Compounds, methods of making and uses thereof

Abstract

The present application belongs to the technical field of natural product separation, and particularly relates to a compound, a preparation method and application thereof. The structure of the compound is shown in formula (I), (II), (III) and (IV). The preparation method of the compound is disclosed, and specifically comprises the following steps: providing a gerbera jackmanii, drying and crushing the gerbera jackmanii, refluxing the gerbera jackmanii with an ethanol aqueous solution to obtain an extract, and concentrating the extract to obtain a first extract; subjecting the first extract to macroporous resin column separation to obtain a first concentrate; and subjecting the first concentrate to one or more of polyamide column chromatography, normal phase column chromatography, reverse phase column chromatography or gel chromatography. The compound can be used for preparing medicines for treating cervical cancer, liver cancer, colon cancer and breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product separation, and particularly relates to a compound, a preparation method and use thereof. BACKGROUND

[0002] Gerbera delavayi Franch. is a plant of Gerbera in Compositae, which is recorded in Yunnan Province Chinese Medicine Standards · Yi Nationality Medicine (III) (2005 edition, the sixth volume), and is recorded in National Chinese Herbal Medicine Compilation, Chinese Medicine Dictionary, Chinese Herbal, Guizhou Chinese Herbal Medicine Resource Research, Guizhou Flora, and other monographs. Gerbera delavayi Franch. is also known as hook-bracket rush grass and fire grass, has the effects of clearing heat and resolving toxin, removing dampness and swelling, and resolving stasis and stopping bleeding, and is used for treating lung heat cough, heat stranguria, lower limb redness and swelling, and external injury bleeding.

[0003] The previous phytochemical research on the plants of Gerbera shows that the plants are rich in chemical components, and contain more coumarins, a small amount of acetophenone, benzopyran, terpenes, flavones, sterols, organic acids and other chemical components. The activity screening experiment shows that the plants have good antibacterial, antitumor, antitussive, expectorant, antiasthmatic, immunological, hypoglycemic and other biological activities. According to the literature reports, Qiang Yin et al. found a new five-ring pyran [3-2c] coumarin structure with certain antitumor activity in G. saxatilis, which is composed of a simple coumarin framework, a pyran ring, a methyl-substituted cyclohexane ring and an epoxy ring. Wu Zhi-li et al. found a new rare 5-methyl coumarin monoterpenes composed of a furan ring in G. anandria, which has a strong neuroprotective effect on PC12 cell damage induced by anisodamine. Li Tuo et al. found a series of isoprenyl and geranyl-substituted coumarin derivatives related to α-glucosidase and protein tyrosine phosphatase 1B (PTP-1B) inhibitory activity in the ethyl acetate fraction of G. anandria, including Diels-Alder cycloaddition to form dimeric coumarin derivatives. Therefore, the research on the chemical components and pharmacological activities of such coumarin components has also attracted widespread attention.

[0004] At present, the researches on Gerbera pterochaeta, Gerbera robusta and Gerbera scapiformis are more, mainly focusing on the chemical components in the small polarity part. The researches on Gerbera delavayi are less, only a small amount of researches on botany and cultivation characteristics are reported, and only one document reports the chemical components, 15 compounds are separated, and no related report on pharmacological activity is reported, and the chemical material basis research is not clear. According to the relationship between plant classification and chemical components, the genetic relationship of plants in the same genus is very similar, and the chemical components with similar structures are often contained. Therefore, taking Gerbera delavayi as the research object, the systematic chemical components and antitumor activity are researched, the medicinal material basis is clarified, and novel potential compounds or candidate drug molecules with novel structures are found, so as to provide a theoretical basis and experimental basis for subsequent research on the structure of active components, research and development of innovative drugs, improvement of quality control standards of medicinal materials and preparations, and reasonable development of resources. SUMMARY

[0005] The present application aims at overcoming the above-mentioned deficiencies of the prior art and providing a compound, a preparation method and uses thereof.

[0006] To achieve the above-mentioned purpose, the present application provides a compound, and the structure of the compound is shown as formula (I),

[0007]

[0008] Further, the present application also provides a compound, and the structure of the compound is shown as formula (II),

[0009]

[0010] Further, the present application also provides a compound, and the structure of the compound is shown as formula (III),

[0011]

[0012] Further, the present application also provides a compound, and the structure of the compound is shown as formula (IV),

[0013]

[0014] Further, the present application also provides a preparation method of the above-mentioned compound, comprising the following steps:

[0015] (1) providing Gerbera delavayi, after drying and crushing treatment, reflux extraction is carried out with ethanol aqueous solution to obtain an extract, and the extract is concentrated to obtain a first extract;

[0016] (2) the first extract is separated by macroporous resin column according to the following steps:

[0017] adding the first extract into a macroporous resin column, wherein the macroporous resin is selected from one of D101, D-201, AB-8 or HP-20 type non-polar macroporous adsorption resin; the ratio of the volume of the macroporous adsorption resin in the macroporous resin column to the weight of the gerbera is 0.5-5:1;

[0018] eluting the macroporous resin column with water as an eluent to remove impurities;

[0019] eluting the macroporous resin column with an ethanol aqueous solution with a concentration of 30-70% v / v as an eluent, collecting the eluate, and concentrating to obtain a first concentrate;

[0020] (3) separating the first concentrate by one or more of polyamide column chromatography, normal phase column chromatography, reverse phase column chromatography or gel chromatography.

[0021] In an embodiment of the present application, the step (3) is specifically:

[0022] eluting the first concentrate by polyamide column chromatography with an ethanol aqueous solution with a concentration of 30-70% v / v as an eluent, collecting the eluate, and concentrating to obtain a second concentrate;

[0023] eluting the second concentrate by normal phase column chromatography with dichloromethane-methanol as an eluent for gradient elution, collecting the eluate, detecting each eluate by thin layer chromatography, and according to the detection results, obtaining 8 fractions in sequence, which are named as Fr.1-Fr.8, respectively.

[0024] In an embodiment of the present application, the fraction Fr.6 is taken, concentrated under reduced pressure, the concentrated solution is eluted by normal phase column chromatography with ethyl acetate-methanol as an eluent for gradient elution, the eluate is collected, each eluate is detected by thin layer chromatography, and according to the detection results, 7 fractions are obtained in sequence, which are named as Fr.6.1-Fr.6.7, respectively.

[0025] In an embodiment of the present application, the fraction Fr.6.4 is taken, concentrated under reduced pressure, the concentrated solution is eluted by reverse phase column chromatography with methanol-water as an eluent for gradient elution, the eluate is collected, each eluate is detected by thin layer chromatography, and according to the detection results, 11 fractions are obtained in sequence, which are named as Fr.6.4.1-Fr.6.4.11, respectively.

[0026] In one embodiment of the present application, the fraction Fr.6.4.6.2 is concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with methanol-water with a concentration of 10%-80% v / v as eluent by reversed-phase column chromatography, the eluate is collected and concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with dichloromethane-methanol as eluent by normal-phase column chromatography, the eluate is collected, each eluate is detected by thin layer chromatography, the eluate containing the target component is combined, dried after being concentrated under reduced pressure, to obtain the compound I.

[0027] In one embodiment of the present application, the fraction Fr.6.4.6.2 is concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with methanol-water with a concentration of 10%-80% v / v as eluent by reversed-phase column chromatography, the eluate is collected and concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with dichloromethane-methanol as eluent by normal-phase column chromatography, the eluate is collected, each eluate is detected by thin layer chromatography, the eluate containing the target component is combined, dried after being concentrated under reduced pressure, to obtain the compound I.

[0028] In one embodiment of the present application, the fraction Fr.6.4.6.2 is concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with methanol-water with a concentration of 10%-80% v / v as eluent by reversed-phase column chromatography, the eluate is collected and concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with dichloromethane-methanol as eluent by normal-phase column chromatography, the eluate is collected, each eluate is detected by thin layer chromatography, the eluate containing the target component is combined, dried after being concentrated under reduced pressure, to obtain the compound I.

[0029] In one embodiment of the present application, the fraction Fr.6.4.6.2 is concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with methanol-water with a concentration of 10%-80% v / v as eluent by reversed-phase column chromatography, the eluate is collected and concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with dichloromethane-methanol as eluent by normal-phase column chromatography, the eluate is collected, each eluate is detected by thin layer chromatography, the eluate containing the target component is combined, dried after being concentrated under reduced pressure, to obtain the compound I.

[0030] In one embodiment of the present application, the fraction Fr.6.4.6.2 is concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with methanol-water with a concentration of 10%-80% v / v as eluent by reversed-phase column chromatography, the eluate is collected and concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with dichloromethane-methanol as eluent by normal-phase column chromatography, the eluate is collected, each eluate is detected by thin layer chromatography, the eluate containing the target component is combined, dried after being concentrated under reduced pressure, to obtain the compound I.

[0031] In one embodiment of the present application, the fraction Fr.6.4.6.2 is concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with methanol-water with a concentration of 10%-80% v / v as eluent by reversed-phase column chromatography, the eluate is collected and concentrated under reduced pressure, the concentrated solution is subjected to gradient elution with dichloromethane-methanol as eluent by normal-phase column chromatography, the eluate is collected, each eluate is detected by thin layer chromatography, the eluate containing the target component is combined, dried after being concentrated under reduced pressure, to obtain the compound I.

[0032] Further, the present application also provides a use of the above compound in the preparation of a drug for treating cervical cancer, liver cancer, colon cancer or breast cancer.

[0033] The present application has the following beneficial effects:

[0034] Studies on the antitumor activity of the isolated compounds revealed that *Hypericum esculentum* contains a variety of antitumor active compounds, exhibiting multiple targets and pathways of action. These compounds can be used to prepare drugs for treating cervical cancer, liver cancer, colon cancer, or breast cancer. The novel compounds or derivatives provided by this invention can serve as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity studies. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0036] Figure 1a The image shows the HR-ESI-MS chromatogram of compound I, m / z: 523.1405 [M+Na]. + ;

[0037] Figure 1 b The image shows the HR-ESI-MS chromatogram of compound I, m / z: 499.1444 [MH]. - ;

[0038] Figure 2 The HR-ESI-MS chromatogram of compound II is shown, m / z: 563.1364 [M+Na]. + ;

[0039] Figure 3a The HR-ESI-MS chromatogram of compound III is shown, m / z: 533.1260 [M+Na]. + ;

[0040] Figure 3b The HR-ESI-MS chromatogram of compound III is shown, m / z: 555.1356 [M+HCOO]. - ;

[0041] Figure 4a The HR-ESI-MS chromatogram of compound IV is shown, m / z: 473.1649 [MH]. - ;

[0042] Figure 4b The HR-ESI-MS chromatogram of compound IV is shown, m / z: 497.1610 [M+Na]. + ;

[0043] Figure 5 For compound I 1 H-NMR spectrum;

[0044] Figure 6 for compound II 1 H-NMR chart

[0045] Figure 7 for compound III 1 H-NMR chart

[0046] Figure 8 for compound IV 1 H-NMR chart

[0047] Figure 9 for compound I 13 C-NMR chart

[0048] Figure 10 for compound II 13 C-NMR chart

[0049] Figure 11 for compound III 13 C-NMR chart

[0050] Figure 12 for compound IV 13 C-NMR chart

[0051] Figure 13 HMQC chart for compound I

[0052] Figure 14 HMQC chart for compound II

[0053] Figure 15 HMQC chart for compound III

[0054] Figure 16 HMQC chart for compound IV

[0055] Figure 17 HMBC chart for compound I

[0056] Figure 18 HMBC chart for compound II

[0057] Figure 19 HMBC chart for compound III

[0058] Figure 20 HMBC chart for compound IV DETAILED DESCRIPTION

[0059] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described in conjunction with specific examples.

[0060] According to one aspect of the present application, the present application provides four compounds with the following structural formulae, respectively:

[0061]

[0062]

[0063] According to an aspect of the present application, the present application provides a method for preparing the above-mentioned compound, comprising the following steps:

[0064] (1) providing a hook-bracket large dengcai, after drying and crushing treatment, reflux extraction with ethanol aqueous solution to obtain an extract, and concentrating the extract to obtain a first extract;

[0065] In an embodiment of the present application, the hook-bracket large dengcai is first weighed, and then reflux extracted with ethanol aqueous solution for 3 times, each time for 1.5 h, the first time with 8 times the amount of ethanol aqueous solution, and the second and third times with 6 times the amount of ethanol aqueous solution, respectively, filtered, and combined to obtain an extract, which is concentrated under reduced pressure to obtain a first extract;

[0066] In an embodiment of the present application, the concentration of the above-mentioned ethanol aqueous solution is 50% v / v;

[0067] (2) the first extract is separated by macroporous resin column according to the following steps:

[0068] The first extract is added to the macroporous resin column, wherein the macroporous resin is selected from one of D101, D-201, AB-8 or HP-20 type non-polar macroporous adsorption resin; the ratio of the volume of the macroporous adsorption resin in the macroporous resin column to the weight of the hook-bracket large dengcai is 1:1;

[0069] The macroporous resin column is eluted with water as an eluent to remove impurities;

[0070] The macroporous resin column is eluted with ethanol aqueous solution with a concentration of 30-70% v / v as an eluent, and the eluate is collected and concentrated to obtain a first concentrate;

[0071] (3) the first concentrate is separated by one or more of polyamide column chromatography, normal phase column chromatography, reverse phase column chromatography or gel chromatography.

[0072] In an embodiment of the present application, the column chromatography further comprises using Sephadex LH-20 or Toyopearl HW-40F column chromatography for separation and purification.

[0073] In an embodiment of the present application, when normal phase column chromatography is used, silica gel with a particle size of 100-300 is selected as the filler;

[0074] In one embodiment of the present application, dichloromethane-methanol, petroleum ether-ethyl acetate, petroleum ether-dichloromethane, dichloromethane, methanol, ethyl acetate-methanol are selected as eluents for normal phase column chromatography.

[0075] In one embodiment of the present application, the first concentrate is subjected to polyamide column chromatography, eluted with an aqueous ethanol solution with a concentration of 30-70% v / v as eluent, the eluate is collected and concentrated to obtain a second concentrate;

[0076] The second concentrate is subjected to normal phase column chromatography, gradient elution is performed with dichloromethane-methanol with a volume ratio of 49:1-1:1 as eluent, the eluate is collected, each eluate is detected by thin layer chromatography, and according to the detection results, eight fractions are obtained in turn according to the elution order, which are named Fr.1-Fr.8, respectively.

[0077] In one embodiment of the present application, the fraction Fr.6 is taken, concentrated under reduced pressure, and the concentrated solution is subjected to normal phase column chromatography, gradient elution is performed with ethyl acetate-methanol with a volume ratio of 49:1-1:49 as eluent, the eluate is collected, each eluate is detected by thin layer chromatography, and according to the detection results, seven fractions are obtained in turn according to the elution order, which are named Fr.6.1-Fr.6.7, respectively.

[0078] In one embodiment of the present application, the fraction Fr.6.4 is taken, concentrated under reduced pressure, and the concentrated solution is subjected to reverse phase column chromatography, gradient elution is performed with methanol-water with a concentration of 10%-80% v / v as eluent, the eluate is collected, each eluate is detected by thin layer chromatography, and according to the detection results, eleven fractions are obtained in turn according to the elution order, which are named Fr.6.4.1-Fr.6.4.11, respectively, wherein the reverse phase column is an MCI reverse phase column.

[0079] In one embodiment of the present application, the fraction Fr.6.4.6 is taken, concentrated under reduced pressure, and the concentrated solution is subjected to normal phase column chromatography, gradient elution is performed with dichloromethane-methanol with a volume ratio of 9:1 as eluent, the eluate is collected, each eluate is detected by thin layer chromatography, and according to the detection results, three fractions are obtained in turn according to the elution order, which are named Fr.6.4.6.1-Fr.6.4.6.3, respectively.

[0080] In one embodiment of the present invention, fraction Fr.6.4.6.2 is taken, concentrated under reduced pressure, and the concentrate is subjected to reversed-phase column chromatography with gradient elution using methanol-water at a concentration of 10%-80% v / v as the eluent. The eluent is collected, concentrated under reduced pressure, and then subjected to normal-phase column chromatography with gradient elution using dichloromethane-methanol at a volume ratio of 9:1 as the eluent. The eluent is collected, and each eluent is detected by thin-layer chromatography. The eluents containing the target component are combined, concentrated under reduced pressure, and dried to obtain compound I. The reversed-phase column is an ODS reversed-phase column.

[0081] In one embodiment of the present invention, fraction Fr.6.4.9 is taken, allowed to stand to precipitate, washed, and filtered to obtain compound II. The washing is performed using methanol, dichloromethane, or ethyl acetate, preferably methanol.

[0082] In one embodiment of the present invention, fraction Fr.6.4.8 is taken, concentrated under reduced pressure, and the concentrate is subjected to reversed-phase column chromatography with gradient elution using methanol-water at a concentration of 5%-80% v / v as the eluent. The eluent is collected, and each eluent is detected by thin-layer chromatography. According to the detection results, six fractions are obtained in sequence according to the elution order, and named Fr.6.4.8.1-Fr.6.4.8.6 respectively. Fraction Fr.6.4.8.5 is taken, allowed to stand to precipitate, washed, and filtered to obtain compound III. The reversed-phase column is an ODS reversed-phase column.

[0083] In one embodiment of the present invention, fraction Fr.6.4.5 is taken, concentrated under reduced pressure, and the concentrate is subjected to normal phase column chromatography with gradient elution using dichloromethane-methanol at a volume ratio of 9:1. The eluent is collected, and each eluent is detected by thin-layer chromatography. According to the detection results, two fractions are obtained in sequence according to the elution order, and named Fr.6.4.5.1-Fr.6.4.5.2 respectively.

[0084] In one embodiment of the present invention, fraction Fr.6.4.5.2 was taken, concentrated under reduced pressure, and the concentrate was subjected to gel column chromatography and reversed-phase column chromatography in sequence. The eluent was collected, concentrated under reduced pressure, and then eluted with methanol-water at a concentration of 30% v / v using preparative liquid chromatography. The eluent was collected, concentrated under reduced pressure, and dried to obtain compound IV. The reversed-phase column is an MCI reversed-phase column.

[0085] In one embodiment of the present invention, fraction Fr.6.4.5.2 is taken and concentrated under reduced pressure. First, the concentrate is subjected to Toyopearl HW-40F gel column chromatography with gradient elution using methanol. The eluents are collected, and each eluent is analyzed by thin-layer chromatography. The eluents containing the target component are combined and concentrated under reduced pressure. Then, the concentrate is subjected to Sephadex chromatography. LH-20 gel column chromatography was performed with gradient elution using acetone-water at a concentration of 50% v / v. The eluents were collected, and each eluent was analyzed by thin-layer chromatography. The eluents containing the target component were combined and concentrated under reduced pressure. The concentrate was then subjected to MCI reversed-phase column chromatography with gradient elution using methanol-water at a concentration of 10%–80% v / v. The eluents were collected, concentrated under reduced pressure, and then eluted using methanol-water at a concentration of 30% v / v. The concentrate was then subjected to preparative liquid chromatography, and the eluents were collected, concentrated under reduced pressure, and dried to obtain compound IV. The chromatographic column used in the liquid chromatography was a Megres C18 column.

[0086] In one embodiment of the present invention, the conditions for thin-layer chromatography are as follows:

[0087] Colorimetric reagent a: Observe fluorescence under ultraviolet light (254nm, 365nm); Colorimetric reagent b: Iodine colorimetric reagent; Colorimetric reagent c: 10% sulfuric acid ethanol.

[0088] According to one aspect of the present invention, the present invention also discloses the use of the above-mentioned compound in the preparation of a medicament for treating cervical cancer, liver cancer, colon cancer or breast cancer.

[0089] Example 1

[0090] Weigh 20 kg of the herbal medicine *Houttuynia cordata* and extract it by reflux with a 50% v / v ethanol aqueous solution. Repeat the extraction 3 times, each time for 1.5 hours. For the first extraction, use 8 times the amount of ethanol aqueous solution. For the second and third extractions, add 6 times the amount of ethanol aqueous solution respectively. Filter, combine the filtrates, and recover the ethanol under reduced pressure. Directly heat and concentrate, continuously adding water to evaporate the ethanol until there is no obvious ethanol smell, to obtain the first extract.

[0091] After mixing 3000g of the first extract, it was separated by a D101 macroporous resin column. First, water was used as the eluent to elute the macroporous resin column to remove impurities and obtain an aqueous eluent. Then, a 50% v / v ethanol aqueous solution was used as the eluent to elute the macroporous resin column to obtain an ethanol eluent. The ethanol eluent was concentrated to obtain the first concentrate.

[0092] The first concentrate was subjected to polyamide column chromatography, eluted with a 50% v / v aqueous ethanol solution, and the eluent was collected and concentrated to obtain the second concentrate.

[0093] The second concentrate was subjected to normal phase column chromatography, using 100-200 mesh silica gel as the packing material. The silica gel source information was: Qingdao Ocean Chemical Co., Ltd., batch number: 0180046. Then, gradient elution was performed sequentially using dichloromethane-methanol with volume ratios of 50:1, 25:1, 10:1, 7:1, 5:1, 3:1 and 1:1. The eluents were collected, and each eluent was analyzed by thin-layer chromatography. Based on the test results, eight fractions were obtained in the elution order and named Fr.1-Fr.8.

[0094] Fraction Fr.6 was collected and concentrated under reduced pressure. The concentrate was subjected to normal-phase column chromatography using 200-300 mesh silica gel as the packing material. The silica gel was sourced from Qingdao Ocean Chemical Co., Ltd., batch number: 020116. Gradient elution was performed using ethyl acetate-methanol at volume ratios of 50:1, 25:1, 10:1, 7:1, 5:1, 3:1, 1:1, 1:5, 1:10, and 1:50. The eluents were collected and analyzed by thin-layer chromatography. Based on the analysis results, seven fractions were obtained in the elution order and named Fr.6.1-Fr.6.7.

[0095] Fraction Fr.6.4 was taken and concentrated under reduced pressure. The concentrate was subjected to reversed-phase column chromatography with gradient elution using a 10% v / v methanol-water solution as the eluent. The eluent was collected and analyzed by thin-layer chromatography. Based on the results, 11 fractions were obtained in the order of elution and named Fr.6.4.1-Fr.6.4.11, respectively. The reversed-phase column was an MCI reversed-phase column.

[0096] Fraction Fr.6.4.6 was collected and concentrated under reduced pressure. The concentrate was subjected to normal phase column chromatography using 200-300 mesh silica gel as the packing material. The source information of the silica gel was Qingdao Ocean Chemical Co., Ltd., batch number: 020116. Then, a gradient elution was performed using dichloromethane-methanol at a volume ratio of 9:1. The eluent was collected, and each eluent was analyzed by thin-layer chromatography. According to the test results, three fractions were obtained in the elution order and named Fr.6.4.6.1-Fr.6.4.6.3, respectively.

[0097] The fraction Fr.6.4.6.2 was taken and concentrated under reduced pressure. The concentrate was subjected to ODS reversed-phase column chromatography, followed by gradient elution with methanol-water at concentrations of 10% v / v, 30% v / v, 45% v / v, 65% v / v and 80% v / v. The eluent was collected, concentrated under reduced pressure, and then subjected to normal-phase column chromatography with 300-mesh silica gel as the packing material and dichloromethane-methanol at a volume ratio of 9:1 as the eluent. The eluent was collected, and each eluent was analyzed by thin-layer chromatography. The eluents containing the target component were combined, concentrated under reduced pressure, and dried to obtain compound I.

[0098] Take fraction Fr.6.4.9, let it stand to precipitate, wash with methanol, and filter to obtain compound II;

[0099] Fraction Fr. 6.4.8 was taken and concentrated under reduced pressure. The concentrate was subjected to ODS reversed-phase column chromatography, followed by gradient elution with methanol-water solutions of concentrations of 5% v / v, 25% v / v, 55% v / v, 65% v / v and 80% v / v. The eluents were collected and analyzed by thin-layer chromatography. According to the detection results, six fractions were obtained in the elution order and named Fr. 6.4.8.1-Fr. 6.4.8.6. Fraction Fr. 6.4.8.5 was taken, allowed to stand to precipitate, washed with methanol, and filtered to obtain compound III.

[0100] Fraction Fr.6.4.5 was collected and concentrated under reduced pressure. The concentrate was subjected to normal phase column chromatography using 200-300 mesh silica gel as the packing material. The source information of the silica gel was Qingdao Ocean Chemical Co., Ltd., batch number: 020116. Then, a gradient elution was performed using dichloromethane-methanol at a volume ratio of 9:1. The eluent was collected, and each eluent was analyzed by thin-layer chromatography. According to the test results, two fractions were obtained in the elution order and named Fr.6.4.5.1-Fr.6.4.5.2, respectively.

[0101] The fraction Fr.6.4.5.2 was collected and concentrated under reduced pressure. First, the concentrate was subjected to Toyopearl HW-40F gel column chromatography with gradient elution using methanol. The eluent was collected, and each eluent was analyzed by thin-layer chromatography. The eluents containing the target component were combined and concentrated under reduced pressure. Next, the concentrate was subjected to Sephadex LH-20 gel column chromatography with gradient elution using 50% v / v acetone aqueous solution. The eluent was collected, and each eluent was analyzed by thin-layer chromatography. The eluents containing the target component were combined and concentrated under reduced pressure. Then, the concentrate was subjected to MCI reversed-phase column chromatography with gradient elution using 10% v / v methanol aqueous solution. The eluent was collected, concentrated under reduced pressure, and dried to obtain compound IV. The chromatographic column used in the liquid chromatography was a Megres C18 column.

[0102] The conditions for thin-layer chromatography are:

[0103] Colorimetric reagent a: Observe fluorescence under ultraviolet light (254nm, 365nm); Colorimetric reagent b: Iodine colorimetric reagent; Colorimetric reagent c: 10% sulfuric acid ethanol.

[0104] Structural identification: using 1 H NMR, 13 The structures of the four compounds obtained from the separation were identified by C10 NMR spectroscopy, two-dimensional NMR spectroscopy, and high-resolution mass spectrometry. The specific results are as follows:

[0105] Compound I is a white powder with the molecular formula: C 22 H 28 O 13 HR-ESI-MS m / z: 499.1444 [MH] - ,calcd499.1446,523.1405[M+Na] + The compound was identified as Gerbelavinside A by spectral techniques, and its NMR data are shown in Tables 1 and 2.

[0106] Compound II is a white solid with the molecular formula: C 24 H 28 O 14 Optical rotation is: -331.9(c 1.88,MeOH); UV(MeOH)λ max (logε)222(3.37),266(3.22),308(3.06)nm; IR(KBr)ν max3424,2924,1721,1705,1688,1622,1593,1476,1425,1379,1344,1319,1211,1134,1096,1060cm -1 ;HR-ESI-MSm / z: 541.1549[M+H] + ,calcd 541.1552,563.1364[M+Na] + The calcd value was 563.1371. The compound was identified as Gerbelavinside B by spectroscopic techniques, and its NMR data are shown in Tables 1 and 2.

[0107] Compound III is a white solid with the molecular formula: C 23 H 26 O 13 Optical rotation is: -287.8(c 0.82,MeOH); UV(MeOH)λ max (logε)218(4.56),250(4.53),304(4.32)nm; IR(KBr)ν max 3426,2920,1722,1705,1690,1626,1582,1464,1148,1065cm -1 ;HR-ESI-MS m / z:555.1356[M+HCOO] - ,calcd555.1345,533.1260[M+Na] + ,calcd 533.1256; The compound was identified as Gerbelavinside C by spectroscopic techniques, and its NMR data are shown in Table 1 and Table 2.

[0108] Compound IV is a pale yellow solid with the molecular formula: C 21 H 30 O 12 Optical rotation is: -210.0(c 0.4,MeOH); UV(MeOH)λ max (logε)214(3.69),290(2.96)nm; IR(KBr)ν max 3399,2926,1686,1674,1659,1607,1564,1545,1524,1512,1464,1360,1290,1070,1047cm -1 HR-ESI-MS m / z: 473.1649 [MH] - ,calcd 473.1654,497.1610[M+Na]+ ,calcd 497.1630; the compound was identified as Gerbelavinside F by spectroscopic techniques, and its NMR data are shown in Table 1 and Table 2.

[0109] Table 1. Compounds I-IV 1 1H NMR data (in CD3OD of I, III, IV, DMSO-d6 of II)

[0110]

[0111] Table 2 Compounds I-IV 13 10⁻⁶ NMR data (in CD3OD of I, III, IV, DMSO-d6 of II)

[0112]

[0113]

[0114] Example 2

[0115] In one embodiment of the present invention, the present invention further studies the compounds isolated from Uncaria rhynchophylla, and the specific antitumor experiments are as follows:

[0116] The experimental principle of the antitumor assay is as follows: Cell Counting Kit-8 (CCK-8) reagent can be used for simple and accurate cell proliferation and toxicity analysis. This reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazanye product under the action of the electron carrier 1-methoxy-5-methylphenazineonium sulfate (1-Methoxy PMS). This product produces an absorption peak at 450 nm, and the absorbance value is linearly positively correlated with the cell count. The number of viable cells can be inferred from the optical density (OD) value; therefore, this characteristic can be used to directly analyze cell proliferation and toxicity.

[0117] Step 1: A549, MCF-7, HCT-15, and A2780 cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (100 U / mL penicillin, 100 mg / L streptomycin) and passaged at 37°C in a 5% CO2 incubator. Adherent cells were digested with 0.25% trypsin for passage, and cells in the logarithmic growth phase were used for experiments.

[0118] Step 2: Human lung cancer cells (A549), human breast cancer cells (MCF-7), human colorectal adenocarcinoma cells (HCT-15), and human ovarian cancer cells (A2780) in logarithmic growth phase were collected and the cell number was adjusted to 1×10⁻⁶ cells / year using DMEM culture medium containing 10% fetal bovine serum. 4 Cells were seeded at a density of 100 μL / mL in 96-well cell culture plates. When cells reached 70-80% adherence, the old culture medium was discarded, and 0.2 mL of culture medium containing different concentrations of compounds I-IV (75 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L) was added to each well. Each concentration was repeated in 5 wells, and the experiment was performed three times. After 24 h of culture, 10 μL of CCK-8 solution was added to each well, and the cells were cultured for another 2 h. The absorbance at 450 nm was measured using a microplate reader. The cell proliferation inhibition rate was calculated using the following formula: Cell inhibition rate (%) = 1 - (OD value) 实验组 / OD value 空白组 The half-maximal inhibitory concentration (IC50) was calculated using SPSS 25.0 software, representing a percentage of 100%. 50 ), the specific data is shown in Table 3:

[0119] Table 3. Cytotoxic activity IC50 of compounds I-IV 50 (μM)(X±S,n=3)

[0120]

[0121] Among them, IC 50 (μM) represents the concentration of the compound at which the proliferation inhibition rate is 50%, used to indicate antitumor activity; cisplatin is a positive control drug.

[0122] As shown in Table 3, compounds I-IV all exhibit certain inhibitory activity against human lung cancer cells (A549), human breast cancer cells (MCF-7), human colorectal adenocarcinoma cells (HCT-15), and human ovarian cancer cells (A2780). They can be used as a reference for the use of inhibitors against human lung cancer A549 cells, human breast cancer MCF-7 cells, human colorectal adenocarcinoma HCT-15 cells, and human ovarian cancer A2780 cells, and can also be used in the preparation of related drugs.

[0123] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no designations in the claims should be construed as limiting the scope of the claims.

Claims

1. A compound, characterized in that, The structure of the compound is shown in formula (Ⅰ). 。 2. A compound, characterized in that, The structure of the compound is shown in formula (II). 。 3. A compound, characterized in that, The structure of the compound is shown in formula (Ⅲ). 。 4. A compound, characterized in that, The structure of the compound is shown in formula (Ⅳ). 。 5. A method for preparing the compound according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Provide Uncaria rhynchophylla, after drying and pulverizing, extract by reflux with ethanol aqueous solution to obtain an extract, and concentrate the extract to obtain a first extract; (2) The first extract was separated by macroporous resin column separation according to the following steps: The first extract is added into a macroporous resin column, wherein the macroporous resin is selected from one of the non-polar macroporous adsorption resins D101, D-201, AB-8 or HP-20; the volume ratio of the macroporous adsorption resin in the macroporous resin column to the weight of Uncaria rhynchophylla is 0.5-5:

1. Water was used as the eluent to elute the macroporous resin column and remove impurities. The macroporous resin column was eluted using an ethanol-water solution with a concentration of 30-70% v / v as the eluent, the eluent was collected, and the first concentrate was obtained by concentration. (3) The first concentrate is separated by one or more of polyamide column chromatography, normal phase column chromatography, reverse phase column chromatography or gel chromatography; Step (3) specifically involves: The first concentrate was subjected to polyamide column chromatography, eluted with an ethanol aqueous solution of 30-70% v / v, the eluent was collected and concentrated to obtain the second concentrate; The second concentrate was subjected to normal phase column chromatography with gradient elution using dichloromethane-methanol as the eluent. The eluent was collected, and each eluent was detected by thin-layer chromatography. According to the detection results, eight fractions were obtained in the order of elution and named Fr.1-Fr.8 respectively. Fraction Fr.6 was taken and concentrated under reduced pressure. The concentrate was subjected to normal phase column chromatography with gradient elution using ethyl acetate-methanol as the eluent. The eluent was collected and analyzed by thin-layer chromatography. Based on the results, seven fractions were obtained in the order of elution and named Fr.6.1-Fr.6.

7. Fraction Fr.6.4 was taken and concentrated under reduced pressure. The concentrate was subjected to reversed-phase column chromatography with methanol-water as the eluent for gradient elution. The eluent was collected and each eluent was detected by thin-layer chromatography. According to the detection results, 11 fractions were obtained in the order of elution and named Fr.6.4.1-Fr.6.4.11 respectively. The fraction Fr.6.4.6 was taken and concentrated under reduced pressure. The concentrate was subjected to normal phase column chromatography with gradient elution using dichloromethane-methanol as the eluent. The eluent was collected and each eluent was analyzed by thin-layer chromatography. According to the detection results, three fractions were obtained in the order of elution and named Fr.6.4.6.1-Fr.6.4.6.3, respectively. The fraction Fr.6.4.6.2 was taken and concentrated under reduced pressure. The concentrate was subjected to reversed-phase column chromatography with a gradient elution using methanol-water at a concentration of 10%-80% v / v. The eluent was collected and concentrated under reduced pressure. The concentrate was subjected to normal-phase column chromatography with a gradient elution using dichloromethane-methanol. The eluent was collected and each eluent was analyzed by thin-layer chromatography. The eluents containing the target component were combined, concentrated under reduced pressure, and dried to obtain compound I. Take fraction Fr.6.4.9, let it stand to precipitate, wash and filter to obtain compound II; Fraction Fr. 6.4.8 was taken and concentrated under reduced pressure. The concentrate was subjected to reversed-phase column chromatography with methanol-water as the eluent for gradient elution. The eluent was collected and analyzed by thin-layer chromatography. According to the detection results, six fractions were obtained in the order of elution and named Fr. 6.4.8.1-Fr. 6.4.8.

6. Fraction Fr. 6.4.8.5 was taken, allowed to stand to precipitate, washed, and filtered to obtain compound III. The fraction Fr.6.4.5 was taken and concentrated under reduced pressure. The concentrate was subjected to normal phase column chromatography with gradient elution using dichloromethane-methanol as the eluent. The eluent was collected and each eluent was analyzed by thin-layer chromatography. According to the detection results, two fractions were obtained in the order of elution and named Fr.6.4.5.1-Fr.6.4.5.2, respectively. The fraction Fr.6.4.5.2 was taken and concentrated under reduced pressure. The concentrate was subjected to gel column chromatography and reversed-phase column chromatography in sequence, and the eluent was collected. After being concentrated under reduced pressure, the concentrate was eluted by preparative liquid chromatography with methanol-water as the mobile phase. The eluent was collected, concentrated under reduced pressure, and dried to obtain compound IV.

6. Use of the compound according to any one of claims 1-4 in the preparation of a medicament for treating cervical cancer, colon cancer or breast cancer.

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