A Halogen-Containing Stilbene Derivative from the Leaves of Cajanus cajan that Induces Osteogenic Differentiation of Human Mesenchymal Stem Cells

A new natural halogen-containing stilbene derivative was isolated from the leaves of Cajanus cajan (L.) Millsp. and identified as 3-O-(3-chloro-2-hydroxyl-propanyl)-longistylin A by comprehensive spectroscopic and chemical analysis, and named cajanstilbene H (1). It is the first halogen-containing stilbene derivative found from plants. In human mesenchymal stem cells (hMSC) from bone marrow, 1 did not promote cell proliferation, but distinctly enhanced osteogenic differentiation of hMSC in time- and dose-dependent manners. In six human cancer cell lines, 1 showed a moderate inhibitory effect on cell proliferation, with IC50 values of 21.42–25.85 μmol·L−1.


Introduction
Cajanus cajan (L.) Millsp., commonly known as pigeon pea, is a plant of Fabaceae grown in semiarid tropics in Asia and Africa. Extracts and components from pigeon pea leaves were reported to show a variety of bioactivities, including antimalaria [1], cytotoxicity [2,3], hypolipidemia [4], reducing bone loss and promoting bone-like tissue formation [5][6][7]. A series of phytochemical studies have demonstrated that flavonoids and stilbenoids are main chemical constituents of the leaves [8][9][10][11][12][13][14]. In our previous investigation, a hydrophobic fraction of pigeon pea leaves showed activity in increasing tibial bone density and improving bone metabolism and lipid metabolism in ob/ob mice with osteoporosis and hyperlipidemia symptom [15]. In this paper, we present the identification of a new compound from this extract and its bioactivities in oesteogenic differentiation of human mesenchymal stem cells (hMSC) from bone marrow and cytotoxicity against human cancer cell lines. , and the presence of chlorine assayde by ion chromatography (Supplementary Information, Pages S11). The NMR spectral data of 1 (Table 1) were similar to those of longistylin A, a major stilbene previously isolated from the same plant [1,10], suggesting the existence of a longistylin A moiety. A closer inspection of the NMR spectra revealed the subtle differences between longistylin A and 1: the resonance signal of C-4 shifted downfield from δ 115.0 in longistylin A to δ 118.3 in 1, while the resonance of C-6 shifted upfield from δ 107.1 in longistylin A to δ 102.9 in 1, due to the shift-induced effects by C-5 substitution; consistently, a proton signal at δ 5.29 (1H, s) ascribing to 5-OH of longistylin A disappeared, but instead signals for a three-carbon aliphatic chain at δC 70.0 (CH), 68.9 (CH2) and 45.8 (CH2) respectively, were observed, indicating that 1 was a 5-O-substituted derivative of longistylin A. The 13 C signals for the aliphatic chain correlated with proton signals at δH 4.21 (1H, m), 4.14 (2H, m) and 3.75 (2H, m) by 13 C-1 H COSY respectively, suggest the existence of a 3-chloro-1, 2-propanediol unit linked upon 5-OH group. Thus, the structure of 1 was deduced as 5-O-(3-chloro-2-hydroxylpropanyl)longistylin A ( Figure 1) and confirmed by the HMBC correlations (Table 1 and Figure 1). Compound 1 was the first halogen-containing stilbene derivative isolated from plant, then named as cajanstilbene H.  Chemically, the structure of 1 could be formed by condensation of longstylin A and 3-monochloro-1, 2-propanediol (3-MCPD) with loss of one molecule of water under acidic condition. Compound 1 on earth is a natural product or an artificial product formed in extraction is an interesting question. 3-MCPD usually exists in form of fatty acid ester [16]; however, solvents used in extraction and isolation do not include oil, fatty acid ester, and glycerol or propanediol, which might be contaminated with 3-MCPD. Therefore, 3-MCPD should not be mixed in solvents. Moreover, because a hypothetic condensed product of 3-MCPD with longstylin C, a major stilbene in leaves of pigeon pea with higher quantity [17], less steric hinderance, and more favorable reactivity as compared to longstylin A, was not isolated, implying 1 should be a biosynthesized product of the plant and some stereospecific enzyme(s) may be involved in the biosynthesis.

Osteogenic Differentiation of hMSC
Extracellular calcium deposits are the indication of successful differentiation of MSC into osteoblasts and in vitro bone-formation. Calcium deposits can be detected after being stained bright orange-red with Alizarin Red S [18]. Highly expressed membrane alkaline phosphatase (AP) is another indication of osteoblast [19], which can be easily detected using 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (BCIP/NBT) to stains cells blue-violet when AP is present [20]. In this study, the effect of 1 on osteogenesis was investigated by employing hMSC, the precurcer cell of osteoblast. Results revealed that, incubation of 1 at concentrations of 10-1 μmol·L −1 together with hMSC in MSC growth medium for 48 h did not increase cell viability (Figure 2), but incubation of 1 (4, 2 or 1 μmol·L −1 ) together with hMSC in osteogenic differentiation medium achieved dramatic increases in extracellular calcium deposits ( Figure 3A,B) and membrane AP ( Figure 3C,D), inferring that 1 did not promote proliferation but distinctly promoted osteoblast differentiation in hMSC in dose-and time-dependent manners. Zheng et al. showed that a lipophilic extract from Cajanus cajan could reduce bone loss in ovariectomy-induced bone loss rats [5], and 2-carboxyl-longistilin A, a known stilbene, is able to promote osteoblast cells proliferation and mineralize bone-like tissue formation in human osteoblast-like TE85 cells [6]. Our data provided evidence for the new compound cajanstilbene H (1) in potential use in anti-osteoporosis.

Cytotoxicity of 1 in Six Human Cancer Cell Lines
Inhibitory effects of 1 on human tumor cell proliferation were investigated in lung cancer cell line (NCI-H460), prostate cancer cell line (PC-3), breast cancer cell line MCF-7, cervical cancer cell line (HeLa), colorectal cancer cell line (HCT-15) and P-glycoprotein overexpressing multidrug resistant oral epidermoid carcinoma cell line (KB-V1). Cells were treated with different concentrations of 1 or paclitaxel (positive control) for 48 h. Table 2 shows the measured concentrations of 1 to inhibit 50% of cell viability (IC50), moderate growth inhibitory effects were observed for 1 in all six tested cancer cell lines, with IC50 values of 21.42-25.85 μM.

Proliferation and Osteoblast Differentiation in hMSC
To investigate the effect of 1 on h-MSC proliferation, 3000 cells in 100 μL MSC growth medium were seeded in 96-well plates and incubated for 24 h. Different concentrations of 1 were added and incubated for 48 h. CCK-8 (10 μL per well) was then added to cells to react for 2 h. The amount of water soluble pigment which was positively correlated with the number of living cell was evaluated by the optical density (OD) at 450 nm. Cell viability (CV) under different treatment was calculated by equation CV = ODTreatment/ODControl, in which the control is the well without drug treatment.
To investigate the effect of 1 on osteoblast differentiation, about 3000 hMSC in MSC growth medium were seeded in 96-well plates to grow to 100% confluency. Osteoblast differentiation was induced by incubating cells in MSC osteogenic differentiation medium for 14 or 17 days, with or without the existence of 4, 2 or 1 μmol·L −1 of 1, or 0.16% DMSO. Medium was changed every 3 days. Cells treated with MSC growth medium were set as negative control. Osteoblast detection was performed by staining the extracellular calcium deposits bright orange-red with Alizarin Red S, or by staining membrane AP blue-violet with BCIP/NBT, by following the procedure of Alizarin Red S staining kit or cell AP activity staining kit, respectively.
To investigate the inhibitory effect of 1 on tumor cell proliferation, cells in 100 μL growth medium were seeded in 96-well plates at a density of 5000 cells per well and incubated for 24 h to let cells to adhere. Cells were replaced the medium with fresh medium containing different concentrations of 1 or paclitaxel and incubated for 48 h. Cell viability under different treatment was measured by MTT assay and expressed as expressed as percentage of the control [21].

Conclusions
In our study, cajanstilbene H, a new halogen-containing stilbene derivative was identified from the leaves of pigeon pea. To our knowledge, it is the first naturally existing ether derivative of 3-monochloro-1,2-propanediol (3-MCPD). Importantly, cajanstilbene H strongly promoted osteoblast differentiation in hMSC, exhibiting a potential to alleviate osteoporosis.