Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.

Protective effects of a polymethoxy flavonoids-rich Citrus aurantium peel extract on liver fibrosis induced by bile duct ligation in mice

Objective: To evaluate the possible protective effect of Citrus aurantium peel extract(CAE) against apoptosis in cholestatic liver fibrosis induced by bile duct ligation in mice. Methods: Male ICR mice were divided to 5 groups: 1) Control group(Sham-operated mice), 2) Cholestatic liver injury group induced by bile duct ligation(BDL), 3) BDL mice treated with silymarin(200 mg/kg) for 4 weeks, 4) BDL mice treated with 50 mg/kg CAE for 4 weeks, 5) BDL mice treated with 200 mg/kg CAE for 4 weeks. Mice were sacrificed and liver fibrosis was evaluated by serum and hepatic tissue biochemistry tests and liver histopathological examination. Effects of CAE on inflammation and apoptosis gene regulation were investigated through real-time PCR. CAE effect on lipid metabolism related signaling was determined by western blot analysis. Results: In BDL mice, administration of CAE for 4 weeks markedly attenuated liver fibrosis based on histopathological alteration. Serum and hepatic tissue biochemistry results revealed that CAE(50 and 200 mg/kg) decreased the levels of alanine transaminase, aspartate transaminase, gamma glutamyl transferase, total bilirubin, nitric oxide, and thiobarbituric acid reactive substances. Real-time PCR and western blot analysis showed that CAE regulated inflammation, apoptosis, and lipid metabolism factors increased by BDL. Interleukin family, tumor necrosis factor α, and related apoptosis factors m RNA levels were increased by BDL treatment. However, these increases were suppressed by CAE administration. In addition, CAE effectively increased phosphorylation of AMPactivated protein kinase, nuclear factor E2-related factor 2, and related cytoprotective proteins. Conclusions: CAE can efficiently regulate BDL-induced liver injury with antioxidant, antiinflammatory, and antiapoptotic activities.

Protective Effect of Immaturue Bitter Orange(Citrus aurantium L.)Flavonoids Extracts on PC12 Cell Injury Induced by 6-Hydroxydopamine

To study the neuro protective effect of flavonoids extracts from immature bitter orange(Citrus aurantium L.),the PC12 cells treated with 6-hydroxydopamine(6-OHDA)were used as the Parkinson’s disease(PD)model.To determine the optimal dose of 6-OHDA for constructing a PD model,PC12 cells were incubated with different concentrations of 6-OHDA for 24 h.After 24 h incubation,PC12 cells of drug groups were added 6-OHDA and different concentrations of flavonoids extracts were measured cell viability by CCK8 for selecting effective concentration of flavonoids extracts;the ROS level was determined using flow cytometry;the levels of MDA,CAT,SOD and GSH-Px were assayed by Colorimetric kit for oxidative stress investigation.Compared with the model group,PC12 cell viability was significantly enhanced(P<0.05),the levels of ROS and MDA were reduced significantly(P<0.05),and the activities of SOD,CAT and GSH-Px were significantly enhanced(P<0.05)in drug groups.In conclusion,immature bitter orange flavonoids extracts could protect PC12 cells against 6-OHDA-induced oxidative stress.

Essential Oil from Citrus aurantium Alleviates Anxiety of Patients Undergoing Coronary Angiography: A Single-Blind, Randomized Controlled Trial

Objective To determine the effectiveness and safety of essential oil from Citrus aurantium on anxiety in patients undergoing coronary angiography.Methods A single-blind,randomized controlled trial was conducted in 80 patients experiencing coronary angiography in Imam Ali Hospital in Kermanshah,Iran from April to November in 2016.All patients were randomly divided into intervention and control groups by a random number table,40 cases in each group.The patients in the intervention group inhaled Citrus aurantium essential oil for 15–20 min about 60 min before angiography.Following the same procedure,distilled water was used instead of Citrus aurantium in the control group.Spielbergers State-Trait Anxiety Inventory(STAI)was filled in and vital signs including systolic blood pressure(SBP),diastolic blood pressure(DBP),respiratory and pulse rate were recorded before and 20 min after the intervention.Adverse reactions after intervention were observed.Results In the intervention group,the mean scores of STAI,SBP,DBP,respiratory and pulse rate were 53.30±10.13,134.82±11.75 mm Hg,84.49±6.99 mm Hg,17.87±1.73 times/min,and 76.48±12.55 beats/min at baseline and significantly decreased to 42.37±10.15,124.49±10.48 mm Hg,79.23±6.62 mm Hg,14.54±1.43 times/min,and 70.03±13.66 beats/min respectively 20 min after intervention(all P<0.05);however,in the control group,neither anxiety scores nor vital signs changed significantly(P>0.05).All subjects reported no adverse reactions.Conclusion Inhalation of the essential oil from Citrus aurantium was effective in reducing anxiety and stress levels in patients undergoing coronary angiography.

Effects of lavender and Citrus aurantium on pain of conscious intensive care unit patients: A parallel randomized placebo-controlled trial

Background: Conscious patients admitted to intensive care units(ICUs) suffer from pain for various reasons, which can affect their recovery process.Objective: The present study compared the effects of aromatherapy with Citrus aurantium and lavender essential oils against placebo for reducing pain in conscious intensive care patients.Design, setting, participants and interventions: This study was a parallel randomized placebo-controlled trial. The ICUs of two educational hospitals in Kerman in Southeastern Iran were the study setting.One hundred and fifty conscious intensive care patients were randomly divided into three groups using a stratified block randomization method. Two groups received aromatherapy with essential oils: one with lavender and the other with C. aurantium;these patients received a 30-minute therapy session using their assigned essential oil on the second day of their intensive care stay. The placebo group used 5 drops of normal saline instead of essential oil during their session.Main outcome measures: Patient’s pain was assessed using a visual analog scale before the aromatherapy intervention, as well as immediately after and one and three hours after intervention.Results: The mean pain score of the lavender group was 40.01 before the aromatherapy intervention and fell to 39.40, 30.60 and 23.68 immediately after the intervention, and at hour one and three postintervention, respectively. The mean pain score of the C. aurantium group was 45.48 before the intervention and was reduced to 32.34 at three hours after the intervention. The mean pain of the placebo group decreased from 42.80 before the intervention to 35.20 at three hours after the intervention. Pain scores of all groups decreased during the study(P < 0.001). The mean pain of the lavender group was significantly lower than that of the placebo group at three hours after the intervention.Conclusion: The results of this study showed that aromatherapy with lavender essential oil reduced pain in conscious ICU patients. Our data could not justify the use of C. aurantium for reducing pain in this population.

Two new phenolic glycosides isolated from the fruits of Citrus aurantium

The present study was designed to investigate the chemical constituents of the fruit of Citrus aurantium L.. The compounds were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Two new phenolic glycosides(compounds 1 and 2) were obtained and identified as 1-O-3, 5-dihydroxyphenyl-(6-O-4-hydroxybenzoyl)-β-D-glucopyranoside(1) and 1-O-3, 5-dihydroxyphenyl-(6-O-3-methoxy-4-hydroxy benzoyl)-β-D-glucopyranoside(2), respectively.

PHYTOCHEMICAL STUDY OF CITRUS AURANTIUM BERGAMIA: ANALYSIS OF BIOACTIVE COMPOUNDS PRESENT IN JUICE, TISSUES AND ESSENTIAL OIL OF THE FRUIT

Flavonoids are secondary metabolites from aromatic plants and one of the most present classes in the vegetable kingdom.This is thanks to its antioxidant properties but also through its benefits on the human body that these molecules have become the center of interest of many researchers in recent years.They are found in large quantities in citrus species.The aim of this study is

不同采收期的酸橙叶片形态及药材质量比较

研究不同采收期酸橙(Citrus aurantium L.)药材干燥后枳实和枳壳的性状特点,比较水分、灰分、浸出物含量和主要化学成分的变化规律,确定酸橙药材枳实和枳壳的最佳采收期,并为充分利用酸橙资源、完善质量控制提供科学依据。采用甲苯法测定水分、热浸法测定浸出物含量,按《中华人民共和国药典(2020年版四部)》规定的方法测定灰分,采用高效液相色谱法测定枳实、枳壳中辛弗林和3种黄酮苷的含量。结果表明,酸橙中果、大果性状稳定性较优,与药典规定规格相近。随着采收期推后,酸橙药材柚皮苷含量呈先升高后降低的趋势,新橙皮苷、橙皮苷、辛弗林含量均呈下降趋势。综合考虑劳动力、时间成本、药材性状、药材有效化学成分等,确定中果酸橙的枳实质量为佳,枳实6月中旬采摘为佳,枳壳7月上中旬果皮尚绿时采收为佳。

Strategic Conservation of Orchard Germplasm Based on Indigenous Knowledge and Genetic Diversity:a Case Study of Sour Orange Populations in China

To effectively conserve sour orange （Citrus aurantium L.） germplasm on two islands at the estuary of the Yangtze River in China, we estimated genetic variation and relationships of the known parental trees and their proposed descendents （young trees） using the fingerprints of random amplified polymorphic DNA （RAPD）. Results based on RAPD analyses showed considerable genetic diversity in the parental populations （He = 0.202）. The overall populations including the parental and young trees showed slightly higher genetic diversity （He = 0.298） than the parents, with about 10% variation between populations. An unweighted pair group method with arithmetic mean analysis dendrogram based on cluster analysis of the Jaccard similarity among individuals demonstrated a more complicated relationship of the parental and young trees from the two islands, although the young trees showed a clear association with parental trees. This indicates a significant contribution of parental trees in establishing the sour orange populations on the two islands. According to farmers＇ knowledge, conservation of only one or two parental trees would be sufficient because they believed that the whole populations were generated from a single mother tree. However, this study suggests that preserving most parental trees and some selected young trees with distant genetic relationships should be an effective conservation strategy for sour orange germplasm on the two islands.