Research On Identification And Anti-osteoporosis Activity Of Traditional Chinese Medicine Hippocampus

Hippocampus, belonging to Syngnathidae family,which distributed widely in the shallow waters of tropical or subtropical, is an important small marine bony fish. Modern medical research have proved that hippocampus has anti-tumor, analgesic, anti-aging, anti-fatigue, anti-thrombotic and sex hormone-like effects and so on.It is found by our survey that the market circulation of hippocampal medicinal market is relatively chaos and their quality is uneven.The phenomena of shoddy and varieties ignorance are existd. Hippocampus histris and hippocampus spines is a confusing situation, most of the literature hardly make a clear distinction between them. Based on this, it is necessary to establish a scientific identification method of traditional Chinese medicine (TCM) hippocampus by their chemical or molecular biological information. Accurate identification, quality evaluation and control are of great significance.It is considered that hippocampus possess effects of warming impotence, scattered quarter swelling in Chinese medicine. Hippocampus folk usage was to treat injury or flutter Warming impotence,suggesting that the hippocampus may be related to anti-osteoporosis.Our previous experiments and literature confirms that, so we focused on HPTLC identification based on small molecule sterol compounds of traditional Chinese medicine (TCM) hippocampus and molecular identification research based on mitochondrial COI and Cytb gene, as well as activity of anti-osteoporosis study based on cytology in vitro experiment.1. Identification of the TCM Hippocampus by HPTLCWith 10 different regions of hippocampal medicinal materials as the research object, TLC identified by different extraction methods, extraction solvent, spotting amount, chromogenic agent, developing solvent, TLC plates and other conditions, we finally chose Cholest-4-en-3-one, cholesterol for reference, as wellas five hippocampus for reference drug, we chose HPTLC Silcon Gel 60 TLC plate, developing solvent system petroleum ether:ethyl acetate= 4:1, with 10% sulfuric acid in ethanol as chromogenic reagent, eventually showing the most abundant and clear chromatography strips, provide controls for identification of those hippocampus adulterants.2. Molecular identification of confusable hippocampus based on DNA barcoding① Based on a mitochondrial COI gene traits to identify apparent hippocampus trimaculatus, while clarify the best method for DNA extraction and PCR amplification system and parameters as well as provide scientific basis for easily confused hippocampus.② Select six commercially available hippocampal samples for molecular identification based on Cytb mitochondrial genes,showed that three of them were hippocampus spin, two were hippocampus histrix,one was hippocampus barburi from Philip. So the identification results were the same with previous our batch identification. The real source of Hippocampus histrix sold in the market was Hippocampus spinosissimus Weber.We recommend Pharmacopoeia could revise hippocampus spin for Hippocampus spinosissimus Weber.Based on mitochondrial cytb gene for molecular identification between hippocampus spin and hippocampus histrix combined with the morphological evidence, which support pharmacopoeia revised Hippocampus histrix Kaup that recorded originally for Hippocampus spinosissimus Weber.3. Studies on anti-osteoporosis activities of the TCM HippocampusNew-born SD rats calvarium osteoblast was chosen as anti-osteoporosis screening model in vitro, five spcies hippocampus of the alcohol extract and five different polar parts to screen anti-osteoporosis activity. Through MTT assay influence on osteoblast proliferation, Determination of the role of ALP activity and bone nodule Content, tracking out that the extracts of water fraction from Hippocampus kuda possess antiosteoporosis activity, TRAP activity was detected through this fraction to inhibit osteoclast. So the anti-osteoporosis active ingredient may be water-soluble components, which included protein, polysaccharide, small molecule polypeptide, while what is the concrete anti-osteoporosis active ingredient has yet to be further research.