CHAPTER ONE

1.0 INTRODUCTION
1.1 Background of the Study
Coccidiosis is a parasitic disease caused by seven species of the genus Eimeria with different
localizations within the intestinal tract of chickens. Eimeria acervulina, E. maxima and E.
tenella are the most prevalent species in broilers in the intensive poultry management system
(Haug et al., 2008; Györke et al., 2013). The disease represents a serious threat for the poultry
industry, affecting the production, and causing high morbidity, mortality and significant
economic loss due to the associated costs of treatment and prophylaxis. Global financial losses
due to coccidiosis have been estimated at three billion USD per annum (Dalloul and Lillehoj,
2006).

In-feed anticoccidials have been used for decades for managing avian coccidiosis and they
were very effective until drug resistance emerged. To date, Eimeria strains have gained
resistance to all known coccidiostats, and new anticoccidials are unlikely to be developed,
mainly because of strict legislative regulations on the use of in-feed drugs and growing
concerns in the general population about the chemical residues in poultry products (Chapman,
1997; Blake and Tomley, 2014; Sundar et al., 2017).

Over the past years, the consumption of poultry meat has grown consistently, especially
because it represents a fairly cheap source of food with lower production costs and accepted
by all religions (Stenhouse, 2008). There is also a higher interest from the consumer in
organic poultry production and a great demand for natural and healthier products (Erian and
Phillips, 2017). In this context, the use of natural remedies has become a promising alternative
to anticoccidial drugs (Wunderlich et al., 2014). Numerous plant-based products have been
found to be effective at treating chicken coccidiosis: Artemisia annua and artemisinin (Allen
et al., 1997; Jiao et al., 2018), oregano (Giannenas et al., 2003), garlic (Pourali et al., 2014),
neem (Abbas et al., 2006), different species of Aloe (Marizvikuru et al., 2006), green tea (Jang
et al., 2007), sugar cane (Awais et al., 2011), turmeric (Kim et al., 2013) and many others
(Wunderlich et al., 2014; Abbas et al., 2012; Muthamilselvan et al., 2016; Quiroz-Castaneda
and Dantan-Gonzalez, 2015).

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Additionally, commercially available herbal combinations are already in used in some
countries for coccidiosis control (Abbas et al., 2012). Most of these natural compounds do not
always aim directly at the parasites but have immunomodulatory effects, antioxidative or anti-
inflammatory properties and act on the intestinal tract, thus helping the host organism to fight
against the coccidial infection (Abbas et al., 2012; Wunderlich et al., 2014). Moreover, the
plant extracts can have a direct effect on the parasites, by altering the process of oocyst wall
formation and inhibiting sporulation (del Cacho et al., 2010; Fatemi et al., 2015), or by
destroying the sporozoites (Kim et al., 2013). Furthermore, there is a lower risk of developing
resistance to these natural substances compared to anticoccidial drugs (Quiroz-Castaneda and
Dantan-Gonzalez, 2015). Furthermore, herbal extracts could improve recovery after
coccidiosis (Arczewska-Włosek and Świątkiewicz, 2012, 2013). Flavonoids and other
polyphenols have been reported to be responsible for most of the biological properties of the
herbs, including the anticoccidial potential (Masood et al., 2013).

Adansonia digitata and its related species belong to the family of Malvaceae. The tree is of
African origin known for its medicinal and nutritional value. It has excellent antioxidant and
anti-inflammatory properties; various parts of the tree are used to treat different types of
ailments (Gokila et al., 2014). Recent phytochemical analysis of its leaves revealed that they
contain a rich repertoire of reducing sugars, flavonoids, terpenoids, saponins, tannins,
alkaloids, anthraquinones, steroids, resins, phenols, and cardiac-active glycosides (GreenVet,
2016). Besides, the leaves have an abundant amount of mucilage, carbohydrate (60 – 70%),
protein (13 – 15%), fiber (11%), fat (4 – 10%), and minerals including calcium, iron,
potassium, magnesium, phosphorous, zinc, and manganese (Namratha and Baobab, 2015).
Previously, Talari et al. (2017), had concluded that methanolic extract of various parts of the
tree showed a strong free radical scavenging activity associated with their phytochemical
constituents. This property raises the possibility that these methanolic extracts could be
utilized in the preparation of antioxidant drugs for the treatment of a range of ailments (Talari
et al., 2017).

In many countries, various parts of the tree have been traditionally used in the treatment of
diarrhea and dysentery (Hossam et al., 2019). Besides, different parts of the tree have been

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reported to have analgesic, immunostimulant, anti-inflammatory, insect repellent, and
pesticidal properties (Hossam et al., 2019). Intriguingly, the leaf extract has ten times more
potent antioxidative capacity than vitamin C. Also, the leaf extract inhibits anti-inflammatory
iNOS (inducible nitric oxide synthase) expression, which might be related to the elimination
of peroxyl radicals as well as inhibition of signal transduction mediated by NF-κB. Its leaf
extract has significant antioxidant potential that might prevent malignancies related to
inflammation (Ayele et al., 2013). The methanolic extract of the bark of its stem has
previously been found to have a hypoglycemic activity in streptozotocin (STZ)-induced
diabetes (Tanko et al., 2008). Further, separate studies indicated that the methanolic extract of
the leaf and pulp of the plant also has a hypolipidaemic effect (Geidam et al., 2004; Bako et
al., 2014), making its parts suitable for treatment of different ailments.

1.2 Aim and Objectives of the Study

The purpose of this study was to assess the anticoccidial activity of methanol leaf extacts and
leave powder of Adansonia digitata on experimental infection of field isolates of Eimeria
species in broiler chickens.

The objectives include:

a) To determine the performance of broiler chickens infected with Eimeria species and
treated with Adansonia digitata methanol leaves extract or leaves powder of
Adansonia digitata.
b) To determine the haematological parameters of broiler chickens experimentally
infected with Eimeria species and treated with methanol leaves extract or leaves
powder of Adansonia digitata.
c) To check for occurrence of pathological lesions in the GIT of broiler chickens
experimentally infected with Eimeria species and treated with methanol leaves extract
or leaves powder of Adansonia digitata.
d) To evaluate the histopathology of some segments of the GIT of broiler chicken
induced coccidiosis and treated with methanol leaves extract and/or leaves powder of
Adansonia digitata.

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1.3 Statement of Research Problem
There is need to identify local plant based extracts for the treatment of poultry coccidiosis.
There is little or no documented information on the use of Adansonia digitata leaves in the
treatment of coccidiosis.

1.4 Justification for the Study

In contemporary research, plant-derived drugs are considered less toxic and more compatible
with biological systems because of their low side effects as compared to the synthetic ones.
Thus, a great deal of scientific research is focusing on developing new drugs based on natural
products or compounds of herbal origin for the treatment of diseases (Wilcox and Bodeker,
2004; Chang et al., 2013).

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 CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Coccidiosis
Each year, over 50 billion chickens are raised as a source of meat, accounting for over one-
third of source of protein for humans (Muthamilselvan et al., 2016). However, poultry
production is often confronted by avian coccidiosis, flu, and other infectious diseases (Quiroz-
Castañeda and Dantán-González, 2015). Avian coccidiosis is characterized as an infectious
protozoan disease caused by gut parasites of the genus Eimeria (Coccidia subclass) (Gilbert et
al., 2011).

So far, nine Eimeria species, E. acervulina, E. brunetti, E. maxima, E. necatrix, E. praecox, E.

mitis, E. tenella, E. mivati, and E. hagani, have been identified from chickens (Joyner and
Long, 2008). These parasites can infect and multiply within the mucosal epithelia in different
parts of bird guts via oral route. As a result, they cause gut damage (i.e., inflammation,
hemorrhage, diarrhea, etc.), morbidity, and mortality in poultry (Chapman, 2014). This
disease annually causes a global loss of over 2.4 billion US dollars in the poultry industry,
including poor growth performance, replacement of chicks, and medication (Quiroz-
Castañeda and Dantán-González, 2015).

Current approaches to constrain avian coccidiosis include anticoccidial chemicals, vaccines,

and natural products. Anticoccidial chemicals, coccidiocides, coccidiostats, and ionophores,
have long been used as a mainstream strategy to control avian coccidiosis in modern poultry
production (Chapman et al., 2010). Although this strategy is cost-effective and successful, the
presence of drug resistance and public demands for residue-free meat has encouraged
development of alternative control strategies (Chapman et al., 2010). Moreover, in European
countries, the prophylactic use of anticoccidial chemicals as feed additives has been strictly
limited since 2006 and a full ban has been proposed to be effective in 2021 (Council Directive
of 2011/50/EU of the European Council). To cope with this global situation, vaccination,
composed of one or more strains of wild-type or attenuated Eimeria species is successfully
developed as another approach to prevent coccidiosis, though their cross-species protection

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and efficacy may need to be improved. Natural products are emerging as an attractive way to
combat coccidiosis (Muthamilselvan et al., 2016).

Currently, there are at least four plant products commercially available in the market and they
can be used as anticoccidial feed additives in chickens and/or other animals, including Cocci-
Guard (DPI Global, USA) (Muthamilselvan et al., 2016), a mixture of Quercus
infectoria, Rhus chinensis, and Terminaliachebula (Kemin Industries, USA) (Gokila et al.,
2014), Apacox (GreenVet, 2016), and BP formulation made up of Bidens pilosa and other
plants (Yang et al., 2015). Besides, investigation of the compounds and/or their derivatives
present in anticoccidial plants may inspire the research and development of anticoccidial
chemicals. One successful example is halofuginone, a synthetic halogenated derivative of
febrifugine, which was initially identified from the antimalarial plant, Chang Shan (Dichroa
febrifuga) (Muthamilselvan et al., 2016). Despite the aforementioned merits of natural
products, several challenges in the anticoccidial use of natural products such as anticoccidial
efficacy, identification of active compounds, mechanism, safety, and cost-effectiveness of
plant extracts and compounds need to be overcome prior to further applications.

Several reviews were published on the plant extracts and their phytochemicals for avian
coccidiosis (Abbas et al., 2012; Bozkurt et al., 2013;Quiroz-Castañeda and Dantán-González,
2015).

2.2 Life Cycle of Eimeria Species

Eimeria species have a complex life cycle, consisting of three developmental stages
(sporogony, schizogony/merogony, and gametogony). Oocysts excreted in poultry feces
sporulate in a favourable environment with high humidity at 25 – 30°C. Once sporulating
oocysts are ingested by birds, physical and chemical agents in their digestive tracts are
released and mature infectious sporozoites form sporocysts. The sporozoites enter epithelial
cells in the gut, depending on the specific Eimeria species, and form trophozoites and, later,
schizonts during schizogony/merogony (Blake and Tomley, 2013).

Merozoites, released from the schizont, can penetrate into the epithelial and continue this
merogony stage 2 to 3 times in order to increase the cell number of merozoites at the asexual
stage of reproduction. Alternatively, merozoites may enter the sexual stage of reproduction by

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forming male microgametes, the equivalent of sperm, and female macrogametes, and the
equivalent of oocytes, in host cells. Following fertilization, the zygotes develop into oocysts
and are excreted into poultry stool. Eimeria species may require 4 – 7 days to complete their
entire life cycles (Muthamilselvan et al., 2016). Thus, the asexual and sexual stages of
reproduction in the Eimeria life cycle have been targeted by anticoccidial compounds
(Chapman, 2014).

2.3 Prophylaxis and Therapy for Avian Coccidiosis

Coccidiosis is one of the major problems in intensive poultry farming (Quiroz-Castañeda and
Dantán-González, 2015). In contrast to anticoccidial chemicals and vaccines, the use of
medicinal plants and phytocompounds as natural remedies for avian coccidiosis has become
an alternative strategy that is easily embraced by eco- and health-conscious consumers. This
strategy is also in compliance with the “anticoccidial chemical-free” regulation enacted by the
European Union (Council Directive of 2011/50/EU). Plants produce a broad-spectrum variety
of phytochemicals such as phenolics, polyacetylenes, alkaloids, polysaccharides, terpenoids,
and essential oils with a large number of bioactivities (Xu, 2011; Efferth and Koch, 2011;
Bozkurt et al., 2013). Different reports on the use of medicinal plants for diseases suggest that
they have the potential to “kill several birds with one stone” because they contain multiple
phytochemicals and can intervene in multiple disease-related signalling pathways (Chang et
al., 2013).

2.4 Plants and Compounds for Avian Coccidiosis

Over 300,000 species of flowering plants have been recorded worldwide. So far, less than 1%
of them have been explored for use against protozoan diseases (Willcox and Bodeker, 2004).
In this section, a total of 68 plants and phytocompounds, which were scientifically tested for
suppression of Eimeria species, are described and discussed (Abbas et al., 2012; Bozkurt et
al., 2013; Quiroz-Castañeda and Dantán-González, 2015; Yang et al., 2015).

2.5 Plants and Compounds That Inhibit the Life Cycle of Eimeria
The phytochemicals and plants, which suppress coccidiosis via intervention with the
developmental stages of life cycle in Eimeria species in poultry, are hereby discussed. They

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are summarized and their chemistry and mechanism of action of phytochemicals and plants
are also described below.

2.5.1 Artemisia annua and Artemisinin

Artemisia annua and its constituent active compound artemisinin have been reported to have
anticoccidial action. Mechanistic studies show that this compound generated reactive oxygen
species (ROS) via degradation of iron-implicated peroxide complex and, therefore, induced
oxidative stress. Note, ROS was documented to directly inhibit sporulation and cell wall
formation in Eimeria species, leading to interference with the life cycle of Eimeria (Oh et al.,
1995; Allen, 1997; Allen et al., 1998; Del Cacho, 2010; Almedia et al., 2012). In addition, A.
annua has lots of phytochemicals, flavonoids, and phenolic compounds which can help birds
maintain commensal microflora and take up large amounts of nitrogen. Commensal bacteria
play a significant role in enhancing digestion of food and absorption of nutrients and improve
innate and acquired immune response in poultry (Brisbin et al., 2008).

2.5.2 Condensed Tannins and Pine Bark

The extract from the bark of the pine tree (Pinus radiata), which is rich in condensed tannins,
was reported to inhibit the life cycle of Coccidia as evidenced by decreased sporulation of the
oocysts of E. tenella, E. maxima, and E. acervulina (Molan et al., 2009). The mode of action
of condensed tannins was suspected to be penetration of the wall of the oocyst and damage to
the cytoplasm since the tannins could inactivate endogenous enzymes responsible for the
sporulation process. This was further supported by the appearance of abnormal sporocysts in
oocysts (Muthamilselvanet al., 2016).

2.5.3 Garlic (Allium sativum) and Allicin

Garlic and its sulfur compounds, allicin, alliin, ajoene, diallyl sulfide, dithiin, and
allylcysteine, are reported to have broad antimicrobial activities which can eliminate negative
factors of microbial infections. An in vitro study has shown that allicin inhibits sporulation
of E. tenella effectively (Elkhtam et al., 2014; Pourali et al., 2014; Alnassan et al., 2015). The
anticoccidial mechanism of garlic and its sulfur compounds remains elusive.

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2.5.4 Selenium, Phenolics, and Green Tea (Camellia sinensis)
Green tea extracts have been shown to significantly inhibit the sporulation process of
coccidian oocysts (Jang et al., 2009; Molan and Faraj, 2009). Accordingly, the selenium and
polyphenolic compounds in green tea are thought to be active compounds to inactivate the
enzymes responsible for coccidian sporulation (Muthamilselvan et al., 2016).

2.5.5 N-3 Fatty Acids, Flavonoids, Vernoside, and Their Plant Sources
After the invasion of Eimeria sporozoites into the intestinal epithelium, reactive nitrogen
species (RNS) and reactive oxygen species (ROS) are often produced by host cells, leading to
the death of sporulating oocysts (Allen et al., 1998). Similarly, another study demonstrated
that the extracts from Berberis lyceum, in which berberine was enriched, inhibited the
sporozoites of E. tenella in chickens via induction of oxidative stress. Other studies indicated
that extracts from Linumusitatissimum (Muthamilselvan et al., 2016), Ageratum
conyzoides (Nweze and Obiwulu, 2009) and Vernonia amygdalina (Al-Fifi, 2007) controlled
coccidian infection via induction of oxidative stress. Moreover, N-3 fatty acids, flavonoids,
and vernoside were identified as active compounds present in L. usitatissimum, A. conyzoides,
and V. amygdalina, respectively. These compounds were shown to elicit oxidative stress.
Oxidative stress is known to cause imbalance of oxidant or antioxidant species in the host and
is often observed in a wide range of microbial and parasitic infections including coccidiosis
(Allen, 1997). Moreover, these natural extracts not only enhanced chicken growth but also had
no noticeable toxicity.

2.5.6 Caricapapaya and Papain

Two studies have reported that extracts from C. papaya leaves significantly inhibit coccidiosis
(Al-Fifi, 2007; Nghonjuyi, 2015). Little is known about the anticoccidial mechanism.
Proteolytic destruction of Eimeria by papain and/or inflammatory suppression by vitamin A
were proposed as possible mechanisms by which C. papaya and its active compounds acted to
suppress coccidiosis (Muthamilselvan et al., 2016).

2.5.7 Saponin, Betaine, and Their Plant Sources

Hassan et al. (2008), demonstrated that dietary supplementation of guar bean (Cyamopsis
tetragonoloba) suppressed coccidiosis in chickens. This suppression was proposed to be

9
achieved by saponins, presumably the active compounds, which bind with sterol molecules
present on the cell membrane of the parasites. Another study also reported that the extracts
of M. cordifolia, M. citrifolia, and M. arboreus showed anticoccidial effects in chickens
(Rakhmani et al., 2014). Saponins were presumed to be the active compounds which could
lyse oocysts. In contrast, another report described betaine, an active compound isolated from
beet or other plants, as contributing to the stabilization and protection of the epithelial cells in
which Eimeria multiply (Augustine et al., 1997).

2.5.8 Essential Oils and Their Plant Sources

Essential oils derived from plants showed inhibition of Eimeria species at different
developmental stages. Essential oils are an important natural product resource, which are rich
in many phytocompounds. Both in vitro and in vivo studies reported that essential oils can be
used as feed additives in chickens to control coccidiosis (Abbas et al., 2012; Bozkurt et al.,
2013; Quiroz-Castañeda and Dantán-González, 2015). Bioactive compounds present in the
essential oils extracted from Oreganum. compactum,  A. absinthium, Rosmarinus.
officinalis, Anredera. cordifolia, Morinda. citrifolia, Malvaviscus. arboreus, Syzygium.
aromaticum, Melaleuca alternifolia, Citrus sinensis, and Thymus vulgaris were able to destroy
the parasites, including oocysts and sporozoites.

2.5.9 Maslinic Acid

Maslinic acid, an active compound in the leaves and fruit of the olive tree (Olea europaea),
was originally identified as a novel anticoccidial compound as indicated by the lesion index,
the oocyst index, and the anticoccidial index (De Pablos et al., 2010). However, its
anticoccidial activity remains unknown.

2.5.10 Proanthocyanidin and Grape Seed

Proanthocyanidin is a naturally occurring polyphenolic antioxidant widely distributed in grape
seed and other sources. Grape seed proanthocyanidin extract was shown to reduce E.
tenella infection as shown by gut pathology, body weight, and mortality (Wang et al., 2008).
Accordingly, this extract decreased nitric oxide but increased superoxide dismutases in the
plasma of chickens (Muthamilselvan et al., 2016). These data suggest that proanthocyanidin
from grape seed diminishes coccidiosis via downregulation of oxidative stress.

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2.5.11 Dichroafebrifuga
Dichroa febrifuga, also known as Chang Shan, is a Chinese medicinal herb for protozoan
diseases. Zhang and coworkers showed that crude extract of D. febrifuga was effective
against E. tenella infection in chickens (Zhang et al., 2012). Febrifugine, an alkaloid, was
isolated from this plant and its halogenated derivative, halofuginone, was developed as
anticoccidial chemical (Muthamilselvan et al., 2016).

2.6 Plants and Compounds That Modulate Host Immunity against Eimeria

From an evolutionary point of view, birds have a complete immune system consisting of
innate and adaptive immune responses (Yegani and Korver, 2008). Both immune responses
are responsible for coccidial clearance and vaccine immunization (Abbas et al., 2012; Bozkurt
et al., 2013). Medicinal plants often have immunomodulatory compounds which boost
antimicrobial immune responses to uphold homeostasis of poultry health (Sugiharto, 2014;
Akram et al., 2014). Therefore, immunoregulatory plant extracts and compounds could be
utilized as an alternative method to reinforce immune response against avian coccidiosis.

2.6.1 Arabinoxylans, Wheat (Triticumaestivum), and Sugar Cane

(Saccharumofficinarum)
Akhtar et al. (2012) showed that arabinoxylan, a bioactive compound from wheat bran,
improved coccidiosis in chickens as indicated by body weight, oocyst count, and gut lesions
(Akhtar et al., 2012). In contrast, Awais and Akhtar (2012), reported that different extracts of
sugar cane juice and bagasse protected against coccidiosis in chickens as shown by body
weight gain, oocyst shedding, lesion score, and anticoccidial indices. The data from both
studies revealed that wheat bran arabinoxylan and sugar cane conferred host protection
against Eimeria infection via natural and adaptive immune response. Cell-mediated immunity
seemed to be a key factor in response to coccidiosis in chickens when compared to humoral
immunity (Akhtar et al., 2012; Awais and Akhtar, 2012).

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2.6.2…Polysaccharides.from Astragalus.membranaceus,.Radix, Carthamus.tinctorius Len
tinus edodes, and Tremella fuciformis
Guo et al. (2004) reported that the polysaccharides derived from the herb A. membranaceus
Radix and.the.mushrooms L..edodes and T..fuciformis effectively.controlled E..tenella infection
in chickens. Concurrent with the anticoccidial protection, the polysaccharides could enhance
anticoccidial antibodies and antigen-specific cell proliferation in splenocytes via cellular and
humoral immunity to E. tenella in chickens. Their mechanism appeared to stimulate cell
proliferation of the lymphocytes via regulation of DNA polymerase activity (Muthamilselvan et
al., 2016).

2.6.3 Cinnamaldehyde, Carvacrol, Capsicum oleoresin, and Turmeric oleoresin

Two phytonutrient mixtures, VAC (carvacrol, cinnamaldehyde, and Capsicum oleoresin) and
MC (Capsicum oleoresin and Turmeric oleoresin), were tested for coccidiosis in chickens
(Lee et al., 2011). The data proved that both combination treatments, effectively protected
against E. tenella infection. Moreover, both treatments exhibited an increase in NK cells,
macrophages, CD4+ T cells, CD8+ T cells, and their cytokines (IFN-γ and IL-6) and a decrease
in TNFSF15 and IL-17F, leading to induction and elevation of host immunity to kill E.
tenella in chickens (Muthamilselvan et al., 2016).

2.6.4 Aloe and Acemannan

Gadzirayi et al. (2005), showed that A. excelsa possesses anticoccidial activity in chickens
(Augustine et al., 1997; Kheirabadi et al., 2014; Akhtar et al., 2012), despite lack of
information about its mode of action and its active compound(s). Another study stated that
aqueous and ethanolic extracts from A. vera mounted a cell-mediated immune response as
well as a humoral response against coccidiosis in chickens. The immunomodulatory
compounds could include aloe polysaccharide acemannan, which binds the mannose receptor
on macrophages, stimulating them to produce inflammatory cytokines such as IL-1 through
IL-6 and TNF-α and eventually suppress coccidiosis as shown by greater weight gain and
lower fecal oocyst counts (Babak and Nahashon, 2014; Akhtar et al., 2012).

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2.6.5 Oriental Plum (Prunussalicina) and Phenolics
One report showed that dietary supplementation of plum fruit powder, rich in phenolic
compounds, added to chicken feed significantly diminished E. acervulina infection in
chickens as demonstrated by increased body weight gain and reduced fecal oocyst shedding
(Lee et al., 2008). Accordingly, plum fruit powder greatly augmented the transcription of
IFN-γ and IL-15 and splenocyte proliferation, indicating that plum fruit can boost immune
response to coccidiosis.

2.6.6 Mushroom (Fomitella fraxinea) and Lectin

One study showed that the lectin derived from a mushroom, F. fraxinea, protected chickens
from Eimeria challenge via enhancement of both cellular and humoral immune responses
(Dalloul and Lillehoj, 2006). This work also suggested that this mushroom could enhance
both immune responses to Eimeria species in chickens. This study implies that
immunoregulatory botanicals such as mushroom can improve poultry growth and
development via immune protection from infectious pathogens and toxins. Moreover,
botanicals, containing micro- and macronutrients, can increase growth performance in poultry
(Muthamilselvan et al., 2016).

2.6.7 Propyl Thiosulfinate and Propyl Thiosulfinate Oxide

One study showed that garlic compounds, propyl thiosulfinate (PTS) and propyl thiosulfinate
oxide (PTSO), could alter the expression levels of 1,227 transcripts related to intestinal
intraepithelial lymphocytes (IEL) in chickens. PTSO/PTS was shown to activate transcription
factor, NF-κB, which plays a key role in regulating the immune response upon infection.
Therefore, it seems that a combination of PTSO and PTS rendered chickens more resistant to
experimental E. acervulina infection and augmented adaptive immunity, including a higher
antibody response and greater splenocyte proliferation, compared with control chickens (Kim
et al., 2013). Another in vitro study showed that PTS could stimulate splenocyte proliferation
and directly kill the sporozoites, pointing to the same conclusion (Kim et al., 2013).

2.6.8 Tannins and Chicoric Acid from Emblica officinalis and Echinacea purpurea

Tannins and chicoric acid, isolated from E. officinalis (Kaleem et al., 2014) and E.
purpurea (Zhai et al., 2007), respectively, were reported to effectively elicit humoral immune

13
response against coccidial infection in chickens. However, the mechanism by which both
compounds boost anticoccidial immunity is not clear.

2.7 Plants and Compounds That Possess Prebiotic Properties

Like in humans, gut microbiota are important for health and disease in poultry (Adil and
Magray, 2012). Gut microbiota perform multiple functions involved in nutrient digestion, gut
development and growth, establishment/maintenance of the immune system, suppression of
pathogenic microbes, microbial infections, and so forth (GreenVet, 2016; Ohimain and C,
2012; Castanon, 2007; Serratosa et al., 2006; van der Wielen et al., 2002; Vispo and Karasov,
1997). Thus, promotion of beneficial microbes and reduction of harmful microbes contribute
to growth performance and health in poultry (Adil and Magray, 2012). Several studies have
indicated that probiotics, containing one or multiple species of Lactobacillus, Enterococcus,
and/or Bifidobacterium, can reduce coccidiosis and enhance growth performance in chickens
(Lee et al., 2012; Abbas et al., 2012; Ohimain and Ofongo, 2012; Giannenas et al., 2014;
Ritzi et al., 2014). Currently, little is known about the anticoccidial mechanisms of probiotics.
These modes of action have been proposed: maintaining a healthy balance of bacteria by
competitive exclusion and antagonism, promoting gut maturation and integrity, modulating
immunity and preventing inflammation, altering metabolism by increasing digestive enzyme
activity and decreasing bacterial enzyme activity and ammonia production, improving feed
intake and digestion, and neutralizing enterotoxins and stimulating the immune system (van
der Wielen et al., 2002; Sugiharto, 2014). On the other hand, coccidiosis is frequently
accompanied by secondary bacterial infection (Williams, 2002; Serratosa et al., 2006;
Castanon, 2007; Orengo et al., 2012).

Prebiotics refer to nondigestible feed ingredients that promote the growth of probiotics and
their activities in guts (Bindels et al., 2013). The most common prebiotics, used in poultry,
include inulin, arabinoxylooligosaccharides (AOS), fructooligosaccharides (FOS), mannan-
oligosaccharides (MOS), xylooligosaccharides (XOS), isomaltooligosaccharides (IMOS), soy
oligosaccharides (SOS), and pyrodextrins (Al-Sheraji et al., 2013; Alloui et al., 2013). These
oligosaccharides are derived from the plants such as chicory, onion, garlic, asparagus,
artichoke, leek, bananas, tomatoes, and wheat (Al-Sheraji et al., 2013). Dietary

14
supplementation of these prebiotics to chicken feed has enhanced immune defence against
pathogen infection and reduced the mortality rate (Ganguly, 2013; Sugiharto, 2014). The
mechanism of prebiotics is yet to be revealed, but they selectively stimulate beneficial bacteria
in the intestinal system of the bird. The increasing number of beneficial microbiota excludes
the harmful pathogens from colonization in the intestinal track of the bird. Subsequently,
healthy hosts can produce a wide variety of bacterions and other immunomodulators that can
stimulate macrophages to neutralize the pathogens (Alloui et al., 2013). Thus, prebiotic-
mediated immunological changes may be partially due to direct interaction between prebiotics
and gut immune cells as well as due to an indirect action of prebiotics via preferential
colonization of probiotics and their products that interact with immune cells (Janardhana et
al., 2009). Therefore, prebiotics exert their functions mainly via increasing gut probiotics to
suppress pathogens and boosting immune response in chickens to constrain gut pathogens
(Sugiharto, 2014). Moreover, Bozkurt et al. (2014) reported that prebiotics diminished
coccidial infection in chickens but kept marginal oocyst production that might serve as a
source of live vaccine for uninfected chickens. Two other publications emphasized that
probiotics composed of Bifidobacterium animalis and Lactobacillus salivarius alleviated the
detrimental impact of the Eimeria infection on chickens and improved growth performance
(Giannenas et al., 2014; Ritzi et al., 2014). These findings suggest that prebiotics suppress
coccidiosis plausibly via indirect regulation of increased probiotics and host immunity.
Apparently, prebiotics share many similar anticoccidial mechanisms with probiotics. Overall,
dietary supplementation of prebiotics is emerging as a novel approach to control coccidiosis
(Muthamilselvan et al., 2016).

2.8 Plants and Compounds with Multiple Mechanisms to Inhibit Coccidiosis

2.8.1 Curcumin and Curcuma longa
As described in Table 1, turmeric (C. longa) has long been used as a spice and medicinal herb.
An author stated that C. longa showed anticoccidial activity (Abbas et al., 2010; Kim et al.,
2013). Another reported that curcumin (diferuloylmethane), an active compound in C. longa,
consistently destroyed sporozoites of E. tenella (Khalafalla et al., 2011). Similarly, a
combination of A. annua and C. longa showed anticoccidial efficacy in broilers challenged
with a mixture of E. acervulina and E. maxima (Almeida et al., 2014). In addition, curcumin

15
was shown to enhance coccidiosis resistance as evidenced by increased BW gains and reduced
oocyst shedding and gut lesions. Consistently, curcumin elevated host humoral immunity
to Eimeria species and diminished gut damage in poultry (Allen et al., 1998; Kim et al.,
2013).

2.8.2. Polyacetylenes and Bidenspilosa

It has been demonstrated that B. pilosa has exhibited anticoccidial activity in chickens
infected with E. tenella as evidenced by survival rate, fecal oocyst count, gut pathology, body
weight, and bloody stool (Yang et al., 2015). Although the active compounds in B.
pilosa responsible for anticoccidial action are unknown, this plant is a rich source of
phytochemicals, such as 70 aliphatics, 60 flavonoids, 25 terpenoids, 19 phenylpropanoids, 13
aromatics, 8 porphyrins, and 6 other compounds (Bartolome et al., 2013). Interestingly, one
polyacetylene (1-phenyl-1,3-diyn-5-en-7-ol-acetate) and one flavonoid (quercetin-3,3-
dimethoxy-7-0-rhamnoglucopyranose) in this plant have been proposed to be active
compounds against the protozoan parasite, Plasmodium (Daugschies et al., 1998).

However, the identity of the active compounds needs to be further ascertained. The
mechanism of B. pilosa and its active compounds is not clear. It is possible that this plant and
its active compounds intervene in the initial phases of the Eimeria life cycle because the
phases may be liable to chemical attack when compared with the oocyst whose wall is very
resistant to physical and chemical insults. In addition, B. pilosa was shown to modulate host
immunity (Chang et al., 2005), which might have impact on coccidiosis.

Compared to anticoccidial drugs, B. pilosa was shown to produce little or no drug resistance

in Eimeria (Yang et al., 2015). Botanicals developed low resistance in Eimeria probably
because different compounds target multiple pathways related to drug resistance (Bozkurt et
al., 2013; Yang et al., 2015).

It should be noted that the plant- and phytochemical-based remedies can be used per se or in
combination with other anticoccidial agents. This idea was further confirmed by a researcher
indicating that Echinacea, an immunotherapeutic herbal extract, was used to boost the
immunization efficacy in chickens in combination with anticoccidial vaccines (Allen, 2003).

16
2.9 Baobab (Adansonia digitata) 
Baobab (Adansonia digitata) is a large iconic tree indigenous to Africa where it is found in
many countries. It is an emblematic, culturally important and physically majestic sub-tropical
tree. The baobab has been referred to as “arbre a palabre”, meaning the place in the village
where the elders meet to resolve problems. In the past decade, it has attracted the interest of
several pharmaceutical companies and researchers due to its various traditional uses
(medicinal, nutritional and cosmetic). Recently, the European Commission authorised the
import of baobab fruit pulp as a novel food (Buchmann et al., 2010) and it was approved in
2009 by the Food and Drug Administration as a food ingredient in the United States of
America (Addy, 2009). Due to the high demand for commercial baobab products in EU and
United States, this tree with its edible fruits needs to be conserved and treasured (Sanchez et
al., 2010).

Baobab products (e.g. fruits, seeds, leaves, bark) contribute to the livelihood of many

populations in Africa as it is a source of food, fibre and medicine (Wickens, 1982; Codjia et
al., 2001; Sidibe and Williams, 2002; Chadare et al., 2009; De Caluwé et al., 2009). More
than three hundred traditional uses have collectively been documented in Benin, Mali,
Zimbabwe, Cameroon, the Central African Republic, Kenya, Malawi, South Africa and
Senegal (Buchmann et al., 2010). Various plant parts (e.g. leaves, bark, fruit pulp), have
traditionally been used for immuno-stimulant, anti-inflammatory, analgesic, insect repellent
and pesticidal properties, in the treatment of diarrhoea and dysentery in many African
countries, and have been evaluated as a substitute for imported western drugs (Ramadan et al.,
1994; El-Rawy et al., 1997 ). Some of the traditional medicinal uses of baobab are presented
in Table 1. Baobab products (e.g. seed oil, fruit pulp) are increasingly being commercialised
and exported around the world leading to increased pressure on this resource (Sidibe and
Williams, 2002).

Table 1. Selected traditional medicinal uses of the A. digitata tree in Africa

17
Therapeutic uses Plant part Country Preparation Reference
used

Fever, diarrhoea Seeds South Africa Mixed with Watt and Breyer-
water Brandwijk (1962)

18
Therapeutic uses Plant part Country Preparation Reference
used

Dysentery, fever Seeds, Cameroon, Decoction Watt and Breyer-

fruits Central African Brandwijk (1962)
Republic

Malaria, fever Leaves Sierra Leone – Watt and Breyer-

Brandwijk (1962)

Coughs Powdered South Africa – Watt and Breyer-

seeds Brandwijk (1962)

Diarrhoea, fever, Leaves South Africa Infusion Van Wyk and Gericke
inflammation, kidney (2000)
and bladder diseases,
blood clearing, asthma

Fever, dysentery Leaves, Sudan – Gebauer et al. (2002)

roots

Anaemia Bark Nigeria Aqueous extract Adesanya et al.

(1988)

Malaria Bark, Nigeria Powdered bark Ajaiyeoba et al.

leaves mixed with (2004)
porridge

Diarrhoea, fever, Leaves Tanzania Decoction, Brendler et al. (2003)

inflammation, kidney infusion
and bladder diseases,
blood clearing, asthma

Dysentery, fever, Fruits, Tanzania Decoction FAO (1993)

haemoptysis, diarrhoea seeds

Refreshing, tonic, Flesh with Burkina Faso Decoction Kerharo and Adam,

19
Therapeutic uses Plant part Country Preparation Reference
used

diuretic, cystitis, peel (1974); Nacoulma,

dysentery, hepatic (1999)
disorders, hypogalactia

Toothache, gingivitis Leaves Burkina Faso – Tapsoba and

Deschamps (2006)

Diarrhoea, worms Leaves, Côte d'Ivoire – Aké Assi (1992)

seeds, fruit
pulp

Wound healing Stem bark Mali Decoction Inngjerdingen et al.

(2004)

Diaphoretic, fever Leaves Kenya Decoction Abbiw (1990)

remedy

Diaphoretic, kidney Leaves – – Wickens (1982)

and bladder diseases,
asthma, insect bites

Microbial diseases Fruits Nigeria and – Hostettmann et al.

Senegal (2000)

2.10.1 Botanical aspects

2.10.1.1 Origin of the name of the plant
The origin of the vernacular name “baobab” is uncertain. However, most scientists believe it
is derived from the Arabic name buhibab meaning fruit with many seeds (Diop et al., 2005).
The genus name Adansonia is used in honour of Michel Adanson (1727–1806) who brought
the seeds to Paris in 1754 and was the first person to provide a comprehensive description
accompanied by a drawing of the plant (Esterhuyse et al., 2001), after a trip to West Africa
(Senegal). The species name digitata (hand-like) was selected in reference to the shape of the
leaves. Several names are used to describe the baobab depending on its geographical location

20
and include “magic tree”, “chemist tree”, “symbol of the earth”, “upside-down tree” and
“monkey bread of Africa” amongst numerous others (Wickens, 1982; Diop et al.,
2005; Vermaak et al., 2011).

2.10.1.2 Botanical description, habitat and distribution in Africa

The baobab is found in many African countries. Eight baobab species have been identified
globally and six species found on the island of Madagascar are endemic to that region
(Wickens and Lowe, 2008). It is postulated that the centre of evolutionary origin of the
genus Adansonia is Madagascar (Drake, 2006). The African species A. digitata is indigenous
to, and widely distributed throughout the savannas and savanna woodlands of sub-Saharan
Africa (Wickens and Lowe, 2008). The only species which is not endemic to the African
continent is A. gibbosa (A.Cunn.) Guymer ex D.A.Baum native to Australia (Drake,
2006; Wickens and Lowe, 2008). In southern Africa, A. digitata is commonly found in
Malawi, Zimbabwe, Mozambique and South Africa especially in the warm parts of the
Limpopo Province, while in West Africa, it is found in Mali, Benin, Senegal, the Ivory Coast,
Cameroon and Burkina Faso. In East Africa, the plant is found in countries such as Kenya,
Uganda and Tanzania (Watt and Breyer-Brandwijk, 1962; Adesanya et al., 1988; UNCTAD
(United Nations Conference on Trade and Development), 2005; Lamien-Meda et al., 2008).

The baobab is a massive deciduous tree easily distinguishable by its huge trunk. It is regarded

as the largest succulent plant in the world with a diameter of 10–12 m and a height of 23 m or
more (Wickens, 1982; Chadare et al., 2009). Some baobab trees bear leaves only for three
months per year. During the leafless period physiological processes such as photosynthesis
take place in the trunk and branches, utilising water stored in the trunk (Gebauer et al., 2002).
The large egg-shaped fruit capsule is covered with velvety hairs, can reach 12 cm and
contains many seeds (Wickens, 1982). It is estimated that it takes between eight and twenty-
three years before the baobab produces seeds and the mature plant (over 60 years) can
produce more than 160 – 250 fruits per year (UNCTAD, 2005). The baobab produces large
pendulous white flowers from October to December (Watson, 2007). Assogbadjo et al.
(2005), showed correlation between soil minerals and/or soil type and fruit production. For
instance, the higher the pH–KCl, the percentage of total nitrogen, organic carbon and organic

21
matter, the higher the number of seeds produced by an individual baobab. It was also noted
that the higher the clay and crude silt content of the soil, the better the fruit production.

Baobab is restricted to hot, semi-arid regions, dry woodland and stony places with low rainfall
(less than 1500 mm annually) (Gebauer et al., 2002) and grows on a wide range of well-
drained soils, from clays to sands, but not on deep unconsolidated sands, where it is unable to
obtain sufficient moisture or anchorage (Wickens and Lowe, 2008). In Africa, the plant grows
at a latitude of 16° N and 26° S in areas not receiving more than one day of frost per year. The
tree grows very slowly probably due to low amount of rainfall received (Venter and Venter,
1996).

2.10.1.3 Pollination, dispersion and cultivation

The white flowers are pollinated by fruit bats that feed on the nectar at night (Fujita,
1991; Watson, 2007). A. digitata is widely spread over the African savanna through natural
reproduction (seeds). Many animals will eat the fruit contents once the outer shell has
withered and broken, and may at the same occasion assist in seed dispersal (Wickens and
Lowe, 2008). Dormancy is broken when the seeds pass through the digestive tract of animals
consuming the fruit. Esenowo (1991), found that the most effective method to break the
dormancy was scarification, and cultivation requires that the seeds be treated before sowing.
The seeds generally take three to five weeks to germinate (Diop et al., 2005), while plants
grown from seed start flowering after eight to twenty-three years. The flowering period of
baobab which is very long can be reduced to less than five years by grafting (Sidibe and
Williams, 2002). Young trees grafted from elite trees with desirable characteristics develop
faster than trees grown from seed. Therefore, good possibilities for future vegetative
cultivation and commercialisation of baobab products can be expected. The probability
of seed germination of baobab is very low (10%) and studies have shown that the probability
of germination will increase up to 85% if the seeds are soaked before sowing (Diop et al.,
2005). In Burkina Faso, people have started planting baobab trees (Ouedraogo, 2004).

In the past few years, consumer products derived from the seed oil and fruit pulp have been
exported to European and USA markets and the demand for these products are increasing
(UNCTAD, 2005). An increased demand can lead to overexploitation of the plant therefore it

22
is important to determine the factors that could lead to the successful cultivation of this
commercially important tree. Ecological niche modelling studies were undertaken to
determine factors that are crucial to the cultivation of baobab and results indicated that annual
precipitation and seasonal temperature fluctuations were two key factors (Sanchez et al.,
2010). The results of the ecological modelling also predict that baobab could be widely
cultivated in most countries in southern Africa and in the Sudano-Sahelian zone of West
Africa from Senegal to Sudan (Sanchez et al., 2010). Furthermore, the modelling prediction
showed that Angola and Somalia are highly suited for cultivating baobab in Africa, while
India was determined to be the most suitable country for baobab cultivation outside Africa
(Sanchez et al., 2010). However, many other factors such as pollinator agents, radiant energy,
soil aeration and structure, soil reaction, biotic factors (allelopathy, heavier fertilisation),
mineral nutrient supply and pollination agents should be incorporated in the model in order to
determine more accurately the potential area of baobab cultivation.

Assogbadjo et al. (2008) investigated the perception and preferences of baobab products in

Benin, Burkina Faso, Ghana, and Senegal and the survey included women and men of
different ages. According to this study, the easier the bark harvesting, the tastier the pulp and
leaves; the slimier the pulp, the less tasty it is; the more closely longitudinally marked the fruit
capsules, the tastier the pulp. This study shows that farmers are able to use preferred
combinations of traits as a guide in collecting germplasm from trees. The indigenous
knowledge can therefore be used to select an appropriate tree candidate for propagation, and
planning for a domestication programme.

23
Picture of Adansonia digitata tree

2.10.2 Traditional and modern uses

Throughout Africa the baobab is regarded with awe by most indigenous people; some even
consider it bewitched (Wickens and Lowe, 2008). Almost all parts of the tree are used in
traditional medicine in Africa although this varies from one country to another. The
traditional medicinal uses of baobab in Africa are summarised in Table 1. The various parts of
the plant (leaves, bark, seeds) are used as a panacea, that is, to treat almost any disease but
specific documented uses include the treatment of malaria, tuberculosis, fever, microbial
infections, diarrhoea, anaemia, dysentery, toothache, etc. (Watt and Breyer-Brandwijk,
1962; Adesanya et al., 1988; Abbiw, 1990; Van Wyk and Gericke, 2000; Brendler et al.,
2003; Tapsoba and Deschamps, 2006; Wickens and Lowe, 2008; De Caluwé et al.,
2009; Nguta et al., 2010). The leaves and fruit pulp are used as febrifuge as well as
an immunostimulant (El-Rawy et al., 1997; De Caluwé et al., 2009). In Nigeria, the leaves of
baobab are used to overcome fever (Wickens, 1982). In India, it is reported that baobab pulp
is used externally with buttermilk for the relief of diarrhoea and dysentery, while the young
leaves are crushed and used to treat painful swellings (Sidibe and Williams, 2002).
Furthermore, it is reported that the leaves of baobab are used to treat various conditions
including internal pains, disease of the urinary tract, otitis, as a tonic and for insect bites
and Guinea worms (Sidibe and Williams, 2002). Watt and Breyer-Brandwijk (1962), reported
that the leaves of baobab are used against excessive sweating and as astringent. The dried

24
leaves are used in many West African countries as an insect repellent (Denloye et al., 2006).
In Ghana, the bark is used as substitute for quinine to relieve fever (Shukla et al., 2001). The
oil extracted from the seeds is used against diarrhoea and hiccough (De Caluwé et al., 2009).
In Africa, people infected with malaria parasites consume a mash containing dried baobab
bark as a febrifuge in order to treat the fever associated with this illness (Wickens and Lowe,
2008). In Mali, it is reported that swollen joints are treated by rubbing a paste made with
baobab fruit into the affected area (Wickens and Lowe, 2008).

The baobab is extremely important to humans and animals in the dry areas of Africa because
it offers shelter, a source of nutrition, clothing as well as raw material for many useful items.
In Benin, the bark has been used for making ropes (De Caluwé et al., 2009). In some villages
in West Africa, people store water in the tree trunk and it is estimated more than 120,000 L of
water can be stored (Royal Botanic Gardens, Kew, 1999). Baobab is used for several purposes
and these include: fruit for food; oil from the seeds; rope, cordage and cloth from the bark
fibre; tannin for curing leather from the tree bark; glue from the pollen grain of the flowers;
pulp for making paper from the harvested tree (although of low quality), seasoning and as
an appetiser (Wickens, 1982; Sidibe and Williams, 2002; Nhukarume et al., 2008). The fresh
leaves are cooked as vegetables (Van Wyk et al., 1997). In Madagascar, baobab seeds (A.
digitata) have been used for the production of vegetable oil (Bianchini et al., 1982).

In some countries in West Africa, the leaves, fruit pulp and seeds are the main ingredient in
sauces, porridges and beverages (Chadare et al., 2009; De Caluwé et al., 2009; Yusha'u et al.,
2010). Recently, baobab has been referred to as a “superfruit” based on its nutritional profile
(e.g. vitamin, fatty acid, mineral) (Gruenwald, 2009). The nutritional value of baobab is only
briefly discussed since a comprehensive report on the nutritive aspects is already available
(Chadare et al., 2009; De Caluwé et al., 2009). The major interest in baobab products is as a
result of its ascorbic acid and dietary fibre content. The level of vitamin C contained in fruit
pulp is high and can range from 2.8 to 3 g/kg (Vertuani et al., 2002). It was noted that baobab
fruit pulp has a very high vitamin C content (280–300 mg/100 g), which is seven to ten times
more than oranges (51 mg/100 g) (Manfredini et al., 2002; Täufel et al., 1993). One study
demonstrated that the consumption of 40 g of baobab pulp provides 100% of the

25
recommended daily intake of vitamin C in pregnant women (19 – 30 years) (Chadare et al.,
2009). The ascorbic acid content was evaluated in the fruit of A. digitata (Diop et al., 1988),
and it was found to contain 337 mg/100 g of ascorbic acid (Eromosele et al., 1991; Gebauer et
al., 2002). Sidibe and Williams (2002) recommended that baobab leaves should be stored as
whole leaves rather than ground leaf powder in order to preserve the high vitamin content.

The calcium content found in the fruit pulp varies according to authors and the origin of the
samples tested. Brady (2011) reported a calcium content of 344.2 mg/100 g sample which
differs from the value of 295.0 mg/100 g reported by Osman (2004). Other authors have
found different levels of calcium in the fruit (211 to 2160 mg/100 g) (Sena et al.,
1998; Lockett et al., 2002). Similarly, the level of potassium in the fruit pulp was found to be
1578.5 mg/100 g sample (Brady, 2011) and 1240.0 mg/100 g (Osman, 2004). The leaves are
particularly rich in calcium (307 to 2640 mg/100 g dw), they are known to contain good
quality proteins and it is estimated that it contains three to five times more calcium than milk.
The seeds have a relatively high lipid content of 11.6 to 33.3 g/100 g dw (Brady, 2011).

Several studies have revealed that baobab is rich in fibre (approximately 44%) which can
balance the intestinal flora. However, the fibre content varied considerably between studies
which could be due to several factors such as the origin of the plant material and the
extraction procedure used. Additional information on the nutritional value of baobab products
can be obtained in other works (e.g. Nordeide et al., 1996; Barminas et al., 1998; Soloviev et
al., 2004; De Caluwé et al., 2009).

Several studies have demonstrated a significant correlation between the intake of fruits and
vegetables and the occurrence of inflammation and diseases such as cardiovascular disease
and cancer (Aruoma, 1998; Willet, 2001). Natural anti-oxidants, including polyphenolic
compounds from plants, vitamins E and C, and carotenoids, are believed to be effective
nutrients in the prevention of these oxidative stress-related diseases (Besco et al., 2007).

The oil obtained from seeds is used to treat skin ailments, thus it may have some cosmetic
applications (Sidibe and Williams, 2002). Fixed oils are important ingredients in cosmetic
products and amongst these is baobab seed oil. It is suitable for use on the skin as it is non-

26
irritating and non-allergenic (Wren and Stucki, 2003). Other properties of
pharmaceutical/cosmetic importance include that it is excellent for restoring and
remoisturising the skin due to its high penetrability and nourishing properties. It can also be
used to treat eczema and psoriasis (PhytoTrade Africa, 2009).

Baobab oil or fruit pulp contains several vitamins (Nkafamiya et al., 2007), that are essential
for skin care. These include vitamins A and F (rejuvenation and cell renewal); vitamin E
(anti-oxidant and anti-ageing effects) (Nyam et al., 2009) and vitamin D3 which increases
calcium absorption and decreases blood pressure in the elderly (Wasserman, 2004). The oil is
said to alleviate pain from burns and regenerates the epithelial tissues in a short time, thereby
improving skin tone and elasticity (Murunga Products, 2006).

The oil can be used as a protecting, nourishing, moisturising, soothing and regenerating agent.
Studies have also shown that the oil contains anti-oxidants (Nkafamiya et al., 2007), can
protect the skin against premature ageing and prevents the appearance of wrinkles (Africajou).
Alone or in combination with other ingredients, it is also used to aid in skin healing (small
cuts, chapping) or as a mask for hair care (dry, brittle hair, split ends) (Africajou). Linoleic
acid (found in baobab seed oil) is the most frequently used fatty acid in cosmetic products as
it moisturises the skin, aids in the healing process of dermatoses and sunburns and is used for
the treatment of Acne vulgaris (Lautenschläger, 2003).

2.10.3 Phytochemistry
Several classes of compounds have been identified from various parts of baobab (fruit
pulp, seed oil, leaves, roots) including terpenoids, flavonoids, sterols, vitamins, amino acids,
carbohydrates and lipids (Chauhan et al., 1984; Chauhan et al., 1987; Shukla et al., 2001).

Ten aromatic compounds including isopropyl myristate and nonanal were identified in

the fruit pulp using GC–MS (Cisse et al., 2009). Several compounds have been isolated from
the pericarp using column chromatography and include: (–)-epicatechin, epicatechin-(4β→8)-
epicatechin (B2), epicatechin-(4β→6)-epicatechin (B5), epicatechin-(2β→O→7, 4β→8)-
epicatechin (A2), and epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin (C1) (Shahat,
2006). Epicatechin is a flavonol (flavonoid) found in many plants such as grapes, cocoa and

27
tea. This class of compound may prevent coffee berry disease by inhibition of
appressorial melanisation. Epicatechin is known to exhibit strong anti-oxidant activity (Lee et
al., 2003) and can also promote survival in diabetic mice (Si et al., 2011). Other compounds
such as 3,7-dihydroxy-flavan-4-one-5-O-β-D-galactopyranosyl (1→4)-β-D-glucapyroside and
a flavonone 3,3′,4′-trihydroxy flavan-4-one-7-O-α-L-rhamnopyranoside and quercetin-7-O-β-
D-xylopyranoside were isolated from the roots of A. digitata (Chauhan et al., 1984; Chauhan
et al., 1987; Shukla et al., 2001).

Compounds such as campesterol, cholesterol, isofucosterol, β-

sitosterol, stigmasterol and tocopherol (α, β, γ, and δ) have been detected in the seed oil. Bianchini
et al. (1982) investigated the lipid composition of the seed oil using GC–MS. The major
hydrocarbons in the seed oil were n-alkanes (57.3%) and squalene (39.5%). Fatty acids present in
the seed oil include linoleic and oleic acids in high concentration as well as lesser amounts of
palmitic, linolenic, stearic and arachidic acids (Yazzie et al., 1994; Glew et al., 1997; Codjia et al.,
2001; Sidibe and Williams, 2002; Osman, 2004; Nkafamiya et al., 2007).

The presence of organic acids such as citric, tartaric, malic, succinic and ascorbic acid in the
fruit pulp was first highlighted in the early fifties. The pulp represents 14 to 28% of the total
fruit weight and the pulp water content is low (less than 15%) (Soloviev et al., 2004). Studies
have shown that the fruit pulp contains high amounts of carbohydrate (≈ 70%), crude fibre
(≈ 11.2%), a low amount of ash (≈ 5.7%) and protein (≈ 2.2%), and a very low amount of fat
(≈ 0.4%) (Lockett et al., 2002). Several amino acids such as alanine, arginine, glycine,
lysine, methionine, proline, serine, valine (from fruit pulp) (Glew et al., 1997), vitamins (B1,
B2, B3, A, C) (from fruit pulp and/or leaves) (Sidibe et al., 1996, UNCTAD (United Nations
Conference on Trade and Development), 2005) and minerals (Cu, Fe, K, Mg, Mn, Na, P, Zn)
(from fruit pulp) (Glew et al., 1997) have also been identified.

2.10.4 Biological activity
2.10.4.1 Analgesic and antipyretic activities
The analgesic effect of the fruit of A. digitata extracted with hot water was tested in
vivo (mice). It was noted that the extract exhibited analgesic activity 2 h after administration.
At 800 mg/kg, the reaction time was 15.4 min in comparison to the negative control

28
(10.2 min) (Ramadan et al., 1994). The petroleum ether extract containing seed oil of A.
digitata was investigated for analgesic activity. The extract exhibited analgesic activity with
the tail flick response in 6.1 s which was not statistically different from aspirin used as
positive control (Khan et al., 2006). The antipyretic activity of A. digitata extract was
evaluated on twenty rats. Hyperthermia was induced by subcutaneous injection of a 12%
yeast suspension and the temperature of each rat was monitored. After a 4 h treatment period,
at a concentration of 800 mg/kg, the rectal temperature of the rats showed a slight decrease
(37.3 °C) in comparison to the initial temperature of 38.6 °C, suggesting that baobab extract
exhibited antipyretic activity (Ramadan et al., 1994). A clinical investigation was conducted
to compare the efficacy of a baobab fruit solution to the WHO standard solution in the
treatment of children (161 children, 6 months old) with acute diarrhoea. The results
demonstrated that the WHO solution was superior to the solution made from A. digitata in
terms of duration of diarrhoea, weight gain and rehydration. However, the difference was not
statistically significant suggesting that the baobab solution could be used as treatment in
children with acute diarrhoea (Tal-Dia et al., 1997).

2.10.4.2 Antibacterial activity
Baobab plant parts have been used for centuries to treat microbial infections both in humans
and animals and many scientific studies have been carried out in order to validate its
traditional antimicrobial uses. Kubmarawa et al. (2007) found that A. digitata extract was not
active against Candida albicans (ATCC 10231), Staphylococcus aureus (ATCC
13709), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 9637)
and Bacillus subtilis (NCTC 8236). This was in contrast to a study carried out by Yagoub
(2008) who investigated the antibacterial activity of a solvent extract of A. digitata against E.
coli isolated from urine and water. The results clearly indicated that the solvent extract
inhibited bacterial growth with the inhibition zone ranging from 20 to 30 mm depending on
the concentration at which the sample was tested (Yagoub, 2008). In a recent study, Masola et
al. (2009) investigated the antimicrobial activity of baobab plant parts (stem and root barks)
against Gram-positive bacteria, Gram-negative bacteria and yeast. The results indicated that
the aqueous and ethanolic root and stem bark extracts inhibited the growth of various micro-
organisms with the MIC values ranging from 1.5 to 6 mg/ml. The antibacterial activity of the

29
plant could be attributed to the presence of tannins, phlobatannins, terpenoids and saponins in
the stem bark (Masola et al., 2009).

2.10.4.3 Anti-inflammatory activity
The anti-inflammatory activity of the fruit extracted with hot water was tested in vivo using
the rat paw formalin-induced oedema test. The extract tested at a dose of 400 and 800 mg/kg
inhibited formalin-induced oedema. After 24 h administration of the aqueous extract, the
mean swelling of the foot was 1.81 and 1.75 mm for 400 mg/kg and 800 mg/kg, respectively,
in comparison to the negative control (6.35 mm) (Ramadan et al., 1994). The DMSO fruit
pulp extract and aqueous leaf extract showed significant inhibition against cytokine IL-
8 (Vimalanathan and Hudson, 2009).

2.10.4.4 Anti-insecticidal activity and repellency

Smoke from pellets of A. digitata leaves was assayed for toxicity and repellency against adult
(0 – 7 days old) Anopheles gambiae (African malaria mosquito), Musca domestica (housefly)
and Periplaneta americana (American cockroach) insects. The results indicated that the
smoke caused the death of A. gambiae and M. domestica, but not P. americana. The
median lethal dose for A. gambiae was 0.47, 0.50 and 0.20 g for the pellets containing 0, 0.01
and 0.05 g of d-allethrin, respectively, while it was 0.46, 0.52 and 0.46 g for M.
domestica (Denloye et al., 2006).

2.10.4.5 Phenolic and flavonoid content and anti-oxidant activity

Phenolic and flavonoid compounds are well known for their good anti-oxidant activity. The
total phenolic levels in fresh ripe fruits were significantly higher in the aqueous methanol than
in the aqueous acetone extracts (4057.5 and 3518.3 mg GAE (gallic acid equivalents)/100 g of
fruit for total phenolics, respectively). Similarly, the flavonoid level was higher in the aqueous
methanol extract of the fresh ripe fruit than in the aqueous acetone extract (42.7 and 31.7 mg
QE (quercetin equivalents)/100 g of fruit for total flavonoids, respectively). The anti-oxidant
activity of fresh ripe fruit of A. digitata was 1000 mg AEAC/100 g (ascorbic acid equivalent
anti-oxidant content) (Lamien-Meda et al., 2008). In a separate investigation, Nhukarume et
al. (2008) showed that phenolic content of baobab fruit pulp (≈ 52 mg/100 ml GAE) was not
statistically different from that of Citrus sinensis Pers. (Nhukarume et al., 2008) suggesting

30
the use of baobab fruit pulp as an anti-oxidant botanical dietary supplement. Brady
(2011) found that the anti-oxidant capacity of the hydrophilic extract of fruit pulp was higher
(7.65 mg ascorbic acid equivalent/g) compared to the lipophilic extract of fruit pulp (3.32 mg
ascorbic acid equivalent/g). Carlsen et al. (2010) summarised the anti-oxidant activity of
various fruits and vegetables. The anti-oxidant activity of the baobab varies according to the
plant part used. The anti-oxidant content of dried leaves was 48.1 mmol/100 g which was
almost five times higher than that of fruit pulp. The anti-oxidant content of fruit or dried
leaves was higher than that of orange (0.9 mmol/100 g); mango (1.7 mmol/100 g); apple
(3.8 mmol/100 g) and papaya (0.6 mmol/100 g). Vertuani et al. (2002) showed that the fruit
pulp exhibited strong anti-oxidant activity corresponding to 6–7 mmol/g of Trolox®, in
comparison to the fruit pulps of orange (0.1 mmol/g), strawberry (0.90 mmol/g), apple
(0.16 mmol/g) and kiwi (0.34 mmol/g). Nhukarume et al. (2008) investigated the ability of
solvent extracts of various fruits including baobab to inhibit the peroxidation of lipids. The
results showed a significant difference in the activity of the beverage extracts with A.
digitata having the greatest activity. The order of activity was A. digitata > ascorbic acid > C.
sinensis Pers. (orange) > Strychnos spinosa Lam. > Parinari curatellifolia Planch. ex
Benth. > baobab nectar.

Besco et al. (2007) evaluated the anti-oxidant activity of baobab red fibre. The lipid soluble
anti-oxidant capacity, the water-soluble anti-oxidant content and the ascorbic acid anti-
oxidant capacity were determined. The results obtained clearly demonstrated the lipid soluble
anti-oxidant capacity of baobab red fibre was high (508.0 ± 0.008 μmol/g, Trolox®
equivalent). Similarly, the water-soluble anti-oxidant capacity, corresponding to the activity
expressed as μmol/g equivalents of ascorbic acid for each gram showed the same pattern with
the ascorbic acid equivalents of baobab red fibre particularly high (386.0 μmol/g). The same
study also showed that the integral anti-oxidant capacity (sum of the anti-oxidant capacity of
hydrophilic and lipophilic anti-oxidants) of baobab red fibre (1617.3 μmol/g) was 66 times
higher than that of orange pulp (24.3 μmol/g) (Besco et al., 2007).

31
2.10.4.6 Inhibitory effects of Trypanosoma by baobab products
The capacity of A. digitata to reduce the mobility of Trypanosoma brucei, which causes
sleeping sickness, was evaluated using four different extracts (petroleum ether, chloroform,
water and methanol) obtained from the leaves and the bark. The time at which mobility stops
ranged between 10 and 45 min for the root bark, while with the leaves, the mobility seized
between 25 and 45 min, when various extracts were tested at 2 mg/ml (Atawodi, 2005). In
another study, the in vitro trypanocidal activity of baobab was microscopically investigated
against T. brucei brucei and T. congolense, which causes nagana in animals. The methanol
root extract exerted a noticeable effect on motility after 50 and 55 min for T. brucei
brucei and T. congolense, respectively (Atawodi et al., 2003).

2.10.4.7 Antiviral activity
Several in vitro and in vivo studies have been carried out to determine the antiviral activity of
various baobab plant parts. Ananil et al. (2000) investigated the antiviral activity of several
plants against the herpes simplex, sindbis and polio viruses. The baobab extract from leaves
was found to have the most potent effect. Vimalanathan and Hudson (2009) investigated the
antiviral activity of A. digitata leaves, fruit-pulp and seed extracted with water, DMSO and
methanol. The study was conducted using the minimum inhibitory concentration method
against influenza virus, herpes simplex virus and the respiratory syncytial virus. The influenza
virus was very susceptible, while the respiratory syncytial virus was resistant. The leaf extract
exhibited the most promising activity against the influenza virus with the MIC value ranging
from 0.12 μg/ml (DMSO) to 2.8 μg/ml (water). The activity of the leaf extract was promising
against the herpes simplex virus (MIC value: 1.0 to 11.7 μg/ml), while the pulp and the seed
exhibited much lower activity (MIC value > 72.5 μg/ml). The study clearly demonstrated
variation in biological activity when different plant parts are investigated. Furthermore, the
anti-HSV activity was considerably enhanced by light, especially long wavelength UV
although they all showed ‘dark’ antiviral activity as well. Thus, all the extracts contained
antiviral photosensitisers (Hudson et al., 2000; Vimalanathan and Hudson, 2009).

32
2.10.4.8 Hepatoprotective activity
The hepatoprotective activity of a water extract of the fruit pulp was evaluated in vivo against
chemical-induced toxicity with CCL4 in rats. The results clearly showed that the water extract
exhibited significant hepatoprotective activity. The liver protective ability of A.
digitata extract was 76, 77, and 87% for alanine transferase, aspartate transferase and alkaline
phosphatase activity, respectively when the plant extract was administered at the start of
CCl4 toxicity. The results suggest that consumption of A. digitata fruit may play an important
part in resistance to liver damage in areas where baobab is consumed (Al-Qarawi et al., 2003).
Although the mechanism of action of liver protection could not be established, this protective
activity could be due to triterpenoids, β-sitosterol, β-amyrin palmitate, and/or α-amyrin and
ursolic acid present in the fruit (Al-Qarawi et al., 2003). The acute toxicity of baobab fruit
pulp extract was tested in vivo on rats and the results showed that the LD 50 was 8000 mg/kg
following parenteral administration suggesting low toxicity (Ramadan et al., 1994).

The effects of baobab seed oil on drug metabolising enzymes (cyclopropenoid fatty acids)
were evaluated in vivo in rats fed either with baobab seed oil (1.27% cyclopropenoid fatty
acids) or heated baobab seed oil (0.046% cyclopropenoid fatty acids in the diet). The rats fed
baobab oil showed retarded growth when compared to other groups of animals. Furthermore,
the relative liver weights were markedly increased whereas cytochrome P-450 content and
NADPH cytochrome C reductase and NADH cytochrome C reductase activities were
decreased (Andrianaivo-Rafehivola et al., 1995).

2.10.4.9 Use as drug permeation enhancer (excipient)

The mechanism through which A. digitata exerts its effect, the kinetics of drug delivery and
the use of A. digitata mucilage in the formulation of matrix tablets were investigated in
vitro using aminophylline as positive control. The drug release retardation efficiency of A.
digitata mucilage at equal polymer concentrations was higher than those of plasma
concentration but less than that of hydroxypropyl methylcellulose in simulated intestinal fluid
and simulated gastrointestinal fluid. Furthermore, it was found that the mechanism of release
of aminophylline from A. digitata mucilage in hydroxypropyl methylcellulose was by
diffusion (Builders et al., 2007).

33
2.10.4.10 Other biological activities
The growth performance of guinea fowl keets that were feed with baobab seed cake diets was
investigated. The diets consisted of different amounts of the baobab seed cake (0, 5, 10 and
15%). The fed intake and body weight gain of the keets was found to be high when the
amount of baobab seed cake added to their diet was between 0 and 5% (Mwale et al., 2008).
The ethanolic extract of the bark was investigated for antihyperglycaemic and antilipidaemic
activities in alloxan-induced diabetic female rats. Baobab bark extract was administered orally
for seven days in doses of 250 and 500 mg/kg bw to diabetic rats, while the antidiabetic
drug glipizide (500 μg/kg bw) was administered to the control group. The bark extract
exhibited hypoglycaemic activity as they decreased plasma glucose levels by 26.7% and
35.9% and increased glycogenesis by 11.3% and 32%, respectively. Furthermore, the extracts
significantly decreased plasma and hepatic lipid profiles suggesting that the solvent extract of
the stem bark of A. digitata exhibited antidiabetic and hypolipidaemic effects on type I
diabetic animals (Bhargav et al., 2009). The methanol extract of A. digitata was investigated
for in vitro antimalarial properties using the lactate dehydrogenase technique, with a
multiresistant strain of Plasmodium falciparum K1. However, the study did not have
significantly positive results (IC50 value: 429.9 μg/ml) (Ajaiyeoba et al., 2004). Köhler et al.
(2002) investigated the antiplasmodial activity of aqueous extract of baobab pulp against the
chloroquine-sensitive and resistant strains of P. falciparum. Poor activity was noted
(IC50 value > 50 μg/ml). The results of an investigation on the cytotoxicity of the baobab plant
varied according to the plant parts (seed, leaves, fruits) and solvents used. The toxicity against
Vero monkey kidney cells ranged from 130 μg/ml (leaves) to ≈ 1900 μg/ml (fruit pulp)
(Vimalanathan and Hudson, 2009).

2.10.5 Commercialisation
Interest in non-timber forest products (NTFPs) is increasing rapidly and the use of these
products constitute a source of income of many rural people in Africa. NTFPs of baobab
(seeds oil, leaves and fruits) are beneficial not only for rural communities but also because
NTFPs are natural products that can be used in pharmaceutical and cosmetic industries.
Baobab is highly sought after in several market segments such as; food and beverages
(Germany, France and The Netherlands), botanical remedies (Germany, France and The

34
Netherlands) and nutraceuticals as well as natural cosmetics (EU, USA and Japan)
(UNCTAD, 2005). Baobab fruit is an ideal candidate in the functional food market as it is
very high in vitamin C and the powder may be used as a thickener due to its high pectin and
fibre content. The import value of the product class of rare edible dried fruit, which includes
baobab fruit pulp, grew by 13% in 2003 (Eurostat, European Statistics, 2004).

A survey revealed that 73% of the German public would buy food and drinks with anti-
oxidant properties (UNCTAD, 2005). Several biological properties have been proven for
baobab products (e.g. seed oil, fruit pulp, leaves) as described above lending credence to its
use as a botanical remedy with possible medical applications.

Natural health and cosmetic products are in great demand especially in North America,
Europe and Japan (UNCTAD, 2005). The turnover of botanical remedies and dietary
supplements almost doubled from US$12.4 billion in 1994 to US$20.3 billion in 2003
(UNCTAD, 2005). The oils extracted from baobab seeds have a history of use in skin and
healthcare recognised by cosmetic industries that incorporate this oil into their products
(Sidibe and Williams, 2002, UNCTAD (United Nations Conference on Trade and
Development), 2005). Anti-oxidant substances, such as baobab fruit pulp, are in significant
demand due to anti-ageing properties (UNCTAD (United Nations Conference on Trade and
Development), 2005, IFAD, 2008).

Baobab is one of the important commercial non-timber forest products (NTFPs). NTFPs are
of major significance in international trade. In 2008, 150 important NTFPs were recognised
including essential oils, medicinal plants, bamboo, wild nuts and seeds, various types of fibres
and mushrooms amongst others. These NTFPs have received much attention and the emphasis
has been on developing the value chain to contribute towards income generation for rural
households. For women especially, the gathering of NTFPs (Fig. 1A) represents the sole
source of food and income for their families (IFAD, 2008; Le Breton and Nemarundwe,
2009).

Non-profit community-based organisations (such as PhytoTrade Africa, 2009) play a major

role in the baobab market in terms of marketing and supply chain development of NTFPs.

35
Baobab fruits are harvested in rural areas, especially in Malawi and Zimbabwe. Processing
then takes place where the seed is separated from the pulp. The pulp is graded by particle size
and the raw material is sold as such. The seeds are retained and processed into oil which is
sold to cosmetic companies (UNCTAD (United Nations Conference on Trade and
Development), 2005; IFAD, 2008). These community-based organisations and businesses aim
to deliver high quality products to global markets. In 2008, close to 20,000 primary producers
sold raw or value-added NTFPs worth US$774,353, a 42% increase from 2007 (IFAD,
2008; Le Breton and Nemarundwe, 2009). PhytoTrade Africa (2009) submitted a Generally
Recognised As Safe (GRAS) notification concerning baobab fruit pulp to the FDA during
2008. According to the Federal Food, Drug and Cosmetic Act (sections 201(2) and 409) any
substance that is intentionally added to food as an additive is subject to premarket review and
approval by the FDA. On 25 July 2009, the FDA's decision was that baobab (A. digitata)
dried fruit pulp (BDFP) is GRAS, through scientific procedures, for use as an ingredient in
blended fruit drinks and fruit cereal bars at levels of up to 10% and 15%, respectively (FDA,
U.S. Food and Drug Administration, 2009).

The properties and phytochemical composition of baobab has led to its inclusion in various

patent applications in recent years. However, the first patent application concerning baobab
titled “Process for manufacturing plastic mass” was filed on 5 July 1904 and granted on 26
September 1905. It described the process of producing a pulp-like mass by alkali treatment of
the fibre or inner bark of the monkey-bread tree. This mass can then be cast and pressed into
any shape before drying to manufacture objects such as buttons, pulleys, handles for tools,
pedestals and so forth (Geipel, 1905). Since then, patents involving baobab have mostly
focused on dermatological as well as nutritional applications. One patent on the use of baobab
as an additive in cosmetic, skincare and dermatological emulsions describes the use of fruit
pulp, leaves, bark, seeds, flowers and roots or a mixture of these plant parts to produce
cosmetic and dermatological emulsions for skin protection and skin care which have a high
stability of the UV filters incorporated in the preparations (Engels, 2009a). Redness and
burning of the skin is alleviated and regeneration of the skin is promoted. Baobab itself has a
photoprotective effect and used in combination with other UV filters, the stability of the UV
filters were improved (dibenzoylmethane derivatives, benzophenone derivatives) (Engels,

36
2009a). Another patent claims that a component or extract from the baobab plant can be used
for the treatment, care, and prophylaxis of skin problems or skin diseases, particularly of
allergic and/or inflammatory diseases in humans and animals (Engels, 2009b). It is also
claimed that baobab used on the skin, hair, eyelashes or nails produces an effect which can be
softening, insulating, hydrating, soothing, emulsifying, regenerative, anti-free radical, anti-
inflammatory, protective, photoprotective, anti-pollutant, anti-toxic or anti-sensitising (Pauly,
2001).

In addition, baobab is included as an ingredient in a nutraceutical combination also

containing Borojoa patinoi Cuatrec. (borojo), Cyclanthera pedata (L.). Schrad. (caigua)
and Aframomum melegueta K. Schum. (grains of paradise) for human consumption to
promote well-being, provide nutritional value and to aid in preventative health management.
According to the patent, it has been established that each of the plants has a nutritional and
medicinal value and it may be useful as a dietary supplement to be sprinkled over food as a
dry powder, as a capsule, a ready to drink juice or a food additive. Baobab is described as a
good source of vitamin C, micronutrients and soluble fibres with a pre- and probiotic effect
thereby serving as an intestinal regulator in the case of gastric disorders (Shatkina and
Gurevich, 2010). The use of baobab is not restricted to humans; another invention is intended
to market baobab as an animal food or animal food additive to provide a high-value food or
food supplement which is tasty and counteracts malnutrition (Engels, 2009c). Baobab is also
listed in various patent documents as a possible ingredient in various cosmetic and other
formulations (Engels, 2009a). Scientific studies are not available to support most of these
patent applications, especially related to cosmetic uses.

CHAPTER THREE
3.0 MATERIALS AND METHODS

37
3.1 Experimental Location
The study was conducted at the research pens of the Veterinary Teaching Hospital, University
of Agriculture Makurdi, Benue State. Makurdi is located on latitude 7o 45 North and
longitude 8o 31 East, which lies within the Southern Guinea Savannah region of Nigeria. The
daily temperature ranges between 21.6oC in December/January and 42.6oC in February
/March. The annual rainfall (April - October) ranges from 1,105 – 1,600mm and relative
humidity is highest (69%) between August and September and lowest (39%) between January
and March (worldweatheronline, 2021).

3.2 Source and Processing of Adansonia digitata leaves

Fresh Adansonia digitata leaves were harvested from Garaku Local Government Area in
Nasarawa State. The leaves were washed and air-dried in a shade. Air drying was done to
prevent the loss of active components if sundried. The dried Adansonia digitata leaves were
ground into powder and leaf extract was done using methanol extraction.

3.3 Extraction Procedure

One hundred (100) grams of leaf powder was mixed with a litre (1000ml) of methanol (ratio
1: 10 w/v), i.e. 800 grams of the powder mixed with 8 litres and agitated every 30 – 40
minutes to facilitate the process of extraction. The mixture was allowed to stand for 48 hours
before filtering the supernatant using filter paper and funnel. The filtrate was then poured into
beakers and allowed to stand in a warm water bath to facilitate evaporation of methanol
leaving a pastry substance (the extract) which was then stored in the freezer to be used when
the need arise (Eibad et al., 2019). The solid ground and extracted leaf material were dried
and also used for the experiment.

3.4 Determination of LD50

Lethal dose (LD50) was 200mg and 500mg for the lower and upper limit methods. However,
200mg/kg was used in the treatment.

3.5 Experimental Animals and Housing

38
A total of thirty (30) day – old broiler chicks of mixed sexes were purchased from a reputable
hatchery and used for the study. The chicks were brooded using the conventional brooding
method as outlined by Dafwang and Ogundipe, (1987). During the study, water and feed were
provided ad libitum. All birds were fed with a standard commercial feed (Chikum Feeds).
Strict and good hygenic practices were maintained in the pen before and during the course of
experiment.

The birds were reared in deep litter half-walled open-sided pen with the upper half covered
with wire mesh. All the birds were routinely vaccinated and medications were administered
accordingly.

3.6 Experimental Design

The experimental birds were randomly divided into five (5) groups of six (6) birds each. The
groups were treated as;

G1- Not infected, not treated.

G2- Infected and treated with a conventional anticoccidiostat (Toltrazuril at a dose rate of
1ml/lit for 48 hours).

G3- Infected and treatedwith 200mg/kg orally of Adansonia digitata leaf extract for 5 days.

G4- Infected and treated with Adansonia digitata leaf powder in the feed at the dose rate of
2g/kg of feed for 5 days.

G5- Infected, not treated.

3.7 Source of Parasite and Infection

Unidentified Eimeria species from the feaces of infected chickens was obtained from the open
market and introduced to a few birds for natural infections to occur. After infections of the
donor broiler chickens, their feaces containing occysts were sporulated and used in infecting
the experimental birds (G2, G3, G4 and G5).

39
3.8 Experimental Monitoring
After exposure of the chickens to the oocysts, they were monitored for clinical signs of the
disease. Few days after exposure, most of the birds developed anorexia, weakness and bloody
feaces. The treated groups were treated immediately the clinical signs started manifesting as
stated above. Anticoccidial effect of Adansonia digitata leaf (extract and powder) was
evaluated on the basis of presence or absence of oocysts in the feaces, body weight, feed
conversion ratio (FCR), haematological indices and gross lesions in the gastrointestinal tract
(GIT). The live body weights of each bird in all the groups were taken weekly throughout the
period of the experiment. Feed intake and their FCR were calculated weekly until the end of
the experiment.

At the end of the experiment, blood samples were collected from three (3) chickens in each
group for haematological analysis. The haematological indices determined were; packed cell
volume, serum total protein, red blood cells and white blood cells.

Two birds were randomly selected and sacrificed from each group for examination of the GIT
and other vital organs (lungs, liver, spleen and gizzard). Liver, lungs, kidney, spleen and
segments of the GITwere collected and fixed in 10 % buffered formalin solution for
histopathological evaluation.

3.7 Statistical Analysis

The data generated were analyzed using computerized statistical program (SPSS version
16.0). Differences in mean body weight were tested by ONE-WAY ANOVA. Mean
separation was accomplished using the Duncan’s Multiple Range Test. P < 0.05 was
considered as significant level.

40
 CHAPTER FOUR
4.0 RESULTS
The result on anticoccidial activity of methanol leaf extract of Adansonia digitata on
experimental infection of field isolates of Eimeria species in broiler chickens is as presented
in the tables below.

41
4.1 Oocyst count per microscopic field
The result showed significant reduction in oocysts output when compared with infected untreated group which has the highest at
the end of the experiment with 13 oocysts per microscopic field. The infected groups that where treated with Toltrazuril,Adansonia
digitata methanol leaf extracts and leaf powder had high oocyst count per microscopic field of 10 which reduced to 1 – 3. The
effect of different treatments on Eimeria infections in broiler chickens shown in table 4.1 below;

Table 4.1: Oocyst count per microscopic field of broiler chickens infected with Eimeria species and treated with Toltrazuril (G 2),
methanol leaf extracts ofAdansonia digitata (G3), leaf powder of Adansonia digitata (G4) and infected, untreated (G5)
Parameters G1 (Control G2 (Infected, treated with G3 (Infected, treated with G4 (Infected, treated with G5 (Infected,
Not infected) Toltrazuril) methanol Leaf extracts) methanol Leaf powder) untreated)
Preinfec infection Post Preinfec Post Post Preinfec Post Post
tion infection tion infection infection tion infection infection
Oocyst count (per - 10+ 3+ 1+ 10+ 4+ 3+ 10+ 3+ 1+ 13+
microscopic field)

42
4.2 Performance parameters
After infection of the birds with Eimeria species and treatment with a conventional
(Toltrazuril) drug, leaf extracts and powder of Adansonia digitata, feed intake, body weight
gain and feed conversion rates were investigated. In all the different treated groups, all the
parameters dropped slightly compared to the control group that was not infected. The
uninfected, untreated feed intake was highest (4.4kg), with a feed conversion ratio of 1.93 as
shown in Table 4.2.

43
Table 4.2: Performance of broiler chickens experimentally infected with Eimeria
species,treated with Toltrazuril,Adansonia digitata methanol leaf extracts and powder and
the infected, untreated.
Before treatment
Parameters G1 (Not G2 G3 (Leaf G4 (Leaf G5
Infected) (Toltrazuril) extract) powder) (Infected,
untreated)
Feed intake (g) 3.79 3.79 3.79 3.79 3.79
Weight gain (g) 1.91 1.91 1.91 1.91 1.91
Feed conversion ratio 1.98 1.98 1.98 1.98 1.98
Presence or absence of - +++ +++ +++ +++
oocysts in the feaces

During treatment
Parameters G1 G2 G3 G4 G5
Feed intake (g) 0.65 0.43 0.44 0.45 0.25
Weight gain (g) 0.39 0.19 0.19 0.21 0.44
Feed conversion ratio 0.32 2.18 2.29 2.10 0.51
Presence or absence of - + + + +++
oocysts in the feaces

After treatment
Parameters G1 G2 G3 G4 G5
Feed intake (g) 4.44 4.22 4.36 4.45 3.54
Weight gain (g) 2.3 2.1 1.92 2.09 1.47
Feed conversion ratio 1.93 2.01 2.27 2.13 2.41
Presence or absence of - + + + +++
oocysts in the feaces

44
4.3 Hematological parameters
The haematology of broiler chickens experimentally infected with field isolate ofEimeria
species, treated with Toltrazuril,Adansonia digitatamethanol leaf extractsand powder is
presented in table 4.3. The infected,untreated group showed the lowest values of PCV (21.83),
Serum Total Protein (6.58), RBC (2.12). However, the value of WBC was higher (2.27) than
other groups that were treated.

45
Table 4.3: Haematology(PCV, TSP, RBC and WBC) of broiler Chickens Experimentally infected with Field isolate ofEimeria
species, treated with Toltrazuril (G2), Adansonia digitatamethanol leaf extracts (G3), Adansonia digitataleaf powder (G4), and
infected, untreated (G5)
Parameters G1 G2 (Infected, treated with G3 (Infected, treated with G4 (Infected, treated G5 (Infected
(Control) Toltrazuril) Adansonia digitatamethanol withAdansonia digitata Leaf but not
Not infected Leaf extracts) powder) treated)
Pre- infection Post - Pre- infection Post- Pre- infectio Post-
infection infection infection infectio infection n infectio
n n
PCV (%) 30.82 31.70 23.8 25.25 31.70 25.12 26.10 31.70 24.03 26.50 21.83
Serum total 7.84 7.80 6.84 7.08 7.80 6.98 7.15 7.82 6.86 7.80 6.58
protein (g/dl)
RBC (x109/l) 2.45 2.59 1.90 2.01 2.59 2.10 2.24 2.59 2.12 2.54 2.12
WBC 2.08 2.09 2.56 2.12 2.09 2.96 2.22 2.09 2.87 2.13 2.27
(x1012/l)

46
4.4 Gross lesions
The pathological lesions observed at the end of the experiment were ballooned caeca,
haemorrhagic intestinal mucosa, thickened intestinal wall, blood clots in the intestinal lumen
and pinpoint heamorrhages in the intestine. From results obtained, the infected, untreated
group showed all the gross lesions, while toltrazuril and Adansonia digitatamethanol leaf
extracts did not show any gross lesions. Meanwhile the control group (uninfected, untreated)
showed ballooned caeca and Adansonia digitata leaf powder had thickened intestinal wall.
Table 4.4 shows the pathological lesions that were observed.

Table 4.4: Gross lesions of GIt of broiler chickens experimentally infected with field isolate
ofEimeria species, treated with Toltrazuril (G2), methanol leaf extracts of Adansonia digitata
(G3), leaf powder of Adansonia digitata (G4), infected, untreated (G5) and uninfected,
untreated (G1)
Parameters G1 G2 G3 G4 G5
Ballooned caeca + – – – +
Haemorrhagic intestine – – – – +
Thickened intestine – – – + +
Blood clots in intestinal lumen – – – – +
Pinpoint haemorrhages in intestine – – – – +

47
 CHAPTER FIVE
5.0 DISCUSSION
The study revealed that coccidian infection adversely affects weight gain of infected,
untreated birds since it interfere with their feed consumption. The depressed immune system
of the infected birds was due to effect of the parasitic infection on feed conversion ratio and
poor absorption from the GIT. Similar observations have been earlier reported (Calnck et al.,
1991; Kettunen et al., 2001; Akhtar et al., 2015). The excellent performance recorded from
Adansonia digitata leaf powder in terms of weight gain (2.09kg) and feed conversion ratio
(2.13), agree with the works of Rafiu et al. (2017) who reported that birds on 50% baobab leaf
meal performed better.

The dropped in the haematological parameters is in agreement with Jain (1993).

Haematological values are good indicators of the well being orhealth status of an animal
including chickens and are predictive of physiological changes resulting from various stress
factors (Kilgas et al., 2006). Both the positive and negative control groups showed remarkable
differences in the heamatology. The high value of WBC and low values of other haemograms
in the infected, untreated birds is in agreement with Jain (1993). This high level of WBC is an
indication of ongoing infection in the birds as previously reported by Kwari et al. (2017), who
also observed highe levels of white blood cells as a result ofleucocytosis which serve as first
line of defensive mechanism against diseases. This also signifies that phagocytes were at the
peak in the birds that were infected anduntreated.

The reduction in feacal oocysts output in all the birds infected and treated with methanol leaf
extract of Adansonia digitata is in agreement with Youn and Noh, (2001) who investigated
infected broiler chickens with E. tenella then treated with herbal extracts and observed
improvement in almost 15 treated groups in terms of survival rates, lesion scores, body weight
gains, bloody diarrhea, and oocysts excretions in comparison to the control group.

Some of the gross pathological lesions observed like haemorrhages, thickening of cecal wall
and mucoid discharge have been earlier reported by Xu et al. (2020). During an infection,
infective sporozoites enter the cecum mucosa by penetrating villus epithelial cells, this leads
to extensive destruction of the cecum epithelium, bloody stools, and a large amount of oocyst

48
excretion (Kawazoe and Fabio, 1994; Dutta et al., 1990), thus making such aforementioned
gross lesions visible.

5.1 CONCLUSION
The study showed that Adansonia digitata leaf methanol leaf extracts was effective against
coccidiosis in broiler chickens. The Adansonia digitata leaf powder was found to be more
potent than the extracts with regards to live body weight gain which is the ultimate aim of
farmers.  The use of herbal extracts could be beneficial in the control of coccidiosis but this
need to undergo more detail investigations to conclude on their effectiveness against the
disease.

5.2 RECOMMENDATION
1. More investigations are required to make available more information on the use of
Adansonia digitata leaf and powder in broilers production.
2. The use of herbal extracts should be encouraged to produce antibiotic free or promote
production of organic broiler.
3. More investigations on the lethal doses (LD50) of some of these herbal extracts are
requiredto establishthe exact LD50since some of the constituents of these herbs are
toxic to humans and animals.

49
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 APPENDIX
Plate 1: Balloned intestine

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Plate 2: Blood clot in intestine

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Plate 3: Haemarrhogic caeca

68
Plate 4: Haemorrhagic intestine

69
Plate 5: Haemorrhagic intestine

70