Comparative pharmacokinetics of icariin in plasma after oral administration of Er-Xian decoction or pure icariin in rats

The present study was to investigate the pharmacokinetics of icariin in rat after oral administration of single dose of Er-xian decoction (EXD) and pure icariin. Blood samples were collected by orbital vein at time intervals after drug administration and the plasma concentrations of the studied components were determined  by high  performance  liquid  chromatography  (HPLC)  after  the  plasma  protein  was precipitated directly with acetonitrile. Icariin was successfully separated using a C18 column with a UV detection at 270 nm and a mobile phase of acetonitrile–water with 0.1% H3PO4 (35:65, v/v) pumped at 1.0 ml/min. The method had a lower limit of quantitation (LOQ) of 0.60 μg/ml for  icariin  with  the  limit  of detection  (LOD)  0.35 μg/ml,  based  on  a  signal-to-noise  ratio  of  3.  The  assay  was  linear  over  a  range 0.667 to 42.67 μg/ml with  coefficient  of  determination  greater  than  0.99  for  analytes.  The  extraction recoveries  was  >80%.  The  method  has  shown  tremendous  reproducibility,  with  intra-  and  inter-day precision  <4.9%  (RSD),  and  has  proved  to  be  highly  reliable  for  the  analysis  of  plasma  samples. The main pharmacokinetic parameters of icariin in rats after administration, EXD and icariin were processed by  Das  2.0,  while  calculating  the  pharmacokinetic  parameters  of  one  compartment  model. From  the experimental  results,  the  method  had  been  applied  successfully  to  pharmacokinetics  of  icariin  in  rat plasma after oral administration of pure icariin or EXD. 

Key words: Icariin, Er-xian decoction, pharmacokinetics.

INTRODUCTION

Er-Xian decoction (EXD),  a  traditional  Chinese  medicine formula  comprised  of Curculi  Rhizoma,  Epimedii  Folium, Morindae  Officinalis  Radix,  Anemarrhenae  Rhizoma, Phellodendri  Cortex  and  Angelicae  sinensis  Radix, has long  been  used  widely for the treatment of osteoporosis disorders (Wang et al., 1998), menopausal syndrome (Liu et  al.,  2005),  and  aging  diseases  for  several  decades (Shen et al., 1995). We previously have reported that EXD has positive effects on bone in ovariectomized rats (Nian et al.,  2006),  the  flavonoids,  saponins,  alkaloids  and anthraquinines  present  in  EXD  are  responsible  for  its antiosteoporotic  activity  (Qin  et  al.,  2008),  stimulate osteoblastic  UMR-106  cell  proliferation,  decrease tartrate-resistant  acid  phosphatase  (TRAP)  activity  and bone  resorption  of  osteoclasts  derived from RAW264.7 cells.  Proteomic  investigate  indicated  that  EXD modulates  bone  metabolism  through  regulation  of

osteoblastic  proliferation,  apoptosis,  cell  activation, osteoclastic  protein  folding  and  aggregation  (Zhu  et  al., 2010).

Icariin  is  one  of  the  most  important  bioactive ingredients  extracted  from Epimedii  Folium, which have the  biological activities  on promotion  of  bone  formation (Wei  et  al.,  2011),  improvement  of  the  learning  and memory  abilities  (Nie  et  al.,  2010;  Guo  et  al.,  2010), induction  of  osteoblasts  proliferation  and  differentiation, resulting in bone formation (Hsieh et al., 2010).

Pharmacokinetic  study  of  active  ingredients  in  natural product  and  Chinese  herbs  is  important to illustrate their action  mechanism  for  the  development  of  traditional Chinese  medicine  (Ma  et  al.,  2007).  Pharmacokinetics studies on icariin (Cheng et al., 2007; Zou et al., 2002) in some herb extracts have been carried out. However, the pharmacokinetics  of icariin  in EXD  has  not  been  studied further.  The  high  performance  liquid  chromatography (HPLC) method  with  high  specificity  and  sensitivity  has been  employed  to  determine  icariin  in various  extraction of icariin. However, method for determination of icariin in plasma  after  administration  of  EXD  has  not  been reported. It is necessary to develop an analytical method for  its  determination  in  biological  samples, for  example, plasma.  In  this  paper,  a  simple,  rapid,  sensitive  and accurate  method  was  applied  to  determine  the  plasma levels  of  icariin  and  has  been  successfully  applied  to pharmacokinetic investigation after the oral administration of icariin and EXD.

In  this  study,  a  simple,  rapid,  accurate  and  reliable method was established for determination of icariin in rat plasma.  The  author  had  comparative  evaluated  the pharmacokinetics of icariin in rats after oral administration of  EXD  which  have  been  used  to  treat  osteoporosis  for years,  or  pure  icariin.  The  pharmacokinetics  parameters were  analyzed  by  software  DAS  2.0  (Chinese Pharmacological  Society,  China).  Differences  among pharmacokinetics  of  icariin  after  oral  administration  of EXD  or  pure  icariin  in  rats  are  illustrated.  It  is  good  for answering  the  combination  of  EXD  and  clinical  research of  icariin  in  osteoporosis.  The  validated  method  is successfully  applied  to  control  the  quality  of  icariin  and investigate  inter  action  among  other  herbals  of  EXD  in osteoporosis disorders and menopausal syndrome.

MATERIALS AND METHODS

Chemicals and reagents

Preparation  of  EXD  consists  of  six  plant  materials,  including          Curculi  Rhizoma,  Epimedii  Folium,  Morindae  Officinalis  Radix, Anemarrhenae  Rhizoma,  Phellodendri  Cortex  and  Angelicae sinensis  Radix,in  a  compositional  ratio  of  12:12:9:9:10:10.  The aforementioned six medicinal materials were obtained from Hua Yu Pharmaceutical  Company  and  identified  by  Professor  H.C.  Zheng of  the  Department  of  Pharmacognosy,  School  of  Pharmacy  of  the Second  Military  Medical  University,   Shanghai,   China.   Voucher specimens (numbered  as  2007091901;  2007091902;  2007091903; 2007091904;  2007091905  and  2007091906,  respectively)  are available at the herbarium of this department.

 The six medicinal materials in the mixture (250 g) were powdered and extracted with distilled water [3000 ml, 12:1 (v/w)] at 100°C for 2  h.  The  extraction  was  repeated  twice.  The  filtrates  were concentrated to 1.5 L under reduced pressure, and lyophilized in a freeze  drier,  and  kept  at  4°C  for  quality  control  and  molecular studies. The  yield  of  dried  extract from the starting crude  materials was 17.8%.  Icariin and benzoic acid was purchased from National Institute for The  Control  of Pharmaceutical  and  Biological  Products (Beijing, China). Methanol and acetonitrile (all of HPLC grade) were provided  by  Fisher  Scientific  International,  Inc.  (Fair  Lawn,  NJ, USA).  Purified  water  was  prepared  in  a  water  purification  system (Mili-Q  Biocel., USA).  All  other reagents were  analytical  grade  and purchased  from  Tianjin  Kermel  Chemical  Reagents  Co.  Ltd. (Tianjin, China).

Chromatographic conditions

The reversed phase-high performance liquid chromatographic (RP-HPLC)  analysis  was  carried  out  on  an  Agilent  high  performance liquid  chromatography  system  (series  1200,  Agilent  Technology, Palo  Alto,  CA, USA)  including  a  quaternary  pump,  vacuum degasser,  thermostatic  column  compartment  and  a  diode  array detector  (DAD).  The  chromatography  data  were  recorded  and processed with Agilent chemstation software. An Agilent Extend C-18  analytical column (5 μm, 4.6 × 250 mm) was used. The mobile phase  consisted  of  acetonitrile  and  water  with  0.1%  H3PO4 in volumetric ratio of 35/65. The injection volume was 20 μl. The flow rate  was  set  to  1.0  ml/min  throughout  the  elution,  and  the temperature was set to 30°C and wavelength to 270 nm.

Preparation of stock solutions

Accurately weighed 10.06 mg  of standard sample icariin (structure shown in Figure 1) was transferred to a 10 ml volumetric flask which was  then  filled  to  mark  with methanol. Final  concentration  of  the resulted stock solution was 1.006 mg/ml.  Standard curve solutions were  freshly  prepared  based  on  diluting  the  above  stock  solution with  methanol  at  appropriate ratios to  yield concentrations  of  2.00, 4.00,  8.00,  16.01, 31.02, 64.04, 128.08 μg/ml of  icariin.  The  stock solution  and  working  standard  solutions  consisting of  icariin  were stored  at -4  and 4°C, respectively.  Internal standard stock solution of  benzoic  acid  (0.313  mg/ml)  was  also  prepared  in  methanol  and diluted with methanol for a working solution of 3.13 μg/ml.

Extraction procedure

An  aliquot  of  0.8 ml acetonitrile  with  0.1 ml hydrochloric  acid  was added  to  150 μl plasma  sample  to  precipitate  proteins  with  50 μl internal standard in a centrifuge tube and the mixture was vortexed for 3 min. After centrifuged at 10000 rpm for 5 min, the supernatant was  transferred  into  another  tuber  and  evaporated  to  dryness  at 35°C. The residue was dissolved by 50 μl methanol. 

HPLC validation procedures

Calibration curves and linearity

The  standard  calibration  curves  in  plasma  were  prepared  daily  by spiking the blank rat plasma (150 μl) with 50 μl of the standard solutions  and  50  μl  internal standard prepared earlier and making

Precision, accuracy and recovery 

The  intra-day  accuracy  and  precision  of  five  replicates  quality control samples were determined  within  one  day  at concentrations of 1.33, 21.7 and 36.3 μg/ml for icariin. The inter-day accuracy and precision  of  the  quality  control  samples  were  determined  on  three different days within one week. The relative standard deviation (%, RSD)  of  each  concentration  was  calculated  to  determine  the precision of the method. The recovery included extraction recovery and  assay  recovery.  The  extraction  recovery  was  determined  for three different concentrations of icariin at 1.33, 21.7 and 36.3 μg/ml by comparing the peak area ratio of icariin to I.S in plasma with that in  methanol  at  same  concentration.  The  assay  recovery  was determined  in  the  same  method,  but  calculated  by  comparing  the concentrations of the analyte from the calibration curves with that in the  prepared  sample.  The  mean  assay  recovery  value  (%, mean±S.D) represented the accuracy of the method.

Stability

The  quality  control  plasma  samples  in  triplicates  at  low,  medium and high concentration were used, which were prepared by spiking the  blank  rat  plasma  (150 μl)  with  50 μl of  the  appropriate  icariin solution and mixing them to yield the following concentrations 1.33, 21.7 and 36.3 μg/ml. Short-term temperature stability was assessed at room temperature (25°C) for 12 h. Long-term stability evaluations involved an analysis of quality control samples that were stored at -20°C for  30  days.  Freeze-thaw  stability  was  evaluated  after  three cycles  of thawing  at room temperature followed by re-freezing to –20°C.  Post-preparation  stability  was  also  determined  using  the extracted samples that were kept at room temperature for 24 h prior to injection and analysis by HPLC.

Applicability of the method in pharmacokinetic studies

Animals 

Ke  Experimental  Animal  Ltd.  (Shanghai  China),  n = 12, weighing 180±10  g.  All  these  animals  were  housed  in  an  air-conditioned room  (temperature,  25°C;  relative  humidity,  55%),  and  allowed  to freely  access  to  food  and  water.  The  rats  were  fasted  for  12  h before dosing.

Pharmacokinetics

The  rats  were  divided  into  two  groups  (six  rats  per  group), respectively.  The  dosing  icariin  and  EXD  were  all  dissolved  in water. One  group was  assigned  to  receive  icariin  solution  by oral administration at  a  single dose  of  4.5  mg/kg  of  icariin,  the  other group  was  assigned  to  receive  4.8  g/kg  of  EXD  solution.  Serial blood  samples  (0.5 ml)  were  obtained  via  the  rats  orbital  vein  at 0.25,  0.5,  1,  2,  4,  6,  8,  12,  24  h after  administration  and  collected into  heparinized  centrifuge  tubes.  The  blood  samples  were immediately  centrifuged  at  4000 g  for  5  min  at  room  temperature. The  plasma  sample  was  extracted  and  analyzed according  to extraction procedure. The data analysis of plasma concentration of icariin versus time and calculation of pharmacokinetic parameters in rats  were  executed  by  the  statistics  software  DAS  2.0.  The pharmacokinetic  parameters  were  estimated  using  compartmental and statistical moment (that is, compartmental) analysis methods. 

RESULTS AND DISCUSSION

Specificity

Typical  chromatograms  obtained  from  blank  plasma, spiked  rat  plasma  and  plasma  samples  were  shown  in Figure 2. The  peaks  of  the  analyte  in  the  plasma  were identified  by  comparing  their  retention  time  with  that  of the standard  sample. In this study, the retention times of icariin varied  from  17.19  to  17.22  min. The  specificity  of the assay was investigated by injecting blank plasma. No peaks  were  observed  that  would  interfere  with  the analysis in HPLC chromatograph.

In  this  study,  several  organic  solvents  were  tested  for precipitating  protein  and  extracting  the  analyte  from  rat plasma. They included methanol with 0.1 ml hydrochloric acid,  acetonitrile  with  0.1 ml hydrochloric  acid  and methanol-ethyl  acetate  (1:1,  v/v)  and  their volume  were one to  four  times  that  of  plasma  sample.  The  result showed  that  the  effect  of  precipitating  protein  and extraction recoveries were best when acetonitrile with 0.1 ml  hydrochloric acid  was  chosen.  The  extraction recoveries  of  the  analyte  at  low,  middle  and  high  were 86.2±1.58%, 85.8±1.80% and 89.65±0.20%, respectively. In  addition,  10%  salicylsulfonic  acid  was  used  to precipitate  protein  in  the  experiment.  It  has  a conspicuous effect on precipitating protein, but defects of which showed a bad peak shape.

Linearity

The  peak  areas  of  the  analyte  were  plotted  against standard concentrations to establish calibration curves for icariin.  The  curves  were  best fitted using a least square

linear  regression  model  y = mx+b,  weighted  by 1/x2, in which y is the peak area ratio, m is slope of  the  calibration  curve, b is  intercept  of  the calibration  curve  and  x  is  the  analyte concentration.  The  slopes  of  the  calibration graphs  were  0.138±0.29  (n  =  6)  with  RSD  of 1.68%, and the intercepts were -0.0470±0.20 (n = 6)  with  RSD  of  2.20%  throughout  the  study.  The correlation  coefficients  (r  >  0.99)  showed  good linearities.  This  result  showed  the  usefulness  of the present HPLC method in the assays  of icariin from low to high plasma levels. The limit of quantitation (LOQ) was achieved as the lowest point on the standard curve, 0.60 μg/ml for  icariin  with  RSD  of  5.8%  (n =  5).  The  limit  of detection  (LOD)  was  defined  as  the  lowest concentration  of  the  drug  resulting  in  a  signal-to-noise  ratio  of  3:1.  The  limit  of  detection  (LOD)  of the  analyte in  plasma  was  determined  to  be  0.35 μg/ml for icariin.

Precision, accuracy and recovery

Quality  control  samples  (n =  5)  representing  low, medium  and  high  concentration  contained  1.33, 21.7 and 36.3 μg/ml  for  icariin.  The  maximum intra- and  inter-day precision was 4.9% for icariin. The  assay  showed  that  the  intra-  and  inter-day precisions for icariin with a RSD of less than 15% were  obtained  at  the  lowest  concentrations.  Data on  accuracy  about  the  recovery  of  icariin  was 86.2±1.58% (n = 5) at the lowest concentration of 1.33 μg/ml in spiked rat plasma samples (Table 1). The mean extraction recovery of the analyte (n =  5)  from  spiked  rat  plasma  (Table  1)  was satisfactory  at  low,  medium  and  high concentrations,  which  varied  from  80.4±3.97  to 89.7±0.20%.  High  recovery  of  icariin  from  rat  plasma suggested  that  there  was  negligible  loss  during drug extraction.

Stability

Acceptable analyte stability was demonstrated for all  phases  of  storage  and  processing  was  shown in  Table  2.  The  accuracy  of  low  (1.33 μg/ml), medium  (21.7  μg/ml)  and  high  (36.3  μg/ml) concentrations  of icariin  in  rat  plasma  for  12  h room  temperature  stability  (88  and  98%),  three cycle freeze-thaw stability (94 and 108%), 30 days at -20°C, stability  (97  and  101%)  and  24  h  post-preparative  stability  (95  and  100%)  were  all acceptable. The RSD (%) for each of the stability experiments varied between 0.9 and 4.9%. Application  of  the  HPLC  method  for pharmacokinetic studies

The debris  assessment software  (DAS)  program

as  validated  methods  has  been  successfully applied for pharmacokinetic studies of icariin in rat plasma  after  oral  administration  of  an  icariin solution.  The  mean  plasma  concentration  versus time profiles of icariin was shown in Figure 3. The pharmacokinetic  parameters  were  listed  in  Table 3. With minimum akaike information criterion (AIC) values, a one-compartment open pharmacokinetic model  was  proposed.  Icariin  appeared  to  be distributed  most  rapidly  into  a  highly  perfused central  compartment.  The  phenomenon  with  a rapid  distribution  and  a  relatively  slow  distribution followed  by  a  slower  elimination  phase  was observed from the compartment model parameters t1/2. The values of t1/2, ke, V1, CL, ka and Cmax of icariin in rat plasma. 

Statistical analysis

The  author  had  compared  pharmacokinetics  of icariin between pure icariin and Er-Xian decoction. The  differences  were  showed  in  two  aspects:  the absorption peak of iacriin and absorption amount. It  was  noted  that  other  ingredients among EXD

influenced  the  pharmacokinetics  of  icariin.  As  shown  in Figure  3  after  administration  of  pure  Icariin,  the absorption  peak  was  larger  than  administration  of  EXD, and  absorption  amount  AUC0−t:  (40.8±5.73)  vs. (30.04±3.28)  (mg/L)·  h  in  rat.  The  similar  absorption result had happened in Pengs study (Peng et al., 2010). The author analyzed that the reasons were enterohepatic circulation  and  effect of other herbs. Under the influence of  enterohepatic  circulation,  icariin  had  been  already metabolized  which  would  excrete  and  re-absorbed.  In addition  to  enterohepatic  circulation,  the  other  reason was  that there  were  interactions  among different  herbs. EXD  was  a  combination  of Curculi  Rhizoma,  Epimedii Folium,  Morindae  Officinalis  Radix,  Anemarrhenae Rhizoma,  Phellodendri  Cortex  and  Angelicae  sinensis Radix,    each     of     which     could     produce     certain

pharmacological  effects,  and  may have  impacts  on  the absorption  and  metabolism  of  icariin.  The  author suggests  that Curculi  Rhizoma,  Anemarrhenae  Rhizoma and Phellodendri Cortex had antiosteoporotic activity and would  block  the  enterohepatic  circulation.  Thereby  they could  weaken  the  absorption  peak  of  icariin.  It  could surmise that multi-componet in EXD and their interactions resulted in the difference of the pharmacokinetics of EXD and pure icariin.

Conclusions

In  conclusion,  a  simple  HPLC  assay  method  has  been used  for  determination of icariin  in  rat  plasma  with  an internal  standard.  Treatment  with  acetonitrile  and hydrochloric acid for icariin results in chromatograms free of  interference.  The  procedures  for  the  determination  of icariin  by  HPLC  method  have  been  validated,  and  the results demonstrate that the standard curve is linear over the concentration of 0.667 to 42.67 μg/ml. 150 μl plasma was  required  for  analysis  of  the  compound.  The  method is suitable for pharmacokinetic study of icariin in rats after oral administration. In summary, results of the present study indicated that disease  and  other  herbs  in  decoction  influenced  the potential pharmacokinetic of icarrin.

ACKNOWLEDGEMENT

This work was supported by the National Natural Science

Foundation of China, no. 90709023.

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Chlorogenic Acid       corosolic acid       Ursolic acid

HoneySuchle Flowers Extract        Eucommia Leaf Extract            Icariin

Amygdalin      10-hydroxycamptothecin        Dihydromyricetin DMY

7-Ethyl-10-Hydroxycamptothecin