Inhibition of squalene synthase of rat liver by abietane diterpenes derivatives

Article abstract of DOI:10.1080/14786419.2019.1678614

In the current study, chemical composition of cultivated Salvia canariensis L was determined. Carnosol was the main product isolated. We prepared more lipophilic derivatives from carnosol, and both isolated and semisynthetic abietane diterpenes were evaluated in?vitro as inhibitors of squalene synthase. Among the compounds tested, carnosol was the most potent inhibitor (IC₅₀ = 17.6 μM). These results highlight the great potential of this species for the production of new ingredients in nutritional supplements for the treatment of hyperlipidemia.

Full text of DOI:10.1080/14786419.2019.1678614

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Inhibition of squalene synthase of rat liver by
abietane diterpenes derivatives
Mariana Macías-Alonso, Lucía S. Andrés, Iván Córdova-Guerrero, Arturo
Estolano-Cobián, Laura Díaz-Rubio & Joaquín G. Marrero
To cite this article: Mariana Macías-Alonso, Lucía S. Andrés, Iván Córdova-Guerrero, Arturo
Estolano-Cobián, Laura Díaz-Rubio & Joaquín G. Marrero (2019): Inhibition of squalene
synthase of rat liver by abietane diterpenes derivatives, Natural Product Research, DOI:
10.1080/14786419.2019.1678614
To link to this article: https://doi.org/10.1080/14786419.2019.1678614
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1678614
SHORT COMMUNICATION
Inhibition of squalene synthase of rat liver by abietane
diterpenes derivatives
a
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Mariana Macıas-Alonso , Lucıa S. Andres , Ivan Cordova-Guerrero ,
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c
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Arturo Estolano-Cobian , Laura Dıaz-Rubio and Joaquın G. Marrero
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Instituto Politecnico Nacional, UPIIG, Silao de la Victoria, Guanajuato, Mexico; Instituto Universitario
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de Bioorganica “Antonio Gonzalez”, Departamento de Quımica Organica, Universidad de La Laguna,
Tenerife, Spain; Facultad de Ciencias Quımicas e Ing, Universidad Autonoma de Baja California,
c
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Tijuana, B. C, Mexico
ABSTRACT
ARTICLE HISTORY
Received 29 July 2019
Accepted 23 September 2019
In the current study, chemical composition of cultivated Salvia can-
ariensis L was determined. Carnosol was the main product isolated.
We prepared more lipophilic derivatives from carnosol, and both
isolated and semisynthetic abietane diterpenes were evaluated
in vitro as inhibitors of squalene synthase. Among the compounds
tested, carnosol was the most potent inhibitor (IC50 ¼ 17.6 lM).
These results highlight the great potential of this species for the
production of new ingredients in nutritional supplements for the
treatment of hyperlipidemia.
KEYWORDS
Carnosol; Hyperlipidemia;
Salvia; Cholesterol;
Squalene synthase
1. Introduction
Hyperlipidemia is a well-established risk factor for the development of cardio and
cerebrovascular diseases. For this reason, lipid lowering is one of the major
approaches used for their primary prevention. A widely used strategy to decrease
plasma cholesterol concentration is the use of HMG-CoA inhibitors such as statins.
However, this enzyme plays a fundamental role in the biosynthesis of many other
nonsteroidal molecules, so statins have potential adverse effects (Balaz and Wolfrum
2019). The inhibition of squalene synthase (SQS), an enzyme essential in the
biosynthesis of the steroids, can prevent the cholesterol biosynthesis without
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CONTACT Joaquın Gonzalez Marrero
jgonzalezm@ipn.mx
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1678614.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

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M. MACIAS-ALONSO ET AL.
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disturbing the nonsterol pathway. Several SQS inhibitors have been developed such
as zaragozic acid and quinuclidine (Kourounakis et al. 2010).
The genus Salvia consists of some 500 species, some of which have shown
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interesting biological activity (Jakovljevic et al. 2019). In previous studies of the aerial
part of Salvia canariensis L., an endemism of the Canary Islands, carnosic acid 1 and
carnosol 2 were isolated as the major phenolic diterpenes (Marrero et al. 2009), which
have been reported to have broad biological activities like neuroprotective and hypoli-
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pidemic properties (Gonzalez 2015; Alonso et al. 2019).
As a continuation of our work on the isolation and synthesis of diterpenes to iden-
tify bioactive components, and due to the great importance to discover potential SQS
inhibitors, the purpose of this study was to provide the chemical profile of cultivated
S. canariensis, obtain synthetic derivatives from the isolated products and evaluate
their ability to inhibit SQS in vitro. These results make this plant to be considered as a
crop that could have commercial importance as useful ingredient in nutritional
supplements.
2. Results and discussion
The acetonic extract of cultivated S. canariensis was fractionated and purified following
the protocol designed by Marrero et al. (2009). The analysis of the extract revealed carno-
sol 2 as the main product. The concentration of rosmanol 3 was also significant, with 7a-
methoxyrosmanol 4, 7a-ethoxyrosmanol 5 and galdosol 6 as minority constituents
(Table S1). These compounds have been previously obtained from S. canariensis (Marrero
et al. 2009). We also reported here, for the first time from this species, the isolation of ros-
madial 7, epirosmanol 8 and isorosmanol 9 previously found in Rosmarinus officinalis
(Nakatani and Inatani 1984); isogaldosol 10 and demetilsalvicanol 11 previously isolated
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from Salvia mellifera (Gonzalez et al. 1992); and 7b-methoxyrosmanol 12 identified previ-
ously in Salvia dorrii (Ahmed et al. 2006). On the other hand, in our study we could not
detect carnosic acid 1, probably due to the harvest time of the plant (Luis and Johnson,
2005). Although 16-acetoxycarnosol was isolated from in vitro culture of S. canariensis
(Luis et al. 1992), we could not detect any abietane derivative oxidized at C-16.
All compounds were tested for SQS inhibitory activity at a concentration of 10 lM
(Amin et al, 1992) (Table S1). Compounds 8, 10, 11 and 12 were not evaluated due to
the small amount isolated. Most compounds exhibited weak inhibition activity of SQS,
except carnosol 2 (IC50 ¼ 17.6 lM). At this point, we decided to prepare some
semisynthetic derivatives of carnosol 2, where we change the hydroxyl groups for
more lipophilic substituents (Scheme 1). The biological evaluation of these derivatives
showed that 11,12-di-O-methylcarnosol 15 was weaker than carnosol 2. However,
the oxidation of rosmanol 3 to rosmaquinone 14, improves the biological activity
(Table S1).
To obtain more information about the mechanism as well as the mode of interactions
of these phytochemicals with SQS, the three compounds with the best in vitro results
were selected to perform a docking analysis (Table S2). Regularly, squalene inhibitors
have been found interacting with Ala176, Met207, Gly208 and Leu76 residues and the
ones nearby, mainly by Van der Waals and hydrophobic interactions, present in a large

NATURAL PRODUCT RESEARCH
3
Scheme 1. Semisynthesis of Carnosol derivatives.
hydrophobic pocket that the SQS receptor possess (Figure S3) (Ichikawa et al. 2012). The
hydroxyl groups of carnosol and rosmaquinone may not interact with the R group of the
residues, but with their skeletons. This would be the case for one of carnosol phenolic
hydroxyl with the skeletal N of Gly208; a weak hydrogen or dipole interaction among
these atoms could be possible, as their distance is 2.5 Å. This same hydroxyl has a 3.08 Å
distance to the carbonyl of Met207. The second phenolic hydroxyl also has a 2.96 Å dis-
tance to the carbonyl of Ala176, so similar interactions can take part (Figure S4). The
hydroxyl of rosmaquinone has Leu76 carbonyl at a distance of 4.35 Å. Thus, we can
assume a weak dipole interaction from this group may be favouring rosmaquinone.
These additional interactions, other than the hydrophobic ones, would partially explain
why 11,12-dimethoxycarnosol (which have none of them) has the lowest docking score,
even though it shares a very similar position in space with rosmaquinone. Even though
carnosol and rosmaquinone have the best docking results, in vitro we see how carnosol
has the lowest IC50 (17.6 lM), followed by 11,12-di-O-methylcarnosol (18.4 lM). Although
this can look as a disagreement, we can observe that the docked poses give the correct
insight for the in silico-in vitro relationship. The docked compounds are very close to
each other in the cavities where is well known inhibitors bind (Ichikawa et al. 2012), but
carnosol can be seen deeper in such cavity. Different studies for SQS inhibitors have
made the observation that, the better the in vitro inhibition shown, the deeper such com-
pound goes interacting in the hydrophobic cavity in the docking analysis (Kourounakis
et al. 2010). In such way, the docking poses allows to explain why carnosol has a better
interaction with the receptor by means of its spatial position.
3. Conclusions
We describe the majority diterpenes present in the extract of cultivated S. canariensis,
which was confirmed to be a taxon strongly characterized by the presence of carnosol
2. We isolated eleven abietane-type compounds. The present study portrays the first
report of the isolation of six of these compounds from this species. We prepared

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M. MACIAS-ALONSO ET AL.
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more lipophilic derivatives from carnosol, and both isolated and synthetic compounds
were evaluated as SQS inhibitors. It was observed that carnosol 2, rosmaquinone 14
and 11,12-di-O-methylcarnosol 15 showed in vitro SQS inhibitory effect. In order to
obtain data about the interactions between these molecules and SQS, we performed
in silico docking studies. To our knowledge, this is the first report of the inhibition
of SQS by abietanes. Application of the findings from this docking study could
led to the development of more potent SQS inhibitors aimed against atherosclerosis.
We think that this species could be cultivated and used as a new useful ingredient
in nutritional supplements with antioxidant and anti-atherosclerosis function.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by CONACYT and Research and Postgraduate Secretary of the National
Polytechnic Institute (SIP- IPN) are gratefully acknowledged.
ORCID
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Joaquın G. Marrero
http://orcid.org/0000-0003-3720-4528
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