The fertility research of "Huajin 6", a new variety of honeysuckle.

The aim of this study was to investigate the fertility of "Huajin 6" and the effect of exogenous methyl jasmonate on its fertility. In this study, "Huajin 6", "Huajin 6" treated with methyl jasmonate and "Damaohua" were used as the research objects, the stamen phenotypes and the shape of pollen grains were observed, pollen viability and stigma receptivity were measured. The results showed that the pistil structure and function were normal, and although the stamen anthers did not dehisce, they were still capable of producing pollen with a certain amount of vigor. Methyl jasmonate could promote the opening of the flowers of "Huajin 6" and improve the development of pollen grains to a certain extent, but it could not promote anthers dehiscence of "Huajin 6". This study can provide theoretical guidance for the cultivation of new honeysuckle varieties using "Huajin 6".

Plant metabolomics for studying the effect of two insecticides on comprehensive constituents of Lonicerae Japonicae Flos.

Pesticides' overuse and misuse have been reported to induce ingredient variations in herbal medicine, which is now gaining attention in the medicinal field as a form of alternative medicine. To date, available studies on pesticide-induced ingredient variations of herbal medicine are limited only on a few compounds and remain most others unexamined. In this study, a plant metabolomics-based strategy was performed to systematically explore the effects of two frequently used insecticides on the comprehensive constituents of Lonicerae Japonicae Flos (LJF), the flower buds of Lonicera japonica Thunb. Field trials were designed on a cultivating plot of L. japonica with controls and treatments of imidacloprid (IMI) and compound flonicamid and acetamiprid (CFA). Unbiased metabolite profiling was conducted by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometer. After data pretreatment by automatic extraction and screening, a data matrix of metabolite features was submitted for statistical analyses. Consequently, 29 metabolic markers, including chlorogenic acids, iridoids and organic acid-glucosides were obtained and characterized. The relative quantitative assay was subsequently performed to monitor their variations across flowering developments. This is the first study that systematically explored the insecticide-induced metabolite variations of LJF while taking into account the inherent variability of flowering development. The results were beneficial for holistic quality assessment of LJF and significant for guiding scientific use of pesticides in the large-scale cultivation.

Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo.

Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain to be elucidated. Here, four honeysuckle extracts, including acids extract, flavonoids extract, total extract and acids-flavonoids mixture, were prepared to clarify the main active antiviral components. The cytopathic effect reduction assay showed that all the four extracts inhibited the replication of influenza viruses H1N1, H3N2 and the oseltamivir-resistant mutant strain H1N1-H275Y. The acids-flavonoids mixture had the strongest inhibitory effects in vitro with EC50 values of 3.8, 4.1, and > 20 µg/mL against H1N1, H3N2 and H1N1-H275Y, respectively, showing competitive antiviral activity with oseltamivir and ribavirin. Honeysuckle acids extract also showed the most significant antiviral activity in vivo. Oral administration of the acids extract at a dosage of 600 mg/kg/d effectively alleviated viral pneumonia, maintained body weight and improved the survival rate to 30% of the mice infected with a lethal dose of H1N1. The results of time-of-drug addition experiment and neuraminidase (NA) inhibition assay showed that honeysuckle extracts had a broad-spectrum inhibitory effect against influenza virus NAs. The flavonoid extract showed the strongest inhibitory effect on the NA of influenza virus H7N9 with an IC50 of 24.7 µg/mL. These results suggested that these extracts might exert their antiviral activity by suppressing the release of influenza viruses. Briefly, our findings demonstrate that acids and flavonoids extracts of honeysuckle are the major antiviral active components, and the acids extract has the potential to be developed into an antiviral agent against influenza virus, especially for oseltamivir-resistant viruses.

[Regulation of exogenous calcium on photosynthetic system of honeysuckle under salt stress].

Exogenous calcium can enhance the resistance of certain plants to abiotic stress. However,the role of calcium insaltstressed honeysuckle is unclear. The study is aimed to investigate the effects of exogenous calcium on the biomass,chlorophyll content,gas exchange parameters and chlorophyll fluorescence of honeysuckle under salt stress. The results showed that the calcium-treated honeysuckle had better photochemical properties than the salt-stressed honeysuckle,such as PIABS,PItotal,which represents the overall activity of photosystemⅡ(PSⅡ),and related parameters for characterizing electron transport efficiency φP0,ψE0,φE0,σR,and φR are significantly improved. At the same time,the gas exchange parameters Gs,Ci,Trare also maintained at a high level. In summary,exogenous calcium protects the activity of PSⅡ,promotes the transmission of photosynthetic electrons,and maintains a high Ci,therefore enhances the resistance of honeysuckle under salt stress.

Drought Alleviated the Negative Effects of Elevated O3 on Lonicera maackii in Urban Area.

Open top chambers were used to study the changes in photosynthesis, physiology and stomata characteristics in 1-year-old Lonicera maackii seedlings exposed to drought (DT, 30%-35% soil saturated water content) or/and elevated ozone (EO, 80 ppb). The results showed that DT or/and EO significantly decreased net photosynthetic rate (Pn), stomatal conductance (gs), maximum photochemical efficiency (Fv/Fm), but increased the activity of superoxide dismutase (SOD), and malondialdehyde content (p < 0.05). Compared with EO alone, the combination of EO and DT caused higher values in Pn, Fv/Fm, SOD activity (p < 0.05), and smaller stomata size and lower visible injury rate. DT alleviated the adverse impact of EO on the shrub by increasing enzyme activity and decreasing stomatal size, particularly stomatal width. The study provided increasing evidence that moderate drought might exert a beneficial effect on the tested plants to adapt to the future climate change, particularly in high ozone regions.

Saline stress enhanced accumulation of leaf phenolics in honeysuckle (Lonicera japonica Thunb.) without induction of oxidative stress.

Honeysuckle (Lonicera japonica Thunb.) is a traditional medicinal plant in Chinese, and chlorogenic acid and luteolosid are its specific bioactive phenolic compounds. This study was to investigate leaf antioxidant responses in honeysuckle to saline stress with emphasis on phenolics through hydroponic experiments and field trials. NaCl stress did not stimulate antioxidant system including superoxide dismutase, ascorbate peroxidase, catalase and ascorbate, and had no significant effect on lipid peroxidation in the leaves. Consistently, no inhibition on photochemical capacity of photosystems suggested that reactive oxygen species (ROS) was maintained at a normal level under NaCl stress. However, leaf phenolic synthesis was activated by NaCl stress, indicated by elevated genes transcription and activity of phenylalanine ammonia-lyase and increased phenolics concentration. Specifically, leaf chlorogenic acid concentration was increased by 67.43% and 48.86% after 15 days of 150 and 300 mM NaCl stress, and the increase of luteolosid concentration was 54.26% and 39.74%. The accumulated phenolics hardly helped detoxify ROS in vivo in absence of oxidative stress, but the elevated phenolic synthesis might restrict ROS generation by consuming reduction equivalents. As with NaCl stress, soil salinity also increased concentrations of leaf phenolics including chlorogenic acid and luteolosid without exacerbated lipid peroxidation. In conclusion, leaf phenolics accumulation is a mechanism for the acclimation to saline stress probably by preventing oxidative stress in honeysuckle; leaf medicinal quality of honeysuckle can be improved by saline stress due to the accumulation of bioactive phenolic compounds.

Can arbuscular mycorrhizal fungi reduce Cd uptake and alleviate Cd toxicity of Lonicera japonica grown in Cd-added soils?

A greenhouse pot experiment was conducted to study the impact of arbuscular mycorrhizal fungi--Glomus versiforme (Gv) and Rhizophagus intraradices (Ri) on the growth, Cd uptake, antioxidant indices [glutathione reductase (GR), ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), ascorbate (ASA), glutathione (GSH) and malonaldehyde (MDA)] and phytochelatins (PCs) production of Lonicera japonica in Cd-amended soils. Gv and Ri significantly increased P acquisition, biomass of shoots and roots at all Cd treatments. Gv significantly decreased Cd concentrations in shoots and roots, and Ri also obviously reduced Cd concentrations in shoots but increased Cd concentrations in roots. Meanwhile, activities of CAT, APX and GR, and contents of ASA and PCs were remarkably higher in Gv/Ri-inoculated plants than those of uninoculated plants, but lower MDA and GSH contents in Gv/Ri-inoculated plants were found. In conclusion, Gv and Ri symbiosis alleviated Cd toxicity of L. japonica through the decline of shoot Cd concentrations and the improvement of P nutrition, PCs content and activities of GR, CAT, APX in inoculated plants, and then improved plant growth. The decrease of shoot Cd concentrations in L. japonica inoculated with Gv/Ri would provide a clue for safe production of this plant from Cd-contaminated soils.

The role of silicon in physiology of the medicinal plant (Lonicera japonica L.) under salt stress.

Silicon(Si) is the only element which can enhance the resistance to multiple stresses. However, the role of silicon in medicinal plants under salt stress is not yet understood. This experiment was conducted to study the effects of silicon addition on the growth, osmotic adjustments, photosynthetic characteristics, chloroplast ultrastructure and Chlorogenic acid (CGA) production of Honeysuckle plant (Lonicera japonica L.) under salt-stressed conditions. Salinity exerted an adverse effect on the plant fresh weight and dry weight, whilst 0.5 g L(-1) K2SiO3 · nH2O addition obviously improved the plant growth. Although Na(+) concentration in plant organs was drastically increased with increasing salinity, higher levels of K(+)/Na(+) ratio was obtained after K2SiO3 · nH2O addition. Salinity stress induced the destruction of the chloroplast envelope; however, K2SiO3 · nH2O addition counteracted the adverse effect by salinity on the structure of the photosynthetic apparatus. K2SiO3 · nH2O addition also enhanced the activities of superoxide dismutase and catalase. To sum up, exogenous Si plays a key role in enhancing its resistance to salt stresses in physiological base, thereby improving the growth and CGA production of Honeysuckle plant.

Influence of sulfur fumigation on the chemical constituents and antioxidant activity of buds of Lonicera japonica.

Lonicera japonica flos is widely used as a pharmaceutical resource and a commonly-employed ingredient in healthy food, soft beverages and cosmetics in China. Sometimes, sulfur fumigation is used during post-harvest handling. In this study, a comprehensive comparison of the chemical profile between sun-dried and sulfur-fumigated samples was conducted by HPLC fingerprints and simultaneous quantification of nine constituents, including secologanic acid, along with another eight usually-analyzed markers. Secologanic acid was destroyed, and its sulfonates were generated, whereas caffeoylquinic acids were protected from being oxidized. The residual sulfur dioxide in sulfur-fumigated samples was significantly higher than that in sun-dried samples, which might increase the potential incidence of toxicity to humans. Meanwhile, compared with sun-dried samples, sulfur-fumigated samples have significantly stronger antioxidant activity, which could be attributed to the joint effect of protected phenolic acids and flavonoids, as well as newly-generated iridoid sulfonates.

[Investigation on formation mechanism of secologanic acid sulfonates in sulfur-fumigated buds of Lonicera japonica].

To investigate formation mechanism of secologanic acid sulfonates in sulfur-fumigated buds of Lonicera japonica, secologanic acid was enriched and purified from the sun-dried buds of L. japonica by various column chromatography on macroporus resin HPD-100, silica gel and ODS. The stimulation experiments of sulfur-fumigation process were carried out using secologanic acid reacted with SO2 in the aqueous solution. The reaction mechanism could be involved in the esterification or addition reaction. The present investigation provides substantial evidences for interpreting formation pathway of secologanic acid sulfonates in sulfur-fumigated buds of L. japonica.

[Hormesis effect of cadmium on Lonicera japonica].

A hydroponic experiment was conducted to study the growth characteristics of Lonicera japonica under the stress of different concentrations (0, 0.5, 2.5, 5, 10, and 25 mg L-1 ) cadmium (Cd), aimed to explore the hormesis effect of low concentrations Cd on L. japonica. At < or =5 mg L-1 of Cd, the maximum root length, plant dry biomass, and the contents of chlorophyll a, chlorophyll b, and carotenoid of L. japonica increased significantly, with the increment being 13. 6%, 11.7%, 14. 0% ,10. 8%, and 54. 5%, respectively, as compared with the control. <5 mg L-1 of Cd also had a definite positive effect on the leaf water content. At> or = 10 mg L-1 of Cd, the growth of L. japonica was inhibited significantly. When exposed to 25 mg L-1 of Cd, the shoot Cd concentration of L. japonica reached 622. 93 mg kg-1 , suggesting that L. japonica had a strong capacity of Cd-hyperaccumulation. The dose range of the hormesis effect of Cd on L. japonica was 0.5-5 mg L-1 of Cd.

Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance.

Hormesis phenomena under Cd stress in a hyperaccumulator--Lonicera japonica Thunb.

A hydroponic experiment was carried out to investigate possible hormetic response induced by cadmium (Cd) in a potential hyperaccumulator-Lonicera japonica Thunb. The results showed that Cd at low concentrations induced a significant increase in plant growth, leaf water content and content of photosynthetic pigments in L. japonica, but decreased them at high concentrations, displayed inverted U-shaped dose response curves, confirming a typical biphasic hormetic response. The U-shaped dose response curves were displayed in malondialdehyde (MDA) and electrolyte leakage in leaves at low doses of Cd, indicating reduce oxidative stress and toxic effect. The increase of superoxide dismutase (SOD) and catalase (CAT) activities was observed along with the increased Cd concentration, indicative of increase in anti-oxidative capacity that ensures redox homeostasis is maintained. After 28 days exposure to 10 mg L(-1) Cd, stem and leaf Cd concentrations reached 502.96 ± 28.90 and 103.22 ± 5.62 mg kg(-1) DW, respectively and the plant had high bioaccumulation coefficient (BC) and translocation factor (TF'). Moreover, the maximum TF value was found at 2.5 mg L(-1) Cd treatment, implying that low Cd treatment improved the ability to transfer Cd from medium via roots to aerial structures. Taking together, L. japonica could be considered as a new plant to investigate the underlying mechanisms of hormesis and Cd tolerance. Our results suggest that hormetic effects should be taken into consideration in phytoremediation of Cd-contaminated soil.

Profiling and characterization of volatile components from non-fumigated and sulfur-fumigated Flos Lonicerae Japonicae using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry coupled with chemical group separation.

Flos Lonicerae Japonicae (FLJ) is a popular herb used for many centuries in Traditional Chinese Medicine as a treatment of fever and inflammation. Non-fumigated processing of FLJ has been the traditional approach used in post-harvest preparation of the commodity for commercial use. However, in recent years, natural drying processing of FLJ has been replaced by sulfur-fumigation for efficiency and pest control. Sulfur-fumigation can induce changes in the volatile compounds of the herb, altering its medicinal properties. A comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF/MS) method was established for the resolution and determination of volatile components in non-fumigated and sulfur-fumigated FLJ. In this paper, analysis of the volatile oils in non-fumigated and sulfur-fumigated (including lab-prepared sulfur-fumigated and industrial sulfur-fumigated) FLJ was performed using GC×GC-TOF/MS. Seventy-three representative volatile components were identified, including furans, alkalies, acids, aldehydes, ketones, alcohols, terpenes, esters, and others, as the main components of FLJ volatile oils. The proposed method was successfully applied for rapid and accurate quality evaluation of FLJ and its related medicinal materials and preparations.

[Effect of abscisic acid on physiological characteristics in Lonicera macranthoides seedlings under salt stress].

OBJECTIVE: To improve salt resistance of Lonicera macranthoides seedlings and provide reference for its cultivation of salt tolerance. METHODS: Pretreatment of seedlings with abscisic acid (ABA) of different concentrations were used to study the effect of ABA on their physiological and biochemical characteristics under 200 mmol/L NaCl for 7 days. RESULTS: Exogenous ABA significantly decreased the content of malondialdehyde (MDA), increased the contents of chlorophyll, soluble sugar, soluble protein and activities of protected enzymes such as peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD). CONCLUSION: It is proposed that exogenous ABA as chemical activator can induce salt resistance and decrease alleviate damage degree of salt stress of Lonicera macranthoides seedlings in a dose-dependent manner.

Effects of cadmium hyperaccumulation on the concentrations of four trace elements in Lonicera japonica Thunb.

Hyperaccumulators are important in the phytoremediation of cadmium (Cd)-contaminated soil. In this study, Cd accumulation and the interactions between Cd and four other trace elements (Fe, Mn, Cu, and Zn) in Lonicera japonica Thunb. were investigated. As a result of exposure to soil containing 50 mg kg(-1) Cd, stem and shoot Cd concentrations reached 344.49 ± 0.71 and 286.12 ± 9.38 µg g(-1) DW respectively, without showing symptoms of visible damage to the plants. This suggests that L. japonica has a strong tolerance to Cd. It is proposed that trace metal elements are involved in the Cd-detoxification mechanisms shown by hyperaccumulators. There is a synergistic interaction in accumulation and translocation between Cd and Fe and a significantly negative correlation between Cd and Cu or Zn concentrations in L. japonica plant tissues. The imbalanced trace element concentrations influences detoxification processes to Cd, therefore, L. japonica could be considered as a new Cd-hyperaccumulator model to investigate the metal tolerance strategies of plants.

[Effects of cadmium stress on the growth and physiological characteristics of Lonicera japonica].

By using hydroponics, the growth and physiological characteristics of Lonicera japonica at different concentrations (0, 5, 10, 25, and 50 mg x L(-1)) of cadmium (Cd) were studied. The results showed that compared with the control, Cd stress had lesser effects on the growth of L. japonica. Within the range of test Cd concentrations, L. japonica biomass had less difference (P > 0.05) with the control, and at low concentration of Cd (5 mg x L(-1)), the total biomass and the biomasses of leaf and root were increased by 1.25%, 2.88 and 2.33%, respectively, illustrating that L. japonica had stronger resistance against Cd. Under low concentration Cd stress, the water content and soluble protein content in plant organs had some decrease, while the malondialdehyde (MDA) content in root and leaf increased by 51.90% and 23.07%, respectively, leaf chlorophyll and carotenoid contents increased by 15.87% and 24.89%, respectively, and superoxide dismutase (SOD) activity increased significantly. With increasing Cd concentration, the chlorophyll and carotenoid contents and SOD activity decreased to some extent.

[Effects of irrigation volume on growth and quality of Lonicera japonica].

OBJECTIVE: To study the effects of irrigation volume on the growth and quality of Lonicera japonica. METHOD: Different volume of irrigation water was applied to the potted L. japonica before the first florescence of honeysuckle in order to keep the relative moisture content of the soil as 100%, 80%, 50%, 30%, and observe the growth and development of shoot and leaves, yield and quality of honeysuckle. RESULT: As the of irrigation volume was reduced, the internodal elongation of shoot and the increase of the knot were restrained, and the yield of honeysuckle dropped. But the content of chlorogen acid in the buds treated by 80% irrigation volume was not affected, while that of the other treatment all decreased. The moisture contents of the leaves treated by 30% irrigation volume was significantly lower than that of the CK, while the specific leaf weight (SLW) increased significantly comparing to the CK. The content of chlorophyl in the leaves of 50% and 30% increased, while the ratio of chlorophyl A and B fell significantly. The content of dissoluble suger in the leaves of 80% and 50% irrigation volume was much higher than that in the CK. The results also showed that, the content of dissoluble protein in the leaves of 30% irrigation volume increased comparing with that in 50%, and the content of proline in leaveas of 30% irrigation volume increased significantly. CONCLUSION: Using less irrigation volume (keeping 80% of relative moisture content of the soil) does not affect the quality of honeysuckle, but decreased dry weight of honeysuckle. It is necessary to take irrigation management during cultivation of honeysuckle.