[Herbalogical study on Cervi Colla].

Cervi Colla, deer's gelatin, had two kinds of original sources historically, including the skin and antler of deer, known as Cervi Corii Colla(Lupijiao, LPJ) and Cervi Cornus Colla(Lujiaojiao, LJJ) respectively.LJJ is the mainstream of the market, while LPJ is only used by common people in Guizhou and Jilin etc. This article sorted out the ancient and modern literature(since Rites of the Zhou in Zhou Dynasty) on Cervi Colla and conducted the herbalogical study. The results of the study include:① In ancient China, there were six types of commonly-used Colla derived from six animals, including deer, horse, cow, rat, fish and rhinoceros. Cervi Colla was ranked the most top among them, and it was often used as adhesive to make bow and Chinese inksticks and more commonly used as a medicine.Cervi Cornus Colla was first described as a medicinal by the name "Bai Jiao"(white gelatin)in The Divine Husbandman's Classic of Material Medica(Shen Nong Ben Cao Jing).② Initially, both the skin and antler were used as raw materials to make Cervi Colla, but antler became the only raw material, and deer skin disappeared from the mainstream of raw materials for Cervi Colla. This can be attributed to other diverse and luxurious uses of the skin, such as making dress and hats, etc., and the easy accessibility of deer antlers. ③ The sources of Cervi Colla were not limited to Cervus elaphus(red deer) or C. nippon(sika deer), and it also included animal from the family Cervidae, such as Elaphurus davidianus(elk) and C. unicolor(sambar). ④ The processing method was passed down from ancient times to the present, and no significant changes had occurred. ⑤ LPJ and LJJ had many similar effects, and their nature was both warm. The effect of LJJ was to warm the liver and kidney, replenish vital essence and blood, and to reinforce Yang. While the effect of LPJ was to reinforce both Yin and Yang, replenish blood, and stop bleeding. It has a unique advantage for both reinforcing Yin and Yang. The findings of this paper can provide support for the promotion of LPJ and the development of its medicinal value.

[Research on chemical compositions in different processed products of Cervi Cornu Pantotrichum].

To investigate the chemical compositions of "antler powder" and "antler slice", two types of processed products of Cervi Cornu Pantotrichum (CCP) documented in Chinese Pharmacopoeia. With polysaccharides, crude protein, amino acids, fatty acids, mineral elements, biogenic amines, nucleosides and nucleobases as the evaluating indicators, the antler powder and antler slice processed with methods documented in Chinese Pharmacopoeia were compared in this study. The results showed that as compared with the antler powder by directly "chopping into pieces, and grinding into fine powder", the crude protein, amino acids, biogenic amines, nucleosides and nucleobases contents were reduced by 5.01%, 4.35%, 5.90%, 27.62% respectively in antler slices processed with 40% ethanol; the polysaccharides and nucleosides contents were reduced by 24.53% and 21.07% respectively in antler slices processed with 50% ethanol; and the crude protein and nucleosides contents were reduced by 1.65% and 20.52% in antler slices processed with 60% ethanol. While the contents of fatty acids and mineral elements were not decreased in these three methods. Polysaccharide, crude protein, amino acids, and nucleosides contents in "antler slices" were less than those in "antler powder", most notably in polysaccharides and nucleosides. According to the comprehensive scores of principal component analysis (PCA), the decrease of active ingredient determined in this study was lowest in antler slice processed with 50% ethanol.

[Research progress on identification and quality control of Cervi Cornu Pantotrichum].

Cervi Cornu Pantotrichum, as a traditional Chinese medicine, has great potential for development. However, the identification and quality control system is not perfect, leading to the market chaos and chronic slow growth in deep processing of Cervi Cornu Pantotrichum. This paper gives an overview of present situation in identification and quality control system of the Cervi Cornu Pantotrichum, and analyzes present problems. Based on these results, the feasibility study scheme in identification and quality control system for Cervi Cornu Pantotrichum would be then put forward, providing ideas to establish its comprehensive evaluation system.

[Comparative study on the protein maps of different parts of Cervus nippon antler by two-dimensional electrophoresis].

OBJECTIVE: To optimize the two-dimensional electrophoresis (2-DE) method for the proteome analysis of the Cervus nippon antler, and compare the protein maps of different parts of Cervus nippon antler. METHODS: The total proteins of Cervus nippon antler were extracted by protein lysate containing 7 mol/L Urea, 4% CHAPS, 2 mol/L Thiourea, 65 mmol/L DTT, 1 mmol/L PMSF and 0.2% Bio-Lyte. The proteins were separated by immobilized pH 3 - 10 linear gradient (IPG), 7 cm strips as the first dimension. Isoelectric focusing conditions were optimized. 12% SDS-PAGE was used as the second dimension electrophoresis. The gels were stained with Coomassie brilliant blue and analyzed by PDQuest analysis software. RESULTS: The contents of total protein and the numbers of protein points of three different parts of Cervus nippon antler reduced gradually from the top to the bottom. Comparing three maps of different parts of Cervus nippon antler, there were 18 different protein points. Isoelectric point, molecular weight and gray value of each different protein point were calculated. CONCLUSION: An optimized two-dimensional electrophoresis method for the proteome analysis of the Cervus nippon antler is established. The 2-DE profiles of different parts of Cervus nippon antler exist obvious differences. The different protein points can be used as reference for Cervus nippon antler quality control and evaluation.

[In vitro study on gastrointestinal absorption of FITC labeled pilose antler protein extraction].

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.

[Nourishing-yin effect and mechanism of different parts of Cornu Elaphuri Davidiani in rats].

OBJECTIVE: To investigate nourishing-yin effect and mechanism of different parts of Cornu Elaphuri Davidiani in rats. METHOD: The model of yin asthenia rats was built by thy roxine. The substance metabolism, pain threshold, hormone levels and biochemical indicators in serum were measured. RESULTS: The ethanol extract of Cornu Elaphuri Davidiani could regulate the substance metabolism and raise the pain threshold in yin asthenia model rats. Furthermore, it could regulate the hormone levels, biochemical indicators in serum and it could improvte the antioxidant ability. CONCLUSION: The ethanol extract of Cornu Elaphuri Davidiani showed significant nourishing-yin effect in rats and the possible mechanism is correlated with regulating the neuroendocrine network.

Anti-resorptive effect of pilose antler blood (Cervus nippon Temminck) in ovariectomized rats.

Anti-bone resorption activity of pilose antler blood (Cervus nippon Temminck) were evaluated in ovariectomized Wistar rats. The rats were randomly divided into sham operated group (SHAM), ovariectomized group (OVX) and pilose antler blood treated group. The ovariectomized rats were treated with pilose antler blood orally in 4000 microl/kg daily doses for 10 weeks. Compared with SHAM group, serum 17 beta-estradiol level decreased significantly and osteocalcin level increased significantly in OVX group, indicating successful model of osteoporosis. The experiments showed that the bone mineral density of the lumbar spine and left femur in OVX group decreased remarkably compared to SHAM group but normalized by treatment with pilose antler blood. Additionally, serum levels of insulin-like growth factor-land testosterone were lower obviously in OVX group than those in SHAM group but preserved by pilose antler blood treatment. However, no obvious changes in serum levels of calcium, phosphorus, total alkaline phosphatase and osteoprotegerin were observed among three groups. These results suggested that administration of pilose antler blood was effective in alleviating osteoporosis in ovariectomized rats.

[Effects of pilose antler and antler glue on osteoporosis of ovariectomized rats].

OBJECTIVE: To observe the effects of pilose antler and antler glue on osteoporosis of ovariectomized rats. METHODS: 60 6-month-old female rats were divided randomly into sham-operated group, model group, the positive group, pilose antler high-dose group, pilose antler low-dose group and antler glue group. We extracted bilateral ovaries to establish osteoporosis model and treated at two weeks postoperationally for consecutively 13 weeks, then observed the effects of pilose antler and antler glue on rat bone mineral density, bone mineral content, serum biochemical indicators, bone tissue morphology and other indicators. RESULTS: After 13 weeks, the high-dose group and antler glue group could significantly improve the BMD and bone mineral content of the ovariectomized rats, reduce BGP and ALP content, and remarkably increase the width of trabecula bone and bone trabecula area percentage, increase osteoblasts significantly, and decrease osteoclast cells significantly. Pilose antler low-dose also had the phenomenon above, but the result was not so good as high-dose group. CONCLUSION: Pilose antler and antler glue can antagonize osteoporosis of the ovariectomized rats.

[Comparison of protein composition and activities of pilose antler processed by different methods].

To elucidate the influence of processing conditions on pilose antlers therapic effects, the protein composition and activities were compared on three kinds of pilose antler processed by lyophilization, freezing and traditional short-time heating, respectively. The concentration of the water soluble protein in freeze-dried pilose antler was 126.54 mg/g (Folin-Phenol assay), which was 13.1 times higher than that of heating processed antler. These proteins distributed widely in SDS-PAGE electrophoresis and the protein band between 50.0 kDa approximately 60.0 kDa achieved the highest concentration. The water extract of freeze-dried antler promoted the proliferation and IGF-I secretion of rat osteogenic-like cell UMR-106 by 245.25% ( MTT assay) and 66.36 ng/ml, which was respectively 2.2 times and 1.2 times of those of heating processed antler. The same candidate inhibited the growth of human hepatic carcinoma cell BEL-7402 by the highest rate of 47.64% , which was 1.4 times of heating processed antler. The activities of frozen fresh pilose antler were lower than those of its freeze-dried counterpart, but were much higher than those of heating processed antler. The results indicated that lyophilization help to remain the activity of pilose antlers proteins as much as possible and improve its efficacy.

[Extraction of sex hormone from antler velvet with supercritical CO2].

OBJECTIVE: To study the method of extraction of sex hormone from antler velvet with supercritical CO2. METHOD: Supercritical fluid extraction (SFE) was used to extract sex hormone from antler velvet and radioimmunoassay (RIA) was used to analyze the extracts. The chemical compositions in extracts were identified by GC-MS, TLC and HPLC, respectively. RESULT: The experimental results indicated that the extraction yield was 1.56% when 85% ethanol was used as co-solvent at temperature of 65 degrees C and extraction pressure of 30 MPa. Estradiol and progesterone in the extracts were 3.07, 776.18 ng x g(-1) respectively. CONCLUSION: It is feasible to extract hormone from antler velvet with supercritical CO2.

[Purification and characterization of the proliferation of rat osteoblast-like cells UMR-106 from pilose antler].

OBJECTIVE: The activity of deer serum albumin on proliferation of rat osteogenic-like cells UMR-106 and the IGF-I secretion were investigated in order to elucidate pilose antler's bone-strengthening mechanism. METHOD: Deer serum albumin was isolated from freeze-dry pilose antler powder extract. The methods were Sephacryl S-200HR gel filtration, POROS 20QE ion-exchange and TSK G3000SW chromatographies. The effect of deer serum albumin on proliferatio of UMR-106 cells was assaied by MTT, and the secretion of IGF-I of UMR-106 cells was assaied by RIA. RESULT: Deer serum albumin, with the molecular weight of 56.3 kDa, significantly increased the proliferation rate of the osteoblast-like UMR-106 cell and IGF-I secretion. When concentration of deer serum albumin reached 0.149 microg x mL(-1), UMR-106 cell proliferation rate was 241.03% and IGF-I secretion was 66.89 ng x mL(-1). CONCLUSION: The concentration of deer serum albumin, from 14.9 ng x mL(-1) to 14.90 microg x mL(-1), significantly increased the proliferation rate of the osteoblast-like UMR-106 cell and IGF- I secretion.

[Influence of the active compounds isolated from pilose antler on syntheses of protein and RNA in mouse liver].

The polyamines of pilose antler (PASPA) consist of putrescine (PU, 70.9%), spermidine (SPD, 26.3%) and spermine (SP, 2.8%). The incorporations of [3H] leucine into protein and [3H] uridine into RNA in mouse liver tissue were increased when PASPA was given orally to mice at the dose of 30 mg/kg for 4 successive days. The incorporations of [3H] leucine into liver protein and [3H] uridine into the cytosolic and nuclear RNA were also increased by treatment with PU (21 mg/kg). In addition, the RNA polymerase activity in the solubilized liver nuclear fraction of PU (21 mg/kg)-treated mice was increased. SPD only promoted the synthesis of protein in mouse liver tissue at the dose of 8 mg/kg. However, SP showed no effect on the synthesis of protein and RNA polymerase activity under the used dose (1 mg/kg). The results suggest that PASPA is the main active substance responsible for the promotion of the synthesis of protein and RNA in mouse liver.

Purification and partial characterization of anti-inflammatory peptide from pilose antler of Cervus nippon Temminck.

An anti-inflammatory compound was purified and isolated from pilose antler of Cervus nippon Temminck by dialysis, gel filtration and ion-exchange chromatography techniques. HPLC and N-terminal amion acid analysis identified the compound as a homogeneous peptide. The peptide is composed of 68 amino acids and its molecular weight as determined by amino analysis, is about 7200.

Analysis on the therapeutic effect of combined use of acupuncture and mediation in 297 cases of male sterility.

Of the 279 cases of male sterility treated by the combination of acupuncture, pilose antler essence injection to acupoints and oral administration of Chinese materia medica, 142 cases (47.8%) were cured, 81 cases (27.3%) markedly effective, 53 cases (17.8%) effective and 21 cases (7.1%) ineffective. The therapeutic effect of the combination of these three treatments was satisfactory.

[Relation of Fourier transform infrared spectroscopic characteristics of pilose antler and its traditional quality grade].

The relationship between FTIR characteristics of Pilose Antler and its traditional quality grade was studied and a rule governing its quality value "Z" was found. We have thus advanced a new objective target for preparing Pilose Antler tablets and powder.

[Character identification of 12 kinds of pilose antler medicinal materials].

In this paper, commercial medicinal materials of 12 kinds of pilose antler, Cervus nippon, C. elaphus, C. albirostris, C. unicolor, C. eldihainanus, C. timorensis C. porinus, Dama dama, Rangifer farandus, Alces alces, Elaphurus davidianus, Capreolus capreolus were compared and identified. A key and simple character illustration were listed.

[The modulation of pilose antler extract (PAE) on the proliferation of rat osteogenic cells UMR-106].

OBJECTIVE: To investigate the modulation of pilose antler extract (PAE) on rat osteogenic cells UMR-106 in vitro. METHOD: Component P2 of PAE was isolated by Sephacryl S-200HR gel filtration chromatography. The proliferative effects of P2 and other components isolated by Sephacryl S-200HR on UMR-106 cells were investigated by MTT assay. RESULT: The P2 could significantly increase the proliferation rate of osteogenic cells. When the protein concentration of P2 was between 0.972 mg x L(-1) and 97.2 mg x L(-1), it could inhibit the proliferation of UMR-106 cells. But while the concentration was equal to or greater than 97.2 mg x L(-1), the P2 could increase the proliferation rate of cells, especially 477.92% at 9.72 g x L(-1), which was approximated to 499.62% of PAE. The molecular weight of the P2 was about 59 kDa determined by SDS-PAGE. On the other hand, inhibition was also observed in the sample of the P3, P4 and P5. CONCLUSION: Those regulative factors in PAE which have different molecular weight can affect the proliferation of UMR-106 cells two-wayly. And this adjustment also relies on the dose of those factors. This finding may help us to understand the possible mechanism of Chinese traditional medicine from animal materials.

[Inhibitory effects of the extract of pilose antler on monoamine oxidase in aged mice].

It was demonstrated that the water extract of Pilose Antler (WEPA) showed a higher inhibitory effect on MAO-B activities in the liver and brain tissues of aged mice, but nearly no effect on NAO-A. WEPA could significantly increase the contents of 5-HT, NE and DA in the brain tissues of aged mice. In vitro experiments revealed that the inhibition of WEPA on MAO-B was competitive, but on MAO-A was of mixed-type.

Hematopoietic effect of Bacillus subtilis-fermented antler extract on phenylhydrazine-induced hemolytic anemia in Sprague-Dawley rats.

This study examined the effect of fermentation on the ability of antler to act as a stimulator of hematopoietic activity. Hemolytic anemia was induced by phenylhydrazine (PHZ) in female Sprague-Dawley rats. The vehicle or antler extract (nonfermented or fermented) mixed in drinking water was administered from Days 2 to 15 after PHZ injection. On Day 15, red blood cell counts in the fermented antler group (6.33×106/µL) were significantly higher than those in the nonfermented antler group (5.90×106/µL) (P<.05), and rats treated with fermented antler extract tended to have higher hemoglobin compared with rats treated with nonfermented antler extract, but not significantly. In addition, rats treated with fermented antler extract had slightly lower serum erythropoietin levels compared with nonfermented antler extract, which were not statistically different from serum erythropoietin levels of nonanemic rats. We conclude therefore that the hematopoietic activity of antler might be increased by the fermentation process.

[Simultaneous determination of eleven sex hormones in antler velvet health products by gas chromatography-tandem mass spectrometry].

A method for the simultaneous determination of 11 sex hormones in antler velvet health products by gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed. The sex hormones in antler velvet were enriched and purified by solid phase extraction and derivatized with heptafluorobutyric acid anhydride (HFBA). A DB-5 column (30 m x 0.25 mm, 0.25 microm) with nonlinear gradient program was used in GC separation. The sex hormones were determined in the multiple reaction monitoring mode. The method realized the complete separation of 11 sex hormones. The limits of detection of this method were from 1.0 to 5.0 microg/kg for the 11 sex hormones. The correlation coefficients were between 0.991 6 and 0.999 9. The recoveries were in the range of 67.4% - 99.1% with relative standard deviations (RSDs) of 2.6% - 13%. This method is accurate and reliable for the determination of the sex hormones in antler velvet health products.