New compound in pteris multifida as well as extraction, separation and purification method and application thereof

Abstract

The invention discloses a novel compound in pteris multifida as well as an extraction, separation and purification method and application thereof. The compound is prepared from the following raw materials: sweet myrrh 10-ene-9, 15-lactone-14-acid (wallichiana A) and 2, 3-dihydro-2-[(beta-D-xylosyl) methyl]-5, 7-dimethyl-6-(2-chloroethyl)-2 (S)-1H-indene-1-ketone (wallichiana B), and the compound is obtained by extraction and crude separation of medicinal materials as well as separation and purification of an ethyl acetate part. An anti-tumor activity screening experiment is carried out on the compounds obtained through separation and purification, and the result shows that the two compounds have half inhibitory activity on in-vitro tumor growth of breast cancer MCF-7, colon cancer HCT-116, cervical cancer Hela and lung cancer A549, can be used for treating anti-tumor diseases, and have good application prospects. Reference is provided for further strengthening research on chemical components and activity screening of the plant and research on structural modification of compounds with good activity in the plant.

Description

Technical Field

[0001] The invention relates to the field of pharmacy, and in particular to a new compound in Pteris crenata and an extraction, separation and purification method and application thereof. Background Art

[0002] Pteris wallichiana Agardh grows at an altitude of 800 to 2000 meters, and the plant height is about 1.5 meters [1] , which is currently mainly distributed in Yunnan, Guizhou, Guangxi and other places [2] . Mainly used to treat dysentery, infantile convulsions, etc. [3] . Currently, there are few studies on Pteris cretica. Most of the studies on Pteris cretica are on its chemical composition, and it is mainly found that the plant has terpenoids and flavonoids. There are few studies on its pharmacological activity. Some scholars have isolated sesquiterpenes from Pteris cretica and tested their anti-tumor activity, but did not find that it has anti-tumor activity; other scholars have found that the ethanol extract of Pteris cretica has the effect of inhibiting ulcerative colitis by studying it. [4-5] . In addition, our research group has also conducted in-depth research on the chemical components of other plants in this genus, such as Pteris semipinnata, Centipede grass, and Pteris integrifolia, and isolated a large number of biologically active monomer compounds, especially ant-kaurane-type diterpenes and their glycosides. Based on the principle that plants of the same genus have similar components, in order to further explore the chemical composition and pharmacological material basis of Pteris southwesternensis, the research group has carried out research on the chemical components of Pteris southwesternensis. Through preliminary experimental research on its chemical components, it was found that the plant may contain chemical components such as glycosides, flavonoids, coumarins and phenols.

[0003] The research group of the present invention studied the chemical composition of methanol extract of Pteris wallichiana and isolated a number of compounds from it, including two new compounds, wallichiana A (1) and wallichiana B (2), which were isolated from the plant for the first time. The anti-tumor activity screening experiment of the new compounds showed that the two compounds had half-inhibitory activity on the in vitro tumor growth of breast cancer MCF-7, colon cancer HCT-116, cervical cancer Hela, and lung cancer A549, and can be used to treat anti-tumor diseases. At the same time, it provides a reference for further strengthening the chemical composition and activity screening research of the plant and the research on the structural modification of compounds with good activity in the plant. Summary of the invention

[0004] The present invention aims to provide a new compound, wallichiana A and wallichiana B, from Pteris wallichiana.

[0005] Another object of the present invention is to provide a method for extracting, separating and purifying new compounds in Pteris crenata.

[0006] Another object of the present invention is to provide a new compound from Pteris crenata for use in antibacterial and / or anti-inflammatory applications.

[0007] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions and steps:

[0008] The new compounds in the southwestern pteris var. cymoides described in the present invention are: wallichiana A, 2,3-dihydro-2-[(β-D-xylosyl)methyl]-5,7-dimethyl-6-(2-chloroethyl)-2(S)-1H-inden-1-one (wallichiana B), and the structural formulas thereof are as follows:

[0009] (1) Structural formula of wallichiana A:

[0010]

[0011] (2) Structural formula of wallichiana B:

[0012]

[0013] The method for extracting, separating and purifying the new compound in Pteris fasciata of the present invention comprises the following steps:

[0014] 1) Extraction and crude separation of medicinal materials

[0015] Southwestern pteris fern medicinal material is naturally dried in the shade, crushed into coarse powder, and passed through a 80-100 mesh sieve; the sieved coarse powder is first extracted twice by soaking in 95% industrial methanol at room temperature with 2 times / times, then extracted twice by soaking in 85% methanol at room temperature with 2 times / times, and finally extracted once by soaking in 70% industrial methanol at room temperature, the extract is concentrated under reduced pressure at 55°C to an extract with a density of 1.06, and then diluted with 1.25 times warm water, and extracted with petroleum ether, ethyl acetate, and n-butanol in turn by liquid-liquid extraction method, with the ratio of organic phase to aqueous phase being 1:1, and after the extraction is completed, the extracts are combined and concentrated under reduced pressure at 55°C to an ethyl acetate extract with a density of 1.06;

[0016] 2) Separation and purification of ethyl acetate fraction

[0017] The ethyl acetate fraction was dissolved with a small amount of methanol, and the polyamide sample was taken for gradient elution using an MCI column with a methanol-water system of 1:1, 7:3, and 9:1, and finally eluted with pure methanol to obtain 4 fractions A to D;

[0018] Separation of sample of part B: add appropriate amount of methanol to dissolve the sample of part B, take 100-200 mesh silica gel to mix the sample, and separate it by silica gel column chromatography; first elute with dichloromethane, then gradient elute with dichloromethane-methanol system of 50:1, 25:1, 10:1, 7:1, 5:1, 2:1, and finally elute with methanol; after TLC detection, combine similar fractions to obtain six parts from B-1 to B-6, and then use silica gel column chromatography to elute part B-1 with petroleum ether-ethyl acetate system of 50:1, 30:1 to obtain four parts from B-1-1 to B-1-4. Crystals are found to precipitate in B-1-2, and compound wallichiana A is obtained after repeated recrystallization;

[0019] Separation of part B-4 sample: Add appropriate amount of methanol to part B-4 sample to dissolve it, weigh 100-200 mesh silica gel to mix the sample, and separate it by silica gel column chromatography; first elute with dichloromethane, then gradient elute with a dichloromethane-methanol system of 50:1, 30:1, 10:1, 5:1, 1:1, and finally elute with methanol. After TLC detection, combine similar fractions to obtain 4 parts, namely B-4-1 to B-4-4; repeatedly subject part B-4-1 to silica gel column chromatography to obtain compound wallichiana B.

[0020] Preferably, in step 1) of the extraction, separation and purification method of the present invention, the sieved coarse powder is first extracted twice by soaking in 2 times / times 95% industrial methanol at room temperature, then extracted twice by soaking in 2 times 85% methanol at room temperature, and finally extracted once by soaking in 70% industrial methanol at room temperature.

[0021] Preferably, in the extraction, separation and purification method of the present invention, the time for the immersion extraction in step 1) is: each immersion extraction is 4 to 6 days.

[0022] Further preferably, in the extraction, separation and purification method of the present invention, the time for the immersion extraction in step 1) is: each immersion extraction is 5 days.

[0023] The invention discloses an application of the new compound in Pteris dasyphylla or the compounds wallichiana A and wallichiana B obtained by the extraction, separation and purification method in the preparation of anti-tumor drugs.

[0024] The invention discloses an application of the compounds wallichiana A and wallichiana B in preparing medicines for treating human breast cancer, human colon cancer, human cervical cancer and human lung cancer.

[0025] The medicine of the present invention is prepared by adding compounds wallichiana A and wallichiana B to pharmaceutically acceptable excipients to form a pharmaceutically acceptable solid preparation or liquid preparation.

[0026] The solid preparations of the present invention are granules, capsules, tablets, pills, powders, and freeze-dried powder injections; the liquid preparations are injection preparations and oral liquids.

[0027] Beneficial effects of the present invention:

[0028] 1. The present invention extracts, separates and purifies two new compounds from Pteris wallichiana, namely wallichiana A and wallichiana B, both of which are isolated from the plant for the first time.

[0029] 2. The present invention extracts, separates and purifies two new compounds from Pteris wallichiana and conducts anti-tumor activity screening studies. The results show that the compounds wallichiana A and wallichiana B have half-inhibitory activity on the in vitro tumor growth of breast cancer MCF-7, colon cancer H CT-116, cervical cancer Hela and lung cancer A549, can be used to treat anti-tumor diseases, and have potential medicinal value. This provides a basis for further strengthening the chemical composition and activity screening research of the plant and the structural modification research of the compounds with good activity in the plant.

[0030] 3. The present invention studied the inhibitory activity of all compounds on breast cancer cells MCF-7. The IC values ​​of compounds wallichiana A and wallichiana B were 50 They are 0.425±0.071 and 0.567±0.333 respectively.

[0031] 4. The present invention studied the inhibitory activity of all compounds on colon cancer cells (HCT-116). The IC of compound wallichiana A 50 It is 3.001±0.629.

[0032] 5. The present invention studied the inhibitory activity of all compounds on cervical cancer cells (Hela), and the IC of wallichianaB 50 It is 8.246±0.175.

[0033] 6. The present invention studied the inhibitory activity of all compounds on human lung cancer cells (A549), and the IC 50 It is 0.278±0.088. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 :Structural formula of compound 1

[0035] Figure 2 :Unit cell diagram of compound 1

[0036] Figure 3 :HMBC of compound 1 (→), 1 H- 1 H COSY Related signals

[0037] Figure 4 :NOESY of compound 1 Related signals

[0038] Figure 5 :Structural formula of compound 2

[0039] Figure 6 :Unit cell diagram of compound 2

[0040] Figure 7 :HMBC(→) of compound 2, 1 H- 1 H COSY Related signals

[0041] Figure 8 :NOESY of compound 2 Related signals

[0042] Fig. 9 : HR-ESI-MS spectrum of compound 1;

[0043] Fig.10 :Compound 1 1 H-NMR spectrum;

[0044] Fig.11 :Compound 1 13 C-NMR spectrum;

[0045] Fig.12 : DEPT spectrum of compound 1;

[0046] Fig.13 : HSQC spectrum of compound 1;

[0047] Fig.14 : HMBC spectrum of compound 1;

[0048] Fig.15 :Compound 1 1 H- 1 H COSY spectrum;

[0049] Fig.16 : NOESY spectrum of compound 1.

[0050] Fig.17 : HR-ESI-MS spectrum of compound 2;

[0051] Fig.18 :Compound 21 H-NMR spectrum;

[0052] Fig.19 :Compound 2 13 C-NMR spectrum;

[0053] Fig. 20 : DEPT spectrum of compound 2;

[0054] Fig.21 : HSQC spectrum of compound 1;

[0055] Fig. 22 : HMBC spectrum of compound 2;

[0056] Fig.23 :Compound 2 1 H- 1 H COSY spectrum;

[0057] Fig.24 : NOESY spectrum of compound 2.

[0058] Fig.25 :IC of the tested compounds against human breast cancer cells (MCF-7) 50 Value Intuition Diagram

[0059] Fig.26 :IC of the tested compounds against human colon cancer cells (HCT-116) 50 Value Intuition Diagram

[0060] Fig. 27 :IC of the tested compounds against human cervical cancer cells (Hela) 50 Value Intuition Diagram

[0061] Fig.28 :IC of the tested compounds against human lung cancer cells (A549) 50 Value Intuition Diagram DETAILED DESCRIPTION

[0062] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. For those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

[0063] Example 1: Extraction, separation and purification of compounds from Pteris dasyphylla

[0064] 1) Extraction and crude separation of medicinal materials

[0065] Southwestern pteris fern medicinal material is naturally dried in the shade, crushed into coarse powder, and passed through a 80-100 mesh sieve; the sieved coarse powder is first extracted twice by soaking in 95% industrial methanol at room temperature with 2 times / times, then extracted twice by soaking in 80% methanol at room temperature with 2 times / times, and finally extracted once by soaking in 70% industrial methanol at room temperature, the extract is concentrated under reduced pressure at 55°C to an extract with a density of 1.06, and then diluted with 1.25 times warm water, and extracted with petroleum ether, ethyl acetate, and n-butanol in sequence by liquid-liquid extraction method, with the ratio of organic phase to aqueous phase being 1:1, and after the extraction is completed, the extracts are combined and concentrated under reduced pressure at 55°C to an ethyl acetate extract with a density of 1.06;

[0066] 2) Separation and purification of ethyl acetate fraction

[0067] The ethyl acetate fraction was dissolved with a small amount of methanol, and the polyamide sample was taken for gradient elution using an MCI column with a methanol-water system of 1:1, 7:3, and 9:1, and finally eluted with pure methanol to obtain 4 fractions A to D;

[0068] Separation of part B samples: Add an appropriate amount of methanol to dissolve the part B samples, take 100-200 mesh silica gel to mix the samples, and separate them by silica gel column chromatography; first elute with dichloromethane, then gradient elute with a dichloromethane-methanol system of 50:1, 25:1, 10:1, 7:1, 5:1, 2:1, and finally elute with methanol; after TLC detection, combine similar fractions to obtain six parts from B-1 to B-6, and then use silica gel column chromatography to elute part B-1 with a petroleum ether-ethyl acetate system of 50:1, 30:1 to obtain four parts from B-1-1 to B-1-4. Crystals were found to precipitate in B-1-2, and compound 1 (wallichiana A) was obtained after repeated recrystallization;

[0069] Separation of the B-4 portion of the sample: Add an appropriate amount of methanol to dissolve the B-4 portion of the sample, weigh 100-200 mesh silica gel to mix the sample, and separate it by silica gel column chromatography; first elute with dichloromethane, then elute with a dichloromethane-methanol system gradient of 50:1, 30:1, 10:1, 5:1, 1:1, and finally elute with methanol. After TLC detection, combine similar fractions to obtain 4 portions, namely B-4-1 to B-4-4; Repeated silica gel column chromatography on portion B-4-1 to obtain compound 2 (wallichiana B);

[0070] Example 2: Structural formula of compound

[0071] (1) Structural formula of wallichiana A:

[0072]

[0073] (2) Structural formula of wallichiana B:

[0074]

[0075] Example 3: Crystal structure of wallichiana A:

[0076]

[0077] Example 4: Crystal structure of wallichiana B:

[0078]

[0079] Example 5: Characteristics of wallichiana A

[0080] Colorless transparent crystals (chloroform), white crystals (chloroform), show fluorescence under ultraviolet light at 254nm, use dichloromethane / methanol system 15:1, color developer: 10% H 2 SO 4 -EtOH solution turns purple-red when heated (its R f value is 0.28); mass spectrum (HR-ESI-MS): [M+Na] + m / z 289.14026 (calcd for C 15 H 22 O 4 Na, 289.14103); Infrared spectrum IR (KBr): V max 1749.5cm -1 、1704.0cm -1 cm -1 ; 1 H-NMR (400 MHz, CDCl 3 ), 13 C-NMR (100 MHz, CDCl 3 )δ C (ppm), see Table 2.

[0081] Crystal parameters: C 15 H 22 O 4 ,monoclinic system,M=266.32, α=90.00°, β=90.00°, γ=90.00°, T=273(2)K,Z=4.

[0082] Example 6: Characteristics of wallichiana B

[0083] White crystals (pyridine), showing fluorescence under UV light at 254nm; dichloromethane / methanol system 5:1; 10% H 2 SO 4 -EtOH solution turns orange-red when heated (R f value 0.59); mass spectrum (HR-ESI-MS): [M+Na] + m / z 407.12295 (calcd for C 15 H 22 O 4 Na, 407.123187); Infrared spectrum IR (KBr): V max 1665.1cm -1 cm -1 ; 1 H-NMR (400 MHz, C 5 D 5 N), 13 C-NMR (100 MHz, C 5 D 5 N)δ C (ppm), see Table 2.

[0084] Crystal parameters: C 19 H 26 C1O 6.5 ,monoclinic system,M=393.85, α=90.00°, β=108.898(2)°, γ=90.00°, T=273(2)K,Z=4.

[0085] Example 7: Take any one of wallichiana A and wallichiana B as the raw material, add 20% microcrystalline cellulose and 20% powdered sugar to granulate, and obtain granules.

[0086] Example 8: Take any one of wallichiana A and wallichiana B as the raw material, add 10% starch and 3% magnesium stearate, mix well, put into capsules, and obtain capsules.

[0087] Example 9: Take any one of wallichiana A and wallichiana B as the raw material, add 10% starch and 15% honey, mix well, and prepare pills.

[0088] Example 10: Take any one of wallichiana A and wallichiana B as the raw material, add 10% starch slurry and 0.3% magnesium stearate, granulate, press and prepare tablets.

[0089] Example 11: Take any one of wallichiana A and wallichiana B as the raw material, add 12 times the amount of injection water, filter, sterilize, and obtain an injection.

[0090] Example 12: Take any one of wallichiana A and wallichiana B as the raw material, add 8 times the amount of water for injection, filter, and freeze-dry to obtain a freeze-dried powder.

[0091] Example 13: Take any one of wallichiana A and wallichiana B as the raw material, add 12 times the amount of purified water and 0.05% sodium benzoate, mix well, filter, sterilize, and obtain an oral solution. In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0092] (I) Isolation and identification of compounds

[0093] 1. Instruments, reagents and materials

[0094] 1.1 Instruments and reagents

[0095] See Table 1.

[0096] Table 1 Instrument and reagent names, models and sources

[0097]

[0098] There are three color developers involved in the experiment, namely 10% sulfuric acid-anhydrous ethanol solution, 5% phosphomolybdic acid-anhydrous ethanol solution and 2% iodine-silica gel.

[0099] 1.2 Materials

[0100] The experimental medicinal materials were collected from Datianba Township, Changning County, Baoshan City, Yunnan Province. After identification by Professor Zhao Junhua of Guizhou University of Traditional Chinese Medicine, it was determined that the plant was Pteriswallichiana Agardh, a plant of the genus Pteris L (Pteridaceae). The plant voucher specimens are stored in the Pharmaceutical Laboratory of Guizhou University of Traditional Chinese Medicine.

[0101] MCF-7 breast cancer cells, HCT116 colorectal cancer cells, Hela cervical cancer cells, and A549 lung cancer cells were all purchased from Shanghai Cell Bank; fetal bovine serum (batch number: SA211028), high-glucose DMEM basal medium (batch number: G4511-500 ML), and PBS buffer (batch number: G4202-500 mL) were all purchased from Wuhan Pronocell Life Science Technology Co., Ltd.; 0.25% trypsin cell digestion solution (batch number: C0207) was purchased from Shanghai Biotech Biotechnology Co., Ltd.; sorafenib (batch number: S828599) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0102] 2. Extraction and separation

[0103] 1) Extraction and crude separation of medicinal materials

[0104] The medicinal material of Southwest Pteris fasciata weighs 46.7kg after natural shade drying, and is crushed into coarse powder (80-100 mesh), soaked twice with 95% methanol at room temperature, then extracted twice with 2 times 80% methanol at room temperature, and finally extracted once with 70% industrial methanol at room temperature, each time for 5 days, and the 5 soaking solutions are combined and recovered under reduced pressure to obtain about 10.3kg of thick extract. The total extract is dissolved in warm water (aqueous phase), and extracted with petroleum ether, ethyl acetate, and n-butanol in turn by liquid-liquid extraction (the ratio of organic phase to aqueous phase is 1:1), and the solvents are recovered under reduced pressure after extraction to obtain 304g of ethyl acetate fraction.

[0105] 2) Separation and purification of ethyl acetate fraction

[0106] The ethyl acetate portion was dissolved with a small amount of methanol, 300 g of polyamide was weighed and mixed with the sample, and MCI column chromatography was used for gradient elution with methanol-water as the elution system (1:1, 7:3, 9:1), and finally eluted with pure methanol to obtain 4 parts A to D.

[0107] Separation of part B samples: Add an appropriate amount of methanol to dissolve the part B samples, take 100-200 mesh silica gel to mix the samples, and separate them by silica gel column chromatography; first elute with dichloromethane, then gradient elute with a dichloromethane-methanol system of 50:1, 25:1, 10:1, 7:1, 5:1, 2:1, and finally elute with methanol; after TLC detection, combine similar fractions to obtain six parts from B-1 to B-6, and then use silica gel column chromatography to elute part B-1 with a petroleum ether-ethyl acetate system of 50:1, 30:1 to obtain four parts from B-1-1 to B-1-4. Crystals were found to precipitate in B-1-2, and compound 1 (wallichiana A) was obtained after repeated recrystallization;

[0108] Separation of part B-4 sample: add an appropriate amount of methanol to dissolve the part B-4 sample, weigh 100-200 mesh silica gel to mix the sample, and separate it by silica gel column chromatography; first elute with dichloromethane, then elute with a dichloromethane-methanol system gradient of 50:1, 30:1, 10:1, 5:1, 1:1, and finally elute with methanol. After TLC detection, combine similar fractions to obtain 4 parts, namely B-4-1 to B-4-4; repeatedly perform silica gel column chromatography on part B-4-1 to obtain compound 2 (wallichiana B);

[0109] 3. Structural identification of two chemical components from Pteris dasyphylla

[0110] Compound 1: white crystals (chloroform), showing fluorescence under UV light at 254 nm, using a dichloromethane / methanol system of 15:1, color developer: 10% H 2 SO 4 -EtOH solution turns purple-red when heated (its R f value is 0.28).

[0111] IR shows CO single bond (1179.3cm -1 ), carbonyl (1749.5cm -1 、1704.0cm -1 ).according to 1 H-NMR (400 MHz, CDCl 3 ) in the main characteristic spectrum data: δ H 1.77 (3H, s), 1.73 (3H, s) are speculated to be methyl signals (see Fig.10 ); 13 C-NMR (100 MHz, CDCl 3 )(See Fig.11 ) spectrum data and DEPT spectrum (see Fig.12 ) shows two methyl groups δ C 25.9 and 18.6, five methylene δ C 32.4, 30.1, 28.6, 28.5, 27.8, five methine delta C 123.0, 75.3, 46.2, 42.8, 36.7, three quaternary carbon delta C 181.3, 177.9, 140.2; among which δ C 181.3, 177.9 suggest that there may be two carbonyl carbon signals, δ C 140.2, 123.0 is a double bond signal, δ C 75.3 is a signal of an oxygen-linked carbon. According to mass spectrometry (HR-ESI-MS) data, [M+Na] +m / z 289.14026 (calcd for C 15 H 22 O 4 Na, 289.14103), the molecular formula of the compound is determined to be C 15 H 22 O 4 , unsaturation is 5 (see Fig. 9 ).

[0112] In the HMBC spectrum (such as Fig.14 ) shows that δ H 1.73(H-8) and δ C 123.9(C-5), 140.2(C-6), 25.9(C-7) related, δ H 1.77(H-7) and δ C 123.9 (C-5), 140.2 (C-6), 18.6 (C-8) related, δ H 5.19 (H-5) is related to 18.6 (C-8), 25.9 (C-7), 75.3 (C-4), δ H 5.01(H-4) and δ C 123.9(C-5), 140.2(C-6) related, δ H 2.56(H-2) and δ C 177.8 (C-1), 32.4 (C-3), 36.7 (C-1′), 30.1 (C-2′), 27.8 (C-6′) related, δ H 2.28(H-3) and δ C 177.8(C-1), 46.2(C-2), 75.3(C-4), 123.0(C-5), 140.2(C-6) related.

[0113] exist 1 H- 1 H-COSY spectrum (such as Fig.15 ), δ H 5.19 (1H, d, J = 8.5 Hz, H-5) is related to 5.01 (1H, m, H-4), δ H 5.01 (1H, m, H-4) and δ H 5.19 (1H, d, J = 8.5 Hz, H-5), 2.28 (1H, m, H-3), 1.70 (1H, m, H-3), it can be deduced that C 3 —C 4 —C 5 —C 7 fragment; δ H 2.28 (1H, m, H-3) and δH 2.56 (1H, m, H-2), δ H 1.70 (1H, m, H-3), δ H 1.49 (1H, m, H-6′), δ H 5.01 (1H, m, H-4) related.

[0114] In the NOESY spectrum (such as Fig.16 ) in the hydrogen configuration, H-2 is found to be correlated with H-4α, so it is speculated that H-2 is α-configuration; similarly, H-1′ is correlated with H-2, so it is speculated that H-1′ may be α-configuration; H-4′ has correlation signals with H-3′β and H-2′β, so it is speculated that H-4′ may be β-configuration. See the structural formula Figure 1 , 1 H-NMR, 13 C-NMR and HSQC data are shown in Table 2. A small amount of sample was dissolved in methanol and crystal cultured at room temperature. The structure of the crystal was studied by X-ray single crystal diffractometer (molybdenum target), and the parameters were obtained: it was determined to be a monoclinic system (monoclinic), α=90.00°, β=90.00°, γ=90.00°, T=273(2)K,Z=4,Molecular formula C 15 H 22 O 4 etc. The unit cell diagram is shown in Figure 2 , further supporting the structure of the compound, searching through the SciFinder database and consulting the literature, it was finally determined that compound 1 was a new compound and named wallichiana A (bisabol-10-ene-9,15-lactone-14-acid). 1 H- 1 H COSY and NOESY spectrum related signals are shown in Figure 3 to Figure 4 , 1 H-NMR, 13 The C-NMR spectrum data are shown in Table 2.

[0115] Table 2 Compound 1 1 H-NMR, 13 C-NMR data (δ in ppm)

[0116]

[0117] The sample was dissolved in deuterated chloroform solvent, and the nuclear magnetic resonance hydrogen and carbon spectra were measured at 400 MHz and 100 MHz respectively.

[0118] Compound 2: white crystals (pyridine), showing fluorescence under UV light at 254 nm; dichloromethane / methanol system 5:1; 10% H2 SO 4 -EtOH solution turns orange-red when heated (R f value 0.59).

[0119] IR showed that there was a carbonyl group (1665.1 cm -1 ).according to 1 H-NMR (400 MHz, C 5 D 5 N) in the main characteristic spectrum data: δ H 6.97 (1H, s) is speculated to be the hydrogen signal on the benzene ring, δ H 2.70 (3H, s), δ H 2.29 (3H, s) is the hydrogen signal of the two methyl groups δ H 4.01-4.67 (5H, m) may be the hydrogen signal of sugar (see Fig.18 );according to 13 C-NMR (100 MHz, C 5 D 5 N) spectrum (see Fig.19 ) and DEPT spectra (see Fig. 20 ) data confirmed that compound 2 has 19 carbon signals, of which two methyl δ C 21.4 and 14.1; five methylene groups (δ C 64.5, 43.4, 41.6, 34.7, 32.7); five methines (δ C 126.7, 77.8, 77.0, 72.5, 44.9) seven quaternary carbons (δ C 211.4, 154.8, 144.9, 138.0, 135.3, 133.1, 99.6); among which δ C 211.4 suggests that there may be a carbonyl carbon signal, δ C 154.2, 144.7, 137.2, 134.9, 132.0, 126.7 are carbon signals on a benzene ring, δ C 99.3, 75.5, 74.8, 70.7, 62.8 are a sugar signal, δ C 21.1, 13.6 suggest that there are two methyl signals. According to the mass spectrometry (HR-ESI-MS) data (see Fig.17 ), [M+Na] + m / z 407.12295 (calcd for C 19 H 25 O 6 ClNa, 407.123187), the molecular formula of the compound is determined to be C 19 H 25 O6 Cl, unsaturation is 8; the above data are consistent with the literature [6] It is similar to pterosin F in the sesquiterpenoids and is preliminarily inferred to be a sesquiterpenoid.

[0120] HMBC displays (such as Fig. 22 ), δ H 6.97(H-4) and δ C 134.9 (C-6), 21.4 (C-11), 34.7 (C-3) related, δ H 2.29(H-11) and δ C 144.9 (C-5), 135.3 (C-6), 126.7 (C-4), from which it can be inferred that C 4 -C 5 -C 11 fragment; δ H 3.67(H-13) and δ C 31.9(C-12), 135.3(C-6) related, δ H 3.11 (H-12) is related to 144.8 (C-5), 135.3 (C-6), 43.4 (C-13), and 137.2 (C-7), from which it can be inferred that C 6 -C 12 -C 13 fragment; δ H 2.70(H-14) and δ C 138.0(C-7), 135.3(C-6), 154.8(C-8) related, δ H 3.41(H-3) and δ C 44.3 (C-2), 133.1 (C-9), 154.8 (C-8), from which it can be inferred that C 7 -C 8 -C 9 -C 3 -C 2 fragment; δ H 2.23(H-10) and δ C 99.6 (C-1′), 34.7 (C-3), 44.3 (C-2), from which it can be inferred that sugar is related to C 10 Bit connected; δ H 4.56(H-5′) and δ C 99.6 (C-1′), 72.5 (C-4′), 77.0 (C-3′) correlation, δ H 4.01(H-2′) and δ C 41.6 (C-10) and 77.0 (C-3′) are related, from which it can be inferred that C2′ -C 3′ -C 4′ -C 5′ Fragment. 1 H- 1 H COSY spectrum (such as Fig.23 ), δ H 3.52(H-2) and δ H 2.23(H-10),δ H 3.06(H-3) related, δ H 3.67(H-13) and δ H 3.11(H-12), related, δ H 4.68(H-3′) and δ H 4.01(H-2′),δ H 4.32(H-4′) related, δ H 4.32(H-4′) and δ H 4.24(H-5′),δ H 4.68(H-3′) related.

[0121] In the NOESY spectrum (such as Fig.24 ) in the hydrogen configuration, H-2′ is found to be related to H-10α, so it is speculated that H-2′ is α-configuration; similarly, H-10 is related to H-2β, so it is speculated that H-2 may be β-configuration. The structure and related signals are shown below Figures 5 to 8 Based on the above data, the structure of the compound is determined as Figure 5 In addition, a small amount of sample was dissolved in methanol and crystal cultured at room temperature, and then the structure of the crystal was studied by X-ray single crystal diffractometer (molybdenum target). The unit cell diagram is shown in Figure 6 , get the parameters: determined to be monoclinic, α=90.00°, β=108.898(2)°, γ=90.00°, T=273(2)K,Z=4,Molecular formula is C 19 H 25 O 6 Cl. Finally, the structure of the compound was further determined. Through the SciFinder database and literature search, it was finally found that compound 2 was a new compound, named wallichiana B (2,3-dihydro-2-[(β-D-xylosyl)methyl]-5,7-dimethyl-6-(2-chloroethyl)-2(S)-1H-inden-1-one). This compound HMBC, 1 H- 1 H COSY and NOESY spectrum related signals are shown in Figures 7 and 8 , 1 H-NMR,13 The C-NMR spectrum data are shown in Table 3.

[0122] Table 3 Compound 2 1 H-NMR, 13 C-NMR data (δ in ppm)

[0123]

[0124]

[0125] The sample was dissolved in deuterated pyridine solvent, and the H NMR and C NMR data were measured at 400 MHz and 100 MHz, respectively.

[0126] (II) Anti-tumor activity screening

[0127] After consulting the literature, it was found that the plants of this genus have good anti-tumor activity [7-8] Therefore, the isolated compounds 1 and 2 were screened for their anti-tumor activity.

[0128] In this experiment, the CCK-8 method was used to screen Hela cervical cancer cells, HepG2 liver cancer cells, A549 lung cancer cells, MCF-7 breast cancer cells and HCT116 colon cancer cells. Sorafenib was used as a positive control in each experiment.

[0129] 1. Experimental Principle

[0130] The CCK-8 method is a rapid and highly sensitive detection kit based on WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt) that is widely used in cell proliferation and cytotoxicity. The principle is that under the action of the electron coupling reagent 1-Methoxy PMS (1-methoxy-5-methylphenazine dimethyl sulfate), the WST-8 reagent can be reduced by the dehydrogenase in the mitochondria of living cells to a highly water-soluble orange-yellow formazan product.

[0131] 2. Experimental Methods

[0132] The cells in the logarithmic growth phase were digested with trypsin and prepared at a density of 3×10 3 cells / mL single cell suspension was inoculated in a 96-well plate (100 μL / well), and 100 μL PBS buffer was added to the outer cell culture wells to reduce the influence of edge effects on the experimental results. 2Incubate at 37°C in an incubator for 24 hours, and after the cells adhere to the wall, dilute the sample to be tested to different concentrations with culture medium, add 100 μL of culture medium containing the tested part and compound, set up blank (culture medium without cells) and negative control (culture medium with only DMSO). After 72 hours of culture, discard the supernatant, add 100 μL of CCK-8 working solution of 10% CCK-8, and culture in the incubator for 2 hours, and measure its absorbance value (OD). Set up 3 replicates for each treatment.

[0133] 3. Experimental results and discussion

[0134] The CCK-8 method was used to screen the anti-tumor activity of compounds 1 and 2. The experimental data showed that compounds 1 and 2 had inhibitory activity against MCF-7 breast cancer cells, and their IC 50 The values ​​were 0.425±0.071μM and 0.567±0.333μM, respectively; Compound 1 had inhibitory activity against HCT-116 colon cancer cells, and its IC 50 The value is 3.001±0.629μM; Compound 2 has inhibitory activity against Hela cervical cancer cells, and its IC 50 The value was 8.246±0.175μM; Compound 2 had inhibitory activity against lung cancer A549 cells, and its IC 50 The value was 0.278±0.088μM; the inhibitory effects of compounds 1 and 2 on MCF-7 were comparable to those of the positive control sorafenib, and the inhibitory effect of compound 2 on A549 was comparable to that of the positive control sorafenib. 50 The values ​​are in the same order of magnitude, which is worth further study. The specific experimental data are shown in Table 5. Figure 25 to Figure 28 .

[0135] Table 5 In vitro cytotoxic activity study of compounds against four tumor cells

[0136]

[0137] (III) Results

[0138] Chemical components of methanol extracts of Pteris chinensis at room temperature and its application 1 H-NMR, 13 C-NMR, 2D-NMR and other related spectral data were analyzed and identified, and two compounds were isolated from Pteris wallichiana, namely wallichiana A (1) and wallichiana B (2). The two compounds were screened for antitumor activity, and compounds 1 and 2 had inhibitory activity against MCF-7 breast cancer cells, and their IC 50 The values ​​were 0.425±0.071μM and 0.567±0.333μM, respectively; Compound 1 had inhibitory activity against HCT-116 colon cancer cells, and its IC50 The value is 3.001±0.629μM; Compound 2 has inhibitory activity against Hela cervical cancer cells, and its IC 50 The value was 8.246±0.175μM; Compound 2 had inhibitory activity against lung cancer A549 cells, and its IC 50 The value was 0.278±0.088μM; In summary, the inhibitory effects of compounds 1-2 on SW480 were comparable to those of the positive control sorafenib, and the inhibitory effect of compound 2 on A549 was comparable to that of the positive control drug. 50 The values ​​are in the same order of magnitude and are worthy of further study.

[0139] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0140] References:

[0141] [1] Flora of China Editorial Committee, Chinese Academy of Sciences. Flora of China [M]. Volume 3, Part 1. Beijing: Science Press, 1990: 10.

[0142] [2] Zeng Xianfeng, Qiu Heyuan. New data on the fern flora of South China[J]. Fujian Forestry Science and Technology, 2015, 42(02): 166-168.

[0143] [3] China National Medicinal Materials Corporation. A Summary of Chinese Medicinal Resources[M]. Science Press: 1994.

[0144] [4] Wang Ruotong. Study on the chemical components of Pteris dasyphylla and Russula edulis[D]. Kunming University of Science and Technology, 2017.

[0145] [5]Tao JH, Huang ZH, Wang YD, et al. Ethanolic extract from Pteriswallichiana alleviates DSS-induced Intestinal Inflammation and intestinal barrier dysfunction by inhibiting the TLR4 / NF-κB pathway and regulating tightjunction proteins [J]. Molecules, 2022, 27(10): 3093-3093.

[0147] [6] Mao Xiaoxian, He Kang, et al. Study on the chemical components of Pteris integrifolia[J]. Guangxi Botany, 2021, 41(07): 1046-1053.

[0149] [7] Wang Jingbin, Liang Nianci, and Mo Lier. Effects of diterpenoid compound 5F from Pteris semipinnata on MAPK activity and expression in K562 cells[J]. Chinese Pharmacological Bulletin, 2002(3):294-297.

[0151] [8] Wang Jiana, Zhang Yan, Song Liyan, et al. Study on the chemical constituents of Pteris spinulosa and its in vitro antitumor activity[J]. China Journal of Traditional Chinese Medicine, 2017, 42(21): 4159-4164.

Claims

1. A new compound from Pteris fasciata, characterized in that: The new compounds are: bisabolol-10-ene-9,15-lactone-14-oic acid (wallichiana A), 2,3-dihydro-2-[(β-D-xylosyl)methyl]-5,7-dimethyl-6-(2-chloroethyl)-2(S)-1H-inden-1-one (wallichiana B), and their structural formulas are as follows: (1) Structural formula of wallichiana A: (2) Structural formula of wallichiana B:

2. The method for extracting, separating and purifying the new compound in Pteris fasciata as claimed in claim 1, characterized in that: The steps include: 1) Extraction and crude separation of medicinal materials Southwestern pteris fern medicinal material is naturally dried in the shade, crushed into coarse powder, and passed through a 80-100 mesh sieve; the sieved coarse powder is first extracted twice by soaking in 95% industrial methanol at room temperature with 2 times / times, then extracted twice by soaking in 85% methanol at room temperature with 2 times / times, and finally extracted once by soaking in 70% industrial methanol at room temperature, the extract is concentrated under reduced pressure at 55°C to an extract with a density of 1.06, and then diluted with 1.25 times warm water, and extracted with petroleum ether, ethyl acetate, and n-butanol in sequence by liquid-liquid extraction method, with the ratio of organic phase to aqueous phase being 1:1, and after the extraction is completed, the extracts are combined and concentrated under reduced pressure at 55°C to an ethyl acetate extract with a density of 1.06; 2) Separation and purification of ethyl acetate fraction The ethyl acetate fraction was dissolved with a small amount of methanol, and the polyamide sample was taken for gradient elution using an MCI column with a methanol-water system of 1:1, 7:3, and 9:1, and finally eluted with pure methanol to obtain 4 fractions A to D; Separation of sample of part B: add appropriate amount of methanol to dissolve the sample of part B, take 100-200 mesh silica gel to mix the sample, and separate it by silica gel column chromatography; first elute with dichloromethane, then gradient elute with dichloromethane-methanol system of 50:1, 25:1, 10:1, 7:1, 5:1, 2:1, and finally elute with methanol; after TLC detection, combine similar fractions to obtain six parts from B-1 to B-6, and then use silica gel column chromatography to elute part B-1 with petroleum ether-ethyl acetate system of 50:1, 30:1 to obtain four parts from B-1-1 to B-1-4. Crystals are found to precipitate in B-1-2, and compound wallichiana A is obtained after repeated recrystallization; Separation of part B-4 sample: Add appropriate amount of methanol to part B-4 sample to dissolve it, weigh 100-200 mesh silica gel to mix the sample, and separate it by silica gel column chromatography; first elute with dichloromethane, then gradient elute with a dichloromethane-methanol system of 50:1, 30:1, 10:1, 5:1, 1:1, and finally elute with methanol. After TLC detection, combine similar fractions to obtain 4 parts, namely B-4-1 to B-4-4; repeatedly subject part B-4-1 to silica gel column chromatography to obtain compound wallichiana B.

3. The extraction, separation and purification method according to claim 2, characterized in that: In step 1), the sieved coarse powder is first extracted twice with 2 times / times 95% industrial methanol at room temperature, then extracted twice with 2 times 85% methanol at room temperature, and finally extracted once with 70% industrial methanol at room temperature.

4. The extraction, separation and purification method according to any one of claims 2 or 3, characterized in that: The time for the leaching and extraction in step 1) is: each leaching and extraction lasts 4 to 6 days.

5. The extraction, separation and purification method according to claim 4, characterized in that: The time for leaching and extraction in step 1) is: 5 days for each leaching and extraction.

6. Use of the new compound from Pteris crenata as claimed in claim 1 or the compounds wallichiana A and wallichiana B obtained by the extraction, separation and purification method as claimed in claim 2 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The compounds wallichiana A and wallichiana B are used in preparing medicines for treating human breast cancer, human colon cancer, human cervical cancer and human lung cancer.

8. The use according to any one of claims 6 or 7, characterized in that: The drug is prepared by adding compounds wallichiana A and wallichiana B to pharmaceutically acceptable excipients to form a pharmaceutically acceptable solid preparation or liquid preparation.

9. The use according to claim 8, characterized in that: The solid preparations are granules, capsules, tablets, pills, powders, and freeze-dried powder injections.

10. The use according to claim 8, characterized in that: The liquid preparation is an injection preparation or an oral solution.

Images