通脉方中异黄酮类化合物在人源肠Caco-2细胞模型的吸收转运

Absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro

通脉方是由葛根、丹参和川芎3味药按质量1∶1∶1组成的复方。该文研究通脉方中异黄酮类化合物大豆苷元、芒柄花素、5-羟基芒柄花苷、芒柄花苷、大豆苷、3'-甲氧基葛根素、染料木苷、葛根素、芒柄花素-8-C-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷、芒柄花素-7-O-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷、澳白檀苷、葛花宁、大豆苷元-7,4'-二-O-β-D-吡喃葡萄糖苷、泰国野葛根素、3'-羟基葛根素、3'-甲氧基大豆苷、芒柄花素-8-C-β-D-吡喃木糖基-(1→6)-O-β-D-吡喃葡萄糖苷、染料木素-8-C-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷、染料木素-7-O-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷、3'-羟基泰国野葛根素、6″-O-β-D-木糖基葛根素、鹰嘴豆芽素A-8-C-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷、3'-甲氧基大豆苷元-7,4'-二-O-β-D-吡喃葡萄糖苷、大豆苷元-7-O-β-D-吡喃葡萄糖基-(1→4)-O-β-D-吡喃葡萄糖苷和大豆苷元-7-O-α-D-吡喃葡萄糖基-(1→4)-O-β-D-吡喃葡萄糖苷在肠的吸收转运。采用人源肠Caco-2细胞单层模型,研究通脉方中上述25个异黄酮类化合物由绒毛面(AP侧)到基底面(BL侧)或从BL侧到AP侧2个方向的转运过程。应用高效液相色谱法分离、紫外检测法对化合物进行定量分析,计算表观渗透系数(P_(app)),并与阳性对照药普萘洛尔和阿替洛尔比较。大豆苷元和芒柄花素由AP侧到BL侧的P_(app)分别为(2.55±0.03)×10^(-5),(3.06±0.01)×10^(-5)cm·s^(-1);由BL侧到AP侧的P_(app)分别为(2.62±0)×10^(-5),(2.65±0.11)×10^(-5)cm·s^(-1)。与本试验中在Caco-2细胞单层模型上呈良好吸收的阳性对照药普萘洛尔的P_(app)(2.66±0.32)×10^(-5)cm·s^(-1)和呈难吸收的阳性对照药阿替洛尔的P_(app)(2.34±0.10)×10-7cm·s^(-1)比较,大豆苷元和芒柄花素与普萘洛尔在同一数量级;其他化合物与阿替洛尔在同一数量级。大豆苷元和芒柄花素的P_(app AP→BL)/P_(app BL→AP)分别为0.97,1.15。可以预测,大豆苷元和芒柄花素可以通过小肠上皮细胞被动吸收进入体内,属于良好吸收的化合物;其他化合物属于吸收不良的化合物。5-羟基芒柄花苷、染料木苷、澳白檀苷、葛花宁和染料木素-7-O-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷的P_(app AP→BL)/P_(app BL→AP)分别为0.18,0.28,0.45,0.38,0.49,推测它们在Caco-2细胞单层模型中的转运可能存在外流机制。

Tongmai formula（ TMF） is a drug combination of three components including Puerariae Lobatae Radix [roots of Pueraria lobata],Salviae Miltiorrhizae Radix（ roots of Salvia miltiorrhiza） and Chuanxiong Rhizoma（ rhizomes of Ligusticum chuanxiong） in a weight ratio of 1∶ 1∶ 1. The absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial（ Caco-2） cells in vitro were studied in this paper. The assay isoflavonoid compounds include daidzein,formononetin,5-hydroxylononin,ononin,daidzin,3＇-methoxypuerarin,genistin,puerarin,formononetin-8-C-β-D-apiofuranosyl-（ 1 →6）-O-β-D-glucopyranoside,formononetin-7-O-β-D-apiofuranosyl-（ 1 →6）-O-β-D-glucopyranoside,lanceolarin,kakkanin,daidzein-7,4＇-di-O-β-D-glucopyranoside,mirificin, 3＇-hydroxypuerarin, 3＇-methoxydaidzin, formononetin-8-C-β-D-xylopyranosyl-（ 1 → 6）-O-β-D-glucopyranoside,genistein-8-C-β-D-apiofuranosyl-（ 1 → 6）-O-β-D-glucopyranoside, genistein-7-O-β-D-apiofuranosyl-（ 1 → 6）-O-β-D-glucopyranoside（ ambocin）,3＇-hydroxymirificin,6″-O-β-D-xylosylpuerarin,biochanin A-8-C-β-D-apiofuranosyl-（ 1→6）-O-β-D-glucopyranoside,3＇-methoxydaidzein-7,4＇-di-O-β-D-glucopyranoside,daidzein-7-O-β-D-glucopyranosyl-（ 1 → 4）-O-β-D-glucopyranoside,and daidzein-7-O-α-D-glucopyranosyl-（ 1 → 4）-O-β-D-glucopyranoside. By using human Caco-2 monolayer as an intestinal epithelial cell model in vitro,the permeability of above-mentioned 25 isoflavonoids in TMF were studied from the apical（ AP） side to basolateral（ BL） side or from the BL side to AP side. The assay compounds were determined by reversed phased high-performance liquid chromatography（ HPLC） coupled with UV detector. Transport parameters and apparent permeability coefficients（ P（app）） were then calculated and and compared with those of propranolol and atenolol,which are the transcellular transport marker and as a control substance for high and poor permeability,respectively. The P（app）values of daidzein and formononetin were（ 2. 55 ± 0. 03） ×10^-5,（ 3. 06 ± 0. 01） ×10^-5cm·s^-1from AP side to BL side,respectively,and（ 2. 62 ± 0. 00） ×10^-5,（ 2. 65 ± 0. 11） ×10^-5cm·s^-1from BL side to AP side,respectively. Under the condition of this experiment,the P（app）value was（ 2. 66 ± 0. 32） ×10^-5cm·s^-1for propranolol and（ 2. 34 ±0. 10） × 10-7cm·s^-1for atenolol. The P（app）values of daidzein and formononetin were at a same magnitude with those of propranolol.And the P（app）values of other 23 isoflavonoid compounds were at a same magnitude with those of atenolol. On the other hand,the rats of P（app AP→BL）/P（app BL→AP）of daidzein and formononetin on the influx transport were 0. 97 and 1. 15,respectively. It can be predicted that daidzein and formononetin can be absorbed across intestinal epithelial cells to go to the body circulation by the passive diffusion mechanism and they were assigned to the well-absorbed compounds. Other 23 isoflavonoid compounds were assigned to the poorly absorbed compounds. Because of the rats of P（app AP→BL）/P（app BL→AP）of 5-hydroxylononin,genistin,lanceolarin,kakkanin,and genistein-7-O-β-Dapiofuranosyl-（ 1→6）-O-β-D-glucopyranoside were 0. 18,0. 28,0. 45,0. 38,0. 49,they may have been involved in the efflux mechanism in Caco-2 cells monolayer model from the BL side to AP side direction.