Compound with anti-osteoporosis effect as well as preparation method and application thereof

Abstract

The invention discloses a compound with an anti-osteoporosis effect and a preparation method of the compound with the anti-osteoporosis effect. The preparation method comprises the following steps: separating from a fermentation solution of a fungus Talaromyces pinophilus (CICC 2707), so as to obtain new compounds, i.e., acetosellin B and acetosellin C. The invention further discloses a preparation method of the compound with the anti-osteoporosis effect and a preparation method of the compound with the anti-osteoporosis effect. Experiments show that prednisolone-induced zebra fish osteoporosis models are selected to carry out experiments on the prepared compounds acetosellin B and acetosellin C, and the compounds are found to have relatively high in-vivo osteogenic activity of zebra fish and can be used for preparing medicines for preventing and treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to two natural products derived from microorganisms, and specifically relates to two compounds with anti-osteoporosis effects, as well as their preparation methods and applications. Background Art

[0002] Osteoporosis (OP) is an abnormal bone metabolic disease, the main feature of which is the imbalance between osteoblast bone formation and osteoclast bone resorption, resulting in the osteoclast bone resorption ability being stronger than the osteoblast bone formation ability, and further leading to bone mass loss and bone mass reduction. Osteoporosis is a common bone disease. In patients, the bone tissue structure degenerates, bone mass decreases, brittleness increases, and the risk of fracture increases, affecting millions of people worldwide. Especially in the elderly population, sarcopenia is often accompanied, and there is a high correlation between sarcopenia and osteoporosis. In China, the situation of severe population aging is severe, and the understanding of the degeneration of the motor system should be emphasized, which is beneficial to improving the physical fitness and quality of life of the elderly. With the intensification of the aging trend of the whole society and people's pursuit of a healthy life in old age, the future demand for anti-osteoporosis drug treatment is huge.

[0003] Anti-osteoporosis drugs mainly include basic supplements, bone resorption inhibitors, and bone formation promoters according to the occurrence of the disease. The basic drugs for drug treatment are mainly calcium agents and vitamin D. Clinically, inhibiting osteoclast bone resorption is the main treatment measure, and the drugs are mainly bisphosphonates, estrogen and its receptor modulators, and calcitonin. There are certain limitations and side effects in the long-term use and dose maintenance of these drugs in clinical treatment, and it is still necessary to further research anti-osteoporosis drugs with high efficacy and low side effects for clinical application.

[0004] Fungi are one of the most biodiverse groups and an important source of a series of small molecule active compounds. In recent years, more than 50% of the secondary metabolites with new structures and new activities have been found to be derived from fungi. Therefore, deeply exploring the natural active compounds in fungi is not only a research hotspot but also will provide a necessary basis for drug research and development and has important scientific significance. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to solve the deficiencies of the prior art and isolate new compounds with anti-osteoporosis effects from the secondary metabolites of fungi.

[0006] The compounds with anti-osteoporosis effects isolated by the present invention have molecular formulas of C 21 H 18 O8, C 21 H 20 O7 and the structures are as follows:

[0007] Compounds 1 and 2 are named acetosellin B and acetosellin C, respectively.

[0008] The preparation method of the compound with anti-osteoporosis efficacy according to the present invention includes the following steps: a. Take the fermentation broth of the fungus Talaromyces pinophilus (CICC 2707), extract it with an organic solvent, obtain the organic phase, and recover the organic phase to obtain an extract.

[0009] b. Separate the extract by chromatographic column chromatography to obtain the target compound.

[0010] As a preferred embodiment, for the preparation method of the compound with anti-osteoporosis efficacy described above, the organic solvent used for extraction in step a is ethyl acetate; the materials for chromatographic column chromatography separation are silica gel and C 18 ODS.

[0011] As a preferred embodiment, the preparation method of the compound with anti-osteoporosis efficacy described above includes the following steps: a. Take the fungus alaromyces pinophilus (CICC 2707), inoculate it on a PDA plate, culture it in an incubator at 28 °C for 2 - 3 days, and then transfer it to a wort liquid medium and continue to culture it on a shaker for 14 days; b. After the fermentation is completed, filter to obtain the fermentation broth, extract it with ethyl acetate 1 - 3 times, combine the ethyl acetate solutions, and recover the solvent to obtain an ethyl acetate extract; c. Perform column chromatography separation on the ethyl acetate extract with normal-phase silica gel, elute it with a gradient of ethyl acetate - petroleum ether with different volume ratios, collect the fractions to obtain fractions F1 - F28; according to TLC thin-layer analysis, combine fractions F16 - 28, and then perform separation with a medium-pressure preparative chromatography ODS column, elute it with a gradient of methanol - water, detect it at 254 and 310 nm, obtain fractions F29 - F47, take fraction F39 among them for preparative liquid-phase HPLC separation, the mobile phase is CH3CN:H2O by volume ratio, the chromatographic column is Eclipse XDB-C18column, with a specification of 9.4 × 250 mm, 5 µm, and compound 1 is separated; Perform preparative liquid-phase HPLC separation on fraction F38, the mobile phase is CH3CN:H2O, the chromatographic column is Eclipse XDB-C18 column, with a specification of 9.4 × 250 mm, 5 µm, and compound 2 is separated.

[0012] As a preferred embodiment, for the preparation method of the compound described above, in step c, it is eluted with a gradient of ethyl acetate - petroleum ether with a volume ratio of 1:0 - 0:1.

[0013] As a preferred embodiment, in the preparation method of the above-mentioned compound, in step c, separation is carried out using a medium-pressure preparative chromatography ODS column, gradient elution is performed with a methanol aqueous solution having a volume concentration of 10%-100%, detection is carried out at 254 and 310 nm, fractions F29-F47 are obtained, and fraction F39 among them is subjected to preparative liquid-phase HPLC separation. The mobile phase is CH3CN:H2O with a volume ratio of 23:77, the chromatographic column is an Eclipse XDB-C18 column with a specification of 9.4 × 250 mm, 5 µm, the flow rate is 2.0 mL / min, and compound 1 with t R = 18 min is separated and obtained.

[0014] Fraction F38 is subjected to preparative liquid-phase HPLC separation. The mobile phase is CH3CN:H2O with a volume ratio of 28:72, the chromatographic column is an Eclipse XDB-C18 column with a specification of 9.4 × 250 mm, 5 µm, the flow rate is 2.0 mL / min, and compound 2 with t R = 15 min is separated and obtained.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following advantages: Through a large number of experiments, the present invention prepares new compounds acetosellin B and acetosellin C. The results of anti-osteoporosis activity experiments show that acetosellin B and acetosellin C prepared by the present invention have strong osteogenic activity, and compound acetosellin B and acetosellin C and their salts or derivatives can be used to prepare drugs for preventing and treating osteoporosis. Description of the Drawings

[0016] Figure 1 It is the HR-ESI-MS diagram of acetosellin B.

[0017] Figure 2 It is the 1 1H NMR diagram of acetosellin B (DMSO- d 6 , 500 MHz).

[0018] Figure 3 It is the 13 13C NMR diagram of acetosellin B (DMSO- d 6 , 125 MHz).

[0019] Figure 4DEPT spectrum of acetosellin B (DMSO- d 6 , 125 MHz).

[0020] Figure 5 For the 1 H- 1 H-COSY spectrum of acetosellin B (DMSO- d 6 , 500 MHz).

[0021] Figure 6 HSQC spectrum of acetosellin B (DMSO- d 6 , 500 MHz).

[0022] Figure 7 HMBC spectrum of acetosellin B (DMSO- d 6 , 500 MHz).

[0023] Figure 8 NOESY spectrum of acetosellin B (DMSO- d 6 , 500 MHz).

[0024] Figure 9 HR-ESI-MS spectrum of acetosellin C.

[0025] Figure 10 For the 1 1H NMR spectrum of acetosellin C (DMSO- d 6 , 500 MHz).

[0026] Figure 11 For the 13 13C NMR spectrum of acetosellin C (DMSO- d 6 , 125 MHz).

[0027] Figure 12 DEPT spectrum of acetosellin C (DMSO- d 6 , 125 MHz).

[0028] Figure 13 For the 1 H- 1 H-COSY spectrum of acetosellin C (DMSO-d 6 , 500 MHz).

[0029] Figure 14 is the HSQC spectrum of acetosellin C (DMSO- d 6 , 500 MHz).

[0030] Figure 15 is the HMBC spectrum of acetosellin C (DMSO- d 6 , 500 MHz).

[0031] Figure 16 is the NOESY spectrum of acetosellin C (DMSO- d 6 , 500 MHz).

[0032] Figure 17 are the structures of acetosellin B and acetosellin C, COSY (bold lines), and key HMBC correlations.

[0033] Figure 18 are the experimental and calculated ECD spectra of acetosellin B and acetosellin C.

[0034] Figure 19 is the DP4+ analysis of the carbon at position 3 of acetosellin B.

[0035] Figure 20 is the regression analysis of the experimental and calculated chemical shifts of the carbon at position 3 of acetosellin B.

[0036] Figure 21 is the DP4+ analysis of the carbon at position 3 of acetosellin C.

[0037] Figure 22 is the regression analysis of the experimental and calculated chemical shifts of the carbon at position 3 of acetosellin B.

[0038] Figure 23 is the anti-osteoporosis activity graph of acetosellin B (1) and acetosellin C (2) (n = 6). Detailed implementation mode

[0039] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, those skilled in the art's various equivalent forms of modification of the present invention fall within the scope defined by the appended claims of this application.

[0040] In the following examples Talaromyces pinophilus (CICC 2707) is the preservation number of the standard strain publicly sold by the China Center for Industrial Culture Collection.

[0041] Example 1 Preparation of the new compound acetosellin B and the compound acetosellin C Take the fungus Talaromyces pinophilus (CICC 2707) and inoculate it on a PDA plate. After culturing in an incubator at 28 °C for 2 - 3 days, transfer it to 100 bottles of wort liquid medium (400 mL per bottle) and continue to culture on a shaker at 28 °C for 14 days. After fermentation, filter the fermentation broth with double-layer gauze. Extract it 3 times with ethyl acetate, combine the ethyl acetate extracts, and recover the solvent to obtain 11.5 g of ethyl acetate extract.

[0042] Perform column chromatography separation on the ethyl acetate extract with normal-phase silica gel, elute with an ethyl acetate - petroleum ether (volume ratio 1:0 - 0:1) gradient, collect the fractions to obtain fractions F1 - F28; according to TLC thin-layer analysis, combine fractions F16 - 28, and perform separation using medium-pressure preparative chromatography ODS column. Elute with a methanol aqueous solution with a volume concentration of 10% - 100% gradient, detect at 254 and 310 nm to obtain 19 components F29 - F47. Perform preparative liquid-phase HPLC separation on fraction F39, with the mobile phase being CH3CN:H2O with a volume ratio of 23:77, the chromatographic column being Eclipse XDB-C18 column, with a specification of 9.4 × 250 mm, 5 µm, and a flow rate of 2.0 mL / min. Separate to obtain R Compound 1 with t = 18 min, named acetosellin B.

[0043] Perform preparative liquid-phase HPLC separation on fraction F38, with the mobile phase being CH3CN:H2O with a volume ratio of 28:72, the chromatographic column being Eclipse XDB-C18 column, with a specification of 9.4 × 250 mm, 5 µm, and a flow rate of 2.0 mL / min. Separate to obtain R Compound 2 with t = 15 min, named acetosellin C.

[0044] Structural analysis of acetosellin B (1): Pale yellow solid,1 1H NMR and 13 13C NMR data are shown in Table 1, Figure 2 and Figure 3 ; High-resolution electrospray ionization mass spectrometry (HR-ESI-MS): m / z 399.1034 (C 21 1 19 8 + O, Calcd. 399.1080), and the mass spectrometry data are shown in Figure 1 . The relative molecular weight was determined to be 398, and the molecular formula was C 21 1 18 8 1 O with thirteen degrees of unsaturation. The 1H-NMR spectrum of 1 showed one methyl δ H 1.78 (s, H3-16), five methylenes δ H 4.28 (dt, J J = 16.9, 3.0 Hz, H-1a), 4.57 (dd, J J = 16.9, 1.5 Hz, H-1b), 2.64 (d, J J = 18.2 Hz, H-4a), 2.76 (ddt, J J = 18.2, 10.0, 3.0 Hz, H-4b), 3.53 (m, H2-17). The 13C-NMR spectrum of 1 showed twenty-one carbon resonances, including six aromatic carbons [δ 13 117.2 (C-6), 156.3 (C-7), 109.8 (C-12), 146.8 (C-13), 117.2 (C-14), and 143.1 (C-15)], four olefinic carbons [δ C -137.9 (C-1'), 132.2 (C-2'), 143.6 (C-3'), and 124.7 (C-4')], and three carbonyls [δ C 195.7 (C-9), 165.8 (C-11), and 167.6 (C-5')]. The 1H and 13C NMR spectral data of 1 were similar to those of acetosellin, and the side chain was no longer heptene but pentadienoic acid. The HMBC correlation of δ C 6.13 (H-4') with δ 1 167.6 (C-5') further confirmed the carboxyl group at the C-5' position in 1. 13 13 H 6.13 (H-4') with δ C 167.6 (C-5') further confirmed the carboxyl group at the C-5' position in 1. Figures 2 - 8 for H-1' / H-2' / H-3' / H-4' of1 H- 1 H COSY correlations and δ H 7.61 (H-1') and δ C 143.1 (C-15), δ C 117.2 (C-14), δ H 7.45 (H-3') and δ C 137.9 (C-1'), δ C 132.2 (C-2'), δ C The HMBC correlations of 124.7 (C-4') with 7.61 (H-1'), 143.1 (C-15), 117.2 (C-14), 7.45 (H-3'), 137.9 (C-1'), and 132.2 (C-2') indicate that the pentadienoic acid is attached to the benzene ring at C-15.

[0045] Compound acetosellin C (2): Pale yellow solid. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS): m / z 383.1132 (C 21 H 19 O7 - , Cacl. 383.1131), and the mass spectrometry data are shown in Figure 9 . A careful comparison of the 1D NMR data of 2 and 1 indicates that they are analogues with similar structures. Compound 2 has one less carbonyl group than 1, which is attributed to the replacement of the C-5' carbonyl group in 1 by the methylene signal H-5' (δ H 4.06, d, J = 4.9 Hz) in 2.

[0046] To determine the absolute configurations of compounds 1 and 2, the electronic circular dichroism (ECD) spectra of all possible stereoisomers of 1 and 2 were calculated using time-dependent density functional theory (TD-DFT) of quantum mechanics ( Figure 18 ). Figures 12 - 18 By comparing the calculated ECD spectra of the isomers (3 S , 8 S ), (3 R , 8 S ), (3 S , 8 R ), and (3 R , 7 R ), their absolute stereochemistries were determined to be 8 S . Interestingly, the chiral carbon (C-3) on the side chain has little effect on the overall ECD spectrum. First, the observed 13 C NMR data were compared with the calculated values of the two isomers by using carbon spectrum calculation and DP4+ probability analysis to determine the absolute configuration of C-3 in compounds 1 and 2. The experimental 13 C NMR data of 1 and 2 were compared with those of 3 SThe calculated data of the isomers matched, with DP4+ probabilities of 80.05% and 68.10% respectively ( Figure 19 and Figure 21 ), confirming that their absolute configurations are 3 S and 8 S .

[0047] Table 1. 1H and 1 13C NMR and HMBC data of Compounds 1 and 2 (500 MHz, DMSO- 13 d 6 )

[0048] Table 2. Boltzmann distribution optimization of acetosellin B at 298.13 K Configuration Conformer E (Hartree) E (kcal / mol) Population (%) 3R 1 -1412.615406870 -886430.293964994 0.98% 2 -1412.618462050 -886432.211120996 25.04% 3 -1412.619069080 -886432.592038391 47.62% 4 -1412.614987030 -886430.030511195 0.63% 5 -1412.614817070 -886429.923859596 0.53% 6 -1412.618467920 -886432.214804479 25.19% 3S 1 -1412.618924350 -886432.501218869 19.37% 2 -1412.619372690 -886432.782556702 31.14% 3 -1412.618282680 -886432.098564527 9.82% 4 -1412.618704440 -886432.363223144 15.34% 5 -1412.618748580 -886432.390921436 16.08% 6 -1412.618120230 -886431.996625527 8.26% Table 3. Boltzmann distribution optimization of acetosellin C at 298.13 K Configuration Conformer E (Hartree) E (kcal / mol) Population (%) 3R 1 -1338.585718080 -839975.923952381 22.40% 2 -1338.582131300 -839973.673212063 0.50% 3 -1338.582727970 -839974.047628455 0.94% 4 -1338.583010110 -839974.224674126 1.27% 5 -1338.586097660 -839976.162142627 33.48% 6 -1338.586298280 -839976.288033683 41.41% 3S 1 -1338.585502010 -839975.788366295 10.47% 2 -1338.586074220 -839976.147433792 19.18% 3 -1338.585739500 -839975.937393645 13.46% 4 -1338.585935970 -839976.060680535 16.57% 5 -1338.584937050 -839975.433848246 5.75% 6 -1338.586630140 -839976.496279151 34.57% .

[0049] Example 2 Evaluation of anti-osteoporosis activity Wild-type AB strain zebrafish at 3 dpf were randomly selected and placed in 24-well plates. All tested zebrafish were treated with 25 μM prednisolone to establish an osteoporosis model. Meanwhile, the test groups were given 0.5, 1, 2, 4, and 8 μM of acetosellin B (1) and acetosellin C (2), and the positive group was given 60 μM of the positive drug etidronate disodium. After culturing at 28 °C for 5 days, they were sacrificed, fixed, decolorized, stained with alizarin red, photographed, and data were collected. The average optical density value of the zebrafish skull was statistically analyzed, and the statistical results were expressed as mean ± SD.

[0050] Figure 23 The results showed that acetosellin B (1) showed strong osteogenic activity at a concentration of 4 μM, with P < 0.05 compared with the model group; acetosellin C (2) showed strong osteogenic activity at a concentration of 2 μM, with P < 0.05 compared with the model group.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.​

Claims

1. A compound with anti-osteoporosis efficacy, characterized in that: The structural formula of the compound is as follows: 。 2. The preparation method of the compound with anti-osteoporosis efficacy according to claim 1, characterized in that, It includes the following steps: a. Take the fungus Talaromyces pinophilus (CICC 2707). After extraction with an organic solvent, an organic phase is obtained, and the organic phase is recovered to obtain an extract. b. Subject the extract to chromatographic separation to obtain the target compound.

3. The method for preparing the compound according to claim 2, wherein The organic solvent used in step a for extraction is ethyl acetate; the materials for chromatographic separation in step b are silica gel and C 18 ODS.

4. The method for preparing the compound according to claim 2, wherein, It includes the following steps: a. Take the fungus alaromyces pinophilus (CICC 2707) and inoculate it on a PDA plate. After culturing in an incubator at 28 °C for 2 - 3 days, transfer it to a wort liquid medium and continue culturing on a shaker for 14 days; b. After the fermentation is completed, filter to obtain the fermentation broth, extract it with ethyl acetate 1 - 3 times, combine the ethyl acetate solutions, and recover the solvent to obtain the ethyl acetate extract; c. Subject the ethyl acetate extract to column chromatography separation with normal-phase silica gel, elute it with an ethyl acetate - petroleum ether gradient with different volume ratios, collect the fractions to obtain fractions F1 - F28; according to TLC thin-layer analysis, combine fractions F16 - 28, and then separate it with a medium-pressure preparative chromatography ODS column, elute it with a methanol - water gradient, detect it at 254 and 310 nm to obtain fractions F29 - F47, and take fraction F39 among them for preparative liquid-phase HPLC separation. The mobile phase is CH3CN:H2O by volume ratio, the chromatographic column is Eclipse XDB-C18 column, with a specification of 9.4 × 250 mm, 5 µm, and compound 1 is separated; Take fraction F38 among them for preparative liquid-phase HPLC separation. The mobile phase is CH3CN:H2O, the chromatographic column is Eclipse XDB-C18 column, with a specification of 9.4 × 250 mm, 5 µm, and compound 2 is separated.

5. The method for preparing the compound according to claim 4, characterized in that, Step c is eluted with an ethyl acetate - petroleum ether gradient with a volume ratio of 1:0 - 0:

1.

6. The method for preparing the compound according to claim 4, characterized in that, In step c, separation is performed using a medium-pressure preparative chromatography ODS column, gradient elution is carried out with an aqueous methanol solution having a volume concentration of 10 % - 100 %, detection is performed at 254 and 310 nm, fractions F29 - F47 are obtained, and fraction F39 among them is taken for preparative liquid-phase HPLC separation. The mobile phase is CH3CN:H2O with a volume ratio of 23:77, the chromatographic column is an Eclipse XDB-C18 column, the specifications are 9.4 × 250 mm, 5 µm, the flow rate is 2.0 mL / min, and compound 1 with t R = 18 min is separated.

7. The method for preparing the compound according to claim 4, characterized in that, In step c, fraction F38 was taken for preparative liquid phase HPLC separation. The mobile phase was CH3CN:H2O with a volume ratio of 28:

72. The chromatographic column was Eclipse XDB-C18 column, with a specification of 9.4 × 250 mm, 5 µm, and the flow rate was 2.0 mL / min. Compound 2 with t R = 15 min was separated.

8. Use of the compound as claimed in claim 1 or 2 and its salts or derivatives in the preparation of a drug for preventing and treating osteoporosis.

9. The application according to claim 8, characterized in that, The salts described include organic acid salts or inorganic acid salts.

10. The application according to claim 7, characterized in that, Prepare the compound into pills, tablets, capsules, granules, powder injections, mixtures, powders, sprays with a pharmaceutically acceptable carrier.

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