蜂膠(翻譯(共14頁)

1、天然產(chǎn)物巴西(b x)蜂膠加強吞噬細胞殺真菌(zhnjn)的活性AbstractNatural product incorporation into microcarriers increases the bioavailability of these compounds, consequently improving their therapeutic properties. Natural products, particularly those from bees such as propolis, are widely used in popular medicine. Propol

2、is is a powerful treatment for several diseases. In this context, the present study evaluated the eect of propolis Scaptotrigonasp. and its fractions, alone or adsorbed to polyethylene glycol (PEG) microspheres, on the activity of human phagocytes against Candida albicans.The results show that propo

3、lis exerts a stimulatory eect on these cells to assist in combating the fungus, especially as the crude extract is compared with the fractions. However, when incorporated into microspheres, these properties were significantly potentiated. These results suggest that propolis adsorbed onto PEG microsp

4、heres has immunostimulatory eects on phagocytes in human blood. Therefore, propolis may potentially be an additional natural product that can be used for a variety of therapies.天然產(chǎn)物包埋在載體中能夠增加這些化合物的生物藥效，因此能夠提高它們的治療功效。天然產(chǎn)物，特別是些來自蜜蜂的，例如(lr)蜂膠，正被廣泛的應(yīng)用到流行藥中。對于一些疾病，蜂膠具有非常好的治療功效。鑒于此，現(xiàn)在研究評估Scaptotrigona sp.

5、的蜂膠及其餾分在單獨的或吸附到聚乙二醇微粒上時，對人類吞噬細胞作用白色念珠菌的效果。結(jié)果表明蜂膠對這些細胞對抗真菌具有輔助的促進作用，值得注意的是粗提物的效果要比餾分的促進效果更好。但是包埋到PEG微粒中的蜂膠的這些功效要更強。這些結(jié)果表明：吸附到PEG微粒上的蜂膠在人類血液中對吞噬細胞具有免疫調(diào)節(jié)功效。因此，蜂膠有可能作為一種天然產(chǎn)物添加，用在各種各樣的治療中。前言The current focus on natural products and alternative medicines has renewed interest in bee products such as honey,

6、 royal jelly, pollen, and propolis. Propolis is a sticky dark-coloured material collected by worker bees from the leaf buds or exuded from numerous tree species. Oncecollected, this material is enriched with salivary and enzy-matic secretions and used in the construction, adaptation, and protection

7、of their nests 3,4. In this way propolis chemical composition is a direct reflex of the vegetable flora and bee species 5,6.目前關(guān)于天然產(chǎn)物和可替代性藥品的研究又重新集中于像蜂蜜、蜂王漿、花粉、蜂膠等蜜蜂產(chǎn)品上，蜂王漿是一種黑色粘稠的物質(zhì)，是由雄蜂采集自葉芽或許多樹種的滲出液。一旦采集完，這種物質(zhì)又富加以唾液和酶促分泌物，然后用來筑造、改建、和保護它們的蜂巢。這樣一來，蜂膠的化學(xué)成分就能直接反映出蔬菜的種類和蜜蜂的種類。In Brazil, besides wide va

8、riety of flora there are also several bees species, among which stand out those belonging to Apidae family and Meliponinae subfamily, better known as indigenous stingless bees, and which produce propolis from the resinous material of plants with wax and soil. In this group are found bees of the Scap

9、totrigona genus with twenty four species described and eight of which already identified in Brazil 4, 79.在巴西，除了廣泛種類的植物群，還有一些密封種類，他們(t men)中有代表性的屬于蜜蜂科和Meliponinae亞科，比較有名的土著無刺蜂它們產(chǎn)的蜂膠來自于含蠟植物的樹脂和土壤。在這個(zh ge)群體中，Scaptotrigona屬的24個種的蜜蜂都有所描述，其中(qzhng)有8個種都已經(jīng)在巴西發(fā)現(xiàn)了。In particular, the propolis shows potenti

10、al because of its therapeutic properties and possible applications in the pharmaceutical industry 1012. A range of biological activities have been attributed to propolis, including immunomodulatory 13,14, antibacterial 15, fungicidal 16, 17, anti-inflammatory, healing 18, anesthetic 19, and anticarc

11、inogenic eects 20.一般說來，由于其治療特性，蜂膠具有可能應(yīng)用于醫(yī)藥行業(yè)的潛在價值。蜂膠有一系列的生物活性，包括免疫調(diào)節(jié)、抗菌、殺真菌、消炎、治愈、麻醉、抗癌的效果。On the other hand, many natural products are not therapeutically eective when used without modification. In many cases, this failure is attributed to low concentrations at the therapeutic targets. One factor t

12、hat influences the bioavailability of natural products is the extensive metabolism that they undergo in vivo by commensals or probiotics during their passage through the intestine and liver, significantly changing the exact species that is found in systemic circulation 21. Susceptibility to chemical

13、 hydrolysis at physiological pH has been demonstrated for natural substances with high biological activity 22.另一方面，許多天然產(chǎn)物如果不經(jīng)修飾是沒有治療效果的。在許多案例中，它們沒有療效的原因是在治療的靶位點濃度很低。影響天然產(chǎn)物的生物利用率的一個原因是廣泛代謝的存在，廣泛代謝是在體循環(huán)中發(fā)現(xiàn)的，當體內(nèi)的共生體或益生菌在通過腸道和肝臟時，受廣泛代謝的影響，微生物的種類會發(fā)生顯著改變的現(xiàn)象。生理pH環(huán)境中易發(fā)生的水解反應(yīng)已經(jīng)證明了天然產(chǎn)物具有高的生物活性。One alternative

14、 to these problems is the use of polymeric microparticle systems, which have shown promise for the adsorption of phytopharmaceuticals. these systems also promote the controlled release of drugs or biologically active hydrophilic or hydrophobic substances 23, 24. Among the polymers used in the prepar

15、ation of microcarriers，polyethylene glycol (PEG) has excellent properties such as solubility in both water and organic solvents and the absence of toxicity and antigenicity, which are essential for biomedical applications. there have been multiple studies of the eect of pairing natural or synthetic

16、drugs with PEG microparticle systems 25-33. these combinations present numerous advantages, such as prolonging residence in the body, decreasing metabolic degradation by enzymes, and reducing or eliminating the immunogenicity of proteins 27 It is likely that the development of drugs that incorporate

17、 natural materials will be able to reduce side eects, decrease costs, and maximize the benefits of natural product formulations to avoid the aforementioned problems.這些問題(wnt)的另一個原因(yunyn)是聚合物微粒(wil)體系的應(yīng)用，在植物藥物學(xué)中，聚合物微粒體系是吸附的前提。這些系統(tǒng)也在藥物或生物活性物質(zhì)的親水性或疏水性的控釋方面起到促進作用。在聚合物用于微載體的制備中，聚乙二醇（PEG）具有極好的性質(zhì)，例如既溶于水又溶

18、于有機溶劑，以及毒性缺失和抗原性，其中抗原性是生物醫(yī)學(xué)應(yīng)用的本質(zhì)。聚乙二醇（PEG）微粒體系在天然藥物或合成藥物的配對影響方面也有一些研究25-33。這些制品表現(xiàn)出了許多優(yōu)勢，比如延長體內(nèi)的滯留期，降低代謝過程中酶的降解，減少或消除蛋白質(zhì)的免疫原型。隨著藥物包括天然產(chǎn)物的發(fā)展，副作用的減小和費用的降低，以及增大天然產(chǎn)物劑型效果而避免前面提到的問題發(fā)生，這些是很有可能做到的。In this context, the aim of this study was to evaluate the immunomodulatory and fungicidal eects of propolis ads

19、orbed to PEG microspheres on human phagocytes in the blood.在這個背景下，本研究的目的在于評估人類血液中吞噬細胞對吸附有蜂膠的聚乙二醇微球的免疫調(diào)節(jié)效果和殺真菌效果。材料與方法2.1蜂膠 蜂膠樣品(yngpn)2011年2月收集(shuj)，直接(zhji)采集于Barra Garas城市（南緯155219.4和西經(jīng)521027.03)的Scaptotrigona sp蜂房，巴西馬托格羅索州的東部塞拉多地區(qū)。這兒年平均氣溫是25.5，有兩個良好的季節(jié):雨季(10月至4月)和旱季(5月至9月)。年降雨量是1750毫米。蜂膠樣品含有樹脂、香

20、脂，呈暗棕色，具有典型的蔬菜氣味和固體雜質(zhì)。收集完應(yīng)及時存儲在有蓋子的容器內(nèi)運輸。2.2蜂膠中粗提取物的制備 蜂膠乙醇提取物的生產(chǎn)采方法采自Miorin 4，其中30克的蜂膠和碎塊放在有蓋的容器，加100毫升無水乙醇。室溫下定時攪拌一周。隨后，將溶液用3號瓦特曼濾紙過濾在培養(yǎng)皿（預(yù)稱重）中。將19.3克粗提取物放在40電爐上將乙醇蒸發(fā)，使其與糖蜜一樣外觀呈暗褐色。2.3蜂膠提取物的化學(xué)篩選 根據(jù)Harbone對蜂膠醇提取物進行化學(xué)定性篩選，驗證了有氰甙，酚類，花青素，單寧，黃酮類化合物，無色花色素，兒茶素，二氫黃酮黃酮醇，酮類，類固醇，三萜皂苷和生物堿的存在。所用的試劑和化學(xué)物質(zhì)有生物堿與Dr

21、agendorff試劑，黃酮類化合物與金屬鎂和鹽酸, 能夠產(chǎn)生泡沫的皂苷,還原糖與費林試劑、苷類和利伯曼試驗,單寧與氯化鐵,多糖與碘溶液。2.4蜂膠提取物的分餾 通過改良桑托斯等人描述的經(jīng)典液相色譜法獲得蜂膠提取物的組分。用60230目硅膠為固定相，有機溶劑（己烷，二氯甲烷，乙酸乙酯，甲醇）作為流動相。固定相的色譜柱的準備：將二氧化硅懸浮在正己烷中充滿填充柱。接下來，將5克的蜂膠溶解在正己烷并添加到固定相中。當樣品通過柱后每個移動相用三百毫升進行洗脫，除了最后一列用450毫升甲醇洗脫。這些餾分用正己烷，二氯甲烷，乙酸乙酯，甲醇洗脫后基于相關(guān)的分光光度分布匯集，然后放入40C烘箱三天，以除去溶劑

22、。樣品干燥后進行稱量，這樣可以制備已知濃度的溶液。2.5聚（乙二醇）（PEG）微球的制備 微球的生產(chǎn)方法依照史葛等人描述和Scherer等人所改進的進行制備。簡要介紹，20克的聚乙二醇6000（合成器）懸浮在100毫升的磷酸鹽緩沖鹽水（PBS）中，在PBS中加入體積分數(shù)（VV）為2%的硫酸鈉溶液混合，然后在37C下孵育45分鐘。孵育后將混合物用PBS稀釋至3：1。將溶液加熱至95C 保溫5分鐘進行熱誘導(dǎo)形成微球。粗提物溶液（VV）、餾分和微球在37C孵育30分鐘使其吸附。用Dylight 熒光染料-488溶液（10微克/毫升，皮爾斯）對有或沒有吸附粗提物、餾分的微球PEG染色，后在PEG：Dy

23、light為100：1摩爾比的二甲基甲酰胺溶液中室溫下過夜，然后用熒光顯微鏡觀察和分析。2.6 血液樣本 一個15毫升的血液樣本是從200個臨床健康男性志愿者中采集的，其年齡在18至35年間。進入研究前由當?shù)氐膫惱砦瘑T會批準，所有的志愿者都簽署了知情同意書。2.7 血細胞分離 血液樣本收集到肝素管（25 U /毫升）中。細胞經(jīng)Ficol-Paque梯度分離（Pharmacia，烏普薩拉，瑞典）后生產(chǎn)純度為98%的單個核細胞制劑，然后用光顯微鏡分析。純化的巨噬細胞獨立懸浮培養(yǎng)在199無血清培養(yǎng)基中至終濃度2106細胞/毫升。2.8白色(bis)念珠菌培養(yǎng) 白色念珠菌ATCC10231標準(bio

24、zhn)菌株被用于該研究。在測試(csh)之前的24小時，將真菌樣品懸浮在腦心浸液肉湯（BHI）37C下孵育24小時。成熟后，用PBS溶液清洗真菌兩次，調(diào)整濃度至2107酵母細胞/毫升。2.9 濃度響應(yīng)曲線的測定 確定濃度響應(yīng)曲線，0g/ml，10ugml，50ugml，100 ug/ml蜂膠和蜂膠組分的影響如第2.9節(jié)所描述的由吞噬細胞釋放的超氧陰離子決定。所有的實驗都做兩次或三次。2.10 細胞毒性分析 細胞毒性試驗用的是如2.10節(jié)所述的吖啶橙法。在這個實驗中，采用蜂膠或蜂膠餾分對吞噬細胞活力性進行評價。所有的實驗都做兩次或三次。2.11超氧陰離子的釋放 一種用來評價蜂膠和蜂膠餾分（獨立

25、的或吸附的PEG微球）處理吞噬細胞功能的方法，是用來分析白色念珠菌中的氧化代謝是否在進行，可由超氧陰離子釋放實驗測量。由米澤爾、弗蘭發(fā)明和奧諾里奧等人改進的細胞色素C還原方法。簡要來說，在真菌存在或不存在的情況下，用獨立的或吸附在PEG微球上的蜂膠和蜂膠餾分，在37處理單核巨噬細胞30分鐘。作為對照，用PBS處理單核吞噬細胞。處理后，將細胞洗滌，懸浮在含有2.6 mM的氯化鈣，2mM氯化鎂，和2mg /ml細胞色素C的PBS中。將懸浮液（100l）置于培養(yǎng)皿于37C下培育60分鐘。用微孔板分光光度計在550 nm下測定減少的細胞色素C。超氧陰離子濃度根據(jù)以下關(guān)系計算：O2濃度（nmol）100

26、 =光密度/ 6.3，結(jié)果表示為nmol / O2。所有的實驗都要重復(fù)兩到三次。2.12 Phagocytosis and Fungicide Activity. In addition to the method described above, phagocytosis and fungicidal activity tests to evaluate the function of cells treated with propolis or propolis fractions, either alone or adsorbed to PEG microspheres, were pe

27、rformed. The acridine orange method proposed by Bellinati-Pires et al.was used for this purpose. Briefly, equal volumes of mononuclear phagocytes and fungal suspensions were treated with propolis or propolis fractions, either alone or adsorbed to PEG microspheres, and incubated for 30 minutes at 37C

28、 with continuous shaking. PBS-treated mononuclear phagocytes and the fungal suspension were used as the control.吞噬作用與真菌活性。 除了上述方法，用來評估被蜂膠和蜂膠餾分(獨立的或者是吸附于PEG微球的)處理過的細胞的功能的吞噬性和真菌活性實驗，都有人做過。Bellinati-Pires等人提出的吖啶橙法就用于這樣做的。簡而言之，等體積的單核吞噬細胞和真菌懸浮液用蜂膠或者蜂膠餾分（單獨或者吸附于PEG微球的）處理，然后在37C下持續(xù)搖晃30分鐘，對照組就用PBS處理的單核吞噬細胞和

29、真菌懸浮液。 after incubation, phagocytosis was stopped by incubation on ice. To remove the extracellular fungal cells, the suspensions were washed and centrifuged twice (160 g, 10 min, 4C). Then, the cells were resuspended in serum-free medium 199 and centrifuged. The supernatant was discarded and the pe

30、llet was stained with 200 uL of acridine orange (14.4 g/L of PBS) for 2 minutes. 溫育之后(zhhu)，冰浴使吞噬作用(tn sh zu yn)停止。為了除去細胞外的真菌細胞，懸浮液得清洗并且離心兩次（160 xg,10 min, 4C )。然后，細胞(xbo)重新懸浮于無血清的199培養(yǎng)基并離心。棄去上清液并且將沉淀物用200 L的吖啶橙（PBS 14.4克/升） 染色2分鐘。 The pellet was then resuspended in cold culture 199 medium, washed t

31、wice, and observed under a fluorescence microscope at 400 x and 1000 x magnification. The phagocytosis index was calculated by counting the number of cells ingesting at least 3 yeast cells within a pool of 100 cells. To determine the fungicide ratio, 100 phagocytes that had ingested yeast cells were

32、 counted,taking into account the number of live yeast (green) and dead yeast (orange) 100 41. All experiments were performed in duplicate or triplicate. 將沉淀物再懸浮于冷的199培養(yǎng)基，洗滌兩次，并在熒光顯微鏡下用400倍和1000倍的放大倍率觀察。吞噬指數(shù)是通過數(shù)出100個吞噬細胞中至少吞下了3個酵母細胞的數(shù)量來計算的。要決定殺菌劑的分數(shù)，得將100個吞噬細胞吞下的酵母數(shù)量進行計數(shù)，考慮到活的酵母細胞（綠色）和死的酵母細胞（橘黃色）的數(shù)量乘

33、以100。所有的實驗都要重復(fù)兩到三次。2.13. Statistical Analysis. Analysis of variance (ANOVA) was used to evaluate the viability, superoxide release, phagocytosis, and the phagocytes fungicide activity. Statistical significance was defined by a P value lower than 0.05 (P 0.05).統(tǒng)計分析。 方差分析可用來評估生存能力，超氧化物的釋放，吞噬性和吞噬細胞的滅菌活

34、力。統(tǒng)計顯著性由小于0.05的P值來定義。3、結(jié)論3.1蜂膠的粗提物的化學(xué)成分分析。對蜂膠的粗提物的進行化學(xué)分析表明，里面有單寧，酚類，黃酮類，類黃酮，和氧雜蒽酮的存在。該混合物的化學(xué)組成還包含少量的黃烷酮和樹脂（表1）。3.2. PEG微球的表觀(bio un)特征用熒光顯微鏡分析PEG微球的形態(tài)，所有的PEG微球表現(xiàn)出相似的幾何形狀(xngzhun)和尺寸并呈現(xiàn)出光滑表面（圖（a）。而（圖(b)）顯示出蜂膠中沿微球表面上的非均相方式(fngsh)進行吸附。這種吸附被認為是在該小區(qū)域上的空心微球存在而導(dǎo)致的。3.3. 蜂膠和蜂膠餾分的濃度反應(yīng)曲線以確定濃度 - 效應(yīng)的關(guān)系，以4種不同劑量的進

35、行實驗（0g/mL,10g/mL,50g/mL,and 100g/mL) 通過暴露于蜂膠和蜂膠餾分中的吞噬細胞釋放的超氧化物進行估計，并觀察濃度與反應(yīng)之間的關(guān)系，隨著蜂膠和蜂膠各餾分的劑量的增加，超氧化物釋放量也相應(yīng)增加?；谶@個結(jié)果，我們使用50g/mL作為實驗濃度（圖2）3.4. 蜂膠和蜂膠組分(zfn)對單核吞噬細胞的毒性(d xn)作用(zuyng)。PEG微球和蜂膠均不會影響吞噬細胞的生存能力。在吞噬細胞的孵育過程中，二氯甲烷分數(shù)，使得細胞利用率小于90，并且其細胞活性在進一步的檢測中并沒有檢測到。然而其它組分則不會影響吞噬細胞的生存力和測試其功能活性(表2)3.5單獨或吸附PEG微

36、球的蜂膠和蜂膠餾分對超氧陰離子釋放的影響。與巨噬細胞相比較，自由釋放的PEG微球并不會改變超氧化物的釋放。在有真菌的存在(cnzi)的條件下，巨噬細胞釋放的O2-與自然界自發(fā)釋放的氧氣(yngq)類似。用蜂膠或蜂膠餾分單獨處理(chl)過的細胞，其速率相似于自發(fā)性的超氧化物的釋放。與吸附PEG蜂膠或蜂膠的乙酸乙酯部分處理過的吞噬細胞與在真菌的存在下釋放的超氧化物歧化物比較，表現(xiàn)出超氧化物歧物的釋放量顯著增加。(P 0.05).當用蜂膠吸附在微球表面培養(yǎng)時與吞噬細胞只暴露蜂膠相比，細胞的超氧化物歧化物釋放量呈增加趨勢，對于吸附有蜂膠乙酸乙酯微球體的部分巨噬細胞培養(yǎng)時，觀察到類似的效果（表3）3.

37、6.單獨或吸附PEG微球的蜂膠和蜂膠餾分對吞噬功能的影響。細胞的吞噬功能表現(xiàn)于白色念珠菌細胞的吞噬活性。此活性并沒有因為PEG微球的存在而增加。當吞噬細胞受到蜂膠或蜂膠餾分的刺激時，觀察到被吞噬的數(shù)目增加，除了在有己烷餾分的情況下，其吞噬能力會顯著減少。與只用PEG微球或單獨的蜂膠相比較，將蜂膠吸附在PEG微球表面有利于增加的吞噬細胞的活性(圖3).3.7.單獨(dnd)或吸附PEG微球的蜂膠和蜂膠餾分(lifn)對吞噬細胞(tn sh x bo)的抗菌性的影響。吞噬細胞對白色念珠菌具有較弱的抗真菌性。當細胞與蜂膠和蜂膠的甲醇餾分接觸時，其抗真菌性能回增強。然而具有正己烷餾分存在時，其抗真菌性

38、能會顯著下降。用帶有PEG微球的吞噬細胞培養(yǎng)，則其抗真菌性能會增強(P0.05,圖4)4. Discussion（討論）In the present study, we determined the effect of propolis and the propolis fractions obtained by a polarity gradient and adsorbed to PEG microspheres on the functional activity of phagocytes（吞噬細胞(tn sh x bo)） from human blood.在目前的研究中，我們(w

39、men)測定蜂膠和蜂膠組分的功效是通過一個極性梯度和吸附PEG微球?qū)ρ褐型淌杉毎墓δ芑钚缘玫降摹EG microspheres are a type of copolymer used for the clinical administration of therapeutics(治療學(xué)） because of their capacity to incorporate drugs , their ability to increase the duration of drug exposure or the production of other substances such as

40、enzymes , and their role as an important signaling vehicle in immunity .PEG微球是一種共聚物，由于它具有結(jié)合藥物的容量而被用于臨床治療學(xué)，它們(t men)的作用是增大藥物的暴露面積或者是提高其他物質(zhì)的產(chǎn)生，比如酶，它們在免疫系統(tǒng)中是重要的信號載體。In this study, analysis by fluorescence microscopy showed the PEG microspheres to be of regular size, be easily separated from a suspensio

41、n, and to readily adsorb propolis. The characterization of PEG microspheres using flow cytometry has been previously reported and has shown that this polymer is approximately 5.8 um of diameter .在這項研究中，通過熒光顯微鏡分析表明， PEG微球為常規(guī)尺寸的，易于從懸浮液中分離，并且容易吸附蜂膠。運用流式細胞儀已經(jīng)表明了PEG微球的結(jié)構(gòu)，這種共聚物直徑約為8.5 umThe use of a polym

42、eric microsphere delivery system for natural products has been described in the literature, and this system is a method that may be useful to deliver a variety of medicinal natural products that could provide additional protection against infection .使用聚合物微球運送天然產(chǎn)物的系統(tǒng)在文獻中已經(jīng)描述過了，這種系統(tǒng)是一種可能對運送各種可能能提供額外保護

43、對抗感染的藥用天然產(chǎn)物的有用方法。The literature describes the importance of natural products in discovering new drugs 45. One of the numerous medicinal properties of natural products is the ability to modulate the immune system, by either stimulating or suppressing certain immune response events.文獻描述了天然產(chǎn)物在新藥發(fā)現(xiàn)中的重要性

44、，天然產(chǎn)物其中一個巨大的藥用特性就是調(diào)節(jié)免疫系統(tǒng)，通過刺激或者抑制某些免疫應(yīng)答的能力。Most studies have been based on the action of propolis without determining the scientific basis of their cytotoxic properties. Propolis is a natural product produced by bees from resinous material collected from a variety of plant species, and it is a compl

45、ex mixture 52. In this study the main chemical constituents of this propolis showed were tannins, phenols, flavones,flavonoids and xanthones,flavanones, resins, and to a lesser degree,flavanones and resins. Because it is a mixture, the isolation of the active components that cause this effect is a v

46、ery long and complex process. Alternatively, fractionation performed with solvents of different polarities allows partial purification of the substances in the mixture, and the examination of the fractions obtained via biological assays enables the determination of some of the metabolites that show

47、therapeutic activity .大多數(shù)研究都是在沒有確定蜂膠細胞毒性的基礎(chǔ)上進行的，蜂膠是通過樹脂中的蜜蜂收集各種各樣的植物產(chǎn)生的一種天然產(chǎn)物，而且它是一種復(fù)雜的化合物。在這項研究中，蜂膠的主要(zhyo)化學(xué)成分顯示為單寧酸，酚類，黃酮類，類黃酮和呫噸酮，黃烷酮，樹脂，以及含量較少的黃烷酮和樹脂。由于它是一種混合物，要分離出引起這種效應(yīng)的有效成分是一個長期且復(fù)雜的過程。另外，具有不同極性的溶劑能夠使混合物最大化的純化，并通過生物測定法得到各成分的性能，并測量出其有治療性的代謝產(chǎn)物。Here, we demonstrated that the dichloromethane（二氯甲

48、烷） fraction is toxic to phagocytes（吞噬細胞(tn sh x bo)）. This may be caused by the presence of toxic metabolite(s)（代謝(dixi)產(chǎn)物） at higher concentrations in this fraction because, according to Cechinel-Filho and Yunes 53, some compounds exhibit cytotoxic（細胞毒性） effects in high doses. On the other hand, th

49、e fractions obtained from elution with hexane（正己烷）,ethyl acetate（乙酸乙酯）, and methanol（甲醇） contain important substances that are able to modulate the activity of phagocytes.這里，我們表明，二氯甲烷餾分對吞噬細胞是有毒的，這可能是由于毒性代謝產(chǎn)物在餾分中濃度較高引起的，根據(jù)Cechinel-Filho和Yunes，有些化合物在高劑量時具有細胞毒性，另一方面，在用己烷、乙酸乙酯和甲醇洗脫的餾分中含有能調(diào)節(jié)吞噬細胞活性的重要物質(zhì)。T

50、he various types of metabolites（代謝產(chǎn)物） obtained by the fractionation of propolis by hexane, dichloromethane（二氯甲烷）, ethyl acetate（乙酸乙酯）, and methanol（甲醇） are well known. The hexane（正己烷） fraction concentrates steroids（甾體）, terpenes（萜烯）, and acetophenones（苯乙酮）.The dichloromethane extract contains lignan

51、s（木酚素）, flavonoids（黃酮）, desmethoxy sesquiterpenes（倍半萜）, triterpenes（三萜酸）, and coumarins（香豆素）. Ethyl acetate isolates flavonoids, tannins（丹寧）, xanthones（氧雜蒽酮）, triterpeneacids（三萜類物質(zhì)）, saponins（皂甙）, and phenolic compounds（酚類化合物） and the methanol extract concentrates glycosylated flavonoids（糖基化的類黃酮）, t

52、annins,saponins, and carbohydrates. 利用正己烷、二氯甲烷、乙酸乙酯和甲醇從蜂膠中分級獲得各種代謝產(chǎn)物的方法是很常見的，正己烷濃縮了餾分中的甾體、萜烯和苯乙酮，二氯甲烷提取的包含了木酚素、黃酮、倍半萜、三萜酸和香豆素。乙酸乙酯能分離黃酮、丹寧、氧雜蒽酮、三萜類物質(zhì)、皂甙和酚類化合物，而甲醇能提取濃縮糖基化的類黃酮、丹寧、皂甙和碳水化合物。Mononuclear（單核）phagocytes play an important role in host defense. They produce phagocytic NADPH oxidase, which fo

53、rms superoxide, and this process is necessary to microbicidal (殺菌)activity and for the success of immune and inflammatory （發(fā)炎）reactions 。單核吞噬細胞在宿主防御中扮演了重要角色，它們產(chǎn)生了能形成超氧化陰離子的吞噬細胞NADPH氧化酶，且它對于殺菌活性以及(yj)免疫和發(fā)炎反應(yīng)都是必須的。During oxidative stress, cells generate high levels of superoxide radicals. Free radical

54、 generation has been reported as an important mechanism for body protection from infections, mainly intestinal infections在氧化應(yīng)激中，細胞生成了高濃度超氧自由基，自由基的產(chǎn)生認為是避免(bmin)身體被感染，主要是腸道感染的重要機制。Phagocytosis is an important defense mechanism, especially for bacterial and fungal(真菌(zhnjn) infections.The microbicidal

55、activity of phagocytes is mediated by the production of reactive oxygen species (ROS) and the release of lysosomal enzymes 59. ROSs are highly reactive molecules as a result of their unpaired electrons.These molecules rapidly react with various biomolecules, leading to DNA damage and the deteriorati

56、on of membranes through lipid peroxidation（過氧化反應(yīng)） and eventually causing cell death 60.The ROS superoxide anion is a major component of this process, as it is a precursor（前驅(qū)體） to other oxygen radicals and essential for effective phagocytosis.吞噬作用是一種重要的防御機制，尤其是對細菌和真菌感染，吞噬細胞的殺菌活性是由活性氧（ ROS）和溶酶體酶的釋放介導(dǎo)產(chǎn)生的，ROS是不成對電子作用而形成的高活性分子，這些分子與各種生物分子快速發(fā)