| Stroke, also known as apoplexy, is defined as an acute neurologic dysfunction of vascular origin with sudden or rapid occurrence of symptoms and signs corresponding to the involvement of focal areas in the brain, with the character of high occurrence, high disability and high mortality. Cerebral ischemia is its main type accounting for 85%. It can lead to coma, paralysis, headache, delirium, and headache and so on, bringing great suffering to the patient. Xing-Nao-Jing (XNJ), based on the famous traditional Chinese medicine prescription "AnGongNiuHuangWan", is composed of musk, gardenia, borneol and curcumae. It could detoxification, eliminate heat, cool blood, refresh and resuscitate, and is used as injection widely in clinic to treat acute stroke. However, for the disadvantage of injection, XNJ has been developed into oral preparations. Based on the previous pharmaceutical research, this study will further research the pharmacokinetics (PK) and pharmacodynamics (PD) characteristic of XNJ oral formulations.The existed researches about PK of XNJ is processed in normal animals, but the real fact is that the stroke will lead to a series of physiological state change and further influence the drug absorption and metabolism. Therefore, we studied the PK of borneol (Bor) and geniposide (Gen) in stroke-afflicted (S A) rats after oral administration of borneol, geniposide or different set of XNJ, compared with the sham-operated rats, the influence of pathology and drug factors could be conducted. Besides, for the lesion site is in brain which stroke could induce serious blood-brain barrier (BBB) structural damage, we further researched the brain PK and plasma PK of the main components in XNJ in SA and SO rats to observe the penetrate processing change in different state. Lastly, PD can show the direct effect of drugs, this study selected the indicators of antioxidant and anti-inflammatory activity, to research the PD of XNJ and discussed the relationship of PK-PD according to our previous study. The research points are shown in detail as following:1 The determination of the main components in XNJ in plasma and brainThe method to evaluate borneol in plasma and brain were established with GC-FID instrument, following the sample treatment of solvent-solvent extraction. The chromatographic conditions are as follows:TM-1701 capillary column (30 m×0.32 mm id.,0.25 μm film, Tianmei, China); FDI detector; carrier gas was nitrogen at a flow rate of 28 mL/min; Injection volume was 1 μL; the injector temperature and detection temperature were 250℃ and 300℃, respectively. The oven temperature was programmed to rise from an initial temperature of 105℃ (remaining 8 min) to 170℃ at a rate of 50℃·min-1 and then held at 250℃ for 4 min. The calibration curves of borneol in plasma was Y=0.1386X+0.0195, r=0.9983, and good linear in the range of 0.1128～18.04 μg·mL-1. The calibration curves in brain was Y= 0.1286X+ 0.0704, r= 0.9997, and good linear in the range of 0.1128～18.04 μg·mL-1.The HPLC method to detected geniposide in rat plasma was performed on an Agilent 1200 series HPLC system with UV detector. Chromatographic separation was accomplished on a Merck C18 (4.6×250 mm I.D.,5.0 μm) analytical column under the following conditions: acetonitrile-water (13:87) at a flow rate of 1.0 mL/min; sample injection,20 μL; column temperature,25℃ and detection wavelength,238 nm. The calibration curves in brain was Y=57.711X+1.1508 (r=0.9998) with a good linear in the range of 0.1074～10.74 μ/mL.The method to determination the geniposide in brain was established with UPLC-MS/MS system and an Acquity UPLC BEH C18 column (100 mm×2.1 mm I.D.,1.7 μm). The column temperature was maintained at 40℃ and the autosampler was conditioned at 4 ℃; mobile phase composed of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.4 mL/min with a gradient program; Injection volume was 5μ. Mass experiment was performed in the negative mode with multiple reaction monitoring (MRM). Nitrogen was the desolvation gas (800 L-h-1), Argon was used as collision gas. Good linearity was achieved for geniposide, Genipin 1-O-beta-D-gentiobioside and Geniposidic acid, the extraction recovery was between 99.6% to 114.3%, the intra-and inter-day precisions and the accuracy were good, and the stability and the matrix effects met the requirements.2 The PK of Bor and Gen after SA and SO rats orally given different drugsWith the reference of Zea Longa method, we improve the middle cerebral artery occlusion (MCAO) to prepara stroke state, and establish the sham-operated group to compare. In the following PK study, the drugs were given at 22 h after operation, the blood samples were collected punctually and determined and the parameters were analyzed using non-compartmental methods with kinetica.2.1 The PK of Bor after SA and SO rats orally given Bor and XNJSA and SO rats were orally given XNJ and borneol (162.0 mg·kg-1 borneol), the blood samples were collected and determined. After data process, results showed that after administration of borneol, the maximum plasma concentration (Cmax) and area under the curve (AUC0-360) values in stroke rats significantly increased by 302% and 275% compared with the sham-operated rats, the same phenomenon appeared after administration of XNJ. In the rats with the same physiological conditions, the Cmax and AUC had higher values in the borneol subgroups. These suggested that the pathological damage of stroke can improve borneol absorption and some components in XNJ inhibit this process.2.2 The PK of Gen after SA and SO rats orally given different sets of XNJComparative pharmacokinetic study of geniposide in stroke and sham-operated rats after administration of XNJ and geniposide was proceeded to evaluate the effect of stroke on pharmacokinetics of geniposide, while the influence of other components in XNJ was determined by using gardenia extract and geniposide-borneol compounds in rats with stroke to compare. The results showed that the Cmax and AUC0-360in stroke after administration of XNJ were 5.97±3.82μg·mL-1, and 570.06±274.32μg-min·mL01, respectively, which were 5 times compared with SO rats or the SA rats given geniposide. In stroke, the Cmax and AUC0-360 of geniposide-borneol group and gardenia extraction group were close to XNJ group and geniposide group, respectively. The geniposide-borneol group had a higher value. We can conducted that stroke improved the absorption of geniposide in XNJ. Borneol may be the key ingredient in XNJ improving the absorption of geniposide.3 The PK study of borneol and geniposide in XNJ in SA and SO rats3.1 The brain and plasma PK study of Bor in XNJ in SA and SO ratsBorneol, as a fat-soluble ingredient, can pass through the blood-brain barrier (BBB) easily, will stroke change this course? After oral administration of XNJ, the blood and brain samples were both collected and detected, the data were analyzed and we defined Te=AUCbrain/AUCblood×100%. Results showed that in stroke group, the Cmax and AUCo-360 of brain and plasma are 1.82±0.825, 1.35±0.43μg·mL-1 and 123.39±55.82,87.91139.81 μg-min mL-1, Te was 70.93%, which were all larger than those in sham-operated group. It can be inferred that the pathological state of stroke can increase the amount of borneol permeating into brain.3.2 The brain and plasma PK study of Gen in XNJ in SA and SO ratsGeniposide, as a water-soluble ingredient, is hard to penetrate BBB, will stroke influence the process? The plasma and brain samples were handled and determined by HPLC and UPLC-MS/MS, respectively. The results indicated that after administration of XNJ, though the plasma Cmax and AUC0-t in stroke were higher than SO rats. In brain, the SA rats had more significant higher Cmax, AUC0-t and Te values (142.40±45.75 ng-mL-1,17907.3616736.61 μg-min-mL-1) with double-peak phenomenon. It can be concluded that the stroke enhance the distribution of geniposide in XNJ into brain through blood-brain barrier.Besides, the other iridoid glycoside, Genipin 1-O-beta-D-gentiobioside and Geniposidic acid will pass though BBB while their low concentration? We analyzed the UPLC-MS/MS data to find that the 3 iridoid glycoside have similar t1/2 and MRT, geniposide and Genipin 1-O-beta-D-gentiobioside has double-peak while Geniposidic acid has one peak at about 120 min.4 The PD study of XNJ and the relationship between PK and PDIn the XNJ PD research, the antioxidant indicators (SOD, MDA) and anti-inflammatory indicators (IL-6, TNF-a) were selected, three group (SA, SA with drug and SO) were set to detect blood samples as S,which showed the indicators trend. We define a effect parameter E%= (SSA-SsA+drug)/(Sso-SsA) to evaluate the XNJ efficacy. The relationship between PK and PD was further discussed. The results showed that XNJ exhibited significant antioxidant and anti-inflammatory capabilities in stroke, but trends of effects vary. The antioxidant effect showed multi-peak over time, but its trend is consistent with drug concentration, and their relationship comply with E=Emax-C/(EC50+C) model. At the same time, the anti-inflammatory efficacy index measured within 360 min rise in different trend with a single peak, and there was a significant hysteresis phenomenon between this effect and drug concentration.5 The influence of XNJ and stroke on the gastric and duodenal mucosaPK study showed that stroke and XNJ components will promote the absorption of drugs, which means those two factors could influence the gastrointestinal mucosal barrier. In this part we research the possible change of gastric and duodenal mucosa, by HE staining and comparative observation. Results showed that in stroke gastric and duodenal mucosa is damaged, loosely arranged, and inflammatory cell infiltration and bleeding occur, the use of XNJ could deteriorate the mucosa lesion. Above indicated that the injuries induced by stroke relate to the gastrointestinal mucosa barrier damage, which may be the pathological basis to promote drug absorption; the components in XNJ aggravate the injury in some degree. |
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