Safety Evaluation of Supercritical Carbon Dioxide Extract of Aloe Vera Gel

Miyuki Tanaka, Muneo Yamada, Tomohiro Toida, and Keiji Iwatsuki

Abstract: The gel of the Aloe vera plant has been used safely for oral and external applications. Previously, we found phytosterols derived from an extract of Aloe vera gel obtained with an organic solvent to have hypoglycemic and antiobesity effects. While developing of functional foods using Aloe vera gel, we produced an active Aloe vera gel extract (AVGE) using a supercritical carbon dioxide (CO2 ) extraction procedure. In this study, we tested the safety of AVGE in vitro and in vivo. In an acute oral toxicological test in which AVGE was administered to rats at a dose of 150 mg/kg body weight, there were no deaths or apparent abnormalities at necropsy. In a 90-d toxicity test in which rats were continuously administrered AVGE at 30 or 150 mg/kg, euthanized, and subjected to pathological examinations, no abnormalities attributable to the AVGE were found. AVGE was nonmutagenic in the Ames test and a 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 up to 150 mg/kg/d. Keywords: Aloe vera gel, acute test, mutagenicity, subchronic-toxicity test, supercritical carbon dioxide extract

Practical Application: AVGE can be safely used as a functional food material.

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
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T: Toxicology & Chemical Food Safety

2011 Institute of Food Technologists R doi: 10.1111/j.1750-3841.2011.2452.x

Further reproduction without permission is prohibited

Efcacy and safety evaluation of Aloe . . .

The anthraquinones in Aloe vera include aloe-emodin (1,8dihydroxy-3-hydroxymethyl-9,10-anthracenedione), barbaloin (10-Glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)anthracenone), and aloesin (7-hydroxy-5-methy-2-(2-oxopropyl) -8-(glucopyranosyl)chromen-4-one) (El-Shemy and others 2010). Anthraquinones are usually extracted not from the gel but from the skin of the plant (El-Shemy and others 2010). The latex of Aloe vera contains approximately 80 phenolic anthraquinones, such as aloe-emodin and aloin (Andersen 2007). The sap included in the pericyclic cell of the latex is different from the liquid in the Aloe vera gel (Williams and others 2010). AVGE has been extracted from Aloe vera gel in which anthraquinones were removed by washing. Therefore, the concentration of anthraquinones in AVGE was 5ppm or less (data not shown). The safety of the supercritical CO2 extract of Aloe vera gel (AVGE) has not been reported. Therefore, we conducted bacterial reverse mutation test, chromosomal aberration test, micronucleus test, single oral toxicity test, and 90-d repeated toxicity tests to investigate the potential toxicity of an AVGE preparation derived from Aloe vera gel that has very low levels of anthraquinones. cal Industries), sodium azide (Wako Pure Chemical Industries), 9-aminoacridine hydrochloride (Sigma-Aldrich Co., St. Louis, Mo., U.S.A), and 2-aminoanthracene (Wako Pure Chemical Industries), which were used in each assay. Two independent mutation tests were performed in the presence or absence of S9 mix. In the dose-nding study, neither mutagenicity nor a cell growth inhibitory effect was observed for any of the strains treated with AVGE. Therefore, the main study was performed using the following 7 doses (78.1, 156, 313, 625, 1250, 2500, and 5000 g/plate) as specied in the guidelines. The results were considered as positive when the revertant colony number increased 2 fold or more compared with the concurrent negative control and the increase was reproducible or dose-dependent. No statistical analysis was performed for evaluation. This study was carried out by Biosafety Research Center (Iwata, Shizuoka, Japan).

Materials and Methods

AVGE obtained by supercritical uid extraction Leaf skins of Aloe vera were peeled, and mesophyll parts were collected. The mesophyll parts were dried and an Aloe vera gel dry powder was prepared. To obtain the extract, including hydrophobic Aloe-sterols, we studied a technique of producing an AVGE without using an organic solvent. Supercritical uid is used in the extraction of natural products, and CO2 is considered clean, safe, and nontoxic. A squalene and phytosterol extract as a byproduct of rice bran oil production was obtained by supercritical CO2 extraction under optimal operative conditions (Sugihara and others 2010). The supercritical uid extraction was carried out using CO2 as a solvent, at 34 MPa and 60 C. The specications of AVGE are summarized in Table 1. Phytosterol was measured by LC-MS using brassicasterol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as an internal standard substance. Bacterial reverse mutation test (Ames test) The assays were performed using 4 histidine-dependent auxotrophic mutants of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and 1 strain of tryptophan-dependent Escherichia coli (WP2uvrA) 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). Positive control compounds were 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (Wako Pure Chemi-

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).

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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

Efcacy and safety evaluation of Aloe . . .

observations and body weight measurements were conducted, and bone marrow specimens were prepared at 24 h after the nal administration. The presence of micronuclei in 2000 polychromatic erythrocytes/animal was examined, and the incidence of micronucleated polychromatic erythrocytes and ratio of polychromatic erythrocytes to total erythrocytes (1000 erythrocytes/animal) were calculated. This study was carried out by Fuji Biomedix Co., Ltd. (Kitakoma, Yamanashi, Japan). This study was performed according to the Rules for Approval of Animal Experiments of Fuji Biomedix Co., Ltd. (Approval Nr 2007156). day. The following parameters were examined in half of the males and females in each group (animals with an even animal number) at Week 13. Fresh urine and 24-h stock urine were used to examine the parameters. The fresh urine that remained after the examination was added to the 24-h stock urine. Blood was collected from the abdominal aorta of all animals under ether anesthesia at necropsy, and the following parameters were examined. The blood anticoagulated with EDTA-2K was used for measurements, except those for prothrombin time and activated partial thromboplastin time, which were examined using the plasma obtained from the blood anticoagulated with 3.2% sodium citrate. WrightGiemsa-stained smears were prepared for the examination of differential leukocyte counts. The serum thus obtained was used for examination except for aspartate aminotransferase and lactate dehydrogenase that were examined using the plasma obtained by centrifugation of the blood anticoagulated with sodium heparin (Novo Heparin Injection 1000; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan). Protein fractions were not examined because no abnormality was found in any parameter among total protein, albumin, and the albumin/globulin ratio. The following organ weights were measured in all the sacriced animals and the weight relative to body weight at necropsy was calculated: brain , pituitary gland, thyroid gland (including parathyroid gland), submandibular gland (including sublingual gland), heart, thymus, spleen, lungs (including bronchus) , liver , kidneys , adrenals, testes , prostate, ovaries, and uterus. Two paired organs were weighed together. The pituitary gland and thyroid gland were xed in neutral buffered 10% formalin and weighed the day after necropsy. The other organs were weighed at necropsy. The weights of organs with an asterisk ( ) are shown in grams (g), the others in milligrams (mg). Organs and tissues were xed and preserved in all the sacriced animals. The eyeballs (including the optic nerve) were xed in Davidsons xative. The other organs and tissues were xed in neutral buffered 10% formalin: cerebrum, cerebellum, pituitary gland, thyroid gland, parathyroid gland, submandibular gland, sublingual gland, heart, thymus, spleen, mesenteric lymph nodes, submandibular lymph nodes, lung, trachea, bronchus, aorta (thoracic), liver, pancreas, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidney, adrenal, testes, epididymis, prostate, seminal vesicle, ovary, uterus, vagina, mammary glands, urinary bladder, spinal cord (thoracic), sciatic nerve, skin (on the back), sternum (including bone marrow), femur (including bone marrow), femoral muscle, eyeball, optic nerve, and Harders gland. Quantitative data were analyzed for homogeneity of variance by Bartletts test. The signicance of any difference between the control and treatment groups was determined by Dunnetts multiple comparison test for homogeneous data or Steels test for heterogeneous data. The level of signicance was set at 5% for Bartletts test and 5% (2-tailed) for the other tests. A signicant result was indicated at either the 5% or the 1% level. Statistical analysis was not performed on data for clinical signs, ophthalmology, urinalysis (qualitative data), necropsy, or histopathology.

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
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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.

T: Toxicology & Chemical Food Safety

Efcacy and safety evaluation of Aloe . . .

At the other concentrations (78.1, 156, 313, 625, 1250, 2500), (Table 3). AVGE did not produce chromosomal aberrations or no growth inhibition was observed. AVGE was nonmutagenic in polyploidy. S. typhimurium TA98, TA100, TA1535, and TA1537 and E. coli WP2uvrA. Mouse micronucleus test The incidence of micronuclei in the 150 mg/kg AVGE group In vitro chromosomal aberration test with CHL cells (0.13 0.03%) was not increased compared with that (0.11 The incidence of cells with numerical and structural aber0.06%) in the negative control group (Table 4). However, the rations induced by AVGE was less than 5% by all methods value (5.81 2.34%) in the positive control group (mitomycin C) was signicantly increased. The proportion of polychromatic eryTable 2 Reverse mutation assay with AVGE in the absence or throcytes in the 150 mg/kg Aloe vera extract group (47.8 6.7%) presence of S9 mix. was not decreased compared with that in the negative control
Dose (g/ plate) TA98 TA100 +S9 TA1535 TA1537 WP2uvrA + S9 19 22 22 22 17 17 20 18 Table 4The micronucleus test of AVGE in mice. Test substrate Negative controla AVGE Positive controlb Dose MN-PCE/2000PCE PCE/1000RBC (mg/kg/day) Mean SD (%) Mean SD (%) 0 150 4 0.11 0.06 0.13 0.03 5.81 2.34 43.9 3.2 47.8 6.7 37.9 4.7 S9 +S9 S9 S9 +S9 S9 +S9 S9

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

Visible precipitation was observed at the end of the exposure period.

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.

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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.

Efcacy and safety evaluation of Aloe . . .

group (43.9 3.2%). However, the value in the positive control and no intergroup difference was found. There was no signicant group (37.9 4.7%) was signicantly decreased. difference in the amount of food consumed by males or females compared to the control. Results of the urinalysis are shown in Single oral dose toxicity study of Aloe vera extract in rats Table 6. There was no signicant difference in both males and None of the animals died during the observation period. There females between the treatment groups and control group. Results were no abnormal signs for any of the animals at any time. Body of the hematological examination are shown in Table 7. In the weight data are presented in Table 5. Body weight in the AVGE- 150 mg/kg group, there was a signicant increase in RBC and a treated groups exhibited changes almost equal to those in the signicant decrease in MCH and MCV in males, and also a sig4 control group throughout the observation period. There were no nicant increase in Ret (10 /L) in females compared with the abnormalities elicited by the test substance in any of the animals. control group. However, the differences between the 150 mg/kg Pathological abnormalities observed in each organ are shown in and control groups were within 5% in red blood cell (RBC), mean Supplementary Table 1. There was no signicant difference in corpuscular hemoglobin (MCH), and mean corpuscular volume these ndings between the control and AVGE-treated groups, and (MCV). In the 30 mg/kg group, there was a signicant increase in eosinophils (%, 102 /L) for the differential leukocyte counts these changes were not related to the test substance. in males and a signicant decrease in MCH, MCV, and monocytes (102 /L) for the differential leukocyte counts in females. Ninety-day repeated dose oral toxicity study of AVGE However, there was no signicant difference between the 150 in rats In the AVGE-treated groups, animals of both sexes exhibited the mg/kg and control groups, and these changes were not dosesame degree of body-weight change as those in the control group, related. Results of the blood chemical examination are shown in Table 8. There was a signicant decrease in P in females of the 150 mg/kg group compared with the control group. In addition, Table 7Hematology in rats treated orally with Aloe vera extract a signicant increase in K was observed in females of the AVGE 30 for 90 d. mg/kg group compared with the control group. However, there AVGE (mg/kg/day) was no signicant difference between the AVGE 150 mg/kg and
Control Male 30 150 RBC (104 /L) 930 27 945 39 962 25 WBC (102 /L) 103.1 26.9 104.5 21.2 108.2 23.5 Ht (%) 44.6 1.3 44.8 0.8 44.1 0.9 Hb (g/dL) 16.5 0.5 16.6 0.3 16.4 0.4 MCH (pg) 17.7 0.7 17.6 0.6 17.1 0.2 MCV (fL) 48.0 1.9 47.5 1.8 45.9 0.9 MCHC (g/dL) 36.9 0.6 37.1 0.5 37.2 0.5 Ret (%) 3.21 0.46 3.05 0.32 2.98 0.46 Ret (104 /L) 29.80 4.02 28.80 3.55 28.67 4.52 Plt (104 /L) 110.4 9.7 110.9 13.6 117.7 14.8 PT (sec) 12.4 0.7 12.9 0.8 12.8 1.1 APTT (sec) 20.3 1.8 21.1 1.2 20.4 1.8 Differential leukocyte counts Baso (%) 0.1 0.1 0.1 0.1 0.1 0.1 Eosi (%) 1.3 0.5 1.8 0.6 1.5 0.4 Neut (%) 19.9 9.0 23.0 8.8 20.2 6.0 Lym (%) 75.7 9.2 72.2 9.2 75.6 5.9 Mono (%) 3.1 1.0 2.9 0.8 2.7 0.8 Baso (102 /L) 0.1 0.1 0.1 0.1 0.1 0.1 Eosi (102 /L) 1.2 0.3 1.8 0.5 1.6 0.6 Neut (102 /L) 20.1 10.5 23.5 8.3 21.1 4.9 Lym (102 /L) 78.6 25.4 76.2 20.7 82.3 20.9 Mono (102 /L) 3.2 1.3 2.9 0.9 3.0 1.6 4 Female RBC (10 /L) 839 27 856 38 863 24 2 WBC (10 /L) 67.5 24.5 46.7 5.3 51.8 17.9 Ht (%) 43.0 1.2 42.4 1.2 43.7 0.8 Hb (g/dL) 15.7 0.4 15.5 0.5 16.0 0.3 MCH (pg) 18.7 0.5 18.1 0.4 18.5 0.5 MCV (fL) 51.3 1.1 49.6 1.7 50.7 1.5 MCHC (g/dL) 36.5 0.5 36.6 0.8 36.6 0.4 Ret (%) 2.87 0.46 2.83 0.43 3.36 0.63 4 Ret (10 /L) 24.03 3.79 24.13 3.21 28.99 5.24 Plt (104 /L) 111.2 14.2 115.6 12.4 111.5 7.2 PT (sec) 12.3 0.3 12.1 0.4 12.2 0.3 APTT (sec) 16.5 1.0 15.6 1.5 15.9 1.6 Differential leukocyte counts (%) Baso (%) 0.0 0.0 0.0 0.0 0.0 0.0 Eosi (%) 1.4 0.5 1.6 0.4 1.3 0.4 Neut (%) 18.4 7.2 20.7 5.3 19.4 5.0 Lym (%) 78.7 7.6 76.5 5.6 78.0 4.8 Mono (%) 1.6 0.6 1.2 0.7 1.2 0.3 2 Baso (10 /L) 0.0 0.0 0.0 0.0 0.0 0.0 2 Eosi (10 /L) 0.9 0.3 0.7 0.2 0.7 0.5 Neut (102 /L) 11.1 1.9 9.7 2.8 9.6 2.7 2 Lym (10 /L) 54.5 24.3 35.7 4.4 40.8 16.2 Mono (102 /L) 1.0 0.4 0.6 0.4 0.7 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. P < 0.05: Signicantly different from the control by Steels test.

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.

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control groups, and these changes were not dose-related. Organ weights are shown in Table 9. There was no signicant difference in males or females compared with the control group. Results of the histopathological examination are shown in Table 10. In a male of the 150 mg/kg group, moderate atrophy of the seminiferous tubules and hyperplasia of the Leydig cells were observed, and also moderate cell debris and a decrease in the number of spermatozoa were observed in the lumen of the epididymis. In addition, a slight aggregation of mononuclear cells in the heart was observed in a male of the control group and in a male and a female of the 150 mg/kg group. Slight eosinophilic body in the kidney was observed in 5 males of the control group and 7 males of the 150 mg/kg group, slight pyelonephritis in the kidney was observed in a male and a female of the control group, and slight inltration of mononuclear cells in the prostate was observed in
Table 9Organ weights in rats treated orally with AVGE for 90 d. AVGE (mg/kg/day) Control Male Brain (g) Pituitary gland (mg) Thyroid glands (mg)a Submandibular glands (mg)b Thymus (mg) Heart (mg) Lung (g)c Liver (g) Spleen (mg) Kidneys (g) Adrenal glands (mg) Testes (g) Prostate (mg) Brain (g) Pituitary gland (mg) Thyroid glands (mg)a Submandibular glands (mg)b Thymus (mg) Heart (mg) Lung (g)c Liver (g) Spleen (mg) Kidneys (g) Adrenal glands (mg) Ovaries (mg) Uterus (mg) 2.13 0.07 15.5 1.7 23.3 3.6 721 57 276 64 1421 114 1.50 0.10 14.37 2.38 759 107 2.97 0.34 59 7 3.48 0.40 884 145 1.97 0.09 18.4 2.3 20.1 2.6 440 67 267 71 874 63 1.11 0.08 6.93 0.62 468 73 1.71 0.13 71 9 118 22 559 130 30 2.09 0.10 15.2 1.8 22.9 3.5 713 90 283 63 1436 118 1.52 0.09 14.27 2.10 783 153 3.07 0.25 60 7 3.44 0.36 955 177 1.96 0.07 17.8 2.9 19.8 3.4 458 43 232 52 888 77 1.12 0.13 6.75 0.62 477 70 1.68 0.21 70 12 118 18 637 138 150 2.14 0.07 14.6 1.0 24.9 3.1 676 66 330 85 1402 124 1.48 0.09 13.24 1.63 735 129 2.94 0.29 62 13 3.14 0.75 954 218 1.95 0.08 17.5 2.0 17.2 2.9 442 61 224 57 905 59 1.08 0.08 6.72 0.29 448 42 1.70 0.08 68 9 112 10 632 107

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

a With parathyroid gland. b With sublingual gland. c

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

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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.

Efcacy and safety evaluation of Aloe . . .

250 g/plate in S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, with or without metabolic activation (Paes-Leme and others 2005). In contrast to the in vitro results, aloe-emodin was shown to be nongenotoxic in assays that included the mouse micronucleus test, chromosomal aberration test in rats, unscheduled DNA synthesis test in rats, and a mouse spot test at doses up to 2000 mg/kg (Heidemann and others 1993, 1996). In this Ames test, no genotoxicity was observed at concentrations ranging from 78.1 to 5000 g/plate in S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA. We also observed nongenotoxic effects of AVGE in vitro chromosomal aberration test with CHL cells in the mouse micronucleus test. These results may contribute to the low concentration of anthraquinones in supercritical CO2 extract of Aloe vera gel (AVGE). AVGE contains phytosterols that are a part of the active ingredient in terms of the antiobesity and antihyperglycemia effects (Tanaka and others 2006). Phytosterols are natural constituents of the human diet and are commonly found in edible vegetable oils. They are structurally similar to cholesterol but reduce intestinal cholesterol absorption (Normen and others 2000) and serum LDL-cholesterol concentrations (Piironen and others 2000; Ostlund and others 2002). Both phytosterols and phytostanol esters have been assessed for their activity in bacterial mutation assay and chromosome aberration assay in human peripheral blood lymphocytes. No evidence of mutagenic activity was observed in any assays. Several studies have investigated the potential toxicity of various preparations of Aloe vera in vivo. An earlier study by Herlihy and others (1998a) examined the long-term effects (1.5 and 5.5 mo) resulting from ingestion of 2 kinds of Aloe vera powder on growth, food intake, serum chemistry, and metabolic parameters in rats. The two substances were designated Process A and Process B. Process A was a preparation of aloe lets that had been homogenized, lyophilized, and frozen. Process B had been prepared the same way as Process A, however, the homogenate was charcoal ltered prior to lyophilization (removal of barbaloin). At 1% of the diet, there was no effect of either Process A or Process B on rat growth. However, intake of a high (>1%) concentration of Process A resulted in diarrhea and restricted growth rates. The cathartic effects of Aloe leaf are known and the compound, barbaloin, is thought to be responsible for the catharsis (Ishii and others 1990). Intake of 10% Process B from which barbaloin was removed by ltration had no cathartic effects. Further, they reported that intake of either 1% process A or 10% process B for 5.5 mo reduced the plasma concentration of parathyroid hormone, calcitonin, and serum lipid peroxide below the level in control rats (Herlihy and others 1998b). Ikeno and others (2002) studied the effects of long-term ingestion of Aloe vera on age-related diseases in Fischer 334 rats. The rats fed a diet containing 1% powder of Aloe vera let had a signicantly lower incidence of multiple causes of death than control rats. The results of the study also showed that long-term intake had no apparent harmful effects, which demonstrates the safety of Aloe vera in the rat. In our recent study using a double-blind method, 80 males consumed a placebo beverage or test beverage containing a dose (0.5, 2, or 8 mg) of AVGE for 12 wk. There were no marked differences in the incidences of adverse reactions, abnormal laboratory data, or side effects between each AVGE-treated group and the control group. However, compared to Aloe vera gel, AVGE is rarely eaten, therefore, additional research on the safety of AVGE is needed.
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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
Andersen FA. 2007. Final report on the safety assessment of Aloe andongensis extract, Aloe andongensis leaf juice, Aloe arborescens leaf extract, Aloe arborescens leaf juice, Aloe arborescens leaf protoplasts, Aloe barbadensis ower extract, Aloe barbadensis leaf, Aloe barbadensis leaf extract, Aloe barbadensis leaf juice, Aloe barbadensis leaf polysaccharides, Aloe barbadensis leaf water, Aloe ferox leaf extract, Aloe ferox leaf juice, and Aloe ferox leaf juice extract. Int J Toxicol 84:150. Badria FA. 1994. Is man helpless against cancer? An environmental approach: antimutagenic agents from Egyptian food and medicinal preparations. Cancer Lett 84:15. Boudreau MD, Beland FA. 2006. An evaluation of the biological and toxicological properties of Aloe barbadensis (miller), Aloe vera. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 1:10354. Davis RH, Donato JJ, Hartman GM, Haas RC. 1994a. Anti-inammatory and wound healing activity of a growth substance in Aloe vera. J Am Podiatr Med Assoc 84:7781. Davis RH, DiDonato JJ, Johnson R Stewart CB. 1994b. Aloe vera, hydrocortisone, and W, sterol inuence on wound tensile strength and anti-inammation. J Am Podiatr Med Assoc 84:61421. El-Shemy HA, Aboul-Soud MA, Nassr-Allah AA, Aboul-Enein KM, Kabash A, Yagi A. 2010. Antitumor properties and modulation of antioxidant enzymes activity by Aloe vera leaf active principles isolated via supercritical carbon dioxide extraction. Curr Med Chem 17:12938. Heggie S, Bryant GP, Tripcony L, Keller J, Rose P, Glendenning M, Heath J. 2002. A Phase III study on the efcacy of topical aloe vera gel on irradiated breast tissue. Cancer Nurs 25:44251. Heidemann A, Miltenburger HG, Mengs U. 1993. The genotoxicity status of senna. Pharmacology 47:17886. Heidemann A, Volkner W, Mengs U. 1996. Genotoxicity of aloeemodin in vitro and in vivo. Mutat Res 367:12333. Herlihy JT, Bertrand HA, Kim JD, Ikeno Y, Yu BP. 1998a. Effects of Aloe vera ingestion in the rat. I. Growth, food and uid intake and serum chemistry. Phytother Res 12:1838. Herlihy JT, Kim JD, Kalu DN, Nelson JF, Ward WF, Ikeno Y, Yu BP. 1998b. Effects of Aloe vera ingestion in the rat. II. Hormonal and metabolic characteristics. Phytother Res 12:35560. Ikeno Y, Hubbard GB, Lee S, Yu BP, Herlihy JT. 2002. The inuence of long-term Aloe vera ingestion on age-related disease in male Fischer 344 rats. Phytother Res 16:7128. Ishii Y, Tanizawa H, Takino Y. 1990. Studies of aloe. III. Mechanism of cathartic effect. (2). Chem Pharm Bull (Tokyo) 38:197200. Im SA, Oh ST, Song S, Kim MR, Kim DS, Woo SS, Jo TH, Park YI, Lee CK. 2005. Identication of optimal molecular size of modied Aloe polysaccharides with maximum immunomodulatory activity. Int Immunopharmacol 5:2719. Logarto Parra A, Silva Yhebra R, Guerra Sardi as I, Iglesias Buela L. 2001. Comparative study n of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. Phytomedicine 8:395400. Misawa E, Tanaka M, Nomaguchi K, Yamada M, Toida T, Takase M, Iwatsuki K, Kawada T. 2008. Administration of phytosterols isolated from Aloe vera gel reduce visceral fat mass and improve hyperglycemia in Zucker diabetic fatty (ZDF) rats. Obesity Res Clin Pract 2:23945. Montaner JS, Gill J, Singer J, Raboud J, Arseneau R, McLean BD, Schechter MT, Ruedy J. 1996. Double-blind placebo-controlled pilot trial of acemannan in advanced human immunodeciency virus disease. J Acquir Immune Dec Syndr Hum Retrovirol 12:1537. M ller SO, Eckert I, Lutz WK, Stopper H. 1996. Genotoxicity of the laxative drug components u emodin, aloe-emodin and danthron in mammalian cells: topoisomerase II mediated? Mutat Res 20:16573. Normen L, Dutta P, Lia A, Andersson H. 2000. Soy sterol esters and beta-sitostanol ester as inhibitors of cholesterol absorption in human small bowel. Am J Clin Nutr 71:90813. Ostlund RE Jr. 2002. Phytosterols in human nutrition. Annu Rev Nutr 22:53349. Paes-Leme AA, Motta ES, De Mattos JC, Dantas FJ, Bezerra RJ, Caldeira-de-Araujo A. 2005. Assessment of Aloe vera (L.) genotoxic potential on Escherichia coli and plasmid DNA. J Ethnopharmacol 102:197201. Piironen V, Lindsay DG, Miettinen TA, Toivo J, Lampi A-M. 2000. Plant sterols: biosynthesis, biological function and their importance to human nutrition. J Sci Food Agric 80:93966. Pugh N, Ross SA, ElSohly MA, Pasco DS. 2001. Characterization of Aloeride, a new highmolecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity. J Agric Food Chem 49:10303. Rowe TD,Parks LM. 1941. A phytochemical study of Aloe vera leaf. J Am Pharmaceut Assoc 30:2626. Shah AH, Qureshi S, Tariq M, Ageel M. 1989. Toxicity studies on six plants used in traditional Arab system of medicine. Phytother Res 3:259. Shelton RM. 1991. Aloe vera. Its chemical and therapeutic properties. Int J Dermatol. 30: 67983. Sugihara N, Kanda A, Nakano T, Nakamura T, Igusa H, Hara S. 2010. Novel fractionation method for squalene and phytosterols contained in the deodorization distillate of rice bran oil. J Oleo Sci 59:6570. Tanaka M, Misawa E, Ito Y, Habara N, Nomaguchi K, Yamada M, Toida T, Hayasawa H, Takase M, Inagaki M, Higuchi R. 2006. Identication of ve phytosterols from Aloe vera gel as anti-diabetic compounds. Biol Pharm Bull 29:141822.

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Efcacy and safety evaluation of Aloe . . .

Vogler BK, Ernst E. 1999. Aloe vera: a systematic review of its clinical effectiveness. Br J Gen Pract 49:8238. Williams LD, Burdock GA, Shin E, Kim S, Jo TH, Jones KN, Matulka RA. 2010. Safety studies conducted on a proprietary high-purity aloe vera inner leaf llet preparation, Qmatrix. Regul Toxicol Pharmacol 57:908. Yagi A, Kabash A, Mizuno K, Moustafa SM, Khalifa TI, Tsuji H. 2003. Radical scavenging glycoprotein inhibiting cyclooxygenase-2 and thromboxane A2 synthase from aloe vera gel. Planta Med 69:26971. Yongchaiyudha S, Rungpitarangsi V, Bunyapraphatsara N, Chokechaijaroenporn O. 1996. Antidiabetic activity of Aloe vera L. juice. I. Clinical trial in new cases of diabetes mellitus. Phytomedicine 3:2413.

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.

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