In vivo and in vitro safety evaluation of fermented Citrus sunki peel extract: acute and 90-day repeated oral toxicity studies with genotoxicity assessment.

BACKGROUND: Citrus sunki Hort. ex Tanaka peel has been traditionally used as an ingredient in folk medicine due to its therapeutic effects on promotion of splenic health and diuresis as well as relief of gastrointestinal symptoms. Although a growing interest in health-promoting natural products and the development of highly concentrated products have facilitated consumption of C. sunki peel, its safety assessment has not been explored, posing a potential health risk. In this study, we carried out a series of systemic and genetic toxicity tests on fermented C. sunki peel extract (FCPE) to provide the essential information required for safe use in human. METHODS: We conducted acute and 90-day repeated oral toxicity studies in Sprague-Dawley rats to evaluate systemic toxicity, and three genotoxicity assays to measure bacterial mutation reversion, cellular chromosome aberration and in vivo micronucleus formation. RESULTS: Single oral administration of FCPE did not cause any clinical signs and lethality in all animals, establishing LD50 to be over 2000 mg/kg BW. Repeated administration of up to 2000 mg/kg BW FCPE for 90 days revealed no test substance-related toxicity as demonstrated in analysis of body weight gain, food/water intake, blood, serum biochemistry, organ weight and histopathology, collectively determining that the no-observable-adverse-effect-level of FCPE is over 2000 mg/kg BW. In addition, we detected no mutagenicity and clastogenicity in FCPE at 5000 µg/plate for the in vitro assays and 2000 mg/kg BW for the in vivo micronucleus test. CONCLUSION: FCPE did not cause systemic and genetic toxicity in our model systems at the tested dose levels. These results suggest a guideline for safe consumption of C. sunki peel in human.

Label-free quantitative proteomic profiling of colon cancer cells identifies acetyl-CoA carboxylase alpha as antitumor target of Citrus limon-derived nanovesicles.

We have previously isolated exosome-like nanoparticles from Citrus-limon juice, able to inhibit in vitro and in vivo tumor cell growth. In order to deeply understand the mechanism underlying nanovesicle effects, we performed a proteomic profile of treated colorectal cancer cells. Among the proteins differentially expressed after nanovesicle treatment, we found a significant downregulation of the Acetyl-CoA Carboxylase 1 (ACACA) and we demonstrated that silencing ACACA in cancer cells leads to a reduction of cell growth. Our study proved that the anti-tumor effects of Citrus-limon nanovesicles is partly mediated by lipid metabolism inhibition, in particular via ACACA downregulation. SIGNIFICANCE: This study represents the attempt to achieve, by a proteomic approach, a better understanding of the role of lemon nanovesicles in affecting colorectal cancer cell growth.

Toxic potentials of ten herbs commonly used for aphrodisiac effect in Turkey.

BACKGROUND/AIM: Sexual dysfunction is a serious problem worldwide. In Turkey, herbal products are used by some people suffering from sexual dysfunction. Despite their therapeutic advantages, some constituents of herbs are potentially toxic and pose health risks because they can be bought from the market without a prescription. Therefore, we aimed to determine the safety of herbs possessing aphrodisiac effects, chosen on the basis of their frequency of medicinal use and commercial importance in Turkey. MATERIALS AND METHODS: Ten herbs (Anethum graveolens, Carthamus tinctorius, Citrus aurantium, Cocos nucifera, Glycyrrhiza glabra, Melissa officinalis, Nigella arvensis, Pinus pinea, Prunus mahaleb, and Zingiber officinale) were extracted with water, methanol, and chloroform. The cyto- and genotoxic potentials of the extracts were assessed using an MTT test on a rat kidney cell line and an Ames assay in Salmonella typhimurium strains, respectively. RESULTS: In the cytotoxic evaluation, IC50 values were 1.51-31.4 mg/mL for the methanol and chloroform extracts, while the water extracts were not cytotoxic. In the genotoxic evaluation, it was revealed that the water extracts had more mutagenic activity than the chloroform and methanol extracts. Water extract of M. officinalis was shown to have the most genotoxic activities to TA100 (±S9) and TA98 (-S9). CONCLUSION: These results might be useful in determining the toxic effects of herbs and lead to precautions being taken in regards to their consumption.

Hepatoprotective effect of limonin, a natural limonoid from the seed of Citrus aurantium var. bigaradia, on D-galactosamine-induced liver injury in rats.

Toll-like receptors have been implicated in inflammation and injury in various tissues and organs including the liver. We have investigated the effects of limonin isolated from the dichloromethane fraction of the seeds of bittersweet orange (Citrus aurantium var. bigaradia) in two dose levels (50 and 100 mg/kg) against D-galactosamine (D-GalN)-induced liver toxicity in comparison with standard silymarin treatment on Toll-like receptors expression and hepatic injury, using a well-established rat model of acute hepatic inflammation. The limonoids in the seeds of bittersweet orange were identified. Oral administration of limonin before D-GalN injection, significantly attenuated markers of hepatic damage (elevated liver enzyme activities and total bilirubin) and hepatic inflammation (TNF-α, infiltration of neutrophils), oxidative stress and expression of TLR-4 but not TLR-2 in D-GalN-treated rats. Limonin effects were similar in most aspects to that of the lignan silymarin. The higher dose of limonin (100 mg/kg) performed numerically better for AST and bilirubin, and both doses yielded similar results for ALT and GGT. While the lower dose of limonin (50 mg/kg) performed better against oxidative stress and liver structural damage as compared to the higher dose. Limonin exerts protective effects on liver toxicity associated with inflammation and tissue injury via attenuation of inflammation and reduction of oxidative stress.

Cardiovascular toxicity of Citrus aurantium in exercised rats.

When safety concerns forced the removal of ephedra from the market, other botanicals, including Citrus aurantium or bitter orange (BO) were used as replacements. A major component of the BO extract is synephrine, a chemical that is structurally similar to ephedrine. Because ephedrine has cardiovascular effects that may be exacerbated during physical exercise, the purpose of this study was to determine whether extracts containing synephrine produced adverse effects on the cardiovascular system in exercising rats. Sprague-Dawley rats were dosed daily by gavage for 28 days with 10 or 50 mg of synephrine/kg body weight from one of two different extracts; caffeine was added to some doses. The rats ran on a treadmill for 30 min/day, 3 days/week. Heart rate, blood pressure, body temperature, and QT interval were monitored. Both doses of both extracts significantly increased systolic and diastolic blood pressure for up to 8 h after dosing. Effects on heart rate and body temperature appeared to be due primarily to the effects of caffeine. These data suggest that the combination of synephrine, caffeine, and exercise can have significant effects on blood pressure and do not appear to be effective in decreasing food consumption or body weight.

Physiological effects following administration of Citrus aurantium for 28 days in rats.

BACKGROUND: Since ephedra-containing dietary supplements were banned from the US market, manufacturers changed their formulations by eliminating ephedra and replacing with other botanicals, including Citrus aurantium, or bitter orange. Bitter orange contains, among other compounds, synephrine, a chemical that is chemically similar to ephedrine. Since ephedrine may have cardiovascular effects, the goal of this study was to investigate the cardiovascular effects of various doses of bitter orange extract and pure synephrine in rats. METHOD: Female Sprague-Dawley rats were dosed daily by gavage for 28 days with synephrine from two different extracts. One extract contained 6% synephrine, and the other extract contained 95% synephrine. Doses were 10 or 50mg synephrine/kg body weight from each extract. Additionally, caffeine was added to these doses, since many dietary supplements also contain caffeine. Telemetry was utilized to monitor heart rate, blood pressure, body temperature and QT interval in all rats. RESULTS AND CONCLUSION: Synephrine, either as the bitter orange extract or as pure synephrine, increased heart rate and blood pressure. Animals treated with 95% synephrine showed minimal effects on heart rate and blood pressure; more significant effects were observed with the bitter orange extract suggesting that other components in the botanical can alter these physiological parameters. The increases in heart rate and blood pressure were more pronounced when caffeine was added. None of the treatments affected uncorrected QT interval in the absence of caffeine.

Developmental toxicity of Citrus aurantium in rats.

BACKGROUND: Ephedra was commonly used in herbal products marketed for weight loss until safety concerns forced its removal from products. Even before the ban, manufacturers had begun to replace ephedra with other compounds, including Citrus aurantium, or bitter orange. The major component in the bitter orange extract is synephrine which is chemically similar to ephedrine. The purpose of this study was to determine if relatively pure synephrine or synephrine present as a constituent of a bitter orange extract produced developmental toxicity in rats. METHOD: Sprague-Dawley rats were dosed daily by gavage with one of several different doses of synephrine from one of two different extracts. Caffeine was added to some doses. Animals were sacrificed on GD 21, and fetuses were examined for the presence of various developmental toxic endpoints. RESULTS AND CONCLUSION: At doses up to 100 mg synephrine/kg body weight, there were no adverse effects on embryolethality, fetal weight, or incidences of gross, visceral, or skeletal abnormalities. There was a decrease in maternal weight at 50 mg synephrine/kg body weight when given as the 6% synephrine extract with 25 mg caffeine/kg body weight; there was also a decrease in maternal weight in the caffeine only group. This decrease in body weight may have been due to decreased food consumption which was also observed in these two groups. Overall, doses of up to 100 mg synephrine/kg body weight did not produce developmental toxicity in Sprague-Dawley rats.

Subchronic toxicity of Citrus aurantium L. (Rutaceae) extract and p-synephrine in mice.

Extracts of Citrus aurantium L. (Rutaceae) unripe fruits have gained popularity for the treatment of obesity. Due to the wide use of C. aurantium/p-synephrine-containing products, this research was undertaken to evaluate its subchronic toxicity in mice and their actions in oxidative stress biomarkers. Groups of 9-10 mice received for 28 consecutive days a commercial C. aurantium dried extract (containing 7.5% p-synephrine) 400, 2000 or 4000 mg/kg and p-synephrine 30 or 300 mg/kg by oral gavage. There was a reduction in body weight gain of animals treated with both doses of p-synephrine. Organs relative weight, biochemical and hematological parameters were not altered in all treated mice. There was an increase in reduced glutathione (GSH) concentration in groups treated with C. aurantium 4000 mg/kg and p-synephrine 30 and 300 mg/kg. In glutathione peroxidase (GPx), there were an inhibition of the activity in C. aurantium 400 and 2000 mg/kg and p-synephrine 30 and 300 mg/kg treated animals, respectively, and was no alteration in malondialdehyde (MDA) levels. Thus, the results indicate a low subchronic toxicity of the tested materials in mice and a possible alteration in the oxidative metabolism. However, further tests are required to better elucidate the effects of these compounds in the antioxidant system.

Bioactivity of citrus seed for mosquito-borne diseases larval control.

This study aimed to determine the activity of citrus-seed extract against Ae. aegypti and Cx. quinquefasciatus larvae. The results indicated that ethanol citrus-seed extract showed the best killing effect on Ae. aegypti larvae, followed by local liquor, and water, with LC50 of 2,267.71, 6,389.22, and 135,319.40 ppm, respectively, whereas against Cx. quinquefasciatus larvae, the LC50 were 2,639.27, 5,611.66, and 127,411.88 ppm, respectively. Temephos was tested against Ae. aegypti larvae; the LC50 was 0.00057 ppm, which was nearly 4,000,000 times less than ethanol citrus-seed extract. When ethanol citrus-seed extract and temephos were tested with Nile tilapia (Oreochromis niloticus), a standard environmental organism, using LC50 of Ae. aegypti larvae at 2,267 and 0.00057 ppm, respectively, fish mortality was 0%. The results suggested that ethanol citrus-seed extract had no harmful effect on the fish, and that temephos, which is recommended by WHO, was safe for use in drinking water. However, when the LC50 dose that killed Ae. aegypti larvae for local liquor (6,389 ppm) and water extract (135,319 ppm) were tested with fish, the mortality rates were 35% and 100%, respectively. On the whole, the results suggested that ethanol citrus-seed extract is environmentally friendly and can be used in the control of Ae. aegypti and Cx. quinquefasciatus larvae.

Experimental phytophotodermatitis.

BACKGROUND/AIMS: Phytophotodermatitis (PPD) is defined as a phototoxic reaction of the skin after contact with substances derived from plants and subsequent exposure to sunlight. It is a frequent disease in our outpatient clinics during summer because of contact with Tahitian lemon. Our objectives were to experimentally reproduce PPD in rats, to identify whether PPD is induced by minimal exposure periods to sunlight, to find what kinds of lemons and which parts of the lemon (the fruit juice or the peel juice) may trigger the disease; to know whether the use of sunblock prevents the reaction; and to perform light microscopy of the lesions to describe their histology. METHODS: Adult rats (Rattus norwegicus), three in each experiment, were used. After painting the rats with the fruit juice or the peel juice they were exposed to sunlight for 2.5, 5, 7.5, and 10 min. Tahitian and Sicilian lemons were used in the experiments. Biopsies with 3-mm punches of different times of exposure were performed. RESULTS: The peel juice of both lemons reproduced PPD, which was clinically evident after 48 h. When the peel juice was alone applied there was no reaction; moreover, exposure to sunlight alone triggered no reaction. Two and a half minutes of exposure time was sufficient to induce phototoxic reaction, which was time dependent (the longer the exposure the more intense the reaction). Histopathological studies showed epithelial time-dependent vacuolar degeneration. The use of sunblock diminished the intensity of the reaction but did not prevent it. CONCLUSION: PPD can be reproduced in an animal model. It may be caused by the peel juice of Tahitian and Sicilian lemon. Because of an extremely short time of exposure (2.5 min) is sufficient to induce PPD it is necessary to alert the population, of the need for caution when handling lemons, especially outdoors despite using sunblock.

Hemograma, bioquímica sérica e histologia da biópsia hepática de bovinos após administração de polpa cítrica / Hemogram, serum biochemistry and hepatic histologic features in cattle after administration of citrus pulp

Hemogram and serum biochemistry (aspartate aminotransferase, alkaline phosphatase and gamma glutamiltransferase, total protein, urea, creatinine, calcium and phosphorus) were performed weekly in five crossbreed bovine after consumption of a diet containing citrus pulp pellets (40 percent), for 43 days. Percutaneous hepatic biopsy and histologic evaluation were performed in each animal before and after consumption of the citrus pulp diet. Hemogram, the enzymes aspartate aminotransferase and gamma glutamiltransferase, urea and creatinine had normal levels at the end of the experiment. No histologic lesions were observed in liver samples before or after citrus pulp consumption. However, there was an increase of serum phosphorus and reduction of serum calcium (p<0.05), without hypercalcemia, after consumption of citrus pulp diet. There was also an increase in serum alkaline phosphatase (p<0.05), probably induced by bone isoenzyme.

Toxicity study of an antidipsotropic Chinese herbal mixture in rats: NPI-028.

OBJECTIVES: To determine the potential toxicity and safety of the Chinese herbal medicine NPI-028 in rats following subchronic (3-month) exposure via daily oral consumption. DESIGN: Subchronic toxicity was evaluated in four groups of rats (n = 10 per group) receiving NPI-028 orally at a dose of either 0.0 (normal diet control), 0.5, 1.0, or 2.0 g/kg, ingested as part of their daily diet for 3 months. NPI-028 was incorporated into powdered rat chow diet as a specific percent of the total diet provided each day. The primary active isoflavone content of NPI-028 (puerarin) used in the rat diet was also determined. OUTCOME MEASURES: Subchronic toxicity was assessed over a 3-month period by biweekly measurement of water and food intake, weight gain, and visual inspection for maintenance of grooming and normal behavior. At the end of the study period rats were euthanized and blood was obtained for hematologic and chemical analysis. Organs were removed for histopathologic examination. RESULTS: Rats in all three NPI-028 dose groups were similar to the control group in weight gain, food intake, and water intake over the study period. Hematology, blood chemistries, and organ histology in rats at all three NPI-028 doses did not significantly differ from control rats. Minor exceptions were elevated urea nitrogen values at all NPI-028 doses, and increased triglyceride and thyroid-stimulating hormone values in the lowest NPI-028 dose-treated group. Puerarin (used as a dietary isoflavone marker) content of NPI-028 was 26 mg/g dry weight. CONCLUSIONS: NPI-028 ingested orally at doses up to 2.0 g/kg per day in the rat diet for up to 3 months resulted in normal growth with no changes in hematologic or hepatic parameters, and only minor alterations in renal and blood chemistry parameters. There was no evidence of abnormal histology. These data suggest the long-term daily oral consumption of NPI-028 as a part of the daily diet for 3 months, at the doses studied, is safe in rats. Thus, NPI-028 may potentially be safe for clinical use as an antidipsotropic agent.

Genetic toxicity of a standardized mixture of citrus polymethoxylated flavones.

Flavonoids are a ubiquitous family of phytochemicals that display a variety of biological effects, both beneficial and adverse depending on the individual compound. Certain flavonoids are genotoxic while others inhibit the genotoxicity of other mutagens. In the present studies, the mutagenicity of a mixture of polymethoxylated flavones (PMFs) purified from citrus peel oil was evaluated. The mixture consisted of nobiletin (32.5%), 3,3',4',5,6,7,8-heptamethoxyflavone (25.0%), tangeretin (14.0%), trimethylscutellarein (9.1%), sinensetin (3.9%), 5-demethyl-nobiletin (2.8%), hexa-O-methylquercetagetin (3.3%), 5-demethyl-tetramethylscutellarein (0.7%), 5-hydroxy-3,3',4',6,7,8-hexamethoxyflavone (0.7%), and a small quantity of unidentified flavonoid compounds (3.9%). In vitro addition of the PMF mixture over a concentration range that spanned four log doses (0.0005-5.0 mg/plate) did not reveal any evidence of mutagenicity in five bacterial tester strains (Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537) either in the absence or presence of S9 activation. The PMF mixture exhibited a statistically significant increase in mutagenicity of L5178Y tk(+/-) mouse lymphoma cells at 0.05 (38.5 x 10(-6); P<0.05) and 0.1 mg/ml (61 x 10(-6); P<0.01) compared with vehicle-treated controls (mutation frequency=19.7 x 10(-6)). However, these responses were within historical values observed in negative control cultures and extremely small compared to the positive control (EMS 0.5 microl/ml; 1685.3 x 10(-6)). Furthermore, in the presence of S9 there was no indication of genetic toxicity in L5178Y tk(+/-) cells. These results demonstrate that the PMF mixture is not genotoxic in in vitro assay systems.

Estudio genotóxico "In Vitro" de una tintura al 50 por ciento de citrus sinensis (L.) Osbeck

Se presentan los resultados obtenidos, al evaluar la actividad genotóxica "in vitro" de una tintura al 5o por ciento de Citrus sinensis (L.) Osbeck empleando como sistema de ensayo el hongo Aspergillus nidulans. Los resultados permiten concluir que la tintura fue tóxica al interferir en el crecimiento lineal del hongo y además genotóxica en el ensayo de segregación mitótica al producir un elevado número de sectores segregantes (AU)

Estudio genotóxico "In Vitro" de una tintura al 50 por ciento de citrus sinensis (L.) Osbeck / Study genotóxic \"in Vitro\" of a tincture to 50 sinensis percent of citrus (L.)Osbeck

Se presentan los resultados obtenidos, al evaluar la actividad genotóxica "in vitro" de una tintura al 5o por ciento de Citrus sinensis (L.) Osbeck empleando como sistema de ensayo el hongo Aspergillus nidulans. Los resultados permiten concluir que la tintura fue tóxica al interferir en el crecimiento lineal del hongo y además genotóxica en el ensayo de segregación mitótica al producir un elevado número de sectores segregantes

[Hygienic examination of oranges subjected to electro-ion treatment for the prolongation of preservation time]. / Gigienicheskoe izuchenie apel'sinov, podvergshikhsia élektroionnoi obrabotke s tsel'iu udlineniia srokov khraneniia.

On the basis of large experimental material (study of organoleptic properties, chemical composition of electro-antiseptic + oranges, microbiological studies of microbic cenosis during their storage, biological experiments to find out the possible general toxic effect of the pretreated fruit on animal organism) safety of oranges subjected to electro-ion++ treatment to prolong the term of preservation has been stated. The possibility to use electro-atiseptic oranges by the people in their nutrition has been proved.

Phototoxic and contact toxic reactions of the exocarp of sweet oranges: a common cause of cheilitis?

Irritant skin reactions were produced within 1 h after application of the exocarp of sweet oranges or alcoholic extracts therefrom. Such reactions faded within 48 h. The exocarp, or extracts thereof, induced phototoxic reactions which were strongest at 72 h after exposure. The phototoxic reactions were only induced in natural blondes and only with some oranges. The in vivo phototoxic reactions were confirmed in vitro, causing a slight but clear photo-inhibition of Candida albicans. Only some oranges inhibited growth.