[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.

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.

Phenolic acids from Prunella vulgaris alleviate cardiac remodeling following myocardial infarction partially by suppressing NLRP3 activation.

Acute myocardial infarction (MI) is one of the leading causes of mortality around the world. Prunella vulgaris (Xia-Ku-Cao in Chinese) is used in traditional Chinese medicine practice for the treatment of cardiovascular diseases. However, its active ingredients and mechanisms of action on cardiac remodeling following MI remain unknown. In this study, we investigated the cardioprotective effect of P. vulgaris on MI rat models. MI rats were treated with aqueous extract of P. vulgaris or phenolic acids from P. vulgaris, including caffeic acid, ursolic acid or rosmarinic acid, 1 day after surgery and continued for the following 28 days. Then the cardioprotective effect, such as cardiac function, inflammatory status, and fibrosis areas were evaluated. RNA-sequencing (RNA-seq) analysis, real-time polymerase chain reaction (PCR), western blotting, and ELISA were used to explore the underlying mechanism. In addition, ultra-high performance liquid chromatography/mass spectrometer analysis was used to identify the chemicals from P. vulgaris. THP-1NLRP3-GFP cells were used to confirm the inhibitory effect of P. vulgaris and phenolic acids on the expression and activity of NLRP3. We found that P. vulgaris significantly improved cardiac function and reduced infarct size. Meanwhile, P. vulgaris protected cardiomyocyte against apoptosis, evidenced by increasing the expression of anti-apoptosis protein Bcl-2 in the heart and decreasing lactate dehydrogenase (LDH) levels in serum. Results from RNA-seq revealed that the therapeutic effect of P. vulgaris might relate to NLRP3-mediated inflammatory response. Results from real-time PCR and western blotting confirmed that P. vulgaris suppressed NLRP3 expression in MI heart. We also found that P. vulgaris suppressed NLRP3 expression and the secretion of HMGB1, IL-1ß, and IL-18 in THP-1NLRP3-GFP cells. Further studies indicated that the active components of P. vulgaris were three phenolic acids, those were caffeic acid, ursolic acid, and rosmarinic acid. These phenolic acids inhibited LPS-induced NLRP3 expression and activity in THP-1 cells, and improved cardiac function, suppressed inflammatory aggregation and fibrosis in MI rat models. In conclusion, our study demonstrated that P. vulgaris and phenolic acids from P. vulgaris, including caffeic acid, ursolic acid, and rosmarinic acid, could improve cardiac function and protect cardiomyocytes from ischemia injury during MI. The mechanism was partially related to inhibiting NLRP3 activation.

Combined treatment with Prunella vulgaris and Radix bupleuri activated the Bax/Bcl-2 and Caspase-3 signal pathways in papillary thyroid carcinoma cells.

To explore the effect of Prunella vulgaris (PV) combined with Radix bupleuri (RB) on apoptosis of papillary thyroid carcinoma cells. Our study was divided into four groups: the control group, the PV group, the RB group, and the PV combined with the RB group. The viability of cells from different treatment groups was assessed by the CCK-8 assay. Cell migration and invasion were assessed by healing wounding and the transwell assay, respectively. Cell apoptosis rate and cell cycle arrest were detected by a flow cytometry assay. The protein expression of Bcl-2, Bax, Caspase-3, CyclinA1, CyclinB1, and CDK1 was detected using a western blot assay. Our results indicated that, compared with the control group, PV combined with RB group could significantly alter the cell morphology, inhibit cell migration and invasion, decrease the number of cells in the G0/G1 phase and increase the number of cells in the G2/M phase, and promote the cell apoptosis. Moreover, PV combined with RB treatment also obviously increased the expression of Bax/Bcl2 and caspase-3 proteins and decreased the expression of Cyclin A1, Cyclin B1, and CDK1 proteins. Overall, our results indicated that PV combined with RB could activate the Bax/Bcl-2 and Caspase-3 signal pathways to induce cell apoptosis in papillary thyroid carcinoma cells; this also provides a new way to treat thyroid cancer.

Integrative transcriptomic, proteomic and metabolomic analysis reveals the dynamic regulation of secondary metabolism upon development of Prunella vulgaris L.

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.

Screening of potent α-glucosidase inhibitory and antioxidant polyphenols in Prunella vulgaris L. by bioreaction-HPLC-quadrupole-time-of-flight-MS/MS and in silico analysis.

Prunella vulgaris L. is a well-known traditional Chinese medicine for blood glucose homeostasis and antioxidant potential. Ethyl acetate fraction of P. vulgaris L. demonstrated higher phenolic content (85.53 ± 6.74 mg gallic acid equivalents per gram dry weight), α-glucosidase inhibitory (IC50 , 69.13 ± 2.86 µg/ml), and antioxidant (IC50 , 8.68 ± 1.01 µg/ml) activities. However, the bioactive polyphenols responsible for the beneficial properties remain unclear. Here, bioreaction-HPLC-quadrupole-time-of-flight-MS/MS method was developed for rapid, accurate, and efficient screening and identification of polyphenols with α-glucosidase inhibitory and antioxidant activities from P. vulgaris L. Bioactive polyphenols can specifically bind with α-glucosidase or react with 1,1-diphenyl-2-picryl-hydrazyl radical, which was easily discriminated from nonactive compounds. Subsequently, 20 bioactive polyphenols (16 phenyl propionic acid derivatives and four flavonoids) were screened and identified. Furthermore, molecular docking analysis revealed that screened 20 polyphenols bind with the active sites of α-glucosidase through hydrogen bonding and π-π stacking. Density functional theory calculations demonstrated their electron transport ability and chemical reactivity. The in silico analysis confirmed the screened results. In summary, this study provided a valuable strategy for rapid discovering bioactive compounds from complex natural products and offered scientific evidence for further development and application of P. vulgaris L.

Effect of Prunella vulgaris polysaccharides on cultured orbit fibroblasts in vitro from patients with thyroid-associated ophthalmopathy.

To observe the effect of Prunella vulgaris polysaccharides (PVP) on cultured orbit fibroblasts in vitro from patients with thyroid-associated ophthalmopathy (TAO). PVP at different concentrations were used to treat different groups of fibroblasts from TAO patients and normal persons. Dexamethasone (Dex) was used as a positive control drug, and interferon-γ (IFN-γ) was used as a positive stimulant. The effects of PVP on the proliferation of orbital fibroblasts, the secretion of hyaluronic acid (HA), the expression of intercellular adhesion molecule-1 (ICAM-1/CD54) and apoptosis in orbital fibroblasts were determined. The experimental results showed when the concentration of PVP was greater than 400 µg/mL, it could significantly inhibit the proliferation of orbital fibroblasts from patients with TAO (P < 0.05). However, no definite inhibitory effect was observed in the orbital fibroblasts from the normal people. Dex could significantly inhibit the proliferation of orbital fibroblasts from patients with TAO and the normal people (P < 0.05). In contrast, every concentration of IFN-γ could promote the orbital fibroblasts from patients with TAO and the normal people proliferation. No groups had statistically significant stimulatory effect on HA secretion by orbital fibroblasts from normal people (P > 0.05). But the Dex group, IFN-γ+PVP-1600 group and IFN-γ+Dex group could significantly inhibit the secretion of HA from orbital fibroblasts of TAO patients. And there were no groups had statistically significant stimulatory effect on the expression of ICAM-1/CD54 in orbital fibroblasts from TAO patients (P > 0.05). PVP and Dex at all concentrations could significantly promote orbital fibroblast co-cultured with IFN-γ apoptosis (P < 0.05). But without IFN-γ, PVP and Dex at all concentrations could only significantly promote orbital fibroblast from TAO patients apoptosis (P < 0.05). These results suggest that PVP exerts its therapeutic effect by inhibiting the proliferation of orbital fibroblasts and promoting the apoptosis of orbital fibroblasts in TAO patients. In addition, in this process, HA secretion is suppressed. But the participation of IFN-γ is required. This effect is similar to that of Dex. And in the MTT experiment, the efficacy of PVP showed selectivity for TAO patients. This is different from Dex. This may be a feature of PVP that deserves attention.

Biosynthesis of antioxidative enzymes and polyphenolics content in calli cultures of Prunella vulgaris L. in response to auxins and cytokinins.

Prunella vulgaris L. is one of the therapeutic herbs containing various polyphenolics, which is used for multiple medicinal purposes. In this study, plant growth regulators (PGRs)-induced calli cultures from seed-derived leaf explants were exploited for the production of stress enzymes and polyphenolics. A growth curve was plotted for each PGR for 49 days period, which showed a distinct lag, log and decline phases. Here, the combination of naphthalene acetic acid (NAA) and 6-benzyleadenine (BA; 0.5 and 2.0 mg l-1) produced maximum fresh (6.32 FW-g/100 ml) and dry biomass (0.75 DW-g/100 ml) in contrast to control. The maximum synthesis of SOD (0.0154 FW-nM/min/mg) was detected on media comprising mixture of NAA and BA (1.5 mg l-1), while POD enzyme (0.366 FW-nM/min/mg) was higher at 0.5 mg l-1 NAA and 2, 4-dichlorophenoxy acetic acid. Further, NAA and BA (1.5 and 2.0 mg l-1) boosted up the synthesis of phenolics (18.83 GAE-mg/g-DW) and flavonoids content (18.05 RE-mg/g-DW) than control. Moreover, NAA of 1.0 and 2.0 mg l-1 were found supportive for maximum antioxidant activity (87.4%) and total protein (716 µg BSAE/mg-DW). This study will contribute in the development of cell culture in fermenter and synthesis of antioxidant secondary metabolites for commercial uses.

GC-MS-based metabolomics research on the anti-hyperlipidaemic activity of Prunella vulgaris L. polysaccharides.

Prunella vulgaris polysaccharides (PVPs) have a variety of biological activities, but the mechanism and extent of their anti-hyperlipidaemic effect remain unclear. In vitro, PVPs had a significant inhibitory effect on angiotensin (Ang II)-induced vascular smooth muscle cell (VSMC) proliferation. A metabolomics approach based on gas chromatography-mass spectrometry (GC-MS) and chemometrics was established in this study to evaluate the anti-hyperlipidaemic activity of PVPs in a high-fat Sprague-Dawley rat model. In vivo, PVPs could significantly reduce the weight gain and the increases in serum total cholesterol (TC), low-density lipoprotein (LDL)-C and non-high-density lipoprotein (HDL)-C levels observed in rats fed a high-fat diet; they could also significantly increase serum GSH-Px activity, reduce the content of MDA and TNF-α and decrease abdominal fat volume in rats. Furthermore, PVPs exerted a repairing effect on morphological and structural damage in liver tissue cells in hyperlipidaemic rats fed a high-fat diet. PVPs improved lipid metabolism disorder in rats. Alanine, threonine, succinic acid, proline, inositol and arachidonic acid levels in the serum were considered potential biomarkers involved in amino acid, glucose, energy and lipid metabolism. Therefore, PVPs may interfere with hyperlipidaemia through anti-lipid peroxidation effects, attenuation of inflammation and regulation of glucose, amino acid, energy and lipid metabolism.

iTRAQ-based quantitative proteomic analysis of salt stress in Spica Prunellae.

Spica Prunellae is an important Chinese herbal medicine. Because of its good curative effect on various diseases, this herb is consumed in large quantities in clinical applications. The metabolites of Spica Prunellae are known to change under salt stress; however, the difference in protein levels of Spica Prunellae between saline and normal conditions is unclear. In this study, we used proteomics techniques to identify differentially expressed proteins in Spica Prunellae under different saline conditions. (iTRAQ) MS/MS was used to detect statistically significant changes in protein between salt stress and normal conditions. Ultimately, we detected 1,937 proteins, 89 of which were detected in two different comparison. Based on GO, STRING and KEGG analyses, 35 significantly differentially expressed proteins were selected for further analysis. The results of functional and signal pathway analyses indicated that the cellular protein and carbohydrate metabolism of Spica Prunellae was weaker, calcium ion transport was higher, photosynthesis was higher, and protein production was faster under saline conditions than under normal conditions. This study provides useful information for studying the causes of differences in secondary metabolites in Spica Prunellae under salt stress and the protein mechanisms related to their quality.

Sustainable production of biomass and industrially important secondary metabolites in cell cultures of selfheal (Prunella vulgaris L.) elicited by silver and gold nanoparticles.

Elicited artificial in vitro cultures are gaining more interest due to their uniform biosynthesis of industrially valuable secondary metabolites. In this study, a unique methodology was applied, in which different ratios of gold (Au) and silver (Ag) nanoparticles (NPs) were supplemented to submerge cultures to investigate sustainable production of biomass and antioxidant secondary metabolites. Cell suspension cultures were exposed to Ag and AuNPs alone or different ratios of AgAuNPs (1:2; 1:3; 2:1; 3:1) in combination with NAA. The combination of AgAuNPs (3:1) with NAA enhanced fresh (9.25 g/100 ml) and dry biomass (0.64 g/100 ml) of suspended cells than control (6.67; 0.233 g/100 ml). AuNPs with NAA-augmented media enhanced biomass accumulation in lag, log and stationary phases in a period of 49 days. Furthermore, AgAu (3:1) and AgAuNPs (2:1; 1:2) with NAA enhanced protein contents, peroxidase and superoxide dismutase enzymes. However, maximum phenolics (TPC; 10.61 mg/g-DW) and flavonoids (7.62 mg/g-DW) were observed in cell cultures exposed to a combination of AgAuNPs (1:3) and NAA than control (6.27, 5.49 mg/g-DW). The combination of AgAuNPs (2:1) with NAA enhanced antioxidant activity (87.85%) in cell cultures. This study will help in illuminating the impact of NPs on cultures development and production of natural antioxidants.

Plant morphology, physiological characteristics, accumulation of secondary metabolites and antioxidant activities of Prunella vulgaris L. under UV solar exclusion.

BACKGROUND: Prunella vulgaris L. has been an important medicinal plant for the treatment of thyroid gland malfunction and mastitis in China for over 2000 years. There is an urgent need to select effective wavelengths for greenhouse cultivation of P. vulgaris as light is a very important factor in P. vulgaris growth. Here, we described the effects of natural light (control) and UV solar exclusion on the morphological and physiological traits, secondary metabolites contents and antioxidant activities of P. vulgaris. RESULTS: The results showed that UV solar exclusion resulted in remarkable alterations to morphological and biomass traits; significantly reduced the chlorophyll a, chlorophyll b and total chlorophyll contents; significantly enhanced the ratio of chlorophyll a to b; and significantly increased the carotenoid and anthocyanin contents in P. vulgaris. UV solar exclusion significantly increased the catalase (CAT) and peroxidase (POD) activities, increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and slightly decreased the glutathione (GSH) content. UV solar exclusion significantly increased the soluble sugar and H2O2 contents and increased the soluble protein content but significantly decreased the proline content and slightly decreased the MDA content. The secondary metabolite contents (total phenolics, rosmarinic acid, caffeic acid, hyperoside, ursolic acid and oleanolic acid) and in vitro antioxidative properties (DPPH· and ABTS·+scavenging activities) were significantly increased in P. vulgaris spicas under UV solar exclusion. Additionally, the total polysaccharide and total flavonoids contents were slightly increased by UV solar exclusion. The salviaflaside content was significantly reduced by UV solar exclusion. CONCLUSION: Our study demonstrated that P. vulgaris activates several antioxidant defence systems against oxidative damage caused by UV solar exclusion.

Plant morphology, physiological characteristics, accumulation of secondary metabolites and antioxidant activities of Prunella vulgaris L. under UV solar exclusion

BACKGROUND: Prunella vulgaris L. has been an important medicinal plant for the treatment of thyroid gland malfunction and mastitis in China for over 2000 years. There is an urgent need to select effective wavelengths for greenhouse cultivation of P. vulgaris as light is a very important factor in P. vulgaris growth. Here, we described the effects of natural light (control) and UV solar exclusion on the morphological and physiological traits, secondary metabolites contents and antioxidant activities of P. vulgaris. RESULTS: The results showed that UV solar exclusion resulted in remarkable alterations to morphological and biomass traits; significantly reduced the chlorophyll a, chlorophyll b and total chlorophyll contents; significantly enhanced the ratio of chlorophyll a to b; and significantly increased the carotenoid and anthocyanin contents in P. vulgaris. UV solar exclusion significantly increased the catalase (CAT) and peroxidase (POD) activities, increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and slightly decreased the glutathione (GSH) content. UV solar exclusion significantly increased the soluble sugar and H2O2 contents and increased the soluble protein content but significantly decreased the proline content and slightly decreased the MDA content. The secondary metabolite contents (total phenolics, rosmarinic acid, caffeic acid, hyperoside, ursolic acid and oleanolic acid) and in vitro antioxidative properties (DPPH· and ABTS·+scavenging activities) were significantly increased in P. vulgaris spicas under UV solar exclusion. Additionally, the total polysaccharide and total flavonoids contents were slightly increased by UV solar exclusion. The salviaflaside content was significantly reduced by UV solar exclusion. CONCLUSION: Our study demonstrated that P. vulgaris activates several antioxidant defence systems against oxidative damage caused by UV solar exclusion.

A tyrosine aminotransferase involved in rosmarinic acid biosynthesis in Prunella vulgaris L.

Rosmarinic acid (RA) and its derivants are medicinal compounds that comprise the active components of several therapeutics. We isolated and characterised a tyrosine aminotransferase of Prunella vulgaris (PvTAT). Deduced PvTAT was markedly homologous to other known/putative plant TATs. Cytoplasmic localisation of PvTAT was observed in tobacco protoplasts. Recombinantly expressed and purified PvTAT had substrates preference for L-tyrosine and phenylpyruvate, with apparent K m of 0.40 and 0.48 mM, and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate. In vivo activity was confirmed by functional restoration of the Escherichia coli tyrosine auxotrophic mutant DL39. Agrobacterium rhizogenes-mediated antisense/sense expression of PvTAT in hairy roots was used to evaluate the contribution of PvTAT to RA synthesis. PvTAT were reduced by 46-95% and RA were decreased by 36-91% with low catalytic activity in antisense transgenic hairy root lines; furthermore, PvTAT were increased 0.77-2.6-fold with increased 1.3-1.8-fold RA and strong catalytic activity in sense transgenic hairy root lines compared with wild-type counterparts. The comprehensive physiological and catalytic evidence fills in the gap in RA-producing plants which didn't provide evidence for TAT expression and catalytic activities in vitro and in vivo. That also highlights RA biosynthesis pathway in P. vulgaris and provides useful information to engineer natural products.

Elicitation of Medicinally Important Antioxidant Secondary Metabolites with Silver and Gold Nanoparticles in Callus Cultures of Prunella vulgaris L.

Prunella vulgaris L. (P. vulgaris) is an important medicinal plant with a wide range of antiviral properties. Traditionally, it is known as self-heal because of its faster effects on wound healing. It is commonly known as a natural antiseptic due to the presence of various polyphenols. There is lack of research efforts on its propagation and production of bioactive compounds under field and in vitro conditions. In this study, the effects of different ratios (1:2, 1:3, 2:1, and 3:1) of silver (Ag) and gold (Au) nanoparticles (NPs) alone or in combination with naphthalene acetic acid (NAA) were investigated for callus culture development and production of secondary metabolites. The Ag (30 µg l-1), AgAu (1:2), and AgAu (2:1) NPs in combination with NAA (2.0 mg l-1) enhanced callus proliferation (100 %) as compared to the control (95 %). Among the different NPs tested, AuNPs with or without NAA produced higher biomass in log phases (35-42 days) of growth kinetics. Furthermore, AgAu (1:3) and AuNPs alone enhanced total protein content (855 µg-BSAE/mg-fresh weight (FW)), superoxide dismutase (0.54 nM/min/mg-FW), and peroxidase (0.39 nM/min/mg-FW) enzymes in callus cultures. The AgAuNPs (1:3) in combination with NAA induced maximum accumulation of phenolics (TPC 9.57 mg/g-dry weight (DW)) and flavonoid (6.71 mg/g-DW) content. Moreover, AgAuNPs (3:1) without NAA enhanced antioxidant activity (87.85 %). This study provides the first evidence of NP effect on callus culture development and production of natural antioxidants in P. vulgaris.

Correlation of different spectral lights with biomass accumulation and production of antioxidant secondary metabolites in callus cultures of medicinally important Prunella vulgaris L.

Light is one of the key elicitors that directly fluctuates plant developmental processes and biosynthesis of secondary metabolites. In this study, the effects of various spectral lights on biomass accumulation and production of antioxidant secondary metabolites in callus cultures of Prunella vulgaris were investigated. Among different spectral lights, green light induced the maximum callogenic response (95%). Enhanced fresh biomass accumulation was observed in log phases on day-35, when callus cultures were exposed to yellow and violet lights. Yellow light induced maximum biomass accumulation (3.67g/100ml) from leaf explants as compared to control (1.27g/100ml). In contrast, violet lights enhanced biomass accumulation (3.49g/100ml) from petiole explant. Maximum total phenolics content (TPC; 23.9mg/g-DW) and total flavonoids content (TFC; 1.65mg/g-DW) were observed when cultures were grown under blue lights. In contrast, green and yellow lights enhanced total phenolics production (TPP; 112.52g/100ml) and total flavonoids production (TFP; 9.64g/100ml) as compared to control. The calli grown under green, red and blue lights enhanced DPPH-free radical scavenging activity (DFRSA; 91.3%, 93.1% and 93%) than control (56.44%) respectively. The DFRSA was correlated either with TPC and TFC or TPP and TFP. Furthermore, yellow lights enhanced superoxide dismutase (SOD), peroxidase (POD) and protease activities, however, the content of total protein (CTP) was higher in control cultures (186µg BSAE/mg FW) as compared to spectral lights. These results suggest that the exposure of callus cultures to various spectral lights have shown a key role in biomass accumulation and production of antioxidant secondary metabolites.

Optimization of adventitious root culture for production of biomass and secondary metabolites in Prunella vulgaris L.

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l(-1)) and naphthalene acetic acid (NAA; 1.5 mg l(-1)). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5-2.0 mg l(-1)). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l(-1) NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l(-1) NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l(-1) NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.

Oleanolic acid regulates NF-κB signaling by suppressing MafK expression in RAW 264.7 cells.

Oxidative stress and inflammation are common to many pathological conditions. Defense mechanisms protect cells from oxidative stress, but can become over-activated following injury and inflammation. NF-κB and Nrf2 transcription factors regulate proinflammatory and antioxidant gene expression, respectively. Studies have shown that many natural dietary compounds regulate NF-κB and Nrf2, preventing inflammation and oxidative stress. Here, we report major compounds of Prunella vulgaris var. lilacina such as rosmarinic acid, oleanolic acid, ursolic acid and caffeic acid as a potential therapeutic for oxidative stress and inflammation. The major compounds exhibited high anti-inflammatory activity, inhibiting NO, PGE2 production, NF-κB expression and activating Nrf2 expression. In addition, we examined the effect of major compounds on MafK expression. Among the compounds, oleanolic acid significantly decreased MafK expression and MafK-mediated p65 acetylation. These findings suggest that oleanolic acid as NF-κB inhibitors can potentially be used in therapeutic applications for the treatment of oxidative stressnduced diseases.

Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

Changes in bioactive components related to the harvest time from the spicas of Prunella vulgaris.

CONTEXT: Prunella vulgaris L. (Labiatae) is a perennial plant common in China and Europe and is rich in rosmarinic acid (RA), ursolic acid (UA), and oleanolic acid (OA). The dried spica of P. vulgaris has been used as traditional medicine in China for over a hundred years. To our best knowledge, no study has been conducted to determine the influence of harvesting time on concentrations of bioactive compounds of P. vulgaris. OBJECTIVE: In the current study, changes in the bioactive compounds present in spicas were investigated at five harvest times over 2 months. MATERIALS AND METHODS: Plant material were collected at five fixed dates: 5th May, 20th May, 7th June, 15th June, and 25th June and assayed for chemical contents by high-performance liquid chromatography (HPLC). RESULTS: Among the different harvest times, the highest levels of RA (56.81 mg·g⁻¹), UA (2.77 mg·g⁻¹), and OA (0.91 mg·g⁻¹) were found on 5th May, whereas the lowest levels of RA (1.66 mg·g⁻¹), UA (2.27 mg·g⁻¹), and OA (0.43 mg·g⁻¹) were observed on 25th June. DISCUSSION AND CONCLUSION: As each medicinal product has its own content requirement for different bioactive components, the optimum harvest time might be determined according to the accumulation dynamics of target compound in dried spicas of P. vulgaris. These results may be useful for determining the optimal harvest time when bioactive components are at the maximum level, which is in early May.