PHYTOCHEMICAL STUDIES ON THE MEDICINAL PLANT Leonotis ocymifolia.

MOSES OTIENO ODEDE

ANC/012/18

ANCC 413: RESEARCH PROJECT

A RESEARCH PROJECT SUBMITTED IN PARTIAL FULFILMENT OF AWARD OF

BACHELOR OF SCIENCE DEGREE IN ANALYTICAL CHEMISTRY WITH

COMPUTING, UNIVERSITY OF ELDORET, SCHOOL OF SCIENCE,

DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY

MAY 2022
 DECLARATION

I Moses Odede registration number ANC/ 012 /18 do hereby declare that this research

project is my original work.

Sign…………………… Date……………………………

SUPERVISOR`S APPROVAL

This research project has been submitted for examination with approval as the project

supervisor.

MR. JOHN M NDEGWA

SIGN………………………… DATE…………………………..

ii
 DEDICATION

This research project is dedicated to my mother Margaret Odede and guardian

Lawrence Oboo for their financial support during my work. I also dedicate this work to

my classmates (Calvin Osida, Lawrence Ayodo, Elkanah Juma and Dennis Kipkirui) for

the encouragement. I hope this work will be of great importance to them.

iii
 ACKNOWLEDGEMENT

I wish to acknowledge the following people who provide guidance, contribution,

encouragement and support both physically and spiritually which enabled my research

to produce a meaningful outcome. Special thanks to my supervisor Mr. John Ndegwa

for his guidance. Thanks to my laboratory technicians Mr. Odero, Mr. Dennis and Mr.

Sang for the instrumentation, their time and patience to remain cooperative throughout

the research work.

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 TABLE OF CONTENTS

DECLARATION..........................................................................................................................ii

DEDICATION............................................................................................................................iii

ACKNOWLEDGEMENT..........................................................................................................iv

LIST OF FIGURES.....................................................................................................................ix

LIST OF TABLES.........................................................................................................................x

ABSTRACT..................................................................................................................................xi

CHAPTER ONE...........................................................................................................................1

INTRODUCTION.......................................................................................................................1

1.1 General background...............................................................................................................1

1.2 Statement of the problem.......................................................................................................1

1.3 Objectives of the study...........................................................................................................2

1.3.1 General objective..................................................................................................................2

1.3.2 Specific objective..................................................................................................................2

1.4 Hypothesis...............................................................................................................................2

1.3 The significance of the study.................................................................................................2

1.5 Scope of the study...................................................................................................................2

1.6 Limitation of the study...........................................................................................................3

CHAPTER TWO...........................................................................................................................4

LITERATURE REVIEW..............................................................................................................4

2.1 Introduction.............................................................................................................................4

2.2 Description of Leonotis ocymifolia..........................................................................................4

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2.3 Habitat and leaf morphology of Lenotis ocymifolia.............................................................5

2.3.1 General habitat.....................................................................................................................5

2.3.2 Leaves morphology.............................................................................................................5

2.4 Uses of the plant......................................................................................................................6

2.5 Essential oil composition and antibacterial activity of Lenotis ocymifolia........................6

2.6 Trypanocidal and antileukemic effects of Leonotis ocymifolia’s essential oil...................7

2.7 Antibacterial and antidiarrheal activities of 80% methanol leaf of Lenotis ocymifolia...7

2.8 Phytochemistry of Leonotis ocymifolia...................................................................................7

2.9 Volatile oils..............................................................................................................................8

2.10 Terpenoids and Steroids......................................................................................................9

CHAPTER THREE.....................................................................................................................10

RESEARCH METHODOLOGY..............................................................................................10

3.1 Introduction...........................................................................................................................10

3.2 Collection of research materials..........................................................................................10

3.3 Preparation of Leonotis ocymifolia for phytochemical screening.....................................10

3.4 Materials.................................................................................................................................10

3.5 Concentration of the methanol extract..............................................................................11

3.6 Thin layer chromatography.................................................................................................11

3.7 Column sample preparation...............................................................................................12

3.8 Setting up a column..............................................................................................................12

3.9 Isolation of the compound...................................................................................................12

3.10 Thin layer chromatography...............................................................................................13

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3.11 Concentration of fractions.................................................................................................14

3.11.1 Thin layer chromatography of the fractions................................................................14

3.12 Smaller column...................................................................................................................15

3.12.1 Thin layer chromatography............................................................................................15

3.13 Qualitative phytochemical screening..............................................................................15

3.13.1 Test for tannins.................................................................................................................15

3.13.2 Test for flavonoids...........................................................................................................16

3.13.3 Test for alkaloids (Mayer’s test).....................................................................................16

3.13.4 Test for sapponins............................................................................................................16

3.14 Validity and reliability.......................................................................................................16

3.15 Data analysis and presentation.........................................................................................16

CHAPTER FOUR.......................................................................................................................17

RESULTS AND DISCUSSIONS............................................................................................17

4.1 Results....................................................................................................................................17

4.1.1 phytochemical in leaf extract of Leonotis ocymifolia.......................................................17

4.1.2 Calculation of retardation Factor.....................................................................................17

4.1.3 Yielding of Leonotis ocymifolia leaf’s essential oil...........................................................18

4.2 Discussions............................................................................................................................19

4.2.1 Phytochemical identification of Alkaloids.....................................................................19

4.2.2 Identification of sapponins...............................................................................................20

4.2.3 Identification of terpenoids..............................................................................................20

4.2.4 Identification of tannins....................................................................................................20

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4.3 Retardation factor.................................................................................................................21

CHAPTER FIVE.........................................................................................................................22

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS.......................................22

5.1 Summary................................................................................................................................22

5.2 Conclusions............................................................................................................................22

5.3 Recommendations................................................................................................................22

REFERENCES.............................................................................................................................24

APPENDICES.............................................................................................................................26

Appendix I: Work Plan..............................................................................................................26

viii
 LIST OF FIGURES

Figure 1: Leonotis ocymifolia plants' habitat................................................................................5

Figure 2: Leonotis ocymifolia leaves..............................................................................................6

Figure 3: structure of Labda-8(17),12E,14-triene-2R,18-diol...................................................8

Figure 4: TLC spotting after isolation from column..............................................................13

Figure 5: Rotary evaporator (for concentration)....................................................................14

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 LIST OF TABLES

Table 4.1.1 phytochemical in leaf extract of Leonotis ocymifolia............................................17

x
 ABSTRACT

The researches carried out the phytochemical studies of the medicinal plant Leonotis
ocymifolia. Methanolic extract of Leonotis ocymifolia was found to contain terpenoids,
alkaloids, tannins and sapponins. The leaves of Leonotis ocymifolia was found to contain
essential oils. Fractions were obtained from the extract when column chromatography
was carried out to determine purity of the compounds. The main methods that were
used include steam distillation to extract essential oils, vacuum distillation to
concentrate the methanolic extract, column chromatography to isolate and fractionate
compounds and chemical test to test for the phytochemicals present. The research was
conducted to provide information that can be used in the manufacturing of herbal
drugs which are very essential in treatment of malaria, neck swelling, skin irritation,
cancer and ulcers to the Moiben natives and even to the entire country.

xi
 CHAPTER ONE

INTRODUCTION

1.1 General background

Leonotis Ocymifolia, occasionally referred to as a minaret flower, is a flowering plant of

the mint family, Lamiaceae. The plant is found in Eastern Africa spanning from Sudan to

South Africa. The plant is reasonably drought - resistant and wind - tolerant (Baudry &

Alvaro, 2015).

Phytochemicals studies is the study of chemical composition of the medicinal plant. In

this study the medicinal plant of the interest is the Leonotis ocymifolia whose genus name

Leonotis is derived from Greek "Leon" meaning "lion" and "Otis" meaning "ear" and

alludes to the resemblance of the corolla to a lion’s ear. The species name of ocymifolia

means with leaves like basil plants. Leonotis ocymifolia was traditionally used especially

in Ethiopia for the treatment of diseases (Van wyk et al., 2008).

1.2 Statement of the problem

Leonotis ocymifolia had been used as traditional medicine in Ethiopia to cure headache,

ulcers of the neck and swelling, diarrhea and bacterial infection. This clearly show that

Leonotis ocymifolia has bioactive compound which are medically valued to human

health. These bioactive compound can be isolated and identified then used in

pharmaceutical industries to manufacture medicine which treat the related diseases.

This will make availability so easy to patient since Leonotis ocymifolia is a perennial

shrub, it grows only in certain region in east Africa and Africa at large. Acquisition of

bioactive compound will improve human health thus improving production of people

1
due to good health and reduced mortality rate to infants as children are very prone to

the above mentioned diseases.

1.3 Objectives of the study

1.3.1 General objective

To determine the phytochemical present in Leonotis ocymifolia

1.3.2 Specific objective

To determine the yield of essential oil in Leonotis ocymifolia

1.4 Hypothesis

Leonotis ocymifolia has phytochemical constitutes.

1.3 The significance of the study

This research was carried out partly as an exercise to gain relevant skills and knowledge

on sample preparation, extraction, fractionation and purification of medicinal plants.

The acquired skills can be used in the manufacture of medicinal drugs to help in

fighting diseases in the current world including fungal and bacterial diseases. Drug

manufacturers using medicine will also help in solving and reducing infant death rates

as they are used in treating infant prone ailments mainly in Africa. In addition, it will

also help in increasing the lifespan of the adults since medicinal plants are very useful

in treatment of fungal and bacterial diseases which are very dangerous health wise.

1.5 Scope of the study

This research used the medicinal plant Leonotis ocymifolia. The species of the plant was

collected at Elgeyo Marakwet in Rift Valley province. Vacuum distillation was used to

2
concentrate methanol extract and column chromatography used to isolate and

fractionate the compounds of the plant.

1.6 Limitation of the study

Time consuming: the study consumes a lot of time especially in the column

chromatography where isolation, fractionation and purification of the plant compounds

was done.

Lack of reagents: the study was to use acetone as a solvent in extracting the extract but

it was not available in the laboratory and there this affected the expectation of the

study.

3
 CHAPTER TWO

LITERATURE REVIEW

2.1 Introduction

In this chapter, the relevant literature information was reviewed. This section is very

important as it determines the information that links the past study of the plant with the

current study and what future studies will need to employ so as to gain vast knowledge

about the medicinal plant, Leonotis ocymifolia.

2.2 Description of Leonotis ocymifolia

Leonotis ocymifolia commonly known as minaret plant. Flower is a high drought tolerant

and wind tolerant. Leonotis ocymifolia has the same growth habit with the well-known

Leonotis leonurus. Species of genus Leonotis are widely distributed in Africa as herbal

remedies for medicinal purposes especially for bronchial illness and epilepsy. In the

eastern cape, where it is usually found, it is used for the treatment of cough, chest

problems, bronchial asthma, stomachaches, skin diseases, hemorrhoids and elipsys.

Leonotis ocymifolia (burm F.) lwarsson (Lamiaceae) is known with vernacular names

Klipdagga, lion’s ear and Umewili. It is indigenous to Eastern and Southern Africa.

Subshrub or shrub 1.5-2.5m high, not very aromatic, commonly cultivated in the colder

places of New Zealand and is occasionally cultured for its medicinal uses, which

include acting as an ascaricide, an anticancer drug and as a treatment for ulcers.

Leaves of the plant are smoked for its anti-epileptic effects (d'Avigdor et al., 2014).

Aqueous leaf preparation have been reported to possess anticonvulsant, anticeptive,

anti-inflammation and ant diabetic properties in rodents (Yineger et al., 2008).

4
In addition to folkloric uses mentioned, Lenotis ocymifolia reputedly produces

marijuana-like effects (d'Avigdor et al., 2014).

2.3 Habitat and leaf morphology of Lenotis ocymifolia

2.3.1 General habitat

Stems, lanky shrub 0.6-5m high, sparsely branched from woody swollen rootstock 20-

150mm diameter, shoots up to 10cm in diameter and appearing rounded in section at

base(Kebede et al., 2019).

Figure 1: Leonotis ocymifolia plants' habitat

2.3.2 Leaves morphology

leaves usually petiolate blades ovate to narrowly obovate, lanceolate to narrowly

spathlate, 0.9-19 (-23), 0.3-9cm, crenate almost to base with 7-65 teeth, apex acute to

accumulate, base chordate, truncate to attenuate, both surface, laxly pubescent to

voluminous with short white to yellow hairs and sessile glands, with (4-) 10-16 lateral

veins per leaf, venation covered with slightly longer hairs, occasionally appressed;

petiole 0.55(-110)in south Africa)mm antrorse or retrorse hairs, eglandular or with

sessile gland(Kebede et al., 2019).

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Figure 2: Leonotis ocymifolia leaves

2.4 Uses of the plant

Leonotis ocymifolia is used for the treatment of diarrhea. By tradition, Lenotis ocymifolia

dried leaf and fruit mixed with honey given orally to treat diarrhea.

In treatment of ocymifolia is also used in the treatment of micro bacteria, headache,

neck ulcers and swelling (Twala, 2019).

2.5 Essential oil composition and antibacterial activity of Lenotis ocymifolia

Species of Leonotis are among the frequently used herbal remedies to treat various

bronchial illness and epilepsy (Afolayan et al., 2005). Analysis of the essential oil of

leaves and flowers of the Lenotis ocymifolia from the Eastern Cape of South Africa was

conducted by the GC-MS. Major constituents of the Lenotis ocymifolia oils were(Z)-β-

ocimene (13.0-15.2%), nonanal (5.5%), β-caryophyllene (21.4-30.8%), - humulene (9.1-

11.6%), germacrene D (21.5-21.7%) and ℽ-muurolol (4.6%)as the prominent compounds.

6
The oils exhibited a broad spectrum activity antibacterial activity against gram-positive

(Staphylococcus epidermidis) and gram-negative (Escherichia coli)

2.6 Trypanocidal and antileukemic effects of Leonotis ocymifolia’s essential oil

Essential oil from Ethiopian medicinal remedies, Lenotis ocymifolia plant was

investigated for its chemical compositions, trypanocidal and cytotoxic activities (Nibret

& Wink, 2010). 68 Components Were Identified from the essential oil of Leonotis

Ocymifolia Aerial Part, Caryophyllene Oxide (12.06%)being the major component. The

oils of Leonotis ocymifolia exhibited trypanocidal activities with 1C50 value of 42.30g/ml

and 15.41g/ml respectively.

2.7 Antibacterial and antidiarrheal activities of 80% methanol leaf of Lenotis

ocymifolia

Leonotis ocymifolia (Burm. F) Iwarisson (lamiaceae) is among the medical plants that that

are claimed to have various pharmacological activities study was carried out to

investigate the anti-diarrheal and antibacterial activities of this plant (Mengie et al.,

2021). They found out that Leonotis ocymifolia’s leaf extract when mixed with honey and

then given orally is a good treatment of diarrhea. Moreover, the plant`s various fraction

of leaf extract showed the presence of a variety of secondary metabolites such as

labdane diterpenoids.

2.8 Phytochemistry of Leonotis ocymifolia

Phytochemistry of particularly non-volatile volatile constituents of Leonotis ocymifolia

has been comprehensively investigated due to interest generated as a result of the wide

variety of biological effects reported for this plant (Mengie et al., 2021). More than 50

7
compounds have been isolated and characterized. Leonotis ocymifolia contains mainly

terpenoids particularly labdane diterpenes.

Below shows chemical structure;

Figure 3: structure of Labda-8(17),12E,14-triene-2R,18-diol

2.9 Volatile oils

Volatile oils have been extracted (mainly through steam distillation) from 63 species in

23 genera of Southern Africa Lamiacae (Gampe & Carreira, 2012). Twelve of the 23

genera have had all member investigated and the remaining eleven genera have had

oils frim some members studied. Major compounds were defined as any essential oil

constituents present at a level of at least 10% of the total oil composition. A total of 133

major compound were identified across the 63 species (Gampe & Carreira, 2012).

8
Β- caryophyllene is by far the most common major compound, followed at some

distance by germacrene-D,1,8-cineole, limonene, -pinene and -bisabolol. It is no

surprise that β-Caryophyllene is the most common major compound in the essential oil

of Southern African Lamiaceae as it widely distributed throughout the plant kingdom.

It contributes to the unique aromas of essential oils and plays a pivotal role in the

evolution and survival of higher plants. Furthermore, studies have provided evidence

that support β-Caryophyllene as a potential therapeutic tool based on the protective

roles it exhibits on animal cells (Arumugam et al., 2020). Moreover, experimental result,

have noted the ability of this molecule to reduce effects of chronic pathologies

characterized by inflammation and oxidative stress, especially metabolic and

neurological diseases (Arumugam et al., 2020). β-Caryophyllene has exhibited beneficial

effects on diabetes, cardiovascular diseases, obesity, some liver diseases, pain and other

nervous system disorders (Abdel-Mogib, Albar & Batterjee, 2002).

2.10 Terpenoids and Steroids

Terpenoids, more specifically diterpenoids are abundant secondary metabolites present

in Lamiaceae (Gampe & Carreira, 2012). Many species contain labdane diterpenoids, a

multitude of which demonstrate a broad spectrum of biological activities, including

anti-inflammatory, antimicrobial, antiviral, lytotoxic, antioxidant, antihypertensive and

hepatoprotective activities (Gampe & Carreira, 2012).

9
 CHAPTER THREE

RESEARCH METHODOLOGY

3.1 Introduction

This chapter describes the methods that was used in the study. It forms the framework

of the study. This chapter discusses various steps in medicinal plant phytochemical

screening, isolation and fractionation. Validity, reliability and data analysis and

presentation was also discussed in this chapter.

3.2 Collection of research materials

Leonotis ocymifolia plant samples were collected at Elgeyo Marakwet with the help of

supervisor who assisted in the identification and transport. Samples were collected by

plucking the leaves and flowers of the medicinal plant, after which the leaves and

flowers were taken for research purposes.

3.3 Preparation of Leonotis ocymifolia for phytochemical screening

Fresh green leaves of Leonotis ocymifolia were spread over a flat board then left to air dry

under the shade at room temperature for one week. Thereafter, they were crushed into

fine particles using hands since they were very soft.

3.4 Materials

1. Erlenmeyer flask

2. Beakers

3. Filter flask

4. Graduated cylinder

5. Conical funnel

10
6. Hirsch funnel

7. Hot plate

8. Methanol

9. Ethyl acetate

10. Hexane

11. stainless steel spatula

12. Round bottomed flask

13. Condenser

14. Claissen connecting

15. Vacuum adapter

16. Plastic joint clip

17. TLC plate.

3.5 Concentration of the methanol extract

Concentration process was done using Rotary evaporator through vacuum distillation.

Methanol extract was placed in the round bottom flask and then connected to the

Rotary evaporator set up. 200 ml of the methanol extract was concentrated to 20 ml of

the crude extract. The concentration process took about 30 minutes to complete.

3.6 Thin layer chromatography

Before compound isolation and fractionation was done, a test to determine good

solvents ratio to be used in extraction was carried out. This was done by TLC to know

the ratio that give large retardation factor. 8 ml of ethyl acetate and 2 mL of hexane

were measured and put in a 250 ml measuring cylinder. TLC of the crude extract was

11
spotted and put in the developed solvent for observations. Another crude spot-on TLC

was immersed in another ratio of hexane to ethyl acetate in the ratio of 6:4 and then last

was immersed in the same solvent at a ratio of 4:6, observations were made and

retardation factors of the TLCs was calculated and found out that the ratio of the hexane

to ethyl acetate in 6:4 was the best.

3.7 Column sample preparation

18 ml of the concentrated crude extract was measured using a 50 ml graduated cylinder

and then placed in a motor. 3 g of silica gel was then added. The mixture was dried by

grinding the extract together with the added silica. The process took one hour to

ground liquid extract into fine powder.

3.8 Setting up a column

Column was washed with the liquid soap and running tap water then rinsed with

distilled water and then left to dry. After drying a small piece of cotton wool was placed

at the bottom. Cotton wool was to prevent the statistic stationary phase from flowing

out. After setting up a column as stationary phase was introduced using a crane final.

The stationary phase silica gel was hit by a rubber band to ensure it is well packed and

types to prevent it from cracking and prevent airspace as this could result to poor

movement of compounds thus consuming a lot of time. The column was clamped in a

clamp stand.

3.9 Isolation of the compound

After setting up the column, column chromatography was carried out to help in

isolating the compounds from the extract. 50g of silica gel was poured into the column

12
using a funnel. Cotton wool was then placed on the extract to prevent the extract from

escaping. 100 mL of hexane was poured into the column with the help of a clean funnel.

The clean and dry test tubes were well arranged in the test tube rack so as to be used to

collect isolated compounds.

3.10 Thin layer chromatography

From the isolated compounds in the test tubes, a selection of representative tubes was

done. The selection process was guided by the concentration of the color of compounds.

The sample of each of test tube was spotted in TLC against crude and the result

obtained were used to make fractions according to the color shown by the compounds.

The test tubes that gave a uniform color from TLC sporting were placed together in the

same reagent bottle simultaneously.

13
Figure 4: TLC spotting after isolation from column

14
3.11 Concentration of fractions

Each fraction’s extract was then placed in a round bottomed flask then to rotary

evaporator setup one at a time to remove the solvent in each fraction.

3.11.1 Thin layer chromatography of the fractions

After the concentration, the TLC spotting of each fraction was carried out where 6 ml

of hexane to 4ml of ethyl acetate was used as a development solvent and then Erlich’s

reagent was used as a visualization solvent. To prevent the TLC from being charred it

was not directly placed on the hot plate but was placed of the HCL fumes which was

more efficient for spot visualizing.

After TLC was carried out, fraction two and fraction three was found to contain three

compounds and four compounds respectively. Fraction one contained only two

compounds hence it was easy to isolate fraction one compared to fraction three and

two.

Fraction one was chosen to proceed for purification through smaller column.

15
Figure 5: Rotary evaporator (for concentration)

16
3.12 Smaller column

After, isolation and fractionation, fraction one was found to contain two compounds

only. Therefore, it was chosen for further purification using the smaller column.

Smaller column was first cleaned with liquid soap and running tap water and then

dried. Silica gel which was the stationery phase was packed and then hit by a rubber

band to pack tightly without any air space. After it was tightly packed, the hexane was

run through it to make it wet. The concentrated, ground and finely crushed extract of

fraction one was then introduced into the smaller column followed by cotton wool then

hexane solvent which was the mobile phase. Clean and dry test tubes were used to

collect the fraction. Hexane was used throughout as the mobile phase.

3.12.1 Thin layer chromatography

From the isolated compounds in the test tubes a selection of representative tubes was

done. The selection process was guided by the concentration of the color of compounds.

The sample of each of the test tube was supported with TLC and then result was used

to make fractions according to the color shown by the compounds. The test tube that

give a uniform color from TLC sporting a place together reagent bottle simultaneously.

3.13 Qualitative phytochemical screening

The test to identify the phytochemical constituents of Leonootis ocymifolia extract

involved standard producers outlined as follows.

3.13.1 Test for tannins

To 1ml of Leonotis ocymifolia's crude extract, 2mL of water was added followed by four

drops of ferric chloride solution.

17
3.13.2 Test for flavonoids

To ml of extract, three drops of dilute sodium hydroxide was added and followed by

two drops of dilute hydrochloric acid.

3.13.3 Test for alkaloids (Mayer’s test)

To 2ml of extract, two drops of Mayer’s reagent was added.

3.13.4 Test for sapponins

To two milliliters of crude extract, two milliliters of water was added.

3.13.5 Test for Terpenoids (Salkowaski test)

To 2ml of extract, 2ml of chloroform was added, chloroform was then evaporated by

passing the solution through the hot plate.2ml of concentrated Sulphuric acid was then

added to the dried sample.

3.14 Validity and reliability

This was done in order to evaluate the quality of the research and gain knowledge on

how to and where to use a certain instrument. It also focuses on checking whether there

might be any ambiguity in the instrument. Availability and accuracy of the instruments

are encountered.

3.15 Data analysis and presentation

The study involved both qualitative and quantitative data. Quantitative was based on

the amount t of yield of the analyzed sample, for example in this research we focused

on the yielding of essential oil. On the other hand, qualitative was based on the

composition and test of the analyzed sample, for example the test for phytochemical in

Leonotis ocymifolia.

18
 CHAPTER FOUR

RESULTS AND DISCUSSIONS

4.1 Results

4.1.1 phytochemical in leaf extract of Leonotis ocymifolia

Table 4.1.1 phytochemical in leaf extract of Leonotis ocymifolia

Phytochemical Type of Test Results Appearance

Tannins Ferric Chloride + Green precipitate was observed

Sapponins Frothing + Formation of stable foam

Flavonoid Lead Acetate - yellow precipitate was not observed

Terpenoid Salkowaski + The reddish brown colour was observed

Alkaloid Mayer's Test + Creamywhite precipitate was observed

4.1.2 Calculation of retardation Factor

When calculating the retardation Factor there are two measurements that are taken into

account the distance moved by the analyte component and the distance moved by the

solvent.

RF of analyte of compound one

Distance moved by the solvent =5.8cm

Distance moved by compound one= 2.6cm

RF=distance moved by analyte

Distance moved by solvent

19
 2.6 cm
=
5.8 cm

= 0.45

RF of analyte compound two

Distance moved by analyte

RF=
Distance moved by solvent

Analyte distance=3.2 cm

Solvent distance=5.8 cm

3.2 cm
=
5.8 cm

¿ 0.55

Rf of analyte compound three

Distance moved by analyte

RF=
Distance moved by solvent

Analyte distance=4.6 cm

Solvent distance=5.8 cm

4.6 cm
= 5.8 cm

¿ 0.79

4.1.3 Yielding of Leonotis ocymifolia leaf’s essential oil

After measuring 390 g of fresh green leaves of Leonotis ocymifolia was placed onto a

setup of steam distillation where the essential oil was extracted and the yield recorded.

Yield of the essential oil=5mL.

20
4.2 Discussions

4.2.1 Phytochemical identification of Alkaloids

The precipitation in Mayer’s identification ensure the presence of alkaloid compounds

in the given extract. The aim of adding sulfuric acid is because of the properties of

alkaloid which is base. Therefore, it should be extracted in the acid solvents. The

positive result of the Mayer's test was confirmed by a yellow precipitate. It was

expected as a complex of potassium-alkaloid. In the formation of Mayer's reagent, the

solution of mercury chloride was added by potassium iodide and produced a red

precipitate of mercury iodide. The excess of potassium iodide edition introduced to

potassium hydrate formation. Alkaloids consist of nitrogen atoms which have lone pair

electrons. The lone pair electrons are examined to form covalent coordinate bonding

with metal ion. In alkaloid identification with Meyer reagent the nitrogen in alkaloids

was predicted to react with metal ion of potassium from potassium tetrahydroborate

producing complex of potassium-alkaloid precipitating. The reaction is as follows;

HgCl2+2KI →HgI2+Kcl

Hgl2+2KI →K2(HgI4)

+ K2(HgI4) + K(HgI4)-
K+

21
4.2.2 Identification of sapponins

The foam introduced in sapponins test proved the presence of glycoside that have an

ability to produce foam in water hydrolyzed in glucose and other compounds.

Sapponins contain of glycosyls as polar groups while steroids and triterpenoids as

nonpolar groups. The compound containing polar and nonpolar group are surface

active compounds. When they are shake strongly with the water, sapponins would

form miscellanea. In the miscellanea, polar groups face to the outside and nonpolar

groups face in the inside. This phenomenon is called as foam.

4.2.3 Identification of terpenoids

Terpenoid screening is based on the ability of compounds to form concentrated H 2SO4

colors in solvents of acetic acid anhydride. Terpenoids react to give red orange or

purple colour. Reaction for terpenoid test with methanol extract gave a positive result

with a reddish color change

4.2.4 Identification of tannins

Tannins create a light yellow to dark brown discoloration in water. The tannin

compound s is widely distributed in many species of plant, where they play a role in

protection from predation (including pesticides) and might help in regulating plant

growth. Tannin has most polyphenols were proved to have a potent antioxidant

effect. Studies on the antitumor effect of the tannins proved that a strong activity is

obtained with allagitannins having galloyl groups at 0-2 and 0-3 positions of the

glucose core(s), as in the tellimagrandins.

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4.3 Retardation factor

Retardation factor values can be compared to identify the components in the

mixture and this is mainly attributed to by the relative affinity of that particular

component. Relative affinity describes how well a component is attracted to either the

stationary phase or mobile phase. It determines how quickly the components

moves through the stationary phase.

Components with greater affinity to the mobile phase like compound three move up

the plate than those with a greater affinity to the stationery phase like compound one.

Compound one is polar and contain chemical groups that can form hydrogen bonds

this is because they have greater affinity with stationery phase and therefore bond

strongly to the polar cellulose-water structure than to the nonpolar solvent. This finally

makes them to travel more slow up the chromatogram plate.

Compound three is nonpolar and therefore bond more strongly to the nonpolar

solvent than to then polar paper. They are non-soluble. They, therefore have a greater

affinity to the mobile phase and will travel more quickly up the paper. These

components gave higher RF values followed by compound two.

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 CHAPTER FIVE

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

5.1 Summary

Phytochemical screening of the extract gave a positive result of tannins, sapponins,

terpenoids and alkaloids. Test for flavonoids gave a negative result, it was absent.

Extraction of essential oils from the fresh leaves of Leonotis ocymifolia was successful. It

yielded a good amount of essential oils.

Isolation and fractionation of the components of Leonotis ocymifolia plant extract was

successful.

5.2 Conclusions

Medicinal plant Leonotis ocymifolia is a source of the secondary metabolite including

alkaloids, sapponins, terpenoids and tunnins. The mentioned secondary metabolites

play a vital role in treatment of various diseases; neck swelling, malaria, cancer,

inflammation and many other ailments.

The phytochemical screening of the plant plays an important role for pharmaceutical

studies especially in discovering new potential drugs for treatment of various diseases.

Further purification, identification and characterization of the bioactive chemical

constituents would be priority in future studies especially for quantitative analysis.

5.3 Recommendations

1. Importation of digitalized hyphenated instruments for structure elucidation of

more complex compounds.

2. Publication of catalogue for crude drugs.

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3. Public educational talks should be conducted to teach and talk to people across

the entire country on the importance and significance of the medicinal plant so

that people should mainly focus on herbal treatment and then stop relying on

imported drugs from the western countries.

4. Research lessons should be conducted to all the universities in Kenya about the

natural products especially medicinal plants. This will equip students with vast

knowledge of herbal drugs thus improving our pharmaceutical skills on drug

manufacturing.

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 REFERENCES

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Plectranthus. Molecules, 7(2), 271-301.

Arumugam, G., Sinniah, U. R., Swamy, M. K., & Lynch, P. T. (2020). Micropropagation

and essential oil characterization of Plectranthus amboinicus (Lour.) Sprengel, an

aromatic medicinal plant. In Vitro Cellular & Developmental Biology-Plant, 56(4),

491-503.

d’Avigdor, E., Wohlmuth, H., Asfaw, Z., & Awas, T. (2014). The current status of

knowledge of herbal medicine and medicinal plants in Fiche, Ethiopia. Journal of

Ethnobiology and ethnomedicine, 10(1), 1-33.

Gampe, C. M., & Carreira, E. M. (2012). Arynes and cyclohexyne in natural product

synthesis. Angewandte Chemie International Edition, 51(16), 3766-3778.

Kebede, A., Ayalew, S., Mesfin, A., & Mulualem, G. (2017). Assessment on the use,

knowledge and conservation of medicinal plants in selected kebeles of dire dawa

administration, eastern Ethiopia. Journal of Plant Sciences, 5(2), 56-64.

Mengie Ayele, T., Chekol Abebe, E., & Bogale Kassie, A. (2021). Investigation of

Antibacterial and Anti-Diarrhoeal Activities of 80% Methanol Leaf and Fruit

Extract of Leonotis ocymifolia (Burm. F) Iwarsson (Lamiaceae). Journal of

experimental pharmacology, 13, 613–626.

Nibret, E., & Wink, M. (2010). Trypanocidal and antileukaemic effects of the essential

oils of Hagenia abyssinica, Leonotis ocymifolia, Moringa stenopetala, and their

26
 main individual constituents. Phytomedicine : international journal of phytotherapy

and phytopharmacology, 17(12), 911–920.

Nsuala, B. N., Enslin, G., & Viljoen, A. (2015). “Wild cannabis”: a review of the

traditional use and phytochemistry of Leonotis leonurus. Journal of

ethnopharmacology, 174, 520-539.

Oyedeji, O. A., Afolayan, A. J., & Eloff, J. N. (2005). Comparative study of the essential

oil composition and antimicrobial activity of Leonotis leonurus and L. ocymifolia

in the Eastern Cape, South Africa. South African Journal of Botany, 71(1), 114-116.

Twala, T. C. (2019). The impact of natural and simulated herbivory on compensatory leaf

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Van Wyk, B. E., De Wet, H., & Van Heerden, F. R. (2008). An ethnobotanical survey of

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 APPENDICES

Appendix I: Work Plan

Date/event 23th 29th 10th 1st 2ed 1st Feb- 10th 4th
Sep- Oct- Nov- Jan- Jan- Feb to May
10th
29th 10th 21st 2ed 1st
Feb 12th
Oct Nov Nov Jan Feb
March
Identification of
supervisor
Proposal writing

Collection of the
plant sample,
drying and
grinding
Sample
extraction
TLC,
phytochemical
test, vacuum
distillation,
column
chromatography
Structure
elucidation and
determination
Report writing

presentation

28