Zhe-Ming Fang

Simultaneous quantification of 19 ginsenosides in black ginseng developed from Panax ginseng by HPLC–ELSD

A high-performance liquid chromatographic method with evaporative light scattering detection (HPLC-ELSD) has been developed to identify and quantify 19 ginsenosides (Rg(1), Re, Rf, Rb(1), Rc, Rb(2), Rd, F(4), Rg(6), Rk(3), Rh(4), 20(S)-, 20(R)-Rg(3), 20(S)-, 20(R)-Rs(3), Rk(1), Rg(5), Rs(4), and Rs(5)) in black ginseng (BG, Korean white ginseng that was subjected to nine cycles of steam treatment). Ultrasonication is employed for sample preparation, and the analysis is achieved on a Discovery C(18) column using gradient elution of CH(3)CN-H(2)O-CH(3)COOH without buffer in 40min. The method was validated by linearity (r(2)> or =0.9994), precision (92.0-107.5%), intra- and inter-day accuracy (R.S.D.<3.21%), and limit of detection (LOD< or =93ng). The quantification method was applied to analyze the composition of ginsenosides in Korean white, red, and black ginsengs. During the preparatory process of BG, ginsenosides transform into constituents of low polarity by hydrolysis, isomerization, and dehydration at C-20, and hydrolysis also occurs at C-3 or C-6. The validated HPLC method is expected to provide the basis for the quality assessment of ginseng products.

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S)-->Rh2(S)-->PPD(S) and Rg3(R)-->Rh2(R)-->PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.

Expression and localization of insulin-like growth factor-I in four parts of the red deer antler

The expression and localization of insulin-like growth factor-I (IGF-I) in the four parts (tip, upper, mid and base) of the red deer antler has been extensively investigated. We used reverse transcriptase polymerase chain reaction (RT-PCR) and real-time reverse transcriptase polymerase chain reaction (real time RT-PCR), in situ hybridization, immunohistochemistry and Western blot techniques to localize IGF-I messenger ribonucleic acid (mRNA) and IGF-I peptide in the four parts of the antler. The specific sequence encoding IGF-I was detected by RT-PCR in all of the four specimens, and the 395 bp IGF-I sequence from the red deer antler was shown to have very high homology with human, goat and mouse IGF-I. In situ hybridization and immunohistochemistry results demonstrated that the expression of IGF-I occurred in chondrocytes and osteoblasts in the tip and upper parts of the antler. However, IGF-I was only detectable in osteoblasts around the bone in the mid and base parts. There were significant differences in the intensity of the signal obtained with the IGF-I probe in the tip, upper, mid and base tissues. The Western blot analysis also provided evidence that IGF-I expression was localized differentially in the four parts of the deer antler. This study indicates that antler tissue is an essential part of the IGF system, which is involved in the regulation of the growth of red deer antlers. The specific expression of IGF-I in the four parts of the deer antler suggests that the IGF-I molecule is present at significantly different levels throughout the deer antler development and regeneration processes. Localization of IGF-I in chondrocytes and osteoblasts suggests that IGF-I may play an important role in cartilage and bone formation. In addition, it may have a variety of biophysical effects that influence the rapid growth of deer antlers.

Partial Purification and Quantification of Insulin-like Growth Factor-I from Red Deer Antler

Deer antler tissue contains the most rapidly growing bone in the animal kingdom. Thus, it is likely that growing antler tissue is a rich source of local paracrine bone-stimulating factors. Growth factors, at least the insulin-like growth factor (IGF), control the bone-remodelling process. In this study, we tried to isolate and purify IGF-I from fresh antler tissue by the routine isolation and purification of protein. The purification involved ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, CM-Sepharose CL-6B ion-exchange chromatography, and Sephadex G-50 chromatography. Purified fractions from each step were analyzed by high-performance liquid chromatography (HPLC), SDS polyacrylamide gel electrophoresis (SDS-PAGE), Dot-blot, and Western-blot methods. Furthermore, the quantification of partially purified IGF-I was calculated by enzyme-linked immunosorbent assays (ELISA) using antibody to human recombinant IGF-I. SDS-PAGE analysis of the final fraction yielded two molecular bands and the signal band was at 12 kDa on the Western-blot film. This purified IGF-I fraction showed a peak at retention time of eight min. The quantity of IGF-I in 20 g deer antler tissue as starting weight was calculated using a standard curve to be 2910 ng/㎖, and total IGF-I amount is 0.291 g. The results show that IGF-I, which can be found in deer antler, can be partially purified and quantified by classic protein isolation methods.