[Platycladi Semen oil ameliorates Aß_(25-35)-induced brain injury in mice based on network pharmacology and gut microbiota].

The present study aimed to investigate the protective effect and underlying mechanism of Platycladi Semen oil(SP) on Aß_(25-35)-induced brain injury in mice to provide a theoretical basis for the clinical treatment of Alzheimer's disease(AD). Male Kunming(KM) mice were randomly divided into a control group, a model group(brain injection of Aß_(25-35), 200 µmol·L~(-1), 0.15 µL·g~(-1)), a positive drug group(donepezil, 10 mg·kg~(-1)), and low-and high-dose SP groups(0.5 and 1 mL·kg~(-1)). Learning and memory ability, neuronal damage, levels of Aß_(1-42)/Aß_(1-40), p-Tau, related indicators of apoptosis and oxidative stress, and immune cells, and protein and mRNA expression related to the sphingosine kinase 1(SPHK1)/sphingosine-1-phosphate(S1P)/sphingosine-1-phosphate receptor 5(S1PR5) signaling pathway of mice in each group were determined. In addition, compounds in SP were analyzed by gas chromatography-mass spectrometry(GC-MS). The mechanism of SP against AD was investigated by network pharmacology, 16S rDNA gene sequencing for gut microbiota(GM), and molecular docking techniques. The results showed that SP could improve the learning and memory function of Aß_(25-35)-induced mice, reduce hippocampal neuronal damage, decrease the levels of Aß_(1-42)/Aß_(1-40), p-Tau, and indicators related to apoptosis and oxidative stress in the brain, and maintain the homeostasis of immune cells and GM. Network pharmacology and sequencing analysis for GM showed that the therapeutic effect of SP on AD was associated with the sphingolipid signaling pathway. Meanwhile,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid, the components with the highest content in SP, showed good binding activity to SPHK1 and S1PR5. Therefore, it is inferred that SP exerts anti-apoptosis and antioxidant effects by regulating GM and inhibiting SPHK1/S1P/S1PR5 pathway, thereby improving brain injury induced by Aß_(25-35) in mice. Moreover,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid may be the material basis for the anti-AD effect of SP.

Integration of non-targeted metabolomics with network pharmacology deciphers the anxiolytic mechanisms of Platycladi Semen extracts in CUMS mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Platycladi Semen was recorded in Shen Nong's Herbal Classic and was considered a herbal medicine with low toxicity after long-term medication. Multiple traditional Chinese medicine prescriptions containing Platycladi Semen have been used to treat insomnia. Modern clinical practitioners commonly use Platycladi Semen to treat anxiety disorders, but there are few studies on its composition and anxiolytic mechanisms. AIM OF THE STUDY: To describe the main components of Platycladi Semen and investigate its anxiolytic effects and mechanisms. MATERIALS AND METHODS: The main components of Platycladi Semen were characterized by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). The anxiolytic effects of oral Platycladi Semen were evaluated in chronic unpredictable mild stress (CUMS) induced mice. To explore the anxiolytic mechanisms of Platycladi Semen, serum non-targeted metabolomics combined with network pharmacology and molecular docking was performed. RESULTS: Fourteen compounds were identified in the 50% methanol extract and 11 fatty acid derivatives were identified in the methyl-esterified fatty oil of Platycladi Semen. In CUMS mice, both the aqueous extract and fatty oil of Platycladi Semen had anxiolytic effects, which were shown by the increase in the time and frequency of mice entering the open arm in the elevated plus maze (EPM) experiment. Through serum non-targeted metabolomics, 34 differential metabolites were identified, and lipid metabolic pathways such as sphingolipid metabolism, steroidogenesis, alpha-linoleic acid, and linoleic acid metabolism were enriched. Through network pharmacology, 109 targets of the main components in Platycladi Semen were identified, and the 'neuroactive ligand-receptor interaction' and 'lipid metabolism' were enriched. The molecular docking results showed that the main components in Platycladi Semen could bind to the key targets such as peroxisome proliferator-activated receptor delta (PPARD), peroxisome proliferator-activated receptor alpha (PPARA), fatty acid binding protein 5 (FABP5), fatty acid binding protein 3 (FABP3), peroxisome proliferator-activated receptor gamma (PPARG), arachidonate 5-lipoxygenase (ALOX5) and fatty acid amide hydrolase (FAAH). CONCLUSION: This study indicated that Platycladi Semen has anxiolytic effects, and the anxiolytic mechanisms may be the regulation of lipid metabolism and the neuroactive ligand-receptor interaction.

Platycladi Semen oil ameliorates Aβ_(25-35)-induced brain injury in mice based on network pharmacology and gut microbiota / 中国中药杂志

The present study aimed to investigate the protective effect and underlying mechanism of Platycladi Semen oil(SP) on Aβ_(25-35)-induced brain injury in mice to provide a theoretical basis for the clinical treatment of Alzheimer's disease(AD). Male Kunming(KM) mice were randomly divided into a control group, a model group(brain injection of Aβ_(25-35), 200 μmol·L~(-1), 0.15 μL·g~(-1)), a positive drug group(donepezil, 10 mg·kg~(-1)), and low-and high-dose SP groups(0.5 and 1 mL·kg~(-1)). Learning and memory ability, neuronal damage, levels of Aβ_(1-42)/Aβ_(1-40), p-Tau, related indicators of apoptosis and oxidative stress, and immune cells, and protein and mRNA expression related to the sphingosine kinase 1(SPHK1)/sphingosine-1-phosphate(S1P)/sphingosine-1-phosphate receptor 5(S1PR5) signaling pathway of mice in each group were determined. In addition, compounds in SP were analyzed by gas chromatography-mass spectrometry(GC-MS). The mechanism of SP against AD was investigated by network pharmacology, 16S rDNA gene sequencing for gut microbiota(GM), and molecular docking techniques. The results showed that SP could improve the learning and memory function of Aβ_(25-35)-induced mice, reduce hippocampal neuronal damage, decrease the levels of Aβ_(1-42)/Aβ_(1-40), p-Tau, and indicators related to apoptosis and oxidative stress in the brain, and maintain the homeostasis of immune cells and GM. Network pharmacology and sequencing analysis for GM showed that the therapeutic effect of SP on AD was associated with the sphingolipid signaling pathway. Meanwhile,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid, the components with the highest content in SP, showed good binding activity to SPHK1 and S1PR5. Therefore, it is inferred that SP exerts anti-apoptosis and antioxidant effects by regulating GM and inhibiting SPHK1/S1P/S1PR5 pathway, thereby improving brain injury induced by Aβ_(25-35) in mice. Moreover,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid may be the material basis for the anti-AD effect of SP.

[Aflatoxins and fungal community distribution during harvesting and processing of Platycladi Semen].

This study aimed to analyze aflatoxins content and fungal community distribution in the harvesting and processing of Platycladi Semen, and explore the key link that affects aflatoxins contamination. The related Platycladi Semen samples of different maturity periods(cone non-rupture period, early rupture, and complete rupture period) and different processing periods(before drying, during 2-d drying, during 7-d drying, before and after seed scale removal, before and after peeling, 1 d after color sorting, and 7 d after color sorting) were collected for identifying the fungal community composition on sample surface by ITS amplicon sequencing. Then the content of aflatoxins B_1, B_2, G_1 and G_2 was determined by HPLC-MS/MS. The results showed that during the harvesting of Platycladi Semen from cone non-rupture to complete rupture, aflatoxins were only detected in the seed scale and seed coat, with aflatoxin G_2 in the seed scale and aflatoxin B_1 in the seed coat. During the drying, with the prolongation of drying time, aflatoxins B_1 and G_2 were detected simultaneously in the seed scale, aflatoxin B_1 in the seed coat, and low-content aflatoxin B_1 in the seed kernel. During subsequent processing, the aflatoxin content in seed kernel during subsequent processing was slighted increased. As demonstrated by fungal detection, Aspergillus flavus was not present during the harvesting of Platycladi Semen, but present during the drying and processing. Its content in the seed coat during the drying process was relatively higher. In short, Platycladi Semen should be harvested as soon as possible after it becomes fully mature. Drying process is the key link of preventing aflatoxin contamination. It is advised to build a sunlight room or adopt similar settings, standardize the operations in other processes, and keep the surrounding environment clean to minimize aflatoxin contamination.

Aflatoxins and fungal community distribution during harvesting and processing of Platycladi Semen / 中国中药杂志

This study aimed to analyze aflatoxins content and fungal community distribution in the harvesting and processing of Platycladi Semen, and explore the key link that affects aflatoxins contamination. The related Platycladi Semen samples of different maturity periods(cone non-rupture period, early rupture, and complete rupture period) and different processing periods(before drying, during 2-d drying, during 7-d drying, before and after seed scale removal, before and after peeling, 1 d after color sorting, and 7 d after color sorting) were collected for identifying the fungal community composition on sample surface by ITS amplicon sequencing. Then the content of aflatoxins B_1, B_2, G_1 and G_2 was determined by HPLC-MS/MS. The results showed that during the harvesting of Platycladi Semen from cone non-rupture to complete rupture, aflatoxins were only detected in the seed scale and seed coat, with aflatoxin G_2 in the seed scale and aflatoxin B_1 in the seed coat. During the drying, with the prolongation of drying time, aflatoxins B_1 and G_2 were detected simultaneously in the seed scale, aflatoxin B_1 in the seed coat, and low-content aflatoxin B_1 in the seed kernel. During subsequent processing, the aflatoxin content in seed kernel during subsequent processing was slighted increased. As demonstrated by fungal detection, Aspergillus flavus was not present during the harvesting of Platycladi Semen, but present during the drying and processing. Its content in the seed coat during the drying process was relatively higher. In short, Platycladi Semen should be harvested as soon as possible after it becomes fully mature. Drying process is the key link of preventing aflatoxin contamination. It is advised to build a sunlight room or adopt similar settings, standardize the operations in other processes, and keep the surrounding environment clean to minimize aflatoxin contamination.

Analysis of Quality Changes of Platycladi Semen Before and After Deterioration Based on HS-SPME/GC-MS and Electronic Sensory Technology / 中国实验方剂学杂志

ObjectiveTo analyze the quality changes of Platycladi Semen before and after the deterioration of moth-eaten and rancidity during storage. MethodFour types samples of Platycladi Semen， including normal， moth-eaten， oxidative rancidity and hydrolytic rancidity， were determined for volatile components， odor， and taste based on headspace solid phase microextraction/gas chromatography-mass spectrometry （HS-SPME/GC-MS） and electronic sensory techniques such as electronic nose and electronic tongue. Volatile components were identified by searching the database and manual comparison， the odor and taste were determined by the response values of the electronic nose and electronic tongue sensors， and the difference between samples before and after deterioration was studied by multivariate statistical analysis. ResultA total of 85 compounds were identified in Platycladi Semen samples. Compared with the normal samples， the number of volatile compounds in samples after hydrolytic rancidity decreased by 5， the number of volatile compounds in samples after moth-eaten and oxidative rancidity increased by 1 and 21， respectively. Aldehydes and acids accounted for majority of types. Among them， the contents of N-hexanoic acid， hexanal and propionic acid in the samples of oxidative rancidity reached 11.49%， 10.21% and 7.52%， which became the key indicators of rancidity. There was significant variance among the odor components corresponding to W1W， W2W and W1S sensors by electronic nose analysis. It was indicated that the value of sourness in deteriorated samples generally increased by mean of electronic tongue analysis. Compared with normal samples， the moth-eaten samples had changed slightly and rancidity samples had changed significantly especially oxidative rancidity samples of volatile components， odor and taste by multivariate statistical analysis. ConclusionIn terms of Platycladi Semen， the oxidative rancidity caused by nature storage for 12 months has the greatest impact on the quality. Therefore， it should be mainly to prevent oxidative rancidity to ensure the quality of Platycladi Semen.

Antifungal Activity of Essential Oil From Zanthoxylum armatum DC. on Aspergillus flavus and Aflatoxins in Stored Platycladi Semen.

The major objective of this study was to evaluate the inhibitory effect of essential oil (EO) from Zanthoxylum armatum DC. on Aspergillus flavus. The chemical composition of the EO was identified by gas chromatography-mass spectrometer. The minimum inhibitory concentration (MIC) of EO was investigated by liquid fermentation. The morphology, colony number, and aflatoxin content of A. flavus in platycladi semen were investigated by stereomicroscopy, scanning electron microscopy, plate counting, and high-performance liquid chromatography. The results indicated that the MIC of EO was 0.8 µL⋅mL-1, and the main components were ß-phellandrene (7.53%), D-limonene (13.24%), linalool (41.73%), terpinen-4-ol (5.33%), and trans-nerolidol (6.30%). After the EO fumigated the platycladi semen, the growth of A. flavus slowed, and the mycelium shrank considerably. The number of colonies after EO treatment at room temperature and cold storage was significantly reduced, the inhibition effect was better under cold storage, and the aflatoxin B1 content did not exceed the standard within 100 days. Therefore, this study demonstrated the good potential of A. flavus growth inhibition during the storage of platycladi semen.

[Investigation and research on fungal diversity of Platycladi Semen on Chinese market].

In the process of harvesting, production and processing, storage, and transportation, the traditional Chinese medicine Platycladi Semen is prone to mildew due to its own and environmental factors, which can nourish the production of toxic or pathogenic fungi, and even produce mycotoxins, which affects the safety of clinical medication. The 2020 edition of Chinese Pharmacopoeia limits the highest standard of aflatoxin content in Platycladi Semen. However, there are few studies on the fungal contamination of Platycladi Semen, and it is difficult to prevent and control it in a targeted manner. Therefore, based on the Illumina NovaSeq6000 platform, this article uses ITS sequence amplicon technology to analyze the distribution and diversity of fungi in 27 batches of commercially available Platycladi Semen in the Chinese market. A total of 10 phyla, 35 classes, 93 orders, 193 families, 336 genera, and 372 species of fungi were identified in China. Among them, Aspergillus, Alternaria spp. were dominant, 20 batches of samples were detected for A. flavus, 10 batches of samples were detected for A. nidulans, and all samples were detected for potential pathogenic fungi such as A. fumigatus and A. niger. According to diversity analysis, the diversity of the fungal communities in the samples from Gansu province was high, the samples in Shandong province contain the largest number of fungal species, and the samples in Guangxi province had the lo-west diversity and the least number of species. In most samples, pathogenic fungi such as A. fumigatus, A. niger, A. flavus, A. parasiticus were detected in varying degrees. This study systematically investigated the fungal contamination of Platycladi Semen from the markets in the last link of the its industrial chain, and clarified the distribution of Platycladi Semen fungi, especially toxin-producing fungi, and provided theoretical basis for the targeted prevention and control of fungal contamination in Platycladi Semen.

Investigation and research on fungal diversity of Platycladi Semen on Chinese market / 中国中药杂志

In the process of harvesting, production and processing, storage, and transportation, the traditional Chinese medicine Platycladi Semen is prone to mildew due to its own and environmental factors, which can nourish the production of toxic or pathogenic fungi, and even produce mycotoxins, which affects the safety of clinical medication. The 2020 edition of Chinese Pharmacopoeia limits the highest standard of aflatoxin content in Platycladi Semen. However, there are few studies on the fungal contamination of Platycladi Semen, and it is difficult to prevent and control it in a targeted manner. Therefore, based on the Illumina NovaSeq6000 platform, this article uses ITS sequence amplicon technology to analyze the distribution and diversity of fungi in 27 batches of commercially available Platycladi Semen in the Chinese market. A total of 10 phyla, 35 classes, 93 orders, 193 families, 336 genera, and 372 species of fungi were identified in China. Among them, Aspergillus, Alternaria spp. were dominant, 20 batches of samples were detected for A. flavus, 10 batches of samples were detected for A. nidulans, and all samples were detected for potential pathogenic fungi such as A. fumigatus and A. niger. According to diversity analysis, the diversity of the fungal communities in the samples from Gansu province was high, the samples in Shandong province contain the largest number of fungal species, and the samples in Guangxi province had the lo-west diversity and the least number of species. In most samples, pathogenic fungi such as A. fumigatus, A. niger, A. flavus, A. parasiticus were detected in varying degrees. This study systematically investigated the fungal contamination of Platycladi Semen from the markets in the last link of the its industrial chain, and clarified the distribution of Platycladi Semen fungi, especially toxin-producing fungi, and provided theoretical basis for the targeted prevention and control of fungal contamination in Platycladi Semen.

Assessment of the Microbiome and Potential Aflatoxin Associated With the Medicinal Herb Platycladus orientalis.

Platycladi Semen, which is derived from the dried ripe seed of Platycladus orientalis, has been used for the treatment of insomnia and constipation in China for 2000 years. However, it is susceptible to fungal and aflatoxin contamination under proper humidity and temperature during storage. Although aflatoxin contamination in Platycladi Semen has been reported preliminarily, few studies have been conducted on fungal infection and aflatoxin contamination simultaneously. Thus, this work aims to provide an in-depth understanding of fungal contamination in Platycladi Semen, and information on aflatoxin contamination. We focused on a comparison of the difference in fungal diversity between aflatoxin-contaminated and aflatoxin-free Platycladi Semen samples. First, aflatoxin levels in 11 Platycladi Semen samples, which were collected from local herbal markets in Shandong, Anhui, and Hebei provinces throughout China, were determined by IAC-HPLC-FLD, and positive confirmation of detected samples was performed by LC-MS/MS. The samples were divided into two groups, based on production or non-production of aflatoxin. We then used the Illumina MiSeq PE250 platform, and targeted the internal transcribed spacer two sequences to analyze the diversity and composition of the fungal microbiome, as well as to assess the presence of potential mycotoxin-producing fungi. Results showed that five samples were contaminated with aflatoxins, one of which exceeded the legal limits of Chinese Pharmacopeia Commission (2015). At the phylum level, the Ascomycota was the most dominant in all tested samples, with a relative abundance of 83.04-99.46%. Aspergillus (27.88-97.28%), Xerochrysium (0-28.49%), and Xeromyces (0-22.24%) were the three predominant genera. Furthermore, differences in fungal composition between the aflatoxin-contaminated and aflatoxin-free groups, as well as between different provinces were observed. A total of 74 species were identified, and four potential mycotoxin-producing fungi were detected in all samples, namely Aspergillus flavus, Aspergillus fumigatus, Fusarium poae, and Penicillium steckii. In conclusion, we report the great diversity of fungi associated with Platycladi Semen, highlight the risk to consumers of ingesting potent aflatoxin, and provide a reference for the safe application and quality improvement of Platycladi Semen.

Study on Inhibitory Effect of Volatile Oils in 16 Plants on Aspergillus flavus of Platycladi Semen / 中国实验方剂学杂志

Objective::To select volatile oils from 16 species of plants (Cymbopogon citratus, Pelargonium graveolens, Pinus tabulieformis, Litsea cubeba, Mentha haplocalyx, Zingiber officinale, Syzygium aromaticum, Curcuma longa, Zanthoxylum bungeanum, Cinnamomum cassia, Ocimum basilicum, Rosmarinus officinalis, Zanthoxylum schinifolium, Zanthoxylum armatum, Illicium verum, Myristica fragrans) that have good inhibitory effect on the growth of Aspergillus flavus. Method::Aspergillus flavus was isolated from the surface of Platycladi Semen medicinal materials by plate culture method. The volatile oils of 16 plants were extracted by steam distillation. The colony diameter of Aspergillus flavus was determined by fumigation of filter paper, and the effect of volatile oils on the growth of Aspergillus flavus was systematically studied. Result::Aspergillus flavus was successfully isolated from Platycladi Semen by means of morphological, microscopic and DNA barcoding identification methods, the bacteriostatic rates of the above 16 kinds of volatile oils against Aspergillus flavus were 2.93%, 0.05%, 0.37%, 76.07%, 0.34%, 0.15%, 50.05%, 8.51%, 1.43%, 58.20%, 0.07%, 2.60%, 8.73%, 100.00%, 52.62%, 0.07%, respectively. Conclusion::The volatile oils of 16 plants all have different degrees of antibacterial activities for Aspergillus flavus, and volatile oils of Zanthoxylum armatum, Litsea cubeba and Cinnamomum cassia have good inhibitory effect. This study can provide a theoretical basis for the prevention and control of Aspergillus flavus in the growth and storage of Platycladi Semen, and provide a basis for further research on plant volatile oil as bacteriostatic agents in the storage process of traditional Chinese medicine.

Study on the surface fungus diversity of commercially Platycladi semen based on high-throughput sequencing technology / 药学学报

The structural composition of the surface fungal community of commercially Platycladi semen was analyzed to reveal the surface fungal biodiversity and structural differences. Platycladi semen was collected from Henan, Shandong and Hong Kong, their DNA was extracted, ITS fragments in DNA were amplified by PCR. Miseq was sequenced on Illumina Hiseq 2500 platform after the PCR products were qualified for quality inspection. The sequence OTU cluster was obtained and bioinformatics analysis was carried out. Microbial communities were not observed in the eyes of the Platycladi semen in the three regions. Sequencing results showed that the surface microbial community had high biodiversity, but there were significant differences in species composition. Seven samples o Platycladi semen obtained 345 947 valid sequences, which were divided into 267 OTUs, 3 phylums. 18 classes, 40 orders, 82 families, 120 genus, 191 species fungi. At the genus level, Aspergillus is the dominant species, accounting for the highest proportion, reaching (93.36 ± 6.01)%. Seven samples were contaminated by Aspergillus flavus, and the pollution levels were 14.58%, 15.98%, 17.64%, 16.44%, 0.97%, 23.39% and 18.86%. Except sample No. 5, Aspergillus cibarius was the most abundant, the other six samples were Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus as the core microflora. By analyzing the diversity of fungi distribution in different habitats, we can fully understand the fungi on the surface of Platycladi semen, lay a foundation for early risk warning of Aspergillus flavus contamination and its aflatoxin contamination, and provide a theoretical basis for the quality and safety of Platycladi semen.

Investigation of aflatoxins contamination in herbal materia medica in a Taiwan pharmaceutical factory.

During the years 2005-2016, a total of 1067 samples for 24 types of herbal materia medica were investigated for the presence of aflatoxins (AFs) using immunoaffinity column cleanup and HPLC-coupled to a fluorescence detector after post-column derivatization. AFs were detected in 373 (35%) out of the total samples. Among them, Platycladi Semen (65% for total AFs and 79% for AFB1), Corydalis Rhizoma (53% for total AFs and 32% for AFB1), Corni Fructus (3% for total AFs), Coicis Semen (3% for total AFs and AFB1), Nelumbinis Semen (6% for total AFs and 9% for AFB1), Arecae Semen (18% for AFB1), Polygalae Radix (5% for total AFs and AFB1), and Cassiae Semen (25% for total AFs and 38% for AFB1) exceeded the official limits of 5 and 10 µg/kg, for AFB1 and total AFs (the sum of AFB1, AFB2, AFG1, and AFG2), respectively, set by the Taiwan government. We concluded that Platycladi Semen, Corydalis Rhizoma, and Cassiae Semen are the most commonly contaminated by AFs.