CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of Study

Tuberculosis, often shortened as TB, is an infectious disease that is caused by bacteria

Mycobacterium tuberculosis. Every year, nearly 8 million people suffer from TB and 2 million

patients die because of improper treatment (Bisht et al., 2019). Tuberculosis is a potentially

serious infectious disease that mainly affects the lungs. Bacteria that cause tuberculosis are

spread from one person to another through tiny droplets released into the air via coughs and

sneezes (Tiwari, 2010). Tuberculosis puts stress on public health because of its high mortality

rates after HIV/AIDS. Tuberculosis is preventable, and the World Health Organization’s (WHO)

End TB Strategy is targeting significant reductions in TB incidence and mortality by 2030 (Pooja

et al., 2021).

Mycolic acids are alpha-alkyl, beta-hydroxy fatty acids that are produced by all mycobacteria

and are the signature lipid of the hydrophobic mycobacterial cell wall. Mycolic acid biosynthesis

has been intensely studied due to the unique structure of these lipids and their importance for

tuberculosis antibiotic therapy and M. tuberculosis pathogenesis (Barkan et al., 2009).

Mycobacteria have an unusual cell wall in which mycolic acids play a critical role in its structure

and function. The inhibition of mycolic acid cyclopropane synthase is critical to the management

and treatment of tuberculosis.

In Africa, many patients resort to medicinal plants and herbs as firstline treatment in disease

management. The belief that herbs have minimal side effects and its cultural acceptability

contribute significantly to the persistent and significant use of herbal medicine (Prasathkumar et

1
al., 2021). One of such important medicinal plants is Bridelia ferruginea, a commonly growing

shrub that belongs to the family, Euphorbiaceae. Known widely in many African countries, this

knotted shrub appears to be one of the well-studied species with regards to its folklore

application and pharmacological properties (Ngueyem et al., 2009). Traditionally, Bridelia

ferruginea is used for managing arthritis, dysentery, constipation, diarrhoea, chronic diabetes,

skin diseases, bladder and intestinal disorders, oral infections, contusion, thrush, bites and as an

arrow poison antidote.

1.2 Justification

Tuberculosis has been implicated to cause nearly 2 million deaths every year. In Nigeria, it

remains a public health issue having been ranked as the second cause of death from single

infectious disease after HIV/AIDS. The use of conventional drugs for the treatment of TB is

associated with a number of side effects; therefore, the use of herbs becomes plausible. Bridelia

ferruginea has been established to treat different infections locally but the potential actions of it

phytocompounds against mycolic acid cyclopropane synthase protein in silico has not been

explored.

1.3 Aim

The aim of this research is to determine the virtual screening of natural compounds derived from

Bridelia ferruginea as potential inhibitors of mycolic acid cyclopropane synthase for tuberculosis

drug discovery.

1.4 Specific Objectives

The specific objectives are to:

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 i. determine the compounds of Bridelia ferruginea that can be potential inhibitors of

mycolic acid cyclopropane synthase and;

ii. determine the molecular interaction of these compounds with the target protein (mycolic

acid cyclopropane synthase)

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Tuberculosis Overview

Tuberculosis is a chronically infectious and notifiable disease produced by Mycobacterium

tuberculosis (Bisht et al., 2019). Tuberculosis occurs most often in crowded, inner city,

economically disadvantaged environments and among people with others medical risk factors

(Fasoranti, 2016). TB is spread from person to person through airborne transmission by droplet

nuclei. When people with pulmonary or laryngeal tuberculosis cough, sneeze, speak, laugh or

sing, they can send droplet nuclei into the air and other people who inhale the infectious air bone

droplet nuclei can become infected (Tiwari, 2010). Baykan et al. (2022), state that the lungs are

primarily involved, but the infection can spread to other organs. Close contacts (people with

prolonged, frequent, or intense contact) are at the highest risk of becoming infected (typically 22

percent infection rate but studies have reported, even up to 100%). A person with untreated,

active tuberculosis can infect an estimated twenty other people per year. Others at risk include

foreign-born from areas where TB is common, immunocompromised patients, residents and

employees of high-risk congregate settings, health care workers who serve high-risk clients,

medically underserved, low-income populations, high-risk racial or ethnic minority populations,

and children exposed to adults in high-risk categories. Transmission can only occur from people

with active TB disease not latent TB infection (Kendall et al., 2021). The probability of

transmission depends on infectiousness of the person with TB (quantity expelled), environment

of exposure, duration of exposure, and virulence of the organism.

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2.2 Epidemiology of Tuberculosis

It is reported that 1/3rd of the entire world's population is infected with tuberculosis (De Martino

and Galli, 2014). From latent infection, the infection can arise to change in active state (Cruz-

Knight and Blake-Gumbs, 2013). About 5 to 10% of LTBI cases are at very high risk due to

evolving from normal infection to active (primary) TB. Those with HIV and other

immunocompromised patients, such as a patient with cancer or currently taking medication of

immunosuppressive drug have a very higher risk of developing active TB. (Diel, 2012).

Tuberculosis is much fatal than cholera as about 9 million people from all over the world were

infected with TB and about 1.5 million stops fighting to TB in 2013. In 2004, only TB was

responsible for more than 2.5% of all deaths in the world (De Martino and Galli, 2014), the

infection rates are very higher in areas such as hospitals or prisons (Goldman, 2011). Expansion

of TB in such areas depends on virulence, innate immunity and sensitivity (Berry, 2013). While

TB can occur in any area in any country, about 95% of the majority of deaths reported occurr in

poor countries where resources are finite (WHO, 2014). Patients who are HIV positive are very

sensitive to getting TB infection and 80% of HIV positive patients live in sub-Saharan Africa

and have TB (Sia, 2011). While diagnostic advancements have been made in the past four years,

80% of TB cases worldwide are concentrated in more than twenty-two countries which include

India, Pakistan, Nigeria, Bangladesh, China, Indonesia, South Africa and Russias (Goldman,

2011).

2.3 Classifications of Tuberculosis

2.3.1. Classification Based on Site of the Diseases and Bacteriological StatusTuberculosis

can be classified based on the site of the diseases and bacteriological status (Afriyie-Mensah

et al., 2023):

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 1. Pulmonary tuberculosis, sputum smear positive (PTB+): This refers to a patient with at

least two sputum specimen positive for acid-fast bacilli (AFB) by microscopy, or a

patient with only one sputum specimen positive for AFB by microscopy plus

radiographic chest abnormalities consistent with active pulmonary TB, or a patient with

only one sputum specimen positive for AFB by microscopy and a culture positive for

Mycobacterium tuberculosis.

2. Pulmonary tuberculosis, sputum smear negative (PTB-): This refers to a patient with

symptoms suggestive of TB, with at least two sputum specimens negative for AFB by

microscopy and with radiographic chest abnormalities consistent with active pulmonary

TB, or patient with at least two sputum specimens negative for AFB by microscopy and

culture positive for Mycobacterium tuberculosis, or a patient with two sets of at least two

sputum specimens taken at least two weeks apart negative for AFB by microscopy and

radiographic abnormalities consistent with pulmonary TB, and lack of clinical response

to one week of broad-spectrum antibiotic and decision by a clinician to treat with full

course of anti-tuberculosis chemotherapy.

3. Extra pulmonary tuberculosis: This refers to a patient with tuberculosis of organs other

than lungs; pleura, lymph nodes, abdomen, genito-urinary tract, skin, joints and bones,

and meninges (Pooja, et al. 2021). Diagnosis should be based on one culture positive

specimen, or histological evidence or strong clinical evidence consistent with active extra

pulmonary tuberculosis, followed by a decision by a clinician to treat with a full course of

anti-tuberculosis chemotherapy.

2.3.2. Classification Based on Type of Cases

World Health Organization (WHO) in 2014 describes TB into 7 types:

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 1. New case: A patient who has never taken anti tuberculosis treatment or has taken it for

less than one month.

2. Relapsed case: A patient declared cured of TB by a physician but who reports back to the

health service and is found to be bacteriologically positive.

3. Transferred-in case: A patient who has been received into a tuberculosis unit/district

hospital after starting treatment in another unit where he has been recorded.

4. Default case: A patient who has received anti-tuberculosis treatment for one month from

any source and who has interrupted treatment for more than two months.

5. Failure case: An initial smear positive patient who remains smear positive at five months

or more after starting treatment or an initial smear negative patient who becomes smear

positive during the course of treatment.

6. Chronic case: A patient who remains smear positive after completing a re-treatment

regimen.

7. Others: A patient who does not fit into any of the above categories, e.g. a relapse patient

may be smear negative or an extra-pulmonary TB patient who has not responded to

treatment.

2.3.3. Classification Based on Severity of Illness

World Health Organization (WHO) in 2014 also described the tuberculosis based on the severity

of illness:

1. Seriously ill / not seriously ill smear negative pulmonary TB: Smear negative pulmonary

TB cases should be clinically ascertained for the severity of illness.

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 2. Seriously ill / not seriously ill extra-pulmonary TB: Seriously ill extra-pulmonary TB

includes meningitis, disseminated TB, tuberculosis pericarditis, peritonitis, bilateral or

extensive pleurisy, spinal disease with neurological complications, intestinal and

genitourinary TB.

2.4 Transmission of Tuberculosis

Tuberculosis infection transmitted by inhalation of infectious particles released by the TB patient

through cough or sneezing (Goldman, 2011). A majority of persons who intake MTB bacteria

through air, can cause an effective response in the lungs which lead to inhibition in the growth of

MTB, result in the bacteria becoming dormant; this condition is known as latent tuberculosis or

LTBI; immuno-competent latent patient is infected with MTB but do not shows signs &

symptoms and do not transmit the disease to other person (Berry, 2013). There are certain

persons that are more sensitive to getting infected which include: adults (commonly in males),

those in developing countries, health care workers who are around this disease and those which

have a very weak immune system, in those who have AIDS. In fact, TB is the main cause of

death in those patients who are infected with HIV and HIV-TB combination has been widely

observed. Additionally, foreign-born persons and those who live in poor areas or where

malnutrition is common are more likely to get TB infection (Berry et al., 2013). TB also

transmitted through droplet aerosolization from an individual who has active TB. AFB positive

patient has the highest chance to easily get infected with TB. However, patients with negative

smears but positive cultures may still transmit the disease (Tostmann et al., 2008). There are

other conditions that may arise a high-risk for susceptibility to MTB infection such as diabetes,

long term use of corticosteroids, TNF-alpha blockers, polymorphism in vitamin D receptors,

polymorphism in IL-12 and IFN-g genes (Cruz-Knight and Blake-Gumbs, 2013).

8
Figure 2.1: Transmission of tuberculosis from an infected person

Figure 2.2: General Symptoms of Tuberculosis

9
Table 2.1: Factors that determine the probability of transmission of M. tuberculosis (ATS

et al., 2005)

Factors Description

Susceptibility Susceptibility (immune status) of the exposed individual.

Infectiousness Infectiousness of the person with TB disease directly related to the number

of tubercle bacilli that he or she expels into the air.

Environment Environment factors that affect the concentration of M. tuberculosis

organisms

Exposure Proximity, frequency and duration of exposure

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2.5 Diagnosis of Tuberculosis

Hence research on new diagnostic and screening tools and standards has become very necessary

in planning to control TB (Berry, 2013). LTBI is diagnosed with the help of Interferongamma

release assays (IGRAs) but the tuberculin skin test (TST) is always cost effective for poor

peoples (De Martino and Galli, 2014). The mechanism behind TST and IGRA is by analyzing

the response of immune T cells to the TB antigens.

2.5.1 Tuberculin Skin Test

In the TST test, tuberculin protein derivative from TB is injected intradermally into the patient

(John, 2001). This will cause a delayed hypersensitivity skin reaction (Type 4), if the patient has

mycobacteria infection (Thillai, 2014). To determine the infection of TB, the size of the skin

reaction is measured; the usual standard is between 2 to 3 days and value from 0.74 at 5 mm to

0.40 at 15 mm. However, the TST gives a false report that is positive responses in the patient

who are BCG vaccinated and negative in immunosuppressed persons

2.5.2 Interferon-Gamma Release Assays (IGRAs)

The IGRAs is a more sensitive and specific diagnostic test for TB (81-88% compared to 70%

sensitivity for the TST) (Diel, 2012), but IGRAs are costly and specific technique is used

(Goldman, 2011). In IGRAs the release of cytokine IFN-g from T cells that react to antigens not

available in the BCG vaccine (Diel, 2012). A blood sample is collected from an individual and

the release of cytokine IFN-g is measured. IGRAs have different Guidelines and constantly

changing. In Canada and in some European countries, it has even been suggested that IGRAs and

TST be used together to detect LTBI, but these tests are not definitive (Thillai, 2014). How the

disease develops in individuals from a latent to active TB is a heavy task and to improve

11
diagnostic tools we have to identify risk factors associated with high and low burden countries

and will improve our understanding of the immune response in TB (Gopinath, 2009).

2.5.3 Chest Radiography

Chest radiography is indicated for all persons being evaluated for latent tuberculosis infection

(LTBI) or active TB. Pulmonary TB as a result of endogenous reactivation of latent infection

classically presents with infiltrates in the apical and posterior segments of the right upper lobe,

the apical-posterior segment of the left upper lobe, and the superior segment of the lower lobe

(Aziza, 2015).

2.5.4 Smear Microscopy

Smear microscopy for the detection of acid-fast bacilli (AFB) is the most rapid and cheap

method for TB diagnosis (Mehraj, 2016). The aura mine fluorescence staining method that

requires fluorescence microscopy is more sensitive than AFB staining as the fluorescence is

easier to see. Smear microscopy has long been the gold standard of TB diagnosis and is posted

under the DOTS (direct observed therapy) for TB control. It is internationally advised by the

WHO (Pooja et al., 2021).

2.6 Overview of Mycobacterium tuberculosis

2.6.1 Cell Wall Structure of Mycobacterium tuberculosis

The cell wall fabrication of Mycobacterium tuberculosis is a key solidify of malevolence of the

bacterium. Cell wall of M. tuberculosis cell wall is composed of three major components;

Mycolic acids, cord factor, and wax. Mycolic acids is dictatorial alpha –divaricate lipids founds

in cell walls of Mycobacterium and Corynebacterium. They embellish 50% of the dry weight of

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the mycobacterial cell envelope. Acids is presumption to be a prominent determinant of

virulence in M. tuberculosis. Cord factor is most abundantly produced in virulent strains of

Tuberculosis. Chains of cells in smears constituted form in vitro-grown colonies (parallel

growth) frequently form Egregious of serpentine cords.

2.6.2 Optimum pH and Temperature

Tuberculosis normally grows at 37oC; growth stops below 25oC and above 40oC. The optimum

PH range is 6.4 - 7.0. M. tuberculosis is more susceptible to acid PH than M. magmatism is as

M. magmatism has more efficient internal PH homeostasis (Pooja, et al. 2021).

2.6.3 Effect of Physical and Chemical Agents

Mycobacterium survives for 20-30 hours in sputum. These bacilli are killed at 80oC in 15 - 20

minutes and instantly at 100oC. Cultures of M. tuberculosis may be killed by exposure at to

direct sunlight for two hours. These bacilli are sensitive to UV but relatively resistant to chemical

disinfectants, can survive exposure to 5% phenol, 15% sulphuric acid, 3% nitric acids, 5% oxalic

acid and 4% sodium hydroxide. They are sensitive to formaldehyde and glutaraldehyde. They are

destroyed by tincture of iodine in 5min, and 80% ethanol in 2-10 minutes.

2.6.4 Pathogenesis

TB infection the mycobacteria reach the pulmonary alveoli, where they invade and replicate

within endosomes of alveolar macrophage. Macrophages elucidate the bacterium as "foreign" to

gesture by deletion of phagocytosis. The growth of all bacteria is counterpane by the macrophage

and provided in a membrane-bound vesicle called phagosomes. The phagosomes are bounded

with a lysosome to build a phagolysosome.

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Figure 2.3: M. tuberculosis Mechanism of Action

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2.7 Treatment for Tuberculosis Patients

Treatment regimens are divided into the initial or intensive phase and the continuation phase

(Fasoranti, 2016).

1. Intensive /initial phase: during the intensive/initial phase, the bactericidal effect of

treatment leads to rapid bacteriological sputum conversion and improvement of clinical

symptoms, the pulmonary TB patient must take the medicines every day for 3-4 months.

2. Continuation phase: during the continuation phase, the patient takes the medicines 3

times a week for 5 months. The sterilizing effect of the therapy eliminates remaining

bacilli and prevents relapse.

There are five essential anti-TB drugs used for treating TB which include; Isonoazid (H),

Rifampicin (R), Pyrazinamide (Z), Streptomycin (S), and Ethambutol (E), and there are three

main properties of anti-TB drugs: bactericidal ability, sterilizing ability and the ability to prevent

resistance. Isoniazid and Rifampicin are the most powerful bactericidal drugs, active against all

types of TB bacilli. Pyrazinamide and Streptomycin are also bactericidal effective against only

certain types of TB bacilli. Pyrazinamide is active in an acid environment against TB bacilli

inside macrophages while Streptomycin is active against rapidly multiplying TB bacilli (WHO,

2014). Ethambutol is a bacteriostatic drug used in association with more powerful bactericidal

drugs to prevent the emergence of resistant bacilli (Fasoranti, 2016).

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Table 2.2: Side Effects of Tuberculosis Medications (Fasoranti, 2016)

Tuberculosis drugs Side Effects

Isoniazid (INH) tingling or pain in hands and feet (neuropathy),

nausea and vomiting, skin rash, fever, may

cause liver problems (hepatitis), psychosis

Ethambutol (E) skin rashes, eye problems such as blurred

vision nausea and vomiting, headaches,

dizziness joint pain

Rifampicin (R) nausea and vomiting, diarrhea, skin rash,

anemia, liver problems (jaundice), fever, flu

like symptoms, body fluids orange

Streptomycin (S) vestibular and auditory nerve damage, renal

damage, coetaneous hypersensitivity, pain,

skin rash, indurations at injection site

Pyrazimamide (Z) joint pain, arthralgia, hepatitis, coetaneous

reactions sideroblastic anemia, gastrointestinal

problem

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2.8 Mycolic Acid Cyclopropane Synthase

Mycolic acids are major components of the cell wall of Mycobacterium tuberculosis. Several

studies indicate that functional groups in the acyl chain of mycolic acids are important for

pathogenesis and persistence (Huang et al., 2002). There are at least three mycolic

acid cyclopropane synthases (PcaA, CmaA1, and CmaA2) that are responsible for these site-

specific modifications of mycolic acids. Mycobacteria have an unusual cell wall in which

mycolic acids play a critical role in its structure and function. Important characteristics conferred

by this structure are resistance to chemical injury, low permeability to antibiotics, resistance to

dehydration, and ability to thrive within the hostile environment of the macrophage

phagolysosome (Barry et al., 1998). Mycolic acids of mycobacteria are long chain α-alkyl, β-

hydroxy fatty acids containing 70–90 carbons in total (Daffe and Draper, 1997). The α-branch is

a saturated alkyl chain, typically 24 carbons long. The meromycolate chain contains 40–60

carbons with two positions of modification, the distal position (close to the ω-end) and the

proximal position (close to the β-hydroxy end). There are three classes of mycolic acids

synthesized by M. tuberculosis called α-, keto-, and methoxymycolates that are classified

according to their modifications at these two positions. The α-mycolates have a cis cyclopropane

ring at both positions, whereas keto- and methoxymycolates have oxygenated groups at the distal

position and a cyclopropane ring at the proximal position that can be in either

a cis or transconformation (Huang et al., 2002).

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Figure 2.4: Chemical structures of the major mycolic acids of M. tuberculosis

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The first protein to be identified from M. tuberculosis involved in carrying out these

modifications at either the distal or proximal position of mycolic acids was a cyclopropane

mycolic acid synthase (cmaA1) (Yuan et al., 1995). It was identified based on its homology to

the Escherichia coli enzyme cyclopropane fatty acid synthase. The enzyme from E. coli had

previously been shown to be a SAM-dependent methyltransferase, which catalyzed the addition

of a methyl group to a double bond of an acyl chain of the phospholipid bilayer, resulting in the

formation of a cyclopropane ring (Huang et al., 2002). When CmaA1 was expressed

in Mycobacterium smegmatis, it was shown that a cyclopropane ring was introduced at the distal

position of the mycolic acids (Yuan et al., 1995).

Since then at least seven other homologous genes have been identified in the genome sequence

of H37Rv. They are cmaA2 (cyclopropane mycolicacid synthase 2), mmaA1–

4(methoxymycolic acid synthase), and umaA1-2 (unknown mycolicacid methyltransferase).

These genes share between 50 and 75% identity with each other and are all thought to be SAM-

utilizing methyltransferases. cmaA2 encodes a protein that cyclopropanates at the proximal

position of mycolic acids when heterologously expressed in M. smegmatis (Cole et al., 1998).

Further studies in M. tuberculosis have confirmed that CmaA2 modifies the proximal position,

specifically catalyzing the formation of a trans cyclopropane ring of keto- and

methoxymycolates (Glickman et al., 2001). UmaA2 was shown to be required for the synthesis

of the proximal cyclopropane ring of the α-mycolates and was so renamed PcaA

(proximal cyclopropanation ofalpha-mycolates) (Glickman et al., 2000). It is thought that

MmaA2, which also synthesizes a proximal cis cyclopropane ring, acts on the methoxymycolates

(Dubnau et al., 1997). MmaA1 is believed to catalyze the conversion of a cis to trans double

19
bond at the proximal position (Yuan et al., 1997), whereas MmaA3 and MmaA4 are required for

the formation of oxygen-containing mycolates at the distal position (Dubnau et al., 2000).

Cyclopropanation of mycolic acids is a modification associated with

pathogenic mycobacteria and is not common in the cell wall of saprophytic species such as M.

smegmatis (Huang et al., 2002). In addition to the recent demonstration of the importance of a

cyclopropane synthase, PcaA, in virulence and persistence of tuberculosis, other studies show

that cyclopropane ring modifications have profound effects on the resistance of the mycobacteria

to the oxidative stress (Yuan et al., 1995) and the fluidity and permeability of the cell wall

(Dubnau et al., 2000). In E. coli and other bacteria that cyclopropanate their plasma membrane,

this modification happens during the transition from active growth to stationary phase (Grogan

and Cronan, 1997). Increased levels of cyclopropanation in E. coli have been shown to be a

response to environmental conditions such as low pH, high incubation temperatures, and low

aeration rates (Chang and Cronan, 1999). Among Helicobacter isolates, those identified as

gastric colonizers tend to make large amounts of cyclopropanated fatty acids, whereas those

identified as intestinal colonizers generally do not (Grogan and Cronan, 1997).

Mycobacteria make an extremely wide diversity of lipids and are unusual in that they possess

both a multifunctional fatty acid synthase (type I), usually found in eukaryotes and a

multicomponent multifunctional fatty acid synthase (type II), and usually found

in prokaryotes (Huang et al., 2002). Type I multifunctional fatty acid synthase catalyzes de

novosynthesis of short chain acyl-CoA esters up to 24–26 carbons long, then type II

multifunctional fatty acid synthase, by steps of fatty acid chain elongation, desaturation, and

cyclopropanation of the olefin, produces long chain fatty acids ranging from 24 to 56 carbons in

length. Finally, it is thought that Claisen-type condensation leads to the biosynthesis of mycolic

20
acids. It is believed that acyl carrier protein (AcpM) functions as the carrier of intermediates

between the dissociated multifunctional fatty acid synthase type II enzymes (Yuan et al., 1998).

The details of the order of the steps in biosynthesis are not clear; however, at least two scenarios

for the biosynthesis of the intact meromycolate chain exist. First, chain elongation produces 48–

56 carbon meromycolates, and then this chain is functionalized by desaturases and cyclopropane

synthases or, second, that desaturation and modifications of the acyl chain occur in a stepwise

manner during elongation (Barry et al., 1998). Intermediates shorter than full-length

meromycolates, which are unsaturated or cyclopropanated, have been identified from cell-

free extracts (Huang et al., 2002), which may favor the suggestion that modifications are coupled

to elongation, although it is unclear if this occurs in vivo. It does seem likely, however, that

desaturation and cyclopropanation do occur before condensation with the α-branch.

To derive information on the specificity and enzyme mechanism of the family of proteins, the

crystal structures of CmaA1, CmaA2, and PcaA were solved to 2-, 2-, and 2.65-Å resolution,

respectively. All three enzymes have a seven-stranded α/β fold similar to

other methyltransferases with the location and interactions with the cofactor S-adenosyl-l-

methionine conserved. The structures of the ternary complexes demonstrate the position of the

mycolic acid substrate binding site. Close examination of the active site reveals electron density

that we believe represents a bicarbonate ion. The structures support the hypothesis that

these enzymes catalyze methyl transfer via a carbocation mechanism in which the bicarbonate

ion acts as a general base. In addition, comparison of the enzyme structures reveals a possible

mechanism for substrate specificity. These structures provide a foundation for rational-drug

design, which may lead to the development of new inhibitors effective against persistent bacteria

(Huang et al., 2002).

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2.9 Bridelia ferruginea

Bridelia ferruginea Benth. commonly known as Bridelia belongs to the family Euphorbiaceae

(Pettit et al., 2016). It is synonymous to Bridelia micrantha var. ferruginea (Benth) Müll

(WAHP, 2013). Several species of Bridelia, about 60–70 are distributed from Africa to Asia

(Ngueyem et al., 2009). About 50 of these species are distributed in Madagascar, Tropical

Africa, Yemen and in Asia ranging from South China, India and Malaysia throughout Indochina

to North Australia, Vanuatu Islands and the Solomons (Ngueyem et al., 2009). In Africa, this

shrub commonly occurs in the Guinea savannah and coastal plains predominantly Burkina Faso,

Cote d'Ivoire, Togo, Nigeria and Ghana (Mshana, 2000). Examples of some species in this genus

include Bridelia atroviridis Muell. Arg., Bridelia crenulata Roxb., Bridelia cathartica Bertol. f.,

Bridelia glauca Bl. f. balansae Tucht., Bridelia balansae Tucht., Bridelia grandis (Pierre ex

Hutch), Bridelia moonii Thw., Bridelia monoica (L.) Merr., Bridelia ndellensis Beille., Bridelia

micrantha (Hochst) Baill., Bridelia ovata Decne., Bridelia scleroneuroides Pax., Bridelia

scleroneura Mull-Arg. and Bridelia stipularis Blume (Ngueyem et al., 2009).

22
Figure 2.5: Image of B. ferruginea

23
2.9.1 Plant Description

B. ferruginea is a shrub or small non-laticiferous scaly tree that develops up to 15 m tall

(Mshana, 2000). The plant develops up to 1.5m in its girth with crooked bole branching down.

The stem bark of B. ferruginea is dark grey, cracked, rough and slash thin. It is characterized by

branches that are long and thin and sometimes (usually when young) equipped with short spines.

Leaves have slightly wavy edges, in a size range of small to medium, simple, petiolate with

stipules, alternate, spiral or distichous in leaf arrangement. It has a broadly elliptic lamina, with

entire margin and an apex that is acute or acuminate. Each flower cluster typically consists of

male and female. The male flowers yellowish-green, pedicellate and the female flowers

subsessile. It bears fruits that are drupe-shaped, oblong, unilocular or sometimes subglobulose.

The fruits have a green pericarp, red then black-blue colour at maturity. The fruits, sometimes

are obovoid, 0.8 cm in length, more usually ellipsoid, 0.6 cm in length and especially persistent

on its branches (WAHP, 2013; Mshana, 2000).

2.9.2 Traditional and Ethnomedicinal Uses of B. ferruginea

The stem bark prepared decoction of Bridelia ferruginea is employed in oedema, epilepsy and

infant irritability treatments. It is also useful in the treatment of gastralgias, dysentery, anaemia

and rheumatisms (Lagnika et al., 2012). An extract from the bark is used as a mouth wash

(combined with lime juice to form traditional gargle “Ogun efu”), milk coagulant, vermifuge and

purgative (Cimanga et al., 1999). Traditionally, diabetes, arthritis and boils have been managed

using B. ferrugenia (Njamen et al., 2012). In western Nigeria, the stem and stem bark are useful

in managing various oral infections such as oral candidiasis while the bark is used in northern

Nigeria as a cure for infections caused by poisoned arrow wounds (Irobi et al., 1994). The roots,

24
bark and leaves are constituents of an infusion by the Yorubas (Nigeria) mostly given to children

(Burkill, 1994). Ethanolic stem bark extracts’ activity against Salmonella typhi (Dada and

Akinyele, 2020) may justify its traditional application in the management of enteric fever. The

leaves and bark decoctions are used as malaria therapy in some parts of Nigeria (Odugbemi et

al., 2007). In Southeastern Nigeria, some local healers prepare this remedy by soaking and

squeezing the material in water and a cupful given as a daily dose for 3–7 days. Hausa and

Fulani tribes (Northern Nigeria) also use the stem bark as a skin cancer medication (Abubakar et

al., 2007). It is also reported to have water purification properties (Kolawole and Olayemi,

2003). In Togo, the root bark is a remedy for intestinal and skin disorders (Bruyne et al., 1997).

Additional activities of the bark extract reported are trypanocidal (Ekanem et al., 2008),

antimicrobial (Owoseni et al., 2010) and anti-inflammatory (Olajide et al., 2003). In Congo, the

stem bark decoction is a remedy for toothache, cystitis, intestinal disorders, roundworm

infestation, diarrhoea and female sterility (Oliver-Bever, 1986). In Cote D'Ivoire, the stem back

decoction is for managing gonorrhoea, diarrhoea and dysentery or as a purgative (Gill, 1992).

The bark extract is combined with Costus for managing minor epilepsy (Akubue and Mittal,

1982). Skin conditions are managed using the stem and root barks. Tea made from the pulped

bark is used for treating fevers, stiffness, headaches and rheumatic pains and also as a local

application treatment for oedemas (Addae-Mensah, 1992). The leaves of B. ferruginea are for

managing dysentery in Cameroon (Talla et al., 2002), whereas the fruits, for mycotic stomatitis

(Ampofo, 1979). In Guinean traditional medicine, B. ferruginea is used to treat infectious

diseases such as sexually transmitted diseases (Magassouba et al., 2007). Pharmacological

studies on various extracts of B. ferruginea supports its use as an antidiabetic in different parts of

West Africa (Onyenibe and Udogadi, 2019). Extracts of the stem bark have shown antimicrobial

25
activity against some of the causative microorganisms of secondary upper respiratory tract and

enteric infections (Jose and Kayode, 2009). Other traditional uses of various parts of B.

ferruginea include epilepsy, rashes, cough, diuretic, asthma, analgesic, gout and impotence

(Olajide et al., 2000).

26
Figure 2.6: Some of the diseases managed traditionally with B. ferruginea

27
2.9.3 Phytochemistry of B. ferruginea

Different phytochemicals reported to be found in various parts and extracts of B. ferruginea

include phenolics, phytosterols, cardiac glycosides, triterpenes, tannins, flavonoids, saponins and

alkaloids (Abubakar et al., 2018). Quinones, catechic tannins, gallic acid, sterols, alkaloids,

polyterpenes, reducing sugars, polyphenols, flavonoids and saponosides are some

phytochemicals identified in the aqueous stem bark extract (Nene-Bi et al., 2009). In a bioassay

guided fractionation, Cimanga et al. (1999) and Bruyne et al. (1997) isolated from the 80%

acetone stem bark extract gallocatechin-(40 -O-7)-epigallocatechin, 3,5-dicaffeoylquinic acid,

1,3,4,5-tetracaffeoylquinic acid in addition to some derivatives of 3-methoxyflavone, which

include quercetin 3-methyl ether, quercetin 3,7,30 ,40 -tetramethyl ether, myricetin 30 ,40 ,50 -

trimethyl ether (ferrugin), myricetin 3,30 ,40 ,50 -tetramethyl ether, myricetin and finally

quercetin 3-O-glucoside. These constituents have been screened against both the classical and

alternative pathways of the compliment system, with the biflavanol and the two caffeoyl ester

quinic acids showing the strongest inhibitory activity on the classical pathway with reference to

rosmarinic acid. In addition, biflavanol, the two derivatives of quinic acids and three derivatives

of 3-methoxyflavones had a better inhibitory effect on the alternative pathway than the standard

rosmarinic acid (Cimanga et al., 1999).

Specific flavonoids and biflavonoids isolated and characterized from the methanolic plant extract

include apigenin, kaempferol and glycosides of both (Oliver-Bever, 1986). Most medicinal

plants containing apigenin are used in the management of gastrointestinal inflammation, bacterial

infections and muscle spasticity. Apigenin is also known to inhibit the secretion of histamine and

hence reduces allergic reactions. Kaempferol is known to regulate blood sugar levels especially

through the inhibition of the aldolase reductase activity and help prevent neuropathy and

28
retinopathy which are diabetic complications (DuPont et al., 2004). It controls lipid metabolism

and can significantly decrease the risk of atherosclerosis and related disorders (DuPont et al.,

2004). Due to its antioxidant activity, kaempferol is well noted for protecting cells from

oxidative stress, stabilizing connective tissues and strengthening blood vessels. It also regulates

the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) and inhibits monocyte chemotactic

protein-1 (MCP-1) (DuPont et al., 2004). Other compounds isolated from B. ferrugenia include

epigallocatechin, rutin, gallocatechin, quercetin, quercetin-3-neohesperidoside and myricetin-3-

glucoside, myricetin-3- rhamnoside (myricitrin) (Ngueyem et al., 2009). These compounds are

mostly present in the leaf extract. Gallocatechin-(4 -O-7)-epigallocatechin, epigallocatechin and

gallocatechin are prodelphinidin, a subclass of the pro-anthocyanidins or condensed tannins

which are part of the flavonoid family. The name prodelphinidin is due to their ability to liberate

delphinidin on hydrolysis by an acid. Pro-anthocyanidins have numerous biological activities

both in epidemiological and in vitro studies. The tendency of these flavonoids to complex

metallic ions and proteins hence acting as an antioxidant may explain their anti-inflammatory,

anti-diabetic, anti-bacterial and anti-cancer effects (Semwal et al., 2016). The presence of

gallocatechin-(4-O-7)-epigallocatechin and its isomeric pro-anthocyanidins in an extract or diet

can be beneficial for the prevention of some chronic diseases particularly those related to

cardiovascular health (Hertog et al., 1993). Cytotoxic lignan derivatives 50 -demethoxy-β-

peltatin-5-O-β-D-glucopyranoside and β-peltatin-5-O-β-D-glucopyranoside, which are related

structurally to podophyllotoxin have been isolated from 50% (v/v) dicloromethane methanolic

root extract of B. ferrugenia and are reported to show similar cytotoxic and cytostatic effects

against NCI's 60 human tumor cell panel (Rashid et al., 2000). Further analysis by Rashid et al.

(2000) indicates that both compounds bind to tubulin, which is quite consistent with many

29
podophyllotoxin-like lignans (MacRae and Towers, 1984). Teniposide and etoposide are tubulin-

interactive anti-mitotic drugs clinically used to manage tumors but are semi-synthetic derivatives

of podophyllotoxin (Rashid et al., 2000). This is an indication that, depending on the

concentrations of 50 -demethoxy-β-peltatin-5-O-β-D-glucopyranoside and β-peltatin-5-O-β-D-

glucopyranoside in the roots, standardized and partially purified or modified extracts of the roots

of B. ferrugenia could be used as a potential anti-tumour agent clinically. However, factors

bordering on the selectivity index, acute and chronic toxicities and further efficacy evaluations of

the extracts should be carefully considered. The methanolic dried leaf extract also yielded 14

compounds in a study by Afolayan et al. (2019). The authors reported for the first time the

isolation of a type of stearic acid composed of a fatty acid monoester of 2-O-β-D-

glucosylglycerol, 6β-hydroxy-(20R)-24-ethylcholest -4-en-3-one, 6β-hydroxy-(20R)-24-

ethylcholest-4,22-dien-3-one, lutein, vomifoliol, corilagin, kaempferide-3-O-β-D-glucoside,

isomyricetin and quercitrin from the methanolic leaf extract in addition to myricetin,

isoquercetin, myricitrin, rutin and β-sitosterol glucoside. It was noted that, Lutein acted strongly

on CB2 receptor and against leishmania, while myricitrin inhibited E. coli.

2.9.4 Pharmacological Activities

Pharmacological studies carried out on different extracts of Bridelia ferruginea have revealed

various pharmacological properties of the plant. These include anti-inflammatory, anti-diabetic,

antioxidant, antimicrobial, anti-infective, analgesic, antipyretic, repellent, insecticidal, fibroblast

growth stimulation, diurectic and natriuretic activities

30
 [Link] Anti-inflammatory Properties

There was significant inhibition of carrageenan-induced oedema in the rat paw of aqueous B.

ferruginea stem bark extract treated rats (Olajide et al., 1999). 3 hours post carrageenan

inhibitory values of oedema were 22, 22, 57 and 58% for doses of 10, 20, 40 and 80 mg/kg of

aqueous stem bark extract, respectively whiles indomethacin (5 mg/kg) gave a 72% inhibition. In

this test, extract ID50 was 36 mg/kg. For the mouse paw oedema test, a significant inhibition of

oedema was not observed in comparison to the control group. Again, it showed a low activity as

compared to that of the rat model although over a period of 5 hours, a reduction of paw size was

observed (Olajide et al., 1999). B. ferruginea extract showed a significant as well as

concentration related inhibition of the dry weight of cotton pellet granuloma with values of

inhibition of 20, 27, 34 and 43% for 10, 20, 40 and 80 mg/kg doses respectively. 80 mg/kg B.

ferruginea extract exhibited similar degree of granuloma tissue formation inhibition as

Hydrocortisone (Olajide et al., 1999). Evaluation of the anti-inflammatory property of aqueous

stem bark extract of B. ferruginea was carried out using tumor necrosis factor-alpha (TNFα)

mediated models. The group of mice pre-treated with 10–80 mg/ kg B. ferruginea extract

demonstrated an inhibition of septic shock syndrome in a dose-dependent manner, with extract

dose 80 mg/kg producing activity comparable to pentoxifylline (100 mg/kg). Animals pretreated

with B. ferruginea or pentoxifylline caused statistically significant (P < 0.05) reduction in serum

enzyme activity of alanine and aspartate aminotransferases. In the skin of mice, there was

suppression of LPS-induced dye leakage at doses 10–80 mg/kg of B. ferruginea extract (Olajide

et al., 2003). The mechanism(s) underlying B. ferruginea's anti-inflammatory activity was

further determined using the extract's effect on vascular permeability. Aqueous extract of B.

ferruginea reduced vesical oedema and vascular permeability increase by cyclophosphamide.

31
The intensity of peritoneal inflammation by acetic acid in mice was also reduced demonstrating

the ability of the extract to inhibit small blood vessels' permeability. The amount of dye leakage

at highest concentration of extract, 80 mg/kg was 29.5μg and that of indomethacin 5 mg/kg, 21.0

μg (Olajide et al., 2000).

[Link] Anti-diabetic Activity

Semi ethanolic extract of B. ferruginea exhibited a dose dependent α-glucosidase

inhibitory activity with a value of 1.4 0,04 μg/mL as IC50. A higher α-glucosidase

inhibitory activity was showed by the extract in comparison to acarbose the reference

drug (IC50 726 15 μg/ mL) (Bothon et al., 2012). The methanolic leaf extract of B.

ferruginea was tested by Onyenibe & Udogadi (2019) for its antidiabetic property

using male wistar rats (140–160g). The rats were grouped into four sets. Three of the

groups received I.P. Streptozocin (50 mg/kg) for induction of hyperglycemia and the

last served as the normal control. The level of blood sugar was measured employing

ACCU- CHEK Glucometer and glycated hemoglobin (HBA1c) was analyzed using

HPLC. The plant extract (50 mg/kg) significantly (P < 0.05) lowered the levels of

blood sugar similar to glibenclamide (6 mg/kg). It was also observed that, the HBA1c

(%) ranged from 4.6±0.2 (Normal Control) to 11.8±0.1 (Negative Control- Untreated

diabetics). Treatment with B. ferruginea leaf extract (7.25±1.6) and control drug,

glibenclamide (7±0.3) lowered significantly (p < 0.05) the levels of HBA1c (%) in

relation to the negative control (11.8±0.1). An observation made between treated and

normal control groups was not significantly (p > 0.05) different (Onyenibe and

Udogadi, 2019). In the oral glucose tolerance test (2 g/kg), sugar levels determined

between 15-120 minutes later, showed a rapid clearance of blood sugar levels in the

32
 B. ferruginea extract (50 mg/kg) administered group in relation to the negative

control (Onyenibe and Udogadi, 2019). A single administered dose of B. ferruginea

methanolic leaf extract significantly (p < 0.05) reduced Fasting blood sugar levels in

sucroseinduced, glucose-intolerant rats. Again, blood glucose was significantly

reduced by the extract (from 167±23 mg/dL to 126±5 mg/dL) in glucose intolerant

rats after a 6-day treatment. The proposed mechanism of action of B. ferruginea was

suggested as relating to insulin sensitivity improvement (Njamen et al., 2012).

Aqueous Bridelia ferruginea leaves extract effect on alloxan induced diabetic rats was

significantly (P < 0.05) reduced blood glucose level on day 7, of treatment at highest

dose (800 mg/kg). Bioactive compounds like flavonoid, terpenoids, glycosides and

alkaloids present may be contributing to its hyperglycaemic effects (Aja, 2013).

[Link] Antioxidant Activity

DPPH radical scavenging assay of the semi ethanolic B. ferruginea bark extract

showed an IC50 value of 5±0.3 μg/mL whiles the IC50 of Lascorbic acid, the positive

control was 1.25±0.07 μg/mL. B. ferruginea had 4.4±0.06 μmol Fe II/g DW for the

FRAP value. B. ferruginea had 5133±161 μmol Trolox/g DW for the ORAC value

(Bothon et al., 2012). Antioxidant properties of the ethanol leaves extract of B.

ferruginea were assessed using the lipid peroxidation and iron chelation assays. The

extracts (3.3 μg/mL-39.6 μg/mL) had different antioxidant potential, with 36.9 μg/mL

of the extract being most potent with inhibition at 54.16% for the brain and 82.46%

for the liver. The ability for the extract to chelate iron was assessed using extract

concentration ranging from 1- 10 μg/mL. At dose 2 μg/mL, the extract was most

potent and very effective against iron (Oloyede and Babalola, 2012). A beta-amyrin

33
 acetate fraction obtained from the successive extraction of the dried powdered leaves

was used for an antioxidant assay. A higher antioxidant potential with an IC50 value

of 158.2 μg/mL compared to gallic acid with an IC50 of 201.1 μg/mL and α-

Tocopherol which had IC50 of 4.577 μg/mL was obtained for B. ferruginea (Fabiyi et

al., 2012). The ethanolic, aqueous and ethyl acetate stem bark extracts of B.

ferruginea exhibited a strong antioxidant activity in the order of decreasing degree;

Aqueous > Ethanolic > Ethyl Acetate. A higher IC50 was exhibited by the aqueous

extract (0.85 mg/mL) compared to 1.34 and 1.41 mg/mL for the ethanolic and ethyl

acetate extract respectively hence conferring highest antioxidant activity to the

aqueous extract (Oloyede et al., 2014). The ethanolic leaf extract revealed the

maximum protection against FS 5 cells damage by H2O2 which was similar to

catalase (82% at 250 μg/ mL). The ethanolic extract of the leaves possessed

antioxidant activity with an IC50 of 12.5±0.3 μg/mL comparable to L-ascorbic acid

with 7.3±0.1 μg/mL using DPPH assay (Adetutu et al., 2011).

[Link] Antimicrobial and Anti-infective Activity

The methanolic extracts of B. ferruginea were screened for its antimicrobial

properties. The extracts had a varied range of activity on Escherichia coli, Proteus

mirabilis, Staphylococcus aureus, Salmonella typhi and Candida albicans. At

different concentrations of 40, 100, 60, 60, and 80 mg/mL, the root extracts caused

inhibition of growth of E. coli, S. aureus, S. typhi, P. mirabilis and C. albicans

respectively. An MIC of 60 mg/ mL on S. typhi as well as 10 mg/mL on C. albicans

was recorded for the stem bark. The methanolic extract of the leaves exhibited no

antimicrobial activity against the organisms (Adebayo and Ishola, 2009). The

34
 methanolic stem bark extract was investigated for efficacy against dental

microorganisms to validate folklore use of B. ferruginea in oral infections (Orabueze

et al., 2016). At concentrations ranging from 50 mg/mL to 400 mg/mL, B. ferruginea

extract showed a dose dependent antibacterial sensitivity against clinical oral isolates

of Streptococcus spp, Lactobacillus acidophilus, Staphylococcus aureus and

Pseudomonas aeruginosa with inhibitory zones ranging from 25.00 – 34.00; 19.50–

27.00; 16.00–22.50; and 15.25–22.25 mm respectively. The extracts however showed

no activity against the tested strains of fungi (Aspergillus fumigatus and Candida

albicans). Chlorhexidine gluconate (standard) at concentrations ranging from 0.625 to

5% showed antibacterial and antifungal effect with zones of inhibition ranging from

16.00 – 26.50; 12.00–18.50; 28.00–35.00; 33.00–38.00; 32.00–38.00 and 31.00–

39.00 for Candida albicans, Aspergillus fumigatus, Pseudomonas aeruginosa,

Staphylococcus aureus, Streptococcus spp and Lactobacillus acidophilus

respectively. The MIC of B. ferruginea against Streptococcus spp was calculated as

0.1 mg mL1 and 25.6 mg mL1 for S. aureus, L. acidophilus and P. aeruginosa

(Orabueze et al., 2016).

[Link] Analgesic Activity

Aqueous B. ferruginea stem bark extract (25, 50 and 100 mg/kg) reduced reasonably

the duration of writhing. 100 mg/kg of extract and aspirin gave a percentage writhing

inhibition of 88.35 and 82.55% respectively. With the tail immersion test, the highest

nociceptic activity occurred at dose 100 mg/kg comparable to morphine at 10 mg/kg

(Akuodor et al., 2011). Aqueous B. ferruginea stem bark extract produced notable

35
 effect for writhing test in mice induced by acetic acid which indicates analgesic

activity (Olajide et al., 2000).

[Link] Antipyretic Activity

In yeast-provoked rise of body temperature in rats, B. ferruginea had a significant

antipyretic effect. However, this was not comparable to drugamol (standard drug)

(Akuodor et al., 2011). 40 and 80 mg/kg of B. ferruginea extract demonstrated some

antipyretic activity. Lowering of rectal temperatures of yeast induced hyperthermic

mice was observed (Olajide et al., 2000).

[Link] Repellent and Insecticidal Activities

B. ferruginea powder has been demonstrated to possess non-dose dependent repellent

activity against D. porcellus. Powder concentration of 2 and 10%w/w belong to the

repellency class II while 5 and 7%w/w belong to repellency class III. The repellent

activity of Antouka (synthetic insecticide) on D. porcellus was greater compared to

the plant powders after the first- and twelfth-hour post treatment. Their recorded

RD50 values were 5.4% and 5.9% (w/w) respectively. The level of the repellent

activity of the B. ferruginea leaf extracts increased with time. Aqueous extract

(7.5%), acetone extract (5%), ethanol extract (2%, 5% and 7.5%), methanol extract

(5%) and propanol extract (5% and 7.5%) showed D. porcellus repellent activities

(Loko et al., 2017).

[Link] Cytotoxicity and Fibroblast Growth Stimulation

β-Amyrin acetate fraction of B. ferruginea leaf extract revealed cytotoxic potential.

The LC50 value for acute dose was 319 μg/mL and that for lethal dose was 5.86

μg/mL. Compared to the standard (cyclophosphamide LC50 value of 2506 μg/mL), β-

36
 Amyrin acetate fraction showed extreme toxicity indicating a potential source of

cytotoxic agents in cancer chemotherapy (Fabiyi et al., 2012).

Stimulating fibroblast cell growth can be used to test for wound healing activity.

Bridelia ferruginea ethanolic leaf extract on fibroblasts proliferation of human dermal

skin presented a significant response (p < 0.001) that was biphasic with increase in

growth at concentrations up to 5 μg/mL (28% increase). The growth of fibroblast

reduced to levels comparable to that of cells grown in minimal growth factors

between 15 and 60 μg/mL. An increase in toxic constituents may possibly account for

this occurrence. The possibility of cytotoxicity shown by the crude ethanolic extract

at concentrations higher than 5 μg/mL implies that the use of higher concentrations in

disease management should be done with caution (Adetutu et al., 2011).

[Link] Diuretic and Natriuretic Activity

B. ferruginea used traditionally for managing hypertension and as a diuretic was

evaluated for its diuretic and natriuretic activity. 24 hours after intraperitoneal

administration in the rat, the aqueous stem bark extract showed a significant water

overload elimination. The extract increased natriuresis and kaliuresis respectively by

55.71% and of 49.80. There was a significant increase in plasma sodium level

(natremy) (Nene-Bi et al., 2012).

37
 CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1 Protein Preparation

The crystal structure of mycolic acid cyclopropane synthase (PDB ID: 1KPI) was added to the

Maestro 12.5 database. According to (Iwaloye et al. 2020), protein preparation errors, such as

missing hydrogen atoms, could be fixed in X-ray crystallography using the Glide software

(Schrodinger Suite). Ions were removed, and the formation of a hydrogen bond took place. In the

refine tab, the H-bond network was optimized with PROPKA, and water molecules that had

fewer than three H-bonds to non-waters were removed (Olsson et al., 2011). The retrained

minimization was carried out using an OPLS3 force field, and the RMSD value was set to 0.3A

3.2 Preparation of the Phyto-compounds

Bridelia ferruginea compounds were drawn in Marvin's sketch (version 19.26) and then

uploaded to the Schrödinger suite's Maestro molecular Interface (version 12.5) workspace. The

LigPrep was used to prepare the compounds. The Ligprep panel enables the conversion of

structures, the generation of structural variations, and the deletion of structures with an OPLS3

force field at pH 7.0 +/- 2.0 utilizing Epik after stereoisomer computation was allowed to

produce a maximum of 32 per ligand, and the output format was allowed to remain as a maestro

(Schrödinger Suite).

3.3 Receptor grid generation

The receptor grid file, which represents the active sites of the receptor and is necessary for glide

ligand docking jobs, was generated with the help of a receptor grid generation panel. The ligand-

38
binding site was defined by selecting the co-crystallized ligand that was associated with the

protein structure on the workspace. Glide takes into account the location and dimensions of the

ligand to establish the default center and dimensions of the region in which grids will be

generated. The receptor grid box resolution was centered at x, y, and z-axis coordinates which

are -3.46, 2.19, and -27.51 respectively.

3.4 Glide extra precision docking

Docking the prepared compound library into the active site of the protein crystal with increased

accuracy was accomplished with the help of the flexible ligand sampling set. The model energy

score, also known as the e-model, was used to choose the structure that best docked with each

ligand.

3.5 ADMET Properties Prediction

The prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) is

required to assess the hit compounds' safety and drug-ability. Any compound's pharmacological

importance is determined by how closely it resembles a drug based on its physicochemical

characteristics (Kikiowo et al., 2020). One of the rules created to filter compounds is Lipinski's

rule of five (RO5), and all compounds were assessed for their respective drug-like properties.

The rule of 5 indicates that poor absorption is more likely to occur when there are more than

(i) 5 hydrogen-bond donors,

(ii) (ii) 10 (5 × 2) hydrogen-bond acceptors,

(iii) (iii) a molecular weight greater than 500 (5 × 100), and

(iv) (iv) a calculated n-octanol-water partition coefficient (cLogP) greater than 5.

39
 Therefore, for a compound to be termed as having good drug-like properties,

(i) hydrogen-bond donors must not be more than 5,

(ii) (ii) hydrogen-bond acceptors must not be more than 10,

(iii) (iii) molecular weight must not be greater than 500, and

(iv) (iv) a calculated Log P (cLogP) must not be greater than 5.

3.6 Ligand Interaction Prediction

Ligand interaction prediction details how individual compounds will bind to the protein at it

active site. From the ligand interaction images, information such as the type of charges (positive

or negative), number of hydrogen bonds, hydrophobic interactions and the amino acids involved

are obtained.

40
 CHAPTER FOUR

4.0 RESULTS

In this study, phytocompounds of Bridelia ferruginea were discovered through the analysis

results to have very good binding affinity with the protein as the docking score had values

ranging from -11.133 kcal/mol to -14.93 kcal/mol for the first 20 compounds (Table 4.1). The hit

potential phytocompounds discovered are Coatline, Myricetin, Quercetin 3-methyl ether,

Luteolin and kaempferol with the following docking scores -14.04 kcal/mol, -12.018 kcal/mol, -

11.553 kcal/mol, -11.53 kcal/mol, and, -11.063 kcal/mol respectively. The 2D structure of these

potential compounds and the well-known ligand are shown. Table 3.2 presents the drug-likeness

and ADMET characteristics of the potential phytocompounds and the well-known compound.

41
Table 4.1: The compounds of Bridelia ferruginea and its docking scores against the protein

of target (mycolic acid cyclopropane synthase)

Compounds of Bridelia ferruginea Docking Score

Isorhamnetin-3-O-rutinoside (Narcissin) -14.93

Kaempferol-3-O-rutinoside (Nicotiflorin) -14.618

quercetin-3-O-glucoside -14.192

Coatline A -14.04

Epigallocatechin-3-O-gallate (Teatannin II) -13.879

Rutin -13.84

Ellagic acid hexoside -13.832

Isoquercetin -13.603

Ellagic acid-O-pentoside -13.278

Isorhamnetin-3-O-rutinoside (Narcissin) -12.713

Eschweilenol C (Ellagic acid-4-O-rhamnoside) -12.595

quercetin-3-O-glucoside -12.504

Salcolin B -12.47

Vitexin (Apigenin-8-C-glucoside) -12.445

Kaempferol-3-O-rutinoside (Nicotiflorin) -12.356

Myricetin -12.018

Isovitexin (Apigenin-6-C-glucoside) -11.727

Quercetin 3-methyl ether -11.553

Luteolin -11.53

42
Myricitrin -11.297

Ducheside A (3-O-Methylellagic acid-40-O- -11.294

xyloside)

(+)-Epigallocatechin -11.235

Ellagic acid hexoside -11.133

Kaempferol -11.063

Eschweilenol C (Ellagic acid-4-O-rhamnoside) -11.012

3,5-Dicaffeoylquinic acid -10.889

Ellagic acid hexoside -10.865

N-trans-Feruloyltyramine -10.782

Isoquercetin -10.535

3-O-Methylellagic acid -10.472

Ellagic acid hexoside -10.448

Tricin -10.398

(-)-Epigallocatechin -10.369

Ellagic acid -10.303

myricetin-3-glucoside -10.238

Catechin -10.226

Myricetin 3,3',4',5'-tetramethyl ether -10.184

Ellagic acid-O-pentoside -10.105

Ellagic acid-O-pentoside -10.043

Apigenin -10.018

Quercitrin -9.971

43
(+)-Gallocatechin -9.919

Quercetin-3,7,3',4'-tetramethyl ether -9.758

Myricetin -9.649

myricetin-3-rhamnoside -9.479

Eschweilenol C (Ellagic acid-4-O-rhamnoside) -9.458

Vitexin (Apigenin-8-C-glucoside) -9.397

Rutin -9.378

Epicatechin-3-O-gallate -9.272

Ellagic acid-O-pentoside -9.215

Myricitrin -9.21

N-trans-Feruloyltyramine -8.716

Quercetin 3-methyl ether -8.698

Coatline A -8.622

Ducheside A (3-O-Methylellagic acid-40-O- -8.567

xyloside)

Ducheside A (3-O-Methylellagic acid-40-O- -8.363

xyloside)

Coatline A -8.152

Ellagic acid -7.955

Luteolin -7.875

Isovitexin (Apigenin-6-C-glucoside) -7.864

(+)-Epigallocatechin -7.781

Quercitrin -7.626

44
Apigenin -7.601

Tricin -7.518

Salcolin B -7.459

Kaempferol -7.379

Ellagic acid -7.3

3-O-Methylellagic acid -7.271

Eschweilenol C (Ellagic acid-4-O-rhamnoside) -7.239

(-)-Epigallocatechin -7.195

Dihydroactinidiolide -7.056

3-O-Methylellagic acid -6.889

Myricetin 3,3',4',5'-tetramethyl ether -6.872

(+)-Gallocatechin -6.703

Epicatechin-3-O-gallate -6.575

Vomifoliol -6.521

3-O-Methylellagic acid -6.398

Gallic acid (3,4,5-Trihydroxybenzoic acid) -6.342

myricetin-3-glucoside -6.25

Ferulic acid -5.748

Epigallocatechin-3-O-gallate (Teatannin II) -5.364

Pantothenic acid -4.952

Kynurenic acid -4.864

Epicatechin-3-O-gallate -4.083

Epigallocatechin-3-O-gallate (Teatannin II) -3.58

45
myricetin-3-rhamnoside -3.258

Gallic acid (3,4,5-Trihydroxybenzoic acid) -2.418

Chlorogenic acid (3-O-Caffeoylquinic acid) -1.45

5-O-Feruloylquinic acid -1.098

46
Table 4.2: Outcome of the ADMET Properties Prediction of the compounds of Bridelia

ferruginea with protein of target (mycolic acid cyclopropane synthase)

Compounds of Bridelia ferruginea MW DonorHB AccptHB QPlogPo/w Rule of 5

Isorhamnetin-3-O-rutinoside (Narcissin) 624.551 8 20.55 -1.642 3

Kaempferol-3-O-rutinoside (Nicotiflorin) 594.525 8 19.8 -1.89 3

quercetin-3-O-glucoside 464.382 7 13.75 -1.142 2

Coatline A.1 436.415 7 13.45 -0.773 1

Epigallocatechin-3-O-gallate (Teatannin 458.378 8 8.75 -0.039 2

II)

Rutin 610.524 9 20.55 -2.691 3

Ellagic acid hexoside 464.339 7 16.5 -2.979 2

Isoquercetin 464.382 7 13.75 -1.352 2

Ellagic acid-O-pentoside 434.312 6 14.8 -2.561 2

Isorhamnetin-3-O-rutinoside (Narcissin) 624.551 8 20.55 -1.785 3

Eschweilenol C (Ellagic acid-4-O- 448.339 6 14.8 -2.262 2

rhamnoside)

quercetin-3-O-glucoside 464.382 7 13.75 -1.405 2

Salcolin B 526.496 4 10.15 2.474 2

Vitexin (Apigenin-8-C-glucoside) 432.383 6 12.25 -0.881 1

Kaempferol-3-O-rutinoside (Nicotiflorin) 594.525 8 19.8 -1.934 3

Myricetin 318.239 5 6 -0.315 1

Isovitexin (Apigenin-6-C-glucoside) 432.383 6 12.25 -0.403 1

47
Quercetin 3-methyl ether 316.267 3 5.25 1.112 0

Luteolin 286.24 3 4.5 0.919 0

Myricitrin 464.382 7 12.8 -1.089 2

Ducheside A (3-O-Methylellagic acid-40- 448.339 5 14.8 -1.84 1

O-xyloside)

(+)-Epigallocatechin 306.271 6 6.2 -0.226 1

Ellagic acid hexoside 464.339 7 16.5 -3.014 2

Kaempferol 286.24 3 4.5 1.021 0

Eschweilenol C (Ellagic acid-4-O- 448.339 6 14.8 -2.303 2

rhamnoside)

3,5-Dicaffeoylquinic acid 516.457 7 11.45 0.976 3

Ellagic acid hexoside 464.339 7 16.5 -3.028 2

N-trans-Feruloyltyramine 313.352 3 4.75 2.985 0

Isoquercetin 464.382 7 13.75 -1.011 2

3-O-Methylellagic acid 316.223 3 8 -0.543 0

Ellagic acid hexoside 464.339 7 16.5 -2.949 2

Tricin 330.293 2 5.25 1.92 0

(-)-Epigallocatechin 306.271 6 6.2 -0.225 1

Ellagic acid 302.197 4 8 -1.288 0

myricetin-3-glucoside 480.381 8 14.5 -1.899 2

Catechin 290.272 5 5.45 0.471 0

Myricetin 3,3',4',5'-tetramethyl ether 374.346 1 6 3.186 0

Ellagic acid-O-pentoside 434.312 6 14.8 -2.555 2

48
Ellagic acid-O-pentoside 434.312 6 14.8 -2.55 2

Apigenin 270.241 2 3.75 1.605 0

Quercitrin 448.382 6 12.05 -0.816 2

(+)-Gallocatechin 306.271 6 6.2 -0.197 1

Quercetin-3,7,3',4'-tetramethyl ether 358.347 0 5.25 3.072 0

Myricetin 318.239 5 6 -0.321 1

myricetin-3-rhamnoside 464.382 7 12.8 -1.065 2

Eschweilenol C (Ellagic acid-4-O- 448.339 6 14.8 -2.264 2

rhamnoside)

Vitexin (Apigenin-8-C-glucoside) 432.383 6 12.25 -0.782 1

Rutin 610.524 9 20.55 -2.557 3

Epicatechin-3-O-gallate 442.378 7 8 0.269 1

Ellagic acid-O-pentoside 434.312 6 14.8 -2.572 2

Myricitrin 464.382 7 12.8 -1.252 2

N-trans-Feruloyltyramine 313.352 3 4.75 2.619 0

Quercetin 3-methyl ether 316.267 3 5.25 1.046 0

Coatline A 436.415 7 13.45 -0.958 1

Ducheside A (3-O-Methylellagic acid-40- 448.339 5 14.8 -1.826 1

O-xyloside)

Ducheside A (3-O-Methylellagic acid-40- 448.339 5 14.8 -1.853 1

O-xyloside)

Coatline A 436.415 7 13.45 -0.933 1

Ellagic acid 302.197 4 8 -1.298 0

49
Luteolin 286.24 3 4.5 0.94 0

Isovitexin (Apigenin-6-C-glucoside) 432.383 6 12.25 -0.432 1

(+)-Epigallocatechin 306.271 6 6.2 -0.237 1

Quercitrin 448.382 6 12.05 -0.673 2

Apigenin 270.241 2 3.75 1.595 0

Tricin 330.293 2 5.25 1.906 0

Salcolin B 526.496 4 10.15 2.559 2

Kaempferol 286.24 3 4.5 1.023 0

Ellagic acid 302.197 4 8 -1.296 0

3-O-Methylellagic acid 316.223 3 8 -0.544 0

Eschweilenol C (Ellagic acid-4-O- 448.339 6 14.8 -2.274 2

rhamnoside)

(-)-Epigallocatechin 306.271 6 6.2 -0.226 1

Dihydroactinidiolide 180.246 0 3 1.567 0

3-O-Methylellagic acid 316.223 3 8 -0.543 0

Myricetin 3,3',4',5'-tetramethyl ether 374.346 1 6 3.01 0

(+)-Gallocatechin 306.271 6 6.2 -0.205 1

Epicatechin-3-O-gallate 442.378 7 8 0.141 1

Vomifoliol 224.299 2 4.45 1.618 0

3-O-Methylellagic acid 316.223 3 8 -0.542 0

Gallic acid (3,4,5-Trihydroxybenzoic 170.121 4 4.25 -0.578 0

acid)

myricetin-3-glucoside 480.381 8 14.5 -1.781 2

50
Ferulic acid 194.187 2 3.5 1.373 0

Epigallocatechin-3-O-gallate (Teatannin 458.378 8 8.75 -0.213 2

II)

Pantothenic acid 219.237 3 6.9 -0.491 0

Kynurenic acid 189.17 2 4.5 0.743 0

Epicatechin-3-O-gallate 442.378 7 8 0.244 1

Epigallocatechin-3-O-gallate (Teatannin 458.378 8 8.75 -0.339 2

II)

myricetin-3-rhamnoside 464.382 7 12.8 -1.244 2

Gallic acid (3,4,5-Trihydroxybenzoic 170.121 4 4.25 -0.577 0

acid)

Chlorogenic acid (3-O-Caffeoylquinic 354.313 6 9.65 -0.467 1

acid)

5-O-Feruloylquinic acid 368.34 5 9.65 0.597 0

51
Figure 4.1: 2D and 3D Image of mycolic acid cyclopropane synthase-coatline interaction

complex. Coatline's molecular interaction with amino acid residues in the binding pocket of the

protein structure (Panel A), as well as its binding position and site with mycolic acid

cyclopropane synthase (Panel B).

52
Figure 4.2: 2D and 3D Image of mycolic acid cyclopropane synthase-myricetin interaction

complex. Myricetin's molecular interaction with amino acid residues in the binding pocket of the

protein structure (Panel A), as well as its binding position and site with mycolic acid

cyclopropane synthase (Panel B).

53
Figure 4.3: 2D and 3D Image of mycolic acid cyclopropane synthase-quercetin 3-methyl

ether interaction complex. Quercetin 3-methyl ether's molecular interaction with amino acid

residues in the binding pocket of the protein structure (Panel A), as well as its binding position

and site with mycolic acid cyclopropane synthase (Panel B).

54
Figure 4.4: 2D and 3D Image of mycolic acid cyclopropane synthase-luteolin interaction

complex. Luteolin's molecular interaction with amino acid residues in the binding pocket of the

protein structure (Panel A), as well as its binding position and site with mycolic acid

cyclopropane synthase (Panel B).

55
Figure 4.5: 2D and 3D Image of mycolic acid cyclopropane synthase-kaempferol

interaction complex. Kaempferol's molecular interaction with amino acid residues in the

binding pocket of the protein structure (Panel A), as well as its binding position and site with

mycolic acid cyclopropane synthase (Panel B).

56
 CHAPTER FIVE

5.0 DISCUSION AND CONCLUSION

5.1 DISCUSSION

Drug-like compounds are highly susceptible to drug-able targets, making them an intriguing

therapeutic possibility in the drug development process (Batool et al., 2019). However, a crucial

computational technique in the planning and launch of the drug discovery process is the

molecular docking analysis (Torres et al., 2019). Ligand (compound) performance against a

target biological molecule (protein) can be evaluated by molecular docking analysis, which also

determines the orientation of the ligand within the binding pocket (Meng et al., 2011). Molecular

docking was carried out utilizing Maestro Schrodinger Suites software, to analyze

phytocompounds of Bridelia ferruginea against mycolic acid cyclopropane synthase protein in

this current investigation. The ligand complex from the analysis outcome was subjected to

further filtering.

The formation of a ligand-protein complex depends significantly on non-covalent interactions

like hydrogen bonding and hydrophobic interaction (Ferreira de Freitas and Schapira, 2017). In

the interaction profiles (figure 4.1 – 4.5), it was possible to see how many hydrogen bonds

were established between the interconnected amino acid residues and the hydrophobic

interaction in the ligand-protein complexes. Amino acid residues present in the active site of

these protein-ligand complexes are the main contributors, as they help stabilize the interactions

(Kumar et al., 2020).

Rapid assessment of ADMET properties significantly reduces pharmacokinetics-related

failure during the clinical stage of drug research (Daina et al., 2017). One of the criteria that must

57
be met when a substance is considered a drug potential is Lipinski RO5 (rule of 5). So, utilizing

RO5 and other standards, the compounds' drug-likeness and pharmacokinetics were evaluated.

The findings revealed that some of the compounds with high docking score were in line with

RO5; therefore they can be seen as potential drug targets.

5.2 CONCLUSION

This study suggests that Bridelia ferruginea phytocompounds are potential inhibitors of mycolic

acid cyclopropane synthase in tuberculosis treatment and Coatline, Myricetin, Quercetin 3-

methyl ether, Luteolin and kaempferol could be potential drug candidates for tuberculosis.

58
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