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. 2021 Feb 3;26(4):784.
doi: 10.3390/molecules26040784.

Ethanol Extract of Amomum tsao-ko Ameliorates Ovariectomy-Induced Trabecular Loss and Fat Accumulation

Ki-Shuk Shim  1 , Youn-Hwan Hwang  1   2 , Seon-A Jang  1 , Taesoo Kim  1 , Hyunil Ha  1
Affiliations

Ethanol Extract of Amomum tsao-ko Ameliorates Ovariectomy-Induced Trabecular Loss and Fat Accumulation

Ki-Shuk Shim et al. Molecules. .

Abstract

In Asia, Amomum tsao-ko has long been used as a spice or seasoning in food to stimulate digestion. In the present study, we evaluated the effects of ethanol extract of Amomum tsao-ko (EEAT) on menopausal osteoporosis and obesity. After the administration of EEAT in ovariectomy (OVX) mice models for five weeks, microcomputed tomography and a histological analysis were performed to assess, respectively, the trabecular structure and the fat accumulation in adipose, liver, and bone tissues. We also examined the effects of EEAT on a bone marrow macrophage model of osteoclastogenesis by in vitro stimulation from the receptor activator of nuclear factor-kappa Β ligand (RANKL) through real-time PCR and Western blot analysis. In addition, ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with authentic standards was applied to characterize the phytochemical profiling of EEAT. We found that EEAT significantly decreased OVX-induced body weight gain and fat accumulation, significantly prevented OVX-induced deterioration of bone mineral density and microstructure of trabecular tissues, and significantly inhibited osteoclast differentiation by downregulating NF-κB/Fos/NFATc1 signaling in osteoclasts. Furthermore, UHPLC-MS/MS identified eight beneficial phytochemicals in EEAT. Collectively, these results suggest that EEAT might be an effective nutraceutical candidate to attenuate menopausal osteoporosis by inhibiting osteoclastogenesis and to prevent obesity by suppressing fat accumulation.

Keywords: Amomum tsao-ko; osteoclast differentiation; osteoporosis; ovariectomy.

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Conflict of interest statement

There are no conflict of interest to declare.

Figures

Figure 1
Figure 1
Ethanol extract of Amomum tsao-ko (EEAT) inhibits osteoclastogenesis in bone marrow-derived macrophage cells (BMMs) stimulated with receptor activator of nuclear factor-kappa Β ligand (RANKL). (A) BMMs were differentiated to osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL for four days. Vehicle or EEAT were treated in the culture medium at day 0. Generated osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP) staining solution and photographed under a microscope (×4). (B) After TRAP staining, multinuclear osteoclasts were enumerated under the microscope (×4). (C) BMMs were incubated with M-CSF and the indicated concentrations of EEAT for 24 h and then assayed with CCK-8 solution to measure cell viability. (D) Mature osteoclasts were incubated with EEAT on the artificial bone surface to evaluate its effect on resorption activity. Resorption pits were photographed and analyzed. ** p  <  0.01 versus vehicle.
Figure 2
Figure 2
Ethanol extract of Amomum tsao-ko (EEAT) inhibits receptor activator of nuclear factor-kappa Β ligand (RANKL)-induced NF-κB/Fos/NFATc1 signaling pathway. Bone marrow-derived macrophage cells (BMMs) were pre-incubated with the vehicle or EEAT (33.3 µg/mL) for 3 h and then simulated with RANKL (50 ng/mL) for four days. (A) The protein levels of c-Fos, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), and β-actin were determined by Western blot with specific antibodies. (B) mRNA expression levels of c-Fos, NFATc1, dendritic cell–specific transmembrane protein (DC-STAMP), and ATPv0d2 were examined by Real-Time Polymerase Chain Reaction (RT-PCR). Day 0 represents the day of plating BMMs untreated with RANKL. Artificial fold change from day 0 to day 3 were represented in comparison with RANKL-untreated vehicle. * p  <  0.05 or *** p  <  0.01 versus vehicle. (C) BMMs were pre-incubated with EEAT for 3 h and then stimulated with RANKL for 5, 15, and 30 min. Protein levels of each target were analyzed by Western blot analysis with the indicated antibodies.
Figure 3
Figure 3
Ethanol extract of Amomum tsao-ko (EEAT) inhibits ovariectomy (OVX)-induced trabecular loss. After oral administration of the vehicle, low concentration EEAT (EEAT-L, 30 mg/kg), or high concentration EEAT (EEAT-H, 100 mg/kg) for five weeks, mouse right femur and serum were collected. (A) The femur was scanned and analyzed by micro-CT. The radiological images were constructed from distal femora. Five bone morphometric parameters were analyzed in the trabecular area below the lower end of the growth plate: bone mineral density (BMD), trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). (B) Serum levels of C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) were measured. * p  <  0.05, ** p  < 0.01 versus OVX group.
Figure 4
Figure 4
Ethanol extract of Amomum tsao-ko (EEAT) attenuates ovariectomy (OVX)-induced fat accumulation. Sham or OVX mice were orally received the vehicle or different doses of EEAT (30 or 100 mg/kg). After five weeks of administration, (A) body weight gain during EEAT administration period was assessed. Mice were euthanized, and then weight change of (B) uterus or (C) gonadal fat was quantified. (D) Isolated tissues were fixed and stained by hematoxylin and eosin staining solution (scale bar, 50 µm). Histological image of the adipocyte area or lipid droplets in each tissue was analyzed, and representative images are shown. * p  <  0.05, ** p  < 0.01 versus OVX group.
Figure 5
Figure 5
Ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) chromatograms of ethanol extract of Amomum tsao-ko (EEAT). (A) Ultraviolet chromatograms at 210 nm and base peak chromatograms with positive and negative modes of EEAT. (B) Individual ion chromatograms of eight components in EEAT.
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