doi.org/10.1002/cbdv.
202300864 Research Article
Seasonal and Circadian Evaluation of the Essential Oil of
Dizygostemon riparius (Plantaginaceae), a Wild Flavoring
Herb from East Maranhão, Brazil
Francielton S. Galvão,[a] Cláudia Q. da Rocha,[a] Odair S. Monteiro,[a]
Pablo Luis B. Figueiredo,[b, c] Paulo Vinicius L. Santos,[c] and José Guilherme S. Maia*[a, c]
Dizygostemon riparius (Plantaginaceae) is an aromatic herbal
species known as “Melosa”, endemic to the Municipality of São
Benedito do Rio Preto, East Maranhão state, Brazil. It has a
refreshing and pleasant aroma and is used for food flavoring
and controlling domestic animal parasites. This work aimed to
evaluate the seasonal and circadian influence on the composi-
tion and production of essential oils (EOs) from D. riparius. The
plant aerial parts were hydrodistilled, and the oils were
analyzed by GC and GC/MS. The seasonal study was conducted
from August 2019 to July 2020, and the circadian study in dry
(November) and rainy (May) periods, at 6 am, 12 am, and 6 pm.
Introduction
The Brazilian autochthonous flora is one of the richest on the
planet, with about 15 to 25 % of its plant species, many of
which have a high rate of endemism and are dispersed in
unique biomes, such as the Brazilian Cerrado. The Brazilian
plants’ biological diversity has been widely studied with
obtaining a significant number of micro- and macromolecular
metabolites, in which several constituents have presented
biological and pharmacological properties with the potential for
developing new drugs, cosmetics, agrochemicals, and food
supplements of natural origin.[1–3] The Brazilian Cerrado is the
second largest vegetation formation in the world, comprising
24 % of the country’s land area and harboring more than
11,000 species. Of these, about 4,400 are endemic, used in folk
medicine, and of great pharmacological importance, contribu-
ting significantly to the knowledge and discovery of new
bioactive compounds.[4,5]
Molecular studies have reshaped the understanding of
angiosperm evolution, which has resulted in new taxonomic
classifications. Systematic botany is faced now with families
whose circumscriptions have sharply changed. Phylogeny works
[a] F. S. Galvão, C. Q. da Rocha, O. S. Monteiro, J. G. S. Maia
Programa de Pós-Graduação em Química, Universidade Federal do
Maranhão, 65080-040 São Luis, MA, Brazil
E-mail: gmaia@ufpa.br
[b] P. L. B. Figueiredo
Laboratório de Química de Produtos Naturais, Universidade do Estado do
Pará, 66087-662 Belém, PA, Brazil
[c] P. L. B. Figueiredo, P. V. L. Santos, J. G. S. Maia
Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade
Federal do Pará, 66075-900 Belém, PA, Brazil
Chem. Biodiversity 2023, 20, e202300864 (1 of 9)
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The results showed that the medium EOs yield was 2.8 %, and
the primary constituents (> 2 %) were endo-fenchyl acetate
(30.5–42.1 %) and endo-fenchol (31.6–37.4 %), (E)-caryophyllene
(2.8–7.6 %), α-fenchene (3.3–6.5 %), p-cymene (0.7–4.5 %), and
caryophyllene oxide (1.4–2.7 %). Yield and composition of EOs
did not significantly correlate with the climatic parameters, but
their seasonal percentages influenced their two main compo-
nents due to precipitation and solar radiation environmental
factors. Quantitative variability in the EOs composition during
the dry and rainy seasons was observed in the circadian period.
These data may be significant for the plant’s economic use.
revealed that the Scrophulariaceae family is not monophyletic
and has a more restricted concept. The genus Dizygostemon
(Benth.) Radlk. ex Wettst., before in Scrophulariaceae, now
belongs to Gratioleae, the mainly tropical tribe of the
Plantaginaceae family, comprising ca. 25 genera and over
300 species.[6–8] Dizygostemon was previously described as a
monotypic section of the genus Beyrichia Cham. & Schltdl.
Subsequently, their distinct morphologies separated them from
other Beyrichia species. Then, the section Dizygostemon was
elevated to the genus category and, until recently, was
represented only by D. floribundum (Benth.) Radlk. ex Wettst.
(syn. D. angustifolium Giul.), a species restricted to the Cerrado
and Caatinga areas of Piauí, Ceará, Pernambuco, and Bahia
states in Northeastern Brazil. It is characterized by herbaceous
habit, with subsessile flowers or short pedicels and pubescent
leaves on the dorsal side, puberulent on the ventral side, and
glandular-pointed on both sides.[7,8] Dyzygostemon riparius
Scatigna & Colletta is a sticky and aromatic sub-shrub, up to
50 cm tall, occurring in the East Maranhão state, Brazil, which
had its botanical description recently reported.[9] It is an
endemic species known as “melosa” by the local population of
the municipality of São Benedito do Rio Preto, MA, Brazil. Its
leaves and thin stems have a refreshing and pleasant aroma,
are used as a food flavoring, control parasites of domestic
animals, and prevent mosquitoes.[9,10]
In Brazil, the representative genera of Plantaginaceae are
Achetaria Cham. & Schltdl., Dizygostemon (Benth.) Radlk. ex
Wettst., Stemodia L., and Tetraulacium Turcz. According to the
taxonomists,[8] there is a close botany relationship between
Achetaria, Dizygostemon, and Tetraulacium. In the Brazilian
herbaria, specimens of Achetaria, Dizygostemon, and Tetraula-
cium have been identified as Stemodia, the largest genus of the
© 2023 Wiley-VHCA AG, Zurich, Switzerland
 Research Article
doi.org/10.1002/cbdv.202300864
group, due to their resemblances, which suggest a close
phylogenetic relationship between them.[11,12] Also, the genus
Otacanthus Lindl. is considered very close to the Achetaria due
to the typical characteristics of their stamens and anthers.[13]
Chemical variability and the relative proportion of secon-
dary metabolites in plants generally result from circadian and
seasonal factors and their interspecific/intraspecific relationship.
Genetic heredity is significant in this context, but plant
expression can change due to the interaction of biochemical,
physiological, and ecological processes. Therefore, secondary
metabolites represent a chemical interface between the plant
and the surrounding environment, with their biosynthetic
pathway affected by factors such as circadian rhythm, season-
ality, and plant development. The time of collection of the
plants is of great importance due to the variability and nature
of the constituents concerning the year’s season. Therefore,
plant seasonal and circadian variations significantly contribute
to essential oils’ chemical composition.[14–16]
The present work aimed to analyze the circadian and
seasonal variability of a Dizygostemon riparius population
sampled in São Benedito do Rio Preto, MA, Brazil, based on the
analysis of yield and composition of their essential oils, using
chemometric tools.
Results and Discussion
Seasonal effect on essential oil yield of D. riparius
The area of occurrence of D. riparius in São Benedito do Rio
Preto (Maranhão, Brazil) belongs to Bioma Cerrado, with
basically two seasons: rainy from January to May, when more
than 90 % of the rains fall, and dry from June to December, with
the almost total absence of rain. Climatic factors such as
insolation, precipitation, temperature, and relative humidity
were monitored from August 2019 to July 2020 to evaluate
their influence on the production and composition of the
essential oils of D. riparius. The insolation values varied between
Figure 1. Relationship between climatic factors and essential oils yield of D. riparius in the seasonal study.
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134.4 h (March) and 303.8 h (August), the monthly precipitation
from almost zero (July, August, September, and October) to
442.4 mm (April), the temperature from 26.3 °C (January) to
29.0 °C (November), and the relative air humidity from 64.0 %
(August) to 89.6 % (March). The dry period in the region was
comprised of the months from August to December 2019 and
June and July 2020, with an average precipitation of 37.8 �
53.9 mm, and the rainy period occurred from January to
May 2020, with an average precipitation of 147.2 � 11.1 mm
(Figure 1). Indeed, May 2020 was considered a transition period
between these two seasons, with an average precipitation of
190.1 + 0.0 mm. In the seasonal study, the essential oil yields of
D. riparius ranged from 1.4 % (June) to 3.8 % (January),
averaging 2.8 � 0.7 % for the annual period (Figure 1). Essential
oil yield showed no significant difference during the dry (2.6 �
0.7 %) and rainy (3.1 � 0.5 %) seasons (Tukey test, p < 0.05).
Regarding the climatic factors, no significant correlation
(p > 0.05) was observed between the essential oil content and
temperature, relative humidity, insolation, and precipitation
(see Table 1). Also, the main volatile constituents that showed
significant correlation with climatic factors were: α-fenchene
with temperature (r = 0.88) and relative humidity (r = 0.79),
endo-fenchol with temperature (r = 0.71) and relative humidity
(r = 0.66), and caryophyllene oxide with temperature (r =
0.79) and relative humidity (r = 0.67) (see Table 1 and Table 2).
The major volatile constituents that did not show a significant
correlation with climatic factors were endo-fenchyl acetate and
(E)-caryophyllene. In the same way, the classes of compounds
identified in the D. riparius essential oil did not correlate with
climatic factors.
Seasonal and circadian effect on essential oil composition of
D. riparius
Table 2 exhibits forty-six chemical constituents identified by GC
and GC/MS in the essential oils of the seasonal study of D.
riparius, in ascending order of their respective retention indices,
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Table 1. Correlation between yield, principal constituents, and classes of

compounds from D. riparius oil vs. climatic factors.
Oil data/Climatic Temperature Humidity Insolation Precipitation
factors

Oil yield 0.12 0.16 0.07 0.22

α-Fenchene 0.88* 0.79* 0.52 0.55
endo-Fenchol 0.71* 0.66* 0.42 0.45
endo-Fenchyl 0.49 0.39 0.25 0.27
acetate
(E)-Caryophyllene 0.45 0.46 0.19 0.14
Caryophyllene 0.79* 0.67* 0.41 0.40
oxide
Monoterpene hy- 0.23 0.27 0.38 0.32
drocarbons
Oxygenated mono- 0.17 0.02 0.01 0.05
terpenes
Figure 3. HCA analysis of main volatiles from D. riparius oils in the seasonal
Sesquiterpene hy- 0.26 0.43 0.38 0.42
study.
drocarbons
Oxygenated ses- 0.12 0.28 0.50 0.43
quiterpenes

*Significance correlation (p > 0.05).
comprising about 96.3 % of the analyzed oils. The predominant
classes of compounds in the oil samples were oxygenated
monoterpenes (69.3 to 77.7 %), followed by monoterpene
hydrocarbons (6.3 to 20.8 %), sesquiterpene hydrocarbons (3.1
to 8.3 %), and oxygenated sesquiterpenes (1.6 to 6.8 %). The
primary chemical constituents (above 2.0 %) identified in oils
were endo-fenchyl acetate (30.5 to 41.3 %), endo-fenchol (31.3
to 37.4 %), (E)-caryophyllene (2.8 to 7.6 %), α-fenchene (3.3 to
6.5 %), p-cymene (0.9 to 4.5 %), caryophyllene oxide (1.0 to
2.7 %), limonene (1,1 to 2.0 %), and γ-terpinene (0.2 to 2.0 %).
In the same way, Table 3 lists the chemical constituents
(> 2.0 %) found in the oils of the circadian study from
November 2019 (dry season) to May 2020 (rainy/transition
season) at 6 am, 12 pm, and 6 pm. The major components were
endo-fenchyl acetate (36.0 to 40.7 %), endo-fenchol (31.7 to
36.6 %), α-fenchene (3.1 to 6.1 %), (E)-caryophyllene (1.9 to
6.1 %), p-cymene (2.3 to 4.1 %), and caryophyllene oxide (1.3 to
2.3 %). The chemical structures of the main compounds
identified in the essential oils of D. riparius are shown in
Figure 2.
Multivariate analysis of D. riparius oils in the seasonal study
Hierarchical cluster analysis (HCA) and principal component
analysis (PCA) were plotted with the constituents above 2 % of
the D. riparius oils. Applying the HCA analysis, the dendrogram
shown in Figure 3 presents the volatiles in three groups with
zero similarity. Group I comprised oils from March, June, July,
August, and September. Group II included the oils of January,

Figure 2. Primary constituents identified in the essential oils of D. riparius.

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Table 2. Yields and constituents of the essential oils of Dizygostemon riparius relating the seasonal study.
Oil Yields (%) 3.7 3.3 2.9 3.0 2.6 3.8 2.9 3.1 2.7 2.2 1.4 2.0
Oil Constituents (%) RICalc RILit Aug Set Out Nov Dec Jan Fev Mar Apr May Jun Jul

2E-Hexenal 846 846a 0.1 0.1 0.1 0.1

3Z-Hexenol 849 850a 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
2E-Hexenol 858 854a 0.1 0.1 0.1 0.1
Santolina triene 906 906a 1.2 1.5 0.7 0.9 0.6 0.8 0.5 1.4 0.9 0.7 1.4 1.3
α-Thujene 925 924a 0.9 1.9 0.9 1.3 0.8 0.8 0.1 1.1 0.9 0.6 1.8 0.2
α-Pinene 932 932a 0.3 0.5 0.2 0.3 0.2 0.2 0.1 0.4 0.3 0.2 0.4 0.3
α-Fenchene 945 945a 6.0 6.1 3.8 4.6 3.6 4.5 3.6 6.5 4.9 3.3 6.3 6.2
Camphene 950 946a 0.2 0.2
6-methyl-Heptan-2-ol 955 958a 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Sabinene 972 969a 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
β-Pinene 976 974a 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Myrcene 989 988a 0.1 0.1 0.1 0.1
α-Terpinene 1016 1014a 0.4 0.7 0.4 0.4 0.4 0.4 0.5 0.4 0.5 0.4 0.9 0.2
p-Cymene 1023 1020a 3.2 4.5 3.6 4.2 3.4 2.8 0.9 3.2 3.1 2.3 4.2 0.7
Limonene 1027 1024a 1.6 2.0 1.3 1.6 1.3 1.3 1.6 1.5 1.1 1.7 1.6
Z-β-Ocimene 1035 1032a 0.5 0.3 0.1 0.3 0.1 0.2 0.2 0.4 0.2 0.3 0.8 1.0
Benzene acetaldehyde 1040 1036a 0.1 0.1 0.2 0.1 0.1
E-β-Ocimene 1045 1044a 0.1 0.1 0.1 0.1 0.1
γ-Terpinene 1057 1054a 1.0 2.0 1.7 1.5 1.6 1.4 0.2 1.2 1.6 1.1 1.8 0.5
Terpinolene 1087 1086a 0.9 0.7 0.5 0.7 0.6 1.0
trans-Sabinene hydrate 1099 1098a 0.2 0.3 0.1 0.3 0.1 0.1 0.1 0.2 0.1 0.1 0.4 0.2
endo-Fenchol (α-Fenchol) 1119 1114a 35.9 32.7 32.2 34.5 31.9 31.8 31.6 35.8 31.3 34.3 37.4 36.7
trans-p-mentha-2,8-dien-1-ol 1120 1119a 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
trans-Pinene hydrate 1122 1119a 0.1 0.1 0.1
Terpinen-4-ol 1150 1144 0.5 0.6 0.6 0.7 0.5 0.5 0.6 0.5 0.4
p-Cymen-8-ol 1182 1179a 0.2 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2
α-Terpineol 1190 1186a 0.2 0.2 0.3 0.2 0.3 0.2 0.2 0.3 0.2 0.2 0.2
endo-Fenchyl acetate 1224 1218a 37.5 34.1 40.7 33.1 41.3 39.7 42.1 36.7 38.7 37.8 30.5 40.1
Nerol 1227 1227a 0.3 0.2 0.1 0.1 0.1 0.1 0.1 0.3 0.1 0.1 0.2 0.3
Ascaridol 1237 1234a 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.2
cis-Piperitone epoxide 1253 1250a 0.1 0.1 0.1
trans-Piperitone epoxide 1255 1252a 0.1 0.1 0.1 0.1 0.1 0.1
Geraniol 1268 1264a 0.1 0.1 0.1
Thymol 1294 1289a 0.1 0.1 0.1 0.1 0.1 0.1
Carvacrol 1300 1298a 0.1 0.1 0.1 0.1 0.1
Isoascaridol 1308 1306b 0.1 0.1 0.1 0.1 0.2
(E)-Caryophyllene 1419 1417a 2.8 3.5 3.5 5.9 3.6 5.7 6.2 2.9 5.4 7.6 4.0 4.1
α-Humulene 1451 1452a 0.3 0.3 0.3 0.6 0.3 0.5 0.5 0.3 0.5 0.7 0.4 0.4
E-Nerolidol 1562 1561a 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Caryophyllene oxide 1582 1582a 1.4 1.6 2.0 2.6 2.1 2.0 2.7 1.4 2.0 2.2 1.4 1.0
Humulene epoxide II 1608 1608a 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Caryophylla-4(12),8(13)-dien-5α-ol 1632 1639a 0.2 0.2 0.3 0.2 0.3 0.3 0.7 0.2 0.3 0.2 0.1 0.1
Caryophylla-4(12),8(13)-dien-5β-ol 1635 1639a 0.6 0.5 0.9 0.7 0.9 0.8 2.2 0.6 0.8 0.7 0.3 0.3
Pogostol 1653 1651a 0.2 0.1 0.2 0.3 0.2 0.2 0.4 0.2 0.2 0.3 0.2
14-hydroxy-9-epi-E-Caryophyllene 1670 1668a 0.2 0.2 0.2 0.6 0.2 0.2 0.1
Abietatriene 2054 2055a 0.1 0.2 0.1 0.2 0.1 0.1 0.3 0.1 0.1 0.2 0.1 0.1
Monoterpene hydrocarbons 15.4 20.8 12.8 16.0 12.1 13.3 6.3 17.2 14.1 10.8 20.7 12.3
Oxygenated monoterpenes 75.0 69.3 74.3 69.7 75.3 73.1 74.0 74.1 71.8 73.3 70.0 77.7
Sesquiterpene hydrocarbons 3.1 3.8 3.8 6.5 3.9 6.2 6.7 3.2 5.9 8.3 4.4 4.5
Oxygenated sesquiterpenes 2.8 2.6 3.6 4.0 3.8 3.7 6.8 2.8 3.5 3.8 2.2 1.6
Others 0.6 0.6 0.2 0.6 0.2 0.2 0.3 0.5 0.3 0.3 0.4 0.6
Total (%) 96.9 97.1 94.7 96.8 95.3 96.5 94.1 97.8 95.6 96.5 97.7 96.7

RIcalc. = calculated retention time (on Rxi-5 ms column); RILit = literature retention time; a = Adams (2007);[x] b = Mondello (2011);[y] bold values = main
constituents.

April, May, October, November, and December. Group III
exhibited only the oil of February.
PCA analysis clarified 90.54 % of the data variability (see
Figure 4). The PCA1 component explained 50.54 % presenting a
negative correlation with endo-fenchyl acetate (r = 4.18), (E)-
caryophyllene (r = 2.98), and caryophyllene oxide (r = 3.40).
The PCA2 component explained 24.91 % exhibiting a negative
correlation with α-fenchene (r = 2.54), endo-fenchol (α-fen-
chol) (r = 2.72), and endo-fenchyl acetate (r = 1.58). The
PCA3 component explained 14.89 % of the data showing a
negative correlation with p-cymene (r = 0.73), limonene (r =
0.61), γ-terpinene (r = 1.08), and endo-fenchyl acetate (r =
1.26). As for HCA, the PCA analysis confirmed the formation of
three distinct groups. Group I was characterized by the highest
content of endo-fenchyl acetate (30.5 to 40.1 %), endo-fenchol
(32,7 to 37.4 %), α-fenchene (6.0 to 6.5 %), and p-cymene (0.7 to
4.5 %). Group II was characterized by the highest content of
endo-fenchyl acetate (33.1 to 41.3 %), endo-fenchol (31.3 to
34.5 %), (E)-caryophyllene (3.3 to 7.6 %), and α-fenchene (3.3 to
4.9 %). Group III was formed by the highest content of endo-

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Table 3. Yields and constituents of the essential oils of Dizygostemon riparius relating the circadian study.
Month/Time November 2019 May 2020
Oil Constituents (%) RICalc RILit 6am 12am 6 pm 6am 12am 6 pm
a
Santolinatriene 906 906 0.6 0.7 0.6 1.3 1.3 0.9
α-Thujene 925 924a 0.8 1.0 0.8 0.9 0.7 0.7
α-Pinene 932 932a 0.2 0.2 0.2 0.3 0.3 0.3
α-Fenchene 945 945a 3.9 4.2 3.1 6.1 5.7 4.8
Camphene 950 946a 0.1 0.1 0.1 0.3 0.1 0.1
Sabinene 972 969a 0.1 0.1 0.1 0.1 0.1 0.1
β-Pinene 976 974a 0.1 0.1 0.1 0.1 0.1 0.1
α-Terpinene 1016 1014a 0.3 0.4 0.4 0.4 0.3 0.4
p-Cymene 1023 1020a 3.1 4.1 3.6 2.3 2.3 2.6
Limonene 1027 1024a 1.1 1.5 1.2 1.2 1.1 1.2
Z-β-Ocimene 1035 1032a 0.2 0.3 0.3 0.2 0.3
E-β-Ocimene 1045 1044a 0.3 0.1
γ-Terpinene 1057 1054a 1.0 1.4 1.3 1.3 1.2 1.4
Terpinolene 1087 1086a 0.9 0.7 0.5
cis-Sabinene hydrate 1065 1065a 0.1 0.1 0.1 0.1 0.1 0.1
trans-Sabinene hydrate 1099 1098a 0.5 0.3 0.2 0.1 0.2 0.1
endo-Fenchol (α-Fenchol) 1119 1114a 35.7 35.3 34.7 31.7 36.6 34.4
trans-p-mentha-2,8-dien-1-ol 1120 1119a 0.1 0.1 0.1 0.1 0.1
trans-Pinene hydrate 1122 1119a 0.1 0.1 0.1 0.1
Terpinen-4-ol 1150 1144 0.4 0.5 0.3 0.4
p-Cymen-8-ol 1182 1179a 0.2 0.2 0.2 0.1 0.1 0.2
α-Terpineol 1190 1186a 0.2 0.2 0.3 0.2 0.2 0.2
endo-Fenchyl acetate 1224 1218a 40.7 36.0 37.7 39.8 40.2 39.3
Nerol 1227 1227a 0.1 0.1 0.1 0.1 0.1 0.1
Ascaridol 1237 1234a 0.1 0.1 0.1 0.1 0.2 0.1
cis-Piperitone epoxide 1253 1250a 0.1 0.1 0.1 0.1
trans-Piperitone epoxide 1255 1252a 0.1 0.1 0.1
Thymol 1294 1289a 0.1 0.1 0.1 0.1 0.1 0.1
Carvacrol 1300 1298a 0.1 0.1 0.1 0.1 0.1 0.1
Isoascaridol 1308 1306b 0.1 0.1 0.1 0.1 0.1 0.1
(E)-Caryophyllene 1419 1417a 4.8 4.7 6.1 2.9 1.9 3.2
α-Humulene 1451 1452a 0.4 0.4 0.5 0.2 0.1 0.3
E-Nerolidol 1562 1561a 0.1 0.1 0.1 0.1
Caryophyllene oxide 1582 1582a 1.8 2.1 2.3 1.4 1.3 1.6
Humulene epoxide II 1608 1608a 0.1 0.1 0.1 0.1 0.1 0.1
Caryophylla-4(12),8(13)-dien-5α-ol 1632 1639a 0.2 0.2 0.2 0.3 0.2 0.2
Caryophylla-4(12),8(13)-dien-5β-ol 1635 1639a 0.5 0.6 0.7 0.8 0.6 0.7
Pogostol 1653 1651a 0.2 0.3 0.3 0.3 0.2 0.3
14-hydroxy-9-epi-E-Caryophyllene 1670 1668a 0.1 0.2 0.2 0.1 0.2
Eremophilone 1737 1734a 0.1 0.7 0.2 0.9
Abietatriene 2054 2055a 0.1 0.1 0.2 0.1 0.1
Monoterpene hydrocarbons 11.5 15.0 11.8 15.3 13.5 13.4
Oxygenated monoterpenes 78.5 73.4 73.9 73.1 78.3 75.5
Sesquiterpene hydrocarbons 5.2 5.1 6.6 3.1 2.0 3.5
Oxygenated sesquiterpenes 3.1 3.4 3.9 3.9 2.7 4.0
Diterpenes 0.1 0.1 0.2 0.1 0.1
Total (%) 98.4 97.0 96.4 95.5 96.5 96.5
a [x]
RIcalc. = calculated retention time (on Rxi-5 ms column); RILit = literature retention time; = Adams (2007); bold values = main constituents.

fenchyl acetate (42.1 %), endo-fenchol (31.6 %), and (E)-caryo-
phyllene (6.2 %).
The analysis of the mean contents and standard deviations
of the constituents present in the oils of D. riparius in the
seasonal study (see Figure 5) showed that the formed Group I
was statistically different (Tukey, p < 0.05) from Groups II and III,
according to the contents of α-fenchene (I = 6.2 � 0.2 %; II =
4.1 � 0.6 %; III = 2.7 � 0.0 %), endo-fenchol (I = 35.7 � 1.7 %; II =
32.7 � 1.4 %; III = 31.6 � 0.0 %), and (E)-caryophyllene (I = 3.5 �
0.6 %; II = 5.3 � 1.5 %; III = 6.2 � 0.0 %); that Group II differed from
the other groups, according to the caryophyllene oxide
contents (I = 1.4 � 0.2 %; II = 2.1 � 0.2 %; III = 2.8 � 0.0 %); and in
addition, Group III was distinguished from the other groups by
the levels of p-cymene (I = 3.7 � 1.4 %; II = 3.2 � 0.5 %; III = 2.7 �
0.0 %) and endo-fenchyl acetate (I = 35.8 � 3.6 %; II = 38.5 �
3.0 %; III = 2.7 � 0.0).
Multivariate analysis of D. riparius oils in the circadian study
The chemical variability of D. riparius oils, sampled during the
circadian period, was also evaluated by multivariate statistical
analysis (HCA and PCA) using constituents’ values greater than
1.0%.
In the HCA, the volatile components were plotted in two
groups with zero similarity (see Figure 6). Group I comprised
the oils from the botanical material sampled in November 2019

Chem. Biodiversity 2023, 20, e202300864 (5 of 9) © 2023 Wiley-VHCA AG, Zurich, Switzerland
 Research Article
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Figure 4. PCA of main volatile compounds from D. riparius oils in the
seasonal study.
Figure 5. The analysis of the mean contents and standard deviations of the
oil constituents of D. riparius in the seasonal study.
at 6 am, 12 am, and 6 pm, and Group II included the oils from
May 2020 at 6 am, 12 am, and 6 pm.
The PCA accounted for 91.81 % of the data variability (see
Figure 7). The PCA1 component explained 56.81 %, showing a
negative correlation with santolina triene (r = 1.13), α-fen-
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Figure 6. HCA of main volatile compounds from D. riparius oils in the
circadian study.
Figure 7. PCA of main volatile compounds from D. riparius oils in the
circadian study.
chene (r = 1.11), and endo-fenchyl acetate (r = 1.12). The
PCA2 component accounted for 25.54 %, exhibiting a negative
correlation with p-cymene (r = 0.02), endo-fenchol (α-fenchol)
(r = 1.11), endo-fenchyl acetate (r = 0.88), (E)-caryophyllene
(r = 0.61), and caryophyllene oxide (r = 0.32). The PCA3
component explained 9.46 % of the data, showing a negative
correlation with α-thujene (r = 0.56), endo-fenchyl acetate (r =
0.46), (E)-caryophyllene (r = 0.58), and caryophyllene oxide
(r = 0.15). The PCA confirmed the formation of two distinct
groups similar to the HCA.
The analysis of the mean contents and standard deviations
of the constituents present in the D. riparius oils in the circadian
study (see Figure 8) showed that the formed Group I was
© 2023 Wiley-VHCA AG, Zurich, Switzerland
 Research Article
doi.org/10.1002/cbdv.202300864
Figure 8. The analysis of the mean contents and standard deviations of the
oil constituents of D. riparius in the circadian study.
statistically different (Tukey, p < 0.05) from Group II by the
contents of santolina triene (I = 0.6 � 0.1 %; II = 1.2 � 0.2 %), α-
fenchene (I = 3.7 � 0.6 %; II = 5.5 � 0.7), p-cymene (I = 3.6 � 0.5 %;
II = 2.4 � 0.2), (E)-caryophyllene (I = 5.2 � 0.8 %; II = 2.7 � 0.7 %),
endo-fenchyl acetate (I = 38.1 � 2.4 %; II = 39.8 � 0.5 %), and
endo-fenchol (α-Fenchol) (I = 35.2 � 0.5 %; II = 34.2 � 2.5 %).
The yield and composition of the essential oil of Dizygoste-
mon riparius were previously reported, with an oil content of
1.5 % and exhibiting endo-fenchyl acetate (48.0 %), endo-fenchol
(35.0 %), (E)-caryophyllene (6.8 %), and caryophyllene oxide
(3.3 %) as its main constituents.[10] Newly, the ethyl acetate and
methanol extracts from D. riparius showed the presence of
polymethoxyflavones and coumarins, such as isorhamnetin-3-
galactoside-7-rhamnoside, 3’,5-dihydroxy-4’,6,7-trimeth-
oxyflavone, and 5,7-dihydroxy-3-(3-hydroxy-4,5-dimeth-
oxyphenyl)-6-methoxy-4-benzopyrone.[17]
The oil of D. riparius has a refreshing and pleasant scent,
similar to that of lemon and camphor, due to the presence of
endo-fenchyl acetate and endo-fenchol, with a percentual of
83 %. These two C10-skeletal oxygenated monoterpenes were
arranged following their biosynthetic pathway, in that case, a
fenchane of the bicyclic compound class. The literature
previously described other essential oils presenting a high
percentage of endo-fenchyl acetate and endo-fenchol. The
essential oils of Alpinia galanga (L.) Willd., A. calcarata Rosc.,
and A. speciosa K. Schum. [syn. A. zerumbet (Pers.) Burtt & Smith]
(Zingiberaceae) have shown a significant percentage of endo-
fenchyl acetate, varying from 40 % to 54 %.[18–21] Also, the volatile
concentrate from inflorescences and shoots of Chaerophyllum
hirsutum L. (Apiaceae) exhibited significant endo-fenchyl
acetate and endo-fenchol content.[22] Similarly, essential oils of
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Tarchonanthus camphoratus L. (Asteraceae) and Ocimum for-
skolei Benth. (Lamiaceae) have shown high content of endo-
fenchol, with 21 % and 31 %, respectively.[23,24]
Conclusions
The main constituents (above 2.0 %) identified in D. riparius oils
of seasonal and circadian studies were endo-fenchyl acetate,
endo-fenchol, (E)-caryophyllene, α-fenchene, p-cymene, and
caryophyllene oxide. Regarding the climatic factors, no signifi-
cant correlation was observed between the oil content with the
temperature, relative humidity, insolation, and precipitation. In
contrast, for the main constituents, observed correlation of α-
fenchene with temperature and relative humidity, endo-fenchol
with temperature and relative humidity, and caryophyllene
oxide with temperature and relative humidity. The major
constituents that did not correlate with climatic factors were
endo-fenchyl acetate and (E)-caryophyllene. In the same way,
the classes of compounds identified in the D. riparius oils did
not correlate with climatic factors. The dendrogram formed by
HCA exhibited three groups: Group I with March, June, July,
August, and September; Group II with January, April, May,
October, November, and December; and Group III with Febru-
ary. The PCA also confirmed the formation of three distinct
groups: Group I was characterized by the highest content of
endo-fenchyl acetate, endo-fenchol, α-fenchene, and p-cymene;
Group II by endo-fenchyl acetate, endo-fenchol, (E)-caryophyl-
lene, and α-fenchene; and Group III by endo-fenchyl acetate,
endo-fenchol, and (E)-caryophyllene. Thus, the present study
contributes to the knowledge of the chemical variability of D.
riparius oils.
Experimental Section
Plant material and climatic data
The botanical material (whole plant, except root) of a Dizygostemon
riparius population was sampled in the Rio Preto banks (geographic
coordinates: 03°19’27.9’’ S; 43°31’02.6’’ W), city of São Benedito do
Rio Preto, State of Maranhão, Brazil, in the period from August 2019
to July 2020. The plant was collected during the reproductive adult
phase, at permanent flowering, following the Brazilian laws for the
protection of biodiversity (SisGen n° A0B272D). For the seasonal
study sampling was done at 12:00 pm. For the circadian study, the
plant was collected in November (dry season) and May (rainy
season) at 6:00 am, 9:00 am, 12:00 pm, 3:00 pm, and 6:00 pm.
Dizygostemon riparius is an herbaceous plant, about 50 cm tall, with
long sepals or slightly longer sepals than corolla tube, posterior
sepal 1.5 times as wide as any of the four other, ovary glandular
villous, large leaves, 1.5–8.0 × 0.5–4.0 cm, stems sparsely to densely
tomentose, and corolla tube with 5.2–6.2 mm long, whose bota-
nical data were recently described.[9] The climatic parameters
(insolation, relative air humidity, rainfall precipitation, and temper-
ature) of the mentioned area were obtained for each month from
the website of the Instituto Nacional de Meteorologia (INMET,
http://www.inmet.gov.br/portal/, accessed on 12 June 2022), of the
Brazilian Government.[25] The meteorological data were recorded
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 Research Article
doi.org/10.1002/cbdv.202300864
through the automatic station A-206 (Vaisala system, model MAWS
301) in Chapadinha, Maranhão state, Brazil.
Essential oil distillation
The aerial part of the plant (100 g) was subjected to hydro-
distillation using a Clevenger-type glass apparatus (3 h). Before,
separately, the plant samples were dried in air at room temperature
for three days and then cut with scissors into small pieces. After the
extraction (in duplicate), the oils were dried over anhydrous sodium
sulfate, and their yields were calculated based on the plant dry
weight (v/w). The moisture content of the samples was calculated
using an infrared moisture balance for water loss measurement.
Oil composition analysis
Analysis of the oils was performed on a GCMS-QP2010 Ultra system
(Shimadzu Corporation, Japan), equipped with an AOC-20i auto-
injector and the GCMS-Solution software containing the NIST and
FFNSC 2 libraries.[26,27] A Rxi-5 ms (30m × 0.25 mm; 0.25 μm film
thickness) silica capillary column (Restek Corporation, Bellefonte,
PA, USA) was used. The conditions of analysis were: injector
temperature of 250 °C; Oven temperature programming of 60–
240 °C (3 °C min 1); Helium as the carrier gas, adjusted to a linear
velocity of 32 cm s 1 (1.0 mL/min); split mode injection for 1.0 μL of
the sample (oil 5.0 μL: hexane 500 μL); split ratio 1 : 20; ionization by
electronic impact at 70 eV; ionization source and transfer line
temperatures of 200 and 250 °C, respectively. The mass spectra
were obtained by automatic scanning every 0.3 s, with mass
fragments in 35–400 m/z. The retention index was calculated for all
volatile components using a homologous series of C8-C40 n-
alkanes (Sigma-Aldrich, USA), according to Van Den Dool and
Kratz’s linear equation.[28] The quantitative data regarding the
volatile constituents were obtained by peak-area normalization
using a GC 2010 Series, coupled with FID Detector, which operated
under similar conditions of the GC/MS system. The components
were identified by comparing their retention indices and mass
spectra (molecular mass and fragmentation pattern) with those
existing in the GCMS-Solution system libraries.[26,27]
Statistical analysis
Statistical significance was evaluated by a Tukey test (p < 0.05), and
the Pearson correlation coefficients (r) were calculated to determine
the relationship among the parameters analyzed (insolation,
relative air humidity, rainfall precipitation, and temperature), using
the software GraphPad Prism, version 5.0. The principal component
analysis (PCA) (OriginPro trial version, OriginLab Corporation, North-
ampton, MA, USA) was applied to verify the interrelation of the oils’
components (> 2 %) in seasonal and circadian studies. The
hierarchical cluster analysis (HCA), considering the Euclidean
distance and complete linkage, was used to verify the similarity of
the oil samples based on the distribution of the constituents
selected in the previous PCA analysis.
Author Contributions
F. S. Galvão, P. L. B. Figueiredo, P. V. L. Santos – carrying out the
experiments, data analysis, writing, and reviewing; O. S.
Monteiro – reviewing; C. Q. da Rocha, J. G. S. Maia – data
analysis, writing, and reviewing.
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Acknowledgements
The authors are grateful to the Coordenação de Aperfeiçoa-
mento de Pessoal de Nível Superior (CAPES) of the Brazilian
Government for providing scholarships to J.G.S.M and P.V.L.S, as
well to the family of Ubiraci and Graça, residents of São
Benedito do Rio Preto, MA, Brazil, for their support in the
systematic collection of plants.
Conflict of Interests
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
Keywords: Dizygostemon riparius · new flavoring herbal plant ·
essential oils · endo-fenchyl acetate and endo-fenchol · circadian
and seasonal study
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Manuscript received: June 13, 2023
Accepted manuscript online: July 17, 2023
Version of record online: July 31, 2023
© 2023 Wiley-VHCA AG, Zurich, Switzerland