FOLUSO-ADEDAPO JOY BUKUNMI

TOPIC : Antitoxic effects of Petiveria alliacea on bone marrow of NMU induced Mus
musculus (ALBINO MICE).

ABSTRACT
Medicinal plants have long served as foundational components of traditional medicine and are
increasingly recognized for their pharmacological potential in modern therapeutics. This study
evaluated the genoprotective effect of Petiveria alliacea extract on NMU (N-nitroso-N-
methylurea)-induced bone marrow toxicity in female mice. Twenty-five (25) mice were divided
into five groups (A–E), based on initial body weights. Over a period of eight weeks, the groups
received the following treatments: Group A (800 mg/kg extract + NMU), Group B (400 mg/kg
extract + NMU), Group C (200 mg+/kg extract + NMU), Group D (NMU only, positive control),
and Group E (distilled water, negative control).
Following treatment, bone marrow cells were harvested and evaluated for genotoxicity using
micronucleus (MN) assay. Nuclear abnormalities were scored in both polychromatic erythrocytes
(PCEs) and normochromatic erythrocytes (NCEs), with specific attention to indicators such
as micronuclei, binucleated cells, nuclear buds, vacuolated nuclei, segmented nuclei, kidney-
shaped nuclei, blebbed nuclei, and apoptotic cells.
Results revealed that the NMU-only group (D) exhibited the highest frequencies of nuclear
abnormalities in both PCEs and NCEs, indicating severe bone marrow genotoxicity. In contrast,
groups treated with P. alliacea extract (A–C) showed a dose-dependent reduction in these
nuclear abnormalities, with Group A (800 mg/kg) demonstrating the most significant protective
effect (p < 0.05). The control group (E) presented minimal or no nuclear abnormalities, reflecting
intact bone marrow function. The results suggest that P. alliacea contains bioactive compounds
capable of preserving bone marrow integrity and reducing chromosomal instability. Further
studies are recommended to isolate its active constituents to isolate its active phytoconstituents
may supports its development as an adjunct-in chemotherapy-induced cytotoxicity management.
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION:
Medicinal plants have an unbelievable history in terms of serving humanity in almost all
continents of the world (Khan, 2014). Medicinal plants have served as the cornerstone of
healthcare systems across diverse cultures for millennia, representing an invaluable repository of
bioactive compounds with profound therapeutic potential (Daniel et al., 2023). For thousands of
years, medicinal plants have been used in various cultures of the world as a safe therapeutic
modality. The operation of medicinal plants is based on the rich experiences of innumerable
healers over centuries, inherited from ancestors, healer-to-healer transfer, or developed through
personal experiences over time (Khan, 2014). Medicinal plants are resources of traditional
medicines and many of the modern medicines are produced indirectly from plants.
The World Health Organization (WHO) recognizes the significance of traditional medicine,
which remains a vital healthcare source for many communities worldwide, particularly in regions
where modern medical infrastructure is limited (Ahmed et al., 2025). It has also been confirmed
by World Health Organization (WHO) that herbal medicines serve the health needs of about 80
percent of the world’s population; especially for millions of people in the vast rural areas of
developing countries (Saleh et al., 2015). Contemporary research has witnessed an
unprecedented surge in scientific investigation of medicinal plants, with publications steadily
increasing since 2015, reaching peak productivity in recent years (Lei et al., 2025).
The interdisciplinary field of ethnopharmacology has emerged as a critical bridge between
traditional knowledge and modern scientific validation, encompassing anthropology,
pharmacology, botany, and chemistry to study various cultures' medicinal plant applications
(Assimopoulou et al., 2024). This multidisciplinary approach has proven essential for
documenting, evaluating, and conserving medicinal plants and their therapeutic applications
across different cultures (Aleixandre et al., 2024). The rapid advancement in phytochemical
analysis and pharmacological screening has enabled researchers to identify and characterize
numerous bioactive compounds responsible for the therapeutic efficacy of traditional
remedies (Refaz et al., 2023). Besides having great therapeutic potential bioactive substances
from medicinal plants are often preferred due to their natural origin, which is perceived as safer
and more environmentally friendly compared to synthetic drugs (Refaz et al., 2023).
Medicinal plants constitute a remarkable source of diverse phytoconstituents, including
alkaloids, flavonoids, terpenoids, phenolics, saponins, tannins, and essential oils (Refaz et al.,
2023). These secondary metabolites serve multiple functions in plant defense mechanisms while
simultaneously offering tremendous therapeutic potential for human health applications (Toi et
al., 2025). Recent phytochemical investigations have revealed that plants produce approximately
one million secondary metabolites, virtually all synthesized in response to environmental stress
factors (Toi et al., 2025).
The therapeutic properties of medicinal plants depend fundamentally upon the biochemical
compounds present in different plant parts employed in medicine preparation (Kavitha,
2021). Phenolic compounds, including flavonoids, tannins, and phenolic acids, have
demonstrated significant antioxidant, anti-inflammatory and antimicrobial properties (Yu et al.,
2021). Terpenoids, another major class of secondary metabolites, exhibit diverse biological
activities ranging from antimicrobial and anti-inflammatory effects to anticancer and
neuroprotective properties (Khan et al., 2025).
Advanced analytical techniques, particularly Gas Chromatography-Mass Spectrometry (GC-MS)
and Fourier-Transform Infrared Spectroscopy (FTIR), have revolutionized the identification and
quantification of bioactive compounds in medicinal plant extracts (Sandra et al., 2025). These
sophisticated methodologies enable precise characterization of phytochemical profiles,
facilitating quality control and standardization of herbal medicines (Anurag et al., 2023).
Contemporary research has provided substantial scientific evidence supporting the traditional
uses of medicinal plants, particularly their antioxidant, anti-inflammatory, antibacterial,
antifungal, antiviral, anticancer, antidiabetic, neuroprotective, and gastroprotective effects
(Airish et al., 2023). The pharmacological versatility of plant extracts encompasses diverse
medical conditions, including wound management, respiratory ailments, gastrointestinal
disorders, and cardiovascular diseases (Ansari et al., 2025).
Medicinal plants demonstrate remarkable therapeutic potential in treating major global health
challenges, including diabetes mellitus, cancer, infectious diseases, cardiovascular disorders, and
inflammatory conditions (Ansari et al., 2025). Active phytomolecules such as cucurbitane
triterpenoids, diosgenin, and limonoids exhibit significant antidiabetic properties through
mechanisms including GLUT4 translocation activation (Ansari et al., 2025). Compounds like
capsaicin and curcumin demonstrate potent anticancer activity by deactivating NF-κB pathways
and inducing cell cycle arrest (Ansari et al., 2025).
The antimicrobial properties of plant extracts have garnered particular attention in the context of
rising antibiotic resistance (Ferreira et al., 2022). Plant-derived compounds including piperine,
reserpine, berberine, dictamnine, chelerythrine, and allitridin have shown promising
antimicrobial activities, with some compounds triggering bacterial cell lysis (Ansari et al.,
2025). The synergistic effects observed when plant extracts are combined with conventional
antibiotics suggest potential applications in combating antibiotic-resistant bacterial
strains (Donkor et al., 2024). In addition to the synthesizing beneficial phytochemicals,
medicinal plants also produce harmful substances that act as a defense mechanism against
infections and may be toxic to certain consumers, such as humans (Oloyede et al., 2020).
Ethnobotanical studies continue to reveal the rich diversity of medicinal plants utilized by
indigenous communities worldwide (Kristiana et al., 2025). Recent investigations across various
geographical regions have documented extensive traditional knowledge systems, with
communities employing hundreds of plant species for treating diverse ailments (Beressa et al.,
2024). The Moi Tribe in Southwest Papua, Indonesia, utilizes traditional medicinal plants for
malaria treatment, with Arcangelisia flava demonstrating the highest relative frequency of
citation (Hutami et al., 2025).
Traditional healers and indigenous communities possess profound knowledge regarding
medicinal plant preparation methods, dosage protocols, and administration routes (O.O et al.,
2025). Studies indicate that oral administration represents the most common route of medicinal
plant consumption, with boiling being the predominant preparation method (Hardiman et al.,
2025). The preservation and documentation of this traditional knowledge have become
increasingly urgent as younger generations show declining familiarity with medicinal plant
applications (Silalahi et al., 2014).
The merging of ethnobotanical knowledge with modern scientific methodologies represents a
promising approach for drug discovery and development (Hao et al.,
2020). Ethnopharmacological approaches coupled with high-throughput screening have
enhanced the hit rate of bioactivity identification, making traditional knowledge an effective
search engine for natural drug discovery (Nasim et al., 2020).
According to Global Plant Based API Market Insights, the pharmaceutical industry has
witnessed renewed interest in plant-based active pharmaceutical ingredients (APIs), with the
global market valued at $30.08 billion in 2024 and projected to reach $52.08 billion by 2034. It
highlights consumer demand for natural preservatives over synthetics, stimulating new
formulations and sustainable alternatives in pharma (Geetha, 2025).
Clinical applications of medicinal plants have expanded significantly, with numerous traditional
Chinese medicine formulations undergoing rigorous clinical trials for various therapeutic
indications (Wu et al., 2023). These investigations encompass phase II and III clinical studies for
conditions including coronary heart disease, cancer, inflammatory disorders, and metabolic
diseases (Wu et al., 2023). The development of standardized plant extracts and bioactive
compounds has facilitated the transition from traditional remedies to evidence-based
pharmaceutical products (Nasim et al., 2022).
Plant-based biomanufacturing has emerged as a growing trend in biopharmaceutical production,
offering considerable advantages in terms of cost-effectiveness, scalability, and safety profiles
compared to traditional manufacturing approaches. The FDA approval of plant-derived
therapeutics, including Elelyso manufactured using carrot cells, demonstrates the viability of
plant-based pharmaceutical production systems (Sheety et al., 2019; Zhou et al.,2025).
Sustainable cultivation and post-harvest management are increasingly important in ensuring the
consistent quality and availability of medicinal plants. Innovative approaches such as hydroponic
cultivation allow for the controlled production of high-quality plant material, while advanced
drying and storage technologies help preserve the integrity of bioactive compounds (Neeharika et
al., 2025). The yield and quality of medicinal plants depend on environmental factors, such as
nutrients, pH, electrical conductivity, temperature, light, nanoparticles, phytohormones, and
microorganisms (Neeharika et al., 2025).
1.2 EXAMPLES OF MEDICINAL PLANTS
Some medicinal plants and their uses
1.2.1 Camellia sinensis (Green Tea):
Camellia sinensis, is an evergreen shrub from the Theaceae family, the source of green tea,
which is widely recognized for its polyphenolic content and health promoting effects, especially
in oncology and dermatology (Hussein, 2021). Hussein (2021) states that green tea (Camellia
sinensis) extracts inhibits both the metastasis and osteolytic components of cancer progression.
The activities are primarily due to the catechins, especially epigallocatechin gallate (EGCG),
which exhibit potent anti-proliferative, pro-apoptotic and antioxidant properties in various cancer
models. Regassa et al. (2022) report that Camellia sinensis polyphenols have demonstrated anti-
cancer, anti- inflammatory and anti-microbial activities, making it one of the most extensively
studied medicinal plants in cancer and dermatological therapies. It inhibits 5-LOX and COX-2
enzymes, reducing inflammation in both acute and chronic animal models (Parvinet et al., 2025).
Green tea promotes healing of UV-induced skin wounds via antioxidative and anti-inflammatory
mechanisms (Lee et al., 2018). Unfermented green tea leaves act as a ‘natural chemotherapy’,
inhibiting angiogenesis and modulating immune responses in tumor microenvironments (Kumar
et al., 2025).

1.2.2 Podophyllum hexandrum (Himalayan Mayapple)

Podophyllum hexandrum or Himalayan Mayapple which belongs to Berberidaceae family is a
rhizomatous plant native to the Himalayas. It is a source of podophyllotoxin, a key component, a
key compound in anticancer drug development. A review highlighted that “Podophyllotoxin
extracted from P. hexandrum is a precursor of anticancer drugs like etoposide and teniposide
used in lung and testicular cancers (Regassa et al., 2022). Its ethanolic extracts demonstrated
potent inhibition of proliferation in melanoma and cervical cancer cells (Lee et al., 2018). For
instance, a 2025 study reported that the ethanolic root extract exhibited strong inhibitory effects
on the proliferation of HT-29 colon cancer cells, with IC₅₀ values of 38.2 µg/mL (24 h) and 32.5
µg/mL (48 h), indicating significant antiproliferative activity (Mohmmad et al., 2025). Recent
reviews and experimental studies confirm the ongoing clinical relevance of these derivatives and
highlight their effectiveness against various cancer types, including non-Hodgkin lymphoma,
glioblastoma, and leukemia (Shah et al., 2021). The anticancer effect is primarily attributed to
the ability of podophyllotoxin and its derivatives to inhibit DNA topoisomerase II and disrupt
microtubule assembly, leading to cell cycle arrest and apoptosis in cancer cells (Khamal et al.,
2015).
1.2.3 Curcuma longa (Turmeric)

Curcuma longa Linn. (C. longa), popularly known as turmeric, belongs to the Zingiberaceae
family and has a long historical background of having healing properties against many diseases
(Fuloria et al., 2022). It is a rhizomatous herbaceous perennial widely cultivated across South
Asia. Its rhizomes are the source of the vibrant yellow spice used in culinary and medicinal
traditions for centuries (Fuloria et al., 2022). The principal bioactive constituent of turmeric is
curcumin, a polyphenolic compound renowned for its potent antioxidant, anti-inflammatory, and
anticancer activities. Other curcuminoids, such as demethoxycurcumin and
bisdemethoxycurcumin, also contribute to its therapeutic effects (Bhurke et al.,2025). Curcumin
triggers programmed cell death and halts the growth of various cancer cell lines, including
breast, colon, prostate, and lung cancers (Selene et al., 2025). Curcumin can induce cell cycle
arrest at different phases (G0/G1, S, G2/M) depending on the cancer type, often through
regulation of cyclins and cyclin-dependent kinases (Tuorkey, 2014). It influences on DNA
methylation, histone modification, and microRNA expression, contributes to its anticancer
properties (Imtiaz et al., 2025). Curcumin has shown synergistic effects when combined with
chemotherapeutic agents (e.g., docetaxel, cisplatin), increasing cancer cell sensitivity and
reducing required drug dosages (Perrone et al.,2015). Although curcumin generally refers to 1,7-
bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, the compound is also known as
“curcumin I”. In brief, curcumin is a diferuloylmethane with a crystalline yellow-orange colour,
molecular weight of 368.39 g/mol, melting temperature of 183°C, and with the chemical formula
C21H20O6 (Sharifi-Rad et al., 202

Figure 1: Curcumin chemical structure

Source: Sharifi-Rad et al., 2020
1.2.4 Elaeis guineensis (African oil palm)
Elaeis guineensis the oil palm or African oil palm is a monoecious plant from the Arecaceae
family, native to equatorial Africa, although the only other species in the genus (E. oleifera) is
indigenous to South and Central America. E. guineensis, however, is the major economic species
fruits of E. oleifera have a much lower oil content and are used only locally (Pasiecznik, 2022).
The plant demonstrates a wide range of anticancer properties, including the ability to induce
apoptosis (cell death), halt the cell cycle, inhibit angiogenesis, and regulate inflammatory
responses. It contains a variety of phytochemicals, such as flavonoids, tannins, terpenoids,
alkaloids, and organosulfur compounds, which contribute to their anticancer effects (Adeoye et
al., 2025). Odion et al. (2021) conducted preliminary screenings and reported high flavonoid and
phenolic content in aqueous extracts, which may play roles in anticancer pathways, including
oxidative stress regulation. In addition to anticancer activities, E. guineensis demonstrates
hepatoprotective, anti-inflammatory, and wound-healing effects (Sulistiarini et al., 2022),
supporting its holistic medicinal value.
1.2.5 Securidaca longipedunculata (violet tree)
The plant Securidaca longipedunculata Fresen (violet tree) belonging to the family
Polygalaceae is known as the ‘mother of all medicines’ in Hausa language in Northern Nigeria
(Ngulde et al., 2019). Its extract is used by Traditional Medicine Practitioners (TMPs) in the
management of many ailments including cancer in African countries. It is the most frequently
used plant in the management of cancer by TMPs in Northeastern Nigeria (Ngulde et al., 2019).
Securidaca longipedunculata has antibacterial, anticonvulsant, antidepressant, anti-diarrheal,
anti-inflammatory, analgesic, anticancer, antiparasitic, antioxidant, antivenomous, antiviral,
cardiovascular and neuromuscular properties, hypoglycemic, pesticidal and immunostimulant
(Kporvie, 2024). In addition, different plant parts secrete highly valuable phytochemicals
including the phenolic compounds, alkaloids, diterpenoids, steroids, alkaloids, and other
complex compounds that are used to combat infections (Shai et al., 2024).
1.2.6 Vernonia amygdalina (Bitter Leaf)
Vernonia amygdalina is a perennial shrub that belongs to the family Asteraceae. The herb is an
indigenous African plant that grows in most parts of sub-Saharan Africa. The plant is
predominantly cultivated in the tropical regions of Africa, especially in the West African (Tekou
et al., 2018). In Igbo, Yoruba, and Hausa tribes of Nigeria, it is called as “Olugbu”, “Ewuro”
and “Fetefete” respectively (Ugbogu et al., 2021). It is probably the most used medicinal plant
in the genus Vernonia (Sileshi et al., 2024). Research from multiple researchers has shown a
wide range of phytochemical and bioactive compounds present in Vernonia amygdalina;
examples are terpenoids, tannins, alkaloids, saponins, flavonoids, steroids, glycosides, etc.,
which have contributed to the medicinal properties of Vernonia amygdalina (Oseghale et al.,
2024). The plant parts are used in traditional medicine for many human and animal healthcare
purposes, including diarrhea, diabetes, wound healing, tonsillitis, evil eye, retained placenta,
headache, eye disease, intestinal parasite, bloating, hepatitis, toothache, anthrax, malaria, urine
retention, gastritis, stomach disorders, and snake bites (Sileshi et al., 2024).
Various in vivo and in vitro studies revealed that V. amygdalina and its bioactive components
possess pharmacological activities such as antioxidant, anti-inflammatory, anticancer,
antimicrobial, hepatoprotective, antidiarrheal, anti-diabetic, and neuroprotective activities
(Ugbogu et al., 2021). The efficacy of V. amygdalina in ameliorating diseases is attributed to its
antioxidant activity and ability to improve the antioxidant system (Ugbogu et al., 2021).
1.2.7 Cola acuminata (Kola Nut)
Cola acuminata, is an evergreen tree of the family Malvaceae, native to tropical Africa;
They are native to the rainforests of tropical West Africa and common names are kola nut
(both) or bitter kola (Cola nitida). They are native to the rainforests of tropical West
Africa and common names are kola nut (both) or bitter kola (Cola nitida) (Lateef, 2023).
Several phytochemical compounds have been isolated from C. nitida, such as catechin,
caffeine, epicatechin, procyanidin B1, procyanidin B2, theobromine, polyphenols,
alkaloids, tannins, saponins, and so on (Akinoso et al., 2014; Dah-Nouvlessounon et al.,
2015). Because of its very high caffeine content, C. acuminata is a significant source in
the production of beverages such as Coca-Cola and Pepsi-Cola. In addition, it is also used
for the production of drugs, soft drinks, and wines (Dah-Nouvlessounon et al., 2015).
Because of its different secondary metabolites, C. acuminata has a variety of biological
properties, including anti-malarial activity (Zailani et al., 2020). The secondary
metabolites are responsible for the various pharmacological activities such as antioxidant,
antibacterial, antifungal, antimalarial, anti-inflammatory, antidiabetic, antidiarrheal,
antiviral, anticancer, antimycobacterium, and antiatherosclerotic and hypolipidaemic
(Ekalu et al., 2020).
1.2.8 Moringa oleifera (Moringa)
Moringa oleifera (M. oleifera), the “miracle tree”, plant belonging to family moringaceae
thrives globally in almost all tropical and subtropical regions, but it is believed to be native to
Afghanistan, Bangladesh, India, and Pakistan (Pareek et al.,2025). Different parts of the plant
such as bark, leaves, seeds, flowers, roots, and immature pods, contains large number of
important phytoconstituents such as terpenoids, alkaloids, tannins, steroidal aglycones and
reducing sugars (Paikra et al., 2017). In recent years, M. oleifera, a versatile and nutritionally
rich plant, has garnered significant attention for its potential therapeutic benefits in combating
inflammatory diseases. The description of bioactive compounds found in M. oleifera and the
approach to intricate mechanisms of action are important in addressing inflammatory conditions,
providing promising insights for future health interventions (Chis et al., 2024). In recent
years, M. oleifera, a versatile and nutritionally rich plant, has garnered significant attention for its
potential therapeutic benefits in combating inflammatory diseases. The description of bioactive
compounds found in M. oleifera and the approach to intricate mechanisms of action are
important in addressing inflammatory conditions, providing promising insights for future health
intervention (Chis et al., 2024).

1.2.9 Dioscorea bulbifera (Air Yam)

Dioscorea bulbifera (Family: Dioscoreaceae) also referred to as ′Air Yam, ′ carries significant
importance in the traditional medicine of China, West Africa and India. It is a common
ingredient in numerous herbals and Ayurvedic formulations used to treat a variety of ailments
(Narzary et al., 2024). Its traditional usage is well-recognized in the Chinese medicinal systems
as well as in West Africa. Previous studies have demonstrated that D. bulbifera is effective
against several therapeutic diseases such as cancer, goiter, skin infections, pharyngitis and
orchitis. Its traditional usage is well-recognized in the Chinese medicinal systems as well as in
West Africa. Previous studies have demonstrated that D. bulbifera is effective against several
therapeutic diseases such as cancer, goiter, skin infections, pharyngitis and orchitis (Kundus et
al., 2021). Tubers of D. bulbifera are roasted and cooked as vegetable and serves to cure cough,
dysentery, piles, ulcers, diabetes, leprosy and syphilis (Dutta, 2015). In Uganada, tubers of D.
Bulbifera are boiled by the local people and eaten to cure HIV. Patients (Nabatanzi, 2016).
Research work has revealed that some of the phytochemicals found within the species of
Dioscorea bulbifera include diosgenin and flavonoids such as quercetin and kaempferol, giving
the plant anti-cancer, anti-inflammatory, and antioxidant activities (Dupare et al., 2025)

1.2.10 Mangifera indica (Mango tree)

Mangifera indica L. (Mango), a member of the Anacardiaceae family, is native to the tropical
and subtropical regions of the world. Leaves of M. indica exhibit pharmacological potential as a
panacea (Mehmood et al., 2024). Various parts of plant are used as a dentrifrice, antiseptic,
astringent, diaphoretic, stomachic, vermifuge, tonic, laxative and diuretic and to treat diarrhea,
dysentery, anaemia, asthma, bronchitis, cough, hypertension, insomnia, rheumatism, toothache,
leucorrhoea, haemorrhage and piles. All parts are used to treat abscesses, broken horn, rabid dog
or jackal bite, tumour, snakebite, stings, datura poisoning, heat stroke, miscarriage, anthrax,
blisters, wounds in the mouth, tympanitis, colic, diarrhea, glossitis, indigestion, bacillosis,
bloody dysentery, liver disorders, excessive urination, tetanus and asthma (Shah et al., 2010).
Some of the main bioactive compounds identified in mango fruit include phenolic
acids (coumaric acid, ferulic acid and hydroxybenzoic acid), polyphenols
(quercetin, mangiferin, catechins,
tannins, kaempferol, anthocyanins, gallic-acid, ellagic acid), carotenoids, which are the most
abundant, and the vitamins ascorbic acid, thiamine, riboflavin, and niacin (Burton-freeman et al.,
2017)

1.3 STUDY AIM AND OBJECTIVE

1.3.1 AIM
The study aims to evaluate the potential protective and restorative effects of Petiveria alliacea on
the bone marrow of Mus musculus following induction with N-Methyl-N-Nitrosourea (NMU).
1.3.2 OBJECTIVE
To assess the effects of N-Methyl-N-Nitrosourea (NMU) exposure on the histological
architecture of the bone marrow in Mus musculus with the use of ethanolic extract of Petiveria
alliacea.
1.4 Petiveria alliacea
Petivera alliacea, commonly known as Guinea hen weed, is a wild and perennial shrub
belonging to the Phytolaccaceae family (Lawal et al., 2024)

Table 1: Taxonomic classification of Petiveria alliacae

Domain Eukaryota
Kingdom Plantae
Subkingdom Tracheobionta
Superdivision Spermatopyhta

Division Magnoliophyta
Class Magnoliopsida
Subclass Caryophyllidae
Order Caryophyllales
Family Petiveriaceae

Genus Petiveria L
Species Petiveria alliacea L.
Source: United State Department of Agriculture (USDA, 2020)

Figure 2: Petivera alliacea Herb garden at FRIN

Source: Forestry Research Institute of Nigeria, 2024

1.5 LITERATURE REVIEW

1.5.1 DESCRIPTION AND MEDICINAL IMPORTANCE
Petiveria alliacea L. (Phytolaccaceae) Petiveria alliacea L., commonly referred to as guinea hen
weed or anamu, is a perennial herb belonging to the Petiveriaceae (often grouped under
Phytolaccaceae) family. Indigenous to the Amazon and widely distributed across the Caribbean,
Central, and South America, as well as regions in Africa, this plant holds a prominent place in
traditional medicine and has attracted considerable scientific interest in recent years, especially
regarding its ethnobotanical, phytochemical, pharmacological, and toxicological properties (Cal
et al., 2022). The strong garlicky odour that is released following tissue damage is connected to
this medicinal plant.
In the 17th century, African slaves used to use preparations obtained from P. alliacea to make
their masters lethargic, and for this reason, P. alliacea is widely known in Brazil as the herb to
“tame the master. The plant is also called mucuracaá, tipi, guiné, pipi, apacin, herbe aux Poules,
anamu, and embayayendo (de Andrade et al., 2018). P. alliacea is known as Guinea– Hen weed
in English, it is called Awogba or Ojusaju by the Yoruba speaking people of Southwest Nigeria,
kanunfari in Hausa, Northern part of Nigeria and Akwa-Ose in Igbo, South– Eastern Nigeria.
Petiveria alliacea is used in herbal medicine in South Western Nigeria for the treatment of
various ailments such as diabetes, arthritis, toothache, skin infection etc (Ayodele et al., 2021).
P. alliacea is a perennial subshrub, sub Woody, erect, and branched with long branches, which
are delicate and ascending, measuring up to 1 m in height. The leaves are 5–10 cm long and 2–
6 cm in width, discolor, oblonglanceolate, acuminate, with a cuneiform base and short petioles,
its s texture ranges from membranous to herbaceous, with prominent midrib in the abaxial face
and secondary argued veins, texture ranges from membranous to herbaceous, with prominent
midrib in the abaxial face and secondary argued veins (de Andrade et al., 2018). P. alliacea roots
are pivoting type and may reach 30 cm in length and 1 cm in diameter in the base; it has a
yellowish–brown surface, tortuous, pale externally, and bright whiteness internally, with an acre
flavor and a garliclike odor (de Andrade et al., 2018).
In traditional medicine, P. alliacea finds application in various therapeutic approaches, among
which stand out antispasmodic, diuretic, abortive, hypoglycemic, anti-inflammatory, and
anticancer activity, in addition to central nervous system activities, such as anxiolytic effects, and
use in the treatment of dementia and nervous disorders (Kelly et al., 2022). The herb is rich in
sulfurcontaining compounds that possess a broadspectrum of in vitro antimicrobial activity
against pathogenic fungi and bacteria at low concentrations. P. alliacea also showed cytotoxicity
and antiproliferative activity against cancer cell lines through sophisticated machinery of cellular
damage in vitro. Other compounds such as flavonoids, terpenoids, and benzenoids are commonly
identified in P. alliacea extracts, and they may also justify these activities (de Andrade et al.,
2018). Linoleic acid and allantoin, also found in P. alliacea leaf extract, increase insulin
secretion (Gunawan et al., 2020).
Although dibenzyl trisulfide (DTS) has been considered the major active ingredient responsible
for much of its antitumor activity, a drug containing this metabolite has not been developed due
to its high toxicity and instability and its possible role as an inhibitor of intracellular
cytochromes, suggesting that Anamu is the ideal candidate for the development of a
polymolecular medicine (Murray et al., 2016). The alcoholic extract of Petiveria
alliacea (Esperanza) has a significant in vitro antitumor effect on other tumor cells and also the
ability to regulate energy metabolism (Murillo et al., 2024). The potential of Petiveria alliacea to
modulate the metabolism of K562 cell lysates was evaluated by non-targeted metabolomics.
Initially, in vitro findings showed that P. alliacea reduces K562 cell proliferation; subsequently,
alterations were observed in the endometabolome of cell lysates treated with the extract, mainly
in glycolytic, phosphorylative, lipid, and amino acid metabolism (Rojas et al., 2023). The anti-
tumor activity of the aqueous extract on the K562 cell line is attributed to the decrease in
metabolites related to cell proliferation and/or growth, such as nucleotides and nucleosides,
leading to cell cycle arrest shows a preliminary part of the mechanism for the anti-tumor and
antiproliferative effects of P. alliacea on cancer (Rojas et al., 2023).

Petiveria alliacea could inhibit glycolysis and OXPHOX modulating tumor metabolism, making
it a potential treatment for tumors with altered metabolism (Ballesteros-Ramírez et al., 2023).
Furthermore, Petiveria alliacea has been shown to accumulate nitrates and has caused nitrate
poisoning in cattle. Therefore, these plants can be grown in the rural communities as well in the
urban community to mitigate the incidence of mosquitoes in the environments. Scientifically,
there is paucity of literature on these plants to control mosquitoes (Akintan et al., 2021)

Although P. alliacea is considered toxic by people, its toxicity remains a concern since it is
strongly dependent on the extraction method and the part of the plant used during tests. Even if
some healers prefer to use the aerial parts in a liquefied form or by chewing them, instead of
decoctions or infusions, no toxicological studies exist using whole dried stems and leaves
(Garcia- Perez et al., 2018). Furthermore, the use of this species for religious ceremonies has
been reported since the era of slavery in the Americas (luz et al., 2016).
 CHAPTER TWO

2.0 MATERIALS AND METHODOLOGY

2.0 MATERIALS

2.1.1 PLANT COLLECTION

The plant of Petiveria alliacea was sourced from Bariga Market, a popular market in Lagos
state, southwest Nigeria. The plant was identified at the National Centre for Genetics and
Biotechnology (NACGRAB), Moor Plantation, Ibadan, Oyo State, Nigeria, with a voucher
number PET/PA/NACG/007 of the plant deposited in the herbarium.

2.1.2 CHEMICALS AND REAGENTS

Cisplatin, N-Nitroso-methyl urea (NMU), absolute ethanol, fetal bovine serum (FBS), May-
Grunwald stain, Giemsa stain, Xylene, DPX mountant, 10% formaldehyde, methanol.

2.1.3 EQUIPMENT AND SUPPLIES

EDTA bottles, cotton wool, beakers, handkerchiefs, normal saline, hand gloves, razor blade, test
tubes, autoclave, foil paper, universal bottles, Eppendorf tubes, vacuum oven, light microscope,
dissecting kit, mice cages, digital weighing balance, sterile bottles, ethanol, distilled water, paper
tape, spatula, marker, 1ml syringe, coplin jar, pipette tips, frosted glass slide, petri dish, funnel,
cannula, sawdust, plant material of Petiveria alliacea

2.2 METHODOLOGY

2.2.1 PLANT EXTRACT PREPARATION

1022g (1.022kg) of Petiveria alliacea plant material was carefully weighed using a precise
weighing balance. The plant material was soaked in 2500ml of ethanol at a concentration of 90%
ethanol and mixed. This mixing process continued until the plant material was saturated and had
achieved a homogenous mixture. The container was sealed with aluminum foil and the main
plastic cover to prevent contamination of the extract and the sample was left to soak for
ethanolic-extraction for 72 hours. At the end of the maceration period, the ethanolic extract was
carefully filtered using a funnel and cotton wool. This was done to remove impurities and other
matter present in the extract that could affect the validity of the result. The filtrate from the
ethanolic extract was obtained by collecting the extract that passed through the filter (the cotton
wool in the funnel acted as the filter in this experiment). The filtrate was then concentrated by
o
evaporating the solvent with a temperature-regulated oven that was set at 45±1 C. The
concentration process continued until the extract had completely turned to a gel form (not
completely dried but thick) which took days. After drying the ethanolic extract, the recovered
extracts were carefully transferred from the beaker into air-tight sample containers. The container
o
was stored at a room temperature of 4 C until it was ready for use in the study. When the extracts
were required for use, they were reconstituted with sterile water at various concentrations,
-1 -1 -1
specifically at doses of 200 mg kg , 400 mg kg ,800mg kg .
2.2.2 EXPERIMENTAL DOSAGE PREPARATION
To prepare the treatment for the study, different concentration of the plant extract and a single
-1
dose of Nitroso-Methyl Urea (NMU) were used. To prepare the concentration of 200mg kg of
the plant extract, 2g of the extract was dissolved in 10ml of distilled water. The mixture was
stirred vigorously in a sterile bottle and then sieved out using cotton wool to filter out any
particulate matter.
-1
The same procedure was repeated for 400mg kg , but in this scenario, 4g of the extract was used
in 10ml of the distilled water and it was mixed vigorously in a sterile bottle, sieved using a
cotton wool and a clear solution of the extract without impurities achieved.
-1
For 800mg kg , 8g was used in 10ml of distilled water and after mixing, it was sieved using
cotton wool and the clear solution without impurities was stored in a sterile bottle.
After these preparations of the extract for dosing, it was well labelled with different
concentration written on the sterile bottle used to preserved them.
The N-Methyl-Urea (NMU) was purchased from Bristol Scientific Company Limited. It was
prepared at University of Lagos Botanical garden by measuring 1g of NMU on a weighing
balance and dissolved in 10ml of distilled water. The solution was mixed in a sample bottle until
the crystals of the NMU dissolved completely in water forming a homogenous solution.
 Figure 3: Chemical structure of NMU (N-Methyl-Urea)
Source: National Center for Biotechnology Information (2025).

2.2.3 ANIMAL PREPARATION AND GROUPING

Twenty-five (25) Mus musculus weighing between 17-27g were purchased from Topo Badagry
o o
Farm, Badagry, Lagos State (Latitude 6 25’0” N, Longitude 2 55’59” E). They were left to
acclimatize for 2(two) week and were housed at the Botanical Gardens of the Faculty of Science,
University of Lagos before bioassay for them to adjust to their new environment so as to ensure
they were ready for the dosing to be effectively and appropriately carried out. Afterward, the
mice were weighed and randomly sorted into five (5) groups A to E, based on their body weight.
They were kept in plastic cages with saw-dust to be provided with warmth and prevent the cage
from being messy under standard temperature and humidity, fed with growers’ pellet and were
given water ad libitum.
2.2.4 NMU INDUCTION AND TREATMENT
Proper dosing and administration protocols are important in scientific studies involving animal
models to ensure reproducibility and reliability of results. In this study, the mice were randomly
assigned to five experimental groups due to their body mass (n= 5 per group) in labelled cages.
Mice in cage A and B received 0.3ml of NMU, cage C received 0.25 ml of NMU, cage D
received 0.2ml of NMU while cage E received 0.1ml of NMU. All were induced
intraperitoneally to initiate systemic toxicity and mimic a carcinogenic condition.
Following induction, groups A to C received an oral administration of the ethanolic extract of
Petiveria alliacea using cannula at different concentrations. Specifically, Group A received
800mg/kg body weight of the extract, Group B received 400mg/kg and Group C was
administered 200mg/kg. The dosing for these groups was carried out daily over a 56-days period.
Group D served as the standard drug group and was treated with cisplatin, a chemotherapeutic
agent known to target rapidly dividing cancerous cells. Mice in this group received 0.1ml of
cisplatin intraperitoneally three times a week specifically on Mondays, Wednesdays and Fridays
for a duration of eight (8) weeks.
Group E functioned as the negative control group. Mice in this group were not administered any
treatment post-induction but were maintained on standard pellet feed and water ad libitum
throughout the experimental period.
Throughout the 8-weeks duration, all animals were weighed weekly and inspected for
irregularities. This was essential for assessing the general health status of the animals and for
adjusting the dosing regimen if necessary. Changes in the body weight served as an indirect
marker for toxicity, therapeutic efficacy and overall physiological response to treatment. The
mice were sacrificed via cervical dislocation at the expiration of treatments. The femur was
surgically harvested, cleaned and stored in normal saline for bone marrow assays.
2.2.5 BONE MARROW ASSAY (MICRONUCLEUS ASSAY PROCEDURE AND
STAINING TECHINQUE)
This was executed according to Oloyede et al., (2020). In the course of this study, bone marrow
micronucleus assay was carried out to evaluate genotoxic damage in animal model. The
procedure involved the use of May-Grunwald and Geimsa stains to visualize micro-nucleated
polychromatic erythrocytes. Following cervical dislocation, both femurs of the animals were
carefully excised and cleaned. The epiphyses were cut off, and the bone marrow was flushed
from each femur using 1 ml of fetal bovine serum (FBS), which was collected into sterile 1.5 ml
Eppendorf tubes. The flushed bone marrow suspension was gently mixed using a micropipette to
ensure homogeneous dispersion of cells.
The cell suspensions were centrifuged at 1000 rpm for 10 minutes. After centrifugation, the
supernatant was discarded and the cell pellet was resuspended in 1 ml of fresh FBS. This was
again mixed thoroughly and subjected to a second round of centrifugation at the same speed.
Once the supernatant was discarded, 0.5 ml of FBS was added to the resulting pellet and mixed
thoroughly. A drop of this final cell suspension was then placed on a clean, grease-free
microscope slide and spread to form a uniform smear. The prepared smears were allowed to air-
dry before proceeding to fixation and staining.
The air-dried slides were fixed in 70% methanol for three minutes to preserve the cellular
morphology. After fixation, differential staining was performed using May-Grunwald Stain I
(MG I) and May-Grunwald Stain II (MG II), both of which were freshly prepared in the
laboratory. MG I was prepared by dissolving 0.2 g of May-Grunwald powder in 50 ml of
absolute methanol to make a 0.4% solution. This served as the primary stain. MG II, on the other
hand, was prepared by mixing equal volumes (15 ml each) of MG I and distilled water,
producing a 1:1 dilution that served as the secondary stain to enhance cytoplasmic detail.
The fixed slides were immersed in 0.4% MG I for 3 minutes, followed by transfer into MG II for
another 3 minutes. The slides were then rinsed thoroughly with distilled water and air-dried.
After drying, they were counter-stained with 5% Geimsa stain for 5 minutes, rinsed again in
distilled water, and allowed to dried at room temperature for microscopic examination.

2.2.6 MICROSCOPIC EXAMINATION AND CELL COUNTING

The prepared slides were examined under a light microscope. Initial observations were made
under low magnification to locate regions with a uniform distribution of non-overlapping cells.
Once appropriate fields were identified, the magnification was increased to 100× oil immersion
for detailed scoring. Each slide was carefully scanned, and cells were counted only when fully
within the focal plane to avoid errors in classification.
A minimum of 1000 polychromatic erythrocytes (PCEs) and normochromatic erythrocytes
(NCEs) were examined per animal to assess the frequency of micronucleated cells (MnPCEs).
The identification of PCEs and normochromatic erythrocytes (NCEs) was based on
morphological and staining characteristics. PCEs were identified by their bluish-purple
coloration, whereas NCEs appeared pinkish-orange. Their relative sizes and staining intensity
further facilitated distinction. The ratio of PCEs to NCEs (PCE: NCE) was also determined,
serving as an indicator of bone marrow toxicity. A decrease in this ratio was interpreted as
evidence of cytotoxic effects induced by the administered substances.
In addition to micronuclei, nuclear abnormalities (NA) were evaluated as supplementary
indicators of cytotoxicity. These abnormalities included binucleated cells (BN), cells with
notched nuclei (NT), and budding nuclei (NBud). Binucleated cells were characterized by the
presence of two distinct nuclei. Notched nuclei contained invaginations or vacuoles with
appreciable depth that lacked nuclear material, nuclear budding involved the presence of small
nuclear protrusions resembling micronuclei still attached to the main nucleus, Encleated cells
with a clear, round or biconcave cell without nuclear material, Necrotic cells with irregular cell
shape or ruptured cell membrane while
Counts were recorded across multiple fields per slide to ensure accuracy and reproducibility. The
mean values obtained from different fields were used for subsequent statistical analysis and
comparison among treatment groups.
PLATE 1: Embeded cells dipped in coplin jar

PLATE 2: Air drying of the stained slides

PLATE 3: Mounting of slides on microscope
 PLATE 4: Visualization of stained slide

2.2.7 STATISTICAL ANALYSIS

The data generated from the micronucleus assay and other experimental parameters were
subjected to statistical analysis using Microsoft Excel. Results were expressed in the format of
Mean ± Standard Error of the Mean (SEM) to represent the central tendency and variability
within each experimental group.
To determine whether there were statistically significant differences between the control and
treated groups, a Student’s t-test was employed. This test was used to compare the means of the
measured parameters such as the frequency of micro-nucleated polychromatic erythrocytes
(MnPCEs), PCE:NCE ratio, and incidence of nuclear abnormalities between the two groups.
The p-values were computed directly using Microsoft Excel’s built-in statistical functions.
Differences were considered statistically significant when p < 0.05, indicating a less than 5%
probability that the observed differences occurred by chance.
This statistical approach was adopted to validate the potential genotoxic or cytotoxic effects of
the test substance(s) and to confirm whether the observed variations in cellular parameters were
biologically meaningful.
 CHAPTER THREE
RESULTS
3.1 Effect of Ethanolic Petiveria alliacea Extract on Body Weight of Mus musculus.
GROUP A (NMU + 800mg/kg extract)
The treatment demonstrated a steady and significant increase in body weight from baseline
through week 8. This suggests a dose-dependent protective or anabolic effect of the high dose
extract in mitigating NMU-induced toxicity. The mean body weight increased progressively from
28.2g in week 1 to 34.8g in week 8, with an overall steady upward trend.
GROUP B (NMU + 400mg/kg extract)
exhibited a general upward trend in body weight, with the mean rising from 27 g in Week 1 to a
peak of 31.78 g by Week 7. A slight decline to 31.28 g was observed in Week 8, suggesting that
while the extract was initially effective at promoting weight gain at this dose, its efficacy may
have begun to plateau toward the end of the experimental period.
Group C (200 mg/kg extract + NMU)
The treatment also showed a moderate increase in weight over time. Starting at 25 g, the mean
body weight rose steadily despite some fluctuations between Weeks 5 and 7, eventually reaching
31.15 g in Week 8. This indicates that the extract retained a positive, though less stable, effect on
body weight at the lower dosage.
Group D (Cisplatin )
The treatment displayed notable fluctuations in weight across the 8-week period. Although there
were intermittent decreases, the group ended with a mean weight of 31.79 g. The inconsistency
in weight progression may be attributed to the cytotoxic effects of Cisplatin, which likely
induced physiological stress, impacting weight maintenance and recovery.
Group E ( Water + NMU)
The treatment maintained a steady and consistent weight gain, increasing from an initial mean of
22.4 g to 31.79 g by Week 8. This predictable pattern highlights the natural growth trajectory of
untreated mice and serves as a stable reference point for evaluating treatment effects in the other
groups.
The tables presents the body weight measurements of the experimental groups over the 8-week
treatment period, from week 1 to week 8.

Table 2: Weekly weight of the Mus musculus

WEEK 800kg/mg 400kg/mg 200kg/mg CISPLATIN CONTROL

Week 1 28.2 27 25 23.7 22.8

29.1 25.8 25.3 23.9 22.7

28.5 25.3 24.4 24.8 23.1

27.5 26 25.1 24.5 21

28.8 26.8 25.6 23.7 -

MEAN 28.42 26.18 25.08 24.12 22.4

SEM 0.165529454 0.504579032 0.180554701 0.361939221 0.286865241

Week 2

800kg/mg 400kg/mg 200kg/mg Cisplatin Control

29.9 26.8 26.3 25.8 22.5

29.8 27.2 25.8 25 23.8

30.3 28.1 26.1 23.9 23.6

29.6 25.9 25.3 23.9 23.2

30.5 25.2 26.2 24.4

MEAN 30.2 26.64 25.94 24.6 23.275

SEM 0.165529454 0.504579032 0.1805547 0.361939 0.286865

Week 3 800kg/mg 400kg/mg 200kg/mg Cisplatin Control

31.3 26.3 27.6 26.6 24.2

31.8 26.5 28.5 24.8 23.8

32.2 28.5 26.6 25.4 23.9

29.3 26.7 25.4 26.2 24.3

31.7 27.2 26.8 - -

MEAN 31.6 27.04 26.98 25.75 24.05

SEM 0.510489961 0.394461658 0.518073354 0.403112887 0.119023807

Week 4 800kg/mg 400kg/mg 200kg/mg Cisplatin Control

28.2 26.9 25.9 24.4 23.9

29.1 27.5 26.8 26.8 24.4
 28.5 28.4 27.7 27.1 25

27.5 28 29.5 26.8 24.3

28.8 27.3 27.2

MEAN 28.42 27.62 27.42 26.275 24.4

SEM 0.274590604 0.263438797 0.597829407 0.628987281 0.227303028

Week 5 800kg/mg 400kg/mg 200kg/mg Cisplatin Control

35.3 31.5 30.4 28.9 26.1

34.8 30.3 28.9 29.3 27.1

33.7 28.8 29.1 27.5 25.4

31.2 27.8 - -
MEAN 34.6 30.45 29.05 28.56666667 26.2

SEM 0.472581563 0.606217783 0.533072853 0.545690185 0.493288286

Week 6 800kg/mg 400kg/mg 200kg/mg Cisplatin Control

32.3 30.5 31.6 32.2 29.3

34.7 33.3 30.4 30 28.6

37.4 32.5 32.7 28.6 27.5

- 30.8 31.1 - -
MEAN 34.8 31.775 31.45 30.26666667 28.46666667

SEM 1.473091986 0.672526332 0.483907705 1.04774891 0.523874455

Week 7 800kg/mg 400kg/mg 200kg/mg Cisplatin Control

33.6 34.2 33.3 32.4 28.8

35.6 31.3 33.5 30.7 31.6

34.9 33.8 30.7 30.8 30

- 32.6 32.6 - -
MEAN 34.7 32.975 32.525 31.3 30.13333333

SEM 0.585946528 0.653675506 0.638194067 0.550757055 0.811035004

Week 8 800kg/mg 400kg/mg 200kg/mg Cisplatin Control

34.8 30.3 31.6 30.8 29.6

35.7 34.8 32.9 31.4 33

34.4 34.5 33.8 33 32.6

- - 33.9 - -

MEAN 34.96666667 33.2 33.05 31.73333333 31.73333333

SEM 0.384418753 1.452583905 0.533072853 0.65659052 1.072898463
WEEKS 800mg/kg 400mg/kg 200mg/kg cisplatin control

WEEK 1 28.42±0.27c 26.18±0.31c 25.08±0.19 c 24.12±0.22b 22.40±0.47

WEEK2 30.02±0.17c 26.64±0.50b 25.94±0.18c 24.60±0.36a 23.28±0.28

WEEK 3 31.26±0.51c 27.04±0.3c 26.98±0.52b 25.75±0.40b 24.05±0.12

WEEK4 32.3±0.97c 27.62±0.26c 27.42±0.59b 26.28±0.63a 24.40±0.23

WEEK5 34.6±0.47c 30.45±0.60b 29.05±0.53a 28.57±0.55a 26.20±0.49

WEEK6 34.82±1.47a 31.77±0.67a 31.45±0.48b 30.27±1.05 28.47±0.52

WEEK7 34.7±0.59a 32.98±0.65a 32.53±0.64 31.30±0.55 30.13±0.81

WEEK8 34.97±0.67a 33.20±1.45 33.05±0.53 31.73±0.66 31.73±1.07

Table 3: The average mean weight ± SEM of Mus musculus

Values are expressed as Mean± SEM (N =5 )
The superscripts a, b and c represent a different level of significance where a= p ≤ 0.05, b= p <
0.01 and c= p < 0.001

3.1.1 Effect of Petiveria alliacae Ethanolic Extract on the Bone Marrow of Mus musculus
Polychromatic erthrocytes (PCE) observed in bone marrow of Mus musculus administered with
ethanolic Petiveria alliacae for 56days.
Table 4 presents the frequency of nuclear abnormalities in polychromatic erythrocytes (PCEs) of
mice treated with various doses (800 mg/kg, 400 mg/kg, and 200 mg/kg) of F. platyphylla
ethanolic extract, compared with a cisplatin-treated group and a control.
 Table 4: Effect of PCE parameter on experimented Mus musculus
NUCLEAR 800mg/kg+ 400mg/kg 200mg/kg+ Cisplatin Water + NMU
ABNORMA- NMU +NMU NMU (control)
LITIES

NM 352.75±55.18a 397.50±50.86a 303.00±39.31c 329.50±38.82b 570.25±21.31

PM 5.25±2.56 13.25±6.38 17.25±4.44 25.00±13.09 5.50±1.93
a
SM 14.00±4.60 22.75±5.29 26.00±7.11 15.75±2.95a 5.50±1.93

KN 1.00±0.71b 6.50±6.50b 6.00±2.45b 6.00±1.47b 30.25±4.89

BLN 9.75±3.82 23.75±12.24 27.75±5.79b 23.50±10.49 1.00±0.41

BN 17.25±5.98 21.25±13.93 63.00±12.49b 16.00±5.34 11.75±2.95

MN 6.75±2.29 16.00±9.35 19.00±1.87a 14.00±2.67 7.25±2.93

VC 2.00±1.68 11.50±6.44 32.50±22.16 42.75±22.95 16.00±7.24

NB 4.75±2.32 5.50±2.10 28.50±12.39

12.00±4.60 13.25±5.49

EN 4.50±3.23 10.25±2.29 12.50±3.92 8.00±2.92 7.00±3.19

NE 17.50±3.07 27.50±8.01 33.00±7.71 23.75±5.34 14.25±4.64

AP 8.50±2.50 11.50±4.29 90.00±54.19 23.75±8.12 8.25±3.68
Values are expressed as Mean ± SEM (N= 5)
The superscript a, b and c represent a different level of significance where a= p≤ 0.05, b= p<
0.01 and c= p< 0.001. The superscript letters a, b and c are determined by the level of
significance when comparing the treated group to the control.
Note:
Superscript a= Slight difference
Superscript b= Large difference
Superscript c= Very large difference
KEY:
NM: Normal PM: Polymorphic Nucleus SM: Segmented Nucleus KN: Kidney Shaped Nucleus BLN:
Blebbed, Lobed or Notched Nucleus BN: Binucleated MN: Micronucleus VC: Vacuolated Nucleus NB:
Nuclear Bud EN: Encleated NE: Necrotic AP: Apoptotic

3.1.2 Effects of Petiveria alliacae Ethanolic Extract on Nuclear Abnormalities in

Normochromatic Erythrocytes (NCEs) of Mus musculus
Table 5 presents the frequency of various nuclear abnormalities in normochromatic erythrocytes
(NCEs) of mice following treatment with Petiveria alliacae ethanolic extract at 800 mg/kg, 400
mg/kg, and 200 mg/kg, as well as a cisplatin-treated group and a control group.
 Table 5: Effect of NCE parameters on experimented Mus musculus
NUCLEAR 800mg/kg + 400mg/kg + 200mg/kg + Cisplatin H2O + NMU
NMU NMU NMU (control)
ABNORMALITES

NM 399.00±19.34 447.75±41.59 336.5±20.98a 523.00±52.65 472.25±30.16

PM 7.75±1.25 8.25±1.70 20.75±4.94a 7.25±0.63 5.25±3.94

SM 4.75±1.55 16.25±2.43 13.50±5.17 20.25±6.37 10.00±1.87

KN 2.00±1.23 1.25±0.25 4.25±2.03 8.00±4.45 0.50±0.50

BLN 4.00±2.71 26.50±15.54 20.25±3.09b 13.00±5.78 3.00±1.78

BN 9.25±2.39 6.00±5.02 18.00±11.92 15.00±5.87 3.00±1.29

MN 3.75±1.18 13.25±4.13 9.25±2.18a 10.50±1.85b 2.50±0.29

VC 141.00±18.09 127.50±46.96 193.00±67.78 225.25±13.85b 142.25±8.70

NB 3.25±1.97 5.75±1.79 16.50±6.31 9.75±3.09a 1.75±0.75

EN 2.25±0.95 3.25±0.75 2.50±0.87 3.00±0.41 1.75±0.85

NE 1.75±0.85 4.75±3.77 4.75±1.38 6.25±5.29 5.00±2.12

AP 1.25±0.63 2.75±0.48 28.50±22.21 4.25±1.84 2.25±1.11

Values are expected as Mean ± SEM (N=5)

The superscript a, b and c represent a different level of signifance where a= p ≤ 0.05, b=p < 0.01
and c= p < 0.001. The superscript letters a, b and c are determined by the level of significance
when comparing the treated group to the control.
Note:
Superscript a= Slight difference
Superscript b= Large difference
Superscript c= Very large difference
KEY:
NM: Normal PM: Polymorphic Nucleus SM: Segmented Nucleus KN: Kidney Shaped Nucleus BLN:
Blebbed, Lobed or Notched Nucleus BN: Binucleated MN: Micronucleus VC: Vacuolated Nucleus NB:
Nuclear Bud EN: Encleated NE: Necrotic AP: Apoptotic

PLATE 5: Microscopic image of Genotoxicity test on the femur cell of Mus musculus treated
-1
with 800mg kg Petiveria alliacea (x 400)
PLATE 6: Microscopic image of Genotoxicity test on the femur cell of Mus musculus treated
-1
with 400mg kg Petiveria alliacea (x 400)
PLATE 7: Microscopic image of Genotoxicity test on the femur cell of Mus musculus treated
-1
with 200mg kg Petiveria alliacea (x 40)
PLATE 8: Microscopic image of Genotoxicity test on the femur cell of Mus musculus treated
with Cisplatin (x 400)

BLN

NM
 PLATE 9: Microscopic image of Genotoxicity test on the femur cell of Mus musculus treated
with (H2O + NMU) Control (x 400)
EN

CHAPTER FOUR
KN
DISCUSSION AND CONCLUSION
DISCUSSION
Body weight is a pertinent criterion in evaluating toxic effects of treatment and drugs or toxic
substances (Ferreira et al., 2014). Variation in weights can be one of the first critical evidence of
toxicity (Ajayi et al., 2019). Evaluation of growth in animals using weight index is a routine in
toxological compound related effects (Murbach et al., 2017). Assessing growth in animals
through weight index is a customary practice in toxicological reaserch (Oloyede et al.,
Moreover, increase or decrease in body weight could be due to the adverse effects of drugs
(Oloyede et al., 2018). This evaluation helps in interpreting the effects related to the compound
SM
being studied. In this study, after 56 days of treatment administration, Mus musculus treated with
Petiveria alliacea extract showed dose dependent weight gain, with 800mg/kg and 400mg/kg
groups recording the highest increases. The 200mg/kg group showed moderate improvement ,
indicating partial protective effects. Compared to the cisplatin group, which gained less
pronounced weight gain demonstrated the superior protective effect of P. alliacea compared to a
standard cytotoxic agent. Meanwhile, the control group, which received no treatment, recorded
steady increase ,consistent with natural growth progression in the absence of NMU-induced
stress.
The bone marrow micronucleus (MN) assay is a cytogenetic technique used to evaluate the
genotoxic potential of various substances by detecting the formation of micronuclei in bone
marrow cells, particularly erthrocytes precursor cells (Sommer et al., 2020). The choice of MN
assay against other genotoxicity tests is because it provides a reliable measure of genotoxic
damage and, crucially, any reduction thereof due to the herbal intervention . MN has been
identified as a biomaker for chromosomal damage in both young and new erthrocytes (Oloyede
et al., 2020). These MNs primarily form from fragments of chromosomes that are not
incorporated within the daughter nuclei during the mitosis process of erythropoietic blast cells
(Oloyede et al., 2020). The ethanolic extract of Petiveria alliacea contains bioactive constituents
such as flavonoids, sulfur-containing derivatives (e.g benzaldehyde and cysteine sulfoxides) and
terpenoids, which contribute to its robust antioxidant and antimicrobial activity (Silva et al.,
2018). It also exhibit strong anti-inflammatory effects, highlighted by reduced leukocyte and
neutrophil migration in animal models of inflammation (Lopes-Martins et al., 2002).
Toxicological evaluations suggest the extract can be relatively safe depending on the dose and
extraction method (Garcia-Perez et al., 2018). Furthermore, its cytotoxic and
immunomodulatory potential has been documented in reviews emphasizing its activity against
cancer cell lines and immune regulation (Silva et al., 2018).
Several abnormalities were observed in polychromatic erthrocytes (PCEs) as seen in the result in
table 3 were highest in the NMU-only groups, confirming its genotoxic effect. Binucleated cells
(BI) were the most frequent abnormality, suggesting cytokinesis disruption, likely induced by
NMU (Bakare et al., 2013). Vacuolated nuclei (VC) and necrotic cells (NE) were also prominent,
indicating oxidative damage and cell death. Segmented nuclei (SM), blebbed or lobed nuclei
(BLN) and apoptotic cells (AP) reflect nuclear stability and genetoxic stress (Oloyede et al.,
2020). Treatment with ethanolic extract, especially at 800mg/kg, reduced these abnormalities
significantly, showing a dose-dependent protective effect. Cisplatin also reduced abnormalities
but showed some toxicity. The control group had minmal abnormalities, supporting the extract’s
potential in protecting bone marrow cells from NMU-induced damage.
The NMU-only control group exhibited the highest total abnormalities in normochromatic
erythrocytes (NCEs), indicating significant genotoxic damage. The presence of micronucleated
cells (MN) was the most prominent abnormality, reflecting chromosomal breakage or mitotic
spindles disruption, commonly induced by genotoxic agents like NMU (Bakare et al., 2013).
Other observed abnormalities include binucleated cells (BI) and vacuolated nuclei (VC),
indicative of impaired cytokinesis and oxidative stress, respectively. Additional lesions such as
blebbed nuclei (BLN), nuclear buds (NB), necrotic (NE) and apoptotic cells (AP) suggests
nuclear fragmentation and programmed cell death (Oloyede et al). Treatment with the ethanolic
extract of P. alliacea led to dose-dependent reduction in abnormalities, with the 200mg/kg group
showing the lowest total nuclear abnormalities among treated groups. Although the 400mg/kg
recorded slightly elevated abnormalities, the 800mg/kg amd 200mg/kg were more effective in
reducing genotoxic effects. Cisplatin, while therapeutic, still caused substantial nuclear damage,
confirming its cytotoxic side effects. Overall, the extract demonstrated protective effects against
NMU-induced NCE abnormalities, likely due to its antioxidant and anti-inflammatory properties.
CONCLUSION
The observed nuclear abnormalities in both polychromatic and normochromatic erythrocytes
reflect significant bone marrow genotoxicity following NMU exposure. The increased frequency
of binucleated cells, micronuclei, nuclear buds, and apoptotic cells indicates disrupted
erythropoiesis and chromosomal instability within the bone marrow microenvironment.
Treatment with the ethanolic extract of Petiveria alliacea resulted in a dose-dependent reduction
in these abnormalities, suggesting a protective and possibly reparative effect on bone marrow
NM
BLN
integrity. The highest dose (800 mg/kg) conferred the most substantial amelioration. Conversely,
the NMU-only and cisplatin-treated groups displayed persistent marrow damage, while the
control group showed intact nuclear architecture, affirming normal bone marrow function dose-
dependent reduction in these abnormalities, suggesting a protective and possibly reparative effect
on bone marrow integrity. The highest dose (800 mg/kg) conferred the most substantial
amelioration. Conversely, the NMU-only and cisplatin-treated groups displayed persistent
marrow damage, while the control group showed intact nuclear architecture, affirming normal
bone marrow function.
NE