Preparation method and application of two linear diaryl heptane compounds in walnut green seedcase
By isolating and preparing linear diarylheptane compounds Juglanin M and Juglanin L from walnut husks, the problems of high toxicity and low safety of walnut husk extracts were solved, and significant inhibitory effects on liver cancer, lung cancer and colorectal cancer cells were achieved, providing a novel source of compounds for the development of anti-tumor drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-04-07
AI Technical Summary
The existing technology for using walnut green peel extract for anti-tumor treatment has problems such as high toxicity and low safety, making it difficult to effectively develop it into a clinical anti-tumor drug.
Two new linear diarylheptane compounds, Juglanin M and Juglanin L, were isolated and prepared from walnut husks. They were purified by 95% ethanol extraction, macroporous resin enrichment and purification, normal-phase silica gel column chromatography and ODS column chromatography to obtain compounds with significant antitumor activity.
The prepared compounds showed significant inhibitory effects on human liver cancer, lung cancer, and colorectal cancer cells, exhibiting good selective toxicity to tumor cells and possessing the potential to be developed into highly effective and low-toxicity anti-tumor drugs.
Smart Images
Figure CN121800645A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation of two linear diarylheptane compounds with antitumor activity from the green husk of walnuts. Background Technology
[0002] The green husk of walnuts, also known as the green dragon skin, is from the jujube tree (Juglans mandshurica) of the genus Juglans in the family Juglandaceae. Juglans mandshurica The green husk of walnuts (Maxim.) is abundant and widely distributed in Northeast my country. According to the *Compendium of Materia Medica*, walnut husk can clear heat and detoxify, relieve rheumatic pain, treat tinea, relieve pain, and treat dysentery. *Shaanxi Traditional Chinese Medicine* records that walnut husk can treat psoriasis, ichthyosis, lotus leaf tinea, and alopecia areata. Particularly noteworthy is the long-standing folk practice in my country of using walnut husk to treat cancer pain. For example, in Anyang, Henan, it is used as an adjunct therapy for esophageal and gastric cancer; clinical findings at the General Hospital of the Beijing Military Region show that it can improve symptoms related to esophageal and gastric cardia cancer; and the compound Qinglongyi capsule developed by the Heilongjiang Provincial Academy of Traditional Chinese Medicine is used to treat digestive tract tumors such as liver and gastric cancer, significantly improving the quality of life for cancer patients. Modern pharmacological research further demonstrates that walnut husk extract and its main active ingredients (such as juglone) have significant inhibitory effects on tumor cells, but due to its high toxicity and low safety, clinical translation and rational application still face challenges.
[0003] Diarylheptane compounds are a class of chemical components with a unique core structure found in the green husk of walnuts. They generally refer to a series of compounds formed by a seven-carbon heptane oxide chain (this hydrocarbon chain may be saturated or contain alkene bonds, carbonyl groups, etc.), with two aromatic rings of different degrees of oxidation or substitution connected to the 1st and 7th positions, respectively. Based on whether they form rings and the connection mode of the two benzene rings, they can be classified into three types: linear, macrocyclic biphenyl, and cyclic diphenyl ether. The compounds involved in this invention belong to the linear diarylheptane class. This class of compounds possesses rich pharmacological activities, covering anti-inflammatory, pro-apoptotic, antiemetic, and neuroprotective effects. This invention has discovered two new linear diarylheptane compounds from the green husk of walnuts. The preparation steps are simple, making industrial production feasible. Preliminary in vitro antitumor experiments have also revealed significant antitumor activity, indicating potential for development into antitumor drugs. Summary of the Invention
[0004] The purpose of this invention is to provide two linear diarylheptane compounds isolated from the green skin of walnuts, their preparation methods and applications, so as to provide new pharmacodynamic substances or novel lead compounds for antitumor drugs in the future.
[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses two linear diarylheptanes, the molecular formula of which is C0. 22 H 28 O5, C 20 H 26 O5, named Juglanin M Juglanin L Its structural formula is as follows:
[0006] This invention also provides a method for preparing them: using walnut green husks as raw material, the analytes are obtained through 95% ethanol extraction, macroporous resin enrichment and purification, normal-phase silica gel column chromatography, ODS column chromatography, and preparative HPLC purification. The specific preparation steps are as follows: (1) Ethanol extraction: Take the green walnut skin dried in stages, crush it appropriately, and extract it three times by reflux with 95% ethanol at a material-to-liquid ratio of 1:8. Combine the resulting extracts, concentrate and recover the solvent to obtain the total extract. (2) Macroporous resin enrichment and purification: The 95% ethanol total extract obtained in step (1) was dispersed in water to a certain concentration and enriched and purified by D-101 macroporous resin column chromatography. It was eluted with water, 25% ethanol and 75% ethanol in sequence, and the 75% ethanol elution fraction was collected. The solvent was recovered to obtain the extract of the 75% ethanol elution fraction. (3) Normal phase silica gel column chromatography: The extract eluted by 75% ethanol in step (2) was separated by normal phase silica gel column chromatography. It was eluted sequentially with pure dichloromethane and dichloromethane-methanol system with different volume ratios to obtain two components, Fr.1 and Fr.2. (4) ODS column chromatography combined with preparative HPLC purification of the target compound: The Fr.1 and Fr.2 fractions obtained in step (3) were further purified by ODS column chromatography, eluted with a methanol-water system, and then separated by preparative high performance liquid chromatography to obtain compound 1 (Juglanin) from the Fr.1 fraction. M Compound 2 (Juglanin) was obtained from the Fr.2 component. L ).
[0007] The present invention also provides the use of the compound in the preparation of antitumor drugs, and preferably in the preparation of drugs for treating liver cancer, lung cancer, and colorectal cancer.
[0008] The beneficial effects and significance of this invention are as follows: This study explores and develops the active ingredients of walnut green peel. Through a systematic separation strategy, compounds with significant anti-tumor activity were successfully screened. Not only are the separation steps simple and easy to prepare, but the prepared compounds also have novel heptane chain structures after structural analysis, providing a potential source of natural products for the development of innovative anti-tumor drugs. Attached Figure Description
[0009] Figure 1 Here is the chemical structural formula of compound 1 of the present invention; Figure 2 The HR-ESI-MS spectrum of compound 1 of the present invention is shown below. Figure 3 Compound 1 of the present invention 1 H-NMR spectrum; Figure 4 Compound 1 of the present invention 13 C-NMR spectrum; Figure 5 The DEPT spectrum of compound 1 of this invention is shown below. Figure 6 The HSQC spectrum of compound 1 of this invention is shown below. Figure 7 The HMBC spectrum of compound 1 of the present invention is shown below. Figure 8 Compound 1 of the present invention 1 H- 1 H COSY spectrum; Figure 9 The ECD spectrum of compound 1 of this invention is shown below. Figure 10 This is a two-dimensional spectral structure diagram of compound 1 of the present invention; Figure 11 Here is the chemical structural formula of compound 2 of this invention; Figure 12 Here is the HR-ESI-MS spectrum of compound 2 of the present invention; Figure 13 Compound 2 of the present invention 1 H-NMR spectrum; Figure 14 Compound 2 of the present invention 13 C-NMR spectrum; Figure 15 The DEPT spectrum of compound 2 of this invention is shown below. Figure 16 The HSQC spectrum of compound 2 of this invention is shown below. Figure 17 The HMBC spectrum of compound 2 of this invention is shown below. Figure 18 Compound 2 of the present invention 1 H- 1 H COSY spectrum; Figure 19 The ECD spectrum of compound 2 of this invention is shown below. Figure 20 This is a two-dimensional spectral structure diagram of compound 2 of the present invention. Detailed Implementation
[0010] Based on the technical content disclosed in this invention, those skilled in the art will clearly understand other embodiments of this invention. The following embodiments are merely examples. Various adjustments and improvements can be made to this invention without violating its spirit and scope. These changes should be within the protection scope of this invention. The invention will be described in detail below with reference to specific embodiments. Example
[0011] The product was obtained from green walnut husks through extraction with 95% ethanol, enrichment and purification with macroporous resin, normal-phase silica gel column chromatography, ODS column chromatography, and preparative HPLC to refine the target analyte. The specific preparation steps are as follows: (1) Ethanol extraction: Take 20.0 kg of green walnut skin, dry it in a vacuum drying oven at 35℃ for 3 h, then raise the temperature to 45℃ and dry it for 1 h. After appropriate crushing, pass it through a No. 2 sieve, and extract it by reflux with 95% ethanol 3 times, each time for 2 h, with a material-liquid ratio of 1:8. Combine the obtained extracts, concentrate and recover the solvent to obtain 2.57 kg of extract. (2) Macroporous resin enrichment and purification: The crude ethanol extract obtained in step (1) was dispersed in water to form a solution with a concentration of 0.45±0.05 g / mL. The concentrated solution was enriched and purified by passing it through a D-101 macroporous resin column (the inner diameter of the column is 14 cm, the height is 180 cm, and the effective height of the resin is 130 cm). The column was eluted with water for 5 column volumes, 25% ethanol for 3.5 column volumes, and 75% ethanol for 5.5 column volumes. The 75% ethanol elution fraction was collected, and the solvent was recovered under reduced pressure to obtain 352.2 g of extract. (3) Normal phase silica gel column chromatography: The extract obtained in step (2) eluted with 75% ethanol was separated by chromatographic separation on a 200-300 mesh silica gel column (the inner diameter of the column is 12 cm, the height is 160 cm, and the effective height of the resin is 80 cm). The extract was eluted with pure dichloromethane for 3 column volumes and discarded. The extract was then eluted with a dichloromethane-methanol gradient of 5:1 for the first 4 column volumes and discarded. The extract was then eluted for another 1.5 column volumes and collected to obtain Fr.1 3.2 g. The extract was then eluted with a dichloromethane-methanol gradient of 2:1 for 2 column volumes and discarded. The extract was then eluted for another 1 column volume and collected to obtain Fr.2 0.9 g. (4) ODS column chromatography combined with preparative HPLC purification of target analytes: The Fr.1 and Fr.2 fractions obtained in step (3) were separated and purified by ODS column chromatography with methanol-water elution (the column selected was a Buchi Pump Module C-601 medium-pressure column with an inner diameter of 30 mm, an effective height of 450 mm for reversed-phase silica gel, a flow rate of 4 mL / min, and an ODS particle size of 50 μm): ①Fr.1 fraction was eluted for 2 column volumes using a methanol-water system at a volume ratio of 10:90 and discarded. Then, it was eluted for 1.5 column volumes using a methanol-water system at a volume ratio of 20:80. The eluent was collected and recovered to obtain crude compound 1. Further purification was performed using preparative high-performance liquid chromatography (HPLC) with a methanol-water mobile phase at a volume ratio of 75:25 and an elution rate of 3 mL / min. R =The fraction was collected within the time period of 51.0 to 51.4 min to obtain compound 1 (7.11 mg); ② Fr.2 fraction was eluted for 3 column volumes using a methanol-water system at a volume ratio of 10:90 and discarded. Elution was then continued for another 1.5 column volumes. The eluent was collected, and the solvent was recovered to obtain crude compound 2. Further purification was performed using preparative high-performance liquid chromatography (HPLC) with a methanol-water mobile phase at a volume ratio of 55:45 and an elution rate of 3 mL / min. R Compound 2 (5.05 mg) was obtained by collecting the fraction within a time period of 31.9–32.1 min. Example
[0012] Identification of Compound 1: The prepared compound 1 of this invention is a colorless amorphous powder, soluble in methanol, and its specific rotation at 26°C is +54.4 (c 0.0974, MeOH); CD(MeOH)λ max (Δ ε): 206 (1.13), 269 (16.81), 319 (-3.87) nm; IR ν max : 3424, 2981, 2950, 2924, 2843, 1644,1471, 1381, 1033, 506, 472 cm -1 ; (-)-HRESIMS: m / z 371.1848 [MH] - (calcd forC 22 H 27 O5 - 371.1858). 1 H-NMR, 13 C-NMR data are shown in Table 1. Based on the physicochemical properties and combined with modern spectroscopic and wave-based data such as UV, CD, mass spectrometry, and NMR spectroscopy, the structure was analyzed and identified as a new compound by searching the SciFinder database. The structure and designation of compound 1 are as follows:
[0013] Table 1. Compound 1 1 H-NMR, 13C-NMR Main Signal Assignment Table
[0014] Identification of Compound 2: The prepared compound 2 of this invention is a yellow amorphous powder, soluble in methanol, and its specific rotation at 25°C was +34.4 (c 0.0823, MeOH); CD(MeOH)λ max (Δ ε): 220 (6.00),275 (19.75) nm; IR ν max : 3441, 2981, 2973, 2865, 2844, 1637, 1555, 1477, 1412,1054, 1032, 1015, 498, 467, 452 cm -1 ; (-)-HRESIMS: m / z 345.1708 [MH] - (calcdfor C 20 H 25 O5 - 345.1702). 1 H-NMR, 13 C-NMR data are shown in Table 2. Based on physicochemical properties and combined with modern spectroscopic and wave-based data such as UV, CD, mass spectrometry, and NMR spectroscopy, the structure was analyzed and identified as a new compound by searching the SciFinder database. The structure and designation of compound 2 are as follows:
[0015] Table 2. Compound 2 1 H-NMR, 13 C-NMR Main Signal Assignment Table
[0016] Example of effect: The effects of two linear diarylheptane compounds on normal human cells and their cytotoxic effects on three types of human tumor cells were determined using the CCK-8 assay.
[0017] (1) Cell types: human normal hepatocytes LO2, human normal lung epithelial cells BEAS-2B, human liver cancer cells HepG-2, human lung cancer cells A549, and human colorectal adenocarcinoma cells HCT-15.
[0018] (2) Specific experimental procedures: LO2 and BEAS-2B normal cells and HepG-2, A549, and HCT-15 tumor cells in logarithmic growth phase and in good growth condition were selected, and 5 × 10⁶ cells were used per well. 4Cells were seeded at a density of 100 μL per well in 96-well plates. After seeding, the tumor cells were placed in DMEM / F-12 medium containing 10% fetal bovine serum and 1% antibiotic and cultured at 37°C with 5% CO2 for 24 h before drug treatment. Drug concentration gradients were set at 128, 64, 32, 16, 8, and 4 μM, with three replicates per group. Blank and control wells were also included (blank wells were unseeded, and control wells contained no drug). After 24 h of drug treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for another 2 h. The absorbance (OD value) of each well at 450 nm was then measured using a microplate reader, and cell viability was calculated based on this value. The experiment was repeated three times, and the average value was used, with cisplatin as a positive control.
[0019] (3) Experimental results: At different detection concentrations, only compound 1 at 128 μM showed a certain inhibitory effect on human normal hepatocytes LO2, with an inhibition rate of 32.5%; the inhibition rates of compound 1 at 64, 32, 16, 8, and 4 μM and compound 2 at 128, 64, 32, 16, 8, and 4 μM on human normal hepatocytes LO2 and human normal lung epithelial cells BEAS-2B were very small, less than 5%; and the experimental data of the half-maximal inhibitory concentrations of the two compounds 1 and 2 and the positive control drug cisplatin on tumor cells at different concentrations are shown in Table 3.
[0020] Table 3. Inhibitory effects of the compounds of this invention on HepG-2, A549, and HCT-15 cells.
[0021] The results in the table above show that the IC calculated by linear regression... 50 The values show that the two linear diarylheptane compounds involved in this invention, compound 1, has an IC50 value against human hepatocellular carcinoma cells HepG-2. 50 The effective concentrations were (39.81 ± 1.46) μM and the IC50 against human lung cancer cells A549 were (39.81 ± 1.46) μM, respectively. 50 The effective concentrations were (46.24 ± 1.54) μM and the IC50 against human colorectal adenocarcinoma cells HCT-15 were respectively. 50 The concentrations were (50.83 ± 1.83) μM, respectively; the IC50 of compound 2 against human hepatocellular carcinoma cells HepG-2 was (50.83 ± 1.83) μM. 50 The effective concentrations were (54.69 ± 1.91) μM and the IC50 against human lung cancer cells A549 were respectively. 50 The effective concentrations were (42.83 ± 1.04) μM and the IC50 against human colorectal adenocarcinoma cells HCT-15 were respectively. 50The concentrations were (46.37 ± 1.39) μM, respectively. This indicates that compounds 1 and 2, two linear diarylheptane compounds, have a certain inhibitory effect on the growth of human hepatocellular carcinoma cells HepG-2, human lung cancer cells A549, and human colorectal adenocarcinoma cells HCT-15, and this effect is dose-dependent. Cisplatin, as a positive control, showed an IC50 of (46.37 ± 1.39) μM on human hepatocellular carcinoma cells HepG-2, human lung cancer cells A549, and human colorectal adenocarcinoma cells HCT-15. 50 The values were (6.21 ± 0.67) μM, (7.11 ± 1.17) μM, and (13.17 ± 0.94) μM, respectively.
[0022] In summary, the two linear diarylheptane compounds isolated from walnut husks described in this invention exhibit strong inhibitory effects on the growth of human liver cancer cells HepG-2, lung cancer cells A549, and human colorectal adenocarcinoma cells HCT-15 without significantly affecting normal cells, demonstrating good selective toxicity to tumor cells and showing promise for development into highly effective and low-toxicity targeted antitumor drugs.
Claims
1. A method for preparing two linear diarylheptane compounds from walnut husk, characterized in that: The product was obtained from green walnut husks through extraction with 95% ethanol, enrichment and purification with macroporous resin, normal-phase silica gel column chromatography, ODS column chromatography, and preparative HPLC to refine the target analyte. The specific preparation steps are as follows: (1) Ethanol extraction: Take the green walnut skin dried in stages, crush it appropriately, and extract it three times by reflux with 95% ethanol at a material-to-liquid ratio of 1:
8. Combine the resulting extracts, concentrate and recover the solvent to obtain the total extract. (2) Macroporous resin enrichment and purification: The 95% ethanol total extract obtained in step (1) was dispersed in water to a certain concentration and enriched and purified by D-101 macroporous resin column chromatography. It was eluted with water, 25% ethanol and 75% ethanol in sequence, and the 75% ethanol elution fraction was collected. The solvent was recovered to obtain the extract of the 75% ethanol elution fraction. (3) Normal phase silica gel column chromatography: The extract eluted by 75% ethanol in step (2) was separated by normal phase silica gel column chromatography. It was eluted sequentially with pure dichloromethane and dichloromethane-methanol system with different volume ratios to obtain two components, Fr.1 and Fr.
2. (4) ODS column chromatography combined with preparative HPLC purification of the target compound: The Fr.1 and Fr.2 fractions obtained in step (3) were further purified by ODS column chromatography, eluted with a methanol-water system, and then separated by preparative high performance liquid chromatography to obtain compound 1 (Juglanin) from the Fr.1 fraction. M Compound 2 (Juglanin) was obtained from the Fr.2 component. L ); The two linear diarylheptane compounds 1 and 2 described above are characterized by having molecular formulas of C1, C2, and C3, respectively. 22 H 28 O5, C 20 H 26 O5, and has the structure shown in the following formula:
2. The use of the two linear diarylheptane compounds of claim 1 in the preparation of antitumor drugs.
3. The application as described in claim 2, characterized in that, The tumors are liver cancer, lung cancer, and colorectal cancer.