Lignan Glycosides and Flavonoid Glycosides from the Aerial Portion of Lespedeza cuneata and Their Biological Evaluations

Lespedeza cuneata (Fabaceae), known as Chinese bushclover, has been used in traditional medicines for the treatment of diseases including diabetes, hematuria, and insomnia. As part of a continuing search for bioactive constituents from Korean medicinal plant sources, phytochemical analysis of the aerial portion of L. cuneata led to the isolation of two new lignan glycosides (1,2) along with three known lignan glycosides (3–7) and nine known flavonoid glycosides (8–14). Numerous analysis techniques, including 1D and 2D NMR spectroscopy, CD spectroscopy, HR-MS, and chemical reactions, were utilized for structural elucidation of the new compounds (1,2). The isolated compounds were evaluated for their applicability in medicinal use using cell-based assays. Compounds 1 and 4–6 exhibited weak cytotoxicity against four human breast cancer cell lines (Bt549, MCF7, MDA-MB-231, and HCC70) (IC50 < 30.0 μM). However, none of the isolated compounds showed significant antiviral activity against PR8, HRV1B, or CVB3. In addition, compound 10 produced fewer lipid droplets in Oil Red O staining of mouse mesenchymal stem cells compared to the untreated negative control without altering the amount of alkaline phosphatase staining.


Introduction
Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae), known as Chinese bushclover, is a warm-season, perennial legume that is widely distributed in Korea, China, and India [1]. This plant has been used in folk medicine for the treatment of diseases, including diabetes, hematuria, and insomnia, as well as for the protection of the kidneys, liver, and lungs [2,3]. Previous pharmacological studies of this medicinal plant have revealed that extracts of L. cuneata exhibit inhibition of inflammatory mediators in Lipopolysaccharide (LPS)-activated RAW264.7 cells and paw edema in carrageenan-stimulated inflammatory mediators in Lipopolysaccharide (LPS)-activated RAW264.7 cells and paw edema in carrageenan-stimulated rats [4], as well as hepatoprotective and antidiabetic effects [1,2,5,6]. A recent study of L. cuneata extract reported its in vitro cytotoxic effects against several cancer cell lines including HeLa, Hep3B, A549, and Sarcoma180 [7]. In terms of phytochemical components, it is a rich source of various compounds such as steroids, flavonoids, phenolics [3,6,8], phenylpropanoids [2,9], lignans [5,9], and phenyldilactones [10]. Among the constituents, lignans, and flavonoids are the main components of L. cuneata, and the lignans were found to have hepatoprotective [5] and antiulcerative colitis activities [9], and the flavonoids were reported to show hepatoprotective [6] and NO-inhibitory effects [11].
As part of a continuing search for bioactive constituents from Korean medicinal plant sources [12][13][14], the methanol (MeOH) extract of the aerial portion of L. cuneata was found to exhibit cytotoxic effects on human ovarian carcinoma cells [15]. In our recent study, bioassay-guided fractionation and repeated chromatography of the MeOH extract of L. cuneata resulted in isolation of (−)-9′-O-(α-Lrhamnopyranosyl)lyoniresinol, which suppresses the proliferation of A2780 human ovarian carcinoma cells through induction of apoptosis [15]. In the current study investigating bioactive compounds from the aerial portion of L. cuneata, further phytochemical analysis was carried out, which led to the isolation of two new lignan glycosides (1,2) along with three known lignan glycosides (3)(4)(5)(6)(7) and nine known flavonoid glycosides (8)(9)(10)(11)(12)(13)(14). Numerous analysis techniques, including 1D and 2D NMR spectroscopy, CD spectroscopy, HR-MS, and chemical reactions, were utilized for structural elucidation of the new compounds (1,2). Subsequently, we investigated the possible therapeutic effects of the isolated compounds using various cell-based assays. In this paper, we describe the isolation and structural characterization of compounds 1-14 ( Figure 1), as well as the evaluation of their applicability to medicinal use including their cytotoxicity, antiviral activity, and their effects on the regulation of adipocyte and osteoblast differentiation.

Antiviral Activity of the Isolated Compounds against PR8, HRV1B, and CVB3 Infection
Recently, many studies exploring antiviral natural products and organic synthetic compounds have reported that a variety of flavonoids exhibit potent antiviral activity by inhibiting the early stages of viral infection, viral protein expression, and neuraminidase activity [35][36][37]. Therefore, we assessed the isolated compounds (1-14) for their antiviral activity against PR8, HRV1B, and CVB3 infection in A549, Vero, and HeLa cells, respectively. Less than 10% of the cells survived in the positive-control group (cells with virus only) after 48 hours of infection. In addition, cells treated with compounds 1-14 (10 µM) also had less than 10% survival. Because we could not identify any significant differences between the control and test groups, these results suggest that the compounds do not show significant antiviral activity against PR8, HRV1B, or CVB3.

Regulatory Effects of Compound 10 on Differentiation into Adipocytes and Osteoblasts
Mesenchymal stem cells (MSCs) in the bone marrow are pluripotent cells, which differentiate into osteocytes as well as adipocytes. Since microenvironmental changes such as hormones, immune responses, and metabolism cause alterations in the regulation of MSC differentiation, where alterations in the expression of the related genes might disturb the balance between osteoprogenitor and adipocyte progenitor cells in osteoporosis patients [38], natural products that are able to suppress MSC differentiation toward adipocytes and/or promote osteogenic differentiation of MSC would be promising in the management of postmenopausal osteoporosis. The biological activity of compound 10 was additionally tested regarding its effects on the differentiation of mouse MSCs into adipocytes or osteoblasts, since large amounts of compound 10 was isolated among the isolated compounds. Compound 10 was added to the MSC culture media during adipocyte differentiation. Compound 10 slightly reduced the formation of lipid droplets and resulted in somewhat fewer Oil Red O (ORO)-stained cells compared to the normally differentiated adipocytes ( Figure 3A). However, ALP staining and ALP activity in the compound 10-treated cells did not increase during the MSC differentiation into osteoblasts, in contrast to the positive control group treated with oryzativol A ( Figure 3B). These results demonstrate that compound 10 marginally suppressed adipogenesis of MSCs but did not influence osteogenesis. responses, and metabolism cause alterations in the regulation of MSC differentiation, where alterations in the expression of the related genes might disturb the balance between osteoprogenitor and adipocyte progenitor cells in osteoporosis patients [38], natural products that are able to suppress MSC differentiation toward adipocytes and/or promote osteogenic differentiation of MSC would be promising in the management of postmenopausal osteoporosis. The biological activity of compound 10 was additionally tested regarding its effects on the differentiation of mouse MSCs into adipocytes or osteoblasts, since large amounts of compound 10 was isolated among the isolated compounds. Compound 10 was added to the MSC culture media during adipocyte differentiation. Compound 10 slightly reduced the formation of lipid droplets and resulted in somewhat fewer Oil Red O (ORO)stained cells compared to the normally differentiated adipocytes ( Figure 3A). However, ALP staining and ALP activity in the compound 10-treated cells did not increase during the MSC differentiation into osteoblasts, in contrast to the positive control group treated with oryzativol A ( Figure 3B). These results demonstrate that compound 10 marginally suppressed adipogenesis of MSCs but did not influence osteogenesis.

Plant Material
The aerial portions of L. cuneata were collected from Mt. Bangtae, Inje, Kangwon Province, Republic of Korea, in October 2016. The plant materials were identified by one of the authors, Prof. S. Lee. A voucher specimen (YKM-2016) was deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Extraction and Isolation
The dried aerial portions of L. cuneata (4.2 kg) were extracted three times with 4.2 L of 80% MeOH for three days at room temperature and filtered. The resultant filtrate was evaporated under reduced , and the number of stained lipid droplets was quantitatively evaluated (A). After osteoblast differentiation, the cells were stained for ALP levels, and the ALP activity was measured (B). Ctrl represents untreated negative control. For the positive controls, 40 micrograms of resveratrol (Res) was used for adipogenesis and 5 µM of oryzativol A (OryA) was added for osteogenesis. * denotes 0.01 ≤ p ≤ 0.05 and *** denotes p < 0.001.

Plant Material
The aerial portions of L. cuneata were collected from Mt. Bangtae, Inje, Kangwon Province, Republic of Korea, in October 2016. The plant materials were identified by one of the authors, Prof. S. Lee. A voucher specimen (YKM-2016) was deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Extraction and Isolation
The dried aerial portions of L. cuneata (4.2 kg) were extracted three times with 4.2 L of 80% MeOH for three days at room temperature and filtered. The resultant filtrate was evaporated under reduced pressure using a rotavap to obtain the MeOH extract (401.8 g), which was suspended in distilled H 2 O (2 L) and successively solvent-partitioned with hexane, CH 2 Cl 2 , EtOAc, and n-BuOH (2.0 L × 3 for each) to yield the hexane-(20.6 g), CH 2 Cl 2 -(0.7 g), EtOAc-(12.7 g), and n-BuOH-soluble (69.3 g) fractions.

Enzymatic Hydrolysis of Compounds 1,2
A solution of each compound (1.0 mg) in H 2 O (1 mL) was individually hydrolyzed with naringinase (10 mg, from Penicillium sp.; ICN Biomedicals Inc., Irvine, CA, USA) at 40 • C for 36 h. Each reaction mixture was extracted with CH 2 Cl 2 to yield the individual CH 2 Cl 2 extract and a water phase. The CH 2 Cl 2 extracts from compounds 1 and 2 were chromatographically separately with a Phenomenex Strata ® C18-E column (2 g) using a gradient solvent system from 100% H 2 O to 100% MeOH to give aglycones 1a (0.  [16]. After drying the water phase in vacuo, the residue was dissolved in anhydrous pyridine (200 µL) followed by the addition of L-cysteine methyl ester hydrochloride (0.6 mg). The reaction mixture was incubated at 60 • C for 1 h, then O-tolyl isothiocyanate (15 µL) was added and the mixture was incubated at 60 • C for 1 h. The reaction product was directly analyzed using LC/MS (0−35% MeCN for 30 min, flow rate: 0.3 mL/min) with an analytical Kinetex column (2.1 × 100 mm, 5 µm) (Agilent Technologies, Santa Clara, CA, USA). The L-rhamnose in compounds 1 and 2 was identified through comparison of the retention times with those of authentic sample (t R = L-rhamnose 25.6 min).

Cytotoxicity Assay
A sulforhodamine B (SRB) bioassay was used to determine the cytotoxicity of each isolated compound against four cultured human tumor cell lines [12,34]. The assays were performed at the Korea Research Institute of Chemical Technology. All the cell lines used, Bt549, MCF7, MDA-MB-231, and HCC70, are human breast cancer cells. Etoposide (purity ≥ 98%, Sigma, St. Louis, MO, USA) was used as a positive control. The half maximal inhibitory concentrations (IC 50 ) of cancer cell growth are expressed as the mean from three distinct experiments.

Oil Red OStaining
At 6-8 days after differentiation, the adipocytes were fixed with 10% neutral buffered formalin (NBF) and stained with 0.5% Oil Red O (Sigma, St. Louis, MO, USA). To stop the reaction, cells were washed with distilled water three times. Stained cells were resolved with 1 mL of isopropanol and the colorimetric changes was measured at 520 nm to evaluate intra-cellular triglyceride content.

Alkaline Phosphatase (ALP) Staining and Activity
At 7-9 days after osteogenic differentiation, the medium was removed, and the cells were washed with 2 mM MgCl 2 solution. After incubation with AP buffer (100 mM Tris−HCl, pH 9.5, 100 mM NaCl, and 10 mM MgCl 2 ) for 15 min, the cells were treated in AP buffer containing 0.4 mg/mL of nitro-blue tetrazolium (NBT, Sigma) and 0.2 mg/mL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) for 15 more minutes. To stop the reaction, the cells were exposed to 5 mM EDTA (pH 8.0) and fixed with 10% NBF for 1 h.
The differentiation into osteoblast was evaluated regarding ALP activity. The ALP activity was determined using an Alkaline Phosphatase Assay Kit (ab83369; Abcam, Cambridge, MA, USA). Briefly, the cell lysates were incubated with p-nitrophenyl phosphate (p-NPP) solution at RT for 1 h in the dark. After stopping the reaction, the optical density was measured at 405 nm using a SpectraMax M2/M2e Microplate Readers (Molecular Devices, San Jose, CA, USA).

Conclusions
In the present study, phytochemical analysis of the aerial portion of L. cuneata led to the isolation of two new lignan glycosides (1,2) along with three known lignan glycosides (3-7) and nine known flavonoid glycosides (8)(9)(10)(11)(12)(13)(14). All the isolated compounds were evaluated for their applicability for medicinal use using cell-based assays. Compounds 1 and 4-6 exhibited weak cytotoxicity against the breast cancer cell lines (Bt549, MCF7, MDA-MB-231 and HCC70) (IC 50 < 30.0 µM), while none of the isolated compound showed significant antiviral activity against PR8, HRV1B, or CVB3. In a mouse mesenchymal stem cell line, treatment with compound 10 resulted in fewer lipid droplets compared to the untreated negative without altering the amount of alkaline phosphatase staining.  Table S1 are available free of charge on the Internet.