Antiviral Potentialities of Chemical Characterized Essential Oils of Acacia nilotica Bark and Fruits against Hepatitis A and Herpes Simplex Viruses: In Vitro, In Silico, and Molecular Dynamics Studies.

Acacia nilotica (synonym: Vachellia nilotica (L.) P.J.H.Hurter and Mabb.) is considered an important plant of the family Fabaceae that is used in traditional medicine in many countries all over the world. In this work, the antiviral potentialities of the chemically characterized essential oils (EOs) obtained from the bark and fruits of A. nilotica were assessed in vitro against HAV, HSV1, and HSV2. Additionally, the in silico evaluation of the main compounds in both EOs was carried out against the two proteins, 3C protease of HAV and thymidine kinase (TK) of HSV. The chemical profiling of the bark EOs revealed the identification of 32 compounds with an abundance of di- (54.60%) and sesquiterpenes (39.81%). Stachene (48.34%), caryophyllene oxide (19.11%), and spathulenol (4.74%) represented the main identified constituents of bark EO. However, 26 components from fruit EO were assigned, with the majority of mono- (63.32%) and sesquiterpenes (34.91%), where trans-caryophyllene (36.95%), Z-anethole (22.87%), and γ-terpinene (7.35%) represented the majors. The maximum non-toxic concentration (MNTC) of the bark and fruits EOs was found at 500 and 1000 µg/mL, respectively. Using the MTT assay, the bark EO exhibited moderate antiviral activity with effects of 47.26% and 35.98% and a selectivity index (SI) of 2.3 and 1.6 against HAV and HSV1, respectively. However, weak activity was observed via the fruits EO with respective SI values of 3.8, 5.7, and 1.6 against HAV, HSV1, and HSV2. The in silico results exhibited that caryophyllene oxide and spathulenol (the main bark EO constituents) showed the best affinities (ΔG = -5.62, -5.33, -6.90, and -6.76 kcal/mol) for 3C protease and TK, respectively. While caryophyllene (the major fruit EO component) revealed promising binding capabilities against both proteins (ΔG = -5.31, -6.58 kcal/mol, respectively). The molecular dynamics simulation results revealed that caryophyllene oxide has the most positive van der Waals energy interaction with 3C protease and TK with significant binding free energies. Although these findings supported the antiviral potentialities of the EOs, especially bark EO, the in vivo assessment should be tested in the intraoral examination for these EOs and/or their main constituents.

Biorational Control of Callosobruchus maculatus (Coleoptera: Buchidae) in Stored Grains with Botanical Extracts.

Globally, around 2000 plant species are used against pest control. The utilization of botanicals is considered the most economic and biodegradable methods for the control of stored grains pests. Therefore, the current study was carried out to investigate the repellency potential of five botanicals against Callosbruchus maculatus F. in Haripur, Pakistan. The concentrations of Azadirachta indica L., Nicotiana tabacum L., Melia azedarach L., Nicotiana rustica L., and Thuja orientalis L. were, i.e., 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% in four replicates to establish contact effects. The data were recorded after 1, 2, 3, 6, 24, 48, 72, and 96 hours. The repellency effect of these plant species against C. maculatus were increased in both the time- and dose-dependent manner, and highest effect was observed at 72 h. In addition, the repellency effect was 91% for A. indica (class: V), 86% M. azedarach, 82%, N. tabacum (class: V), 79% N. rustica (class: IV), and 75% T. orientalis (class: IV) at 3% concentration against C. maculatus. Furthermore, following 96 hours' exposure to treatment the sensitivity response of insects decreases as the time interval increases, i.e., 86% A. indica (class: V) was followed by 71% M. azedarach (class: IV), 65% N. tabacum (class: IV), 61% N. rustica (class: IV), and T. orientalis 57% (class: III) repellency at highest concentration of 3%. The current study concluded that A. indica and M. azedarach can be incorporated for the management of C. maculatus and these plant species might be helpful in the productions of new biopesticides.

Enhanced photodegradation of rifampicin and co-trimoxazole by ZnO/ZnMn2O4/ZnS-PVA and its genotoxicity studies on Allium cepa.

Oxygen vacancies and its associated defect states have a great influence on the electronic and structural aspects of semiconductor photocatalysts, yet there is paucity of investigations about the influence of the defect states on their photocatalytic properties. Herein, this study reports the hierarchical fabrication of oxygen vacancy enriched ZnO/ZnMn2O4/ZnS-PVA nanocomposite (NCs) for the enhanced photodegradation of rifampicin and co-trimoxazole. The formation of lattice expansion induced oxygen vacancies and its associated Urbach tail energy, and n-p-n heterojunction-based S-scheme charge transfer path synergistically contributed to the boosted photocatalytic performance of the as prepared NCs. The photocatalytic performance of the nanomaterial towards rifampicin and co-trimoxazole has been determined to be 80% and 90% under visible light irradiation, respectively. Furthermore, various operating parameters including the concentration of NCs and drug, pH and interference of various ions have been evaluated. The degraded product intermediates have been elucidated by GC-MS analysis. The toxicity of the as-prepared nanomaterials has been evaluated by treating the samples with root tips of Allium cepa, where the NCs was found to be non-toxic. The study provides a new-fangled insight on the preparation and fabrication of non-toxic and defect rich nanomaterials which may help stimulate this area of research.

Novel Bioengineered Antibacterial and Anticancer ZnO Nanoparticles.

Transition metal oxide NPs have delivered wide applications in various fields. Therefore, in this study, a novel fungus, Alternaria sp. (NCBI Accession No: MT982648) was isolated and characterized from the vicinity of medicinal plants. Eventually, in this method extracted proteins from isolated fungus were utilized to synthesize highly biocompatible zinc nanoparticles (ZnO NPs). The various physical techniques including UV-visible spectroscopy, TEM, HR-TEM, XRD, DLS, zeta potential, and FTIR were used to characterize particles. The UV-visible absorption (λMax) and binding energy for the as-synthesized particles were found to be 329 nm and 3.91 eV, respectively. Further, the polydispersed particles were revealed to have regular crystallinity with hexagonal wurtzite phase of ZnO with the spacing of ~2.46 Å under XRD and HR-TEM. The average size of a particle under TEM was found to be ~18 nm. The evaluation of various surface functional groups of particles was done by FTIR. The average hydrodynamic diameter of particles was found to be ~57 d. nm with 0.44 particle distribution index whereas the nanoemulsion stability was explained by Zeta potential (-9.47 mV). These particles were found to exhibit potential antibacterial and anticancer activities. They were found to be bactericidal against S. abony (MIC 5.73 µg/mL); B. pumilis (MIC 6.64 µg/mL); K. pneumonia (MIC 14.4 µg/mL); E. coli (MIC 8.7 µg/mL); B. subtilis (MIC 5.63 µg/mL) and S. aureus (MIC 12.04 µg/mL). Further, they are also found to be concentration-dependent anticancer and inhibited the growth of A549 cells (IC50-65.3 µg/mL) whereas they were found to demonstrate no any cytotoxicity against NRK normal kidney cell line. The internalization of particles into the nucleus (i.e., nuclear fragmentation and DNA damage) was confirmed by DAPI staining. The intracellular particles were found to generate excessive ROS. Further, the anticancer potential was also estimated by noticing a hike in oxidative stress parameters, cell viability, cell morphology, and change in mitochondrial membrane potential. We effectively synthesized potentially potent antibacterial and anticancer novel bioengineered ZnO NPs.

Biodegradation of PAHs by Bacillus marsiflavi, genome analysis and its plant growth promoting potential.

The biodegradation of hazardous petroleum hydrocarbons has recently received a lot of attention because of its many possible applications. Bacillus marsiflavi strain was isolated from oil contaminated soil of Rawalpindi, Pakistan. Initial sequencing was done by 16s rRNA sequencing technique. Bac 144 had shown 78% emulsification index and 72% hydrophobicity content. Further, the strain displayed production of 15.5 mg/L phosphate sloubilization and 30.25 µg/ml indole acetic acid (IAA) in vitro assay. The strain showed 65% biodegradation of crude oil within 5 days by using Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Whole Genome analysis of Bac 144 was performed by PacBio sequencing and results indicated that Bacillus marsiflavi Bac144 strain consisted of size of 4,417,505bp with closest neighbor Bacillus cereus ATCC 14579. The number of the coding sequence was 4662 and number of RNAs was 141. The GC content comprised 48.1%. Various genes were detected in genome responsible for hydrocarbon degradation and plant defense mechanism. The toxic effect of petroleum hydrocarbons in soil and its mitigation with Bac 144 was tested by soil experiment with three levels of oil contamination (5%, 10% and 15%). Soil enzymatic activity such as dehydrogenase and fluorescein diacetate (FDA) increased up to 49% and 40% with inoculation of Bac 144, which was considered to be correlated with hydrocarbon degradation recorded as 46%. An increase of 20%, 14% and 9% in shoot length of plant at 5%, 10% and 15% level of oil was recorded treated with Bac 144 as compared to untreated plants. A percent increase of 14.89%, 16.85%, and 13.87% in chlorophyll, carotenoid, and proline content of plant was observed by inoculation with Bac 144 under oil stress. Significant reduction of 14% and 18%, 21% was recorded in the malondialdehyde content of plant due to inoculation of Bac 144. A considerable increase of 21.33%, 19.5%, and 24.5% in super oxide dismutase, catalase, and peroxidase dismutase activity was also observed in plants inoculated with strain Bac 144. These findings suggested that Bac-144 can be considered as efficient candidate for bioremediation of hydrocarbons.

Fruit Derived Potentially Bioactive Bioengineered Silver Nanoparticles.

INTRODUCTION: Protein-derived biogenic syntheses of inorganic nanoparticles have gained immense attention because of their broad spectrum of applications. Proteins offer a reducing environment to enable the synthesis of nanoparticles and encapsulate synthesized nanoparticles and provide them temporal stability in addition to biocompatibility. METHODS: In the present study, Benincasa hispida fruit proteins were used to synthesize silver nanoparticles (AgNPs) at 37 °C over five days of incubation. The synthesis of AgNPs was confirmed by UV-Vis spectroscopy, TEM, zeta potential, and DLS analyses. Further, these NPs depicted antibacterial and antibiofilm effects. Additionally, the anticancer activities of nanoparticles were also tested against the lung cancer cell line (A549) with respect to the normal cell line (NRK) using MTT assay. Further, the estimation of ROS generation through DCFH-DA staining along with a reduction in mitochondrial membrane potential by Mito Tracker Red CMX staining was carried out. Moreover, nuclear degradation in the AgNPs treated cells was cross-checked by DAPI staining. RESULTS: The average size of AgNPs was detected to be 27 ±1 nm by TEM analysis, whereas surface encapsulation by protein was determined by FTIR spectroscopy. These NPs were effective against bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella enteric, and Staphylococcus epidermis with MICs of 148.12 µg/mL, 165.63 µg/mL, 162.77 µg/mL, and 124.88 µg/mL, respectively. Furthermore, these nanoparticles inhibit the formation of biofilms of E. coli, S. aureus, S. enteric, and S. epidermis by 71.14%, 73.89%, 66.66%, and 64.81%, respectively. Similarly, these nanoparticles were also found to inhibit (IC50 = 57.11 µM) the lung cancer cell line (A549). At the same time, they were non-toxic against NRK cells up to a concentration of 200 µM. DISCUSSION: We successfully synthesized potentially potent antibacterial, antibiofilm and anticancer biogenic AgNPs.

Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line.

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.

Stemphylium Leaf Blight of Welsh Onion (Allium fistulosum): An Emerging Disease in Sanxing, Taiwan.

Welsh onion (Allium fistulosum L.) is one of the main and oldest vegetable crops grown in Taiwan. A severe epidemic of leaf blight in Welsh onion caused by a Stemphylium-like pathogen was found in Sanxing, Taiwan, from 2018 to 2020. However, correct species identification, biology, and control of Stemphylium leaf blight (SLB) of Welsh onion are not well-established. Therefore, the main objective of this study was to investigate the causal agent of SLB in Sanxing and evaluate the in vitro sensitivity of Stemphylium-like pathogen to commonly used fungicides. A phylogenetic analysis based on combining the internal transcribed spacer (ITS) region and glyceraldedyhe-3-phosphate dehydrogenase (gapdh) and calmodulin (cmdA) gene sequences together with morphological features identified that S. vesicarium is associated with SLB in Sanxing. When inoculated onto Welsh onion leaves, the isolates caused symptoms identical to those observed in the field; therefore, S. vesicarium was reisolated and Koch's postulates were confirmed. We observed a higher incidence of SLB symptoms on the oldest leaves compared with younger leaves. The maximum and minimum temperatures for in vitro mycelial growth and conidial germination (%) of S. vesicarium were 20 to 30°C and 5°C, respectively. Sixteen fungicides were tested for their effectiveness to reduce the mycelial growth and conidial germination of S. vesicarium in vitro. Boscalid plus pyraclostrobin, fluopyram, fluxapyroxad, and fluxapyroxad plus pyraclostrobin were highly effective at reducing mycelial growth and conidial germination in S. vesicarium. However, strobilurin fungicides (azoxystrobin and kresoxim-methyl) commonly used in Welsh onion production in Sanxing were ineffective. This study discusses the emergence of SLB caused by S. vesicarium in the foliar disease complex affecting Welsh onion and the management of the disease using fungicides with different modes of action in Taiwan. The research will support the sustainable management of SLB in Sanxing, Taiwan; however, further field assessments of the fungicides are warranted.

Biogenic Synthesis of NiO Nanoparticles Using Areca catechu Leaf Extract and Their Antidiabetic and Cytotoxic Effects.

Nanoworld is an attractive sphere with the potential to explore novel nanomaterials with valuable applications in medicinal science. Herein, we report an efficient and ecofriendly approach for the synthesis of Nickel oxide nanoparticles (NiO NPs) via a solution combustion method using Areca catechu leaf extract. As-prepared NiO NPs were characterized using various analytical tools such as powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and UV-Visible spectroscopy (UV-Vis). XRD analysis illustrates that synthesized NiO NPs are hexagonal structured crystallites with an average size of 5.46 nm and a hexagonal-shaped morphology with slight agglomeration. The morphology, size, and shape of the obtained material was further confirmed using SEM and TEM analysis. In addition, as-prepared NiO NPs have shown potential antidiabetic and anticancer properties. Our results suggest that the inhibition of α-amylase enzyme with IC 50 value 268.13 µg/mL may be one of the feasible ways through which the NiO NPs exert their hypoglycemic effect. Furthermore, cytotoxic activity performed using NiO NPs exhibited against human lung cancer cell line (A549) proved that the prepared NiO NPs have significant anticancer activity with 93.349 µg/mL at 50% inhibition concentration. The biological assay results revealed that NiO NPs exhibited significant cytotoxicity against human lung cancer cell line (A549) in a dose-dependent manner from 0-100 µg/mL, showing considerable cell viability. Further, the systematic approach deliberates the NiO NPs as a function of phenolic extracts of A. catechu with vast potential for many biological and biomedical applications.

Vincamine, a safe natural alkaloid, represents a novel anticancer agent.

Vincamine, a well-known plant alkaloid, has been used as a dietary supplement and as a peripheral vasodilator to combat aging in humans. In this study, for the very first time, we demonstrated that vincamine can function as an anticancer agent in a human alveolar basal epithelial cell line A549 (IC50 = 309.7 µM). The anticancer potential of vincamine in A549 cells was assessed by molecular assays to determine cell viability, generation of intracellular ROS, nuclear condensation, caspase-3 activity and inhibition, and change in mitochondrial membrane potential (ΔΨm). In silico studies predicted that the anti-proliferative potential of vincamine is enhanced by its interaction with the apoptotic protein caspase-3, and that this interaction is driven by two hydrogen bonds and has a high free energy of binding (-5.64 kcal/mol) with an estimated association constant (Ka) of 73.67 µM. We found that vincamine stimulated caspase-3-dependent apoptosis and lowered mitochondrial membrane potential, which ultimately led to cytochrome C release. Vincamine was also found to quench hydroxyl free radicals and deplete iron ions in cancer cells. As a dietary supplement, vincamine is almost non-toxic in BEAS-2B and 3T3-L1 cells. Therefore, we propose that vincamine represents a safe anticancer agent in lung cancer cells. Its role in other cancers has yet to be explored.

The potential of Allium sativum and Cannabis sativa extracts for anti-tick activities against Rhipicephalus (Boophilus) microplus.

The efficacy of Allium sativum and Cannabis sativa against Rhipicephalus microplus ticks was evaluated using the adult immersion and the larval packet test. In addition, an in silico approach was utilized by performing a docking study in order to identify the active ingredients from both plants. Results showed a comparatively high lethal effect of A. sativum and C. sativa on egg laying (index of egg laying = 0.26 and 0.24, respectively), egg hatching (33.5 and 37.1, respectively), and total larval mortality (100%, both), at 40 mg/mL. When applied to cattle which had been inoculated with larvae ticks, it was observed that a 45% solution of both herbal extracts significantly reduced the number of ticks by 96 h post treatment. We analyzed in silico 27 known active molecules from both plants and identified in the PubChem database to explore the hypothesis that the effect found on ticks was based on inhibition of acetylcholinesterase (AChE). Vitamin E and cannabidiol are the most potent AChE inhibitors with docking scores of -15.85 and -14.38, respectively. Based on these findings, we conclude that A. sativum and C. sativa may potentially be used for the control of R. microplus, and should be further investigated as a potential supplement to or replacement of synthetic acaricides.

Green synthesized zinc oxide nanoparticles regulates the apoptotic expression in bone cancer cells MG-63 cells.

Management of degenerative spine pathologies frequently leads to the need for bone growth. Rehmanniae Radix (RR), a Chinese herbal formulation was found to exhibit numerous therapeutic properties including its potent effect against cancer cell lines. However, the underlying mechanism through which the Zinc oxide nanoparticles (ZnONPs) synthesized from Rehmanniae Radix exerts its anti-cancer activity against osteosarcoma cell line MG-63 needs to be explored. Therefore, the study was performed to evaluate the anticancer, cytotoxicity and apoptotic effectiveness of ZnONPs from RR against MG-63 cells. Characterization studies such UV-vis spectroscopy, FTIR, TEM and XRD analysis were performed. Cytotoxicity assay, mitochondrial membrane potential (MMP), morphological examination of cells and formation of reactive oxygen species (ROS), and apoptosis inducing ability of RR were evaluated by various procedures. Western blot analysis of apoptotic markers such as Bax, caspase-3 and caspase-9 were also performed. RR was found to inhibit growth of MG-63 cells at increasing dose. AO/EB staining confirmed the apoptotic efficacy of ZnONPs induced by RR in MG-63 cells. ZnONPs was also found to initiate increased generation of ROS and decreased MMP. Decreased MMP has resulted in increased levels of apoptotic proteins Bax, caspase-3 and caspase-9 and induction of apoptosis was substantiated by western blot analysis. The outcomes of the work propose that ZnONPs from RR exhibits strong anticancer action and inducing apoptosis on MG-63 cells via stimulating increased generation of ROS. Thus, ZnONPs from RR might be used as a hopeful drug target against several types of cancer cell lines.

Zinc oxide nanoparticles synthesized from Fraxinus rhynchophylla extract by green route method attenuates the chemical and heat induced neurogenic and inflammatory pain models in mice.

Fraxinus rhynchophylla belongs to the family of Oleaceae and also called as Chinese ash wood possesses various pharmacological properties such as neuroprotective, antimicrobial, anti-inflammatory, etc. Therefore we synthesized ZnO nanoparticles using Fraxinus rhynchophylla wood extract as reducing and capping agent. The synthesized nanoparticles were characterized with the aid of UV-Spec, DLS, FT-IR and TEM analysis. Green synthesized ZnO nanoparticles were then assessed for anti-nociceptive property by using various nociception models such as thermal stress-induced, acetic acid, glutamate, capsaicin, and formalin-induced nociception. The sedative effect of synthesized ZnO nanoparticles was evaluated with an open field test. UV-Spectroscopic analysis confirms the formation of ZnO nanoparticles and the characterization studies DLS, FT-IR, and TEM analysis prove it has ideal nanoparticle can be used as a nano-drug. Results of both thermal stress-induced methods hot plate and tail immersion nociception test verified the synthesized ZnO nanoparticles are a potent antinociceptive drug. ZnO nanoparticles effectively reduced the abdominal writhes in acetic acid-induced nociception and it also significantly decreased the nociception activity in another glutamate, capsaicin, and formalin-induced nociception models. Open field experiment proved that synthesized ZnO nanoparticles are less sedative compared to the standard antinociceptive drug morphine. Overall our findings authentically confirm ZnO nanoparticles synthesized from Fraxinus rhynchophylla wood extract is a novel drug that persuasively reduces nociception in different nociceptive induced mice models and can be the best alternative for allopathic drugs which renders severe side effects.