Improvement and prediction of the extraction parameters of lupeol and stigmasterol metabolites of Melia azedarach with response surface methodology.

BACKGROUND: Melia azedarach is known as a medicinal plant that has wide biological activities such as analgesic, antibacterial, and antifungal effects and is used to treat a wide range of diseases such as diarrhea, malaria, and various skin diseases. However, optimizing the extraction of valuable secondary metabolites of M. azedarach using alternative extraction methods has not been investigated. This research aims to develop an effective, fast, and environmentally friendly extraction method using Ultrasound-assisted extraction, methanol and temperature to optimize the extraction of two secondary metabolites, lupeol and stigmasterol, from young roots of M. azedarach using the response surface methodology. METHODS: Box-behnken design was applied to optimize different factors (solvent, temperature, and ultrasonication time). The amounts of lupeol and stigmasterol in the root of M. azedarach were detected by the HPLC-DAD. The required time for the analysis of each sample by the HPLC-DAD system was considered to be 8 min. RESULTS: The results indicated that the highest amount of lupeol (7.82 mg/g DW) and stigmasterol (6.76 mg/g DW) was obtained using 50% methanol at 45 °C and ultrasonication for 30 min, and 50% methanol in 35 °C, and ultrasonication for 30 min, respectively. Using the response surface methodology, the predicted conditions for lupeol and stigmasterol from root of M. azedarach were as follows; lupeol: 100% methanol, temperature 45 °C and ultrasonication time 40 min (14.540 mg/g DW) and stigmasterol 43.75% methanol, temperature 34.4 °C and ultrasonication time 25.3 min (5.832 mg/g DW). CONCLUSIONS: The results showed that the amount of secondary metabolites lupeol and stigmasterol in the root of M. azedarach could be improved by optimizing the extraction process utilizing response surface methodology.

Rheological and physical properties of yogurt enriched with phytosterol during storage.

Phytosterols enriched products are innovative types of functional foods, in which dairy products, like low fat yogurt are ideal vehicles for this functional component. In this study, phytosterol dispersions were prepared using an oil/water (O/W) emulsion. The emulsion was added to yogurt milk. pH, titratable acidity (TA), syneresis, firmness and apparent viscosity of enriched yogurt were determined during storage. Moreover, phytosterols distribution in different parts of enriched yogurt was studied. Results indicated that in enriched yogurt, apparent viscosity and syneresis were lower and firmness was higher compared to the control. Addition of phytosterol to the yogurt had significant effect on acidity. Distribution of phytosterols in different parts of one sample was not uniform. Sensory results showed that there was no significant difference between enriched and control on texture, appearance, flavor and overall acceptance.

Cloning, identification and expression analysis of ACC oxidase gene involved in ethylene production pathway.

1-aminocyclopropane-1-carboxylic acid oxidase (ACO) enzyme is a member of the Fe II-dependent family of oxidases/oxygenases which require Fe(2+) as a cofactor, ascorbate as a cosubstrate and CO(2) as an activator. This enzyme catalyses the terminal step in the plant signaling of ethylene biosynthetic pathway. A 948 bp fragment of the ACO1 gene cDNA sequence was cloned from tomato (Lycopersicon esculentum) fruit tissues by using reverse transcriptase-polymerase chain reaction (RT-PCR) with two PCR primers designed according to the sequence of a tomato cDNA clone (X58273). The BLAST search showed a high level of similarity (77-98 %) between ACO1 and ACO genes of other plants. The calculated molecular mass and predicted isoelectric point of LeACO1 were 35.8 kDa and 5.13, respectively. The three-dimensional structure studies illustrated that the LeACO1 protein folds into a compact jelly-roll motif comprised of 8 α-helices, 12 ß-strands and several long loops. The cosubstrate was located in a cofactor-binding pocket referred to as a 2-His-1-carboxylate facial triad. Semi-quantitative RT-PCR analysis of gene expression revealed that the LeACO1 was expressed in fruit tissues at different ripening stages.

Comprehensive RNA-Seq-based study and metabolite profiling to identify genes involved in podophyllotoxin biosynthesis in Linum album Kotschy ex Boiss. (Linaceae).

Linum album is a well-known rich source of anticancer compounds, i.e., podophyllotoxin (PTOX) and other lignans. These compounds play an important role in the plant's defensive system. The RNA-Seq data of flax (L. usitatissimum) were analyzed under various biotic and abiotic stresses to comprehend better the importance of lignans in plant defense responses. Then, the association between the lignan contents and some related gene expressions was experimented with HPLC and qRT-PCR, respectively. Transcriptomic profiling showed a specific expression pattern in different organs, and just the commonly regulated gene EP3 was detected with a significant increase under all stresses. The in silico analysis of the PTOX biosynthesis pathway identified a list of genes, including laccase (LAC11), lactoperoxidase (POD), 4-coumarate-CoA ligase (4CL), and secoisolariciresinol dehydrogenase (SDH). These genes increased significantly under individual stresses. The HPLC analysis showed that the measured lignan contents generally increased under stress. In contrast, a quantitative expression of the genes involved in this pathway using qRT-PCR showed a different pattern that seems to contribute to regulating PTOX content in response to stress. Identified modifications of critical genes related to PTOX biosynthesis in response to multiple stresses can provide a baseline for improving PTOX content in L. album.

Isolation, identification and sequence analysis of a thioredoxin h gene, a member of subgroup III of h-type Trxs from grape (Vitis vinifera L. cv. Askari).

Thioredoxins (Trxs) are small ubiquitous proteins which play a regulatory role in a variety of cellular processes. In contrast to other organisms, plants have a great number of Trx types, consisting of six well-defined groups: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A full-length cDNA, designated VvCxxS2, encoding Trx h polypeptide was isolated and cloned from grape (Vitis vinifera L. cv. Askari) berries organ by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was 381 bp nucleotides in length with a deduced amino acid of 126 residues, possessing a WCIPS active site, which belongs to the subgroup III of h-type Trxs based on phylogenetic analysis. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptide are 14.25 kDa and 4.68, respectively. Nucleotide sequence analysis of genomic DNA fragment of VvCxxS2 gene revealed that this gene possesses two introns at positions identical to the previously sequenced Trx h genes. A modeling analysis indicated that VvCxxS2 shares a common structure with other Trxs, and is preferably reduced by Grx rather than NADPH-dependent thioredoxin reductase (NTR). The deduced protein sequence showed a high similarity to Trx h from other plants, in particular from castor bean (Ricinus communis), Betula pendula and sweet orange (Citrus sinensis). Semiquantitative RT-PCR experiments indicated that the transcripts of VvCxxS2 gene are present in all plant organs and different developmental stages. In addition, the higher expression of the VvCxxS2 gene was observed in berry organ as compared to the other organs.

Improvement and prediction of secondary metabolites production under yeast extract elicitation of Azadirachta indica cell suspension culture using response surface methodology.

Neem is a medicinal plant used as antimalarial, antibacterial, antiviral, insecticide, and antimicrobial drug. This study aimed to investigate and predict the effect of yeast extract and sampling time on cell growth, secondary metabolites synthesis, SQS1 and MOF1 genes expression by response surface methodology. The highest fresh and dry cell weights were 580.25 g/L and 21.01 g/L, respectively obtained 6 days after using 100 mg/L yeast extract. The highest azadirachtin accumulation and production were 16.08 mg/g DW and 219.78 mg/L obtained 2 and 4 days, respectively after using 25 mg/L yeast extract. Maximum mevalonic acid accumulation (1.75 mg/g DW) and production (23.77 mg/L) were observed 2 days after application of 50 mg/L yeast extract. The highest amount of squalene accumulation (0.22 mg/g DW) and production (4.53 mg/L) were achieved 4 days after using 50 mg/L yeast extract. Prediction results exhibited the highest azadirachtin accumulation (13.61 mg/g DW) and production (190.50 mg/L), mevalonic acid accumulation (0.50 mg/g DW) and production (5.57 mg/L), and squalene accumulation (0.30 mg/g DW) by using 245 mg/L yeast extract for 2 days, 71 mg/L yeast extract for 2 days, 200 mg/L yeast extract for 4.96 days, without yeast extract for 6.54 days and 4 days, respectively. Also, it was predicted that the highest squalene production is achieved by long-term exposure to high concentrations of yeast extract. The qRT-PCR analysis displayed the maximum relative gene expression of SQS1 and MOF1 by using 150 and 25 mg/L yeast extract for 4 and 2 days treatment.

New biological trends on cell and callus growth and azadirachtin production in Azadirachta indica.

Azadirachtin is an important secondary metabolite from Azadirachta indica used as a natural biopesticide. This study is the first comprehensive report concerning the influence of plant growth regulators on callus induction, cell suspension growth, and azadirachtin accumulation and production in cell suspension cultures of A. indica. We investigated the effect of plant growth regulators including different types of auxins and cytokinins and their combinations on callus induction, cell suspension growth, and azadirachtin accumulation and production. The highest percentage of callusing (100%) obtained at different combinations of plant growth regulators on MS medium supplemented with 1 mg/L picloram and 2 mg/L kinetin and the highest fresh weight of callus (264.50 mg) was observed in MS medium containing 1.5 mg/L NAA and 3 mg/L kinetin. In cell suspension cultures, the maximum cell density, SCV, and PCV were 2.44 × 106 cells per mL, 97.95%, and 81.46%, respectively, obtained in the MS medium containing 1.5 mg/L 2,4-D and 3 mg/L zeatin riboside. The highest average growth rate (0.25 days) was on MS medium containing 1.5 mg/L NAA and 3 mg/L zeatin riboside. The MS medium supplemented with 1 mg/L picloram and 2 mg/L kinetin produced the highest amount of fresh cell weight (493.02 g/L), dry cell weight (77.27 g/L), azadirachtin accumulation (3.69 mg/gDW), and azadirachtin production (285.64 mg/L). The results showed that all measured indices had positive correlation with together except FCW and DCW with azadirachtin accumulation.

Computer-based tools provide new insight into the key factors that cause physiological disorders of pistachio rootstocks cultured in vitro.

During the in vitro culture of plants some physiological disorders caused major problems that have been associated with culture media composition. The objective of this study was to better understand the abnormal physiological response of two pistachio rootstocks to changes in culture media ingredients. On this purpose, two computer-based tools were employed: design of experiment (DOE) and neurofuzzy logic. DOE was employed to generate a five-dimensional IV-design spaces allowing to reduce the number of treatments from 6,250 to 61. The second one, an artificial intelligence (AI) tool, neurofuzzy logic, was used to understand the cause-effect relationships between the factors studied (25) and seven physiological disorders including shoot-tip necrosis (STN), leaf necrosis (LN), leaf color (LC), basal callus (BC) formation, shoot fasciation (SF), hyperhydricity and epinasty, typically described during pistachio in vitro culture. Four out of the seven disorders were successfully modeled, being significantly affected by a limited number of factors. STN and BC were significantly affected by the concentration of EDTA-. However, while a low concentration of EDTA- reduces the STN, promotes BC. LN and LC were strongly alleviated by high amounts of thiamine-HCl. Undoubtedly, the results demonstrate the importance of recording and using data related to physiological disorders along with growth parameters when developing suitable culture media for plant tissues. The computer-based tools have been useful to: i) well sample experimental design; ii) reduce the final number of treatments and the experimental work; iii) identify the key factors affecting each disorder; iv) get insight about the causes that promote the appearance of physiological disorders. Our findings demonstrate that the recently AI designed POM media, although not optimal, is the most suitable (favouring growth and limiting physiological abnormalities) media for in vitro culture of pistachio compared to those media, currently used.

Combining DOE With Neurofuzzy Logic for Healthy Mineral Nutrition of Pistachio Rootstocks in vitro Culture.

The aim of this study was to determine the effects of Murashige and Skoog (MS) salts on optimal growth of two pistachio rootstocks, P. vera cv. "Ghazvini" and "UCB1" using design of experiments (DOE) and artificial intelligence (AI) tools. MS medium with 14 macro-and micro-elements was used as base point and its concentration varied from 0 to 5 × MS concentrations. Design of experiments (DOE) software was used to generate a five-dimensional design space by categorizing MS salts into five independent factors (NH4NO3, KNO3, mesos, micros and iron), reducing the experimental design space from 3,125 to just 29 treatments. Typical plant growth parameters such as shoot quality (SQ), proliferation rate (PR), shoot length (SL), and some physiological disorders including shoot-tip necrosis (STN) and callus formation at the base of explants (BC) were evaluated for each treatment. The results were successfully modeled using neurofuzzy logic software. The model delivered new insights, by different sets of "IF-THEN" rules, pinpointing the key role of some ion interactions ( SO 4 2 - × Cl-, K+ × SO 4 2 - × EDTA-, and Fe2+ × Cu2+ × NO 3 - ) for SQ, PR, and SL, whilst physiological disorders (STN and BC) were governed mainly by independent ions as Fe2+ and EDTA-, respectively. In our opinion, the methodology and results obtained in this study is extremely useful to understand the effect of mineral nutrients on pistachio in vitro culture, through discovering new complex interactions among macro-and micro-elements which can be implemented to design new media of plant tissue culture and improve healthy plant micropropagation for any plant species.

Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides.

Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR.