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ISOLATION, PURIFICATION AND IDENTIFICATION OF β-CAROTENE FROM CARROT AND ITS

ANTIOXIDANT PROPERTY

DISSERTATION REPORT

From January 4, 2003 to March 31, 2003

Subject

MASTER OF SCIENCE IN

Biochemistry

SUBMITTED BY

HARIPRASAD PEDDl

Department of Biochemistry

J.J. College of Arts and Science

Bharathidasan University



Tiruchirappalli

WORK DONE AT

Department of Biochemistry and Nutrition

Central Food Technological Research Institute

Mysore -570 013

March 2003

DEPARTMENT Of BIOCHEMISTRY AND NUTRITION

31st March 2003

Dr. V. BASKARAN, M.Sc., M.Phil., M.Ed., Ph.D.

Scientist

CERTIFICATE

This is to certify that Mr. Hari Prasad Peddi, student of the final year Master degree in

Biochemistry, J.J. College of Arts and Science, Pudukkottai, Tamil Nadu, has completed his

dissertation successfully in the department of Biochemistry and Nutrition, Central Food

Technological Research Institute, Mysore for the partial fulfillment of the award of the degree of

M.Sc., in Biochemistry.

He worked under my guidance on the research topic "Isolation, purification and identification of

ß -carotene from carrot and its antioxidant property" during the period 4th January 2003 to 31st

March 2003.



(DR. .B KARAN)

ACKNOWLEDGEMENT It gives me an immense pleasure to express my gratitude to my guide dr. V. Baskaran, Scientist,

Dept. of Biochemistry and Nutrition, CFTRI, Mysore for his valuable guidance, suggestion,

support and encouragement throughout the investigation work.

I am grateful to C. Parasuraman, Principal, J. J. College, Pudukkottai and T. Malarvilli,

Head Dept. of Biochemistry, J. J. College, Pudukkottai for providing me this opportunity of having

educative and pleasant project work at CFTRI.

I wish to express my profound gratitude to Dr. V. Prakash, Director, CFTRI, Mysore, Dr. S. G.

Bhat, Head, Dept. of Biochemistry and Nutrition and Dr. M. C. Varadaraj, Head, HRD Dept,

CFTRI, for providing an opportunity to under take this work.

My sincere thanks to Dr. R. P. Singh, Scientist, HRD Dept, CFTRI and Mr. K. Rathina Raj,

Mr. S. Vishwanth staffs of animal house facility, CFTRI, for their co-operation and help during my

work. I am very much thankful to Mr. K. N. Chadrashekar, Mr. T. R. Ramaprasad, Mr. M. Raju,

Mr. R. Lakshmi Narayana and Ms. S. Anitha, Research scholars for their constant help.

I am indebted to my parents for their constant encouragement, love and moral support

in all my endeavours. Last but not least, I would like to thank all my classmates who were

associated with me for successful completion of this work.

Above, all I express my gratitude to the great almighty for showing me the right path.

Hari Prasad, Peddi

CONTENTS



SI. No. Chapter Page no.

1. Introduction 1

2. Materials and methods 14

3. Results and discussion 31

4. Summary 43

5. Conclusion 44

6. References 45

INTRODUCTION In the Indian subcontinent developed and developing countries in the world vitamin-A

deficiency and the accompanying secondary complications pose a major health problems. Fruitsand vegetables have been identified as rich in pro-vitamins and used in the control of vitamin-A

deficiency. Since, dietary factors (lipids, protein, dietary fiber etc) have been implicated as

primary and secondary contributors to the poor or enhances bio-availability of pro-vitamin-A

carotenoids, namely l3-carotene, a-carotene etc (Figure 1). Among dietary factors mentioned

above, lipids, in specific, polyunsaturated fatty acids may influence the intestinal absorption of ß -

carotene, though PUFA are highly prove to oxidation. Further, the intestinally absorbed ß -

carotene may be a good source of antioxidant in vivo. ß -carotene is used primarily by food

industry as colorants and antioxidants. Hence, antioxidant effect of ß -carotene in vitro and in

vivo should be studied in detail. Studies have shown that under specific conditions, ß-carotene

can act as antioxidant or pro-oxidant by scavenging or activating free radical both in vitro and in

vivo.

Pro-vitamin A carotenoids, such as ß -carotene, are values in the diet of many mammals

for their contribution as precursors of vitamin A (Table 1). Sources, recommended daily

allowance and benefits of carotenoids are given in Table 2 and 3 and Figure 2. Carotenoids also

function in the prevention of some chronic disease by improving cellular communication,

enhancing cell-mediated immune responses. β-carotene increases the number of T –helper cellsand expression of interleukin-2 receptors on natural killer cells in humans.



Figure 1. Carotenoids Structure

Table 1. Pro-vitamin A carotenoids: Prepotencies (Vitamin -A Activity)

Carotenoid Relative triopotency (%)

β-carotene 100

α-carotene 50 –54

γ-carotene 42 –50

β-zeacarotene 20 –40

β-apo-8'-carotenal 57

Lycopene Inactive

Lutein Inactive

Canxanthin Inactive

Source: Bauernfeird (1972) J. Agri. Fd. Chern. 20: 456

Table 2. β-carotene concentration in natural sources

Source β-carotene µg/100g fresh weight

Green leafy (4 types) 330 -5,030

Green, non-leafy (6 types) 217 –763

Leafy vegetable (32 types) 1,000 -44,400

Tuberous vegetables and beans (16

types)

40 -1,700



Carrot (Raw) 4,600 -12,500

Banana 40 –100

Mango 63 –615

Orange, mandarin 25 –80

Papaya, watermelon 228 –324

Berries, grapes, black currant 6 –150

Table 3 .Recommended daily allowance of β-carotene

# Has not been established

# Five portions of fruits and vegetables a day

# provide approx 6 to 12 mg/day

# upper safe level = 20 mg I day

WHO / FAO recommendation

# Males (11+) = 1000 RE = 1000 µg retinal = 6 mg β-carotene

# Female (11 +1) 800 RE= 800 µg retinal = 4.8 mg β-carotene

# additional 200 RE or 400 RE during Pregnancy or lactation

# Ratio β-carotene I vitamin A in diet = 9: 1 (USDA)

Figure 2. Carotenoid and their benefits

However, the potential use of carotenoids as modulators of disease and the prevention of

vitamin-A deficiency has been hindered by the limited work in the understanding carotene

absorption and its antioxidant property. Hence, currently carotenoids are under intense scruting



regarding their potential to modulate chronic disease and emphasis has been placed on achieving

better understanding of the metabolic fate of these compounds in humans. The development of

in vitro models has greatly improved the understanding of r3-carotene absorption (Figure 3).

However, understanding the metabolic fate of carotenoids under in vivo remains unresolved.

Carotenoids absorption in vivo involves several steps: breakdown of the food matrix to

release the carotenoids, dispersion in lipid emulsion particles, solubilization into mixed bile salt

micelles, movement across the unstirred water layer adjacent to the microvilli, uptake by the cells

of intestinal mucosa and incorporation into the lymphatic lipoproteins (Olson, 1994; Van het Hof

et al. 1999; Furr and Clark, 1997).

Figure 3. Carotenoids absorption

The processes up to solubilization in mixed micelles are dependent mostly on the

physicochemical properties of food and carotenoids and on the micelles formation from bile and

lipid hydrolysates (Garret et al. 1999). Cellular uptake of carotenoids is mediated by a simple

diffusion mechanism, as previously shown in per fused rat intestinal cells (Hollander and Ruble,

1978). Several studies in vitro have evaluated the cellular absorption of carotenoids solubilized in

micelles under conditions simulating those in the intestinal lumen (Garret et al. 1999). Mixed

micelles formed in intestinal lumen play an essential role not only in the digestion and absorption

of triacylglycerols but also in the uptake of other lipophilic compounds. Recent studies have

indicated that digestion, absorption and conversion of carotenoids into vitamin -A (Flow chart 1)

may be influenced by various dietary factors (Van Vliet et al. 1996; Tyssandier et al. 2001;

Sugawara et al. 2001). One such factor concerns that may modulate the intestinal absorption of

carotenoids are dietary lipids, in specific, polyunsaturated fatty acids and phospholipids.



Flow chart 1: Path of β-carotene to retinal (in brief)

Source: Int. J. Vitam. Nuts. Res. 72 : 2002

Fats are important sources of energy in the diet (Burr and Burr, 1929) and in India

contributed a mean of 14.7% energy (Achaya, 1987). Common edible oils and fats consumed in

their percentage content of linoleic (18:2), linolenic (18:3), eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) are not synthesized in the body and hence are essential nutrients,

which must be supplied in the diet. The percentage of these acids present in the various oils andfats are given in the Table-4.

Table 4. Sources of essential fatty acids

Fatty Acids Source

18:2 linoleic

(%)

18:3 Linolenic (%) EPA 25:5 (%) DHA 22:6 (%)

Groundnut oil 27 - - -

Coconut oil 2 - - -

Safflower oil 78 Trace - -

Corn oil 61 1 - -

Canola oil 26 10 - -

Soybean oil 54 7 - -

Ghee / Butter 2 1.2 - -



Fish (salmon) - 0.1 0.6 1.2

Source: Achaya (1987).

The balance between n-6 and n-3 in the diet is important (Table 2 & 3). FAO / WHO Committee

has recommended n-6 and n-3 ratio between 5:1 and 10: 1 and advised for consuming diets with

a higher ratio and to take more foods containing n-3 fatty acid such as.

Table 5. Recommended dietary intake for n-6 and n-3 fatty acids

(Simmopoulos et al. 1999)

Fatty acids Grams/day (2000 Kcal ) Energy (%)

Linoleic acid 4.44 2.2

Linolenic acid 2.22 1.0

DHA +EPA 0.65 0.3

EPA 0.22 0.1

Table 6. Recommendation for fat intake (% of total energy)

Fat (%) Saturated fat (%) PUFA

Canadaa

Recommended

Actual

<30

<35

<10

12.6

3% n-6, 0.5%

n-3

United Statesb

Recommended

Actual

<30

38

<10

16

<10 %

7%

United Kingdomc

Recommended

Actual

WHO/FAO

<30

39

15-30

<10

17.2

-

7 %

6.4 %

3.7 %



a b

Health and Welfare, Canada, Recommended Daily Allowance, USA

British Nutrition Foundation Task Force (1992)

The beneficial effects of polyunsaturated fatty acids (PUFA) seem to be confined to EFA.

The term essential fatty acids was introduced by Burr and Burr in 1930 for linoleic acid. They

found it to be essential for the growth and health of young rats. Later studies by other workers

showed that linolenic acid and arachidonic acid, were also effective in promoting growth of rats

and curing fat deficiency syndrome. Biological activities of EFAs are listed in Table 7. The two

main functions of EFAs relate to their roles in the structure of membranes and their metabolic

transformation in the animal body by chain elongation and desaturation is by two series of fatty

acids, the n-6 series derived from linoleic acid and n-3 series derived from linolenic acid (Flowchart 2).

Table 7. Biological activities of essential fatty acids

Sl.

No.

Fatty acids Growth effect Cure of dermal

symptoms

1 Linoleic family

(a) Linoleic acid (C 18:2)

(b) Gamma linoleic acid (C 20:4)

+

+

+

+

2 Linolenic family

(a) Linolenic acid (C 18:3 )

(b) Eicosapentanoic acid (C 20:5)

Docosahexaenoic acid (C 22:6)

+

+

+

-

-

-

Flow Chart 2 : Metabolism of Essential Fatty Acids (n-3 and n-6 series).



Studies indicated that rats fed fat free diets fail to grow and reproduce and also developed renal

and vascular disease. Later studies identified the deficient component of the diet as

polyunsaturated fatty acids (Holman, 1968). The two fatty acids proved as essential dietary

nutrients for humans are linoleic acid (18:2n-6) and linolenic acid (18:3 n-3). Once obtained from

the diet, these fatty acids undergo sequential desaturation and elongation to a series of longer

chain polyunsaturated fatty acids with 3 to 6 double bonds (Innis, 1992) (Flow Chart 2). Of

these, arachidonic acid (22:6n-3) and docosahexaenoic acid (22:6n-3) are of particular interest

because they are found in very high concentrations in membrane structural lipids of central

neNous system and visual elements of the retina (Fliesler and Anderson, 1983; Sastry, 1985) and

spermatozoa (Connor et al 1992). It has been known for many years that deficiency of dietary n-

3 fatty acids during development may have long term consequences on the functions of central

nervous system (Bourre et al 1989; Galli et.al 1975) and cardiovascular system (Innis, 1991).

Long chain fatty acids of n-6 series are essential for synthesis of complex lipids, leukotriens,



thromboxanes and prostoglandins. These are required for normal functioning of developing

tissues and for maintenance of a variety of physiological functions ( Chapman et al 1986). Fish

and marine mammals can provide the direct source of n-3 fatty acids in human diets.

Beneficial effects of n-3 fatty acids

In brief, beneficial effects of n-3 fatty acids are listed below.

1. n-3 fatty acids increases bleeding time, decreases platelet aggregation, blood viscosity

and increase erythrocyte deformability, thus decreasing the tendency to thrombus

formation (Rodger and Levin, 1990).

2. n-3 fatty acids decreases low density lipoprotein-cholesterol in the case of patients with

hyperlipidemia.

3. It reduces serum triglycerides in the normal subjects and with hypertriglyceridemia

condition (Harris, 1989).

4. EPA and DHA in fish oil improve joint pain and in patients with rheumatoid arthritis

(Krener et aI1989).

5. EPA and DHA are essential for normal growth and development.

Use of n-fatty acids against common heart diseases Arteriosclerosis: Arteries in which fatty material is exposed on the vessel wall, resulting in

narrowing and eventual impairment of blood flow.

Arrhythmogenic right ventricular dysplasia: Muscle of right ventricle is replaced by fat and

fibrosis, which causes abnormal heart rhythms.

Congenital heart disease: Abnormality of cardiac structure and function that develops during

gestation.

Cardiomyopathies: Caused by viral infections, heart attacks, alcoholism, long term severe

hyper tension and usually result in inadequate heart pumping.

Heart attack: Occurs when an area of heart muscle dies or damaged because of inadequate

supply of oxygen to that area.

Congestive heart failure: Inability to pump blood efficiently, there by failing to meet the

demands of the body.



PUFA in the prevention of heart disease:

Atherosclerosis is a major cause for heart disease, is a complex disease of arterial wall. Many

cellular, biochemical and physical components interact within the arterial wall. The first step in

the formation of atherosclerosis is a non specific injury to endothelium followed by an

accumulation of monocytes and macrophages, foam cell formation and platelet aggregation. The

platelets release growth factor that leads to smooth muscle migration and proliferation. At this

point cholesterol is deposited in the smooth muscle cells and monocytes, macrophages in the

vessel wall. These events further lead to plaque formation. Many nutritional studies have shown

that the dietary fat is an important factor for the modulation of heart disease. Evidences suggest

that PUFA help to reduce plasma concentration of LDL - cholesterol. However, n-3 fatty acids

alone will not lead to the complete prevention of atherosclerosis. There are increasing evidence

that dietary fish oil supplementation may help in the prevention of atherosclerosis. Fatty acids

predominantly found in fish (EPA and DHA) have demonstrated significant inverse association

with cardiovascular disease. Small doses of n-3 fatty acids over long periods of time may have

beneficial effects by reducing blood pressure and other risk factors. Research workers have under

taken many clinical and epidemiological studies regarding n-3 fatty acids and health benefits.

In the recent past, biological importance of marine oils rich in n-3 PUFA have been

studied extensively, as they playa protective role against cardiovascular disease. On the other

hand, high or long -term consumption of PUFA may likely alter membrane lipid composition

leading to lipid peroxidation (Lpx). Although, the potential benefits from fish oil intake remains

high, the possibility of its effects on the induction of Lpx in non-target tissue organs like blood

and testis needs to be studied. There are increasing evidence that carotenoids may help in

reduction prevention of Lpx in vivo. Hence, it is reasonable to consider that elevated level of lipid

peroxidation may contribute to the biochemical alterations in these tissue. Thus, the usage of

dietary n-3 PUFA as a therapeutic agent, their long term efficacy and safety are to be studied.

Our approach and rational of using (3-carotene as antioxidant in the present study is shown in

Figure 4.



Rational of using carotenoids Natural no side effect

Figure 4. Our approach and rational of using (3-carotene from carrot

Objectives: The present work is aimed to find out

1. Influence of fish oil rich in PUFA on intestinal absorption of (3-carotene in rats

2. Influence of (3-carotene on the lipid peroxidation in vitro and in vivo

3. Influence of fish oil on the lipid peroxidation and other lipid profile in serum and testis of rats.

M

A

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M

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T

H

O

D

S

Materials

Materials used in this study are given below

Fresh carrot was purchased from local market. Acetone, methanol hexane, THF, of HPLC

grade were obtained from Merck, Mumbai. Tocopherol, NaCI, Na2S04, silica gel, MgCO3, KOH,

purchased from SRL, Mumbai, p-carotene, fish oil, Thiobarbutaric acid, sodium louryl sulphate,

borontrifloride were purchased from Ranbaxy, Mumbai. BaCI3, copper sulphate, ammoniumthiocyanate, FeCI3, isopropanol, chloroform were obtained from Qualigens, Mumbai. Acetic acid,

HCI, H2SO4, butanol were purchased from Merck, Acetylacetone was obtained from

Vebloborchemic,

Apolda, USA.

Methods

Solubility test for p-carotene

Solubility of ß-carotene (dietary grade) was tasted using different solvent systems, viz.,

THF, Methanol, acetone, dichloromethane and hexane. In brief, varying concentration of ß -

carotene (0.1 to 1 mg 1m L) was dissolved separately in known volume of solvents mentioned

above, to find out solubility of ß -carotene in specific solvent system.

Isolation of ß-carotene from carrot

Fresh carrot was purchased from the local market. They were chopped into small pieces,

ground and homogenized (potter Elvehjem) using methanol: THE (1:1 v/v) containing 0.1 mL of

a-tocopherol in methanol (2 mM). The mixture was centrifuged at 3000 rpm for 10 minutes and

the upper organic phase was transferred into a clean stoppered conical flask. To this, 50 mL of

10% NaCI solution was added and allowed to stand for few minutes for phase separation.



The organic layer was collected and this step was repeated three times. Three extractions were

pooled and evaporated by rotatory evaporator. The concentrated extract was re-dissolved in

known volume of hexane (HPLC grade) and stored at 4°C until analyzed.

The stepwise procedure adopted for extraction and purification of ß-carotene from carrot was

given in Flow chart 3 and Figure 5.

Flow chart 3. Isolation of β-carotene from carrot

Purification of β-carotene from carrot

The crude solvent extract from carrot may contain other classes of carotenoids other

than ß-carotene. Hence, ß -carotene was purified by employing column chromatography



technique (Figure 5). In brief, a clean column (size 7-10 cm) was packed with activated (300°C I

2 Hrs) silica gel. To remove moisture from the extract, a layer of Na2S04 (0.5 cm) was loaded

above the silica gel bed, followed by ß-carotene was eluted (0.5 mL/minute) using acetone:

hexane (4:96 v/v) and concentrated using flash evaporator (Buchi Rotavaport). Then the

concentrate was reloaded to the column and eluted with methanol (to remove carotenoids otherthan ß-carotene). Methonal and hexane fraction were checked for their spectrum by UV-VS

spectrophotometer (Shimadzu).

Figure 5. Purification of f3-carotene through column chromatography

Handling samples, homogenization and extraction was carried out on ice under dim lightin order to minimize isomerization and oxidation of β-carotene by light irradiation.

Identification of β-carotene

β-carotene spectrum: The spectra of purified ß-carotene was checked by UV-VIS

spectrophotometer at 300 to 700AM.

Quantification of β-carotene by HPLC

J3-carotene extracted from carrot was separated and quantified by HPLC (Shimadzu)

equipped with SPD 10A V UV-VIS detector operating at 450 nm. TSK gel ODS-80 TS (Tosoh)

column (4.6x155 CM) attached to a pre-column (2x20 mm) of pelliguard LC-18 (Supelco Ine,

Bellefonte, PA) was used. The column was kept in an oven (Shimadzu, Japan) at 20°c. The

column eluant was a mixture of ethyl acetate: methanol (30:70v/v) containing 0.1 % ammonium

acetate. Isocratic analysis was performed at 1 mL / minute. The samples were evaporated and



dissolved in Dichloromethane: methanol (2: 1 v/v) and the volume of sample injected was 10µL.

The concentration of J3-carotene was quantified from the peck area by using its respective

standard.

Selection of β-carotene concentration

Based on the recommended level of ß -carotene in the diet, concentration at a level of 20

µ mol was selected to study its antioxidant proper1y in vitro. Later the concentration was

increased to 4 folds (80 µ mol) since no antioxidant proper1y was notices at 20 µ mole level

using n-3 PUFA (fish oil) as a model system at 40°c for varying time intervals. In case of in vivo

studies; higher concentration 200 11 mol of ß -carotene was selected due to its bio-availability.

Antioxidant activity of β-carotene in vitro

The purified β-carotene was checked for its antioxidant property using fish oil rich in EPA and

OHA as a model systems, by measuring generation of oxidation products (PV) after heating fish

oil at 400 C with or without added ß-carotene. PV in heated oil was estimated as per the method

of peroxide

value (Stine et al, 1954) 4.8 ml of chloroform: methanol (3:5v/v) and 25ml ammonium

thiocyanate was added to standard ferric ion solution of different concentration (20, 40, 60, 80,

100 µL)-, and mixed well. The optical density of colour developed was measured at 505nm.

Standard curve was drawn plotting fe+3 concentration versus O.D. values obtained.

To find out the antioxidant property of ß -carotene in vitro, ß -carotene was added to fish

oil rich in n-3 PUFA and incubated at 400 C. The concentration of ß-carotene used was 20 I1mole

and 80 I1mol as mentioned above. Samples were drawn at different time intervals, (for 20 µmol

0.5 hr to 6 hr) and measured for PV as per the procedure mentioned elsewhere. PV was

calculated using the formula mentioned below and expressed as meq O2 / kg fat.

Net conc. of fe+3(µg)

PV = = meq O2 / kg fat

gm of fat used x 55.84

Table 8. 5tep wise procedure for estimation of peroxide value using standard ferric chloride

Sl. Vol of Fe+3 Concentration Vol. of solvent Vol. of Na4SCN OD at 505 nm



No. (µl) (mg) (mL) (µL)

1 00 00 4.8 25 00

2 20 2 4.8 25 0.0847

3 40 4 4.8 25 0.1456

4 60 6 4.8 25 0.2173

5 80 8 4.8 25 0.2954

6 100 10 4.8 25 0.3630

Figure 6. Standard curve of peroxide value

Absorption and Antioxidant activity of ß- carotene in vivo

Aim of the experiment was to study the influence of PUFA on the intestinal absorption of

β-carotene and antioxidant activity of β-carotene extracted from carrot in v/\..o. Growing rats

were randomly divided into four groups (n =4/group). Animals were housed individually in cages.

Group 2, 3 and 4 were fed either fish oil (0.5 mL) or fish oil (0.5 mL) containing ß -carotene (200

~mol) or J3-carotene alone by gavage and considered as experimental groups. The group 1 was

considered as control. The experiment was run for 10 days. Food and water was made available

to animals ad. libitum. Initial and final body weight were recorded to find out, food consumption

and growth rate. At the termination of the experiment, animals were sacrificed, blood was drawn

and processed for plasma separation, Liver was removed, washed with ice cold saline (0.9%),



weighed. Plasma and liver homogenate was used for analysis of lipid peroxidation and β-

carotene.

Extraction of β-carotene from plasma and liver

β-carotene (BC) was extracted according to the method of Baura et al., (1998) with slight

modification. Briefly, plasma was removed from 4°C, thawed at 4°C and 0.4 mL from each rat

was placed into a glass test tubes and made up to 0.8 mL with ice -cold de-ionized water

followed by the addition of 0.1 mL of a-tocopherol in methanol (2 mM) and 3 mL of

dichloromethane: methanol (1:2 v/v). After vortex for a minute, 1 mL of hexane was added and

the content was mixed vigorously on a vortex mixer for 10 minutes. Then the mixture was

centrifuged at 3000 rpm for 5 min. and the upper phase was collected into a clean glass test

tubes. The extraction procedure was repeated three times using dichloromethane: hexane (1.5: 1

v/v) and collected the upper phase. The three extractions were pooled and evaporated to

dryness under nitrogen. The residue was re-dissolved (0.1 mL) in dichloromethane: methanol

(2:1 v/v) subjected to HPLC analysis.

Liver samples were removed from -4°C thawed at 4°C, homogenized (10%) with ice-cold

isotonic saline with a potter Elvehjem homogenizer and 0.8 mL was used for extraction of BC.

Otherwise, the extraction procedure was same as described for plasma.

Handling samples, homogenization and extraction was carried out on ice under dim

yellow light in order to minimize isomerization and oxidation by light irradiation.

Analysis of β-carotene by HPLC

β-carotene from the extracts of plasma and liver were separated with HPLC system consisted of

an LC -10 AMP pump (Shimadzu, Kyoto, Japan), an SPD -16 A UV-VS absorbance detector

(Shimadzu, Japan) an C-R3A integrator (Shimadzu, Japan) and a personal computer with EZ

chrome chromatography data system software (Scientific software Inc. Pleasantion, CA). All the

components were separated on an TSK gel ODS -80TS (Tosoh), 4.6 x 150 nm, attached to a pre

column (2x20 mm) of pelliguard LC- 18 (Supelco, Bellefonte, PA). The column was kept in an

oven (Shimadzu, Japan) at 20°C. The column eluant was a mixture of ethylacetate : methanol

(30 : 70 v/v) containing 0 1 % ammonium acetate. Isocratic analysis was preformed at 1 mL /

minute. ß -carotene was analyzed by the same HPLC system. The concentration of these

components was quantified from their peak area by use of respective standard. The peak identity

of these components was further confirmed from their characteristic UV.



Estimation of lipid peroxidation

Lipid peroxidation in liver and testis was measured by the method of Yagi (1987). In

brief, 1 gm of tissue was homogenized in 1.15% kCI (10% homogenate). To the 200 µL

homogenate, 200 ~L of sodium louryl sulphate, 1.5 mL of acetic acid (20%, pH 3.5) and 1.5 mL

of TBA reagent (0.8%) was added and mixed well. Then the mixture was heated at 90°C for 1

hr. Cooled and the colour developed was extracted using 5 mL of n-butanol. The colour intensity

was read at 532 nm using spectrophotometer.

Lipid peroxidation products were expressed as MDA equivalents. Freshly diluted 1, 1, 3,

3, tetra methoxypropane was used for preparing standard curve for lipid peroxidation. Step wise

procedure and standard curve for Lpx is given in Table 9 and Figure 7.

Table 9. Step wise procedure for estimation of lipid peroxidation

Sl.

No.

Vol

TMP

(mL)

Vol H2O

(ML)

Con.

(mole)

Vol 20%

acetic

acid

(mL)

Vol SDS

(µL)

Vol. TBA

(mL)

O.D. at 532

nm

1 00 1.0 00 0.000

2 0.2 0.8 20 0.120

3 0.4 0.6 40 0.250

4 0.6 0.4 60 0.310

5 0.8 0.2 80 0.416

6 1.0 0.0 100 0 ←

1

. 5

→

←

2 0 0

→

←

1 . 5

→ Boil for

60 min

0.532



Figure 7. Standard curve of Lipid peroxidation

Influence of fish oil on serum and testicular lipid profile

Aim of this experiment was to study the effect of fish oil on testicular and serum lipid profile of in

rats. Growing male Wistar rats (40:t2g) were randomly grouped into four of 5 animals. Rats were

housed individually in steel cages and maintained at room temperature. After 7 days of

acclimatization, rats were deprived of the regular diet for 12 hours before fish oil administration.

Rats in group 1, 2 and 3 were fed menhaden fish oil (Sigma) by gavage at a dose of 0.5, 1.0 and

2.0 mU100 g bwt/day for 20 days and considered as experimental groups. The group that did not

receive fish oil served as control. Food and water was made available to animals ad libitum. The

animals were thoroughly examined for any signs of symptoms. During the feeding trial feed

efficiency and gain in body weight was measured. At 20 days, rats were sacrificed, blood and test

were sampled for biochemical analysis. Fatty acid composition of fish oil was analyzed by gas

chromatography to ascertain the percentage composition of individual fatty acids (Figure 8).

Table 10. Fatty acid composition (%) of fish oil used for gavage studies

FA 14:0 16:0 16:1 18:0 18:1' 18:2 18:3 20:0 20:4 20:5 22:5 22:6

% 10.9 30.5 15.1 4.65 17.0 1.7 0.7 1.65 0.45 5.38 13.2 13

14:0 -Myristic, 16:0 -palmitic, 16:1 -palmitoleic, 18:0 -stealic, 18:1 -



oleic, 18:2 -linoleic, 18:3 -linolenic, 20:0 -arachidic, 20:4 -arachidonic,

20:5 -eicosapentanoic, 22:5 -docosapentaenoic, 22:6 -docosahexaenoic.

Figure 8. Chromatogram showing fatty acid profile in fish oil Biochemical analysis

Before being estimating lipid profile (phospolipid, triglyceride and cholesterol) in serum and

testis of fish oil fed rats samples, respective reference standards were run and standard, curves

were made plotted. Stepwise procedure for estimating of standard cholesterol, phospholipid and

triglycerides and their respective standard curves are given in Table 11-13 and Figure 9-11.

Total lipid extraction from serum and testis of fish oil fed rats (20 days)

Total lipid was extracted from serum and testis by the method of Folch et al (1957). In brief, 1

gm of tissue was homogenized with 1.0 mL of 0.74% potassium chloride in a potter Elehjem

homogenizer. To the extract, 20 mL of chloroform; methanol (2:1 v/v) was added and left over

night. To the filterate, three mL of 0.74 % potassium chloride was added and mixed well. The

solution was allowed to stand, till the chloroform and aqueous layers were separated. Upper layer

was removed carefully, washed with 3 mL of 0.74% potassium chloride and then twice, with 3

mL of chloroform; methanol water (3: 48 : 47 v/v). The chloroform layer was pooled and used

for lipid analysis. In case of serum, total lipid was extracted using ethanol; acetone (1: 1 v/v).

After addition of this mixture the tubes were heated and supernatant was collected for further

lipid analysis.

Total lipid analysis



Clean, dry test tubes were weighed thrice until concordant values obtained. Known

amount of lipid extract was taken into previously weighed test tubes and evaporated to dry ness.

The weight of the tubes were noted until a concordant value obtained. Total lipid content was

calculated as per the following formula

Total lipid gms = W 1-W 2

V

Where,

W1 = Dry weight of test tube with lipid extract

W2 = Empty weight of test tube

V = Volume of sample used.

Phospholipid Estimation

Phospholipid was estimated by ferrous ammonium thiocyanate method (Stewart, 1980)

using dipalmitylcholine (10 to 100 µg) as reference standard. In case of serum and testis of fish

oil fed rats, 250 µL of lipid extract (Folch et al 1957) was taken and evaporated to dryness using

steam of nitrogen. The residue was dissolved in 2 mL of chloroform. To this 2 mL ammonium

thiocyanate was added in and centrifuged at 2500 rpm for 15 minutes. The lower chloroform

layer was read spectrophotometrically at 488 nm. Step wise procedure and standard curve for

phospholipids is given in Table 11 and Figure 9.

Table 11. Stepwise procedure for estimation of phospholipids

Sl. No. Vol. Std

(mL)

Conc.

Std

(µg)

Vol.

CHCl3

(mL)

Vol.regent

(mL)

O.D. at 488

nm

1 00 00 00

2 0.2 20 0.136 3 0.4 40 0.254

4 0.6 60 0.359

5 0 8 80 0.655

6 1.0 1 00 0.620

7 1.2 120

E v a p o r a t i o n

b y N 2

↑

2

↓

↑

2

↓

Voltex and

centrifuge

for 10

minutes

2500 rpm

0.733



8 1.4 140 0.831

9 1.6 160 0.935

10 1.8 180 1.081

11 2.0 200 1.137

• Ammonium ferrothiocyanate

Figure 9. Standard curve of Phospholipid

Cholesterol Estimation

Total cholesterol was estimated by Fecl3: CH3COOH method (Zlatkis and Zak,1969) using

cholesterol (Sigma) as reference standard. In brief, 250 µL of lipid extract from serum and testis

of fish oil fed rats, were taken and evaporated to dryness using steam of nitrogen. To this, 1.5

mL ferrichloride : acetic acid reagent was added and allowed to stand for 10 minutes, followed by

1 mL concentrated H2SO4 was added, vortexed and kept in dark for 45 minutes. The optical

density of the colour developed was measured spectrophotometrically at 540 nm step wise

procedure and standard curve for cholesterol is given in Table 12 and Figure 10.

Table 12. Step wise procedure for estimation of cholesterol

Sl. Vol. Conc. Solvent Vol. O.D. at 540



No. Std

(mL)

Std

(µL)

* (mL) H2SO4

(mL)

nm

1 00 00 0.000

2 20 20 0.280

3 30 30 0.290

4 40 40 0.330

5 50 50 0.410

6 60 60 0.504

7 70 70 0.577

8 80 80 0.591

9 90 90 0.799

10 100 100

E v a p o r a t i o n

b y

N 2

↑

1.5

↓

Allowed

to stand

for 10

minutes

↑

1

↓

Keep in dark

for 45

minutes

0.832

* FeCI3 : CH3 GOOH (Saturated)

Figure 10. Standard curve of cholesterol

Triglycerides Estimation

Triglycerides was estimated as per the method of Fletcher (1968), using triolein as

reference standard. In brief, 250 µL of lipid extract from the fish oil fed rats of serum and testis,

3mL of isopropanol and 0.5g of TG purifier were added and vortexed. The content was then

centrifuged to the supernatant, 5% KOH (O.6mL) was added, incubated at 60°C for 15 minutes,

cooled, 1 mL of sodiummetaperiodate (O.025m) was added and mixed well. To the reaction



mixture, distilled acetyl acetate (0.5mL) was added and incubated at 50° C for 30 minutes.

Optical density of the colour developed was measured spectrophotometrically at 405 nm. Step

wise procedure and standard curve for triglycerides in given in Table. 13 and Figure. 11.

Table 13 : Step wise procedure for estimation of triglycerides

Sl.No. Vol

std

(mL)

Conc.

Std

(µg)

Isopr

opano

l (mL)

TG purifier

(g)

Supe

rnate

nt

(mL)

5% KOH

(mL)

Reag

ent*

(mL)

Acetyl

Acetate

(mL)

OD

at

405

nm

1 0 0 0.5 0.6 0.5 0.0

2 0.2 60 0.5 0.6 0.5 0.13

3 0.4 120 0.5 0.6 0.5 0.22

4 0.6 180 0.5 0.6 0.5 0.35

5 0.8 240 0.5 0.6 0.5 0.50

6 1.0 300

↑

3

↓

0.5

Vor

tex

and

cen

trifu

ge

↑

1

↓

0.6

Incub

ation

at 60’

for 15

min

↑

1

↓

0.5

Incu

batio

n at

50’

for

30

min

0.59

* Sodiummetaperiodate.

Fig.11 Standard curve of triglycerides



RESULTS AND DISCUSSION Solubility test

Table 14 shows results for the solubility of ß-carotene (1 mg) in various organic solvents (1 ml).

ß-carotene was dissolved in organic solvents viz., methanol. THF, ethanol, pertrolium ether,

hexane and benzene separately. It was found that ß -carotene dissolved completely in THF and

hexane and

partially in ethanol, petrolium ether and benzene while ß -carotene is not dissolved in methanol.

The use of THF as a solvent for carotenoid solubilization as reported by coonely et al (1993) can

be toxic hence, in this study hexane was used to solubilization l3-carotene for in vivo studies.

Table 14. Solubility of l3-carotene in organic solvents

Solvent Soluble Partial soluble Inso

Methanol - -

THF* + -

Ethanol - +

Petrolium ether - +

Hexane + -

Benzene - +

* tetrahydrofurran

Isolation of β-carotene

β-carotene was extracted from fresh carrot, after diluting the extract with hexane into a known

ratio, β-carotene spectra was read spectrophotometrically β-carotene spectra shown in Figure 12-

14. UV-VIS spectra of standard β-carotene is shown in Figure 15, the spectra of ß - carotene

extracted from carrot was found to be similar as the spectra of standard. The average amounts

of total β-carotene from one portion (100 g) of fresh carrot relishes are presented in Table 15and varied between 1200 and 1210/lg.



Figure 12. UV -Vis absorption spectra of β-carotene extracted from carrot

Figure 13. UV- VIS absorption spectra of β-carotene extracted from carrot

Figure 14. UV- VIS absorption spectra of β-carotene extracted from carrot

Purification of β-carotene



β-carotene extracted from carrot was purified by column chromatography. The purified extract

was checked for its spectra and is shown in Figure 15. The spectra was found to be similar as

standard β-carotene spectra. After purification, concentration of β-carotene obtained was

calculate and given in Table 15.

Table 15. Weight of carrot used and concentration of β-carotene obtained.

Raw material Vol. extract1(mL) Vol extract2 (mL) Conc. β-caro

Carrot (100 g) 140 135 1220±10

1Before purification; 2 After purification

Figure 15. UVNIS absorption spectra of standard β-carotene and its ester

Table 15 shows the weight of fresh carrot used volume of extraction and concentration

of β-carotene obtained from 100 g carrot was 1220 µg which is very much lower that that of

reported value (1500 µg) in the literature. The low concentration of p-carotene obtained in thepresent study may be due to the loss during processing and extraction.

Identification of p-carotene

The purified β-carotene was separated by HPLC method. Identification of β-carotene peak was

identified comparing with standard p-carotene peak. Chromatogram for the standard and p-



carotene extracted from carrot are shown in figure 16 and 17. An additional peak appeared at

18.417 and 18.517 minute in both standard and sample may be isomer of p-carotene. White et al

1993 reported that an additional peak appeared in α -carotene. They have extracted p-carotene

from carrot juice and identified the compound by HPLC.

Figure 16. HPLC chromatogram of standard p-carotene in hexane

Figure 17. HPLC chromatogram of ß -carotene extracted, and purified from carrot

To find out the concentration of ß -carotene in carrot extract, standard graph for ß -carotene

was plotted using concentration of ß -carotene (2 to 8 p mol) against their peak area (Figure

18).



Figure 18. Standard curve of ß -carotene assay by HPLC

Antioxidant activity of β-carotene in vitro

The peroxide values for fish oil and fish oil containing β-carotene (20 µ mol) heated at 40°C for 6

h is given in Table 16. No significant change was observed in the level of PV in fish oil heated

upto 5 hrs (33.88 meq O2 / kg fat) compared to its level at 0 hr (33.6 meq O2 / kg fat). While,

there was slight increase in its level (12.82%) after 6 hrs of heating. In case of fish oils

containing β-carotene (20 mol) treated in a similar condition, there was 5.6% decline in PV.

While, its level was found to be increased as similar as PV in fish oil alone. The results indicates

that the concentration of β-carotene added as an antioxidant was not sufficient to protect the fish

oil against oxidation. Hence, the concentration of β-carotene was increased four fold (80 µmol,

Table 17) and found the level of PV was significantly (18.53%) decreased compared to PV in fish

oil (12.15) after two hrs of heating at 40°C.

Table 16. Effect of β-carotene (20 µ mole) on oxidation of fish oil

Peroxide value meq O2 / kg fat Time (Hours)

Fish oil Fish oil + β-carotene

0 33.60 33.60

0.5 32.87 30.04

1 31.44 30.69

2 33.00 30.69



3 33.47 30.44

4 33.80 34.4

5 33.88 35.2

6 37.64 35.47

Table 17. Effect of β-carotene (80 µ mol) on oxidation of fish oil at 40°C

Peroxide value meq O2 / kg fat Time (Hours)

Fish oil Fish oil + β-carotene

0 12.30 12.30

1 13.13 10.78

2 11.16 9.01

Antioxidant activity of β-carotene in vivo

In this study, grouping male rats were fed either fish oil (0.5 mL 1100 g bwt) or fish oil

containing p-carotene (0.5 mL to 200 µmol 100g bwt) for 10 days to find out antioxidant

property of J3-carotene. The level of Lpx in liver and testis of rats is given in Table 18. The

results showed that lipid peroxidation level was significantly higher by 163.9 % (FO) and 115.8%

(FO + βC) in liver compared to that of control. Incase of testis, the level of lipid peroxidation in

fish oil fed group was slightly higher (5.4%) in OF fed group compared to control. From the

results it could be seen that feeding of β-carotene along with fish oil (200 µ mol) to fish oil

moderately reduced the lipid peroxidation in liver and testis.

Table 18. Lipid peroxidatlon of fish oil with or without β-carotene fed rats

10 days Group

Liver Testis

Control 0.906 ± 0.07 0.205 ± 0.01

FO 2.39 ± 0.08 0.216 ± 0.02

FO + βC 1.954 ± 0.09 0.195 ± 0.05

The present study has shown that addition of p-carotene to fish oil results in reduced level of PV

(in vitro) and lipid peroxidation (in vivo). Adding p-carotene in the fish oil incubated at 40°C for 6

h resulted in significant decrease of PV in vitro compared to control (fish oil system). It is

reported that heating fish oil rich in polyunsaturated fatty acids are prone to oxidation under heat



treatment. Oxidation of oils leads to the generation of oxygen radicals according to the Haber -

weiss reaction (Kellogg III and Fridovich, 1975).

The result obtained in the present study further indicates that the generation of lipid

peroxidation products in vivo could be minimized by addition of p-carotene along with fish oil.

Fish oil supplemented with p-carotene showed lower level of lipid peroxidation in testis and liver.

Absorption of p-carotene in plasma and liver

Tolerance of rats for consumption of fish oil (0.5 mL) containing β - carotene (200 µmol) was

confirmed in a preliminary study. A standard daily intake of 0.5 mL of fish oil per rat was chosen

for the current study on the basis of the preliminary ad libitum consumption data. This volume

was well tolerated and completely consumed by each animal throughout the 10 days feeding

period. There was no significant difference of the body weights or weight gain between groups atthe end of the treatment period (data not shown). HPLC elusion profile of p-carotene in plasma

of rat fed β-carotene in fish oil is shown in figure 19. No β-carotene detected in the plasma and

liver of rat before gavages of fish oil containing p-carotene. After a 10days period compare with

those of rats that received β carotene alone and control which did not receive either β-carotene

in fish oil or β-carotene (Table 19).

Similarly, the results of containing β-carotene or β-carotene alone showed higher level of β-

carotene (189.47%) than those of rats fed β-carotene alone and the control group (Figure 20).

The results further indicates that level of β-carotene both in plasma and liver are significantly

higher than that of base line group (Table 19).



Figure 19. HPLC elusion profile of β-carotene in plasma

Figure 20. Percentage increase from control of serum and liver β-carotene over 10 d feeding

Table 19. Serum and liver β-carotene concentration of rats after 10 days of β-carotene injestion

Group Plasma (µ mol / mL) Liver (n moll g)

Control ND ND

β-carotene 0.41 2.85

*FO + β-carotene 0.97 8.25



FO = Fish oil,

ND = Not detected

Values are mean of 2 animals

In each column values with astric are significantly different

Effect of fish oil on growth parameters

The amount of FO administered did not change, the food intake, gain in body weight of rats

compared to rats in control group. However, rats fed on 2 mL / 100 g bwt, which may be food

spillage showed a slight decrease (20%) in the food intake which may be due to food spillage.

Testicular weight of rats fed on FO are comparable with control group.

Table 20. Growth parameters of n-3 PUFA fed rats for 20 days

Fish oil (mL /100 9 bwt / day) 20 d Parameter

Control 0.5 1.0 2.0

Gain in body

weight (g)

55.2 ±19.6 68.2 ± 12.2 65.7±8.7 52.2±11.36

Diet in take1

(g)

235±15.9 233±32 217±7.0 174.1±23

FER Testis

weight (g)

1.332 ± 0.35 1.779±0.39 1.93±0.29 1.313±0.29

Values are mean ± SD of 5 animals.

1 Average food consumption per rat for 20 days.

Influence of fish oil on serum and testicular lipid profile

The serum lipid profile of rats fed on fish oil for 20 days is given in Table 21. Results showed that

feeding FO at doses of 0.5, 1.0 and 2.0 mL / day, significantly brought down the level of serum

cholesterol by 76.9% (0.5 mL). 83.2 % (1.0 mL) and 63.10% (2.0 mL) compared to that of

control group. Similarly, the level of phospholipid in serum was also reduced by 36.14% (0.5

mL), 46.78 (1.0 mL) and 38.7 % (2.0 mL) respectively compared to control.



Table 21. Serum lipid profile of fish oil fed rats for 20 days

Fish oil (mL /100 g bwt / day) Parameter

Control 0.5 1.0

Cholesterol (mg /dL)

984 ± 16 22.7 ± 7.6 16.5 ± 8.4 36.3 ± 6.7

Phospholipid (mg

/ dL)

54.5 ± 3.2 34.8 ± 10.0 29 ± 5.0 33.44 ± 19.6

Values are mean ± SO of 5 animals.

Fish oil administration significant by reduced testicular lipid profile .The testicular lipid

profile (cholesterol and phospholipids) of rats fed on fish oil for 20 days is given in Table 22.

Results showed that feeding fish oil significantly reduced the cholesterol level in testis in a dose

dependent manner. While, no change was observed in the level of phopholipids and total lipid

content of fish oil fed animals compared to control animals.

Table 22. Testicular lipid profile of rats fed fish oil over 20 days

Fish oil (mL / 100 g bwt / day) Parameter

Control 0.5 1.0 2

Triglycerides (mg

/ 9 tissue)

2.1±0.02 2.4±0.015 2.4 ± 0.04 2.1 ± 0.047

Phospholipids (mg

/ g tissue)

7.8 ± 0.10 6.91± 0.02 7.5±0.12 7.0±0.02

Cholesterol (mg /

g tissue)

5.6 ± 0.02 4.3 ± 0.39 3.6 ± 0.10 3.6 ± 0.22

Values are mean ± SD of 5 animals

SUMMARY Investigation deals mainly with isolation purification and identification of β-carotene from

carrot and influence of polyunsaturated fatty acids (fish oil) on intestinal absorption of β-carotene

and its antioxidant property in vivo the report is presented with brief introduction, materials and

methods adopted and results obtained followed by a brief literature referred.



Introduction describes the importance of β-carotene as pro-vitamin-A and as an antioxidant,

source daily recommendation, deficiency and health effects.

Material and methods describes various experimental procedures followed viz., solubility

test for β-carotene, isolation of β-carotene from carrot, purification by column chromatography,

identification by HPLC and its antioxidant activity in vitro and in vivo, estimation of biochemical

parameters in serum and testis of rats fed fish oil. The results describes the findings/ out come of

each experiment mentioned above.

CONCLUSION • The investigation work was carried out with the objective to isolate, purify and identify β-

carotene from the vegetable -carrot and to study its anti oxidant property in vitro and in

vivo.

• Among the organic solvent system studied, THF and haxane was found to be suitable

solvents to dissolve β-carotene.

• β-carotene was successfully extracted from carrot and its spectra was compared with the

respective standard.

• β-carotene extracted from carrot was purified by column chromatography and it was

separated and quantified by HPLC.

• Antioxidant property of p-carotene in vitro (Fish oil, PUFA model system) and in vivo (rat

model) was studied and found β-carotene significantly decreased generation of PV (in

vitro) and lipid peroxidation (in vivo).

• The absorption study showed that feeding β-carotene with PUFA (Fish oil) significantly

enhanced its level in serum and liver.

• Feeding fish oil alone to growing rats showed an induction of oxidative stress by

enhancing the level of lipid peroxidation in testes and liver.

• Fish oil could be used as a source of n-3 PUFA to enhance intestinal absorption of

bioactive molecule β-carotene.

• β-carotene could be used in vitro and in vivo to minimize the lipid peroxidation.

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