Antiplasmodial Effect ofAnthocleista vogeliion Albino Mice Experimentally Infected with伯氏疟原虫疟原虫（NK 65）

抽象

本研究的目的是调查的乙醇干树皮提取物的抗疟原虫效应Anthocleista vogeliiat different doses in albino mice infected with伯氏疟原虫疟原虫（NK 65）。三十六个小鼠分为6组，每组六只小鼠。五组（B₁–B₃, D, and G) were infected with伯氏疟原虫疟原虫parasitized red blood cells. Groups D, H, and G served as the controls. Six days after infection, mice in groups B₁, B₂, and B₃were treated orally with 100, 200, and 400 mg/kg body weight ofAnthocleista vogelii，respectively, for six executive days. Group D was treated with 5 mg/kg body weight of chloroquine while Group G was given distilled water. Group H was not infected and was not treated. It served as the normal control. The extracts exhibited significantdose-dependent chemosuppression ofP. berghei。The extract exhibited average chemosuppressive effects of 48.5%, 78.5%, and 86.6% at dose levels of 100, 200, and 400 mg/kg body weight, respectively. Phytochemical screening of the plant extract revealed the presence of saponins, cardiac glycosides, flavonoids, terpenes, alkaloids, and steroid. The acute toxicity (LD₅₀) of the plant was estimated to be 3162 mg/kg body weight. It showed that the stem bark ofA. vogelii具有抗疟原虫财产。

1.简介

Malaria, an infectious disease associated with fever, anaemia, and other pathologies, is caused by species ofPlasmodium。本属感染哺乳动物，鸟类和蜥蜴，由雌蚊叮咬（传输Anophelesspecies in mammals or库蚊species in birds and lizards) in which part of the life cycle is spent. Worldwide, clinical cases of malaria were observed in about 270 million people annually resulting in at least 1.5–2.7 million deaths a year [1]。There were approximately 3.3 billion people at risk of developing malaria each year, with at least 500 million cases, and nearly a million deaths annually worldwide [2]。This averages one person dying of malaria every 30 seconds. Over 90% of deaths occur within the continent of Africa, mainly among young children [3]。Despite efforts to reduce transmission and increase treatment, there has been little change in areas at risk of this disease [4]。Precise statistics are unknown about increasing prevalence of malaria because many cases occur in rural areas where people do not have access to hospitals or the means to afford health care; hence, majority of cases are undocumented [5]。

In Nigeria, the burden of malaria is well documented and has been shown to be a big contributor to the economic burden of disease in communities where it is endemic and is responsible for annual economic loss of 132 billion Naira [6,7]。It is estimated that 300,000 deaths occur each year, and 60% of outpatient visits and 30% hospitalizations are all attributed to malaria [8]。关于人口的50％有疟疾至少一个情节每年造成高生产力损失[8,9]。The disease is particularly virulent among pregnant women and children under 5 years of age due to their low levels of immunity [1]。的趋势正在迅速由于目前疟疾电阻增加至第一线的抗疟疾药物[10]。它负责在尼日利亚[热带疾病的报告病例超过90％11,12]。

The efficacy of these conventional drugs against malaria parasite has been reported with variable successes [13,14]。The toxic effects of these chemicals on humans [15,16], the development of resistance to it by target parasites [17]，和药品成本高[18]有草药作为合理的替代铺平了道路。

Numerous plants indigenous to Nigeria have been found with amazing antimalarial properties. It is therefore highly essential that indigenous plants used by the local people to treat malaria be scientifically investigated to prove their ethnotherapeutic use.

属的许多物种Anthocleistaare used in the treatment of malaria in South-South Nigeria.Anthocleista vogelii(family: Loganiaceae) is a tree, about 15–20 m in height, found mostly in the tropical rainforest. The stem bark decoctions are used traditionally in the treatment of malaria [19]，作为一种驱虫尤其对于蛔虫[20], as antidiarrhoea [21], and for treatment of epilepsy [22]。

The present study seeks to evaluate the antiplasmodial activity of the ethanolic extract of the stem bark ofAnthocleista vogeliiat different dose levels in albino mice infected with伯氏疟原虫疟原虫。

2. Materials and Methods

2.1。植物材料的采集

的茎皮A. vogeliiwas obtained from Awi forest, Akamkpa Local Government Area of Cross River State, Nigeria. The plants were properly identified using appropriate identification keys. Voucher specimen of the plant was deposited in the herbarium of the Department of Botany, Faculty of Science, University of Calabar, Calabar, Nigeria.

2.2。粉末的制备茎皮和乙醇的干树皮提取物Anthocleista vogelii

Fresh stem bark ofA. vogeliiwas washed with clean water and air dried under shade for five days to reduce the water content. The dried stem bark was ground into powder in a mortar with pestle before being pulverized into powder form using an electric blender. About 300 g of the powdered sample was macerated in 80% ethanol in a plastic bottle. The mixture was allowed to stand overnight and the supernatant was carefully drained into a small cleaned stainless basin and evaporated to dryness in a water bath at 45°C overnight.

2.3. Phytochemical Analysis of the Plant Extracts

的乙醇树皮提取物的植物化学分析A. vogeliiwas carried out using the procedure described by [23–25]。The phytochemical analysis examined the presence of the following chemical parameters in the plant extracts: tannins, saponins, flavonoids, cardiac glycosides, phenols, and alkaloids. About 0.5–2 g of powdered/aqueous extracts ofA. vogelii煮沸或具有取决于化学参数的各种试剂混合，以使用由所述方法进行研究[23–25]。在着色的变化来确定研究的特定参数的存在或不存在。

2.4。急性毒性（LD₅₀)

The median lethal dose (LD₅₀）的干树皮提取物的A. vogelii将被determinedorally使用该方法通过[描述杀死一个群体中的50％动物19]。The mice were divided into five groups of four mice eachweighing between 18 g and 20 g. The mice were subjected to 24 hours fasting (with only water) before administration of extracts. The powdered stem bark extract was dissolved in 20% Tween-80 and administered in doses of  500, 1,000, 2,000, 3,000, and 4,000 mg/kg body weight orally. The sixth group served as the control and received only 20% Tween-80. The mice were then observed for toxicity and fatalities within 72 hours. The LD₅₀使用下式计算[26]： where =至少耐受剂量; = maximum tolerable dose.

2.5. Ethical Consideration

批准使用动物的这一研究中的应用到了医学科学院基础医学乌约大学的学院，阿夸伊博姆州，尼日利亚道德委员会作出。该委员会直接授予批准这项研究的目的是热带非洲大陆非常关键。

2.6。收购伯氏疟原虫疟原虫和Mice

Mice already parasitized with伯氏疟原虫疟原虫（NK 65）由国家医学研究所（NIMR），拉各斯买了，维持活着。从基础医学科学院乌约大学的学院，乌约，尼日利亚的动物之家，获得了研究小鼠。将小鼠圈养在标准实验室笼子并稳定从品牌粮油米尔斯有限公司，布库鲁，乔斯，尼日利亚和清洁的饮用水获得7天期间，他们分别饲喂标准饲料（饲料重要种植者）。The study was conducted in the animal house and in the Department of Biochemistry, University of Uyo, Uyo. The mice were handled in accordance with the guidelines for the care and use of laboratory animals by [27]。

2.7. Inoculation of the Mice with the Parasites

The mice parasitized with伯氏疟原虫疟原虫（NK 65）的六天后处死，已经被观测到已经记录所示的67.2％寄生虫血症一个疟疾的临床症状。将小鼠在含有在氯仿中浸泡药棉玻璃罐麻醉。血液通过使用无菌注射器和针头心脏穿刺从处死小鼠收集。The blood was diluted in normal saline in the ratio of 1 : 10 (1 mL of blood in 10 mL of normal saline). The parasitized erythrocyte in volume of 0.3 mL was used to infect each of the experimental mice intraperitoneally six days before treatment.

At the commencement of the experiment, 36 albino mice weighing between 13 and 23 g were divided into 6 groups of 6 mice each. These were labelled as Groups B₁, B₂, and B₃组和对照组d，G和H.

Groups B₁, B₂, and B₃were treated for 6 consecutive days with 100, 200, and 400 mg doses of extract ofA. vogelii分别为每公斤体重口服和日常。Three control groups were used. Control Group D was inoculated with the parasite and treated with 5 mg chloroquine/kg weight orally and daily. The chloroquine was obtained from Sigma-Aldrich Company, St Louis M O, USA. Group G was infected with the parasite but was not treated with any extract. Group H was neither infected with the parasite nor treated with the extract. The extract was administered for 6 days and, on the first day after administration, the mice were sacrificed, and blood was collected from each mouse in all the groups by cardiac puncture using sterile syringes and needles. Fresh blood from the sacrificed mice was used to make thin and thick blood films for parasite count and determination of parasitaemia.

2.8. Determination of Parasitaemia

Six days after inoculation of parasite,blood was collected from the tail of each mouse in the various groups before administration of extracts. This was used to make thin and thick blood smears to determine the baseline parasitaemia.

Percentage of parasitaemia was determined by counting the number of parasitized erythrocytes out of 200 erythrocytes in random fields of the microscope. Percentage parasitaemia and average percentage parasitaemia were calculated according to the following formula adopted by [28]： where PP = percentage parasitaemia, PRBC = parasitized red blood cells, and RBC = red blood cells.

平均百分比寄生虫是 where APP = average percentage parasitaemia, APPC = average percentage parasitaemia in the control, and APPT = average percentage parasitaemia in the test group.

2.9. Determination of Percentage Average Suppression

The percentage average chemosuppression (AS) was determined using the method of [29]。It was calculated by subtracting the average percentage parasitaemia in the test group (APT) from average percentage parasitaemia in control Group G (infected untreated group) (APC). The value obtained was expressed as a percentage of the average percentage parasitaemia in the control Group G:

2.10. Data Analysis

All tests were performed at statistical significance ofusing SPSS version 18.0 software package and values were expressed as mean ± SEM (standard error of mean) and comparisons were made using one-way ANOVA.

3. Results

3.1. Phytochemical Composition ofAnthocleista vogelii

初步植物化学测试结果上的乙醇干树皮提取物进行A. vogelii显示皂苷，强心苷，类黄酮，萜烯，生物碱的存在，和类固醇（表1)。

3.2. Acute Toxicity (LD₅₀) ofA. vogelii

The acute toxicity study ofA. vogeliishowed behavioural signs of toxicity at doses above 2000 mg/kg body weight. The percentage mortality of the mice ranged between 75 and 100% at doses of 3000–6000 mg/kg body weight (Table2)。劳工处₅₀的A. vogeliiwas 3162 mg/kg body weight (Table2)。

3.3。的乙醇茎提取物的抗疟原虫活动A. vogelii

该提取物显示显著剂量依赖性（) antiplasmodial activity at the various concentrations (100, 200, and 400 mg/kg body weight) administered with average chemosuppression of 48.5%, 78.5%, and 86.6%, respectively (Table3)。提取400毫克/公斤体重similarly well (86.6%) as the standard drug, chloroquine (5 mg/kg/day), which produced 100% chemosuppression (Table3)。

4. Discussions

Studies of the antiplasmodial effect ofA. vogeliiwere carried out on albino mice experimentally infected withP. berghei berghei。The choice of these plants was based on previous reports of their antiplasmodial property [30,31]。Again, the remarkable activity of quinine and other related drugs and the success of artemisinin stimulated the search for new plant derived antimalarial drugs [19]。然而，耐药性，这些药物的病例报告作出的搜索和替代抗疟疾药物必然[发展32]。

Phytochemical analysis of the stem bark extract ofA. vogeliishowed the presence of alkaloids, cardiac glycosides, flavonoids, terpenes, steroid, and saponins. These phytochemicals were previously reported by [19,33,34]。另据报道，另一个物种。这些植物化学化合物，A. djalonensis[31]。The presence of similar phytochemicals was recorded inA.玉兰[35]。These constituents have been found in other natural products which exhibited antimalarial activity [36]。包含有生物活性，包括生物碱和黄酮类植物化学物质，很多植物可以作为抗疟疾药物来源[37]。因此，的抗疟原虫活性A. vogelii可以归因于这些植物化学化合物的存在[30,31]。

The ethanolic stem bark extract ofA. vogeliiwas well tolerated by the mice up to the dose level of 2000 mg/kg body weight within 24–72 hours. However, physical signs of toxicity were noticed in mice administered with 3000 mg/kg body weight of the extract 24–72 hours after administration. The LD₅₀的ethanolic stem bark ofA. vogeliiwas estimated to be 3,162 mg/kg body weight far above the highest administered dose level of 400 mg/kg body weight. This indicated that the mice were safe with the different doses of the ethanolic stem extracts administered to them. Similar result was recorded using the methanolic stem bark extract of another species of the plant,A.玉兰[35]。类似的结果所报告的植物的另一物质的乙醇叶提取物的给药后30分钟至1小时内，A. djalonensis,at the dose level of 5 g/kg [31]。The results implied that the ethanolic stem bark ofA. vogeliiwas toxic at doses above 3000 mg/kg body weight causing toxic effects and eventual death of the animals [38,39]。

寄生虫血症在感染的小鼠中的所有使用从小鼠的尾静脉制成厚和薄血膜的基团进行监控。百分比平均寄生虫感染表现出高水平的各组后的接种五天P. berghei。This result is consistent with previous reports of high percentage parasitaemia inP. bergheiinfected mice after five days and death of infected mice after seven days of inoculation [40,41]。The high level of parasitaemia is an important feature ofPlasmodiuminfection which could result in severe anaemia.

The starting parasitaemia for the three replicates B₁, B₂, and B₃before administration of ethanolic stem bark extracts ofA. vogeliiwas 10.34%, 5.5%, and 7.3% as indicated in Table3。After administration of extract, the results obtained from the present study showed that the ethanolic stem bark extract ofA. vogeliiexhibited significant () chemosuppressive effect against伯氏疟原虫疟原虫infection in mice. At the dose level of 100, 200, and 400 mg/kg body weight, the extracts showed remarkable average chemosuppressive effect of 48.5%, 78.5%, and 86.6%, respectively. This result suggested that the extract possesses some active phytochemical compounds that have direct effects on the parasites and the effect is dose-dependent [19]。

该显著（）剂量依赖性的效果chemosuppressiveA. vogelii在研究中观察到的是由[早先的报告一致19,31]。P. bergheiparasite densities of 12,000 μL and 19,520 μL were recorded in mice before treatment, but, with the administration of ethanolic leaf extract ofA. vogeliiat the dose levels of 250 mg/kg and 100 mg/kg, parasite density reduced to 2,350 μL and 10,000 μL, respectively [19]。The chemosuppressive activity of 68.20% was recorded forA.玉兰in mice at a dose level of 700 mg/kg/day [35]。These results are indication that plants of the genusAnthocleistapossess phytochemical constituents that have antiparasitic property.

In this study, chloroquine, used as a positive control, was observed to significantly (）减少在感染小鼠的寄生虫血症在比的干树皮提取物更高的速率（100％）A. vogelii。The mechanism of action of these extracts ofAnthocleistaspecies was however said to be similar to that of chloroquine, a standard antimalarial drug which induced the destruction of the asexual forms of thePlasmodiumparasite [42]。记录在本研究中氯喹的100％chemosuppressive效果表明，它仍然是首选药，对疟疾寄生虫之一[43]。然而，这并不排除对氯喹抗性Plasmodium哺乳动物物种。而且，事实是，在非洲一些农村居民点仍然更多地依靠利用药用植物的疟疾导致的识别处理A. vogelii这被证明是非常有效的对抗P. berghei bergheiat the dose level of 400 mg/kg recording 86.6% average chemosuppression.

5. Conclusion

The result obtained in this study showed that ethanolic extract of the stem bark ofA. vogeliishowed a dose-dependent antiplasmodial activity. It was most effective at the dose level of 400 mg/kg body weight. This plant can be recommended for use since it possessed a high chemosuppressive effect against the malaria and can be obtained at relatively no cost from the forest.

利益冲突

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

作者感谢工人（Inemesit Udofia，Mberebong Umoren和Akaninyere阿塔赫）在动物的房子，基础医学科学学院，乌约大学，阿夸伊博姆州，尼日利亚。

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