In vitro elicitation and detection of apigenin, catalpol and gallic acid in hairy root culture of Plantago major L. and assessment of cytotoxicity and anti-bacterial activity of its methanolic extract.

The aim of this study was to establish the hairy root (HR) culture of Plantago major to evaluate the accumulation of apigenin, catalpol and gallic acid after elicitation and investigate the biological activity of its methanolic extraction. The highest transformation frequency was obtained by Agrobacterium rhizogenes strain A4, 0.5 mg/L 6-Benzylaminopurine in pre-cultivation medium, 150 µM acetosyringone in co-cultivation medium (1/2 MS), and immersion method for inoculation of leaf explants. The production of apigenin, catalpol and gallic acid compounds were significantly affected by treatment of 1.18 mM AgNO3 at 24 h which yielded 4.30, 8.24 and 2.89-fold increase, respectively. The assessment of anti-bacterial activity showed that the methanolic extracts of the HRs elicited with 1.18 mM AgNO3 were significantly active against Proteus vulgaris (PTCC 1182) (MIC = 25 mg/mL and MBC = 25 mg/mL). Furthermore, the MTT assay revealed that the methanolic extracts of the HRs were cytotoxic on the SW-480 cell (IC50=337.56 ± 1.82 µg/mL).

HPLC-DAD-ESI/MSn analysis of phenolic components of Scutellaria araxensis, S. bornmuelleri and S. orientalis.

Scutellaria araxensis, S. bornmuelleri and S. orientalis are traditional herbs widely used by native Iranians for the treatment various ailments. In the current study, we used HPLC-DAD-ESI/MSn to investigate the presence of phenolic compounds of root and shoot methanolic extracts. The flavonoid composition were identified based on mass of pseudomolecular [M - H]- ions and fragmentation spectra and comparisons with literature data. A total of 26, 25, 17, 23, 19 and 26 flavonoids were identified from roots and shoots of S. araxensis, S. bornmuelleri and S. orientalis, respectively. The obtained phenolic profiles revealed the presence of the major flavonoid components of Scutellaria genus including baicalein, wogonin, scutellarein and their glycosides, as well as chrysin, tricin, skullcapflavon II, pinocembrin and phenylethanoid glycosides such as acteoside and verbascoside. This study, in addition to reveal the potency of chromatographic profiling for identification of Scutellaria flavonoids, introduces this species as a potential source of bioactive metabolites.

High-frequency adventitious shoot organogenesis from in vitro stem explants of Scutellaria araxensis Grossh.

The present study introduced an in vitro shoot organogenesis protocol for the medicinal plant Scutellaria araxensis (Lamiaceae). Stem, leaf, and petiole explants were cultured in half-strength Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP) alone or in combination with thidiazuron (TDZ), indole-3-butyric acid (IBA), or α-naphthalene acetic acid. Callus formation occurred from stem and petiole explants in most cultures; however, in leaf explants, it was observed only in cultures containing 0.5 mg/l BAP supplemented with TDZ at all concentrations. The highest frequency of indirect shoot induction (100 and 90%) with an average of 20.33 and 12 shoots per explant was observed in stem-derived calli cultured on half-strength MS medium containing 2.0 mg/l BAP plus 0.5 and 1.5 mg/l TDZ, respectively. The best direct shoot organogenesis (40%) was observed in stem explants cultured on half-strength MS medium containing 0.5 mg/l BAP and 0.5 mg/l IBA with a mean of 18 shoots per stem explant. The regenerated micro-shoots were elongated on a medium fortified with 0.5 mg/l gibberellic acid and then successfully rooted in half-strength MS medium supplemented with 0.5 mg/l IBA. The obtained plantlets were acclimatized in a growth chamber with a survival rate of 100%. This study is the first report of a simple and efficient in vitro shoot organogenesis and regeneration protocol for S. araxensis by using stem explants, which could be useful for the conservation, genetic manipulation, and exploitation of biological molecules of this valuable genetic source.

Fractional analysis of dichloromethane extract of Scutellaria araxensis Grossh root and shoot by HPLC-PDA-ESI-MSn.

Scutellaria araxensis is a well-known ethnobotanical herb for the treatment of various diseases in Iranian traditional medicine. In this study, dichloromethanolic fraction of partitioned methanol root and shoot extract was analysed by silica gel column chromatography, thin layer chromatography and high-performance liquid chromatography combined with photodiode-array detector, and coupled to electrospray ionization with Q-Exactive Orbitrap mass spectrometry (HPLC-PDA-ESI-MSn) in positive ion mode. Metabolites were tentatively characterized by comparing their mass spectrometry spectra with those of bibliographic data. A total of 11 flavonoids were identified from different fractions. Results showed that root and shoot produced very similar flavone patterns characterized by the presence of baicalein, tricin, tenaxin I, tenaxin II, skullcapflavon II, chrysin, wogonin and isorhamnetin. Norwogonin and apigenin-7-glucoside were identified only from shoot. Remarkably, Tenaxin I, II, tricin, apigenin-7-glucoside and norwogonin were tentatively profiled as new compounds for the first time from S. araxensis formula.

Impact of COVID-19 Infection on Neonatal Birth Outcomes.

INTRODUCTION: There is limited data on newborns born to mothers with COVID-19 infection. This multicenter cohort study aimed to investigate the clinical characteristics and outcomes of neonates born to mothers with and without COVID-19 infection to fill a gap in the literature review. METHODS: The medical records of all neonates in Isfahan, Iran, between October 2020 and March 2021, were retrospectively reviewed. RESULTS: Among the 600 neonates in this study, 255 (42.5%) were in the infected group and 345 (57.5%) were assigned to the control group as they were born to non-infected mothers. In the infected group, sepsis, fever and pneumothorax were detected in 3 (1.2%), 3 (1.2%) and 4 (1.6%) neonates, respectively, compared with no case in the control group. In the infected group, neonatal respiratory distress (NRDS) (32, 12.5%) was significantly higher than the control group (27, 10.6%). Asphyxia in the infected group was 22(6.4%), compared with 19 (5.5%), in the control group. Preterm labor (PTL) (55, 21.65%), premature rupture of membranes (PROMs) (24, 9.4%) and intra-uterine growth retardation (IUGR) (15, 5.9%) were significantly higher in women with COVID-19 (45, 13.0%, 4, 1.2% and 7, 2.0%, respectively). Low birth weight (LBW) accounted for 42 (16.5%) neonates in the infected group and 25 (7.2%) in the control group (p < 0.05). Of the 255 neonates born to infected mothers, 38 (14.9%) were admitted to the Neonatal Intensive Care Unit (NICU), compared with 31 out of 345 (9.0%) in the control group (p < 0.05). RT-PCR test results were positive in two newborns (0.8%), one of whom died of necrotizing enterocolitis. CONCLUSION: As a result of maternal COVID-19 infections, neonates experienced higher rates of sepsis, fever, pneumothorax, asphyxia and NRDS in addition to PTL, PROMs, IUGR, and LBW.

Contradictory results have been reported on the impact of COVID-19 infection on neonatal outcomes. We conducted a review of 600 cases of neonates, with 255 (42.5%) in the COVID-19 infected and 345 (57.5%) in the control group. The results indicated that neonates born to women with COVID-19 showed higher rates of sepsis, fever, pneumothorax, asphyxia and neonatal respiratory distress in addition to preterm labor, premature rupture of membranes, intra-uterin growth retardation and low birth weight.

Anti-Proliferative Properties, Biocompatibility, and Chemical Composition of Different Extracts of Plantago major Medicinal Plant.

Background: To study the anticancer activity of Plantago major, we assessed the effect of ethanolic, methanolic and acetonic extracts of this plant on HCT-116, SW-480, and HEK-293 cell lines as control. Methods: The cytotoxic activity, biocompatibility, and toxicity were evaluated by MTT assay, hemolysis, and Artemia salina-LD50 (on mice) tests, respectively. The analysis of the extracts was performed by GC-MS analysis. Results: The results showed that all the extracts had the most antiproliferative properties on the HCT-116 cell line. The P. major root extract was more effective than the aerial parts, and IC50 values for ethanolic, methanolic and acetonic root extracts were 405.59, 470.16, and 82.26 µg/mL, respectively on HCT-116 cell line at 72 h. Hemolysis degree of the ethanolic extract of aerial and root parts were approximately 1% at 400 µg/mL.. Using the ethanolic extracts, the Artemia survived every concentration, and no toxicity was observed. One week after the oral administration of different parts of P. major extracts, none of the mice died, even those were administered 2000 mg/kg. The results of GC/MS analysis showed that P. major extracts contain potential anticancer compounds, such as stearic acid (8.61%) in aerial parts of methanolic extract and 1,2- Benzenedicarboxylic acid, mono(2-ethylhexyl)ester (88.07% and 40.63%) in aerial and root parts of acetonic extract of P. major. Conclusion: Our findings suggest that the P. major is a source of potential compounds with antiproliferative properties.

Establishment and elicitation of transgenic root culture of Plantago lanceolata and evaluation of its anti-bacterial and cytotoxicity activity.

Hairy root induction in Plantago lanceolata was optimized to take advantage of transformed root cultures. The highest frequency of transformation was achieved using leaf explant, A4 strain, pre-cultivation of explant, 150 µM Acetosyringone, 5 min inoculation, half-strength Murashige and Skoog basal medium as co-cultivation, and half-strength Gamborg's basal medium as a selective medium with 3% sucrose. Among the studied compound encompassing gallic acid, catalpol and apigenin, only the production of gallic acid in hairy roots was affected by 20 mg L-1 AgNO3 and 100 mg L-1 chitosan at 24 hr which yielded 7.63, 4.76-fold increase in its content, respectively. The methanolic extracts of hairy roots elicited by 20 mg L-1 AgNO3 exhibited anti-bacterial activity (MIC and MBC = 25 mg mL-1) against Klebsiella pneumoniae, Proteus vulgaris and Salmonella typhi and anti-bacterial potential of non-elicited hairy roots of P. lanceolata (MIC = 25 mg mL-1 and MBC = 35 mg mL-1) were more active against Klebsiella pneumoniae and P. vulgaris than other bacteria. The methanolic extracts of the P. lanceolata hairy roots demonstrated significant cytotoxic activity on colorectal carcinoma cell line (SW-480) with IC50 = 250.65 ± 6.8 µg mL-1 in comparison to human embryonic kidney (HEK-293) with IC50 = 5263.65 ± 4.6 µg mL-1. Plantago lanceolata hairy roots showed important biological activity explaining its role in traditional medicine.

Enhanced flavonoid production in hairy root cultures of Scutellaria bornmuelleri by elicitor induced over-expression of MYB7 and FNSП2 genes.

For the purpose of the current study, hairy root induction in S. bornmuelleri, which is an important medicinal plant, was examined using a particular protocol. Accordingly, some factors such as four strain types of Agrobacterium rhizogenes (A4, A13, MSU440 and ATCC15834), three different explants, namely stem, petiole and leaf, two co-cultivation media, i.e. full and half-MS were studied. Besides, two inoculation methods including injection and immersion as well as three inoculation times (5, 7 and 10 min) were closely taken into account. Utilizing injection method by MSU440 strain, hairy root induction took place in stem explants, and a remarkable increase in transformation frequency (100%) was observed in half-strength MS medium. Methyl jasmonate (MeJA, 100 µM), methyl-b-cyclodextrin (b-CD, 0.7, 7 and 14 mM) and Chitosan (Chi, 50, 100 and 200 mg/l) were used either individually or in a combined way to elicitation. Based on the HPLC results, production of chrysin, wogonin and baicalein increased 9.15, 10.56 and 13.25 times after elicitation of hairy roots by MeJA + Chi. In addition, transcripts of FNSП2 and MYB7, two important genes involved in the flavonoid biosynthesis pathway, were studies. By applying Chi and MeJA + Chi elicitor, the expression of both genes increased noticeably. It can be concluded that the mentioned hairy root culture system of S. bornmuelleri can be an alternative to flavonoids production. Moreover, there is a direct and positive relationship between the expression of FNSП2 and MYB7 genes as well as the level of three flavonoids.

Comparison and evaluation of two diagnostic methods for detection of npt II and GUS genes in Nicotiana tabacum.

To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and also a visual detection protocol on the basis of npt II and GUS genes in transgenic tobacco plants were used. Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum leaf discs was performed with plant transformation vector of pBI 121. From kanamycin-resistant plants selected by their antibiotic resistance, four plants were selected for DNA isolation. Presence of the transgene was confirmed in the transformants by PCR and LAMP. In this regard, all LAMP and PCR primers were designed on the basis of the gene sequences of npt II and GUS. The LAMP assay was applied for direct detection of gene marker from plant samples without DNA extraction steps (direct LAMP assay). Also, a novel colorimetric LAMP assay for rapid and easy detection of npt II and GUS genes was developed here, its potential compared with PCR assay. The LAMP method, on the whole, had the following advantages over the PCR method: easy detection, high sensitivity, high efficiency, simple manipulation, safety, low cost, and user friendly.