Biomedical Potential of Silver and Gold Nanoparticles Synthesized From Daphne Mucronata And Monotheca Buxifolia As New Precursors

Abstract

Traditional treatment of diseases is primarily based on empirical learning without scientific proofs. Monotheca buxifolia (M. buxifolia) and Daphne mucronata (D. mucronata) has been used traditionally across the globe in different communities including Pakistan, for the treatment of arthritis, tooth ache, rheumatism, flue like conditions, inflammations, ulcers, urinary track diseases,fevers and vermifuge. The present study was designed for synthesis and characterization of biogenic nanoparticles (NPs) and their biomedical potential comparison to that of plant extracts. From the plant D. mucronata (leaves, bark and roots) and M. buxifolia (leaves, seeds and fruits) crude methanolic extracts and fractions were obtained. Phytochemical analysis for the presence of carbohydrates, phenols/tannins, flavonoid, saponins and glycosides was performed. Elemental analysis through atomic absorption spectroscopy (AAS) was performed for determination of various trace and heavy metals. Nutritional analysis of the plants was also determined. Synthesis of NPs included silver nitrate (AgNO3) and gold chloride (AuCl4) that were reduced using aqueous extracts of both plants. The biogenic NPs were then characterized by UV Visible spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infra-Red (FTIR) Spectroscopy, X-Ray Diffraction(XRD), Thermo gravimetric-Differential Thermal Analysis (TG-DTA) and Energy Dispersive X-Ray Detection (EDX) techniques. The synthesized NPs and methanolic, n-hexane, chloroform, ethyl acetate and aqueous fractions of both plants were tested for biological activities including antioxidant, anti-bacterial, anti-fungal, haemagglutination, phytotoxic, insecticidal, anti-termite and cytotoxic activities. The aqueous extracts and synthesized silver nanoparticles (AgNPs) of both plants and gold nanoparticles (AuNPs) mediated by M. buxifolialeaves were screened for DNA damage, hemolytic and anti-thrombolytic activities. Crude methanolic extracts and synthesized NPs of both plants were screened for acute toxicity assay, anti-analgesic, anti-pyretic, GIT motility and anti-inflammatory activities. Hematological parameters were also evaluated using male Guinea pigs and Rabbits. All data were then analyzed and interpreted using Microsoft Excel, Origin Pro 8.5 and Graph Pad Prism 6. Phytochemical screening oftest samplesrevealed the presence of reducing sugars and carbohydrates. Saponins were detected in D. mucronata leaves, roots and M. buxifolialeaves and fruits. Phenols and tannins were present in all parts except D. mucronata roots. Glycosides were present in D. mucronate bark, leaves and roots and M. buxifolia leaves. Flavonoids were present in D. mucronata bark and all parts of M. buxifolia. Elemental analysis revealed that D. mucronata contains Fe (0.316), Zn (0.176), Ca (42.26), Cr (0.001), Ni (0.018), Pb (0.404), Mn (0.285), Co (0.051) and Cu (0.035). The M. buxifolia contains Fe (0.169), Zn (0.060), Ca (20.24), Cd (0.022), Pb (0.119), Mn (0.150), Co (0.012) and Cu (0.016). Nutritional analysis determined that D. mucronata contains carbohydrates (61.56), proteins (4.12), fat (2.733), fibers (23.58), ash (9.94) and moisture (3.85). M. buxifolia contains carbohydrates (56.53), proteins (3.15), fat (0.87), fibers (24.08), ash (10.71) and moisture (2.54%). Synthesized NPs were first detected by change in color i.e. Brown for AgNPs and cherry red for AuNPs. The UV-Vis spectral patterns of D. mucronataleaves derived AgNPs were observed with corresponding Surface Plasmon Resonance (SPR) peak at 425 nm, whereas M. buxifolia leaves derived AgNPsand AuNPs at 405 and 540 nm peaks. The FTIR results demonstrated that -CH and -OH groups were involved in synthesis of D. mucronataleaves derived AgNPs as reducing agents. The OH, -CH and COOH groups were involved in synthesis of M. buxifolialeaves derived AgNPs. In case of M. buxifolialeaves derived AuNPsthe -OH, -CH and -CO groups were involved in synthesis of AuNPs. Crystal size through XRD analysis observed in D. mucronataleaves derived AgNPswas 94.779°A whereas M. buxifolialeaves derived AgNPsand AuNPs was 96.18°A and 109.94°A. The EDX analysis confirmed D. mucronataleaves derived AgNPs by containing 42.90% silver (Ag), whereas in M. buxifolialeaves derived AgNPs 53.68% Ag and in AuNPs 15.43% gold (Au) along some other elements in different amounts. The SEM and TEM analysis revealed AgNPs derived from D. mucronata and M. buxifolialeaves possess well dispersed spherical shape with size range 8-30 nm and 8 20 nm respectively. Nano prisms, Nano rods and hexagonal shaped structures were observed in case of M. buxifolialeaves derived AuNPs with approximate size of 10-60 nm. The TGA profile of D. mucronataleaves derived AgNPswas observed with weight loss at 320 and 440°C, whereas M. buxifolialeaves derived AgNPs at 320, 500 and 900 °C, and AuNPs at 320, 480 and 906 °C. The DTA graphs showed endothermic and exothermic reactionsoccurrence of both plants at various degrees. D. mucronate andM. buxifolia extracts and D. mucronataleaves derived AgNPs (86.4%), M. buxifolialeaves derivedAgNPs (84.58%) and AuNPs (86.31%) showed significant antioxidant activity at 600 μg/ml. Potential anti-bacterial activity of both plants extracts and synthesized NPs was observed against Acenatobacter baumanni, Morganella morganii, Escherichia coli, Pseudomonas aeruginosa, Vancomycin-Resistant Staphylococcus aureus (VRSA) and Proteus vulgaris. Both plants mediated AgNPs showed moderate activity against Candida albicans and low activity against Aspergillus niger. These samples were inactive against Fusariumoxysporum, Aspergillus flavus, Aspergillus parasiticus and Penicillium digitatum. The tested samples did not show heameagglutination activity which means phytolectins were absent in them. Highest phytotoxic activity was observed for ethyl acetate extract with 80% inhibitory effect in D. mucronata at 20 mg/ml against Lemna minor. Resultant insecticidal and anti-termite activity of tested samples showed 100% activity against Tribolium castaneum, Rhyzopertha dominica, Callosobruchus analis and Heterotermes indicola. Brine shrimp lethality assay revealed that plants leaves extract derived AgNPs and AuNPs exhibited higher cytotoxic activity than plants extracts alone. Mild thrombolytic activity was observed that ranges from 15.9 to 25.8% clot lysis by the tested samples. Hemolytic and mutagenic activity showed negative results for the tested samples. All extracts were found safe as no lethality was observed. D. mucronataleaves derived AgNPs showed higher percent of writhihng inhibition than both the plants alone and M. buxifolia leaves derived NPs. Anti-pyretic activity observed in tested samples showed that D. mucronataleaves derived AgNPs exhibited better antipyretic activity as compared to M. buxifolialeaves derived AgNPs and AuNPs at both the doses tested. Gastro intestinal track motility (GIT motility) showed that with increase in sample concentration, decrease in% GIT motility was observed. Both plants mediated AgNPs and AuNPs were observed to possess significant anti-inflammatory properties. Biochemical parameters analyzed fortest samples revealed a slight increase and decrease in all hematological parameters, liver function tests, body weight, feed and water intake which did not affect the normal health.