Key words
Centella asiatica
- Apiaceae - ECa 233 - madecassoside - asiaticoside - pharmacokinetics
Abbreviations
ALT: alanine aminotransferase
AST: aspartate aminotransferase
AUC0–24
: area under the curve from time zero to 24 h
AUC0–inf
: area under the curve from time zero to infinity
Cmax
: maximal plasma concentration
Clapp
: apparent clearance
ECa 233: standardized extract of Centella asiatica
IS: internal standard
IV: intravenous
Kp: tissue to plasma ratio
MRT: mean resident time
NSS: normal saline solution
PO: per oral
T1/2
: elimination half-life
Tmax
: time to reach maximal plasma concentration
Vapp
: apparent volume of distribution
XlogP: partition coefficient
Introduction
Traditional medicine serves as an alternative choice for disease prevention and treatment
for people in developing countries. Centella asiatica (L.) Urb., a tropical herb belonging to the Apiaceae family, is commonly used in
alternative and traditional medicines in Asia [1]
[2]
[3]. C. asiatica from different sources showed significant variations in the quality and quantity
of its bioactive compounds, leading to unpredictable pharmacological activities. Using
a strictly controlled method, we developed a standardized extract of C. asiatica, ECa 233 [4]
[5], which was defined as a white to off-white extracted powder of C. asiatica that contained triterpenoid glycosides, namely, madecassoside and asiaticoside, in
an amount of at least 80% and the ratio between them was maintained at 1.5±0.5:1 ([Fig. 1]). ECa 233 exhibited pharmacological profiles that corresponded to the traditional
use of C. asiatica in humans; that is, wound healing properties, neuroprotective properties, and memory-enhancing
effects [6]
[7]
[8]
[9]. In parallel with the favorable pharmacological profiles, ECa 233 demonstrated minimal
toxicity in both acute and subchronic toxicity tests in rodents [10].
Fig. 1 Chemical structures of triterpenes from C. asiatica (Glu: glucose, Rha: rhamnose).
Recently, our laboratory reported pharmacokinetic profiles following single and multiple
oral dosings of ECa 233 in rodents [11]
[12]. This standardized extract showed dose linearity within a pharmacologically effective
dose range of 50–200 mg/kg PO in male Wistar rats. Madecassoside and asiaticoside,
in a clear solution of ECa 233, rapidly reached the maximum concentration in plasma
within 1 h following oral dosing and were prominently detected in most pharmacologically
relevant organs (e. g., skin and brain). Negligible amounts of the proposed active
metabolites, madecassic and asiatic acids, were detected in rat plasma, and, unexpectedly,
we found a bidirectional interconversion between madecassoside and asiaticoside [13]. Both triterpenoid glycosides were mainly excreted via the hepatobiliary system,
and were extensively biotransformed into their respective triterpenic acids, madecassic
and asiatic acids, likely by normal flora in the gastrointestinal tract [14]
[15]. Interestingly, in relation to the pharmacokinetics of its respective single compounds,
an increased exposure of the two triterpenoid glycosides present in ECa 233 was observed. Questions on whether other minor constituents in ECa 233 may underlie
increased exposure to its major bioactive constituents, madecassocide and asiaticoside,
therefore, emerged and required clarification. To obtain more insight into the results
previously observed, we compared the pharmacokinetic behavior of ECa 233 compared
it with that exhibited by a mixture of madecassoside and asiaticoside at equivalent
amounts. The pharmacologically active dose of ECa 233 at 100 mg/kg PO, containing
51% madecassoside and 38% asiaticoside, was administered to male Wistar rats. Accordingly, a mixture of 51 mg/kg
madecassoside and 38 mg/kg asiaticoside was prepared for the comparative pharmacokinetic
study. In addition, a study of the pharmacokinetics following intravenous administration
of ECa 233 at 10 mg/kg and a mixture of madecassoside and asiaticoside at an equivalent
amount was also conducted. In addition to madecassoside and asiaticoside, other minor
constituents are also present in ECa 233 and their impact on the major constituents
remains elusive. Information obtained from this study could illustrate the significance
of interactions among the different chemical constituents that are present in ECa
233.
Results
All rats had a normal physical appearance both before and after receiving ECa 233
or the mixture of madecassoside and asiaticoside. There were no significant changes
in the creatinine level, reflecting normal kidney function after dosing for all test
groups at 24 h. Similarly, stable AST and ALT levels were observed after dosing in
all of the test groups. There were no significant alterations in liver markers for
any of the animals tested, either via IV or PO routes of administration ([Table 1]).
Table 1 Physical appearance and biochemical profiles at time zero and 24 h after dosing.
|
Parameters
|
ECa 233 10 mg/kg IV
|
Mixture (MD+AS) IV
|
ECa 233 100 mg/kg PO
|
Mixture (MD + AS) PO
|
|
Pre-dose
|
Post-dose
|
Pre-dose
|
Post-dose
|
Pre-dose
|
Post-dose
|
Pre-dose
|
Post-dose
|
|
Physical appearance
|
Normal
|
Normal
|
Normal
|
Normal
|
Normal
|
Normal
|
Normal
|
Normal
|
|
Creatinine (mg/dL)
|
0.23±0.06
|
0.20±0.00
|
0.23±0.04
|
0.23±0.06
|
0.20±0.00
|
0.23±0.02
|
0.22±0.04
|
0.20±0.00
|
|
AST (U/L)
|
71.50±19.36
|
68.25±13.48
|
73.83±9.80
|
58.00±2.96
|
48.75±3.30
|
55.00±21.22
|
57.83±7.00
|
61.17±15.34
|
|
ALT (U/L)
|
21.50±12.24
|
21.75±8.88
|
25.00±6.60
|
20.17±6.84
|
14.75±12.94
|
37.75±30.22
|
20.17±5.20
|
13.50±9.42
|
Data are presented as the mean±S.D. (n=4). AS: asiaticoside, MD: madecassoside
Significant differences in the plasma concentration-time profiles between madecassoside
and asiaticoside present in ECa 233 and their respective counterparts at equivalent
doses in the mixture groups were demonstrated ([Fig. 2], [3]). After IV dosing, rats in the ECa 233 group exhibited significantly higher plasma
concentrations of madecassoside and asiaticoside compared with their respective counterparts
in the mixture group. Similarly, oral administration of ECa 233 could prolong exposure
of both madecassoside and asiaticoside compared to the results observed with the mixtures.
Determination of pharmacokinetic parameters from the plasma concentration-time curves
by non-compartmental analysis is presented in [Table 2], [3]. In comparison with the pharmacokinetic profiles of the mixture of madecassoside
and asiaticoside, an increase of approximately 400% of the madecassoside area under
the curve (AUC) and an increase of 50% of the asiaticoside AUC were detected following
IV dosing of ECa 233. Similarly, a significant increase of both the madecassoside
and asiaticoside AUCs were also observed following oral dosing with ECa 233. The MRT
of both madecassoside and asiaticoside following dosing with ECa 233 showed a tendency
to prolong in comparison to these components in the mixture.
Fig. 2 Plasma concentration-time profiles of madecassoside (a) and asiaticoside (b) following intravenous administration of ECa 233 (10 mg/kg) and a mixture of madecassoside
and asiaticoside at an equivalent dose. Data are presented as the mean±S.D. (n=4);
*p<0.05 for ECa 233 vs. mixture.
Fig. 3 Plasma concentration-time profiles of madecassoside (a) and asiaticoside (b) following oral administration of ECa 233 (100 mg/kg) and a mixture of madecassoside
and asiaticoside at an equivalent dose. Data are presented as the mean±S.D. (n=4);
*p<0.05 for ECa 233 vs. mixture.
Table 2 Pharmacokinetic parameters of madecassoside and asiaticoside after intravenous administration
of ECa 233 10 mg/kg and a mixture of madecassoside and asiaticoside at equivalent
doses.
|
Pharmacokinetic parameters
|
Madecassoside
|
Asiaticoside
|
|
ECa 233 10 mg/kg
|
Mixture MD + AS
|
ECa 233 10 mg/kg
|
Mixture MD + AS
|
|
AUC0-24 (µg.h/L)
|
86571.91±40136.51
|
22034.21±7637.14*
|
20139.85±7173.22
|
14707.54±18920.30*
|
|
AUC0-inf (µg.h/L)
|
88907.03±41437.76
|
22362.07±7655.40*
|
22558.42±7997.22
|
15174.02±18848.86*
|
|
Vapp (L/kg)
|
0.55±0.13
|
2.27±0.97*
|
1.99±0.72
|
4.45±3.26
|
|
MRT (h)
|
3.00±1.42
|
1.19±1.07*
|
7.29±5.20
|
3.41±4.33
|
|
Elimination half-life (h)
|
6.21±1.47
|
6.14±2.58
|
7.80±3.08
|
5.59±2.48
|
|
CLapp (L/hr/kg)
|
0.07±0.02
|
0.25±0.07
|
0.19±0.08
|
0.52±0.29
|
Data are presented as the mean±S.D. (n=4); *p<0.05 for ECa 233 vs. mixture. AS: asiaticoside, MD: madecassoside
Table 3 Pharmacokinetic parameters of madecassoside and asiaticoside after oral administration
of ECa 233 100 mg/kg and a mixture of madecassoside and asiaticoside at equivalent
doses.
|
Pharmacokinetic parameters
|
Madecassoside
|
Asiaticoside
|
|
ECa 233 100 mg/kg
|
Mixture MD + AS
|
ECa 233 100 mg/kg
|
Mixture MD + AS
|
|
Cmax (µg/L)
|
3474.63±1969.04
|
618.99±307.64*
|
509.05±213.46
|
697.95±445.78
|
|
Tmax (h)
|
0.08±0.00
|
0.19±0.20
|
0.19±0.21
|
0.08±0.00
|
|
AUC0-24 (µg.h/L)
|
3,572.14±1,001.28
|
747.37±270.94*
|
1409.40±652.63
|
749.35±331.33*
|
|
AUCo-inf (µg.h/L)
|
4241.76±1005.12
|
1131.52±840.01*
|
1787.54±737.60
|
888.50±438.71*
|
|
MRT (h)
|
8.52±3.21
|
7.21±6.77
|
12.72±4.04
|
6.48±4.17
|
|
Elimination half-life (h)
|
7.80±2.24
|
6.77±4.99
|
7.13±1.75
|
5.07±2.01
|
Data are presented as the mean±S.D. (n=4); *p<0.05 for ECa 233 vs. mixture. AS: asiaticoside, MD: madecassoside
The tissue to plasma values (Kp) of madecassoside and asiaticoside following IV dosing
of ECa 233 or the mixture at an equivalent dose are presented in [Fig. 4]. Madecassoside and asiaticoside could be concentrated in and distributed to several
organs, including the skin and brain, which were target sites of pharmacological actions.
The accumulation of madecassoside and asiaticoside in the spleen was observed from
1 to 4 h after dosing with ECa 233 or the mixture. Interestingly, the Kp values of
both madecassoside and asiaticoside increased significantly from 1 to 4 h following
IV dosing of both test formulae. ECa 233 showed higher Kp values of both madecassoside
and asiaticoside in some organs at 1 and 2 h compared with the mixture.
Fig. 4 Tissue to plasma ratio of madecassoside (a, b, c) and asiaticoside (d, e, f) in internal organs at 1, 2, and 4 h after intravenous administration of ECa 233
(10 mg/kg) and a mixture of madecassoside and asiaticoside at an equivalent dose.
Data are presented as the mean±S.D. (n=4).
Negligible amounts of unchanged madecassoside and asiaticoside were found in excreta
following the IV administration of all test formulae ([Table 4]). After dosing for 24-48 h, it was likely that most of the administered madecassoside
and asiaticoside were extensively biotransformed into madecassic and asiatic acids,
before being excreted in feces. There were no significant differences in the concentrations
of madecassic and asiatic acids in feces between the ECa 233 and mixture groups.
Table 4 Percent recovery of madecassoside and asiaticoside via urine and feces after administration
of ECa 233 and a mixture of madecassoside and asiaticoside at an equivalent dose.
|
Percent recovery
|
Intravenous administration
|
Oral administration
|
|
ECa 233 10 mg/kg
|
Mixture (MD + AS)
|
ECa 233 100 mg/kg
|
Mixture (MD + AS)
|
|
0–24 h
|
24–48 h
|
0–24 h
|
24–48 h
|
0–24 h
|
24–48 h
|
0–24 h
|
24–48 h
|
|
Urine
|
Madacassoside
|
1.95±1.62
|
0.07±0.04
|
1.73±2.08
|
0.08±0.02
|
0.02±0.00
|
0.00±0.00
|
0.01±0.00
|
0.01±0.00
|
|
Asiaticoside
|
0.28±0.24
|
0.02±0.04
|
0.23±0.36
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
0.00±0.00
|
|
Feces
|
Madacassoside
|
0.04±0.02
|
0.04±0.04
|
0.08±0.04
|
0.03±0.00
|
0.02±0.00
|
0.01±0.02
|
0.01±0.00
|
0.01±0.00
|
|
Asiaticoside
|
0.05±0.02
|
0.05±0.04
|
0.13±0.06
|
0.04±0.02
|
0.06±0.04
|
0.02±0.00
|
0.04±0.02
|
0.03±0.02
|
|
Madacassic acid
|
25.44±5.02
|
9.80±2.42
|
22.19±6.58
|
10.45±3.68
|
8.84±3.20
|
9.11±5.16
|
6.45±2.42
|
6.03±1.98
|
|
Asiatic acid
|
32.45±7.20
|
17.99±13.44
|
35.56±2.94
|
25.76±2.60
|
11.19±6.98
|
7.68±2.62
|
15.76±4.76
|
20.15±7.40
|
Data are presented as the mean±S.D. (n=4). AS: asiaticoside, MD: madecassoside
The compounds of the ECa 233 extract were analyzed using HPLC ESI-QTOF-MS/MS and two
standards (madecassoside and asiaticoside). The first major peak of the mass chromatogram
was detected at 7.0 min with a molecular weight of 997.4995 daltons (Da). This molecular
weight was deduced to be a sodium adduct [M+Na]+ of madecassoside. The second major peak detected at 7.95 min exhibited a molecular
weight of 981.5034 Da ([Fig. 5]). This molecular weight was deduced to be a sodium-adduct [M+Na]+ of asiaticoside. These two major peaks were confirmed using standards that had the
same retention time eluted with the same solvent systems and molecular weight. MS/MS
fragmentation data provided further evidence for the presence of madecassoside and
asiaticoside in the ECa 233 extract. In addition, a minor peak at 7.38 min with a
molecular weight of 981.5047 Da was detected. This compound was considered to likely
be a sodium adduct [M+Na]+ of centellasaponins, which had an exact molecular weight corresponding to 958.51 Da
plus sodium 22.99 Da. However, the specific type of centellasaponins (A, C, D) remained
undetermined due to the lack of commercially available centellasaponins. Overall,
by matching retention times, mass spectra of sodium ion adducted molecular ions, and
fragmentation patterns of madecassoside and asiaticoside, it was evident that the
minor components of ECa 233 extracts contain a centellasaponin.
Fig. 5 MS chromatogram and spectra from high-resolution LC-MS/MS identifying major and minor
components of ECa 233.
Additionally, two small peaks of minor components at 13.0 and 15.0 min were found.
These signals were confirmed to be madecassic and asiatic acids, consistent with their
retention times and molecular weights compared with analytical standards. These triterpenic
acids accounted for less than 1.0% of ECa 233 using quantitative analysis with a triple
quadrupole LC-MS/MS system.
Discussion
Unlike other commercially available extracts of C. asiatica, ECa 233 is a white to off-white natural extract of C. asiatica, with known and standardized consistent amounts of its bioactive markers [4]. In agreement with traditional uses, ECa 233 exerts numerous pharmacological activities
with minimal toxicity [6]
[7]
[8]
[9]
[16]. Some pharmacokinetic studies of ECa 233 at its effective dose range have been conducted
in recent years [12]. In the present study, comparative pharmacokinetics between ECa 233 and a mixture
of madecassoside and asiaticoside at equivalent amounts were conducted in male Wistar
rats. All animals showed good tolerability following IV or PO dosing of the test formulae.
No significant changes were observed in physical appearance or kidney and liver markers,
which indicates a good safety profile of all test formulae. The results of the present
study corresponded well with previous pharmacokinetic studies and toxicity tests of
ECa 233 [10].
As shown in the plasma concentration-time profiles, all rats in the ECa 233 group
demonstrated significantly higher levels of madecassoside in absorption, distribution,
and elimination phases compared with those of the mixture group. Similar response
profiles were also exhibited by asiaticoside in the ECa 233-treated group where prolonged
plasma levels, especially during the elimination phase, were observed. These results
were similar to those reported by Hengjumrut et al. [13] in which longer exposure of madecassoside and asiaticoside was observed following
administration of ECa 233 compared with the single compounds administered separately.
Oral bioavailability of both madecassoside and asiaticoside was increased approximately
2- to 4-fold following administration in the form of ECa 233, suggesting that other
minor components present in ECa 233 might play a substantial role as a bioenhancer
of madecassoside and asiaticoside [17]
[18]
[19]. Therefore, increases of madecassoside and asiaticoside levels following dosing
with ECa 233 were observed from the absorption through the elimination phase.
Determination of the minor components in ECa 233 was conducted using high-resolution
LC-MS/MS analysis. It appears likely that ECa 233 contains a certain amount of centellasaponin(s)
and is known to contain a small amount of triterpenic acids. In general, appropriate
concentrations of minor components to act as bioenhancers should constitute 10% of
the major components. Therefore, it is possible that centellasaponin(s) could act
as bioenhancers of the two triterpenoid glycosides in ECa 233. Centellasaponins have
structures similar to madecassoside and asiaticoside, which consist of pentacyclic
triterpenes and glucose-glucose-rhamnose. These saponins in ECa 233 might have competitive
or inhibitory activities toward efflux transporters of madecassoside and asiaticoside.
Therefore, improvement of the pharmacokinetic profiles of the two major bioactive
components in ECa 233 was observed from the absorption, distribution, and elimination
phases. The MRTs of both madecassoside and asiaticoside were improved by intravenous
or oral administration of ECa 233 compared with the mixture of pure madecassoside
and asiaticoside. Further studies of the bioenhancer activity of centellasaponins
could be developed if pure chemicals become commercially available in the future.
The Kp of both madecassoside and asiaticoside increased over time from 1 to 4 h following
IV dosing of both test formulae. This result correlated well with our previous finding
that continuous dosing of 100 mg/kg PO of ECa 233 for 7 days could significantly increase
tissue levels of both triterpenoid glycosides [11]. Madecassoside and asiaticoside are large hydrophilic molecules with molecular weights
of more than 900 Da. These large triterpenoid molecules might have limited channels
to enter tissue compartments compared with small lipophilic molecules. In general,
small lipophilic molecules enter tissues by simple diffusion, depending on the concentration
gradient of the diffusing molecules [20]. It is likely that the distribution patterns of both triterpenoid glycosides are
time dependent rather than concentration dependent. Madecassoside and asiaticoside
were able to reach the brain and skin, two major targeted organs of pharmacological
activities. The Kp values in these organs increased from 1 to 4 h following IV dosing
of both test formulae, suggesting the potential for ECa 233 to be used as a neuroprotective
substance for brain injuries.
In the present study, madecassic and asiatic acids, which have previously been proposed
to be the active metabolites [15], were not detected either in plasma or tissue compartments. Therefore, it is likely
that administered madecassoside and asiaticoside were absorbed and distributed as
unchanged triterpenoid glycosides, not triterpenic acids. Subsequently, the triterpenoid
glycosides in the systemic circulation were excreted into the gut lumen via the hepatobiliary
system [21]. From there, the unchanged madecassoside and asiaticoside were further biotransformed
by the gut flora into triterpenic acids before being excreted in feces [14]
[15]. We found abundant amounts of the two triterpenic acid metabolites, madecassic and
asiatic acids, in feces; the percent of recovery was greater than 50% of the administered
doses of triterpenoid glycosides. These two triterpenic acids had very high lipophilic
properties, with XlogP ranging from 4.4 to 5.7. Therefore, excretion of these lipophilic
molecules should occur via the biliary system rather than the urinary system. P-glycoprotein
and multidrug resistance-associated protein 2 are two major transporters responsible
for the excretion of madecassoside and asiaticoside into the biliary system [21]. Further information on the hepatobiliary transport and biotransformation of the
two triterpenoid glycosides in the gastrointestinal tract will be essential for the
development of phytopharmaceutical products from C. asiatica.
Overall, ECa 233, the standardized extract of C. asiatica containing mainly madecassoside and asiaticoside, demonstrated superior pharmacokinetic
profiles compared with the mixture of madecassoside and asiaticoside at an amount
equivalent to their counterparts presented in ECa 233. Higher oral bioavailability
and more rapid tissue distribution of triterpenoid glycosides were observed in rats
treated with ECa 233. There were no significant differences between the metabolism
and excretion profiles of the two triterpenoid glycosides in ECa 233 and the mixture.
Pharmacokinetic information obtained from this study clearly demonstrated prolonged
exposure of the two major bioactive substances, madecassoside and asiaticoside, of
the standardized extract of C. asiatica ECa233 compared with a mixture of the two pure compounds. The significant role of
the minor constituents on the pharmacokinetic profiles of the major constituents calls
for precaution in the interpretation and extrapolation of the data of different extracts.
Materials and Methods
Chemicals
ECa 233, madecassoside, and asiaticoside were provided by Siam Herbal Innovation Co.,
Ltd. The standardized extract (batch number MRA1214004) contained 51% madecassoside
and 38% asiaticoside, and was analyzed by LC-MS/MS. Analytical standards of asiaticoside
(>98.5%), madecassoside (>95.0%), and asiatic acid (>95.0%) were purchased from Sigma-Aldrich
Corp. Madecassic acid (>98.9%) and glycyrrhetinic acid (>99.0%) used for LC-MS/MS
experiments were purchased from Wako Pure Chemical Industries.
Animals and treatments
Male Wistar rats were obtained from the National Laboratory Animal Center, Mahidol
University, Thailand. The rats were housed under 12-h light-dark cycles, 24±2+°C,
50±10% humidity, and ad libitum access to food and water. Rats aged 16–20 weeks that
weighed more than 400 g were used in the pharmacokinetic experiments; they were placed
into metabolic cages and fasted overnight with free access to water. The animal protocols
were approved by the Institutional Animal Care and Use Committee of the Faculty of
Pharmaceutical Sciences, Chulalongkorn University, Thailand (approval number 15-33-002,
approval date April 22, 2015).
Pharmacokinetic experiments
The rats were divided into four groups (n=4): ECa 233 10 mg/kg IV, ECa 233 100 mg/kg
PO, a mixture of madecassoside and asiaticoside (5.1 and 3.8 mg/kg, respectively)
IV, and a mixture of madecassoside and asiaticoside (51 and 38 mg/kg, respectively)
PO. All formulations were freshly prepared as a clear solution in 20% DMSO/NSS before
administration via the lateral tail vein or oral gavage. The rats were anesthetized
with isoflurane during drug administration and blood or organ collections. We collected
300 μL of blood via the lateral tail vein at 0, 5, 15, and 30 min, and 1, 2, 4, 8,
16, and 24 h after dosing. Blood samples were collected from these rats and centrifuged
at 1500×g for 15 min to collect 150 µL of plasma. Plasma samples at 0 and 24 h were used to
determine creatinine, AST, and ALT levels. Tissue samples were collected at 1, 2,
and 4 h after IV dosing, and washed with cold saline solution. Rat excreta, urine,
and feces were collected separately from the metabolic cages 24–48 h after dosing
to determine the excretion routes of drugs and metabolites. All biological samples
were stored at −80°C until analysis.
Sample preparation
Methanol was used as a precipitating agent in the protein precipitation method. Fifty
microliters of plasma or urine samples were thawed at room temperature and vigorously
mixed with 200 µL of methanol containing 10 ng of glycyrrhetinic acid as the IS. The
mixture was centrifuged at 3000×g for 15 min, and the supernatants (10 µL) were analyzed by LC-MS/MS. Fifty milligrams
of feces or tissues were mixed with 200 µL of methanol that contained 10 ng of IS,
homogenized in an ice bath, and processed in the same manner as the liquid samples.
In the case that the drug or metabolite levels exceeded the linearity of the calibration
curves, blank matrices were used to dilute the samples prior to protein precipitation.
LC-MS/MS analysis
LC-MS/MS analysis was conducted under conditions and methods previously described
in a pharmacokinetic study of ECa 233 [12]. In brief, an LC-MS/MS system was conducted with an Eksigent UPLC 100 and a QTRAP
6500 mass spectrometer and Analyst software version 1.6 (AB Sciex Pte., Ltd.). The
stationary phase was a Synergi Fusion-RP C18 column (Phenomenex, Inc.) with a 40°C
oven temperature. The retention times of madecassoside, asiaticoside, madecassic acid,
asiatic acid, and glycyrrhetinic acid were 1.79, 1.82, 1.93, 1.99, and 2.12 min, respectively.
The standard curves of the triterpenoid glycosides and triterpenic acids showed good
linearity, with R2>0.99 for 0.5–300 µg/L. The lower limit of detection was 0.1–0.5 µg/L, and intra-
and inter-day precision and accuracy were within±10%. The calculated percent recoveries were more than 70% for all analytes.
Minor components identification
In this study, the identification of the minor components of ECa 233 was conducted
with high-resolution LC-MS/MS analysis. LC-MS/MS analysis was performed on an Agilent
6530 QTOF mass spectrometer with an Agilent HPLC 1260 binary pump. A Kinetex Phenomenex
C18 column (3.5 μm, 2.1×150 mm) was used as the stationary phase. The mobile phase was
run with a gradient of 0.1% formic acid in water and acetonitrile (20-80%) at a flow
rate of 0.2 mL/min. The mass spectrometer was equipped with an Agilent Jet Stream
ESI source. LC-MS/MS was performed in the positive ionization mode over a mass range
of m/z 100-1700 at a scan rate of 3 spectra/second. ECa 233 (10 µL) at a concentration of
0.1 mg/mL was injected into the LC system, with the column oven maintained at 25+°C.
The elution gradient of the mobile phase started with 20% acetonitrile at time zero
and increased to 80% acetonitrile after 20 min, was maintained at 80% acetonitrile
for 3 min, decreased to 20% acetonitrile over 5 min, and was equilibrated with 20%
acetonitrile for 5 min. Analysis of the eluates was conducted in the positive ion
mode of ionization, and employed a QTOF mass spectrometer (QTOF 6530, Agilent Technologies).
Qualitative analysis of mass spectra was performed with MassHunter software B.07.00
(Agilent Technologies).
Data analysis
The pharmacokinetic parameters of madecassoside and asiaticoside were calculated by
non-compartmental analysis using PK solution software (Summit Research Service). The
Cmax and Tmax were directly observed from real experimental data. The AUC0-24, AUC0–inf, Vapp, T1/2, Clapp, and MRT were reported. The Kp values were calculated from the drug concentrations
in the tissue divided by the drug concentration in the plasma at the same time point.
The percent of recovery of the drugs and metabolites in the excreta was calculated
as the total drugs or metabolites found in the excreta divided by the administered
dose based on molarity. All parameters were reported as the mean±standard deviation.
Statistical differences in pharmacokinetic parameters between the two experimental
groups were analyzed using a nonparametric test (p<0.05). SPSS statistical analysis
(version 16) was used for all data analyses (SPSS, Inc.).
Supporting information
A representative LC-MS/MS chromatogram from a plasma sample and MS/MS fragments of
major and minor components of ECa 233 are available as Supporting Information.
