Effects of R/B Ratio and Light Intensity of the LED Lamp on the Growth and Iridoid Glycoside Content of <i>Hedyotis diffusa</i>

Efficient and targeted screening and isolation of bioactive compounds from complex natural products is still a challenging work. Herein, diagnostic ion filtering based high-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry was firstly developed to screen six main iridoid glycosides from Hedyotis diffusa. Then, online extraction-high-speed counter current chromatography was proposed for targeted enrichment and preparative isolation using ethyl acetate/n-butanol/water (4.5:0.5:5, v/v/v) as solvent system. After that, Sephadex LH-20 column chromatography using methanol as solvent system was selected for further purification of six iridoid glycosides with purities over 98%. They were finally identified as monotropein, desacetylasperuloside acid, asperuloside, 6-O-(Z)-p-coumaroyl scandoside methyl ester, 6-O-(Z)-feruloyl scandoside methyl ester, and 6-O-(E)-p-coumaroyl scandoside methyl ester. And their anti-inflammatory activities were evaluated and confirmed by lipopolysaccharide activated RAW 264.7 macrophages. Obviously, the results provide a scientific basis for the potential applications of H. diffusa, and the developed methodology is efficient and reliable for targeted screening and isolation of bioactive compounds from natural products.

Development of Environmental Control Method for Rapid Production of High Quality Hedyotis diffusa

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity>95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.

The analysis of chemical constituents in Chinese herbal medicines (CHMs) is a challenge because of numerous compounds with various polarities and functional groups. Liquid chromatography coupled with quadrupole time-of-flight (QTOF) mass spectrometry (LC/MS) is of particular interest in the analysis of herbal components. One of the main attributes of QTOF that makes it an attractive analytical technique is its accurate mass measurement for both precursor and product ions. For the separation of CHMs, comprehensive two-dimensional chromatography (LCxLC) provides much higher resolving power than traditional one-dimensional separation. Therefore, a LCxLC-QTOF-MS system was developed and applied to the analysis of flavonoids and iridoid glycosides in aqueous extracts of Hedyotis diffusa (Rubiaceae). Shift gradient was applied in the two-dimensional separation in the LCxLC system to increase the orthogonality and effective peak distribution area of the analysis. Tentative identification of compounds was done by accurate mass interpretation and validation by UV spectrum. A clear classification of flavonol glycosides (FGs), acylated FGs, and iridoid glycosides (IGs) was shown in different regions of the LCxLC contour plot. In total, five FGs, four acylated FGs, and three IGs were tentatively identified. In addition, several novel flavonoids were found, which demonstrates that LCxLC-QTOF-MS detection also has great potential in herbal medicine analysis.

When a discharge lamp is run from an alternating supply, the intensity of light from it varies between maximum and minimum values at twice the supply frequency. This so-called intensity “ripple” can, if such lamps are used as light sources in motion-picture film work, lead to a cyclic variation in frame-to-frame exposure. This in turn can cause the overall luminance of the displayed picture to fluctuate. In this paper, an investigation of the visibility of such luminance fluctuations over the frequency range 0.1–12.5 Hz is described. Limits of film exposure variation are recommended, below which these picture luminance fluctuations should not be visible, taking into account a nonlinear transfer characteristic. The magnitude of film exposure variation which can occur in practice is discussed, based on the assumption that the variation in lamp intensity is sinusoidal in character, as is the frequency of the luminance fluctuations which occur when the film is replayed. Using these factors, theoretical relationships are derived between camera frame frequency and shutter angle, and lamp supply frequency and intensity ripple magnitude, such that picture luminance fluctuations due to the presence of the ripple component are not perceptible. Camera shutter angles in the range 135°-235°, camera frame frequencies of 24 Hz and 25 Hz, and lamp supply frequencies both in the range covered by conventional public electricity supplies and also of values much higher than this are included. In this latter case a “safe” lamp supply frequency is assessed, above which the effects of intensity ripple in causing film exposure variation may be discounted. The effect of ripple asymmetry (successive maxima of the ripple waveform having alternately higher and lower magnitudes) is discussed. The characteristics of two currently available metal-halide discharge lamps are examined, and experimental techniques for measuring intensity ripple under standardized conditions are described.

Iridoids and sfingolipids from Hedyotis diffusa

Two new iridoid glycosides, named productasperulosidic acid butyl ester (1) and E-6-O-3-hydroxy-p-methoxycinnamoyl scandoside methyl ester (2), along with nine known ones (3–11), were isolated from Hedyotis diffusa Willd. The structures of them were elucidated by extensive 1D, 2D NMR and HR-ESI-MS spectral data. Compounds 1–11 showed no significant cytotoxic activity against HeLa cells.

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS(n)). The optimized separation condition was obtained using an Agilent ZorBax SB-C18 column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC-ESI-MS(n) method.

Previous studies have reported that Hedyotis diffusa Willdenow extract shows various biological activities on cerebropathia, such as neuroprotection and short-term memory enhancement. However, there has been a lack of studies on the inhibitory activity on neurodegenerative diseases such as Alzheimer’s disease (AD) through enzyme assays of H. diffusa. Therefore, H. diffusa extract and fractions were evaluated for their inhibitory effects through assays of enzymes related to AD, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and on the formation of advanced glycation end-product (AGE). In this study, ten bioactive compounds, including nine iridoid glycosides 1–9 and one flavonol glycoside 10, were isolated from the ethyl acetate and n-butanol fractions of H. diffusa using a bioassay-guided approach. Compound 10 was the strongest inhibitor of cholinesterase, BACE1, and the formation of AGEs of all isolated compounds, while compound 5 had the lowest inhibitory activity. Compounds 3, 6, and 9 exhibited better inhibitory activity than other compounds on AChE, and two pairs of diastereomeric iridoid glycoside structures (compounds 4, 8, and 6, 7) showed higher inhibitory activity than others on BChE. In the BACE1 inhibitory assay, compounds 1–3 were good inhibitors, and compound 10 showed higher inhibitory activity than quercetin, the positive control. Moreover, compounds 1 and 3 were stronger inhibitors of the formation of AGE than aminoguanidine (AMG), the positive control. In conclusion, this study is significant since it demonstrated that the potential inhibitory activity of H. diffusa on enzymes related to AD and showed the potential use for further study as a natural medicine for AD treatment on the basis of the bioactive components isolated from H. diffusa.

Evaluation of human neutrophil elastase inhibitory effect of iridoid glycosides from Hedyotis diffusa

Effect of Air Temperature during Light and Dark Periods on the Iridoid Glycoside Content of <i>Hedyotis diffusa</i>

Variation in concentrations of leaf nitrogen and iridoid glycosides was examined in replicate plants of five genotypes ofPlantago lanceolata (Plantaginaceae) grown in an experimental garden. Nitrogen concentration and iridoid glycoside concentration were affected by leaf age. New leaves had nitrogen concentrations 1.7 to 2.7 times higher than mature leaves. Catalpol concentration was highest in new and intermediate-aged leaves. The concentration of aucubin, the biosynthetic precursor to catalpol, was higher in intermediate-aged leaves than in mature leaves, in three of five genotypes. Consequently, the proportion of aucubin relative to total iridoid glycosides increased as leaves aged. Concentration of iridoid glycosides was not correlated with plant size. Plant genotype significantly affected concentration of nitrogen and iridoid glycosides, as well as plant size. Thus, major indicators of hostplant quality for insect herbivores varied considerably both within and among plant genotypes and individuals.

As a TCM, Hedyotis diffusa Willd. has been using to treat malignant tumors, and many studies also showed that the extracts from Hedyotis diffusa Willd. possessed evident antitumor activities. Therefore, we carried out chemical study on Hedyotis diffusa Willd. and investigated the cytotoxicity of the obtained compounds on a panel of eight tumor cell lines. As a result, four new compounds were isolated from Hedyotis diffusa Willd., including three iridoid glycosides of Shecaoiridoidside A–C (1–3) and a cerebroside of shecaocerenoside A (4). Also, six known iridoid compounds (5–10) were also obtained. The cytotoxicity of all compounds against human tumor cell lines of HL-60, HeLa, HCT15, A459, HepG2, PC-3, CNE-2, and BCG-823 were also evaluated in vitro. New compound 3 exhibited evident cytotoxicity to all tumor cell lines except the Hela, and the IC50 values are from 9.6 µM to 62.2 µM, while new compound 4 showed moderate cytotoxicity to all the cell lines, and the IC50 values are from 33.6 µM to 89.3 µM. By contrast, new compound 1 and known compound 9 showed moderate cytotoxicity to HCT15, A459, and HepG2 selectively. Known compound 7 also exhibited moderate cytotoxicity to HCT15 and A459 selectively.

Effect of Light Intensity and Light/Dark Period on Iridoids in <i>Hedyotis diffusa</i>

Plant performance and chemistry may vary due to a variety of factors, such as plant genotype, environmental conditions, presence of herbivores, timing of herbivory, and species of herbivore. The relative importance of these factors, and how the plants respond to them, may affect the dynamics of the plant population, as well as the insect herbivores feeding on those plants. To understand the relative importance of some of these factors on plant performance and chemistry, we used Plantago lanceolata L. (Plantaginaceae). In an experimental garden at Binghamton, New York, we examined the effects of plant age, plant genotype, and herbivory by generalist or specialist caterpillars on P. lanceolata. There were two parts of this experiment. In the first, we examined variation in nutritional quality (nitrogen) and defensive chemistry (the iridoid glycosides aucubin and catalpol) as a consequence of plant age, plant genotype, and leaf age class. We compared these parameters for a set of four pairs of plants of each of five genotypes that were not exposed to herbivores, and were harvested in early July (control—start plants) with another set harvested 6 wk later in mid—August (control—end plants). The older plants harvested in August (control—end plants) had concentrations of aucubin, catapol, total iridoid glycosides, and nitrogen approximately one—half that of plants harvested 6 wk earlier. Leaf age affected all of these variables, and plant genotype influenced the iridoid glycoside variables but not the nitrogen concentration. Overall, leaf explained twice as much of the variation in iridoid glycosides as did plant age, which accounted for twice as much variation as did plant genotype. The second part of the experiment compared the effects of herbivory, genotype, and leaf age on plant performance (leaf biomass, scape [flower stalk] biomass, and total biomass) and chemistry (nitrogen, protein, and iridoid glycoside concentration). We compared these measures for a set of four pairs of replicate plants of each of five genotypes, exposed to one of three herbivory treatments: no herbivory, herbivory by specialist caterpillars, and herbivory by generalist caterpillars. The results of this experiment showed that herbivory had little effect on plant performance, and there was no difference due to herbivory by the specialist Junonia coenia Hubner (Nymphalidae), compared to the generalist Spilosoma congrua Wlk. (Arctiidae). However, plant chemistry was significantly affected by herbivory. Herbivory by both caterpillar species induced iridoid glycosides and resulted in an increased concentration of catalpol and an increase in the proportion of catalpol relative to total iridoid glycosides in those plants exposed to herbivores compared to controls. In addition, plants exposed to specialist caterpillars had higher concentrations of catalpol and a higher proportion to total iridoids that was catalpol than those exposed to generalist caterpillars. Overall, in this experiment, leaf age explained three times as much of the variation in iridoid glycoside concentration in P. lanceolata leaves decreases over time, and that exposure to herbivores induces increased concentrations of iridoid glycosides.