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.

Value of Chuanjin Qinggan decoction in improving the depressive state of patients with herpes zoster combined with depression.

BACKGROUND: Western medicine is beneficial for the recovery of neurological function in patients with depression, but some patients experience side effects such as headaches, dizziness, nausea, gastrointestinal symptoms, insomnia, and cardiac dysfunction. In recent years, integrative medicine has achieved positive results in the treatment of various diseases. AIM: To study Chuanjin Qinggan decoction combined with selective serotonin reuptake inhibitors (SSRIs) in patients with herpes zoster complicated by depression. METHODS: Patients with herpes zoster complicated by depression who were treated at the Yantai Hospital of Traditional Chinese Medicine from January 2021 to December 2022 were retrospectively selected as research participants. Among them, 43 patients with herpes zoster complicated by depression who received SSRI treatment between January and December 2021 were assigned to the Western medicine group, while those who received combined treatment of traditional Chinese and Western medicine between January and December 2022 were assigned to the combined group. Both groups were treated for eight weeks. The degree of pain, effect of herpes zoster treatment, degree of improvement in depressive symptoms, serum neurotransmitter levels, sleep quality, and occurrence of adverse reactions were compared between the two groups. RESULTS: We found that after eight weeks of drug treatment, the two treatment schemes achieved differing efficacy. In further comparison, we found that, compared with patients treated with SSRIs alone, patients treated with Chuanjin Qinggan decoction combined with SSRIs showed more significant improvement in depression and a greater increase in dopamine and 5-hydroxytryptamine levels after treatment (P < 0.05). Patients treated with Chuanjin Qinggan decoction combined with SSRIs also experienced lower pain, better treatment efficacy for herpes zoster, better sleep quality, and a lower incidence of adverse reactions compared to those treated with SSRIs alone (P < 0.05). All minor adverse reactions occurring during treatment were resolved after symptomatic treatment. CONCLUSION: The treatment scheme of Chuanjin Qinggan decoction combined with SSRIs can improve the psychological state of patients with herpes zoster complicated by depression and alleviate adverse reactions.

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.

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.

The complete chloroplast genome of Atractylodes japonica Koidz. ex Kitam. and its phylogenetic inference.

Atractylodes japonica Koidz. ex Kitam. is a perennial herbal plant, and its dried rhizomes have been widely used as traditional medicine in China and Japan. In this study, we assembled and annotated the complete chloroplast (cp) genome sequence of A. japonica using the high-throughput sequencing approach. The cp genome of A. japonica is 153,208 bp in length with the overall GC content of 37.7%, including two inverted repeat (IR) regions of 25,147 bp, which was separated by a large single-copy (LSC) region of 84,255 bp and a small single-copy (SSC) region of 18,659 bp. 113 unique genes were annotated in the genome, including 80 protein-coding genes, 29 represented tRNA genes, and four denoted rRNA genes. A maximum-likelihood phylogenetic analysis with 38 complete cp sequences showed that Atractylodes formed a monophyletic clade, and A. japonica and A. koreana formed a subclade in Atractylodes. This study provides the chloroplast genome structure features and phylogenetic relationship of A. japonica.

Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) turn out to be a promising source of cell-free therapy. Here, we investigated the biodistribution and effect of nebulized human adipose-derived MSC-EVs (haMSC-EVs) in the preclinical lung injury model and explored the safety of nebulized haMSC-EVs in healthy volunteers. DiR-labelled haMSC-EVs were used to explore the distribution of nebulized haMSC-EVs in the murine model. Pseudomonas aeruginosa-induced murine lung injury model was established, and survival rate, as well as WBC counts, histology, IL-6, TNF-α and IL-10 levels in bronchoalveolar lavage fluid (BALF) were measured to explore the optimal therapeutic dose of haMSC-EVs through the nebulized route. Twenty-four healthy volunteers were involved and received the haMSC-EVs once, ranging from 2 × 108 particles to 16 × 108 particles (MEXVT study, NCT04313647). Nebulizing haMSC-EVs improved survival rate to 80% at 96 h in P. aeruginosa-induced murine lung injury model by decreasing lung inflammation and histological severity. All volunteers tolerated the haMSC-EVs nebulization well, and no serious adverse events were observed from starting nebulization to the 7th day after nebulization. These findings suggest that nebulized haMSC-EVs could be a promising therapeutic strategy, offering preliminary evidence to promote the future clinical applications of nebulized haMSC-EVs in lung injury diseases.

The complete chloroplast genome of Atractylodes koreana (Nakai) Kitam and its phylogenetic analysis.

Atractylodes koreana (Nakai) Kitam is a perennial herb of Asteraceae, mainly distributed in China and Korea, which is the main adulterant of traditional herbal medicine 'Cangzhu'. In the present study, we reported the complete chloroplast (cp) genome of A. koreana with the total length of 153,232 bp, which is consisted of four regions, including one large single copy (LSC) region of 84,250 bp, one small single copy (SSC) region of 18,690 bp, and two inverted repeat regions (IRa and IRb) of 25,146 bp. The GC content of the complete cp genome is 37.7%. A total of 110 unique genes were annotated, comprising 79 protein-coding genes, 27 transfer RNA (tRNA) genes and four ribosome RNA (rRNA) genes. Moreover, nine protein-coding genes contained one intron and three protein-coding genes (clpP, ycf3, and rps12) contained two introns. The phylogenetic analysis indicated that A. koreana is a sister group of A. chinensis and A. lancea.

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.

The Effects of Taoren-Honghua Herb Pair on Pathological Microvessel and Angiogenesis-Associated Signaling Pathway in Mice Model of CCl4-Induced Chronic Liver Disease.

Chronic liver disease is one of the most common diseases that threaten human health. Effective treatment is still lacking in western medicine. Semen Persicae (Taoren) and Flos Carthami (Honghua) are known to relieve acute hepatic injury and inflammation, improve microcirculation, and reduce tissue fiber. The aim of our study is to investigate the potential mechanisms of Taoren-Honghua Herb Pair (THHP) in murine model of chronic liver disease caused by Carbon Tetrachloride (CCl4). Mice were randomly divided into seven groups: (1) blank, (2) model, (3) control (colchicine, 0.1 mg/kg), (4) THHP (5.53, 2.67, and 1.33 g/kg), and (5) Tao Hong Siwu Decoction (THSWD) (8.50 g/kg). Histological change and microvessels density were examined by microscopy. Hepatic function, serum fibrosis related factors, and hepatic vascular endothelial growth factor (VEGF) were measured with ELISA. VEGF, kinase insert domain-containing receptor (KDR), Flt-1, and Akt mRNA expression in hepatic tissue were determined with PCR. Tissues of Akt, pAkt, KDR, and Flt-1 were measured with western blotting. Data from this study showed that THHP improved hepatic function and restrained the hepatic inflammation and fibrosis. Its role in inhibiting pathological angiogenesis and hepatic fibrogenesis may be through affecting the angiogenesis-associated VEGF and its upstream and downstream signaling pathways.

The combination effects of Shen-Ling-Bai-Zhu on promoting apoptosis of transplanted H22 hepatocellular carcinoma in mice receiving chemotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen-Ling-Bai-Zhu Powder (SLBZP) is a classic traditional Chinese medical formula that has been used for several decades in the treatment of patients with gastrointestinal malignancies. Whether SLBZP is best employed as single agent or adjunctive therapy has yet to be determined as does the mechanism whereby SLBZP exerts its anti-tumor effects. AIM OF THE STUDY: To investigate the effects of SLBZP alone and in combination with Cytoxan (CTX) on tumor growth, malignant cell apoptosis and Akt/Nuclear Factor kappa B (NF-КB) signaling in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty-four adult mice developed HCC following subcutaneous inoculation with H22 hepatocellular carcinoma cells. Seven days later, all received chemotherapy with CTX (200mg/kg) once. Mice were then randomized into eight study groups (N=8/group). Three groups were treated with different concentrations of SLBZP alone (6.00, 3.00, 1.5g/kg), three with SLBZP (6.00, 3.00, 1.5g/kg) plus CTX (20mg/kg), one with CTX (20mg/kg) alone (positive control), and one with physiologic saline (untreated, negative control). All groups were treated for 14 days. Tumor size, histology and serum or tissue levels and/or mRNA expression of PDGF-BB, VEGF, Ang-1, Ang-2, NF-КB, B-cell lymphoma-2 (Bcl-2); B-cell lymphoma-extra large (Bcl-xL); X-linked inhibitor of apoptosis (XIAP), Survivin, Caspase-3, Caspase-9, Caspase-7, Akt and phosphorylated Akt expression were documented at the end of treatment. RESULTS: Compared to untreated negative controls, tumor sizes were decreased in the CTX alone, SLBZP (M)+CTX and SLBZP (H)+CTX groups (-52%,-53% and -58% respectively). Tumor cell density was decreased in all treated groups but most apparent in the SLBZP (H)+CTX group. Electron microscopic evidence of apoptosis was also most apparent in this group. Serum and/or tissue levels and expression of PDGF-BB, VEGF, Ang-1, Ang-2, their downstream signaling proteins and anti-apoptotic markers were lowest and pro-apoptotic markers highest in SLBZP (H)+CTX treated mice. CONCLUSIONS: In this chemotherapy-induced animal model of HCC, SLBZP was most efficacious as adjunctive therapy and appears to act by inhibiting tumor growth promoters and anti-apoptotic proteins while enhancing pro-apoptotic proteins.

The effects of Tao-Hong-Si-Wu on hepatic necroinflammatory activity and fibrosis in a murine model of chronic liver disease.

BACKGROUND: Tao-Hong-Si-Wu decoction (THSWD) is a traditional Chinese herbal medicine that has been used for centuries in the treatment of Chinese patients with chronic liver disease. Recently, THSWD has been reported to alter vascular endothelial growth factor (VEGF) induced angiogenesis, raising the possibility that in addition to its anti-inflammatory properties; THSWD might also inhibit hepatic blood flow associated fibrosis. AIM: To document the effects of THSWD on hepatic necroinflammatory disease activity, fibrosis and VEGF signaling in a murine model of chronic liver disease. METHODS: Sixty adult mice were equally divided into six study groups. Five groups were exposed to subcutaneous carbon tetrachloride (0.1 ml/10 g BW) for six weeks. Three of the five groups were treated with different concentrations of THSWD (4.25, 8.50, 17.00 g/kg), one with 0.1mg/kg of Colchicine (positive control), and one with physiologic saline (negative control). Mice in the sixth group were not exposed to CCl4 and remained untreated (healthy controls). Liver enzymes/function tests, hyaluronic acid and laminin levels were measured in serum, and hepatic histology, VEGF, Flt-1 and kinase insert domain-containing receptor (KDR), Akt and phosphorylated Akt (pAkt) expression were documented in liver tissue at the end of treatment. RESULTS: Hepatic necroinflammatory disease activity and fibrosis were significantly attenuated in THSWD treated mice in a dose dependent manner. These beneficial results were similar and often exceeded those achieved with Colchicine. In addition, VEGF, Flt-1, KDR, Akt and pAkt mRNA and protein expression were reduced in TSHWD treated mice. CONCLUSIONS: In this animal model of chronic liver disease, THSWD decreased hepatic necroinflammatory disease and fibrosis. Inhibition of VEGF expression and downstream signaling were associated with these findings. Further studies with this and other TCMs as treatment for chronic liver disease are warranted.