Abstract
Vegetative growth, chemical compositions, biological and antioxidant activities of plants are widely affected by several environment parameters and conditions, therefore, this study was designed to determine the effects of Light intensity, soil moisture and benzyl adenine hormone on the growth parameters of M.vulgare L. medicinal plant. The study consisted of six experiments, the first experiment was a factorial pot experiment (3x3x2) applied as Randomized Complete Block Design(RCBD), with three replications, to determine the effects of light intensity (100%, 120% and 80%) of the natural light intensity (4400 lux), benzyl adenine (0, 150 and 300 ppm) and soil moisture contents (100% and 60% of the field capacity) on the growth, development and chemical constituents of M.vulgare L. plants conducted in the greenhouse of the Department of Biology, College of Science, Salahaddin University- Erbil, Iraq. For the rest experiments the construction and analyses of the experimental design, the response surface and determination of optimum conditions were carried out using Design-Expert 0.0.07 software (Minneapolis, USA). This part conducted in the laboratories of Shahid Beheshti University -Tehran, Iran. Experiment one comprised the effects of light, hormone and soil water on vegetative growth of M.vulgare L. plants, Experiment two was green gold nanoparticle biosynthesis of different treatments to determine the best plant sample for nanoparticle synthesis. The third experiment used various methanol extraction, of M.vulgare L. plant leaves to measure some phytochemicals and metabolites (Chlorophyll a, Chlorophyll b, Total Chlorophyll, Total Carotenoids, Total phenol, Total protein, Flavonoids, Alkaloids and Carbohydrates). And Liquid Chromatography-Mass Spectrometry for identification of chemical compounds.
Experiment fourth was antioxidant activity and enzymatic pathways in plant defense system against different stresses, enzymes such as (Catalase, Superoxid Dismutase, Phenylalanine Ammonia-lyase, Malondialdehyde and Glutathione Reductase) were determined for this part. Fifth experiment was the effects of different treatments methanol extracts on regulation of female hormones including (testosterone, estradiol, progesterone, and estrogen). The last experiment was using synthesized nanoparticles alone and combined with two anticancer drugs to perform MTT test for three female cancer cell lines (MCF-7, OVAR-3 and HeLa).
Results of experiment one shows high intensity light (L 120%), decreased total plant leaf area, leaf fresh weight, leaf dry weight and root parameters. Low light intensity (L80%) increased total plant leaf area, leaf dry weight, root fresh weight and root dry weight, while decreased other root parameters. Benzyl adenine hormone increased total plant leaf area, moisture content, root diameter and root mass density. While L80% decreased root dry weight, root mass density, dry root mass density and root tissue density. Soil moisture (FC 60%) increased dry matter, root diameter and root volume.
Results of experiment two indicated that combination treatment L(120%)M(100%)BA(300)ppm was recorded as best sample to green synthesis of gold nanoparticles and characterized nanoparticles showed size in the range 19.98 –39.69 nm that clearly imaged by Scanning Electron Microscope (SEM). Dynamic Light Scattering (DLS) results indicated 95% of the synthesized gold nanoparticles (AuNPs) have a size of20 nm. Zeta potential for gold nanoparticle (AuNPs) was 42.36. This value indicates a relative stability in the dispersion of nanoparticles. X-Ray Diffraction (XRD) results showed three main peaks are located at 38.1, 44.3, 64.6 and 77.6 equal 2theta degrees, respectively, which correspond to (111), (200), (220) and (311) planes in the cubic structure (FCC) of gold. UV-Vis results showed specific absorption peak around 558.3 nm.
Experiment three results illustrate that L120% increase all pigments amount, and flavonoids, alkaloids, carbohydrate amount while L80% decreased all these parameters but increased total phenol and total protein. Benzyl Adenine hormone (150 and 300ppm), increased pigments and phytochemicals except carbohydrate. FC 60% (M2) increased total phenol and total proteins while decreased pigments amount flavonoids, alkaloids and carbohydrate amount. Liquid Chromatography-Mass Spectrometry results detection of three compounds such as vulgarole, marrubiin, and marrubenol.
Results of experiment four showed increased activity of enzymes and antioxidant activity by L120%, except malondialdehyde enzyme. L3, also, increased enzyme activity while decreased antioxidant properties of extracts. Benzyl Adenine hormone (150 and 300ppm), increased antioxidant activity and enzyme activity except malondialdehyde.
Experiment five results show L120% decreased inhibitory effects of female hormones; in contrast, L80% increased hormone inhibition effects. Benzyl Adenine hormone (150 and 300ppm), increased estrogen inhibitory effect but decreased testosterone and progesterone. FC 60%(M2) increased all hormones inhibitory effects.
Experiment six results showed that M.vulgare L. extracts show best effects on HeLa cell lines with IC50= after 24h. at concentration of 10 mg/ml. AuNPs effects on cancer cell lines show best growth inhibition of OVAR-3 cell line after 72h, in concentration of 5 mg.ml-1. Drug-delivery properties of NPs were examined by Cyclophosphamide drug and nanoparticles that showed the best effects on MCF-7 cell lines after 24h, in concentration of 0.312 mg.ml-1. Taxol plus Au-NPs showed the best growth inhibition of HeLa cell lines after 24h in concentration of 0.625 mg.ml-1.
