Ajuga 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. Aerial 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. AbMA 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. Based 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.

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.

Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug. In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC–MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined. Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4–12-fold increase in artesunate and 1.2–3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant. Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.