Effects of honokiol combined with resveratrol on bacteria responsible for oral malodor and their biofilm

Figures & data

Table 1. Primer sequence of target genes.

Strains	Genes	Primer sequence(5’−3)	Product Length (bp)
Pg	fimA	F: 5’-CAGCAGGAAGCCATCAAATC-3’	140
		R: 5’-CAGTCAGTTCAGTTGTCAAT-3’
	hagA	F: 5’-GAAATGACGGAATGCCAAGT-3’	125
		R: 5’-ACAAACCTGCTCCGATGAAC-3’
	kgp	F: 5’-TTGGAGATCATCACCGCATA-3’	125
		R: 5’-GGAATGGCTTTGAATCCTGA-3’
	luxS	F: 5’-CCTCACGCAGGTATTTCTCG-3’	138
		R: 5’-CCCTCATGCAGGAGACTTTTC-3’
	16s rRNA	F: 5’- TGTAGATGACTGATGGTGAAA −3’	138
		R: ACTGTTAGCAACTACCGATGT −3’
Fn	fadA	F: CACAAGCTGACGCTGCTAGA-3’	108
		R: GTTACCAGCTCTTAAAGCTTG-3’
	fomA	F: CTCCAGCTTGGAGACCAAAT −3’	100
		R: CAAGGACTTGCTGGGTCTTC −3’
	fap2	F: AACAGCTTCAGCTGCAACAA-3’	123
		R: GCATTTTGTCCCAACATTCC −3’
	luxS	F: TGTAGATGACTGATGGTGAAA −3’	108
		R: ACTGTTAGCAACTACCGATGT −3’
	16s rRNA	F: AAGAACAGCGGTTTTTCCAC-3’	110
		R: TAATTGCCCAAGATGCTTCA-3’

Table 2. The MIC results of drugs against tested strains (n = 3).

drugs\tested strains	Pg (μg/mL)	Fn (μg/mL)
honokiol	30.00 ± 11.55	22.50 ± 12.58
resveratrol	160.00 ± 0.00	160.00 ± 0.00
chlorhexidine	1.88 ± 0.63	0.94 ± 0.31

Pg means Porphyromonas gingivalis; Fn means Fusobacterium nucleatum.

Table 3. The FICI results of drugs against tested strains (n = 3).

drugs\tested strains (μg/mL)	honokiol		resveratrol		FICI	effect
	MICsingle	MICcombination	MICsingle	MICcombination
Pg	30.00	8.33	160.00	33.33	0.45 ± 0.07	synergistic
Fn	22.50	5.00	160.00	26.67	0.38 ± 0.00	synergistic

Pg means Porphyromonas gingivalis; Fn means Fusobacterium nucleatum.

Figure 1.  Effect of honokiol combined with resveratrol on the growth curve of Pg and Fn (n=5).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination: honokiol combined with resveratrol.

Table 4. MBIC of drugs on Pg and Fn (n = 3).

tested strains	honokiol (g/mL)		resveratrol (g/mL)		chlorhexidine (g/mL)
	MBICsingle	MBICcombination	MBICsingle	MBICcombination	MBICsingle
Pg	60 ± 23.09	20.00 ± 0.00	280.00 ± 80.00	80.00 ± 0.00	10.00 ± 4.08
Fn	60 ± 23.09	16.67 ± 5.77	240.00 ± 92.37	53.33 ± 23.09	5.62 ± 3.14

Figure 2. Effect of drugs on bacterial metabolic activity at each stage of Pg and Fn biofilm formation (n=3).

Note: (A1), (B1) and (C1) mean effect of drugs on Pg metabolic activity at 36 h (adhesion phase), 60 h (aggregation phase) and 84 h (maturation phase), respectively. (D1), (E1) and (F1) mean effect of drugs on Fn metabolic activity at 24 h (adhesion phase), 42 h (aggregation phase) and 60 h (maturation phase), respectively. Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination for Pg: 20μg/mL honokiol + 80 μg/mL resveratrol. Combination for Fn: 16.67 μg/mL honokiol + 53.33 μg/mL resveratrol. *means compare with growth control group, ** P < 0.01, *** P < 0.001, **** P < 0.0001; #means compare with combination group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001.

Figure 3. Effect of drugs on the Pg and Fn biomass at each stage of biofilm formation (n=3).

Note: (A2), (B2) and (C2) mean effect of drugs on Pg metabolic activity at 36 h (adhesion phase), 60 h (aggregation phase) and 84 h (maturation phase), respectively. (D2), (E2) and (F2) mean effect of drugs on Fn metabolic activity at 24 h (adhesion phase), 42 h (aggregation phase) and 60 h (maturation phase), respectively. Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination for Pg: 20μg/mL honokiol + 80 μg/mL resveratrol. Combination for Fn: 16.67 μg/mL honokiol + 53.33 μg/mL resveratrol. *means compare with growth control group, *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; #means compare with combination group, # P < 0.05, ### P < 0.001, #### P < 0.0001.

Figure 4. Observe the effect of drugs on the Pg biofilm morphological structure at different stages by SEM (×10000).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination: 20μg/mL honokiol + 80 μg/mL resveratrol. 36 h (adhesion phase), 60 h (aggregation phase), 84 h (maturation phase).

Figure 5. Observe the effect of drugs on the Fn biofilm morphological structure at different stages by SEM (×20000).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine. Combination: 16.67 μg/mL honokiol + 53.33 μg/mL resveratrol. 24 h (adhesion phase), 42 h (aggregation phase), 60 h (maturation phase)

Figure 6. Expression of Pg biofilm formation related genes after drug intervention (n=3).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination: 20μg/mL honokiol + 80 μg/mL resveratrol; *means compare with growth control group, * P < 0.05, ** P < 0.01; #means compare with combination group, # P < 0.05.

Figure 7. Expression of Fn biofilm formation related genes after drug intervention (n=3).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination: 16.67μg/mL honokiol + 53.33 μg/mL resveratrol; *means compare with growth control group, * P < 0.05, ** P < 0.01; #means compare with combination group, # P < 0.05.

Figure 8. Effect of drugs on biofilm extracellular polysaccharide of Pg and Fn (n = 3).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination for Pg: 20 μg/mL honokiol +80 μg/mL resveratrol. Combination for Fn: 16.67 μg/mL honokiol +53.33 μg/mL resveratrol. *means compare with growth control group, *P < 0.05, **** P < 0.0001; #means compare with combination group, ## P < 0.01.

Figure 9. Effect of drugs on biofilm extracellular proteins of Pg and Fn (n=3).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination for Pg: 20 μg/mL honokiol + 80 μg/mL resveratrol. Combination for Fn: 16.67 μg/mL honokiol + 53.33 μg/mL resveratrol. *means compare with growth control group, *P < 0.05, ** P < 0.01, *** P < 0.001; #means compare with combination group, # P < 0.05, ## P < 0.01.

Figure 10. Effect of drugs on biofilm extracellular DNA of Pg and Fn (n=3).

Note: Control: growth control group; HKL: honokiol; RES: resveratrol; CHX: chlorhexidine; Combination for Pg: 20μg/mL honokiol + 80 μg/mL resveratrol. Combination for Fn: 16.67 μg/mL honokiol + 53.33 μg/mL resveratrol. *means compare with growth control group, ** P < 0.01, *** P < 0.001, **** P < 0.0001; #means compare with combination group, # P < 0.05, ## P < 0.01, #### P < 0.0001.