Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent Chlorogenic acid in vitro

Objective:To examine the acanthamoebicidal effects of ethyl acetate,aqueous and butanol fractions of dried flower buds of Lonicera japonica(L.japonica) Thunb.(Flos Lonicerae) in vitro.Methods:Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR.They were exposed to ethyl acetate,water and butanol fractions of L.japonica Thunb.at concentrations ranging from 0.5 mg/m L to 1.5 mg/m L.The extracts were evaluated for growth inhibition at 24,48 and 72 h,respectively.Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment.Results:Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h,49.2% after 48 h and 33.7% after 72 h Chlorogenic acid,the major active constituent of L.japonica Thunb.at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h,84.0% after 48 h and 72.3% after 72 h.This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h.Conclusions:Results obtained from this study show that ethyl acetate fraction at 1.5 mg/m L is the most potent fraction of L.japonica Thunb.and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Review of Factors Influencing Active Components of Lonicera japonica Thunb.

Lonicera japonica Thunb. is a typical medicinal and edible plant in China. It has antibacterial, anti-oxidant, anti-endotoxin, and anti-inflammatory effects, etc. Its pharmacological action is closely related to its active components. Based on the results of previous studies, this paper summarized the effects of variety, origin, cultivation conditions, pruning, harvesting period and processing methods on the active components of L. japonica , in the hope of providing theoretical guidance for high-yield and high-quality cultivation of L. japonica.

Analysis of Volatile Constituents in Lonicera japonica Thunb. from Different Origins by GC-MS

[Objective] This study aimed to analyze the volatile constituents in Lonicera japonica Thunb. from different origins. [Method] HP-5MS capillary columns were used and column temperature was controlled by a program. MS analysis was performed with EI and quadruple mass analyzer. The volatile constituents in L. japonica Thunb. were identified by NIST02 and Wiley275 libraries, and their relative contents were determined with chromatographic peak area normalization method. [Result] According to GC-MS total ion-current chromatograms, 35 volatile constituents were identified in L. japonica Thunb. from Guangxi Zhuang Autonomous Region, mainly including methyl linolenate, n-hexadecanoic acid and ζ-muurolene; 18 volatile constituents were identified in L. japonica Thunb. from Hunan Province, mainly including n-hexadecanoic acid, linoleic acid and α-curcumene. [Conclusion] Main volatile constituents in L. japonica Thunb. from two different origins varied significantly.

Effect of Different Factors on Rooting Characteristics and Survival of Lonicera japonica Thunb

[Objective] The aim was to explore the effects of different factors on rapid propagation and seedling survival of Lonicera japonica Thunb. [Method] The cuttings of 1, 2 or 3 years old, with different length, were soaked in different hormones at different concentrations for different time and planted at different dates. Finally, their rooting rate and survival rate in different treatments were measured and compared. [Result] The rooting rate and survival rate of one-year old cuttings with length of 10-13 cm and planted on September 20 and October 20, 2012 were relatively higher among all the treatments. Hormone treatment could significantly promote the callus formation and accelerate the rooting of the cuttings, and improve their survival rate. The cuttings treated with 120-200 mg/L ABT1 or NAA for 35-45 min had higher rooting rate and survival rate. Among them, the survival rate of cuttings treated with 160 mg/L ABT1 for 35 min was the highest, up to 92.5%. [Conclusion] The study provided basis for improving the survival rate of L. japonica by cuttage, and for optimizing the rapid propagation technique.

Protective effect of different medicinal parts of Lonicera japonica Thunb. on myocardial ischemic injury induced by isoproterenol in rats

Background:To study the protective effect of different medicinal parts of honeysuckle on myocardial ischemia injury in rats.Method:SD mice were randomly divided into 6 groups:the normal group,model group,compound salvia miltiorrhiza group,Lonicera japonica Thunb.of flower bud group,flower group,and the stem leaf group.There were 8 rats in each group,including 4 males and 4 females.The normal group and model group were given equal amount of distilled water,whereas the other dosage groups were given the appropriate drug by intragastric administration,continuous gavage for 14 days.From the 12 days,in addition to the normal group were injected of the same amount of normal saline,the other groups were injected with 5g/kg.Injection of isoproterenol to establish the mouce mycardial ischemia model,its continuously 3d.To detect the serum of AST,CK,CK-MB,LDH and the myocardial issue of Chinese super oxideismutase and malondialdehyde level.Results:Compared with normal group,the levels of AST,CK,CK-MB,cTn-I,LDH and malondialdehyde were significantly increased in the model group(P<0.01)and the level of super oxideismutase decreased significantly(P<0.01)the levels of AST,CK,CK-MB,cTn-I,LDH and malondialdehyde in the serum of flower bud group,flower group and stem leaf group were significantly decreased(P<0.05),and the decreasing level of flower bud group,flower,leaf group of AST,CK,CK-MB,cTn-I,LDH and malondialdehyde was flower bud group>flower>leaf group,the activity of super oxideismutase was obviously increased,and the activity level of super oxideismutase of flower bud group,flower group and stem leaf group was flower bud group>flower>leaf group.Conclusion:In the myocardial ischemic injury model of rats induced by isoproterenol,Lonicera japonica Thunb.had a protective effect in all parts,and Lonicera japonica Thunb.bud had the greatest protective effect.

金银花(Lonicera japonica Thunb.)愈伤组织的诱导和分化

以金银花的茎、叶片、芽为外植体,在70个不同浓度IAA、NAA、6-BA组合的MS培养基上进行愈伤组织诱导,分析了金银花的快速繁殖体系的培养条件,发现芽是金银花组织培养的最佳外植体,IAA和NAA的最佳浓度均为1mg·L-1,6-BA的最适浓度为0.1mg·L-1,确定适宜的初代培养基为:MS+6-BA0.1mg·L-1+NAA1mg·L-1,诱芽培养基为:MS+6-BA0.1mg·L-1+NAA0.02mg.L-1.关键词愈伤组织,金银花,快速繁殖。

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.

Structural Characterization of Chloroplast Genome in Alpinia japonica(Thunb.)Miq.,a Medicinal Plant of the Genus Alpinia

The analysis of chloroplast gene characteristics in Alpinia japonica(Thunb.)Miq.is of great significance for developing relevant genetic resources.The high-throughput sequencing and bioinformatic research were performed to analyze the chloroplast genome characteristics of A.japonica.The total chloroplast genome length of A.japonica was 161,906 bp,with a typical circular tetrameric structure.And 133 genes were annotated,comprising 87 protein-coding,38 tRNA,and 8 rRNA genes.Furthermore,22 genes contained two copies,and 18 genes owned introns.Repeat sequence analysis showed that it contains 321 simple sequence repeats(SSRs)and 37 long segment repeats.Compared with the chloroplast genomes of eight representative plants in the genus Alpinia,the gene structure,type,and quantity were relatively conservative.Rps12 was the highest variation site in the entire chloroplast gene.A phylogenetic tree showed that the genus Alpinia was the most closely related to the genus Amomum.Meanwhile,A.japonica is the most closely related to Alpinia chinensis belonging to the genus Alpinia.Overall,the chloroplast genome of a new species was reported in the genus Alpinia,and a basis was provided for the utilization of Alpinia plants as a medical resource.

Chemical constituents and pharmacological effects of Lonicerae japonicae

Lonicera japonica,also known as honeysuckle,is an evergreen shrub in the family of Syzygium.By consulting Sciencedirect databases and Web of Science databases,79 related articles were found,of which 22 were related to chemical composition and pharmacological activity.These articles show that L.japonica has a wide range of pharmacological activities,including antiviral,anti-tumor,and antioxidant effects.These activities have important applications in the pharmaceutical,food,and fragrance industries.This review focuses on the chemical composition and pharmacological effects of L.japonicae,which is of great significance to the development of new drugs and therapeutic methods.

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.

Leaf epidermal characters of Lonicera japonica and Lonicera confuse and their ecology adaptation

The leaf epidermis of Japanese honeysuckle （Lonicera japonica Thunb.） and Wild Honeysuckle （Lonicera confusa） in the genus of Flos Lonicerae were mainly observed by scanning electron microscopes （SEM） to study the characteristics of stomata, trichomes and dermal cell, etc.. The results showed that stoma exists only on the lower epidermis and its distribution is irregular, and leaf epidermis consist of epidermis cells, stoma complexes and bushy trichomes including glandular hair and non-glandular hair. On the upper epidermis, anticlinal wall caves in sinuous groove to countercheck the transpiration. Evidences from leaf morphological structures serve as another proof on drought-resistant mechanisms. Some strumaes distributing regularly are hypothesized as oxalic calcium on the lower epidermis under laser scanning confocal microscopy （LSCM） with Fluo-3/AM, which can increase their endurance to drought stress. Therefore, the above characteristics of Flos Lonicerae can reduce the loss of water and make Japanese honeysuckle and Wild Honeysuckle adapt to the droughty environment at Karst area in southwest China. However, there is some difference of the two species. From the SEM （Scanning Electron Microscopy） result, it is shown that on the upper epidermis, some glandular hair regularly present along the midrib of Japanese honeysuckle, but Wild Honeysuckle has no glandular hair on the upper epidermis, which can verify the relationships of Flos Lonicerae species and provide the significance for classification of Flos Lonicerae.

Study on Tissue Culture of Japanese Honeysuckle ( Lonicera japonica )

[Objectives]This study was conducted to increase the reproduction coefficient of Japanese honeysuckle(Lonicera japonica)to keep the character of optimal benign.[Methods]The young leaves of medicinal Japanese honeysuckle were selected as explants,and MS was used as the basic culture medium.Suitable culture concentrations and conditions were screened through different concentration gradients of growth regulators and cytokinin.[Results]As the concentration of 6-BA in the culture medium increased,the browning rate increased,and the browning phenomenon occurred earlier.On the contrary,a lower concentration of 6-BA was suitable for the differentiation and growth of young leaves,and the browning response was slow.However,if the cultivation time was too long and the materials were not transferred in a timely manner,browning would also occur.The optimal combination of levels was obtained through a 3×3 orthogonal experiment(three parallel groups for each of 6-BA and NAA).The culture conditions included a constant temperature of 26℃and light intensity of 1200 lx.The optimal medium for inducing callus proliferation was MS+6-BA 0.5 mg/L+NAA 0.5 mg/L;and the optimal medium for inducing bud differentiation was MS+6-BA 1.0 mg/L+NAA 0.1 mg/L.[Conclusions]This study provides a theoretical basis for accelerating the development of the honeysuckle industry.

In comparison with vitamin C and butylated hydroxytoluene, the antioxidant capacity of aqueous extracts from buds and flowers of Lonicera japonica Thunb.

OBJECTIVE: To assess the antioxidant capacity of aqueous extracts of buds and flowers of Lonicera japonica Thunb.(BLJ and FLJ) using in vitro assays.METHODS: We assessed the in vitro antioxidant activities of aqueous extracts of BLJ and FLJ and compared with that of classical antioxidants vitamin C and butylated hydroxytoluene, using several well-established methods including the 1,1-diphenyl-2-picryl-hydrazyl assay, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assay, reducing power assay, fluorescence recovery after photobleaching assay, β-carotene bleaching assay, ferric thiocyanate assay, and thiobarbituric acid method.RESULTS: The aqueous extracts of both BLJ and FLJ had similarly potent antioxidant capacity. There were no significance differences between BLJ and FLJ in all the assays.CONCLUSION: The aqueous extracts of both BLJ and FLJ have antioxidant activity with comparable efficacy. These findings suggest that both BLJ and FLJ may have the potential as natural antioxidants.

青荚叶〔Helwingia japonica(Thunb.)Dietr.〕种下新组合

Helwingia japonica (Thunb.) Dietr. var. papillosa Fang et Soong was reduced as a new synonym of H. japonica (Thunb.) Dietr. var. hypoleuca Hemsl. ex Rehd. Helwingia zhejiangensis Fang et Soong was combined into H. japonica (Thunb.) Dietr. and renamed H. japonica (Thunb.) Dietr. var. zhejiangensis (Fang et Soong) M. B. Deng et Y. Zhang.

基于R语言的专利中药复方防治呼吸道传染病的用药规律研究

【目的】挖掘专利中药复方防治呼吸道传染病的遣方用药规律,为呼吸道传染病的防治提供参考。【方法】在国家专利数据库中收集防治呼吸道传染病的中药复方,经数据筛选与规范化后,利用R语言进行数据挖掘。【结果】共纳入429首中药专利复方,涉及中药846味;高频中药有26味,使用频次居前10位的药物分别为甘草、金银花、黄芩、连翘、桔梗、百部、苦杏仁、黄芪、柴胡、薄荷。高频中药主要为清热解毒药和补气药;所涉中药药性以寒为主,药味多为苦、甘、辛,主归肺经;关联规则分析得到19条核心关联规则、多个药物组合;聚类分析得到3个药物聚类。【结论】专利中药复方防治呼吸道传染病时注重寒温相配、宣降相伍,着重宣散肺邪、清热解毒,同时不忘益气和中、滋阴润燥,使祛邪不伤正、扶正不敛邪。

Main Diseases and Pests of Lonicerae japonica in Guangxi Province and Fluctuation Pattern of Their Natural Enemies

[ Objective ] The paper was to study the main diseases and pests in Lonicerae japonica in Guangxi Province and fluctuation pattern of their natural ene- mies. [Method] The population change of main diseases and pests, and main predatory naturaI enemies of pests in different species of Lonicerae japonica in Xincheng County of Guangxi Province were systematically investigated during 2008 -2010. [ Result ] The variation cycle of population number of Semiaphis heraclei （Takahashi） belongs to single peak type, and it has large occurrence in April. The variation cycle of population number of Franklinlella sp. and Haptonchus luteo- ius （Erichson） belongs to double peak type, and they have large occurrence at April and September. The variation cycle of disease severity of Microsphaera lonicerae （ Wint. ） belongs to double peak type, and the disease index will be larger at April and September. The variation cycle of diseased shoot rate belongs to single peak type; diseased shoot rote of Lonicera dasystyla Rehd. and Loniccra hvpolzlauca Miquel is relatively high during April to June, while the index of Lonice- ra confusa （Sweet） DC. is relatively high during February to May. The variation cycle of population number of main natural enemies such as Lemnia biplagiata （ Swartz）, Coccinella septcmpunctata （ Linnaeus ） and Harmonia axyridis （Pallas） belongs to double peak type, and they have large occurrence at April and Sep- tember. [ Conclusion] The results could provide scientific basis for determination of optimum control period against major diseases and pests in Loniceraejaponica and development of integrated control programs.

金银花中绿原酸的分离纯化

目的建立金银花中绿原酸的色谱分离纯化方法.方法采用亲脂性吸附树脂与葡聚糖凝胶色谱纯化技术,将其水提物经亲脂性吸附树脂柱色谱预分离后,0.1mol?L-1盐酸调节pH到4.0左右,葡聚糖凝胶sephadexLH-20精制纯化,洗脱溶剂为50%丙酮水溶液.结果产品经MS、红外、紫外和HPLC图谱鉴定,并与对照品比较对照,结果显示所得的绿原酸纯度>98%.结论该方法简单,重复性较好,可用于绿原酸单体成分的制备.

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.

Lonicera japonica-induced inhibition of interleukin-1 beta thermogenesis and E-type prostaglandin receptor-3 expression in the preoptic area of rabbits

BACKGROUND： It has been shown that interleukin-1β（IL-1β） can induce fever by activating vascular endothelial cells and macrophages of the supraoptic crest to generate prostaglandin E2, which binds with receptors of the thermo-sensitive hypothalamic neurons. Lonicera japonica is one of the medicinal plants used widely in Asia for its antipyretic properties. However, these mechanisms have not yet been intensively studied. OBJECTIVE： To investigate the antipyretic effect and mechanisms of Lonicera japonica on IL-1β- induced febrile New Zealand rabbits by observing expression changes of E-type prostaglandin receptor-3 （EP3） mRNA in the preoptic anterior hypothalamus （POAH）. DESIGN： A randomized controlled study. SETTING： Electrophysiological Laboratory at the Department of Pathophysiology, Medical College of Jinan University; Department of Orthopaedics, First Hospital Affiliated to Medical College of Jinan University. MATERIALS： The experiment was performed from April to December 2005, using a total of 32 New Zealand white rabbits of both sexes, weighing 1.5 2.0 kg. All the animal experiments were performed according to the internationally accepted ethical guidelines. Lonicera japonica injection was purchased from Huanghe pharmaceutical factory of Xi＇an, China. IL-1βwas purchased from Sigma, USA. METHODS： A total of 32 rabbits were divided randomly into four groups： ① Normal saline （NS） control group;② Lonicerajaponica treatment group; ③ IL-1βtreatment group; and ④Lonicerajaponica plus IL-1βtreatment group. In the first 3 groups, the rabbits were given separate intravenous （i.v.） injections of l mL NS, l mL Lonicera japonica, and 100 ng IL-l β （dissolved in 0.9% NS without pyrogen）. In the Lonicerajaponica plus IL-1βgroup each rabbit was given i.v. injections of l mL NS and, 30 minutes later, 100 ng IL-1 β. MAIN OUTCOME MEASURES： Colonic temperature of each rabbit was measured at 0, 10, 20, 30, 40, 50, 60, and 70 minutes after injection and the maximum temperature rise （ A T） and the temperature response index after l hour （TRII） was calculated. Subsequently, in situ hybridization （ISH） was done with an ISH kit, EP3 mRNA expression in the POAH of all groups was measured （number of positive cells and average gray scale value）. RESULTS： A total of 32 rabbits were included in the result analysis, without any loss, （i） A T and TRII： there was no significant difference between the Lonicera japonica group and NS group （P 〉 0.05）. The IL-1β group was significantly greater compared to NS group （P 〈 0.01）. The Lonicera japonica plus IL-1β group was significantly less than the IL-1β group （P 〈 0.05）. ② In the NS and Lonicera japonica groups, the EP3 mRNA expression was negative （no coloration） or only weakly positive （only a few brown yellow particles in the cytoplasm cells could not be identified）. The number of positive cells and the average gray scale value were not significantly different between the two groups （P 〉 0.05）. In the IL-1β group, the number of positive cells was remarkably higher and the average gray scale value was lower than the NS group （P 〈 0.0 l）. In the Lonicera japonica plus IL-1β group, the number of positive cells was significantly less than the IL-1β group （P 〈 0.05）. However, the average gray scale value was greater than the IL-1β group （P 〈 0.05）. CONCLUSION： Lonicera japonica has obvious antipyretic effects on IL-1β-induced febrile rabbits and acts by inhibiting expression of EP3 mRNA in the POAH.

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.