Indole alkaloid compounds in Folium Isatidis and their extraction and separation methods and applications

Abstract

The present invention provides an indole alkaloid compound in Folium Isatidis and an extraction and separation method and application thereof, and belongs to the fields of extraction and separation of traditional Chinese medicine, phytochemistry and pharmaceutical technology. The present invention provides an indole alkaloid compound extracted from Folium Isatidis, and its chemical structural formula is shown in formula (II) or formula (III), wherein formula (II) is named 4-hydroxyindole-3-acetonitrile, and formula (III) is named 3-acetamido-indolesulfonamide; and the present invention also provides a simple and rapid extraction and separation method for the compound. The extraction and separation process of the present invention is simple to operate, easy to control, and suitable for industrial production; the obtained indole alkaloid compound has anti-inflammatory and anti-tumor effects, and has good application prospects in the preparation of anti-inflammatory and therapeutic drugs for gastric cancer, liver cancer and lung cancer.

Description

Technical Field

[0001] The invention relates to the technical fields of traditional Chinese medicine extraction and separation, plant chemistry and medicine, and in particular to indole alkaloid compounds in isatis indigofera and extraction and separation methods and applications thereof. Background Art

[0002] The traditional Chinese medicinal material Isatis indigotica Fort. is the dried leaves of Isatis indigotica Fort., a plant of the genus Isatis in the family Cruciferae. It is cold in nature and bitter in taste, and has the effects of clearing away heat and detoxifying, cooling blood and removing spots. It is widely used for fever, high fever, thirst, coma, macules, vomiting blood, epistaxis, jaundice, diarrhea, erysipelas, throat paralysis, mouth ulcers, mumps and other symptoms, and has always been an important medicine for clearing heat and detoxifying.

[0003] Modern pharmacological studies have shown that Folium Isatidis has antiviral, antibacterial, anti-endotoxin, anti-tumor, antipyretic, and anti-inflammatory effects. Folium Isatidis contains complex and diverse chemical components, mainly alkaloids, organic acids, flavonoids, terpenes, etc. Among them, alkaloids and organic acids have strong biological activities. The discovery of new anti-inflammatory and anti-tumor active ingredients in Folium Isatidis can further promote the clinical application of Folium Isatidis in anti-inflammatory and anti-tumor.

[0004] In view of this, the development of new compounds with anti-inflammatory and anti-tumor activities in Folium Isatidis is of great significance. Summary of the invention

[0005] Based on the background technology, one of the purposes of the present invention is to provide an indole alkaloid compound extracted and separated from Folium Isatidis, which has a novel structure and pharmacological activity, and provides a material basis for the research on the pharmacological effects of Folium Isatidis.

[0006] The second object of the present invention is to provide a method for extracting and separating the compound from Folium Isatidis. The entire extraction and separation process is simple to operate, easy to control, and suitable for industrial production.

[0007] The third purpose of the present invention is to provide the anti-inflammatory and anti-tumor effects of the compound, providing a strong basis for discovering potential new anti-inflammatory and anti-tumor drugs.

[0008] To achieve the above purpose, the present invention specifically adopts the following technical solutions:

[0009] The present invention provides a class of indole alkaloid compounds extracted and separated from Folium Isatidis. The indole alkaloid compounds are shown in the general formula (I):

[0010]

[0011] Among them, when R1 is hydroxyl, R2 is cyano and R3 is hydrogen; when R1 is hydrogen, R2 is amide and R3 is sulfonamide.

[0012] Preferably, the chemical structural formula of the indole alkaloid compound is as shown in the following formula (II) or formula (III):

[0013]

[0014] The present invention also provides a method for extracting and separating the above-mentioned indole alkaloid compound, comprising the following steps:

[0015] Step 1, reflux extraction of the isatis indigo leaf with an ethanol solution, and concentrating the extract under reduced pressure to obtain an alcohol-free dry extract;

[0016] Step 2, diluting the alcohol-free dry extract with water, and then extracting with petroleum ether, ethyl acetate and n-butanol in sequence, concentrating the n-butanol extract under reduced pressure, subjecting the obtained concentrate to macroporous resin column chromatography, gradient elution with ethanol-water as the eluent, taking the 10% ethanol elution part and subjecting it to silica gel column chromatography, gradient elution with dichloromethane-methanol as the eluent, and combining them to obtain 16 fractions after thin layer chromatography inspection;

[0017] Among them, the volume ratio of eluent ethanol-water is 0:100, 10:90, 30:70, 50:50, 95:5, and the volume ratio of eluent dichloromethane-methanol is 15:1, 13:1, 11:1, 9:1, 7:1, 5:1, 3:1, 1:1;

[0018] Step 3: Take one of the fractions obtained in step 2 and subject it to silica gel column chromatography with dichloromethane-methanol as the eluent for gradient elution. Combine and obtain 4 fractions after thin layer chromatography. Take one of the fractions and subject it to reverse phase preparative HPLC with methanol-0.05% trifluoroacetic acid as the mobile phase to obtain the compound of formula (II);

[0019] Among them, the volume ratio of the eluent dichloromethane-methanol is 15:1, 13:1, 11:1, 10:1, 9:1, 8:1;

[0020] Step 4: Take one of the fractions obtained in step 2 and subject it to silica gel column chromatography with dichloromethane-methanol as the eluent for gradient elution, and combine it after thin layer chromatography to obtain 3 fractions. Take one of the fractions and subject it to Sephadex LH-20. Combine it after thin layer chromatography to obtain 3 fractions, and take one of the fractions and subject it to reverse phase preparative HPLC with methanol-0.01% trifluoroacetic acid as the mobile phase to obtain the compound of formula (III);

[0021] Among them, the volume ratio of eluent dichloromethane-methanol is 8:1, 6:1, and 4:1.

[0022] Preferably, in step three, the volume ratio of methanol-0.05% trifluoroacetic acid as the mobile phase is 10:90, and the volume flow rate is 10 mL / min.

[0023] Preferably, in step 4, the volume ratio of methanol-0.01% trifluoroacetic acid as the mobile phase is 6:94, and the volume flow rate is 10 mL / min.

[0024] The present invention also provides a pharmaceutical composition, which comprises at least one of the compounds of formula (II) and formula (III) as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0025] The present invention also provides the use of the indole alkaloid compound in the preparation of anti-inflammatory drugs.

[0026] The present invention also provides the use of the indole alkaloid compound in preparing a medicine for treating cancer.

[0027] Preferably, the cancer is gastric cancer, liver cancer or lung cancer.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention extracts and separates a class of indole alkaloid compounds from the medicinal material of Isatis indigotica, and also determines their molecular configurations based on relevant data such as hydrogen spectrum and carbon spectrum. According to their molecular configurations, they are named 4-hydroxyindole-3-acetonitrile and 3-acetamido-indolesulfonamide.

[0030] 2. The present invention uses the medicinal material of Isatis indigotica as raw material, and obtains indole alkaloid compounds through the steps of ethanol reflux extraction, reduced pressure concentration, extraction, chromatographic separation and the like. The entire extraction and separation process is simple to operate, easy to control, and suitable for industrial production.

[0031] 3. In vitro biological activity experiments show that 4-hydroxyindole-3-acetonitrile and 3-acetamido-indolesulfonamide both exhibit strong anti-inflammatory effects, and have significant inhibitory effects on gastric cancer, liver cancer, and lung cancer cells. They have good application prospects in the preparation of anti-inflammatory drugs and drugs for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the structural diagram of the compound of formula (II) and formula (III).

[0033] Figure 2 It is the HR-Q-TOF-MS chart of the compound of formula (II).

[0034] Figure 3 It is the UV pattern of the compound of formula (II).

[0035] Figure 4 The compound of formula (II) 1 H-NMR spectrum.

[0036] Figure 5 The compound of formula (II) 13C-NMR spectrum.

[0037] Figure 6 is the HSQC spectrum of the compound of formula (II).

[0038] Figure 7 It is the HMBC spectrum of the compound of formula (II).

[0039] Figure 8 The compound of formula (II) 1 H- 1 HCOSY diagram.

[0040] Fig. 9 It is the HR-Q-TOF-MS chart of the compound of formula (III).

[0041] Fig.10 It is the UV pattern of the compound of formula (III).

[0042] Fig.11 The compound of formula (III) 1 H-NMR spectrum.

[0043] Fig.12 The compound of formula (III) 13 C-NMR spectrum.

[0044] Fig.13 is the HSQC spectrum of the compound of formula (III).

[0045] Fig.14 is the HMBC spectrum of the compound of formula (III).

[0046] Fig.15 The compound of formula (III) 1 H- 1 HCOSY diagram. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0049] The equipment and instruments used in the present invention include: UltiMate 3000 high performance liquid chromatograph; Varian UNITY INOVA 600MHz superconducting nuclear magnetic resonance instrument; ACQUITY UPLC I-Class PLUS-XEVO G2-XS QTOF ultra-high performance liquid chromatography quadrupole ultra-high resolution mass spectrometer; FDU-2110 freeze dryer; Agilent 1260infinity IIPrep preparative high performance liquid chromatograph; BUCHI R-300 rotary evaporator; ME204TE electronic balance; UV-260 ultraviolet spectrophotometer; and Perkin-Elmer 341 polarimeter.

[0050] Example 1 Extraction and separation of indole alkaloid compounds

[0051] 1. Take 18 kg of dried medicinal materials of Folium Isatidis, crush them, and extract them with 75% ethanol solution by volume for 3 times, each time for 2 hours. Combine the extracts and concentrate under reduced pressure to obtain alcohol-free dry extract. Dilute the alcohol-free dry extract with appropriate amount of water, extract with petroleum ether, ethyl acetate, and n-butanol in turn, extract 4 times each, combine the extracts and concentrate to dryness under reduced pressure to obtain 515 g of petroleum ether extract concentrate, 330 g of ethyl acetate extract concentrate, and 620 g of n-butanol extract concentrate.

[0052] 2. Take the n-butanol extract concentrate and subject it to D101 macroporous resin column chromatography, using ethanol-water (volume ratios of 0:100, 10:90, 30:70, 50:50, 95:5) as the eluent for gradient elution for 26 h (the corresponding elution times for the five gradient volume ratios are 6 h, 6 h, 5 h, 5 h, and 4 h, respectively); take the 10% ethanol elution portion (30.8 g) and subject it to silica gel column chromatography, using dichloromethane-methanol (volume ratios of 15:1, 13:1, 11:1, 9:1, 7:1, 5:1, 3:1, and 1:1) as the eluent for gradient elution for 12 h (the elution time for the eight gradient volume ratios is 1.5 h). Collect the eluate, collect one portion per 1 L and number them sequentially, examine the collected eluate by thin layer chromatography in turn, combine similar components, and obtain 16 fractions recorded as Fr.1 to Fr.16; among them, 1.25g of fraction Fr.8 was obtained, and fraction Fr.8 was obtained by combining the eluates from the 22nd to the 25th portions; 1.08g of fraction Fr.12 was obtained, and fraction Fr.12 was obtained by combining the eluates from the 33rd to the 35th portions.

[0053] 3. Take the obtained fraction Fr.8 and subject it to silica gel column chromatography, use dichloromethane-methanol (volume ratio 15:1, 13:1, 11:1, 10:1, 9:1, 8:1) as the eluent for gradient elution for 9h (the elution time of the 6 gradient volume ratios is 1.5h), collect the eluent, collect one portion of 50mL and number them in sequence, examine the collected eluents in turn by thin layer chromatography, combine similar components, and obtain 4 fractions recorded as Fr.8-1 to Fr.8-4; among them, 172.0mg of fraction Fr.8-1 is obtained, and fraction Fr.8-1 is obtained by combining the eluents of the 6th to 13th portions. Take the obtained fraction Fr.8-1 and subject it to reverse phase preparative HPLC, use methanol-0.05% trifluoroacetic acid (volume ratio 10:90, flow rate 10mL / min) as the mobile phase, and prepare the compound of formula (II) (18.2mg).

[0054] 4. Take the obtained fraction Fr.12 and subject it to silica gel column chromatography, use dichloromethane-methanol (volume ratio of 8:1, 6:1, 4:1) as eluent for gradient elution for 4.5 h (the elution time for the three gradient volume ratios is 1.5 h), collect the eluate, collect one portion of 50 mL and number them in sequence, examine the collected eluate by thin layer chromatography in sequence, combine similar components, and obtain three fractions recorded as Fr.12-1 to Fr.12-3; among them, 120 mg of fraction Fr.12-3 is obtained, and fraction Fr.12-3 is obtained by combining the eluates from the 15th to the 18th. The obtained fraction Fr.12-3 was passed through Sephadex LH-20 (eluted with methanol), and the eluent was collected. Each 10 mL fraction was collected and numbered in sequence. The collected eluents were examined by thin layer chromatography in sequence, and similar components were combined to obtain three fractions recorded as Fr.12-3-1 to Fr.12-3-3; 47.9 mg of fraction Fr.12-3-3 was obtained, and fraction Fr.12-3-3 was obtained by combining the eluents of the 34th to 39th fractions. The obtained fraction Fr.12-3-3 was subjected to reverse phase preparative HPLC, with methanol-0.01% trifluoroacetic acid water (volume ratio 6:94, flow rate 10 mL / min) as the mobile phase to prepare the compound of formula (III) (7.1 mg).

[0055] Example 2 Structural analysis and identification of indole alkaloid compounds

[0056] The compound of formula (II) obtained in Example 1, which is a light brown powder, is dissolved in DMSO, [α] 20 D -0.480(c0.06, MeOH), UV(MeOH)λ max (logε): 194.7 (1.94) nm, 265.31 (1.44) nm, 280.46 (1.02) nm. By HR-Q-TOF-MS m / z 173.0713 [M+H]+ ,(calcd for C 10 H 9 N 2 O, 173.0715), indicating that its molecular weight is 172.0715, and then through 1 H. 13 C determines its molecular formula to be C 10 H 8 N 2 O.

[0057] 1 H-NMR spectrum shows a phenolic hydroxyl proton signal δ H 9.54 (1H, s, -OH); signal of an amino proton on the indole ring δ H 10.92 (1H, s, NH); three aromatic ring proton signals δ H 6.87 (1H, t, J = 7.8 Hz, H-6), 6.81 (1H, d, J = 8.1 Hz, H-7), 6.35 (1H, d, J = 7.5 Hz, H-5); one unsaturated olefin proton signal δ H 7.14 (1H, d, J = 2.7 Hz, H-2); one methylene proton signal δ H 4.04(2H,s,H-8). 13 C-NMR spectrum shows a group of aromatic ring carbon signals δ C 151.2 (C-4), 102.9 (C-5), 122.4 (C-6), 102.9 (C-7), 138.2 (C-7a), 115.5 (C-3a); a group of unsaturated olefin carbon signals δ C 103.4 (C-3), 121.9 (C-2), a group of acetonitrile carbon signals δ C 14.8 (C-8), 119.8 (C-9). Based on the above, it is speculated that the compound is an indole alkaloid.

[0058] HMBC spectrum shows that NH(δ H 10.92) and C-3(δ C 103.4), C-3a(δ C 115.5), C-2(δ C 121.9) and C-7a (δ C 138.2) related; H-2(δ H 7.14) and C-7a(δ C 138.2), C-3(δ C 103.4) and C-3a (δ C 115.5) related; H-8 (δ H4.04) and C-3(δ C 103.4), C-3a(δ C 115.5), C-2(δ C 121.9) and C-9 (δ C 119.8) related; and 1 H- 1 The H-5 / H-6 / H-7 in the HCOSY spectrum were correlated, and the compound of formula (II) was finally named 4-hydroxyindole-3-acetonitrile according to its structure.

[0059] Figure 1 It is a structural diagram of the compound of formula (II) and the compound of formula (III).

[0060] HR-Q-TOF-MS and UV images of the compound of formula (II) 1 H-NMR spectrum, 13 C-NMR spectrum, HSQC spectrum, HMBC spectrum, 1 H- 1 HCOSY diagrams are shown in Figure 2-Figure 8 .

[0061] The compound of formula (III) obtained in Example 1, which is a white powder, is dissolved in DMSO, [α] 20 D -3.797(c0.07, MeOH), UV(MeOH)λ max (logε): 194.1 (2.85) nm, 215.6 (1.84) nm, 260.8 (1.23) nm. By HR-Q-TOF-MS m / z 254.0593 [M+H] + ,(calcd for C 10 H 12 N 3 O 3 S, 254.0599), indicating that its molecular weight is 253.0599. 1 H. 13 C determines its molecular formula to be C 10 H 11 N 3 O 3 S.

[0062] 1 H-NMR spectrum shows two terminal amino proton signals δ H 9.01(2H,s,NH 2 ),8.78(2H,s,NH 2 ); Four aromatic ring proton signals δ H7.81 (1H, d, J = 8.4 Hz, H-7), 7.62 (1H, d, J = 7.8 Hz, H-4), 7.16 (1H, t, J = 7.8 Hz, H-6), 7.08 (1H, t, J = 7.8 Hz, H-5); a sharp unsaturated olefin proton signal δ H 7.58 (1H, s, H-2); one methylene proton signal δ H 3.78(2H,s,H-8). 13 C-NMR spectrum shows a group of aromatic ring carbon signals δ C 127.6 (C-3a), 118.1 (C-4), 119.8 (C-5), 122.0 (C-6), 113.8 (C-7), 134.7 (C-7a); a group of unsaturated olefin carbon signals δ C 127.0 (C-2), 105.9 (C-3); minus one methylene carbon signal δ C 28.5 (C-8) and a carbonyl carbon signal δ C 169.6 (C-9), it can be clearly inferred that the parent nucleus of this compound is an indole ring.

[0063] HSQC and HMBC spectra showed that H-8(δ H 3.78) and C-3a(δ C 127.6) and C-2(δ C 127.0) related; indicating that the methylene (δ H 3.78) is connected to the 3rd position of the mother nucleus; the mother nucleus H-2 (δ H 7.58) and C-3a(δ C 127.6), C-7a(δ C 134.7) and C-8 (δ C 28.5) related, we know that δ H 7.58(δ C 127.0) is C-2; -NH(δ H 8.78) and C-8(δ C 28.5) related, we know that δ H 8.78 is connected to the carbonyl group at position 9. The above proves that the compound has a 3-indoleacetamide fragment, and the molecular formula given by HR-Q-TOF-MS can be judged that in addition to the fragment, there is also a sulfonamide group, which is connected to the N at position 1. Finally, according to the structure of the compound of formula (III), it is named 3-acetamido-indolesulfonamide.

[0064] HR-Q-TOF-MS and UV images of the compound of formula (III) 1 H-NMR spectrum, 13C-NMR spectrum, HSQC spectrum, HMBC spectrum, 1 H- 1 HCOSY diagrams are shown in Figure 9-Figure 15 .

[0065] Table 1 1 H-NMR, 13 C-NMR data

[0066]

[0067] Example 3 Anti-inflammatory activity assay

[0068] 1 Materials and methods

[0069] 1.1 Experimental animals

[0070] 40 SPF SD rats, male, weighing 180-220 g, were purchased from Changsha Tianqin Biotechnology Co., Ltd., animal qualification certificate number: NO.430726231100180112. They were kept in a barrier-level environment of our hospital at 20-25°C and relative humidity of 50-60%. The experimental animal use license number is SYXK (Gan) 2019-0001.

[0071] 1.2 Experimental instruments and reagents

[0072] Animal balance, microplate reader, constant temperature incubator, lipopolysaccharide were purchased from Addison, rat tumor necrosis factor α (TNF-α) ELISA kit, rat interleukin 6 (IL-6) ELISA kit, rat interleukin-1β (IL-1β) ELISA kit were purchased from Addison.

[0073] 1.3 Experimental methods

[0074] Before the formal experiment, the rats were fed adaptively for 5 days, and they were fasted but not water-deprived before the experiment. The rats were randomly divided into 5 groups, namely the negative control group, the model control group, the positive control group, the 4-hydroxyindole-3-acetonitrile group, and the 3-acetamido-indolesulfonamide group, with 8 rats in each group, and were gavaged for 7 consecutive days. The positive control group was given aspirin 125 mg / kg, the 4-hydroxyindole-3-acetonitrile group was given a dose of 5 mg / kg, and the 3-acetamido-indolesulfonamide group was given a dose of 5 mg / kg. The negative control group and the model control group were given the same volume of purified water for gavage. 30 minutes after the last administration, each group of rats was intraperitoneally injected with 10 mg / kg of LPS solution to establish the model (the negative control group was given the same volume of normal saline). 6 hours after the modeling, the rats were anesthetized with 3% sodium pentobarbital, blood was collected from the abdominal aorta, and serum was obtained by centrifugation at 3500rpm. The TNF-α, IL-6, and IL-1β levels in the serum of each group of rats were determined according to the instructions of the ELISA kit.

[0075] 2 Statistical methods

[0076] SPSS 17.0 statistical software was used to process the data. The differences among the groups were statistically significant. The Dunnett-t test was used for the comparison of the variances, and the Tamhane's T2 test was used for the comparison of the variances. P < 0.05 indicated that the differences were statistically significant.

[0077] 3 Results

[0078] After 6 hours of modeling, the levels of serum inflammatory factors TNF-α, IL-6, and IL-1β in the model control group were higher than those in the negative control group, and the difference was statistically significant (P < 0.05). The levels of TNF-α, IL-1β, and IL-6 in the 4-hydroxyindole-3-acetonitrile and 3-acetamido-indolesulfonamide groups were lower than those in the model control group, and the difference was statistically significant (P < 0.05). The details are shown in Table 2.

[0079] Table 2 Effects of each experimental group on the levels of serum inflammatory factors in the LPS-induced rat inflammation model

[0080]

[0081] Note: Compared with negative control group *P<0.05, **P<0.01; compared with model control group #P<0.05, ##P<0.01

[0082] 4 Discussion

[0083] TNF-α, IL-6 and IL-1β are the most common pyrogenic inflammatory factors. In animal experiments, the levels of the above inflammatory factors showed an upward trend in all groups of rats (except the negative control group) after intraperitoneal injection of LPS. 4-Hydroxyindole-3-acetonitrile and 3-acetamido-indolesulfonamide both significantly reduced the levels of TNF-α, IL-1β and IL-6, showing a strong anti-inflammatory effect.

[0084] Example 4 In vitro antitumor activity assay

[0085] 1 Materials and methods

[0086] 1.1 Cell lines

[0087] BGC-823 (human gastric cancer cells), 7721 (human liver cancer cells) and A549 (human lung cancer cells) were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0088] 1.2 Experimental instruments and reagents

[0089] ELISA reader, CO 2Incubator, clean bench, cell counter, MTT, paclitaxel, DMEM complete medium, trypsin.

[0090] 1.3 Test methods

[0091] Take cells in logarithmic growth phase, digest them, and blow them thoroughly into single cell suspension. Count them and dilute them into 1×10 5 Each well of a 96-well culture plate was inoculated with 100 μL of cell suspension and placed at 37°C and 5% CO 2 After culturing in an incubator for 24 hours, the original culture medium was discarded. Six concentration gradients were designed for each sample, and then 100 μL of culture medium containing samples of each concentration gradient and paclitaxel (positive control) was added to the test wells, with 6 parallel wells for each concentration; an equal volume of solvent was added to the control group. The 96-well culture plate was placed at 37°C and 5% CO 2 After culturing in a saturated humidity incubator for 72 hours, 20 μL of freshly prepared serum-free medium containing 5 mg / mL MTT was added to each well. After continuing to culture at 37°C for 4 hours, the supernatant was removed. 150 μL of DMSO was added to each well for dissolution, and the mixture was shaken for 5 minutes. The absorbance at 490 nm was measured on an ELISA reader, which can reflect the number of living cells. The calculation formula is as follows: Tumor cell growth inhibition rate (%) = (1-test well measurement value / control well measurement value) × 100%. Finally, the inhibitory concentration (IC) of the drug was calculated using SPSS software. 50 )value.

[0092] 1.4 Test results

[0093] Table 3 Hepatoma cell line 7721

[0094]

[0095]

[0096] Table 4 Gastric cancer cell BGC-823

[0097]

[0098] Table 5 Lung cancer cell A549

[0099]

[0100]

[0101] 1.5 Conclusion

[0102] Experiments have shown that 4-hydroxyindole-3-acetonitrile and 3-acetamido-indolesulfonamide have significant inhibitory effects on human gastric cancer cells, human liver cancer cells and human lung cancer cells, and can be used to develop drugs for treating cancer or tumors.

[0103] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Extract and separate indole alkaloid compounds from Folium Isatidis. It is characterized in that The chemical structural formula of the indole alkaloid compound is shown in the following formula (III): 。 2. A method for extracting and separating the indole alkaloid compound as claimed in claim 1, It is characterized in that The steps include: Step 1, reflux extraction of the isatis indigo leaf with an ethanol solution, and concentrating the extract under reduced pressure to obtain an alcohol-free dry extract; Step 2, diluting the alcohol-free dry extract with water, and then extracting with petroleum ether, ethyl acetate and n-butanol in sequence, concentrating the n-butanol extract under reduced pressure, subjecting the obtained concentrate to macroporous resin column chromatography, using ethanol-water as the eluent for gradient elution, taking the 10% ethanol elution part and subjecting it to silica gel column chromatography, using dichloromethane-methanol as the eluent for gradient elution, and combining and obtaining 16 fractions after thin layer chromatography inspection; Among them, the volume ratio of eluent ethanol-water is 0:100, 10:90, 30:70, 50:50, 95:5, and the volume ratio of eluent dichloromethane-methanol is 15:1, 13:1, 11:1, 9:1, 7:1, 5:1, 3:1, 1:1; Step 3: Take one of the fractions obtained in step 2 and subject it to silica gel column chromatography, using dichloromethane-methanol as the eluent for gradient elution, and combine it to obtain 4 fractions after thin layer chromatography inspection. Take one of the fractions and subject it to reverse phase preparative HPLC, using methanol-0.05% trifluoroacetic acid as the mobile phase to obtain a compound of formula (II). The chemical structure of the compound of formula (II) is as follows: ； The volume ratio of dichloromethane-methanol for the eluent was 15:1, 13:1, 11:1, 10:1, 9:1, and 8:1, the volume ratio of methanol-0.05% trifluoroacetic acid for the mobile phase was 10:90, and the volume flow rate was 10 mL / min; Step 4: Take one of the fractions obtained in step 2 and subject it to silica gel column chromatography, using dichloromethane-methanol as the eluent for gradient elution, and combine it after thin layer chromatography to obtain 3 fractions. Take one of the fractions and subject it to Sephadex LH-20, and combine it after thin layer chromatography to obtain 3 fractions. Take one of the fractions and subject it to reverse phase preparative HPLC, using methanol-0.01% trifluoroacetic acid as the mobile phase to obtain the compound of formula (III); The volume ratios of dichloromethane-methanol of the eluent were 8:1, 6:1, and 4:1, the volume ratio of methanol-0.01% trifluoroacetic acid of the mobile phase was 6:94, and the volume flow rate was 10 mL / min.

3. A pharmaceutical composition, It is characterized in that It comprises the compound of formula (III) as claimed in claim 1 as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

4. Use of the indole alkaloid compound of claim 1 in the preparation of anti-inflammatory drugs.

5. Use of the indole alkaloid compound of claim 1 in the preparation of a medicament for treating gastric cancer, liver cancer or lung cancer.

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