Study on the Marker Steroids of New Zealand Deer (Cervus elaphus var. scoticus) Velvet Antler by UPLC-MS/MS and HPLC-PDA Methods

Eleven steroid hormones (SHs: androstene-3,17-dione, estrone, β-estradiol, α-estradiol, testosterone, dehydroepiandrosterone, 17á-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, progesterone, and androsterone) were detected from New Zealand deer (Cervus elaphus var. scoticus) velvet antler (NZA, 鹿茸). A method for the quantification of eleven SHs was established by using ultraperformance liquid chromatography (UPLC)-MS/MS. The linearities (R² > 0.991), limits of quantification (LOQ values, 0.3 ng/mL to 23.1 ng/mL), intraday and interday precisions (relative standard deviation: RSD 0.999), LOQ values (30 ng/mL to 350 ng/mL), intraday and interday precisions (RSD < 1.93%), and recovery rates (97.2% to 103.5%) for the three 7-O-CSs were determined. These quantitative methods are accurate, precise, and reproducible. As a result, it is suggested that the five steroid compounds of androstene-3,17-dione, androsterone, 7-ketocholesterol, 7α-hydroxycholesterol, and 7β-hydroxycholesterol could be marker steroids of NZA. These methods can be applied to quantify or standardize the marker steroids present in NZA.

Analysis of Differential Proteins of Sika Deer Antler at Rapid Growth and Ossification Stages / 分析化学

Differential proteomics analysis of Sika deer antlers at rapid growth stage (60 d) and ossification stage ( 90 d) was performed by isobaric tags for relative and absolute quantitation ( iTRAQ ) , ultra high performance liquid chromatography and mass spectrometry technologies. A total of 127 differential proteins were identified. Compared with the ossification stage, 80 differential proteins were significantly up-regulated and 47 differential proteins were significantly down-regulated at the rapid growth stage. These differential proteins were mainly distributed in the regions of extracellular matrix, nucleosome, haptoglobin-hemoglobin complex, actin filament, endoplasmic reticulum-Golgi intermediate compartment, endoplasmic reticulum lumen, and endometrium, etc. The up-regulated differential proteins were mainly involved in the regulations of oxygen transport in the blood, nerve growth and regeneration, cartilage and bone development and ATP synthesis compared with ossification stage, and the down-regulated differential proteins were mainly involved in the endochondral ossification process. The changes of protein expression at different growth stages were closely related to antler rapid growth and ossification. Therefore, the results of this study provided a basic data for discovering the molecular mechanisms of antler rapid growth and ossification, and it was of great significance for further study of the pharmacological basis and clinical application of antlers.

Identification of Peptide Profiling of Deer Antler by Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry and Bioactivities in Simulated Gastrointestinal Digestion / 分析化学

Reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) was utilized to investigate peptide profiling and bioactivities of antler aqueous extract(AAE),digested antler aqueous extract (AED) and powder(PD). A total of 23,417 and 389 peptides,as well as 15,146 and 75 collagen peptides were identified from AAE, AED and PD, respectively. Angiotensin converting enzyme (ACE) inhibitory activity,dipeptidyl peptidase IV(DPP-IV) inhibitory activity,prolyl endopeptidase(PEP) inhibitory activity and antioxidant activity were used to evaluate the bioactivities of AAE,AED and PD,and it was found that the sequence of their bioactivities was AAE<AED<PD. All the results above proved that AED released more collagen peptides and PD possessed better biological activities. It suggests that the two edible ways of antler are complemented each other and have their own advantages.

Characterization and fungal community of antler dermophytosis in Jilin and Liaoning provinces / 中国人兽共患病学报

Dermophytosis is a common fungal disease that affects fast-growing antlers of sika deer (Cervus nippon)and red deer (Cervus elaphus),causing the so-called 'white-skin antlers'and 'crusted antlers'.Here we described the features of dermophytosis in deer antler observed from 20 affected deer from 8 farms in Jilin and Liaoning province by clinical findings,he-matology,pathological examination and fungal species distribution.The fungal infection in the antlers as indicated by HE stai-ning,affected only epidermis and the dermis layers,with the main lesion of necrosis of the dermis tissue and inflammatory in-filtrate.Hematologic profile suggested the insignificant cell count change of lymphocyte,neutrophil,white blood cell between dermophytosis and healthy deer(n=10).A total of 68 fungi isolates were then recovered from the antlers with dermophytosis, of which 64.7% (44/68)were identified as members within Deuteromycotina,the rest 35.3% (24/68)belonged to the Saccha-romycotina.Notably,the well-known opportunistic pathogen,including species within Trichophyton,Epidermophyton as well as Candida albican,might account for the dermophytosis of deer antler.In conclusion,'white-skin antlers'and 'crusted antlers'are high likely caused by opportunistic fungi.

Protective effect of Sika deer velvet antler protein on cisplatin-induced kidney injury / 中国药理学与毒理学杂志

OBJECTIVE To investigate the protective effect of Sika deer velvet antler protein (SVPr) against renal toxicity in mice and its mechanism.METHODS Forty ICR mice were randomly divided into 5 groups:normal control group (ig distilled water),model group (ig distilled water for 7 d,on the 7th day,ip cisplatin 25 mg·kg-1 to establish the model,afterwards ig distilled water for 3 d) and SVPr 5,10 and 20 mg· kg-1 groups (ig SVPr for 7 d,cisplatin 25 mg· kg-1 was provided 2 h after the last administration,then ig SVPr for 3 d).Testing kits were adopted for the measurement of renal indexes in mice,such as blood urea nitrogen (BUN) and serum creatinine (SCr);oxidative stress indictors of super oxide dismutase (SOD),catalase (CAT),glutathione (GSH) and malondialdehyde (MDA);inflammation indictor levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).Caspase 3,Bax and Bcl-2 were detected via Western blotting,and renal pathological changes were observed by HE staining.RESULTS SVPr (5,10 and 20 mg·kg-1) significantly reduced the levels of SCr,BUN,MDA,TNF-α and IL-6,and the expressions of caspase 3 and Bax (P＜0.05),but increased the activities of SOD,CAT and GSH,and the expression of Bcl-2 (P＜0.05).The renal pathological changes were improved.CONCLUSION SVPr can reduce renal toxicity induced by cisplatin in mice,and the mechanism is probably related to inhibiting oxidative stress or inflammatory reaction and improving cell apoptosis.

Purification, identification and hypoglycemic activity in vitro of a novel peptide from red deer antler / 中国药科大学学报

@#This study was aimed at isolating a peptide with hypoglycemic activity from dried red deer antler. A peptide from dried red deer antler of Tarim in Xingjiang was extracted with acetate buffer(pH 3. 5), followed by precipitation with ethanol, ultrafiltration and separation via ion exchange chromatography, gel filtration and reversed phase chromatography. The molecular mass of the peptide was detected via MALDI TOF/MS and the peptide was identified by LC-MS/MS. The activities of the peptide to promote islet β cells proliferation and recovery the damage induced by streptozotocin(STZ)were detected by MTT, and the effect of the peptide on glucose consumption of hepatocyte was determined. The peptide, named as CAP, was obtained from dried red deer antler through purification with the molecular mass of 6 804 via MALDI TOF/MS. The result of LC-MS/MS and the comparison in relative database showed that CAP was a novel peptide. CAP stimulated the proliferation of pancreatic cells and recovery of STZ-damaged pancreatic cells, as well as increased glucose consumption of HepG2.

Effects of Deer Antler Extract on Serum IGF-I, Bone Growth and Splenocyte Proliferation in Growing Rats

Although it has traditionally known that deer antler and medicinal herbs extract contain some functional components for health promotion, the nutritional significance remains to be elucidated. This study examined the efficacy of deer antler extract (DA) , medicinal herbs extract (MH) and their mixture (DAMH) on serum IGF-I, bone growth with growing rats in vivo and splenocyte proliferation with spleen cells in vitro. Three week-old young female rats (Sprague-Dawley) were divided into 4 groups and then fed basal diet (AIN-93G) or experimental diets containing DA, MH, DAMH, respectively, for 7 weeks. We collected blood, liver, kidney, spleen, femur and tibia from rats. There was no significant difference in weight gain, but food intake increased in DA- and MH-fed groups. There were no signs of liver and kidney damage in the DA, MH and DAMH-fed groups compared to basal diet group. In femur and tibia, wet weights, breaking forces and bone minerals (Ca, Mg and Zn) were significantly higher in the DA-fed group than in the other groups. Serum alkaline phosphatase (ALP) , bone-specific alkaline phosphatase (BALP) activities were significantly lower in the DA, MH, DAMH-fed groups than in basal diet group. Also, serum insulin-like growth factor-I (IGF-I) concentrations were significantly increased in DA-fed group compared to the other groups. Therefore DA was shown to have an activity of bone growth promotion by increasing the IGF-I, a major bone growth factor. The deer antler extract showed an enhanced immune action on the primary cultured-cells from spleen of rats, representing that splenocytes were proliferated by lipopolysaccharide (LPS) , but not by concanavalin A (Con A) . These results indicate that deer antler extract has beneficial effects on bone growth via IGF-I and on splenocyte activation.

Effect of Deer Antler Drink Supplementation on Blood Pressure, Blood Glucose and Lymphocyte DNA Damage in Type 2 Diabetic Patients

Deer Antler has been known for its traditional oriental medicinal properties and has been widely used to promote growth, boost immune function, treat blood loss and chronic joint pain. Recent study showed imported (New Zealand) Deer Antler was beneficial in reducing the side effects of cancer treatments. However, there was no intervention study conducted on the effect of Korean Deer Antler on reducing the oxidative stress to patients with diabetes. One of the sensitive ways to measure endogenous oxidative stress is by measuring cellular DNA damage using single cell gel electrophoresis (COMET assay). This study was conducted to investigate the possible beneficial effect of commercial Deer Antler drink (provided by Chung-yang Deer Farm) on lymphocyte DNA damage and blood glucose of diabetic patients. Ten patients (4 men, 6 women) participated in the study and consumed 2 pouches of Deer Antler drink every day for 20 days. Blood was collected on the morning before and after the intervention for lymphocyte isolation and blood glucose analysis. Both systolic and diastolic blood pressure showed a tendency to decrease but did not reach statistical significance after the trial. Blood glucose level was not affected by the supplementation. After the intervention, over 50% reduction were noted in the cellular DNA damage, expressed as tail length (TL) and tail moment (TM; tail length x percent tail DNA). Although we did not obtain beneficial effect on lowering blood glucose levels in the patients, this results suggest that Deer Antler may initially act in protecting endogenous DNA damage in short-term experiment.

The Effects of Deer(Cervus nippon) Antler Extracts on Differentiation of MC3T3 Cells / 대한치주과학회지

Deer antler has been widely prescribed in Chinese and Korean pharmacology. Although there have been several reports concerning the effects of deer antler, such as anti-aging action, anti-inflammatory activity, antifungal action and regulatory activity of the level of glucose, the effect on bone has not determined yet. The purpose of this study was to examine the effect of deer antler on osteoblast differentiation. Hexane extract(CNH) and chloroform extract(CN-C) were acquired from deer antler(Cervus nippon) and MC3T3-E1 preosteoblasts were cultured in the presence or absence of each extract. Osteoblast differentiation was estimated with the formation of mineralized nodules and the mRNA expression of alkaline phosphatase(ALP), osteocalcin(OC) and bone sialoprotein(BSP) which are markers of osteoblast differentiation. Non-treated group did not show mineralized nodule. CN-C or CN-H-treated group showed minerlaized nodules in 16 days. In northern blot analysis, CN-C or CN-H-treated group showed the elevated expression of ALP, BSP and OC in 16 days. These results suggest the possibility to develop deer antler as a bone regenerative agent in periodontal therapy by showing the stimulating activity of deer antler on differentiation of osteoblast.