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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. To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. 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. 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-1β, 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. 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. [Display omitted] • Phenolic acids and flavonoids in P vulgaris may be effective ingredients of Prunella vulgaris L for anti-mastitis. • Experimental evidence supports P vulgaris as a potential remedy for acute mastitis. • UPLC-Q-TOF-MS/MS confirms phenolic acids and flavonoids in P vulgaris extract. • P vulgaris extract can improve the inflammatory reaction in the mammary gland tissue. [ABSTRACT FROM AUTHOR]

Prunella vulgaris L. (P. vulgaris , Labiatae) is a perennial medicinal and edible plant widely used in China, Korea, Japan and Europe. The reddish brown spica of P. vulgaris (Prunellae Spica), which is collected in summer, has been commonly used in traditional medicine and food industry, while it is also used with whole grass in Europe and Taiwan. To clarify the regulatory pathways and mechanism of quality formation in P. vulgaris , targeted metabolomic, transcriptomic, and proteomic analyses of Prunellae Spica samples from five consecutive developmental stages were carried out. The results showed that terpenoids were mainly synthesized in the maturity stage of Prunellae Spica, with the key enzymes and coding genes in downstream pathways being mainly expressed during ripening, while related enzymes in the upstream pathway showed the opposite pattern. Flavonoids mainly accumulated before ripening, with highly expressed pathway enzymes and coding genes. The accumulation of phenylpropanoids was relatively active throughout the development process. Rosmarinic acid (RA) and its synthetic intermediate products mainly accumulated via more active pathway enzymes and coding genes before ripening. The regulatory factors and metabolites related to RA synthesis were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, plant pathogen interaction, oxidative phosphorylation, and endoplasmic reticulum protein processing pathways. [Display omitted] [ABSTRACT FROM AUTHOR]

Prunella vulgaris L., better known as 'self-heal', has been extensively used in the traditional system of medicines. To reveal the regulatory mechanism of its development, TMT-based quantitative proteome analysis was performed in the Prunella vulgaris L. spica before and during ripening (Group A and Group B, respectively). This analysis resulted in the identification of 7655 proteins, of which 1910 showed differential abundance between the two groups. Pronounced changes in the proteomic profile included the following: 1) Stress-responsive proteins involved in protecting cells and promoting fruit ripening and seed development were highly abundant during ripening. 2) The degradation of chlorophyll, inhibition of chlorophyll biosynthesis and increased abundance of transketolase occurred simultaneously in the spica of Prunella vulgaris L., resulting in the spica changing color from green to brownish red. 3) The abundance of protein species related to phenylpropanoid biosynthesis mainly increased during ripening, while flavonoid and terpenoid backbone biosynthesis mostly occurred before ripening. This study establishes a link between protein profiles and mature phenotypes, which will help to improve our understanding of the molecular mechanisms involved in the maturation of Prunella vulgaris L. at the proteome level and reveal the scientific connotation for the best time to harvest Prunella vulgaris L. This work provides a scientific basis for the production of high-quality medicinal Prunella vulgaris L., as well as a typical demonstration of molecular research used for the harvest period of traditional Chinese medicine. This work provided a comprehensive overview on the functional protein profile changes of Prunella vulgaris L. spica at different growing stages, as well as the scientific rationale of Prunella vulgaris L. harvested in summer after brownish red, thus laid an intriguing stepping stone for elucidating the molecular mechanisms of quality development. Unlabelled Image • Prunella vulgaris L. spica was sampled at five developmental stages (from heading to physiological maturity). • LC-MS matched 7655 proteins, of which 6405 can be quantified and 1910 proteins showed differences in abundance. • High abundance of stress-responsive protein species involved in fruit maturity and seed development during spica ripening. • The up-representation of transketolase, the degradation and inhibition synthesis of chlorophyll cause the spica changing color. • Increased levels of protein related to phenylpropanoid biosynthesis were observed during spica ripening [ABSTRACT FROM AUTHOR]