In vitro synergistic effect of Hibiscus sabdariffa aqueous extract in combination with standard antibiotics against Helicobacter pylori clinical isolates

Abstract

Context The increasing problem of drug-resistant strains has led to the failure of current treatment regimens of Helicobacter pylori (HP) infection. Recently, a new treatment strategy has been developed to overcome the problem by using natural products in combination with antibiotics to enhance the treatment efficacy.

Objective The antimicrobial combinatory effect of the aqueous extract of Hibiscus sabdariffa L. (Malvaceae) (AEHS) with antibiotics (clarithromycin, CLA; amoxicillin, AMX; metronidazole, MTZ) has been evaluated in vitro against HP strains.

Materials and methods Hibiscus calyces (35 g) were brewed in 250 mL of boiled water for 30 min, and minimum inhibitory concentrations (MICs) were determined by agar dilution method. The checkerboard assay was used to evaluate the antimicrobial combinatory effect according to the sum of fractional inhibitory concentration (∑FIC) indices.

Results In this study, AEHS exerted remarkable bacteriostatic effect against all HP strains tested with MICs values ranging from 9.18 to 16.68 μg/mL. Synergy effect of AEHS with CLA or MTZ was obtained against four of seven HP strains tested with ∑FIC ranging from 0.21 to 0.39. The additive effect of AEHS with AMX was obtained against five of seven HP strains tested with ∑FIC ranging from 0.61 to 0.91.

Conclusion This study presents AEHS as a potent therapeutic candidate alone, or in combination with antibiotics for the treatment of HP infection.

Keywords:

• Antimicrobial agents
• combinatory effect
• drug-resistant strains
• H. pylori infection
• natural products

Introduction

Helicobacter pylori (HP) infection is one of the most common causes of many diseases of the gastrointestinal tract, including non-ulcer dyspepsia, peptic ulcer, gastritis and gastric cancer (Jung et al. 2015; Mansour-Ghanaei et al. 2015; Paydas 2015). Numerous treatment strategies containing a proton pump inhibitor and combination of two or more antibiotics such as clarithromycin (CLA), amoxicillin (AMX) and metronidazole (MTZ) or tetracycline (TET) have been successfully used to eradicate HP infection (Nagahara et al. 2000; Chaabane & Al-Adhba 2015). Although the efficacy of such strategies in the therapy, the increasing use of antibiotics has led to the problem of drug-resistant strains (Essa et al. 2009; Gisbert & Calvet 2011). Resistance rates have been reported vary from 0 to 45% for CLA, from 0 to 33% for AMX, from 10% to 90% for MTZ, from 6% to 21% for levofloxacin and from 5% to 59% for TET (Karamanolis et al. 2014; O'Connor et al. 2014; Song et al. 2014). In recent years, the combination effect of common antibiotics with natural products has been applied as a new strategy to enhance the treatment of bacterial infections and overcome the complications of drug-resistant strains (Nostro et al. 2006; Hassan et al. 2015).

For decades, plants play an essential role in drug discovery development as a rich source of biologically active compounds that exhibited significant antimicrobial properties (Castillo-Juárez et al. 2009; Njume et al. 2011). Hibiscus sabdariffa L. (Malvaceae) (HS; roselle) is a medicinal plant, which has a long history of herbal and edible uses worldwide (Alshami & Alharbi 2014; Wang et al. 2014). Numerous studies have reported the antibacterial activity of H. sabdariffa, and its phytochemicals against Gram-negative and Gram-positive bacteria (Liu et al. 2005; Chao & Yin 2009). This study aims to evaluate the in vitro antimicrobial combinatory effect of aqueous extract of Hibiscus sabdariffa L. (Malvaceae) (AEHS) with standard antibiotics (CLA, AMX and MTZ) against HP strains (six clinical isolates and one standard control of HP).

Materials and methods

Bacterial strains, cultures and antibiotics

HP (ATCC 6583) standard strain was obtained from the American Type Culture Collection (ATCC) (Rockville, MD) as well as six clinical isolates of HP (HP01, HP02, HP03, HP04, HP05 and HP06; isolated from patients with duodenal ulcers) were obtained from The Motol University Hospital, Prague, Czech Republic. For antimicrobial assay, the strains were grown in Mueller-Hinton agar–7% horse blood (Sigma Aldrich, Prague, Czech Republic), and incubated under micro-aerobic conditions (5% O₂, 10% CO₂ and 85% N₂) at 37 °C for 3 days. The identification was based on micro-aerophilic growth requirement, morphology, Gram’s stain, and oxidase, catalase, and urease activities. In addition, the effects of aging, temperature, aerobiosis, starvation and antibiotics on the morphologic conversion rate to coccoid forms, and culturability of HP were determined. HP strains were kept in trypticase soy broth supplemented with 20% glycerol at −80 °C until further use. CLA, AMX and MTZ were purchased from Sigma-Aldrich.

Preparation of plant material

Plant collection

Hibiscus sabdariffa was collected from the northern part of Aswan, Egypt in June 2014, and identified by the authors in the Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic. A voucher specimen of the plant was deposited with the number EGHS5 at the herbarium of the department.

Extraction procedure

Air-dried Hibiscus calyces were prepared as a form of tea extract, which is in common use in traditional folk medicine, especially in tropical and subtropical regions. Hibiscus calyces (35 g) were brewed in 250 mL of boiled water and allowed to stand for 30 min. The mixture was filtered, and the solution evaporated to dryness. The extract was stored at 4 °C.

Determination of total flavonoid content in AEHS

Total flavonoid content (TFC) of AEHS was determined as previously described (Fernandes et al. 2012). Briefly, 5 g of air-dried Hibiscus calyces were extracted in 100 mL of boiled water for 1 h by sonication extraction. The mixture was filtered by Sartorius 388 filter paper. One milliliter of the extract was transferred to a 25-mL volumetric flask, 2 mL of AlCl₃ (5% w/v) was added and the volume was completed with distilled water (probe solution, PS). The same procedure was repeated without the addition of AlCl₃ for preparation of contrast solution (CS). The absorbance of PS against CS was determined in spectrophotometer at 410 nm. The percentage of TFC was calculated as rutin according to the following equation: , where A,  absorbance (AU); DF,  dilution factor; w,  mass of plant material (g); ld,  loss on drying plant material (8%, w/w); , specific absorption for rutin–AlCl₃ complex (259.4).

Determination of total anthocyanins in AEHS

Total anthocyanins (TAC) of AEHS were determined by a pH-differential method as previously described (Jakobek et al. 2007). In brief, 5 g of air-dried Hibiscus calyces was extracted in 100 mL of boiled water for 1 h by sonication extraction. The mixture was filtered by Sartorius 388 filter paper. The extract (0.4 mL) was transferred to a 25-mL volumetric flask, and two dilutions of the extract were prepared (ratio = 1:62.5), one with potassium chloride buffer (pH 1.0) (1.86 g KCI in 1 L of distilled water, pH value adjusted to 1.0 with concentrated HCI) and the other with sodium acetate buffer (pH 4.5) (54.43 g CH₃CO₂Na·3H₂O in 1 L of distilled water, pH value adjusted to 4.5 with concentrated HCI). After 15 min of incubation at room temperature, absorbance was measured simultaneously at 520 and 700 nm. The content of TAC was calculated in mg of cyaniding-3-O-glucoside equivalent per 100 g of dry weight using a molar extinction coefficient (ɛ) of cyaniding-3-O-glucoside of 26 900 L/mol/cm and molecular weight (449.4 g/mol).

Antimicrobial assay

For antibiotic susceptibility testing, HP (ATCC 6583) was used as a control strain. CLA, AMX and MTZ were used as positive control drugs. Minimum inhibitory concentrations (MICs) were determined by agar dilution method according to the recommendation of Clinical and Laboratory Standards Institute (2009) using Mueller-Hinton agar supplemented with 7% horse blood. Briefly, serial dilutions of CLA, AMX and MTZ ranging from 0.5 to 5 μg/mL and AEHS at concentrations ranging from 0.5 to 5 μg/mL were added to Mueller-Hinton agar supplemented with 7% horse blood in a 5% O₂, 10% CO₂ and 85% N₂. The bacteria were sub-cultured on Mueller-Hinton agar supplemented with 7% horse blood under the same micro-aerobic conditions (5% O₂, 10% CO₂ and 85% N₂) at 37 °C for 3 days. The bacterial suspension in Mueller–Hinton broth was adjusted to a final concentration of a McFarland No. 0.5 standard, 2 μL of the adjusted inoculum was delivered to the agar plates. After 72 h of incubation under the micro-aerobic conditions (5% O₂, 10% CO₂ and 85% N₂) at 37 °C, the MICs of CLA, AMX, MTZ and AEHS were determined. MICs were considered as the lowest concentration of drugs inhibiting visible growth. MICs were obtained from three independent experiments that performed in triplicate.

Combination effect of AEHS with standard antibiotics

The combination effect of antibiotics and AEHS was determined by checkerboard assay and evaluated algebraically based on the sum of the fractional inhibitory concentration (∑FIC) indices as previously described (Vuuren & Viljoen 2011; Rondevaldova et al. 2015). In brief, twofold serial dilutions of each CLA, AMX and MTZ prepared in horizontal rows of micro-titer plate were subsequently cross-diluted vertically by twofold serial dilutions of AEHS. The one-half MIC of each tested compound was used as a starting concentration in combinations. For evaluation of synergistic effect of AEHS (A) with antibiotic tested (B), the ∑FIC was calculated based on the following equation: ∑FIC = FIC_A+ FIC_B, where FIC_A=MIC_A (in the presence of B)/MIC_A (alone), and FIC_B=MIC_B (in the presence of A)/MIC_B (alone). The MICs used in this equation are the averages of MICs obtained from three independent experiments performed in triplicate. The interpretation of the in vitro antibacterial interactions was determined as follows: synergistic effect if ∑FIC ≤ 0.5; additive if ∑FIC > 0.5 and ≤ 1; no interaction if ∑FIC > 1 and ≤ 4 and antagonistic if ∑FIC > 4.

Results

The results indicated that AEHS showed remarkable inhibition of the growth of all HP strains tested (bacteriostatic effect) with MICs values ranging from 9.18 to 16.68 μg/mL (Table 1), while demonstrated synergistic effects when combined with CLA or MTZ against four of seven HP strains tested (Table 2). The synergistic effect of AEHS in combination with CLA was obtained at a concentration of 1 μg/mL against HP (ATCC 6583) (∑FIC = 0.28) and HP06 (∑FIC = 0.21) strains, causing 46- and 29-fold reductions in CLA MICs, respectively. The synergistic effect of AEHS when combined with MTZ was determined (at a concentration of 0.5 μg/mL) against HP01 (∑FIC = 0.34), and HP03 (∑FIC = 0.39) strains, and caused 40- and 30-fold reductions in MTZ MICs, respectively. The additive antibacterial effect of AEHS with AMX (at concentrations of 0.5, 1 and 3 μg/mL) was obtained against HP (ATCC 6583), HP02, HP04, HP05 and HP06 strains (Table 3), while in few cases ∑FIC values were close to 0.61, which can be considered as strong additive effect. The percentage of TFC in AEHS was determined as rutin (0.247%), while the content of TAC was estimated as mg of cyaniding-3-O-glucoside equivalent (1465.8 mg/100 g dry weight of Hibiscus calyces) (Table 4).

Table 1. In vitro antibacterial activity of CLA, AMX, MTZ and AEHS against HP strains.

	MIC (μg/mL)
HP strains	CLA	AMX	MTZ	AEHS
HP (ATCC 6583)	16.21	13.50	2.24	12.25
HP01^a	12.56	12.41	13.54	16.68
HP02^a	9.15	13.85	4.59	15.24
HP03^a	10.56	16.89	11.99	9.18
HP04^a	10.98	13.69	10.17	12.81
HP05^a	14.23	12.88	10.97	13.56
HP06^a	10.49	14.73	13.12	14.17

MIC, minimum inhibitory concentration; the values are expressed as an average from three independent experiments, each performed in triplicate. HP, Helicobacter pylori; ATCC, American Type Culture Collection; AEHS, the aqueous extract of H. sabdariffa; CLA, clarithromycin; AMX, amoxicillin; MTZ, metronidazole.

^a Clinical isolates.

Table 2. In vitro combinatory antibacterial effect of AEHS with CLA or MTZ against HP strains.

	AEHS with CLA at following concentrations (μg/mL)								AEHS with MTZ at following concentrations (μg/mL)
	5		3		1		0.5		5		3		1		0.5
HP strains	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC
HP (ATCC 6583)	22.23	1.27	19.23	3.13	0.35	0.28	9.45	2.93	13.56	3.12	16.34	2.12	12.34	1.99	12.23	1.27
HP01^a	21.45	2.13	16.24	3.78	12.34	1.72	9.23	3.15	13.32	3.18	12.43	2.23	13.12	3.15	0.34	0.34
HP02^a	17.34	1.98	19.34	2.45	16.34	2.14	15.63	1.98	14.26	2.15	7.43	1.16	9.32	2.45	9.45	1.36
HP03^a	18.56	3.24	20.23	2.16	16.23	3.45	14.65	2.43	17.23	2.87	8.45	1.13	17.32	3.14	0.40	0.39
HP04^a	19.34	2.89	18.45	2.13	13.78	2.45	18.45	3.15	8.34	1.98	8.34	2.84	17.32	2.36	9.45	2.26
HP05^a	17.43	3.14	17.34	1.13	14.67	3.19	19.43	3.48	7.23	1.92	17.43	2.36	16.43	2.73	10.21	1.99
HP06^a	18.34	2.75	18.45	1.78	0.36	0.21	12.45	2.41	8.98	1.23	21.34	2.73	12.98	2.83	21.32	1.78

∑FIC: sum of fractional inhibitory concentrations – the combination interaction is evaluated as follows: synergy ∑FIC ≤ 0.5 (bold font indicates synergistically acting combinations); additive ∑FIC >0.5 and ≤1; no interaction ∑FIC > 1 and ≤4; antagonistic if ∑FIC >4; MIC – bold font indicates fold reductions. MIC, minimum inhibitory concentration; HP, Helicobacter pylori; ATCC, American Type Culture Collection; AEHS, the aqueous extract of H. sabdariffa; CLA, clarithromycin; MTZ, metronidazole.

^a Clinical isolates.

Table 3. In vitro combinatory antibacterial effect of AEHS with AMX against HP strains.

	AEHS with AMX at following concentrations (μg/mL)
	5		3		1		0.5
HP strains	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC	MIC	∑FIC
HP (ATCC 6583)	12.43	1.36	13.12	0.73	13.23	0.62	15.32	0.71
HP01^a	14.25	1.36	12.34	2.13	14.23	2.13	13.24	3.13
HP02^a	13.65	2.36	18.34	0.81	17.44	0.79	14.56	0.92
HP03^a	13.45	2.34	17.34	1.68	17.34	1.95	10.89	1.98
HP04^a	10.89	3.15	15.34	0.78	18.34	0.96	12.34	0.82
HP05^a	15.25	3.98	14.28	0.91	17.36	0.84	8.45	0.87
HP06^a	16.34	3.57	9.78	0.63	16.98	0.61	7.14	0.82

∑FIC: sum of fractional inhibitory concentrations – the combination interaction is evaluated as follows: synergy ∑FIC ≤ 0.5; additive ∑FIC >0.5 and ≤1 (bold font indicates additive acting combinations); no interaction ∑FIC > 1 and ≤4; antagonistic if ∑FIC > 4. MIC, minimum inhibitory concentration; HP, Helicobacter pylori; AEHS, the aqueous extract of H. sabdariffa; ATCC, American Type Culture Collection; AMX, amoxicillin.

^a Clinical isolates.

Table 4. Chemical characterisation of TFC and TAC in AEHS.

TFC (%) calculated as rutin	TAC as mg of cyaniding-3-O-glucoside equivalent (mg/100 g DW)
0.247%	1465.8 mg/100 g DW

TFC, total flavonoid content; TAC, total anthocyanins; DW, dry weight of Hibiscus calyces; AEHS, the aqueous extract of H. sabdariffa.

Discussion

Hibiscus sabdariffa, consumed by people worldwide in the form of tea extract, has a wide range of antimicrobial activities (Rukayadi et al. 2008; Jung et al. 2013). Beside this fact, the present study suggests that AEHS could be a useful agent acting in combination with antibiotics to enhance the treatment efficacy of HP infection. Numerous studies have reported that compounds such as flavonoids and anthocyanins are responsible for the antimicrobial properties of H. sabdariffa (Yin & Chao 2008; Alarcón-Alonso et al. 2012; Camelo-Méndez et al. 2013). The antibacterial activity of AEHS may be related to its ability to inhibit bacterial protein synthesis (Higginbotham et al. 2014). The bacteriostatic activity of CLA depends on its capacity to inhibit protein synthesis by binding to the 50S bacterial ribosomal subunit. CLA resistance is mainly due to point mutations in the 23S ribosomal RNA gene, and nucleotides A2142G and A2143G are the most frequent mutations (Gerrits et al. 2006; Dolapcioglu et al. 2014; Ferreira & Moss 2014). MTZ resistance among HP strains has been related to alterations in gene products having MTZ nitroreductase activities, mainly including oxygen-insensitive NAD(P)H nitroreductase (RdxA) and NAD(P)H flavin oxidoreductase (FrxA) (Jenks et al. 1999; Jenks & Edwards, 2002). Based on these facts, we suggest that AEHS in combination with CLA or MTZ inhibit bacterial protein synthesis, and thus can be considered as a useful alternative therapeutic agent in the development of anti-HP drugs.

Conclusion

New antimicrobial combination drugs which include plant products combinations have recently gained a great attention in research field. This approach has financial implications as reformulation of existing drugs or combinations may prove to be a more viable option, rather than developing a new drug which will require extensive clinical trials for verification. In this study, the susceptibility of HP strains to AEHS in combination with antibiotics (CLA, AMX and MTZ) was examined. In addition, the chemical characterization of TFC and TAC in AEHS was determined. The results demonstrated that AEHS can enhance the growth inhibitory activity of CLA, AMX and MTZ against HP strains tested. Furthermore, no antagonistic interactions were observed. Although the synergistic effect of AEHS in combination with CLA or MTZ against four of seven HP strains tested as well as the additive effect of AEHS in combination with AMX against five of seven HP strains tested were investigated, further studies should be carried out to confirm these activities as well as the mechanisms of action in vivo and in clinical trials.

Declaration of interest

The authors have declared that there is no conflict of interest. This study was funded by Internal Grant Agency (IGA) of the Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Czech Republic. Project No. 20154247/2015.