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20heilongjiang astragalus membranaceus, namely north astragalus membranaceus, wood ray cell, smaller screen and bast fiber bundle interaction, near cambium screen nest of tubes in the foreign fiber bundle is relatively dense, arrangement is neat, form a band in an intermittent, cork seriously. Shanxi astragalus , wood ray is broadly fusiform, long near the cambium screen irregular polygon. Cambium scattered visible outside the stone cell, the main distribution and phloem and cambium. In this paper provide the basis for the root of astragalus class student origin and source identification. Reference
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Study on the HPLC Fingerprint of Celosia cristata L.
ZHANG Hai-Jing 1, JIN-Lan 1*,CHENG Da-zhong 1**
1.Heilongjiang University of Chinese Medicine, Harbin 150040, China 1*Heilongjiang University of Chinese Medicine, Harbin 150040, China 1**Research Institute of Traditional Chinese Medicine,Heilongjiang University of Chinese Medicine, Harbin 150040, China
Abstract: Objective: To establish the method to analyze HPLC fingerprint of Celosia cristata L., provide a reliable method for the science appraisal and active control its quality.Methods: HPLC with Dikma C18 chromatobar column ( 250mm*4.6mm,5p,m) , using gradient elution with acetonitrile -0.1% phosphoric acid solution ,volume flow rate 1.0mL/min, under the detection wave length of 257nm, and record time 90 min. Data was analyzed by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2004A) to compare the similarity of the samples for identifying the main chromatographic peaks furthermore. Results: The chromatographic fingerprint of 10 batches of Celosia cristata L. showed 16 characteristic peaks,with the similarity above 0.9. The qualities of Celosia cristata L. in different batches were in a good similarity. Conclusion: The method can be applied to quality of Celosia cristata L.
Key words: Celosia cristata L.; fingerprint spectrum; HPLC
Celosia cristata L. is the dry inflorescence of genus Celosia in the family Amaranthaceae[1], which is widely distributed in the world,rich in resources. It has long history of medicinal, which is one of China's traditional Chinese medicineinhemostasis,anti-diabetes[2],anti-vaginal trichomoniasis[3],anti-osteoporosis[4], improveimmunity[5], anti-tumors[6], anti-aging, liver protection[7]and anti-atherosclerosis[8], and so has a good pharmacological activity. However, for the quality control of medicinal reported rare, or even none. Fingerprint is the use of modern technology to characterize and describefor Chinese chemical information in a graphical way , it has become an internationally recognized most effective means ofquality standardscontrol for Chinese medicine or natural medicineby virtue of its accuracy and operability. HPLC with the features of high separation efficiency, analysis speed, high sensitivity, stability and reproducibility, the mobile phase selectivity wide variety , is a ideal for building fingerprint spectrum. HPLC fingerprint is an internationally recognized type of traditional Chinese medicine quality evaluation model, you can grasp the fingerprintcharacteristics of traditional Chinese medicine to evaluate the authenticity of Chinese herbal medicines, good and stability , and it iscore technology to establish quality standards systemfor modern medicine. In this paper, HPLC fingerprint of Celosia cristata L. is discussed, and its methodologyto be inspected, so that to provide a more viable basisfor the quality control of medicines .
Materials and methods
1.Materials
Waters 2695 HPLC (Waters 2996 diode array detector, Waters Empower Pro chromatographic workstation), Dikma C18 column (250mm x 4.6mm, 5p,m); LA204 electronic analytical balance (Mettler - Toledo Instruments Co., Ltd. ); KQ-2500E ultrasonic cleaning machine. Acetonitrile and phosphoric acid are HPLC grade , water for ultra-pure water and other reagents are of analytical grade; kaempferolreference substance (MUST-10102801, National Instisutes for Food and Drug Control ), quercetinreference substance(100081-200406, National Instisutes for Food and Drug Control , luteolin reference substance (111520-200504, National Instisutes for Food and Drug Control ).
Celosia cristata L. were collected in Heilongjiang Province, Hunan, Hebei, Jilin and other places, a total of 10 batches, identifiedas Amaranthaceae genus Celosia cristata L. seeds byProfessorSun Huifeng who from Department of Pharmacognosy, School of Pharmacy,Heilongjiang University of Chinese Medicine.Specimen (20110912) are kept at Research Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine.
2 Test Methods 2.1 Chromatographic Conditions
Column: Dikma C18 (250mm x 4.6mm, 5p,m) ,Mobile phase: acetonitrile-0.1% phosphoric acid gradient elution, flow rate: 1.0mL/min, column temperature: 25 °C, detection wavelength: 260nm, analysis time: 90min, injection volume: 20p,L.
2.2 Preparation of Standard Solutions
Defined amount ofkaempferol, quercetin, luteolin standard were accurately weighed, and were fully dissolved in methanol, fixed volume to 10mL volumetric flask, served as reference solution through 0.45p,m filter membrane .
2.3 Preparation of the Test Solution
AftersmashingCelosia cristata L.with grinding mill,take it through a 60 mesh sieve. 5.0gdry powder was weighed ,adding 95% ethanol 50mL, which was weighed accurately, ultrasonic extraction 60min. Let cool, weigh again, then supplementthe lossquality with solvent, shake, filtration, fixed volume to25mL volumetric flaskwith methanol. Through 0.45p,m microporous membrane, the solutionwas the test solution.
2.4 MethodologicalStudy
2.4.1 Precision Test
Take the same test solution, continuous injectsix timesaccording to the above chromatographic conditions , record the characteristic peak's relative retention time and relative peak area, respectively, Results showed that the relative retention time RSD was 0.02% ~ 0.76%, RSD <1%, relative peak area RSD was 0.07% ~ 1.38%, RSD <3%, indicating that the instrument performance is good, in linewith the requirements of fingerprint.
2.4.2 Stability Test
Take the same test solution,take sample testing at 0,3,6,9,12,24 haccording to the above chromatographic conditions , and record the characteristic peak's relative retention time and relative peak area. Results showed that relative retention time RSD was 0.02% ~ 0.39%, RSD <1%, relative peak area RSD was 0.14% ~ 1.84%, RSD <3%, indicating that the sample solution was stable within 24h .
2.4.3 Reproducibility Test
Weigh the same batch of samples 6 copies for the test solutionaccurately, prepare for analysis, the same way as the above experiment. Results showed therelative retention time RSD was 0.03% ~ 0.53%, RSD <1%, relative peak area RSD was 0.41% ~ 2.24%, RSD <3%, showing that the method has better reproducibility results, in line with the requirements of fingerprint spectrum .
Results and Discussion
1 Results
1.1 Establishment of the HPLC Fingerprint of Celosia cristata L.
Reference solutionand 10 batches of samplesolution 20^L solution inject into the HPLC instrument,respectively,according to established above chromatographic conditions, and record the chromatogram within 90min. In the fingerprints of 10 batches of samples, there are 16 peaks with the stablerelative retention times and relativepeak, which were identified as common peak, No. 1 to 16. By comparison with the reference substance, identify three peaksin them. That is, the 9th peak is luteolin; the 10th peak is quercetin; the 13th peak is kaempferol.
1.2 Similarity Evaluation of Chromatographic Fingerprint
According to the HPLC chromatogram of 10 batches of Celosia cristata L., matchthe 1st herb spectra which as a reference spectrum , S peak as internal reference to calculate the relative retention time and peakarea. The resulting chromatograms of AIA format import "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2004A)"(Study Edition) software in sequence.Output the chromatographic fingerprint of 10 batches of Celosia cristata L. and reference fingerprint , and use it as the basis for similarity calculation. With the result that the similarityof 10 batches of different region's herbs are greater than 0.9.
2 Discussion
2.1 Selection of Detection Wavelength
To make a full spectrum scan to samples in 220 to 400 nm wavelength by using the Waters2695-2996. In line with the principlesof chromatographic information-rich, the appropriate number of peaks and good resolution betweeneach peak, 260nm was determinedas detection wavelength .
2.2 Determination of the Mobile Phase
4 kinds of solvent system of methanol - water, acetonitrile - water, methanol -0.1% phosphoric acidsolution, acetonitrile -0.1% phosphoric acid solution wre investigated in this experiment, and were elutedwith different gradient, to record the chromatograms. The result showed that there are more peak's information, good separationand stable baseline with acetonitrile(A) -0.1% phosphoric acid solution (B)gradient elution . The linear gradient program was set as follows: 0 ~ 15min, 5% ~ 20% A; 15 ~ 35min, 20% ~ 30% A; 35 ~ 45min, 30% ~ 40% A; 45 ~ 60min, 40% ~ 65% A; 60 ~ 70min, 65% ~ 85% A; 70 ~ 90min, 85% ~ 100% A.
2.3 Investigation of Extraction Solvent
There are 5extraction solventsto compare by ultrasonic extractionin this experiment, including 50% methanol, 100% methanol, water, 65% ethanol, 95% ethanol. The results show that chromatogram of different solvent extraction has large differences, that is the waterextract obtained fewer chromatographic peak, 50% methanol and 65% ethanol extractobtained basically similarchromatogram, 100% methanol and 95% ethanol extract also obtainedbasically similarchromatogram. But the chromatogram of the 95% ethanol extract obtained better separation, and more stable baseline, so making the choice of 95% ethanol as extraction solvent.
HPLC fingerprint of different regions
Characteristic fingerprint spectrum of Celosia cristata L.
Conclusion
In this study, conduct fingerprint of Celosia cristata L. from different regions by HPLC. Researching retention time and peak area ofa variety of chemical constituents as the parameters to investigate overalland calculate its similarity. the establish fingerprintsof Celosia cristata L. of different regions and generate characteristic fingerprint spectrum. Through the fingerprint's comparison analysis by "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2004A)" software on 10 batches Celosia cristata L. of different regions to determine 16 characteristic fingerprint peaks in the 10 batches of samples.The
relative retention time of commom peaks is stable, but relative peak area is different, which means that the species ofcontainedchemical composition of different region'sherbs is similar, but the content of each component is different. Similarityof 10 batches of samples and common pattern were greater than 0.9, indicating that this experiment established fingerprint havemarked characteristic and good stability, for the quality control of Celosia cristata L. provide a scientific basis and effective method of identifying .The results are described in the following diagram. References
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[8] Tian Yu-hui, Li Wan -li, Xue Ying-chun, et al. The impact on Zn, Cu ,Fe,Ca of Celosia cristata L ethanol extract with rats fed high-fat[J]. Modern Rehabilitation, 1998, 2 (2): 92-93.
The research progress of osteoporosis treated with traditional Chinese medicine
Wu Xinyu, Li Ji
(Heilongjiang University Of Chinese Medicine,Harbin150040,China) [Abstract]This article summarized the etiology and pathogenesisclinical diagnosis,medical treatment and other therapies of osteoporosis,to help further investigation. [Key words]Osteoporosis;traditional Chinese Medicine
There's no disease or syndrome named as osteoporosis in classics of traditional Chinese medicine,but it could be differentiated as lumbago,bone atrophy or bone arthralgia. 1 Etiology and pathogenesis
There are a large number of discuss in the classics,and kidney is the primary viscera involved.Plain Questions said,"the kidney is concerned with the bone and produces bone marrow."Danxi Xinfa,"limbs witherjoints aching,refined essence and marrow disappear when kidney deficiency."
It's also involved with liver and spleen.As said in Li Gao's Treatise of Spleen and Stomach,"Spleen disease downgoing to kidney cause deficiency of bone marrow".Liu Yuansu's Su Wen Xuan Ji Yuan Bing Shi said,organs,channels limbs and body all depend on Spleen and Stomach for nourishing."Plain Questions said,the tendons can't move if liver qi deficiency.Zheng
