Iranian Journal of Pharmaceutical Research (2018), 17 (3): 1057-1067 Received: March 2017 Accepted: July 2017

Original Article

Ethyl Acetate Extract of Licorice Root (Glycyrrhiza glabra) Enhances Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

Arezou Azizsoltania, b, Khosro Piria*, Sahar Behzadc, d, Masoud Soleimanie, Mina Nekouei f, Zahra Mahmoudig and Asad Kazemih

aDepartment of Biotechnology, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran. bDepartment of Medical Biotechnology, Faculty of Advanced Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. cDepartment of Pharmacognosy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. dEvidence-based Phytotherapy and Complementary Medicine Research Center, Alborz University of Medical Sciences, Karaj, Iran. eDepartment of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. fDepartment of Phytochemistry, Medicinal Plants and Drug Research Institute, Shahid Beheshti University, Tehran, Iran. gDepartment of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran, Tehran, Iran. hDepartment of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran.

Abstract

Glycyrrhiza glabra (G. glabra) has been used as a flavoring and sweetener agent, in addition to its therapeutic properties. It is rich in phytoestrogen and may prevent osteoporosis caused by estrogen deficiency; however, there is no evidence for its effects on proliferation and osteogenesis in mesenchymal stem cells. So, we were encouraged to investigate whether the ethyl acetate extract of licorice root as a source of phytoestrogen can act similar to estrogen in cell culture. Furthermore, the analysis of the licorice extract (LE) based on HPLC-DAD-ESI- MS indicated that LE comprises phytoestrogen compounds, such as glabridin and . In this study, the effects of LE on proliferation of human bone-marrow mesenchymal stem cells (hBM-MSCs) were investigated using MTT assay. In addition, its effects on the osteogenesis were evaluated using alkaline phosphatase activity (ALP), calcium deposition, and bone specific gene expression such as ALP, osteocalcin, Runx2, and BMP-2. The quantitative gene expression was studied by real-time RT-PCR. Our results showed a significant increase in proliferation in presence of LE in concentration 10-50 µg/mL. The differentiation of hBM- MSCs increased in doses of LE (10-25 µg/mL) compared to the control group. The effects of LE were similar to those of 17β-estradiol (E2) (10-8 M) and were abolished by ICI 182,780 an antagonist of estrogen receptor (ER) (10-7), indicating that the stimulatory effects of LE occur through estrogen receptor-mediated mechanism . Taking these into account, LE may be a potential candidate for prevention of osteoporosis in menopausal women.

Keywords: Glycyrrhiza glabra; Fabaceae; Phytoestrogen; Osteoporosis; Mesenchymal stem cell; Differentiation.

* Corresponding author: E-mail: [email protected] Azizsoltani A et al. / IJPR (2017), 17 (3): 1057-1067

Introduction yeast estrogen bioassay; so we chose the ethyl acetate extract of licorice root (20). Although the Osteoporosis is a common disease of bone stimulatory effect of glabridin and glabrene were in elderly people, which causes a reduction examined on bone metabolism and was similar to in bone density and consequently increased E2, the mechanism of action an licorice extract risk of fracture. Osteoporosis is traced back to on osteogenic differentiation and proliferation of an imbalance of osteoclastic bone resorption mesenchymal stem cells have not been evaluated vs. osteoblastic bone formation in the bone (21-23). BM-MSCs can be differentiated remodeling process (1). Estrogen is known as an into multiple lineages such as osteoblasts, important regulator in bone remodeling playing a chondrocytes, and adipocytes (24). To screen significant role in the differentiation of osteoblast the phytochemical compounds in our study, progenitor cells into mature osteocytes. we applied HPLC-DAD-MS analysis and then In women, age-related decline in estrogen investigated the ability of Ethyl-acetate extract following menopause can lead to a reduction of licorice root on proliferation and osteoblastic in bone mineral density (2). The most common differentiation of hBM-MSCs. Moreover, we remedy for osteoporosis is hormone replacement used ICI 182,780, a specific estrogen receptor therapy (HRT) which is not recommended alpha (ERα) antagonist to determine whether according to world health organization (WHO) licorice extract could act through ER-dependent investigations (3), because HRT is associated mechanism (25). with increased risk of breast, endometrial and ovarian cancers and cardiovascular disease Experimental (4-6). Recently, most studies have tended to discover alternate treatments such as natural Plant extract compound application (7). Phytoestrogens The root and stolon of the licorice was are natural substances that mimic estrogen collected in autumn at Hamadan, Iran and were and can replace hormone therapy to inhibit identified by the department of botany (Bu-Ali menopausal symptoms and osteoporosis (8). Sina University). A voucher specimen of this These substances contain lignans and flavonoids plant was deposited at the Bu-Ali Sina University (, and ) which are known herbarium (BASU) for future reference. The as selective estrogen receptor modulators air-dried and powdered root of licorice was (SERM) due to acting as antagonists of estrogen macerated in Hexane (1:4) and shaked for 24 in breast tissue and agonists in bone (9). h at room temperature to remove lipids. Then, Glycyrrhiza glabra which is known as licorice the residues were extracted with ethyl acetate. belonging to the Fabaceae, is native to southern The extract was concentrated with a rotary Europe and parts of Asia including Iran (10). evaporator at temperature less than 40 °C and Licorice roots have been used as a remedy for then freeze dried (yielding 12.3% of dry residue). cough, laxative, menopausal hot flashes, peptic The extract was dissolved in dimethyl sulfoxide ulcer, and viral diseases (11-13). Besides, the (DMSO) at a final concentration of 500 µg/mL prevention of LE on proliferation of breast, colon and filtered through a Millipore filter (0.22 µm). and prostate cancer cell-line and also diverse The stock solution was serially diluted with the pharmacological effects such as antimicrobial, medium. For all experiments final concentration antioxidant, and anti-inflammatory activity of DMSO was less than 0.1%. were investigated (14-17). There are several compounds in LE which have phytoestrogen HPLC-DAD-ESI-MS analysis properties such as hispaglabridin A, HPLC-DAD-ESI-MS was carried on an hispaglabridin B, licochalcon-A, licochalcon-B Agilent HPLC system (Waldbronn, Germany) and formononetin and phyto-SERM such as equipped with a binary pump, a diode array glabridin and glabrene (18, 19). Simons et detector and coupled to a Finnigan TM LCQ al. showed that the ethyl acetate extract of TM DECA instrument equipped with an ion licorice root displayed estrogenic activity in trap mass spectrometer and an electrospray

1058 The effect of licorice extract on osteogenic differentiation ionization (ESI) source (electrospray voltage: with Alizarin Red 2% at pH 7.2. For adipogenesis 4.5 kV, sheath gas and auxiliary gas: nitrogen, the cells were cultured in adipogenic medium, capillary temperature: 200 °C). RPLC analysis including DMEM supplemented with 10% FBS, was carried out on a Macherey-Nagel Nucleodur indomethacin (50 µg/mL), dexamethasone (1 C18ec (5 µm, 4.6 × 250 mm, Germany) column. µM), and ascorbate-2-phosphate (50 µg/mL). Mobile phase composed of water (with 0.1% The culture medium was changed tow times formic acid, A) and acetonitrile (with 0.1% per week for up to 21 days. The cells were fixed formic acid, B). The gradient elution was from in methanol for 45 min and visualized with 45% B to 60% B within 50 min, then isocratically Oil Red staining (all substances from Sigma- for further 10 min and after that increased to Aldrich®). The cells were observed under 80% in 20 min. the flow rate was 0.5 mL/min. LB290 inverted microscope (Labomed Inc., HPLC chromatograms were recorded at 254 nm, USA) with magnification 10X. The isolated 283 nm, and 330 nm. The mass spectrometer cells were characterized using flow cytometry, data were collected over a m/z range of 150- which analyzed the expression surface markers 2000 in both negative ion (NI) and positive ion such as CD31, CD44, and CD73. The cells were (PI) modes. Data dependent MS2 analysis was detached using trypsin/EDTA and incubated performed with collision energy of 35%. Data with the specific antibodies or isotype control acquisition and processing were performed antibodies, in 100 µL 3% BSA/PBS for 1h with the Xcalibur 2.0 SR2 software (Thermo at 4 °C. Then, the cells were fixed with 1% Scientific). paraformaldehyde and analyzed using flow cytometry (BD FACSCalibur™) and FlowJo® Cell culture software (Tree Star, Ashland, USA). The human bone marrow mesenchymal stem cells were gifted by Dr Soleimani and were MTT assay isolated as described previously (Yazdani et The effect of different concentration of LE on al., 2013). The cells were grown in DMEM-LG the proliferation rate of hMSCs was evaluated (Gibco®, grand island, NY, USA) supplemented via MTT assay (26). The cells were plated at the with 10% fetal bovine serum (FBS) (Gibco®), density of 104 cell/well in 96-well culture plate. 2 mM L-glutamin (Gibco®), Penicillin (100 After 24 h incubation, the cells were treated U/mL, Gibco®) and streptomycin (100 µg/ with medium plus different doses of LE (0.1, 10, mL, Gibco®) and incubated at 37 °C under 5% 25, 50 and 100 µg/mL) and 0.1% DMSO as the

CO2 atmosphere. The medium was changed control group. Cells were incubated at 37 °C for twice a week. When the adherent cells reached 48 and 72 h and then 20 µL of MTT solution confluence (P0) they were trypsinized by 1 mL (5 mg/mL in DMEM) was added to each well of 0.05% trypsin (Sigma-Aldrich®, USA) and and incubated for 4 h at 37 °C. The supernatant subcultured into new flasks. For most of the was discarded and 200 µL DMSO was added to experiments, we used the hBM-MSCs at the 3rd dissolve the formazan salt. The optical density passages. was measured at 570-630 nm

Differentiation and Flow cytometry analysis ALP activity of hBM-MSCs To study the ALP activity, hMSCs were For osteogenesis assay, the cells were grown seeded in 24-well plates at a density of 105 cells/ in osteogenic induction medium consisting well and after 24 h cells were exposed to test of (DMEM+10%FBS) supplemented with 10 agent (osteogenic medium (OS) + LE, OS + nM Dexamethasone, 0.2 mM ascorbic acid 0.1% DMSO, OS + E2). After 6 and 12 days of 2-phosphate, and 10 mM β-glycerol phosphate treatment total protein of cells was extracted using (all from Sigma-Aldrich®, St. Louis, USA) cell lysis buffer. The cells were washed twice and the medium was changed twice per week with PBS and lyzed with RIPA and centrifuged for up to 21 days. The cells were fixed with at 14000×g for 5 min. Aliquots of supernatant paraformaldehyde for 20 min at 4 °C and stained were used for ALP activity. Enzyme activity was

1059 Azizsoltani A et al. / IJPR (2017), 17 (3): 1057-1067 measured with the ALP assay kit according to the osteogenic medium with LE (25 µg/mL) or E2 manufacturer’s instructions (Parsazmun, Tehran, in the presence or absence ICI182,780 (Cayman Iran) using ρ-nitrophenyl phosphate as substrate. chemical, USA). After 6 and 12 of treatment, the ALP activity was normalized according to the cells were collected for ALP activity, calcium total protein. deposition, and real-time PCR.

Measurement of calcium content Statistical analysis To analyze the amount of calcium deposition The results are expressed as mean ± SD. in our study, cultures were decalcified with Significant levels among different groups were 0.6 N Hcl (Merck), previously described analyzed using GraphPad Prism 7.01, Two- (27). Calcification was evaluated by calcium way ANOVA. P-value < 0.05 was regarded as content assay kit (Parsazmun,Tehran,Iran) statistically significant. using the cresophthalein compelexon method. The absorbance at 570 nm was measured. The Results calcium deposited of culture was normalized to serial dilution of various concentrations of Identification of Compounds in the G. glabra calcium versus optical density (OD). extract In order to identification of some Real-time RT PCR phytochemical compounds in the ethyl acetate The expression of osteogenic mRNA was extract of G. glabra root, HPLC-ESIMS analysis evaluated using real-time RT-PCR (27). The was applied in both positive and negative ion cells were cultured in 6-well plate and after modes. As a preliminary experiment, the peak 24 h exposed to experimental treatment. The of glabridin in the extract was assigned by medium was replaced every 3 days. After 6 comparing its retention time, UV spectra, as well and 12 days total RNA was isolated by RNeasy as parent and production ions to the standard. The kit according to manufacturer’s instructions RP-HPLC-DAD profile of the extract (Figure 1) (Qiagen, Germantown, MD, USA). cDNA was and the total ion current (TIC) chromatogram synthesized by revert Aid first strand cDNA obtained in the NI mode are displayed (Figure synthesis kit (Fermentase, Burlington, Canada). 2). The compounds were numbered according to Amplification was performed by Rotor-Gene Q the peak elution order in the chromatogram as real-time analyzer (Corbett, Australia) and SYBR shown in Table 1. Premix Ex Taq (Takara Bio Inc., Japan). Our results were analyzed by Rotor-Gene Q Software Characterization of BM-MSCs (Corbett) and melting curve analysis was used To characterize isolated mesenchymal to confirm PCR specificity and threshold cycle stem cells, we used the osteogenesis and the average. The primer sequences are emerged in adipogenesis potential of these cells and the Table S1 (Supplementary File). Fold change expression of mesenchymal-related surface was calculated according to ΔΔCT method. marker. Figure 3A shows the characteristic GAPDH was used as internal control and target spindle shape and plastic adherent properties of genes were calibrated to MSC P3. Two-step MSCs. In Figure 3B, red spots illustrate calcium amplification protocol was chosen, consisting of nodules and mineralization in MSCs which initial denaturation at 95 °C for 3 min followed confirmed undergoing osteogenic differentiation by 40 cycles with 5 sec denaturation at 95 °C and in Figure 3C. Lipid droplets in MSCs which and 20 sec annealing/extension at 60 °C. Each were cultured in appropriate medium exhibited reaction was duplicated and repeated twice. adipogenic differentiation potential. In addition, flow cytometry analysis showed that MSCs Osteogenic analysis after the addition of ICI passage 2 cells were positive for CD44, CD73 182,780 and were negative for CD31 which confirmed The MSCs were plated in 6-well plate, after their non-hematopoietic origin (Figure S1, incubation for 24 h, the medium changed into Supplementary File).

1060 The effect of licorice extract on osteogenic differentiation

Table 1. Compounds assigned in the G. glabra extract by LC-MS. MS2 product ions No Rt. (min) UV (nm) Identification [M-H]- [M+H]+ (relative intensity)

1 13.6 230, 280 Dihydroxy-glabridin 357 135, 235, 339 341

2 17 230, 280, 340 Hydroxy-glabrene 337 161, 268, 293, 309, 322 339

3 18 220,280 Hydroxy-4’-O-methylglabridin 353 165, 201, 321, 309, 338 355

217, 250, 285, 4 19.8 Glabrene 321 175, 293, 306 323 325

5 25.5 280 Hydroxy-glabridin 339 135,203,221,321 341

6 26 230, 280 Glabridin 323 121, 135, 201 325

7 27 222, 250, 302 Glabrone 335 213, 291, 292, 307, 320 337

8 36 230, 280, 290 Hispaglabridin A 391 335 393

9 46 230, 280, 320 Hispaglabridin B 389 373 391 161, 268, 293, 309, 2 17 230, 280, 340 Hydroxy-glabrene 337 339 322

Hydroxy-4’-O- 165, 201, 321, 309, 3 18 220,280 353 355 methylglabridin 338 Effect of licorice extract on the proliferation inhibited in a 100 µg/mL concentration as this of hBM-MSCs 4 19.8 217, 250, 285, 325 Glabreneconcentration 321 exhibited 175, 293, 306 toxic effects 323 on cell

The proliferation5 of25.5 BM-MSCs 280 in variousHydroxy-glabridin proliferation. 339 135,203,221,321 341 concentrations of LE (1-100 µg/mL) was evaluated via 6 MTT after 26 48 230, and 280 72 h of Glabridin Alkaline 323 phosphatase 121, 135, 201 activity assay 325 treatment (Figure 4). LE significantly increased We evaluated the quantitative ALP activity 213, 291, 292, 307, cell proliferation7 in concentrations 27 222, 250, 10, 302 25, and Glabroneas a biochemical 335 marker of osteogenesis337 in BM- 320 50 µg/mL after 48 h of treatment as compared MSC in the presence or absence (control) of LE to control (P 8 ˂ 0.05) 36 and a 230, higher 280, 290 increase Hispaglabridin and AE2 on 391 day 6 and335 12 of treatment.393 LE (10- was achieved after 72 h exposure to 50 µg/mL 25 µg/mL) caused a significant enhancement in of plant extract.9 The proliferation 46 230, 280, of 320 cells was Hispaglabridin the Benzyme 389 activity of373 stem cells391 on day 6 and

Figure 1. The RP-HPLC-DAD chromatogram of the G. glabra extract at 283 Figure 1. The RP-HPLC-DAD chromatogram of the G. glabra extract at 283 nm. nm.

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Azizsoltani A et al. / IJPR (2017), 17 (3): 1057-1067

FigureFigure 2. The 2. TheTIC TIC chromatogram chromatogram of ofthe theG. glabra G. glabra extract obtainedextract inobtained NI mode. in NI mode.

The proliferation of BM-MSCs in various concentrations of LE (1-100 µg/mL) was evaluated

via MTT after 48 and 72 h of treatment (Figure 4) LE significantly increased cell proliferation in concentrations 10, 25, and 50 µg/mL after 48 h of treatment as compared to

control (P ˂ 0.05) and a higher increase was achieved after 72 h exposure to 50 µg/mL of plant extract. The proliferation of cells was inhibited in a 100 µg/mL concentration as this Figureconcentration 3. (A) Morphology exhibited of undifferentiated toxic effects Mesenchymal on cell proliferation. Stem Cells. (B) Osteogenic differentiation of MSCs was visualized by Alizarin Red which stained calcium deposited in extracellular matrix . (C) adipogenic differentiation was confirmed by lipid vacuoles stained with Oil Red staining. (Magnification 10X). Characterization of BM-MSCs

To characterize isolated mesenchymal stem cells, we used the osteogenesis and the adipogenesis potential of these cells and the expression of mesenchymal-related surface marker. Figure 3A shows the characteristic spindle shape and plastic adherent properties of

Figure 4. Effect of LE on the proliferation of hBM-MSCs. Cells were exposed to LE (1-100 µg/mL) and 0.1% DMSO for 48 and 72 h. Data represent mean ± SD and expressed as an Optical density. *P ˂ 0.05 significantly different compared to control (n = 4). Figure 4. Effect of LE on the proliferation of hBM-MSCs. Cells were exposed to LE (1-100

µg/mL) and 0.1% DMSO for 48 and 72 h. Data1062 represent mean ± SD and expressed as an Optical density. *P 0.05 significantly different compared to control (n = 4).

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Alkaline phosphatase activity assay We evaluated the quantitative ALP activity as a biochemical marker of osteogenesis in BM- MSC in the presence or absence (control) of LE and E2 on day 6 and 12 of treatment. LE (10- 25 µg/mL) caused a significant enhancement in the enzyme activity of stem cells on day 6 and 12 of the treatment. As shown in Figure 5 the maximum effectThe of effect LE of was licorice observed extract in on 25 osteogenic differentiation µg/mL on day 12 compared to the control.

* Figure 5. Effect of Figure LE on Alkaline 5. Effect phosphatase of LE on activityAlkaline of phosphatase hBM-MSCs. ac Datativity shown of hBM-MSCs. are mean ± SD.Data Pshown ˂ 0.05 are vs. control (n = 5). mean ± SD. *P 0.05 vs. control (n = 5).

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12 of the treatment. As shown in Figure 5 the the amount of calcium deposition increased over maximum effect of LE was observed in 25 µg/ time in all treatments and a significant increase mL on day 12 compared to the control. was observed on day 12 at concentration of 10-25 µg/mL. The maximum effect was at the Effect of LE on calcium deposition concentration of 25 µg/mL (Figure 6). Calcium depositionEffect of LE ason calcium a late deposition marker of osteogenesis was measured to assess the effect Effect of LE on mRNA expression of bone- of LE on osteoblastic Calcium differentiationdeposition as a late marker of MSCs of osteogen relatedesis was measured genes to assess the effect of LE on days 6 and 12.on Asosteoblasticwas observed observed differentiation on from day 12the atof concentration results,MSCs on days of 6 10-25 andQuantitative 12. µg/mL. As observed The maximum real-timefrom the effect results, PCRwas the at the was utilized amountconcentration of calcium of deposition 25 µg/mL increased (Figure 6). over time in all treatments and a significant increase

Figure 6. Effects of LE on calcium deposition of MSCs. Data shown are mean ± SD. *P ˂ 0.05 vs. control. Figure 6. Effects of LE on calcium deposition of MSCs. Data shown are mean ± SD. *P

0.05 vs. control. ˂ 1063

Effect of LE on mRNA expression of bone-related genes

Quantitative real-time PCR was utilized to study the effect of LE on the expression of osteoblast specific genes, including Runx2, ALP, osteocalcin, and BMP2 on day 6 and 12 of osteogenic culture. RT-PCR analysis showed that Runx2 and ALP mRNA were up-regulated on day 6 followed by a decrease on day 12 and had a 2-fold and 2.4-fold increase respectively, compared to control at concentration of 25 µg/mL. Results showed a significant Azizsoltani A et al. / IJPR (2017), 17 (3): 1057-1067 to study the effect of LE on the expression of of hBM-MSCs. Moreover, BM-MSCs have osteoblast specific genes, including Runx2, recently widespread attention due to their ALP, osteocalcin, and BMP2 on day 6 and 12 potential use in tissue engineering application of osteogenic culture. RT-PCR analysis showed and cell therapy; we encourage investigating the that Runx2 and ALP mRNA were up-regulated effect of LE on differentiation and proliferation on day 6 followed by a decrease on day 12 and of BM-MSC and compared with that of the E2 as had a 2-fold and 2.4-fold increase respectively, a positive control. The results of our experiment compared to control at concentration of 25 µg/ demonstrated that the LE has stimulatory effects mL. Results showed a significant increase in on proliferation and osteogenic differentiation of osteocalcin gene expression in the presence mesenchymal stem cells. The proliferation rate of LE on days 6 and 12 and reached a peak at of MSCs is consequential in that involvement concentration of 25 µg/mL. Notably BMP2 gene of MSCs in bone-forming osteoblast and expression increased and had a 4.5-fold increase osteogenesis. Cell proliferation results indicate compared to control and a greater level of BMP2 that LE was able to accelerate MSCs proliferation expression was achieved on day 12 of culture in a dose and time dependent manner (32). Our (Figure 7). We observed in gene expression findings were in line with previous studies pattern that E2 (10-8M) group is similar to showing a stimulatory effect of phytoestrogens, LE group (25 µg/mL), therefore, the optimal genistein and diadzein, on the proliferation and concentration which was similar to E2 was differentiation of osteoblastic bone marrow selected for evaluating the effect of ICI 182,780 stromal cell (33). The beneficial effect was (10-7) in complementary experiment (Figure 7). accomplished at a concentration of 10-50 µg/mL As can be seen in the figures, the stimulatory and reached a peak at 50 µg/mL after 72 h. LE effect of LE and E2 on ALP activity, calcium diminished proliferation at concentration of 100 deposition, and gene expression was declined by µg/mL and this biphasic pattern may be related to ICI 182,780. phytoSERM, glabrene, and glabridin, as Hu et al., has noted the biphasic effect of LE on the growth Discussion of MCF-7 breast cancer cells (34). We evaluated the effect of LE on differentiation by monitoring As shown in Table 1, Peaks No. 1-9 were the ALP activity and calcium deposition of identified in licorice extract by comparing MSCs. ALP activity is an early-stage biomarker with metabolites of G. glabra whose structural enzyme that has an important role in osteogenic characteristic, including m/z values, UV spectra, differentiation of MSCs and calcium deposition and retention times can be found in the published that is a late marker of osteogenesis (35). In data (28). Also, further mass fragmentation comparison to control a significant increase patterns (HPLC-ESIMS2) in NI mode have in ALP activity was observed on day 6 in the to be considered for decisive confirmation of presence of 10-25 µg/mL of LE and reached a compound structures in the G. glabra extract. peak in 25 µg/mL. In addition, LE accelerated In previous studies several constituents of the calcium deposition on day 12 at concentration LE such as Hydroxy-4’-O-methylglabridin, of 25 µg/mL. These data were in agreement hispaglabridin A, and hispaglabridin B were with previous studies investigating the effect identified as a phytoestrogen and glabridin and of flavonoids of Herba epimedii and petroleum glabrene as a phyto-SERM. Components of the ether extract of Cissus quadrangularis on MSCs extract were in agreement with the previous (3, 36). Park et al. reported that a hot-water findings (19, 29 and 30). Hence, the importance extract of Allium hookeri roots has a stimulatory of estrogen level in pathogenesis of osteoporosis effect on proliferation, ALP activity, and mineral in women and the effect of E2 in osteoblastogenic deposition of osteoblast–like MG-63 cells (37). differentiation of hBM-MSCs were identified The changes in ALP activity and deposition of (31), we hypothesized the LE have the potential calcium nodule were accompanied by the up- to prevent osteoporosis through the control over regulation of the osteogenic marker genes. The the osteogenic differentiation and proliferation elevation in the expression of the bone-related

1064 increase in osteocalcin gene expression in the presence of LE on days 6 and 12 and reached a peak at concentration of 25 µg/mL. Notably BMP2 gene expression increased and had a 4.5- fold increase compared to control and a greater level of BMP2 expression was achieved on day 12 of culture (Figure 7). We observed in gene expression pattern that E2 (10-8M) group is similar to LE group (25 µg/mL), therefore, the optimal concentration which was similar to E2 was selected for evaluating the effect of ICI 182,780 (107-) in complementary experiment increase in osteocalcin gene expression in the presence of LE on days 6 and 12 and reached a (Figure 7). As can be seen in the figures, the stimulatory effect of LE and E2 on ALP activity, increasepeak at in concentration osteocalcin gene of 25expression µg/mL. inNotably the presence BMP2 of gene LE on expression days 6 and increased 12 and reached and had a a 4.5- calcium deposition, and gene expression wasincreaseincrease declined in osteocalcin in by osteocalcin ICI 182,780. gene gene expression expression in the in thepresence presence of LE of LEon dayson days 6 and 6 and 12 and12 and reached reached a a peakfold at increaseconcentration compared of 25 toµg/mL. control Notably and a BMP2 greater gene level expression of BMP2 increased expression and washad aachieved 4.5- on peakpeak at concentration at concentration of 25of µg/mL.25 µg/mL. Notably Notably BMP2 BMP2 gene gene expression expression increased increased and and had had a 4.5- a 4.5- fold increase compared to control and a greater level of BMP2The effect expressionof licorice extract on was osteogenic achieved differentiation-8 on day 12 of culture (Figure 7). We observedfold infold increase gene increase expression compared compared topattern controlto control that and E2 and a (10greater a greaterM) level group level of isBMP2of BMP2 expression expression was was achieved achieved on on day 12 of culture (Figure 7). We observed in gene expression pattern that E2 (10-8M) group is similar to LE group (25 µg/mL), therefore,dayday 12the of 12optimal culture of culture concentration(Figure (Figure 7). We7). Weobservedwhich observed was in genesimilar in gene expression toexpression E2 pattern pattern that that E2 (10E2 -8(10M)-8 M)group group is is similar to LE group (25 µg/mL), therefore, the optimal concentration7- which was similar to E2 was selected for evaluating the effect similarof similarICI to 182,780 LE to LEgroup group (10 (25 (25)µg/mL), in µg/mL), complementary therefore, therefore, the theoptimalexperiment optimal concentration concentration which which was was similar similar to E2 to E2 7- was(Figure selected 7). forAs canevaluating be seen the in theeffect figures, of ICIwas the was182,780 selected stimulatory selected (10for forevaluating) effectin evaluating complementary of LEthe theandeffect effect E2 ofexperiment on ICIof ALP ICI182,780 activity,182,780 (10 7-(10) in7-) complementaryin complementary experiment experiment (Figure 7). As can be seen in the figures, the stimulatory effect of LE and E2 on ALP activity, calcium deposition, and gene expression(Figure was(Figure declined 7). As7). canAs by canbe ICI seenbe 182,780. seen in the in thefigures, figures, the thestimulatory stimulatory effect effect of LE of LEand and E2 onE2 ALPon ALP activity, activity, calcium deposition, and gene expression wascalcium declinedcalcium deposition, by deposition, ICI 182,780. and and gene gene expr expressionession was was declined declined by ICIby ICI182,780. 182,780.

Figure 7. The real time RT-PCR analysis of osteoblastic genes. Changes in expression of Runx2, ALP, BMP-2 and osteocalcin were analyzed in cells, which were grown in osteogenic medium (control) plus different doses of LE or E2 with or without ICI 182,780 on day 6 and 12. Data represent mean ± SD expressed as a percentage of control. *P ˂ 0.05 significantly different compared to control (n = 4).

1065 Azizsoltani A et al. / IJPR (2017), 17 (3): 1057-1067 genes, including Runx-2, osteocalcin, ALP, and for technical advice. BMP-2 compared to control were observed. Our results are in accordance with those of Refrences others who reported that another phytoestrogen, , has direct stimulatory effects (1) Zaidi M. Skeletal remodeling in health and disease. on bone development in cultured MC3T3-E1 Nat. Med. (2007) 13: 791-801. osteoblast cell (36, 38). Lin et al. indicating that (2) Khosla S, Melton LJ and Riggs BL. The unitary model for estrogen deficiency and the pathogenesis of the combination of licorice extract and quercetin osteoporosis: Is a revision needed? J. Bone Miner. Res. increased in the expression of BMP2 mRNA and (2011) 26: 441-51. protein level of the new bone area in cultured (3) Potu BK, Bhat KM, Rao MS, Nampurath GK, mouse calvaria (39). Also Choi et al. indicated Chamallamudi MR, Nayak SR and Muttigi MS. that glabridin, an of licorice, increases Petroleum ether extract of Cissus quadrangularis (Linn.) enhances bone marrow mesenchymal stem cell osteocalcin secretion and ALP activity in proliferation and facilitates osteoblastogenesis. Clinics osteoblast MC3T3-E1 (21). Moreover, glabridin (2009) 64: 993-8. and glabrene also increased creatine kinase (4) Hsu A, Card A, Lin SX, Mota S, Carrasquillo O and activity in epiphyseal cartilage and diaphyseal Moran A. Changes in postmenopausal hormone bone in prepubertal female rats (30). In this study, replacement therapy use among women with high the inductions in osteoblastic differentiation cardiovascular risk. Am. J. Public Health (2009) 99: 2184-67. were mediated through an estrogen like action, (5) Kim JK, Alley D, Hu P, Karlamangla A, Seeman T and Not only was the effect of LE on differentiation Crimmins EM. Changes in postmenopausal hormone similar to that observed with E2, but also these therapy use since 1988. Womens Health Issues (2007) effects were diminished by a pure ER antagonist 17: 338-41. ICI 182, 780. Consistent with our finding, Choi (6) Santen RJ. Menopausal hormone therapy and breast cancer. J. Steroid Biochem. Mol. Biol. (2014) 142: 52- reported that the elevation of ALP activity and 61. collagen synthesis by glabridin in osteoblastic (7) Mahmud K. Natural hormone therapy for menopause. MC3T3-E1 cells was abolished completely by Gynecol. Endocrinol. (2010) 26: 81-5. the anti-estrogen tamoxifen (21). (8) Kronenberg F and Fugh-Berman A. Complementary In conclusion, this is the first evidence that and alternative medicine for menopausal symptoms: LE can increase the proliferation and osteogenic A review of randomized, controlled trials. Ann. Intern. Med. (2002) 137: 805-13. differentiation of BM-MSCs and having the (9) Moreira AC, Silva AM, Santos MS and Sardão VA. pattern as well as estradiol. Accordingly LE Phytoestrogens as alternative hormone replacement can be a useful candidate for the prevention of therapy in menopause: What is real, what is unknown. osteoporosis in menopausal women. Further J. Steroid Biochem. Mol. Biol. (2014) 143: 61-71. studies are required for evaluating the effect of (10) Vibha J, Choudhary K, Singh M, Rathore M and Shekhawat N. A study on pharmacokinetics and LE and the components on signaling pathways therapeutic efficacy of Glycyrrhiza glabra: A miracle in bone formation in-vitro and in-vivo. medicinal herb. Bot. Res. Int. (2009) 2: 157-63. (11) Parvaiz M, Hussain K, Khalid S, Hussnain N, Iram N, Supplementary File Hussain Z and Ali MA. A review: Medicinal importance The identified compounds in LE and the of Glycyrrhiza glabra L. (Fabaceae family). Global J. RP-HPLC chromatogram at 283 nm and TIC Pharmacol. (2014) 8: 8-13. (12) Yeh CF, Wang KC, Chiang LC, Shieh DE, Yen MH chromatogram in NI mode as well as the flow and Chang JS. Water extract of licorice had anti-viral cytometry of CD31, CD44, and CD73 in hBM- activity against human respiratory syncytial virus in MSCs available as Supplementary File. human respiratory tract cell lines. J. Ethnopharmacol. (2013) 148: 466-73. Acknowledgment (13) Nahidi F, Zare E, Mojab F and Alavi-majd H. Effects of licorice on relief and recurrence of menopausal hot flashes. Iran. J. Pharm. Res. (2012) 11: 541. This study was accomplished at the Stem (14) Chu XT, de la Cruz J, Hwang SG and Hong H. Cell Technology Research Center, Tehran, Iran. Tumorigenic effects of Endocrine-disrupting chemicals We thank Dr Ehsan Seyedjafari and Dr Amir are alleviated by Licorice (Glycyrrhiza glabra) root Atashi at Stem Cell Technology Research Center extract through suppression of AhR expression in

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