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.

[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.

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.

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.

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.

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.

[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.

[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.

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.

Determination of astragaloside III in rat plasma by liquid chromatography-tandem mass spectrometry and its application to a rat pharmacokinetic study.

Astragaloside III (AST III), a naturally occurring saponin compound isolated from Radix Astragali, has been demonstrated to have anti-gastric ulcer, immunomodulatory and antitumor effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method has been developed and validated for the quantification of astragaloside III in rat plasma. Samples were pretreated using a simple protein precipitation with methanol-acetonitrile (50:50, v/v) and the chromatographic separation was performed on a C18 column by a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Astragaloside III and the internal standard (buspirone) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range of 5.00-5000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that astragaloside III spiked into rat plasma was stable for 24 h at 20°C temperature, for up to 30 days at -80°C, and during three freeze-thaw cycles. The method was successfully used to investigate the pharmacokinetic profile of AST III after oral (10 mg/kg) and intravenous (1.0 mg/kg) administration in rats. The oral absolute bioavailability of AST III was calculated to be 4.15 ± 0.67% with an elimination half-life value of 2.13 ± 0.11 h, suggesting its poor absorption and/or strong metabolism in vivo.

Artemisinin and Its Derivatives as a Repurposing Anticancer Agent: What Else Do We Need to Do?

Preclinical investigation and clinical experience have provided evidence on the potential anticancer effect of artemisinin and its derivatives (ARTs) in the recent two decades. The major mechanisms of action of ARTs may be due to toxic-free radicals generated by an endoperoxide moiety, cell cycle arrest, induction of apoptosis, and inhibition of tumor angiogenesis. It is very promising that ARTs are expected to be a new class of antitumor drugs of wide spectrum due to their detailed information regarding efficacy and safety. For developing repurposed drugs, many other characteristics of ARTs should be studied, including through further investigations on possible new pathways of anticancer effects, exploration on efficient and specific drug delivery systems-especially crossing biological barriers, and obtaining sufficient data in clinical trials. The aim of this review is to highlight these achievements and propose the potential strategies to develop ARTs as a new class of cancer therapeutic agents.

Application of a sensitive and specific LC-MS/MS method for determination of eriodictyol-8-C-ß-d-glucopyranoside in rat plasma for a bioavailability study.

This study is the first to detail the development and validation of a rapid, sensitive and specific LC-ESI-MS/MS method for the determination of eriodictyol-8-C-ß-d-glucopyranoside (EG) in rat plasma. A simple protein precipitation method was used for plasma sample preparation. Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C18 column (2.1 × 50 mm, 3.5 µm) using a step gradient program with the mobile phase of 0.1% formic acid aqueous solution and acetonitrile with 0.1% formic acid. EG and the internal standard (IS) were detected using an electrospray negative ionization mass spectrometry in the multiple reaction monitoring mode. This method demonstrated good linearity and did not show any endogenous interference with the active compound and IS peaks. The lower limit of quantification of EG was 0.20 ng/mL in 50 µL rat plasma. The average recoveries of EG and IS from rat plasma were both above 80%. The inter-day precisions (relative standard deviation) of EG determined over 5 days were all within 15%. The present method was successfully applied to a quantification and bioavailability study of EG in rats after intravenous and oral administration. The oral absolute bioavailability of EG in rats was estimated to be 7.71 ± 1.52%.

[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.

[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.

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.

LC-ESI-MS/MS analysis and pharmacokinetics of plantainoside D isolated from Chirita longgangensis var. hongyao, a potential anti-hypertensive active component in rats.

Plantainoside D (PD) is a potential anti-hypertensive active ingredient newly isolated from the dried plants of Chirita longgangensis var. hongyao. A sensitive and specific LC-ESI-MS/MS method was first developed and validated for the analysis of PD in rat plasma using genistein as the internal standard (IS). The plasma samples were pretreated with methanol-acetonitrile (50:50, v/v) to precipitate protein, and then chromatographed on a reverse-phase Agilent Zorbax XDB C18 column (50 mm × 2.1 mm, 3.5 µm). Gradient elution was utilized, with a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid, and the flow rate was set at 0.50 mL/min. The analytes were monitored by tandem-mass spectrometry with negative electrospray ionization. The precursor/product transitions (m/z) in the negative ion mode were 639.2 → 160.9 Thomson (Th) and 268.9 → 158.9 Thomson (Th) for PD and IS, respectively. Linearity was achieved in the 0.10-200 ng/mL range, with a lower limit of quantification of 0.10 ng/mL. The precision and accuracy for both intra- and inter-day determination of the analyte were all within ±15%. The present method has been applied for pharmacokinetic study of PD after oral and intravenous administration in rats. The oral absolute bioavailability (F) of PD in rats was estimated to be 1.12% ± 0.46% with an elimination half-life (t1/2) value of 1.63 ± 0.19 h, suggesting its poor absorption and/or strong metabolism in vivo.

[Artemisinin resistance in Plasmodium falciparum: global status and basic research].

Artemisinin-resistant Plasmodium falciparum has been identified by WHO in the Greater Mekong subregion. While there is no report on artemisinin resistance in Africa and South America by now, related surveillance measures have been taken place. The genes related artemisinin-resistance has been identified and the molecular markers will be used for large-scale surveillance efforts to contain artemisinin resistance. The emergence and spread of artemisinin resistance worldwide is a present danger and needs more attention. This article reviews the progress of artemisininresistance malaria parasites and artemisinin-based combination therapies.

[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.

[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.

Transformation profiles of the isoflavones in germinated soybean based on UPLC-DAD quantification and LC-QTOF-MS/MS confirmation.

Germinated soybean is one kind of food and a medicine. In the actual process of producing a large amount of naturally germinated soybean, it is difficult to strictly control the germination process conditions. However, sprout length may be more suitable as the terminal judgment indicator for naturally germinated soybean. An UPLC-DAD method was developed and validated to explore the transformation profiles of soybean isoflavones in germinated yellow or black soybean with different sprout lengths. Moreover, an LC - QTOF-MS/MS method was used to avoid false positive results. The contents of daidzein, glycitein, and genistein almost reached their corresponding maximum values when the sprout length ranged from 1.0 cm to 1.5 cm (P < 0.05). Therefore, yellow soybean is suggested to be the processing raw material with higher contents of those isoflavones, and the optimal sprout length for germinated soybean may be in the range of 1.0-1.5 cm.