A new compound in southwest pteridium aquilinum, extraction, separation and purification method and application

Abstract

The application discloses a new compound in Pteris wallichiana and an extraction, separation and purification method and application of the new compound. The compound is wallichiana A, 2,3-dihydro-2-[(beta-D-xylosyl) methyl]-5,7-dimethyl-6-(2-chloroethyl)-2(S)-1H-inden-1-one (wallichiana B), which is obtained through extraction and crude separation of medicinal materials, separation and purification of an ethyl acetate part. The compounds obtained through the separation and purification are subjected to anti-tumor activity screening experiments, and the results show that the two compounds have half-inhibition activity on in-vitro tumor growth of breast cancer MCF-7, colon cancer HCT-116, cervical cancer Hela and lung cancer A549, and can be used for treating anti-tumor diseases, and provide a reference for continuing to strengthen the chemical component and activity screening research of the plant and the research on structure modification of the compounds with good activity in the plant.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical science, and in particular to a novel compound from the Southwest Fern, its extraction, separation, purification method, and application. Background Technology

[0002] The southwestern maidenhair fern (Pteris wallichiana Agardh) grows at altitudes of 800–2000 m and reaches a height of about 1.5 meters. [1] Currently, it is mainly distributed in Yunnan, Guizhou, Guangxi and other places. [2] It is mainly used to treat dysentery, infantile convulsions, and other ailments. [3] Currently, research on *Pteris vittata* is limited. Most studies focus on its chemical composition, primarily identifying terpenoids and flavonoids. Pharmacological activity studies are scarce. Some researchers isolated sesquiterpenes from *Pteris vittata* and tested them for antitumor activity, but no antitumor activity was found. Other researchers, through studies on the ethanol extract of *Pteris vittata*, found that it has an inhibitory effect on ulcerative colitis. [4-5] In addition, our research group has previously conducted in-depth studies on the chemical constituents of other plants in this genus, such as *Pteris vittata*, *Pteris multifida*, and *Pteris integrifolia*, isolating a number of bioactive monomeric compounds, especially enantio-kaurane 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 basis of *Pteris vittata*, our research group has carried out research on the chemical constituents of *Pteris vittata*. Through preliminary experimental studies on its chemical constituents, we found that this plant may contain glycosides, flavonoids, coumarins, and phenols.

[0003] Our research group conducted a chemical composition study on the methanol extract of *Pteris vittata*, isolating several compounds, including two new compounds: *wallichiana* A(1) and *wallichiana* B(2), both of which were isolated from this plant for the first time. Screening experiments on the antitumor activity of these new compounds showed that they exhibited half-maximal inhibitory activity against the in vitro tumor growth of breast cancer MCF-7, colon cancer HCT-116, cervical cancer HeLa, and lung cancer A549. These compounds could be used to treat antitumor diseases and provide a reference for further research on the chemical composition and activity screening of this plant, as well as for structural modification of compounds with good activity from this plant. Summary of the Invention

[0004] The purpose of this invention is to provide a new compound, wallichiana A, and wallichiana B, from the Southwest Fern.

[0005] Another objective of this invention is to provide a method for the extraction, separation, and purification of a new compound from the Southwest Fern.

[0006] Another object of the present invention is to provide the use of a novel compound from the Southwest Fern in antibacterial and / or anti-inflammatory applications.

[0007] To achieve the objectives of this invention, the following technical solutions and steps are employed:

[0008] The novel compounds from *Pteris vittata* described in this invention are: bisabolol-10-ene-9,15-lactone-14-acid (wallichiana A) and 2,3-dihydro-2-[(β-D-xylosyl)methyl]-5,7-dimethyl-6-(2-chloroethyl)-2(S)-1H-inden-1-one (wallichiana B), with the following structural formulas:

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

[0010]

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

[0012]

[0013] The method for extraction, separation, and purification of novel compounds from *Pteris vittata* of the present invention comprises the following steps:

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

[0015] The medicinal material of Southwest China's Pteris vittata was naturally air-dried, pulverized into coarse powder, and passed through an 80-100 mesh sieve. The sieved coarse powder was first extracted twice with 2 times the amount of 95% industrial methanol at room temperature, then twice with 2 times the amount of 85% methanol at room temperature, and finally once with 70% industrial methanol at room temperature. The extract was concentrated under reduced pressure at 55°C to a paste with a density of 1.06, and then diluted with 1.25 times the amount of warm water. Liquid-liquid extraction was then performed sequentially with petroleum ether, ethyl acetate, and n-butanol, with an organic phase to aqueous phase ratio of 1:1. After extraction, the extracts were combined and concentrated under reduced pressure at 55°C to a paste of ethyl acetate with a density of 1.06.

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

[0017] The ethyl acetate fraction was dissolved in a small amount of methanol, and the polyamide sample was mixed. MCI column chromatography was performed with gradient elution using methanol-water elution systems of 1:1, 7:3, and 9:1. Finally, pure methanol was used as the elution solution to obtain four fractions, A to D.

[0018] Separation of Part B Sample: Part B sample was dissolved in an appropriate amount of methanol, mixed with 100-200 mesh silica gel, and separated by silica gel column chromatography. First, dichloromethane was used as the eluting agent, followed by gradient elution with dichloromethane-methanol systems at ratios of 50:1, 25:1, 10:1, 7:1, 5:1, and 2:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain six fractions, B-1 to B-6. Fraction B-1 was then subjected to silica gel column chromatography, eluted with petroleum ether-ethyl acetate systems at ratios of 50:1 and 30:1, yielding four fractions, B-1-1 to B-1-4. Crystals were observed to precipitate from B-1-2. After repeated recrystallization, compound wallichiana A was obtained.

[0019] Separation of sample B-4: For sample B-4, an appropriate amount of methanol was added to dissolve it, and 100-200 mesh silica gel was weighed and mixed in. Separation was performed using silica gel column chromatography. First, dichloromethane was used as elution, followed by gradient elution with dichloromethane-methanol systems of 50:1, 30:1, 10:1, 5:1, and 1:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain four fractions, B-4-1 to B-4-4. Fraction B-4-1 was subjected to repeated 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 with 2 times of 95% industrial methanol at room temperature, then extracted twice with 2 times of 85% methanol at room temperature, and finally extracted once with 70% industrial methanol at room temperature.

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

[0022] In a further preferred embodiment, the extraction, separation, and purification method of the present invention, in step 1), involves an extraction time of 5 days per extraction.

[0023] The application of the novel compounds from the Southwest Fern or compounds wallichiana A and wallichiana B obtained by the extraction, separation and purification methods described in this invention in the preparation of antitumor drugs.

[0024] The application of the compounds wallichiana A and wallichiana B described in this invention in the preparation of drugs for treating human breast cancer, human colon cancer, human cervical cancer, and human lung cancer.

[0025] The drug described in this invention is prepared by adding compounds wallichiana A and wallichiana B to pharmaceutically acceptable excipients to form a pharmaceutically acceptable solid or liquid formulation.

[0026] The solid preparations described in this invention are granules, capsules, tablets, pills, powders, and lyophilized powder injections; the liquid preparations are injectable preparations and oral liquids.

[0027] Beneficial effects of this invention:

[0028] 1. This invention extracts, isolates, and purifies two new compounds from the Southwest Fern, namely wallichiana A and wallichiana B, both of which are isolated from this plant for the first time.

[0029] 2. This invention extracted, isolated, and purified two new compounds from *Pteris vittata*, and screened them for antitumor activity. The results showed that the compounds wallichiana A and wallichiana B had half-maximal inhibitory activity against the in vitro tumor growth of breast cancer MCF-7, colon cancer HCT-116, cervical cancer HeLa, and lung cancer A549. They can be used to treat antitumor diseases and have potential medicinal value. Further research can be conducted to strengthen the screening of chemical components and activities of this plant and to modify the structure of compounds with good activity in this plant.

[0030] 3. This invention investigated the inhibitory activity of all compounds against MCF-7 breast cancer cells, and the IC50 values ​​of compounds wallichiana A and wallichiana B. 50 The values ​​were 0.425±0.071 and 0.567±0.333, respectively.

[0031] 4. This invention investigated the inhibitory activity of all compounds against colon cancer cells (HCT-116), and the IC50 of compound wallichiana A was measured. 50 It is 3.001±0.629.

[0032] 5. This invention investigated the inhibitory activity of all compounds against cervical cancer cells (HeLa), and the IC50 of wallichiana B. 50 It is 8.246±0.175.

[0033] 6. This invention investigated the inhibitory activity of all compounds against human lung cancer cells (A549), and the IC50 of wallichiana B. 50 It is 0.278±0.088. Attached Figure Description

[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 The 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] Figure 9 HR-ESI-MS spectrum of compound 1;

[0043] Figure 10 Compound 1 1 H-NMR spectrum;

[0044] Figure 11 Compound 1 13 C-NMR spectrum;

[0045] Figure 12 DEPT spectrum of compound 1;

[0046] Figure 13 : HSQC spectrum of compound 1;

[0047] Figure 14 HMBC spectrum of compound 1;

[0048] Figure 15 Compound 1 1 H- 1 H COSY spectrum;

[0049] Figure 16 NOESY spectrum of compound 1.

[0050] Figure 17 HR-ESI-MS spectrum of compound 2;

[0051] Figure 18 Compound 21 H-NMR spectrum;

[0052] Figure 19 Compound 2 13 C-NMR spectrum;

[0053] Figure 20 DEPT spectrum of compound 2;

[0054] Figure 21 : HSQC spectrum of compound 1;

[0055] Figure 22 HMBC spectrum of compound 2;

[0056] Figure 23 Compound 2 1 H- 1 H COSY spectrum;

[0057] Figure 24 NOESY spectrum of compound 2.

[0058] Figure 25 IC50 of the test compound against human breast cancer cells (MCF-7) 50 Value Visualization

[0059] Figure 26 IC50 of the test compound against human colon cancer cells (HCT-116) 50 Value Visualization

[0060] Figure 27 The IC50 of the test compound against human cervical cancer cells (HeLa) 50 Value Visualization

[0061] Figure 28 IC50 of the test compound against human lung cancer cells (A549) 50 Value Visualization Detailed Implementation

[0062] The present invention will be described in detail below with reference to 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 way. For those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0063] Example 1: Extraction, separation and purification of compounds from *Pteris vittata*.

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

[0065] The medicinal material of Southwest China's Pteris vittata was naturally air-dried, pulverized into coarse powder, and passed through an 80-100 mesh sieve. The sieved coarse powder was first extracted twice with 2 times the amount of 95% industrial methanol at room temperature, then twice with 2 times the amount of 80% methanol at room temperature, and finally once with 70% industrial methanol at room temperature. The extract was concentrated under reduced pressure at 55°C to a paste with a density of 1.06, and then diluted with 1.25 times the amount of warm water. Liquid-liquid extraction was then performed sequentially with petroleum ether, ethyl acetate, and n-butanol, with an organic phase to aqueous phase ratio of 1:1. After extraction, the extracts were combined and concentrated under reduced pressure at 55°C to a paste of ethyl acetate with a density of 1.06.

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

[0067] The ethyl acetate fraction was dissolved in a small amount of methanol, and the polyamide sample was mixed. MCI column chromatography was performed with gradient elution using methanol-water elution systems of 1:1, 7:3, and 9:1. Finally, pure methanol was used as the elution solution to obtain four fractions, A to D.

[0068] Separation of Part B Sample: Part B sample was dissolved in an appropriate amount of methanol, mixed with 100-200 mesh silica gel, and separated by silica gel column chromatography. First, dichloromethane was used as the eluting agent, followed by gradient elution with dichloromethane-methanol systems at ratios of 50:1, 25:1, 10:1, 7:1, 5:1, and 2:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain six fractions, B-1 to B-6. Fraction B-1 was then subjected to silica gel column chromatography, eluted with petroleum ether-ethyl acetate systems at ratios of 50:1 and 30:1, yielding four fractions, B-1-1 to B-1-4. Crystals were observed to precipitate from B-1-2. Repeated recrystallization yielded compound 1 (wallichiana A).

[0069] Separation of sample B-4: Sample B-4 was dissolved in an appropriate amount of methanol, mixed with 100-200 mesh silica gel, and separated using silica gel column chromatography. First, dichloromethane was used as the eluting agent, followed by gradient elution with dichloromethane-methanol systems at ratios of 50:1, 30:1, 10:1, 5:1, and 1:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain four fractions: B-4-1 to B-4-4. Fraction B-4-1 was repeatedly subjected to silica gel column chromatography to obtain compound 2 (wallichiana B).

[0070] Example 2: Structural formula of the 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), fluorescent under 254 nm UV light, using a dichloromethane / methanol system of 15:1, colorimetric reagent: 10% H₂SO₄-EtOH solution, turns purple-red upon heating (its R... f Value 0.28); Mass spectrometry (HR-ESI-MS): [M+Na] + m / z 289.14026 (calcd for C) 15 H 22 O4Na, 289.14103); Infrared spectrum IR (KBr): V max 1749.5cm -1 1704.0cm -1 cm -1 ; 1 H-NMR (400MHz, CDCl3), 13 C-NMR (100MHz, CDCl3)δ C (ppm), see Table 2.

[0081] Crystal parameters: C 15 H 22 O4, 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) fluoresce under 254 nm UV light; a 5:1 dichloromethane / methanol system and a 10% H₂SO₄-EtOH solution show an orange-red color upon heating (R₂O₃). fValue 0.59); Mass spectrometry (HR-ESI-MS): [M+Na] + m / z 407.12295 (calcd for C) 15 H 22 O4Na, 407.123187); Infrared spectrum IR (KBr): V max 1665.1cm -1 cm -1 ; 1 H-NMR (400MHz, C5D5N), 13 C-NMR (100MHz, C5D5N)δ C (ppm), see Table 2.

[0084] Crystal parameters: C 19 H 26 ClO 6.5 Monoclinic crystal 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 the compounds, wallichiana A and wallichiana B, as the active pharmaceutical ingredient, add 20% microcrystalline cellulose and 20% sugar powder to granulate, and obtain granules.

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

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

[0088] Example 10: Take any one of wallichiana A and wallichiana B as the active pharmaceutical ingredient, add 10% starch paste and 0.3% magnesium stearate, granulate, compress into tablets.

[0089] Example 11: Take any one of the compounds, wallichiana A or wallichiana B, as the active pharmaceutical ingredient, add 12 times the amount of water for injection, filter, sterilize, and obtain the injection.

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

[0091] Example 13: Using either wallichiana A or wallichiana B as the active pharmaceutical ingredient, add 12 times the volume of purified water and 0.05% sodium benzoate, mix thoroughly, filter, and sterilize to obtain an oral liquid. To verify the effectiveness of this invention, the inventive team conducted a series of experiments, as follows:

[0092] (I) Investigation of the separation and identification of compounds

[0093] 1. Instruments, reagents, and materials

[0094] 1.1 Instruments and Reagents

[0095] See Table 1.

[0096] Table 1. Names, Models, and Sources of Instruments and Reagents

[0097]

[0098] The experiment involved three colorimetric reagents: 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 material was collected from Datianba Township, Changning County, Baoshan City, Yunnan Province. After identification by Professor Zhao Junhua of Guizhou University of Traditional Chinese Medicine, the plant was confirmed to be Pteris wallichiana Agardh, a plant belonging to the genus Pteris L. of the family Pteridaceae. The plant certificate specimen is stored in the University Pharmacy 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 the 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 Pronosei Biotechnology Co., Ltd.; 0.25% trypsin cell digestion solution (batch number: C0207) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and sorafenib (batch number: S828599) was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0102] 2. Extraction and Separation

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

[0104] 46.7 kg of *Pteris vittata* (a type of fern) was naturally air-dried and then pulverized into coarse powder (80-100 mesh). The powder was then soaked twice in 95% methanol at room temperature, followed by two more extractions in twice more 80% methanol at room temperature, and finally one more extraction in 70% industrial methanol at room temperature. Each extraction lasted 5 days. The five extraction solutions were combined and recovered under reduced pressure to obtain approximately 10.3 kg of thick extract. The total extract was dissolved in warm water (aqueous phase) and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol using a liquid-liquid extraction method (organic phase to aqueous phase ratio of 1:1). After extraction, the solvents were recovered under reduced pressure, yielding 304 g of the ethyl acetate fraction.

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

[0106] The ethyl acetate fraction was dissolved in a small amount of methanol, and 300g of polyamide was weighed and mixed. MCI column chromatography was performed with gradient elution using methanol-water elution system (1:1, 7:3, 9:1). Finally, pure methanol was used to elute, resulting in four fractions, A to D.

[0107] Separation of Part B Sample: Part B sample was dissolved in an appropriate amount of methanol, mixed with 100-200 mesh silica gel, and separated by silica gel column chromatography. First, dichloromethane was used as the eluting agent, followed by gradient elution with dichloromethane-methanol systems at ratios of 50:1, 25:1, 10:1, 7:1, 5:1, and 2:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain six fractions, B-1 to B-6. Fraction B-1 was then subjected to silica gel column chromatography, eluted with petroleum ether-ethyl acetate systems at ratios of 50:1 and 30:1, yielding four fractions, B-1-1 to B-1-4. Crystals were observed to precipitate from B-1-2. Repeated recrystallization yielded compound 1 (wallichiana A).

[0108] Separation of sample B-4: Sample B-4 was dissolved in an appropriate amount of methanol, mixed with 100-200 mesh silica gel, and separated using silica gel column chromatography. First, dichloromethane was used as the eluting agent, followed by gradient elution with dichloromethane-methanol systems at ratios of 50:1, 30:1, 10:1, 5:1, and 1:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain four fractions: B-4-1 to B-4-4. Fraction B-4-1 was repeatedly subjected to silica gel column chromatography to obtain compound 2 (wallichiana B).

[0109] 3. Structural identification of two chemical components in *Pteris vittata*.

[0110] Compound 1: White crystals (chloroform), fluorescent under 254 nm UV light, using a dichloromethane / methanol system of 15:1, with 10% H₂SO₄-EtOH solution as the colorimetric reagent, turns purple-red upon heating (its R...f The value is 0.28.

[0111] IR display of CO single bond (1179.3cm) -1 ), carbonyl (1749.5cm) -1 1704.0cm -1 ).according to 1 Key characteristic spectral data from H-NMR (400MHz, CDCl3): δ H 1.77 (3H, s) and 1.73 (3H, s) are presumed to be methyl signals (see...). Figure 10 ); 13 C-NMR (100MHz, CDCl3) (see...) Figure 11 Spectral data and DEPT spectra (see) Figure 12 ) shows two methyl δ C 25.9 and 18.6, five methylene δ C 32.4, 30.1, 28.6, 28.5, 27.8, five methine δ C 123.0, 75.3, 46.2, 42.8, 36.7, three quaternary carbon δ values C 181.3, 177.9, 140.2; where δ C The values ​​of 181.3 and 177.9 suggest the possible presence of two carbonyl carbon signals, δ C 140.2, 123.0 is a double bond signal, δ C 75.3 represents a carbon-ionized oxygen signal. Based on mass spectrometry (HR-ESI-MS) data, [M+Na] + m / z 289.14026 (calcd for C) 15 H 22 O4Na, 289.14103), the molecular formula of this compound was determined to be C 15 H 22 O4, with an unsaturation degree of 5 (see...) Figure 9 ).

[0112] In HMBC spectrum (such as Figure 14 As shown in the figure, δ H 1.73(H-8) and δ C 123.9 (C-5), 140.2 (C-6), 25.9 (C-7) correlation, δ H 1.77(H-7) and δ C 123.9 (C-5), 140.2 (C-6), 18.6 (C-8) correlation, δ H 5.19(H-5) correlates with 18.6(C-8), 25.9(C-7), and 75.3(C-4), δH 5.01(H-4) and δ C 123.9 (C-5), 140.2 (C-6) correlation, δ H 2.56(H-2) and δ C The correlations are 177.8 (C-1), 32.4 (C-3), 36.7 (C-1′), 30.1 (C-2′), and 27.8 (C-6′), with 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) are relevant.

[0113] exist 1 H- 1 H-COSY spectrum (e.g.) Figure 15 ), δ H 5.19 (1H, d, J = 8.5 Hz, H⁻⁵) is correlated with 5.01 (1H, m, H⁻⁴), δ H 5.01(1H, m, H-4) and δ H Correlation between 5.19 (1H, d, J = 8.5 Hz, H⁻⁵), 2.28 (1H, m, H⁻³), and 1.70 (1H, m, H⁻³) leads to the derivation of the C3-C4-C5-C7 segment; δ H 2.28 (1H, m, H-3) and δ H 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 NOESY spectra (such as) Figure 16 The hydrogen configurations in the sample show a correlation between H-2 and H-4α, suggesting that H-2 is α-configured; similarly, H-1′ is correlated with H-2, suggesting that H-1′ may be α-configured; H-4′ shows correlations with H-3′β and H-2′β, suggesting that H-4′ may be β-configured. See the structural formula below. 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 crystallized at room temperature. The structure of the crystal was then studied using X-ray single-crystal diffraction (molybdenum target), and the parameters were determined: it was identified as a monoclinic crystal system. α=90.00°, β=90.00°, γ=90.00°, T = 273(2)K, Z = 4, molecular formula C 15 H 22 O4, etc. See the unit cell diagram. Figure 2 Further evidence was provided regarding the structure of this compound. A search of the SciFinder database and a review of relevant literature confirmed that compound 1 was a new compound, named wallichiana A (bisabolol-10-ene-9,15-lactone-14-acid). This compound HMBC, 1 H- 1 The related signals of H COSY and NOESY spectra are shown in […]. Figures 3 to 4 , 1 H-NMR, 13 The C-NMR spectral data are shown in Table 2.

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

[0116]

[0117] The sample was dissolved in deuterated chloroform solvent, and the proton and carbon NMR spectra were measured at 400 M and 100 M, respectively.

[0118] Compound 2: White crystals (pyridine), fluorescent under 254 nm UV light; turns orange-red upon heating in a 5:1 dichloromethane / methanol system and a 10% H₂SO₄-EtOH solution (R₂O₃). f (Value 0.59).

[0119] IR spectroscopy revealed the presence of carbonyl groups (1665.1 cm⁻¹). -1 ).according to 1 Key characteristic spectral data from H-NMR (400MHz, C5D5N): δ H 6.97 (1H, s) is presumed to be a hydrogen signal on the benzene ring, δ H 2.70 (3H, s), δ H 2.29 (3H, s) represents the hydrogen signal δ on the two methyl groups. H 4.01-4.67 (5H, m) is speculated to be a hydrogen signal on the sugar (see...) Figure 18 );according to 13 C-NMR (100MHz, C5D5N) spectrum (see...) Figure 19 ) and DEPT spectrum (see Figure 20 Data analysis confirms that compound 2 has 19 carbon signals, including two methyl δ-carbons. C 21.4 and 14.1; five methylene groups (δ C 64.5, 43.4, 41.6, 34.7, 32.7); five methines (δC Seven quaternary carbons (δ) of 126.7, 77.8, 77.0, 72.5, and 44.9 C 211.4, 154.8, 144.9, 138.0, 135.3, 133.1, 99.6); where δ C 211.4 suggests the possible presence of a carbonyl carbon signal, δ C 154.2, 144.7, 137.2, 134.9, 132.0, and 126.7 represent carbon signals on a benzene ring, δ C 99.3, 75.5, 74.8, 70.7, and 62.8 represent a sugar signal, δ C 21.1 and 13.6 indicate two methyl signals. Based on mass spectrometry (HR-ESI-MS) data (see...), Figure 17 ), [M+Na] + m / z 407.12295 (calcd for C) 19 H 25 O6ClNa, 407.123187), the molecular formula of this compound was determined to be C 19 H 25 O6Cl, with an unsaturation degree of 8; the above data is consistent with the literature. [6] Similar to the compound pterosin F, it is preliminarily inferred that this compound is a sesquiterpene compound.

[0120] HMBC display (e.g.) Figure 22 ), δ H 6.97(H-4) and δ C 134.9 (C-6), 21.4 (C-11), 34.7 (C-3) correlation, δ H 2.29(H-11) and δ C The correlations of 144.9 (C-5), 135.3 (C-6), and 126.7 (C-4) suggest that C4-C5-C 11 Fragment; δ H 3.67(H-13) and δ C 31.9 (C-12), 135.3 (C-6) correlation, δ H 3.11(H-12) is correlated with 144.8(C-5), 135.3(C-6), 43.4(C-13), and 137.2(C-7), from which we can infer C6-C 12 -C 13 Fragment; δ H 2.70(H-14) and δ C Correlation between 138.0 (C-7), 135.3 (C-6), and 154.8 (C-8), δ H3.41(H-3) and δ C Correlation exists at 44.3 (C-2), 133.1 (C-9), and 154.8 (C-8), from which the C7-C8-C9-C3-C2 segment can be deduced; δ H 2.23(H-10) and δ C The correlations with 99.6 (C-1′), 34.7 (C-3), and 44.3 (C-2) suggest that sugars are related to C. 10 Position connected; δ H 4.56(H-5′) and δ C Correlation between 99.6 (C-1′), 72.5 (C-4′), and 77.0 (C-3′), δ H 4.01(H-2′) and δ C The correlation between 41.6 (C-10) and 77.0 (C-3′) suggests that C... 2′ -C 3′ -C 4′ -C 5′ Fragment. In 1 H- 1 H COSY spectrum (e.g.) Figure 23 In ), δ H 3.52(H-2) and δ H 2.23(H-10), δ H 3.06(H-3) correlation, 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′) correlation, δ H 4.32(H-4′) and δ H 4.24(H-5′), δ H 4.68(H-3′) related.

[0121] In NOESY spectra (such as) Figure 24 The hydrogen configurations of H-2′ and H-10α were found to be correlated, so it is inferred that H-2′ is α-configuration; similarly, H-10 is correlated with H-2β, so it is inferred 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 was determined as follows: Figure 5 In addition, a small amount of sample was dissolved in methanol and crystallized at room temperature. The structure of the crystal was then studied using X-ray single-crystal diffraction (molybdenum target). The unit cell diagram is shown below. Figure 6 The parameters obtained indicate that the crystal system is monoclinic. α=90.00°, β=108.898(2)°, γ=90.00°, T = 273(2)K, Z = 4, molecular formula is C 19 H 25 O6Cl. The structure of this compound was further determined. A search of the SciFinder database and literature revealed compound 2 to be 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 The related signals of H COSY and NOESY spectra are shown in […]. Figures 7 to 8 , 1 H-NMR, 13 The C-NMR spectral data are shown in Table 3.

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

[0123]

[0124]

[0125] The sample was dissolved in deuterated pyridine solvent, and the proton and carbon NMR spectra were measured at 400 M and 100 M, respectively.

[0126] (II) Screening for antitumor activity

[0127] Literature review revealed that plants in this genus possess good antitumor activity. [7-8] Therefore, compounds 1 and 2 obtained by isolation were screened for antitumor activity.

[0128] This experiment used the CCK-8 assay 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 assay 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-tetrazole monosodium salt), widely used for 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 dehydrogenases in the mitochondria of living cells to a highly water-soluble orange-yellow formazan product.

[0131] 2. Experimental Methods

[0132] After digesting logarithmic growth phase cells with trypsin, the cells were prepared into a density of 3 × 10⁻⁶. 3 Single-cell suspensions (100 μL / well) were seeded into 96-well plates. 100 μL of PBS buffer was added to the outer cell culture wells to minimize edge effects. Cells were incubated statically at 37°C for 24 h in a 5% CO2 incubator until adherence. The test samples were diluted to different concentrations with culture medium, and 100 μL of culture medium containing the test site and compound was added. Blank (cell-free culture medium) and negative control (culture medium with only DMSO) were prepared. After 72 h of incubation, the supernatant was discarded, and 100 μL of 10% CCK-8 working solution was added. After incubation for 2 h, the absorbance (OD) was measured. Each treatment was performed in triplicate.

[0133] 3. Experimental Results and Discussion

[0134] Compounds 1 and 2 were screened for antitumor activity using the CCK-8 assay. Experimental data showed that compounds 1 and 2 had inhibitory activity against MCF-7 breast cancer cells, with an IC50 value of [missing information]. 50 The values ​​were 0.425±0.071μM and 0.567±0.333μM, respectively; compound 1 showed inhibitory activity against HCT-116 colon cancer cells, with an IC50 value of 0.425±0.071μM and 0.567±0.333μM, respectively. 50 The value was 3.001±0.629 μM; compound 2 had inhibitory activity against HeLa cervical cancer cells, and its IC50 value was 3.001±0.629 μM. 50 The value was 8.246±0.175 μM; compound 2 had inhibitory activity against lung cancer A549 cells, and its IC50 value was 8.246±0.175 μM. 50 The value was 0.278±0.088 μM; the inhibitory effects of compounds 1 and 2 on MCF-7 were comparable to those of the positive control sorafenib, and the inhibitory effects of compound 2 on A549 were comparable to those of the positive control sorafenib. Their IC50 values ​​were... 50 The values ​​are on the same order of magnitude, warranting further investigation. Specific experimental data are shown in Table 5. Figures 25 to 28 .

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

[0136]

[0137] (III) Results

[0138] Chemical composition and application of methanol extract of *Pteris vittata* at room temperature. 1 H-NMR, 13 C-NMR, 2D-NMR and other related spectroscopic data were analyzed and identified, and two compounds were isolated from *Pteris vittata*, namely wallichiana A(1) and wallichiana B(2). The antitumor activity of the two compounds was screened, and compounds 1 and 2 showed inhibitory activity against MCF-7 breast cancer cells, with an IC50 of [missing information]. 50 The values ​​were 0.425±0.071μM and 0.567±0.333μM, respectively; compound 1 showed inhibitory activity against HCT-116 colon cancer cells, with an IC50 value of 0.425±0.071μM and 0.567±0.333μM, respectively. 50 The value was 3.001±0.629 μM; compound 2 had inhibitory activity against HeLa cervical cancer cells, and its IC50 value was 3.001±0.629 μM. 50 The value was 8.246±0.175 μM; compound 2 had inhibitory activity against lung cancer A549 cells, and its IC50 value was 8.246±0.175 μM. 50 The value was 0.278±0.088 μM; In summary, the inhibitory effects of compounds 1 and 2 on SW480 were comparable to those of the positive control sorafenib, and the inhibitory effects of compound 2 on A549 were comparable to those of the positive control. 50 The values ​​are on the same order of magnitude, making them worthy of in-depth research.

[0139] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0140] References:

[0141] [1] Editorial Committee of Flora of China, 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 flora of ferns in South China [J]. Fujian Forestry Science and Technology, 2015, 42(02):166-168.

[0143] [3] China Pharmaceutical Corporation. A Brief Account of Chinese Medicinal Herbs Resources [M]. Science Press: 1994.

[0144] [4] Wang Ruotong. Study on chemical composition of Southwest China fern and orange-yellow red mushroom [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.

[0146] [6] Mao Xiaoxian, He Kang, et al. Study on chemical constituents of Pteris integrifolia [J]. Guangxi Plants, 2021, 41(07): 1046-1053.

[0147] [7] Wang Jingbin, Liang Nianci, Mo Lier. Effects of diterpenoid compound 5F from *Pistacia chinensis* on the activity and expression of MAPK in K562 cells [J]. Chinese Journal of Pharmacology, 2002(3):294-297.

[0148] [8] Wang Jiana, Zhang Yan, Song Liyan, et al. Study on chemical constituents and in vitro antitumor activity of Pteris argentea [J]. Chinese Journal of Traditional Chinese Medicine, 2017, 42(21):4159-4164.

Claims

1. A compound from the Southwest Pteris vittata, characterized in that, The compounds are: bisabolol-10-ene-9,15-lactone-14-acid (wallichiana A) and 2,3-dihydro-2-[(β-D-xylosyl)methyl]-5,7-dimethyl-6-(2-chloroethyl)-2(S)-1H-inden-1-one (wallichiana B), with the following structural formulas: (1) Structural formula of wallichiana A: ， (2) Structural formula of wallichiana B: 。 2. The method for extracting, separating, and purifying the compounds in *Pteris vittata* as described in claim 1, characterized in that, Includes the following steps: 1) Extraction and crude separation of medicinal materials The medicinal material of Southwest China's Pteris multifida was naturally air-dried, pulverized into coarse powder, and passed through an 80-100 mesh sieve. The sieved coarse powder was first extracted twice with 2 times the amount of 95% industrial methanol at room temperature, then twice with 2 times the amount of 85% methanol at room temperature, and finally once with 70% industrial methanol at room temperature. The extract was concentrated under reduced pressure at 55 ℃ to a paste with a density of 1.06, and then diluted with 1.25 times the amount of warm water. Liquid-liquid extraction was then performed sequentially with petroleum ether, ethyl acetate, and n-butanol, with an organic phase to aqueous phase ratio of 1:

1. After extraction, the extracts were combined and concentrated under reduced pressure at 55 ℃ to a paste of ethyl acetate with a density of 1.

06. 2) Separation and purification of the ethyl acetate fraction The ethyl acetate fraction was dissolved in a small amount of methanol, and the polyamide sample was mixed. MCI column chromatography was performed with gradient elution using methanol-water elution systems of 1:1, 7:3, and 9:

1. Finally, pure methanol was used as the elution solution to obtain four fractions, A to D. Separation of Part B Sample: Part B sample was dissolved in an appropriate amount of methanol, mixed with 100-200 mesh silica gel, and separated by silica gel column chromatography. First, dichloromethane was used as the eluting agent, followed by gradient elution with dichloromethane-methanol systems at ratios of 50:1, 25:1, 10:1, 7:1, 5:1, and 2:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain six fractions, B-1 to B-6. Fraction B-1 was then subjected to silica gel column chromatography, eluted with petroleum ether-ethyl acetate systems at ratios of 50:1 and 30:1, yielding four fractions, B-1-1 to B-1-4. Crystals were observed to precipitate from B-1-2. After repeated recrystallization, compound wallichiana A was obtained. Separation of sample B-4: For sample B-4, an appropriate amount of methanol was added to dissolve it, and 100-200 mesh silica gel was weighed and mixed in. Separation was performed using silica gel column chromatography. First, dichloromethane was used as elution, followed by gradient elution with dichloromethane-methanol systems of 50:1, 30:1, 10:1, 5:1, and 1:1, and finally eluted with methanol. After TLC detection, similar fractions were combined to obtain four fractions, B-4-1 to B-4-4. Fraction B-4-1 was subjected to repeated silica gel column chromatography to obtain compound wallichiana B.

3. The extraction, separation, and purification method according to claim 2, characterized in that, The extraction time described in step 1) is 4 to 6 days for each extraction.

4. The extraction, separation, and purification method according to claim 3, characterized in that, The extraction time described in step 1) is 5 days for each extraction.

5. The use of the compound wallichiana A from the Southwest Fern as described in claim 1 or the compound wallichiana A obtained by the extraction, separation and purification method described in claim 2 in the preparation of drugs for treating human breast cancer and human colon cancer.

6. The use of the compound wallichiana B from the Southwest Fern as described in claim 1 or the compound wallichiana B obtained by the extraction, separation and purification method described in claim 2 in the preparation of drugs for treating human breast cancer, human cervical cancer and human lung cancer.

7. The application according to any one of claims 5 or 6, 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 or liquid dosage form.

8. The application according to claim 7, characterized in that, The solid dosage forms include granules, capsules, tablets, pills, powders, and lyophilized powder injections.

9. The application according to claim 7, characterized in that, The liquid preparation is an injectable preparation or an oral liquid.

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