10-Deacetylbaccatin-III (10-DAB)   中文名称: 10-脱乙酰巴卡亭 Ⅲ

Intermediate for synthesis of paclitaxel

尽管在转录水平上对紫杉醇生物合成的调控尚不清楚，但10-脱乙酰基浆果赤霉素III-10β-O-乙酰基转移酶（DBAT）是紫杉醇生物合成中的关键酶。从红豆杉细胞中克隆了dbat基因的1740 bp片段5'侧翼序列。通过缺失分析，在中华鳖细胞中定位了dbat启动子活性所需的重要调控元件。以重要调控元件为诱饵，利用酵母单杂交系统从中华鳖细胞cDNA文库中分离出一种新的WRKY转录因子TcWRKY1。茉莉酸甲酯（MeJA）特异性诱导TcWRKY1在中华鳖悬浮细胞中的基因表达。生化分析表明，TcWRKY1蛋白与重要调控元件中的两个W-box（TGAC）顺式元件特异性相互作用。TcWRKY1的过表达增强了中华绒螯蟹悬浮细胞中dbat的表达，RNA干扰（RNAi）降低了dbat转录物的水平。这些结果表明，TcWRKY1参与了中国红豆杉细胞紫杉醇生物合成的调控，dbat是该转录因子的靶基因。这项研究还为工程增加紫杉醇在红豆杉细胞培养中的积累提供了一个潜在的候选基因。[2]

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紫杉醇和其他紫杉烷存在时的吸收[2]
为了确定其他紫杉烷对Flutax-2®摄取的影响，使用了聚集的悬浮细胞培养物。悬浮细胞培养物与Flutax-2®在其他紫杉烷（紫杉醇、浆果赤霉素III、头孢甘露碱和10-脱乙酰紫杉醇）存在下孵育。这些紫杉烷在结构上与紫杉醇相似，但有几个不同之处：浆果赤霉素III缺乏苯丙氨酸衍生的侧链；头孢甘露碱在侧链的第三个碳位置具有直链碳链，而不是苯基；10-脱乙酰基紫杉醇在C-10位缺少乙酰基（图3）。只有紫杉醇被发现显著（p<0.05）抑制Flutax-2®的细胞摄取（图4A）。这些结果清楚地表明了紫杉醇摄取的特异性。此外，Flutax-2®的抑制作用与存在的未标记紫杉醇的量呈线性关系（图4B）。在Flutax-2®的恒定浓度下，紫杉醇的加入能够竞争性地抑制荧光标记配体的细胞结合。竞争性抑制表明这两种化合物是通过特定的机制运输的。

醋酸诱导的扭体反应[3]
所有化合物[例如10-脱乙酰基浆果赤霉素III（10-DAB）]均显示出显著的镇痛活性，尤其是与仅用生理盐水治疗的组相比，在40mg/kg剂量下，tasumatrol B将乙酸诱导的扭动减少了72.23%（表1）。

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热板试验[3]
根据热板试验的结果，用紫杉类治疗的动物和用生理盐水治疗的动物之间没有统计学上的显著差异（表2）。

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卡拉胶诱导的水肿[3]
分离的化合物[例如10-Deacetylbaccatin-III (10-DAB)/10-脱乙酰基浆果赤霉素III（10-DAB）]显著减少了卡拉胶引起的水肿。然而，发现tasumatrol B是最具活性的化合物，在20和40mg/kg的试验剂量下显示出显著（p<0.05）和高度显著（p<0.01）的活性。

体外脂肪氧合酶抑制试验[3]
通过使用不同浓度的分离化合物Sieboldogenin进行酶抑制试验。通过稍微修改Tappel（1962）开发的光谱法来测量脂氧合酶抑制活性。使用I-B型脂肪氧合酶（EC 1.13.11.12）（大豆）和亚油酸，无需进一步纯化。160μL磷酸钠缓冲液，0.1毫米（pH 7.0），将10 mL样品溶液（试验化合物）和20μL脂肪氧合酶溶液混合，在258°C下孵育5分钟。通过加入10μL亚油酸底物溶液引发反应，并在10分钟内观察吸收随（9Z，11E）-13S）-13-氢过氧化十八碳-9,11-二烯酸酯的形成而变化。将测试样品和对照品溶解在50%乙醇中。所有反应均进行三次。黄芩素被用作脂肪氧化酶抑制的阳性对照（Khan等人，2009）。使用EZFit酶动力学程序计算抑制浓度（IC50）值。

Acetic acid-induced writhing [3]
The acetic acid abdominal constriction test (Koster et al., 1959) was used with modification according to Nisar et al. (2008b). Mice were divided into various groups of five mice each and starved for 18 h. The negative control group received saline 10 mL/kg, p.o., while test groups received various doses of the test compounds via oral route. Meanwhile, the positive control group received acetylsalicylic acid (ASA); 100 mg/kg, p.o. After half an hour, all mice received a 0.7% aqueous solution of acetic acid 10 mg/kg, i.p. and writhings were counted for 10 min after acetic acid injection.

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Hot-plate test [3]
A hot-plate latency test was performed according to the method described by Zhang et al. (2009). Animals were habituated twice to the hot-plate in advance. For testing, mice were placed on a hot-plate maintained at 55 ± 0.5°C. The time that elapsed until the incidence of either a hind paw licking or a jump off the surface was recorded as the hot-plate latency. Mice with baseline latencies of < 5 s or > 30 s were eliminated from the study. After the determination of baseline response latencies, hot-plate latencies were redetermined at 30, 60 and 120 min after oral administration of test drugs (aspirin as the reference drug).

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Carrageenan-induced oedema [3]
The method of Winter et al. (1962) was utilized to assess the antiinflammatory potential of the test sample via testing its ability to inhibit the carrageenan-induced hind paw oedema, as reported earlier (Khan et al., 2009). Test samples and the control samples were administered orally in groups to rats. After 1 hour, acute inflammation at the desired site was induced by subplantar injection of 1% suspension of carrageenan (0.1 mL) using 2% gum acacia as a suspending agent in normal saline, in the right hind paw of the rats. The paw volume was measured plethysmometrically (Ugo Basile, Italy) at ‘0’ and 3 h after the carrageenan injection. Indomethacin 5 mg/kg, p.o. suspended in 2% gum acacia was used as the positive control. Percentage inhibition of the inflammation was determined by applying statistics on raw data followed by the calculation of percentage inhibition for each group by comparing with the control group. The formula used for comparison was: %I = 1 − (dt/dc) × 100, where dt is the difference in paw volume in the drug-treated group, dc is the difference in paw volume in control group and I stands for inhibition of inflammation.

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Cotton-pellet oedema model [3]
In order to evaluate the antiinflammatory effect of the isolated compounds on the cotton-pellet oedema model, male rats were divided into four groups of five animals. To each animal, two cotton wool pellets weighing 18 ± 1 mg were implanted subcutaneously, one on each side of the abdomen. Animals were kept under light ether anesthesia according to the method reported by Swingle and Shideman (1972). Each compound was administered once daily throughout the experimental period of 7 days. On the day 8 after implantation, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). After induction of anesthesia, pellets were dissected, dried at 55°C for 15 h, and weighed after cooling. The mean granuloma weights as well as the percentage granuloma inhibition of the test compounds were calculated.

Acute toxicity [3]
All the compounds [e.g. 10-Deacetylbaccatin-III (10-DAB)] were found safe after 48 h of administration. Statistically, no considerable difference was observed between the negative control and other treatment groups both in terms of mortality and morbidity.

[1]. Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis. Plant Biol (Stuttg). 2013 Jan;15(1):19-26.

[2]. Paclitaxel uptake and transport in Taxus cell suspension cultures. Biochem Eng J. 2012 Apr 15;63:50-56.

[3]. Analgesic and antiinflammatory activities of taxoids from Taxus wallichiana Zucc. Phytother Res. 2012 Apr;26(4):552-6.

[4]. Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli. PNAS, 2000 , 97(25)：13591-13596.

10-deacetylbaccatin III is a tetracyclic diterpenoid and a secondary alpha-hydroxy ketone. It is functionally related to a baccatin III.
10-Deacetylbaccatin III has been reported in Taxus sumatrana, Taxus cuspidata, and other organisms with data available.

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The transport of paclitaxel in Taxus canadensis suspension cultures was studied with a fluorescence analogue of paclitaxel (Flutax-2(®)) in combination with flow cytometry detection. Experiments were carried out using both isolated protoplasts and aggregated suspension cell cultures. Flutax-2(®) was shown to be greater than 90% stable in Taxus suspension cultures over the required incubation time (24 hours). Unlabeled paclitaxel was shown to inhibit the cellular uptake of Flutax-2(®), although structurally similar taxanes such as cephalomannine, baccatin III, and 10-deacetylbaccatin III did not inhibit Flutax-2(®) uptake. Saturation kinetics of Flutax-2(®) uptake was demonstrated. These results indicate the presence of a specific transport system for paclitaxel. Suspension cells elicited with methyl jasmonate accumulated 60% more Flutax-2(®) than unelicited cells, possibly due to an increased cellular storage capacity following methyl jasmonate elicitation. The presence of a specific mechanism for paclitaxel transport is an important first result that will provide the basis of more detailed studies as well as the development of targeted strategies for increased paclitaxel secretion to the extracellular medium.[2]

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A study was conducted to identify constituents that might be responsible for analgesic and antiinflammatory conditions. Tasumatrol B, 1,13-diacetyl-10-deacetylbaccatin III (10-DAD) and 4-deacetylbaccatin III (4-DAB) were isolated from the bark extract of Taxus wallichiana Zucc. All the compounds were assessed for analgesic and antiinflammatory activities using an acetic acid-induced writhing model, a hot-plate test, a carrageenan-induced paw oedema model, a cotton-pellet oedema model and in vitro lipoxygenase inhibitory assay. All the compounds, especially tasumatrol B, revealed significant analgesic activity in comparison to a saline group based on an acetic acid-induced model. Similarly all of the test compounds, particularly tasumatrol B, showed significant antiinflammatory activity. However, all the compounds failed to exhibit any considerable activity in of the hot-plate test and the in vitro lipoxygenase inhibitory assay. This study has highlighted the potential of tasumatrol B to be further explored as a new lead compound for the management of pain and inflammation, one that has been discovered by scientific validation of the traditional medicinal use of T. wallichiana Zucc.[3]

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A cDNA clone encoding a taxane 2α-O-benzoyltransferase has been isolated from Taxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Escherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC, 1H-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0, Km values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2α-hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl groups at the C2 position.[4]

C29H36O10

544.59

544.23

C, 63.96; H, 6.66; O, 29.38

32981-86-5

154272

White to off-white solid powder

1.4±0.1 g/cm3

716.8±60.0 °C at 760 mmHg

231-236 °C

233.5±26.4 °C

0.0±2.4 mmHg at 25°C

1.624

3.51

159.82

4

10

5

39

1090

9

CC1=C2[C@H](C(=O)[C@@]3([C@H](C[C@@H]4[C@]([C@H]3[C@@H]([C@@](C2(C)C)(C[C@@H]1O)O)OC(=O)C5=CC=CC=C5)(CO4)OC(=O)C)O)C)O

YWLXLRUDGLRYDR-ZHPRIASZSA-N

InChI=1S/C29H36O10/c1-14-17(31)12-29(36)24(38-25(35)16-9-7-6-8-10-16)22-27(5,23(34)21(33)20(14)26(29,3)4)18(32)11-19-28(22,13-37-19)39-15(2)30/h6-10,17-19,21-22,24,31-33,36H,11-13H2,1-5H3/t17-,18-,19+,21+,22-,24-,27+,28-,29+/m0/s1

(2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-acetoxy-4,6,9,11-tetrahydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.59 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。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.59 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。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.59 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。

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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。