Epimedium extract as well as preparation method and application thereof

Abstract

The invention discloses a herba epimedii extract as well as a preparation method and application thereof. The herba epimedii extract is selected from any one or a combination of more of the following structures: # imgabs0. The herba epimedii extract disclosed by the invention has relatively good activity in resisting sarcopenia and has a good application prospect in the aspect of treating diabetic sarcopenia.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an epimedium extract, a preparation method of the epimedium extract and application of the epimedium extract in preparing a medicament for preventing or treating diabetic sarcopenia. Background Art

[0002] Sarcopenia, also known as sarcopenia, is a progressive, systemic skeletal muscle disease characterized by loss of muscle mass and function. With the deepening of research on sarcopenia, it has been found that sarcopenia is divided into two types: primary and secondary, and it is more common in two groups: the elderly without underlying diseases, which can be called elderly sarcopenia; and the group with underlying diseases such as chronic, wasting diseases, such as diabetes, neurodegenerative diseases, cancer, etc. Such underlying diseases are likely to induce the loss of muscle mass and function in patients, thereby causing secondary sarcopenia.

[0003] Diabetes is a chronic metabolic disease. The diabetes rate is increasing year by year and tends to be younger. In recent years, with the in-depth study of diabetes, sarcopenia caused by diabetes has gradually received attention. Although sarcopenia is mostly related to age and occurs in the elderly, as diabetes tends to young people, diabetic sarcopenia has gradually appeared in the young group. With the in-depth study of diabetic sarcopenia, it is found that diabetes and secondary sarcopenia have a strong correlation. The results of community surveys show that the prevalence of sarcopenia in elderly people with diabetes is much higher than that of non-diabetic patients, and their symptoms are more severe, such as muscle mass index (limb skeletal muscle mass / body fat mass index) and muscle strength are relatively lower. Diabetes and sarcopenia promote each other's development. Most studies currently support that insulin resistance (IR) plays an important role in its mechanism. IR is a state in which the insulin-targeted tissue has a reduced responsiveness to the physiological level of insulin, which may be an important mechanism for inducing sarcopenia. The reduction in muscle mass is caused by an imbalance between protein synthesis and decomposition rates. As an anabolic hormone, insulin can stimulate protein synthesis by absorbing amino acids into muscle tissue. However, the reduction of insulin signaling in patients with type 2 diabetes will hinder muscle protein synthesis and promote protein degradation, which will lead to a decrease in muscle mass and ultimately sarcopenia. Similarly, sarcopenia can also cause insulin resistance. The main target organ of insulin is skeletal muscle, which plays a major role in the uptake and metabolism of glucose. Muscle mass begins to gradually decline around the age of 30, and the loss accelerates after the age of 60. This age-related muscle change will reduce insulin sensitivity, induce glucose regulation abnormalities, lead to the occurrence of IR, and promote the development of type 2 diabetes.

[0004] Application No. 202410890766.5 discloses the use of a flavonoid derivative for preventing and treating sarcopenia. Icariin, epimedin A, epimedin A1, epimedin B, epimedin C, icariin I, and icariin II were screened out and it was found that icariin, epimedin A, epimedin A1, and epimedin C can increase the muscle strength and muscle content of SAMP8 sarcopenia model mice, reduce body weight, body fat and BMC content, and delay the aging process. Application No. 202311695764.2 discloses the use of an epimedium extract for preventing and treating sarcopenia, wherein the epimedium extract includes extract I, extract II, extract III, extract IV, extract V and extract VI. The results show that extract V can increase the muscle strength and muscle content of SAMP8 sarcopenia model mice, reduce body weight, body fat and BMC content, delay the aging process, and has no obvious effect on food intake.

[0005] The above patent documents all involve the use of epimedium extracts in the treatment of sarcopenia in the elderly. In view of the difference in the pathogenesis of sarcopenia in the elderly and diabetic sarcopenia, there is no clinical data to prove that the epimedium extracts obtained above can be used in the treatment of diabetic sarcopenia. This application aims to study drugs for preventing and / or treating diabetic sarcopenia. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention provides an epimedium extract, which can be used to prevent and / or treat diabetic sarcopenia; to this end, the present invention also provides a preparation method of the epimedium extract and the use of the epimedium extract in a drug for preventing and / or treating diabetic sarcopenia.

[0007] In a first aspect of the present invention, there is provided an application of an epimedium extract or a composition containing the epimedium extract in the preparation of a medicament for preventing and / or treating diabetic sarcopenia, wherein the epimedium extract is selected from any one or more combinations of the following structures:

[0008]

[0009]

[0010] As a preferred embodiment, the diabetic sarcopenia is sarcopenia caused by type 2 diabetes, and the symptoms are chronic muscle atrophy and gradual apoptosis of muscle cells.

[0011] As a preferred embodiment, the composition further contains pharmaceutically acceptable excipients.

[0012] The second aspect of the present invention provides a pharmaceutical composition for preventing and / or treating diabetic sarcopenia, wherein the pharmaceutical composition comprises an epimedium extract as a main active ingredient, and the epimedium extract is selected from any one or more combinations of the following structures:

[0013]

[0014]

[0015] The third aspect of the present invention provides a method for preparing an epimedium extract, comprising the following steps:

[0016] S1. Take Epimedium leaves, process them, and extract them with ethanol to obtain a thick paste;

[0017] S2, adsorbing the thick paste in S1 with a macroporous resin column, eluting and concentrating with alcohol or acetone at volume concentrations of 20%, 40%, 60%, 80% and 95% in sequence, to obtain the corresponding first extract, second extract, third extract, fourth extract and fifth extract;

[0018] S3, separating the second extract by normal solid phase extraction, eluting with chloroalkane: polar solvent in a volume ratio of 50:1, 25:1, 15:1, 5:1, and 0:1, and detecting by thin layer chromatography to obtain a first fraction, a second fraction, a third fraction, a fourth fraction, a fifth fraction, a sixth fraction, a seventh fraction, and an eighth fraction, wherein the chloroalkane is dichloromethane or chloroform, and the polar solvent is methanol, acetone, or ethyl acetate;

[0019] S4, separating the second fraction by gel column chromatography or reversed phase solid phase extraction, using methanol as the mobile phase to elute, to obtain compound 1;

[0020] S5. Separate the first extract by normal solid phase extraction, elute with dichloromethane:methanol in a volume ratio of 40:1, 25:1, 15:1, 5:1, and 0:1, respectively, and detect by thin layer chromatography to obtain a first separated liquid, a second separated liquid, a third separated liquid, a fourth separated liquid, a fifth separated liquid, a sixth separated liquid, a seventh separated liquid, an eighth separated liquid, and a ninth separated liquid. Separate the ninth separated liquid by high performance liquid chromatography to obtain compound 2;

[0021] S6, separating the seventh fraction by reverse solid phase extraction, and gradient eluting with alcohol or acetone and water in a volume ratio of 60:40, 65:35, 81:19, and 86:14, to obtain a first eluent, a second eluent, a third eluent, and a fourth eluent;

[0022] S7, separating the second eluent by gel column chromatography, reversed phase solid phase extraction or high performance liquid chromatography, using 20% ​​to 40% by volume acetonitrile-water as a mobile phase for elution, to obtain a first solution, a second solution, a third solution and a fourth solution;

[0023] S8, separating the second solution by gel column chromatography, reversed phase solid phase extraction or high performance liquid chromatography, and eluting with 19% to 23% by volume acetonitrile-water as a mobile phase to obtain compound 3;

[0024] S9. Separate the second extract by normal solid phase extraction, and elute with dichloromethane:methanol in a volume ratio of 40:1 to obtain compound 4, wherein compound 1, compound 2, compound 3, and compound 4 are:

[0025]

[0026] As a preferred embodiment, in step S1, after the epimedium leaves are processed, 50% ethanol by volume is added, and the mixture is extracted and concentrated under reflux conditions to obtain a thick paste.

[0027] As a preferred embodiment, the macroporous resin column in step S2 is a LSA-12S macroporous resin column, and the alcohol is methanol or ethanol.

[0028] As a preferred embodiment, the silica gel used in the normal solid phase extraction and the reverse solid phase extraction is spherical silica gel or amorphous silica gel, and the mesh number of the silica gel is 200-300 meshes.

[0029] As a preferred embodiment, the mobile phase used for HPLC separation in step S5 is methanol with a volume concentration of 60%.

[0030] As a preferred embodiment, the alcohol in step S6 is methanol or ethanol.

[0031] As a preferred embodiment, the elution flow rate in step S7 is 19 ml / min and the elution time is 20 min.

[0032] As a preferred embodiment, the elution flow rate in step S8 is 19 ml / min and the elution time is 20 min.

[0033] The fourth aspect of the present invention provides an epimedium extract, the structural formula of which is as follows:

[0034]

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) 5-hydroxy-2R-(4-hydroxyphenyl)-8-methoxy-4-oxobenzopyran-7-carboxylic acid, 5,4′,8′-trihydroxy-3,3′-dimethoxy-9-acetoxy-7,9′-epoxylignan, 3-(α-L-rhamnosylpyranoxy)-3′,5-dihydroxy-4′-methoxy-7-(β-D-pyranose glucoseoxy)-8-(3,3-dimethylallyl)-one and chohodine B in the epimedium extract of the present invention all have protective effects on serum-starved C2C12 myoblasts. 50 The concentrations of 4.49μM, 4.70μM, 3.54μM and 1.49μM, respectively, showed protective effects on C2C12 cells injured by dexamethasone. 50 They are 5.54μM, 5.64μM, 4.30μM and 3.34μM respectively.

[0037] (2) The epimedium extract of the present invention can improve the muscle strength of diabetic sarcopenia model mice, reduce the body fat content of diabetic sarcopenia model mice, and increase their muscle content, and has good application prospects in the treatment of diabetic sarcopenia.

[0038] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the HR-ESI-MS spectrum of compound 1 of the present invention;

[0040] Figure 2 is the IR spectrum of compound 1 of the present invention;

[0041] Figure 3 The compound 1 of the present invention 1 H-NMR spectrum;

[0042] Figure 4 The compound 1 of the present invention 13 C-NMR spectrum;

[0043] Figure 5 is the HMQC spectrum of compound 1 of the present invention;

[0044] Figure 6 is the HMBC spectrum of compound 1 of the present invention;

[0045] Figure 7 is the NOESY spectrum of compound 1 of the present invention;

[0046] Figure 8 is the HR-ESI-MS spectrum of compound 2 of the present invention;

[0047] Fig. 9 The compound 2 of the present invention 1 H-NMR spectrum;

[0048] Fig.10 The compound 2 of the present invention 13 C-NMR spectrum;

[0049] Fig.11 is the HMQC spectrum of compound 2 of the present invention;

[0050] Fig.12 is the HMBC spectrum of compound 2 of the present invention;

[0051] Fig.13 is the NOESY spectrum of compound 2 of the present invention;

[0052] Fig.14 is the compound 3 of the present invention 1 H-NMR spectrum;

[0053] Fig.15 is the compound 3 of the present invention 13 C-NMR spectrum;

[0054] Fig.16 This is a graph showing the EC50 effects of compounds 1, 2, 3, and 4 of the present invention on starvation-induced sarcopenia cells;

[0055] Fig.17 This is a graph showing the EC50 effects of compounds 1, 2, 3 and 4 of the present invention on dexamethasone-induced sarcopenia cells. DETAILED DESCRIPTION

[0056] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0057] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0058] Example 1 Preparation of Compound 1

[0059] S11. Take 25 kg of dried leaves of Epimedium sagittatum Maxim., add 5 kg of sheep oil, and stir-fry the sheep oil according to the processing method of the 2015 edition of the Chinese Pharmacopoeia to obtain a processed product. After drying, grind the processed product into powder, reflux with 50% ethanol at 83°C, extract for 1.5 hours, extract twice to obtain an extract, and concentrate under reduced pressure to obtain 5 kg of total fluid extract;

[0060] S12, the total fluid extract obtained in step S11 is adsorbed by a LSA-12S macroporous resin column, and eluted with solvents with volume concentrations of 20% (10-20mL), 40% (10-20mL), 60% (10-20mL), 80% (10-20mL) and 95% (10-20mL) in sequence, and the eluent is concentrated to obtain a first extract (200g), a second extract (236.5g), a third extract (305g), a fourth extract (105g) part and a fifth extract (175g), wherein the solvent is ethanol, methanol or acetone, and the volume concentration is the volume percentage of ethanol, methanol or acetone dispersed in water, such as ethanol with a volume concentration of 20%, which refers to the volume percentage of ethanol in the ethanol aqueous solution;

[0061] S13, separating the second extract obtained in step S12 by normal solid phase extraction, eluting with chloroalkane:polar solvent in a volume ratio of 50:1, 25:1, 15:1, 5:1, and 0:1, and detecting by thin layer chromatography to obtain a first fraction, a second fraction, a third fraction, a fourth fraction, a fifth fraction, a sixth fraction, a seventh fraction, and an eighth fraction, wherein the chloroalkane is dichloromethane or chloroform, the polar solvent is methanol, acetone, or ethyl acetate, the adsorbent used in the normal solid phase extraction separation is silica gel, the mesh number of the silica gel is 200-300 meshes, the silica gel is spherical silica gel or amorphous silica gel, and the normal solid phase extraction separation time is 30-40 min;

[0062] S14. The second fraction (0.8 g) was separated by gel column chromatography or reverse solid phase extraction with methanol as the mobile phase, and the eluate was collected and concentrated to obtain compound 1 (14 mg, purity was 98% as determined by HPLC), wherein the adsorbent used for reverse solid phase extraction was silica gel, and the particle size of the silica gel was 60 μm or 40 μm.

[0063] By HR-ESI-MS (high resolution mass spectrometry), infrared spectroscopy, 1 HNMR, 13 C NMR, HMQC, HMBC, and NOESY were used to identify the structure of compound 1. The obtained spectra are shown in Figure 1-7 shown.

[0064] Figure 1 The HR-ESI-MS spectrum of compound 1 shows that the quasi-molecular ion peak m / z of compound 1 is 329.0673 [MH] - , C 17 H 12 The calculated value of O7 is 329.0667, combined with 1 HNMR and 13 The molecular formula is inferred from C NMR data to be C 17 H 12 O7, unsaturation is 11.

[0065] Figure 2 The infrared spectrum of compound 1 shows that compound 1 has a -1 (Carboxyl), 1734cm -1 (carbonyl) and 1458cm -1 (benzene ring) shows an absorption band.

[0066] Figure 3 For compound 1 1 HNMR spectrum, 1 HNMR spectra show that in δ H There is a group of A2B2 coupling systems at 7.21 (2H, d, J = 8.6 Hz, H-2' / H-6'), 6.72 (2H, d, J = 6.5 Hz, H-3' / H-5'), which are speculated to be aromatic proton signals on the B ring; δH 5.78-5.80 (1H, m, H-6) is speculated to be an aromatic hydrogen on the A ring; δ H 3.01 (1H, dd, J = 17.1, 13.0 Hz, H-3α) and 2.59 (H, dd, J = 17.1, 3.0 Hz, H-3β) are speculated to be the two methylene proton signals of the C ring. H 3.84 (3H, s, OCH3-8) is speculated to be a methoxy signal.

[0067] Figure 4 For compound 1 13 C NMR spectrum, Figure 6 is the HMBC spectrum of compound 1, 13 C NMR spectrum shows 17 carbon signals, δ C 197.8 (C-4) is speculated to be a keto group. C 57.0 is the methoxy signal, combined with the δ H 3.84(3H,s,OCH3-8) and C-8(δ C 149.6), it is inferred that the methoxy group is located at the C-8 position. Based on the degree of unsaturation and carbon spectrum, it is inferred that there is a carboxyl carbon δ C168.4, H-6(δ H 5.78-5.80) and δ C-6 (96.2),δ C-7 (97.1),δ C 168.4 (carboxyl), δ C-5 (165.9), it is inferred that the carboxyl group is located at C-7 and C-5 is connected to the hydroxyl group.

[0068] The optical rotation value of compound 1 was measured by polarimeter. The absolute configuration of C-2 was determined to be S configuration.

[0069] Compound 1 1 HNMR (600MHz, CD3OD), 13 C NMR (151 MHz, CD3OD) and HMBC NMR data are shown in Table 1.

[0070] Table 1

[0071]

[0072]

[0073] By analyzing the HMBC spectrum of compound 1, the substituent positions of compound 1 are obtained as follows:

[0074]

[0075] In summary, the structural formula of compound 1 is deduced as follows:

[0076]

[0077] Example 2 Preparation of Compound 2

[0078] S21. The first extract obtained in step S12 of Example 1 is separated by normal phase solid phase extraction, wherein silica gel is selected as the adsorbent, the mesh size is 200-300 mesh, the amount of silica gel used is about 1800 g, and the extract is eluted with dichloromethane:methanol in a volume ratio of 40:1, 25:1, 15:1, 5:1, and 0:1, respectively, and detected by thin layer chromatography to obtain a first separated liquid, a second separated liquid, a third separated liquid, a fourth separated liquid, a fifth separated liquid, a sixth separated liquid, a seventh separated liquid, an eighth separated liquid, and a ninth separated liquid;

[0079] S22. The ninth separated liquid (1.0 g) was purified by preparative HPLC (HPLC chromatograph purchased from Beijing Aonuo Technology Co., Ltd., LC2100 preparative liquid chromatograph, C18 chromatographic column, 10 μm, 20×250 nm, flow rate 19 mL / min, mobile phase with 60% volume concentration of MeOH) to obtain compound 2 (41 mg, purity was 98.1% as determined by HPLC).

[0080] By HR-ESI-MS (high resolution mass spectrometry), 1 HNMR, 13 C NMR, HMQC, HMBC, NOESY and rotation spectrum were used to identify the structure of compound 2. The spectrum obtained is shown in Figure 8-13 shown.

[0081] Figure 8 This is the HR-ESI-MS spectrum of compound 2. The HR-ESI-MS spectrum shows that the quasi-molecular ion peak m / z of compound 2 is 441.1519 [M+Na] + , C 22 H 26 The calculated value of NaO8 is 441.1520, combined with 1 HNMR and 13 The molecular formula is inferred from C NMR data to be C 22 H 26 O8, its unsaturation degree is 10.

[0082] Fig. 9 For compound 2 1 HNMR spectrum, 1 HNMR spectra show that in δ H 6.92 (1H, s, H-4) and 6.73 (2H, s, H-2 / 6) suggest the existence of a 1,3,5-trisubstituted benzene ring; δ H 7.09 (1H, d, J = 2.0 Hz, H-2'), 6.85 (1H, dd, J = 8.1, 2.0 Hz, H-6'), 6.75 (1H, d, J = 8.1 Hz, H-5') suggest the presence of a 1,3,4-trisubstituted benzene ring; δ H 4.38 (1H, dd, J = 11.4, 6.0 Hz, H-9a), 4.23 (1H, dd, J = 11.4, 7.5 Hz, H-9b). 3.83 (1H, d, J = 9.4 Hz, H-9'a), 3.63 (1H, d, J = 9.3 Hz, H-9'b). 2.81 (1H, d, J = 13.9 Hz, H-7'a), 2.91 (1H, d, J = 13.9 Hz, H-7'b)) are speculated to be three groups of methylene signals; δ H3.85 (3H, s, OMe-3), 3.85 (3H, s, OMe-3') are two methoxy signals.

[0083] Fig.10 , 11 are respectively 13 C NMR spectrum and HMQC spectrum, combined with compound 2 1 H NMR spectrum showed that 1.91 (3H, s, H-11) and 172.7 (C-10), 20.7 (C-11) indicated acetoxy groups, δ H 4.64 (1H, d, J = 7.9 Hz, H-7), 2.54 (1H, m, H-8), 3.83 (1H, d, J = 9.4 Hz, H-9'a), 3.63 (1H, d, J = 9.3 Hz, H-9'b), 86.4 (C-7), 56.4 (C-8), 82.2 (C-8'), 77.6 (C-9'), which is speculated to be a signal of a tetrahydrofuran ring. HMBC spectrum shows: δ H 4.38 (H-9a) and 56.4 (C-8) are related, δ H 2.91 (H-7a) and 82.2 (C-8') are related, δ H ,6.73(H-2 / 6) and 86.4(C-7) are correlated.

[0084] Fig.13 This is the NOSEY spectrum of compound 2. The NOSEY spectrum shows that 4.64 (1H, d, J = 7.9 Hz, H-7) and 2.54 (1H, m, H-8) are correlated, and it is inferred that H-7 and H-8 are on the same side, so H-7 and H-8 of compound 2 are in cis configuration.

[0085] HPLC chiral column detection showed that compound 2 was a racemic compound (containing a pair of optical isomers). Therefore, the structure of compound 2 was determined to be 5,4′,8′-trihydroxy-3,3′-dimethoxy-9-acetoxy-7,9′-epoxylignan.

[0086] Compound 2 1 H NMR (600 MHz, CD3OD), 13 The C NMR (151 MHz, CD3OD) data are shown in Table 2.

[0087] Table 2

[0088]

[0089]

[0090] After comprehensive analysis, the structural formula of compound 2 is as follows:

[0091]

[0092] Example 3 Preparation of Compound 3

[0093] Step S31, the seventh fraction (64.8 g) in step S13 of Example 1 is separated by reverse phase solid phase extraction, the adsorbent used is silica gel, and gradient elution is performed with alcohol or acetone and water in a volume ratio of 60:40, 65:35, 81:19, and 86:14 to obtain a first eluent, a second eluent, a third eluent, and a fourth eluent, wherein the alcohol is methanol or ethanol;

[0094] Step S32, purifying the second eluent by preparative HPLC (HPLC chromatograph purchased from Beijing Aonuo Technology Co., Ltd., LC2100 preparative liquid chromatograph, C18 chromatographic column, 10 μm, 20×250 nm), using acetonitrile-water with a volume content of 20%-40% as the mobile phase (preferably acetonitrile-water with a volume content of 20% as the mobile phase), the flow rate is 19 ml / min, and the elution is 20 min to obtain a first solution, a second solution, a third solution and a fourth solution;

[0095] Step S33, the second solution was purified by preparative HPLC (HPLC chromatograph purchased from Beijing Aonuo Technology Co., Ltd., LC2100 preparative liquid chromatograph, C18 chromatographic column, 10 μm, 20×250 nm), with acetonitrile-water with a volume content of 19%-23% as the mobile phase (preferably acetonitrile with a volume content of 20% as the mobile phase), and the flow rate was 19 ml / min for 20 min to obtain compound 3 (12 mg, t R =15.4min, purity was 98.3% as determined by HPLC).

[0096] Compound 3 was a yellow powder, and the hydrochloric acid-magnesium powder reaction was positive, and was determined to be a flavonoid compound.

[0097] pass 1 H NMR, 13 C NMR was used to identify the structure of compound 3, and the obtained spectrum is as follows Fig.14 and 15 shown.

[0098] Fig.14 For compound 3 1 H NMR spectrum, 1H NMR spectrum showed: δH: 7.46 (1H, dd, J = 8.5 Hz, 2.1 Hz, H-6'), 7.40 (1H, d, J = 2.1 Hz, H-2'), 7.10 (1H, d, J = 8.5 Hz, H-5'), 6.67 (H-6, 1H, s), 5.40 (1H, d, J = 1.7 Hz, Rha H-1'), 5.22 (1H, t, J = 6.9 Hz, H-12), 5.09 (1H, d, J = 7.1 Hz, Glu H-1”),4.25(1H,dd,J=3.4,1.7Hz,Rha-2”’),3.97(3H,s,4’-OCH3),3.93(1H,d,J=2.2Hz,H-6”),3.42-3.57(4H,m,Glu-2”,3”,4”,5”),3. 33-3.71(3H,m,Rha-3"',4"',5"'),1.76(3H,s,H3-15),3.53-3.61(2H,m,H-12a / 12b),1.67(3H,s,H3-14),0.96(3H,d,J=5.6Hz,H3-6"').

[0099] Fig.15 For compound 3 1 C NMR spectrum (101MHz, CD3OD), 13 C NMR spectrum shows: δC: 180.2 (C-4), 162.0 (C-7), 162.0 (C-5), 159.5 (C-2), 155.0 (C-9), 151.8 (C-4'), 147.8 (C-3'), 136.5 (C-3), 132.8 (C-13), 124.3 (C-1'), 123.5 (C-12), 116.8 (C-2'), 110.6 (C-8), 107.7 (C-10), 107.5 (C-1"'), 103.5 (C-1"') ,102.0(C-1”),101.9(C-6),78.3(C-3”),78.3(C-5””),78.3(C-5”),75.0(C-4”’),73.2(C-4”’),,72.1(C-3”’),72.1 (C-5”'),71.9(C-2”’),71.2(C-4”’),62.4(C-6”),56.5(OCH3),25.9(C-14),22.7(C-11),18.3(C-15),17.7(C-6”’).

[0100] Comprehensive analysis combined with existing literature data comparison, the structure of compound 3 was determined as follows:

[0101]

[0102] Example 4 Preparation of Compound 4

[0103] The second extract in step S12 of Example 1 was separated by normal phase solid phase extraction, with silica gel as the adsorbent, and eluted with dichloromethane:methanol in a volume ratio of 40:1 to obtain compound 4 (2.93 g, purity of 98% as determined by HPLC).

[0104] Compound 4 was a yellow powder, and the hydrochloric acid-magnesium powder reaction was positive, and was determined to be a flavonoid compound.

[0105] Compound 4 was subjected to 1 H NMR, 13 C NMR detection.

[0106] 1 H NMR (600MHz, CD3OD) shows: δH:7.88(2H,d,J=8.6Hz,H-2' / H-6'),7.11(2H,d,J=8.6Hz,H-3' / H-5'),6.65(1H,s,H-6),5.45(1H,d,J=1.5Hz,Rha H-1”),5.07(1H,d,J=7.4Hz,Glu H-1””),5.20(H,t,J=6.8Hz,H-12),4.29(1H,d,J=7.7Hz,Xyl H-1”'),3.90(3H,s,4'-OCH3),1.73(3H,s,H3-15),1.65(3H,s,H3-14),0.98(3H,d,J=6.3Hz,H3-6”).

[0107] 13C NMR (151 MHz, CD3OD) showed: δC: 180.2 (C-4), 163.6 (C-4'), 162.1 (C-5), 161.0 (C-7), 159.3 (C-9), 155.0 (C-2), 137.0 (C-3), 132.7 (C-13), 131.9 (C-2', 6'), 123.8 (C-12), 123.6 (C-1'), 115.3 (C-3', 5'), 110.6 (C-8), 107.7 (C-10), 107.5 (C-1"'), 103.1 (C-1"), 101.9 (C-1""), 99.4 (C-6), 82.6 (C-2”), 78.3 (C-3””), 78.2 (C-5””), 77.8 (C-3”'), 75.2 (C-2”'), 75.2 (C-2””), 73.6 (C-4”), 71.9 (C-3”), 71.9 (C-5”), 71.1 (C-4””), 70.9 (C-4”’), 67.1 (C-6”’), 62.4 (C-6””), 56.1 (OCH3), 25.9 (C-14), 22.7 (C-11), 18.3 (C-6”), 17.7 (C-15). By comparing with the literature data, compound 4 was determined to be Chaohuodine B.

[0108] The structural formula of compound 4 is as follows:

[0109]

[0110] Example 5

[0111] To detect the effects of compounds 1, 2, 3, and 4 on the activity of mouse myoblast C2C12, the following cell activity experiments were performed:

[0112] 1 Materials and Methods

[0113] 1.1 Experimental Materials

[0114] 1.1.1 C2C12 cells were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0115] 1.1.2 Main reagents and instruments

[0116] DMEM medium (AJ30740638, HyClone), fetal bovine serum (SH30406.05, HyClone), 0.25% trypsin (P2847398, Adamas life), PBS (23205634, Biosharp). White transparent bottom 96-well cell culture plate (FCP963, Beyotime), 15 mL and 50 mL centrifuge tubes (17122124O, Labselect). Dexamethasone (23030973, Tmsstandard), CellTiter-Lumi TM Plus II luminescent cell viability detection kit (C0057M, Beyotime). Spark multifunctional microplate reader (Tecan).

[0117] 1.2 Experimental methods

[0118] 1.2.1 Viability measurement of serum starved cell model

[0119] Step 1, seeding C2C12 myoblasts on a cell culture dish and culturing them in proliferation medium (DMEM + 10% fetal bovine serum, 1% penicillin / streptomycin [P / S]), changing the medium at least every 2 days;

[0120] Step 2: C2C12 cells (0.5×10 5 cells / well) were seeded on a 96-well cell culture dish and cultured overnight in proliferation medium;

[0121] Step 3, the C2C12 cells were divided into a normal group, a starvation group and an experimental group, wherein the C2C12 cells in the normal group were cultured in a proliferation medium, the C2C12 cells in the starvation group were cultured in a serum-free medium (DMEM, 1% penicillin / streptomycin [P / S]), and the C2C12 cells in the experimental group were cultured in a serum-free medium, and different concentrations of compound 1 (60, 20, 6.66, 2.22, 0.74, 0.25 μM), compound 2 (60, 20, 6.66, 2.22, 0.74, 0.25 μM), compound 3 (60, 20, 6.66, 2.22, 0.74, 0.25 μM), and compound 4 (60, 20, 6.66, 2.22, 0.74, 0.25 μM) were added and cultured for 48 hours.

[0122] Step 4: Add 100 μl CellTiter-Lumi TMPlus II luminescence detection reagent, the chemiluminescence value of each well was detected by a multifunctional microplate reader. Each experiment was repeated 3 times, with 6 replicate wells each time. The formula for calculating cell viability is: cell viability = (chemiluminescence value of experimental group - chemiluminescence value of starvation group) / (chemiluminescence value of normal group - chemiluminescence value of starvation group) × 100%; Gragpad Prism 10.1.2 software was used to plot the promoting effect of the compound on cell proliferation and determine the EC value of each compound 50 Value (EC 50 The EC refers to the half effective concentration of the drug on the cell. 50 The value was determined by constructing a curve of the concentration of Epimedium extract and cell viability).

[0123] 1.2.2 Viability measurement of dexamethasone cell injury model

[0124] Step 1, seeding C2C12 myoblasts on a cell culture dish and culturing them in proliferation medium (DMEM + 10% fetal bovine serum, 1% penicillin / streptomycin [P / S]), changing the medium at least every 2 days;

[0125] Step 2: C2C12 cells (0.125×10 5 cells / well) were seeded on a 96-well cell culture dish and cultured in proliferation medium for 24 h;

[0126] Step 3, the C2C12 cells were divided into a control group, a dexamethasone group and an experimental group, wherein the C2C12 cells in the control group were cultured in a proliferation medium without dexamethasone treatment; the C2C12 cells in the dexamethasone group were treated with 200 μM dexamethasone; the C2C12 cells in the experimental group were treated with 200 μM dexamethasone, and different concentrations of compound 1 (60, 20, 6.66, 2.22, 0.74, 0.25 μM), compound 2 (60, 20, 6.66, 2.22, 0.74, 0.25 μM), compound 3 (60, 20, 6.66, 2.22, 0.74, 0.25 μM), and compound 4 (60, 20, 6.66, 2.22, 0.74, 0.25 μM) were added to the culture medium and cultured for 48 hours.

[0127] Step 4: Add 100 μl CellTiter-Lumi TMPlus II luminescence detection reagent, the chemiluminescence value of each well was detected by a multifunctional microplate reader. Each experiment was repeated 3 times, with 6 replicate wells each time. The formula for calculating cell viability is: cell viability = (chemiluminescence value of the experimental group - chemiluminescence value of the dexamethasone group) / (chemiluminescence value of the control group - chemiluminescence value of the dexamethasone group) × 100%. Gragpad Prism 10.1.2 software was used to plot the promoting effect of the compound on cell proliferation and determine the EC value of each compound. 50 value.

[0128] 1.2.3 Statistics and Analysis

[0129] The results are as follows Fig.16 , 17 As shown in Table 3, Fig.16 The chemiluminescence values ​​corresponding to the normal group, starvation group and experimental groups of each concentration under the serum starvation cell model; Fig.17 These are the chemiluminescence values ​​corresponding to the control group, dexamethasone group and experimental groups of each concentration under the dexamethasone cell injury model.

[0130] Table 3

[0131] serial number <![CDATA[Serum starvation model EC 50 (μM)]]> <![CDATA[Dexamethasone model EC 50 (μM)]]> Compound 1 4.49μM 5.54μM Compound 2 4.70μM 5.64μM Compound 3 3.54μM 4.30μM Compound 4 1.49μM 3.34μM

[0132] As shown in Table 3, compounds 1, 2, 3, and 4 all have certain protective activities against serum-starved and dexamethasone-damaged C2C12 cells. Among them, compound 4 (epimedin B) has the best activity, with an EC 50 are 1.49 μM and 3.34 μM, compound 3 has better activity, EC 50 were 3.54 μM and 4.30 μM, while the EC of compound 2 50 The EC values ​​of compound 1 were 4.70 μM and 5.64 μM. 50 4.49 μM and 5.54 μM.

[0133] In summary, all four compounds can significantly improve the survival rate of C2C12 cells under serum starvation and dexamethasone injury, indicating that compounds 1-4 have good therapeutic activity.

[0134] Example 6

[0135] In order to investigate the effects of compounds 1, 2, 3, and 4 on diabetic sarcopenia model mice, the following experiment was performed.

[0136] 1. Materials

[0137] 1.1 Experimental Animals

[0138] Male KM mice, 6-8 weeks old, weighing 26-30 g, were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0139] The mice were grouped in cages of 5 and fed in an environment with a room temperature of 20-25°C and a humidity of 30-60% for 7 days before the experiment began. During the feeding process, the mice's diet, activity, coat color, defecation, mental state, etc. were observed at any time during the feeding process. The weight was measured at the same time every day. All animals were operated in accordance with the "Regulations on the Management of Experimental Animals" formulated by the National Science and Technology Commission.

[0140] 1.2 Experimental drugs

[0141] Compound 1, Compound 2, Compound 3 and Compound 4 were prepared by the methods of Examples 1 to 4. Dexamethasone sodium phosphate injection was purchased from Shanghai Pharmaceutical Group Co., Ltd.

[0142] 1.3 Experimental instruments

[0143] Dual-energy X-ray absorptiometry (iNSiGHT VET DXA, OsteoSys, KOREAN); YLS-13A muscle strength tester, Jinan Yiyan Technology Co., Ltd.

[0144] 2. Methods

[0145] 2.1 Modeling

[0146] After adaptive feeding, the male KM mice were randomly divided into groups, weighed and numbered. The negative control group (control) was given normal saline, and the (diabetic) sarcopenia model group was given dexamethasone sodium phosphate injection. The administration method was subcutaneous injection. The modeling and administration process lasted for 12 days (2 weeks) (20 mg / kg / day). During this period, food and water were supplied without distinction, and the bedding was changed twice a week to keep the living environment clean and hygienic.

[0147] 2.2 Grouping and Dosing

[0148] After 2 weeks of dexamethasone sodium phosphate injection, 72 male KM mice were randomly divided into a model group, a low-dose group (5 mg / kg) and a medium-dose group (10 mg / kg) of compound 1, a low-dose group (5 mg / kg) and a medium-dose group (10 mg / kg) of compound 2, a low-dose group (5 mg / kg) and a medium-dose group (10 mg / kg) of compound 3, and a low-dose group (5 mg / kg) and a medium-dose group (10 mg / kg) of compound 4, with 8 mice in each group; 8 KM mice of the same age were used as a negative control group.

[0149] Epimedium extract compounds 1, 2, 3 and 4 were dissolved in 0.3% sodium carboxymethylcellulose solution. Each administration group was given different concentrations of epimedium extract solution by gavage for 6 consecutive weeks, once a day, with a gavage volume of 0.1 mL / 10 g. The negative control group and the model group were given 0.3% sodium methylcellulose solution by gavage for 6 consecutive weeks, once a day, with a gavage volume of 0.1 mL / 10 g.

[0150] 2.3. Detection indicators and methods

[0151] 2.3.1. Blood glucose measurement in mice

[0152] Two weeks after subcutaneous injection of dexamethasone sodium phosphate, mice in the negative control group and the diabetic sarcopenia model group fasted for 12 hours, and blood was collected from the tail vein to measure fasting blood glucose using a blood glucose meter.

[0153] 2.3.2. Mouse muscle strength measurement

[0154] The forelimb grip strength of mice was measured using a YLS-13A muscle strength tester (Jinan Yiyan Technology Co., Ltd., Jinan, China). The mice were lifted and fixed by their tails so that their limbs could grasp a wire mesh. Then, the mice were gently pulled back by their tails, with their posture parallel to the surface of the table, until they released the mesh. The peak force exerted by the mouse limbs was recorded in grams (g). Evaluations were performed every 0, 2, 4, and 6 weeks, and each mouse was tested three times, and the average was taken and weighted for statistical analysis.

[0155] 2.3.3. Measurement of body composition of mice

[0156] Three days before the mice were killed, dual-energy X-ray absorptiometry (DEXA) was used to measure body composition, including body fat content, muscle content, bone mineral content, etc. The data were analyzed using the software provided by the manufacturer.

[0157] 2.3.4 Statistical methods

[0158] All data were expressed as mean ± SD, and the data were subjected to one-way ANOVA, and multiple comparisons were performed using Tukey post-hoc test. In the results, P < 0.05 indicated that the two groups of data had significant differences, P < 0.01 indicated that the two groups of data had very significant differences, and P < 0.001 indicated that the two groups of data had extremely significant differences. Graphpad Prism 10.1.2 was used as the statistical software.

[0159] 3. Results

[0160] 3.1. General observation of experimental animals

[0161] The negative control group was in good general condition, with smooth and shiny fur and no obvious irritability. Eating and drinking were normal, defecation was normal, weight increased steadily, and reaction was quick. Before the injection of dexamethasone, the mice in each group were in good condition, with shiny fur and agile activities. After the injection of dexamethasone began, the subcutaneous injection model group gradually showed irritability, slow reaction, listlessness, fluffy and dull fur, and a surge in weight. Some mice had necrosis at the tip of their tails and fell off automatically.

[0162] 3.2. Blood glucose measurement of model mice

[0163] The results are shown in Table 4. Two weeks after modeling, the fasting blood glucose level of mice injected subcutaneously with dexamethasone sodium phosphate was significantly higher than that of mice in the negative control group (P < 0.01), indicating that the diabetic mice were successfully modeled.

[0164] Table 4 is a comparison of fasting blood glucose levels between the negative control group and diabetic sarcopenia model mice (X±SD).

[0165] Table 4

[0166]

[0167] Note: Compared with the control group, **P<0.01 in the model group.

[0168] 3.3 Effects of the above four epimedium extracts on muscle strength in diabetic sarcopenia model mice

[0169] The results are shown in Table 5. Before administration, compared with the control group, the muscle strength of each group decreased significantly (P < 0.01); there was no significant difference in the muscle strength of the model mice between the groups, indicating that the experimental system is reliable and the groups are comparable. After 2 weeks of administration, compared with the control group, the muscle strength of the model group decreased significantly (P < 0.01); compared with the model group, the muscle strength of compound 4 and compound 1 in the medium-dose group increased significantly (P < 0.01), and the muscle strength of compound 2 and compound 3 increased significantly (P < 0.05). The muscle strength of each low-dose group showed an upward trend, and there was no significant difference between them; after 4 weeks of administration, compared with the control group, the muscle strength of the model group decreased significantly (P < 0.01); compared with the model group, the muscle strength of compound 4 and compound 1 in the medium-dose group increased significantly ( P < 0.01), the muscle strength of compound 2 and compound 3 increased significantly (P < 0.05), and the muscle strength of each low-dose group showed an increasing trend, and there was no significant difference between them; after 6 weeks of administration, compared with the control group, the muscle strength of the model group decreased significantly (P < 0.01); compared with the model group, the muscle strength of compound 4 and compound 1 in the medium-dose group increased significantly (P < 0.01), the muscle strength of compound 2 and compound 3 increased significantly (P < 0.05), and the muscle strength of each low-dose group showed an increasing trend, and there was no significant difference between them.

[0170] Table 5 shows the effects of the above four epimedium extracts on the muscle strength of dexamethasone-induced diabetic sarcopenia model mice (X±SD)

[0171] Table 5

[0172]

[0173] Note: Compared with the control group, the model group *P<0.05, **P<0.01; compared with the model group, the drug administration group #P<0.05, ##P<0.01.

[0174] 3.4 Effects of the above four epimedium extracts on body fat content and muscle content in diabetic sarcopenia model mice

[0175] The results after 6 weeks of administration are shown in Table 6. Compared with the control group, the body fat content of the model group was significantly increased (P < 0.05); compared with the model group, the body fat content of compound 4 and compound 1 in the medium-dose group was significantly reduced (P < 0.01), the muscle content of compound 4 and compound 1 was significantly increased (P < 0.01), and the muscle content of compound 2 and compound 3 was significantly increased (P < 0.05). The body fat content of the low-dose group tended to decrease, and the muscle content tended to increase.

[0176] Table 6 shows the effects of the above four epimedium extracts on body fat content and muscle content in diabetic sarcopenia model mice (X±SD)

[0177] Table 6

[0178]

[0179] Note: Compared with the control group, the model group *P<0.05, **P<0.01; compared with the model group, the drug administration group #P<0.05, ##P<0.01

[0180] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Use of an epimedium extract or a composition containing an epimedium extract in the preparation of a medicament for preventing and / or treating diabetic sarcopenia, wherein the epimedium extract is selected from any one or more combinations of the following structures:

2. The use according to claim 1, wherein the diabetic sarcopenia is sarcopenia caused by diabetes, and the symptoms are chronic muscle atrophy and gradual apoptosis of muscle cells.

3. The use according to claim 1, wherein the composition further contains a pharmaceutically acceptable excipient.

4. A pharmaceutical composition for preventing and / or treating diabetic sarcopenia, characterized in that: The pharmaceutical composition uses the epimedium extract as a main active ingredient, and the epimedium extract is selected from any one or more combinations of the following structures:

5. A method for preparing an epimedium extract, characterized in that: The steps include: S1. Take Epimedium leaves, process them, and extract them with ethanol to obtain a thick paste; S2, adsorbing the thick paste in S1 with a macroporous resin column, eluting and concentrating with alcohol or acetone at volume concentrations of 20%, 40%, 60%, 80% and 95% in sequence, to obtain the corresponding first extract, second extract, third extract, fourth extract and fifth extract; S3, separating the second extract by normal solid phase extraction, eluting with chloroalkane: polar solvent in a volume ratio of 50:1, 25:1, 15:1, 5:1, and 0:1, and detecting by thin layer chromatography to obtain a first fraction, a second fraction, a third fraction, a fourth fraction, a fifth fraction, a sixth fraction, a seventh fraction, and an eighth fraction, wherein the chloroalkane is dichloromethane or chloroform, and the polar solvent is methanol, acetone, or ethyl acetate; S4, separating the second fraction by gel column chromatography or reversed phase solid phase extraction, using methanol as the mobile phase to elute, to obtain compound 1; S5. Separate the first extract by normal solid phase extraction, elute with dichloromethane:methanol in a volume ratio of 40:1, 25:1, 15:1, 5:1, and 0:1, respectively, and detect by thin layer chromatography to obtain a first separated liquid, a second separated liquid, a third separated liquid, a fourth separated liquid, a fifth separated liquid, a sixth separated liquid, a seventh separated liquid, an eighth separated liquid, and a ninth separated liquid. Separate the ninth separated liquid by high performance liquid chromatography to obtain compound 2; S6, separating the seventh fraction by reverse solid phase extraction, and gradient eluting with alcohol or acetone and water in a volume ratio of 60:40, 65:35, 81:19, and 86:14, to obtain a first eluent, a second eluent, a third eluent, and a fourth eluent; S7, separating the second eluent by gel column chromatography, reversed phase solid phase extraction or high performance liquid chromatography, using 20% ​​to 40% by volume acetonitrile-water as a mobile phase for elution, to obtain a first solution, a second solution, a third solution and a fourth solution; S8, separating the second solution by gel column chromatography, reversed phase solid phase extraction or high performance liquid chromatography, and eluting with 19% to 23% by volume acetonitrile-water as a mobile phase to obtain compound 3; S9. Separate the second extract by normal solid phase extraction, and elute with dichloromethane:methanol in a volume ratio of 40:1 to obtain compound 4, wherein compound 1, compound 2, compound 3, and compound 4 are:

6. The method for preparing the epimedium extract according to claim 2, characterized in that: In step S1, after the epimedium leaves are processed, 50% ethanol by volume is added, and the leaves are extracted and concentrated under reflux conditions to obtain a thick paste.

7. The method for preparing the epimedium extract according to claim 2, characterized in that: The macroporous resin column is a LSA-12S macroporous resin column, and the alcohol is methanol or ethanol.

8. The method for preparing the epimedium extract according to claim 2, characterized in that: The adsorbent used in the normal solid phase extraction and the reverse solid phase extraction is silica gel, and the silica gel is spherical silica gel or amorphous silica gel, and the mesh number of the silica gel is 200-300 meshes.

9. The method for preparing the epimedium extract according to claim 2, characterized in that: The mobile phase used for the HPLC separation in step S5 is methanol with a volume concentration of 60%.

10. An epimedium extract, characterized in that: The structural formula is as follows:

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