INTRODUCTION

It is generally accepted that stratum corneum(SC),the outermost layer of the epidermis,is the principal barrier to drug permeation following transdermal drug delivery(TDD).In order to improve the percutaneous permeation rate of the drug,lots of strategies are utilized to alter the barrier function of SC.1One of the most widely used strategies is the use of penetration enhancers(PEs)which ideally cause a temporary and reversible reduction in the barrier function of SC.2

Essential oils(EOs)which possess diverse and relevant biological activities have been widely used for several applications in pharmaceutical,cosmetic,agricultural,and food industries.EOs and their volatile constituents have been used as PEs for decades of years.3They can facilitate the delivery of small drug compounds acrossing SC by interacting with the intercellular lipids through physical processes including extraction,fluidization,increased disorder,and phase separation.At present,natural PEs including EOs3and terpenes4have been increasingly used due to their better safety profile compared to chemically synthetic PEs.Moreover,it was also proved that the penetration enhancement capacities of the whole EOs were significantly higher than its main terpene components.5,6

In Traditional Chinese Medicine(TCM),about 300 kinds of Chinese Herbal Medicines(CHMs)contain EOs.The association between penetration enhancement activities of EOs7and drug properties of CHMs containing EOs was significant,especially the four properties defined in terms of TCM theory.Furthermore,most EOs are from CHMs with warm or hot nature.Consequently,it is proposed that the screening of EOs as PEs can be performed based on drug characteristics of CHMs containing EOs.

It is found that EOs from warming the interior medicinal(WIM)from TCM are usually applied as PEs.The addition of 3%clove oil showed a significant permeation enhancement activity on ibuprofen and the enhancement ratio was determined to be 7.3in vitroand 2.4in vivo.9The skin permeation of trazodone hydrochloride was also found to be remarkably enhanced by EOs and the effect of fennel oil was significantly better than that of mentha oil,one of the most commonly used PEs.10However,to our knowledge,there is currently no information available about the comparison of different EOs from WIM.

Despite most PEs display fairly excellent performance for TDD,only a few of them have been approved for clinical application due to their skin toxicity or irritation issue.It is challenging to maintain an optimal balance between the safety and the potency of PEs in drug permeation.Therefore,in the present study,both cytotoxicity and penetration enhancement activity were evaluated and compared for eight different EOs from four WIM[Bichengqie(Litseae Fructus),Dingxiang(Flos Syzygii Aromatici),Huajiao(Pericarpium Zanthoxyli Bungeani),and Xiaohuixiang(Fructus Foeniculi)]with warm property and another four WIM[Ganjiang(Rhizoma Zingiberis),Gaoliangjiang(Rhizoma Alpiniae Officinari),Rougui(Cortex Cinnamomi Cassiae),and Wuzhuyu(Fructus Evodiae Rutaecarpae)]with hot property.The influence of the WIM properties with pungent flavor on the cytotoxicity and penetrate enhancement activity of extracted EOs was further investigated and compared.

MATERIALS AND METHODS

Instruments

An Agilent 7890A gas chromatograph interfaced to an Agilent 5975C inert MSD with Triple-Axis Detector(Agilent Technologies,Palo Alto,CA,USA)was employed for the analyses of EOs.A Chromate-4300 microplatespectrophotometer(AwarenessTechnology Inc,Palm City,FL,USA)and a flow cytometer(Becton Dickinson,San Jose,CA,USA)were used for cytotoxicity studies.A Shimadzu HPLC system(Kyoto,Japan)consisting of a LC-20AT pump and a SPD-20A UV-VIS detector and a modified Franz diffusion cell device(Shanghai Kai Kai Technology,Shanghai,China)were employed for permeation studies.

尽管完成任务的过程困难重重，但是肖建国觉得一旦接受了任务就一定要完成。当时国内排版系统落后，要依靠输入命令进行排版，版面输出结果要凭想象，如果要实现交互式组版系统，就需要从头研发。在天生好强和不落后精神的推动下，肖建国完成了方正第一代报纸排版系统——交互式报纸组版系统的开发工作，实现了从屏幕所见即所得的排版能力，大大提高了组版改版的效率。

Plant materials and reagents

Bichengqie(Litseae Fructus),Dingxiang(Flos Syzygii Aromatici),Gaoliangjiang(Rhizoma Alpiniae Officinari),Ganjiang(Rhizoma Zingiberis),Huajiao(Pericarpium Zanthoxyli Bungeani),Rougui(Cortex Cinnamomi Cassiae),Wuzhuyu(Fructus Evodiae Rutaecarpae),and Xiaohuixiang(Fructus Foeniculi)were all purchased from the Nanjing Medicinal Material Company(Nanjing,China).All the crude herbs were authenticated by Prof.Yue Wei from School of Pharmacy,Nanjing University of Chinese Medicine.The voucher specimens were kept in the Herbarium of School of Pharmacy,Nanjing University of Chinese Medicine(Nanjing,China).

Ibuprofen,propylene glycol(PG),isopropyl alcohol(IPA),dimethylsulfoxide(DMSO),and 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)were obtained from Sigma-Aldrich Inc.(St Louis,MO,USA).Azone was obtained from Sinopharm Chemical Reagent Co.,Ltd.,(Shanghai,China).Acetonitrile with HPLC-grade obtained from Tedia Company Inc.,(Fairfield,OH,USA)and deionized water was purified by an EPED super purification system(EPED,Nanjing,China).Dulbecco's modified Eagle's medium(DMEM)and Fetal bovine serum(FBS)were purchased from Gibco BRL(GrandIsland,NY,USA).AnnexinV-FITC/PI Apoptosis Detection Kit,propidium iodide(PI),and DNA content Quantitation Assay were obtained from Nanjing KeyGen Biotech Co.,Ltd.,(Nanjing,China).All other reagents were used commercially available and of analytical grade.

Animals

Male Sprague-Dawley rats(180-220)g were obtained from Shanghai Jiesijie Laboratory Animal Co.,Ltd.,(Shanghai,China)with the license number of SCXK(Shanghai)2013-0006.The animals were acclimatized for at least 1 week in a 12 h light/dark cycle with free access to standard chow and water.They were fasted for 12 h before experiments with the exception of water.Animal experiments were performed in accordance to the Principles of Laboratory Animal Care and Use in Research(Ministry of Health,Beijing,China).The protocols of animal experiments were approved by the Animals Ethics Committee of Nanjing University of Chinese Medicine.

Cell lines

HaCaT(human epidermal keratinocytes)cell lines were obtained from Nanjing KeyGen Biotech Co.,Ltd.,(Nanjing,China).The cells were incubated in DMEM supplemented with 10%heat-inactivated fetal bovine serum(FBS)and 100 U/mL penicillin/streptomycin in a humidified incubator at 37℃and 5%CO2.Culture medium was replaced every other day.For all experiments,cells were seeded in a culture dish until adhere to 60%-80%confluency.In addition,the control cells were treated with 1%(v/v)DMSO only.

家长要为孩子营造温馨、宽松、自由的阅读氛围。阅读时要安静，没有打扰，家长要带领孩子开展形式新颖、内容丰富的阅读活动。阅读时，家长要给予孩子想象的空间，鼓励孩子说出自己的想法，使孩子在愉悦的环境中快乐阅读。

Extraction of EOs

Briefly speaking,the dried pericarp or rootstock of those CHM samples was ground into powders with a mixer.For each sample,the pulverized herb was accurately weighed and then transferred into a 1000-mL round-bottomed flask.A certain multiple(mL/g)of distilled water was added,and the sample was soaked for 1 h and subjected to 5-9 h of steam distillation using a Clevenger-type apparatus.Then the oil layer cooled to room temperature was transferred to a 5-mL measuring flask.A little anhydrous sodium sulfate was added to the sample to remove moisture.After centrifuge to remove sodium sulfate,the resultant EO was stored in an air tight bottle and kept refrigerated until use.EOs extracted from Bichengqie(Litseae Fructus),Dingxiang(Flos Syzygii Aromatici),Huajiao(Pericarpium Zanthoxyli Bungeani),and Xiaohuixiang(Fructus Foeniculi)with warm property were named cubeb oil,clove oil,zanthoxylum oil,and fennel oil,respectively.EOs extracted from Ganjiang(Rhizoma Zingiberis),Gaoliangjiang(Rhizoma Alpiniae Officinari),Rougui(Cortex Cinnamomi Cassiae),and Wuzhuyu(Fructus Evodiae Rutaecarpae)with hot property were named ginger oil,galange oil,cinnamon oil,and evodia oil,respectively.

EOs were extracted by the steam distillation method.

Gas chromatography coupled to mass spectrometry(GC-MS)analysis

13 Charoo NA,Shamsher AA,Kohli K,Pillai K,Rahman Z.Improvement in bioavailability of transdermally applied flurbiprofen using tulsi(Ocimum sanctum)and turpentineoil.ColloidsSurfBBiointerfaces2008;65(2):300-307.

Cytotoxicity assay

The MTT assay was used to monitor the toxicity of EOs on human skin epidermal keratinocytesin vitro.HaCaT cells were plated in 96-well plate at a density of 7000 cells in a 100 μL medium per well.After adhering,the cells were incubated with varying concentrations of EOs or azone(positive control)in a culture medium with 1%DMSO for 24 h at 37℃.The cells treated with culture medium containing 1%DMSO were used as the control.Then the medium was replaced by a fresh medium containing 20 μL MTT solution(5 mg/mL in PBS)and the cells were incubated again for 4 h.Subsequently,the medium was removed and 150 μL DMSO was added to dissolve the formazan crystals.The plate was incubated for 10 min while shaking.The absorbance was measured at 570 nm.Cell viability was calculated according to the following equation:

在监管工作中，赣州市局作出“把赣州打造成为全省乃至全国审批项目最少、办事效率最高、服务质量最好”的承诺。为落实这一承诺，赣州市局积极推进简政放权，审批时限压缩至平均5个工作日；把支持生物制药产业发展作为重要工作，寓服务于监管之中，发挥部门优势，积极帮扶企业，辖区生物制药产业发展保持良好势头，培育了青峰药业、海欣制药等优势龙头企业。2015年青峰药业产值达到38.65亿元，销售收入37.37 亿元，在全省药品生产企业中排名第一。

Cell viability=B/A×100%

where A was the absorbance of the control and B was the absorbance of the cells incubated with different EOs or azone,respectively.All samples were evaluated at least five times.Half maximal(50%)inhibitory concentration(IC50)values were evaluated by SPSS 17.0 statistical software(SPSS Inc.,Chicago,IL,USA).

Cell cycle analysis with PI staining

For cell cycle analysis,HaCaT cells were incubated in 6-wells plate,being exposed to different concentrations of zanthoxylum oil and ginger oil for 24 h.The cells were then harvested by centrifugation and fixed gently with ice-cold 70%ethanol at 4℃overnight.Prior toanalysis,cells were washed twice with ice-cold PBS and treated with 100 μL RNase at 37 ℃ for 30 min,followed by staining with 400 μL PI at 4 ℃ for 30 min.Cells were then analyzed on the flow cytometer.

Table 1 Parameters of GC-MS analysis

Essential oil Inlet pressure(psi)5.466 Spilt ratio Temperature of inlet(℃)Cubeb oil 1∶30250 Clove oil Zanthoxylum oil Fennel oil Ginger oil Galange oil 10.523 4.960 6.478 4.701 4.960 1∶20 1∶20 1∶20 1∶20 1∶10 200 250 250 250 250 Cinnamon oil 7.6251∶20250 Evodia oil 5.9741∶20250 Temperature of column(℃)60℃for 2 min,then 6℃/min to 80℃for 6 min,then 2℃/min to 100℃,then 6℃/min to 170℃for 5 min,then 20℃/min to 250℃for 5 min 100℃for 3 min,then 6℃/min to 200℃,then 30℃/min to 250℃for 5 min 50℃for 2 min,then 15℃/min to 250℃for 5 min 80℃for 2 min,then 5℃/min to 120℃for 5 min,then 1℃/min to 130℃for 2 min,then 50℃/min to 250℃for 5 min 45℃for 2 min,then 5℃/min to 120℃,then 15℃/min to 180℃for 10 min,then 20℃/min to 250℃for 5 min 2℃/min from 50℃ to 90℃,then 7℃/min to 145℃ for 6 min,then 10℃/min to 250℃for 5 min 100℃for 1 min,then 5℃/min to 120℃for 3 min,then 2℃/min to 130℃,then 20℃/min to 150℃for 5 min,then 20℃/min to 200℃for 5 min 70℃for 5 min,then 5℃/min to 150℃,then 2℃/min to 200℃,then 20℃/min to 250℃for 5 min

Apoptosis analysis with Annexin-V and PI double staining

The induction of apoptosis and necrosis caused by EOs was analyzed by double staining with Annexin-V-FITC and PI.HaCaT cells were incubated in 6-wells plate with different concentrations of ginger oil and zanthoxylum oil for 24 h.Then the processed cells were resuspended in annexin binding buffer and then incubated with Annexin-V conjugated with FITC as well as PI for 15 min at room temperature in the dark.Cells were analyzed on a flow cytometer and the data were analyzed by FlowJO7.6.5 software(Becton Dickinson,San Jose,CA,USA)

In vitro skin permeation studies

In vitroskin permeation studies are the classic methods to evaluate the penetration enhancement activity of PEs.9-12The studies were carried out according to our previous methods.11,12After sacrificing rats with excess diethyl ether inhalation,the abdomen skin fragment used for the experiment was excised from the rats and the adhering fat and other tissues were carefully removed.The prepared full thickness skin was subsequently washed with physiological saline solution three times and stored at－20℃(used in two weeks).The hydrated skin sample was clamped between the donor and the receptor chambers of the modified Franz diffusion cell with an effective permeation area of 3.14 cm2and a receiver cell volume of 8 mL.Physiological saline solution containing 35%ethanol12was used as the receptor solution and incubated at(37.0±0.2)℃using a water bath with a magnetic stirrer at 500 r/min.The donor compartments containing 1 mL(0.32%,w/v)ibuprofen solution with various 3%(w/v)EOs or azone were dissolved in PG∶IPA(30∶70,v/v)solvent mixture.13Negative control samples were obtained in a similar way without addition of any enhancers.Samples(0.2 mL)were withdrawn from the receptor chambers at predetermined time intervals(8,10,22,24,26,28,32,36,and 48 h)and then replaced with an equal volume of fresh medium.The receptor fluid samples were then analyzed by HPLC for ibuprofen content.

The cumulative amount of drug permeating through a unit area of skin was plotted against time.Steady state flux values were calculated from the slope of the linear portion of the plot(between 8 and 26 h).The cumulative amount of drugs permeating through the skin at 48 h(Q48)was calculated from the drug concentration in the receiver compartments.To compare the permeation enhancement capacity of each EO,the enhancement ratio(ER)was determined as follows:ER=(flux for skin treated with EO)/(flux for negative control).

HPLC analysis of ibuprofen

HPLC system was used for the assay of ibuprofen.The mobile phase consisted of acetonitrile and water(adjusted pH to 3.0 with phosphonic acid)(65∶35,v/v).Separation was carried out at 25℃using a reverse-phase C18column(Inertsil ODS-3,5 μm,6 mm×250 mm,Hanbang Corp.,China).The detection wavelength was 220 nm and a flow rate of 1.0 mL/min was employed.A sample volume of 10 μL was injected.

现代企业固定资产投资行为管理信息化需要一支专业的管理团队开负责各项工作，各司其责，避免推诿造成了不必要的浪费，归根结底就是按照现在企业管理制度的要求来组建管理团队，按照团队协作、分类管理、高效运作的原则，组建出高层决策、中层监控、基层执行的管理团队，以适应管理信息系统的运作需要，让这套系统发挥出相应的管理效应，达到预期的投资目的。

Statistical analysis

Data were expressed as the mean±standard deviation(±s)from at least three independent experiments.One-way analysis of variance was performed to test the differences between groups using SPSS 17.0(SPSS Inc.,Chicago,IL,USA).A P value of less than 0.05 was considered statistically significant.

Table 2 Extraction yield(%)of EOs from CMs(±s,n=3)

Notes:CHM:Chinese herbal medicine;EOs:essential oils.Listed original Chinese herbal medicines are all belonged to warm interior medicinal(WIM).

Essential oil Cubeb oil Clove oil Zanthoxylum oil Fennel oil Ginger oil Galange oil Cinnamon oil Evodia oil Original CHM Bichengqie(Litseae Fructus)Dingxiang(Caryophylli Flos)Huajiao(Zanthoxyli Pericarpium)Xiaohuixiang(Foeniculi Fructus)Ganjiang(Zingiberis Rhizoma)Gaoliangjiang(Alpiniae Officinarum Rhizoma)Rougui(Cinnamomi Cortex)Wuzhuyu(Evodiae Fructus)Property of CHM Warm Warm Warm Warm Hot Hot Hot Hot Extraction yield 2.42±0.17 13.80±0.31 3.70±0.17 1.76±0.16 1.35±0.16 0.90±0.07 3.57±0.40 0.57±0.04

RESULTS

Extraction and analysis of EOs

11 Jiang QD,Wu YM,Zhang H,et al.Development of essential oils as skin permeation enhancers:penetration enhancement effect and mechanism of action.Pharm Biol 2017;55(1):1592-1600.

Cytotoxicity of EOs on HaCaT cells

The results of HaCaT cytotoxicity treated with different concentrations of EOs were presented in Table 3.The IC50values showed that the cellular toxicities of the examined agents were in the following decreasing order:zanthoxylum oil＞fennel oil＞clove oil＞cubeb oil＞evodia oil＞galange oil＞ginger oil＞cinnamon oil＞azone.It was obvious that the IC50values of EOs were markedly higher in HaCaT cells in comparison to azone,indicating that EOs derived from natural products exhibited lower skin irritation potential compared with chemically synthetic PEs.

In the range of WIM,the property of CHMs significantly affected the cytotoxicity of extracted EOs.Cubeb oil,clove oil,zanthoxylum oil,and fennel oil were extracted from WIM with warm property.Their IC50values were in the range of 69.80-333.69 μg/mL.Ginger oil,galange oil,cinnamon oil,and evodia oil were extracted from WIM with hot property.Their IC50values were in the range of 12.47-49.22 μg/mL.By statistical comparison,the cytotoxicity of EOs from WIM with hot property were significantly(P=0.020)higher than that of EOs from WIM with warm property.

目前，绝大多数烟气制酸生产厂家均采用动力波技术净化烟气，但会产生大量含酸烟气洗涤液，该液中含有较高的H2SO4、Cu等，多数厂家对该液的处理采用常规硫化-石膏沉淀法处理［3-4］。为中和其中硫酸，产出大量石膏废渣。随着我国对固体危废的严格控制，石膏渣将给企业带来极大的危废处置压力。

Table 3 Comparison of IC50values of eight EOs on cultured HaCaT cells(ˉ±s,n=5)

Note:EOs:essential oils.

Essential oil Azone Cubeb oil Clove oil Zanthoxylum oil Fennel oil Ginger oil Galange oil Cinnamon oil Evodia oil IC50(μg/mL)5.4±0.4 69.9±5.4 192.3±30.0 333.7±51.4 332.8±70.3 19.7±2.8 42.7±9.6 12.5±2.0 49.2±7.7

Cytotoxic mechanism studies

The changes of the cell cycle distribution were shown in Figure 1.The cell population of the G0/G1 phase was effectively increased in a dose-dependent manner after treatment with ginger oil and zanthoxylum oil.There were no significant differences in the HaCaT cells population analysis between the treatment with low dose of EOs and the control group.Treatment with high dose of ginger oil and zanthoxylum oil resulted in a remarkable accumulation of cells in the G0/G1 phase(87.6%±2.3%,89.6%±2.1%)and a concomitant decrease of S and G2/M phase,while compared with the control(G0/G1:75.8%±3.8%).Taken together,these results revealed that ginger oil and zanthoxylum oil induced the same HaCaT cell cycle arrest in the G0/G1 phase in a dose-dependent manner.

Figure 1 Cell cycle arrest effects of ginger oil and zanthoxylum oil on HaCaT cells

Control group:cells treated with 100 μL culture medium containing 1%DMSO for 24 h at 37 ℃;ginger oil groups:cells incubated with 6,20,35 μg/mL of ginger oil in a culture medium with 1%DMSO for 24 h at 37 ℃;zanthoxylum oil groups:cells incubated with 200,350,500 μg/mL of zanthoxylum oi in a culture medium with 1%DMSO for 24 h at 37 ℃.Data are mean±standard deviaton from three independent representative experiments.aP＜0.01vscontrol group.EOs:essential oils;DMSO:dimethylsulfoxide.

As illustrated in Figures 2 and 3,the great majority of cells were intact when exposed to vehicle control and lower dose of those EOs.With the increase of the concentration of ginger oil from WIM with hot property,the proportion of necrotic cells was significantly enhanced compared with control group(P＜0.001).For zanthoxylum oil from WIM with warm property,the percentage of apoptotic cells was increased obviously with the dose.However,for ginger oil,the induction effect of cell apoptosis was no longer obvious.

In vitro skin permeation studies

The activities of different EOs on thein vitroskin permeation profiles of ibuprofen through excised rat skin were shown in Figure 4.The corresponding permeation parameters were summarized in Table 4.It was found that almost all tested EOs showed remarkable penetration enhancement activities on ibuprofen.Both thecumulativeamountofibuprofen permeating through the skin at 48 h and flux values were significantly(P＜0.05)increased in the presence of galange oil,ginger oil,and fennel oil compared with azone,one of the most commonly used PEs.The enhancement ratio(ER)values showed that the efficacies of the examined agents were in the following decreasing order:galange oil＞fennel oil＞ginger oil＞cinnamon oil＞cubeb oil＞evodia oil＞azone＞zanthoxylum oil＞clove oil.The ER values of cubeb oil,clove oil,zanthoxylum oil,and fennel oil extracted from WIM with warm property were in the range of 1.20-2.01.The ER values of ginger oil,galange oil,cinnamon oil,and evodia oil extracted from WIM with hot property were in the range of 1.69-2.21.It seemed that hot property slightly helped to the penetration enhancement activity of EOs.However,no statistical significance(P=0.18)was found between EOs from WIM with warm and hot properties.

DISCUSSION

Since SC is a lipid barrier,the penetration drugs must have some lipophilicity.15The lipophilic drugs such as ibuprofen are transferred.Natural terpenes as penetration enhancers for transdermal drug deliveryed around the corneocytes in the lipid-rich extracellular regions in the SC.It should be noted that the mechanisms of action of EOs are mainly based on changing the structure of the SC barrier and interaction with intercellular SC lipids to increase diffusivity of drugs.3Consequently,the skin permeation of ibuprofen can be enhanced by lots of EOs.9,16

According to TCM theory,the CHMs should be applied based on their characteristics,including four properties,five flavors,channel tropism,and so on.Pungent flavor can disperse and promote the circulation ofQiand blood,which may facilitate the diffusion of drugs across the skin barrier.In addition,pungent flavor can also enter lung channel which determines the function of skin and fur.Accordingly,it is logical to hypothesize that the effect of EOs might be related with the characteristics of CHMs containing EOs.8The results of our previous studies have revealed that four properties of CHMs are one of the key factors affecting penetration enhancement activity of extracted EOs.7Except mentha oil,most EOs used as PEs are from CHMs with warm or hot property.Therefore,in the present study,the role of warm or hot property on the cytotoxicity and penetration enhancement activity was intensively investigated.

Figure 2 Ginger oil and zanthoxylum oil induced apoptosis in HaCaT cells

A:control group(cells treated with 100 μL culture medium containing 1%DMSO for 24 h at 37 ℃).B-D:ginger oil groups(cells incubated with 6,20,35 μg/mL of ginger oil in a culture medium with 1%DMSO for 24 h at 37 ℃).E-G:zanthoxylum oil groups(cells incubated with 200,350,500 μg/mL of zanthoxylum oi in a culture medium with 1%DMSO for 24 h at 37 ℃).HaCaT cells were incubated with different concentrations of ginger oil and zanthoxylum oil for 24 h.Q1 represents necrosis(AV-,PI+),Q2 shows late-apoptotic cells(AV+,PI+),Q3 describes early-apoptotic cells(AV+,PI-),and Q4 expresses alive cells(AV-,PI-).All cell uptake experiments were performed in triplicate.DMSO:dimethylsulfoxide.

6 Lan Y,Li H,Chen Y,et al.Essential oil from Zanthoxylum bungeanum Maxim.and its main components used as transdermal penetration enhancers:a comparative study.J Zhejiang Univ-Sci B(Biomed&Biotechnol)2014;15(11):940-952.

The chemical components of EOs have also been proved to be associated with the characteristics of CHMs.18The chemical components can also be applied to explain the penetration enhancement activity of EOs.In the present study,galange oil had been found to possess the best penetration enhancement activityamongtheinvestigated EOs.Revealed by GC-MS analysis,the most abundant constituent in galange oil was 1,8-cineole,the most commonly used terpene PE.4Thein vivostudies revealed that incorporation of 1,8-cineole in gel formulation can enhance the permeation of valsartan significantly.19

In conclusion,EOs from WIM with warm property should be regarded as potential PEs due to their cytotoxicity results.Moreover,fennel oil was found to possess low cytotoxicity and high penetration enhancement activity simultaneously.

3 Herman A,Herman AP.Essential oils and their constituents as skin penetration enhancer for transdermal drug delivery:a review.J Pharm Pharmacol 2015;67(4):473-485.

Figure 3 Bar chart presentation of distribution of viable,apoptotic,and necrotic HaCaT cells in population

HaCaT cells were incubated with different concentrations of ginger oil and zanthoxylum oil for 24 h.Control group:cells treated with 100 μL culture medium containing 1%DMSO for 24 h at 37 ℃.Gginger oil groups:cells incubated with 6 L,20,35μg/mL of ginger oil in a culture medium with 1%DMSO for 24 h at 37 ℃.zanthoxylum oil groups:cells incubated with 200,350,500 μg/mL of zanthoxylum oi in a culture medium with 1%DMSO for 24 h at 37℃.Values were expressed as mean±standard deviation(n=3),and were statistically significant(aP＜0.05;bP＜0.01;cP＜0.001)compared with control.EOs:essential oils;DMSO:dimethylsulfoxide.

Figure 4 Permeation profiles of ibuprofen with 3%w/v EOs through excised rat skin(n=5)

EOs extracted from Bichengqie(Litseae Fructus),Dingxiang(Caryophylli Flos),Huajiao(Zanthoxyli Pericarpium),and Xiaohuixiang(Foeniculi Fructus)with warm property were named cubeb oil,clove oil,zanthoxylum oil,and fennel oil,respectively.EOs extracted from Ganjiang(Zingiberis Rhizoma),Gaoliangjiang(Alpiniae Officinarum Rhizoma),Rougui(Cinnamomi Cortex),and Wuzhuyu(Evodiae Fructus)with hot property were named ginger oil,galange oil,cinnamon oil,and evodia oil,respectively.EOs:essential oils.

1 Trommer H,Neubert RHH.Overcoming the stratum corneum:the modulation of skin penetration.Skin Pharmacol Physiol 2006;19(2):106-121.

2 Parhi R,Suresh P,Mondal S,Kumar PM.Novel penetration enhancers for skin applications:a review.Curr Drug Deliv 2012;9(2):219-230.

REFERENCES

4 Chen J,Jiang QD,Chai YP,Zhang H,Peng P,Yang XX.Natural terpenes as penetration enhancers for transdermal drug delivery.Molecules 2016;21(12):e1709.

5 Monti D,Chetoni P,Burgalassi S,Najarro M,Saettone MF,Boldrini E.Effect of different terpene-containging essential oils on permeation of estradiol through hairless mouse skin.Int J Pharm 2002;237(1-2):209-214.

Eight kinds of CHMs belonging to WIM with pungent flavor were compared.Their channel tropism characteristics were mainly related with liver and spleen channels.As shown in Table 2,four of them belong to warm property and four of them belong to hot property.For the first time,the warm or hot property of CHMs was demonstrated to significantly affect the cytotoxicity of EOs from WIM.However,the penetration enhancement activity seemed to be a little related with the warm or hot property.

7 Yang WG,Chen J,Liu P,et al.Association between penetration enhancement effect of essential oils and drug properties of Traditional Chinese Medicine by data mining method.Zhong Guo Zhong Yao Za Zhi 2015;40(23):4609-4615.

Table 4 Effect of EOs on percutaneous permeation parameters of ibuprofen through excised rat skin(ˉ±s,n=5)

Notes:negative control:ibuprofen solution without EOs.aP＜0.05,bP＜0.01,cP＜0.001vsthe control group(ibuprofen only);dP＜0.05,eP＜0.01vsthe azone group.gQ48:cumulative amount of permeated ibuprofen at 48 h/added ibuprofen×100%.EOs:essential oils;ER:enhancement ratio;Q48:cumulative amount of drugs permeating through skin at 48 h.

Essential oil Negative control Azone Cubeb oil Clove oil Zanthoxylum oil Fennel oil Ginger oil Galange oil Cinnamon oil Evodia oil Flux(μg·cm－2·h－1)18±2 27±6a32±4b22±14 25±8 37±3cd35±6cd40±3ce33±3b31±8aER-1.49 1.76 1.20 1.39 2.01 1.92 2.21 1.82 1.69 Q48(μg/cm2)14213±2906.0 19218±3345a21581±3106b14238±7333 17389±1936 26574±2246ce25749±3041cf27340±105ce22097±2829b24515±4932cfPermeation ratiog(%)44.41 60.06 67.44 44.49 54.34 83.04 80.47 85.44 69.05 76.61

8 Chen J,Liu P,Jiang QD,et al.Idea and method of regularity knowledge of Chinese materia medica on essential oils as potential enhancers based on drug property characteristics.Zhong Cao Yao 2016;47(24):4305-4312.

9 Shen Q,Li WJ,Li WY.The effect of clove oil on the transdermal delivery of ibuprofen in the rabbit byin vitroandin vivomethods.Drug Dev Ind Pharm 2007;33(12):1369-1374.

10 Das MK,Bhattacharya A Ghosal SK.Effect of different terpene-containing essential oils on percutaneous absorption of trazodone hydrochloride through mouse epidermis.Drug Deliv 2006;13(6):425-431.

The extraction yield values of different EOs were shown in Table 2.Revealed by the GC-MS analysis,the most abundant constituents in cubeb oil,clove oil,zanthoxylum oil,and fennel oil from WIM with warm property were determined to be D-limonene(38.30%),eugenol(79.96%),linalool(22.17%),and anethole(84.65%),respectively.From WIM with hot property,the most abundant constituents in ginger oil,galange oil,cinnamon oil,and evodia oil were determined to bezingiberene(25.66%),1,8-cineole(44.53%),trans-cinnamaldehyde(62.79%),and myrcene(37.21%),respectively.The detailed results of GC-MS analysis could be found in Supplementary materials.

徐渭从小居住在大云坊观桥弄大乘庵东边的榴花书屋(今青藤书屋)，距张岱高祖张天复和曾祖张元忭居住的车水坊状元府，大约只有10分钟的步程。徐渭生于明正德十六年(1521)，比生于明正德八年(1513)的张天复小8岁，又较生于明嘉靖十七年(1538)的张天复的大儿子张元忭大17岁。因此，徐渭与张氏父子可能从小就熟，但由于年龄的原因又不可能有太深的交往。

通过调查结果可得出，设计时要合理保留自然生境，遵循“师法自然”原则。景观要素越复杂的环境，越能激发起游人的探索欲，美感度也越高。灵活的空间变化、因地制宜营造地势、丰富的植被种类和配置方式、特色功能和标志性建筑等，能够提高新奇度，带给游人耳目一新的感受，也能提升景观的美感度。

12 Chen J,Jiang QD,Wu YM,et al.Potential of essential oils as penetration enhancers for transdermal administration of ibuprofen to treat dysmenorrhoea.Molecules 2015;20(10):18219-18236.

1.1.2 生态地理资料 以《云南农业地理》《云南省种植业区划》和《云南省农业气候资料集》[7-9]等资料为基础，分析整理云南128个县(市、区)的生态气候类型和稻作种植业区划。

EOs were analyzed with GC-MS.A NIST library was used for identifying the components.EO of 1 μL was injected into a HP-5 MS capillary column using a helium as gas carrier at flow rate of 1 mL/min.Detailed parameters are described in Table 1.Mass spectra were recorded from 30 to 650 m/z.Individual components were identified by matching their 70 eV mass spectra with those of the spectrometer database as well as by comparison of the fragmentation pattern with those reported in the published literature.

14 Li K,Zhou R,Wang JW,et al.Zanthoxylum bungeanum essential oil induces apoptosis of HaCaT human keratinocytes.J Ethnopharmacol 2016;186:351-361.

15 Bartosova L,Bajgar J.Transdermal drug deliveryin vitrousing diffusion cells.Curr Med Chem 2012;19(27):4671-4677.

随着科学技术的发展，露天矿山边坡已经成为困扰矿山安全生产的重要问题，其稳定性是保证矿山正常生产的先决条件。在社会经济迅速发展的今天，人们对资源的需求量越来越多，露天矿山的挖掘面积也在逐渐增加，挖掘深度随之逐渐加大，因此一旦发生滑坡事故，将会造成重大人身伤亡与财产损失。因此，对高陡露天矿山边坡稳定性进行分析，尽早发现存在的安全隐患，及时制定有关的安全防范措施，确保矿山安全。

3.4 DPP-4抑制剂(DPP-4i) GLP-1与GLP-2体内半衰期极其短暂，其降解主要是通过DPP-4。绝经后骨质疏松患者及糖尿病患者血清DPP4水平均升高[50-52]。在糖尿病患者中，DPP4i与二甲双胍联合应用较二甲双胍单药能降低发生骨折的风险[53]。研究[54]发现，DPP-4i西格列汀可改善糖尿病大鼠及非糖尿病去卵巢大鼠骨质疏松，且存在剂量依赖性[54]。因此，DPP4i在骨质疏松中具有广阔的应用前景。

16 Luo XQ,Gu YH,Wu ZY.Comparison of the effect of eight kinds of volatile oil of Chinese material medica on percutaneous absorption of ibuprofen in vitro.Zhong Yao Cai 2007;30(5):571-573.

首先，拓展实验是以教材实验为起点，建立在对教材实验充分掌握的基础之上，可以促进教材实验教学目标的达成。其次，相对教材实验探究，拓展实验具有自主性、开放性和未知性的特点，给学生留有广阔的思维空间和探究视域。拓展实验需要学生的全程参与和自主探究，包括问题的提出、实验的设计、材料的准备、具体的实施和结果的讨论分析等，对学生的能力要求上升到了一个新高度。对照上述的科学探究能力的内涵，不难发现，拓展实验的开展可以对科学探究能力进行全方位的训练，促进学生能力的提升。再次，拓展实验的开展，为学生创造了更多进入实验室进行实践的机会。实践出真知，学生需要在实践中去验真和证伪。

17 Sun YP,Zhang TJ,Cao H,et al.Expression of pungent-taste herbs and their applications in clinical compatibility.Zhong Cao Yao 2015;46(6):785-790.

18 Jiang QD,Yang WG,Cai H,et al.Association between chemical composition of essential oil with penetrationenhancement effect and drug properties of Traditional Chinese Medicine.Zhong Guo Zhong Yao Za Zhi 2016;41(13):2500-2505.

19 Ahad A,Aqil M,Ali A.Investigation of antihypertensive activity of carbopol valsartan transdermal gel containing 1,8-cineole.Int J Biol Macromol 2014;64:144-149.