Title: Antinociceptive activity and compounds from the aqueous extract

of Melampodium divaricatum

Authors: Araceli Pérez-Vásquez, Sofía Padilla-Mayne, Ana Laura

Martínez, José S Calderón, Martha Lidia Macias-Rubalcava,
Rafael Torres-Colín, Manuel Rangel-Grimaldo, and Rachel
Mata

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To be cited as: Chem. Biodiversity 10.1002/cbdv.202100369

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Chemistry & Biodiversity 10.1002/cbdv.202100369

Chem. Biodiversity

Antinociceptive activity and compounds from the aqueous extract of

Melampodium divaricatum

Araceli Pérez-Vásquez,a Sofía Padilla-Mayne,a Ana Laura Martínez,a José S. Calderón,b Martha Macías-
Ruvalcaba,b Rafael Torres-Colín,c Manuel Rangel-Grimaldo,a and Rachel Mata*,a
a
Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, México. e-mail: rachel@unam.mx
b
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, México.
c
Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad de México 04510, México.

Accepted Manuscript
A decoction prepared from the aerial parts of Melampodium divaricatum showed antinociceptive and antihyperalgesic responses when tested
in the formalin model in mice. From the CH2Cl2 fraction of the decoction, two non-previously reported secondary metabolites, 3-O-β-D-
glucopyranosyl-16α-hydroxy-ent-kaurane (1) and melampodiamide (2) [(2′R*,4′Z)-2′-hydroxy-N-[(2S*,3S*,4R*)-1,3,4-trihydroxyoctadec-2-
yl]tetracos-4-enamide] were separated and characterized by spectroscopic, spectrometric, and computational techniques. The flavonoids
isoquercitrin and hyperoside, which possessed noted antinociceptive properties, were obtained from the active EtOAc fraction of the
decoction. The chemical composition of the essential oil of the plant was also analyzed by gas chromatography-mass spectrometry. The major
constituents were (E)-caryophyllene, germacrene D, β-elemene, δ-elemene, γ-patchoulene, and 7-epi-α-selinene. Headspace solid-phase
microextraction analysis detected (E)-caryophyllene as the main volatile compound of the plant.

Keywords: Melampodium divaricatum • Asteraceae • essential oil • antinociceptive activity • antihyperalgesic action

Introduction

Melampodium divaricatum (Rich. ex Rich.) DC. (Asteraceae) is an annual aromatic herb distributed from the South of the United States of
America, through Mexico, Central America, Colombia, to Brazil as well as the Caribbean and Africa.[1,2] In Mexico, a decoction prepared from the
aerial parts of M. divaricatum is used as a traditional remedy for treating dysentery, fever,[3] embolism,[4] infections, pain,[5] emesis, and stomach
complaints. In Guatemala, the leaves are taken to treat stomach pain, and the whole plant is used against influenza.[6] In Brazil, the leaves are
employed as diuretics and to alleviate malaria, rheumatism, joint or muscle pain, vertigo, and stomachache.[7,8] Earlier biological testing showed
the antimutagenic[9] and repellent action against Atta cephalotes[10] of extracts prepared from the aerial parts of the plant.[10] The essential oil, on
the other hand, demonstrated anticariogenic and antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus
mirabilis, Pseudomonas aeruginosa, Shigella sonnei, Serratia marcescens,[11] Streptococcus mitis, and Streptococcus mutans.[12] M. divaricatum has
been previously investigated phytochemically. These studies resulted in the isolation and characterization of thymol type of
monoterpenoids,[13-15] sesquiterpenes, a diterpenoid (kolavenol),[10] flavonoids (kaempferol and two quercetin glycosides),[16] and coumarins.[17]
In addition, the chemical analysis of the essential oil of the Brazilian species revealed the presence of three major components, (E)-
caryophyllene (56.0 %), germacrene D (12.7 %), and bicyclogermacrene (9.2 %).[12]

Continuing with our systematic research to determine the preclinical efficacy of selected Mexican Medicinal plants, [18] this work aimed: (i)
To assess the potential antinociceptive effect of an aqueous extract of M. divaricatum to provide scientific support to its traditional use to treat
pain. (ii) To establish some of the compounds responsible for the pharmacological action. (iii) To find out the volatile composition of this
aromatic herb. Altogether, these results will be helpful to promote the rational use of M. divaricatum.

Results and Discussion

Pharmacological Effects

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Acute toxicity testing in rodents is usually the first stage during the evaluation of the efficacy of plant preparations. The data from an
acute toxicity trial is useful for choosing suitable doses for pharmacological investigations and detecting initial signs of toxicity. In this work,
acute toxicity was examined in mice according to the Lorke procedure,[19] which gives trustworthy outcomes using lowest number of animals.
Administration of a dried decoction made from the aerial parts of M. divaricatum did not cause conduct alterations, macroscopic tissue

damage, or weight loss for the period of 14-day observation; the likely LD50 was higher than 5 g/kg. Thus, the traditional preparation of M.
divaricatum devoid of acute toxic effects for mice, according to the Lorke method (Table S1, Supporting Information).
Next, the potential antinociceptive effect of the traditional preparation (decoction) of M. divaricatum was determined using the formalin
test, a consistent model of nociception susceptible to many types of analgesic drugs.[18, 20] The harmful stimulus was an injection of dilute
formalin (2 % in saline) put under the skin of the dorsal side of the right hind paw. The response analyzed was the extent of time the animals
expended licking the inoculated paw. In this test, two periods of licking action are identified, an initial phase enduring 10 min and a later phase
happening from 10 to 30 min following the injection of formalin.[20] The early phase is caused largely by peripheral C-fiber stimulation

Accepted Manuscript
(neurogenic activity) which are directly activated by TRPA1 and TRPV1 ionic channels in the presence of formalin; whereas the second phase is
due to a combination of an inflammatory response in the peripheral tissue and functional changes in the dorsal horn of the spinal cord; this
pain can be inhibited by anti-inflammatory drugs.[21] The positive control was diclofenac (DIC, 31.6 mg/kg). Mice treated with the decoction
(31.6, 100 or 316.2 mg/kg, p.o.) showed a significant reduction of the response due to the chemical stimulation during both phases of the test
(Figures 1A-1C) as compared with the vehicle (VEH) and the diclofenac (DIC). Though the extract was active in both stages, the most important
antinociceptive action was achieved during the inflammatory phase (10−30 min, Figure 1C). Shibata et al.,[22] described that the second phase
of the formalin test takes place when inflammatory mediators, such as bradykinin and prostaglandins, are released. Furthermore, it has been
studied that central sensitization is mediated by the activation of glutamate and substance P receptors.[23] The overall effect suggested that the
aqueous extract of M. divaricatum exhibited both neurogenic and inflammatory antinociceptive effects. It is important to point out that the
doses selected for testing followed a pharmacological conventional logarithmic scheme. [18, 20]

Figure 1. Antinociceptive effect of the aqueous extract of Melampodium divaricatum at 31.6, 100, and 316.2 mg/kg (p.o.), in the formalin test in mice. (A) Temporal

course curves. (B) AUC from the time course curve of phase 1. (C) AUC from the time course curve of phase 2. VEH: vehicle (saline solution). DIC: diclofenac (31.6

mg/kg, p.o.). Each measurement represented as the mean ± SEM with 6 mice per group. Significantly different from VEH group (*p < 0.05, **p < 0.01, and ***p <

0.001) was determined by ANOVA followed by Dunnett’s test.

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The hyperalgesic action of the aqueous extract was also analyzed in hyperglycemic mice [NAD (nicotinamide)/STZ (streptozotocin),
50/130 mg/kg]). After two-weeks of hyperglycemia induction, mice developed chemical hyperalgesia.[24] Under these conditions, administration
of 1 % formalin solution to hyperglycemic mice enhances the nociceptive response in comparison to healthy mice, showing a decreased pain
threshold due to chemical stimulation.[25] The aqueous extract of M. divaricatum reduced the hyperalgesic response in hyperglycemic mice in

both phases of the test (Figure 2A). During the first phase (Figure 2B), the effect was dose-dependent and comparable to gabapentin (GBP)
employed as positive control. Moreover, this effect was also observed in the second phase at all doses administered (Figure 2C). Thus, M.
divaricatum shows also promise for the treatment of neuropathic pain.

Accepted Manuscript
Figure 2. Antihyperalgesic effect of the aqueous extract of Melampodium divaricatum at 31.6, 100, and 316.2 mg/kg (p.o.), in the formalin test in hyperglycemic

mice. (A) Temporal course curves. (B) AUC from the time course curve of phase 1. (C) AUC from the time course curve of phase 2. VEH: vehicle (saline solution). GBP:

gabapentin (31.6 mg/kg, p.o.). Each measurement represented as the mean ± SEM with 6 mice per group. Significantly different from VEH group (*p < 0.05, **p <
0.01, and ***p < 0.001) was determined by ANOVA followed by Dunnett’s test.

Isolation and Characterization of Compounds 3 and 4 from the Active Fraction of the Decoction

To find the active principles responsible for the pharmacological activity, the decoction of was initially fractionated by partitioning with
CH2Cl2 and AcOEt. The most active fraction was the AcOEt which inhibited the licking time by 56.7 % at 100 mg/kg (data not shown). The
UPLC-chromatographic profile of this fraction (Figure 1S, Supporting Information) showed the presence of two major compounds, which were
isolated and characterized as isoquercitrin and hyperoside; their identification was accomplished by comparing their spectroscopic and
spectrometric properties with those of authentic samples and reported data.[26] This is the first report of these products in Melampodium
divaricatum. The finding of these compounds in the decoction is consistent with is antinociceptive and antihyperalgesic properties. Quercetin
and it glycosides demonstrated antinociceptive effect in the formalin test and PIFIR model (Pain Induced Functional Impairment Model in Rats)
at a dose of 100 mg/kg.[26-30] Anjaneyulu and Chopra[31] demonstrated that quercetin relieves neuropathic pain in STZ-induced diabetic mice.
This effect was attributed to the modulation of opioidergic mechanisms. Besides, hyperoside demonstrated dose-dependent antinociceptive
effect in the hot plate model, mild anti-inflammatory effect in the croton oil induced edema, and suppressed the production of prostaglandin

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Chemistry & Biodiversity 10.1002/cbdv.202100369

Chem. Biodiversity
E2 (PGE2), and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated macrophages.[32-34] In other investigations, it was found that the mixture
of hyperoside and isoquercitrin showed potent antinociceptive activity against p-benzoquinone induced writhes.[35]

Isolation and Characterization of Compounds 1 and 2

The CH2Cl2 fraction was also phytochemically investigated to yield compounds 1 and 2. Compound 1 was obtained as an optically active
glassy solid. The HRESI-MS revealed a [M+Na]+ molecular sodiated ion peak at m/z 491.2977 which agreed to a molecular formula C26H44O7,
implying five indices of hydrogen deficiency. The IR spectrum indicated absorptions for hydroxy group (3328, 1210, and 1050 cm-1) (Figure S2,
Supporting Information). Examination of the NMR data (Table 1, Figures S3-S8, Supporting Information) led to the conclusion that 1 was a
glucoside of a kaurene-3,6-diol type of diterpenoid.[36-40] Thus, the 13
C-NMR data (Table 1, Figure S4, Supporting Information) indicated the
presence of twenty-six carbons, six of them corresponding to a glucopyranosyl moiety (δC 61.3, 70.3, 76.6, 73.5, 77.0, 100.7), and the remaining

Accepted Manuscript
to the kaurane backbone, including four methyl (δC 16.6, 17.5, 28.3, 24.4), eight methylenes, and four methines groups as well as four no
protonated carbons. The chemical shift of the signals at δC 76.8 (C-16) and 84.0 (C-3) indicated that there were two oxygenated carbons in the
kaurene nucleus. The chemical shift and coupling pattern of the anomeric proton (H-1', δH 4.14, d, J = 7.7 Hz) suggested its axial orientation.
The D configuration of the β-glucopyranosyloxy moiety was demonstrated by the hydrolysis of compound 1 with β-glucosidase; the aqueous-
soluble fraction showed identical optical rotation ([α]20 +30) and Rf (co-TLC) to those of a standard sample of β-D-glucopyranose. The HMBC
correlations (Figure S6, Supporting Information) from H-1', H-18 and H-19 to C-3 (δC 84.0) confirmed the position the β-D-glucopyranosyloxy
moiety at C-3. The other HMBC correlations shown in Table 1 corroborated the assignment of H-3, H-5, H-20, H-9, H-14, and the hydroxy
group at C-16. Moreover, the coupling pattern observed for H-3 (dd, J = 12.9, 4.8 Hz) in the 1H-NMR spectrum of 1, revealed that it was axially
(β) oriented. The NOESY correlations (Table 1) corroborated the relative orientation of the substituents on the kaurane core (Figure 3). In
particular, the correlations H-3/H-5; H-5/H-1, H-9; H-14b/H-20; 14a/H-12b, H-13; and H-15a/ H-9 indicated the relative disposition of the
substituents along the diterpenoid nucleus. Comparison of the optical rotation of compound 1 ([α]D20 −31.1) with those of many ent-kaurenes
and biogenetic considerations, suggested that compound 1 is an ent-kaurane. Indeed, calculation of the optical rotation for the normal and
ent-isomers using a DFT method at B3LYP/6-311G+(2d,p) level in methanol (Tables S1 and S2, Supporting Information), with the PCM solvation
model,[41, 42] revealed that the calculated value for the ent-kaurane ([α]D20 −27.12) was closer to the experimental value than that calculated for
the normal isomer ([α]D20 +13.15).

A B
OH
1
3
OH 5' O O 9
HO 10 17
H HO 5 15
OH
3' OH 1' 19
O O H 8
12 OH
HO 20
HO H
OH 18
H 14
13

Figure 3. Structure of compound 1; A) plane projection; B) chair conformation.

To confirm the most plausible biogenetically absolute configuration[43] at C-16 of compound 1, NMR chemical shifts of the
diastereoisomers (16R and 16S) were calculated, using the GIAO method at the B3LYP/6-31G+(d,p) level with the conductor polarizable
calculation model (PCM) in CHCl3.[44, 45] The isotropic shielding tensors for each compound were subjected to DP4+ statistical analysis, which
predicted that the configuration 16R was the most probable, 85.1 % at B3LYP/6-31G+(d,p) level. The assigned stereochemistry was endorsed
by J-DP4 probability analysis (Tables S3 and S4, Supporting Information), which mixes 3JH-H couplings constants and DP4 analysis for
stereochemical designation.[46] This analysis confirmed that the most probable configuration (94.34 %) at C-16 was R, according to recent
biogenetic demonstration.[47] Thus, compound 1 is 3-α-O-β-D-glucopyranosil-16-α-hydroxy-ent-kaurane. It is important to point out that
Castro and collaborators reported a similar glycoside from M. cinereum var hirtellum,[40] however there are many differences in their chemical
shift values and coupling pattern. Thus, their structure must be revised.

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Melampodiamide (2) was obtained as an optically active white solid ([α]D20= 13, c = 0.1, pyridine). The IR spectrum (Figure S9, Supporting
Information) showed bands for amide (1635 and 1545 cm-1), hydroxy (3330 cm-1) and for aliphatic groups (2490, 2859 and 1453 cm-1). The
molecular formula, C42H84NO5, established from HR-ESI-MS data (Figure S10, Supporting Information), indicated two indices of hydrogen
deficiency. Analysis of the 1H and 13
C-NMR data (Table 2, Figure S11-S-16, Supporting Information) was consistent with a ceramide.[48-51] The

typical 2-amino-1,3,4-triol moiety was evidenced by the NMR signals at δH/δC 4.44 and 4.53( H-1)/63.3 (C-1); 5.13 (H-2)/54.2 (C-2); 4.38 (H-
3)/78.1 (C-3) and 4.30 (H-4)/74.2 (C-4), and the analysis of the COSY and HMBC cross-peaks (Figure 4A). The 1H-NMR signals for five
exchangeable protons were observed, including one for the amide at δH 8.60 (d, J = 9.0 Hz), and those for the hydroxy groups of at δH 7.64,
6.72, 6.71, and 6.27. The NMR spectra also exhibited signals corresponding to several methylene groups between δH 1.27–2.50 and two
terminal methyl groups, supporting the presence of two long hydrocarbon chains, one of these belonging to an α-hydroxy γ,δ-unsaturated
fatty acid [δC 131.9 (C-4′) and 132.1 (C-5′), 176.4 (C-1′) and 73.7 (C-2′) and δH 4.96 (OH-2′)], which was further supported by the HMBC
correlations between H-2′/C-1′and C-4′ andH-5′/C-2′ (Figure 4A) The second chain belonged to the 2-amino-1,3,4-triol moiety. The

Accepted Manuscript
configuration of the C-4′-C-5′ double bond was deduced to be Z from the J value (13.5 Hz) between H-4′ and H-5′. The length of the
unsaturated hydroxy fatty acid chain was determined by the peaks at m/z 665 and 316 due to the loss of water, and a fragment resulting from
an α-cleavage of the amide group, respectively (Figure 4B). Therefore, the amino chain was characterized as 2-amino-octadecosan-1,3,4-triol
while the long chain fatty acid precursor was characterized as 2-hydroxy-4Z-en-tetradecanoic acid.

A) B)
4' 6' 665

15
HO 3' HO
1'

O NH OH
O NH OH

2 4
1
316
9
OH OH
OH OH

HMBC COSY

Figure 4. A) Important HMBC and COSY correlations; B) mass fragmentation pattern (DART) for compound 2.

Acetylation of 2 with acetic anhydride in pyridine resulted in a tetra acetylated derivative (2a; Figure 5) corroborating the presence of
four hydroxyl groups. The NMR data is included in Table S5 and Figures 19S and S20 (Supporting information).

5' 7' 9' 11' 13' 15' 17' 19' 21' 23'

RO 3'

1'
O NH OR

1 3
5 7 9 11 13 15 17
OR OR
2 R =H
2a R = Ac

Figure 5. Structure of 2 and its acetylated derivative 2a.

The comparison of the NMR data of the 2-amino-1,3,4-triol fragment with those reported for other ceramides previously described for
plants, allowed to propose that the stereogenic centers at C-2, C-3, and C-4 as 2S,3S,4R.[51] The positive value for the optical rotation seems to
support this idea. Biogenetically, the configuration for C-2' is proposed to be R, because it is likely for the hydroxyl group at this position to
have this configuration as in other plant ceramides.[50, 51] Thus, all of the information above mentioned resulted in the structure of compound 2,
which was determined as (2′R*,4′Z)-2-hydroxy-N-[(2S*3S*,4R*)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide, and given the common name
of melampodiamide (Figure 5).

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Table 1. 1H (700 MHz) and 13C (175 MHz) NMR Data of compound 1 in DMSO-d6 (δ in ppm).

Position δC Type δH, mult. (J in Hz) HMBC 1H→13C NOESY

1a 0.75-0.78 (m) 10 9
38.0 CH2
1b 1.75‒1.77 (m) 10, 5, 3 7a

2 26.6 CH2 1.50‒1.53 (m) 1

3 84.0 CH 3.14 (dd, J= 11.8, 5.0) 18, 19, 1' 5,1a

4 37.9 C ‒

5 55.1 CH 0.68−0.72 (overlapped) 1, 4, 9, 18 3, 9

6a 19.9 CH2 1.26−1.31 (m) 7, 8

Accepted Manuscript
6b 1.46−1.49 (m)

7a 1.36‒1.38 (m) 8, 14 1b, 9

41.7 CH2
7b 1.52‒1.55 (m) 20

8 44.5 C ‒

9 56.3 CH 0.-87-0.88 (overlapped) 8, 14 1a, 5, 15a

10 38.4 C ‒

11 17.8 CH2 1.50‒1.53 (m) 9, 12

12a 1.47‒1.51 (m) 14

22.9 CH2
12b 1.67 (d, J= 9.5) 14a

13 47.8 CH 1.71−1.74 (m) 9, 16 14a

14a 37.0 CH2 1.52−1.55 (m) 8, 9, 13 12b, 13

1.72−1.75 (m)
14b 15, 16 20

15a 1.38 (d, J= 13.9) 7, 8, 9, 13, 14, 16 20, 9

57.5 CH2
15b 1.44 (d, J= 13.9) 20

16 76.8 C ‒

17 24.4 CH3 1.21 (s) 13, 15, 16

18 28.3 CH3 0.71 (s) 3, 4, 5, 6, 19

19 16.6 CH3 0.93 (s) 3, 4, 5,18

20 17.5 CH3 0.98 (s) 9,1 7b, 14b

1' 100.7 CH 4.14 (d, J= 7.7) 3, 5' 3'

2' 73.5 CH 2.9 (dd, J= 9.0, 7.7) 1', 5'

3' 76.6 CH 3.10‒3.12 (overlapped) 2', 4' 1'

4' 70.3 CH 3.02−3.05 (overlapped) 3', 5'

5' 77.2 CH 3.02‒3.05 (overlapped) 4', 6'

6'a 3.40 (dd, J= 11.5, 5.3) 3', 4'

61.3 CH2
6’b 3.63 (dd, J= 11.5, 1.7) 4'

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Chemistry & Biodiversity 10.1002/cbdv.202100369

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Table 2. 1H and 13C-NMR Data (700 and 175 MHz) of compound 2 in pyridine-d5 (δ in ppm).

Position δC Type δH, mult. (J in Hz) HMBC 1H→13C

1a 4.44 (dd, J= 10.8, 4.7) 2, 3

63.3 CH2
1b 4.53 (dd, J= 10.8, 4.7)

2 54.2 CH 5.13 (dd, J= 9.1, 4.6) 1, 1′, 3

3 78.1 CH 4.36–4.40 (overlapped) 1, 2, 4, 5

4 74.2 CH 4.28–4.33 (overlapped)

5a 1.94–2.00 (m) 3, 4
34.3 CH2
5b 2.15–2.19 (m)

Accepted Manuscript
6‒16 30.7‒31.4 CH2 1.22−1.39 (m)

17 24.2 CH2 1.26 (br s) 18

18 15.6 CH3 0.88 (t, J= 6.8) 17

NH 8.60 (d, J= 8.9) 1, 1′, 2, 3

1' 176.5 C ‒

2' 73.7 CH 4.62–4.67 (overlapped) 1′, 3′

3'a 2.05–2-08 (m) 1′, 2′

37.0 CH2
3'b 2.21–2.28 (m) 1′, 2′

4' 131.9 CH 5.52 (dt, J= 13.5, 6.4) 9'-22'

5' 132.1 CH 5.57 (dt, J= 13.5, 6.4) 6'

6' 35.1 CH2 2.21–2.28 (m) 4', 5'

7' 34.6 CH2 2.15–2.19 (m) 5'

8'‒22' 30.7‒31.4 CH2 1.22−1.39 (br s)

23' 24.2 CH2 1.26 (br s) 24'

24' 15.6 CH3 0.88 (t, J= 6.8) 23'

OH-1 ― 6.71 (br s)

OH-3 ― 6.72 (br s)

OH-4 ― 6.27 (br s)

OH-2' ― 7.64 (br s)

Volatile Composition
The essential oil (EO) from M. divaricatum (0.091 % yield) was obtained by hydrodistillation of the aerial parts of the plant. CG-MS
analysis of the oil indicated the presence of fourteen compounds representing 100 % of its total composition (Table 3, Figure 21S, Supporting
Information). All the compounds identified were sesquiterpenes, being the major ones (E)-caryophyllene (32.9 %), germacrene D (21.6 %), β-
elemene (7.39 %), δ-elemene (12.6 %), γ-patchoulene (6.63 %), and 7-epi-α-selinene (5.78 %). This result is consistent with that of Duarte-
Moreira et al.[12] who analyzed a specimen collected in Brazil.
The volatile compounds from M. divaricatum were also obtained by Head Space-Solid Phase Micro extraction using a DVB/CAR/PDMS
fiber. In this case GC-MS analysis detected (E)-caryophyllene as the major compound (60.5 %; Table 3, Figure 22S, Supporting Information),
followed by δ-elemene (15.1 %), β-elemene (10.0 %), aromandendrene (8.73 %), and cis-α-bisabolene (5.69 %).

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Chemistry & Biodiversity 10.1002/cbdv.202100369

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Table 3. Volatile constituents from M. divaricatum identified by GC-MS obtained by hydrodistillation and HS-SPME using a DVB/CAR/PDMS fiber.

%
No. Compound IR
EO DVB/CAR/PDMS

5 δ-Elemene 1327 12.58 15.09

6 α-Cubebene 1345 1.10 ‒

7 β-Elemene 1389 7.39 10.04

8 (E)-Caryophyllene 1414 32.92 60.45

9 trans-α-Bergamotene 1432 3.83 ‒

10 Aromadendrene 1435 ‒ 8.74

Accepted Manuscript
11 cis-β-Farnesene 1440 0.56 ‒

12 α-Humulene 1452 3.29 ‒

13 Germacrene D 1454 21.59 ‒

14 γ-Elemene 1470 3.59 ‒

15 γ-Patchoulene 1502 6.63 ‒

16 β-Bisabolene 1505 0.75 ‒

17 cis-α-Bisabolene 1506 ‒ 5.69

18 7-epi-α-Selinene 1520 5.78 ‒

Total % 100 100

Conclusions

A decoction M. divaricatum demonstrated antinociceptive and antihyperalgesic activities when tested in the formalin test, revealing its
potential as pain reliever and for treating neuropathic pain. These outcomes offer scientific support to the use of M. divaricatum in Mexican folk
medicine. The major active principles of the aqueous extract were characterized as isoquercitrin and hyperoside, which possessed noted
antinociceptive and antihyperalgesic effects.[32−35] From the decoction were also isolated 3-O-β-D-glucopyranosyl-16-α-hydroxy-ent-kaurane
(1) and (2R*,4Z)-2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide (2), two non-previously reported secondary
metabolites. Their involvement in the antinociceptive action of the decoction remained an open question. From the chemotaxonomic point of
view, the presence ceramides and ent-kauranes has been previously documented In the Asteraceae family. GC-MS analysis showed that the
essential oil composition from the Mexican and Brazilian[12] species is similar; in both cases the main components were (E)-caryophyllene,
germacrene D, β-elemene, δ-elemene, γ-patchoulene, and 7-epi-α-selinene.

Experimental Section

General Section

IR spectra were recorded using a PerkinElmer 400 (Waltham, MA, USA) spectrophotometer. Optical rotation analyses were recorded at
the sodium D-line wavelength using a PerkinElmer model 343 polarimeter at 25 °C (PerkinElmer, Waltham, MA, USA). Analysis by High-
Resolution Electrospray Mass Spectrometry (HRESI-MS) were acquired with a Thermo QExactive Plus mass spectrometer (ThermoFisher, San
Jose, CA, USA) combined with an electrospray ionization source in positive mode, or by High-Resolution Direct Analysis in Real Time (HRDART-
MS) data using a Jeol AccuTOF JMS-T100LC (JEOL Ltd., Tokyo, Japan) mass spectrometer in positive mode. The 700 and 175 MHz (1H and 13C)

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Chemistry & Biodiversity 10.1002/cbdv.202100369

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NMR spectra were recorded on a Bruker Ascend III HD with a 5 mm TCI CryoProbe 700 H–C spectrometer (Bruker, Hamburg, Germany). The
400 and 100 MHz (1H and 13
C) NMR spectra were recorded on a Varian VNMRS spectrometer (Palo Alto, California, USA). In all analyzes was
used tetramethylsilane as an internal standard. Flash chromatography was accomplished on a CombiFlash Rf+Lumen system (Teledyne
Technologies, Inc., Lincoln, NE, USA) with an integrated evaporative light scattering detector (ELSD), using a RediSep Rf GOLD silica gel column
(Teledyne) eluted with a gradient of n-hexane, EtOAc, MeOH, at a flow rate of 5 mL min-1 and 55.0 column volumes over 230.0 min. Column
chromatography (CC) was carried out on silica gel 60 (70–230 mesh, Merck, Darmstadt, Germany) or Sephadex LH-20 (GE Healthcare, Chicago,
IL, USA).

Plant Material

Melampodium divaricatum (Rich. ex Rich.) DC. was collected on April 2018 in, Cuernavaca, Morelos, Mexico (18°54'24.6"N 99°12'32.0"W),
and taxonomically authenticated by Rafael Torres Colín. A voucher specimen (No. 1465411) has been deposited at the National Herbarium

Accepted Manuscript
(MEXU), Instituto de Biología, UNAM, Mexico City. The dried aerial parts were finely ground by a mechanical mill (Thomas Model 4 Wiley Mill,
NJ, USA) prior to extraction.

Preparation of the Aqueous Extracts, Organic Fractions, and Essential Oil

Dried aerial parts (flowers, leaves and stems, crude drug) of M. divaricatum (10 g) were boiled with 1000 mL of boiling water for 10 min.
The resulting decoction was left to stand for 30 min, filtered and concentrated under reduced pressure to yield 88 mg of a solid extract. This
process was repeated as needed. On the other hand, in other set of experiments, 100 g of the crude drug was boiled with 10 L of water during
10 min; the resulting decoction was filtered and sequentially partitioned with CH2Cl2 (10 L × 5) and EtOAc (10 L × 5). The organic phases were
dried over anhydrous sodium sulphate (Na2SO4), filtrated in Whatman No. 1 and concentrated under reduced pressure to obtain 1.080 g and
188 mg, respectively (yield: 10.8 % and 1.88 %). Both fractions were tested at a single dose (100 mg/kg, data not shown), and only the EtOAc
fraction inhibited the extent of time the animals expended licking the inoculated paw.
Essential oil was obtained from 200 g of fresh plant material and 1.5 L of H2O by hydrodistillation using a Clevenger apparatus for 4 h. The
collected oil samples were partitioned with CH2Cl2 (100 mL × 2), the organic phases were dried (Na2SO4), filtrated in Whatman No. 1 and
concentrated under reduced pressure, and stored at 4 °C until analysis (yield: 0.091 %).

Isolation of Compounds 1 and 2 from the CH2Cl2 Fraction

One g of CH2Cl2 fraction was subjected to flash chromatography on a silica gel column (80 g), using a gradient solvent system of n-
hexane, EtOAc and MeOH. Fractions were pooled according to UV and ELSD profiles to obtain 23 fractions (F1–F23). From factions F11 and F12 8.0
mg of 1 were obtained. From the fractions F21–F23 was obtained 10 mg of 2. The NMR spectra (Tables 1 and 2, Supporting Information) and
chromatographic profiles indicated that the compounds were pure.

3-α-O-β-D-glucopyranosil-16-α-hydroxy-ent-kaurane (1): White solid. [α]D25 -31. 11 (c 0.09, MeOH). IR (ATR) νmax 3348, 2925, 2866, 1072
cm-1. 1H-NMR (700 MHz, DMSO-d6) and 13
C-NMR (175 MHz, DMSO-d6) see Table 1. HRESI-MS m/z 491.2977 [M+Na]+ (C26H44O7Na, calcd.
491.2985).

(2R*,4Z)-2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide (2): White solid. [α]D25 + 13 (c 0.1, Pyridine). IR (ATR)

νmax: 3331, 3211, 2917, 2849, 1619, 1610 cm-1. 1H-NMR (700 MHz, C5D5N) and 13C-NMR (175 MHz, C5D5N), see Table 2. DART-MS m/z (rel. int.
in %): 665 (40), 637 (20), 316 (100), 298 (45 %); HR-ESI-MS m/z 682.6327 [M + H]+ (C42H84NO5 calcd. 682.6349).

Acetylation of (2R*,4Z)-2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide (2)

Compound 2 (3.1 mg, 4.54 x 10-6 mol) was treated with acetic anhydride (0.2 mL, 2.12 x 10-3 mol) and pyridine (0.2 mL, 2.48 x 10-3 mol).
The mixture was left in agitation at room temperature for 16 h; then, an aqueous solution of HCl 2 % (v/v) was added to the reaction mixture
and extracted with CH2Cl2. The organic layer was successively washed firstly with a saturated solution of Na2CO3 (5 mL × 5) and then with

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Chemistry & Biodiversity 10.1002/cbdv.202100369

Chem. Biodiversity
distilled H2O (5 mL × 5). Finally, it was dried over Na2SO4, and concentrated under reduced pressure. The crude reaction mixture was purified
by silica gel CC using CHCl3 to obtain 5.6 mg of product (2a).

(2R*,4Z)-2-acetoxy-N-[(2S,3S,4R)-1,3,4-triacetoxyoctadec-2-yl]tetracos-4-enamide (2a): Vitreous solid. IR (ATR) νmax: 2917, 2849, 1720,

1619, 1611 cm-1. 1H-NMR (700 MHz, CDCl3) and 13C-NMR (175 MHz, CDCl3), see Supporting Information. DART-MS m/z (rel. int. in %): 682 (52),
499 (60), 271 (100), 229 (68); HRDART-MS m/z 850.67491 [M+H]+ (C50H92NO9 calcd. 850.677).

Isolation of the Active Compounds from the EtOAc Fraction

The active EtOAc fraction (100 mg) was fractionated by Sephadex LH-20 CC (CH2Cl2-MeOH, 2:8) to afford eleven secondary fractions (A1–
A4). From fraction A3, 5 mg of hyperoside were obtained. From fraction A4 crystallized 4 mg of isoquercitrin. These compounds were identified

Accepted Manuscript
by comparison with authentic samples.

GC-MS Analysis

The volatile compounds from SPME fiber and essential oil were analyzed in a Gas Chromatographer Agilent 6890N apparatus (Agilent
Technology, Santa Clara, CA) equipped with a DB-5 column (Agilent HP, 20 m × 0.18 mm; 0.18 µm) and coupled to a LECO Pegasus 4D Time-
of-Flight mass spectrometer (Agilent, Santa Clara, CA). The injector port was heated to 300 °C and the samples were injected using a split ratio
of 1:20, with helium as the carrier gas at a constant flow rate of 1 mL min-1. The oven temperature program was set at 40 °C for 3 min,
increasing by 20 °C min-1 to 300 °C for 50 min. All mass spectra were acquired in Electron Impact (EI) ionization (70 eV). Compounds were
identified by comparison of their retentions indices (IR) with those from the literature data,[52] and by comparison of their MS fragmentation
patterns with those of compounds contained in the spectral database of the National Institute of Standards and Technology (NIST,
Gaithersburg, MD, USA). [53] The IR was determined relative to a series of n-alkanes (C8 – C27).

Headspace-Solid Phase Microextraction (HS-SPME)

A manual holder and a 50/30 µm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was used for HS-SPME procedure. Twenty mg of M.
divaricatum, 7.5 mg of NaCl and 5 mL of distilled water, were sealed in a 25 mL vial for 5 min, at room temperature under continuous magnetic
stirring (600 rpm). The fiber was exposed to the headspace of the sample for the 5 min and after this time, the SPME fiber was directly inserted
into the GC injector port and the fiber thermally desorbed. A desorption time of 2 min at 250 °C was used.

Antinociceptive and Anti-hyperalgesic Activity

Diclofenac and gabapentin were diluted in saline solution (s.s.) and used as reference drugs. Streptozotocin (STZ), nicotinamide (NA),
diclofenac and gabapentin were administered orally (p.o.) in a volume of 0.2 mL/10 g body weight. Control animals received the same volume
of vehicle alone by the same route of administration. Drugs were freshly prepared on the day of the experiment. Streptozotocin, nicotinamide,
diclofenac, gabapentin, and formaldehyde were purchased from Sigma-Aldrich (Darmstadt, Germany).

Animals

Male ICR mice (25–30 g) were obtained from the Animal Center UNAM-ENVIGO (ENVIGO RMS, SA de CV). Animals were housed under
standard laboratory conditions (25 ± 2 °C, 12 h light-dark cycle). The animals had free access to pellet food and water ad libitum. All animal
treatments were in accordance with the Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999) and in compliance with
international rules of care and use of laboratory animals. The protocols to evaluate antinociceptive and antihyperalgesic effects were carried
out under standards marked by the Ethical Guidelines for Investigations of Experimental Pain[54] and with the approval of the Institutional
Committee for the Care and Use of Laboratory Animals. (CICUAL), Facultad de Química, UNAM (FQ/CICUAL/391/19). Each animal was used only
once, and they were euthanized at the end of the experiment.

Experimental Hyperglycemia Induction

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Chemistry & Biodiversity 10.1002/cbdv.202100369

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Hyperglycemia was induced by i.p. administration of NA (50 mg/kg) dissolved in 0.9 % NaCl aqueous solution, after 15 min STZ (130
mg/kg) dissolved in 0.1 M citrate buffer, pH 4.5, was administered. Control groups received the vehicle. Immediately after the administration,
animals had free access to food and water. One week later, the blood glucose levels of each mouse were measured by the enzymatic glucose
oxidase method using a commercial glucometer. Blood samples were collected from the tail vein by means of a small incision at the end of the
tail. Mice with glucose levels higher than 200 mg/dl were included in the study.

The Formalin Test

Each mouse was placed into a plexiglass cylinder for 30 min habituation period. To induce nociception, mice were injected into the
plantar surface of the right hind paw with 30 mL of formalin (2 %), hyperglycemic mice received 40 mL of formalin (1 %). The accumulated time
spent in licking the injected paw was taken as nociceptive response. Two phases were considered: the first phase was obtained immediately
after injection and lasted 10 min; this was known as neurogenic phase. The second phase was observed 11–30 min after injection and

Accepted Manuscript
denominated as inflammatory phase. Animals were treated with M. divaricatum aqueous extract (see Extraction and Isolation) (31.6, 100 or
316.2 mg/kg, p.o.), diclofenac (31.6 mg/kg, p.o.), gabapentin (31.6 mg/kg, p.o.), or vehicle (p.o.).

Statistical Analysis

Results are expressed as the mean ± standard error of the mean (SEM) of six animals in each group. One-way analysis of variance (one-
way ANOVA) was used to analyze changes in time spent in licking the paw administered with formalin followed by Dunnett’s multiple
comparisons test using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. p <
0.05 or less was considered statistically significant.

Computational Section

Minimum energy structures for the enantiomers were built with Spartan'10 software (Wavefunction Inc., Irvine, CA, USA). Conformational
analysis was performed with the Monte Carlo search protocol under the MMFF94 molecular mechanics force field. The conformers with an
energy cutoff of 5 kcal/mol were submitted to Gaussian 09 program (Gaussian Inc., Wallingford, CT, USA)[55] calculation for their geometry
optimization performed using DFT B3LYP/6-311G+(2d,p) level of theory and all of which were subjected to specific optical rotation calculations
at the B3LYP/6-311+G(2d,p) level in methanol with the PCM model. The calculated specific optical rotation data of these conformers were
averaged according to the Boltzmann distribution and their relative Gibbs free energy.[44-46]

Supplementary Material

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number. 1D and 2D NMR spectra
and computational details of compound 1 and 2. TIC off the essential oil and HS-SPME from M. divaricatum. Theoretical calculations for
compound 1.

Acknowledgements

This work was supported by grants from CONACyT CB A1-S-11226, DGAPA-UNAM IN217320, and PAIP-UNAM 5000-9140. S. P-M

(771856) acknowledges fellowship support from CONACyT to pursue graduate studies. A.L.M acknowledges fellowship support from DGAPA-

UNAM to pursue a postdoctoral stay. We thank Isabel Rivero-Cruz, Gina Duarte-Lisci, Ramiro del Carmen, Marisela Gutiérrez-Franco, Rosa Isela

del Villar and Nayeli López-Balbiaux, from Facultad de Química; and Beatriz Quiroz García, Carmen García, and Rocío Patiño,†, from Instituto de

Química for their valuable technical assistance. We are indebted to Dirección General de Cómputo y de Tecnologías de Información y

Comunicación (DGTIC), UNAM, for the resources to carry out computational calculations through the Miztli supercomputing system (LANCAD-

UNAM-DGTIC-313).

11

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Chemistry & Biodiversity 10.1002/cbdv.202100369

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Author Contribution Statement

A.P.-V. and S. P.-M. equally contributed to this work performing most of the phytochemical work, analyzing spectroscopic data, and with

the writing process. A.L.M designed and conducted, with S.P.-M., the pharmacological studies. R.T.-C identified the plant. M.L. M-R.

contributed with analysis tools and data analysis. M.R.-G. performed the theoretical calculations. J.S.-C. partially conceived the study and

collected the plant material. R. M. conceived the study, analyzed the data, and wrote the manuscript.

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N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam,

M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R.
L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski,

and D. J. Fox, Gaussian, Inc., Wallingford CT, 2013.

Accepted Manuscript

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Twitter Text

Melampodium divaricatum showed antinociceptive and antihyperalgesic activity due to the presence of hyperoside and
isoquercetrin. Two new metabolites, a kaurane derivative and the ceramide melampodiamide, were also separated and
characterized. By A. Pérez-Vásquez et al., @Rachel1M88859857

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