Pharmacokinetic and tissue distributions study of adenosine, 4-hydroxybenzyl alcohol and Parishin C from Gastrodia elata extract in rats.

The plant Gastrodia elata is a type of the orchid plant Gastrodia elata Bl. which contains glycosides, phenols, polysaccharides, sterols, and organic acids and a variety of active ingredients are proved to have certain pharmacological activities. To understand the process in the body of Gastridua elata, we used HPLC to study pharmacokinetics and tissue distributions of adenosine, 4-hydroxybenzyl alcohol and Parishin C in rats. The results showed that the three ingredients could be detected in plasma and different organizations at various time points. There was no significant difference in systemic clearance at three ingredients and it may be show that the three ingredients distributed (0.475±0.025, 0.518±0.033, 0.699±0.051) quickly and eliminated (5.37±0.87, 4.54±0.69, 5.34±0.82) slowly in plasma. There was the highest content of adenosine in spleen, followed by liver and lung. The highest content of 4-hydroxybenzylacohol in liver, and was higher in spleen. Parishin C was highest in heart, followed by liver and spleen. It is obvious that the contents of three ingredients are all higher in liver. The trends of the three ingredients' contents in G. rhizome extract were consistent with the contents in the plasma after intravenous administration.

Determination of the Main Nucleosides and Nucleobases in Natural and Cultured Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis, a recently described species of Ophiocordycepsthat is associated with the larvae of Phassusnodus (Hepialidae) in the living root or trunk of the medicinal plant Clerodendrumcyrtophyllum, isthe largest known Cordycepsspecies and is recognized as a desirable alternative for natural Ophiocordycepssinensis. This study investigated the main nucleosides and nucleobases in natural and cultured Ophiocordycepsxuefengensis. The contents of the nucleosides and nucleobases in the natural and cultured samples were determined by reverse phase HPLC. The highest concentration of adenosine was found in the natural fruit body and the cultured stroma, with almost no adenosine in the cadaver of Phassusnodus. The contents of adenine, guanosine, uridine and uracil in the cultured mycelium were significantly higher than those in the natural sample. Inosine was only detected in the natural samples. Thymidine and 2-deoxyadenosine were only found in the cadaver of Phassusnodus. Cordycepin was not detected in the five samples examined. These results suggested that the cultured mycelium and cultured stroma of Ophiocordycepsxuefengensis might be a promising substitute for natural O. xuefengensis.

Acidified Hot Water Extraction of Adenosine, Cordycepin, and Polysaccharides from the Chinese Caterpillar Mushroom, Ophiocordyceps sinensis CS1197 (Ascomycetes): Application of an Artificial Neural Network and Evaluation of Antioxidant and Antibacterial Activities.

The effects of process variables (temperature, time, and pH) on the extraction of adenosine, cordycepin, and polysaccharides from Ophiocordyceps sinensis CS1197 were studied using response surface methodology (RSM) and an artificial neural network (ANN). The ANN model resulted in low root mean square errors (0.022, 0.079, 0.018) and high R2 values (0.995, 0.934, 0.997) for adenosine, cordycepin, and polysaccharide yields, respectively, which implied good agreement between the predicted and actual data. An overall desirability of 0.86 suggested optimal extraction conditions (temperature, 70°C; time, 1 hour; and pH ~4) for adenosine (0.205%) and cordycepin (0.246%) yields. For polysaccharide yield (6.34%), an overall desirability of 0.93 suggested extraction conditions: temperature, 87°C; time, 3.4 hours, and pH ~4. The water extract exhibited better antioxidant activity. The antibacterial activity of the water extract was assayed against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, and larger amounts of the extract (30 and 50 mg) exhibited antibacterial activity (<35%). The predictive ability of an ANN is superior to RSM and resulted in the best agreement between experimental and predicted values.

Ultrasensitive and universal fluorescent aptasensor for the detection of biomolecules (ATP, adenosine and thrombin) based on DNA/Ag nanoclusters fluorescence light-up system.

We report here an ultrasensitive strategy based on the recognition-induced conformational alteration of aptamer and fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequence in proximity to silver nanoclusters for adenosine triphosphate (ATP), adenosine (A) and thrombin (TB) detection. Herein, we designed two tailored DNA sequences noted as complementary DNA (abbreviated as c-DNA) and signal probe DNA (abbreviated as s-DNA), respectively. c-DNA is designed as a special structure consisting of a sequence complementary to aptamer at the 3'-end and a guanine-rich DNA sequence at the 5'-end; s-DNA contains a cytosine-rich sequence responsible for Ag NCs templated synthesis at the 3'-end and a link sequence (part of aptamer) complementary to partial of the c-DNA at the 5'-end. In the presence of target, the aptamer associated with the target, resulting in the formation of duplex DNA (dsDNA), the DNA-Ag NCs thereafter could close to the guanine-rich sequence, leading to enhanced fluorescence signal readout. The widespread application of the sensing system is achieved success in the detection of three biomolecules. ATP, adenosine and thrombin in the range of 0.5-8.0 µM, 0.5-7.0 µM and 50-900 nM could be linearly detected with the detection limits of 91.6 nM, 103.4 nM and 8.4 nM, respectively. This label-free and turn-on fluorescent sensing system employing the mechanism proposed here turns out to be sensitive, selective, and convenient for the detection of biomolecules without washing and separation steps.

Cordycepin and N6-(2-hydroxyethyl)-adenosine from Cordyceps pruinosa and their interaction with human serum albumin.

Cordyceps pruinosa (CP) is often used as Traditional Chinese Medicine, but the substance basis of its medicinal properties is unclear. In this study, two compounds were isolated from CP cultures by column chromatography, and identified as cordycepin and N6-(2-hydroxyethyl)-adenosine (HEA) by Nuclear Magnetic Resonance. In order to understand the efficacy of these two substances as potential therapeutic agents, it is necessary to explore their binding with proteins. The molecular mechanisms of interaction between cordycepin, HEA and human serum albumin (HSA) were studied using UV and fluorescence spectroscopy. The bingding constants between HSA and cordycepin were 4.227, 3.573 and 3.076 × 10(3)·at 17, 27 and 37°C respectively, and that of HSA and HEA were 27.102, 19.409 and 13.002 × 10(3)·at the three tempretures respectively. Both cordycepin and HEA can quench the intrinsic fluorescence of HSA via static quenching, and they can bind with HSA to form complexes with a single binding site. The interaction forces between cordycepin and HSA were determined as electrostatic and hydrophobic, and those of HEA and HSA were hydrogen bonding and van der Waals forces. Using Foster's equation, the distance between fluorophores of cordycepin and HSA, and HEA and HSA are estimated to be 5.31 nm and 4.98 nm, respectively. In this study, cordycepin was isolated for the first time from CP, and will provide a new source of cordycepin and expand the use of this taxon. The interaction mechanisms between cordycepin and HSA was studied for the first time, which will provide a useful guide for the clinical application of cordycepin. The pharmacological importance of this study is to understand the interaction of HSA with cordycepin and HEA, which will be essential for the future designing of drugs based on the two compounds.

[Alkaloids from cervi cornu pantotrichum and its effect on murine splenocytes proliferation].

OBJECTIVE: To study the alkaloids of Cervi Cornu Pantotrichum and its effect on murine splenocytes proliferation. METHODS: The constituents isolation and purification from Cervi Cornu Pantotrichum was carried out by reported column chromatography including Sephadex LH-20 and MCI (CHP20P) and their structures were elucidated on the basis of spectral compounds. The method of MTT was used to examine the effects of eight alkaloids and total alkaloids content (TAC) of Cervi Cornu Pantotrichum on murine splenocytes proliferation. RESULTS: Eleven compounds were isolated from Cervi Cornu Pantotrichum, and their structures were identified as follows: uracil (1), hypoxanthine (2), uridine (3) inosine (4), guanosine (5), 2'-deoxyguanosine (6), guanine (7), thymidine (8), thymine (9), cytidine (10) and adenosine (11). By the experiment of murine splenocytes proliferation activity in vitro, the results showed that the total alkaloids, uracil and adenosine had significantly promoted the proliferation of mouse spleen cells. CONCLUSION: Compounds 4 - 11 are isolated from Cervi Cornu Pantotrichum for the first time. The total alkaloids is one of the material basis of immunomodulatory effects of Cervi Cornu Pantotrichum, and uracil and adenosine are the most active.

[Study on the chemical constituents of the PTP 1B inhibitory effective parts of Paeoniae Rubra Radix].

OBJECTIVE: To study the chemical constituents of the PTP 1B inhibitory effective parts of Paeoniae Rubra Radix. METHODS: The effective part of Paeoniae Rubra Radix was enriched by Sephadex LH-20. Compounds were isolated by various column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS: Thirteen compounds were identified as: benzoylpaeoniflorin(1), albiflorin(2), paeoniflorigenone(3), methyl gallate(4), adenosine(5),2-amino adenosine(6), 3,3'-Di-O-methylellagic acid(7), 3,3',4-trimethyl ellagic acid(8), dihydrokaempferol(9), glycerol(10), dibutyl phthalate(11). CONCLUSION: Compounds 5-11 are obtained from this plant for the first time.

[Constituents from the leaves of Aquilaria sinensis].

OBJECTIVE: To study the chemical constituents of the leaves of Aquilaria sinensis. METHOD: The compounds were isolated and purified by the methods of solvent extraction and chromatographic technique, and their structures were identified on the basis of the analyses of spectral data. RESULT: Thirty-three compounds were obtained. Among them, twelve compounds were identified as 5-hydroxyl-7,4'-dimethoxyflavone (1), acacetin (2), luteolin (3), genkwanin (4), yuankanin (genkwanin-5-O-beta-D-primeveroside, 5), adenosine (6), genkwanin-5-O-beta-D-glucopyranoside (7), hypolaetin-7-O-beta-D-glucopyranoside (8), hypoxanthine (9), uracil (10), 8-C-beta-D-galactopyranosylisovitexin (11), and 4-(1,2,3-trihydroxypropyl) -2,6-dimethoxyphenyl-1-O-beta-D-glucopyranoside (12), respectively. CONCLUSION: All compounds except for 1, 3 and 4 were isolated from the leaves of A. sinensis for the first time.

[Chemical constituents from Corydalis yanhusuo].

OBJECTIVE: To investigate the chemical constituents of Corydalis yanhusuo. METHOD: The compounds were isolated and purified by column chromatography over macroporous absorption resin, silica gel, and Sephadex LH-20. Their structures were elucidated on the basis of physicochemical properties and spectral data. RESULT: 22 compounds were isolated and identified as corydaline (1), tetrahydropalmatine (2), protopine (3), tetrahydrocorysamine (4), tetrahydrocoptisine (5) , tetrahydroberberine (6), tetrahydrocolumbamine (7), noroxyhydrastine (8), dehydrocorydaline (9), glaucine (10), columbamine (11), 8-oxocoptisine (12), 13-methyl-columbamine (13), coptisine (14), palmatine (15), herberine (16), oxoglaucine (17), 13-methyl-palmatrubine (18), dehydrocorybulbine (19), stepharanine (20), adenosine (21), and N5 -acetylornithine (22). CONCLUSION: Compounds 13, 20, 21, and 22 were isolated from this plant for the first time.

Hypotensive and cardio-chronotropic constituents of Tinospora crispa and mechanisms of action on the cardiovascular system in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for the control of blood pressure. We previously found that an extract of Tinospora crispa stems decreased the mean arterial blood pressure (MAP) with a transient decrease, followed by an increase in the heart rate (HR) in rats. AIM OF THE STUDY: To identify the active components of the Tinospora crispa extract and investigate the mechanisms of action on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: The active components of Tinospora crispa extract were separated by column chromatography and a preparative HPLC. The effects and mechanisms of the active compounds on blood pressure and heart rate were studied in anesthetized, normal and reserpinized rats in vivo. RESULTS: 5 active compounds: adenosine, uridine, salsolinol, higenamine and tyramine were isolated. Adenosine decreased MAP and HR and this effect was inhibited by DMPX (A(2A) adenosine receptor antagonist). Uridine increased MAP and decreased HR and this was inhibited by suramin but not by DMPX. Salsolinol decreased the MAP and HR and this was inhibited by phentolamine but not by ICI-118,551 (ß(2)-adrenoceptor antagonist) or atropine. In reserpinized rats, salsolinol had a hypertensive effect that was inhibited by prazosin and phentolamine, but not by atenolol, and caused an increase in HR that was inhibited by atenolol, but not by prazosin or phentolamine. Higenamine decreased the MAP with an increase in HR. The hypotensive effect was inhibited by ICI-118,551 or atenolol, whereas the increase in HR was not inhibited by ICI-118,551. Atenolol inhibited the increase in HR at a small dosage of higenamine but potentiated it at a higher dosage. In reserpinized rats, a small dosage of higenamine tended to potentiate the effect but at a higher dosage it caused inhibition. ICI-118,551 significantly inhibited this hypotensive effect. Tyramine caused an increase in MAP and HR and these effects almost disappeared in reserpinized rats. CONCLUSIONS: The results demonstrate that these 5 compounds from Tinospora crispa acted in concert on the cardiovascular system of anesthetized rats. Salsolinol, tyramine and higenamine acted via the adrenoreceptors, whereas uridine and adenosine acted via the purinergic adenosine A(2) and P(2) receptors to decrease blood pressure with a transient decrease of HR followed by an increase.

Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method.

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA(3)), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75, 3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35°C) and flow rate (1ml/min). This method presented an excellent linearity (0.2-100µg/ml) with limit of detection (LOD) as 0.2µg/ml for ABA, 0.5µg/ml for KR and salicylic acid, and 1µg/ml for IAA, IBA and GA(3). The precision and accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80-120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized in this study determined IBA along with IAA for the first time in the seaweed species investigated except Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample preparation and analysis time were substantially reduced while allowing determination of more classes of PGRs simultaneously.

[Polar constituents of Mosla chinensis].

OBJECTIVE: To study polar constituents of Mosla Chinensis. METHOD: The constituents were isolated and purified by various chromatographic techniques and their structures were elucidated by physico-chemicalproperties and spectroscopic data. RESULT: Seven glucosides were obtained and their structures were identified as 4-hydroxy-2,6-dimethoxyphenyl-beta-D-glucopyranoside (1), 4-hydroxy-3,5-dimethoxyphenyl-beta-D-glucopyranoside (2), 3, 4, 5-trimethoxypheyl-beta-D-glucopyranoside (3), 3-hydroxyestragole-beta-D-glucopyranoside (4), (6S,9R) -roseoside (5), adenosine (6), and p-hydroxybenzoicacid glucoside (7). CONCLUSION: All compounds were isolated from the genus Mosla for the first time.

[Studies on the chemical constituents of the herb of Antenoron filiforme].

OBJECTIVE: To study the chemical constituents of the herb of Antenoron filiforme. METHODS: The constituents were separated by column chromatography and their structures were elucidated by spectral data analyses. RESULTS: Eleven compounds were isolated from the ethanol extract of A. filiforme and identified as, bronane-5-hydroxy-2-O-beta-D-glucopyranoside (I), adenosine (II), bonaroside (III), rhamnetin (IV), hyperoside (V), rhamnetin-3-O-beta-D-galactopyranoside (VI), kaempferol-3, 7-O-bis-alpha-L-rhamnopyranoside (VII), stigmasterol (VIII), nonacosanoic acid (IX), daucosterol (X), 3beta-sitosterol (XI). CONCLUSION: All compounds are obtained from A. filiforme for the first time.

Tyrosinase inhibition by water and ethanol extracts of a far eastern sea cucumber, Stichopus japonicus.

BACKGROUND: Tyrosinase plays a key role in hyperpigmentaion and enzymatic browning. The present study was aimed at investigating the inhibitory effects of water and 70% aqueous ethanol extracts of Stichopus japonicus, a sea cucumber long consumed as a tonic food and traditional medicine, on the diphenolase activity of tyrosinase. RESULTS: In the tyrosinase inhibition study, high-performance liquid chromatography completely separated L-3,4-dihydroxyphenylalanine and dopachrome from other compounds present in the extracts, and provided more reliable results than the commonly used spectrophotometry. The ethanol extract (IC(50)=0.49-0.61 mg mL(-1)) showed higher inhibitory activity than the water extract (IC(50)=1.80-1.99 mg mL(-1)). Enzyme inhibition by the extracts was reversible and of mixed type. For both extracts, the dissociation constants for binding to free enzyme were significantly smaller than those for binding to enzyme-substrate complex. Ethyl-α-D-glucopyranoside (IC(50)=0.19 mg mL(-1)), isolated for the first time from sea cucumber, and adenosine (IC(50)=0.13 mg mL(-1)), were identified as key tyrosinase inhibitors. CONCLUSION: The sea cucumber extracts were demonstrated to possess considerable inhibitory potency against the diphenolase activity of tyrosinase, suggesting that the sea cucumber may be a good source of safe and effective tyrosinase inhibitors.

Isolation of adenosine, iso-sinensetin and dimethylguanosine with antioxidant and HIV-1 protease inhibiting activities from fruiting bodies of Cordyceps militaris.

According to previous studies, a close relationship between oxidative stress and AIDS suggests that antioxidants might play an important role in the treatment of AIDS. Cordyceps militaris was selected from nine edible mushrooms by assay of inhibition of erythrocyte hemolysis. Macroporous adsorption resin and HPLC were used to purify three micromolecular compounds named L3a, L3b and L3c. L3a was identified to be adenosine with the molecular formula C(10)H(13)N(5)O(4); L3b was 6,7,2',4',5'-pentamethoxyflavone with the molecular formula C(20)H(20)O(7), and L3c was dimethylguanosine with the molecular formula C(12)H(17)N(5)O(5). The compound 6,7,2',4',5'-pentamethoxyflavone was first isolated from C. militaris. The assay of inhibition of HIV-1 protease (HIV-1 PR) was based on the fact that the expression of this enzyme can inhibit the growth of E. coli. This is a new screening system for HIV-1 PR inhibitors. Both L3a and L3b showed high inhibition to HIV-1 PR. These compounds could be new anti-HIV-1 PR drugs.

[Studies on the chemical constituents from aerial parts of Gynura divaricata].

OBJECTIVE: To study the chemical constituents from aerial parts of Gynura divaricata. METHODS: The constituents were isolated on silica gel column chromatography, preparative TLC and Sephadex LH-20 column chromatography, identified by physicochemical properties and the structures were elucidated by spectral analysis. RESULTS: 10 compounds were isolated and identified as 2-(1', 2', 3', 4'-tetrahydroxybutyl)-6-(2", 3", 4"-trihydroxybutyl)-pyrazine (1), 2-(1', 2', 3', 4'-tetrahydroxybutyl)-5-(2", 3", 4"-trihydroxybutyl) -pyrazine (2), nicotinic acid (3), 5-hydroxy-picolinic acid(4), methyl-5-hydroxy-2- pyridinecarboxylate (5), adenosine (6), uridine (7), stigmasterol-5-O- beta-D-glucoside (8), dibutyl terephthalate (9), methyl chlorogenate (10). CONCLUSION: Compounds 1, 2, 5, 9, 10 are obtained from this genus for the first time, Compounds 3, 4 are obtained from this plant for the first time.

[Study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products].

OBJECTIVE: To study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products. METHODS: To determinate 13 samples of Cordyceps sinensis and its similar products by HPLC, and analyze the HPLC results with similar appraisal method and graphical methods of multivariate sample in two dimensional plane such as the methods of profile, radar chart and constellation graph. RESULTS: The similar appraisal method might synthesize the similar degree in quantification, while the graphical methods such as profile graph, radar chart and constellation graph could show more details about the classification and the characteristic of varieties directly. CONCLUSIONS: We recommend the combined application of similar appraisal method and the graphical methods due to its advantages on the judgment and characteristic analysis of fingerprint.

Immunomodulatory effect of Antrodia camphorata mycelia and culture filtrate.

AIM OF THE STUDY: Antrodia camphorata, a precious folkloric medicinal mushroom, has been used to treat tumorigenic diseases in Taiwan. This study was to investigate the innate immunity augmentation effects of different fractions prepared from hot water extracts of submerged cultured Antrodia camphorata (AC). MATERIALS AND METHODS: The cytokine induction potency of AC fraction in diluted peripheral blood culture was measured by ELISA. The effects of AC fraction on phagocytic activity and CD11b expression were measured by the ingestion of FITC-labeled Escherichia coli and by labeling with PE-labeled CD11b monoclonal antibody, respectively, using flow cytometry. The molecular mass of hot water-soluble polysaccharides and content of adenosine in AC fraction were determined by gel permeation chromatography (GPC) and HPLC, respectively. RESULTS: The mycelia fraction, Fr. M II, and culture filtrate fractions, Fr. E II and Fr. E III, showed the strongest TNF-alpha and IL-6 induction effect as a function of their concentration. These fractions (20mug/ml) also showed marked activity in enhancing phagocytosis in human polymorphonuclear neutrophils (PMN) and monocytes. In parallel, the expression of CD11b, an early marker of PMN activation, was also up-regulated dose-dependently. Composition analysis suggested that immunomodulatory effect of mycelia is mainly attributed to the 10-20kDa polysaccharides and adenosine. CONCLUSIONS: These results provide evidences that Antrodia camphorata can modulate innate immunity and may serve as an adjuvant for tumor treatment.

[Studies on chemical constituents of the seeds of Allium cepa].

OBJECTIVE: To study the chemical constituents from the seeds of Allium cepa L., the constituents of the seeds of Allium cepa L. METHODS: To isolate and purify by silica gel, macroporous resin HP-20, Sephadex LH-20, RP-18 column. RESULTS: Seven compounds were isolated from the EtOH extract of the seeds of Allium cepa., their structures were elucidated by physico-chemical properties and spectroscopic analysis as tianshic acid (I), N-trans-feruloyl tyramine (II), beta-sitosterol-3 beta-glucopyranoside-6'-palmitate (III), sitosterol (IV), daucosterol (V), tryptophane (VI), adenine riboside (VI). CONCLUSION: Compounds V-VIII are obtained from this plant for the first time, compounds I-IV are isolated from the genus Allium for the first time.

[Studies on the chemical constituents of the roots of Anemone altaica].

OBJECTIVE: To investigate the chemical constituents of the roots of Anemone altaica Fisch. ex C. A. May. METHODS: The constituents of n-BuOH-soluble portion were isolated and purified by means of chromatography. Compounds were identified by their physical characteristics and spectral features. RESULTS: Six compounds were isolated and identified as cimigenol-3-O-beta-D-xylopyranoside (1), cimigenol-3-O-beta-D-xylopyranol (1 -->3)-beta-D-xylopyranoside (2), isolariciresinol-9-O-beta-D-glucopyranoside (3), adenosine (4), uridine (5) and methyl-beta-D-glucopyranoside (6). CONCLUSION: All compounds are isolated from this genus for the first time.