Abstract
In a previous study, we discovered that the ethanolic extract of sea buckthorn (Hippophae rhamnoides) fruits exhibited anti-osteoporosis effects both in vitro and in vivo. Through bioassay-guided fractionation, we identified the hexane fraction (HRH) as the active fraction, which was further fractionated using preparative HPLC. Among the resulting six fractions, HRHF4 showed significant activity. In the present study, we focused on the bioassay-guided isolation of bioactive compounds from the HRHF4 fraction. We successfully identified the active HRHF43 fraction, which led us to the isolation of potential bioactive compounds (1–6). The chemical structures of these compounds were determined using NMR data, LC-MS analysis, and HR-ESI-MS data as four triterpenes, ursolic acid (1), uvaol (2), oleanolic aldehyde (3), and ursolic aldehyde (4), together with two fatty acids, methyl linoleate (5) and ethyl oleate (6). To evaluate the efficacy of promoting osteoblast differentiation and the expression of mRNA biomarkers related to osteogenesis, we tested the isolated compounds in the mouse mesenchymal stem cell line, C3H10T1/2. Alkaline phosphate staining demonstrated that triterpenes (1–4) displayed osteogenic activity. Particularly noteworthy, ursolic aldehyde (4) exhibited the most potent effect, showing an 11.2-fold higher activity at a concentration of 10 μg/mL compared to the negative control. Moreover, ursolic aldehyde (4) upregulated the gene expression of bone formation-related biomarkers, including Runx2, Osterix, Alp, and Osteopontin. These findings suggest that the fruit extract of H. rhamnoides may have potential as a nutraceutical for promoting bone health, with ursolic aldehyde (4) identified as an active constituent.
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The data used to support the findings of this study are available from the corresponding author upon request.
References
Agirrezabal I, Cabases JM, Di Tanna GL, Sanchez-Iriso E (2020) Inequalities in prescription rates of anti-osteoporosis drugs in primary care in England: a practice-level prescribing data analysis in 2013–2018. Bone 130:115125. https://doi.org/10.1016/j.bone.2019.115125
Bano Z, Begum S, Ali SS, Kiran Z, Siddiqui BS, Ahmed A, Khawaja S, Fatima F, Jabeen A (2022) Phytochemicals from Carissa carandas with potent cytotoxic and anti-inflammatory activities. Nat Prod Res 36:1587–1592. https://doi.org/10.1080/14786419.2021.1886101
Carbonare LD, Valenti MT, Zanatta M, Donatelli L, Lo Cascio V (2009) Circulating mesenchymal stem cells with abnormal osteogenic differentiation in patients with osteoporosis. Arthritis Rheumatol 60:3356–3365. https://doi.org/10.1002/art.24884
Chen Q, Shou P, Zheng C, Jiang M, Cao G, Yang Q, Cao J, Xie N, Velletri T, Zhang X (2016) Fate decision of mesenchymal stem cells: adipocytes or osteoblasts? Cell Death Differ 23:1128–1139. https://doi.org/10.1038/cdd.2015.168
Cheng J, Kondo K, Suzuki Y, Ikeda Y, Meng X, Umemura K (2003) Inhibitory effects of total flavones of Hippophae rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation. Life Sci 72:2263–2271. https://doi.org/10.1016/S0024-3205(03)00114-0
Cho H, Kim KH, Han SH, Kim HJ, Cho IH, Lee S (2022) Structure determination of heishuixiecaoline a from Valeriana fauriei and its content from different cultivated regions by HPLC/PDA analysis. Nat Prod Sci 28:181–186. https://doi.org/10.20307/nps.2022.28.4.181
Dalle Carbonare L, Innamorati G, Valenti MT (2012) Transcription factor Runx2 and its application to bone tissue engineering. Stem Cell Rev Rep 8:891–897. https://doi.org/10.1007/s12015-011-9337-4
Filleul O, Crompot E, Saussez S (2010) Bisphosphonate-induced osteonecrosis of the jaw: a review of 2400 patient cases. J Cancer Res Clin Oncol 136:1117–1124. https://doi.org/10.1007/s00432-010-0907-7
Ganju L, Padwad Y, Singh R, Karan D, Chanda S, Chopra MK, Bhatnagar P, Kashyap R, Sawhney RC (2005) Anti-inflammatory activity of Seabuckthorn (Hippophae rhamnoides) leaves. Int Immunopharmacol 5:1675–1684. https://doi.org/10.1016/j.intimp.2005.03.017
Guliyev VB, Gul M, Yildirim A (2004) Hippophae rhamnoides L.: chromatographic methods to determine chemical composition, use in traditional medicine and pharmacological effects. J Chromatogr B 812:291–307. https://doi.org/10.1016/j.jchromb.2004.08.047
Gupta A, Kumar R, Pal K, Singh V, Banerjee PK, Sawhney RC (2006) Influence of sea buckthorn (Hippophae rhamnoides L.) flavone on dermal wound healing in rats. Mol Cell Biochem 290:193–198. https://doi.org/10.1007/s11010-006-9187-6
Haynes B, Dowsett M (1999) Clinical pharmacology of selective estrogen receptor modulators. Drugs Aging 14:323–336. https://doi.org/10.2165/00002512-199914050-00001
Hu Y, He Y, Niu Z, Shen T, Zhang J, Wang X, Hu W, Cho JY (2022) A review of the immunomodulatory activities of polysaccharides isolated from Panax species. J Ginseng Res 46:23–32. https://doi.org/10.1016/j.jgr.2021.06.003
Ito T, Sasaki M, Taguchi T (2015) Enhanced ALP activity of MG63 cells cultured on hydroxyapatite-poly (ethylene glycol) hydrogel composites prepared using EDTA-OH. Biomed Mater 10:015025. https://doi.org/10.1088/1748-6041/10/1/015025
Jeong Y, Yang W, Ko H, Kim M (2014) The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells. Restor Dent Endod 39:187–194. https://doi.org/10.5395/rde.2014.39.3.187
Kallio H, Yang B, Peippo P, Tahvonen R, Pan R (2002) Triacylglycerols, glycerophospholipids, tocopherols, and tocotrienols in berries and seeds of two subspecies (ssp. sinensis and mongolica) of Sea Buckthorn (Hippopha> ë rhamnoides). J Agric Food Chem 50:3004–3009. https://doi.org/10.1021/jf011556o
Kim DH, Han KM, Chung IS, Kim DK, Kim SH, Kwon BM, Jeong TS, Park MH, Ahn EM, Baek NI (2005) Triterpenoids from the flower of Campsis grandiflora K. Schum. as human acyl-CoA: cholesterol acyltransferase inhibitors. Arch Pharm Res 28:550–556. https://doi.org/10.1007/BF02977757
Kim MK, Park G, Hong S-P, Jang YP (2021) Analytical quality by design methodology approach for simultaneous quantitation of paeoniflorin and decursin in herbal medicine by RP-HPLC analysis. Nat Prod Sci 27:264–273. https://doi.org/10.20307/nps.2021.27.4.264
Lee B, Thirunavukkarasu K, Zhou L, Pastore L, Baldini A, Hecht J, Geoffrey V, Ducy P, Karsenty G (1997) Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nat Genet 16:307–310. https://doi.org/10.1038/ng0797-307
Lee SU, Park SJ, Kwak HB, Oh J, Min YK, Kim SH (2008) Anabolic activity of ursolic acid in bone: stimulating osteoblast differentiation in vitro and inducing new bone formation in vivo. Pharmacol Res 58:290–296. https://doi.org/10.1016/j.phrs.2008.08.008
Lee DY, Lee MH, Jung TS, Kwon BM, Baek NI, Rho YD (2010) Triterpenoid and lignan from the fruits of Cornus kousa inhibit the activities of PRL-3 and LDL-oxidation. J Korean Soc Appl Biol Chem 53:97–100. https://doi.org/10.3839/jksabc.2010.016
Lee KH, Kim JK, Yu JS, Jeong SY, Choi JH, Kim JC, Ko YJ, Kim SH, Kim KH (2021a) Ginkwanghols A and B, osteogenic coumaric acid-aliphatic alcohol hybrids from the leaves of Ginkgo biloba. Arch Pharm Res 44:514–524. https://doi.org/10.1007/s12272-021-01329-3
Lee SY, Kim MH, Kim SH, Ahn T, Kim SW, Kwak YS, Cho I-H, Nah SY, Cho SS, Park KM (2021b) Korean red ginseng affects ovalbumin-induced asthma by modulating IL-12, IL-4, and IL-6 levels and the NF-κB/COX-2 and PGE2 pathways. J Ginseng Res 45:482–489. https://doi.org/10.1016/j.jgr.2020.10.001
Lee BS, So HM, Kim S, Kim JK, Kim JC, Kang DM, Ahn MJ, Ko YJ, Kim KH (2022a) Comparative evaluation of bioactive phytochemicals in Spinacia oleracea cultivated under greenhouse and open field conditions. Arch Pharm Res 45:795–805. https://doi.org/10.1007/s12272-022-01416-z
Lee SR, Lee BS, Yu JS, Kang H, Yoo MJ, Yi SA, Han JW, Kim S, Kim JK, Kim JC (2022b) Identification of anti-adipogenic withanolides from the roots of Indian ginseng (Withania somnifera). J Ginseng Res 46:357–366. https://doi.org/10.1016/j.jgr.2021.09.004
Li Q, Fan Y-s, Gao Z-q, Fan K, Liu Z-j (2015) Effect of Ursolic acid and oleanolic acid on osteoblastic like cell-line MC3T3-E1. Pak Vet J 35:414–419. https://doi.org/10.3748/wjg.v8.i3.493
Lin RC, Ng SF, Morris MJ (2014) Gene expression in rat models for inter-generational transmission of islet dysfunction and obesity. Genom Data 2:351–353. https://doi.org/10.1016/j.gdata.2014.09.013
Murata S (1984) A facile method for activation of carboxylic acids. Bull Chem Soc Jpn 57:3597–3598. https://doi.org/10.1246/bcsj.57.3597
Nyamsambuu A, Ahmed A, Khusbu FY, Oidovsambuu S, Khan MA, Zhou X, Fu JJ, Chen H-C (2021) Anti-oxidant and antiproliferative activities of Mongolian medicinal plant extracts and structure isolation of gnetin-h compound. Med Chem 17:963–973. https://doi.org/10.2174/1573406416666201106110117
Panossian A, Wagner H (2012) From traditional to evidence-based use of Hippophae rhamnoides L.: chemical composition, experimental, and clinical pharmacology of sea buckthorn berries and leaves extracts. Evidence Rational Based Res Chin Drugs. https://doi.org/10.1007/978-3-7091-0442-2_5
Park SY, Kim SH, Kim TY, Lee YK, Ha YC, Jang S, Ahn SH, Kim HY (2021) Incidence and risk of venous thromboembolism in bisphosphonates and selective estrogen receptor modulators treatment in Korea. J Korean Med Sci. https://doi.org/10.3346/jkms.2021.36.e186
Park B, Yu SN, Kim SH, Lee J, Choi SJ, Chang JH, Yang EJ, Kim KY, Ahn SC (2022a) inhibitory Effect of Biotransformed-fucoidan on the differentiation of osteoclasts induced by receptor for activation of nuclear factor-κB ligand. J Microbiol Biotechnol 32:1017. https://doi.org/10.4014/jmb.2203.03001
Park KH, Hong JH, Kim SH, Kim JC, Kim KH, Park KM (2022b) Anti-osteoporosis effects of the fruit of sea buckthorn (Hippophae rhamnoides) through promotion of osteogenic differentiation in ovariectomized mice. Nutrients 14:3604. https://doi.org/10.3390/nu14173604
Rachner T, Khosla S, Hofbauer L (2011) Osteoporose: nå og fremtiden. Lancet 377:1276–1287. https://doi.org/10.1016/S0140-6736(10)62349-5
Reher G, Buděšínský M (1992) Triterpenoids from plants of the Sanguisorbeae. Phytochem 31:3909–3914. https://doi.org/10.1016/S0031-9422(00)97552-6
Rosen CJ, Bouxsein ML (2006) Mechanisms of disease: is osteoporosis the obesity of bone? Nat Clin Pract Rheumatol 2:35–43. https://doi.org/10.1038/ncprheum0070
Rushdey El-Seedi H (2005) Antimicrobial triterpenes from Poulsenia armata miq. standl. Nat Prod Res 19:197–202. https://doi.org/10.1080/14786410410001730724
Siddalingaswamy M, Khanum F (2010) Hypolipidemic and antioxidant effects of seabuckthorn leaf based herbal formulation. Int J Pharm Biol Sci Arch 1:457–466. https://doi.org/10.3390/ijerph18178986
Sipos W, Pietschmann P, Rauner M, Kerschan-Schindl K, Patsch J (2009) Pathophysiologie der Osteoporose. Wien Med Wochenschr 159:230–234. https://doi.org/10.1007/s10354-009-0647-y
Upadhyay NK, Kumar MY, Gupta A (2010) Antioxidant, cytoprotective and antibacterial effects of Sea buckthorn (Hippophae rhamnoides L.) leaves. Food Chem Toxicol 48:3443–3448. https://doi.org/10.1016/j.fct.2010.09.019
Wei J, Zhao J, Su T, Li S, Sheng W, Feng L, Bi Y (2023) Flavonoid extract from seed residues of Hippophae rhamnoides ssp. sinensis protects against alcohol-induced intestinal barrier dysfunction by regulating the Nrf2 Pathway. Antioxidants 12:562. https://doi.org/10.3390/antiox12030562
Weitzmann MN, Pacifici R (2006) Estrogen regulation of immune cell bone interactions. Ann N Y Acad Sci 1068:256–274. https://doi.org/10.1196/annals.1346.030
Yogesh H, Chandrashekhar V, Katti H, Ganapaty S, Raghavendra H, Gowda GK, Goplakhrishna B (2011) Anti-osteoporotic activity of aqueous-methanol extract of Berberis aristata in ovariectomized rats. J Ethnopharmacol 134:334–338. https://doi.org/10.1016/j.jep.2010.12.013
Yoshino T, Imori S, Togo H (2006) Efficient esterification of carboxylic acids and phosphonic acids with trialkyl orthoacetate in ionic liquid. Tetrahedron 62:1309–1317. https://doi.org/10.1016/j.tet.2005.09.147
Yu JS, Jeong SY, Li C, Oh T, Kwon M, Ahn JS, Ko SK, Ko YJ, Cao S, Kim KH (2022) New phenalenone derivatives from the Hawaiian volcanic soil-associated fungus Penicillium herquei FT729 and their inhibitory effects on indoleamine 2, 3-dioxygenase 1 (IDO1). Arch Pharm Res 45:105–113. https://doi.org/10.1007/s12272-022-01372-8
Zhai YK, Pan YL, Niu YB, Li CR, Wu XL, Fan WT, Lu TL, Mei QB, Xian CJ (2014) The importance of the prenyl group in the activities of osthole in enhancing bone formation and inhibiting bone resorption in vitro. Int J Endocrinol. https://doi.org/10.1155/2014/921954
Zhang Y, Jayaprakasam B, Seeram NP, Olson LK, DeWitt D, Nair MG (2004) Insulin secretion and cyclooxygenase enzyme inhibition by cabernet sauvignon grape skin compounds. J Agric Food Chem 52:228–233. https://doi.org/10.1021/jf034616u
Zheng H, Feng H, Zhang W, Han Y, Zhao W (2020) Targeting autophagy by natural product ursolic acid for prevention and treatment of osteoporosis. Toxicol Appl Pharmacol 409:115271. https://doi.org/10.1016/j.taap.2020.115271
Zhang T, Qin X, Cao Y, Zhang J, Zhao J (2020) Sea buckthorn (Hippophae rhamnoides L.) oil enhances proliferation, adipocytes differentiation and insulin sensitivity in 3T3-L1 cells. Food Sci Biotechnol 29:1511–1518. https://doi.org/10.1007/s10068-020-00817-4
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This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT; grant numbers 2019R1A5A2027340 and 2021R1A2C2007937).
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Lee, D.E., Park, K.H., Hong, JH. et al. Anti-osteoporosis effects of triterpenoids from the fruit of sea buckthorn (Hippophae rhamnoides) through the promotion of osteoblast differentiation in mesenchymal stem cells, C3H10T1/2. Arch. Pharm. Res. 46, 771–781 (2023). https://doi.org/10.1007/s12272-023-01468-9
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DOI: https://doi.org/10.1007/s12272-023-01468-9