New anti-aging iridoid components in eucommia ulmoides oliver leaf and preparation method and application thereof

Abstract

The present invention relates to a novel anti-aging active iridoid component extracted from Eucommia ulmoides leaves, as well as its preparation method and application. Through advanced extraction and separation technology, we successfully isolated a novel iridoid compound from Eucommia ulmoides leaves, which showed significant antioxidant and anti-aging properties. The preparation method of the present invention includes precise solvent selection, temperature control and purification steps, ensuring high purity and high activity of the target compound. In addition, we also explored the potential applications of this new compound in cosmetics, pharmaceuticals and health foods, especially in anti-aging and skin care. The invention not only expands the application range of iridoid compounds, but also provides new raw materials for natural anti-aging products.

Description

Technical Field

[0001] This invention relates to the field of plant extract technology, and specifically discloses a method for extracting and applying anti-aging active iridoids from Eucommia ulmoides leaves. Background Technology

[0002] Eucommia ulmoides is a traditional Chinese medicinal herb that has long been used to improve physical strength and endurance. Recent studies have found that Eucommia ulmoides leaves contain a variety of bioactive components, including iridoids, which exhibit potential antioxidant and anti-aging properties. However, current research on these compounds in Eucommia ulmoides leaves is relatively limited, particularly regarding their extraction methods and specific applications.

[0003] Traditional plant extraction techniques, such as water extraction and alcohol extraction, are often inefficient and may not retain all active ingredients. Furthermore, many naturally derived anti-aging ingredients exhibit poor stability during purification and application, limiting their widespread use in the pharmaceutical and cosmetic fields. Summary of the Invention

[0004] In view of the above shortcomings, this invention discloses a novel anti-aging active iridoid ether compound extracted from Eucommia ulmoides leaves. This compound exhibits high stability, high extraction efficiency, and excellent effects in the production of anti-aging products. The anti-aging products are pharmaceuticals or cosmetics.

[0005] This invention is achieved through the following technical means:

[0006] This invention first discloses a novel anti-aging active iridoid ether compound extracted from Eucommia ulmoides leaves, which is formed in Eucommia ulmoides leaves through a specific biosynthetic pathway. We performed structural identification on this compound and found that it has a unique chemical structure (Formula I) and significant anti-aging properties.

[0007] (Formula I).

[0008] This invention also discloses a method for preparing a novel anti-aging active iridoid ether compound extracted from Eucommia ulmoides leaves, comprising the following steps:

[0009] Eucommia ulmoides leaves are extracted under controlled conditions using a specific solvent mixture, followed by separation and purification of the target compounds using advanced chromatographic techniques. This method offers advantages in terms of high efficiency, high purity, and excellent retention of the active ingredients.

[0010] Furthermore, the preparation method includes:

[0011] 12 kg of fresh Eucommia ulmoides leaves were dried in the shade, crushed, and passed through a 180-mesh sieve to obtain Eucommia ulmoides leaf powder for later use.

[0012] The sample was extracted three times with 50 L of 75%~80% ethanol by heating, each time for 3 h, and then concentrated to 25 L under vacuum.

[0013] Then, the extract was extracted three times with ethyl acetate at a volume ratio of 1:1. The filtrates were combined and concentrated under reduced pressure to obtain 3.9 kg of ethyl acetate extract.

[0014] After dissolving the obtained extract, an equal mass of silica gel was weighed and mixed with the extract. The mixture was then separated by silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate (10:1-1:10). During the elution process, TLC was used to track and combine the fractions, resulting in 5 fractions Fr. AE.

[0015] 13.6 g of Fr. A was separated by gradient chromatography on an MCI column using dichloromethane-water (70:30-100:0, v / v) to obtain three fractions: Fr. A1-A3.

[0016] 300 mg of fraction Fr.A2 was eluted with dichloromethane-water (1:1, v / v) and identified by high performance liquid chromatography (HPLC, methanol:water = 7:3, v / v) to obtain a new iridoid terpene component with a purity of 98%, as shown in Formula I, namely DT.

[0017] The present invention also discloses an application of the novel anti-aging active iridoid ether compound extracted from Eucommia ulmoides leaves, which is used in the preparation of cosmetics and pharmaceuticals.

[0018] Furthermore, in cosmetics, this compound can be used to create products that reduce wrinkles, moisturize, and improve skin elasticity.

[0019] Furthermore, in the pharmaceutical field, it can be used to develop anti-aging and performance-enhancing products.

[0020] The beneficial effects of this invention are as follows:

[0021] The invention provides a novel drug candidate molecule derived from natural plants; the isolation and preparation steps are short and can be scalable; it has higher efficacy and fewer side effects. Attached Figure Description

[0022] Figure 1 This is a high-resolution mass spectrum of DT (cyclohexene ethers);

[0023] Figure 2 The following are the proton and carbon NMR spectra of DT, where Figure 2 A is the DT hydrogen spectrum. Figure 2 B is the DT carbon spectrum;

[0024] Figure 3The DEPT spectrum of DT;

[0025] Figure 4 The HSQC spectrum of spectrum DT;

[0026] Figure 5 The HMBC spectrum of spectrum DT;

[0027] Figure 6 For the spectrum DT 1 H- 1 H COSY spectrum;

[0028] Figure 7 The NOESY spectrum of spectrum DT;

[0029] Figure 8 The figure shows the effect of continuous DT administration for 9 weeks on D-galactose-induced body weight in mice; where NC is the blank group, MC is the model group, PC is the NMN positive group, DT is the DT administration group of Eucommia ulmoides glycoside derivative, and WSW is the microbial administration group.

[0030] Figure 9 The results of SOD and MDA detection in mice induced by D-galactose after 9 consecutive weeks of DT administration. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] To better understand the present invention, the following embodiments further illustrate the invention in detail, but these should not be construed as limiting the invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description are also considered to fall within the protection scope of the present invention. The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

[0033] The instruments and reagents used in the following examples are:

[0034] Instruments: Hewiett Pakard 110 mass spectrometer (Hewlett-Packard, Inc., USA), Bruker AM-600MHz nuclear magnetic resonance spectrometer (Bruker Corporation, Inc., USA).

[0035] Reagents and reagents: Eucommia ulmoides leaves (origin: Baiyun District, Guiyang City, Guizhou Province); ethanol, petroleum ether, and ethyl acetate were of analytical grade; water was ultrapure water; 200-300 mesh silica gel (Qingdao Marine Biological Company).

[0036] Example 1

[0037] Preparation method of novel anti-aging iridoid ether terpenoid components from Eucommia ulmoides leaves:

[0038] Fresh Eucommia ulmoides leaves (12 kg) were air-dried, pulverized, and passed through a 180-mesh sieve. They were extracted three times (3 h each time) with 75%–80% ethanol (50 L) under heat, and concentrated to 25 L under vacuum. Then, they were extracted three times with ethyl acetate (1:1 v / v). The filtrates were combined and concentrated under reduced pressure to obtain 3.9 kg of ethyl acetate extract. The extract was dissolved, and an equal volume of silica gel was weighed and mixed. Separation was performed by silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate (10:1–1:10). TLC was used to monitor and combine the elutions, yielding five fractions (Fr. AE). Fr. A (13.6 g) was separated by gradient chromatography on an MCI column using dichloromethane-water (70:30–100:0, v / v) to obtain three fractions (Fr. A1–A3). The fraction Fr.A2 (300 mg) was eluted with dichloromethane-water (1:1, v / v), and high performance liquid chromatography (HPLC, methanol:water = 7:3, v / v) was used to obtain a new iridoid terpene, DT, as shown in Formula I, with a purity of 98%.

[0039] (Formula I).

[0040] Structure determination and derivation of the prepared Formula I compounds:

[0041] Its structure is mainly identified by nuclear magnetic resonance (NMR) spectroscopy, and its molecular weight and molecular formula are then precisely determined by high-resolution mass spectrometry (e.g., Figure 1 (As shown).

[0042] Its spectral data is as follows:

[0043]

[0044]

[0045] Experimental Example 1

[0046] Anti-aging activity test:

[0047] (1) Grouping of animals:

[0048] Preparation of aging mouse model: Healthy adult female ICR mice aged 6-8 weeks were selected and injected subcutaneously into the neck and back with D-galactose 500mg / kg and physiological saline injection 0.1ml / 10g once a day for 9 consecutive weeks to prepare aging mouse model.

[0049] D-galactose saline injection: Prepare D-galactose saline injection according to the dosage of 400 mg / kg*d and the volume of administration of 0.1 ml / 10 g, that is, prepare a D-galactose solution of 40 mg / ml.

[0050] NMN saline solution: Prepare NMN saline solution according to the dosage of 500mg / kg*d and the administration volume of 0.1ml / 10g; that is, prepare an NMN saline solution of 50mg / ml.

[0051] DT group: 0.9% saline solution was prepared and administered at a dose of 2 mg / kg*d. A total of 10 animals were given the drug for 42 days. The dosage was measured at 0.1 ml / 10 g, and the required monomer was approximately 25.2 mg.

[0052] Mouse treatment: After the last gavage, mice were fasted and deprived of water for 12 hours. Blood was collected from their eyes, taking care to ensure cleanliness during collection to prevent hemolysis of red blood cells. After equilibration at room temperature for 1-2 hours, the mice were centrifuged at 4°C and 3500 r / min for 15 min. The supernatant was collected, aliquoted, and stored at -80°C. Mice were euthanized by cervical dislocation, and the liver, kidneys, and heart were dissected and removed. The organs were washed with 4°C physiological saline to remove bloodstains and other impurities and stored at -80°C.

[0053] (2) Detection indicators

[0054] Weight recording: Mouse weight was recorded weekly.

[0055] Compared with the blank control group and the model group, the DT-treated group, i.e., the new compound group, did not show a decrease in body weight from the start of the experiment to the end of week 9 (e.g., Figure 8 (As shown in the figure); and combined with actual observation of the mouse activity status, the DT administration group significantly delayed the aging effect of mice.

[0056] Growth status record: Take photos weekly to record the appearance, behavior and other status of the mice.

[0057] Mouse organ index determination: Blood, thymus, spleen, liver, and feces were collected from mice and weighed using an electronic balance. The organ coefficient of each organ and tissue was calculated using the following formula: Organ coefficient / (mg / g) = Organ mass / Mouse body mass. Thymus and spleen indices: The thymus is an important lymphatic organ in animals, playing a vital role in immunity. It is also one of the first organs to age in animals. The proportion of the thymus in an animal's body can indirectly indicate the degree of aging, and the thymus index can indirectly reflect the body's immune function.

[0058] Table 1. Effects of DT on organ indices induced by D-galactose in mice.

[0059]

[0060] Note: Compared with the MC group control group, "*" indicates a significant difference (P<0.05), and "**" indicates an extremely significant difference (P<0.01); compared with the NC experimental group, "#" indicates a significant difference, and "##" indicates an extremely significant difference (P<0.01).

[0061] Table 1 shows the effects of oral administration of the DT group on organ indices in mice. The intervention was able to slow down or improve age-related changes in organ indices, which may indicate that the novel iridoid ether component has potential anti-aging effects.

[0062] Serum SOD and MDA levels (blood was collected from decapitated aging mice at week 7 of intervention, incubated at 4°C for 2 hours, centrifuged at 3500 r / min for 15 minutes, and the supernatant was collected. The procedure was performed according to the instructions of the micro-malondialdehyde test kit).

[0063] Compared with the blank control group and the model group, the DT-treated group (i.e., the new compound group) showed better serum SOD activity than the model group and was close to the blank control group (NC), indicating that the new compound enhances SOD activity in mice, thereby delaying aging. Similarly, compared with the model group, the DT-treated group showed a significant decrease in serum MDA, and was equal to the blank control group (NC), indicating that the new compound can reduce serum MDA and achieve the purpose of delaying aging (see results). Figure 9 ).

[0064] Application Example 1

[0065] Application of DT in the preparation of anti-aging drugs:

[0066] Based on the experimental results, the newly extracted iridoid compounds were applied to the development of anti-aging products. These anti-aging products include cosmetics and pharmaceuticals. For example, in cosmetics, the compound can be added at appropriate concentrations to lotion or cream formulations to enhance their anti-wrinkle and moisturizing effects. In the pharmaceutical field, the compound can be used to manufacture oral supplements or drugs to support the body's anti-aging efforts and enhance physical performance.

[0067] DT is used as a single active ingredient and added to a pharmaceutical carrier or excipient (one or more solid, semi-solid, liquid diluents, fillers, and pharmaceutical excipients) to form a pharmaceutical preparation for treating anti-aging (the dosage form can be tablets, capsules, drops, or granules), with an effective dose of 25 mg / kg.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a novel anti-aging active iridoid ether terpenoid component from Eucommia ulmoides leaves, comprising: (1) 12 kg of fresh Eucommia ulmoides leaves were dried in the shade, crushed, and passed through a 180-mesh sieve to obtain Eucommia ulmoides leaf powder for use. (2) Extract with 50 L of 75%~80% ethanol by heating and concentrate under vacuum to obtain the first extract for later use; (3) The first extract was extracted three times with ethyl acetate at a volume ratio of 1:

1. The filtrates were combined and concentrated under reduced pressure to obtain 3.9 kg of ethyl acetate extract. (4) After dissolving the ethyl acetate extract, weigh an equal mass of silica gel and mix it with the extract. Separate the sample by silica gel column chromatography, using petroleum ether-ethyl acetate gradient elution. During the elution process, use TLC to track and combine the fractions to obtain 5 fractions Fr.AE; (5) 13.6 g of Fr. A was separated by gradient chromatography on an MCI column using dichloromethane-water solution to obtain three fractions, namely Fr. A1, Fr. A2 and Fr. A3; (6) Take 300 mg of fraction Fr.A2 and elute with dichloromethane-water. After high performance liquid chromatography, the new iridoid ether terpene component with a purity of 98% as shown in Formula I is obtained, namely DT; (Equation I).

2. The preparation method according to claim 1, wherein: The heating and extraction temperature in step (2) is 50℃, the extraction time is 3h, and the extraction is performed 3 times.

3. The preparation method according to claim 1, wherein: The vacuum concentration in step (2) is 25 L.

4. The preparation method according to claim 1, wherein: The volume ratio of petroleum ether to ethyl acetate in step (4) is 10:1 to 1:

10.

5. The preparation method according to claim 1, wherein: In step (5), the volume ratio of dichloromethane to water in the dichloromethane-water solution is 70:30-100:

0.

6. The preparation method according to claim 1, wherein: In step (6), the volume ratio of dichloromethane to water in the dichloromethane-water solution is 1:

1.

7. The application of a novel anti-aging active iridoid ether terpene component from Eucommia ulmoides leaves in the preparation of anti-aging products, wherein: The anti-aging products mentioned are: cosmetics and pharmaceuticals; The structural formula of the novel anti-aging active iridoid ether terpenoid component in Eucommia ulmoides leaves is as follows: (Equation I).

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