Scopolamine causes delirium-like brain network dysfunction and reversible cognitive impairment without neuronal loss

Delirium is a severe acute neuropsychiatric syndrome that commonly occurs in the elderly and is considered an independent risk factor for later dementia.However,given its inherent complexity,few animal models of delirium have been established and the mechanism underlying the onset of delirium remains elusive.Here,we conducted a comparison of three mouse models of delirium induced by clinically relevant risk factors,including anesthesia with surgery(AS),systemic inflammation,and neurotransmission modulation.We found that both bacterial lipopolysaccharide(LPS)and cholinergic receptor antagonist scopolamine(Scop)induction reduced neuronal activities in the delirium-related brain network,with the latter presenting a similar pattern of reduction as found in delirium patients.Consistently,Scop injection resulted in reversible cognitive impairment with hyperactive behavior.No loss of cholinergic neurons was found with treatment,but hippocampal synaptic functions were affected.These findings provide further clues regarding the mechanism underlying delirium onset and demonstrate the successful application of the Scop injection model in mimicking delirium-like phenotypes in mice.

Kai Xin San ameliorates scopolamine-induced cognitive dysfunction

Kai Xin San(KXS, containing ginseng, hoelen, polygala, and acorus), a traditional Chinese herbal compound, has been found to regulate cognitive dysfunction; however, its mechanism of action is still unclear. In this study, 72 specific-pathogen-free male Kunming mice aged 8 weeks were randomly divided into a vehicle control group, scopolamine group, low-dose KXS group, moderate-dose KXS group, high-dose KXS group, and positive control group. Except for the vehicle control group and scopolamine groups(which received physiological saline), the doses of KXS(0.7, 1.4 and 2.8 g/kg per day) and donepezil(3 mg/kg per day) were gastrointestinally administered once daily for 2 weeks. On day 8 after intragastric treatment, the behavioral tests were carried out. Scopolamine group and intervention groups received scopolamine 3 mg/kg per day through intraperitoneal injection. The effects of KXS on spatial learning and memory, pathological changes of brain tissue, expression of apoptosis factors, oxidative stress injury factors, synapse-associated protein, and cholinergic neurotransmitter were measured. The results confirmed the following.(1) KXS shortened the escape latency and increased residence time in the target quadrant and the number of platform crossings in the Morris water maze.(2) KXS increased the percentage of alternations between the labyrinth arms in the mice of KXS groups in the Y-maze.(3) Nissl and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that KXS promoted the production of Nissl bodies and inhibited the formation of apoptotic bodies.(4) Western blot assay showed that KXS up-regulated the expression of anti-apoptotic protein Bcl-2 and inhibited the expression of pro-apoptotic protein Bax. KXS up-regulated the expression of postsynaptic density 95, synaptophysin, and brain-derived neurotrophic factor in the cerebral cortex and hippocampus.(5) KXS increased the level and activity of choline acetyltransferase, acetylcholine, superoxide dismutase, and glutathione peroxidase, and reduced the level and activity of acetyl cholinesterase, reactive oxygen species, and malondialdehyde through acting on the cholinergic system and reducing oxidative stress damage. These results indicate that KXS plays a neuroprotective role and improves cognitive function through reducing apoptosis and oxidative stress, and regulating synapse-associated protein and cholinergic neurotransmitters.

Neuroprotection of N-benzylcinnamide on scopolamine-induced cholinergic dysfunction in human SH-SY5Y neuroblastoma cells

Alzheimer＇s disease, a progressive neurodegenerative disease, affects learning and memory resulting from cholinergic dysfunction. Scopolamine has been employed to induce Alzheimer＇s disease-like pathology in vivo and in vitro through alteration of cholinergic system. N-benzylcinnamide （PT-3）, purified from Piper submultinerve, has been shown to exhibit neuroprotective properties against amyloid-β-induced neuronal toxicity in rat cortical primary cell culture and to improve spatial learning and memory of aged rats through alleviating oxidative stress. We proposed a hypothesis that PT3 has a neuroprotective effect against scopolamine-induced cholinergic dysfunction. PT-3 （125-200 nM） pretreatment was performed in human neuroblastoma SH-SY5Y cell line following scopolamine induction. PT-3 （125-200 nM） inhibited scopolamine （2 mM）-induced generation of reactive oxygen species, cellular apoptosis, upregutation of ace- tylcholinesterase activity, downregulation of choline acetyltransferase level, and activation of p38 and JNK signalling pathways. These findings revealed the underlying mechanisms of scopolamine-induced Alzheimer＇s disease-like cellular dysfunctions, which provide evidence for developing drugs for the treatment of this de- bilitating disease.

Anti-Amnesic Activity of Vitex Negundo in Scopolamine Induced Amnesia in Rats

In the present study we investigated the anti-amnesic activity of Vitex negundo in scopolamine induced amnesia in rats. Wistar rats (180-200 g) were trained on active avoidance task. Each animal received session of 15 trials with inter trial duration of 15 s for 5 days. Scopolamine (3 mg/kg, i.p) was administered at different time periods on the basis of stages of memory i.e acquisition, consolidation and retention in different groups (n = 6). Effect of Vitex negundo extract was evaluated and compared to a standard drug, Donepezil. Significant (p 【0.05) increase in the avoidance response on the 5th session has been observed as compared to 1st session in control group. Scopolamine treatment significantly (p 【0.05) reduced the avoidance response compared to control. Extract treated groups shown significant (p 【0.05) increase in number of avoidance responses as compared to scopolamine treated groups. Increased oxidative stress in brain after scopolamine treatment, as observed by increase in MDA &decrease in GSH &SOD, was lowered in the groups treated with extracts. AChE activity was also improved after V. negundo treatment. Results of the study have shown that V. negundo treated groups decrease the phenomenon of amnesia by increasing learning of memory through antioxidant effect and decreasing AChE activity.

Long-term administration of scopolamine interferes with nerve cell proliferation, differentiation and migration in adult mouse hippocampal dentate gyrus, but it does not induce cell death

Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperito- neal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen （NeuN; a neuronal marker） and Fluoro-]ade B （a marker for the localization of neuronal degeneration） was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67 （a marker for proliferating cells）-immunoreactive cells were reduced in number and reac hed the lowest level at 4 weeks. Doublecortin （DCX; a marker for newly generated neurons）-im- munoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2＇-deoxyuridine （BrdU）-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature BrdU/NeuN double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death.

PROLONGED PARALYSIS OF CHILDREN TREATED WITH SCOPOLAMINE ACUPOINT INJECTION

Authors reported 209 cases of childish paralysis. Of them 104 cases were treatedwith scopolamine acupoint injection, and another 105 cases were divided into 3 groups and treatedwith scopolamine intramuscular injection, Salviae miltiorrhizae acupoint injection and acupuncture re-spectively as control. The results show that the therapeutic effect of scopolamine acupoint injection ismuch better than that of all other groups.

SYNTHESIS OF A NEW TYPE OF SCOPOLAMINE ANALOGUES

A series of compounds,which are structurally analogous to scopolamine and also in accordance with the general formula of neuroleptic benzamides,were synthesized and tested for their potential antipsychotic activity.

Novel insights on acetylcholinesterase inhibition by Convolvulus pluricaulis,scopolamine and their combination in zebrafish

Acetylcholinesterase(AChE)inhibitors increase the retention of acetylcholine(ACh)in synapses.Although they allevi-ate cognitive deficits in Alzheimer’s disease,their limited benefits warrant investigations of plant extracts with similar properties.We studied the anti-AChE activity of Convolvulus pluricaulis(CP)in a zebrafish model of cognitive impair-ment induced by scopolamine(SCOP).CP is a perennial herb with anti-amnesiac and anxiolytic properties.It contains alkaloid,anthocyanin,coumarin,flavonoid,phytosterol and triterpenoid components.Isoxazole(ISOX)was used as a positive control for AChE inhibition.CP-treated 168 hpf larvae showed a similar pattern of AChE inhibition(in the myelencephalon and somites)as that of ISOX-treated larvae.CP was superior to ISOX as evidenced by the retention of avoidance response behavior in adult zebrafish.Molecular docking studies indicated that ISOX binds Ser203 of the catalytic triad on the human AChE.The active components of CP-scopoletin and kaempferol-were bound by His447 of the catalytic triad,the anionic subsite of the catalytic center,and the peripheral anionic site.This suggested the ability of CP to mediate both competitive and non-competitive modes of inhibition.Surprisingly,SCOP showed AChE inhibition in larvae,possibly mediated via the choline-binding sites.CP+SCOP induced a concentration-dependent increase in AChE inhibition and ACh depletion.Abnormal motor responses were observed with ISOX,CP,ISOX+SCOP,and CP+SCOP,indicative of undesirable effects on the peripheral cholinergic system.Our study proposes the examination of CP,SCOP,and CP+SCOP as potential AChE inhibitors for their ability to modulate cognitive deficits.

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-1 a) 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-1 a.

Anti-Radical and Neuroprotective Potential of <i>Ficus infectoria</i>in Scopolamine Induced Memory Impairment in Mice

Ficus infectoria has a wide distribution in Bangladesh, Nepal, Pakistan, Sri Lanka, Southwest China and Indochina and is an enrich source of phytochemicals thereby possess antibacterial, antifungal and hyperglycaemic activities. In this study, we attempted to examine the cognitive ability of methanolic and ethanolic extract of F. infectoria fruit extract in scopolamine induced memory impairment in mice by using preliminary phytochemical and antioxidant tests, and the cognitive ability of the methanolic and ethanolic fruit extract of F. infectoria. Fruit extract was analyzed in scopolamine amnesia mice using passive avoidance approach. Piracetam was used as a reference drug (200 mg/Kg). Further confirmation was provided by means of mice brain homogenate biochemical tests. Maximum phytochemical, antioxidant activity and nootropic ability were observed in the ethanolic fruit extract of F. infectoria. Plant extract was used at three doses i.e. 75 mg/Kg, 150 mg/Kg and 300 mg/Kg and exhibited nootropic abilities in all tests used. Enhanced SDL value i.e. (291.2 ± 0.33+++###) was observed by the administration of plant extract at all dose range in comparison to reference drug i.e. piracetam (252.8 ± 1.60###) used in the study. The plant extract utilization has showed increase in total protein (25.08 ± 0.26+++### mg/g of tissue) and reduced glutathione content (33.0 ± 0.46+++### nmoles/mg of protein) and vice versa while low malondialehyde (MDA) (9.18 ± 0.17+++### nmoles/mg of protein) and AChE activity (0.067 ± 0.009+++### M/min/g protein). However, opposite situation was observed in the scopolamine amnesia mice. Hence it was concluded the plant extract possessed neuroprotective activity in the scopolamine induced cognitive decline in mice thereby used as cost effective natural medicines in near future.

Liquid Chromatographic Determination of Scopolamine in Hair with Suspended Drop Liquid Phase Microextraction Technique

Hair analysis is used in some branches of alternative medicine as a method of investigation to assist diagnosis. It is very useful when a history of drug use is difficult or impossible to obtain. In this re-search suspended droplet liquid phase microextraction (SDLME) coupled with high-performance liquid chromatography and photodiode array detection (HPLC-DAD) was used for preconcentration and analysis of scopolamine in hair samples. Therefore scopolamine was extracted from 2.0 g hair sample incubated in methanol (5 h, 50°C) and adjusted to pH 7.4 with, Na2HPO4–H3PO4 buffer solution (donor phase, P1) into an organic phase (P2) 350 μl n-octanol and then back extracted into a micro drop of aqueous acceptor phase (P3), adjusted at pH 3, with HCL. The extraction time, T1 (from P1 to P2) was 2 min and T2 (from P2 to P3) was 30 min. Optimum instrumental conditions were included;A C18 reverse phase column with water-acetonitrile-methanol (80:10:10) as the mobile phase was used and wavelength for UV detec-tion was 205 nm. The linear range was 10 to 10000 ng●mL–1, enrichment factor, detec-tion limit and relative standard deviation were 77, 0.1 ng●mL–1 and 5.4 respectively.

Effects of Scopolamine on Blood Vessels in Rabbit Ear after Sympathetic and Sensory Denervation

Objectives To investigatethe effects and involved mechanisms of scopolamine(Scop) on rabbit ear blood vessels. Methods Rabbitear blood vessels were desympathetic and desensoryinnervation with surgical operation. Diameters of dor-sal auricular arterial trunks in vivo were measuredwith a pair of compasses and the ruler in a dissectingmicroscope, and effluents from isolated ear underconstant perfusion pressure were recorded with a digi-tal drop-recorder. Results Intramuscular injectionof Scop 0.1 mg/kg made the diameter of denerveddorsal auricular arterial trunks, as well as that of in-nerved ones, significantly increased. Scop by itself,atthe maximal concentration (Cmax) of 3μM, 30μMand 300μM, did not alter the effluent flow from theisolated denervated rabbit ear, but chlorpromazine(CPZ), at Cmax of 1μM, acetylcholine (ACh), 0.25μM, all significantly increased the effluent flow, andnorepinephrine (NE), 0.1μM, significantly decreasedthe effluent. Scop, 3μM, did not affect ACh (0.25μM)-induced the increase of effluent flow, but Scop,30μM, alleviated the increase. Scop, 3μM, did notaffect NE (0.1μM)-induced the decrease of effluentflow, but Scop, 10, 30 and 100μM, significantly alle-viated the decrease. Conclusions The study sug-gests that Scop has no direct vasodilator effect. Thevasodilator effect of Scop is not due to the blockade ofmuscarinic receptor. However, Scop can dilate bloodvessels contracted by α_1-adrenoceptor activation.

Clinical Assessment of the Use of Propinox Hydrochloride and Scopolamine Hydrochloride in the Treatment of Abdominal Colic: A Retrospective, Comparative Study

Objectives: The purpose of this study was to evaluate and compare the use of propinox hydrochloride and scopolamine hydrochloride in patients presenting abdominal colic (abdominal pain), in terms of treatment efficacy and tolerability. Material & Methods: This was an analytical, retrospective, comparative study based on hospital records of outpatients treated at Serviço de Clínica Médica do Hospital das Clínicas Costantino Otaviano (HCTCO) and at Santa Casa de Misericórdia do Rio de Janeiro, from 1988-1998. Subjects were divided into two groups: patients from Group 1 were treated with propinox hydrochloride, while patients from Group 2 were treated with scopolamine hydrochloride. Statistical analysis was performed using GraphPad Prism version 5.0. For comparison of categorical variables, we used the chi-squared or Fisher’s test, while continuous variables were analyzed using ANOVA or the Student’s T test. Results: A total of 1042 subjects were included, of which 525 were allocated to Group 1 and 517 to Group 2. Mean treatment duration was 9.166 days (±4.208) in Group 1 and 8.795 days (±5.052) in Group 2, with no statistically significant difference in treatment duration between the two groups (p = 0.198). All subjects in Group 1 were treated with propinox 10 mg (2 coated tablets, three times per day) while all subjects in Group 2 were treated with scopolamine hydrochloride 10 mg (2 coated tablets, three times per day). There were no statistically significant between-group differences in weight, BMI, heart rate, and respiratory rate at pre- and post-treatment;with the exception of higher post-treatment systolic blood pressure in Group 1, blood pressure measures also remained homogenous. Adverse events were reported among both treatment groups with no significant between-group difference in incidence (p = 0566). At pretreatment, pain intensity was more severe in Group 1 (p = 0.0257), while at post-treatment, there was no statistically significant difference between the two treatment groups (p = 0.895). There was a statistically significant improvement in pain intensity within both treatment groups (χ2 = 631.4;df = 3;p < 0.0001 for Group 1 and χ2 = 554.3;df = 3;p < 0.0001 for Group 2). Conclusion: The results obtained in this study indicate a therapeutic equivalence between propinox hydrochloride and scopolamine hydrochloride. Both treatments demonstrated good efficacy and tolerability in the treatment of abdominal colic pain, in the population evaluated.

Low dose transdermal scopolamine increases cardiac vagal tone in patients after acute myocardial infarction

OBJECTIVE: To investigate whether transdermal scopolamine increased cardiac vagal activity in patients during the acute phase of myocardial infarction. METHODS: 30 patients with a first acute myocardial infarction and preserved sinus rhythm who were on no drug that could influence the sinus node were randomly assigned to either treatment group or placebo group. Measures of heart rate variability (HRV) in patients given drug or placebo were obtained by digital 24 hour Holter recording before and after treatment. Baroreflex sensitivity was performed using the phenylephrine method. RESULTS: No significant differences was found in the indices of the time domain and the frequency domain in both groups before treatment. Patients with transdermal scopolamine showed a significant increase in the standard deviation of normal RR intervals (SDNN), standard deviation of all five min mean normal RR intervals (SDANN), root mean square of differences of successive normal RR intervals (rMSSD), total power (TP, 0.000. - 0.40 Hz), low frequency peak (LF, 0.040 - 0.15 Hz), high frequency peak (HF, 0.15 - 0.40 Hz), and Baroreflex sensitivity after treatment (P

The mixture of procyanidins extracted from the lotus seed pod and bilobalide ameliorates scopolamine-induced memory impairment in mice

Objective To study the co-effect of procyanidins extracted from the lotus seed pod （LSPC） and bilobalide （BIL） on ameliorating scopolamine-induced learning and memory impairment in young mice. Methods Fifty male Kunming mice with similar learning and memory capabilities were selected by Morris water maze test and were randomized into 5 groups （n=10 in each group）： control group, scopolamine group, L-（LSPC＋BIL） group （50 mg/kg LSPC＋10 mg/kg BIL）, M-（LSPC＋BIL） group （100 mg/kg LSPC＋20 mg/kg BIL）, H-（LSPC＋BIL） group （150 mg/kg LSPC＋30 mg/kg BIL）. Scopolamine model with impaired learning and memory was established by scopolamine treatment （1 mg/kg）, and after 10 min mice were tested. In L-, M-, and H- （LSPC＋BIL） groups, mice were treated with LSPC and BIL ig. for 30 days, while mice in the other 2 groups were treated with normal saline ig. instead. After the 30-day＇s treatment, the co-effect of LSPC and BIL on learning and memory was tested by Morris water maze and the step-down avoidance tests. Results The memory impairment caused by scopolamine in young mice could be ameliorated by co-treatment of LSPC and BIL, as indicated by significantly shorter escape latency and swimming distance in the Morris water maze test, when compared with those in the scopolamine group. In the step-down avoidance test, mice in all the 3 dose groups showed significantly smaller number of errors and longer latency than mice in the scopolamine group did. Conclusion Co-treatment of LSPE and BIL can ameliorate scopolamine-induced learning and memory impairment in young mice.

Effect of Co-administration of Morphine and Cholinergic Antagonists on Y-maze Spatial Recognition Memory Retrieval and Locomotor Activity in Mice

The interaction of morphine and cholinergic system was shown in previous studies. In the present study, we investigated whether morphine would interact with the cholinergic antagonists, scopolamine and atropine in a Y-maze spatial recognition memory. Pre-test treatments of morphine （5, 1.5, 0.5 mg/kg）, scopolamine （1, 0.1 mg/kg）, atropine （0.5, 0.1 mg/kg） were used in the experiments, relatively high or low doses were paired respectively as co-administration measures. The results showed that co-administration of morphine 0.Smg/kg ~ scopolamine 0.1 mg/kg and morphine 0.5 mg/kg ＋ atropine 0.1 mg/kg disturbed the inspective exploratory behavior （percent of arm duration） but not the inquisitive behavior （percent of arm visits） of the spatial memory retrieval, while the drugs didn＇t cause amnesia when single administered of the concerned low doses. Distinct interaction was found between scopolamine and morphine on increasing locomotor activity.

ACOUSTIC STARTLE RESPONSE AFFECTED BY AGING AND CHOLINERGIC NEUROTRANSMITTERS

The acoustic startle response has been used to evaluate tinnitus and hyperacusis in animal models. Gap induced prepulse in- hibition of the acoustic startle reflex （gap-PPI） is affected by tinnitus and loudness changes. Since tinnitus and reduced sound tolerance are commonly seen in elderly, we measured gap-PPI in Fischer 344 rats, an aging related hearing loss model, at dif- ferent agcs： 3-5 months, 9-12 months, and 15-17 months. The startle response was induced by three different intensity of sound： 105, 95 and 85 dB SPL. Gap-PPI was induced by different duration of silent gaps from 1 to 100 ms. When thc startle was induced by 105 dB SPL sound intensity, the gap-PPI induced by 50 ms silent gap was significantly lower than those in- duced by 25 or 100 ms duration, showing a ＂notch＂ in the gap-PPI function. The ＂notch＂ disappeared with the reduction of startle sound, suggesting the ＂notch＂ may be related with hyper-sensitivity to loud sound. As the intensity of the stimulus de- creased, the appearance of the hyperacusis-like effect decreased more quickly for the youngest group of rats. We also tested scopolamine, a muscarinic acetylcholine receptor antagonist, and mecamylamine, a nicotinic acetylcholine receptor antago- nist, on the effect of gap-PPI. When scopolamine was administered, the results indicated no addition effect on the hyperacu- sis-like phenomenon in the two older groups. Mecamylamine, the nicotinic antagonist also showed effects on the appearance of hyperacusis on rats in different ages. The information derived from the study will be fundamental for the further research in determining the cause and treatment for hyperacusis.

Twenty-five Cases of Obstinate Hiccup Treated by Point Injection at Tianding

From 1997 to 2000, the author treated 25 cases of obstinate hiccup by injecting chlorpromazine and 654-2 injection at Tianding (LI 17), with satisfactory therapeutic results as reported in the following.

Suggested Two Hypotheses on Dementia (“Anticholinergic Hypothesis” and “Cranial Skeletal Muscles Hypothesis”) and the Therapeutic Agent

The present study was conducted with the objective of further developing the cholinergic hypothesis and not using the prevalent amyloid beta plaque hypothesis or the tau protein hypothesis on dementia. The experiment was conducted on mice using anticholinergic drugs scopolamine and biperiden to investigate the root cause of dementia. First, we measured the mice serum for liquid chromatography-tandem mass spectrometry (LC-MS/MS) after administration of scopolamine and biperiden and found an accumulation of anticholinergic drugs metabolites in the body. The Y-maze test and measurement of LC-MS/MS in the cranial skeletal muscle cells showed that the Scopolamine metabolites have a significant effect on the cranial skeletal muscles, leading to the conclusion that Methocarbamol is an effective treatment for dementia.

Further Insights on the <i>Datura innoxia</i>Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.