Development of a UHPLC-PDA method for the simultaneous quantification of 4-phenylcoumarins and chlorogenic acid in Exostema caribaeum stem bark

Article abstract of DOI:10.1021/np400785z

Potential toxic effects in mice of an infusion prepared from the stem bark of Exostema caribaeum was assessed by means of the Lorke procedure. The preparation was not found to be toxic, with the LD₅₀ value estimated to be more than 5 g/kg. This preparation at 100, 300, and 500 mg/kg also caused a significant hypoglycemic effect and a reduction in the postprandial glycemia peak in both normal and nicotinamide/streptozotocin (NA/STZ)-diabetic mice in an oral sucrose tolerance test. Phytochemical analysis of the infusion revealed that the major active principles are 4-phenylcoumarins (2-8) and chlorogenic acid (1). During this process, a new 4-phenylcoumarin was isolated along with several known analogues. The structure of the new compound was established as 5-O-[β-d-xylopyranosyl-(1-6)-β-d-glucopyranosyl]-7,3′, 4′-trihydroxy-4-phenylcoumarin (2) by spectroscopic means. A simple, efficient, fast, and reliable UHPLC-PDA analytical method for quantifying 4-phenylcoumarins and chlorogenic acid (1) was developed and validated. Parameters assessed for the method validation were selectivity, linearity, the limits of detection (LOD) and quantification (LOQ), precision, and accuracy. It was found that all calibration curves showed good linearity (R² > 0.9931), within the range of concentrations tested.

Full text of DOI:10.1021/np400785z

Article
pubs.acs.org/jnp
Development of a UHPLC-PDA Method for the Simultaneous
Quantification of 4‑Phenylcoumarins and Chlorogenic Acid in
Exostema caribaeum Stem Bark
†
†
́ ́
Araceli Perez-Vasquez, Erika Castillejos-Ramírez, Sol Cristians, and Rachel Mata*
́ ́ ́ ́
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autonoma de Mexico, Mexico D.F., 04510, Mexico
*
S Supporting Information
ABSTRACT: Potential toxic effects in mice of an infusion
prepared from the stem bark of Exostema caribaeum was
assessed by means of the Lorke procedure. The preparation
was not found to be toxic, with the LD value estimated to be
5
0
more than 5 g/kg. This preparation at 100, 300, and 500 mg/
kg also caused a significant hypoglycemic effect and a
reduction in the postprandial glycemia peak in both normal
and nicotinamide/streptozotocin (NA/STZ)-diabetic mice in
an oral sucrose tolerance test. Phytochemical analysis of the
infusion revealed that the major active principles are 4-phenylcoumarins (2−8) and chlorogenic acid (1). During this process, a
new 4-phenylcoumarin was isolated along with several known analogues. The structure of the new compound was established as
5
-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl]-7,3′,4′-trihydroxy-4-phenylcoumarin (2) by spectroscopic means. A simple,
efficient, fast, and reliable UHPLC-PDA analytical method for quantifying 4-phenylcoumarins and chlorogenic acid (1) was
developed and validated. Parameters assessed for the method validation were selectivity, linearity, the limits of detection (LOD)
2
and quantification (LOQ), precision, and accuracy. It was found that all calibration curves showed good linearity (R > 0.9931),
within the range of concentrations tested.
Exostema caribaeum (Jacquin) Roemer & Schultes (Rubiaceae),
commonly known as “copalchi” or “copalche”, among other
names, belongs to the so-called “copalchi” complex, which
includes several Rubiaceae species and a few Euphorbiaceae
species. The characteristics of these plants are their extremely
bitter stem bark and their use as antidiabetic and antimalarial
agents. The stem bark of E. caribaeum is also valued today in
Mexico for treating gastrointestinal ailments and dengue
composition test useful for quantifying the active principles of
E. caribaeum stem bark. For this investigation, an ultra-high-
performance liquid chromatography−photodiode array
(UHPLC-PDA) analytical method was established and
validated for the simultaneous quantification of chlorogenic
acid (1) and seven 4-phenylcoumarins (2−8) in two batches of
the stem bark of E. caribaeum. The potential toxicity and
antidiabetic properties of the infusion of this plant part, not
previously described, were also assessed.
1
−3
fever.
bark resulted in the isolation and characterization of nine 4-
Previous phytochemical studies of E. caribaeum stem
4
−6
phenylcoumarins including compounds 5, 6, and 8. Some of
these 4-phenylcoumarins as well as the plant stem bark
methanol extract showed hypoglycemic activity when tested in
RESULTS AND DISCUSSION
■
Pharmacological and Toxicological Testing. The
potential toxic effects in mice of the E. caribaeum stem bark
aqueous extract was assessed by means of the Lorke
7
,8
rats using well-known pharmacological procedures.
The
9
organic extract also displayed moderate toxicity to mice.
Finally, Noster and Kraus demonstrated that different extracts
prepared from E. caribaeum stem bark possess moderate in vitro
11
procedure. After 14 days, the treated mice did not exhibit
any change in their behavior or body weight or in the
macroscopic morphology of the heart, liver, kidney, or lung.
The LD50 value was more than 5 g/kg. Thus, when compared
10
anti-Plasmodium falciparum activity.
A major problem with medicinal plants that are commer-
cialized widely in Mexico, including E caribaeum, is the
adulteration or substitution of herbal drugs with less efficacious
if not dangerous products. As a consequence, the prevalence of
low-quality raw material reduces the potential for clinical
efficacy and raises the risk of adverse reactions. Furthermore, in
the context of the global market in medicinal plants, it is very
important to establish quality-control procedures for the most
widely internationally distributed crude drugs of Mexico. With
this in mind, the present study was undertaken to develop a
9
with the organic-soluble extract (LD50 of 0.7 g/kg), the
infusion can be regarded as nontoxic according to the Lorke
11
criteria. These results endorsed the preclinical safety testing of
this traditional preparation.
Special Issue: Special Issue in Honor of Otto Sticher
Received: September 25, 2013
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 1. NMR Spectroscopic Data (125 MHz 13C, 500 MHz
1
H, CD OD) of the 4-Phenylcoumarin 2
3
position
δC
δH (J in Hz)
HMBC
2
3
4
161.9
111.0
157.0
103.1
156.0
99.4
5.86 (s)
C-2, C-4a, C-1′
4
5
6
7
8
8
1
2
3
4
5
6
a
6.64 (d, 1.8)
6.47 (d, 1.8)
C-4a, C-5, C-7, C-8
C-4a, C-6, C-7, C-8a
162.2
97.3
a
′
′
′
′
′
′
156.8
131.6
114.9
144.2
145.6
114.3
119.7
100.2
73.3
6.80 (d, 1.8)
C-4, C-1′, C-3′, C-4′
6.80 (d, 8.0)
6.71 (dd, 7.8, 2.2)
4.73 (d, 7.5)
2.71 (dd, 8.6, 7.4)
3.26
C-1′, C-3′
C-4, C-4′
C-5
The antidiabetic activity of the infusion was determined using
1
2
1″
a known method. As shown in Figure S7 and Table S1
Supporting Information), the aqueous extract of E. caribaeum
2″
3″
4″
5″
6″
C-3″, C-1″
(
76.1
stem bark (500 mg/kg) caused a significant hypoglycemic effect
in nicotinamide/streptozotocin (NA/STZ)-diabetic mice in
comparison to the vehicle-treated group (p < 0.05). The effect
of this aqueous extract (500 mg/kg) was similar to that exerted
by glibenclamide (10 mg/kg) when used as the positive control.
In the oral sucrose tolerance test, this aqueous extract (100,
69.6
3.21 (m)
C-3″
76.4
3.50 (m)
68.2
4.05 (brd, 10.9)
3
.73 (dd, 11.3, 6.5)
1‴
2‴
3‴
4‴
5‴
103.8
73.7
76.2
69.8
65.5
4.31 (d, 7.4)
C-6″
C-1‴
C-4‴, C-2‴
3.21 (m)
3
00, and 500 mg/kg) caused a significant decrease in the
3.26
postprandial glycemia peak in both normal and NA/STZ-
diabetic mice when compared with the vehicle-treated group (p
<
3.46 (ddd, 10.1, 9.9, 5.3)
3.82 (dd, 11.5, 5.3)
0.05) (Figure S8 and Tables S2 and S3, Supporting
3
.08 (dd, 11.5, 10.3)
Information).
Compound Isolation and Identification. For validation
of analytical methods, it is mandatory to employ reference
standards. However, due to the lack of commercially available
analytical 4-phenylcoumarin standards, in this study the
isolation of suitable standards from the stem bark of E.
caribaeum was performed initially. For this purpose, an aqueous
extract was subjected to conventional phytochemical isolation.
This process led to the purification of chlorogenic acid (1), a
H-2″, due to the protection exerted by the phenyl group at C-4
of the phenylcoumarin core, were consistent with the
attachment of the glucose moiety at the C-5 phenolic group.
On the other hand, the HMBC correlation between the
anomeric hydrogen of xylose (H-1‴; δ 4.31) and C-6″ (δ
H
C
68.2) was in agreement with the 1→6 linkage of the two
monosacharides. The nature of the sugars was confirmed by
acid hydrolysis of 2 with 2 N HCl to yield an aglycone, together
with D-xylose and D-glucose identified by GC-MS analysis of
13
well-known hypoglycemic agent, and seven 4-phenylcoumar-
ins, including the new natural product 2. Compounds 1, 3, 4,
and 7 are described for the first time from this plant.
14
their trimethylsilyl derivatives.
The structure of compound 2 was elucidated using
spectroscopic and spectrometric methods including one- and
two-dimensional NMR techniques (Table 1). The molecular
formula was established as C H O by HRESIMS. The NMR
The remaining 4-phenylcoumarins were identified by
comparison of their spectroscopic data with those previously
5
,6,15−17
reported,
and chlorogenic acid (1) was identified by
2
6
28 15
comparison with an authentic sample obtained commercially.
Optimization of Extraction Time. For the plant sample
investigated, an infusion was chosen as the extraction method
by considering the mode in which the crude drug is used in
traditional medicine. In addition, no attempt was made to use
an organic solvent, because it was demonstrated previously that
spectra (CD OD, Table 1) were very similar to those of the 4-
3
phenylcoumarins 3−8. The NMR spectra of compounds 2 and
4
were almost identical, with the only difference between them
being the absence of the signals for the methoxy group at C-7
in the spectra of compound 2. Thus, the aromatic region of the
1
9
H NMR (Table 1) spectrum of 2 showed signals due to two
such extracts are toxic to mice. Although the infusion is usually
meta-coupled hydrogens (H-6 and H-8), an ABX system
prepared by treatment with boiling water for 30 min, in this
study the influence of time on the extraction efficiency was
monitored at 10, 20, 30, and 40 min. In general, the results
indicated that the active principles are extracted with the best
yield within 30 min (Figure 1).
formed by H-2, H-5, and H-6, and a typical singlet (δ 5.86) for
H
the proton of the α-pyrone (H-3) unit. This spectrum showed
1
3
also resonances for a disaccharide unit. The C NMR
resonances (Table 1) of compound 2 were assigned through
the analysis of HSQC and HMBC experiments and indicated
clearly the presence of one xylopyranosyl unit and one
glucopyranosyl moiety in the molecule. The HMBC
UHPLC Method Validation and Application. Ultra-high-
performance liquid chromatography was selected for the quality
control of E. caribaeum stem bark, because this relatively new
technique offers a potential decrease in time of analysis and
correlations of the anomeric hydrogen of glucose (H-1″; δ
H
18
4
.73) to C-5 (δ 156.0) as well as the upfield shifts of H-1″ and
reduced solvent consumption. Several mobile phases and two
C
B
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employed to quantify compounds 5 and 6 simultaneously, the
curve of 4-phenylcoumarin 7 was used for 2, and the curve of
compound 4 was employed to quantify 8 considering that they
have similar structural properties and their UV absorption
profiles are identical. Calibration curves were constructed by
plotting the peak areas (y) against the corresponding standard
concentration (x, μg/mL), using a linear least-squares fit
regression model. The data in Table 2 indicate that the
calibration curves showed good linearity with a high correlation
coefficient within the concentration ranges tested: 10 to 150
μg/mL for 1 and 7, 50 to 350 μg/mL for 3, and 10 to 300 μg/
2
mL for 4. In all cases, correlation coefficient (R ) values were
found to exceed 0.99.
Figure 1. Influence of time on the extraction efficiency of 4-
phenylcoumarins 3, 4, and 7 and chlorogenic acid (1).
The limit of detection and limit of quantification values were
.13 μg/mL and 0.38 μg/mL for compound 1, 0.34 μg/mL and
.05 μg/mL for compound 3, 0.03 μg/mL and 0.09 μg/mL for
0
1
stationary phases (UHPLC BEH Shield-C and UHPLC-BEH
1
8
compound 4, and 0.08 μg/mL and 0.23 μg/mL for compound
. The precision of the method was determined, and the
C ) were examined. The former stationary phase was regarded
8
7
as the more appropriate. Acetonitrile and water were selected
for the mobile phase. Since chromatographic separation of
analytes was much more efficient after acidification of the
mobile phase, two different acids were tested: trifluoroacetic
acid (TFA) and formic acid. The optimal UHPLC separation
conditions were achieved with a mixture of water containing
relative standard deviations (%RSD) for intraday and interday
repeatability values were less than 1.86% and 1.91%,
respectively (Table 2). The recovery rates of all analytes
ranged from 100.51% to 99.94%, which demonstrated sufficient
accuracy of the developed method. The results of the stability
study indicated that the samples are very stable in water
solution and the % RSDs of the four analytes were all less than
0.1% formic acid and acetonitrile. To achieve good sensitivity
and accuracy for quantification, different UV λmax values of the
four standards were considered. Thus, the PDA detection
wavelength (λ) was set at 327 nm for the 4-phenylcoumarins
and 248 nm for chlorogenic acid (1). Under these optimized
conditions, effective baseline resolution was achieved for
compounds 1−8, and coelution was detected only for 4-
phenylcoumarins 5 and 6, while the rest of components were
separated appropriately. A representative chromatogram of the
major components in the E. caribaeum stem bark infusion is
illustrated in Figure 2. Compounds 1−8 were selected for
2
.0%.
The validated method was applied successfully for the
quantification of 4-phenylcoumarins (2−8) and chlorogenic
acid (1) in two different geographical batches (EC-1 and EC-2)
of the crude drug. The results are presented in Table 3 and
show that the content of marker active compounds was similar
in the two batches. In both cases, the 4-phenylcoumarins 3, 5,
and 6 were found in higher amounts, whereas the
concentrations of chlorogenic acid (1) and 4-phenylcoumarins
2
, 4, 7, and 8 were lower.
In summary, phytochemical analysis of an infusion of E
caribaeum stem bark led to the isolation of a new 4-
phenylcoumarin (2). A simple, efficient, fast, and reliable
UHPLC-PDA analytical method for quantifying 4-phenyl-
coumarins and chlorogenic acid (1) was developed and
validated. This method was applied successfully to quantify
these components in two different batches of the plant.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Infrared spectra were
−1
obtained in the range 4000−400 cm using a Perkin-Elmer Spectrum
1
4
00 FT-IR spectrometer (Perkin-Elmer, San Jose, CA, USA). H and
1
3
C NMR spectra were acquired on a JEOL ECA-500 NMR
1
spectrometer (JEOL, Tokyo, Japan) operating at 500 MHz for H
and 125 MHz for 13C, and chemical shifts are given on a δ (ppm) scale
with tetramethylsilane (TMS) as internal standard. Mass spectra were
recorded on a Thermo Scientific LTQ Orbitrap XL mass spectrometer
Figure 2. UHPLC-PDA chromatogram of E. caribaeum stem bark
aqueous extract (EC-2) under optimized conditions; detection
wavelength 327 nm. For chromatographic conditions, see the
Experimental Section.
(
(
Thermo Scientific, Rochester, NY, USA) using electrospray ionization
ESI).
Reagents and solvents were analytical or HPLC grade; chlorogenic
acid (1), acarbose, glybenclamide, nicotinamide, streptozotocin,
sucrose, and Tween 80 were purchased from Sigma-Aldrich (Toluca,
Mexico); HPLC grade water, methanol, acetonitrile, formic acid
(88.0%), and ethyl acetate were purchased from Tecrom (Mexico City,
Mexico). The purity of the isolated compounds used as standards were
determined by UHPLC analysis using a normalization method and
were calculated to be ≥97%. Open column chromatography was
carried out on silica gel 60 (Merck, 70−230 mesh; Darmstadt,
Germany) or Sephadex LH-20 (Sigma-Aldrich Quimica, Toluca,
quantitative determination because some of these possess
8
hypoglycemic effects, and several were obtained in good yields
5
,6,8
from E. caribaeum and other species.
18
For assay validation, compounds 1, 3, 4, and 7 were
included as standards and were selected due to the fact that
they are soluble in the mobile phase employed and were
obtained in good yields. The curve for compound 3 was
Mex
́
ico). All separations with semipreparative RP-HPLC were
C
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Table 2. Validation Report of the Method for Simultaneous Determination of Selected Constituents in the Stem Bark of E.
caribaeum
precision
linear range
(μg/mL)
LOD
(μg/mL)
LOQ
(μg/mL)
intraday
(% RSD)
interday
(% RSD)
recovery
(% mean)
stability
(% RSD)
compound
calibration equation
R2
a
1
3
4
7
10−150
50−350
10−300
10−150
y = 58 101x + 228 029
y = 43 610x + 747 088
y = 27 482x + 7413.7
y = 40 569x + 18 862
0.9955
0.9931
0.9998
0.9987
0.13
0.34
0.03
0.08
0.38
1.05
0.09
0.23
0.63
0.36
1.86
0.63
1.20
0.62
1.91
1.46
99.98
99.94
0.60
0.13
0.32
0.11
100.07
100.51
a
R2 correlation coefficient for five data points in the calibration curves (n = 6).
mg) as a residual solid; the MeOH-soluble fraction was purified
using semipreparative RP-HPLC to obtain 5-O-β-D-glucopyranosyl-
Table 3. Contents (mg/g) of Eight Major Compounds in
Two Batches of the Stem Bark of E. caribaeum (n = 3)
7
,3′,4′-trihydroxy-4-phenylcoumarin (3, 16 mg). From fraction F6
(132 mg) precipitated 72 mg of 5-O-β-D-galactopyranosyl-7-methoxy-
′,4′-dihydroxy-4-phenylcoumarin (5). Fraction F8 (58 mg) was
content (mg/g) (mean ± sd)
3
compound
tR (min)
EC-1
EC-2
subjected to semipreparative RP-HPLC to obtain 5-O-[β-D-xylopyr-
anosyl-(1→6)-β-D-glucopyranosyl]-7,3′,4′-trihydroxy-4-phenylcou-
marin (2, 15 mg) and 5-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyr-
anosyl]-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin (4, 16 mg).
Finally, RP-HPLC of fraction F9 yielded 16 mg of chlorogenic acid
(1).
1
1.45
11.46 ± 0.29
16.62 ± 0.35
100.24 ± 1.63
21.09 ± 0.17
359.41 ± 5.58
43.37 ± 1.12
8.82 ± 0.07
27.5 ± 0.78
a
2
1.61
3
2.12
83.88 ± 1.39
28.25 ± 0.27
350.64 ± 5.33
35.75 ± 0.73
25.67 ± 0.47
4
2.76
b
5
7
8
+ 6
3.74/3.80
5.85
2
5
Compound 2: brown solid; mp >320 °C; [α]
−49 (c 1.0,
D
c
MeOH); UV λmax (log ε) 356 (3.98), 328 (4.05) nm; IR ν 3279,
906, 1684, 1598, 1357, 1037 cm ; H and C NMR data, see Table
; HRESIMS (positive-ion mode) m/z 581.1482 [M + H] (calcd for
max
8.25
27.70 ± 0.59
−1
1
13
2
1
a
b
c
Quantified as 7. Quantified as 3. Quantified as 4.
+
C H O , 581.1497).
26
28 15
performed using a Waters Dual HPLC system equipped with a
quaternary pump (Waters, model 600), a UV/vis dual detector (model
Acid Hydrolysis of Compound 2 and Sugar Analysis. A 4 mg
amount of 2 in 5 mL of 2 N HCl was refluxed for 1 h. Then, the
reaction mixture was extracted with EtOAc (3 × 8 mL). The aqueous
phase was neutralized with 1 N KOH, extracted with n-butanol (8
2
487), and a Phenomenex Nucleosil C18 column (250 × 10 mm, i.d., 5
μm). Elution was conducted in the isocratic mode with acetonitrile−
water (80:20) at a flow rate of 4.0 mL/min. The UV detector was set
at a monitoring wavelength of 327 nm.
mL), washed with H
colorless residue. The mixture was treated with chlorotrimethylsilane
O (2 × 4 mL), and concentrated to yield a
2
1
4
Quantitative analysis was performed on a Waters Acquity UHPLC-
H class system (Waters Co., Milford, MA, USA) equipped with a
photodiode array detector, sample manager, and quaternary solvent
manager. System control, data collection, and data processing were
accomplished using Waters Empower 3 chromatography software.
Plant Material. A first batch of the stem bark of E. caribaeum was
collected at Tuzantlan (EC-1), and a second was collected at Temalac
(Sigma Sil-A) and then analyzed by GC-MS, using the following
conditions: HP-5 MS column (30 m × 0.25 mm × 0.25 μm); He at a
constant flow rate of 1.2 mL/min; the GC column temperature
program applied was 65 °C isothermal for 2 min, linear gradient to 150
at 6 °C/min for 5 min, followed by an isothermal hold at 300 °C for
15 min. The retention times for TMS derivatives of common sugars
were used as standards for GC identification; only D-xylose (t
and D-glucose (t 45.0) were detected.
33 min)
R
(
EC-2), Atenango del Rıo
́
Municipality, Guerrero State, Mexico, both
R
in July 2010. The plant specimens were authenticated by Dr. Sol
Cristians and deposited at the Faculty of Sciences Herbarium UNAM
Preparation of Standard and Sample Solutions. The
accurately weighed powder (12 mg) of the dried aqueous extract of
E. caribaeum stem bark was redissolved in 10 mL of water, and then
the solution was filtered through a 0.2 μm membrane filter before
being injected into a UHPLC setup for analysis. For the specific
calibration plot, a stock solution of 1.0 mg/mL of each standard was
(
vouchers: FCME 131339 and 131340, respectively). Batch 2 was used
to validate the analytical method and for isolating chlorogenic acid (1)
and seven 4-phenylcoumarins (2−8).
Extraction and Isolation. Thirty grams of fine powder (particle
size <2000 μm, mesh size, 2 mm) of the dried stem bark of E.
caribaeum was extracted with boiled distilled water (5000 mL) for 30
min. After extraction, the resulting infusion was filtered through filter
paper (Whatman No. 1) to remove macroparticles and was
concentrated under reduced pressure to obtain 10.74 g of dried
aqueous extract.
prepared in H O, stored at 4 °C, and brought to room temperature
2
before use. Five working standard solutions were prepared by serial
dilution of the stock solution with H O, in appropriate concentrations.
2
Chromatographic Conditions for UHPLC-PDA Analysis.
Separations were performed at 35 °C using an Acquity UHPLC
BEH Shield-RP18 column (100 × 2.1 mm, 1.7 μm) at a flow rate of
0.4 mL/min. The mobile phase consisted of acetonitrile (A) and water
containing 0.1% (v/v) formic acid (B). The following gradient elution
program was used: 16% A for 0−5 min, 16−30% A for 5−7 min, 30%
A for 7−10 min, and 30−16% A for 10−12 min. The injection volume
was 10 μL. The UV detector was set at a monitoring wavelength of
327 nm.
In order to isolate the 4-phenylcoumarins, 7.11 g of the aqueous
extract was dissolved in 30 mL of MeOH, and the soluble fraction was
filtered, evaporated (6.41 g), and separated by silica gel column
chromatography, eluting with hexane−EtOAc (100:0 → 0:100)
followed by EtOAc−MeOH (100:0 → 50:50), to obtain nine fractions
(
F1−F9). Fraction F2 (74 mg) was submitted to repeated washing
with MeOH to give 5-O-(6″-acetyl-β-D-galactopyranosyl)-7-methoxy-
Method Validation. The UHPLC-PDA method was validated
according to the International Conference on Harmonization (ICH)
Guidelines and included a determination of selectivity, linearity,
3
′,4′-dihydroxy-4-phenylcoumarin (8, 18 mg). The remaining solution
generated was filtered and evaporated to obtain 52 mg of a methanol
fraction (F2-M), which was chromatographed on a Sephadex LH-20
column (CH Cl −MeOH, 5:95), to yield eight fractions (F2-M1−F2-
1
9
precision, accuracy, LOD, and LOQ. The determination of peak
purity to improve the selectivity was assessed using PDA detection
wavelengths of 327 nm for 4-phenylcoumarins 3, 4, and 7 and 248 nm
for 1. LOD and LOQ were determined based on the standard
deviation (σ) of the response and the slope (S) [LOD = 3.3 × σ/S and
LOQ = 10 × σ/S, respectively]. The precision was evaluated using
2
2
M8). From fraction F2-M6, 9 mg of 5-O-(6″-acetyl-β-D-glucopyr-
anosyl)-7,3′,4′-trihydroxy-4-phenylcoumarin (7) was precipitated.
Fraction F4 (106 mg) was washed with MeOH to obtain 5-O-β-D-
glucopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin (6, 61
D
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Journal of Natural Products
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repeatability (intraday) and intermediate precision (interday). Intraday
and interday variations were established using six replicates within the
same day and three consecutive days (a total of 18 determinations),
respectively. Recovery experiments were performed in order to study
the accuracy of the method. The samples were spiked with known
amounts of the standards, at different concentrations levels (low,
medium, and high): 1 (10, 50, and 150 μg/mL), 3 (50, 150, and 350
μg/mL), 4 (10, 100, and 300 μg/mL), and 7 (10, 60, and 150 μg/mL).
The average recoveries were calculated by the following formula:
recovery (%) = [(amount found − original amount)/amount spiked]
Author Contributions
†
A. Perez-Vasquez and E. Castillejos-Ramır
́
́
́
ez contributed
equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by grants from Consejo Nacional de
■
Ciencia y Tecnologıa
́
(CONACyT, Proyecto99395) and
×
100%. The stability was tested by analyzing the sample solution at
DGAPA-UNAM PAPIIT IN212913. The technical assistance
of I. Rivero-Cruz, R. I. del Villar, M. Gutierrez, G. Duarte-Lisci,
different time points (0, 12, 24, and 48 h), and peak areas of all
standards were recorded and compared. The % RSD ≤ 2.0 was taken
as a measure of precision and stability.
́
and R. del Carmen-Lezama is also recognized. E.C. and S.C.
acknowledge scholarships awarded by CONACyT to carry out
graduate studies.
Acute Toxicity. The toxicity studies and antidiabetic assays were
performed on male ICR mice (body weight range, 20−25 g). The
animals were purchased from Centro UNAM-Harlan (Mexico City,
Mexico). Mice were housed in a climate- and light-controlled room
with a 12 h light/dark cycle and maintained on a standard pellet diet
and water ad libitum. Procedures involving animals and their care were
conducted in conformity with the Mexican Official Norm for Animal
Care and Handling (NOM-062-ZOO-1999) and in compliance with
international rules on care and use of laboratory animals. The
Institutional Committee for Care and Use of Laboratory Animals
DEDICATION
■
Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich,
Switzerland, for his pioneering work in pharmacognosy and
phytochemistry.
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ASSOCIATED CONTENT
■
9
* Supporting Information
S
(
UV and selected NMR spectra for compound 2. Full tables and
figures of hypoglycemic and antihyperglycemic effects of the
infusion of the plant material. This information is available free
of charge via the Internet at http://pubs.acs.org.
Procedures: Text and Methodology Q2(R1). Available at: http://
www.ich.org/fileadmin/Public_Web_Site/ICH_Products/
Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf (accessed
on 03.06.2012).
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AUTHOR INFORMATION
■
Corresponding Author
*
Tel: 525-556225289. Fax: 525-556225329. E-mail: rachel@
unam.mx.
E
dx.doi.org/10.1021/np400785z | J. Nat. Prod. XXXX, XXX, XXX−XXX