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.

Accumulation of main alkaloids composition in growth process of different parts of Phellodendron chinense / 中草药

Objective: To analyze the content variation of phellodendrine and berberine from Phellodendron chinense in different years, different seasons, and different parts, and to provide the basis for increasing the planting benefit of Chinese herbal medicine. Methods: High performance liquid chromatography (HPLC) analysis method was used to determine the contents. Results: The contents of phellodendrine and berberine from different parts of P. chinense showed as the following order: root barks > barks > roots. The contents of the two kinds of alkaloids changed each year. In first three years, the content change trend was obvious, and the content in barks was close to 5% in the third year. The young P. chinense had development value. After the third year, the contents of the two kinds of alkaloids changed slowly. The contents of phellodendrine and berberine in October were higher. Conclusion: The metabolic accumulation rules of berberine and phellodendrine are summarized, in order to provide the basis for the sustainable utilization of P. chinensis resources.

Studies on biopharmacy of ultra-fine particles of Atractylodes lancea, Phellodendron chinense and a pill composed of the two drugs Ⅰ. Characterization by scanning electron microscopy / 中草药

To characterize the ultra-fine particles of Atractylodes lancea (Thunb.) DC., Phellodendron chinense Schneid and the ERMIAO PILL*, a compounded preparation of the above two drugs, for the purpose to observe a deeper view on their physico-chemical properties. Methods　By comparing their particle size, porosity and specific surface area with light and scanning electron microscope. Results　The size of the ultra-fine particles were more uniform in size, 90% of which were under 20 μm, their specific area were increased by 60% ～ 190% and bulk density were around 0.42 g/cm3, and the great majority of the plant cells were broken as compared with the conventional coarse powder. Conclusion　Both A. lancea and P. chinense together with their combined preparation became smaller in particle size, more uniformly distributed with increased specific surface area and broken cell wall.

Studies on biopharmacy of ultra-fine particles of Atractylodes lancea, Phellodendron chinense and a pill composed of the two drugs Ⅰ. Characterization by scanning electron microscopy / 中草药

Object To characterize the ultra fine particles of Atractylodes lancea (Thunb ) DC., Phellodendron chinense Schneid and the ERMIAO PILL *, a compounded preparation of the above two drugs, for the purpose to observe a deeper view on their physico chemical properties Methods By comparing their particle size, porosity and specific surface area with light and scanning electron microscope Results The size of the ultra fine particles were more uniform in size, 90% of which were under 20 ?m, their specific area were increased by 60% ～ 190% and bulk density were around 0 42 g/cm 3, and the great majority of the plant cells were broken as compared with the conventional coarse powder Conclusion Both A. lancea and P chinense together with their combined preparation became smaller in particle size, more uniformly distributed with increased specific surface area and broken cell wall

Study on extracting purified active components of Picria fel-terrae, Scutellaria baicalensis and Phellodendron chinense with macroporous resins / 中草药

Object To study the application of macroporous resins to the purified active components of Picria fel-terrae Lour., Scutellaria baicalensis Georgi, and Phellodendron chinense Schneid. Methods With the purified active components of Chinese materia medica (CMM) as a standard, we selected the suitable macroporous resins and studied the optimum technological parameters of the adsorption and elution. Results The suitable three types of macroporous resins which were used to the purified active components of CMM were HPD500 for P. fel-terrae., HPD300 for S. baicalensis, and HPD100 for P. chinense. In the absorption course, when the ratios between the medicinal materials amount and the volume of macroporous resins were 1.5 for P. fel-terrae, 0.5 for S. baicalensis and 1 for P. chinense, the purified active components of CMM appeared leaking, and were no more absorbed when the ratios were 11 for P. fel-terrae, 2 for S. baicalensis and 4.75 for P. chinense. In the elution course, the optimum alcohol concentrations were 50% for P. fel-terrae, 30% for S. baicalensis and 50% for P. chinense. Conclusion It is obviously different to refine the active components of CMM, while using the diverse types of macroporous resins.

Reversed-phase ion pair high performance liquid chromatography in determination of berberine and palmatine in Phellodendron chinense Schneid.and its granules / 第二军医大学学报

Objective:To determine berberine and palmatine in Phellodendron chinense Schneid.and its granules.Methods: The reversed-phase ion pair high performance liquid chromatography(RP-HPLC) was used and the validation of the method was tested.The chromatography condition was with Lichrospher C18 column(4.6 mm?250 mm,5 ?m), mobile phase was ACN∶25 mmol/L NaH 2PO 4∶25 mmol/L SDS(2∶1∶1),flow speed was 1.0 ml/min,detection wavelength was 345 nm,and temperature of column was 25℃,Phellodendron chinense Schneid.and its granules were extracted with methanol solution of hydrochloric acid.Results: The theoretical plate number of berberine and palmatine were 14 906 and 14 847,the resolution were 2.33 and 2.86,the tailing factor were 1.09 and 1.06,respectively; all the parameters were suitable for determination.The calibration curves were linear in the range of 40-500 ng,Y=698 278X-3 846,r=1.000(berberine) and 20-250 ng, Y= 536 632X- 7 738, r=0.999 9,r=0.999 4(palmatine).The intra-day and inter-day precision(RSD) at low,middle and high injection amount was all less than 2.5%(berberine) and 1.5%(palmatine).The stability(RSD) was 0.66%(berberine) and 0.70% (palmatine) in 48 h.The recurrence(RSD,n=5) was 0.11%(berberine) and 0.12%(palmatine).The limits of detection was 2.0 ng(berberine) and 1.0 ng(palmatine).The recoveries were 100.4% (RSD=0.12%,n=3) for berberine and 99.80%(RSD=0.22%,n=3) for palmatine.The contents of berberine and palmatine in 3 batch of Phellodendron chinense Schneid.and 5 batch of its granules were determined.Conclusion: Our method can be used for determination of berberine and palmatine in Phellodendron chinense Schneid.and its granules, which is simple and reliable.