Embryology of Clitoria ternatea (Fabaceae)

ABSTRACT

The embryology of Clitoria ternatea was studied. Anthers contain four sporangia. The anther wall comprises an epidermis, an endothecium, a middle layer and a glandular tapetum. Microspore tetrads are tetrahedral and pollen grains are shed at the 2-cell stage. The ovule is campylotropous, bitegmic and crassinucellate. The micropyle is formed by both the integuments. The megaspore tetrad is linear or T-shaped. The chalazal megaspore is functional and embryo sac development follows the monosporic Polygonum type. Endosperm development is of the nuclear type. The chalazal part of the endosperm forms a haustorium. Embryo development follows the Onagrad type.     

Micropropagation through excised root culture of Clitoria ternatea and comparison between in vitro–regenerated plants and seedlings

An efficient protocol has been developed for high‐frequency shoot regeneration and plant establishment of Clitoria ternatea – a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6‐benzyladenine (BA), kinetin, α‐naphthalene acetic acid (NAA) or 2,4‐dichlorophenoxy acetic acid (2,4‐D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μm BA and 2.0 μm NAA. Organogenic calli were produced on a medium containing 2,4‐D (10 or 20 μm ) and BA (5.0 μm ). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5–10 μm BA and 2.0 μm NAA. The microshoots were rooted on half‐strength MS medium supplemented with 5.0 μm indole‐3‐butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo–grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro–regenerated and in vivo–grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).     

Malonylated flavonol glycosides from the petals of Clitoria ternatea

Three flavonol glycosides, kaempferol 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, quercetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, and myricetin 3-O-(2",6"-di-O-alpha-rhamnosyl)-beta-glucoside were isolated from the petals of Clitoria ternatea cv. Double Blue, together with eleven known flavonol glycosides. Their structures were identified using UV, MS, and NMR spectroscopy. They were characterized as kaempferol and quercetin 3-(2(G)- rhamnosylrutinoside)s, kaempferol, quercetin, and myricetin 3-neohesperidosides, 3-rutinosides, and 3-glucosides in the same tissue. In addition, the presence of myricetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was inferred from LC/MS/MS data for crude petal extracts. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.     

Cross-genera amplification of Cajanus spp. specific SSR markers in Clitoria ternatea (L.) and their application in genetic diversity studies

Clitoria ternatea (L.) is a medicinal leguminous plant and is cultivated to cater the need of herbal industries and asthetic purposes. The unavailability of steady molecular marker impedes the genetic improvement of C. ternatea. In the present study, transferability of 98 pairs of Cajanus spp. specific SSR primers were assessed among 14 genotypes of C. ternatea, varied for their flower color, floral architecture and bio-metabolite (taraxerol and delphinidin) content, and out of them 43 had successfully amplified the fragments. Among them, 36 pairs of primers showed 100% transferability, whereas rest seven varied from 42.86 to 92.85% transferability. The transferable 43 pairs of SSR primers generated 196 alleles across the 14 genotypes and the AMOVA analysis showed moderate genetic variation (55.1%) among the genotypes of C. ternatea, which was also reinforced by Nei’s genetic distance and gene identity estimates derived haplotype matrix. Similarly, both the principal coordinate analysis and dendrogram grouped these 14 genotypes of C. ternatea into two major clusters based on SSR allele distribution and frequency, and the clustering pattern is in accordance with petal color but in contrast to floral architecture. MCheza based outlier analysis revealed 16 alleles for balancing selection, which are putatively involved in the maintenance of genetic polymorphism in C. ternatea. Moreover, the estimates of molecular diversity and bio-metabolite content revealed the possible use of these genotypes in future breeding programme of this species.Electronic supplementary materialThe online version of this article (10.1007/s12298-020-00907-x) contains supplementary material, which is available to authorized users.     

A HPTLC method for the determination of the mycotoxin zearalenone in cereal products

An HPTLC method for the quantification of zearalenone (ZEA) in cereals and cereal products (wheat flour and malt) has been developed. ZEA was extracted with 50 ml of acetonitrile-purified water (9+1) with addition of 2 g NaCl. The extracts were further purified on VICAM ZearalaTest^(tm) immunoaffinity columns, then analysed by instrumental high-performance thin-layer chromatography (HPTLC) on silica gel plates with fluorescence detection. Ethyl acetate - n-hexane (1+1) was used as the mobile phase. The chromatogram was scanned in fluorescence mode after excitation at λ=254 nm with λ=400 nm measuring filter: SENS and SPAN parameters were 195 and 20, respectively. TheR _F of ZEA under these conditions was 0.43. The recovery was 95% in the range 15–65 μg/kg cereal products; the mean relative standard deviation of repeatability (RSDr) was 7.6%. The limit of quantification (LoQ) of ZEA was 10 μg/kg. Validation of the method was performed according to the principles of the ICH for pharmaceutical analysis. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low‐cost, high‐throughput analytical methods for quality control. Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Results – Δ⁹‐THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r² = 0.9968. Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of Δ⁹‐THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars. Copyright © 2009 John Wiley & Sons, Ltd.     

New rapid validated HPTLC method for the determination of galanthamine in Amaryllidaceae plant extracts

The work reported in this paper aims at developing an accurate, specific, repeatable and robust HPTLC method for the determination of galanthamine in different Amaryllidaceae plant extracts.     

Simultaneous determination of cinnamaldehyde, eugenol and piperine by HPTLC densitometric method

An HPTLC densitometric method for the simultaneous determination of cinnamaldehyde and eugenol as well as trace amounts of piperine in pepper-contaminated cinnamon was developed. The applicability of the method was tested with cinnamon bark powder adulterated with pepper powder, cinnamon oil, clove powder, clove oil and a commercial preparation containing cinnamaldehyde and eugenol. The method was validated for specificity, precision, accuracy and robustness. The method was found to be precise for different concentrations of cinnamaldehyde, eugenol and piperine. The accuracy of the method was checked by conducting a recovery study at three different levels. The linearity was found to be in the ranges 52.54-735.56, 533.2-8531.2 and 50-300 ng/spot, respectively, with correlation coefficients of 0.9985 +/- 0.04, 0.9982 +/- 0.06 and 0.9937 +/- 0.11 for cinnamaldehyde, eugenol and piperine.     

Biosynthesis of malonylated flavonoid glycosides on the basis of malonyltransferase activity in the petals of Clitoria ternatea

The crude malonyltransferase from the petals of Clitoria ternatea was characterized enzymatically to investigate its role on the biosynthetic pathways of anthocyanins and flavonol glycosides. In C. ternatea, a blue flower cultivars (DB) and mauve flower variety (WM) accumulate polyacylated anthocyanins (ternatins) and delphinidin 3-O-(6'-O-malonyl)-beta-glucoside which is one of the precursors of ternatins, respectively. Moreover, WM accumulates minor delphinidin glycosides - 3-O-beta-glucoside, 3-O-(2'-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2'-O-alpha-rhamnosyl-6'-O-malonyl)-beta-glucoside of delphinidin. These glycosidic patterns for minor anthocyanins in WM are also found among the minor flavonol glycosides in all the varieties including a white flower variety (WW) although the major flavonol glycosides are 3-O-(2'-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(6'-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2',6'-di-O-alpha-rhamnosyl)-beta-glucoside of kaempferol, quercetin, and myricetin. How do the enzymatic characteristics affect the variety of glycosidic patterns in the flavonoid glycoside biosynthesis among these varieties? While the enzyme from DB highly preferred delphinidin 3-O-beta-glucoside in the presence of malonyl-CoA, it also has a preference for other anthocyanidin 3-O-beta-glucosides. It could use flavonol 3-O-beta-glucosides in much lower specific activities than anthocyanins; however, it could not utilize 3-O-(2'-O-alpha-rhamnosyl)-beta-glucosides of anthocyanins and flavonols, and 3,3'-di- and 3,3',5'-tri-O-beta-glucoside of delphinidin - other possible precursors in ternatins biosynthesis. It highly preferred malonyl-CoA as an acyl donor in the presence of delphinidin 3-O-beta-glucoside. The crude enzymes prepared from WM and WW had the same enzymatic characteristics. These results suggested that 3-O-(2'-O-alpha-rhamnosyl-6'-O-malonyl)-beta-glucosides of flavonoids were synthesized via 3-O-(6'-O-malonyl)-beta-glucosides rather than via 3-O-(2'-O-alpha-rhamnosyl)-beta-glucosides, and that malonylation proceeded prior to glucosylation at the B-ring of delphinidin in the early biosynthetic steps towards ternatins. It seemed that the substrate specificities largely affected the difference in the accumulated amount of malonylated glycosides between anthocyanins and flavonols although they are not simply proportional to the accumulation ratio. This enzyme might join in the production of both malonylanthocyanins and flavonol malonylglycosides as a result of broad substrate specificities towards flavonoid 3-O-beta-glucosides.     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

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 (R_f = 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.     

高效薄层色谱测定湛江等鞭金藻培养过程脂质变化

【目的】研究湛江等鞭金藻(Isochrysis zhangjiangensis)由氮元素丰富至限制培养过程中脂质成分的变化。【方法】利用高效薄层色谱(HPTLC)分离分析微藻中脂质。【结果】随着培养基中营养物质的消耗,细胞逐渐处于胁迫状态,在这种状态下,细胞大量积累贮存脂质—甘油三脂(TAG),组成生物膜系统的单半乳糖甘油二脂(MGDG)、硫代异鼠李糖甘油二脂(SQDG)、磷脂酰甘油(PG)和磷脂酰胆碱(PC)含量降低,而游离脂肪酸(FFA)、甘油二脂(DAG)、双半乳糖甘油二脂(DGDG)、磷脂酰肌醇(PI)和磷脂酰乙醇胺(PE)则相对稳定。【结论】HPTLC可作为一种简便、可靠的微藻中脂质分离分析方法,为研究微藻油脂代谢途径以及甘油三脂(TAG)的调控积累提供有效手段。     

Chemical fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and densitometry

Introduction –  Lawsonia inermis L. is a natural red colouring agent, commonly named “Henna”, which is used to dye skin and hair. The aim of this study was to evaluate the quality of L. inermis that is commercially available as a raw plant material or preparation in order to guarantee good quality products. Objective ?  To develop a simple protocol for the qualification of different samples labelled as L. inermis by using the HPTLC densitometry method and to identify possible adulterations with other plants. Methodology ?  Samples of leaves of L. inermis were extracted with methanol. Two chromatographic methods were developed to determine the chemical fingerprinting of L. inermis. The first was based on HPTLC identification followed by densitometric measurements at 337 nm. The second was based on RP‐HPLC separation with gradient elution and photodiode array detection at 337 nm. Samples of Cassia obovata Collad., and Indigofera tinctoria L., were treated in the same way. Results –  The simplicity of the sample preparation, and the possibility of analysing several samples of herbal products simultaneously in a short time, make HPTLC the method of choice. The HPTLC method was feasible for the comprehensive quality evaluation of herbal products. From the comparison of their “fingerprint”, it was possible to detect substitution of plants that are different from those declared on the label. Conclusion ?  The HPTLC may be used as a rapid method by which to control the quality of raw plant materials and formulations based on the title plant. Copyright © 2008 John Wiley & Sons, Ltd.     

HPTLC determination of vasicine and vasicinone in Adhatoda vasica

A simple high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of the pharmacologically important quinazoline alkaloids vasicine and vasicinone in Adhatoda vasica. The assay combines the separation and quantification of the analytes on silica gel 60 GF254 HPTLC plates with visualisation under UV and scanning at 270 and 281 nm. Using this technique, the alkaloidal content of different parts of the title plant have been determined.     

Qualitative and Quantitative One-step Analysis of Lipids and Encapsulated Bioactive Molecules in Liposome Preparations by HPTLC/FID (IATROSCAN)

Liposomes are widely used vehicles for the delivery of bioactive molecules. They are composed mainly from acyl-phosphatidylcholines, cholesterol, and charged lipids (e.g., stearylamine, dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine).

The incorporation efficiencies of the bioactive molecule and the drug to lipid molar ratio are important factors for the assessment of the liposomal formulation. In order to successfully characterize a liposomal formulation, it is necessary to be able to accurately measure the lipids and the encapsulated molecule, using the smallest possible sample.

The present work describes an analytical methodology on qualitative and quantitative determination of all the lipid ingredients that are involved in the liposome formulation, as well as the drug incorporation and the drug–lipid ratio, by a simultaneous measurement of all the liposomal ingredients using thin-layer chromatography coupled with a flame ionization detector (HPTLC/FID).

The procedure requires only one measurement per sample, and it can be applied even in very small or much diluted samples.

The proposed analytical method can be applied in general on all steps of the development of liposomal formulations. The purity and stability of the raw materials can also be easily evaluated. In addition the preparation procedure can be tracked in order to locate possible losses of raw material and errors of the preparation method resulting in the amelioration of the method.     

A new furanoditerpenoid marker for the distinction between the seeds of two species of Caesalpinia

A new cassane furanoditerpene, 17-methylvouacapane-8(14),-9(11)-diene (1), has been isolated from the seed kernels of Caesalpinia crista and its structure determined by mass, IR and NMR spectrometry. The 1H- and 13C-NMR spectra were assigned using a combination of 1H-1H COSY, HSQC and HMBC two-dimensional NMR experiments. An HPTLC method has been developed to quantify 1 in seed material. The furanoditerpene can serve as a marker chemically to differentiate C. crista from the synonymous C. bonduc.     

Comparative HILIC/ESI-QTOF-MS and HPTLC studies of pyrrolizidine alkaloids in flowers of Tussilago farfara and roots of Arnebia euchroma

The utility of HPTLC and HILIC/ESI-QTOF-MS for the determination of pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) was compared in the selected plant species: Tussilago farfara L. (TF, flower) and Arnebia euchroma (Royle) I.M. Johnst. (AE, root). HPTLC confirmed the postulated presence of PAs (saturated and unsaturated) or PANOs in the tested extracts. In accordance with previous studies, HILIC/ESI-Q-TOF-MS confirmed the presence of the toxic PA senkirkine and the saturated otonecine-type PAs, tussilagine and isotussilagine in the TF extract and 7-angeloylretronecine and 9-angeloylretronecine in AE extract. Moreover, the following alkaloids were identified in AE root: intermedine, intermedine-N-oxide, leptanthine-N-oxide, echimidine-N-oxide (or their corresponding stereoisomers) and traces of 7-angeloylretronecine and 9-angeloylretronecine-N-oxide. The study demonstrates the HILIC/ESI-Q-TOF-MS method to be a very useful tool for monitoring PAs and PANOs in the test samples, even when not all of the necessary standards are available. Quantitative analysis of senkirkine in TF flower by HILIC/ESI-QTOF-MS featured high resolution, high precision, high mass accuracy, and very high sensitivity with limit-of-detection (LOD) of 27.50 fg/μL and limit-of-quantitation (LOQ) of 91.60 fg/μL. The results from both methods may be used for the development or rejection of European Pharmacopoeia (X) monographs of both investigated species.     

RETRACTED ARTICLE: Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05–2.5 μM) was used as a supplement to the Murashige and Skoog’s (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.