Evaluation of the cytotoxic effect and antibacterial, antifungal, and antiviral activities of Hypericum triquetrifolium Turra essential oils from Tunisia
© Rouis et al.; licensee BioMed Central Ltd. 2013
Received: 4 April 2012
Accepted: 23 January 2013
Published: 29 January 2013
A number of bio-active secondary metabolites have been identified and reported for several Hypericum species. Many studies have reported the potential use of the plant extracts against several pathogens. However, Hypericum triquetrifolium is one of the least studied species for its antimicrobial activity. The aim of the present study was to evaluate the cytotoxic effect of the essential oils of Hypericum triquetrifolium as well as their antimicrobial potential against coxsakievirus B3 and a range of bacterial and fungal strains.
The essential oils of Hypericum triquetrifolium harvested from five different Tunisian localities (Fondouk DJedid, Bou Arada, Bahra, Fernana and Dhrea Ben Jouder) were evaluated for their antimicrobial activities by micro-broth dilution methods against bacterial and fungal strains. In addition, the cytotoxic effect and the antiviral activity of these oils were carried out using Vero cell lines and coxsakievirus B3.
The results showed a good antibacterial activities against a wide range of bacterial strains, MIC values ranging between 0.39-12.50 mg/ml and MBC values between 1.56-25.0 mg/ml. In addition, the essential oils showed promising antifungal activity with MIC values ranging between 0.39 μg/mL and 12.50 μg/mL; MFC values ranged between 3.12 μg/mL and 25.00 μg/mL; a significant anticandidal activity was noted (MIC values comprised between 0.39 μg/mL and 12.50 μg/mL). Although their low cytotoxic effect (CC50 ranged between 0.58 mg/mL and 12.00 mg/mL), the essential oils did not show antiviral activity against coxsakievirus B3.
The essential oils obtained from Hypericum triquetrifolium can be used as antimicrobial agents and could be safe at non cytotoxic doses. As shown for the tested essential oils, comparative analysis need to be undertaken to better characterize also the antimicrobial activities of Hypericum triquetrifolium extracts with different solvents as well as their purified fractions and their pure secondary metabolites.
KeywordsHypericum triquetrifolium Coxsakievirus B3 Essential oils Bacteria Fungi
Essential oils are aromatic extracts which have been used since ancient times as flavouring agents and constituents of several commercial products. The chemical composition of essential oils is often variable among different plants and even between different plant parts. In addition, the composition may also differ according to the site of collection (geographical provenance), as their components play a major role in the plant adaptation to the ecology and the environment, including biotic and abiotic factors [1, 2]. Currently, the use of essential oils is more common today than ever before due to their increasing demand for food, cosmetics and pharmaceutical industries. In addition, the interest in essential oils has increased as potential alternatives for therapeutic purposes against common microbes. Bacterial resistance is spreading throughout the world primarily due to the excessive use of antibiotics and poor infection control practices in hospitals, making it one of our times biggest issues . Scientific literature revealed the antimicrobial, antifungal and antioxidant potentials of several essential oils [4, 5]. In addition, the antiviral potential of essential oils has been well documented [6, 7].
Microorganisms such as Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Enterococcus faecalis (E. faecalis), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) are frequently isolated from skin wounds in humans and animals. In addition, S. epidermidis infections are commonly acquired in hospitals as a result of contamination of surgical cuts with microorganisms from the patients themselves or from the hospital personnel . Infection with P. aeruginosa is one of the most serious complication in burn patients [9, 10], followed by infections with E. coli, S. aureus and other microorganisms . Infection with Bacillus cereus has been well documented in the literature for over a century and it is generally associated with gastroenteritis caused by the consumption of infected food. Vibrio alginolyticus is ubiquitous in seawater and tends to cause superficial wound and ear infections (otitis media and otitis externa) ; this infection can progress to bacteraemia and necrotising fasciitis, particularly in the immunocompromised patients . Vibrio cholerae (V. cholerae), a Gram (−) bacterium and the causative agent of cholera, has caused several pandemics since 1816, as well as sporadic inter-epidemic outbreaks. V. cholerae is autochthonous in a region of the world where cholera never occurs and that the human body is not an obligate environment for the presence and dispersal of this organism . Salmonella typhimurium causes typhoid fever associated with gastroenteritis; the infection is caused by consuming contaminated food or drinks. Aeromonas hydrophila (A. hydrophila) has been receiving increasing attention both as an opportunistic and as a primary pathogen of humans, aquatic and terrestrial animals . A. hydrophila inhabits aquatic environments and the gastrointestinal tract of healthy fish. It also commonly occurs in foods, milk, red meats and poultry [15–17]. It causes disease and mortality mainly in freshwater fish but sometimes in marine fish . The bacterium also infects humans and causes lesions ranging from gastroenteritis to septicaemia .
The genus Hypericum is a member of the Hypericaceae family [19, 20]. A number of bio-active secondary metabolites have been identified and reported for several Hypericum species [21–23]. Essential oils extracted from Hypericum species are well documented for their antimicrobial activities [4, 24–33].
Hypericum triquetrifolium Turra (H. triquetrifolium), native to Eastern Europe and the Mediterranean area, has been traditionally used for its sedative, antihelminthic, anti-inflammatory, and antiseptic effects [24, 34]. In addition, several studies have reported the potential use of its essential oil and crude extracts as therapeutic substances, mainly in the treatment of burns, gastroenteritis, antinociceptive and antioxidant drugs [35–37]. However, H. triquetrifolium is one of the least studied species for its antimicrobial activity. According to literature data, only a previous study using the growth inhibition assay for a number of bacterial and candidal strains  is reported for H. triquetrifolium.
In the present study, the antimicrobial, cytotoxic effect and the antiviral activities of the essential oils extracted from H. triquetrifolium from five different Tunisian localities were evaluated in vitro. The variation in their activities was discussed according to their chemical compositions previously reported .
Plant material and essential oil extraction
Voucher specimens identified by Prof. Mohammed El Hedi El Ouni (Department of Biology, Faculty of Sciences of Bizerte, Tunisia) have been deposited in the Herbarium of the Laboratory of Transmissible Diseases and Biological Active Substances (Faculty of Pharmacy of Monastir, Tunisia), under the following accession codes: H. tri. 1, H. tri. 2, H. tri. 3, H. tri. 4, and H. tri. 5 for Hypericum triquetrifolium from Bou Arada, Bahra, Dhrea Ben Jouder, Fernana and Fondouk Djedid, respectively.
Aerial parts (the top 25 cm) of the plant have been collected during full blooming from five different Tunisian localities between June and July 2008. In brief, plant samples were air-dried in darkness at room temperature for one week. Then, samples (500 g) were cut in small pieces and subjected to hydro-distillation for 3 h, using the standard apparatus recommended by the European Pharmacopoeia. The obtained oils were stored at +4°C in glass vials until analysis. The resultant oils were studied for their chemical variability using Gas Chromatography – Electron Ionization Mass Spectrometry (GC-EIMS) and GC coupled with Chemical Ionization Mass Spectrometry (GC/CIMS). The results are reported in a previous work .
Cells and tested microorganisms
The Vero cells were derived from the kidney of a normal, adult, African green monkey (Cercopithecus) in 1962, by Yasumura and Kawakita at the Chiba University in Japan. This cell line has been extensively used for virus replication studies and plaque assays. Vero cells (kindly provided by Pr. Bruno Pozzetto, Laboratory of Bacteriology-Virology, Saint-Etienne, France) were used for culturing enterovirus strains. Vero cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), L-Glutamin (2 mM), penicillin (100 U/mL), and streptomycin (100 μ g/mL). Cells were incubated at 37°C in a 5% CO2 humidified atmosphere.
Bacterial and fungal strains
Bacterial reference strains and their pathological effects
Bacillus cereus a
Escherichia coli b
Vibrio alginolyticus b
Intestinal diseases, wound and ear infections
Vibrio cholerae b
Pseudomonas aeruginosa b
Salmonella typhimurium b
Aeromonas hydrophila b
Gastroenteritis and Cellulitis
Enterococcus faecalis a
Staphylococcus aureus a
Foodborn, scalded skin syndrome
Staphylococcus epidermidis a
Fungal and candidal strains and their effects
Fungal and yeast strains
Catalogue number/ isolates
Black mold on certain fruits and vegetables, contaminant of food, aspergillosis, otomycosis, damage to the ear canal and tympanic membrane.
Isolated from Tomato plants
Damping off on certain fruits and vegetables, keratitis, endophthalmitis, cutaneous infections, burn patients, mycetoma, onychomycosis, sinusitis, pulmonary disease, endocarditis, catheter infections, and septic arthritis
Isolated from strawberry fruit
Winegrower’s lung, Hypersensitivity pneumonitis, Grey mould affects many plant species
Candidiasis, opportunistic oral and genital infections
Pathogen for the urogenital tract, and for the bloodstream (fungemia)
Fungemia, nosocomial pathogen
Coxsakievirus B3 Nancy strain (kindly provided by Pr. Bruno Pozzetto, Laboratory of Bacteriology-Virology, Saint-Etienne, France) was propagated in Vero cells. In brief, 100 μ L of the virus suspension were used to infect a confluent monolayer of Vero cells in 75 cm2 culture flask and adsorbed for 1 h to allow viruses to adhere onto the cells. Non-adherent particles were washed off using 2% RPMI 1640 medium and the infected cells overlaid with 20 mL of 2% RPMI 1640 and incubated again until full cytopathic effect was observed in five to six days. The harvested virus was stored at −70°C until used.
Minimum inhibitory and minimum bactericidal concentrations (MIC and MBC)
The minimum inhibitory concentration (MIC) values for each essential oil against the tested bacterial strains and environmental isolates were determined according to the standard protocols . The bacterial strains were cultured in tryptic soy broth (TSB) or agar (Sigma, Tunis, Tunisia) at the appropriat temperature for the strain (30°C or 37°C). Inocula were prepared by adjusting the turbidity of each bacterial culture to reach an optical density of 0.5 McFarland standards, corresponding to approximately 1 – 5 × 108 CFU/mL. The concentration of spore suspensions was determined using a haematocytometer (Thoma cell) and adjusted to 1 – 5 × 107 spores/mL. The broth dilution method was carried out in 96-well microtitre plates using microbial reference strains and field isolates. The essential oils were prepared aseptically and transferred to sterile 96-well microtitre plates by two-fold serial dilutions using 5% dimethylsulfoxide (DMSO) and then diluted in TSB. The resultant doses of the tested essential oils ranged between two and 250 μ g/mL. Eighty microliters of the prepared oil suspension were added to each well, followed by 10 μ L of each oil dose and 10 μ L of resazurin indicator solution (7-Hydroxy-3H-phenoxazin-3-one 10-oxide). The latter reagent allows the detection of microbial growth in extremely small volumes of solution in microtitre plates without using a spectrophotometer. Two control wells were used for each plate: one well containing microorganism and resazurin and a second well containing only medium and resazurin (in order to check the sterile conditions of the experiment). The plates were incubated anaerobically at 37°C for 24 h. After incubation, bacterial growth was evaluated by color change from blue to pink. The lowest dose indicating inhibition of growth was recorded as the MIC.
To determine the MBC, 10 μ L of each culture medium with no visible growth were removed from each well and inoculated in TSB plates. The CFU values of surviving organisms were determined after aerobic incubation at the appropriated temperature during 16 – 20 hours .
Minimium inhibitory and minimium fungicidal concentrations (MIC and MFC)
The fungicidal activity was evaluated as discussed above. The only differences consisted of the culture of fungi and the yeast strains on malt extract broth (MEB) or agar (Fluka, Madrid, Spain) and incubation at 28°C. The essential oils (diluted in 5% DMSO) at different doses were mixed with MEB and the plates were incubated anaerobically at 25°C for 48 hours.
The evaluation of the cytotoxic effect of the essential oils is based on the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), by the mitochondrial dehydrogenase of viable cells, to give a blue formazan product that can be measured spectrophotometrically . Cells were seeded in 96-well plates at a concentration of 5 × 104 cells per well and incubated at 37°C for 24 h in a 5% CO2 humidified atmosphere. After treatment with various doses of the essential oils (0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, and 25.00 mg/mL), the cells were incubated at 37°C for an additional 48 hours. The cells were examined daily under a phase-contrast microscope to determine the minimum dose of the tested essential oil that induced alterations in cell morphology. At this stage, the medium was removed and cells in each well were incubated for 3–4 hours at 37°C with 100 μ L of MTT solution (5 mg/mL). MTT solution was then discarded and 50 μ L DMSO were added to dissolve insoluble formazan crystals. Optical density (OD) was measured at 540 nm using a standard microplate reader (BIO-TEK® EL×800™ Universal Microplate Reader, NY, USA). Cell viability was expressed with respect to the absorbance of the control wells (untreated cells), which were considered 100% of absorbance. The percentage of cytotoxicity was calculated as [(A-B)/A] × 100; where A and B are the OD540 of untreated and treated cells, respectively. The 50% cytotoxic concentration (CC50) was defined as the dose of the essential oil required for the reduction of cell viability by 50%, which were calculated by regression analysis.
Virus inhibition assay
In this assay, essential oils were tested for their possible use either to cure infected cells or to protect them from infection. The experiment is simple, and relies on a cell culture system able to support virus growth. Confluent Vero cells were treated with the essential oils at three different doses (CC50, ½ CC50, ¼ CC50) during and after virus infection in two sets of experiments as follows: (Experiment 1) 5 × 104 TCID50 of the virus were exposed to three doses (CC50, 1/2 CC50, 1/4 CC50) of each essential oil for one hour at 37°C. Then 100 μ L of the mixture were added to the cells cultured fluently in 96-well flat-bottom microtiter plate; (Experiment 2) Cells were treated with three doses (CC50, 1/2 CC50, 1/4 CC50) of each essential oil (100 μ L) for one hour at 37°C. Then, 5 × 104 TCID50 of the virus (100 μ L) were added.
Altogether, the experiment aims to test the mode of action of the essential oils and to evaluate any effect of the essential oils on the virus (Experiment 1) or on the cells before the infection (Experiment 2).
Where (ODT) V, (ODC) V and (ODC) M indicate the absorbance of the sample, the virus-infected control (no compound) and mock-infected control (no virus and no compound), respectively .
Data of antibacterial and antifungal activities were subjected to statistiical analysis using Principal Components (PCA) and Hierarchical Clusters Analysis (HCA). Statistical tests were performed using STATISTICA-Pc Software 9.0 (Stat Soft Inc, http://www.statsoft.com).
Antibacterial activity of the essential oils of H. triquetrifolium (MIC / MBC; mg/mL)
H. tri. B.A.*
H. tri. Bah.*
H. tri. D.B.J.*
H. tri. Fer.*
H. tri. F.Dj.*
Bacillus cereus ATCC 11778 a
Enterococcus feacalis ATCC 29212 a
Staphylococcus aureus ATCC 25923 a
Staphylococcus epidermidis CIP 106510 a
Vibrio alginoliticus ATCC 17749 b
Escherichia coli ATCC 35218 b
Vibrio cholerae ATCC 39315 b
Pseudomonas aeruginosa ATCC 27853 b
Salmonella typhimirium CIP 104 b
Aeromonas hydrophila ATCC 7966 b
The essential oil of H. triquetrifolium from Fondouk DJedid (F.DJ.) showed a more potent antibacterial activity against the tested strains (MIC range: 0.39 – 1.56 mg/mL; MBC range: 1.56 – 6.25 mg/mL), with the exception of Vibrio cholerae (MIC = MBC = 25 mg/mL).
The essential oil of H. triquetrifolium collected in Bou Arada (B.A.) was comparatively more bacteriostatic against Gram (−) bacterial strains (MIC range: 0.39 – 12.50 mg/mL; MBC range: 6.25 – 25.00 mg/mL). However, the highest bactericidal effect was detected against S. aureus (MIC = MBC = 3.12 mg/mL).
The essential oils obtained from western regions, Bahra (Bah.) and Fernana (Fer.), were lesser active. Essential oils from Bahra showed a good activity against E. feacalis and A. hydrophila (MIC = 0.39 mg/mL for both strains; MBC range: 0.39 – 3.12 mg/mL, respectively).
The essential oils from Dhrea Ben Jouder (D.B.J.) exhibited a weaker antimicrobial activity against the tested strains (MIC range: 12.50 – 25.00 mg/mL; MBC range: 12.50 – 25.00 mg/mL). On the contrary, a significant activity (MIC = 0.78 mg/mL; MBC = 3.12 mg/mL) was detected against Vibrio cholerae.
Altogether, with the exception of the essential oils from F.DJ., all the remaining samples showed a bacteriostatic effect against V. cholerae, with MIC values ranging between 0.39 mg/mL and 12.50 mg/mL. MBC values were 3.12 mg/mL and 25.00 mg/mL for the essential oils from Fer./D.B.J. and B.A./Bah., respectively. In addition, B. cereus, S. epidermidis, E. coli and V. alginoliticus were resistant to all the essential oils, but sensitive to the essential oils from F.DJ., with MIC and MBC values ranging between 6.25 and 25.00 mg/mL.
Group II was represented by the oil of H. triquetrifolum obtained from plants harvested in Fondouk Djedid. This oil was characterized for its strong activity against all the tested strains, except Vibrio cholerae that was sensitive for the majority of the other oils.
Group III was limited to the essential oil of plant harvested in Dhrea Ben Jouder and it is distinguished from all the other groups by its week activity against all the tested strains, with the exception of Vibrio cholerae.
Group C was characterized by its potent activity against Enterococcus feacalis which was resistant to the action of the other oils.
Contrary to all other groups, group D was characterized by a moderate bactericidal activity against two or more strains (Aeromonas hydrophila, Pseudomonas aeruginosa, and Staphylococcus epidermidis for Bou Arada samples, and against Vibrio cholerae and Pseudomonas aeruginosa for Fernana essential oil).
Antifungal activity of essential oils of H. triquetrifolium against fungal and yeast strains (MIC / MFC; μg/mL)
H. tri. B.A.*
H. tri. Bah.*
H. tri. D.B.J.*
H. tri. Fer.*
H. tri. F.Dj.*
Candida krusei ATCC 6258
Candida albicans ATCC 90028
Candida glabrata ATCC 90030
The essentials oil from B.A. showed the most potent fungistatic activity, with MIC values of 0.39 μ g/mL and 3.12 μ g/mL for candidal and filamentous strains (Aspergillus niger, Fusarium solani and Botrytis cinerea), respectively. The best antifungal activity was exerted against C. glabrata (MFC = 1.56 μ g/mL).
The essential oils from the Estern regions of Tunisia (F.DJ. and D.B.J.) showed a more potent antifungal activity against the tested candidal strains, with MIC values ranging between 1.56 μ g/mL and 6.25 μ g/mL. The essential oils from F.DJ. showed a more potent antifungal activity against filamentous fungal strains (MIC = 3.12 μ g/mL, MFC = 3.12 μ g/mL) than the essential oils from D.B.J. (MIC = 6.25 μ g/mL, MFC = 6.25 μ g/mL). In addition, the essential oils from F.DJ. (MIC range: 1.56 – 6.25 μ g/mL, MFC = 6.25 μ g/mL) showed slightly higher anticandidal activity than the essential oils from D.B.J. (MIC range: 1.56 – 3.12 μ g/mL, MFC = 25.00 μ g/mL).
Compared to the eastern regions, essential oils from the western localities (Bah. and Fer.) were less fungistatic, with MIC values ranging between 3.12 μ g/mL and 12.50 μ g/mL, while similar fungicidal activity, with MFC values ranging between 3.12 μ g/mL and 12.50 μ g/mL.
Statistical analysis based on MFC values against fungi strains showed that while essential oils were classified according to their activities against mycetes and yeast strains respectively using the PCA analysis, they were discriminated according to the total of their activities with the HCA analysis.
Cytotoxicity test and antiviral activity
50% cytotoxic doses (CC50) of Hypericum triquetrifolium essential oils collected from five different localities of Tunisia
Essential oils from different localities
CC50 (mg/mL) ± SD
H. tri. B.A.
12.00 ± 0.25
H. tri. Bah.
2.50 ± 0.01
H. tri. D.B.J.
0.58 ± 0.00
H. tri. Fer.
1.12 ± 0.07
H. tri. F.Dj.
4.17 ± 0.09
The antibacterial activity of the essential oils of Hypericum species is well documented in the literature for H. calycinum L. , H. kouytchense H. Lév. , H. coris L. , H. barbatum Jacq., H. richeri Vill. (published as H. alpinum WK.) , H. rumeliacum Boiss. , H. hyssopifolium ssp. elongatum Chaix. (syn: H. elongatum Ldb) , H. Hyssopifolium ssp. hyssopifolium Chaix. , H. Hyssopifolium ssp. microcalycinum Chaix., H. Lysimachioides Boiss var. lysimachioides, H. Scabroides Robson & Poulter, H. triquetrifolium Turra , H. maculatum Crantz , H. perforatum L. , H. hirsutum L.  and H. cordatum Vell. .
The antibacterial activity of H. triquetrifolium was previously reported only against Bacillus brevis, Bacillus cereus, Escherichia coli PBR 322, Escherichia coli PUC 9, Pseudomonas aeruginosa and Staphylococcus aureus. However, the antimicrobial activity are generally influenced by the type of assay used .
The major components of the essential oils are found to reflect quite well their biophysical and biological features . Among the main compounds detected in these essential oils, antimicrobial activities of α-pinene, camphene, β-pinene, myrcene, ƥ-cymene, limonene, Ɣ-terpinene, borneol, 1-terpinen-4-ol, α-terpineol, geraniol, caryophyllene oxide, longiborneol, and sclareol have been well-documented [28, 57–63]. The percent values of the above compounds were 26.7, 22.8, 19.6, 19.5 and 18.5 in the H. triquetrifolium essential oil from B.A., F.DJ., Fer., Bah. and D.B.J., repectively . These values may explain the good antimicrobial activities of the essential oils from B.A. and F.DJ.
It’s a challenge to determine which components in an essential oil are responsible on its antimicrobial activity. Although extensive research have been done within this field [63–65], an essential oil contain different identifiable components which makes it difficult to attribute this activity to one or more components without consideration of synergistic and antagonistic effects of this components. Further research is still required.
The antifungal and anticandidal activity observed in this study were higher than those obtained for antibacterial activity for all studied essential oils. The essentials oil from B.A. showed more potent fungistatic activity against candidal strains, with MIC values ranging between 0.39 μg/mL and 3.12 μg/mL, followed by the essential oils from F.DJ. and D.B.J., with MIC values ranging between 1.56 μ g/mL and 6.25 μg/mL. The essential oils from Bah. and Fer. were endowed with the least fungistatic effectiveness, with MIC values ranging between 3.12 μ g/mL and 12.50 μg/mL.
The best fungicidal effect of the essential oil from B.A. was shown against C. glabrata (MFC = 1.56 μ g/mL). The essential oil from F.DJ. had better fugicidal activity against filamentous strains (MFC = 3.12 μ g/mL) than the one from D.B.J. (MFC = 6.25 μ g/mL). The essential oils from the western localities (Bah. and Fer.) had similar fungicidal activity, with MFC values ranging between 3.12 μ g/mL and 12.50 μ g/mL. A similar study reported the antifungal activity of H. triquetrifolium against Candida albicans using disk diffusion assay .
Unfortunately, the tested essential oils of the Tunisian H. triquetrifolium did not show any clear anti-enteroviral activity. However, their activity against other viral agents cannot be excluded, as previously reported for Hypericum connatum, Hypericum caprifoliatum and Hypericum polyanthemum against lentiviruses .
Antibiotic-resistant bacteria and fungi continue to be of major health concern worldwide. Bacteria have progressively developed resistance. Consequently, scientific efforts have been made to study and develop new compounds to be used beyond conventional antibiotic and antifungal therapy.
To the best of our knowledge, the present work is the first study reporting the antimicrobial activity of the essential oils of H. triquetrifolium from Tunisia.
These essential oils obtained from different Tunisian localities showed promising activity against bacterial and fungal strains at non-cytotoxic doses and merit worth consideration in future evaluation of Tunisian natural products for their antimicrobial potential. However, it was not possible to determine the mechanism(s) underlying these activities.
Unfortunately, these essential oils did not show any antiviral activity against coxsakievirus B3 Nancy strain, known to be resistant in the environment. However, the tested essential oils may exhibit antiviral activities against other viral strains, possibly the enveloped viruses such as herpes virus.
The authors thank Mr Ben Salah Mohamed for his technical help.
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