Beruflich Dokumente
Kultur Dokumente
and others 1996; Yongchaiyudha and others 1996). Aloe vera can also be useful for reducing lipid levels in patients with hyperlipidemia (Yongchaiyudha and others 1996). Aloe vera gel (the mesophyll part of Aloe vera) contains about 98.5% water (Rowe and Parks 1941). Its major components are polysaccharides (pectins, hemicelluloses, glucomannan, acemannan, and mannose derivatives) but it also has amino acids, lipids, sterols (lupeol, campesterol, and -sitosterol), tannins, and enzymes (Shelton 1991; Vogler and Ernst 1999). Mannose 6phosphate is a major sugar component (Davis and others 1994a). Lupeol was found to be the most active ingredient and reduced inammation in a dose-dependent manner (Davis and others 1994b). In our previous study, Aloe-sterols (lophenol, 24-methyllophenol, 24-ethyl-lophenol, cycloartanol, and 24-methylenecycloartanol) were isolated from Aloe vera gel and found to reduce the hemoglobin A1c (HbA1c) levels of type 2 diabetic db/db mice (Tanaka and others 2006). In another study, the oral administration of lophenol and cycloartanol reduced visceral fat accumulation and improved hyperglycemia and hyperlipidemia in Zucker Diabetic Fatty (ZDF) rats independent of changes in food intake (Misawa and others 2008). In a previous acute toxicity study, 500 mg/kg, 1 g/kg, and 3 g/kg of Aloe vera (extracted in ethanol) exhibited no signs of toxicity (Shah and others 1989). These authors also conducted a study in which 20 male Swiss albino mice were administered Aloe vera in drinking water at a dose of 100mg/kg for 3 mo. Body and organ weights in the treated animals did not differ from control values. No abnormalities of the viscera were observed in treated or control animals. The mortality rate as compared to the control was signicant and hematological studies revealed a considerable decrease in RBC (Shah and others 1989). MS 20110340 Submitted 3/17/2011, Accepted 9/16/2011. Authors are with Logarto Parra and others (2001) administered an Aloe extract to Functional Food Research Dept., Food Science & Technology Inst., Morinaga Milk Industry Co., Ltd., 183, Higashihara 5-chome, Zama-City, Kanagawa 2528583, Swiss albino mice. The extracts (concentrations not given) were given orally. The estimated LD50 for aloe 24 h after dosing was Japan. Direct inquiries to author Tanaka (E-mail: m_tanaka@morinagamilk.co.jp). 120.65 mg/kg. Aloe vera (L.) Burm. f. (synonym = Aloe baradensis Miller) is a plant belonging to the family Liliaceae (Grindlay and Reynolds). Aloe vera gel, obtained from inner thin-walled parenchyma cells, has been used as a health food and has been used traditionally in Indian Ayurvedic medicine. There have been numerous reports of Aloe vera having diverse biological activities. Aloe is known for its topical use for treating wounds and burns. A clinical trial demonstrated the usefulness of Aloe vera for the prophylaxis of radiation-induced dermatitis (Heggie and others 2002). Polysaccharides isolated from the gel of Aloe species have various biological activities, including immunomodulatory effects. Polysaccharides of between 400 Da and 5 KDa were reported to exhibit potent antitumor activity in vivo (Im and others 2005). An immunostimulatory polysaccharide called Aloeride (4 and 7 million Da) increased NF-kappa B-directed luciferase expression in THP-1 human monocytic cells to levels 50% of those achieved by maximal concentrations (10 g/mL) of Lipopolysaccharide (LPS) (Pugh and others 2001). Yagi and others (2003) isolated a glycoprotein from aloe that inhibited cyclooxygenase and reduced thromboxane synthesis. In fact, Aloe vera gel has been used in nutritional supplements in clinical trials for the treatment of acquired immune deciency syndrome (AIDS) because of its antiviral and immunological properties (Montaner and others 1996). Two nonrandomized clinical studies found that 2 tablespoons of Aloe vera gel juice a day signicantly reduced fasting blood glucose levels in diabetic patients after 6 wk of intake (Bunyapraphatsara
T2 Journal of Food Science r Vol. 71, Nr. 1, 2012
C
Introduction
In vitro chromosomal aberration test with CHL cells A chromosomal aberration test of AVGE was performed with cultured mammalian cells from the lung of a Chinese hamster (CHL/IU) (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) in both a nonactivated and an activated system in accordance with On Guidelines for Genotoxicity Studies of Drugs (Notication Nr 1604 of the Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare, November 1, 1999). The potential mutagenicity of AVGE was evaluated in 2 experiments in which the cells were exposed to the test material in the short term (6 h) at concentrations of 200, 400, 800, and 1600 g/mL in the presence and absence of S9 mix, and for a long-term (24 h) exposure period at concentrations of 100, 200, 400, and 800 g/mL. The positive control group for the short-term treatment in the presence of S9 mix was treated with dimethylnitrosamine (Wako Pure Chemical Industries). That for the short-term and long-term treatments in the absence of S9 mix was treated with mitomycin C (Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan). The nal judgment on the outcome of each test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5%, the result was assessed as negative; from 5% to 10% (10% excluded) as equivocal; and 10% or higher with a concentration-dependent rise as positive. The incidence of cells having structural aberrations with gaps and the incidence of those without gaps were calculated separately, and cells without gaps were assessed for chromosomal aberrations. No signicance tests were performed since the incidence of cells with chromosomal aberrations was assessed according to the methods described in the evaluation criteria. It is considered that Aloe vera extract does not induce numerical or structural aberrations since the incidence of cells with numerical and structural aberrations induced by Aloe vera extract was less than 5% by all methods. This study was carried out by Nihon Bioresearch Inc. (Hashima, Gifu, Japan).
Mouse micronucleus test AVGE was evaluated for mutagenic potential in mouse bone Table 1Specications of Aloe vera gel extract (AVGE). marrow in accordance with the Guidelines for Genetic Toxicity Description Specication Studies (Notication Nr 1604 of Pharmaceutical Affairs BuBotanical source Aloe barbadensis (Miller) reau, November 1, 1999). The negative control group and AVGE (150 mg/kg) were set. Each group consisted of 6 animals. AVGE Water 26.98% Protein 0.64% was administered intragastrically using a probe twice to Inst. of Fat 67.0% Cancer Research (ICR) male mice at the age of 8 wk at 24-h Ash 0.05% intervals. In the negative control group, 15% propylene glycol was Carbohydrate 5.32% administered. In the positive control group, 4 mg/kg of mitoPhytosterols Mi n.5% Phyosterols are beta-sitosterol, lophenol, 24-methyl-lophenol, 24-ethyl-lophenol, mycin C was intraperitoneally administered once on the day of cycloartanol, and 24-methylene-cycloartanol. the nal administration at a dosing volume of 10 mL/kg. Clinical
Single oral dose toxicity study of Aloe vera extract in rats Male and female Crl:CD(SD) rats (10 males and 10 females/ group), at 5 wk of age, were purchased from Charles River Laboratory Japan, Inc., and 150 mg/kg of AVGE, considered the technical maximum level for administration, was prepared, taking into account the viscosity of the dosing solution. Dosing was performed once by gavage, using a plastic stomach tube, to animals that had been fasted for approximately 17.5 h. The control group received vehicle via the same method. Feeding was restarted 3 h after dosing. The observation period was from the day of dosing (Day 0) to Day 14. On Day 0, the physical condition (clinical signs) of the animals was observed individually once within the rst 30 min and every hour until 6 h after the dosing. During the period from Day 1 to 14 the animals were observed once a day. On Day 14, all animals were euthanized by exsanguination under ether anesthesia and then necropsied. The necropsy consisted of observations of the bodys surface and all orices as well as all organs and tissues including the abdominal, thoracic, pelvic, and cranial cavities. All gross abnormalities were recorded with regard to location, size, and color tone. Carcasses were incinerated immediately after necropsy. This study was carried out by Biosafety Research Center (Iwata, Shizuoka, Japan). This study was conducted in compliance with the Law Concerning the Protection and Control of Animals, Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain, and Guidelines for Animal Experimentation, Biosafety Research Center, Foods, Drugs, and Pesticides (BSRC Experimental Animal Ethics Committee Authorization Nr 08203A). Ninety-day repeated dose oral toxicity study of AVGE in rats This study was carried out by Fuji Biomedix Co., Ltd. It was performed according to the Rules for Approval of Animal Experiments of Fuji Biomedix Co., Ltd. (Approval Nr 2007138). Male and female Crl:CD(SD) rats, at 4 wk of age, were purchased from Charles River Laboratory Japan, Inc. During the acclimation period, they were observed for clinical signs once a day, weighed on the nal day of acclimation, and measured for food consumption once. Since no abnormal changes in clinical signs, body weight, food consumption, or ophthalmological features were found, it was considered that all the animals could be used. Animals were assigned to 3 groups, namely a control group and 2 groups treated with Aloe vera extract at concentrations of 30 and 150 mg/kg. Each group consisted of 10 animals of both genders. The duration and frequency of administration were set. Administration via gavage was carried out once daily for 90 d to investigate repeated dose toxicity of the test substance. All animals were observed for mortality and clinical signs twice daily (before dosing and within approximately 1 h after dosing) during the administration period, and once before necropsy on the necropsy
T4 Journal of Food Science r Vol. 71, Nr. 1, 2012
Results
Bacterial reverse mutation test (Ames test) No obvious increase in the number of revertant colonies was observed for any of the strains treated with AVGE compared with the negative control, with or without metabolic activation (Table 2). Precipitation of the test substance restricted the use of a stereomicroscope at 5000 g/plate for all strains in both assays.
0a 25 30 133 133 12 11 8 20 24 78.1 23 33 132 134 11 9 10 19 19 156 26 37 135 133 14 11 11 18 22 313f 20 39 131 138 11 10 9 19 24 625f 19 38 119 140 9 12 4 15 15 1250f 21 31 134 124 9 12 6 15 18 2500f 19 34 113 123 10 11 5 15 19 5000f 21 31 116 128 10 10 6 15 18 Positive 0.01 717b 141b 0.1 711b 0.5 443c 705d 1.0 1075c 2.0 341c 221c 10.0 80.0 364e
a Negative control was dimethyl sulfoxide. b Positive control was 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide. c Positive control was 2-aminoanthracene. d Positive control was sodium azide. e Positive control was 9-aminoacridine hydrochloride. f
PCE = polychromatic erythrocyte; MN-PCE = micronucleated polychromatic erythrocyte. The total counts of MN-PCE were compared for the value in the negative control with that in each treated group by the Kastenbaum and Bowman method, and a signicant difference was found ( P < 0.01). The PCE counts were compared for the mean in the negative control with that in each treated group by t-test, and a signicant difference was found ( P < 0.05). a A total of 104 negative controls were propylene glycol. b A total of 105 positive controls were mitomycin C.
Table 5Body weight in rats singly treated with AVGE. 853c Days after oral administration Male 0 7 14 Female 0 7 14 Control n = 10 136 3 210 7 270 16 121 3 166 6 195 9 Body weight (g) AVGE n = 10 136 3 211 8 272 12 121 3 169 7 198 11
Table 3 Chromosomal aberration test of AVGE with cultured CHL cells. Aberrant cells (%) Aberrations (Mean number)
Dose (g/mL)
PE ER Survival +gap gap +gap gap (Mean) (Mean) (%) 0 1.5 1.5 0.5 0 53.5 1.0 1.5 0 0.5 1.0 44.5 0 0.5 0.5 0 0 37.0 0 3 3 1 0 107 2 3 0 1 2 89 0 1 1 0 0 74 0 3 3 1 0 107 2 3 0 1 2 89 0 1 1 0 0 74 1 1 2 1 3 1 1 0 1 1 1 2 2 2 2 3 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 96 91 76 46 83 100 94 90 75 43 84 100 98 92 80 44 83
Values are the mean standard deviation. No signicant differences were found between the control and each treated group.
6-h treatment S9 0a + 0 200 + 1.5 400 + 1.5 800e + 0.5 e + 0 1600 Positiveb + 53.5 6-h treatment S9 0a 1.0 200 1.5 400 0 800e 0.5 1.0 1600e Positivec 44.5 24-h treatment 0a 0 100 0.5 200 0.5 400 0 800e 0 Positived 37.0
Table 6 Urinalysis in rats treated orally with Aloe vera extract for 90 d. AVGE (mg/kg/day) Control Male Volume (mL) Specic gravity Na (mEq/L) K (mEq/L) Cl (mEq/L) Total excretion Na (mg/day) K (mg/day) Cl (mg/day) Female Volume (mL) Specic gravity Na (mEq/L) K (mEq/L) Cl (mEq/L) Total excretion Na (mg/day) K (mg/day) Cl (mg/day) 155 48 1.063 0.014 155 48 312.9 72.8 202 64 47 4 164 18 95 14 10.5 2.0 1.048 0.006 108 24 232.3 36.0 152 40 26 5 95 17 56 14 30 117 10 1.054 0.004 117 10 276.6 25.3 170 19 43 3 174 7 97 3 13.1 10.5 1.053 0.024 109 36 248.1 97.2 147 50 26 12 98 39 54 23 150 117 34 1.050 0.011 117 34 265.3 65.9 168 5 43 8 167 29 96 16 13.2 5.6 1.047 0.017 105 43 236.8 96.9 150 59 28 3 106 14 61 7
Each group consisted of 10 animals of both sexes. Values are the mean standard deviation. No signicant differences were found between the control and each treated group.
Following Ishidates method1, 100 cells in metaphase per plate (200 cells per concentration), with well-spread chromosomes, were observed. PE was polyploid cells. ER was endo-reduplication. a Negative control was dimethyl sulfoxide. b Positive control was dimethylnitrosamine (500 g/mL). c Positive control was mitomycin C (0.16 g/mL). d Positive control was mitomycin C (0.05 g/mL). e Visible precipitation was observed at the end of the exposure period.
Table 8 Blood chemistry in rats treated orally with Aloe vera extract for 90 d.
AVGE (mg/kg/day) Control Male 30 150 AST (IU/L) 60.0 8.0 62.2 7.5 63.5 6.4 ALT (IU/L) 27.8 4.1 28.0 4.6 27.3 2.8 ALP (IU/L) 93.3 14.2 97.7 24.1 90.1 27.5 LDH (IU/L) 139.2 29.0 129.4 21.2 124.9 24.2 -GTP (IU/L) 2.02 0.61 1.72 0.69 1.73 0.32 Glu. (mg/dL) 170 21 164 12 162 18 T.Cho. (mg/dL) 60.7 9.9 52.4 9.2 61.9 12.8 TG (mg/dL) 61.6 24.6 55.3 15.5 49.3 12.0 PL (mg/dL) 101 14 91 9 98 15 TP (g/dL) 6.1 0.3 6.1 0.3 6.0 0.3 Alb. (g/dL) 2.2 0.1 2.1 0.1 2.1 0.1 A/G 0.54 0.04 0.53 0.03 0.55 0.05 BUN (mg/dL) 16.8 1.8 17.6 2.1 17.1 1.7 Crea. (mg/dL) 0.59 0.09 0.62 0.05 0.57 0.08 T.Bil. (mg/dL) 0.39 0.09 0.37 0.13 0.29 0.12 Na (mEq/L) 140 2 141 1 141 2 K (mEq/L) 4.8 0.5 4.5 0.2 4.5 0.3 Cl (mEq/L) 110 2 109 1 109 1 P (mg/dL) 5.8 0.9 5.3 0.5 5.8 0.5 Ca (mg/dL) 9.5 0.2 9.4 0.2 9.4 0.2 Female AST (IU/L) 68.5 24.3 72.2 16.7 62.2 4.1 ALT (IU/L) 29.6 9.5 30.8 7.5 22.4 2.4 ALP (IU/L) 46.1 17.3 46.5 11.6 43.2 12.9 LDH (IU/L) 129.0 38.7 139.1 34.2 121.1 20.9 -GTP (IU/L) 2.15 0.50 2.12 0.49 2.01 0.57 Glu. (mg/dL) 140 23 142 19 146 7 T.Cho. (mg/dL) 68.0 15.3 69.0 12.3 68.0 14.7 TG (mg/dL) 20.9 10.7 18.1 9.2 17.3 4.5 PL (mg/dL) 131 26 129 20 131 21 TP (g/dL) 6.7 0.4 6.5 0.3 6.6 0.4 Alb. (g/dL) 2.6 0.2 2.6 0.2 2.6 0.2 A/G 0.64 0.04 0.66 0.04 0.64 0.03 BUN (mg/dL) 20.6 2.8 19.8 2.5 19.1 2.1 Crea. (mg/dL) 0.64 0.07 0.68 0.06 0.68 0.10 T.Bil. (mg/dL) 0.35 0.12 0.40 0.11 0.31 0.16 Na (mEq/L) 142 1 142 2 142 1 K (mEq/L) 4.0 0.3 4.4 0.4 4.1 0.4 Cl (mEq/L) 113 2 114 2 113 2 P (mg/dL) 4.8 1.0 5.2 1.2 3.7 0.8 Ca (mg/dL) 9.7 0.2 9.6 0.3 9.6 0.3 Each group consisted of 10 animals of both sexes. Values are the mean standard deviation. P < 0.05: Signicantly different from the control by Dunnetts test.
3 males each of the control group and 150 mg/kg group. There was no signicant difference between the control and 150 mg/kg groups, and these changes were not related to the test substance.
Discussion
An earlier study by Badria (1994) conducted with S. tryphimurium strains TA98 and TA100, found that an extract of the crushed leaves of A. barbadensis did not exhibit toxic effects with or without metabolic activation. There have been genotoxicity studies conducted with anthraquinones found in the peel of Aloe vera, including aloin (barbaroin) and aloe-emodin. In in vitro studies, aloe-emodin exhibited positive effects in bacterial and mammalian cell assays at concentrations ranging from 50 to 250 g/plate (Heidemann and others 1996; M ller and u others 1996). In an Ames test conducted with aloin (barbaroin), there was no reported genotoxicity at concentrations of 50 to
Female
With bronchus. Each group consisted of 10 animals of both sexes. Values are the mean standard deviation. No signicant differences were found between the control and each treated group.
Table 10Histopathological ndings in rats treated orally with AVGE for 90 d. Grade Male Control Findings Mononuclear cell aggregation Eosinophilic body Pyelonephritis Atrophy, seminiferous tubule Hyperplasia, Leydig cell Cell debris in lumen Spermatozoa in lumen, decreased Mononuclear cell inltration 0 9 5 9 10 10 10 10 7 1 1 5 1 0 0 0 0 3 2 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 9 3 10 9 9 9 9 7 AVGE 1 1 7 0 0 0 0 0 3 2 0 0 0 1 1 1 1 0 3 0 0 0 0 0 0 0 0 0 10 10 9 Control 1 0 0 1 2 0 0 0 3 0 0 0 0 9 10 10 Female AVGE 1 1 0 0 2 0 0 0 3 0 0 0
Numerals represent the number of animals with the ndings. Each group consisted of 10 animals of both sexes. Grades were 0 = no change, 1 = slight, 2 = moderate, and 3 = marked. No signicant changes were detected in cerebrum, cerebellum, spinal cord, sciatic nerve, eyeball, optic nerve, Harders gland, pituitary gland, thyroid gland, parathyroid gland, adrenal gland, thoracic aorta, thymus, spleen, mesenteric lymph node, submaxillary lymph node, trachea, bronchus, lung, liver, pancreas, submaxillary gland, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, seminal vesicles, ovary, uterus, vagina, mammary gland, skin, femoral muscle, sternum and bone marrow, and femur and bone marrow.
Conclusion
AVGE was nonmutagenic in the Ames test and chromosomal aberration test at concentrations of up to 5000 g/plate and 1600 g/plate, respectively, and in an in vivo bone marrow micronucleus test at to 150 mg/kg/d. In the acute toxicity test and the 90-d subchronic toxicological test, there were no treatmentrelated adverse effects of AVGE at 150 mg/kg. In conclusion, the AVGE prepared by supercritical CO2 extraction, which has a very low level of anthraquinones, is nongenotoxic and does not cause any obvious side effects.
References
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Supporting Information
The following supporting information is available for this article: Table S1. Gross ndings in rats singly treated with AVGE. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.