Journal of Ethnopharmacology, 28 (1990) 235-241 Elsevier Scientific Publishers Ireland Ltd.
235
I’HARMACOKINETICS OF THE TOTAL TRITERPENIC FRACTION OF CEiVTEUA ASJATICA AFTER SINGLE AND MULTIPLE ADMINISTRATIONS TO HEALTHY VOLUNTEERS. A NEW ASSAY FOR ASIATIC ACID
R. GRIMALDI”, F. DE PONTI*, L. D’ANGELO’, LECCHINIb, G.M. FRIGO* and A. CREMA*
M. CARAVAGGIb,
G. GUIDI”,
S.
“Sharper SpA, Dire&one Medico, Rodano, Milan and bDepartment of Internal Medicine and Therapeutics, Section of Pharmacology and Toxicology, University of Pavia, Piazza Botta 10, Z-27100 Pavia (Italy) (Accepted September 28, 1989)
Summary A new HPLC assay method was used to investigate the pharmacokinetics of asiatic acid after oral administration of the total triterpenic fraction of Centella asiatica in single doses (30 or 60 mg) and after a 7-day treatment (30 or 60 mg twice daily). Twelve healthy volunteers received each treatment following a randomized cross-over design with trials separated by a 3-week interval. The time of peak plasma concentration was not affected by dosage difference or by treatment scheme. Differences in peak plasma concentration and area under the concentration vs. time curve from 0 to 24 h (AUC, _ & calculated after 30 or 60 mg administration (single dose) were accounted for by the different dose regimen. However, after chronic treatment with both 30 and 60 mg, peak plasma concentrations, AU& _ 24 and halflife were significantly higher than those observed after the corresponding single dose administration. This phenomenon could be explained by a metabolic interaction between asiatic acid and asiaticoside, which is transformed into asiatic acid in vivo.
Introduction The total triterpenic fraction (TTF) of Centella asiatica (L.) Urban. (family Umbelliferae) is a natural product which has been used for many years in the treatment of venous ulcers and venous hypertension for its activity on connective Correspondence to: Dr. F. De Ponti. 0378-8741/90/$2.80 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
236
tissue metabolism (Sasaki et al., 1972; Spicer and Urwin, 1972; Tenni et al., 1988) and endothelial integrity (Montecchio et al., 1989). The three principal components of the TTF of C. asiatica are asiatic acid, madecassic acid and asiaticoside. Asiatic and madecassic acids together account for approximately 60% and asiaticoside for 40% of the product. There is some evidence that asiatic acid is the most therapeutically active of the three components (Marquart et al., 1989) and that the therapeutic effects of asiaticoside are mediated through its conversion into asiatic acid (unpublished data). Despite its long time use, studies on the pharmacokinetics of the TTF of C. asiuticu have been hampered by the lack of suitable analytical techniques, as often happens with extractives. In the clinical field, on the contrary, the development of new techniques, such as laser-Doppler flowmetry, has provided new insights into the physiological alterations which characterize venous hypertension and has allowed objective and reliable measurements of drug effects in this condition. Thus, it has been shown that the TTF of C. usiuticu can antagonize some of the pathologic changes, such as baseline resting flow and venivasomotor reflex, as measured by laser-Doppler flowmetry (Belcaro, 1987). An increase in transcutaneous oxygen tension and a decrease in transcutaneous carbon dioxide tension have also been reported during treatment with TTF (Allegra et al., 1987). Both symptomatological and instrumental parameters have shown greater improvement during treatment with TTF 60 mg twice daily than with TTF 30 mg twice daily. Although the difference of the doses may account for the better results, it is also possible that pharmacokinetic factors, such as dose-dependent pharmacokinetics, may play a role. This laboratory has recently developed a new HPLC assay method to measure plasma concentrations of asiatic acid. The aims of the present study were to apply this new technique to assess the pharmacokinetics of asiatic acid after oral administration of different doses of TTF, both after single administration and after chronic treatment, and to investigate whether pharmacokinetic factors, beyond the simple dosing difference, may contribute to the explanation of the better results obtained with higher doses. Methods Subjects and protocol Twelve healthy male volunteers, aged 22-31 years, were recruited for the study after full clinical examination. Routine blood and urine laboratory tests were performed. All subjects signed an informed consent form according to the provisions of the Helsinki and Tokyo declarations. The protocol was approved by the local ethics committee. Each subject received, according to a randomized crossover design, the following treatments separated by a three-week interval: (i) 30 mg of TTF in a single dose after an overnight fast, followed by a treatment with TTF (30 mg twice daily) for 6 days, starting on the second day, with the last dose of 30 mg given on the morning of the seventh day after an overnight fast; (ii) 60 mg of TTF in single dose after an overnight fast, followed by a treatment with TTF (60 mg twice daily) for 6 days, starting on the second day, with the last dose of 60 mg given on the morning of the seventh day after an overnight fast.
237
On days 1 and 7 of both treatments, no food or drink were allowed for 3 h after drug administration and blood samples were taken into heparinized tubes at the following times: 0 (predose on day 1 or before the last dose on day 7), 1, 1.5,2,3,4, 6, 8, 10, 12, 14 and 24 h after dosing. Samples were centrifuged immediately at 5000 rev./min for 10min and plasma was separated and stored at -20°C until analysis. Assay method Plasma (2 ml) was added to 200 ~1 of HJO, (85%) and extracted twice with 5 ml chloroform. The chloroformic phase was then dried under a nitrogen stream. The dried residue was added to 400 ~1 methanol and centrifuged at 5000 rev./min for 5 min. The final solution (100 ~1) was injected into the column of a high-performance liquid chromatograph (Perkin Elmer Corp., Pomona, CA). Assays were performed using a fixed loop (100 pl), a Perkin Elmer C,, precolumn and a Perkin Elmer Cyanosil-X-10 column. The mobile phase was a mixture of (a) acetonitrile/methanol (700 : 200) and (b) distilled water/H,PO, (85%); the two solutions were premixed by a Perkin Elmer SE 410 pump at the ratio of 42 : 58. The flow rate was 1 ml/min; readings were performed with a Perkin Elmer LC-95 detector at 200nm wavelength. Values of asiatic acid concentrations were obtained by fitting the areas of asiatic acid peaks from the sample to a calibration curve obtained by standard samples containing known concentrations of asiatic acid and undergoing the same extraction and determination procedures. Kinetic and statistical analysis The following parameters have been calculated both after a single administration and at the end of the two treatment periods: time of peak plasma concentration (t,,,) and peak plasma concentration (C,,,), directly from the experimental data; area under the concentration vs. time curve from time 0 to 24 h (AUC,_,,) according to the linear trapezoidal rule; terminal elimination rate constant (p) by linear least-squares regression analysis of the log concentration vs. time curve; halflife (t& according to the relationship: tljz = 0.69318. Statistical analysis was performed by means of the Student’s t-test for paired data. Results
Figures 1 and 2 show mean plasma concentrations of asiatic acid after single administration and at the end of chronic treatment with 30 and 60 mg of TTF of C. asiatica, respectively. Mean pharmacokinetic parameters are given in Table 1. After single administration of 30 mg, values below the limit of detection were observed in most subjects at the first sampling time and from the tenth hour after administration onwards. In no subject could asiatic acid be detected 24 h after dosing (Fig. 1). After single administration of 60 mg, asiatic acid levels were detectable from the first sampling time and, in most subjects, up to the 12-h sampling time. As with the
238 Plasma level OWW
1.5
1
30 Time (h)
Fig. 1. Mean (+ S.E.M.) plasma concentrations of asiatic acid after administration of 30 mg of TTF Centella asiatica; 0, single administration; n , chronic treatment.
of
Plasma level (wml)
30 Tlme (h)
Fig. 2. Mean ( f S.E.M.) plasma concentrations of asiatic acid after administration of 60 mg of TTF Centella asiatica: 0, single administration; n , chronic treatment.
of
lower dose, in no subject were asiatic acid levels detectable 24 h after dosing (Fig. 2). Although large inter-subject variability was found, plasma levels generally increased to 3-6 h after administration for both 30mg and 60mg and then decreased rapidly. As shown in Table 1, mean t,,, was not significantly different between doses, while the differences in mean C,,, and AUCo-24 were accounted for by dosing difference. No significant difference was found in mean half-life, although a somewhat higher value was obtained after 60mg than after 30mg (Table 1).
239 TABLE
1
PHARhUCOKINETIC Parameter
t,, (h> C,, @g/ml) tl/z (h) AU%a., fpgfmllh) -_.
PARAMETERS Chronic
Single dose
--
treatment
30 mg
6Omg
30 mg
6omg
4.5 50.4 0.703c0.11 2.20f0.30 4.16f1.10
4.2 +0.3 1.36f0.13” 3.4OkO.68 9.39 * 1.02”
4.1 +0.3 1.03 * 0.05* 6.33+ 1.82b 10.47 f 1.09b
4.2 +0.4 1.69f0.07b.’ 10.28+ 1.8d 20.83f l.27b-C
-
Values are means + S.E.M. Significance: “P < 0.05 vs. 30mg single dose; ‘P < 0.05 vs. corresponding single dose; 'P -c0.05vs.30 mg chronic treatment.
AUC0_24 values after chronic treatment with both doses were consistently higher than AUC, _ 24 values after single dosing. A highly significant increase was found with both the lower dose and the higher dose (Table I>. These findings were accompanied by an increase, although to a lesser extent, in peak plasma concentrations, which was more marked for the lower than for the higher dose (Table 1). The time of peak plasma concentration remained unchanged after chronic treatment. Half-life values showed a significant increase as compared to the values obtained after the corresponding single administration with both the lower and the higher dose (Table 1). Half-life after chronic treatment with 60mg twice daily was somewhat higher than after chronic treatment with 30mg twice daily, but this difference was not statistically significant. Discussion The most striking result of our study is the increase in AUC, _ 24, peak plasma concentrations and half-life values after multiple administrations of the TTF of C. usiutica. As far as AUC, _ 24 is concerned, the different values obtained with the two doses both after single administration and at the end of chronic treatment are completely accounted for by dosing difference. Yet, within-dose comparison (AU& _ 24 after chronic treatment vs. AUC after single dose) showed a signi~c~t increase in AU& _ 24 values with both treatment schemes. This phenomenon was unexpected in consideration of the short half-life obtained after single administration. moreover, the plasma half-life increased at the end of chronic treatment with both doses, but especially with the higher one, as if this parameter was doseand time-dependent. Interpretation of these results for practical significance is difficult especially because data about the pharmacokinetics of asiaticoside and madecassic acid are lacking. However, a recent cross-over study (unpublished data) investigating the pharmacokinetics of asiatic acid and asiaticoside after administration of equimolar
240
doses of the two pure compounds helps one to interpret the present data. Briefly, asiaticoside administration contributes to the plasma levels of asiatic acid because of the in vivo transformation of asiaticoside into asiatic acid. Since there is no difference between asiatic acid AUCs obtained after the administration of the two pure substances independently, the biotransformation of asiaticoside into asiatic acid is probably complete, but needs some time to take place, as indicated by a significant delay in the time of peak plasma concentrations of asiatic acid after asiaticoside administration. Thus, one can assume that both the dose and the number of administrations may interfere with the equilibrium of the metabolic reaction generating asiatic acid from asiaticoside. If this is the case, during chronic treatment, especially with higher doses, the metabolic reaction will be slowed in a manner proportional to blood levels of asiatic acid, either because of delayed absorption or because of delayed metabolism. This hypothesis may help to interpret our results. While between-dose differences in pharmacokinetic parameters are completely accounted for by dosing differences, within-dose comparisons give evidence of accumulation phenomena during chronic treatment, as shown by AUCs and, to a lesser extent, by peak plasma concentrations. The accumulation may be explained by a delayed transformation of asiaticoside into asiatic acid, because of the higher plasma levels of the latter during chronic treatment. Asiaticoside can be considered as a prodrug, whose transformation rate is determined by asiatic acid levels: the higher the latter, the slower the rate of transformation. A delay in the biotransformation of asiaticoside may interfere with the half-life calculation. Biases in half-life calculation, due to overlapping of processes of asiatic acid generation with elimination phenomena, may thus be expected. As a matter of fact, the identification of the terminal phase of elimination has been difficult in many subjects after chronic treatment, especially with the higher dose. These observations may also explain the apparent time- and dose-dependency of the halflife. The clinical relevance of this possible metabolic interaction between asiaticoside and asiatic acid deserves some consideration. As a consequence of delayed biotransformation, the time-course of plasma asiatic acid levels could be more stable during treatments with higher doses of TTF. This may help to explain the better results obtained in clinical trials with higher doses, suggesting that pharmacokinetic factors may also be involved, besides the mere dose difference. In conclusion, the development of a new method to determine plasma levels of asiatic acid has been the first step for a better understanding of the pharmacokinetics of the TTF of Centella asiatica. Further studies, including pharmacokinetic data about all the components of TTF, are required to confirm our hypothesis on the different pharmacokinetic parameters found after single and multiple dosing. Acknowledgement The authors thank Mr. M. Pica for his skilful technical assistance.
241
References Allegra, C., Antonini, V., Carlizza, A. and Tonelli, V. (1987) L’estratro titolato triterpenico di Centella asiarica nella insufficienza venosa. Minetva Angiologica 12, 107- 116. Belcaro, G. (1987) Microvascular evaluation by Laser-Doppler flowmetry of the effects of Centellase in the treatment of severe venous hypertension and leg ulcers. Phlebology 2, 189-195. Marquart, F.X., Bellon, G., Gillery, P., Randoux, A., Brieu, M.A. and Borel, J.P. (1989) Stimulation of collagen synthesis in fibroblasts cultures by an extract of Cenrella asiatica: specific effect on the free proline pool. Biochimica et Biophysics Acta, in press. Montecchio, G.P., Della Moretta, A. and Piovella, F. (1989) Effect of treatment with Centella asiarica total tritepenic fraction (Centellasea) on the number of circulating endothelial cells in subjects with venous insufficiency. Thrombosis Research, in press. Sasaki, S., Shinkai, H., Akashi, Y. and Kishihara, Y. (1972) Studies of the mechanism of action of asiaticoside (Madecassol@) on experimental granulation tissue and cultured fibroblasts and its clinical application in systemic scleroderma. Acta Dermatonenereologica 52, 141-150. Spicer, E.J.F. and Urwin, C. (1972) Effect of Madecassol on fibroblasts in tissue culture. Antenne Medicale 7 (Suppl. 3), 23-25. Tenni, R., Zanaboni, G., De Agostini, M.P., Rossi, A., Bendotti, C. and Cetta, G. (1988) Effect of the triterpenoid fraction of Centella asiatica on macromolecules of the connective matrix in human skin fibroblast cultures. The Italian Journal of Biochemistry 37, 69-77.
235
I’HARMACOKINETICS OF THE TOTAL TRITERPENIC FRACTION OF CEiVTEUA ASJATICA AFTER SINGLE AND MULTIPLE ADMINISTRATIONS TO HEALTHY VOLUNTEERS. A NEW ASSAY FOR ASIATIC ACID
R. GRIMALDI”, F. DE PONTI*, L. D’ANGELO’, LECCHINIb, G.M. FRIGO* and A. CREMA*
M. CARAVAGGIb,
G. GUIDI”,
S.
“Sharper SpA, Dire&one Medico, Rodano, Milan and bDepartment of Internal Medicine and Therapeutics, Section of Pharmacology and Toxicology, University of Pavia, Piazza Botta 10, Z-27100 Pavia (Italy) (Accepted September 28, 1989)
Summary A new HPLC assay method was used to investigate the pharmacokinetics of asiatic acid after oral administration of the total triterpenic fraction of Centella asiatica in single doses (30 or 60 mg) and after a 7-day treatment (30 or 60 mg twice daily). Twelve healthy volunteers received each treatment following a randomized cross-over design with trials separated by a 3-week interval. The time of peak plasma concentration was not affected by dosage difference or by treatment scheme. Differences in peak plasma concentration and area under the concentration vs. time curve from 0 to 24 h (AUC, _ & calculated after 30 or 60 mg administration (single dose) were accounted for by the different dose regimen. However, after chronic treatment with both 30 and 60 mg, peak plasma concentrations, AU& _ 24 and halflife were significantly higher than those observed after the corresponding single dose administration. This phenomenon could be explained by a metabolic interaction between asiatic acid and asiaticoside, which is transformed into asiatic acid in vivo.
Introduction The total triterpenic fraction (TTF) of Centella asiatica (L.) Urban. (family Umbelliferae) is a natural product which has been used for many years in the treatment of venous ulcers and venous hypertension for its activity on connective Correspondence to: Dr. F. De Ponti. 0378-8741/90/$2.80 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
236
tissue metabolism (Sasaki et al., 1972; Spicer and Urwin, 1972; Tenni et al., 1988) and endothelial integrity (Montecchio et al., 1989). The three principal components of the TTF of C. asiatica are asiatic acid, madecassic acid and asiaticoside. Asiatic and madecassic acids together account for approximately 60% and asiaticoside for 40% of the product. There is some evidence that asiatic acid is the most therapeutically active of the three components (Marquart et al., 1989) and that the therapeutic effects of asiaticoside are mediated through its conversion into asiatic acid (unpublished data). Despite its long time use, studies on the pharmacokinetics of the TTF of C. asiuticu have been hampered by the lack of suitable analytical techniques, as often happens with extractives. In the clinical field, on the contrary, the development of new techniques, such as laser-Doppler flowmetry, has provided new insights into the physiological alterations which characterize venous hypertension and has allowed objective and reliable measurements of drug effects in this condition. Thus, it has been shown that the TTF of C. usiuticu can antagonize some of the pathologic changes, such as baseline resting flow and venivasomotor reflex, as measured by laser-Doppler flowmetry (Belcaro, 1987). An increase in transcutaneous oxygen tension and a decrease in transcutaneous carbon dioxide tension have also been reported during treatment with TTF (Allegra et al., 1987). Both symptomatological and instrumental parameters have shown greater improvement during treatment with TTF 60 mg twice daily than with TTF 30 mg twice daily. Although the difference of the doses may account for the better results, it is also possible that pharmacokinetic factors, such as dose-dependent pharmacokinetics, may play a role. This laboratory has recently developed a new HPLC assay method to measure plasma concentrations of asiatic acid. The aims of the present study were to apply this new technique to assess the pharmacokinetics of asiatic acid after oral administration of different doses of TTF, both after single administration and after chronic treatment, and to investigate whether pharmacokinetic factors, beyond the simple dosing difference, may contribute to the explanation of the better results obtained with higher doses. Methods Subjects and protocol Twelve healthy male volunteers, aged 22-31 years, were recruited for the study after full clinical examination. Routine blood and urine laboratory tests were performed. All subjects signed an informed consent form according to the provisions of the Helsinki and Tokyo declarations. The protocol was approved by the local ethics committee. Each subject received, according to a randomized crossover design, the following treatments separated by a three-week interval: (i) 30 mg of TTF in a single dose after an overnight fast, followed by a treatment with TTF (30 mg twice daily) for 6 days, starting on the second day, with the last dose of 30 mg given on the morning of the seventh day after an overnight fast; (ii) 60 mg of TTF in single dose after an overnight fast, followed by a treatment with TTF (60 mg twice daily) for 6 days, starting on the second day, with the last dose of 60 mg given on the morning of the seventh day after an overnight fast.
237
On days 1 and 7 of both treatments, no food or drink were allowed for 3 h after drug administration and blood samples were taken into heparinized tubes at the following times: 0 (predose on day 1 or before the last dose on day 7), 1, 1.5,2,3,4, 6, 8, 10, 12, 14 and 24 h after dosing. Samples were centrifuged immediately at 5000 rev./min for 10min and plasma was separated and stored at -20°C until analysis. Assay method Plasma (2 ml) was added to 200 ~1 of HJO, (85%) and extracted twice with 5 ml chloroform. The chloroformic phase was then dried under a nitrogen stream. The dried residue was added to 400 ~1 methanol and centrifuged at 5000 rev./min for 5 min. The final solution (100 ~1) was injected into the column of a high-performance liquid chromatograph (Perkin Elmer Corp., Pomona, CA). Assays were performed using a fixed loop (100 pl), a Perkin Elmer C,, precolumn and a Perkin Elmer Cyanosil-X-10 column. The mobile phase was a mixture of (a) acetonitrile/methanol (700 : 200) and (b) distilled water/H,PO, (85%); the two solutions were premixed by a Perkin Elmer SE 410 pump at the ratio of 42 : 58. The flow rate was 1 ml/min; readings were performed with a Perkin Elmer LC-95 detector at 200nm wavelength. Values of asiatic acid concentrations were obtained by fitting the areas of asiatic acid peaks from the sample to a calibration curve obtained by standard samples containing known concentrations of asiatic acid and undergoing the same extraction and determination procedures. Kinetic and statistical analysis The following parameters have been calculated both after a single administration and at the end of the two treatment periods: time of peak plasma concentration (t,,,) and peak plasma concentration (C,,,), directly from the experimental data; area under the concentration vs. time curve from time 0 to 24 h (AUC,_,,) according to the linear trapezoidal rule; terminal elimination rate constant (p) by linear least-squares regression analysis of the log concentration vs. time curve; halflife (t& according to the relationship: tljz = 0.69318. Statistical analysis was performed by means of the Student’s t-test for paired data. Results
Figures 1 and 2 show mean plasma concentrations of asiatic acid after single administration and at the end of chronic treatment with 30 and 60 mg of TTF of C. asiatica, respectively. Mean pharmacokinetic parameters are given in Table 1. After single administration of 30 mg, values below the limit of detection were observed in most subjects at the first sampling time and from the tenth hour after administration onwards. In no subject could asiatic acid be detected 24 h after dosing (Fig. 1). After single administration of 60 mg, asiatic acid levels were detectable from the first sampling time and, in most subjects, up to the 12-h sampling time. As with the
238 Plasma level OWW
1.5
1
30 Time (h)
Fig. 1. Mean (+ S.E.M.) plasma concentrations of asiatic acid after administration of 30 mg of TTF Centella asiatica; 0, single administration; n , chronic treatment.
of
Plasma level (wml)
30 Tlme (h)
Fig. 2. Mean ( f S.E.M.) plasma concentrations of asiatic acid after administration of 60 mg of TTF Centella asiatica: 0, single administration; n , chronic treatment.
of
lower dose, in no subject were asiatic acid levels detectable 24 h after dosing (Fig. 2). Although large inter-subject variability was found, plasma levels generally increased to 3-6 h after administration for both 30mg and 60mg and then decreased rapidly. As shown in Table 1, mean t,,, was not significantly different between doses, while the differences in mean C,,, and AUCo-24 were accounted for by dosing difference. No significant difference was found in mean half-life, although a somewhat higher value was obtained after 60mg than after 30mg (Table 1).
239 TABLE
1
PHARhUCOKINETIC Parameter
t,, (h> C,, @g/ml) tl/z (h) AU%a., fpgfmllh) -_.
PARAMETERS Chronic
Single dose
--
treatment
30 mg
6Omg
30 mg
6omg
4.5 50.4 0.703c0.11 2.20f0.30 4.16f1.10
4.2 +0.3 1.36f0.13” 3.4OkO.68 9.39 * 1.02”
4.1 +0.3 1.03 * 0.05* 6.33+ 1.82b 10.47 f 1.09b
4.2 +0.4 1.69f0.07b.’ 10.28+ 1.8d 20.83f l.27b-C
-
Values are means + S.E.M. Significance: “P < 0.05 vs. 30mg single dose; ‘P < 0.05 vs. corresponding single dose; 'P -c0.05vs.30 mg chronic treatment.
AUC0_24 values after chronic treatment with both doses were consistently higher than AUC, _ 24 values after single dosing. A highly significant increase was found with both the lower dose and the higher dose (Table I>. These findings were accompanied by an increase, although to a lesser extent, in peak plasma concentrations, which was more marked for the lower than for the higher dose (Table 1). The time of peak plasma concentration remained unchanged after chronic treatment. Half-life values showed a significant increase as compared to the values obtained after the corresponding single administration with both the lower and the higher dose (Table 1). Half-life after chronic treatment with 60mg twice daily was somewhat higher than after chronic treatment with 30mg twice daily, but this difference was not statistically significant. Discussion The most striking result of our study is the increase in AUC, _ 24, peak plasma concentrations and half-life values after multiple administrations of the TTF of C. usiutica. As far as AUC, _ 24 is concerned, the different values obtained with the two doses both after single administration and at the end of chronic treatment are completely accounted for by dosing difference. Yet, within-dose comparison (AU& _ 24 after chronic treatment vs. AUC after single dose) showed a signi~c~t increase in AU& _ 24 values with both treatment schemes. This phenomenon was unexpected in consideration of the short half-life obtained after single administration. moreover, the plasma half-life increased at the end of chronic treatment with both doses, but especially with the higher one, as if this parameter was doseand time-dependent. Interpretation of these results for practical significance is difficult especially because data about the pharmacokinetics of asiaticoside and madecassic acid are lacking. However, a recent cross-over study (unpublished data) investigating the pharmacokinetics of asiatic acid and asiaticoside after administration of equimolar
240
doses of the two pure compounds helps one to interpret the present data. Briefly, asiaticoside administration contributes to the plasma levels of asiatic acid because of the in vivo transformation of asiaticoside into asiatic acid. Since there is no difference between asiatic acid AUCs obtained after the administration of the two pure substances independently, the biotransformation of asiaticoside into asiatic acid is probably complete, but needs some time to take place, as indicated by a significant delay in the time of peak plasma concentrations of asiatic acid after asiaticoside administration. Thus, one can assume that both the dose and the number of administrations may interfere with the equilibrium of the metabolic reaction generating asiatic acid from asiaticoside. If this is the case, during chronic treatment, especially with higher doses, the metabolic reaction will be slowed in a manner proportional to blood levels of asiatic acid, either because of delayed absorption or because of delayed metabolism. This hypothesis may help to interpret our results. While between-dose differences in pharmacokinetic parameters are completely accounted for by dosing differences, within-dose comparisons give evidence of accumulation phenomena during chronic treatment, as shown by AUCs and, to a lesser extent, by peak plasma concentrations. The accumulation may be explained by a delayed transformation of asiaticoside into asiatic acid, because of the higher plasma levels of the latter during chronic treatment. Asiaticoside can be considered as a prodrug, whose transformation rate is determined by asiatic acid levels: the higher the latter, the slower the rate of transformation. A delay in the biotransformation of asiaticoside may interfere with the half-life calculation. Biases in half-life calculation, due to overlapping of processes of asiatic acid generation with elimination phenomena, may thus be expected. As a matter of fact, the identification of the terminal phase of elimination has been difficult in many subjects after chronic treatment, especially with the higher dose. These observations may also explain the apparent time- and dose-dependency of the halflife. The clinical relevance of this possible metabolic interaction between asiaticoside and asiatic acid deserves some consideration. As a consequence of delayed biotransformation, the time-course of plasma asiatic acid levels could be more stable during treatments with higher doses of TTF. This may help to explain the better results obtained in clinical trials with higher doses, suggesting that pharmacokinetic factors may also be involved, besides the mere dose difference. In conclusion, the development of a new method to determine plasma levels of asiatic acid has been the first step for a better understanding of the pharmacokinetics of the TTF of Centella asiatica. Further studies, including pharmacokinetic data about all the components of TTF, are required to confirm our hypothesis on the different pharmacokinetic parameters found after single and multiple dosing. Acknowledgement The authors thank Mr. M. Pica for his skilful technical assistance.
241
References Allegra, C., Antonini, V., Carlizza, A. and Tonelli, V. (1987) L’estratro titolato triterpenico di Centella asiarica nella insufficienza venosa. Minetva Angiologica 12, 107- 116. Belcaro, G. (1987) Microvascular evaluation by Laser-Doppler flowmetry of the effects of Centellase in the treatment of severe venous hypertension and leg ulcers. Phlebology 2, 189-195. Marquart, F.X., Bellon, G., Gillery, P., Randoux, A., Brieu, M.A. and Borel, J.P. (1989) Stimulation of collagen synthesis in fibroblasts cultures by an extract of Cenrella asiatica: specific effect on the free proline pool. Biochimica et Biophysics Acta, in press. Montecchio, G.P., Della Moretta, A. and Piovella, F. (1989) Effect of treatment with Centella asiarica total tritepenic fraction (Centellasea) on the number of circulating endothelial cells in subjects with venous insufficiency. Thrombosis Research, in press. Sasaki, S., Shinkai, H., Akashi, Y. and Kishihara, Y. (1972) Studies of the mechanism of action of asiaticoside (Madecassol@) on experimental granulation tissue and cultured fibroblasts and its clinical application in systemic scleroderma. Acta Dermatonenereologica 52, 141-150. Spicer, E.J.F. and Urwin, C. (1972) Effect of Madecassol on fibroblasts in tissue culture. Antenne Medicale 7 (Suppl. 3), 23-25. Tenni, R., Zanaboni, G., De Agostini, M.P., Rossi, A., Bendotti, C. and Cetta, G. (1988) Effect of the triterpenoid fraction of Centella asiatica on macromolecules of the connective matrix in human skin fibroblast cultures. The Italian Journal of Biochemistry 37, 69-77.
