Phenolic contents and antioxidant potential of desert prickly pear

Phenolic contents and antioxidant potential of desert prickly pear (Opuntia engelmannii) fruit extracts
Thesis submitted as partial fulfillment of the requirement for the degree of M. Phil in Analytical / Inorganic Chemistry
BY
WARDA JABEEN
2015-2017
Student ID: PCHF15E009

DEPARTMENT OF CHEMISTRY
UNIVERSITY OF SARGODHA
SARGODHA

52070012827000NAME OF
IN THE NAME OF ALLAH,
THE MOST MERCIFUL, THE MOST BENEFICENT
“The first step in knowledge is to listen then to be quiet and attentive, then to preserve it, then to put it into practice and then to spread it”.

(Sufyan Bin’Uyainah)

A thesis submitted
toUNIVERSITY OF SARGODHA
inPartial fulfillment of the requirement for the degree
ofMASTER OF PHILOSOPHY
inANALYTICAL/ INORGANIC CHEMISTRY
By
WARDA JABEEN
Session: 2015-2017
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF SARGODHA
SARGODHA

“To My Beloved MOTHER”
Who is the symbol of my strength, confidence, guidance, and her prayers and love enabled me to succeed in achieving my M. Phil degree

“And to All My Teachers and to Those”
Special persons who are near and dear to me

APPROVAL CERTIFICATE
This is to certify that we have read this thesis titled “Phenolic contents and antioxidant potential of desert prickly pear (Opuntia engelmannii) fruit extracts” submitted by Warda Jabeen Student I.D PCHF15E009 in partial fulfillment of requirements for the degree Master of Philosophy in Chemistry and here by approve it for submission.

Supervisor: ______________
Dr Shahid Iqbal
Assistant Professor,
Department of Chemistry,
University of Sargodha,
Sargodha.
Chairman:
__________________
Dr. Farooq Anwar
Chairman,
Departmentof Chemistry,
University of Sargodha,
Sargodha.

DECLARATION
It is certified that the research work mentioned in the thesis entitled “Phenolic contents and antioxidant potential of desert prickly pear (Opuntia engelmannii) fruit extracts” is original and nothing has been stolen / copied / plagiarized from any source.

Supervisor:
_____________
Dr. Shahid Iqbal
Assistant Professor,
Department of Chemistry,
University of Sargodha,
Sargodha.

DECLARATION
It is submitted that the research work mentioned in this thesis entitled “Phenolic contents and antioxidant potential of desert prickly pear (Opuntia engelmannii) fruit extracts” by Warda Jabeen is original and nothing has been stolen/ copied/ plagiarized from any source.
_____________________
Warda Jabeen
M.Phil Research Scholar,
Department of Chemistry,
University of Sargodha,
Sargodha.

ACKNOWLEDGEMENT
All praises to Almighty ALLAH, the creator of whole universe, and the most merciful who taught me by pen. Whose blessings flourished my thoughts and thrived my ambitions to have the fruit of my modest effort in the form of this manuscript. After ALMIGHTY ALLAH, all praises and thanks for the Holy Prophet Muhammad (Peace Be Upon Him) who is forever a torch of guidance and light of the knowledge for mankind.
I wish to express my sincerest gratitude and a deep sense of obligation to a very hardworking and personalized man Dr. Shahid Iqbal, my supervisor, for giving me this opportunity to pursue my Master of Philosophy degree at the University of Sargodha. I sincerely appreciate his guidance, advice, and patience throughout the period of my study in the Department of Chemistry, particularly in this research work.
The most sincere appreciation belongs to my mother. Her love, care, patience and moral support were the main sources of power that upheld me to accomplish the study. I wish to express my great gratitude to my all family members for their cooperation, moral support and especially patience to allow me to complete my work with full concentration.

Friendship is blessing ; miracle of God, I am highly grateful to all my friends for their cooperation and good wishes for me.
Thanks to all non-teaching staff of Department of Chemistry, University of Sargodha, Sargodha.

Warda Jabeen

ABSTRACTNatural antioxidants are preferred over synthetic antioxidants because natural antioxidants have no side effects and synthetic antioxidants are carcinogenic. Therefore researchers focused on natural origin to identify the new antioxidants. Plants are the vital source of antioxidants. Each plant is enriched with various bioactive compounds that differ in their structures and reactivity. Extraction of these bioactive compounds depends on nature of extraction solvent and extraction technique. Therefore, unique extraction strategies are used to extract the maximum yield of these bioactives. Opuntia engelmannii is a therapeutic fruit which have particular antioxidants.

Phenolic contents and antioxidant activity of Opuntia engelmannii fruit extracts was determined by utilizing distinctive extraction methods (maceration, microwave-assisted extraction and orbital shaker) and extraction media (distilled water, methanol, ethanol, n-hexane, acetone and chloroform). Different solvents are used because antioxidant compounds have different solubility in solvents. Solubility of antioxidant compounds varies due to polarity difference of solvents. Mostly antioxidants compounds are dissolved in polar solvents. Besides the solvent polarity, extraction techniques have given different results for the recovery of bioactive compounds because each extraction technique is based on different extraction mechanisms. All the extraction techniques are used and have given good results for the recovery of bioactives. Solvents polarity and extraction technique effect the extraction of antioxidant components. To make extraction process efficient solvent polarity and extraction technique should be optimized. Extracts were subjected for determination of different bioactive contents, i.e. total phenolic content, total flavonoids content, and total tannin content employing spectrophotometric assays. Antioxidant potential was assessed by employing different assays like DPPH radical scavenging assay, and ferric ion reducing assay. For quantification of selected phenolic acids, HPLC was utilized. Results showed that Opuntia engelmannii fruit is a good source of natural antioxidants.
List of Abbreviations
Sr No. Complete Name Abbreviations used
1 Reactive Oxygen Species ROS
2 Oxidative stress OS
3 Antioxidant Capacity AOC
4 Total Phenolic Content TPC
5 Total Flavonoid Content TFC
6 Total Tannin Content TTC
7 Total Carotenoids Content TCC
8 Ferric Reducing Antioxidant Power FRAP
9 Folin-Ciocalteu FC
10 2, 2-diphenyl-1-picrylhydrazyl DPPH
11 High Performance Liquid Chromatography HPLC
12 Gallic Acid Equivalents GAE
13 Catechin Equivalents CE
14 Ascorbic acid equivalent AAE
Table of Contents
Contentspage number
CHAPTER: 1
INTRODUCTION
Bioactive compounds1
1.1.1 Phenolic compounds1
1.1.2 Polyphenols2
1.13 Phenolic acids2
1.14 Flavonoids2
1.15 Tannins3
1.2 Reactive oxygen species3
1.2.1 Generation of ROS4
1.2.2 Effects of ROS on Biological Systems5
1.2.3 Advantages of ROS in living organisms5
1.2.3 Disadvantages of ROS in living organisms5
1.3 Oxidative stress5
1.4Antioxidants6
1.4.1 Sources of Antioxidants6
1.4.2 Significance of Antioxidants7
1.4.3 Characteristics of Antioxidants8
1.4.4 Classification of Antioxidants8
1.4.4.1 Classification of antioxidants on the basis of Catalytic Activity9
1.4.4.1.1 Enzymatic Antioxidants 9
1.4.4.1.2 Non-Enzymatic Antioxidants9
1.4.4.2 Classification of antioxidants on the basis of mechanism of action9
1.4.4.2.1 Primary Antioxidants9
1.4.4.2.2 Secondary Antioxidants 10
1.4.4.3 Classification of antioxidants on the basis of Solubility10
1.4.4.3.1 Hydrophilic Antioxidants10
1.4.4.3.2 Lipophilic Antioxidants11
1.4.4.3 Classification of antioxidants on the basis of origin of Antioxidants11
1.4.4.3 Natural Antioxidants 11
1.4.4.3.2 Synthetic Antioxidants12
1.4.5 Mode of action of Antioxidants12
1.4.5.1 Free radical scavenging activity12
1.4.5.2 Metal chelating13
1.4.5.3 Singlet oxygen quenchers13
1.4.5.4Photosensitizer inactivation13
1.5 Extraction techniques used for recovery of Antioxidants14
1.5.1 Maceration extraction14
1.5.2 Orbital shaker extraction14
1.5.3 Microwave-assisted extraction15
1.6 Factors affecting extraction of antioxidants15
1.7 Valuation of Antioxidant capacity16
1.7.1 Hydrogen atom transfer (HAT)-based assays16
1.7.2 Single electron transfer (SET)-based assays16
1.7.2.1 DPPH radical scavenging assay17
1.7.2.2 Ferric ion reducing antioxidant power (FRAP) assay17
1.7.2.3 Total phenol assay by Folin-Ciocalteu (FC) reagent18
1.8 Opuntia engelmannii fruit18
1.8.1 Growth of Opuntia engelmannii 18
1.8.2 Phytochemicals19
1.8.3 Benefits of Opuntia engelmannii 19
1.8.4 Side effects19
Objectives of study20
Scope of work 20
CHAPTER: 2
LITERATURE REVIEW21
CHAPTER: 329
MATERIALS AND METHODS29
3.1 Chemicals and Reagents29
3.2 Instrumentation and Apparatus29
3.3 Collection of fruit29
3.4 Preparation of Extracts30
3.5 Phytochemical Screening of Crude Extracts Procedure30
3.5.1 Phenolics30
3.5.2 Flavonoids30
3.5.3 Anthraquinones31
3.5.4 Carbohydrate31
3.5.5 Amino acids31
3.5.6 Sterol31
3.5.7 Glycosides31
3.5.8 Tannins31
3.5.9 Alkaloids32
3.5.10 Phlobatannins 32
3.5.11 Terpenoids32
3.5.12 Saponins32
3.5.13 Proteins32
3.5.14 Steroids32
3.5.15 Caumarin33
3.6 Proximate composition analysis33
3.7 FT-IR analysis34
3.8 DPPH radical scavenging assay34
3.9 Total carotenoids content34
3.10 Total phenolic content 35
3.11 Total Flavonoid Content35
3.12Total Tannin Content 35
3.13 Ferric ion Reducing antioxidant Power (FRAP) Assay36
3.14 Phenolic acids determination by HPLC 36
CHAPTER: 4 37
RESULTS AND DISCUSSIONS
4.1 Phytochemical Analysis37
4.2 Proximate Composition Analysis38
4.3 FT-IR Analysis38
4.4 Determination of Total Phenolic Content38
4.5 Determination of Total Flavonoid Content39
4.6 Determination of Total Tannin Content 40
4.7 Total carotenoids content41
4.8 DPPH free radical scavenging assay41
4.9 Ferric ion reducing antioxidant power assay43
4.10 Quantification of Phenolic Acids by HPLC44
Conclusion45
References
INDEX OF TABLES
Table 4.1: Preliminary Phytochemical Analysis of Opuntia engelmannii fruit Extract in different Extraction Media
Table 4.2: Preliminary Phytochemical Analysis of Opuntia engelmannii fruit Extract in different Extraction Media
Table 4.3: Proximate Composition Analysis of Opuntia engelmannii fruit
Table 4.4: FT-IR spectral peak values and functional groups obtained for the Opuntia engelmannii fruit Extract
List of Figures
Figure: 4.1 Percentage extraction yields of Opuntia engelmannii fruit
Figure: 4.2 Determination of total phenolic content from Opuntia engelmannii fruit extracts by applying different extraction techniques in different extraction media
Figure: 4.3 Determination of total flavonoids content from Opuntia engelmannii fruit extracts by applying different extraction techniques in different extraction media
Figure: 4.4 Determination of total tanin content from Opuntia engelmannii fruit extracts by applying different extraction techniques in different extraction media
Figure: 4.5 Determination of total carotenoids content from Opuntia engelmannii fruit
Figure: 4.6 DPPH Radical Scavenging Activity of Opuntia enelmannii fruit extract in concentration 0.1
Figure: 4.7 DPPH Radical Scavenging Activity of Opuntia enelmannii fruit extract in concentration 0.2
Figure: 4.8DPPH Radical Scavenging Activity of Opuntia enelmannii fruit extract in concentration 0.05
Figure: 4.9FRAP assay for Opuntia engelmannii fruit extract in different extraction media by using different extraction techniques
Figure: 4.10Phenolic Acids determination of Opuntia engelmannii fruit extract prepared in distilled water extracted by Maceration
Figure: 4.11Phenolic Acids determination of Opuntia engelmannii fruit extract prepared in ethanol extracted by Maceration
Figure: 4.12Phenolic Acids determination of Opuntia engelmannii fruit extract prepared in methanol extracted by Maceration
Figure: 4.13Standard curve of various concentration of Gallic acid for TPC
Figure: 4.14Standard curve of various concentration of Ascorbic acid
Figure: 4.15Standard curve of various concentration of Catechin for TFC

CHAPTER: 1
INTRODUCTION
Phenolic contents and antioxidant potential of desert prickly pear (Opuntia engelmannii) fruit extracts
For many centuries, to treat human diseases plants, are used because of the presence of many health promoting effects. It was proved through research that diet rich with fruits and vegetables have good impact on health. Recently, researchers work on edible plants to explore the antioxidant potential of these bioactive compounds 1.

1.1 Bioactive compounds:
Bioactive compounds give strength to defensive system in human beings against oxidative stress caused by reactive oxygen species (ROS). Bioactive compounds are categorized into various groups on the basis of chemical structure and function. Phenolic compounds, along with their subgroups, are almost exist in all plants and mostly determined in cereals, nuts, legumes, tea, olive oil, fruits, vegetables, and red wine 2. Various phenolic compounds exhibit antioxidant activity and provide protection from different chronic diseases like auto-immune maladies, aging, inflammation, rheumatoid arthritis, cancer, Parkinson’s disease, , cataract, atherosclerosis, cardiovascular and neurodegenerative diseases. These bioactive compounds also protect plant cells from risks of UV exposure, trauma, drought, pollution, and pathogenic attack. 1, 3.
1.1.1 Phenolic compounds:
Dominant group of bioactive compounds is the phenolics. Aromatic ring is substituted with hydroxyl group called a phenolic compound. Phenolics and polyphenolics (polymeric phenolics) protect from different diseases, like the reduction of cardiovascular disease and cancers 4.
 Phenolic compounds are responsible for the color and flavor of natural material (shrubs, herbs, vegetables, fruits) based foods; they give red color to fruits, juices and wines, and also give flavor to fruits and vegetables 5.

1.1.2 Polyphenols:
Polyphenols work as natural antioxidants. Damage produced by reactive oxygen species (ROS) in body is prevented by polyphenols. Enzymes and substances which are concerned with the development of cancer can be blocked by polyphenols. They are mostly found in tea and in dark fruits 6. Polyphenols existence gives taste, color and aroma to food. Polyphenols must be taken through diet because they cannot be prepared by our body.
Compounds contain an aromatic ring which has one or greater than one hydroxyl groups known as polyphenols. Different groups are included in polyphenols which vary in structure due to the presence of different substances. Variation occurs from simple molecule (phenolic acids) to complex molecules (condensed tannins). Diverse subgroups are formed by different substitutions of the heterocyclic compounds 7.

1.13 Phenolic acids:
Phenolic acids are the simplest type of polyphenols. Phenolic acids containe at least one carboxylic acid. Phenolic acids are slightly different in chemical structure from polyphenols. Phenolic acids that exist in nature comprise of diverse group of organic acids, having two unique carbon frameworks i.e., hydroxycinnamic and hydroxybenzoic structures 2.

1.14 Flavonoids:
The dominant group of the polyphenols is the flavonoid group. They are present in fruits, vegetables, red wine, soybean, cocoa and in green tea. They are responsible for different colour of plant parts (vegetables, grains, seeds, bark, leaves, fruits and flowers). Flavonoids play an important role in protection from different diseases in humans 10. They are classified into different subgroups on the basis of their chemical structure e.g. anthocyanins, flavonols, flavones, chalcones, dihydrochalcones, isoflavones and flavan-3-ols. Each of these groups contains two aromatic rings which are connected by an oxygenated heterocycle (C) and influence their antioxidant activity.
Some flavonoids are: quercetin, rutin, hesperidin, limonene and naranjin.

1. Quercetin is a flavonoid found in apples, grapes, green-yellow onions, broccoli, cherries and red cabbage. Hesperidin is present in the peels of lemons and oranges. Narangin results in the bitter taste of many fruits like orange, lemon, and grapefruit. Limonene found in lime and lemon.
2. Isoflavones (genistein and daidzein) are isolated from soy foods like tempeh, tofu, soy milk, beans, vegetable protein, miso and flour.

3. Anthocyanidins are known as plant pigments which are responsible for the blue, red and purple color of fruits and vegetables. Anthocyanidins are used in treatment of eye and heart diseases.

4. Proanthocyanidins are isolated from grape seeds, red wine, pine bark and sea extract.

5. Ellagic acid is a flavonoid found in vegetables and fruits such as grapes.

6. Catechins are present in green and black tea.

7. Kaempferol is found in radish, broccoli, endive, leek and red beets.

1.15 Tannins:
Tannins are the high molecular weight polyphenolic compounds that exist in nature. Tannins are catagorized into two groups on the basis of structure i. e., condensed tannins and hydrolysable tannins. Condensed tannins exist in nature as oligomers and polymers of flavonoids. Hydrolysable tannins contain a catechin unit which is glycosidically attached to an ellagitannin or gallotannin unit. Tannins are found in grapes, tea, legumes, blueberry and grasses. Medicinal plants rich in tannins are used in the treatment of many diseases 11.
1.2 Reactive oxygen species (ROS):
Oxygen (O2) is required in a number of biochemical reactions occurring in the respiratory chain, such as for the production of adenosine triphosphate (ATP), which provides the energy for different cellular actions. Molecular oxygen can take four electrons and then on attachment with corresponding number of protons produces two molecules of water. This process leads to the generation of different by-products that are generally ROS and reactive nitrogen species (RNS). Intracellular production of ROS consists of oxygen radicals, like superoxide, peroxide, which are present in cell in the form of hydrogen peroxide (H2O2), and the hydroxyl radical 10. Superoxide, peroxide, and the hydroxyl radical are considered as the primary ROS. Only about 2-3% of the O2 used in the respiratory chain is changed to ROS 11. In the biological systems harmful effects of oxygen are oxidation of lipids, inactivation of enzymes, mutations in the DNA, and damage of cell membranes and finally, cells are responsible for the reduction of O2 to ROS 12. Reactive oxygen species (ROS) term is used for both oxygen derivative free radicals and oxygen derivative non radicals which are highly reactive. Accumulation of ROS produces a phenomenon called oxidative stress (OS) 13. This process is responsible for different chronic diseases like auto-immune maladies, cataract, aging, inflammation, rheumatoid arthritis, Parkinson’s disease, atherosclerosis, cancer, cardiovascular and neurodegenerative diseases. In aerobic metabolism, a great number of free radicals and non-radicals are generated. Different external aspects are accountable for the generation of ROS e.g. pollution, cigarette smoke, radiation and, medication 14.

1.2.1 Generation of ROS:
ROS are generated endogenously and exogenously
ROS are generated exogenously by UV light irradiation and by using X-rays and gamma rays, in metal-catalyzed reactions, generated from pollutants existing in the atmosphere, by neutrophils, eosinophil, macrophage, and in inflammation and generated as by product in mitochondrial-catalyzed electron transport reactions 10.

ROS are generated endogenously by mitochondrial respiratory chain, Cytochrome P-450 enzyme mixed function oxidases, by using oxidative enzymes present in cells, such as xanthine oxidase, other sources of ROS generation are two types of immune cells called neutrophils and macrophages.

Types of radical form:
Superoxide O2•, Hydroxyl OH•, Alkoxyl RO•, Peroxyl ROO•, Nitric oxide NO•, Thiyl radical R-S•
Types of ROS in non-radicals form:
Hydrogen peroxide H2O2, Hypocholorous acid HOCl, Ozone O3, Singlet oxygen 1O2, Peroxy nitrile ONOO, Lipid peroxide LOOH
But mostly ROS specie existing in living cells are superoxide (O2 •-), hydrogen peroxide (H2O2), hydroxyl radical (OH•) and nitric oxide (NO•).

1.2.2 Effects of ROS on Biological Systems:
ROS has following advantages and disadvantages on biological systems of human beings
1.2.3 Advantages of ROS in living organisms:
Nitric oxide is vital in cell communication and redox control systems. It performs defensive functions against different diseases. Correspondingly, hydrogen peroxide (H2O2) and superoxide also play the same functions 15.

1.2.3 Disadvantages of ROS in living organisms:
Because of great reactivity of free radicals, they cause oxidation of biomolecules. In biological systems, hydroxyl radical (OH•) can react with any molecule in the cell. Hydroxyl radical (OH•) also causes mutation in DNA and then damages double stranded DNA by reacting with bases of DNA and then lead to structural changes. These structural changes cause cytotoxic effects, chromosomal aberrations which in turn lead to cancer like diseases 16. ROS also cause oxidation of amino acid residues which cause denaturing of enzymes. ROS also cause lipid peroxidation, through autoxidation; lipids reaction with oxygen can cause degradation. Lipids, present in foods, can be oxidized by free radicals which cause rancidity, damages proteins and cause oxidation of pigments that permits the loss of nourishing value, sensory pigments and shelf-life of food products 17.

1.3 OXIDATIVE STRESS (OS):
The idea of oxidative stress (OS) was first given by Sies in 1986. All ROS types are tremendously dangerous to all organisms in their great quantity. When the level of ROS overcomes human antioxidant defense system a cell is said to be in a condition of “oxidative stress.” As a result of the great production of ROS, environmental stress occurs which leads a threat to cells by causing peroxidation of lipids, oxidation of proteins, damage to nucleic acids, enzyme inhibition, activation of programmed cell death (PCD) pathway and ultimately leading to death of the cells. Oxidative stress causes diseases like cancer, aging, and cardiovascular and neurodegenerative diseases 29.In the cells of human beings, oxidative stress causes rise in intracellular levels of free Ca 2+ and iron, and extreme rising of intracellular free Ca 2+ may cause DNA fragmentation 18.

1.4Antioxidants:
ROS can have adverse effects on molecules like DNA, RNA, lipid and protein. ROS attacking on macromolecules is sometimes known as oxidative stress. Thus, oxidative stress causes various diseases like cardiovascular disease, diabetes, cancer, autism, Alzheimer’s disease, cataracts, and aging 19. To neutralize these radicals, human body and other organisms have developed an antioxidant protection system which comprises of enzymatic, metal chelating and free radical scavenging activities. But in times of environmental stress and cell dysfunction, ROS levels can increase, and cause major cellular damage in the body. In this case dietary antioxidants aid to sustain sufficient antioxidant status in the body.

Antioxidants are any substance that, by taking or giving electron(s), can neutralize free radicals.

Antioxidant is defined by Halliwell in 2007:
“Any molecule that can interrupt, avoid or eradicate oxidative damage from target molecule” 20.

Antioxidants work in 2 ways:
1. The antioxidant molecules react with free radicals and distort them.

2. Antioxidant molecules may react with free radicals and convert them into new radical forms which are less reactive, longer-lived and less harmful than those radicals which have been neutralized. They may be neutralized by other antioxidants or other mechanisms to terminate their radical status.

Some molecules like vitamin E, vitamin C, uric acid and glutathione work as cellular antioxidants by reacting with free radicals. These antioxidants may help to prevent from chronic diseases. Such as, ginseng consists of steroid-like compounds, ginsenosides, which exhibit antioxidant activities in the vascular endothelium. Ginkgo has strong antioxidant activities because of flavone glycosides that can neutralize free radicals. Flavonoids such as catechin and epicatechin in green tea and grape seed extracts can be able to perform antioxidant activities by distorting free radicals 21.

Second function of antioxidants is to normalize ROS related enzymes. Antioxidants may decrease the free radicals in cells either by obstructing the activities of free radical producing enzymes like NAD(P)H oxidase and xanthine oxidase (XO) or by improving the activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) 22. These antioxidant enzymes, produced in the body, provide an important defense against free radicals. These remarks of inhibition or improvement of enzyme activities by antioxidants have encouraged us to recognize the molecular mechanisms of action of antioxidants. In 1990s, the research in antioxidant has extended, the antioxidant activity have been studied in different biological systems (cell cultures, animal models) 23. Different research models have been proposed for determining the antioxidant activity of biological systems. Usually, the antioxidant activity was determined in the form of total antioxidant activity, total antioxidant capacity, total antioxidant potentials, total radical absorption abilities, ferric reducing/antioxidant power and oxygen radical absorption capacity 24.

1.4.1 Sources of Antioxidants:
Antioxidants can be supplied to body through food to work against free radicals and provide protection from different ailments. Many plants, fruits, vegetables, grains, nuts etc. contain different antioxidant compounds, which provide protection from hazards of free radicals.

Tocopherols, tocotrienols, phospholipids present in olive oil and in oil seeds. Lignin-derived compounds are present in oats. Ascorbic acid, hydroxycarboxylic acids, flavonoids, carotenoids are present in citrus fruit. Phenolic compounds are present in spices, herbs, tea. Flavonoids, carotenoids are present in vegetables. Flavonoids, P-coumaricaud are present in spinach. Vitamin C, vitamin A and phenolic compounds are present in jackfruit fruits. Vitamin C, Vitamin E, carotenoids (alpha and beta carotene, chlorophylls, poly phenols are present in algae. Folic acid and folate are present in red and brown algae.

1.4.2 Significance of Antioxidants:
Antioxidants protect the cell from oxidative stress (OS) and, lipid peroxidation. They work against ROS. In the cell, they guard from degenerative ailments like cancer, cardiovascular diseases (CVD), neurological maladies, and oxidative stress dysfunctions. Sometime they also increase the quality of food item because they provides health improving dietary supplements and also increase shelf-life of products. Antioxidants have great applications other than food industry; antioxidants are used in fuels, beauty care products, pharmaceutical industry, horticultural food, rubber, elastomers and plastic 64. In food industry, antioxidants may be used in the form of fine droplets or aerosols which is sprinkled over food products, like nuts and oats to increase the energy of food products.

1.4.3 Characteristics of Antioxidants:
Antioxidants used in food products, are having monohydroxy or polyhydroxy phenolic compounds which contain different ring substitutions. Mostly these compounds need low activation energy for donation of hydrogen. Therefore, antioxidants radical does not initiate another free radical due to the stabilization of delocalized radical electron. So, antioxidant free-radical do not undergo quick oxidation process due to its stability. Furthermore, antioxidant free radicals can react with lipid free radicals to form a stable complex compound.

Antioxidant must have following qualities for use in food industry:
It should be free from biological values. It should not offer any bad flavors, textures to food on prolonged storage or on exposure to heat. It should be active at minute concentrations (0.01-0.001%). It should not be expensive and toxic. It should be thermally and artificially stable. It should not to be used up by the body. Identification and control of antioxidant in food industry should be a simple and easy process 25.

1.4.4 Classification of Antioxidants:
Antioxidants classification is based on their source, mechanism of action and nature.

1.4.4.1 Classification of antioxidants on the basis of Catalytic Activity:
Antioxidants are categorized as enzymatic and non-enzymatic due to their catalytic action. Enzymes, vitamins, flavonoids, metals, are biological and chemical species which work against harmful effects produced by ROS in biological systems 26.

1.4.4.1.1 Enzymatic Antioxidants:
The enzymatic antioxidant is specific against a distinct ROS, due to specificity of its cell and use of different metals like Zn, Cu, Fe, Mn and Se as catalysts. Enzymatic antioxidant based on copper-zinc super oxide dismutase (CuZn SOD) changes super oxide radicals to hydrogen peroxide (H2O2). After this Glutathione peroxidase (GSH-Px) and catalase (CAT) changes H2O2 to a water molecule. These metal based antioxidants offer the primary defense mechanism 27.

1.4.4.1.2 Non-Enzymatic Antioxidants:
The non-enzymatic antioxidants are based on water soluble vitamin ascorbic acid (vitamin C), glutathione (GSH), lipid soluble vitamins, ?- tocopherol (vitamin E) (?-Carotene, and vitamin A). The non-enzymatic antioxidant defense system works against ROS 26.

1.4.4.2 Classification of antioxidants on the basis of mechanism of action:
Antioxidants just not work against ROS but they are also used to remove lethal products formed in various reactions occuring in biological system. On the basis of their mechanism of action, antioxidants are classified as primary and secondary antioxidants.

1.4.4.2.1 Primary Antioxidants:
Primary antioxidants called chain breaking or disrupting antioxidants because they can scavenge radicals, and break chain propagations 28. Primary antioxidants give an electron or a hydrogen atom on reaction with free radicals. Primary antioxidants are effective when present in small quantity. Examples of primary antioxidant are eugenol, rosemary, vanillin and vitamins (like vitamin C and vitamin E) etc.

1.4.4.2.2 Secondary Antioxidants:
Secondary antioxidants are also known as preventive antioxidants because they hinder the generation of free radicals 28. Reaction between secondary antioxidants and lipid peroxides results in non-radical processes. Sulfur, thiols, sulfides, and disulfides act as preventive antioxidants because they restrict autoxidation. They do variety of actions
1. Chelate pro-oxidant metals and deactivate them.

2. Replace hydrogen to essential antioxidants.

3. Cause degeneration of hydroperoxide to non-radical species.

4. Free singlet oxygen.

5. Integrate bright radiation.

6. Function as oxygen scavengers.

Such antioxidants are regularly mentioned to as ‘synergists’ because they can validate the antioxidant activity of primary antioxidants (e.g. ascorbic acid, citric acid, lecithin, and so forth) 29.

1.4.4.3 Classification of antioxidants on the basis of Solubility:
Antioxidants are classified on the basis of their solubility in lipids and water.

1.4.4.3.1 Hydrophilic Antioxidants:
These antioxidants are soluble in water and have ability to neutralize reactive oxygen species which are present in cell cytoplasm and in blood plasma. Examples of hydrophilic antioxidants are: Vitamin C,flavonoids, thiols and uric acid.
1.4.4.3.2 Lipophilic Antioxidants:
These antioxidants are soluble in lipids and safe cell membrane from lipid peroxidation. Examples of Lipophilic antioxidants are: Vitamin E, carotenoids ubiquinols 30.

1.4.4.3 Classification of antioxidants on the basis of origin:
Antioxidants which are obtained from natural sources have high contents of phenolics, flavonoids, ascorbic acid, tannins, which work more efficiently against ROS. Due to toxicological effects of synthetic antioxidants, they are not favored for pharmacologic use. Due to this reason plant based antioxidants are focused to use as dietary antioxidants. So, antioxidants are classified as natural and synthetic antioxidants 31.

1.4.4.3.1 Natural Antioxidants:
These antioxidants which we get from natural sources mostly consist of polyphenols (phenolic acids, anthocyanins, flavonoids, lignans and stilbenes), carotenoids (carotenes and xanthophylls) and vitamins (vitamin C and E). Usually, polyphenols and carotenoids show biological effects like anti-inflammatory, antiviral, antibacterial, anti-aging, and anticancer 4.

Enzymes (superoxide dismutase, catalase, glutathion peroxidase, etc.) are present in plasma which acts as antioxidants convert ROS to stable substance.

High molecular weight compounds (proteins such as albumin, ceruplasmin, transferrin,) which oppose the generation of metal catalyzed free radicals.

Low molecular weight compounds are classified as lipid soluble antioxidants (tocopherol, bilirubin, quinines and some polyphenols) and water soluble antioxidants (uric acid, ascorbic acid, and some polyphenols).

Minerals (Se, Cu, Mn, and Zn) act as antioxidants.

Vitamins (Vitamin A, C and E) act as antioxidants.

Antioxidants present in algae work against ROS. It is reported that the brown, red, green algae, and cyanobacteria, show high radical scavenging activities 32.

1.4.4.3.2 Synthetic Antioxidants:
These are manufactured artificially in the laboratory or industry. Generally, these are phenolic compounds and shows similarity with other phenolic compounds of natural origin in terms of their reaction with free radicals. In most of the cases, they function as chain disrupting compounds when they donate an electron or hydrogen atom to the free radicals framing stable quinines. Different synthetic antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ter-butyl hydroquinone (TBHQ), propyl gallate (PG), octyl gallate (OG), and dodecyl gallate (DG) are utilized as food additives 33. Within synthetic antioxidants, BHA and BHT are most favored and are generally utilized as a part of oil-in-water emulsions because of their characteristic solid lipophilic nature. Additionally, they are considered applicable for thermally treated food articles since they are reasonably thermostable. They diffuse effortlessly into lipid layers because of their steam volatile nature. Due to exceptional thermo-stability of TBHQ, it is considered appropriate for frying purposes. Hence its usage is regarded as ideal in case of vegetable oils when compared with BHA and BHT 34. Because of lower stability of PG, it is not employed in vegetable oils. Literature has not only revealed that synthetic antioxidants are acceptable at concentrations below 0.02% but in high concentration they are carcinogenic and toxic. This has augmented the interest of consumers to look for some other options that have to be safe, economical, and must be of natural origin.

1.4.5 Mode of action of Antioxidants:
Different chemical phenomena are used for considering oxidation of fats and oils. Mostly there are two types of oxygen (singlet and triplet) which can react with fats and oils. Triplet oxygen can react with radicals and cause autoxidation and singlet oxygen that cannot react with radicals but can react with fats and oils on their unsaturated position. Antioxidant based phenomenon mentioned below are used for considering oxidation of foods 35.

1.4.5.1 Free radical scavenging activity:
Free radicals can be scavenged by antioxidants from food products by donating hydrogen atom. This method efficiency depends on the bond dissociation energy (among oxygen and phenolic hydrogen), pH related with acid dissociation constant, Reduction potential and delocalization of free radicals 36.

1.4.5.2 Metal chelating:
Scavenging of free radicals is done by using metal chelators to avoid oxidation of food products. Frequently steric hindrance is offered by metal chelators, among metals and food products 37. Mostly metal chelators used in food products are citrus acid, phospholipids, and EDTA. Maximum metal chelators are dissolved in water but now a days citrus acid can be used by mixing with oils.

1.4.5.3 Singlet oxygen quenchers:
In comparison of triplet oxygen, singlet oxygen has greater energy which is 93.9kJ so it can react quickly with lipids. Many antioxidants have capacity to quench singlet oxygen which can be carried out by two ways i.e. physical or chemical way. In physical quenching, by providing energy, deactivation of unsteady singlet oxygen is done to stable ground state triplet oxygen. For energy exchange, carotenoids are used as vital singlet oxygen quenching agents. Because the carotenoid’s quenching ability depends on the totality of conjugated double bonds existing in its chemical structure. And chemical quenching depends on a chemical reaction through which oxidation products are formed by oxidation of quencher. ?-Carotene, tocopherols, ascorbic acid, amino acids (like histidine, tryptophan, cysteine, and methionine), peptides, and phenolics can be oxidized by singlet oxygen 38.

1.4.5.4Photosensitizer inactivation:
Sensitizers such as chlorophyll and riboflavin exist in each food product which can be stimulated by light. Sensitizers are transformed into singlet oxygen (reactive ROS) by photo stimulation. Free radicals are produced due to chain reaction which occurs as a result of singlet oxygen reaction with food products. Carotenoids with fewer than 9 conjugated double bonds, may assist in deactivation of photosensitizers while carotenoids with 9 or greater conjugate double bonds indicate to singlet oxygen extinguishing 39.

1.5 Extraction techniques used for recovery of Antioxidants:
Many bioactive compounds are present in each plant which varies in chemical structure and reactivity. So different extraction techniques such as orbital shaker, maceration, and microwave-assisted extraction (MAE) are employed. Each extraction technique depends on different interaction of solute and solvent 40.

1.5.1 Maceration extraction:
Maceration is the easiest and low-cost extraction technique. In this technique sample’s powder is taken in a volumetric flask with desired amount of the specific solvent. Then aluminum foil is used to cover this volumetric flask. The mixture (sample and solvent) is kept for specific time at room temperature. After this specific time period this sample mixture passed through filter paper Whatmann No. 1 then the liquid is separated from the solid material (marc). Diffusion and solubility affect the efficiency of this technique.

Advantages:
This technique is suitable for thermo labile compounds because no heat treatment is required.

Disadvantages:
Longer extraction time is required. Sometimes mechanical shaking is used 41.

1.5.2 Orbital shaker extraction:
In this technique sample is taken in a volumetric flask with some particular solvent and kept in an electric device (called orbital shaker) for a specific time. This device shakes the sample and solvent mixture. This volumetric flask is kept in this device by using metal clamps. This device works under some parameters such as specific speed (rpm) and time. Antioxidant’s extraction from botanical matrix is based upon the sample solubility in extracting solvent according to the law of solubility, “like dissolves like”. Mostly antioxidants are polar species, that’s why for their efficient extraction polar solvents are used 40.

Advantages:
This technique is suitable for thermolabile compounds because no heat treatment is required. No mechanical shaking is required.

Disadvantages:
Efficiency of extraction is reduced when nonpolar solvents are used.

1.5.3 Microwave-assisted extraction (MAE):
In this extraction technique microwave energy wavelength ranges from 0.001 m to 1 m is used. Mixture (sample and solvent) is kept in microwave oven for specific time due to this heating sample gets the high energy of the microwaves then as a result internal temperature of the sample (plant extract) increases. Plant cell wall becomes ruptured, due to increase in internal temperature. By rupturing of cell good extraction occurs because solvent enters into the plant matrix and secondary metabolites from plant into the solvent. Usually different solvents are used for effective extraction. Mostly those solvents will be chosen which have high dielectric constant for good absorption of microwave energy.

Advantages:
Small extraction time (approx. 10 min) is required. Multiple extractions occur. Minimum sample handling is required. Use the small quantity of extraction media. No mechanical shaking is required.

Disadvantages:
Efficiency of extraction is reduced when nonpolar solvents are used 42.

1.6 Factors affecting extraction of antioxidants:
Each plant is enriched naturally with botanical matrix. Extraction of Antioxidants from botanical matrix in plant is based on different factors:
1. Nature of solvent like polar sample dissolves in polar solvents and nonpolar sample dissolves in nonpolar solvents 43. Extracting solvent affects the mechanism of antioxidant capacity (AOC), rate of hydrogen atom transfer (HAT) reactions and single electron transfer (SET) reactions.

2. Extraction time like specific time is required for good extraction.

3. Mostly high temperature gives good result in less time. But temperature also depends on the nature of solvents
4. Quantity of extraction media
5. Extraction technique which give good result in less time
1.7 Valuation of Antioxidant capacity (AOC):
Antioxidant capacity (AOC) is defined as “the ability of a compound (or a mixture) to obstruct oxidative decay. Diverse terms such as antioxidant activity (AOA) and antioxidant potential (AOP) are used for same perspective. For antioxidant capacity evaluation two dissimilar sorts of tests are used which depend on reactions and reaction mechanism: i. Hydrogen atom transfer (HAT) based test ii. Single electron transfer (SET) based test. Both of these tests give similar results but they are different in kinetics and potential of side reactions 44.

1.7.1 Hydrogen atom transfer (HAT)-based assays:
This test is used to estimate the ability of antioxidants to produce stable compounds by donation of hydrogen atoms to free radicals. These assays are used to determine the potential of antioxidant to scavenge free radicals by donating hydrogen atom. These tests depend on free radical initiator, an oxidizable molecular probe with an antioxidant. In decomposition of azo compounds thermally produced peroxyl radical (ROO•) compete with the antioxidant along with oxidizable molecular probe. These tests do not depend on solvent and pH and for their completion; less time is required 45.
1.7.2 Single electron transfer (SET)-based assays:
This test is used to estimate the ability of antioxidants to produce stable compounds by transfer of electron to free radicals (oxidant), metals, and carbonyls. In these tests, one determines the ability of antioxidant to retain an oxidant which alters color 45 when reduced. Changing in color is associated with concentration of sample. SET-based tests are dependent on pH and solvent, relatively slow, and time taking test. Tests in which single electron transfer occurs are:
ABTS (2, 2´-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid)
FRAP (Ferric Ion Reducing Parameter)
DPPH (1, 1´-dipehnyl-2-picrylhydrazyl)
TPC (Total phenolic content, with Foiln-Ciocalteu reagent) 46.

1.7.2.1 DPPH radical scavenging assay:
Blois in 1958 established this method and after this various scholars worked on it. Due to its suitability, it is used to find out antioxidants from plants and plant based drugs. DPPH free radical (DPPH•) is not reactive and solvable in organic extraction media such as methanol, ethanol, and acetone. This test depends on the disappearance of radical’s color when treated with antioxidants. It displays maximum absorption at 514 nm. DPPH test depends on pH, solvent, concentration of sample, and reaction time. Though, it works simply, rapidly, and economical method for determination of antioxidants ability to scavenge free radicals. Demerits of DPPH are; dual behavior of DPPH, i.e. it acts as oxidant and as radical probe. It is only solvable in organic extraction media such as methanol, ethanol, and acetone while it can’t evaluate the ability of hydrophilic antioxidants 47.

1.7.2.2 Ferric ion reducing antioxidant power (FRAP) assay:
This test was first represented by Benzie and Strain in 1996 to check the reducing power in plasma. After this it was used to check the reducing capacity of various substrates. This test is used to evaluate compound’s ability to reduce yellow ferric tripyridyltriazine complex Fe(III)(TPTZ) (TPTZ = 2,4,6-tripyridyl-s-triazine) to blue ferrous complex Fe(II)-TPTZ). Mostly electron-donating antioxidants in an acidic medium give response to this test. It is easy, faster and economical test that even can be applied manually or automatically. This test depends on transfer of electron instead of incorporation of SET and HAT method. For polyphenols determination it takes more time 48.

1.7.2.3 Total phenol assay by Folin-Ciocalteu (FC) reagent:
Mostly this test is used in food research laboratories. This test is based on oxidation-reduction reaction between the Folin-Ciocalteu (FC) reagent with molybdenum (Mo) and a phenolic compound, so the reducing potential of a sample is determined as mentioned in the given Equation. Phenolic proton dissociation to phenolate anion, have ability to reduce FC reagent. Proton dissociation occurs at pH ~10; for attaining this pH sodium carbonate solution is used 46.

MoVI (yellow) + e- ? MoV (blue)
At start this test was used for evaluation of proteins, because of reagent’s potential for tyrosine (an amino acid containing phenol groups). But now a days this method is used for the study of total phenolic content (TPC) in natural products by small change in composition of the FC reagent 49.
1.8 Opuntia engelmannii fruit:
Therapeutic plants are recognized as vital sources of antioxidant. Various species of the genus Opuntia of the cactus family (cactaceae) are used to harvest cactus pear fruit, and are grown in several countries 50.

Opuntia genus of 200 species belongs to the kingdom: Plantae, phylum: Anthophyta, class: Dicotyledoneae, order: Caryophyllales, family: Cactaceae, genus: Opuntia developed all through Mexico and in America. Its natural product are known as desert cactus pear, desert prickly pear, cow tongue prickly pear, discus prickly pear, cow’s tongue cactus, Engelmann’s prickly pear and Texas prickly pear in the US, and in Mexico named as nopal, joconostle, vela de coyote and abrojo. There are different varieties of fruiting Opuntia prickly plant, one of them is Opuntia engelmannii. It is used as a source of bioactive compounds.

1.8.1 Growth of Opuntia engelmannii:
Opuntia engelmannii have stem of yellow green to blue green color, mostly growing up to length of 1 to 3(3.5) m, and have a small trunk. Flowers color is yellow, but sometimes reddish. Flowers blooming season is from April to May. The purple fleshy fruits are 3–7 cm long 51.

1.8.2 Phytochemicals:
Opuntia’s species have flavonoids (quercetin, kaempferol, taxifolin, phenolics and narcissin,), lactones named as alpha-pyrones (opuntiosides); terpenoids (freideline, lupenone etc.) and alkaloids (tyramine, hordenine, mescaline, etc.). Betalain pigments (indicaxanthin, betanin) are at least found in the fruits and act as anti-oxidants 52.

1.8.3 Benefits of Opuntia engelmannii:
The fruit, as well as cactus stem are used to prepare value-added products, such as squash, jam, pickle, wine, shampoo, body lotions, creams, etc. It also has several medicinal and industrial uses. Prickly pear juice is utilized in candies and jellies. Its seeds can be used as flavouring agents 53. Opuntia species act as anti-inflammatory, anti-diabetic, galactogogue, analgesic, antiviral, hypoglycemic, and anti-oxidant. That’s why Opuntia species are utilized to blood sugar, rise fiber intake, regulate weight, and are utilized for the cure of asthma, liver injury, fatigue, corns, alcohol abuse, dysentery, diarrhea, gastritis, dyspnea, colitis and gastrointestinal disorders, measles, gonorrhea and syphilis, hypercholesterolemia, obesity, nosebleed, snakebite, sore throat, vaginitis, and inflammation of the eyes 54. The pad’s pulp utilized for dressing of burns, wounds, cuts, and fractures and is used as pain killer 55. Now a days prickly pear is used to cure hyperglycemia, alcohol hangover, benign prostatic hyperplasia, artheriosclerosis, diabetes, acidosis and urinary system’s problems.

1.8.4 Side effects:
Prickly pear can cause minor diarrhea, greater stool volume, nausea, headache and abdominal fullness 56.

Objectives of study:
To study the effect of different solvents (distilled water, methanol, acetone, chloroform, n-hexane and ethanol) and different extraction techniques (maceration, microwave-assisted extraction and orbital shaker) on yield of bioactive compounds in extracts. To determine the phenolic contents and antioxidant activity of different extracts of Opuntia engelmannii fruit employing evaluation assays based on different working principles. To identify and quantify selected phenolic acids from Opuntia engalmannii fruit extracts by HPLC. To determine contents of phenolics, flavonoids, tannins, carotenoids, and minerals in fruit extracts prepared in different solvent systems employing different extraction techniques.

Scope of work:
Antioxidants work against free radicals in biological system. Antioxidants may be prepared by our body or can be obtained through diet. Plants are considered important sources of antioxidants because of phenolic compounds which work against ROS causing ailments. Opuntia engelmannii is a medicinal fruit full of bioactive compounds such as phenolics, tannins, flavonoids, lignans, alkaloids, gallic acid, amino acids, terpenoids etc. To get maximum yield of bioactives different antioxidant capacity (AOC) assays will be employed.

CHAPTER: 2
LITERATURE REVIEW:
Butera et al (2002) studied red, white and yellow fruits of sicilian cultivars of prickly pear (Opuntia ficus indica) which is because of combination of two betalain pigments like the purple-red betanin and the yellow-orange indicaxanthin. It was concluded from spectrophotometric results that the betalains contents was higher in yellow cultivar then in red and white. In red fruit polyphenol components were insignificant. The methanolic fruit extracts give positive results for antioxidant activity when 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox) was measured equivalents per gram of pulp. In scavenging the 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt cation radical betanin worked more efficiently as compare to Trolox. It was revealed from results that antioxidant activity of prickly pear fruits was due to betalains 52.

Galati et al (2003) analyzed sicilian variety of Opuntia (Opuntia ficus indica) for determination of total phenolic, flavonoid and ascorbic acid contents. Total phenolic contents in the Opuntia ficus indica was 746 ?g/mL. High-performance liquid chromatography (HPLC) ?diode array detector was employed to determine flavonoid contents, isorhamnetin and rutin derivatives were determined. DPPH• test was used for determination of antioxidant activity. Phenolic compounds are responsible for antioxidant activity in Opuntia ficus indica, because they are highly radical scavengers 57.

Stintzing et al (2005) studied clones of cactus pear ( Opuntia ficus-indica and one O. robusta Wendl) of different color for determination of ascorbic acid, phenolics, and betalain contents and related to antioxidant potential measured by oxygen radical absorbance capacity (ORAC) and Trolox-equivalent antioxidant capacity (TEAC) tests. Phenolics had the higher contribution for TEAC and ORAC values. Measurement of cactus pear juices by high-performance liquid chromatography (HPLC)?diode array detector (DAD)?tandem mass spectrometry (MS/MS) determined that difference in clones was due to change in pigment patterns and concentration of betalain. In cactus fruits of lime green color, yellow and red betalains were not present. Different colors of the clone red, orange, yellow, and purple were due to difference in ratio and concentration of pigments. New food products could grow by the different combinations of red-purple betacyanins with yellow betaxanthins 58.

Moßhammer et al (2006). analyzed cactus pear fruits and identified different compounds like taurine, betalains, minerals etc. presence of these compounds showed high antioxidant activity and worked against various diseases. Due to the presence of these compounds it can be used in food industry. It can be beneficial for health of the consumer 59.

Mahattanatawee et al (2006). studied fourteen tropical fruits (white guava, red guava, carambola, white pitaya (white dragon), red pitaya (red dragon), lychee, mamey sapote, sapodilla, longan, ripe mango, green mango, ripe papaya and green papaya) for determination of antioxidant activity by using following assays: total dietary fiber (TDF), total soluble phenolics (TSP), DPPH (1,1-diphenyl-2-picrylhydrazyl), radical scavenging activity, pectin ORAC (oxygen radical absorbance capacity) and total ascorbic acid (TAA). Ranged of ORAC, TSP, and DPPH from 205.4 to 2316.7 g gallic acid equiv/g puree respectively. TSP compounds (r) 0.96 are highly related with antioxidant activity and ascorbic acid (r) 0.35 and 0.23 less related to DPPH and ORAC data, respectively). Antioxidant activity was determined by two tests DPPH and ORAC and which exhibited same trend. Results showed that ellagic acid conjugates, hydrolyzable tannins, and flavone glycosides were present in various tropical fruits 60.

Osorio et al (2011). studied sour prickly pears (xoconostles) from Opuntia joconostle cactus for the determination of phenolic and pigment content . DPPH+ method was used for the determination of antioxidant activity of methanolic extract and some semi-purified fractions. Every part of fruit (pericarp, mesocarp and endocarp) was analyzed. Phenolic and flavonoids content were higher in pericarp as compared to endocarp and mesocarp. In pericarp phenolic and flavonoids content were 2.07 mg Gallic acid equivalents (GAE)/g fresh weight (FW) and 0.46 mg (+)-catechin equivalents (CE)/g FW respectively. Different phenolic compounds were determined for example protocatechuic, caffeic, 4-hydroxybenzoic, vanillic and syringic acids rutin and quercetin. betacyanins are responsible for the color in fruit. Concentration of betacyanin (23.03 mg betanin equivalents/100 g fresh weight) was higher in endocarp as compared to pericarp and mesocarp. HPLC-PDA-MS method was used for the evaluation of Betacyanins as betanin, betanidin, isobetanin, phyllocactin and isobetanidin. Results showed that many antioxidants compounds like phenolic compounds and betacyanins were present in xoconostle 61.

Moussa et al (2011). studied Opuntia macrorhiza fruits and compared with fruits of Opuntia ficus-indica in respect of betacyanins flavonols, content and antioxidant activity. Flavonol contents were determined by HPLC-DAD after enzymatic hydrolysis procedure (glycosides vs. aglycons). Purple to red color of cactus pear fruits due to betacyanins was also analysed and compared with Beta vulgaris ssp. (roots) and red O. ficus-indica fruits. But in result no flavonols content were determined in cactus fruit pulpand different compounds like isorhamnetin glycosides were determined in peel of O. ficus-indica fruit, isorhamnetin-3-O-rutinoside was determined in O. macrorhiza fruit. Both peel and pulp of O. macrorhiza fruit exhibited high antioxidant activity. Due to betacyanins, peel and pulp of O. macrorhiza fruit showed a dark red-purple color, which is about 8-fold greater in impact as compared to red fruits of O. ficus-indica 62.

Jamuna et al (2011). determined the antioxidant activity of eleven different fruits in vitro.  Ananas comosus, Carica papaya, Artocarpus heterophyllus, Citrus sinensis,  Citrullus vulgaris, Manilkara zapota,  Malus paradisiaca,domestica, Psidiumguajava  Musa Phyllanthusemblica  and Pyruscommunis. Three  different  assays  like DPPH,  total  antioxidant capacity, reducing power  and  are used for evaluation of antioxidant activity at different concentrations of methanol  extract  of  the  fruits. From all assays, the highest antioxidant activity was shown by P. emblica from the different fruits studied 63.

Almeida et al (2011). analyzed fresh exotic fruits for determining antioxidant activity. Two assays: 2,2?-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were used for determination of antioxidant activity, VCEAC (Vitamin C Equivalent Antioxidant Capacity) and TEAC (Trolox Equivalent Antioxidant Capacity) values were used to express the results of these tests. According to results mangaba and murici showed good antioxidant activity. The values of antioxidant activity determined by ABTS (R = 0.94, P ? 0.001) and DPPH (R = 0.88, P ? 0.001) tests were absolutely correlated with phenolic contents. These tests gave positive results for antioxidant activity in exotic fruits 64.
Dhaouadi et al (2013). investigated four cactus varieties O. ficus-indica (green-skinned), O. streptacantha (red-skinned), O. stricta var. stricta (yellow-skinned) and  O. lindheimeri (purple-skinned),  for the evaluation of antioxidant compounds. He seperated conjugated flavonoids (isorhamnetin, kaempferol and quercetin,), carotenoids and ascorbic acid, and from the extracts of Opuntia species. In all Opuntia species, quercetin was present in highest quantity. Kaempferol was present in red-skinned, green-skinned and purple-skinned, species and isorhamnetin exists in purple-skinned and green-skinned species. Mostly carotenoids (23.7 ?g/g fw) were present in yellow-skinned fruits and ascorbic acid (815 ?g/g fw) was present in red-skinned fruits. Purple-skinned fruit extract showed highest antioxidant activity as compared to other species supported result by total flavonoid content. The results showed that natural antioxidants are present in cactus fruits 65.
Cha et al (2013). investigated Korean cactus Opuntia humifusa (OH) fruit for the evaluation of chemical composition and antioxidant activity. Major minerals in the fruit were Mostly Ca, P, and Mg minerals were present in OH. 22.8% total dietary fiber was present and the ratio between soluble dietary fiber and insoluble dietary fiber was 1:1.3. For the evaluation of antioxidant activity of 80% ethanol extract, DPPH radical scavenging activity, flavonoid and total phenolic assays were used. Fractions of OH fruit extract obtained from 80% ethanol were made according to solvent’s polarity. By the comparison of all fractions, ethyl acetate fraction exhibited highest antioxidant activity, flavonoid and total phenolic contents then the other fractions. Phenolic acid in the form of ferulic acid was present in high quantity in ethyl acetate fraction, followed by protocatechuic acid and flavonoid in the form of taxifolin which were present in great quantity, followed by myricetin. It was clear from results that OH fruit showed high antioxidant activity66.

Yeddes et al (2013). studied peel and pulp of two Opuntia species Opuntia stricta and Opuntia ficus indica (thornless and spiny forms) in methanol extract to determine antioxidant activity. DPPH method was used to determined antioxidant activity. Folin–Ciocalteu and colorimetric methods were used for the determination of total flavonoid contents and total phenolic compound (TPC). For qualitatively and quantitative determination of phenolic compounds, high-performance liquid chromatography (HPLC) along with an electrospray ionization mass spectrometry (ESI-MS) were used. It was concluded from results that O. stricta fruits have high antioxidant activities as compared to O. ficus indica, whereas the TPC was in abundance in O. ficus indica as compared to O. stricta fruits. Flavonoids content were higher in peels as compared to pulp and higher in thornless species as compared to spiny 67.

Mahadkar et al (2013). determined antioxidant activity of five species from wild edible fruits like Oroxylum indicum (L.) Vent, Gmelina arborea Roxb, Bauhinia recemosa Lam. Zanthoxylum rhetsa (Roxb.) and Caryota urens L and DC from Kolhapur district, (India) by employing DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging activity, chelating activity on ferrous ion, ferric reducing antioxidant property(FRAP), total antioxidant capacity and reducing power ability. The range of total phenolic content from 0.061±0.29 (Zanthoxylum rhetsa ) to 0.500±0.012 g/100g FW(Caryota urens). These results showed that natural antioxidant could be used in food industry as well as in pharmaceutical industry 68.

Miletic et al (2014). investigated total phenolics and antioxidant capacity of dried fruits (plums, figs, apricots, grapes (amber dark and amber light), bilberries and chokeberries, and candied fruits (cherries, dates and cranberries) and compared their results. The order of increasing total phenolics contents of candied and dried fruits were given: candied dates, dried grapes (amber dark) ,dried figs, candied cranberries <dried grapes (amber light) < dried apricot< candied cherries < dried plums < dried bilberries <dried chokeberries. Antioxidant capacity presented the same trend as the total phenolics content. Phenolic compounds were determined by HPLC 69.

Ghazil et al (2015). investigated seeds oils and fruit juices of two Opuntia species (Opuntia dillenii and Opuntia ficus-indica) from Morocco for evaluation of antioxidant activity. Minerals identified in dry seeds of Opuntia ficus indica and Opuntia dillenii were given: potassium 304.51 and 201.96, calcium 480.93 and 408.28, sodium 48.33 and 18.18, phosphorus 1417.59 and 970.15 zinc 70.77 and 78.26 and magnesium 316.59 and 240.30 mg/100g respectively. Two tests (ascorbic acid and 2, 2-diphenyl-1- picrylhydrazyl (DPPH) radical-scavenging) were used for determination of antioxidant activity of fruit juices and seed oils of both fruits (Opuntia dillenii and Opuntia ficus- indica). It was concluded from results that antioxidant activities of both fruits (Opuntia dillenii and Opuntia ficus indica) and seed oil of (IC50 = 19.79 ± 0.023 and 27.21 ± 0.075 µL/mL) were greater than as compared to reference ascorbic acid (IC50 = 16.56 ± 0.019 µg/mL) 70.

Rahman et al (2015). analyzed freeze-dried and fresh mango for determining total phenolic content and the antioxidant activity. The antioxidant activity of sample was evaluated by DPPH assay and phenolic contents were evaluated by Folin-Ciocaltue method. They experienced that fresh green mango extract in methanol at concentration 50mg/ml has exhibited high scavenging activity (98.72 ± 0.88%) as compared to Green freeze dried mango (97.26 ± 1.8%) at 50mg/ml. They determinant total phenolic contents by taking sample’s absorbance at different concentrations and plotted their values against standard Gallic acid curve. Results revealed that phenolic contents were greater in fresh ripe mango 71.

Mabrouki et al (2015). studied two Opuntia species Opuntia streptacantha (OS) and Opuntia ficus-indica (OFI) to determine flavonoid and phenolic contents. temperature was maintained 4°C until determination of phenolic contents. Phenolics contents were evaluated by Folin-Ciocalteu method. Flavonoids evaluation depends on the synthesis of ?avonoid- aluminium complex. Antioxidant activity was determined by three assays: DPPH radical scavenging activity, ?-carotene bleaching (BCB) and the reducing power tests. More phenolics and flavonoids contents determined in O. streptacantha fruit extract as compared to O. ficus-indica. Extraction of OS fruit by using DPPH method BCB and the reducing power showed greatest activity with the lowest EC50 values (5.76 ± 0.46, 2.01 ± 0.03 and 2.93 ± 0.77 mg/ml, respectively 72.

Dantas et al (2015). Studied some cacti species are source of compounds like phenolics and Betalains which effect human health. In Northeastern Brazil the genus Opuntia has been highly cultivated. Purpose of this study was to determine betalain content and antioxidant activity of different fruits of Opuntia stricta, Opuntia ficus-indica and tacinga inamoena. The range of betaxanthin contents from 1.39 to 46.90 mg/100 g FW, T. inamoena and O. stricta, respectively. The betacyanin’s content for T. inamoena and O. ficus-indica pulp was given respectively 17.36 mg/100 g and 0.64 to 1.94 mg/L FW, and the maximum content presented by O. stricta of 182.27 mg/100 g FW. O. stricta fruits gave higher antioxidant activity with 1730.44 g pulp/g DPPH 73.

Alam et al (2015). studied Opuntia elatior aqueous extract for the evaluation of antioxidant activity. Methanol was used for the extraction of Opuntia elatior and then partitioned with petether, ethyl acetate, DMSO. Brine lethality bioassay shrimp was used to measure cytotoxic activity. In brine shrimp lethality test result showed LC50 value of Opuntia elatior aqueous extract was 12.5µg/ml. The fraction contained total reducing power content (30.5 mg) AAE/g, total phenolic content (12.315 mg) AAE/g, and total flavonoid content (56 mg) AAE/g. The results showed that aqueous extract have cytotoxic and poor antioxidant properties. It was clear from results that this plant is used in pharmaceutical industry 74.

Anwar et al (2016). improved quality of pan bread by using prickly pear peels. To make pan beard prickly pear peels powder was mixed with wheat flour (72 %) at levels 1.0 and 2.0%. Results exhibited that prickly pear peels had higher content of antioxidant components, pectin (14.25 %), ascorbic acid (87.82 %) and fiber (32.67 %). It also contains flavonoids (35.2) mg/100 g, total phenols (441.11 mg/100 g), and DPPH radical-scavenging (62.14%), oil holding capacity (2.35) ml oil/g, water holding capacity (1.8 ml) H2O / g , and foam stability (7.15%) 75.

Zou et al (2016). studied Citrus  fruit for determination of antioxidant activity. The antioxidant activity of Citrus fruits was determined by DPPH free radical assays and ABTS. Results suggested that antioxidant compounds were present in Citrus fruit and due to antioxidant activity it can be used for the cure of many chronic diseases 76.

Hwang et al (2016). analyzed three varieties of Opuntia (O. humifusa, O. humifusa f. jeollaensis, and O. ficus-indica (Baiknyuncho; OFI), for evaluation of antioxidant activity. Antioxidant activity of different plant parts was confirmed by using five assays in 70% methanol extract. The results were 13.32±2.47, 38.87±3.01, and 22.22±2.53 mg GAE/100g for total polyphenol contents of extracts of these fruits OH, OHJ and OFI respectively and the results for total flavonoid contents were 78.67±0.61, 98.11±0.58, and 2.61±0.13 mg QE/100g, respectively. The average IC50 values of OH, OHJ, and OFI alcoholic extracts against on DPPH radical scavenging activities, in testing 0.25, 0.125, 0.5, and 1 µg/ml concentrations in order, were 0.90, 0.85, and 1.18 µg/ml, respectively (ascorbic acid, 10.37 µg/ml). Results showed that O. humifusa f. jeollaensis has high contents of polyphenols and flavonoids than OH and OFI 77.

Sanjeevkumar et al (2017). studied a climber named as Bryonopsis laciniosa with red fruits for the determination of antioxidant activity. Chloroform extract of B. laciniosa fruits were used to evaluate in vitro antioxidant study. DPPH, H2O2, ABTS, and FRAP assays were used to determine antioxidant activity and chloroform extract was used for this purpose. All assays give positive results for antioxidant activity of chloroform extract. Due to the antioxidant activity this fruit can be used in food industry. It can improve health of consumer 78.

Chintu et al (2017). analyzed extract of Opuntia elatior fruit and quercetin in hydro alcohol for determination of antidiabetic and antioxidant activity. Carbohydrate, alkaloids, phenols, flavonoids, sterol, saponin, protein and tannins were present in Opuntia elatior according to phytochemical screening results. DPPH inhibition test was used for determining in-vitro antidiabetic and antioxidant activity. According to DPPH inhibition, the antioxidant activity of extract of Opuntia elatior fruit and quercetin in hydro-alcohol exhibited results as 38.14 ± 1.07 and 37.74 ± 1.06 % respectively. It was concluded from results that Opuntia elatior fruit and quercetin exhibited antioxidant and antidiabetic activities 79.

. 
CHAPTER: 3
MATERIALS AND METHODS:
3.1 Chemicals and Reagents:
These Chemicals and reagents were used in research work; methanol, ethanol, distilled water, n-Hexane, chloroform, acetone, sodium carbonate, FC-reagent, calcium chloride, sodium hydroxide, sodium nitrite, trichloroacetic acid, dihydrogen potassium phosphate, phosphoric acid, ?-Naphthol, ascorbic acid, catechin, gallic acid, hydrocholric acid, sulphuric acid, n-butanol, nitric acid, ammonium hydroxide, ammonia, ninhydrin, ferric chloride, potassium ferrycyanide, picric acid, DPPH, deionized water, olive oil, copper sulphate and glacial acetic acid. Chemicals and reagents used in research work were of analytical grade and obtained from E. Merck and Sigma Chemical Co. (St Louis, MO, USA).
3.2 Instrumentation and Apparatus:
UV-visible spectrophotometer (CECIL CE 7200), FTIR, Rotary evaporator (Heidolph Laborota 4002), Muffle furnace (Incu safe MCO-15AC), Centrifuge machine (Rotofix 32 A-Hettich Zentrifugen), pH meter (HI 2211), Analytical balance (Shimadzu Ay 220), HPLC system (Shimadzu 10A), FTIR spectrometer (Shimadzu), Orbital shaker, Microwave-assisted extraction assembly, Burette, Conical flasks, Volumetric flasks, Measuring cylinder, Beakers, Test tubes, Glass stirrer, Crucibles, Petri dish, Glass pipette, and Micropipette. Glassware used was made of pyrex.

3.3 Collection of fruit:
Fruit of the Opuntia engelmannii was collected from the surroundings of University of Sargodha, Sargodha. Then the plant sample was identified and authenticated by some taxonomist in Department of Botany, University of Sargodha, Sargodha.

Collected fruit sample was washed thoroughly with tap water and grinded to paste form and was stored in polythene at -4 C for further analysis
3.4 Preparation of Extracts:
Three extraction methods like maceration, microwave-assisted extraction (MAE), and orbital shaking were utilized for maximum recovery of bioactive compounds, among the solvents distilled water, methanol, acetone, chloroform, n-hexane and ethanol were employed for extraction. For each method sample was soaked into solvent in 1:5.

In maceration technique paste of sample was taken in a glass holder (cup) with known amount of solvent, the sample mixture was left for 48hours at ambient temperature. After this, the fluid was isolated from strong material (marc) by filtration through channel paper.
In orbital shaker, paste of sample and solvent was taken in volumetric flask then kept in orbital shaker assembly (an electrical device) for 6 h almost at 200 rpm. Then fluid was obtained by filtration process.

In microwave-assisted extraction (MAE), paste of sample and solvent was taken in volumetric flask then kept in microwave oven for 1 min. In this process fluid was obtained by filtration process. Each process was performed two times by re-extracting the residue. Fluid was evaporated by using rotary evaporator. The concentrated extract was weighed and stored in an air tight sample bottles. Then percentage yield was calculated.

Yield (%) = (amount of extract/ dry weight of sample) × 100 80.

3.5 Phytochemical Screening of Crude Extracts Procedure:
For the presence of bioactive compounds the fruit was tested by using standard procedures.

3.5.1 Phenolics:
One drop of ferric chloride solution (5%) was added in fruit extract (1ml). Formation of greenish precipitates was considered as indication of presence of phenolics.

3.5.2 Flavonoids:
Ammonium hydroxide solution (10%, 2 mL) was added in the fruit extract (3 ml). Intense yellow color was considered the signal that test results were positive.
3.5.3 Anthraquinones:
Mixture of extract (0.5 g) and sulphuric acid (10 mL) was filtered. Then the filtrate was treated with chloroform (5 mL) and chloroform layer was taken out in another test tube and remaining part was treated with dilute ammonia (1 mL). The result was considered positive by color change 81.

3.5.4 Carbohydrate:
Solvent extract (1ml) was added in water (10ml), shaken forcefully and then filtered. Molisch reagent (1ml) was treated with the filterate then some drops of conc. H2SO4 were added. Formation of two junctions is the indication of positive result of carbohydrates.

3.5.5 Amino acids:
Solvent extract (1ml) was added in Ninhydrin reagent (0.1ml, 1ml), after shaking violet color appeared which indicated the presence of free amino acids 82.

3.5.6 Sterol:
Chloroform (1ml) and concentrated H2SO4 (1ml) was added in solvent extract (1ml). After shaking 2 (chloroform and acid) layers formed, Chloroform layer appeared red while acid layer appeared greenish yellow in color. This result was considered positive for sterols.

3.5.7 Glycosides:
Glacial acetic acid (1ml), one drop of 5% FeCl3 and conc. H2SO4 (1ml) were added in solvent extract (2ml). Then junction formed between 2 liquid layers, upper layer of junction showed bluish green and junction showed reddish brown color which indicated the occurrence of glycosides 83.

3.5.8 Tannins:
When 45% solution of ethanol (5ml) was boiled with extract of fruit prepared in different solvents for 5 minutes. Then every mixture cooled and filtered. Then every filtrate (1ml) was diluted with distilled water and treated with ferric chloride (2 drops). Change in color from greenish to black indicated the occurrence of tannins.

3.5.9 Alkaloids:
Methanolic extract was treated with 2% H2SO4 and then warmed for 2 minutes. Then filtration occurred. Few drops of 1% picric acid were added to filtrate and yellow color was taken as evidence for the presence of alkaloids 84.

3.5.10 Phlobatannins:
When 1 % aqueous hydrochloric acid was boiled with an aqueous extract then deposition of a red precipitate was taken as indication for the phlobatannins.

3.5.11 Terpenoids:
When 1 ml of chloroform was added in 2.5ml of extract pepared in different solvents. A layer formed when treated with 1.5 ml of concentrated H2SO4. At the interface reddish brown precipitate appeared which was taken as evidence for the presence of terpenoids.

3.5.12 Saponins:
When 1 g powdered sample was boiled with 10ml of distilled water for 10 minutes. The boiled mixture was filtered and cooled. Then shake forcefully the mixture of 2.5 ml of filtrate and 10ml of distilled water for few minutes. Stable emulsion obtained when added 2 drops of olive oil to the solution. Emulsion was taken as evidence for the presence of saponins 85.

3.5.13 Proteins:
Solvent extract was treated with equal volume of 5% NaOH and 1% CuSO4 and then heated. Purple/blue color was taken an evidence for proteins.

3.5.14 Steroids:
Solvent extract was treated with acetic acid. 1 drop of H2SO4 was added after warming and cooling. Appearance of green color indicated the existence of steroids.

3.5.15 Caumarin:
Aqueous extract of 1ml was treated with 1.5 ml of 10% NaOH. Yellow color was taken as evidence for caumarin 86.

3.6 Proximate composition analysis:
The parameters used for determination of proximate analysis were moisture content, total ash, acid-insoluble ash, water-soluble ash, and pH.

pH:
pH determination of 1% aqueous solution of opuntia engelmannii fruit powder was carried out by digital pH meter.

Moisture Content determination:
After taking dried powdered sample (5 g) in a crucible was put in an oven at 110?C for 4 h. then removed crucible from oven and kept in a desiccator for some time and then weighed. The procedure was tried many times till constant weight was attained.

Moisture content was determined by using this formula:
Moisture content (%) = Loss in weight/ initial weight of sample x 100
Total Ash Content:
After taking dried powdered sample (5 g) in a crucible was put in furnace at 550?C till it became grey white. Appearance of grey white color indicated the oxidation of organic matter of the sample. Then removed crucible from furnace and kept in a desiccator for some time and then weighed. Determination of total ash content (mg) depends upon dried sample (g).

Water-Soluble Ash:
Total ash from crucible was mixed with deionized water (25 mL) and boiled for 5 min. After this mixture was filtered and insoluble matter was put in furnace for 15 min at 450?C. Then, crucible was kept in the desiccator for some time. Then weight of residue (mg) was abstracted from weight of total ash. Water-soluble ash content (mg) was calculated with reference to air-dried material (g).

This formula was used for calculating ash content:
Ash content (%) = Loss in weight (mg) W × 100
Here ‘W’ is weight of air-dried material in grams 81.

3.7 FT-IR analysis:
Fourier Transform Infrared (FT-IR) analysis (Shimadzu FT-IR Spectrometer) of Opuntia engelmannii fruit was carried out in Hi-Tec laboratory Pharmacy Department, University of Sargodha, Sargodha for the identification of different functional groups.

3.8 DPPH radical scavenging assay:
DPPH radical scavenging assay was utilized to evaluate antioxidant activity by slightly modified method of Zhuang Y. et al (2012) 87. Briefly, 80% solution of DPPH in methanol was made by dissolving DPPH (the 2, 2-diphenyl-1-picrylhydrazyl) 20g in 400ml methanol and 100ml water, and made 3 fractions such as (2ml of DPPH, 0.05ml extract and 0.95ml methanol), ( 2ml of DPPH,0.1ml extract and 0.9ml methanol), (2ml of DPPH, 0.2ml extract and 0.8ml methanol) and methanol was taken as blank. Each solution was left in dark for 30 min, after this absorbance was measured at 517nm spectrophotometerically (CECIL CE 7200). Decrease in the absorbance of DPPH solution was taken as indication of increase in DPPH radical scavenging activity.
3.9 Total carotenoids content:
Total carotenoids were evaluated by method described by Lichtenthaler (1987) 88. In 50ml 80% acetone solution, 5g powdered sample was added and put in refrigerator for 3days at 4 degree centigrade. Then filtration was carried out and filtrate centrifuged for 10-15 min at 2500 rpm and recorded its absorbance by using UV-Visible spectrophotometer at 663 nm, 646 nm and 470 nm respectively.
3.10 Total phenolic content:
The Total phenolic content of plant extracts was evaluated spectrophotometrically by Folin-Ciocalteu reagent. Firstly Prepared mixture of 0.2ml of extract, 0.8ml of diluted Folin-Ciocalteu reagent and 2ml of 7.5% sodium carbonate. The mixture was made up to 7ml with deionized water. Then mixture was incubated in oven at 32 degree centigrade. After that noted the absorbance at 765 nm, by using UV-Vis spectrophotometer. Gallic acid was used as standard and results were determined as Gallic acid equivalents (g/100g) of dry matter 89.

3.11 Total Flavonoid Content (TFC):
Total flavonoid content was determined spectrophotometrically by the method of Zhishen et al (1999) 90. Concisely, 1ml of fruit extract was taken in a sample vial and by using deionized water (4ml) made volume up to 5ml then treated with 0.3ml of sodium nitrite solution (5%). After this mixture was left for 5 min, then 0.3ml aluminium chloride (10%) was added. Again incubated for 6 min, then added 2ml of sodium hydroxide solution (1M) and made volume up to 10 ml with deionized water. By using spectrophotometer (CECIL CE 7200) absorbance was recorded at 510nm. TFC was expressed as catechin equivalents (mg CE/g dry sample).
3.12Total Tannin Content (TTC):
The Total Tannin content of plant extracts were assessed spectrophotometrically by Folin-Ciocalteu reagent by using the method of Tambe V.D. et al (2014) 91. 0.1ml of each sample was diluted with deionized water (7.5 mL) in the test tubes and 0.5ml of FC reagent was added. Then mixture left untouched for 15 min, then mixture was treated with 1ml of sodium carbonate solution (35 %) and made volume up to 10 ml by using deionized water. This mixture was again incubated for few minutes. . By using spectrophotometer (CECIL CE 7200) absorbance was recorded at 725nm. TTC was expressed as gallic acid equivalents (mg GAE/g of dry sample). 2
3.13 Ferric ion Reducing antioxidant Power (FRAP) Assay:
FRAP of plant extracts were determined by using the method of Zhuang Y. et al (2012) 87. Briefly, 10mg of extract (10 mg) was treated with 3.5ml of phosphate buffer (0.2 M; pH 6.6) then 2ml of potassium ferricyanide (1%) was added. And put for incubation for 20 min, then mixture was treated with 2.5ml of trichloroacetic acid (10%). After this mixture was centrifugated for 10 min at 3000 rpm, 2.5 ml of upper layer was taken in another test tube and treated with 2.5ml of deionized water and 0.5ml of ferric chloride solution (0.1%) . Absorbance was measured at 700 nm spectrophotometrically (CECIL CE 7200). Results were shown as ascorbic acid equivalent (mg AAE/g extract).
3.14 Phenolic acids determination by HPLC:
The sample solutions were obtained by mixing 25mg extract with 12ml of HPLC grade methanol. After this 8ml of deionized water and 5ml of hydrochloric acid (6 M) were added. Then kept for incubation for 2 h, at 95?C, then samples were passed through HPLC system (Shimadzu 10A), at a flow rate of 1 ml/min, and utilizing of 100 mm length of column (Shim-Pack CLC-ODS (C-18), 25 cm × 4.6 mm, 5 µm). The mobile phase contained H2O: acetic acid (94:6, pH 2.27) and acetonitrile (100%). Then recorded the individual absorption peaks of phenolic acids. Then compared the retention times of both peaks in sample chromatograms and standards, each phenolic acids were evaluated qualitatively and quantitatively by using the peak area of every phenolic acid. The quantity of each phenolic acids was expressed as mg/g extract.

CHAPTER: 4
RESULTS AND DISCUSSIONS:
Fruit extracts of Opuntia engelmannii prepared in six solvents (ethanol, methanol, distilled water, acetone, chloroform and n-hexane) by using three techniques (maceration, microwave oven and orbital shaker). Extracts were prepared by dissolving 15g sample paste in 75ml solvents and apply three techniques. In maceration technique maximum yield was given by ethanol then followed by methanol, distilled water, acetone, chloroform and minimum yield was given by n-hexane. It is showed that high yield was given by ethanol, methanol and distilled water due to high polarity of solvents. All the solvents which given less yield are used because they are less toxic. However, Distilled water is more polar as compare to other solvents given less yield then ethanol and methanol because of less compatibility between solute and solvent and acetone, chloroform and n-hexane given less yield due to less extracting ability. From all the three extraction technique the yield order was microwave oven > orbital shaker > maceration. Each technique has different principle that’s why each technique given different yield.
4.1 Phytochemical Analysis:
Opuntia engelmannii fruit extracts give positive results for bioactive compounds like phenolics, saponins, terpenoids, anthraquinones, cardiac glycosides, flavonoids, etc. Tannins are predicted used as anti-inflammatory and anticancer agent. Flavonoids are used as health improving agent. Preliminary study revealed its importance for the treatment of malaria, headache, cold, acne, and bacterial diseases. Phenolic compounds present in it used as antioxidants and phenolic compounds also used in some antimicrobial agents like cresol and dettol. The presence of saponins which act as cardioprotective agent and the occurrence of alkaloids play a role as anti-inflammatory agent and anti-hyperglycaemic 82.

4.2 Proximate Composition Analysis:
Moisture content at 110?C, total ash content showed their solidity used as herbal plant material. Less moisture content is required for prevention bacterial and yeast growth. The pH of fruit was in acidic region. Higher ash content indicated the existence of carbonate, phosphate, oxides, silicates, and silica material in higher amount 92.

4.3 FT-IR Analysis:
For determination of functional groups FT-IR analysis was used. The sample was analyzed at room temperature among 4000 to 400 cm-1 spectral range. FT-IR analysis of Opuntia engelmannii fruit given number of peaks among the region 3298.28 cm-1 , 3278.99 cm-1 , 3007.02cm-1 , 2927.94 cm-1 , 1712.79 cm-1 , 1616.35 cm- 1 , 1602.85 cm-1 , 1390.65 cm-1 , 1352.10 cm-1 , 1240.23 cm-1 , and 1056.99 cm-1 , 648.08 cm- 1 , 540.07 cm-1 93.

4.4 Determination of Total Phenolic Content (TPC):
The purpose of Phenolic compounds to work as antioxidants by reducing free radicals, scavenging singlet oxygen and chelating with metals. For determination of phenolic compounds in fruit extract (Opuntia engelmannii) Folin-Ciocalteu (FC) reagent was used. The formation of Folin-Ciocalteu (FC) reagent is the result of combination of phosphotungstic (H3PW12O40 ) and phosphomolybdic (H3PMo12O40) acids. When phenol oxidation takes place than it reduced from yellow to navy blue oxides of molybdene (Mo8O23) and tungsten (W8O23). Folin-Ciocalteu (FC) reagent reacts with penolic compounds in the presence of sodium carbonate. Follin-Ciocalteau reagent was non- specific and can reduce non-phenolic groups for example ascorbic acid and can cause interference in results. Change in color depend on concentration of phenolics and spectrophotometer was used to detect it at 765nm 94.

In maceration technique, maximum phenolic content was observed in ethanol and then decrease in the order, distilled water; methanol; acetone; chloroform; n-hexane. It was concluded from results that TPC increases by increasing solvent’s polarity. Difference in results of phenolic content’s determination in different solvents is due to soil conditions, growth and development, polarity of solute and solvent and solubility of phenolics in solvents 95. Phenolic compounds determined maximum in ethanol due to suitability of polarity between solvent and phenolic compounds. Ethanol has given highest value because of its ability to dissolve maximum amount of bioactives. n-hexane has given lowest value because of less suitability in polarity between n-hexane and phenolic compounds. In orbital shaker technique and in microwave assisted extraction same trend was obtained as in mecration technique. Ethanol was observed as a good solvent for good determination of phenolics in all techniques. Microwave assisted extraction was given good results as compare to orbital shaker and maceration extraction. Because of, microwave energy increase the temperature as a result secondary plant metabolites dissolve in solvent and increase the yield of extraction. TPC has given good results in all techniques. Results were compared with Mabrouki et al (2015) 72. Our results show variation with Mabrouki results who determined (TPC) of Opuntia streptacantha and Opuntia ficus indica fruits pulp.

4.5 Determination of Total Flavonoid Content (TFC):
Flavonoids are known as secondary polyphenols and mostly present in flowering tissues,leaves and in woody parts. These compounds work as antioxidants by reducing free radical and chelating with metals. The flavonoids which obtain from plants are work as natural antioxidants and also showed anti-mutagenic, anti-cancerogenic , cardioprotective, anti-inflammatory and antimicrobial activity.

Flavonoids comprises of 15-carbon skeleton having two benzene ring linked with one carbon heterocyclic pyrane ring contain C6-C3-C6 arrangement. Spectrophotometric technique preffered over other analytical techniques for determination of flavonoids content because only aluminium chloride form complex with flavonoids even in the presence of other phenolic compounds.

Spectrophotometer was used for determination of total flavonoid content in Opuntia engelmannii fruit extracts. Three methods was used for extraction maceration, orbital shaker, and microwave-assisted extraction (MAE) by using 6 solvents (ethanol, methanol, distilled water, acetone, chloroform and n-hexane) and results were expressed as catechin equivalent (CE). In maceration technique, maximum flavonoids content has given by methanol then remaining solvents extracts in this order, ethanol ; distilled water ; acetone ; chloroform ; n-hexane. These results are due to compatibility in polarity between solute and solvent 96. In orbital shaker extraction, maximum flavonoids content has given by ethanol then remaining all in the decreasing order as given distilled water ; methanol ; acetone ; n-hexane ; chloroform. In microwave-assisted extraction (MAE), maximum flavonoids content has given by ethanol and all other in this order methanol ; distilled water ; acetone ; chloroform ; n-hexane. All the techniques have given different results due to compatibility in polarity between solute and solvent. On comparing these three extraction techniques for determination of flavonoids, microwave-assisted extraction (MAE) showed good result as compare to orbital shaker and maceration extraction techniques due to microwave energy, internal heating phenomena occur and secondary plant metabolites mixes with solvent and increase the yield of extraction. Maceration showed poor result due to less compatibility between sample components and solvent. TFC has given good results in all techniques. Results were compared with Mabrouki et al (2015) 72. Our results show variation with Mabrouki results who determined (TFC) of Opuntia streptacantha and Opuntia ficus indica fruits pulp
4.6 Determination of Total Tannin Content (TTC):
Tannins are known as high molecular weight compounds (500-3000 g/mol) and have ability of binding with proteins. According to chemical nature, tannins are classified as hydrolysable and condensed tannins. Hydrolysable tannins are further categorized as:
Gallotannins: contain gallic acid subunits esterified to glucose.

Ellagitannins: polymers of ellagic acid and gallic acid.

It is named as Hydrolysable tannins because it can easily hydrolyze in the presence of acid and alkali. Condensed tannins also named as proanthocyanidins or “PACs” because on heating in the presence of acid gives anthocyanidin monomers 44.

They work as antioxidants and also as metal ion chelators. These are natural antioxidants, present in vegetables, fruits, and seeds. These are sometimes used in wine industry as color stabilizing agent. Spectrophotometer was used for determination of Tannin content. For determination of tannin contents in fruit extract (Opuntia engelmannii) Folin-Ciocalteu (FC) reagent was used.
In maceration technique, tannin content was evaluated maximum in ethanol and remaining in the following order distilled water ; methanol ; acetone ; chloroform ; n-hexane. The sequence in results was due to difference in polarities of tannin compounds present in Opuntia engelmannii fruit. The lowest yield given by n-hexane and chloroform was due to non-polar nature, because mostly bioactive compounds are polar in nature due to occurrence of many hydroxyl groups in their structure 97. Similar trend in results was observed in all extraction techniques. On comparision of all the three extraction techniques, microwave-assisted extraction (MAE) showed good results for the recovery of tannins then orbital shaker and maceration technique because each extraction technique work on distinctive principle.
4.7 Total carotenoids content:
Carotenoids founds in fruits and vegetables of more than 600 types. The structural formula of most of the carotenoids consists of 40-carbon polyene chain and is polyisoprenoid compounds. The reactive nature and specific molecular shape of carotenoids is due to resonance of electron over the polyene chain. Carotenoids work as antioxidants by scavenging free radicals, quenching singlet oxygen and provide protection from skin cancer 98. Spectrophotometer was used for determination of carotenoids content. Results were varied from the results of Thaipong et al (2006) 99. Who determined antioxidant activity of guava fruit.4.8 DPPH free radical scavenging assay:
The DPPH test is used for determining antioxidant compounds. This test is popular because of its sensitivity. The principle of this test is that antioxidants are Hydrogen donor. It measures radical scavenging activity of compounds. Antioxidant donates hydrogen to DPPH•. DPPH• is available as organic nitrogen radicals. Antioxidant activity is directly related to the disappearance of DPPH• in test samples. UV spectrometer was used for the determination of DPPH• because of its easiness and accuracy. Maximum absorption showed by DPPH• at 517 nm (purple). Color change from purple to yellow when DPPH absorbed hydrogen from an antioxidant and converts into diphenyl hydrazine. This reaction is stoichiometric depend upon the number of absorbed hydrogen atoms. So, antioxidant activity can be simply determined by decrease in UV absorption at 517 nm DPPH assay is considered simple faster and cheaper method to determine antioxidant potential 100.

The DPPH test is used for determination of antioxidant potential in Opuntia engelmannii fruit extract by employing three extraction techniques and six different solvents at three different concentrations. At concentration (0.2) by using maceration technique, percentage inhibiting potential of extracts was more prominent in chloroform and remaining all in the decreasing order n-hexane ; acetone ; ethanol ; methanol ; distilled water. Results showed that antioxidant activity depend upon concentration of extracted phenolics by different solvents.

RH(antioxidant)R?Hydrogen atom transfer and single electron transfer are involved in determining antioxidant activity, are influenced by solvent polarity. Distilled water showed lowest radical scavenging activity that may be due to the formation of intermolecular hydrogen bond between polar solvent and hydroxyl groups of antioxidant components, which create hindrance for free radicals to take hydrogen atom from antioxidant molecule, therefore decreasing the antioxidant activity.
In orbital shaker technique, percentage inhibition of extracts was highly determined in ethanol and remaining all in the following order distilled water ; chloroform ; methanol ; acetone ; n-hexane. In microwave extraction technique, percentage inhibiting potential of extracts was more pronounced in ethanol then followed by decreasing order of solvent extracts acetone ; n-hexane ; methanol ; distilled water ; chloroform.

Antioxidant potential was determined at concentration (0.1) by using all the techniques. In maceration technique highest radical scavenging activity by ethanol then remaining in the following order methanol ; distilled water ; acetone ; chloroform ; n-hexane. In orbital shaker technique highest radical scavenging activity was showed by distilled water then remaining in this order ethanol ; methanol ; acetone ; chloroform ; n-hexane. In microwave extraction technique, percentage inhibiting potential of extracts was more pronounced in ethanol then followed by methanol ; distilled water ; n-hexane ; acetone ; chloroform.

Antioxidant potential was also determined at concentration (0.05) by using all the techniques. In maceration technique highest radical scavenging activity by methanol then remaining in the following order ethanol ; distilled water ; chloroform ; n-hexane ; acetone. In orbital shaker technique highest radical scavenging activity was showed by distilled water then remaining in this order methanol ; ethanol ; chloroform ; n-hexane ; acetone. In microwave extraction technique, percentage inhibiting potential of extracts was more pronounced in distilled water then followed by ethanol ; acetone ; chloroform ; methanol ; n-hexane. Results showed that DPPH scavenging activity (%) was increased by increasing the concentration of extracts. Results were related to Ghazi et al (2015) 70. who determined antioxidant activity of Opuntia ficus-Indica and Opuntia dillenii.
The irregularity in results was due to the interference of non-antioxidants compounds which can be co-extracted with antioxidant compounds, which are solublize in different solvents and show different antioxidant potential 101. On comparing all extraction techniques, microwave-assisted extraction (MAE) showed highest percent inhibition potential, beyond other techniques that is due to the interferences of thermo-labile components in the reaction among antioxidant and free radical 102.
4.9 Ferric ion reducing antioxidant power assay:
Some scientist suggests that antioxidant activities of bioactive compounds are directly related with reducing power. Due to the occurrence of reducing components in plant extracts, ferric ions are reduced to ferrous ions by changing the color from yellow to bluish green. The transforming in color from yellow to bluish green related with reducing potential of sample’s compounds. Sharp bluish green color shows high absorption and reducing power. Iris and Strain in 1996 recognized the FRAP assay which measures the potential of extracts to reduce ferric ions 76. The complete evaluation was performed spectrophotometer was used for the determination of reducing power and absorbance was recorded at 700 nm. The difference between FC assay and FRAP assay is that pH less than 7 is suitable for FRAP assay and pH greater than 7 is suitable for FC assay 103.

In maceration technique, highest reducing potential was showed by distilled water then followed by decreasing order of solvent extracts, ethanol > methanol > acetone > chloroform > n-hexane. In orbital shaker technique, maximum reduction potential was showed by methanol then followed by order of different solvent extracts, distilled water > ethanol > acetone > chloroform > n-hexane. In microwave-assisted extraction (MAE), highest reducing potential was observed in distilled water then followed by decreasing order of different solvent extracts, ethanol > methanol > acetone > chloroform > n-hexane. From comparison all the extraction techniques, microwave-assisted extraction (MAE) showed higher reducing potential than orbital shaker and maceration techniques. It was concluded from results that all extracts prepared in different solvents and studied in different extraction techniques, showed significant reducing potential under acidic conditions. Results were varied from the results of Thaipong et al (2006) 99. Who determine antioxidant activity of guava.4.10 Quantification of Phenolic Acids by HPLC:
Quantification of Phenolic acids was determined by using HPLC. For recognition of different phenolic acids High performance liquid chromatography (HPLC) are used.

In the present study, HPLC analysis has been applied to separate and identify individual phenolic acids from Opuntia engelmannii fruit extracts.
Conclusion:
Opuntia engelmannii was analyzed for the determination of phenolic compounds and antioxidant potential. For the identification of phenolic compounds, three extraction techniques were used orbital shaker, microwave-assisted extraction and maceration. Six solvents was used for extraction are distilled water, methanol, ethanol, n-hexane, acetone and chloroform. From all the extraction techniques microwave-assisted extraction gave good results as compared to other techniques, in this order: microwave-assisted extraction > orbital shaker > maceration). In most cases, ethanol was considered good solvent then other solvents for recovery of phenolic compounds from Opuntia engelmannii fruit, and showed results in this order (ethanol > methanol > distilled water > acetone > chloroform > n-hexane). Different tests were performed for the determination of phenolic compounds. For the qualitatively determination of phenolic compounds, phytochemical tests such as salkowski, keller killani, biuret, molish test etc. were performed. For quantitatively determination of phenolic compounds, TPC, TFC, TTC, were performed which gave results spectrophotometrically. Folin-Ciocalteu (FC) reagent was used for the determination of Total phenolic content (TPC) and Total tannin content (TTC). Aluminum chloride colorimetric method was used for the determination of Total flavonoid content (TFC). Antioxidant potential was measured by performing different antioxidant capacity (AOC) evaluation assays like DPPH, FRAP and showed results spectrophotometrically . For the individual quantification of phenolic acids isolated from the fruit extract, HPLC was employed.
The results showed that extraction techniques and solvents effect the extraction and antioxidant potential of antioxidant components. It was concluded from results that Opuntia engelmannii is a potential source of antioxidants, which was confirmed by FTIR and HPLC analysis, which showed the presence of many natural antioxidants in matrix of this fruit.

Tables and Figures
Table 4.1: Preliminary Phytochemical Analysis of Opuntia engelmannii fruit Extract in different Extraction Media
Opuntia
engelmannii extract Phytoconstituent and test
Saponins
(Frothing test) Anthraquinones Terpenoids
(Salkowski test) Phlobatannins
(HCl test) Tannins
(1% FeCl3 solution) Cardiac glycosides
(Keller-Killiani test) Alkaloids
(1% picric acid)
Distilled
water (M) + _ + _ + _ +
(O) + _ + _ + _ +
(µ) + _ + _ + _ +
Ethanol
(M) + _ + _ + + +
(O) + _ + _ + + +
(µ) + _ + _ + + +
methanol
(M) + _ + _ + _ +
(O) + _ + _ + _ +
(µ) + _ + _ + _ +
Acetone
(M) + _ + _ + + +
(O) + _ + _ + + +
(µ) + _ + _ + + +
chloroform
(M) + _ + _ + + +
(O) + _ + _ + + +
(µ) + _ + _ + + +
n-hexane
(M) + _ + _ + + +
(O) + _ + _ + + +
(µ) + _ + _ + + +
Table 4.2: Preliminary Phytochemical Analysis of Opuntia engelmannii fruit Extract in different Extraction Media
Opuntia
engelmannii extract Phytoconstituent and test
Flavonoids
(NH4OH test)
Protein
(Biuret test) Carbohydrate
(Molish reagent test) Sterol
(Salkowski test) Phenolics
5% FeCl3 solution
Caumarin
(10% NaOH)
Amino acids
(Ninhydrin reagent’s)
Steroid
(Acetic acid)
Distilled
Water (M) + _ + + + + _ _
(O) + _ + + + + _ _
(µ) + _ + + + + _ _
ethanol
(M) + _ + + + + _ _
(O) + _ + + + + _ _
(µ) + _ + + + + _ _
methanol
(M) + _ + + + + _ __
(O) + _ + + + + _ _
(µ) + _ + + + + _ _
acetone
(M) + _ + + + + _ _
(O) + _ + + + + _ _
(µ) + _ + + + + _ _
chloroform
(M) + _ + _ + + _ _
(O) + _ + _ + + _ _
(µ) + _ + _ + + _ _
n-hexane
(M) + _ + _ + + _ _
(O) + _ + _ + + _
_
(µ) + _ + _ + + _
_
Proximate Composition Analysis of Opuntia engelmannii fruit
Experimental studies* Observation for Opuntia engelmannii fruit
Moisture content (% w/w) 24.65
Total ash (% w/w) 23.4583
Water-soluble ash (%w/w) 3.03
Ph 3.59
Peak
(cm-1 ) Intensity Group(s) Identified Vibrational Mode Remarks
3298.28 (w)
(m)
(s)
Carboxylic acid
2908309334400 AcO H, H-bonded
Amides
Secondary, free H-bonded
Alcohols and Phenols
4064007873900 O H polymer OH stretching
NH stretching
OH stretching Broad band
Amide I
Broad
3278.99 (w)
(m)
(s) Carboxylic acid
2908309334400 AcO H, H-bonded
Amides
Secondary, free H-bonded
Alcohols and Phenols
4064007873900 O H polymer OH stretching
NH stretching
OH stretching Broad band
Amide I
Broad
3007.02 (w)
Carboxylic acid
2908309334400 AcO H, H-bonded

OH stretching Broad band
2927.94 (w)
(w)
Aldehydes
Ac-H
Carboxylic acid
2908309334400 AcO H H-bonded
CH stretching
OH stretching Usually two bands
Broad band
1712.79 (s)
(s)
(s)
Ketones
Acyclic, sat (alkyl aryl)
Cyclic, 6-membered and higher
Aldehydes
Aromatic C=O stretching
C=O stretching
C=O stretching Modified by substituents
Modified by substituents
1616.35 (s)

(m) Amides
Primary, solid soln
Amines
Primary
Secondary
NH def
NH def Amide II
Aliphatic amines
Aromatic amines
1602.85 (v)
(v)
Imines
Azo compounds C=N stretching
N=N stretching 1390.68 (m) Alkanes
-CH3 Sym def 1352.10 (s)
(s)
Nitro compounds
Aliphatic
Amines
Tertiary
NO2 stretching
C-N stretching 1240.23 (s)
(s)
Esters
Ac-O
Ethers
Alkyl aryl
C-O stretching

Ar-O stretching 1056.99 (s)
Anhydrides
C-O-C, acyclic C-O stretching One or two bands
648.08 (m) Alkynes
?C-H C-H def Broad overtone near 1250 cm-1
540.07 (s) Halogen compounds
Bromo C-Br stretching Two or more bands in solution

Sr. No. Phenolic Acids Identified Quantity (mg/g) Extract
Detection and Quantification of Individual Phenolic acids by HPLC of Opuntia engelmannii fruit Extract prepared in methanol by maceration
Detection and Quantification of Individual Phenolic acids by HPLC of Opuntia engelmannii fruit Extract prepared in ethanol by maceration
Sr. No. Phenolic Acids Identified Quantity (mg/g) Extract
Detection and Quantification of Individual Phenolic acids by HPLC of Opuntia engelmannii fruit Extract prepared in distilled water by maceration
Sr. No. Phenolic Acids Identified Quantity (mg/g) Extract
Figure: 4.1 Percentage extraction yield of Opuntia engelmannii fruit

Figure: 4.2 Determination of total phenolic content from Opuntia engelmannii fruit extracts by applying different extraction techniques in different extraction media

Figure: 4.3 Determination of total flavonoids content from Opuntia engelmannii fruit extracts by applying different extraction techniques in different extraction media

Figure: 4.4 Determination of total tannin content from Opuntia engelmannii fruit extracts by applying different extraction techniques in different extraction media

Figure: 4.5 Determination of total carotenoids content from Opuntia engelmannii fruit

Figure: 4.6 DPPH Radical Scavenging Activity of Opuntia enelmannii fruit extract in concentration 0.1

Figure: 4.7 DPPH Radical Scavenging Activity of Opuntia enelmannii fruit extract in concentration 0.2

Figure: 4.8DPPH Radical Scavenging Activity of Opuntia enelmannii fruit extract in concentration 0.05

Figure: 4.9FRAP assay for Opuntia engelmannii fruit extract in different extraction media by using different extraction techniques

Figure 4.10 Phenolic Acids determination of Opuntia engelmannii fruit extract prepared in distilled water extracted by Maceration
yFigure 4.11 Phenolic Acids determination of Opuntia engelmannii fruit extract prepared in ethanol extracted by Maceration
xFigure 4.12 Phenolic Acids determination of Opuntia engelmannii fruit extract prepared in methanol extracted by Maceration
Figure 4.13 Standard curve of various concentration of Catechin for TFC

Figure 4.14 Standard curve of various concentration of Gallic acid for TPC

Figure 4.15 Standard curve of various concentration of Ascorbic acid

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