EVALUATION OF SUB-CHRONIC TOXICITY AND HEAVY METAL TOXICITY OF KAPPAPHYCUS ALVAREZII IN- VIVO

EVALUATION OF SUB-CHRONIC TOXICITY AND HEAVY METAL TOXICITY OF KAPPAPHYCUS ALVAREZII IN- VIVO
Xin Qi Chuah and Swee Sen Teo*
Department of Biotechnology, Faculty of Applied Science, UCSI University Kuala Lumpur, Jalan Menara Gading, UCSI Heights, Cheras, 56000, Malaysia.

ABSTRACT: Seaweed consumption is becoming more popular in Asian population especially the red seaweed, Kappaphycus alvarezii (K. alvarezii). However, there are few reported studies on the safety of K. alvarezii for consumption in Malaysia. This study focuses on the in vivo evaluation of toxicity and heavy metals of K. alvarezii performed in albino rats. K. alvarezii were sent for heavy metal analysis while methanolic extracts of K. alvarezii were administered orally to albino rats (2000mg/kg) daily for a total of 28 days according to Organization for Economic Co-operation and Development (OECD) guidelines. The water level and feed consumption of both groups were observed daily. At day 28th the control and experimental group of rats were sacrificed, blood and organs such as kidney and liver were collected and sent for haematology and biochemical analysis. There was low to no detectable concentration of heavy metals such as arsenic, iron and zinc in K. alvarezii. In addition, the low levels of heavy metals in the K. alvarezii samples did not cause toxicity in rats due to the results obtained from haematological test and biochemical parameters showed no significant different between experimental and control groups of rats under statistical analysis. It is therefore concluded that K. alvarezii which was proven to be rich in nutrients and antioxidants is safe for human consumption

K. alvarezii, sub-chronic, heavy metal, haematological, biochemica

INTRODUCTION: Kappaphycus alvarezii (K. alvarezii) are literally multicellular organism that belongings to Rhodophyta, a red algae. It has been shown that K. alvarezii is unique with its photosynthetic pigment, phycoerythin that characterized it under group of Rhodophyta ¹. They are marine algae that were found abundantly in Southeast Asia with favorable temperature from 21oC to 24ºC ².

Furthermore, Kappaphycus are fast growing and abundantly present in the subtidal zone of the reef and sandy-coral areas. In Philipines, Kappaphycus are largely cultivated in conjunction with their commercial significance in the market.

Over the past few decades, K. alvarezii has been significantly introduced into global markets and widely consumed by people all around the world. The importance of this species is remarkable together with the increasing of health consciousness of people towards the natural therapeutic products. K. alvarezii is suggested as healthy and nutraceutical food. It is well known that K. alvarezii are antioxidant-rich in addition with high dietary fibres, minerals and vitamins. This specie is attractive for many researchers and could be exploited for their uses in medicines and also food industry ³. Since ancient time, seaweed had been introduced to people as food source. On top of that, K. alvarezii are reported as the largest group of seaweed consumed by people from all around the world ⁴. Unfortunately, systemic studies on sub-chronic toxicity of K. alvarezii are not available in Malaysia. Furthermore, rapid industrialization and economic development are reported in Malaysia leading to an increase in water pollution around coastal areas ⁵. Moreover, seaweeds have high metal accumulation capacity as reported by Bersada et al. ⁶.

Toxicity study is necessary to investigate the potential risk of K. alvarezii upon ingestion. This experiment is able to provide information about the possible hazards to human health after prolonged exposure to this species of seaweed. In the present study, we employed Sparague Dawley rats as experimental model to test for the sub chronic toxicity and heavy metal toxicity of the K. alvarezii. Haematological and biochemical parameters were investigated with respect to the significance of K. alvarezii toxicity.

MATERIAL AND METHOD:

Assessment of heavy metal in K. alvarezii:

1. alvarezii:
2. alvarezii were drained and soaked in water to remove excess salts. The clean seaweed were placed in 60^oC oven for drying. The weights of the seaweed were measure daily until consistent weight achieved. After that, dried samples of K. alvarezii were sent to DXN Holding Berhad, Laboratory Department for heavy metal screening.

Preparation of seaweed extraction:

1. alvarezii was purchased and collected from the cultivation site in Sabah, Malaysia. The seaweeds were soaked and washed thoroughly with water to remove excessive salt and contaminant before the extraction began. Initially, the cleaned seaweed was dried until a constant weight achieved. It was continued by grinding the seaweed coarsely into powder form with the aid of liquid nitrogen. 25g of seaweed powder was preweighed and 250 ml of 70% methanol by MERCK KGaA (Darmstadt, Germany) were added into the conical flask. This methanol extraction was placed on a shaker at 1200 rpm at room temperature. After 2 hours, the filtrate of the methanol extraction was concentrated by rotary evaporator at 45^oC. The crude extract was kept in falcon tube and stored in -20^oC deep freezer for further use.

Animal preparation: 

Male and female Sprague Dawley rats, purchased from University Kebangsaan- Malaysia (UKM), were chosen to become experimental model in this study. All the rats were acclimatized in animal holding area for a week under standard environmental condition according to OECD guidelines ⁷. Each group of rats consisted of 6 male and female rats respectively. They were housed in stainless steel cage and provided with standard pellet diet and water ad libitium.

Sub-chronic toxicity study:

The rats were randomly picked and grouped as testing and control groups for each male and female category. Weight of rats was taken into account during the preparation of the extract supplement for the rats. 200mg of K. alvarezii extract per kg of body weights was prepared and administered orally to the experimental groups. This single dosage administration was performed daily for 28 days. Animals models were handled with care throughout this study period. Control group was fed with standard pellet and water ad libitium meanwhile treating them with identical care as the experimental one. Appearance and daily performance of the rats were tightly monitored throughout the 28 days.

Abnormalities and any other clinical signs or findings such as mortality or distress pattern were observed and recorded daily. In response to the sub-chronic toxicity study, body weight of each rat was assessed before the commencement of the extract, daily before the administration of supplement and on the day of sacrifice with the aid of weighing scale. On day 29th, rats were sacrificed and blood samples were collected using cardiac puncture technique for both experimental and control group of rats. The blood samples mainly for haematological analysis were collected in EDTA tube whereas, the blood for biochemistry tests were contained in plain tube without any anticoagulant.

Haematological parameters:

Haemoglobin, RBC count, WBC count, Packed Cell Volume (PCV), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC) were examined by UCSI Path Lab.

Biochemical parameters:

Glucose, Cholesterol, Total protein, Alkaline phosphatase (ALP), Alanine transaminase (ALT), Aspartate transaminase (AST), Urea, Creatinine, Calcium, Phosphorus, Sodium, Potassium and Chloride were assessed by UCSI Path Lab.

Statistical analysis:

The data obtained from haematological and biochemical tests were interpreted and examined to access the sub-chronic and heavy metal toxicity studies. One-Way ANOVA statistical test was performed using SPSS Statistic Software to illuminate the significant difference of the data between groups. Data were accepted as significant different at ((P>0.05))

RESULTS:

Screening of heavy metals in dried sample of K. alvarezii: To determine the potential toxicity that caused by supplementation of K. alvarezii extract on the experimental group, heavy metal contents in the seaweed were the first attempt. There were three heavy metals which were Arsenic, Iron and Zinc detected with a level of 3.9, 14.9 and 3.0 ppm respectively. On top of that, the concentration of Cadmium, Chromium, Manganese, Lead, Mercury, Nickel and Selenium were lower than 0.1 ppm. Thus, these heavy metals were considered as non-detectable.

Effect of seaweed extract on body weight of rats:

At the beginning of this study, both male and female rats had initial weight ranging from 250-330 g and 190-230 g, respectively. It was found that the body weight of male rats was always higher than the females one among the groups. At the end of the study period, all groups of rats were found to increase in their mean weight. In conjunction with that, mean weight of supplemented groups had discovered to be 372.06 g and 241.70 g which is not significantly ((P>0.05)) similar as compared to control groups. This might be due to the presence of secondary metabolite, caretonoids in K.alvarezii which facilitate β-oxidation activity in fat tissues 8. Further investigation should be done for detail explanation. The mean weight of all groups was tabulated in Table 2.

TABLE 2: BODY WEIGHT OF RATS FOR EXPERIMENTAL AND CONTROL GROUPS ON 29^TH DAY (g)

Values are expressed as mean ± SEM (n=6) in each group, n- number of animals, SEM- Standard error of mean

Effect of seaweed extracts on haematological parameters:

Rats were sacrificed on 29^th day and haematological tests were performed by using the withdrawn blood. No significant ((P>0.05)) difference can be observed from the mean values of Haemoglobin (Hb), Red Blood Cell (RBC), Packed Cell Volume (PCV), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC) between experimental and control groups. The results for haematological parameters were presented in Table 3.

Effect of seaweed extract on Biochemical parameters:

Results for serum biochemical parameters were tabulated in Table 4. It was clearly revealed from the table that there was no significant ((P>0.05)) difference for the level of Alkaline Phosphatase (ALP), Alanine Transaminase (ALT), Aspartate Transaminase (AST), glucose, cholesterol, total protein, urea, creatinine, calcium, phosphorus, sodium, potassium and chloride in the serum between the experimental and control groups.

TABLE 3: MEAN VALUES OF HAEMATOLOGICAL PARAMETERS FOR EXPERIMENTAL AND CONTROL GROUPS

Values are expressed as mean ± SEM (n=6) in each group, n- number of animals, SEM- Standard error of mean

TABLE 4: MEAN VALUES OF BIOCHEMICAL PARAMETERS FOR EXPERIMENTAL AND CONTROL GROUPS

Values are expressed as mean ± SEM (n=6) in each group, n- number of animals, SEM- Standard error of mean

Effect of seaweed extract on organ weight of rats: Organs like kidney and liver were removed from all rats and weighed. The readings of those weights were illustrated in Table 5 below. Similarly, there was no significant (P>0.05) difference of organ weight between experimental and control groups.

TABLE 5: MEAN WEIGHT OF ORGAN FOR BOTH EXPERIMENTAL AND CONTROL GROUPS (g)

Values are expressed as mean ± SEM (n=6) in each group, n- number of animals, SEM- Standard error of mean

DISCUSSION: In this study, some heavy metals like arsenic, iron and zinc were found present in the seaweed. This result indicated that K. alvarezii which originate from marine may cause toxicity due to heavy metal accumulation. It was believed that heavy metal that entered body system tend to bond strongly with biomolecules which eventually lead to various bio-dysfunction ^{9, 10}. K. alvarezii extract was introduced to Sprague Dawley rats for sub-chronic toxicity and heavy metal toxicity. The rats were under observation along with supplementation of K. alvarezii extract for 28 days. During this study period, no mortality case was observed and manifestation signs like diarrhea and abnormal stool colour were not occured. In this respect, experimental group tolerated the dosage given in this study.

Throughout the study period, the mean weight of both experimental and control groups increased. Meanwhile, there was no significant (P>0.05) increase in weight of rats which consumed seaweed extract as compared to the control. This may be due to the presence of fucoxanthin, one of the caretonoid produced by K. alvarezii. According to Maye ⁸, fucoxanthin has down-regulating potential of lipogenic enzyme activities and facilitating β-oxidation activity in fat tissues. Due to less reference of explanation, further studies need to be explored in this matter.

In haematological perspective, no significant (P>0.05) difference was observed between experimental and control group. All values obtained from hematological analysis placed between the standard range for rats except White Blood Cell count for male rats. This might be explained since fighting scenario was observed among the male albino rats during the study period. Cell damage due to injuries might elicit immune system, hence increase the cell count of white blood cell. Thus, K. alvarezii did not disclose any toxicity and was proved to be non toxic as observation can be compared with the control group.

By referring to Table 4, some parameters such as ALT, AST, urea, calcium, phosphorus and potassium were found out of their standard range. Potassium level revealed from rats serum was explainable because all seaweeds offer high amount of potassium especially to the supplemented group in this study ¹¹. Moreover, the value obtained for urea was under wise consideration since the source of urea might originate from the diet ¹². Besides, calcium and phosphorus level might be interfered by anesthetic agent used during dissection because this agent tend to obstruct the airway and body electrolyte level ^{13, 14}.

On top of that, the level of ALT and AST exceeded the normal range that they should be present in rat body. In fact, animal models are often under stress during the experiment period. These enzymes were often induced by emotional stress as reported by Olga et al. ¹⁵. Nevertheless, it can be deduced that the consumption of seaweed extracts did not elicit any toxicity that might be due to heavy metal and repeated dosage since no significant difference was observed in biochemical analysis.

CONCLUSION: In short, no adverse effect reflected from the experimental group after comparison with the control group. Haematological and biochemical parameters showed no significant difference between treated and control group. Furthermore, organ weight of rats were found compatible between groups indicated that consumption of K. alvarezii extract did not alter normal body function. Thus, K. alvarezii can be presented as safe for consumption. Further studies on nutraceutical properties of K. alvarezii are encouraging to support more in-depth studies on its properties such as anticancer. Mechanism of K. alvarezii to defense against heavy metals should be studied to enhance the understanding of society regarding the safety of seaweed consumption.

ACKNOWLEDGEMENT: Special thanks to my supervisor, Dr. Teo Swee Sen for her excellent guidance and support throughout my final year project. Gratitude to all seniors and laboratory staffs of UCSI University Kuala Lumpur throughout my study periods, not forgetting UCSI Path Lab who help me to process my heavy metal analysis.

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    How to cite this article:

    Chuah XQ and Teo SS: Evaluation of Sub-Chronic Toxicity and Heavy Metal Toxicity of Kappaphycus Alvarezii in- Vivo . Int J Pharm Sci Res 2016; 7(2): 573-78.doi: 10.13040/IJPSR.0975-8232.7(2).573-78.

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