UNIVERSITY OF NIGERIA, NSUKKA

FACULTY OF BIOLOGICAL SCIENCE

DEPARTMENT OF PLANT SCIENCE AND BIOTECHNOLOGY

TOPIC: NUTRITIONAL COMPOSITION of Mallotus subulatus LEAF

A RESEARCH PROJECT SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENT FOR THE AWARD OF BACHELOR OF SCIENCE
(B.SC.) DEGREE IN PLANT SCIENCE AND BIOTECHNOLOGY

BY

OGBONNA, PRECIOUS UGOCHUKWU

REG. NO: 2018/242248

SUPERVISOR: MR. EZE, N.M.

OCTOBER , 2023.

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 TITLE PAGE

NUTRITIONAL COMPOSITION of M. subulatus LEAF.

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 DEDICATION

I would like to dedicate this work to my family. I am very much appreciative,

thankful and blessed to have the ultimate support from my family. Lastly, I
dedicate this work to God almighty for his guidance and strength.

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 ACKNOWLEDGEMENT

I wish to extend my sincere gratitude to my project guide, Mr. Eze, N.M., for his valuable
guidance and encouragement which has been absolutely helpful in successful completion of
this seminar.

I am grateful to my family and friends for their timely aid without which i would not have
finished my seminar successfully. I extend my thanks to all my well-wishers and all those
who have contributed directly and indirectly for the completion of this work.

And last but not least; I thank God for his blessings without which the completion of this
seminar would not have been possible.

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TABLE OF CONTENTS

TITLE PAGE

APPROVAL PAGE

DEDICATION

ACKNOWLEDGEMENT

LIST OF TABLES

LIST OF PLATES

ABSTRACT

CHAPTER ONE

1.0. Introduction

1.1 Background of the study

1.2 Statement of the problem

1.3 Justification of study

1.4 Aim and objectives

CHAPTER TWO

2.0 Literature review

2.1 Botanical description of Mallotus subulatus

2.2. Ethnomedicinal uses of Mallotus subulatus

2.3. Modern scientific research on the medicinal properties of Mallotus subulatus

2.4. Distribution of Mallotus subulatus

2.5. Factors affecting the distribution of Mallotus subulatus

2.6. Conservation and management of Mallotus subulatus populations

2.7. Ecological requirements of Mallotus subulatus

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2.8. Role of Mallotus subulatus in Ecosystems

2.9. Phytochemistry of Mallotus subulatus

2.9.1. Flavonoids

2.9.2. Alkaloids

2.9.3. Phenols

2.9.4. Tannins

2.10. Nutritional Composition of Mallotus subulatus Leaves

2.10.1. Vitamins

2.10.2. Minerals

2.10.3. Dietary fiber

2.11. Pharmacological activities of Mallotus subulatus

2.11.1 Antimicrobial and Antioxidant Activities of Mallotus subulatus

2.11.2. Antiinflammatory and Cytotoxic Activities of Mallotus subulatus

2.12. Methods of administration and safety considerations for Mallotus subulatus

2.13. Key studies on the biological activities of Mallotus subulatus

2.14. Potential applications of Mallotus subulatus in medicine and industry

CHAPTER THREE

3.0. Materials and Methods

3.1. Plant collection

3.2. Sample preparation

3.3. Extraction

3.4. Proximate composition analysis

3.4.1. Determination of ash content

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3.4.2 Determination of crude protein content

3.4.3 Determination of crude fat content (Soxhlet method)

3.4.4 Determination of crude fiber

3.4.5 Determination of moisture content

3.5 Mineral analysis

3.5.1. Determination of Calcium Content

3.5.2. Determination of Potassium and Sodium Content

3.5.3. Determination of Magnesium

3.5.4. Determination of Phosphorus

3.6 Vitamin analysis

3.6.1. Determination of Vitamin C Content

3.6.2 Determination of Beta Carotene

3.6.3 Determination of Vitamin E Content

3.6.4. Determination of Vitamin B6

CHAPTER FOUR

4.0. Result

4.1. Proximate analysis

4.2. Mineral content analysis

4.3. Vitamin constituent analysis

CHAPTER FIVE

5.0. Discussion

5.1. Conclusion

REFERENCES

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 ABSTRACT

Mallotus subulatus is a shrub or tree that belongs to the genus Mallotus and the
Euphorbiaceae family of spurges that is native to tropical Africa and Asia.

Traditional medicine is relied upon by 80% of the world's population. Mallotus

subulatus is a plant that has been used as a traditional medicine in a number of
traditional medical systems. The nutritional composition of the leaves
of M.subulatus was investigated in this study. Proximate analysis revealed that
the leaves are a good source of protein, fiber, and minerals. The average
proximate composition of Mallotus subulatus leaves on a dry basis is as
follows: moisture 9.290.05%, protein 14.38 0.04%, fat 4.68 0.04%,
fiber 13.450.41%, ash 8.56 0.04%, and carbohydrates 49.66 0.26%. The leaves
are also a good source of vitamins and minerals, including vitamin A, vitamin
C, calcium, iron, and potassium. The mineral content of the leaves is as follows,
per 100 grams of dried leaves: calcium 194.73 0.38mg, iron 13.06 0.19mg,
potassium 432.49 0.05mg, magnesium 383.69 0.27 mg,
phosphorus 76.79 0.45mg, and sodium 22.11 0.24 mg. The vitamin content of
the leaves is as follows, per 100 grams of dried leaves: vitamin C
8.28 0.04mg, vitamin A 12.39 0.03mg, vitamin E 3.84 0.18 mg and vitamin
B6 0.22 0.01 mg. The leaves are a particularly good source of calcium, iron, and
potassium. This study aims to provide a comprehensive understanding of the
nutritional value of this plant, which can be used to inform dietary
recommendations and potential uses in the food industry. The plant has
cytotoxic and anti-inflammatory characteristics, which point to possible medical
uses. The results of this study highlight the importance of promoting and
investing in research on underutilized crops to address issues of food security
and nutrition.

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 Chapter one

Introduction

1.1 Background of the study

Medicinal plant species are used to alleviate and treat a myriad of ailments in most countries
of the world (Chen et al., 2016). It has been reported that 80% of the world’s population are
completely reliant on traditional medicine for their primary health care (Chen et al., 2016).

Plant serve various functions such as sources of food, phytochemicals, dietary supplements,
and crude drugs (Onyeka et al., 2021). Phyto-nutrients including Proteins, Fibre, Vitamins,
and various specialized functional additives will remain the top-selling group of nutraceutical
ingredients globally due to their health benefits (Onyeka et al., 2021). Proteins, carbohydrates
and vitamins remain the most important nutraceutical for food and beverage industries
throughout the world due to their leading role in solving problems of malnutrition (Onyeka et
al., 2021). A nutraceutical is a food or part of a food that provides medical or health benefits
including the prevention and/or treatment of disease.

Secondary metabolites in plants are the key drivers of the resulting biological activities and
drug - like properties associated with several medicinal plants (Adetunji et al., 2022). These
phytochemicals have become valuable resources for developing new drugs (Yuan et al.,
2016). For example, quinine, which was the first drug for the treatment of malaria, and
quinidine, a drug for cardiac arrhythmias were originally derived from Cinchona officinalis
(Gutiérrez-Grijalva et al., 2020). The synthetic drugs digitoxin and digoxin used in treating
atrial fibrillation and heart failure were derived from the foxglove plant, Digitalis purpurea
(Whayne, 2018). Herbal formulations with phytochemicals possess an excellent alternative to
orthodox drugs in the treatment of cancer and other diseases because of the fact that plant has
synthesized in nature more nutrients and therapeutic moiety than man has ever synthesized
(Onyeka et al., 2021).

The genus Mallotus is a large genus of shrubs and trees and belongs to the spurge family
(Euphorbiaceae) (Gangwar et al., 2014). The genus comprises c. 150 species occurring in
many parts of tropical and sub-tropical regions in Asia, while a few species have been found
in the Pacific-Ocean Archipelago and the North and East of Australia. Only two species, viz.
M. oppositifolius (Geiseler) Müll.Arg. and M. baillonianus Müll. Arg. are found in Africa and
Madagascar, the latter being endemic to Madagascar (Rivière et al., 2010). Species in this

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genus have been reported to contain important phytochemicals that exhibit a wide range of
biological activities (Gangwar et al., 2014; Rivière et al., 2010). Polyphenols, terpenoids and
benzopyrans are considered the chemotaxonomic markers of this genus (Rivière et al., 2010).

One of the less well-known species of legumes growing in south-west Nigeria is Mallotus
subulatus. White and brown variants are the two that are grown. Peasant farmers plant and
use these types as backup crops that are harvested for consumption, particularly during the
dry season when other crops are scarce. Their difficult-to-cook character restricts their
cultivation to specific regions of the nation, despite the fact that they are simple to grow and
have a high level of disease resistance. Information on their anti-nutritional and nutritional
qualities is scarce. Therefore, sharing knowledge about their components would help these
lesser-known native legumes adapt, improve dietary diversity, and make vital nutrients
accessible at reasonable prices.

1.2 Statement of the problem

Mallotus subulatus is a species of shrub or tree that belongs to the genus Mallotus and the
Euphorbiaceae family of spurges that is native to tropical Africa and Asia. It is a popular food
source in many countries, and its leaves are also used in traditional medicine. However, there
are some gaps in the knowledge about the nutritional composition of Mallotus subulatus leaf.

One problem is that the nutritional composition of the leaves can vary depending on factors
such as the climate and soil conditions in which the plant is grown. This can make it difficult
to provide accurate nutritional information to consumers.

Another problem is that there is a lack of research on the bioavailability of the nutrients in
Mallotus subulatus leaf. Bioavailability refers to the proportion of a nutrient that can be
absorbed and used by the body. It is important to know the bioavailability of nutrients in
order to assess their nutritional value.

Finally, there is a need for more research on the long-term health effects of consuming
Mallotus subulatus leaf. While some studies have shown that the leaves are safe to eat, more
research is needed to confirm this and to identify any potential health risks associated with
long-term consumption.

These gaps in the knowledge can make it difficult for consumers to make informed choices
about whether or not to consume Mallotus subulatus leaf. Additionally, the lack of

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information about the long-term health effects of consuming the leaves is a concern for
consumers and public health officials alike.

1.3 Justification of study

Studying the nutritional composition of Mallotus subulatus is important for several reasons.
Firstly, it can provide valuable information on the potential use of the plant as a food source.
With the increasing demand for alternative food sources to alleviate malnutrition in
developing countries, studying the nutritional composition of underutilized plants like
Mallotus subulatus can provide valuable insights (Ade-Omowaye et al., 2015). Additionally,
understanding the nutritional composition of the plant can help in identifying any potential
health benefits or risks associated with its consumption.

Several studies have been conducted to investigate the nutritional composition of Mallotus
subulatus. For instance, Senguttuvan et al. conducted a study in 2014 to examine the physical
and functional properties of two species of Mallotus subulatus. The study found that the
brown species had a higher mean seed weight compared to the white species (Senguttuvan et
al., 2014). Another study conducted by Ali et al. in 2020 investigated the effects of
hydrothermal processing methods on the nutrients and anti - nutritional components of
Mallotus subulatus. The study found that the plant is a good source of protein, fat, fiber, and
minerals (Ojo et al., 2017). These studies provide valuable insights into the nutritional
composition of Mallotus subulatus and highlight its potential as a food source.

1.4 Aim and objectives

The study aims to provide a comprehensive understanding of the nutritional value of this
plant, which can be used to inform dietary recommendations and potential uses in the food
industry. The specific objectives of the study are as follows:

I. To determine the proximate composition of Mallotus subulatus leaf, including its moisture,
protein, fat, ash, and fiber content;
II. To identify and quantify the vitamins and minerals present in Mallotus subulatus leaf;
III. To assess the bioavailability of the nutrients in Mallotus subulatus leaf, and
IV. To investigate the potential health benefits of consuming Mallotus subulatus leaf.

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 CHAPTER TWO

2.0. LITERATURE REVIEW

2.1. Botanical description of Mallotus subulatus

Mallotus subulatus is a species of shrub or tree that belongs to the genus Mallotus and the
Euphorbiaceae family of spurges. It was originally reported in 1790. A tiny tree or shrub
native to forests, Mallotus subulatus can reach heights of up to 5 metres (Burkill., 1994). The
plant's native habitat is the tropical, forested region of Africa, which includes Sierra Leone,
Cameroon, and the Democratic Republic of the Congo (Baker &Wright., 1913). Mallotus
subulatus has leaves that are opposite, petioled, and membranous, and twigs that are
subscabridly stellate-pubescent. The leaves are broadly rounded at the base, oblong or, less
frequently, oval, and sharp (Baker & Wright., 1913) (plate 1). The majority of the leaves are
leathery and have two glands at the base. They are oblong to lanceolate and cuneate to
rounded. At the tip, the leaves are primarily acute or acuminate (Gangwar et al., 2014). Disc-
to-globose-shaped glandular hairs are reasonably obvious on the leaf's upper surface (Sierra
et al., 2010). The cream-colored blossoms of the dioecious Mallotus subulatus shrub or small
tree grow in forests. Another distinctive aspect of the plant is its stellate-pubescent branchlets
and racemes. Male and female Mallotus subulatus blooms are located on different plants
since the flowers are unisexual. Insects are the plant's pollinators (Burkill., 1994). Mallotus
subulatus produces a fruit that is a capsule with one to three seeds within. The 2 - 3 cm -
diameter capsule is stellate-pubescent (Sierra et al., 2007). Mallotus subulatus seeds are
disseminated by the wind or by animals. The kind and effectiveness of seed dissemination
can be impacted by the presence of glandular hairs on the fruit, according to a 2010 study by
Sierra et al.

Botanical classification of M. subulatus (WFO., 2023)

Kingdom: Plantae

Order: Malpighiales Juss. ex Bercht & J. Presl

Family: Euphorbiaceae Juss

Genus: Mallotus Lour.

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Species: Mallotus subulatus Müll.Arg.

Plate. 1. Mallotus subulatus; leaves.

https://plants.jstor.org/stable/history/10.5555/al.ap.specimen.k000252573. 2010.

2.2. Ethnomedicinal uses of Mallotus subulatus

In many regions of the world, the plant species Mallotus subulatus has long been valued for
its therapeutic benefits. The therapy of skin disorders is one of this plant's most popular
applications. Scabies, ringworm, and herpes are examples of parasitic skin illnesses that have
been treated using the glandular hair powder of M. philippinensis, a close relative of M.
subulatus (Gangwar et al., 2014). One plant used to treat skin conditions in Northern Pakistan
is M. subulatus (Malik et al., 2019). Mallotus species are another treatment option used by
traditional healers in Thailand for skin conditions (Saising et al., 2022). According to a study,
M. subulatus is one of the plants used to cure skin conditions in Nigeria. Due to its medicinal

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qualities, Mallotus subulatus has historically and culturally been employed in traditional
medicine. Every portion of the plant has unique therapeutic characteristics that are utilised in
Ayurveda to treat intestinal worms in grazing and domestic animals (Gangwar et al., 2014).
Mallotus subulatus leaves are also used in traditional medicine to cure fever, colds, coughs,
and other illnesses (Adeleye et al., 2021).

2.3. Modern scientific research on the medicinal properties of Mallotus subulatus

Traditional medicine has employed the medicinal herb Mallotus subulatus for many years to
treat a variety of illnesses. There hasn't been much recent scientific study on the plant's
therapeutic effects, though (Gangwar et al., 2014). In the plant, bioactive substances such as
flavonoids, alkaloids, and terpenoids have been found by recent investigations (Sierra et al.,
2007). These substances have been discovered to have antibacterial, anticancer, and
antioxidant effects (Iñiguez-Luna et al., 2021). To fully comprehend these chemicals'
potential for medical use, more study is required. Mallotus subulatus has the potential to be a
source of novel medications, according to pharmacological investigations on the plant.
Antihelmintic, antibacterial, anticancer, purgative, and wound healing properties of the herb
have been discovered (Mital & Acharya., 2020). Mallotus subulatus has also historically been
used to cure a number of ailments, including epilepsy, convulsions, pain, dysentery, and
diarrhoea (Olatunji et al., 2022). The potential of the plant's components for the creation of
novel medications for various illnesses requires further research. Mallotus subulatus has been
used as a traditional medicine in a number of traditional medical systems. The herb has been
used to treat tuberculosis, intestinal worms, and other diseases (Sabran et al., 2016). It has
also been investigated whether the plant's components have any potential for treating parasite
infections (Romero-Benavides et al., 2017). To completely comprehend the effectiveness and
safety of the plant's components for medical application, more study is necessary. In
conclusion, Mallotus subulatus has the potential to be an important source of fresh
medications for a variety of ailments, but more investigation is required to completely
understand its therapeutic characteristics.

2.4. Distribution of Mallotus subulatus

Mallotus subulatus can be found throughout tropical Africa, especially in Cameroon, Central
African Republic, Congo, Equatorial Guinea, Gabon, Ghana, Gulf of Guinea Island, Ivory
Coast, Liberia, Nigeria, Sierra Leone, Togo, Zaïre (Burkill., 1913) (plate 2). The plant is
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collected from the wild for usage locally as food, fuel, and medicinal. Additionally, the white
and brown forms of Mallotus subulatus, which are available worldwide (Ayodele et al.,
2014) are underutilised hard-to-cook legumes (plate 3&4).
Understanding the ecological and commercial value of Mallotus subulatus requires
knowledge about its spread. Mallotus and Macaranga, a closely related genus, are found
throughout the world, from Madagascar and Central Africa to India and Southeast Asia (Van
Welzen et al., 2014). Researchers looking in the phylogeny of Mallotus and similar taxa are
likewise interested in the origin and spread of the plant (Saising et al., 2022). Additionally,
Mallotus subulatus has minerals and phytochemicals that make it a viable source of food and
medicine, making its distribution and use crucial for sustainable development (Ayodele et al.,
2014; Ojo et al., 2017).

Table 1
Vernacular names of M. subulatus in different countries (Burkill., 1994).
Countries Vernacular name

Sierra Leone LOKO; haka (NWT)

Nigeria EDO; ebuhosa (AHU; JMD) uhosa (JRA)

EFIK; nne okpokri inyang (AJC)
IGBO (Asaba); kpọkọwa (NWT)
IGBO (Uburubu); ifie ogume (NWT), odoji
(NWT)

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Plate.2. Distribution of M. subulatus, https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:351693-
1#source-KBD. N.D.

Plate. 3. Mallotus subulatus (Pepelupe funfun)—- white species . https://www.researchgate.net/figure/a-

Mallotus-subulatus-Pepelupe-funfun-white-specie_fig1_320197918. 2017.

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Plate. 4. Mallotus subulatus (Pepelupe pupa)—- brown species . https://www.researchgate.net/figure/a-
Mallotus-subulatus-Pepelupe-funfun-white-specie_fig1_320197918. 2017.

2.5. Factors affecting the distribution of Mallotus subulatus

Mallotus subulatus is mostly found in the lowland evergreen forests of tropical Africa, from
Sierra Leone through Cameroon and the Central African Republic, and farther south to
Gabon, and the Democratic Republic of the Congo. Climate, soil type, and biotic factors are
only a few of the variables that have an impact on this species' spread. Mallotus subulatus
distribution is significantly influenced by climatic factors. This species is primarily found in
the wet tropical biome, where it is well-suited to conditions with high humidity and
precipitation. Mallotus subulatus range in tropical Africa is further impacted by the existence
of endemic species (Van Welzen et al., 2014).
Another significant factor that influences the distribution of Mallotus subulatus is the type
and quality of the soil. Mallotus species have been seen to flourish on a range of soil types,
including limestone (SEC., 2007). The development and distribution of this species can both
be impacted by the soil's quality. For instance, Kadiri et al.'s study discovered that different
soil types had an impact on the stomatal type and leaf structure of Mallotus species (Kadiri et
al., 2019). As a result, the distribution of Mallotus subulatus can be significantly influenced
by the quantity and quality of suitable soil types.
Mallotus subulatus distribution may also be influenced by biotic factors like competition and
predation. For instance, the survival and development of this species may be impacted by the
presence of predators, herbivores, pollinators, parasites, and diseases. According to a study
by Martini et al. herbivory and the density of conspecific and heterospecific seedlings both

9
had an impact on where Mallotus subulatus was found (Martini et al., 2019). Another study
(De França-Beltrão et al., 2023) discovered that macroclimatic biotic factors like interspecific
competition and predation as well as abiotic factors like temperature had an impact on the
spread of Mallotus subulatus. Therefore, the distribution of Mallotus subulatus in its native
habitat might be considerably impacted by the presence of biotic variables.

2.6. Conservation and management of Mallotus subulatus populations

Mallotus subulatus is a shrub or tree that is mostly found in West and Central Africa's wet
tropical biome. There are still a number of hazards to its populations, despite the fact that it is
at least locally common and not in danger from genetic erosion. These dangers include
habitat loss resulting from deforestation, excessive grazing, and increased agricultural
production ( Yan et al., 2019). Additionally, because of the species' high economic and
medicinal value, it is frequently harvested, which can cause overexploitation and population
decline. Therefore, it is essential to put conservation measures in place to save Mallotus
subulatus populations.
Several tactics can be used to protect Mallotus subulatus populations. These include the
creation of protected areas, the restoration and protection of habitat, and rules governing
harvesting methods. In order to preserve the species' long-term existence, it is also critical to
promote sustainable usage of the species, such as through cultivation and controlled
harvesting (Ojo et al., 2017). Studies on genetic variety can also help with the creation of
efficient conservation strategies (Yan et al., 2019). We can ensure the survival of Mallotus
subulatus populations and their ongoing contribution to the ecosystem and economy by
putting these strategies into practise.
Mallotus subulatus is crucial to the ecology and to the economy. It is employed medicinally
to treat conditions like respiratory infections, fever, and malaria (Gangwar et al., 2014). The
plant also has commercial worth because its seeds can be used to make fuel and oil for
creating soap from its seeds (Gangwar et al., 2014). Mallotus subulatus is also a significant
source of food for some West African populations since its leaves are used to prepare the dish
amala, which is a traditional cuisine there (Ojo et al., 2017). Therefore, maintaining healthy
populations of Mallotus subulatus helps not just the species, but also the ecosystems and
communities that depend on it.

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2.7. Ecological requirements of Mallotus subulatus

Mallotus subulatus requires particular needs for the soil and environment in order to flourish.
It often grows in lowland woods that are evergreen. The species has evolved to survive in a
hot, humid, tropical climate with distinct dry and wet seasons. The dry season runs from
November to February, whereas the rainy season lasts from March to October. Additionally,
the plant needs soils with a pH range of 5.5 to 7.5 that are well-drained. From sandy loam to
clay loam, it can endure a variety of soil types (Edwin-Wosu, 2022).

Mallotus subulatus needs enough water and nutrients to grow and develop appropriately. The
species is known to have a substantial amount of protein and is used as food in some regions
(Ade-Omowaye et al., 2015). Underutilised legumes like Mallotus subulatus, according to a
2018 study by Ojo et al. can have their nutritional content boosted through hydrothermal
processing and soaking (Ma., 2022; Ojo., 2018). The plant can thrive in a variety of
environments, and the ability to adapt to the environment is a prerequisite for the
reproduction of its life forms (Edwin-Wosu, 2022).

The internal energy state of an ecosystem is essential for Mallotus subulatus to flourish and
survive. Three interrelated concepts are enthalpy, entropy, and Gibbs free energy (Wu et al.,
2017). The species is similar to important Euphorbiaceae genera like Macaranga in terms of
morphology, geography, and ecology and thrives in lowland evergreen woods (Sierra et al.,
2010). According to a 2017 study by Ademoh et al, Mallotus subulatus is one of the
functional groups in a secondary rainforest (Ademoh et al., 2017). The management and
protection of Mallotus subulatus in their natural habitats relies on an understanding of the
biological requirements of the species.

2.8. Role of Mallotus subulatus in Ecosystems

Mallotus subulatus, a species of tree found in lowland evergreen forests, is a key component
in food webs as it provides food and habitat for a variety of organisms. An ecosystem's
functional diversity, which includes the variety of plant species, is recognised to improve
ecosystem functioning and resource utilisation(Gravel et al., 2016; Kéfi et al., 2012).
According to research, reduced litter, high tree densities, and species richness in plantations
are all related to the functional diversity of soil food webs (SEC., 2007). Mallotus subulatus

11
can therefore assist the survival and well-being of a variety of creatures by improving the
resilience and efficiency of food webs in ecosystems.

Mallotus subulatus has a considerable impact on soil health and erosion control in addition to
its function in food webs. It has been demonstrated that the vast root system of bamboo, a
relative of Mallotus subulatus, enhances the physical, chemical, and biological characteristics
of soil and controls soil erosion (Kaushal et al., 2020). On the other hand, logging can hasten
soil erosion, deteriorate the physical characteristics of the soil, and cause nutrient loss (Xiao
et al., 2020). Common agricultural practises such as tillage can also alter soil structure and
hasten surface runoff and soil erosion. Mallotus subulatus can improve soil health and
prevent erosion, which can assist to lessen the detrimental effects on ecosystems.

Additionally, Mallotus subulatus is employed in ecological preservation and restoration

projects. Ecosystems have deteriorated as a result of human activities such as land use
changes and timber harvesting that eliminate native plant species (BOWLER., 2000).
Degraded habitats frequently require active restoration of plant diversity, including the
purposeful reintroduction of native species (De Vitis et al., 2022). Mallotus subulatus can be
a useful plant for ecological restoration and conservation efforts because of its capacity to
enhance soil health and limit erosion(Atkinson et al., 2022).

2.9. Phytochemistry of Mallotus subulatus

Mallotus subulatus leaves are rich in phytochemicals, which are natural compounds found in
plants that have been shown to have various biological activities. A study conducted by
Gangwar et al. (2014) analyzed the phytochemical composition of Mallotus subulatus leaves
and found the presence of several bioactive compounds, including flavonoids, alkaloids,
phenols, and tannins. These compounds have been associated with antioxidant, anti-
inflammatory, and anticancer properties.
2.9.1. Flavonoids
Flavonoids are a group of phytochemicals that are widely distributed in the plant kingdom
and have been recognized for their potential health benefits. The study by Gangwar et al.
(2014) identified several flavonoids in Mallotus subulatus leaves, including quercetin,
kaempferol, and rutin. These flavonoids have been shown to possess antioxidant activity,
which helps protect the body against oxidative stress and reduce the risk of chronic diseases
such as cardiovascular disease and cancer.

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2.9.2. Alkaloids
Alkaloids are a class of nitrogen-containing compounds that are known for their diverse
biological activities. The study by Gangwar et al. (2014) revealed the presence of alkaloids in
Mallotus subulatus leaves, including berberine and palmatine. These alkaloids have been
reported to have antimicrobial, antidiabetic, and anticancer properties. They may also have
potential as therapeutic agents for the treatment of various diseases.
2.9.3. Phenols
Phenols are a group of aromatic compounds that have been widely studied for their
antioxidant and anti-inflammatory properties. The study by Gangwar et al. (2014) found the
presence of phenols in Mallotus subulatus leaves, including gallic acid and ellagic acid.
These phenols have been shown to scavenge free radicals, reduce inflammation, and protect
against oxidative damage. They may also have potential in the prevention and treatment of
chronic diseases.
2.9.4. Tannins
Tannins are polyphenolic compounds that are commonly found in plants and have been
recognized for their antioxidant and antimicrobial activities. The study by Gangwar et al.
(2014) identified tannins in Mallotus subulatus leaves, including tannic acid and catechins.
These tannins have been shown to have antimicrobial properties, which may help protect
against certain bacterial and fungal infections. They may also contribute to the antioxidant
activity of Mallotus subulatus leaves.
2.10. Nutritional Composition of Mallotus subulatus Leaves
In addition to phytochemicals, Mallotus subulatus leaves also contain a variety of essential
nutrients that are important for overall health and well-being. A study conducted by Ayodele
et al. (2014) analyzed the nutritional composition of Mallotus subulatus leaves and found
them to be a rich source of vitamins, minerals, and dietary fiber.
2.10.1. Vitamins
Mallotus subulatus leaves are particularly high in vitamins A, C, and E. Vitamin A is
essential for maintaining healthy vision, supporting the immune system, and promoting
proper growth and development. Vitamin C is a powerful antioxidant that helps protect
against oxidative damage and boost the immune system. Vitamin E is also a potent
antioxidant that plays a crucial role in protecting cells from free radical damage.
2.10.2. Minerals
Mallotus subulatus leaves are a good source of minerals such as calcium, iron, and
potassium. Calcium is essential for maintaining strong bones and teeth, regulating muscle
13
contractions, and supporting nerve function. Iron is necessary for the production of red blood
cells and the transport of oxygen throughout the body. Potassium is important for maintaining
proper fluid balance, regulating blood pressure, and supporting heart health.
2.10.3. Dietary Fiber
Mallotus subulatus leaves are rich in dietary fiber, which is important for digestive health and
maintaining a healthy weight. Fiber helps regulate bowel movements, prevent constipation,
and promote the growth of beneficial gut bacteria. It also helps control appetite, promote
satiety, and reduce the risk of obesity and related diseases.

2.11. Pharmacological activities of Mallotus subulatus

Mallotus subulatus has been researched for its therapeutic characteristics and is a valuable
source of natural chemicals, primarily phenols (Gangwar et al., 2014). Regarding its
phytochemistry and biological function, current information of this endangered species of
medicinal plant is still restricted (Gangwar et al., 2014). Two different varieties of the plant,
Mallotus subulatus, have been examined for their nutrients and phytochemicals, and research
has also been done on the plant's physical and functional characteristics (Ayodele et al.,
2014; Ojo et al., 2017).

Mallotus subulatus has potential applications in a number of disciplines, making it crucial to

investigate its biological processes. Numerous biological and pharmacological effects of the
plant have been discovered, including anti-inflammatory, antioxidant, and antibacterial
characteristics (Arisawa., 1994). Due to the plant's widespread local distribution and lack of
genetic erosion, Mallotus subulatus has also been investigated for its potential as a source of
genetic resources. In order to comprehend the evolutionary relationships within the genus
better, the phylogeny of Mallotus subulatus has also been researched (Sierra et al., 2010; Van
Welzen et al., 2014).

Mallotus subulatus has potential uses in a number of industries, including biotechnology,

agriculture, and medicine. Rottlerin, a significant natural chemical identified in Mallotus
subulatus and containing a number of biological actions, has been examined for its potential
as a medicine (Gangwar et al., 2014). Mallotus subulatus has also been investigated for its
potential as a neglected difficult-to-cook legume, with two different types of the plant being
examined for their nutritional content and phytochemical content (Ayodele et al., 2014). For
the creation of novel medications, the advancement of agricultural methods, and the

14
development of biotechnological applications, knowledge about Mallotus subulatus and its
biological activity can be very useful.

2.11.1. Antimicrobial and Antioxidant Activities of Mallotus subulatus

It has been discovered that the Euphorbiaceae plant species Mallotus subulatus has potent
antibacterial activities ( Ogundipe et al., 2000). According to studies, Mallotus subulatus
methanol extract includes bioactive phenolics such chicoric acid and caftaric acid, which may
be the cause of its antibacterial activity (Chassagne et al., 2021). Another investigation
discovered that the high concentrations of thymol and carvacrol in essential oils from related
plant species, including Thymus capitata and Satureja montana, also have antibacterial
characteristics ( Valerio et al., 2021). Mallotus subulatus may therefore provide a promising
natural source of antibacterial compounds for use in therapeutic procedures and other fields.

Additionally, Mallotus subulatus has been discovered to have strong antioxidant

characteristics ( Ade-Omowaye et al., 2015). The white type of Mallotus subulatus has been
discovered to have significant amounts of total phenolic compounds, which support its
antioxidant action, according to a study by Ade-Omowaye et al. (Ade-Omowaye et al.,
2015). It has also been discovered that the plant contains anti-inflammatory, antiradical, and
anti - H. pylori properties (Gangwar et al., 2014). These characteristics might result from the
existence of several bioactive substances, like rottlerin, which has been discovered to have a
variety of biological functions (Gangwar et al., 2014). As a result, Mallotus subulatus may
one day be used therapeutically to treat a variety of illnesses.

Mallotus subulatus has antibacterial and antioxidant capabilities, although the exact methods
by which they work are still being studied. According to certain research, the plant's bioactive
substances might affect bacterial cell membranes or oxidative stress - related enzyme
activities (Xu & Chang., 2009). To completely comprehend the mechanisms of action of
Mallotus subulatus and its potential applications in medicine and other disciplines, more
study is nonetheless required. Nevertheless, the plant is a valuable resource for local
communities in Nigeria and other places because of its therapeutic and nutritional qualities
(Ademoh et al., 2017).

15
2.11.2. Anti - inflammatory and Cytotoxic Activities of Mallotus subulatus

It has been discovered that the plant Mallotus subulatus, which has long been utilised in folk
medicine, possesses anti-inflammatory qualities (Gangwar et al., 2014). Alkaloids, cardiac
glycosides, and phenolic chemicals may all be responsible for Mallotus subulatus' ability to
reduce inflammation (Farombi et al., 2001). The plant has also been used to treat hepatitis,
fever, rheumatoid arthritis, and muscle discomfort (Hasan et al., 2014). According to these
results, Mallotus subulatus may be useful in the creation of fresh anti-inflammatory
medications.

Additionally the plant, Mallotus subulatus has also been discovered to have cytotoxic effects
(Jain et al., 2013). The plant contained natural substances such phenols, diterpenoids,
flavonoids, and triterpenoids, according to phytochemical study (Gangwar et al., 2014). It
has been demonstrated that these substances have cytotoxic effects on cancer cells (Anh et
al., 2022). Mallotus subulatus thus has the potential to serve as a natural source for the
creation of fresh anticancer medications.

Mallotus subulatus has cytotoxic and anti-inflammatory characteristics, which point to

possible medical uses. To fully comprehend the phytochemistry and biological activities of
this plant, however, more investigation is required (Gangwar et al., 2014). Mallotus
subulatus is an underutilised legume whose nutrients and phytochemicals have been
studied( Ayodele et al., 2014; Ma., 2022). A natural harvest of the plant is made for local
usage as a therapeutic plant. Mallotus subulatus has the potential to be utilised in the creation
of novel medications for a range of medical uses.

2.12. Methods of administration and safety considerations for Mallotus subulatus

Ayurvedic medicine has long employed the medicinal plant Mallotus subulatus to treat
intestinal worms in grazing and domestic animals (Gangwar et al., 2014). The use of dried
leaves and seeds, which can be eaten as tea or powder, is one of the traditional methods of
administration (Hasan et al., 2014). Liquid chromatographic techniques are used in
contemporary administration techniques to identify specific plant components, such as
sennosides B and A (Srivastava et al., 2006). Although the conventional methods of
administration have been in use for many years, it is crucial to remember that additional study
is still required to identify the best route of administration for particular circumstances.

16
When utilising Mallotus subulatus, safety precautions and any negative effects should also be
taken into mind. The plant contains tannins, which may have a negative impact on nutrition
and may need to be removed through hydrothermal processing (Ma., 2022). Furthermore, it
has been discovered that the herb has harmful effects on reproductive parameters in female
rats (Gangwar et al., 2014). As a result, it's crucial to get medical advice before using
Mallotus subulatus and to adhere to suggested dosages and administration techniques.

Mallotus subulatus may have medicinal uses, but it's crucial to remember that further study is
required to completely grasp its pharmacology. According to a study done by Gangwar et al.
Mallotus subulatus will only continue to be significant locally as a medicinal plant unless
chemical and pharmacological research yields noteworthy results (Gangwar et al., 2014).
Additionally, it has been discovered that the plant may be poisonous and detrimental to aphid
populations. As a result, it's crucial to utilise Mallotus subulatus with prudence and give
safety a high priority.

2.13. Key studies on the biological activities of Mallotus subulatus

Mallotus subulatus is a plant whose antibacterial properties have been researched. The fruit
extract of Mallotus subulatus has antibacterial action, but no antifungal potential, according
to a 2014 study by Gangwar et al. (Gangwar et al., 2014). As a result, it might be possible to
cure bacterial illnesses with the plant. These results demonstrate the potential for using
natural substances obtained from plants like Mallotus subulatus to create novel antibacterial
chemicals.

Mallotus subulatus has been investigated for its anti - inflammatory qualities in addition to its
antibacterial capabilities. Mallotus subulatus leaf extract was tested for its ability to reduce
inflammation in a 2014 study by Hasan et al. and they discovered that it did so significantly
(Hasan et al., 2014). As a result, it might be possible to treat inflammatory diseases with the
plant. These results demonstrate the potential for using natural substances produced from
plants like Mallotus subulatus to create novel anti-inflammatory drugs.

Antioxidant qualities of Mallotus subulatus have also been researched. According to

Senguttuvan et al (2014), the plant has the potential to be a potent antioxidant. The plant may
be helpful in avoiding the oxidative damage brought on by free radicals, according to this

17
indication. The potential of natural substances obtained from plants like Mallotus subulatus
for the creation of novel antioxidant agents is highlighted by these findings.

2.14. Potential applications of Mallotus subulatus in medicine and industry

Mallotus subulatus may be used as an antibacterial agent because it has been discovered to
have antimicrobial qualities. The fruit extract of Mallotus subulatus has been shown to have
antibacterial activity by a study done by Gangwar et al. in 2014 (Gangwar et al., 2014). The
Mallotus genus may also be a viable source of biological activity chemicals, particularly
those with antibacterial properties, according to a review of the genus undertaken by
Jesumani et al. in 2019 (Anh et al., 2022). According to these results, Mallotus subulatus
may be used in the creation of fresh antibacterial compounds that could be effective in the
management of infectious disorders.

Mallotus subulatus has been discovered to offer potential in the treatment of inflammatory
illnesses in addition to its antibacterial capabilities. Mallotus subulatus is traditionally used to
alleviate muscle soreness, itching, and rheumatoid arthritis, according to a 2014 study by
Hasan et al. (Hasan et al., 2014). Due to their anti-inflammatory qualities, marine algae,
particularly those belonging to the genus Mallotus, have the potential to be used in the
pharmaceutical and cosmetic industries, according to a subsequent study by Thiyagarasaiyar
et al. from 2020 (Thiyagarasaiyar et al., 2020). These findings imply that Mallotus subulatus
may be employed in the creation of fresh anti-inflammatory medications.

Mallotus subulatus may also find use in the food and cosmetics sectors. Mallotus subulatus
was discovered to have aesthetic potential in a study by Genva et al. in 2022 that examined
the biological activities of plants from Mayotte for their prospective application as skin care
agents.These results imply that Mallotus subulatus may be utilised in the creation of new
foods and cosmetics.

18
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Plant Collection

Two varieties of Dehulled seeds of Mallotus subulatus Müll.Arg. were purchased directly

from farmers at “Ikpa” market, Nsukka L.G.A. of Enugu State, South - East Nigeria. They

were properly identified and authenticated by Mr Felix Nwafor, a plant taxonomist in the

Department Plant Science and Biotechnology, University of Nigeria, Nsukka. Voucher

samples () were prepared and deposited in the Herbarium of the same institution.

3.2 SAMPLE PREPARATION

All of the samples were washed in running tap water to remove any traces of soil dirt and air

dried at room temperature (25 ± 2 ºC) to a constant weight. The two samples were ground

into fine powder (flour) using a heavyweight blender (Kenwood 6000 BT, China) and stored

separately in labelled airtight containers until needed for further analysis.

3.3 EXTRACTION

The powdered materials from the two varieties were extracted separately. Five (50) grams

each of dried and ground samples were macerated with 95% methanol for 48 hours at room

temperature and filtered. The solvent was evaporated in a water bath 45 ºC and the dried

extract was used for analysis.

3.4 PROXIMATE COMPOSITION ANALYSIS

Proximate composition (ash content, crude protein content, crude fat content, crude fibre and

moisture content) was determined using modified methods of the Association of Official

Analytical Chemist (AOAC, 2005).

19
3.4.1 Determination of ash content

Two grams (2 g) of the ground sample was weighed out into a previously weighed silica dish.

The silica dish was placed in a muffle furnace and ashed (heated) at 600 oC for 3 hours,

allowed to cool in a desiccator and weighed with a weighing balance. The difference in

weight gave the weight of the ash. The percentage ash content was calculated using the

formula below:

Ash = weight of ash x 100 (AOAC, 2005).

Weight of sample

3.4.2 Determination of crude protein content

The micro-Kjeldahl procedures (1979) were used to determine the crude protein content. One

gram of the dried ground material was weighed into a 50 ml Kjeldahl flask. Twenty millilitres

of concentrated H2SO4 was carefully added, with a pinch of catalyst (mixture of selenium

oxide, K2SO4 and CuSO4). The mixture was heated continuously on digestion under a fume

hood until a clear greenish solution appeared. After the digest had cleared, the mixture was

heated for another 2 minutes and allowed to cool. 10 ml of distilled water was added to avoid

caking and the sample made up to 50 ml of distilled water. 10 ml of the digested sample was

transferred into the Kjeldahl apparatus and a receiver flask containing 20 ml of boric acid

indicator solution was placed under the dispenser of the distillation apparatus. 10 ml of 40%

NaOH solution was added through a funnel stop cork and distillation commenced. The

distillate (35 ml) was collected through the condenser tip and titrated with 0.01 M of HCL

until a pink colour was observed. The concentration of the protein was calculated using the

formula below:

Nitrogen = titre value x 14.1 x 0.01 x 100 x 50

1000 x 1 g x 10 ml

Protein = % Nitrogen x 6.25 (where 6.25 is a constant) (AOAC, 2005).

20
3.4.3 Determination of crude fat content (Soxhlet method)

An extraction flask was washed and dried over hot air oven for 30 minutes and allowed to

cool in desiccators and marked (A). Two grams of the ground sample (A) was transferred into

an extraction thimble and dropped inside Soxhlet extractor. Two hundred millilitres of

petroleum ether was added. The apparatus was set up and allowed to run for 4 hours. The

thimble was removed and the ether recovered at the end of the extraction. The oil collected in

the flask was dried at 100 oC in an oven and then the flask with oil (C) was weighed.

The percentage (%) fat content was calculated using the formula below:

Fat = C – B x 100

A 1

Where B = Weight of empty flask

A = weight of sample

C = weight of flask + oil after drying (AOAC, 2005).

3.4.4 Determination of crude fiber

To 150 ml of 0.218 M pre-heated H2SO4 in 400 ml beaker, 2 g (W1) of the ground sample

was added and then heated to boiling for 30 minutes, cooled and filtered. The residue was

washed three times with hot water. To this, 128 moles of pre-heated KOH was added and

heated to boiling. Some drops of antifoaming agent were added. It was allowed to boil for 30

minutes and filtered. The residue was washed three times with hot water, and another three

times with acetone and dried at 103 oC with crucible in an oven for 1 hour and weighed (W 2).

It was ashed at 500 oC and weighed again (W3).

The percentage fiber was calculated using the formula below

%Fiber =W2 – W3 x 100

21
 W1 1

Where W1 = weight of the ground sample used

W2 = Weight of residue after drying at 103oC

W3 = Weight of ash (AOAC, 2005).

3.4.5 Determination of moisture content

Five grams (5 g) of the ground sample was dried to a constant weight at 600 oC in a hot air

circulating oven. The moisture content was calculated as the difference in weight after drying

as shown below:

W2 – W3 x 100

W1 1

Where W1=initial weight of empty crucible

W2 = Weight of crucible + sample before drying

W3 = Final weight of crucible + sample after drying (AOAC, 2005).

3.5 MINERAL ANALYSIS

The analysis of mineral composition of the two varieties of Mallotus subulatus seeds was

carried out according to the methods of Association of Official Analytical Chemists AOAC

(2005). A portion (1 g) of each sample was measured into a digestion flask, 20 mls of acid

mixture (65 mls of conc. HNO3; 80 ml Phosphoric acid; 20 ml conc. H2SO4) was then added

and the flask was heated until a clear digest was obtained. The digest was diluted with

distilled water to the 500 ml mark. Appropriate dilution was then made for each element

standards.

3.5.1. Determination of Calcium Content:

For determination of calcium, SrCl2 solution, containing 10,000 mg/ml to yield a 1,500

mg/ml of Sr2+ in the final solution, was added. A calibration curve was prepared for each

element using standard solution (Akpana et al., 2017).

22
3.5.2. Determination of Potassium and Sodium Content

Each sample digest (5 mls) was pipette into separate test tubes in duplicates, 2 mls of cobalt

nitrite was then added, shaken vigorously and allowed to stand for 45 minutes then

centrifuged for 15 minutes a 3000 rpm. The supernatant was drained-off and 2 mls of ethanol

was added to the residues. The individual solution was shaken vigorously and centrifuged for

another 15 minutes. The supernatants were drained off and 2 mls of distilled water was added

to the residue. The individual solution was boiled for 10 minutes with frequent shaking to

dissolve the precipitate, 1 ml of 1% choline hydrochloride and 1 ml of 2% sodium ferric

cyanide was added. Then 2 mls of distilled water was also added and the solutions were

shaken to mix well. The absorbance was taken at 620 nm against the blank (Akpana et al.,

2017).

3.5.3. Determination of Magnesium

Five millilitres (5 mls) of each sample digest was pipetted into a test tube in duplicate, 1 ml

of 0.67 N sulphuric acid (H2SO4) and 1 ml of 0.05% titan yellow was added. Then 1 ml of

0.01% gum acacia and 2 mls of 10% sodium hydroxide (NaOH) were also added. The

solution was mixed and the absorbance was taken at 520 nm against the blank (Akpana et al.,

2017).

3.5.4. Determination of Phosphorus

The molybdate colorimetric method was used to determine phosphorus. Two (2) ml of dry

ash extract was mixed with equal volume of Vanodo-molybdate colour reagent. The mixture

was then diluted with 50 ml distilled water. A standard phosphorus solution was also

prepared. The absorbances of the standard and sample were measured at 660 nm after fifteen

minutes (Akpana et al., 2017).

3.6 VITAMIN ANALYSIS

23
3.6.1. Determination of Vitamin C Content:

The vitamin C content was determined by the method of A.O.A.C. (2005). A measured

volume (20 mls) of 1% acetic acid and 20 mls of 1% oxalic acid were added to about 2 g of

each dried samples. The mixture was allowed to stand for two hours before it was filtered.

Then, 10 mls of the filtrate was pipetted into a conical flask and titrated with 2, 4-

dinitrophenol-hydrazine and the volume noted. A portion of 10 mls standard ascorbic acid of

analytical grade was also titrated with indophenol dye solution. The weight of ascorbic acid

oxidized by 1 ml of the dye was calculated in mg. A blank titration was also done in the same

manner without the sample. The value obtained was used to calculate vitamin C

concentration using the formula:

Vitamin C mg /100 g = 100 ×Vt ( V 1−V 2 )

W −V 3 (Vt −Vo)

Where,

W = weight of sample

V1 = value for standard ascorbic acid

V2 = value for sample

Vo = value for blank

V3 = volume of extract and for titration

VT = total extract

3.6.2 Determination of Beta Carotene:

Content Beta carotene content was determined using spectrophotometric method described by

Kirk and Sawyer (1991). Each sample was weighed (2 g) into a flat bottom reflux. Distilled

water (10 ml) was added and shaken carefully to form a paste; 25 ml of alcoholic KOH

solution was added and a reflux condenser attached. The mixture was heated in boiling water

bath for one hour with frequent shaking, cooled rapidly and 30 ml of distilled water was

added. The hydrolyzed product obtained was transferred into a separator funnel and the

24
solution was extracted three times with 20 ml of ether; 20 g of anhydrous Na 2SO4 was added

to the extract to remove any traces of water. The mixture was then filtered into a 100 ml

volumetric flask and made up to mark with ether. A standard solution of β-carotene of range

0-50 μg/ml was dissolved in 100 ml of ether. The gradients of different standard solutions

were determined with reference to their absorbance from which the average gradient was

taken to calculate β-carotene in μg/100 g. Absorbance of sample and standards was read on a

spectrophotometer (Metrohn Spectronic 21D Models) at a wavelength of 328 nm.

Calculations to determine actual beta carotene content was done using the formula:

Vitamin A (mg /100 g) ¿ Absorbance of sample × dilution factor × gradient factor

weight of sample

3.6.3 Determination of Vitamin E Content:

Beta carotene content was determined using spectrophotometric method described by

A.O.A.C. (2005). One gram (1 g) of each sample was weighed into a 250 ml conical flask, 10

mls of absolute alcohol and 20 mls of 1 M alcoholic sulphuric acid were added. The

condenser and flask were wrapped in aluminum foil and refluxed for 45 minutes and then

cooled for 15 minutes. A volume of 50 mls distilled water was added to the mixture and

transferred to a 250 ml separating funnel covered with aluminum foil. The unsaponifiable

matter in the mixture was extracted with 30 mls of dimethyl ether. The combined extracts

were washed free of acid and dry-evaporated at a low temperature and the residues obtained,

immediately dissolved in 10mls of absolute alcohol. Aliquots of solutions of each sample and

standards (0.3-3.0 mg vitamin E) were transferred into a 20 ml volumetric flask and 5 ml

absolute alcohol added, followed by a careful addition of 1 ml concentrated HNO 3. The flask

was placed on a water bath and temperature set at 90°C for 3 minutes. The flask was allowed

to cool rapidly under running water and volume was adjusted with absolute alcohol.

Absorbance was measured at 470 nm against a blank solution containing 5 ml absolute

25
alcohol and 1 ml concentrated HNO3 treated in a similar manner. Vitamin content was then

calculated using the formula:

Vitamin A (mg /100g) ¿ Absorbance of sample × dilution factor × gradient factor

weight of sample

3.6.4. Determination of Vitamin B6:

Vitamin B6 was determined according to the method described by A.O.A.C. (2010). A

weighed (2 g) portion of sample was placed in 25 mL of H 2SO4 (0.1 N) solution and

incubated for 30 minutes at 121°C. Then, the contents were cooled and adjusted to pH 4.5

with 2.5 M sodium acetate, and 50 mg Takadiastase was added. The preparation was stored at

35°C overnight. The mixture was then filtered through a Whatman No. 4 filter, and the

filtrate was diluted with 50 mL of pure water and filtered again through a micropore filter

(0.45 μm). Twenty microliters (20 µl) of the filtrate were injected into the HPLC system.

Quantification of vitamin B6 content was accomplished by comparison to vitamin B 6

standards. Standard stock solutions for pyridoxine was prepared as reported previously

(Aslam et al., 2008; Ringling et al., 2013). Chromatographic separation was achieved on a

reversed phase (RP) HPLC column (Agilent ZORBAX Eclipse Plus C18; 250 × 4.6 mm i.d.,

5 μm) through the isocratic delivery mobile phase (A/B 33/67; A: MeOH, B: 0.023 M

H3PO4, pH = 3.54) at a flow rate of 0.5 mL/min. Ultraviolet (UV) absorbance was recorded

at 270 nm at room temperature (Marzougui et al., 2009).

26
 CHAPTER 4
4.0 Result
4.1. Proximate composition
Proximate composition (ash content, crude protein content, crude fat content, crude fiber and
moisture content) was determined using modified methods of the Association of Official
Analytical Chemist (AOAC, 2005). The micro - Kjeldahl procedures (1979) were used to
determine the crude protein content. The Soxhlet method were used to determine the crude
fat content.
Table 2
Proximate composition of the leaf of Mallotus subulatus

Proximate composition Mallotus subulatus leaf(%)

Moisture content 9.29 ± 0.05

Crude fat content 4.68 ± 0.04

Ash content 8.56 ± 0.03

Crude fibre 13.45 ± 0.41

Protein 14.38 ± 0.04

Carbohydrate 49.66 ± 0.26

Values expressed as mean ± standard error of duplicate analysis

4.2 Mineral content analysis of Mallotus subulatus

27
The analysis of mineral composition of the leaf of M. subulatus was carried out according to
the methods of Association of Official Analytical Chemists AOAC (2005). This showed the
presence of various essential minerals like sodium, potassium, magnesium, iron, phosphorus
and calcium.

Table 3
Mineral elements composition of the leaf of Mallotus subulatus

Minerals Mallotus subulatus leaf (mg/100g)

Sodium 22.11 ± 0.24

Potassium 432.49 ± 0.05

Magnesium 383.69 ± 0.27

Iron 13.06 ± 0.19

Calcium 194.73 ± 0.38

Phosphorus 76.79 ± 0.45

Values expressed as mean ± standard error of duplicate analysis

4.3 Vitamin constituent analysis of Mallotus subulatus

This showed the presence of Vitamin A, C, E and B6. Beta carotene content was determined
using spectrophotometric method described by Kirk and Sawyer (1991). Vitamin B6 was
determined according to the method described by A.O.A.C. (2010). Vitamin E content was
determined using spectrophotometric method described by A.O.A.C. (2005). The vitamin C
content was determined by the method of A.O.A.C. (2005).
Table 4
Vitamin constituents of Mallotus subulatus leaf.

Vitamin Mallotus subulatus leaf (mg/100g)

28
 Vit C 8.28 ± 0.04

Vit A 12.38 ± 0.03

Vit E 3.84 ± 0.18

Vit B6 0.22 ± 0.01

Values expressed as mean ± standard error of duplicate analysis.

CHAPTER FIVE
5.0 Discussion
Mallotus subulatus, a legume seed commonly found in Nigeria, has been evaluated for its
proximate content, including protein, carbohydrates, and fats and oils (Ade-omowaye et al.,
2015). In a study conducted by James et al. in 2020, the red variety of Mallotus subulatus
was found to have the highest protein content compared to other legumes such as Vigna and
Sphenostylis sterocarpa (Ade-omowaye et al., 2015). Hydrothermal processing of the seeds
had a significant effect on the proximate composition, with the protein content of the raw
sample being 0% hydration (Ojo et al., 2017). The high protein content of Mallotus subulatus
makes it a valuable source of plant - based protein for individuals looking to increase their
protein intake.
In addition to protein, Mallotus subulatus also contains carbohydrates, which are an essential
source of energy for the body (Ade-omowaye et al., 2015). The high protein content of
Mallotus subulatus leaves makes them a good food source for people who are looking to
build muscle mass or lose weight. The study conducted by James et al. found that Vigna
racemosa and Sphenostylis sterocarpa had high starch contents, while Cajanus cajan rated
best for its sugar content (Ade-omowaye et al., 2015). Mallotus subulatus contains a
significant amount of carbohydrates, which can provide sustained energy throughout the day
and support physical activity.

Mallotus subulatus also contains fats and oils, which are essential for various bodily
functions, including hormone production and nutrient absorption(Ade-omowaye et al., 2015).
Proximate analysis revealed that the leaf contains 4.68% crude fat, indicating that it is a
relatively low-fat food source. The study by Zubair et al. in 2020, found that the fat content

29
of Mallotus subulatus was determined using the Soxhlet solvent extraction method and was
present in appreciable quantities (James et al., 2020). The fatty acid profile of Mallotus
subulatus has not been extensively studied, but fatty acids play a significant role in the
development of fat products with enhanced nutritional value(Ade-omowaye et al., 2015).
While the fat content of Mallotus subulatus may not be as high as other sources of fats and
oils, it can still contribute to a balanced and healthy diet. The low fat content of Mallotus
subulatus leaf suggests that it could be a healthy addition to a balanced diet, particularly for
individuals who are seeking to manage their fat intake.

Proximate analysis revealed that the leaf contains 13.45% fiber. The fiber content of
Mallotus subulatus leaves can help to regulate digestion and prevent constipation.

Proximate analysis revealed that the leaf contains 9.29% moisture, indicating that it is
relatively dry and could be stored for longer periods without spoilage. This is an important
finding for communities in developing countries who may rely on locally available food
sources to alleviate malnutrition (Ade-omowaye et al., 2015). The low moisture content of
Mallotus subulatus leaf suggests that it could be a viable option for food preservation and
storage, particularly in areas with limited access to refrigeration or other preservation
methods.

Mallotus subulatus is a rich source of essential minerals such as calcium, iron, magnesium,
phosphorus, potassium, and sodium. These minerals play a crucial role in maintaining healthy
bones, muscle function, and nerve function. The presence of these minerals in Mallotus
subulatus makes it an excellent addition to a balanced diet.
The nutritional makeup of these underutilised legumes is of relevance since calcium, a
mineral element, is essential for the growth and development of bones and teeth. Mineral
analysis revealed that the leaf contains 194.73 mg/100g of calcium.
The legume included sodium, a prevalent ingredient in many food plants, in amounts safe for
human health (Sunetra, 2009). Hypertension has been linked to a high sodium intake (Apata
and Ologbobo., 1994).
Iron was present in the legumes. Iron is essential for carrying oxygen throughout the body.
The magnesium content of the leaf was notable. The potassium content was also notable. The
main intracellular cation that controls pH balance and osmotic pressure is potassium.
In addition to minerals, Mallotus subulatus is also a good source of vitamins. It contains
essential vitamins such as vitamin A, vitamin C, vitamin E, and vitamin B6. Vitamin A is
30
essential for maintaining healthy vision, while vitamin C is crucial for boosting the immune
system and promoting wound healing. Vitamin E is a potent antioxidant that helps protect the
body against oxidative stress, while vitamin B6 supports brain function, the immune system,
and the transportation of oxygen around the body. The presence of these vitamins in Mallotus
subulatus makes it a valuable addition to a healthy and balanced diet (Definitions of Health
Terms: Minerals: MedlinePlus, 2022).
Studies have shown that consuming a diet rich in minerals and vitamins can have significant
health benefits (Sh., 2018). For instance, a study conducted by Akpana et al. in 2017 found
that magnesium, one of the minerals found in Mallotus subulatus, can help reduce the risk of
developing type 2 diabetes. Another study conducted by Ali et al. in 2020 found that vitamin
E may help reduce the risk of developing age - related cataracts (Ali et al., 2020). These
studies demonstrate the importance of incorporating nutrient - rich foods such as Mallotus
subulatus into one's diet to promote overall health and well-being (Idris et al., 2019).

5.1 Conclusion and implications of the study

In conclusion, the leaf of Mallotus subulatus has been found to be a rich source of protein
(Ade-omowaye et al., 2015). Additionally, other underutilized crops such as Vigna racemosa
and Sphenostylis sterocarpa were found to have high starch contents, while Cajanus cajan
rated best in terms of overall nutritional value (SSRG | Open Access Engineering Science
and Technology Research Journals, n.d.). Efforts have been made to investigate the effects of
hydrothermal processing methods on the nutrients and anti - nutritional components of
Mallotus subulatus (Ojo et al., 2017). These findings highlight the potential of underutilized
crops as sources of nutrition and the importance of further research in this area.
The implications of this study for nutrition and health are significant. The high protein
content of Mallotus subulatus, in particular, makes it a valuable source of nutrition for
individuals who may have limited access to other protein-rich foods (Ojo et al., 2017). This
can be especially important in areas where malnutrition and protein deficiency are prevalent.
Additionally, the potential of underutilized crops to provide a diverse range of nutrients can
contribute to improved overall health and well-being, especially in regions where access to a
variety of foods may be limited (Gangwar et al., 2014). The findings of this study, therefore,
have important implications for food security and nutrition policy.
Future directions for research on Mallotus subulatus and other underutilized crops are
numerous. Further investigation into the effects of processing methods on the nutrient
composition of these crops can help to identify the most effective methods of preparation and

31
preservation (Ojo et al., 2017). Additionally, research on the sensory qualities and consumer
acceptance of these crops can help to promote their wider use and incorporation into local
diets (Ma., 2022). Further studies can also explore the potential of these crops to provide
other health benefits, such as reducing the risk of chronic diseases (Gangwar et al., 2014).
Overall, the findings of this study highlight the importance of promoting and investing in
research on underutilized crops to address issues of food security and nutrition.

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