Transcriptome and Metabolome Profiling Provide Insights into Flavonoid Synthesis in Acanthus ilicifolius Linn.

Acanthus ilicifolius is an important medicinal plant in mangrove forests, which is rich in secondary metabolites with various biological activities. In this study, we used transcriptomic analysis to obtain differentially expressed genes in the flavonoid metabolic pathway and metabolomic methods to detect changes in the types and content in the flavonoid metabolic synthesis pathway. The results showed that DEGs were identified in the mature roots vs. leaves comparison (9001 up-regulated and 8910 down-regulated), mature roots vs. stems comparison (5861 up-regulated and 7374 down-regulated), and mature stems vs. leaves comparison (10,837 up-regulated and 11,903 down-regulated). Furthermore, two AiCHS genes and four AiCHI genes were up-regulated in the mature roots vs. stems of mature A. ilicifolius, and were down-regulated in mature stems vs. leaves, which were highly expressed in the A. ilicifolius stems. A total of 215 differential metabolites were found in the roots vs. leaves of mature A. ilicifolius, 173 differential metabolites in the roots vs. stems, and 228 differential metabolites in the stems vs. leaves. The metabolomic results showed that some flavonoids in A. ilicifolius stems were higher than in the roots. A total of 18 flavonoid differential metabolites were detected in the roots, stems, and leaves of mature A. ilicifolius. In mature leaves, quercetin-3-O-glucoside-7-O-rhamnoside, gossypitrin, isoquercitrin, quercetin 3,7-bis-O-ß-D-glucoside, and isorhamnetin 3-O-ß-(2″-O-acetyl-ß-D-glucuronide) were found in a high content, while in mature roots, di-O-methylquercetin and isorhamnetin were the major compounds. The combined analysis of the metabolome and transcriptome revealed that DEGs and differential metabolites were related to flavonoid biosynthesis. This study provides a theoretical basis for analyzing the molecular mechanism of flavonoid synthesis in A. ilicifolius and provides a reference for further research and exploitation of its medicinal value.

α-Amylase inhibitory potential of Thunbergia mysorensis leaves extract and bioactive compounds by in vitro and computational approach.

In this study, we aim to evaluate the anti-diabetic potential of Thunbergia mysorensis leaves methanolic extract (MeL) using inhibitory assays for α-glucosidase (AG), α-amylase (AM) (carbohydrate digestive enzymes) and aldose reductase (AR) (an enzyme involved in the polyol pathway responsible for glycation). In addition to antidiabetic studies, antioxidant studies were also performed due to the fact that reactive oxygen species (ROS) are produced by various pathways under diabetic conditions. Hyperglycemia induces ROS by activating the glycation reaction and the electron transport chain in mitochondria. The MeL effectively inhibited the enzymes (AG IC50: 27.86 ± 1.0, AM IC50: 12.00 ± 0.0, AR IC50: 4.50 ± 0.09 µg/mL) and showed effective radical ion scavenging activity during the antioxidant assay (DPPH EC50: 30.10 ± 0.75, ABTS EC50: 27.25 ± 1.00, Superoxide EC50: 35.00 ± 1.50 µg/mL). Using activity-guided repeated fractionation on a silica gel column chromatography, two compounds including 3,4-dimethoxy benzoic acid (DMBA) (101 mg) and 3,4-dimethoxy cinnamic acid (DMCA) (87 mg) with potent anti-diabetic activity were extracted from the MeL of T. mysorensis leaves. Both DMBA (IC50 AG: 27.00 ± 1.05, IC50 AM: 12.15 ± 0.10, IC50 AR: 4.86 ± 0.30 µg/mL) and DMCA (IC50 AG: 27.25 ± 0.98, IC50 AM: 12.50 ± 0.20, IC50 AR: 5.00 ± 1.00 µg/mL) were subjected for enzyme inhibition. Since both compounds significantly inhibited AM, enzyme kinetics for AM inhibition was performed. The compounds also showed effective antioxidant potential (DPPH EC50: 30.50 ± 0.99, ABTS EC50: 27.86 ± 0.16, Superoxide EC50: 36.10 ± 0.24 µg/mL), and DMCA (DPPH EC50: 31.00 ± 1.00, ABTS EC50: 28.00 ± 0.25, Superoxide EC50: 36.25 ± 0.37 µg/mL). Further, to elucidate the role of DMBA and DMCA in enzyme inhibition and stability at the molecular level, both compounds were subjected for in silico enzyme inhibitory studies using molecular docking simulation, molecular dynamics (MD) simulation, and binding free energy calculations. Compared to AR and AG, AM was the most significantly inhibited enzyme (DMBA: -6.6 and DMCA: -7.8 kcal/mol), and compounds combined with AM were subjected to MD simulation. Both compounds were stable in the binding pocket of AM till 100 ns and chiefly use Van der Waal's energy to bind. Compared to the controls, both DMBA and DMCA had a higher efficiency in the inhibition of target enzymes in vitro and in silico. The presence of DMBA and DMCA is more likely to be associated with the potential of MeL in antihyperglycemic activity. This bio-computational study indicates DMBA and DMCA as potential lead inhibitors of AM and could be used as effective anti-diabetic drugs in the near future.

Expression of TNF-α on Wistar Rat (Rattus norvegicus L.) with Extract of Pletekan Leaves (Ruellia tuberosa L.).

<b>Background and Objective:</b> Alcoholic drink produced traditionally by Maluku-Indonesia is called sopi, if consumed in excessive doses, it causes kidney damage. The ability of pletekan leaf extract (<i>Ruellia tuberosa</i> L.) is used as an alternative pharmacy because it has strong antioxidant activity. This study aimed to determine the expression of TNF-α in the kidney of Wistar rats (<i>Rattus norvegicus </i>L.) exposed to sopi alcoholic beverages after being treated with a pletekan herbal extract (<i>Ruellia tuberosa</i> L.). <b>Materials and Methods:</b> Thirty Wistar rats with an average weight of 200 g, divided by group I control (-) were given demineralized water, group II control (+) only given sopi, group III-V were given sopi 2.5 mL/head/body weight and pletekan leaf extract with a concentration of 5.04 mg/100 mL water, 10.08 mg/100 mL water and 12.12 mg/100 mL water for each group. This treatment is done for 24 days by giving sopi 3 times a week, then taking the kidney organ to make kidney histology. Observation of TNF-α expression by immunohistochemistry methods. <b>Results:</b> Wistar rats (<i>R. norvegicus </i>L.) exposed to sopi had decreased creatinine levels then were treated with ethanol extract of pletekan leaf (<i>R. tuberosa </i>L.) at a dose of 6% (12.12 mg/100 mL water) which was more effective in reducing creatinine levels and decreased the expression of TNF-α kidney Wistar rats. <b>Conclusion:</b> Pletekan leaf (<i>Ruellia tuberosa</i> L.) ethanol extract has the potential to prevent/repair kidney damage which is characterized by a decrease in the expression of TNF-α kidney Wistar rats (<i>Rattus norvegicus</i> L.) in line with an increase in the extract dosage in all treatments of sopi.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Adhatoda vasica (Nees.): A Review on its Botany, Traditional uses, Phytochemistry, Pharmacological Activities and Toxicity.

BACKGROUND: Adhatoda vasica (Nees.) of the family Acanthaceae has been used in the Southeast tropical zone as it is efficacious against headache, colds, cough, whooping cough, fever, asthma, dyspnea, phthisis, jaundice, chronic bronchitis, and diarrhea. It exhibits commendable pharmacological activities. OBJECTIVE: The aim of the review is to provide a systematic overview of pharmacological activities with toxicity and clinical assessment, phytochemistry of A. vasica along with its characterization, geographical observation, phenology, traditional uses, as well as an organized representation of the findings. METHOD: The overall information of A. vasica was collected from various resources, including books, review papers, research papers, and reports which were obtained from an online search of globallyaccepted scientific databases. ChemDraw software was used to draw the compound's structure. RESULTS: Phytochemical review on A. vasica has led to the collection of 233 compounds of different types such as alkaloids, flavonoids, essential oils, terpenoids, fatty acids, phenols, etc. It is a promising source of potential phytopharmaceutical agent that exhibits diverse pharmacological activities, including antibacterial, antifungal, hepatoprotective, anti-ulcer, abortifacient, antiviral, antiinflammatory, thrombolytic, hypoglycemic, anti-tubercular, antioxidant, and antitussive activities. CONCLUSIONS: Sufficient number of studies on ethnopharmacology, traditional uses, and pharmacological activities of A. vasica are conducted. Furthermore, it is necessary to study the activity of chemical constituents for new drug design and discovery from natural products.

Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63.

Strobilanthes cusia (Nees) Kuntze is a Chinese herbal medicine used in the treatment of respiratory virus infections. The methanol extract of S. cusia leaf contains chemical components such as ß-sitosterol, indirubin, tryptanthrin, betulin, indigodole A, and indigodole B that have diverse biological activities. However, the antiviral action of S. cusia leaf and its components against human coronavirus remains to be elucidated. Human coronavirus NL63 infection is frequent among immunocompromised individuals, young children, and in the elderly. This study investigated the anti-Human coronavirus NL63 (HCoV-NL63) activity of the methanol extract of S. cusia leaf and its major components. The methanol extract of S. cusia leaf effectively inhibited the cytopathic effect (CPE) and virus yield (IC50 = 0.64 µg/mL) in HCoV-NL63-infected cells. Moreover, this extract potently inhibited the HCoV-NL63 infection in a concentration-dependent manner. Among the six components identified in the methanol extract of S. cusia leaf, tryptanthrin and indigodole B (5aR-ethyltryptanthrin) exhibited potent antiviral activity in reducing the CPE and progeny virus production. The IC50 values against virus yield were 1.52 µM and 2.60 µM for tryptanthrin and indigodole B, respectively. Different modes of time-of-addition/removal assay indicated that tryptanthrin prevented the early and late stages of HCoV-NL63 replication, particularly by blocking viral RNA genome synthesis and papain-like protease 2 activity. Notably, tryptanthrin (IC50 = 0.06 µM) and indigodole B (IC50 = 2.09 µM) exhibited strong virucidal activity as well. This study identified tryptanthrin as the key active component of S. cusia leaf methanol extract that acted against HCoV-NL63 in a cell-type independent manner. The results specify that tryptanthrin possesses antiviral potential against HCoV-NL63 infection.

RETRACTED: Biosynthesis of Clinacanthus nutans Lindau leaf extract mediated ag NPs, au NPs and their comparative strong muscle relaxant, analgesic activities for pain management in nursing care for using in intensive nursing care unit.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor. After a thorough investigation, the Editor has concluded that the acceptance of this article was partly based upon the positive advice of one illegitimate reviewer report. The report was submitted from an email account which was provided by the corresponding author as a suggested reviewer during the submission of the article. Although purportedly a real reviewer account, the Editor has concluded that this was not of an appropriate, independent reviewer. This manipulation of the peer-review process represents a clear violation of the fundamentals of peer review, our publishing policies, and publishing ethics standards. Apologies are offered to the reviewer whose identity was assumed and to the readers of the journal that this deception was not detected during the submission process.

The Blepharis persica seed hydroalcoholic extract synergistically enhances the apoptotic effect of doxorubicin in human colon cancer and gastric cancer cells.

The goal of this survey is to evaluate the anti-proliferative effects of the hydroalcholic extract of Blepharis persica seeds and its synergic effect on doxorubicin (DOX) in human colon cancer (HT-29) and gastric cancer cell (AGS) lines. 70% Ethanol was used for extraction of B. persica seed. Aluminum-chloride colorimetric and Folin-Ciocalteu reagent methods were used to measure total flavonoid and total phenolic contents of the extract respectively. Gas chromatography-mass spectrometry (GC-MS) analysis of the B. persica extract was performed on GC-MS equipment after silylation. HT-29, AGS, and human fibroblast (SKM) cell lines were treated by different concentration of the B. persica extract, (DOX) and the combination of extraction and DOX. The cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay while the apoptosis induction was monitored using flowcytometry by annexin-V FITC/PI double-staining. The changes in expression levels of BAX and BCL-2 were determined using Real-Time RT-qPCR. GC-MS analysis of the hydroalcoholic extract from B. persica seeds revealed 24 major components. The MTT assay revealed the cytotoxicity against three cell lines and also it was shown that 125 ng/mL of DOX and 0.625 mg/mL of B. persica extract had synergistic behavior against HT29 cell line. These results showed B. persica extract induced apoptosis in AGS and HT29 cells and its extract caused dose-dependent increase in up-regulation of BAX level (p < 0.05) and down-regulation of BCL2 (p < 0.05). B. persica showed the synergistic effect in combination with DOX on HT29 cell line. These findings demonstrated a basis for further studies on the characterization and mechanistic evaluation of the bioactive compounds of B. persica extract which had antiproliferative effects on cancer cell lines.

De novo characterization of the Baphicacanthus cusia(Nees) Bremek transcriptome and analysis of candidate genes involved in indican biosynthesis and metabolism.

Baphicacanthus cusia (Nees) Bremek is an herb widely used for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine. The roots, stems and leaves can be used as natural medicine, in which indigo and indirubin are two main active ingredients. In this study, quantification of indigo, indirubin, indican and adenosine among various tissues of B. cusia was conducted using HPLC-DAD. Leaves have significantly higher contents than stems and roots (380.66, 315.15, 20,978.26, 4323.15 µg/g in leaves, 306.36, 71.71, 3,056.78, 139.45 µg/g in stems, and 9.31, 7.82, 170.45, 197.48 µg/g in roots, respectively). De novo transcriptome sequencing of B. cusia was performed for the first time. The sequencing yielded 137,216,248, 122,837,394 and 140,240,688 clean reads from leaves, stems and roots respectively, which were assembled into 51,381 unique sequences. A total of 33,317 unigenes could be annotated using the databases of Nr, Swiss-Prot, KEGG and KOG. These analyses provided a detailed view of the enzymes involved in indican backbone biosynthesis, such as cytochrome P450, UDP-glycosyltransferase, glucosidase and tryptophan synthase. Analysis results showed that tryptophan synthase was the candidate gene involved in the tissue-specific biosynthesis of indican. We also detected sixteen types of simple sequence repeats in RNA-Seq data for use in future molecular mark assisted breeding studies. The results will be helpful in further analysis of B. cusia functional genomics, especially in increasing biosynthesis of indican through biotechnological approaches and metabolic regulation.

Surface active gold nanoparticles biosynthesis by new approach for bionanocatalytic activity.

In the present day, nanotechnology is one of the most promising leading scientific and potentials areas in modern key technology development toward to the humankind. The synthesis of noble metal nanoparticles (NPs) is an expanding research area due to the possible applications for the development of bio-medical applications. Eco-friendly approach for the biosynthesis of gold nanoparticles (AuNPs) using the aqueous extract from Ruellia tuberosa and Phyllanthus acidus (leaf and twig) for the first time. Surface active AuNPs were characterized by UV-Vis spectroscopy, FTIR (Fourier transform infrared) spectroscopy, DSC (differential scanning colorimetry), DLS (dynamic light scattering) and environmental SEM (scanning electron microscope) analysis at room temperature (RT). Enhanced surface plasmon resonance (SPR) absorbance UV visible optical spectra were detected in the range of 552, 548, 558 and 536 nm. SEM and DLS (transmission mode) analysis confirmed the morphology of the nanoparticles to be spherical with the average size in the range of 88.37, 94.31, 82.23 and 81.36 nm. Further they have enhanced the enzyme activity on α-amylase, cellulase, and xylanase. The results suggest that the phyto-fabricated AuNPs from R. tuberosa and P. acidus is simple, less expensive, eco-friendly, green synthesis and also can be exploited for the potential future industrial and bio-medical applications.

Facile biosynthesis of silver nanoparticles using Barleria cristata: mosquitocidal potential and biotoxicity on three non-target aquatic organisms.

Mosquitoes (Diptera: Culicidae) act as vectors of important pathogens and parasites, such as malaria, dengue, chikungunya, Japanese encephalitis and lymphatic filariasis. The use of synthetic mosquitocides often leads to high operational costs and adverse non-target effects. Recently, plant-borne compounds have been proposed for rapid extracellular biosynthesis of mosquitocidal nanoparticles. However, the impact of these nanomosquitocides against biological control agents of mosquito larval populations has been poorly studied. In this research, we biosynthesized silver nanoparticles (Ag NP) using the Barleria cristata leaf extract as a reducing and stabilizing agent. The biosynthesis of Ag NP was confirmed analyzing the excitation of surface plasmon resonance using ultraviolet-visible (UV-vis) spectrophotometry. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed the clustered and irregular shapes of Ag NP. The presence of silver was confirmed by energy-dispersive X-ray (EDX) spectroscopy. Fourier transform infrared (FTIR) spectroscopy investigated the identity of secondary metabolites, which may also act as Ag NP capping agents. The acute toxicity of B. cristata leaf extract and biosynthesized Ag NP was evaluated against larvae of Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus. Compared to the leaf aqueous extract, biosynthesized Ag NP showed higher toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with lethal concentration (LC)50 values of 12.46, 13.49, and 15.01 µg/mL, respectively. Notably, biosynthesized Ag NP were found safer to non-target organisms Diplonychus indicus, Anisops bouvieri, and Gambusia affinis, with respective LC50 values ranging from 633.26 to 866.92 µg/mL. Overall, our results highlight that B. cristata-fabricated Ag NP are a promising and eco-friendly tool against young instar populations of mosquito vectors of medical and veterinary importance.

Selenopeptides and elemental selenium in Thunbergia alata after exposure to selenite: quantification method for elemental selenium.

Three month old Thunbergia alata were exposed for 13 days to 10 µM selenite to determine the biotransformation of selenite in their roots. Selenium in formic acid extracts (80 ± 3%) was present as selenopeptides with Se-S bonds and selenium-PC complexes (selenocysteinyl-2-3-dihydroxypropionyl-glutathione, seleno-phytochelatin2, seleno-di-glutathione). An analytical method using HPLC-ICPMS to detect and quantify elemental selenium in roots of T. alata plants using sodium sulfite to quantitatively transform elemental selenium to selenosulfate was also developed. Elemental selenium was determined as 18 ± 4% of the total selenium in the roots which was equivalent to the selenium not extracted using formic acid extraction. The results are in an agreement with the XAS measurements of the exposed roots which showed no occurrence of selenite or selenate but a mixture of selenocysteine and elemental selenium.

Secondary metabolites isolated from Castilleja rubra exert anti-inflammatory effects through NF-κB inactivation on lipopolysaccharide-induced RAW264.7 macrophages.

8-Epiloganin (1), mussaenoside (2), and 5-O-caffeoylshikimic acid (3) have been isolated from Castilleja rubra, and the anti-inflammatory properties of these metabolites in a cell culture system were investigated. Compounds 1-3 suppressed not only the production of nitric oxide (NO) and prostaglandin E2, but also the expression of inducible NO synthase and cyclooxygenase-2 induced by lipopolysaccharide (LPS) in the RAW264.7 murine macrophage cell line. Compounds 1-3 also inhibited the release of pro-inflammatory cytokines induced by LPS, namely, tumor necrosis factor-α and interleukin-1ß. The underlying mechanism of the anti-inflammatory action of compounds 1-3 was associated with downregulation of nuclear factor-κB.

Rhinacanthus nasutus extracts prevent glutamate and amyloid-ß neurotoxicity in HT-22 mouse hippocampal cells: possible active compounds include lupeol, stigmasterol and ß-sitosterol.

The Herb Rhinacanthus nasutus (L.) Kurz, which is native to Thailand and Southeast Asia, has become known for its antioxidant properties. Neuronal loss in a number of diseases including Alzheimer's disease is thought to result, in part, from oxidative stress. Glutamate causes cell death in the mouse hippocampal cell line, HT-22, by unbalancing redox homeostasis, brought about by a reduction in glutathione levels, and amyloid-ß has been shown to induce reactive oxygen species (ROS) production. Here in, we show that ethanol extracts of R. nasutus leaf and root are capable of dose dependently attenuating the neuron cell death caused by both glutamate and amyloid-ß treatment. We used free radical scavenging assays to measure the extracts antioxidant activities and as well as quantifying phenolic, flavonoid and sterol content. Molecules found in R. nasutus, lupeol, stigmasterol and ß-sitosterol are protective against glutamate toxicity.

Effect of medium composition and light on root and rhinacanthin formation in Rhinacanthus nasutus cultures.

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) has long been used in Thai traditional medicine for treatment of tinea versicolor, ringworm, pruritic rash, and abscess. The active constituents are known as a group of naphthoquinone esters, rhinacanthins. This work focused on establishment of R. nasutus root cultures and determination of rhinacanthin production. Induction of R. nasutus root formation was accomplished on solid Gamborg's B5 (B5) medium, supplied with 0.1 mg/L indole-3-butyric acid (IBA) and 20 g/L sucrose. The effects of explants (whole leaf explants and four-side excised leaf explants), light and medium composition on root and rhinacanthin formation were investigated. The root formation from the whole leaf explants was 10 times higher than that from the four-side excised leaf explants. In addition, light possessed an inhibitory effect on the root and rhinacanthin formation of R. nasutus. Medium manipulation found that Murashige and Skoog (MS) medium supplied with 3 mg/L IBA and 30 g/L sucrose was the most suitable for induction of the root formation. Unfortunately, the obtained root cultures produced only rhinacanthin-C in very low amount, 0.026 mg/g dry weight (DW), when they were transferred into the same MS liquid medium. With semisolid medium (4 g/L agar) of the same MS composition, however, the root cultures appeared to produce higher content of rhinacanthin-C, -D and -N (3.45, 0.07 and 0.07 mg/g DW, respectively). Our finding suggests that culturing in semisolid medium is capable of improving of rhinacanthin production in R. nasutus root cultures.

Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) Nees--a medicinal herb.

High frequency plant regeneration in A. longifolia (L.) was achieved from leaf explant implanted on MS basal medium supplemented with NAA (0.5 mg/l) + BA (2.0 mg/l) through intervening callus phase. Well-developed shoots (>3cm) were successfully rooted on MS medium supplemented with NAA (0.1 mg/l). Protein and total soluble sugar contents were maximum during organogenesis and multiple shoot induction phase compared with non-organogenic callus and root induction phase. Esterase and catalase activities were maximum during organogenic differentiation, while activities were minimum at non-differentiated callus stages. Peroxidase activities were higher during rhizogenesis. Contradiction to peroxidase activity, acid phosphatase activities were high during organogenesis and declined during rhizogenesis. SDS-PAGE analysis of total soluble proteins revealed expression of non-organogenic callus (97.9 kDa), organogenic callus (77.2, 74.1, 21.9 kDa), multiple shoot induction phase (106.6, 26.9, 11.6 kDa) and root induction phase (15.9 kDa) specific polypeptides. Esterase zymogram revealed one band (Rm 0.204) appeared in both organogenic callus and multiple shoot induction phase. Peroxidase zymogram detected two stage specific bands, one band (Rm 0.42) was specific to root induction phase, while another (Rm 0.761) was specific to multiple shoot induction. Catalase and acid phosphatase zymogram resolved one band (Rm 0.752 and 0.435, respectively) in differentiated stages including both multiple shoot induction phase and root induction phase, but absent in undifferentiated phases.

Complete assignments of 1H and 13C NMR data for 10 phenylethanoid glycosides.

Ten phenylethanoid glycosides, including two new ones, isolated from the aerial parts of the mangrove plant Acanthus ilicifolius were identified. The first complete assignments of the 1H and 13C NMR chemical shifts for these glycosides were achieved by means of 2D NMR techniques, including 1H-1H COSY, TOCSY, HSQC and HMBC spectra.