Comparative transcriptomics of two Salvia subg. Perovskia species contribute towards molecular background of abietane-type diterpenoid biosynthesis.

Tanshinones, are a group of diterpenoid red pigments present in Danshen - an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography-Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy.

Electrostimulation improves plant growth and modulates the flavonoid profile in aeroponic culture of Scutellaria baicalensis Georgi.

Scutellaria baicalensis Georgi is a valuable medicinal plant of the Lamiaceae family. The roots, Scutellariae baicalensis radix, are valued in the traditional medicine of East Asia and are also listed in several pharmacopeias, such as the Chinese and European versions. The roots contain a high amount of flavones, such as baicalein, wogonin and their glucuronides, baicalin and wogonoside, respectively, with rare structures of unsubstituted B-ring. These major constituents are responsible for its pharmacological activity, mainly anti-inflammatory, antiviral, and antitumor, as well as BDZ-receptor modulating. There is a fast-growing demand for both the crude drug and the individual flavonoids obtained from it. However, the variability of content and composition of flavonoids in the roots is significant and affects pharmaceutical use, and little is known about the influence of various factors on root quality. In our experiments, we use aeroponics to determine the effect of electroporation as an abiotic stressor on plant growth, development, and root mass, as well as on its metabolic profile. Results: Electroporation significantly impacted plant growth and the content of flavonoids, especially baicalein and wogonin, depending on the treatment parameters. Concentrations of aglycones were increased in at least half of the treatment conditions. The greatest amounts (a 2.5-fold increase compared to controls) were recorded after applying an electrical field characterized by the following parameters: E = 3 kV/cm, t = 100 µs, and N = 10. In conclusion, electrostimulation is an innovative and efficient way to increase plant growth and yield in an aeroponic system, as well as modulate the profile and content of bioactive flavones in the roots. However, the fine-tuning of these parameters, such as the electrical field strength (E), length (t), and number (N) of impulses delivered, is of great importance. It was also shown that cultivation of the experimental plants in aeroponics had a positive impact on their survival and development while being a sustainable and efficient horticultural practice.

A first approach for the micropropagation of the medicinal halophyte Polygonum maritimum L. and phenolic profile of acclimatized plants.

Polygonum maritimum L. (sea knotgrass) belongs to a genus commonly used in folk medicine to treat inflammation-related disorders. In vitro pharmacological studies have confirmed these properties that were ascribed to bioactive flavonoids, such as myricetin and quercetin glycosides. Therefore, this study aimed at establishing a micropropagation procedure for sea knotgrass for obtaining standardized materials for its potential commercial cultivation. For that, a complete plant regeneration protocol was developed by improving shoot multiplication from nodal explants, rooting and acclimatization procedures, followed by the assessment of the phenolic profile of the in vitro-produced plants. The combination of 3 mg/L 6-benzylaminopurine (BA) + 0.1 mg/L indole-3-acetic acid (IAA) induced the maximum shoot formation (10.3), which was significantly increased from the first to the second cycle (18.3). The best rooting capacity was observed on shoots derived from the control medium (100%), followed by 2 mg/L kinetin (KIN) (97%) and 3 mg/L BA + 0.1 mg/L IAA (90%); however, the shoot number at the end of the rooting phase was higher on shoots derived from 3 mg/L BA + 0.1 mg/L IAA (6.16). The plant growth regulators used in the multiplication phase influenced survival in the acclimatization process, and plants derived from the control medium had the highest survival percentage (63.1%). Acetone extracts made from aerial organs of micropropagated sea knotgrass showed a predominance of the flavonoid myricetin-3-O-rhamnoside (8.135 mg/g). Overall, the halophyte sea knotgrass was successfully micropropagated showing its potential as a medicinal crop for the extraction of bioactive molecules.

The effect of ensiled paulownia leaves in a high-forage diet on ruminal fermentation, methane production, fatty acid composition, and milk production performance of dairy cows.

BACKGROUND: The use of industrial by-products rich in bioactive compounds as animal feeds can reduce greenhouse gas production. Paulownia leaves silage (PLS) was supplemented to dairy cows' diet and evaluated in vitro (Exp. 1; Rusitec) and in vivo (Exp. 2, cannulated lactating dairy cows and Exp. 3, non-cannulated lactating dairy cows). The study investigated the PLS effect on ruminal fermentation, microbial populations, methane production and concentration, dry matter intake (DMI), and fatty acid (FA) proportions in ruminal fluid and milk. RESULTS: Several variables of the ruminal fluid were changed in response to the inclusion of PLS. In Exp. 1, the pH increased linearly and quadratically, whereas ammonia and total volatile fatty acid (VFA) concentrations increased linearly and cubically. A linear, quadratic, and cubical decrease in methane concentration was observed with increasing dose of the PLS. Exp. 2 revealed an increase in ruminal pH and ammonia concentrations, but no changes in total VFA concentration. Inclusion of PLS increased ruminal propionate (at 3 h and 6 h after feeding), isovalerate, and valerate concentrations. Addition of PLS also affected several populations of the analyzed microorganisms. The abundances of protozoa and bacteria were increased, whereas the abundance of archaea were decreased by PLS. Methane production decreased by 11% and 14% in PLS-fed cows compared to the control in Exp. 2 and 3, respectively. Exp. 3 revealed a reduction in the milk protein and lactose yield in the PLS-fed cows, but no effect on DMI and energy corrected milk yield. Also, the PLS diet affected the ruminal biohydrogenation process with an increased proportions of C18:3 cis-9 cis-12 cis-15, conjugated linoleic acid, C18:1 trans-11 FA, polyunsaturated fatty acids (PUFA), and reduced n6/n3 ratio and saturated fatty acids (SFA) proportion in milk. The relative transcript abundances of the 5 of 6 analyzed genes regulating FA metabolism increased. CONCLUSIONS: The dietary PLS replacing the alfalfa silage at 60 g/kg diet can reduce the methane emission and improve milk quality with greater proportions of PUFA, including conjugated linoleic acid, and C18:1 trans-11 along with reduction of SFA. Graphical abstract of the experimental roadmap.

Changes in the Antioxidant and Mineral Status of Rabbits After Administration of Dietary Zinc and/or Thyme Extract.

This study was aimed at determining the impact of organic zinc (Zn) and thyme extract (TE) administration, given alone or together for 6 weeks, on the antioxidant and mineral status (Zn, Cu, Fe, and Mn) in the plasma and tissues of growing rabbits. A total of 96 rabbits of age 35 days were randomly assigned to one of four treatment groups: a control group (C), a Zn group supplemented with dietary zinc (50 mg/kg), a TE group receiving thyme extract applied in drinking water (1 ml/L), and a Zn + TE group treated with both additives. Lipid peroxidation in the plasma was influenced by Zn intake and in the kidney was affected by both the Zn and TE treatment (P < 0.05). Zn supplementation led to a significant increase in glutathione peroxidase activity (P = 0.017), total antioxidant capacity (P = 0.009) and total thiol groups level (P = 0.047) in the kidney, with the highest values occurring in rabbits receiving the combination Zn + TE. Administration of TE influenced Zn content in the kidney (P < 0.001), while zinc intake decreased Cu concentration in muscle (P = 0.021). In conclusion, the simultaneous administration of organic Zn and TE positively affected the antioxidant response of kidneys and can be used for improving the antioxidant status of growing rabbits.

Greener Is Better: First Approach for the Use of Natural Deep Eutectic Solvents (NADES) to Extract Antioxidants from the Medicinal Halophyte Polygonum maritimum L.

In this study, natural deep eutectic solvents (NADES) formed by choline chloride (ChCl), sucrose, fructose, glucose, and xylose, were used to extract antioxidants from the halophyte Polygonum maritimum L. (sea knotgrass) and compared with conventional solvents (ethanol and acetone). NADES and conventional extracts were made by an ultrasound-assisted procedure and evaluated for in vitro antioxidant properties by the radical scavenging activity (RSA) on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC), and copper chelating activity (CCA). Samples were profiled by liquid chromatography (LC)-electrospray ionization (ESI)-QTOF-MS analysis. ChCl:fructose was more efficient in the DPPH assay, than the acetone extract. ChCl:sucrose and ChCl:fructose extracts had the highest ORAC when compared with the acetone extract. NADES extracts had higher CCA, than the acetone extract. The phenolic composition of the NADES extracts was less complex than the conventional extracts, but the proportions of major antioxidants, such as flavonols and flavan-3-ols, were similar in all the solvents. Myricitrin was the major flavonoid in all of the samples, while gallic acid was the main phenolic acid in the conventional extracts and present in a greater amount in ChCl:fructose. Results suggest that NADES containing ChCl and sucrose/fructose can replace conventional solvents, especially acetone, in the extraction of antioxidants from sea knotgrass.

Seasonal Variations of Rosmarinic Acid and Its Glucoside and Expression of Genes Related to Their Biosynthesis in Two Medicinal and Aromatic Species of Salvia subg. Perovskia.

Salvia abrotanoides Kar. and Salvia yangii B.T. Drew are medicinal and aromatic plants belonging to the subgenus Perovskia and used as herbal medicines in Asia. Derivatives of caffeic acid, mainly rosmarinic acid (RA), are the major phenolic compounds identified in these plants. Understanding the factors and molecular mechanisms regulating the accumulation of pharmacologically and ecologically relevant phenolic metabolites is essential for future biotechnological and medical applications. Up to date, no studies of phenylpropanoid biosynthetic pathway at the transcriptional level has been performed in the Perovskia subgenus. Using a combined qRT-PCR transcriptional activity analysis with LC-MS based metabolic profiling of roots and leaves at the beginning, in the middle and at the end of vegetation season, we have identified the following gene candidates with properties correlating to phenolic acid biosynthesis in S. abrotanoides and S. yangii: PAL, C4H, 4CL, TAT, HPPR, RAS1, RAS2 and Cyp98A14. A comparison of phenolic acid profiles with gene transcript levels revealed the transcriptional regulation of RA biosynthesis in the roots but not the leaves of the studied species. Additionally, RAS1 and Cyp98A14 were identified as rate-limiting steps regulating phenylpropanoid biosynthesis on a transcription level. In the future, this will facilitate the gene-based metabolic enhancement of phenolic compounds production in these promising medicinal herbs.

Corrigendum: Impact of Zinc and/or Herbal Mixture on Ruminal Fermentation, Microbiota, and Histopathology in Lambs.

[This corrects the article DOI: 10.3389/fvets.2021.630971.].

Impact of Zinc and/or Herbal Mixture on Ruminal Fermentation, Microbiota, and Histopathology in Lambs.

We investigated the effect of diets containing organic zinc and a mixture of medicinal herbs on ruminal microbial fermentation and histopathology in lambs. Twenty-eight lambs were divided into four groups: unsupplemented animals (Control), animals supplemented with organic zinc (Zn, 70 mg Zn/kg diet), animals supplemented with a mixture of dry medicinal herbs (Herbs, 100 g dry matter (DM)/d) and animals supplemented with both zinc and herbs (Zn+Herbs). Each lamb was fed a basal diet composed of meadow hay (700 g DM/d) and barley (300 g DM/d). The herbs Fumaria officinalis L. (FO), Malva sylvestris L. (MS), Artemisia absinthium L. (AA) and Matricaria chamomilla L. (MC) were mixed in equal proportions. The lambs were slaughtered after 70 d. The ruminal contents were used to determine the parameters of fermentation in vitro and in vivo and to quantify the microbes by molecular and microscopic methods. Samples of fresh ruminal tissue were used for histopathological evaluation. Quantitative analyses of the bioactive compounds in FO, MS, AA, and MC identified 3.961, 0.654, 6.482, and 12.084 g/kg DM phenolic acids and 12.211, 6.479, 0.349, and 2.442 g/kg DM flavonoids, respectively. The alkaloid content in FO was 6.015 g/kg DM. The diets affected the levels of total gas, methane and n-butyrate in vitro (P < 0.046, < 0.001, and < 0.001, respectively). Relative quantification by real-time PCR indicated a lower total ruminal bacterial population in the lambs in the Zn and Zn+Herbs groups than the Control group (P < 0.05). The relative abundances of Ruminococcus albus, R. flavefaciens, Streptococcus bovis, and Butyrivibrio proteoclasticus shifted in the Zn group. Morphological observation found a focally mixed infiltration of inflammatory cells in the lamina propria of the rumen in the Zn+Herbs group. The effect of the organic zinc and the herbal mixture on the parameters of ruminal fermentation in vitro was not confirmed in vivo, perhaps because the ruminal microbiota of the lambs adapted to the zinc-supplemented diets. Long-term supplementation of a diet combining zinc and medicinal herbs, however, may negatively affect the health of the ruminal epithelium of lambs.

Metabolomics and DNA-Based Authentication of Two Traditional Asian Medicinal and Aromatic Species of Salvia subg. Perovskia.

Subgenus Perovskia of the extended genus of Salvia comprises several Central Asian medicinal and aromatic species, of which S. yangii and S. abrotanoides are the most widespread. These plants are cultivated in Europe as robust ornamentals, and several cultivars are available. However, their medicinal potential remains underutilized because of limited information about their phytochemical and genetic diversity. Thus, we combined an ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) based metabolomics with DNA barcoding approach based on trnH-psbA and ITS2 barcodes to clarify the relationships between these two taxa. Metabolomic analysis demonstrated that aerial parts are more similar than roots and none of the major compounds stand out as distinct. Sugiol in S. yangii leaves and carnosic acid quinone in S. abrotanoides were mostly responsible for their chemical differentiation, whereas in roots the distinction was supported by the presence of five norditerpenoids in S. yangii and two flavonoids and one norditerpenoid in S. abrotanoides. To verify the metabolomics-based differentiation, we performed DNA authentication that revealed S. yangii and S. abrotanoides to be very closely related but separate species. We demonstrated that DNA barcoding coupled with parallel LC-MS profiling constitutes a powerful tool in identification of taxonomically close Salvia species.

Norditerpenoids with Selective Anti-Cholinesterase Activity from the Roots of Perovskia atriplicifolia Benth.

Inhibition of cholinesterases remains one of a few available treatment strategies for neurodegenerative dementias such as Alzheimer's disease and related conditions. The current study was inspired by previous data on anticholinesterase properties of diterpenoids from Perovskia atriplicifolia and other Lamiaceae species. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition by the three new natural compounds-(1R,15R)-1-acetoxycryptotanshinone (1), (1R)-1-acetoxytanshinone IIA (2), and (15R)-1-oxoaegyptinone A (3)-as well as, new for this genus, isograndifoliol (4) were assessed. Three of these compounds exhibited profound inhibition of butyrylcholinesterase (BChE) and much weaker inhibition of acetylcholinesterase (AChE). All compounds (1-4) selectively inhibited BChE (IC50 = 2.4, 7.9, 50.8, and 0.9 µM, respectively), whereas only compounds 3 and 4 moderately inhibited AChE (IC50 329.8 µM and 342.9 µM). Molecular docking and in silico toxicology prediction studies were also performed on the active compounds. Natural oxygenated norditerpenoids from the traditional Central Asian medicinal plant P. atriplicifolia are selective BChE inhibitors. Their high potential makes them useful candidate molecules for further investigation as lead compounds in the development of a natural drug against dementia caused by neurodegenerative diseases.

Anemarrhenae asphodeloides rhizoma Extract Enriched in Mangiferin Protects PC12 Cells against a Neurotoxic Agent-3-Nitropropionic Acid.

The rhizome of Anemarrhena asphodeloides Bunge, used in Traditional Chinese Medicine as a brain function-improving herb, is a promising source of neuroprotective substances. The aim of this study was to evaluate the protective action of xanthones from A. asphodeloides rhizomes on the PC12 cell line exposed to the neurotoxic agent-3-nitropropionic acid (3-NP). The xanthone-enriched fraction of the ethanolic extract of A. asphodeloides (abbreviated from now on as XF, for the Xanthone Fraction), rich in polyphenolic xanthone glycosides, in concentrations from 5 to 100 µg/mL, and 3-NP in concentrations from 2.5 to 15 mM, were examined. After 8, 16, 24, 48, and 72 h of exposure of cells to various combinations of 3-NP and XF, the MTT viability assay was performed and morphological changes were estimated by confocal fluorescence microscopy. The obtained results showed a significant increase in the number of cells surviving after treatment with XF with exposure to neurotoxic 3-NP and decreased morphological changes in PC12 cells in a dose and time dependent manner. The most effective protective action was observed when PC12 cells were pre-incubated with the XF. This effect may contribute to the traditional indications of this herb for neurological and cognitive complaints. However, a significant cytotoxicity observed at higher XF concentrations (over 10 µg/mL) and longer incubation time (48 h) requires caution in future research and thorough investigation into potential adverse effects.

Effect of dry medicinal plants (wormwood, chamomile, fumitory and mallow) on in vitro ruminal antioxidant capacity and fermentation patterns of sheep.

The objective of this study was to determine the effect of dry medicinal plants (wormwood, chamomile, fumitory and mallow) and dietary substrates containing a mix of the plants on the end products of in vitro ruminal and intestinal fermentation, rumen protozoan population and ruminal antioxidant capacity of sheep. The experiment consisted of fermentations with the four plants used individually as the sole substrate and fermentation of a mix of medicinal plants (Plants): meadow hay:barley grain (MH:B), 700/300 w/w and Plants:MH:B, 100/600/300 w/w/w. The experiment was conducted using the in vitro gas production technique (IVGPT) with 35 ml of buffered inocula and approximately 250 mg (DM basis) of substrate incubated for 24 hr at 39°C in anaerobic conditions. Quantitative analyses of the bioactive compounds by ultra-high-resolution mass spectrometry in Plants identified three main groups: flavonoids (22 mg/g DM), phenolic acids (15 mg/g DM) and alkaloids (3 mg/g DM). The total antioxidant capacity (TAC) of the plant extracts and rumen fluid was analysed using a ferric reducing antioxidant power assay. The values of total and individual short-chain fatty acids, acetate:propionate ratio, pH and total gas production were significantly affected by the single plant substrates and inocula (p < .001). Apart from these parameters, the values of ammonia N, methane production and total gas produced were decreased in Plants:MH:B in comparison with MH:B (p < .001). A positive correlation was recorded between total polyphenols content and TAC of plant extracts (R2  = .778, p < .001). The counts of the total ciliate protozoan population in rumen fluid after 24 hr of fermentation were not significantly different (p > .05). Results suggest that the dietary substrate containing the medicinal plant mix possessed strong ruminal antioxidant capacity, had the potential to reduce methane emission and ammonia concentration and caused desirable changes in the gastrointestinal ecosystem.

Natural chemotherapeutic alternatives for controlling of haemonchosis in sheep.

BACKGROUND: Parallel in vitro and in vivo experiments were designed to evaluate promising chemotherapeutic alternatives for controlling haemonchosis in ruminants. In vitro anthelmintic activities (egg hatch test - EHT; larval development test - LDT) of aqueous and methanolic herbal extracts Mix1 and Mix2 were investigated. The in vivo effects of dietary supplementation with Mix1 and Mix2 on the parasitological status, inflammatory response, antioxidant parameters and microbial community of the lambs infected experimentally with Haemonchus contortus were investigated. Lambs were divided into four groups for the in vivo study: uninfected control lambs (C), infected lambs (I), infected lambs supplemented with Mix1 (I + Mix1) and infected lambs supplemented with Mix2 (I + Mix2). The experimental period was 70 days. RESULTS: The number of eggs per gram (EPG) of feces was quantified 22, 30, 37, 44, 51, 58, 65 and 70 days post-infection, and mean abomasal worm counts were assessed 70 days post-infection. Quantitative analyses identified 57.3 and 22.2 mg/g phenolic acids, 41.5 and 29.5 mg/g flavonoids and 1.4 and 1.33 mg/g protoberberine-type alkaloids in Mix1 and Mix2, respectively. The methanolic extracts of the herbal mixtures in both in vitro tests had higher anthelmintic effects (P < 0.01) than the aqueous extracts, but the effects did not differ significantly between Mix1 and Mix2 (P > 0.05). I + Mix1 and I + Mix2 lowered mean EPGs between 44 and 70 d by 58.1 and 51.6%, respectively. The level of IgG antibodies against H. contortus increased significantly after infection in each infected group. CONCLUSION: These results represent the first monitoring of the in vitro anthelmintic effects of herbal mixtures on H. contortus. The in vivo experiment indicated that the anthelmintic effect was not sufficient for the elimination of parasites, but this herbal treatment may affect the host over a longer term, reducing the parasitic infection in the host.

Phytochemical Diversity in Rhizomes of Three Reynoutria Species and their Antioxidant Activity Correlations Elucidated by LC-ESI-MS/MS Analysis.

The rhizome of Reynoutria japonica is a well-known traditional herb (Hu zhang) used in East Asia to treat various inflammatory diseases, infections, skin diseases, scald, and hyperlipidemia. It is also one of the richest natural sources of resveratrol. Although, it has been recently included in the European Pharmacopoeia, in Europe it is still an untapped resource. Some of the therapeutic effects are likely to be influenced by its antioxidant properties and this in turn is frequently associated with a high stilbene content. However, compounds other than stilbenes may add to the total antioxidant capacity. Hence, the aim of this research was to examine rhizomes of R. japonica and the less studied but morphologically similar species, R. sachalinensis and R. x bohemica for their phytochemical composition and antioxidant activity and to clarify the relationship between the antioxidant activity and the components by statistical methods. HPLC/UV/ESI-MS studies of three Reynoutria species revealed 171 compounds, comprising stilbenes, carbohydrates, procyanidins, flavan-3-ols, anthraquinones, phenylpropanoids, lignin oligomers, hydroxycinnamic acids, naphthalenes and their derivatives. Our studies confirmed the presence of procyanidins with high degree of polymerization, up to decamers, in the rhizomes of R. japonica and provides new data on the presence of these compounds in other Reynoutria species. A procyanidin trimer digallate was described for the first time in, the studied plants. Moreover, we tentatively identified dianthrone glycosides new for these species and previously unrecorded phenylpropanoid disaccharide esters and hydroxycinnamic acid derivatives. Furthermore, compounds tentatively annotated as lignin oligomers were observed for the first time in the studied species. The rhizomes of all Reynoutria species exhibited strong antioxidant activity. Statistical analysis demonstrated that proanthocyanidins should be considered as important contributors to the total antioxidant capacity.

Coleus amboinicus (Lour.) leaves as a modulator of ruminal methanogenesis and biohydrogenation in vitro.

Active plant metabolites (APM) are recognized as modifiers of ruminal microbial fermentation including methanogenesis and biohydrogenation of fatty acids (FA). Coleus amboinicus Lour. leaves (CAL) are rich in several APM, which could serve as ruminal fermentation modulators. A phytochemical analysis showed that CAL contain phenolic acids (10.4 mg·g-1 dry matter [DM]; high in rosmarinic acid), flavonoids (2.6 mg·g-1 DM), diterpenes (2 mg·g-1 DM), and linolenic acid (35.4 g (100 g)-1 FA). This study aimed to investigate the effect of CAL on ruminal methanogenesis and biohydrogenation as well as basic fermentation characteristics and microbial populations. The in vitro experiment was carried out using Hohenheim gas test system with 40 mL of buffered ruminal fluid incubated for 24 h at 39 °C in anaerobic conditions. Approximately 400 mg (DM basis) of total mixed ration (TMR) was used as a control substrate and the CAL substrate was used at doses of 10, 20, 40, and 80 mg DM replacing equal amounts of TMR. Addition of CAL decreased methane production up to 30% linearly as the amount of CAL increased (P < 0.05). In vitro dry matter digestibility and ammonia tended to increase with increasing doses of CAL. Concentration of total volatile fatty acids was not affected by the CAL although there appeared to be a minor positive linear trend; however, acetate, butyrate, and isobutyrate proportion increased quadratically (P < 0.001). CAL tended to linearly increase α-linolenic acid and conjugated linoleic acid as well as increased stearic acid concentration in buffered ruminal fluid. CAL particularly increased total protozoa and bacterial populations during fermentation, but inhibited methanogens. It is concluded that the CAL may be promising to be used as a feed additive to decrease methanogenesis as well as biohydrogenation of FA in the rumen.

Comparison of Polyphenol Profile and Antimutagenic and Antioxidant Activities in Two Species Used as Source of Solidaginis herba - Goldenrod.

European Pharmacopoeia accepts two equivalent species Solidago canadensis L. and S. gigantea Aiton as goldenrod (Solidaginis herba). We compared phytochemical profile of both species from invasive populations in Poland. Further, we compared in vitro antimutagenic and antioxidant activities of solvent extracts from aerial (AP) and underground parts (UP). In S. gigantea, flavonoid profile was dominated by quercetin glycosides, with quercitrin as the major compound. In S. canadensis, quercetin and kaempferol rutinosides were two major constituents. Caffeoylquinic acids (CQAs) were less diverse with 5-CQA as a main compound. In UP, over 20 putative diterpenoids were detected, mostly unidentified. Several CQAs were present in higher amounts than in AP. Antioxidant and antimutagenic activities were different between species and organs, with the strongest inhibition of lipid peroxidation by Et2 O and AcOEt fractions from AP of both species (IC50 13.33 - 16.89 µg/mL) and BuOH fraction from S. gigantea UP (IC50  = 13.32 µg/mL). Chemical mutagenesis was completely inhibited by non-polar fractions, but oxidative mutagenesis was inhibited up to 35% only by S. canadensis. No clear relationship was found between chemical profiles and antimutagenic activity. In conclusion, both species have diverse activity and their phytochemical profiles should be considered in quality evaluation. UP of these weeds can also provide potential chemopreventive substances for further studies.

Effects of herbal nutraceuticals and/or zinc against Haemonchus contortus in lambs experimentally infected.

BACKGROUND: The gastrointestinal parasitic nematode Haemonchus contortus is a pathogenic organism resistant to several anthelmintics. This study assessed the efficacy of a medicinal herbal mixture (Herbmix) and organic zinc, as an essential trace element for the proper functioning of both unspecific and specific immune defensive mechanisms, against experimental infections with H. contortus in lambs. All lambs were infected orally with approximately 5000 third-stage larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Twenty-four female lambs 3-4 months of age were divided into four groups: unsupplemented animals (control), animals supplemented with Herbmix (Hmix), animals supplemented with organic zinc (Zn) and animals supplemented with Herbmix and organic zinc (Hmix+Zn). Eggs per gram (EPG) of faeces were quantified 20, 28, 35, 42, 49, 56, 62 and 70 d post-infection and mean abomasal worm counts were assessed 70 d post-infection. Samples of blood were collected from each animal 7, 35, 49 and 70 d post-infection. RESULTS: Quantitative analyses of the bioactive compounds in Herbmix identified three main groups: flavonoids (9964.7 µg/g), diterpenes (4886.1 µg/g) and phenolic acids (3549.2 µg/g). Egg counts in the lambs treated with Hmix, Zn and Hmix+Zn decreased after 49 d. The EPGs in the Zn and Hmix+Zn groups were significantly lower on day 56 (P < 0.05 and P < 0.01, respectively), and the EPGs and mean worm counts were significantly lower on day 70 in all supplemented groups (P < 0.05 and P < 0.01). Hemograms of complete red blood cells of each animal identified clinical signs of haemonchosis after day 35. Serum calprotectin concentrations and IgA levels were significantly affected by treatment. The treatment influenced serum malondialdehyde concentrations (P < 0.05) and sulfhydryl groups (P < 0.01) of antioxidant status. The mineral status was unaltered in all lambs. CONCLUSION: A direct anthelmintic impact on the viability of nematodes was not fully demonstrated, but the treatments with herbal nutraceuticals and zinc likely indirectly contributed to the increase in the resistance of the lambs to nematode infection.

Inhibition of Glycation-induced Cytotoxicity, Protein Glycation, and Activity of Proteolytic Enzymes by Extract from Perovskia atriplicifolia Roots.

BACKGROUND: Protein glycation and glycotoxicity belong to the main oxidative-stress related complications in diabetes. Perovskia species are used in Asian folk medicine as antidiabetic herbs. OBJECTIVE: The aim of this study was to verify the ability of the methanolic extract from Perovskia atriplicifolia Benth. roots to diminish glycation of albumin and to prevent cell damage in vitro. Furthermore, we tested the extract for in vitro antioxidant activity and inhibition of elastase and collagenase. MATERIAL AND METHODS: The aqueous methanol extract was analyzed by UHPLC-MS for the content of polyphenols and terpenoids. The prevention of glycated albumin-induced cell damage was tested in four mammalian cell lines (peripheral blood mononuclear cells, human embryonic kidney cells - HEK293, normal human fibroblasts, and Chinese hamster ovary cells) with the 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl) tetrazolium assay. RESULTS: Glycated albumin is significantly more toxic than native human serum albumin (LC50 from 35.00 to 48.34 µg/mL vs. 5.47-9.10 µg/mL, respectively). The extract, rich in rosmarinic acid (344.27 mg/g dry mass), mitigated the glycated albumin toxicity, and increased glycated albumin-treated cell survival by more than 50%. The inhibition of advanced glycation endproduct formation was confirmed by monitoring conformational changes. The free radical scavenging activity was higher than Trolox and metal reducing power was one-third to half that of ascorbic acid. The activity of elastase and collagenase was inhibited by 54.75% ± 6.87% and 60.03% ± 7.22%, respectively. CONCLUSIONS: The results confirm antiglycative and antiglycotoxic potential of Perovskia root and its traditional antidiabetic use. The high activity can be attributed to rosmarinic acid abundance. SUMMARY: Perovskia is a small genus of aromatic shrubby plants growing in arid regions of Central and South Asia. Different parts are used in folk medicine as antiparasitic, anti-infectious and antidiabetic remedy. Here, we have studied the extract from roots for inhibition of: glycation-induced cytotoxicity, human serum albumin glycation, inflammation-related enzymes, as well as for antioxidant activity. Result: the extract from P. atriplicifolia roots inhibited protein glycation and AGE-induced toxicity in cell cultures. The mechanism is likely to rely on the antioxidant activity of high content of rosmarinic acid. Abbreviations used: AGE: advanced glycation end-products; DPPH: 2,2-diphenyl-1-picrylhydrazyl; HSA: human serum albumin.

Selective in vitro and in silico butyrylcholinesterase inhibitory activity of diterpenes and rosmarinic acid isolated from Perovskia atriplicifolia Benth. and Salvia glutinosa L.

Cholinesterase inhibition is one of the most treatment strategies against Alzheimer's disease (AD) where metal accumulation is also strongly associated with pathology of the disease. In the current study, we assessed inhibitory effect against acetyl- (AChE) and butyrylcholinesterase (BChE) and metal-chelating capacity of twelve diterpenes: arucadiol, miltirone, tanshinone IIa, 1-oxomiltirone, cryptotanshinone, 1,2-didehydromiltirone, 1,2-didehydrotanshinone IIa, 1ß-hydroxycryptotanshinone, 15,16-dihydrotanshinone, tanshinone I, isotanshinone II, 1(S)-hydroxytanshinone IIa, and rosmarinic acid, isolated from Perovskia atriplicifolia and Salvia glutinosa. The compounds were tested at 10 µg/mL using ELISA microtiter assays against AChE and BChE. QSAR and molecular docking studies have been also performed on the active compounds. All of the compounds showed higher [e.g., IC50 = 1.12 ± 0.07 µg/mL for 1,2-didehydromiltirone, IC50 = 1.15 ± 0.07 µg/mL for cryptotanshinone, IC50 = 1.20 ± 0.03 µg/mL for arucadiol, etc.)] or closer [1,2-didehydrotanshinone IIa (IC50 = 5.98 ± 0.49 µg/mL) and 1(S)-hydroxytanshinone IIa (IC50 = 5.71 ± 0.27 µg/mL)] inhibition against BChE as compared to that of galanthamine (IC50 = 12.56 ± 0.37 µg/mL), whereas only 15,16-dihydrotanshinone moderately inhibited AChE (65.17 ± 1.39%). 1,2-Didehydrotanshinone IIa (48.94 ± 0.26%) and 1(S)-hydroxytanshinone IIa (47.18 ± 5.10%) possessed the highest metal-chelation capacity. The present study affords an evidence for the fact that selective BChE inhibitors should be further investigated as promising candidate molecules for AD therapy.