Bruceine D   中文名称: 鸦胆子素 D

Natural product; anticancer; anthelmintic; Notch

治疗 48 小时后，马钱子碱 D 对 H460 和 A549 细胞活力产生剂量依赖性影响，IC50 值分别为 0.5 和 0.6 μM [1]。在 H460 和 A549 细胞中，马钱子碱 D (0.125-1.0 μM) 剂量调节可增强染色质凝聚、膜联蛋白 V 诱导的细胞聚集和 caspase 细胞死亡 [1]。

---

马钱子碱D（BD）显示出对DBM的强效抗饲料活性[1]
鉴定了Bruceine D (BD)，并通过根和叶施用评估了其保护开花大白菜免受DBM幼虫取食的能力。据报道，印楝素是一种具有系统作用的卓越的喂养威慑剂；因此，采用印楝素进行比较。

---

通过根施用进行无选择饲喂试验[1]
在24和48小时与100μg/mL的马钱子碱D/Bruceine D (BD)或印楝素孵育后，评估了对DBM三龄幼虫的拒食活性（图2A）。在根上施用马钱子碱D后，大白菜叶片表现出优异的拒食活性，分别减少了93.80%和96.83%的消耗。与马钱子碱D相比，印楝素通过这种应用是无效的，在治疗24小时后没有显示出进食抑制作用，在治疗48小时后的拒食率为22.60%。然后，通过HPLC-HRESIMS定量叶片中马钱子碱D和印楝素的含量（图2C和D）。马钱子碱D和印楝素都具有显著的系统行为，并且随着时间的推移不断转移到叶片中（图2B）。根系施用24和48小时后，马钱子碱D的浓度分别为38.69μg/g（鲜重，FW）和108.45μg/g（FW），显著高于印楝素21.43μg/g（FWs）和36.88μg/g（FWD）的浓度（p<0.05）。显然，在48小时内，马钱子碱D比印楝素表现出更好的全身特性。叶片中马钱子素D的剂量足以防止DBM的摄食行为，而印楝素对摄入量的影响很小。

---

通过叶面施用进行无选择饲喂试验[1]
Bruceine D（BD）显示出强烈的拒食作用。为了评估对DBM三龄幼虫的拒食活性，使用2.5至160μg/mL的一系列浓度覆盖开花的大白菜叶盘，并使用印楝素作为阳性对照，不同浓度为20、40、80、160、320、640μg/mL。饲喂24小时后，这两种化合物表现出剂量依赖的拒食活动行为。马钱子碱D和印楝素的AFC50值分别为0.11和0.68μg/cm2，这表明在我们的实验条件下，马钱子素D的拒食活性比印楝素强约6.2倍。

---

Bruceine D（BD）在体外抑制HCC细胞活力并诱导细胞凋亡[2]
为了鉴定HCC的治疗药物，我们试图在HEK293T细胞中发现一种Notch抑制剂，该细胞含有基于萤光素酶的RBP-Jκ-Luc报告子，该报告子具有ICN（Notch的细胞内结构域）驱动的萤光素酶，以响应Notch信号活性（图S1A）。在1600种天然产物的筛选中，BD对RBP-Jκ-Luc活性的抑制作用最强（图S1B）。为了确定BD是否是HCC抑制剂，将Huh7和Hep3B细胞用不同浓度的BD处理48小时。CCK8检测显示，这两种细胞类型的增殖都受到强烈的剂量和时间依赖性抑制，并且HCC细胞的敏感性高于人肝细胞L-02（图S2A，B）。与对细胞存活的影响类似，BD还以浓度依赖性方式抑制Huh7和Hep3B的癌症细胞集落形成（图S2C）。此外，与对照细胞相比，BD诱导Huh7和Hep3B细胞凋亡的剂量依赖性增加，尤其是早期凋亡（图S2D）。这些结果证实，BD对Notch信号的抑制是通过抑制HCC细胞的增殖和诱导凋亡来实现的。

---

Bruceine D (BD)抑制HCC细胞中Jagged1的蛋白质合成[2]
为了了解BD抑制Notch信号传导的潜在机制，我们接下来确定了BD对Huh7和Hep3B细胞系中Notch信号相关蛋白的影响。值得注意的是，虽然我们观察到Notch 1、2或3没有变化，但Notch配体Jagged1、切割的Notch受体NICD和Notch特异性下游蛋白Hes1都明显受到BD的下调（图4A）。

---

Jagged1是β-catenin靶基因，是Bruceine D (BD)介导的HCC增殖抑制所必需的 Jagged1的抑制与恶性HCC中肿瘤负荷的减少有关。为了说明Jagged1与BD作用的关系，我们在HCC细胞中进行了Jagged1的功能获得和丧失测定（图5A）。台盼蓝排斥试验表明，尽管BD具有细胞毒性作用，但含有丰富Jagged1的Huh7细胞仍显示出增殖能力（图5B）。与BD抑制的效果一致，与对照组相比，Jagged1的敲除导致细胞存活率降低和凋亡增加（图5B）。此外，接受BD联合shJagged1治疗的组与单独接受shJagged2治疗的组没有差异。这些结果表明，BD的作用需要Jagged1。

---

Bruceine D（BD）诱导β-catenin的蛋白酶体依赖性降解[2]
我们进一步推测BD是否通过Wnt通路下调Jagged1的表达。为了研究这一假设，我们测试了BD对HCC细胞中β-catenin/Tcf4转录复合物的影响。Huh7细胞与含有3个串联Tcf共有结合位点（TOP）或突变Tcf结合位点（FOP）的萤光素酶报告基因和作为正常转染对照的Renilla萤光素酶报道基因共转染。根据我们的假设，BD以剂量依赖的方式有效地抑制了TOP萤光素酶的活性，但没有抑制FOP萤光素酶（图6A）。

在鸦胆子中鉴定出一种类Bruceine D (BD)。对小菜蛾（DBM，Plutella xylostella L.）、甜菜夜蛾（Spodoptera exigua Hübner）和棉花叶虫（Spodopera litura Fabricius）表现出优异的系统性和拒食活性。其对DBM三龄幼虫的拒食作用比印楝素强约6.2倍。当在根部以100μg/mL的浓度施用Bruceine D 24和48小时时，其在开花大白菜（Brassica campestris L.ssp.chinensis var.utiliz Tsen et Lee）叶片中的浓度分别为38.69μg/g（鲜重，FW）和108.45μg/g（FW）。这些浓度可以达到93.80%和96.83%的拒食效果，明显高于印楝素。与印楝素相似，Bruceine D对昆虫幼虫也有强烈的生长抑制作用。在喂食20μg/g的Bruceine D后，没有观察到蛹。结果表明，Bruceine D是一种有效的植物性昆虫拒食剂，具有出色的系统性，具有强大的害虫生长抑制活性[1]。

---

本研究旨在确定鸦胆子干果的驱虫活性，并分离和表征其活性成分。从B.javanica果实中提取的甲醇提取物对Dactylogyrus intermedius显示出显著的驱虫活性（EC（50）（中效浓度）值=49.96 mg l（-1））。基于这一发现，甲醇提取物在硅胶柱色谱法上进行生物测定指导的分级，得到两种已知的具有强效活性的奎西诺，即Bruceine a和Bruceine D (BD)。Bruceinea和D对中间型D.表现出显著的活性，EC（50）值分别为0.49 mg l（-1）和0.57 mg l（-1），比阳性对照甲苯咪唑更有效（EC（50”值=1.25 mg l（-1-））。此外，BruceineA和D对宿主（鲫鱼）的48小时中位致死浓度（LC（50））比中间D.的EC（50）高10.6倍和9.7倍。这些结果提供了证据，表明分离的化合物可能是控制Dactylogyrus的新型抗寄生虫药物的潜在来源。这是首次报道爪哇小蠊对间日疟原虫的体内驱虫研究。

肝细胞癌（HCC）以高死亡率和有限的治疗方法而闻名。Wnt和Notch信号通路的异常激活对肝癌的发生和进展至关重要。在这里，我们使用RBP-Jκ依赖的萤光素酶报告系统鉴定了一种小分子，即马钱子碱D（BD），作为Notch抑制剂。BD显著抑制了肝肿瘤的生长，并增强了索拉非尼在各种小鼠HCC模型中的治疗效果。从机制上讲，BD促进β-catenin的蛋白酶体降解及其核积累的耗竭，这反过来又破坏了HCC中Notch配体Jagged1的Wnt/β-catenin-dependent转录。我们的研究结果提供了关于一种新型Wnt/Notch串扰抑制剂的重要信息，该抑制剂与索拉非尼协同治疗HCC，因此具有很高的临床影响[2]。

细胞活力测定[2]
将细胞在96孔板中培养过夜（3000个细胞/孔），然后用指定浓度的Bruceine D（BD）或索拉非尼处理24、48或72小时。然后，在每个孔上用10μl细胞计数试剂盒-8（CCK8）处理细胞1小时。在450nm处测量光密度，并将其归一化为无细胞培养基的背景吸光度。所有样品均进行了三次化验。

---

软琼脂集落形成试验和克隆形成试验[2]
将6孔板中的细胞（1000/孔）在含有10%FBS的1 ml 0.33%基础培养基鹰琼脂或含有10%FBS、浓度为3.5 ml 0.5%基础培养基的3.5 ml鹰琼脂中与或不与Bruceine D（BD）（0-1μM）一起孵育。在5%CO2的培养箱中，将培养物在37°C下保持10-14天。使用Image Pro Plus软件程序在显微镜下计数细胞集落。

---

流式细胞术分析细胞凋亡 用Annexin V-FITC凋亡检测试剂盒检测细胞凋亡。在用膜联蛋白V-FITC和碘化丙啶（PI）染色之前，用马钱子碱D（BD）或索拉非尼单独或联合处理细胞24小时。在室温下在黑暗中孵育5分钟后，通过流式细胞术分析细胞凋亡。

---

台盼蓝排斥试验[2]
Huh7细胞在6孔板中培养过夜，与或不与Bruceine D（BD）一起孵育24小时。根据推荐的方案用台盼蓝染料对细胞进行染色。

---

免疫细胞化学（ICC）和免疫组织化学（IHC）[2]
细胞被放置在35毫米的玻璃板上，并在用Bruceine D（BD）（0.5-1μM）处理24小时之前生长过夜。DMSO用作未处理的对照。将第一代β-catenin抗体稀释1:100，并在4°C下孵育过夜。然后，将1:200稀释的Alexa Fluor 594标记的山羊抗兔IgG二抗加入固定的Huh7细胞中。然后将带有细胞的盖玻片安装在含有DAPI染色（CST）的Vectashield安装介质的载玻片上。荧光成像由LAS AF Lite软件和Leica TCS SP8 X显微镜捕获

---

细胞转染[2]
使用X-tremeGENE HP DNA转染试剂在Opti-MEM培养基中用萤光素酶报告质粒和pcDNA4/TO-N100-GV瞬时转染T-REx-HeLa细胞。转染24小时后，在受试化合物存在下用1μg/ml四环素处理细胞。孵育24小时后，将细胞在Glo裂解缓冲液中裂解5分钟，并通过在96孔Nunc MaxiSorp平板中混合裂解物和Bright Glo萤光素酶测定试剂来测量萤光素酶活性。在室温下孵育5分钟后，在微孔板读数器中监测单个孔中的发光。
在Opti-MEM培养基中，用Notch1-NEXT质粒或pcDNA3质粒（对照）以及2μl X-tremeGENE HP DNA转染试剂转染HEK293细胞。转染24小时后，将HEK293细胞与DAPT（GSI-IX）或Bruceine D（BD）一起孵育24小时，提取全细胞进行蛋白质印迹分析。
根据制造商的建议，用shCTNNB1和shJAG1瞬时转染Huh7细胞，并用lipo2000转染。转染24小时后，用DMSO或Bruceine D（BD）处理细胞24小时，提取全细胞进行蛋白质印迹。

---

萤光素酶报告物测定[2]
简而言之，将细胞接种到96孔板中，并与pTOPflash或pFOPflash载体以及肾荧光素酶报告载体pRL-TK共转染，作为转染效率的对照。转染12小时后，将细胞与Bruceine D（BD）一起孵育24小时。根据推荐的方案测量萤光素酶和肾菌的活性并使其标准化。为了进行药物筛选，将含有RBP-JκReporter的HEK293细胞系接种到96孔板中。孵育过夜后，将1600种10μM的天然产物加入每个孔中24小时，使用Promega E1500试剂盒检测萤光素酶活性。

Insect Growth Regulatory Assay[1]
Bruceine D was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5000 μg/mL and then added to the DBM larvae diet at final concentrations of 2.5, 5, 10, and 20 μg/g. Freshly molted third instar larvae (n = 100 per group) were placed in insect rearing containers. Sufficient food was supplied after starvation for 3 h. Larvae fed diets with DMSO were used as controls. The weight of larval body and diet consumption were measured after treatment with 10, 20 μg/g bruceine D diets at 6, 12, 24, 48 h. The number of individuals who underwent pupation and eclosion was counted at 12 h intervals for the treatment and control groups. Simultaneously, the development processes and phenotypes of the larvae were observed.

---

In vivo anthelmintic efficacy assay [3]
The anthelmintic activity of fractions and the pure compounds such as Bruceine D (BD) was determined against D. intermedius. Tests were conducted in glass tank of 5-l capacity, filled with 2 l aerated groundwater, each containing the test samples and five previously infected fish. The water pH ranged from 7.0 to 7.5, dissolved oxygen between 6.2 and 7.8 mg l−1 (72–85% saturation) and the water temperature was constant at 24 ± 1 °C. Initial tests were conducted to get moderate concentration boundaries to avoid the mortality of fish at high concentrations. The derived fractions and pure compounds were dissolved in 1 ml dimethysulphoxide (DMSO) and made up to 100 ml with distilled water, which were used for the preparation of the different concentrations of the test solutions. The methanol extract and isolated fractions were conducted at a different series of concentrations based on the initial tests, respectively. All the experiments were repeated independently in duplicate, except for the pure compounds 1 and 2, which were conducted in triplicate, following the same protocol for the B. javanica fractions, using five concentrations, 0.5, 1.0, 1.5, 2.0 and 2.5 mg l−1. To discard the possible effects of DMSO on the parasites, a negative control was included using distilled water containing the corresponding percentage of DMSO as the test sample; Mebendazole was used as a positive control with a different series of concentrations of 0.6, 1.0, 1.5, 2.0 and 2.5 mg l−1. After 48 h, all the surviving goldfish in all the treatment and control groups were euthanised by a spinal severance for necropsy. The lamella branchialis were placed on glass slides, and the numbers of parasites on the gills were counted under a light microscope at 4 × 10 magnification to determine the mean number of parasites per infected goldfish.

---

Acute toxicity assay [3]
Acute toxicity of the pure compounds (1 and 2)/Bruceine D (BD) was assayed using the healthy goldfish. The tests were conducted in duplicate using 10 individuals in a tank of 5 l capacity, containing 2-l test solutions at 24 ± 1 °C during the experiment. The pure compounds were conducted at a different series of concentrations based on the initial tests. Control groups were set under the same test conditions without chemicals. The death of fish was recorded when there was no opercula movement and the goldfish no longer responded to mechanical stimulus (touch by glass rod). Any observed dead fish was removed from the medium in time to avoid deterioration of the water quality. After a 48-h exposure, fish mortalities in the treatment and control groups were recorded.

---

Subcutaneous HCC xenograft model study [2]
Four-week-old male Balb/c nude mice were housed and maintained under specific pathogen-free conditions. For in vivo studies, 5 × 106 Huh7 cells in 100 μl PBS were injected subcutaneously into the right flanks of the mice. Based on the data from a pilot study, the mice were randomly divided into 3 groups (n = 6 per group) and were treated with saline with 0.9% sodium chloride (vehicle) or Bruceine D (BD) at doses of 0.75 mg/kg and 1.5 mg/kg body weight by tail vein injection once daily for 10 consecutive days. Tumor volume was calculated according the formula: tumor volume (mm3) = 1/2 × (tumor length) × (tumor width)

[1]. Bruceine D Isolated from Brucea Javanica (L.) Merr. as a Systemic Feeding Deterrent for Three Major Lepidopteran Pests. J Agric Food Chem. 2019 Apr 17;67(15):4232-4239.
[2]. Bruceine D inhibits hepatocellular carcinoma growth by targeting β-catenin/jagged1 pathways. Cancer Lett. 2017 Sep 10;403:195-205.
[3]. In vivo anthelmintic activity of bruceine A and bruceine D from Brucea javanica against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus). Vet Parasitol. 2011 Apr 19;177(1-2):127-33.

Bruceine D is a quassinoid that is 13,20-epoxypicras-3-ene substituted by hydroxy groups at positions 1, 11, 12, 14 and 15 and oxo groups at positions 2 and 16. Isolated from the ethanol extract of the stem of Brucea mollis, it exhibits cytotoxic activity. It has a role as a metabolite, an antineoplastic agent and a plant metabolite. It is a delta-lactone, a pentol, a quassinoid, an organic heteropentacyclic compound and a secondary alpha-hydroxy ketone. It derives from a hydride of a picrasane.
Bruceine D has been reported in Brucea mollis, Samadera indica, and Brucea javanica with data available.

---

Systemicity is a desirable property for insecticides. Many phytochemicals show good systemic properties and thus are natural sources of novel systemic insecticidal ingredients. Bruceine D, a quassinoid, was identified in Brucea javanica (L.) Merr. and displayed outstanding systemic properties and excellent antifeedant activity against the diamondback moth (DBM, Plutella xylostella L.), beet armyworm ( Spodoptera exigua Hübner), and cotton leafworm ( Spodoptera litura Fabricius). Its antifeedant effect on third instar larvae of DBM was approximately 6.2-fold stronger than that of azadirachtin. When bruceine D was applied to roots at a concentration of 100 μg/mL for 24 and 48 h, its concentration in flowering Chinese cabbage ( Brassica campestris L. ssp. chinensis var. utiliz Tsen et Lee) leaves was 38.69 μg/g (fresh weight, FW) and 108.45 μg/g (FW), respectively. These concentrations could achieve 93.80% and 96.83% antifeedant effects, which were significantly greater than those of azadirachtin. Similar to azadirachtin, bruceine D also posed a potent growth inhibition effect on insect larvae. After feeding with 20 μg/g bruceine D, no pupae were observed. The results demonstrated that bruceine D is an effective botanical insect antifeedant with outstanding systemic properties, causing potent pest growth inhibitory activity. [1]

---

Hepatocellular carcinoma (HCC) is known for high mortality and limited available treatments. Aberrant activation of the Wnt and Notch signaling pathways is critical to liver carcinogenesis and progression. Here, we identified a small molecule, bruceine D (BD), as a Notch inhibitor, using an RBP-Jκ-dependent luciferase-reporter system. BD significantly inhibited liver tumor growth and enhanced the therapeutic effects of sorafenib in various murine HCC models. Mechanistically, BD promotes proteasomal degradation of β-catenin and the depletion of its nuclear accumulation, which in turn disrupts the Wnt/β-catenin-dependent transcription of the Notch ligand Jagged1 in HCC. Our findings provide important information about a novel Wnt/Notch crosstalk inhibitor that is synergistic with sorafenib for treatment of HCC, and therefore have high clinical impact. [2]

---

The present study was designated to ascertain the anthelmintic activity of the dried fruits of Brucea javanica and to isolate and characterise the active constituents. The methanol extract from the fruits of B. javanica showed significant anthelmintic activity against Dactylogyrus intermedius (EC(50) (median effective concentration) value=49.96 mg l(-1)). Based on this finding, the methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording two known quassinoids showing potent activity, bruceine A and bruceine D. Both bruceine A and D exhibited significant activity against D. intermedius with EC(50) values of 0.49 mg l(-1) and 0.57 mg l(-1), respectively, which were more effective than the positive control, mebendazole (EC(50) value=1.25 mg l(-1)). In addition, the 48-h median lethal concentration (LC(50)) for bruceine A and D against the host (Carassius auratus) was 10.6-fold and 9.7-fold higher than the EC(50) for D. intermedius. These results provide evidence that the isolated compounds might be potential sources of new anti-parasitic drugs for the control of Dactylogyrus. This is the first report on an in vivo anthelmintic investigation for B. javanica against D. intermedius. [3]

C20H26O9

410.4150

410.157

C, 58.53; H, 6.39; O, 35.08

21499-66-1

21499-66-1

122784

White to off-white solid powder

1.6±0.1 g/cm3

661.3±55.0 °C at 760 mmHg

235.8±25.0 °C

0.0±4.5 mmHg at 25°C

1.670

-0.5

153.75

5

9

0

29

855

11

O1C([H])([H])[C@@]23C4([H])C([H])([H])C5([H])C(C([H])([H])[H])=C([H])C(C([H])([C@]5(C([H])([H])[H])C2([H])C([H])(C([H])([C@]1(C([H])([H])[H])[C@@]3(C([H])(C(=O)O4)O[H])O[H])O[H])O[H])O[H])=O

JBDMZGKDLMGOFR-KQSRGDCESA-N

InChI=1S/C20H26O9/c1-7-4-9(21)13(23)17(2)8(7)5-10-19-6-28-18(3,14(24)11(22)12(17)19)20(19,27)15(25)16(26)29-10/h4,8,10-15,22-25,27H,5-6H2,1-3H3/t8-,10+,11+,12+,13+,14-,15-,17-,18+,19+,20+/m0/s1

(1R,2R,3R,6R,8S,12S,13S,14R,15R,16S,17R)-2,3,12,15,16-pentahydroxy-9,13,17-trimethyl-5,18-dioxapentacyclo[12.5.0.01,6.02,17.08,13]nonadec-9-ene-4,11-dione

Bruceine-D; Bruceine D; 21499-66-1; Brucein D; CHEBI:68931; C08752; (1R,2R,3R,6R,8S,12S,13S,14R,15R,16S,17R)-2,3,12,15,16-Pentahydroxy-9,13,17-trimethyl-5,18-dioxapentacyclo[12.5.0.01,6.02,17.08,13]nonadec-9-ene-4,11-dione; (1R,2S,3R,3aR,3a1R,4R,6aR,7aS,11S,11aS,11bR)-1,2,3a,4,11-pentahydroxy-3,8,11a-trimethyl-1,2,3,3a,4,7,7a,11,11a,11b-decahydro-5H-3,3a1-(epoxymethano)dibenzo[de,g]chromene-5,10(6aH)-dione; AC1L9BNP; Bruceine D

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 82~100 mg/mL (199.8~243.7 mM)
H2O: ~5 mg/mL (~12.2 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.09 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (6.09 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (6.09 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。