High-throughput extraction and automatic purification of alternariol from edible and medicinal herbs based on aptamer-functionalized magnetic nanoparticles.

Mycotoxin contamination is widespread in plants and herbs, posing serious threats to the consumer and human health. Of them, alternariol (AOH) has attracted great attention as an "emerging" mycotoxin in medicinal herbs. However, a specific and high-throughput extraction method for AOH is currently lacking. Thus, developing an efficient pre-treatment technique for AOH detection is extremely vital. Here, a novel automated magnetic solid-phase extraction method was proposed for the highly efficient extraction of AOH. Combining the aptamer-functionalized magnetic nanoparticles (AMNPs) and the automatic purification instrument, AOH could be extracted in medicinal herbs in high throughput (20 samples) and a short time (30 min). The main parameters affecting extraction were optimized, and the method was finally carried out by incubation AMNPs with 3 mL of sample solution for 10 min, and then desorption in 75% methanol for liquid-phase detection. Under optimal conditions, good reproducibility, stability, and selectivity were realized with an adsorption capacity of 550.84 ng/mg. AOH extraction in three edible herbs showed good resistance to matrix interference with recovery rates from 86% to 111%. In combination with AMNPs and the automatic purification instrument, high-throughput and labor-free extraction of AOH in different complex matrices was achieved, which could be extended in other complex matrices.

Validation of the shotgun metabarcoding approach for comprehensively identifying herbal products containing plant, fungal, and animal ingredients.

Identifying plant, fungal, and animal ingredients in a specific mixture remains challenging during the limitation of PCR amplification and low specificity of traditional methods. Genomic DNA was extracted from mock and pharmaceutical samples. Four type of DNA barcodes were generated from shotgun sequencing dataset with the help of a local bioinformatic pipeline. Taxa of each barcode was assigned by blast to TCM-BOL, BOLD, and GenBank. Traditional methods including microscopy, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC) were carried out according to Chinese pharmacopoeia. On average, 6.8 Gb shotgun reads were sequenced from genomic DNA of each sample. Then, 97, 11, 10, 14, and one operational taxonomic unit (OTU) were generated for ITS2, psbA-trnH, rbcL, matK, and COI, respectively. All the labeled ingredients including eight plant, one fungal, and one animal species were successfully detected in both the mock and pharmaceutical samples, in which Chebulae Fructus, Poria, and Fritilariae Thunbergia Bulbus were identified via mapping reads to organelle genomes. In addition, four unlabeled plant species were detected from pharmaceutical samples, while 30 genera of fungi, such as Schwanniomyces, Diaporthe, Fusarium were detected from mock and pharmaceutical samples. Furthermore, the microscopic, TLC, and HPLC analysis were all in accordance with the standards stipulated by Chinese Pharmacopoeia. This study indicated that shotgun metabarcoding could simultaneously identified plant, fungal, and animal ingredients in herbal products, which has the ability to serve as a valuable complement to traditional methods.

Phylogenomic analyses based on the plastid genome and concatenated nrDNA sequence data reveal cytonuclear discordance in genus Atractylodes (Asteraceae: Carduoideae).

Atractylodes species are widely distributed across East Asia and are cultivated as medicinal herbs in China, Japan, and Korea. Their unclear morphological characteristics and low levels of genetic divergence obscure the taxonomic relationships among these species. In this study, 24 plant samples were collected representing five species of Atractylodes located in China; of these, 23 belonged to members of the A. lancea complex. High-throughput sequencing was used to obtain the concatenated nrDNA sequences (18S-ITS1-5.8S-ITS2-28S) and plastid genomes. The concatenated nrDNA sequence lengths for all the Atractylodes species were 5,849 bp, and the GC content was 55%. The lengths of the whole plastid genome sequences ranged from 152,138 bp (A. chinensis) to 153,268 bp (A. lancea), while their insertion/deletion sites were mainly distributed in the intergenic regions. Furthermore, 33, 34, 36, 31, and 32 tandem repeat sequences, as well as 30, 30, 29, 30, and 30 SSR loci, were detected in A. chinensis, A. koreana, A. lancea, A. japonica, and A. macrocephala, respectively. In addition to these findings, a considerable number of heteroplasmic variations were detected in the plastid genomes, implying a complicated phylogenetic history for Atractylodes. The results of the phylogenetic analysis involving concatenated nrDNA sequences showed that A. lancea and A. japonica formed two separate clades, with A. chinensis and A. koreana constituting their sister clade, while A. lancea, A. koreana, A. chinensis, and A. japonica were found based on plastid datasets to represent a mixed clade on the phylogenetic tree. Phylogenetic network analysis suggested that A. lancea may have hybridized with the common ancestor of A. chinensis and A. japonica, while ABBA-BABA tests of SNPs in the plastid genomes showed that A. chinensis was more closely related to A. japonica than to A. lancea. This study reveals the extensive discordance and complexity of the relationships across the members of the A. lancea complex (A. lancea, A. chinensis, A. koreana, and A. japonica) according to cytonuclear genomic data; this may be caused by interspecific hybridization or gene introgression.

Fungal communities in Nelumbinis semen characterized by high-throughput sequencing.

For a long period, Nelumbinis semen has been widely used as a medicinal and edible product. However, it is susceptible to contamination with toxigenic fungi and aflatoxins during the growth, collection, transportation, and storage processes, causing serious health threats to humans and huge economic losses. Effectively monitoring the fungal communities is of great importance to avoid aflatoxins contamination in Nelumbinis semen. High-throughput sequencing (HTS) is a new technology to evaluate fungal communities so as to overcome the limitations of the traditional cultural ways. In this study, the ITS2 based Illumina-MiSeq platform was developed to evaluate the fungal communities in normal and moldy Nelumbinis semen by using the HTS technology. Two different primer pairs were introduced to compare their performance in amplifying the target gene. The primer pair that produced more reads was selected to analyze the results. In all the nine tested Nelumbinis semen samples, 2 phyla, 5 classes, 6 orders, 8 families, 9 genera and 4 species were detected. A total of 9 genera were spotted, of which, Aspergillus (0.04%-72.93%) and Rhizopus (0.002%-48.12%) were the most dominant. ANOISM analysis showed no significant differences in the normal and moldy groups. The use of HTS technology can detect the fungal communities in complex Nelumbinis semen samples, providing an early warning for toxigenic fungi and aflatoxins contamination to warrant their quality and safety.

Biological Ingredient Analysis of Traditional Herbal Patent Medicine Fuke Desheng Wan Using the Shotgun Metabarcoding Approach.

With the widespread use of traditional medicine around the world, the safety and efficacy of traditional herbal patent medicine have become an increasing concern to the public. However, it is difficult to supervise the authenticity of herbal materials in mixed herbal products according to the current quality standards, especially for traditional herbal patent medicine, with a distinct variance in the dosage of herbal materials. This study utilized the shotgun metabarcoding approach to analyze the biological ingredients of Fuke Desheng Wan (FKDSW), which is an effective traditional herbal product for the treatment of dysmenorrhea. Six herbal materials were collected, and a lab-made mock FKDSW sample was produced to establish a method for the authentication assessment of biological ingredients in traditional herbal patent medicine based on shotgun metabarcoding. Furthermore, four commercial FKDSW samples were collected to verify the practicality of the shotgun metabarcoding approach. Then, a total of 52.16 Gb raw data for 174 million paired-end reads was generated using the Illumina NovaSeq sequencing platform. Meanwhile, 228, 23, and 14 operational taxonomic units (OTUs) were obtained for the ITS2, matK, and rbcL regions, respectively, after bioinformatic analysis. Moreover, no differences were evident between the assembly sequences obtained via shotgun metabarcoding and their corresponding reference sequences of the same species obtained via Sanger sequencing, except for part of the ITS2 and matK assembly sequences of Paeonia lactiflora Pall., Saussurea costus (Falc.) Lipsch. and Bupleurum chinense DC. with 1-6 different bases. The identification results showed that all six prescribed ingredients were successfully detected and that the non-authentic ingredient of Bupleuri Radix (Chaihu, Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd.) was found in all the commercial samples, namely Bupleurum falcatum L. Here, 25 weed species representing 16 genera of ten families were detected. Moreover, 26 fungal genera belonging to 17 families were found in both lab-made and commercial FKDSW samples. This study demonstrated that the shotgun metabarcoding approach could overcome the biased PCR amplification and authenticate the biological ingredients of traditional herbal patent medicine with a distinct variance in the dosage of the herbal materials. Therefore, this provides an appropriate evaluation method for improving the safety and efficacy of traditional herbal patent medicine.

Precise Species Detection in Traditional Herbal Patent Medicine, Qingguo Wan, Using Shotgun Metabarcoding.

As one of the high-incidence diseases in the world, pharyngitis seriously affects the lives of those with the condition. Qingguo Wan is a herbal medicine used for treating pharyngitis, and its quality evaluation is currently only accomplished via traditional identification. However, precise identification becomes challenging with fake products on the market or fungal contamination during the production process. This study used the Illumina NovaSeq platform for targeting the ITS2, psbA-trnH, matK, and rbcL sequences to survey the species composition of lab-made and commercial samples. The results showed that a total of 34.56 Gb of raw data that was obtained represented more than 0.23 billion reads. After assembly, annotation, and operational taxonomic unit clustering, 103, 12, 10, and 12 OTUs were obtained, which belonged to the ITS2, psbA-trnH, matK, and rbcL sequences of the mock lab-made and commercial samples. The analytical results indicated that the sequences of all the prescription ingredients were successfully obtained in the two lab-made samples. The positive control medicinal Panax quinquefolius L. sequence was obtained in HSZY175, while Scutellaria baicalensis Georgi, Lonicera japonica Thunb. Menispermum dauricum DC. and Paeonia lactiflora Pall. were detected in the three commercial samples. The detection results of the other four herbs in different fragments were not all the same. In addition, a total of 28 fungi OTUs, representing 19 families and 20 genera, were obtained from both the commercial and mock lab-made samples. Aspergillus, Cladosporium, and Penicillium dominated among the 20 genera. This study demonstrated that the shotgun metabarcoding method is a powerful tool for the molecular identification of the biological ingredients in Qingguo Wan. It can be used to effectively supplement traditional methods while providing a new technique for the quality evaluation of Qingguo Wan.

The Species Identification in Traditional Herbal Patent Medicine, Wuhu San, Based on Shotgun Metabarcoding.

Traditional herbal patent medicine typically consists of multiple ingredients, making it challenging to supervise contamination by impurities and the improper use of raw materials. This study employed shotgun metabarcoding for the species identification of biological ingredients in traditional herbal patent medicine, Wuhu San. The five prescribed herbal materials found in Wuhu San were collected, and their reference sequences were obtained by traditional DNA barcoding using Sanger sequencing. Two lab-made and three commercial Wuhu San samples were collected, and a total of 37.14 Gb of shotgun sequencing data was obtained for these five samples using the Illumina sequencing platform. A total of 1,421,013 paired-end reads were enriched for the Internal Transcribed Spacer 2 (ITS2), psbA and trnH intergenic spacer region (psbA-trnH), maturase k (matK), and ribulose-1, 5-bisphosphate carboxylase (rbcL) regions. Furthermore, 80, 11, 9, and 8 operational taxonomic units were obtained for the ITS2, psbA-trnH, matK, and rbcL regions, respectively, after metagenomic assembly, annotation, and chimeric detection. In the two lab-made mock samples, all labeled ingredients in the Wuhu San prescription were successfully detected, and the positive control, Panax quinquefolius L., was detected in the HSZY172 mock sample. Three species, namely Angelica sinensis (Oliv.) Diels, Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., and Carthamus tinctorius L., belonging to three labeled ingredients, Angelicae Sinensis Radix (Danggui), Saposhnikoviae Radix (Fangfeng), and Carthami Flos (Honghua), were detected in the three commercial samples. Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav., the original Angelicae Dahuricae Radix (Baizhi) species, was only detected in WHS003. Arisaema erubescens (Wall.) Schott, Arisaema heterophyllum Blume, or Arisaema amurense Maxim., the original Arisaematis Rhizoma (Tiannanxing) species, were not detected in any of the commercial samples, which could be attributed to the fact that this medicinal material underwent extensive processing. In addition, the Saposhnikovia divaricata adulterant was detected in all the commercial samples, while 24 fungal genera, including Aspergillus, were identified in both the lab-made and commercial samples. This study showed that shotgun metabarcoding provided alternative strategy and technical means for identifying prescribed ingredients in traditional herbal patent medicine and displayed the potential to effectively complement traditional methods.

Tracing the Edible and Medicinal Plant Pueraria montana and Its Products in the Marketplace Yields Subspecies Level Distinction Using DNA Barcoding and DNA Metabarcoding.

Increased public awareness of nutritional and health issues has resulted in the increasing consumption of food and herbal products made from the root of Pueraria montana var. lobata (Willd.) Maesen & S. M. Almeida ex Sanjappa & Predeep (kudzu vine) and P. montana var. thomsonii (Benth.) M. R. Almeida. The famous herbal medicine Yufeng Ningxin, which is used to treat cardiovascular diseases, can be legally produced only using P. montana var. lobata. However, precise identification at the subspecies level is usually challenging when these products' ingredients lose their morphological characteristics after deep processing. Here, six herbarium specimens, 21 expert-identified original plant samples, 30 raw material samples, 10 food products and 12 herbal products were collected to test the subspecies-level authentication abilities of ITS2 sequences. The results showed that ITS2 sequences can distinguish P. montana var. lobata from P. montana var. thomsonii with stable single nucleotide polymorphism (SNP) sites. A total of 93.3% of raw material samples were consistent with the markings on their labels, but only 50% of Gegen Powder samples were made from P. montana var. lobata. High-quality ITS2 sequences were successfully obtained from nine of the 12 herbal products using Sanger sequencing. Substitution and fungal contamination were detected in 3 herbal products by further DNA metabarcoding, even though thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) tests verified that the products met existing quality standards. This study demonstrated that DNA barcoding is a powerful tool for the identification of P. montana var. lobata and P. montana var. thomsonii at the subspecies level, and we conclude that DNA barcodes can be broadly applied to trace the raw materials of food and herbal products.

Development of a ZnCdS@ZnS quantum dots-based label-free electrochemiluminescence immunosensor for sensitive determination of aflatoxin B1 in lotus seed.

In this study, we designed a ZnCdS@ZnS quantum dots (QDs)-based label-free electrochemiluminescence (ECL) immunosensor for sensitive determination of aflatoxin B1 (AFB1). A Nafion solution assembled abundant QDs on the surface of a Au electrode as ECL signal probes, with specially coupled anti-AFB1 antibodies as the capturing element. As the reduction reaction between S2O82- in the electrolyte and QDs on the electrode led to ECL emission, the decreased ECL signals resulting from target AFB1 in the samples were recorded for quantification. We evaluated electrochemical impedance spectroscopy and ECL measurements along each step in the construction of the proposed immunosensor. After systematic optimization of crucial parameters, the ECL immunosensor exhibited a good sensitivity, with a low detection limit of 0.01 ng/mL for AFB1 in a wide concentration range of 0.05-100 ng/mL. Testing with lotus seed samples confirmed the satisfactory selectivity, stability, and reproducibility of the developed ECL immunosensor for rapid, efficient, and sensitive detection of AFB1 at trace levels in complex matrices. This study provides a powerful and universal analytical platform for a variety of analytes that can be used in broad applications for real-time analysis, such as food and traditional Chinese medicine safety testing, environmental pollution monitoring, and disease diagnostics. Graphical abstract Development of a ZnCdS@ZnS quantum dots based label-free electrochemiluminescence immunosensor for sensitive detection of aflatoxin B1 in lotus seed.

Development of a high-performance liquid chromatography with tandem mass spectrometry method for identifying common adulterant content in saffron (Crocus sativus L.).

OBJECTIVES: This study aimed to develop an efficient and reliable method for estimating common adulterants in saffron by detecting their characteristic components to warrant its efficacy and regular use as a highly valuable medicinal herb. METHODS: A selective and sensitive high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method was developed to estimate the common adulterants in saffron from corn stigma, chrysanthemum and safflower through the simultaneous determination of specific constituents including allantoin, chlorogenic acid (ChA) and hydroxysafflor yellow A (HSYA). Peak identification of each target compound was confirmed from product ions obtained using multiple reaction monitoring triggered enhanced product ions mass chromatogram. Method validation in terms of linearity, sensitivity, reproducibility, accuracy and stability was systematically performed according to official guidelines. KEY FINDINGS: Satisfactory separation of the three components was achieved on a C18 column (4.6 × 250 mm, 5 µm) with methanol-acetonitrile-ammonium acetate (3.0 mm) as the mobile phase at gradient elution. The identification of these specific constituents was accomplished using the multiple reaction monitoring mode in combination with enhanced product ion supplementary confirmation. The established method was validated in terms of linearity, sensitivity, reproducibility, accuracy and recovery, which were found satisfactory for sensitive detection of the three target compounds. CONCLUSIONS: By detecting the specific constituents allantoin, ChA and HSYA in one run, the adulterants of corn stigma, chrysanthemum and safflower can be effectively identified and estimated in saffron. This is the first report on developing a simple, sensitive and operational method for the identification and estimation of common adulterants of saffron, that was forwarded for broaden application.

Regeneration and Reuse of Immunoaffinity Column for Highly Efficient Clean-Up and Economic Detection of Ochratoxin A in Malt and Ginger.

Immunoaffinity columns (IACs) are most popularly used for mycotoxin clean-up in complex matrices prior to chromatographic analysis. But, their high cost has limited their wide application and the regeneration of IACs for multiple instances of reuse is important. This study aimed to investigate the feasibility of regeneration and reuse of IACs for purification of ochratoxin A (OTA) in spiked raw malt and dried ginger samples followed by high performance liquid chromatography-fluorescence detection. After each use, the IACs were filled with phosphate buffer saline (PBS) as the preservation solution and stored at 8 °C overnight for regeneration and reuse until the recovery rate was <70%. The results showed that matrix type, preparation procedure, and pH value of sample extraction exhibited major effects on the reuse of IACs for OTA clean-up. While, after modifying the sample preparation procedure using water as the diluent and the solution at a pH of 7 to 8, the IACs could be used eight and three times for the spiked raw malt and dried ginger samples with OTA after regeneration. Regarding the traditional procedure recommended in Chinese Pharmacopoeia (2015 edition), the IACs could be used for three and two times for the spiked raw malt and dried ginger samples with OTA, respectively. Therefore, the corresponding experimental cost could be reduced to one-eighth and one-third of the original cost. This is the first study on the regeneration and reuse of IACs for OTA clean-up in complex Chinese herbal medicines, providing a green and economical tool for a large number of samples analysis with low cost.

[Research progress of pretreatment technology for mycotoxin detection in Chinese materia medica and complex matrices].

In the processes of planting, harvest, transport and storage, improper treatment of Chinese materia medica (CMM) and foodstuffs and agricultural products will result in fungal growth and mycotoxins contamination, which will not only directly affect the quality, safety and efficacy of these complex matrices, but also seriously threaten the consumers' health and lives. Therefore, the establishment of high-throughout analytical methods with high sensitivity for the determination of mycotoxins in CMM and foodstuffs and agricultural products at trace levels will provide reliable references for reducing the risk of mycotoxin exposure in humans. Due to the matrix complexity of CMM and foodstuffs and agricultural products, highly-effective pretreatment technologies are necessary for the establishment of such analytical techniques. In this review, the current extraction and purification methods commonly used for the detection of mycotoxins were summarized, the importance of pretreatment techniques for the precise quantification of mycotoxins in complex matrices such as Chinese herbal medicines was highlighted, as well as the development tendency about the pretreatment techniques for mycotoxins in complex matrices in the future was proposed.

Multiclass mycotoxins in lotus seeds analysed by an isotope-labelled internal standard-based UPLC-MS/MS.

OBJECTIVES: This study aimed to explore the residue levels of multiclass mycotoxins in medicinal and edible lotus seeds. METHODS: A rapid and reliable isotope-labelled internal standard-based UPLC-MS/MS method was developed and validated for sensitive and accurate analysis of multiclass mycotoxins including aflatoxins (AFB1 , AFB2 , AFG1 and AFG2 ), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FB1 and FB2 ), T-2 and HT-2 toxins in lotus seeds. Some critical conditions such as extract solution with the addition of isotope-labelled internal standard, type of mobile phase and the elution condition were scientifically optimized. The 11 mycotoxins obtained satisfactory resolution and sensitive detection in multiple reactions monitoring scanning mode combined with the ion switching technology in positive and negative ion switching mode. KEY FINDINGS: The developed isotope-labelled internal standard-based UPLC-MS/MS method exhibited an approving linearity (r ≥ 0.9984), high sensitivity (limit of detection in the range of 0.015-30.05 µg/kg), acceptable precision (RSDs ≤6.3%) and good recovery (76.0-116.0%) for 11 analytes, respectively. Ten batches of real lotus seed samples were tested, and three batches out of which were contaminated with AFB1 , FB2 , T-2 and ZEN. AFB1 showed the highest occurrence rate (30%) with contents of 10.50 and 8.32 µg/kg in two samples over the official limit (5.0 µg/kg). CONCLUSIONS: The monitoring of multiclass mycotoxins in Chinese herbal medicines is in great urgency to ensure the security of consumers. The proposed method could be further utilized for simple, sensitive and rapid detection of more mycotoxins in other complex matrices to compensate for matrix effects.

Contamination Parts and Residue Levels of Multi-Mycotoxins in Medicinal and Edible Locust.

Locust is esteemed as a traditional Chinese medicine, as well as one of the most important nutritional foods especially in Asian countries. However, some toxic secondary metabolites such as mycotoxins are usually found in different parts of locust to affect its quality and safety. This study aimed to investigate the aflatoxins (AFs) contaminated parts by observing Aspergillus flavus, spores' diameter, amount and distribution on head, tentacle, wing, belly and shank parts of the locust with scanning electron microscopy (SEM). Furthermore, to assess the residue levels of multi-mycotoxins in the locust, the high performance liquid chromatography with fluorescence detection (HPLC-FLD) was adopted. The technique was used to determine the contents of AFs, zearalenone (ZON) and α-zearalenol (α-ZOL) in locust and the positive samples were confirmed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The chromatographic conditions, MS/MS parameters and the method of sample extraction were carefully optimized. Results revealed that obvious differences of Aspergillus flavus strains and spores were found, while the spores' diameter ranged from 3.0 to 13.0 µm in different contaminated parts of the locust samples. The HPLC-FLD method for multi-mycotoxins analysis showed good selectivity, linearity, recovery and precision. Limits of quantification (LOQs) were lower than 27.6 µg/kg, while limits of detection (LODs) were in the range of 0.02-8.6 µg/kg. The accuracy of the developed method was validated regarding recoveries of 80.1-118.1% with relative standard deviation (RSD) ≤ 11.4%. Finally, the developed multi-mycotoxin method was applied for screening of these mycotoxins in 11 commercial locust samples. Only AFB1 and AFB2 were found in six samples, and the contamination levels ranged from 0.12 to 4.4 µg/kg, which were lower than the maximum residue limit and can be used safely. This is the first report on the exploration of contamination parts and levels of multi-mycotoxins in medicinal and edible locust. The combined method of SEM and HPLC-FLD exhibited advantages of low cost, high sensitivity, rapid determination, convenience and especially intuitive judgment, which is proposed for contamination parts observation, for the large-scale quantification of multi-mycotoxins in other medicinal animal matrices.

[Contamination distribution and control measures of aflatoxins in Chinese herbal pieces].

As an important part of traditional medicine in China, traditional Chinese medicine(TCM) plays a significant role because of its unique medical efficiency, less adverse reactions and extensive resources. However, in recent years, the aflatoxins in medicinal herbs have been detected excessive both at home and abroad, seriously affecting the reputation and credibility of traditional Chinese medicine. In this paper, the current status of aflatoxins contamination in medicinal herbs was analyzed, and the internal and external factors of aflatoxins contamination in traditional Chinese medicine were also summarized. In view of the high toxicity of aflatoxins, it is proposed to strengthen the mildew prevention and control from the early planting to storage stage, and the reasonable detoxification mode should also be considered. This review aims to provide a reference in guaranteeing the clinical safe administration of medicinal herbs and reducing the risk of being poisoned by aflatoxins.

Transfer rates of aflatoxins from herbal medicines to decoctions determined by an optimized high-performance liquid chromatography with fluorescence detection method.

OBJECTIVES: This study aimed to explore the transfer rates of aflatoxins from several contaminated herbal medicines by fungi to their decoctions. METHODS: Five types of commonly used herbal medicines including Lilii Bulbus, Hordei Fructus Germinatus, Nelumbinis Semen, Polygalae Radix and Bombyx Batryticatus were selected as the examples. Raw herbal medicine samples were treated by ultrasonication-assisted extraction with 70% methanol and immunoaffinity column clean-up, and the decoctions were prepared following the commonly used boiling method with water for 2 h. Then, the optimized high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was validated for the quantitative analysis of four aflatoxins (AFG2 , AFG1 , AFB2 and AFB1 ) after postcolumn photochemical derivatization, which was proved to be reliable and sensitive. KEY FINDINGS: Aflatoxins were detected to be transferred from the herbal medicines to decoctions with significantly different transfer rates in the five types of herbal medicines. Quietly high transfer rates of 7.26-115.36% for AFG2 , 4.37-26.37% for AFB1 and 9.64-47.68% for AFB2 were obtained. AFB1 as the most toxic aflatoxin expressed the lowest transfer rate, but still exhibited high amount in the samples. CONCLUSIONS: Therefore, the monitoring of aflatoxins in herbal medicines and their decoctions is in great urgency to ensure the security of consumers taking decoctions.

[Research progresses and formulation development of natural anti-mildew agents based on Chinese herbal medicines].

Owing to the intrinsic factors and some extrinsic environmental conditions, many foods, agricultural products and Chinese materia medicas (CMMs), if not handled properly in the processes of growth, harvesting, processing and storage, can be easily contaminated by all kinds of molds to produce mycotoxins of serious toxicity, which will not only affect the quality, safety and effectiveness of CMMs, but also result in potential threatens to human and animal's health and life. Therefore, in recent decades, it has become the focus on how to prevent and control the foods, agricultural products and CMMs from being moldy and producing toxicity for scientific preservation. Many Chinese herbal medicines (CHMs) especially those with high content of volatile oils with strong antifungal activities have been applied for the scientific preservation of foods, agricultural products and CMMs. Based on these situations, natural anti-mildew agents have been further developed and made into some useful dosage forms, such as tablets, aerosol, liposomes and inclusion, which will not only greatly expand the application scope of CHMs to make the use of anti-mildew agents more convenient, but also achieve the sustained or controlled release of the antifungal effect for scientific preservation of foods, agricultural products and CMMs.

Evaluation and quantitative analysis of 11 compounds in Morinda officinalis using ultra-high performance liquid chromatography and photodiode array detection coupled with chemometrics.

Morinda officinalis (Rubiaceae) is a traditional Chinese medicine widely used for the treatment of impotence and osteoporosis in clinical therapy. In the present study, a rapid and simple ultra-high performance liquid chromatography with photodiode array detection method was developed and validated for the simultaneous determination of 11 bioactive compounds in M. officinalis. This assay method was validated with respect to linearity (R2  > 0.9991), precision, repeatability, limit of detection, limit of quantification, and accuracy (with observed recovery rates between 94.21 and 100.38%). The quantitative results revealed significant differences in the concentrations of the selected compounds. Additionally, chemometric methods, including hierarchical clustering analysis, principal component analysis, and partial least-squares discriminate analysis, were applied to compare and sort the 25 batches of M. officinalis samples based on the quantitative data of the analytes. All of the samples were clearly divided into two groups: the Hainan samples were successfully discriminated from the samples from other origins. Simultaneous determination of multiple compounds using the proposed method combined with chemometrics could be a viable strategy to compare and evaluate the quality of M. officinalis.

Combination of chemical fingerprint and bioactivity evaluation to explore the antibacterial components of Salvia miltiorrhizae.

The aim of this study was to explore the possible antibacterial components of Salvia miltiorrhizae on Pseudomonas aeruginosa using a combination of chemical fingerprint and bioactivity evaluation. The chemical fingerprints of 32 batches of S. miltiorrhizae samples from different sources were developed using high-performance liquid chromatography with diode array detection, and then were evaluated by similarity analysis and hierarchical clustering analysis. Anti-P. aeruginosa activity was determined by microcalorimetry. Some crucial thermokinetic parameters obtained from the heat-flow power-time curves of P. aeruginosa growth in the absence or presence of these S. miltiorrhizae samples were evaluated using principal component analysis. Thereafter, multiple linear regression analysis was used to analyze the fingerprint-activity relationship between the chemical fingerprints and anti-P. aeruginosa activity. This established the related equation between the inhibition ratio (I, %) of S. miltiorrhizae samples on P. aeruginosa and the peak areas of the common peaks. The results showed that the 32S. miltiorrhizae samples could be grouped into three clusters according to their chemical fingerprints and anti-P. aeruginosa activities. Protocatechualdehyde, salvianolic acid B, together with three unidentified compounds might be the major components that contributed largely to the antibacterial properties of S. miltiorrhizae and should be the focus of S. miltiorrhizae quality control. Thus, this study provided a preferred way for exploring the bioactive components of medicinal plants.

Assessment of critical points and development of a practical strategy to extend the applicable scope of immunoaffinity column cleanup for aflatoxin detection in medicinal herbs.

Although extraction methods based on immunoaffinity column (IAC) cleanup have been used to detect aflatoxins in medicinal herbs, they do not yield satisfactory results for all sample matrices. The difficulty arises from the chemical complexity of the herbs, and there is a pressing need to determine which steps in IAC cleanup limit the scope of aflatoxin detection in many different kinds of medicinal herbs. In this work, we found that there were two main factors that severely decreased antibody-antigen recognition and led to serious nonspecific adsorption: (1) high extract acidity and (2) high co-extraction of interfering compounds. We therefore carried out a systematic study to optimize extraction efficiency. We found that dilution of samples in 0.1M phosphate buffer solution (pH 7.8, 2% Tween-20) at a 1:8 dilution ratio mitigated the effect of high acidity, decreased co-precipitation of compounds and nonspecific adsorption, and ameliorated the matrix effect. To validate this finding, and test if our method is widely applicable to in different kinds of herbal materials, we analyzed several representative complex sample matrices including fructus, cortex, and radix with varying extract pH values. The recovery efficiency was generally higher than 70%. We further validated our method by testing a certified reference material, and found that our approach accurately quantified aflatoxin concentration. After validation, we successfully used this method to determine the aflatoxin concentration of real samples. The approach described here could potentially be used to extract aflatoxin from other complex matrices with varying acidity.