Bioactive components in prunella vulgaris for treating Hashimoto's disease via regulation of innate immune response in human thyrocytes.

Background: Hashimoto's thyroiditis (HT) is a thyroid autoimmune disease characterized by lymphocytic infiltration and thyroid destruction. Prunella vulgaris (PV) is a traditional Chinese herbal medicine with documented clinical efficacy in treating HT. We previously reported an immunoregulatory effect of PV in thyrocytes; however, the bioactive components of PV remained unclear. This study aimed to elucidate key components of PV for treating HT and their acting mechanisms. Methods: Network pharmacology was used to predict key PV components for HT. The predicted components were tested to determine whether they could exert an immunoregulatory effect of PV in human thyrocytes. Limited proteolysis-mass spectrometry (Lip-MS) was used to explore interacting proteins with PV components in human thyrocytes. Microscale thermophoresis binding assay was used to evaluate the affinity of PV components with the target protein. Results: Eleven PV components with 192 component targets and 3415 HT-related genes were gathered from public databases. With network pharmacology, a 'component-target-disease' network was established wherein four flavonoids including quercetin, luteolin, kaempferol, morin, and a phytosterol, ß-sitosterol were predicted as key components in PV for HT. In stimulated primary human thyrocytes or Nthy-ori-31 cells, key components inhibited gene expressions of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interferon-ß (IFN-ß), cellular apoptosis, and activation of nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3). Heat shock protein 90 alpha, class A, member 1 (HSP90AA1), was identified to interact with flavonoids in PV by Lip-MS. Morin had the highest affinity with HSP90AA1 (KD = 122.74 µM), followed by kaempferol (KD = 168.53 µM), luteolin (KD = 293.94 µM), and quercetin (KD = 356.86 µM). Conclusion: Quercetin, luteolin, kaempferol, morin, and ß-sitosterol reproduced an anti-inflammatory and anti-apoptosis effect of PV in stimulated human thyrocytes, which potentially contributed to the treatment efficacy of PV in HT.

Heavy metals and probabilistic risk assessment via Prunella vulgaris (food and medicine homology) consumption in Guangdong Province, China.

Prunella vulgaris is widely used as the main ingredient of herb tea in Southeast Asia, as well as a traditional Chinese medicine. However, the heavy metal contaminations such as arsenic, cadmium, mercury and lead in P. vulgaris may be a cause for concern due to the environment pollution around, plantation and processing contamination. Thus, this study intented to assess both non-carcinogenic risks and carcinogenic risks attributed to cumulative exposure to the four heavy metals in P. vulgaris. The contaminations levels of heavy metals were determined in 90 batches of P. vulgaris. And the consumption level was obtained through a questionnaire survey among a total of 6,235 adult participants in Guangdong province. This study estimated the probabilistic health risks using Monte Carlo simulation, and found that the estimated mean and the 95th percentile values for cumulative noncarcinogenic risk (HI value) and carcinogenic risk (TCR value) of P. vulgaris were all within the acceptable risk. And the assessment results indicated that arsenic was the primary contributors to both noncarcinogenic and carcinogenic risks through P. vulgaris consumption. These findings and continuing the surveillance of heavy metals in P. vulgaris will be particularly relevant to both consumers and policy makers.

Construction and anti-pancreatic cancer activity of selenium nanoparticles stabilized by Prunella vulgaris polysaccharide.

Selenium nanoparticles (SeNPs), as a potential cancer therapeutic agent, have attracted extensive attention due to their high anticancer activity and low toxicity. Polysaccharides could be the modifiers and stabilizers to improve the stability and dispersibility of SeNPs in aqueous solution. This study aimed to investigate the physicochemical characterization, stability, and anti-pancreatic cancer cell activities of SeNPs stabilized by a heteroxylan PVP3-1 extracted from the clusters of Prunella vulgaris Linn. Our results showed that PVP3-1 with Mw of 154 kDa was composed of →4)-ß-D-Xylp(1→, →2, 4)-ß-D-Xylp(1→, t-α-L-Araf(1→ and 4-MeO-α-D-GlcpA(1→. Red, zero-valent, and uniform spherical SeNPs with an average diameter of about 60 nm and high stability in aqueous solution were constructed successfully by polysaccharide PVP3-1. Anti-pancreatic cancer cell activity assays showed that PVP3-1-SeNPs could inhibit the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, PVP3-1-SeNPs induced apoptosis and autophagy of pancreatic cancer cells through inhibiting mTOR signaling pathway. In conclusion, these results indicated that PVP3-1-SeNPs could be potential anti-tumor nanoparticles for treating pancreatic cancer.

Study on chemical characterization and sleep-improvement function of Prunella vulgaris L. based on the functional components.

Prunella vulgaris L. (P. vulgaris) has great application value and development prospects in improving sleep. In this study, we continued to evaluate the sleep-improvement function and mechanism of P. vulgaris from both chemical characterization and function based on sleep-improvement functional ingredients, rosmarinic acid and salviaflaside, screened out in the previous stage as the index components. The chemical constituents of P. vulgaris and its phenolic acid fraction were characterized by the UPLC-MSn technology. The quality of the sleep-improvement phenolic acid fraction of P. vulgaris was scientifically evaluated by fingerprints combined with quantitative analysis of rosmarinic acid and salviaflaside. The function of phenolic acid parts of P. vulgaris in improving sleep was verified by different insomnia models including the PCPA-induced insomnia model and surface platform sleep deprivation model. HE staining was used to observe the effect of P. vulgaris on the morphology of nerve cells in different brain regions. In vivo experiments and molecular docking explored the sedative-hypnotic effects of functional ingredients of P. vulgaris. All these results investigated the material basis and mechanism of P. vulgaris to improve sleep from multiple perspectives, which contribute to providing a basis for the development of functional food to improve sleep.

Characterization and identification of the wide-polarity multicomponents from Prunella vulgaris by offline two-dimensional liquid chromatography and hydrophilic interaction chromatography coupled to ion mobility-quadrupole time-of-flight mass spectrometry.

Metabolites identification is crucial to develop functional foods or perform quality control. Prunella vulgaris (Xia-Ku-Cao) is a medicinal and edible plant used as the herbal medicine or main additive in functional beverage. However, current analytical strategies can only on-line characterize tens of compounds, restricted by insufficient chromatographic resolution and low coverage of the mass spectrometric scan methods. This work was designed to characterize the wide-polarity components from the ear of P. vulgaris. The total extract was fractionated by semi-preparative high-performance liquid chromatography into the retained medium-polarity fraction and unretained polar fraction, which were further analyzed by offline two-dimensional liquid chromatography (2D-LC) and hydrophilic interaction chromatography, respectively. Data-independent high-definition MSE of the Vion™ ion mobility time-of-flight mass spectrometer was utilized enabling the high-coverage acquisition of collision-induced dissociation-MS2 data. The offline 2D-LC, configuring the XBridge Amide and HSS T3 columns, gave high orthogonality (0.81) and effective peak capacity (1555). Automatic peak annotation facilitated by the UNIFI™ bioinformatics platform and comparison with 62 reference compounds achieved the efficient and more reliable structural elucidation. We could characterize 255 compounds from P. vulgaris, with numerous phenylpropanoid phenolic acids and triterpenoid O-glycosides newly reported. Especially, collision cross section (CCS) prediction and targeted isolation of three compounds assisted in the identification of 39 groups of isomers. Additionally, 17 hydrophilic compounds, involving oligosaccharides and organic acids, were characterized from the unretained polar fraction. Conclusively, the in-depth metabolites identification of P. vulgaris was accomplished, and the results can benefit the development and better quality control of this valuable plant.

Mechanism of Prunella vulgaris L. and luteolin in restoring Tfh/Tfr balance and alleviating oxidative stress in Graves' disease.

BACKGROUND: The pathophysiology of Graves' disease (GD) involves imbalances between follicular helper T (Tfh) and follicular regulatory T (Tfr) cells, as well as oxidative stress (OS). Prunella vulgaris L. (Xia Ku Cao, XKC) and its primary bioactive compound, luteolin, are recognized for their potential in treating GD. Yet, the mechanism accounting for the immune-modulatory and antioxidant effects of XKC remains elusive. PURPOSE: This study aims to evaluate the pharmacological effects and elucidate the underlying mechanism of XKC and luteolin in a GD mouse model induced by recombinant adenovirus of TSH receptor A subunit (Ad-hTSHR-289). METHODS: High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (HPLC-QTOF MS) was used to detect the constituents of XKC. The GD model was established through inducing female BALB/c mice with three intramuscular injections of Ad-TSHR-289. Thyroid function, autoantibody and OS parameters were measured by ELISA. Changes of Tfh cells and Tfr cells were detected by flow cytometry. RT-qPCR, Western Blotting, immunohistochemistry were used to explore the related molecular mechanisms. RESULTS: A total of 37 chemical components from XKC were identified by HPLC-QTOF MS, represented by flavonoids, steroids, terpenoids, and luteolin. XKC and luteolin reduced T4, TRAb levels and facilitated the recovery from thyroid damage in GD mice. Meanwhile, XKC and luteolin effectively alleviated OS by decreasing the levels of MDA, NOX2, 4-HNE, 8-OHdG, while increasing GSH level. Flow cytometry showed that XKC and luteolin restored the abnormal proportions of Tfh/Tfr and Tfh/Treg, and the mRNA levels of IL-21, Bcl-6 and Foxp3 in GD mice. In addition, XKC and luteolin inhibited PI3K, Akt, p-PI3K and p-Akt, but activated Nrf2 and HO-1. CONCLUSION: XKC and luteolin could inhibit the development of GD in vivo by rebalancing Tfh/Tfr cells and alleviating OS. This therapeutic mechanism may involve the Nrf2/HO-1 and PI3K/Akt signaling pathways. Luteolin is the main efficacy material basis of XKC in countering GD. For the first time, we revealed the mechanism of XKC and luteolin in the treatment of GD from the perspective of autoimmune and OS.

The anti-hyperplasia effect of polysaccharide from Prunella vulgaris L. on mammary gland hyperplasia in rats through regulation of the AKT-FOXO3a signaling pathway and intestinal flora.

BACKGROUND: Prunella vulgaris L., a medicinal and edible homologous plant, is often used to treat conditions such as breast hyperplasia, thyroid enlargement and lymphatic tuberculosis. Research has demonstrated that it is particularly effective in the treatment of mammary gland hyperplasia (MGH). However, the material basis and mechanism of its efficacy are still unclear. RESULTS: Our results showed that in rats with MGH, polysaccharide from Prunella vulgaris L. (PVP) led to a reduction in the levels of estradiol, prolactin and malondialdehyde, while simultaneously increasing the concentrations of progesterone (P), superoxide dismutase (SOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) in the serum. In addition, results obtained from 16S rRNA sequencing demonstrated that PVP had the capacity to increase the richness and diversity of the intestinal microbiota in MGH rats, as well as modify the structure of the microbiota. Correlation analysis revealed that the levels of P, SOD, MnSOD and CAT were positively associated with Allobaculum, Romboutsia, Faecalibaculum and Clostridium, while negatively correlated with Turicibacter. CONCLUSIONS: The mechanism of PVP in treating MGH might be through inhibiting the phosphorylation of the AKT-FOXO3a signaling pathway and then activating the expression of downstream antioxidant enzymes, such as MnSOD and CAT. At the same time, PVP could restore intestinal flora homeostasis in rats with MGH by regulating the flora changes of Allobaculum, Romboutsia, Clostridium and Faecalibaculum, thereby reducing oxidative stress in rats with MGH. © 2024 Society of Chemical Industry.

Systematic characterization of gene families and functional analysis of PvRAS3 and PvRAS4 involved in rosmarinic acid biosynthesis in Prunella vulgaris.

Prunella vulgaris is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses showed that 17 of them could be involved in RA biosynthesis. In vitro enzymatic assay showed that PvRAS3 catalyzed the condensation of p-coumaroyl-CoA and caffeoyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was higher than caffeoyl-CoA. PvRAS4 catalyzed the condensation of p-coumaroyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was lower than PvRAS3. UPLC and LC-MS/MS analyses showed the existence of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid in P. vulgaris. Generation and analysis of pvras3 homozygous mutants showed significant decrease of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid and significant increase of DHPL and pHPL. It suggests that PvRAS3 is the main enzyme catalyzing the condensation of acyl donors and acceptors during RA biosynthesis. The role of PvRAS4 appears minor. The results provide significant information for quality control of P. vulgaris medicinal materials.

Green and Efficient Extraction of Phenolic Components from Plants with Supramolecular Solvents: Experimental and Theoretical Studies.

The supramolecular solvent (SUPRAS) has garnered significant attention as an innovative, efficient, and environmentally friendly solvent for the effective extraction and separation of bioactive compounds from natural resources. However, research on the use of a SUPRAS for the extraction of phenolic compounds from plants, which are highly valued in food products due to their exceptional antioxidant properties, remains scarce. The present study developed a green, ultra-sound-assisted SUPRAS method for the simultaneous determination of three phenolic acids in Prunella vulgaris using high-performance liquid chromatography (HPLC). The experimental parameters were meticulously optimized. The efficiency and antioxidant properties of the phenolic compounds obtained using different extraction methods were also compared. Under optimal conditions, the extraction efficiency of the SUPRAS, prepared with octanoic acid reverse micelles dispersed in ethanol-water, significantly exceeded that of conventional organic solvents. Moreover, the SUPRAS method demonstrated greater antioxidant capacity. Confocal laser scanning microscopy (CLSM) images revealed the spherical droplet structure of the SUPRAS, characterized by a well-defined circular fluorescence position, which coincided with the position of the phenolic acids. The phenolic acids were encapsulated within the SUPRAS droplets, indicating their efficient extraction capacity. Furthermore, molecular dynamics simulations combined with CLSM supported the proposed method's mechanism and theoretically demonstrated the superior extraction performance of the SUPRAS. In contrast to conventional methods, the higher extraction efficiency of the SUPRAS can be attributed to the larger solvent contact surface area, the formation of more types of hydrogen bonds between the extractants and the supramolecular solvents, and stronger, more stable interaction forces. The results of the theoretical studies corroborate the experimental outcomes.

Phytochemical Analysis and Antioxidant Effects of Prunella vulgaris in Experimental Acute Inflammation.

Prunella vulgaris (PV) is one of the most commonly used nutraceuticals as it has been proven to have anti-inflammatory and antioxidant properties. The aim of this study was to evaluate the phytochemical composition of PV and its in vivo antioxidant properties. A phytochemical analysis measuring the total phenolic content (TPC), the identification of phenolic compounds by HPLC-DAD-ESI, and the evaluation of the in vitro antioxidant activity by the DPPH assay of the extract were performed. The antioxidant effects on inflammation induced by turpentine oil were experimentally tested in rats. Seven groups with six animals each were used: a control group, the experimental inflammation treatment group, the experimental inflammation and diclofenac sodium (DS) treatment group, and four groups with their inflammation treated using different dilutions of the extract. Serum redox balance was assessed based on total oxidative status (TOS), nitric oxide (NO), malondialdehyde (MDA), total antioxidant capacity (TAC), total thiols, and an oxidative stress index (OSI) contents. The TPC was 0.28 mg gallic acid equivalents (GAE)/mL extract, while specific representatives were represented by caffeic acid, p-coumaric acid, dihydroxybenzoic acid, gentisic acid, protocatechuic acid, rosmarinic acid, vanillic acid, apigenin-glucuronide, hesperidin, kaempferol-glucuronide. The highest amount (370.45 µg/mL) was reported for hesperidin, which is a phenolic compound belonging to the flavanone subclass. The antioxidant activity of the extracts, determined using the DPPH assay, was 27.52 mmol Trolox/mL extract. The PV treatment reduced the oxidative stress by lowering the TOS, OSI, NO, and MDA and by increasing the TAC and thiols. In acute inflammation, treatment with the PV extract reduced oxidative stress, with lower concentrations being more efficient and having a better effect than DS.

[Analysis of differential metabolites of spikes of Prunella vulgaris at different stages based on UPLC-MS/MS].

Prunella vulgaris, aptly named for its withering at the summer solstice, displays significant variation in quality arising from differing harvest time. However, research on the chemical composition changes of its spikes at various stages is limited, and the specific metabolites remain unclear. In order to elucidate the metabolites and metabolic pathways of the spikes of P. vulgaris, the current study deployed ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) and targeted metabolomics to characterize the compound variability in the spikes of P. vulgaris across different periods. Multivariate statistical techniques such as principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to identify the differences in metabolites, and relevant metabolic pathways were analyzed. A total of 602 metabolites were identified by metabolomics, of which organic acids and their derivatives were the most abundant, followed by flavonoids. Multiple differential metabolites, including p-hydroxybenzoic acids and gallic acids were identified based on variable importance in projection(VIP)>1 and P<0.05. The results of enrichment analysis suggested that isoflavonoids biosynthesis, aminobenzoate degradation, benzoate degradation, anthocyanins biosynthesis, metabolic pathways, microbial metabolism in different environments, secondary plant metabolite biosynthesis, tryptophan metabolism, and phenylpropanoid synthesis were the main metabolic pathways. These results intend to elucidate the dynamic changes of differential metabolites of P. vulgaris and provide a theoretical basis for further study of the harvesting mechanism of spikes of P. vulgaris.

Anti-Tumor Effects and Toxicity Reduction Mechanisms of Prunella vulgaris: A Comprehensive Review.

PURPOSE: To investigate and systematically describe the mechanism of action of Prunella vulgaris (P. vulgaris) against digestive system tumors and related toxicity reduction. METHODS: This study briefly describes the history of medicinal food and the pharmacological effects of P. vulgaris, focusing on the review of the anti-digestive tumor effects of the active ingredients of P. vulgaris and the mechanism of its toxicity reduction. RESULTS: The active ingredients of P. vulgaris may exert anti-tumor effects by inducing the apoptosis of cancer cells, inhibiting angiogenesis, inhibiting the migration and invasion of tumor cells, and inhibiting autophagy. In addition, P. vulgaris active ingredients inhibit the release of inflammatory factors and macrophages and increase the level of indicators of oxidative stress through the modulation of target genes in the pathway to achieve the effect of toxicity reduction. CONCLUSION: The active ingredients in the medicine food homology plant P. vulgaris not only treat digestive system tumors through different mechanisms but also reduce the toxic effects. P. vulgaris is worthy of being explored more deeply.

Exploring Information Exchange between Thesium chinense and Its Host Prunella vulgaris through Joint Transcriptomic and Metabolomic Analysis.

BACKGROUND: Thesium chinense known as the "plant antibiotic" is a facultative root hemi-parasitic herb while Prunella vulgaris can serve as its host. However, the molecular mechanisms underlying the communication between T. chinense and its host remained largely unexplored. The aim of this study was to provide a comprehensive view of transferred metabolites and mobile mRNAs exchanged between T. chinense and P. vulgaris. RESULTS: The wide-target metabolomic and transcriptomic analysis identified 5 transferred metabolites (ethylsalicylate, eriodictyol-7-O-glucoside, aromadendrin-7-O-glucoside, pruvuloside B, 2-ethylpyrazine) and 50 mobile genes between T. chinense and P. vulgaris, as well as haustoria formation related 56 metabolites and 44 genes. There were 4 metabolites (ethylsalicylate, eriodictyol-7-O-glucoside, aromadendrin-7-O-glucoside and pruvuloside B) that are transferred from P. vulgaris to T. chinense, whereas 2-ethylpyrazine was transferred in the opposite direction. Furthermore, we inferred a regulatory network potentially involved in haustoria formation, where three metabolites (N,N'-Dimethylarginine/SDMA, NG,NG-Dimethyl-L-arginine, 2-Acetoxymethyl-anthraquinone) showed significant positive correlations with the majority of haustoria formation-related genes. CONCLUSIONS: These results suggested that there was an extensive exchange of information with P. vulgaris including transferred metabolites and mobile mRNAs, which might facilitate the haustoria formation and parasition of T. chinense.

Exploring the Antibacterial Potential and Underlying Mechanisms of Prunella vulgaris L. on Methicillin-Resistant Staphylococcus aureus.

Prunella vulgaris L. (PV) is a widely distributed plant species, known for its versatile applications in both traditional and contemporary medicine, as well as in functional food development. Despite its broad-spectrum antimicrobial utility, the specific mechanism of antibacterial action remains elusive. To fill this knowledge gap, the present study investigated the antibacterial properties of PV extracts against methicillin-resistant Staphylococcus aureus (MRSA) and assessed their mechanistic impact on bacterial cells and cellular functions. The aqueous extract of PV demonstrated greater anti-MRSA activity compared to the ethanolic and methanolic extracts. UPLC-ESI-MS/MS tentatively identified 28 phytochemical components in the aqueous extract of PV. Exposure to an aqueous extract at ½ MIC and MIC for 5 h resulted in a significant release of intracellular nucleic acid (up to 6-fold) and protein (up to 10-fold) into the extracellular environment. Additionally, this treatment caused a notable decline in the activity of several crucial enzymes, including a 41.51% reduction in alkaline phosphatase (AKP), a 45.71% decrease in adenosine triphosphatase (ATPase), and a 48.99% drop in superoxide dismutase (SOD). Furthermore, there was a decrease of 24.17% at ½ MIC and 27.17% at MIC in tricarboxylic acid (TCA) cycle activity and energy transfer. Collectively, these findings indicate that the anti-MRSA properties of PV may stem from its ability to disrupt membrane and cell wall integrity, interfere with enzymatic activity, and impede bacterial cell metabolism and the transmission of information and energy that is essential for bacterial growth, ultimately resulting in bacterial apoptosis. The diverse range of characteristics exhibited by PV positions it as a promising antimicrobial agent with broad applications for enhancing health and improving food safety and quality.

Effect and mechanism of Prunella vulgaris L. extract on alleviating lipopolysaccharide-induced acute mastitis in protecting the blood-milk barrier and reducing inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.

Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

Prunella vulgaris polysaccharide inhibits herpes simplex virus infection by blocking TLR-mediated NF-κB activation.

BACKGROUND: Prunella vulgaris polysaccharide extracted by hot water and 30% ethanol precipitation (PVE30) was reported to possess potent antiviral effects against herpes simplex virus (HSV) infection. However, its anti-HSV mechanism has not yet been fully elucidated. PURPOSE: This study aimed to investigate the potential mechanisms of PVE30 against HSV infection. METHODS: Antiviral activity was evaluated by a plaque reduction assay, and the EC50 value was calculated. Immunofluorescence staining and heparin bead pull-down assays confirmed the interactions between PVE30 and viral glycoproteins. Real-time PCR was conducted to determine the mRNA levels of viral genes, including UL54, UL29, UL27, UL44, and US6, and the proinflammatory cytokines IL-6 and TNF-α. The protein expression of viral proteins (ICP27, ICP8, gB, gC, and gD), the activity of the TLR-NF-κB signalling pathway, and necroptotic-associated proteins were evaluated by Western blotting. The proportion of necroptotic cells was determined by flow cytometric analysis. RESULTS: The P. vulgaris polysaccharide PVE30 was shown to compete with heparan sulfate for interaction with HSV surface glycoprotein B and gC, thus strongly inhibiting HSV attachment to cells. In addition, PVE30 downregulated the expression of IE genes, which subsequently downregulated the expression of E and L viral gene products, and thus effectively restricted the yield of progeny virus. Further investigation confirmed that PVE30 inhibited TLR2 and TLR3 signalling, leading to the effective suppression of NF-κB activation and IL-6 and TNF-α expression levels, and blocked HSV-1-induced necroptosis by reducing HSV-1-induced phosphorylation of MLKL. CONCLUSION: Our results demonstrate that the P. vulgaris polysaccharide PVE30 is a potent anti-HSV agent that blocks TLR-mediated NF-κB activation.

Multi-level chemical characterization and anti-inflammatory activity evaluation of the polysaccharides from Prunella vulgaris.

Prunella vulgaris (PV) is a medicine and food homologous plant, but its quality evaluation seldom relies on the polysaccharides (PVPs). In this work, we established the multi-level fingerprinting and in vitro anti-inflammatory evaluation approaches to characterize and compare the polysaccharides of P. vulgaris collected from the major production regions in China. PVPs prepared from 22 batches of samples gave the content variation of 5.76-24.524 mg/g, but displayed high similarity in the molecular weight distribution. Hydrolyzed oligosaccharides with degrees of polymerization 2-14 were characterized with different numbers of pentose and hexose by HILIC-MS. The tested 22 batches of oligosaccharides exhibited visible differences in peak abundance, which failed to corelate to their production regions. All the PVPs contained Gal, Xyl, and Ara, as the main monosaccharides. Eleven batches among the tested PVPs showed the significant inhibitory effects on NO production on LPS-induced RAW264.7 cells at 10 µg/mL, but the exerted efficacy did not exhibit correlation with the production regions. Conclusively, we, for the first time, investigated the chemical features of PVPs at three levels, and assessed the chemical and anti-inflammatory variations among the different regions of P. vulgaris samples.

Exploring the mechanism of bioactive components of Prunella vulgaris L. in treating hepatocellular carcinoma based on network pharmacology.

In traditional Chinese Medicine, Prunella vulgaris L. (PVL) is potentially effective in the treatment of some human malignancies including hepatocellular carcinoma (HCC). However, the detailed mechanism of action remains unclear. The purpose of this study was to decipher the constitutes of the bioactive ingredients of PVL, and its mechanism against HCC using network pharmacology and in vitro experiments. The bioactive components of PVL were obtained by Traditional Chinese Medicine System Pharmacology Database and Analysis platform database, and the targets of bioactive components of PVL was investigated by Swiss Target Prediction database. HCC related targets were obtained from GEO database, GeneCards database and DisGeNET database, and the gene ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted for annotating the biological function of gene targets. A protein-protein interaction network was constructed using STRING database. Molecular docking of key bioactive ingredients was performed using AutoDock Vina. Cell proliferation and apoptosis were detected by cell counting kit-8 and flow cytometry, respectively. The expression level of the target genes of PI3K/Akt pathway were detected by qPCR. In the present work, 11 bioactive components of PVL were screened out, which acted on 177 potential targets. In addition, 13,517 genes were strongly associated with HCC pathogenesis, of which 158 targets are overlapped with PVL's targets. KEGG results identified 39 signaling pathways closely associated with the 158 targets. Molecular docking showed that the main bioactive components of PVL, kaempferol, morin, quercetin, luteolin, and spinasterol, had good binding activity with the core proteins in cancer biology such as AKT1, EGFR, SRC, ESR1, and PPARG. In vitro assays showed that quercetin, one of the main components of PVL extracts effectively inhibited HCC cell proliferation, and promoted apoptosis, which may be associated with PI3K/AKT signaling pathway. In summary, PVL may regulate HCC progression by regulating core targets such as AKT1, EGFR, SRC, ESR1, and PPARG, and acting on PI3K-Akt signaling pathway.

Analysis of the phytochemical components of Prunella vulgaris using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with molecular networking and assessment of their antioxidant and anti-α-glucosidase activities.

Prunella vulgaris has long been used in traditional medicine and is consumed as a tea in China. Here, the total phenolic and flavonoid concentrations of plants from different geographical regions were measured. It was found that the total phenolic acid concentration ranged from 4.15 to 8.82 g of gallic acid equivalent per 100 g of dry weight (DW), and the total flavonoid concentration was 4.67-7.33 g of rutin equivalent per 100 g DW. Antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and the results ranged from 73.47% to 94.43% and 74.54% to 93.39%, respectively, whereas α-glucosidase inhibition was between 75.31% and 95.49%. Correlation analysis showed that the total flavonoids in P. vulgaris had superior antioxidant and anti-α-glucosidase activities compared to the total phenolic compounds. The active components of P. vulgaris were analyzed using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with both classical molecular networking and feature-based molecular networking on the Global Natural Products Social platform, identifying 32 compounds, namely 14 flavonoids, 12 phenolic compounds, and 6 other chemical components. These results could provide useful information on the use of P. vulgaris as a functional tea.