A newly developed UPLC-MS/MS method for simultaneous quantitative analysis of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-acetylharpagide derived from Ajuga plants in mice blood and the in vivo pharmacokinetics.

OBJECTIVE: To develop a sensitive and fast detection method via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to assess the concentration of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-Acetylharpagide primarily derived from Ajuga plants in mice blood and their pharmacokinetics. METHODS: Single protein precipitation with high-proportioned acetonitrile is chosen for sample clean-up. The UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column with a mobile phase in gradient elution mode at the flow rate of 0.4 mL/min was used for sample separation. Acetonitrile was selected as the organic phase solution and water containing 0.1% formic acid was chosen as the aqueous solution. A tandem mass spectrometer containing an electrospray ionization (ESI) source in the positive ionization mode was used to detect four compounds via multiple reaction monitoring (MRM). RESULTS: The calibration curves (5-1000 ng/mL) of four compounds were linear with correlation coefficients > 0.997. The matrix effects, accuracy, precision, and recovery were all within permissible scope. CONCLUSIONS: In this approach, the corresponding pharmacokinetic parameters were successfully clarified in mouse for the first time, which provided a theoretical basis for the improvement of the standard of Ajuga plants and the safety of clinical medication. Furthermore, this method may provide the UPLC-MS/MS evidence for the differentiation of the main close relative varieties of genus Ajuga according to these plants contain different mixtures of the four marker compounds.

Phytochemical, acute toxicity and renal protective appraisal of Ajuga parviflora hydromethanolic leaf extract against CCl4 induced renal injury in rats.

BACKGROUND: Degenerative kidney diseases are mostly associated with oxidative stress. Natural products are considered as the antioxidants enrich food that can restrict the progress of oxidative stress induced disorders. Therefore, the present study was aimed to evaluate the renal protective effect of Ajuga parviflora leaf extract in carbon tetrachloride intoxicated rats. METHODS: The hydromethanolic extract of A. parviflora leaves was obtained by extracting twice in 60% methanol. The principal bioactive constituents were detected by LC/MS analysis. Toxicity of plant extract was assessed using brine shrimp lethal toxicity test and acute toxicity model on healthy Sprague-Dawley male rats. Nephroprotective effects of plant extract were also evaluated on rats by inducing CCl4 renal toxicity in comparison with positive control and naïve groups. The dose of A. parviflora administered to animal was 100, 200 and 300 mg/kg. All administrations were given orally on an alternate day basis for 30 days. Urine and serum biomarkers were analyzed, along with antioxidant enzymes. Finally, the DNA damages, lipid peroxides, hydrogen peroxides and nitrites were assessed in rat's renal tissue. The histopathology alterations in renal tissues were further studied for kidney damages. RESULTS: The LC/MS analysis confirmed the presence of different important pharmacological compounds in A. parviflora methanolic leaf extract. The key bioactive compounds include pyocyanin, zonisamide, D Saccharic acid, altretamine, carbocyclic thromboxane A2, Sinapyl alcohol, and vitamin C. The important polypeptides identified include Lys-Tyr-Lys, His-His-Lys, Met-Asp-Arg, Phe-Val-Arg, and PyroGlu-Val-Arg. The LD50 of A. parviflora was found to be > 1000 µg/mL. A. parviflora administration significantly subsides CCl4 toxicity in rats, reduced the elevated level of RBCs, pus and epithelial cells. The abnormal elevated level of specific gravity, creatinine, urobilinogen, urea and albumin were also reduced to normal physiological level. The reduced urinary protein and pH were also normalized. The serum urobilinogen, urea and total bilirubin levels were also reversed to normal levels while the diminished albumin and total protein levels also came to normal. The important phase I and II enzyme levels were also reversed in A. parviflora administered rats. The H2O2, thiobarbituric acid reactive substance (TBARS) and nitrite levels were significantly decreased. Furthermore, the damaged DNA and histopathological changes in CCl4 exposed rats were also highly significantly reversed after the administration of A. parviflora. All effects were significant (P < 0.05) and highly significant (P < 0.005) at 100 and 300 mg/kg respectively. CONCLUSION: The restored urine and serum profile of various parameters to normal physiological levels suggests that the A. parviflora has potential antioxidant and repairing potential in renal disorders.

Diterpenoids from the Whole Plants of Ajuga nipponensis and Their Inhibition of RANKL-Induced Osteoclastogenesis.

Two new diterpenoids, ajudecunoid A (1) and ajudecunoid B (14), along with thirteen known diterpenoids, were isolated from the whole plants of Ajuga nipponensis Makino. Their structures were elucidated by the extensive spectroscopic analysis (UV, IR, MS, and NMR). The absolute configurations of ajudecunoid A (1) and ajudecunoid B (14) were defined through analysis of X-ray crystallography. Fifteen compounds were evaluated for inhibition of the formation of osteoclasts in bone marrow-derived macrophages (BMM) cells. Two neo-clerodane diterpenoids ajuganipponin B (5) and (12S)-6α,19-diacetoxy-18-chloro-4α-hydroxy-12-tigloyloxy-neo-clerod-13-en-15,16-olide (12) showed significant inhibition of osteoclastogenesis with IC50 values of 0.88 and 0.79 µM, respectively. Here we firstly reported diterpenoids with anti-osteoclastogenesis activity from A. nipponensis.

Production of biomass and medicinal metabolites through adventitious roots in Ajuga bracteosa under different spectral lights.

Ajuga bracteosa an important medicinal herb, is getting endangered worldwide due to destructive harvesting by pharmaceutical industries in its different habitats. It is in dire need for protection and demands conservation and sustainable utilization. In the present study, effects of α-naphthalene acetic acid (NAA) under different spectral lights were estimated on the growth, secondary metabolism and biosynthesis of phenolic acids in adventitious roots (AR) cultures of A. bracteosa. Among the different spectral lights, highest AR induction frequency (88%) and formation of biomass (72 g/L FW and 22 g/L DW) were recorded in explants incubated in the presence of 1.5 mg/L NAA under yellow light. Maximum production of poly phenols (TPC;44.2 mg) and flavonoids (TFC;2.51 mg) were recorded in the AR cultures grown in the presence of blue light. Further, highest total protein content of (401.6 µg) was detected in the AR in response to normal white light. Blue spectral light induced maximum superoxide dismutase (SOD; 2.5 nM) and peroxidase activity (POD;0.85 nM) respectively, in AR cultures. Compared with other monochromatic lights, red light significantly enhanced the antioxidant potential of the AR cultures. Analysis through High performance liquid chromatography (HPLC-DAD) revealed significant variations in the levels of important phenolic acids such as gallic acid, catechin, rutin, caffeic acid, myricetin and apigenin in the AR samples treated with the lights of different spectra.

Impacts of hormonal elicitors and photoperiod regimes on elicitation of bioactive secondary volatiles in cell cultures of Ajuga bracteosa.

Light is an important physical factor necessary for the growth, morphogenesis and production of bioactive compounds in plants. In this study, effects of different photoperiod regimes and hormonal elicitors were investigated on the accumulation of biomass, antioxidant potential and biosynthesis of secondary volatiles in the cell cultures of Ajuga bracteosa. Maximum accumulation of biomass (13.2 g/L) was recorded in cell cultures established at 1.0 mg/L benzylaminopurine (BA) in exposure to continuous dark. Biochemical assays showed that in the presence of 0.5 methyl jasmonate (Me-J), cell cultures grown under continuous dark had the higher activities of superoxide dismutase (SOD: 4.5 U/mg), peroxidase (POD: 3.1 U/mg), total phenolic content (TPC: 8.1 mg GAE/g of DW) and total flavonoid content (TFC: 5.2 mg QE/g of DW) respectively. Nonetheless, the free radical scavenging activity (FRSA) was found correlated with the phenyl ammonia lyase (PAL) activity in the dark grown cell cultures. Analysis through gas chromatography-mass spectrometry (GC-MS) showed, biosynthesis of 29 compounds in the in vitro raised cell cultures. The major identified compounds consisted of monoterpene hydrocarbons such as ß-pinene (2.1-9.5%), ß-ocimene (1.4-8.3%), 1-terpinene-4-ol (5.8-9.6%), caryophyllene (1.3-6.2%), ß-farnesene (0.82-7.8), oxygenated monoterpenes including myrtenal (2.2-8.4%), citronellyl acetate (2.1-7.3%) and sesquiterpenes such as caryophyllene oxide (1.5-5.5) and ß-elemene (2.2-8.8%). This protocol has the potential for commercial production of important secondary volatiles.

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol.

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ(24(28))-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ(24(25))intermediate. In this study, we characterized theArDWF1gene from an expression sequence tag library ofAjuga reptansvar.atropurpureahairy roots. The gene was functionally expressed in the yeast T21 strain. Thein vivoandin vitrostudy of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-(2)H3,28-(13)C)-24-methylenecholesterol, followed by(13)C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ(24(25))intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes,Oryza sativa DWF1(OsDWF1) was similarly expressed in yeast. Anin vivoassay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in theAjugahairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.

Evaluation of in vitro antiprotozoal activity of Ajuga laxmannii and its secondary metabolites.

Context Some Ajuga L. (Lamiaceae) species are traditionally used for the treatment of malaria, as well as fever, which is a common symptom of many parasitic diseases. Objective In the continuation of our studies on the identification of antiprotozoal secondary metabolites of Turkish Lamiaceae species, we have investigated the aerial parts of Ajuga laxmannii. Materials and methods The aerial parts of A. laxmannii were extracted with MeOH. The H2O subextract was subjected to polyamide, C18-MPLC and SiO2 CCs to yield eight metabolites. The structures of the isolates were elucidated by NMR spectroscopy and MS analyses. The extract, subextracts as well as the isolates were tested for their in vitro antiprotozoal activities against Plasmodium falciparum, Trypanasoma brucei rhodesiense, T. cruzi and Leishmania donovani at concentrations of 90-0.123 µg/mL. Results Two iridoid glycosides harpagide (1) and 8-O-acetylharpagide (2), three o-coumaric acid derivatives cis-melilotoside (3), trans-melilotoside (4) and dihydromelilotoside (5), two phenylethanoid glycosides verbascoside (6) and galactosylmartynoside (7) and a flavone-C-glycoside, isoorientin (8) were isolated. Many compounds showed moderate to good antiparasitic activity, with isoorientin (8) displaying the most significant antimalarial potential (an IC50 value of 9.7 µg/mL). Discussion and conclusion This is the first report on the antiprotozoal evaluation of A. laxmannii extracts and isolates. Furthermore, isoorientin and dihydromelilotoside are being reported for the first time from the genus Ajuga.

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture.

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 µL L(-1), 10 mg L(-1), and 80 µmol L(-1).

Odintifier--A computational method for identifying insertions of organellar origin from modern and ancient high-throughput sequencing data based on haplotype phasing.

BACKGROUND: Cellular organelles with genomes of their own (e.g. plastids and mitochondria) can pass genetic sequences to other organellar genomes within the cell in many species across the eukaryote phylogeny. The extent of the occurrence of these organellar-derived inserted sequences (odins) is still unknown, but if not accounted for in genomic and phylogenetic studies, they can be a source of error. However, if correctly identified, these inserted sequences can be used for evolutionary and comparative genomic studies. Although such insertions can be detected using various laboratory and bioinformatic strategies, there is currently no straightforward way to apply them as a standard organellar genome assembly on next-generation sequencing data. Furthermore, most current methods for identification of such insertions are unsuitable for use on non-model organisms or ancient DNA datasets. RESULTS: We present a bioinformatic method that uses phasing algorithms to reconstruct both source and inserted organelle sequences. The method was tested in different shotgun and organellar-enriched DNA high-throughput sequencing (HTS) datasets from ancient and modern samples. Specifically, we used datasets from lions (Panthera leo ssp. and Panthera leo leo) to characterize insertions from mitochondrial origin, and from common grapevine (Vitis vinifera) and bugle (Ajuga reptans) to characterize insertions derived from plastid genomes. Comparison of the results against other available organelle genome assembly methods demonstrated that our new method provides an improvement in the sequence assembly. CONCLUSION: Using datasets from a wide range of species and different levels of complexity we showed that our novel bioinformatic method based on phasing algorithms can be used to achieve the next two goals: i) reference-guided assembly of chloroplast/mitochondrial genomes from HTS data and ii) identification and simultaneous assembly of odins. This method represents the first application of haplotype phasing for automatic detection of odins and reference-based organellar genome assembly.

Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans (Lamiaceae).

MAIN CONCLUSION: In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of assimilates.

Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different tissue types of wild Ajuga bracteosa Wall. ex Benth.

Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeutic importance like 20-hydroxyecdysone (20-HE). As geography and habitat play a crucial role in the metabolism and morphology of a plant, the present study was aimed at evaluating the impact of phytogeography, season and tissue type on morphology and 20-HE content of A. bracteosa. The results revealed large morphological variations in various ecotypes of A. bracteosa. However, plants from the same altitude, regardless of their phytogeography, represented similar morphology. Effect of habitat on 20-HE content remained non-significant except for Karot (1608µg/g) and Kahuta (728µg/g). Effect of tissue types was significant (p value <0.016) for 20-HE content and followed ascending order: rootspring (1071µg/g)>summer (617µg/g). The aerial tissue types contained more 20-HE content in all seasons; especially during winter its amount radically rose in flowers (µ=2814µg/g). The aerial portion of Karot ecotype harvested in winter offers a valuable source of 20-HE. To confirm the effect of low temperature on 20-HE content, profiling of A. bracteosa raised in vitro at different temperature regime was carried out. On the basis of these results we hypothesize that chilling cold hampers vegetative growth and triggers stress induced 20-HE accumulation as a defense response.

Antiplasmodial activity of Ajuga bracteosa against Plasmodium berghei infected BALB/c mice.

BACKGROUND & OBJECTIVES: The present work was undertaken to evaluate antiplasmodial activity of ethanolic leaves extract of traditional medicinal plant Ajuga bracteosa in Plasmodium berghei infected BALB/c mice along with its phytochemical screening and acute toxicity test to support its traditional use as a remedy for malaria. METHODS: Plant extract (ethanolic) 250, 500, 750 mg/kg/day was evaluated in the early and established infection along with repository activity in P. berghei infected BALB/c mice through suppressive, curative and preventive test. The phytochemical screening was carried out by employing standard procedures. The acute toxicity was checked through limit test. RESULTS: The ethanolic leaves extract of A. bracteosa (250, 500 and 750 mg/kg/day) demonstrated a dose-dependent chemosuppression during early and in established infections, along with significant (P<0.05) repository activity. At a concentration of 750 mg/kg/day maximum 77.7 per cent chemosuppression during early infection and 68.8 per cent chemosuppression in repository activity were found. This dose enhanced significant mean survival period up to 27.4 +/- 0.46 days in established infection. ELEAB was found to be safe up to 5 g/kg weight when administrated orally in the female BALB/c mice, which is upper limit for oral administration of the test material to rodents. ED(50) of ELEAB was 300 mg/kg body weight of mice. INTERPRETATION & CONCLUSION: ELEAB inhibited parasitaemia and enhanced mean survival time in a dose- dependent manner upto 750 mg/kg/day dose in treated mice. Further studies need to be done to isolate and characterize active constituents of extract and to study their mechanism of action.

Raffinose in chloroplasts is synthesized in the cytosol and transported across the chloroplast envelope.

In chloroplasts, several water-soluble carbohydrates have been suggested to act as stress protectants. The trisaccharide raffinose (alpha-1,6-galactosyl sucrose) is such a carbohydrate but has received little attention. We here demonstrate by compartmentation analysis of leaf mesophyll protoplasts that raffinose is clearly (to about 20%) present in chloroplasts of cold-treated common bugle (Ajuga reptans L.), spinach (Spinacia oleracea L.) and Arabidopsis [Arabidopsis thaliana (L.) Heynh.] plants. The two dedicated enzymes needed for raffinose synthesis, galactinol synthase and raffinose synthase, were found to be extra-chloroplastic (probably cytosolic) in location, suggesting that the chloroplast envelope contains a raffinose transporter. Uptake experiments with isolated Ajuga and Arabidopsis chloroplasts clearly demonstrated that raffinose is indeed transported across the chloroplast envelope by a raffinose transporter, probably actively. Raffinose uptake into Ajuga chloroplasts was a saturable process with apparent K(m) and v(max) values of 27.8 mM and 3.3 micromol mg(-1) Chl min(-1), respectively.

Frost tolerance in excised leaves of the common bugle (Ajuga reptans L.) correlates positively with the concentrations of raffinose family oligosaccharides (RFOs).

Mass increases in raffinose family oligosaccharides (RFOs, alpha1,6-galactosyl extensions of sucrose) are well documented in the generative tissues of many plants upon cold acclimation, and they (i.e. mainly the two shortest RFO members, raffinose and stachyose) have been suggested as frost stress protectants. Our focus here was on the longer RFO members as they commonly occur in the frost-hardy evergreen labiate Ajuga reptans in its natural habitat, and accumulate to their highest concentrations in winter when the plant is faced with sub-zero temperatures. We examined the effects of RFO concentration and chain length on frost tolerance using excised leaves which accumulate long-chain RFOs under both cold and warm conditions, thereby uncoupling the acclimation temperature from RFO production. We demonstrated that frost tolerance in excised A. reptans leaves correlates positively with long-chain RFO accumulation under both acclimation temperatures. After 24 d post-excision in the warm, the leaves had increased their RFO concentrations (mainly long-chain RFOs) 22-fold to 78 mg g(-1) fresh weight, and decreased their EL(50) values (temperature at which 50% leakage occurred) from -10.5 to -24.5 degrees C, suggesting a protective role for these oligosaccharides in the natural frost tolerance of A. reptans.