Paris polyphylla steroidal saponins, their preparation methods and applications

Abstract

This invention provides steroidal saponins such as Paris polyphylla saponin A, Paris polyphylla saponin B, Xuefengshan saponin A, Xuefengshan saponin B, and Xuefengshan saponin C, their preparation methods, and their applications in the cosmetics and pharmaceutical fields. It belongs to the field of cosmetics and pharmaceutical technology. Paris polyphylla saponin VII, protodiospin, protodiospin, protodiospin, Xuefengshan saponin B, Xuefengshan saponin C, and methyl protosaponin Pb are prepared at extremely low concentrations (0.1%). m At concentrations of M, it exhibits activity in promoting collagen secretion in adult dermal fibroblasts (HDFa), with an increase in collagen secretion exceeding 50%; furthermore, at this concentration, it is not cytotoxic and can be used to prepare anti-aging drugs or skincare products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics and medicine, specifically to the technical field of plant medicine, more specifically, to heavy building steroidal saponins, which have collagen secretion promoting activity, as well as their preparation method and application. BACKGROUND

[0002] Collagen is a kind of main structural protein in mammalian extracellular matrix, which widely exists in tissues such as skin, bone, muscle, etc., mainly participates in physiological and biochemical behaviors such as cell proliferation, differentiation, migration and signal transmission, and plays a supporting, repairing and protecting role for tissues and cells [Ye T.,Xiang Q.,Yang Y.,Huang Y.D.Development and application research progress of collagen.Bioengineering Bulletin,2023,39(3):942-960]. Among them, type I collagen is the most abundant protein in human skin, accounting for about 70% of the dry weight of the skin. Type I collagen can increase skin elasticity and delay skin aging [Zhang Y.,Zhou J.,Zhang N.,Zhao L.,Zhang L.,Zhou F.Research progress of collagen peptide and elastin peptide on improving photoaging skin.Food Research and Development,2023,44(11):208-215]. In addition, on the wound surface, type I collagen promotes wound healing by providing structural support for cell attachment and migration. However, animal-derived type I collagen may have immunogenicity, and problems related to processing technology, degradation rate and disinfection process of type I collagen limit its application [Cheng Y.,Li Y.,Huang S.,Yu F.,Bei Y.,Zhang Y.,Tang J.,Huang Y.,Xiang Q.Hybrid freeze-dried dressings composed of epidermal growth factor and recombinant human-like collagen enhance cutaneous wound healing in rats.Front.Bioeng.Biotechnol.,2020,8:742]. Therefore, screening of plant extracts and natural products with collagen secretion promoting activity is a current research hotspot.

[0003] Steroidal saponins are the main chemical components of Paris L. plants, which have anti-cancer, hemostatic, anti-inflammatory, antibacterial and other biological activities [Ding Y.-G., Zhao Y.-L., Zhang J., Zuo Z.-T., Zhang Q.-Z., Wang Y.-Z. The traditional uses, phytochemistry, and pharmacological properties of Paris L. (Liliaceae): A review. J. Ethnopharmacol., 2021, 278: 114293]. The main types of steroidal saponins of Paris L. include spirostane and furostane, and when the aglycone of spirostane steroidal saponin contains three or more than three hydroxyl groups, it is called polyhydroxyl spirostane steroidal saponin. Generally, polyhydroxyl spirostane steroidal saponin and furostane have weak cytotoxicity. Although there are many reports on the biological activity of steroidal saponins of Paris L., there are few reports on their research on promoting collagen secretion. Therefore, the present application evaluates the activity of 24 steroidal saponin compounds in promoting collagen secretion. SUMMARY

[0004] The present application aims to provide a class of steroidal saponin compounds PFV92, PFV93, PGA4, PGA5 and PGA6, their pharmaceutical compositions and cosmetics, their preparation methods, and their applications in preparing collagen secretion promoting active agents, in preparing anti-aging drugs, and in preparing cosmetics, together with the following steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PGA27.

[0005] In order to achieve the above-mentioned purpose of the present application, the present application provides the following technical solutions:

[0006] The steroidal saponin compounds PFV92, PFV93, PGA4, PGA5 and PGA6 are shown in the following structural formula,

[0007]

[0008] The preparation method of the steroidal saponin compounds PFV92, PFV93, PGA4, PGA5 and PGA6 comprises the following steps:

[0009] The dried rhizomes of *Paris polyphylla* were pulverized and extracted three times with 70% ethanol (50 L × 3) at 78–80 °C for 4 h, 3 h, and 3 h respectively. The filtrate was filtered and concentrated to obtain a crude extract. This crude extract was suspended in 16 L of distilled water and extracted three times with an equal volume of ethyl acetate. After recovering the solvent from the organic phase, the ethyl acetate extract was obtained. The aqueous phase was then extracted four times with an equal volume of n-butanol. After recovering the solvent from the organic phase, the n-butanol extract was obtained. The n-butanol extract was then subjected to gradient elution using a 200–300 mesh normal-phase silica gel column chromatography with a 10:1 eluent. The eluent of CH2Cl2-MeOH in a 1–0:1 ratio was analyzed by thin-layer chromatography and the eluent was combined into seven fractions: Fr.I, Fr.II, Fr.III, Fr.IV, Fr.V, Fr.VI, and Fr.VII. Fr.VII was eluted by gradient chromatography using normal-phase silica gel column chromatography with EtOAc-MeOH in a 10:1–0:1 ratio. The eluent was analyzed by thin-layer chromatography and the eluent was combined into four fractions: Fr.VII-1, Fr.VII-2, Fr.VII-3, and Fr.VII-4. Fr.VII-4 was further analyzed by reversed-phase (RP) chromatography. 18 Silica gel column chromatography (MeOH-H2O, 10–100%) was used to divide the sample into four fractions: Fr.VII-4-1, Fr.VII-4-2, Fr.VII-4-3, and Fr.VII-4-4. Fr.VII-4-1 was further purified by Sephadex LH-20 gel column chromatography (MeOH) to obtain Fr.VII-4-1-1. Fr.VII-4-1-1 was then purified by semi-preparative HPLC to obtain Paris polyphylla saponin B, i.e., PFV93. 10.0 g of Fr.VII-3 was then subjected to RP-C... 18 Silica gel column chromatography (MeOH-H2O, 10:90~100:0) was divided into 5 parts: Fr.VII-3-1, Fr.VII-3-2, Fr.VII-3-3, Fr.VII-3-4 and Fr.VII-3-5. Fr.VII-3-3 was purified by semi-preparative HPLC after Sephadex LH-20 gel column chromatography to obtain Paris polyphylla saponin A, i.e., PFV92.

[0010] The dried rhizomes of *Paris polyphylla* from Xuefeng Mountain were pulverized and extracted three times with 70% ethanol aqueous solution (4L, 3L, and 3L) at 60℃ using ultrasound, each extraction lasting 0.5h. The extracts were filtered, and the filtrates were concentrated to dryness using a rotary evaporator at 60℃ to obtain a crude extract. The crude extract was added to distilled water to form a suspension, which was then extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The organic phase and the aqueous phase after the n-butanol extraction were concentrated under reduced pressure using a rotary evaporator at 60℃ to obtain four fractions: petroleum ether extract, ethyl acetate extract, n-butanol extract, and aqueous phase. The n-butanol extract was subjected to gradient elution using normal-phase silica gel column chromatography to obtain Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, and Fr.G. Fr.C was further eluted by RP-C. 18 Silica gel column chromatography (MeOH-H2O, 10-100%) was used to obtain ten fractions (Fr.C-1 to Fr.C-10); Fr.C-5 was purified by Sephadex LH-20 gel column chromatography and semi-preparative HPLC to obtain Xuefengshan saponin B (PGA5), Xuefengshan saponin C (PGA6), and Xuefengshan saponin A (PGA4).

[0011] The application of the Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5, and PGA6 in the preparation of collagen secretion activators.

[0012] The application of the Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5, and PGA6 in the preparation of anti-aging drugs.

[0013] The application of the Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5 and PGA6 in the preparation of cosmetics.

[0014] A pharmaceutical composition comprising any one or any combination of the Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5, and PGA6, and at least one pharmaceutically acceptable carrier.

[0015] Cosmetics, comprising any one or any combination of the Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5, and PGA6, and conventional cosmetic excipients.

[0016] Application of Paris polyphylla steroidal saponins PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, and PGA27 in the preparation of collagen secretion activators.

[0017] Application of Paris polyphylla steroidal saponins PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, and PGA27 in the preparation of anti-aging drugs.

[0018] Application of Paris polyphylla steroidal saponins PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, and PGA27 in the preparation of cosmetics.

[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. The Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5 and PGA6 are new compounds.

[0023] 2. At extremely low concentrations (0.1 μM), Paris saponin VII, protodiospin, prototrifolio saponin, protodiospin, Xuefengshan saponin B, Xuefengshan saponin C, and methyl protosaponin Pb have the activity of promoting collagen secretion in adult dermal fibroblasts, with an increase in collagen secretion rate of over 50%.

[0024] 3. At the concentration at which the drug exerts its effect (0.1 μM), Paris saponin VII, protodiospin, prototrifolio saponin, protodiospin B, Xuefengshan saponin C, and methyl protosaponin Pb have no toxicity to adult dermal fibroblasts. Attached Figure Description

[0025] Figure 1 Schematic diagram of the chemical structures of the new Paris polyphylla steroidal saponin compounds PFV92, PFV93, PGA4, PGA5 and PGA6.

[0026] Figure 2 A schematic diagram of the known chemical structure of Paris polyphylla steroidal saponins (I).

[0027] Figure 3 Schematic diagram of the known chemical structure of Paris polyphylla steroidal saponins (II).

[0028] Figure 4 Schematic diagram of the known chemical structure of Paris polyphylla steroidal saponins (III).

[0029] Figure 5 Two-dimensional NMR correlation diagram of the compound. Detailed implementation method:

[0030] 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.

[0031] Example 1:

[0032] The source and preparation of compounds.

[0033] A total of 24 Paris polyphylla steroidal saponins were tested. Figures 1-4 The following are PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27, and their sources or preparation methods are as follows.

[0034] 1. Preparation of PFV70, PFV72, PFV77, PFV78, PFV92 and PFV93.

[0035] Paris rugosa H.Li & Kurita were purchased from a Paris rugosa cultivation base in Shiyueliang Township, Fugong County, Yunnan Province in October 2020. The dried rhizomes (4.1 kg) were pulverized and extracted three times with 70% ethanol (50 L × 3) at 78–80 °C for 4 h, 3 h, and 3 h respectively. The filtrate was filtered and concentrated to obtain a crude extract (1.4 kg). The crude extract was suspended in 16 L of distilled water and extracted three times with an equal volume of ethyl acetate. After recovering the solvent from the organic phase, the ethyl acetate extract (123.4 g) was obtained. The aqueous phase was further extracted four times with an equal volume of n-butanol. After recovering the solvent from the organic phase, the n-butanol extract (219.3 g) was obtained.

[0036] The n-butanol extract (209.2 g) was subjected to gradient elution using normal-phase silica gel (200–300 mesh) column chromatography with CH2Cl2-MeOH (10:1–0:1) as the eluent. After thin-layer chromatography analysis, the eluent was combined into seven fractions: Fr.I, Fr.II, Fr.III, Fr.IV, Fr.V, Fr.VI, and Fr.VII. Fr.VII (162.0 g) was subjected to gradient elution using normal-phase silica gel (200–300 mesh) column chromatography with EtOAc-MeOH (10:1–0:1) as the eluent. After thin-layer chromatography analysis, the eluent was combined into four fractions: Fr.VII-1 (0.3 g), Fr.VII-2 (6.1 g), Fr.VII-3 (22.2 g), and Fr.VII-4 (112.0 g). Fr.VII-4 (112.0g) was processed by reverse-phase (RP)C 18 Silica gel column chromatography (MeOH-H2O, 10-100%) was divided into four fractions: Fr.VII-4-1 (0.8 g), Fr.VII-4-2 (18.6 g), Fr.VII-4-3 (13.3 g) and Fr.VII-4-4 (56.8 g).

[0037] Fr.VII-4-1 (0.8 g) was purified by Sephadex LH-20 gel column chromatography (MeOH) to obtain Fr.VII-4-1-1 (9.1 mg, t) by semi-preparative high performance liquid chromatography (HPLC; Agilent Zorbax RX-C8, φ9.4×250 mm; MeCN-H2O, 20:80; v=2 mL / min). R =9.3 min). Fr.VII-4-1-1 was purified by semi-preparative HPLC (Agilent Zorbax RX-C8, φ9.4×250mm; MeCN-H2O, 16:84; v = 2 mL / min) to obtain Paris polyphylla saponin B (PFV93; 4.1 mg, t) R=43.1min).

[0038] Fr.VII-4-2 (18.6 g) was recrystallized from methanol to obtain methyl protocellulose diosgenin (PFV72; 10.6 g).

[0039] Fr. VII-4-3 (13.3 g) was subjected to normal-phase silica gel (300-400 mesh) column chromatography (CH2Cl2-MeOH, 8:1-0:1) to obtain Fr. VII-4-3-1 (8.3 g) and Fr. VII-4-3-2 (0.3 g). 100 mg of crystals from Fr. VII-4-3-1 were crystallized and subjected to Sephadex LH-20 gel column chromatography (MeOH) followed by semi-preparative HPLC (Agilent Zorbax SB-C) 18 (φ9.4×250mm; MeOH-H2O, 85:15; v=2mL / min) After purification, proto-fibrous diosgenin (PFV70; 5.1mg, t) was obtained. R =9.7 min). Fr.VII-4-3-2 (0.3 g) was subjected to Sephadex LH-20 gel column chromatography (MeOH) followed by semi-preparative HPLC (Agilent Zorbax SB-C) 18 After purification, methyl protobarbital saponin I (PFV77; 18.7 mg, t) was obtained (φ9.4×250mm; MeOH-H2O, 70:30; v=2mL / min). R =17.5min) and methylprotobiose saponin (PFV78; 23.3mg,t) R =21.0min).

[0040] Take 10.0g from Fr.VII-3 and process it through RP C 18 Silica gel column chromatography (MeOH-H2O, 10:90~100:0) was used to separate the sample into five fractions: Fr.VII-3-1 (63.4 mg), Fr.VII-3-2 (312.3 mg), Fr.VII-3-3 (334.0 mg), Fr.VII-3-4 (56.0 mg), and Fr.VII-3-5 (7.6 g). Fr.VII-3-3 (334.0 mg) was further purified by Sephadex LH-20 gel column chromatography (MeOH) using a semi-preparative HPLC system (Agilent Zorbax SB-C). 18 (φ9.4×250mm; MeCN-H2O, 43:57; v=2mL / min) The purified Paris polyphylla saponin A (PFV92; 4.5mg, t) was obtained. R =19.4min).

[0041] 2. Preparation of PGA4, PGA5, PGA6 and PGA27.

[0042] Paris xuefengshanensis H.Li, Z.Wang & G.W.Hu, sp. nov.ined. were purchased in January 2021 from a Paris xuefengshanensis cultivation base in Malin Yao Township, Xinning County, Hunan Province. The dried rhizomes (1.4 kg) were pulverized and ultrasonically extracted three times at 60°C with 70% ethanol aqueous solution (4 L, 3 L, and 3 L), each extraction lasting 0.5 h. The extract was filtered, and the filtrate was concentrated to dryness using a rotary evaporator at 60°C to obtain a crude extract (319.5 g). The crude extract was added to distilled water (1 L) to form a suspension, and then extracted three times each with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The organic phase and the aqueous phase after n-butanol extraction were concentrated under reduced pressure using a rotary evaporator at 60°C to obtain four fractions: petroleum ether extract (315.2 mg), ethyl acetate extract (1.7 g), n-butanol extract (79.8 g), and aqueous phase (237.1 g).

[0043] The n-butanol extract (66.2 g) was subjected to normal-phase silica gel (100-200 mesh) column chromatography (CH2Cl2-MeOH, 15:0-0:1) gradient elution to obtain Fr.A (619.9 mg), Fr.B (416.4 mg), Fr.C (3.6 g), Fr.D (1.3 g), Fr.E (6.1 g), Fr.F (10.0 g) and Fr.G (9.2 g).

[0044] Fr.C (3.6g) via RP C 18 Silica gel column chromatography (MeOH-H2O, 10–100%) was used to obtain ten fractions (Fr.C-1 to Fr.C-10). Fr.C-5 (60% methanol eluent, 65.2 mg) was subjected to Sephadex LH-20 gel column chromatography (MeOH) and then semi-preparative HPLC (Agilent Zorbax SB-C) was used. 18 (φ9.4×250mm; CH3CN-H2O, 28:72; v=2mL / min) Xuefeng Mountain saponin B (PGA5; 1.6mg, t) was purified to obtain Xuefeng Mountain saponin B (PGA5; 1.6mg, t) R =20.7min), Xuefeng Mountain saponin C (PGA6; 1.5mg,t) R =28.1min) and Xuefeng Mountain saponin A (PGA4; 27.4mg,t) R =32.4min).

[0045] Fr.G (9.2g) via RP C 18Silica gel column chromatography (MeOH-H2O, 10–100%) yielded nine fractions (Fr.G-1–Fr.G-8). Fr.G-6 (60% methanol eluent, 4.3 g) was subjected to gradient elution on a normal-phase silica gel (200–300 mesh) column (CH2Cl2-MeOH, 7:1–1:1) to obtain six fractions (Fr.G-6-1–Fr.G-6-6). Fr.G-6-4 (CH2Cl2:MeOH = 1:1 eluent, 2.5 g) was subjected to Sephadex LH-20 gel column chromatography (MeOH) to obtain three fractions (Fr.G-6-4-1–Fr.G-6-4-3). Fr. G-6-4-2 (2.2 g) was subjected to Sephadex LH-20 gel column chromatography (MeOH) to obtain G-6-4-2-1 (843.6 mg), which was then purified by semi-preparative HPLC (Reprospherical Carbohydrate, φ4.6×250 mm; MeOH-H2O, 98:2; v = 1 mL / min) to obtain methyl prostaglandin Pb (PGA27; 36.2 mg, t R =3.8min).

[0046] 3. The sources of PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73 and PFE75.

[0047] Paris dulongensis saponin B (PFE52), Paris dulongensis saponin A (PFE53), Paris lancifolium saponin B (PFE54), Paris dulongensis saponin D (PFE56), Paris dulongensis saponin I (PFE57), Paris dulongensis saponin C (PFE59A), prodiosgenin (PFE62A), Paris dulongensis saponin II (PFE66), Paris dulongensis saponin III (PFE67), Paris dulongensis saponin VII (PFE73), and Yunnan Paris dulongensis saponin I (PFE75) were extracted and isolated from the dried rhizomes of Paris dulongensis H. Li & Kurita. This plant material was purchased in September 2021 from a Paris dulongensis planting base in Dulongjiang Township, Gongshan County, Yunnan Province. The preparation methods of the compounds are described in the references [Jia J.-K., Yang J., Yang X.-Z., Luo J.-F., Duan X.-Y., Yang Y.-L., Wan]. J.-F., WangY.-H. Polyhydroxylated spirostanol saponins from the rhizomes of Parisdulongensis. Chem. Biodivers., 2024, e202400980. https: / / doi.org / 10.1002 / cbdv.202400980].

[0048] 4. The origins of PFV50, PFV51 and PFV53.

[0049] The original prodiosgenin (PFV50) was purchased from Chengdu Pusi Biotechnology Co., Ltd.; the original triangular leaf saponin (PFV51) was purchased from Chengdu Purifa Technology Development Co., Ltd.; and the methyl prodiosgenin (PFV53) was purchased from Chengdu Zhibiao Chemical Biotechnology Co., Ltd.

[0050] Example 2:

[0051] Spectroscopic data and structural analysis of the compound.

[0052] 1. Structural analysis of the new compounds PFV92, PFV93, PGA4, PGA5 and PGA6.

[0053] Paris polyphylla saponin A (PFV92): white amorphous powder. (c 0.2,MeOH); UV(MeOH)λ max (logε)323(2.03),274(2.51),216(3.54)nm; ECD(c,0.08,MeOH)λ max (Δε)217(+10.04)nm; ESI-MS m / z 935[M+Na]+ ;HR-ESI-MS m / z 935.4245[M+Na] + (C 45 H 68 NaO 19 (Calculated value: 935.4247); 1 H and 13 C NMR data are shown in Table 1.

[0054] In the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) of compound PFV92, a quasi-molecular ion peak at m / z 935.4245 [M+Na] can be observed. + (Calculated value C) 45 H 68 NaO 19 ,935.4247), combined 13 The 10⁻¹⁴ NMR data (Table 1) suggest that its molecular formula is C₁⁻¹. 45 H 68 O 19 The degree of unsaturation is 12. According to... 1 H and 13 The 10⁻¹⁴ NMR data (Table 1) indicate that this compound contains 5 methyl groups [δ¹⁴]. H 1.73 (3H, d, J = 6.2 Hz), 1.21 (3H, s), 1.05 (d, J = 6.8 Hz), 0.89 (3H, s), 0.67 (d, J = 5.4 Hz)], 1 disubstituted double bond [δ H 6.67(1H,d,J=8.5Hz) and 6.33(1H,d,J=8.5Hz); δ C 136.6(CH) and 130.9(CH)], one trisubstituted double bond [δ H 5.48(1H,dd,J=5.3,1.7Hz); δ C 144.0 (C) and 119.4 (CH)] and 3 sugars [δ H 6.39(1H,br s),5.09(1H,d,J=7.6Hz),4.80(1H,d,J=7.5Hz); δ C [104.5(CH), 102.1(CH), 100.6(CH)]. After acid hydrolysis, D-glucose pyranose and L-rhamnose pyranose were detected in compound PFV92. Combined with NMR data, it was determined that compound PFV92 contains two β-D-glucose β-p-glucose groups [δ]. H 5.09 (1H,d,J = 7.6 Hz) and 4.80 (1H,d,J = 7.5 Hz)] and 1 α-L-pyranoside [δ H6.39 (1H, br s)]. By comparing with the NMR data of Parisvanioside A [Yan H., Ni W., Yu L.-L., Xiao L.-G., Ji Y.-H., Liu H.-Y. Parisvaniosides A–E, five new steroidal saponins from Paris vaniotii. Steroids, 2022, 177: 108949], it is speculated that compound PFV92 is a steroidal saponin with the same aglycone.

[0055] according to 1 H- 1 H COSY related ( Figure 5 From this, we can deduce fragments a(H-1 / H2-2 / H-3 / H2-4), b(H-6 / H-7), c(H-11 / H2-12), d(H-14 / H2-15 / H-16 / H-17 / H-20 / H3-21), e[H2-23 / H2-24 / H-25 / (H2-26) / H3-27], f(H-1′ / H-2′ / H-3′ / H-4′ / H-5′ / H2-6′), g(H-1″ / H-2″ / H-3″ / H-4″ / H-5″ / H3-6″) and h(H-1″′ / H-2″′ / H-3″′ / H-4″′ / H-5″′ / H2-6″′). In the HMBC correlation spectrum ( Figure 5 We can see that H3-19 is associated with C-1, C-5, C-9, and C-10; H2-4 with C-10; H-6 with C-4 and C-10; H-7 with C-9; H3-18 with C-12, C-13, C-14, and C-17; and H3-21 and H2-26 with C-22, indicating that the double bond is located at C-6. H2-15 is associated with C-8, and H3-19 with C-5, indicating that C-5 and C-8 are oxidized. In the ROESY correlation spectrum ( Figure 5 We can see correlations between H-1β / H3-19, H3-19 / H-6, H-7 / H3-18, H3-18 / H-20, H-20 / H2-23, and H3-27 / H2-26, indicating that these hydrogen atoms are in the same plane. Assuming a β orientation, the orientation of the peroxide bridge and H-17 is an α orientation. The correlations between H-17 / H-14, H-14 / H-16, and H-1α / H-3 indicate that these hydrogen atoms are α-oriented, and H-26... ax / H3-27 and H-26 eqThe correlation of / H3-27 indicates that 25-Me is located on the equatorial bond of this six-membered ring chair conformation, thus confirming that the aglycone of compound PFV92 is (25R)-Spirost-6,11-diene-5α,8α-epidioxy-3β-ol. Based on the HMBC correlations of H-3 with C-1′, H-1′ with C-3, H-2′ with C-1″, H-1″ with C-2′, H-3′ with C-1″′, and H-1″′ with C-3′, it indicates that β-D-pyranose [4.80 (1H, d, J = 7.5 Hz)] is located at C-3, and β-D-pyranose [δ...]... H 5.09 (1H, d, J = 7.6 Hz) is located at C-2′, α-L-pyranorhamnetose [δ H [6.39(1H,br s)] is located at C-3′, therefore, the sugar chain is determined to be α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranosyl, located at the 3-OH of the aglycone. Therefore, the chemical structure of PFV92 is determined to be (25R)-Spirost-6,11-diene-5α,8α-epidioxy-3β-ol3-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside, named rugosaroside A.

[0056] Table 1. PFV92 in deuterated pyridine 1 H (500MHz) and 13 C (126MHz) NMR data (δin ppm, J in Hz)

[0057]

[0058]

[0059] Paris polyphylla saponin B (PFV93): white amorphous powder. (c 0.2,MeOH); UV(MeOH)λ max (logε):279(2.19),253(2.58)nm; ESI-MS m / z 1249[M+Na] + ;HR-ESI-MS m / z 1225.5497[M–H] – (C 56 H 89 O 29(Calculated value: 1225.5495); 1 H and 13 CNMR data are shown in Table 2.

[0060] In the high-resolution electrospray ionization mass spectrum of compound PFV93, a quasi-molecular ion peak at m / z 1225.5497 [M–H] can be observed. – (Calculated value C) 56 H 89 O 29 (1225.5495). Combined 13 Based on the 10⁻¹⁴ NMR data (Table 2), its molecular formula is inferred to be C₁⁻¹. 56 H 90 O 29 The degree of unsaturation is 12. According to... 1 H NMR spectrum and 13 Based on the 10⁻¹⁴ NMR data (Table 2), it can be inferred that the compound contains 5 methyl groups [δ¹⁴]. H 1.71(3H,d,J=6.1Hz),1.48(3H,d,J=6.4Hz),1.35(3H,s),1.08(3H,s),1.00(3H,d,J=6.8Hz); δ C 19.3(CH3),17.3(CH3),16.9(CH3),15.1(CH3),13.2(CH3)], 5 sugars [δ H 6.36(1H,br s),5.08(1H,d,J=7.9Hz), 4.90(1H,d,J=7.6Hz), 4.88(1H,d,J=7.6Hz), 4.74(1H,d,J=7.6Hz); δ C [106.0(CH),105.4(CH),105.2(CH),101.8(CH),100.2(CH)], and one trisubstituted double bond [δ H 5.54 (1H, br d, J = 5.8Hz); δ C [139.5(C), 124.8(CH)]. After acid hydrolysis, compound PFV93 showed the detection of D-glucose pyranopyranose, L-rhamnose pyranopyranose, D-xylpyranopyranose, D-galactopyranopyranose, and D-fucopyranopyranose. Based on the chemical shifts and coupling constants in the NMR spectrum, the presence of β-D-glucose pyranopyranose [δ] in the structure of compound PFV93 was confirmed. H 4.74 (1H, d, J = 7.6 Hz)], α-L-pyranorhamnetosyl [δ H 6.36(1H,br s)], β-D-xylanose[δ H 4.90 (1H, d, J = 7.6 Hz)], β-D-galactopyranosyl [δH 4.88 (1H, d, J = 7.6 Hz)] and β-D-pyranofucose [δ H 5.08 (1H, d, J = 7.9 Hz)] One each. The NMR data of compound PFV93, except for the presence of an additional sugar signal, are very similar to the known polyhydroxyspirostanol type steroidal saponin Padelaoside B [Zhang T., Liu H., Liu X.-T., Chen X.-Q., Wang Q. Steroidal saponins from the rhizomes of Paris delavayi. Steroids, 2009, 74: 809–813].

[0061] According to compound PFV93 1 H- 1 H COSY related ( Figure 5 From this, we can deduce fragments a(H-1 / H2-2 / H-3 / H2-4), b[H-6 / H2-7 / H-8 / (H-9 / H2-11 / H2-12 / )H-14 / H2-15 / H-16 / H-17 / H-20 / H2-21], c(H-23 / H-24 / H-25 / H2-26 / H3-27), d(H-1′ / H-2′ / H-3′ / H-4′ / H-5′ / H2-6′), e( H-1″ / H-2″ / H-3″ / H-4″ / H-5″ / H3-6″), f(H-1″′ / H-2″′ / H-3″′ / H-4″′ / H2-5″′), g(H-1″″ / H-2″″ / H-3″″ / H-4″″ / H2-5″″ / H2-6″″), and h(H-1″″′ / H-2″″′ / H-3″″′ / H-4″″′ / H-5″″′ / H3-6″″′). In the HMBC correlation spectrum ( Figure 5 We can see that H3-19 is associated with C-1, C-5, C-9, and C-10; H2-4 with C-10; H-6 with C-4 and C-10; H3-18 with C-12, C-13, C-14, and C-17; and H2-21, H-24, and H2-26 with C-22. Combining these with fragments a to c, we can obtain the planar structure of the aglycone as Spirost-5-ene-1,3,21,23,24-pentol. The ROESY correlation between H-1 / H-3 and H-1 / H-9 (…) Figure 5 It is inferred that 1-OH and 3-OH are β-oriented; the ROESY correlation between 23-OH / H-20 and H-23 / H-25 indicates that H-23 and H-25 are both axially bonded in the F ring, suggesting that the configuration of C-23 and C-25 is S; the coupling constant between H-23 and H-24 is relatively small (J23,24 =2.7Hz), indicating that H-24 is in an equatorial bond, suggesting that C-24 is in the S configuration. In the HMBC correlation spectrum, we can see that H-1 is correlated with C-1′, H-1′ with C-1, H-2′ with C-1″, H-1″ with C-2′, H-3′ with C-1″′, H-1″′ with C-3′, H-21 with C-1′, H-1′ with C-21, H-24 with C-1″″, and H-1″″ with C-24, indicating that β-D-glucopyranose [δ H 4.74 (1H, d, J = 7.6 Hz)] located at C-1, α-L-pyranorhamnetose [δ H 6.36(1H,br s)] located at C-2′,β-D-xylanose[δ H 4.90 (1H, d, J = 7.6 Hz)] located at C-3′, β-D-galactopyranose [δ H 4.88 (1H, d, J = 7.6 Hz)] located at C-21 and β-D-pyran-fucose [δ H [5.08 (1H, d, J = 7.9 Hz)] is located at C-24. In summary, the chemical structure of compound PFV93 was identified as (1β,3β,23S,24S,25S)-21-(β-D-galactopyranosyloxy)-24-(β-D-fucopyranosyloxy)-3,23-dihydroxyspirost-5-en-1-ylO-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside, and named it rugosaroside B.

[0062] Table 2. PFV93 in deuterated pyridine 1 H (500MHz) and 13 C (126MHz) NMR data (δin ppm, J in Hz)

[0063]

[0064] Xuefengshanoside A (PGA4), white amorphous powder; (c0.1, MeOH); 1 H and 13 C10 NMR data are shown in Table 3; ESI-MS m / z 777 [M+Na] + ;HR-ESI-MS m / z 777.4037[M+Na]+ (C 39 H 62 NaO 14 (Calculated value 777.4032).

[0065] High-resolution electrospray ionization mass spectrometry of compound PGA4 showed a cation signal of m / z 777.4037 [M+Na]. + (C 39 H 62 NaO 14 The calculated value is 777.4032. Based on the nuclear magnetic resonance data (Table 3), its molecular formula is determined to be C. 39 H 62 O 14 Its degree of unsaturation is 9. 1 H NMR data showed six methyl signals [δ] H 1.55 (3H,d,J=6.4Hz), 1.50 (3H,d,J=6.4Hz), 1.21 (3H,s), 1.08 (3H,d,J=7.1Hz), 1.07 (3H,d,J=6.9Hz), 0.98 (3H,s)]. Hydrolysis of compound PGA4 yielded D-fucose. (c 0.5,MeOH)[Xu Tunhai, Chen Ping, Xu Yajuan, Hao Lingzhu, Xie Shengxu, Xu Dongming. Isolation and identification of new furostrosinases from Ophiopogon japonicus. Journal of Chemical Research in Chinese Universities, 2007, 28(2):286–288], combined with two sugar-terminal proton signals [δ H [5.10 (1H,d,J=7.9Hz), 4.71 (1H,d,J=7.6Hz)], suggesting that this compound may contain two β-D-pyranofucose groups. PGA4 13 The 10⁻⁶ C NMR spectrum showed a signal at 39 carbons, suggesting that the compound contains a double bond (δ¹⁸). C 139.8, 124.6), 6 methyl groups (δ) C The NMR data show that PGA4 contains 17.5, 17.4, 17.0, 14.9, 14.9, and 13.3 NMR groups, 7 methylene groups (1 of which is oxygen-bound), 21 methine groups (15 of which are oxygen-bound), and 3 quaternary carbon groups (1 of which is oxygen-bound). Based on these NMR characteristics, it is inferred that PGA4 is a polyhydroxyspirostanol-type steroidal saponin with two β-D-pyranofucose groups.

[0066] According to PGA4 1 H- 1 H COSY related ( Figure 5 ), which yields 5 connection segments (a~e, Figure 5 In HMBC related ( Figure 5We can see that H2-4 is associated with C-10, H-6 with C-4 and C-10, H3-19 with C-1, C-5, C-9 and C-10, H3-18 with C-12, C-13, C-14 and C-17, and H3-21, H-23 and H2-26 with C-22. Thus, fragments a to c can be linked together to obtain the aglycone structure, namely spirost-5-ene-1,3,23,24-tetraol. The HMBC correlations between H-1 and C-1′, H-1′ and C-1, H-24 and C-1″, and H-1″ and C-24 indicate that the two β-D-pyranofucose fragments are attached to the 1-OH and 23-OH groups of the aglycone, respectively.

[0067] The relative configuration of the PGA4 aglycone moiety can be correlated with ROESY ( Figure 5 This can be determined by: First, assuming H3-19 is β-oriented, the ROESY correlations of H3-19 / H-2β, H-2β / 3-OH, H3-19 / H-8, H-8 / H3-18, H3-18 / H-12β, and H3-18 / H-20 indicate that these hydrogens are β-oriented; the correlations of H-12α / H-14, H-14 / H-17, H-14 / H-16, H-14 / H-9, and H-9 / H... The ROESY correlation between -1 indicates that these hydrogens are α-oriented; the ROESY correlation between H-20 and 23-OH and H-23 indicates that the C-22-C-23 bond is β-oriented; the ROESY correlation between H-23 and H3-21 and H-25 indicates that H-23 and H-25 are axial bonds, while 27-Me is an equatorial bond; the ROESY correlation between 23-OH and H-1″ indicates that H-24 is an equatorial bond.

[0068] After hydrolysis of PGA4, in addition to fucose, aglycone (1β,3β,23S,24S,25S)-spirost-5-ene-1,3,23,24-tetraol was also obtained [Chen Z.,Xue X.,Zhang S.,Zhang R.,Zhang X.,Guo Z.,Zhang X.Steroidal components from the roots and rhizomes of Smilacina henryi and their cytotoxic activities.Rec.Nat.Prod.,2020,14(3):225–230]. Thus, the structure of PGA4 was determined, as follows: Figure 1 As shown, it was named Xuefengshan saponin A.

[0069] Table 3. Compounds PGA4 and PGA5 in deuterated pyridine 1 H and 13C NMR data (δin ppm, J in Hz).

[0070]

[0071] Xuefengshanoside B (PGA5), white amorphous powder; (c0.2,MeOH); ECD(c0.044,MeOH)λ max (Δε)202(-6.19)nm; 1 H and 13 C10 NMR data are shown in Table 3; ESI-MS m / z 775 [M+Na] + HR-ESI-MS m / z 751.3910 [MH] - (C 39 H 59 O 14 , calculated value 751.3910).

[0072] High-resolution electrospray ionization mass spectrometry of PGA5 showed an anion signal of m / z 751.3910 [MH]. - (C 39 H 59 O 14 The calculated value is 751.3910. Based on the nuclear magnetic resonance data (Table 3), its molecular formula is determined to be C. 39 H 60 O 14 Its degree of unsaturation is 10. 1 HNMR data showed five methyl signals [δ] H 1.55 (3H,d,J=6.4Hz), 1.47 (3H,d,J=6.4Hz), 1.19 (3H,s), 1.03 (3H,d,J=7.0Hz), 0.92 (3H,s)], a terminal double bond signal [δ H 5.24(1H,s), 5.10(1H,s)], a trisubstituted double bond signal [δ H 5.56 (1H, br d, J = 5.3 Hz)], and two β-pyran-fucose terminal proton signals [δ H 5.15(1H,d,J=7.9Hz), 4.71(1H,d,J=7.7Hz)]. PGA5 13The 1 / 32-2000 NMR spectrum showed 39 carbon signals, suggesting the presence of two double bonds, five methyl groups, seven methylene groups (one of which is oxygen-bound), 20 methine groups (15 of which are oxygen-bound), and three quaternary carbons (one of which is oxygen-bound). Comparing the NMR data of compounds PGA5 and PGA4, it is speculated that PGA5 is a structural analogue of PGA4, differing in that PGA5 has one more double bond and one less methyl group than PGA4. Based on the NMR data of PGA5... 1 H- 1 H COSY and HMBC are related ( Figure 5 It was deduced that the aglycone of PGA5 is spirost-5,25(27)-diene-1,3,23,24-tetraol, meaning the extra terminal double bond is located at C-25, while the sugar linkage position is consistent with that of PGA4. PGA5 loses its chirality at C-25 due to the formation of a double bond. This was determined through ROESY correlation (… Figure 5 This proves that the relative configurations of the remaining chiral carbons in PGA5 are consistent with those in PGA4. Therefore, the structural formula of PGA5 is determined as follows: Figure 1 As shown, it was named Xuefengshan saponin B.

[0073] Xuefengshanoside C (PGA6), a white amorphous powder; (c 0.1,MeOH); 1 H and 13 C10 NMR data are shown in Table 4; ESI-MS m / z 647 [M+Na] + HR-ESI-MS m / z 623.3439 [MH] - (C 33 H 51 O 11 (Calculated value: 623.3437).

[0074] High-resolution electrospray ionization mass spectrometry of PGA6 showed an anion signal of m / z 623.3439 [MH]. - (C 33 H 51 O 11 The calculated value is 623.3437. Based on the nuclear magnetic resonance data (Table 4), its molecular formula is determined to be C. 33 H 52 O 11 Its degree of unsaturation is 8. 1 HNMR data showed four methyl signals and one trisubstituted double bond signal [δ] H 5.55 (1H, br d, J = 5.6Hz)], and one β-pyran-fucose terminal proton signal [δ H 4.71 (1H,d,J=7.5Hz)]. PGA613 C10 NMR showed 33 carbon signals, suggesting the presence of one double bond, four methyl groups, eight methylene groups (two of which are oxygen-bound), sixteen methine groups (ten of which are oxygen-bound), and three quaternary carbons (one of which is oxygen-bound). Comparing the NMR data of PGA6 and PGA4, it is speculated that PGA6 is a structural analog of PGA4 with one less sugar group. Furthermore, because the aglycone of PGA6 lacks one methyl signal but has an additional oxygen-bound methylene signal, it is presumed that this methyl group is oxidized. Analysis of PGA6... 1 H- 1 H COSY related and HMBC related ( Figure 5 ), revealing that the glycosyl group is attached to 1-OH, and C-21 is a hydroxymethyl group. Analysis of ROESY related ( Figure 5 It was found that the relative configuration of the PGA6 aglycone moiety is identical to that of PGA4. Therefore, PGA6 is identified as... Figure 1 As shown, it is named Xuefengshan saponin C.

[0075] Table 4. PGA6 in deuterated pyridine 1 H(800MHz) and 13 C(201MHz) NMR data (δin ppm, J in Hz)

[0076]

[0077] 2. The spectral data of compounds PFV70, PFV72, PFV77, PFV78 and PGA27 are known.

[0078] Protogracillin (PFV70): White amorphous powder, C 51 H 84 O 23 ; (c0.2,MeOH); 1 ¹H NMR (pyridine-d5, 500MHz) δ H 6.39(1H,br s,H-1″),5.31(1H,br d,J=5.1Hz,H-6).5.10(1H,d,J=7.7Hz,H-1″′),4.81(d,J=7.8Hz,H-1′),1.75(3H,d,J=6.2Hz,H3-6″) ,1.33(3H,d,J=6.9Hz,H3-21),1.05(3H,d,J=5.4Hz,H3-27),0.97(3H,s,H3-19),0.88(3H,s,H3-18); 13 C-NMR (pyridine-d5, 126MHz) δC 140.8(C-5),121.9(C-6),110.7(C-22),105.0(glc-1″″),104.6(glc-1″′),102.2(rha -1″),100.0(glc-1′),89.6(glc-3′),81.1(C-16),78.8(glc-3″″),78.7(glc-5″″),77 .9(glc-5′),77.7(glc-2′),77.0(glc-3″′,5″′),75.3(glc-2″″),75.0(glc-2″′),74. 2(rha-4″),72.8(rha-3″),72.5(rha-2″),71.7(glc-4″″),71.5(glc-4′),69.6(rha-5 ″),69.6(glc-4″′),63.9(C-17),62.8(glc-6″″),62.5(glc-6′,6″′),56.6(C-14),50. 3(C-9),40.8(C-13),40.5(C-20),40.0(C-12),38.7(C-4),37.5(C-1),37.2(C-10),34 .3(C-25),32.5(C-7),32.4(C-15),31.7(C-8),30.8(C-23),30.1(C-2),28.4(C-24),2 1.1(C-11),19.4(C-19),18.7(rha-6″),17.2(C-27),16.3(C-18),16.3(C-21); ESI-MS m / z 1087[M+Na] + Its nuclear magnetic resonance spectroscopy data are basically consistent with the data reported in the literature [Hu K.,Dong A.,Yao X.,Kobayashi H.,Iwasaki S.Antineoplasticagents II:four furostanol glycosides from rhizomes of Dioscorea collettiivar.hypoglauca.Planta Med.,1997,63(2):161–165].

[0079] Methyl protogracillin (PFV72): white needle crystals (70% MeOH), mp 287–289℃, C 52 H 86 O 23 ; (c 0.3,MeOH); 1H NMR(pyridine-d5,500MHz)δ H 6.40(1H,br s,H-1″),5.33(1H,br d,J=5.0Hz,H-6),5.11(1H,d,J=7.8Hz,H-1″′),4.85(1H,d,J=7.7Hz,H-1′),3.83(1H,m,H-3),3.26(3H,s,22-OCH3),1.76(3H,d,J=6.3Hz,H3-6″),1.19(3H,d,J=6.9Hz,H3-21),1.05(3H,s,H3-19),1.00(3H,d,J=6.7Hz,H3-27),0.81(3H,s,H3-18); 13 C-NMR(pyridine-d5,126MHz)δ C 140.8(C-5),121.9(C-6),112.7(C-22),105.0(glc-1″″),104.6(glc-1″′),102.3(rha-1″),100.0(glc-1′),89.6(glc-3′),81.3(C-16),78.7(glc-3″″),78.7(glc-5″″),77.9(glc-5′),77.7(glc-2′),77.0(glc-3″′,5″′),75.2(glc-2″″),75.0(glc-2″′),74.2(rha-4″),72.8(rha-3″),72.5(rha-2″),71.5(glc-4″″),71.5(glc-4′),69.6(rha-5″),69.6(glc-4″′),64.2(C-17),62.9(glc-6″″),62.4(glc-6′,6″′),56.6(C-14),50.3(C-9),47.3(22-OCH3),40.8(C-13),40.5(C-20),39.8(C-12),38.7(C-4),37.5(C-1),37.2(C-10),34.2(C-25),32.3(C-7),32.2(C-15),31.7(C-8),30.8(C-23),30.1(C-2),28.2(C-24),21.0(C-11),19.4(C-19),18.7(rha-6″),17.2(C-27),16.3(C-18),16.3(C-21);ESI-MS m / z 1101[M+Na] +Its nuclear magnetic resonance spectroscopy data are basically consistent with the data reported in the literature [Hu K.,Dong A.,Yao X.,Kobayashi H.,Iwasaki S.Antineoplastic agentsII:four furostanol glycosides from rhizomes of Dioscorea collettiivar.hypoglauca.Planta Med.,1997,63(2):161–165].

[0080] Methyl protopolyphyllin I (PFV77): white amorphous powder, C 51 H 84 O 22 ; (c 0.3,MeOH); 1 ¹H NMR (pyridine-d5, 500MHz) δ H 6.99(1H,br s,ara-1″′),6.40(1H,br s,rha-1″),5.30(1H,br d,J=5.1Hz,H-6),4.95(d,J=7.1Hz,glc-1′),3.25(3H,s,22-OCH3),1.76(3H,d,J=6.2Hz,H3-6″),1 .18(3H,d,J=6.8Hz,H3-21),1.03(3H,s,H3-19),0.99(3H,d,J=6.7Hz,H3-27),0.80(3H,s,H3-18); 13 C-NMR (pyridine-d5, 126MHz) δ C:140.8(C-5),121.8(C-8),112.7(C-22),109.6(ara-1″′)105.0(glc-1″″),101.9(rha-1″) ,100.2(glc-1′),86.7(ara-4″′)82.7(ara-2″′),81.3(C-16),78.6(glc-5″″),78.5(glc-2″ ′),78.1(C-3),77.9(glc-3″″),77.9(glc-2″′),77.7(ara-2″′),77.4(glc-5′)77.0(glc-3′ ),76.7(glc-4′),75.2(C-26),74.1(rha-4″),72.8(rha-2″),72.5(rha-3″),71.8(glc-4″″) ,69.5(rha-5″),64.2(C-17),62.9(glc-6″″)62.5(rha-5″),61.4(ara-5″′),56.6(C-14),50 .3(C-9),47.3(OCH3-22),40.8(C-20),40.5(C-13),39.7(C-12),39.0(C-4),37.5(C-1),37. 1(C-10),34.2(C-25),32.3(C-15),32.2(C-7),31.7(C-8),30.8(C-23),30.2(C-2),28.2(C- 24),21.1(C-11),19.4(C-19),18.7(rha-6″),17.2(C-27),16.4(C-18),16.3(C-21); ESI-MS m / z 1071[M+Na] + Its nuclear magnetic resonance spectroscopy data are basically consistent with the data reported in the literature [Miyamura M., Nakano K., Nohara T., Tomimatsu T., Kawasaki T. Steroid saponins from Paris polyphylla Sm.-Supplement. Chem. Pharm. Bull., 1982, 30(2): 712–718].

[0081] Methyl protobioside (PFV78): white amorphous powder, C 46 H 76 O 18 ; (c 0.3,MeOH); 1 ¹H NMR (pyridine-d5, 500MHz) δH 6.42(1H,br s,H-1″),5.30(1H,brd,J=5.0Hz,H-6),4.84(1H,d,J=7.8Hz,H-1′),3.25(3H,s,22-OCH3),1.77(3H,d,J=6.3Hz,H3-6″),1.18(3H,d,J=6.9Hz,H3-21),1.03(3H,s,H3-19),0.99(3H,d,J=6.7Hz,H3-27),0.80(3H,s,H3-18); 13 C NMR(pyridine-d5,126MHz)δ C 140.9(C-5),121.8(C-6),112.7(C-22),105.0(rha-1″′),102.1(glc-1″),100.4(glc-1′),81.3(C-16),79.7(glc-2′),78.7(glc-3″′),78.6(glc-5″′),77.9(glc-3′),77.9(C-3),77.9(glc-5′),75.3(glc-2″′),75.2(C-26),74.2(rha-4″),72.9(glc-4″′),72.6(rha-3″),71.8(rha-2″),71.7(glc-4′),69.5(rha-5″),64.2(C-17),62.9(glc-6″′)62.7(glc-6′),56.6(C-14),50.3(C-9),47.3(OCH3-22),40.8(C-20),40.5(C-13),39.7(C-12),39.0(C-4),37.5(C-1),37.2(C-10),34.2(C-25),32.3(C-15),32.2(C-7),31.7(C-8),30.2(C-23),30.2(C-2),28.2(C-24),21.1(C-11),19.4(C-19),18.7(rha-6″),17.2(C-27),16.5(C-18),16.5(C-21);ESI-MS m / z 939[M+Na] +Its nuclear magnetic resonance spectroscopy data are basically consistent with the data reported in the literature [Shen P.,Wang S.L.,Liu XK,Yang CR,Cai B.,Yao XSA new steroidal saponin from Dioscoreadeltoidea Wall var. orbiculata.Chin.Chem.Lett.,2002,13(9):851–854].

[0082] Methyldichotomin (PGA27), C 58 H 96 O 26 White amorphous powder; (c 0.2,MeOH); 1 H NMR and 13 C NMR data are shown in Table 5; ESIMS m / z 1231 [M+Na] + Its NMR data are consistent with those reported in the literature [Yang D.-J., Lu T.-J., Hwang LSIsolation and identification of steroidalsaponins in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto). J. Agric. Food Chem., 2003, 51(22): 6438–6444].

[0083] Table 5. PGA27 in deuterated pyridine 1 H (500MHz) and 13 C NMR (126MHz) data (δin ppm, J inHz).

[0084]

[0085]

[0086] Example 3:

[0087] Experiments on compounds that promote collagen secretion.

[0088] 1. Cells

[0089] Adult dermal fibroblasts (HDFa) were purchased from Beina Biotechnology.

[0090] 2. Reagents

[0091] 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.

[0092] 3. Experimental Methods

[0093] HDFa cells were mixed with the test compound in 96-well cell culture plates. A blank control without the compound and a TGF-β positive control were also 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.

[0094] Collagen secretion increase rate (%) = (OD of experimental wells) 450 nm / Cell viability / Octopic pore OD 450 nm -1)×100%

[0095] 4. Experimental Results

[0096] This invention evaluated the activity of 24 Paris polyphylla steroidal saponins in promoting HDFa collagen secretion. The test results are shown in Table 6. Some compounds showed good activity, among which, Paris polyphylla saponin VII (PFE73; increase rate 56.5%), protodiospin (PFV50; increase rate 65.6%), protodiospin (PFV51; increase rate 56.1%), protodiospin (PFV70; increase rate 70.9%), Xuefengshan saponin B (PGA5; increase rate 53.8%), Xuefengshan saponin C (PGA6; increase rate 61.2%), and methyl protosaponin Pb (PGA27; increase rate 74.0%) showed extremely strong activity. At very low concentrations (0.1 μM), they promoted collagen secretion in adult dermal fibroblasts, with an increase in collagen secretion rate exceeding 50%; and at this concentration, they showed no cytotoxicity (cell viability greater than 100%).

[0097] Table 6. The promoting effect of Paris polyphylla steroidal saponins on HDFa collagen secretion

[0098]

[0099] *Positive control

[0100] Formulation Examples

[0101] In the following formulation examples, conventional reagents were selected and formulations were prepared according to existing conventional methods. This application example only demonstrates that the Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6, and PGA27 described in this invention can be prepared into different formulations. Specific reagents and operations are not specifically limited.

[0102] 1. The Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27, or any combination thereof, are added to water for injection according to conventional methods, filtered, filled and sterilized to prepare an injection solution with a concentration of 0.1 mg / mL.

[0103] 2. Dissolve any one or any combination of Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27 in sterile water for injection, stir until dissolved, filter using a sterile suction funnel, then perform sterile fine filtration, dispense into ampoules, freeze-dry at low temperature, and then sterilely seal to obtain a powder for injection.

[0104] 3. One or any combination of Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27, are added to the excipient at a weight ratio of 9:1 to prepare a powder.

[0105] 4. Add any one or any combination of Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27, at a weight ratio of 5:1 to the excipient, and granulate and compress into tablets.

[0106] 5. Prepare an oral liquid by taking any one or any combination of Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27, according to conventional oral liquid preparation methods.

[0107] 6. One or any combination of Paris polyphylla steroidal saponin compounds PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27, are added to an excipient at a weight ratio of 5:1 to prepare capsules.

[0108] 7. Any one or any combination of several of the following Paris polyphylla steroidal saponin compounds: PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6, and PGA27.

[0109] Cosmetic Formulation Examples

[0110] 1. A cream formulation (W%) containing any one or any combination of the following Paris polyphylla steroidal saponin compounds of the present invention: PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27:

[0111] Paris polyphylla steroidal saponins PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6, and PGA27.

[0112]

[0113]

[0114] The cosmetic product of the above-described formula of the present invention is prepared using conventional methods for manufacturing cosmetics.

[0115] 2. An emulsion formulation (W%) containing any one or any combination of the following Paris polyphylla steroidal saponin compounds of the present invention: PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6 and PGA27:

[0116] Paris polyphylla steroidal saponins PFE52, PFE53, PFE54, PFE56, PFE57, PFE59A, PFE62A, PFE66, PFE67, PFE73, PFE75, PFV50, PFV51, PFV53, PFV70, PFV72, PFV77, PFV78, PFV92, PFV93, PGA4, PGA5, PGA6, and PGA27.

[0117] The cosmetic product of the above-described formula of the present invention is prepared using conventional methods for manufacturing cosmetics.

[0118] 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. The following structural formulas represent the Paris polyphylla steroidal saponin compounds PFV93, PGA4, PGA5, and PGA6. 。 2. A process for preparing the Paris saponin compounds PFV93, PGA4, PGA5 and PGA6 according to claim 1, characterized in that, The method includes the following steps: The dried rhizome of Paris fargesii was crushed, extracted with 70% ethanol (50 L x 3) at 78-80 °C for 4 h, 3 h and 3 h, respectively, and filtered to obtain a filtrate. The filtrate was concentrated to obtain a crude extract, which was suspended in 16 L distilled water and extracted with an equal volume of ethyl acetate for 3 times. The organic phase was recovered to obtain an ethyl acetate extraction part. The water phase was extracted with an equal volume of n-butanol for 4 times. The organic phase was recovered to obtain a n-butanol extraction part. The n-butanol extraction part was subjected to gradient elution using a 200-300 mesh normal phase silica gel column, and the eluent was 10:1-0:1 CH2Cl2-MeOH. After the eluent was detected by thin layer chromatography, 7 parts were combined: Fr. I, Fr. II, Fr. III, Fr. IV, Fr. V, Fr. VI and Fr. VII. Fr. VII was subjected to gradient elution using a normal phase silica gel column, and the eluent was 10:1-0:1 EtOAc-MeOH. After the eluent was detected by thin layer chromatography, 4 parts were combined: Fr. VII-1, Fr. VII-2, Fr. VII-3 and Fr. VII-4. Fr. VII-4 was subjected to gradient elution using a reversed phase (RP) C 18 silica gel column (MeOH-H2O, 10-100%) to obtain 4 parts: Fr. VII-4-1, Fr. VII-4-2, Fr. VII-4-3 and Fr. VII-4-4. Fr. VII-4-1 was subjected to Sephadex LH-20 gel column chromatography (MeOH) and purified by semi-preparative high performance liquid chromatography to obtain Fr. VII-4-1-1. Fr. VII-4-1-1 was purified by semi-preparative HPLC to obtain Paris saponin B, i.e., PFV93. The dried Xuefengshan Paris polyphylla rootstock was crushed, and extracted with 70% ethanol aqueous solution (4 L, 3 L and 3 L) at 60 ℃ for 3 times, 0.5 h each time. The extract was filtered, and the filtrate was concentrated and evaporated at 60 ℃ using a rotary evaporator to obtain a crude extract. The crude extract was added with distilled water to form a suspension, which was extracted with petroleum ether, ethyl acetate and n-butanol, each for 3 times. The organic phase and the n-butanol extracted water phase were concentrated and evaporated at 60 ℃ using a rotary evaporator under reduced pressure to obtain petroleum ether extraction, ethyl acetate extraction, n-butanol extraction and water phase. The n-butanol extraction was subjected to normal phase silica gel column chromatography gradient elution to obtain Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F and Fr. G. Fr. C was subjected to RP C 18 silica gel column chromatography (MeOH-H2O, 10~100%) to obtain ten fractions (Fr. C-1~Fr. C-10). Fr. C-5 was subjected to Sephadex LH-20 gel column chromatography, and then purified by semi-preparative HPLC to obtain Xuefengshan saponin B (PGA5), Xuefengshan saponin C (PGA6) and Xuefengshan saponin A (PGA4).

3. The use of the Paris polyphylla steroidal saponin compounds PFV93, PGA4, PGA5 and PGA6 as described in claim 1 in the preparation of a collagen-promoting active agent.

4. The use of the Paris polyphylla steroidal saponin compounds PFV93, PGA4, PGA5 and PGA6 as described in claim 1 in the preparation of anti-aging drugs.

5. The use of the Paris polyphylla steroidal saponin compounds PFV93, PGA4, PGA5 and PGA6 as described in claim 1 in the preparation of cosmetics.

6. A pharmaceutical composition comprising any one or any combination of the Paris polyphylla steroidal saponin compounds PFV93, PGA4, PGA5, and PGA6 as described in claim 1, and at least one pharmaceutically acceptable carrier.

7. A cosmetic product comprising any one or any combination of the Paris polyphylla steroidal saponin compounds PFV93, PGA4, PGA5, and PGA6 as described in claim 1, and conventional cosmetic excipients.

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