Chapter III: Safety and toxicity evaluation of...

Chapter III: Safety and toxicity evaluation of Botryococcus biomass in albino rats

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Chapter III

Safety and toxicity evaluation of Botryococcus biomass

in albino rats


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Summary

The safety of B. mahabali biomass after oral administration to short and long term was

studied in rats. In short term study (acute oral toxicity), by administration of single dose

of B. mahabali at the maximum level (5g Kg -1

BW) and toxicity symptoms, if any, were

monitored for 14 days. In long term toxicological study, the effects of 90 days oral

administration of B. mahabali (20g of algal biomass per Kg diet) was assessed and

compared with the control rats. Acute and subchronic toxicity studies in rat models

revealed that the B. mahabali biomass at the given doses did not induce any treatment

related observable toxic effects, when compared to control group of animals devoid of

biomass. Haematological and histopathological examinations did not reveal any adverse

effects of the algal biomass feeding on male and female rats and the other clinical

observations have also not shown any significant toxicological affects in both the sexes.

Rather, its feeding has resulted in serum and tissue lipid lowering and cholesterol

lowering benefits. Hence Botryococcus mahabali biomass was found to be safe at the

tested levels in animal models.


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3.1. Introduction

Algae have recently received a lot of attention as a new biomass source for various

applications form food to fuel. Some of the major characteristics which placed algae apart

from other biomass sources are that algae can have a high biomass yield per unit of light

and area and can have a high content of lipid, starch, protein and other bioactive

molecules like carotenoids etc. In contrast, the global food crisis and steady increase in

food prices is hitting with alarming speed and force and thus posing challenges on the

key international scientific organizations to respond with a strategic and long term

approach (USDA NASS news release March 30, 2007).

Many species of the green microalgae Botryococcus have been found to produce

large amount of lipids. Their lipid profile is reported to be as good as olive oil and their

amino acids composition is also found to be having substantial amounts of all the

essential amino acids as well. Apart from this, few strains of the genus Botryococcus are

also reported to produce considerable amounts of specialty lipids like, methyl branched

fatty acids, lutein, beta–carotene, PUFA etc. (Metzger and Largeau, 2005; Banerjee et al.,

2002; Dayananda et al., 2006a, 2006b). Solvent extracts of Botryococcus species are also

being reported for high degree of antioxidant activities. The new Indian isolate

Botryococcus mahabali was found to contain considerable amounts of protein,

carbohydrate, and lipids. In view of utilizing Botryococcus mahabali biomass as a source

of food or feed or nutraceutical or pharmaceutical use, the present work intended to

assess safety of biomass for consumption using acute and subchronic toxicity studies in

rat models. Similar studies have been conducted for various prospective microalgal

species like Dunaliella, Haematococcus, Chlorella and Spirulina etc. (Vanitha et al.,

2007; Kamath et al., 2008, Son et al., 2009; Hutadilok-Towatana et al., 2008).

3.2. Materials and methods

3.2.1. Source of Botryococcus mahabali

Botryococcus mahabali cultures were grown for 14 days in modified Chu 13 medium.

The culture was harvested by online centrifuge at 5000 rpm, lyophilized and analyzed for

total lipid, fatty acid profiles, and chlorophyll and carotenoid contents (detailed in chapter

III) and used for the animal feeding experiments.


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3.2.2. Study design

The subchronic toxicity study was conducted according to a well-designed protocol. The

study was conducted at Animal House facility of the Department of Biochemistry and

Nutrition at Central Food Technological Institute, Mysore according to the guidelines of

Organization for Economic Co-operation and Development (OECD) on good laboratory

practices. The subchronic study was designed to meet the known requirements of EC

Directive 94/40/EC 1994 and institutional ethical committee norms.

3.2.3. Chemicals and reagent kits

Glucose assay kit was procured from AccuDx Technologies Pvt. Ltd., New Delhi; assay

kits for triglycerides (TG), aspartate amino transferase (AST), alanine amino transferase

(ALT) and alkaline phosphatase (ALP) from Aspen laboratories Pvt. Ltd., New Delhi,

cholesterol, creatinine and urea reagent kits from Span diagnostics Ltd., Surat, India and

lactate dehydrogenase (LDH) from Teco diagnostics, U.S.A. All other chemicals and

solvents were of the highest purity obtained from E Merck India, Pvt. Ltd.

3.2.4. Test material

Stock cultures of Botryococcus mahabali (CFTRI isolate) maintained routinely on both

liquid and agar slants of modified Chu 13 medium by regular sub-culturing at 2-week

intervals. Cultures were maintained at 25 ± 1 °C temperature with 1.2 ± 0.2 klux light

intensity under 16:8 light dark cycles. The algal biomass was scaled up in prototype open

raceway ponds and thus obtained biomass was lyophilized and used for the animal

experiments.

3.2.5. Animals and treatment design

Animal care and handling conformed to the guidelines of the Committee for the purpose

of Control and Supervision of Experiments on Animals (CPCSA), Government of India

and the protocols were approved by the Institutional Animal Ethical Committee (IAEC).

The animals were housed in individual stainless steel cages and were provided food and

water ad libitum. They were maintained under specifically controlled environmental

conditions viz temp 26 ± 2ºC under light and dark cycle (12–12 h) and a relative

humidity of 60 –70 percent.


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3.2.6. Acute oral exposure in rats

In acute study a total of 10 healthy adult male albino Wistar rats (260 ± 10 g) were

randomly assigned to two groups of 5 each and were given a dose of algal biomass at 0.0

and 5.0 g per kg b.w. mixed with diet and monitored for a period of 14 days. No females

were used for acute study. Daily feed and water intake was recorded throughout the

experimental period. Body weights were obtained on the day of test administration and

on 2nd, 7th and 14th day. On completion of the experiment, the animals were weighed,

sacrificed by ether anesthesia, and blood samples were collected for haematological and

serum biochemical investigations. The vital organs were weighed and relative organ

weights (g/100 g body weight) were calculated. Complete histological analysis was done

for any treatment- related anomalies.

3.2.7. Subchronic oral exposure study

Three week old male and female (36–40 g) Wistar rats bred in Animal House Facility of

the Institute were randomly assigned to 2 groups, each comprising of six males and six

females and were fed Botryococcus mahabali biomass at, 20 g/kg of the diet for 13

weeks. The administration of test diet containing lyophilized Botryococcus mahabali

biomass was commenced at the weanling stage. The animals were observed twice in a

day for clinical signs. Weekly bodyweight was recorded throughout the treatment period.

The amount of feed intake was recorded on daily basis and the mean daily feed intake for

each week was calculated. Test substance intake was calculated from the feed intake and

concentration of the test substance in the diet admixture. Test diet was available ad

libitum for 13 weeks except for one-night fasting prior to sacrifice.

3.2.8. Diet preparation

The lyophilized Botryococcus mahabali biomass (proximate composition is given in

Table 23 of Chapter I) was added with the diet to prepare the experimental diet. Diet for

the subchronic study, was prepared once in every 10 days. The stability of the test

substance in the experimental diet was ensured by storage at 4ºC. The composition of

standard rat diet is given in Table 25.


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3.2.9. Haematology and serum biochemical indices

Haematological parameters were analyzed by using a K-4500 automated haematology

analyzer (Sysmex Corp, Japan). Aliquots of whole blood samples were mixed with a 4-

fold volume of suppliers buffer containing 0.5% ethylene diamine tetracetic acid and

applied to the analyzer for the following parameters: haemoglobin (Hb), red blood cells

(RBC), white blood cells (WBC), packed cell volume (PCV), mean corpuscular

haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin

concentration (MCHC), platelet count (PC). For measuring differential leukocyte count,

blood samples were mixed with 1= 4 volume of 5.0% EDTA.2K, analyzed with a Microx

HEG-120A (Omron Tateishi Electronics Co., Ltd, Tokyo, Japan). The serum levels of

glucose, triglycerides, cholesterol, urea, creatinine and activities of key marker enzymes

namely alkaline phosphatase (ALP),alanine amino transferase (ALT), aspartate amino

transferase (AST) and lactate dehydrogenase (LDH) were assayed by using standard kits.

3.2.10. Relative organ weights

At necropsy all the organs/tissues were carefully observed macroscopically for any

lesions. The fresh organ weights were noted for liver, lungs, kidneys, heart, spleen,

testis/ovary, brain and adrenals. The organ weights were recorded as absolute values and

their relative values were calculated on the basis of the final body weights of the rats.

Table 25. Composition of AIN-76 rat diet

Casein 20.00%

Corn Starch 50.00%

Sucrose 15.00%

Cellulose 5.00%

Vegetable Oil 5.00%

AIN mineral mix 3.50%

AIN vitamin mix 1.00%

DL-Methionine 0.30%

Choline 0.20%


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3.2.11. Histopathology

At autopsy the vital organs were surgically removed from the rats, washed with normal

saline, fixed and preserved in 10% neutral buffered formalin. All major tissues were

further processed and trimmed, embedded in paraffin, sectioned to a thickness of

approximately 4µm and stained with hematoxylin and eosin for complete

histopathological evaluations. Liver, lungs, kidneys, heart, spleen, testis/ovary, brain and

adrenals were examined microscopically for any histological anomalies.

3. 3. Results and Discussion

3.3. 1. Acute oral exposure study

The 14 days acute study of Botryococcus mahabali biomass on male Wistar rats caused

no deaths or treatment related anomalies. Occurrence of any clinical signs of toxicity

attributed to algal biomass ingestion namely, abnormal behavior, ill health, changes in

locomotor activity, ataxia and gastrointestinal intolerance were not noticed either

immediately or during the post treatment even at the high dosage of 5g /kg BW. Thus it

indicated that the Botryococcus mahabali biomass was not toxic according to the criteria

for acute toxic classifications. No abnormal clinical signs or gross pathological

abnormalities were manifested in any organ or tissue in the external cephalic, thoracic or

abdominal regions of the tested animals. On autopsy no remarkable internal gross

abnormalities were observed. Water, fecal and urine output were found to be normal. No

significant differences (p<0.05) were observed in feed intake, the body weight (gain/loss)

pattern of B. mahabali fed animals when compared to control (Table 26 & 27). No

significant (p<0.05) changes were noticed in relative organ weights and in

haematological parameters of the control and the treated animals (Table 28 & 29). This

study is an initial step in establishing a dosage regime of B. mahabali biomass for further

studies. This will provide information on the health hazards likely to arise from a short-

term exposure by the oral route.


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Table 26. Diet intake of male Wistar rats of oral acute toxicity studies

Days Control 5g biomass /kg B.W

1 15.53 ± 0.76 15.98 ± 1.49

7 15.27 ± 1.29 14.77 ± 1.00

14 14.58 ± 0.46 14.97 ± 0.51

Table 27. Body weights of male Wistar rats of oral acute toxicity studies

Days Control 5g biomass /kg B.W

1 273 ± 11.01 272 ± 8.29

7 285 ± 9.54 286 ± 9.62

14 292 ± 9.02 295 ± 10.22

Table 28. Relative organ weight of male Wistar rats of oral acute toxicity studies

Organs Control 5g biomass /kg B.W

liver 3.92 ± 0.05 3.92 ± 0.05

lungs 0.49 ± 0.06 0.49 ± 0.06

Kidney 0.58 ± 0.05 0.58 ± 0.05

Testis 0.97 ± 0.09 0.97 ± 0.09

Adrenal 0.013 ± 0.0007 0.013 ± 0.0007

Heart 0.31 ± 0.01 0.31 ± 0.01

Spleen 0.23 ± 0.03 0.23 ± 0.03

Brain 0.56 ± 0.02 0.56 ± 0.02

Group of rats (n=5) were administered with single dose of B. mahabali biomass

(5g kg-1 BW) and toxicity symptoms were monitored for 14 days. The values are

expressed as mean ± standard deviation


6

Table 29. Haematological profiles of male Wistar rats of oral acute toxicity studies

Parameter Control 5g biomass /kg B.W

Hb (g/dl) 13.92 ± 0.54 13.77 ± 0.06

RBC (106/µL) 8.61 ± 0.24 8.48 ± 0.08

WBC (103/µL) 11.7 ± 4.46 10.73 ± 1.95

PCV (%) 46.88 ± 1.94 44.7 ± 0.44

MCV (fl) 54.42 ± 0.98 52.7 ± 1.01

MCH (pg) 16.16 ± 0.40 16.23 ± 0.21

MCHC (%) 29.7 ± 0.94 30.8 ± 0.17

PLC (105/µL) 7.89± 0.78 8.37± 1.07

Differential count (%)

Neutrophils 13.8 ± 3.89 18 ± 7.93

Lymphocytes 81 ± 5 79.3 ± 7.64

Eosinophils 2 ± 0.71 1.33 ± 0.58

Monocytes 3.2 ± 1.3 1.33 ± 0.58

Group of rats (n=5) were administered with single dose of B. mahabali biomass (5g kg-1 BW) and toxicity

symptoms were monitored for 14 days. The values are expressed as mean ± standard deviation

3.3.2. Subchronic oral exposure study

3.3.2.1. Clinical signs and mortality

All the rats survived the experimental period. During the administration period, animals

in their cages appeared normal with no overt clinical signs of toxicity or allergenic

reactions. There were no incidences of diarrhea, constipation or other gastrointestinal

disorders. General condition and behavior were not adversely affected by the algal

biomass during the study period. On autopsy, macroscopic observation of the animals

revealed no alterations in the external surface and the orifices of the cranial, thoracic and

abdominal cavities were normal. Hence the study revealed that the rats fed on

Botryococcus mahabali biomass did not develop any clinical signs of toxicity either

immediately or during the post treatment at 20g /kg diet.


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3.3.2.2. Feed intake, body weight and organ weight

In general, observance with the experimental diet was excellent, and all the animals

tolerated the dietary changes well, as assessed by 24 h feed intake (Table 30 & 31). Algal

biomass fortification did not show any adverse effect on the palatability of food. The

daily intakes of B. mahabali biomass by both male and female rats are detailed in Tables

32 & 33. There was no significant change in the feed intake of animals in the

Botryococcus mahabali fed rats with their respective control animals was observed. No

treatment-related body weight loss was manifested in both male and female animals

during the 14 week experimental period (Figures 40 & 41).

Table 30. Daily feed intake of male Wistar rats

daily feed intake (g/rat/day)

Weeks Control 20g of biomass /kg of diet

1 7.09 ± 0.58 6.98 ± 0.66

2 11.82 ± 0.31 11.58 ± 0.10

3 13.34 ± 0.83 13.51± 0.08

4 15.88 ± 0.16 16.02 ± 1.32

5 17.92 ± 0.99 17.50 ± 0.41

6 18.91 ± 1.25 17.94 ±0.63

7 18.59 ± 0.33 17.92 ± 0.72

8 19.28 ± 0.14 17.46 ± 0.81

9 18.39 ± 1.28 18.18 ± 0.96

10 18.68 ± 0.27 18.22 ± 0.75

11 19.62 ± 1.34 19.26 ± 0.23

12 20.44 ± 0.30 20.61 ± 1.29

13 21.18 ± 1.03 21.48 ± 0.95


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Table 31. Daily biomass intake of male Wistar rats

daily biomass intake (mg/rat/day)

Weeks Control 20 g of biomass /kg of diet

1 _ 13.97 ± 1.07

2 _ 23.16 ± 1.63

3 _ 26.77 ± 0. 16

4 _ 32.05 ± 0.33

5 _ 35.01 ± 1.19

6 _ 35.89 ± 1.55

7 _ 35. 84 ± 1.98

8 _ 34.91 ± 0.28

9 _ 36.37 ± 0.28

10 _ 36.46 ± 0.62

11 _ 38.52 ± 2.77

12 _ 41.21 ± 0.72

13 _ 42.08 ± 2.06

Table 32. Daily feed intake of female Wistar rats

daily feed intake (g)

Weeks Control 20g/kg of diet

1 7.78 ± 1.01 6.98 ± 0.48

2 10.34 ± 1.03 9.93 ± 0.87

3 11.48 ± 0.75 11.19 ± 0.63

4 12.28 ± 0.32 12.31 ± 0.17

5 12.83 ± 0.49 13.26 ± 0.47

6 12.44 ± 0.22 12.87 ±0.28

7 12.35 ± 0.36 12.66 ± 0.30

8 12.77 ± 0.59 12.96 ± 0.68

9 13.87 ± 0.66 14.17 ± 0.31

10 14.08 ± 0.44 13.89 ± 0.26

11 14.15 ± 0.34 14.21 ± 0.59

12 14.53 ± 0.42 14.73 ± 0.9

13 14.85 ± 0.48 15.43 ± 1.09


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Table 33. Daily biomass intake of female Wistar rats

daily biomass intake (g)

Weeks Control 20g/kg of diet

1 _ 15.57 ± 2.26

2 _ 23.34 ± 2.06

3 _ 22.96 ± 1.50

4 _ 24.48 ± 0. 65

5 _ 25.65 ± 0.99

6 _ 24.89 ± 0.44

7

25.07 ±1.19

8 _ 25.53 ± 1.19

9 _ 27.73 ± 1.33

10 _ 28.16 ± 0.89

11 _ 28.29 ± 0. 68

12 _ 29.06 ± 0.85

13 _ 29.70 ± 0.96

Figure 40. Body weight curves of male Wistar rats fed with B. mahabali biomass

0

50

100

150

200

250

300

350

400

1 2 3 4 5 6 7 8 9 10 11 12 13

Control 20g biomass /kg diet

Bo

dy

we

igh

t (g

)

Week


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Figure 41. Body weight curves of female Wistar rats fed with B. mahabali biomass

3.3.2.3. Relative organ weights and histopathology

There were no statistically significant differences in the group mean relative weights of

various vital organs such as liver lungs kidney heart brain spleen adrenals, testis, and

ovary in the biomass treated rats compared to that of control group (Table 34). There

were no adverse histopathology deviations in spleen, liver or kidneys of both male and

female rats compared to their respective control sex animals. Liver of treated animals

showed no shrunken hepatocytes or congestions in portal tracts and sinusoids. Sections

revealed well formed structure of parenchyma and portal triads. Lungs showed organized

alveolar spaces and no thickening of inter alveolar septa or cellular infiltrations were

observed. Kidneys of treated animals were also normal and did not present any

glomerular or vascular congestion. Other vital organs like brain and heart also showed

normal in their structure. Histopathological examination of ovaries revealed different

stages of follicular development and no abnormalities were observed in germinal

epithelium, stages of follicular development, maturation and corpus luteum. There were

no microscopic or macroscopic lesions in any organs that could be attributed to algal

biomass ingestion. Histopathological investigations failed to reveal any incidence of

organ toxicity in the study (42 & 43).

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10 11 12 13

Control 20g biomass /kg diet

Bo

dy

we

igh

t (g

)

Week


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Figure 42. The histology of various vital organs (H & E stain, 100 X) of animals fed on

control diet


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Figure 43. The histology of various vital organs (H & E stain, 100 X) of animals fed on

B. mahabali biomass supplemented diet


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3.3.2.4. Haematological and serum biochemical indices

Haematological investigations revealed a decrease in haemoglobin concentrations in

female rats with respective to their control counterparts. Significant increase in

lymphocyte count was found in female rats whereas no such significant alteration was

found for eosinophhils, monocytes or basophils. No significant differences were found in

RBC counts, PVC, platelets, MCV, MCH and MHC counts in both the sexes (Table 35).

Serum biochemical analysis revealed that there were no statistically significant alterations

in glucose, urea, Ck-Nac, ALT, AST, LDH, creatinine, direct bilirubin and total bilirubin

levels in both the sexes were found to be in the normal range. However, cholesterol and

triglycerides levels were found to be decreased in both the male and female rats

administered with algal biomass. Phospholipids contents were found to be increased in

treated animals. Alkaline phosphatase activity showed an increment in Botryococcus

mahabali fed rats of both sexes (Table 36).

Table 34. Relative organ weights of Wistar rats fed with B. mahabali biomass for 13 weeks

Control 20g of B. mahabali /kg of diet

Organ Male Female Male Female

Liver ( g%) 3.22 ± 0.06 3.01 ± 0.02 3.02 ± 0.16 3.10 ± 0.029

Lungs (g%) 0.54 ± 0.06 0.53 ± 0.01 0.52 ± 0.08 0.58 ± 0.06

Kidney (g%) 0.79 ± 0.07 0.77 ± 0.05 0.74 ± 0.04 0.75 ± 0.03

Heart (g%) 0.33 ± 0.016 0.34 ± 0.01 0.34 ± 0.02 0.35 ± 0.01

Brain (g%) 0.89 ± 0.06 0.91 ± 0.03 0.91 ± 0.01 0.93 ± 0.03

Spleen (g%) 0.20 ± 0.008 0.24 ± 0.03 0.23 ± 0.01 0.20 ± 0.008

Adrenal (g%) 0.03 ± 0.004 0.03 ± 0.007 0.03 ± 0.004 0.03 ± 0.005

Testies (g%) 1.02 ± 0.013

1.18 ± 0.02

Ovary (g%)

0.08 ± 0.002

0.08 ± 0.005

Group of rats (n = 6) were administered with repeated doses of B. mahabali biomass for 90 days.

The Toxicity symptoms were monitored for 90 days. The values are expressed as mean ± standard

deviation


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Table. 35. Haematological profile of male and female Wistar rats fed with B. mahabali

biomass for 13 weeks

Control 20g biomass /kg of diet

Parameter Male Female Male Female

Hb (g/dl) 14.7 ± 0.26 14.5 ± 0.27 15.4 ± 0.38 14.2 ± 0.19

RBC (106/µL) 8.79 ± 0.18 8.34 ± 0.04 8.37± 0.19 8.93 ± 0.20

WBC (103/µL) 7.5 ± 0.40 6.2 ± 0.08 7.2 ± 0.18 6.75 ± 0.35

PCV (%) 46.5 ± 1.97 45.35 ± 1.20 45.9 ± 0.42 48.3 ± 2.26

MCV (fl) 52.4 ± 0.63 55.1 ± 0.98 54.3 ±1.27 56 ± 2.75

MCH (pg) 16.1 ± 0.28 16.7 ± 0.70 16.4 ±0.28 17.3 ± 1.27

MCHC (%) 29.9 ± 0.28 30.3 ± 0.77 29.5 ±0.21 30.9 ± 0.78

PLT (105/µL) 9.89 ± 1.15 10.89 ± 0.44 10.75±0.34 9.87 ± 55

Differential count (%)

Neutrophils 14.5 ± 4.5 15 ± 5 16.5 ± 4.5 14 ± 2

Lymphocytes 79 ± 7 75 ±6 78 ± 4 82 ± 5

Eosinophils 2 ± 1 1 ± 1 2±1 1 ± 0

Monocytes 2 ± 0 1 ± 1 2±2 1 ± 0

Basophiles 0 ± 0 1 ± 0 2 ± 0 3 ± 0

Group of rats (n=6) were administered with repeated doses of B. mahabali

biomass for 90 days. The Toxicity symptoms were monitored for 90 days. The

values are expressed as mean ± standard deviation


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Group of rats (n=6) were administered with repeated doses of B. mahabali biomass for 90 days. The

Toxicity symptoms were monitored for 90 days. The values are expressed as mean ± standard

deviation

Table 36. Effect of dietary B. mahabali on serum biochemistry of male and female Wistar rats

Parameter Male Female Male control Female control

LDH (IU/L) 174.6 ± 29.72 204.3 ± 27.96 259.28 ± 10.06 261.4 ± 21.19

Creatinine Kinase (IU/L) 142.3 ± 23.58 160.4 ± 31.15 169.19 ± 23.15 160.4 ± 33.17

ALT (U/L) 14.83 ± 8.36 10.90 ± 9.05 17.45 ± 7.40 27.92 ± 8.89

AST (U/L) 19.19 ± 7.40 17.45 ± 8.66 27. 04 ± 6.12 20.35 ± 6.60

ALP(U/L) 94.3 ± 8.87 75.2 ± 16.32 117.5 ± 8.71 109.3 ± 11.29

Glucose (mg/dL) 96.9 ± 9.03 99.4 ± 8.20 102.1 ± 2.84 97.9 ± 11.90

Urea (mg/dL) 23.4 ± 7.69 25.2 ± 7.53 32.61 ±4.91 30.5 ± 3.73

Creatinine (mg/dL) 1.28 ± 0.10 0.72 ± 0.17 0.64± 0.29 0.79 ± 0.16

Direct bilirubin (mg/dL) 0.29 ± 0.02 0.26 ± 0.05 0.33 ± 0.02 0.32 ± 0.02

Total bilirubin ( mg/dL) 0.87 ± 0.04 0.89 ± 0.05 0.92 ± 0.04 0.93 ± 0.03

Triglycerides (mg/dL) 91.40 ± 6.38 77. 86 ± 7.11 133.07 ± 07 117.38 ± 9.63

Cholesterol (mg/dL) 54. 65 ± 8.24 53. 39 ± 13.62 75.19 ± 3.4 77.90 ± 6.4

Phospholipids (mg/dL) 143.41 ± 12.4 134.9 ± 6.43 119.2 ± 8.2 100.9 ± 6.6


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B. mahabali fed animals were also evaluated for their possible influence on hepatic lipid

profiles. Interestingly the animals administered with algal biomass have shown lowered

levels of triglycerides and cholesterol and consequently there were marginal increases in

phospholipids were noticed with the tested animals of both the sexes (Table 37).

Table 37. Hepatic lipid profile of male and female Wistar rats fed with B. mahabali for 13 weeks

Animal group Total lipids Cholesterol Triglycerides Phospholipids

Control male rats 65.3 ± 1.81 6.93 ± 0.62 9.84 ± 0.8 22.95 ± 3.26

Control female rats 71.1 ± 1.70 7.62 ± 0.54 10.19 ± 0.75 21.52 ± 2.52

Male rats fed with B. mahabali 57.4 ± 1.08 5.45 ± 0.32 8.65 ± 1.61 31.11 ± 0.84

Female rats fed with B. mahabali 62.8 ± 2.16 5.67 ± 0.45 7.58 ± 1.39 30.95 ± 1.49

Group of rats (n=6) were administered with repeated doses of B. mahabali biomass for 90 days. The

Toxicity symptoms were monitored for 90 days. The values are expressed as mean ± standard

deviation

In order to explore the utilization of Botryococcus mahabali biomass as

nutritional and nutraceutical supplement, safety evaluations of the freeze dried biomass

was studied. There were no reports available on safety of Botryococcus sp. whole

biomass) and hence the study was conducted on safety of the green colonial microalgae

Botryococcus mahabali. The safety of blue green algae, Spirulina (Becker and

Venkataraman, 1982; Krishnakumari et al, 1981) and its coloring pigment, the

phycocyanin has been well established in rats (Naidu et al, 1999). Krishnakumari et al.

(1981) showed that Spirulina and Scenedesmus were safe up to 800mg Kg-1

b.w in albino

rats. Spirulina when supplemented in the diet as 10% protein supplement showed less

growth and body weight compared to rats treated with 10% casein. However the relative

organ weights of vital organs were higher compared to casein treated groups (Becker and

Venkataraman, 1982). Concerning the toxin of nucleic acids, the safe level of algae is

known to be 20g of algae per day or 300mg kg-1

body weight (Becker, 2007).

A considerable interest has developed in recent years for production of algal

biomass for various applications from nutraceuticals, pharmaceuticals and as animal and

aquaculture feed etc. The fortification of biomass as such could evade the solvent

extraction of oil which made the PUFAs prone to oxidative damage. Furthermore in

addition to PUFAs, B. mahabali biomass could be a source of protein and carbohydrate.


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In the present study a stimulatory effect of biomass fortification on body weight gain in

male and female rats was observed (Figures 40 & 41). It was reported earlier that organ

weight changes have long been accepted as a sensitive indicator of chemically induced

changes to organs and in toxicological experiments comparison of organ weights between

control and treated groups have conventionally been used to predict toxic effect of a test

article (Peters and Boyd, 1966; Pfeiffer, 1968). Algal biomass fortified feed associated

morphological changes and no evidence of toxicity was found. Feed supplementation of

algal biomass did not induce any statistically significant alteration in the haematological

profile of rats except for a reduced haemoglobin concentration. Other haematological

parameters had no significant differences between the control and treatment groups

which indicated that haematological values were not significantly affected by fortification

of algal biomass at the tested dose level. These findings also suggest that the biomass of

algae may not be toxic as they do not significantly affect the circulating red blood cells

nor the haematopoiesis or leucopoiesis that could otherwise have caused a megaloblastic

anemia, or significant changes in packed cell volume (PCV) and eosinophils. These also

indicate that the normal metabolism of the animals was not affected. The observed

diminution in triglycerides found in the present study can be attributed to the lowering

effects of PUFAs on blood lipids (Harris, 1989; Herzberg, 1989; Merritt et al., 2003).

Similar reduction of serum triglycerides has been reported in humans following

administration of fish oil (Saynor and Gillot, 1992). This reduction in triglycerides may

be due to increased fatty acid oxidation or reduced lipogenesis (Rodriguez-Cruz et al.,

2005). The results of the present study reveal no elevated activity of any of the marker

enzymes (LDH, ALT and AST). Elevated levels of lactate dehydrogenase are found in

pathologic situations like myocardial infraction, liver diseases, renal disease, and certain

forms of anemia, malignant diseases and progressive muscular dystrophy. Leakage of

liver enzyme lactate dehydrogenase is very commonly used for measuring cytotoxicity of

test reagents. Absence of any elevated activity of these diagnostic marker enzymes

suggest the safety of B. mahabali augmentation at levels used in the present study.

Absence of any significant histological findings namely cellular infiltrations,

inflammation and lesions in the vital organs emphasizes the safety aspect of algal

biomass at levels given in this experiment.


Page 108

The fundamental issue in this study was the safety of algal biomass as a source of

oil. In conclusion the subchronic oral administration of B. mahabali biomass at 20g/kg of

diet to albino rats, revealed no treatment related observable toxic effects, when compared

to control group of animals devoid of biomass. Rather there was a considerable decrease

in hepatic and serum cholesterol and triglycerides. However evaluation should be done

regarding its possible use as feed and for other nutraceuticals for aquaculture, animal,

poultry feed and other applications by experimentation with this algal biomass at higher

levels using long term feeding studies with rat, rodents and other model systems.

Chapter III: Safety and toxicity evaluation of...

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