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.

A comparative evaluation of neuroprotective activity of perindopril and valsartan in rats

Background: Angiotensin II receptor blockers and Angiotensin-converting enzyme inhibitors are widely used drugs for cardiovascular disorders, renal disease, and diabetes. Recently, they have been recognized for neuroprotective activity and are used in many brain disorders. Aim and Objective: The present study was done to explore effects of perindopril and valsartan on experimentally induced learning and memory impairment in Wistar rats. Materials and Methods: 40 Wistar rats were divided into 5 groups, eight rats in each group, namely normal control, disease control, positive control (Piracetam 600 mg/kg), test group I (Perindopril 4 mg/kg), and test group II (Valsartan 15 mg/kg). Except normal control group, all animals received intraperitoneal injection of Scopolamine 1 mg/kg for 21 days to induce memory impairment. Piracetam and Test drugs were administered once daily orally for 21 consecutive days. On day 0, 7th, 14th, and 21st of the experiment, muscle grip strength (Wire hanging grip test) and memory functions Elevated plus maze (EPM) of all the animals were assessed. On 8th, 15th, and 22nd day of the experiment, retention memory functions (EPM) were assessed. Results: Animals treated with Scopolamine showed significant reduction in grip strength and significant rise in transfer latency (TL) (EPM model). Rats treated with piracetam and test drugs showed significant increase in grip strength. The animals treated with piracetam and test drugs showed significant reduction in TL (EPM model) when compared with disease control group. Similar results were seen in retention memory test. Conclusion: Perindopril and valsartan demonstrated neuroprotective effect in scopolamine-induced memory impairment in rats. Memory improvement by these test drugs was comparable with positive control piracetam.

Improvement on Quality Standard of Belladonnae Herba / 中国实验方剂学杂志

ObjectiveTo improve the current standard of Belladonnae Herba in the 2020 edition of Chinese Pharmacopoeia. MethodTaking hyoscyamine sulfate， atropine sulfate and scopoletin as reference substances， and ethyl acetate-methanol-concentrated ammonia（17∶4∶2）as developing solvent， thin layer chromatography （TLC） was applied in the qualitative identification of Belladonnae Herba. The moisture， total ash and ethanol-soluble extract of Belladonnae Herba were determined based on the general principles in the 2020 edition of Chinese Pharmacopoeia （volume Ⅳ）. The contents of hyoscyamine sulfate and scopolamine hydrobromide were analyzed by high performance liquid chromatography （HPLC） with mobile phase of acetonitrile-54 mmol·L-1 phosphate buffer solution （14∶86）， flow rate of 1.0 mL·min-1 and detection wavelength at 210 nm. ResultThe spots in the TLC were clear with good separation and specificity. Hyoscyamine sulfate and scopolamine hydrobromide showed a good linearity with peak area in the range of 0.024 7-0.789 6 g·L-1 （r=0.999 9） and 0.003 9-0.124 0 g·L-1 （r=0.999 9）， the average recoveries of these two ingredients were 100.29% （RSD 1.6%） and 99.04% （RSD 1.4%）， respectively. The limits for moisture， total ash in Belladonnae Herba should be less than 13.0% and the limit for the ethanol-soluble extract should be more than 10.0%. Due to the low content and wide variation of scopolamine hydrobromide， the content of hyoscyamine sulfate should not be less than 0.098%. ConclusionThe established method is simple， specific and reproducible， which can be used to improve the quality control standard of Belladonnae Herba.

Effects and mechanism of the ethanol extract of Tiarella polyphylla on learning and memory impairment in mice / 中国药房

OBJECTIV E To investigate the effect s and mechanism of the ethanol extract of Tiarella polyphylla （“TPE”）on learning and memory impairment in mice. METHODS Male Kunming mice were randomly divided into normal group ，model group，positive group （donepezil hydrochloride 4 mg/kg）and TPE low-dose ，medium-dose and high-dose groups （150，300，600 mg/kg），with 10 mice in each group. Drug administration groups were given relevant medicine intragastrically once a day ，and normal group and model group were given water intragastrically once a day ，for consecutive 22 d. On the 17th day ，administration groups and model group were intraperitoneally injected with scopolamine hydrobromide （3 mg/kg）to establish a model of learning and memory impairment. The learning and memory ability of the mice were evaluated by the Morris water maze. Hematoxylin-eosin （HE） staining was used for morphological observation of hippocampus cells of the mice. The levels of acetylcholinesterase （AChE），choline acetyltransferase （ChAT），superoxide dismutase （SOD），malondialdehyde（MDA），interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in cerebral tissue as well as the relative expression of phosphorylated Tau protein （p-Tau），β-site amyloid precursor protein cleaving enzyme 1（BACE1）and amyloid precursor protein （APP）in hippocampus tissue were all detected. RESULTS The escape latency of mice in positive group ，TPE medium-dose and high-dose groups were all significantly shortened than the model group on the 4th to 5th day of training ，while the times of crossing platform and the percentage of movement distance in target quadrant were significantly increased （P＜0.05）. Compared with model group ，the neurons in the hippocampal CA 1 region of mice were increased to var ying degrees in administration groups ，the ne urons in solidified and atrophic state decreased ，and the arrangement of neurons tended to be close；the levels of ChAT and SOD in cerebral tissue were significantly increased in positive group and TPE medium-dose and high-dose groups ；the levels of AChE ，MDA，IL-6，the levels of TNF-α and relative expression of p-Tau ，BACE1 and APP in hippocampus tissue were decreased significantly （P＜ 0.05）. CONCLUSIONS TPE can improve the learning and memory impairment induced by scopolamine in mice ，and the mechanism may be related to balancing the brain cholinergic system ，alleviating oxidative stress injury ，improving inflammatory response，and inhibiting the overexpression of p-Tau ，BACE1 and APP .

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.

Mechanism of Tibetan medicine Ershiwuwei Shanhu Pills on scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway / 中国中药杂志

This study aims to investigate the mechanism of the Tibetan medicine Ershiwuwei Shanhu Pills(ESP) in improving scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway. ICR mice were randomized into blank group, model group, low-dose(200 mg·kg~(-1)), medium-dose(400 mg·kg~(-1)), and high-dose(800 mg·kg~(-1)) ESP groups, and donepezil hydrochloride group. The learning and memory impairment was induced in mice by intraperitoneal injection of scopola-mine. The learning and memory abilities of mice were detected by Morris water maze test, and the damage of hippocampal neurons and cortical neurons was detected based on Nissl staining. The expression of neuron specific nuclear protein(NeuN) in hippocampus and cortex of mice was determined by immunofluorescence assay, and the content of acetylcholine(Ach) and the activity of acetylcholines-terase(AchE) in hippocampus of mice by kits. Moreover, the content of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in serum of mice was detected. The content of Kelch-like ECH-associated protein 1(Keap1), nuclear factor erythroid 2-related factor 2(Nrf2), and heme oxygenase 1(HO-1) in hippocampus was determined by Western blot. The results showed that there were significant differences in the trajectory map of mice among different groups in the behavioral experiment. Moreover, the latency of ESP groups decreased significantly compared with that in the model group. The hippocampal neurons in the high-dose ESP group were significantly more than those in the model group and the cortical neurons in the high-dose and medium-dose ESP groups were significantly more than those in the model group. The expression of NeuN in the model group was significantly decreased compared with that in the blank group, and the expression in the ESP groups was significantly higher than that in the model group. The AchE activity and MDA level were significantly decreased, and Ach content and levels of SOD, CAT, and T-AOC in the ESP groups were significantly increased in the ESP groups compared with those in the model group. The expression of Keap1 in the model group was significantly increased compared with that in the blank group, and the Keap1 expression increased insignificantly in ESP groups compared with that in the model group. The expression of Nrf2 and HO-1 was significantly lower in the model group than in the blank group, and the expression was significantly higher in the medium-dose ESP group than in the model group. In conclusion, ESP protected mice against the scopolamine-induced learning and memory impairment by regulating the Keap1/Nrf2/HO-1 signaling pathway.

Total flavonoids of Drynariae Rhizoma regulates ER-p38 MAPK signaling pathway to improve scopolamine-induced learning and memory impairments in model mice / 中国中药杂志

This study intended to explore the effect and mechanism of total flavonoids of Drynariae Rhizoma in improving scopola-mine-induced learning and memory impairments in model mice. Ninety four-month-old Kunming(KM) mice were randomly divided into six groups. The ones in the model group and blank group were treated with intragastric administration of normal saline, while those in the medication groups separately received the total flavonoids of Drynariae Rhizoma, Kangnaoshuai Capsules, donepezil, as well as total flavonoids of Rhizoma Drynariae plus estrogen receptor(ER) blocker by gavage. The mouse model of learning and memory impairments was established via intraperitoneal injection of scopolamine. Following the measurement of mouse learning and memory abilities in Morris water maze test, the hippocampal ERβ expression was detected by immunohistochemistry, and the expression levels of ERβ and phosphorylated p38(p-p38) in the hippocampus and B-cell lymphoma 2(Bcl-2), Bcl-2-associated death promoter(Bad), and cysteinyl aspartate-specific protease-3(caspase-3) in the apoptotic system were assayed by Western blot. The contents of malondia-ldehyde(MDA), superoxide dismutase(SOD), and nitric oxide(NO) in the hippocampus were then determined using corresponding kits. Compared with the control group, the model group exhibited significantly prolonged incubation period, reduced frequency of cros-sing the platform, shortened residence time in the target quadrant, lowered ERβ, Bcl-2 and SOD activity in the hippocampus, and increased p-p38/p38, Bad, caspase-3, MDA, and NO. Compared with the model group, the total flavonoids of Rhizoma Drynariae increased the expression of ERβ and SOD in the hippocampus, down-regulated the expression of neuronal pro-apoptotic proteins, up-re-gulated the expression of anti-apoptotic proteins, and reduced p-p38/p38, MDA, and NO. The effects of total flavonoids of Drynariae Rhizoma on the above indexes were reversed by ER blocker. It has been proved that the total flavonoids of Drynariae Rhizoma obviously alleviate scopolamine-induced learning and memory impairments in mice, which may be achieved by regulating the neuronal apoptotic system and oxidative stress via the ER-p38 mitogen-activated protein kinase(ER-p38 MAPK) signaling pathway.

Cloning and functional analysis of AbCYP80F1 gene in Atropa belladonna / 中草药

Objective: To clone AbCYP80F1 gene and its promoter, analyze the Cis-acting elements related to control signals and transcription factors in AbCYP80F1 gene promoter and study the effect of AbCYP80F1 overexpression on scopolamine accumulation in Atropa belladonna. Methods: CYP80F1cDNA from Hyoscyamus niger was used as query sequence to do blastn search in A. belladonna transcriptome database to retrieve homologous unigenes. Full-length of AbCYP80F1 cDNA and gene promoter were acquired by RACE and hiTAIL-PCR, respectively. Agrobacterium rhizogenes-dipping method was adopted to induce AbCYP80F1- overexpressed A. belladonna hairy roots, and scopolamine content in hairy roots was detected by HPLC. Results: A full-length of 1 675 bp of AbCYP80F1 gene was obtained and the deduced protein had a closed homology with CYP80F1 from Anisodus luridus. A length of 2 000 bp promoter contained Cis-acting elements related to light, anaerobic induction, auxin, MeJA, gibberellins and low temperature, as well as recognition sites of transcription factor of MYB, AP2/ERF, WRKY and bHLH. Overexpression of AbCYP80F1 gene increased the scopolamine content in hairy roots of A. belladonna by an average of 1.8 times. Conclusion: AbCYP80F1 is a secondary root-specific gene in scopolamine biosynthetic pathway and its overexpression can enhance scopolamine accumulation. Expression of AbCYP80F1 may be regulated by WRKY and bHLH transcription factors.

Efecto de Petiveria alliacea y drogas colinérgicas sobre la habituación / Effects of Petiveria alliacea and cholinergic drugs on the habituation cognitive behavior

Petiveria alliacea (PA) have anxiolytic, antidepressant and cognitive effects. In the present paper the effect of PA water infusion and cholinergic drugs on cognitive behavior were studied. For that, 40 male NMRI mice were divided in 4 groups: Control (n=10), Drug Control (n=10), PA (n=10) and PA plus Drug (n=10). PA 1% was administered orally (7.59±1.39 ml/day); while scopolamine (2 mg/Kg), galantamine (1 mg/Kg) and nicotine (0.1 mg/Kg) were administered intraperitoneally. Behavioral tests included: anxiety maze (AM), open field (OF) and marble burying (MB). Habituation cognitive behavior was evaluated in 4 sessions, one week each session. PA had anxiolytic and antidepressant effect effect in AM, combined with nicotine potentiated an anxiogenic effect in AM, galantamine favored habituation in OF. Scopolamine potentiated the habituation in LA and decreased the obsessive-compulsive behavior in OF. In conclusion; PA had an anxiolytic effect and favored deshabituation, combined with nicotine induced an anxiogenic effect, galantamine favored habituation and scopolamine decreased obsessive-compulsive behavior and favored motor habituation indicated a possible anxiolytic effect.

La Petiveria alliacea (PA) está relacionada con efectos ansiolíticos, antidepresivos y cognitivos. El presente trabajo estudió el efecto de la infusión de PA y drogas colinérgicas sobre la habituación. 40 ratones NMRI machos fueron divididos en 4 grupos: Control (n=10), Control Drogas (n=10), PA (n=10) y PA plus Drogas (n=10). La PA (1%) fue administrada vía oral (7.59±1.39 ml/día); escopolamina (2 mg/Kg), galantamina (1 mg/Kg) y nicotina (0.1 mg/Kg) fueron administrados vía intraperitoneal. Los ensayos conductuales incluyeron: laberinto de ansiedad (LA), campo abierto (CA) y enterramiento aversivo (EA). La habituación fue evaluada en 4 sesiones con duración de una semana cada una. PA mostró un efecto ansiolítico en el LA, combinada con nicotina potenció un efecto ansiogénico en el LA. Galantamina favoreció la habituación en CA, y escopolamina potenció el fenómeno de habituación en LA y disminuyó la conducta obsesivo-compulsiva en CA. En conclusión, la PA mostró un efecto ansiolítico y antidepresivo que potencia la deshabituación, combinada con nicotina indujo un efecto ansiogénico, galantamina favoreció la habituación y escopolamina disminuyó la conducta obsesivo­ compulsiva y favoreció la habituación motora indicando un posible efecto ansiolítico.

Modafinil and Learning and Memory- Is there a Connection? Evaluation of the Effect of Modafinil on Learning and Memory in Wistar Rats

Background: Modafinil, a drug approved for use innarcolepsy, has shown conflicting effects on cognition.This study was conducted to observe the effects ofModafinil on learning and memory following acute andchronic administration in Wistar rats. Aim andObjectives: To observe the effects of Modafinil onlearning and memory following acute and chronicadministration. Material and Methods: The studyconducted in 42 male Wistar rats, had seven groups:Group I: Control, Group II: Negative Control(Vehicle), Group III: Standard Control (Donepezil),Group IV: Chronic Modafinil 10 mg/kg, Group V:Chronic Modafinil 20 mg/kg, Group VI: AcuteModafinil 10 mg/kg, Group VII: Acute Modafinil 10mg/kg. All drugs were administered for 15 days.th Scopolamine was used to induce amnesia on the 15day in all groups except Group I. Using the HebbWilliam maze, baseline learning score was recorded onday 1, and post-treatment learning scores wererecorded on days 15 and 16. Results: On days 15 and16, the learning scores significantly decreased in GroupI, while it significantly increased in group II, comparedto baseline, indicating induction of amnesia byscopolamine. In Group III the learning scores on days15 and 16 (8.66 ± 2.63, 9.66 ± 2.75, in seconds) weredecreased significantly compared to baseline (18.83 ±2.65), indicating a reversal of scopolamine-inducedamnesia. All doses of Modafinil (Acute 10 mg and 20mg/kg, Chronic 10 mg and 20 mg/kg) showed astatistically significant increase in learning scores ondays 15 and 16, compared to baseline, indicating noreversal of scopolamine-induced amnesia. Conclusion:Modafinil in doses of 10 mg/kg and 20 mg/kg, giveneither as a single dose or over a period of time, does notreverse amnesia induced by scopolamine in rats.

Study on the Improvement Effects of Lanqian Buccal Tablet on Scopolamine-induced Learning and Memory Impairment of Mice / 中国药房

OBJECTIVE： To study the improvement effects of Lanqian buccal tablets on scopolamine-induced learning and memory impairment of mice. METHODS： The mice were randomly divided into blank group （normal saline）， model group （normal saline）， positive group （Donepezil hydrochloride tablets， 1.52 mg/kg） and Lanqian buccal tablets high-dose， medium-dose and low-dose groups （800， 400， 200 mg/kg）， with 14 mice in each group. Once a day， 30 days after continuous intragastric administration， except for blank group， other groups were intraperitioneal injected scopolamine hydrobromide 3 mg/kg to induce learning and memory impairment model. After modeling， the learning and memory ability of mice were evaluated with step through test （latency， mistake times of entering darkroom as indexes） and Morris water maze （the time of going up on the platform， the times of crossing the platform， swimming time in target quadrant as indexes）. The levels of Ach， ChAT， AchE， SOD and MDA in cerebral tissue of mice were determined. RESULTS： Compared with blank group， latency， the times of crossing the platform， swimming time in target quadrant， the levels of ChAT， Ach and SOD were shortened or decreased significantly in model group （P＜0.05 or P＜0.01）， while mistake times of entering darkroom， the time of going up on the platform， the levels of AchE and MDA were extended or rised significantly （P＜0.01）. Compared with model group， latency （except for Lanqian buccal tablet low-dose group）， the times of crossing the platform， swimming time in target quadrant， the levels of ChAT， Ach and SOD were extended or rised significantly in positive group and Lanqian buccal tablet groups （P＜0.05 or P＜0.01）. The mistake times of entering darkroom （except for Lanqian buccal tablet low-dose group）， the time of going up on the platform （except for Lanqian buccal tablet low-dose group） and the levels of AchE and MDA （except for Lanqian buccal tablet medium-dose and low-dose group） were shortened or decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Lanqian buccal tablet can improve scopolamine-induced learning and memory impairment of mice by increasing the levels of ChAT， Ach and SOD and decreasing the levels of AchE and MDA.

Effect of scopolamine butylbromide injection combined with tamsulosin oral auxiliary extracorporeal shock wave lithotripsy in the treatment of urinary calculi and influence on serum inflammatory factors / 中国基层医药

Objective To study the effect of scopolamine butylbromide injection combined with tamsulosin oral auxiliary extracorporeal shock wave lithotripsy(ESWL) in the treatment of urinary calculi,and to investigate its effect on serum inflammatory factors.Methods From January 2016 to May 2017,96 patients with urinary calculi in the People's Hospital of Lanxi were selected and randomly divided into control group(n =42) and observation group(n =54).The control group received scopolamine butylbromide assisted ESWL treatment,the observation group treated with tamsulosin on the basis of the control group.The VAS score,clinical efficacy,adverse reactions and serum C reactive protein(CRP),tumor necrosis factor alpha(TNF-alpha) and other inflammatory factors were compared between the two groups.Results Before treatment,no statistically significant difference in VAS score was found between the two groups (t =0.274,P ＞ 0.05).After treatment,the VAS score of the observation group was (2.21 ± 0.28) points,which was significantly lower than that of the control group[(4.75 ± 0.63) points] (t =7.935,P ＜ 0.05).The stone clearance rate in the observation group was 96.30％ (52/54),which was significantly higher than that in the control group[71.43％ (30/42)] (x2 =6.712,P ＜ 0.05).The time of disappearance of pain in the observation group was (10.86 ± 1.54) d,which was significantly lower than that in the control group [(7.02 ± 1.25) d] (t =7.468,P ＜0.05).The lithecbole time of the observation group was (9.01 ± 1.53) h,which was significantly shorter than that of the control group[(13.25 ± 2.21) h] (t =8.035,P ＜ 0.05).Before treatment,there were no statistically significant differences in serum CRP and TNF-levels between the two groups (t =0.152,0.185,all P ＞0.05).After treatment,the serum level of CRP in the observation group was (11.64 ± 1.75) mg/L,which was significantly lower than that in the control group [(18.94 ± 2.03) mg/L] (t =7.008,P ＜ 0.05).After treatment,the serum TNF-level in the observation group was (13.95 ± 2.01)mg/L,which was significantly lower than that in the control group[(20.76 ±3.18) mg/L] (t =7.008,P ＜0.05).The incidence rate of adverse reactions of the observation group was 5.56％,which was significantly lower than 26.19％ of the control group (x2 =9.572,P ＜ 0.05).Conclusion Urinary calculi underwent intravenous infusion of scopolamine butylbromide combined with tamsulosin oral adjuvant ESWL therapy,can effectively reduce the pain of patients,reduce the serum levels of inflammatory cytokines,improve the stone clearance rate,and with less complications,it is worthy of clinical popularization and application.

Fagodisuasión de extractos deBrugmansia candida(Solanaceae) en larvas deHypsipyla grandella(Lepidoptera: Pyralidae) / Phagodeterrence of extracts ofBrugmansia candida(Solanaceae) onHypsipyla grandella(Lepidoptera: Pyralidae) larvae

Resumen Hypsipyla grandellaes una plaga forestal clave en América Latina y el Caribe, donde ha impedido todo intento de establecer plantaciones comerciales de caobas (Swieteniaspp.) y cedros (Cedrelaspp.). En la búsqueda de métodos de manejo de sus larvas, que sean preventivos y rentables, así como basados en recursos de la flora tropical, fue evaluada la actividad fagodisuasiva del extracto crudo y de cuatro particiones fitoquímicas (agua, hexano, diclorometano y acetato de etilo) de la flor de reina de la noche,Brugmansia candida(Solanaceae). Se efectuaron bioensayos de laboratorio con concentraciones crecientes del extracto crudo (0.1; 0.3; 1.0; 3.2 y 10.0 % m/v) y con cada una de las particiones (según el rendimiento del proceso de particionamiento). Se utilizó un diseño de bloques completos al azar, con cuatro repeticiones, y se expusieron larvas de instar III de H. grandellaa discos de cedro amargo (Cedrela odorata) impregnados con el respectivo tratamiento, por 24 h. Se midió el porcentaje de consumo de cada disco. El extracto crudo y la partición de hexano, mostraron un evidente efecto fagodisuasivo a partir de concentraciones de 1.0 % y 0.122 % m/v, respectivamente. También se realizó un bioensayo para comparar una sola concentración (0.38 % m/v) del alcaloide escopolamina, el cual redujo en 90% el consumo con respecto a los testigos. Asimismo, se evaluó la mortalidad y los efectos subletales en larvas expuestas al extracto crudo, la partición de hexano y la escopolamina, pero no hubo evidencias de ellos, lo que confirmó su acción fagodisuasiva. Además, se realizó un tamizaje fitoquímico del extracto crudo, y mediante pruebas cualitativas se determinó que los alcaloides, taninos, triterpenos y cumarinas fueron los metabolitos secundarios más importantes. Finalmente, se confirmó la presencia de la escopolamina tanto en el extracto crudo como en la partición de hexano, mediante el método de cromatografía de gases acoplado a un espectrómetro de masas (GC-MS). Se recomienda preparar una formulación de escopolamina más algunos coadyuvantes, y evaluar su eficacia en condiciones de invernadero, como un primer paso para avanzar en el desarrollo de un fagodisuasivo comercial.

Abstract Hypsipyla grandella is a key forest pest in Latin America and the Caribbean, where it has precluded attempts to establish commercial plantations of mahoganies (Swieteniaspp.) and cedars (Cedrelaspp.). In the search for methods to manage its larvae, being both preventative and cost-effective, as well as based upon resources from tropical flora, the crude flower extract of angel's trumpet, Brugmansia candida, as well as four partitions thereof (water, hexane, dichlorometane, and ethyl acetate), were tested for phagodeterrence. Laboratory bioassays involved increasing concentrations of the crude extract (0.1, 0.3, 1.0, 3.2 and 10.0 % w/v) as well as each one of the partitions (in accordance to the yield obtained from the partitioning process). A randomized complete block design, with four replicates, was used. H. grandellainstar III larvae were exposed for 24 h to Spanish cedar (Cedrela odorata) leaf discs dipped into the respective treatment, after which disc consumption was measured. Strong phagodeterrence was detected at concentrations as low as 1.0 % y 0.122 % w/v for the crude extract and the hexane partitions, respectively. In addition, a single bioassay was performed to compare a single concentration (0.38 % w/v) of the alkaloid scopolamine, which reduced consumption in 90 % with respect to the controls. Also, mortality and sublethal effects were assessed in larvae exposed to the crude extract, the hexane partition and scopolamine, with no evidence for them, which confirmed their phagodeterrence. Moreover, the crude extract was submitted to a phytochemical screening by means of a number of qualitative tests, which showed that alkaloids, tannins, triterpenes and cumarins were the most important secondary metabolites. Finally, the presence of scopolamine was confirmed in both the crude extract and the hexane partition, by means of the gas chromatography-spectrometry (GC-MS) analytical method. It is recommended to prepare a formulation of scopolamine plus some adjuvants, in order to test its effectiveness under greenhouse conditions, as a first step to advance in the development of a commercial phagodeterrent. Rev. Biol. Trop. 66(1): 58-69. Epub 2018 March 01.

Comparison of scopolamine-induced cognitive impairment responses in three different ICR stocks / 한국실험동물학회지

Cognitive impairment responses are important research topics in the study of degenerative brain diseases as well as in understanding of human mental activities. To compare response to scopolamine (SPL)-induced cognitive impairment, we measured altered parameters for learning and memory ability, inflammatory response, oxidative stress, cholinergic dysfunction and neuronal cell damages, in Korl:ICR stock and two commercial breeder stocks (A:ICR and B:ICR) after relevant SPL exposure. In the water maze test, Korl:ICR showed no significant difference in SPL-induced learning and memory impairment compared to the two different ICRs, although escape latency was increased after SPL exposure. Although behavioral assessment using the manual avoidance test revealed reduced latency in all ICR mice after SPL treatment as compared to Vehicle, no differences were observed between the three ICR stocks. To determine cholinergic dysfunction induction by SPL exposure, activity of acetylcholinesterase (AChE) assessed in the three ICR stocks revealed no difference of acetylcholinesterase activity. Furthermore, low levels of superoxide dismutase (SOD) activity and high levels of inflammatory cytokines in SPL-treated group were maintained in all three ICR stocks, although some variations were observed between the SPLtreated groups. Neuronal cell damages induced by SPL showed similar response in all three ICR stocks, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Nissl staining analysis and expression analyses of apoptosis-related proteins. Thus, the results of this study provide strong evidence that Korl:ICR is similar to the other two ICR. Stocks in response to learning and memory capacity.

HPLC fingerprint of Hyoscyamus Extract Tablets and quantitative analysis of tropane alkaloids / 中草药

Objective: To establish a fingerprint of Hyoscyamus Extract Tablets (HET) by high performance liquid chromatography and determine the content of atropine, anisodamine, scopolamine, and anisodine. Methods: The separation was performed on a GL Sciences Inc C18 (250 mm × 4.6 mm, 5 μm) column with gradient elution of methanol and 0.05% phosphoric acid solution. The detection wavelength was set at 218 nm, the flow rate was 1.0 mL/min, and the column temperature was 30℃. Similarity evaluation system for chromatographic fingerprint of TCM was used for establishing the reference fingerprint and similarity evaluation, and clustering analysis was carried out by SPSS 19.0. Results: There were 14 marked common peaks, and the similarity of S1-S4, S11 and the control fingerprint was over 0.98, others were above 0.995. It was shown that these 12 batches of samples were of good similarity. Cluster analysis results showed that the S1-S6 were grouped into one class, and the remaining samples were grouped into one class. The results of assay showed that alkaloids in different batches of HET varied a lot in contents but kept consistent in compositions. Conclusion: The method could well display the chemical information of HET. It has simple operation and good repeatability, which provides more comprehensive and scientific data for quality evaluation of HET.

Nacre extract prevents scopolamine-induced memory deficits in rodents

Objective:To investigate whether the extract from the nacreous layer of pearl oysters (nacre extract) improves impairments in memory caused by scopolamine administration in rodents.Methods:Nacre extract was prepared from the inner shell layer of pearl oyster. Effects of nacre extract on scopolamine-induced memory impairment were estimated using novel object recognition test, Y-maze test, and Barnes maze test. Effect of nacre extract on mRNA expressions which are genes associated with memory in the hippocampus was investigated using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis.Results:Administration of nacre extract led to the protection against scopolamine-induced impairments in object recognition, short-term memory, and spatial memory. Treatment with nacre extract reversed the mRNA expression of brain-derived neurotrophic factor (BDNF) and Homer protein homolog 1 (Homer-la) in the hippocampus, which decreased with the treatment of scopolamine.Conclusions:These results suggest that nacre extract has attenuating effects on memory impairments induced by scopolamine through the increase in mRNA expression of BDNF and Homer-1a.

Effects of methyl jasmonate on metabolism of topical alkaloids and expression of relate genes in Atropa belladonna / 中国中药杂志

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 μmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 μmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 μmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.

Effects of Shenqi Xingnao Prescription on Learning and Memory Ability, Contents of Choline Acetyltransferase and Acetylcholine Esterase in Brain Tissue of Mice Models with Alzheimer Disease / 中国中医药信息杂志

Objective To observe the effects of Shenqi Xingnao Prescription on learning and memory ability, contents of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) in brain tissue in mice models with scopolamine-induced Alzheimer disease (AD); To investigate its mechanism for prevention and treatment for AD. Methods Totally 110 ICR mice were randomly divided into control group, control+Shenqi Xingnao Prescription high-dose group,model group,donepezil group,model+Shenqi Xingnao Prescription high-,medium-,and low-dose groups. The control and model group were given distilled water for gavage, and the other groups were given the corresponding medicine for gavage, once a day, for 14 days. On the 15th day, Morris water maze test and object recognition test were used to evaluate the learning and memory ability of each group. The model mice of memory impairment induced by intraperitoneal injection of scopolamine was established 20 minutes before the behavioral test. The expressions of ChAT and AChE in cortex and hippocampus were detected by Western blot. Results The results of Morris water maze test showed that compared with the control group, the model group had significant longer escape latency(P<0.05);Compared with the model group,Shenqi Xingnao Prescription medium-and high-dose groups could shorten the escape latency (P<0.05). The results of the object recognition test showed that compared with the control group, the ability of the model group to explore new things decreased and the discrimination index (DI) decreased (P<0.001);Compared with the model group,Shenqi Xingnao Prescription groups could increase the DI of model mice (P<0.05, P<0.01, P<0.001). The results of Western blot showed that the expression of AChE protein in the cortex and hippocampus of the model group was significantly higher than the control group (P<0.01); Compared with the model group, Shenqi Xingnao Prescription low- and medium-dose groups could decrease the expression of AChE in the cortex in different degrees(P<0.01);Shenqi Xingnao Prescription groups could decreaed the expression of AChE in the hippocampus (P<0.001); There was no significant statistical significance in the expression of ChAT in the cortex and hippocampus in each group.Conclusion Shenqi Xingnao Prescription can significantly improve the learning and memory ability of AD model mice induced by scopolamine, which may be related to the descent expression of AChE protein in the cortex and hippocampus of the model mice.

Analysis of Clinical Application and Adverse Reactions of Scopolamine Butylbromide Injection / 中国药师

Objective:To investigate and analyze the clinical application and adverse reactions of scopolamine butylbromide injection to promote the rational use in clinics. Methods:Totally 3695 effective cases treated with scopolamine butylbromide injection were collected from January 2016 to July 2017, and the age, sex, cause of drug use, medication days, combined use, high risk factors, and names, types and clinical manifestations of adverse reactions induced by scopolamine butylbromide were classified and statistically analyzed. Results:The overall incidence of adverse reactions of scopolamine butylbromide was 0.22%. The drug use rate was 44.36% in high-risk patients. The incidence of adverse drug reactions in high-risk patients was 1.83%,and that in low-risk patients was 0.24%, and the difference was 7.63 times. Combined drug use was common, and 1.98% of the combined use had potential drug interactions. Conclusion:The requirements of drug instructions should be strictly followed,and high-risk patients and drug combination should be paid much attention in order to reduce the risk of adverse drug reactions and ensure the drug safety.

Protective effects of cultured and fermented ginseng extracts against scopolamine-induced memory loss in a mouse model / 한국실험동물학회지

This study was performed to investigate the effect of a concentrate of fermented wild ginseng root culture (HLJG0701) on memory improvement in the scopolamine (SPL)-induced memory-deficient mouse model. Eight-week-old male ICR mice were used to evaluate the protective effect of HLJG0701 against the SPL-induced memory loss animal model. The Morris water maze test, which measures hippocampus-dependent learning ability, and the Y-maze test, a short-term memory assessment test, were performed and related markers were analyzed. HLJG0701-treated groups displayed significantly reduced acetylcholinesterase activity and increased acetylcholine level compared with the SPL-administered group (SPL-G) (P < 0.05). In the Y-maze test, the spontaneous alternation in al HLJG0711-treated groups was significantly increased compared with that in SPL-G (P < 0.05). In the Morris water maze test, the escape latency and time spent in the target quadrant in all HLJG0701-treated groups were significantly decreased and increased, respectively, compared with those in SPL-G (P < 0.05). In addition, the brain-derived neurotrophic factor level in groups treated with HLJG0701 300 and 600 mg/kg body weight was significantly increased compared with that in SPL-G (P < 0.05). These results suggest that the HLJG0701 may protect against memory loss by inhibiting acetylcholinesterase activity and preventing acetylcholine deficiency.