anthocyanin – study of extraction from basella rubra l and verifying antioxidant activity, application in identificating di sodium tetra borate in food

The anthocyanin content was calculated by mean of pH differential method at pH 4.5 and 12.0, with light absorbance at maximum wave length and 700nm.. Extracted juice from fruits were use

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY

ANTHOCYANIN – STUDY OF EXTRACTION FROM BASELLA RUBRA L AND VERIFYING ANTIOXIDANT ACTIVITY,

APPLICATION IN IDENTIFICATING DI SODIUM TETRA

APPROVAL

SUPERVISOR STUDENT

Can Tho, December , 2014

PRESIDENT OF EXAMINATION COMMITTEE

Study of extracting anthocyanins from Basella rubra L and antioxidant

activity, identifying of Di Sodium Tetra Borate in food Through the experiment with different solvents, temperatures and compared the total anthocyanin content among products, the most effective method with the highest outcome was recorded for further experiment The anthocyanin content was calculated by mean of pH differential method at pH 4.5 and 12.0, with light absorbance at maximum wave length and 700nm Extracted juice from fruits were used for estiamating antioxidant activity and verifying the ability of identifying borax in food due to the specific color change in reaction with borax solution pH from 1

to 8 In this research, the treatment was carried out with distilled water as solvent and temperature of 30 o C was recorded as the most effective extraction method, the total anthocyanin content yield was 3.33%.Moreover, the IC50 index from antioxidant activity experiment was 1 8 µg/mL and lower when compared with ascorbic acid and garlic acid 8.4 µg/mL and 2.6 µg/mL respectively, this result demonstrated the high potential of antioxidant activity

from Basella rubra L Finally, the identification of Di Sodium Tetra Borate

experiment resulted in a positive abilities from anthocyanins: both 20% anthocyanins indicator kits (pH =2) and indicator papers gave clear purple color and high contrast displays with the minimum concentration of borax at 0.05% for kits and 0.1% for papers The extend experiment of the two indicators also achieved the further ability that could identify Sodium hydroxide with high and low concentration

Key word: anthocyanin, antioxidant, borax, di sodium tetra borate,

CONTENT

APPROVAL i

ABSTRACT ii

CONTENT iii

CHAPTER I INTRODUCTION 1

1.Introduction 1

2.Objective 2

CHAPTER II MATERIALS AND METHODS 3

1.Materials 3

1.1.Sample 3

1.2.Apparatus 3

1.3.Equipment 3

1.4.Chemicals 3

2 Methods 3

2.1.Sample preparation 3

2.2.Anthocyanin extraction method 3

2.3.Moisture determination 4

2.4.Total anthocyanin content analysis 4

2.5.Verification of antioxidant activity and IC50 index 5

2.6.Preparation of indicator solution: 5

2.7.Data analysis 5

3 Experiment arrangement 5

3.1.Experiment 1: Verification of color change among the pH range 5

3.2.Experiment 2: Verification of maximum absorbance wave length 6

3.3.Experiment 3: The effect of solvent and temperature on the total anthocyanin content 6

3.3.1.Determination of moisture 6

3.3.2.The effect of solvent and temperature on the extraction process 6

3.4.Experiment 4: Antioxidant activity verification 7

3.5.Experiment 5: Application in Identification of di sodium tetra borate 8 3.5.1.Identification of Borax 8

3.5.2.Producing indicator solution 8

3.5.3.Producing indicator papers 9

3.5.4.Identification of sodium hydroxide in food 9

CHAPTER III RESULT AND DISCUSSION 10

1.Verification of color change among the pH range 10

2.Verification of maximum absorbance wave length 11

3.The effect of temperature and solvent on total Anthocyanin content 12

3.1.Moisture content 12

3.2.Total Anthocyanin content 12

4.Experiment 4: Antioxidant activity verification 14

5.Application in Identification of Di Sodium Tetra Borate 16

5.1.Identification of Borax 16

5.2.Possibilities of indicator solution 17

5.3.Possibilities of indicator papers 19

5.4.Identification of sodium hydroxide 20

5.4.1.Identification of sodium hydroxide by indicator solution: 20

5.4.2.Identification of sodium hydroxide by indicator papers: 21

CONCLUSIONS AND SUGGESTIONS 23

1.Conclusions 23

2.Suggestions 24

REFFERENCES 25

CHAPTER I INTRODUCTION

1 Introduction

Anthocyanin are water-soluble vacuolar pigments occur in tissues of higher plans, play an important role beside Chlorophyll and Carotenoid This polyphenol act as main constituent of natural color on flowers, fruits, seeds that can not be replaced by artificial colored products In fact, those man – made colorant only adapt to coloring function but still existed problems in food safety like cancer, neurological disorder, intestinal or poisoned lead to lethal situations

In contrast, anthocyanins – polar organic components are safe, soluble in polarized solvents with characteristic of antioxidant, anti-cancer, anti-inflammatory and prevent cardiovascular diseases (Joseph,1999; Pawlowicz, 2000) Thanks to the natural qualities and valuable functions, anthocyanins have been leading scientists and companies to study and exploit this natural pigment

in food consumption to make sure the safety in society

Basella rubra L are vegetable that evaluated as easy crops because of

short time growth, simple cultivate methods, suitable for many kinds of soil; they have been using as high nutrient vegetable in daily dishes due to both delicious and medicinal properties Farmers usually use only stems and leaves for sale, the left over fruits after harvest are kept as breeding purpose in few amount, the rest are useless The flesh contain red or purple – red color, these pigments can soluble in water easily, which are high potential for food colorants Pigment content inside fruit can supply for food color, food preservatives or

functional food By mean of using Basella rubra L fruits for extraction

anthocyanins purpose, the problem with leftover fruits after harvested can be solved among with produce natural colorant safe for health

Problems in food safety are not only cause by chemical colorant but also food preservative There is a fact that some banned preservatives that still have been using, are threatening consumers health Di Sodium Tetra Borate or Borax

are using as an anti-fungal compound in food preservation, that can help fishes and meat become tough and elongate the storage period The color of anthocyanins can change along with the pH range, this property can be applied

as an indicator to identify the present of borax in food through the color change

In total, this thesis aim to: study of extraction from Basella rubra L and

verifying antioxidant activity, application in identifying disodium tetra borate in food

2 Objective

‒ Study of extracting anthocyanins from fruits of Basella rubra L

‒ Verifying antioxidant activity of anthocyanin from Basella rubra L

‒ Determining the identical possibility of borax in food by anthocyanin

from Basella rubra L fruits

CHAPTER II MATERIALS AND METHODS

1 Materials

1.1 Sample

Fruits of Basella rubra L were harvested from Biotechnology R&D

Institute, Can Tho University and Dong Thap province, Vinh Long province, Tien Giang province

1.2 Apparatus

Beaker, buret, crucible and cover, Erlenmeyer flask, filter paper, funnel, graduated cylinder, micropipette, mortar and pestle, test tube, crack, thermal metter, volumetric flask, volumetric pipet, vacuum bottle, vacuum filter bottle, wash bottle

‒ Antioxidant activity: DPPH, methanol

‒ Borax identification: Di Sodium Tetra Borate, pH buffers (1-8)

2.2 Anthocyanin extraction method

Moisture = (M1-M2) x100/M1 (%)

In which: M1: The weight of eggfruit powder sample before drying M2: The final unchanged weight of eggfruit powder sample

after drying This experiment was repeated 3 times

2.4 Total anthocyanin content analysis

Total anthocyanins content was calculated by pH differential method The principle of method: anthocyanins have flavium form at pH 4.5 and carbinol form at pH 12

Method: Measuring light absorbance at maximum wave length and 700nm within pH 4.5 and pH 12.0

Equations:

l

K M

A

a

.

.

M: molecular weight of Anthocyanin, g/mole;

l: path length of cuvette, cm;

K: dilution factor;

ɛ = 26900 L/mole

Total Anthocyanin content:

% Total Anthocyanin content =

.

w m

V a



V: volume of extracted liquid, mL

m: mass of sample, g

w: moisture

2.5 Verification of antioxidant activity and IC50 index

The Antioxidant activity was calculated by:

Abscontrol

Abssample -

Abscontrol

AA%: antioxidant activity

Abssample : Light absorbance of sample at 517nm

Abscontrol: Light absorbance of control sample at 517nm

IC50 value (mg/ml) is the effective concentration at which DPPH radicals were scavenged by 50% IC50 was calculate from trend line that built

up by the result of AA%

2.6 Preparation of indicator solution:

Indicator solution were produced by diluted centrifuged extracted juice with pH buffer solution and store in 2.2mL tubes within dark cabinet

the main ingredient of anthocyanins from Basella rubra L fruits and their effect

to the result of lateral experiment

Objective: anthocyanins extracted juice

Method: 1mL extracted juice was diluted with 24mL distilled water in each test tube The adjustment of pH in each tube was carried out by the addition

of HCl 1N or NaOH 1N The color change were observed and recorded at each grade of pH from 1 to 14

3.2 Experiment 2: Verification of maximum absorbance wave length Purpose: Define the maximum absorbance wave length

Object: Extracted juice from fruits

Method: Diluted 1mL juice with 24mL pH = 1 buffer solution in a 25mL volumetric flask Absorbance spectra were recorded by continuous scanning range from 450 nm to 650 nm

3.3 Experiment 3: The effect of solvent and temperature on the total anthocyanin content

3.3.1 Determination of moisture

Aim: determining the moisture content of fruits, then used for calculating total anthocyanins content

Object: fresh fruits of Basella rubra L

Method: dried sample at 70oC for 48 hours, experiment was repeated 3 times

3.3.2 The effect of solvent and temperature on the extraction process The experiment was carried out for the aim of verifying the effect of solvent and temperature on the same object when extracted anthocyanins from fruits There were two factors in this experiment, solvent and temperature; solvent with 3 treatments: distilled water added 0.1% HCl, Ethanol: H2O (1:1) added 1% HCl, Acetone: H2O (1:1) added 0.01% HCl; temperature with 2 treatments: 30oC and 35oC Total 3x2 = 6 treatments and repeat 3 times

3.4 Experiment 4: Antioxidant activity verification

Aim: Verifying Antioxidant activity

Object: extracted juice from Basella rubra L fruits

Method:

Extracted juice was centrifuged 13000 spins/minute and diluted into different concentration Sample was mix with DPPH and measure light absorbance at 517 nm Repeat 3 times for each dilution (Brand-Williams et al., 1995; Huang et al., 2005)

‒ Dilution range: 10-1, 10-2, 10-3, 10-4 times

H2O

Vacuum filter Dark cabinet Solvent added

‒ Tested sample included 1mL DPPH, 1mL sample for each dilution in one 2.2 mL tube The sample was incubated in dark cabinet for 60 minutes and measured the light absorbance

‒ Control sample included 1mL DPPH and 1mL Methanol

3.5 Experiment 5: Application in Identification of di sodium tetra borate Aim: identify borax in different concentration due to the color change of Anthocyanin when reacted with borax

Object: Anthocyanin from Basella rubra L

3.5.1 Identification of Borax

Anthocyanin extract from fruits was diluted into 20%, 10% and 5% in pH buffer solutions from 1 to 8 and reacted with Di Sodium Tetra Borate in 5 concentration 2%, 1%, 0.1%, 0.05% and 0.005% The results with clear and visible color change were used for the next part of experiment

3.5.2 Producing indicator solution

Aim: producing indicator solution that could quickly identify the present

of borax

Object: anthocyanin solution and di sodium tetra borate solution

From the color change of Anthocyanin within borax solution at specific

pH, the indicator kits were produced Anthocyanin was diluted into 20% and 10% solution with pH buffer solution and kept in dark cabinet with room temperature The possibilities were test by the change color of indicator kits with borax in 5 concentration 2%, 1%, 0.1%, 0.05% and 0.005%

3.5.3 Producing indicator papers

Aim: producing indicator pappers that could quickly identify the present

of borax

Object: di sodium tetra borate solution

Filter papers were cut into 0.5x5cm sheets and preserved in extracted

juice from Basella rubra L fruits with different dilutions: 20%, 30%, 40%,

50%, and 60% in 24 hours Then these papers were dried under room temperature and used for identifying borax solution: 2%, 1%, 0.1%, 0.05% and 0.005%

3.5.4 Identification of sodium hydroxide in food

Base on the most clear and high contrast result at specific pH from identification experiment, the indicator kits and papers were used to identify NaOH with concentration of 1%, 0.5% and 0.05% Sodium hydroxide is representative of alkaline food preservatives, which are added as anti fungus and antibacterial, these preservatives can threaten consumers‘ health The result were compared with the other indicators that were used in market to estimate the possibility of indicator products from this research

CHAPTER III RESULT AND DISCUSSION

1 Verification of color change among the pH range

Anthocyanin change color due to the transfer of ion H+ and OH- that produce colored salt contain flavium, quinonoidal, chalcone and colorless salt

as carbinol

Figure 1 The colored change of Anthocyanin among pH range

The color of Anthocyanin solution changed through the pH range from 1

to 14 as shown on the figure 1 The rising of pH from an initial point of 1 to 7 witnessed the stable color of Anthocyanin with pink and red-purple color At

pH nearly 8, Anthocyanin remained purple color and then changed to violet, blue respectively when pH rose from 9 to 11 due to the present of quinoidal ion Solution gradually lose blue color and became colorless of carbinol salt at pH = 12.0 The rest pH range gave result of yellowish color due to the present of chalcome salt In comparison with the color range of Anthocyanin from berry,

Anthocyanin from Basella rubra L had a specific difference that it could

maintain pink color among pH range from 1 to 7

Base on the result of colored change, flavium salt could be the main

contributions of Anthocyanin from Basella rubra L Those flaivium salt affect

strongly to the result of extraction experiment

2 Verification of maximum absorbance wave length

The light absorbance was at 450nm then rose gradually and peaked at high of 539nm as the maximum absorbance wave length The light absorbance stably reduced when wave length rose from 540nm to 650nm

Figure 2 The light absorbance of anthocyanin from 450nm – 650nm

Base on the research out come of Sullivan in 1998, the maximum absorbance wave length of anthocyanin ranged fluctuated between 510 to 540 For instance, maximum wave length of mulberry is 527 (Nhan, 2011), strawberry is 520 nm (Francisco et al., 1998) Due to the outcome of reported

paper, the pigments from extracted juice from Basella rubra L fruits were

anthocyanins

0 0.5 1 1.5 2 2.5

Wave length (nm)