Use, preparation method and application of saponin derivatives in the preparation of drugs for treating and preventing ulcerative colitis

Abstract

The present invention belongs to the field of biomedicine, and provides a use, preparation method and application of a saponin derivative in the preparation of a drug for treating and preventing ulcerative colitis. The present invention conducts in vivo anti-ulcerative colitis activity studies on the obtained triterpenoid saponins and different pharmacodynamic components composed of active saponins. Pharmacological experimental studies show that the 3,28-bis-oligosaccharide hederagenin compounds 3 to 12 show weak anti-UC activity; the 3,28-bis-oligosaccharide oleanolic acid triterpenoid saponin compounds 1, 2, 13 to 15 and the pharmacodynamic component P70 (the anti-UC active Pulsatilla chinensis extract) composed thereof have a significant therapeutic effect on DSS-induced ulcerative colitis mice, and their activity is significantly better than that of the 3,28-bis-oligosaccharide hederagenin compounds. Therefore, it can be used to treat ulcerative colitis and is used in the preparation of drugs for treating and preventing ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the use, preparation method and application of saponin derivatives in the preparation of drugs for the treatment and prevention of ulcerative colitis. The 28 oleanane-type saponin derivatives are isolated from the dried roots of Pulsatilla chinensis (Bunge) Regel by phytochemical means, and the application of such compounds in inhibiting the activation of the MLCK pathway and preventing and treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease with main symptoms of abdominal pain, diarrhea and rectal bleeding, which seriously affects the working ability and quality of life of patients. Although great progress has been made in the treatment of ulcerative colitis, its pathogenesis is still unclear, including the accumulation of genetic, environmental or immune factors. Current drug therapies for UC include aminosalicylates (such as mesalazine, MLZ), glucocorticoids and immunosuppressive agents, but the above treatments cannot help patients get rid of the pain and serious side effects brought by long-term medication. In addition, the high recurrence rate and severe complications of UC are associated with a high risk of colorectal cancer.

[0003] The onset of UC is caused by damage to the colonic mucosal epithelial barrier, increased permeability of the epithelial cell layer, and the entry of immunogenic substances into the intestinal wall, triggering a series of excessive immune response reactions. The DSS-induced mouse UC model is a classic model for the study of the pathogenesis and drugs of UC. Typical pathological changes in the UC animal model include weight loss, shortening of the colon tissue, increased DAI score, damage to the long tissue epithelial barrier and abnormally high expression of TNF-α in the colon tissue. The intestinal epithelial barrier includes a single layer of epithelial cells and tight junction structures that regulate barrier leakage between cells, and plays a key role in the intestinal homeostasis environment. Studies have shown that epithelial cell myosin light chain kinase (MLCK) is closely related to the regulation of epithelial barrier permeability. After MLCK is activated, on the one hand, it phosphorylates myosin light chain (MLC), triggering muscle cell contraction and increasing epithelial barrier permeability; on the other hand, MLCK also participates in the regulation of tight junction structures such as claudin-2, occludin, and ZO-1. It has been found that in UC intestinal tissue, long-chain MLCK is upregulated, initiating the transcription of downstream long-chain MLCK and upregulating the expression of claudin-2, which destroys the tight junction structure between cells and the intestinal epithelial barrier, and exacerbates the process of UC. Therefore, inhibiting the MLCK signaling pathway is a key target for discovering new UC treatment drugs.

[0004] The traditional Chinese medicine Pulsatilla Root is the dried root of Pulsatilla chinensis (Bunge) Regel, a plant of the genus Pulsatilla in the Ranunculaceae family. The oleanane-type saponins in Pulsatilla Root can be divided into two types according to the aglycone: oleanolic acid and hederagenin; according to whether there are oligosaccharide side chains on C-3 and C-28, they can be divided into three types: 3-oligosaccharide saponins, 28-oligosaccharide saponins, and 3,28-bis-oligosaccharide saponins. In addition, the diversity of the types, connection positions, and sugar chain lengths of monosaccharide units on the oligosaccharide side chains further enriches the structural diversity of Pulsatilla saponins. The differences between the aglycone and the glycosyl side chain make different types of Pulsatilla saponins have different activities. For example, Pulsatilla saponin A shows good anti-cancer activity (CN102133220A), but does not show anti-UC activity.

[0005] The therapeutic activity of compounds 1-9, 11-16 involved in the present invention against UC was discovered for the first time, and compounds 1, 2, and 3 are new compounds.

[0006] The present invention discovers for the first time that 3,28-bis-oligosaccharide oleanolic acid saponins are the main pharmacodynamic components of Pulsatilla Root in treating UC, which can relieve the symptoms of colitis in UC mice, down-regulate the MLCK signaling pathway, and repair the intestinal epithelial barrier. Summary of the Invention

[0007] The oleanane-type saponin derivatives of the present invention are extracted and isolated from the dried root of Pulsatilla chinensis (Bunge) Regel. Pharmacological experimental studies have shown that these compounds have a significant therapeutic effect on UC. One of the purposes of the present invention is to provide oleanane-type saponin derivatives with anti-UC activity and their preparation methods. Another purpose of the present invention is to provide a Pulsatilla Root extract with an active ingredient content of 50.0-99.9% (weight percentage) and its preparation method based on the anti-UC active ingredient. Another purpose of the present invention is to provide the uses of the oleanane-type saponin derivatives and / or Pulsatilla Root extract, including the application of the oleanane-type derivatives and / or Pulsatilla Root extract in the preparation of drugs for preventing and treating UC.

[0008] At present, regarding the research on the active ingredients of Pulsatilla chinensis in treating ulcerative colitis (UC), only Pulsatilla saponin B4 (CN112107586A) and Pulsatilla saponin B5 (CN108451964A) have been publicly reported. The nuclear skeleton of Pulsatilla saponin B4 is different from that of the compounds involved in this patent. Pulsatilla saponin B4 has a lupane-type triterpene skeleton, while the compounds involved in this patent have an oleanane-type triterpene skeleton. Pulsatilla saponin B5 is a compound with hederagenin as the aglycone, and its aglycone structure and glycosyl substituents are different from those of the compounds 1, 2, 13 - 15 involved in this patent, which have oleanolic acid as the aglycone. In addition, a new compound 3 with hederagenin as the aglycone involved in this patent has completely different glycosyl substituents from those of Pulsatilla saponin B5. The differences in the aglycone and glycosyl substituents of Pulsatilla saponins significantly affect the activity of the compounds. For example, Pulsatilla saponin compounds 16 - 28 with hederagenin and oleanolic acid as the aglycones did not show anti-UC activity in this study.

[0009] The technical solution of the present invention is as follows: Use of a saponin derivative in the preparation of a drug for treating and preventing ulcerative colitis, wherein the saponin derivative is an oleanane-type triterpenoid saponin compound, and its structural general formula is the compound shown in Formula I;

[0010]

[0011] Wherein: R1 is a glycosyl or hydrogen, R2 is a glycosyl or hydrogen, R3 is a glycosyl or hydrogen, R5 is a glycosyl or hydrogen, and R1, R2, R3 and R5 are the same or different; the glycosyl is a monosaccharide group or an oligosaccharide group formed by 2 - 4 monosaccharide groups; R4 is a hydroxyl group or hydrogen.

[0012] The above-mentioned oleanane-type triterpenoid saponin compounds are mainly 28 oleanane-type triterpenoid saponin compounds isolated from the dry roots of Pulsatilla chinensis (Bunge) Regel, and their chemical structural formulas can be specifically as follows:

[0013]

[0014]

[0015] The above-mentioned oleanane-type triterpenoid saponin compounds are selected from compounds 1 - 9, 11 - 15 or selected from: compounds 1 - 3, 5, 13 - 15, and their chemical structural formulas are as follows:

[0016] The compounds 1, 2, 13, 14, 15 are included in the Chinese herbal medicine extract of Pulsatilla chinensis. Calculated based on the total weight of the Chinese herbal medicine extract of Pulsatilla chinensis being 100%, the Chinese herbal medicine extract of Pulsatilla chinensis contains any one or more than 2 of the compounds 1, 2, 13, 14, 15, and the weight ratio accounts for 50% to 99.9% of the total weight of the Chinese herbal medicine extract of Pulsatilla chinensis.

[0017] A preparation method of a Chinese herbal medicine extract of Pulsatilla chinensis, comprising the following steps:

[0018] 1) Take the dried roots of Pulsatilla chinensis, extract with an ethanol aqueous solution with a volume concentration of 20-95%, concentrate the extract to obtain a crude extract, subject the crude extract to macroporous resin column chromatography, and the eluent is an ethanol aqueous solution with a volume concentration of 0, 20%, 50%, 70%, and 100%, and elute respectively; discard the pure water eluent, collect the eluents of the remaining parts, and concentrate to obtain the 20% ethanol aqueous solution elution part P20, the 50% ethanol aqueous solution elution part P50, the 70% ethanol aqueous solution elution part P70S, and the ethanol aqueous solution elution part P100;

[0019] 2) Load the P70S component onto an MCI small pore resin chromatographic column, rinse with pure water until the eluent is colorless, rinse with 50% methanol for 2 column volumes, then rinse with 70% methanol for 5 column volumes, collect the 70% methanol eluent, and obtain the Chinese herbal medicine extract P70 of Pulsatilla chinensis after drying under reduced pressure.

[0020] 3) Based on the components obtained in step 1) and step 2), further carry out separation and purification

[0021] The above-mentioned component P50 was subjected to gradient elution on a normal-phase silica gel column chromatography to obtain components P50-I, P50-II, and P50-III. Component P50-I was purified by Sephadex gel column chromatography, reverse-phase chromatography column, and semi-preparative liquid chromatography to obtain compounds 11 and 12. Component P50-II was separated by Sephadex gel column chromatography and reverse-phase chromatography column to obtain components P50-IIA and P50-IIB. Component P50-IIA was purified by semi-preparative liquid chromatography to obtain compounds 9 and 10. Component P50-IIB was purified by semi-preparative liquid chromatography to obtain compounds 6, 7, and 8. Component P50-III was purified by Sephadex gel column chromatography, reverse-phase column chromatography, and semi-preparative liquid chromatography to obtain compounds 3, 4, and 5. Component P70S was subjected to gradient elution on a normal-phase silica gel column chromatography to obtain two components, P70S-I and P70S-II. Component P70S-I was purified by semi-preparative liquid chromatography to obtain compounds 1 and 15; Component P70S-II was purified by semi-preparative liquid chromatography to obtain compounds 2, 14, and 13. Component P100 was subjected to gradient elution on a normal-phase silica gel column chromatography to obtain fractions P100-I to V; Component P100-I was separated by normal-phase and reverse-phase column chromatography to obtain compounds 28, 26, and 27; P100-II was purified by normal-phase silica gel column chromatography and semi-preparative liquid chromatography to obtain compounds 23 and a mixture 24 / 25; Component P100-III was purified by reverse-phase column chromatography to obtain compounds 21 and 22. Component P100-IV was separated and purified by Sephadex gel column chromatography and semi-preparative liquid chromatography to obtain compounds 20, 19, 18, and compound 17. Component P100-V was repeatedly purified by semi-preparative liquid chromatography after reverse-phase column chromatography to obtain compound 16.

[0022] The above-mentioned macroporous resin can be a conventional macroporous resin in the art, such as resins like D101, XAD16, XAD1600, etc.

[0023] In the above preparation method, in the separation and purification step, the normal-phase silica gel used was of 100 - 200 mesh and / or 300 - 400 mesh, and gradient elution was carried out using a dichloromethane - methanol - water - formic acid system (volume ratio 4:1:0.1:0.1 to 1:1:0.2:0.1).

[0024] In the above preparation method, in the separation and purification step, the packing material used for reverse-phase chromatography was ODS-18, the packing material for the semi-preparative high-performance liquid chromatography column was ODS-18, and the eluent was a methanol - water system (volume ratio 3:7 to 7:3).

[0025] Advantages of the present invention: The present invention conducts in-vivo anti-ulcerative colitis activity studies on the obtained triterpenoid saponins and different pharmacodynamic components composed of active saponins. Pharmacological experimental studies show that the 3,28-bis-oligosaccharide hederagenin compounds 3-12 exhibit weak anti-UC activity; the 3,28-bis-oligosaccharide oleanolic acid triterpenoid saponin compounds 1, 2, 13-15 and the pharmacodynamic component P70 (anti-UC active Pulsatilla chinensis extract) composed thereof have significant therapeutic effects on DSS-induced ulcerative colitis mice, and their activity is significantly better than that of 3,28-bis-oligosaccharide hederagenin compounds. Therefore, it can be used to treat ulcerative colitis and is used to prepare drugs for the treatment and prevention of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1(A) is the HPLC fingerprint of the pharmacodynamic component P50; Figure 1(B) is the HPLC fingerprint of P70; Figure 1(C) is the HPLC fingerprint of P100.

[0027] Figure 2 : Structural diagrams of compounds 1-28.

[0028] Figure 3 : Of compound 1 1 1H NMR spectrum (pyridine-d5, 600 MHz).

[0029] Figure 4 : Of compound 1 13 13C NMR spectrum (pyridine-d5, 150 MHz).

[0030] Figure 5 : HSQC spectrum of compound 1.

[0031] Figure 6 : HMBC spectrum of compound 1.

[0032] Figure 7 : COSY spectrum of compound 1.

[0033] Figure 8 : TCOSY spectrum of compound 1.

[0034] Figure 9 : Of compound 2 1 1H NMR (pyridine-d5, 600 MHz) spectrum.

[0035] Figure 10 : Of compound 2 13 13C NMR (pyridine-d5, 150 MHz) spectrum.

[0036] Figure 11 : HSQC spectrum of compound 2.

[0037] Figure 12 : HMBC spectrum of Compound 2.

[0038] Figure 13 : COSY spectrum of Compound 2.

[0039] Figure 14 : TCOSY spectrum of Compound 2.

[0040] Figure 15 : For Compound 3 1 1H NMR (pyridine-d5, 600 MHz) spectrum.

[0041] Figure 16 : For Compound 3 13 13C NMR (pyridine-d5, 150 MHz) spectrum.

[0042] Figure 17 : HSQC spectrum of Compound 3.

[0043] Figure 18 : HMBC spectrum of Compound 3.

[0044] Figure 19 : COSY spectrum of Compound 3.

[0045] Figure 20 : TCOSY spectrum of Compound 3

[0046] Figure 21(A)-Figure 21(F) : Effects of each extract component on the physiological changes of UC mice. Figure 21(A) shows the daily body weight change rate of each group of mice; Figure 21(B) shows the body weight change rate of each group of mice on the 9th day, (body weight change rate = current day body weight / body weight on the 0th day * 100%); Figure 21(C) shows the DAI score of each group of mice; Figure 21(D) shows the DAI score of each group of mice on the 8th day; Figure 21(E) shows the colon photos of each group of mice; Figure 21(F) shows the colon length of each group of mice. ***P < 0.001, **P < 0.01, *P < 0.05, n = 8.

[0047] Figure 22(A) and Figure 22(B) show the effects of each extract component on the expression level of TNF-α in the colon tissue of UC mice evaluated by Western blotting; Figure 22(C) shows the protective effect of P70 on the colon epithelial damage of UC mice evaluated by H&E staining, and the legend indicates 100 μm. ***P < 0.001, **P < 0.01, *P < 0.05, n = 8.

[0048] Figure 23(A)-Figure 23(F):Effects of Compounds 1-3, 5, 13-15 on the physiological changes in UC mice. Figure 23(A): Effect of Compound 1 on the weight change rate of UC mice; Figure 23(B): Effect of Compound 1 on the DAI score of UC mice; Figure 23(C): Effect of Compound 1 on the colon length of UC mice, compared with the blank group ### P < 0.001, ***P < 0.001, **P < 0.01 compared with the model group; Figure 23(D): Effects of Compounds 2, 3, 5, 13-15 on the weight change rate of UC mice; Figure 23(E): Effects of Compounds 2, 3, 5, 13-15 on the DAI score of UC mice; Figure 23(F): Effects of Compounds 2, 3, 5, 13-15 on the colon length of UC mice; ***P < 0.001, **P < 0.01

[0049] Figure 24(A)-Figure 24(F) : Figure 24(A) Evaluation of the repair effect of Compound 1 on the colon epithelial injury in UC mice by H&E staining, n = 8. The legend indicates 100 μm Figure 24(B)-Figure 24(F) Regulatory effect of Compound 1 on the MLCK pathway evaluated by Western blotting, compared with the blank group ### P < 0.001; ***P < 0.001, **P < 0.01 compared with the model group Specific implementation manners

[0050] The implementation manners of the present invention will be described in detail below in combination with the implementation manners. However, the following implementation manners and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention

[0051] Example 1 Preparation of the pharmacodynamic components P20, P50, P70 and P100 of Pulsatilla chinensis

[0052] 1. Preparation of the Pulsatilla chinensis extract

[0053] The dried roots of Pulsatilla chinensis (5 kg) were pulverized, cold-soaked with an aqueous ethanol solution with a volume concentration of 70% at 10-30 times the weight of the raw material for 24 hours, and reflux-extracted at 60 °C for 6 hours. This step was repeated 2 times. The extraction solutions were combined and concentrated under reduced pressure to dryness to obtain the total extract PE of Pulsatilla chinensis (3 kg)

[0054] 2. Preparation of the pharmacodynamic components P50, P70 and P100 of Pulsatilla chinensis

[0055] The above extract was suspended in 6 L of pure water, filtered, adsorbed by a macroporous resin chromatographic column, and eluted with gradients of 0, 20%, 50%, 70%, and 100% ethanol. Each gradient was rinsed with about 3 column volumes. The pure water elution part was discarded, and the elution solutions of the remaining parts were collected, concentrated, and dried to obtain the pharmacodynamic components P20 (260 g), P50 (800 g), P70S (500 g), and P100 (220 g)

[0056] Suspend the above-mentioned component P70S in pure water, load it into an MIC chromatographic column (the sample loading amount of every 100 mL of MIC packing is 15 mL of 0.25 g / mL P70S aqueous solution), rinse with pure water until the eluate is colorless, rinse with a methanol aqueous solution with a volume concentration of 50% for 2 column volumes, then rinse with a methanol aqueous solution with a volume concentration of 70% for 5 column volumes, collect the 70% methanol eluate, concentrate it under reduced pressure, and obtain a light yellow powder after drying, which is the anti-UC active Pulsatilla chinensis extract P70 for the following fingerprint and in vivo activity studies.

[0057] The HPLC fingerprints of the pharmacodynamic components P50, P70, and P100 are shown in Figure 1. The numbers in the figure are the peak numbers, and the shaded areas indicate the characteristic compounds in this part. As shown in the fingerprint in Figure 1(A), component P50 is mainly composed of 3,28-bis-oligosaccharide hederagenin saponin compounds 3 - 12, and its content in this component measured by area normalization method is 77.72% (Table 1); as shown in Figure 1(B), P70 is composed of 3,28-bis-oligosaccharide oleanolic acid saponin components 1, 2, 13 - 15, and its content in this component measured by area normalization method is 95.93% (Table 2); as shown in Figure 1(C), component P100 mainly contains 3-mono-oligosaccharide hederagenin saponin and 3-mono-oligosaccharide oleanolic acid saponin components 16 - 28, and its content in this component measured by area normalization method is 88.82% (Table 3).

[0058] Table 1. Attribution and content of each component in the pharmacodynamic part P50 (area normalization)

[0059]

[0060]

[0061] Table 2. Attribution and content of each component in the pharmacodynamic part P70 (area normalization)

[0062]

[0063] Table 3. Attribution and content of each component in the pharmacodynamic part P100 (area normalization)

[0064]

[0065]

[0066] Example 2. Preparation of Pulsatilla saponin derivatives 1 - 28

[0067] 1. Isolation and purification of monomeric compounds

[0068] (1) The above-mentioned component P50 (20 g) was subjected to column chromatography on normal-phase silica gel (100 - 200 mesh), and gradient elution was carried out with a dichloromethane - methanol - water - formic acid system (4:1:0.1:0.1 - 1:1:0.2:0.1), obtaining sub-components P50-I (1.0 g), P50-II (8.0 g) and P50-III (9.0 g).

[0069] (2) After component P50-I (1.0 g) was eluted by Sephadex LH-20 column chromatography (40% methanol) and reverse-phase chromatography column (ODS-18, 53% MeOH), it was purified by semi-preparative liquid chromatography (ODS-18, 25×300 mm, 10 μm) with 60% methanol as the mobile phase to obtain compound 11 (13 mg) and 12 (19 mg).

[0070] (3) Component P50-II (4.0 g) was purified by Sephadex LH-20 column chromatography (40% methanol) and reverse-phase chromatography column (ODS-18, 60% MeOH) to obtain compound 9 (93 mg) and 10 (20 mg); it was purified by semi-preparative liquid chromatography (ODS-18, 25×300 mm, 10 μm) with 55% methanol as the mobile phase to obtain compound 6 (18 mg), 7 (100 mg) and 8 (96 mg).

[0071] (4) After component P50-IV (5.0 g) was eluted by Sephadex LH-20 column chromatography (40% methanol), it was purified by reverse-phase column chromatography (ODS-18, 30% MeOH) to obtain compound 3 (34 mg), 4 (25 mg) and 5 (1.6 g).

[0072] (5) The above-mentioned pharmacodynamic component P70S (10 g) was subjected to column chromatography on normal-phase silica gel column (100 - 200 mesh), and gradient elution was carried out with a dichloromethane - methanol - water - formic acid system (4:1:0.1:0.1 - 4:1:0.2:0.1, v / v), obtaining two sub-components, P70S-I (5.0 g) and P70S-II (4.0 g).

[0073] (6) Component P70S-I was purified by semi-preparative liquid chromatography (ODS-18, 25×300 mm, 10 μm, 60% MeOH) to obtain compound 1 (1.1 g) and 15 (800 mg).

[0074] (7) Component P70S-II was purified by semi-preparative liquid chromatography (ODS-18, 25×300 mm, 10 μm, 60% MeOH) to obtain compound 2 (50 mg), 13 (1.2 g) and 14 (1.3 g).

[0075] (8) The above-mentioned component P100 (500 g) was subjected to normal-phase silica gel column chromatography (100 - 200 mesh), and gradient elution was performed with the C:M:W:A system (4:1:0.1:0.1 - 4:1:0.2:0.1, v / v) to obtain sub-components P100-I to V.

[0076] (9) Component P100-I was separated by normal-phase silica gel column chromatography (5 × 80 cm, 300 - 400 mesh, C:M:W 6:1:0.1) to obtain compound 28 (54 mg), and was purified by reverse-phase ODS-18 column chromatography (5 × 50 cm, 65% MeOH) to obtain compound 26 (53 mg) and 27 (30 mg).

[0077] (10) Component P100-II (3.1 g) was purified by normal-phase silica gel column chromatography (300 - 400 mesh, C:M:W 6:1:0.1), reverse-phase ODS-18 column chromatography (70% methanol), and semi-preparative liquid chromatography (ODS-18, 25 × 300 mm, 10 um, 68% MeOH) to obtain compound 23 (82 mg) and a mixture 24 / 25 (33 mg).

[0078] (11) Component P100-III (800 mg) was purified by ODS-18 column chromatography (70% MeOH) to obtain compound 21 (30 mg) and 22 (106 mg).

[0079] (12) After component P100-IV was separated by Sephadex LH-20 column chromatography, it was purified by semi-preparative liquid chromatography (ODS-18, 65% MeOH) to obtain compound 20 (80 g), 19 (61 mg), 18 (88 mg), and 17 (54 mg).

[0080] (13) Component P100-V (5.0 g) was subjected to ODS-18 reverse-phase column chromatography and eluted with 60% methanol, and then repeatedly purified by semi-preparative liquid chromatography to obtain compound 16 (1.3 g).

[0081] 2. Structural identification of monomeric compounds

[0082] The structures of new compounds 1 - 3 were determined by 1D NMR, 2D NMR, MS, IR, optical rotation, etc. Figure 2 ) and named pulsatilloside P (1), pulsatilloside Q (2), pulsatilloside R (3). The structures of known compounds were identified by 1D NMR and MS data, in combination with physical and chemical properties being consistent with the literature reports. Figure 2) The known compounds are pulsatilloside F (4), pulsatilla saponin H (5), pastuchoside C (6), leonloside D (7), yemuoside YM 34 (8), patrinia saponin H3 (9), pulsatilla saponin F (10), leonticin F (11), hederasaponin D (12), hederacholchiside E (13), raddeanoside 17 (14), beesioside Q (15), pulchinenoside B3 (16), scabioside C (17), macranthoside B (18), macranthoside A (19), pulsatilla saponin A (20), leontoside A (21), hederacolchiside A1 (22), 3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] oleanolic acid (23), scabioside B (24), raddeanin R2 (25), pulsatilla saponin I (26), pulchinenoside B11 (27), and asperosaponin C (28). The specific physicochemical data are as follows:

[0083] Compound 1: White powder; –33.0 (c 1.4, MeOH); IR (film) ν max 3384, 2941, 1736, 1641, 1455, 1387, 1363, 1269, 1231, 1201, 1058, 1031, 913, 814 cm -1 ; ESIMS m / z 1227.7 [M+Na] + HRESIMS m / z 1227.6248 [M+Na] + (calcd for C59H96O25Na + , 1227.6133). 1313C NMR (Pyridine-d5, 150 MHz) δ 176.3 (C-28), 143.9 (C-13), 122.6 (C-12), 104.6 (Glc I-1), 104.6 (Ara-1), 102.5 (Rha I-1), 101.5 (Rha II-1), 95.4 (Glc II-1), 88.5 (C-3), 78.5 (Glc II-3), 77.9 (Glc I-4), 77.8 (Glc II-5), 76.9 (Glc I-5), 76.3 (Glc I-3), 75.7 (Ara-2), 75.1 (Glc I-2), 73.8 (Rha II-4), 73.7 (Rha I-4), 73.6 (Glc II-2), 73.6 (Ara-3), 72.5 (Rha I-3), 72.3 (Rha II-3, Rha I-2), 72.1 (Rha II-2), 70.5 (Glc II-4), 70.1 (Rha I-5), 69.7 (Rha II-5), 68.9 (Glc II-6), 68.5 (Ara-4), 64.5 (Ara-5), 61.0 (Glc I-6), 55.7 (C-5), 47.8 (C-9), 46.8 (C-17), 46.0 (C-19), 41.9 (C-14), 41.4 (C-18), 39.7 (C-8), 39.3 (C-4), 38.7 (C-1), 36.8 (C-10), 33.8 (C-21), 32.9 (C-29), 32.9 (C-7), 32.3 (C-22), 30.5 (C-20), 28.0 (C-15), 27.9 (C-23), 26.3 (C-2), 25.8 (C-27), 23.6 (C-11), 23.4 (C-30), 23.1 (C-16), 18.4 (Rha II-6), 18.3 (C-6), 18.3 (Rha I-6), 17.3 (C-26), 16.8 (C-24), 15.4 (C-25); 1HNMR (Pyridine-d5, 600 MHz) δ 6.22 (1H, d, J = 7.8 Hz, Glc II-1), 6.11 (1H, s, Rha II-1), 5.83 (1H, s, Rha I-1), 5.39 (1H, brs, H-12), 4.98 (1H, d, J = 7.8 Hz Glc I-1), 4.95 (1H, m, Rha I-5), 4.88 (1H, d, J = 4.8 Hz, Ara-1), 4.73 (1H, brs, Rha II-2), 4.66 (1H, m, Rha I-2), 4.61 (1H, dd, J = 9.0, 3.0 Hz, Rha II-3), 4.57 (1H, m, Rha II-5), 4.54 (1H, m, Ara-2), 4.53 (1H, m, Rha I-3), 4.39 (1H, m, Glc I-4), 4.31 (1H, m, Glc II-6a), 4.30 (1H, m, Ara-5a), 4.30 (1H, m, Rha II-4), 4.30 (1H, m, Glc II-4), 4.29 (1H, m, Rha I-4), 4.28 (1H, m, Ara-3), 4.27 (1H, m, Ara-4), 4.20 (1H, m, Glc II-3), 4.18 (1H, m, Glc I-6a), 4.18 (1H, m, Ara-5b), 4.12 (1H, m, Glc I-3), 4.11 (1H, m, Glc II-2), 4.08 (1H, m, Glc II-5), 4.06 (1H, m, Glc I-6), 3.92 (1H, t, J = 8.4 Hz, Glc I-2), 3.81 (1H, m, Glc II-6b), 3.63 (1H, brd, J = 9.6 Hz, Glc I-5), 3.22 (1H, dd, J = 12.0, 4.2 Hz, H-3), 3.16 (1H, dd, J = 13.8, 3.6 Hz, H-18), 2.29 (1H, m, 15a), 2.05 (H, m, 2a, 16a), 2.05 (H, m, 16a), 1.93 (1H, m, 16b), 1.92 (1H, m, 11a), 1.87 (1H, m, 11b), 1.84 (1H, m, 22a), 1.81 (1H, m, 2b), 1.73 (1H, m, 22b), 1.72 (1H, m, 19a), 1.68 (3H, d, J = 6.6 Hz, RhaI-6), 1.61 (3H, d, J = 6.6 Hz, Rha II-6), 1.59 (1H, over, H-9), 1.47 (1H, m, 1a), 1.43 (1H, m, 6a), 1.42 (1H, m, 7a), 1.31(1H,m,H-21a),1.31(3H,m,7b),1.29(3H,m,6b),1.23(3H,s,H-27),1.20(1H,over,19b),1.15(3H,s,H-23),1.13(1H,m,15b),1.09(1H,over,H-21b),1.07(3H,s,H-26),1.06(3H,s,H-24),0.92(1H,m,1b),0.88(3H,s,H-29),0.87(3H,s,H-30),0.86(3H,s,H-25),0.75(1H,brd,J=14.4Hz,H-5); See. Figures 3-8 。

[0084] Compound 2: White powder; –46.0(c 1.1,MeOH); IR(film) ν max 3358,2938,1735,1641,1453,1363,1258,1232,1055,1027,912,812,781 cm -1 ; ESIMS m / z 1713.9[M+Na]+; HRESIMS m / z 1713.7885[M+Na] + (calcd for C77H126O40Na + ,1713.7718); 1313C NMR (Pyridine-d5, 150 MHz) δ 176.3 (C-28), 143.9 (C-13), 122.6 (C-12), 106.3 (Glc III-1), 106.1 (Glc I-1), 105.1 (Ara-1), 104.6 (Glc II-1), 104.5 (Glc V-1), 102.4 (Rha II-1), 101.2 (Rha I-1), 95.3 (Glc IV-1), 88.4 (C-3), 82.8 (Rha I-3), 80.8 (Glc I-4), 79.9 (GlcI-3), 78.4 (Glc III-3), 78.4 (Glc III-5), 78.1 (Glc IV-3, Glc II-3), 77.9 (Glc V-4, GlcII-5), 77.7 (Glc IV-5), 76.8 (Glc V-5), 76.3 (Glc I-5), 76.2 (Glc V-3), 75.7 (Ara-2), 75.2 (Glc I-2), 75.1 (Glc V-2, Glc III-2), 74.5 (Glc II-2), 74.3 (Ara-3), 73.7 (Rha II-4), 73.6 (Glc IV-2), 72.6 (Rha I-4), 72.4 (Rha II-3), 72.3 (Rha II-2), 71.3 (Rha I-2), 71.2 (Glc II-4), 71.0 (Glc III-4), 70.4 (Ara-4), 70.4 (Glc IV-4), 70.0 (Rha II-5), 69.5 (Rha I-5), 68.8 (Glc IV-6), 65.2 (Ara-5), 62.2 (Glc II-6), 62.1 (Glc III-6), 61.5 (GlcI-6), 60.9 (Glc V-6), 55.8 (C-5), 47.8 (C-9), 46.8 (C-17), 46.0 (C-19), 41.9 (C-14), 41.4 (C-18), 39.6 (C-8), 39.3 (C-4), 38.6 (C-1), 36.8 (C-10), 35.4, 33.7 (C-21), 32.9 (C-29), 32.8 (C-7), 32.2 (C-22), 30.5 (C-20), 28.0 (C-15), 27.9 (C-23), 26.5 (C-2), 25.9 (C-27), 23.5 (C-11), 23.4 (C-30), 23.1 (C-16), 21.4, 19.3, 18.3 (C-6), 18.3 (Rha I-6, Rha II-6), 17.2(C-26), 17.0(C-24), 15.4(C-25);. 11H NMR (Pyridine-d5, 600 MHz) δ 6.20 (1H, d, J = 6.6 Hz, Glc IV-1), 6.14 (1H, s, Rha I-1), 5.80 (1H, s, Rha II-1), 5.40 (1H, d, J = 7.8 Hz, GlcI-1, H-12), 5.39 (1H, over, H-12), 5.13 (1H, d, J = 7.2 Hz, Glc II-1), 5.09 (1H, d, J = 6.6 Hz, Glc III-1), 4.97 (1H, d, J = 7.2 Hz, Glc V-1), 4.92 (1H, m, Rha II-5), 4.89 (1H, m, Rha I-2), 4.73 (1H, m, Rha I-3), 4.66 (1H, over, Ara-1), 4.64 (1H, m, Rha II-2), 4.64 (1H, m, GlcIV-6a), 4.60 (1H, m, Rha I-5), 4.52 (1H, m, Rha II-3), 4.48 (1H, m, Glc II-6a), 4.47 (1H, m, Glc III-6a), 4.46 (1H, m, Rha I-4), 4.41 (1H, m, Ara-2), 4.41 (1H, m, Ara-5a), 4.39 (1H, m, Glc I-6b), 4.37 (1H, m, Glc V-4), 4.31 (1H, m, Rha II-4), 4.31 (1H, m, Glc III-6b), 4.30 (1H, m, Glc IV-4), 4.30 (1H, m, Glc IV-6b), 4.28 (1H, m, Glc I-4), 4.24 (1H, m, Glc I-3), 4.21 (1H, m, Glc II-6b), 4.19 (1H, m, Glc IV-3), 4.18 (1H, m, Ara-3), 4.18 (1H, m, Glc II-3), 4.18 (1H, m, Glc III-5), 4.16 (1H, m, Ara-4), 4.16 (1H, m, Glc III-4), 4.16 (1H, m, GlcV-6a), 4.12 (1H, m, Glc II-4), 4.11 (1H, m, Glc V-3), 4.10 (1H, m, Glc IV-2), 4.07 (1H, m, Glc I-2), 4.07 (1H, m, Glc IV-5), 4.05 (1H, m, Glc V-6b), 4.01 (1H, m, Glc II-2), 3.99 (1H, m, Glc III-2), 3.96 (1H, m, Glc II-5), 3.90 (1H, m, Glc I-5), 3.90 (1H, m, Glc V-2), 3.87 (1H, m, Glc III-3), 3.80 (1H, t, J = 6.6 Hz, Glc I-6a), 3.74 (1H, d, J = 11.0 Hz, Ara-5b), 3.61 (1H, d, J = 7.8 Hz, Glc V-5), 3.21 (1H, brs, H-3), 3.15 (1H, d, J = 7.2 Hz, H-18), 2.27 (1H, m, 15a), 2.04 (1H, m, 16a), 1.91 (1H, m, 16b), 1.90 (1H, m, 11a), 1.84 (1H, m, 11b), 1.81 (1H, m, 22a), 1.81 (1H, m, 2a), 1.71 (1H, m, 19a), 1.70 (1H, m, 22b), 1.66 (3H, d, J = 6.0 Hz, RhaII-6), 1.59 (1H, m, H-9), 1.56 (3H, d, J = 6.0 Hz, Rha I-6), 1.44 (1H, m, 1a), 1.43 (1H, m, 6a), 1.40 (1H, m, 7a), 1.31 (1H, over, H-21), 1.27 (3H, s, H-23), 1.25 (1H, over, H-6b), 1.25 (1H, over, 7b), 1.23 (3H, s, H-27), 1.20 (1H, over, H-2b), 1.20 (1H, over, 19b), 1.14 (1H, over, 15b), 1.13 (3H, s, H-24), 1.08 (1H, over, 15b, H-21), 1.05 (3H, s, H-26), 0.88 (1H, over, 1b), 0.88 (3H, over, H-29), 0.86 (3H, over, H-25), 0.86 (3H, over, H-30), 0.76 (1H, brs, H-5); see. Figures 9-14 。

[0085] Compound 3: white powder; –38.0 (c 1.0, MeOH); IR (film) ν max 3358, 2932, 1733, 1642, 1452, 1365, 1259, 1231, 1028, 912, 812, 782 cm -1 ; ESIMS m / z 1729.8 [M+Na] + ; HRESIMS m / z 1729.7842 [M+Na] + (calcd for C77H126O41Na+, 1729.7667); 1313C NMR (Pyridine-d5, 151 MHz) δ 176.3 (C-28), 143.9 (C-13), 122.7 (C-12), 106.6 (Glc I-1), 106.4 (Glc III-1), 104.7 (Glc II-1), 104.6 (Glc V-1), 104.6 (Ara-1), 102.5 (Rha II-1), 101.1 (Rha I-1), 95.4 (Glc IV-1), 82.9 (Rha I-3), 80.9 (Glc I-4, C-3), 80.5 (Ara-4), 78.6 (Glc I-3), 78.5 (Glc I-5), 78.2 (Glc II-3), 78.2 (Glc II-5), 78.0 (Glc V-4), 77.9 (Glc IV-3), 77.8 (GlcIV-5), 76.9 (Glc V-5), 76.5 (Glc III-5), 76.4 (Glc III-3), 76.3 (Glc V-3), 75.4 (Ara-2), 75.3 (Glc III-2), 75.2 (Glc I-2), 75.1 (Glc V-2), 75.0 (Ara-3), 74.5 (Glc II-2), 73.8 (Rha II-4), 73.7 (Glc IV-2), 72.7 (Rha I-4), 72.5 (Rha II-3), 72.4 (Rha II-2), 71.4 (Rha I-2), 71.2 (Glc II-4), 71.0 (Glc IV-4), 70.5 (Glc III-4), 70.1 (Rha II-5), 69.5 (Rha I-5), 68.9 (Glc IV-6), 65.8 (Ara-5), 63.6 (C-23), 62.2 (Glc I-6), 62.1 (GlcIII-6), 61.5 (Glc II-6), 61.0 (Glc V-6), 49.4, 48.0 (C-5), 47.3 (C-9), 46.8 (C-17), 45.9 (C-19), 43.3 (C-4), 41.9 (C-14), 41.4 (C-18), 39.6 (C-8), 38.8 (C-1), 36.6 (C-10), 33.7 (C-21), 32.9 (C-29), 32.5 (C-7), 32.3 (C-22), 30.5 (C-20), 28.1 (C-15), 26.2 (C-2), 25.9 (C-27), 23.6 (C-11), 23.4 (C-30), 23.1 (C-16), 18.3 (Rha I-6), 18.3 (Rha II-6), 17.9 (C-6), 17.3 (C-26), 16.0 (C-25), 14.0 (C-24);... 11H NMR (Pyridine-d5, 600 MHz) δ 6.27 (1H, s, Rha I-1), 6.26 (1H, over, Glc IV-1), 5.89 (1H, s, Rha II-1), 5.48 (1H, d, J = 7.2 Hz, Glc III-1), 5.41 (1H, s, H-12), 5.18 (1H, d, J = 7.8 Hz, Glc II-1), 5.12 (1H, d, J = 7.8 Hz, Glc I-1), 5.01 (1H, d, J = 7.8 Hz, Glc V-1), 4.99 (1H, m, Rha II-5), 4.94 (1H, m, Rha I-2), 4.91 (1H, d, J = 7.2 Hz, Ara-1), 4.84 (1H, brd, J = 9.0 Hz, Rha I-3), 4.74 (1H, m, Rha I-5), 4.70 (1H, m, RhaII-2), 4.69 (1H, m, Glc IV-6a), 4.58 (1H, dd, J = 9.0, 2.4 Hz, Rha II-3), 4.54 (1H, m, Glc I-6a), 4.52 (1H, m, Glc II-6a), 4.52 (1H, m, Rha I-4), 4.52 (1H, s, Glc III-6a), 4.48 (1H, m, Ara-2), 4.45 (1H, m, Glc III-6b), 4.43 (1H, m, Glc V-4), 4.37 (1H, m, Ara-5a), 4.37 (1H, m, Rha II-4), 4.36 (1H, m, Glc IV-6b), 4.36 (1H, m, Glc III-4), 4.35 (1H, m, H-3), 4.32 (1H, m, 23a), 4.29 (1H, m, Glc I-6b), 4.29 (1H, m, Glc III-3), 4.25 (1H, m, Glc IV-4), 4.24 (1H, m, Glc I-4), 4.23 (1H, m, Glc I-5), 4.22 (1H, m, Glc II-5), 4.22 (1H, m, Glc IV-5), 4.21 (1H, m, Glc V-6b), 4.18 (1H, m, Glc II-4), 4.17 (1H, m, Glc V-3), 4.16 (1H, m, Glc II-6b), 4.14 (1H, m, Ara-4), 4.14 (1H, m, Glc IV-2), 4.13 (1H, m, Glc III-2), 4.12 (1H, m, Glc IV-3), 4.11 (1H, m, Glc V-6'), 4.06 (1H, m, Glc II-2), 4.05 (1H, m, Glc I-2), 4.00 (1H, m, Glc II-3), 3.96 (1H, m, Glc III-5), 3.96 (1H, m, Glc V-2), 3.92 (1H, m, 23b), 3.90 (1H, m, Glc I-3), 3.89 (1H, m, Ara-3), 3.66 (1H, d, J = 9.6 9.6Hz, Glc V-5), 3.58 (1H, d, J = 11.4Hz, Ara-5b), 3.17 (1H, brd, J = 12.0Hz, H-18), 2.30 (1H, m, 15a), 2.19 (1H, m, 2a), 2.04 (1H, m, 16a), 2.02 (1H, m, 16a), 1.93 (1H, m, 11a), 1.90 (1H, m, 16b), 1.88 (1H, m, 11b), 1.85 (1H, m, 6a), 1.84 (1H, m, 22a), 1.77 (1H, over, H-5), 1.75 (1H, over, 22b), 1.74 (1H, over, H-9), 1.72 (3H, d, J = 6.0Hz, Rha II-6), 1.68 (1H, m, 19a), 1.62 (1H, over, 7a), 1.58 (3H, d, J = 6.0Hz, Rha I-6), 1.54 (1H, over, 1a), 1.35 (1H, m, 6b), 1.30 (1H, m, 21a), 1.26 (1H, m, 7b), 1.22 (1H, over, 19b), 1.20 (3H, s, H-27), 1.16 (3H, s, H-24), 1.12 (3H, s, H-26), 1.09 (1H, m, H-21b), 1.07 (1H, m, 15b), 1.03 (1H, m, 1b), 0.98 (3H, s, H-25), 0.88 (3H, s, H-29), 0.87 (3H, s, H-30); see. Figures 15-20 。

[0086] Compound 4: 1313C NMR (Pyridine-d5, 150 MHz) δ 176.3, 143.9, 122.7, 106.5, 106.0, 105.1, 104.7, 104.2, 102.4, 101.5, 95.4, 84.4, 80.7, 80.2, 78.6, 78.5, 78.3, 78.3, 77.8, 77.0, 77.0, 76.9, 76.2, 76.1, 76.0, 75.9, 75.2, 75.2, 75.2, 74.8, 73.9, 73.8, 73.6, 72.5, 72.3, 72.0, 71.8, 70.9, 70.5, 70.1, 69.7, 69.4, 68.9, 68.2, 65.2, 63.6, 62.2, 61.1, 60.9, 47.9, 47.5, 46.8, 45.9, 43.2, 41.9, 41.4, 39.6, 38.8, 36.6, 33.7, 32.9, 32.5, 32.3, 30.5, 28.0, 26.1, 25.8, 23.6, 23.4, 23.1, 18.4, 18.4, 18.3, 17.9, 17.3, 15.9, 13.8; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.19 (1H, s), 6.15 (1H, d, J = 8.1 Hz), 5.77 (1H, s), 5.30 (1H, s), 5.12 (1H, d, J = 7.8 Hz), 5.03 (1H, d, J = 7.8 Hz), 1.74 (3H, d, J = 6.0 Hz), 1.62 (3H, d, J = 6.0 Hz), 1.57 (3H, d, J = 6.0 Hz), 1.07 (3H, s), 1.01 (3H, s), 1.01 (3H, s), 0.87 (3H, s), 0.78 (6H, brs).

[0087] Compound 5: 1313C NMR (Pyridine-d5, 150 MHz) δ 176.3, 143.8, 122.7, 106.6, 104.7, 104.2, 102.5, 101.5, 95.4, 80.7, 80.3, 78.6, 78.5, 78.3, 77.9, 77.8, 77.0, 76.3, 76.0, 75.3, 75.1, 74.9, 73.9, 73.8, 73.6, 72.5, 72.4, 72.2, 72.0, 70.9, 70.6, 70.1, 69.4, 68.9, 65.3, 63.6, 62.2, 61.0, 47.9, 47.5, 46.8, 45.9, 43.3, 41.9, 41.4, 39.7, 38.8, 36.6, 33.7, 32.9, 32.5, 32.3, 30.5, 28.1, 26.1, 25.8, 23.6, 23.4, 23.1, 18.4, 18.3, 17.9, 17.3, 16.0, 14.0; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.26 (1H, s), 6.23 (1H, d, J = 7.8 Hz), 5.85 (1H, s), 5.38 (1H, brs), 5.11 (1H, d, J = 7.9 Hz), 4.97 (3H, d, J = 8.1 Hz), 4.96 (3H, d, J = 8.1 Hz), 1.69 (3H, d, J = 6.1 Hz), 1.64 (3H, d, J = 6.1 Hz), 1.15 (3H, s), 1.10 (3H, s), 1.09 (3H, s), 0.95 (3H, s), 0.85 (3H, s), 0.84 (3H, s).

[0088] Compound 6: 13 13C NMR (Pyridine-d5, 150 MHz) δ 176.9, 144.4, 123.2, 107.1, 105.6, 104.7, 102.0, 96.0, 81.3, 80.8, 79.1, 79.0, 78.9, 78.8, 78.7, 78.3, 76.5, 75.8, 75.5, 75.4, 74.4, 74.2, 72.8, 72.6, 71.8, 71.5, 71.2, 70.0, 69.7, 65.8, 64.2, 62.9, 62.8, 48.5, 48.1, 47.3, 46.5, 43.8, 42.4, 42.0, 40.2, 39.3, 37.2, 34.2, 33.4, 33.1, 32.8, 31.0, 28.6, 26.6, 26.4, 24.2, 24.0, 23.7, 19.0, 18.4, 17.9, 16.5, 14.3; 11H NMR (Pyridine-d5, 600 MHz) δ 6.27 (1H, s), 6.26 (1H, s), 5.40 (1H, brs), 5.12 (1H, d, J = 7.9 Hz), 5.04 (1H, d, J = 7.7 Hz), 4.98 (1H, d, J = 6.7 Hz), 3.17 (1H, dd, J = 4.6, 13.9 Hz), 1.66 (3H, d, J = 6.2 Hz), 1.17 (3H, s), 1.12 (3H, s), 1.09 (3H, s), 0.96 (3H, s), 0.85 (3H, s), 0.84 (3H, s).

[0089] Compound 7: 13 13C NMR (Pyridine-d5, 150 MHz) δ 176.9, 144.4, 123.2, 107.1, 106.8, 105.1, 103.0, 95.9, 82.3, 80.2, 79.1, 79.0, 78.7, 78.4, 78.3, 77.4, 76.8, 76.1, 75.6, 75.0, 74.3, 74.1, 74.0, 73.1, 72.9, 71.6, 71.0, 70.6, 69.4, 66.7, 64.7, 62.8, 61.5, 48.5, 47.9, 47.3, 46.4, 43.8, 42.4, 41.9, 40.2, 39.0, 37.2, 34.2, 33.4, 33.0, 32.8, 31.0, 28.6, 26.4, 26.3, 24.2, 24.0, 23.6, 18.8, 18.5, 17.9, 16.5, 13.9; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.26 (1H, d, J = 8.1 Hz), 5.88 (1H, s), 5.42 (1H, brs), 5.27 (1H, d, J = 7.8 Hz), 5.01 (1H, d, J = 8.1 Hz), 4.92 (1H, d, J = 7.3 Hz), 3.18 (1H, dd, J = 4.6, 13.9 Hz), 1.72 (3H, d, J = 6.1 Hz), 1.18 (3H, s), 1.13 (3H, s), 0.99 (3H, s), 0.94 (3H, s), 0.88 (3H, s), 0.87 (3H, s).

[0090] Compound 8: 1313C NMR (Pyridine-d5, 150 MHz) δ 176.8, 144.4, 123.3, 107.1, 105.4, 105.2, 103.1, 101.7, 96.0, 83.8, 81.4, 81.4, 79.1, 78.8, 78.5, 78.4, 78.4, 77.5, 77.1, 77.1, 76.8, 75.8, 75.7, 75.4, 75.1, 74.4, 74.2, 73.4, 73.1, 72.9, 72.2, 71.8, 71.1, 70.6, 70.2, 70.0, 69.5, 66.9, 64.3, 62.7, 62.1, 61.6, 50.0, 48.5, 47.9, 47.3, 46.5, 43.9, 42.4, 41.9, 40.2, 39.4, 37.2, 34.3, 33.4, 33.0, 32.8, 31.1, 28.6, 26.7, 26.4, 24.2, 24.0, 23.6, 18.9, 18.8, 18.4, 17.8, 16.5, 14.6; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.33 (1H, s), 6.26 (1H, d, J = 8.1 Hz), 5.88 (1H, s), 5.47 (1H, d, J = 7.9 Hz), 5.38 (1H, d, J = 3.8 Hz), 5.20 (1H, d, J = 7.9 Hz), 4.94 (1H, over), 1.71 (3H, d, J = 6.2 Hz), 1.54 (3H, d, J = 6.1 Hz), 1.17 (3H, s), 1.15 (3H, s), 1.11 (3H, s), 0.97 (3H, s), 0.87 (3H, s), 0.86 (3H, s).

[0091] Compound 9: 1313C NMR (Pyridine-d5, 150 MHz) δ 176.3, 143.9, 122.7, 106.7, 104.8, 104.6, 102.5, 101.2, 95.4, 82.7, 81.0, 78.5, 78.4, 78.3, 77.9, 77.8, 76.9, 76.3, 75.7, 75.1, 75.1, 75.0, 73.8, 73.6, 72.8, 72.5, 72.4, 71.5, 71.3, 70.5, 70.1, 69.7, 69.5, 68.9, 66.4, 63.7, 62.2, 61.0, 48.0, 47.3, 46.8, 45.9, 43.4, 41.9, 41.4, 39.7, 38.8, 36.6, 33.7, 32.9, 32.5, 32.3, 30.5, 28.0, 26.2, 25.8, 23.6, 23.4, 23.1, 18.3, 18.3, 17.9, 17.3, 16.0, 14.0; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.33 (1H, s), 6.27 (1H, d, J = 8.1 Hz), 5.89 (1H, s), 5.53 (1H, d, J = 7.8 Hz), 5.40 (1H, brs), 4.97 (1H, over), 4.90 (1H, d, J = 9.5 Hz), 3.17 (1H, d, J = 12.9 Hz), 1.72 (3H, over), 1.55 (3H, d, J = 6.4 Hz), 1.19 (3H, s), 1.17 (3H, s), 1.12 (3H, s), 0.98 (3H, s), 0.88 (3H, s), 0.88 (3H, s).

[0092] Compound 10: 13 13C NMR (Pyridine-d5, 150 MHz) δ 176.8, 144.3, 123.2, 105.1, 104.7, 103.0, 101.9, 95.9, 81.2, 79.0, 78.4, 78.3, 77.4, 76.7, 76.0, 75.6, 75.1, 74.4, 74.3, 74.1, 73.0, 72.8, 72.8, 72.6, 71.0, 70.6, 70.0, 69.7, 69.4, 66.1, 64.2, 61.5, 48.4, 47.9, 47.3, 46.4, 43.8, 42.4, 41.9, 40.1, 39.3, 37.1, 34.2, 33.4, 33.0, 32.8, 31.0, 28.5, 26.5, 26.3, 24.1, 23.9, 23.6, 18.8, 18.8, 18.4, 17.8, 16.4, 14.3;1 1H NMR (Pyridine-d5, 600 MHz) δ 6.28 (1H, s), 6.26 (1H, d, J = 8.2 Hz), 5.88 (1H, s), 5.41 (1H, brs), 5.13 (1H, d, J = 6.2 Hz), 5.01 (H, d, J = 7.6 Hz), 3.17 (1H, dd, J = 4.6, 13.8 Hz), 1.72 (4H, d, J = 6.1 Hz), 1.65 (3H, d, J = 6.1 Hz), 1.17 (3H, s), 1.13 (3H, s), 1.09 (3H, s), 0.98 (3H, s), 0.88 (3H, s), 0.87 (3H, s).

[0093] Compound 11: 13 13C NMR (Pyridine-d5, 150 MHz) δ 176.9, 144.4, 123.2, 106.3, 105.6, 105.2, 104.3, 103.1, 96.0, 82.5, 81.7, 79.1, 78.7, 78.6, 78.4, 78.4, 77.5, 77.0, 76.8, 76.6, 75.7, 75.5, 75.1, 74.4, 74.2, 73.1, 72.9, 71.8, 71.7, 71.1, 70.6, 69.5, 68.7, 65.4, 65.1, 62.8, 62.1, 61.6, 48.5, 48.2, 47.3, 46.5, 43.8, 42.4, 42.0, 40.2, 39.1, 37.2, 34.3, 33.4, 33.1, 32.8, 31.1, 28.6, 26.4, 26.4, 24.2, 24.0, 23.6, 18.9, 18.8, 18.5, 17.9, 16.5, 13.8; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.29 (1H, s), 6.25 (1H, d, J = 8.2 Hz), 6.22 (2H, d, J = 8.2 Hz), 5.85 (2H, s), 5.39–5.34 (4H, m), 5.16 (4H, t, J = 6.2 Hz), 4.99–4.96 (3H, m), 3.14 (0H, s), 1.68 (8H, d, J = 6.1 Hz), 1.11 (5H, s), 1.09 (6H, s), 1.01 (3H, s), 0.93 (7H, s), 0.84 (6H, s), 0.83 (7H, s).

[0094] Compound 12: 1313C NMR (Pyridine-d5, 150 MHz) δ 177.5, 145.0, 123.8, 107.7, 105.8, 103.7, 96.5, 82.8, 79.7, 79.0, 79.0, 78.1, 77.4, 76.3, 75.7, 74.9, 74.8, 74.0, 73.7, 73.5, 71.7, 71.2, 70.6, 70.1, 68.0, 65.3, 62.1, 49.1, 48.5, 47.9, 47.1, 44.4, 43.0, 42.6, 40.8, 39.7, 37.8, 34.9, 34.0, 33.7, 33.4, 31.7, 29.2, 27.1, 27.0, 24.8, 24.6, 24.2, 19.5, 19.1, 18.5, 17.1, 14.6; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.26 (1H, d, J = 8.2 Hz), 5.89 (1H, s), 5.42 (1H, brs), 5.01 (1H, over), 5.00 (1H, over), 3.18 (1H, dd, J = 4.9, 14.2 Hz), 1.72 (3H, d, J = 6.0 Hz), 1.17 (3H, s), 1.13 (3H, s), 0.98 (3H, s), 0.94 (3H, s), 0.88 (3H, s), 0.87 (3H, s).

[0095] Compound 13: 13 13C NMR (Pyridine-d5, 150 MHz) δ 176.3, 143.9, 122.6, 106.2, 104.7, 104.6, 102.5, 101.5, 95.4, 88.4, 79.4, 78.5, 78.5, 78.3, 77.9, 77.8, 76.9, 76.3, 76.1, 75.2, 75.1, 73.8, 73.8, 73.6, 72.5, 72.3, 72.2, 72.1, 71.0, 70.5, 70.1, 69.6, 68.9, 64.3, 62.3, 61.0, 55.7, 47.8, 46.8, 46.0, 41.9, 41.4, 39.6, 39.3, 38.7, 36.8, 33.7, 32.9, 32.9, 32.3, 30.5, 28.0, 27.8, 26.4, 25.8, 23.6, 23.4, 23.1, 18.4, 18.3, 17.3, 16.8, 15.4, 14.0; 1HNMR (Pyridine-d5, 600 MHz) δ 6.24 (1H, d, J = 8.2 Hz), 6.18 (1H, s), 5.85 (1H, d, J = 7.4 Hz), 5.39 (1H, brs), 5.14 (1H, s), 4.98 (1H, d, over), 4.75 (1H, d, over), 3.16 (1H, m), 2.28 (1H, m), 1.69 (3H, d, over), 1.64 (3H, d, J = 6.6 Hz), 1.22 (3H, s), 1.15 (3H, s), 1.10 (3H, s), 1.08 (3H, s), 0.87 (9H, brs).

[0096] Compound 14: 13 C NMR (Pyridine-d5, 150 MHz) δ 176.3, 143.9, 122.6, 106.4, 105.2, 104.8, 104.6, 102.5, 101.3, 95.4, 88.4, 83.3, 80.8, 78.5, 78.2, 78.0, 77.9, 77.8, 76.9, 76.5, 76.3, 75.3, 75.1, 75.1, 74.5, 73.8, 73.7, 72.8, 72.5, 72.4, 71.6, 71.2, 70.6, 70.1, 69.4, 69.2, 68.9, 65.7, 62.1, 61.6, 61.0, 55.7, 47.8, 46.8, 46.0, 41.9, 41.4, 39.6, 39.4, 38.7, 36.8, 33.7, 32.9, 32.8, 32.3, 30.5, 28.1, 28.0, 26.5, 25.9, 23.6, 23.4, 23.1, 18.3, 18.3, 17.2, 17.0, 15.4; 1 HNMR (Pyridine-d5, 600 MHz) δ 6.24 (1H, d, over), 6.23 (1H, s), 5.85 (1H, s), 5.44 (1H, d, J = 7.8 Hz), 5.38 (1H, brs), 5.19 (1H, d, J = 7.9 Hz), 4.98 (1H, over), 4.97 (1H, over), 3.26 (1H, dd, J = 4.3, 11.8 Hz), 3.15 (1H, dd, J = 4.7, 13.7 Hz), 1.69 (1H, s), 1.52 (1H, s), 1.28 (3H, s), 1.23 (3H, s), 1.14 (3H, s), 1.06 (3H, s), 0.87 (3H, s), 0.87 (3H, s), 0.86 (3H, s).

[0097] Compound 15:13 13C NMR (Pyridine-d5, 150 MHz) δ 176.3, 143.9, 122.6, 106.6, 105.3, 104.6, 102.5, 101.3, 95.4, 88.4, 83.0, 78.5, 78.4, 78.3, 77.9, 77.8, 76.9, 76.2, 75.7, 75.2, 75.1, 74.5, 73.8, 73.6, 72.8, 72.5, 72.3, 71.5, 71.2, 70.5, 70.1, 69.5, 69.3, 68.9, 65.8, 62.2, 61.0, 55.7, 47.8, 46.8, 46.0, 41.9, 41.4, 39.6, 39.4, 38.7, 36.8, 33.7, 32.9, 32.8, 32.3, 30.5, 28.0, 28.0, 26.5, 25.9, 23.6, 23.5, 23.1, 18.3, 18.3, 18.3, 17.2, 17.0, 15.4; 1 1H NMR (Pyridine-d5, 600 MHz) δ 6.23 (1H, d, J = 8.4 Hz), 6.22 (1H, s, over), 5.84 (1H, s), 5.47 (1H, d, J = 7.8 Hz), 5.38 (1H, brs), 4.98 (1H, d, over), 4.79 (1H, d, over), 3.26 (1H, dd, J = 4.3, 11.7 Hz), 3.15 (1H, dd, J = 4.7, 13.7 Hz), 1.68 (3H, d, J = 6.0 Hz), 1.51 (3H, d, J = 6.0 Hz), 1.31 (3H, s), 1.22 (3H, s), 1.14 (3H, s), 1.06 (3H, s), 0.87 (3H, s), 0.86 (3H, s), 0.85 (3H, s).

[0098] Compound 16: 13 13C NMR (DMSO-d6, 150 MHz) δ 178.6, 143.9, 121.6, 105.4, 102.9, 99.9, 79.2, 79.1, 77.0, 76.3, 74.9, 73.9, 72.9, 72.0, 70.4, 70.4, 70.0, 68.2, 64.3, 62.4, 61.1, 47.1, 46.2, 45.7, 45.5, 42.3, 41.4, 40.9, 38.9, 38.2, 36.0, 33.3, 32.9, 32.1, 32.0, 30.4, 27.2, 25.6, 25.4, 23.4, 22.9, 22.6, 17.8, 17.1, 16.9, 15.6, 13.0;1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 5.12 (1H, s), 4.28 (1H, d, J = 6.7 Hz), 4.24 (1H, d, J = 7.8 Hz), 2.73 (1H, dd, J = 4.9, 13.4 Hz), 1.09 (3H, s), 1.07 (3H, d, J = 6.0 Hz), 0.87 (3H, s), 0.87 (3H, s), 0.86 (3H, s), 0.71 (3H, s), 0.57 (3H, s).

[0099] Compound 17: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.1, 144.1, 121.5, 105.2, 104.9, 79.9, 78.5, 77.0, 76.4, 74.1, 72.6, 71.8, 70.0, 67.2, 64.7, 62.7, 61.1, 47.2, 46.1, 45.9, 45.5, 42.5, 41.4, 40.4, 38.9, 38.0, 36.1, 34.8, 33.5, 33.0, 32.2, 32.1, 30.5, 27.3, 25.7, 25.2, 23.5, 23.0, 22.8, 17.3, 17.0, 15.7, 13.0; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 4.29 (1H, d, J = 7.8 Hz), 4.16 (1H, d, J = 6.9 Hz), 2.75 (1H, dd, J = 4.8, 13.9 Hz), 1.09 (3H, s), 0.89 (3H, s), 0.87 (6H, s), 0.72 (3H, s), 0.59 (3H, s).

[0100] Compound 18: 13 13C NMR (DMSO-d6, 150 MHz) δ 178.5, 143.7, 121.5, 104.4, 103.2, 103.1, 99.8, 81.7, 80.1, 79.3, 76.7, 76.4, 74.6, 74.5, 73.8, 73.6, 73.2, 73.1, 70.7, 69.9, 69.2, 68.1, 67.7, 64.9, 62.2, 60.3, 60.1, 51.5, 47.0, 46.0, 45.6, 45.3, 42.2, 41.3, 40.7, 38.7, 38.1, 35.9, 33.2, 32.7, 32.0, 31.8, 30.3, 27.1, 25.5, 25.3, 23.3, 22.8, 22.5, 17.7, 17.0, 16.8, 15.5, 12.9;1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 5.11 (1H, s), 4.39 (1H, d, J = 7.8 Hz), 4.31 (1H, d, over), 4.26 (1H, d, J = 7.9 Hz), 2.73 (1H, dd, J = 5.2, 12.2 Hz), 1.09 (6H, brs), 0.86 (9H, brs), 0.70 (3H, s), 0.57 (3H, s).

[0101] Compound 19: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.7, 144.5, 120.8, 104.8, 103.2, 99.7, 81.7, 79.4, 76.7, 76.2, 73.9, 73.7, 73.2, 70.8, 69.6, 69.3, 68.1, 67.7, 65.0, 62.3, 60.7, 48.5, 47.1, 46.1, 45.4, 42.2, 41.3, 41.0, 38.7, 38.1, 35.9, 33.6, 32.9, 32.3, 31.9, 30.4, 27.3, 25.5, 25.3, 23.4, 22.8, 22.8, 17.7, 17.1, 16.9, 15.5, 12.9; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, s), 5.11 (1H, brs), 4.33 (1H, over), 4.32 (1H, over), 2.78 (1H, dd, J = 4.6, 14.2 Hz), 1.10 (3H, d, J = 6.1 Hz), 1.08 (3H, s), 0.88 (3H, over), 0.86 (3H, over), 0.85 (3H, over), 0.72 (3H, s), 0.58 (3H, s).

[0102] Compound 20: 13 13C NMR (Pyridine-d5, 150 MHz) δ 179.9, 158.3, 144.6, 122.4, 104.3, 101.5, 80.8, 75.5, 74.7, 73.9, 72.3, 72.2, 69.5, 69.2, 65.6, 63.7, 49.4, 47.9, 47.5, 46.4, 46.1, 43.3, 41.9, 41.7, 39.5, 38.7, 36.6, 34.0, 33.0, 33.0, 32.6, 30.7, 28.1, 26.0, 25.9, 23.6, 23.5, 23.4, 18.3, 17.9, 17.2, 15.8, 13.8; 11H NMR (Pyridine-d5, 600 MHz) δ 6.27 (1H, s), 5.44 (1H, brs), 5.10 (1H, d, J = 6.3 Hz), 3.27 (1H, dd, J = 4.6, 14.3 Hz), 1.62 (3H, d, J = 6.0 Hz), 1.20 (3H, s), 1.05 (3H, s), 1.00 (3H, s), 0.98 (3H, s), 0.92 (3H, s), 0.90 (3H, s).

[0103] Compound 21: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.0, 144.1, 121.7, 105.0, 80.3, 73.0, 71.3, 67.9, 65.3, 62.9, 47.3, 46.3, 45.9, 45.6, 42.6, 41.5, 41.0, 39.0, 38.1, 36.2, 33.6, 33.1, 32.3, 32.1, 30.6, 27.4, 25.8, 25.3, 23.6, 23.2, 22.8, 17.4, 17.1, 15.8, 13.1; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.14 (1H, brs), 4.18 (1H, s), 2.73 (1H, dd, J = 5.1, 13.2 Hz), 1.08 (3H, s), 0.88 (3H, s), 0.87 (3H, s), 0.85 (3H, s), 0.70 (3H, s), 0.58 (3H, s).

[0104] Compound 22: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.2, 144.3, 121.9, 105.5, 104.3, 100.5, 88.2, 78.9, 77.4, 76.7, 75.6, 74.3, 72.8, 72.4, 70.8, 70.8, 70.4, 68.8, 64.1, 61.5, 55.6, 47.5, 46.2, 45.9, 41.8, 41.3, 39.3, 39.1, 38.7, 36.8, 33.8, 33.3, 32.9, 32.6, 30.9, 27.8, 27.6, 26.2, 26.0, 23.8, 23.3, 23.1, 18.3, 18.2, 17.3, 16.7, 15.7; 11H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 5.10 (1H, s), 4.26 (1H, d, over), 4.25 (1H, d, over), 2.74 (1H, dd, J=5.1, 13.3 Hz), 1.08 (3H, d, over), 1.07 (3H, s), 0.94 (3H, s), 0.87 (9H, brs), 0.75 (3H, s), 0.71 (3H, s).

[0105] Compound 23: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.1, 144.3, 122.0, 104.9, 104.6, 103.7, 100.4, 88.3, 82.1, 80.7, 77.3, 76.9, 75.2, 75.0, 74.5, 74.1, 73.7, 73.3, 71.3, 70.5, 69.8, 68.5, 68.4, 65.1, 61.5, 60.7, 55.6, 47.5, 46.1, 45.9, 41.8, 41.3, 39.3, 39.1, 38.7, 36.8, 33.8, 33.3, 32.8, 32.5, 30.9, 27.8, 27.6, 26.3, 26.0, 23.8, 23.3, 23.1, 18.3, 18.3, 17.3, 16.8, 15.7; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.16 (1H, brs), 5.12 (1H, s), 4.39 (1H, d, J=7.8 Hz), 4.28 (1H, d, J=5.7 Hz), 4.26 (1H, d, J=7.9 Hz), 2.74 (1H, dd, J=4.7, 13.9 Hz), 1.10 (3H, d, over), 1.09 (3H, s), 0.95 (3H, s), 0.87 (6H, s), 0.86 (3H, s), 0.76 (3H, s), 0.71 (3H, s).

[0106] Compound 24: 1313C NMR (DMSO-d6, 150 MHz) δ 179.1, 144.3, 122.0, 106.3, 105.6, 88.2, 78.9, 77.4, 76.8, 74.4, 72.9, 72.1, 70.4, 65.1, 61.5, 55.5, 47.5, 46.2, 45.9, 41.8, 41.3, 39.3, 39.2, 38.5, 36.8, 33.8, 33.3, 32.8, 32.6, 30.9, 28.1, 27.7, 26.2, 26.0, 23.8, 23.8, 23.4, 23.1, 18.3, 17.3, 16.9, 15.6; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.16 (1H, brs), 4.30 (1H, d, J = 7.8 Hz), 4.09 (1H, d, J = 6.8 Hz), 2.74 (1H, dd, J = 4.7, 13.9 Hz), 1.09 (3H, s), 0.97 (3H, s), 0.87 (9H, s), 0.76 (3H, s), 0.71 (3H, s).

[0107] Compound 25: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.1, 144.3, 122.0, 104.3, 103.9, 88.4, 79.0, 77.2, 76.8, 75.0, 71.8, 70.3, 66.9, 63.8, 61.3, 55.4, 47.5, 46.2, 45.9, 41.8, 41.3, 39.3, 39.2, 38.5, 36.8, 33.8, 33.3, 32.8, 32.6, 30.9, 28.1, 27.7, 26.2, 26.0, 23.8, 23.8, 23.4, 23.1, 18.3, 17.3, 16.9, 15.6; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 4.39 (1H, d, J = 5.3 Hz), 4.35 (1H, d, J = 7.7 Hz), 2.74 (4H, dd, J = 4.7, 13.9 Hz), 1.09 (3H, s), 0.97 (12H, s), 0.87 (9H, s), 0.76 (3H, s), 0.71 (3H, s).

[0108] Compound 26: 1313C NMR (DMSO-d6, 150 MHz) δ 179.1, 144.3, 122.0, 105.2, 104.7, 100.4, 88.3, 82.1, 77.1, 76.7, 74.6, 74.3, 73.2, 71.2, 70.1, 69.8, 68.4, 65.1, 61.3, 55.6, 47.5, 46.2, 45.9, 41.7, 41.2, 39.3, 39.1, 38.7, 36.8, 33.8, 33.3, 32.8, 32.5, 30.8, 27.8, 27.6, 26.2, 26.0, 23.8, 23.3, 23.1, 18.3, 17.2, 16.7, 15.6; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 5.11 (1H, s), 4.31 (1H, d, J = 7.7 Hz), 4.27 (1H, brs), 2.73 (1H, dd, J = 5.1, 13.2 Hz), 1.10 (3H, d, J = 6.1 Hz), 1.09 (3H, s), 0.95 (3H, s), 0.87 (3H, brs), 0.76 (3H, s), 0.71 (3H, s).

[0109] Compound 27: 13 13C NMR (DMSO-d6, 150 MHz) δ 179.1, 144.3, 122.0, 104.3, 100.5, 88.2, 74.9, 72.8, 72.4, 70.9, 70.8, 68.8, 67.9, 64.3, 55.6, 47.5, 46.2, 45.9, 41.8, 41.3, 39.3, 39.1, 38.7, 36.8, 33.8, 33.3, 32.8, 32.6, 30.9, 27.9, 27.6, 26.2, 26.0, 23.8, 23.3, 23.1, 18.3, 18.2, 17.3, 16.7, 15.7; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 5.04 (1H, s), 4.29 (1H, d, J = 5.6 Hz), 2.74 (1H, dd, J = 4.7, 13.7 Hz), 1.09 (3H, s), 1.07 (3H, d, J = 6.2 Hz), 0.93 (3H, s), 0.87 (6H, s), 0.86 (3H, s), 0.75 (2H, s), 0.71 (2H, s).

[0110] Compound 28: 1313C NMR (DMSO-d6, 150 MHz) δ 179.5, 144.5, 121.8, 106.4, 88.2, 73.2, 71.5, 68.1, 65.7, 55.4, 47.5, 46.3, 45.9, 41.8, 41.3, 39.3, 39.2, 38.5, 36.8, 33.9, 33.3, 32.9, 32.6, 30.9, 28.1, 27.7, 26.1, 26.0, 23.9, 23.4, 23.1, 18.3, 17.4, 16.9, 15.6; 1 1H NMR (DMSO-d6, 600 MHz) δ 5.15 (1H, brs), 4.11 (1H, brs), 2.75 (1H, d, J = 13.9 Hz), 1.09 (3H, s), 0.97 (3H, s), 0.87 (9H, s), 0.76 (3H, s), 0.72 (3H, s).

[0111] Test Example 1 Anti-UC Activity Experiment of Active Components P20, P50, and P70 in Total Extract (PE)

[0112] 1 Experimental Method

[0113] Healthy C57BL / 6J male mice (22 ± 2 g) were randomly divided into groups of 8. Dextran sulfate sodium (3% DSS) was used to induce ulcerative colitis in mice. The therapeutic effects of different drug treatments on ulcerative colitis were evaluated by indicators such as disease activity index (DAI), body weight change, colon length change, pathological changes of colon tissue, and expression level of inflammatory factor TNF-α in colon tissue.

[0114] 1.1 Modeling and Drug Administration

[0115] 1) Blank control (CON): Drink pure water freely throughout the entire experimental period without any drug treatment.

[0116] 2) Model group (DSS): After drinking pure water freely for 3 days, the pure water was replaced with a 3% DSS solution (re-prepared every other day). The start of free drinking of the DSS solution was defined as day 0. After 7 days of free drinking the DSS solution (day 0 - day 7), it was replaced with pure water until the end of the experiment (day 9).

[0117] 3) Treatment groups with active components of Pulsatilla chinensis: Each group of mice was given a dose of 200 mg / kg / day of P20, P50, P70, P100, and PE by gavage until the end of the experiment; Modeling started 3 days after drug administration, and the modeling method and cycle were the same as those of the model group.

[0118] 4) Positive drug group: Mesalazine (MLZ) (purchased from Sigma Chemical Co.) was selected as the positive drug. Mesalazine at a dose of 250 mg / kg / day was administered by gavage until the end of the experiment; the model was established 3 days after drug administration, and the modeling method and cycle were the same as those of the model group.

[0119] 1.2 Evaluation indicators:

[0120] 1) Body weight change rate: After the start of modeling, the body weights of the mice in each group were weighed and recorded at the same time every day; the body weight at the start of modeling (day 0) was used as the initial body weight, and the body weight change rate = (body weight on the current day / initial body weight) × 100% was used as the evaluation index for the change in body weight ratio.

[0121] 2) Disease Activity Index (DAI): From the second day after the start of modeling (day 2), the body weights, blood in feces conditions, and fecal traits of the mice in each group were recorded. Among them, the occult blood score of mouse feces was carried out according to the steps of the Beso Biotech Pyramidon semi-quantitative detection kit, and tested every two days, so the DAI score was calculated every two days. The calculation method of the Disease Activity Index (DAI) is: the body weight loss rate of the mice (body weight per day / body weight at the start of modeling), the occult blood in feces and the fecal traits were scored separately and the average value was calculated. The specific scoring rules are shown in Table 4:

[0122] Table 4. DAI scoring criteria

[0123]

[0124] 3) Colon length: Two days after the end of modeling (day 9), the mice in each group were sacrificed, the colon was quickly removed, photographed, and its length was measured.

[0125] 1.3 H&E staining of colon tissue: Take 0.5 cm of the posterior colon tissue, after fixation with 4% paraformaldehyde and dehydration, the colon tissue was embedded in paraffin, and sections were prepared using a paraffin microtome. After dewaxing and hydration of the paraffin sections, according to the manufacturer's instructions, the paraffin sections were stained with hematoxylin and eosin (H&E). After sealing the slides, a microscopic imaging system was used to observe and record the pathological changes of the colon tissue.

[0126] 1.4 Western blotting analysis: Two days after the establishment of the model (day 9), the mice in each group were sacrificed, and the colon was quickly removed. After measuring the length, an appropriate amount of colon tissue (about 30 mg) was taken from about 1 cm at the end of the colon and washed clean with normal saline. A certain amount (700 μL) of protein lysate was added, and homogenization was performed at 4 °C for 90 s using a high-speed homogenizer and an ultrasonic crusher; the homogenate was left to lyse statically at 4 °C for 30 min. The homogenate was centrifuged at 12,000 rpm for 30 min, and the supernatant was aspirated as the protein; the protein concentration was measured by the BCA method, an appropriate amount of 5×SDS Loding buffer was added, and it was boiled in water for 10 min. After cooling to room temperature, Western blotting analysis was carried out. After transferring the protein to the PVDF membrane, it was incubated with anti-TNF-α as the primary antibody; after incubation with the corresponding secondary antibody, ELC luminescent solution was added, and image acquisition was performed using a gel imager. Finally, the protein bands were quantitatively analyzed using Image Lab software.

[0127] 2 Experimental results

[0128] The anti-UC activities of the total extract PE and the fractions P20, P50, P70, and P100 were evaluated using a DSS-induced UC model mouse. As shown in Figure 21, compared with the control group, the body weight of the mice in the DSS group was significantly reduced (Figure 21(A), Figure 21(B)), the disease activity index (DAI) was significantly increased (Figure 21(C), Figure 21(D)), and the colon was significantly shortened (Figure 21(E), Figure 21(F)). Compared with the DSS group, the body weight and colon length of the mice in the PE, P50, and P70 fractions were significantly increased, and the DAI scores were also reduced to varying degrees; the P20 and P100 groups did not significantly improve the above indicators ( Figure 21(A)-Figure 21(F) ); the P70 treatment group was significantly superior to the PE treatment group or the P50 treatment group in the treatment of ulcerative colitis (p < 0.05), and the PE and P50 treatment groups only showed weak anti-UC activities.

[0129] High expression of TNF-α is one of the pathological indicators of ulcerative colitis. The expression level of TNF-α in colon tissues was determined by Western blotting. As shown in Figure 22, compared with the control group, the expression level of TNF-α in the colon tissues of DSS-induced UC mice was significantly increased. At the same time, treatment with P50 or P70 significantly inhibited the expression of TNF-α in the colon tissues of DSS-induced UC mice. It is worth noting that in UC mice, the inhibitory effect of P70 on TNF-α expression was significantly better than that of the P50 treatment group (p<0.05); the effects of PE, P20, and P100 on TNF-α expression were not statistically significant (p>0.05, Figure 22(A), Figure 22(B)). These results indicate that the P70 fraction is the key active substance of Pulsatilla chinensis extract in the treatment of UC.

[0130] To further verify the anti-UC ability of P70, we detected the pathological changes of colon tissues by H&E staining. The colon mucosa of the control group was intact, the glands were regularly distributed, the crypt structure was complete, and goblet cells were abundant (Figure 22(C)). Induction with DSS severely damaged the colon mucosa, resulting in muscle edema and inflammatory infiltration (Figure 22(C)), while treatment with P70 significantly improved the above pathological changes.

[0131] 3 Experimental conclusions

[0132] The research results of this test example show that there are obvious differences in anti-UC activities between the total extract PE and each extract fraction. At the same dosing dose, P100 did not show anti-UC activity, while PE, P50, and P70 showed anti-UC activity. The activity of P70 was significantly better than that of P50 and PE, and the activities of P50 and PE were close. This indicates that different compound fractions have different activities. As can be seen from Example 1, fraction P50 is mainly composed of 3,28-bis-oligosaccharidyl hederagenin saponin compounds 3-12, P70 is composed of 3,28-bis-oligosaccharidyl oleanolic acid saponin components 1, 2, 13-15, and fraction P100 mainly contains 3-mono-oligosaccharidyl hederagenin saponin and 3-mono-oligosaccharidyl oleanolic acid saponin components 16-28. The research results of this test example show that the anti-UC activity of 3,28-bis-oligosaccharidyl oleanolic acid saponin components is significantly better than that of 3,28-bis-oligosaccharidyl hederagenin saponin components; saponins with a free carboxyl group at the 28th position did not show anti-UC activity. The above activity results indicate that the differences in aglycone types and oligosaccharide substituents significantly affect the anti-UC activity of this type of compound. Therefore, the P70 fraction of the Pulsatilla chinensis extract of the present invention can be used for anti-UC purposes and can be used in the preparation of drugs for preventing and treating UC.

[0133] Test example 2 Anti-UC experiment of monomeric compounds

[0134] 1 Experimental method

[0135] The experimental protocol and evaluation indicators are referred to Test Example 1. The administration methods of the blank control (CON), model group (DSS), and positive drug group are referred to Test Example 1. The treatment group of active monomers of Pulsatilla chinensis: Each group of mice was given the active monomer compounds 1, 2, 3, 5, 13, 14, and 15 at a dose of 150 mg / kg / day by gavage until the end of the experiment. Three days after administration, the modeling was started, and the modeling method and cycle were the same as those of the model group.

[0136] 2 Experimental results

[0137] The anti-UC activities of monomer compounds 1, 2, 3, 5, 13, 14, and 15 were evaluated using DSS-induced UC model mice. As shown in Figure 23, compared with the control group, the body weights of the mice in the DSS group were significantly decreased (Figure 23(A), Figure 23(D)), the disease activity index (DAI) was significantly increased (Figure 23(B), Figure 23(E)), and the colon was significantly shortened (Figure 23(C), Figure 23(F)) (P<0.01). Compared with the DSS group, the body weights and colon lengths of the mice in the treatment groups of compounds 1, 2, 13, 14, and 15 were significantly increased, and the DAI scores were significantly decreased (P<0.01). Compounds 3 and 5 showed only weak anti-UC activities. The anti-UC activities of compounds 1, 2, 13, 14, and 15 were significantly better than those of compounds 3 and 5 (P<0.01).

[0138] 3 Experimental conclusions

[0139] Compounds 1, 2, 13, 14, and 15 can all significantly improve the pathological indexes of UC mice, can be used for the prevention and treatment of UC, and can be used to prepare drugs for the treatment of UC. Combining the active effects of P70 and the types of compounds contained therein, it can be determined that its activity mainly stems from compounds 1, 2, 13, 14, and 15.

[0140] Test Example 3 Effects of Compound 1 on Intestinal Tissue Pathology and TNFα / NF-κB / MLCK Signaling Pathway in UC Mice

[0141] 1 Experimental methods

[0142] 1.1 The experimental protocol and H&E staining of colon tissue are referred to Test Example 1. The administration methods of the blank control (CON), model group (DSS), and positive drug group are referred to Test Example 1. The treatment group of monomer compound 1: Each group of mice was given the active monomer compound 1 at doses of 75 mg / kg / day and 150 mg / kg / day by gavage until the end of the experiment. Three days after administration, the modeling was started, and the modeling method and cycle were the same as those of the model group.

[0143] 1.2 Western blotting analysis: The method is shown in Test Example 1. After electrophoresis, the proteins were transferred onto PVDF membranes and incubated with anti-claudin-2, anti-TNF-α, anti-GAPDH (Cell Signaling Technology, Beverly, MA), anti-long MLCK and anti-p-MLC (Abcam, Cambridge, MA) as primary antibodies; after incubation with the corresponding secondary antibodies, ELC luminescent solution was added, and image acquisition was performed using gel imaging. Finally, the protein bands were quantitatively analyzed using Image Lab software.

[0144] 2 Experimental results

[0145] We detected the pathological changes of colon tissues by H&E staining. The colon mucosa of the control group was intact, the glands were regularly distributed, the crypt structure was complete, and goblet cells were abundant (Figure 24(A)). DSS induction severely damaged the colon mucosa, showing muscle edema and inflammatory infiltration (Figure 24(A)), while treatment with Compound 1 significantly improved the above pathological changes (Figure 24(A)). Subsequently, the expression of claudin-2 protein in colon tissues was detected by Western blotting to evaluate the effect of Compound 1 on the tight junction structure of the colon epithelium. As shown in Figures 24(B) and 24(C), compared with the DSS group, Compound 1 significantly downregulated the expression of claudin-2. Given the pathological characteristics of TNF-α in ulcerative colitis and the important role of the long-chain myosin light chain kinase (MLCK) signaling pathway in the intestinal epithelial barrier disorder in UC, we detected the expression levels of key proteins in the MLCK pathway. Western blotting experiments showed that treatment with Compound 1 significantly downregulated the expression levels of TNF-α and MLCK in the colon tissues of UC mice and inhibited the phosphorylation of MLC (Figure 24(B), Figure 24(D)-Figure 24(F) ).

[0146] 3 Experimental conclusions

[0147] These in vivo experimental results indicate that Compound 1 can significantly inhibit the pathological damage of colon tissues in UC mice and inhibit the expression of TNF-α; by inhibiting the activation of the MLCK signaling pathway, downregulating the expression of claudin-2, restoring the abundance of goblet cells, and improving the intestinal epithelial barrier function of UC mice.

Claims

1. Use of a pulchinenoside derivative in the preparation of a medicament for treating and preventing ulcerative colitis, characterized in that, The pulchinenoside derivative is a 3,28-bis-oligosaccharide oleanolic acid saponin compound, and its structural general formula is the compound shown in Formula I; wherein R1, R2, and R3 are glycosyl groups or hydrogen, and R5 is a glycosyl group; the glycosyl group is a monosaccharide or an oligosaccharide group formed by 2 to 4 monosaccharides.

2. The use according to claim 1, wherein The 3,28-bis-oligosaccharide oleanolic acid saponin compounds are selected from compounds 1, 2, 13, 14, and 15, and their chemical structural formulas are as follows:

3. The use according to claim 1 or 2, characterized in that, The compounds 1, 2, 13, 14, and 15 are contained in the traditional Chinese medicine extract of Pulsatilla chinensis. Based on the total weight of the traditional Chinese medicine extract of Pulsatilla chinensis being 100%, the traditional Chinese medicine extract of Pulsatilla chinensis contains any one or more of the compounds 1, 2, 13, 14, and 15, and the weight ratio accounts for 50% to 99.9% of the total weight of the traditional Chinese medicine extract of Pulsatilla chinensis.

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