Geissoschizine methyl ether   中文名称: 缝籽嗪甲醚

5-HT_1A Receptor

Geissoschizine methyl ether与5-HT1A受体的激动性结合[2]
图6显示了七种UH衍生生物碱对[3H]8-OH-DPAT与5-HT1A受体结合的体外竞争结合试验结果<与其他六种生物碱相比，GM（0.01-100μM）以浓度依赖的方式强烈抑制[3H]8-OH-DPAT与5-HT1A受体的结合。GM的50%抑制浓度（IC50）和抑制常数（Ki）分别估计为0.904μM和0.517μM。

为了阐明Geissoschizine methyl ether/GM对5-HT1A受体[35S]GTPγS的激动作用，进行了体外结合试验（图7）。GM（0.1-100μM）或5-HT（1-300 nM）以浓度依赖的方式增加[35S]GTPγS结合。然而，GM的结合率约为5-HT的40%。

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YKS中的GM/Geissoschizine methyl ether浓度[2]
表1显示了YKS中含有的七种UH衍生生物碱的浓度。转基因约占YKS提取物的0.014%。因此，可以确认1.0 g YKS中含有约140μg GM。

GM/Geissoschizine methyl ether对社交孤立小鼠攻击行为增加和社交行为减少的影响[2]
单次给予转基因（75-300μg/kg）并没有改变增加的攻击行为（图8A），而转基因在300μg/kg时显著改善了减少的社会行为（P<0.01）（图8B）。

连续14天重复服用GM/Geissoschizine methyl ether显著降低了150μg/kg（P<0.01）和300μg/kg（P<0.01）的攻击行为（图9A），并增加了150μ/kg（P<0.01）、300μg/kg的社会行为（P<0.01）（图9B）。连续14天给予GM（300μg/kg）的这些改善作用在GM给药后第14天30分钟单次给予WAY-100635（0.1mg/kg）时被抵消（P<0.01）（图9A，B），YKS（图3）和UH（图5）也是如此。

WAY-100635联合给药对反复给药盖氏嗪甲醚/GM[2]
改善攻击行为和社会行为的影响 为了研究重复给药WAY-100635对重复给药GM/Geissoschizine methyl ether改善相互作用行为的影响，口服GM（300μg/kg）15天，WAY-100638（0.1mg/kg）与GM联合给药14天（图10）。在第15天的社交互动测试中，连续15天给予GM对攻击行为（图10A）和社交行为（图10B）的改善作用被连续14天给予WAY-100635抵消（P<0.05）。

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通过反复服用Geissoschizine methyl ether/GM[2]
，维持攻击行为和社会行为的改善效果 对离体小鼠口服GM/Geissoschizine methyl ether（300μg/kg）14天，然后在第15天进行社交互动测试（图11）。在第15天，也就是在行为测试日未服用GM的情况下，观察到对攻击行为（P<0.05）和社会行为（P<0.01）的改善作用（图11A，B）。

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单独使用WAY-100635对社交孤立小鼠攻击行为增加和社交行为减少的影响[2]
如图3、图5、图9、图10所示，WAY-100635与YKS、UH或Geissoschizine methyl ether/GM的联合给药抵消了这些受试物质对攻击性和社会行为的改善作用。在这里，我们研究了单独使用WAY-100635对隔离诱导的攻击性和社交行为的影响（图12）。单次（0.1和1.0 mg/kg）或重复（0.1 mg/kg）服用WAY-100635 14天不会影响隔离小鼠的攻击性（图12A，D）和社交行为（图12B，E）。所有组的运动活动也没有显著差异（图12C，F）。

5-HT1A受体的竞争性结合试验 [2]
通过将各种浓度的钩藤碱、异钩藤碱，Corynoxein，异Corynoxine，hirsuteine，hirsutine和Geissoschizine methyl ether/GM溶解在0.5%的二甲亚砜（DMSO）中制备。 使用先前描述的方法进行5-HT1A受体的竞争性结合测定（Terawaki等人，2010）。简而言之，将5.25μl试验化合物溶液或载体与500μl CHO-h5-HT1A细胞膜溶液（60-92μg蛋白质/mL）和20μl 40 nM[3H]8-OH-DPAT在pH 7.4的50 mM Tris-HCl缓冲液中，在25°C下孵育60分钟，该缓冲液含有0.1%抗坏血酸、0.5 mM EDTA和10 mM MgSO4。通过添加10μM甲草啉来测定非特异性结合。孵育后，使用细胞采集器通过Whatman GF/B过滤器快速过滤分离5-HT1A受体配体复合物。用3ml冰冷的50mM Tris-HCl缓冲液冲洗被捕获的放射性复合物四次并干燥。使用液体闪烁计数器测量干燥过滤器上捕获的放射性（每分钟计数，cpm）。 特异性结合是通过从总结合中减去非特异性结合来确定的，并使用以下公式表示为抑制百分比：抑制率（%）=[1-（c-a）/（b-a）]×100，其中“a”是非特异性结合的平均cpm，“b”是总结合的平均cpm，“c”是受试物质存在时的平均cpm。

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[35S]GTPγS与5-HT1A受体的结合分析 [2]
使用之前描述的方法检查了Geissoschizine methyl ether/GM对[35S]GTPγS与5-HT1A受体结合的影响（Terawaki等人，2010）。简而言之，将溶解在50%DMSO或载体中的0.42μlGeissoschizine methyl ether/GM溶液在30°C下与50μl CHO-h5-HT1A膜溶液（25-30μg蛋白质/mL）和25μl 10μM GDP在pH 7.4的20 mM HEPES缓冲液中预孵育20分钟，该缓冲液含有100 mM NaCl、10 mM MgCl2、1 mM DTT和1 mM EDTA，在加入小麦胚芽凝集素包被的闪烁邻近测定珠后，在相同温度下进一步孵育60分钟。通过向混合物中加入10μl 3.3 nM[35S]GTPγS引发结合反应。孵育30分钟后，使用液体闪烁计数器测量放射性。通过添加100μM GTPγS确定非特异性结合。将受试物质诱导的[35S]GTPγS结合率与300 nM 5-HT（5HT1A受体的完全激动剂）诱导的结合率进行比较。

Effects of a single administration of YKS, UH, and Geissoschizine methyl ether/GM on aggressive and social behaviors in socially isolated mice [2]
YKS (0.5 and 1.0 g/kg), UH (75 and 150 mg/kg), or Geissoschizine methyl ether/GM (75, 150, and 300 μg/kg) was orally administered in a single dose to the mice that had been isolated for 4 weeks. Distilled water (10.0 ml/kg) for the tests of YKS and UH, or 0.5% Tween-80 (10.0 ml/kg) for the test of GM was orally administered to the corresponding isolated control or group-housed mice by the same schedule. The social interaction test was performed 60 min after the drug administration.

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Effects of repeated administration of YKS, UH, and Geissoschizine methyl ether/GM on aggressive and social behaviors in socially isolated mice [2]
YKS (0.5 and 1.0 g/kg), YKS-UH (1.0 g/kg), UH (75 and 150 mg/kg), or Geissoschizine methyl ether/GM (150 and 300 μg/kg) was orally administered once a day for 14 days from the 4th week to 6th week to the isolated mice. Distilled water (10.0 ml/kg) for the tests of YKS and UH or 0.5% Tween-80 (10.0 ml/kg) for the test of GM was orally administered to the corresponding isolated control or group-housed mice by the same schedule. In this experiment, WAY-100635 (0.1 mg/kg) or saline (10.0 ml/kg) was administered i.p. once 30 min after administration of YKS (1.0 g/kg), UH (150 mg/kg), or GM (300 μg/kg) on the 14th day. The social interaction test was performed 60 min after the final administration of test substance on the 14th day.

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Coadministration effects of WAY-100635 on repeated administration effects of Geissoschizine methyl ether/GM [2]
GM/Geissoschizine methyl ether (300 μg/kg) was orally administered once a day for 15 days from the 4th to 6th week to the isolated mice, and WAY-100635 (0.1 mg/kg) or saline (10.0 ml/kg) was i.p. coadministered with GM once a day for 14 days; WAY-100635 was not administered on the 15th day. On the social interaction test day (the 15th day), GM was administered 60 min before the behavioral test.

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On sustention of repeated administration effects of Geissoschizine methyl ether/GM [2]
GM/Geissoschizine methyl ether (300 μg/kg) or 0.5% Tween-80 (10.0 ml/kg) was orally administered once a day for 14 days from the 4th to 6th week to the isolated mice, and then the social interaction test was performed on the 15th day; GM was not administered on the 15th day.

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Effects of WAY-100635 alone on increased aggressive behavior and decreased social behavior in socially isolated mice [2]
To evaluate single administration effect of WAY-100635 alone, WAY-100635 (0.1 and 1.0 mg/kg) or saline (10.0 ml/kg) was i.p. administered to mice isolated for 4 weeks. Social interaction test was performed 30 min after the administration of WAY-100635. To evaluate repeated administration effect of WAY-100635 alone, WAY-100635 (0.1 mg/kg) or saline (10.0 mg/kg) was i.p. administered once a day for 14 days from the 4th to 6th weeks to the isolated mice. On the 15th day of the next day, social interaction test was performed without administration of WAY-100635.

1. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. 2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. 3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes. 4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS. [1]

[1]. In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan. Xenobiotica. 2016;46(4):325-34.

[2]. Geissoschizine methyl ether, an alkaloid in Uncaria hook, is a potent serotonin ₁A receptor agonist and candidate for amelioration of aggressiveness and sociality by yokukansan. Neuroscience. 2012 Apr 5;207:124-36.

Geissoschizine methyl ether has been reported in Uncaria sinensis and Uncaria rhynchophylla with data available.

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okukansan (YKS), a traditional Japanese medicine, is composed of seven kinds of dried herbs. It is widely prescribed in clinical situation for treating psychiatric disorders such as aggressiveness in patients with dementia. We previously demonstrated that YKS and Uncaria hook (UH), which is a constituent herb of YKS, had a partial agonistic effect to 5-HT(1A) receptors in vitro. However, it has still been unclear whether this in vitro effect is reflected in in vivo, and what the active ingredients are. The purpose of the present study is to find the active ingredient in YKS and to demonstrate the effect in in vivo. In the present study, we first studied the effect of YKS and UH on aggressiveness and sociality in socially isolated mice. YKS and UH ameliorated the isolation-induced increased aggressiveness and decreased sociality, and these ameliorative effects were counteracted by coadministration of 5-HT(1A) receptor antagonist WAY-100635, or disappeared by eliminating UH from YKS. These results suggest that the effect of YKS is mainly attributed to UH, and the active ingredient is contained in UH. To find the candidate ingredients, we examined competitive binding assay and [(35)S] guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding assay of seven major alkaloids in UH using Chinese hamster ovary cells expressing 5-HT(1A) receptors artificially. Only Geissoschizine methyl ether(GM) among seven alkaloids potently bound to 5-HT(1A) receptors and acted as a partial agonist. This in vitro result on GM was further demonstrated in the socially isolated mice. As did YKS and UH, GM ameliorated the isolation-induced increased aggressiveness and decreased sociality, and the effect was counteracted by coadministration of WAY-100635. These lines of results suggest that GM in UH is potent 5-HT(1A) receptor agonist and a candidate for pharmacological effect of YKS on aggressiveness and sociality in socially isolated mice.[1]

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Therefore, we screened the ingredients in UH to determine the candidate binding to 5-HT1A receptors. That is, we examined the in vitro competitive binding of seven UH-derived alkaloids. The result of the binding assay showed that only GM/Geissoschizine methyl ether strongly bound to 5-HT1A receptors and that it acted as a partial agonist as well as YKS and UH (Fig. 6, Fig. 7). This result is supported by other in vitro results previously reported: several UH-derived alkaloids, such as GM, corynantheine, and dihydrocorynantheine, have been demonstrated to possess partial agonistic effects on 5-HT receptors in guinea-pig ileum, although the subtype of 5-HT receptors was not determined (Kanatani et al., 1985). Pengsuparp et al. (2001) also demonstrated that GM is a 5-HT1A receptor agonist by an in vitro binding assay. However, these in vitro results were still not verified in vivo, and no one has demonstrated the relative importance of GM in psychiatric disorder model. Thus, in this study, we further evaluated the effects of GM on aggressive and social behaviors.

As shown in Fig. 9, we newly demonstrated that repeated administration of GM (150 and 300 μg/kg) for 14 days ameliorated aggressiveness and sociality in a dose-dependent manner, as did YKS and UH (Fig. 3, Fig. 5). A liquid chromatographic assay indicated that the concentration of GM in YKS extract was 0.014%, that is, 1.0 g of YKS is considered to contain approximately 140 μg of GM (Table 1). Therefore, the dosage of 150 μg/kg of GM is almost the same as the amount included in YKS. These results suggest that the ameliorating effect of YKS is mainly attributed to UH-derived ingredient GM. In addition, the ameliorating effects by GM administration for 14 days were counteracted by single coadministration of a 5-HT1A receptor antagonist WAY-100635 as well as YKS and UH. These results also suggest that the ameliorating effects of aggressive and social behaviors by GM, at least, are mediated by 5-HT1A receptors. Although the ameliorating effects of GM as well as YKS and UH were observed by repeated administration, their effects were not observed by a single administration. These results suggest the possibility of neuroadaptation such as hypersensitivity or upregulation to the repeated administration effect. In the present study, the involvement of 5-HT1A receptors on the ameliorative effect by repeated administration was examined by using active compound GM, on behalf of YKS, UH, and GM. As shown in Fig. 10, the ameliorative effects of aggressive and social behaviors by GM administration for 15 days were counteracted by coadministrating WAY-100635 for 14 days. This result suggests that neuroadaptation against 5-HT1A receptors by repeated administration of GM is involved in the amelioration of aggressive and social behaviors. In addition, the ameliorating effect by repeated administration of GM for 14 days was observed even on the next day that GM was not administered, as shown in Fig. 11. Therefore, such sustained effect presumably attributed to neuroadaptation mainly contributed to the amelioration of aggressiveness by GM.

It has been demonstrated that GM has several inhibitory effects on spontaneous motor activity (Sakakibara et al., 1999), convulsion (Mimaki et al., 1997), and head-twitching behavior (Pengsuparp et al., 2001). However, these in vivo responses are induced by doses 100- or 1000-fold higher (several dozen mg to several hundred mg) than the amount contained in YKS. Here, we revealed that real dose of GM contained in YKS ameliorated aggressive and social behavior in socially isolated mice. This is a first finding that GM in YKS ameliorates aggressiveness and sociality. In addition, we recently demonstrated that GM was detected in the plasma and brain of rats after oral administration of YKS and also demonstrated that GM was able to cross BBB in the in vitro BBB assay (Imamura et al., 2011). These results suggest that GM in YKS administered orally is absorbed into the blood and then reaches the brain through BBB, and effects of GM on aggressive and social behaviors in YKS is central action.

Although we need further studies to confirm the details of the amelioration mechanism of YKS, our results show the possibility that YKS has two different effects: an acute effect on sociality and a chronic effect on aggressiveness and sociality. Deficits in social contacts were ameliorated by a single treatment with the test substances, YKS, UH, and GM (Fig. 2, Fig. 4, Fig. 8), whereas chronic treatment was needed to ameliorate aggressiveness (Fig. 3, Fig. 5, Fig. 9). Thus, the ameliorating effects on social behavior by YKS, UH, and GM are thought to be mainly due to its direct partial agonistic effect on 5-HT1A receptors. Social behaviors such as the sniffing, following, and contacting observed between two rats have been often evaluated as an index of anxiety or anxiolytic effects because benzodiazepine- and 5-HT-related anxiolytic drugs increase social behaviors, whereas anxiogenic agents decrease them (File and Seth, 2003). Kuribara and Maruyama (1996) and Kamei et al. (2009) demonstrated the anxiolytic effect of YKS, and Jung et al. (2006) demonstrated anxiolytic effects of the aqueous extract of UH. Therefore, our present data regarding social behavior might suggest that YKS, UH, and GM have an anxiolytic effect. In contrast, chronic effect on aggressiveness may be thought to be due to neuroadaptation against 5-HT1A receptors, as already described. Furthermore, it will be necessary to clarify the detailed mechanisms for the interaction between 5-HT1A receptors and other receptors such as 5-HT2A receptors to induce the neuroadaptation, in future studies. For example, Egashira et al. (2008) reported the involvement of 5-HT2A receptors in the effect of YKS: a 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head-twitch response was reduced by 14 days administration of YKS but not by a single administration. Thus, it is possible that downregulation of the 5-HT2A receptor protein by repeated administration of YKS is closely related to the ameliorative effect on aggressive behavior.

Although the molecular and neuronal mechanism for amelioration of YKS will be performed in the future, the present study demonstrated that GM is candidate of active ingredient for the psychotropic effects such as aggressiveness and sociality of YKS.

Conclusion
YKS and its component UH significantly ameliorated the isolation-induced decrease in social behavior and increase in aggressive behavior. Screening of seven alkaloids in UH for binding to 5-HT1A receptors revealed that GM bound to 5-HT1A receptors and acted as a partial agonist. Furthermore, GM ameliorated both abnormal behaviors as well as YKS and UH. In addition, the effects of GM were attenuated by coadministration of WAY-100635. These results suggested the possibility that GM is the active ingredient responsible for amelioration by YKS, and mainly 5-HT1A receptors are associated with the ability.[1]

C22H26N2O3

366.4534

366.194

60314-89-8

6443046

White to off-white solid powder

1.2±0.1 g/cm3

Uncaria sinensis and Uncaria rhynchophylla

3.674

54.56

1

4

4

27

629

2

O(C([H])([H])[H])C(/C(=C(/[H])\OC([H])([H])[H])/[C@]1([H])/C(=C(/[H])\C([H])([H])[H])/C([H])([H])N2C([H])([H])C([H])([H])C3C4=C([H])C([H])=C([H])C([H])=C4N([H])C=3[C@]2([H])C1([H])[H])=O

VAMJZLUOKJRHOW-XEASWFAXSA-N

InChI=1S/C22H26N2O3/c1-4-14-12-24-10-9-16-15-7-5-6-8-19(15)23-21(16)20(24)11-17(14)18(13-26-2)22(25)27-3/h4-8,13,17,20,23H,9-12H2,1-3H3/b14-4-,18-13-/t17-,20-/m0/s1

methyl (Z)-2-[(2S,3E,12bS)-3-ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo[2,3-a]quinolizin-2-yl]-3-methoxyprop-2-enoate

Geissoschizine methyl ether; Geissoschizine methyl ether; 60314-89-8; methyl (Z)-2-[(2S,3E,12bS)-3-ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo[2,3-a]quinolizin-2-yl]-3-methoxyprop-2-enoate; TNN2THT2NX; (Z)-Methyl 2-((2S,12bS,E)-3-ethylidene-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate; methyl (Z)-2-((2S,3E,12bS)-3-ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo(2,3-a)quinolizin-2-yl)-3-methoxyprop-2-enoate; UNII-TNN2THT2NX; SCHEMBL22615910;

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: ~100 mg/mL (~272.9 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。