A Discovery-Based Metabolomic Approach Using UPLC-Q-TOF-MS/MS Reveals Potential Antimalarial Compounds Present in Artemisia annua L.

In 1972, Nobel laureate Youyou Tu's research team conducted clinical trials on the dried material of Artemisia annua L. from Beijing extracted by ether and then treated with alkali (called "ether neutral dry"), which showed that artemisinin was not the only antimalarial component contained. The biosynthesis of sesquiterpenoids in A. annua has increased exponentially after unremitting cultivation efforts, and the plant resources are now quite different from those in the 1970s. In consideration of emerging artemisinin resistance, it is of great theoretical and practical value to further study the antimalarial activity of A. annua and explore its causes. The purpose of this study is to clarify scientific questions, such as "What ingredients are synergistic with artemisinin in A. annua?", and "Are there non-artemisinin antimalarial ingredients in A. annua?". In this paper, Beijing wild A. annua was used as a control and two representative cultivation species of A. annua were selected to evaluate the antimalarial activity of the herbal medicine. The antimalarial activity of different extracts on mice was studied using the Peters' four-day suppressive test. UPLC-Q-TOF-MS was used to obtain mass spectrum data for all samples, and a UNIFI platform was used for identification. A multivariate statistical method was used to screen the different compounds with positive correlations. The antimalarial activity of different components from the ether extract and alkali treatments was determined and antimalarial components other than artemisinin were obtained. A total of 24 flavonoids, 68 sesquiterpenoids and 21 other compounds were identified. Compounds associated with differential antimalarial activity were identified. The material basis for the antimalarial activity of A. annua was clarified. The antimalarial components of A. annua include two categories: first, artemisinin and non-artemisinin antimalarial active components, of which the non-artemisinin antimalarial active components may include 5α-hydroperoxy-eudesma-4(15),11-diene; second, several antimalarial synergistic ingredients in A. annua, including arteanniun B, arteanniun B analogues and polymethoxy flavonoids.

Casticin and chrysosplenol D from Artemisia annua L. induce apoptosis by inhibiting topoisomerase IIα in human non-small-cell lung cancer cells.

BACKGROUND: Artemisia annua L. (A. annua) and its active components exhibit antitumour effects in many cancer cells. However, the biological processes and mechanisms involved are not well understood, especially for the treatment of non-small-cell lung cancer (NSCLC). PURPOSE: This study aimed to comprehensively explore the biological processes of A. annua and its active components in NSCLC cells and to identify the mechanism by which these compounds induce apoptosis. STUDY DESIGNS/METHODS: Cell viability and flow cytometry assays were used to evaluate the cytotoxicity of A. annua active components casticin (CAS) and chrysosplenol D (CHD) in A. annua in NSCLC cells. After treatment with CAS and CHD, A549 cells were subjected to RNA sequencing (RNA-seq) analysis, differentially expressed genes (DEGs) were screened and subjected to functional enrichment analysis (KEGG and GO analysis) as well as protein interaction network analysis. The key targets associated with apoptosis induction in A549 cells were screened by Cytoscape, and the screened DEGs were validated by qRT-PCR. Immunoblotting, immunofluorescence, and molecular docking assays were used to determine whether CAS and/or CHD could induce apoptosis in NSCLC cells by inducing DNA damage through down-regulation of topoisomerase IIα (topo IIα) expression. The same experiments were verified again in the H1299 lung cancer cell line. RESULTS: CAS and CHD inhibited NSCLC cells proliferation in a time- and dose-dependent manner, and significantly induced apoptosis. A total of 115 co-upregulated DEGs and 277 co-downregulated DEGs were identified in A549 cells following treatment with CAS and CHD. Comprehensive and systematic data about biological processes and mechanisms were obtained. DNA damage pathways and topo IIα targets were screened to study the apoptosis effects of CAS and CHD on NSCLC cells. CAS and CHD may be able to induce DNA damage by binding to topo IIα-DNA and reducing topo IIα activity. CONCLUSION: This study suggested that CAS and CHD may reduce topo IIα activity by binding to topo IIα-DNA, affecting the replication of DNA, triggering DNA damage, and inducing apoptosis. It described a novel mechanism associated with topo IIα inhibition to reveal a novel role for CAS and CHD in A. annua as potential anticancer agents and/or adjuvants in NSCLC cells.

Affinity ultrafiltration and UPLC-HR-Orbitrap-MS based screening of thrombin-targeted small molecules with anticoagulation activity from Poecilobdella manillensis.

This study aims to screen potential anticoagulant components from leeches, a representative animal-sourced traditional Chinese medicine using thrombin (THR)-targeted ultrafiltration combined with ultrahigh performance liquid chromatography and high-resolution Orbitrap mass spectrometry (UPLC-HR-Orbitrap-MS). As a result, five small molecules in leech extract were discovered to interact with THR for the first time. Among them, two new compounds were isolated and their structures were identified by IR, HR-MS and NMR data. Furthermore, their THR inhibitory activity was confirmed with IC50 values of 4.74 and 8.31 µM, respectively. In addition, molecular docking analysis showed that the active (catalytic) site of THR might be the possible binding site of the two hits. Finally, reverse screening analysis indicated that LTA4-H, ACE and ALOX5AP were potential anticoagulant targets of the two new compounds. This study will broaden our understanding of the medicinal substance basis in leeches and further contribute to the discovery and development of clinical anticoagulant drugs from leeches.

[Research advances of biosynthesis, in vivo analysis and pharmacokinetics of chemical constituents in Artemisiae Annuae Herba].

Artemisiae Annuae Herba is a traditional Chinese medicine for clearing deficiency and heat. It is the only natural source of artemisinin, which is a specific antimalarial drug, and has been widely concerned all over the world. In addition to artemisinin, Artemisiae Annuae Herba also contains many sesquiterpenes, coumarins, flavonoids, volatile oils, polysaccharides and other chemical components, which show antipyretic, anti-inflammatory, antiviral microorganisms, anti-asthma, anti-oxidation, anti-tumor and other pharmacological activities. In addition to their own pharmacological activities, some components could enhance the antimalarial activity of artemisinin through different mechanisms at absorption and metabolism in vivo. In order to understand the pharmacokinetic characte-ristics of the chemical constituents contained in Artemisiae Annuae Herba and provide reference for the full development and clinical utilization of Artemisiae Annuae Herba resources in China, this present paper systematically collated the modern research literatures, and summarized the biosynthesis, in vivo analysis and pharmacokinetics of the chemical constituents in Artemisiae Annuae Herba.

Simultaneous Quantification of Five Sesquiterpene Components after Ultrasound Extraction in Artemisia annua L. by an Accurate and Rapid UPLC⁻PDA Assay.

Objective: To develop an accurate and rapid ultra-performance liquid chromatography (UPLC) coupled with a photodiode array (PDA) method for the simultaneous determination of artemisinin (Art), arteannuin B (Art B), arteannuin C (Art C), dihydroartemisinic acid (DHAA) and artemisinic acid (AA) in Artemisia annua L. Methodology: Chromatography separation was performed on an ACQUITY UPLC BEH C18 Column with isocratic elution; the mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B) (A:B = 40:60, v/v). Data were recorded at an ultraviolet (UV) wavelength of 191 nm for Art, Art C, DHAA and AA, and 206 nm for Art B. Results: The calibration curves of the five sesquiterpene components were all linear with correlation coefficients more than 0.9990. The linear ranges were 31.44-1572 µg/mL, 25.48-1274 µg/mL, 40.56-2028 µg/mL, 31.44-1572 µg/mL and 26.88-1396 µg/mL for Art, Art B, Art C, DHAA and AA, respectively. The precision ranged from 0.08% to 2.88%, the stability was from 0.96% to 1.66%, and the repeatability was all within 2.42% and had a mean extraction recovery of 96.5% to 100.6%. Conclusion: The established UPLC-PDA method would be valuable for improving the quantitative analysis of sesquiterpene components in Artemisia annua L.

Safety evaluation of Semen Sojae Preparatum based on simultaneous LC-ESI-MS/MS quantification of aflatoxin B1 , B2 , G1 , G2 and M1.

Semen Sojae Preparatum (SSP) is one of the most widely used traditional Chinese medicines, and is also a functional food. However, contamination with aflatoxins may occur in the fermentation process. To evaluate its safety, an accurate and rapid LC-ESI-MS/MS analytical method was developed and validated for the simultaneous determination of AFB1 , AFB2 , AFG1 , AFG2 and AFM1 in SSP. After a simple ultrasonic extraction of SSP samples, chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (2.1 × 50 mm, 3.5 µm) with a flow rate of 0.50 mL/min. The gradient elution program was performed using a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. Detection of five aflatoxins was based on triple quadrupole mass spectrometry using a multiple reaction monitoring mode with an electrospray ionization source. SSP is likely to be contaminated by aflatoxins in the processes of fermentation, storage, transportation and usage, and it is necessary to strictly monitor it. Artemisia annua L. and Morus alba L. may inhibit the production and growth of AFB1 - and AFB2 -producing fungi, which has a certain detoxification effect on contamination with aflatoxins in SSP.

[Construction of artesunate nanoparticles modified by hyaluronic acid and cell-penetrating peptides and its inhibitory effect on cancer cells in vitro].

Hyaluronic acid (HA) and cell-penetrating peptide (CPP) R6H4-SA modified artesunate nanostructured lipid carrier (HA-R6H4-NLC/ART) for anti-tumor therapy was prepared. The physicochemical properties and in vitro drug release of HA-R6H4-NLC/ART were evaluated, and the uptake and cytotoxicity of liver cancer HepG2 cells were studied. The results showed that HA-R6H4-NLC/ART was spherical like in appearance, and the average particle size was about 160 nm. In vitro release experiments showed that the drug delivery system had sustained release characteristics. Cell results showed that, in slightly acidic environment, pH sensitive CPP R6H4-SA mediated cellular uptake of nanoparticles was significantly higher than that of non-sensitive peptide R8-SA. Meanwhile, HA-R6H4-NLC/ART had a targeting effect on HepG2 cells, and the HA receptor saturation experiment showed that the endocytosis of HA-R6H4-NLC/ART was mediated by the HA receptor on the cell surface. As compared with the unmodified or R6H4-SA single modified group, HA and R6H4-SA co-modified HA-R6H4-NLC/ART significantly improved the cell uptake and had a stronger anti-tumor effect under the conditions of the slightly acid environment and hyaluronidase degradation. The above results showed that hyaluronic acid and CPP R6H4-SA co-modified artesunate nanostructured lipid carrier, which can effectively identify and penetrate the tumor cell membrane into the cell, is a potentially efficient targeting delivery system for anti-tumor drugs.

Simultaneous high-performance liquid chromatography with diode array detection and time-of-flight mass spectrometric confirmation of the ten bioactive compounds in Semen Sojae Preparatum.

Semen Sojae Preparatum is one of the most widely used traditional Chinese medicines. A reliable and accurate high-performance liquid chromatography with diode array detection method has been developed and validated for the quantitative determination of the ten bioactive compounds contained in Semen Sojae Preparatum. The samples were first extracted by pressurized liquid extraction using 80% ethanol at 100°C for 15 min and three static extraction cycles. Chromatographic separation was conducted on a C18 column using a mobile phase consisting of water and acetonitrile under gradient elution, and the detection wavelength was set at 210 nm. The samples were further analyzed on a high-performance liquid chromatography with time-of-flight mass spectrometry system to confirm the determination results. All the ten analytes were well separated, and the calibration curves showed good linearity. The intra- and interday precisions were evaluated in terms of relative standard deviation values within the ranges of 0.20-1.43% and 0.40-4.78%, respectively. The recoveries for the ten analytes were all in the ranges of 96.2-104.3%, with relative standard deviation values < 3.85%. The established high-performance liquid chromatography method could serve as a reliable and accurate method for the quality evaluation of Semen Sojae Preparatum from different origins.

[History of processing of Sojae Semen Praeparatum].

Sojae Semen Praeparatum (SSP) is commonly used as a type of dietetic Chinese herb. By collecting and analyzing ancient and recent literatures, a textual criticism was conducted on the historical evolution of the processing of SSP. Fermented soybean was recorded in Shijing, and relevant rational processing was described in Qimin Yaoshu. In the early time, fermented soybean included the type of "salty" and "light". After the Ming Dynasty, "light" fermented soybean or SSP was recognized as a better medicinal matter than salty fermented soybean, and the fermentation processing was recorded more clearly. In modern time, many characteristic methods for processing SSP have been developed. Today, the processing of SSP is mainly based on the Chinese Pharmacopoeia, which records soybean as a main ingredient and Artemisiae Annuae Herba, Mori Folium as excipients.

Combination of artemisinin-based natural compounds from Artemisia annua L. for the treatment of malaria: Pharmacodynamic and pharmacokinetic studies.

Currently, the most effective antimalarial is artemisinin, which is extracted from the leaves of medicinal herb Artemisia annua L. (A. annua). Previous studies showed that the complex chemical matrix of A. annua could enhance both the bioavailability and efficacy of artemisinin. The present study aims to evaluate the efficacy and pharmacokinetic properties of a combination therapy based on artemisinin and 3 components from A. annua with high content (arteannuin B, arteannuic acid, and scopoletin). In vivo antimalarial activity was assessed following a 4-day treatment in murine malaria models (Plasmodium yoelii and Plasmodium berghei). Results showed that a much sharper reduction in parasitemia (~93%) was found in combination therapy compared with pure artemisinin (~31%), indicating pharmacodynamic synergism occurring between artemisinin and arteannuin B, arteannuic acid, and scopoletin. Multiple-dose pharmacokinetics further demonstrated that combination therapy results in increased area under the curve (AUC0→∞ ), Cmax , and t1/2 by 3.78-, 3.47-, and 1.13-fold in healthy mice, respectively, and by 2.62-, 1.82-, and 1.22-fold in P. yoelii-infected mice, respectively. The calculated oral clearance of combination therapy in healthy and P. yoelii-infected mice was also reduced. These findings imply that specific components in A. annua might offer a possibility to develop new artemisinin-based natural combination therapy for malaria treatment.

Two new guaiane-type sesquiterpenoids from Valeriana hardwickii and their cytotoxicity.

Two new guaiane-type sesquiterpenoids, named 4α,5α-epoxy-8ß-hydroxy-1α-hydro-α-guaiene (1) and 4α,5α-epoxy-1-hydroxy-α-guaiene (2), were isolated from the whole plants of Valeriana hardwickii. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1 and 2 showed weak cytotoxicity against the lung adenocarcinoma (A549) and hepatoma (Bel7402) cell lines with IC50 values of 9.2 and 8.5 µM, respectively.

Identification of active ingredients in Wuzhuyu decoction improving migraine in mice by spectral efficiency association.

Wuzhuyu decoction is a traditional Chinese medicine used for the effective treatment of migraines, termed 'Jueyin headache', in China. However, there have been few investigations to clarify the composition of Wuzhuyu decoction for the treatment of migraines. In the present study, 10 types of Wuzhuyu decoction were analyzed by chromatograms. 5-hydroxytryptamine (5-HT)-depletion mouse models of migraine were prepared by subcutaneous injection of reserpine and placement of autologous blood clots in the cerebral cortex. The levels of 5-HT, noradrenaline (NE), dopamine (DA), nitric oxide (NO) and nitric oxide synthase (NOS) in the brain tissues and sera of the mice were determined. The ingredients and pharmacodynamic indices of the Wuzhuyu decoctions were analyzed using spectral efficiency association by partial least squares regression. The levels of 5-HT, NE and DA in the mouse brain tissues were reduced to 337.785 ± 84.504, 171.173 ± 65.172 and 242.075 ± 158.621 mg/g brain tissue, respectively. The level of NO in the brain tissues increased to 0.425 ± 0.184 µmol/g protein and the activities of NOS in the brain tissues and sera increased to 0.719 ± 0.477 U/mg and 50.688 ± 8.132 U/ml, respectively. Regarding the ingredients of the Wuzhuyu decoction, those with significant regression coefficients were ginsenoside-Rg1, Re, Rb1, rutaevine (Rv), limonin (Li), evodiamine (Ev), rutaecarpine (Ru) and substance X (awaiting identification). Rg1, Re, Rb1, Rv, Li, Ev, Ru and X in the Wuzhuyu decoction were observed to yield the pharmacological effects, whereas Rb1, Rv and Ev were important in index improvement.

[Scientific connotation of processing Bombyx Batryticatus under high temperature].

The aim of this study was to elucidate the scientific connotation of Bombyx Batryticatus processing with wheat bran under high temperature. The contents of soluble protein extracted from Bombyx Batryticatus and its processed products and the limited content of AFT in Bombyx Batryticatus and the processed one were compared. The concentration of protein was measured with the Bradford methods and the difference of protein between Bombyx Batryticatus and its processed products was compared by SDS-PAGE analysis. Aflatoxin B1, B2, G1, and G2 were determined by reversed-phase HPLC. The results showed that the soluble protein content of Bombyx Batryticatus and its processed products were (47.065 +/- 0.249), (29.756 +/- 1.961) mg x g(-1), correspondingly. Analysis of protein gel electrophoresis showed that there were no significant differences between the crude and processed one in protein varieties. 6 bands were detected: 31.90, 26.80, 18.71, 15.00, 10.18, 8.929 kDa. Below 10 kDa, the color of bands of the processed one was deeper than the crude one, which demonstrate that macromolecular protein was degradated into micromolecule. The content of AFG1, AFB1, AFG2, AFB2 were 0.382, 0.207, 0.223, 0.073 g x kg(-1), not exceeded 5 microg x kg(-1) while the processed one was not detected. Through processing with wheat bran under high temperature, the content of soluble protein in Bombyx Batryticatus decreased, the processing purpose for alleviating drug property was achieved. Meanwhile, the limited content of aflatoxins were reduced or cleared by processing procedure or absorbed by processing auxillary material, adding the safety of the traditional Chinese Medicine. In conclusion, as a traditional processing method, bran frying Bombyx Batryticatus was scientific and reasonable.

[Chemical constituents from whole plants of Valeriana hardwickii].

Chemical investigation of the whole plants of Valeriana hardwickii has led to the isolation of 11 flavones and 2 monoterpe- noids by using various chromatographic techniques including column chromatography on silica gel and Sephadex LH-20, preparative TLC, and preparative HPLC. Their structures were identified by spectroscopic data analysis as syzalterin (1), 6-methylapigenin (2), 5-hydroxy-7,4'-dimethoxyflavone (3), genkwanin (4), acacetin (5), apigenin (6), quercetin (7), tricin (8), (-)-farrerol (9), sosakuranetin (10), 5,3',4'-trihydroxy-7-methoxyflavanone (11), (-)-bornyl ferulate ( 12) , and (-)-bornyl caffeate ( 13). All compounds were isolated from this plant for the first time, while compounds 1, 9-13 were obtained from this genus for the first time.

[Simultaneous quantitation of artemisinin, arteannuin B, artemisic acid, and scopoletin in mice plasma by HPLC-MS].

The objective of this study is to develop a sensitive and reliable high-performance liquid chromatography mass spectrometry (LC-MS) method for simultaneous determination of artemisinin, arteannuin B, artemisic acid, and scopoletin, and study the pharmacokinetics of the four constituents in mouse serum after oral administration of the four components to mice. The analytical column used was Agilent Zorbax SB-C18 (2.1 mm x 150 mm, 5 mm). The mobile phase was acetonitrile: 0.5% acetic acid (60: 40) and the flow rate was 0.3 mL x min(-1). The temperature of the column was 40.0 degrees C. In this condition, we established an analysis method to simultaneously determine the four components. A sensitive and specific liquid chromatography-mass spectrometric (LC-MS) method was developed and validated for the determination of artemisin in derivatives in mice plasma. The method we established has a linear range of 5-3 000 µg x L(-1) with a good sensitivity and specificity for all of the four components. This method is simple, rapid, accurate and suitable for the determination of the content of the four compounds.

Simultaneous quantitative analysis of nine triterpenoid saponins for the quality control of Stauntonia obovatifoliola Hayata subsp. intermedia stems.

Stauntonia obovatifoliola Hayata subsp. intermedia is used in China to treat rheumatic arthralgia, hernia pain, and traumatic pain. An accurate and reliable method based on high-performance liquid chromatography with diode array detection has been developed and validated for the quantitative determination of nine triterpenoid saponins in this herb. By using a Kromasil 100-5 C18 column (250 mm × 4.6 mm, 5 µm), nine analytes were separated by gradient elution over a running time of 45.0 min. All standard calibration curves demonstrated satisfactory linearity (R(2) ≥ 0.9995) within a relatively wide range. The precision was evaluated by intra- and interday tests, which revealed relative standard deviation values within the ranges of 0.20-2.83 and 0.51-2.79%, respectively. The recoveries for the nine target compounds were between 84.6 and 103% with relative standard deviation values less than 2.67%. The samples were also analyzed on a linear trap quadrupole Orbitrap Velos Pro mass spectrometer equipped with an electrospray ionization source in negative mode to confirm the quantification results. In conclusion, the present high-performance liquid chromatography with diode array detection method could serve as an accurate and reliable method for the quality evaluation of Stauntonia obovatifoliola Hayata subsp. intermedia stems.

[Triterpenoids from Stauntonia obovatifoliola Hayata subsp. intermedia stems].

In the current study, a total of nineteen triterpenoids (1-19) from 60% EtOH extracts of Stauntonia obovatifoliola Hayata subsp. intermedia stems were separated and purified by solvent extraction and chromatographic methods including silica gel, ODS as well as preparative HPLC. According to the results of chemical reactions and spectral data, compounds were identified as: lupeol (1), betulinonic acid (2), betulinic acid (3), 3-epi-betulinic acid (4), quinatic acid (5), 24-O-acetyl quinatic acid (6), 3-O-α- L-arabinopyranosyl-30-nor-hederagenin-28-O-α-L-rhamnopyranosyl-(1 --> 4) -ß-D-glucopyranosyl-(1 --> 6) -ß-D-glucopyranosyl ester (7), Stauntoside A (8), kalopanax saponin A (9), kalopanax saponin J (10), Kizuta saponin K10 (11), 3-O-α-L-rhamnopyranosyl (1--> 2) -α-L-arabinopyranosyl-hederagenin-28-O-ß-D-xylopyranosyl-(1 --> 6) -ß-D-glucopyranosyl ester (12), kalopanax saponin B (13), 3-O-α-L-rhamnopyranosyl-(1 --> 2) -α-L-arabinopyranosyl-hederagenin-28-O-ß-D-glucopyranosyl-(1 --> 6) -ß-D-glucopyranosyl ester (14), sieboldianoside A (15), septemoside A (16), kalopanax saponin K (17), septemloside I (18), and 3-O-α-L-arabinopyranosyl (1 --> 2)-ß-D-glucuronopyranosyl- hederagenin (19). Among them, compounds 4, 6, 10, 12, 14, and 16-19 were isolated from the Stauntonia genus for the first time, and compound 6 was a new natural product.

[Study on intervention effect of Danggui Shaoyao San on rats with cirrhotic ascites].

OBJECTIVE: To investigate the intervention effect of Danggui Shaoyao San on rats with cirrhotic ascites, and discuss the effect of arginine vasopressin (AVP) on cirrhotic ascites. METHOD: Male SD rats were randomly divided into the control group, the model group, Danggui Shaoyao San low, middle and high dose groups. The cirrhotic ascites rat model was established by CCl4 combined with phenobarbital. Their urines were collected at 24 h to observe urine excretion of each group. Filter papers were used to determine the amount of ascites. The levels of serum alanine aminotransferasa (ALT) , aspartate aminotransferase (AST) were detected by the automatic biochemistry analyzer. Plasma prothrombin time (PT) was evaluated by the blood coagulation analyzer. The concentration of AVP in plasma was detected by enzyme-linked immunosorbent assay (ELISA). Pathological changes in livers were observed by HE staining. RESULT: Compared with the model group, the Danggui Shaoyao San group showed significant improvement in live indexes, with notable decrease in serum ALT and AST and the time of PT, improvement in liver pathological changes. Simultaneously, the amount of ascites decreased to varying degrees, with notable increase in urine in 24 h and decrease in AVP concentration in plasma. CONCLUSION: Danggui Shaoyao San can notably improve liver functions of rats with cirrhotic ascites, reduce the generation of ascites and delay the progress of liver pathological changes. Its mechanism may be related to AVP.

A new beta-naphthalenecarboxylic acid biglycoside from Chirita longgangensis var. hongyao.

To investigate the chemical constituents in the stems of Chirita longgangensis var. hongyao, methanol extract of the stems was subjected to column chromatography with various chromatographic techniques. One new beta-naphthalenecarboxylic acid biglycoside, 1, 4-dihydroxy-2-naphthalenecarboxylic acid methyl ester-4-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (1) was isolated, along with two known compounds: isotaxiresinol 4-O-methyl ether (2) and (R)-7-hydroxy-alpha-dunnione (3). Compound 2 was first obtained from Chirita genus and compound 3 was isolated from this plant for the first time. All structures were elucidated on the basis of spectral and chemical evidence, and the NMR spectroscopic data of compound 2 was published for the first time.

Simple HPLC evaluation of lipoamidase activity in tissue using a newly synthesized fluorescent substrate, dansyl-α-lipoyllysine.

α-Lipoic acid (LA) is a naturally occurring disulfide-containing compound used as an antioxidant supplement which also has been used as a medicine for diabetic neuropathy in Europe. Physiologically LA acts as a coenzyme of mitochondrial multienzyme complex in its protein bound form but it is not yet clear how the externally administrated LA is incorporated into other proteins in the same protein-bound form or why the bound form is active as an antioxidant. The binding and cleavage of LA to or from the protein is mediated by lipoamidase and thus determines LA distribution in tissues. We have developed a simple sensitive assay for lipoamidase using a fluorescent substrate, dansyl-α-lipoyllysine (DLL). Lipoamidase in tissues cleaves the amide bond between LA and the ε-amino-lysine moiety to release dansylated lysine (DL). A HPLC comparison of the fluorescence intensity between DLL and DL was used to quantify the enzyme activity. The hydrolytic reaction did not occur when the tissue was heat-treated before incubation with DLL and was inhibited by free LA, especially by the R-enantiomer of LA (physiologically active form). N(ε)-Acetyl-L-lysine did not compete with DLL in the cleavage reaction. The method was applied for the determination of lipoamidase activity levels in various rat tissues. It was revealed the spleen had the highest activity followed by the kidney, heart, lung and liver. The activity in the brain was below the detection limit of the assay.