[Study on quality control of Holotrichia diomphalia larvae by TLC and HPLC].

The TLC method was established for identification of Holotricha diomphalia larvae and the HPLC method was used to determine the content of inosine and guanosine in H. diomphalia larvae. The HPLC analysis was performed on a Waters HSS T3（4.6 mm×250 mm, 5 µm） column of with mobile phase consisting of acetonitrile (A) and 0.08% trifluoroacetic acid (B) in gradient elution. The detection wavelength was 260 nm. The flow rate was 1.0 mL·min⁻¹. The column temperature was 30 °C. As a result, TLC identification method had a good reproducibility and highly specificity. The linear equations of inosine and guanosine were in good linear range (r>0.999 8). The average recovery of inosine and guanosine was 96.53% (RSD=1.6%), 99.71% (RSD=2.7%). The method is simple, accurate and reproducible, which can provide a basis for quality standard improvement H. diomphalia larvae.

Rapid analysis of traditional Chinese medicine Pinellia ternata by microchip electrophoresis with electrochemical detection.

Traditional Chinese herbal medicine has long enjoyed the reputation of the world's most advanced system of natural medicine. Pinellia ternata is one of the most commonly used herbs in the traditional Chinese medical science. In this study, five representative ingredients of Pinellia ternata guanosine, methionine, glycine, 3,4-dihydroxybenzaldehyde, and homogentisic acid, were assayed using simple derivatization procedures. Under optimized experimental condition, five analytes in Pinellia ternata were rapidly separated and detected using microchip electrophoresis, affording the benefits of speed, minimal sample requirements, and sensitive on-the-chip electrochemical detection, in 5 min with linearity over a concentration of 20-500 µM (R2  = 0.994) with nearly complete recovery (95.6-98.5%).

Development and Validation of LC-MS/MS Method for Quantitative Determination of Adenosine, Guanosine, Xanthine and Uric acid in Widely Consumed Vegetables in Thailand.

In this study, a triple quadrupole LC-MS/MS electrospray ionization method was developed and validated for quantitative determination of adenosine, guanosine, xanthine and uric acid in fifteen widely consumed Thai vegetables. The method was successively developed by using caffeine as internal standard. The lower limit of quantitation (LLOQ) was 0.2 µg/g for adenosine and guanosine, and 1.0 µg/g for uric acid and xanthine. The method was fully validated according to USFDA guidelines and all performance characteristics were found acceptable. Subsequently, the developed and validated LC-MS/MS method was applied to determine the four interest substances in fifteen widely consumed vegetables in Thailand. The results showed that all vegetables included in the study could be classified as low adenosine, guanosine, xanthine and uric acid containing foods since the concentrations of these substances were less than 50 mg per 100 g. This finding was enormously valuable information for hyperuricemia and gouty patients.

[Determination of Nucleosides and Nucleobases in Natural, Cultured and Tissue Culture Anoectochilus roxburghii Using LC-MS].

OBJECTIVE: To establish a method for simultaneous determination of nucleosides and nucleobases in natural, cultured and tissue culture Anoectochilus roxburghii by high performance liquid chromatography-electrospray ionization/ion trap mass spectrometry (HPLC-ESI/MS). METHODS: The separation was performed on a Welch Ultimate XB-C18 column (250 mm x 4.6 mm,5 µm). 20 mmol/L ammonium acetate solution and methanol were adopted as the mobile phase with gradient elution. The flow rate was 1.0 mL/min. The injection volume was 20 µL. The column temperature and UV wavelength were set at 30 degrees C and 260 nm, respectively. RESULTS: Cytosine, uracil, cytidine, uridine, hypoxanthine, adenine, inosine, guanosine,fl-thymidine and adenosine were identified in natural, cultured and tissue culture Anoectochilus roxburghii. The total content of nucleosides and nucleotides in Anoectochilus roxburghii were 1.6639, 1.8568 and 2.2013 mg/g,respectively. CONCLUSION: The contents of nucleosides and nucleobases in herb are affected by its growth pattern. The total content of nucleosides and nucleotides was tissue culture herb > cultured herb > natural herb. This investigation would provide the theoretic basis for quality standards and applications of Anoectochilus roxburghii in clinical research.

Wounding stress causes rapid increase in concentration of the naturally occurring 2',3'-isomers of cyclic guanosine- and cyclic adenosine monophosphate (cGMP and cAMP) in plant tissues.

3',5'-Cyclic guanosine monophosphate (cGMP) and 3',5'-cyclic adenosine monophosphate (cAMP) are well reported second messenger molecules involved in cellular signal transduction, in physiological functions such as neurotransmission in animals and in the modulation of cell growth and differentiation. In plants, 3',5'-cyclic nucleotides have been implicated in the regulation of ion homeostasis, hormone and stress responses. The behavior of the 2',3'-cyclic nucleotide variants is also known in animal tissue but no quantitative information is available about 2',3'-cAMP and 2',3'-cGMP in plant material. A recently developed HILIC-SPE/LC-MS/MS method for the analysis of cyclic nucleotides in blood and animal tissue was therefore adapted to measure 2',3'-cAMP and 2',3'-cGMP concentrations in plant material. Cyclic nucleotide concentrations were measured in Arabidopsis thaliana (Col-0) leaves before and after the application of wounding stress. A significant (∼5-fold) up-regulation of 2',3'-cAMP and 2',3'-cGMP was measured in Arabidopsis leaves compared to the control samples. The results indicate a thus far unreported strong correlation between plant stress and both 2',3'-cAMP and 2',3'-cGMP levels in plant material, and may open new avenues towards understanding the role of cyclic nucleotides in plants.

[New processing procedure for Croton tiglium with study on comparison of Croton tiglium and processed product].

OBJECTIVE: To build a new processing procedure for Croton tiglium, providing a more simple, efficient and safe way of processing. METHODS: Used the contents of isoguanosine and toxic protein in Croton tiglium as the indexes to investigate the effect of different temperature, thickness and baked time on processing for Croton tiglium. After established all factors and levels, processed a batch of Croton tiglium under optimum processing conditions and compared it with raw Croton tiglium in the test of acute toxicity and gastrointestinal propulsive motility. RESULTS: The parameters of optimum processing were as follows:the temperature was set at 180 degrees C, the thickness of placement was 3 cm and baked time was 90 min. The LD50 value of raw Croton tiglium and the processed Croton tiglium was 888 mg/kg and 2139 mg/kg respectively. CONCLUSION: The processing procedure is simple, affordable, safe and efficient, deserved to promote for application.

Hydrogen gas ameliorates oxidative stress in early brain injury after subarachnoid hemorrhage in rats.

OBJECTIVE: Hydrogen gas has been demonstrated to neutralize free radicals and reduce oxidative stress recently. Our objective was to determine the therapeutic effect of H2 inhalation and its antioxidative activity on early brain injury after subarachnoid hemorrhage. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: One hundred thirty-seven adult male Sprague-Dawley rats weighing 280-350 g. INTERVENTIONS: Subarachnoid hemorrhage was induced by endovascular perforation method in rats. Subarachnoid hemorrhage rats were treated with 2.9% hydrogen gas inhaled for 2 hrs after perforation. At 24 and 72 hrs, mortality, body weight, neurologic deficits, and brain water content were assessed. Blood-brain barrier permeability and apoptosis were also measured at 24 hrs. To investigate the antioxidative activity of hydrogen gas, the expression of malondialdehyde, nitrotyrosine, and 8-hydroxyguanosine, which are oxidative markers of lipid, protein, and DNA damage, respectively, were measured at 24 hrs. MEASUREMENTS AND MAIN RESULTS: Hydrogen gas significantly alleviated brain edema and blood-brain barrier disruption, reduced apoptosis, and improved neurologic function at 24 hrs but not 72 hrs after subarachnoid hemorrhage. These effects were associated with the amelioration of oxidative injury of lipid, protein, and DNA. CONCLUSIONS: Hydrogen gas could exert its neuroprotective effect against early brain injury after subarachnoid hemorrhage by its antioxidative activity.

Determination of the nucleosides and nucleobases in Tuber samples by dispersive solid-phase extraction combined with liquid chromatography-mass spectrometry.

A simple, fast and inexpensive method based on dispersive solid phase extraction (DSPE) combined with LC-MS was developed for simultaneous determination of 7 nucleosides and nucleobases (i.e., adenine, hypoxanthine, uridine, adenosine, guanine, guanosine, and inosine) in Tuber fruiting-bodies and fermentation mycelia. The DSPE procedure was firstly introduced to remove the protein interference from sample solutions, and D3520 macroporous resin was chosen as the DSPE sorbent because of its high removal capability on protein interferences, but low adsorption rate on analytes. Besides, key parameters on DSPE procedure (i.e., macroporous resin type, macroporous resin amount, methanol concentration, and vortex time) were optimized, and the protein removal efficacy could achieve about 95% after the process optimization. Though the method validation test, the DSPE-LC-MS method was confirmed to be precise, accurate and sensitive, and the column blinding problem was solved successfully. By using this established method, the total amount of nucleosides and nucleobases in the fermentation mycelia was determined to range from 4881.5 to 12,592.9µgg⁻¹, which was about 2-25 times higher than the fruiting-bodies (from 498.1 to 2274.1µgg⁻¹). The formulation of nucleosides and nucleobases in the fermentation mycelia maintained relatively constant, while the formulation in Tuber fruiting-bodies varied significantly with their species. Hierarchical cluster analysis (HCA) showed the formulation similarity of nucleosides and nucleobases between Tuber fermentation mycelia and the fruiting-bodies of Tuber indicum and Tuber himalayense. From the viewpoint of nucleosides and nucleobases, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting-bodies.

Simultaneous determination of five nucleosides and nucleobases of Rehmannia glutinosa Libosch. by high performance liquid chromatography.

This study is to establish a method for simultaneously determination of five nucleosides and nucleobases, including hypoxanthine, uridine, adenine, guanosine and adenosine in Rehmannia glutinosa Libosch. which was collected from different regions in China. A Diamonsil C18 column (250 mm x 4.6 mm, 5 microm) was used. Acetonitrile and 0.04 mol L(-1) potassium dihydrogen phosphate solution were adopted as mobile phase with gradient elution. The flow rate was 1 mL min(-1) and column temperature was 30 degrees C. The detection wavelength was at 254 nm. The method had good linearity over the range of 1.0 - 16.0 microg mL(-1) (r2 = 0.999 8), 5.0 - 80.0 microg mL(-1) (r2 = 0.999 8), 1.0 - 16.0 microg mL(-1) (r2 = 0.999 5), 1.25 - 20.0 microg mL(-1) (r2 = 0.999 8) and 1.0 - 16.0 microg mL(-1) (r2 = 0.999 8) for hypoxanthine, uridine, adenine, guanosine and adenosine, respectively. The average recoveries were between 98.8% and 100.7%. The content of hypoxanthine, uridine, adenine, guanosine and adenosine in Rehmannia glutinosa Libosch. from different regions was significantly different. This established method was sensitive and reliable for the quantification of five chemical constituents in Rehmannia glutinosa Libosch.

Nucleotides and nucleosides in ovine and caprine milk during lactation.

The aim of this study was to determine the nucleoside and nucleotide content in ovine and caprine milks at the colostral, transitional, and mature stages of lactation. Samples from 18 dairy sheep and 18 dairy goats were collected at 1, 2, 3, 4, 5, and 15 d postpartum. Separation and quantitation of the 5'-nucleotides (NT) and the nucleosides (NS) was performed by reverse phase HPLC. For each compound measured, considerable interindividual variation was recorded in both species of milk. The total NS content ranged from 57 to 132 micromol/L and from 54 to 119 micromol/L in ovine and caprine milk, respectively. The major NS identified in both species of milk was uridine, representing more than 60% of the total NS pool. The mean levels of inosine and guanosine were comparable between ewe and goat milk. Instead, the mean level of cytidine across the sampling period was much higher in ewe milk (11.9 micromol/L compared with 4.5 micromol/L in goat milk) and exhibited a peak value on the fourth day of lactation. The adenosine content was at least 3-fold higher in caprine milk compared with its ovine counterpart. The total NS and orotic acid contents did not differ significantly between the 2 species. However, in the case of total NT content, interspecies differences were significant, with NT levels ranging from 294 to 441 micromol/L in ovine milk and from 166 to 366 micromol/L in caprine milk. The NT content in colostrum (1-3 d) of both species was higher than in mature milk (15 d), and uridine monophosphate was the dominant NT in all samples.

[Determination of isoguanosine in Semen Crotonis Tiglii by high-performance liquid chromatography].

OBJECTIVE: To develop a high-performance liquid chromatographic method for the determination of isoguanosine in Semen Crotonis Tiglii. METHODS: The determination was done by using reverse-phase high-performance liquid chromatography with a Hedera ODS-2 column (4.6 mm x 250 mm, 5 microm). Elution was employed with the mobile phase of acetonitrile-methanol-water (1:4:95) at flow rate of 1.0 mL/min. The detection wavelength was set at 292 nm and the column temperature was 25 degrees centigrade. Injection volume was 10 microL. RESULTS: The linear range of isoguanosine was from 0.161 to 0.967 mg. The correlation coefficient of the calibration curves was 0.999 8. The average recovery rate was 98.78% with relative standard deviation of 0.02%. CONCLUSION: The results show that the method is simple, accurate, and repeatable, and it is suitable for quality control of Semen Crotonis Tiglii.

Qualitative evaluation and quantitative determination of 10 major active components in Carthamus tinctorius L. by high-performance liquid chromatography coupled with diode array detector.

Flavonoids in the water extract of Carthamus tinctorius L. exhibit potent biological activities such as anti-coagulant, vasodilation, anti-oxidant, neuroprotection and immunosuppressant. A high-performance liquid chromatographic method was established to evaluate the quality of Carthamus tinctorius through a simultaneous quantitation of eight flavonoids, hydroxysafflor yellow A (2), 6-hydroxykaempferol 3,6-di-O-beta-glucoside-7-O-beta-glucuronide (3), 6-hydroxykaempferol 3,6,7-tri-O-beta-glucoside (4), 6-hydroxykaempferol 3-O-beta-rutinoside-6-O-beta-glucoside (6), 6-hydroxykaempferol 3,6-di-O-beta-glucoside (7), 6-hydroxyapigenin 6-O-glucoside-7-O-glucuronide (8), anhydrosafflor yellow B (9), and kaempferol 3-O-beta-rutinoside (10), together with two other compounds named guanosine (1) and syringin (5). Among them, compound 8 was identified as a new compound. The compounds were separated on an Alltech Alltima-C(18) column with gradient elution of acetonitrile and 0.01% trifluoroacetic acid. The detection wavelength was 280 nm. All the compounds showed good linearity (r(2) >or= 0.9989). The recoveries, measured at three concentration levels, varied from 94.9% to 105.2%. This method was also validated with respect to precision, repeatability and accuracy, and was successfully applied to quantify the 10 components in 46 batches of C. tinctorius samples from different areas. Significant variations were found in the contents of these compounds in these samples. Compared with the reported analytical methods of C. tinctorius, this simple and reliable method provided a new basis for overall assessment on quality of C. tinctorius and should be considered as a suitable quality control method.

[Study on processing for Rhizoma Pinelliae Praeparatum by comprehensive evaluation of multiple mark].

OBJECTIVE: To investigate the best processing technology of Rhizoma Pinelliae Praeparatum. METHODS: L9(3(4)) orthogonal design was used with four factors: quantities of quick lime and Glycyrrhiza urallensis, processing time and temperature, the contents of Calcium oxalate crystals, Guanosine and Glycyrrhizic acid were determined by RP-HPLC, the irritation test was detected by swollen rabbits eyes with processing products. RESULTS: The optimized processing technology was satisfied with some conditions as follows: the processing time was 48 hours, the processing temperature was 30 degrees C, quantities of quick lime and Glycyrrhiza uralensis were 10 g and 15 g. CONCLUSION: The optimized processing method by orthogonal design has achieved the goal to reduce toxicity, the processing time and the processing temperature are confirmed respectively, and the processing time is significantly shortened comparing with Pharmacopoeia.

Effects of sample preparation methods on the quantification of nucleosides in natural and cultured Cordyceps.

Sample preparation is the first and very important step, which can greatly influence the repeatability and accuracy of the analysis. To date, several sample preparation methods with different solvents have been used for quantitative determination of nucleosides in Cordyceps, but their data are greatly various. In this study, five nucleosides, including adenosine, guanosine, inosine, uridine and cordycepin, in Cordyceps were determined using three extraction methods i.e. organic solvent pressurized liquid extraction, boiling water extraction and ambient temperature water extraction and high performance liquid chromatography (HPLC)-diode array detection (DAD). The similar results were obtained when organic solvent pressurized liquid extraction and boiling water extraction were applied. However, the amounts of nucleosides in natural C. sinensis and cultured C. militaris extracted with ambient temperature water were greatly increased except those of adenosine in natural C. sinensis and cordycepin in cultured C. militaris. In addition, the amount of investigated nucleosides in cultured C. sinensis had no obvious variation among the three extraction methods. The results suggest that sample preparation has significant effect on the quantification of nucleosides in Cordyceps.

[Comparative study on main economic characters of different populations of Pinellia ternata in China].

OBJECTIVE: To analyze the content of guanosine, total alkaloid and individual yield of Pinellia ternata from different populations in China and evaluate its quality. METHOD: Reversed-phase high-performance liquid chromatography (HPLC) was used to determine the content of guanosine. The content of alkaloid was determined by ultra violet spectrophometry. The results were analyzed by SPSS software. RESULT: The contents of guanosine and total alkaloid in P. ternata were 0.0136% -0.0264% and 0.0155% -0.0652% respectively. Individual yield was 0.5536-2.9740 g. All of the populations could be classified into 3 types through hierarchical cluster analysis. CONCLUSION: There exist significant differences in the content of guanosine, total content of alkaloid and individual yield of P. ternata from different populations. It is suggested that breeding and selection for type II of P. ternata should be strengthened.

[Quantitative analysis of the nucleosides in Cordyceps sinensis with capillary zone electrophoresis].

OBJECTIVE: To establish a quantitative analysis method for analyzing the nucleosides in Cordyceps sinensis with capillary electrophoresis, and compare the difference between natural and the cultured C. mycelia. METHOD: Capillary zone electrophoresis method was employed to quantitate the adenosine, uridine, guanosine and inosine in C. sinensis, with 0.25 mg x L(-1) boric acid-sodium hydroxide buffer, pH 9.5. The working voltage was 20 kV, the temperature was 25 degrees C, and the detection wavelength was 260 nm. RESULT: With the capillary zone electrophoresis method, the average recovery of the above 4 nucleosides was 98.9%, 95.1%, 97.8% and 98.8% respectively, with the RSD 0.4%, 1.7%, 1.3% and 5.0%. There was no adenosine in natural C. sinensis and no inosine in the cultured C. mycelia detected. CONCLUSION: This method can be used to determine the adenosine, uridine, guanosine and inosine in C. sinensis. The nucleosides in C. sinensis produced from Qinghai province and cultured C. mycelia are obviously different.

[Identification of 5 constituents of the aqueous extract of Isatis indigotica by HPLC-MS2].

OBJECTIVE: To identify five constituents in the aqueous extract of Isatis indigotica Fort. METHODS: After separation of the aqueous extract of Isatis indigotica Fort. by HPLC, the eluates of five peaks were collected separatively and analysed by MS2. UV spectra and MS2 were compared with those of reference standards of cytidine, uridine, guanosine xanthine and hypoxanthine. RESULTS: Each HPLC elute of the aqueous extract had same retention time, UV spectra and fragment pattern in the MS2 spectrum as the corresponding standards. CONCLUSION: Five constituents of the aqueous extract of Isatis indigotica Fort. are identified as cytidine, hypoxanthine, uridine, xanthine and guanosine.

[Determination of four nucleosides in Banlangen injections using capillary zone electrophoresis].

The contents of cytidine, adenosine, guanosine and uridine in Banlangen Injections were determined by capillary zone electrophoresis. Shorter fused silica capillary (32.5 cm x 50 microm i. d. with effective length of 23.5 cm) was used. The samples were analyzed with 60 mmol/L borate-10% (v/v) 2-propanol-20% (v/v) acetonitrile running buffer at 20 kV voltage (25 degrees C capillary temperature). Pressure injection of 1 kPa x 10 s was employed, and the detection wavelength was 254 nm. Factors of the electrophoresis condition were investigated, such as the kinds and concentration of the electrolyte, buffer pH value, the type and concentration of organic modifier, separation voltage and temperature. The samples were filtered through 0.45 microm membrane, then direct injection was employed. External calibration of peak area versus concentration was used in the determination. Linear calibrations of four nucleosides were obtained within 12.5 - 250 mg/L (r > 0.999 1). The limits of detection (mg/L) were 6.2 of cytidine, 4.6 of adenosine, 6.7 of guanosine, and 9.0 of uridin. The average recoveries of the four nucleosides were between 95.1% - 102.3% with RSDs of 0.3% - 4.9%. The method is simple, rapid, reproducible and accurate. It can be used for the routine analysis of the four nucleosides in Banlangen Injections.

[Determination of nucleosides in siweilingzhi mixture by HPCE].

OBJECTIVE: To establish a method for determining nucleosides (adenoside and guanoside) in Siweilingzhi Mixture by HPCE. METHOD: Adenoside and guanoside were separated within 25 min using an 20 mmol.L-1 borate buffer with 30 mmol.L-1 SDS and 5% Ethanol (adjusted to pH 10.0 with sodium hydroxide solution), with an operation voltage of 10 kV, temperature of 20 degrees C and a hydrodynamic injection time of 15 s. Seperations were carried out in a fused-silica capillary 75 microns id x 57 cm (effective length 50 cm) with peak detection by direct UV at 254 nm. RESULT: Regression equation of adenoside and that of guanoside were Y = 0.0705 + 0.01707X (r = 0.9995) and Y = 0.0232 + 0.01864X (r = 0.9999) respectively. The average recovery rate was 99.22% (RSD = 3.66%) and 104.3% (RSD = 1.91%) respectively. Nine samples were determined with the method. CONCLUSION: The method is simple, rapid and accurate with good repeatability and it can be used to determine nucleosides.

Determination of nucleosides in natural Cordyceps sinensis and cultured Cordyceps mycelia by capillary electrophoresis.

Cordyceps sinensis is a well-known traditional Chinese medicine, and some of the active components are nucleosides. The analysis of nucleosides in Cordyceps material has been performed by reversed-phase high-performance liquid chromatography (HPLC) with gradient elution or by spectrometry. Here, we have explored the possibility of using capillary electrophoresis to determine the content of three major nucleosides (adenosine, guanosine and uridine) in Cordyceps. Capillary electrophoresis needs no gradients, and it provides a better separation due to its higher efficiency. In order to optimize the resolution, the separation of adenosine, guanosine and uridine was determined in Cordyceps with respect to the variation of buffer concentration, pH, temperature, and voltage. By using the calibrated electrophoresis system, the separation was achieved for the three nucleosides in less than 10 min with a background electrolyte consisting of 0.2 M boric acid-sodium hydroxide buffer, pH 8.5. The nucleoside contents of various types of natural Cordyceps and cultured Cordyceps mycelia were determined and compared. There was a great variation of nucleoside content in different sources of Cordyceps; the cultured Cordyceps mycelia, however, contains a much higher concentration than the natural Cordyceps.