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Grapefruit Seed Extract is a Powerful in vitro Agent Against Motile and Cystic Forms of Borrelia burgdorferi sensu lato
Abstract
Lyme borreliosis (1), caused by B. burgdorferi sensu lato, may lead to long-term tissue infection, which may be dif- ficult to cure. The outcome of Lyme borreliosis is highly dependant on the antibiotic treatment (2). The observation of the ability of B. burgdorferi sensu lato to convert (and reconvert) to cystic forms (3-5) may explain why the infec- tion sometimes is persistent and reactivating. Therefore, it might be important to eradicate all germative forms (not only the motile form) of the bacterium to obtain a proper treatment for Lyme borreliosis. Grapefruit-seed extract (GSE) contains bioactive flavenoids (e.g., hesperitin, res- veratrol, and naringenin) and has been shown to possess anti-microbiological effect against bacteria and fungus (6, 7). Many studies indicate that GSE is a substance whose therapeutic effect ranks equal to or better than other known anti-bacterial agents. Positive effects of GSE are decreased levels of TNF-α, Nuclear factor Kb, NO, protec- tion of the gastrointestinal tract against mechanical stress, and has anti-allergic and other antioxidative properties (8, 9). Naringenin, hesperidin and other citrus flavones have been found in plasma and tissue after ingestion (10). Lacto- bacillus and bifidobacteria in the gut seems to be insignifi- cantly affected by GSE (6), and no severe side effects have been observed. B. burgdorferi sensu lato has a gene for ef- flux mechanism which may be responsible for antibiotic resistance (11). GSE is an efflux inhibitor, which can be used to enhance the activity of antibacterial agents (12). For the reasons mentioned above it is reasonable to test the hypothesis that motile and cystic forms of B. burgdorferi sensu lato will be susceptible to GSE, and this is the aim of our study.
... According to a hypothesis prevail-ing in the literature, the mechanism of fungicidal action of polyenes is based on their binding to ergosterol in the fungal cell membrane (Gray et al., 2012) and impairment of its permeability due to the formation of ion-conductance pores (Andreoli, 1974;Ermishkin et al., 1976). The possible binding of polyene antibiotics to cholesterol in human cell membranes determines their high toxicity of (Wilcock et al., 2013), most often manifested in nephropathy (Sawaya et al., 1995) and significantly limits their use in clinical practice (Zotchev, 2003;Laniado-Laborín and Cabrales-Vargas, 2009;Bagnis and Deray, 2013). ...
... Grapefruit seeds are known to be active against Staphylococcus sp. (in particular, Staphylococcus aureus), Klebsiella sp., Escherichia coli, Pseudomonas aeruginosa, and Legionella pneumophila, as well as against some species of spirochetes (Ionescu et al., 1990;Heggers et al., 2002;Fukuyama et al., 2003;Oyelami et al., 2005;Brorson, O. and Brorson, S.H., 2007). Different varieties of sea buckthorn (e.g., Hippophae rhamnoides) are an important source of antimicrobial agents. ...
... In recent years, the effects of several phytochemicals on Borrelia have been studied and found to be effective against the different pleomorphic forms of the bacteria [8][9][10][16][17][18]. For instance, Stevia whole leaf extract has been found to reduce the size and number of Borrelia biofilms [10]. ...
... For instance, Stevia whole leaf extract has been found to reduce the size and number of Borrelia biofilms [10]. Additionally, grapefruit seed extract has been demonstrated to work against Borrelia spirochetes and RBs [16]. Recently though, oregano oil was found to be highly active against persistent (7day growth) forms of Borrelia even at low concentrations [17]. ...
... There are several reports demonstrating the ability of various bioactive plants to act as antibacterial agents. For example, grape (Vitis vinifera) seed extract [8], sugar leaf (Stevia rebaudiana) [9] and cat's claw (Uncaria tomentosa) [6] have shown potential activity against acute Borrelia spirochetes and the nongrowing stationary phase. This is further supported by the use of treatments where phytochemicals are used in combination with antibiotics [10] or micronutrients such as vitamin D [11], enabling lower LD 50 (median lethal dose) and MIC (minimum inhibitory concentration) concentrations against latent rounded forms of B. burgdorferi. ...
Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi. Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi. This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti-Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives—sylvestrosides III and IV, that showed a considerable anti-Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.
... 5,11,12 The efficacy of naturally occurring and biologically active substances as anti-borreliae agents is still not well explored, although the number of research investigations with such agents has been growing. [13][14][15][16] Our previous in vitro study showed that a specific combination of polyphenols with fatty acids and iodine has significant bactericidal effect against two species of Borrelia that have been recognized as a pathogenic factor of LD in the USA and Europe. Moreover, this defined composition of phytochemicals worked synergistically and was shown to affect the membrane but not the DNA of the bacteria, demonstrating significant anti-oxidative and antiinflammatory properties at the same time. ...
Background:
Lyme disease (LD) is a tick-borne infection caused by Borrelia burgdorferi sensu lato. The current therapeutic approach to this disease is limited to antibiotics. However, after their administration, about 20% of patients experience delayed onset of this illness manifesting as lingering persistent symptoms.
Methods:
To determine a suitable approach that would help reduce this number, we examined the efficacy of a composition of polyphenolic compounds (baicalein, luteolin, and rosmarinic acid) with fatty acids (monolaurin and cis-2-decenoic acid), and iodine/kelp in a Lyme disease animal model and volunteers.
Results:
The results showed that 4 weeks of dietary intake of this composition reduced the spirochete burden in animal tissues by about 75%. Basic and differential blood parameters did not show significant differences between control animals and the animals fed with this composition. Also, hepatic and renal toxicity markers were not changed and apoptosis was not observed. Relevant inflammatory cytokines such as IL-6, IL-17, TNF-α, and INF-γ, were elevated in infected animals but normalized in infected and treated animals. A small observational study revealed that after administration of this composition to 17 volunteers three times per day for 6 months, 67.4% of the volunteers with late or persistent LD, and not receptive to previous antibiotic application, responded positively, in terms of energy status as well as physical and psychological wellbeing to supplementation with this composition, while 17.7% had slight improvement, and 17.7% were none responsive.
Conclusion:
We concluded that this specific composition revealed feasible benefits in late or persistent LD management, although double-blind controlled clinical trials are warranted.
... Grapefruit seed extract (GSE) was previously reported to have in vitro activity against motile and cystic morphologic forms of borrelia bacteria in an in vitro model (125). In contrast, the current study did not demonstrate meaningful activity against B. burgdorferi. ...
Lyme disease is the most common vector-borne disease in the US and Europe. Although the current recommended Lyme antibiotic treatment is effective for the majority of Lyme disease patients, about 10–20% of patients continue to suffer from persisting symptoms. There have been various anecdotal reports on the use of herbal extracts for treating patients with persisting symptoms with varying degree of improvements. However, it is unclear whether the effect of the herb products is due to their direct antimicrobial activity or their effect on host immune system. In the present study, we investigated the antimicrobial effects of 12 commonly used botanical medicines and three other natural antimicrobial agents for potential anti-Borrelia burgdorferi activity in vitro. Among them, 7 natural product extracts at 1% were found to have good activity against the stationary phase B. burgdorferi culture compared to the control antibiotics doxycycline and cefuroxime. These active botanicals include Cryptolepis sanguinolenta, Juglans nigra (Black walnut), Polygonum cuspidatum (Japanese knotweed), Artemisia annua (Sweet wormwood), Uncaria tomentosa (Cat's claw), Cistus incanus, and Scutellaria baicalensis (Chinese skullcap). In contrast, Stevia rebaudiana, Andrographis paniculata, Grapefruit seed extract, colloidal silver, monolaurin, and antimicrobial peptide LL37 had little or no activity against stationary phase B. burgdorferi. The minimum inhibitory concentration (MIC) values of Artemisia annua, Juglans nigra, and Uncaria tomentosa were quite high for growing B. burgdorferi, despite their strong activity against the non-growing stationary phase B. burgdorferi. On the other hand, the top two active herbs, Cryptolepis sanguinolenta and Polygonum cuspidatum, showed strong activity against both growing B. burgdorferi (MIC = 0.03–0.06% and 0.25–0.5%, respectively) and non-growing stationary phase B. burgdorferi. In subculture studies, only 1% Cryptolepis sanguinolenta extract caused complete eradication, while doxycycline and cefuroxime and other active herbs could not eradicate B. burgdorferi stationary phase cells as many spirochetes were visible after 21-day subculture. Further studies are needed to identify the active constituents of the effective botanicals and evaluate their combinations for more effective eradication of B. burgdorferi in vitro and in vivo. The implications of these findings for improving treatment of persistent Lyme disease are discussed.
... The antimicrobial activity of GSE has been attributed to the antioxidant activity of bioactive flavonoids such as naringin, naringenin, hesperidin, and hesperitin. The organic acids, including ascorbic acid and citric acid also involved in antimicrobial activity of the GSE 10,11 . Among the components of the GSE, naringin is a major constituent found in the seed of ripe grapefruits and is associated with a bitter taste 12 . ...
In the present study, polycaprolactone (PCL) composite films incorporated with various concentrations of grapefruit seed extract (GSE) as an antimicrobial agent were prepared using a twin-screw extruder. Physical characteristics as well as antimicrobial properties of the PCL/GSE composite films were analyzed. The results showed that the surface color of the films gradually changed with increasing GSE concentration. Fourier transform infrared spectra indicated no significant structural changes such as chemical bond formation between PCL and GSE. Thermal properties were slightly affected due to GSE incorporation. Crystallinity of the composite films decreased as the amount of GSE increased. In vitro analysis indicated that the antimicrobial activity of the PCL/GSE composite films increased as the GSE concentration increased, with a 5% concentration showing the strongest inhibitory activity against Listeria monocytogenes, with 5.8-log reduction in bacterial count. Application testing of the films was carried out for cheese packaging, and biodegradation of the samples was assessed via soil burial testing. Our findings confirmed the potential use of PCL/GSE composite films as biodegradable food packaging material with antimicrobial activity.
... One of the reasons might be the rather small number of compounds that have been studied thus far against Borrelia sp. [22][23][24][25][26][27]. However, naturally-derived substances, either in the form of the isolated active compounds, their metabolites, or plant-based extracts, have been a valuable source of non-synthetic and non-modified agents for human and animal health longer than the synthetic therapeutics [28][29][30][31][32]. Their potential as antimicrobials has been tested against an abundance of bacteria species. ...
Background
Borrelia sp. is a causative pathogen of Lyme disease which has become a worldwide health concern. Non-toxic approaches especially directed toward latent persistent forms of this pathogen are desired. Lipids in the form of volatile and non-volatile oils, and fatty acids with proven anti-borreliae efficacy could become an additional support or an alternative for consideration in treatment approaches.
Methods
In this study we investigated 47 lipids (30 volatile and non-volatile oils, and 17 fatty acids) of plant and animal origin against typical motile, knob/round-shaped persisters, and biofilm-like aggregates of Borrelia burgdorferi s.s. and Borrelia garinii, which are identified as pathogenic factors of Lyme disease in the USA and Europe, using direct microscopic counting and spectrofluorometric measurements.
Results
Out of all examined lipids, 5 oils (Bay leaf oil, Birch oil, Cassia oil, Chamomile oil German, and Thyme oil) at or below 0.25%, and 3 fatty acids (13Z,16Z Docosadienoic acid, erucic acid, and petroselinic acid) at or below 0.75 mg/ml, showed bactericidal activity against typical motile spirochetes and knob/round-shaped persisters. Only Bay leaf oil and Cassia oil, including their major constituents, eugenol and cinnamaldehyde, showed to target biofilm-like aggregates of both tested Borrelia spp. at the same concentration, although with 20–30% eradication mark.
Conclusion
Based on obtained results, volatile oils were more potent than non-volatile oils, and unsaturated fatty acids were more effective than saturated fatty acids. Among all tested oils, Bay leaf oil and Cassia oil, with their major components eugenol and cinnamaldehyde, seem to have the highest anti-borreliae efficacy.
... There are large quantities of polyphenolic compounds in it [8,9]. Bioflavonoids with bioactive properties are present in grapefruit seed extract e.g., hesperitin and naringenin [10]. Grapefruit seeds contain 17-beta-D-glucopyranosides of limonin, nomilin, obacunone, deacetylnomilin, nomilinic acid and deacetylnomilinic acid. ...
Several natural compounds deriving from plants are known to be efficient anti-amyloid aggregation agents. In this study, anti-aggregation activity of grapefruit seed extract was investigated using trypsin as a model protein in aqueous ethanol at pH 7.0. Using turbidity measurement, Congo red (CR) binding assay, electronic circular dichroism (ECD) and transmission electron microscopy (TEM), we found that grapefruit seed extract has ability to inhibit trypsin amyloidlike fibril formation in vitro, and effectiveness increases with growing concentration of grapefruit seed extract. The total phenolic content of it was determined. The results showed that in addition to the polyphenolic compounds some other compounds are also responsible for the fibril formation inhibitory effect. We indicated it for the first time that limonin has anti-fibrillation effect.
... [41][42][43]. Despite a rather small pool of available data on this subject, Bronson and Bronson study exhibited grapefruit seed extract as a powerful in vitro agent against spirochetes and their cystic forms of Borrelia afzelii [44]. Dipsacus sylvestris extract against Borrelia burgdorferi sensu stricto revealed growth inhibiting activity as well [45], whereas Sapi, et al., reported significant efficacy of leaf extracts from Stevia rebaudiana on all forms of Borrelia burgdorferi sensu stricto [46]. ...
Phytochemicals and micronutrients represent a growing theme in antimicrobial defense; however, little is known about their anti-borreliae effects of reciprocal cooperation with antibiotics. A better understanding of this aspect could advance our knowledge and help improve the efficacy of current approaches towards Borrelia sp. In this study, phytochemicals and micronutrients such as baicalein, luteolin, 10-HAD, iodine, rosmarinic acid, and monolaurin, as well as, vitamins D3 and C were tested in a combinations with doxycycline for their in vitro effectiveness against vegetative (spirochetes) and latent (rounded bodies, biofilm) forms of Borrelia burgdorferi and Borrelia garinii. Anti-borreliae effects were evaluated according to checkerboard assays and supported by statistical analysis. The results showed that combination of doxycycline with flavones such as baicalein and luteolin exhibited additive effects against all morphological forms of studied Borrelia sp. Doxycycline combined with iodine demonstrated additive effects against spirochetes and biofilm, whereas with fatty acids such as monolaurin and 10-HAD it produced FICIs of indifference. Additive anti-spirochetal effects were also observed when doxycycline was used with rosmarinic acid and both vitamins D3 and C. Antagonism was not observed in any of the cases. This data revealed the intrinsic anti-borreliae activity of doxycycline with tested phytochemicals and micronutrients indicating that their addition may enhance efficacy of this antibiotic in combating Borrelia sp. Especially the addition of flavones balcalein and luteolin to a doxycycline regimen could be explored further in defining more effective treatments against these bacteria.
... Interestingly, the authors observed the abnormal protrusion of membranes and their eventual disruption with the contents leaking at lower applied concentrations of GSE, while the highest GSE concentrations made the bacteria and cysts disappear completely. The MBC was strongly dependent on the length of the incubation, but even short incubation with GSE showed to be very effective against active and latent rounded forms of Borrelia afzelii [Brorson and Brorson, 2007]. Effect of GSE on biofilm was not performed in the study. ...
Naturally occurring substances have been used for centuries to fight against various pathogens. They serve as a source for new chemical entities or provide options to already existing therapeutics. While there is an increasing interest in studying antimicrobial properties of naturally derived agents, little is known about their effects against Borrelia burgdorferi sensu lato, the causative pathogens of Lyme disease. A better understanding of this aspect could advance knowledge about pathophysiology of these bacteria and help improve the efficacy of current approaches against Lyme disease. Here, we review all naturally occurring substances scientifically evaluated to date, including plant extracts, their metabolites, and micronutrients, against vegetative (spirochetes) and latent (rounded bodies, biofilm) forms of Borrelia sp. This summary reveals the potent anti-borreliae activity of several of these natural compounds indicating their potential in enhancing the efficacy of current treatments for Lyme disease, and offering new options to already existing therapeutic regiments.
... To date, only a few plant metabolites have undergone extensive scientific evaluation for antimicrobial activity against Borrelia sp., e.g. grape seed extract and teasel root extract (Brorson and Brorson 2007;Liebold et al. 2011). There is an enormous potential in exploring anti-borreliae properties of natural substances as they are generally ascribed as safe with promising outcomes, and they might be effective as a substitution or adjunct treatment to the standard antibiotic based therapies (Patil and Saraogi 2014;Takeuchi et al. 2014). ...
AimsLittle is known about the effects of phytochemicals against Borrelia sp. causing Lyme disease. Current therapeutic approach to this disease is limited to antibiotics. This study examined the anti-borreliaea efficacy of several plant-derived compounds and micronutrients.Methods and ResultsWe tested the efficacy of 15 phytochemicals and micronutrients against three morphological forms of Borrelia burgdoferi and Borrelia garinii: spirochetes, latent rounded forms and biofilm. The results showed that the most potent substances against the spirochete and rounded forms of Borrelia burgdorferi and Borrelia garinii were cis-2-decenoic acid, baicalein, monolaurin, and kelp (iodine); whereas, only baicalein and monolaurin revealed significant activity against the biofilm. Moreover, cis-2-decenoic acid, baicalein, and monolaurin did not cause statistically significant cytotoxicity to human HepG2 cells up to 125 μg ml−1, and kelp up to 20 μg ml−1.Conclusions
The most effective antimicrobial compounds against all morphological forms of the two tested Borrelia sp. were baicalein and monolaurin. This might indicate that the presence of fatty acid and phenyl groups is important for comprehensive antibacterial activity.Significance and Impact of the StudyThis study reveals the potential of phytochemicals as an important tool in the fight against the species of Borrelia causing Lyme disease.This article is protected by copyright. All rights reserved.
... The latter 2 examples, however, cannot be assumed to approximate growth characteristics in vivo, in which the organism would face the evolving biological conditions of tissue injury and inflammation with innate and adaptive immune responses. A number of additional in vitro studies have reported that such forms arise after exposure to antibiotics or (more generally) that antibiotics induce pathologic effects on cell morphology; still others have evaluated their susceptibility to a variety of antibiotics and other compounds [97][98][99][100][101][102][103][104][105][106][107][108][109]. Tested compounds have included vancomycin, tigecycline, telithromycin, tinidazole, metronidazole, ranitidine bismuth sulfate, hydroxychloroquine, and grapefruit seed extract. ...
Background:
Much of the controversy that surrounds Lyme disease pertains to whether it produces prolonged, treatment-refractory infection, usually referred to as chronic Lyme disease. Some have proposed that round morphologic variants of Borrelia burgdorferi, known variably as "cyst forms" and "L-forms," are responsible for the pathogenesis of chronic Lyme disease. We have undertaken a systematic review of the literature to determine if there is a documented role of these variants in Lyme disease pathogenesis or in syndromes compatible with chronic Lyme disease.
Methods:
Two systematic literature searches were performed to identify studies in which round morphologic variants of B. burgdorferi have been described in situ in human specimens.
Results:
Our primary literature search identified 6 studies that reported round morphologic variants of B. burgdorferi in specimens obtained from 32 total patients. No study described these forms in patients who had purely subjective symptom complexes (eg, fatigue or pain). No study investigated a causal relationship between morphologic variants and clinical disease or evaluated treatment of morphologic variants in vivo. Of 29 additional studies that described the morphology of B. burgdorferi from patients with Lyme disease, the organism was invariably described as having spirochetal morphology.
Conclusions:
In the context of the broader medical literature, it is not currently possible to ascribe a pathogenic role to morphologic variants of B. burgdorferi in either typical manifestations of Lyme disease or in other chronic disease states that are often labeled chronic Lyme disease. There is no clinical literature to justify specific treatment of B. burgdorferi morphologic variants.
... From the view of phytotherapy, solely grapefruit seed extract has been tested against Borrelia burgdorferi s.s. (Bbss) in vitro, but without any relation to therapeutical use (Brorson and Brorson 2007). Here, the activity of C. creticus leaf preparations of different polarities against mobile forms of Bbss in vitro is reported for the first time. ...
Borreliosis patients from self-help groups reported considerable pain relief after ingestion of Cistus creticus leaf preparations. C. creticus leaf extracts of different polarities such as aqueous, ethyl acetate, hexane extracts as well as the volatile oil fraction obtained by steam distillation were tested for their antibacterial activity against Borrelia burgdorferi sensu stricto (Bbss) in vitro using the antibiotic amoxicilline as standard and polysorbate 80 as solubilizer for lipophilic extracts. Comparison of the four plant preparations shows that the volatile oil exerts the strongest growth inhibitory effect. Even concentrations of 0.02% (w/v) volatile oil in cultivation media reduced the total number of bacteria to 2% in comparison to a growth control after an eight-day cultivation period. While the aqueous extract did not reduce bacterial growth, incubation with hexane and ethyl acetate extracts clearly inhibited microbial growth. The main volatile components of the three active extracts tested were analyzed by GC-MS. The number of different labdane-type diterpenes as well as the total relative amount of diterpenes in the samples tested was highest in the essential oil of C. creticus. Identification of ten different volatile labdane-type diterpenes was assigned to the essential oil of C. creticus. Among these, manoyl oxide, 13-epi-manoyl oxide, 3-acetoxy-manoyl oxide and the monoterpene carvacrol were determined to be major constituents, accompanied by minor amounts of 3-hydroxy-manoyl oxide, all of which are known to exert antimicrobial activity.
... (Wood 1997;Storl 2007), Dipsacaceae, although antibacterial effects of the European teasel have not been described so far. In this context, solely grapefruit seed extract without any relation to therapeutical use (Brorson and Brorson 2007), and due to patient's reports Cistus creticus extracts by bioguided fractionation in our lab (Hutschenreuther et al. 2010) were tested against Borrelia burgdorferi in vitro. The biennial Dipsacus sylvestris, introduced to Europe in antiquity, grows to a basal rosette with a strong tap root in its first year of cultivation, followed by the flowering period in the second year. ...
Fresh first year roots from Dipsacus sylvestris HUDS. were extracted with 70% ethanol, ethyl acetate as well as dichloromethane. Extracts were solubilized in water (lipophilic extracts with addition of polysorbate 80) and tested for their activity against Borrelia burgdorferi sensu stricto in vitro during an eight-day period using amoxicillin as standard. The hydroethanolic extract showed no growth inhibition whereas significant
growth inhibiting activity could be shown in the two less polar fractions for the first time. Strongest inhibition was found in the ethyl acetate extract. The effect of polysorbate 80 on bacterial growth was examined and found to be negligible. As the nature of bioactive constituents has not been clarified yet, a micellar electrokinetic capillary chromatography fingerprint analysis for a methanolic extract was applied including loganin, chlorogenic acid, cantleyoside and caffeic acid as marker substances.
... (Wood 1997;Storl 2007), Dipsacaceae, although antibacterial effects of the European teasel have not been described so far. In this context, solely grapefruit seed extract without any relation to therapeutical use (Brorson and Brorson 2007), and due to patient's reports Cistus creticus extracts by bioguided fractionation in our lab (Hutschenreuther et al. 2010) were tested against Borrelia burgdorferi in vitro. The biennial Dipsacus sylvestris, introduced to Europe in antiquity, grows to a basal rosette with a strong tap root in its first year of cultivation, followed by the flowering period in the second year. ...
Fresh first year roots from Dipsacus sylvestris HUDS. were extracted with 70% ethanol, ethyl acetate as well as dichloromethane. Extracts were solubilized in water (lipophilic extracts with addition of polysorbate 80) and tested for their activity against Borrelia burgdorferi sensu stricto in vitro during an eight-day period using amoxicillin as standard. The hydroethanolic extract showed no growth inhibition whereas significant growth inhibiting activity could be shown in the two less polar fractions for the first time. Strongest inhibition was found in the ethyl acetate extract. The effect of polysorbate 80 on bacterial growth was examined and found to be negligible. As the nature of bioactive constituents has not been clarified yet, a micellar electrokinetic capillary chromatography fingerprint analysis for a methanolic extract was applied including loganin, chlorogenic acid, cantleyoside and caffeic acid as marker substances.
... From the view of phytotherapy, solely grapefruit seed extract has been tested against Borrelia burgdorferi s.s. (Bbss) in vitro, but without any relation to therapeutical use (Brorson and Brorson 2007). Here, the activity of C. creticus leaf preparations of different polarities against mobile forms of Bbss in vitro is reported for the first time. ...
Borreliosis patients from self-help groups reported considerable pain relief after ingestion of Cistus creticus leaf preparations. C. creticus leaf extracts of different polarities such as aqueous, ethyl acetate, hexane extracts as well as the volatile oil fraction obtained by steam distillation were tested for their antibacterial activity against Borrelia burgdorferi sensu stricto (Bbss) in vitro using the antibiotic amoxicilline as standard and polysorbate 80 as solubilizer for lipophilic extracts. Comparison of the four plant preparations shows that the volatile oil exerts the strongest growth inhibitory effect. Even concentrations of 0.02% (w/v) volatile oil in cultivation media reduced the total number of bacteria to 2% in comparison to a growth control after an eight-day cultivation period. While the aqueous extract did not reduce bacterial growth, incubation with hexane and ethyl acetate extracts clearly inhibited microbial growth. The main volatile components of the three active extracts tested were analyzed by GC-MS. The number of different labdane-type diterpenes as well as the total relative amount of diterpenes in the samples tested was highest in the essential oil of C. creticus. Identification of ten different volatile labdane-type diterpenes was assigned to the essential oil of C. creticus. Among these, manoyl oxide, 13-epi-manoyl oxide, 3-acetoxy-manoyl oxide and the monoterpene carvacrol were determined to be major constituents, accompanied by minor amounts of 3-hydroxy-manoyl oxide, all of which are known to exert antimicrobial activity.
The primary objective of this paper is to provide an evidence-based update of the literature on the use of bioactive phytochemicals, nutraceuticals, and micronutrients (dietary supplements that provide health benefits beyond their nutritional value) in the management of persistent cases of Borrelia burgdorferi infection (Lyme disease) and two other tick-borne pathogens, Babesia and Bartonella species. Recent studies have advanced our understanding of the pathophysiology and mechanisms of persistent infections. These advances have increasingly enabled clinicians and patients to utilize a wider set of options to manage these frequently disabling conditions. This broader toolkit holds the promise of simultaneously improving treatment outcomes and helping to decrease our reliance on the long-term use of pharmaceutical antimicrobials and antibiotics in the treatment of tick-borne pathogens such as Borrelia burgdorferi, Babesia, and Bartonella.
Valorization of food byproducts has attracted recently considerable attention. Citrus fruits provide considerable non-edible residues reach 80% in juice production. They are considered agri-wastes to comprise peel, pulp and seeds. Previous investigations have focused on peel and pulp to recover value-added products. The review presents for the first-time phytochemical composition of Citrus seeds’ products, i.e., oil and extracts. Fatty acids, phytosterols and tocopherols amounted as the major bioactives in Citrus seeds, in addition to limonoids, dietary fibers and flavonoids. Besides their nutritional values, these chemicals have promising applications including production of biodiesel, food enhancers and antioxidants, especially from mandarin and grapefruit seeds. Optimum conditions of the different Citrus seeds' valorization are discussed to improve extraction yield and lessen environmental hazards of solvent extraction. This review presents the best utilization practices for one of the largest cultivated fruit seeds worldwide and its different applications.
La commercialisation des extraits de pépins de pamplemousse (EPP) connaît, depuis quelques années, un essor considérable, avec un nombre de spécialités sur le marché en constante augmentation. Bien que soumis à la réglementation des compléments alimentaires, le mode de fabrication et la composition chimique exacte des EPP restent très floues.
Il nous est apparu, que les plantes à l'origine des EPP appartenaient à plusieurs espèces (dont hybrides) du genre Citrus, incluant pamplemousse (Citrus grandis (L.) Osbeck syn. Citrus maxima (Burm.) Merr.) et pomelo (Citrus x paradisi Macfad.). Nous avons dans un premier temps analysé en détails la composition chimique de ces extraits, puis nous avons étudié leurs propriétés d'action, afin de comprendre leur intérêt au sein des complémentes alimentaires.
Nous avons ensuite établi un comparatif entre les différents EPP présents sur le marché français, et avons ainsi mis en évidence une grande variabilité chimique d'un extrait à l'autre (teneurs en flavonoïdes différant d'un facteur 12,5 selon les spécialités). Il est à noter par ailleurs que les laboratoires leur accordent des propriétés similaires.
Il apparaît donc nécessaire d'approfondir les études sur les extraits de pépins de pamplemousse, afin de pouvoir les évaluer sur un plan thérapeutique et offrir une information claire aux professionnels de santé. Le Pharmacien d'officine sera alors à même de pouvoir réaliser un conseil adapté.
Aims:
Borrelia spp., a causative pathogenic factor of Lyme disease, has become a major public health threat. Current treatments based on antibiotics often lead to relapse after their withdrawal. Naturally derived substances that could work synergistically to display higher efficacy compared to the individual components may serve as a resource for the development of novel approaches to combat both active and latent forms of Borrelia spp.
Methods and results:
Using checkerboard assay, we investigated the anti-borreliae reciprocal cooperation of phytochemicals and micronutrients against two species of Borrelia selected as prevalent causes of Lyme disease in the US and Europe. We tested 28 combinations of phytochemicals such as polyphenols (baicalein, luteolin, rosmarinic acids), fatty acids (monolaurin, cis-2-decenoic acid), and micronutrients (ascorbic acid, cholecalciferol, and iodine). The results showed that the combinations of baicalein with luteolin as well as monolaurin with cis-2-decenoic acid expressed synergistic anti-spirochetal effects. Moreover, baicalein and luteolin, when combined with rosmarinic acid or iodine, produced additive bacteriostatic and bactericidal effects against typical corkscrew motile spirochetes and persistent knob/round-shaped forms, respectively. An additive anti-biofilm effect was noticed between baicalein with luteolin as well as monolaurin with cis-2-decenoic acid. Finally, application of the combination of baicalein with luteolin increased cytoplasmic permeability of Borrelia spp. but did not cause DNA damage.
Conclusions:
These results show that a specific combination of flavones might play a supporting role in combating Borrelia spp. through either synergistic or additive anti-borreliae effects.
Significance and impact of study:
Presented here in vitro results might help advancing our knowledge and improving the approach to target Borrelia spp. This article is protected by copyright. All rights reserved.
Flavonoids are a class of plant constituents that have received increasing interest over the last decades. This chapter deals with the antimicrobial activity of some natural flavonoids or extracts rich in these constituents reported in the literature during the last 5 years. An introduction explains briefly the chemical structure of this class of natural compounds, their biosynthesis, plant sources and health benefits. Then the most significant articles from the scientific literature are reported, divided into two sections: studies on flavonoids with antibacterial and antifungal activities, respectively. In each paragraph the papers are listed according to the chemical complexity of the flavonoid structures, from the simplest to the most complex ones, both aglicones and glycosides and often gathering together the articles according to the main microbial target. A paragraph on the antimicrobial activity of combination of different flavonoids or between flavonoids and antibiotics (synergic effect) is also present. For many of the flavonoids cited the MIC values of their activity were also reported. The chemical structure of the majority of the compounds cited in the chapter are pictured in figures; tables have been compiled summarising the most important information reported in this chapter.
Serial dilutions of a citrus seed extract (ParaMycrocidin) were highly effective at concentrations ranging from 0.5 to 2 p.c. against a large number of Gram positive, Gram negative and yeast strains in vitro. The oral administration of the same agent in atopic patients with intestinal dysbiosis (3 x 150 mg daily) resulted in a significant inhibition of Candida Sp., Geotrichum sp. and hemolytic E. Coli growth. Staph, aureus, aerobic spore formers and lacto-bacilli were only slightly inhibited and no activity could be noticed against Klebsiella sp. and Bifidobacteria.
As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated from antibiotically treated and untreated patients with the clinical diagnosis of definite and probable Lyme borreliosis and from patient specimens contaminated with bacteria were investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron microscopy revealed diverse alterations. Persisters isolated from a great number of patients (60-80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation. Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells to replicate, the long period of adaptation to growth in MKP-medium and the mycoplasma-like colonies after growth in solid medium (PMR agar) suggest that B. burgdorferi produce spheroplast-L-form variants. With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion.
The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.
Difficulties in diagnosis of late stages of Lyme disease include low sensitivity of serological testing and late inclusion of Lyme disease in the differential diagnosis. Longer treatment modalities may have to be considered in order to improve clinical outcome of late disease stages. These difficulties clinical cases of Lyme borreliosis. The different clinical cases illustrate several aspects of late borreliosis: false negative serology due to narrow antigen composition of the used ELISA format, the need for prolonged antibiotic treatment in chronic or recurrent forms and typical presentations of late Lyme disease, such as lymphocytic meningo-encephalitis and polyradiculoneuritis.
Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [3H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2h post-gavage. After 18h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18h post-gavage. Total identified metabolites detected after 18h in most tissues were only 1-5% of the levels detected after 2h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18 h versus 2 h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18 h are retained as smaller decomposition molecules which cannot yet be identified.
The purpose of this paper is to overview the relations between plant-originated substances and their bioactivity measured in terms of antioxidant, cytoprotective and antiulcer activities. In addition, we assessed whether these compounds are capable of affecting the gastric mucosal lesions induced by absolute ethanol applied intragastrically (i.g.). The following plant-originated flavonoid substances were considered; Solon (Sophoradin extract), Amaranth seed extract, grapefruit-seed extract (GSE) and capsaicin (extract of chilly pepper). The area of gastric mucosa lesions and gastric blood flow were measured in rats with ethanol-induced lesions without (control) and with one of the tested substances without and with capsaicin denervation of afferent nerves or administration of L-nitro-arginine (L-NNA), an inhibitor of nitric oxide synthase (NOS). Male Wistar rats, weighing 180-220 g fasted for 24 h before the study where used 100% ethanol was applied i.g. to induce gastric lesions, whose area was determined by planimetry. Gastric blood flow was assessed using electrolytic regional blood flowmeter. All tested plant-originated substances afforded gastroprotection against ethanol-induced damage and this was accompanied by increase in gastric microcirculation, both changes being reversed by pretreatment with neurotoxic dose of capsaicin or by pretreatment with L-NNA. We conclude that plant-originated flavonoid substances are highly gastroprotective probably due to enhancement of the expression of constitutive NOS and release of NO and neuropeptides such as calcitonin gene related peptide (CGRP) released from sensory afferent nerves increasing gastric microcirculation.
To investigate dermal and epidermal involvement in the presence of Borrelia burgdorferi and to analyze the role of Langerhans cells and keratinocytes, 14 cases of erythema chronicum migrans and two controls were studied by means of electron microscopy, using negative staining and sectioning techniques. Using immunoelectron microscopy and histochemistry, positive results for B. burgdorferi were disclosed in 5 cases of erythema chronicum migrans and 3 cases of neuroborreliosis which were confirmed by cultivation. We cultured 4 stains of B. burgdorferi from the skin, 1 from blood and 2 from cerebrospinal fluid in BSK medium. Near to the centre of erythema chronicum migrans with focal necrosis were both a dissolved basal membrane and keratinocyte desmosomes surrounding damaged B. burgdorferi cells in the epidermis. Markedly oedematous keratinocytes and Langerhans cells with B. burgdorferi were released into lymphocyte infiltrates. At the periphery of all erythema chronicum migrans lesions, keratinocytes were well preserved while all dendritic cells seemed to be vacuolated. Above foci of B. burgdorferi located perivascular or among collagen fibers, Langerhans cells were frequent and more granulated. The possible role of Langerhans cells in the identification and elimination of B. burgdorferi is discussed.
Resveratrol, naringenin and naringin are naturally occurring flavonoids in grapes and grapefruits. The anti-inflammatory effects of these flavonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in inflammation, and the prevention of the expression of iNOS may be an important anti-inflammatory mechanism. In this study, the effects of these flavonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml−1) were investigated.
Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible.
In electrophoretic mobility shift assays, the activation of NFκB induced by LPS for 1 h was inhibited by resveratrol (30 μM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IκBα, and a reduced nuclear content of NFκB subunits.
The flavonoids may be of value for inhibiting the enhanced expression of iNOS in inflammation through down-regulation of NFκB binding activity.
British Journal of Pharmacology (1999) 126, 673–680; doi:10.1038/sj.bjp.0702357
The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole. Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test. For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC > or = 512 microg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ. When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC > or = 4 microg/ml, but the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees C. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was > or = MBC. Some cysts were also ruptured. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts. Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.
Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.
Methicillin-resistant Staphylococcus aureus (MRSA) and MSSA strains were treated with: (a) grapefruit oil (GFO) components, isolated by chromatography and characterised by NMR and mass spectroscopy; (b) antimicrobial agents, or (c) a combination of both to evaluate (MIC determination) intrinsic antibacterial activity and to determine whether GFO components could modulate bacterial sensitivity to the anti-bacterial agents. Preliminary data suggested that the grapefruit component 4-[[(E)-5-(3,3-dimethyl-2-oxiranyl)-3-methyl-2-pentenyl]oxy]-7H-furo[3,2-g]chromen-7-one (2) enhances the susceptibility of test MRSA strains to agents, e.g., ethidium bromide and norfloxacin, to which these micro-organisms are normally resistant.
Lyme borreliosis — a review of the late stage and treatment of four cases Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo
- M Petrovic
- Van D Vogelaers
- L Renterghem
- D Carton
- J Reuck
- M Afschrift
- T Malovrh
- R Murgia
- Cinco
Petrovic M, Vogelaers D, Van Renterghem L, Carton D, de Reuck J, Afschrift M: Lyme borreliosis — a review of the late stage and treatment of four cases. Acta Clin Belg 1998; 53: 178–183. 3. Gruntar I, Malovrh T, Murgia R, Cinco M: Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo. APMIS 2001; 109: 383–388.