A class of chromone derivatives and their preparation method and application

Abstract

The present invention relates to the field of utilization of marine medicinal biological resources, and in particular to a class of chromone derivatives, a preparation method and an application thereof. The present invention obtains chromone derivatives SonnerachromoneA-G (1-7) by extracting and separating the fruit of Sonneratia apetala, a mangrove tree. The chromone derivatives SonnerachromoneA-G are natural active ingredients and are new compounds. It is found that the chromone derivatives SonnerachromoneA-G all have good nematode aging activity, and some compounds have better aging activity than a positive control group. The chromone derivatives can be used alone or in combination as medicinal or non-medicinal anti-aging products, and have good medicinal and dietary supplement prospects.

Description

Technical field

[0001] The present invention relates to the field of utilization of marine medicinal biological resources, and in particular to a class of chromone derivatives and preparation methods and applications thereof. [Background Technology]

[0002] At present, the aging population and the high incidence of aging-related diseases are major social problems faced by the whole world.

[0003] With the advancement of life sciences, our understanding of the mechanisms of longevity has advanced from the holistic and organ-level to the cellular and molecular levels. Caenorhabditis elegans has become a classic model organism for lifespan extension research. The use of mutant nematodes has also matured and is widely used in research on lifespan extension mechanisms.

[0004] Natural medicines are an important component in the search for life-extending ingredients. With the increasing depletion of terrestrial resources, the development of the ocean and the extraction of marine medicines are becoming increasingly urgent. Due to their more specialized living environments than terrestrial organisms, marine organisms are able to produce more active natural products with novel structures and specific effects. Sonneratia apetala, a plant of the genus Sonneratia in the family Rhizophoraceae, has a non-toxic, edible and medicinal fruit that is highly valuable and is commonly used by the public to treat bleeding, diarrhea, and coughs. However, there is currently little research on the medicinally active ingredients in Sonneratia apetala fruit, and even less on the ingredients that have anti-aging effects. Furthermore, no chromone compounds with anti-aging activity have been isolated from the fruit. [Summary of the invention]

[0005] In view of the above, it is necessary to provide a new type of compound that can effectively delay aging and increase the healthy lifespan of nematodes, providing new ideas for the development of anti-aging drugs or dietary supplements.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] Chromone derivatives, including any of the following compounds and combinations thereof:

[0008]

[0009] The present invention also includes a method for extracting the chromone derivatives, which are extracted from Sonneratia apetala fruits.

[0010] Furthermore, the method comprises: crushing the Sonneratia apetala fruit and extracting with ethanol, concentrating the extract under reduced pressure, and then extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain an extraction portion;

[0011] The n-butanol extract was separated by silica gel chromatography using a CHCl3-MeOH system gradient elution. A total of 200 fractions were collected, and fractions with similar polarity were combined and divided into 12 components (Z1-Z12); the 10th component Z10 was collected;

[0012] Fraction Z10 was separated using a silica gel column using a chloroform-methanol solvent system. A total of 120 fractions were collected and combined into nine subfractions (a1-a9). The third fraction, a3, was collected.

[0013] Component a3 was separated using a chloroform-methanol solvent system and a gel chromatography column; a total of 180 fractions were collected and combined into 8 sub-fractions (k1-k8); Sonnerachromone A and Sonnerachromone B were identified from component k3; Sonnerachromone C, Sonnerachromone D and Sonnerachromone E were identified from component k4; Sonnerachromone F and Sonnerachromone G were identified from component k6.

[0014] The present invention also includes the use of the chromone derivative or the chromone derivative prepared by the extraction method in preparing anti-aging related medicines or foods.

[0015] The present invention also includes the use of the chromone derivative or the chromone derivative prepared by the extraction method in extending the lifespan of nematodes.

[0016] The present invention also includes a culture medium for prolonging the lifespan of nematodes. The culture medium is prepared by adding the chromone derivative or the chromone derivative prepared by the extraction method to NGM culture medium.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention obtains chromone compounds (Sonnerachromone A-G) extracted from the fruits of the medicinal mangrove Sonnera apetala. These compounds are natural active ingredients with safe ingredients. These compounds are new compounds and have not been reported in the literature. Among them, the chromone derivatives Sonnerachromone A, Sonnerachromone B and Sonnerachromone C are chromones with a spirocyclic carbon glycoside skeleton. The structural skeleton is relatively novel and has not been reported from natural products.

[0019] 2. The chromone compounds described in this invention (Sonnerachromone A-G) exhibit strong anti-aging activity in nematodes, with some compounds exhibiting superior anti-aging activity compared to the positive control group. They can be used alone or in combination as medicinal or dietary supplements, demonstrating promising drug development prospects. [Specific implementation method]

[0020] Figure 1 This figure shows the effects of chromone derivatives Sonnerachromone A-G (1-7) on the lifespan of Caenorhabditis elegans under normal culture conditions;

[0021] Figure 2 This figure shows the effects of chromone derivatives Sonnerachromone A-G (1-7) on the lifespan of Caenorhabditis elegans under heat stress conditions;

[0022] Figure 3 This is a graph showing the effects of chromone derivatives Sonnerachromone A-G (1-7) on the bending ability of Caenorhabditis elegans;

[0023] Figure 4 This figure shows the effects of chromone derivatives Sonnerachromone A-G (1-7) on the pharyngeal pumping ability of Caenorhabditis elegans.

[0024] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0025] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely one example of a series of equivalent or similar features.

[0026] Example 1:

[0027] This example is a method for preparing chromone compounds Sonnerachromone A-G (1-7), which is as follows:

[0028] (1) Fresh Sonneratia apetala fruits (wet weight approximately 106 kg) were returned to the laboratory on the day of sampling, cleaned, weighed, and crushed. Extraction was performed three times with 95% ethanol, each for 7 days, and concentrated under reduced pressure to obtain an extract. The extract fraction was extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, yielding 1300 g of the n-butanol extract.

[0029] (2) Add appropriate amount of silica gel powder to 1300.0 g of n-butanol extract, stir, dry, grind, and pack with normal phase gel wet method (silica gel 200-300 mesh, solvent: CHCl3, V effective = 15000 cm3 ), a CHCl3-MeOH system (CHCl3:MeOH = 10:1, 10:2.5, 5:2, 0:10 / V:V) was used for gradient elution, with 4-5 effective column volumes of solvent per gradient elution. A total of 200 fractions were collected. According to the TLC analysis results, fractions with similar polarity were combined and roughly divided into 12 components (Z1: 1-6; Z2: 7-23; Z3: 24-35; Z4: 36-49; Z5: 50-78; Z6: 79-92; Z7: 93-110; Z8: 111-135; Z9: 136-152; Z10: 153-167; Z11: 168-189; Z12: 190-200).

[0030] (3) Take component Z10 and separate it using a chloroform-methanol solvent system and a silica gel column (silica gel particles 200-300 mesh, column solvent: chloroform, V effective = 900 cm 3 ) was eluted with a chloroform-methanol system (chloroform:methanol = 10:0, 10:1, 10:1.5, 20:7, 10:5, 0:10 / V:V) for gradient elution. A total of 120 fractions were collected. After TLC analysis, the fractions were combined into 9 subfractions (a1-a9).

[0031] (4) Separate the a3 components using gel column chromatography (methanol column, V effective = 1000 cm 3 ), with methanol:chloroform in a ratio of 1:1 as the elution solvent, a flow rate of 4-7 s / d, and 9-10 mL per fraction. A total of 180 fractions were collected, which were combined into 8 sub-fractions (k1-k8) after TLC analysis.

[0032] (5) Sonnerachromone AB (1-2) (102.2, 78.5 mg, t R =15.7, 16.1min), Sonnerachromone CE (3-5) (15, 20, 5.2 mg, t R =16.3, 16.6, 17.1min), Sonnerachromone FG (6-7) (7.9, 5.7 mg, t R =17.6, 18.1min).

[0033] The structure of the compound Sonnerachromone AG obtained by the above extraction was analyzed as follows.

[0034] The chemical structure of the chromone compounds of the present invention was determined by nuclear magnetic resonance detection and mass spectrometry analysis, and their physicochemical properties and spectral data are as follows:

[0035] Sonnerachromone A(1): Pale yellow needle-shaped crystals, slightly soluble in methanol, freely soluble in DMSO; 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S361.05261 (calcd.for C 15 H 14 O9+Na, 361.05300).

[0036] Sonnerachromone B(2): colorless feather-like crystals, soluble in DMSO. 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S361.05310 (calcd.for C 15 H 14 O9+Na, 361.05300).

[0037] Sonnerachromone C(3): Pale yellow oil, slightly soluble in methanol, freely soluble in DMSO; 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S339.07105 (calcd.for C 15 H 14 O9+H, 361.05300).

[0038] Sonnerachromone D(4): Pale yellow oil, slightly soluble in methanol, freely soluble in DMSO; 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S379.10043 (calcd.for C 15 H 16 O 10 +Na, 339.07117).

[0039] Among them, the compound Sonnerachromone AD (1-4) 1 H (500MHZ) and 13 The relevant data of CNMR (125MHZ) are shown in Table 1:

[0040] Table 1 Sonnerachromone A-D (1-4) 1 H (500MHZ) and 13 CNMR (125MHZ) related data

[0041]

[0042] Compound Sonnerachromone E(5): dark green oil, soluble in methanol, easily soluble in DMSO; 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S225.03972 (calcd.for C 10 H8O6+Na, 225.03936).

[0043] Compound Sonnerachromone F(6): yellow oil, soluble in methanol, easily soluble in DMSO; 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S321.05820 (calcd.for C 12 H 12 O8+Na, 321.05809).

[0044] Among them, the compound Sonnerachromone EF (5-6) 1 H (500MHZ) and 13 The relevant data of CNMR (125MHZ) are shown in Table 2:

[0045] Table 2 Sonnerachromone EF (5-6) 1 H (500MHZ) and 13 CNMR (125MHZ) related data

[0046]

[0047] Compound Sonnerachromone G (7): yellow oil, soluble in DMSO; 1 H(DMSO, 500MHZ), 13 C (DMSO, 125MHZ), HRESIMS: M / S 479.15277 (calcd.for C 21 H 28 O 11+Na, 479.15238).

[0048] Among them, the compound Sonnerachromone G (7) 1 H (500MHZ) and 13 The relevant data of CNMR (125MHZ) are shown in Table 3:

[0049] Table 3 Sonnerachromone G(7) 1 H (500MHZ) and 13 CNMR (125MHZ) related data

[0050]

[0051]

[0052] Among them, the structural formula of the above compounds 1-7 is as follows:

[0053]

[0054] Example 2:

[0055] The compound Sonnerachromone A-G (1-7) obtained by extraction in Example 1 was subjected to an experiment to test the activity of delaying the growth of Caenorhabditis elegans, as follows:

[0056] 1. Preparation of culture medium and buffer: Prepare culture medium and buffer according to the culture medium and buffer formula in Table 4:

[0057] Table 4 Culture medium and buffer formulations

[0058]

[0059]

[0060] (2) Cultivation and synchronization of nematodes: N2 wild-type adults in the egg-laying stage were rinsed with M9 buffer, and the rinse solution was collected in a centrifuge tube. The solution was precipitated in a 4°C refrigerator for 10 min. Part of the supernatant was aspirated, and 1.5 ml of lysis buffer (1 ml of 5M NaOH and 0.5 ml of 5% Naclo were mixed before use) was added. The solution was vortexed for 1-3 min until the worms were lysed. The solution was centrifuged at 1100 rpm for 1 min, and the supernatant was poured off. After washing three times with 1 ml of M9 buffer, the solution was removed. Finally, 0.5 ml of M9 was added to the precipitate, and 100 μL / plate was added to a culture dish containing OP50. The culture was cultured in a 20°C incubator for 48 h until the nematodes reached the L4 stage.

[0061] (3) Preparation of OP50E.coli fermentation broth: A single colony of OP50E.coli was inoculated into LB liquid medium, cultured in a shaker at 37°C for 12 h, diluted with M9 buffer to OD ≈ 0.6, and dispensed into 50 ml EP tubes and stored in a refrigerator at 4°C.

[0062] (4) Sample treatment: The prepared compound Sonnerachromone A-G (1-7) was solubilized with DMSO and diluted to 100 μM with E. coli fermentation broth. 100 μL / plate was added to NGM culture medium and set aside.

[0063] The prepared compound SonnerachromoneA-G (1-7) was used as the experimental group, rapamycin as the positive control, and 0.1% DMSO as the negative control. Three plates were set up for each drug, with 20 nematodes on each plate. The nematodes cultured to the L4 stage were transferred to NGM culture medium supplemented with the corresponding drug at a rate of 60 per group for culture. The number of days of culture at this time was recorded as day 0. Thereafter, the nematodes on the culture medium were counted once a day, and the number of nematodes that survived, died, and were eliminated was observed and recorded (during the lifespan experiment, nematodes were considered dead when they did not respond to external stimuli and had no pharyngeal pumping movement; nematodes that escaped from the surface of the culture medium and died of dryness, whose eggs hatched in the body to form bag-like worms, or burrowed into the culture medium were eliminated). The experiment was repeated three times.

[0064] Acute heat stress experiment: Heat stress is often used to assess the effects of drugs or other factors on the environmental adaptability and health of C. elegans under high temperature. At least 50 nematodes were randomly selected from NGM plates incubated with the corresponding compound for 4 days and transferred from a 20°C to a 37°C incubator. Live and dead nematodes were counted every two hours. Dead nematodes were considered dead if they showed no response to probing with a needle or pharyngeal pumping. The experiment was repeated three times.

[0065] Pharyngeal twitching and body bending ability monitoring: Body bending and pharyngeal pumping are important indicators of C. elegans locomotion and can also provide a direct reflection of the nematode's health span. Bending is a key parameter reflecting nematode locomotion. The pumping function of the C. elegans pharyngeal muscles is similar to that of the human heart. These parameters can directly reflect the aging process. Aging characteristics such as bending and pharyngeal twitching are used as parameters for evaluating health span. Body bending assay: 10 nematodes are divided into groups and transferred to a 96-well plate containing 200 μL M9 buffer. After acclimating for at least 2 minutes, the number of body bends per minute is counted. A complete change in the direction of the entire body bend is considered a single bend. The body bending assay is monitored every 3 days and monitoring ends when bending ability decreases by 50% below optimal levels. Pharyngeal twitching assay: 10 nematodes are transferred to a new NGM plate containing OP50 E. coli and allowed to recover for at least 5 minutes. The number of pharyngeal twitchings per minute was observed every two days, and monitoring was terminated when the pharyngeal twitching capacity decreased by 50% from the optimal level. The experiment was repeated three times.

[0066] Results of the anti-aging activity of compound SonnerachromoneA-G (1-7): In the experiment on the effect of compound SonnerachromoneA-G (1-7) on nematode lifespan, the drug concentration in each group was 100 μM. Compared with the blank group, all compounds could significantly prolong the lifespan of nematodes (P≤0.01). After the administration of compound Sonnerachromone AG (1-7), the average lifespan of nematodes was 16.600±0.544day(d), 15.900±0.432d, 15.200±0.479d, 16.100±0.555d, 14.200±0.390d, 16.500±0.411d, 16.263±0.550d, respectively, which were extended by 40.7%, 34.7%, 28.8%, 36.4%, 20.3%, 39.8%, 37.2% compared with the blank group. Among them, compound Sonnerachromone A (1) had the most significant effect on prolonging the lifespan of nematodes. Figure 1In the heat stress test, the drug concentration of each group was 100μM. The average lifespan of the nematodes in the blank group was 7.754±0.431hour(h). After the administration of the positive drug rapamycin and the compound Sonnerachromone AG(1-7), the average lifespan of the nematodes was 10.846±0.412h, 10.526±0.449h, 9.836±0.454h, 10.300±0.339h, 9.400±0.396h, 8.983±0.347h, 9.705±0.302h, and 9.933±0.359h, respectively. Except for the compound Sonnerachromone E(5), the other compounds showed very significant differences compared with the blank group (P≤0.01). Among them, the compound Sonnerachromone A(1) had the most significant effect, extending the lifespan of the nematodes by 35.7%. Figure 2 .

[0067] In the experiment on the effect of compound Sonnerachromone AG (1-7) on the number of bending times of nematodes, compared with the blank group, Sonnerachromone A-G (1-7) can significantly increase the number of bending times of nematodes on the 6th and 9th days (P ≤ 0.01), and can significantly improve the bending movement ability of nematodes on the 3rd, 12th and 15th days (P ≤ 0.05). Figure 3 In the experiment on the effect on the pharyngeal pumping frequency of nematodes, compared with the blank group, the chromone compound Sonnerachromone AG (1-7) can slow down the decrease in the number of pharyngeal pumping of N2 nematodes after administration (P≤0.05), among which the compound Sonnerachromone A (1) has the most significant activity. The number of pharyngeal pumping of nematodes on the 12th and 14th days increased by 109% and 125% respectively compared with the blank group. Figure 4 The results showed that the compounds of the present invention have an enhancing effect on the pharyngeal pumping and bending of nematodes.

[0068] In summary, the present invention obtains chromone derivatives, Sonnerachromone A-G (1-7), by extracting and isolating the fruit of Sonnera apetala. These derivatives are natural active ingredients, safe ingredients, and novel compounds not previously reported in the literature. Furthermore, experimental verification revealed that the compounds, Sonnerachromone AG (1-7), exhibit strong anti-aging activity, with some exhibiting anti-aging activity far exceeding that of the blank control group. These compounds can be used alone or in combination as medicinal or non-medicinal antioxidants or anti-aging products, demonstrating promising medicinal applications.

[0069] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of the present invention. Therefore, the scope of the present invention shall be determined by the appended claims.

Claims

1. A chromone derivative, characterized in that The structural formula of the chromone derivative is any one of Sonnerachromone A, Sonnerachromone B and Sonnerachromone C: 。 2. The method for extracting chromone derivatives according to claim 1, wherein The chromone derivatives are extracted from the Sonneratia apetala fruit by the following method: crushing the Sonneratia apetala fruit and extracting with ethanol, concentrating the extract under reduced pressure, and then extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain an extraction fraction; The n-butanol extract was separated by silica gel chromatography using a CHCl3-MeOH system gradient elution. A total of 200 fractions were collected, and fractions with similar polarity were combined and separated into 12 components Z1-Z12; Collect the 10th component Z10; Component Z10 was separated using a silica gel column using a chloroform-methanol solvent system; a total of 120 fractions were collected and combined into 9 sub-components a1-a9; Collect the third component a3; Component a3 was separated using a chloroform-methanol solvent system and a gel chromatography column; a total of 180 fractions were collected and combined into 8 sub-fractions k1-k8; Sonnerachromone A and Sonnerachromone B were identified from component k3; Sonnerachromone C, Sonnerachromone D and Sonnerachromone E were identified from component k4; Sonnerachromone F and Sonnerachromone G were identified from component k6; 。 3. Use of the chromone derivative according to claim 1 in the preparation of anti-aging drugs.

4. A culture medium for prolonging the lifespan of nematodes, characterized in that The culture medium is prepared by adding the chromone derivative according to claim 1 to NGM culture medium.

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