Two novel cyclic peptide compounds extracted from radix stellariae as well as extraction method and application thereof

Abstract

The invention relates to two new cyclic peptide compounds extracted from radix stellariae and a preparation method and application thereof, can effectively solve the problems of extraction and application of radix stellariae, and adopts the technical scheme that the chemical structural formulas of the two new cyclic peptide compounds are shown as follows: the two new cyclic peptide compounds are separated and identified from # imgabs0 # radix bupleuri; experimental results show that the two compounds have an obvious improvement effect on mice with acute liver injury, and have important practical significance on full excavation of effective components of radix stellariae.

Description

1. Technical Field

[0001] The invention relates to the field of drug extraction, in particular to two new cyclic peptide compounds extracted from Bupleurum chinense and an extraction method and application thereof. 2. Background Technology

[0002] Acute liver injury refers to liver tissue damage caused by factors such as viruses, drugs, alcohol, and hypoxia. It is one of the common severe diseases in clinical practice. Its pathogenesis includes loss of liver detoxification function, massive death of hepatocytes in a short period of time, and uncontrollable inflammation. When it develops to the late stage, it manifests as acute liver failure with a high mortality rate. It has a huge impact on human health. Therefore, it is crucial to study drugs and methods for the prevention and treatment of liver diseases. Lipopolysaccharide is a common inflammatory stimulus that can cause endotoxin damage, produce inflammatory factors, and then lead to apoptosis and necrosis of liver cells; D-galactosamine is a sensitizer of lipopolysaccharide, which can competitively inhibit uridine nucleotides in the liver, inhibit the synthesis of mRNA and protein, and further aggravate liver damage; currently, lipopolysaccharide combined with D-galactosamine has been widely used in the induction of acute liver injury models and the screening of related therapeutic drugs. Traditional Chinese medicine has the characteristics of multiple components, multiple targets, and multiple pathways. Therefore, it is very important to find the material basis and mechanism of action for the treatment of acute liver injury from traditional Chinese medicine and develop effective therapeutic drugs.

[0003] Bupleurum chinense is the dried root of Gypsophila oldhamiana Mip and Gypsophila licentiana Hand-Hazz, both of which are plants of the Caryophyllaceae family. It is used for hepatitis, asthenia and bone steaming, yin deficiency and chronic malaria, and infantile malnutrition. Bupleurum chinense has a wide history of folk medicine use, but there are few reports on basic research on Bupleurum chinense in China. A large amount of Bupleurum chinense has been discarded because its medicinal and health value has not been fully utilized. Therefore, how to extract new compounds from Bupleurum chinense and develop its medicinal value is a technical problem that needs to be solved urgently. III. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide two new cyclic peptide compounds extracted from Bupleurum chinense and their extraction method and application, which can effectively solve the problems of extraction and application of Bupleurum chinense.

[0005] The technical solution provided by the present invention is that the chemical structural formulas of the two new cyclopeptide compounds are as follows:

[0006]

[0007] The extraction method of the two new cyclopeptide compounds comprises the following steps:

[0008] 1) The roots of the fine-leaved stone flower (with residual stems above the ground) are broken into coarse segments, and refluxed and extracted three times with 70%-80% ethanol, each time for 1-3 hours, with the amount of solvent being 6-10 times, and the extract is concentrated by rapid centrifugation to a volume of 40-60L, and concentrated under reduced pressure until there is no alcohol taste, and equal volumes of petroleum ether, ethyl acetate, and n-butanol are added in sequence to extract repeatedly until the color of the upper phase solution no longer becomes lighter, and after reduced pressure concentration, the extract is transferred to an evaporating dish and dried to obtain extracts of various parts;

[0009] 2) The ethyl acetate extract of the fine-leaved stone flower obtained in step 1) is purified by macroporous resin (DM-130), activated in 95% ethanol for 24 hours before use to wash away impurities in the filler, suspended in 95% ethanol, wet-loaded on a column, 1BV=20L, and then rinsed with 95% ethanol. When the washed ethanol is mixed with an equal amount of water and does not become turbid, the ethanol is then successively transferred from 95% ethanol to 10% ethanol, the ethyl acetate extract is dissolved in 10% ethanol, slowly added to the column bed, the lower end opening of the column is closed or reduced, and the sample is slowly adsorbed on the macroporous resin, and gradient elution is performed using 10%, 30%, 50%, 70%, and 95% ethanol solutions, each gradient washing four column volumes, and each gradient eluate is collected and concentrated under reduced pressure to obtain five fractions Fr1-Fr5;

[0010] 3) Purify the fraction Fr4 obtained in step 2) by ODS column chromatography: wet column packing, dry sample loading, and the peak times according to high performance liquid chromatography (HPLC) analysis are: 10min, 30min, 43min, and 60min, respectively; select 20%, 50%, 80%, and 100% methanol water for gradient elution, 1BV=500mL, and collect 1 / 2 column volume once, and obtain a total of 51 fractions, which are transferred to 1.5ml centrifuge tubes, placed on a evaporator to evaporate, and then spot plated for inspection under ultraviolet light, and merged to obtain 4 fractions Fr4-1 to Fr4-4;

[0011] 4) Purify the fraction Fr4-2 obtained in step 3) by silica gel column chromatography: wet column loading, dry loading, dual elution system of dichloromethane-methanol = 5:1, ethyl acetate-methanol = 10:1-1:1, 1BV = 467mL, collect 1 / 2 column volume once, and obtain a total of 43 fractions. The spot plate was inspected under ultraviolet light and combined to obtain 4 fractions Fr4-2-1 to Fr4-2-4;

[0012] 5) Purify the fraction Fr4-2-2 obtained in step 4), wet-pack the ODS column, dry-load the sample, and analyze it according to high performance liquid chromatography (HPLC). Select 10%, 21%, 45%, and 50% methanol-water gradient elution; 1BV = 228mL, collect 1 / 2 column volume once, and obtain 30 fractions in total. The plate is inspected under ultraviolet light to obtain 2 fractions Fr4-2-2-1 to Fr4-2-2-2;

[0013] 6) Purify the fraction Fr4-2-2-2 obtained in step 5), silica gel column chromatography: wet column loading, dry loading, dichloromethane-methanol = 4:1, ethyl acetate-methanol = 20:1-4:1 dual system elution, 1BV = 227mL, 1 / 3 column volume collected once, a total of 52 fractions were obtained, the spot plate was inspected under ultraviolet light, and 4 fractions Fr4-2-2-2-1 to Fr4-2-2-2-4 were combined;

[0014] 7) Purify the fraction Fr4-2-2-2-3 obtained in step 6) by ODS column chromatography: wet column packing, dry sample loading, and high performance liquid chromatography (HPLC) analysis, using 45% and 50% methanol-water gradient elution, 1BV = 246mL, 1 / 2 column volume was collected once, and a total of 30 fractions were obtained. The plate was inspected under ultraviolet light and three fractions Fr4-2-2-3-1 to Fr4-2-2-3-3 were combined;

[0015] 8) The fraction Fr4-2-2-3-1 obtained in step 7) was semi-preparatively purified (36%, isocratic elution with acetonitrile and water, flow rate 3 mL / min, dual channel wavelength: 210 nm, 254 nm) to obtain compound Sanguisorba officinalis cyclotide new A (CyA).

[0016] 9) The fraction Fr4-2-2-3-2 obtained in step 8) was semi-preparatively purified (50%, isocratic elution with methanol and water, flow rate 3 mL / min, dual channel wavelength: 210 nm, 254 nm) to obtain compound Sanguisorba officinalis cyclotide new B (CyB).

[0017] The invention discloses an application of two new cyclic peptide compounds extracted from Bupleurum chinense in the preparation of drugs for acute liver injury.

[0018] The present invention separates and identifies two new cyclic peptide compounds from Bupleurum chinense, namely, new cyclic peptide A from Bupleurum chinense and new cyclic peptide B from Bupleurum chinense. Experimental results show that the two compounds have a significant improvement effect on mice with acute liver injury, and can provide an experimental basis for the pharmacological research of Bupleurum chinense and the development of anti-acute liver injury drugs, which has important practical significance for fully exploring the effective ingredients of Bupleurum chinense. IV. Description of the drawings

[0019] Figure 1 The molecular structure diagram of the compound of the present invention, new shanyin bupleurum cyclic peptide A (CyA) and new shanyin bupleurum cyclic peptide B (CyB); Note: the left side of the figure is new shanyin bupleurum cyclic peptide A; the right side of the figure is new shanyin bupleurum cyclic peptide B.

[0020] Figure 2 This is the HMBC correlation diagram of the compound of the present invention, Sanyin Chaihu Cyclic Peptide New A.

[0021] Figure 3This is the 1HNMR spectrum of the compound Shanyin Chaihu Cyclic Peptide New A of the present invention.

[0022] Figure 4 The compound of the present invention is a new A of the shanyin bupleurum cyclic peptide 13 CNMR spectrum.

[0023] Figure 5 This is the DEPT spectrum of the compound Shanyin Chaihu cyclotide Xin A of the present invention.

[0024] Figure 6 This is the HSQC spectrum of the compound Sanyin Chaihu cyclotide new A of the present invention.

[0025] Figure 7 This is the HMBC spectrum of the compound Sanyin Chaihu cyclotide new A of the present invention.

[0026] Figure 8 This is the NOE spectrum of the compound Shanyin Chaihu cyclotide Xin A of the present invention.

[0027] Fig. 9 This is the HH COSY spectrum of the compound Sanyin Chaihu cyclotide new A of the present invention.

[0028] Fig.10 This is the IR spectrum of the compound Shanyin Chaihu Cyclic Peptide New A of the present invention.

[0029] Fig.11 This is the UV spectrum of the compound Shanyin Chaihu Cyclic Peptide New A of the present invention.

[0030] Fig.12 This is the mass spectrum of the compound Sanyin Chaihu cyclotide Xin A of the present invention.

[0031] Fig.13 This is the key HMBC correlation diagram of the compound Sanyin Chaihu cyclic peptide new B of the present invention.

[0032] Fig.14 The compound of the present invention is the new B of the shanyin bupleurum cyclic peptide 1 H-NMR spectrum.

[0033] Fig.15 This is the 13C-NMR spectrum of the compound Sanyin Chaihu Cyclic Peptide New B of the present invention.

[0034] Fig.16 This is the DEPT spectrum of the compound Sanyin Chaihu cyclotide new B of the present invention.

[0035] Fig.17 This is the HSQC spectrum of the compound of the present invention, Sanyin Bupleurum Cyclotide New B.

[0036] Fig.18 This is the HMBC spectrum of the compound of the present invention, Sanyin Bupleurum cyclotide new B.

[0037] Fig.19 This is the NOE spectrum of the compound Sanyin Chaihu cyclotide new B of the present invention.

[0038] Fig. 20 This is the HH COSY spectrum of the compound Sanyin Chaihu cyclotide new B of the present invention.

[0039] Fig.21 This is the IR spectrum of the compound Sanyin Chaihu cyclotide new B of the present invention.

[0040] Fig. 22 This is the UV spectrum of the compound Sanyin Chaihu cyclotide new B of the present invention.

[0041] Fig.23 This is the mass spectrum of the compound Sanyin Chaihu cyclotide new B of the present invention.

[0042] Fig.24 This is the effect of two cyclic peptide compounds of Bupleurum chinense on the liver coefficient of the present invention.

[0043] Fig.25 The invention discloses the effects of two cyclic peptide compounds of Bupleurum chinense on pathological sections of liver tissue.

[0044] Fig.26 This is the effect of two cyclic peptide compounds of Bupleurum chinense on liver function indicators of the present invention.

[0045] Fig. 27 This is the effect of two cyclic peptide compounds of Bupleurum chinense on F4 / 80 and Ly6G in liver tissue. V. Specific implementation methods

[0046] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples.

[0047] Example 1

[0048] In the specific implementation of the present invention, the extraction method of the two new cyclopeptide compounds comprises the following steps:

[0049] 1) 20 kg of the roots of the fine-leaved stone flower (with the residual stems above the ground) were broken into coarse segments, and refluxed and extracted with 70% ethanol for three times, each time for 2 hours, with the amount of solvent being 10, 8, and 6 times, respectively. The extract was concentrated to 50 L by rapid centrifugation, and concentrated under reduced pressure until there was no alcohol taste. An equal volume of petroleum ether, ethyl acetate, and n-butanol were added in sequence and extracted repeatedly until the color of the upper phase solution no longer became lighter. After concentrated under reduced pressure, the extract was transferred to an evaporating dish and dried to obtain extracts of various parts;

[0050] 2) 266.57 g of the ethyl acetate extract of the fine-leaved stone flower obtained in step 1) was purified by macroporous resin (DM-130), activated in 95% ethanol for 24 hours before use to wash away impurities in the filler, suspended in 95% ethanol, wet-loaded on a column, 1BV=20L, and then rinsed with 95% ethanol. When the washed ethanol was mixed with an equal amount of water and remained turbid, it was successively transitioned from 95% ethanol to 10% ethanol, the ethyl acetate extract was dissolved in 10% ethanol, slowly added to the column bed, the lower end opening of the column was closed or reduced, and the sample was slowly adsorbed on the macroporous resin, and gradient elution was performed using 10%, 30%, 50%, 70%, and 95% ethanol solutions, each gradient washing was about four column volumes, and each gradient eluate was collected and concentrated under reduced pressure to obtain five fractions Fr1-Fr5;

[0051] 3) The fraction Fr4 (25.12 g) obtained in step 2) was purified by ODS column chromatography: wet column loading, dry sample loading, and the peak times according to high performance liquid chromatography (HPLC) analysis were: 10 min, 30 min, 43 min, and 60 min, respectively; 20%, 50%, 80%, and 100% methanol-water gradient elution was selected, 1BV=500 mL, and 1 / 2 column volume was collected once, and a total of 51 fractions were obtained, which were transferred to 1.5 ml centrifuge tubes, placed on a evaporator to evaporate, and then the plate was inspected under ultraviolet light, and 4 fractions Fr4-1 to Fr4-4 were obtained by merging;

[0052] 4) Purify the fraction Fr4-2 (8.54 g) obtained in step 3) by silica gel column chromatography: wet column loading, dry loading, elution with a dual system of dichloromethane-methanol (5:1) and ethyl acetate-methanol (10:1-1:1), 1BV=467mL, collect 1 / 2 column volume once, and obtain a total of 43 fractions. The spot plate was inspected under ultraviolet light and combined to obtain 4 fractions Fr4-2-1 to Fr4-2-4;

[0053] 5) The fraction Fr4-2-2 (2.786 g) obtained in step 4) was purified, wet-packed on an ODS column, and dry-loaded. According to high performance liquid chromatography (HPLC), 10%, 21%, 45%, and 50% methanol-water gradient elution was selected; 1BV = 228 mL, 1 / 2 column volume was collected once, and a total of 30 fractions were obtained. The plate was inspected under ultraviolet light to obtain 2 fractions Fr4-2-2-1 to Fr4-2-2-2;

[0054] 6) The fraction Fr4-2-2-2 (2.648 g) obtained in step 5) was purified by silica gel column chromatography: wet column loading, dry loading, elution with a dual system of dichloromethane-methanol (4:1) and ethyl acetate-methanol (20:1-4:1), 1BV=227 mL, 1 / 3 column volume was collected once, and a total of 52 fractions were obtained. The spot plate was inspected under ultraviolet light and combined to obtain 4 fractions Fr4-2-2-2-1 to Fr4-2-2-2-4;

[0055] 7) The fraction Fr4-2-2-2-3 (845.2 mg) obtained in step 6) was purified by ODS column chromatography: wet column loading, dry loading, and analyzed by high performance liquid chromatography (HPLC). 45% and 50% methanol-water gradient elution was selected, 1BV = 246 mL, and 1 / 2 column volume was collected once to obtain a total of 30 fractions. The plate was inspected under ultraviolet light and combined to obtain 3 fractions Fr4-2-2-3-1 to Fr4-2-2-3-3;

[0056] 8) The fraction Fr4-2-2-3-1 (103 mg) obtained in step 7) was semi-preparatively purified (36%, isocratic elution with acetonitrile and water, flow rate 3 mL / min, dual channel wavelength: 210 nm, 254 nm) to obtain the compound Sanyin Bupleurum Cyclic Peptide New A (CyA) (15.6 mg, T = 23 min).

[0057] 9) The fraction Fr4-2-2-3-2 (410 mg) obtained in step 8) was semi-preparatively purified (50%, isocratic elution with methanol and water, flow rate 3 mL / min, dual channel wavelength: 210 nm, 254 nm) to obtain compound Sanyin Bupleurum Cyclotide New B (CyB) (15.6 mg, T = 26 min).

[0058] The present invention separates and identifies two new cyclopeptide compounds from Bupleurum chinense, namely, new cyclopeptide A from Bupleurum chinense and new cyclopeptide B from Bupleurum chinense, and conducts activity experiments on the two compounds. The experimental results show that the two compounds have a significant improvement effect on acute liver injury in mice. The relevant specific determination and experimental data are as follows: 1. Instruments, test drugs and animals

[0059] Nuclear magnetic resonance was performed using a Bruker AVANCEⅢ500 NMR instrument (TMS internal standard) (Bruker), infrared spectroscopy was performed using a Nicolet is 10 Microscope Spectrometer (Thermo Scientific, USA), ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry was performed using an Agilent 6546 UPLC-Q-TOF-MS system (Agilent, USA), and ultraviolet spectroscopy was performed using a Shimadzu UV-2401PC apparatus, FDU-2110 freeze dryer (Shanghai Ailang Instrument Co., Ltd.), 3020 multifunctional microplate reader (Thermo Fisher Scientific Inc.), AE2000 inverted microscope (Motic Industrial Group Co., Ltd.), superconducting nuclear magnetic resonance instrument (Bruker, Switzerland, DPX-500), semi-preparative high performance liquid chromatography (Beijing Cypress Technology Co., Ltd., LC-52), fully preparative high performance liquid chromatography (Shanghai Wufeng Science Co., Ltd., LC100), rotary evaporator (EYELA Tokyo Rika, Japan, N-1000, BSA124S-CW), condensed water circulation device (EYELA Tokyo Rika, Japan, N-1111), Agilent high performance liquid chromatograph (Agilent Technologies, USA, Agilent 1260Infinity II).

[0060] Alanine aminotransferase (ALT) (Cat. No.: C009-2-1), aspartate aminotransferase (AST) (Cat. No.: C010-2-1), lactate dehydrogenase (LDH) (Cat. No.: A020-2-2) were purchased from Nanjing Jiancheng Bioengineering Research Institute, D-galactosamine hydrochloride (Cat. No.: G115554) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and lipopolysaccharide (Batch No.: 0000081275) was purchased from SIGMA. Silybin capsules (approval number: National Medicine Standard H20040299) were purchased from Tianjin Tasly Saint Pharmaceutical Co., Ltd. Shanyin Bupleurum was collected in Tianshui County, Gansu Province in August 2022, and was identified as the underground root of Gypsophila licentiana Hand.-Mazz. by Professor Chen Suiqing of Henan University of Traditional Chinese Medicine, and was preserved in the Key Laboratory of Traditional Chinese Medicine Resources and Traditional Chinese Medicine Chemistry of Henan Province.

[0061] 40 male BALB / c mice, SPF grade, weight 18-22 g, purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd., license number SCXK (Zhe) 2024-0001. The animal husbandry and experimental operations conducted in this study were reviewed by the Experimental Animal Ethics Committee of Henan University of Traditional Chinese Medicine, in accordance with the principles of animal protection, welfare and ethics, and the ethics review approval number was IACUC-202402006. The animals were housed in the Animal Experiment Center of Henan University of Traditional Chinese Medicine, with 12 h of light, constant temperature of 24 ° C, free food and water.

[0062] 2 Structure identification

[0063] 2.1 New A of Sanyin Bupleurum Cyclotide: light yellow crystals, soluble in methanol. ESI-MS m / z: 670.3928 [M+H] + , the molecular formula may be: C 34 H 51 O7N7. In the IR spectrum, 1525 and 1640 are carbonyl infrared signals, and 3312.17 is the secondary amine infrared signal. Observing its full spectrum, it is found that it has the characteristics of cyclic peptide compounds: 8 α hydrogen proton signals (4.69, 4.65, 4.50, 4.19, 4.10, 4.03, 3.90, 3.50); ​​7 α carbon signals (62.85, 60.74, 58.83, 56.32, 50.94, 49.69, 44.73); 7 carbonyl carbon signals (175.74, 174.33, 173.37, 173.07, 172.72, 172.40, 171.54); from the DEPT spectrum, it is found that the above 7 α carbon signals except δ C :44.73 is the methylene hydrogen signal, and the rest are methine carbon signals. Combined with HSQC, it is found that they are related to δ H : 3.90, 3.50 correlation, it is speculated that the compound may be a seven-membered cyclic peptide; 1 In H-NMR (500MHz, CD3OD), there is a group of hydrogen signals in the aromatic region, δ H : 7.26 (1H, m) is the hydrogen signal of the monosubstituted benzene ring; 13 C-NMR (125MHz, CD3OD) showed δ C : The monosubstituted benzene carbon signals at 137.99, 130.24, 129.68, and 128.06 were speculated to be a substituent on the cyclic peptide; after consulting the literature, it was found that the compound was similar in structure to Cyclo-(Phe-Asn-Ala-Met-Ala-Ile-Pro), but the substituents were different.

[0064] In summary, the new A structure of Sanyin Bupleurum cyclotide is Cyclo-(Phe-Ala-Ile-Ile-Ala-Gly-Pro), which has not been reported before and is determined to be a new compound. 13 C-NMR, 1 The assignments of H-NMR and HMBC related peaks are shown in Table 1.

[0065] Table 1 NMR data attribution table of the compound of the present invention, Sanyin Bupleurum cyclotide new A

[0066]

[0067]

[0068] 2.2 Sanyin Bupleurum Cyclotide New B: light yellow crystals, soluble in methanol. ESI-MS m / z: 655.3426 [M+Na] + , the molecular formula may be: C 31 H 48 O8N6. In the IR spectrum, 1539.84-1675.27 are infrared signals of carbonyl groups; 3313.65-3425.34 are infrared signals of secondary amines. It is inferred that the compound contains nitrogen atoms. Observation of its full spectrum shows that it has the characteristics of cyclic peptide compounds: 7 α hydrogen proton signals (4.76, 4.57, 4.45, 4.43, 4.36, 4.34, 3.46); 6 α carbon signals (61.67, 60.89, 59.12, 57.84, 56.65, 43.64); 6 carbonyl carbon signals (174.46, 174.18, 172.93, 173.45, 172.29, 171.45); from the DEPT spectrum, it is found that the above 6 α carbon signals except δ C :43.64 is the methylene hydrogen signal, and the rest are methine carbon signals. Combined with HH COSY, it is found that δ H : 4.36 is related to 3.46, suggesting that the compound may be a six-membered cyclic peptide; 1 In H-NMR (500MHz, CD3OD), there is a group of hydrogen signals in the aromatic region, δ H : 7.30 (5H, m) is the hydrogen signal of the monosubstituted benzene ring; 13 C-NMR (125 MHz, CD3OD) C:128.15, 129.79, 129.95, 137.99 are monosubstituted benzene carbon signals. From the DEPT spectrum, we can see that δC: 37.56 is a methylene carbon signal. In the HSQC spectrum, it is correlated with 3.19 (1H, m) and 3.11 (1H, m). In the HMBC spectrum, it is BC-correlated with 59.12 (α carbon), 174.18, 172.93 (carbonyl carbon signals) and benzene ring carbon signals. It is speculated that this partial structure is phenylalanine (Phe); in the HSQC spectrum, 57.84 is correlated with 4.76 (1H, d, J=3.0HZ), and it is correlated with 172.93, 173.45 (carbonyl carbon signals), 70.42 (-CH2-carbon signal), 20.21 (-CH3 carbon signal). BC correlation, it is speculated that the partial structure is threonine (Thr); in the HSQC spectrum, 61.67 is correlated with 4.43 (1H, d, J = 5.3HZ), which is correlated with 173.45, 171.45 (carbonyl carbon signal), 39.49 (-CH), 25.91 (-CH2-), and 15.80 (-CH3), and it is speculated that the partial structure is isoleucine (Ile); in the HSQC spectrum, 56.65 is correlated with 4.57 (1H, m), which is correlated with 171.45, 174.46 (carbonyl carbon signal), 37.67 (-CH), 25.91 (-CH2-), and 15.97 (-CH3), and it is speculated that the partial structure is isoleucine (Ile); δ C : 43.64 is BC-correlated with 174.46 and 172.29 (carbonyl carbon signals), and it is speculated that this partial structure is glycine (Gly); the remaining carbon signals are threonine (Thr).

[0069] In summary, the new B structure of Sanyin Bupleurum cyclotide is Cyclo-(Phe-Thr-Ile-Ile-Gly-Thr), which has not been reported before and is determined to be a new compound. 13 C-NMR, 1 The H-NMR and HMBC related peak assignments are shown in Table 2.

[0070] Table 2 NMR signal assignments of the new cyclotide B of Bupleurum chinense

[0071]

[0072]

[0073] 3 In vivo experimental methods

[0074] 3.1 Animal experiments

[0075] After 7 days of adaptive feeding, 40 male BALB / c mice were randomly divided into a normal group (CON), a model group (M), a positive drug silybin group (Y, 48 mg / kg), a Sanyin Chaihu Cyclic Peptide New A group (CyA, 10 mg / kg) and a Sanyin Chaihu Cyclic Peptide New B group (CyB, 10 mg / kg), 8 mice in each group, and the drugs were administered orally for 7 consecutive days. One hour after the drug administration on the 7th day, except for the normal group, which was intraperitoneally injected with the same dose of normal saline, the other four groups were intraperitoneally injected with 10 μg / kg lipopolysaccharide and 700 mg / kg D-galactosamine for modeling. After 6 hours of modeling, the samples were collected for analysis and stored at -80℃ for future use.

[0076] 3.2 Detection of mouse liver index

[0077] After modeling, each mouse was weighed, blood was collected from the eyeball, and liver tissue was removed to calculate the liver index, which was liver index = liver weight (g) / body weight (g) × 100%.

[0078] Test results such as Fig.24 As shown in the figure, CyA and CyB can significantly reduce the liver coefficient (P<0.01). Note: Compared with the CON group, ## P<0.01, compared with group M, * P<0.01, ** P<0.01.

[0079] 3.3 Observation of pathological morphological changes of mouse liver tissue by HE staining

[0080] The blood stains on the surface of the liver tissue were washed off with physiological saline, and the same part of the liver tissue of each group of mice was taken and immersed in 4% paraformaldehyde fixative for 24 h, dehydrated with gradient ethanol, transparentized with xylene, embedded in paraffin, cut into 5 μm thin slices, dewaxed with xylene, eluted with gradient ethanol, and HE staining was performed.

[0081] The test results are as follows Fig.25 As shown, the liver tissue of mice in the CON group was clearly stained and had normal structure; inflammatory cell infiltration was observed in mice in the M group, and the liver tissue structure of mice in the CyA and CyB groups was significantly improved.

[0082] 3.4 Microplate assay for changes in mouse liver function

[0083] After blood was collected from the mouse eyeball, it was placed in a 4°C refrigerator for 2 hours and centrifuged at 3000 r / min for 10 minutes. The supernatant was taken to determine the levels of ALT and AST. The operating steps were carried out according to the instructions of the kit.

[0084] The test results are as follows Fig.26 As shown in the figure, compared with the CON group, the AST and ALT levels in the M group were significantly increased (P<0.01); compared with the M group, the ALT and AST levels in the CyA and CyB groups were significantly decreased (P<0.01).## P<0.01, compared with group M, * P<0.01, ** P<0.01.

[0085] 3.5 Immunohistochemical determination of F4 / 80 and Ly6G content in mouse liver tissue

[0086] After liver tissue is dehydrated, embedded, sliced, then dewaxed and rehydrated, the slices are immersed in EDTA repair solution, microwave repair, repair for 20 minutes, cooled, washed 3 times with PBS, 5 minutes each time, slices are placed in 3% hydrogen peroxide solution, incubated at room temperature in the dark for 25 minutes, the slides are placed in PBS (PH7.4) and washed 3 times on a decolorizing shaker, 5 minutes each time, blocked with bovine serum albumin, room temperature for 1 hour; gently shake off the blocking solution, add PBS to the slices according to a certain ratio of primary antibodies (F4 / 80, Ly6G), and lay the slices flat in a wet box and incubate overnight at 4°C. Wash 3 times with PBS, 5 minutes each time; after the slices are slightly dried, add the secondary antibody (HRP labeled) of the corresponding species to the primary antibody to cover the tissue, and incubate at room temperature for 50 minutes. Prepare fresh DAB color developing solution, add it to the tissue, color at room temperature, control the color development time under the microscope, positive is brown-yellow, wash the slices with distilled water to stop color development; counterstain with hematoxylin for 3 minutes, wash with tap water, differentiate with clear water to turn blue, and then rinse with running water; soak the slices in 75%, 85%, 95%, anhydrous ethanol, and xylene for 10 minutes respectively, and seal with neutral gum. Collect slice images, measure the absorbance of all collected images, and analyze the expression of F4 / 80 and Ly6G.

[0087] The test results are as follows Fig. 27 As shown in the figure, compared with the CON group, the levels of F4 / 80 and Ly6G in the M group were significantly increased (P<0.01); compared with the M group, the levels of F4 / 80 and Ly6G in the CyA and CyB groups were significantly decreased (P<0.01). ## P<0.01, compared with group M, * P<0.01, ** P<0.01.

[0088] 4 Conclusion

[0089] In summary, the present invention has abundant raw materials and the extraction method is easy to operate. Two new cyclic peptide compounds, namely, new cyclic peptide A of Bupleurum chinense and new cyclic peptide B of Bupleurum chinense, are separated and identified from Bupleurum chinense. The results of relevant in vivo activity experiments show that the two compounds have obvious improvement effects on mice with acute liver injury, and can provide an experimental basis for the pharmacological research of Bupleurum chinense and the development of anti-acute liver injury drugs. It has important practical significance for fully exploring the effective ingredients of Bupleurum chinense, expands the medicinal value of Bupleurum chinense, has huge development and application prospects, and has huge economic and social benefits.

[0090] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. Two new cyclic peptide compounds extracted from Bupleurum chinense, characterized in that: They are respectively Shanyin Bupleurum Cyclic Peptide New A and Shanyin Bupleurum Cyclic Peptide New B, and the chemical structural formulas are as follows:

2. The method for extracting two new cyclic peptide compounds from Bupleurum chinense according to claim 1, characterized in that: The following steps are involved: 1) The roots of the fine-leaved stone flower are broken into coarse segments, and refluxed and extracted with 70%-80% ethanol for three times, each time for 1-3 hours, with the amount of solvent being 6-10 times, and the volume of the extract is concentrated by rapid centrifugation to 40-60L, and concentrated under reduced pressure until there is no alcohol taste, and equal volumes of petroleum ether, ethyl acetate, and n-butanol are added in sequence and extracted repeatedly until the color of the upper phase solution no longer becomes lighter, and after reduced pressure concentration, the extract is transferred to an evaporating dish and dried to obtain extracts of various parts; 2) The ethyl acetate extract of the fine-leaved stone flower obtained in step 1) is purified by macroporous resin DM-130, activated in 95% ethanol for 24 hours before use to wash away impurities in the filler, suspended in 95% ethanol, wet-loaded into a column, 1BV=20L, and then rinsed with 95% ethanol. When the washed ethanol is mixed with an equal amount of water and does not become turbid, it is then successively transitioned from 95% ethanol to 10% ethanol, the ethyl acetate extract is dissolved in 10% ethanol, slowly added to the column bed, the lower end opening of the column is closed or reduced, and the sample is slowly adsorbed on the macroporous resin, and gradient elution is performed using 10%, 30%, 50%, 70%, and 95% ethanol solutions, each gradient washing four column volumes, and each gradient eluate is collected and concentrated under reduced pressure to obtain five fractions Fr1-Fr5; 3) Purify the fraction Fr4 obtained in step 2) by ODS column chromatography: wet column packing, dry sample loading, and the peak times according to HPLC analysis are: 10min, 30min, 43min, and 60min, respectively; select 20%, 50%, 80%, and 100% methanol water for gradient elution, 1BV=500mL, and collect 1 / 2 column volume once, to obtain a total of 51 fractions, which are transferred to 1.5ml centrifuge tubes, placed on a evaporator to evaporate, and then spot-plate inspected under ultraviolet light, and four fractions Fr4-1 to Fr4-4 are obtained by merging; 4) Purify the fraction Fr4-2 obtained in step 3) by silica gel column chromatography: wet column loading, dry loading, dual elution system of dichloromethane-methanol = 5:1, ethyl acetate-methanol = 10:1-1:1, 1BV = 467mL, collect 1 / 2 column volume once, and obtain a total of 43 fractions. The spot plate was inspected under ultraviolet light and combined to obtain 4 fractions Fr4-2-1 to Fr4-2-4; 5) Purify the fraction Fr4-2-2 obtained in step 4), wet-pack the ODS column, dry-load the sample, and analyze by HPLC, using 10%, 21%, 45%, and 50% methanol-water gradient elution; 1BV = 228 mL, 1 / 2 column volume was collected once, and a total of 30 fractions were obtained. The plate was inspected under ultraviolet light to obtain 2 fractions Fr4-2-2-1 to Fr4-2-2-2; 6) Purify the fraction Fr4-2-2-2 obtained in step 5), silica gel column chromatography: wet column loading, dry loading, dichloromethane-methanol = 4:1, ethyl acetate-methanol = 20:1-4:1 dual system elution, 1BV = 227mL, 1 / 3 column volume collected once, a total of 52 fractions were obtained, the spot plate was inspected under ultraviolet light, and 4 fractions Fr4-2-2-2-1 to Fr4-2-2-2-4 were combined; 7) Purify the fraction Fr4-2-2-2-3 obtained in step 6) by ODS column chromatography: wet column packing, dry sample loading, and HPLC analysis, using 45% and 50% methanol-water gradient elution, 1BV=246mL, 1 / 2 column volume was collected once, and a total of 30 fractions were obtained. The plate was inspected under ultraviolet light and three fractions Fr4-2-2-3-1 to Fr4-2-2-3-3 were combined; 8) The fraction Fr4-2-2-3-1 obtained in step 7) was semi-preparatively purified, isocratically eluted with 36% acetonitrile water, a flow rate of 3 mL / min, and dual-channel wavelengths of 210 nm and 254 nm to obtain compound Sanguisorba officinalis cyclotide new A. 9) The fraction Fr4-2-2-3-2 obtained in step 8) was semi-preparatively purified, isocratically eluted with 50% methanol water, a flow rate of 3 mL / min, and dual-channel wavelengths of 210 nm and 254 nm to obtain compound Sanguisorba officinalis cyclotide new B.

3. Use of the two new cyclic peptide compounds extracted from Bupleurum chinense as claimed in claim 1 in the preparation of drugs for acute liver injury.

4. Use of two new cyclic peptide compounds prepared by the extraction method of two new cyclic peptide compounds extracted from Bupleurum chinense as claimed in claim 2 in the preparation of acute liver injury drugs.

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