molecules Multidisciplinary Investigations on Galphimia glauca: A Mexican Medicinal Plant with Pharmacological Potential

Abstract

Galphimia glauca (Cav.) Kuntze is an important endemic plant species, which possesses many medicinal properties and has been used in the Mexican traditional medicine for its sedative, anxiolytic, anticonvulsant, antiasthmatic and antiallergic properties. The therapeutic properties of this plant are mainly due to the presence of diverse bioactive compounds such as flavonoids, triterpenoids, and phenolics. Several triterpenoids and flavonoids compounds have been isolated and identified. Modern studies have demonstrated many biological activities such as anti-inflammatory, antidiarrheal, gastroenteritis, antimalarial and cytotoxic activities. Nevertheless, many studies are restricted to the crude extract, and many bioactive compounds are yet to be identified and validated according to its traditional use. However, its commercial exploitation and use are highly limited due to the non-availability of enough plant material and lack of knowledge about its agronomical practices. Moreover, the misinterpretation and mislabeling of closely related species of the genus Galphimia Cav. as G. glauca or G. gracilis is a common problem for its rigorous scientific study and commercial exploitation. The present review provides comprehensive knowledge based on the available scientific literature. To the best of our knowledge, this is the first review on G. glauca. This comprehensive information will certainly provide a guide for the better understanding and utilization of G. glauca for its scientific and industrial exploitation.

Full text

molecules
Review
Multidisciplinary Investigations on Galphimia glauca: A
Mexican Medicinal Plant with Pharmacological Potential
Ashutosh Sharma 1, *, Paola Isabel Angulo-Bejarano 1, Alfredo Madariaga-Navarrete 2,
Goldie Oza 3, Hafiz M. N. Iqbal 4, Alexandre Cardoso-Taketa 5and Maria Luisa Villarreal 5
1
Tecnologico de Monterrey, School of Engineering and Sciences, Campus Queretaro, Av. Epigmenio González
No. 500, Fracc. San Pablo, Queretaro CP 76130, Mexico; pangulobe@tec.mx
2Área Académica de Ciencias Agrícolas y Forestales, Instituto de Ciencias Agropecuarias, Universidad
Autónoma del Estado de Hidalgo, Tulancingo CP 42000, Mexico; alfredomadariaga60@gmail.com
3Centro de Investigación y Desarrollo Tecnológico en Electroquímica (CIDETEQ), Parque Tecnológico,
Querétaro S/N, Sanfandila. Pedro Escobedo, Querétaro CP 76703, Mexico; goza@cideteq.mx
4Tecnologico de Monterrey, School of Engineering and Sciences, Campus Monterrey, Ave. Eugenio Garza
Sada 2501, Monterrey CP 64849, Mexico; hafiz.iqbal@itesm.mx
5Centro de Investigación en Biotecnología (CEIB), Universidad Autónoma del Estado de Morelos (UAEM),
Cuernavaca CP 62209, Mexico; ataketa@uaem.mx (A.C.-T.); luisav@uaem.mx (M.L.V.)
*Correspondence: asharma@tec.mx; Tel.: +52-(442)-238-3322
Received: 19 October 2018; Accepted: 14 November 2018; Published: 15 November 2018


Abstract:
Galphimia glauca (Cav.) Kuntze is an important endemic plant species, which possesses
many medicinal properties and has been used in the Mexican traditional medicine for its sedative,
anxiolytic, anticonvulsant, antiasthmatic and antiallergic properties. The therapeutic properties
of this plant are mainly due to the presence of diverse bioactive compounds such as flavonoids,
triterpenoids, and phenolics. Several triterpenoids and flavonoids compounds have been isolated and
identified. Modern studies have demonstrated many biological activities such as anti-inflammatory,
antidiarrheal, gastroenteritis, antimalarial and cytotoxic activities. Nevertheless, many studies are
restricted to the crude extract, and many bioactive compounds are yet to be identified and validated
according to its traditional use. However, its commercial exploitation and use are highly limited due
to the non-availability of enough plant material and lack of knowledge about its agronomical practices.
Moreover, the misinterpretation and mislabeling of closely related species of the genus Galphimia
Cav. as G. glauca or G. gracilis is a common problem for its rigorous scientific study and commercial
exploitation. The present review provides comprehensive knowledge based on the available scientific
literature. To the best of our knowledge, this is the first review on G. glauca. This comprehensive
information will certainly provide a guide for the better understanding and utilization of G. glauca
for its scientific and industrial exploitation.
Keywords: Galphimia glauca; phytochemicals; anxiety; biotechnology
1. Introduction
Galphimia glauca Cav. also known as “calderona amarilla”, “flor estrella”, “hierba del desprecio” “hierba
del cuervo”, “ojo de gallina”, among other names, is an important Mexican plant species for the treatment
of anxiety and depression among other ailments since pre-Hispanic times [
1
,
2
]. Its distribution is
limited to only a few states in Mexico [
3
], and only two main zones generate cultivars that are associated
with the highest concentrations of its main anxiolytic compound: galphimine B [
4
]. Besides its use
for its effects on the central nervous system (CNS) [
5
], it has also been employed for the treatment
of asthma, allergies, diarrhea, gastroenteritis, and malaria, among others [
6
]. These uses are tightly
Molecules 2018,23, 2985; doi:10.3390/molecules23112985 www.mdpi.com/journal/molecules

Molecules 2018,23, 2985 2 of 22
linked with the presence of phytochemical compounds, mainly a triterpenoid family of nine known
compounds named galphimines, isolated for the first time in this plant [
4
,
7
]. Also, they contain high
levels of antioxidant compounds such as flavonoids, namely quercetin [8].
This review intends to provide comprehensive information on Galphimia glauca, which includes
its botanical, phytochemical, biotechnological aspects and their beneficial effects on human health.
In addition, we address the fact that some Galphimia spp. are incorrectly identified with different
names. The provided data will certainly work as a guide for the researchers which will support adequate
scientific and commercial exploitation of this important plant species. This review includes the information
collected from various scientific databases such as PubMed, Scopus, EBSCO, Science Direct, and ProQuest.
Even though Galphimia glauca has been intensively studied over the past 25 years, only 43
articles were found to be strictly related with Galphimia glauca which were included in this review.
Articles other than English language were not considered and several articles or reviews that mention
Galphimia glauca in studies related with homeopathy or molecular taxonomy of Malpighiaceae family
were also excluded. In addition, several research articles book chapters, and reviews related with
medicinal plants, nutraceuticals, ethnobotanical uses, and taxonomical features were also revised.
Finally, one patent related with the use of galphimine B is also documented in the present review.
2. Botanical Description, Taxonomy, Distribution, and Ecosystems
The Malpighiaceae family includes approximately 71 genera, which spans in 1250 species.
The majority of these species are climbers; however, trees and shrubs are also found [
9
,
10
]. Plants
within the Malpighiaceae family are considered economically important. The Barbados cherry
(Malpighia emarginata) is high in vitamin C content [
11
]. Also, hallucinogenic compound production is
found in some species of both the Banisteriopsis and Diplopterys genera [
12
]. On the other hand, some
species of the Banisteriopsis,Malpighia,Peixotoa,Stigmaphyllon, and Galphimia genera are cultivated as
ornamental plants [13].
As is the case with many plants belonging to the Malpighiaceae family, most of the species
comprised in the Galphimia genus are shrubs which can reach up to 4 m, while others form treelets
up to 6 m in height. Also, depending on their morphology they can grow as small shrubs up to 1.5 m
high [
3
] (Figure 1). The Malpighiaceae family present high chromosome number variability, and in the
case of the Galphimieae tribe, it ranges from 12 to 24 chromosomes, where the last value is the most
represented one (62.5%) [14].
Molecules 2018, 23, x 2 of 22
for the treatment of asthma, allergies, diarrhea, gastroenteritis, and malaria, among others [6]. These
uses are tightly linked with the presence of phytochemical compounds, mainly a triterpenoid family
of nine known compounds named galphimines, isolated for the first time in this plant [4,7]. Also,
they contain high levels of antioxidant compounds such as flavonoids, namely quercetin [8].
This review intends to provide comprehensive information on Galphimia glauca, which includes
its botanical, phytochemical, biotechnological aspects and their beneficial effects on human health. In
addition, we address the fact that some Galphimia spp. are incorrectly identified with different names.
The provided data will certainly work as a guide for the researchers which will support adequate
scientific and commercial exploitation of this important plant species. This review includes the
information collected from various scientific databases such as PubMed, Scopus, EBSCO, Science
Direct, and ProQuest.
Even though Galphimia glauca has been intensively studied over the past 25 years, only 43 articles
were found to be strictly related with Galphimia glauca which were included in this review. Articles
other than English language were not considered and several articles or reviews that mention
Galphimia glauca in studies related with homeopathy or molecular taxonomy of Malpighiaceae family
were also excluded. In addition, several research articles book chapters, and reviews related with
medicinal plants, nutraceuticals, ethnobotanical uses, and taxonomical features were also revised.
Finally, one patent related with the use of galphimine B is also documented in the present review.
2. Botanical Description, Taxonomy, Distribution, and Ecosystems
The Malpighiaceae family includes approximately 71 genera, which spans in 1250 species. The
majority of these species are climbers; however, trees and shrubs are also found [9,10]. Plants within
the Malpighiaceae family are considered economically important. The Barbados cherry (Malpighia
emarginata) is high in vitamin C content [11]. Also, hallucinogenic compound production is found in
some species of both the Banisteriopsis and Diplopterys genera [12]. On the other hand, some species
of the Banisteriopsis, Malpighia, Peixotoa, Stigmaphyllon, and Galphimia genera are cultivated as
ornamental plants [13].
As is the case with many plants belonging to the Malpighiaceae family, most of the species
comprised in the Galphimia genus are shrubs which can reach up to 4 m, while others form treelets
up to 6 m in height. Also, depending on their morphology they can grow as small shrubs up to 1.5 m
high [3] (Figure 1). The Malpighiaceae family present high chromosome number variability, and in
the case of the Galphimieae tribe, it ranges from 12 to 24 chromosomes, where the last value is the
most represented one (62.5%) [14].
Figure 1. Galphimia glauca at different stages of development (photo courtesy of Ashutosh Sharma).

Molecules 2018,23, 2985 3 of 22
The natural distribution of Galphimia is mostly in dry habitats. The genus Galphimia comprises 26
species, and 22 of these species are concentrated in Mexico; some are considered endemic [
3
]. In fact,
G.speciosa extends from Mexican territories into Central America, and G.angustifolia distributes into the
United States (Texas). Some Mexican species are found typically in dry environments within pine-oak
forests and shrublands, and just one of them (G.grandiflora) is found in mesic sites in oak, pine and fir
forests. Other species can yet be found in really dry environments such as dunes in the Pacific coast of
the Tehuantepec Isthmus [3] (Figure 2).
Molecules 2018, 23, x 3 of 22
Figure 1. Galphimia glauca at different stages of development (photo courtesy of Ashutosh Sharma).
The natural distribution of Galphimia is mostly in dry habitats. The genus Galphimia comprises
26 species, and 22 of these species are concentrated in Mexico; some are considered endemic [3]. In
fact, G. speciosa extends from Mexican territories into Central America, and G. angustifolia distributes
into the United States (Texas). Some Mexican species are found typically in dry environments within
pine-oak forests and shrublands, and just one of them (G. grandiflora) is found in mesic sites in oak,
pine and fir forests. Other species can yet be found in really dry environments such as dunes in the
Pacific coast of the Tehuantepec Isthmus [3] (Figure 2).
Figure 2. Geographical distribution of the 22 species of the genus Galphimia.
Interestingly, Galphimia glauca, one of the most studied species for its medicinal properties, is not
widely represented in Mexico, in fact, its distribution is limited to the following states:
Aguascalientes, Guanajuato, Hidalgo, Jalisco, Nuevo Leon, Queretaro, Tamaulipas and Zacatecas
(Figure 3). These states comprise part of the central and northeast zones of the country. This
distribution pattern can be attributed mainly to their adaptation to the shrubland and pine-oak forest
present in these zones [3]. Some species like Galphimia amambayensis, Galphimia australis, and Galphimia
platyphylla are found in Paraguay and Galphimia brasiliensis found in Brazil [2].
Figure 2. Geographical distribution of the 22 species of the genus Galphimia.
Interestingly, Galphimia glauca, one of the most studied species for its medicinal properties,
is not widely represented in Mexico, in fact, its distribution is limited to the following states:
Aguascalientes, Guanajuato, Hidalgo, Jalisco, Nuevo Leon, Queretaro, Tamaulipas and Zacatecas
(Figure 3). These states comprise part of the central and northeast zones of the country. This distribution
pattern can be attributed mainly to their adaptation to the shrubland and pine-oak forest present in
these zones [
3
]. Some species like Galphimia amambayensis,Galphimia australis, and Galphimia platyphylla
are found in Paraguay and Galphimia brasiliensis found in Brazil [2].
Molecules 2018, 23, x 3 of 22
Figure 1. Galphimia glauca at different stages of development (photo courtesy of Ashutosh Sharma).
The natural distribution of Galphimia is mostly in dry habitats. The genus Galphimia comprises
26 species, and 22 of these species are concentrated in Mexico; some are considered endemic [3]. In
fact, G. speciosa extends from Mexican territories into Central America, and G. angustifolia distributes
into the United States (Texas). Some Mexican species are found typically in dry environments within
pine-oak forests and shrublands, and just one of them (G. grandiflora) is found in mesic sites in oak,
pine and fir forests. Other species can yet be found in really dry environments such as dunes in the
Pacific coast of the Tehuantepec Isthmus [3] (Figure 2).
Figure 2. Geographical distribution of the 22 species of the genus Galphimia.
Interestingly, Galphimia glauca, one of the most studied species for its medicinal properties, is not
widely represented in Mexico, in fact, its distribution is limited to the following states:
Aguascalientes, Guanajuato, Hidalgo, Jalisco, Nuevo Leon, Queretaro, Tamaulipas and Zacatecas
(Figure 3). These states comprise part of the central and northeast zones of the country. This
distribution pattern can be attributed mainly to their adaptation to the shrubland and pine-oak forest
present in these zones [3]. Some species like Galphimia amambayensis, Galphimia australis, and Galphimia
platyphylla are found in Paraguay and Galphimia brasiliensis found in Brazil [2].
Figure 3.
Galphimia glauca distribution in Mexico according to Anderson [
2
]. Gray color indicates
the corresponding states. 1. Jalisco; 2. Zacatecas; 3. Aguascalientes; 4. Guanajuato; 5. Queretaro;
6. San Luis Potosi; 7. Nuevo Leon; 8. Tamaulipas.

Molecules 2018,23, 2985 4 of 22
Due to the overall aesthetic characteristics of Galphimia glauca plants (catchy flower colors)
collectors have taken samples and helped in its dispersion outside of its endemic site [
3
], which makes
this plant exotic in other parts of the world. According to certain studies, the presence of
Galphimia glauca in places such as Cuba and the Virgin Islands has also been reported [
15
]. In fact,
Galphimia glauca is also considered as an invasive plant in Cuba [16].
3. History and Uses
The use of plants as part of traditional medicine is tightly linked to human civilization
development. Different historical documents highlight the use of plants in many cultures [
17
].
In Mexico, the use of traditional medicine is widely documented. The earliest information is found
in “Libellus de Medicinalibus Indorum Herbis” or the Codex de la Cruz-Badiano wrote in 1552 which
concentrates the information in Aztec medicine available at the time and translated to Latin [
18
].
This codex includes thorough information as to which plants, their parts and how they were prepared
to be used as medicine. Since Mexico is a megadiverse country, the list of plants with potential medical
use is large. Many of them have been extensively used since pre-Hispanic times up to our days. Such is
the case of plants belonging to the Malpighiaceae family. The use of Galphimia glauca in Mexican
traditional medicine can be tracked up to the 1500s as described by Hernández [
19
]. The Aztecs
designated this plant as “Totoncapatli” a name formed by “totonqui”: hot, and “patli”: medicine [
1
].
Nowadays, this plant is also known as “calderona amarilla”, “flor estrella”, among others [20].
The principal uses of this plant are related mainly to its tranquilizing and sedative properties.
Historical records include its utilization during certain civil war movements in Mexico, were an
infusion was made out of leaves and stems and delivered to soldiers suffering from anxiety episodes [
1
].
Mexican traditional medicine relates the use of Galphimia glauca, to the treatment of “nervios” a folk term
for an illness whose symptoms are closely related with those of depression and anxiety disorders [
20
].
This effect is due to various phytochemical compounds that have been identified in this plant species.
In addition, several studies have described the use of Galphimia glauca in different formulations
for homeopathic remedies [
21
]. Thus, in 1985 Wiesenauer and Gaus, [
22
] described the use of a
homeopathic product called Galphimia D6, (a six times potentiation in 90% ethanol), and evaluated its
effects in patients suffering from pollinosis. As a result, the use of this D6 formulation was proven to be
better than the use of regular Galphimia glauca dilutions. Further on, preparations of Galphimia D4 on
saccharose globules were studied in patients suffering from pollinosis, the overall effect was a reduction
in symptoms versus the placebo population analyzed [
23
]. Mixtures involving Galphimia glauca have
also been described. In 2004, a study evaluated the use of a nasal spray containing Luffa operculata,
Galphimia glauca, histamine and sulphur against hay fever symptoms [
24
]. In brief, the use of this nasal
spray was as efficient as the use of commercial cromolyn solution. Homeophatic studies have also
been conducted in Galphimia glauca-based formulations. Accordingly, the patients experienced several
symptoms when using the product such as relaxing, sedative, anxiolytic and antiallergenic effects [
25
].
4. Phytochemistry
Galphimia glauca is a rich source of different types of natural products, with a variety of structural
patterns. The presence of several of these compounds has been reported in the different plant organs
of Mexican “calderona amarilla”.
4.1. Phenolic Compounds
The phenolic compounds along with alkaloids and terpenoids constitute one of the most
studied groups in phytochemistry [
26
]. The term “phenolics” comprises a large group of chemical
compounds derived from the phenylpropanoid pathway. Their biological activities are highly related
with their structure, participating in metabolic and cell signaling pathways and present antioxidant,
antiproliferative, pro-apoptotic, anti-angiogenic and anti-inflammatory properties. Along with these
properties, they can affect the enzyme and protein function [27].

Molecules 2018,23, 2985 5 of 22
Gallic acid (3,4,5-trihydroxybenzoic acid), one of the simplest phenolic compounds present
in plants, is found in grapes, berries, and tea, and it presents antioxidant, anti-inflammatory and
anticancer properties [
27
,
28
]. The presence of gallic acid, as well as some other complex forms such
as tetragalloylquinic acid (1,3,4,5-tetra-O-galloylquinic acid), methyl gallate, and ellagic acid, have
been reported in Galphimia glauca methanolic and ethyl acetate extracts [
8
,
29
]. In fact, Dorsch et al. [
8
],
analyzed the chemical composition of leaves and stems of Galphimia glauca by quantitative assays and
determined that 1 g of dried methanolic extract contains 6 mg of gallic acid, 12 mg methyl gallate
and 125 mg of tetragalloylquinic acid. Tetragalloylquinic acid is considered a very strong antioxidant
which is highly related to some of the biological activities attributed to Galphimia glauca [30].
Flavonoids are very important phytochemicals, recognized mainly for their contribution to
the flavor and color of fruits and vegetables. They are distributed in different plant parts such as
fruits, leaves, flowers, stems, roots, and seeds [
31
] and are normally ingested directly from fruits,
meals, tea and wine [
32
]. However, it is their widely studied antioxidant properties and their positive
health effects such as anti-inflammatory, anti-carcinogenic and neuroprotective that accounts for
their importance. Among them, the flavonol group is the most abundant in Nature, specifically
quercetin which is the most characteristic of this group [
33
]. This flavonoid acts as a potent scavenger
of reactive oxygen and nitrogen species (ROS and RNS), superoxide and hydroxyl radicals, nitric oxide,
and peroxynitrite. It can also quench free radicals and lipid peroxides [
31
]. Quercetin is considered
a dietary flavonoid due to its wide distribution among plant foods [
34
]. Accordingly, some reports
indicate the presence of quercetin in the methanolic and ethyl acetate extracts of Galphimia glauca
leaves; however, the exact concentration was not described [
8
,
29
,
35
]. Additionally, a synergic role for
quercetin along with various polyphenols against asthma and bronchial reactions to allergens have
been proposed [8].
4.2. Terpenoids
Secondary metabolites comprise a large group of diverse molecules, which include the terpenoids.
These chemicals are derived from the isoprene (C
5
) units that are arranged in a “head to tail” or “tail
to head” fashion. They can be classified in: hemiterpenes (C
5
), monoterpenes (C
10
), sesquiterpenes
(C
15
), diterpenes (C
20
), sesterpenes (C
25
), triterpenes (C
30
), tetraterpenes (C
40
) and polyterpenes (more
than C
40
) [
36
]. Terpenoids exert different biological activities which include anticancer, analgesic,
antiinflammatory, antimicrobial, antifungal, antiviral and antiparasitic [37].
Galphimia glauca contains various compounds belonging to the terpenoid division of polyphenols,
that mainly include “nor-seco-triterpenes,” which in this case are named the galphimine series (
1
–
9
) [
4
]
(Figure 4). The first report of the presence of such compounds dates back to 1998 [
7
] when the
galphimine B was discovered. Moreover, the isolation of galphimines A, B, D and the exocyclic forms
for galphimines F to I has been reported [
38
], along with galphimine J [
39
]. Additionally, one of the
galphimines detected, galphimine C was found to be a double bond isomer of galphimines B and F,
being the only galphimine with a double bond at C-19 and C-20. Galphimine B is the major sedative
compound in Galphimia glauca [
38
]. Other nor-seco-triterpenoids reported in Galphimia glauca include
galphins A, B, and C along with galphimidin [
40
]. However, the most studied galphimines include
galphimine B [7,41] galphimine A and galphimine E [42,43].
Interestingly, the content of galphimines in Galphimia glauca leaves can vary depending on their
natural distribution as was demonstrated in a study where two main locations were tested: Dr. Mora,
Guanajuato, and Jalpan de Serra, Queretaro. Thus, the major galphimine concentration was found
in Dr. Mora derived plants, with a mean value for total galphimines of 6.58 mg/g DW leaves [
4
,
44
].
Furthermore, new triterpenoids glaucacetalins A–C (
1
–
3
) were reported in transformed roots [
45
] and
gauccacetalin D in transformed cell suspension cultures of Galphimia glauca [46].

Molecules 2018,23, 2985 6 of 22
Molecules 2018, 23, x 6 of 22
Figure 4. List of galphimines A–I identified in Galphimia glauca.
5. Pharmacological Activities
5.1. Anti-Asthmatic and Anti-Allergenic Activities
Studies on active components of extracts from Galphimia glauca and their mechanism of action
were first published in 1992. Accordingly, the potential use of Galphimia glauca methanolic extract in
the treatment of acute bronchial reactions to allergens, and its anti-asthmatic potential in guinea pigs,
was demonstrated. The main compounds responsible for this effect were found to be gallic acid,
methyl gallate, and quercetin which showed a significant effect at a 45 mg/kg oral dose, while
tetragalloylquinic acid displayed significant effects at a 5 mg/kg oral dose. Also, when a mixture of
compounds was used, a reduction in allergen and bronchial reactions was observed [8] (Table 1).
Furthermore, a different research group reached the same observations when comparing various
phenolic compounds derived from Galphimia glauca leaves. In this study, the methanolic and ethyl
acetate extracts revealed the presence of methyl gallate and tetragalloylquinic acid, respectively.
These extracts were used to analyze its effect versus bronchial hyperreactivity and allergic reactions
also. The compound that exerted the major activity against these illness markers was
tetragalloylquinic acid [29]. Moreover, the effect of Galphimia glauca extracts on patients suffering
“pollinosis” or “hay fever” has been evaluated with promising results; however, the exact mechanism
and the chemical compounds associated with this effect were not reported [23] (Table 1). Also, the
effectiveness of the ethyl acetate extract of Galphimia glauca in treating asthma-related symptoms has
been analyzed. In this sense, male guinea pigs were treated with various concentrations of the extract
and subjected to leukotriene (LTD4) induced bronchoconstriction. As a result, the Galphimia glauca
extract was capable of reducing these symptoms in a similar way than the one reported for other
chemicals that block leukotriene (LTD4) activity [47]. The effect of flavonoids from the aerial parts of
Galphimia glauca in the reduction of complement induced hemolysis has been described. In this sense,
isoquercetin hyperoside, gallic acid, and ellagic acid were the main compounds related to the
hemolysis inhibition activity (Table 1) [35].
Figure 4. List of galphimines A–I identified in Galphimia glauca.
5. Pharmacological Activities
5.1. Anti-Asthmatic and Anti-Allergenic Activities
Studies on active components of extracts from Galphimia glauca and their mechanism of action
were first published in 1992. Accordingly, the potential use of Galphimia glauca methanolic extract
in the treatment of acute bronchial reactions to allergens, and its anti-asthmatic potential in guinea
pigs, was demonstrated. The main compounds responsible for this effect were found to be gallic
acid, methyl gallate, and quercetin which showed a significant effect at a 45 mg/kg oral dose, while
tetragalloylquinic acid displayed significant effects at a 5 mg/kg oral dose. Also, when a mixture of
compounds was used, a reduction in allergen and bronchial reactions was observed [8] (Table 1).
Furthermore, a different research group reached the same observations when comparing various
phenolic compounds derived from Galphimia glauca leaves. In this study, the methanolic and ethyl
acetate extracts revealed the presence of methyl gallate and tetragalloylquinic acid, respectively.
These extracts were used to analyze its effect versus bronchial hyperreactivity and allergic reactions
also. The compound that exerted the major activity against these illness markers was tetragalloylquinic
acid [
29
]. Moreover, the effect of Galphimia glauca extracts on patients suffering “pollinosis” or “hay
fever” has been evaluated with promising results; however, the exact mechanism and the chemical
compounds associated with this effect were not reported [
23
] (Table 1). Also, the effectiveness of
the ethyl acetate extract of Galphimia glauca in treating asthma-related symptoms has been analyzed.
In this sense, male guinea pigs were treated with various concentrations of the extract and subjected to
leukotriene (LTD
4
) induced bronchoconstriction. As a result, the Galphimia glauca extract was capable
of reducing these symptoms in a similar way than the one reported for other chemicals that block
leukotriene (LTD
4
) activity [
47
]. The effect of flavonoids from the aerial parts of Galphimia glauca in the
reduction of complement induced hemolysis has been described. In this sense, isoquercetin hyperoside,
gallic acid, and ellagic acid were the main compounds related to the hemolysis inhibition activity
(Table 1) [35].

Molecules 2018,23, 2985 7 of 22
Table 1. Phytochemical compounds with beneficial health effects found in Galphimia glauca.
Type of
Compound Compound Plant Part Used Extract Dose Model Effect Reference
Phenolic
Gallic acid
Methyl gallate
Tetragalloyl quinic acid
Leaves and stems Methanolic 2 mg/kg body
weight Guinea pigs Inhibition of acute
bronchial reactions
[8]
Tetragalloyl quinic acid
Gallic acid
Methylgallate
Ellagic acid
Aerial parts Ethanolic 5 mg/kg body
weight Guinea pigs
Bronchial
hyperreactivity
and allergic
reactions
[29]
Tetragalloyl quinic acid Aerial parts Ethyl acetate 100 µg/mL Guinea pigs Asthma-related
symptoms [47]
Flavonoids
Quercetin Leaves and stems Methanolic 2 mg/kg body
weight Guinea pigs Inhibition of acute
bronchial reactions
[8]
Quercetin Aerial parts Ethyl acetate 5 mg/kg body
weight Guinea pigs
Bronchial
hyperreactivity
and allergic
reactions
[29]
Quercetin 3-O-(2
00
-galloyl)-
β
-D-glucoside
Quercetin-3-O-(6
00
-galloyl-)-
β
-D-glucoside
Aerial parts Ethyl acetate 100 µg/mL Guinea pigs
Reduction of
complement
induced hemolysis
[35]

Molecules 2018,23, 2985 8 of 22
5.2. Anti-Depressive and Anxiolytic Effects
Depression and anxiety disorders are present in 4.4% and 3.6% of the world population,
respectively. These mental conditions are more prevalent in females than in males [
48
]. Medical
treatments for these disorders rely on a plethora of pharmaceuticals which ameliorate the conditions
but have a long recorded history of negative side effects [
49
]. Nowadays, the search for therapeutic
phytochemicals is seen as a promising alternative [49,50].
5.2.1. Anxiolytic and Other Effects on the CNS Evaluated in Animal Models
Galphimia glauca is the most studied species for the treatment of anxiety disorders in Mexico [
20
,
44
].
Accordingly, the methanolic extract derived from the aerial parts of the plant was used to evaluate
the anxiolytic effect in mice through a neuro-pharmacological test [
51
] (Table 2). In a further study,
this effect was found to be caused by the presence of galphimine B [
52
]. Additionally, further analysis
revealed that this nor-secotriterpene compound could be exerting this anxiolytic effect due to an
inhibition in dopaminergic activity [
7
,
41
] along with possible interactions with the serotonergic
system [53] (Table 2).
Furthermore, the effect of galphimines A and E on anxiety was also evaluated; however, their
action is less potent than that of galphimine B. Nevertheless, a study was conducted to analyze the
effect of chronic administration of three main types of Galphimia glauca extracts in mice: aqueous,
ethanolic, and methanolic. All extracts were standardized in the content of the tree main galphimines.
In brief, after 56 days of administration, no changes in liver function biochemical parameters were
found, and none of the three extracts was found to be genotoxic. In fact, no deaths were caused by the
use of these extracts, nor any histopathological symptoms were registered [54] (Table 2).
Since galphimines have proven activity on the CNS, a study conducted in 2009 described its
effects on treating insomnia in mice. When comparing different Mexican plants used in traditional
medicine for insomnia treatment, the most active extract was the methanolic extract of Galphimia glauca,
where the effective dose was registered as 22.06 mg/kg. Also, this ED
50
was slightly lower than the
one registered for the Cympobogon citratus hexane extract, which is known as “téde limón” and is used
in Mexican traditional medicine for insomnia treatment [55] (Table 2).
The capacity of galphimine A, one of the most abundant triterpenoids with anxiolytic activity
found in Galphimia glauca, to act on the CNS has also been described [
56
]. In this study conducted
in ICR Male mice, 200 mg/kg of galphimine A were used to analyze the pharmacokinetics of this
triterpenoid. Since most studies over the years have been focused on the action and anxiolytic effects
of galphimine B, the novelty of this study was to understand if galphimine A, which also has anxiolytic
properties is capable of reaching the CNS and address its pharmacological target [
56
]. Chemical
conversion of galphimine E to A was also conducted to give extra use to galphimine E since this
compound is more abundant, but it is also an inactive triterpene. By making this chemical change,
they could harness this anxiolytic compound.
Recently, Garige et al. [
57
] reported the depressant effects of a stem methanol extract from
Galphimia glauca during an
in vivo
study. Accordingly, Swiss albino mice were subjected to a different
set of behavior analysis, all of them with the aim to elucidate the effects of the methanolic extract
on CNS. In general, the authors propose that the Galphimia glauca extract exerts depressant effects in
a similar way as with diazepam which is a drug normally used in patients suffering from anxiety,
panic disorder, GAD, phobia disorder, obsessive-compulsive disorder (OCD) among others [
58
].
The mechanism for this drug is based in its capacity to bind to molecular subunits (
α
and
γ
) of
GABA
A
(gamma amino butyric acid type A) receptors located in the neuronal membranes of the
central nervous system, which allows the opening of chloride ion channels. During all the analysis,
the Galphimia-treated animals developed behaviors which were very similar to the ones exerted when
diazepam is used.

Molecules 2018,23, 2985 9 of 22
Table 2. Pharmacological effects of galphimines and related compounds from Galphimia glauca tested in vivo in animal models.
Compound Extract Plant Part
Used Model Analysis Disorder Dosage Controls Result Ref.
Galphimine B Crystallized
galphimine B Aerial parts 79 male
Wistar rats.
Effects on cerebral
activity
Central
nervous
system
diseases
0.5, 1.0 and 2.5 mg/kg 10%
polyethyleneglycol
Administration (systemic and
localized) of Galphimine B,
demonstrated excitatory effects in
neurons localized mainly in the
Ventral Tegmental Neurons that is
a target for antipsychotic drugs
[7]
Galphimine B Purified
Galphimine B Aerial parts Wistar rats Effects on VTA neurons
through patch clamps 1µM–5 mM Not reported
Action upon dopaminergic VTA
neurons in a
nonGABAergic mechanism
[41]
Galphimines
A, B, and E
Fractionation
of the
methanolic
extract
Aerial parts Male
ICR mice Elevated Plus Maze Anxiety
15 mg/kg of purified
galphimines or Galphimine
Rich Fraction (GRF)
5% Tween 20
Anxiolytic effect induced not
significant differences
with diazepam
[42]
Galphimines Methanolic Aerial parts ICR
albino mice
Open arms in elevated
plus maze, light dark
paradigm test, forced
swimming test
Anxiety and
depression
125, 250, 500 and 2000 mg/kg
Not reported Anxiolytic like effect [43]
Galphimines Ethylacetate Aerial parts Male
guinea pigs
Leukotriene D4 (LTD4)
induced
bronchoconstriction
Asthma 10, 31.6, 56.2 and 100 µg/mL Not reported Similar than SK&F 104353 an
LTD4 antagonist. [47]
Galphimines Methanolic Aerial parts 10 Wistar Rats Strictine induced
convulsions Convulsions 1, 10, 50, and 100 mg/b g of
methanolic extract 10% Tween 80
Decrease in seizures and reduction
in mortality at the
50 mg/100 g dose
[51]
Galphimines Methanolic Aerial parts 10
Albino mice
Protection against
leptazol-induced
convulsions
Convulsions 50 mg/I 00 g i.p. 10% Tween 80
Decrease in seizures and reduction
in mortality at the
50 mg/100 g dose
[51]
Galphimines Methanolic Aerial parts 10 Male
albino mice Barbiturate potentation Convulsions 1, 10, 50 mg/b g 10% Tween 80
Increase of sleeping time induced
by sodium pentobarbital in a
dose-dependent manner, higher
effect at 50 mg/100 g
[51]
Galphimine B Crystallized
galphimine B Aerial parts Male
albino mice
Strychnine-induced
convulsions Convulsions 10, 40, 80 mg/kg 10% Tween 80 Not a significant effect [52]
Galphimine B Crystallized
galphimine B Aerial parts Male
albino mice
Leptazol-induced
convulsions Convulsions 10, 40, 80 mg/kg 10% Tween 80 Not a significant effect [52]

Molecules 2018,23, 2985 10 of 22
Table 2. Cont.
Compound Extract Plant Part
Used Model Analysis Disorder Dosage Controls Result Ref.
Galphimine B Crystallized
galphimine B Aerial parts Male
albino mice
Potentiation of general
anesthetics Convulsions 10, 40, 80 mg/kg 10% Tween 80
Significant increase in narcosis
time induced by sodium
pentobarbital.
Highest effects found at 80 mg/kg
[52]
Galphimines Methanolic
Hexane Aerial parts Male
ICR mice Exploratory cylinder test Insomnia 22.06 mg/kg
0.05% Tween 80 in
saline solution Sedative effects in mice. [55]
Galphimine A Ethyl acetate Aerial parts Male
ICR mice Pharmacokinetic study Anxiety 200 mg/kg Not reported Anxiolytic effect of galphimine A
in the CNS [56]
Galphimines
A, B and E
Methanolic
fraction,
Galphimine
Rich Fraction
Galphimine A,
B and E
Male
ICR Mice
Open Field Test
Passive Avoidance Test
Forced Swimming Test
Behavioural
changes
Metanol extract: 25, 100,
250 and 500 mg/kg p.o
GRF: 5, 15, and 30 mg/kg p.o,
GA, GB and GE (5, 10 and
30 mg/kg p.o
1% Tween 20
The effect caused partially by their
interaction with dopaminergic and
glutaminergic systems in vivo.
Protection against hallucinations
and psychosis.
[59]
Galphimines
N-hexane,
ethyl acetate,
dichloromethane
and methanol
Leaves Male
ICR mice
Acute inflammation
with TPA Inflammation 3.2 mg/ear Indomethacin
Antiinflammatory principles were
attributed to Galphimine A and
Galphimine E
[60]

Molecules 2018,23, 2985 11 of 22
Furthermore, Galphimia glauca methanolic extract can induce a delay in the onset of seizures
induced by picrotoxin and pentylenetetrazole (PTZ) as well as to eliminate the deaths of animals due
to convulsions. Also, a reduction of anxiety observed in the behavior and locomotor activity tests.
Finally, muscle relaxing effects were detected in the plant extract treated mice. Therefore, the results of
this study reinforce the role of Galphimia glauca in the treatment of anxiety-related disorders and the
generation of calming effects on the nerves [57] (Table 2).
In recent findings, the use of either the methanolic extract of Galphimia glauca, a galphimine rich
fraction (GRF) or purified galphimines (G-A, G-B, and G-E) in the behavior of mice was analyzed.
In this study, different extracts were evaluated for their effects on acute symptoms associated with
the onset of schizophrenia, induced by ketamine in mice using the Haloperidol-induced catalepsy.
Furthermore, the methanolic extract and the GRF showed positive results in treating the symptoms
associated with psychosis [59].
5.2.2. Human Clinical Trials for Anxiolytic Effects
Medicinal herbal products have been designed using Galphimia glauca extracts. In 2007, the use of
the capsules containing 310 mg of dried aqueous extract of Galphimia glauca (0.348 mg of galphimine B)
in comparison to lorazepam in patients with a generalized anxiety disorder (GAD) was evaluated [
49
].
Patients were evaluated with the use of the Hamilton Anxiety Rating Scale (HAM-A) also other
symptoms such as tolerability, the absence of excessive sedation and safety (absence of pathological
alterations in renal and hepatic functions) were evaluated (Table 3). In conclusion, the effectiveness
of this plant versus anxiety was demonstrated, and its behavior was similar to the one observed
when lorazepam is used. The major difference between the use of lorazepam and Galphimia glauca
(galphimine B) relied on tolerability since lorazepam induced excessive sedation than the plant extract.
Furthermore, the anxiolytic effect caused by Galphimia glauca was noticed during the first week of
administration, which is not the case for other drugs (azapirones and selective serotonin reuptake
inhibitors, SSRIs) that require at least 3 to 4 weeks to start with this effect, therefore this is an advantage
for patients that require a rapid sensation of wellbeing with a concomitant reduction of anxiety
levels [49].
In a further study, the effect of a standardized dose of 0.175 mg of galphimine B in a
formulation during a 15 weeks clinical trial was evaluated in patients with GAD (double-blind
study) [
61
]. The results showed the greater effectiveness of Galphimia glauca over lorazepam (Table 3).
The maximum anxiolytic effect was observed after the first week of administration for both groups
(Galphimia glauca and lorazepam). However, the GB extract maintained this effect even after four
weeks of administration. Furthermore, in the last weeks of the study, the plant extract exceeded the
therapeutic effectiveness versus the benzodiazepine, with an overall anxiolytic effectiveness >90%.
In addition, the use of GB extract was found to be better than other types of anxiolytics, namely:
tricyclic antidepressants and buspirone regarding the anxiolytic effect and these pharmaceuticals
started showing anxiolytic activity after the third week of administration. Finally, the use of the
phytopharmaceutical presented fewer side effects than the use of lorazepam (i.e., disabling somnolence)
and did not generate tolerance, intoxication, dependence or suppression syndrome during its use [
61
].
Recently, the effect of galphimine B in the treatment of social anxiety in young individuals was
also evaluated (Table 3) [
62
]. In this study, purified galphimine B was administered to a group of
patients from both sexes with ages ranging from 18 to 35 years old for 10 weeks, while sertraline was
administered to the control group. In general, the effect of galphimine B was not significantly different
from using sertraline and patients experienced a significant reduction in anxiety according to the
value obtained from the Brief Social Phobia Scale (BSPS) demonstrating efficacy and safety in patients
suffering from social anxiety disorder [62].

Molecules 2018,23, 2985 12 of 22
Table 3. Human clinical trials to treat anxiety related disorders reported for Galphimia glauca.
Clinical Trial
Extract
(Alone or in
Combination)
Plant Parts
Used Dosage Duration of
the Study Controls
Sample
(Number of
Patients)
Scale Result
Tolerability,
Security or
LD50
Reference
Double blind study,
randomized,
lorazepam
controlled
Aqueous Leaves
and stems
310 mg of dried
aqueous
extract capsules
4 weeks Lorazepam 152 HAM
Same anxiolytic
effect than
Lorazepam. Side
effects reduction
Well
tolerated [49]
Double blind study,
randomized,
lorazepam
controlled
Purified
galphimine B
Leaves
and stems
0.175 mg of
galphimine B
12 weeks
12 weeks Lorazepam 191 HAM
Anxiolytic effect
superior to
Lorazepam
Well
tolerated [61]
Double blind
randomized study
Purified
galphimine B Aerial parts 0.374 mg/dose
galphimine B 10 weeks Sertraline 34 BSPS
No significant
difference with the
use of sertraline
Well
tolerated [62]
HAM-A: Hamilton Anxiety Scale; BSPS: Brief Social Phobia Scale.

Molecules 2018,23, 2985 13 of 22
5.3. Antimicrobial Activity
Antimicrobial compounds in plants are normally produced for protection and defense purposes,
some of them occur in the pre-infectious while others are produced as a post-infection defense
system. Some of these compounds are also a product of the plant’s response to biotic stress conditions.
Currently, food industry applications for natural sources of antimicrobials such as the ones found in
plants is increasing [
63
]. In this sense, studies regarding the antimicrobial activity of plants belonging
to the Galphimia genus are scarce.
The first report regarding the antimicrobial potential of this plant was described in 2002,
where the antiprotozoal activity of Galphimia glauca was evaluated [
40
]. Thus, the n-butanol and
chloroform fractions of Galphimia glauca were found to exert moderate antiprotozoal activity against
Plasmodium falciparum K1, Trypanosoma brucei brucei,Leishmania donovani. These fractions contained
galphins A, B and C, galphimidin, quercetin, stigmasterol, and sitosterol 3-O-
β
-D-glucoside which
could correlate with the activities displayed against protozoans. Nevertheless, the authors explained
that quercetin exhibited a weak activity versus plasmodium,trypanosoma, and leishmania when compared
to the standard drugs. Finally, stigmasterol and sitosterol 3-O-
β
-D-glucoside were not effective versus
any of the microorganisms analyzed.
In 2003, García et al. [
64
] analyzed the antifungal activities of nine Mexican medicinal plants,
including Galphimia glauca. Plant extracts were obtained through sequential extraction with n-hexane
and methanol. The action of these extracts was analyzed against diverse microorganisms such as
Aspergillus niger,Trichophyton mentagrophytes,Trichophyton rubrum, and Candida albicans. Even though
the minimum inhibitory concentration (MIC) of Galphimia glauca was not amongst the most effective
plants analyzed in this study, it did show inhibition versus all fungal microorganisms analyzed,
and the MIC values were above 8 mg/mL except for Trichophyton rubrum (4.0 mg/mL). Therefore,
these activities can rely on the plethora of phytochemical compounds found in these plant species,
namely flavonoids and terpenes, among others [64].
Other studies focused on the Galphimia genus include the analysis of antioxidant, brine shrimp
lethality and antimicrobial activity in Galphimia gracilis Bartl. in 2015 [
65
]. The antimicrobial activity
was evaluated against Bacillus subtilis,Staphylococcus aureus,Escherichia coli,Salmonella typhi,Salmonella
abony, and Pseudomonas aeruginosa. Furthermore, three different extracts were utilized: methanol,
ethyl acetate, and petroleum ether. The results of the antimicrobial activity analysis revealed that the
methanol extract was more effective against E. coli,S. typhi, and P. aeruginosa, while the ethyl acetate
extract was more effective against S.typhi and P.aeruginosa. Ethyl acetate extract exhibited the highest
antimicrobial activity. However, the authors consider that all the extracts showed poor activity when
compared to the control (azithromycin).
5.4. Antiinflammatory Activity
As stated previously, Galphimia glauca has been utilized since pre-Hispanic times to treat diverse
illness which includes gastric ulcers, traumatic blows, wounds, scars, kidney, and uterus inflammation
as well as rheumatism. All these medical conditions have the inflammatory process in common. In
addition, this plant contains a high amount of polyphenol compounds that can address these conditions
and serve for their treatment.
As part of a major study, an antiinflammatory test was done in mice using the methanolic
extract of different cultivars of Galphimia glauca. As a result, this plant extract was capable of
reducing the inflammation in mouse ears which was previously induced by tetradecanoyl phorbol
acetate [
44
]. Furthermore, González-Cortázar et al. [
60
] analyzed the antiinflammatory properties of
Galphimia glauca crude extracts. To do this, n-hexane, ethyl acetate, dichloromethane, and methanol
extracts were analyzed. This study revealed that the galphimine A and galphimine E were the main
anti-inflammatory principles present in the methanolic extract [60] (Table 2).

Molecules 2018,23, 2985 14 of 22
5.5. Antiproliferative Activity
The use of plants as main sources of chemical compounds with antiinflammatory, antiproliferative
or cytotoxic potential has long been reported. This constitutes the basic studies when linking a
particular set of compounds or fractions and its effects on
in vitro
analysis with their potential health
benefits. [
66
]. In some cases, these results are applied by the phytopharmaceutical or biopharmaceutical
industries in their formulations.
In the particular case of Galphimia glauca, a report indicates the evaluation of different extracts
versus several cancer cell lines. In 2007, Aguilar-Santamaria et al. [
54
] analyzed the toxicological
effects of Galphimia glauca aqueous, methanolic and ethanolic extracts regarding its effects on different
parameters. For the hepatoxicity analysis, the enzymatic profiles of alkaline phosphatase (ALP), alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) were evaluated. The results showed no
hepatotoxic effects of these extracts in mice. Also, they developed cytotoxicity studies in different cell
lines such as HCT-15 (colon), UISO (uterus), KB (nasopharyngeal) and OVCAR-5 (ovarian cancer); as a
result, all the extracts analyzed exhibited an ED
50
higher than 20
µ
g/mL on nasopharyngeal, UISO
and OVCAR-5 cell lines. However, a cytotoxic effect in the colon cell line was found, where the ED
50
for all extracts was lower than 2
µ
g/mL. Finally, the genotoxicity analysis revealed that the evaluated
plant extracts (50, 100, and 250
µ
g/mL) had no genotoxic effects. Therefore, the authors suggest that
the use of Galphimia glauca extracts possess very low risks of toxicity and genotoxicity that is in line
with the therapeutic safety guidelines for phytopharmaceuticals [54].
5.6. Vasoactive Effect and Spasmolytic Activity
The infusions of flowers of Galphimia glauca to treat heart pain has also been reported in the
traditional medicine system of Mexico [
67
]. In this sense, a study regarding the potential use of
Galphimia glauca as source of compounds with vasoactive effect has been reported [
68
]. An aqueous
extract was prepared from leaves and flowers of Galphimia glauca which was evaluated in thoracic
aorta from adult male Wistar rats. This analysis revealed that leaves extract of Galphimia glauca was
more active than the flowers extract in a concentration dependent manner.
Other traditional uses of Galphimia glauca indicate its application for the treatment of stomach
related problems [
6
], in this sense the methanolic extract of Galphimia glauca, which is known for
its sedative properties was used to evaluate its spasmolytic effect in the ileum of guinea pigs [
39
].
Accordingly, several fractions were tested to evaluate their capacity to reduced contractions that
were induced artificially. As a result, the use of the methanolic extract reduced the contractions by
80.42%. Among all the fractions evaluated, galphimine F showed four times higher inhibition than
galphimine B.
6. Toxicity Tests
The use of plants in the formulation of phytopharmaceuticals is increasing day by day. Due to the
importance of this plant species in the treatment of mental disorders, such as anxiety the analysis of
toxicological effects is relevant.
To the best of our knowledge, the Lethal Dosage (LD
50
) for Galphimia glauca has not been
determined yet. A closely related species Galphimia gracilis was evaluated with brine shrimp
(Artemia salina Leach) through bioassay analysis [
65
]. In this study, three different plant extracts
were used: methanol, ethyl acetate, and petroleum ether. Among them, methanol extract was found to
be the most toxic to brine shrimp nauplii with a LC
50
of 64.46
µ
g/mL, while the ethyl acetate extract
exhibited a LD
50
of 131.88
µ
g/mL, and the petroleum ether extracts a LD
50
of 225.42
µ
g/mL. According
to the authors, LD
50
values below 500
µ
g/mL are considered toxic [
69
]. Therefore, the results observed
for the different Galphimia gracilis extracts are promising versus further studies involving cancer
cell lines. Furthermore, the observed LD
50
values can easily correlate with the different types of
polyphenolic compounds found within the Galphimia genus [65].

Molecules 2018,23, 2985 15 of 22
7. Biotechnological Studies
The use of phytochemical compounds from a natural origin is tightly linked to human civilization
and development. Many of these compounds present in plants drive the increasing demand for
pharmaceuticals, without them more than half of the drugs from which we benefit today might not
have existed [70].
The use of biotechnology to improve yields of medicinal plants such as in the case of plant tissue
culture is a promising tool to avoid germoplasm extinction. Therefore, the first studies related to a
tissue culture of Galphimia glauca reported in 1999 [
71
]. The authors described a methodology for
callus formation, where the best callus induction medium consisted of Murashige and Skoog (MS)
supplemented with 2 m/L of 2,4-dichlorophenoxyacetic acid (2,4-D). The cellular growth of these calli
was observed when 2 mg/L naphthaleneacetic acid (NAA) plus 1 mg/L kinetin (KN) were employed.
The subculture of these callus structures in 2,4-D (4 mg/L) medium resulted in increased production
of galphimine B and 6-acetoxygalphimine B [71].
In a further study, this same research group described the
in vitro
production of galphimine B in
cell suspension cultures [
72
]. Thus, a cell suspension batch culture was employed, and the effect of the
main parameters (inoculum size, plant growth regulators and various sucrose, nitrate, and phosphate
concentrations) were analyzed. As a result, a major cellular growth was found when 2 mg/L NAA +
1 mg/L KN was utilized. Finally, when 4 mg/L 2,4-D was used a marked increase in galphimine B
accumulation was observed (36% higher concerning calli, and wild plants).
Also, Rojas et al. [
73
] developed a micropropagation procedure for Galphimia glauca and the
establishment of plantlets in the field. In addition, they also evaluated the contents of galphimine B in
both wild and micropropagated plants. Various concentrations of both IAA and KN were evaluated.
However, the best treatment for shoot induction was found to be MS supplemented with 4 mg/L
KN. Still, the combination of 4 mg/L KN + 0.1 mg/L IAA produced the highest shoot length and the
highest pair of leaves per explant. The formation of roots was observed 20 days after ex vitro indol
butyric acid application (IBA) and is consequent transplantation to soil conditions. The plantlets were
transferred to greenhouse conditions for two months, and after this period they were moved to field
conditions, where they exhibited a 90% survival with the first flowering event taking place only four
months after they were transplanted. The content of galphimine B in methanol extracts from either,
wild and micropropagated plants was very similar (7.83 and 7.39 mg/g DW, respectively). In brief,
the authors suggest that the use of this micropropagation technique can be used for the conservation
of this important medicinal plant [73].
Since galphimines are very important compounds to achieve the anxiolytic effect in
Galphimia glauca, experiments using the
in vitro
tissue culture techniques to increase their yield and
production have also been done. A cell suspensions culture for Galphimia glauca were developed
specifically for galphimine B production [
74
]. In this study, a two-stage culture system was used,
first immobilization in Ca
2+
alginate beads and scale up from flask to bioreactors (stirred and airlift).
As a result, cells immobilized in the matrix excreted up to 100% of the galphimine B produced. In this
study a stirred type bioreactor showed promising results, generating galphimine B content up to
1381 µg/L after 24 days of culture.
Plant genetic transformation has also been achieved for Galphimia glauca. A report describing the
production of galphimines using Agrobacterium rhizogenes was described by [
45
]. The transformation
process was done on cotyledons and hypocotyls of Galphimia glauca seedlings grown in Gamborg
B5 solid medium devoid of plant growth regulators. Then, the explants were transferred into a new
medium containing 200 mg/L timentin and 2 g/L polyvinylpyrrolidone (PVP) for hairy root induction.
The main objective of this research, besides generating a genetic transformation protocol, was to assess
the production of nor-friedelanes via hairy root production. Hence, the hairy root nutrient medium
was used to analyze the yield of these compounds. The transformed roots generated three major
nor-friedelanes, namely glaucacetalins A–C that were found in the nutrient medium [45].

Molecules 2018,23, 2985 16 of 22
The same research group described the production of triterpenoids in liquid cultivated hairy
roots of “calderona amarilla” [
75
]. Thus, galphimine E, glaucacetalin A, and mascalinic acid were
obtained from this process. Glaucacetalin A was excreted to the culture media presenting a maximum
concentration of 2.14 mg/L after 21 days of culture. On the other hand, galphimine E and mascalinic
acid were obtained in the root biomass with concentrations as high as 0.11 and 0.43 mg/g, respectively
after 39 days in culture [
75
]. Another study involving cell suspension cultures from Galphimia glauca
(GgBa) led to the discovery of a novel triterpenoid compound related with galphimines: glaucacetalin
D with an overall yield of 2.9 mg/L. The reported glaucacetalin D exerted a sedative effect similar to
galphimine B [46].
Plants respond to abiotic or biotic stress by inducing the expression of certain metabolic pathways,
most of them are also related with the production of secondary metabolites. Jasmonic acid or methyl
jasmonate [the methyl ester form (MeJA)] are part of this defense response in plants [
76
]. The role
for these compounds in the induction of triterpenes and sterols has been analyzed in several plants,
including Galphimia glauca [
77
]. Thus, the effect of 100
µ
M MeJA on roots of Galphimia glauca was
evaluated. Root growth inhibition was observed, which resulted in plantlet premature death. However,
the exposure of roots to MeJA led to the increase in galphimine B production (
≈
17
µ
g/g DW) as well
as the cholesterol levels (15% of all total free sterol content). This constitutes the first report regarding
the use of elicitor molecules in the nor-seco friedelane galphimine metabolic pathway [77].
8. Taxonomical Misinterpretations in the Galphimia Genus
Showy inflorescences and morphological similarity have led to the incorrect labeling of the species
of this genus which was formerly known as Thryallis L, now, Galphimia. Also, the morphological
resemblance among various members of the Galphimia genus has misled collectors to incorrectly assign
names for the specimens collected, mainly labeled as Galphimia glauca and Galphimia gracilis [3].
Further studies have demonstrated disagreements at the metabolomic and genetic level between
plants allegedly classified as Galphimia glauca in various regions of Mexico. Accordingly, a study
discovered that only certain regions of Mexico produce Galphimia glauca specimens that contain high
levels of “galphimines” which are responsible for its anxiolytic and sedative effects, as well as other
important medicinal properties. This was confirmed through a metabolomics analysis done in plants
collected in different localities of Central Mexico. This analysis revealed that plants collected from only
two regions (Dr. Mora, Guanajuato, and Jalpan, Queretaro) were capable of producing galphimines,
while the others fail to do so, or produced them at a considerable lower rate [3].
On a further study, Sharma et al. [
44
] analyzed four years later the metabolomic profile of
these same localities plus two new ones and found that populations collected from Dr. Mora and
Jalpan were the only ones that produce galphimines and labeled them as active after evaluating them
through mouse models to test anxiety and sedative effects. These results suggest that the galphimine
production in these plants can be related to environmental factors that could help increase or decrease
their production, in fact, most of the plants analyzed grow in contrasting environments.
A second study utilizing DNA barcoding as a molecular marker confirmed the clear differences
among the plant populations used in the former study [78]. The results found in this study indicated
that the plants from all seven populations analyzed do not belong to the one Galphimia glauca species
as labeled in the respective herbarium, on the contrary, there could be at least three different species
of the genus Galphimia. Also, TLC results were consistent with the expected galphimine production
profile in the two main “producing” populations: Dr. Mora Guanajuato, and Jalpan Queretaro.
9. Patents
In 2007, Tortoriello et al. registered a patent for the dry extract production from Galphimia glauca
and use of a pharmaceutical composition in the treatment of anxiety [
79
]. Accordingly, the authors
described that their invention is related mainly with the pharmaceutical formulation comprising a dry
extract from “calderona amarilla” which is obtained after various solvent extraction procedures. Thus,

Molecules 2018,23, 2985 17 of 22
the formulation generated is to be used for treatment, prevention and possible cure of anxiety in all
its forms. To the best of our knowledge, this is the only patent existing for Galphimia glauca extracts
until now.
10. Current Needs
The present data demonstrate the pharmacological potential of Galphimia glauca to treat mental
disorders such as anxiety, insomnia, and depression. Millions of people worldwide suffer from mental
problems, and the treatment of these ailments is restricted to some synthetic drugs which induce
from mild to severe side effects, including dependence. Galphimia glauca has shown comparatively
superior medicinal properties, but its uses are still limited to traditional preparation. Development of
phytomedicines with standardized extracts would be of great support for the treatment of patients
with such a disorder. The available data on Galphimia glauca represents a wide range of traditional as
well as potentially new health applications especially anti-inflammatory, cytotoxic and antimalarial
properties. Limited data that correlates the traditional/therapeutics uses of Galphimia glauca and
phytochemical profile of the extract used are available.
Most of the literature used to prepare this review is mainly focused on the type of extract and
part of the plant but, do not identify or quantify bioactive compounds. Modern metabolomics tools
could be very useful to study and identify novel compounds and to develop a complete phytochemical
profile. The functions of galphimines are not known in the plant, so further research is required to
understand their importance in nature. Exploring their role in the plant may open new research
areas. Special emphasis is needed in the correct identification of the plant material because of many
closely related species of the genus share very similar morphological appearances which disoriented
collector, taxonomist, and researcher. Scientists inside or outside of Mexico should identify their plant
material of Galphimia glauca or related species as reported by the authors [
3
,
69
] for reliable scientific
production, so DNA barcoding using matK and rpoc1, as well as TLC analysis could be useful for the
correct identification to develop authentic research data or even a reliable final product. The natural
habitat for Galphimia glauca is limited to several states of Mexican territories such as Aguascalientes,
Guanajuato, Hidalgo, Jalisco, Nuevo Leon, Querétaro, Tamaulipas, San Luis Potosíand Zacatecas
but most of the research data presented in this review have utilized the samples collected from
Guanajuato and Queretaro. Local researchers should identify and declare the new localities within
the Mexican territory. In our own experience, the collection of this plant species is not an easy task
because very little or no information is available about its geographical distribution. Many reported
sites in the literature are too old and have been converted into agricultural farms or urban areas.
The local herbal market is depending on the availability of the plant material in restricted locations,
and it is completely sessional. There is very little, or no information available on the plant material
collected from other states. Constant collection of the plant material from its natural habitat may put
the species on the risk of overexploitation. More studies with a focus on conservation and agronomical
practices are highly required. Micropropagation has been reported, but its scientific or commercial
exploitation is not known. More studies to develop fast and scale-up micropropagation protocols will
be highly helpful to produce the plant for research and commercial purposes. To be able to develop
phytomedicine or synthetic drug for the treatment of CNS disorders, multidisciplinary kind of research
on Galphimia glauca is highly required. Genomics and transcriptomics are the promising areas that can
help in understanding the biosynthesis pathways that lead the production of important triterpenoid
family. Bioreactor-based investigations have been reported, but no significant advances are achieved
for the further scale-up production of desired galphimines.
11. Conclusions
Plants of the genus Galphimia are mainly distributed throughout Mexico, and some species
have been extended to Texas, USA and Central America including Guatemala and Brazil. In this
review, we have emphasized on the species Galphimia glauca because most of the research has been

Molecules 2018,23, 2985 18 of 22
centralized on this species especially that is correlated with the therapeutic uses for the CNS disorders.
With regards to 26 species of the genus, there are many species that have received very little of
no attention. In this review, we have condensed the biological activities and reported bioactive
compounds that represent most of the scientific literature published on Galphimia glauca. The present
information helps in filling the gap between scientific studies and traditional use of this plant.
The present review also suggests the current needs of multidisciplinary research such as agronomical
practices, tissue culture protocols, phytochemical studies, bioactive compound identification, bioreactor
scale-up studies, recognitions of genes involved in the biosynthesis, and molecular taxonomy for
Galphimia glauca.
Author Contributions:
A.S. initiated the project, designed the manuscript, wrote, prepared and edited figures
and the final version of the manuscript. P.I.A.-B. contributed in writing the draft, preparing tables and figures
and formatting; A.M.-N. edited figures and wrote part of Section 7; G.O. and A.C.-T. revised text and formatting;
H.M.N.I. and M.L.V. participated in editing and critical analysis of the information in the paper. All authors have
read the manuscript before submission.
Funding: This research received no external funding.
Acknowledgments:
The literature facilities provided by Tecnologico de Monterrey, Queretaro are
thankfully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CNS: central nervous system; ROS: reactive oxygen species; DW: dry weight; GAD: generalized anxiety disorder;
HMA: Hamilton Anxiety Rating Scale; SSRI: serotonin reuptake inhibitors; GB: galphimine B, OCD: Obsessive
Compulsive Disorder; GABA: γ-aminobutyric acid type A; PTZ: pentylenetetrazole.
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