Quality Evaluation of Leontopodium franchetii Beauv. from Different Localities Based on Moisture,Ash and Extract Contents and Thin-layer Chromatography

[Objectives] The aim was to determine the moisture,ash and extract contents in Leontopodium franchetii Beauv. from different localities. [Methods] The contents of moisture,total ash,acid-insoluble ash and extract in L. franchetii from different localities were determined according to the methods described in Chinese Pharmacopoeia( 2015 Edition). [Results] In the 10 samples,the contents of moisture were all less than or equal to 15. 0%; the contents of total ash were all below 12. 0%; and the contents of acid-insoluble ash were all less than 3. 0%. The contents of water-soluble extract( by cold-soaked extraction method) in the samples were all below 12. 0% except those from Hongyuan Prairie of Sichuan Province and Xinglong Mountain in Lanzhou City,Gansu Province. [Conclusions]The contents of the four indicators measured in this experiment varied by small margins,indicating that the quality was relatively stable. This study provides theoretical data for the revision of the quality standards for L. franchetii.

Separation of Amino Acids Based on Thin-Layer Chromatography by a Novel Quinazoline Based Anti-Microbial Agent

A newly designed quinazoline based compound, 6-pyridin-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c] quinazoline (PDBIQ) has shown the ability for the easy detection of nineteen amino acids on thin-layer chromatography plates as a spray reagent. This new reagent enabled to produce various distinguishable colors with amino acids with different RF values. The detection limits and the binding ability of PDBIQ with amino acids have been calculated. PDBIQ is also able to detect aminoacids from hydrolised seed protein. The title compound also exhibited profound inhibitory action against some gm (+ve) and gm (-ve) bacterial organisms. This paper deals with synthesis, spectroscopic application and biological evaluation of the organic moity.

ANALYSIS OF HUMAN PLACENTA BY ^(31)P NUCLEAR MAGNETIC RESONANCE AND THIN-LAYER CHROMATOGRAPHY SCANNING COMBINED WITH THE CORRECTIVE METHOD OF ABSORBANCE PROPORTIONAL COEFFICIENT

Five phospholipids in human placenta were determined by phosphorus 31 nuclear magnetic resonance(^(31)P NMR)spectroscopy and thin-layer chromatography(TLC) scanning combined with the corrective method of absorbance proportional coefficient. The NMR spectrometer used this investigation was a Bruker AM-500 spectrometer operating at 202.4 MHz for ^(31)P chemical shifts are relative to 85% phosphoric acid. TIC was carried out by silica gel H plate developed in chloroform-methanol-glacial acetic acid-ethanol-water(25:4:6:2:0.5),with Vaskovsky reagent as colour -developing agent of phospholipids.

Bio-screening and quantification of methyl paraben in vinegar and coconut juice separated by HPTLC

As a widely used food preservative,methyl paraben was experimentally evidenced with serious hormonelike adverse effects.Herein,a high performance thin-layer chromatography platformed bioluminescent bioautography and image analysis for the selective quantification and confirmation of methyl paraben was proposed and validated in vinegar and coconut juice.First,the detectability of the bioautography to the analyte on different layer materials was estimated,revealing that normal silica gel was the best choice.After that,the liquid of sample extract and working solution were separated to overcome the background noises due to co-extracted matrices.The separation result was then coupled to the optimized bioautography,enabling instant and straightforward screening of the targeted conpound.For accurate quantification,bioluninescent inhibition pattern caused by the analyte was processed by image analysis,giving useful sensitivity(LOD>16 mg/kg),precision(RSD<10.1%)and accuracy(spike-recovery rate 76.9%-112.2%).Finally,the suspected result was confirmed by determining its MS fingerprint,further strengthening the reliability of screening.

TLC Identification and Extraction Process of Rubiasin-1-methyl Ether from Yao Medicine Chuanlianzhu

[Objectives]To establish a thin-layer chromatography(TLC)method for the determination of rubiadin-1-methyl ether in Yao Medicine Chuanlianzhu(Damnacanthus giganteus).[Methods]A silica gel G thin-layer plate was adopted for TLC.Petroleum ether(60-90℃)-chloroform-methanol-water(7:15:3:1)was used as the developing solvent and inspected under ultraviolet lamp(365 nm).The content was determined by Inertsil ODS-3 C 18 column(4.60 mm×250 mm,5μm),mobile phase:acetonitrile-0.2%phosphoric acid gradient elution,detection wavelength 277 nm,flow rate 1.0 mL/min,column temperature 30℃,injection volume 10μL.[Results]The spots of 10 Chuanlianzhu samples from different origins showed the same color at the same position as the control,and the spots were clear and specific.The injection volume of rubiadin-1-methyl ether showed a good linear relationship in the range of 2.90-145μg(R=0.9996).The average recovery rate of rubiadin-1-methyl ether in the low,medium and high dose groups of Yao Medicine Chuanlianzhu was 98.72%,and RSD=1.78%.[Conclusions]This method can effectively identify Yao Medicine Chuanlianzhu medicinal materials and accurately determine the content of rubiadin-1-methyl ether in the medicinal materials.It provides a scientific basis for the development and utilization of Yao Medicine Chuanlianzhu medicinal resources.

Separation of liquefied product of Salix psammophila by column chromatography and structure analysis of its components

The liquefied product of Salixpsammophila wood was separated by thin-layer chromatography （TLC） and column chromatography, and its structure was identified by nuclear magnetic resonance （NMR） spectra in our study. The separation result indicates that the sample of liquefied S. psammophila contained at least two categories of components. The structure of the main components was guaiacyl C-1, C-2 of the hydroxyphenyl propane, i.e., the aromatic nucleus protons of lignin. Degradation and polycondensation reactions occurred when the S. psammophila wood was liquefied in phenol. Polycondensation reactions occurred among the depolymerization products from cellulose, the aromatic depolymerization products from lignin and the products of the displacement reactions between phenoxide ion and cellulose.

Comparison of liquid-liquid extraction-thin layer chromatography with solid-phase extraction-high-performance thin layer chromatography in detection of urinary morphine

Liquid-liquid extraction-thin layer chromatography （LLE-TLC） has been a common and routine combined method for detection of drugs in biological materials. Solid-phase extraction （SPE） is gradually replacing the tra- ditional LLE method. High performance thin layer chromatography （HPTLC） has several advantages over TLC. The present work studied the higher efficiency of a new SPE-HPTLC method over that of a routine LLE-TLC method, in extraction and detection of urinary morphine. Fifty-eight urine samples, primarily identified as mor- phine-positive samples by a strip test, ＇were re-screened by LLE-TLC and SPE-HPTLC. The results of LLE-TLC and SPE-HPTLC were then compared with each other. The results showed that the SPE-HPTLC detected 74% of total samples as morphine-positive samples whereas the LLE-TLC detected 48% of the same samples. We further discussed the effect of codeine abuse on TLC analysis of urinary morphine. Regarding the importance of morphine detection in urine, the present combined SPE-HPTLC method is suggested as a replacement method for detection of urinary morphine by many reference laboratories.

Chromatographic fingerprinting and free-radical scavenging activity of ethanol extracts of Muntingia calabura L. leaves and stems

Objective: To determine the thin-layer chromatography(TLC) fingerprint profiles and to evaluate the in vitro antioxidant activity of ethanol extracts of Muntingia calabura(M. calabura) leaves and stems.Methods: The leaves and stems were extracted using ethanol as solvent. The TLC separation of the phytochemical constituents of the leaf and ethanol extracts was carried out in ethyl acetate: n-hexane and chloroform: ethyl acetate mobile phase systems.Distinct spots were visualized under visible light, UV 254 nm, UV 366 nm and after spraying with vanillin-sulfuric acid. The 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging assay was used to evaluate the antioxidant activity of the extracts.Results: Both the leaf and stem ethanol extracts at 4 mg/mL exhibited 2,2-diphenyl-1-picrylhydrazyl inhibition of more than 90%, relative to gallic acid. The results of TLC showed that the degree of resolution between the constituent spots was comparable between the two mobile phase systems using the different visualization wavelengths. Under the 254 nm visualization, few spots were observed in leaf and stem extracts. Visualization at 366 nm yielded the greatest number of observable spots of various colors in both leaf and stem extracts. More spots were visualized upon post-derivatization with vanillinsulfuric acid in the TLC chromatograms using chloroform: ethyl acetate mobile phase,compared to those in ethyl acetate: n-hexane mobile phase.Conclusions: M. calabura exhibited very high antioxidant activity in its leaves and stems ethanol extracts, both of which are used in traditional medicine. The TLC results demonstrated the presence of diverse secondary metabolites in the leaf and stem ethanol extracts, indicating that the antioxidant activity, including other bioactivities may be attributed to these phytochemical constituents. This paper has reported for the first time the TLC fingerprinting of M. calabura using visible light, UV 254 nm, UV 366 and postderivatization with vanillin-spray to visualize separate spots on TLC plates.

Assessment of Poultry Feed Contamination Level by Aflatoxin B1: Quantification by Two Chromatographic Analysis Methods

Aflatoxin B1 is a mycotoxin that can contaminate a wide feedstuffs variety. Ingestion of contaminated feed by poultry can lead to impaired health and zootechnical performances but also a human diet safety problem related to residues presence in animal origin products. Aflatoxin B1 contamination of poultry feed samples marketed in Dakar city and in peri-urban areas (Gorom, Sangalkam) was studied. A total of 15 samples were collected from Dakar city markets as well as from poultry farms in Gorom and Sangalkam areas. Aflatoxin B1 quantification was performed by high performance liquid chromatography and thin-layer chromatography. HPLC results showed that all samples were contaminated with levels ranging from 0.15 to 22 ppb, 0.099 to 2.05 ppb and 0.099 to 4.95 ppb respectively for Gorom, Sangalkam and Dakar. Only the finishing feed from Gorom had an aflatoxin B1 level above the maximum limit set by regulations. TLC is a suitable method for aflatoxins detection. However, it was associated with overestimation for aflatoxin B1 quantification. Results suggest that poultry feed represents a real source of human diet contamination. In addition, HPLC remains the most reliable quantification technique for quality control.

Analysis of the Nucleoside Content of Cordyceps sinensis Using the Stepwise Gradient Elution Technique of Thin-Layer Chromatography

Nucleoside is the main class of active components in Cordyceps sinensis. Thin-layer chromatography (TLC) is one of the most commonly used methods in pharmacopoeias for analyzing chemical components of herbal medicine. Since the isocratic elution method cannot be applied successfully in TLC analysis for separating all the nucleoside components, the stepwise gradient elution has been developed in this work to separate eight nucleoside standards with success. In this way, quantitative analyses of the samples of Cordyceps sinensis were achieved via the pro-posed TLC procedure coupled with the scanning densitometric techniques of CAMAG and TLCQA methods for qualitative and quantitative analysis.

Simple and robust differentiation of Ganoderma species by high performance thin-layer chromatography coupled with single quadrupole mass spectrometry QDa

In this study,a high performance thin-layer chromatography/single quadrupole mass spectrometry QDa(HPTLC-QDa)method for robust authentication of Ganoderma lucidum,a popular and valuable herbal medicine,has been developed.This method is simple and practical,which allows direct generation of characteristic mass spectra from the HPTLC plates automatically with the application of in situ solvent desorption interface.The HPTLC silica gel plates were developed with toluene-ethyl formate-formic acid(5:5:0.2,V/V)and all bands were transferred to QDa system directly in situ using 80%methanol with 0.1%formic acid as desorption solvent.The acquired HPTLC-QDa spectra showed that luminous yellow band b3,containing ganoderic acid B/G/H and ganodeneric acid B,the major active components of Ganoderma,could be found only in G.lucidum and G.lucidum(Antler-shaped),but not in G.sinense and G.applanatum.Moreover,bands b13 and b14 with m/z 475/477 and m/z 475/491/495,respectively,could be detected in G.lucidum(Antler-shaped),but not in G.lucidum,thus allowing simple and robust authentication of G.lucidum with confused species.This method is proved to be simple,practical and reproducible,which can be extended to analyze other herbal medicines.

Differentiation of Belamcandae Rhizoma and Iridis Tectori Rhizoma by Thin-Layer Chromatography and High-Performance Liquid Chromatography

Objective:Belamcandae Rhizoma and Iridis Tectori Rhizoma are easily confused with each other.The main objective of this study is to distinguish them using chemical analysis.Materials and Methods:Thin-layer chromatography(TLC)and high-performance liquid chromatography(HPLC)fingerprint methods were established to compare the chemical profile,while HPLC quantitation was used to determine the contents of three isoflavones in thirty batches of Belamcandae Rhizoma and Iridis Tectori Rhizoma samples.Results:The two herbs could be distinguished by TLC using acetic acid-n hexane-ethyl acetate(1:90:80 v/v/v)as the mobile phase,according to the fluorescent band under 366 nm at R_(f) 0.2.In total,12 compounds were identified in the 24-min HPLC fingerprint.The similarity coefficient between the two herbs was 0.54±0.01.Mangiferin(1),tectoridin(2),iridin(3),irigenin(5),irisflorentin(6),and iristectorin A(9)were the main peaks in Belamcandae Rhizoma,while tectoridin(2)and tectorigenin(4)were the major peaks in Iridis Tectori Rhizoma.The contents of 2 in Iridis Tectori Rhizoma(2.50±0.20%)were 8.93 times higher than that of Belamcandae Rhizoma(0.28±0.08%),while the ones of 5 and 6 were slightly lower in Iridis Tectori Rhizoma.Conclusions:The study established fast and effective methods to distinguish Belamcandae Rhizoma from Iridis Tectori Rhizoma.

Methods for in vitro evaluating antimicrobial activity： A review

In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cy- tofluorometric and bioluminescent methods are not widely used because they require specified equip- ment and further evaluation for reproducibility and standardization, even if they can provide rapid re- sults of the antimicrobial agent＇s effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limita- tions are reported.

In vitro Safety Evaluation and Anticlastogenic Effect of BacoMind^(TM) on Human Lymphocytes

Objective BacoMind^TM （BM） is a standardized extract of Bacopa monnieri, which belongs to the family Scrophulariaceae and is a creeping annual plant found throughout the Indian subcontinent. It has been used by Ayurvedic medicinal practitioners in India for almost 3000 years and is classified as a medharasayana, a substance which improves memory and intellect. With the widespread traditional use as well as scientific validation of Bacopa monnieri for nootropic activity, a bioactive-rich unique phytochemical composition-BacoMind^TM was developed from B. monnieri for use as a cognition and memory enhancing agent. The present study aimed to investigate the in vitro toxicity of this formulation of BacoMind^TM on human lymphocytes and to rule out its possible contribution to mutagenicity. Methods In the present investigation the active ingredients present in BM were identified and quantified by high performance liquid chromatography （HPLC） and high performance thin-layer chromatography （HPTLC）. Antioxidant and anticlastogenic properties of BM were studied in vitro with and without metabolic activation. Doses of BM were chosen on the basis of mitotic index （MI） and cytokinesis-block proliferation index （CBPI）. Clastogenicity assays were performed at 31.2 μg/mL, 62.5 μg/mL, and 125 μg/mL, while the Salmonella reverse mutation assay （Ames test） was performed at doses of 61.72, 185.18, 555.55, 1666.67, and 5000.00 μg/plate. Results HPLC and HPTLC analysis of BM revealed the presence of bacoside A3. bacopaside Ⅰ, bacopaside Ⅱ, jujubogenin isomer of bacopasaponin C, bacosine, luteolin, apigenin, bacosine, and β-sitosterol D glucoside. BM demonstrated significant antioxidant activity. The number of chromosomal aberrations and the frequency of micronuclei induced by BM were not statistically significant up to a dose of 62.5 μg/mL. A subsequent dose of 125 μg/mL prior to metabolic activation induced mild clastogenicity, but it was found to be biologically insignificant as this effect was not seen post metabolic activation. BM also demonstrated a dose-dependent protection against the clastogens used in this study using the above tests for clastogenicity. Maximum protection was observed in presence of metabolic activation. Moreover, BM demonstrated no mutagenic effect on the tested strains, as observed in the Ames test. Conclusion BM protected human lymphocytes against various clastogens. BM also exhibited high antioxidant activity which might be responsible for the observed protective effects against the clastogens since the used clastogens are known to induce their clastogenic effects via production of oxidative radicals.

Preparation of Red Component in Secondary Metabolites of Recombinant Hansenula anomala N6076 and Its GC-MS Detection

[ Objective] This study aimed to investigate the preparation of red component in secondary metabolites of recombinant Hansenula anomala strain N6076 and its GC-MS detection. [ Method] Thin-layer chromatography method was applied for large-scale preparation of red component in the secondary metabolites of re- combinant H. anomala strain N6076. The red component was dissolved in anhydrous ethanol for GC-MS detection and chemical structure comparison in the data- base to identify its type. [ Resultl The red component is preliminarily identified as a quinane compound, while no compound with exactly the same structure as the red component has been found in WILEY, Nist and Nbs compound libraries. [ Conclusion] This study laid the foundation for further NMR structure identification of the obtained red component and investigation of the relationship between its structure and biolo#cal effects.

Pharmacognosy Identification of Xiaohuangcao (Dendrobium loddigesi Rolfe)

[Objectives]Pharmacognosy identification was performed on Xiaohuangcao ( Dendrobium loddigesi Rolfe).[Methods]The medicinal materials were identified through original plants,characters,microscopic structure and thin-layer identification characteristics.[Results]D.loddigesi has obvious plant morphology,characters,microscopic structure and thin-layer identification characteristics.The stem of Xiaohuangcao is slender and cylindrical,and the surface is golden yellow;and the fiber bundles outside the vascular bundles are crescent-shaped or semi-circular in the stem transection.For the powder,crystal fiber can be observed;the vascular bundles are embedded with siliceous block cells;and there are more starch grains.In the thin layer chromatography,petroleum ether-ethyl acetate-butanone-glacial acetic acid (8.5∶ 3.5∶ 1.5∶ 5 d) was used as a developing solvent,and 10% sulfuric acid ethanol solution was used as a color developing agent.[Conclusions]The research results provide reference for the application of the medicinal material and the formulation of its related quality standards.

Qualitative Identification and Content Determination of Psoralen in Ficus Pandurata Hance

[Objectives]This study aimed to establish a qualitative identification and content determination method for psoralen in Ficus pandurata Hance and compare the psoralen contents in roots,stems and leaves of F.pandurate Hance and Ficus pandurata Hance var.holophylla Migo.[Methods]Thin-layer chromatography(TLC)was used for qualitative identification,and the content of psoralen was determined by high-performance liquid chromatography.The chromatographic conditions were as follows:column,Agilent ZORBAX Eclipse Plus C18(250 mm×4.6 mm,5μm);mobile phase,methanol-water(55∶45);flow rate,1 mL/min;detection wavelength,246 nm;column temperature,30℃;and injection volume,10μL.[Results]The TLC chromatogram of F.pandurate Hance showed clear spots and good separation.The concentration of psoralen detected in the range of 2.06-41.20μg/mL had a good linear relationship with the peak area(r=0.9999).The RSD values of the precision,stability and reproducibility tests were all less than 2%.The average recovery rate was 100.2%(RSD=1.13%,n=6).[Conclusions]The established method is simple,easy,accurate and reproducible.It can be used for quality control of F.pandurate Hance.

Analysis on Quality Control of Argyreia acuta Lour

[Objectives]To establish a method for caffeic acid content determination and thin-layer chromatography identification of the Zhuang medicine Argyreia acuta Lour.[Methods]Silica gel GF254(thin-layer plate),toluene-formic acid-ethyl acetate(V∶V∶V=5∶1.2∶3)(developing agent)and 365 nm ultraviolet light were used for thin-layer chromatography identification.Under the chromatographic conditions of column of Thermo SCIENTIFIC Hypersil C18(5μm,4.60 mm×250 mm),mobile phase of methanol-0.1%phosphoric acid(23∶77),detection wavelength of 320 nm,column temperature of 30℃,sample size of 10μL and flow rate of 1.0 mL/min,the content of caffeic acid in A.acuta Lour was determined.[Results]Caffeic acid can be detected in thin layer chromatography,with strong specificity and clear spots.When the sample size of caffeic acid is within the range of 0.0151-0.4530μg(R^(2)=0.9999),the content of caffeic acid showed a relatively good linear relationship with the peak area.The average recovery rate of A.acuta Lour was 99.94%(RSD=2.68%),97.56%(RSD=1.57%)and 99.79%(RSD=2.05%),respectively.[Conclusions]This method can effectively identify A.acuta Lour,and can accurately determine the content of caffeic acid in A.acuta Lour.It has characteristics of high accuracy,high precision,good color rendering stability,good reproducibility and fast analysis speed.

Quality Standard of Zijinbiao

[Objectives]To establish the quality standard for Zijinbiao.[Methods]Microscopic identification,physical and chemical identification,and thin-layer chromatography(TLC)were used to qualitatively identify Zijinbiao.The moisture,total ash,acid-insoluble ash,and alcohol-soluble extract content were determined.The content of Plumbagin was determined by high performance liquid chromatography(HPLC).[Results]Microscopic identification,physical and chemical identification and thin layer identification features were remarkable.The moisture,total ash,acid-insoluble ash,and extract content of the 15 batches of samples were 7.49%-11.84%,2.43%-5.81%,0.59%-3.18%and 13.80%-20.45%,respectively.The linear equation of Plumbagin was Y=38094X,R^(2)=0.9996.Plumbagin had a good linear relationship in the range of 0.01-0.53 mg/mL.[Conclusions]This method is specific and reproducible,and can be used for quality control of Zijinbiao.

Quality Standard of Haleniae Herba

[Objectives]The research aimed to establish qualitative and quantitative detection methods of Haleniae Herba,and provide basis for improving its quality standard.[Methods]Using thin-layer chromatography(TLC),qualitative identification of Haleniae Herba was conducted.Using high-performance liquid chromatography,luteolin content of Haleniae Herba was determined.[Results]TLC identification method had good specificity,with clear spots and good degree of separation,and reference medicinal material of Haleniae Herba was prepared.Content determination results displayed that luteolin had good linear relationship in the range of 0.088-1.760μg.The RSD values of precision,repeatability,stability and sample recovery of the instrument met the requirements.[Conclusions]The established method is simple and reproducible,and can be used for the quality control of Haleniae Herba.