ C 82.4 (C-14), 80.5 (C-13), and 82.5 (C-17); Based on this, it is inferred that the compound is a physalin-type compound, with a structural change. It is speculated that C-14 and C-16 are directly linked, while C-15 and C-17 are linked through an ester bond, suggesting that this compound is a new type A physalin. To verify this conclusion, it was demonstrated using HMBC, δ... H2.77, 2.69 (H-2) and δ C 68.5 (C-3), δ C 127.5 (C-4), δ C 51.5 (C-10) correlation, δ H 4.47 (H-3) and δ C 141.9 (C-5), δ C 127.5 (C-4) and δ C 131.0 (C-6) related, δ H 5.70 (H-4) and δ C 47.5 (C-2), δ C 131.0 (C-6) and δ C 50.1 (C-10) correlation, δ H 6.45 (H-6) and δ C 127.5 (C-4), δ C 141.9 (C-5), δ C 50.1 (C-8) and δ C 51.5 (C-10) correlation, δ H 6.19 (H-7) and δ C 141.9 (C-5), δ C 50.1 (C-8) and δ C Based on 51.5 (C-10), the positions of the olefinic double bonds and hydroxyl substitutions on rings A and B of this compound can be deduced; δ H 1.97, δ H 1.49 (H-11) and δ C 50.1 (C-8), δ C 38.5 (C-9), δ C 51.5 (C-10), δ C 29.2 (C-12), δ C 80.5 (C-13) and δ C 82.4 (C-14) related, δ H 2.46, δ H 2.12 (H-12) and δ C 38.5 (C-9), δ C 58.2 (C-16), δ C 80.5 (C-13), δ C 82.5 (C-17) and δ C 83.1 (C-20) related, δH 2.96 (H-16) and δ C 29.2 (C-12), 80.5 (C-13), δ C 82.4 (C-14), δ C 177.6 (C-15), δ C 82.5 (C-17), δ C Related to 35.7 (C-24), and 41.0 (C-28), δ H 1.37 (H-19) and δ C 141.9 (C-5), δ C 38.5 (C-9), δ C Related to 51.5 (C-10), δ H 1.88 (H-21) and δ C 83.1 (C-20) and δ C Related to 78.7 (C-22), δ H 4.52 (H-22) and δ C 82.5 (C-17), δ C 30.6 (C-23), δ C 35.7 (C-24) and δ C Related to 176.9 (C-26), δ H 2.13, 1.66 (H-23) and δ C 58.2 (C-16), δ C 20.5 (C-21), δ C 78.7 (C-22), δ C [[ID=4)5]] 35.7 (C-24) and δ C Related to 41.0 (C-25), δ H 3.63 (H-25) and δ C J 58.2 (C-16), δ[[ID=5))2]] C 30.6 (C-23), δ C 41.0 (C-25), δ C 35.7 (C-24) and δ C Related to 17.4 (C-25), δ H 1.32 (H-27) and δ C 35.7 (C-24), δ C Related to 41.0 (C-25) and δ C Related to 176.9 (C-26), δ H1.37 (H-28) and δ C 58.2 (C-16), δ C 30.6 (C-23), δ C 41.0 (C-25), δ C 35.7 (C-24) and δ C 17.4 (C-25) correlation confirms that C-8 and C-14 are linked, C-14 and C-16 are directly linked by carbon-carbon bonds, and C-17 and C-15 are linked by ester bonds, thus identifying this compound as a type A neophytic acid physalin. NOESY and HMBC spectra, δ H 6.19 (H-7) and δ H 2.90 (H-8) correlation, δ H 5.70 (H-4) and δ H 4.47 (H-3) correlation, δ H 4.47 (H-3) and δ H 2.69 (H-2), and δ H The correlation with 1.18 (H-19) suggests that the 3-OH configuration is 3-OH(α). Based on the above data, the structure of compound 1 is further confirmed as a novel physalin compound. Comparing the 1H NMR spectra of compound 1 with those of Physalin II, the main difference is that compound 1 lacks a methoxy group signal, and its δ 68.5 (C-3) is 6 ppm lower than that of Physalin II (δ 74.5 (C-3). It is speculated that the substitution at C-3 in compound 1 is an oxidation of methoxy to hydroxyl substitution, which is verified by high-resolution mass spectrometry. In summary, the absolute configuration of compound 1 can be obtained. Important related signals include... Figure 3 As shown.

[0049] Compound 2

[0050]

[0051] Physalin Ⅷ (2) is a colorless needle-like crystal, readily soluble in methanol and chloroform. It appears dark black under 254 nm UV-Vis light and pale lemon yellow under 10% sulfuric acid in ethanol. High-resolution mass spectrometry (HR-ESIMS) shows that the quasi-molecular ion peak of compound 2 is 581.1613 [M + Na]. + (Calculated value 581.1629), the molecular formula of compound 2 is inferred to be C. 28 H 30 O 12 Its unsaturation degree was calculated to be 14. The IR spectrum at 3356 cm⁻¹ -1 1654cm -1and 1033cm -1 The presence of an absorption peak indicates the presence of hydroxyl and carbonyl groups in compound 2. 1 The 1H NMR spectrum shows that this compound has three methyl groups: δ H 1.37 (3H, s, H-19), 1.88 (3H, s, H-21), and 1.24 (3H, s, H-28); it can also be inferred that this compound contains two olefinic double bonds: δ H 6.19 (1H, d, J = 5.9 Hz, H⁻⁴), 7.01 (1H, dd, J = 9.7, 5.9 Hz, H⁻³), and 5.95 (1H, d, J = 9.7 Hz, H⁻²), three proton signals of the hydrogen bond: δ H 4.21 (1H, d, J = 3.6 Hz, H-6), 4.43 (1H, dd, J = 3.6, 1.7 Hz, H-7) and 4.58 (1H, d, J = 3.6, 2.0 Hz, H-22); via 13 C NMR (CDCl 3, 150MHz) and DEPT spectra revealed 28 carbon signals, including two ketone carbonyl carbon signals: δ C 209.6 (C-1) and 209.5 (C-15); two groups of olefin double bond carbon signals: δ C 156.2 (C-5), 141.7 (C-3), 126.7 (C-2) and 122.8 (C-4); carbon signals of the four hydroxyl groups: δ C 77.3 (C-6), 70.4 (C-7), 80.5 (C-13), and 75.4 (C-25); 3 methyl carbon signals: δ C Based on the carbon-hydrogen correlations in DEPT and HSQC spectra, the following links were initially identified as steroidal structures: -CH2(2)-CH(3)-CH(4)-, CH(6)-CH(7)-CH(8)-CH(9)-CH2(11)-CH2(12)-, and -CH(22)-CH2(23)-. These links preliminarily confirmed the aliphatic hydrocarbon structural segments of the compound. 13 C NMR data yielded three oxygen-related quaternary carbon signals: δ C 82.6 (C-17), 80.5 (C-13), and 79.4 (C-22) and double oxygen-carbon signals: δ C108.3 (C-14); this suggests that the compound is a physalin-type compound, the target compound. In the HMBC spectrum, δ... H 5.95 (H-2) and δ C 156.2 (C-5), δ C 122.8 (C-4),δ C 54.9 (C-10) correlation, δ H 7.01 (H-3) and δ C 156.2 (C-5), δ C 122.8 (C-4) and δ C 209.6 (C-1) related, δ H 6.19 (H-4) and δ C 126.7 (C-2), δ C 141.7 (C-3), δ C 54.9 (C-10), δ C 20.1(C-19) related, δ H 4.21 (H-6) and δ C 209.6 (C-1), δ C 156.2 (C-5), δ C 122.8 (C-4), δ C 70.4 (C-7), δ C 44.3 (C-8) and δ C 54.9 (C-10) correlation, δ H 4.43 (H-7) and δ C 156.2 (C-5), δ C 77.3 (C-6), δ C 44.3 (C-8), δ C 33.7 (C-9), δ C Based on 108.3 (C-14), infer the positions of the olefinic double bonds and hydroxyl substitutions on rings A and B of this compound; δ H 2.74, δ H 1.16 (H-11) and δ C 44.3 (C-8),δ C 33.7 (C-9), δ C 54.9 (C-10), δ C 26.5 (C-12) and δ C 80.5 (C-13) related, δ H 3.26 (H-9) and δC 209.6 (C-1), δ C 33.7 (C-9), δ C 54.9 (C-10), δ C 22.6 (C-11), δ C 26.5 (C-12), δ C 108.3 (C-14), δ C 20.1 (related to C-19), δ H 1.49, δ H 2.12 (H-12) related to δ C 33.7 (C-9), δ C 22.6 (C-11), δ C 80.5 (C-13), δ C 82.6 (C-17), δ C 173.5 (related to C-18 and 20.1 (C-19)), δ H 2.70 (H-16) related to δ C 80.5 (C-13), δ C 108.3 (C-14), δ C 209.5 (C-15), δ C 82.6 (C-17), δ C 29.8 (C-23), δ C 37.3 (C-24), δ C 75.4 (related to C-25 and 20.1 (C-28)), δ H 1.39 (H-19) related to δ C 156.2 (C-5), δ C 209.6 (C-1),δ C 54.9 (C-10), δ C related to 33.7 (C-9), δ H 1.88 (H-21) related to δ C 82.6 (C-17), δ C 81.3 (C-20) and δ C related to 79.4 (C-22), δ H 4.58 (H-22) related to δ C 82.6 (C-17), δ C 81.3 (C-20), δ C 22.7 (C-21), δC 29.8 (C-23), δ C 37.3 (C-24) and δ C 172.1 (C-26) related, δ H 2.50, 1.76 (H-23) and δ C 55.8 (C-16), δ C 81.3 (C-20), δ C 79.4 (C-22), δ C 29.8 (C-23), δ C 37.3 (C-24), δ C 75.4 (C-25) and δ C 20.1(C-28) related, δ H 4.13, 3.60 (H-27) and δ C 108.3 (C-14), δ C 37.3 (C-24), δ C 75.4 (C-25) and δ C 172.1 (C-26) related, δ H 1.24 (H-28) and δ C 55.8 (C-16), δ C 82.6 (C-17), δ C 79.4 (C-22), δ C 29.8 (C-23), δ C 37.3 (C-24) and δ C 75.4 (C-25) correlation proves that C-8 and C-14 are linked, C-18 and C-14 are linked by an oxygen bridge bond, and C-14 and C-27 are linked by an ether bond. This can be confirmed by NOESY and HMBC spectra, δ H 4.42 (H-7) and δ H 2.66 (H-8) correlation, δ H 6.18 (H-4) and δ H 4.22 (H-6) correlation, δ H 2.66(H-8) and δ H 1.14 (H-11), δ H 1.36 (H-9) and δ HBased on the correlation of 1.82 (H-23), the configurations of 7-OH and 6-OH can be deduced to be 7-OH(β) and 6-OH(α). Using the above data, the structure of compound 2 is further determined, and its absolute configuration is obtained. Important related signals include... Figure 4 As shown.

[0052] Compound 3

[0053]

[0054] Physalin IX(3) is a colorless needle-like crystal, readily soluble in methanol and chloroform. It appears dark black under 254 nm UV-Vis light and pale lemon yellow under 10% sulfuric acid in ethanol. High-resolution mass spectrometry (HR-ESIMS) determined the quasi-molecular ion peak of compound 3 to be m / z 551.1893 [M + Na]. + (Calculated value 551.1898), the molecular formula of compound 3 is inferred to be C. 28 H 32 O 10 Its unsaturation degree was calculated to be 13. The IR spectrum at 3323 cm⁻¹ -1 1769cm -1 1700 cm -1 and 1063cm -1 The presence of an absorption peak indicates the presence of hydroxyl and carbonyl groups in compound 3. 1 The 1H NMR spectrum shows that the compound has four methyl groups: 1.39 (3H, s, H-19), 1.91 (3H, s, H-21), 1.34 (3H, s, H-28), and 1.32 (3H, d, J = 7.2 Hz, H-27); it can also be inferred that the compound contains two olefinic double bonds: 6.17 (1H, dd, J = 10.4, 2.8 Hz, H-6), 6.33 (1H, d, J = 10.4 Hz, H-7), and 5.61 (1H, d, J = 3.4 Hz, H-4), and two proton signals for oxygen bonds: 4.67 (1H, s, H-3) and 4.57 (1H, d, J = 4.0 Hz, H-22); through 13 C NMR (CDCl 3, At 150 MHz, 28 carbon signals were observed, including 2 ketone carbonyl carbon signals: δ C 211.7 (C-1) and 212.5 (C-15); two groups of olefin double bond carbon signals: δ C142.1 (C-5), 128.0 (C-6), 127.9 (C-7) and 126.0 (C-4); carbon signal of a single hydroxyl group: δ C 69.6 (C-3); 4 methyl carbon signals: δ C Based on the carbon-hydrogen correlations in the DEPT and HSQC spectra, the following links can be deduced: -CH2(2)-CH(3)-CH(4)-, CH(6)-CH(7)-CH(8)-CH(9)-CH2(11)-CH2(12)-, and -CH(22)-CH2(23)-. 13 C NMR data yielded three oxygen-related quaternary carbon signals: δ C 83.1 (C-20), 82.5 (C-17), and 80.7 (C-13) and double oxygen-carbon signals: δ C 101.7 (C-14); This suggests the compound is a physalin-type compound, the target compound. In the HMBC spectrum, δ... H 2.86, δ H 3.10 (H-2) and δ C 69.6 (C-3), δ C 126.0 (C-4), δ C 51.5 (C-10) correlation, δ H 6.17 (H-6) and δ C 126.0 (C-4), δ C 142.1 (C-5), δ C 45.2 (C-8), δ C 51.5 (C-10) correlation, δ H 6.33 (H-7) and δ C 128.0 (C-6), δ C 142.1 (C-5), δ C 45.2 (C-8),δ C 31.7 (C-9), δ C 101.7 (C-14) related, δ H 5.61 (H-4) and δ C 44.9 (C-2), δ C 69.6 (C-3), δ C 128.0 (C-6) and δ CBased on 51.5 (C-10), infer the positions of the olefinic double bonds and hydroxyl substitutions on rings A and B of this compound; δ H 1.61, δ H 1.15 (H-11) and δ C 142.1 (C-5), δ C 45.2 (C-8), δ C 31.7 (C-9), δ C 51.5 (C-10), δ C 29.2 (C-12), δ C 101.4 (C-14), δ C 82.5 (C-17) related, δ H 1.81, δ H 2.45 (H-12) and δ C 31.7 (C-9), δ C 51.5 (C-10), δ C 24.8 (C-11), δ C 80.7 (C-13), δ C 82.5 (C-17) related, δ H 1.39 (H-19) and δ C 142.1 (C-5), δ C 31.7 (C-9), δ C 51.5 (C-10) related, δ H 1.91 (H-21) and δ C 83.1 (C-20) and δ C 78.7 (C-22) related, δ H 2.73 (H-11 / H-25) and δ C 82.5 (C-17), δ C 30.6 (C-23), δ C 35.7 (C-24), δ C 176.7 (C-26), δ C 16.3 (C-27) related, δ H 1.32 (H-27) and δ C 35.7 (C-24), δ C 176.7 (C-26), δ C 42.8 (C-25) related, δ H 1.34 (H-28) and δ C54.8 (C-16), δ C The correlation at 30.6 (C-23) proves that C-8 and C-14 are connected, and C-18 and C-14 are connected by oxygen bridges. This can be confirmed by HSQC and HMBC spectra, δ... H 2.86 (H-2) is associated with 3-OH, and 14-OH with δ H 1.61 (H-11), δ H 1.81 (H-12) and δ H 2.72 (H-8) related, 13-OH and δ H 2.19(H-23), δ H 2.99 (H-9) and δ H Based on the correlation of 2.43 (H-12), it can be inferred that 3-OH and 13-OH are α-configurations, and 14-OH is a β-configuration. Using the above data, the structure of compound 3 is further determined, yielding its absolute configuration. Important related signals include... Figure 5 As shown.

[0055] The ethyl acetate and petroleum ether fractions of Physalis perforata were systematically separated using various column chromatography techniques. The monomeric compounds were identified by NMR, HRESIMS, IR, UV, ECD, and optical rotation determination. The chemical structures of the isolated monomeric compounds were also determined. A total of 20 compounds were isolated and identified from Physalis perforata, including three new Physalis glycosides: 3α-hydroxy-2,3,25,27-tetrahydro-4,7-didehydro-7-deoxyneophysalin A (1), Physalin VIII (2), and Physalin IX (3). Seven known Physalis glycosides were also identified: Physalis glycoside L (4), Physalis glycoside D (5), alkekengilin A (6), iso-Physalis glycoside A (7), Physalis glycoside V (8), Physalis glycoside A (9), and Physalis glycoside N (10). Three steroidal compounds: Withaphysalin C (11), Withaminilide B (12), and stigmasterol (13); one flavonoid compound: Rhamnazin (14); five sesquiterpenoid compounds: (+)-Anhydro-β-rotunol (15), Cyperusol C (16), (3S, 5R, 8R)-3,5-dihydroxymegastigma-6,7-dien-9-one (17), Capsidiol (18), and 4,5-dihydroxybruhmol (19); and one fatty acid compound: (9Z, 11E)-13-oxo-9,11-ocatadecadienoic acid (20). Compounds 11, 12, 16, and 18 are known to have been isolated from physalis for the first time.

[0056]

[0057] Three new compounds and 17 known compounds were isolated from the 95% ethanol extract of Physalis alkekengi calyx using solvent extraction and chromatographic separation techniques. Ten of these compounds were physalis-based compounds, and this study primarily focused on physalis-based compounds. All isolated compounds showed UV absorption at 254 nm on silica gel thin-layer chromatography plates and developed as yellow spots with 10% sulfuric acid in ethanol. They exhibited the following characteristics: all physalis-based compounds possessed the basic skeleton of steroidal compounds. Based on whether ether bonds were formed at C-14 and C-27, physalis-based compounds could be further divided into type A and type B physalis-based compounds; characteristic signal of type A physalis-based compounds: 1 H-NMR data at δ H 6.17 ± 0.02 and δ HThere are characteristic hydrogen signals from two alkene double bonds at 6.33 ± 0.11, at δ H 1.39 ± 0.2 (3H, s, H-19), δ H 1.88 ± 0.02 (3H, s, H-21), 1.32 (3H, d, H-27) and δ H The characteristic methyl signal of physalin compounds is located at 1.34 ± 0.12 (3H, s, H-28); 13 In the C-NMR spectrum, the physalin compound appears at δ C There are at least 14 carbon signals in the range of 60 to 210 ppm, among which in δ C Two ketone carbonyl and ester carbonyl carbon signals were observed at 210 ± 1.1 ppm (C-1 / C-15) and 176 ± 2.1 ppm (C-15 / C-26), respectively, with δ C A quaternary carbon signal is present at 101 ± 3.5 ppm (C-14), indicating that these compounds are type A physalin compounds. Characteristic signals of type B physalin compounds: 1 H-NMR data at δ H 6.17 ± 0.02 and δ H There are characteristic hydrogen signals from two alkene double bonds at 6.33 ± 0.11, at δ H 1.39 ± 0.2 (3H, s, H-19), δ H 1.88 ± 0.02(3H, s, H-21) and δ H The characteristic methyl signal of physalin compounds is located at 1.34 ± 0.12 (3H, s, H-28); 13 In the C-NMR spectrum, the physalin compound appears at δ C There are at least 14 carbon signals in the range of 60 ~ 211 ppm, among which in δ C Two ketone carbonyl carbon and ester carbonyl carbon signals were observed at 210 ± 1.1 ppm (C-1 / C-15) and 176 ± 2.1 ppm (C-18 / C-26), respectively, with δ C A quaternary carbon signal at 101 ± 3.5 ppm (C-14) indicates that these compounds are type B physalis compounds. The structural characteristics of the new physalis are that C-14 and C-16 are directly linked by a carbon-carbon bond, and C-15 and C-17 are linked by an ester bond, confirming its new physalis composition. Characteristic signals of the new physalis: 1 H-NMR data at δ H 6.17 ± 0.02 and δ HThere are characteristic hydrogen signals from two alkene double bonds at 6.33 ± 0.11, at δ H 1.39 ± 0.2 (3H, s, H-19), δ H 1.88 ± 0.02 (3H, s, H-21) and δ H The characteristic methyl signal of physalin compounds is located at 1.34 ± 0.12 (3H, s, H-28); 13 In the C-NMR spectrum, the physalin compound appears at δ C There are at least 14 carbon signals in the range of 60 ~ 211 ppm, among which in δ C There is one ketone carbonyl carbon signal and three ester carbonyl carbon signals at 210 ± 1.1 ppm (C-1) and 176 ± 2.1 ppm (C-15 / C-18 / C-26), respectively, at δ C A quaternary carbon signal was observed at 101 ± 3.5 ppm (C-14), indicating that these compounds are novel physalin compounds. Six physalin compounds were identified: 3α-hydroxy-2,3,25,27-tetrahydro-4,7-didehydro-7-deoxyneophysalin A (1), PhysalinⅧ (2) and Physalin IX (3), Physalin L (4), Physalin D (5), and alkekengilin A (6). All samples were monomeric compounds extracted and isolated in this experiment.

[0058] Experimental methods

[0059] Preparation of test sample solution

[0060] Using DMSO as a solvent, the six isolated physalin compounds were dissolved sequentially to form the test sample solution.

[0061] Cell resuscitation

[0062] The prepared culture medium was preheated in a 37°C water bath; the cell cryopreservation tubes preheated in a -80°C freezer were taken out and labeled; the cell cryopreservation solution was then rapidly thawed and transferred to a centrifuge tube containing complete culture medium, centrifuged at 1000 rpm for 5 min to mix, the supernatant was discarded, and 5 mL of complete culture medium was added. The cells were repeatedly pipetted and aspirated until they were completely resuspended and then transferred to a 75 cm² culture flask; the culture flask was carefully moved and placed horizontally in a 37°C, 5% CO2 cell culture incubator.

[0063] Cell culture and passage

[0064] Observe cell fusion density and viability under a microscope. When the cell fusion rate in the culture flask reaches 85% or higher, it can be passaged (dump the old culture medium and wash three times with sterile PBS before passage). Add an appropriate amount of preheated trypsin containing 0.25% EDTA to the culture flask, place it horizontally in an incubator and let it digest for 24 hours. When the cells are observed to lose their polygonal shape and become spherical under a microscope, no longer adhering to the wall and beginning to suspend in the liquid solvent, complete culture medium and trypsin can be added to the culture flask. Then, centrifuge the mixture with culture medium at 1000 rpm for 5 minutes, discard the supernatant, add new culture medium, resuspend the cells, and dilute and re-seed them according to different passage ratios.

[0065] Cell cryopreservation

[0066] Remove the cells to be cryopreserved from the incubator when the cell density reaches approximately 80%, discard the old culture medium, and rinse the cells with sterile PBS solution. Discard the PBS solution, add trypsin to the culture flask, and place it flat in the incubator to allow it to digest. After digestion, add complete culture medium and trypsin, tap the culture flask to allow the cells to fall completely into the liquid, collect the liquid, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1-2 mL of serum-free cell cryopreservation solution, mix well, transfer to cryovials for labeling and sealing, and after gradient cooling of the cryovials, transfer them to a liquid nitrogen tank or low-temperature freezer for storage.

[0067] Live cell workstation for detecting cell proliferation

[0068] Cells in the logarithmic growth phase and in good condition were seeded in 96-well plates with 100 μL of culture medium per well. The drug was added every other day, and then the 96-well plates were placed in a cell culture station and cultured for 48 h.

[0069] Preliminary screening of antitumor activity of physalin compounds

[0070] Single-cell suspensions were prepared using culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum. 3000-15000 cells were seeded per well in 96-well plates at a volume of 100 μL per well. Adherent cells were seeded and cultured 12-24 h in advance, with a final volume of 100 μL per well. Test samples were diluted as needed, with an initial screening concentration of 40 μM. Paclitaxel and cisplatin were used as positive controls, prepared as 1.0 mg / mL solutions using DMSO aqueous solution. -1 The solution is administered at the required concentration during testing.

[0071] After incubation at 37℃ for 48 h, for adherent cells, discard the culture medium in each well and add 20 μL of MTS solution and 100 μL of culture medium to each well; for suspended cells, discard 100 μL of culture supernatant and add 20 μL of MTS solution to each well; set up 3 replicates (a mixture of 20 μL MTS solution and 100 μL culture medium), and continue incubation for 2-4 h to allow the reaction to proceed fully before measuring the absorbance. Each replicate was set up with 3 replicates. The absorbance of each well was read at 492 nm using a multi-functional microplate reader (MULTISKAN FC), and the experimental results were recorded.

[0072] Secondary screening of antitumor activity of physalin compounds

[0073] Under the initial screening condition of 40 μM, compounds with a tumor cell growth inhibition rate greater than 50% were included in a gradient rescreening at five concentrations (using the same method as the initial screening). Compound 5 was used for rescreening the in vitro tumor growth inhibition activity against HL-60, A-549, SMMC-7221, MDA-MB-231, and SW480. The negative control group was physiological saline, and the positive control compounds were cisplatin (DDP) and paclitaxel (Taxol). The sample addition volume was 10 μl / well. Cell growth curves were plotted with concentration on the x-axis and cell viability on the y-axis. The IC50 of the compounds was calculated using the Reed and Muench method. 50 value.

[0074] Experimental results

[0075] Preliminary screening results of the antitumor activity of physalin compounds

[0076] Cytotoxicity test results of physalin compounds (inhibition rate %)

[0077]

[0078] Six physalin compounds were initially screened for antitumor activity. The initial screening results showed that compounds 1 and 2 had growth inhibition rates of 19.61% and 15.20% against MDA-MB-231 tumor cells, respectively, indicating weak growth inhibition. Compound 3 had a growth inhibition rate of 13.18% against SW480 tumor cells, also indicating weak growth inhibition. Compounds 4 and 6 had growth inhibition rates of 30.17% and 29.71% against SMMC-7221 tumor cells, respectively, indicating weak growth inhibition. Compound 5 showed a growth inhibition rate of more than 50% against all five types of tumor cells. This compound was then used for a gradient secondary screening at five different concentrations.

[0079] Results of secondary screening of antitumor activity of physalin compounds

[0080] Inhibitory effect of physalin compounds on the proliferation of five tumor cell lines (IC50) 50 (μM)

[0081]

[0082] Note: "—" indicates no activity and no rescreening.

[0083] The cytotoxic activity of five cell lines isolated and identified from physalin was determined using the MTT assay, with cisplatin and paclitaxel as positive control agents. The results showed that compound 5 exhibited good inhibitory activity against all five cell lines, with IC50 values ​​of [missing value]. 50 The range is μmol / L.

[0084] The inhibitory activities of the six isolated physalin compounds against five cell lines cultured in vitro were investigated. The results showed that, compared with positive control drugs, the initial screening revealed that, at a concentration of 100 μmol / L, compounds 1 and 2 exhibited weak inhibitory activity against MDA-MB-231 tumor cells, with growth inhibition rates of 19.62% and 15.20%, respectively; compound 3 showed weak inhibitory activity against SW480 tumor cells, with a growth inhibition rate of 13.18%; compounds 4 and 6 showed weak inhibitory activity against SMMC-7221 tumor cells, with growth inhibition rates of 30.17% and 29.71%, respectively; and compound 5 showed an inhibition rate greater than 50% against HL-60, A-549, SMMC-7221, MDA-MB-231, and SW480 cells, indicating that these compounds had good activity against these cells. Therefore, the IC50 of these more active compounds was further determined. 50 The physalin compounds isolated in this study exhibit certain cytotoxic activity against tumor cells. Further research can be conducted to investigate the mechanism by which these physalin compounds inhibit tumor proliferation and the extent of their toxic side effects on normal cells, providing experimental evidence for the development of novel antitumor drugs or lead compounds.

[0085] Since these compounds have many substitution positions and different substituents have a significant impact on their activity, the effects of different substituents and substitution positions on the antitumor activity of these compounds can be further systematically discussed, which can provide a valuable theoretical basis for further molecular design and structural optimization of these compounds.

[0086] The specific embodiments of the present invention have been described in detail above. It should be noted that the present invention is not limited to the specific embodiments described above. Various modifications or alterations can be made by those skilled in the art without departing from the scope of protection defined by the claims, and all such modifications or alterations fall within the scope of the present invention.

Claims

1. A method for separating compounds from the calyx of Physalis alkekengi, characterized in that, Includes the following steps: Step 1: Place the calyx in a dark and ventilated place to dry naturally. After it is completely dry, crush it into a fine powder. Step 2: After soaking in 95% ethanol, heat and extract three times at 60 ℃. Combine the extracts and concentrate them at 35 ℃ and 0.3 MPa until they just flow out of the concentration tank. Then further concentrate under reduced pressure to obtain crude extract. Step 3: Thoroughly mix the extract with warm water at a 1:1 mass ratio to obtain a suspension. Add petroleum ether, dichloromethane, ethyl acetate, and n-butanol sequentially to the suspension for fractional extraction (V... 水 :V 有机相 =1:1), let stand to separate into layers, extract and concentrate, slowly add dichloromethane (CH2Cl2) (V aqueous phase:V organic phase = 1:1) to the suspension to remove PE, seal, shake and mix, let stand to separate into layers, extract the CH2Cl2 layer and concentrate, repeat the extraction multiple times until the CH2Cl2 layer becomes lighter in color and transparent, to obtain petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract and n-butanol fraction extract; Step 4: Separate the ethyl acetate fraction of *Phyllostachys edulis*. Use a 100-200 mesh silica gel column for chromatography, mixing the sample with silica gel, and perform separation on a normal-phase, atmospheric-pressure column. Elute with a mobile phase of dichloromethane:methanol at a ratio of 50:1 → 30:1 → 20:1 → 15:1 → 10:1 → 5:

1. Spot the obtained fractions onto a thin-layer silica gel plate and observe under 254 nm UV light. Combine the colorimetric results with 10% sulfuric acid ethanol. Combine identical fractions to obtain five fractions (Fr-1 to Fr-5). Step 5: Fraction Fr-4 was further separated using a 200-300 mesh normal-phase silica gel column with dichloromethane:methanol as the mobile phase at a ratio of 15:1 → 10:1 → 5:1 → 3:1 → 2:1, yielding four fractions (Fr-4-1 ~ Fr-4-4). Fr-4-4 was then separated by normal-phase silica gel column chromatography, further purified by LH-20 gel column chromatography (MeOH), and then further purified by semi-preparative HPLC under the mobile phase conditions of (MeOH-H2O, 65:35, 2 ml / min, v / v) to obtain compound 1. The t-value of compound 1... R =25.0 min; Step 6: Fraction Fr-5 was further separated using a 200-300 mesh normal-phase silica gel column with dichloromethane-methanol as the mobile phase, eluting at a ratio of 10:1 → 5:1 → 3:1 → 2:1 → 1:1 to obtain three fractions (Fr-5-1 ~ Fr-5-3). Fr-5-3 was further purified by normal-phase silica gel column separation, followed by LH-20 gel column (MeOH) purification, and then further purified by semi-preparative HPLC with a mobile phase of MeOH-H2O (50:50, 2 ml / min, v / v) to obtain compound 2. The t of compound 2... R = 30.0 min; Step 7: Separate the petroleum ether fraction of the calyx of *Phyllostachys edulis*. Take 373.46 g of the petroleum ether fraction extract and separate it by silica gel column chromatography using a 100-200 mesh screen. Elute with petroleum ether-ethyl acetate as the mobile phase at a ratio of 8:1 → 5:1 → 3:1 → 2:1 → 1:

1. Combine the eluents to obtain 10 fractions (Fr-1 to Fr-10). Fr-10 was further separated by silica gel column chromatography (200-300 mesh), using dichloromethane-methanol as the mobile phase with a ratio of 80:1→50:1→30:1→20:1→15:1→10:

1. The fractions were then combined to obtain five fractions (Fr-10-1~Fr-10-5). Fr-10-4 was subjected to LH-20 dextran gel column chromatography and eluted with methanol-dichloromethane (1:1) to obtain three components (Fr-10-4-1~Fr-10-4-3); Fr-10-4-3 was further separated by 200-300 mesh silica gel column chromatography with gradient elution of (100:1, 80:1, 50:1) to obtain two fractions (Fr-10-4-3-1 and Fr-10-4-3-2); Fr-10-4-3-2 was separated by high performance liquid chromatography (MeCN-H2O, 34:66, v / v, 2 ml / min) to obtain compounds 9 and 10 and Fr-10-4-3-2-1; Fr-10-4-3-2-1 was purified by semi-preparative high performance liquid chromatography (MeCN-H2O, 32:68, v / v, 2 ml / min) to obtain compound 3 (t R = 20.0min).

2. The method according to claim 1, characterized in that, In step 2, the solid-liquid ratio for soaking is 1:10, and the extraction time is 8 hours each time.

3. The method according to claim 1, characterized in that, The structural formula of compound 1 is 。 4. The method according to claim 1, characterized in that, The structural formula of compound 2 is 。 5. The method according to claim 1, characterized in that, The structural formula of compound 3 is 。

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