A norlignan compound and its extraction method and its application in preparing anti-postmenopausal osteoporosis drug

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a norlignan compound and an extraction method thereof and an application thereof in a drug for treating postmenopausal osteoporosis (PMOP). The present invention discloses a norlignan compound, the structural formula of which is:; A method for extracting a norlignan compound, the extraction method mainly comprising taking the dried rhizome of Curculigo macrophylla, extracting twice with 95% ethanol under 60°C, each time for 3 hours, combining the extracts and performing reduced pressure distillation until no extract remains, to obtain an ethanol extract of Curculigo macrophylla. The present invention provides a preparation method and an identification process of three novel norlignan compounds from Curculigo macrophylla for treating PMOP, showing that the three compounds have good effects of promoting bone formation and regulating bone metabolism.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a norlignan compound and an extraction method thereof, and application of the compound in preparing an anti-postmenopausal osteoporosis drug. Background Art

[0002] Postmenopausal osteoporosis (PMOP) is a chronic metabolic bone disease that mainly affects postmenopausal women. With the increasingly obvious trend of population aging in my country and the large base of the elderly population, the number of postmenopausal women suffering from osteoporosis has reached more than 180 million, and it is predicted that by 2030, the number of patients with postmenopausal osteoporosis in my country will reach 300 million; at the same time, PMOP brings a series of complications, such as spinal fractures, hip fractures, distal radius fractures and humeral fractures. It is estimated that by 2035, the number of patients with osteoporotic fractures in my country will reach 4.83 million, and by 2050 it is expected to reach 5.99 million; it can be seen that PMOP has become a global public health issue that seriously threatens the bone health of postmenopausal women, and the prevention and treatment of PMOP is urgent.

[0003] The main treatment drugs for PMOP are bisphosphonates, denosumab, selective estrogen receptor modulators and anabolic therapy. Long-term use of these drugs may lead to serious complications such as renal insufficiency and pulmonary edema, posing a threat to the overall health of patients. On the other hand, menopausal hormone therapy (MHT) can relieve menopausal symptoms by using estrogen or in combination with progesterone. However, the use of MHT is accompanied by some potential risks and side effects, including an increased risk of cardiovascular events, thromboembolic diseases and breast cancer. In view of the poor efficacy of existing clinical drugs for the treatment of PMOP, various adverse reactions and the many defects of MHT therapy, it is of great significance to develop new anti-osteoporosis drugs that are efficient, fast-acting and safe.

[0004] Traditional medicines are the product of human beings' understanding and utilization of nature in long-term medical practice. They have mild effects and few side effects. Many traditional Chinese medicines for kidney tonification and classic kidney-tonifying prescriptions have shown good results and unique advantages in improving and treating PMOP, such as Epimedium, Eucommia, Liuwei Dihuang Decoction, etc. Studies have pointed out that the Drynaria fortunei in the kidney-tonifying and blood-activating prescription contains flavonoids, which can play a role similar to estrogen, improve hormone levels, regulate bone metabolism, inhibit osteoclast activity, promote bone formation, reduce bone loss, and increase bone density.

[0005] Curculigo capitulata (Lour.) Kuntze is a perennial herb of the genus Curculigo (Curculigo Gaertn) of the family Curculigoaceae. It is native to southern China and distributed in southern, central, southeastern and southwestern China. It is rich in resources. Curculigo is a traditional Chinese medicine for tonifying the kidney and strengthening yang. It was first recorded in Leigong Paozhi Lun written by Lei Xiao in the Southern Dynasty. It has the effects of "strengthening kidney yang, strengthening tendons and bones, and removing cold and dampness". Plants belonging to the genus Curculigo also have the effect of warming and tonifying kidney yang, such as Curculigo capitulata. According to the theory of "kidney deficiency leading to impotence", Curculigo capitulata, which has the effect of tonifying the kidney, is very likely to contain ingredients with anti-PMOP activity, but no reports have been seen so far. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a norlignan compound and an extraction method thereof and an application thereof in the preparation of an anti-postmenopausal osteoporosis drug, which can effectively solve the problems mentioned in the prior art.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] A norlignan compound, the structural formula of the compound is:

[0009]

[0010] The compound is extracted from Curculigo macrophylla, which is a Curculigo macrophylla of the Curculigo macrophylla group of the genus Curculigo.

[0011] Preferably, a method for extracting norlignan compounds comprises the following steps:

[0012] Take the dried rhizome of Curculigo macrophylla, extract it with 95% ethanol under reflux for 2 times at 60°C, each time for 3 hours, combine the extracts and distill under reduced pressure until no extract remains, to obtain the ethanol extract of Curculigo macrophylla;

[0013] The ethanol extract of Curculigo orchioides is extracted with weak acid water, the obtained extract is mixed with an equivalent amount of weak alkaline water, and then extracted with ethyl acetate for 3 times, and the extract is concentrated to obtain an ethyl acetate extract;

[0014] The ethyl acetate extract is separated by chromatography and eluted to obtain fractions Fr.2.1 and Fr.2.2. The Fr.2.2 column is separated into fractions Fr.2.2.1 and Fr.2.2.2. Fraction Fr.2.2.1 is prepared and purified to obtain compounds 1 and 3. Fr.2.2.2 is separated and purified to obtain compound 2.

[0015] Preferably, the ethyl acetate extract is separated by silica gel column chromatography and eluted with dichloromethane:methanol in a volume ratio of 50:1 to 10:1 to obtain fractions Fr.2.1 and Fr.2.2; fraction Fr.2.2 is separated by a medium pressure preparative chromatography column and a methanol:water solvent in a volume ratio of 100:1 to 10:1 to obtain fractions Fr.2.2.1 and Fr.2.2.2; fraction Fr.2.2.1 is purified by high pressure liquid chromatography at a flow rate of 2.5 ml / min using an acetonitrile:water solvent in a volume ratio of 40:60 to obtain compounds 1 and compound 3; fraction Fr.2.2.2 is separated by a dextran gel-based chromatography column and a dichloromethane:methanol volume ratio of 1:1 to obtain compound 2.

[0016] Preferably, a norlignan compound is used in the preparation of an anti-postmenopausal osteoporosis drug.

[0017] The spectral data of the norlignan compound described in the present invention are determined by modern spectral means such as high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, and the identification results are as follows:

[0018] Compound 1, amorphous powder; UV(MeOH)λmax(logε)190(4.04),204.5(4.18),279(3.19)nm; IR νmax:3442.2,1631.4,1592.2,1384.2,1350.3,765.9cm-1; 1H(600MHz,MeOH)and 13C(175MHz,MeOH)NMR; (+)-HRESIMS m / z 339.0880[M+Na]+(calcd.for C17H16NaO6339.0845);

[0019] Compound 2, amorphous powder; UV(MeOH)λmax(logε)191(3.98),203(4.13)nm,283(3.10)nm; IR νmax:3434.3,2951.6,1652.1,1581.7,1518.7,1453.2,1293.6,1092.6,964.4,879.6,814.7,783.1,639.5cm-1; 1H (600MHz, MeOH)and 13C(175MHz,MeOH)NMR; (+)-HRESIMS m / z 329.1030[MH]-(calcd.for C18H17O6-329.1025);

[0020] Compound 3, amorphous powder; UV(MeOH)λmax(logε)191.5(4.11),203.5(3.99)nm,279.5(2.98)nm; IR νmax:3406.8,1649.4,1580.5,1518.6,1452.1,1258.4,1092.8,880.2,815.0,784.25cm-1; 1H (600MHz, MeOH) and 13C (175MHz, MeOH) NMR; (+)-HRESIMS m / z 329.1030[MH]-(calcd.for C18H17O6-329.1025).

[0021] Compared with the known prior art, the technical solution provided by the present invention has the following beneficial effects:

[0022] The present invention obtains the compound of the present invention from the large-leaf Curculigo plant for the first time, finds a new way to obtain the nolignin compounds, and identifies three nolignin compounds. At the same time, the three nolignin compounds extracted from the large-leaf Curculigo are tested for in vivo and in vitro efficacy. The test results show that the three nolignin compounds significantly enhance the alkaline phosphatase activity of MC3T3-E1 cells, promote the expression of ERα, the expression of bone-related proteins OSX, Runx2 and OCN, and promote the expression of calcium and phosphorus levels, and have a good effect on promoting bone formation and regulating bone metabolism. Therefore, the nolignin compounds extracted from the large-leaf Curculigo of the present invention provide new compounds for the development of new drugs for treating postmenopausal osteoporosis, and have a good development and application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 Compounds 1-3 of the present invention promote the proliferation of MC3T3-E1;

[0025] Figure 2 Compounds 1-3 of the present invention enhance the alkaline phosphatase activity of MC3T3-E1 cells;

[0026] Figure 3 Compounds 1-3 of the present invention promote the proliferation of MCF-7;

[0027] Figure 4 Compounds 1-3 of the present invention promote the increase of ERα expression;

[0028] Figure 5 Compounds 1-3 of the present invention promote the expression of MC3T3-E1 osteogenic-related proteins;

[0029] Figure 6 Compounds 1-3 of the present invention promote the expression of MC3T3-E1 osteogenesis-related gene mRNA;

[0030] Figure 7 Compounds 1-3 of the present invention promote the expression of ERα in PMOP mice;

[0031] Figure 8 Compounds 1-3 of the present invention promote the expression of calcium and phosphorus in PMOP mice;

[0032] Fig. 9 Compounds 1-3 of the present invention promote the expression of osteogenesis-related proteins in PMOP mice;

[0033] Fig.10 Compounds 1-3 of the present invention promote the expression of osteogenesis-related mRNA in PMOP mice. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The present invention will be further described below in conjunction with the embodiments.

[0036] Embodiment 1:

[0037] A method for extracting norlignan compounds comprises the following steps:

[0038] Take the dried rhizome of Curculigo macrophylla, extract it with 95% ethanol under reflux for 2 times at 60°C, each time for 3 hours, combine the extracts and distill under reduced pressure until no extract remains, to obtain the ethanol extract of Curculigo macrophylla;

[0039] The ethanol extract of Curculigo orchioides is extracted with weak acid water, the obtained extract is mixed with an equivalent amount of weak alkaline water, and then extracted with ethyl acetate for 3 times, and the extract is concentrated to obtain an ethyl acetate extract;

[0040] The ethyl acetate extract is separated by chromatography and eluted to obtain fractions Fr.2.1 and Fr.2.2. The Fr.2.2 column is separated into fractions Fr.2.2.1 and Fr.2.2.2. Fraction Fr.2.2.1 is prepared and purified to obtain compounds 1 and 3. Fr.2.2.2 is separated and purified to obtain compound 2.

[0041] The ethyl acetate extract was separated by silica gel column chromatography and eluted with dichloromethane:methanol in a volume ratio of 50:1 to 10:1 to obtain fractions Fr.2.1 and Fr.2.2; fraction Fr.2.2 (9.8 g) was separated by medium pressure preparative chromatography column with methanol:water solvent in a volume ratio of 100:1 to 10:1 to obtain fractions Fr.2.2.1 and Fr.2.2.2; fraction Fr.2.2.1 (6.7 g) was purified by high pressure liquid chromatography with acetonitrile:water solvent in a volume ratio of 40:60 at a flow rate of 2.5 ml / min to obtain compound 1 (168 mg, tR 17.3min) and compound 3 (250mg, tR28.5min); fraction Fr.2.2.2 (1.8g) was separated using a dextran gel-based chromatographic column with a volume ratio of 1:1 dichloromethane:methanol to obtain compound 2 (150.0mg).

[0042] Embodiment 2:

[0043] A norlignan compound, the spectral data were determined by modern spectral methods such as high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, and the identification results are as follows:

[0044] Compound 1, amorphous powder; UV(MeOH)λmax(logε)190(4.04),204.5(4.18),279(3.19)nm; IR νmax:3442.2,1631.4,1592.2,1384.2,1350.3,765.9cm-1; 1H(600MHz,MeOH)and 13C(175MHz,MeOH)NMR; (+)-HRESIMS m / z 339.0880[M+Na]+(calcd.for C17H16NaO6339.0845);

[0045] Compound 2, amorphous powder; UV(MeOH)λmax(logε)191(3.98),203(4.13)nm,283(3.10)nm; IR νmax:3434.3,2951.6,1652.1,1581.7,1518.7,1453.2,1293.6,1092.6,964.4,879.6,814.7,783.1,639.5cm-1; 1H (600MHz, MeOH)and 13C(175MHz,MeOH)NMR; (+)-HRESIMS m / z 329.1030[MH]-(calcd.for C18H17O6-329.1025);

[0046] Compound 3, amorphous powder; UV(MeOH)λmax(logε)191.5(4.11),203.5(3.99)nm,279.5(2.98)nm; IR νmax:3406.8,1649.4,1580.5,1518.6,1452.1,1258.4,1092.8,880.2,815.0,784.25cm-1; 1H (600MHz, MeOH) and 13C (175MHz, MeOH) NMR; (+)-HRESIMS m / z 329.1030[MH]-(calcd.for C18H17O6-329.1025);

[0047] The H NMR and C NMR data of compounds 1-3 are shown in Table 1:

[0048] Table 1: H NMR and C NMR data of compounds 1-3

[0049]

[0050] Example 3: In vitro efficacy test of compounds 1-3 against PMOP:

[0051] (1) Effects of compounds 1-3 on MC3T3-E1 cell proliferation:

[0052] In this example, five different compound concentration gradients (0.001, 0.01, 0.1, 1, 10 μM) were set up in the experiment, 1 μM estradiol (E2) was used as a positive control, and the untreated Normal group was used as a negative control;

[0053] The experimental results are as follows Figure 1As shown, the results showed that compound 1 significantly promoted the proliferation of MC3T3-E1 cells at concentrations of 0.1 μM and above, which was statistically significant compared with the Normal group (p<0.05); its effect reached a peak at 10 μM, which was significantly different from the E2 treatment (p<0.05); compound 2 showed a cell proliferation promoting effect at 0.01 μM, and the effect was most significant at 1 μM (p<0.001), which was significantly different from E2 (p<0.05), and the proliferation effect was weakened at a concentration of 10 μM; compound 3 had no obvious effect on cell proliferation at 0.001 μM, but began to show a significant proliferation promoting effect at 0.1 μM, and reached the maximum effect at 10 μM (p<0.001), which was better than the positive control E2; these results showed that compounds 1-3 all have the ability to significantly promote the proliferation of MC3T3-E1 cells, and the effect shows a certain dose dependence.

[0054] (2) Effects of compounds 1-3 on alkaline phosphatase activity in MC3T3-E1 cells:

[0055] In this example, an alkaline phosphatase (ALP) kit was used to evaluate the effects of compounds 1-3 on the ALP activity of MC3T3-E1 cells. MC3T3-E1 cells were cultured in an osteogenic induction medium containing 50 μg / mL ascorbic acid and 5 mM sodium β-glycerophosphate. The experimental groups included a Control group (untreated), an E2 group (1 μM, positive control), and three different concentration groups of compounds 1-3 (0.1, 1, 10 μM);

[0056] The experimental results are as follows Figure 2 As shown, the results showed that compound 1 significantly enhanced the alkaline phosphatase activity of MC3T3-E1 cells at concentrations of 1 μM and 10 μM, which was statistically significant compared with the Control group (p<0.01), among which the activity improvement was most obvious at 10 μM, close to the positive control E2; compound 2 showed an alkaline phosphatase activity-promoting effect at all concentrations, especially at 1 μM, the effect was significant (p<0.001), and was comparable to the E2 group; compound 3 showed a significant alkaline phosphatase activity-promoting effect at 1-10 μM, and reached a peak at 10 μM (p<0.001); these results showed that compounds 1-3 can significantly enhance the alkaline phosphatase activity of MC3T3-E1 cells, and show a certain dose dependence.

[0057] (3) Effects of compounds 1-3 on promoting ERα expression in MC3T3-E1:

[0058] As women age, their ovarian function gradually declines, and their estrogen levels also decrease accordingly. The complex pathological mechanism of PMOP is closely related to this. The significant decrease in estrogen levels is the dominant factor in bone loss in PMOP. The decrease in estrogen not only leads to perforation of trabecular bone and rapid apoptosis of osteoclasts, but also reduces the function of osteoblasts, causing a gradual decrease in bone wall width. The decrease in estrogen levels in menopausal women will reduce the expression of osteoprotegerin (OPG) and increase the expression of receptor activator of nuclear factor kappa B ligand (RANKL), thereby increasing osteoclasts and decreasing osteoblasts, and ultimately causing bone loss and PMOP. Therefore, it is very important to detect whether compounds 1-3 have estrogen-like effects to confirm whether natural products can treat PMOP.

[0059] In this example, the experiment first used the CCK8 method to detect whether the MCF-7 cells treated with compounds 1-3 proliferated; then the classic E-Screen experiment was used, and the results were as follows. Figure 3 As shown, it was found that compounds 1-3 (0.1, 1 and 10 μM) significantly promoted the proliferation of the estrogen-dependent estrogen receptor-positive (ER+) breast cancer cell line MCF-7, and showed a clear dose-time dependent trend;

[0060] Next, Western blot was used to detect the expression of ERα in MC3T3-E1 cells after treatment with compounds 1-3. Figure 4 As shown, compared with E2 (1 μM), compounds 1-3 can effectively promote ERα expression, and the results are statistically significant (p < 0.05); the most effective concentrations of compounds 1 and 3 are 10 μM (p < 0.001), and the most effective concentration of compound 2 is 1 μM (p < 0.001).

[0061] (4) Effects of compounds 1-3 on promoting osteogenic differentiation of MC3T3-E1:

[0062] OCN is a protein specifically expressed in osteoblasts. It participates in the mineralization of bone matrix and is one of the markers of osteogenic differentiation. Runx2 is the main transcription factor for osteogenic differentiation. It regulates the expression of multiple osteogenic-related genes and is essential for the development and maintenance of bone tissue. In addition, OSX is another key osteogenic differentiation transcription factor. It acts downstream of Runx2, regulates the synthesis and mineralization of matrix proteins during bone formation, and plays an important role in promoting osteocyte differentiation. The synergistic effect of Runx2 and OSX is essential for bone development and bone maintenance.

[0063] In this example, Western blot was used to detect the expression of osteogenic-related proteins (OSX, Runx2, and OCN), and RT-qPCR was used to detect the expression levels of SP7, Runx2, and Bglap genes and quantitatively analyze them; the results are shown in Figure 5 and Figure 6 As shown, compounds 1-3 can effectively promote the expression of osteogenic markers OCN (Bglap), Runx2 and OSX (SP7). On the other hand, ICI 182,780 can inhibit this promotion, suggesting that this osteogenic effect is related to estrogen receptors.

[0064] Example 4: In vivo efficacy test of compounds 1-3 against PMOP:

[0065] In this example, the PMOP mouse model was constructed by inducing bilateral ovariectomy (OVX) to simulate osteoporosis caused by decreased estrogen levels after menopause; the groups were: Sham (sham operation group), OVX (bilateral ovariectomy group), E2 (1 mg / kg estradiol), A1 and A2 (2 mg / kg and 4 mg / kg of compound 1), B1 and B2 (2 mg / kg and 4 mg / kg of compound 2), C1 and C2 (2 mg / kg and 4 mg / kg of compound 3).

[0066] (1) Effects of compounds 1-3 on promoting ERα and calcium phosphate expression:

[0067] In this example, the effects of compounds 1-3 on bone metabolism were evaluated by measuring the expression levels of ERα, calcium and phosphorus in serum; the results are shown in FIG. Figure 7 As shown in the figure, the expression of ERα in OVX mice was significantly reduced, while the expression of ERα in the E2 treatment group and compounds 1-3 at various concentrations was significantly increased, especially at a concentration of 4 mg / kg, where the effect of compounds 1-3 was the most significant, and the ERα level was close to or even slightly higher than that in the control group. Figure 8 As shown, the serum calcium and phosphorus concentrations of the OVX group mice were significantly lower than those of the control group, however, the E2 and compound 1-3 treatment groups significantly improved the calcium and phosphorus levels at concentrations of 2 mg / kg and 4 mg / kg, especially compound 3 at 4 mg / kg.

[0068] These results indicate that compounds 1-3 can effectively promote the expression of ERα and restore calcium and phosphorus levels, showing their potential application value in the treatment of osteoporosis.

[0069] (2) Effects of Compounds 1-3 on the Expression of Osteogenesis Marker Proteins and Genes:

[0070] In this example, Western Blot and qPCR analysis were performed on mice in different treatment groups (sham, OVX, A2, B2, C2) to evaluate the effects of compounds 1-3 on the expression of related proteins and genes in bone tissue; the Western Blot results are shown in Fig. 9 As shown in Figure 2, the protein expressions of OSX, Runx2 and OCN in the OVX group were significantly lower than those in the sham group (p<0.05), while in the A2, B2 and C2 treatment groups, the expression levels of OSX, Runx2 and OCN were significantly increased (p<0.001); qPCR results are shown in Figure 2 Fig.10 As shown, the expression of Sp7, Runx2 and Bglap genes in mice of the OVX group was significantly decreased (p<0.05), while those in the A2, B2 and C2 groups showed significant upregulation (p<0.01);

[0071] These results indicate that compounds 1-3 can significantly improve the expression of osteoblast-related proteins and mRNA in the bone tissue of OVX mice, showing their potential role in promoting bone formation.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

1. A norlignan compound, characterized in that: The structural formula of this class of compounds is:

2. Use of a norlignan compound according to claim 1 in the preparation of an anti-postmenopausal osteoporosis drug.

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