Application of near-infrared spectroscopy for the rapid analysis of Lonicerae Japonicae Flos solution extracted by water

A rapid quantitative analytical method for three components of Lonicerae Japornicae Flos solution(Lonicera Japonica Thumb.)extracted by water was developed using near-infrared(NIR)spectroscopy and the partial least-squares(PLS)method.The NIR spectra of 81 samples collected from a production line were obtained.The concentrations of secologanic acid,chlorogenicacid and galuteolin were detemmined by using high-performance liquid chromatography-diodearray detection as the reference method.Several pretreatment methods for the NIR spectra wereusedi during PLS calibration.The most appropriate latent variable number of the PLS factor wasselected based on the standard error of cross-validation(SECV).The performance of the finalPLS models was evaluated according to SECV,standard error of predliction(SEP)and deter-mination coeficient(R^(2)).The compounds secologanic acid,chlorogenic acid and galuteolin hadSEP values of 0.030,0.061 and 1.668μg/mL,respectively and R^(2) values over 0.85.This workshows that NIR spectroscopy is a rapid and convenient method for the analysis of LoniceraeJaponicae Flos solution extracted by water.The proposed method can help in the application ofprocs analytical technology in the pha maceutical industry,particularly in tra ditional Chinesemedicine injections.

Comparative analysis of volatile constituents between herbal pair flos lonicerae-caulis lonicerae and its single herbs

Gas chromatography-mass spectrometry(GC-MS) and the chemometric resolution method(alternative moving window factor analysis,AMWFA) were used for comparative analysis of volatile constituents in herbal pair(HP) flos lonicerae-caulis lonicerae(FL-CL) and its single herbs.The temperature-programmed retention index(PTRI) was also employed for the identification of compounds.In total,44,39,and 50 volatile chemical components in volatile oil of FL,CL and HP FL-CL were separately determined qualitatively and quantitatively,accounting for 87.22%,94.54% and 90.08% total contents of volatile oil of FL,CL and HP FL-CL,respectively.The results show that there are 32 common volatile constituents between HP FL-CL and single herb FL,33 common volatile constituents between HP FL-CL and single herb CL,and 10 new constituents in the volatile oil of HP FL-CL.

Quality Analysis and High-Performance Liquid Chromatographic Fingerprint Analysis of New Cultivated Kind of <i>Lonicerae japonicae</i>Flos “Hua Jin 6” from Different Harvest Times

Lonicerae japonicaeFlos (LJF) is widely used in traditional Chinese medicines for the treatment of various diseases, which is now in great demand every year and has a broad development prospect. However, the flowering phase of common LJF varieties is so short, which seriously restricts the development of LJF industry. As a new cultivated kind of Lonicerae japonicae Flos, “Hua Jin 6” has characteristics in long flowering phase and conveniently picking, which makes it have a broad development prospect. The aim of this study is to provide scientific guidance for its suitable harvest period by measuring yield and quality of “Hua Jin 6” from different harvest time. Studies show that flower size had a slowly rising trend from the first day to the seventh day, and then slowly declined or kept stable. There were no significant differences of total phenolic acid contents in different samples from different days, but contents of total flavonoids were on the rise and up to maximum in the ninth day. The contents of total iridoids had an increasing tendency from the first day to the fifth day and then kept relatively stable in other days. We demonstrated that the quality of “Hua Jin 6” is relatively stable and suitable for harvesting in all flower buds white stage in term of HPLC fingerprints. Our findings can make it possible to select the suitable time for different harvest purpose.

Screening hepatoprotective effective components of Lonicerae japonica Flos based on the spectrum-effect relationship and its mechanism exploring

Lonicerae japonicae Flos(LF)is a kind of healthcare food with hepatoprotective function.This study was designed to explore the spectrum-effect relationships between UPLC fingerprints and the hepatoprotective effects of LF.Fingerprints of ten batches of LF were established by UPLC-PDA.The inhibitory levels of AST and ALT were used as pharmacological indexes,and secoxyloganin,isochlorogenic acid A and isochlorogenic acid C were screened as hepatoprotective active compounds by grey relational analysis(GRA)and partial least squares regression analysis(PLSR).Caspase-3 was obtained by network pharmacology as a key target of hepatoprotective active compounds.Molecular docking is used to explore the interaction between small molecules and proteins.This work provided a general model of the combination of UPLC-PDA and hepatoprotective effect to study the spectrum-effect relationship of LF,which can be used to considerable methods and insight for the fundamental research of the material basis of similar healthcare food.

Comparative Analysis of the Complete Chloroplast Genome Sequences of Four Origin Plants of Lonicerae Flos(Lonicera;Caprifoliaceae)

Lonicerae Flos(LF)derived from the dried flower buds or opening flowers of four Lonicera plants(Lonicera macranthoides,L.hypoglauca,L.confusa,and L.fulvotnetosa),is a popular traditional Chinese medicine.Because the four origin plants are very similar in morphology,it is difficult to control the quality of LF in actual production.Over the past decade,many reports have pointed out the differences among them,including the botanical characteristics and active ingredients.However,there is still a lack of rapid methods that can be applied to the identification of the four origins.In this study,comparative analysis of the four chloroplast genomes was performed,and they showed low diversity(Pi=0.00267),three variation hotspots regions(rbcL-accD,rps12-ndhF and rps12-trnN-trnG)were identified as potentially molecular marker of highly informative.Meanwhile,the most obvious difference in SSR comparative analysis is reverse and complement repeats were only identified in L.confusa and L.hypoglauca,respectively.Lastly,the phylogenetic tree showed that L.confusa is more closely related to L.fulvotnetosa,while L.macranthoides is closer to L.hypoglauca.This study systematically revealed the differences among the four chloroplast genomes,and it provides valuable genetic information for identifying the origin of LF.

Chemical compositions and biological activities of Flos lonicerae

Flos lonicerae is the dry flower bud of Lonicera japonica Thunb,and it is a very important Chinese herbal medicine in China.In ancient times,it was widely used to treat the exopathogenic wind-heat,epidemic febrile diseases,sores,carbuncles and some infectious diseases.Modern pharmacological research has indicated that it has anti-inflammatory,anti-bacterial,anti-viral and anti-oxidation activities.This paper reviews the chemical compositions and biological activities of Flos lonicerae in order to provide reference for its application in clinical practice in the future.

Short-term Prediction of Occurrence Period of Main Diseases and Pests of Flos lonicerae using Phenoecology

The occurrence periods of Semiaphis heraclei Takahashi,Frankliniella sp.,Haptonchus luteolus and Microsphara linicerae Enchson wint.in Rabenh.causing damage on Flos lonicerae were investigated in F.lonicerae planting area in XinCheng county of Guangxi Province during 2008-2010,which were coincided with the occurrence periods of related phenology of local Prunus persica Rootstock.With P.persica Rootstock as indicator plant,the occurrence periods of three species of pests and one species of disease were predicted,respectively,and the method was simple and accurate,which could be the foundation for preventing these pests and diseases in the local field.

Separation and Purification of Macranthoidin B and Dipsacoside B from Flos Lonicerae by HP-20 and HP-SS Macroporous Resin

[Objective] This study was conducted to develop a method for rapidly separating macranthoidin B and dipsacoside B from Flos Lonicerae. [Method] HP-20 and HP-SS macroporous resin were applied to separate and purify macranthoidin B and dipsacoside B from Flos Lonicerae. The extract of Flos Lonicerae was first loaded onto an HP-20 column to enrich saponins, which were then separated by an HP-SS macroporous resin column to get pure macranthoidin B and dipsacoside B.[Result] The optimal HP-20 purification conditions included： a concentration of sample liquid at 4.8 mg/ml, a sample volume of 2 BV, an adsorption flow rate at 1.5BV/h, an ethanol concentration for desorption at 60%, a desorption volume of 3 BV,and a desorption flow rate at 1.5 BV/h. Total saponins were then separated by an HP-SS macroporous resin column which was eluted sequentially by water, 20%ethanol, 30% ethanol, 40% ethanol and 50% ethanol. Two purified compounds were obtained in fractions eluted by 40% ethanol and 50% ethanol, respectively. The two compounds were identified as macranthoidin B and dipsacoside B by13 C and1H nuclear magnetic resonance spectroscopy. [Conclusion] The combination of HP-20 and HP-SS macroporous resin could efficiently separate macranthoidin B and dipsacoside B from Flos Lonicerae.

Effect of chlorogenic acid on antioxidant activity of Flos Lonicerae extracts

Flos Lonicerae is a medically useful traditional Chinese medicine herb. However, little is known about the antioxidant properties of Flos Lonicerae extracts. Here the antioxidant capacity of water, methanolic and ethanolic extracts prepared from Flos Lonicerae to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and reduce Fe3+ to Fe2+ is examined. Chlorogenic acid, a major component of Flos Lonicerae, is identified and further purified from 70% ethanolic extract with high performance liquid chromatography (HPLC) and its antioxidant capacity is characterized. The total phenolic compounds and chlorogenic acid con-tents in Flos Lonicerae are determined. The present results demonstrate that the Flos Lonicerae extracts exhibit antioxidant ac-tivity and chlorogenic acid is a major contributor to this activity.

Extraction process of chlorogenic acid in flos lonicerae by enzymatic treatment

A new method of extracting chlorogenic acid from flos lonicerae, and treating the materials with enzyme before being extracted by ethanol is developed, and the optimum conditions are also investigated in detail. Three important factors, enzyme dosage, treatment time and treatment temperature are adapted to optimize the extraction process. The experimental results show that the extract yield of flos lonicerae and chlorogenic acid can be obviously increased by the cellulase treatment, 61.5 mg chlorogenic acid is obtained from 1.00 g flos lonicerae at most. The optimal temperature of enzymatic treatment is 40 50 ℃. Compared with the use of single cellulase, the combined treatment of cellulase and pectinase increase the extract yield obviously but fail to improve that of chlorogenic acid.

Lonicerae Flos:A Review of Chemical Constituents and Biological Activities

Lonicerae flos,a widely used traditional Chinese medicine(TCM),has been used for several thousand years in China.As a famous traditional Chinese herbal medicine,it was used as heat-clearing drug and alexipharmic agent and was widely cultivated in Hunan province.L.flos mainly contains biologically active compounds such as caffeic acid derivatives,essential oil,flavonoids,iridoid glycosides and terpenoids.A range of biological activities has been reported from plant extracts including anti-inflammatory,antitumor,antioxidant,antiallergy,immunomodulating and antibacterial activity.In this study,the author investigated ancient books of TCM and nowadays reports,summarized the chemical constituents,biological activity of L.flos to provide a comprehensive systematic review.

In vivoand in vitroanti-arthritic efficacy of a herbal formula consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos

OBJECTIVE Rheumatoid arthritis(RA)is the most common inflammatory autoimmune disease,affecting around 1% of the world population.Toll-like receptor 4(TLR4)signalling has been found to be involved in the pathogenesis of RA.It is a potential therapeutic target for RA treatment.A herbal formula(RL)consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos has traditionally been used in treating various inflammatory disorders.In this study,we would evaluate the anti-arthritic effect of RL on collagen-induced arthritis(CIA)in rats and investigate the involvement of TLR4 signaling in the mode of action of RL in vivo and in vitro.METHODS In vivo anti-arthritic efficacy was evaluated using CIA rats induced by bovine typeⅡ collagen.The treatment groups were treated with various concentrations of RL or positive control indomethacin for 35 d.Clinical signs(hind paw volume and arthritis severity scores),changes in serum inflammatory mediators,histological and radiographic changes of joints were investigated.Spleens and peritoneal macrophages were used to determine the effects of RL on innate and adaptive immune responses in CIA rats.The involvement of TLR4 signalling pathways in the anti-arthritic effect of RL was examined in cartilage tissue of CIA rats,murine RAW264.7macrophages and human THP-1 monocytic cells.RESULTS The severity of arthritis in the CIA rats was significantly attenuated by RL.Histological score and radiographic score were efficiently improved by RL.RL could also dose-dependently inhibit pro-inflammatory cytokines in serum of CIA rats.RL significantly inhibited the production of various pro-inflammatory mediators,the expression and/or activity of the components of TLR4 signalling pathways in animal tissue and cell lines.CONCLUSION RL possesses anti-arthritic effect on collagen-induced arthritis in rats.The therapeutic effect of RL may be related to its inhibition on pro-inflammatory cytokines in serum.The inhibition of the TLR4/TAK1/NF-κB and TLR4/TAK1/MAPK pathways participate in the anti-arthritic effects of RL.This provides a pharmacological justification for the use of RL in the control of various arthritic diseases.Further investigation should be done to develop RL into a modern anti-arthritic agent.

Optimization of Extraction Process of Total Flavonoids and Chlorogenic Acid from Flos Lonicerae by Orthogonal Experiment

[Objectives]To explore the process of simultaneous extracting total flavonoids and chlorogenic acid from Flos Lonicerae by pressure assisted extraction.[Methods]Based on the single factor experiment,orthogonal experiments were carried out to investigate the effects of solvent concentration,solid-to-liquid ratio,extraction pressure,extraction time and extraction temperature on the extraction rate of total flavonoids and chlorogenic acid.[Results]The results showed that the extraction pressure had a significant effect on the extraction of two kinds of chemical constituents from F.Lonicerae.The optimum process conditions were as follows:solvent concentration was 60%,solid-liquid ratio was 1∶20(g/mL),extraction pressure was 2 MPa,extraction time was 30 min,extraction temperature was 70℃.The process conditions are stable,and the extraction efficiency is higher than that of ultrasonic assisted extraction method.Under the test conditions,the extraction rates of total flavonoids and chlorogenic acids were respectively 15.66%and 3.89%.[Conclusions]This study provides a new theoretical basis for the development and utilization of F.Lonicerae.

Content Determination of Chlorogenic Acid and Luteoloside in Flos Lonicerae

The contents of chlorogenic acid and luteoloside in Flos Lonicerae from Binhai New Area and Jinnan District of Tianjin were determined to provide basis for the quality identification of this medicinal material. The content of chlorogenic acid was determined by HPLC. In Flos Lonicerae from Binhai New Area and in Flos Lonicerae harvested at different stages from Jinnan District,the contents of chlorogenic acid were 3. 804%,5. 507%( three green stage),4. 855%( silver flower stage) and 4. 220%( golden flower stage),respectively,and the contents of luteoloside were 5. 53%,12. 405%( three green stage),14. 370%( silver flower stage) and 0. 917%( golden flower stage),respectively. The contents of chlorogenic acid in Flos Lonicerae from Jinnan District were higher than that from Binhai New Area. Among different stages,the content of chlorogenic acid was highest in three green stage,followed by that in silver flower stage. As the flowers bloomed,the content of chlorogenic acid in the medicinal material showed a significant downward trend. In Flos Lonicerae from Jinnan District,the content of luteoloside was highest in silver flower stage and lowest in golden flower stage.

Determination of Chlorogenic Acid,Macranthoidin B and Dipsacoside B in Flos Lonicera by HPLC-ELSD

[Objective] A method for the determination of chlorogenic acid, macranthoidin B and dipsacoside B in Flos Lonicera by high performance liquid chromatography（HPLC） with evaporative light scattering detection（ELSD） was established.[Method] The separation was carried out using a ZORBAX SB-C18 chromatographic column（4.6 mm ×250 mm, 5 μm）, with a simple mobile phase of acetonitrile and0.1% formic acid. Evaporative light scattering detector was employed. [Result] The result showed that chlorogenic aicd, macranthoidin B and dipsacoside B showed good linearities in the ranges of 3.00-6.40, 4.24-9.33 and 0.40-2.00 μg with correlation coefficient of 0.999, 0.993 and 0.999, respectively. [Conclusion] The method has the advantages of simple operation, good accuracy, repeatability and separation effect and high specificity and is suitable for overall quality control of Flos Lonicera.

Extraction Condition Optimization and Identification of Phenols from Lonicerae flos

Polyphenols were obtained from the natural dried Lonicerae flos by ultrasound-assisted extraction with ethanol as the solvent.Single factor experiment and response surface methodology were employed to optimize the extraction conditions.Ultra-performance liquid chromatrography(UPLC)-tandem mass spectrometry(MS/MS)was employed to identify polyphenols based on the plant widely targeted metabolomics database in a qualitative and quantitative manner.The results showed that the optimal extraction conditions for total phenols from Lonicerae flos were ultrasound-assisted extraction with a solid-to-liquid ratio of 10∶1 g/mL and 57%ethanol at 70 W and 60°C for 11 min.The yield of total phenols extracted under the optimal conditions reached 71.08 mg/g.The phenols in Lonicerae flos were mainly chlorogenic acid isomers,and the flavonoids were mainly nobiletin,galuteolin,and homoarbutin.

Comprehensive analysis of the chemical constituents in sulfur-fumigated Lonicerae Japonicae Flos using UHPLC-LTQ-Orbitrap mass spectrometry

Lonicerae Japonicae Flos(LJF),a kind of traditional Chinese medicines(TCMs),has functions of detoxifying and evacuating heat.In the study,a method based on ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS)was developed for the chemical constituent analysis of organic acids,flavonoids,iridoids and new-generated compounds in sulfur-fumigated LJF(SF-LJF).Based on the accurate mass measurement(<±5 ppm),chromatographic behavior and diagnostic product ions(DPIs),113 constituents were unambiguously or tentatively characterized from SFLJF extract,including 46 chlorogenic acids,19 flavonoids,29 iridoid glycosides and 19 newly-generated compounds(including 17 sulfur-containing derivatives).In addition,5-CQA(5-caffeoylquinic acid,chlorogenic acid)was chosen to be sulfur-fumigated for the result validation.It was found that the most significant change of LJF after sulfur fumigation was the occurrence of sulfate or sulfite esterification reactions,which resulted in the emergence of many new sulfur-containing components.Our results demonstrated that the established method was a useful and efficient analytical tool to comprehensively characterize the material basis of SF-LJF,and also an excellent guidance of quality control about LJF.

Qualitative and quantitative evaluation of Flos Puerariae by using chemical fingerprint in combination with chemometrics method

In order to better control the quality of Flos Puerariae(FP),qualitative and quantitative analyses were initially performed by using chemical fingerprint and chemometrics methods in this study.First,the fingerprint of FP was developed by HPLC and the chemical markers were screened out by similarity analysis(SA),hierarchical clustering analysis(HCA),principal components analysis(PCA),and orthogonal partial least squares discriminant analysis(OPLS-DA).Next,the chemical constituents in FP were profiled and identified by HPLC coupled to Fourier transform ion cyclotron resonance mass spectrometry(HPLCFT-ICR MS).Then,the characteristic constituents in FP were quantitatively analyzed by HPLC.As a result,31 common peaks were assigned in the fingerprint and 6 of them were considered as qualitative markers.A total of 35 chemical constituents were detected by HPLC-FT-ICR MS and 16 of them were unambiguously identified by comparing retention time,UV absorption wavelength,accurate mass,and MS/MS data with those of reference standards.Subsequently,the contents of glycitin,genistin,tectoridin,glycitein,genistein,and tectorigenin in 13 batches of FP were detected,ranging from 0.4438 to 11.06 mg/g,0.955 to 1.726 mg/g,9.81 to 57.22 mg/g,3.349 to 41.60 mg/g,0.3576 to 0.989 mg/g,and 2.126 to 9.99 mg/g,respectively.In conclusion,fingerprint analysis in combination with chemometrics methods could discover chemical markers for improving the quality control standard of FP.It is expected that the strategy applied in this study will be valuable for further quality control of other traditional Chinese medicines.

灰毡毛忍冬bZIP25基因的克隆及其表达模式分析

目的克隆灰毡毛忍冬bZIP25基因序列全长,并进行生信分析及表达模式分析,以初步探索其在灰毡毛忍冬中的生物学功能。方法通过逆转录PCR技术克隆bZIP25基因的序列全长,采用生物信息学分析方法对bZIP25及其编码的蛋白进行分析。运用实时荧光定量PCR(qRT-PCR)技术测定其在茎、叶及七个花期花中的表达水平。结果克隆得到LmbZIP25基因(OR551766),其编码191个氨基酸,具有典型的bZIP家族结构,在bZIP结构域中与其他植物同源性较高。LmbZIP25基因具有组织特异性,在茎中的表达量显著高于花和叶,在七个花期中黄色花蕾期的表达量最高。结论克隆得到LmbZIP25基因全长,分析其在不同器官和不同花期的表达差异,为进一步探索其在灰毡毛忍冬中花发育中的功能奠定了研究基础。

忍冬不同部位提取液抑菌活性及其对酸土脂环酸芽孢杆菌的抑菌特性

本研究探究了忍冬(Lonicera japonica Thunb.)不同部位提取液的生物活性物质含量及其对12株供试菌株的抑菌活性。首先,分别测定了忍冬叶、金银花和忍冬藤中的总酚、总黄酮和绿原酸含量,并分析了它们的提取液对12株菌株的抑菌效果。然后,进一步研究了金银花和忍冬叶提取液对酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris)的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)、生长曲线、生物膜形成和细胞形态的影响。结果显示,忍冬叶和金银花中的总酚、总黄酮和绿原酸含量均显著高于忍冬藤(P<0.05)。此外,忍冬叶的总黄酮含量显著高于金银花(P<0.05)。三种提取液对A.acidoterrestris、金黄色葡萄球菌、无乳链球菌、单增李斯特氏菌、枯草芽孢杆菌、大肠埃希菌和沙门氏杆菌均具有一定的抑菌效果,且忍冬叶和金银花提取液的抑菌效果优于忍冬藤。忍冬叶和金银花提取液对A.acidoterrestris的抑菌效果最显著,MIC值均为3.91 mg/mL,MBC值均为31.25 mg/mL。此外,忍冬叶和金银花提取液能有效抑制A.acidoterrestris生长和生物膜的形成,破坏其菌体形态,抑菌效果随提取液浓度的增加而增强。本研究为阐明忍冬抑制A.acidoterrestris的机制奠定了坚实基础,同时也为开发安全高效的新型食品抑菌剂提供了新思路。