Quality control of Cordyceps sinensis, a valued traditional Chinese medicine

Cordyceps sinensis, a well-known and valued traditional Chinese medicine, is also called DongChongXiaCao (winter worm summer grass) in Chinese. It is commonly used to replenish the kidney and soothe the lung for the treatment of fatigue, night sweating, hyposexualities, hyperglycemia, hyperlipidemia, asthemia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease, and liver disease. As the rarity and upstanding curative effects of natural Cordyceps, several mycelial strains have been isolated from natural Cordyceps and manufactured in large quantities by fermentation technology, and they are commonly sold as health food products in Asia. In addition, some substitutes such as Cordyceps militaris also have been used and adulterants also confused the market. Therefore, quality control of C. sinensis and its products is very important to ensure their safety and efficacy. Herein, markers and analytical methods for quality control of Cordyceps were reviewed and discussed.     

Analysis of sterols and fatty acids in natural and cultured Cordyceps by one-step derivatization followed with gas chromatography–mass spectrometry

Ten free fatty acids namely lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, docosanoic acid and lignoceric acid and four free sterols including ergosterol, cholesterol, campesterol and β-sitosterol in natural (wild) Cordyceps sinensis, Cordyceps liangshanensis and Cordyceps gunnii, as well as cultured C. sinensis and Cordyceps militaris were first determined using pressurized liquid extraction (PLE), trimethylsilyl (TMS) derivatization and GC–MS analysis. The conditions such as the amount of reagent, temperature and time for TMS derivatization of analytes were optimized. Under the optimum conditions, all calibration curves showed good linearity within the tested ranges. The intra- and inter-day variations for 14 investigated compounds were less than 3.4% and 5.2%, respectively. The results showed that palmitic acid, linoleic acid, oleic acid, stearic acid and ergosterol are main components in natural and cultured Cordyceps which could be discriminated by hierarchical clustering analysis based on the contents of 14 investigated compounds or the 4 fatty acids, where the contents of palmitic acid and oleic acid in natural Cordyceps are significantly higher than those in the cultured ones.     

冬虫夏草繁育品质量控制和药理活性研究进展

冬虫夏草为传统的名贵中药材,在中国有悠久的药用历史,具有补肾、益肺、止血、化痰的功效。近年来,冬虫夏草繁育品的研究获得突破,实现了冬虫夏草的大规模产业化繁育。冬虫夏草繁育品基原符合中国药典规定,与野生冬虫夏草相比,冬虫夏草繁育品不仅具有相似的化学成分和药理活性,还具有较低的重金属含量,安全性更容易控制。     

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.     

冬虫夏草人工繁育品、野生品及亚香棒虫草中氨基酸比较

目的：比较冬虫夏草人工繁育品、野生品与亚香棒虫草中水解和游离氨基酸的含量。方法：采用氨基酸分析仪测定冬虫夏草人工繁育品、野生品以及亚香棒虫草中17种氨基酸的含量。结果：冬虫夏草人工繁育品的水解氨基酸含量为16.776%~19.080%,野生冬虫夏草为14.857%~21.959%,亚香棒虫草为13.043%~14.933%。冬虫夏草人工繁育品的游离氨基酸含量为1.767%~2.373%,野生冬虫夏草为1.753%~2.521%,亚香棒虫草为2.856%~3.197%。结论：冬虫夏草人工繁育品和野生品中氨基酸含量基本一致,和亚香棒虫草有显著性差异。本研究为冬虫夏草的鉴别及人工繁育品的进一步开发利用提供科学依据。     

基于代谢组学技术的虫草鉴别研究

目的 采用超高效液相色谱-飞行时间质谱（UPLC-Q-TOF-MS）对不同产地虫草化学成分进行鉴定与比较分析。方法 采用超高效液相-飞行时间质谱（UPLC-Q-TOF-MS）及多元统计分析技术对实验结果进行分析,并对差异化合物进行鉴定。结果 对不同产地的虫草进行了成分鉴定,核苷类物质含量高。不同产地虫草药材在组分种类和相对量上存在着一定的差异。湖南、湖北、丽江的虫草含有其他成分,丽江虫草含有大量的虫草素,湖南虫草含有一些黄酮类物质。采用主成分分析（PCA）将不同产地药材分别聚成不同组别,其中西藏（那曲）、四川、青海之间聚集紧密,而凉山、湖北、湖南及丽江虫草散布在四周,差别非常显著。结论 UPLC-Q-TOF-MS结合多元统计分析方法可为虫草药材的质量评价提供依据。     

Composition of the endophytic filamentous fungi isolated from the tea plant <Emphasis Type="Italic">Camellia sinensis</Emphasis>

It has been found by ribosomal DNA analysis that the endophytic filamentous fungi isolated from the tea plant Camellia sinensis (Theaceae) are composed of six groups; one Fusarium sp., one Penicillium sp., two Schizophyllum sp., and two Diaporthe sp..     

Determination of nucleosides and nucleobases in different species of Cordyceps by capillary electrophoresis–mass spectrometry

In present study, a capillary electrophoresis–mass spectrometry (CE–MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2′-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 μL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 °C, respectively. The optimized CE–MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138 ± 4823 μg/g for cultured C. sinensis; 3722 ± 1446 μg/g for C. militaris) than in natural ones (2167 ± 412 μg/g). However, the hypoxanthine (131 ± 47 μg/g) and inosine (335 ± 90 μg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5 ± 842.6 μg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.     

Stability of Lidocaine in Aqueous Solution: Effect of Temperature,pH, Buffer,and Metal Ions on Amide Hydrolysis

The degradation of lidocaine in aqueous solution obeys the expression k _obs = (k _H+[H ⁺] + k _o ) [H⁺]/([H ⁺ ] + K _a + k_o K _a([H ⁺ ] + K _a) where k _H+ is the rate constant for hydronium ion catalysis, and k _o and k_o are the rate constants for the spontaneous (or water-catalyzed) reactions of protonated and free-base lidocaine. At 80°C, the rate constants for these processes are 1.31 × 10^–7 M ^–l sec^–1, 1.37 × 10^–9 sec^–1, and 7.02 × 10^–9sec^–1; the corresponding activation energies are 30.5, 33.8, and 26.3 kcal mol^–1, respectively. It was found that the room temperature pH of maximum stability is 3–6 and that lidocaine is more reactive in the presence of metal ions such as Fe²⁺ and Cu²⁺. The dissociation constant, K _a, for lidocaine at 25–80°C was also measured at 0.1 M ionic strength and a plot of pK _a versus 1/T gave a slope of (1.88 ± 0.05) × 10³ K^–1 and intercept 1.56 ± 0.16.     

Anti-staphylococcal activity of ent-kaurane-type diterpenoids from Croton tonkinensis

Ent-kaurane-type diterpenpoids 1–11, isolated from the dried leaves of the endemic Vietnamese medicinal plant Croton tonkinensis Gagnep. (Euphorbiaceae), were evaluated for inhibitory activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) strains. The most active diterpenoids, 2, 3, and 8, exhibited minimum inhibitory concentrations (MICs) of 32, 500, and 125 g/ml, respectively, against MRSA strains.     

In vitro antimicrobial activity of a chitooligosaccharide mixture against Actinobacillus actinomycetemcomitans and Streptococcus mutans

The purpose of this study was to evaluate the in vitro antibacterial activity of a chitooligosaccharide mixture (MW 2000–30 000 Da) with a deacetylation degree of 91.5% against two representative oral pathogens, Actinobacillus actinomycetemcomitans and Streptococcus mutans. A 0.1% concentration of the chitooligosaccharides (derived from the exoskeletons of marine crustaceans) was used to estimate antibacterial activity. Approximately 2 log colony forming units (CFU)/ml of A. actinomycetemcomitans were inactivated by 0.1% chitosan after 30 min, while 120 min exposure inactivated about 4.5 log CFU/ml of this organism. In contrast, the level of inactivation against S. mutans was less than 0.5 log CFU/ml after an exposure of up to 120 min. Electron microscopy showed that the exposure of A. actinomycetemcomitans to the chitooligosaccharides resulted in the disruption of cell membranes and that it could be considered for the treatment of periodontal diseases associated with A. actinomycetemcomitans.     

Analysis of three lupane type triterpenoids in Helicteres angustifolia by high-performance liquid chromatography

Kang-chih-ma is the dried roots and stems of Helicteres angustifolia (Sterculiaceae) and a commonly used folk herbal drug in Taiwan. It possesses anti-dotal, analgesic, anti-inflammatory and anti-bacterial effects and is also known as a kind of tumor inhibitory plant. To evaluate the quality of H. angustifolia, a simple, rapid and accurate high-performance liquid chromatographic (HPLC) method was developed for the assay of three lupane type triterpenes: 3β-acetoxy-27-benzoyloxylup-20(29)-en-28-oic acid methyl ester (methyl helicterate), 3β-acetoxy-27-benzoyloxylup-20(29)-en-28-oic acid, and 3β-acetoxy-27-(p-hydroxyl) benzoyloxylup-20(29)-en-28-oic acid methyl ester. The present HPLC system used an Inertsil ODS-2 column by gradient elution with acetonitrile and water as the mobile phase and detected at UV 230 nm. Regression equations revealed good linear relationships (correlation coefficients: 0.9922–0.9997). The relative standard deviations of these three constituents ranged between 1.05–3.14% (intraday) and 2.12–4.38% (interday). The contents of these three constituents of the heartwood and the bark of the roots of H. angustifolia in five different samples have also been determined.     

Separation and determination of coumarins in Fructus cnidii extracts by pressurized capillary electrochromatography using a packed column with a monolithic outlet frit

The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15 min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at −6 kV applied voltage. The calibration curves were linear in the range of 10.0–100.0 μg/mL for bergapten, 20.0–200.0 μg/mL for imperatorin, 5.0–400.0 μg/mL for osthole, 10.0–100.0 μg/mL for 2′-acetylangelicin, 10.0–200.0 μg/mL for oroselone, and 10.0–200.0 μg/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.     

Effects of Rhodocodon madagascariensis extracts on embryo–larval development of medaka fish, Oryzias latipes

Rhodocodon madagascariensis, also named Urginea mascarenensis, is a malagasy plant belonging to the Hyacinthaceae family. As for the other members of the endemic malagasy genus Rhodocodon, the chemical and toxicological properties of this species have not yet been studied. The present study concerns the analysis of the toxicity of R. madagascariensis to medaka embryo–larval development. The incubation of medaka fish embryos or larvae in a medium containing R. madagascariensis extract resulted in a dose dependent reduction in development of embryos leading to lethality and a drastic reduction in survival rate of exposed larvae. Survival rates were reduced up to 100% with an extract concentration of 4 mg mL⁻¹. The LD₅₀ was estimated to be 1 mg mL⁻¹. Anatomopathological studies did show some neuro-embryonal modifications in the encephalic region. The data presented in this paper thus extends the use of medaka embryos as a valuable model to detect and analyse the effects of plant toxins.     

Toxicity of Lithium to Three Freshwater Organisms and the Antagonistic Effect of Sodium

Lithium (Li) is the lightest metal and occurs primarily in stable minerals and salts. Concentrations of Li in surface water are typically <0.04mg l^–1 but can be elevated in contaminated streams. Because of the general lack of information concerning the toxicity of Li to common toxicity test organisms, we evaluated the toxicity of Li to Pimephales promelas (fathead minnow), Ceriodaphnia dubia, and a freshwater snail (Elimia clavaeformis). In the laboratory, the concentration of Li that inhibited P. promelas growth or C. dubia reproduction by 25% (IC25) was dependant upon the dilution water. In laboratory control water containing little sodium (2.8mg l^–1), the IC25s were 0.38 and 0.32mg Li l^–1 and in ambient stream water containing 17mg Na l^–1, the IC25s were 1.99 and 3.33, respectively. A Li concentration of 0.15mgl^–1 inhibited the feeding of E. clavaeformis in laboratory tests. Toxicity tests conducted to evaluate the effect of sodium on the toxicity of Li were conducted with fathead minnows and C. dubia. The presence of sodium greatly affected the toxicity of Li. Fathead minnows and Ceriodaphnia, for example, tolerated concentrations of Li as great as 6mg l^–1 when sufficient Na was present. The interaction of Li and Na on the reproduction of Ceriodaphnia was investigated in depth and can be described using an exponential model. The model predicts that C. dubia reproduction would not be affected when animals are exposed to combinations of lithium and sodium with a log ratio of mmol Na to mmol Li equal to at least 1.63. The results of this study indicate that for most natural waters, the presence of sodium is sufficient to prevent Li toxicity. However, in areas of historical disposal or heavy processing or use, an evaluation of Li from a water quality perspective would be warranted.     

High-performance liquid chromatography—Two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis

A simple and sensitive HPLC–DAD method has been developed for the first time to simultaneously determine 10 triterpenoid acids (ceanothic acid, alphitolic acid, zizyberanal acid, zizyberanalic acid, epiceanothic acid, ceanothenic acid, betulinic acid, oleanolic acid, ursonic acid and zizyberenalic acid) in the dried fruit of Ziziphus jujuba (called Dazao) which has been widely used as one of the traditional Chinese medicines (TCMs). This HPLC assay was performed on a reversed-phase C₁₈ column (250 mm × 4.6 mm, 5 μm) with the column temperature at 35 °C. The mobile phase was composed of A (acetonitrile) and B (0.05% aqueous phosphoric acid, v/v). The flow rate was 1.0 ml/min and the detection wave length was set at 205 nm for reference compounds 1–9 and 238 nm for reference compound 10. All calibration curves showed good linear regression (r² > 0.9999) within the test range. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.43–1.72% and 0.53–2.45%, respectively, and overall recoveries of 94.98–104.09% for the 10 compounds analyzed. The validated method was successfully applied for the simultaneous determination of the 10 triterpenoid acids in 42 batches of Dazao which contained 36 cultivars from 22 cultivation regions, and were investigated and authenticated as Z. jujuba. Hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to differentiate and classify the samples based on the contents of the 10 triterpenoid acids. The presented HPLC–DAD method conjugated with chemometrics approach was demonstrated to be very helpful in using Dazao resources, and was possibly useful in chemotaxonomic characterization.     

Group 1 metabotropic glutamate receptors contribute to slow-onset potentiation in the rat CA1 region in vivo

It has been demonstrated in the CA1 region of the hippocampus in vitro, and in the dentate gyms and CA1 region in vivo, that application of the metabotropic glutamate receptor (mGluR) agonist, 1S, 3R-amino cyclopentane 2,3-dicarboxylic acid triggers a slow-onset potentiation of synaptic transmission in the hippocampus. This study examined the involvement of group 1 and 2 mGluRs in this phenomenon in the CA1 region of freely moving rats. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. The group 1 mGluR agonists, 3,5-dihydroxyphenylglycine (DHPG, 20–100 nmol/5 μl) and trans-azetidine-2,4-dicarboxylic acid (ADA, 100 nmol-1 μmol/5 μl) induced a dose-dependent potentiation of basal synaptic transmission. The mGluR antagonist R,S-α-methyl-carboxyphenylglycine (MCPG, 1 μmol), and the group1 mGluR antagonist, S-4- carboxyphenylglycine (4CPG, 100 nmol) competely inhibited the effects of both DHPG and ADA. The group 2 mGluR agonist, (S)-4-carboxy-3-hydroxy phenylglycine (4C3H-PG, 50–200 nmol/5 μl) induced a dosedependent decrease of basal synaptic transmission. These results suggest that in the CA1 region in vivo, slowonset potentiation may be mediated by group 1 mGluRs.     

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M_r = 34,000 under reducing conditions and pI  6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (−70 to 37 °C), pH values (3–9) or in the presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺ and Mn²⁺). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.     

Cytochrome P 450 2C9 phenotyping using low-dose tolbutamide

Objectives The hypoglycaemic drug tolbutamide is used for assessment of CYP2C9 activity in vivo. However, therapeutically active doses of 500 mg bear the risk of hypoglycaemia, and a tolbutamide-derived parameter based on a single plasma or urine concentration reflecting CYP2C9 activity accurately is lacking.Methods We examined tolbutamide and its metabolites 4-hydroxy-tolbutamide and carboxytolbutamide in plasma and urine of 26 healthy, male volunteers up to 24 h after intake of 125 mg tolbutamide using liquid chromatography–tandem mass spectrometry. CYP2C9 genotypes were determined by sequencing of exons 3 and 7. Raw plasma and urine data were compared with pharmacokinetic parameters, CYP2C9 genotypes, and data from a study in 23 volunteers with all six CYP2C9*1–*3 combinations who received 500 mg tolbutamide.Results Plasma clearance and tolbutamide plasma concentrations 24 h after drug intake reflected the genotypes: 0.85 l/h and 1.70 µg/ml (95% confidence interval, CI, 0.80–0.89 l/h and 1.50–1.90 µg/ml) for CYP2C9*1 homozygotes (n=15), 0.77 l/h and 2.14 µg/ml (95%CI, 0.67–0.88 l/h and 1.64–2.63 µg/ml) for *1/*2 genotypes (n=7), 0.60 l/h and 3.13 µg/ml (95%CI, 0.58–0.62 l/h and 2.68–3.58 µg/ml) for *1/*3 genotypes (n=3), and 0.57 l/h and 3.27 µg/ml in the single *2/*2 carrier. Natural logarithms of tolbutamide plasma concentrations 24 h after intake correlated to plasma clearance (r²=0.84, P<0.0000001). This correlation was confirmed in the comparison data set (r²=0.97, P<0.0000001).Conclusions A low dose of 125 mg tolbutamide can safely and accurately be used for CYP2C9 phenotyping. As a simple metric for CYP2C9 activity, we propose to determine tolbutamide in plasma 24 h after drug intake.     

Design and in vivo evaluation of an indapamide transdermal patch

The aim of the present study was to develop and evaluate a novel drug-in-adhesive transdermal patch system for indapamide. Initial in vitro experiments were conducted to optimize formulation parameters prior to transdermal delivery in rats. The effects of the type of adhesive and the content of permeation enhancers on indapamide transport across excised rat skin were evaluated. The results indicated that DURO-TAK^® adhesive 87-2852 is a suitable and compatible polymer for the development of transdermal drug delivery systems for indapamide. The final formulation contained 4% N-dodecylazepan-2-one, 6% l-menthol and 3% isopropyl myristate. For in vivo studies patch systems were administered transdermally to rats while orally administered indapamide in suspension was used as a control. The PK parameters, such as the maximum blood concentration (C_max), time to reach the peak blood concentration (T_max), mean residence time (MRT), area under the curve (AUC_0–t) and terminal elimination half-life (T_1/2) were significantly (p < 0.05) different following transdermal administration compared with oral administration. In contrast to oral delivery, a sustained activity was observed over a period of 48 h after transdermal administration. This sustained activity was due to the controlled release of drug into the systemic circulation following transdermal administration.