Abstract
Phase2®, which has been reported to reduce body weight by its inhibition of α-amylase, was evaluated for toxicity in young adult male and female Wistar rats (10 animals/dose group). Evaluations included mortality, change in body weight, food consumption pattern, organ weight, and other adverse side reactions as well as hematological, biochemical, and histopathological analyses. Acute toxicity was determined after a single dose of Phase2 by oral gavage at doses of 5.0, 1.0, and 0.5 g/kg body weight. Animals were sacrificed on fourteen days after Phase2 administration. Subchronic toxicity was determined by administering Phase2 daily for 90 days to rats, at doses of 1.0, 0.5, and 0.2 g/kg body weight. These animals were sacrificed on day 90. Acute and subchronic administration of Phase2 did not produce any adverse reactions or any significant change in the loss of body weight as compared to untreated controls, organ weight, and mortality. Administration of Phase2 did not alter the hepatic and renal function, and did not produce any change in the hematological parameters and in lipid profile. Subchronic administration produced a reduction in the food consumption after 77 days (1.0 g/kg body weight). These data indicate that acute and subchronic administration of Phase2 did not produce any toxicity to rats as evident from weight change, mortality, and limited biochemical and histopathological analyses.
Obesity is a major health problem in Western developed countries (Kopelman 2000). This is a highly complex chronic condition with multifactorial etiology. The derivation and continuation of the chronic disorder prevails from a web of genetic, social, cultural, behavioral, physiologic, and metabolic factors (Linda 2002). In addition, the modern societies have been impacted by industrialized food systems, which often supply a high-calorie, high-fat diet with low fiber content. Health care professionals are highly concerned about people who are obese because of the well-established relationship between excess body weight and medical conditions such as cardiovascular diseases, hypertension, and osteoarthritis (National Task Force on the Prevention and Treatment of Obesity 2000). The increasing prevalence of obesity is also accompanied by an increasing prevalence of type 2 diabetes (Maggio and Pi-Sunyer 2003). One recently reported work concluded that increased body weight was associated with increased death rates from cancer (Calle et al. 2003). Obesity may also produce infertility (Nicholas et al. 2003).
A number of herbal and botanical products have been marketed as weight loss agents (Brudnak 2002). One of the mechanisms through which these products act is by inhibiting the enzyme α-amylase that inhibits the breakdown of carbohydrates present in food. The major dietary source of carbohydrate is starch and digestion of starch begins with the enzymatic action of α-amylase in the mouth followed by further enzymatic actions in duodenum. Specific inhibitors of animal α-amylase were discovered in plants especially from tepary bean (Yamada, Hattori, and Ishimoto 2001) and wheat (O’Donnell and McGreeney 1976). Such inhibitors may inhibit carbohydrate breakdown and thereby produce a reduction in body weight.
Phaseolamine, a proteinaceous inhibitor of α-amylase, was purified from Phaseolus vulgaris (white kidney bean). Phaseolamine is specific for animal α-amylase and has no activity towards the corresponding plant, bacterial, and fungal enzymes or any other hydrolytic enzymes tested (Marshal and Lauda 1975). Partially purified preparations inhibited starch digestion in vitro and also reduced the α-amylase activity after intraduodenal perfusion (Layer, Carlson, and DiMagno 1985). Administration of partially purified inhibitor was also found to reduce carbohydrate digestion in normal individuals and reduced the blood sugar in type 2 diabetic patients (Boivin et al. 1987; Layer, Zinsmeister, and DiMagno 1986). Amylase inhibition delayed the gastric emptying of carbohydrates in human volunteers (Jain et al. 1989). Pharmachem Laboratories Inc., USA, has recently introduced Phase2, which is the first standardized, all natural, starch neutralizer with no effect on cardiovascular and central nervous system. There are no reported studies available on the safety of Phase2 in experimental animals and the present investigation was undertaken to evaluate the biosafety of this preparation in young adult Wistar rats.
MATERIALS AND METHODS
Animals
Young adult Wistar rats (both males and females) were purchased from National Institute of Nutrition, Hyderabad, India. The age of the animal was approximately 7 to 8 weeks with weights in the range of 180 to 200 g for both males and females. They were housed at the animal house facility of Amala Cancer Research Centre in well-ventilated polypropylene cages under controlled temperature (22°C to 25°C) and humidity (60% to 80%), and a light-dark cycle of 12 h. They were provided with normal pelleted rat chow (Sai Durga Feeds and Foods, Banglore, India) and water ad libitum. The animal experiments were conducted after getting prior permission from Institutional Animal Ethics Committee (IAEC) and as per the instructions prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India.
Phase2
Phase2 was provided by Pharmachem Laboratories Inc., Kearny, USA as a fine powder. This was suspended in double distilled water at the required concentration. Fresh homogenous preparation was given to the animals. Control animals received 1.0 ml double-distilled water.
Acute Toxicity of Phase2
Wistar rats (20 males and 20 females) were randomly divided into following groups.
This dosage was calculated from ‘effective’ human dose (Udani, Hardy, and Madsen 2004), which is 25 mg/kg body weight. Conversion of human dosage to rat dosage was done by multiplying with a factor of 10. Hence the effective rat dosage was calculated as 250 mg/kg body weight. We had given Phase2 at 2 times, 4 times, and 20 times higher than the effective dose.
The drug was administered orally by gavage single dose in 1.0 ml of the vehicle on the first day of the experiment. Rats were monitored for 14 days for clinical or behavioral changes, mortality, and any adverse reactions. Food consumption and body weight were determined every third day. Food consumption was determined by placing a known quantity of the feed in the cage and after 24 h, remaining feed was collected and weighed. No effort was done to find out the spillage. On the 14th day animals were sacrificed by vertebral dislocation.
Subchronic Toxicity of Phase2
Wistar rats (20 males and 20 females) were randomly divided into the following groups.
The drug was administered daily by oral gavage in 1.0 ml of double-distilled water for 90 days. Dosage of Phase2 for rats was calculated and adjusted from the ‘effective’ human dosage (Udani, Hardy, and Madsen 2004). As given above it was administered at 2 times and 5 times higher than the effective dose. During this period rats were monitored for clinical or behavioral changes such as diarrhea, immobility, neuromuscular problems, and mortality. Food consumption and body weight were determined every third day. Animals were sacrificed on day 90 by vertebral dislocation.
In both experiments blood was collected during necropsy into heparinized and nonheparinized tubes through heart puncture for assaying hematological and serum chemistry. Necropsies were performed and the organs were examined visibly for any type of abnormalities. Selected organs (liver, spleen, and kidney) were dissected out and rinsed in ice-cold physiologic saline (0.89%) and weights were recorded.
Parameters Assayed
Total white blood cell (WBC), red blood cell (RBC), differential, hemoglobin (Hb), and platelet counts in blood were determined using a hematology analyzer (Swelab AC920 E.O Plus). Liver function markers, such as aspartate aminotransferase and alanine aminotransferase, alkaline phosphatase (ALP), albumin, globulin, and bilirubin, and kidney function markers, creatinine and blood urea nitrogen (BUN), were determined in serum with commercially available kits (Roche Diagnostics, Mannheim, Germany) using a Hitachi 704 fully automated analyzer. Cholesterol and triglycerides were estimated by calorimetric procedures using kits supplied by Roche Diagnostics. High-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol were estimated by immunoturbidimetric method (Roche Diagnostics). The levels of electrolytes such as sodium, potassium, bicarbonate, and chloride were estimated using a Biolyte 2000 (Version 3.2) counter.
Histopathological Analysis
A portion of the selected tissues (liver and kidney) was fixed in 10% neutral-buffered formalin for sectioning followed by staining with hematoxylin-eosin for histopathological evaluation.
Statistical Analysis
Data were expressed as mean ± standard deviation (SD). Significant levels for comparison of differences compared to control were determined using Student’s t test and p values ≥.05 were considered to be significant.
RESULTS
Acute Toxicity
The single dose administration of Phase2 up to 5.0 g/kg body weight did not produce any mortality or adverse reactions.
There was no statistically significant body weight change in animals (male and female) treated with Phase2. Weight change in animals treated with 5.0 g/kg body weight is given in Figure 1. Control males animals gained an average of 22.0 g during the 14-day observation period, whereas females gained a weight of 23.0 g during this period. Male animals treated with 5.0 g/kg body weight lost an average weight of 8.30 g and females gained 13.7 g. However, these changes were not significant. There was no significant difference in the food consumption of male and female rats treated with different doses of Phase2. Food consumption data of controls and animals treated with Phase2 at a dose of 5.0 g/kg body weight is given in Figure 2.
There were no significant differences in the hematology values as compared to that of controls (Table 1).
Acute administration of Phase2 at doses up to 5.0 g/kg body weight did not produce any significant change in the hepatic function as seen from the serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase as well as bilirubin, albumin, and globulin when compared to the untreated controls (Table 2).
Similarly acute administration of Phase2 (up to 5.0 g/kg body weight) did not produce any change in renal function as seen from the BUN, serum creatinine, and electrolytes (Tables 3 and 4).
A gross examination of the tissues during necropsy did not indicate any visible changes. Organ weights of the animals (liver, kidney, and spleen) did not differ significantly from that of untreated animals (data not shown).
Subchronic Toxicity
There was one mortality (male rat) after 21 days of treatment with Phase2 in group II (1.0 g/kg body weight). Phase2 administration produced no adverse side reactions or weight change in any animals, including the dead animal, indicating that the death may be due to reasons not related with its toxic effect.
There was no significant body weight change in Phase2 treated as compared to controls. Among control animals, males gained an average of 64.3 g during the 3 months of the study whereas females gained an average of 2.8 g. In animals treated with Phase2 (1.0 g/kg body weight), male rats gained a weight of 133.05 g and females gained 34.1 g (Figure 3).
Administration of Phase2 did not produce significant change in the food consumption of animals in any of the group up to 77 days. However, there was a consistent decrease in the food consumption of animals treated with 1.0 g and 0.5 g/kg body weight at 77 days and thereafter indicating Phase2 may decrease the food consumption. This needs further validation (Figure 4).
Subchronic administration of Phase2 up to 90 days did not produce any change in WBC, RBC, platelets, hemoglobin, and differential counts. Values were comparable to untreated animals. This indicates that Phase2 did not produce any hematological toxicity (Table 5).
Subchronic administration of Phase2 did not produce any significant change in the lipid profile as seen from serum levels of cholesterol, triglycerides, HDL, LDL, and very-low-density liproprotein (VLDL) (Table 6). Serum cholesterol was found to be increased in male rats treated with Phase2 but it was not statistically significant.
The effect of subchronic administration of Phase2 on different marker enzymes of hepatic function is shown in Table 7. The level of alanine aminotransferase and was slightly elevated in the group receiving 1.0 g/kg body weight of Phase2, but the elevation was not statistically significant. The levels of aspartate aminotransferase, ALP, albumin, globulin, and bilirubin did not show any change compared to controls.
Phase2 did not produce any toxicity to renal system as seen from the levels of BUN and serum creatinine, which were almost similar to controls (Table 8). The electrolyte levels were found to be unaltered in treated animals as compared to the normals (Table 9).
There was no significant differences in the organ weight of animals treated with Phase2 and nontreated animals (Table 10). The histological architecture of the livers of Phase2-treated animals (acute and subchronic) were similar to controls. The hepatocytes, structure of portal areas, central vein, sinusoidal spaces, and Kupffer cells were normal. The kidneys of Phase2-treated animals showed unaltered renal architecture. Glomerulii are normal with normal Bowman’s capsule and arterioles and sinuses. Renal tubules were also normal. Thus the histopathology of the liver as well as of the kidney after Phase2 treatment were almost similar to untreated animals.
DISCUSSION
Currently prescribed medications for obesity are divided into two broad categories; (a) appetite suppressants and (b) medications that reduced nutrient absorption (Yanovski and Yanovski 2002). Phentermine and phendimetrazine falls in the first category whereas orlistat (Xenical) is a candidate for second category. Both these medicines are approved by the Food and Drug Administration (FDA). However, long-term administration results in several side effects such as constipation, insomnia, euphoria, steatorrhea, palpitations, etc. (Heck, Yanovski, and Calis 2000).
Due to side effects of these commonly available antiobesity medications, attention was diverted to compounds of herbal origin. Phase2 works as an inhibitor of the enzyme α-amylase. Comprehensive experimental investigations revealed that many of the commercially available amylase inhibitors have failed to influence starch digestion owing to their low amylase inhibitor activity in humans.
A double-blind, placebo-controlled cross-over pilot study in healthy adult human volunteers demonstrated that Phase2 decreased the starch absorption by about 66%. In another double-blind study in 60 adult human beings, it was reported that Phase2 administration for a period of 30 days produced 11.63% reduction of adipose membrane as revealed by echography, whereas while in the placebo-treated group only 1.39% adipose membrane reduction was observed. Administration of Phase2 also produced a reduction in the waistline, hip, and thigh circumferences (Meiss and Ballerini 2003). Another recently reported double-blind, placebo-controlled study in 50 obese adults revealed that after 8 weeks of Phase2 administration the treated group demonstrated a loss of an average 3.79 pounds, whereas in the placebo-treated group weight loss was 1.65 pounds. Triglycerides levels of Phase 2-treated group were reduced by an average of 26.3 mg/dl as compared to the placebo-controlled group, which was 8.2 mg/dl (Udani, Hardy, and Madsen 2004). In the present study no change was observed in triglyceride values after Phase2 administration. No human toxicity of Phase2 has been reported so far.
The present preliminary study clearly revealed that Phase2 did not produce any toxicity to animals at doses of 5.0 g/kg body weight given acutely and 1.0 g/kg body weight given subchronically for 90 days. These data indicate that Phase2 is not toxic several times higher than its effective dose. No impairment in hepatic, renal, or hemopoetic function was observed throughout the study.
Some of the available literature (Wato et al. 2000; Nahoum et al. 2000) suggested that P. vulgaris contains two kinds of α-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). Alpha AI-s has recently been revealed to be a tetrameric complex, α 2 β 2, with two active sites. The structure of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, αβγ. The inhibition of Al-u by increasing amounts of porcine pancreatic α-amylase (PPA) indicated that an inactive 1:1 complex is formed between alpha Al-u and PPA. As evident from Lineweaver-Burk plots, the nature of inhibition was a mixed type noncompetitive mechanism (Anton et al. 1997). Purified α-amylase inhibitor had a molecular weight of 49 kDa and it demonstrated an in vitro inhibition corresponding to 3000 units/gram (Meiss and Ballerini 2003).
The present study indicates that at the limited dosage and animals studied here Phase2 did not produce any toxicity in rats as seen by clinical evaluation, biochemical, and histopathological analyses.
Footnotes
Figures and Tables
Acknowledgements
The authors are thankful to Dr. Radha. K. Maheswari, Department of Pathology, Uniformed Service University, Bethesda, MD, and Dr. R. C. Srimal, former Director, Industrial Toxicology Research Centre, Lucknow, for their keen interest in the work and for helpful suggestions and to Ms. N. V. Babitha for correcting the manuscript. The work was funded by a grant from Pharmachem Laboratories Inc., USA.