Andrographolide tablet_A4 - CiteSeerX

Original Article

Pharmacokinetic Study of Andrographis paniculata Tablets in Healthy Thai Male Volunteers Jinda Wangboonskul, Supawadee Daodee*, Kanokwan Jarukamjorn and Bung-orn Sripanidkulchai Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand * Corresponding author: [email protected]

ABSTRACT Pharmacokinetics of andrographolide, main active compound contained in Andrographis paniculata product, was investigated in healthy Thai male volunteers. The bio-analytical method using HPLC for the determination of andrographolide in plasma was developed and validated following US FDA and Thailand guidelines for the bioavailability and bioequivalence studies. Six healthy Thai male volunteers were enrolled in the study. Each was administered with 3 doses (3, 6 and 9 tablets) of A. paniculata tablet which is equivalent to 29.55, 59.10 and 88.65 mg of andrographolide, respectively. Blood samples were pretreated by using C-18 solid phase cartridges before injecting into the HPLC. The result of this study demonstrated the individual variation of bioavailability among subjects. The concentration-time profile of andrographolide in plasma was obtained from a volunteer taking the highest dose. The peak concentration (Cmax) was 335.85 ng/mL and reached the peak time (Tmax) at 1 hour. Andrographolide was neither detected in plasma samples from the lower doses nor detected from other volunteers. However, an individual peak was detected in all plasma samples obtained from the volunteers taking A. paniculata products. A characteristic profile demonstrated the possibility of this peak being a metabolite of andrographolide. The chromatography assumed to be that of a compound polar than andrographolide. Tmax of this compound was also longer than that of andrographolide. The peak in the chromatogram did not appear in the blank plasma or in the extract of A. paniculata. Moreover, the peak response-time profiles of this compound were correspondent to the pattern of the concentration-time profile in pharmacokinetic study. Thus it can be concluded that the saturated metabolism process of andrographolide possibly occurred at high dose. Keyword: Andrographis paniculata, andrographolide, pharmacokinetic study Thai Pharm Health Sci J 2006;1(3):209-218§

Introduction Andrographis paniculata (Burm.f.) Nees, also known as King of Bitters, is a member of the plants in Acanthaceae family. It has been used for centuries to treat infections in gastrointestinal tract and upper respiratory tract, fever, herpes, sore throat, and a variety of other chronic infectious diseases.1 Moreover, Kan Jang, the extract from A. paniculata and Eleutherococcus senticosus, has been used as a non-herbal medicinal §

§

product for treatment of non-complicated upper respiratory tract infection.2-7 Andrographolide, the main active component in A. paniculata8, enhances functionalities of the immune system and inhibits the proliferation of tumor cell lines including various human cancers.9,10 Significant anti-inflammatory, anti-allergic and antipyretic effects have also been demonstrated by using the pure andrographolide, as well as the extracts of A. paniculata.11,12

11th year of Srinakharinwirot Journal of Pharmaceutical Sciences

Thai Pharmaceutical and Health Science Journal, Vol. 1 No. 3, Sep. – Dec. 2006

PDF created with pdfFactory Pro trial version www.pdffactory.com

209

From the study of Panossian13, the validated analytical methods for determining the amount of andrographolide in the blood plasma of rats and human volunteers following the oral administration of A. paniculata extract were developed and used for the pharmacokinetic study. Andrographolide was quickly and almost completely absorbed into blood following the oral administration of A. paniculata extract at a dose of 20 mg/kg body weight in rats. Following the oral administration of four Kan Jang tablets (a single therapeutic dose, equal to 20 mg of andrographolide) to humans, maximum plasma levels of approximately 393 ng/mL were reached after 1.5 - 2 hours, as quantified by a UV diode-array detection method. It had been found that the pharmacokinetics of andrographolide in humans were highly variable among individuals. Pharmacokinetic profiles of andrographolide had been documented. However, ones applicable to Thai population are lacking. Thus, it is necessary to study the pharmacokinetics of A. paniculata particularly andrographolide in Thai people. The results from this study will provide the information of the oral bioavailability and pharmacokinetic profiles of andrographolide. Such information is extremely important not only for determining the right dosage regimen, but also for lessening the possibility of side effects due to overdosing.

Materials and methods Chemicals and reagents All solvents were HPLC-grade and all reagents were analytical reagent grade. Andrographolide and internal standard (naproxen sodium) were purchased from Sigma® (USA) and Fluka® (Switzerland), respectively. Sep-Pak® (1 cc, 100 mg) C18 cartridges were purchased from Waters® (Ireland). HPLC quality water was purified using a Millipore Milli-Q purification system (USA).

210

HPLC apparatus and chromatographic conditions The chromatographic separation was performed using a Hewlett Packard LC-1100 gradient liquid chromatography instrument, equipped with an autosampler system and a photodiode array with a Hypersil ODS column (250x40 mm, 5 µm). The mobile phase system was optimized to give a good resolution of andrographolide from other endogenous substances in plasma sample. A gradient method was used, where the percentage of solvent A (0.025% trifluoroacetic acid in acetonitrile) and solvent B (0.025% trifluoroacetic acid in water) was varied from 0 - 90% of solvent A in 50 minutes at a flow rate of 1 mL/min. The detection was performed with a diode array detector at a wavelength of 230 nm. The structure of the metabolite was identified by ion-trap liquid chromatography-mass spectrometer (Bruker®). The mobile phase condition was the gradient system of 2% acetonitrile in 0.1% formic acid and 80% acetonitrile in 0.1% formic acid. The run time was within 90 minutes.

Sample preparation

The standard stock solutions of andrographolide (9750 ng/mL) were prepared in acetonitrile separately for calibration standards and for quality-control (QC) samples. A 10,000 ng/mL internal standard solution (naproxen sodium) was prepared in methanol and acetonitrile (1:9). All standard stock solutions were stored at 2 - 8 °C. The standard stock andrographolide solutions (5 - 50 µL) were added into drug free plasma to give the final volume of 1,000 µL. To each calibration standard, 10 µL of internal standard solution was added and vortexed. The calibration standards were prepared to yield final respective concentrations of 48.75, 97.50, 146.25, 195.00, 292.50 and 487.5 ng/mL of andrographolide in plasma. The sample pretreatment method was developed by solid-phase extraction (SPE). Sep-Pak C18 solid-phase cartridge was selected. The cartridge was conditioned with 4 mL methanol and 4 mL water. Internal standard solution with amount of 10 µL was added into 1,000 µL



of plasma sample before applying to the SPE column. The SPE column was washed once with 1 mL water and 100 µL methanol and dried in a low vacuum for 1 minute. The sample was eluted directly from the SPE column into a tube with 2 mL methanol and dried over nitrogen gas. The extracted solution was reconstituted with 200 µL of acetonitrile:water (50:50) and transferred to an autosampler vial before injected into HPLC system.

Bio-analytical method validation The method was adopted from Bioanalytical method validation, provided by the US FDA (2001) and from Thailand guidelines for the conduct of bioavailability and bioequivalence studies.14,15 The specificity of the analytical method was determined by comparison of the chromatogram obtained from the plasma blank and spiked plasma sample. The blank and spiked plasma samples should show no interference from the endogenous substance. Lower limit of quantification (LLOQ) was evaluated by determining the lowest concentration of andrographolide spiked in plasma in which the analyte response at the LLOQ should be at least 5 times as much as the response from the blank sample. Moreover, the analyte peak response should be identifiable, discrete and reproducible with a precision of 20% and accuracy of 80 - 120%. Accuracy was assessed by means of replicate analysis (n = 5) of the spiked samples containing andrographolide at three concentrations (97.50, 195.00 and 487.50 ng/mL). The mean value should be within 15% of the actual value except at the LLOQ, where the deviation should not be more than 20%. Recovery of andrographolide in plasma should be close to 100%, or not less than 50 - 60%, and precise and consistent. Within-day precision was evaluated from replicate analysis (n = 5) of quality control samples containing andrographolide with different concentrations (97.50, 195.00 and 487.50 ng/mL) on the same day. Between-day precision was also assessed from the same concentrations of quality control samples on five days.

The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV. The linearity of the method was evaluated using freshly prepared spiked plasma samples with a concentration range of 48.75 - 487.50 ng/mL. Calibration curve consisted of a blank sample, a zero sample and six calibrator concentrations. The minimum concentration of the calibration curve must be the concentration at LLOQ. Samples were quantified using the ratio of peak area of analyte to that of internal standard. Linearity can be justified by regression analysis which is shown as coefficient of determination. The coefficient of determination should be more than 0.99 and the calculated value at each concentration should not deviate from the spiked value more than 15% except for the LLOQ, where it should not exceed 20% of the CV. Stability procedures were carried out during sample collection and handling, both after long term storage (frozen at the intended storage temperature) and short term storage (room temperature), and after going through freeze-thaw cycles and the analytical process. Conditions used in stability experiments were to reflect situations likely to be encountered during actual sample handling and analysis. Percent deviation of the concentrations comparing with the initial concentration before storage condition should be in the range of ±15%. In the study-phase validation, calibration curves were prepared for each of the analytical runs and used in the calculation of volunteer’s plasma concentration in each run. Two aliquots of each of the low, medium and high quality control samples were analyzed during the analytical run. At least 4 in 6 of quality control samples must have the concentrations in a range of ±15% comparing with the spiked concentration.

Experimental procedures The study was applied and submitted for the approval by the Ethical Committee of Khon Kaen University following the Declaration of Helsinki and accepted on December 17, 2005 (No. HE471113). Study



211

information, risks and benefits were explained to the subjects, and written consent forms to participate the study were obtained. Three periods crossover design using six subjects was used. A batch of A. paniculata tablets which was confirmed for its consistency of andrographolide content was selected for administering to the subjects.16 The amount of andrographolide per tablet was 9.8521 ± 0.3436 (%RSD = 3.49). Subjects were fasted for 10 hours and allowed for water as desired except for one hour before and after drug administration. Three doses of 3, 6 and 9 tablets which were equivalent to 29.55, 59.10 and 88.65 mg of andrographolide, respectively were given in separate treatment periods to the subjects with 250 mL of water. Each treatment was separated by a 7-day washout period. Three hours after drug administration, hot drink and juice were served. Standard meal (rice and boiled chicken) was not provided until four hours after drug administration. All subjects were required to remain

within the investigation unit for the day of the study. Blood samples (5 mL each) were taken before administration and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0 and 8.0 hrs thereafter. Each blood sample was centrifuged and the prepared plasma samples were stored at -80 °C prior to analysis.

Results and discussions Bio-analytical method validation Figure 1 showed the chromatograms of blank plasma (A) and internal standard plasma spiked with andrographolide (B) after samples pretreatment procedure. No interference with an adequate separation of andrographolide from the endogenous substances in human plasma was observed. This indicated the specificity of the sample pretreatment and chromatographic procedure.

Figure 1 Chromatograms of blank plasma (A) and internal standard plasma (800 ng/mL) spiked with andrographolide (200 ng/mL) (B) after sample pretreatment procedure

212



Table 1 summarized the validation values for determination of andrographolide in human plasma which indicated that this HPLC method and sample pretreatment procedure using C-18 SPE was appropriate and reliable to determine andrographolide in human plasma. The assay was linear over a range of 48.75 487.5 ng/mL with the lower limit of quantification at 48.75 ng/mL. The accuracy of the three concentrations (97.50, 195.0, 487.5 ng/mL) was 82.08 - 89.18%. Percentage coefficient of variation and the deviation of recovery were less than 11.16 and 8.84, respectively which were less

than 15% of the acceptance criteria. The coefficients of variation of within-day and between-day precision were less than 10.24% in all tested concentrations (97.50, 195.0, 487.5 ng/mL) which were less than 15% of the acceptance criteria indicating good precision of this bioanalytical method. Table 2 illustrated the stability of andrographolide in plasma under storage conditions which were less than 4.06% deviation. Based on the acceptable range of ±15% deviation, stability of andrographolide under storage conditions was thus established.

Table 1 The validation values for determination of andrographolide in human plasma Validation criteria

Value

1. Accuracy (n = 5) %Deviation from actual three concentrations (97.50, 195.0, 487.5 ng/mL) 2. Precision (within-day and between-day) (n = 5) %Coefficient of variation of three concentrations (97.50, 195.0, 487.5 ng/mL) 3. Recovery of extraction (n = 5) %recovery of andrographolide from three concentrations (97.50, 195.0, 487.5 ng/mL) %recovery of internal standard (naproxen sodium) 4. Calibration curve (n = 5) %Deviation from actual value of spiked sample in six concentrations r from regression analysis 5. LLOQ (n = 5) %CV %Deviation

2.59 - 8.84% 2.76 - 10.24% (within-day) 2.86 - 9.14% (between-day) 82.08 - 89.18% (%CV = 6.29 - 11.16) 64.40% (%CV = 13.22) 2.10 - 10.07% 0.9991 48.75 ng/mL 5.34% 9.48%

Table 2 Stability testing in bio-analytical method validation for andrographolide quantification Stability testing (n=3) 1. Freeze-thaw stability 2. Short-term temperature stability 3. Post-preparative stability 4. Long-term stability in freezer

% Deviation found in high and low concentrations Low conc. (97.5 ng/mL) High conc. (487.5 ng/mL) 3.01% 4.06% 0.17% 0.93%

From these findings, we concluded that this method was able to determine andrographolide level. Therefore in the following pharmacokinetic study, the peaks of

0.88% 3.01% 0.33% 2.52%

andrographolide, naproxen sodium and a metabolite are adequately separated from endogenous compounds (Figure 2).



213

Figure 2 Chromatogram of plasma sample obtained from subject No. 1 at 1 hour after administered 9 tablets of A. paniculata product

Pharmacokinetic study

Only one concentration-time profile was obtained from subject No. 1 who took the highest dose (9 tablets) of A. paniculata product (Figure 3). Therefore the peak response of andrographolide in plasma could be quantified although the peak appeared in a short period of time. The tiny peaks of andrographolide in conce ntration (ng/ml)

chromatograms were also observed in subjects No. 2 and No. 6 at the highest dose, but the level was less than LLOQ. The peak of andrographolide in plasma from other samples was not detected. This suggested a high individual variation of absorption of andrographolide among subjects.

400 350 300 250 200 150 100 50 0 0

0.5

1

1.5

2

2.5 ti me (hrs)

Figure 3 Concentration-time profile of andrographolide in plasma of Subject No. 1 taking 9 tablets of A. paniculata product (equivalent to 88.65 mg andrographolide) From all volunteers and at all doses, an obvious peak suspected to be that of the metabolite of A. paniculata was detected. The likelihood of being the metabolite is supported by 3 findings. First, the polarity of

214

this compound was higher than that of andrographolide as this compound was less retained in the reversed phase column of HPLC than was andrographolide. The retention time of this compound peak was about 23



minutes while the one of andrographolide was at 27 minutes. Second, the peak response-time profile of this compound fitted a pattern of a given active compound and its metabolite profile. In other words, Tmax of this compound (2 – 2.5 hrs) was longer than that of andrographolide (1 hr) (Figure 4). This phenomenon is

common since a metabolite of a given compound always appears after its parent one. Third, the peak of this compound in the chromatogram was not found either in the blank plasma (Figure 1A) or in the extract solution of A. paniculata product (Figure 5).

peak area ratio

1.5 metabolite

1

andrographolide

0.5 0 0

5

10

time (hrs)

Figure 4 Peak response ratio-time profile of andrographolide and its metabolite obtained from subject No. 1 at 1 hour after administering 9 tablets of A. paniculata product

Figure 5 Chromatogram of the extract solution of A. paniculata product Figure 6 shows the mean peak response ratio-time profiles of the metabolite obtained from three doses of A. paniculata product from six volunteers. Andrographolide was likely to possess linear pharmacokinetic profile at a certain dose. This means that the peak response ratio depended on the doses. When the metabolism was over the limitation or saturated, the nonlinear pharmacokinetic then occurred.

This was supported by two observations from the peak response ratio-time profiles of the metabolite from subjects No.1, 2 and 6 (Figure 7). First, the peak of andrographolide metabolite in plasma could be detected only at the highest dose. Second, for the administered doses of 6 and 9 tablets, the similar levels and patterns of the metabolite were observed.



215

mean area ratio 2.5

peak area ratio

mean area ratio (3 tabs) 2

mean area ratio (6 tabs) 1.5

mean area ratio (9 tabs)

1

0.5

0 0

2

4

6

8

10

time (hrs)

Figure 6 The mean peak area ratio-time profiles of the metabolite from six volunteers who had taken three doses of A. paniculata product.

peak area ratio

peak area ratio

1

A

1.5 1 0.5 0

B

0.5

0 0

2

4

6

8

10

0

peak area ratio

C

2

4

6

8

10

time (hrs)

time(hrs)

2.5

2

Figure 7 Peak area ratio-time profiles of the metabolite from Subject No.1 (A), 2 (B) and 6(C) who had taken three doses of A. paniculata product (—— = 3 tablets, −··− = 6 tablets, and ······ = 9 tablets)

1.5 1 0.5 0 0

2

4

6

8

10

time (hrs)

The concentration-time profile is generally expressed for the pharmacokinetic study. The level of metabolite in this study can be shown as only the peak response ratio-time profile because the pure compound

216

was not available and the separation or the synthesis to get the pure compound of this metabolite is out of the scope of this study. However, the structure of the metabolite was identified by using ion-trap liquid



chromatography-mass spectrometer (Bruker®). High possibility of this peak was a glucuronide conjugate metabolite. Its molecular mass ion was at m/z 527 which was equal to the molecular mass ion of andrographolide-glucuronide metabolite. Figure 8 shows

the mass spectrum of this metabolite. More techniques such as Nuclear magnetic resonance (NMR) and Infrared spectroscopy (IR) may be needed to confirm the structure.

Figure 8 Mass spectrum of the metabolite from Ion-Trap liquid chromatography-mass spectrometer (Bruker®) One of the problems in this study was the dosage contained single compound of andrographolide which has not been registered as the medicine in Thailand. So A. paniculata product was used instead of the pure andrographolide product. The efficacy and safety data of using pure andrographolide is lacking because almost of the clinical trials were tested in A. paniculata product but not in the single active compound of andrographolide.

Conclusions It can be concluded that andrographolide is rapidly absorbed and extensively metabolized. The saturated metabolism process of andrographolide occurred at high dose. The individual variation of absorption among subjects was observed which may lead to the variation of therapeutic efficacy. The mechanism of andrographolide metabolism should be further investigated which the processes to get the pure compound of the metabolite is needed. This would be beneficial in the use of this plant and allow more confidence in their efficacy and safety.

Acknowledgements

The authors are indebted to the Center of Research and Development of Herbal Health Product, the Graduate School of Khon Kaen University and the Faculty of Pharmaceutical Sciences, Khon Kaen University for financial support and facilities, and thank Prof. Minoru Isobe for the kind assistance in training of LC-MS technique.

References 1. Medicinal Plant Research Institute. Standard of Thai herbal medicine: Andrographis paniculata (Burm.f.) Nees. Thailand: The War Veterans Organization Press, 1999. 2. Caceres DD, Hancke JL, Burgos RA. Double blind study with a new monodrug Kan Jang: decrease of symptoms and improvement in the recovery from common colds. Phytother Res 1995;9:559-562. 3. Caceres DD, Hancke JL, Burgos RA, Wikman GK. Prevention of common colds with Andrographis paniculata dried extract: a pilot double blind study. Phytomedicine 1997;4(2):101-104.



217

4. Melchior J, Spasov AA, Ostrovskij OV, Bulanov AE, Wikman G. Double-blind, placebo-controlled pilot and phase III study of activity of standardized Andrographis paniculata fixed combination Kan Jung in rats and human. Phytomedicine 2000;7(5):341-364. 5. Melchior J, Palm S, Wikman G. Controlled clinical study of standardized Andrographis paniculata extract in common cold-a pilot trial. Phytomedicine 1996/97;3:315-318. 6. Kulichenko LL, Kireyeva LV, Malyshkina EN, Wikman G. A randomized, controlled study of Kan Jang versus amantadine in the treatment of influenza in Volgograd. J Herb Pharm Ther 2003;3(1):77-93. 7. Spasov AA, Ostrovskij OV, Chernikov MV, Wikman G. Comparative controlled study of Andrographis paniculata fixed combination, Kan Jang and an Echinacea preparation as adjuvant, in the treatment of uncomplicated respiratory disease in children. Phytother Res 2004;18:47-53. 8. Sharma A, Lal K, Handa SS. Standardization of Indian crude drug Kalmegh by high pressure liquid chromatographic determination of andrographolide. Phytochem Anal 1992;3:29. 9. Sriram R, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata. J Exp Ther Onc 2003;3(3):147. 10. Kumar RA, Sridevi K, Kumar NV, Nanduri S, Rajagopal S. Anticancer and immunostimulatory compounds from Andrographis paniculata. J Ethnopharm 2004;92:291295. 11. Madav S, Tandan SK, Lal J, Tripathi HC. Antiinflammatory activity of andrographolide. Fitotherapia 1996;67:452-458. 12. Gupta PP, Tandon JS, Patnaik GK. Antiallergic activity of andrographolides isolated from Andrographis paniculata (Burm.F) Wall. Pharm Bio 1998;36(1):72-74. 13. Panossian A, Ovhannisyan A, Mamikonyan G. Pharmacokinetic and oral bioavailability of andrographolide from Andrographis paniculata fixed combination Kan Jang in rats and human. Phytomedicine 2000;7(5):351-364. 14. U.S. Department of Health and Human Services. Bioanalytical method validation. (Accessed on March 7, 2003, at http://www.fda.gov/cder/guidance)

218

15. Drug Control Division, Food and Drug Administration, Ministry of Public Health. Thailand guidelines for the conduct of bioavailability and bioequivalence studies. Thailand. 16. Daodee S, Wangboonskul J, Jarukamjorn K, Sripanidkulchai B. The consideration for the quality control of Andrographis paniculata products. KKU Res J (in press).

