Industrial Crops and Products 77 (2015) 391401

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Comparing the phenolic prole of Pilocarpus pennatifolius Lem. by

HPLCDADESI/MSn with respect to authentication and enzyme
inhibition potential
Federico Ferreres a, , Clara Grosso b , Angel Gil-Izquierdo a , Andreia Fernandes b ,
Patrcia Valento b , Paula B. Andrade b,
a
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus
University Espinardo, Murcia, Spain
b
REQUIMTE/LAQV, Laboratrio de Farmacognosia, Departamento de Qumica, Faculdade de Farmcia, Universidade do Porto, Rua de Jorge Viterbo Ferreira,
no. 228, 4050-313 Porto, Portugal

a r t i c l e

i n f o

Article history:
Received 10 April 2015
Received in revised form 27 August 2015
Accepted 3 September 2015
Available online 21 September 2015
Keywords:
Pilocarpus pennatifolius Lem.
HPLCDADESI/MSn
Phenolic compounds
Authenticity
Enzyme inhibition

a b s t r a c t
Pilocarpus pennatifolius Lem. is a neotropical species with economic importance, whose phenolic prole
is poorly described. A comparative HPLCDADESI/MSn analysis between herbal teas prepared from a
commercial sample (CS) and an authenticated one (BGS) was carried out. From a qualitative point of view,
45 compounds were identied, 42 being reported for the rst time in Pilocarpus genus; the samples only
shared the presence of 18 compounds. From a quantitative perspective, BGS contained higher amounts
of phenolics than CS (44.94 and 37.20 mg/g extract d.w., respectively) and both samples exhibited
different major compounds, namely hesperetin-7-O-rutinoside (BGS) and 6-methoxy-quercetin-3-Orobinobioside (CS). Despite being one of the markers of Pilocarpus genus and previously described in
this species, pilocarpine was not found in none of the samples.
This is also the rst report on the inhibition of enzymes implicated in Alzheimers disease
(cholinesterases) and diabetes mellitus type II (-glucosidase) by P. pilocarpus. BGS and CS were more
effective against -glucosidase, displaying IC50 values of 604.34 g/mL (BGS) and 573.48 g/mL (CS).
These results can provide a basis of future studies on the application of these herbal teas as nutraceutical
ingredients.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Rutaceae encompasses many members of economic importance, including species of different genera: Citrus, Pilocarpus,
Boronia, Choisya, Poncirus, and Skimmia (Chase et al., 1999). Pilocarpus pennatifolius Lem. is a neotropical medicinal plant native to
South America (Sawaya et al., 2011). Its main commercial value
relies on the imidazole alkaloids found only in Pilocarpus genus,
such as pilocarpine. This alkaloid possesses CNS activity and is
used for the treatment of glaucoma and xerostomy (Santos and
Moreno, 2004). Nonetheless, this plant is still traded to treat other
illnesses, since its dried leaves possess gastrostimulant, peristaltic
and secretagogue activities, which are suitable for the treatment of

Corresponding authors. Fax: +351 226093390.

E-mail addresses: federico@cebas.csic.es (F. Ferreres), pandrade@ff.up.pt
(P.B. Andrade).
http://dx.doi.org/10.1016/j.indcrop.2015.09.006
0926-6690/ 2015 Elsevier B.V. All rights reserved.

urinary and gastrointestinal tracts disorders, pulmonary disorders,

etc. However, scientic proofs are scarce and available information
is mainly related with pilocarpine rather than with other classes
of compounds (Santos and Moreno, 2004; Gruenwald et al., 2000;
Duke et al., 2002; Skidmore-Roth, 2009). The in vivo antidiabetic
activity of other species, Pilocarpus microphyllus Stapf ex Holm., has
already been scientically evaluated (Devi et al., 2010). Although
pilocarpine has shown to inuence insulin secretion in normal and
obese hyperglycaemic mice (Atkins et al., 1975), Devi et al. (2010)
suggested that the antidiabetic activity of P. microphyllus could be
due to the presence of avonoids. Unfortunately, no identication
of bioactive compounds was undertaken by the authors.
Type 2 diabetes mellitus (DM) is a chronic disorder that leads to
a decient production of insulin and a decreased sensitivity of the
organs to the secreted insulin. This condition results in hyperglycaemia and changes on the metabolism of lipids, ketones and amino
acids (Lin et al., 2011). Hyperglycaemia may be treated with drugs
that delay and/or prevent the absorption of glucose into the blood-

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F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

stream, such as -amylase and -glucosidase inhibitors (Wang

et al., 2012; Nouri et al., 2014; Custdio et al., 2015). Patients with
type 2 DM have higher risk to develop Alzheimers disease (AD),
which is recently recognized as a brain form of diabetes (Kroner,
2009; Custdio et al., 2015). The cholinergic hypothesis assumes
that the reduction of acetylcholine (ACh) in the synaptic cleft is
responsible for AD symptoms. By inhibiting the enzymes responsible for the breakdown of synaptic ACh, acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE), ACh level increases and a slowdown of the disease progression occurs (Pohanka, 2011; Custdio
et al., 2015).
Phenolic compounds, avonoids in particular, are regarded as
multi-target drugs since they have already shown to affect different
enzymes and receptors. Some studies already reported their ability
to inhibit -glucosidase and AChE and BuChE (Vinholes et al., 2011;
Ferreres et al., 2012; Wang et al., 2012; Custdio et al., 2015).
Considering that Pilocarpus species can act as cholinergic modulators, through the imidazole alkaloids, and have also demonstrated
antidiabetic potential, the goals of this study were to assess for the
rst time the capacity of P. pennatifolius to inhibit enzymes related
with the pathologies mentioned above, namely -glucosidase,
AChE and BuChE. Moreover, for authenticity purposes, the presence
of pilocarpine was determined and a systematic characterization
of the phenolic prole of this species by HPLCDADESI/MSn was
undertaken, since no previous exhaustive study was performed.
Finally, a putative relationship between the observed bioactivities
and the chemical composition is discussed.
2. Material and methods
2.1. Plant material
Dried leaves of Pilocarpus pennatifolius Lem. (commercial sample (CS)) were purchased in a local herbal store, the technical sheet
attesting its identity as P. pennatifolius. The authenticated sample
(leaves) was provided by the Botanical Garden of Lisbon University. Leaves were dried in an oven at 30 C, during 2 weeks. Part
of the sample was stored under the identication of Pp-L-032014
and was deposited at the Laboratory of Pharmacognosy of the Faculty of Pharmacy of Porto University. The remaining sample (BGS)
was used for subsequent studies. Dried material was ground to a
mean particle size below 910 m and stored at room temperature,
protected from light.

2.3. Extraction
Herbal teas were prepared by mixing 3 g of plant material with
500 mL of distilled water. The extraction was performed at 100 C,
during 20 min. The extracts were then ltered under reduced pressure and submitted to freeze drying. Extraction yields for CS and
BGS were 16.3 and 28.2%, respectively.
2.4. HPLCDADESI/MSn qualitative analyses
Chromatographic analyses were carried out on a Kinetex column (5 m, C18, 100 A, 150 4.6 mm; Phenomenex, Maccleseld,
UK). The mobile phase consisted of two solvents: formic acid
in water (1%) (A) and acetonitrile (B), starting with 15% B and
using a gradient to obtain 20% B at 25 min and 70% B at 27 min.
The ow rate was 800 L/min, and the injection volume was
5 L. Spectral data from all peaks were accumulated in the range
of 240400 nm, and chromatograms were recorded at 340 and
280 nm. The HPLCDADESI/MSn analyses were carried out in an
Agilent HPLC 1200 series equipped with a diode array detector
and mass detector in series (Agilent Technologies, Waldbronn,
Germany). The HPLC consisted of a binary pump (model G1376A),
an autosampler (model G1377A) refrigerated at 4 C (G1330B),
a degasser (model G1379B), and a diode array detector (model
G1315D). The HPLC system was controlled by ChemStation software (Agilent, v. B.01.03-SR2). The mass detector was a Bruker ion
trap spectrometer (model HCT Ultra) equipped with an electrospray ionisation interface and was controlled by LCMSD software
(Agilent, v. 6.1). The ionisation conditions were adjusted at 350 C
and 4.0 kV for capillary temperature and voltage, respectively. The
nebulizer pressure and ow rate of nitrogen were 65.0 psi and
11 L/min, respectively. The full scan mass covered the range from
m/z 100 up to m/z 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as the collision
gas, with voltage ramping cycles from 0.3 up to 2 V. Mass spectrometry data were acquired in the negative ionisation mode. MS2 was
carried out in the automatic mode on the more abundant fragment
ion in MS.
Compounds were numbered following the order of elution in
Fig. 1, without taking into account their presence or absence in
each of the extracts. Table 1 shows the fragmentation pattern of
the identied phenolic acid derivatives. Moreover, in Table 2, compounds were grouped by their similarities in terms of glycosylation
patterns ((2-rhamnosyl) rutinosides, (rhamnosyl) hexosides and
mono-glycosides).

2.2. Standards and reagents

-Glucosidase (type I from bakers yeast), 4-nitrophenyl
-d-glucopyranoside (PNP-G), acarbose, galantamine, AChE
(from electric eel), acetylthiocholine iodide (ATCI), BuChE
(from equine serum), S-butyrylthiocholine iodide (BTCI), bovine
serum albumin (BSA), 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB),
Trizma hydrochloride (TrisHCl), pilocarpine hydrochloride and
quercetin-3-O-rutinoside were obtained from SigmaAldrich (St.
Louis, MO, USA). Potassium di-hydrogen phosphate and acetonitrile were from Merck (Darmstadt, Germany). Magnesium chloride
hexahydrate (MgCl2 6H2 O) was purchased from Fluka (Steinheim,
Germany). Sodium chloride and diethyl ether were purchased from
VWR (Geldenaakse, Belgium). Formic acid, HCl 37% and ammonium
25% were from VWR (Fontenay-sous-Bois, France). Quercetin-3-Oglucoside, quercetin-3-O-galactoside, kaempferol-3-O-glucoside,
kaempferol-3-O-rutinoside,
isorhamnetin-3-O-glucoside,
isorhamnetin-3-O-rutinoside, apigenin-7-O-glucoside, apigenin7-O-neohesperidoside, 5-O-caffeoylquinic acid, sinapic acid,
chrysoeriol and hesperetin-7-O-rutinoside were from Extrasynthse (Genay, France).

2.5. HPLCDAD quantitative analyses

For phenolic compound quantication 20 L portion of each
extract (CS and BGS) was analysed on an analytical HPLC unit
(Gilson), using a C18 Spherisorb ODS2 (25.0 0.46 cm; 5 m
particle size) column from Waters (Ireland). The mobile phase
consisted of two solvents: formic acid in water (1%) (A) and
acetonitrile (B), starting with 15% B and using a gradient to
obtain 20% B at 60 min and 70% B at 62 min. The ow rate
was 1.2 mL/min. Detection was achieved with a Gilson diode
array detector. Spectral data from all peaks were collected in
the range of 200400 nm, and chromatograms were recorded at
280, 320 and 340 nm. Data were processed on Unipoint System
software (Gilson Medical Electronics, Villiers le Bel, France). Peak
purity was checked by the software contrast facilities. Phenolic compounds quantication was achieved by the absorbance
recorded in the chromatograms at 280 nm (for avanone), 320 nm
(for hydroxycinnamic acids) and 340 nm (for the remaining
avonoids) relative to calibration curves (Table 3). Compounds
13, 4 + 5, 69 were quantied as 5-O-caffeoylquinic acid, com-

F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

393

Fig. 1. HPLC phenolic prole of BGS and CS of Pilocarpus pennatifolius (340 nm). Peaks: 110 (see Table 1); 1145 (see Table 2).

Table 1
Rt, UV and MS: [MH] and MS2 [MH] data of phenolic acids from BGS and CS.a
Compounds
1
2
3
4
5
6
7
8
9
10

Cinnamoyl quinic acid derivative

Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Sinapoyl quinic acid
a

Rt (min)

UV (nm)

[MH] , m/z

MS2 [MH] , m/z (%)

2.8
3.0
3.2
3.5
3.6
3.9
4.2
4.4
4.8
6.9

298sh, 312
298sh, 312
298sh, 312
298sh, 312
300sh, 326
300sh, 326
298sh, 312
298sh, 312
300sh, 326
300sh, 324

355
355
355
355
385
385
355
355
385
397

173(5), 191(100). 209(65)

173(5), 191(100), 209 (40)
173(5), 191(100), 209(20)
191(100), 209(50)
173(5), 191(100), 209(20)
191(100)
173(15), 191(100), 209(40)
173(5), 191(100), 209(50)
173(3), 191(100), 209(10)
191(100), 223(40)

Main observed fragments. Other ions were found but they have not been included.

pound 10 as sinapic acid, compounds 11, 12, 16, 17 and 18

as quercetin-3-O-rutinoside, compound 13, 19, 26 and 27 or
27 + 28 as kaempferol-3-O-rutinoside, compounds 14 + 15 + 16, 25,
30, 34 + 35 and 41 + 42 as isorhamnetin-3-O-rutinoside, compounds 20 and 21 or 21 + 22 as quercetin-3-O-galactoside,
compounds 29 and 31 + 32 as kaempferol-3-O-glucoside, compounds 23 and 24 as quercetin-3-O-glucoside, compounds 33 + 34
and 39 as apigenin-7-O-neohesperidoside, compound 37 as
isorhamnetin-3-O-glucoside, compound 36 as hesperetin-7-Orutinoside, compounds 38, 40 and 44 + 45 as chrysoeriol and
compound 43 as apigenin-7-O-glucoside.
2.6. Alkaloids detection
One gram of plant material was heated until boiling with 10 mL
of HCl 10%. After ltration the resulting extract was alkalinized
with ammonia (1:1) and mixed with 20 mL of diethyl ether. The
organic phase was separated and 10 mL of HCL 10% were added. The
organic phase was then discarded and the presence of alkaloids in
the aqueous acidic fraction was checked by positive/negative precipitation tests with Dragendorffs, Mayers and Bertrands reagents
(Bruneton, 2009). Pilocarpine hydrochloride was taken as positive
control. Pilocarpine hydrochloride was also injected in HPLCDAD
using the same chromatographic conditions as for the phenolic
compounds.
2.7. Inhibition of -glucosidase
-Glucosidase inhibitory activity was assessed by a previously
reported procedure (Vinholes et al., 2011; Ferreres et al., 2012).
Briey, each well contained 100 L of 2.5 mM PNP-G dissolved

in 10 mM potassium phosphate buffer (pH 7.0), 130 L of phosphate buffer and 50 L of the extract or buffer (negative control).
The reaction was initiated by adding 20 L of the enzyme solution
(0.28 U/mL). The plates were incubated at 37 C for 10 min. The rate
of release of 4-nitrophenol from PNP-G at 405 nm was measured
in a Multiskan Ascent plate reader (Thermo Electron Corporation)
from T = 0 min to T = 10 min. Acarbose was taken as positive control.
2.8. Inhibition of cholinesterases
AChE and BuChE were inhibited according to a methodology
described before (Vinholes et al., 2011). Briey, 25 L of extract
dissolved in 50 mM TrisHCl buffer (buffer A, pH 8) or just buffer
(negative control) were added to each well, together with 25 L
of ATCI or of BTCI (15 mM), 125 L of DTNB and 50 L of buffer
B (50 mM TrisHCl, with 0.1% BSA, pH 8). The absorbance was
measured at 405 nm in a Multiskan Ascent plate reader (Thermo
Electron Corporation). The rates of reactions were calculated after
addition of 25 L of AChE (0.44 U/mL in buffer B) or of BuChE
(0.1 U/mL in buffer B). Galantamine was used as positive control.
2.9. Statistical analysis
The phenolic content of both extracts was compared using
unpaired t-test (GraphPad Prism 6 Software, Inc., San Diego, CA,
USA). For the biological activities, six different concentrations of
each extract were tested. Analysis of variance (one-way ANOVA,
post-test Bonferroni) was carried out to compare IC50 and IC25 values of the extracts with those of the positive controls. Differences
at p < 0.05 were considered statistically signicant (p > 0.05n.s.;
p < 0.05*; p < 0.01**; p < 0.001***; p < 0.0001****).

394

Table 2
Rt, UV and MS: [MH] and MS2 [MH] data of avonoids from BGS and CS.a
Compoundsb

Rt (min)

UV (nm)

[MH] , m/z

Querct-3-O-(2-rhmn) rut
Kaempf-3-O-(2-rhmn) rut
6-OMe-Isorhmn-3-O-(2-rhmn) rut
Isorhmn-3-O-(2-rhmn) rut

7.2
9.5
10.8
10.9

Flavonoid-3-O-(2-rhamnosyl) rutinosides
256, 267sh, 302sh, 356
755
739
267, 293sh, 348
c
799
769
255, 266sh, 300sh, 354

12
16
17
18
19
25
26
27
30
33
34
36
38
39
40
41

Querct-3-O-(2-rhmn) glc
Querct-3-O-rut
6-OMe-Querct-3-O-rob
6-OMe-Querct-3-O-rut
Kaempf-3-O-(2-rhmn) glc
Isorhmn-3-O-rob
Kaempf-3-O-rut
6-OMe-Kaempf-3-O-rut
Isorhmn-3-O-rut
Apig-7-O-rut
6-OMe-Isorhmn-3-O-rob
Hespt-7-O-rut
Chrys-7-O-rob
6-OMe-Apig-7-O-rut
Chrys-7-O-rut
6-OMe-Isorhmn-3-O-rut

9.2
11.1
11.6
12.1
12.4
13.8
14.1
15.2
17.0
17.7
17.9
18.5
19.3
20.4
21.2
21.8

Flavonoid-O-(rhamnosyl) hexosides
256, 267sh, 300sh, 355
609
609
256, 266sh, 300sh, 354
258, 270sh, 352
639
258, 270sh, 346
639
267, 290sh, 345
593
255, 268sh, 348
623
593
266, 295sh, 345
c
623
255, 266sh, 300sh, 352
623
268, 338
577
c
653
284, 332
609
250, 266, 290sh, 345
607
273, 335
607
252, 266, 292sh, 346
607
c
653

20
21
22
23
24
28
29
31
32
35
37
42
43
44
45

Querct-3-O-gal
6-OMe-Querct-3-O-gal
Kaempf-3-O-gal
Querct-3-O-glc
6-OMe-Querct-3-O-glc
6-OMe-Kaempf-3-O-gal
Kaempf-3-O-glc
6-OMe-Querct-3-O-pent
6-OMe-Kaempf-3-O-glc
6-OMe-Isorhmn-3-O-glc
Isorhmn-3-O-glc
6-OMe-Apig-7-O-gal
6-OMe-Apig-7-O-glc
Chrys-7-O-glc
6-OMe-Chrys-7-O-glc

12.6
12.9
13.0
13.4
13.7
15.3
16.6
17.2
17.3
18.0
18.9
21.8
22.4
24.1
24.3

Flavonoid-O-monoglycosides
c
463
258, 270sh, 348
493
447
c
256, 266sh, 354
463
258, 268sh, 346
493
c
477
c
447
c

463
c

477
507
c
c
477
c
461
272, 334
461
461
c
c

491

15

146

(146 + 18)

(146 + 120)

[AglcH/2H]

[AglcH-15]

784(30)
754(7)

609(20)
593(13)
653(5)
623(20)

591(25)
575(47)
635(35)
605(37)

489(45)
473(12)
533(10)
503(15)

300(100)
285(100)
345(100)
315(100)

330(30)
300(30)

463(5)

445(10)

624(10)
624(5)
447(15)

300(25)

638(10)

478(3)

478(4)
462(10)
448(7)
462(25)
492(20)
446(90)
446(60)
476(100)

429(70)

300(100)
301(100)
331(100)
331(100)
284(100)
315(100)
285(100)
315(100)
315(100)
269(100)
345(100)
301(100)
299(100)
299(100)
299(100)
345(100)
301(100)
331(100)
285(100)
301(100)
331(100)
315(100)
285(100)
331(100)
315(100)
345(100)
315(100)
299(100)
299(100)
299(100)
329(80)

316(40)
316(50)
300(40)
300(60)
300(23)
330(50)
284(30)
284(45)
284(55)
330(20)

316(30)

316(20)
300(70)
316(20)
300(60)
330(30)
284(50)
284(90)
284(10)
314(70)

Main observed fragments. Other ions were found but they have not been included.
b
Querct: quercetin; Kaempf: kaempferol; Isorhmn: isorhamnetin; Apig: apigenin; Chrys: chrysoeriol; Hespt: hesperetin; hex: hexoside; rhmn: rhamnoside; glc: glucoside; gal: galactoside; pent: pentoside; rut: rutinoside;
rob: robinobioside.
c
Coelutes with other compounds or are in trace amounts and its spectrum cannot be properly observed.

F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

11
13
14
15

MS2 [MH] , m/z (%)

F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

395

Table 3
Regression equations, R2 , limits of quantication and limits of detection of the phenolic compounds.
Compoundsa

Regression equation

5-CQA
Sinapic acid
Querct-3-O-rut
Querct-3-O-gal
Querct-3-O-glc
Hespt-7-O-rut
Kaempf-3-O-rut
Kaempf-3-O-glc
Apig-7-O-neoh
Apig-7-O-glc
Isorhmn-3-O-rut
Isorhmn-3-O-glc
Chrys

4.54 10 x + 3.28 10
1.03 109 x + 9.37 105
3.47 108 x 8.68 105
4.65 108 x 2.22 104
4.78 108 x 7.05 105
3.25 108 x + 8.66 105
3.69 108 x + 1.14 106
4.23 108 x + 6.14 105
3.69 108 x 2.55 106
5.48 108 x 2.06 106
3.84 108 x 5.80 105
4.62 108 x + 4.00 105
5.77 108 x + 2.18 106
8

R2
5

0.9969
0.9971
0.9992
0.9972
0.9991
0.9902
0.9955
0.9976
0.9930
0.9977
0.9996
0.9990
0.9865

Range of concentrations (mg/mL)

3

7.95 10 3.95 10
6.20 103 1.24 101
3.20 103 4.86 101
4.10 103 8.20 102
6.05 103 2.41 101
4.90 103 2.00 101
3.53 103 2.22 101
1.78 103 1.78 101
6.30 103 6.30 101
2.68 103 2.68 101
1.85 103 4.10 101
6.50 103 6.50 101
2.00 103 2.00 101

LODb (mg/mL)
4

1.85 10
1.33 103
6.75 104
5.31 104
1.34 104
1.10 103
3.20 104
7.91 105
1.45 103
9.63 105
2.88 104
2.10 103
5.69 104

LOQc (mg/mL)
5.61 104
4.04 103
2.04 103
1.61 103
4.07 104
3.35 103
9.70 104
2.40 104
4.39 103
2.92 104
8.73 104
6.36 103
1.73 103

a
5-CQA: 5-O-caffeoylquinic acid; Querct: quercetin; Kaempf: kaempferol; Isorhmn: isorhamnetin; Apig: apigenin; Chrys: chrysoeriol; Hespt: hesperetin; glc: glucoside;
gal: galactoside; rut: rutinoside; neoh: neohesperidoside.
b
LODlimit of detection.
c
LOQlimit of quantication.

3. Results and discussion

Several classes of compounds have been identied in Pilocarpus genus, including alkaloids, coumarins, avonoids, terpenes,
lignans, hydrocarbons and their alcohols, amides, carboxylic acids,
esters and ketones derivatives (Santos and Moreno, 2004). Only
the alkaloids pilocarpine and isopilocarpine, the monoterpenes
E--ocimene and Z--ocimene, the sesquiterpenes -cadinene,
-cubebene, -muurolene, selinene, -elemene, germacrene B,
bicyclogermacrene, -humulene, -copaene, -borbonene, caryophyllene and spathulenol, the phenylpropanoid eugenol, the
methyl ester of hexadecanoic acid, the alcohols hexanol and undecanol and the hydrocarbons tridecane and pentadecane were
already characterized in P. pennatifolius (Santos and Moreno, 2004).
However, its phenolic prole was not explored yet.
3.1. HPLCDADESI/MSn analysis of phenolic compounds
The HPLCDADESI/MSn screening of the herbal teas from two
samples of P. pennatifolius Lem., BGS and CS, revealed UV chromatograms (340 nm) (Fig. 1) in which 39 compounds (10 phenolic
acids and 29 avonoids) and 24 compounds (9 phenolic acids and
15 avonoids) were identied, respectively. The structures of the
identied avonoids are displayed in Figs. 2 and 3. The comparative study indicates that both samples share the presence of 9
phenolic acids and 9 avonoids. However, the relative proportion
of the most abundant avonoids in both extracts is different. Thus,
in BGS, compounds 13, 36 and 40 are abundant and compound
17 is in low amount, while in CS the opposite occurs. Moreover,
other compounds that are abundant in BGS (15, 30, 33, 39 and
4145) are absent from the CS. Nonetheless, the two extracts
present compounds with similar structures, corresponding to
avonoid derivatives (mainly avonols), many of them 6-methoxy
derivatives and usually O-rutinosyl glycosylated. Bertrand et al.
(2001) isolated and identied 6-methoxy-kaempferol/ quercetin/
isorhamnetin-3-O-rutinoside from another Pilocarpus species, Pilocarpus trachylophus Holmes. All these differences between both
samples seem to indicate that CS is a distinct species or, at least,
a different subspecies of P. pennatifolius, being known that there
are several varieties, forms and subforms of P. pennatifolius (Anon,
1912; Kaastra, 1982).
3.1.1. Phenolic acids derivatives
In the rst part of the chromatogram of both extracts a series
of peaks displaying UV spectra characteristic of cinnamoyl derivatives was observed, which MS spectra showed the base peak at m/z

191 amu ([quinic acid-H] ). Therefore, compounds 14, 7 and 8, at

Rt 2.8, 3.0, 3.2, 3.5, 4.2 and 4.4 min, respectively, exhibited the same
UV spectrum (298sh, 312 nm) and the same deprotonated molecular ion (355 amu). Moreover, in their MS fragmentation, besides the
ion at 191, another abundant ion at 209 amu was observed, hence
they can be characterized as dihydrocaffeoyl quinic acid isomers
(Table 1).
Compounds 5, 6 and 9 (Rt: 3.6, 3.9 and 4.8 min, respectively) also
presented a similar UV spectrum (300sh, 326 nm) and the deprotonated molecular ion ([MH] , m/z 385) was 30 amu higher than
that of compounds 14, 7 and 8; therefore, compounds 5, 6 and
9 could be considered as the methoxylated derivatives of 14, 7
and 8 (Table 1). Compound 10 (Rt: 6.9 min; UV: 300sh, 324 nm;
MS: 397 [MH] ; MS2 (397): 191 (100%, [(MH)-206] , 223(40%,
[sinapic acid-H] ), other less abundant cinnamoyl quinic acid, was
observed only in BGS, and could be labelled as sinapoyl quinic acid
(Table 1).

3.1.2. Flavonoid-O-monoglycosides
Excepting compound 31, the other compounds from this group
(2024, 28, 29, 32, 35, 37 and 4245) presented the loss of 162 amu
(hexosyl radical) in their MS fragmentation, to give place to the
deprotonated ion of the aglycone, which, excepting that of compound 45, was the base peak (Table 2). Thus, these compounds are
avonoid-monohexosides.
The isomeric compounds 20 and 23 presented quercetin as aglycone ([MH] , m/z 301) and, due to their RP chromatographic
mobility (Schieber et al., 2002), can be labelled as quercetin3-O-galactoside (20) and quercetin-3-O-glucoside (23). Similarly,
compounds 22 and 29 can be kaempferol-3-O-galactoside (22) and
kaempferol-3-O-glucoside (29) (Table 2).
The deprotonated aglycone of the isomeric compounds 28, 32 and 37 at m/z 315 indicated that they are
tetrahydroxy-methoxy-avones, which could be isorhamnetin
(3,5,7,4 -tetrahydroxy-3 -methoxy-avone)
or
6/8-methoxykaempferol (3,5,7,4 -tetrahydroxy-6/8-methoxy-avone). It was
not possible to observe the UV spectrum of these and other
compounds because some co-elute and others were present
in trace amounts, precluding their identication. Nonetheless,
abundant ions [Aglycone-H-15] , as well as [(MH)-15] , were
observed in the MS of compounds 28 and 32, which were absent in
compound 37, this last compound being probably an isorhamnetin
derivative, namely isorhamnetin-3-O-glucoside. As already indicated, the avonoids isolated from P. trachylophylus by Bertrand
et al. (2001) were 6-methoxy derivatives. This fact and the differentiation performed above with the pairs 20/23 and 22/29

396

F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

Fig. 2. Structure of the avonoid-O-monoglycosides.

(galactoside/glucoside) leads to the tentative identication of

compounds 28 and 32 as 6-methoxy derivatives, differing in the
sugar moiety: 6-methoxy-kaempferol-3-O-galactoside (28) and
6-methoxy-kaempferol-3-O-glucoside (32).
Similarly, compounds 21 and 24, possessing pentahydroxymethoxy-avone aglycones ([MH] , m/z 331) and showing
the loss of methyl radicals and UV spectra characteristic of
quercetin derivatives (Table 2), can be considered as 6-methoxyquercetin derivatives and, therefore, compound 21 can be
labelled as 6-methoxy-quercetin-3-O-galactoside and compound
24 as 6-methoxy-quercetin-3-O-glucoside. Compound 35 is a
methoxylated derivative of compound 32 and can be labelled as
6-methoxy-isorhamnetin-3-O-glucoside. In the same way, as compounds 4244 showed a trihydroxy-methoxy-avone aglycone
([AglcH] , m/z 299) and as compounds 42 and 43 displayed more
abundant losses of methyl radicals than 44 (Table 2), they can
be considered 6-methoxy-apigenin derivatives (42 and 43, which
is conrmed by the UV spectrum of 43: 272, 334 nm), and 44
could be 3 -methoxy-apigenin (chrysoeriol) derivative. Thus, these
compounds could be tentatively labelled as 6-methoxy-apigenin-

7-O-galactoside (42), 6-methoxy-apigenin-7-O-glucoside (43) and

chrysoeriol-7-O-glucoside (44). Accordingly, compound 45 can
be considered as 6-methoxy derivative of compound 44 and be
labelled as 6-methoxy-chrysoeriol-7-O-glucoside.
Compound 31 was the only non-hexoside monoglycoside in
which the loss of a fragment of 132 amu to give place to a deprotonated ion of methoxy-quercetin (m/z 331) was observed in its
MS fragmentation. So, compound 31 can be labelled as 6-methoxyquercetin-3-O-pentoside.
3.1.3. Flavonoid-O-(rhamnosyl)hexosides
All of the compounds belonging to this group showed in
their MS fragmentation a loss of 308 amu (rhamnohexosyl radical) to give rise to the ion of the deprotonated aglycone as
base peak. With the exceptions of compounds 12 and 19, in
the MS fragmentation of all compounds from this group (1618,
2527, 30, 33, 34, 36 and 3841) the ions due to the interglycosidic linkage were not observed, indicating a 1 6 linkage,
since this kind of linkage is hard to break. In compounds 12 and
19, the loss of fragments of 146 (rhamnosyl radical) and 164

F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

397

Fig. 3. Structure of avonoid-O-hexosides and of avonoid-3-O-(2-rhamnosyl)rutinosides.

(146 + 18) amu due to the break of the interglycosidic linkage was
observed (Table 2), 1 2 being the most common one. Therefore,
the pairs 12/16 and 19/26 can be labelled as quercetin-3-O-(2rhamnosyl) glucoside (12), quercetin-3-O-(6-rhamnosyl) glucoside
(quercetin-3-O-rutinoside, rutin) (16) (this last one conrmed by
the co-injection of the commercial standard), kaempferol-3-O(2-rhamnosyl) glucoside (19) and kaempferol-3-O-(6-rhamnosyl)
glucoside (kaempferol-3-O-rutinoside) (26). The pair of isomers
17/18 showed UV spectra of quercetin derivatives; their aglycone
was pentahydroxy-methoxy-avone ([AglcH] , m/z 331), and loss
of methyl radicals (15) was observed in their MS fragmentations;
thus, they could be considered as 6-methoxy-quercetin derivatives.

As already indicated, Bertrand et al. (2001) isolated 6-methoxyquercetin-3-O-rutinoside in P. trachylophus and taking into account
what was explained for monoglycosides in relation to the possible isomers 6/8 or galactoside/glucoside, these compounds
could be tentatively characterized as 6-methoxy-quercetin-3-O-(6rhamnosyl) galactoside (6-methoxy-quercetin-3-O-robinobioside)
(17) and 6-methoxy-quercetin-3-O-rutinoside (18). The isomers
25, 27 and 30 showed a tetrahydroxy-methoxy-avone aglycone
([AglcH] , m/z 315) in their MS fragmentation. Compounds 25 and
30 exhibited UV spectra of B-ring di-substituted avonoids and ions
[MH-15] were not observed in their MS fragmentation (Table 2),
so that they may be isorhamnetin derivatives, and similarly to

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F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

the indicated above, they can be labelled as isorhamnetin-3O-robinobioside (25) and isorhamnetin-3-O-rutinoside (30). The
ion [MH-15] was observed in the MS of compound 27, which
can coincide with 6-methoxy-kaempferol-3-O-rutinosde, already
described in P. trachylophus by Bertrand et al. (2001). The pair 34/41
displayed a deprotonated molecular ion 30 amu higher than that
of the previous tetrahydroxy-methoxy-avone derivatives (25/30)
and may be 6-methoxy-isorhamnetin-derivatives, although the ion
[MH-15] was not abundant in compound 34 and was absent
from compound 41. For those reasons, they can be labelled as
6-methoxy-isorhamnetin-3-O-robinobioside (34) and 6-methoxyisorhamnetin-3-O-rutinoside (41), which were already isolated
from P. trachylophus by Bertrand et al. (2001). Compounds 3840
are trihydroxy-methoxy-avones ([AglcH] , m/z 299), with UV
spectra of di-substituted B-ring (Table 2) and, therefore, they can
correspond to chrysoeriol-7-O-robinobioside (38) and chrysoeriol7-O-rutinoside (40). Compound 39, possessing a UV spectrum of
apigenin derivative, may be 6-methoxy-apigenin-7-O-rutinoside.
Compound 33 is apigenin-7-O-rutinoside. Compound 36 has a UV
spectrum characteristic of avanone and was chromatographically
coincident, as well as in terms of MS spectrum, with the standard of hesperidin (hesperetin-7-O-rutinoside), already described
by Andrade-Neto et al. (1996) in P. trachyllophus.
3.1.4. Flavonoid-3-O-(2-rhamnosyl) rutinosides
Compounds 11 and 1315 presented deprotonated molecular
ions of avonoid-triglycosides (two rhamnoses and one hexose)
and the base peaks of their deprotonated aglycones were observed
in their MS spectra (11: quercetin, m/z 301; 13: kaempferol, m/z
285; 14: 6-methoxy-isorhamnetin, m/z 345 and 15: isorhamnetin,
m/z 315). The loss of the fragment of 266 amu (146 + 120) was
observed in the MS of all of these compounds, which is produced by the internal cleavage of a hexose by positions 0, 2
(120 amu) and involves a rhamnose (146 amu) at position 6,
this interglycosidic linkage being hard to be broken. Moreover, the
loss of 146 (radical rhamnosyl) and 164 (146 + 18, radical rhamnosyl+water) was observed, indicating a new glycosylation with
other rhamnose, this one being situated in the position 2 of the
hexose, a structure that is corroborated by the above mentioned
120 amu fragment (Fig. 4). Compounds 11 and 13 result from the
rhamnosylation of the 6 -position of compounds 12 and 19 or
of the 2 -position of compounds 16 and 26 and can be labelled
as quercetin-3-O-(2-rhamnosyl) rutinoside (11) and kaempferol3-O-(2-rhamnosyl) rutinoside (13). Similarly, compounds 14 and
15 derive from the rhamnosylation in 2 -position of the glucose
of compounds 41 and 30, respectively, and can be labelled as
6-methoxy-isorhamnetin-3-O-(2-rhamnosyl) rutinoside (14) and
isorhamnetin-3-O-(2-rhamnosyl) rutinoside (15).
3.2. Quantication of phenolic compounds
Besides qualitative differences between BGS and CS, as revealed
by the presence of compounds 10, 12, 14, 15, 19, 20, 22, 25, 26,
2830, 33, 3739 and 4145 only in BGS and of compounds 18,
23, 24, 31, 32 and 35 only in CS, their total phenolic content is
also quantitatively different (Fig. 1, Table 4). BGS (44.94 mg/g of
extract d.w.) contained more amount of phenolic compounds than
CS (37.20 mg/g of extract d.w.; p < 0.05). All compounds were found
in higher amounts (p < 0.05) in BGS, excepting compounds 11 (similar content) and compounds 17, 21 and 27 (in higher amounts in
CS).
Concerning phenolic acids, dihydrocaffeoyl quinic acid
4 + methoxylated dihydrocaffeoyl quinic acid 5 are the major
dihydrocaffeoyl quinic acid isomers in both samples, accounting
to 33.63 and 25.52% of total phenolic acids in BGS and CS, respectively. In BGS, other dihydrocaffeoyl quinic acid, compound 7, is

Fig. 4. Scheme of MS fragmentation of compounds 11 and 1315.

also found in high amount (23.02% of total phenolic acids). On

the other hand, the major avonoid in both samples is different.
Hesperetin-7-O-rutinoside (compound 36) corresponds to 50.82%
of total avonoids determined in BGS, while the same compound
accounts to half this value in CS (22.38%). In CS, 6-methoxyquercetin-3-O-(6-rhamnosyl) galactoside (compound 17) is the
major avonoid (46.80%).
3.3. Imidazole alkaloids
The presence of alkaloids was checked according to the precipitation tests with Dragendorffs, Mayers and Bertrands reagents.
In contrast to what happened with the positive control used
(pilocarpine hydrochloride), no simultaneous precipitation was
observed with all reagents for CS and BGS, indicating that, if present,
alkaloids were in low amount since negative results were obtained
with pilocarpine at concentrations below 0.1 mg/mL. Additionally,
pilocarpine hydrochloride was analysed by HPLCDAD, displaying
a UV spectrum with a maximum at 247 nm; nevertheless, no compound with this characteristic was found in the chromatograms
of CS and BGS. These results are not in accordance with previous
studies that reported the presence of imidazole alkaloids, including pilocarpine, in P. pennatifolius (Salles et al., 2004; Santos and
Moreno, 2004; Sawaya et al., 2011). Sawaya et al. (2011) reported
the presence of pilocarpine in dried leaves of P. pennatifolius (10%
humidity), but no reference on the age of the leaves was done.
However, Salles et al. (2004) quantied pilocarpine in callus and
cell suspension cultures established from young leaves of P. pennatifolius. This probably means that pilocarpine could be already
degraded or altered in BGS and CS leaves. This compound is unstable and if present in the sample will probably be degraded. It is
known that although well preserved, the folioles of P. pennatifolius
loose half of the content of alkaloids at the end of the rst year and,

F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

399

Table 4
Phenolic composition of GBS and CS (mg/g of extract d.w.).a
Compoundsb

GBS

CS

0.95 0.04
0.45 0.01
1.03 0.03
2.47 0.08
1.14 0.06
1.81 0.11
1.13 0.10
0.70 0.16

0.70 0.02

0.70 0.08
1.45 0.25
(only 16)
17
6-OMe-Querct-3-O-rob
0.40 0.16
12.88 0.40
18
6-OMe-Querct-3-O-rut

1.47 0.05
19
Kaempf-3-O-(2-rhmn) glc
0.54 0.06

20
Querct-3-O-gal
0.02 0.00

21 + 22
6-OMe-Querct-3-O-gal + Kaempf-3-O-gal
0.03 0.00
1.71 0.11
(only 21)
23
Querct-3-O-glc

0.59 0.14
24
6-OMe-Querct-3-O-glc

0.16 0.05
25
Isorhmn-3-O-rob
0.12 0.01

26
Kaempf-3-O-rut
0.55 0.04

27 + 28
6-OMe-Kaempf-3-O-rut + 6-OMe-Kaempf-3-O-gal
0.10 0.04
1.08 0.18
(only 27)
29
Kaempf-3-O-glc
0.05 0.01

Isorhmn-3-O-rut
1.17 0.07

30
6-OMe-Querct-3-O-pent + 6-OMe-Kaempf-3-O-glc

0.16 0.05
31 + 32
33 + 34 or 34 + 35 Apig-7-O-rut + 6-OMe-Isorhmn-3-O-rob or 6-OMe-Isorhmn-3-O-rob + 6-OMe-Isorhmn-3-O-glc 1.69 0.21(33 + 34) 0.43 0.17
(34 + 35)
36
Hespt-7-O-rut
12.11 0.66
6.16 0.07
37
Isorhmn-3-O-glc
0.04 0.03

Chrys-7-O-rob
0.08 0.04

38
39
6-OMe-Apig-7-O-rut
0.40 0.09

40
Chrys-7-O-rut
0.21 0.12
0.03 0.00
6-OMe-Isorhmn-3-O-rut + 6-OMe-Apig-7-O-gal
0.05 0.00

41 + 42
43
6-OMe-Apig-7-O-glc
0.05 0.01

44 + 45
Chrys-7-O-glc + 6-OMe-Chrys-7-O-glc
0.21 0.00

1
2
3
4+5
6
7
8
9
10
11
12
13
14 + 15 + 16

Cinnamoyl quinic acid derivative

Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivatives
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Cinnamoyl quinic acid derivative
Sinapoyl quinic acid
Querct-3-O-(2-rhmn) rut
Querct-3-O-(2-rhmn) glc
Kaempf-3-O-(2-rhmn) rut
6-OMe-Isorhmn-3-O-(2-rhmn) rut + isorhmn-3-O-(2-rhmn) rut + querct-3-O-rut

Total

1.15 0.02
0.70 0.08
2.49 0.11
7.10 0.67
1.52 0.12
4.86 0.44
2.08 0.13
1.13 0.06
0.08 0.04
0.68 0.09
0.37 0.04
2.29 0.17
2.67 0.13

44.94

37.20

P Valuec
**
**
****
***
**
***
***
*
n.s.
***
**
****

****

***

**
****

n.s.

Results are expressed as mean standard deviation of three determinations.

b
Querct: quercetin; Kaempf: kaempferol; Isorhmn: isorhamnetin; Apig: apigenin; Chrys: chrysoeriol; Hespt: hesperetin; hex: hexoside; rhmn: rhamnoside; glc: glucoside;
gal: galactoside; pent: pentoside; rut: rutinoside; rob: robinobioside.
c
P value: p > 0.05n.s.; p < 0.05*; p < 0.01**; p < 0.001***; p < 0.0001****.
a

after two years there are no alkaloids (Santos and Moreno, 2004;
Cunha, 2005). Although it is not possible for us to know the age of
the CS leaves, the BGS leaves collected were one year old, i.e., were
produced in the previous season.

3.4. Biological activities

The ability of CS and BGS to inhibit AChE, BuChE and glucosidase was evaluated in vitro. Since pilocarpine is absent
from both samples, the activities may only be related to the phenolic content. In all cases, the inhibition was dose-dependent
(Fig. 5AC). Due to solubility issues, it was not possible to calculate the IC50 value for AChE inhibition, being presented the
IC25 value. The two samples were similarly active against AChE
(IC25 of 872.02 and 1056.03 g/mL for BGS and CS, respectively;
p > 0.05) and -glucosidase (IC50 of 604.34 and 573.48 g/mL
for BGS and CS, respectively; p>0.05). On the other hand, a
stronger capacity (p < 0.01) to inhibit BuChE was observed for
BGS (IC50 = 1112.14 g/mL) than for CS (IC50 = 1743.96 g/mL).
Nevertheless, both extracts showed weak activity (p < 0.001 for
AChE and p < 0.0001 for BuChE) when compared to galantamine
(IC25 = 0.79 g/mL for AChE and IC50 = 4.71 for BuChE) and half the
capacity of acarbose (IC50 = 357.45 g/mL, p < 0.01), the positive

controls for cholinesterases and -glucosidase inhibition assays,

respectively.
Studies focusing structure-activity relationships showed that
the mandatory structural features for anti-AChE activities are the
presence of methoxylation (OMe) at C4 , O-glycosylation at C7 and a
double bond between C2C3. For BuChE inhibition, OMe at C4 is not
so important (Fan et al., 2008; Sawasdee et al., 2009). Substitutions
at C3 diminish cholinesterases inhibition (Jung and Park, 2007;
Khan et al., 2009). Taking into account these features, both samples contain some compounds O-glycosylated at C7 (33, 36, 3840
and 4245), one compound without double bond between C2-C3
but with 4 -OMe (compound 36, one of the major compounds) and
several compounds with 3-O-glycosylation (1132, 34, 35, 37 and
41). In fact, compounds with 3-O-glycosylation account to 37.89
and 77.51% of the avonoids of BGS and CS, respectively. For this
reason, it was not expected both samples to display a remarkable
activity against cholinesterases.
Regarding -glucosidase inhibition, avonoids possessing
hydroxylation in positions 3 , 5 and 6 and a double bond
between C2 and C3 are strongly active, while those possessing Oglycosylations at C3 and C7 are not (Ochir et al., 2010). All avonoids
described present 3- or 7-O-glycosylation; however, IC50 values
displayed by both sample are only twice the value obtained for
acarbose, the positive control. Therefore, we cannot exclude the

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F. Ferreres et al. / Industrial Crops and Products 77 (2015) 391401

-glucosidase observed with both samples, the herbal tea could be

used as nutraceutical ingredient for the food and the pharmaceutical industries.
Acknowledgments
The authors would like to acknowledge Professor Doctor Ana
Isabel Dias Correia and Professor Doctor Teresa Antunes from
Jardim Botnico, Museu Nacional de Histria Natural e da Cincia
(MUHNAC), Universidade de Lisboa, for providing the authenticated sample of Pilocarpus pennatifolius Lem.
This work received nancial support from the European
Union (FEDER funds through COMPETE) and National Funds
(FCT, Fundaco para a Cincia e Tecnologia) through project
UID/QUI/50006/2013. The work also received nancial support
from the European Union (FEDER funds) under the framework of
QREN through Project NORTE-07-0124-FEDER-000069 and project
AGL2011-23690 (CICYT). To all nancing sources the authors are
greatly indebted. Clara Grosso thanks FCT for the FCT Investigator
(IF/01332/2014).
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Fig. 5. In vitro inhibition of AChE (A), BuChE (B) and -glucosidase (C) by CS ()
and BGS (). The results are shown as mean SEM of three assays performed in
triplicate.

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is of extreme importance since one of the taxonomic markers,
pilocarpine, is unstable and was not found even in the authenticated sample. Moreover, based on the moderate activity against

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