Development of innovative structural components of Chinese medicine by multidisciplinary crossing.

"Prescription embodied in Preparation", Chinese medicine preparation, aims to study the specific form of Chinese medicine from raw materials to preparation for acting on patients directly. Its development has gone through three stages according to the characteristics of raw materials pretreatment, including "direct smash and initial extraction for Chinese materia medica", "Extensive extraction and preliminarily impurity for Chinese materia medica" and "Refining and purification for Chinese materia medica". With the development of new technologies and new theories, Chinese medicine preparation emerged in a new stage: structural components of Chinese medicine, with the characteristics of definited material basis, clear mechanisms, determined ADME/T properties, reasonable drug release system designs and scientific productions quality controls. This requires multidisciplinary to solve systemly the problems of Chinese medicine preparation. In this article, we reviewed the development of Chinese medicine preparation in different times, and analyzed the development and the characteristics of Chinese medicine preparation; and mainly focused on a fact that multidisciplinary promoted the study and development of Chinese medicine preparation, especially in structural components of Chinese medicine. It provides development direction and theoretical basis for Chinese medicine preparation.

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes.

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes.

[Alkaloids from cervi cornu pantotrichum and its effect on murine splenocytes proliferation].

OBJECTIVE: To study the alkaloids of Cervi Cornu Pantotrichum and its effect on murine splenocytes proliferation. METHODS: The constituents isolation and purification from Cervi Cornu Pantotrichum was carried out by reported column chromatography including Sephadex LH-20 and MCI (CHP20P) and their structures were elucidated on the basis of spectral compounds. The method of MTT was used to examine the effects of eight alkaloids and total alkaloids content (TAC) of Cervi Cornu Pantotrichum on murine splenocytes proliferation. RESULTS: Eleven compounds were isolated from Cervi Cornu Pantotrichum, and their structures were identified as follows: uracil (1), hypoxanthine (2), uridine (3) inosine (4), guanosine (5), 2'-deoxyguanosine (6), guanine (7), thymidine (8), thymine (9), cytidine (10) and adenosine (11). By the experiment of murine splenocytes proliferation activity in vitro, the results showed that the total alkaloids, uracil and adenosine had significantly promoted the proliferation of mouse spleen cells. CONCLUSION: Compounds 4 - 11 are isolated from Cervi Cornu Pantotrichum for the first time. The total alkaloids is one of the material basis of immunomodulatory effects of Cervi Cornu Pantotrichum, and uracil and adenosine are the most active.

[Anti-hepatic fibrosis effects research on serum containing extracts of Periplaneta americana in vitro].

OBJECTIVE: To study the anti-hepatic fibrosis effect of serum containing extracts of Periplaneta americana. METHODS: The serum contained extracts of Periplaneta americana was prepared with serologic pharmacological method. MTT method was used to observe the effect of serum containing extracts from periplaneta americana on hepatic stellate cells (HSC), and Elisa method was used to detect the contents of TGF-beta1 and collagen I in supernatant. RESULTS: Serum containing extracts I and II (15%) of Periplaneta americana had inhibitory effect on HCS (P < 0.05) after HSC were cultured with serum containing extracts of different concentration of Periolaneta americana for 24, 48 and 72 h. At 24 and 48 h, serum containing extracts I and II of Periplaneta americana decreased the content of collagen I in supernatant without significant difference (P < 0.05). Serum containing extracts I (15%, 9%, 5.4%) of Periplaneta americana could reduce generation of TGF-beta1 in supernatant for 24 h (P < 0.05). As for 48 h, only high concentration serum containing extracts I (15%) deceased the content of TGF-beta1 in supernatant. For 24 and 48 h,serum containing extracts II couldn't reduce the content of TGF-beta1 in supernatant (P < 0.05). CONCLUSION: It has definite effect on anti-hepatic fibrosis with serum containing extracts of Periplaneta americana in vitro. The mechanism may be related to inhibiting HSC propagation and reducing the production of TGF-beta1.

[Determination of trace elements in mylabris before and after being processed].

OBJECTIVE: To discuss the processing principle of Mylabris by comparing the differences between the contents of 11 trace elements in Mylabris before and after being processed. METHODS: Used Flame AAS, Graphite Furnace AAS and Hydride generation AAS to determine the content of Cu, Mn, Zn, Fe, Mg, Ca, K, Pb, Cd, As and Hg elements in Mylabris samples. RESULTS: The sequence of 11 trace elements contents in the Mylabris samples from high to low was: K > Mg > Fe > Ca > Zn > Mn > Cu > Pb > As > Hg > Cd. In Mylabris after being processed, the contents of Pb, As, Hg, Cd and Fe elements decreased, Cu, Mg and Ca increased; In the head, legs and wings of Mylabris, the contents of Hg and Pb elements were higher than those of the other parts. The content of As in the inner wings was the highest. CONCLUSION: The traditional processing method, stir-frying with rice and removing head, legs and wings is scientific.

[Extraction of fatty constituents from Tabanus bivittatus and their analysis by GC-MS].

OBJECTIVE: To optimize the extracting condition of fatty constituents from Tabanus bivittatus and analyse by GC-MS. METHODS: Taking fatty yield as index, the extraction technology parameters of fatty constituents of Tabanus bivittatus ere optimized by single factor test and orthogonal test. RESULTS: The optimum extraction parameters were as follows: the ratio of acetone to petroleum ether 1: 2, extraction time 2 h, solid-liquid ratio 1: 50. Twenty-one fatty acids were identified by GC-MS. CONCLUSION: The process is reasonable and with good reproducibility. The main components of fatty acids in Tabanus bivittatus are palmitoleic acid, palmitic acid, linoleic acid, oleic acid and stearic acid.

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

Preclinical toxicological evaluations of the sclerotium of Lignosus rhinocerus (Cooke), the Tiger Milk mushroom.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerus (Tiger Milk mushroom) is distributed in South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. In Malaysia, it is the most popular medicinal mushroom used by the indigenous communities to relieve fever, cough, asthma, cancer, food poisoning and as a general tonic. In China, this mushroom is an expensive traditional medicine used to treat liver cancer, chronic hepatitis and gastric ulcers. The sclerotium of the mushroom is the part with medicinal value. This rare mushroom has recently been successfully cultivated making it possible to be fully exploited for its medicinal and functional benefits. The present study was carried out to evaluate the chronic toxicity of the sclerotial powder of Lignosus rhinocerus cultivar (termed TM02), its anti-fertility and teratogenic effects as well as genotoxicity. MATERIALS AND METHODS: Sprague Dawley rats (10 rats/group/sex) were fed orally with 250, 500 and 1000 mg/kg of sclerotial powder of TM02. The sclerotial powder was orally administered once daily and consecutively for 180 days. At the completion of the oral feeding period, analysis of hematological and clinical biochemical parameters, urine profiles, organ weight as well as histopathological analysis were carried out. The effect of the sclerotial powder on fertility and its possible teratogenicity were examined by feeding rats orally with 100 mg/kg sclerotial powder consecutively for 7-8 weeks. Genotoxicity was evaluated by Ames test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. RESULTS: The results showed that oral administration of the sclerotial powder of the Lignosus rhinocerus cultivar at daily dose of up to 1000 mg/kg for 180 days had no adverse effect on the general clinical observations, body weight, hematology, clinical biochemistry, urinalysis, absolute organ weight as well as relative organ weight, nor induced histological changes in the organs. Oral administration of 100 mg/kg sclerotial powder of the Lignosus rhinocerus for 7-8 weeks did not affect the fertility of the rats nor induce teratogenic effect on their offspring. Lignosus rhinocerus sclerotial powder up to 5000 µg/plate in the presence and absence of metabolic activation did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. CONCLUSION: Our results showed that the no-observed-adverse-effect level (NOAEL) dose of the sclerotial powder of Lignosus rhinocerus in 180-day chronic toxicity study is more than 1000 mg/kg. Oral feeding of the sclerotial powder at 100mg/kg did not induce adverse effect on rats' fertility nor causing teratogenic effect on their offspring. In the reverse mutation Ames test, the sclerotial powder at all tested concentration did not show any genotoxicity.

[Antitumor effects of fresh gecko hydrolysate on H22 transplanted tumor in mice].

OBJECTIVE: To compare the inhibitory effects of fresh gecko crude extract and its hydrolysate on H22 transplanted tumor in mice. METHODS: The content of soluble nitrogen (SN-TCA index) was used to determine the degree of enzymolysis. The hydrolysate of gecko was obtained from fresh gecko crude extract by pepsin and papain hydrolyzing. H22 transplanted tumor mouse models were established and divided into negative group, positive group,crude extract group and hydrolysate group. RESULTS: The inhibition rate of the H22 tumor-bearing mice was 29.17%, 48.99% respectively for the crude extract group and the hydrolysate group. The inhibition rate of hydrolysate group and the negative group were significantly different (P < 0.05). The spleen and thymus index for the crude extract group and the hydrolysate group didn't show different compared with the negative group. CONCLUSION: The crude extract of the fresh gecko and the hydrolysate can inhibit the growth of the H22 transplanted tumor. The enzymolysis by pepsin and papain can increase the antitumor activity of the crude extract of fresh gecko.

[Comparison of the effects of traditional and modern processing methods on antibacterial and anti-inflammatory effects of Musca domestica].

OBJECTIVE: To investigate the different effects of traditional and modern processing methods onantibacterial and anti-inflammatory effects of Musca domestica. METHODS: Antibacterial and anti-inflammatory effects of traditional and modem processing products were carried out on Staphylococcus aureus, Escherichia coli and macrophage RAW264.7 which activated by LPS. RESULTS: The antibacterial and anti-inflammatory effects were more pronounced in modern processing product treatment group than those of traditional processing product treatment group. CONCLUSION: Modern processing technology can protect the substances in Musca domestica which have antibacterial and anti-inflammatory effects.

Pearl extract enhances the migratory ability of fibroblasts in a wound healing model.

CONTEXT: For 2000 years, traditional Chinese medicine has been used as a remedy for general health improvement, including the fight against aging. Pearl powder has recently been used as a health food that has antioxidant, antiaging, antiradioactive, and tonic activities for cells; it is also applied to cure aphthous ulcer, gastric ulcer, and duodenal ulcer on clinical therapy. In addition, the mother of pearl, nacre, could enhance the cell adhesion and tissue regeneration of skin fibroblasts. OBJECTIVE: Fibroblast is regarded as indispensable in the processes of wound healing. Therefore, the effect of pearl extract (PL) on fibroblasts is investigated in this study. MATERIALS AND METHODS: PL is produced by a room temperature super extraction system (Taiwan patent no. I271 220). DMEM medium containing PL (300 µg/mL) was used to examine the effect of migration-promoting potential on human fibroblast cell line or human primary fibroblast cells in a wound healing model in vitro. RESULTS: Medium containing PL (300 µg/mL) demonstrated that the migratory cell numbers of fibroblasts were three times more than that without PL, and mRNA expression of collagen type III was higher than in collagen type I in fibroblasts. It revealed a migration-promoting potential of human fibroblasts in a wound healing model in vitro. DISCUSSION AND CONCLUSION: The present study found that the migration-promoting effect in PL, which could be a supplement in cell culture. These data suggest PL could be useful for enhancing the wound healing of fibroblasts.

[Research on TLC identification and anti-coagulant activity quantitatively methods of Scolopendra subspinipes mutilans].

OBJECTIVE: To improve the quality standard of Scolopendra subspinipes mutilans by researching the methods of the TLC identification and anti-coagulant activity quantitatively. METHODS: Identified the free arginine (Arg) and serine (Ser) in scolopendra by TLC, screened the samples preparation process and developed solvent systems; Determined the anti-coagulant activity by method of titration with thrombin and screened the pretreatment methods. RESULTS: When medicinal materials was extracted by formic acid and 95% ethanol (1:1) with ultrasonic method and developed by n-butanol-acetic acid-water (12:5:4), the spots of Arg and Ser were well separated. Ultrasonic method was suitable for preparation of the anti-coagulant components in Scolopendra subspinipes mutilans and their anti-coagulant activity was determined by method of titration with thrombin could get a well reproducibility, the anti-thrombin activity of testing sample was (14.00 +/- 1.53) U/g and those of three different batch were (13.00 +/- 0.58) U/g, (17.00 +/- 1.15) U/g, (15.67 +/- 1.53) U/g respectively. CONCLUSION: The methods of TLC identification and anti-coagulant activity quantitatively could be used as a basis for improving the quality standard of Scolopendra subspinipes mutilans.

[Analysis of liposoluble constituents in Holotrichia diomphalia by GC-MS and investigation their anti-inflammatory and analgesic activities].

OBJECTIVE: To analysis liposoluble constituents of Holotrichia diomphalia by GC-MS and measure their anti-inflammatory and analgesic activities. METHODS: The composition of liposoluble constituents were determined by GC-MS. The dimethylbenzene-induced mice inflammatory models were established. The pain models were obtained by hot plate and acetic acid in mice. RESULTS: Twenty-two components were identified from the petroleum ether extract of Holotrichia diomphalia. The major components were oleic acid, palmitic acid and palmitoleic acid. The petroleum ether extract was able to significantly inhibit the mice ear edema induced by dimethyl-benzene. The pain in mice caused by acetic acid and hot plate were evidently suppressed by the petroleum ether extract. CONCLUSION: The petroleum ether extract of Holotrichia diomphalia has obvious anti-inflammation and analgesic effects.

[Study on differentiation and extract determination of Porcellio scaber and Armadillidium vulgare].

OBJECTIVE: To identify consistency of the composition and efficacy of Porcellio scaber and Armadillidium vulgare and provide reference for the safe and scientific use of the medicinal material. METHODS: Identified the Porcellio scaber and the Armadillidium vulgare with the method of characteristics; Compared the two chemical composition by thin layer method; Determined the extract by water and ethanol using the pharmacopoeia extract method. RESULTS: Porcellio scaber and Armadillidium vulgare were two varieties, they had similar chemical composition, the water and ethanol extract were the same. CONCLUSION: The compostion and efficary of Porcellio scaber and Armadillidium vulgare are the same, and these two varieties can be mixed or used alone.

[Effect of centipede extract on cervical tumor of mice and its mechanism].

UNLABELLED: To study the anti-tumor activity of centipede extract on cervical tumor of mice and its mechanism. METHODS: The tumor-bearing mice were treated with centipede extract from two solvents [ether (CE) and alcohol (CA)] at different comcentration. The mice' life span, tumor inhibition rate and immune function were estimated. RESULTS: No mice died in CE and CA treatment groups and the tumor inhibition rate was 52.85% and 33.65% respectively. Observed the tumor tissue slices with light microscope and found infiltration of tumor cells in striated muscle in the control group but centipede treatment groups had massive necrosis and apoptosis. Karyopyknosis and apoptotic tumor cells were observed in the treatment groups under transmission electron microscopy. Compared with control group, the expression of Bax increased, the expressions of Bcl-2 and Survivin decreased, but the content of VEGF, the indexes of thymus and spleen had no significant change in treatment groups. The number of CD3+ T lymphocytes had no significant change while the ratio of CD4+ and CD8+, the number of CD19+ B lymphocytes decreaed in the CE group. The numbers of CD3+ and CD4+ lymphocytes decreased in the CA group. The pathological examine indicated no obvious change in the tissue slices of mice's liver and kidney, manifested the concentrations of CE and CA between the article's had no visible side effect. CONNCLUSION: The two extracts (CE and CA) can suppress the growth of cervical tumor and its mechanism may be related to Bax and Caspase-3 medicated the mitochondrial signal transit pathway.

[Study on fibrolytic and hypotensive activity of earthworm homogenate of ultrafiltration separation from enzymolysis].

OBJECTIVE: To study the fibrolytic and hypotensive activity of earthworm homogenate of ultrafiltration separation from simulated enzymolysis of gastrointestinal tract system. METHODS: The before and after enzymolysis homogenate of fresh earthworm was seperated with different pore size PVDF ultrafiltration membrane and its fibrolytic and hypotensive activity was assayed. RESULTS: The fibrolytic activity of the total homogenate after enzymolysis overall changed little, but decreased in the the site of higher molecular weight and increased in the lower site of molecular weight; The ACE inhibitory activity improved, especially in the filtrate of the MW 4000 membrane. CONCLUSION: The fibrolytic activity of earthworm homogenate was not reduced by the digestive simulated enzymolysis, and the retention site of MW 10 000 membrane have more fibrolytic activity; The hypotensive activity of earthworm homogenate is enhanced by the digestive simulated enzymolysis. So the stronger activity could be obtained from enzymolysis.

[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.

[Research on HPCE fingerprint of eupolyphaga steleophaga].

OBJECTIVE: To set up the fingerprint of HPCE for medicinal material Eupolyphaga Steleophaga. METHOD: Separation was performed at 25 degrees C on an Agilent uncoated silica capillary column (40 cm x 75 microm) with 20 mmol x L(-1) borax buffer solution (pH 9.44) as CE buffer. The isolating voltage was 13 KV, and the DAD detection was set at 265 nm. RESULT: The characteristic peak of fingerprint of Eupolyphaga Steleophaga was consisted of 6 common peaks. CONCLUSION: The method can be used for the quality control of Eupolyphaga Steleophaga.

[The research of thrombolytic and hemolysis effect from Eupolyphage fibrinolyric protein and its inhibitory effect on S180 ascites tumor of mice].

OBJECTIVE: Eupolyphage fibrinolyric protein (EFP) was isolated and purified from Eupolyphage sineses, and its thrombolytic effect, hemolysis effect and inhibitory effect on S180 ascites tumor were investigated. METHODS: EFP was isolated and purified by ammonium precipitation and DEAE ion exchange chromatography. It's thrombolytic and hemolysis effect were determined. MTT method and Colony-forming method were used to determine the inhibitory effect on S180 ascites tumor. RESULTS: the EFP was proved to have the effect of Thrombolytic and Hemolysis, and both increased dose-dependently, however at a lower concentration, the EFP had no hemocytolysis. The EFP was also proved the effect of inhibitory on cell proliferation and Colony-forming on S180 ascites tumor of Mice. CONCLUSION: EFP has a strong thrombolytic activity and weak hemolytic, and has inhibitory effect on S180 ascites tumor of mice.

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.