Medicinal plants Melissa officinalis (M. officinalis), Atropa belladonna (A. belladonna), and Artemisia siberia (A. siberia) have been widely used in traditional medicine in Iran and other countries due to their numerous ethno-pharmacological effects. Therefore, the purpose of this research was to compare the sedative and pre-anesthetic effects of, M. officinali, A. belladonna, A. siberia and diazepam in rats. For this purpose, during an experimental study, 150 female Wistar rats which were allocated into different experimental groups received intra-peritoneal injections of 150, 300 and 450 mg per kilogram of  A. belladonna extract, 50, 200 and 350 mg per kilogram of M. officinalis extract, 100, 200 and 400 mg per kilogram of A. siberia extract, 1.2 mg per kilogram of the drug diazepam and equal volumes of the chemical substance dimethyl sulfoxide as placebo. The obtained results showed a significant increase in the duration of sleep induced by ketamine, as well as a significant increase in the treatment groups with higher doses of the extract (p<0.01). The highest sedative and pre-anesthetic effects were observed in the doses of 450, 400, and 200 mg/kg of A. belladonna, A. siberia, and M. officinalis plant extracts respectively. According to the results, extracts of A. belladonna, A. siberia, and M. officinalis have significant sedative and pre-anesthetic effects.

Atropine and scopolamine are two important tropane alkaloids, widely use as anticholinergic drugs. Such as most solanaceae plants, Atropa belladonna produces these two tropane alkaloids. Carbohydrates are not only act as carbon sources for solanaceus plants in vitro cultures, but also have some effects as signal molecules on tropane alkaloids metabolism. Effect of sorbitol, mannitol and sucrose on growth, chlorophyll, hyoscyamine and scopolamine contents, and hyoscyamine 6- beta hydroxylase (H6H) gene expression, investigated. These treatments decreased the growth of plantlets but increased chlorophyll, hyoscyamine and scopolamine contents. The treatments increase h6h gene expression in different organs of Atropa belladonna plantlets. There was an agreement between h6h expression pattern and scopolamine contents.

Atropa belladonna is one of the most important medicinal plants in Solanaceae family. It contains tropani alkaloids that often are synthesised in root. Present study was conducted in order to induction of hairy root in A.belladonna using gene transfer via Agrobacterium rhizogenes method. The stem explants with Agrobacterium suspensions harbouring rolB gene were inoculated. Then, in order to direct regeneration, five compounds (MS, MS + 3 mg/l Kin, B5, MS+ 12 mg/l Kin, B5+ 5 mg/l Kin)) in a completely randomized design with three replications was used. The statistical analysis indicated that among treated hormone, 12 mg /l KIN level in MS medium showed the most shoots direct regeneration. Two methods for the analysis of transgenic plants, phenotypic (observed in hair roots of transgenic seedlings), gene amplification of the rolB (using PCR) was used.

Atropa belladonna، a member of Solanaceae family، is a very important pharmaceutical plant. Its roots are composed of significant amount of important alkaloids atropine and scopolamin. In this study the effect of different concentrations (0، 0. 01 and 0. 1 mM) of salicylic acid as a signal molecule was evaluated on gene expression levels of hyocyamin 6-β hydroxylase (H6H) and putrescinN-methyl transferase (PMT) in the hairy roots and plantlet. Expression level of these genes was evaluated by semi quantitative RT-PCR. The results showed that different concentrations of salicylic acid have different effects on gene expression levels. Isozymes of PMT and H6H in different organs and in response to the concentrations of salicylic acid were differentially expressed. H6H and PMT2 genes expression were increased at 0. 01mM in the hairy roots. Expression of PMT2 in the roots was increased at 0. 01mM. But the level of gene expression in shoot was not significantly affected.

Symbiotic fungus Piriformospora indica has useful and unique features to enhance growth and resistance of plants against biotic and abiotic stresses. In the present study the effects of this endophyte fungus on the growth and activities of antioxidant enzymes in medicinal plant Atropa belladonna were studied. After co-existence, the effect of fungus on plant growth, total protein content and the activities of ascorbate peroxidase (APX, EC 1,11,1,11), catalase (CAT, EC 1,11,1,6) and peroxidase (POX, EC 1,11,1,7) enzymes were determined using a spectrophotometer. The results showed an increase on the growth parameters, and total protein content in root, stem and leaf of inoculated plants in compare to control. Inoculation by endophte fungus increased the total fresh weight of plantlets by 1.87-fold and shoot height by 2.11-fold. The presence of P. indica decreased all three enzyme activities in the stems and leaves, whereas it increased enzyme activities of CAT, APX and POX in roots by 2.2-, 2.3- and 2.5-fold, respectively, in comparison with control plants. We conclude that this fungus can be used to increase plant growth and as bio-material protection tool via increasing root antioxidant enzyme activities in important medicinal plant Atropa belladonna.

Belladonna (Atropa belladonna), from Solanaceae, is an annual plant and has an important tropane alkaloids called hyoscyamine. Considering the pharmacological and economic importance of A. belladonna and priority in its domestication and cultivation, advances in its tissue culture and regeneration methods of this plant under in vitro conditions is important to facilitate methods of gene transfer to this medicinal herb. So, in this research, optimization of cultivation conditions for callogenesis and regeneration of Atropa belladonna was investigated. For callogenesis, belladonna sterilized seeds were initially sown in MS medium. After seed germination and seedlings growth for two months, stem and leaf explants were cultured in MS medium including a combination of different hormonal concentrations of 2.4-D (0, 0.5 and 1 mg/l) and Kin (0, 1 and 2 mg/l).To evaluate the regeneration percentage of callus from shoot explants, Kin hormone was used in four levels (0, 3, 5 and 8 mg/l) using completely randomized design with three replications. These experiments indicated that, shoot explants had the highest callogenesis in terms of explant type. In terms of hormonal concentrations, combination of two hormones at concentrations of 2.4-D 1 mg/l + Kin 2 mg/l+MS and also, 2.4-D 1mg/l +Kin 1mg/l +MS showed the highest rate of callogenesis. Increase in the concentration of Kin hormone in MS medium was directly correlated with increase in regeneration percentage and the highest regeneration rate of 36.66% was observed at 8 mg/l.

Atropa Belladonna L. is the most important medicinal plant of Solanaceae family, due to important commercial sources of atropine and scopolamine as tropane alkaloids. Applications of hairy root cultures are advised because of their special properties such as fast growth, short doubling time, ease of maintenance, and ability to synthesize and continuous production of a range of chemical compounds. Effects of ozone depletion and UV irradiation have been studied by most of the investigators. In the present study, the effect of different levels of UV-B irradiation was studied on atropine production and activity of some antioxidant enzymes. UV-B irradiation during 3 days was treated for 4, 8 and 12 min that was equal to 13, 26 and 39 Kj/m2 respectively. Results showed that increase of UV-B lending to decrease hairy roots dry weight. This irradiation increased atropine content in comparison of control treat and the highest was recorded in 26 Kj/m2. The activity of antioxidant enzymes including catalase, guaiacol peroxidase, and ascorbate peroxidase was also elevated in treated hairy roots rather than of the control at 0.01% of probability. Based on obtained results in this work UV-B irradiation can be considered as one of the useful methods for Atropine and antioxidant enzymes in A. belladonna hairy roots.

The rapid and mass production of secondary metabolites through chemical methods is mostly expensive, difficult, or impossible. Considering the economic importance of these metabolites and their small production in medicinal plants, some methods such as the cultivation of hairy roots and the use of biotic and abiotic stimuli have been suggested to increase these metabolites. The accumulation of the tropane alkaloid atropine in Atropa belladonna L. is of great importance in the pharmaceutical industry. In this research, transgenic hairy roots of A. belladonna was produced using an A7 strain of Agrobacterium rhizogenes. The transformation of hairy roots was confirmed by PCR test. The results of the PCR test showed the correctness of the amplification of the bp780 fragment for the rolB gene in hairy roots. In addition, the effect of different concentrations (0, 125, 250, and 500 μM) of salicylic acid in two times of 24 and 48 hours on the amount of atropine content, the plant growth rate and the activity of antioxidant enzymes of A. belladonna hairy roots was investigated. The data statistically analyzed using factorial experiment based on a completely random design with three replications. The results of the analysis of variance showed significant effect of salicylic acid on all the traits (P<0.01). The effect of time was significant for the amount of atropine and ascorbate peroxidase (APX). The salicylic acid by time interaction effects were significant for the amount of atropine, catalase (CAT), and guaiacol peroxidase (GPX) (P<0.01, 0.05). The results showed that by increasing salicylic acid concentrations the growth of hairy roots significantly decrease compared to the control and the lowest values of fresh and dry weight of the hairy roots (2.79 and 0.148 g) were obtained in 500 μM of salicylic acid compared to the control (6.76 and 0.273 g). However, based on GC/MS analysis, the highest amount of atropine (11 times more than the control) was obtained in the 250 μM salicylic acid in a period of 24 hours. The highest catalase and guaiacol peroxidase activities were obtained at concentrations of 250 and 125µM salicylic acid for 24 hours, respectively. The highest level of ascorbate peroxidase enzyme activity was obtained at the concentration of 250 µM salicylic acid compared to the control. It was concluded that the salicylic acid stimulant at low concentrations is useful for cell signaling and stimulating the production of secondary metabolites, but at high concentrations, it disrupts the growth of hairy roots.

Most Solanaceae plants produce a range of biologically important alkaloids including nicotine and tropane alkaloids, such as hyoscyamine (atropine) and scopolamine. These alkaloids are used for their medicinal properties. Atropa belladonna is a medicinally important herbaceous plant that produces high amount of tropane alkaloids in roots. In this research, Atropa belladonna explants were obtained from sterilized seedlings in the modified Murashige and Skoog (MS) solid medium. The seeds were collected from Vaz and Garmestan regions of Mazandaran Province, Iran. Explants were cultured for four weeks on a modified MS solid medium, containing different concentrations of salicylic acid (0, 0.01, 0.1 and 1 mM). In addition, hairy roots of Atropa belladonna that was established by transformation with Agrobacterium rhizogenes, were cultured in MS medium for two weeks. Then the production of two tropane alkaloids, atropine and scopolamine, in different parts of neoformed plants were assayed by HPLC method. Generally, tropan alkaloids content in hairy roots was considerably higher compared to those contents in plants. Although Garmestan population was better than Vaz group, the amount of atropine in hairy roots was influenced by salicylic acid as high as 5 to 35 times of produced atropine in different parts of plants. The scopolamine content in the hairy roots was 2-30 times higher than in plant organs. In conclusion, it is suggested that, hairy root lines can be used as a replacement of plants in further studies and for tropane alkaloids production in economical and commercial scales.

The parasympatholytic tropane alkaloids, Atropine and Scopolamine, accumulated in Atropa belladonna, are of great interest for the pharmaceutical industry. In this research, Atropa belladonna transformed hairy roots were produced by Agrobacterium rhizogenes strain A4 mediated transformation. Effects of different concentrations of methyl jasmonate (0, 150 and 300 Micro molar) were investigated on production of Atropine and Scopolamine in 24 hours treatment. Then the contents of two tropane alkaloids, Atropine and Scopolamine, were assayed by HPLC method. The tropane alkaloids contents were shown to be significantly increased in transformed hairy roots. Results showed Atropine and Scopolamine content in transformed hairy roots reached respectively from 0.12 to 1.715 and 0.0195 to 0.43 of mg in 100 mg of dry weight. Methyl jasmonate dramatically enhanced both tropane alkaloids, Atropine and Scopolamine. Tropane alkaloids content increased in 300 Mµ concentration of methyl jasmonat in comparison with 150 Mµ concentration and control treatment. In conclusion, it is suggested that, hairy root can be used as a replacement of plants in further studies and for tropane alkaloids production in economical and commercial scales.