Preparation method and application of yunnan rhizoma paridis saponin G

Abstract

This invention provides a method for obtaining ingredients from Dulong Paris polyphylla (… Paris dulongensis A method for preparing parisyunnanoside G (PFE55) in H. Li & Kurita and its application in cosmetics and pharmaceuticals. This research belongs to the field of cosmetics and pharmaceutical technology. PFE55 is used at extremely low concentrations (0.1... μ At a concentration of g / mL, it exhibits activity in promoting collagen secretion in adult dermal fibroblasts (HDFa) and is non-cytotoxic at this concentration, making it suitable for the preparation of anti-aging drugs or skincare products.

Description

Technical Field

[0001] This invention belongs to the fields of cosmetics and pharmaceutical technology, specifically relating to the field of herbal medicine technology, and more specifically, to Yunnan Paris saponin G, which has collagen-promoting activity, as well as its preparation method and application. Background Technology

[0002] With the improvement of living standards and the development of medical technology, while meeting basic living needs, delaying aging has become a new pursuit for people. Skin aging is the result of various factors working together, mainly divided into two types: one is endogenous aging, mainly influenced by genetic factors, which is a natural aging process; the other is exogenous aging caused by external environmental factors such as ultraviolet radiation, smoking, gravity, and chemicals. Maintaining youthful skin, solving skin problems, and restoring skin health have become new research hotspots. 75% of the skin is composed of collagen, and skin growth, repair, and nutrition all depend on collagen. The two key aspects of healthy skin—moisturizing and anti-wrinkle—are both related to collagen. The effects of anti-aging active substances include scavenging free radicals, increasing cell proliferation rate, slowing down the degradation rate of the extracellular matrix, and replenishing collagen in the skin, restoring and rebuilding the collagen layer. Currently, common methods of supplementing collagen include oral intake of collagen-rich foods, subcutaneous injections, and the use of collagen-rich skincare products, but their effects are limited, the time required is long, they are prone to causing allergies and infections, and the absorption effect is limited. Adding active ingredients that stimulate the skin's own collagen synthesis to skincare products or medications to replenish collagen has become a new focus in anti-aging drug and skincare product research and development. Therefore, finding natural anti-aging products that promote the skin's own collagen secretion has become a new research trend.

[0003] Yunnan Paris saponin G, Figure 1 () is a polyhydroxyspirostane alcohol compound, which was previously isolated from the rhizome of Paris rugosa H. Li & Kurita [Duan Xiaoyan, Yue Meicen, Yang Jun, Bai Xue, Luo Jifeng, Li Heng, Wang Yuehu. Study on chemical constituents and antibacterial activity of rhizome of Paris rugosa. Chinese Journal of Traditional Chinese Medicine 2023, 48 (11), 2981–2988], but there were no reports of its activity in promoting collagen secretion. Summary of the Invention

[0004] The purpose of this invention is to provide Yunnan Paris saponin G (PFE55), its preparation method, and its application in the preparation of anti-aging drugs or skin care products.

[0005] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0006] The preparation method of Yunnan Paris saponin G compound PFE55, as shown in the following structural formula, is described.

[0007]

[0008] The method includes the following steps:

[0009] 1.4 kg of dried Paris polyphylla rhizomes were crushed and extracted with 70% ethanol at 60 °C using ultrasound-assisted extraction. The filtrate was then filtered. The residue was extracted again under the same conditions for a total of 3 extractions. The filtrates were combined and the solvent was recovered under reduced pressure using a rotary evaporator to obtain a crude extract. The crude extract was then mixed with water to prepare a suspension, which was extracted with n-butanol for a total of 3 extractions. The extracts were combined and the solvent was recovered under reduced pressure to obtain the n-butanol extract.

[0010] The n-butanol extract was separated by normal-phase silica gel column chromatography with a mesh size of 200-300. Gradient elution was performed using EtOAc-MeOH at a ratio of 15:1 to 0:1. After thin-layer chromatography, the extracts were combined into six fractions, namely Fr. 1-Fr. 6.

[0011] Using inverted C 18 Fr. 6 was separated by silica gel column chromatography using a gradient elution of MeOH-H2O at a ratio of 10:90 to 100:0. After detection by thin-layer chromatography, the fractions were combined into three fractions: Fr. 6-1 to Fr. 6-3.

[0012] Fr. 6-1 was separated by normal-phase silica gel column chromatography using 300-400 mesh, with gradient elution using EtOAc-MeOH 6:1-0:1. After thin-layer chromatography detection, the fractions were combined into two parts, namely Fr. 6-1-1 and Fr. 6-1-2.

[0013] Fr. 6-1-1 was separated by MeOH using Sephadex LH-20 gel column chromatography, and further purified by semi-preparative high-performance liquid chromatography (HPLC) using an Agilent Zorbax RX-C8 column (Ø 9.4 × 250 mm), with MeCN-H2O as the eluent (20:80) and a flow rate of 2 mL / min. After semi-preparative HPLC separation and purification, Yunnan Paris saponin G compound PFE55 was obtained.

[0014] Application of Yunnan Paris saponin G compound PFE55 in the preparation of anti-aging drugs.

[0015] Application of Yunnan Paris saponin G compound PFE55 in the preparation of skin care products.

[0016] According to the application described, the concentration of the compound PFE55 is 0.1 μg / mL.

[0017] An anti-aging pharmaceutical composition, wherein Yunnan Paris saponin G compound PFE55 is the active ingredient, and the concentration of PFE55 is 0.1 μg / mL.

[0018] The cosmetic uses Yunnan Paris saponin G compound PFE55 as its active ingredient, and the concentration of PFE55 is 0.1 μg / mL.

[0019] In this invention, when the pharmaceutical composition is used to prepare a drug, the content of the compound or its combination in the drug is preferably 0.1% to 99%; in the pharmaceutical composition, the content of any one or any combination of the active ingredients is preferably 0.5% to 90%. The pharmaceutical composition of this invention is preferably used in the form of a dose per unit body weight. In this invention, the prepared drug is preferably administered by both injection (intravenous injection, intramuscular injection) and oral administration. The pharmaceutical preparations include tablets, capsules, nasal sprays, pills, drops, etc., including but not limited to the above-mentioned preparations.

[0020] In this invention, when the compound PFE55 is used to prepare cosmetics, the type of cosmetic formulation is not limited. It can be prepared using conventional methods for preparing cosmetics.

[0021] Compared with the prior art, the present invention has the following advantages.

[0022] 1. PFE55 can promote collagen secretion even at very low concentrations (0.1 μg / mL).

[0023] 2. PFE55 is not toxic to adult dermal fibroblasts at the effective concentration (0.1 μg / mL). Attached Figure Description

[0024] Figure 1 A schematic diagram of the chemical structure of Yunnan Paris saponin G (PFE55). Detailed Implementation

[0025] The following embodiments of the present invention will further illustrate the substantive content of the present invention, but are not intended to limit the present invention.

[0026] Example 1:

[0027] Preparation of Yunnan Paris saponin G (PFE55).

[0028] 1.4 kg of dried rhizomes of *Paris dulongensis* H. Li & Kurita were pulverized and extracted with 10 L of 70% ethanol at 60 °C using ultrasound-assisted extraction for 1 h. The filtrate was then filtered, and the residue was extracted again under the same conditions. This process was repeated three times. The filtrates were combined, and the solvent was recovered under reduced pressure using a rotary evaporator to obtain 526.0 g of crude extract. A suspension was prepared by adding 1 L of water to the crude extract and then extracted with 1 L of n-butanol. This process was repeated three times, and the extracts were combined. The solvent was recovered under reduced pressure to obtain 71.8 g of n-butanol extract.

[0029] The n-butanol extract (66.6 g) was separated by normal-phase silica gel column chromatography (200-300 mesh) using gradient elution with EtOAc-MeOH (15:1-0:1). After thin-layer chromatography, the fractions were combined into six fractions: Fr. 1 (3.2 g), Fr. 2 (3.6 g), Fr. 3 (16.8 g), Fr. 4 (18.3 g), Fr. 5 (10.8 g), and Fr. 6 (3.8 g).

[0030] Using inverted C 18 Fr. 6 was separated by silica gel column chromatography using gradient elution with MeOH-H2O (10:90~100:0). After detection by thin-layer chromatography, the fractions were combined into three fractions: Fr. 6-1 (0.8 g), Fr. 6-2 (0.6 g), and Fr. 6-3 (1.8 g).

[0031] Fr. 6-1 (0.8 g) was separated by normal-phase silica gel column chromatography (300-400 mesh) using gradient elution with EtOAc-MeOH (6:1-0:1). After detection by thin-layer chromatography, the fractions were combined into two parts: Fr. 6-1-1 (0.4 g) and Fr. 6-1-2 (0.3 g).

[0032] Fr. 6-1-1 (0.4 g) was separated by Sephadex LH-20 gel column chromatography (MeOH), and further purified by semi-preparative high-performance liquid chromatography (HPLC) using an Agilent Zorbax RX-C8 column (Ø 9.4 × 250 mm) and eluent (MeCN-H2O, 20:80) at a flow rate of 2 mL / min. After semi-preparative HPLC purification, Yunnan Paris saponin G compound PFE55 (21.2 mg, t) was obtained. R = 22.7 min).

[0033] Example 2:

[0034] Spectroscopic data of Yunnan Paris saponin G (PFE55).

[0035] Yunnan Paris saponin G (PFE55): White powder, C 56 H 88 O 29 CAS No. 1369542-76-6;

[0036] .

[0037] By comparing the spectral data in the literature [Duan Xiaoyan, Yue Meicen, Yang Jun, Bai Xue, Luo Jifeng, Li Heng, Wang Yuehu. Study on chemical composition and antibacterial activity of rhizome of Paris polyphylla. Chinese Journal of Traditional Chinese Medicine 2023, 48 (11), 2981–2988], PFE55 was identified as parisyunnanoside G.

[0038] Example 3:

[0039] Experiments on the promotion of collagen secretion by PFE55.

[0040] 1. Cell

[0041] Adult dermal fibroblasts (HDFa) were purchased from BeiNa Bio.

[0042] 2. Reagents

[0043] DMEM (high glucose) medium, PBS, Hank's balanced salt solution (HBSS), penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Hyclone; 0.25% trypsin (containing EDTA) was purchased from Thermo Fisher Scientific; transforming growth factor beta (TGF-β) was purchased from Peprotech; collagen ELISA kit was purchased from TaKaRa; and MTS reagent was purchased from Promega.

[0044] 3. Experimental Methods

[0045] HDFa cells were mixed with the test compound in 96-well cell culture plates, with a drug-free blank control and a TGF-β positive control included. Cells were cultured at 37 °C and 5% CO2 for 3 days. The cell culture supernatant was collected and stored at –80 °C. MTS was added, and the OD value at 490 nm was measured using the MTS colorimetric method. Collagen secretion was detected according to the method provided in the collagen ELISA kit, with the OD value measured using a microplate reader at a wavelength of 450 nm. The collagen secretion increase rate was calculated.

[0046] Collagen secretion increase rate (%) = (OD of experimental wells) 450 nm / Cell viability / Octopic pore OD 450 nm –1)×100%

[0047] 4. Experimental Results

[0048] Table 1. The promoting effect of PFE55 on HDFa collagen secretion

[0049]

[0050] Formulation Examples

[0051] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. This application example only demonstrates that Yunnan Paris saponin G (PFE55) described in this invention can be prepared into different formulations, and does not specifically limit the specific reagents and operations:

[0052] 1. The Yunnan Paris saponin G (PFE55) of the present invention is mixed with water for injection according to conventional methods, filtered, filled and sterilized to prepare an injection solution with a concentration of 0.1 mg / mL.

[0053] 2. Dissolve Yunnan Paris saponin G (PFE55) of the present invention in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature and then seal aseptically to obtain powder for injection.

[0054] 3. Add Yunnan Paris saponin G (PFE55) of the present invention to the excipient at a weight ratio of 9:1 to prepare a powder.

[0055] 4. Add Yunnan Paris saponin G (PFE55) of the present invention to the excipient at a weight ratio of 5:1, and granulate and compress into tablets.

[0056] 5. Prepare an oral liquid using the Yunnan Paris saponin G (PFE55) of this invention according to conventional oral liquid preparation methods.

[0057] 6. Add Yunnan Paris saponin G (PFE55) of the present invention to the excipient at a weight ratio of 5:1 to make capsules.

[0058] 7. Add Yunnan Paris saponin G (PFE55) of the present invention to the excipient at a weight ratio of 5:1 to prepare granules.

[0059] Cosmetic Formulation Examples

[0060] 1. Cream formulation (W%) containing Yunnan Paris saponin G (PFE55) of this invention:

[0061]

[0062] The cosmetic product of the above-described formula of the present invention is prepared using conventional methods for manufacturing cosmetics.

[0063] 2. Emulsion formulation (W%) containing Yunnan Paris saponin G (PFE55) of this invention:

[0064]

[0065] The cosmetic product of the above-described formula of the present invention is prepared using conventional methods for manufacturing cosmetics.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Yunnan Paris saponin G compound PFE55, as shown in the following structural formula. ； Its features are: The method includes the following steps: After the dried rhizomes of Paris polyphylla were pulverized, they were extracted with 70% ethanol at 60 °C using ultrasound-assisted extraction. The filtrate was then filtered. The residue was extracted again under the same conditions for a total of 3 extractions. The filtrates were combined, and the solvent was recovered under reduced pressure using a rotary evaporator to obtain a crude extract. The crude extract was then mixed with water to prepare a suspension, which was extracted with n-butanol for a total of 3 extractions. The extracts were combined, and the solvent was recovered under reduced pressure to obtain the n-butanol extract. The n-butanol extract was separated by normal-phase silica gel column chromatography with a mesh size of 200-300. Gradient elution was performed using EtOAc-MeOH at a ratio of 15:1 to 0:

1. After thin-layer chromatography, the extracts were combined into six fractions, namely Fr. 1-Fr.

6. Using inverted C 18 Fr. 6 was separated by silica gel column chromatography using a gradient elution of MeOH-H2O at a ratio of 10:90 to 100:

0. After detection by thin-layer chromatography, the fractions were combined into three fractions: Fr. 6-1 to Fr. 6-3. Fr. 6-1 was separated by normal-phase silica gel column chromatography using 300-400 mesh, with gradient elution using EtOAc-MeOH 6:1-0:

1. After thin-layer chromatography detection, the fractions were combined into two parts, namely Fr. 6-1-1 and Fr. 6-1-2. Fr. 6-1-1 was separated by Sephadex LH-20 gel column chromatography with MeOH, and further purified by semi-preparative high performance liquid chromatography (HPLC) using an Agilent Zorbax RX-C8 column (Ø 9.4 × 250 mm), with MeCN-H2O as the eluent (20:80) and a flow rate of 2 mL / min. After semi-preparative HPLC separation and purification, Yunnan Paris saponin G compound PFE55 was obtained.

2. The application of Yunnan Paris saponin G compound PFE55, as shown in the following structural formula, in the preparation of anti-aging drugs. 。 3. The application of Yunnan Paris saponin G compound PFE55, as shown in the following structural formula, in the preparation of skin care products. 。 4. The application according to claim 2 or 3, characterized in that: The concentration of the Yunnan Paris saponin G compound PFE55, as shown in the following structural formula, is 0.1 μg / mL. 。 5. An anti-aging pharmaceutical composition, characterized in that: Its active ingredient is Yunnan Paris saponin G compound PFE55, as shown in the following structural formula, and the concentration of PFE55 is 0.1 μg / mL. 。 6. Cosmetics, characterized in that: Its active ingredient is Yunnan Paris saponin G compound PFE55, as shown in the following structural formula, and the concentration of PFE55 is 0.1 μg / mL. 。

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