Abstract
It has been shown that water hyacinth leaf protein concentrate (WHLPC) is nutritionally and economically available for applications in food and feed, such as biscuits or seasonings industries, but, its dietary safety has never been studied. The dietary safety of WHLPC was therefore evaluated by analyzing the contents of total alkaloids, phenolic compounds, and heavy metals, followed by laboratory animal feeding test. The total alkaloid and phenolic contents of WHLPC were 18.7 mg/kg and 5.2 mg/kg, respectively. WHLPC contained non-detectable Cd, 0.04 mg/kg Cr, 0.001 mg/kg Pb, 0.002 mg/kg Pt, 0.001 mg/kg Pd, 0.003 mg/kg Sn, 0.002 mg/kg Hg, 0.01 mg/kg Ba, 0.001 mg/kg Ag, 0.006 mg/kg Sd, and 0.03 mg/kg Al. The LD50 of WHLPC in mice was more than 20.5 g/kg body weight (bw). After feeding mice for 7, 30, 60 or 90 days, either on diet containing WHLPC or a control diet of equivalent protein content, there were no significant differences in absolute body weight or weight gain of WHLPC-treated mice. The results of haematological analysis, histopathological evaluation, general dissection, and investigations of internal organs did not show any adverse effects from diet containing WHLPC. It was concluded that WHLPC is not acutely toxic and does not show sub-chronic in mice.
Introduction
High yield, or low cost, and high nutritional quality of a new material are not sufficient to demonstrate its suitability for use as an ingredient in various foods or feedstuffs. It must first be shown to be non-toxic and this has never been studied before in relation to water hyacinth leaf protein concentrate (WHLPC). The dietary safety evaluation of any newly developed foods or food ingredients is an important and necessary step before putting them on the market. Water hyacinth (Eichhornia crassipes Mart. Solms) is superior to soybean in terms of the production of protein per hectare. Its leaves typically contain 20%−25% protein on a dry matter basis, and its essential to non-essential amino acid ratio is very good. 1 Some attempts have been made to utilize water hyacinth leaves because of this. For example, much research has concentrated on chemical evaluation of proteins and method of isolating proteins from water hyacinth leaves and trying to use them as food ingredients. 2–5
WHLPC may be practically applied in supplements of high quality proteins and dietary fiber together with very small amount of fat present. This could be beneficial to manufacturers who intend to produce high-quality foods for people intending to lose weight. Because of the enrichment of lysine, WHLPC could also be used as a high-quality ingredient of nutritional foods suitable for people whose staple foods are cereals or grains, and as a material for making seasonings.
The objective of the present study was to evaluate the dietary safety of WHLPC for human or animal consumption by analyzing total alkaloids, phenolic compounds, and heavy metals followed by animal feeding test.
Materials and methods
Sample preparation
Fresh water hyacinth leaves were taken from a pond on the campus of Southwest University, PRC, in the spring when plants were at the flower-bud stage. The cleaned leaves were blanched in boiling 0.5% acetic acid solution for 5 minutes, and then washed with de-ionized water several times until the washings were neutral. After the surface of the acid-treated leaves was leached to dryness, they were soaked in 95% ethanol for 6 hours. Then, the leaves treated with acetic acid followed by ethanol were taken out from the container and put on a piece of clean and dry filter cloth; and the ethanol in the leaves was pressed out by twisting and squeezing the cloth. The residue was washed with 95% ethanol several times and the operating process was the same as the former step. After the residual ethanol in the residue was completely removed by air flow at about 45°C, it was ground and its nitrogen content was measured by Kjeldahl procedures. The dried sample was named water hyacinth leaf protein concentrate (WHLPC). The crude protein (N × 6.25) content of WHLPC was 40.00% on a dry-matter basis.
Chemical analysis of WHLPC
Determination of total alkaloids
WHLPC (20.00 g) was firstly treated with 10 mL concentrated aqueous formic acid, and then extracted with 350 mL 95% ethanol for 3 hours. The crude ethanol extract was concentrated in vacuo to remove all the ethanol on a rotary evaporator. Hydrochloric acid (1%, 10 mL) was added to the concentrated extract, and they were mixed thoroughly. The mixture was filtered and the filtrate was collected. After the filtrate was adjusted to pH 8−9 by adding aqueous ammonia, it was re-extracted 5 times with chloroform (10 mL, 10 mL, 10 mL, 8 mL, and 8 mL, respectively). All the chloroform extracts were mixed, and the aqueous layer was discarded. The chloroform solution was extracted with 1% hydrochloric acid, and the resulting aqueous acidic extract (alkaloid containing) was basified (pH 8-9) with concentrated aqueous ammonia. The resulting aqueous basic solution was extracted with chloroform. The chloroform solutions were collected and mixed. After the removal of all the chloroform in vacuo, 10 mL 95% ethanol was added to the residue. Standard sulphuric acid (0.01 mol/L, 10 mL) and two drops methyl red were added to the ethanol solution. The excess acid was titrated with 0.02 mol/L NaOH. The amount of acid neutralized by the alkaloid in the ethanol solution was obtained by calculation. C15H23N2O was employed as a reference alkaloid. The total alkaloid content of WHLPC was represented by equivalent to the amount of C15H23N2O.
Determination of total phenolic compounds
WHLPC (5.0000 g) was extracted with 50 mL 50% aqueous acetone solution for 30 minutes in an ultrasonic bath. The mixture was then filtered and the filtrate was collected. The residue was washed twice, respectively, using 50 mL 50% aqueous acetone solution. The filtrate and washings were mixed for each sample, and then after the removal of all the acetone in vacuo on a rotary evaporator, 10 mL 1% HCl was added. The acidified mixture was heated at 45°C for 3 hours in a flask with a reflux funnel put on its top. After cooling, the heated acidified mixture was extracted with ethyl acetate (4 × 50 mL). The ethyl acetate extracts were mixed and, after removal of the ethyl acetate in vacuo, on a rotary evaporator, an appropriate amount of methanol was added. This step was repeated several times. The methanol solutions were filtered into a 100 mL volumetric flask and an appropriate amount of methanol was added to make up the volume. The methanol extract solution (1 mL) was then pipetted into another volumetric flask (100 mL) and diluted with distilled water (20 mL). Folin-Ciocalteu reagent (5 mL) was added, and the contents of the flask were mixed thoroughly. After 5 min, 15 mL 20% Na2CO3 solution was added and finally quantified to 100 mL with distilled water; the mixture was allowed to stand for 2 hours with intermittent shaking. The absorbance of the blue colored solution was measured at 760 nm with a spectrophotometer. The concentration of total phenolic compounds in WHLPC was determined by comparison with the absorbance of standard catechin at different concentrations. The total phenolic compound content of WHLPC was represented by equivalent to the amount of catechin.
Determination of Cd, Cr, Pb, Pt, Pd, Sn, Hg, Ba, Ag, Sd, and Al
Dry mineralization 6 was employed in preparing the sample solution (HNO3 used for dissolving metals from ash) for measuring Cd, Cr, Pb, Pt, Pd, Sn, Hg, Ba, Ag, Sd, and Al by atomic absorption spectrometry. Three replicates of each sample were performed. The determination of Hg in the prepared solution was the same as that reported by Hintelmann and Wilken. 7
Animal feeding test
The experimental protocol followed the main features of OECD Guideline for Testing of Chemicals, No.420 (2001): Acute Oral Toxicity-Fixed Dose and 408 (1998): Health Effects (1995), Repeated Dose 90-day Oral Toxicity Study in Rodents, with the exceptions that total numbers of animals studied were 64 instead of 20, and that visual observation, tissue histopathology, general dissection, internal organ investigation, and hematology analysis were carried out after 7-day, 30-day, 60-day, and 90-day feeding, respectively, instead of after 90-day feeding alone.
Ingredient composition (g/100g) of the diets for7-day, 30-day, 60-day, and 90-day animal feeding tests
Two kinds of diets were prepared in accordance with the composition of source materials and the daily nutrient requirements, which were named Control and WHLPC, respectively. Details of each feedstuff formula are presented in Table 1 . The assay diets were set at a protein and oil level of 15.46% and 8%, respectively. All the components of the formulated feedstuffs of each group were well mixed together and pressed into sticks by employing a screw extrusion presser.
Ingredient composition (g/100 g) of the experimental diets
a Protein content in WHLPC group was adjusted to 15.46% (12.07% from WHLPC and 3.39% from wheat bran).
b Wheat bran (100 g) contains 14.3 g water, 15.7 g protein, 4.0 g fat, 31.2 g dietary fiber, 4.2 g ash, 400 IU retinol (VA), 6.7 IU α-tocopherol (VE), 0.3 μg thiamine (VB1), 0.3 mg riboflavin (VB2), 9.8 mg Fe, 2.1 mg Cu, 6.0 mg Zn, 10.8 mg Mn, 7.15 μg Se, 681.0 mg P, 207.1 mg Ca, 384.1 mg Mg, 12.3 mg Na, and 6.8 mg K.
c The Control (100 g) for the animal feeding tests consists of 35 g corn powder, 5 g soybean powder, 15 g de-oiled soya powder, 15 g wheat flour, 2 g yeast powder, 2.5 g bone powder, 3 g de-oiled sesame powder, 4 g fish powder, 2 g milk powder, 0.44 g salt, 0.06 g Chinese Viduo (vitamin + mineral mixture), 1 g salad oil, and 15 g wheat bran.
d WHLPC-water hyacinth leaf protein concentrate.
e 1.33g Chinese Viduo contributes 5000 IU retinol (VA), 400 IU cholecalciferol (VD), 30 IU α-tocopherol (VE), 1.5 mg thiamine (VB1), 1.7 mg riboflavin (VB2), 3.13 mg pyridoxine (VB6), 60 mg ascorbic acid (VC), 6 μg cobalamin (VB12), 25 μg phylloquinone (VK1), 30 μg biotin (VH), 400 μg folic acid (VB11), 20 mg niacin (VB5), 10 mg pantothenic Acid (VB3), 17.75 mg Fe, 2 mg Cu, 6.24 mg Zn, 2.5 mg Mn, 150 μg I, 25 μg Cr, 25 μg Mo, 25 μg Se, 5 μg Ni, 10 μg Si, 4 μg Li, and 10 μg V to the experimental diet (100 g)
Animals for 7-day, 30-day, 60-day, and 90-day feeding tests
Mice prepared for the experiments included 32 male Kunming mice weighing 17−18 g and 32 female Kunming mice weighing 17−18 g. They were randomized into 2 groups. Each group included 16 male mice and 16 female mice, respectively. Mice were housed individually in stainless steel cages in an air-conditioned room at 22°C (±3°C) and 50%−55% relative humidity. Each cage only kept ≤4 mice.
Observations on 7-day, 30-day, 60-day, and 90-day animal feeding tests
Each group (32 mice) was fed either control diet or diet containing WHLPC, from 4 weeks of age. Water was provided ad libitum. After the mice were fed for 7 days, 2 male and 2 female mice were selected randomly from each group, respectively. Body weight was measured, and blood samples were taken. The mice were then sacrificed, and their livers, kidneys, and spleens were taken out and weighed. Tissue samples were collected at necroscopy for histopathological evaluation following OECD Test Guideline 408. During the experiment, the visual investigation of the behavior and any signs of illness of the mice was also undertaken every day. The rest of the mice in two groups (Control and WHLPC testing group) were continuously fed for 30-day, 60-day, and 90-day observation. After they were fed for 30 days, another 2 male and 2 female mice were picked out from each group, respectively. The following steps were the same as those for a 7-day observation. The rest of the mice in two groups (Control and WHLPC testing group) were continuously fed for 60-day and 90-day observations. After they were fed for 60 days, another 2 male and 2 female mice were picked out from each group, respectively. The following steps were the same as those for 7-day observation. The rest of the mice in two groups (Control and WHLPC testing group) were continuously fed for a 90-day observation. After they were fed for 90 days, all mice of each group were picked out from housing cages, respectively. The following steps were the same as those for 7-day observation. In the experiment, the visual investigation of the behavior and illness of the mice was also undertaken every day.
Red blood cell count
Blood samples were taken at the retro-orbital sinus, an area near to eyes, after the body weighing prior to killing the mice and put into tubes. Blood sample (10 μL) was diluted with 1.99 mL 0.9% sodium chloride solution. After mixing well, an appropriate aliquot of the diluted blood filled up the blood cell count pool (0.1 mm, 1/400 mm2). The red blood cells in the pool were then counted through a microscope.
White blood cell count
Blood samples were the same as those prepared for Red blood cell count described above. Blood sample (20 μL) was diluted with 0.38 mL 1% hydrochloric acid solution. After mixing well, the blood cell count pool (0.1 mm, 1/400 mm2) was filled with an appropriate aliquot of the diluted blood. The white blood cells in the pool were then counted through a microscope.
Determination of LD50
The control diet was the same as for the repeated dose study (see Table 1, footnote c, for the composition). All diet components were mixed together and pressed into sticks by employing a screw extrusion presser. Mice prepared for the experiments included 20 male mice weighing 17−20 g and 20 female mice weighing 17−20 g. They were randomized into 4 groups. Each group included 5 male mice and 5 female mice. After stopping to supply the normal feedstuff for 12 hours, four groups of the mice were fed with 3.25, 8.37, 17.51, and 21.50 g/kg body weight WHLPC by using an intra-gastric syringe. For the dosage of ≥17.51 g/kg body weight, the tested material was prepared by mixing 1 part WHLPC with 2 parts salad oil and the mice were fed in 2 times in 2 hours by using an intra-gastric syringe. Observations were made to check for any deaths within 24 hours of acute dosing, with special attention given during the first 4 hours, and daily thereafter, for a total of 14 days. Then, the normal feedstuff sticks and water were fed ad libitum to mice throughout the experimentation.
Results and discussion
Chemical analysis of WHLPC
Total alkaloid content in WHLPC was 18.7 mg/kg. Alkaloids have been reported to have a range of toxic effects on animals and human. 8,9 For example, alkaloids relate to neurotic, other reproductive, and birth abnormalities. 10–13 Therefore, total alkaloid content in WHLPC was thought to be one of safety indicators in this study. Although a guideline limiting glycoalkaloid content to 200 mg/kg (equivalent to the amount of C15H23N2O) in a given potato cultivar was implemented, 14 the toxicity of alkaloids is usually dependent on their structures. Since alkaloids in WHLPC have not been studied before and their properties remain unknown, the dietary safety of a level of 18.7 mg/kg in WHLPC needs to be further evaluated by animal feeding.
For preliminary dietary safety evaluation, the total phenol contents of WHLPC were quantitatively determined by Folin-Ciocalteu method. The value was 5.2 mg/kg of dry weight. Biological effects of polyphenolic compounds in animals and humans vary considerably depending upon their chemical structures. Although some polyphenolic compounds are reported to possess antimutagenic, anticarcinogenic, antiglycemic, and antioxidative beneficial properties, 15–21 most of them also adversely affect protein nutritional quality, 22 and some of them are acutely toxic. Since phenolic compounds in WHLPC have not been studied before and their properties remain unknown, the dietary safety of a level of 5.2 mg/kg (equivalent to the amount of catechin) needs to be further evaluated by animal feeding.
It was also fund that some individual heavy metal contents in WHLPC were as the following: non-detectable Cd, 0.04 mg/kg Cr, 0.001 mg/kg Pb, 0.002 mg/kg Pt, 0.001 mg/kg Pd, 0.003 mg/kg Sn, 0.002 mg/kg Hg, 0.01 mg/kg Ba, 0.001 mg/kg Ag, 0.006 mg/kg Sd, and 0.03 mg/kg Al. The concentrations of all these metals in WHLPC are within their maximum limits in food additives reported by WHO. 23
Animal feeding tests
LD50 of WHLPC
The results of the determination of LD50 indicated that all of the mice tested were alive at the doses of 3.25, 8.37, 17.51, and 21.50 g/kg body weight of WHLPC, respectively. This means that the LD50 of WHLPC is more than 21.50 g/kg body weight. According to National Standards of PRC, 24 if the LD50 of a chemical compound is >15 g/kg body weight, it should be considered to be acutely non-toxic. Therefore, it is suggested that WHLPC be not acutely toxic.
7-Day, 30-day, 60-day, and 90-day animal feeding tests
The visual observation result of the samples picked out from two testing groups after feeding for 7, 30, 60, and 90 days indicated that all of the mice appeared clinically healthy (neither deaths nor adverse changes in general appearance were observed during the experimental period), respectively. The results of tissue histopathology, general dissection, and internal organ investigation after feeding for 7, 30, 60, or 90 days also showed that no abnormality was found.
The body weight of all mice was increased after feeding for 7, 30, 60, or 90 days. The results of the body weight gain of the mice after 7-day, 30-day, 60-day, or 90-day feeding are given in Table 2 . There were no significant effects of treatment on body weight gain or absolute body weight.
Body weight gain of Kunming mice at the end of 7, 30, 60, or 90 days feeding time
Abbreviations: AIBW: average initial body weight, AFBW: average final body weight, ABWG: average body weight gain, NM: number of mice.
The results of viscera investigation of the mice after 7, 30, 60, or 90 days feeding are shown in Table 3 . Statistical analysis (t-test) of the average liver/body weight ratio values shows no significant differences (p > 0.05) between Control and WHLPC at each feeding time except the Control of 30-day feeding time. Statistical analysis (t-test) shows significant differences (p < 0.05) of the average liver/body weight ratio values between Control and WHLPC after feeding for 30 days. The reasons for this result need to be further investigated. This difference could be due either to differences in chemical composition between the two diets or simply to individual variation, given the small numbers of animals in each group. Since the results of visual observations including behavior of the mice tested, the appearance of liver, and longer feeding time than 30 days indicated no abnormality, it is suggested that no component in WHLPC or Control would be acutely harmful to the liver of the mice. This table also shows that the kidney/body weight and spleen/body weight ratios of the mice of all diet groups fed for 7, 30, 60, and 90 days were not affected by treatment. Statistical analysis (t-test) of the average kidney/body weight and spleen/body weight ratio values indicate no significant differences (p > 0.05) between Control and WHLPC at each feeding time, respectively. Visual observations of the behavior and appearance of the mice did not indicate any evidence of toxicity.
Results of viscera investigation at the end of 7, 30, 60, or 90 days feeding time
Abbreviation: BW-body weight.
The results of hematology analysis of the mice after feeding for 7, 30, 60, and 90 days are shown in Table 4 . From the table, it is obvious that Hb, RBC, and WBC of the mice of all diet groups were normal. It is therefore suggested that there should not be any component in WHLPC acutely harmful to the blood system of mice.
Results of haematology analysis at the end of 7, 30, 60, or 90 days feeding time
Abbreviations: Hb: hemoglobin content, RBC: red blood cell content, WBC: white blood cell content, measured values of blood samples from 30 healthy Kunming mice.
It was therefore concluded that WHLPC prepared by the method described in this study would not be acutely or sub-chronically poisonous to mice.
Footnotes
This research received no specific grant from any funding agency in the public, commercial, or not for-profit sectors.