90 Original Papers

Anti-Inflammatory Activity and Chemical Profile

of Galphimia glauca

Authors Manasés González-Cortazar 1, Maribel Herrera-Ruiz 1, Alejandro Zamilpa 1, Enrique Jiménez-Ferrer 1, Silvia Marquina 2,
Laura Álvarez 2, Jaime Tortoriello 1

1
Affiliations Centro de Investigación Biomédica del Sur (CIBIS), Instituto Mexicano del Seguro Social (IMSS), Argentina No. 1, Col. Centro,
Xochitepec, Morelos, México
2
Centro de Investigaciones Químicas (CIQ), Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México

Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.

Key words Abstract tracts showed an important anti-inflammatory
" Galphimia glauca
l ! effect. Chemical separation of the active methanol
l
" Malpighiaceae
Galphimia glauca, commonly known as “flor de extract allowed us to identify the nor-seco-triter-
l
" galphimine
estrella”, is a plant species used in Mexican tradi- penes galphimine-A (1) and galphimine-E (3) as
l
" nor‑secotriterpene

tional medicine for the treatment of different dis- the anti-inflammatory principles. Analysis of
l
" anti‑inflammatory activity

l
" flavonoid eases that have an acute or chronic inflammatory
l
" organic acids process in common. Aerial parts of this plant con-
tain nor-seco-triterpenoids with anxiolytic prop-
erties, which have been denominated galphi-
mines. Other compounds identified in the plant
are tetragalloyl-quinic acid, gallic acid, and quer-
cetin, which are able to inhibit the bronchial ob-
struction induced by platelet-activating factor.
The objective of this work was to evaluate the
anti-inflammatory effect of crude extracts from
G. glauca and, by means of bioguided chemical
separation, to identify the compounds responsi-
ble for this pharmacological activity. n-Hexane,
ethyl acetate, dichloromethane, and methanol ex-
structure-activity relationships evidenced that
the presence of an oxygenated function in C6 is
absolutely necessary to show activity. In this
work, the isolation and structural elucidation of
two new nor-seco-triterpenes denominated as
galphimine-K (4) and galphimine-L (5), together
with different alkanes, fatty acids, as well as three
flavonoids (17–19), are described, to our knowl-
edge for the first time, from Galphimia glauca.
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica

Introduction 7]. Among these, galphimine-B (G‑B) has been

received March 22, 2013
revised Nov. 6, 2013
accepted Nov. 11, 2013
Bibliography
DOI http://dx.doi.org/
10.1055/s-0033-1360150
Published online December 11,
2013
Planta Med 2014; 80: 90–96
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Maribel Herrera-Ruiz, PhD
Centro de Investigación
Biomédica del Sur (CIBIS)
Instituto Mexicano del Seguro
Social (IMSS)
Argentina No. 1, Col. Centro
62790 Xochitepec, Morelos
México
Phone: + 52 77 73 61 21 55
cibis_herj@yahoo.com.mx
! identified as the most active, followed by galphi-
Galphimia glauca (Malpighiaceae) is a plant spe-
cies widely used in Mexican traditional medicine
for the treatment of different diseases such as gas-
tric ulcers, and as an anti-inflammatory in the
case of traumatic blows, wounds, scars, inflam-
mation of the kidney or uterus, and rheumatism
[1, 2]. It is noteworthy that these conditions have
an acute or chronic inflammatory process in com-
mon. This plant species, popularly known as “flor
de estrella”, “telpinoxochilt” (Nahuatl), “yerba del
desprecio”, “árnica de raíz”, or “árnica roja”, has
been submitted (over the last two decades) to dif-
ferent chemical, toxicological, pharmacological,
and clinical studies in order to analyze its effect
on the central nervous system [3–5]. It has been
demonstrated that the aerial parts of this plant
contain compounds of the nor-seco triterpenoid
type, which possess anxiolytic properties and
which have been denominated galphimines [6,
mine-A (G‑A) [8]. In this regard, scientific re-
search has achieved a high level of knowledge
that has permitted the development of phyto-
pharmaceuticals elaborated with the standard-
ized extract of G. glauca, which has important
anxiolytic effectiveness, as demonstrated by clini-
cal studies. The G. glauca phytopharmaceutical,
administered orally for 4 and for 15 weeks, was
compared with Lorazepam in patients with gen-
eralized anxiety disease (GAD) [9, 10]. There is al-
so scientific background on the pharmacological
effect produced by extracts obtained from G. glau-
ca on bronchial asthma, a disease with an impor-
tant inflammatory process of the airways. In this
case, the tetragalloyl-quinic acid as well as the
gallic acid and quercetin were identified as the ac-
tive suppressor compounds of bronchial obstruc-
tion induced by the platelet-activating factor [11].
Recently, it was reported that a methanolic

González-Cortazar M et al. Anti-Inflammatory Activity and … Planta Med 2014; 80: 90–96
 Original Papers 91

Edema (mg) Edema inhibition (%) Table 1 Effect produced by dif-

Extracts from G. glauca (3.2 mg/ear) ferent extracts, fractions, sub-frac-
" GgHex 1.43 ± 0.78* 87.94 tions, and compounds obtained
" GgAcOEt 1.48 ± 1.43* 87.51 from Galphimia glauca on auricular
" GgCH2Cl2 1.57 ± 1.46* 86.74 edema induced by 12-Ο-tetrade-
" GgMeOH 1.48 ± 1.45* 87.51 canoylphorbol 13-acetate (TPA) in
Fractions from GgMeOH (1.6 mg/ear) ICR mice. Analysis of variance
" C F1 5.98 ± 1.30* 55.74* (ANOVA) with post hoc Bonferroni
1
" C F2 5.80 ± 1.90* 57.04* (n = 7; mean ± standard deviation
1
" C F3 2.65 ± 1.60* 80.37* [SD]). * p < 0.05 in comparison
1
" C F4 2.93 ± 1.50* 78.27* with the negative control group.
1
" C F5 10.48 ± 2.79 11.28
IND = indomethacin.
1
" C F6 7.45 ± 1.93* 44.81*
1
" C F7 1.15 ± 0.61* 91.48*
1
" C F8 9.42 ± 1.66 20.28
1
" C F9 10.88 ± 1.63 7.87
1
" C F10 9.95 ± 3.00 15.77
1
" C F11 11.23 ± 1.73 4.90
1
Subfractions from C1F7 (1.0 mg/ear)

Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.

" C F1 7.55 ± 2.20* 36.08
2
" C F2 7.62 ± 2.32* 35.52
2
" C F3 4.80 ± 1.62* 59.37
2
" C F4 5.08 ± 1.50* 52.41
2
" C F5 10.67 ± 2.30 9.70
2
Galphimines from C2F3 (1.0 mg/ear)
" G‑A 6.18 ± 2.21* 51.75
" G‑B 9.80 ± 1.64 17.04
" G‑E 3.68 ± 1.20* 68.85
" G–K + GL 10.23 ± 1.39 13.40
Positive control group (IND) (1.0 mg/ear) 0.82 ± 0.50* 93.06
Negative control group 11.81 ± 1.74

(MeOH) extract from G. glauca reduced the inflammation in-
duced in mouse ear with tetradecanoylphorbol acetate [12]. Re-
garding the chemical composition of aerial parts from G. glauca,
compounds of the nor-seco triterpene type, known as galphi-
mines (galphimine-B, galphimine-A, galphimine-E, and galphi-
mine-J), have been identified. Some galphimines have shown se-
lective anxiolytic activity, while a structure-activity relationship
has evidenced the importance of a double-bond between C-1 and
C-2 of the A ring in order to exert the pharmacological effect [7].
Other reports showed the presence of terpenoids denominated
galphin-A, galphin-B, galphin-C, and galphimidin, as well as
quercetin, stigmasterol, and sitosterol 3-O-β-D-glucoside [13].
The objective of this work was to evaluate the anti-inflammatory
effect of a crude extract from G. glauca and, by means of a bio-
guided chemical separation (by using the mouseʼs ear edema in-
duced by 12-O-tetradecanoylphorbol 13-acetate (TPA) as the
monitoring test), to identify the compounds responsible for this
pharmacological activity.
Results and Discussion
! pholipids in the cellular membrane with the consequent release
Different extracts from G. glauca (n-hexane, ethyl acetate, dichlo-
romethane, and methanol) at a dose of 3.2 mg/ear were eval-
uated in TPA-induced auricular edema in mice [14]. This study
showed that all of the tested extracts had an important anti-in-
flammatory effect, which was significantly different (p < 0.05)
with respect to the negative control group (l " Table 1), but not
different from that of the positive control group which was treat-
ed with indomethacin (IND). This result suggested the presence
of secondary metabolites capable of diminishing TPA-induced in-
flammation. Thus, we continued with a bioguided chemical frac-
tionation to identify the active anti-inflammatory compounds.
Methanol extract (GgMeOH) was selected for fractionation be-
cause it was that with higher yield.
All fractions and subfractions obtained from GgMeOH, as well as
the purified compounds, were submitted to the pharmacological
anti-inflammatory test. As illustrated in l
" Table 1, the C F7 frac-
1
tion was the most active with 91.48 % inhibition of auricular ede-
ma, while C1F1, C1F2, C1F3, C1F4, and C1F6 were also able to re-
duce the TPA-induced inflammation but at a lesser proportion.
After analyzing the chemical content, it was not surprising to find
that fractions C1F1-C1F6 exhibited anti-inflammatory activity.
For example, gas chromatography-mass spectrometry (GC‑MS)
analysis of the C1F1 fraction allowed identification of the follow-
ing non-polar compounds: triacontane, tricosane, pentacosane,
hexacosane, heptacosane, octacosane, nonacosane, hentriaco-
sane, cyclotetracosane, hexacosene, and hexadecanoic acid. This
latter saturated fatty acid is able to modulate the immune re-
sponse. A previous report demonstrated that this fatty acid inhib-
its the phospholipase A2 enzyme (PLA2) which hydrolyzes phos-
of araquidonic acid (AA), thus initiating the inflammatory re-
sponse [15]. In this respect, TPA is capable of stimulating AA re-
lease in a keranocyte culture through the action of cytosolic PLA2
action [16]. There is no information about the anti-inflammatory
activity of the other compounds identified in the C1F1 fraction.
On the other hand, fraction C1F2 contains α-tocoferol, triaconta-
nol, eicosanol, pentacosanol, phytol, α-amyrin (15), and β-amyrin
(16), and these triterpenes have antinociceptive and anti-inflam-

González-Cortazar M et al. Anti-Inflammatory Activity and … Planta Med 2014; 80: 90–96
92 Original Papers
matory action (at a dose of 30 mg/kg) through activation of can-
nabinoid receptors and by inhibiting cytokine production [17]. α-
Amyrin derivatives, such as α-amyrin acetate and α-amyrin cin-
namate, induce a decrease of TPA-induced inflammation [18]. Re-
garding fractions C1F3 and C1F4, both contain β-sitosterol (13)
which has been demonstrated to possess an anti-inflammatory
activity. For example, a mixture of β-sitosterol, stigmasterol, and
campesterol, isolated from Buddleja globosa, is able to reduce
TPA-induced edema up to 78.2 % at a 1-mg/ear dose [19]. It is im-
portant to point out that these results are similar to those ob-
served in our work, in which C1F3 and C1F4 inhibited edema by
80.37 and by 78.27 %, respectively. Fractions C1F5, C1F8, C1F9,
C1F10, and C1F11 did not induce an anti-inflammatory effect,
and their chemical analyses indicated the presence of organic ac-
ids such as methylgallate (10), 4-methoxy methyl gallate (11),
and gallic acid (12), as well as the sterol β-sitosteryl-3-O-β-D-glu-
copyranoside (14) and the flavonoids kaempferol 3-O-β-D-gluco-
pyranoside (8), quercetin 3-O-β-D-glucopyranoside (9), kaemp-
ferol 3-O-β-D-(2′′-galloyl)-glucopyranoside (17), kaempferol 3-
O-β-D-(2′′-galloyl)-galactopyranoside (18), and quercetin 3-O-β-
D-(2′′-galloyl)-galactopyranoside (19). Although compounds 17,
18, and 19 have already been described for Galphimia glauca
[20], in this paper we report, to our knowledge for the first time,
the experimental evidence by spectroscopic analysis of the pres-
ence of these metabolites.
It has been reported that compounds associated with methylgal-
late (10), such as (−)-epicatechin 3-(3-O-methylgallate) and
(+)-catechin 3-(3-O-methylgallate), exert anti-inflammatory ac-
tivity on auricular edema induced by the TPA model [21]. We
can assume that the active part of these molecules is the epicate-
chin and not the methylgallate. On the other hand, fractions con-
taining kaempferol derivatives were not active, although it was
reported that these substances show activity when administered
orally (50 and 100 mg/kg) in an animal model. This metabolite
was capable of inhibiting the production of nitrites and prosta-
glandin-E2, and it also diminished the cellular infiltration into
rat granuloma air pouch induced by carrageenan by modulation
of the cyclooxygenase pathway via inhibition of nitric oxide (NO)
synthesis [22]. It was also able to reduce the activity of nuclear
factor (NF)-kappaβ and NF-kappaβ-dependent pro-inflamma-
tory gene activity [23] in both experiments in which this flavonol
was administered orally. In our work, the correspondent fraction
did not exhibit activity, probably because of the administration
route employed, or, the presence of more complex molecules as-
sociated with kaempferol.
The C1F7 fraction was selected because it exhibited the highest
activity; subsequently, it was submitted to chemical separation.
Only subfractions C2F3 and C2F4 (these subfractions also con-
tained β-sitosterol) inhibited the inflammation by 59.37 and
52.41 %, respectively. This result was significantly (p < 0.05) dif-
ferent from that of the negative control group (l " Table 1). Sub-
fractions C2F1 and C2F2 inhibited edema by 36 and 35 %, respec-
tively. Even though these effects were significantly different from
those of the control group, they were lower. C2F5 failed to pro-
duce anti-inflammatory activity; however, this fraction was con-
stituted mainly of the flavonols kaempferol (6) and quercetin (7),
as well as galphimine-J (G–J).
Finally, reverse phase chromatography of C2F3 allowed us to ob-
tain the triterpenes galphimine-A (1), galphimine-B (2), and gal-
phimine-E (3), in addition to a new triterpene described as gal-
phimine-K (4) and its isomer galphimine-L (5). Structural eluci-
dation of both compounds (4 and 5) was performed by their
González-Cortazar M et al. Anti-Inflammatory Activity and … Planta Med 2014; 80: 90–96
NMR spectra data analysis (1H, 13C, DEPT) with two-dimension
(2-D) experiments (COSY, HSQC, HMBC). Direct comparison of
1
H and 13C signals from this compound displayed high similarity
with those previously described for galphimine-B (2) [24], except
for the absence of signals assigned to C-4 and C-7. The signal cor-
responding to the hydroxymethine group at C-7, which in G‑B
appears at δH 4.22 and δC 66.5, disappeared in compounds 4 and
5; instead, signals for an aliphatic methylene group were evi-
denced. In compound 4, this methylene signal (δH 1.64; δC 30.4)
showed COSY correlation with H-6 (δ 1.39) and H-8 (δ 2.36–
2.41). In addition, the signal for C-4 hydroxymethine was shifted
downfield (Δδ = 0.22 in 1H and 9.4 in 13C) with respect to 2, due to
the presence of an acetyl group in this position. This was also
confirmed by the 3J correlation between H-4 (δ 4.29, q,
J = 6.9 Hz) and the acetate carbonyl group at δ 167.7 in the HMBC
spectra. Full assignment of proton and carbon signals is described
in the experimental section. This compound has not been, to our
knowledge, previously isolated and represents a new natural
Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.
product from G. glauca; it was denominated galphimine-K (4).
In addition, some samples of G–K are made up of a mixture of G–
K and its Δ20(29) isomer (80 : 20); this was evidenced by the pres-
ence of additional vinyl signals at δ 4.61 (1H, br, d, J = 2.3 Hz, H-
29a) and 4.38 (1H, br, d, J = 2.3 Hz, H-29b) in the 1H NMR spectra,
corresponding to an exocyclic double bond. The remaining sig-
nals in these spectra were very similar to those of compound 4.
This compound is also a new, natural product, and it was de-
nominated galphimine-L (5).
Nor-secofriedeles 1–3 were evaluated (1.0 mg/ear) in TPA-in-
duced auricular edema in mouse model. l " Table 1 illustrates the
anti-inflammatory activity displayed by these compounds. The
natural G‑A (1) inhibited inflammation by 51.75 %, while G‑E (3)
did so by 68.85 %. Both exhibited statistical differences in com-
parison with the negative control group. The known anxiolytic
compound, G‑B (2), as well as the mixture of the new galphi-
mines G–K and G–L, failed to induce any anti-inflammatory ac-
tivity. To date, there have been, to our knowledge, no previous re-
ports on the anti-inflammatory activity of nor-secotriterpenes
from G. glauca. The biological effect of these compounds confirms
and validates the traditional use of G. glauca in Mexican tradi-
tional medicine as an anti-inflammatory agent. The effect of G‑A
(1) and G‑E (3) was analyzed by means of a dose-response curve
(l" Table 2); the results showed an E
max of 99.01 % and an ED50 of
0.81 mg/ear for G‑A, and an Emax of 90.8 % and an ED50 of 0.31 mg/
ear for G‑E (3), indicating that compound 1 is more active than
compound 3, but that compound 3 is more potent. The structural
difference between these two active galphimines (1 and 3) and
the inactive G‑B (2) is the oxidation state in the C6 position of
ring B (l " Fig. 1). In this position, there is a hydroxyl group in
G‑A and an acetate group in G‑E, while in G‑B, these groups are
absent. This fact is in agreement with the absence of activity of
the remaining C6-non-oxygenated galphimines G–K and G–L,
suggesting that the presence of an oxygenated function in C6 is
necessary for anti-inflammatory activity.
In this work, isolation and structural elucidation of different alka-
nes and fatty acids, as well as of three flavonoids (17–19), are de-
scribed, to our knowledge for the first time, for this species. Two
new nor-secotriterpenes were also identified and denominated
galphimine-K (4) and galphimine-L (5).
Chemical separation of active compounds performed by means of
their anti-inflammatory effectiveness allowed us to identify nor-
secotriterpenes G‑A (1) and G‑E (3) as responsible for this activ-
ity. Analysis of the structure-activity relationships of galphimines
 Original Papers 93

Table 2 Dose-response curve of galphimine-A and galphimine-E obtained from Galphimia glauca in auricular edema induced by 12-Ο-tetradecanoylphorbol 13-
acetate (TPA) in ICR mice. Analysis of variance (ANOVA) with post-hoc Bonferroni (n = 7, mean ± standard deviation [SD]); * p < 0.05 in comparison with the neg-
ative control group. a Emax = maximum effect; b DE50 = effective doses to 50%.

Edema (mg) Edema inhibition (%) Emaxa ED50b

Treatment G‑A (mg/ear)
" 0.25 8.81 ± 1.87 25.36 99.01% 0.81 mg/ear
" 0.50 7.02 ± 1.66* 40.53
" 1.0 6.18 ± 2.21* 51.75
" 1.5 2.57 ± 1.01* 78.35
" 2.0 2.01 ± 0.42* 82.93
Treatment G‑E (mg/ear)
" 0.25 7.03 ± 3.04* 40.46 90.80% 0.31 mg/ear
" 0.50 4.91 ± 2.01* 58.52
" 1.0 3.68 ± 1.20* 68.85
" 1.5 2.99 ± 0.91* 74.69
" 2.0 2.36 ± 0.57* 80.02
Positive control group 1.0 0.82 ± 0.50* 93.06
Negative control group 11.81 ± 1.74

Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.

Fig. 1 Chemical structure of secondary metabo-
lites from Galphimia glauca.

González-Cortazar M et al. Anti-Inflammatory Activity and … Planta Med 2014; 80: 90–96
94 Original Papers
(1–5) evidenced that the presence of an oxygenated function in
C6 is absolutely necessary for exhibiting activity.
Anxiolytic effectiveness, previously identified in galphimines
from G. glauca, has already been demonstrated by preclinical
and clinical studies and, together with anti-inflammatory activ-
ity, suggests that the standardized extracts or fractions obtained
from this plant could be effective for the treatment of degenera-
tive disorders that express an inflammatory component, such as
anxiety and Alzheimer disease [25]. It is also important to con-
clude that, considering the yield of galphimines (0.2 %) in the
methanol extract, it is evident that the anti-inflammatory activ-
ity displayed by the extract is due to the pharmacologic interac-
tion among the different compounds.
Material and Methods
! 60 (Merck, mesh 70–230). A dichloromethane/methanol gradient
Drugs and general procedure
12-O-tetradecanoylphorbol 13-acetate (TPA, ≥ 99 %, TLC) and in-
domethacin (IND, ≥ 99 %, TLC) were purchased from Sigma Chem-
ical Co. Melting points were obtained on a Thermo Scientific
IA9000 series melting point apparatus and were uncorrected. All
NMR spectra were recorded on Varian INOVA-400 at 400 MHz for
1
H NMR, 1H-1H COSY, HSQC, and HMBC, and at 100 MHz for 13C
NMR in CDCl3. NOESY experiment was used for the characteriza-
tion of galphimine K (4). Chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane (TMS).
Plant material
Leaves from Galphimia glauca (10 kg) were collected from a con-
trolled crop developed in Morelos State, Mexico. Taxonomic
identification was performed by Abigail Aguilar-Contreras, M.
Sc., the Herbarium Head. A voucher sample was deposited in the
Mexican Institute of Social Security Herbarium with the code
number IMSSM-8646. Plant material was dried under dark condi-
tions at room temperature for 2 weeks. Afterwards, in order to
obtain particles 3–5 mm in size, the plant material was ground
in an electric grinder mill.
Preparation of extracts
The dried and ground material (4.6 kg) was organized into four
samples of 1.15 kg, which were individually extracted by macer-
ation with n-hexane, dichloromethane, ethyl acetate, or metha-
nol for a 2-day period and repeated three times. All extractions
were performed by using 1 : 5 plant material/solvent proportions.
Solvent was eliminated under reduced pressure distillation with
a Heidolph-brand rotary evaporator, obtaining total yields of 1.1,
2.2, 4.1, and 12.2 % of the n-hexane, dichloromethane, ethyl ace-
tate, and methanol extracts, respectively.
Methanolic extract fractionation
GgMeOH extract (50.0 g) was fractionated by chromatographic
open column (600 × 70 mm) previously packed with silica gel 60
(Merck, mesh 70–230, 500 g). An n-hexane/EtOAc/MeOH gra-
dient system was used as mobile phase, starting with 100 % of n-
hexane with successive increments of EtOAc (the volume of all
samples was 250 mL). When the gradient system consisted of
50 : 50 % mixture (n-hexane/ethyl acetate), ascendant concentra-
tions of MeOH were incorporated into this mobile phase. One
hundred and fifty six fractions were obtained, which were
grouped into the following 11 final fractions according to their
chemical composition: C1F1 (100 : 0 : 0, 4 g, fatty acids); C1F2
González-Cortazar M et al. Anti-Inflammatory Activity and … Planta Med 2014; 80: 90–96
(90 : 10 : 0, 3.2 g); C1F3 (80 : 20 : 0, 4.4 g); C1F4 (70 : 30 : 0, 4.1 g);
C1F5 (60 : 40 : 0, 5.2 g); C1F6 (50 : 50 : 0, 7.2 g); C1F7 (47.5 : 47.5 : 5,
2.9 g); C1F8 (45 : 45 : 10, 3.8 g); C1F9 (40 : 40 : 20, 2.6 g); C1F10
(35 : 35 : 30, 5.3 g,); and C1F11 (0 : 0 : 100, 3.7 g). C1F1 and C1F2
fractions were subjected to a GC‑MS analysis. All of the remain-
ing compounds were elucidated through their 1H and 13C NMR
spectra analysis.
Recrystallization of C1F8, C1F9, and C1F10 allowed us to obtain
compounds 17 (0.120 g, 97 %), 18 (0.070 g, 99 %), and 19 (0.170 g,
99 %), respectively. All of these flavonoids were identified by their
1
H and 13C NMR spectra analysis as well as their two-dimensional
experiments (COSY, HSQC, and HMBC).
Flavonoid derivatives isolation
The C1F7 fraction (5 g) was submitted to a chromatographic open
column (500 × 30 mm) previously packed with 100 g of silica gel
system was used as mobile phase, starting with 100% of the sol-
Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.
vent of least polarity and finalizing with 100 % methanol (volume
of all samples was 75 mL). Forty fractions were obtained that
were grouped in five final fractions according to their chemical
composition: C2F1 [100 : 0, 0.4 g); C2F2 (95 : 5, 1.1 g); C2F3
(90 : 10, 0.8 g, β-sitosterol (13) and galphimines (1, 2, 3)]; C2F4
(70 : 30, 1.8 g); C2F5 (60 : 40, 0.8 g). Crystallization of C2F3 (ace-
tone) gives uncolored needles. GC‑MS analysis of this crystalline
compound indicated that it corresponds to 13. C2F4 and C2F5
were subjected to an HPLC method which revealed that these
fractions contain the flavonols: kaempferol (6, 98 %) and querce-
tin (7, 97%), as well as the triterpene galphimine-J (comparison
with authentical samples previously isolated from this plant).
Galphimines purification
The C2F3 fraction (1.5 g) was purified in a chromatographic open
column (500 × 21 mm) previously packed with 30 g of reverse
phase silica gel 60 (Merck, 40–63 µm, RP-18) and stabilized with
methanol. A water/acetonitrile gradient system was used as mo-
bile phase, starting with 100 % water and finalizing with 100 %
acetonitrile (the volume of all samples was 30 mL). Forty eight
fractions were obtained and grouped into the following eight fi-
nal fractions according to their chemical composition. C3F1
(100 : 0, 0.03 g); C3F2 (70 : 30, 0.12 g, galphimine-A [1, 89 %]);
C3F3 (40 : 60, 0.13 g); C3F4 (40 : 60, 0.26 g, galphimine-B [2,
95 %]); C3F5 (30 : 70, 0.48 g, galphimine-E [3, 99%]); C3F6 (30 : 70,
0.24 g); C3F7 (20 : 80 : 0, 0.15 g), and C3F8 (0 : 100, 0.04 g). These
nor-secofriedelanes were identified by direct comparison by
HPLC with authentical samples previously isolated from this
plant [8].
Chromatographic purification of C3F7 (0.15 g) by silica-gel open
column (21 × 300 mm, 70–230 mesh; Merck) and a mixture of
the n-hexane/EtOAc gradient system as mobile phase allowed us
to obtain a yellow precipitate which was crystallized (acetone) to
give both compound 4 (30 mg, 95 %) and compound 5 (1 mg,
95 %). These last compounds were identified, to our knowledge
for the first time, in this plant species.
GC‑MS analysis
The chemical composition of the C1F1 and C1F2 fractions was an-
alyzed on a gas chromatograph equipped with a quadrupole
mass detector in electron impact mode at 70 eV. Volatile com-
pounds were separated on a HP 5MS capillary column (25 m long,
0.2 mm i. d., 0.3-µm film thickness). Oven temperature was set at
40 °C for 2 min, then programmed from 40–260 °C at 10 °C/min

and maintained for 20 min at 260 °C. Mass detector conditions
were as follows: interphase temperature 200 °C, and mass acqui-
sition range, 20–550. The temperatures of the injector and detec-
tor were set at 250 and 280 °C, respectively. The splitless injection
mode was carried out with 1 µL of each fraction (3 mg/mL solu-
tion). The carrier gas was helium at a flow rate of 1 mL/min. Iden-
tification of volatiles was performed comparing their mass spec-
tra with those of the National Institute of Standards and Technol-
ogy (NIST) 1.7 Library. In addition, a standard solution of C7-C40
alkanes was used to obtain the retention index of compounds and
to compare these with data in the literature [26].
Animals
Male albino ICR mice weighing around 35 g were used (Harlan).
All animals were housed eight per cage, maintained under labo-
ratory conditions at 25 °C, under 12-h light/12-h dark cycles, with
lights turned on at 7 : 00 a. m. and free access to water and stan-
dard food pellets (Harlan). The mice were allowed at least 3
weeks to adapt to the laboratory environment prior to initiating
the experiments. All experimental procedures were carried out
according to a protocol approved by the Institutional Research
Committee in compliance with the Official Mexican Regulation
that dates from 1999 (NOM-062-ZOO-1999). Minimum number
of animals (n = 7) and duration of observation required to obtain
consistent data were employed. The experimental protocol was
approved by the local Ethics Committee on April 13th, 2010, and
received registration number R-2010-1701-23.
Acute inflammation in mice with TPA
Animal inflammation was induced following the method previ-
ously described by Gábor [14]. The mice were grouped (into
groups of seven individuals), and TPA (2.5 µg) dissolved in ace-
tone (20 µL) was topically administered on the internal and exter-
nal surfaces of the mouseʼs right ear to cause edema.
Doses of 3.2 mg/ear of different polarity extracts were adminis-
tered topically on the ear of each individual mouse. In case of
fractions, 1.6-mg/ear doses were employed, while for subfrac-
tions and isolated compounds (galphimines), doses were 1.0 mg/
ear. The reference anti-inflammatory drug indomethacin (IND)
was administered at 1.0 mg/ear [14]. Treatments were dissolved,
according to their solubility, in acetone, methanol, and a mixture
of methanol-acetone, and subsequently administered topically
onto both ears. Ten min later, the TPA solution was administered,
and 6 h after administration of the inflammatory agent, the ani-
mals were sacrificed by cervical dislocation. Circular sections,
6 mm in diameter, were taken from both treated (t) and non-
treated (nt) ears and were weighed to determine the inflamma-
tion.
Percentage of inhibition was obtained by using the expression as
follows:
Inhibition % = [Δ w control – Δw treatment/Δw] [100],
where Δw = wt-wnt, while wt is the weight of the section of the
treated ear and wnt is the weight of the section of the non-treat-
ed ear.
Statistical analysis
The results obtained from the pharmacological test were submit-
ted to ANOVA, followed by a post hoc Bonferroni test. Values of
p < 0.05 were considered significantly different.
González-Cortazar M et al. Anti-Inflammatory Activity and …
Original Papers 95
Isolated compounds
Kaempferol 3-O-β‑D-(2′′-galloyl)-glucopyranoside (17): White
amorphous solid; 1H NMR (400 MHz, CD3OD); δ 6.16 (1H, s, H-
6), 6.33 (1H, d, J = 1.6 Hz, H-8), 7.94 (1H, d, J = 8.8 Hz, H-2′, H-6′),
6.84 (2H, d, J = 9.2 Hz, H-3′, H-5′), 7.12 (2H, s, H-2′′′, H-6′′′), 5.73
(1H, d, J = 8.4 Hz, H-1′′), 5.10 (1H, dd, J = 8, 9.6 Hz, H-2′′), 3.64–
3.66 (1H, m, H-3′′), 3.43 (1H, dd, J = 8.4, 8,4 Hz, H-4′′), 3.32–3.36
(1H, m, H-5′′), 3.5–3.6 (1H, m, H-6′′a), 3.7–3.8 (1H, m, H-6′′b);
13
C NMR (100 MHz, CD3OD); δ 158.3 (C, C-2), 134.9 (C, C-3),
179.3 (C, C-4), 163.2 (C, C-5), 99.8 (CH, C-6), 167.8 (C, C-7), 94.7
(CH, C-8), 158.4 (C, C-9), 106.0 (C, C-10), 122.8 (C, C-1′), 132.2
(CH, C-2′), 116.3 (CH, C-3′), 161.4 (C, C-4′), 116.3 (CH, C-5′),
132.2 (CH, C-6′), 100.6 (CH, C-1′′), 76.1(CH, C-2′′), 77.5 (CH, C-3′
′), 71.8 (CH, C-4′′), 78.8 (CH, C-5′′), 62.7 (CH2, C-6′′), 121.7 (C, C-1′
′′), 110.7 (CH, C-
2′′′), 146.4 (C, C-3′′′), 139.9 (C, C-4′′′), 146.4 (C, C-5′′′), 110.7 (CH,
C-6′′′), 167.8 (C, C-7′′′).
Kaempferol 3-O-β‑D-(2′′-galloyl)-galactopyranoside (18): White
Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.
amorphous solid; 1H NMR (400 MHz, CD3OD); δ 6.16 (1H, s, H-
6), 6.33 (1H, d, J = 1.6 Hz, H-8), 7.92 (1H, d, J = 8.8 Hz, H-2′, H6′),
6.8 (2H, d, J = 8.8 Hz, H-3′, H-5′), 7.13 (1H, s, H-2′′′, H-6′′′), 5.63
(1H, d, J = 7.6 Hz, H-1′′), 5.41 (1H, dd, J = 8, 9.6 Hz, H-2′′), 3.8 (1H,
dd, J = 2.4, 10 Hz, H-3′′), 3.90 (1H, br, d, J = 3.2 Hz, H-4′′), 3.5–3.6
(1H, m, H-5′′), 3.6–3.7 (2H, m, H-6′′); 13C NMR (100 MHz, CD3OD);
δ 158.4 (C, C-2), 135.0 (C, C-3), 179.4 (C, C-4), 163.2 (C, C-5), 99.8
(CH, C-6), 168.1 (C, C-7), 94.7 (CH, C-8), 158.3 (C, C-9), 105.9 (C, C-
10), 122.7 (C, C-1′), 132.2 (CH, C-2′), 116.4 (CH, C-3′), 161.4 (C, C-
4′), 116.4 (CH, C-5′), 132.2 (CH, C-6′), 101.1 (CH, C-1′′), 74.6 (CH,
C-2′′), 73.5 (CH, C-3′′), 70.6 (CH, C-4′′), 77.5 (CH, C-5′′), 62.2 (CH2,
C-6′′), 121.6 (C, C-1′′′), 110.7(CH, C-2′′′), 146.4 (C, C-3′′′), 139.9 (C,
C-4′′′), 146.4 (C, C-5′′′), 110.7 (CH, C-6′′′), 168.1 (C, C-7′′′).
Quercetin 3-O-β‑D-(2′′-galloyl)-galactopyranoside (19): Yellow
needles; 1H NMR (200 MHz, CD3OD); δ 6.17 (1H, d, J = 1.8 Hz, H-
6), 6.30 (1H, d, J = 1.8 Hz, H-8), 7.55 (1H, d, J = 2.2 Hz, H-2′), 6.66
(1H, d, J = 8.8 Hz, H-5′), 7.28 (1H, dd, J = 2.2, 8.8 Hz, H-6′), 7.04
(2H, s, H-2′′′, H-6′′′), 5.39 (1H, d, J = 8 Hz, H-1′′), 5.11 (1H, dd,
J = 8.8, 9.6 Hz, H-2′′), 3.92 (1H, br, d, J = 2.6 Hz, H-4), 3.5–3.7 (5H,
m, H-3′′, H-5′′, H-6a’’, H6b’’); 13C NMR (50 MHz, CD3OD); δ 157.5
(C, C-2), 135.0 (C, C-3), 178.6 (C, C-4), 162.1 (C, C-5), 99.5 (CH, C-
6), 167.5 (C, C-7), 94.4 (CH, C-8), 157.5 (C, C-9), 105.4 (C, C-10),
122.2 (C, C-1′), 115.7 (CH, C-2′), 145.0 (CH, C-3′), 148.8 (C, C-4′),
116.7 (CH, C-5′), 122.4 (CH, C-6′), 100.9 (CH, C-1′′), 74 (CH, C-2′′),
72.4 (CH, C-3′′), 69.8 (CH, C-4′′), 76.4 (CH, C-5′′), 61.4 (CH2, C-6′′),
120.7 (C, C-1′′′), 110.3 (CH, C-2′′′, C-6′′′), 145.0 (C, C-3′′′), 148.8 (C,
C-4′′′), 145.3 (C, C-5′′′), 167.6 (C, C-7′′′).
13α-Carbomethoxy-4R-acetoxy-18β-hydroxy-30-nor-3,4-seco-
friedela-1,20-dien-3,24-olide, galphimine K (4): mp 212–215 °C;
UV λmax: 198 nm; 1H NMR (400 MHz, CDCl3); δ 6.32 (1H, dd,
J = 8.8, 12 Hz, H-1), 5.97 (1H, d, J = 12.5 Hz, H-2), 4.29 (1H, q,
J = 6.9 Hz, H-4), 1.39 (2H, m, H-6), 1.64 (2H, m, H-7a, H-7b), 2.68
(1H, d, J = 8.8 Hz, H-10α), 2.36–2.41 (1H, m, H-8α), 2.58 (2H, m, H-
16, H-22), 5.08 (1H, br, s, H-21), 0.96 (3H, d, J = 6.4 Hz, H-23), 4.25
(2H, br, s, H-24a, H-24b), 0.9 (3H, s, H-25), 1.31 (3H, s, H-26), 0.91
(3H, s, H-28), 1.51 (3H, s, H-29), 3.46 (3H, s, OCH3), 1.46 (3H, s,
CH3COO); 13C NMR (100 MHz, CDCl3); δ 144.1 (CH, C-1), 123.5
(CH, C-2), 168.8 (C, C-3), 72.9 (CH, C-4), 50.7 (C, C-5), 38.4 (CH2,
C-6), 30.4 (CH2, C-7), 53.7 (CH, C-8), 38.7 (C, C-9), 49.5 (CH, C-
10), 37.2 (CH2, C-11), 23.5 (CH2, C-12), 53.3 (C, C-13), 42.4 (CH,
C-14), 37.2 (CH2, C-15), 22.9 (CH2, C-16), 35.8 (C, C-17), 77.2 (C,
C-18), 42.6 (CH2, C-19), 130.9 (C, C-20), 119.2 (CH, C-21), 40.4
(CH2, C-22), 15.4 (CH3, C-23), 65.5 (CH2, C-24), 13.5 (CH3, C-25),
Planta Med 2014; 80: 90–96
96 Original Papers
20.6 (CH3, C-26), 174.9 (C, C-27), 26.5 (CH3, C-28), 21.2 (CH3, C-
29), 51.2 (OCH3, C-27), 22.9 (CH3, CH3COO), 167.7 (C, CH3COO).
13α-Carbomethoxy-4R-acetoxy-18β-hydroxy-30-nor-3,4-seco-
friedela-1,29-dien-3,24-olide, galphimine-L (5): 1H NMR
(400 MHz, CDCl3); δ 6.27(1H, br, H-1), 5.98 (1H, d, J = 12 Hz, H-
2), 4.61 (1H, br, d, J = 2.3 Hz, H-29a), 4.38 (1H, br, d, J = 2.3 Hz, H-
29b).
Supporting information
1
H and 13C NMR for compounds (4), (5), and (17), as well as two
dimensions experiments, HSQC and HMBC, for 4, are available as
Supporting Information.
Acknowledgements
! inflammatory property of n-hexadecanoid acid: structural evidence
This work was supported by grants from CONACyT-México (CON-
ACYT‑CB-2012–01–181180) and the Mexican Institute of Social
Security (IMSS) (FIS/IMSS/PROT/G11/928).
Conflict of Interest
! 18 Akihisa T, Kojima N, Kikuchi T, Yasukawa K, Tokuda H, Masters E, Man-
All authors contributed to and have approved the final manu-
script. The authors declare no conflict of interest.
References
1 Monroy-Ortiz C, Castillo-España P. Plantas medicinales utilizadas en el
Estado de Morelos, 2nd edición. México: Editorial Universidad Autóno-
ma del Estado de Morelos; 2007: 182–183
2 Argueta VA. Atlas de las Plantas Medicinales de México, 1a edición. Mé-
xico. D.F.: Instituto Nacional Indigenista; 1992: 169
3 Tortoriello J, Lozoya X. Effect of Galphimia glauca methanolic extract on
neuropharmacological tests. Planta Med 1992; 58: 234–236
4 Tortoriello J, Ortega A. Sedative effects of Galphimine B, a nor,seco-tri-
terpenoid from Galphimia glauca. Planta Med 1993; 59: 398–400
5 Herrera-Ruiz M, Jiménez-Ferrer JE, De Lima TC, Avilés-Montes D, Pérez-
García D, González-Cortazar M, Tortoriello J. Anxiolytic and antidepres-
sant-like activity of a standardized extract from Galphimia glauca. Phy-
tomedicine 2006; 13: 23–28
6 Toscano RA, Ortega A, Maldonado E, Gaviño R, Lozoya X, Tortoriello J.
Structure of Galphimine B. Acta Crystallogr 1993; C49: 774–776
7 González-Cortazar M, Tortoriello J, Álvarez L. Norsecofriedelanes as
spasmolytics, advances of structure-activity relationships. Planta Med
2005; 71: 711–716
8 Herrera-Ruiz M, González-Cortazar M, Jiménez-Ferrer E, Zamilpa A, Ál-
varez L, Ramírez G, Tortoriello J. Anxiolytic effect of natural galphimines
from Galphimia glauca and their chemical derivatives. J Nat Prod 2006;
69: 59–61
9 Herrera-Arellano A, Jiménez-Ferrer E, Zamilpa A, Morales-Valdéz M, Gar-
cía-Valencia CE, Tortoriello J. A randomized, double-blind clinical trial
controlled with lorazepam. Planta Med 2007; 73: 713–717
10 Herrera-Arellano A, Jiménez-Ferrer JE, Zamilpa, A, García-Alonso G, He-
rrera-Álvarez S, Tortoriello J. Therapeutic effectiveness of Galphimia
glauca vs. Lorazepam in generalized anxiety disorder. A controlled 15-
week clinical trial. Planta Med 2012; 78: 1529–1535
González-Cortazar M et al. Anti-Inflammatory Activity and … Planta Med 2014; 80: 90–96
11 Dorsch W, Bittinger M, Kaas A, Müller A, Kreher B, Wagner H. Antiasth-
matic effects of Galphimia glauca, gallic acid, and related compounds
prevent allergen- and platelet- activating factor-induced bronchial ob-
struction as well as bronchial hyperreactivity in guinea pigs. Int Arch
Allergy Immunol 1992; 97: 1–7
12 Sharma A, Cardoso-Taketa A, Hae Choi Y, Verpoorte R, Villarreal ML. A
comparison on the metabolic profiling of the Mexican anxiolytic and
sedative plant Galphimia glauca four years later. J Ethnopharmacol
2012; 141: 964–974
13 Camacho MR, Phillipson JD, Croft SL, Marley D, Kirby GC, Warhurst DC.
Assessment of the antiprotozoal activity of Galphimia glauca and the
isolation of new nor-secofriedelanes and nor-friedelanes. J Nat Prod
2002; 65: 1457–1461
14 Gábor M. 12-O-tetradecanoylphorbol-13-acetate ear oedema (TPA). In:
Gábor M, editor. Mouse ear inflammation models and their pharmaco-
logical applications. Budapest: Hungarian Academy of Sciences; 2000:
28–33
15 Aparna V, Dileep KV, Mandal PK, Karthe P, Sadasivan C, Haridas M. Anti-
and kinetic assessment. Chem Biol Drug Des 2012; 80: 434–439
16 Kast R, Fürstenberger G, Marks F. Phorbol ester TPA- and bradykinin-in-
Downloaded by: Universidad Autonoma del Estado de Morelos. Copyrighted material.
duced arachidonic acid release from keratinocytes is catalyzed by a cy-
tosolic phospholipase A2 (cPLA2). J Invest Dermatol 1993; 101: 567–
572
17 da Silva KA, Paszcuk AF, Passos GF, Silva ES, Bento AF, Meotti FC, Calixto
JB. Activation of cannabinoid receptors by the pentacyclic triterpene
α,β-amyrin. Pain 2011; 152: 1872–1887
osroi A, Manosroi J. Anti-inflammatory and chemopreventive effects of
triterpene cinnamates and acetates from Shea fat. J Oleo Sci 2010; 50:
273–280
19 Backhouse N, Rosales L, Apablaza C, Goity L, Erazo S, Negrete R, Theodoluz
C, Rodríguez J, Delporte C. Analgesic, anti-inflammatory and antioxi-
dant properties of Buddleja globosa, Buddlejaceae. J Ethnopharmacol
2008; 116: 263–269
20 Neszmély A, Kreher B, Müller A, Dorsh W, Wagner H. Tetragalloylquinic
acid, the major antiasthmatic principle of Galphimia glauca. Planta
Med 1993; 59: 164–167
21 Lijima T, Mohri Y, Hattori Y, Kashima A, Kamo T, Hirota M, Kiyota H, Ma-
kabe H. Synthesis of (−)-epicatechin 3-(3-O-methylgallate) and (+)-cat-
echin 3-(3-O-methylgallate), and their anti-inflammatory activity.
Chem Biodivers 2009; 6: 520–526
22 Mahat MY, Kulkarni NM, Vishwakarma SL, Khan FR, Thippeswamy BS,
Hebballi V, Adhyapk AA, Banade VS, Ashfaque SM, Tubachi S, Patil BM.
Modulation of cyclooxygenase pathway via inhibition of nitric oxide
production contributes to the anti-inflammatory activity of kaempfer-
ol. Eur J Pharmacol 2010; 642: 169–176
23 Kim JM, Lee EK, Kim DH, Yu BP, Chung HY. Kaempferol modulates pro-
inflammatory NF-kappaB activation by suppressing advanced glyca-
tion endproducts-induced NADPH oxidase. Age (Dordr) 2010; 32:
197–220
24 Cardoso-Taketa AT, Lozada-Lechuga J, Fragoso-Serrano M, Villarreal ML,
Pereda-Miranda R. Isolation of nor-secofriedelanes from the sedative
extracts of Galphimia glauca. J Nat Prod 2004; 67: 644–649
25 OʼDonovah A, Slavich GM, Epel ES, Neylan TC. Exaggerated neurobiolog-
ical sensitivity to threat as a mechanism linking anxiety with increased
risk for diseases of aging. Neurosci Biobehav 2013; 37: 96–108
26 Adams RP. Identification of essential oils components by gas chroma-
tography/mass spectrometry, 4th edition. Carol Stream: Allured Busi-
ness Media; 2007: 1–804