Phytochemistry

Inhibition of multidrug-resistant MCF-7 breast cancer cells with combinations of clinical

drugs and resin glycosides from Operculina hamiltonii
--Manuscript Draft--

Manuscript Number: PHYTOCHEM-D-23-00836

Article Type: Full Length Article

Section/Category: Chemistry and Bioactive Products (Full Length Article)

Keywords: Operculina hamiltonii; Convolvulaceae; Cytotoxicity; Cocktail chemotherapy;

Glycolipids; Oligosaccharides; Potentiation effects; Recycling HPLC

Corresponding Author: jhon Castaneda, Ph.D.

South Colombian University Faculty of Health
Neiva, COLOMBIA

First Author: Jhon Fredy Castañeda-Gómez, Ph.D.

Order of Authors: Jhon Fredy Castañeda-Gómez, Ph.D.

Armando Moreno-Velasco, Ph. D.

Mabel Fragoso-Serrano, Ph. D.

Pedro de Jesús Flores-Tafoya

Sebastian Carrillo-Rojas

Elihu Bautista, Ph. D.

Suzana Guimarães Leitão, Ph. D.

Rogelio Pereda-Miranda, Ph. D.

Abstract: The jalap roots, Operculina hamiltonii D.F. Austin & Staples (Convolvulaceae), are
extensively commercialized as a depurative and laxative remedy in traditional medicine
of the north and northeast regions of Brazil. The purification by recycling HPLC and
structure elucidation of four undescribed acyl sugars or resin glycosides are described
here from a commercial product made of powdered roots. Three macrocyclic structures
of a tetrasaccharide of (11S)-hydroxyhexadecanoic acid, operculinic acid C (1), named
as hamiltonins II-IV (3-5), presented a diastereoisomeric relationship as one residue of
n-dodecanoic acid esterified the oligosaccharide core on a different position in each
compound. Additionally, hamiltonin V (6) was characterized as an ester-type
homodimer of acylated operculinic acid C with the same substitution pattern identified
in hamiltonins II (3) and III (4) for each of the dimer subunits. All the isolated resin
glycosides did not display any intrinsic cytotoxicity (IC50 >25 μg/mL). However, a
combination of the individual isolated compounds 3-6 (1-50 μM) demonstrated an
enhancement of cytotoxic effects with sublethal doses of vinblastine and
podophyllotoxin (0.003 µM) in multidrug-resistant breast carcinoma epithelial cells
(MCF-7/Vin).

Suggested Reviewers: Muriel Cuendet, Ph. D.

Professor, University of Geneva
muriel.cuendet@unige.ch
Isolation and antiproliferative activity in cancer cells of resin glycosides

Norberto Peporine Lopes, Ph. D.

Professor, University of Sao Paulo
npelopes@fcfrp.usp.br
Evaluation of Biologically Active Natural Products and Mass Spectrometry, HPLC, and
NMR Analytical Studies
Cited in reference Demarque, D.P.; Crotti, A.E.; Vessecchi, R.; Lopes, J.L.; Lopes,
N.P. Nat. Prod. Rep. 2016, 33, 432-455.

Toshio Miyase, Ph. D.

Professor, University of Shizuoka
miyase@ys7.u-shizuoka-ken.ac.jp

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 Isolation and structure elucidation of plant oligosaccharides

Zi-Hua Jiang, Ph. D.

Professor, Lakehead University
zjiang@lakeheadu.ca
Isolation of resin glycosides from the Indian Jalap, Operculina turpethum

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Cover Letter

Licenciatura en Ciencias Naturales: Fisica, Química y Biología

Facultad de Educación
Universidad Surcolombiana
Avenida Pastrana Borrero, Cra 1ª
Neiva-Huila
Tel: (57) (8) 875 4753 Extensión: 1067
E-mail: jhon.castaneda@usco.edu.co

Dr. Helen Skaltsa

Editor-in-Chief
Phytochemistry July 27, 2023.

Dear Dr. Helen Skaltsa,

We ask you to consider the following revised manuscript entitled “Inhibition of multidrug-
resistant MCF-7 breast cancer cells with combinations of clinical drugs and resin glycosides
from Operculina hamiltonii” by Armando Moreno-Velasco, Mabel Fragoso-Serrano, Pedro
de Jesús Flores-Tafoya, Sebastian Carrillo-Rojas, Elihu Bautista, Suzana Guimarães Leitão,
Jhon F. Castañeda-Gómez, and Rogelio Pereda-Miranda, as a Full Paper for possible
publication in the Phytochemistry.

This article highlights the isolation and structural characterization of resin glycosides from
roots of Operculina hamiltonii, a valuable treatment in traditional medicine for enteric
disorders due to its purgative activity in Brazil. The procedures for isolation, purification by
recycling HPLC, and structural elucidation of four undescribed resin glycosides are
presented. The isolation of the structurally related novel compounds demonstrated that resin
glycosides are not easy to purify for they are always present as complex mixtures of
homologues. Recycling HPLC provides a maximal resolution in a short-term analysis. All
the isolated compounds were found to be inactive as cytotoxic agents. However, when they
were evaluated in combination with a sublethal concentration of the anticancer agents
vinblastine and podophyllotoxin produced a significant enhancement of the cytotoxicity,
specially to the multidrug resistant MCF-7/Vinblastine. Such combined potentiation effect
against breast cancer epithelial cells may be beneficial for coktail chemotherapy, making
resin glycosides potential candidates for drug repurposing of conventional chemotherapeutic
drugs to reduce side effects.

I thank you for your time and attention.

Cordially,

____________________________
Dr. Jhon Fredy Castañeda Gómez
Professor of Medicinal Chemistry and Pharmacognosy
Universidad Surcolombiana, Colombia
Highlights

Highlights

 Purification by recycling HPLC of resin glycosides was accomplished from commercial

powdered roots of Operculina hamiltonii.

 Hamiltonin V represents the first resin glycoside with an ester-type dimer structure

assembled by two units of operculinic acid C.

 In combination assays, non-cytotoxic resin glycosides enhanced the intracellular

concentration of partner clinical drugs resulting in cytotoxicity potentiation.

 Macrocyclic amphiphilic resin glycosides produced the inhibition of drug efflux and

contributed to the accumulation of cytotoxic partner agents.

Graphical Abstract

Combination of resin glycosides demonstrated a potentiation of cytotoxicity with sublethal doses

of vinblastine and podophyllotoxin in multidrug-resistant breast carcinoma cells.

Manuscript File Click here to view linked References

1
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5 1 Inhibition of multidrug-resistant MCF-7 breast cancer cells
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2 with combinations of clinical drugs and resin glycosides from
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3 Operculina hamiltonii
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13 4
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15 5 Armando Moreno-Velasco,a Mabel Fragoso-Serrano,a Pedro de Jesús Flores-Tafoya,a Sebastian
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17 6 Carrillo-Rojas,a Elihu Bautista,b Suzana Guimarães Leitão,c Jhon F. Castañeda-Gómezd*, and
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19 7 Rogelio Pereda-Mirandaa,*
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21
a
22 8 Departamento de Farmacia, Facultad de Química and Programa de Maestría y Doctorado en
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24 9 Ciencias Químicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico
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26 10 City 04510, Mexico
27
28 b
29 11 Consejo Nacional de Ciencia y Tecnología, División de Biología Molecular, Instituto Potosino
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31 12 de Investigación Científica y Tecnológica A.C., San Luis Potosí, S.L.P, Mexico.
32
33 13
34 c
35 14 Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, CCS, Bloco A, Ilha do Fundão,
36
37 15 21941-902, Rio de Janeiro, Brazil
38
39
16
40 d
17 Grupo Químico de Investigación y Desarrollo Ambiental. Programa de Licenciatura en Ciencias
41
42 18 Naturales y Educación Ambiental, Facultad de Educación. Universidad Surcolombiana, Neiva,
43
44 19 Colombia.
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46 20
47
48 21 *Corresponding authors
49
22 E-mail address: jhon.castaneda@usco.edu.co (J.F. Castañeda-Gómez);
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51 23 pereda@unam.mx (R. Pereda-Miranda)
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53 24
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55 25
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57 26
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 1
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27 ABSTRACT
6
7 28 The jalap roots, Operculina hamiltonii D.F. Austin & Staples (Convolvulaceae), are extensively
8
9
10 29 commercialized as a depurative and laxative remedy in traditional medicine of the north and
11
12 30 northeast regions of Brazil. The purification by recycling HPLC and structure elucidation of four
13
14 31 undescribed acyl sugars or resin glycosides are described here from a commercial product made of
15
16
17 32 powdered roots. Three macrocyclic structures of a tetrasaccharide of (11S)-hydroxyhexadecanoic
18
19 33 acid, operculinic acid C (1), named as hamiltonins II-IV (3-5), presented a diastereoisomeric
20
21
22 34 relationship as one residue of n-dodecanoic acid esterified the oligosaccharide core on a different
23
24 35 position in each compound. Additionally, hamiltonin V (6) was characterized as an ester-type
25
26
27 36 homodimer of acylated operculinic acid C with the same substitution pattern identified in
28
29 37 hamiltonins II (3) and III (4) for each of the dimer subunits. All the isolated resin glycosides did
30
31
32 38 not display any intrinsic cytotoxicity (IC50 >25 g/mL). However, a combination of the individual
33
34 39 isolated compounds 3-6 (1-50 μM) demonstrated an enhancement of cytotoxic effects with
35
36
37
40 sublethal doses of vinblastine and podophyllotoxin (0.003 µM) in multidrug-resistant breast
38
39 41 carcinoma epithelial cells (MCF-7/Vin).
40
41 42
42
43 43
44
45 44 Keywords
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47
48
45 Operculina hamiltonii; Convolvulaceae; Cytotoxicity; Cocktail chemotherapy; Glycolipids;
49
50 46 Oligosaccharides; Potentiation effects; Recycling HPLC
51
52 47
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48 1. Introduction
6
7 49 The Brazilian jalap root, Operculina hamiltonii D.F. Austin & Staples (Convolvulaceae),
8
9
10 50 is widely marketed in the northern region of Brazil as a raw drug in powders named as “jalapa em
11
12 51 pó” (jalap powder), “batata-de-purga”, "batatão", or "jalapa" (Montiel-Ayala et al., 2021), in
13
14 52 addition to numerous products made with the roots and content of its resins, such as syrup, alcoholic
15
16
17 53 extracts, and pills (Fig. S1). Among the principal medicinal properties of jalap root are its action
18
19 54 as depurative, diuretic, febrifuge, laxative, and purgative herbal medicine. The laxative folk
20
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22 55 medicine is used, especially for intestinal congestion (Cunha et al., 2011).
23
24 56 Resin glycosides or acyl sugars represent mixtures of diverse glycolipids generated by
25
26
27 57 monohydroxy and dihydroxy long-chain fatty acids (C14-C18) bonded to linear or branched
28
29 58 heteropolysaccharides (Fan et al., 2022; Pereda-Miranda et al., 2010). These plant principles
30
31
32
59 provoke the purgative action of all the species from the Convolvulaceae or morning glory family
33
34 60 used in folk medicinal practices (Lira-Ricárdez et al., 2019; Montiel-Ayala et al., 2021; Pereda-
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36 61 Miranda et al., 2006). These active glycolipids increase the membrane permeability of intestinal
37
38
39 62 cells to cations and anions (Zhu et al., 2019b), and, therefore, acting as osmotic agents by inducing
40
41 63 water elimination and intestinal peristalsis. This action was also recognized to be associated with
42
43
44 64 the activation of nuclear factor-kappa B in the colon, which enhanced cyclooxygenase-2-mediated
45
46 65 secretion of prostaglandin E2, resulting in a reduced aquaporin-3 expression and the inhibition of
47
48
49
66 absorption of water from the intestine to the blood vessel (Zhu et al., 2019a).
50
51 67 Recently, the procedures for the isolation, purification by recycling HPLC, and structure
52
53 68 elucidation of a tetrasaccharide of (11S)-hydroxyhexadecanoic acid were described from a
54
55
56 69 commercial sample of powdered roots of O. hamiltonii, which was found to be a macrocyclic
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58 70 structure of operculinic acid C (1), diacylated by n-decanoic acid and the unusual n-hexadecanoic
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 1
2
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4 71 acid moiety, named as hamiltonin I (2). This isolated resin glycoside was inactive as a cytotoxic
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7 72 agent (IC50 > 50 µM). However, when it was evaluated (1-50 μM) in combination with a sublethal
8
9 73 concentration of the clinical agent vinblastine (0.003 M), a noteworthy improvement of the
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11
12 74 resultant cytotoxicity was produced (Moreno-Velasco et al., 2022), particularly for multidrug-
13
14 75 resistant breast carcinoma epithelial cells with IC50 values of 5.0 ± 0.4 µM (MCF-7) and 10.9 ± 0.6
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17 76 µM (MCF-7/Vin) for this non-cytotoxic compound 2.
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19 77
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48 78
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79 1
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53 80
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55 81
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60 83
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4 84
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28 85
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30 86 2
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32 87
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35 88 The inhibition of cell growth for a non-cytotoxic resin glycoside in combination with an
36
37 89 antitumor agent is in line with the modulatory activity of efflux pumps responsible for the
38
39
40
90 multidrug resistance (MDR) phenotype in prokaryotic (Pereda-Miranda et al., 2006; Corona-
41
42 91 Castañeda and Pereda-Miranda, 2012) and eukaryotic (Figueroa-González et al., 2012; Castañeda-
43
44 92 Gómez et al., 2013) cells previously described for this class of acyl sugars. In vitro assessments
45
46
47 93 have revealed the potential of resin glycosides as coadjutants to elude drug resistance and
48
49 94 reestablish the clinical utility of conventional drugs for treating refractive infections and cancer
50
51
52 95 (Lira-Ricárdez and Pereda-Miranda, 2020).
53
54 96 In this work, it was hypothesized that sublethal concentrations of podophyllotoxin in
55
56
57 97 combination with resin glycosides could act with all undescribed resin glycosides (3-6), from a
58
59 98 commercialized powder sample of the Brazilian jalap root (Fig. S2A), in the same fashion as
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 1
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4 99 hamiltonin I (2) and vinblastine against multidrug resistant MCF-7 breast cancer cells (MCF-
5
6
7 100 7/Vin) (Moreno-Velasco et al., 2022). This strategy of drug combination or co-administration of
8
9 101 two agents would certainly produce a higher inhibitory effect than the two drugs alone
10
11
12 102 (monotherapy) to avoid toxicity in therapeutical practices by targeting multiple molecular pathways
13
14 103 (Hu et al., 2016). Similarly, the cytotoxicity enhancements of sublethal concentrations of both
15
16
104 antitumoral agents, vinblastine and podophyllotoxin, in combination with all purified non-
17
18
19 105 cytotoxic resin glycosides to human tumor cells, namely sensitive and MDR MCF-7 (breast cancer
20
21 106 cells), HCT-116 (colon carcinoma), and Hela (cervix carcinoma) are described. In addition,
22
23
24 107 extensive work was accomplished by NMR, ESI-Q-TOF, and MALDI-TOF analyses to complete
25
26 108 the structural characterization of the undescribed isolated glycosidic acids, named as hamiltonins
27
28
29 109 II-V (3-6), which were purified by reserved-phased recycling HPLC.
30
31 110
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52 111
53
54 112 3 R1 = n-C12H23O; R2 = R3 = H
55 113 4 R1 = R3 = H; R2 = n-C12H23O
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57
114 5 R1 = R2 = H; R3 = n-C12H23O
58 115 9 R1 = R2 = R3 = H
59 116 Rha = Rhamnose; Fuc = Fucose
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117 2. Results and discussion
6
7 118 2.1 Isolation of resin glycosides
8
9
10 119 The powdered roots of Operculina hamiltonii were purchased under the name of “jalapa
11
12 120 em pó” or “batata-de purga” (Mercadão Natural; Fig. S2A) and submitted to a previously described
13
14 121 HPLC-ESIMS fingerprinting as quality control to specifically dereplicate operculinic acids A and
15
16
17 122 C (1), as the oligosaccharide cores for the resin glycosides of the analyzed commercial sample.
18
19 123 Therefore, this preliminary analytical procedure helped to confirm the authenticity of the plant
20
21
22 124 material and the reproducibility of our chemical results (Montiel-Ayala et al., 2021). The crude
23
24 125 CH2Cl2-MeOH soluble extract from the commercial powdered root was fractionated on Si gel CC
25
26
27 126 with mixtures of CH2Cl2 and Me2CO as elution solvents to afford twelve combined fractions (Fig.
28
29 127 S2B). Fractions F9 and F10 concentrated all resin glycosides and were analyzed by analytical
30
31
32
128 reversed-phase HPLC under refractive index detection (RI), since resin glycosides are poor-UV
33
34 129 absorbing compounds (Alsanea and Gamal, 2022), coupled off-line to electrospray mass
35
36 130 spectrometry. The identification of novel acyl sugars was achieved by recognizing mass values for
37
38
39 131 the ions [M + Na]+ and [M – H] (Table S1), as previously described (Castañeda-Gómez et al.,
40
41 132 2019; Moreno-Velasco et al., 2022). Surprisingly, ESI-MS analysis of the eluates from fraction F9
42
43
44 133 showed that these subfractions were populated by the same ions at m/z 1043 [M + Na]+ and 1019
45
46 134 [M – H] which suggested the presence of diastereoisomeric undescribed compounds, as the major
47
48
49 135 constituents (Table S1 and Fig. S3). Semipreparative HPLC of fraction F10 was used to collect
50
51 136 two major peaks with tR = 14.5 min (F10-1) and 21.7 min (F10-2) (Fig. S4). UHPLC-ESI-MS in
52
53
54 137 negative ion mode permitted the identification of batatinoside VI (7) from subfraction F10-1 (tR =
55
56
57
138 14.5 min) through the detection of the deprotonated molecule with m/z 1153 [M  H] and the
58
59 139 adduct with formic acid at m/z 1199 [M  H + formic acid (CH2O2)] (Fig. S5A and S5B)
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 1
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4 140 (Demarque et al., 2016; Huang et al., 1999); in addition, a coelution experiment in HPLC-refractive
5
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7 141 index with an authentic sample (Fig. S5C) corroborated the identity for this pentasaccharide
8
9 142 macrolactone (Moreno-Velasco et al., 2022). Recycling HPLC of subfraction F10-2 (recycled
10
11
12 143 eluate: tR = 21.7 min; Fig. S4) allowed for the purification of batatinoside IX (8), which was
13
14 144 identified by HPLC coelution experiments with an authentic sample and by comparison of its
15
16
17
145 physical constants: mp 118-120 °C; []22D 51.0 (c 1.0, MeOH); HRESIMS m/z 1205.6601 [M
18
19 146 a]Fig. S6); and NMR data (Fig. S7) with published values (Rosas-Ramírez and Pereda-
20
21
22 147 Miranda, 2013).
23
24 148 Saponification of fraction F9 afforded a single water-soluble glycosidic acid and an organic
25
26
27 149 solvent-soluble acidic fraction. The liberated acylating fatty acid mixture was identified by GC-
28
29 150 MS analysis as only constituted by n-dodecanoic acid (m/z 200 [M]+.) (Moreno-Velasco et al.,
30
31
32
151 2022). The glycosidic acid was characterized as the previously described tetrasaccharide
33
34 152 operculinic acid C (1) by HPLC coelution experiments with an authentic sample and by comparison
35
36
153 of its physical constants: mp 118 °C; []D 49.0 (c 1.0, MeOH) ([α]22589 48.3); HRESIMS m/z
37
38
39 154 855.4606 [M ]Fig. S8); and NMR data (Fig. S9) with published values (Moreno-Velasco et
40
41
42 155 al., 2022). These results confirmed the absolute configuration for the sugar units in the
43
44 156 oligosaccharide core of the undescribed metabolites 3-6 as well as the glycosylation sequence
45
46
47
157 according to the procedures previously described for the characterization of hamiltonin I (2)
48
49 158 (Moreno-Velasco et al., 2022). Therefore, for the description of undescribed hamiltonins II-V (3-
50
51 159 6), a comparison with known operculinic acid (1) and hamiltonin I (2) will be used to highlight
52
53
54 160 structural similitudes and differences among them to avoid reiterative lengthy descriptions.
55
56 161 Fraction F9, rich in resin glycosides (Fig. S2), was independently fractionated by
57
58
59 162 semipreparative HPLC with a C-18 column and CH3CN:H2O (4:1) as a mobile phase. Three eluates
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 1
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4 163 were collected with tR = 22.5 min (F9-1), tR = 33.1 min (F9-2), and tR = 38.5 min (F9-3) from
5
6
7 164 fraction F9 (Fig. S3). Each isolated peak was reinjected in the same column and further recycled
8
9 165 with the help of a recycling valve and a mobile phase of CH3CN-H2O (5:1). For subfraction F9-1,
10
11
12 166 resolution was achieved for two peaks with tR = 18.5 min and tR = 28.0 min after the first cycle
13
14 167 (Fig. S3A). Further recycling of the first peak allowed to obtain an eluate with a 100% purity by
15
16
168 application of peak shaving and heart cutting for elimination of trace impurities (Pereda-Miranda
17
18
19 169 and Hernández-Carlos, 2002) after seven consecutive cycles to yield compound 3 (15.3 mg).
20
21 170 Collection of the second eluate afforded pure compound 4 (16.0 mg). Subfraction F9-2 (tR = 33.1
22
23
24 171 min) was treated with the same approach and pure compound 5 (20.5 mg) was obtained after nine
25
26 172 consecutive cycles (Fig. S3B). Finally, F9-3 (tR = 38.5 min; 30 mg) was recycled using CH3CN-
27
28
29 173 H2O (5:1) to yield pure compound 6 (12 mg) after seven successive cycles (Fig. S3C).
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58 174
59
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175 6
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 1
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4 176 2.2 Characterization of compounds 3-6
5
6
7 177 In all hamiltonins II-IV (3-5), a peak corresponding to the adduct [M + Na]+ with a value
8
9 178 of m/z 1043 Da for a molecular formula of C52H92O19Na was detected by ESI-Q-TOF in the positive
10
11
12 179 ionization mode (Fig. S13 and S25). Also, for the deprotonated molecule in the negative mode, the
13
14 180 anion [M H] of m/z 1019 Da was observed for the three isomers (Table S1 and Fig. S19). These
15
16
17 181 results confirmed the suspected isomerism between these three compounds and their structural
18
19 182 difference lied in the position where the acylating residue functionalized the oligosaccharide
20
21
22 183 nucleus when considering a difference in weight of 182 Da (C12H24O2 m/z 200 [M]+. H2O) for
23
24 184 esterification by n-dodecanoic acid of a postulated lactonized operculinic acid C (9) ([879 Da
25
26
27 185 (C40H72O19Na)  H2O]+). It was deduced that the variation between the retention time between
28
29 186 these acyl sugars was a consequence of their structural isomerism. For hamiltonin V (6), a peak
30
31
32 187 with the same value of m/z 1043 Da (Fig. S33) was detected for a sodium adduct in ESI-MS.
33
34 188 However, taking into account the retention time and duplicity of the signals observed in both 1H
35
36 189 and 13C NMR spectra when compared with those registered for 3-5 (Fig. S11 and Fig. S12), it was
37
38
39 190 assumed that this adduct corresponded to the cation [M/2 + Na]+ (Fig. S33), a species that is
40
41 191 commonly detected when analyzing ester-type dimers produced by intermolecular condensations
42
43
44 192 between two individual resins (Bah and Pereda-Miranda, 1997; Castaneda-Gómez and Pereda-
45
46 193 Miranda, 2011; Castañeda-Gómez et al., 2013; Escalante-Sánchez and Pereda-Miranda, 2007;
47
48
49 194 Rosas-Ramírez et al., 2011).
50
51 195 The NMR spectra in 1D (H1 and C13) and 2D (homonuclear 1H-1H and hetero 1H-13C
52
53
54
196 correlations) were analyzed and the first examination consisted in the comparison of the 1H NMR
55
56 197 signals of the purified compounds 3-5 (Fig. S11). The region in the range centered at δH 5.0-6.5,
57
58 198 i.e., the signals most downfield shifted, corresponded to the anomeric hydrogens and the positions
59
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 1
2
3
4 199 of acylation and lactonization of the oligosaccharide core. Their multiplicity and coupling constants
5
6
7 200 were compared with those previously described for operculinic acid C (1) (Moreno-Velasco et al.,
8
9 201 2022), which was identified as the oligosaccharide core for compounds 3-5. Three anomeric
10
11
12 202 hydrogens corresponding to rhamnose (J = 0.8-1.2 Hz) and one hydrogen for fucose (J = 7.0-7.4
13
14 203 Hz) were observed in the spectra corresponding to hamiltonins II (3; Fig. S14), III (4; Fig. S20),
15
16
204 and IV (5; Fig. S26). Comparison of the HSQC spectrum for operculinic acid C (1; Fig. S10) with
17
18
19 205 those registered for the undescribed hamiltonins II-IV (3-5) helped to easily identify the
20
21 206 lactonization position. The spectra shared a signal with a multiplicity for a doublet of doublets (J3-
22
23
24 207 4= 9.0 and J3-2 = 3.0 Hz) centered between δH 5.5-5.7 (Table 1), which corresponded to the signal
25
26 208 at position C-3 (δC 77-79) of the second monosaccharide unit, the first internal rhamnose (Rha).
27
28
29 209 For glycolipid 3, the signals for position C-3 of rhamnose unit was centered at δH 5.60/δC 77.8 (Fig.
30
31 210 S18); in compound 4, these signals were at δH 5.69/δC 78.7 (Fig. S24); and for 5, the position of
32
33
34
211 lactonization was identified at δH 5.58/δC 78.2 (Fig. S30). The observed multiplicity for this
35
36 212 position in 3-5, as a diagnostic doublet of doublets (J3-4 = 9.0 and J3-2 = 3.0 Hz), was different with
37
38
39
213 the one observed for the C-2 signal of the same Rha unit centered at H 5.29 (δC 72.7) as a doublet
40
41 214 of doublets (J2-3 = 2.9 and J2-1 = 1.5 Hz) for the macrolactonization in hamiltonin I (2) (Moreno-
42
43 215 Velasco et al., 2022) and similar to the same signal in known compounds 7 and 8 with a
44
45
46 216 macrolactonization at Rha-3 (Fig. S7 and Fig. S14).
47
48 217
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22 218
23
24
25 219 7 R = n-C10H19O
26 220 8 R = n-C12H23O
27
28 221
29
30 222 Consequently, the remaining signal in this low-field zone in the 1H NMR spectra was
31
32
33 223 assigned to the position of acylation of each oligosaccharide nucleus. As illustrated in the piled
34
35 224 spectra of 3-5 (Fig. S11 and Fig. S12), the signals centered between δH 5.70-5.85, except for those
36
37
38 225 described for the anomeric protons, differed in their chemical shifts in each of the samples as
39
40 226 follows: in compound 3, a doublet of doublets (J2-3 = 3.3, J2-1 = 0.8 Hz) corresponding to the C-2
41
42
43
227 position (δH 5.81/δC 74.1) of the second internal rhamnose (Rha') was observed as a result of one
44
45 228 axial-equatorial interaction with the adjacent H-3 proton and a second one equatorial-equatorial
46
47 229 with the anomeric proton; in compound 4, a doublet of doublets (J3-4 = 9.0 and J3-2 = 3.0 Hz) was
48
49
50 230 identified, which corresponds to position C-3 (δH 5.75/δC 76.1) of the second internal rhamnose
51
52 231 (Rha'), and showed trans-diaxial and axial-equatorial vicinal interactions; and for compound 5, a
53
54
55 232 doublet of doublets (J4-3 = 9.9 and J4-5 = 9.8 Hz) was observed in the form of a triple-like signal
56
57 233 due to two trans-diaxial couplings corresponding to position C-4 (δH 5.84/δC 75.3) of the terminal
58
59
60 234 rhamnose (Rha''). Finally, the use of TOCSY experiments helped to easily identify all signals in
61
62
63
64
65
 1
2
3
4 235 each pyranose and by inspection of their multiplicity all signals were assigned (Table 1). Rha' H-2
5
6
7 236 (δH 5.81) for hamiltonin II (3), where the position of esterification was identified, generated
8
9 237 correlations in the TOCSY spectrum for all protons in the second rhamnose unit at δH 5.58 (d, 1.7
10
11
12 238 Hz, Rha'-1), 4.62 (dd, 9.1, 3.3 Hz, Rha'-3), 4.32 (dd, 9.3, 9.3 Hz, Rha'-4), and 4.34 (m, Rha'-5),
13
14 239 which were used to identify this pyranoside spin system. In addition, fucose H-4 (3.95 d), Rha-
15
16
17 240 1 (δH 6.37), Rha-5 (δH 5.01), and Rha''-2 (δH 4.81) were also selected to recognize all their TOCSY
18
19 241 correlation as illustrated in Fig. S17. This combined approach for a preliminary analysis of HSQC
20
21
22
242 followed by detailed examination of the TOCSY was used for all undescribed hamiltonins to
23
24 243 differentiate all the individual pyranoside spins systems in each undescribed compound. Tables 1-
25
26 244 3 summarize all chemical shift assignments after a careful analysis of 2D experiments such as
27
28
29 245 COSY, TOCSY, ROESY, HSQC, and HMBC (Castañeda-Gómez et al., 2023; Pereda-Miranda et
30
31 246 al., 2010).
32
33
34
35 247 For this type of macrocylic acyl sugars, the carbonyl resonance for the lactone is always
36
37 248 centered at ca. 172-173 ppm (Castañeda-Gómez et al., 2013; Moreno-Velasco et al., 2022) (Table
38
39
40 249 2; Fig. S12), which is undoubtedly recognized through the correlations (2JCH) with the non-
41
42 250 magnetically equivalent diastereotopic C-2 methylene protons (alpha to the carbonyl C-1) of the
43
44
45 251 aglycone; for example, the carbonyl center at δ 173.3 in compound 5 displayed a correlation (3JCH)
46
47 252 with H-4 (δ 5.84) of the terminal rhamnose (Rha'') and a vicinal interaction (2JCH) with each the C-
48
49
50 253 2 methylene protons at 2.28 and 2.13 (Fig. S31). Therefore, the additional carbonyl signal in each
51
52 254 individual spectrum was assigned to the C-1 position of the n-dodecanoyl residue and found around
53
54
55 255 174 ppm (Fig. S12) (Moreno-Velasco et al., 2022).
56
57
58
59
60
61
62
63
64
65
 1
2
3
4 256 The retention of pure acylated sugars in a C-18 reversed-phase is mainly determined by
5
6
7 257 their hydrophobicity. Esterified resin glycosides with a large hydrophobic area (esterifying
8
9 258 residues) infiltrate deeper in the stationary phase than those analytes with a smaller area. For
10
11
12 259 compounds 3-5, the furthest the aliphatic chain of the acylating residue was with respect to the
13
14 260 macrocyclic aglycone, the interactions of the eluates with the C-18 support were superior, because
15
16
261 there is less steric hindrance imposed by the macrolactone. Consequently, hamiltonin V (6), as a
17
18
19 262 diacylated ester-type dimer, presents the largest hydrophobic area and the elution sequence for
20
21 263 compounds 3-6 as a function of their acylation was hamiltonin II (3, tR = 18.5 min) < hamiltonin
22
23
24 264 III (4, tR = 28.0 min) < hamiltonin IV (5, tR = 33.1 min) < hamiltonin V (6, tR = 38.5 min).
25
26 265 For hamiltonin V (6), low resolution or high-resolution exact-mass ESI-MS analyses were
27
28
29 266 not able to produce protonated/deprotonated molecules ([M + H]/ [M H] ) or the cluster adduct
30
31
32
267 formation with alkaline salts in significant abundance (Demarque et al., 2026; Huang et al., 1999),
33
34 268 such as the cationized molecule at m/z 2065 [M + Na] in the positive mode or the chloride-
35
36
37 269 attachment ion at m/z 2077/2079 of the form [M Cl]− in negative mode (Castañeda-Gómez et al.,
38
39
40
270 2013) in contrast, the ions at m/z 1043 [M/2 + Na]+ and 1019 [M/2  H] were always obtained as
41
42 271 the single product ions despite the use of different solvent combinations or salt solutions for the
43
44 272 sample introduction into the spray needle/capillary, even upon decreasing the cone voltage settings
45
46
47 273 from 150 to 15 V. These detected ions indicated that the fragmentation pathway for hamiltonin V
48
49 274 (6) resulted from the easy cleavage of a symmetrically glycolipid ester-type homodimer. For the
50
51
52 275 previously described dimeric structures of resin glycosides (Bah and Pereda-Miranda, 1997;
53
54 276 Castaneda-Gómez and Pereda-Miranda, 2011; Castañeda-Gómez et al., 2013; Escalante-Sánchez
55
56
57 277 and Pereda-Miranda, 2007; Rosas-Ramírez et al., 2011), this cleavage produced the diagnostic
58
59 278 [M/2  H]and [M/2  H H2O]ions, corresponding to each of the monomeric units A and B,
60
61
62
63
64
65
 1
2
3
4 279 where the [M/2  H H2O]ion indicated a macrocyclic moiety for unit A and the other
5
6
7 280 represented the linear second unit B [M/2  H]where the ester-type linkage is stablished and,
8
9
10
281 therefore, a linear oligosaccharide is characterized for this unit (Bah and Pereda-Miranda, 1997;
11
12 282 Castañeda-Gómez et al., 2013; Escalante-Sánchez and Pereda-Miranda, 2007; Rosas-Ramírez et
13
14 283 al., 2011). The presence of only one high-mass fragment ions in ESI-MS (Fig. S33), at both positive
15
16
17 284 (m/z 1043 [M/2 + Na]+) and negative (m/z 1019 [M/2  H]) modes, for compound 6 indicated that
18
19 285 both units A and B were connected through an ester-type linkage with each other as linear
20
21
22 286 oligosaccharides and confirmed by the highly symmetric substitution pattern for both
23
24 287 tetrasaccharide units of the dimeric structure in NMR.
25
26
27 288 To avoid the difficulties found with the ESI-MS analysis for the detection of a cluster
28
29 289 adduct, positive ion mode LR- and HR-MALDI-TOF-MS spectra of hamiltonin V (6) with a matrix
30
31
32 290 of 2,5-dihydroxybenzoic acid (DHB) (Teearu et al., 2014) supplemented with AgTFA were
33
34 291 recorded (Habumugisha et al., 2022), as illustrated in Fig S32A. This combination of the polar
35
36 292 matrix DHB with the silver salt produced a silver-matrix adduct ion of very low abundance at m/z
37
38
39 293 2302.1777 [M + Matrix (C7H6O4) + Ag]+ with the isotopic distribution and relative abundances
40
41
42 294 for the molecular formula of C111H190O42Ag (Mass accuracy: = 0.5 ppm; Fig. S32B) and the
43
44 107 109
295 characteristic mass difference from isotope ratios between Ag and Ag (Fig. S32C). The
45
46
47 296 addition of Ag+ increased ionization efficiency and thus detectability of the silver-matrix adduct
48
49 297 ion. As expected, the adduct at m/z 1127/1129 [M/2 + 107
Ag/109Ag]+ was detected with a high
50
51
52 298 relative abundance (Fig. S32D).
53
54 299 The registered values for NMR spectra for compounds 3 and 4 were used as the starting
55
56
57 300 points to compare these macrocyclic tetrasaccharides with the signals recorded for each dimeric
58
59 301 unit of hamiltonin V (6), which were arbitrarily designated as units A and B (Fig. S11 and Fig.
60
61
62
63
64
65
 1
2
3
4 302 S12). A combination of 1H NMR spectra (Table 3) and 2D homo-nuclear techniques, COSY (Fig.
5
6
7 303 S36), and TOCSY (Fig. S37) permitted all C-bonded protons to be recognized within each pyranose
8
9 304 system (Table 3). The analysis of the 1H-detected {1H, 13C} one-bond correlation experiment by
10
11
12 305 HSQC (Fig. S38) allowed to assign all the resonances in the 13C NMR spectra (Table 3), where
13
14 306 eight diagnostic signals in the anomeric region (ca. 99 to 103 ppm) were registered. Six resonances
15
16
307 observed in the downfield region δ 5.50-6.40 (Table 1) were assigned to the anomeric protons for
17
18
19 308 the rhamnose unit because their multiplicity for each signal is a broad singlet (J = 0.8-1.2 Hz) as
20
21 309 follows (HSQC; Fig. S38): for unit A: Rha-1 δH 6.40 (δC 99.7), Rha'-1 δH 5.92 (δC 101.8), and Rha''
22
23
24 310 δH 5.74 (δC 102.9), while for unite B: Rha-1 δH 6.38 (δC 99.7), Rha'-1 δH 5.58 (δC 100.1), and Rha''
25
26 311 δH 6.25 (δC 103.0). Two fucose anomeric protons as doublets (J = 7.8 Hz) were recognized at 4.79
27
28
29 312 (δC 101.2; unit A) and 4.78 (δC 101.1; unit B). Furthermore, four paramagnetically shifted non-
30
31 313 anomeric protons indicated the sites of acylation at Rha-3 (δH 5.69/δC 78.5) and Rha'-3 (δH 5.74/δC
32
33
34
314 72.1) for the first pair of signals for monomeric unit A, as well as Rha-3 (δH 5.56/ δC 77.5) and
35
36 315 Rha'-2 (δH 5.70/ δC 73.9) for unit B. The convergence of all the and 1H and 13C NMR signals (Table
37
38 316 3) for hamiltonin V (6) with those registered for hamiltonins II (3) and III (4) (Table 1 and 2)
39
40
41 317 revealed the structural complexity resulting from an intramolecular condensation between the two
42
43 318 linear tetrasaccharides to yield an almost symmetrical homodimer by establishing the ester-type
44
45
46 319 bond at position Rha-3 of both units and only differing in the position of esterification by the n-
47
48 320 dodecanoic acid at the third saccharide unit in both units A (Rha'-3) and B (Rha'-2) of compound
49
50
51 321 6. Hamiltonin V (6) represents the first resin glycoside with an ester-type dimer structure that
52
53 322 consists of two units of operculinic acid C (1).
54
55
323
56
57
58 324
59
60 325
61
62
63
64
65
 1
2
3
4 326 2.3 Cytotoxicity enhancement of clinical drugs
5
6
7 327 Cytotoxicity of all isolated compounds was evaluated against a panel of human cancer cell
8
9 328 lines, namely, breast cancer (sensitive MCF-7 and multidrug resistant MCF-7/Vin), cervical
10
11
12 329 carcinoma (Hela), and colorectal cancer (HCT-116), using the sulforhodamine B (SRB)
13
14 330 colorimetric assay (Vichai and Kirtikara, 2006). The MDR MCF-7/Vin cell line was developed
15
16
331 through uninterrupted exposition to vinblastine during three consecutive years and maintained in
17
18
19 332 vinblastine-supplemented medium for over 10 years (Figueroa-González et al., 2012). The MDR
20
21 333 phenotype in the MCF-7/Vin cell line was demonstrated to be preserved as a cross-resistance to
22
23
24 334 multiple structurally and functionally different drugs (Lopes-Rodrigues et al., 2017) such as
25
26 335 vinblastine, podophyllotoxin, colchicine, and ellipticine (Table 4). As expected, the developed
27
28
29 336 MDR cell lines MCF-7/Vin demonstrated a higher vinblastine susceptibility value (IC50) up to 41-
30
31 337 fold than that of MCF-7 drug-sensitive cells (Table 4). All tested resin glycosides did not display
32
33
34
338 any intrinsic cytotoxicity (IC50 >25 μM), which is an important requirement for performing the
35
36 339 proposed combination testing with clinical drugs, such vinblastine and podophyllotoxin, to be able
37
38
39 340 to specifically identify any potentiation effect of the resin glycosides with the combined antitumor
40
41 341 drugs in this MDR MCF-7/Vin line (Moreno-Velasco et al., 2022).
42
43
44
342 The potentiation effects by resin glycosides 1-6 at the concentrations of 1-50 M to enhance
45
46 343 vinblastine and podophyllotoxin cytotoxicity at sublethal doses (0.003 M) was investigated and
47
48
49 344 the results of the SRB assay for both cytotoxic agents are given in Table 5. Based on these results,
50
51 345 a dose-response curve was obtained and the IC50 value for the combination of compound 1-6 was
52
53
54 346 calculated (Fig. S40). The number of surviving cells was compared with an untreated control after
55
56 347 48 h. All these resin glycosides showed a remarkable enhancement for both vinblastine and
57
58
59 348 podophyllotoxin cytotoxicity even at 5 μM, except for compound 5 which only displayed an
60
61
62
63
64
65
 1
2
3
4 349 important increase after 10 μM (Fig. 1). For instance, while vinblastine and podophyllotoxin at
5
6
7 350 0.003 M induced no cell death, supplementation of 10 μM of hamiltonin V (6) increased this
8
9 351 value to 55% (IC50 10.6 μM) and 15% (IC50 14.3 μM), respectively, in MCF-7/Vin cells (Fig. 2),
10
11
12 352 in a similar manner as previously described for hamiltonin I (2) with an IC50 10.9 μM for the
13
14 353 combination with vinblastine (Moreno-Velasco et al., 2022). Table 5 summarizes the half maximal
15
16
17 354 inhibitory concentration (IC50) values as the enhancement of cytotoxic potency of the partner active
18
19 355 drugs at a sublethal dose (0.003 M) in relation to the tested non-cytotoxic hamiltonins II-V (3-6)
20
21
22 356 concentrations. Microscopic visualization of MCF-7/Vin cells (Fig. 3A, control) after 48 h of
23
24 357 incubation with a sub-inhibitory concentration of vinblastine (Fig. 3B) and podohyllotoxin (Fig.
25
26
27 358 3C) at 0.003 M in combination with halmitonin V (6) at a concentration of 30 M illustrates the
28
29 359 potentiation effects for the combination with non-cytotoxic resin glycosides which enhanced the
30
31
32 360 efficacy of the clinical drugs, while treatment with each of the resin glycosides alone did not cause
33
34 361 cell death.
35
36
37 362 Hamiltonins II-V (2-6), in combination with clinical antitumoral agents, restored the
38
39 363 chemotherapeutic susceptibility in MCF-7/Vin cells; therefore, these highly amphiphilic
40
41
42
364 compounds appear to interact with their targets, the efflux pumps that confer resistance to the MCF-
43
44 365 7/Vin cells, as previously demonstrated for some individual resin glycosides by rhodamine-123
45
46 366 efflux experiments and flow cytometry after incubating with the UIC2 anti-MDR1 mouse
47
48
49 367 monoclonal antibody, specific for P-glycoprotein (P-gp) (Figueroa-González et al., 2012;
50
51 368 Castañeda-Gómez et al., 2013; Lira-Ricárdez and Pereda-Miranda, 2020). Therefore, this
52
53
54 369 mechanism of potentiation by non-cytotoxic resin glycosides appears to modulate the regulation of
55
56 370 drug internalization (Hu et al., 2016), which would enhance intracellular drug concentration of
57
58
59
371 vinblastine and podophyllotoxin. Therefore, the inhibition of drug efflux and the resulting
60
61
62
63
64
65
 1
2
3
4 372 accumulation of cytotoxic agents inside the cells helped to maximize the efficacy of the partner
5
6
7 373 cytotoxic drugs (Duarte and Vale, 2020; Duarte et al., 2021).
8
9 374 The lipophilicity/hydrophilicity balance, previously described as an important
10
11
12 375 consideration for the bioactivity of resin glycosides (Lira-Ricárdez and Pereda-Miranda, 2020),
13
14 376 was demonstrated from the stronger potentiation effect displayed for the hamiltonins I (2) and V
15
16
377 (6) that could be a consequence of their larger hydrophobic area (the number and position of the
17
18
19 378 lipophilic alkylating substituents), which may facilitate an irreversible binding between these resin
20
21 379 glycosides with their biological targets, the multidrug and xenobiotic extrusion pumps of the ABC
22
23
24 380 family. This conclusion is sustained by the inactivity verified as a cytotoxicity agent by operculinic
25
26 381 acid C (1), the glycosidic core of all isolated hamiltonins I-V (2-6), which did not achieve an
27
28
29 382 inhibition of the proliferation of MDR MCF-7 breast cancer cells in combinations with both clinical
30
31 383 tested drugs (vinblastine or podophyllotoxin: IC50 > 50 μM; Table 5).
32
33
34
384 The macrocyclic structure is an essential requirement for the biological activity of resin
35
36 385 glycosides (Lira-Ricárdez and Pereda-Miranda, 2020). All properties are totally lost, including
37
38 386 cytotoxicity and purgative action, in the glycosidic acid derivatives prepared by saponification of
39
40
41 387 intact resin glycosides, and suggested both the importance of the overall lipophilicity to the
42
43 388 molecule’s potency and the need for a macrocyclic structure as the pharmacophore, which
44
45
46 389 effectively constrains the oligosaccharide core into bioactive conformations (Rencurosi et al.,
47
48 390 2004).
49
50
51 391
52
53 392 3. Conclusions
54
55
56 393 The accumulation of cytotoxic agents and their regulation inside the malignant cells through
57
58 394 enhancement of their intracellular concentrations by the action of efflux pumps inhibitors, such as
59
60
61
62
63
64
65
 1
2
3
4 395 non-cytotoxic resin glycosides, is a clear example of a potentiation effect of drug combinations.
5
6
7 396 Macrocyclic resin glycosides with a certain degree of amphiphilicity seems to be an important
8
9 397 requirement for the inhibition of drug efflux to contribute to the accumulation of cytotoxic agents,
10
11
12 398 such as vinblastine and podophyllotoxin, to destabilize microtubules by binding tubulin and thus
13
14 399 preventing cell division. The potentiation effects demonstrated by the combinations of
15
16
400 chemotherapeutical drugs and the non-cytotoxic oligosaccharides in cocktail chemotherapy could
17
18
19 401 be further investigated in vivo and may be helpful for repurposing of conventional antineoplastic
20
21 402 drugs.
22
23
24 403
25
26 404 4. Experimental
27
28
29 405 4.1 General experimental procedures
30
31
32
406 Melting points were determined on a Fisher-Johns 220 VAC apparatus (Thermo Scientific)
33
34 407 and are uncorrected. Optical rotations were measured with a PerkinElmer model 341 polarimeter
35
36 408 using methanol as solvent. NMR spectra were recorded in C5D5N solution, using micro-NMR
37
38
39 409 sample tubes (2.5 mm o.d. stem; sample volume 115 μL at 30 mm height), on a Varian VNMRS
40
41 410 instrument at 400 MHz and a Bruker Avance III HD spectrometers at 900 MHz, using
42
43
44 411 tetramethylsilane as an internal standard. Low-resolution ESIMS data were measured using a
45
46 412 Waters SQD2 system (Waters) equipped with an electrospray ionization source (ESI) by direct
47
48
49
413 infusion of the purified compounds according to the analytical conditions previously described
50
51 414 (Montiel-Ayala et al., 2021; Moreno-Velasco et al., 2022). High resolution ESIMS of intact
52
53 415 isolated compounds were carried out using a quadrupole time-of-flight mass spectrometer (UPLC-
54
55
56 416 ESI-QTOF-MS Xevo G2 XS, Waters). The following operating ESI source conditions were used:
57
capillary voltage, 1.5 kV; sample cone, 25 V; source temperature, 100 ◦C; desolvation temperature,
58
59 417
60
61
62
63
64
65
 1
2
3
250 ◦C; cone gas flow rate 50 L/h; desolvation gas (N2) flow rate 850 L/h. Leucine-enkephalin (5
4
5 418
6
7 419 ng/mL) in CH3CN-H2O (1:1, 0.1% formic acid), introduced by a lock spray at 10 μL/min for
8
9
10
420 accurate mass acquisition, was used as lock mass, generating for positive-ion mode m/z 556.2771
11
12 421 [M + H]+ and for negative-ion mode m/z 554.2615 [M – H]–. For the ESIMs analysis of compound
13
14 422 6, cesium iodide (50 ng/μl) in 2-propanol/water (1:1) was used and the cluster ions m/z 2465.1936
15
16
17 423 (CsI)9I and m/z 2471.1946 (CsI)9Cswere used for accurate mass detection in negative and
18
19 424 positive modes, respectively (Mochizuki, 2015). Data acquisition was achieved using UNIFI
20
21
22 425 Scientific Information System (Waters, Milford, MA, USA). For MALDI-TOF-MS of compound
23
24 426 6, a solution (1 L) of 2,5-dihydroxybenzoic acid in 50% acetonitrile/2.5% trifluoroacetic acid in
25
26
27 427 ultrapure water (Milli-Q Advantage A 10, Millipore) at the concentration of 10 mg/ml and the
28
29 428 cationized reagent (AgTFA) (Teearu et al., 2014) at a concentration of 0.01 M in THF was added
30
31
32 429 to each spot of a stainless-steel sample disc and allowed to evaporate at room temperature, leaving
33
34 430 the solid sample mixture for analysis. LR-MALDI-TOF-MS was performed with the MicroFlex
35
36
37 431 LT MS as instructed by the manufacturer: operating in linear positive mode with following
38
39 432 operational conditions: laser frequency, 60 Hz; laser attenuator, 20-30 %; gain detector, 1.0x;
40
41
42
433 spectrum range, 100-700 Da; ion source 1: 18 kV, ion source 2: 15 kV, lens: 6 kV, pulsed ion
43
44 434 extraction: 0 ns. Spectra were measured randomly by 500 laser shots. The spectra were analyzed
45
46 435 using the Flex Control 3.0 software (Bruker Daltonics, Inc., Billerica, MA, USA). HR-MALDI-
47
48
49 436 TOF-MS mass spectra for compound 6 was obtained with Varian 930 FT-ICR-MS instrument with
50
51 437 7 Tesla superconducting magnet. It has an intermittent pressure (103 Torr) MALDI source with a
52
53
54 438 New Wave Orion 50083 Nd:YAG laser (355 nm, 4-mJ energy output, pulse length 4–5 ns)
55
56 439 according to procedures previously described. Applied Biosystems stainless-steel 192 + 6 spot
57
58
59 440 sample plate was used. Varian OMEGA 9.1.21 version software was used to operate the instrument,
60
61
62
63
64
65
 1
2
3
4 441 collect, and process mass spectra. GC-MS was performed on a Thermo-Electron equipment with a
5
6
7 442 DB-5MS column, 5%-phenyl-methylpolysiloxane (30 m × 0.25 mm, 0.1 µm) as stationary phase
8
9 443 and He as mobile phase as previously described (Montiel-Ayala et al., 2021).
10
11
12 444 4.2 Plant material
13
14 445 The powdered roots of Operculina hamiltonii (five bags containing 200 g each) were
15
16
446 purchased under the name of “jalapa em pó” and/or “batata-de-purga” (lot # 1111010010473,
17
18
19 447 Mercadão Natural, São Paulo, Brazil) through the internet on July 11, 2019 (Fig. S2A). Application
20
21 448 of a previously described HPLC-ESIMS fingerprinting methodology to dereplicate operculinic
22
23
24 449 acids A and C (1) was used for quality control of the pulverized dried roots to guarantee
25
26 450 reproducibility of our chemical results (Montiel-Ayala et al., 2021). The commercial powders of
27
28
29 451 jalap roots were also identified by co-authors S.G. Leitão and R. Pereda-Miranda by comparing
30
31 452 with the microscopic anatomical characteristics described for Operculina species (Michelin et al.,
32
33
34
453 2010).
35
36 454 4.3 Extraction and isolation
37
38 455 Powdered roots (950 g) were extracted by successive maceration phases (3 × 4 L) with a
39
40
41 456 mixture of CH2Cl2-CH3OH (1:1). The extracts were evaporated to dryness under reduced pressure
42
43 457 to obtain a deep brownish wax-like residue. The total extract (49 g) was fractionated by CC over
44
45
46 458 Si gel (500 g) and solvent mixtures of increasing polarity (CH2Cl2, Me2CO and MeOH). This
47
48 459 procedure afforded sixty eluates (250 mL) that were monitored by TLC and combined into twelve
49
50
51 460 fractions (Fig. S2B). The fractions were analyzed by NMR spectroscopy and those eluates rich in
52
53 461 resin glycosides were labeled as F9 (7.1 g; Fig. S2C), eluted with CH2Cl2-Me2CO (1:1), and F10
54
55
462 (7.9 g), eluted with Me2CO-CH2Cl2, (4:1), were chosen for their resolution. The following analytic
56
57
58 463 conditions were established for the best resolution of both fractions using a C18 column (4.6 × 250
59
60 464 mm, 5 μm; Waters Symmetry); mobile phase: CH3CN-H2O (4:1), flow rate: 0.4 mL/min. These
61
62
63
64
65
 1
2
3
4 465 parameters were extrapolated to the following preparative conditions: column C18 (19 × 300 mm,
5
6
7 466 7 μm; Waters Symmetry), mobile phase: CH3CN-H2O (4:1), flow rate: 8.0 mL/min; sample
8
9 467 injection: injection volume, 500 μL (50 mg/500 μL in MeOH). From F9, two eluates were collected,
10
11
12 468 F9-1 (40.1 mg; tR = 22.5 min) and F9-2 (30 mg; tR =31.1 min). Each peak was reinjected in the
13
14 469 same column and further recycled with a mobile phase of CH3CN-H2O (5:1) (Huang et al., 1999).
15
16
470 Subfraction F9-1 was resolved into two peaks after the first cycle (Fig. S3A). The first peak was
17
18
19 471 additionally recycled to obtain after seven consecutive cycles pure compound 3 (15.3 mg; tR = 18.5
20
21 472 min). Collection of the second eluate afforded pure compound 4 (16.0 mg: tR = 28.0 min).
22
23
24 473 Subfraction F9-2 (tR = 33.1 min) was purified by the same methodology after nine consecutive
25
26 474 cycles to afford pure compound 5 (20.5 mg; Fig. S3B). Subfraction F9-3 (tR = 38.5 min;) was
27
28
29 475 individually recycled through a C-18 column with CH3CN-H2O (5:1) to yield after seven
30
31 476 successive cycles pure compound 6 (12 mg; Fig. S3C). Finally, from fraction F10, semipreparative
32
33
34
477 HPLC was used to collect two major peaks with tR = 14.5 min (F10-1) and 21.7 min (F10-2) (Fig.
35
36 478 S4). UHPLC-ESI-MS in negative ion mode permitted the identification of batatinoside VI (7) from
37
38 479 subfraction F10-1; in addition, a coelution experiment in HPLC-refractive index with an authentic
39
40
41 480 sample (Fig. S5C) corroborated the identity for this pentasaccharide (Moreno-Velasco et al., 2022).
42
43 481 Recycling HPLC of subfraction F10-2 (recycled eluate: tR = 21.7 min; Fig. S4) allowed for the
44
45
46 482 purification of batatinoside IX (8), which was identified by HPLC coelution experiments with an
47
48 483 authentic sample (Rosas-Ramírez and Pereda-Miranda, 2013).
49
50
51 484 4.3.1 Hamiltonin II (3)
52
53 485 White solid; mp 105-107 °C; ORD (c 1.8, MeOH) [α]22589 55.0, [α]578 57.8, [α]546 63.9,
54
55
56 486 [α]436 104.4, [α]365 157.8; 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data,
57
58
59
60
61
62
63
64
65
 1
2
3
4 487 see: Tables 1 and 2 (Fig. S14 and Fig. S15); HRESIMS m/z 1043.6161 [M + Na]+ (calcd for
5
6
7 488 C52H92O19Na requires 1043.6125,  ppm) (Fig. S13).
8
9 489 4.3.2 Hamiltonin III (4)
10
11
12 490 White solid; mp 105-106 °C; ORD (c 0.6, MeOH) [α]22589 46.7, [α]578 48.1, [α]546 54.3,
13
14 491 [α]436 90.0, [α]365 131.9; 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data,
15
16
17 492 see: Tables 1 and 2 (Fig. S20 and Fig. S21); HRESIMS m/z 1019.6140 [M  H] (calcd for
18
19
20 493 C52H91O19 requires 1019.6160, 1.9 ppm) (Fig. S19).
21
22 494 4.3.3 Hamiltonin IV (5)
23
24
25 495 White solid; mp 106-108 °C; ORD (c 3.0, MeOH) [α]22589 50.3, [α]578 52.3, [α]546 59.0,
26
27 496 [α]436 97.3, [α]365 146.7; 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data,
28
29
30 497 see Tables 1 and 2 (Fig. S26 and Fig. S27); HRESIMS m/z 1043.6128 [M  Na]+ (calcd for
31
32
498 C52H92O19Na requires 1043.6125,  ppm) (Fig. S25).
33
34
35 499 4.3.4 Hamiltonin V (6)
36
37
38
500 White solid; mp 110-112 °C; ORD (c 4.1, MeOH) [α]22589 68.5, [α]578 71.7, [α]546 80.7, [α]436
39
40 501 132.2, [α]365 198.5; 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see
41
42
43 502 Tables 3 and 4 (Fig. S34 and Fig. S35); HRESIMS m/z 2302.1782 [M  Matrix (C7H6O4) + Ag]+
44
45 503 (calcd for C111H190O42Ag requires 2302.17777, ppm) (Fig. S32).
46
47
48 504 4.3.5 Batatinoside VI (7)
49
50 505 White amorphous solid; mp 140-142 °C; ORD (c 0.3, MeOH) [α]22589 16.7, [α]578 20.0, [α]546
51
52
53 506 20.1, [α]436 30.0, [α]365 40.0; HRESIMS m/z 1177.6293 [M  Na]+ (calcd for C56H98O24Na
54
55 507 requires 1177.6340). This compound was identified by HPLC coelution experiments with an
56
57
58 508 authentic sample and literature data (Moreno-Velasco et al., 2022).
59
60 509 4.3.6 Batatinoside IX (8)
61
62
63
64
65
 1
2
3
4 510 White solid; mp 118-120 °C; ORD (c 1.0, MeOH) [α]22589 51.0, [α]578 54.0, [α]546 61.0, [α]436
5
6
7 511 99.0, [α]365 150.0; HRESIMS m/z 1205.6601 [M  Na]+ (calcd for C58H102O24Na requires
8
9
10
512 1205.6653 (Fig. S6); 1H NMR (400 MHz, C5D5N) and 13
C NMR (100 MHz, C5D5N) data (Fig.
11
12 513 S7). This compound was identified by HPLC coelution experiments with an authentic sample and
13
14 514 literature data (Moreno-Velasco et al., 2022; Rosas-Ramírez and Pereda-Miranda, 2013).
15
16
17 515 4.4 Alkaline Hydrolysis
18
19
20 516 Fraction F9 (10 mg) was dissolved in 2.5 mL of 5% KOH and refluxed at 95 °C for 3 h.
21
22 517 The solution was acidified to pH 5 with 1 N HCl and extracted with CH2Cl2 (3 × 10 mL). The
23
24
25 518 organic layers were washed with H2O, dried over anhydrous Na2SO4, and evaporated under
26
27 519 reduced pressure. The residue was analyzed by GC-MS under the conditions previously described
28
29
30 520 (Montiel-Ayala et al., 2021) to only detect a major peak with tR 9.1 min: n-dodecanoic acid [M]+.
31
32
33 521 200 (9), 183 (2), 171 (15), 157 (38), 143 (9), 129 (45), 115 (22), 101 (15), 85 (30), 73 (100), 60
34
35 522 (68), 57 (30), 55 (30).
36
37
38 523 The aqueous layers from the saponification were individually extracted with n-BuOH (3 ×
39
40
41
524 50 mL) and concentrated to give a solid which was analyzed by coelution experiments in HPLC
42
43 525 with an authentic sample of operculinic acid C (1). HPLC conditions: NH2-column for
44
45 526 carbohydrate analysis (Waters; 10 m, 3.9 × 300 mm), isocratic elution of CH3CN–H2O (1:1), and
46
47
48 527 a flow rate of 0.4 mL/min, this procedure allowed to identify operculinic acid C (1, tR = 8.3 min)
49
50
528 as the glycosidic acid constituent of the resin glycoside cores in fraction F9 (6.8 mg). Comparison
51
52
53 529 of its physical constants and NMR data was performed with published values (Moreno-Velasco et
54
55 530 al., 2022).
56
57
58 531
59
60 532
61
62
63
64
65
 1
2
3
4 533 4.4.1 Operculinic acid C (1)
5
6
7 534 White solid; mp 118 °C; [α]22589  49.2 (c 0.2, MeOH) ([]D 48.3); HRESIMS m/z
8
9
10
535 855.4606 [M  (Fig. S8). 1H NMR (900 MHz, C5D5N) and 13C NMR (225 MHz, C5D5N) data
11
12 536 (Fig. S9). This compound was identified by comparison with literature data (Moreno-Velasco et
13
14 537 al., 2022).
15
16
17 538 4.5 Cytotoxicity and Combination Assays
18
19 539 Human colorectal (HCT-116 ATCC CCL-247), cervical carcinoma (Hela ATCC CCL-2),
20
21
22 540 and breast cancer (MCF-7 ATCC HTB-22) cell lines were obtained from the American Type
23
24 541 Culture Collection (Manassas, VA, USA) and maintained according to ATCC’s guideline at 37 °C
25
26
27 542 and 5% CO2 in an appropriate medium supplemented with 10% fetal bovine serum, 100 U/mL
28
29 543 penicillin G, and 100 μg/mL streptomycin. The MDR MCF-7/Vin cell line was established through
30
31
32 544 exposition to vinblastine during three consecutive years and continued for a decade (Figueroa-
33
34 545 González et al., 2012). In order to preserve the drug resistance phenotype, MCF-7/Vin cells were
35
36
37 546 cultured in medium containing 0.192 μg/mL vinblastine. A stock of MCF-7/Vin− cells was
38
39 547 conserved in vinblastine-free medium. All cancer cells were maintained in the logarithmic growth
40
41
42 548 phase. The medium was changed every two days and trypsinized (0.25% trypsin-EDTA). A total
43
44 549 of 200 μL of Hela (5000 cells/well), HT-29 cells (7500 cells/well) or MCF-7 cells (5000 cells/well)
45
46
47 550 were seeded in 96-well plates and allowed to adhere overnight before test compound exposure.
48
49 551 After 24 h, the medium was replaced with 200 μL of drug-containing medium at the appropriate
50
51
52
552 drug dose: solution of tested compound (30, 25, 20, 15, 10, 5 and 1 mM) and vinblastine or
53
54 553 podophyllotoxin at sublethal concentrations of 0.003 mM. Cells were exposed to test compounds
55
56 554 for 48 h, followed each by a SRB assay (Figueroa-González et al., 2012). Results are expressed as
57
58
59 555 the concentration that inhibited 50% control growth after the incubation period (IC50). The values
60
61
62
63
64
65
 1
2
3
4 556 were estimated from a semilog plot of the drug concentration (μM) against the percentage of growth
5
6
7 557 inhibition. Vinblastine, podophyllotoxin, and colchicine were included as positive-control
8
9 558 compounds (Moreno-Velasco et al., 2022).
10
11
12 559
13
14 560 Acknowledgements
15
16
561 Financial support was provided by the Dirección General de Asuntos del Personal Académico,
17
18
19 562 UNAM (DGAPA: IN202123), Conselho Nacional de Desenvolvimento Científico e Tecnológico,
20
21 563 and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (314591/2021-4 and E-
22
23
24 564 26/201.003/2021, Brazil). We are indebted to the technical personnel of the Unidad de Servicios
25
26 565 de Apoyo a la Investigación y a la Industria, Facultad de Química, UNAM, especially to N. López
27
28
29 566 Balbiaux and R. I. del Villar Morales for the recording of the 400 MHz NMR spectra, and Margarita
30
31 567 Guzmán for the performing of the UHPLC-ESI-MS analysis. This study made use of the 900 MHz
32
33
34
568 NMR instrument at the National Center for Nuclear Magnetic Resonance of Macromolecules Jiri
35
36 569 Jonas, Universidade Federal do Rio de Janeiro.
37
38 570
39
40
41 571 Author contributions
42
43 572 This article was taken in part from the Ph.D. thesis of Armando Moreno- Velasco in the Programa
44
45
46 573 de Maestría y Doctorado en Ciencias Químicas, UNAM. Sebastian Carrillo-Rojas was a graduate
47
48 574 student at UNAM from the Universidad Industrial de Santander, Facultad de Ciencias, Escuela de
49
50
51 575 Química, Bucaramanga, Colombia.
52
53 576
54
55
577
56
57
58 578
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 1
2
3
4
5
579 References
6
7 580 Al-Sanea, M.M, Gamal, M., 2022. Critical analytical review: rare and recent applications of
8
9
10 581 refractive index detector in HPLC chromatographic drug analysis. Microchem. J. 178:107339.
11
12 582 https://doi.org/10.1016/j.microc.2022.107339.
13
14 583 Bah, M., Pereda-Miranda, R., 1997. Isolation and structural characterization of new glycolipid ester
15
16
17 584 type dimers from the resins of Ipomoea tricolor (Convolvulaceae). Tetrahedron 53, 9007–9022.
18
19 585 https://doi.org/10.1016/S0040-4020(97)00607-8.
20
21
22 586 Castañeda-Gómez, J., Lavias-Hernández, P., Fragoso-Serrano, M., Lorence, A., Pereda-Miranda,
23
24 587 R., 2019. Acylsugar diversity in the resin glycosides from Ipomoea tricolor seeds as
25
26
27 588 chemosensitizers in breast cancer cells. Phytochem. Lett. 32, 77-82.
28
29 589 https://doi.org/10.1016/j.phytol.2019.05.004.
30
31
32
590 Castañeda-Gómez, J., Figueroa-González, G., Jacobo, N., Pereda-Miranda, R., 2013. Purgin II, a
33
34 591 resin glycoside ester-type dimer and inhibitor of multidrug efflux pumps from Ipomoea purga.
35
36 592 J. Nat. Prod. 76, 64-71. https://doi.org/10.1021/np300739y.
37
38
39 593 Castañeda-Gómez, J. F., Leitão, S. G., Pereda-Miranda, R., 2023. Hederifolic acids AD, hepta and
40
41 594 hexasaccharides from the resin glycosides of Ipomoea hederifolia. Phytochemistry 211, 113689.
42
43
44 595 https://doi.org/10.1016/j.phytochem.2023.113689.
45
46 596 Castaneda-Gómez, J., Pereda-Miranda, R., 2011. Resin glycosides from the herbal drug jalap
47
48
49
597 (Ipomoea purga). J. Nat. Prod. 74, 1148–1153. https://doi.org/10.1021/np200080k.
50
51 598 Corona-Castañeda, B., Pereda-Miranda, R., 2012. Morning glory resin glycosides as modulators
52
53 599 of antibiotic activity in multidrug-resistant Gram-negative bacteria. Planta Med. 78, 128-131.
54
55
56 600 https://doi.org/10.1055/s-0031-1280292.
57
58
59
60
61
62
63
64
65
 1
2
3
4 601 Cunha, G.H., Fechine, F.V., Santos, L.K., Pontes, A.V., Oliveira, J.C., Moraes, M.O., Moraes,
5
6
7 602 M.E., 2011. Efficacy of the tincture of jalap in the treatment of functional constipation: a double-
8
9 603 blind, randomized, placebo-controlled study. Contemp. Clin. Trials. 32, 153–159.
10
11
12 604 https://doi.org/10.1016/j.cct.2010.10.011.
13
14 605 Demarque, D.P., Crotti, A.E., Vessecchi, R., Lopes, J.L. and Lopes, N.P., 2016. Fragmentation
15
16
606 reactions using electrospray ionization mass spectrometry: an important tool for the structural
17
18
19 607 elucidation and characterization of synthetic and natural products. Nat. Prod. Rep. 33, 432-455.
20
21 608 https://doi.org/10.1039/c5np00073d.
22
23
24 609 Duarte, D., Cardoso, A., Vale, N., 2021. Synergistic Growth Inhibition of HT-29 Colon and MCF-
25
26 610 7 Breast Cancer Cells with Simultaneous and Sequential Combinations of Antineoplastics and
27
28
29 611 CNS. Drugs. Int. J. Mol. Sci. 22, 7408-7447. https://doi.org/10.3390/ijms22147408.
30
31 612 Duarte, D., Vale, N., 2020. New Trends for Antimalarial Drugs: Synergism between
32
33
34
613 Antineoplastics and Antimalarials on Breast Cancer Cells. Biomolecules 10, 1623-1823.
35
36 614 https://doi.org/10.3390/biom10121623.
37
38 615 Escalante-Sánchez, E., Pereda-Miranda, R., 2007. Batatins I and II, ester-type dimers of acylated
39
40
41 616 pentasaccharides from the resin glycosides of sweet potato. J. Nat. Prod. 70, 1029-1034.
42
43 617 https://doi.org/10.1021/np070093z.
44
45
46 618 Fan, B.-Y., Jiang, X., Li, Y.-X., Wang, W.-L., Yang, M., Li, J.-L., Wang, A.-D., Chen, G.-T., 2022.
47
48 619 Chemistry and biological activity of resin glycosides from Convolvulaceae species. Med. Res.
49
50
51 620 Rev. 2022, 1-42. https://doi.org/10.1002/med.21916.
52
53 621 Figueroa-González, G., Jacobo-Herrera, N., Zentella-Dehesa, A., Pereda-Miranda, R., 2012.
54
55
622 Reversal of multidrug resistance by morning glory resin glycosides in human breast cancer
56
57
58 623 cells. J. Nat. Prod. 75, 93-97. https://doi.org/10.1021/np200864m.
59
60
61
62
63
64
65
 1
2
3
4 624 Habumugisha, T., Zhang, Z., Ndayishimiye, J.C., Nkinahamira, F., Kayiranga, A., Cyubahiro, E.,
5
6
7 625 Rehman, A., Yan, C., Zhang, X., 2022. Evaluation and optimization of the influence of silver cluster
8
9 626 ions on the MALDI-TOF-MS analysis of polystyrene nanoplastic polymers. Anal. Methods. 14, 763-
10
11
12 627 772. https://doi.org/10.1039/D1AY02219A.
13
14 628 Hu, Q., Sun, W., Wang, C., Gu, Z., 2016. Recent advances of cocktail chemotherapy by
15
16
629 combination drug delivery systems. Adv. Drug Deliv. Rev. 98, 19–34.
17
18
19 630 https://doi.org/10.1016/j.addr.2015.10.022.
20
21 631 Huang, N., Siegel, M., Kruppa, G.H., Laukien, F.H., 1999. Automation of a Fourier transform ion
22
23
24 632 cyclotron resonance mass spectrometer for acquisition, analysis, and e-mailing of high-
25
26 633 resolution exact-mass electrospray ionization mass spectral data. J. Am. Soc. Mass Spectrom.
27
28
29 634 10, 1166-1173. https://doi.org/10.1016/S1044-0305(99)00089-6.
30
31 635 Lira-Ricárdez, J., Pereda-Miranda, R., Castañeda-Gómez, J., Fragoso-Serrano, M., Simas, R.C.,
32
33
34
636 Leitão, S.G., 2019. Resin glycosides from the roots of Operculina macrocarpa (Brazilian jalap)
35
36 637 with purgative activity. J. Nat. Prod. 82, 1664-1677.
37
38 638 https://doi.org/10.1021/acs.jnatprod.9b00222.
39
40
41 639 Lira-Ricárdez, J., Pereda-Miranda, R., 2020. Reversal of multidrug resistance by amphiphilic
42
43 640 morning glory resin glycosides in bacterial pathogens and human cancer cells. Phytochem. Rev.
44
45
46 641 19, 1211-1229. https://doi.org/10.1007/s11101-019-09631-1.
47
48 642 Lopes-Rodrigues, V.; Di Luca, A.; Mleczko, J.; Meleady, P.; Henry, M.; Pesic M.; Cabrera D.; van
49
50
51 643 Liempd, S.; Lima R.T.; O’Connor, R.; Falcon-Perez, J.; Vasconcelos, M.H., 2017. Identification
52
53 644 of the metabolic alterations associated with the multidrug resistant phenotype in cancer and their
54
55
645 intercellular transfer mediated by extracellular vesicles. Sci. Rep. 7, 44541.
56
57
58 646 https://doi.org/10.1038/srep44541.
59
60
61
62
63
64
65
 1
2
3
4 647 Michelin, D., Gandolfo, S.C., Silva Sacramento, L.V., Vilegas, W., Nunes Salgado, H., 2010.
5
6
7 648 Controle de qualidade da raiz de Operculina macrocarpa (Linn) Urb., Convolvulaceae. Rev.
8
9 649 Bras. Farmacogn. 20, 18-22. https://doi.org/10.1590/S0102-695X2010000100005.
10
11
12 650 Mochizuki, S., 2015. Enhanced measurement of CsI cluster ions for mass calibration in MALDI-
13
14 651 MS using sugar alcohols. Anal. Methods 7, 2215-2218. https://doi.org/10.1039/C4AY02509A.
15
16
652 Montiel-Ayala, M.E., Jiménez-Barcénas, N.R., Castañeda-Gómez, J., Moreno-Velasco, A., Lira-
17
18
19 653 Ricárdez, J., Fragoso-Serrano, M., Guimarães-Leitão, S., Pereda-Miranda, R. 2021. Glycosidic
20
21 654 acid content from the roots of Operculina hamiltonii (Brazilian Jalap) and some of their
22
23
24 655 phytopharmaceuticals with purgative activity. Rev. Bras. Farmacogn. 31, 698-709.
25
26 656 https://doi.org/10.1007/s43450-021-00190-1.
27
28
29 657 Moreno-Velasco, A., Flores-Tafoya, P.D., Fragoso-Serrano, M., Leitão, S.G., Pereda-Miranda, R.
30
31 658 2022. Resin glycosides from Operculina hamiltonii and their synergism with vinblastine in
32
33
34 659 cancer cells. J. Nat. Prod. 85, 23852394. https://doi.org/10.1021/acs.jnatprod.2c00594.
35
36 660 Pereda-Miranda, R., Hernández-Carlos, B., 2002. HPLC isolation and structural elucidation of
37
38
39 661 diastereomeric niloyl ester tetrasaccharides from Mexican scammony root. Tetrahedron 58,
40
41 662 31453154. https://doi.org/10.1016/S0040-4020(02)00284-3.
42
43
44 663 Pereda-Miranda, R., Fragoso-Serrano, M., Escalante-Sánchez, E., Hernández-Carlos, B., Linares,
45
46 664 E., Bye, R., 2006. Profiling of the resin glycoside content of Mexican jalap roots with purgative
47
48
665 activity. J. Nat. Prod. 69, 1460-1466. https://doi.org/10.1021/np060295f.
49
50
51 666 Pereda-Miranda, R., Kaatz, G.W., Gibbons, S., 2006. Polyacylated oligosaccharides from
52
53 667 medicinal Mexican morning glory species as antibacterials and inhibitors of multidrug
54
55
56 668 resistance in Staphylococcus aureus. J. Nat. Prod. 69, 406-409.
57
58 669 https://doi.org/10.1021/np050227d.
59
60
61
62
63
64
65
 1
2
3
4 670 Pereda-Miranda, R., Rosas-Ramírez, D., Castañeda-Gómez, J., 2010. Resin glycosides from the
5
6
7 671 morning glory family. In: Kinghorn, A. D., Falk, H., Kobayashi, J. (Eds), Progress in the
8
9 672 Chemistry of Organic Natural Products, Vol. 92. Springer-Verlag, New York, pp 77−152.
10
11
12 673 https://doi.org/10.1007/978-3-211-99661-4_2.
13
14 674 Rencurosi, A., Mitchell, E.P., Cioci, G., Pérez, S., Pereda-Miranda, R., Imberty, A. 2004. Crystal
15
16
675 structure of tricolorin A: molecular rationale for the biological properties of resin glycosides
17
18
19 676 found in some Mexican herbal remedies. Angew. Chem. Int. Edit. 43, 5918–5922.
20
21 677 http://dx.doi.org/10.1002/anie.200460327.
22
23
24 678 Rosas-Ramírez, D., Escalante-Sánchez, E., Pereda-Miranda, R., 2011. Batatins III–VI, glycolipid
25
26 679 ester-type dimers from Ipomoea batatas. Phytochemistry 72, 773–780.
27
28
29 680 https://doi.org/10.1016/j.phytochem.2011.03.002.
30
31 681 Rosas-Ramírez, D., Pereda-Miranda, R., 2013. Resin glycosides from the yellow-skinned variety
32
33
34
682 of sweet potato (Ipomoea batatas). J. Agric. Food Chem. 61, 9488–9494.
35
36 683 https://doi.org/10.1021/jf402952d.
37
38 684 Teearu, A., Vahur, S., Haljasorg, U., Leito, I., Haljasorg, T., Toom, L., 2014. 2,5-
39
40
41 685 Dihydroxybenzoic acid solution in MALDI-MS: ageing and use for mass calibration. J. Mass
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43 686 Spectrom. 49, 970-979. https://doi.org/10.1002/jms.3395.
44
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46 687 Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening.
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48 688 Nat. Protoc. 1, 1112–1116. https://doi.org/10.1038/nprot.2006.179.
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51 689 Zhu, D., Chen, C., Bai, L., Kong, L., Lu, J., 2019a. Downregulation of aquaporin 3 mediated the
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53 690 laxative effect in the rat colon by a purified resin glycoside fraction from Pharbitis semen. Evid
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55
691 Based Compl. Alt. Med. 2019, 9406342. https://doi.org/10.1155/2019/9406342.
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58 692 Zhu, D., Chen, C., Xia, Y., Kong. L., Luo, J., 2019b. A purified resin glycoside fraction from
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60 693 Pharbitidis semen induces paraptosis by activating chloride intracellular channel-1 in human
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4 694 colon cancer cells. Integr. Cancer. Ther. 18, 1–13.
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7 695 https://doi.org/10.1177/1534735418822120.
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 1
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4 696 Table 1.
5 1
6
697 H NMR (400 MHz) spectroscopic data of compounds 3-5 in C5D5N (δ in ppm, and J in Hz).
7 698
8 Position 3 4 5
9
10 Fuc-1 4.78 d (7.8) 4.79 d (7.8) 4.77 d (7.8)
11 2 4.54 dd (9.5, 7.8) 4.55 dd (9.2, 7.8) 4.54 dd (9.5, 7.8) *
12
13
3 4.18 dd (9.5, 3.5) 4.20 dd (9.2, 3.8) 4.18 dd (9.5, 3.6)
14 4 3.95 d (3.5) 3.96 d (3.8) 3.93 d (3.6)
15 5 3.82 q (6.4) 3.83 q (6.3) 3.82 q (6.5)
16
17 6 1.53 d (6.4) 1.54 d (6.3) 1.53 d (6.3)
18 Rha-1 6.37 d (1.1) 6.40 d (1.3) 6.38 d (0.8)
19
2 5.29 dd (2.8, 1.1) 5.24 dd (2.8, 1.3) 5.27 dd (2.8, 0.8)
20
21 3 5.60 dd (9.8, 2.8) 5.69 dd (10.0, 2.8) 5.58 dd (9.9, 2.8)
22 4 4.61 dd (9.8, 9.8) 4.73 dd (10.0, 10.0) 4.64 dd (9.9, 9.9)
23
24 5 5.01 dq (9.8, 6.2) 5.07 dq (10.0, 6.2) 5.02 m*
25 6 1.59 d (6.2) 1.58 d (6.2) 1.62 d (6.2)
26 Rha'-1 5.58 d (1.7) 5.92 d (1.1) 5.80 d (0.8)
27
28 2 5.81 dd (3.3, 1.7) 4.73 dd (3.1, 1.1) 4.56 (3.3, 0.8)
29 3 4.62 dd (9.1, 3.3) 5.75 dd (10.0, 3.1) 4.46 dd (9.1, 3.3)
30
31 4 4.32 dd (9.3, 9.3) 4.59 dd (10.0, 10.0) 4.38 dd (9.1, 9.1)
32 5 4.34 m* 4.39 dq (10.0, 6.1) 4.35 dq (9.1, 6.4)
33 6 1.66 d (5.4) 1.57 d (6.1) 1.62 (6.4)
34
35 Rha''-1 6.25 d (1.6) 5.75 d (1.1) * 6.23 d (0.8)
36 2 4.81 dd (3.3, 1.6) 4.53 dd (3.5, 1.1) 4.84 dd (3.2, 0.8)
37
38 3 4.47 dd (9.1, 3.3) 4.38 dd (9.1, 3.5) 4.54 dd (9.8, 3.2)
39 4 4.32 dd (9.1, 9.1) 4.27 dd (9.1, 9.1) 5.84 dd (9.9, 9.8)
40 5 4.39 dq (9.1, 6.4) 4.29 dq (9.1, 5.7) 4.38 dq (9.9, 6.4)
41
42 6 1.70 d (6.0) 1.62 d (5.7) 1.38 d (6.4)
43 Agl-1   
44
45
2a 2.73 ddd (13.5, 10.3, 2.7) 2.31 m 2.28 ddd (13.5, 10.3, 2.7)
46 2b 2.28 ddd (13.5, 7.1, 3.1) 2.19 m 2.13 ddd (13.5, 7.1, 3.1)
47 11 3.84 m 3.92 m 3.84 m
48
49 16 0.99 t (7.0) 0.99 t (7.1) 1.00 t (7.1)
50 Dodeca-
51   
1
52
53 2a 2.33 m 2.35 m 2.47 m
54 2b 2.36 m 2.49 m 2.54 m
55 0.89 t (6.8) 0.89 t (7.0) 0.88 t (6.6)
12
56
57 699 Chemical shifts (δ) are in ppm relative to TMS and J in Hz.
58 700 Chemical shifts marked with an asterisk (*) indicate overlapped signals.
59 701 Abbreviations: Rha, rhamnose; Fuc, fucose; Agl, aglycone; Dodeca, dodecanoyl.
60 702
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 1
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4 703 Table 2.
5 13
6
704 C NMR (100 MHz) spectroscopic data of compound 6 in C5D5N (δ in ppm, and J in Hz).
7 705
8 Position 3 4 5
9
10 Fuc-1 101.4 102 101.4
11 2 72.9 73.3 72.7
12
13
3 76.4 77.2 76.6
14 4 73.4 73.9 * 73.3
15 5 71.0 71.5 70.9
16
17 6 17.0 17.5 17.0
18 Rha-1 100.1 100.5 100
19
2 69.5 70 69.3
20
21 3 77.7 79.2 72.7
22 4 78.5 76.1 77.4
23
24 5 67.7 67.8 67.5
25 6 18.7 19.6 18.5
26 Rha'-1 103.2 102.7 103.2
27
28 2 74.1 70.7 72.7
29 3 70.8 76.1 72.9
30
31 4 80.2 77.5 80.2
32 5 67.6 69.4 67.6
33 6 18.5 18.8 17.7
34
35 Rha''-1 103.3 103.7 103
36 2 72.1 72.9 * 72.1
37
38 3 72.4 72.8 70
39 4 79.5 73.9 * 75.3
40 5 70.4 71.0 68.3
41
42 6 18.2 18.6 18.6
43 Agl-1 173.0 172.4 
44
45
2 34.2 34.9 34.4
46 11 79.2 80.0 79.2
47 16 14.3 14.9 14.3
48
49 Dodeca-1 175.7 174.2 
50 2 34.3 34.7 34.6
51
52 12 14.0 14.6 14.1
53 706 Chemical shifts (δ) are in ppm relative to TMS and J in Hz.
54 707 Chemical shifts marked with an asterisk (*) indicate overlapped signals.
55 708 Abbreviations: Rha, rhamnose; Fuc, fucose; Agl, aglycone; Dodeca, dodecanoyl.
56
57
709
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 1
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4 710 Table 3.
5 1
6
711 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of compound 6 in C5D5N (δ in ppm,
7 712 and J in Hz).
8 713
9 Unit A Unit B
10
11 Position δH δC δH δC
12 Fuc- 1 4.79 d (7.8) 101.2 4.78 d (7.8) 101.1
13
14 2 4.52 dd (9.2, 7.8) 72.7 4.54 dd (9.1, 7.8) 72.5
15 3 4.19 dd (9.2, 3.6) 76.4 4.17 dd (9.5, 3.5) 76.3
16 4 3.94 d (3.6) 73.1 3.93 d (3.5) 73.1
17
18 5 3.81 q (6.5) 70.7* 3.80 q (6.5) 70.7
19 6 1.52 d (6.3) 16.7 1.51 d (6.3) 16.7
20
21
Rha-1 6.40 d (1.7) 99.7 6.38 d (1.7) 99.7*
22 2 5.26 brs 69.2 5.29 brs 69.1
23 3 5.69 dd (10.0, 2.8) 78.5 5.56 dd (9.8, 2.8) 77.5
24
25 4 4.72 dd (10.0, 10.0) 75.3 4.60 dd (9.8, 9.8) 78.2
26 5 5.08 m * 66.9 5.01 dq (9.8, 6.4) 67.2
27 6 1.57 d (6.4) 18.4
28
1.57 d (6.4) 18.8
29 Rha'-1 5.92 d (1.6) 101.9 5.58 d (1.7) 100.0
30 2 4.72 m 69.2 5.70 dd (3.3, 1.7) 73.9
31
32
3 5.74 dd (10.0, 3.0) 72.1 4.61 (10.0, 3.3) 70.5
33 4 4.61 dd (10.0, 10.0) 76.7 4.33 dd (10.0, 10.0) 73.3
34 5 4.37 m 68.6 4.31 m 68.2
35
36 6 1.57 d (5.4) 18.5 1.66 d (5.4) 18.2
37 Rha''-1 5.74 d (1.1) 102.9 6.25 d (1.6) 103.0
38 2 4.53 dd (4.4, 1.1) 72.1
39
4.79 m 71.9
40 3 4.38 m 70.0 4.46 dd (9.2, 3.3) 72.2
41 4 4.28 dd (7.4, 4.4) 73.3 4.31 m 70.1
42
43
5 4.36 m 69.9 4.39 m 72.1*
44 6 1.61 d (5.7) 17.8 1.70 d (6.2) 18.0
45 Agl-1 - 172.2 - 172.7
46
47 2a 2.72 ddd (13.5, 10.3, 2.7) 2.72 ddd (13.5, 10.3, 2.7)
33.9 34.1
48 2b 2.27 ddd (13.5, 7.1, 3.1) 2.27 ddd (13.5, 7.1, 3.1)
49 11 3.83 79.2 3.91 m 80
50
51 16 0.99 t (7.1) * 14.1 0.99 t (7.1) * 13.9
52 Dodeca-1 - 173.9 - 174.4
53
54 2a 2.34 m 2.24 m
34.1 34.2
55 2b 2.49 m 2.11 m
56 12 0.88 t (7.0) 13.9 0.88 t (7.0) 13.9
57
58 714 Chemical shifts (δ) are in ppm relative to TMS and J in Hz.
59 715 Chemical shifts marked with an asterisk (*) indicate overlapped signals.
60 716 Abbreviations: Rha, rhamnose; Fuc, fucose; Agl, aglycone; Dodeca, dodecanoyl.
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13
14
15
16
17
18
19
20 Table 4.
21 Cross-resistance and cytotoxicity for control drugs in standard SRB assays.
22
23
24 Cell line/IC50 (µM)
25 MCF-7 MCF-7 Vin MCF-7 Resistance valuea HeLa HCT-116
26
27 Vinblastine 0.04 ± 0.01 1.63 ± 0.3 41-fold 0.004 ± 2e-4 0.3 ± 2e-2
28 Podophyllotoxin 0.02 ± 7e-4 0.40 ± 7e-3 20-fold 0.062 ± 0.01 0.4 ± 0.06
29 Colchicine 0.02 ± 2e-3 1.57 ± 0.3 78-fold 0.006 ± 2e-4 0.08 ± 2e-2
30 Ellipticine 0.36 ± 4e-2 0.95 ± 7 e-2 2-fold NT NT
31
32 a
33 Resistance value = IC50 MCF-7 Vin/ IC50 MCF-7; Abbreviations: MCF-7 = breast carcinoma; MCF-7/Vin = multidrug resistant breast
34 carcinoma; HeLa = cervix carcinoma, HCT-116 = colon carcinoma. NT = not tested.
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13
14
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18
19
20 Table 5.
21 Half-maximal inhibitory concentration registered for different combinations of a subinhibitory concentration of vinblastine or
22
23 podophyllotoxin with non-cytotoxic resin glycosides in Cancer Cells.
24
25 IC50 (µM)
26
27 Samplea Combination MCF-7 MCF-7/Vinb HeLa HCT-116
28
29
30
1 Vin >50 >50 >50 >50
31 PPT >50 >50 >50 >50
32  
33 2 Vinb 5.0 ± 0.4 10.9 ± 0.6 5.2 ± 0.8 10.7 ± 1.1
34
35
36 3 Vin 18.2 ± 1.4 24.5 ± 1.5 28.1 ± 2.1 26.6 ± 1.4
37 PPT 0.5 ± 4e-2 17.2 ± 1.1 0.4 ± 1e- 0.6 ± 0.2
38
39 4 Vin 19.4 ± 1.6 43.8 ± 2.5 29.8 ± 1.1 28.1 ± 0.6
40
41
PPT 4.1 ± 0.3 23.2 ± 2.7 0.8 ± 9e-2 0.6 ± 2e-2
42
43 5 Vin 20.3 ± 1.1 32.9 ± 1.7 28.8 ± 1.1 25.3 ± 2.2
44 PPT 1.8 ± 0.3 34.6 ± 2.4 2.5 ± 0.41 3.8 ± 0.4
45
46
47 6 Vin 5.0 ± 0.2 T14.3 ± 0.7 10.4 ± 0.4 9.5 ± 1.3
48 PPT 6.1 ± 0.1 12.0 ± 0.3 7.7 ± 0.3
49
50 a
Non-cytotoxic resin glycosides (IC50 > 25 µM) were tested at the concentrations of 50, 30, 25, 20, 15, 10, 5 and 1 M to enhance cytotoxic
51
52 effects tested in combination with 0.003 M of vinblastine (Vin) or podophyllotoxin (PPT). bSee, J. Nat. Prod. 2022, 85, 23852394.
53 Abbreviations: MCF-7 = breast carcinoma; MCF-7/Vin = multidrug resistant breast carcinoma; HeLa = cervix carcinoma, HCT-116 = colon
54 carcinoma.
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Figure Click here to access/download;Figure;Figures.docx

Fig. 1. Viability of multidrug resistant MCF-7/Vin cells after 48 h of combination therapy using a
SRB assay. Cells were exposed to concentrations (50, 30, 20, 15, 10, 5, and 1 M) of halmitonins
II (3; A), III (4; B), IV (5, C), and V (6; D) with a sub-inhibitory concentration (0.003 M) of
vinblastine (blue) or podophyllotoxin (violet). Each experiment was performed six times
independently (n = 6). Values are expressed as the percentage of the control and represent means
± SEM.
Fig. 2. Effect of hamiltonin V (6) in combination with cytotoxic clinical drugs as determined by
the SRB assay to measure cell viability of MCF-7/Vin cells. Dose-response curves for cells
cultured in the presence of increasing concentrations of compound 6 after 48 h of incubation with
a sublethal concentration (0.0032 M) of vinblastine (A, blue circles) and podophyllotoxin (B,
violet squares). Values are express in percentage of control and represent means ± SEM. Each
experiment was done three times independently (n = 3).
Fig. 3. Cytotoxicity enhancement of clinical drugs. Microscopic visualization of MCF-7/Vin cells
(A, control: vinblastine at the sublethal concentration of 0.003 M without resin glycoside
treatment) after 48 h of incubation with a sub-inhibitory concentration of vinblastine (B) and
podohyllotoxin (C) at 0.003 M in combination with isolated halmitonin V (6) at a concentration
of 30 M.
Supplementary Material

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Supplementary Material
Supporting Information.pdf
Declaration of Interest Statement (Pls upload in word format)

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.