ISOLATION AND CHARACTERIZATION OF COMPOUNDS FROM STEM

BARK OF HOLARRHENA FLORIBUDA

COLLEGE OF BASIC AND APPLIED SCIENCES

SCHOOL OF PHYSICAL AND MATHEMATICAL SCIENCES

BY

TWUM DANIEL

(10727430)

A PROJECT REPORT SUBMITTED TO THE UNIVERSITY OF GHANA,

CHEMISTRY DEPARTMENT IN PARTIAL FULFILMENT OF THE
REQUIREMENT
FOR THE AWARD OF BSc IN CHEMISTRY

DEPARTMENT OF CHEMISTRY

SEPTEMBER, 2022
 DECLARATION

I affirm that this project is my original work, based on research conducted under the
supervision of Dr J.J.E.K Harrison and that it has not been submitted for any other
award or degree at this university or elsewhere.

……………………………………………………….

Twum Daniel (10727430)

………………………………………………………

Dr. Jerry Joe Harrison

(Supervisor)

I
 DEDICATION
I devote this work to the glory of God and to the service of mankind

II
 ACKNOWLEDGEMENT

Thanks be to God for seeing me through this work successfully. Also, a big thank you
goes to my supervisor Dr. J. J. Harrison for the guidance and support given to me
throughout this work. I express my my profound gratitude to all those who supported
me in all kinds to ensure this came to successful end.

IVII
 ABSTRACT
The methanol extract of the stem bark of Holarrhena floribunda was found to
contain alkaloids, tannis, steroids, anthraquinone and coumarins through the
phytochemical analysis. Column chromatographic separation from the methanol
extract was carried out using gradient elution, beginning hexane, hexane/ethyl
acetate or chloroform mixtures and finally methanol. A total of 27 fractions were
obtained. Four fractions precipitated semi-solids which were further purified by
repeated washing and recrystallization. IR spectra of these compounds were
obtained, NMR and mass spectrometry data are yet to be obtained.

IV
V
 TABLE OF CONTENT

Declaration…………………………………………………………………...i
Dedication…………………………………………………………………...ii
Acknowledgement…………………………………………………………...iii
Table of content……………………………………………………………...iv
Abstract……………………………………………………………………...v
List of figures………………………………………………………………. viii
List of tables…………………………………………………………………. viii
List of abbreviation…………………………………………………………. viii

CHAPTER ONE INTRODUCTION

1.1 Background information………………………………………………….1

1.2 Description of Holarrhena Floribunda……………………………………1
1.3 Traditional uses…………………………………………………………...2
1.4 Classification of Holarrhena Floribunda………………………………….2
1.5 Common names…………………………………………………………...3
1.6 Problem statement…………………………………………………………3
1.7 Aim………………………………………………………………………...3
1.8 Objectives………………………………………………………………….3

VI
CHAPTER TWO LITERATURE REVIEW
2.1 Compounds isolated………………………………………………………3
2.2 Pharmacological activities…………………………………………………4
2.2.1 Antimalarial activity…………………………………………………….5
2.2.2 Antibacterial activity…………………………………………………….5
2.2.3 Antioxidant and Antimutagenic activity…………………………………5
2.2.4 Anticancer activity……………………………………………………….6

CHAPTER THREE METHODOLOGY

3.1.0 Materials………………………………………………………………….6
3.1.1 General experimentation process…………………………………………7
3.2 Procedure……………………………………………………………………7
3.2.1 Sample collection………………………………………………………….7
3.2.2 Extraction of plant material……………………………………………….8
3.3.0 Phytochemical screening………………………………………………….8
3.3.1 Preparation of reagents……………………………………………………9
3.3.1.1 Alkaloids reagents………………………………………………………9
Wagner’s reagent………………………………………………………...9
Dragendorff’s reagent……………………………………………………9
Mayer’s reagent………………………………………………………….9
Hager’s reagents…………………………………………………………10
Tannic acid reagent……………………………………………………….10
3.3.2 Tannins test…………………………………………………………….….10
3.3.3 Test for flavonoids………………………………………………………...10
3.3.4 Test for Anthraquinone……………………………………………………10
3.3.5 Test for steroids……………………………………………………………10
3.3.6 Test for coumarins…………………………………………………………11
3.4 Column chromatography…………………………………………………….11
CHAPTER FOUR
4.0 Results and discussion……………………………………………………….14

VII
4.1 Summary of experiment………………………………………………….…....14
4.2 Mass of sample used for analysis……………………………………………...14
4.3 Phytochemical screening of crude extract…………………………….….……14
4.4 Chemical composition of Holarrhena floribunda……………………………...15
4.4.1 Characterisation of DT23……………………………………………………15
4.4.2 Characterisation of DT25A………………………………………………….15
4.4.3 Characterisation of DT25……………………………………………………15
4.4.4 Characterisation of DT26……………………………………………………16
CHAPTER FIVE
5.0 Conclusion……………………………………………………………………17
5.1 Recommendation……………………………………………………………...17

REFERENCES…………………………………………………………………….18
APPENDICES……………………………………………………………………...21

VIII
 LIST OF FIGURES
Figure 1. Picture of a separated fraction…………………………………….14
Figure 2. Pictures of some phytochemical results…………………………...16

LIST OF TABLES
Table 1. Classification of Holarrhena floribunda……………………………2
Table 2. Mobile phase used to run TLC for the crude extract…………….…9
Table 3. Mass of sample used for analysis………………………………….14
Table 4. Results for phytochemical screening of crude extract……………...15

LIST OF SCHEMES
Scheme 1. Summary of methodology……………………………………….13

LIST OF ABBREVIATIONS
TLC Thin Layer Chromatography
RF Retention Factor
NMR Nuclear Magnetic Resonance
IR Infra-red spectroscopy
HE Hexane
EA Ethylacetate
MS Mass Spectroscopy

I
 CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND INFORMATION

One of the most important use of plants, is the role it plays in medicine[1]. Plant
medicine is a suitable alternative, synthetic medicines fail to achieve the desired
results[2]. Plants were a major source of medications in the ancient times[3]. Plant
medicine are generally affordable and has low or no side effect on the body since its
components are all natural[4]. Medicinal plants are used most in non-industrialized
societies on the grounds that they are cheaper than modern medicines[5]. Research
has made it clear that plants are major source of bioactive natural products and has
been a primary source of drug discovery[2]. Despite the importance of plant in
medical delivery,only a small percentage of plants have been investigated to know
their components which are responsible for their therapeutic effect. One of such plant
is Holarrhena floribunda which is also known as false rubber tree[2][6].
1.2 DESCRIPTION OF H. floribunda
Holarrhena floribunda could be as long as 17- meter- tall tree with up to a 1-meter-
wide trunk and found mostly in deciduous forests and savannah woodlands[7]. They
have follicles which are up to 60 cm long and slender fruits that are pale grey to dark
brown with seeds having apical tufts of hair[2]. The leaves are shiny mostly,ovate-
acuminate 5-18cm long and2- 8cm broad with 6-12 pairs of lateral nerves[8]. Flowers
are white, scented and in almost umbel-like inflorescences; corollatube 5-9mm long
and lobes3.5-8mm and overlapping to the right[8]. Anthers are fertile to the base. It is
mostly found in Senegal, Nigeria and other regions of West Africa[2]. Holarrhena
floribunda is a member of the Apocynacea family[6].
1.3 TRADITIONAL USES
Holarrhena floribunda contains a lot of chemical components and has been used in
the treatment of malaria, amoebic illness, diarrhea, fever, infertility and diabetes using
`plants parts such as roots, stem bark and leaves are used[4]. Traditional healers make
use of different parts of the plant in the treatment of illnesses[4]. The plant is used to
cure boils, eczema and diabetes in northern Nigeria using the dried leaves. Headaches
can be relieved by applying crushed leaves to the forehead[8]. The leaves are used to

1
cure gonorrhea in Sierra Leone when combined with kola nuts and consumed. The
latex of the plant is used to treat snakebites and stings[9]. The roots of Holarrhena
floribunda are used in the treatment of constipation, colic discomfort and sterility. It is
also used to treat diarrhea and dysentery[8]. The root is boiled in milk and used to
bath boys attaining puberty[10]. The stem bark of H. floribunda can be used to treat
malaria and serve as a substitute for quinine[7]..
1.4 CLASSIFICATION OF H. floribunda
KINGDOM Plantae
SUBPHYLUM Euphylum
INFRAPHYLUM Radiatopes
SUBCLASS Magnoliidae
SUPERORDER Asternae
FAMILY Gentiales
TRIBE apocynaceae
GENUS Malouetieae
SPECIES Floribunda
Table 1 Classification of Holarrhena floribunda
1.5 COMMON NAMES
H. floribunda has common names such as; kurchibark, conessi bark, false rubber
tree[11].
Locally, the Ashanti’s call it slae[6].
1.6 PROBLEM STATEMENT
Most local plant medicines do not have strong scientific evidence to back their
authenticity. Therefore, there is the need to use modern scientific analysis to
investigate various compounds in the plants which enables them to give such good
health benefits and understand their mechanism of action. H. floribunda thus contains
active secondary metabolites that are responsible for the various pharmacological
activities attributed to it.

1.7 AIM
The purpose of this project is to isolate, purify and characterize compounds found in
the stem bark of Holarrhena floribunda.
1.8 OBJECTIVES

2
The objectives of this captured under this project to;
 extract compounds in the stem bark of H. floribunda using cold percolation.
 perform photochemical screening on the crude product of H. floribunda.
 determine the constituent of the crude extracts using column chromatography.
 characterize and elucidate the structures of purified compounds by using
spectroscopic and spectrometric methods.

CHAPTER TWO
2.0 LITERATURE REVIEW
Holarrhena floribunda is a member of the Apocynaceae family. Alkaloids, tannins
conessine, flavonoids and phenolic chemicals are the major classes of chemical
compounds found in the plants. It is known to have many alkaloids of various
structural classes. The plant has rich alkaloid composition which is highly found in
the root, stem and leaves in a decreasing order[2]. Phytochemical analysis done on the
crude extracts of the stem bark revealed the classes of compounds present, this
included alkaloids, saponnins, tannis ,polyphenols, cardiac glucosides triterpenes,
leucoanthocyans and sugars[12]. However, there was the absence of phlobatannins,
flavonoids, lipids, anthraquinones, sterols and glucosides.
Pharmacological studies have shown that it had activities such as antimalarial,
antibacterial, antimicrobial and anticancer effects.
2.1 Compounds isolated from H. floribunda
Holarrhena floribunda has many active phytochemical components which gives its
numerous therapeutic applications and pharmacological activity[13]. Following
chromatographic isolation and purification of compounds, it was revealed that there
were six (1-6) and eleven (7-17) major steroidal alkaloids from leaves and stem bark
respectively and also, one nitrogen-free steroid (18) from stem bark and a non-steroid
alkaloid-like (19) compounds isolated from leaves[7]. The compounds were identified
as follows 3β-holaphyllamine (1), holaphyllamine acetamide (2), N-
methylholaphyllamine (3), 3α-holaphyllamine (4), 3β-dihydroholaphyllamine (5), 3α-
dihydroholaphyllamine (6), holadienine (7), holonamine (8), cona-4,6-dienin-3-one
(9), cona-3,5-dienin-7-one (10), conessimine (11), isoconessimine (12), conessine
(13), holarrhesine (14), holarrhetine (15), holarrheline (16), holarrhenine (17),

3
kurchinin (18) and isopentenyl adenine[7]. (19).

Figure 1. Compounds isolated from leaves (1-6,19) and stem bark (7-17) of
Holarrhena floribunda[7].

2.2 Pharmacological activity of H. floribunda

The numerous therapeutic and pharmacological activities is attributed to the
photochemical components of the plants. Investigation of the pharmacological

4
activities of this plant shows that it has high antimalarial, antibiotic, antioxidant and
antioxidant, and anticancer potential[14].

2.2.1. Antimalarial activity

Ethanolic, aqueous and chloroform extracts of the stem bark of H. floribunda non-
alkaloid compounds showed activity against Plasmodium falciparum[15]. This
indicates that it has a good antimalarial potential. It serves as a substitute for
quinine[13].

2.2.2 Antibacterial activity

Compounds extracted from H. floribunda showed significant activity against many
microorganisms. It showed a good antibacterial against Bacillus stearotherphilus and
all other strains tested against. This potential is attributed to the conessine fraction
of the plant[16].

2.2.3 Antioxidant, antimutagenic activity

Ethylacetate extracts of leaves of H. floribunda shows very strong activity against
cyclophosphamide induced chromosomal aberrations[17]. Both ethylacetate and
methanolic extracts of H. floribunda shows anti-mutagenic activity[9].

5
2.2.4. Anticancer activity
Methanolic extracts of H. floribunda was tested against breast (MCF-7), colorectal
(HT-29) and cervical (HeLa) cancer cell relative to the normal KMST-6 fibroblasts[18].
Results showed that induction of apoptosis, ROS and cell arrest were dose and time
dependent. Also isolated compounds from the leaves showed cytotoxicity against
cancer cell[16].

CHAPTER THREE

3.0 METHODOLOGY

3.1.0 MATERIALS
Pulverised stem bark of Holarrhena floribunda, methanol, silica gel, cotton,
deionised water, chloroform, ethyl acetate, hexane, heptane, iodine, trimethyl
amine, hydrochloric acid, nitric acid, sulphuric acid, ethanol.

6
 3.1.1 GENERAL EXPERIMENTATION PROCESS
The extraction was done by dissolving finely grounded stem bark of Holarrhena
floribunda in a bucket using methanol and distilled water as solvents in the ratio 2:1
v/v. It was left for three days and then the extraction was done. This extraction
method is called cold percolation. The extract was filtered and concentrated to
dryness using a Cole-Palmer rotary vacuum evaporator. Solvents recovered was used
for the next extraction process until a sufficient mass of crude product was obtained.
Phytochemical test was conducted on the crude to know the class of compounds
present this species. Solid-liquid chromatography with silica gel as the stationary
phase was used for the isolation of compounds in the crude.
Gradient elution was performed, beginning with hexane (40 - 60°C) and increasing
the polarity by adding ethyl acetate until the mobile phase system eluted the most
polar elements in the extract. The solvents were continued with a mixture of
chloroform, ethyl acetate, and trimethyl amine in various proportion and then later the
ethyl acetate was substituted for methanol in the later solvents. The progress of the
chromatographic separation was monitored and also the purity of elute was
determined using thin layer chromatography (TLC) on aluminium foil slides pre-135
covered with silica gel (0.2mm thick, Kiesegel 60 F254 Merck type). UV light and
iodine vapor was used to visualize spot on the TLC plate. Recrystallization with a
suitable solvent used to purify compounds. Identification of pure compound isolated
was done by determining their melting point using Stuart Scientific melting point
apparatus. Further identification was done by using spectroscopic techniques. Neat
infrared spectral data was recorded on a Perkin Elmer FTIR spectrometer.

3.2 PROCEDURE

3.2.1 SAMPLE COLLECTION

7
 Stem bark of Holarrhena floribunda was collected at Ajumako in the central
region of Ghana by my supervisor.The sample was air-dried for about a month and
pulverised with a miller.

3.2.2 EXTRACTION OF PLANT MATERIAL

About 3.5Kg of finely grounded stem bark of Holarrhena floribunda was soaked
for three days in methanol and distilled water in the ratio of 2:1 v/v thus 2.5L of 96%
methanol and 5L of distilled water for extraction. The extract was filtered and
concentrated to dryness using a Cole-Palmer rotary vacuum evaporator. This yielded a
mass of 90g of crude extract, the crude was greenish dark in colour. Thin layer
chromatography was done on the crude using varied solvent ratio of mobile phase to
know which solvent system will be best for the separation of the individual
components of the extract and also know the general components of the crude.

8
Table 2. Mobile phase used to run TLC for the crude extract
Solvent system Ratio
HE 100%
HE/EA 8:2
HE/EA 6:4

3.3.0 PHYTOCHEMICAL SCREENING

The crude extract was analysed to ascertain the various classes of compounds in it.
Several tests were done on the methanolic solution of the crude extract to check for
the presence of Alkaloids, Tannins, Steroids, Flavonoids, Anthraquinone and
Coumarins. The procedure below was followed to prepare reagents required for the
test.

3.3.1. PREPARATION OF REAGENTS

3.3.1.1 ALKALOIDS REAGENT

A. Wagner’s Reagent (Harborne, 1998)

In a 100 cm3 volumetric flask, 0.64 g I2and 1.0 g KI was dissolved. The solution was
then filled to the mark with distilled water. When drops of reagent were added to the
acidified extract solution, alkaloids formed a huge granular and flocculent precipitate.

9
B. Dragendorff's Reagent (Harborne, 1998)

In a beaker, 10 cm3 of strong hydrochloric acid was mixed with 0.4 g of hydrated
bismuth nitrate. The resulting solution was mixed in a beaker with an aqueous
solution of 13.6 g potassium iodide in 50 cm3 of distilled water. The precipitated
granular potassium nitrate was filtered. The precipitate is a reddish-brown colour
which forms when two or three drops of the reagent are added to the test solution,
confirming the presence of alkaloid in the extract. This reagent can also be used as a
spray reagent on a TLC plate. It gives off an orange colour.

C. Mayer’s reagent (Harbone, 1998)

In a flask, 1.36 g of HgCl2 was dissolved using 50 cm3 of water. Aqueous potassium
iodide solution was made by dissolving 5.0 g of the solute in 10 cm3 of distilled water
in a beaker. The resultant solution was filled to the 100 cm3 mark with distilled water.
A pale-yellow solution was formed after a few drops of the reagent was added to it
show the presence of alkaloid.

D. Hager's Reagent (Harborne, 1998)

In a reagent bottle, 1.0 g of picric acid was mixed with 100 cm3 of distilled water. A
yellow precipitate develops when two or three drops of this reagent are added to the
test solution, indicating the presence of alkaloids.

E. Tannic acid reagent (Harborne, 1998)

In 10 cm3 of ethanol, tannic acid was dissolved and diluted with 90 cm 3 distilled water
to reach the 100 cm3 mark of the volumetric flask (100 cm 3). A positive test for
alkaloids is the appearance of a yellow precipitate. Tannic acid was dissolved in 10

10
cm3 ethanol and diluted with 90 cm3 distilled water to reach the 100 cm 3 mark of the
volumetric flask (100 cm3).

3.3.2. Tannins Test

Approximately 0.09 g of the crude was dissolved in ethanol separately. This was

placed in two test tubes labeled A and B.  Each solution received a freshly prepared
FeCl3. The formation of a dark greenish color revealed the presence of tannins in
extracts.

3.3.3. Test for Flavonoids

In a test tube, 0.006g of crude was placed with a few drops of dilute NaOH solution.
A bright yellow color formed in the test tube. Due to the presence of flavonoids, it
turned colorless when a few drops of dilute acid were added.

3.3.4. Test for Anthraquinone

Approximately 0.005g of crude was dissolved in methanol, and a few drops of 10%
NH3 were added. The presence of Anthraquinone was detected by the formation of a
red or purple color.

3.3.5. Test for Steroids

The crude extracts (0.009g) were placed in a test tube and dissolved in chloroform (6
mL) before being mixed with an equivalent volume of concentrated sulphuric acid.
The chloroform layer was red, whereas the sulphuric acid layer was yellow with green
fluorescence. This proved the presence of a steroid.

3.3.6. Test for Coumarins

10% of NaOH was added to the 0.05g of the methanolic crude extract of Holarrhena
floribunda. The presence of coumarins is indicated by the production of a yellow
color.

11
 3.4 COLUMN CHROMATOGRAPHY

A glass column was washed, dried and placed on a retort stand in a vertical position.
Cotton wool was placed at the bottom of the column and the column was rinsed
with hexane. An activated silica gel was dissolved in hexane and transferred into the
column. The column was opened to allow the hexane to drain to allow the silica
settle uniformly in the column, the column was filled to about two-third with the
activated silica gel. The column was loaded with a mixture of activated silica gel and
70.2 g of the crude extract by dry packing. The column was first eluted with 100%
hexane and successively increased the polarity with ethyl acetate. It was further
eluted with a mixture of chloroform, ethyl acetate and trimethyl amine in various
proportions. Furthermore, the ethyl acetate in the later mixture was substituted for
methanol. Fractions were collected with 10 mL test tubes and based on the TLC
analysis done, the various fractions collected were combined, elute with similar
retention factors (RF) and similar spot under UV light and iodine vapour were
combined and concentrated. A total of 27 fractions were obtained.

12
 Scheme: Summary of the Methodology

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

13
4.1SUMMARY OF EXPERIMENT

Stem bark of Holarrhena floribunda was analysed to know compounds that are
present in it. The pulverised stem bark was extracted with methanol and distilled
water in the ratio in the ratio 2:1 respectively. The crude extracted was placed in a
column to isolate compounds in it. Fractions collected analysed with TLC and elute
with similar RF and similar spots under UV light and in iodine vapour were combined
and concentrated. A total of 27 fractions were obtained labelled DT1-DT27. Out of
that fractions DT23, DT25, DT25A and DT26 precipitated as semi-solids.

Figure 1: Picture of a separated fraction

4.2 Mass of sample used for analysis
Table 3
Material Mass (Kg) ± 0.001 Percentage yield (%)
Stem bark (pulverised) 3.500
Crude extract 2.100 60
Crude used for analysis 1.470 70

14
4.3 PHYTOCHEMICAL SCREENING OF CRUDE EXTRACT
Table 4. Results for Phytochemical Screening of crude extract
SECONDARY TEST INFERENCE
METABOLITES
ALKALOIDS 1. Mayer’s Reagent +
2. Hager’ Reagent +
3. Wagner’s Reagent +
4. Dragendorff’s Reagent +

TANNINS +
STEROIDS +
FLAVONOIDS -
ANTHRAQUINONE +
COUMARINS +
+ Shows metabolite presence - Shows metabolites absence

Phytochemical analysis of the crude extract showed the presence of alkaloids,

tannins, steroids, anthraquinone, coumarins and the absence of flavonoids.

15
Figure 2: Picture of some of the results shown in the phytochemical analysis

4.4 Chemical composition of Holarrhena floribunda

4.4.1 Characterisation of DT23
The IR spectrum (Appendix I) showed absorption band at 1734.77cm-1 which may
suggests presence of a carbonyl (C=O) stretch. Peaks at 2776.50 2848.76cm -1 may be
characteristic of aldehyde (C-H) stretch. Medium peak at 1662.14cm -1 may suggest
the presence of imine (C=N) stretch. Weak broad peak at centred on 3385.81cm -1
suggest the presence of amine (N-H) stretch, the single spike may show that it is a
secondary amine. Peak at 2916.72 and 2848.76cm -1 may show the presence of a
methyl (C-H) asymmetric and symmetric stretch respectively. Peaks at 1462.73 and

16
1377.49cm-1 shows the bending and rocking vibrations of methyl (C-H) respectively.
However, NMR and MS data are required for full characterisation of compound.
4.4.2 Characterisation of DT25A
From the IR spectrum (Appendix II) strong, broad absorption centred on 3392.13cm -1
suggest the presence of alcohol (O-H) stretch. Strong peaks at 2925.26 and
2853.13cm-1 may show presence of methyl (C-H) asymmetric and symmetric stretch
respectively. Peaks at 1451 and 1379.84cm-1 may show the presence of bending and
rocking vibration of methyl (C-H) stretch respectively. NMR and MS data are required
for full characterisation of compound.

4.4.3 Characterization of DT25

The IR spectrum of (Appendix III), shows peak at 1738.37cm-1 which suggests the
presence of a carbonyl (C=O) stretch. Peaks at 2760.67 and 2848.70cm -1 suggest
aldehyde (C-H) stretch. Strong peak at 2937.46cm-1 suggest asymmetric methyl (C-H)
stretch. Bending and rocking vibrations of methyl (C-H) may be shown by peaks at
1433.64 and 1376.54cm-1 respectively. NMR and MS data are required for full
characterisation of compound.

4.4.4 Characterisation of DT26

The IR spectrum of (Appendix IV) shows medium peak at 1736.49cm-1 which may
show the presence of carbonyl (C=O) stretch. Peaks at 2774.32 and 2850.24cm -1
suggests the presence of aldehyde (C-H) stretch. Broad weak band centred 3400cm -1
may show the presence of amine (N-H) stretch. One spike may suggest a secondary
amine. Peak at 1664.41cm-1 may suggest the presence of imine (C=N) stretch. Peak
at 2932.26cm-1 may show asymmetric (C-H) stretch. Peaks at 1454.76cm -1 and
1377.69cm-1 may suggest bending and rocking vibration of methyl (C-H) respectively.
NMR and MS data are required for full characterisation of compound.

17
 CHAPTER FIVE
5.0 CONCLUSION
Stem bark of Holarrhena floribunda was found to contain alkaloids, tannis, steroids,
anthraquinone and coumarins through the phytochemical analysis. A total of 27
fractions were obtained. Four fractions precipitated as solids and their IR spectrum
was not able to characterize the compounds completely.

5.1 RECOMMENDATIONS
Further work should be done on the isolated compounds to fully characterise and
elucidate their structures. The medicinal properties of the various compounds
should be investigated.

18
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19
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APPENDICES

Appendix I (IR spectrum of DT23)

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Appendix II (IR spectrum of DT25A)

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Appendix III (IR spectrum of DT25)

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Appendix IV (IR spectrum of DT26)

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