Five compounds in rhizoma paridis and preparation method and application thereof

Abstract

The application discloses five new components in aboveground parts of Paris polyphylla, and the structural formulae of the five new components are shown in formula I and formula II: the application also provides a preparation method and application of the five new components. α ,23 α ,27-trihydroxyyamogenin-3 β -yl-O- α -L-rhamnopyranosyl-(1→4)[- α -L-rhamnopyranosyl-(1→2)]- β -D-glucopyranoside (1), 12 α ,23 α ,27-trihydroxyyamogenin-3 β -yl-O- α -L-rhamnopyranosyl-(1→4)- α -L-rhamnopyranosyl-(1→4)- β -D-glucopyranoside (2), 12 α ,23 α ,27-trihydroxyyamogenin-3 β -yl-O- α -L-rhamnopyranosyl-(1→4)- β -D-glucopyranoside (3), (25S)-17 α ,23 α, 27-trihydroxyspirost-5-en-3 β -yl- β -D-glucopyranoside (4), (25S)-3 β ,17 α, 27-trihydroxyspirost-5-en-23- α -yl- β -D-glucopyranoside (5). The compounds 1 and 2 can shorten plasma recalcification time and have hemostatic effect, and the compounds 3-5 can prolong plasma recalcification time and have blood activating effect, and can be developed into new hemostatic / blood activating drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine chemistry and medicine, in particular to a new compound isolated and identified from the aboveground part of Paris, a preparation method and application thereof. BACKGROUND

[0002] Paris is the general name of the plants of the genus Paris of Liliaceae, and the dried rhizomes of Paris polyphylla Smith var. yunnanensis (Franch.) Hand-Mazz. and P. polyphylla Smith var. chinensis (Franch.) Hara are recorded in Chinese Pharmacopoeia 2020 edition, which has the effects of clearing heat and resolving toxins, relieving swelling and pain, and cooling liver and calming shock [1] . The traditional medicinal part of Paris is the underground rhizome, but the resources of Paris are endangered at present, and the yield cannot meet the production needs. The present application takes the non-traditional medicinal part of Paris, the aboveground part of Paris, as the research object, finds new active compounds therefrom, and has great significance for fully exploiting and utilizing the endangered resources of Paris, and promoting the sustainable and rapid and healthy development of Paris industry. SUMMARY

[0003] The purpose of the present application is to provide five new compounds from the aboveground part of Paris, a preparation method and application thereof. In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0004] Five new compounds, the structure is 12α, 23α, 27-trihydroxyyamogenin-3β-yl-O-α-L-rhamnopyranosyl-(1→4)[-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (1), 12α, 23α, 27-trihydroxyyamogenin-3β-yl-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside (2), 12α, 23α, 27-trihydroxyyamogenin-3β-yl-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside (3), (25S)-17α, 23α, 27-trihydroxyspirost-5-en-3β-yl-β-D-glucopyranoside (4), (25S)-3β, 17α, 27-trihydroxyspirost-5-en-23-α-yl-β-D-glucopyranoside (5)

[0005]

[0006] The five new compounds are all isolated from the aboveground part of Paris polyphylla.

[0007] The preparation method of the five new compounds comprises the following steps:

[0008] Step one: the naturally dried aboveground part of Paris polyphylla is cut into small pieces with a length of 6-10 cm, and then is placed in a multifunctional extraction tank, 10 times (volume of solvent / mass of medicinal material) of 70% ethanol is added to soak overnight, and 10 times (volume of solvent / mass of medicinal material) of 70% ethanol is added to reflux extract three times, each time for 2 h, and then the filtrate is combined and concentrated to no alcohol taste in a double-effect concentrator (60-70 ℃) to obtain an extraction concentrated liquid.

[0009] Step two: the extraction concentrated liquid in step one is centrifuged, the centrifugal liquid is separated by HPD-100 macroporous adsorption resin, and 5 times of water, 30% ethanol and 80% ethanol are used for elution respectively, and then the concentrated liquids are dried in a vacuum drying box.

[0010] Step three: 30% ethanol segment immersion paste, using silica gel column for crude separation, eluent is (ethyl acetate: 95% ethanol = 10:1→ 5:3) to obtain 8 fractions, named A-H segment respectively. Take E segment in step two, separate by ODS (EtOH:H2O, 8%→28%), to obtain 5 fractions (E1-E5). E2 fraction is separated by silica gel (CH2Cl2:MeOH:H2O, 7:2:1→7:3:1, after extraction, take the lower layer) to obtain fractions E2.1-E2.5. E2.2 is separated by semi-preparative liquid phase, and the chromatographic conditions are as follows: chromatographic column: YMC-pack C 18 (10×250 mm, 5 μm), mobile phase A: 14% acetonitrile, mobile phase B: 86% water, detection wavelength: 203 nm, flow rate: 3 ml / min, injection volume: 100-200 µml, to obtain compounds 1, 2 and 3. Take C segment in step two, separate by ODS (EtOH:H2O, 15%→25%), to obtain 4 components (C1-C4). C3 fraction is separated and purified by silica gel column chromatography (CH2Cl2:MeOH:H2O, 7:1.5:1→7:2.5:1, after extraction, take the lower layer), recrystallization to obtain compound 4. C4 fraction is separated by Sephadex LH-20 (methanol), and after recrystallization separation and purification, compound 5 is obtained.

[0011] The present application is characterized in that 1 H-NMR, 13 C-NMR, HMBC, HSQC, DEPT and HR-ESI-MS spectra are used to identify the structures of compounds 1-5 in step three.

[0012] The present application provides in vitro hemostatic and blood-activating activities of the five new compounds.

[0013] The five new compounds, the structure, the preparation method and the pharmacological activity of the present application have not been reported in the literature. Compounds 1-3 are the first time to find 12-hydroxyl-substituted diosgenin. In vitro coagulation activity research shows that the paris saponin compounds 1 and 2 provided by the present application have hemostatic effect, and compounds 3-5 have blood-activating effect, which can be developed into new hemostatic / blood-activating drugs. In addition, the preparation of the new compounds described in the present application is from the non-traditional medicinal part of paris above ground part, which has important significance for the comprehensive utilization of paris, an endangered medicinal material. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The H-NMR, 1 H(500MHz) and 13 C NMR(125MHz) data.

[0015] Figure 2 The H-NMR,1 H (500 MHz) and 13 C NMR (125 MHz) data.

[0016] Figure 3 Compound 1-5 of 1 H- 1 H COSY and HMBC correlations. DETAILED DESCRIPTION

[0017] The present application is further illustrated by the following examples, but is not limited to them.

[0018] Example 1: Isolation and preparation

[0019] Step 1: Extraction

[0020] Take 126 kg of dried rhizoma paridis (batch 20190312) and cut into small pieces of 6-10 cm long, and place in a multifunctional extraction tank (3 m 3 ) and add 10 times the amount of 70% ethanol (volume of solvent / mass of medicinal material) and soak overnight, and reflux extract three times, each time adding 10 times the amount of 70% ethanol (volume of solvent / mass of medicinal material) and extract for 2 h, and combine the filtrate and place in a double-effect concentrator (60-70 °C) and concentrate to no alcohol taste, and obtain the extraction concentrate.

[0021] Step 2: Sectioning

[0022] Centrifuge the extraction concentrate again in a tubular centrifuge, and separate the centrifugal liquid (200 L) using HPD-100 macroporous adsorption resin, and elute with 5 times water, 30% ethanol, and 80% ethanol, respectively, and after concentrating, place in a vacuum drying oven and dry, and obtain 3.5 kg of water section, 2.1 kg of 30% ethanol section dry extract, and 6.0 kg of 80% ethanol section.

[0023] Step 3: Separation and purification

[0024] Take 30% ethanol segment immersion paste 1.5 kg, ethanol ultrasonic dissolution, weighing silica gel (80-100 mesh) 2 kg, mix the dissolved sample with silica gel, dry the solvent. Another 20 kg or so silica gel (200-300 mesh) is packed into a column with a volume of about 40 L. The sample is roughly divided into segments after being mixed with silica gel, and the eluent is (industrial ethyl acetate: industrial 95% ethanol = 10:1→ 5:3). Eight crude fraction polarity segments are obtained, which are collected according to the polarity from small to large and named as A-H segments. E segment (58 g) sample is separated by ODS (EtOH:H2O, 8%→28%), to obtain 5 components (E1-E5). E2 is separated by silica gel (CH2Cl2:MeOH:H2O, 7:2:1→7:3:1, after extraction, the lower layer is taken out), Preparation-HPLC (acetonitrile-water, 14:86) to obtain compound 1 (99.2 mg), 2 (32.5 mg), 3 (54.1 mg). C segment sample is separated by ODS (EtOH:H2O, 15%→25%) to obtain 4 components (C1-C4). C3 sample is separated by silica gel column chromatography (CH2Cl2:MeOH:H2O, 7:1.5:1→7:2.5:1, after extraction, the lower layer is taken out), Sephadex LH-20 (methanol), and recrystallization to obtain compound 4 (24.4 mg). C4 is separated by Sephadex LH-20 (methanol), recrystallization to obtain compound 5 (250.0 mg).

[0025] Example Two: Structure Identification

[0026] Compound 1, white amorphous powder, high resolution mass spectrometry shows [M+Na] + Peak 939.4560, the molecule is speculated to be C 45 H 72 O 19。 1 H NMR shows three methyl signals δ H 1.13 (Me-18), 1.02 (Me-19), 1.26 (3H, d, J = 6.8 Hz, H3-21), two oxygen-containing substituted methylene signals [δ H 3.69, 3.73 (1H each, both m, 27-H2), 3.90, 4.09 (1H each, both d, J = 11 Hz, 26-H2)], three oxygen-containing substituted methine signals [δ H3.82 (1H, m, 3-H), 4.01 (1H, m, 12-H), 4.75 (1H, q, J = 7.2 Hz, 16-H), 3.99 (1H, m, 23-H)], a single olefin proton signal δ H 5.31 (d, J = 4.9 Hz, H-6) and three glycosidic proton signals δ H 4.91 (d, J = 7.4 Hz, H-1' ), 6.37 (br s, H-1''), 5.84 (brs, H-1'''). 13 C-NMR showed 45 carbon signals, including 5 methyl groups, 10 methylene groups, and 26 methine groups (including three telomere carbon signals). C 102.2, 102.1, 102.9, a olefinic carbon signal δ C 122.0) and 4 quaternary carbons (including one olefinic carbon signal δ) C 140.9). The C-H signal of compound 1 is similar to that of compound Borassosides B. [2] Except for compound 1, which has an oxygen-substituted methine signal δ C 71.6 (C-12), while Borassosides B shows a methylene signal. In the HMBC spectrum, δ H 1.13 (Me-18) and δ C 71.6 (C-12), δ H 4.01 (H-12) and δ C There is a correlation between 44.5 (C-9) and 48.2 (C-14). 1 H- 1 In the H COSY spectrum, δ H 1.82 (H-11) and δ H The presence of 4.01 (H-12) indicates that the C-12 position in compound 1 is substituted with a hydroxyl group. In the ROESY spectrum, δ H 4.01 (H-12) and δ H The presence of a correlation at 1.13 (H-18) confirms that the hydroxyl group at position 12 is in the α configuration. Compound 1 was identified as 12α,23α,27-trihydroxyyamogenin-3β-yl-O-α-L-rhamnopyranosyl-(1→4)[-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside.

[0027] Compound 2, white amorphous powder. High resolution mass spectrum showed the same molecular weight (940.4572 [M+Na] + ) and molecular formula (C 45 H 72 O 19 ) as compound 1. Comparative study of NMR data of compound 2 and compound 1 found that they have the same parent nucleus, only the difference in sugar linkage part. HMBC spectrum showed δ H 5.84 (H-1' of Rha I) and δ C 78.6 (C-4 of Glc), δ H 6.28 (H-1'' of Rha II) and δ C 80.3 (C-4 of Rha I) were correlated, proving that compound 2 is 12α,23α,27-trihydroxyyamogenin-3β-yl-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside.

[0028] Compound 3, white amorphous powder. High resolution mass spectrum showed the molecular ion peak 794.4003 [M+Na] + . Carbon spectrum and hydrogen spectrum data were similar to compound 1, except that compound 3 had one less rhamnose signal. HBMC spectrum showed δ H 4.92 (H-1' of glucose) and δ C 78.3 (C-3 of aglycone), δ H 5.88 (H-1'' of Rha) and δ C 78.3 (C-4 of glucose) were correlated, so compound 3 was identified as 12α,23α,27-trihydroxyyamogenin-3β-yl-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside.

[0029] Compound 4, white amorphous powder. High resolution mass spectrum showed the molecular ion peak 647.3393 [M+Na] + , the molecular formula is C 39 H 62 O 15 . Carbon spectrum and hydrogen spectrum showed 3 methyl groups [δ H0.85 (3H, s, Me-19), 1.16 (3H, s, Me-18), and 1.32 (3H, d, J = 7.0 Hz, Me-21); δ C 9.4 (C-21), 17.5 (C-18), and 19.4 (C-19)], two oxygen-substituted methylene groups [δ H 3.91, 4.02 (1H each, both m, H2-26), 3.67, 3.73(1H each, both d, J = 5.0 Hz, H2-27); δ C 63.1 (C-26), 64.0 (C-27)], three oxygen-substituted methines [δ H 3.89 (m, H-3), 4.02 (1H, m, H-23), 4.60 (1H, m, H-16); δ C 68.1 (C-23), 78.1 (C-3), 90.0 (C-16)], a set of olefinic carbon signals [δ H 5.25 (1H, d, J = 5.1 Hz, H-6);δ C 140.9 (C-5) and 121.8 (C-6)], a sugar-terminal proton signal [δ H [5.02 (d, J = 7.7 Hz, H-1')], the above information proves that compound 4 is a spirostane compound linked to a sugar. The NMR data of compound 4 is (23S,25S)-17α,23,27-trihydroxyspirost-5-en-3β-yl-O-α-Lrhamno-pyranosyl-(1-4)-β-Dglucopyranoside [3] Very similar, except that compound 4 lacks one rhamnose signal. HMBC spectra show δ H 5.02 (d, J = 7.7 Hz, H⁻¹') and δ C 78.1 (C-3 of aglycone) is relevant. Therefore, compound 4 was identified as (25S)-17α,23α,27-trihydroxyspirost-5-en-3β-yl-β-D-glucopyranoside.

[0030] Compound 5, a white amorphous powder, showed the same molecular ion peak and molecular formula as compound 4 according to high-resolution mass spectrometry. Its proton and carbon spectra were similar to those of compound 4. The difference lay in the chemical shifts at C-3 and C-23, which were δ¹⁸ and δ¹⁸ respectively in compound 5.C 71.3, 77.1, 78.1, 68.1 in compound 4, respectively. It proved that the sugar in compound 5 was linked to C-23, not C-3 as in compound 4. H-1' (δ H 5.02, d, J = 7.7 Hz) and C-23 (δ C 77.1) existed HMBC correlation, which further confirmed the structure. Therefore, compound 5 was identified as (25S)-3β,17α,27-trihydroxyspirost-5-en-23-α-yl-β-D-glucopyranoside.

[0031] Example Three: Activity determination

[0032] Take 2 rats abdominal vena cava blood, 3.8% sodium citrate physiological saline solution anticoagulation, mixed after 3500 r / min centrifugal 10 min, separation of plasma for use. The plasma into 8 groups, each group of 6 plasma, each 0.1 mL, namely physiological saline, vitamin K, aspirin, compound 1-5 group. In the above containing plasma test tube, each tube was added corresponding drug solution 0.1 mL, so that the final concentration of drug in test tube equal to the corresponding group of the indicated concentration, physiological saline group with the same method to add the same volume of physiological saline. The above test tube was put into 37 ℃ water bath for 1 min, then added calcium chloride solution 0.1 mL, mixed, and then put into 37 ℃ water bath, start timing, and every 30 s slowly tilt test tube 1 times, record from the addition of calcium chloride to the time required for the liquid surface, namely the plasma recalcification time. Calculate the mean value of each group of 6 plasma data.

[0033]

Claims

1. Five compounds in the above-ground parts of Paris polyphylla, the structures of which are shown in Formula I and Formula II: Compound 1 when R = S1, Compound 2 when R = S2, Compound 3 when R = S3, Compound 4 when R1= S4, R2= H, Compound 5 when R1= H, R2= S4.

2. The method for preparing five compounds in the aboveground parts of Paris polyphylla according to claim 1, characterized in that The steps are as follows: Step A: extraction: after the above-ground parts of Paris polyphylla are cut into segments, extraction is performed with an appropriate amount of solvent, and the solvent is recovered under reduced pressure to obtain an extract concentrate; Step B: sectioning: after the extract concentrate is filtered, sectioning is performed with macroporous adsorption resin; Step C: separation and purification: after the sample sectioned with macroporous resin is separated and purified with a chromatographic column, monomeric compounds are obtained.

3. The method of claim 2, wherein the five compounds are prepared from the aboveground parts of Paris polyphylla Smith var. chinensis (Franch.) Hand.-Mazz. The extraction method in step A is reflux extraction, the extraction solvent is an ethanol solution, the volume of the extraction solvent is 10 times the mass of the medicinal material, the extraction is performed 3 times, each time for 2 h, the extract is combined, and the solvent is recovered under reduced pressure until there is no alcohol smell.

4. The method of claim 2, wherein the five compounds are prepared from the aboveground parts of Paris polyphylla Smith var. chinensis (Franch.) Hand.-Mazz. The macroporous adsorption resin in step B is HPD-100, which is eluted with water, low-concentration ethanol, and high-concentration ethanol solution in sequence, and the solvents are recovered.

5. The method of claim 2, wherein the five compounds are prepared from the rhizome of Paris polyphylla var. chinensis. The chromatographic column in step C is a normal-phase, reverse-phase, gel column, or preparative high-performance liquid chromatography column. ​ 6. The method of claim 5, wherein the five compounds are prepared from the aboveground parts of Paris polyphylla Smith var. chinensis (Franch.) Hand.-Mazz. The normal-phase chromatographic column is a silica gel column, the reverse-phase chromatographic column is an ODS column, the gel column is Sephadex LH-20, and the preparative high-performance liquid chromatography column is a C18 column.

7. The use of five compounds in the aboveground parts of Paris in the preparation of a medicine according to claim 1, characterized in that The use of compound 1-2 in the preparation of a hemostatic drug and the use of compounds 3-5 in the preparation of a blood-activating drug.

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