Five long-stalk eleutherococcus alkenyl alcohol compounds in long-stalk eleutherococcus, extraction and separation method and application thereof

Abstract

The application discloses five long-stalk acanthopanax alkenyl alcohol compounds in long-stalk acanthopanax, an extraction and separation method and application thereof, and belongs to the technical field of traditional Chinese medicine extraction and separation. Five new compounds, long-stalk acanthopanax alkenyl alcohol A, long-stalk acanthopanax alkenyl alcohol B, long-stalk acanthopanax alkenyl alcohol C, long-stalk acanthopanax alkenyl alcohol D and long-stalk acanthopanax alkenyl alcohol E, are extracted and separated from long-stalk acanthopanax. The new compounds 1-5 obtained in the application have no cytotoxicity at 10-40 muM; at a drug concentration of 40 muM, the compounds 1, 2 and 4 have significant NO inhibitory activity, and can better reduce the content of NO in BV2 cells and RAW264.7 cells induced by LPS, wherein the compound 2 has the best inhibitory effect on the production of NO in the two kinds of cells.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction and separation technology, and in particular relates to five eleutherococcal alcohol compounds from Acanthopanax senticosus and their extraction and separation methods and applications. Background Technology

[0002] Eleutherococcus leucorrhizus var. scaberulus (Harms & Rehder) Nakai, also known as purple eleutherococcus or narrow-leaved eleutherococcus, is recorded in the "Chinese Materia Medica" as having medicinal uses for its root bark and leaves. The root bark is described as pungent, slightly bitter, and warm in nature; it is said to dispel wind and dampness, promote blood circulation, and relieve pain; it is used to treat rheumatic pain, lower back and knee weakness, traumatic injuries, fractures, headaches, abdominal pain, dysmenorrhea, beriberi, and unidentified boils and carbuncles. It is a folk medicine in Yunnan, used locally as eleutherococcus bark. Its fresh leaves are used to stop bleeding, primarily treating external bleeding. It is also a popular local health-promoting vegetable, listed as one of the three treasures of Mojiang County, Yunnan Province, and is widely loved.

[0003] Currently, *Eleutherococcus senticosus* is widely cultivated in Pu'er City, Yunnan Province. Furthermore, numerous *Eleutherococcus senticosus*-related products, such as *Eleutherococcus senticosus* tea, *Eleutherococcus senticosus* wine, *Eleutherococcus senticosus* beverages, *Eleutherococcus senticosus* quick-dissolving powder, and *Eleutherococcus senticosus* crisps, are being developed and utilized. Therefore, *Eleutherococcus senticosus* has significant research value and development potential. However, there are currently no reports on the systematic separation, identification, and application research of the chemical components in *Eleutherococcus senticosus*. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes five eleutherophyllol compounds from *Eleutherococcus senticosus*, along with their extraction, separation, and applications. This invention isolates five eleutherophyllol compounds from *Eleutherococcus senticosus*, and based on this, uses a BV2 / RAW264.7 two-cell model to study the inhibitory effects of these five compounds on NO production. This allows for the screening of chemical components with potential anti-neuritis and anti-inflammatory activities, providing a scientific basis for further in-depth development of *Eleutherococcus senticosus* and the discovery of potential anti-inflammatory lead compounds.

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

[0006] This invention provides five eleutherophyllol compounds from Acanthopanax senticosus, namely eleutherophyllol A (compound 1), eleutherophyllol B (compound 2), eleutherophyllol C (compound 3), eleutherophyllol D (compound 4), and eleutherophyllol E (compound 5).

[0007] The structural formula of the long-stem ginkyne alcohol A is as follows: The structural formula of the long-stemmed ginsenoside B is as follows: The structural formula of the long-stemmed ginsenoside C is as follows: The structural formula of the long-stemmed acetylenol D is as follows: The structural formula of the long-stemmed ginsenoside E is as follows: .

[0008] This invention provides a method for extracting and separating five eleutherophyllol compounds from *Eleutherococcus senticosus* as described in the above-mentioned technical solution, comprising the following steps:

[0009] (1) The roots of Acanthopanax senticosus were extracted by cold soaking in methanol, and the extract was concentrated to obtain an extract. The extract was dispersed in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively.

[0010] (2) The n-butanol extract is loaded onto a macroporous resin column and eluted using a methanol-water solvent system with a gradient elution. The volume ratio of methanol to water in the methanol-water solvent system is 30:70, 50:50, 70:30 and 90:10, respectively, to obtain 30% methanol elution fraction, 50% methanol elution fraction, 70% methanol elution fraction and 90% methanol elution fraction;

[0011] (3) The 30% methanol eluent was loaded onto a normal silica gel column and subjected to gradient elution using a dichloromethane-methanol solvent system to obtain 20 components RB1-20. The components RB1-20 were separated and purified to obtain five long-stemmed pentacene alcohol compounds.

[0012] Furthermore, in step (1), the temperature of the cold soaking extraction is room temperature, and the number of cold soaking extractions is 3 times, 7 days / time.

[0013] Further, in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent system is 30:1 to 0:1.

[0014] Further, in step (3), semi-preparative HPLC is used to separate component RB15. The solvent system used for separation is a methanol-water solvent system with a volume ratio of methanol to water of 35:65, to obtain four subcomponents RB15A-D. Semi-preparative HPLC is used to separate and purify subcomponent RB15B. The solvent system used for separation and purification is a methanol-water solvent system with a volume ratio of methanol to water of 45:55, to obtain long-stemmed acetylenol A.

[0015] Furthermore, the subfraction RB15D was separated and purified by semi-preparative HPLC. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 47:53, to obtain long-stemmed acetylenol B.

[0016] Further, in step (3), semi-preparative HPLC is used to separate component RB18. The solvent system used for separation is a methanol-water solvent system with a volume ratio of methanol to water of 35:65, to obtain 5 subcomponents RB18A-E; semi-preparative HPLC is used to separate and purify subcomponent RB18B. The solvent system used for separation and purification is a methanol-water solvent system with a volume ratio of methanol to water of 40:60, to obtain long-stemmed acetylenol C.

[0017] Furthermore, the subfraction RB18D was separated and purified by semi-preparative HPLC. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 43:57, to obtain long-stemmed pentacene alcohol D.

[0018] Furthermore, the subfraction RB18E was separated and purified by semi-preparative HPLC. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 42:58, to obtain long-stemmed acetylenol E.

[0019] The present invention also provides the application of five eleutherocene alcohol compounds from the above-described eleutherocene spp. in the preparation of drugs for the prevention and / or treatment of inflammatory diseases.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] This invention extracted and isolated five novel compounds—eleutherophyllin A, eleutherophyllin B, eleutherophyllin C, eleutherophyllin D, and eleutherophyllin E—from *Eleutherococcus senticosus*. All five compounds obtained in this invention showed no cytotoxicity at concentrations ranging from 10 to 40 μM. At a drug concentration of 40 μM, compounds 1, 2, and 4 exhibited significant NO inhibitory activity, effectively reducing NO levels in LPS-induced BV2 and RAW264.7 cells. Compound 2 showed the best inhibitory effect on NO production in both cell types. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 The structural formulas of compounds 1-5 are shown.

[0024] Figure 2 For the structure of compound 1 and key HMBC, 1 H- 1 H COSY correlation;

[0025] Figure 3 The image shows the ECD spectrum of compound 1. In the figure, Exp. represents the measured value and Cal. represents the theoretical value, and the same applies below.

[0026] Figure 4 For the structure of compound 2 and key HMBC, 1 H- 1 H COSY correlation;

[0027] Figure 5 The ECD spectrum of compound 2;

[0028] Figure 6 For the structure of compound 3 and key HMBC, 1 H- 1 H COSY correlation;

[0029] Figure 7 The ECD spectrum of compound 3;

[0030] Figure 8 For the structure of compound 4 and key HMBC, 1 H- 1 H COSY correlation;

[0031] Figure 9 The ECD spectrum of compound 4 is shown below.

[0032] Figure 10 For the structure of compound 5 and key HMBC, 1 H- 1 H COSY correlation;

[0033] Figure 11 The ECD spectrum of compound 5 is shown below. Detailed Implementation

[0034] 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.

[0035] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] In this embodiment of the invention, room temperature refers to "25±2℃".

[0037] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0038] Example 1

[0039] The extraction and separation method of five eleutherophyllol compounds from Acanthopanax senticosus is as follows:

[0040] (1) Take 10.25 kg of long-stemmed Acanthopanax senticosus root, dry and pulverize it, and extract it three times with methanol at room temperature, once every 7 days. Combine the concentrates, concentrate them under reduced pressure to 0.025 times the original volume, and then evaporate them to dryness in a water bath until no methanol is present, to obtain 560 g of extract. Take 550 g of extract and disperse it in 10 L of water, and then extract it sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract (50 g), ethyl acetate extract (60 g) and n-butanol extract (90 g) respectively. Attach the above n-butanol extract to a D101 macroporous resin column and perform methanol-water solvent system. Gradient elution was performed in a methanol-water solvent system with volume ratios of methanol to water of 30:70, 50:50, 70:30, and 90:10, yielding fractions of 30%, 50%, 70%, and 90% methanol, respectively. The 30% methanol fraction was then loaded onto a normal silica gel column and subjected to gradient elution in a dichloromethane-methanol solvent system with volume ratios of dichloromethane to methanol of 30:1, 20:1, 15:1, 10:1, 7:1, 5:1, and 0:1, resulting in 20 fractions RB1-20.

[0041] (2) Semi-preparative HPLC was used to separate component RB15. The solvent system used for separation was a methanol-water solvent system with a volume ratio of methanol to water of 35:65, yielding four subcomponents RB15A-D. Semi-preparative HPLC was used to separate and purify subcomponent RB15B. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 45:55, yielding compound 1 (t R =43min, 3.2mg);

[0042] Subfraction RB15D was separated and purified by semi-preparative HPLC using a methanol-water solvent system with a volume ratio of 47:53 (methanol:water), yielding compound 2 (t). R =46min, 5.9mg);

[0043] Semi-preparative HPLC was used to separate component RB18 in a methanol-water solvent system with a volume ratio of methanol to water of 35:65, yielding five subcomponents RB18A-E. Semi-preparative HPLC was also used to separate and purify subcomponent RB18B in a methanol-water solvent system with a volume ratio of methanol to water of 40:60, yielding compound 3 (t).R =36min, 33.7mg);

[0044] Subfraction RB18D was separated and purified by semi-preparative HPLC using a methanol-water solvent system with a volume ratio of methanol to water of 43:57, yielding compound 4 (t). R =43min, 14.9mg).

[0045] Subfraction RB18E was separated and purified by semi-preparative HPLC using a methanol-water solvent system with a volume ratio of methanol to water of 42:58, yielding compound 5 (t). R =35min, 10.4mg).

[0046] Through HR-ESI-MS, 1 H-NMR, 13 C-NMR and DEPT, MS 1 H- 1 The structures of compounds 1-5 were identified by H COSY, HMQC, HMBC, and NOESY. The results are shown in [Figure number missing]. Figures 2-11 .

[0047] Identification of Compound 1

[0048] The structural formula of compound 1 is shown in [reference needed]. Figure 1 Key HMBC 1 H- 1 The correlation between H COSY and other factors can be found in [link to H COSY]. Figure 2 ECD spectrum Figure 3 , 1 H-NMR and 13 C-NMR data are shown in Table 1.

[0049] Compound 1: Yellow powder; 1% vanillin-concentrated sulfuric acid TLC colorimetric reaction shows light blue, 1% indigo-concentrated sulfuric acid TLC colorimetric reaction shows brown. The TLC colorimetric results suggest that compound 1 may be a polyacetylenic compound. –12.50 (c0.1, MeOH); HR-ESI-MS m / z 331.15127 [M+Na] + (Calculate C) 17 H 24 O5Na, 331.15159). The molecular formula of this compound was determined to be C by HR-ESI-MS. 17 H 24 O5, with an unsaturation degree of 6. Infrared spectroscopy shows that this compound contains hydroxyl groups (3289 cm⁻¹). -1 ), methylene (2920, 2878cm) -1), alkynyl group (2200, 2140cm) -1 ), alkenyl (1620cm) -1 UV spectroscopy showed that compound 1 exhibited the characteristic absorption of polyeneyne compounds [λmax: 238, 262, 280, 293, 312 nm]. According to... 1 H-NMR (600MHz, CD3OD) spectra revealed two sets of olefin proton signals in compound 1, namely two internal olefin protons and four terminal olefin protons with δ values ​​of 5.93 (1H, ddd, J = 17.1, 10.2, 5.5 Hz, H-2), 5.86 (1H, ddd, J = 16.9, 10.4, 6.2 Hz, H-16), 5.42 (1H, dt, J = 17.1, 1.4 Hz, H-1a), 5.21 (2H, m, H-1b, H-17a), and 5.08 (1H, dt, J = 10.4, 1.6 Hz, H-17b); and five hydroxymethyl hydrogen signals with δ values ​​of 5.93 (1H, ddd, J = 17.1, 10.2, 5.5 Hz, H-2), 5.86 (1H, ddd, J = 16.9, 10.4, 6.2 Hz, H-16), 5.42 (1H, dt, J = 17.1, 1.4 Hz, H-1a), 5.21 (2H, m, H-1b, H-17a), and 5.08 (1H, dt, J = 10.4, 1.6 Hz, H-17b). H 4.89 (1H, d, H-3), 4.45 (1H, d, J = 7.3 Hz, H-8), 4.04 (1H, q, J = 6.3 Hz, H-15), 3.80 (1H, ddd, J = 7.9, 4.7, 2.6 Hz, H-10), 3.40 (1H, dd, J = 7.3, 2.6 Hz, H-9); according to 13 C-NMR (150MHz, CD3OD) and DEPT spectra indicate that compound 1 has a total of 17 carbon signals. Combined with HSQC spectral analysis, it is inferred that compound 1 contains four typical alkyne carbon signals δ. C 80.0 (C-7), 69.0 (C-5), 68.2 (C-6), and 77.6 (C-4); four olefin carbon signals δ C 115.2 (C-1), 136.7 (C-2), 141.2 (C-16), and 113.1 (C-17); four methylene carbon signals (36.7, 33.1, 25.4, 25.1), and five hydroxymethyl carbon signals δ C 75.3 (C-9), 72.4 (C-15), 69.6 (C-10), 63.3 (C-8), and 62.5 (C-3). From these results, it can be concluded that compound 1 is a C-type compound containing two terminal double bonds. 17 -Diyneyl alcohol. Based on HMBC spectra and... 1 H- 1 Further analysis of the H COSY spectrum, in which H-15 (δ H4.04) / H-16 (δ H 5.86) / H-17 (δ H 5.08, 5.21) have 1 H- 1 H COSY correlation suggests that compound 1 contains a -CH(OH)CH=CH2 segment in its structure, and H-15 (δ) in the HMBC spectrum. H 4.04, q, J = 6.3 Hz) and C-13 (δ C 25.1) / C-14 (δ C 36.7) related, H-17[δ H 5.21 (m, H-17a), δ H 5.08 (dt, J = 10.4, 1.6 Hz, H-17b)] and C-15 (δ C 72.4) / C-16 (δ C Related to 141.2), further verification confirmed that the hydroxyl group is attached to C-15, and the second double bond is attached to C-16 and C-17. Therefore, the chemical structure of compound 1 was initially identified as heptadeca-1,16-dien-4,6-diyne-3,8,9,10,15-pentaol. However, a search of the sciFinder database did not reveal this compound. By comparing the spectral data with those of (3S,8R,9S,10R)-heptadec-1-ene-4,6-diyne-3,8,9,10-tetrol ((3S,8R,9S,10R)-heptadeca-1-ene-4,6-diyne-3,8,9,10-tetrol), the absolute configurations of C-3, C-8, C-9, and C-10 in compound 1 are preliminarily inferred to be 3S, 8R, 9S, and 10R, respectively. Combined with the results of CD spectroscopy and electronic circular dichroism (ECD) spectroscopy, it was found that the experimental data... Since the values ​​are consistent with theoretical calculations, it is further inferred that the absolute configuration of compound 1 may be (3S,8R,9S,10R,15R)-heptadeca-1,16-dien-4,6-diyne-3,8,9,10,15-pentaol. This compound may be a new compound and is named eleuthdiyne A.

[0050] Table 1 Compound 1 1 H-NMR (600 MHz, CD3OD) and 13C-NMR (150 MHz, CD3OD) data

[0051]

[0052] Identification of compound 2

[0053] The structural formula of compound 2 is shown in [reference needed]. Figure 1 Key HMBC 1 H- 1 The correlation between H COSY and other factors can be found in [link to H COSY]. Figure 4 ECD spectrum Figure 5 , 1 H-NMR and 13 C-NMR data are shown in Table 2.

[0054] Compound 2: Pale yellow powder. The 1% vanillin-concentrated sulfuric acid TLC reaction produces a dark blue color, while the 1% indigo-concentrated sulfuric acid TLC reaction produces a brown color. The TLC results suggest that compound 2 may be a polyacetylenic compound. –8.67 (c0.11, MeOH); HR-ESI-MS m / z 333.16700 [M+Na] + (Calculate C) 17 H 26 O5Na, 333.16725). The molecular formula of this compound was determined to be C5Na based on HR-ESI-MS m / z. 17 H 26 O5, degree of unsaturation is 5; infrared spectroscopy shows that this compound contains hydroxyl groups (3309 cm⁻¹). -1 ), methylene (2890, 2878cm) -1 ), alkynyl group (2263, 2200cm) -1 ), alkenyl (1600cm) -1 UV spectroscopy showed that compound 2 exhibited the characteristic absorption of polyeneyne compounds [λmax: 239, 228, 241, 253 nm]. According to... 1 H-NMR (600MHz, CD3OD) spectra revealed the presence of a methyl proton signal δ in compound 2. H 1.01 (3H, t, J = 7.4 Hz, H⁻¹); Two sets of olefin proton signals, one for an internal olefin proton and two for two terminal olefin protons. H 5.86 (ddd, J = 16.9, 10.5, 6.3 Hz, H-16), 5.21 (dt, J = 17.2, 1.7 Hz, H-17a), 5.08 (1H, dt, J = 10.4, 1.5 Hz, H-17b); 5 hydroxymethyl hydrogen signals δ H4.45 (1H, d, J = 7.4 Hz, H-8), 4.32 (1H, t, J = 6.5 Hz, H-3), 4.04 (1H, q, J = 6.3 Hz, H-15), 3.80 (1H, ddd, J= 8.8, 4.5, 2.6 Hz, H-10), 3.40 (1H, dd, J = 7.3, 2.6 Hz, H-9); based on 13 C-NMR and DEPT spectra show that compound 2 has a total of 17 carbon signals. Combined with HSQC spectral analysis, compound 2 contains one methyl signal (δ). C 8.4 (C-1), four typical alkyne carbon signals δ C 79.8 (C-4), 68.4 (C-5) and δ C 67.9 (C-6), 79.3 (C-7); 5 hydroxymethyl carbon signals δ C 75.3 (C-9), 72.4 (C-15), 69.6 (C-10), 63.3 (C-8), and 63.0 (C-3), two olefin carbon signals δ C 141.2 (C-16), 113.1 (C-17) and 5 methylene carbon signals δ C 30.4 (C-2), 33.1 (C-11), 25.4 (C-12), 25.1 (C-13), and 36.7 (C-14); Based on the above analysis, compound 2 is a C-type compound containing a terminal double bond. 17 -Diynyl alcohol, upon comparison of NMR data with compound 1, was found to have a similar structure, with compound 2 lacking only one terminal double bond; further analysis of HMBC spectra and... 1 H- 1 Further analysis using the 1H COSY spectrum confirmed the compound's structure as heptadeca-16-ene-4,6-diyne-3,8,9,10,15-pentaol. A search of the sciFinder database did not reveal this compound. Comparison with the spectral data of compound 1 showed that the coupling constant (J) in compound 2... H2-H3 = 6.5 Hz / J H8-H9 = 7.3 Hz / J H9-H10 = 2.6 Hz / J H15-H16= 6.3 Hz) is consistent with that in 1, suggesting that compound 2 and compound 1 have the same absolute configuration. Combining the CD spectrum and the experimental results of electronic circular dichroism (ECD), the experimental data are consistent with the theoretical calculation value. Therefore, it is further inferred that the absolute configuration of compound 2 may be (3S,8R,9S,10R,15R)-heptadeca-16-ene-4,6-diyne-3,8,9,10,15-pentaol. This compound may be a new compound and is named eleuthdiyne B.

[0055] Table 2 Compound 2 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data

[0056]

[0057] Identification of compound 3

[0058] The structural formula of compound 3 is shown in [reference needed]. Figure 1 Key HMBC 1 H- 1 The correlation between H COSY and other factors can be found in [link to H COSY]. Figure 6 ECD spectrum Figure 7 , 1 H-NMR and 13 C-NMR data are shown in Table 3.

[0059] Compound 3: White powder. The 1% vanillin-concentrated sulfuric acid TLC reaction produces a light purple color, while the 1% indigo-concentrated sulfuric acid TLC reaction produces a brown color. The TLC results suggest that compound 3 may be a polyacetylenic compound. –10.00 (c0.10, MeOH); HR-ESI-MS m / z 351.17852 [M+Na] + (Calculate C) 17 H 28 O6Na, 351.17781). The molecular formula of this compound was determined to be C6Na by HR-ESI-MS. 17 H 28 O6, with an unsaturation degree of 4; infrared spectroscopy shows that this compound contains hydroxyl groups (3298 cm⁻¹). -1 ), methylene (2920, 2878cm) -1 ), alkynyl (2210, 2150cm) -1UV spectroscopy showed that compound 3 exhibited the characteristic absorption of polyyne compounds [λmax: 238, 245, 258 nm]. According to... 1 The 1H-NMR (600 MHz, CD3OD) spectrum shows a methyl proton signal δ. H 1.01 (3H, t, J = 7.4 Hz, H⁻¹); 5 hydroxymethyl hydrogen signals δ H 4.45 (1H,dd, J = 7.3, 0.7 Hz, H-8), 4.32 (1H, t, J = 6.6 Hz, H-3), 3.80 (1H, ddd, J = 7.9, 4.7, 2.6 Hz, H-10), 3.60 (1H, d, J = 6.0 Hz, H-16), 3.40 (1H, dd, J = 7.3, 2.7 Hz, H-9) and two methylene hydrogen signals with hydroxyl groups: 3.49 (1H, dd, J = 11.1, 4.5 Hz, H-17a), 3.44 (1H, dd, J = 11.1, 6.6 Hz, H-17b); according to 13 C-NMR (150MHz, CD3OD) and DEPT spectra indicate that compound 3 has 17 carbon signals. Based on HSQC spectral analysis, it is inferred that compound 3 contains one methyl signal (δ). C 8.4 (C-1); Four typical alkyne carbon signals δ C 79.8 (C-4), 67.9 (C-5) and δ C 68.4 (C-6), 79.3 (C-7); six carbon signals δ with hydroxyl groups C 75.2 (C-9), 71.9 (C-16), 69.7 (C-10), 66.0 (C-17), 63.4 (C-8), and 63.0 (C-3); Based on the above analysis results, compound 3 is C... 17 -Diynyl alcohol, upon comparison of NMR data with compound 2, shows structural similarity; compound 3 has one less terminal double bond and one more hydroxyl group. Further analysis of HMBC spectra and... 1 H- 1 Further analysis of the H COSY spectrum, including 1 H- 1 H COSY spectrum H-16 (δ H 3.60) and H-17 (δ) H 3.49, δ H 3.44) is related, suggesting that compound 3 has a -CH(OH)CH2OH fragment in its structure; H-16 (δ) in the HMBC spectrum H3.60, d, J = 6.0Hz) and C-14 (δ C 25.3) / C-15 (δ C 33.0) / C-17 (δ C 66.0) related, H-17[(δ H 3.49 (dd, J = 11.1, 4.5 Hz, 1H), 3.44 (dd, J = 11.1, 6.6 Hz, 1H)] and C-14 (δ C 25.3) / C-16 (δ C Related to 71.9), it was further determined that both C-16 and C-17 of compound 3 are attached to an OH group. Therefore, the chemical structure of compound 3 is initially speculated to be heptadeca-4,6-diyne-3,8,9,10,16-hexaol. A search of the Sci Finder database did not find this compound. By comparing the spectral data with those of compound 2, the coupling constant (J) in compound 3... H2-H3 = 6.6Hz / J H8-H9 = 7.3 Hz / J H9-H10 = 2.7 Hz / J H15-H16 = 6.0 Hz) is consistent with that of compound 2, suggesting that compound 3 and compound 2 may have the same absolute configuration. Combining the CD spectrum and the experimental results of electronic circular dichroism (ECD), the experimental data are consistent with the theoretical calculation value. Therefore, it is further inferred that the absolute configuration of compound 3 may be (3S,8R,9S,10R,16R)-heptadec-4,6-diyne-3,8,9,10,16-hexaol ((3S,8R,9S,10R,16R)-heptadeca-4,6-diyne-3,8,9,10,16-hexaol). This compound may be a new compound, and it is named eleuthdiyne C.

[0060] Table 3 Compound 3 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data

[0061]

[0062] Identification of compound 4

[0063] The structural formula of compound 4 is shown in [reference needed]. Figure 1 Key HMBC 1 H- 1The correlation between H COSY and other factors can be found in [link to H COSY]. Figure 8 ECD spectrum Figure 9 , 1 H-NMR and 13 C-NMR data are shown in Table 4.

[0064] Compound 4: White powder. The 1% vanillin-concentrated sulfuric acid TLC reaction produces a deep purple color, while the 1% indigo-concentrated sulfuric acid TLC reaction produces a brown color. The TLC results suggest that compound 4 may be a polyacetylenic compound. –9.091 (c0.11, MeOH); HR-ESI-MS m / z 351.17770 [M+Na] + (Calculate C) 17 H 28 O6Na, 351.17781). The molecular formula of this compound was determined to be C6Na by HR-ESI-MS. 17 H 28 O6, with an unsaturation degree of 4; infrared spectroscopy shows that this compound contains hydroxyl groups (3381 cm⁻¹). -1 ), methylene (2920, 2878cm) -1 ), alkynyl group (2251, 2148cm) -1 The UV spectrum showed that compound 4 exhibited the characteristic absorption of polyyne compounds [λmax: 230, 243, 255 nm]. According to... 1 H-NMR (600 MHz, CD3OD) spectra indicate the presence of a methyl proton signal δ in compound 4. H 1.01 (3H, t, J = 7.4 Hz, H⁻¹); 5 hydroxymethyl hydrogen signals δ H 4.49 (1H, d, J = 7.0 Hz, H-8), 4.32 (1H, t, J = 6.5 Hz, H-3), 3.66 (1H, dt, J = 8.0, 4.3 Hz, H-11), 3.64 (2H, m, H-9, H-10) and a hydroxyl-terminated methylene hydrogen signal of 3.57 (2H, t, J = 6.6 Hz, H-17); according to 13 C-NMR and DEPT spectra show that compound 4 has a total of 17 carbon signals. Combined with HSQC spectral analysis, it can be seen that compound 4 has one methyl carbon signal (δ). C 8.4 (C-1), four typical alkyne carbon signals δ C 79.8 (C-4), 68.0 (C-5) and δ C 68.5 (C-6), 79.0 (C-7); six carbon signals δ with hydroxyl groups C74.0 (C-9), 72.3 (C-11), 72.0 (C-10), 63.2 (C-8), 63.0 (C-3), and 61.6 (C-17); Based on the above analysis results, compound 4 is C... 17 -Diynyl alcohol, upon comparison with NMR data of compound 3, shows structural similarity, differing only in the position of the hydroxyl group; further analysis of HMBC spectra and... 1 H- 1 Further analysis of the H COSY spectrum, including 1 H- 1 H-11 (δ) in H COSY spectrum H 3.66) and H-12 (δ) H 2.24) / H-13(δ H 2.11) related, H-16 (δ H 1.83) and H-17 (δ) H The correlation between 3.57, t, J = 6.6 Hz, and 2H suggests that C-11 and C-17 in the structure of compound 4 may be connected to an OH group; H-9 and 10 (δ) in the HMBC spectrum H 3.64) and C-7 (δ) C 79.0) / C-8(δ C 63.2) / C-11 (δ C 72.3) / C-12 (δ C 32.8) related, H-11 (δ) H 3.66, dt, J = 8.0, 4.3 Hz) and C-12 (δ C 32.8) / C-13 (δ C 25.3) related, H-17 (δ) H 3.57 (t, J = 6.6 Hz) and C-15 (δ C 25.5) / C-16 (δ C Related to 32.2), it was further determined that both C-11 and C-17 of compound 4 are connected to a hydroxyl group. Therefore, the chemical structure of compound 4 is initially speculated to be heptadeca-4,6-diyne-3,8,9,10,11-hexaol. A search of the Sci Finder database did not find this compound. By comparing the spectral data with those of compound 3, the coupling constant (J) in compound 4... H2-H3 = 6.5 Hz / J H8-H9 = 7.0 Hz / J H9-H10= 2.7 Hz) is consistent with that of compound 3, suggesting that compound 4 and compound 3 have the same absolute configuration. The absolute configurations of C-3, C-8, C-9, and C-10 in compound 4 are preliminarily determined to be 3S, 8R, 9S, and 10R, respectively. Combining the CD spectrum and the experimental results of electronic circular dichroism (ECD), it was found that the experimental data are consistent with the theoretical calculation values. Therefore, it is further inferred that the absolute configuration of compound 4 may be (3S,8R,9S,10R,11S)-heptadeca-4,6-diyne-3,8,9,10,11-hexaol ((3S,8R,9S,10R,11S)-heptadeca-4,6-diyne-3,8,9,10,11-hexaol). This compound may be a new compound and is named eleuthdiyne D 4.

[0065] Table 4 Compound 4 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data

[0066]

[0067] Identification of compound 5

[0068] The structural formula of compound 5 is shown in [reference needed]. Figure 1 Key HMBC 1 H- 1 The correlation between H COSY and other factors can be found in [link to H COSY]. Figure 10 ECD spectrum Figure 11 , 1 H-NMR and 13 C-NMR data are shown in Table 1.

[0069] Compound 5: White powder. The 1% vanillin-concentrated sulfuric acid TLC reaction produces a light purple color, while the 1% indigo-concentrated sulfuric acid TLC reaction produces a brown color. The TLC results suggest that compound 5 may be a polyacetylenic compound. 0 (c 0.10, MeOH); HR-ESI-MS m / z 351.17834, [M+Na] + (Calculate C) 17 H 28 O6Na, 351.17781). The molecular formula of this compound was determined to be C6Na by HR-ESI-MS. 17 H 28 O6, with an unsaturation degree of 4; infrared spectroscopy shows that this compound contains hydroxyl groups (3381 cm⁻¹). -1 ), methylene (2920, 2878cm) -1 ), alkynyl group (2250, 2142cm)-1 The UV spectrum showed that compound 5 exhibited the characteristic absorption of polyyne compounds [λmax: 231, 242, 255 nm]. According to... 1 The 1H-NMR (600 MHz, CD3OD) spectrum showed that compound 5 contained two methyl proton signals δ. H 1.01 (3H, t, J = 7.4 Hz, H⁻¹), 1.17 (3H, d, J = 6.2 Hz, H⁻¹⁷); 6 hydroxymethyl hydrogen signals δ H 4.49 (1H, d, J = 7.1 Hz, H-8), 4.32 (1H, t, J = 6.5 Hz, H-3), 3.74 (1H, p, J = 6.0 Hz, H-16), 3.67 (1H, dt, J = 8.1, 4.3Hz, H-11), 3.64 (2H, m, H-9, H-10); according to 13 C-NMR (150MHz, CD3OD) and DEPT spectra indicate that compound 5 contains 17 carbon signals. Combined with HSQC spectral analysis, it is inferred that compound 5 contains two methyl signals (δ). C 8.4 (C-1), 22.1 (C-17), four typical alkyne signals δ C 79.8 (C-4), 67.8 (C-5) and δ C 68.5 (C-6), 79.0 (C-7); six carbon signals δ with hydroxyl groups C 74.1 (C-9), 72.3 (C-11), 71.9 (C-10), 67.1 (C-16), 63.2 (C-8), and 63.0 (C-3); Based on the above analysis results, compound 5 is C... 17 -Diynyl alcohol, upon comparison with NMR data of compound 4, was found to have a similar structure, differing only in the position of the hydroxyl group; further analysis of HMBC spectra and... 1 H- 1 Further analysis of the H COSY spectrum revealed H-16 (δ) H 3.74,t, J = 6.0 Hz, 1H) and H-17 (δ) H The correlation between 1.17, t, J = 6.2 Hz, and 3H suggests that compound 5 may have a hydroxyl group attached to C-16 and a methyl group attached to C-17; H-17 (δ) in the HMBC spectrum... H 1.17) and C-15 (δ) C 38.8) / C-16 (δ C67.1) Related; further determined that C-16 and C-17 of compound 5 are respectively connected to a hydroxyl group and a methyl group. Therefore, the chemical structure of compound 5 is initially speculated to be heptadeca-4,6-diyne-3,8,9,10,11,16-hexaol. A search of the Sci Finder database system did not find this compound. By comparing the spectral data with those of compounds 3 and 4, the coupling constant (J) in compound 5... H2-H3 =6.5 Hz / J H8-H9 = 7.1 Hz / J H9-H10 = 2.7 Hz / J H16-H17 = 6.0 Hz) is consistent with that of compounds 3 and 4, suggesting that compound 5 has the same absolute configuration as compounds 3 and 4. The preliminary determination of the absolute configurations of C-3, C-8, C-9, C-10, C-11, and C-16 in compound 5 is 3S, 8R, 9S, 10R, 11S, and 16R, respectively. Combining the CD spectrum and electronic circular dichroism (ECD) experimental results, the experimental data are consistent with the theoretical calculations. Therefore, further inferences are made regarding the absolute configuration of compound 5. The compound with the configuration (3S,8R,9S,10R,11S,16R)-heptadeca-4,6-diyne-3,8,9,10,11,16-hexaol ((3S,8R,9S,10R,11S,16R)-heptadeca-4,6-diyne-3,8,9,10,11,16-hexaol) may be a new compound and has been named eleuthdiyne E.

[0070] Table 5 Compound 5 1 H-NMR (600 MHz, CD3OD) and 13 C-NM (150 MHz, CD3OD) data

[0071]

[0072] Cellular experiments with compounds 1-5

[0073] 1. Experimental Materials

[0074] Test cell lines: RAW264.7 macrophages and BV2 mouse microglia were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0075] Test compounds: Compounds 1-5 isolated from Acanthopanax senticosus were dissolved in DMSO (dimethyl sulfoxide), added to complete culture medium to prepare a 500 μM solution, and then diluted to the required concentration.

[0076] Inducer: Lipopolysaccharide (LPS), 200 ng / mL;

[0077] Positive control drug: Dexamethasone (DEXA).

[0078] 2. Cell Culture and Experimental Grouping

[0079] BV2 and RAW264.7 cells were cultured in high-glucose DMEM medium (complete medium) containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 cell culture incubator. Experimental groups included: a blank control group, an LPS (200 ng / mL) induced model group, a positive control group (dexamethasone), and high-dose (40 μM), medium-dose (20 μM), and low-dose (10 μM) groups of the test compound.

[0080] 3. Determination of cell viability

[0081] Cell viability was detected using the CCK-8 assay, a commonly used method for assessing cell viability. The principle is that the CCK-8 reagent contains WST-8, which is reduced by dehydrogenases in the mitochondria of cells to produce a highly water-soluble, orange-yellow formazan product. This reaction occurs only in living cells; therefore, the amount of formazan produced is directly proportional to the number of living cells. The intensity of the formazan product's color is directly related to cellular metabolic activity. By measuring its absorbance (OD value) at 450 nm using a microplate reader, the number and activity of living cells can be indirectly reflected. Within a certain cell number range, the OD value is directly proportional to the amount of formazan product formed.

[0082] BV2 cells and RAW264.7 cells in logarithmic growth phase were respectively divided into groups of 5 × 10⁻⁶. 5 The microplates were seeded at a density of 100 μL / well to 96 wells and incubated at 37°C with 5% CO2 for 24 hours. Then, the target compound was added at the required concentration and incubated for another 24 hours. Finally, 10 μL of CCK-8 was added to each well, the plate was covered with aluminum foil to protect it from light, and incubated at 37°C with 5% CO2 for another hour. The absorbance (A value) was measured at 450 nm using a microplate reader.

[0083] The formula for calculating cell viability is: Cell viability (%) = (A 给药 -A0) / (A 空白对照 -A0)×100%;

[0084] Among them, A 给药 A represents the absorbance of the well containing cells, CCK8, and the solution of the analyte compound; 空白对照A0 is the absorbance of the well containing cells and CCK8 but not the solution of the test compound; A1 is the absorbance of the well containing culture medium and CCK8 but not the solution of cells and the test compound.

[0085] 4. Determination of NO content in cells

[0086] The Griess method was used to determine the NO content. The Griess method is a commonly used method for detecting NO concentration. Its principle is that NO is easily oxidized to NO2 in aqueous solution. Under acidic conditions, the product formed after NO2 reacts with the Griess reagent has maximum absorption at 540 nm, which is linearly correlated with the NO concentration.

[0087] BV2 cells and RAW264.7 cells in logarithmic growth phase were respectively divided into groups of 5 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to ensure complete adhesion. Then, different concentrations of the test compound and LPS (200 ng / mL) were added to treat the cells. After treatment, the cells were incubated for another 24 hours. 50 μL of the supernatant was transferred to a new 96-well plate, and 50 μL each of Griess reagent I and II were added sequentially. After mixing on a shaker for 5 min, the absorbance (OD value) at 540 nm was scanned on a microplate reader. A standard curve was prepared using the standard, and the corresponding NO concentration was calculated by substituting the OD values ​​of each group into the standard curve.

[0088] 5. Statistical Analysis

[0089] Experimental results are expressed as Means ± SD. Graphpad Prism 8.0 software was used to test normality and perform one-way ANOVA to compare differences within and between groups. p < 0.05 was considered statistically significant. All experiments were repeated three times.

[0090] 6. Experimental Results

[0091] The inhibitory effects of compounds 1-5 on NO production in lipopolysaccharide-induced BV2 cells are shown in Table 6, and the inhibitory effects of compounds 1-5 on NO production in lipopolysaccharide-induced RAW264.7 cells are shown in Table 7.

[0092] Table 6 shows the inhibitory effects of compounds 1-5 on NO production in lipopolysaccharide-induced BV2 cells.

[0093]

[0094] Note: a Blank control group: No drug was administered. bThe LPS concentration in the LPS model group was 200 ng / mL; data are expressed as mean ± SD, n=3; compared with the model group... * p < 0.05 ** p < 0.01, **** p < 0.0001.

[0095] Table 7 shows the inhibitory effects of compounds 1-5 on lipopolysaccharide-induced NO production in RAW264.7 cells.

[0096]

[0097] Note: a Blank control group: No drug was administered. b The LPS concentration in the LPS model group was 200 ng / mL; data are expressed as mean ± SD, n=3; compared with the model group... * p < 0.05 ** p < 0.01, **** p < 0.0001; compared with the blank control group, # p < 0.05 ## p < 0.01, #### p < 0.0001.

[0098] Cellular experiments showed that compounds 1-5 were not cytotoxic at concentrations of 10-40 μM, indicating that the samples were not toxic to BV2 and RAW264.7 cells (cell viability ≥80%) at concentrations below 40 μM.

[0099] The NO inhibition experiment results showed that at a drug concentration of 40 μM, compounds 1, 2, and 4 had significant NO inhibitory activity and could effectively reduce the NO content in LPS-induced BV2 and RAW264.7 cells. Among them, compound 2 had the best inhibitory effect on NO production in both cell types, and the effect was concentration-dependent. Other compounds also showed some NO inhibitory effects.

[0100] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. Five eleutherophyllol compounds from *Eleutherococcus longipes*, characterized in that, The five long-stemmed eleutherool compounds are long-stemmed eleutherool A, long-stemmed eleutherool B, long-stemmed eleutherool C, long-stemmed eleutherool D, and long-stemmed eleutherool E; The structural formula of the long-stem ginkyne alcohol A is as follows: ； The structural formula of the long-stem ginkyne alcohol B is as follows: ； The structural formula of the long-stem ginkyne alcohol C is as follows: ； The structural formula of the long-stem ginkyne diol D is as follows: ； The structural formula of the long-stem ginkyne alcohol E is as follows: 。 2. A method for extracting and separating five eleutherophyllol compounds from *Eleutherococcus senticosus* as described in claim 1, characterized in that, Includes the following steps: (1) The roots of Acanthopanax senticosus were extracted by cold soaking in methanol, and the extract was concentrated to obtain an extract. The extract was dispersed in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively. The cold maceration extraction temperature is room temperature, which is 25±2℃; (2) The n-butanol extract is loaded onto a macroporous resin column and eluted using a methanol-water solvent system with a gradient elution. The volume ratio of methanol to water in the methanol-water solvent system is 30:70, 50:50, 70:30 and 90:10, respectively, to obtain 30% methanol elution fraction, 50% methanol elution fraction, 70% methanol elution fraction and 90% methanol elution fraction; (3) The 30% methanol eluent was loaded onto a normal silica gel column and gradient eluted using a dichloromethane-methanol solvent system to obtain 20 components RB1-20. Components RB15 and RB18 were separated and purified. RB15 was separated to obtain four sub-fractions RB15A-D. From RB15B and RB15D of the four sub-fractions RB15A-D, long-stemmed ginsenoside A and long-stemmed ginsenoside B were separated. RB18 was separated to obtain five sub-fractions RB18A-E. From RB18B, RB18D, and RB18E of the five sub-fractions RB18A-E, the following compounds were obtained: Acanthopanax acetylenol C, Acanthopanax acetylenol D, and Acanthopanax acetylenol E.

3. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 2, characterized in that, In step (1), the cold maceration extraction is performed 3 times, once every 7 days.

4. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 2, characterized in that, In step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent system is 30:1 to 0:

1.

5. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 2, characterized in that, In step (3), semi-preparative HPLC was used to separate component RB15. The solvent system used for separation was a methanol-water solvent system with a volume ratio of methanol to water of 35:65, resulting in four subcomponents RB15A-D. Semi-preparative HPLC was used to separate and purify subcomponent RB15B. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 45:55, resulting in long-stemmed acetylenol A.

6. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 5, characterized in that, The subfraction RB15D was separated and purified by semi-preparative HPLC. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 47:53, to obtain long-stemmed acetylenol B.

7. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 2, characterized in that, In step (3), semi-preparative HPLC was used to separate component RB18. The solvent system used for separation was a methanol-water solvent system with a volume ratio of methanol to water of 35:65, resulting in 5 subcomponents RB18A-E. Semi-preparative HPLC was used to separate and purify subcomponent RB18B. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 40:60, resulting in long-stemmed acetylenol C.

8. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 7, characterized in that, The subfraction RB18D was separated and purified by semi-preparative HPLC. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 43:57, to obtain long-stemmed acetylenol D.

9. The method for extracting and separating five eleutherophyllol compounds from Acanthopanax senticosus according to claim 7, characterized in that, The subfraction RB18E was separated and purified by semi-preparative HPLC. The solvent system used for separation and purification was a methanol-water solvent system with a volume ratio of methanol to water of 42:58, to obtain long-stemmed acetylenol E.

10. The use of five Acanthopanax senticosus alcohol compounds from Acanthopanax senticosus according to claim 1 in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases.

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