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DTT 2024; 3(2): 95-104
Published online September 30, 2024
https://doi.org/10.58502/DTT.24.0007
Copyright © The Pharmaceutical Society of Korea.
Si Hyeon Chae1, Chan Hee Cho1, Seon Hee Kim2, Ki Hyun Kim1 
Correspondence to:Ki Hyun Kim, khkim83@skku.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Thlaspi arvense Linn, known as “Pennycress,” is a plant from the Brassicaceae family that has been traditionally used to treat glomerulonephritis, gastritis, and rheumatoid arthritis. As part of our current projects to discover bioactive compounds from natural resources, three flavonoids (1-3), one diarylheptanoid (4), and two phenolic glycosides (5-6) were isolated from the whole plant of T. arvense via semi-preparative HPLC purification. The chemical structures of compounds 1-6 were elucidated as isoorientin (1), isovitexin (2), isoscoparin (3), oregonin (4), 1,2-disinapoylgentiobiose (5), and 1,2,2’-trisinapoylgentiobiose (6) based on the comparison of their NMR spectroscopic and physical properties with those of previous studies. Notably, this is the first report of the presence of compounds 3-6 in this plant. Compounds 1-6 were then tested to determine their effects on osteogenesis and adipogenesis in the mouse mesenchymal stem cell (MSC) line C3H10T1/2. Among the six compounds, isovitexin (2) was found to promote the osteogenic differentiation of MSCs. As the concentration of 2 increased, the differentiation of MSCs into osteoblasts became more active, as evidenced by the induction of mRNA expression of the osteogenic markers, alkaline phosphatase (ALP) and osteopontin (OPN). Accordingly, our findings demonstrate that isovitexin (2) could potentially serve as a valuable compound for the treatment of menopause-associated syndromes, such as osteoporosis, by promoting MSC osteogenesis.
KeywordsThlaspi arvense, Brassicaceae, phenolic compounds, isovitexin, osteogenesis
As part of our continuous endeavor to discover structurally and biologically novel compounds from diverse natural sources (Cho et al. 2022; Lee et al. 2022a; Lee et al. 2023; Lee et al. 2022b; Yu et al. 2022), we have collected and prepared ethanol (EtOH) extracts of freshwater plants for phytochemical investigation. Among the collected freshwater plants, Thlaspi arvense Linn, also referred to as “Pennycress,” is a plant belonging to the family Brassicaceae, which grows in most districts of Eurasia. Historically considered an agricultural weed, field pennycress is now extensively cultivated due to several desirable agronomic traits, such as high seed yield and high oil content, making it useful as a feedstock (Evangelista et al. 2012). Its aerial parts, including the whole herb, seedlings, and seeds, are utilized in medicine for their heat-clearing, detoxifying, diuretic, and detumescent effects (Ma et al. 2023).
T. arvense is a well-known Chinese herbal medicine with flavonoids as its main chemical components, used for the treatment of glomerulonephritis, gastritis, and rheumatoid arthritis (Hojilla-Evangelista et al. 2013). The herb is also recorded in the Pharmacopoeia of the People’s Republic of China (2010 edition). Shisanwei Thlaspi Capsule, a product derived from this traditional Chinese medicinal extract, has been approved by the National Medical Products Administration (NMPA) for the treatment of cystitis and rheumatoid arthritis. Additionally, Huahong tablet, which contains T. arvense as an ingredient, is known for its heat-clearing and detoxifying effects (Fan et al. 2013).
Previous studies on T. arvense have revealed its pharmacological properties, including acting as a hepatic tonic, improving vision, reducing fever, and promoting diuresis (Ge et al. 2013). Clinically, it is applied in the treatment of conditions such as kidney and urinary disorders (Ballabh et al. 2008). T. arvense mainly contains flavonoids, which are associated with antioxidant, antibacterial, anti-inflammatory, and antitoxic activities (Pang et al. 2013). A recent study on flavonoids from T. arvense reported that these compounds increased receptive and perceptive sexual motivations in the sexual behavior of male rats and improved the physiological functions of spermatogenesis (Pupykina et al. 2022).
Despite several previous reports, there have been few studies describing the biologically active constituents of T. arvense. In this study, we performed an extensive phytochemical analysis of the whole plant of T. arvense. Column chromatographic separation of its EtOH extract followed by high-performance liquid chromatography (HPLC) purification led to the isolation of three flavonoids (1-3), one diarylheptanoid (4), and two phenolic glycosides (5-6) via liquid chromatography/mass spectrometry (LC/MS)-based analysis. Their structures were elucidated by combining both spectroscopic NMR data and LC/MS analysis. The isolated compounds (1-6) were then tested to determine their effects on adipogenesis and osteogenesis in the mouse mesenchymal stem cell (MSC) line C3H10T1/2. Herein, we describe the isolation and structural characterization of the six compounds (1-6) and their potential effects on the reciprocal regulation of adipocyte and osteoblast differentiation.
The optical rotations were obtained using a Jasco P-1020 polarimeter (Jasco, Easton, MD, USA). The nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AVANCE III HD 850 NMR spectrometer at 850 MHz (1H) and 212.5 MHz (13C) with chemical shifts given in ppm (δ). Semi-preparative HPLC was performed on a Agilent 1200 Series with G1311A quaternary pump and diode array detector using Phenomenex Luna C18 column (250 × 10 mm, 5 µm; flow rate: 2 mL/min; Phenomenex, Torrance, CA, USA). The HR-ESI-MS data were obtained with an Agilent 6545 Q-TOF LC/MS spectrometer using an EclipsePlus C18 95 Å column (50 × 2.1 mm, 1.8 μm; flow rate: 0.3 mL/min; Agilent Technologies). LC/MS analysis was carried out on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 Series ESI mass spectrometer by using an analytical Kinetex C18 100 Å column (100 mm × 2.1 mm i.d., 5 μm) (Phenomenex, Torrance, CA).
The whole plant of T. arvense were collected at Namyangju-si, Gyeonggi-do, Korea, in April 2023. The plant material was verified by Dr. Moon Jin Ra, Min Hee Kwon and Jeong Nam Yu at Nakdonggang National Institute of Biological Resources, Sangju, Republic of Korea. A voucher specimen, namely HIMH-2314, was stored at the herbarium of the School of Pharmacy affiliated in Sungkyunkwan University, Suwon, Republic of Korea.
Dried whole plant of T. arvense (701.9 g) were extracted by sonicating three times (2.0 L × 3) for 90 min in 80% EtOH at room temperature and then filtered. The filtrate was subsequently evaporated in vacuo to obtain a crude EtOH extract (40.2 g). The enriched extract was suspended in distilled water (700 mL) and solvent-partitioned with n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) (each 700 mL × 3). Four fractions were obtained: n-hexane-soluble (6.3 g), CH2Cl2-soluble (778.1 mg), EtOAc-soluble (1.5 g), and n-BuOH-soluble fractions (20.4 g). LC/MS analysis of the four solvent-partitioned fractions indicated the presence of the main phenolic compounds exhibiting a characteristic UV spectrum of flavonoids in the EtOAc-soluble fraction. Compounds 1 (tR = 18.9 min, 2.1 mg), 2 (tR = 29.6 min, 11.4 mg), 3 (tR = 31.7 min, 1.3 mg), 4 (tR = 24.3 min, 1.4 mg), 5 (tR = 36.6 min, 0.8 mg), and 6 (tR = 45.6 min, 2.7 mg) were isolated from subfraction EtOAc-soluble fraction (1.5 g) via semi-preparative reversed-phase HPLC (Phenomenex C-18 100 Å column, 250 × 2.1 mm i.d., 5 μm) with 38% MeOH/H2O (isocratic system, flow rate: 2 mL/min).
Isoorientin (1) – Yellow powder,
Isovitexin (2) – Yellow powder,
Isoscoparin (3) – Yellow powder,
Oregonin (4) – Yellow powder,
1,2-Disinapoylgentiobiose (5) – Colorless gum,
1,2.2’-Trisinapoylgentiobiose (6) – Colorless gum,
The LC-MS analysis was carried out using an Agilent 1200 series HPLC system, equipped with a diode array detector and a 6130 Series ESI mass spectrometer. For the analysis, an analytical Kinetex C18 100 Å column (100 × 2.1 mm i.d., 5 μm particle size) from Phenomenex (Torrance, CA, USA), was employed, with a flow rate maintained at 0.3 mL/min. The fraction samples were analyzed using a gradient elution program from 10% MeOH-H2O to 100% MeOH for 52 min [10% MeOH-H2O → 100% MeOH (0-30 min), 100% MeOH (30-41 min), 100% MeOH → 10% MeOH-H2O (41-42 min), 10% MeOH-H2O (42-52 min), and a flow rate of 0.3 mL/min]. For the LC-MS data analysis, the application MZmine 3.2.8 was used to process the 3D plot visualization of the LC/MS data. The parameters were set as follows: MS mass range 100-1000 Da, retention time resolution 500, m/z resolution 500, MS level 1, Polarity –, Spectrum type Centroided.
The C3H10T1/2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin, Hyclone) at 37 °C in a 5% CO2 humidified atmosphere. For adipocyte differentiation, confluent cells were cultured in DMEM supplemented with 10% FBS, 1 μM dexamethasone (Sigma, St. Louis, MO, USA), 0.5 mM isobutyl-1-methylxanthine (Sigma), 5 μg/mL insulin (Sigma), and 10 μM troglitazone for 48 hours. Subsequently, the cells were transferred to DMEM containing 10% FBS, 5 μg/mL insulin, and 10 μM troglitazone and further incubated for an additional 3 days. During adipogenesis, 20 µM of compounds 1–6 was added to the cells, with resveratrol (20 µM) used as a positive control. For osteogenic differentiation, cells reaching confluence were cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, along with 10 mM β-glycerophosphate (Sigma-Aldrich) and 50 μg/mL ascorbic acid (Sigma-Aldrich), for 9 to 12 days. The medium was refreshed every 3 days. During osteogenic differentiation, 20 µM of compounds 1–6 was added to the cells, with 5 µM oryzativol A used as a positive control.
The cultured cells were rinsed with phosphate-buffered saline (PBS) and fixed in 10% neutral-buffered formalin at room temperature for 1 hour. Subsequently, the cells were stained with a 0.5% filtered stock solution of Oil Red O in isopropanol (Sigma, Saint Louis, MO, USA). After staining, the intracellular triglyceride content was assessed by redissolving the stained cells in isopropanol, followed by measurement of absorbance at a wavelength of 520 nm.
The cultured cells were rinsed with a 2 mM MgCl2 solution. Subsequently, they were incubated with ALP buffer, which is composed of 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 10 mM MgCl2, and 0.05% Tween-20, for 15 minutes. Following this, the cells were incubated in ALP buffer supplemented with 0.4 mg/mL of nitro-blue tetrazolium (Merck) and 0.2 g/mL of 5-bromo-4-chloro-3-indolyl phosphate (Merck). After washing with 0.5 mM ethylenediaminetetraacetic acid (EDTA), the cells were fixed with 10% neutral-buffered formalin for 1 hour.
To assess ALP activity, an alkaline phosphatase assay kit (ab83369; Abcam, Cambridge, MA, USA) was employed. Cell lysates obtained from the differentiated osteogenic cells were treated with a p-nitrophenyl phosphate (p-NPP) solution at 25 °C for 1 hour in the dark. Following cessation of the reaction, the absorbance of the samples was measured at 405 nm.
RNA extraction from the cells was performed using the NucleoZOL reagent (NucleoZOL; Macherey-Nagel GmbH & Co., KG, Germany). Subsequently, complementary DNA (cDNA) was synthesized from 0.5 μg of total RNA utilizing a ReverTraAce qPCR reverse transcription (RT) Master Mix Kit (FSQ-201; Toyobo, Japan) with random primers. The resulting cDNA was then combined with the amplification mixture comprising the Thunderbird SYBR qPCR Mix (Toyobo) along with the specified primers. The primers used for the quantitative PCR were:
Alkaline phosphatase (ALP): forward 5’-CAAGGATGCTGGGAAGTCCG-3’ and reverse 5’-CGGATAACGAGATGCCACCA-3’
Osteopontin (OPN): forward 5’-CTGGCAGCTCAGAGGAGAAG-3’ and reverse 5’-CAGCATTCTGTGGCGCAAG-3’
Each sample was tested in triplicate, and the experiment was repeated three times. Data are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to identify statistically significant differences between the control and test groups.
The EtOH extract of the whole plant of T. arvense underwent solvent partitioning using four organic solvents: n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). This process afforded four main fractions: n-hexane, CH2Cl2, EtOAc, and n-butanol soluble fractions (Fig. 1). Each fraction was analyzed by LC-MS in negative ion mode, and the 3D plot visualization of the LC/MS data was processed using the application MZmine 3.2.8. The 3D plot visualization revealed that the EtOAc-soluble fraction contained major peaks in the m/z range of 400-1000 (Fig. 2), which corresponded phenolic compounds as indicated by their characteristic UV spectra in the LC/MS data. This finding indicated that the EtOAc-soluble fraction is a promising fraction for investigating the main phenolic compounds. The chemical analysis of the EtOAc-soluble fraction using semi-preparative HPLC resulted in the purification of six compounds (1-6) (Fig. 1), which include three flavonoid derivatives (1-3), one diarylheptanoid derivative (4), and two phenolic glycosides (5-6). The compounds were identified as isoorientin (1) (Kumarasamy et al. 2004), isovitexin (2) (Pang et al. 2013), isoscoparin (3) (Senatore et al. 2000), oregonin (4) (Kuroyanagi et al. 2005), 1,2-disinapoylgentiobiose (5) (Piao et al. 2005), and 1,2.2’-trisinapoylgentiobiose (6) (Pedras and Zheng 2010) by comparing their spectroscopic data with values reported in previously published studies (Fig. 3).
Figure 1.(A) Separation scheme of compounds 1-6. (B) HPLC-UV chromatograms of EtOAc-soluble fraction (UV wavelengths: 210 nm [blue], 315 nm [red]).
Figure 2.The 3D plot visualization of the LC/MS data from the EtOAc-soluble fraction.
Figure 3.The chemical structures of compounds 1-6 from T. arvense.
To evaluate the effects of the isolated compounds 1-6 on MSC differentiation into adipocytes and osteoblasts, the mouse mesenchymal stem cell line C3H10T1/2 was exposed to concentrations of 20 µM of compounds 1-6 during adipogenic or osteogenic differentiation. While compounds 1-6 showed no significant impact on the adipocyte differentiation process of MSCs, isovitexin (2) exhibited a slight enhancement of osteogenic differentiation at a concentration of 20 μM compared to the effects observed with the other isolated compounds (Fig. 4).
Figure 4.The effects of isolated compounds 1-6 on MSC differentiation into osteoblasts or adipocytes using the mouse MSC line, C3H10T1/2. Adipogenic differentiation was assessed by staining cells with Oil Red O (ORO) (A) and quantifying stained lipid droplets’ absorbance at a red stain wavelength (B). Osteoblast differentiation was evaluated through alkaline phosphatase (ALP) staining (C), with the intensity of stained cells measured (D). The untreated negative control is denoted as NC. Adipogenesis was induced using a 20 μM concentration of resveratrol (Res) as a positive control, while osteogenesis was stimulated using 5 μM oryzativol A (OryA). Each compound was applied at a concentration of 20 μM in adipogenesis- or osteogenesis-inducing media. **p < 0.01, ***p < 0.005.
When isovitexin (2) was introduced into the culture medium of MSCs undergoing osteogenic differentiation, ranging from 1 to 20 μM concentrations, the intensity of alkaline phosphatase (ALP) staining increased proportionally with the increasing concentration of compound 2 (Fig. 5). This observation suggests that the process of osteogenic differentiation is facilitated in a dose-dependent fashion by isovitexin (2), although the activity was very weak. MSCs cultured in osteogenic differentiation medium containing isovitexin (2) exhibited an increase in the expression of osteopontin (OPN) and alkaline phosphatase (ALP), markers typically associated with bone and tooth formation (Fig. 6). Osteogenic differentiation of MSCs was induced in osteogenic differentiation medium, followed by mRNA analysis on the 10th day of culture. This analysis revealed a proportional increase in the expression levels of osteogenic markers ALP and OPN in response to varying concentrations of isovitexin (2). At the highest concentration of 20 μM, the gene expression of ALP and OPN was respectively 2.0-fold and 2.9-fold higher compared to the untreated negative control group.
Figure 5.The stimulatory effects of compound 2 on osteogenic differentiation. The mouse MSC line, C3H10T1/2, was treated with compound 2 in osteogenic differentiation media. ALP staining was conducted to assess osteoblast formation, with untreated cells marked as 0. Fully-differentiated cells were stained with ALP at 10 days post-osteogenic differentiation with varying concentrations of compound 2 (A). ALP activity was measured in osteogenic-differentiated MSCs treated with different concentrations of compound 2 (B). Oryzativol A (OryA, 5 μM) was used as a positive control. *p ≤ 0.05, **p < 0.01, ***p < 0.001.
Figure 6.Gene expression levels of osteogenic markers, ALP, and OPN, by compound 2 in MSCs. Oryzativol A (OryA, 5 μM) was used as a positive control. *p ≤ 0.05, **p < 0.01, ***p < 0.001.
As part of our continued search for natural products with biological properties, three flavonoids (isoorientin, isovitexin, and isoscoparin), one diarylheptanoid (oregonin), and two phenolic glycosides (1,2-disinapoylgentiobiose and 1,2,2’-trisinapoylgentiobiose) were isolated from the whole plant of T. arvense via semi-preparative HPLC purification. Compounds 1 (isoorientin) and 2 (isovitexin), both belong to the class of C-glycosylflavones (6-C-glucosyl luteolin and 6-C-glucosyl apigenin). They have been previously isolated from various sources including Drosophyllum lusitanicum, Cucumis sativus, and Gnidia involucrate (Peng et al. 2005). Previous studies have indicated that isoorientin (1) exhibited significant hepatoprotective effects and caused concentration-dependent inhibition of the amplitude and frequency of phasic contractions of rats and guinea-pig uterus (Orhan et al. 2003). Isovitexin (2) has also been reported to possess antioxidant properties. Compound 3 (isoscoparin), another C-glycosylflavone (isoorientin 3’-O-methyl ether), has been previously isolated from various sources including Citrullus colocynthis, Isatis tinctoria, and Potamogeton natans. Compound 4 (oregonin), a diarylheptanoid, has been isolated from the bark of Alnus species such as Alnus hirsuta var. sibirica (Lee et al. 2000). A previous study indicated that oregonin (4) exhibited inhibitory effects on the B16-F10 mouse melanoma cell line. Compounds 5 and 6, 1,2-disinapoylgentiobiose and 1,2,2’-trisinapoylgentiobiose, respectively, are two hydroxycinnamic acid esters of gentiobiose. They were first reported to be present in the fruits of Boreave orientalis, a member of the Cruciferae family (Price et al. 1997). The literature search revealed that the compounds (3-6) have been isolated and structurally elucidated from T. arvense for the first time.
Understanding the differentiation pathways of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes is crucial for developing treatments for conditions such as joint damage, obesity, and osteoporosis, especially in postmenopausal women (Phetfong et al. 2016). MSCs, found in the bone marrow, have the ability to differentiate into both osteocytes and adipocytes. In joint damage, degenerative changes around the articular cartilage can affect both cartilage and bone, leading to joint inflammation. Research on MSCs can aid in the development of new therapies for joint damage. Adipose-derived stem/stromal cells (ASCs) have been found to induce osteoblast differentiation and hold promise for treating bone tissue damage (Ciuffi et al. 2017). Postmenopausal weight gain is a significant issue, driven by hormonal changes such as decreased estrogen production, which can lead to increased cholesterol levels and visceral fat accumulation, particularly in the abdominal area (Davis et al. 2012). Furthermore, the decline in estrogen inhibitory effect on bone resorption after menopause contributes to significant bone loss and increases the risk of osteoporosis and fractures in postmenopausal women (Ji and Yu 2015). Therefore, there is a pressing need to develop treatments that can regulate the differentiation of adipocytes and osteoblasts to address complications associated with menopause, including obesity and osteoporosis. By understanding the mechanisms underlying MSC differentiation and targeting specific pathways involved, novel therapeutic interventions may be developed to mitigate the adverse effects of menopause on bone health and metabolism.
The findings of this study highlight the potential of compound 2, isovitexin, as a promoter of osteogenic differentiation. The increase in alkaline phosphatase (ALP) staining intensity with higher concentrations of compound 2 suggests its role in facilitating osteogenesis. Moreover, the expression of ALP and osteopontin (OPN) mRNA, important markers of osteogenic differentiation, was significantly upregulated in mesenchymal stem cells (MSCs) treated with compound 2 during osteogenesis. Although the gene expression levels of ALP and OPN induced by compound 2 were lower compared to the active positive control, oryzativol A, these results still underscore the osteogenic potential of isovitexin (2). Further studies may elucidate the precise mechanisms by which compound 2 enhances osteogenic differentiation and explore its therapeutic applications in bone regeneration and treatment of bone-related disorders.
It has been demonstrated that isovitexin (2) possesses a number of properties, including anti-oxidant, anti-inflammatory, and anti-Alzheimer’s disease effects (He et al. 2016). Recently, it was reported that in osteoblasts, isovitexin (2) rapidly phosphorylated AMP-activated protein kinase (pAMPK), which is downstream of AdipoRs and a master regulator of cellular energy metabolism, and upregulated the expression of AdipoRs (Pal et al. 2021). Isovitexin (2) also upregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a mitochondrial biogenesis factor in osteoblasts (Pal et al. 2021). Accordingly, isovitexin (2) was demonstrated to induce osteoblast differentiation that was fueled by mitochondrial respiration. However, to the best of our knowledge, the potential impact of isovitexin (2) on the enhancement of osteoblastic differentiation in MSCs is reported for the first time in this study.
In conclusion, the phytochemical investigation of the EtOH extract from the whole plant of T. arvense led to the isolation and identification of six compounds: isoorientin (1), isovitexin (2), isoscoparin (3), oregonin (4), 1,2-disinapoylgentiobiose (5), and 1,2,2’-trisinapoylgentiobiose (6). To the best of our knowledge, this is the first report of the presence of compounds 3–6 in this plant. Our study evaluated the effects of these isolated compounds on MSC differentiation into adipocytes and osteoblasts, revealing that isovitexin (2) enhanced osteoblastic differentiation. This was demonstrated by its ability to stimulate ALP production in a concentration-dependent manner and promote the mRNA expression of osteogenic markers such as ALP and OPN. These findings are particularly significant, suggesting the potential therapeutic efficacy of isovitexin (2) in preventing and treating conditions such as osteoporosis and other bone loss disorders.
No potential conflict of interest relevant to this article was reported.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2018R1A2B2006879 and 2019R1A5A2027340). This work was also supported by the Korea Environment Industry & Technology Institute (KEITI) through a project to improve the development of multi-ministerial national biological research re-sources, funded by the Republic of Korea Ministry of Environment (MOE) (grant number 2021003420003).
DTT 2024; 3(2): 95-104
Published online September 30, 2024 https://doi.org/10.58502/DTT.24.0007
Copyright © The Pharmaceutical Society of Korea.
Si Hyeon Chae1, Chan Hee Cho1, Seon Hee Kim2, Ki Hyun Kim1
1School of Pharmacy, Sungkyunkwan University, Suwon, Korea
2Sungkyun Biotech Co., Ltd., Anyang, Korea
Correspondence to:Ki Hyun Kim, khkim83@skku.edu
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Thlaspi arvense Linn, known as “Pennycress,” is a plant from the Brassicaceae family that has been traditionally used to treat glomerulonephritis, gastritis, and rheumatoid arthritis. As part of our current projects to discover bioactive compounds from natural resources, three flavonoids (1-3), one diarylheptanoid (4), and two phenolic glycosides (5-6) were isolated from the whole plant of T. arvense via semi-preparative HPLC purification. The chemical structures of compounds 1-6 were elucidated as isoorientin (1), isovitexin (2), isoscoparin (3), oregonin (4), 1,2-disinapoylgentiobiose (5), and 1,2,2’-trisinapoylgentiobiose (6) based on the comparison of their NMR spectroscopic and physical properties with those of previous studies. Notably, this is the first report of the presence of compounds 3-6 in this plant. Compounds 1-6 were then tested to determine their effects on osteogenesis and adipogenesis in the mouse mesenchymal stem cell (MSC) line C3H10T1/2. Among the six compounds, isovitexin (2) was found to promote the osteogenic differentiation of MSCs. As the concentration of 2 increased, the differentiation of MSCs into osteoblasts became more active, as evidenced by the induction of mRNA expression of the osteogenic markers, alkaline phosphatase (ALP) and osteopontin (OPN). Accordingly, our findings demonstrate that isovitexin (2) could potentially serve as a valuable compound for the treatment of menopause-associated syndromes, such as osteoporosis, by promoting MSC osteogenesis.
Keywords: Thlaspi arvense, Brassicaceae, phenolic compounds, isovitexin, osteogenesis
As part of our continuous endeavor to discover structurally and biologically novel compounds from diverse natural sources (Cho et al. 2022; Lee et al. 2022a; Lee et al. 2023; Lee et al. 2022b; Yu et al. 2022), we have collected and prepared ethanol (EtOH) extracts of freshwater plants for phytochemical investigation. Among the collected freshwater plants, Thlaspi arvense Linn, also referred to as “Pennycress,” is a plant belonging to the family Brassicaceae, which grows in most districts of Eurasia. Historically considered an agricultural weed, field pennycress is now extensively cultivated due to several desirable agronomic traits, such as high seed yield and high oil content, making it useful as a feedstock (Evangelista et al. 2012). Its aerial parts, including the whole herb, seedlings, and seeds, are utilized in medicine for their heat-clearing, detoxifying, diuretic, and detumescent effects (Ma et al. 2023).
T. arvense is a well-known Chinese herbal medicine with flavonoids as its main chemical components, used for the treatment of glomerulonephritis, gastritis, and rheumatoid arthritis (Hojilla-Evangelista et al. 2013). The herb is also recorded in the Pharmacopoeia of the People’s Republic of China (2010 edition). Shisanwei Thlaspi Capsule, a product derived from this traditional Chinese medicinal extract, has been approved by the National Medical Products Administration (NMPA) for the treatment of cystitis and rheumatoid arthritis. Additionally, Huahong tablet, which contains T. arvense as an ingredient, is known for its heat-clearing and detoxifying effects (Fan et al. 2013).
Previous studies on T. arvense have revealed its pharmacological properties, including acting as a hepatic tonic, improving vision, reducing fever, and promoting diuresis (Ge et al. 2013). Clinically, it is applied in the treatment of conditions such as kidney and urinary disorders (Ballabh et al. 2008). T. arvense mainly contains flavonoids, which are associated with antioxidant, antibacterial, anti-inflammatory, and antitoxic activities (Pang et al. 2013). A recent study on flavonoids from T. arvense reported that these compounds increased receptive and perceptive sexual motivations in the sexual behavior of male rats and improved the physiological functions of spermatogenesis (Pupykina et al. 2022).
Despite several previous reports, there have been few studies describing the biologically active constituents of T. arvense. In this study, we performed an extensive phytochemical analysis of the whole plant of T. arvense. Column chromatographic separation of its EtOH extract followed by high-performance liquid chromatography (HPLC) purification led to the isolation of three flavonoids (1-3), one diarylheptanoid (4), and two phenolic glycosides (5-6) via liquid chromatography/mass spectrometry (LC/MS)-based analysis. Their structures were elucidated by combining both spectroscopic NMR data and LC/MS analysis. The isolated compounds (1-6) were then tested to determine their effects on adipogenesis and osteogenesis in the mouse mesenchymal stem cell (MSC) line C3H10T1/2. Herein, we describe the isolation and structural characterization of the six compounds (1-6) and their potential effects on the reciprocal regulation of adipocyte and osteoblast differentiation.
The optical rotations were obtained using a Jasco P-1020 polarimeter (Jasco, Easton, MD, USA). The nuclear magnetic resonance (NMR) spectra were obtained using a Bruker AVANCE III HD 850 NMR spectrometer at 850 MHz (1H) and 212.5 MHz (13C) with chemical shifts given in ppm (δ). Semi-preparative HPLC was performed on a Agilent 1200 Series with G1311A quaternary pump and diode array detector using Phenomenex Luna C18 column (250 × 10 mm, 5 µm; flow rate: 2 mL/min; Phenomenex, Torrance, CA, USA). The HR-ESI-MS data were obtained with an Agilent 6545 Q-TOF LC/MS spectrometer using an EclipsePlus C18 95 Å column (50 × 2.1 mm, 1.8 μm; flow rate: 0.3 mL/min; Agilent Technologies). LC/MS analysis was carried out on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 Series ESI mass spectrometer by using an analytical Kinetex C18 100 Å column (100 mm × 2.1 mm i.d., 5 μm) (Phenomenex, Torrance, CA).
The whole plant of T. arvense were collected at Namyangju-si, Gyeonggi-do, Korea, in April 2023. The plant material was verified by Dr. Moon Jin Ra, Min Hee Kwon and Jeong Nam Yu at Nakdonggang National Institute of Biological Resources, Sangju, Republic of Korea. A voucher specimen, namely HIMH-2314, was stored at the herbarium of the School of Pharmacy affiliated in Sungkyunkwan University, Suwon, Republic of Korea.
Dried whole plant of T. arvense (701.9 g) were extracted by sonicating three times (2.0 L × 3) for 90 min in 80% EtOH at room temperature and then filtered. The filtrate was subsequently evaporated in vacuo to obtain a crude EtOH extract (40.2 g). The enriched extract was suspended in distilled water (700 mL) and solvent-partitioned with n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) (each 700 mL × 3). Four fractions were obtained: n-hexane-soluble (6.3 g), CH2Cl2-soluble (778.1 mg), EtOAc-soluble (1.5 g), and n-BuOH-soluble fractions (20.4 g). LC/MS analysis of the four solvent-partitioned fractions indicated the presence of the main phenolic compounds exhibiting a characteristic UV spectrum of flavonoids in the EtOAc-soluble fraction. Compounds 1 (tR = 18.9 min, 2.1 mg), 2 (tR = 29.6 min, 11.4 mg), 3 (tR = 31.7 min, 1.3 mg), 4 (tR = 24.3 min, 1.4 mg), 5 (tR = 36.6 min, 0.8 mg), and 6 (tR = 45.6 min, 2.7 mg) were isolated from subfraction EtOAc-soluble fraction (1.5 g) via semi-preparative reversed-phase HPLC (Phenomenex C-18 100 Å column, 250 × 2.1 mm i.d., 5 μm) with 38% MeOH/H2O (isocratic system, flow rate: 2 mL/min).
Isoorientin (1) – Yellow powder,
Isovitexin (2) – Yellow powder,
Isoscoparin (3) – Yellow powder,
Oregonin (4) – Yellow powder,
1,2-Disinapoylgentiobiose (5) – Colorless gum,
1,2.2’-Trisinapoylgentiobiose (6) – Colorless gum,
The LC-MS analysis was carried out using an Agilent 1200 series HPLC system, equipped with a diode array detector and a 6130 Series ESI mass spectrometer. For the analysis, an analytical Kinetex C18 100 Å column (100 × 2.1 mm i.d., 5 μm particle size) from Phenomenex (Torrance, CA, USA), was employed, with a flow rate maintained at 0.3 mL/min. The fraction samples were analyzed using a gradient elution program from 10% MeOH-H2O to 100% MeOH for 52 min [10% MeOH-H2O → 100% MeOH (0-30 min), 100% MeOH (30-41 min), 100% MeOH → 10% MeOH-H2O (41-42 min), 10% MeOH-H2O (42-52 min), and a flow rate of 0.3 mL/min]. For the LC-MS data analysis, the application MZmine 3.2.8 was used to process the 3D plot visualization of the LC/MS data. The parameters were set as follows: MS mass range 100-1000 Da, retention time resolution 500, m/z resolution 500, MS level 1, Polarity –, Spectrum type Centroided.
The C3H10T1/2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin, Hyclone) at 37 °C in a 5% CO2 humidified atmosphere. For adipocyte differentiation, confluent cells were cultured in DMEM supplemented with 10% FBS, 1 μM dexamethasone (Sigma, St. Louis, MO, USA), 0.5 mM isobutyl-1-methylxanthine (Sigma), 5 μg/mL insulin (Sigma), and 10 μM troglitazone for 48 hours. Subsequently, the cells were transferred to DMEM containing 10% FBS, 5 μg/mL insulin, and 10 μM troglitazone and further incubated for an additional 3 days. During adipogenesis, 20 µM of compounds 1–6 was added to the cells, with resveratrol (20 µM) used as a positive control. For osteogenic differentiation, cells reaching confluence were cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, along with 10 mM β-glycerophosphate (Sigma-Aldrich) and 50 μg/mL ascorbic acid (Sigma-Aldrich), for 9 to 12 days. The medium was refreshed every 3 days. During osteogenic differentiation, 20 µM of compounds 1–6 was added to the cells, with 5 µM oryzativol A used as a positive control.
The cultured cells were rinsed with phosphate-buffered saline (PBS) and fixed in 10% neutral-buffered formalin at room temperature for 1 hour. Subsequently, the cells were stained with a 0.5% filtered stock solution of Oil Red O in isopropanol (Sigma, Saint Louis, MO, USA). After staining, the intracellular triglyceride content was assessed by redissolving the stained cells in isopropanol, followed by measurement of absorbance at a wavelength of 520 nm.
The cultured cells were rinsed with a 2 mM MgCl2 solution. Subsequently, they were incubated with ALP buffer, which is composed of 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 10 mM MgCl2, and 0.05% Tween-20, for 15 minutes. Following this, the cells were incubated in ALP buffer supplemented with 0.4 mg/mL of nitro-blue tetrazolium (Merck) and 0.2 g/mL of 5-bromo-4-chloro-3-indolyl phosphate (Merck). After washing with 0.5 mM ethylenediaminetetraacetic acid (EDTA), the cells were fixed with 10% neutral-buffered formalin for 1 hour.
To assess ALP activity, an alkaline phosphatase assay kit (ab83369; Abcam, Cambridge, MA, USA) was employed. Cell lysates obtained from the differentiated osteogenic cells were treated with a p-nitrophenyl phosphate (p-NPP) solution at 25 °C for 1 hour in the dark. Following cessation of the reaction, the absorbance of the samples was measured at 405 nm.
RNA extraction from the cells was performed using the NucleoZOL reagent (NucleoZOL; Macherey-Nagel GmbH & Co., KG, Germany). Subsequently, complementary DNA (cDNA) was synthesized from 0.5 μg of total RNA utilizing a ReverTraAce qPCR reverse transcription (RT) Master Mix Kit (FSQ-201; Toyobo, Japan) with random primers. The resulting cDNA was then combined with the amplification mixture comprising the Thunderbird SYBR qPCR Mix (Toyobo) along with the specified primers. The primers used for the quantitative PCR were:
Alkaline phosphatase (ALP): forward 5’-CAAGGATGCTGGGAAGTCCG-3’ and reverse 5’-CGGATAACGAGATGCCACCA-3’
Osteopontin (OPN): forward 5’-CTGGCAGCTCAGAGGAGAAG-3’ and reverse 5’-CAGCATTCTGTGGCGCAAG-3’
Each sample was tested in triplicate, and the experiment was repeated three times. Data are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to identify statistically significant differences between the control and test groups.
The EtOH extract of the whole plant of T. arvense underwent solvent partitioning using four organic solvents: n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). This process afforded four main fractions: n-hexane, CH2Cl2, EtOAc, and n-butanol soluble fractions (Fig. 1). Each fraction was analyzed by LC-MS in negative ion mode, and the 3D plot visualization of the LC/MS data was processed using the application MZmine 3.2.8. The 3D plot visualization revealed that the EtOAc-soluble fraction contained major peaks in the m/z range of 400-1000 (Fig. 2), which corresponded phenolic compounds as indicated by their characteristic UV spectra in the LC/MS data. This finding indicated that the EtOAc-soluble fraction is a promising fraction for investigating the main phenolic compounds. The chemical analysis of the EtOAc-soluble fraction using semi-preparative HPLC resulted in the purification of six compounds (1-6) (Fig. 1), which include three flavonoid derivatives (1-3), one diarylheptanoid derivative (4), and two phenolic glycosides (5-6). The compounds were identified as isoorientin (1) (Kumarasamy et al. 2004), isovitexin (2) (Pang et al. 2013), isoscoparin (3) (Senatore et al. 2000), oregonin (4) (Kuroyanagi et al. 2005), 1,2-disinapoylgentiobiose (5) (Piao et al. 2005), and 1,2.2’-trisinapoylgentiobiose (6) (Pedras and Zheng 2010) by comparing their spectroscopic data with values reported in previously published studies (Fig. 3).
Figure 1. (A) Separation scheme of compounds 1-6. (B) HPLC-UV chromatograms of EtOAc-soluble fraction (UV wavelengths: 210 nm [blue], 315 nm [red]).
Figure 2. The 3D plot visualization of the LC/MS data from the EtOAc-soluble fraction.
Figure 3. The chemical structures of compounds 1-6 from T. arvense.
To evaluate the effects of the isolated compounds 1-6 on MSC differentiation into adipocytes and osteoblasts, the mouse mesenchymal stem cell line C3H10T1/2 was exposed to concentrations of 20 µM of compounds 1-6 during adipogenic or osteogenic differentiation. While compounds 1-6 showed no significant impact on the adipocyte differentiation process of MSCs, isovitexin (2) exhibited a slight enhancement of osteogenic differentiation at a concentration of 20 μM compared to the effects observed with the other isolated compounds (Fig. 4).
Figure 4. The effects of isolated compounds 1-6 on MSC differentiation into osteoblasts or adipocytes using the mouse MSC line, C3H10T1/2. Adipogenic differentiation was assessed by staining cells with Oil Red O (ORO) (A) and quantifying stained lipid droplets’ absorbance at a red stain wavelength (B). Osteoblast differentiation was evaluated through alkaline phosphatase (ALP) staining (C), with the intensity of stained cells measured (D). The untreated negative control is denoted as NC. Adipogenesis was induced using a 20 μM concentration of resveratrol (Res) as a positive control, while osteogenesis was stimulated using 5 μM oryzativol A (OryA). Each compound was applied at a concentration of 20 μM in adipogenesis- or osteogenesis-inducing media. **p < 0.01, ***p < 0.005.
When isovitexin (2) was introduced into the culture medium of MSCs undergoing osteogenic differentiation, ranging from 1 to 20 μM concentrations, the intensity of alkaline phosphatase (ALP) staining increased proportionally with the increasing concentration of compound 2 (Fig. 5). This observation suggests that the process of osteogenic differentiation is facilitated in a dose-dependent fashion by isovitexin (2), although the activity was very weak. MSCs cultured in osteogenic differentiation medium containing isovitexin (2) exhibited an increase in the expression of osteopontin (OPN) and alkaline phosphatase (ALP), markers typically associated with bone and tooth formation (Fig. 6). Osteogenic differentiation of MSCs was induced in osteogenic differentiation medium, followed by mRNA analysis on the 10th day of culture. This analysis revealed a proportional increase in the expression levels of osteogenic markers ALP and OPN in response to varying concentrations of isovitexin (2). At the highest concentration of 20 μM, the gene expression of ALP and OPN was respectively 2.0-fold and 2.9-fold higher compared to the untreated negative control group.
Figure 5. The stimulatory effects of compound 2 on osteogenic differentiation. The mouse MSC line, C3H10T1/2, was treated with compound 2 in osteogenic differentiation media. ALP staining was conducted to assess osteoblast formation, with untreated cells marked as 0. Fully-differentiated cells were stained with ALP at 10 days post-osteogenic differentiation with varying concentrations of compound 2 (A). ALP activity was measured in osteogenic-differentiated MSCs treated with different concentrations of compound 2 (B). Oryzativol A (OryA, 5 μM) was used as a positive control. *p ≤ 0.05, **p < 0.01, ***p < 0.001.
Figure 6. Gene expression levels of osteogenic markers, ALP, and OPN, by compound 2 in MSCs. Oryzativol A (OryA, 5 μM) was used as a positive control. *p ≤ 0.05, **p < 0.01, ***p < 0.001.
As part of our continued search for natural products with biological properties, three flavonoids (isoorientin, isovitexin, and isoscoparin), one diarylheptanoid (oregonin), and two phenolic glycosides (1,2-disinapoylgentiobiose and 1,2,2’-trisinapoylgentiobiose) were isolated from the whole plant of T. arvense via semi-preparative HPLC purification. Compounds 1 (isoorientin) and 2 (isovitexin), both belong to the class of C-glycosylflavones (6-C-glucosyl luteolin and 6-C-glucosyl apigenin). They have been previously isolated from various sources including Drosophyllum lusitanicum, Cucumis sativus, and Gnidia involucrate (Peng et al. 2005). Previous studies have indicated that isoorientin (1) exhibited significant hepatoprotective effects and caused concentration-dependent inhibition of the amplitude and frequency of phasic contractions of rats and guinea-pig uterus (Orhan et al. 2003). Isovitexin (2) has also been reported to possess antioxidant properties. Compound 3 (isoscoparin), another C-glycosylflavone (isoorientin 3’-O-methyl ether), has been previously isolated from various sources including Citrullus colocynthis, Isatis tinctoria, and Potamogeton natans. Compound 4 (oregonin), a diarylheptanoid, has been isolated from the bark of Alnus species such as Alnus hirsuta var. sibirica (Lee et al. 2000). A previous study indicated that oregonin (4) exhibited inhibitory effects on the B16-F10 mouse melanoma cell line. Compounds 5 and 6, 1,2-disinapoylgentiobiose and 1,2,2’-trisinapoylgentiobiose, respectively, are two hydroxycinnamic acid esters of gentiobiose. They were first reported to be present in the fruits of Boreave orientalis, a member of the Cruciferae family (Price et al. 1997). The literature search revealed that the compounds (3-6) have been isolated and structurally elucidated from T. arvense for the first time.
Understanding the differentiation pathways of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes is crucial for developing treatments for conditions such as joint damage, obesity, and osteoporosis, especially in postmenopausal women (Phetfong et al. 2016). MSCs, found in the bone marrow, have the ability to differentiate into both osteocytes and adipocytes. In joint damage, degenerative changes around the articular cartilage can affect both cartilage and bone, leading to joint inflammation. Research on MSCs can aid in the development of new therapies for joint damage. Adipose-derived stem/stromal cells (ASCs) have been found to induce osteoblast differentiation and hold promise for treating bone tissue damage (Ciuffi et al. 2017). Postmenopausal weight gain is a significant issue, driven by hormonal changes such as decreased estrogen production, which can lead to increased cholesterol levels and visceral fat accumulation, particularly in the abdominal area (Davis et al. 2012). Furthermore, the decline in estrogen inhibitory effect on bone resorption after menopause contributes to significant bone loss and increases the risk of osteoporosis and fractures in postmenopausal women (Ji and Yu 2015). Therefore, there is a pressing need to develop treatments that can regulate the differentiation of adipocytes and osteoblasts to address complications associated with menopause, including obesity and osteoporosis. By understanding the mechanisms underlying MSC differentiation and targeting specific pathways involved, novel therapeutic interventions may be developed to mitigate the adverse effects of menopause on bone health and metabolism.
The findings of this study highlight the potential of compound 2, isovitexin, as a promoter of osteogenic differentiation. The increase in alkaline phosphatase (ALP) staining intensity with higher concentrations of compound 2 suggests its role in facilitating osteogenesis. Moreover, the expression of ALP and osteopontin (OPN) mRNA, important markers of osteogenic differentiation, was significantly upregulated in mesenchymal stem cells (MSCs) treated with compound 2 during osteogenesis. Although the gene expression levels of ALP and OPN induced by compound 2 were lower compared to the active positive control, oryzativol A, these results still underscore the osteogenic potential of isovitexin (2). Further studies may elucidate the precise mechanisms by which compound 2 enhances osteogenic differentiation and explore its therapeutic applications in bone regeneration and treatment of bone-related disorders.
It has been demonstrated that isovitexin (2) possesses a number of properties, including anti-oxidant, anti-inflammatory, and anti-Alzheimer’s disease effects (He et al. 2016). Recently, it was reported that in osteoblasts, isovitexin (2) rapidly phosphorylated AMP-activated protein kinase (pAMPK), which is downstream of AdipoRs and a master regulator of cellular energy metabolism, and upregulated the expression of AdipoRs (Pal et al. 2021). Isovitexin (2) also upregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a mitochondrial biogenesis factor in osteoblasts (Pal et al. 2021). Accordingly, isovitexin (2) was demonstrated to induce osteoblast differentiation that was fueled by mitochondrial respiration. However, to the best of our knowledge, the potential impact of isovitexin (2) on the enhancement of osteoblastic differentiation in MSCs is reported for the first time in this study.
In conclusion, the phytochemical investigation of the EtOH extract from the whole plant of T. arvense led to the isolation and identification of six compounds: isoorientin (1), isovitexin (2), isoscoparin (3), oregonin (4), 1,2-disinapoylgentiobiose (5), and 1,2,2’-trisinapoylgentiobiose (6). To the best of our knowledge, this is the first report of the presence of compounds 3–6 in this plant. Our study evaluated the effects of these isolated compounds on MSC differentiation into adipocytes and osteoblasts, revealing that isovitexin (2) enhanced osteoblastic differentiation. This was demonstrated by its ability to stimulate ALP production in a concentration-dependent manner and promote the mRNA expression of osteogenic markers such as ALP and OPN. These findings are particularly significant, suggesting the potential therapeutic efficacy of isovitexin (2) in preventing and treating conditions such as osteoporosis and other bone loss disorders.
No potential conflict of interest relevant to this article was reported.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2018R1A2B2006879 and 2019R1A5A2027340). This work was also supported by the Korea Environment Industry & Technology Institute (KEITI) through a project to improve the development of multi-ministerial national biological research re-sources, funded by the Republic of Korea Ministry of Environment (MOE) (grant number 2021003420003).
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