BackgroundAjuga bracteosa has been extensively used traditionally for the treatment of a variety of diseases. The aim of the study was to scientifically validate the wide-scale exploitation of A. bracteosa in folk medicine various in vitro and in vivo assays. Moreover, these activities were related to the intrinsic biologically active phytoecdysteroids of A. bracteosa.MethodsAerial and root parts of A. bracteosa were first extracted separately with chloroform (AbCA and AbCR) and the residue was again extracted with methanol (AbMA and AbMR). Total flavonoid and phenolic contents were assayed as quercetin (QE) and gallic acid equivalents (GAE), respectively. These extracts were analyzed for in vitro antioxidant assessment including DPPH and H2O2 (% inhibition of free radicals), and reducing power and phosphomolybdenum methods (ascorbic acid equivalents AAE mg/g DW). Further, these extracts were assayed in vivo in separate groups of Sprague–Dawley rats for carrageenan induced rat paw edema inhibition, hotplate antinociception, forced swim antidepression and anticoagulation. Dose of each crude extract and standard drug given to rats was 200 mg/Kg- and 10 mg/10 mL/Kg body weight respectively. Plant extracts and standard drugs were administered orally, 60 min prior to the conduction of assays. Moreover, biologically active phytoecdysteroids were screened in A. bracteosa with the help of RP-HPLC.ResultsAbMA represented highest values of flavonoids (QE 1.98 % DW) and phenolic contents (GAE 5.94 % DW), significantly scavenged DPPH radicles (IC50 36.9) and reduced ferric ions with 718.4 mg ascorbic acid equivalent/g (AAE). Highest total antioxidant capacity was expressed by AbMR (927 mg AAE) with an IC50 value 19.1 μg/mL. The extracts which were found potent anti-oxidants, were also good at in vivo activities. AbMA significantly reduced edema in all the three hours of treatment (67.9, 70.3 and 74.3 %). AbMA also showed maximum nociceptor suppression in analgesic assay by delaying the time to start licking of paws in rats (57.7 ± 4.9 s). In addition, maximum anti-coagulation was also exhibited by AbMA (89.3 s), while all extracts were found strong antidepressants (≤15.66 s immobility time). Screening of biologically active phytoecdysteroids revealed the presence of 20-hydroxyecdysone (20-HE), makisterone (MKA), cyasterone (CYP) and ajujalactone (AJL). Total phytoecdysteroid content found in A. bracteosa was 1232.5 μg/g DW and 20-HE was most abundant (1232.5 μg/g DW) as compared to other phytoecdysteroids.ConclusionBased on the tested in vitro and in vivo activities, AbMA was found to be a promising bioactive extract. These activities can be attributed to the intrinsic polyphenols and phytoecdysteroids contents of A. bracteosa.

The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01–0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism.

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An accumulating body of evidence reports the synthesis and biomedical applications of silver nanoparticles. However, the studies regarding the use of maleic acid and citric acid in the synthesis of nano-sized silver particles (AgNPs) and micro-sized silver particles (AgMPs) as well as their antibacterial, antifungal, and anticancer activities have not been reported. In the current study, we synthesized AgNPs and AgMPs using maleic acid and citric acid as capping agents and have characterized them by UV-Vis, energy-dispersive X-Ray spectroscopy (EDS), X-Ray diffraction (XRD), and scanning electron microscope (SEM) analysis. The capped silver particles were examined for their antimicrobial activity and cytotoxicity against bacteria, fungi, and brine shrimp. Additionally, the anticancer activity of these particles was tested against human breast and liver cancer cell lines. The free radical scavenging activity of capped silver particles was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. SEM analysis revealed a round plate-like morphology of maleic acid capped particles with an average size of 39 ± 4 nm, whereas citric acid capped particles display flower-shaped morphology with rough surfaces and an average size of 250 ± 5 nm. The uncapped AgMPs were hexagonal with 500 ± 4 nm size. EDS and XRD analysis confirmed the presence of Ag and face-centered cubic crystalline nature, respectively. Functionally, capped silver particles exhibited antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus) and Gram-negative bacteria (Salmonella setubal, Enterobacter aerogenes, and Agrobacterium tumefaciens). The bactericidal activity was more active against Gram-negative bacteria with minimum inhibitory concentration (MIC) as low as 5 ppm as compared to 25 ppm for Gram-positive. Similarly, the silver particles demonstrated antifungal activity by inhibiting the growth of five fungal strains (Mucor species, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, and Fusarium solani) up to 50% at the concentration of 500 ppm. Additionally, these particles showed substantial toxicity against brine shrimp and also significantly inhibited the proliferation of breast cancer (MCF7) and liver cancer (HePG2) cell lines (IC50 8.9–18.56 µM). Uncapped AgMPs were less effective, inhibiting only the proliferation of MCF7 cells with IC50 46.54 µM. Besides cytotoxicity, these particles acted as potential antioxidants, showing free radical scavenging up to 74.4% in a concentration-dependent manner. Taken together, our results showed that the modifiers affect the shape and size of silver particles and may, in part, contribute to the antimicrobial and antioxidant activity of silver particles. However, the contribution of maleic acid and citric acid in enhancing the antimicrobial, anticancer, and antioxidant potential independent of silver nano and microparticles needs to be studied further. In vivo experiments may determine the therapeutic effectiveness of silver particles capped with these modifiers.

Flavonoids are famous for their antioxidant capacity and redox potential. They can combat with cell aging, lipid peroxidation, and cancer. In the present study, Artemisia annua hybrid (Hyb8001r) was subjected to qualitative and quantitative analysis of flavonoids through HPLC. Rol genes transgenics of A. annua were also evaluated for an increase in their flavonoid content along with an increase in antioxidant and cytotoxic potential. This was also correlated with the expression level of flavonoids biosynthetic pathway genes as determined by real-time qPCR. Phenylalanine ammonia-lyase and chalcone synthase genes were found to be significantly more highly expressed in rol B (four to sixfold) and rol C transgenics (3.8-5.5-fold) than the wild-type plant. Flavonoids detected in the wild-type A. annua through HPLC include rutin (0.31 mg/g DW), quercetin (0.01 mg/g DW), isoquercetin (0.107 mg/g DW) and caffeic acid (0.03 mg/g DW). Transgenics of the rol B gene showed up to threefold increase in rutin and caffeic acid, sixfold increase in isoquercetin, and fourfold increase in quercetin. Whereas, in the case of transgenics of rol C gene, threefold increase in rutin and quercetin, 5 fold increase in isoquercetin, and 2.6-fold increase in caffeic acid was followed. Total phenolics and flavonoids content was also found to be increased in rol B (1.5-fold) and rol C (1.4-fold) transgenics as compared to the wild-type plant along with increased free radical scavenging activity. Similarly, the cytotoxic potential of rol gene transgenics against MCF7, HeLA, and HePG2 cancer cell lines was found to be significantly enhanced than the wild-type plant of A. annua. Current findings support the fact that rol genes can alter the secondary metabolism and phytochemical level of the plant. They increased the flavonoids content of A. annua by altering the expression level of flavonoids biosynthetic pathway genes. Increased flavonoid content also enhanced the antioxidant and cytotoxic potential of the plant.