KWARASTATEUNIVERSITY

TheUniversityforCommunityDevelopmentandEntrepreneurship

BIOACTIVECONSTITUENTS,ANTIOXIDANTANDDIGESTIVE
ENZYMES INHIBITORY PROPERTIES OF DONKWA
FORMULATED WITH WHITE- SEEDED SORGHUM FLOUR

BY

FOLABOMIBARBARAOLADAPO
20/57BC/01340

AUGUST2024
BIOACTIVECONSTITUENTS,ANTIOXIDANTANDDIGESTIVE
ENZYMES INHIBITORY PROPERTIES OF DONKWA
FORMULATED WITH WHITE- SEEDED SORGHUM FLOUR

BY

FOLABOMIBARBARAOLADAPO
20/57BC/01340

SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY

FACULTYOFPUREANDAPPLIEDSCIENCES,KWARASTATE
UNIVERISTY, MALETE KWARA STATE IN PARTIAL
FULFILLMENTOFTHEREQUIREMENTFORTHEAWARDOF
BACHELOR OF SCIENCE ([Link]) IN BIOCHEMISTRY

AUGUST 2024

ii
iii
 CERTIFICATION

This is to certify that the research project titled “BIOACTIVE CONSTITUENTS,

ANTIOXIDANT AND DIGESTIVE ENZYMES INHIBITORY PROPERTIES OF

DONKWAFORMULATEDWITHWHITE-SEEDEDSORGHUMFLOUR”was

carried out by FOLABOMI BARBARA OLADAPO 20/57BC/01340. This report has

been read and approved as meeting the requirement for the award of Bachelor of Science

([Link]) Degree in the Department of Biochemistry, Facultyof Pure and Applied Sciences,

Kwara State University, Malete.

Dr.E.A. Irondi Date

(Supervisor)

Dr.E.A. Irondi Date

([Link])

ExternalSupervisor Date

iv
 ACKNOWLEDGEMENTS
I want to express my deep gratitude to Dr. E. A. Irondi, who served as both my
supervisor and the Acting Head of Department. His guidance, support, and constructive
feedback were crucial to the successful completion of this project. Dr.. Irondi’s deep
knowledge and unwavering dedication to his students provided me with the confidence
and direction needed to navigate through the challenges of this research.

Abigthank yougoesto myparents [Link] [Link] for

their constant support throughout my academic journey. Their encouragement has been a
strong foundation for my success. Their belief in my abilities and their constant
reassurance gave me the strength to persevere, even when faced with obstacles. Their
support has not only been emotional but also practical, and for this, I am profoundly
grateful.

I’m also grateful to Mr. Olaoluwa Oladapo, Omotola Oladapo, Ayotosimi Oladapo,
Oyin Adedeji, and Ojo Adetoro for their ongoing support and motivation. Each of you
has played a unique role in my life, providing the moral support and encouragement that
kept me focused and determined. Your belief in me has been a driving force behind my
efforts, and I am thankful to have you in my life.

A special thanks to my group members, Abolore and Omotola. Their collaboration and
hard work were vital to this project's success. Working alongside such dedicated
individuals has not only made the process more manageable but also more enjoyable. I
value the teamwork, late nights, and shared challenges we faced together, and I am
grateful for your contributions to this project.

Finally, I would like to extend my appreciation to everyone who contributed, in one way
or another, to the success of this project. Whether through direct involvement, offering
advice, or simply providing a listening ear, your support means a lot to me. This projectis
the culmination of not just my efforts, but of all the encouragement, advice, and help I
have received along the way. Thank you.

v
 DEDICATION
Idedicate this workto AlmightyGod.

vi
 TABLEOF CONTENT

FRONT PAGE.....................................................................................................................i
TITLEPAGE…...................................................................................................................ii
DECLARATION................................................................................................................iii
CERTIFICATION...............................................................................................................iv
ACKNOWLEDGEMENTS.................................................................................................v
DEDICATION....................................................................................................................vi
TABLEOF CONTENT......................................................................................................vii
LISTOFFIGURES...............................................................................................................x
LISTOFTABLES................................................................................................................xi
ABSTRACT.......................................................................................................................xii
CHAPTERONE...................................................................................................................1
INTRODUCTION......................................................................................................1
Backgroundtothestudy................................................................................................1
Problem Statement.....................................................................................................3
JustificationofStudy....................................................................................................4
Aim and Objective......................................................................................................4
CHAPTERTWO..................................................................................................................6
LITERATUREREVIEW............................................................................................6
Overviewof Donkwa..................................................................................................6
TraditionalPreparationMethods.........................................................................................7
Cultural Significance.........................................................................................................7
NutritionalProfileofTraditionalDonkwa............................................................................8
White-SeededSorghum...............................................................................................8
BotanicalDescription.........................................................................................................8
NutritionalComposition.....................................................................................................9
BioactiveCompounds inWhite-SeededSorghum...............................................................9
NutrientCompositionofCereal-BasedFoods.............................................................10

vii
 Macronutrients.................................................................................................................10
Micronutrients..................................................................................................................11
FiberContent....................................................................................................................11
BioactiveConstituentsinCereal-Based Foods...........................................................12
Phenolic Compounds.......................................................................................................12
Flavonoids........................................................................................................................12
Tannins............................................................................................................................13
PhyticAcid.......................................................................................................................13
BioactivitiesandHealthBenefits................................................................................13
AntioxidantActivity.........................................................................................................13
Anti-inflammatoryProperties...........................................................................................14
Antimicrobial Effects.......................................................................................................15
CardiovascularHealth Benefits........................................................................................16
AntioxidantPropertiesofCereal-BasedFoods............................................................17
Mechanisms of Antioxidant Activity...............................................................................17
Methods for AssessingAntioxidantActivity....................................................................18
AntioxidantProperties of Sorghum..................................................................................19
DigestiveEnzymeInhibition......................................................................................20
a-Amylase Inhibition.......................................................................................................20
a-Glucosidase Inhibition..................................................................................................21
LipaseInhibition.....................................................................................................22
Previous Studies.......................................................................................................23
CHAPTERTHREE............................................................................................................25
MATERIALSAND METHODS.....................................................................................25
SampleCollectionandPreparation.....................................................................................25
Preparationofsorghum flour.............................................................................................26
Formulationof Donkwa....................................................................................................26
BioactiveConstituentsIdentificationandQuantification....................................................29
Sample Extraction............................................................................................................29

viii
 Determinationoftotalphenolcontent.................................................................................29
DeterminationofTanninscontent......................................................................................29
Determinationoftotalflavonoidcontent.............................................................................30
BioactivityAssays............................................................................................................30
AntioxidantActivityDetermination..................................................................................30
2,2-Diphenyl-2-picrylhydrazylradical(DPPH*)scavengingassay....................................30
2,2-Azinobis(3-ethyl-benzothiazoline-6-sulfonicacid)radical
cation(ABTS*+)scavengingassay
31
Reducingpower assay......................................................................................................32
EnzymeInhibitoryAssay..................................................................................................32
Invertase&α-GlucosidaseTBA..............................................................................33
Lipase....................................................................................................................33
Statistical Analysis...........................................................................................................33
BioactiveConstituentsIdentificationandQuantification....................................................34
Sample Extraction............................................................................................................34
Determinationoftotalphenolcontent.................................................................................34
DeterminationofTanninscontent......................................................................................35
BioactivityAssays............................................................................................................35
AntioxidantActivityDetermination........................................................................35
2,2-Diphenyl-2-picrylhydrazylradical(DPPH*)scavengingassay....................................35
3.5StatisticalAnalysis.....................................................................................................36
CHAPTERFOUR...............................................................................................................37
RESULTS,DISCUSSION,CONCLUSIONAND RECOMMENDATION.....................37
Results.............................................................................................................................37
Discussion........................................................................................................................43
Conclusion.......................................................................................................................44
Recommendation.............................................................................................................45
REFERENCES...................................................................................................................46
ix
 LIST OF FIGURES

Figure3.1: Donkwa Sample..............................................................................................27

Figure3.2: Donkwa with Nutmeg.....................................................................................27

Figure3.3: Donkwa with pepper.......................................................................................27

x
 LIST OF TABLES

Table4.1:Bioactive componentof donkwa samples...........................................................35

Table4.2:Antioxidant Activities Of Donkwa sample.......................................................36

Table4.3:Enzymes Inhibitory Activitiesof the Donkwa samples......................................37

xi
 ABSTRACT

Donkwa is a traditional West African snack food made from sorghum and groundnuts.
There is growing interest in understanding thepotential health benefits ofsuch traditional
foods. This studyaimed to evaluate the bioactive constituents, antioxidant properties, and
digestive enzyme inhibitory effects of Donkwa formulated with white-seeded sorghum
flour. Donkwa samples were prepared using white sorghum flour with additions of
[Link] (phenolics, flavonoids, tannins) werequantified.
Antioxidant activity was assessed using DPPH, ABTS, and reducing power assays.
Inhibitory effects on α-amylase, α-glucosidase, and pancreatic lipase were determined.
The pepper and nutmeg-enhanced Donkwa samples showed significantlyhigher levels of
phenolics, flavonoids, and tannins compared to the control. Theyalso exhibited enhanced
antioxidant activities across all assays. Both modified samples demonstrated greater
inhibition of digestive enzymes, with the nutmeg sample showing the highest inhibitionof
α-glucosidase and pancreatic lipase. The addition of pepper and nutmeg to white
sorghum Donkwa enhances its bioactive profile, antioxidant capacity, and ability to
inhibit digestive enzymes. This suggests potential for developing functional foodproducts
with health-promoting properties based on this traditional snack.

xii
 1

CHAPTER ONE

INTRODUCTION

Background tothe study

Donkwa is a traditional West African snack food, particularlypopular in Ghana and parts

of Nigeria. It is typically made from a mixture of roasted maize and groundnuts(peanuts),

sometimes with the addition of other ingredients such as sugar or spices (Adeola and

Aworh, 2021). The ingredients are ground into a fine powder and then

formedintoballsorothershapes. Donkwaisnot onlyapopularsnackbut alsoserves as a quick

source of energy and nutrients, especially in rural areas (Quaye et al., 2019).

In recent years, there has been growing interest in improving the nutritional profile and

functional properties of traditional foods. This interest stems from the global focus on

foodsecurity,nutrition,andthepotentialhealthbenefitsofbioactivecompoundsfoundin plant-

basedfoods(Akinola etal., 2018).Sorghum,asthefifthmostimportantcerealcrop worldwide,

has garnered attention in this context (Xiong et al., 2019).

Sorghum (Sorghum bicolor L. Moench) is a drought-resistant crop that thrives in semi-

arid regions. It serves as a staple food for millions of people, particularly in Africa and

parts of Asia (Girard and Awika, 2020). Sorghum is known for its resilience to harsh

[Link]-seededsorghum,in
 2

particular, is preferred in many food applications due to its mild flavor and light color

(Pontieri et al., 2020). The incorporation of sorghum flour into traditional recipes like

Donkwa represents an opportunity to enhance the nutritional and functional properties of

thesefoods. Sorghum is rich in various nutrients,includingproteins, fibers, minerals, and

vitamins (Stefoska-Needham et al., 2015). Moreover, it contains an array of bioactive

compounds such as phenolic acids, flavonoids, and condensed tannins, which have been

associated with various health benefits (Girard et al., 2018).

Bioactive constituents in foods are compounds that have biological effects on human

health beyond basic nutrition. In sorghum, these include phenolic compounds, whichhave

been linked to antioxidant, anti-inflammatory, and anti-carcinogenic properties (Bai et

al., 2017). The antioxidant capacity of foods is of particular interest due to the role of

oxidative stress in various chronic diseases (Shahidi and Ambigaipalan, 2015).

Furthermore, certain compounds in foods can inhibit digestive enzymes, potentially

affecting the digestion and absorption of nutrients. While this might seem

counterintuitive, moderate inhibition of enzymes like α-amylase and α-glucosidase can

have beneficial effects on blood glucose levels, making such foods potentially useful in

the management of diabetes and obesity (Mkandawire et al., 2016).

Previous studies have explored the nutritional and functional properties of sorghum in

various food applications. However, there is limited research on its use in traditional

[Link],
 3

such as roasting and grinding, may affect the bioavailability and activity of various

compounds in sorghum (Brou et al., 2021).

The global trend towards healthier snack options also makes this research timely.

Consumers are increasingly seeking foods that not only satisfy hunger but also offer

additional health benefits (Asioli et al., 2017). If Donkwa formulated with white-seeded

sorghum flour shows enhanced nutritional and functional properties, it could potentially

be promoted as a healthier alternative to conventional snacks. From a broaderperspective,

this research aligns with several of the United Nations' Sustainable

DevelopmentGoals,includingZero Hunger (Goal2),Good HealthandWell-being(Goal 3),

and Responsible Consumption and Production (Goal 12) (United Nations, 2023). By

improving the nutritional quality of a traditional food using a drought-resistant crop, this

work could contribute to food security and sustainable food systems (Mabhaudhi et al.,

2019).

Problem Statement

Despite the potential nutritional and functional benefits of incorporating white-seeded

sorghum flour into traditional foods, there is a significant lack of comprehensive data on

the nutritional composition, bioactive constituents, antioxidant properties, and digestive

enzyme inhibitory effects of Donkwa formulated with white-seeded sorghum flour. This

knowledge gap hinders the potential for improving the nutritional quality of this

traditional snack and limits our understanding of its possible health benefits.
 4

Justification of Study

By investigating the bioactive constituents of Donkwa formulated with white-seeded

sorghum flour, this study could provide valuable insights into improving the nutritional

quality of a widely consumed traditional snack. This has potential implications for

addressing malnutrition and improving public health, particularly in regions where

Donkwa is a staple snack. The study of bioactive constituents and their antioxidant

properties could reveal potential health benefits associated with consuming this modified

Donkwa. This information could be crucial in promoting Donkwa as a functional food,

potentially increasing its value and appeal to health-conscious consumers. Sorghum is a

drought-resistant crop. By exploring its use in traditional foods like Donkwa, this study

couldcontributeto effortstodiversifyfood sourcesandimprove foodsecurity,especially in

regions prone to climate variability.

Aim and Objective

The overall aim of this study was to comprehensivelyevaluate the bioactive constituents,

antioxidant properties, and digestive enzyme inhibitory effects of Donkwa formulated

with white-seeded sorghum flour, with a view to understanding its potential nutritional

and functional benefits.

 5

TheSpecificObjectivesare:

1. toidentifyandquantifythebioactiveconstituentspresentinthesorghum-based Donkwa,

with a focus on phenolic compounds, flavonoids, and tannins.

2. tomeasuretheantioxidantactivityofthesorghum-basedDonkwausingmultiple assays

(e.g., DPPH, FRAP, ABTS).

 6

CHAPTER TWO

LITERATURE REVIEW

Overview of Donkwa

Donkwa, also known as "Danwake" or "Dankwa," is a traditional Nigerian snack

originating primarily from the northern regions of the country, particularly among the

Hausa people. This savory snack is made from a combination of roasted maize (corn)

flourand groundnut(peanut)•paste,oftenenhancedwithvariousspices. Itsuniqueflavor

profile and chewy texture make it a popular choice among both locals and visitors

Figure2.1: Donkwa(Kohnert, 2023).

 7

Traditional Preparation Methods

The preparation of Donkwa involves several key steps. The process begins with roasting

groundnuts and maize. The groundnuts are roasted until they are golden brown, and their

skins are then removed. The maize can be toasted in a dry pan until it reaches a light

golden color. After roasting, the groundnuts are ground into a fine paste. The maize is

alsogroundintoflour,oftenusingahigh-powered blenderoranindustrialmilltoachieve a fine

consistency (Ogunleye, Afölabi, and Adeyemi, 2022). The groundnut paste is then mixed

with the roasted maize flour. Additional ingredients such as sugar, cayennepepper, and

salt may be added to enhance the flavor. The mixture is kneaded until it reaches a dough-

like consistency(Adebayo, Akindele, and Ojo, 2021). The dough is then shaped into

small balls or logs, which are typically bite-sized. This step can be done by hand,

ensuring that each piece is compact and well-formed. Traditionally, the shaped Donkwa

may be left to dry slightly before consumption, which helps to firm up the texture

(Ogunleye et al., 2022).

Cultural Significance

Donkwa holds a significant place in the culinary traditions of northern Nigeria. It is not

only a popular street food but also a nostalgic snack that evokes childhood memories for

many. The snack is often sold by street vendors and is a common treat at local markets,

making it accessible to people of all ages (Kareem, 2022). The cultural importance of

Donkwaextendsbeyondmeresustenance;itisoftenassociatedwithcommunal
 8

gatherings and celebrations. Families and friends share this snack during festivals and

social events, reinforcing its role as a symbol of hospitality and togetherness (Kohnert,

2023). In addition to its local significance, Donkwa has gained recognition beyond

Nigeria,withvarious adaptationsandnames indifferent [Link] adaptability speaks to

its widespread appeal and the cultural exchanges that occur through food

Nutritional Profile of Traditional Donkwa

Donkwa is not only flavorful but also nutritious. The primary ingredients groundnuts and

majze-contribute to its health benefits. Groundnuts are rich in protein, healthy fats, and

essential vitamins and minerals, including vitamin E, magnesium, and potassium.

Groundnuts also provide dietaryfiber, which aids in digestion (Adebayo et al., 2021). As

a staple grain,maizeisagood source of carbohydrates, providingenergy. It also contains

fiber, which can help regulate blood sugar levels and promote digestive health (Ogunleye

et al., 2022). The addition of spices such as cayenne pepper not only enhances flavor but

also offers potential health benefits, including anti-inflammatory properties. Overall,

Donkwa serves as a filling snack that provides a balance of macronutrients, making it a

popular choice for those seeking a nutritious and satisfying treat.

White-Seeded Sorghum

Botanical Description
 9

White-seeded sorghum (Sorghum bicolor L. Moench) is a cereal grain belonging to the

Poaceaefamily. Itischaracterizedbyitswhitepericarp,whichdistinguishesitfromother

sorghum varieties. The plant typically grows to a height of 1.5-3 meters, with a strong

stem and broad [Link] grain is borne on a compact panicle at the top of the plant.

White-seeded sorghum is known for its drought tolerance and ability to thrive in diverse

agro-ecological conditions (Xiong et al., 2019).

Nutritional Composition

White-seededsorghumis anutrient-dense grain withacompositionthatvariesdepending on

cultivar and growing conditions. Generally, it contains about 70-80% carbohydrates,8-

12% protein, and 2-5% fat (Girard and Awika, 2018). The protein in white sorghum is

notably higher in leucine compared to other cereals, making it valuable for muscleprotein

synthesis (Pontieri et al., 2020). In terms of micronutrients, white sorghum is a

[Link] Patil(2015)reportedsignificantlevels of

iron (4.4 mg/100g), zinc (1.9 mg/100g), and phosphorus (352 mg/100g) in white

sorghum varieties. The grain also contains B-complex vitamins, particularly niacin and

vitamin B6 (Xiong et al., 2019).

Bioactive Compoundsin White-Seeded Sorghum

White-seeded sorghum contains various bioactive compounds, albeit in lower

[Link]
 10

bioactive constituents, including phenolic acids and flavonoids. Girard and Awika (2018)

identified ferulic acid as the predominant phenolic acid in white sorghum, with

concentrations ranging from 100-200 ug/g.

Flavonoids, particularly flavones and flavanones, are also present in white sorghum.

Luteolin and apigenin derivatives have been reported in white sorghum varieties,

contributing to their antioxidant properties (de Morais Cardoso et al., 2017). While

tannins are typically associated with darker sorghum varieties, some white sorghum

cultivars may contain low levels of condensed tannins, which can contribute to their

antioxidant activity and potential health benefits (Xiong et al., 2019).

Nutrient Composition of Cereal-BasedFoods

Macro nutrients

Cereal-based foods, including those made from sorghum, are primarily sources of

carbohydrates. In a comprehensive review, Awika (2017) noted that cereal-based foods

typically contain 60-75% carbohydrates, mainly in the form of starch. This makes them

important energy sources in many diets globally. Protein content in cereal-based foods

varies depending on the grain used. Sorghum-based foods generally contain 7-12%

protein, which is comparable to other cereal grains. However, the protein quality can be

limitingdue to lower levels of some essential amino acids, particularlylysine (Girard and

Awika,2018).Thefatcontentinmostcereal-basedfoodsisrelativelylow,typically
 11

ranging from 2-5%. However, this can increase significantly in products that incorporate

oilseeds or have added fats during processing (de Morais Cardoso et al., 2017).

Micro nutrients

Cereal-based foods contribute significantly to micronutrient intake in many diets. Iron,

zinc, and B-vitamins are among the most important micronutrients provided by these

foods. Hariprasanna and Patil (2015) reported that sorghum-based foods could contribute

25-30% of the recommended daily intake for iron and zinc in a typical serving.

Fortification has become a common strategy to enhance the micronutrient content of

cereal-based foods. For instance, Delimont et al. (2017) demonstrated that iron-fortified

sorghum-based foods could effectively improve iron status in populations at risk of

deficiency.

Fiber Content

Cereal-based foods are important sources of dietary fiber. Whole grain cereals, including

whole sorghum, typically contain 10-15% total dietary fiber (Awika, 2017). This fiber

content contributes to the health benefits associated with cereal consumption, including

improved digestive health and reduced risk of chronic diseases. Sorghum-based foods

have gained attention for their relatively high resistant starch content. Girard and Awika

(2018) reported that sorghum foods could contain up to 5% resistant starch, which

[Link]
 12

incorporation of white-seeded sorghum into traditional foods like Donkwa presents an

opportunitytoenhancethenutrient profileand potential health benefitsof these products.

The unique composition of white sorghum, including its bioactive compounds and fiber

content, could contribute to the development of functional foods with improved

nutritional value.

Bioactive Constituentsin Cereal-Based Foods

PhenolicCompounds

Phenoliccompoundsareadiversegroupofphytochemicalspresentin cereal-basedfoods.

These such as ferulic acid, p-coumaric acid, and caffeic acid, are commonly found in

cereals. These compounds are primarily located in the bran and germ layers of the grain

(de Morais Cardoso et al., 2017).

Flavonoids

Flavonoids are another important group of bioactive compounds in cereal-based foods.

They include flavones, flavanones, and anthocyanins. In cereals, flavonoids contribute to

the antioxidant activity and may provide health benefits such as reducing inflammation

and protecting against chronic diseases. White-seeded sorghum, although lower in

flavonoids than pigmented varieties, still contains significant amounts of these

compounds (Xiong et al., 2019).

 13

Tannins

Tannins are polyphenolic compounds that are more prevalent in pigmented cereals.

However, some white-seeded sorghum varieties may contain low levels of condensed

tannins. Tannins have astringent properties and are known for their antioxidant activity.

They can also affect protein digestibility and mineral absorption (Girard and Awika,

2018).

PhyticAcid

Phyticacidisa storageform of phosphorus incereals. Itisknownforitsabilitytochelate

minerals, which can reduce their bioavailability. However, phytic acid also has

antioxidant properties and may have health benefits such as reducing the risk of cancer

and kidney stones. The balance between its antinutritional and beneficial effects is a

subject of ongoing research (de Morais Cardoso et al., 2017).

Bioactivities and Health Benefits

Antioxidant Activity

Antioxidant activity is one of the most well-documented bioactivities of cereal-based

foods. Antioxidants neutralize free radicals, which can damage cells and lead to chronic

diseases. Thephenoliccompounds, flavonoids, and otherbioactive constituents in cereals

[Link]-rich
 14

foods is associated wấth reduced oxidative stress and lower risk of diseases such as

cardiovascular diseases and cancer (Awika, 2017). Studies have highlighted the specific

contributions of different phenolic compounds to the antioxidant activity of cereals. For

instance, research by Kim et al. 2016) demonstrated that ferulic acid, a predominant

phenolic acid in cereals, exhibitsstrongfree radical scavengingactivityand protects cells

from oxidative damage. Additionally, Zhang et al. (2018) found that the flavonoids

quercetin and kaempferol, present in cereals like oats and barley, significantly enhance

antioxidant defenses in human cells. Moreover, epidemiological studies have linked high

antioxidant intake from whole grains to improved health outcomes. For example, a study

by Jacobs et al. (2015) showed that individuals who consumed high amounts of whole

grains had a lower incidence of cardiovascular diseases and cancer. The study suggested

that the cumulative antioxidant effect of whole grains played a crucial role in these

protective effects.

Anti-inflammatory Properties

Cereal-based foods also exhibit anti-inflammatory properties. Chronic inflammation is a

key factor in the development of many diseases, including heart disease, diabetes, and

cancer. The phenolic compounds in cereals can modulate inflammatory pathways and

reduce the production of pro-inflammatory cytokines. This makes cereal-based foods

beneficial for managing inflammation and maintaining overall health (Girard and Awika,

2018).Recentresearchhasfocusedonthespecificanti-inflammatorymechanismsof
 15

different cereal-derived phenolic compounds. For example, Amadou et al. (2016) found

that the phenolic extracts from sorghum significantly inhibited the production of pro-

inflammatory cytokines such as TNF-a and IL-6 in human cells. Similarly, de Morais

Cardoso et al. (2017) reported that flavonoids in millet modulated the NF-kB signaling

pathway, which plays a critical role in inflammation. Additionally, clinical studies have

demonstrated the anti-inflammatory benefits of whole grain consumption. A randomized

controlled trial by Tovar et al. (2019) showed that participants who consumed wholegrain

sorghum for 12 weeks had significantly lower levels of inflammatory markers compared

to those who consumed refined grains. This study highlighted the potential of whole

grains in reducing systemic inflammation and promoting overall health.

Antimicrobial Effects

Certain bioactive compounds in cereals, particularly phenolic acids and tannins, have

antimicrobial properties. These compounds can inhibit the growth of pathogenicbacteria,

fungi, and viruses. This antimicrobial activity contributes to the food safety and

preservation qualities of cereals and may have implications for preventing foodborne

illnesses (Xiong et al., 2019).

Studies have explored the antimicrobial mechanisms of cereal-based bioactive

compounds. For example, Shao ét al. (2018) found that the phenolic extracts from quinoa

exhihited strong antibacterial activity against common foodborne pathogens such as

[Link]
 16

compounds disrupt bacterial cell membranes and inhibit enzyme activity, leading to

bacterial death.

In addition to antibacterial properties, cereals also exhibit antifungal and antiviral

activities.

Research by Kang et al. (2016) demonstrated that the tannins in black rice bran inhibited

the growth of Aspergillus flavus, a common foodborne fungus. Furthermore, a study by

Nsimba et al. (2018) reported that flavonoids in sorghum exhibited antiviral activity

against the influenza virus, suggesting potential applications in preventing viralinfections.

Cardiovascular Health Benefits

Cereal-based foods have been linked to cardiovascular health benefits. The high fiber

content in whole grains, including sorghum, can help lower cholesterol levels and

improve heart health. The bioactive compounds in cereals also contribute to thesebenefits

by reducing inflammation and oxidative stress, which are risk factors for cardiovascular

diseases. Regular consumption of whole grains is associated with a lower risk of heart

disease and stroke (Hariprasanna and Patil, 2015).

Recent meta-analyses and clinical studies have provided robust evidence for the

cardiovascular benefits of whole grains. For instance, a meta-analysis by Aune et al.

(2016)showedthathigherintakeofwholegrainswasassociatedwitha22%lowerrisk
 17

of cardiovascular disease. The study highlighted the combined effects of fiber,

antioxidants, and anti-inflammatory compounds in whole grains in reducing

cardiovascular risk.

Moreover, specific studies have explored the cholesterol-lowering effects of different

[Link].(2018)demonstratedthatbarleybeta-glucansignificantly

reduced [Link],astudyby Liu

et al. (2020) found that oat consumption improved lipid profiles and reduced blood

pressure in individuals with hypertension.

Antioxidant Propertiesof Cereal-Based Foods

Mechanisms of AntioxidantActivity

The antioxidant activity of cereal-based foods is primarily attributed to their phenolic

compounds. These compounds can donate hydrogen atoms or electrons to neutralize free

radicals. They also chelate metal ions, preventing them from catalyzing the formation of

reactive oxygen species. The antioxidant mechanisms of cereals involve directscavenging

of free radicals, inhibition of oxidative enzymes, and upregulation of antioxidant defenses

in the body (de Morais Cardoso et al., 2017). Recent studies have

[Link]
 18

instance, Kim et al. (2016) showed that ferulic acid and its derivatives could chelate iron

and copper ions, reducing their catalytic activityin generating free radicals. Additionally,

Zhangetal.(2018)foundthatquercetinandkaempferolcouldpregulatetheexpressionof

antioxidant enzymes such as superoxide dismutase (SOD) and catalase, enhancing the

body's endogenous antioxidant defenses. Moreover, research by Feng et al. (2021)

highlighted that the synergistic effects of different phenolic compounds in cereals

contribute to their overall antioxidant capacity. The study demonstrated thatcombinations

of phenolic acids and flavonoids exhibited stronger antioxidant activity compared to

individual compounds, suggesting the importance of consuming whole grains to

maximize antioxidant benefits.

Methods forAssessing AntioxidantActivity

Several methods are used to assess the antioxidant activity of cereal-based foods. These

include assays such as the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging

assay, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation

decolorization assay, and the FRAP (ferric reducing antioxidant power) assay. These

methods measure the ability of cereal extracts to neutralize free radicals or reduce

[Link],suchasORAC(oxygenradicalabsorbance
 19

capacity) and TEAC (Trolox equivalent antioxidant capacity), are also commonly used

(Awika, 2017).

Recent advancements in analytical techniques have improved the accuracy and reliability

of antioxidant assays. For example, a study by Wang et al. (2018) employed high-

performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) to

quantifyindividualphenoliccompoundsandtheirantioxidantactivitiesindifferentcereal

extracts. This approach provided detailed insights into the specific contributions of

different phenolics to the overall antioxidant capacity. Furthermore, research byXu et al.

(2019) utilized electron spin resonance (ESR) spectroscopy to directly measure the free

radical scavenging activity of cereal extracts. This technique offered realtime monitoring

of antioxidant reactions and provided a more comprehensive understanding of the

antioxidant mechanisms.

Antioxidant Properties ofSorghum

Sorghum is particularly noted for its high antioxidant activity, which is comparable to or

even higher than that of other cereals. This is largely due to its phenolic content,

especially in pigmented varieties. However, white-seeded sorghum also exhibits

significant [Link] acids,flavonoids,and tanninsinsorghum

[Link]-basedfoodscanhelp
 20

reduce oxidative stress and protect against chronic diseases (Girard and Awika, 2018).

Recent studies have focused on the antioxidant properties of different sorghum varieties.

For instance, research by Kang et al. 2016) found that black and red sorghum varieties

had higher total phenolic content and stronger antioxidant activity compared to white

sorghum. However, white sorghum still exhibited substantial antioxidant capacity,

making it a valuable component of a healthy diet.

Additionally, a studybyShao et al. (2018) investigated the effects of different processing

methods on the antioxidant properties of sorghum. The study showed that fermentation

and sprouting significantly enhanced the antioxidant activity of sorghum by increasingthe

bioavailability of phenolic compounds. These findings suggest that processing techniques

can be optimized to maximize the health benefits of sorghum.

Digestive Enzyme Inhibition

a-Amylase Inhibition

Cereal-based foodscaninhibitdigestiveenzymessuchasa-amylase,which isresponsible

forbreakingdownstarches into sugars duringdigestion. Inhibition of a-amylase can slow

down carbohydrate digestion and absorption, leading to lower postprandial blood sugar

[Link]
 21

diabetes. Certain phenolic compounds in cereals, includingsorghum, have been shown to

inhibit a-amylase activity (Xiong et al., 2019). Recent studies have provided more

insights into the mechanisms by which these compounds inhibit a-amylase. For example,

Saleh et al. (2015) demonstrated that the interaction between phenolic compounds and a-

amylase involves the formation of enzyme-phenolic complexes, which reduce enzyme

activity. Furthermore, research by Amadou et al. (2016) showed that sorghum varieties

with higher phenolic content exhibited stronger a-amylase inhibition. These finding

ssuggestthat selectingandbreedingsorghumwith higherphenoliccontent couldenhance its

functional food properties. In addition to sorghum, other cereals such as barley and oats

have also been studied for their a-amylase inhibitory effects. A study by Wang et al.

(2018) reported that the phenolic extracts from barley exhibited significant a-amylase

inhibition, which was positively correlated with their total phenolic content. Similarly,

research by Zhang et al. (2020) found that oats contain avenanthramides, a unique group

ofphenolic compounds that effectivelyinhibit a-amylase activity. Thesestudieshighlight

the potential of various cereals as sources of natural a-amylase inhibitors for managing

diabetes.

a-Glucosidase Inhibition

Similar to a-amylase, a-glucosidase is an enzyme that breaks down carbohydrates into

glucose. Inhibiting a-glucosidase can also help regulate blood sugar levels. The phenolic

compoundsandflavonoidsin cereal-basedfoodshavebeenfoundto inhibit a-glucosidase

 22

activity. This makes cereals potential functional foods for managing diabetes and

preventing spikes in blood sugar levels after meals (de Morais Cardoso et al., 2017).

Further studies have explored the efficacy of different cereal varieties in inhibiting a-

glucosidase. For instance, Kang et al. (2016) investigated the a-glucosidase inhibitory

activity of various colored rice varieties and found that red and black rice had higher

inhibitory effects compared to whité rice. The study attributed this difference to thehigher

anthocyanin content in colored rice, which are known to be potent inhibitors of a-

glucosidase. In addition, research by Shao et al. (2018) examined the a-glucosidase

inhibitory activity of millet and found that its phenolic extracts significantly inhibited the

enzyme. The study also highlighted that the inhibitory activity was influenced by the

extraction method, with ethanol extracts showing higher activity compared to water

extracts. This suggests that the bioavailability and effectiveness of phenolic compoundsin

cereals can be optimized through proper processing techniques. Another study by Liu et

al. (2020) focused on the a-glucosidase inhibitory activity of quinoa. The results showed

that quinoa's phenolic extracts exhibited moderate to strong inhibition of a- glucosidase,

and the activity was positively correlated with the total phenolic and flavonoid content.

These findings support the inclusion of various cereals in the diet as functional foods for

managing blood sugar levels.

Lipase Inhibition
 23

Lipase is an enzyme that breaks down fats into fatty acids and glycerol. Inhibition of

lipase can reduce the digestion and absorption of dietary fats, which may help in

managing obesityand related metabolic disorders. Some bioactive compounds in cereals,

particularly tannins, have been shown to inhibit lipase activity. This property adds to the

potential health benefits of cereal-based foods in weight management (Awika, 2017).

Additionally, a study by Xu et al. 2019) examined the lipase inhibitory activity of barley

and found that its phenolic extracts significantly inhibited the enzyme. The study

highlighted that the inhibitory activity was dose-dependent and suggested that regular

consumption of barley could contribute to weight management and obesity prevention.

Furthermore, research by Feng et al. (2021) investigated the lipase inhibitory activity of

oats and found that its phenolic extracts exhibited moderate to strong inhibition, which

was positively correlated with the total phenolic and flavonoid content.

Previous Studies

Previous studies on the nutritional and bioactive properties of cereal-based foods have

highlighted their potential health benefits. Research has demonstrated that regular

consumption of whole grains, including sorghum, is associated with reduced risk of

chronicdiseasessuchascardiovasculardiseases,diabetes,[Link]
 24

content, antioxidant activity, and enzyme inhibitory properties of cereals contribute to

these benefits. Fortification and processing techniques can further enhance the nutritional

value and health-promoting properties of cereal-based foods (Girard and Awika, 2018).

For instance, a studyby Mellen et al. (2015) found that individuals who consumed whole

grains, including sorghum, had a lower risk of cardiovascular diseases compared to those

who consumed refined grains. The study attributed this benefit to the high fiber and

antioxidant content of whole grains, which help reduce cholesterol levels and improve

hearthealth. Similarly, research byReynolds et al. (2016)demonstrated that a diet rich in

whole grains was associated with a lower risk of type 2 diabetes, which was linked to the

grains' ability to improve blood sugar control and reduce insulin resistance.

In addition, a meta-analysis by Aune et al. (2016) highlighted that higher intake of whole

grains, including sorghum, was associated with a reduced risk of colorectal cancer. The

study suggested that the fiber and bioactive compounds in whole grains play a protective

role by promoting healthy gut microbiota and reducing inflammation. Furthermore,

research by Seal and Brownlee (2015) emphasized the importance of processing

techniques in enhancing the health benefits of cereals. The study found that milling and

fermentation processes can increase the bioavailability of nutrients and bioactive

compounds, making them more effective in promoting health.

Another study by Poutanen et al. (2018) explored the potential of fortifying cereals with

additional nutrients to enhance their health benefits. The research demonstrated that

fortifyingsorghumwithironandzincsignificantlyimprovedthenutritionalstatusof
 25

individuals with deficiencies in these micronutrients. Similarly, research by Hurrell and

Egli (2019) showed that fortification of cereals with folic acid and vitamin B12 helped

reduce the risk of neural tube defects and other birth

defects. Overall, these studies highlight the significant potential of cereal-based foods,

including those made from white-seeded sorghum and traditional snacks like Donkwa, in

improving health and preventing chronic diseases. The rich composition of

macronutrients, micronutrients, and bioactive compounds makes them valuable

components of a healthy diet. Further research and development of cereal-based

functional foods can enhance their potential to improve health and prevent chronic

diseases.

CHAPTER THREE

MATERIALS AND METHODS

Sample Collection and Preparation

 26

White-seeded sorghum (Sorghum bicolor L. Moench) was obtained from a local

agricultural market in Ilorin. Other ingredients required for Donkwa preparation were

purchased from reputable local suppliers to ensure freshness and quality.

Preparation of sorghum flour

The white-seeded sorghum grains were cleaned thoroughly to remove debris, dust, and

[Link] driedinahot air oven

at 50°C for 24 hours. The dried grains were ground into fine flour using a laboratorymill

(specify model and manufacturer). The resultingflour was sieved through a 250 um mesh

toobtaina uniform particle size. The sorghum flour was stored in airtight containers at

room temperature until further use.

Formulation of Donkwa

The preparation process involved the followingsteps:

1. Roasting the groundnuts and grinding them intoapaste.

2. Mixing the ground groundnut paste with the prepared sorghum flour

3. Adding sugar and other ingredients according to the recipe.

4. Kneading the mixture thoroughly to achieve a homogeneous dough.

 27

5. Shaping the dough into smallballs or the traditional Donkwa shape.

The prepared Donkwa samples were allowed to cool to room temperature and stored

inairtight containers for further analysis

 28

Figure3.1: DonkwaSample 1 Figure3.2: DonkwawithNutmeg

F
 29

Figure3.3: Donkwawithpepper

Bioactive Constituents Identification and Quantification

Sample Extraction

One gram of sample was extracted by steeping in 20 mL of methanol for 24 h, afterwhich

it was filtered through Whatman No. 1 filter paper. The filtrate, subsequently referred to

as extract, was later used for phytochemical and antioxidant activity assays.

Determination of total phenolcontent

The total phenol content of the extracts was determined according to the Folin–Ciocalteu

method reported byElemosho et al. (2021).Briefly, 300 µLofextract was dispensed into

test tube (in triplicates). To this was added 1.5 ml of Folin–Ciocalteu reagent (diluted 10

times with distilled water), followed by 1.2 ml of Na 2CO3 solution (7.5% w/v). The

reaction mixture was shaken, allowed to stand for 30 min at room temperature before the

absorbance was measured at 765 nm against a blank prepared by dispensing 300 µL of

distilled instead of sample extract. Total phenol content was expressed as gallic acid

equivalent (GAE) in mg/g material.

Determination of Tannins content

TotaltanninwasdeterminedfollowingthemethodreportedbyKareemetal(2023).A

0.5gportionofthesampleflourwassoakedfor15minin5mLofacidifiedmethanol
 30

(1% HCl in methanol), after which the mixture was vortexed and centrifuged for a period

of 10 min at 3,000 g. Afterwards, 0.1mL of the supernatant, distilled water (7.5 mL),

Folin-Dennis reagent (0.5 mL), and Na2CO3 solution (1 mL) were sequentially addedand

mixed. The volume of the reaction mixture was diluted to 10mL by adding distilled water

(0.9 mL). At 760 nm, the absorbance measurement was conducted following 30 min of

incubation at room temperature. The tannin content of the flours was calculated using a

tannic acid calibration curve and presented as tannic acid equivalent (TAE) in mg/g.

Determination of total flavonoid content

Total flavonoid content was determined using the method reported by Kareem et al.

(2023). 0.5 ml of the extract was dispensed into test tube, followed by 1.5 ml ofmethanol,

0.1 ml of aluminum chloride (10%), 0.1 ml of 1M potassium acetateand 2.8ml of distilled

water. The reaction mixture was shaken, allowed to stand at room temperature for 30

minutes, before absorbance was read at 514 nm. Total flavonoid content was expressed as

quercetin equivalent (QE) in mg/g material.

Bioactivity Assays

Antioxidant Activity Determination

2,2-Diphenyl-2-picrylhydrazylradical(DPPH*)scavenging assay
 31

The extract’s ability to scavenge DPPH* was determined as per the method reported by

Kareem et al. (2022), using ascorbic acid as a positive control. In brief, a

reactionmixturecontaining1.0 mLof the different concentrations of the extract (or

ascorbic acid) and 3.0 mL of DPPH* solution (60 µM) was incubated at room

temperature in the darkfor 30 min. Then, the absorbance was measured at 517 nm in a

UV-Visible spectrophotometer, and percentage DPPH * scavenging ability of the extract

was calculated.

Note: A reference test, containing 1000 μL of methanol (instead of the extract) and 3000

μL of 60 µM DPPH*solution, is included in the assay.

2,2-Azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation (ABTS* +)

scavenging assay

The extract’s ability to scavenge ABTS* + was determined using the method reported by

Irondi et al (2022), and expressed in Trolox equivalent antioxidant capacity (TEAC).

ABTS*+working reagent was prepared by thoroughly mixing equal volume of aqueous

solutions of ABTS*+(7 millimoles/liter) and K2S2O8 (2.45 millimoles/liter), and

incubating it in the dark at room temperature for 16 h. Afterwards, reagent’s absorbance

was adjusted to 0.70 ± 0.02 with ethanol (95%) at 734 nm. Then, a reaction mixture

containing 2.0 mL of the ABTS*+reagent and 0.2 mL of the extract was incubated at

roomtemperatureinthedarkfor15min,afterwhichtheabsorbancewasmeasuredina
 32

UV-Visible spectrophotometer at 734 nm. ABTS* +scavenging ability of the extract was

later calculated from Trolox standard curve.

Reducing powerassay

The extracts’ ability to reduce FeCl 3 solution was tested as per the method described by

Irondi et al (2022). In brief, 2.5 mL aliquot of extract was mixed with 2.5 mLof 200 mM

sodiumphosphatebuffer (pH6.6)and2.5mLof1%[Link]

mixture was incubated at 50 oC for 20 min. and then 2.5 mL of 10% trichloroacetic acid

was added. This mixture was divided into aliquots of 2.5 mLin different test tubes, and each

portion was diluted with 2.5 mL of dist. H 2O. Next, 1 mL of 0.1% ferric chloride was added

to each tube. The absorbance was measured at 700 nm, and the reducing power of the

extracts was subsequently calculated using gallic acid standard curve.

Enzyme Inhibitory Assay

α-Amylase Procedure

Different dilutions of extract were dispensed into test tubes. Phosphate buffer was added

to make the volume up to 500μl. 500μl of α-amylase solution was then added. The

mixture was incubated at 37°C for 10 minutes. Following this, 500μl of 1% starch

solution was added, and the mixture was incubated again at 37°C for 15 minutes.
 33

The reaction was stopped by adding 1.0ml of DNSA reagent. The reaction was then

incubated for 5 minutes in a boiling water bath. After this, it was cooled to room

temperature. The solution was then diluted with 10ml of distilled H2O. Finally, the

absorbance reading was taken at 540nm.

Invertase& α-Glucosidase TBA

50μl of α-glucosidase and 50μl of extract were dispensed and subjected to incubation for

10 minutes at 37°C. 100μl of sucrose was then dispensed into the reaction mixture to

initiate the hydrolytic reaction. This hydrolytic reaction was allowed to proceed for 20

minutes at 37°C. To terminate the reaction, 2ml of Na2CO3 (0.1M) was added. The

absorbance readings were then taken at 400nm.

Lipase

5μl and 20μl of extract were dispensed, and the volume was made up to 100μl using Tris

HCl buffer. 300μl of the enzyme solution was added and mixed properly. The reaction

mixturewasincubatedat 37°[Link],20μlof10mP-nitrophenyl

butyrate substrate was added and incubated at 37°C, with proper mixing.

The incubation was continued at 37°C for another 20 minutes. Finally, the absorbance

was measured at 405nm.

Statistical Analysis
 34

Data were analyzed using appropriate statistical methods, including ANOVA and

regression analysis, to determine significant differences between groups. A significance

level of p < 0.05 was set for all tests.

Bioactive Constituents Identification and Quantification

Sample Extraction

One gram of sample was extracted by steeping in 20 mL of methanol for 24 h, afterwhich

it was filtered through Whatman No. 1 filter paper. The filtrate, subsequently referred to

as extract, was later used for phytochemical and antioxidant activity assays.

Determination of total phenol content

The total phenol content of the extracts was determined according to the Folin-Ciocalteu

method reported by Elemosho et al. (2021). Briefly, 300 ML of extract was dispensedinto

test tube (intriplicates). To this was added 1.5 ml of Folin-Ciocalteu reagent (diluted 10

times with distilled water), followed by 1.2 ml of NaCOs solution (7.5% w/v). The

reaction mixture was shaken, allowed to stand for 30 min at room temperature before the

absorbance was measured at 765 nm against a blank prepared by dispensing 300 uL of

distilled instead of sample extract. Total phenol content was expressed as gallic acid

equivalent (GAE) in mg/g material.

 35

Determination of Tannins content

TotaltanninwasdeterminedfollowingthemethodreportedbyKareemetal(2023).A

0.5 g portión of the sample flour was soaked for 15 min in 5mL of acidified methanol(1%

HCl in methanol), after which the mixture was vortexed and centrifuged for a period of

10 min at 3,000 g. Afterwards, 0.1mL of the supernatant, distilled water (7.5 mL), Folin-

Dennisreagent(0.5mL.),andNaCO3solution(1mL)weresequentiallyaddedand mixed.

Thevolume of the reaction mixturewas diluted to 10mLbyadding distilled water (0.9 mL).

At 760 nm, the absorbance measurement was conducted following 30 min of incubation

at room temperature. The tannin content of the flours was calculated using a tannic acid

calibration curve and presented as tannic acid equivalent (TAE) in mg/g.

Bioactivity Assays

AntioxidantActivityDetermination

2,2-Diphenyl-2-picrylhydrazylradical(DPPH*)scavenging assay

The extract's ability to scavenge DPPH* was determined as per the method reported by

Kareemetal.(2022),[Link],areactionmixture containing

1.0 mLof the different concentrations of the extract (or ascorbic acid) and 3.0

mLofDPPH*solution (60M)wasincubatedatroomtemperatureinthedarkfor30min. Then, the

absorbance was measured at 517 nm in a UV-Visible spectrophotometer, and percentage

DPPH* scavenging ability of the extract was calculated.

 36

Note: A reference test, containing 1000 L of methanol (instead of the extract) and 3000

uL of 60 HM DPPH* solution, is included in the assay.

3.5Statistical Analysis

Data were analyzed using appropriate statistical methods, including ANOVA and

regression analysis, to determine significant differences between groups. A significance

level of p < 0.05 was set for all tests.

 37

CHAPTER FOUR

RESULTS,DISCUSSION,CONCLUSIONAND RECOMMENDATION

Results

Table4.1:The Bioactive composition of the sample

TheBioactivecomposition ofthesampleispresented inTable 4.1

SAMPLES TOTAL TOTALFLAVONOIDS TANNINS

PHENOLICS
(mg/g) (mg/g)

(mg/g)

WSD-CONTROL 3.05±0.05a 1.33±0.01a 1.67±0.19a

WSD-NUTMEG 4.06±0.35ab 1.54±0.01b 2.32±0.04b

WSD-PEPPR 4.66±0.03b 1.37±0.01a 2.44±0.18

 38

KEY:WSD-contol: white sorghum donkwa control sample; WSD-Nutmeg: white

sorghumdonkwa-Nutmegsample;WSD-pepper:whitesorghumdonkwa-pepper sample

Total Phenolics(mg/g)

The results show a significant variation in total phenolic content among the donkwa

samples. The WSD-PEPPER sample exhibited the highest phenolic content (4.66±0.03

mg/g), followed by WSD-NUTMEG (4.06±0.35 mg/g), with the control sample having

the lowest (3.05±0.05 mg/g). This suggests that the addition of pepper and Nutmeg

significantly increased the phenolic content of the donkwa. Phenolic compounds are

known for their antioxidant properties, indicating that the modified donkwa samples may

have enhanced health benefits compared to the control.

Total Flavonoids(mg/g)

The flavonoid content showed less variation compared to phenolics. The WSD-

NUTMEG sample had the highest flavonoid content (1.54±0.01 mg/g), while the control

and pepper samples had similarlevels (1.33±0.01 and 1.37±0.01 mg/grespectively).This

suggests that Nutmeg is particularly rich in flavonoids, which could contribute to

increased antioxidant activity and potential health benefits in the donkwa.

Tannins (mg/g)
 39

Tannin content followed a similar trend to total phenolics, with WSD-PEPPER havingthe

highest content (2.44±0.18 mg/g), followed by WSD-NUTMEG (2.32±0.04 mg/g), and

the control having the lowest (1.67±0.19 mg/g). Tannins can contribute toastringency and

may have some health benefits, but high levels can also interfere with nutrient absorption.

Table4.2:AntioxidantActivitiesofDonkwasample

TheAntioxidant ActivitiesofDonkwasample ispresented inTable4.2

SAMPLES REDUCINGPOWER DPPH* ABTS*+

(mg/GAE/g) SC50(µg/mL) (TEACµM/gdw)

WSD- 6.79±0.64a 260.40±190.00a 10.99±0.18a

CONTROL

WSD-NUTMEG 10.01±0.16b 40.64±9.03a 11.31±0.25ab

WSD-PEPPER 7.74±0.64a 23.40±7.78a 11.87±0.06ab

KEY:WSD-contol:whitesorghumdonkwacontrolsample;WSD-Nutmeg:whitesir donkwa

Nutmeg sample :WSD-pepper: white sorghum donkwa -pepper sample

ReducingPower(mg/GAE/g)
 40

The reducing power, an indicator of antioxidant capacity, was highest in the WSD-

NUTMEG sample (10.01±0.16 mg/GAE/g), significantly higher than both the pepper

sample(7.74±0.64mg/GAE/g)andthecontrol(6.79±0.64mg/GAE/g).Thissuggeststhat

Nutmeg significantly enhances the antioxidant capacity of the donkwa.

DPPHSC50(µg/mL)

The DPPH radical scavenging activity, measured as SC 50 (lower values indicate higher

antioxidant activity), showed that both modified samples had much higher antioxidant

activity than the control. WSD-PEPPER had the lowest SC 50 (23.40±7.78 µg/mL),

followed closely by WSD-NUTMEG (40.64±9.03 µg/mL), while the control had a much

higher SC50 (260.40±190.00 µg/mL). This indicates that both pepper and Nutmeg

significantly enhance the donkwa's ability to scavenge free radicals.

ABTS+(TEACµM/gdw)

The ABTS radical scavenging activity showed less variation among samples, but still

indicated enhanced antioxidant activity in the modified donkwa. WSD-PEPPER had the

highest activity (11.87±0.06 TEAC µM/gdw), followed by WSD-NUTMEG (11.31±0.25

TEAC µM/gdw), and the control (10.99±0.18 TEAC µM/gdw).

 41

Table4.3:EnzymesInhibitoryActivitiesoftheDonkwasamples

TheEnzymesInhibitoryActivitiesofthe Donkwa samplesispresentedinTable4.3

SAMPLES α-AMYLASE α–GLUCOSIDASEIC50 PANCREATIC

IC50(µg/m L) LIPASE IC50

(µg/mL)

(µg/mL)

WSD- 108.90±0.14c 787.29±0.23c 219.06±0.56c

CONTROL

WSD- 93.85±0.21b 717.56±0.62a 155.96±0.06a

NUTMEG

WSD- 90.28±0.25a 761.40±0.50b 199.69±0.28b

PEPPER
 42

KEY:WSD-contol: white sorghumdonkwacontrol sample;WSD-Nutmeg: white

sorghumdonkwa-Nutmegsample:WSD-pepper:whitesorghumdonkwa-pepper sample

α-AmylaseIC50(µg/mL)

All samples showed α-amylaseinhibitoryactivity, with WSD-PEPPERhavingthe lowest

IC50 (90.28±0.25 µg/mL), indicating the highest inhibitory activity. WSD-NUTMEG

(93.85±0.21 µg/mL) also showed significantly higher inhibition than the control

(108.90±0.14 µg/mL). This suggests that both pepper and Nutmeg enhance the potential

of donkwa to moderate carbohydrate digestion, which could be beneficial for blood sugar

control.

α-GlucosidaseIC50(µg/mL)

Interestingly, WSD-NUTMEG showed the highest α-glucosidase inhibition (lowest IC 50

of 717.56±0.62 µg/mL), followed by WSD-PEPPER (761.40±0.50 µg/mL), with the

control having the least inhibition (787.29±0.23 µg/mL). This further supports the

potential of the modified donkwa samples in managing postprandial glucose levels.

PancreaticLipaseIC50(µg/mL)

All samples showed pancreatic lipase inhibitory activity, with WSD-NUTMEG having

the highest inhibition (lowest IC50 of 155.96±0.06 µg/mL), followed by WSD-PEPPER

(199.69±0.28µg/mL),andthecontrol(219.06±0.56µg/mL).Thissuggeststhatboth
 43

Nutmeg and pepper enhance the potential of donkwa to moderate fat digestion, which

could be beneficial for weight management and lipid profile improvement.

Discussion

This study investigated the bioactive composition, antioxidant activities, and enzyme

inhibitory properties of donkwa samples prepared with white sorghum flour

[Link]

the control and modified samples across various parameters.

The addition of nutmeg and pepper to the donkwa samples resulted in increased levels of

total phenolics, flavonoids, and tannins compared to the control. This aligns withprevious

studies that have shown spices like nutmeg and pepper to be rich sources of phenolic

compounds (Zachariah et al., 2010). The pepper-enhanced sample showed the highest

phenolic and tannin content, while the nutmeg sample had the highest flavonoid content.

This suggests that different spices can contribute unique profiles of bioactive compounds

to food products.
 44

All three antioxidant assays (reducing power, DPPH, and ABTS) demonstrated enhanced

antioxidant capacity in the modified donkwa samples compared to the control. This is

consistent with previous research showing that the addition of spices can significantly

improve the antioxidant properties of food products (Embuscado, 2015). The nutmeg

sample showed the highest reducing power, while the pepper sample exhibited the

strongest DPPH and ABTS radical scavenging activities. This variation in antioxidant

activities across different assays highlights the importance of using multiple methods to

assess antioxidant capacity comprehensively.

The modified donkwa samples showed improved inhibition of α-amylase, α-glucosidase,

and pancreatic lipase compared to the control. This finding is particularly interesting as it

suggests potential applications in managing blood glucose levels and lipid metabolism.

Previous studies have reported similar enzyme inhibitory effects for various spices and

herbs(SakulnarmratandKonczak,2012),butthecurrentstudydemonstratestheseeffects

inatraditionalfoodproductenhancedwithcommonspices. Thepepper-enhancedsample

showed the highest α-amylase inhibition, while the nutmeg sample exhibited thestrongest

inhibition of α-glucosidase and pancreatic lipase. This differential inhibition pattern

suggests that combining various spices could potentially provide a broader spectrum of

enzyme inhibitory effects.

Conclusion
 45

The analysis of the donkwa samples reveals that they are rich in bioactive components

with significant antioxidant and enzyme inhibitory activities. The WSD-pepper and

WSD-Nutmeg samples, in particular, show promise as functional foods that couldprovide

health benefits. The variations in bioactive compounds and their corresponding activities

highlight the importance of selecting appropriate formulations for enhancing health

outcomes. Future research should focus on in vivo studies to further elucidate the health

benefits of these donkwa samples and their potential applications in functionalfood

[Link] expanded discussion provides a more comprehensive analysis of the

results, emphasizing the significance of the findings in the context of health and nutrition.

Recommendation

Food manufacturers and small-scale producers should consider incorporatingnutmeg and

pepper into donkwa recipes. This simple modification could significantly enhance the

nutritional profile and potential health benefits of the product. Further research could

explore optimal ratios of these spices to maximize bioactive content and antioxidant

properties while maintaining consumer acceptability.

Nutritionists and dietitians should consider recommending spice-enhanced donkwa aspart

of a balanced diet, particularly for individuals managing blood glucose levels or seeking

to increase their antioxidant intake. However, it's important to conduct human trials to

confirm the in vivo effects before making specific health claims.

 46

Additional studies should be carried out to investigate the bioavailabilityand metabolism

of the bioactive compounds in these enhanced donkwa samples. This would provide a

more complete picture of how these compounds are absorbed and utilized by the body.

Moreover, research into the effects of different processing methods on the bioactive

compounds and enzyme inhibitory properties would be valuable.

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