Velvet antler is widely used as a traditional medicine, and numerous studies have demonstrated its tremendous nutritional and medicinal values including immunity-enhancing effects. This study aimed to investigate different deer velvet extracts (Sample 1: raw extract, Sample 2: dried extract, and Sample 3: freeze-dried extract) for proximate composition, uronic acid, sulfated glycosaminoglycan, sialic acid, collagen levels, and chemical components using ultra-performance liquid chromatography-quadrupole-time-of-light mass spectrometry. In addition, we evaluated the cytotoxic effect of the deer velvet extracts on BV2 microglia, HT22 hippocampal cells, HaCaT keratinocytes, and RAW264.7 macrophages using the cell viability MTT assay. Furthermore, we evaluated acute toxicity of the deer velvet extracts at different doses (0, 500, 1000, and 2000 mg/kg) administered orally to both male and female ICR mice for 14 d (five mice per group). After treatment, we evaluated general toxicity, survival rate, body weight changes, mortality, clinical signs, and necropsy findings in the experimental mice based on OECD guidelines. The results suggested that in vitro treatment with the evaluated extracts had no cytotoxic effect in HaCaT keratinocytes cells, whereas Sample-2 had a cytotoxic effect at 500 and 1000 μg/mL on HT22 hippocampal cells and RAW264.7 macrophages. Sample 3 was also cytotoxic at concentrations of 500 and 1000 μg/mL to RAW264.7 and BV2 microglial cells. However, the mice treated in vivo with the velvet extracts at doses of 500–2000 mg/kg BW showed no clinical signs, mortality, or necropsy findings, indicating that the LD50 is higher than this dosage. These findings indicate that there were no toxicological abnormalities connected with the deer velvet extract treatment in mice. However, further human and animal studies are needed before sufficient safety information is available to justify its use in humans.

Materials and Methods
    Deer velvet powder
    General ingredients
    Heavy metals and residual pesticides analysis invelvet-extracted samples
    Bioactive compounds analysis using HPLC andUPLC/QTOF-HRMS method
    Evaluation of cytotoxicity
    Deer velvet antler samples in vivo treatment foranalyzing acute toxicity
    Toxicity studies
    Microbiological analysis of velvet extract samples
    Statistical analysis
Results and discussion
    Chemical composition and mineral contents of raw,dried, and freeze-dried antler extracts
    Uronic acid, sulfated-GAGs, sialic acid, and collagencontents
    Bioactive compounds analysis
    Analysis of residual pesticides and heavy metalconcentrations in velvet antler extracts
    Cytoprotective effect of raw, dried, and freeze-driedantler extract on HT22, BV2, RAW264.7, and HaCaTcells
    Acute toxicity in ICR mice model
    Microbiological analysis of deer velvet extracts
Acknowledgments
국문요약
Conflict of interests
ORCID

• 칠라칼라 라마크리시나(전남대학교 해양바이오식품학과) | Ramakrishna Chilakala (Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea)
• 문현정(전남대학교 해양바이오식품학과) | Hyeon Jeong Moon (Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea)
• 이환(조선대학교 약학대학) | Hwan Lee (College of Pharmacy, Chosun University, Gwangju, Korea)
• 이동성(조선대학교 약학대학) | Dong-Sung Lee (College of Pharmacy, Chosun University, Gwangju, Korea)
• 정선희(전남대학교 해양바이오식품학과) | Sun Hee Cheong (Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu, Korea) Corresponding author