Enhancing effect of natural borneol on the absorption of geniposide in rat via intranasal administration

Enhancing effect of natural borneol on the absorption of geniposide in rat via intranasal administration 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:dushouying@263.net †E-mail:dushouying@263.net dushouying@263.net 1 2 1 1 1 1 Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China Department of Chinese Pharmacy, Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China Department of Chinese Pharmacy, Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China Both geniposide (Ge) and natural borneol (NB) are bioactive substances derived from traditional Chinese herbs. The effect of NB on the pharmacokinetics of Ge in rat via intranasal administration was investigated. The concentrations of Ge in plasma were determined by reversed-phase high-performance liquid chromatography (HPLC) after intranasal administration of Ge (4 mg/kg) alone and combined with different doses (0.08, 0.8, and 8 mg/kg) of NB. The intravenous administration was given as a reference (4 mg/kg of Ge and 8 mg/kg of NB). Compared with the intravenous administration, the absolute bioavailability of Ge was 76.14% through intranasal administration combined with NB. Compared with the intranasal administration of Ge alone, Ge could be absorbed rapidly in the nasal cavity combined with NB; the peak time of Ge in the plasma became shorter (3–5 min vs. 40 min); the peak concentration became higher (1.32–4.25 μg/ml vs. 0.67 μg/ml); and, the relative bioavailability of Ge combined with NB was 90.3%–237.8%. The enhancing effect was attenuated as the dose of NB decreased. The results indicated that NB can accelerate the absorption of Ge dose-dependently in the nasal cavity. Geniposide Natural borneol Intranasal administration Intravenous administration Pharmacokinetics Introduction 1 Geniposide (Ge), one of the biologically active ingredients of gardenia fruit ( Gardenia jasminoides Ellis, Rubiaceae), is widely used in multi-component injection in the treatment of cerebrovascular and cardiovascular diseases in China for its anti-thrombotic and anti-inflammatory effects (Suzuki et al., 2001 2001 ; Koo et al., 2006 2006 ). Borneol is a monoterpenoid component in medicinal plants. It is widely used in traditional Chinese medicine (TCM) combined with gardenia for stroke treatment, such as ‘Xingnaojing’ injection. Some studies had shown that borneol can improve the nasal, oral, and gastrointestinal bioavailabilities of drugs, accelerate the opening of the blood-brain-barrier (BBB), and enhance the distribution of drugs in brain tissue (Chen et al., 2006 2006 ; Li et al., 2007 2007 ; Cai et al., 2008 2008 ; Dai et al., 2009 2009 ). The structures of geniposide and borneol are shown in Fig. 1 1 . Both synthetic borneol (SB) and natural borneol (NB) are widely used in Chinese medicine. SB consists of d -borneol and isoborneol, while NB only contains d -borneol. It has been reported that isoborneol had more mucosa stimulus and hepatotoxicity (Hu et al., 2005 2005 ). After a comprehensive evaluation, NB ( d -borneol), the safer form of borneol, was used in the current research. As more and more adverse reactions have been reported on multi-component Chinese herbal medicine (MCHM) injection, it is urgent to find a new and safer way for drug administration. The intranasal route can be a hopeful substitution for MCHM injection, as drugs can be absorbed sufficiently and rapidly into blood for systemic administration (Albrecht et al., 2010 2010 ; Zhou et al., 2010 2010 ), then transported from the nasal cavity to the central nervous system (Illum, 2003 2003 ). The intranasal route is safer due to the barricade effect of the nasal mucosa. According to the results of preliminary study (Zhang et al., 2009 2009 ), the absorption percentage of Ge in intestinal tract of rats was only about 15.9% within 3 h, and the apparent partition coefficient (log P ) of Ge between octanol and water was about 0.108. The purpose of this study was to investigate the enhancing effect of NB on the absorption of Ge in rat via intranasal administration. The concentrations of Ge in rat plasma after intranasal administration combined with different amounts of NB were detected and the pharmacokinetic parameters of each group were compared. Materials and methods 1 Materials 2 Ge was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China), and NB was obtained from Tongrentang Co. Ltd. (Beijing, China). Acetonitrile, methanol, and water were of high-performance liquid chromatography (HPLC) grade (Qualigens, China) and all other reagents were of analytical grade. Animals 2 Male Sprague Dawley rats (250–280 g) were obtained from Weitong biotechnology Inc. (Beijing, China). All animals were clinically healthy and biochemically normal throughout the experimental period. In vivo experiments 2 Twenty five male Sprague Dawley rats, weighing (265±16) g, were randomly assigned into five groups. All the animals were fasting for 12 h before experiment, but allowed to take water freely. The rats were anesthetized by intraperitoneal injection of urethane (1.2 g/kg body weight). An incision was made in the neck of rat, the distal end of left carotid artery was ligated, and the proximal end was clamped by a bulldog clamp. An intravenous tube was cannulated before administration. Blood samples were taken by controlling the bulldog clamp. One group was intravenously administered with about 0.5 ml of injection sample (a single dose of 4 mg/kg of Ge and 8 mg/kg NB) via tail vein. The other four groups were administered with 20 μl of nasal solution via nostril by a modified microinjector with a soft polyethylene (PE) tube, which contained the same amount of Ge (4 mg/kg) and 0, 0.08, 0.8, or 8 mg/kg of NB, respectively. The tube was inserted into the nostril about 4 mm and maintained at least 15 s in order to avoid drug loss. A total of 0.25 ml of blood was collected from the left carotid artery at 0.5, 1, 3, 5, 10, 20, 40, 60, 90, 120, 180, and 240 min after drug administration (Lu et al., 2010 2010 ). The pharmacokinetic parameters associated with each animal were estimated by Kinetica 4.4 software. All the experiments on animals were performed according to the Guidelines for Animal Experimentation, University of Traditional Chinese Medicine of Beijing, China. HPLC assay method 2 The concentration of Ge was quantitated by reversed phase (RP)-HPLC (Agilent1100, HP Inc., USA; Diamonsil® C 18 column, 250 mm×4.6 mm, 5 µm, Dikma Technology Co., China), as described by Ye et al. ( 2006 2006 ) with a slight modification. The mobile phase was acetonitrile and water (15:85, v/v) mixture, and the signal was monitored at 238 nm. The flow rate was maintained at 1.0 ml/min. All the samples were centrifuged at 12 000 r/min for 15 min before determination. Plasma sample preparation and validity 2 Blood sample was collected via left carotid artery and placed into heparinized tubes, as described by Ueno et al. ( 2001 2001 ) with a slight modification. After centrifugation, the obtained plasma was stored at −20 °C until determination. An aliquot of 100 µl plasma sample was placed into a centrifuge tube and 200 µl acetonitrile was added. After being vortexed for 1 min, the mixture was centrifuged at 12 000 r/min for 10 min. Then 250 µl of supernatant was placed into another centrifuge tube and evaporated under nitrogen gas. The residue was redissolved with methanol. The methanol solution was centrifuged at 12 000 r/min for 15 min. Then 20 µl of solution was injected into HPLC system. Plasma standards (0.273–13.650 μg/ml) covering the expected sample concentration range were prepared by spiking various quantities of Ge into blank plasma. These calibration standards were used to validate the linearity, recovery, and precision of the analytical method. Statistical evaluation 2 The in vivo pharmacokinetic parameters of the intranasal and intravenous groups were compared by one-way analysis of variance (ANOVA) at the 0.05 significance level, and the statistical differences were carried out by paired t -tests (Statistics Analysis System 8.0). Results 1 Under the chromatographic conditions described above, the retention time of Ge was found at 10.6 min (Fig. 2 2 ). The limit of detection of Ge was 20 ng/ml in plasma (signal-to-noise ratio ( S / N ) ≥3). The calibration curves of Ge were linear in the range of 0.273–13.650 μg/ml. There was a good linearity between concentration ( C ) and peak area ( A ) ( C =0.0094 A +0.2577, r =0.9997). The mean relative recoveries of Ge at low, middle, high concentrations were (97.14±3.78)%, (95.06±2.95)%, and (91.50±1.82)%, respectively. The relative standard deviations (RSDs) of intra-day precision of Ge at low, middle, and high concentrations were 1.68%, 3.87%, and 1.93%, respectively, while inter-day precisions were 4.09%, 0.33%, and 1.08%, respectively. The limit of quantification was 0.27 μg/ml. The validity of plasma sample assay method was satisfying. The concentration-time profiles of Ge (4 mg/kg) with NB (8 mg/kg) in the plasma after intravenous or intranasal administration are shown in Fig. 3 3 . The data of intravenous and intranasal administrations best fitted a two-compartment open model. The main pharmacokinetic parameters of the compartment model were calculated using Kinetica 4.4 software as shown in Table 1 1 . The results showed that the average value of calculated maximum concentration ( c max (calc.)) was 4.696 μg/ml with the time corresponding to c max (calc.) ( t max (calc.)) at about 2 min after intranasal administration of Ge with NB. The total partial areas under the curve (AUC 0–∞ ) of injection and nasal solutions were 471.026 and 452.396 µg/(ml·min), respectively. The Ge in plasma of the intravenous group was eliminated faster than that of the intranasal group. The elimination rate constant from central compartment ( K el ) for injection solution was 0.053 min −1 while for nasal solution was 0.016 min −1 . The total mean residence time (MRT 0–∞ ) for injection and nasal solutions was 59.1 and 167.5 min, respectively. Data are expressed as mean±SD ( n =5) K a : absorption rate constant; A : coefficient in the sum of exponentials; α , β : exponent; B : coefficient in the sum of exponentials; AUC 0–∞ : total partial area under the curve; MRT 0–∞ : total mean residence time; c max (calc.): calculated maximum concentration; t max (calc.): time corresponding to c max (calc.); K el : elimination rate constant from central compartment; K 12 : transfer rate constant from central compartment to the superficial compartment; K 21 : transfer rate constant from superficial compartment to the central compartment; t 1/2, K a : half-life of the absorption phase; t 1/2, α : half-life of α ; t 1/2, β : half-life of β . Data are expressed as mean±SD ( n =5) P < 0.05 vs. the intravenous group P < 0.01 vs. the intravenous group 2 Parameter 2 Value Intravenous Intranasal K a (min −1 ) − 2.912±1.776 A (μg/ml) 22.231±2.668 0.917±0.266 ** ** α 0.106±0.016 0.314±0.443 B (μg/ml) 2.554±1.342 −0.006±0.002 β 0.010±0.002 0.007±0.003 AUC 0–∞ (µg/(ml·min)) 471.026±62.785 452.396±147.478 MRT 0–∞ (min) 59.112±8.122 167.489±53.390 ** ** c max (calc.) (μg/ml) 24.785±1.539 4.696±1.486 ** ** t max (calc.) (min) − 2.006±0.542 K el (min −1 ) 0.053±0.009 0.016±0.012 ** ** K 12 (min −1 ) 0.042±0.015 0.167±0.238 * * K 21 (min −1 ) 0.020±0.009 0.138±0.204 * * t 1/2, K a (min) − 0.327±0.206 t 1/2, α (min) 6.666±0.985 8.802±7.894 t 1/2, β (min) 74.073±14.781 127.867±56.484 * * For the intranasal administrations of Ge (4 mg/kg) with various doses (0, 0.08, 0.8, and 8 mg/kg) of NB, the concentration-time profiles of Ge in the plasma of rats are shown in Fig. 4 4 . Non-compartmental analysis of pharmacokinetic data was performed by Kinetica 4.4 software. The pharmacokinetic parameters are shown in Table 2 2 . The results show that the average values of c max were 0.673, 1.322, 2.324, and 4.249 μg/ml after intranasal administration of 4 mg/kg Ge with 0, 0.08, 0.8, or 8 mg/kg NB, respectively. The t max was at about 40, 6, 3.4, and 3.8 min, respectively. The absolute bioavailability ( F ) of Ge in the plasma was calculated as the ratio of AUC intranasal /AUC intravenous , and the F was 30.54%, 27.59%, 56.89%, and 76.14% for Ge with 0, 0.08, 0.8, and 8 mg/kg NB, respectively. The relative bioavailability ( F r ) of Ge was calculated as the ratio of AUC Ge+NB /AUC Ge , and F r of Ge with 0.08, 0.8, and 8 mg/kg NB by intranasal administration was 90.33%, 186.31%, and 249.34%, respectively. Our present results demonstrate that nasal absorption of Ge is dose-dependent and can be enhanced effectively by NB. Data are expressed as mean±SD ( n =5) i.v.: intravenous; i.n.: intranasal; c max : experimental maximum concentration; t max : time corresponding to c max ; AUC 0–240 : partial area under the curve from 0 to 240 min; MRT 0–240 : mean residence time from 0 to 240 min; F : absolute bioavailability; F r : relative bioavailability. Data are expressed as mean±SD ( n =5) P < 0.05, vs. Group 2 P < 0.01, vs. Group 2 The first peak of Group 2 Group Dose of NB (mg/kg) c max (μg/ml) t max (min) AUC 0–240 (µg/(ml·min)) MRT 0–240 (min) F (%) F r (%) 1 8 (i.v.) 32.342±1.926 0.50 440.59±70.70 43.00±7.76 100.00 − 2 0 (i.n.) 0.673±0.104 40.00 Δ Δ 134.57±13.13 131.34±6.20 30.54 100.00 3 0.08 (i.n.) 1.322±0.378 ** ** 6.00±2.24 ** ** 121.55±27.69 101.39±9.33 * * 27.59 90.33 4 0.8 (i.n.) 2.324±0.197 ** ** 3.40±0.89 ** ** 250.67±17.86 ** ** 112.08±5.60 * * 56.89 186.31 5 8 (i.n.) 4.249±0.785 ** ** 3.80±1.10 ** ** 335.48±59.65 ** ** 81.92±20.22 ** ** 76.14 249.34 Discussion 1 As TCM injections are used widely in China, more and more adverse effects have been reported. Thus, it is important to find a better route of administration. Intranasal administration may be a good choice. In our formulation, Ge is a water-soluble compound with low molecular weight (388.4). Its log P value in octanol/water system was −0.97, suggesting that it was hard for Ge to permeate mucous membrane. Its intestinal absorption coefficient ( K ) was only about 0.056 h −1 (Zhang et al., 2009 2009 ). NB is a small lipophilic compound and can be rapidly absorbed via the mucous membrane because its liposolubility and low molecular weight (154.25). Based on our preliminary experiments, the half-life ( t 1/2 ) of nasal absorption of NB was about 10 min, while the t max of borneol in mice plasma was 3 min after intragastric administration (Yu et al., 2006 2006 ). When passing through the mucous membrane, NB might enhance epithelial junction permeability and promote paracellular drug absorption as SB (Xiao et al., 2007 2007 ). Several methods have been used to measure the concentration of Ge in plasma (Hou et al., 2008 2008 ; Lv et al., 2008 2008 ). We have developed a simple and sensitive RP-HPLC method to measure the concentration of Ge in rat plasma. The pharmacokinetic profiles of Ge (intravenous or intranasal) and the enhancement of absorption of Ge with different amounts of NB in rat plasma after intranasal administration were investigated. The MRT results show that the absorption of Ge combined with NB after intranasal administration was faster, but eliminated slower than that of intravenous administration. The absolute bioavailability of Ge by intranasal administration combined with NB (8 mg/kg) was high, reaching 76.14%. NB did increase the nasal absorption of Ge. With the increase of NB, nasal bioavailability of Ge was increased. After intranasal coadministration of 0.08, 0.8, and 8 mg/kg of NB, nasal bioavailability of Ge in plasma was 0.90, 1.86, and 2.49 times, respectively, higher than that without NB, based on the data of a non-compartment model. There was no significant difference in the bioavailability between groups of 0 and 0.08 mg/kg of NB. The results showed the enhancing effect of NB was in proportion with the log m NB ( m NB : amount of NB), with the equation of AUC Ge =106.97×log m NB +246.27 ( r =0.9929). From Fig. 4 4 , the results showed that the absorption of Ge combined with NB (low or high) after intranasal administration was very rapid and t max was less than 10 min in plasma. The presumption was that the nasal absorption of Ge combined with NB could be divided into two consecutive processes, the rapid absorption phase (with NB) and the slow absorption phase (without NB). With the existence of a high concentration of NB, Ge was absorbed rapidly at the very beginning. Along with the absorption, the concentration of NB reduced rapidly, and its enhancement of absorption weakened greatly. The absorption of Ge got into the slow absorption phase when most of NB had been absorbed by rats. The nasal absorption enhancement of Ge by NB was reversible and dose-dependent. Many researchers have used one of the following methods to study the mechanisms of nasal absorption enhancer: increasing membrane fluidity, inhibiting enzyme activity, reducing mucus viscosity or elasticity, opening up tight junctions, or solubilizing the drug (Merkus et al., 1996 1996 ). However, it is more essential to study whether absorption enhancer can be absorbed and to determine the speed and extent of enhancer absorption. The absorption characteristics of enhancers themselves are closely related to their effects on promoting drug absorption. Conclusions 1 In conclusion, the present study demonstrates that NB can accelerate the transnasal absorption of Ge, which is consistent with our previous findings (Lu et al., 2010 2010 ). The enhancing effect is temporary and closely related to the dose of NB. 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Intranasal administration of CpG DNA lipoplex prevents pulmonary metastasis in mice 10.1631/jzus.B1000121 Project supported by the National Natural Science Foundation of China (No. 81073057), the Key New Drug Creation and Development Programme of China (Nos. 2009ZX09502-008 and 2009ZX09308-003), and the Doctoral Fund of the Ministry of Education of China (No. 20090013110007) 2010-04-01 2010-12-31 2010-08-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 12