Propolis polyphenolic compounds affect the viability and structure of <i>Helicobacter pylori in vitro</i>

Romero, Mario; Freire, José; Pastene, Edgar; García, Apolinaria; Aranda, Mario; González, Carlos

doi:10.1016/j.bjp.2019.03.002

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Original articles • Rev. bras. farmacogn. 29 (3) • Mar-Apr 2019 • https://doi.org/10.1016/j.bjp.2019.03.002 copy

Propolis polyphenolic compounds affect the viability and structure of Helicobacter pylori in vitro

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

To evaluate the anti-Helicobacter pylori activity of the major polyphenol compounds of propolis and their cellular damage, both as single molecule or in combination. Honey bees propolis were fractionated by using CPC and preparative HPLC. Four major polyphenols (chrysin, pinocembrin, galangin and caffeic acid phenethyl ester) were identified by thin layer chromatography–mass spectroscopy and liquid chromatography–mass spectroscopy. These compounds inhibited both ATCC and clinical H. pylori strains, with caffeic acid phenethyl ester being the most active. The four compounds presented minimum inhibitory concentration in the range 256–1024 µg ml⁻¹ and a fractional inhibitory concentration of 64–512 µg ml⁻¹. In mixtures all compounds showed an indifference effect (FIC < 0.15) but chrysin + galangin which was synergistic (FIC = 2.0). Killing curves show a similar behavior as the antibiotic amoxycillin. On the other hand, analyses by transmission electron microscopy at sub inhibitory concentration show vesicle formation and cell lysis after exposition to both individual polyphenol compounds and in mixture. The major compounds of propolis show anti-H. pylori activity both as individual compounds and in mixture. When combined they present mainly indifference but exert a lytic activity upon H. pylori, suggesting a potential bactericidal activity of propolis.

Keywords:
Propolis; Caffeic acid phenethyl ester; Galangin; Indifference; Membrane vesicles; Cellular lysis

Peak	Propolis compound	Retention time (min)	λ_max (nm)	Reference
1	Caffeic acid	3.6	292, 322	^a a Confirmed with standard.
2	p-Coumaric acid	5.2	310	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
3	Ferulic acid	6.2	295sh, 322	^a a Confirmed with standard.
4	3,4-Dimethyl-caffeic acid	10.9	295sh, 322	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
5	Pinobanksin-5-methyl-ether	14.7	286	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
6	Kaempferide	16.4	265, 335, 364	^a a Confirmed with standard.
7	Apigenin	18.8	268, 337	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
8	Kaempferol	20.3	265, 364	^a a Confirmed with standard.
9	Cinnamilidenacetic acid	29.1	256, 349	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
10	Caffeic acid prenyl ester	38.5	298, 325	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
11	Chrysin	39.6	268, 313	^a a Confirmed with standard.
12	Pinocembrin	41.5	289	^a a Confirmed with standard.
13	Galangin	42.6	265, 300sh, 358	^a a Confirmed with standard.
14	Caffeic acid phenylethyl ester	44.7	295, 325	^a a Confirmed with standard.
15	Pinobanksin-3-O-acetate	44.7	292	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
16	p-Coumaric prenyl ester	51.9	294, 310	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
17	p-Coumaric cinnamyl ester	58.5	294, 310	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
18	Pinobanksin-3-O-butyrate	59.1	292	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
19	Pinobanksin-3-O-pentanoate	64.3	292	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
20	Pinobanksin-3-O-hexanoate	65.7	292	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.
21	p-Methoxy cinnamic acid cinnamyl ester	68.5	295, 325	^b b Confirmed by reference (database). Peaks correspond to those compounds shown in Fig. 1.

Polyphenol	Rt (min)	Exact mass	M-H^exp	Standard curve* (regression equation)	R ²	Proportion (%)
CRY	39.6	254.05	253.00	$y = 42.435 x - 95.176$	0.9980	1.85
PIN	41.5	256.07	256.00	$y = 70.748 x - 373.620$	0.9980	4.00
GAL	42.6	270.05	269.05	$y = 44.729 x - 179.980$	0.9986	3.42
CAPE	44.7	284.10	285.10	$y = 31.446 x - 15.007$	0.9993	8.02

Polyphenol assayed	Anti-bacterial activity upon strain
	H. pylori ATCC 43504		H. pylori J99		H. pylori 84C
	AD test	MIC	AD test	MIC	AD test	MIC
CRY	21 ± 1.16	256	20 ± 0.80	ND	18 ± 1.50	512
PIN	22 ± 1.32	512	22 ± 0.51	ND	19 ± 1.80	1024
GAL	21 ± 1.16	256	20 ± 1.04	ND	18 ± 1.90	512
CAPE	25 ± 1.41	256	24 ± 1.03	ND	20 ± 0.81	512

Parameter	Polyphenols combined
	CRY + PIN	GAL + CAPE	CRY + CAPE	GAL + PIN	CRY + GAL	PIN + CAPE
FIC	32 + 8	64 + 256	256 + 128	256 + 512	256 + 256	256 + 128
FIC index	0.14	1.25	1.5	2.0	2.0	1.0

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[1] * Corresponding author. E-mail:carlosg@udec.cl (C. González).