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Lotus (Nelumbo nucifera) is one of the main aquatic vegetables in China. Its seed and rhizome are main edible parts which are rich in starch. Preliminary experiments of starch contents revealed that seed and rhizome expressed great differences in amylose and amylopectin contents. The rhizomes have higher amylopectin content, while the seeds have higher amylose content. In this study, we have estimated 16 varieties of lotus seeds and found that the amylose content of lotus seeds ranged from 30% to 50%, with an average amylose content of 43%, which showed high-amylose content characteristics. Morphological analysis of lotus seed shown that starch rapid accumulated in 20 DAF (day after fertilization) ∼ 26 DAF. Transcriptome of lotus seeds indicated that starch genes played an important role in seed development. Especially in 22 DAF, the genes which controlled amylose synthesis significantly increased, in contrast, the expression of amylopectin-related genes was stable and might limit the synthesis of amylopectin. We further analyzed the expression patterns of 11 key related genes between lotus seeds and rhizomes, and found that the expression of amylose-related genes to were higher in lotus seed, while the expression of genes related to amylopectin synthesis were higher in rhizome. This study provided a comprehensive research of molecular basis for starch in lotus seed and rhizome. Different expression among key genes during starch accumulation might be the principal cause of the differences in starch properties between seed and rhizome. PRACTICAL APPLICATION: Differential expression involved in starch synthesis pathway genes is the main reason for various starch characteristics of seed and rhizome in lotus. High amylose content in lotus seed is a valuable trait for developing functional food.

The study explored the cardioprotective role of the methanolic leaf extract of Nelumbo nucifera and nuciferine against isoproterenol-induced myocardial infarction (MI) in Wistar rats. Pretreatment with leaf extract and nuciferine (200 and 20 mg/kg body weight, respectively) against MI induced by isoproterenol (85 mg/kg body weight) significantly decreased heart weight; levels of cardiac markers such as lactate dehydrogenase and creatine kinase-MB were similar to those in controls. The treatment significantly increased the content of endogenous antioxidants and decreased lipid peroxidation in all treated groups. Treated groups showed a significant reduction in heartbeats per minute as compared with the MI-induced positive control. The MI-induced group showed pathological implications such as tachycardia, left atrial enlargement, and anterolateral ST-elevated MI, which were absent in treated groups. Histology confirmed that the leaf extract and nuciferine prevented structural abnormality and inflammation in heart and liver tissues of treated groups. On in silico analysis, nuciferine showed stronger binding interaction with both β1 and β2 adrenergic receptors than isoproterenol. Hence, the leaf extract of N. nucifera and nuciferine could be used as plant-based cardioprotective agents.

Microwave vacuum drying (MVD) was applied to dehydrate lotus (Nelumbo nucifera Gaertn.) seeds. This paper evaluated the changes of molecular, crystalline, and morphological structural properties of lotus seeds after MVD. The results showed the frequency of δ(CH) bending, β(CH), ν(OH), and carbon-oxygen double bonds (C = O) increased with increasing microwave power density. Moreover, as microwave power density increased, the transition enthalpy of crystallinity gradually increased, which was related to the formation of crystalline starch, re-crystallization, and complexes of starch-lipid structure depending on the hydrogen bonds formed. The MVD process resulted molecular polarity of lotus seeds, whereas the dielectric properties also changed. The dielectric properties interacted with the changes of molecules and crystalline structure. The morphology of lotus seed flour changed because the shape of starch granules was disrupted and the granules became aggregated with nonstarch components as the microwave power density increased. All these results explain the interactions among polymer molecules in lotus seeds dried by MVD.

Expeditious evolution of antimicrobial resistance in recent years has been identified as a growing concern by various health organizations around the world. Herein, facile and environmentally benign production of highly antibacterial carbonaceous nanomaterials from Colocasia esculenta and Nelumbo nucifera leaves is reported. After carbonization and oxidative treatment, smaller graphene domains are formed in Colocasia esculenta derivatives, whereas larger sheetlike structures are observed in the case of Nelumbo nucifera. Smaller particle size makes quantum confinement effects more prominent, as is evident in fine-tuning of the photoluminescence emission after each stage of treatment. The influence of precursor materials on the antibacterial properties of the nanosystems is also demonstrated. When microbiocidal activity was tested against model bacteria Pseudomonas aeruginosa, the nanocomposite derived from Colocasia esculenta leaves showed higher activity than the antibiotic drug clarithromycin (control) with a measured zone of inhibition of 40±0.5 mm. This is one of the highest values reported in comparison with plant-based carbon-silver nanosystems. Quantitative analysis revealed that the nanocomposite obtained from Colocasia esculenta leaves has antimicrobial efficacy equivalent to those of commercial antibiotic drugs and is able to eradicate bacteria at much lower concentrations than that obtained from Nelumbo nucifera leaves.

Doxorubicin (DOX) induced cardiotoxicity is a major problem during chemotherapy of cancers. DOX-mediated suppression of type 1 IGF receptor (IGF-1R) signaling leads to cardiac dysfunction. Neferine, a bisbezylisoquinoline alkaloid from the seed embryos of Nelumbo nucifera Gaertn possesses a distinct range of pharmacological properties. Herewith, the present study attempts to elucidate the protective role of neferine against DOX induced toxicity in H9c2 rat cardiomyoblast cell line model. DOX-treated H9c2 cells significantly increased mitochondrial superoxide generation, depleted cellular antioxidant status, suppressed the activation of IGF-1R signaling via PI3K/Akt/mTOR and induced autophagy by the activation of ULK1, Beclin1, Atg7, and LC3B. Neferine pre-treatment activated IGF-1R signaling, improved cellular antioxidant pool, increased the expression of down-stream targets of IGF-1R, such as PI3K/Akt/mTOR, inhibited mitochondrial superoxide generation and autophagy significantly with the induction of Nrf2 translocation and expressions of HO1 and SOD1. Our study suggests the use of neferine for amelioration of DOX-mediated cardiotoxicity.

Nelumbo nucifera, also known as sacred lotus, has primarily been used as food throughout the Asian continent, and its medicinal values have been described in Ayurvedic and Traditional Chinese Medicine. The purpose of this study is to systematically characterize the chemical profiling and pharmacological activities of N. nucifera. Herein, we critically reviewed and analysed the phytochemical and pharmacological reports of N. nucifera. Our search for the keyword 'Nelumbo nucifera pharmacology' in all databases reported in Web of Science yielded 373 results excluding reviews and abstracts in document types. Two hundred and forty-three spectrum natural compounds from different parts of N. nucifera belonging to diverse chemical groups, including alkaloids, flavonoids, glycosides, terpenoids, steroids, fatty acids, proteins, minerals, and vitamins have been reported. In addition, distinct pharmacological activities, mainly against cancer, microbial infection, diabetes, inflammation, atherosclerosis, and obesity, have been associated with crude extracts, fractions, and isolated compounds. This review highlights potential use of neferine, liensinine, isoliensinine, and nuciferine in clinical trials. In depth, mechanism of the potential chemical entities from N. nucifera via structure activity relationship needs to be explored to guarantee the stability and safety for the clinical use. Copyright © 2016 John Wiley & Sons, Ltd.

Melanoma is the deadliest cancer. We identified 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent that antagonizes melanoma tumor growth in mice xenograft model in vivo. Cell proliferation assay demonstrated strong anticancer effects of 7-HDNF to exhibit a dose-dependent behaviour and displayed minor cytotoxicities on normal human skin cells, including epidermal keratinocytes and melanocytes, and dermal fibroblasts. With acridine orange (AO) staining and flow analysis, we found 7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained to confirm the cellular membrane asymmetry lost. One-dimensional flow cytometric analysis showed 7-HDNF increased the cellular arrest in cell cycle at the G2/M phase. Through Western blot examinations, protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. Finally, 7-HDNF presented a high-quality antimigratory activity in wound-healing assay. Overall, 7-HDNF presented high-quality anticancer bio-functions and inhibited melanoma tumor growth in vivo and in vitro.

Nelumbo Adans. (Nelumbonaceae) is an important member of the early-diverging eudicots. It contains two extant species: N. nucifera Gaertn. (the Sacred lotus), distributed in Asia and Australia and N. lutea Willd. (the American lotus), occurring in North America. This paper reviews the taxonomic, evolutionary and phytogeographic studies of the genus Nelumbo, and also raises scientific questions about it in further paleobotanic research. There are about 30 fossil species of Nelumbo established since the Early Cretaceous. Based on fossil studies, the ancestors of the extant N. nucifera and N. lutea are respectively considered to be N. protospeciosa from the Eocene to Miocene of Eurasia, and N. protolutea from the Eocene of North American. However, molecular systematic studies indicate that N. nucifera and N. lutea are probably split from a common ancestor during the Late Miocene to Early Pliocene, or even the Pleistocene, rather than separate relicts from extinct species on different continents. The characters of lotus stomatal development, seedling morphology as well as its flowering, pollination and fertilization in air reveal that it evolves from the land plants. Fossil data of Nelumbo indicates that the genus first occurs in mid-latitude area of Laurasia in the Early Cretaceous, then becomes widespread in North America and Eurasia and expands into Africa and South America during the Late Cretaceous; the genus probably colonizes the Indian Subcontinent from Asia during the Early Eocene after the collision of India and the Asian plates; the genus becomes extinct in Europe, but survives in Asia and North America during the Quaternary Ice Age, and later forms the present East Asia and North Australia-North America disjunctive distribution.

Floral thermogenesis is an unusual floral trait with a well-documented physiological process, and yet, there is limited understanding of how this trait influences plant reproduction. The current study was undertaken to gain a better understanding of how floral thermogenesis in Nelumbo lutea impacts pollinator attraction and consequent plant reproduction.We conducted field studies on floral thermogenesis and thermoregulation, flower sexual development, floral visitation patterns, breeding system, pollen transfer dynamics, and floral scent production.The most abundant visitors to the thermoregulatory flowers included the Phoridae (Diptera), Chrysomelidae (Coleoptera), and Hymenoptera. Chrysomelid beetles, particularly Diabrotica, were frequent visitors to both first-day female- and second-day bisexual-phase flowers, while phorid flies were most common in bisexual-phase flowers. Pollen transfer experiments indicated that Diabrotica was equally effective in depositing pollen on stigmas, as were the less frequent, but pollen-loaded halictid bees.Flowers received a taxonomically wide assemblage of floral visitors and appear adapted to attract beetles, primarily Chrysomelidae and medium-sized bees. This study is the first to provide strong support that beetles can comprise the dominant portion of floral visitors and are as effective in pollen transfer as bees. Thermogenesis aids in dispersing the main floral scent component-1,4-dimethoxybenzene-attracting both chrysomelids and bees, while thermoregulation causes chrysomelid beetles to actively seek out new flowers for evening residence. This search behavior likely results in chrysomelids affecting cross-pollination.

Sacred lotus (Nelumbo nucifera) is an ornamental plant that is also used for food and medicine. This basal eudicot species is especially important from an evolutionary perspective, as it occupies a critical phylogenetic position in flowering plants. Here we report the draft genome of a wild strain of sacred lotus. The assembled genome is 792 Mb, which is approximately 85-90% of genome size estimates. We annotated 392 Mb of repeat sequences and 36,385 protein-coding genes within the genome. Using these sequence data, we constructed a phylogenetic tree and confirmed the basal location of sacred lotus within eudicots. Importantly, we found evidence for a relatively recent whole-genome duplication event; any indication of the ancient paleo-hexaploid event was, however, absent. Genomic analysis revealed evidence of positive selection within 28 embryo-defective genes and one annexin gene that may be related to the long-term viability of sacred lotus seed. We also identified a significant expansion of starch synthase genes, which probably elevated starch levels within the rhizome of sacred lotus. Sequencing this strain of sacred lotus thus provided important insights into the evolution of flowering plant and revealed genetic mechanisms that influence seed dormancy and starch synthesis.

Nelumbonaceae is a morphologically unique family of angiosperms and was traditionally placed in Nymphaeales; more recently, it was placed in Proteales based on molecular data, or in an order of its own, Nelum- bonales. To determine the systematic position of the family and to date the divergence time of the family and the divergence time of its two intercontinentally disjunct species, we sequenced the entire chloroplast genome of Nelumbo lutea and most of the chloroplast genes of N. nucifera. We carried out phylogenetic and molecular dating analyses of the two species and representatives of 47 other plant families, representing the major lineages of an- giosperms, using 83 plastid genes. The N. lutea genome was 163 510 bp long, with a total of 130 coding genes and an overall GC content of 38%. No significant structural differences among the genomes of N. lutea, Nymphaea alba, and Platanus occidentalis were observed. The phylogenetic relationships based on the 83 plastid genes revealed a close relationship between Nelumbonaceae and Platanaceae. The divergence times were estimated to be 109 Ma between the two families and 1.5 Ma between the two Nelumbo species. The estimated time was only slightly longer than the age of known Nelumbo fossils, suggesting morphological stasis within Nelumbonaceae. We conclude that Nelumbonaceae holds a position in or close to Proteales. We further conclude that the two species of Nelumbo diverged recently from a common ancestor and do not represent ancient relicts on different continents.

PREMISE OF THE STUDY To study population genetics, phylogeography, and hybridization of Nelumbo (Nelumbonaceae), chloroplast microsatellite markers were developed. METHODS AND RESULTS Seventeen microsatellite loci were identified from the chloroplast genomes of N. nucifera and N. lutea. Polymorphisms were assessed in three populations of N. nucifera and one population of N. lutea. Nine loci were found to be polymorphic in N. nucifera, and all 17 loci were found to be polymorphic in N. lutea. In N. nucifera, the number of alleles per locus ranged from two to six, and the unbiased haploid diversity per locus ranged from 0.198 to 0.790. In N. lutea, the number of alleles ranged from two to four, and the unbiased haploid diversity per locus ranged from 0.245 to 0.694. CONCLUSIONS The identified chloroplast simple sequence repeat markers will be useful for the study of genetic diversity, phylogeography, and identification of Nelumbo cultivars.

Silver microcoil is fabricated through a biotemplating process combined with electroless plating. Spiral vessels in Lotus root are employed as a biotemplate because of their left-handed coil structure. The silver microcoil exhibits a solenoidal microcoil showing self-inductance in the level of picohenry, which could be applied for electromagnetic-responsive materials in the high-frequency region such as millimeter waves or terahertz waves.

Thermogenesis is a process of heat production in living organisms. It is rare in plants, but it does occur in some species of angiosperm. The heat is generated via plant mitochondrial respiration. As possible involvement in thermogenesis of mitochondrial factors, alternative oxidases (AOXs) and plant uncoupling mitochondrial proteins (PUMPs) have been well studied. AOXs and PUMPs are ubiquitously present in the inner membrane of plant mitochondria. They serve as two major energy dissipation systems that balance mitochondrial respiration and uncoupled phosphorylation by dissipating the H+ redox energy and proton electrochemical gradient (ΔμH+) as heat, respectively. AOXs and PUMPs exert similar physiological functions during homeothermic heat production in thermogenic plants. AOXs have five isoforms, while PUMPs have six. Both AOXs and PUMPs are encoded by small nuclear multigene families. Multiple isoforms are expressed in different tissues or organs. Extensive studies have been done in the area of thermogenesis in higher plants. In this review, we focus on the involvement and regulation of AOXs and PUMPs in thermogenesis.

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high-performance thin-layer chromatography method for estimation of betulinic acid in hydro-alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin-layer chromatography aluminium plate pre-coated with silica gel 60F254, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (Rf = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra-day and inter-day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.

Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes.

BACKGROUND: Lotus root beverage is commonly made from raw lotus root (RLR). However, RLR production is strictly limited, because it is prone to decomposition and browning after its short harvest season. In this study an innovative beverage was prepared from full lotus root powder (FLRP) as a substitute for RLR in an attempt to solve this problem. RESULTS: The components of FLRP basically corresponded to those of RLR, but there was some loss of heat-labile compounds. Using differential scanning calorimetry, a gelatinisation temperature range of 57.08–67.80 °C was determined for FLRP with an average particle size distribution of 70 µm. The optimal conditions for enzymatic treatment of FLRP beverage were determined by response surface methodology as an enzyme concentration of 2.2 g kg−1 at 53 °C for 86 min. Turbidity decreased from 1082 to 280 nephelometric turbidity units following enzymolysis. Properties of FLRP beverage were also studied and a qualitative comparison of flavour compounds between RLR and FLRP beverages was made by electronic nose. CONCLUSION: Basic flavour compounds were consistent and flavour radar plots had approximately the same shape, area and proportion when all ingredients were identical apart from FLRP and RLR. Therefore, in terms of flavour, FLRP beverage appears to be a feasible substitute for RLR beverage. Copyright © 2010 Society of Chemical Industry

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high-speed counter-current chromatography (HSCCC). Methodology – Following an initial clean-up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC-PAD, ESI-MS, 1H-NMR and 13C-NMR. Results – The separation was performed using a two-phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow-rate of 1.0 mL/min in the head-to-tail elution mode. Ultimately, 5.0 mg syringetin-3-O-β-d-glucoside, 6.5 mg quercetin-3-O-β-d-glucoside, 12.8 mg isorhamnetin-3-O-β-d-glucoside and 32.5 mg kaempferol-3-O-β-d-glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.

A new high-speed counter-current chromatography method for semi-preparative separation and purification of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn was developed by using pH-gradient elution mode. Diethyl ether was used as the stationary phase of the two-phase solvent system and Na(2)HPO(4)/NaH(2)PO(4) buffer solution with pH values of 7.5 and 7.2 in gradient mode as the mobile phase. Consequently, 33 mg of liensinine, 42 mg of isoliensinine, and 67 mg of neferine were obtained from 200 mg of crude extracts. The purities of them were all over 98% as determined by HPLC area normalization method, and the structures were identified by (1)H-NMR and (13)C-NMR.

A preparative high-speed counter-current chromatography method for separation and purification of liensinine, isoliensinine and neferine from seed embryo of Nelumbo nucifera GAERTN was successfully established by using n-hexane-ethyl acetate-methanol-water (5:8:4:5, v/v, containing 0.5% NH(4)OH) as the two-phase solvent system. From 200 mg of crude extract, 18.4 mg of liensinine, 19.6 mg of isoliensinine and 58.4 mg of neferine were obtained with the purity of 96.8, 95.9, and 98.6%, respectively. The identification of the three alkaloids was performed with (1)H NMR and (13)C NMR.

The cholinergic hypothesis of Alzheimer's disease (AD) has provided the rationale for the current pharmaco-therapy of this disease, in an attempt to reduce the cognitive decline caused by cholinergic deficits. Nevertheless, the search for potent and long-acting acetylcholinesterase (AChE) inhibitors that exert minimal side effects in AD patients is still ongoing. AChE inhibitors are currently the only approved therapy for the treatment of Alzheimer's disease; only a limited number of drugs are commercially available. Hydroalcohol extracts of six herbs, Andrographis paniculata, Centella asiatica, Evalvulus alsinoides, Nardostachys jatamansi, Nelumbo nucifera, Myristica fragrans used in Indian systems of medicine, were tested for in vitro acetylcholinesterase inhibitory activity based on Ellman's method in 96-well microplates using AChE obtained from bovine erythrocytes. The results showed that the hydroalcohol extract from Centella asiatica, Nardostachys jatamansi, Myristica fragrans, Evalvulus alsinoides inhibited 50% of AChE activity at concentrations of 100-150 microg/mL. Andrographis paniculata and Nelumbo nucifera extracts showed a weak inhibition of acetylcholinesterase with IC(50) values of 222.41 +/- 19.87 microg/mL and 185.55 +/- 21.24 microg/mL, respectively. Physostigmine was used as a standard and showed inhibition of acetylcholinesterase with an IC(50) value of 0.076 +/- 0.0042 microg/mL.

The application of mass spectrometry in drug discovery, especially in drug metabolites, is very important. This present paper is at first focused on the elucidation of fragmentation patterns of the phenolic bisbenzyltetrahydroisoquinoline alkaloid, neferine, together with its analogues isoliensinine and liensinine with anti-HIV activities using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. All title compounds displayed major diagnostic fragments that formed by the cleavage of the C1'--C9' bond resulting in positive group CD, and the loss of 4-ethyl-1-phenol or 4-ethyl-1-methoxybenzene following rearrangements. Their ESI-MS/MS spectra also showed the relatively stable fragment ions formed by the elimination of H2O, CH3NH2, CH3OH, and CH3-N==CH2. Secondly, the metabolites of neferine from dog hepatic microsomal incubations were analyzed and characterized by high-performance liquid chromatography (HPLC) and data-dependent ESI-MS/MS. Based on fragmentation patterns and compared with their retention times in LC, molecular weights and ultraviolet (UV) absorbances with standard compounds, six metabolites were identified as isoliensinine, liensinine and four novel bisbenzyltetrahydroisoquinoline alkaloids named as 6-O-desmethylneferine, 2'-N-desmethylneferine, 2'-N-6-O-didesmethylneferine, and 6,13-O-didesmethylneferine. All metabolites were desmethyl or didesmethyl products of neferine. The possible metabolic pathways for neferine have been proposed. The results suggest that N-demethylation and O-demethylation are two important metabolic pathways of neferine in dog hepatic microsomal incubations. This is critical for screening and development of phenolic bisbenzyltetrahydroisoquinoline alkaloids with anti-HIV activities such as neferine and its analogues isoliensinine and liensinine.

Neferine and isoliensinine are the two major compositions of the Chinese medicine lotus plumule. They have extensive cardiovascular activity, such as antiarrhythmic, antithrombic and antihypertensive. In order to confirm the structures reported in the literature by explicit 1H and 13C assignments, we used a series of NMR experiments including 1H, 1H-COSY, HSQC and HMBC. The absolute configuration of neferine (C-1 and C-1′) was determined as R and S and that of isoliensinine (C-1 and C-1′) was determined as R and R. Copyright © 2004 John Wiley & Sons, Ltd.

Vessels have been previously reported in the roots (but not rhizomes) of Nelumbo on the basis of light microscopy. We have reinvestigated vessel occurrence in Nelumbo by means of scanning electron microscopy (SEM). On the basis of these studies, vessels are characteristic of root metaxylem. Pores in primary walls of pits of end walls of these vessels are various in size, but feature incomplete lysis of pit membranes, often with residual webs or threads of primary wall material, much as in vessels of primitive woody dicotyledons. In addition, we newly report occurrence of vessels in metaxylem of rhizomes; pores in these vessels are smaller and more confined than in those of roots. The present study offers not only data of possible use in determining the phylogenetic position of Nelumbonaceae, but also contributes evidence that vessel origin in Nelumbonaceae relates to habit and ecology. We conclude that organographic distribution of vessels in Nelumbo follows the patterns seen in monocotyledons, which, like Nymphaeaceae and other aquatics, have sympodial architecture.

SUMMARY A survey of root meristems shows that in general, closed meristems, those with separate regions generating outer and inner tissues, derive the epidermis with the cortex in monocotyledons and with the cap in dicotyledons. Open meristems, where the cells between the stelar pole and cap divide transiently producing an unstable boundary between the cap and the rest of the root, also show a distinction between the classes, monocotyledons displaying greatest affinity between epidermis and cortex, dicotyledons between epidermis and cap. Exceptions in monocotyledons occur where there is a discrete epidermal meristem, an anticlinally dividing, monostromatic sheet of cells between cortex and cap. This condition has been reliably found only in a few floating plants and it is associated with the root being derived from both pericycle and endodermis of the mother organ. This is uncommon as a permanent feature in angiosperms though not confined to those with discrete epidermises. The mother tissue producing the epidermis depends on the species. In many other monocotyledons with closed meristems the epidermis is functionally discrete, for the branching of the epidermal cell files from, the cortical files is seen within the quiescent centre, i.e. it occurred during the meristem's initiation. Exceptions in dicotyledons occur in the Nymphaeales. In their closed meristems the epidermis is part of the cortical complex and in their open meristems its affinity with the cortex is manifest by early separation of the cap creating a cylindrical cleft in some genera. Differentiation of epidermal trichoblasts is also by a mode more characteristic of monocotyledons. Nelumbo roots, previously described as lacking an epidermis, as in gymnosperms, have closed meristems of dicotyledonous type and epidermises without distinctive trichoblasts, supporting Nelumbno's transfer from the Nymphaeales. The survey has corrected many errors of observation and interpretation in the literature enabling better use to be made of the mode of origin of root epidermises in discussing evolution and the new reclassification of angiosperms.

The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera and Nelumbo lutea. Historically, the genus has been considered to be closely related to Nymphaeales, however new systematic work has allied Nelumbo with lower eudicots, particularly Platanus. In recent years, studies of pollen development have contributed greatly to the understanding of phylogenetic relationships, but little has been known about these events in Nelumbo. In this paper, pollen and anther development are morphologically described for the first time in N. lutea. A comprehensive ontogenetic sequence is documented, including the sporogenous tissue, microspore mother cell, tetrad, free spore, and mature pollen grain stages. The deposition of a microspore mother cell coat and callose wall, the co-occurrence of both tetrahedral and tetragonal tetrads, the formation of a primexine in tetrads, and primexine persistence into the late free spore stage are shown. The majority of exine development occurs during the free spore stage with the deposition of a tectate-columellate ectexine, a lamellate endexine, and an unusual granular layer below and intermixed with the endexine lamellae. A two-layered intine forms rapidly during the earliest mature pollen stage. Major events of anther development documented include the degradation of a secretory-type tapetum during the free spore stage and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The majority of mature pollen grains are tricolpate, however less common monosulcate and diaperturate grains also develop. Co-occurring aperture types in Nelumbo have been suggested to be an important transition in angiosperm aperture number. However, aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus, which occurs in the early free spore stage. This character, as well as other details of pollen and anther ontogeny in Nelumbo, are compared to those of Nymphaeales and Platanus in an effort to provide additional insight into systematic and phylogenetic relationships. Although Nelumbo is similar to both groups in several characters, the ontogenetic sequence of the genus is different in many ways.

The floral biology of Nelumbo pentapetala (Walter) Fernald, the American lotus, native to Texas, was investigated. Anthesis occurs over three consecutive days with flowers opening each morning and closing around noon. First-day flowers are protogynous with the perianth parts partially expanded so that pollen-covered insects which are attracted by floral color and the intense "fruity" odor (diffused with the aid of increased floral temperature) are directed on to the flattened receptacle (= carpellary receptacle) from which the receptive stigmas protrude, thus accomplishing pollination. During the second morning anther dehiscence begins and insects which visit and forage within the flower become covered with pollen and typically crawl over the still receptive stigmas achieving "facilitated" self-pollination (indirect autogamy). By mid-morning of the second day the stigmas dry and become non-receptive to pollen. During the third day of anthesis perianth and staminal parts quickly abscise and over the period of a few weeks the receptacle and enclosed fruits mature. In most populations studied, Hymenoptera (e.g., Lasioglossum spp., and Apis mellifera) were the most abundant and effective pollinators. In some populations, however, Coleoptera (e.g., Chauliognathus) were also numerous and effective pollinators. It is suggested that the overall floral structure (e.g., large numbers of stamens, masses of pollen, staminal appendages) are adaptations which facilitate the pollination of Nelumbo by beetles. STUDIES dealing with the floral biology of Nelumbo (Nymphaeaceae, sensu lato) are numerous in the literature. Delpino (1875) regarded the flowers of Nelumbo to be specifically adapted for beetle pollination. Robertson (1889) stated that the flowers of N. lutea Planchon [=N. pentapetala (Walter)

Summary Evolutionary proposals involving pollen grain characters have treated germinal apertures as though they were formed by one universal process and hence derivable from a common type. A review of published data on apertural formation for pollen of Silene, Tradescantia, and Nelumbo and original information for Epilobium indicates that there are several possibly distinct apertural induction processes. It would seem to be an oversimplification and perhaps misleading to ignore apertural genesis in interpretations involving relationships between taxa.

Definition of some vascular plant families is unsatisfactory and, consequently, it is impossible to classify them adequately. The lack of an analytical key to the families based on easily observed, widespread characters sometimes results in surprising misinterpretations. A striking example in this regard is the Casuarinaceae. Engler (1897) considered them to be true Angiospermae (Dicotyledoneae, Order 1. Verticillatae). Melchior (Engler's Syllabus der Pflanzenfamilien, vol. 2. 1964) and Wettstein (1935) concur with Engler, but Hutchinson (1973) placed the Casuarinales (Order 18) between the Juglandales and Urticales. According to Takhtajan (1969, 1973) the Casuarinaceae probably arose from the Hamamelidales. Gaussen (1940) derived the Casuarinaceae from the Articulatae. Hegnauer (1964) recalls that authors have noted similarities between Casuarina, Equisetum, Ephedra, and the Coniferae, but states the chemistry of the family is not well enough known to determine its position in the system. The Nymphaeaceae is another difficult family to place in the system. In Engler's Syllabus, Order 16 Ranunculales includes the suborders Ranunculineae and Nymphaeineae. The latter contains families Nymphaeaceae, and Ceratophyllaceae. The first of these is divided into the subfamilies Cabomboideae, Nymphaeoideae and Nelumbonoideae. Wettstein places the Nymphaeaceae in Order 18, Polycarpicae, following Family 20, Berberidaceae. The Nymphaeaceae contains the same subfamilies as in Engler's Syllabus. The following family is Ceratophyllaceae, which is considered closely related to the Nymphaeaceae. Hegnauer (1969) states that the evidence is not sufficient to form an opinion with respect to the relationships of all the genera included in the Nymphaeaceae. Hutchinson puts the family in the Ranales and it includes Nelumbo, Euryale, Nuphar and Barclaya. He treats Cabombaceae and Ceratophyllaceae as separate families. According to Kubitzki (1972), the Nymphaeaceae, s. str., do not belong in the Polycarpicae. A third example is the Cyclanthaceae. Wettstein placed them in the Order Synanthae, with the Palmae and Araceae but the plants of the three families are very different. The Palmae are mostly derived, the Cyclanthaceae include at least two different plant types, and the Araceae, mostly herbaceous, exhibit distinctive features. In Engler's Syllabus, the Order Synanthae (Cyclanthales) comprises the family Cyclanthaceae with subfamilies Carludovicoideae and Cyclanthoideae. These are herbs of palm-like habit and deeply bilobed leaves. They are very old, reduced and derived relicts. Hegnauer considers the Palmae, Cyclanthaceae and Pandanaceae more closely related to one another than to the herbaceous Araceae, Typhaceae and Sparganiaceae. According to Takhtajan, the Arales, Arecales and Cyclanthales were derived from the immediate ancestors of the Liliales.

A B S T R A C T The examination of the primary xylem of roots of Nelumbo has revealed the presence of vessel elements with intra-face, transversely-rowed to obliquely-rowed bordered pitted side walls and scalariformly perforated end walls. Whole mounts of macerated xylem, serial sections of roots, and cells through which a water suspension of India ink particles under slight or no pressure was passed, were the means of investigation. It is our opinion that this discovery does not support theories which derive monocotyledons from the Nymphaeaceae. A discussion leading to this conclusion is presented. MANY INVESTIGATORS think that Nymphaeaceae (sensu lato) do not have vessel elements; yet, if one searches the older literature, one finds numerous references to vessels in members of the family. We have ot, however, discovered any convincing evidence that a thorough and modern study of the xylem has been conducted. Trecul (1845, 1854) used the word "vaiseaux" numerous times in the descriptions of the vascular systems of Nuphar, Nelumbo, and Victoria. An examination of his papers reveals simple drawings of long, helical-reticulate elements. It is reasonably clear that he recognized no differences between end walls and side walls of the elements and probably did not observe the pitted elements with perforations described in this paper. It appears to us that he was using the word