A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Functional divergence of two arginine decarboxylase genes in tropane alkaloid biosynthesis and root growth in Atropa belladonna.

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2, exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N-methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N-methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to ß-glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis.

Chemical Composition Analysis of Atropa belladonna Grown in Iran and Evaluation of Antibacterial Properties of Extract-loaded Nanofibers.

In the present work, the chemical composition of extract fractions of the Atropa belladonna plant growing in the north of Iran was investigated by HPTLC, HPLC, and GC-MS. Based on HPLC results, atropine, and scopolamine were found to be higher in the fruit and leaf extracts than in other parts of the plant. The comparative GC-MS analysis showed that diacetone alcohol, mesityl oxide, palmitic acid, and linoleic acid were the major bioactive components in the root, stem, leaf, and fruit extracts, respectively. Leaf extract showed the best antioxidant activity in the DPPH test. The antibacterial activity of fractional extracts was determined against Pseudomonas aeruginosa using the MIC method, and the fruit and leaf extracts exhibited the best antibacterial activities. The leaf extract was embedded into nanofibers by electrospinning technique, and its antibacterial activity was determined against Pseudomonas aeruginosa. The morphology and mechanical properties of the nanofibers were studied with SEM, contact angle, and tensile analysis, showing ultrafine fibers with uniform morphology.

Plant Production Protocols from Seeds of Threatened Atropa baetica and Widespread A. belladonna, Both Rich in Alkaloids.

Members of the genus Atropa contain various tropane alkaloids, including atropine ((±)-hyoscyamine) and scopolamine, which possess medicinal properties. Preserving the diverse genetic background of wild populations via optimal plant production from seeds could be essential for avoiding the loss of potential uses. We analyzed the germination ecology of two Atropa species comprising the threatened A. baetica and widespread A. belladonna to determine the: (1) influence of temperature, light, and seed age on germination patterns; (2) effects of cold stratification and gibberellic acid (GA3); (3) phenology of seedling emergence in outdoor conditions; (4) phenology of dormancy break and loss of viability in buried seeds; and (5) ability to form persistent soil seed banks. Freshly matured seeds exhibited conditional physiological dormancy, with germination at high temperatures (32/18 °C) but not at low and cold ones (5, 15/4, 20/7 °C). The germination ability increased with time of dry storage and with GA3, thereby suggesting nondeep physiological dormancy. Under outdoor conditions, no seedlings emerged during the first post-sown autumn, but emergence peaks occurred in late winter-early spring. Both species could form small persistent soil seed banks with short durations (3-5 years). A plant production protocol from seeds was established for both taxa.

Intradetrusor OnabotuliniumtoxinA injection for refractory bladder spasms before vesicovaginal fistula repair.

This is a report of a women who failed 2 surgical repairs of vesicovaginal fistula secondary to refractory bladder spasms. After each surgical procedure - the hysterectomy and subsequent fistula repairs - the patient reported severe bladder spasms refractory to medical management. Our treatment was intradetrusor onabotuliniumtoxinA injections 4 weeks prior to a planned surgical fistula repair. The patient had successful vaginal approach fistula repair and has not required any subsequent overactive bladder (OAB) treatment.

Redirecting tropane alkaloid metabolism reveals pyrrolidine alkaloid diversity in Atropa belladonna.

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna / 药学学报

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Indian traditional medicinal plants in ophthalmic diseases.

Objective: Traditional herbal plants have been in use since ancient times to treat ophthalmic conditions; so, the aim of this study is to evaluate some potent Indian traditional medicinal plants used in ophthalmic diseases in order to summarize their potential effect in ophthalmology along with their mechanism of action. Materials and Methods: Databases PubMed, Google Scholar, and Embase were extensively explored. Additionally, relevant textbooks and literatures were consulted to summarize most of the considerable scientific literature for the review. Search term included ophthalmology, glaucoma, cataract, trachoma, conjunctivitis, traditional medicines, Unani drugs, and ayurvedic drugs were used. Around 80 review articles were consulted from the year 1982 to 2021. Results: The traditional medicinal plants are easily available, cost-effective and have no associated side effects in comparison to current conventional treatments. Moreover, these drugs in oppose to modern medicine, have an inherent potential to accelerate the body's own immunity to fight against any infection. A large volume of scientific studies has reported the beneficial effects of traditional drugs in ophthalmology. Conclusion: This review, therefore, describes the potential benefits and uses of some traditional medicinal plants used in ophthalmic diseases.

Ornithine decarboxylase regulates putrescine-related metabolism and pollen development in Atropa belladonna.

Polyamines, including putrescine, spermidine, and spermine, play critical roles in cell physiology by different forms. As a rate-limiting enzyme that converts ornithine to putrescine, ornithine decarboxylase (ODC, EC 1.1.1.37) has been studied in detail in animals and microorganisms, but its specific functions are poorly understood in plants. In this study, the metabolic and developmental roles of the ODC gene were studied through RNAi-mediated suppression of the ODC gene (AbODC) in A. belladonna. Suppression of AbODC reduced the production of precursors of medicinal tropane alkaloids, including putrescine and N-methylputrescine, as well as hyoscyamine and scopolamine. In AbODC-RNAi roots, the production of putrescine and spermidine in free form was reduced, but in the AbODC-RNAi leaves, the content of free polyamines was not altered. In the roots/leaves of AbODC-RNAi plants, the production of conjugated and bound polyamines was reduced. In addition, suppression of the ODC gene resulted in reduction of polyamines and pollen sterility in AbODC-RNAi flowers. In floral organs, GUS-staining results indicated that AbODC was domainantly expressed in pollen. In summary, ornithine decarboxylase not only plays a key role in regulating the biosynthesis of diverse forms of polyamines and medicinal tropane alkaloids, but also participates in pollen development.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Antibacterial, cytotoxicity and biodegradability studies of polycaprolactone nanofibers holding green synthesized Ag nanoparticles using atropa belladonna extract.

Atropa belladonna is one of the herbs used to treat wounds and prevent inflammation. This study provides a scientific assessment for the wound healing potential of biodegradable nanofibers containing Ag nanoparticles encapsulated with atropa belladonna extract (eAgNPs). Ultraviolet-visible (UV-vis) spectroscopy was used to observe the localized surface plasmon resonance (LSPR) band of AgNPs synthesized from atropa belladonna extract prepared under different conditions. Polycaprolactone (PCL) nanofibers with eAgNPs were fabricated using the electrospinning technique. The distribution of AgNPs and eAgNPs and the size of nanofibers were characterized with scanning and transmission electron microscopy (SEM, TEM) before and after degradation at the end of 18 weeks. Fourier transform infrared (FTIR) spectroscopy showed the surface interactivity between AgNPs, atropa belladonna extract and PCL nanofibers and also approved the modification of PCL nanofibers with eAgNPs. X-ray diffraction analysis (XRD) defined the formation of the crystalline AgNPs and appreciated the orientation of the nanofibers. Results of tension tests revealed that modification of PCL nanofibers with pure AgNPs and eAgNPs significantly increased strength and tensile modulus. Due to the hydrophobic nature of PCL, modification with pure AgNPs and eAgNPs slightly reduced its hydrophobicity. Biodegradation tests of PCL nanofibers with eAgNPs exhibited a higher degradation rate than neat PCL nanofibers. In vitro MTT results revealed that eAgNPs doped PCL samples have better cell viability than AgNPs doped and neat PCL nanofibers. Owing to their antibacterial properties, biodegradation rates, low cytotoxicity, mechanical and surface morphologic properties of AgNPs modified PCL nanofibers containing atropa belladonna are considered to have a great potential for skin regeneration.

The pattern of coding sequences in the chloroplast genome of Atropa belladonna and a comparative analysis with other related genomes in the nightshade family.

Atropa belladonna is a valuable medicinal plant and a commercial source of tropane alkaloids, which are frequently utilized in therapeutic practice. In this study, bioinformaticmethodologies were used to examine the pattern of coding sequences and the factors thatmight influence codon usage bias in the chloroplast genome of Atropa belladonna andother nightshade genomes. The chloroplast engineering being a promising field in modernbiotechnology, the characterization of chloroplast genome is very important. The resultsrevealed that the chloroplast genomes of Nicotiana tabacum, Solanum lycopersicum, Capsicum frutescens, Datura stramonium, Lyciumbarbarum, Solanum melongena, and Solanumtuberosum exhibited comparable codon usage patterns. In these chloroplast genomes, weobserved a weak codon usage bias. According to the correspondence analysis, the genesisof the codon use bias in these chloroplast genes might be explained by natural selection,directed mutational pressure, and other factors. GC12 and GC3S were shown to have nomeaningful relationship. Further research revealed that natural selection primarily shapedthe codon usage in A. belladonna and other nightshade genomes for translational efficiency. The sequencing properties of these chloroplast genomes were also investigated by investing the occurrences of palindromes and inverted repeats, which would be useful forfuture research on medicinal plants.

Acute angle closure glaucoma precipitated by homeopathic eyedrops containing Atropa belladonna.

Acute angle closure glaucoma is a sight-threatening condition that may lead to blindness. This is a case report of a woman who presented to the emergency department (ED) with acute angle closure glaucoma following use of an over-the-counter (OTC) homeopathic eye drop containing atropa belladonna (deadly nightshade). A 55-year-old woman presented to the ED with a 5-day history of left eye redness, swelling, tearing, and foreign-body sensation that had acutely worsened in the last two days. Her exam revealed mild left conjunctival injection with watery tearing and a hazy appearance of her left cornea. Fluorescein staining was negative, while tonometry revealed elevated intraocular pressure on the left, suggestive of acute angle closure glaucoma. She was urgently referred to ophthalmology. The etiology of the acute angle closure glaucoma was initially unclear however, with additional prompting, she revealed that two days prior she had started using homeopathic OTC eye drops. Inspection of the eyedrop's ingredients revealed that atropa belladonna was the primary ingredient and likely precipitated her isolated episode of acute angle closure glaucoma. A high level of clinical suspicion and focused ophthalmic exam including tonometry is essential to identify acute angle closure glaucoma in the ED. We present a case report of acute angle closure glaucoma associated with the use of homeopathic belladonna-containing eyedrops. Our report reinforces the necessity to perform thorough medication and supplement history given the prevalence of physiologically active substances available in OTC medications.

Anticholinergic syndrome after atropine overdose in a supposedly homeopathic solution: a case report.

BACKGROUND: A 53-year-old male with no pre-existing conditions and no permanent medication presented to our emergency department with an anticholinergic syndrome including confusion, anxiety, ataxia and dysarthria after ingestion of a homeopathic solution containing Atropa belladonna extract supposedly in a D4 dilution. METHODS: Atropine sulphate was quantitatively analysed in serum and the homeopathic preparation via liquid chromatography/mass spectrometry. RESULTS: Analysis revealed concentrations of approximately 3 mg/mL atropine sulphate in the homeopathic solution and a serum level of 5.7 ng/mL (±1.4) in the patient's blood proving a 600-fold overdose of atropine due to a production error of the homeopathic dilution. The patient was observed and recovered without further intervention. CONCLUSION: Rare but possibly dangerous manufacturing errors should be considered when faced with symptoms occurring after ingestion of homeopathic or holistic remedies.

Effect of the rolC gene on biosynthesis of tropane alkaloids in Atropa belladonna / 药学学报

Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants, and the enhanced metabolic capacity is driven by the rol gene. We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids (TAs) in Atropa belladonna. In this study, the rolC gene from Agrobacterium rhizogene pRiA4 plasmid, driven by a CaMV35S promoter, was overexpressed in A. belladonna. The phenotypes, TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A. belladonna plants. Results show that transgenic A. belladonna exhibited a well-developed root system, male sterility, higher stamen column length than pistil, early flowering, internode shortening, smaller but more flowers, increased axillary buds and lateral buds, decreased apical dominance, and long and narrow leaves as compared to wild-type plants. Transgenic A. belladonna produced more TAs than wild-type plants, with the content of hyoscyamine, anisodamine and scopolamine reaching 2.58, 3.59 and 15.77-fold that of the control group, respectively. The gene expression of putrescine N-methyltransferase (PMT), tropinone reductase I (TRⅠ) and hyoscyamine 6-β-hydroxylase (H6H), key enzymes in TAs biosynthesis, were up-regulated compared with the control group. The above results indicate that the rolC gene enhances TAs biosynthesis in A. belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway, laying a foundation for genetic manipulation of A. belladonna to increase TAs content by increasing rolC gene expression.

CRISPR/Cas9-mediated disruption of the PYRROLIDINE KETIDE SYNTHASE gene reduces the accumulation of tropane alkaloids in Atropa belladonna hairy roots.

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

A new synthetic biology approach for the production of curcumin and its glucoside in Atropa belladonna hairy roots.

Curcumin has ignited global interest as an elite drugable molecule, owing to its time-honoured pharmacological activities against diverse human ailments. Limited natural accessibility and poor oral bioavailability caused major hurdles in the curcumin-based drug development process. We report the first successful testimony of curcumin and its glucoside synthesis in Atropa belladonna hairy roots (HR) through metabolic engineering. Re-routing the inherent biosynthetic precursors of the phenylpropanoid pathway of A. belladonna by heterologous expression of key curcumin biosynthetic pathway genes (i.e., Diketide-CoA synthase-DCS and Curcumin synthase-CURS3) and glucosyltransferase gene (CaUGT2) resulted in the production of curcumin and its glucoside in HR clones. Under shake-flask cultivation, the PGD2-HR1clone bearing DCS/ CURS3 genes showed the maximum curcumin yield (180.62 ± 4.7 µg/g DW), while the highest content of curcumin monoglucoside (32.63 ± 2.27 µg/g DW) along with curcumin (67.89 ± 2.56 µg/g DW) were noted in the PGD3-HR3 clone co-expressing DCS/CURS3 and CaUGT2 genes. Bioreactor up-scaling showed yield improvements in the PGD2-HR1 (2.3 fold curcumin) and the PGD3-HR3 clone (0.9 and 1.65 folds of curcumin-monoglucoside and curcumin respectively). These findings proved the advantageous use of HR cultures as the production source for curcumin and its glucoside, which remained unexplored so far.

Atropa belladonna (Deadly Nightshade) Poisoning in Childhood

ABSTRACT Objective: A very small amount of Atropa belladonna (AB) can lead to serious symptoms of poisoning and can cause death in children. In this study, demographic, clinic and laboratory results of AB poisoning were evaluated. Methods: A total of 108 cases with belladonna poisoning were retrospectively evaluated. At time of admission, age, age groups, gender, signs and symptoms caused by poisoning, duration of stay in hospital, laboratory data, intensive care needs, and applied treatments were recorded. Results: Approximately 44.4% were females and 55.6% were males. While the most common symptoms were xeroderma and flushing, the most frequent findings were tachycardia and mydriasis. Eight patients complained about astasis and five of them were taken into the intensive care unit. Astasis complaint was relatively higher (p < 0.01) in the patients who needed intensive care than those who did not. Creatine kinase levels were relatively higher (p = 0.06) in the intensive care patients as compared to non-intensive care patients. Neostigmine was given to all patients. Five patients, who failed to respond to therapy, were taken into the intensive care and respond to treatment successfully with physostigmine. Conclusion: Atropa belladonna poisoning may seriously progress in the act of late diagnosis and treatment in childhood. Thus, it is crucial to realize that AB poisoning should be taken into consideration in the patients with flushing, xeroderma with mydriasis, tachycardia, tremor, abdominal pain, and fever symptoms. Patients with astasis complaints should be evaluated carefully in terms of intensive care need. Patients with a Glasgow Coma Scale lower than 12 should be observed in the intensive care.

Antiviral Activity of Belladonna During Japanese Encephalitis Virus Infection via Inhibition of Microglia Activation and Inflammation Leading to Neuronal Cell Survival.

Japanese encephalitis virus (JEV) is the main cause of viral encephalitis resulting in more than 68 000 clinical cases every year with case fatality rate as high as 30-40% for which no specific treatments are available. We have recently exhibited belladonna may be widely applicable for the treatment of various neurological disorders. Therefore, we developed a hydroalcoholic formulation of belladonna (B200) consisting of atropine and scopolamine and showed its antiviral efficacy against JEV infection. B200 treatment increases neuronal cell survival by reducing JEV induced cytopathic effects which were evident from significant reduction in necrotic cell population by flow-cytometry analysis and caspase 3 and 8 enzymatic activities. B200 treatment was found to reduce the intracellular JEV level observed by significant reduction in JEV-fluorescein isothiocyanate (FITC) expression in both neurons and microglia. Because microglia plays a crucial role in JEV pathogenesis, we further investigated the anti-JEV effects of B200 on human microglia cells and elucidated the mechanism of action by performing whole-transcriptome sequencing. Gene expression analysis revealed that B200 reduces the pro-apoptotic and inflammatory gene expression observed by significant reduction in BAD, BAX, CASP3, CASP8, IL1B, and CXCL10 and increase in IL10 responsive gene expression. Interestingly, our molecular docking analysis revealed that atropine and scopolamine interact with the His288 residue of NS3 protein, a crucial residue for RNA unwinding and ATPase activity that was further confirmed by degradation of NS3 protein. Drug likeness, ADME (absorption, distribution, metabolism, and excretion), and toxicity analysis further suggests that atropine and scopolamine both cross the blood-brain barrier, which is crucial for effective treatment of Japanese encephalitis (JE).