KR20220152070A - Composition for preventing or treating of osteoclast differentiation comprising barley grass extracts or alkaloids frction therefrom or alkaloid compounds isolated therefrom - Google Patents

Abstract

The present invention relates to an osteoclast differentiation inhibitory composition comprising a barley sprout extract or an alkaloid fraction thereof or an alkaloid compound isolated therefrom, and specifically, a barley sprout alkaloid extract or an alkaloid compound isolated therefrom is confirmed to inhibit osteoclast formation and inhibit bone loss, and thus it is expected to contribute to the prevention or treatment of osteolytic bone disease by using the barley sprout alkaloid extract or the alkaloid compound isolated therefrom.

Description

Composition for preventing or treating osteoclast differentiation comprising barley grass extracts or alkaloids frction therefrom or alkaloid compounds isolated therefrom}

The present invention relates to a composition for inhibiting osteoclast differentiation comprising barley sprout alkaloid extract or an alkaloid compound isolated therefrom.

Osteoclasts are bone resorbing cells distinct from bone marrow hematopoietic stem cells in the bone marrow (Xu & Teitelbaum, 2013). Excessive osteoclastogenesis and/or activity due to increased nuclear factor-κB ligand receptor activator (RANKL)/RANK signaling leads to osteolytic bone diseases such as osteoporosis and rheumatoid arthritis (Kearns et al., 2008). Therefore, as a strategy for treating and preventing osteolytic bone disease, inhibiting the RANKL/RANK signaling pathway can suppress elevated osteoclast formation and/or activity. Recently, interest in the treatment and prevention of osteolytic bone disease using natural products has increased (An et al., 2016; Kim et al., 2018; Sethi et al., 2007).

On the other hand, barley ( Hordeum vulgare var. Hexastichon (L.) Aschers.) is the world's fourth most important grain crop after wheat, maize and rice. Barley is a species of the genus Hordeum belonging to the family Gramineae. It has been cultivated as a major domestic food crop in Europe, North Africa and several Asian countries since 10,000 years ago and is widely consumed until now. 80-90% of barley production is used as animal feed, malting for beer production and nutritious food (Goupy et al., 1999; Kremer et al., 2009; Qingming et al., 2010). Barley is also used to treat several ailments, including jaundice, various inflammatory and cardiovascular diseases. Previous studies have shown that barley is rich in aminophenolic acid derivatives, benzoic acid and cinnamic acid derivatives, tannins, flavonoids such as flavonols, chalcones, flavones, flavanones and proanthosinidins, alkaloids, and cyanoglycosides. It contains poly/acid amines with various biological activities such as antioxidant, acetylcholine, antidepressant, lipid-lowering and anti-ulcer (Beri et al., 2007; Kremer et al., 2009; Qingming et al., 2010; Yamaura et al., 2012). In particular, young barley sprouts are known as a popular functional food in Korea, China, and Japan, and are also used to treat various inflammatory and cardiovascular diseases (Thatiparthi et al., 2019). Evidence is accumulating that barley sprouts have immune stimulating abilities and are beneficial for several health problems, such as cancer, type 2 diabetes, obesity, excess cholesterol, anemia, circulatory disorders, and kidney disorders (Lahouar et al., 2015). However, currently there is limited information on the osteoclastogenesis inhibitory activity of active compounds isolated from barley sprouts.

Korean Patent Registration No. 2184812, which is a prior art, describes a composition for preventing or treating osteoporosis containing an extract of barley, but the barley of the present invention ( Hordeum vulgare var. Hexastichon (L.) Aschers.) bud alkaloid extract and the alkaloid compound isolated from the osteoclast formation inhibitory effect are not described, so there is a difference. Korean Patent Registration No. 1959731 discloses a composition for preventing or treating menopausal disorders containing an extract of barley sprouts, but also does not disclose an alkaloid extract of barley sprouts or an alkaloid compound isolated therefrom of the present invention.

Korean Patent No. 2184812, composition for preventing or treating osteoporosis containing barley extract, registered on November 24, 2020. Korean Patent Registration No. 1959731, Composition for preventing or treating menopausal disorders containing sprout barley extract, registered on March 13, 2019.

Wilson, S. R., Peters, C., Saftig, P., & Brmme, D. (2009). Cathepsin K activity-dependent regulation of osteoclast actin ring formation and bone resorption. Journal of Biological Chemistry, 284, 2584-2592. Tran, P. T., Dat, N. T., Dang, N. H., Van Cuong, P., Lee, S., Hwangbo, C., Van Minh, C., & Lee, J.-H. (2019). Ganomycin I from Ganoderma lucidum attenuates RANKL-mediated osteoclastogenesis by inhibiting MAPKs and NFATc1. Phytomedicine, 55, 1-8. Franzoso, G., Carlson, L., Xing, L., Poljak, L., Shores, E. W., Brown, K. D., Leonardi, A., Tran, T., Boyce, B. F., & Siebenlist, U. (1997). Requirement for NF-κB in osteoclast and B-cell development. Genes & development, 11, 3482-3496. Grigoriadis, A. E., Wang, Z.-Q., Cecchini, M. G., Hofstetter, W., Felix, R., Fleisch, H. A., & Wagner, E. F. (1994). c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science, 266, 443-448. Karin, M. (1999). How NF-κB is activated: the role of the IκB kinase (IKK) complex. Oncogene, 18, 6867-6874. Abu-Amer, Y. (2013). NF-κB signaling and bone resorption. Osteoporosis International, 24, 2377-2386. Wada, T., Nakashima, T., Hiroshi, N., & Penninger, J. M. (2006). RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends in molecular medicine, 12, 17-25. Boyle, W. J., Simonet, W. S., & Lacey, D. L. (2003). Osteoclast differentiation and activation. Nature, 423, 337-342. Takayanagi, H., Kim, S., Koga, T., Nishina, H., Isshiki, M., Yoshida, H., Saiura, A., Isobe, M., Yokochi, T., & Inoue, J.-i. (2002). Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Developmental cell, 3, 889-901. Goupy, P., Hugues, M., Boivin, P., & Amiot, M.J. (1999). Antioxidant composition and activity of barley (Hordeum vulgare) and malt extracts and of isolated phenolic compounds. Journal of the Science of Food and Agriculture, 79, 1625-1634. Kremer, RJ, & Ben-Hammouda, M. (2009). Allelopathic Plants. 19. Barley (Hordeum vulgare L). Allelopathy Journal, 24. Qingming, Y., Xianhui, P., Weibao, K., Hong, Y., Yidan, S., Li, Z., Yanan, Z., Yuling, Y., Lan, D., & Guoan, L. (2010). Antioxidant activities of malt extract from barley (Hordeum vulgare L.) toward various oxidative stress in vitro and in vivo. Food chemistry, 118, 84-89. Xu, F., & Teitelbaum, S.L. (2013). Osteoclasts: new insights. Bone research, 1, 11-26. Kearns, A.E., Khosla, S., & Kostenuik, P.J. (2008). Receptor activator of nuclear factor κB ligand and osteoprotegerin regulation of bone remodeling in health and disease. Endocrine reviews, 29, 155-192. An, J., Hao, D., Zhang, Q., Chen, B., Zhang, R., Wang, Y., & Yang, H. (2016). Natural products for treatment of bone erosive diseases: the effects and mechanisms on inhibiting osteoclastogenesis and bone resorption. International immunopharmacology, 36, 118-131. Kim, EH, Jo, CS, Ryu, SY, Kim, SH, & Lee, JY (2018). Anti-osteoclastogenic diacetylenic components of Dendropanax morbifera. Archives of pharmacal research, 41, 506-512. Sethi, G., & Aggarwal, BB (2007). Mending the bones with natural products. Chemistry & biology, 14, 738-740. Beri, V., & Gupta, R. (2007). Acetylcholinesterase inhibitors neostigmine and physostigmine inhibit induction of alpha-amylase activity during seed germination in barley, Hordeum vulgare var. Jyoti. Life sciences, 80, 2386-2388. Thatiparthi, J., Dodoala, S., Koganti, B., & Kvsrg, P. (2019). Barley grass juice (Hordeum vulgare L.) inhibits obesity and improves lipid profile in high fat diet-induced rat model. Journal of ethnopharmacology, 238, 111843. Lahouar, L., El-Bok, S., & Achour, L. (2015). Therapeutic potential of young green barley leaves in prevention and treatment of chronic diseases: an overview. The American journal of Chinese medicine, 43, 1311-1329. Tran, PT, Dat, NT, Dang, NH, Van Cuong, P., Lee, S., Hwangbo, C., Van Minh, C., & Lee, J.-H. (2019). Ganomycin I from Ganoderma lucidum attenuates RANKL-mediated osteoclastogenesis by inhibiting MAPKs and NFATc1. Phytomedicine, 55, 1-8.

Wilson, SR, Peters, C., Saftig, P., & Brmme, D. (2009). Cathepsin K activity-dependent regulation of osteoclast actin ring formation and bone resorption. Journal of Biological Chemistry, 284, 2584-2592. Tran, PT, Dat, NT, Dang, NH, Van Cuong, P., Lee, S., Hwangbo, C., Van Minh, C., & Lee, J.-H. (2019). Ganomycin I from Ganoderma lucidum attenuates RANKL-mediated osteoclastogenesis by inhibiting MAPKs and NFATc1. Phytomedicine, 55, 1-8. Franzoso, G., Carlson, L., Xing, L., Poljak, L., Shores, EW, Brown, KD, Leonardi, A., Tran, T., Boyce, BF, & Siebenlist, U. (1997) . Requirement for NF-κB in osteoclast and B-cell development. Genes & development, 11, 3482-3496. Grigoriadis, AE, Wang, Z.-Q., Cecchini, MG, Hofstetter, W., Felix, R., Fleisch, HA, & Wagner, EF (1994). c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science, 266, 443-448. Karin, M. (1999). How NF-κB is activated: the role of the IκB kinase (IKK) complex. Oncogene, 18, 6867-6874. Abu-Amer, Y. (2013). NF-κB signaling and bone resorption. Osteoporosis International, 24, 2377-2386. Wada, T., Nakashima, T., Hiroshi, N., & Penninger, J. M. (2006). RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends in molecular medicine, 12, 17-25. Boyle, WJ, Simonet, WS, & Lacey, D.L. (2003). Osteoclast differentiation and activation. Nature, 423, 337-342. Takayanagi, H., Kim, S., Koga, T., Nishina, H., Isshiki, M., Yoshida, H., Saiura, A., Isobe, M., Yokochi, T., & Inoue, J. -i. (2002). Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Developmental cell, 3, 889-901.

An object of the present invention is to provide a composition for inhibiting osteoclast differentiation comprising barley sprout alkaloid extract or an alkaloid compound isolated therefrom.

The present invention provides a composition for inhibiting osteoclast differentiation comprising barley sprout alkaloid extract or an alkaloid compound isolated therefrom.

The alkaloid extract,

Extracting the crushed barley sprouts with an organic solvent and concentrating under reduced pressure to prepare a crude extract of barley sprouts (Step 1);

The distilled water suspension of the crude barley extract obtained in step 1 was adjusted to pH 2 and then CHCl ₃ removing by extraction with a solution to obtain an acidic aqueous layer (step 2); and

The acidic aqueous layer of step 2 was adjusted to pH 10, then CHCl ₃ extracting with to obtain a concentrated alkaloid extract (step 3);

It may be manufactured by a manufacturing method comprising a.

At this time, the organic solvent is water, C1-4 lower alcohol, acetone, ethyl acetate, diethyl acetate, diethyl ether, benzene, chloroform ) and hexane, or a mixed solvent thereof, preferably C1-4 lower alcohol, more preferably methanol.

The alkaloid compound may include one or more selected from compounds 1 to 1 represented by the following formula [1].

Formula [1]

The alkaloid compounds are Hordeumin A (Compound 1), 3-(1-Pyrrolidiylmethyl)-1H-indole (Compound 2), (1H-indole-3-ylmethyl)(3-methylbutyl)amine (Compound 3), N,N At least one selected from the group consisting of -bis(indol-3-ylmethyl)methylamine (Compound 4) and 3-[2-[(indol-3-yl)methyl]indol-3-yl]methylindole (Compound 5) It may include, and the structures of the compounds 1 to 5 are the same as the formula [1]. As shown in the above structure, the alkaloid compound isolated from the barley sprout extract of the present invention includes an indole alkaloid compound.

The composition for inhibiting osteoclast differentiation of the present invention may suppress a decrease in the number of trabecular lines and a bone volume ratio (BS/BV), or an increase in trabecular separation.

The composition for inhibiting osteoclast differentiation can inhibit expression of p-p38 and c-Fos or degradation of IκBα, and inhibit nuclear translocation of NFATc1.

The composition for inhibiting osteoclast differentiation of the present invention reduces the number of osteoclasts, inhibits the formation of a filament actin ring of osteoclasts, inhibits the maturation of osteoclasts, inhibits the formation of bone resorption pits, or inhibits the formation of osteoclasts. it may be

The composition for inhibiting osteoclast differentiation of the present invention may also inhibit the expression of TRAP, Ctsk, DC-STAMP, OSCAR and MMP-9. Cathepsin K (CtsK) is known as an osteoclast marker, and its expression is induced by RANKL treatment. In addition, RANKL includes NF-κB and MAPK signaling pathways in the early signal transduction process, and activation of the MAPK signaling pathway induces the AP-1 transcription factor. As a heterodimer of the c-Jun and c-Fos transcription factors, AR-5 can inhibit RANKL-induced c-Fos expression and thereby inhibit RANKL-induced early signaling.

The composition for inhibiting osteoclast differentiation of the present invention can inhibit nuclear translocation of NFATc1. Nuclear Factor Of Activated T Cells 1 (NFATc1) is a major transcription factor in the process of osteoclastogenesis, and activation of NFATc1 results in CtsK, MMP-9, It is known to directly regulate the induction of numerous bone genes including OSCAR, TRAP and DC-STAMP. The composition for inhibiting osteoclast differentiation of the present invention can inhibit nuclear translocation of NFATc1 and significantly reduces the expression level of NFATc1 target genes including CtsK, MMP-9, OSCAR, TRAP and DC-STAMP, resulting in osteoclast formation can impede

The present invention provides a composition for inhibiting osteoclast differentiation comprising barley sprout alkaloid extract or an alkaloid compound isolated therefrom. Barley ( Hordeum vulgare var. Hexastichon (L.) Aschers.) is an annual or biennial plant belonging to the genus Barley in the rice family Poaceae. Barley sprout crude extract by extraction with one or more solvents selected from the group consisting of: acetate), diethyl acetate, diethyl ether, benzene, chloroform and hexane It is possible to prepare a barley sprout alkaloid extract by preparing and extracting it with chloroform in an acidic and basic solution.

As a method for extracting the crude barley sprout extract, any one of hot water extraction method, cold brew extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, and compression method may be selected and used. In addition, the desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method. The barley sprout alkaloid extract can be prepared by extracting the barley sprout crude extract extracted by the above solvent extraction method with chloroform in an acidic or basic solution. The prepared barley sprout alkaloid extract may be used as it is while being stored in a refrigerator after centrifugation, filtration and concentration followed by freeze-drying. In addition, a fraction further purified from the primary extract using various chromatography methods such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. Obtaining and purifying it to obtain the alkaloid compound represented by the formula [1].

The composition for inhibiting osteoclast differentiation of the present invention may be prepared as a pharmaceutical composition. The pharmaceutical composition of the composition for inhibiting osteoclast differentiation is preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, and most preferably 0.001 to 30% by weight based on the total weight of the entire pharmaceutical composition. may be added.

The pharmaceutical composition is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidulants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, for example, It is prepared by mixing starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, and acidulants are used. can be included Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base for the suppository, witepsol, macrogol, tween-61, cacao butter, laurin paper, glycerogeratin, and the like may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dose is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dose is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection and dermal application. Since the barley sprout alkaloid extract or the alkaloid compound isolated therefrom of the present invention has little toxicity and side effects, it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

The present invention provides a health functional food for inhibiting osteoclast differentiation, characterized in that it contains a barley sprout alkaloid extract or at least one selected from among compounds 1 to 9 isolated from the extract and represented by the above formula [1].

The health functional food is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, most preferably 0.001 to 10% by weight of barley sprout alkaloid extract or an alkaloid compound isolated therefrom based on the total weight of the total food. % can be added.

The health functional food includes the form of tablets, capsules, pills or liquids, and foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc.

The present invention provides a novel compound 5 represented by the following formula [2].

Formula [2]

The compound 5 was prepared by passing the EtOAc-soluble fraction of the crude barley sprout extract (HVA) onto a silica gel column (60 × 12 cm, _63-200 μM particle size, 63-200 μM particle size, Merck) may be obtained by chromatography and purified.

The present invention relates to an osteoclast differentiation inhibitory composition comprising a barley sprout extract or an alkaloid fraction thereof or an alkaloid compound isolated therefrom, and specifically, a barley sprout alkaloid extract or an alkaloid compound isolated therefrom inhibits osteoclast formation and It was confirmed to suppress bone loss, and it is expected to contribute to the prevention or treatment of osteolytic bone disease using this.

Figure 1 shows the effect of the barley sprout alkaloid extract (HVA) according to the present invention on the formation of osteoclasts. (A) TRAP staining micrographs (× 40) and the number of osteoclasts after treatment of RANKL-induced RAW264.7 cells with HVA at different concentrations. (B) TRAP staining micrographs (× 40) and the number of osteoclasts after treatment of M-CSF and RANKL-induced BMM cells with HVA at different concentrations. (C) Cell viability of BMM as a function of HVA concentration (mean ± SE (*p < 0.05 and **p < 0.01, versus vehicle-treated control, n = 3).
Figure 2 shows the effect of HVA on LPS-induced bone loss in ICR mice. (A) Three-dimensional μCT images of the femurs of LPS-injected mice treated with HVA at different concentrations. (B) Analysis of trabecular number (Tb. N), trabecular bone volume/tissue volume ratio (BS/TV) and trabecular separation (Tb. Sp) (n = 4 (8 legs). *p < 0.05, LPS treatment compared to the control group)
Figure 3 shows the chemical structure (A) of compounds 1-9 isolated from HVA and the selected 1H-1H COZY (blue bold line) and 1H-13C HMBC (red arrow line) correlations (B) of compounds 1-4. will be.
Figure 4 shows the effect of alkaloid compounds 1 to 9 isolated from HVA on osteoclast formation. (A) Number of osteoclasts in RANKL-induced RAW264.7 cells treated with compounds 1 to 9 (1 and 10 μM). (B, C) Photomicrographs (× 40) and osteoclast numbers of TRAP-positive osteoclasts by compound 4 or 5 treatment in RANKL and M-CSF-induced BMMs. (D) Cell viability cultured for 3 days in the presence of each concentration of compounds 4 and 5 in M-CSF-induced BMM (mean ± SE (** p < 0.01)
Figure 5 confirms the effect of compound 5 on actin ring formation and reuptake pit in RANKL-induced mouse BMM. (A) FITC-conjugated phalloidin staining pictures and the number of osteoclasts after culturing RANKL and M-CSF-induced BMMs in the presence of compound 5 at each concentration for 7 days. (B) Photographs of observed resorption pits after culturing M-CSF and RANKL-induced BMMs with Compound 5 at each concentration in a Corning OsteoAssay Surface 24-well plate for 7 days and a graph of pit area measurement thereof. (C) TRAP-positive osteoclasts (mean ± SE (*p < 0.05 , versus solvent-treated control, n = 3)
Figure 6 shows the effect of compound 5 on c-Fos and NFATc1 in RANKL-induced RAW264.7 cells. (A) RAW264.7 cells after stimulation with RANKL for 10 min in the presence of compound 5 at the indicated concentrations. Western blotting results of p-ERK, p-JNK and p-p38 MAPK. (B) After stimulation of RAW264.7 cells with RANKL (100 ng/mL) for 10 min in the presence of the indicated concentrations of compound 5, the expression level of IκBα was obtained by Western blotting and quantification. (C) Western blotting results and quantification results of c-Fos after RAW264.7 cells were stimulated with RANKL in the presence of the indicated concentrations of compound 5 for 24 h (normalized to each tubulin above). (D) NFATc1 and nuclear (DAPI) staining results after BMMs were stimulated with M-CSF and RANKL in the presence of compound 5 (10 μM) for 4 days (scale bar: 10 μm). (E) After RAW264.7 cells were cultured with RANKL for 4 days in the presence of compound 5 at the indicated concentrations, the mRNA reverse transcription qPCR analysis results of TRAP, CtsK, DC-STAMP, OSCAR and MMP-9 (mean ± SE, *p < 0.05 and **p < 0.01, versus solvent-treated control, n = 5).

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Example 1. Preparation of barley sprout alkaloid extract>

Barley ( Hordeum . vulgare var. hexastichon ) sprouts were collected in Jeju Island, Republic of Korea in March 2018, and were identified by Professor Min Byung-seon, one of the inventors of the present invention, and the voucher sample (CUD-373) was obtained from Daegu Catholic University, Korea. deposited in the herbarium of

The dry powder (10.0 kg) of the barley sprout was extracted with methanol 3 times (3h x 15L). The extract was concentrated under reduced pressure by rotary evaporation to obtain a crude extract of barley sprouts.

The crude extract was suspended in distilled water (2L), the pH was adjusted to 2 with 2% HCl solution, and extracted with CHCl ₃ . At this time, an acidic aqueous layer was obtained, adjusted to alkali (pH = 10) with ammonia solution, extracted with CHCl ₃ (5 × 4 L), and the CHCl ₃ extract was concentrated to obtain a crude alkaloid extract (HVA, 50 g).

<Example 2. Confirmation of effect of barley sprout alkaloid extract (HVA) on RANKL-induced osteoclast formation>

To investigate the effect of barley sprout alkaloid extract (HVA) on osteoclast formation, the effect of HVA on RANKL-induced osteoclast formation was confirmed using mouse RAW264.7 cells and BMM. Isolation of bone marrow-derived macrophages (BMM) was previously performed using the method of Tran et al. (2019). Briefly, bone marrow cells were isolated from the femur and tibia of ICR mice (DBL, Negative, Korea) and cultured in α-MEM (Hiclone, UT, USA) containing 10% FBS (Cambrex, Charles City, IA, Logan, UT, USA). cultivated in the USA). 100 U/mL penicillin, 100 μg/mL streptomycin and 10 ng/mL M-CSF (macrophage colony stimulating factor) were incubated overnight at 37°C in a 5% CO ₂ humidified incubator. Thereafter, non-adherent cells were collected and cultured in 30 ng/mL M-CSF for 3 days, and cells attached to the bottom of the culture dish were classified as BMM. These adherent BMMs were washed with PBS, collected, and used in subsequent experiments. RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin (100 U/mL)-streptomycin (100 μg/ml). Cultured in DMEM. Cells were maintained at 37° C. with air humidified with 5% CO ₂ .

BMMs were stimulated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of 0, 10, 30 and 100 μg/mL of HVA, and the culture medium was changed every 2 days for 6 days. . RAW264.7 cells were also stimulated with RANKL (100 ng/mL) in the presence of 0, 10, 30 and 100 μg/mL of HVA, and the culture medium was changed every 2 days for 4 days. At the end of the incubation, each cell was fixed with 3.7% formalin for 15 minutes, permeabilized with 0.1% Triton X-100, and osteoclasts were counted using the acid phosphatase leukocyte kit (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) did At this time, TRAP-positive multinucleated cells (> 5 nuclei) were defined as osteoclasts, and their micrographs and the number of osteoclasts are shown in FIG. 1 .

Figure 1A shows the effect of HVA on RANKL-induced osteoclastogenesis in RAW264.7 cells. As shown in Figure 1A, as a result of treating RAW264.7 cells with HVA, the formation of RANKL-induced TRAP-positive osteoclasts was greatly reduced by HVA in a concentration-dependent manner. At this time, the IC ₅₀ value of HVA was 27 ± 3.5 μg/ml.

Data were expressed as the mean ± standard error (SD) from at least three independent experiments. Statistical significance was assessed with one-way analysis of variance and Turkey's test to examine differences between two groups using SPSS version 14.0 (SPNN Inc., Chicago, IL, USA). A value of p < 0.05 was considered to indicate statistical significance, and subsequent experiments were presented with the same criteria.

Figure 1B shows the effect of HVA on RANKL-induced osteoclastogenesis in BMM. As shown in Fig. 1B, stimulation with M-CSF and RANKL effectively differentiated BMMs into TRAP-positive multinucleated osteoclasts, but treatment with HVA significantly reduced RANKL-induced osteoclastogenesis in a concentration-dependent manner. At this time, the IC ₅₀ value was 9.1 ± 0.8 μg/ml.

To determine if HVA affects cell viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was used to determine the effect of HVA on cell viability. did BMMs (5×10 ⁵ cells/mL) were seeded in 96-well plates and cultured with 30 ng/mL M-CSF in the presence of 0, 10, 30 and 100 μg/mL of HVA. After incubation for 72 hours, MTT (0.5 mg/mL) was added to each well and the plates were incubated for an additional 3 hours. After the incubation was completed, the insoluble formazan product was dissolved in dimethyl sulfoxide, and the absorbance at 540 nm was measured with a microplate reader, as shown in FIG. 1C.

As shown in Figure 1C, HVA did not affect cell viability. Accordingly, it was confirmed that HVA inhibited RANKL-induced osteoclast formation without cytotoxicity.

<Example 3. Confirmation of the effect of HVA on LPS-induced bone loss in vivo>

The anti-osteogenesis effect of HVA was evaluated in vivo using an inflammatory bone loss mouse model. Animal experimental protocols were approved by the Institutional Animal Care Committee (IACUC) of Kangwon National University (IACUC approval number KW-180119-3). 6-week-old ICR mice were randomly divided into 4 groups [vehicle-treated group (Control), LPS-treated group (LPS), HVA (50 mg/kg) + LPS-treated group, and HVA (100 mg/kg) + LPS-treated group]. divided (n = 4/group). Before the first injection of LPS (5 mg/kg), mice were orally administered with HVA (dissolved in corn oil) or control vehicle (corn oil) for 1 hour, then daily for 8 days. LPS was injected on days 2 and 6. On the ninth day, the mice were sacrificed, and the femurs were collected and fixed in 4% paraformaldehyde. The intact left femoral metaphysis region of each mouse was scanned using a NFR-Polaris-S160 device (Nano Focus Ray; Chuncheon Center, Korea Basic Science Institute) with 90 μA current, 45 kVp source voltage, and 7 μm isotropic resolution. Computed tomography (μCT) analysis was evaluated. Femoral scans were performed 2 mm above the growth plate, with a total of 350 sections per scan. After three-dimensional reconstruction, the number of trabeculae (Tb.N), trabecular separation (Tb. Sp) and bone volume/tissue volume ( BV/TV) ratio was analyzed and the results are shown in FIGS. 2A and 2B.

As shown in Figure 2A, as a result of three-dimensional analysis using micro-computed tomography (μCT), LPS administration caused obvious cancellous bone loss in the femur, and oral administration of HVA significantly reduced LPS-induced bone loss. . In addition, according to Fig. 2B, the decrease in trabecular number (Tb.N) and bone volume/tissue volume (BV/TV) ratio after LPS injection was restored in HVA-treated mice. Moreover, HVA administration attenuated the LPS-induced increase in trabecular separation (Tb.Sp). This suggests that HVA exhibited an anti-osteoclastogenic effect in vivo.

<Example 4. Separation of alkaloid compounds from barley sprout alkaloid extract (HVA)>

Specific compounds having an anti-osteoclastogenic effect were identified from the barley sprout alkaloid extract (HVA) having an anti-osteoclastogenic effect confirmed in Example 3.

The barley sprout alkaloid extract (HVA) obtained in Example 1 was suspended in distilled water and fractionated successively with CHCl ₃ and EtOAc to obtain CHCl ₃ (21 g) and EtOAc (16 g) fractions.

The CHCl ₃ fraction was chromatographed on a silica gel column (80 × 12 cm, 63–200 µM particle size, Merck) using a stepwise gradient of n-hexane-EtOAc (40:1 to 0:1) to obtain five fractions (C1-1). C5) was obtained. Of these, fraction C3 (4.5 g) was subjected to column chromatography (CC) on silica gel eluted with a mixture of n-hexane and EtOAc (40:1) to obtain five further fractions (C3.1 to C3.5).

Fraction C3.3 (1.5 g) was separated using a RP-18 silica gel column with MeOH-H ₂ O (1:1) as a mobile phase to obtain 7 small fractions (C3.3.1-C3.3.7), Fraction C3.3.4 (326 mg) was further purified using a semi-preparative Waters HPLC system (isocratic eluent, 45% MeOH in H ₂ O + 0.1% TFA, 5 mL/min, 60 min) to obtain compound 1 (10.5 mg), compound 2 (5.0 mg) and compound 3 (2.5 mg). In addition, fraction C3.3.6 (157 mg) was further purified using semi-preparative Waters HPLC (isocratic eluent, 30% MeOH in H ₂ O + 0.1% TFA, 5 mL/min, 50 min) to obtain compound 4 (6.3 mg), compound 6 (3.6 mg) and compound 7 (4.5 mg).

The EtOAc-soluble fraction (16.0 g) was chromatographed on a silica gel column (60 × 12 cm, 63–200 µM particle size, Merck) using a stepwise gradient of CHCl ₃ -MeOH (10:1 to 0:1) to obtain six fractions. (E1-E6) were obtained, among which, fraction E4 (2.1 g) was separated on silica gel CC (CHCl ₃ -MeOH, 7:1) to obtain 8 further fractions E4.1-E4.8. Among them, fraction E4.6 (0.7 g) was applied to Sephadex LH-20 CC eluted with a mixture of MeOH-H ₂ O (1:1) to obtain five small fractions E4.6.1-E4.6.5, Fraction E4.6.4 (237 mg) was purified by semipreparative Waters HPLC system (isocratic eluent, 50% MeOH in H ₂ O + 0.1% TFA, 5 mL/min, 50 min) to give compound 5 (6.0 mg), compound 8 (2.0 mg) and compound 9 (27.0 mg) were obtained. Silica gel (Merck, 63-200 μm particle size), RP-18 (Merck, 75 μm particle size) and Sephadex LH-20 (Pharmacia) were used for the column chromatography, and Merck silica gel 60 F254 and RP-18 F254 plates TLC was performed using High-performance liquid chromatography (HPLC) was performed using a Water System with UV detector 2996 and YMC-Triart C18 column (10 × 250 mm, 5 μm particle size, YMC Co., Ltd., Japan).

The chemical structures of compounds 1 to 9 of the indole alkaloids isolated above were determined using a wide range of spectroscopic techniques including 1D, 2D NMR and MS, and compared with published data, and are shown in FIG. 3 . And ¹ H and ¹³ C NMR results measured in methanol- d ₄ of compounds 1 to 4 are shown in Table 1. As a result of the above analysis, Compound 1 was identified as a new substance and named Hordemin A ( Hordeumin A ). Among known compounds 2 to 9, compounds 2 to 4 were first isolated from natural products.

UV spectrum for molecular structure determination was recorded using a Thermo spectrometer, and IR spectrum was recorded using a JASCO FT / IR-4100 spectrometer. 1D and 2D NMR spectra were obtained using Varian Unity Inova 400 MHz and Bruker Advance Digital 500 MHz spectrometers using tetramethylsilane (TMS) as an internal standard and chemical shifts were reported as δ values (ppm). Mass spectra were recorded using a JEOL JMS-700 mass spectrometer.

Position One ^a 2 ^b 3 ^b 4 ^b δ _H mult., ( J in Hz) δ _C δ _H mult., ( J in Hz) δ _C δ _H mult., ( J in Hz) δ _C δ _H mult., ( J in Hz) δ _C 2 7.53, s 129.6 7.64, s 128.7 7.52, s 128.0 7.57, s 129.3 3 103.8 104.7 105.9 104.5 3a 137.9 137.6 138.0 138.0 4 7.68, dd(8.0, 0.8) 119.1 7.81, d (8.0) 118.7 7.74, dt(8.0, 1.0) 118.9 7.59 d (8.0) 118.9 5 7.12, dt(8.0, 0.8) 121.4 7.24, t(8.0) 121.1 7.16, dt(8.0, 1.0) 121.0 7.14, dt(8.0, 1.0) 121.4 6 7.17, dt(8.0, 0.8) 123.5 7.29, t(8.0) 123.2 7.22, dt(8.0, 1.0) 123.4 7.22, dt(8.0, 1.0) 123.5 7 7.43, d d (8.0, 0.8) 113.0 7.54, d (8.0) 112.8 7.46, dt(8.0, 1.0) 112.8 7.48, d (8.0) 113.0 7a 128.0 127.8 127.9 128.5 8 4.57, s
4.47, s 52.5 4.63, s 49.9 4.44, s 43.4 4.61, s 51.6 9 3.58 m
3.31 m 53.7 3.09, t(8.0) 46.6 2.80, s 10 2.20 m
2.09 m 23.6 1.61, dd(16.0, 8.0) 36.8 11 2.20 m
2.09 m 23.6 1.68 m 27.3 12 3.58 m
3.31 m 53.7 0.96, d, (6.5) 22.5 13 0.96, d, (6.5) 22.5 One' 128.5 2' 7.02, d (8.4) 130.8 7.57, s 129.3 3' 6.71, d (8.4) 116.7 104.5 3'a 138.0 4' 157.7 7.59 d (8.0) 118.9 5' 6.71, d (8.4) 116.7 7.14, dt(8.0, 1.0) 121.4 6' 7.02, d (8.4) 130.8 7.22, dt(8.0, 1.0) 123.5 7' 2.95 m 30.9 7.48, d (8.0) 113.0 7'a 128.5 8' 3.39 m
3.20 m 57.6 4.61, s 51.6 9' 2.82, s 39.9

^{a 1} H (400 MHz) and ¹³ C (100 MHz) NMR.

^{b 1} H (500 MHz) and ¹³ C (125 MHz) NMR.

compound 1 ( Hordeumin A )

White amorphous powder; UV λ _max (MeOH) (log ε): 230 (2.45) and 270 (1.60) nm; IR (ATR) ν _max : 3266, 1471 and 1189 cm ⁻¹ ; ¹ H and ¹³ C NMR (methanol- d ₄ ) data see Table 1; HR-ESIMS m/z 281.1656 [M+H] ⁺ (C ₁₈ H ₂₁ N ₂ O, 281.1654).

compound 2 ( 3-(1-Pyrrolidiylmethyl)-1H-indole )

White amorphous powder; UV λ _max (MeOH) (log ε): 230 (1.90) and 270 (1.30) nm; IR (ATR) ν _max : 3700, 3006 and 1055 cm ⁻¹ ; ¹ H and ¹³ C NMR (methanol- d ₄ ) data see Table 1; HR-ESIMS m/z 201.1393 [M+H] ⁺ (C ₁₃ H ₁₇ N ₂ , 201.1392).

compound 3 (( 1H-indole-3-ylmethyl )( 3-methylbutyl ) amine )

White amorphous powder; UV λ _max (MeOH) (log ε): 225 (2.39) and 275 (1.69) nm; IR (ATR) ν _max : 3333, 2939 and 1023 cm ⁻¹ ; ¹ H and ¹³ C NMR (methanol- d ₄ ) data see Table 1; HR-ESIMS m/z 217.1705 [M+H] ⁺ (C ₁₄ H ₂₁ N ₂ , 217.1705).

compound 4 ( N,N-bis(indol-3-ylmethyl)methylamine )

White amorphous powder; UV λ _max (MeOH) (log ε): 225 (2.17) and 275 (1.00) nm; IR (ATR) ν _max : 3329, 2942 and 1024 cm ⁻¹ ; ¹ H and ¹³ C-NMR (methanol- d ₄ ) data see Table 1; HR-ESIMS m/z 290.1658 [M+H] ⁺ (C ₁₉ H ₂₀ N ₃ , 290.1657).

<Example 5. Separation of alkaloid compounds from barley sprout alkaloid extract (HVA)>

Specific compounds having an anti-osteoclastogenic effect of the 9 barley sprout alkaloid compounds isolated in Example 4 were identified. In the same manner as in Example 2, the treated samples were treated with 9 compounds instead of HVA.

RAW264.7 cells were cultured in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS) and penicillin (100 U/mL)-streptomycin (100 μg/ml) with 5% CO ₂ in humidified air. It was maintained at 37 °C.

Compounds 1 to 9 obtained in Example 4 were added to each well to be 1 and 10 μM, respectively, and then stimulated with RANKL (100 ng/mL) together with a control, and the culture medium was changed every 2 days for 4 days. . At the end of incubation, each cell was fixed with 3.7% formalin for 15 minutes, permeabilized with 0.1% Triton X-100, and multinucleated cells (> 5 nuclei) osteoclasts were counted and shown in FIG. 4 .

As shown in Fig. 4A, five kinds of compounds 1 to 5 significantly reduced the number of osteoclasts at a concentration of 10 μM. In particular, compound 3 significantly reduced the number of osteoclasts at a concentration of 1 μM. However, the compound 3 exhibited cytotoxicity at a concentration of 10 μM (data not shown). However, in the case of compounds 1, 2, 4, and 5, it was confirmed that the formation of osteoclasts was inhibited without cytotoxicity. The four compounds of Compounds 6 to 9 tended to inhibit osteoclast differentiation in a concentration-dependent manner without cytotoxicity, but, like Compounds 1 to 5, no significant difference was shown at concentrations up to 10 μM. Therefore, subsequent experiments were conducted on Compounds 4 and 5, which exhibited the strongest osteoclast formation inhibitory effect and did not exhibit cytotoxicity.

In the same manner as in Example 2, BMM was stimulated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of 0, 3, and 10 μM of compound 4 or 5, and the culture medium was maintained for 6 days. After culture by replacement every 2 days, each cell was fixed with 3.7% formalin for 15 minutes and permeabilized with 0.1% Triton X-100 using acid phosphatase leukocyte kit (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) TRAP-positive multinucleated cells (> 5 nuclei) were photographed and osteoclasts were counted and shown in FIGS. 4B and 4C.

As shown in FIGS. 4B and 4C , treatment of BMMs with compound 4 or 5 significantly reduced RANKL-induced formation of TRAP-positive multinucleated osteoclasts in a concentration-dependent manner. At this time, the IC ₅₀ values were 2.0 ± 0.6 and 1.3 ± 0.4 μM, respectively. However, it was confirmed that these compounds did not significantly inhibit the viability of BMM at a concentration of up to 10 μM and did not exhibit cytotoxicity (see FIG. 4D ).

<Example 6. Inhibition of RANKL-induced osteoclastogenesis by Compound 5>

mme, 2009).Whether the compound 5 could inhibit F-actin ring formation, which is the most obvious feature of mature osteoclasts, during osteoclastogenesis was investigated (Wilson, Peters, Saftig, & Br

mme, 2009).

BMM (10 ⁶ cells/mL) was seeded on a cover glass and cells were stimulated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of 0, 0.3, 1, 3 and 10 μM of Compound 5 and incubated at 37 °C with humidified air of 5% CO ₂ . Compounds and culture media were changed every 2 days. After 7 days, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, BMM blocked and stained with FITC-conjugated phalloidin for 10 min at room temperature. . Then, after washing twice with PBS, the cells were mounted on a microscope and images were taken using an inverted confocal laser scanning microscope (OLYMPUS FV1000, Shinjuku, Tokyo, Japan) equipped with an external argon laser, HeNe laser green and HeNe laser red. Captured. Images were captured in the middle of colonies using an UPLSAPO 60X NA1.35 oil immersion objective (OLYMPUS) and are shown in Figure 5A.

As shown in Fig. 5A, when RANKL and M-CSF were used to stimulate BMMs, FITC-phalloidin-stained F-actin ring structures were formed. However, when BMMs were treated with compound 5, the number and size of actin ring structures significantly decreased in a concentration-dependent manner, suggesting that compound 5 inhibited the formation of mature osteoclasts.

To confirm whether compound 5 inhibits the bone resorption activity of osteoclasts, a resorption pit formation assay was performed. Osteoclast differentiation analysis was performed by applying the method of Tran et al. (2019). BMMs were cultured in OsteoAssay Surface 96-well plates (Corning Incorporated, Corning, NY, USA) and then plated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of 0, 1, 3, and 10 μM of compound 5. mL) was stimulated. Compound 5 and culture medium were changed every 2 days. After 7 days the cells were removed and the plates were thoroughly washed with distilled water. Resorption pits were captured using a model H550L microscope (Nikon Corporation, Tokyo, Japan) and quantified using ImageJ software (Java 1.6.0_20 (64 bit); NIH, Bethesda, MD, USA) and are shown in Figure 5B. .

As shown in Fig. 5B, compound 5 greatly reduced the formation of RANKL-stimulated bone resorption pits on the OsteoAssay surface in a concentration-dependent manner. These results show that compound 5 can inhibit RANKL-induced formation of mature osteoclasts and bone resorption pits.

In order to determine whether compound 5 inhibits the early or late stages of osteoclast differentiation, BMM (10 ⁶ cells/mL) was seeded on a cover glass and cells were incubated with M-CSF (30 ng/mL) and RANKL (100 cells/mL). ng/mL) and incubated at 37 °C with 5% CO ₂ humidified air. On days 1 and 3 of culture, compound 5 was treated with 10 μM, respectively, and on day 7, TRAP-positive multinucleated cells (> 5 nuclei) were photographed and osteoclasts were counted, as shown in FIG. 5C .

As shown in Figure 5C, compound 5 significantly reduced the number of TRAP-positive osteoclasts when applied on day 1 and day 3. This suggests that compound 5 inhibited RANKL-induced osteoclastogenesis in both early and late stages of differentiation.

<Example 7. Mechanism of compound 5 inhibiting RANKL-induced osteoclastogenesis>

Binding of RANKL to RANK results in activation of downstream signaling pathways including the MAPK and NF-κB pathways that regulate RNAKL-induced osteoclastogenesis (Franzoso et al., 1997; Grigoriadis et al., 1994). Therefore, the effect of compound 5 on the RANKL-induced MAPK and NF-κB signaling pathways was investigated.

RAW264.7 cells were stimulated with RANKL for 10 minutes in the presence of 0, 1, 3 and 10 μM of compound 5, followed by Western blotting to determine the expression levels of p-ERK, p-JNK, p-p38 MAPK, IκBα was performed and the results are shown in Figures 6A and 6B.

As shown in FIG. 6A, phosphorylation levels of ERK, JNK, and p38 MAPK were increased by RANKL stimulation, and compound 5 significantly inhibited RANKL-induced p38 MAPK phosphorylation. In contrast, phosphorylation levels of ERK and JNK were not reduced by compound 5 treatment. Also, as shown in Fig. 6B, stimulation with RANKL induced the degradation of IκBα protein. Degradation of the IκBα protein is known to be an essential step for canonical NF-κB activation (Karin, 1999). Compound 5 significantly inhibited RANKL-induced degradation of IκBα protein in a concentration-dependent manner. MAPK and NF-κB pathways are known to activate the AP-1 transcription factor (Abu-Amer, 2013; Wada et al., 2006), which is a heterodimer of c-Jun and c-Fos transcription.

RAW264.7 cells were stimulated with RANKL (100 ng/mL) in the presence of 0, 1, 3 and 10 μM of Compound 5 for 24 hours. Then, the expression level of c-Fos was confirmed by Western blotting and is shown in FIG. 6C. As shown in Fig. 6C, stimulation with RANKL increased the c-Fos expression level, and compound 5 decreased the RANKL-induced c-Fos expression level in a concentration-dependent manner. This suggests that compound 5 can inhibit RANKL-induced osteoclastogenesis by inhibiting MAPK/c-Fos and NF-κB activation.

c-Fos and NF-κB induce auto-amplification of NFATc1, a master regulator of RANKL-induced osteoclast differentiation, and regulate the expression of osteoclast genes including CtsK, MMP-9, OSCAR, TRAP, and DC-STAMP (Boyle et al., 2003; Takayanagi et al., 2002). Therefore, the effect of compound 5 on RANKL-induced nuclear translocation of NFATc1 and target gene expression was confirmed. After stimulating BMMs with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days in the presence of compound 5 (10 μM), cells were washed with PBS and then incubated in fresh 4% paraformaldehyde. was fixed for 20 min at room temperature. Thereafter, the cells were permeabilized with 0.5% TritonX-100 and incubated with PBS containing 1% goat serum to block non-specific sites. Cells were then incubated overnight at 4°C with an antibody to anti-NFATc1 (1:200 dilution). Then, after washing four times with PBS, the cells were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:250 dilution) at room temperature for 4 hours, washed, and then stained with DAPI. Confocal images were acquired using an OLYMPUS FV1000 inverted laser scanning confocal microscope equipped with an external argon laser, HeNe laser green and HeNe laser red. Images were captured in the middle of colonies using an UPLSAPO 60X NA1.35 oil immersion objective (OLYMPUS) and are shown in Fig. 6D (scale bar: 10 μm). As shown in Figure 6D, compound 5 reduced RANKL-induced nuclear translocation of NFATc1.

Real-time qPCR analysis was performed to confirm the effect of compound 5 on the expression of osteoclast genes including CtsK, MMP-9, OSCAR, TRAP and DC-STAMP. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol, and then first-strand cDNA was prepared from 1 μg of total RNA. Real-time qPCR analysis was performed using TOPreal qPCR 2X PreMIX (SYBR Green; Enzynomics, Inc., Daejeon, Korea) and StepOnePlus™ Real-Time PCR System (Applied Biosystem), using the following specific primers: CtsK, 5′-GAC ACC CAG TGGGAG CTA TG-3′ (sense) and 5′-AGA GGC CTC CAG GTT ATG GG-3′ (antisense); TRAP, 5′-ACT TGC GAC CAT TGT TAG CC-3′ (sense) and 5′-TTC GTT GAT GTC GCA CAG AG-3′ (antisense); MMP-9, 5′-TGG GCA AGC AGT ACT CTT CC-3′ (sense) and 5′-AACAGG CTG TAC CCT TGG TC-3′ (antisense); β-actin, 5′-GGG AAA TCG TGC GTG ACA TCA AAG-3′ (sense) and 5′-AAC CGC TCC TTG CCAATA GT-3′ (antisense). Primers for DC-STAMP and OSCAR were purchased from Origene (Rockville, MD, USA). Each PCR cycle was performed with 40 cycles at 95 °C for 10 min, 95 °C for 10 sec, 60 °C for 15 sec, and 72 °C for 20 sec, all reactions were performed in triplicate and gene expression was performed using the housekeeping gene, β-actin. Normalized to , and shown in FIG. 6E. As shown in Figure 6E, as a result of real-time qPCR analysis, compound 5 significantly increased the expression levels of NFATc1 target genes including TRAP, CtsK, DC-STAMP, OSCAR and MMP-9 in a concentration-dependent manner during RANKL-induced osteoclast differentiation. Reduced. These results suggest that compound 5 can inhibit RANKL-induced osteoclast differentiation by inhibiting NFATc1 activation.

<Formulation Example 1. Pharmaceutical formulation>

Formulation Example 1-1. manufacture of tablets

200 mg of the barley sprout alkaloid extract or an alkaloid compound isolated therefrom of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. After adding 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. It was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of active agent, and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Preparation of injection solution

100 mg of the barley sprout alkaloid extract of the present invention or an alkaloid compound isolated therefrom, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was bottled and sterilized by heating at 20° C. for 30 minutes.

<Formulation Example 2. Manufacture of health functional food>

Formulation Example 2-1. Manufacture of health functional food

Barley sprout alkaloid extract of the present invention or 2 g of an alkaloid compound isolated therefrom, suitable amount of vitamin mixture, vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg , Vitamin C 10mg, Biotin 10μg, Nicotinamide 1.7mg, Folic Acid 50μg, Calcium Pantothenate 0.5mg, Mineral Mixture Appropriate amount, Ferrous Sulfate 1.75mg, Zinc Oxide 0.82mg, Magnesium Carbonate 25.3mg, Potassium Phosphate Monobasic 15 mg, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were prepared into granules, but it can be prepared by transforming into various formulations depending on the use. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above components according to a conventional health functional food manufacturing method.

Formulation example 2-2. Manufacture of health functional beverages

A beverage was prepared by mixing 1 g of the barley sprout alkaloid extract of the present invention or an alkaloid compound isolated therefrom, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water, followed by stirring, heating, filtering, sterilization, and refrigeration according to a conventional beverage manufacturing method. .

Claims (11)

A composition for inhibiting osteoclast differentiation comprising a barley sprout alkaloid extract or an alkaloid compound isolated therefrom. According to claim 1,
The alkaloid extract,
Extracting the crushed barley sprouts with an organic solvent and concentrating under reduced pressure to prepare a crude extract of barley sprouts (Step 1);
The distilled water suspension of the crude barley extract obtained in step 1 was adjusted to pH 2 and then CHCl ₃ removing by extraction with a solution to obtain an acidic aqueous layer (step 2); and
The acidic aqueous layer of step 2 was adjusted to pH 10, then CHCl ₃ extracting with to obtain a concentrated alkaloid extract (step 3);
A composition for inhibiting osteoclast differentiation, characterized in that produced by a manufacturing method comprising a. According to claim 2,
The organic solvent is water, C1-4 lower alcohol, acetone, ethyl acetate, diethyl acetate, diethyl ether, benzene, chloroform and hexane, or a mixed solvent thereof. A composition for inhibiting osteoclast differentiation. According to claim 1,
The alkaloid compound is a composition for inhibiting osteoclast differentiation, characterized in that it comprises one or more selected from compounds 1 to 9 represented by the following formula [1].
formula [1]

According to claim 1,
The alkaloid compounds are Hordeumin A (Compound 1), 3-(1-Pyrrolidiylmethyl)-1H-indole (Compound 2), (1H-indole-3-ylmethyl)(3-methylbutyl)amine (Compound 3), N,N At least one selected from the group consisting of -bis(indol-3-ylmethyl)methylamine (Compound 4) and 3-[2-[(indol-3-yl)methyl]indol-3-yl]methylindole (Compound 5) A composition for inhibiting osteoclast differentiation, characterized in that it comprises. According to any one of claims 1 to 5,
The composition for inhibiting osteoclast differentiation is a composition for inhibiting osteoclast differentiation, characterized in that for suppressing a decrease in trabecular number, bone volume ratio (BS / BV), or an increase in trabecular separation. According to any one of claims 1 to 5,
The composition for inhibiting osteoclast differentiation is characterized in that the composition for inhibiting the expression of p-p38, c-Fos or degradation of IκBα. According to any one of claims 1 to 5,
The composition for inhibiting osteoclast differentiation is a composition for inhibiting osteoclast differentiation, characterized in that inhibits nuclear translocation of NFATc1. According to any one of claims 1 to 5,
The composition for inhibiting osteoclast differentiation is a composition for inhibiting osteoclast differentiation, characterized in that for inhibiting the expression of TRAP, Ctsk, DC-STAMP, OSCAR and MMP-9. A health functional food for inhibiting osteoclast differentiation, characterized in that it contains barley sprout alkaloid extract or at least one selected from compounds 1 to 9 isolated from it and represented by the following formula [1].
Formula [1]

A novel compound 5 represented by the following formula [2].
Formula [2]

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