Sensitive and Rapid Detection of Traditional Chinese Herbs by Loop-Mediated Isothermal Amplification Method and Real-Time Fluorescence Quantitative PCR

ABSTRACT Adulterant herbal materials are threats to import and export trade and consumer safety. In this study, we established a simple and rapid examination system for the detection of Phellodendron chinense Schneid. Two detection methods, real-time fluorescence quantitative PCR (real-time PCR) and loop-mediated isothermal amplification (LAMP), were developed for traditional Chinese medicine detection, and their specificity and sensitivity were compared. The DNA of P. chinense was extracted and its special periods amplified with designed primers. Real-time PCR and LAMP experiments were conducted to test the specificity of primers in contrast to other similar species. The template concentration was diluted from 101 ng/µL to 10-5 ng/µL in order to contrast sensitivity between real-time PCR and LAMP. Real-time PCR and Lamp method has shown specificity because P. chinense was positive as opposed to other negative similar species. The Lamp method could detect a limited DNA concentration of 10-4ng/µL in 60 minutes with same sensitivity to real-time PCR. The results indicate that real-time PCR and LAMP are sensitive, accurate and specific in detection of P. chinense. However, LAMP is more convenient and cast less time. What's more, expensive equipments are not necessary for LAMP detector. For a better detection, we suggest an establishment of a real-time PCR and LAMP method for TCM market supervision which depends on DNA barcode sequences and LAMP.

Numerical taxonomy of corlor in Phellodendron Cortex / 中国中药杂志

Through the investigation of Phellodendron Cortex on the market, and 28 batches of samples were collected. By using spectrophotometer the color values of outer surface, inner surface and cross - section of these samples were measured. These measured color data was translated into 3D structure diagram by using the Lab color space tool. The level difference value, the mean value and the threshold value were calculated based the measured color data of these different batches of samples. All 28 groups measured data was analyzed using the methods of Ward linkage and average Euclidean distance. At the same time, we invited Professor Jin Shiyuan, the "Chinese medicine master", to identify, quality-evaluate and grade these 28 batches of Phellodendron Cortex samples base on the traditional experience, then compared the traditional empirical results with the spectrophotometer measurement results. The result showed that, the Phellodendron Cortex could be divided into Phellodendri Amurensis Cortex and Phellodendri Chinensis Cortex by color numerical clustering, and classified according to quality. The classification result has a high degree of consistency with the traditional experience.

Modified Si-Miao-San inhibits inflammation and promotes glucose disposal in adipocytes through regulation of AMP-kinase / 中国天然药物

Modified Si-Miao-San (mSMS) is composed of Rhizoma Coptidis, Cortex Phellodendri, Rhizoma Coptidis Semen Coicis and Atractylodes Rhizome. The prescription is used for the management of diabetes and insulin resistance in the clinic. This study aims to investigate its regulation of glucose disposal in adipocytes. Differentiated 3T3-L1 adipocytes were stimulated with conditioned medium derived from activated macrophages to induce insulin resistance and observed the effects of Mac-CM on insulin-mediated glucose uptake along the insulin receptor substrate-1/PI3K/Akt signaling pathway. Moreover, its regulation of AMPK phosphorylation was also investigated. mSMS enhanced AMPK phosphorylation and promoted basal glucose uptake in adipocytes; mSMS inhibited NF-κB activation by reducing P65 phosphorylation and improved insulin-stimulated IRS-1 tyrosine and Akt phosphorylation, leading to the restoration of insulin-mediated glucose uptake when cells were exposed to inflammatory stimulation. These beneficial effects were diminished in the presence of the AMPK inhibitor compound C. mSMS positively regulated AMPK activity, and this action contributed to improving insulin PI3K signaling by the beneficial regulation of IRS-1 function through inhibition of inflammation in adipocytes.

ILs-HPLC simultanesous determination of five alkaloids in phellodenddri chinensis cortex / 中国中药杂志

A RP-HPLC method was established for simultaneous determination of phellodendrine hydrochloride (PH1), magnoflorine hydrochloride (MH), jatrorrhizine hydrochloride (JH), palmatine hydrochloride (PH2) and berberine hydrochloride (BH) in Phellodendri Chinensis Cortex by using ionic liquids as mobile phase additives. The separation was performed on a Kromasil C18 (4.6 mm x 250 mm, 5 μm) coupled with ultraviolet (UV) detection. The effect of extraction solvent, detection wavelength, length of alkyl chain on different imidazolium ionic liquids and concentration of ionic liquids on the separation and determination of alkaloids were investigated. Ionic liquid, [BMIm] BF4, can obviously improve the resolution and peak shape. This ILs-HPLC method is simple, rapid, and reliable, which can be used for determination of alkaloids in Phellodenddri Chinensis Cortex.

Research on allelopathic effects of phellamurine / 中国中药杂志

Conducted research on new allelochemicals phellamurine extracted from deciduous of Phellodendron amurense, which worked in allelopathy effect to seed germination and growth process of P. amurense and P. chinense in order to interpret the causes of rare seedlings of wild populations of P. amurense. Extracted and separated phellamurine from P. amurense deciduous, and treated the seeds of P. amurense and P. chinense in after-ripening stage and germination stage with different concentrations of phellamurine solution, then detection of the seed germination rate, germination index, seedling height, root length and seed vigor index to evaluate the allelopathic effect of phellamurine. The results show that: phellamurine solution at 0.30 g x L(-1) produce significant inhibition to seed after-ripening of P. amurense, and also the solution at 0.15 g x L(-1) produce significant inhibition to seeds germination of P. amurense; the solution at 0.15 g x L(-1) produce significant inhibition to seeds after-ripening and seeds germination of P. chinense, inhibition intensity increased with the concentration and enhanced. For both species, the presence of phellamurine can lower the seed germination rate, extend the germination time, reduce the ability of seedlings to adapt to the environment, thus the phellamurine may be one of the causes of rare seedlings in the wild population of P. amurense.

Optimization of technology conditions from Ermiao Wan decoction by semi-bionic extraction method / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize the technology of Ermiao Wan decoction by SBE with uniform design.</p><p><b>METHOD</b>Uniform design was used to optimize the efficient processing conditions of the SBE for Ermiao Wan decoction according to the content of the berberine, total alkaloids, volatile oil and the yield of the total extract.</p><p><b>RESULT</b>The extract condition for Ermiao Wan decoction was set up as follow: the pH value of the water was 6.00, 6.50 and 8.7, respectively,and the extract times were 2, 1 and 1 h,respectively.</p><p><b>CONCLUSION</b>The optimized extraction condition of the SBE for Ermiao Wan decoction is reasonable.</p>

Study on compatibility extraction technology of Rhei Radix et Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the optimum compatibility extraction technology of Rhei Radix et Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex.</p><p><b>METHOD</b>The drugs were combined respectively firstly, then use HPLC to determinate extraction rates of evaluating indicators including emodin, chrysophano, baicalin and berberine hydrochloride. Orthogonal experiment was used to detect the effect on concentration and volume of ethanol, times of extraction.</p><p><b>RESULT</b>The extraction rates of evaluating indicators were relatively high in combined extraction of Rhei Radix et Rhizoma with Scutellariae Radix and single extraction of Phellodendri Chinensis Cortex. The optimized combined extraction were 12 times more than the volume of 60% ethanol solution, by boiling for 3 times, each for 1.0 hour, and that of single extraction were 10 times more than the volume of 60% ethanol solution, by boiling for 3 times, each for 1.0 hour.</p><p><b>CONCLUSION</b>The optimized compatibility extraction technology is reasonable, stable and practical.</p>

Phellodendron amurense and Its Major Alkaloid Compound, Berberine Ameliorates Scopolamine-Induced Neuronal Impairment and Memory Dysfunction in Rats

We examine whether Phellodendron amurense (PA) and its major alkaloid compound, berberine (BER), improved memory defects caused by administering scopolamine in rats. Effects of PA and BER on the acetylcholinergic system and pro-inflammatory cytokines in the hippocampus were also investigated. Male rats were administered daily doses for 14 days of PA (100 and 200 mg/kg, i.p.) and BER (20 mg/kg, i.p.) 30 min before scopolamine injection (2 mg/kg, i.p.). Daily administration of PA and BER improved memory impairment as measured by the passive avoidance test and reduced the escape latency for finding the platform in the Morris water maze test. Administration of PA and BER significantly alleviated memory-associated decreases in cholinergic immunoreactivity and restored brain-derived neurotrophic factor and cAMP-response element-binding protein mRNA expression in the hippocampus. PA and BER also decreased significantly the expression of proinflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha and cyclooxygenase-2 mRNA in the hippocampus. These results demonstrated that PA and BER had significant neuroprotective effects against neuronal impairment and memory dysfunction caused by scopolamine in rats. These results suggest that PA and BER may be useful as therapeutic agents for improving cognitive functioning by stimulating cholinergic enzyme activity and alleviating inflammatory responses.

Study on procedure of seed quality testing and seed grading scale of Phellodendron amurense / 中国中药杂志

<p><b>OBJECTIVE</b>To study the procedure of seed quality testing and seed grading scale of Phellodendron amurense.</p><p><b>METHOD</b>Seed quality testing methods were developed, which included the test of sampling, seed purity, weight per 1 000 seeds, seed moisture, seed viability and germination rate. The related data from 62 cases of seed specimens of P. amurense were analyzed by cluster analysis.</p><p><b>RESULT AND CONCLUSION</b>The seed quality test procedure was developed, and the seed quality grading scale was formulated.</p>

Investigation on pattern and methods of quality control for Chinese materia medica based on dao-di herbs and bioassay - bioassay for Coptis chinensis / 药学学报

Establishment of bioassay methods is the technical issues to be faced with in the bioassay of Chinese materia medica. Taking the bioassay of Coptis chinensis Franch. as an example, the establishment process and application of the bioassay methods (including bio-potency and bio-activity fingerprint) were explained from the aspects of methodology, principle of selection, experimental design, method confirmation and data analysis. The common technologies were extracted and formed with the above aspects, so as to provide technical support for constructing pattern and method of the quality control for Chinese materia medica based on the dao-di herbs and bioassay.

Study on allelopathy effect of pericarp extract of Phellodendron amurense / 中国中药杂志

Through the study of allelopathy of the pericarp of Phellodendron amurense, the role of self-regeneration barriers was investigated in order to find ways and means for the protection of wild populations of P. amurense. Solution preparation: soaked pericarp of P. amurense in distilled water at 4 degrees C to get solution A, and reflux extraction of pericarp with distilled water at 100 degrees C to get solution B. Both of the solution A and solution B were used in the experiment of seed germination and seedling growth with the seeds of cabbage and wheat. The results showed that 20 g x L(-1) concentration of solution A and solution B inhibited significantly seed germination of cabbage and wheat, while 100 g x L(-1) concentration of solution A even completely inhibited the seed germination of wheat. 20 g x L(-1) concentration of solution A significantly inhibited the cabbage and wheat seedling growth, completely inhibited the root growth of cabbage, while 100 g x L(-1) concentrations of solution A completely inhibited seedling growth of cabbage and wheat. Comparing to solution A, the intensity of solution B are diminished on seed germination and seedling growth. It is concluded that the allelopathy of pericarp of P. amurense is multi-material role in the results, some of allelochemicals are easily degradable when exposed to heat. Overall, the allelopathy of pericarp of P. amurense can affect the seed germination and seedling growth. It is supposed that allelochemicals existed in the pericarp of P. amurense is one of the reason leading to difficulties in self-regeneration of its population.

Simultaneous determination of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex by RP-HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To develop a HPLC method to determine the content of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex simultaneously. MEHTOD: The separations were carried out at 25 degrees C on a Phenomenex Gemini C18 column (4.6 mm x 250 mm, 5 microm) eluted with acetonitril and water containing 0.1% phosphoric acid in gradient mode. The flow rate was 1.0 mL x min(-1), detection wavelengthes were 345 nm for jatrorrhizine , palmatine, berberine and 210 nm for obacunone.</p><p><b>RESULT</b>The average recoveries of jatrorrhizine, palmatine, berberine, and obacunone were 98.94%, 101.17%, 96.22% and 98.90%, respectively.</p><p><b>CONCLUSION</b>The method is simple, accurate and repeatable, and can be used in content determination of jatrorrhizine, palmatine, berberine, and obacunone in Phellodendri Amurensis Cortex.</p>

Optimization of combination modes of medical materials in extract of Huanglian Jiedu decoction by semi-bionic extraction / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize the combination modes of medical materials in extract of Huanglian Jiedu decoction.</p><p><b>METHOD</b>Divided the medical materials of Huanglian Jiedu decotion into 15 groups and extracted by semi-bionic estraction (SBE), the contents of berberine, baicalin, geniposide, total alkaloid, total flavonoid, total integral calculus area of HPLC and extract of 1 000 were determined, standardized, weighted and evaluated, respectively.</p><p><b>RESULT</b>The maximum evaluated value Y for the extracting technique was AD + BC.</p><p><b>CONCLUSION</b>The optimal combination mode of the perscription is that Rhizoma Coptidis and Fructus Gardeniae, Radix Scutellariae and Cortex Phellodendri Amurensis are decocted together respectively then the two decoctions are merged.</p>

Water-soluble chemical constituents from fruits of Phellodendron chinense var. glabriusculum / 中国中药杂志

<p><b>OBJECTIVE</b>To study the water-soluble chemical constituents from the fruits of Phellodendron chinense var. glabriusculum.</p><p><b>METHOD</b>Chemical constituents were purified by chromatographic methods and identified by spectroscopic analysis.</p><p><b>RESULT</b>Eight known compounds were obtained, whose structures were elucidated as sinapyl 9-O-beta-D-glucopyranoside (1), betulalbuside A (2), arbutin (3), adenosine (4), 3,5-dihydroxyphenethyl alcohol 3-O-beta-D-glucopyranoside (5), orcinol-3-O-beta-D-glucopyranoside (6), N-trans-p-coumaroyloctopamine (7), berberine (8) respectively.</p><p><b>CONCLUSION</b>Compounds 1-7 were obtained from Phellodendron chinense var. glabriusculum for the first time.</p>

Compatibility chemistry of acid-alkaline pair medicine of Dahuang and Huangbai in Dahuang Xiaoshi decoction / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the change pattern of alkaline and acid components of Dahuang and Huangbai amoung different combinations in Dahuang Xiaoshi decotion.</p><p><b>METHOD</b>The contents of anthraquinones and berberine were determined by HPLC in samples of Dahuang extracts, Huangbai extracts, Dahuang and Huangbai pair medicine extracts, and extracts of whole recipe.</p><p><b>RESULT</b>The contents of anthraquinones and berberine were decreased with the changes of combinations: from component medicinal material alone to pair medicines, and to whole recipe. the change pattern was more apparent in water decoctions than in ethanol extracts.</p><p><b>CONCLUSION</b>The contents of alkaline and acid components are changed with different extract methods and different combinations.</p>

Construction and analysis of subtractive cDNA library of Phellodendron amurense under drought stress / 生物工程学报

With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300-800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.

Ecological evaluation of suitable area for production of Phellodendron amurense based on TCMGIS- I / 中国中药杂志

The TCMGIS- I (Suitability evaluation geographic information system of traditional Chinese medicine producing area ) was developed and used to analyze the appropriate producing area of Phellodendron amurense in China. Taoshan county of Heilongjiang province, one of the main producing areas of the geo-authentic crude drug, was selected as the analytical basal place. The results show that besides traditional producing area in Taoshan county of Heilongjiang province, 131 counties of the provinces of Heilongjiang, Jilin, Liaoning and Innermongolia are also alternative areas according to the eco-soil condition that the medicinal plants needed, and the sum area are 567 989. 3 m2, the results also indicate some condign regions in Innermongolia municipality. The system is much valuable to the division of adaptive area, introduction and acclimatization of medicinal materials.

Genetic diversity evaluation of ex-situ conservation population of Phellodendron amurense detected by AFLP markers / 中国中药杂志

<p><b>OBJECTIVE</b>To evaluate the genetic diversity from ex-situ conservation population of Phellodendron amurense.</p><p><b>METHOD</b>Genetic diversity in 67 individuals from ex-situ conservation population of P. amurense were assessed using AFLP technique by eight fluorescent labeled primer groups.</p><p><b>RESULT</b>The eight primer combinations generated a total of 1,574 bands, of which 1,354 were polymorphic. As for P. amurense at species level, the percentage of polymorphic loci (PPL) was 86.02, the effective number of alleles per locus (Ne) was 1.4275 ,the Nei's gene diversity (H) was 0.2312, and the Shannon's information index (I) was 0.3973. At the population level, the estimates PPL=41.55, H=0.1400, Ne=0.2376 and I=0.2113.</p><p><b>CONCLUSION</b>The effect of present ex-situ conservation was preliminarily assessed.</p>

Hairy roots induction of Phellodendron chinense and production of its active constituents / 中国中药杂志

<p><b>OBJECTIVE</b>To introduce the hairy roots of Phellodendron chinense and determine the content of its active constituents.</p><p><b>METHOD</b>Transformed hairy roots of P. chinense were obtained by the transformation of Agrobacterium rhizogenes A4, R1600, ATCC15834 and R1000.</p><p><b>RESULT AND CONCLUSION</b>It was clearly demonstrated that T-DNA of A. rhizogenes Ri plasmid was integrated into the cells of hairy roots by PCR. The content of berberine hydrochlodride, which was determined by HPLC, was higher in hairy roots than that in the axenic plantet and callus.</p>

Qualitative and quantitative determination of the main components of huanglianjiedu decoction by HPLC-UV/MS / 药学学报

<p><b>AIM</b>To establish a comprehensive HPLC analytical method of Huanglianjiedu decoction.</p><p><b>METHODS</b>This study was performed by HPLC-UV/MS to identify the chemical constituents of the whole and individual herbs of the "Huanglianjiedu decoction". Zorbax Extend C18 (150 mm x 4. 6 mm ID, 5 microm) column was used; the mobile phase was composed of acetonitrile (A) and water (B, with 0.5% acetic acid) with gradient elution; the flow rate was 1.0 mL x min(-1) and the column temperature was setup at 25 degrees C. The detection wavelength was 254 nm.</p><p><b>RESULTS</b>The chromatogram of Huanglianjiedu decoction showed 21 main peaks. Peaks 1, 2, 5 and 18 were from Gardenia jasminoides Ellis, Peaks 8, 13, 14, 15, 16, 17, 19 and 21 from Scutellaria baicalensis Georgi. While 10 from Coptis chinensis Franch and 20 from Phellodendron amurense Rupr., Peaks 3, 4, 6, 9, 11 and 12 came from them together. Peak 7 presented in the chromatograms of the herbs except Gardenia jasminoides Ellis. By comparison of the retention time, the on-line UV spectra and MS spectra, 11 peaks were identified as 5 (geniposide), 9 (jatrorrhizine), 10 (coptisine), 11 (palmatine), 12 (berberine), 13 (baicalin), 15 (oroxin A), 17 (wogonoside), 19 (baicalein), 20 (obaculactone), 21 (wogonin), then eight of them were quantified by HPLC-UV.</p><p><b>CONCLUSION</b>The method could represent the characteristics of Huanglianjiedu decoction, and it could be used to evaluate the quality and quantity of Huanglianjiedu decoction. It distinguished between Coptis chinensis Franch and Phellodendron amurense Rupr. by HPLC for the first time.</p>