Two Ellagic Acids Isolated from Roots of Sanguisorba officinalis L. Promote Hematopoietic Progenitor Cell Proliferation and Megakaryocyte Differentiation

Using a bioassay-directed chromatographic separation, two ellagic acids were obtained from the ethyl acetate extract of the roots of Sanguisorba officinalis L. On the basis of chemical and spectroscopic methods, the two ellagic acids were identified as 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside and 3,3',4-tri-O-methylellagic acid. Stimulation of cell proliferation was assayed in hematopoietic progenitor cells using the Cell Counting kit-8 method. The megakaryocyte differentiation was determined in human erythroleukemia (HEL) cells using Giemsa staining and flow cytometry analysis. The ellagic acids significantly stimulated the proliferation of Baf3/Mpl cells. Morphology analysis and megakaryocyte specific-marker CD41 staining confirmed that the ellagic acids induced megakaryocyte differentiation in HEL cells. This is the first time that 3,3',4-tri-O-methylellagic acid or 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside are reported to induce megakaryopoiesis, suggesting a class of small molecules which differ from others non-peptidyl, and appears to have potential for clinical development as a therapeutic agent for patients with blood platelet disorders.


Introduction
The induction of megakaryocyte differentiation is a potent strategy for the clinical treatment of diseases related to blood platelet disorders. Thrombopoietin (TPO) and its receptor (c-Mpl) are important regulators of platelet production. TPO binds to the c-Mpl on megakaryocytic progenitors and stimulates proliferation and differentiation leading to increased platelet production. Recombinant human TPO have been shown to be potent stimulator of megakaryocyte growth and platelet production in several clinical trials [1,2], but an immune response and the generation of anti-TPO antibodies impede the development of recombinant TPO [3,4]. Current and future interest is focused on the development of TPO mimetics, especially non-peptidyl molecules [5,6].

Compounds 1 and 2 Stimulated Cell Proliferation
To confirm the megakaryopoietic effects, we measured first cell-proliferative activity of two compounds in Baf3/Mpl cells. As shown in Figure 3A,B, compounds 1 and 2 promoted the proliferation of Baf3/Mpl cells in a dose-dependent and time-dependent manner under cytokine-free conditions. These results suggested that compounds 1 and 2 stimulate cell proliferation via TPO-independent or cytokines-independent pathways.

Compounds 1 and 2 Increased Megakaryocyte Ploiy in HEL Cells
We next examined whether the compounds possess the capacity to induce megakaryocyte differentiation. HEL cells were cultured for 12 days in serum-free medium containing 20 μg/mL compounds 1 or 2, DMSO as a negative control. As shown in Figure 4A, Giemsa staining clearly showed a morphological change of polyploidy megakaryocytes (right panel). The polyploidy megakaryocyts were determined by counting the number of endomitotic cells. The mean ploidy was increased from 1.35% to 7.35%, having a tendency to increase the megakaryocytes from 8N to 32N compared with control ( Figure 4B).

Flow Cytometric Analysis
The expression of CD41 (GPIIb/IIIa), which is considered to be an early megakaryocyte-specific marker (MK-specific marker), was analyzed by flow cytometry. Corresponding to the result of Giemsa staining, both compounds 1 and 2 increased the expression of MK-specific marker CD41 ( Figure 5). These results indicate that the compounds have the potential to induce megakaryocyte differentiation and maturation in vitro.

Plant Material
The roots of Sanguisorba officinalis L. were purchased from a local market in Chengdu (China), and were identified at Chengdu University of TCM. The voucher specimen was deposited in the laboratory of Chengdu University of TCM.

Extraction and Isolation
The air-dried roots (500 g) were ground, and then reflux-extracted with 75% ethanol three times (3 × 5,000 mL, 1 h each). After filtration and evaporation, the ethanol extract was suspended in water and incubated at 60 °C for 30 min. The ethanol extract was then extracted using 500 mL ethyl acetate three times continuously. The solvent was evaporated under vacuum to yield the ethyl acetate extract of Sanguisorba offoconalis L. (11.2 g). The ethyl acetate extract (5.0 g) was dissolved in methanol and subjected to AKTA column chromatography, which were separated using Kromasil C 18 column (250 mm × 20 mm, 5 μm) and eluted at 10 mL/min flow rate and wavelength 254 nm using pH 3.0 hydrochloric acid (solvent A) and MeOH (solvent B) with the following gradient of composition: starting with 20% solvent B and changing to 60% during 35 min, followed by a second ramp to 100% B in 10 min, maintained for 10 min, and then was purified by preparative HPLC to yield compound 1 (8.4 mg, purity 98.5%, RT30.71 min) and compound 2 (14.4 mg, purity 98.1%, RT33.77 min). The yields and purity of two compounds are shown in Table 3.

Cell Proliferation Assay
Baf3/Mpl cells were maintained in RPMI 1640 medium supplemented 10% fetal bovine serum and 5 ng/mL human TPO. After 3 days, the cells were washed twice with serum-free medium and were aliquoted into individual well of a 96-well plate. Compound 1 or compound 2 was dissolved in DMSO and diluted in 1640 medium, and added to cells at different concentrations. DMSO (below 1%) and recombinant human TPO (rhTPO) were used respectively as negative and positive controls. All cells were grown in a humidified incubator at 37 °C and 5% CO 2 . After 72 or 96 h, proliferation assay was performed using the Dojindo Cell Counting kit-8 according to the instruction supplied by the manufacturer. Absorbance values (450 nm) were recorded in triplicate using M5 Microplate Reader.

Megakaryocyte Ploidy and Morphological Analysis
HEL cells were incubated in RPMI 1640 medium supplemented 10% fetal bovine serum in the 24-well plate. Compound 1 or compound 2 at final concentrations of 10 μg/mL or 20 μg/mL was added. DMSO (below 1%) was used in the control wells. All cells were grown in a humidified incubator at 37 °C and 5% CO 2 for 14 days. Cells were collected on the 4th day, 8th day and 12th day, and stained with Giemsa to assess megakayocyte ploid and morphological analysis.

Flow Cytometric Analysis
HEL cells were incubated in RPMI 1640 medium supplemented 10% fetal bovine serum in the 24-well plate. Compound 1 or compound 2 at final concentration of 20 μg/mL was added. DMSO (below 1%) was used in the control wells. After 12 day, cells were collected and resuspended in 300 μL of phosphate-buffered saline (PBS). Cells were stained with a CD41-PE-conjugated monoclonal antibody (Life Technologies, Carlsbad, CA, USA) and were analyzed on a flow cytometer.

Statistical Analysis
The results are expressed as the mean ± SD. Statistical differences were determined using a Oneway ANOVA with p < 0.05 considered as statistically significant. Statistical analyses were carried out using the software SPSS 17.0 for Windows.

Conclusions
Two compounds were isolated from the roots of Sanguisorba offoconalis L., and identified as the known 3,3',4-tri-O-methylellagic acid-4'-O-β-D-xyloside and 3,3',4-tri-O-methylellagic acid. In the present study, we report for the first time that these compounds not only promote proliferation of megakaryocyte progenitor cells, but also induce megakaryocyte differentiation, resulting in polyploidy formation, MK-specific marker CD41 expression of HEL cells. These results suggest the megakaryopoietic effect of ellagic acids isolated from the roots of Sanguisorba offoconalis L. The induction of megakaryocyte differentiation is a potent strategy for the clinical treatment of diseases related to blood platelet disorders. Based on the structure analysis, the compounds are a class of small molecules which differ from other non-peptidyl molecules [5,6]. Therefore, it will be necessary to investigate the thrombopoiesis effect of the compounds. Research into the mechanism of megakaryopoiesis may also help guide the clinic application of the compounds and Sanguisorba offoconalis L. as a Traditional Chinese Medicine.

Acknowledgments
This work was supported by National Natural Science Foundation of China (Number: 81073159).

Conflicts of Interest
The authors declare no conflict of interest.