Nutr Res Pract.  2015 Oct;9(5):451-458. 10.4162/nrp.2015.9.5.451.

Hematopoietic effect of deer antler extract fermented by Bacillus subtilis on murine marrow cells

• Park Y
• Choi HS
• Lee HS
• Suh HJ

• KMID: 2313864
• DOI: http://doi.org/10.4162/nrp.2015.9.5.451

Abstract

BACKGROUND/OBJECTIVES
We examined the chemical composition and the effect of fermented deer antler on hematopoietic factors in bone marrow cells.
MATERIALS/METHODS
For the preparation of fermented deer antler extract (FAB), fermentation was carried out using Bacillus subtilis at 30degrees C for 7 days. The hematopoietic effect of FAB was investigated hematopoietic factors in marrow cells.
RESULTS
The contents of total sugar, sulfated glycosaminoglycans, and uronic acid and the dry weight gradually increased with fermentation time. The sialic acid content (from 0.14 mg/mL to 0.54 mg/mL) was the highest on the 4th day of fermentation after which it decreased. The proliferating activity of bone marrow cells increased with fermentation times. The levels of various hematopoietic growth factors were determined to verify the beneficial effect of deer antler extract fermented by B. subtilis on hematopoiesis. FAB increased the number of stem cell factors and granulocyte colony-stimulating factor in bone marrow cells. In addition, FAB augmented the burst-forming unit erythroid and total colonies in splenocyte-conditioned medium compared with non-fermented antler extract (NFA). However, FAB did not affect the mRNA levels of erythropoietin, an important factor for erythropoiesis.
CONCLUSIONS
FAB, like NFA, did not directly affect hematopoiesis, but contributed to hematopoiesis by stimulating the production of hematopoietic factors.

Keyword

Bacillus subtilis; fermented antler; bone marrow

MeSH Terms

Animals
Antlers*
Bacillus subtilis*
Bacillus*
Bone Marrow Cells
Bone Marrow*
Deer*
Erythropoiesis
Erythropoietin
Fermentation
Glycosaminoglycans
Granulocyte Colony-Stimulating Factor
Hematopoiesis
Intercellular Signaling Peptides and Proteins
N-Acetylneuraminic Acid
RNA, Messenger
Stem Cell Factor
Erythropoietin
Glycosaminoglycans
Granulocyte Colony-Stimulating Factor
Intercellular Signaling Peptides and Proteins
N-Acetylneuraminic Acid
RNA, Messenger
Stem Cell Factor

Figure

• 

  Fig. 1 Effect of fermented antler extract on chemical compositions and bone marrow cell proliferation during Bacillus subtilis-mediated fermentation. Cultures were incubated in a 3 L stirred-tank fermenter containing 1.8 L of the antler powder medium after inoculation with 2% (v/v) flask culture for seven days (temperature, 30℃; aeration rate, 1 vvm; agitation speed, 150 rev/min; initial pH 5.6). Total carbohydrate and uronic acid (A), S-GAG and sialic acid (B), protein and solid content (C) were analyzed, and the proliferative activity of bone marrow cell (D) were determined during fermentation of antler extract. Values are means ± SD (n = 6). Means with different superscript letters within a row are significantly different at P < 0.05 by Duncan's multiple range tests. S-GAG: sulfated glycosaminoglycans.

• 

  Fig. 2 Effect of NFA and FAB on hematopoietic growth factors in SCM. Balb/C mice-derived splenocytes were cultured in RPMI 1640 medium for 72 h in the presence or absence of NFA and FAB. Levels of hematopoietic growth factors including SCF (A), G-SCF (B), GM-SCF (C), and IL-3 (D) were analyzed by ELIZA. The data were expressed as the mean ± SEM of three separate experiments. NFA: non-fermented antler extract; FAB: antler fermented extract by B. subtilis; SCF: stem cell factor; G-CSF: granulocyte colonystimulating factors; GM-SCF: granulocyte-macrophage CSF; IL-3: interleukin-3.

• 

  Fig. 3 Effect of EPO and cytokines on colony-forming cells. Bone marrow cells derived from Balb/c mice were cultured in RPMI1640 medium with 10% FCS (base media). For colony formation, IL-3, SCF, and EPO were added to the media (completed media), and evaluated. Colony formation was photographed by microscope (A). After 14 days of incubation, the colony-forming unit-granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) and erythroid (BFU-E) were determined (B). Total colony: (GFU-GEMM+BFU-E). The data were expressed as the mean±SEM of three separate experiments. BFU-E had multicentric (burst) erythroid colonies with red or pink color. IL-3: interleukin-3; SCF: stem cell factor; EPO: erythropoietin; GFU-GEMM: colony-forming unit-granulocyte/erythrocyte/macrophage/megakaryocyte; BFU-E: burstforming unit erythroid.

• 

  Fig. 4 Effect of NFA and FAB on colony formation in splenocyte conditioned media containing EPO. Bone marrow cells derived from Balb/c mice were cultured in RPMI1640 medium with 10% FCS (base media). Colony formation of bone marrow cells was induced with SCM and EPO. PHA, NFA, and FAB were added to stimulate colony formation, and evaluated (A). The numbers of total colonies and BFU-E were determined in NFA or FAB-treated SCMs containing different concentrations (B and C).Data from four separate experiments are expressed as the mean ± SEM. Means with different superscript letters indicated significant differences at P < 0.05 by Duncan's multiple range tests. NFA: non-fermented antler extract;. FAB: antler extract fermented by B. subtilis ; EPO: erythropoietin; SCM: splenocyte conditioned media; BFU-E: burstforming unit erythroid. PHA was positive control and its concentration was 10 µg/mL.

• 

  Fig. 5 Effect of antler extracts on the mRNA expression of EPO in kidney cells. mRNA expressions of EPO was determined in treated kidney cells treated by NFA and FAB by semi-quantitative RT-PCR. Data from four separate experiments are expressed as the mean ± SEM. Control: saline; NFA: non-fermented antler extract; FAB: antler extract fermented by B. subtilis. Data are expressed as contrast of control, EPO: erythropoietin.

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