Natural Product Research

Formerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Quantitative determination of pentacyclic

triterpenic acids in Amazonian species Eugenia
punicifolia DC by ATR-FTIR

Raphael P. Albuquerque, Glauce C. A. Duarte, Nelise Duarte, Débora T.

Ohana, Wilson C. Santos & Thelma B. Machado

To cite this article: Raphael P. Albuquerque, Glauce C. A. Duarte, Nelise Duarte, Débora T.
Ohana, Wilson C. Santos & Thelma B. Machado (2019): Quantitative determination of pentacyclic
triterpenic acids in Amazonian species Eugenia�punicifolia DC by ATR-FTIR, Natural Product
Research, DOI: 10.1080/14786419.2019.1680662

To link to this article: https://doi.org/10.1080/14786419.2019.1680662

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Published online: 01 Nov 2019.

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1680662

SHORT COMMUNICATION

Quantitative determination of pentacyclic triterpenic

acids in Amazonian species Eugenia punicifolia
DC by ATR-FTIR
Raphael P. Albuquerquea, Glauce C. A. Duarteb, Nelise Duarteb, D
ebora T.
Ohanac, Wilson C. Santosb and Thelma B. Machadoa,b
a
Programa de P
os-Graduaç~ao em Engenharia de Biossistemas, Universidade Federal Fluminense,
Nitero
i, RJ, Brazil; bPrograma de P
os-Graduaç~ao em Ci^encias Aplicadas a Produtos de Sa
ude,
Faculdade de Farm
acia, Universidade Federal Fluminense, Niter
oi, RJ, Brazil; cFaculdade de Ci^encias
Farmac^euticas, Universidade Federal Do Amazonas, Manaus, AM, Brazil

ABSTRACT ARTICLE HISTORY

Leaf extracts from Eugenia punicifolia are rich in pentacyclic triter- Received 23 April 2019
penic acids (PTAs), especially barbinervic acid (BA), which is an Accepted 30 September 2019
important biomarker of the species. Dichloromethane extracts of
E. punicifolia leaf samples harvested in Amazonian summer and KEYWORDS
winter seasons were analysed by infrared spectroscopy using ATR- Eugenia punicifolia; Pedra-
ume ca
a; barbinervic acid;
FTIR technique aiming to evaluate barbinervic acid (BA) and its seasonality
PTAs equivalent contents. A validated HPLC-DAD quantification
method was also performed to compare the relationship between
BA and PTAs contents in E. punicifolia extracts with ATR-FTIR tech-
nique. The use of ATR-FTIR allowed a rapid, efficient and environ-
ment-friendly quantification method for total PTAs equivalent
content, showing a significant statistical difference (p< 0.05) in the
production of these metabolites (38.66 lg/mL, summer; 13.62 lg/
mL, winter). A mathematical correction factor between the HPLC-
DAD and ATR-FTIR quantification methods was established.

1. Introduction
Eugenia punicifolia (Kunth) DC (Myrtaceae) is a shrub native from several regions of
Brazil, especially the Amazon region, popularly known as pedra-ume caa
(Silva et al.
2015). It is extensively used in traditional medicine in decoctions or infusions, as a nat-
ural therapeutic agent for the empirical treatment of several diseases such as diabetes

CONTACT Thelma B. Machado thel34@gmail.com

Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1680662.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 R. P. ALBUQUERQUE ET AL.

mellitus. Several studies converge to its putative pharmacological potential, probably

due to the presence of pentacyclic triterpenic acids (PTAs) such as barbinervic acid
(BA), which is found mainly in the leaves of this species (Leite et al. 2014).
Standardised extracts with pharmacologically active amounts of these metabolites
may contribute to the therapeutic arsenal of Brazilian herbal medicines. Therefore,
qualitative and quantitative methods with characteristics, such as high reliability, sensi-
tivity, reproducibility and low cost, contemplating the tendency of the use of green
chemistry are required (Goh et al. 2017).
Infrared spectroscopy is a valuable method for analysing complex natural materials
by means of vibrational bands which are readily identifiable by Fourier transform-infra-
red spectroscopy (Mart
ın-Ramos et al. 2018). The attenuated total reflection–Fourier
transform infra-red (ATR-FTIR) spectroscopy is an attractive alternative to conventional
methods, being a fast and robust analytical solution for sample-to-sample analyses
with a small variation in the user-to-user spectrum (Hifumi et al. 2016). ATR main
advantage is that it allows measurement of dispersions, gels, liquids and pastes, very
fast, with no extra preparation procedures (Rytwo et al. 2015). FTIR spectrometers
have become a common feature of most laboratories and are used in a wide variety
of researches. Over the last years, quantitative analysis of mixtures using FTIR has pro-
gressed from matching a few peaks to matching entire spectra, working with software
to address linear system theory to direct the experiments. Some drawbacks are related
to the software, which may not be available, require a specific knowledge of the user
or be complicated for many analyses. Some mathematical models may be required for
mixtures in which the concentrations of components vary widely, or if another compo-
nent must be added to the mixture. These drawbacks may deter users, reducing adop-
tion of ATR-FTIR as a quantitative technique. In the other hand, simpler ATR-FTIR
quantitative methodologies are already available in the scientific literature to analyse
the component percentages in mixtures of known substances (Mackie et al. 2016).
In view of climatic specificities of the Amazon Region, this study aimed to apply
the ATR-FTIR technique to evaluate BA and PTAs equivalent contents in dichlorome-
thane leaf extracts (DLE) of E. punicifolia using vegetal samples harvested in seasons of
lower and higher convective activity, using BA as a chemical marker.

2. Results and discussion

The region of Manaus (AM) has a very specific environmental characteristic (Neves
et al. 2017), with a very high average rainfall, about 67.95 to 76.91 mm among the dri-
est months and from 237.87 to 315.59 mm among the months with the highest pre-
cipitation (INMET – National Institute of Meteorology, Brazil). According to Pereira and
Cardoso (2012) and Costa (2017), the increase in the production of secondary metabo-
lites is conditioned to the degree of stress and the time when it occurs. Short-term
water stresses may have a direct effect on the increase in production of these metabo-
lites, whereas, in the long term, an opposite effect is usually observed (Gobbo-Neto
and Lopes 2007). To analyse these effects in Eugenia punicifolia extracts, the barbi-
nervic acid and its equivalents were used as chemical markers.
 NATURAL PRODUCT RESEARCH 3

Barbinervic acid (supplementary material Figure S1) has demonstrated its import-
ance as a molecule associated with many distinct pharmacological effects. It is present
in several species in concentrations depending on specific metabolic characteristics
and factors related to seasonality, circadian rhythm and plant development (Morais
2009). Triterpenes with similar molecular structures (supplementary material Figure S2)
have pharmacological activities such as reparative healing action, anti-inflammatory,
antibacterial, antiviral, hepatoprotective and antitumor properties, combined with low
toxicity (Leite et al. 2014; Chudzik et al. 2015).
The quantitative analysis of BA standard and PTAs equivalents showed ATR-FTIR as a
linear, accurate and robust technique (supplementary material Figure S3). The contents
of total PTAs equivalents in summer and winter DLE of E. punicifolia showed significant
statistical differences (p< 0.05) and are presented in supplementary material Table S2. A
substantial increase of 2.8 times in the content of PTAs equivalents was observed in
extracts from plant materials harvested in summer when compared to those of the win-
ter season. The analysis of total PTAs by ATR-FTIR is based on the ability of covalent
bonds to vibrate at frequencies of the same order as infrared radiation waves (Hifumi
et al. 2016). A specific absorption band of the molecule was tracked, the acid carbonyl
absorption band, which has its best absorption between 1716 and 1712 cm
1 (supple-
mentary material Figure S4a). The limit of detection (LOD) for ATR-FTIR technique was
of 2.033 lg, calculated based on the standard deviation of the response and slope.
HPLC-DAD analysis showed a barbinervic acid content about 18% higher in summer
DLE of E. punicifolia when compared to winter leaf extracts (supplementary material
Table S3) and a LOD of 0.06317 lg for BA. This result is in accordance with the detec-
tion range (0.058 lg for the single molecule of BA) established by Fan and He (2006).
It is possible to infer that the high value of LOD in the ATR-FTIR technique is due to
the fact that the technique allows the quantification of not only the BA molecule, but
of total PTAs, not distinguishing the acid carbonyl present in the different molecules
of this class of substances. HPLC-DAD chromatograms of DLE of E. punicifolia and bar-
binervic acid standard are shown in supplementary material Figure S4b. The results
have shown that, as well as the barbinervic acid standard, chromatograms of leaf
extracts presented, in addition to barbinervic acid (tR ¼ 3.978 min), at least two other
pentacyclic triterpenic acids are present, namely, rotungenic acid (tR ¼ 4.490 min) and
24-hydroxy-ursolic acid (tR ¼ 5.10 min). The results are in accordance with Fan and He
(2006), who showed the same chromatographic profile in their study on Diospyros
kaki, with retention times of 2.658, 3.490 and 5.680 min, respectively.
The behaviour of the metabolic productivity of PTAs and BA in E. punicifolia leaves
during the Amazonian summer and winter seasons are shown in supplementary
material Figure S7. The concentration of BA alone showed no significant statistical dif-
ferences between the Amazonian seasons (0.00631%, summer; 0.00517%, winter).
However, when analysing total PTAs contents, a significant statistical difference is
observed between summer (3.86%) and winter (1.36%), clearly reflecting a metabolic
change due to environmental differences.
The study focusing on PTAs carbonyl vibrational deformation band analysis
(1712 cm
1) by ATR-FTIR allowed the quantification of this class of substances in DLE
of E. punicifolia. The quantification of total pentacyclic triterpenic acids by means of
4 R. P. ALBUQUERQUE ET AL.

ATR-FTIR, and using barbinervic acid as a chemical marker, allowed the results to be
expressed as barbinervic acid equivalents. Quantitative assessments of specific sub-
stances classes, such as alkaloids and flavonoids, among others, are present in several
monographs of official compendia, such as pharmacopoeias, which express these
chemical markers as equivalents of a particular substance. Many quantification official
methods for herbal products already established in the pharmaceutical industry are
performed by the same manner. Some examples are herbal medicines of Valerian offi-
cinalis extracts, standardised at Not Less Than (NLT) 0.17% of total sesquiterpene acids,
expressed as valerenic acid equivalents; and Aesculus hippocastanum L., standardised
with triterpene glycoside content, calculated as escin equivalents, among others (USP
41-NF 36 2016; WHO monographs on selected medicinal plants 2006).
HPLC-DAD results of barbinervic acid content in E. punicifolia extracts in comparison
with those obtained for PTAs by ATR-FTIR showed a direct and proportional correl-
ation. To establish a relationship between HPLC-DAD and ATR-FTIR quantification
methods, a standardisation of the analyses by means of a correction factor between
the techniques was proposed (Supplementary material). The results obtained for BA
and PTAs contents expressed as barbinervic acid equivalents present in the seasonal
DLE of E. punicifolia were 0.0632 lg (BA, HPLC-DAD) and 38.66 lg (PTAs, ATR-FTIR) for
Amazonian summer season and 0.0518 lg (BA, HPLC-DAD) and 13.62 lg (PTAs, ATR-
FTIR) for Amazonian winter season. The use of ATR-FTIR technique represents a great
decrease in organic solvent expenses and time of analysis, lower energy expenditure
and less exposure of the analysts to products that are highly volatile and harmful to
their health. To the best of our knowledge, this is the first manuscript that uses ATR-
FTIR for the quantification of total pentacyclic triterpenic acids in plant species.

3. Experimental
See Supplementary material.

4. Conclusion
Due to the great diversity and slight differences between groups of pentacyclic triter-
penic acids present in the analysed extracts, ATR-FTIR is not suitable as a quantitative
method for the distinction of specific molecules and requires auxiliary techniques for
their assay. However, it is possible to propose a correlation between ATR-FTIR and
HPLC methods by calculating a correction factor and using barbinervic acid as a chem-
ical marker. Total pentacyclic triterpenic acids can therefore be quantified in percen-
tages and expressed as barbinervic acid equivalents in leaf extracts of E. punicifolia.
Thus, by establishing a correlation between these two techniques, it is possible to
reduce considerably time and cost of analysis, as well as the use of organic solvents,
considering that most of the solvents can be reused in future extraction processes,
thus adopting the principles of green chemistry.

Disclosure statement
No potential conflict of interest was reported by the authors.
 NATURAL PRODUCT RESEARCH 5

ORCID
Wilson C. Santos http://orcid.org/0000-0001-9971-094X
Thelma B. Machado http://orcid.org/0000-0001-9449-8695

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