Identification of Dyes in Coptic Textiles from the Museum of Faculty of Archaeology, Cairo University

High Performance Liquid Chromatography coupled to a Diode-Array-Detector (HPLC-DAD) is used to investigate samples which were extracted from ancient Egyptian textiles (4th–5th c. AD) of the Museum of Faculty of Archaeology, Cairo University. Madder is identified in several samples. According to semi-quantitative results, which are obtained from HPLC peak areas measured at 254 nm, madder that is rich in purpurin and poor in alizarin is identified in samples which were treated (i) only with madder and (ii) with madder and either indigo/woad (Indigofera species and other/Isatis tinctoria L.) or weld (Reseda luteola L.). The madder dye used in these samples could have been originated from Rubia peregrina L. However, the possible use of Rubia tinctorum L. (or other plants of the Rubiaceae family) by the Egyptian dyers cannot be ruled out, particularly if methods were developed by the ancient dyers to affect and control the relative composition of madder dye. The HPLC peak area ratio of alizarin versus purpurin is very high (>2.2) for samples which were treated with madder (probably originated from R. tinctorum) and a tannin source. Finally, in some samples, only indigoid dyes (indigo/woad) are identified.

Rapid Screening for EGFR Inhibitor in Rhei Radix et Rhizoma by HTRF Assay Coupled with HPLC Peak Fractionation

In this paper, an HPLC peak fractionation approach combined with homogeneous time-resolved fluorescence analysis is proposed for screening epidermal growth factor receptor inhibitors from Rhei Radix et Rhizoma. With this approach, the amount of sample used for a single HPLC run is sufficient for performing a multiple assay due to the miniaturization ability of the homogeneous time-resolved fluorescence technology. This allows for improving the stability and repeatability of the activity assay for each fraction. From a total of 26 fractions collected from the Rhei Radix et Rhizoma extract, 13 fractions exhibit inhibitory activity against the epidermal growth factor receptor. The structures of activity compounds were determined by HPLC-LTQ-Orbitrap MS, revealing the presence of gallic acid, rhein, and emodin with IC50 values of 21.5, 5.29, and 10.2 µM, respectively. The ligand epidermal growth factor receptor interactions were explored by molecular docking simulations, and the inhibitory effects of the three compounds on A549 cell growth were tested in vitro by an MTT assay. This study demonstrates the suitability of the present screening method for drug discovery in natural products.

Markers Assessment in Triphala (Batch-I, II & III) and its Ingredients from Different Geographical Locations of India through TPC, TFC, HPLC & HPTLC

Background: Relations among markers, quality assessment and standardization of classical preparations like Triphala an Ayurvedic potent formulation are necessary for the selectivity as well as acceptability of genuine plant drugs and formulation. Objective: Qualitative and quantitative evaluations of three batches of in-house Triphala along with its ingredients collected from three different locations of India with respect to assess the six active markers. Method: Phytochemical studies, spectrophotometric estimations (TPC & TFC), chromatographic (HPLC & HPTLC) methods were developed for the identification and quantification of active markers in Triphala. Result: Chemical analysis and HPTLC profiles with respect of gallic acid at Rf 0.35 of methanol extracts showed the presence of almost similar phytochemicals in three batches. The highest HPLC peak % area for corrilagin, 1,3,6-Trigalloyl-beta-D-glucose, ellagic acid and chebulinic acid was calculated to be 3.753, 5.27, 24.55 and 29.47, respectively with a majority of markers i.e. four observed in batch-III. The percentage amount of TPC at λ max 720 for batch-III of Phyllanthus emblica L., Terminalia bellirica Roxb., Terminalia chebula Retz. and Triphala was 393.1, 374.81, 628 & 644.5 mg of TAE/g dry weight equivalent, respectively. Similarly, TFC at λ max 510 for the same batch and ingredients was calculated to be 60.27, 40.043, 74.84 and 59.21 QUE/g dry weight equivalent, which were also observed to be maximum in batch-III. Conclusion: Batch-III of Triphala is of the highest quality and up to pharmacopoeial standards (API). It may be used to predict the quality and efficacy of various commercial formulations of Triphala. These outcomes may be utilized in pharmaceuticals for routine batch standardization and quality control.

Isolation and identification of tuliposides D and F from tulip cultivars

Abstract6-Tuliposides A (6-PosA) and B (6-PosB) are major defensive secondary metabolites in tulip cultivars (Tulipa gesneriana), having an acyl group at the C-6 position of d-glucose. Although some wild tulip species produce 1,6-diacyl-glucose type of Pos (PosD and PosF), as well as 6-PosA/B, they have not yet been isolated from tulip cultivars. Here, aiming at verifying the presence of PosD and PosF in tulip cultivars, tissue extracts of 25 cultivars were analyzed by high-performance liquid chromatography (HPLC). Although no HPLC peaks for PosD nor PosF were detected in most cultivars, we found two cultivars giving a minute HPLC peak for PosD and the other two cultivars giving that for PosF. PosD and PosF were then purified from petals of cultivar ‘Orca’ and from pistils of cultivar ‘Murasakizuisho’, respectively, and their identities were verified by spectroscopic analyses. This is the first report that substantiates the presence of 1,6-diacyl-glucose type of Pos in tulip cultivars.

Fast identification of the antibacterial in Aspergillus species

Background and Purpose: Aspergillus genus biosynthesize a large number of metabolites with wide-ranging bioactivities, but how to target the active compounds quickly and accurately? In this work, we provide a strategy to fast identify bioactive metabolites in fungi with similar morphology but different antimicrobial activities. Materials and Methods: Combined with High Performance Liquid Chromatography (HPLC) profiles and bioactivity screening of fungal extracts, three Aspergillus sp. strains YSN038, YSN052 and YSN064 were studied. The bacteria Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, Kocuria rhizophila, Escherichia coli and Ralstonia solanacearum were used for antibiotic assay.Results: Three strains showed the similar morphology, but the crude extracts of YSN038 and YSN064 displayed antibacterial activities against B. pumilus, B. subtilis, S. aureus and K. rhizophila, whereas YSN052 had not. The compound contributed to antibacterial activity in vitro was rapidly identified, isolated and characterized. Compound 3 connected with specific HPLC peak should be the antibiotic substance by analysis of HPLC profiles, and was confirmed after the following antimicrobial tests. The compound was elucidated as butyrolactone I based on nuclear magnetic resonance(NMR) and mass spectrometry (MS) data, and showed various biological activities.Conclusions: Our study has significant scope on targeting antibacterial metabolites. The described method could also be used as a rapid and cost-effective tool for screening other bioactivity products. As such, this article could offer a fast approach to isolate drug-lead compounds from microorganisms.

Combined effect of Bacillus cereus CPOU13 and B. subtilis SPC14 on polycyclic aromatic hydrocarbons degradation in vitro

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) with suitable bacterial strains conveys much interest in recent years. We studied biodegradation of PAHs namely phenanthrene, anthracene and pyrene using two efficient PAHs degrading strains, B. cereus CPOU13 and B. subtilis SPC14 in vitro experiments. Standard HPLC chromatograms for phenanthrene, anthracene and pyrene were plotted separately based on HPLC peak area values and Retension time of known concentrations of the test PAHs and using software, ‘Origin 6.0’. Biodegradation of PAHs mixture containing phenanthrene, anthracene and pyrene was studied in for 13 days. We found that the combination of bacterial strains, B. cereus CPOU13 and B. subtilis SPC14 degraded high amounts of phenanthrene, anthracene and pyrene in 13 days of incubation. The recorded degradation percentages of phenanthrene, anthracene and pyrene were 85.31, 92.82 and 85.70 respectively. Concentration of phenanthrene was degraded from 217µg/5ml to 31.9µg/5ml. Concentration of anthracene was degraded from 211µg/5ml to 16µg/5ml. Concentration of pyrene was degraded from 233µg/5ml to 33µg/5ml in 13 days of incubation. We also observed biodegradation of phenanthrene, anthracene and pyrene from 1st day to 13th day.

Isomeric separation and identification of tetra-, tri-, and di-β-sulphonic phthalocyanine zinc complexes

The synthesis, isomeric separation, and identification of β-sulphonic phthalocyanine zinc complexes were reported. While the sulphonic phthalocyanines have been studied extensively, the development of separation technology may enable deeper insights into their isomeric constitution. An ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) method was developed to separate the sulphonic phthalocyanine isomers. The results showed that the product of the condensation reaction is a mixture of all possible isomers with statistical distribution. Several isomers were obtained and structural determination was undertaken by NMR. Based on the IP-RP-HPLC elution sequence of these well-identified isomers, a relationship between the structure and efficiency was deduced: closely spaced intervals of sulphonic groups lead to higher hydrophobicity and shorter retention times on HPLC. Based on this relationship, each HPLC peak was assigned to the corresponding isomeric structure.

Rapid LC and LC/MS for routine analysis of mycotoxins in foods

Affinity column clean-up of food samples for mycotoxin analysis produces extracts which are free of co-extractives and therefore require little chromatography for separation and quantification of the target analytes. Using such clean extracts, we report rapid chromatographic methods for aflatoxins B1, B2, G1 and G2, aflatoxin M1, ochratoxin A, zearalenone and fumonisins. Using short columns with small particle size packing, HPLC conditions have been developed reducing analysis time typically by 75%, e.g. for aflatoxins from 19.0 min to 4.0 min with full separation of the four toxins and for aflatoxin M1 giving an analysis time of less than 1 min compared to 5 min for conventional analysis. Fumonisins were analysed directly by LC/MS in a run time of 4.1 min, using selected ion monitoring to avoid the need for derivatisation for fluorescence detection by HPLC. Peak purity under rapid analysis conditions has been demonstrated by LC/MS for a variety of food extracts such as pistachios, hazelnuts, paprika, cocoa, coffee, cereals, sesame oil, cheese, maize, animal feed, dried figs, dried vine fruits, cornflakes and bread. These substantial reductions in analysis time offer significant benefits to laboratories undertaking routine screening and quantitative analysis of mycotoxins in foods.

DNA Sequence of Hemoglobin Chico.

We were asked to investigate a 54 year old male who was undergoing evaluation for sleep apnea. The patient had a low resting O2 saturation at 91%, but no signs of cardiac or pulmonary disease as indicated by a normal echocardiogram, normal spirometry, and normal high resolution CT of the chest. Arterial blood gases revealed a pO2 of 79 mm Hg on room air, but pCO2 was normal. The patient’s hemoglobin concentration was 13.7 g/dL. The possibility of a mutant hemoglobin was raised and HPLC revealed HbA2 = 2.6%, HbA = 51.4% and an unidentified mutant hemoglobin of 46%. Oxygen dissociation testing demonstrated a P50 of 39 mm Hg (normal 24–32) consistent with a low affinity hemoglobin. The isopropanol test showed that the hemoglobin variant was unstable. Trypsin digestion of the abnormal hemoglobin resulted in a smaller than normal HPLC peak for the β9 segment of β-globin, which is composed of amino acids 67 through 82, and the appearance of a new peak at 193 min. This peak did not match any of the known mutant hemoglobins contained in our reference bank. We PCR amplified and sequenced the β-globin gene. The patient was heterozygous for an A to C mutation in the second position of codon 66. This mutation changed the lysine to threonine and revealed that the hemoglobin variant was Hb Chico. This result was consistent with the loss of ~50% of the β9 peak in the trypsin digest due to loss of a cleavage site after amino acid 66, and the appearance of a new peak which was a hybrid segment of β8 and β9. This represents the first DNA sequence of hemoglobin Chico a mildly unstable hemoglobin variant with decreased oxygen affinity.