Studies On Quality Standard And Chemical Constituents Of Radix Et Rhizoma Nardostachyos

Objective:Radix et Rhizoma Nardostachyos is dry radix and rhizoma of plant Nardostachys chinensis Bet. or N. jatamansi (D. Don) DC., named as "Gansong"or "Gansongxiang" in traditional Chinese Medicine.It has many efficacies such as regulating qi to alleviate pain, relieving stagnation to activate spleen, calm the nerves, have been used as the common traditional medicinal herb with a long medicinal history in Tibetan Medicine traditional Chinese medicines, contains terpenoids, flavonoids, coumarins, lignans, etc., terpenes is the main components, with sedation, antiepileptic, antidepressant, promote nerve growth, to improve the cognitive ability to protect cardiac cells, lowering blood pressure, antibacterial and anti-malarial, anti-tumor and other pharmacological activities. In addition to medicine, Radix et Rhizoma Nardostachyos in the field of food and cosmetics are also very broad prospects of development. With the development and wide applications of Radix et Rhizoma Nardostachyos in medicine, food and cosmetic fields, need a more comprehensive quality standards to ensure quality. The national pharmacopoeia only stipulate the content of essential oil no less than 2%, target a single, lack precise indicators of composition, can not accurately reflect its quality. For research in various fields Radix et Rhizoma Nardostachyos application and development trends, the topic aims at the study of chemincal constituent from Nardostachyos quality standard and quality standard, establishing HPLC finger print of Nardostachyos which can totally reflect characteristic of its chemical composition, establishing assay of diverse effective constituents, providing the science basic for the establishment of quality evaluation of Nardostachyos., providing material support for the study of pharmacology and pharmacodynamics.Method:Reviewed the recent studies of Nardostachyos DC.; Through the spot investigation and literature study to investigate resource status of medicinal plants of Nardostachys DC. in Gansu, Qinghai and Sichuan provinces, including the species, distribution, and purchase; to study on the isolation and purification of chemical components from radix et rhizome Nardostachyos by a few column chromatogram; to Study on the structures of compounds on the basis of modern spectrum and MS method; to extract volatile oil of Radix et Rhizoma Nardostachyos of different varieties, and the aerial and underground parts of Nardostachys chinensis Bet. with wet distillation, then to analyse the chemical composition of the two extractions with GC-MS; to use the content of nardosinone as assay index, optimize the extraction process by L 9(3) 4 orthogonal design, compare the extraction efficiency of three different methods as reflux extraction, percolation extraction, ultrasonic extraction; adopt thin layer chromatography to set up a method for identifiting Radix et Rhizoma Nardostachyos; to set up the color spectrum-finger printing of Radix et Rhizoma Nardostachyos with HPLC; to test the content of active component with HPLC.Result:The main study and result:1 Literature review Reviewed the recent studies of Nardostachyos, including identification, chemical composition, pharmacology action, clincal application and so on.2 Resource Investigation To develop high value and cost-effective products, and expand the development, thesis group investigated the species, distribution, habitat, purchases of Nardostachyos in main producing areas, such as Sichuan, Gansu, Qinghai.3 The study of Chemical constituents from Nardostachyos(1)Using a variety of the isolation and purification methods (silica gel column chromatography, etc.) and structure identification means (such as UV, IR,1HNMR,13CNMR and other spectroscopic techniques and chemical methods),7 chemical compounds were abstracted from Nardostachyos, identified 4 chemical compound of them.(2) To compare the content and composition of essential oil in Radix et Rhizoma Nardostachyos of different varieties, and the aerial and underground parts of Nardostachys chinensis Bet. The chemical constituents were separated and identified by gas chromatography-mass spectrometry (GC-MS).21 and 36compounds were identified from the essential oils of underground parts of Nardostachys jatamansi (D. Don) DC.and Nardostachys chinensis Bet. The contents were 75.83% and 89.02%, respectively; 40 and 36compounds were isolated and identified from the essential oils of the aerial and underground parts of Nardostachys chinensis Bet. The contents were 77.32% and 89.02%, respectively; the common components were Calarene,β-Maaliene and Aristolene.4 Study on extraction technology of nardosinone from Radix et Rhizoma NardostachyosThe percolation extraction is the optimal extraction method, and the optimal extraction process conditions were as follows:drug powder passing 40 meshes,adding 10 times the volume of ethanol, soaking 24 hours each time, and extracted 2 times. The percolation extraction has such advantages as simple device, safe operation, avoid the effect of temperature, reduce the damage and loss of components, higher content of nardosinone, etc.5 Studies on Quality Standard of Radix et Rhizoma Nardostachyos(1) Study On Botanical and macroscopical identification of Radix et Rhizome Nardostachy The characteristics of original plants N. chinensis Batal and N. jatamansi (D. Don) DC. are almost the same, but there are some differences:the rhizomes of N. chinensis Batal were covered foliated old leaf sheath, after flowering most inflorescence main axis and lateral axis obvious elongated, the fruit bared; the rhizomes of N. jatamansi (D. Don) DC. densely covered with fibrous leaf sheath, after flowering most inflorescence main axis and lateral axis elongation obvious, the fruit hairiness. The main difference between medicinal materials of N. chinensis Batal and is N. jatamansi (D. Don) DC is that the rhizomes of N. chinensis Batal were covered foliated old leaf sheath, while N. jatamansi (D. Don) DC. densely covered with fibrous leaf sheath.(2) Study on Microscopic Identification of Radix et Rhizoma NardostachyosThe microscopic characteristics of powder of N. chinensis Batal and N. jatamansi (D. Don) DC. are almost the same, but there are some differences:the cell wall of stone cell of N. chinensis Batal are quadrangle, while N. jatamansi (D. Don) DC are protracted quadrangle; the structures of N. chinensis Batal and N. jatamansi (D. Don) DC roots are same:outer layer of cork tissue 3-4 series, phloem wide, more cracks, xylem composed of parenchyma cells and duct, the vascular bundleswere divided into 2-6 by interxylary cork; the structures of rhizome are same, stone cell group can be seen in the medulla; the phloem and xylem of stem are narrow, pith hollow, and there are slightly different places:catheter of N. chinensis Batal stem arranged in a discontinuous ring, N. jatamansi (D. Don) DC stem catheter arranged in a ring; both the upper epidermis and lower epidermis of leaf composed of a layer of cell, and the lower epidermal cells are smaller, parenchyma cells which under the epidermal cells contain the fan-shaped crystal and purple inclusions, the cells under the upper epidermis of N. chinensis Batal leaf are horizontal round or irregular shape, and contain less purple inclusions, the cells of N. jatamansi (D. Don) DC are 2 column, vertical stretch or long rectangular strip, and full of purple inclusions.(3) Study on TLC Chromatogram of Radix et Rhizoma Nardostachyos.Adopt thin layer chromatography, a mixed solvent of ether-ethyl acetate-acetic acid (10:3:0.1) was employed as the mobile phase, color developing agent was 10% sulfuric acid and ethanol solution.petroleum as developing agents.The result showed 17 samples have common character, the spots in the TLC were clear and distinguishable.It is a simple, accurate and practical method of identification of Radix et Rhizoma Nardostachyos.(4) Study on the finger print of Radix et Rhizoma Nardostachyos.Set up the finger print of Radix et Rhizoma Nardostachyos with HPLC, make sure the chromatographic condition:Re-C18 Collumn, Acetonitril-0.5% orthophosphoric acid water solution as flow phase, gradient elution; wavelength of detection 288nm; flow rate l.OmL /min. The RSD of relative retention time and relative peak area which belong to each common peak are all smaller than 3% in the study of precision, stability, reproducibility. The result of stability test shows that the solution was stable in 24h 20 common peaks were found after analyzing 17 samples with "the estimate system of semblance in the finger print of Chinese medicinal materials", basic characteristic is at equal pace, the result shows the established method was stable, reliable and accurate.(5) Determination of nardosinone in Radix et Rhizoma Nardostachyos from different places by HPLC HPLC was adopted, a Shiseido MGⅡC18 chromatographic column (250 mm×416 mm, 5μm) was used, acetonitrile-water (65:35) as the mobile phase, the flow rate was 0.8 mL-min-1, and the temperature of the chromatographic column was room temperature, the detective wavelength was 334nm. The nardosinone peak in chromatograms was completely separated from other peaks. The linear range of nardosinone was 1.225 to 14.7μg (r= 0.9992). The average recovery of nardosinone was 98.84% and RSD was 1.77%. The content of nardosinone of all 17 samples ranged from 0.505% to 1.970%.(6) Determination of acaciin in Radix et Rhizoma Nardostachyos from different places by HPLCUsed HPLC, a Shiseido MGC18 chromatographic column (250 mm×4.6 mm,5μm) was used with a mixture of methanol-0.5% orthophosphoric acid water solution (58-42) as the mobile phase; the flow rate was 1.0 mL·min-1 and the column temperature maintained at 30℃; 334nm as the detective wavelength. The acaciin and its peak area shows favourable, filate relation in peak in 0.022 to 0.22μg (r=0.9992), the average recovery of acaciin was 100.59%, the RSD of 2.49%. The content of nardosinone of all 17 samples ranged from 0.0033% to 0.0384%.The method of assaying of nardosinone and acaciin is simple and accurate, with strong specificity. It can be used to provide evidence for establish quality evaluation methods of Radix et Rhizoma Nardostachyos.Novative points:(1)To compare the chemical constituents essential oil from the aerial and underground parts of Nardostachys chinensis Bet. for the first time, and it is first time to compare the chemical constituents essential oil in underground parts of Nardostachys jatamansi (D. Don) DC. and Nardostachys chinensis Bet at equal pace.(2) To optimize the technology of extracting nardosinone from Radix et Rhizoma Nardostachyos for the first time, make sure the optimal extraction method, and the optimal extraction process conditions.(3) It is first time to determine content ofeffective constituent nardosinone in Radix et Rhizoma Nardostachyos from different places by HPLC.