Two Commonly Used Herbal Medicine Multi-component Rapid Analytical Methods

The pharmacodynamic action of Traditional Chinese Medicines are based on the multicomponents existed in them.Rapid,precise and sensitive analytical technology and method are significant for components analysis and quality evaluation for Traditional Chinese Medicines.RRLC(Rapid Resolution Liquid Chromatography) is an advanced rapid resolution liquid chromatography.It has some dominance such as rapid,high resolution and high sensitivity.So it can be used to multiple-component analysis in complicated matrix appropriately.In the first part of the thesis,RRLC methods have been developed and validated for the fingerprint analysis and quantitation of principal constituents simultaneously of two commonly used Traditional Chinese Medicines.Aconite roots have been used widely in folk medicine throughout China and other East Asian countries.However poisoning induced by ingestion of aconite roots are still frequent and numerous.The diester-diterpene alkaloids especially such as mesaconitine, aconitine and hypaconitine are the main active components in aconite roots,however they are highly toxic and showed a narrow margin of safety between the therapeutic and toxic dosage.In the second chapter of the first part,a RRLC method coupled to diode array detection had been developed and validated for the fingerprint analysis of aconite roots and quantitation of mesaconitine,aconitine and hypaconitine simultaneously.The analysis was performed on a Zorbax Extend C₁₈ column and gradient elution.The mobile phase consisted of MeOH and water containing 0.26%ammonium acetate.The analysis run time was just 10 min shortened 6 times than that of traditional HPLC method for each sample of aconite roots.11 major chromatographic peaks were identified by RRLC-DAD/MS.The method showed good linearity within test ranges of 4.93-492.50μg/mL for mesaconitine,5.18-517.50μg/mL for aconitine,4.93-492.50μg/mL for hypaconitine.The RSDs of intra-day and inter-day precisions of the method were in the range of 0.80-1.91%and 0.19-4.93%for the three components.The repeatabilities were good with RSD less than 2.19%.It's also showed good intra-day and inter-day stability by RSD less than 3.23%,3.08%respectively.The recoveries were in the range of 98.37-101.50%with RSD less than 3.41%for the three components.The method is rapid, accurate and suitable for fingerprint analysis and quantitation of the three components.It can provide reference information for quality evaluation of aconite roots. Rhizoma Coptidis is one of the most well-known and widely used herbs in traditional Chinese medicine.Traditional cultivation of Rhizoma Coptidis is carried out under artificial cover,a method that has been used for hundreds years.The deforestation and loss of water and soil that result from this method lead to damages to the natural environment.More recently,several ecological cultivation methods have been developed that shade Rhizoma Coptidis under economic crops.Such crops include fruit trees,Galla chinensis forests,corn,etc.In addition to protecting the environment,these new cultivation methods provide the additional economic benefits.In the third chapter of the first part,we developed and validated a sensitive and specific method using RRLC coupled with an ultra-visible spectrometric detector for analysis of Rhizoma Coptidis samples grown under different cultivation conditions,different habitats and processed with different methods.The method allows simultaneous quantitation of four mian alkaloids:berberine,palmatine,jatrorrhizine,and coptisine as well as fingerprint analysis for multicomponents in Rhizoma Coptidis.The analysis was carried out using a Zorbax Eclipse Plus C₈ reversed-phase column(4.6×50 mm,1.8μm) and gradient elution.The mobile phase consisted of acetonitrile and 20 mmol/L KH₂PO₄.It just needed 3.5 min shortened 7 times than that of traditional HPLC method for each sample of Rhizoma Coptidis.The method showed good linearity within test ranges of 4.75-47.50μg/mL for jatrorrhizine,20.60-164.80μg/mL for coptisine,18.07-180.73μg/mL for palmatine,and 89.70-717.57μg/mL for berberine.The RSDs of the intra-day and inter-day precisions were determined to be in the range of 0.4-1.5%and 0.3-3.7%for the four components. The repeatabilities were good with RSD in the range of 0.6-1.2%.It's also showed good intra-day and inter-day stability by RSD in the range of 0.4-0.5%,0.4-1.3%respectively. The recoveries were in the range of 96.30-104.10%with RSD less than 3.3%for the four components.The lowest limit of detection was 0.19 ng for jatrorrhizine,0.21ng for coptisine,0.15 ng for palmatine,and 0.14 ng for berberine.The lowest limit of quantification was 57 ng for jatrorrhizine,0.82 ng for coptisine,0.55 ng for palmatine, and 0.27 ng for berberine.The method is accurate,rapid,and convenient,and is suitable for use in routine quality control of Rhizoma Coptidis.Oligosaccharides and polysaccharides have significant biological and pathological effects and have attracted more and more public attention.The research of size distribution of the oligosaccharides and monosaccharide composition is very elemental and important for quality evaluation,physico-chemical property investigation and structure investigation.In the first chapter of the second part,we developed a capillary electrophoresis method coupled with laser-induced fluorescence detector(LIF) to detect the size distribution of the oligosaccharides linked to the N-terminal of the glucoprotein in Shuxuetong injection.At first,N-oligosaccharides in glucoprotein were released by peptide N-glycoside F enzyme.Then oligosaccharides were derivatized with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate(APTS),and all of the labeled oligosaccharides were separated well by capillary gel electrophoresis with laser-induced fluorescence detection.The analysis was carried out using an eCAP^TMN-CHO column. The separation buffer was eCAP^TM Carbohydrate separation gel buffer N.The polarity on the instrument was reversed before experiment.The sample was injected by pressure on negative electrode and detected on the positive electrode.The excitation wavelength was 488 nm,and the emission wavelength was 520 nm.The migration time of the labeled oligosaccharides was related to the size of the oligosaccharides and can be detected by the detector one by one.By comparing with the electrophoretogram of the labeled dextran(n=1-20) size marks,the size distribution of the oligosaccharides can be definited. Oligosaccharides linked to the N-terminal of the glucoprotein in Shuxuetong injection may have monosaccharide,disaccharide,trisaccharide and tetrasaccharide according to the results.This method is accurate and effective,can provide useful information for the study of relationship between structure and bioactivities of oligosaccharides.In the second chapter of the seond part,a simple and sensitive high performance liquid chromatographic method for simultaneous determination of five kinds of neutral monosaeeharides(glucose,mannose,galactose,rhamnose,xylose) and two kinds of uronic acid(glucuronic acid,galacturonic acid) by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone(PMP) had been developed.The PMP-derivatives were stable and had no isomerides or by-products.The PMP-derivatives of the five neutral monosaccharides and two uronic acids were well separated by HPLC using a developed gradient elution process on a common C₁₈ column and monitored by DAD detector at 240 nm.We successfully applied this method to monosaccharide composition analysis of polysaccharides prepared from artificial Cordyceps militars.The method is specific, simple,rapid,precise,convenient and can be also used to analyze monosaccharide composition of polysaccharide isolated from Chinese herbal medicine.