1. Introduction

Mosquito-borne diseases remain the biggest health problem for humans worldwide. In Thailand, Aedes aegypti (Ae. aegypti)and Aedes albopictus (Ae. albopictus) are the primary vectors for transmitting dengue and dengue hemorrhagic fever[1], Anopheles minimus (An. minimus) is one of the primary vector for the seasonal outbreaks of malaria[2], and Culex quinquefasciatus (Cx.quinquefasciatus) transmits Japanese encephalitis[3]. In 2017, the Bureau of Epidemiology, Department of Disease Control Ministry of Public Health in Thailand reported that more than 30000 Thai were infected by those mosquitoes-borne diseases.

Insecticides have traditionally been the first option for controlling outbreaks of vector-borne diseases, owing to their outstanding efficacy[4]. Temephos, the most well-known larvicide, is widely used for controlling the mosquito larvae population[5]. However,continuous use of temephos has led to negative effects on humans.Moreover, reports of temephos-resistant mosquitoes are continuously being published[6-8]. Therefore, plant biosubstances have been the focus of replacement insecticides.

Plant extracts have been a challenging subject with regard to vector control because of the abundance of plant species and human safety issues. One potentially safer alternative is Dracaena loureiri Gagnep(D. loureiri), commonly known as “Chan Pha”, “Chan Daeng”,and “Lukka Chan”. D. loureiri is a folkloric medical plant with antipyretic and analgesic properties that is used in Thailand for the treatment of colds, fever, cough, in flammation, and gastrointestinal disturbances[9,10]. We previously reported on the larvicidal efficacy of crude extract from the endocarp of D. loureiri against thirdstage larvae of Ae. aegypti, in which the 24-h and 48-h lethal concentration 50 (LC50) values were 84.00 mg/L and ＜ 50.00 mg/L,respectively[11]. Thus, we aimed to assess the larvicidal efficacy of crude and fractionated extracts of D. loureiri against Ae. aegypti and other mosquito species (i.e., Ae. albopictus, Cx. quinquefasciatus, and An. minimus).

2. Materials and methods

2.1. Crude extracts

Crude extracts of D. loureiri (voucher number: DTNU008) endocarp were prepared according to the method outlined in the previous study[11]. Brie fly, the fruits were collected from naturally growing trees and cleaned with tap water. Their endocarps (2.36 kg) were completely dried in a hot air oven at 45 ℃. The dried endocarps(586.33 g) were ground with an electric blender at 22000 r/min,and the resulting dried powder was macerated with absolute ethanol at a ratio of 1:10 (powder:solvent, w/v) with 24 h of continuous shaking (180 r/min) on a rotary shaker. The suspension was then filtered through a WhatmanTM No.1 filter paper (GE Healthcare UK Limited, UK) via a Büchner funnel. Afterward, the extracts were evaporated to dryness under reduced pressure to yield crude extract(26.29 g), which was stored in a desiccator.

针对目前我国病理学教学的普遍问题，基础病理学教学所安排的学时，对于病理学这一涉及到所有临床专业疾病的重要学科是远远不够的，医学生病理专业素质不高；临床病理教学难以培养高质量的病理专科医生，并且病理专科医生存在巨大的缺口等困境。结合目前慕课平台的高速发展，认真调研慕课平台的开放原则、注册对象，培养模式、运营情况等等，在病理学教学中引入慕课平台，将大有作为。但是，如何让慕课平台在病理学教学中发挥更大作用也有待于病理学教研工作者进行深入的调查和研究。

2.2. Column chromatographic fractionation

The authors acknowledge the Naresuan University Research Fund (Reference Number: R2560B057) for the financial support and Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University for the laboratory facilities.We would like to thank Asst. Prof. Dr. Anuluck Junkum, the staff of Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand, and Dr. Danita Champakaew for their laboratory assistance.

2.3. Mosquito colonization

The protocol for testing larvicidal activity followed that of our previous study[11]. Brie fly, a stock solution of crude and fractionated extracts (1%,w/v) were prepared with dimethyl sulfoxide as the diluent. From the stock solutions, a series of crude and fractionated extract concentrations were prepared (30–190 mg/L and 2–110 mg/L, respectively). Afterward,200 mL of each concentration of extract was placed into plastic bowls.Twenty-five of the late third-stage larvae were transferred into the extract solutions. Mortality rates were determined after 24 h and 48 h of exposure. Larvae confirmed dead when they were pricked by a needle and not moved. This experiment was performed in quadruplicate (total of 100 larvae for each concentration). Dimethyl sulfoxide in distilled water was used as the control.

2.4. Larvicidal bioassay

Ae. aegypti and Ae. albopictus colonies were obtained from laboratory strains from the Department of Microbiology and Parasitology,Faculty of Medical Science, Naresuan University, Thailand. Cx.quinquefasciatus and An. minimus were obtained from laboratory colonies from the Department of Parasitology, Faculty of Medicine,Chiang Mai University, Chiang Mai, Thailand. The larvae were reared in tap water under laboratory conditions: (25±2) ℃, 70%–80%relative humidity, and 10:14 (light:dark) photoperiod. Larval food consisted of powdery dog biscuits (for Aedes and Culex) and fish food (for Anopheles). After pupation, the larvae were transferred into plastic cups filled with tap water that were placed in mosquito cages (30 cm × 30 cm × 30 cm). After emergence, the adults were provided solutions of 5% sugar mixed with 5% multivitamin syrup.After 5 d, the females were provided blood meal through an artificial membrane feeding method. After blood-feeding, female Aedes and Culex were reared until gravid and permitted to lay eggs. Meanwhile,blood-fed female Anopheles were mated though an artificial mating method[12], after which they were permitted to lay eggs. After the eggs hatched, the larvae were reared according to the above conditions until they were required for bioassays.

2.5. Data analysis

[35]鲁塞尔·发菲尔德：《美国政策中的东南亚》，北京：世界知识出版社，1965年，第13、28-29页。

氧化铋为一次性投入，可长期使用，长期运行后除氯效果有所下降，这是因为有其他杂质超标所致，经过除杂工序可进行回收再次投入系统，效果依然很好。

3. Results

The larvicidal activities of D. loureiri crude endocarp extract against Ae. aegypti, Ae. albopictus, Cx. quinquefasciatus, and An. minimus mosquitoes were presented in Table 1. At 24 h, An. minimus larvae had the highest susceptibility to crude extract (LC50 77.88 mg/L). Its 24-h LC50 was significantly lower than that of Ae. aegypti (224.73 mg/L), Ae. albopictus (261.75 mg/L), and Cx. quinquefasciatus (282.86 mg/L). At 48 h, An. minimus was so highly susceptible to crude extract (＞ 90% mortality rate at 30 mg/L) that we did not calculate the 48-h LC50 value, although it was estimated to be ＜ 30 mg/L.

Fractionated extraction by column chromatography produced 188 eluted fractions from the crude extract. The fractions were classified into six groups: RC-DT 009 to RC-DT 014 (Figure 1). All groups were preliminarily screened for larvicidal ability. One concentration(110 mg/L) from each group was tested against the third-stage Ae.aegypti larvae. After 24 h of exposure, the RC-DT 012 and RC-DT 013 fractions produced ＞ 90% mortality rates, while the remaining fractions produced 0%–3% mortality rates. For that reason, RC-DT 012 and RC-DT 013 were selected for the bioassays.

The results of larvicidal activity experiments on RC-DT 012 and RCDT 013 were presented in Tables 2 and 3, respectively. In contrast to results from crude extract, Cx. quinquefasciatus (as opposed to An.minimus) was extremely susceptible to both fractions. For RC-DT 012,the 24-h LC50 and LC90 values were 0.66 and 3.29 mg/L, respectively.For RC-DT 013, those values were 0.94 and 2.77 mg/L, respectively. An.minimus, Ae. aegypti, and Ae. albopictus larvae had minor susceptibility to the fractions. However, the mortality rates of all mosquito species were significantly higher for those exposed to fractionated extracts than for those exposed to crude extract.

The LC50 and LC90 values of the crude and fractionated extracts for each mosquito species were compared and statistically analyzed.Results showed that the larvicidal activities of fractionated extracts were statistically greater than that of the crude extract for all mosquito species. In fact, the only values that were not statistically significant were the 48-h LC90 values for Ae. albopictus (crudeextract: 279.89 mg/L; and RC-DT 012: 224.29 mg/L). According to the results in this study, fractionated extracts were more effective than crude extract against all tested mosquito species.

Table 1 Larvicidal activities of crude ethanolic D. loureiri extracts against the third-stage larvae of 4 mosquito vectors.

Values of mortality rate were expressed as mean±SD. χ2 chi-square test, P＜0.05 represented significant difference. *The mortality rates were very high, so the parameters could not be calculated.

Mosquito Concentration(mg/L)24-hour exposure time 48-hour exposure time Mortality rate(%)LC50 (mg/L)(LCL-UCL)(LCL-UCL) χ2 Mortality rate (%)LC90 (mg/L)LC50 (mg/L)(LCL-UCL)(LCL-UCL) χ2 LC90 (mg/L)Ae. aegypti Control 0224.73(204.19-267.17)4.77050 0 5.00±3.8370 026.00±7.6690 045.00±6.00110 2.00±2.3164.00±3.2713010.00±4.0083.00±5.0315015.00±3.8390.00±7.6617021.00±2.8389.00±6.0019034.00±6.9397.00±3.83 Ae. albopictus Control 0261.75(220.28-369.95)367.97(299.11-545.42)1.532 093.37(89.03-97.58)156.52(147.59-167.88)7.40450 0 1.00±2.0070 014.00±5.1690 5.00±2.0028.00±8.6411013.00±2.0042.00±6.9313015.00±3.8348.00±8.6415024.00±3.2750.00±8.3317028.00±3.2765.00±3.8319030.00±5.1674.00±2.31 Cx. quinquefasciatus Control 0282.86(228.79-426.04)648.75(434.17-1502.71)1.582 0134.40(127.15-142.73)279.89(247.85-328.88)9.9298.050 0 -* -* -*3031.00±2.0093.00±5.035036.00±3.2795.00±5.037042.00±5.1692.00±4.629050.00±8.3395.00±3.8311056.00±3.2794.00±2.3113063.00±6.0096.00±2.0015069.00±6.8398.00±2.3117071.00±8.2596.00±3.2719082.00±8.3597.00±2.0070 7.00±5.03 49.00±10.009010.00±5.1641.00±8.8711021.00±5.0363.00±7.5713017.00±5.0370.00±7.6615028.00±7.30 63.00±10.5217032.00±5.66 67.00±11.9419031.00±6.0071.00±8.87 An. minimus Control 077.88(67.84-87.73)974.88(583.32-2741.42)3.800 082.55(58.94-97.65)541.33(342.47-1633.50)462.98(344.62-720.30)

Table 2 Larvicidal activities of RC-DT 012 fractionated D. loureiri extract against the third-stage larvae of 4 mosquito vectors.

Values of mortality rate were expressed as mean±SD. χ2 chi-square test, P＜0.05 represented significant difference. *The mortality rates were very high, so the parameters could not be calculated.

Mosquito Concentration(mg/L)24-hour exposure time 48-hour exposure time Mortality rate (%) LC50 (mg/L)(LCL-UCL)(LCL-UCL) χ2 Mortality rate(%) LC50 (mg/L)(LCL-UCL)LC90 (mg/L)(LCL-UCL) χ2 LC90 (mg/L)Ae. aegypti Control 026.45(21.39-30.58)3.00210 0 6.00 ± 2.3130 61.00±10.5291.00 ± 6.8350 77.00±10.0098.00 ± 4.0070 96.00±3.2710090 97.00±2.00100110 99.00±2.00100 Ae. albopictus Control 065.98(56.55-82.81)60.87(54.78-69.45)4.999018.43(16.65-20.28)31.17(27.96-35.57)3.63410 5.00±2.0027.00±5.032011.00±3.8341.00±8.233012.00±3.2742.00±5.164033.00±6.8358.00±7.6650 43.00±10.52 65.00±11.946047.00±8.8770.00±6.93 Cx. quinquefasciatus Control 1.00±2.000.66(0.09-1.19)234.53(160.97-431.73)7.862029.54(24.86-34.91)224.29(141.96-495.02)3.29(2.50-4.62)0.454510.20±3.2737.23±8.871023.47±2.00 77.66±10.521526.53±5.6696.81±2.002040.82±7.6696.81±3.832554.08±8.8798.94±2.003059.18±9.80 1000.606 1.00±2.00 -* -* -*281.82±9.5297.98±2.31490.91±3.8396.97±2.00696.97±3.83100810010010100100 An. minimus Control 2.00±2.3124.57(21.41-29.48)109.72(75.38-199.29)3.609 6.00±4.006.13(5.35-6.83)13.49(12.08-15.51)

Figure 1. Thin-layer chromatography spots of organic compounds from isolated fractions (RC-DT 009-014) of D. loureiri.

4. Discussion

Surprisingly, the crude ethanol endocarp extract of D. loureiri had lower activity against Ae. aegypti at 24 h (LC50 224.73 mg/L) and 48 h (LC50 93.37 mg/L) than in the previous study (24-h LC50 84.00 mg/L and 48-h LC50 ＜ 50 mg/L)[11]. Both studies utilized the same protocol for producing crude extract, so the differences in larvicidal efficacy could be attributed to climate and seasonal difference. That is, the previous study used plants harvested in October 2013[11];this study used the same plants, but the plants were harvested in September 2016.

（3）要闭环控制。企业要把标准的制定、标准的实施、实施过程的跟踪检查、数据资料收集、信息反馈，直到评价验收，看成一个完整的工作流程。标准体系和其中的每一个标准都要经受这个流程的检验，凡是不符合总目标要求的标准都必须改进。标准体系在运行过程中还会随着情况的变化调整其总目标，相应的标准也要及时调整、改进。在这个标准化过程循环中，每一个标准都整体地、协同地在总目标的引领下向前发展。

Of all mosquito species tested, An. minimus showed the greatest susceptibility to D. loureiri crude extract. Other species (Ae. aegypti, Ae.albopictus, and Cx. quinquefasciatus) demonstrated a significant, threefold greater tolerance than that of An. minimus. Similarly, other studies have found that Anopheles larvae are more susceptible to plant extracts than other mosquitoes. For example, Govindarajan et al. discovered that Anopheles stephensi is more susceptible (LC50 61.65 μg/mL) to Origanum scabrum essential oil than Ae. aegypti (LC50 67.13 μg/mL),Cx. quinquefasciatus (LC50 72.45 μg/mL), and Culex tritaeniorhynchus(LC50 78.87 μg/mL)[14]. In addition, Anopheles stephensi is more susceptible to Terminalia chebula extract than Ae. aegypti and Cx.quinquefasciatus, with LC50 values of 87.13, 93.24, and 111.98 ppm,respectively[15].

Table 3 Larvicidal activities of RC-DT 013 fractionated D. loureiri extract against the third-stage larvae of 4 mosquito vectors.

Values of mortality rate were expressed as mean±SD. χ2 chi-square test, P＜0.05 represented significant difference. *The mortality rates were very high, so the parameters could not be calculated.

Mosquito Concentration(mg/L)24-hour exposure time 48-hour exposure time Mortality rate (%) LC50 (mg/L)(LCL-UCL)(LCL-UCL) χ2 Mortality rate (%) LC50 (mg/L)(LCL-UCL)LC90 (mg/L)(LCL-UCL) χ2 LC90 (mg/L)6.735 0 -* -* -*1023.00±3.8389.00±8.253085.00±6.8399.00±2.005097.00±3.831007098.00±2.311009099.00±2.0010011099.00±2.00100 Ae. albopictus Control 034.62(30.77-39.23)Ae. aegypti Control 016.53(14.20-18.85)43.62(37.83-51.88)4.94010 9.00±3.8326.00±2.3120 35.00±11.49 76.00±11.7830 48.00±11.3182.00±6.934064.00±5.6693.00±6.005055.00±9.4595.00±6.006066.00±9.52100 Cx. quinquefasciatus Control 1.00±2.000.94(0.37-1.36)143.85(108.29-220.16)8.940014.52(12.73-16.16)35.56(31.64-41.21)2.77(2.25-3.43)1.5935 1.01±2.3118.09±9.451010.10±3.8372.34±8.331521.21±9.5290.43±5.032038.38±7.5796.81±6.0025 65.66±10.0797.87±2.313080.81±3.831000.235 2.00±2.31 -* -* -*281.82±8.3390.82±3.83494.95±5.03100698.99±5.03100810010010100100 An. minimus Control 1.00±2.0020.99(19.72-22.42)40.74(36.10-48.20)8.274 6.00±4.007.73(7.03-8.39)14.95(13.63-16.71)

While An. minimus was the species most susceptible to crude extract,this did not hold true for fractionated extracts. On the contrary, the mosquitoes most susceptible to RC-DT 012 (LC50 0.66 mg/L) and RC-DT 013 (LC50 0.94 mg/L) were Cx. quinquefasciatus, which had the lowest LC50 values. Furthermore, Cx. quinquefasciatus had the highest tolerance (LC50 282.86 mg/L) to crude extract compared to other species: Ae. aegypti (LC50 224.73 mg/L), Ae. albopictus (LC50 261.75 mg/L), and An. minimus (LC50 77.88 mg/L). This outcome could not be explained because of the data limitations of this study.However, we hypothesize that both fractions (RC-DT 012 and RCDT 013) must contain compounds that are highly toxic only to Culex larvae.

许诺见过前女友的事，并没有瞒丁小慧，她对许诺的行为挑不出错，却忍不住揣测他们见面聊了什么，他看向她的眼神，是不是一如几年前那样的炽热，只不过是在这样的想象里，丁小慧就伤了心。

The fractionated extracts of D. loureiri provided much better larvicidal efficacy against mosquito vectors than crude extract, which concurs with studies on Sphaeranthus indicus Linn. (Asteraceae)extracts. In those studies, steam-distilled crude extract of leaves were compared with the most effective fractionated ethyl acetate extract of the whole plant[16,17], revealing that fractionated extract is more effective than crude extract against Ae. aegypti (24-h LC50 36.76 ppm vs 140 ppm, respectively) and Cx. quinquefasciatus (24-h LC50 32.60 ppm vs 130 ppm, respectively).

Larval mortality data from the larvicidal bioassays were analyzed using a computerized probit analysis for determination of LC50 and lethal concentration 90 (LC90)[13]. The chi-square values and 95%fiducial confidence intervals [lower confidence limit (LCL) and upper confidence limit (UCL)] were calculated. A commercial LdP Line® software (Plant Protection Research Institute, Egypt) was used.

Our findings suggest that the larvicidal activity of crude extract was not a synergistic action of all compounds in the extract, echoing another recent study that reported the same[18]. In that study, only two of seven groups of fractionated extracts of Acacia pennata (L.)Willd. subsp. insuavis shoot tips contained compounds active against Ae. aegypti larvae. The LC50 values of the Fr-G2 and Fr-G3 fractions were 50.75 and 39.45 mg/L, respectively, while the LC50 values of the other fractions (Fr-G1 and Fr-G4–Fr-G7) were ＞ 100 mg/L.Similarly, our study found that the active substances in D. loureiri extract were contained only in RC-DT 012 and RC-DT 013, which had the lowest LC50 and LC90 values for all tested mosquito species.Phytochemical studies have revealed several flavonoids isolated from stems of D. loureiri, including homoiso flavans[9], dihydrochalcone[19],and stilbene[20]. Of those, (2S)-pinocembrin, (3S)-7,4′-dihydroxy-3-(4-hydroxybenzyl)-chromane, and loureirin D have antibacterial activity against Staphylococcus aureus and Bacillus subtilis; and 7,4′-dihydroxyflavan is fungitoxic against Botrytis cinerea and Cladosporium herbarum[9]. Studies by Meksuriyen and Cordell and Ichikawa et al. have reported that retrodihydrochalcones and homoiso flavones isolated from stem wood are estrogen agonists[19,21].In addition, stilbenoids, isolated from stem wood are potent inhibitors of cyclooxygenase (COX)-1 and COX-2 enzymes[20]. Although some phytochemical constituents and their activities have been studied,the phytochemical compounds in the fruit endocarp of D. loureiri have never been investigated. Moreover, until our previous study of crude extract[11], the larvicidal activity of D. loureiri has never been elucidated. Thus, the results of this study could not be compared to the results of other studies. Further studies on the larvicidal activity of D. loureiri extract, phytochemical constituent analysis (e.g., gas chromatography-mass spectroscopy)[22], purification, and mosquito larvicide evaluation of substances purified from the RC-DT 012 and RC-DT 013 groups must be performed.

第一，我国受国内外经济、政治的变化影响，中国在参与国际财经合作的形式和手段更加多元化、合作内容也更为丰富，中国对于世界经济长期稳定可持续发展起到了越来愈重要的作用，中国政府一直在积极调整宏观经济政策，加强宏观调控，实现全球经济长期、稳定、快速增长。

Conflict of interest statement

The authors declare that there is no conflict of interest.

高效教学有两个基本维度：目标和时间。单位时间的达标率，才是效率问题。每堂课上，教师要把学习目标明确地告诉学生，这样师生才能心往一处想、劲往一处使，提高目标达成率。在上课的过程中，教师要紧扣目标，落实目标，下课前要回扣目标。

Article history:

Acknowledgements

The crude extract was fractionated by column chromatography(Merck silica gel 60 PF254, 250 g) using a gradient solvent system of CH2Cl2, CH2Cl2–MeOH, and MeOH, with increasing amounts of the more polar solvent (mobile phase: 10% MeOH in dichloromethane).After heating at 90–110 ℃ for 4 min, the developing reagent(anisaldehyde reagent, consisting of 3 mL p-methoxybenzaldehyde,2 mL concentrated sulfuric acid, 2 mL water, and 90 mL absolute ethanol) caused organic compounds to emit specific colors, which were examined by thin-layer chromatography. From there, six groups of fractionated extracts were obtained: RC-DT 009 (1.23 g),RC-DT 010 (0.59 g), RC-DT 011 (0.75 g), RC-DT 012 (0.70 g),RC-DT 013 (3.80 g) and RC-DT 014 (1.31 g).

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