Toxicity Profile of Lutein and Lutein Ester Isolated From Marigold Flowers ( Tagetes erecta )

Abstract

Lutein is a carotenoid with antioxidant properties and is commonly present in many fruits, vegetables, and egg yolk. Lutein affords protection against the development of the two common eye diseases of aging: cataract and macular degeneration. As the dietary lutein concentration is much lower compared to the actual requirement to reduce macular degeneration, supplementation of lutein is under consideration. There are very few data on the toxicity of lutein. In the present study, the authors have evaluated the short-term and long-term toxicity profile of lutein and its esterified form isolated from marigold flowers (Tagetes erecta) in young adult male and female Wistar rats. Lutein and its ester form administered orally at doses of 4, 40, and 400 mg/kg body weight for 4 weeks for short-term toxicity study and 13 weeks for a subchronic toxicity study did not produced any mortality, change in body weight, food consumption pattern, organ weight, and other adverse side reactions. Administration of lutein and ester form did not alter the hepatic and renal function, and did not produce any change in the hematological parameters and in lipid profile. Histopathological analysis of the organs supported the nontoxicity of lutein and its ester form.

Lutein belongs to the class of xanthophylls (carotenoids that contain one or more polar functional groups) and is the second most prevalent carotenoid present in the serum (Mares-Perlman et al. 2002; Alves-Rodrigues and Shao 2004). Out of the several carotenoids present in the serum, lutein and zeaxanthin (a type of xanthophylls) are localized in the retina, particularly dense in the foveal region or macula, where they are the main components of the macular pigment (Ribaya-Mercado and Blumberg 2004; Whitehead, Mares, and Danis 2006). Both lutein and zeaxanthin are isomers of one another and differ only by the presence of a single allylic hydroxyl group (Figure 1). They are more polar than many other carotenoids, mainly due to the presence of hydroxyl groups in the cyclic ring structure (Mares-Perlman et al. 2002). Lutein is not synthesized in the human and ingestion of plant products is the source of lutein. Lutein is a common carotenoid found in most fruits and vegetables as well as in egg yolk, whereas zeaxanthin is present only in minute quantities (Calvo 2005). There is a positive correlation between dietary intake of lutein and macular pigment density (Beatty et al. 2004; Stringham and Hammond 2005).

Like other carotenoids, lutein also possess several biological functions. Lutein act as antioxidants and thereby protect the macular retina and retinal pigment epithelium from light-initiated oxidative damage (Krinsky, Landrum, and Bone 2003). Lutein affords protection against chromatic aberrations, cataract, and age-related macular degeneration (AMD). AMD is the leading cause of irreversible vision loss in the elderly population. It is estimated that 1.6% of the population in the 50- to 65-year-old age group is affected with AMD, rising to 30% in the >75-year-old age group. Several studies showed that intake of lutein is associated with decrease in AMD (Goldberg et al. 1998). Lutein is also reported to possess anticarcinogenic activity. Lutein inhibited the N-nitrosourea–mediated aberrant crypt foci, an intermediate biomarker of colon cancer, in the colonic epithelium of rats (Narisawa et al. 1996). Very low amount of dietary lutein (0.002%) was found to decrease the incidence and growth of mammary tumor in BALB/c mice (Park et al. 1998). Lutein also inhibited the growth of rat ascites hepatoma cells in culture (Kozuki, Miura, and Yagasaki 2000). Epidemiological studies have shown inconsistent associations between high intake or serum levels of lutein and lower risk for developing cardiovascular disease (Granado, Olmedilla, and Blanco 2003). The possible role of lutein as a dietary supplement is gaining interest now mainly because of its protective role in human health (Hadden et al. 2002). It has been reported that the free form of lutein is more bioavailable than the ester form. There is only one study on the short-term and long-term toxicities of lutein supplementation in rats (Kruger et al. 2002). However, these authors did not undertake any study using esterified form of lutein.

In the present study, we have evaluated the toxicity profile of lutein isolated from marigold flowers (Tagetes erecta) in Wistar rats using concentrations nearly 2000 times higher than the suggestion daily intake of lutein. Moreover, we have compared the results with that of esterified form of lutein which is commonly present in nature.

MATERIALS AND METHODS

Animals

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.

Young adult male and female Wistar rats in the weight range of 180 to 200 g and age 7 to 8 weeks were purchased from Small Animal Breeding Station, Kerala Agricultural University, Thrissur, India. 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, Bangalore, India) and water ad libitum. The diet was rich in protein and broken sugars. The fat content was 4% to 5%. The composition of the rat chow given was as follows: Crude protein: 20% to 21%; ether extract: 4% to 5%; crude fiber: 4%; ash: 8%; calcium: 1% to 2%; phosphorus: 0.6%; non-fatty extracts (NFE): 54%; mineral extracts (ME) (kcal/g): 3600; pellet size: 12.

Lutein and Lutein Ester

Lutein and lutein ester were prepared from marigold flowers in which it is present at a concentration of 0.12% to 0.2%. Lutein and ester were extracted by hexane and lutein was prepared after saponification and crystallization, purity 85%. Lutein (5%, 0.5%, and 0.05% prepared in sunflower oil) and lutein ester (5%, 0.5%, and 0.05% of molar equivalent of lutein prepared in sunflower oil) were obtained from Omni Active Health Technologies, Mumbai. These products have been prepared from marigold flowers by solvent extraction (US Patent Nos. 6,743,953 (2004) and 6,737,535 (2004)). Sunflower oil does not have any detectable lutein.

Lethal Dose 50 (LD50) Studies of Lutein and Lutein Ester

Seventy female Wistar rats were divided into following groups.

Lutein and lutein ester were dissolved in sunflower oil. The lutein was given as four equal doses (2 ml) at intervals of 2 h. Lower doses of lutein ester were given as four equal doses (2 ml) at intervals of 2 h. Higher concentrations of lutein ester (4 g/kg) were given as six equal doses (2 ml) at intervals of 2 h. The controls animals received six equal doses of 2 ml sunflower oil at every 2 h.

The lutein and its ester were administered through oral gavage and rats were monitored for 12 days for mortality, clinical, and behavioral symptoms and any adverse reaction. The food consumption and body weight were recorded on 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 calculate the spillage.

Short-Term Toxicity Studies of Lutein and Lutein Ester

Seventy young adult Wistar rats of both sex (35 males and 35 females) were divided into seven groups; each group consists of 5 male and 5 female rats. Lutein and lutein ester were dissolved in the vehicle, sunflower oil. Controls were given 2 ml of vehicle/day. The animals were divided as follows:

The lutein and its ester were administered once daily by oral gavage and continued for 4 weeks. The animals were monitored for mortality, clinical, and behavioral symptoms and any adverse reaction of the lutein and its ester. The food consumption and body weight were recorded on every fifth day. After 4 weeks, the animals were sacrificed under light ether anesthesia, and a necropsy was conducted.

Subchronic Toxicity Study of Lutein and Lutein Ester

Seventy young adult Wistar rats of both sex (35 males and 35 females) were divided into seven groups; each group consisted of 5 male and 5 female rats. The animals were divided as follows:

The lutein and its ester were administered once daily by oral gavage and continued for 13 weeks. The animals were monitored for clinical and behavioral symptoms such as diarrhea, immobility, neuromuscular problems, and mortality and any adverse reaction of the lutein and its ester was recorded. The food consumption (spillage was not accounted) and body weight were recorded on every fifth day. After 13 weeks, the animals were sacrificed under light ether anesthesia.

Blood was collected by direct heart puncture method. A part of the blood was used for the estimation of hematological parameters and other part was used for serum chemistry. Necropsy of the animal was performed and observations were recorded. Selected organs such as the liver, lungs, thymus, spleen, kidney, brain, and eyes were dissected out and weights were recorded.

Parameters Assessed

Blood was analyzed for hematological parameters such as red blood cell (RBC), hemoglobin, and platelet using a Hematology Analyzer (Swelab AC 920; E. O. Plus). Hemoglobin was measured by Drabkin’s method using kit from Agappe Diagnostics, Thane, India. Total white blood cells were measured after diluting the blood in Turk’s fluid and counting them using a hemocytometer. For lymphocyte count, blood was spread on a clean slide and treated with Leishman’s stain and various types of cells were counted manually using a microscope.

Liver function markers, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), alkaline phosphatase, albumin, globulin, and bilirubin, and kidney function markers such as 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, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and very low-density lipoprotein (VLDL) cholesterol were estimated by cholesterol oxidase–para-aminophenazone method using kits supplied by Roche Diagnostics. The serum sodium and potassium were estimated using Biolyte 2000 (Version 3:2) ion selective electrolyte analyzer. Bicarbonate was determined by total CO₂ detection by Randox phosphoenol pyruvate carboxylase–malate dehydrogenase method. Chloride was estimated colorimetrically using a kit of Raichem.

Histopathological Analysis

A portion of the selected tissues (liver, kidney, spleen, and brain) was fixed in 10% neutral-buffered formalin and ocular tissues were fixed in Bouin solution. Sections (4 μm) were taken and stained with hematoxylineosin and observe under oil immersion microscope (100×). Photographs were taken.

Statistical Analysis

The values were expressed as mean ± SD. The significant levels for comparison of differences compared to that of the control was determined by one-way analysis of variance (ANOVA) followed by appropriate post hoc test (Dunnet multiple comparison test) using Graph Pad in Stat 3 software. p < .05 was considered as significant.

RESULTS

LD₅₀ Study

The single-dose administration of lutein and lutein ester up to a concentration of 4 g/kg did not produce any mortality. The body weight of the animals did not differ much during the period of study. The food consumption was found to be low initially, probably due to the high quantity of sunflower oil administered. On the third day onwards, the food consumption was found to be similar to that of the controls. Diarrhea was observed in all the animals for the first 2 days, and the reason can be attributed to the administration of sunflower oil and from third day onwards diarrhea was decreased. The results indicated that lutein and lutein ester did not produce any mortality even up to a concentration of 4 g/kg.

Short-Term Toxicity Study of Lutein and Lutein Ester

Short-term administration of lutein and lutein ester at doses 4, 40, and 400 mg/kg for 4 weeks did not produce any change in the body weight of the animals and any differences in the food consumption of male and female rats when compared to controls. No significant change was noticed during necropsy and there was no change in organ weight.

Short-term administration of lutein and its ester up to a concentration of 400 mg/kg did not produce any change in the hepatic function parameters in serum such as ALT, AST, alkaline phosphatase as well as in albumin/globulin ratio. A slight increase was observed in the case of total bilirubin in animals treated with 400 mg/kg body weight (bw) of lutein and its ester, whereas it was absent at lower concentrations. However, bilirubin content fell within the limits of normal range.

The renal function test such as blood urea and serum creatinine did not show any variation when compared to controls. There was also no significant change in serum electrolytes sodium, potassium, chloride, and bicarbonate, indicating that lutein and its ester did not produce any change in renal function.

Short-term administration of lutein and its ester did not produce any significant change in the levels of cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, and VLDL cholesterol. A small but significant increase in HDL cholesterol was seen in animals treated with lutein in some groups of animals.

Administration of lutein and its ester did not produce any change in RBC count, platelet count, and hemoglobin levels. There was no change in total WBC and lymphocyte count, indicating that lutein and lutein ester did not produce any change in hematological parameters in these animals.

Histopathological analysis of the brain, spleen, kidney, liver, and eyes did not show any pathological lesions in the organs of animals treated with lutein and lutein ester.

The above observations concluded that lutein and lutein ester did not produce any toxicity to Wistar rats when given for 4 weeks.

Subchronic Toxicity Study of Lutein and Lutein Ester

No clinical signs of any adverse or toxic symptoms were noticed throughout the period of study. Subchronic administration of lutein and its ester for 13 week at doses 4, 40, and 400 mg/kg for 13 weeks did not produce any change in body weight of the animals (Figure 2). The gains in body weight were comparable across the groups. There was no mortality reported in any of the groups.

Administration of these compounds did not produce any difference in the food consumption of male and female rats when compared to controls (Figure 3). The average food intake was nearly 10 g/day/ animal.

Administration of lutein at 4 to 400 mg/kg did not show any change during necropsy and in the weight of the liver, lungs, thymus, spleen, kidney, and eyes. A reduction was seen in the weight of the liver of animals treated with 4 mg lutein ester, but not at other higher concentrations (Table 1).

When the serum biochemistry profiles were evaluated, the subchronic administration of lutein and its ester to a concentration of 400 mg/kg did not produce any change in hepatic function parameters in serum such as ALT, alkaline phosphatase, total bilirubin, and albumin/globulin ratio. A slight decrease in AST was observed in case of 400 mg lutein ester male group and 40 mg lutein and 400 mg lutein female groups (Table 2).

No significant change was noticed in any of the renal function markers (Table 3) as well as in the levels of electrolytes such as sodium, potassium, and bicarbonate. A small increase in chloride was seen in animals treated with 400 mg/kg of lutein ester (Table 4), indicating that lutein and its ester did not produce any changes in renal function which has any biological or toxicological relevance.

There was no change in cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and VLDL cholesterol, indicating that administration of lutein and lutein ester for 13 weeks did not produce any change in lipid profile (Table 5). These results indicated that lutein did not impair the functions of hepatobiliary system, which is mainly involved in the fat metabolism.

Administration of lutein and its ester did not produce any change in RBC count, platelet count, and hemoglobin levels. No change has been observed in WBC and lymphocyte count, indicating that administration of these compounds did not produce any change in hematological parameters in these animals (Tables 6 and 7).

Histopathological analysis of the brain, spleen, kidney, liver, and eyes did not show any pathological lesions in the organs of animals treated with lutein and lutein ester. In case of eyes, the histopathological evaluation showed normal cornea and retina. The retinal epithelium retained its normal structure. The foveal and macular region also appeared normal. So it can be concluded that the lutein and its ester form did not produce any kind of pathological alterations to eye. The findings were generally consistent with the expected pattern for Wistar rats of this particular age. Histopathological observations further support the safety of the lutein and its esterified form (Figure 4).

DISCUSSION

Lutein is known as “the eye-protective nutrient.” This reputation came from the observational studies showing an inverse relationship between intakes of lutein with macular degeneration. According to U.S. Department of Agriculture, the average daily intake of lutein by Americans is about 1.7 mg per day, whereas in Europe, 2.3 mg per day. These values are below the levels of 6 to 14 mg per day recommended to reduce the risk of macular degeneration and cataract. Autopsy of patients with macular degeneration have found that retinas contain lutein and zeaxanthin, which is nearly 30% lower than that of normal. Monkeys raised on a xanthophil-free diet were characterized by absence of yellow pigment in the macula (Malinow et al. 1980). These animals had more frequent occurrence of drusen like bodies, which possibly convert to AMD. Lutein supplementation produced significant enhancement of macular pigment 70 to 80 days after supplementation (Landrum et al. 1997). Other than their role in AMD, experimental and epidemiological evidences have also found that lutein and other hydroxy carotenoids have a protective role in many other diseases such as heart diseases, stroke, and cancer. Hydroxy carotenoids such as lutein have also been found to improve the immune system (Kim et al. 2000).

There are very few reports on the toxicity studies of lutein. Kruger et al. (2002) has determined the nontoxicity of unesterified lutein (FloraGLO) isolated from marigold. Safety evaluation of the product for a 4-week pilot study and a 13-week study indicated that no clinical signs or adverse effect was noted due to the consumption of lutein throughout the course of the study. Lutein was also found to be nonmutagenic to Salmonella typhimurium (Ames test) (Wang et al. 2006). Similarly, human studies indicated that dietary supplementation of lutein, 30 mg/day for 1 year, did not produce any adverse side effects in human subjects while the concentration of lutein in serum as well as macular pigmentation increased. (Landrum et al. 1997).

In the present study, we have checked the toxicity of isolated lutein and its ester from marigold separately for the toxicity using short-term (4 weeks) and long-term (13 weeks) supplementation. Both lutein and its ester are present in the natural sources. Moreover, interconversion of lutein and its ester may happen during the absorption. Compared to lutein, ester-free form is more absorbed within the intestinal villi and may be more bioavailable. A comparison of toxicity of lutein and the ester has not been undertaken before and the current work describes a detailed investigation on the toxicity of the two forms using biochemical and histopathological analysis. Our studies using different concentrations (4 to 400 mg/kg) of these two forms indicated that both lutein and its ester do not produce any toxicity to rats. These concentrations we have used are much higher (20 to 2000 times) than the suggested daily dose, thus supporting of their supplementation in the diet may not produce any possible toxicity to organs in the body.