A new compound and its preparation method and application

Abstract

The application discloses a new compound and a preparation method and application of the compound. The compound is extracted and separated from a Chinese herb, has anti-aging activity, and can be used for preparing food, medicine and cosmetics with the anti-aging activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a novel compound extracted and isolated from *Gynostemma pentaphyllum*, its preparation method, and its application. Background Technology

[0002] *Vicatia thibetica* H. Boissieu, also known as wild angelica or single-footed angelica, is the root of a plant in the Apiaceae family. It is mainly distributed in northwestern Yunnan, western Sichuan, and Tibet. As a commonly used folk medicine, *Vicatia thibetica* is often used as a substitute for the traditional Chinese medicine *Angelica sinensis* and has a long history of use among the Bai ethnic group. The *Illustrated Handbook of Bai Medicinal Plants* records that *Vicatia thibetica* can strengthen the spleen and kidneys, and is used for blood deficiency or related conditions; the root can be stewed with pig's trotters, or combined with *Angelica sinensis* and mutton. The *Commonly Used Bai Herbal Medicines* records that *Vicatia thibetica* can be used for blood deficiency, wind-cold-dampness syndrome, stomach pain due to cold, lower back pain, and itching. Modern pharmacological studies have shown that *Vicatia thibetica* has antioxidant, anti-fatigue, immune-enhancing, and memory-improving effects. Current research on the pharmacodynamic material basis of *Vicatia thibetica* is insufficient; existing studies have isolated 21 chemical components from it, mainly steroids, coumarin derivatives, and polysaccharides. Summary of the Invention

[0003] The purpose of this invention is to provide a new compound, which is extracted and isolated from Astragalus membranaceus. This compound has anti-aging activity and can be used to prepare food, medicine and cosmetics with anti-aging activity.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A compound with the following structural formula:

[0006] .

[0007] The method for preparing the compound includes the following steps:

[0008] (1) Dry and pulverize the angelica sinensis, extract it by soaking in ethanol, and concentrate it to obtain an alcohol extract. Disperse the alcohol extract in water, and extract it in sequence with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water fraction, respectively.

[0009] (2) The ethyl acetate extract was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate gradient to obtain 6 fractions (Fr.1~Fr.6).

[0010] (3) Take Fr.2 and separate it again by silica gel column chromatography, and elute with petroleum ether-ethyl acetate gradient to obtain 9 fractions (Fr.A~Fr.I).

[0011] (4) The fraction Fr.B was purified by semi-preparative high performance liquid chromatography to obtain the compound.

[0012] Preferably, the ethanol used for soaking and extraction in step (1) is 95% ethanol.

[0013] Preferably, in step (2), the volume ratio of petroleum ether to ethyl acetate is 60:1 to 0:100.

[0014] Preferably, in step (3), the volume ratio of petroleum ether to ethyl acetate is 50:1 to 0:100.

[0015] Preferably, the conditions for semi-preparative high performance liquid chromatography in step (4) are: mobile phase is ACN:H2O (volume ratio) = 28:72, and flow rate is 6 mL / min.

[0016] The compound can be added with food and / or pharmaceutically acceptable excipients and / or carriers to form compositions and formulations, and can be further formulated into food, pharmaceuticals, cosmetics, etc.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This invention is the first to isolate a new compound, Vicatia A, from Astragalus membranaceus, enriching the research foundation of active ingredients in Astragalus membranaceus, clarifying the material basis for Astragalus membranaceus to exert its anti-aging effects, and providing a new theoretical basis for the high-value development and utilization of Astragalus membranaceus resources.

[0019] 2. This invention provides a method for preparing Vicatia A. Through steps such as ethanol extraction, solvent extraction, silica gel column chromatography separation, and semi-preparative liquid chromatography purification, the target compound with high purity can be obtained. The preparation process is stable, the operation method is clear, and it has good reproducibility and application promotion value.

[0020] 3. The Vicatia A obtained by this invention has significant anti-aging activity, can prolong the lifespan of model organisms, improve the body's antioxidant stress capacity, and improve aging-related physiological functions. It has potential application value as an active ingredient in anti-aging drugs, functional foods, and cosmetics.

[0021] 4. This invention expands the application scope of Astragalus membranaceus in the field of anti-aging and health products, and provides a new technical approach for the modernization of traditional Chinese medicine resources and the research and development of related products. Attached Figure Description

[0022] Figure 1 This is the HR-ESI-MS mass spectrum of Vicatia A obtained in this invention.

[0023] Figure 2 The HSQC spectrum (CD3OD) of Vicatia A obtained in this invention is shown.

[0024] Figure 3 The HMBC spectrum (CD3OD) of Vicatia A obtained in this invention is shown.

[0025] Figure 4 The Vicatia A obtained by this invention 1 H- 1 H COSY spectrum (CD3OD).

[0026] Figure 5 The NOESY spectrum (CD3OD) of Vicatia A obtained in this invention is shown.

[0027] Figure 6 For Vicatia A's key HMBC, 1 H- 1 H COSY and NOESY, where A is the key HMBC and 1 H- 1 HCOSY, B is NOESY (in the diagram: Indicates HMBC, express 1 H- 1 H COSY, (This means NOESY).

[0028] Figure 7 The effect of Vicatia A on the lifespan of nematodes (in the figure, KB is the control group, and Vicatia A is the Vicatia A group).

[0029] Figure 8 The effect of Vicatia A on the body wiggling ability of nematodes (in the figure, the first of each color is the blank group, and the second is the Vicatia A group).

[0030] Figure 9 The effect of Vicatia A on the swallowing frequency of nematodes (in the figure, the first color of each group is the blank group, and the second color is the Vicatia A group).

[0031] Figure 10 The effect of Vicatia A on the UV stress resistance of nematodes (in the figure, KB is the control group and Vicatia A is the Vicatia A group). Detailed Implementation

[0032] The present invention will be further described in detail below with reference to embodiments. These embodiments are merely illustrative of the technical features, objectives, and effects of the present invention, and are not intended to limit the scope of the invention.

[0033] Example 1

[0034] The astragalus root was naturally air-dried, and 14.80 kg was weighed, crushed, and soaked in 95% ethanol for one month. The extract was collected and repeated twice. The extracts were combined and concentrated under reduced pressure to obtain an alcohol extract. The weight of the extract was 3.07 kg, and the yield was calculated to be 20.74%.

[0035] The obtained zeaxanthin extract was dispersed in distilled water and then extracted stepwise with petroleum ether, ethyl acetate and n-butanol, each extraction was performed 4 times, each time for 4 hours; 167g of petroleum ether fraction (VTE-PE), 20g of ethyl acetate fraction (VTE-EA), 137g of n-butanol fraction (VTE-n-BuOH) and 1900g of the final raffinate fraction (VTE-RA) were obtained.

[0036] The ethyl acetate extract of *Syzygium aromaticum* was subjected to silica gel column chromatography (chromatographic silica gel, 200-300 mesh, purchased from Qingdao Ocean Chemical Plant) for separation. Gradient elution was performed with petroleum ether-ethyl acetate (60:1-0:100) (v / v). Based on thin-layer chromatography, after combining the same fractions, a total of 6 fractions (Fr.1-Fr.6) were obtained.

[0037] Fr.2 from the obtained 6 fractions was further separated by silica gel column chromatography, eluted with a PE-EtOAc (v / v) gradient of 50:1 to 0:100, and identified by thin-layer chromatography and high-performance liquid chromatography (mobile phase: methanol-water, 0–30 min, methanol volume fraction 10%–100%, flow rate: 0.8 mL / min). After combining the same fractions, 9 fractions Fr.AI were obtained. Fraction Fr.B was purified by semi-preparative high-performance liquid chromatography (ACN:H2O (v / v) = 28:72, flow rate: 6 mL / min) to obtain the new compound (1.5 mg), named Vicatia A.

[0038] Example 2 – Structural Identification of Compound Vicatia A

[0039] The compound Vicatia A obtained in Example 1 was a colorless gel with an HRESIMS m / z of 305.1006 [M+Na]. + (calcd. for C) 14 H 18 O6Na, 305.1001 (e.g.) Figure 1 As shown), combined with its 1H-NMR and 13 C10-NMR data confirmed that the molecular formula of this structure is C10. 14 H 18 O6, the calculated degree of unsaturation is 6.

[0040] 1 1H-NMR (600MHz, CD3OD) showed that Vicatia A contains two methyl signal δ groups. H 3.67 (3H, s, H-15), 3.23 (3H, s, H-16); 3 methylene proton signals δ H 4.19 (1H, dd, J = 12.0, 8.4, H-8) / 4.10 (1H, dd, J = 12.0, 4.2, H-8), 2.61 (2H, overlap, H-11), 2.62 (2H, overlap, H-12); 1 methylene proton signal δ H 4.36 (1H, dd, J = 7.8, 3.6, H-7); 4 aromatic proton signals δ H 6.79 (2H, d, J = 8.4, H-3 / H-5), 7.16 (2H, d, J = 8.4, H-2 / H-6).

[0041] 13 C-NMR (150 MHz, CD3OD) showed 14 carbon signals: δ C 174.5 (C-13), 173.9 (C-10), 158.8 (C-4), 129.8 (C-2 / 6), 129.4 (C-1), 116.4 (C-3 / 5), 82.4 (C-7), 68.9 (C-8), 56.9 (C-16), 52.2 (C-15), 29.9 (C-11), 29.7 (C-12). The directly linked carbons and hydrogens were assigned using HSQC spectra, as shown in Table 1.

[0042] Table 1. Vicatia A 1 H-NMR and 13 C-NMR data

[0043] Vicatia A with 4-hydroxyphenethyl methyl succinate 1 D、 2 D-NMR data comparison suggests that Vicatia A has a similar structure to 4-hydroxyphenethyl methyl succinate, with the main difference being an additional substituent at C-7.1 In D-NMR, Vicatia A and similar compounds show δ-ray diffraction. C The displacement at 82.4 (C-7) changes; in the QC spectrum, δ C 82.4 only with δ H 4.36 is directly correlated, and Vicatia A has one more carbon signal δ compared to similar compounds. C 56.9, it is speculated that Vicatia A has an additional substituent at C-7.

[0044] In HSQC spectra (such as) Figure 2 In the figure shown, δ C 56.9 and δ H 3.23 (3H, s) is directly related. From the HMBC spectrum (e.g. Figure 3 δ was observed in (as shown) H 3.23 (3H, s) and C-7 (δ) C 82.1) shows a relevant signal, indicating that C-7 is substituted with a methoxy group. The HMBC spectrum shows relevant signals at H 4.19 (1H, dd, H-8) and 7.16 (2H, d, H-2 / H-6) / C-7 (δC 82.14), combined with... 1 H- 1 HCOSY chart (e.g.) Figure 4 In the (as shown), H2 / 6 is correlated with H3 / 5; H7 / H8, as shown in the NOESY spectrum (e.g. Figure 5 As shown), H2 / 6 has correlated signals with H8, H7, and H3 / 5, and H7 has correlated signals with H8. ECD is being fitted based on the CD spectrum data (e.g., Figure 6 As shown in the figure, Vicatia A is confirmed to have an S configuration. In summary, the structural formula of Vicatia A can be determined as follows:

[0045] .

[0046] Example 3 – Anti-aging Activity Experiment

[0047] 1. Experimental Procedure

[0048] 1.1 Caenorhabditis elegans lifespan experiment: A senescence model was established using synchronized L4-stage hermaphroditic nematodes. A single-generation population was maintained in NGM medium supplemented with ampicillin (to prevent bacterial contamination) and pentafluorodeoxyuridine (FudR, to inhibit reproduction). Vicatia A was prepared as a 50 μM stock solution with M9 buffer + 50 μL of E. coli OP50 and added to NGM medium. The control group was prepared as a 50 μM M9 buffer solution + 50 μL of E. coli added to NGM medium, serving as the administration plates. Synchronized L4 larvae (20-25 larvae / plate) were inoculated onto the administration plates. The number of survivors was counted daily (death was defined as no response to platinum wire stimulation), and individuals with vulvar rupture or those that dried up and climbed the wall were removed until all larvae died.

[0049] 1.2 Experiment on the healthy lifespan of *C. elegans*: A senescence model was established using synchronized L4-stage hermaphroditic nematodes. A single-generation population was maintained in NGM medium supplemented with ampicillin (to prevent bacterial contamination) and pentafluorodeoxyuridine (FudR, to inhibit reproduction). Vicatia A was prepared as a 50 μM stock solution with M9 buffer + 50 μL of *E. coli* OP50 and added to NGM medium. For the control group, 50 μM M9 buffer solution + 50 μL of *E. coli* was added to NGM medium to prepare the administration plates. Synchronized L4 larvae (20-25 larvae / plate) were inoculated onto the administration plates.

[0050] (1) Body swinging experiment: On odd-numbered days from day 1 to day 11, nematodes (20-25 nematodes per group) in the blank group and the drug-treated group were transferred one by one to an NGM plate without OP50 (50 μL of M9 buffer was added). After acclimatization for 30 seconds, the number of times the body flexed on one side within 30 seconds was recorded under a stereomicroscope.

[0051] (2) Pharyngeal pump frequency experiment: Under the same conditions, observe and record the number of pharyngeal suction movements of nematodes within 30 seconds.

[0052] 1.3 Experiment to determine the UV stress resistance of *C. elegans*: The regulatory effect of Vicatia A on the stress resistance of nematodes was evaluated using a UV stress model. Synchronized L4-stage nematodes were exposed to NGM medium containing 50 μM Vicatia A (M9 buffer) stock solution (incubated at 20°C for 5 days). The control group's NGM medium did not contain Vicatia A. Three biological replicates were set up for each group (20-30 nematodes per plate). Before stress treatment, the nematodes were transferred to blank NGM plates.

[0053] UV stress model: The tablets were exposed to a UV lamp (254nm, 15W) with the lid off, and the mortality rate was recorded every 0.5 hours. Nematode mortality was determined using the triple negative criterion: ① no spontaneous movement (cessation of body swaying); ② disappearance of pharyngeal pump movement; ③ no avoidance response to platinum filament tactile stimulation.

[0054] 2. Experimental Results

[0055] 2.1 Effects on the lifespan of *Caenorhabditis elegans*

[0056] like Figure 7 As shown in Table 2, Vicatia A increased the median survival time, average lifespan, and maximum lifespan of nematodes compared to the control group. Furthermore, Vicatia A exhibited superior life-extending activity, exceeding 30.00%.

[0057] Table 2. Effects of Vicatia A on the lifespan of Caenorhabditis elegans.

[0058] 2.2 Effects on the healthy lifespan of *C. elegans*

[0059] 2.2.1 Effects on the body wiggling ability of *Caenorhabditis elegans*

[0060] This study evaluated the effect of Vicatia A on the body bending frequency of nematodes. The number of body swings within 30 seconds was measured on days 1, 3, 5, 7, 9, and 11 of the adult stage. The results are as follows: Figure 8 As shown: Vicatia A group showed an increasing trend of vitality from day 3, and the frequency of swinging increased significantly from day 5 to day 7. The difference between the two groups was statistically significant (*P<0.05).

[0061] 2.2.2 Effects on the swallowing frequency of *C. elegans*

[0062] This study evaluated the effect of Vicatia A on the swallowing frequency of nematodes. The number of swallowings within 30 seconds was measured on days 1, 3, 5, 7, 9, and 11 of the adult stage. The results are as follows: Figure 9 As shown, the swallowing frequency of nematodes within 30 seconds gradually decreased over time. In particular, the Vicatia A group showed a significant decreasing trend in swallowing frequency starting from day 3, and the difference between the two groups reached statistical significance on day 5 (*P<0.05).

[0063] 2.2.3 Effects on the UV stress resistance of *Caenorhabditis elegans*

[0064] The results are shown in Table 3 and Figure 10 As shown, the mean survival time of the control group was 3.90 h, while the Vicatia A group exhibited better enhancement of nematode resistance to ultraviolet stress, with the mean survival time extended to 5.53 h (an increase of 41.79%). These results suggest that the life-extending mechanism of Vicatia A may be related to enhancing the organism's resistance to ultraviolet stress.

[0065] Table 3 Effects of Vicatia A on UV stress resistance in nematodes

[0066]

Claims

1. A compound, characterized in that, The structural formula is: 。 2. A composition comprising the compound of claim 1 and food- and / or pharmaceutically acceptable excipients and / or carriers.

3. The use of the compound according to claim 1 in the preparation of anti-aging foods, pharmaceuticals, and cosmetics.

4. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: (1) Dry and pulverize the angelica sinensis, soak it in ethanol for extraction, and concentrate it to obtain an alcohol extract. Disperse the alcohol extract in water and extract it in sequence with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water fraction respectively. (2) The ethyl acetate extract was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate gradient to obtain 6 fractions Fr.1~Fr.6; (3) Fr.2 was separated again by silica gel column chromatography and eluted with petroleum ether-ethyl acetate gradient to obtain 9 fractions of Fr. AI; (4) The fraction Fr.B was purified by semi-preparative liquid chromatography to obtain the compound.

5. The preparation method according to claim 4, characterized in that, The ethanol used for soaking and extraction in step (1) is 95% ethanol.

6. The preparation method according to claim 4, characterized in that, In step (2), the volume ratio of petroleum ether to ethyl acetate is 60:1 to 0:

100.

7. The preparation method according to claim 4, characterized in that, In step (3), the volume ratio of petroleum ether to ethyl acetate is 50:1 to 0:

100.

8. The preparation method according to claim 4, characterized in that, In step (4), the mobile phase for the semi-preparative high performance liquid chromatography is ACN:H2O with a volume ratio of 28:

72.

9. The preparation method according to claim 4, characterized in that, In step (4), the flow rate of the semi-preparative high performance liquid chromatography is 6 mL / min.

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