PHYTOCHEMICAL INVESTIGATION AND EXTRACTION OF

ADHATODA SCHIMPERIANA

BY: HAJI KEMAL

A PROPOSAL SUBMITTED TO THE DEPARTMENT OF

CHEMISTRY, FACULTY OF NATURAL AND COMPUTATIONAL
SCIENCE IN PARTIAL FULFILLMENT OF THE REQUIREMENT
FOR THE DEGREE OF MASTER OF CHEMISTRY

GONDER
UNIVERSITY
GONDER,
ETHIOPIA
JUNE,

23
 2020

TABLE OF CONTENTS

Table OF Contents................................................................................................................................................2
CHAPTER ONE......................................................................................................................................................3
1. Introduction...................................................................................................................................................3
1.2. Problem of the Research...........................................................................................................................4
1.3. Objectives..................................................................................................................................................8
1.3.1. General objective...................................................................................................................................8
1.3.2. Specific objectives.................................................................................................................................8
1.4. Significancy of the study...........................................................................................................................8
CHAPTER TWO...................................................................................................................................................10
2. Litreture Riview..............................................................................................................................................10
2.1. Botanical background..................................................................................................................................10
2.2. Ethnopharmacological use of Justicia schimperiana..............................................................................10
2.3. Chemicals, drugs and media...................................................................................................................11
2.4. . EXTRACTION OF PHENOLIC COMPOUNDS USING SOLVENTS .....................................11
2.5. MICROWAVE-Assisted Extraction (MAE)...............................................................................................12
2.6. Preparation of Extracts............................................................................................................................13
CHAPTER THREE...............................................................................................................................................14
3. Materials and Methods....................................................................................................................................14
3.1. Study Area...............................................................................................................................................14
3.2. Extraction and Isolation...............................................................................................................................16
3.1.1. Extraction.................................................................................................................................................16
3.1.2. Isolation....................................................................................................................................................16
3.2. Fractionation...............................................................................................................................................16
3.3.5. Test for alkaloids:..............................................................................................................................18
3.3.6. Test for polyphenol:..............................................................................................................................18
3.3.7. Test for flavonoids................................................................................................................................18
4. Time schedule.................................................................................................................................................20
5. Budget schedule..............................................................................................................................................20
6. References.......................................................................................................................................................22

23
 CHAPTER ONE

1. INTRODUCTION
Medicinal plants have a long history of use in most communities throughout the world. It has
been confirmed by WHO that herbal medicines serve the health needs about 80% of the world’s
population, especially for millions of people in the vast rural areas of developing countries
[(ziyad, 2018)]. In Africa, the use of traditional medicine has persisted over the years and the last
few decades have witnessed an upsurge of interest in traditional medicine and other alternative
forms of healthcare in the developing and developed countries

The family Acanthaceae is a taxon of dicotyledonous flowering plants containing almost 250
genera and 2500 species. Most are tropical herbs, shrubs or twining vines, some are epiphytes.
Only a few species are distributed in temperate region. The four main centers of distribution are
Indonesia, Malaysia, Africa, Brazil and Central America (Reddy et al., 2013). Adhatoda is the
largest genus of Acanthaceae, with approximately 600 species that are found in pantropical and
tropical regions (Corrêa and Alcântara, 2012; Hedberg et al., 2006). The species of Adhatoda can
be easily recognized by their bilabial corolla, with a posterior lip that is generally two-lobed, an
anterior lip that is three lobed, two stamens, a capsule with four seeds, and a basal sterile portion
(Corrêa and Alcântara, 2012). The presence of compounds with diverse chemical class including
alkaloids, lignans, flavoinoids and terpenoids will be reported (Corrêa and Alcântara, 2012).
Other chemical class of chemicals like essential oils, vitamins, fatty acids and salicylic acid,
steroids also reported to reside in the Adhatoda (Corrêa and Alcântara, 2012)

Local name ‗‟dhumuugaa„ in Afan Oromo, ‗‟Sensel„ or ‗‟simiza‟ in Ahmaric,It is common

shrub growing in moist mountain forest, usually near streams and rivers, in evergreen scrub on
hill slopes, forest clearings, different plantations, will be teground or planted. It belongs to the
family of Acanthaceae. It is a leafy shrub with much branched easily breakable stems, simple
and opposite, long oval and tip pointed leaves and white or yellow white flowers. It is up to 4 m
high, with slightly unpleasant smell (Hedberg et al., 2006).

23
In Northern and East Ethiopia the plant alone or in combination with other plants is will be use
for various diseases such as epilepsy, mental illness, eye diseases, jaundice, malaria, leprosy,
syphilis, gonorrhea, rabies, measles, relapsing fever, vitiligo, gout and acute febrile illness In
Southwest Ethiopia, it is will be use for malaria, scabies, where the fresh leaves are crushed and
macerated in water and then the affected area is will behed with the macerate [8]. The
determination of the phytochemical constituents of plant extracts are essential in order to ensure
the reliability and repeatability of pharmacological and clinical research, to understand their
bioactivities and possible side effects of active compounds and to enhance product quality
control. Thus, with regard there is much to be explored.

1.2. PROBLEM OF THE RESEARCH

The clinical use of antibacterial agents may have very different effects on bacterial agents due to
the differences in the mechanisms by which antibacterial agents affect bacteria: leading to an
endpoint of either inactivation or actual death of the bacteria. The antibacterial agents that inhibit
multiplication of bacteria are called bacteriostatic and other antibacterial agents which lead to
death of bacteria are called bactericidal. Most protein synthesis inhibitors and anti-metabolites
antibacterial agents are bacteriostatic, while cell wall synthesis inhibitors, membrane disrupting
agents, nucleic acid inhibitors and aminoglycosides are bactericidal. The bactericidal effects of
cell wall synthesis inhibition involve interference in the cell's osmotic defenses and causing it to
absorb excess water and burst. Cell membrane disrupting agents cause bacterial death probably
by resulting loss of vital metabolites through disrupted membrane (Crofton, 1969). In addition to
their specific lethal effects, most bactericidal drugs have been suggested to result death of
bacteria by producing highly deleterious hydroxyl radicals as an end product of oxidative cellular
damage pathway (Kohanski et al., 2007). Protein synthesis inhibitors usually interfere with the
synthesis of protein at an initiation phase and result in insufficient rather than distorted proteins
and prevent the growth and proliferation of the bacteria without actually destroying them. Unlike
other protein synthesis inhibitors, aminoglycosides are bactericidal as they cause mistranslation
by acting both at initial and later phase of protein synthesis and result in distorted proteins in
addition to the decrease in the rate of the protein synthesis (Crofton, 1969; Davis, 1987).

Among the different groups of fungi, there are three major groups of fungi that cause disease in
humans. Moulds (like Dermatophytes) are filamentous fungi that grow as long filaments that

23
intertwine to form a mycelium. The other group is true yeasts which are unicellular round or oval
fungi like Candidia neoformans. The remaining group is yeast like fungi which are similar to
yeasts but also form long non-branching filaments. Among them Candidia albicans is a common
commensal organism in gut, mouth and vagina. It causes wide range of disease including oral
thrush, vaginitis, endocarditis and septicemia (Mahmoud et al., 1999).

The antifungal agents can be classified as antifungal antibiotics and synthetic antifungals.
Antifungal antibiotics are antifungals usually extracted from natural sources. One of such drugs
is amphotericin B which is a naturally occurring polyene macrolide antibiotic, produced by
Streptomyces nodosus and produce its antifungal effect by binding with ergosterol forming a
pore on the membrane of susceptible fungi. Griseofulvin is also an antifungal antibiotic which is
a narrow-spectrum antifungal agent isolated from cultures of Penicillium griseofulvum and
causes a fungistatic action by interacting with fungal microtubules and interfering with mitosis.

There are other antifungals antibiotics like nystatin and echinocandins.

The other group of antifungal drugs consists of synthetic antifungals, which are not extracted
from natural source. Azole antifungal drugs are a group of synthetic fungistatic agents with a
broad spectrum of activity based on the imidazole or triazole nucleus. Their mechanism of action
is inhibition of 14-α-sterol demethylase which resulting in impairment biosynthesis of ergosterol
and lead to the accumulation of 14-α-methylsterols which disrupt the close packing of acyl
chains of phospholipids, impairing the functions of certain membrane-bound enzyme systems
such as ATPase and enzymes of the electron transport system. Flucytosine is also a synthetic
orally active antifungal agent which is effective against a limited range (mainly yeasts) of
systemic fungal infections, when converted to the antimetabolite 5-fluorouracil by fungal
enzyme, 5-Fluorouracil inhibits thymidylate synthetase and thus DNA synthesis. The other
synthetic antifungals include terbinafine and naftifine.

The development of cellular resistance occurs as a result of mutations to endogenous genes or

via lateral gene transfer of resistance determinants from other microorganisms. Recent advances
in genomics have revealed that many natural ecosystems, including diverse environments such as
the human gut and soil, contain large number of genes whose functions can be co-opted to confer
resistance to antimicrobials (Perry, 2014; Martinez et al., 2015). These genes are collectively

23
known as the resistome.

The resistome concept is anthropocentric, since the original functions of the genes that comprise
the resistome will probably not to confer antimicrobial resistance phenotypes. The recovery of
genes that can confer resistance phenotypes from extreme environments that have not been in
contact with humans, such as the deep subsurface (Brown& Balkwill be, 2009) and permafrost
(D‟Costa et al., 2011), further suggests that these genes have natural roles other than conferring
antibiotic resistance. Resistance mechanisms such as multidrug transporters might have evolved
as transporters for naturally occurring substrates, serving as mechanisms to pump toxins from
cells, and their ability to also transport antimicrobials may be fortuitous (Paulsen et al., 1996).
“Resistance” genes during the pre-antibiotic period will probably chromosomal, and encoded
functions of physiological importance. In the post antibiotic period, resistome genes will
laterally transferred to a new host where they lacked their original biochemical and genetic
context, and their functions became limited to antimicrobial resistance (Debabov, 2013).

Over the last fifty years research into resistance has mainly focwill be use on clinical aspects of
antibiotic resistance, while the possible original functions of resistance genes have been largely
overlooked. Understanding the original roles of these resistome elements may aid the
development of successful strategies to fight infections cawill be use by antibiotic resistant
pathogens.

The antimicrobial resistance is a natural process in microorganism, which is will be use to defend
themselves from any pressure against their survival. As long as, antimicrobials are being will be
use, the eradication of antimicrobial resistance is unachievable and it is not possible to totally
avoid the use of antimicrobials as long as pathogenic microbes are present.

The antimicrobial resistance prevention ways mentioned above can slow down the occurrence of
antimicrobial resistance, not totally prevent antimicrobial resistance. Therefore, the development
of new antimicrobial agents is not an option. There is need for new groups of antimicrobials and
newer compounds from the old groups of antimicrobial drugs (Paudel, 2008; Ozkan, 2015). One
of the areas for search for effective antimicrobial agents is looking for natural products that can
serve as antimicrobial agent or lead compound for development of antimicrobial agent from
plants that are traditionally will be use as remedy for microbial infection related diseases (Baker

23
et al., 1995).

23
Fig 1:-Adhatoda schimperianaHochst. Ex in its
natural habitat

1.3. OBJECTIVES

1.3.1. GENERAL OBJECTIVE

 To evaluate the in-vitro antibacterial and antifungal activity of crude extract and solvent
fractions of leaves of Adhatoda schimperianaon selected bacterial and fungal species.

1.3.2. SPECIFIC OBJECTIVES

 To determine zone of inhibition of the crude extract and solvent fraction of the leaves of
Adhatoda schimperianaon selected bacterial and fungal species using agar well diffusion
method.

 To determine the minimum inhibitory concentration of the crude extract and solvent
fraction of the leaves of Adhatoda schimperianaagainst selected bacterial and fungal
species using broth dilution method.

 To determine the minimum bactericidal and fungicidal concentration of the crude extract
and solvent fractions of the leaves of Adhatoda schimperianaagainst the selected bacterial
and fungal species.

 To qualitatively determine the phytochemical constituents of the crude extract and

solvent fractions of leaves of Adhatoda schimperiana.

1.4. SIGNIFICANCY OF THE STUDY

Infectious diseases kill about 15 million people every year, which means 25% of total annual
death (WHO, 2013). Effectiveness of antimicrobial chemotherapy is highly threatened by the
wide spreading antimicrobial resistance (Rashmi et al., 2005). Most common infections are now
becoming resistant to the usual treatment. In addition, new antimicrobial drugs are not being

23
released to the market in the rate comparable to occurrence of antimicrobial resistance (Joseph,
2013). So, there is a continuous and urgent need to discover new antimicrobial compounds with
diverse chemical structures and novel mechanisms of action(Rojas et al., 2003).To fill this gap
there should be search for new antimicrobials with effective and novel antimicrobial mechanism.
Natural products are among the main area for search of new antimicrobial agents (Rashmi et
al.2005).

Plants which are traditionally will be use for medicinal purpose are candidates for search of new
antibacterial agent and usually they are safe for human use because, they have long history of use
by humans without major adverse effect (Vermani and Garg, 2002). In most of the cases, these
practices are handed down from generation to generation empirically without knowing the
plausible mechanisms, safety, and efficacy of herbal treatments (Mujumdar et al., 2001). In order
to get scientific ground for the traditional use of herbal medicine and to search for new, effective
and safe antimicrobial agent, scientific studies have to be conducted on the herbal medicines.

The leaf of Adhatoda schimperianais traditionally will be use for management of wound,
gonorrhea (Habtamu et al., 2014) and skin burn (Muthuswamy and Solomon, 2009). Extracts
obtained from different parts of Adhatoda schimperianahad been tested for antimicrobial activity.
Murthy et al. (1993) studied the antibacterial activity of aqueous, methanol and chloroform
extracts of different parts (leaf, bark, stem and root) of Adhatoda schimperianaand reported the
plant to have significant activity against S. aureus, E. coli, B. cereus, P. mirabilis, K. pneumoniae
and P. aeruginosa. By contrast, Tamirat et al. (2015) did not detect any antimicrobial activity
with the ethanol extract of root of the plant. As the methanol extract will be the one with the
highest activity

(Murthy et al., 1993), the present study attempted to further investigate the antimicrobial activity
(determination of zone of inhibition, MIC and MBC) of the 80% methanol leaf extract and
different solvent fractions against a wider microbial species, including bacteria and fungi.

23
 CHAPTER TWO
2. Litreture Riview

2.1. BOTANICAL BACKGROUND

The family Acanthaceae is a taxon of dicotyledonous flowering plants containing almost 250
genera and 2500 species. Most are tropical herbs, shrubs or twining vines, some are epiphytes.
Only a few species are distributed in temperate region. The four main centers of distribution are
Indonesia, Malaysia, Africa, Brazil and Central America (Reddy et al., 2013). Justicia is the
largest genus of Acanthaceae, with approximately 600 species that are found in pantropical and
tropical regions (Corrêa and Alcântara, 2012; Hedberg et al., 2006). The species of Justicia can
be easily recognized by their bilabial corolla, with a posterior lip that is generally two-lobed, an
anterior lip that is three lobed, two stamens, a capsule with four seeds, and a basal sterile portion
(Corrêa and Alcântara, 2012). The presence of compounds with diverse chemical class including
alkaloids, lignans, flavoinoids and terpenoids was reported (Corrêa and Alcântara, 2012). Other
chemical class of chemicals like essential oils, vitamins, fatty acids and salicylic acid, steroids
also reported to reside in the Justicia (Corrêa and Alcântara, 2012)

2.2. ETHNOPHARMACOLOGICAL USE OF JUSTICIA SCHIMPERIANA

The traditional use of Justicia schimperiana varies in different geographical area and culture. It is
being will be use for management of wound, gonorrhea, malaria, rabies and headache by Hadiya
people of southern Ethiopia for centuries (Habtamu et al., 2014). The leaf paste of Justicia
schimperiana topically applied over areas affected by burning and arthrithis and the leaf juice
given orally for jaundice by traditional healers of Bahirdar zuria, Ethiopia (Muthuswamy and
Solomon, 2009). The dried root of Justicia schimperiana is traditionally will be use for
management of seizure in some areas of Ethiopia (Tamrat et al., 2015). The traditional healers
use this plant also as treatment agent for patient with rheumatism (Haile et al., 2008). The leaves
of Justicia schimperiana traditionally will be use for management of malaria and coccidiosis
(Getaneh et al., 2014). In addition to the above aliments, Justicia schimperiana is traditionally
will be use for treatment of bilirubinemia (Ivo et al., 2014) and intestinal parasites (Fisseha et al.,
2009)
 2.3. CHEMICALS, DRUGS AND MEDIA
The following chemicals, drugs and media (bacteriological and mycological media) were will be
use in the present study: absolute methanol and n- butanol (Carlo Erba reagents, France), ethyl
acetate (HiMedia Laboratories Pvt. Ltd., India), distilled water, sterile sheep blood
(Ethiopian Public Health Institute, Ethiopia), 0.5 McFarland equivalence/standards (Remel,
Lenexa Kansas 66215, USA), Dimethyl sulfoxide (DMSO) (Uni-Chem, India), resazurin
sodium salt (Serva Feinbiochemica Heidelberg, New York), Mueller-Hinton Agar (MHA),
Mueller-Hinton Broth (MHB), brain heart infusion agar, blood agar base, manitol salt agar and
XLD medium (Oxoid Ltd, Basingstoke, Hampshire, England), brain heart infusion broth
(Becton Dickinson and , Cockeysville), violet red Bile glucose agar (Research-lab Fine Chem.
Industries, India), Potato Dextrose Broth (PDB) (HiMedia Laboratories Pvt. Ltd. India),
Potato Dextrose Agar (PDA) (Sisco Research Laboratories Pvt. Ltd. India), ketoconazole
50µg/disc, nystatin 100 units/disc, ceftriaxone 10ug/disc ampicillin 10 μg/disc (Oxoid Ltd,
Basingstoke, Hampshire, England) and ciprofloxacin 5 μg/disc (Becton, Dickinson and
Company, USA). All the chemicals and solvents were of analytical grade.

2.4. . EXTRACTION OF PHENOLIC COMPOUNDS USING SOLVENTS

Scientists have studied and analyzed the impact of different types of solvents, such as methanol,
hexane, and ethyl alcohol, for the purpose of antioxidant extraction from various plants parts,
such as leaves and seeds. In order to extract different phenolic compounds from plants with a
high degree of accuracy, various solvents of differing polarities must be will be use. Moreover,
scientists have discovered that highly polar solvents, such as methanol, have a high effectiveness
as antioxidants.

Anokwuru et al. reported that acetone and N,N dimethylformamide (DMF) are highly effective
at extracting antioxidants, while Koffi et al. found that methanol was more effective in at a large
amount of phenolic contents from walnut fruits when compared to ethanol (Koffi, et al 2010, 5,
550–558) It has been reported that ethanolic extracts of Ivorian plants extracted higher
concentrations/amount of phenolics compared to acetone, water, and methanol (Koffi, et al 2010,
5, 550–558) Multiple solvents have been commonly will be use to extract phytochemicals, and
scientists usually employed a dried powder of plants to extract bioactive compounds and
eliminate the interference of water at the same time. Solvents will be use for the extraction of
biomolecules from plants are chosen based on the polarity of the solute of interest. A solvent of
similar polarity to the solute will properly dissolve the solute. Multiple solvents can be will be
use sequentially in order to limit the amount of analogous compounds in the desired yield. The
polarity, from least polar to most polar, of a few common solvents is as follows: Hexane <
Chloroform < Ethylacetate < Acetone < Methanol < Water.

2.5. MICROWAVE-ASSISTED EXTRACTION (MAE)

MAE has attracted the attention of researchers as a technique to extract bioactive compounds
from a wide variety of plants and natural residues (Anokwuru, et al 2011, 9, 53–61.).
Microwaves have electromagnectic radiation that occurs at frequencies between 300 MHz to 300
GHz, and wavelengths between 1 cm and 1 m. These electromagnetic waves consist of both an
electrical field and a magnetic field. These are described as two perpendicular fields. The first
application of microwaves was to heat up objects that can absorb a part of the electromagnetic
energy and convert it into heat. Commercial microwave instruments commonly use the
frequency 2450 MHz, which corresponds to an energy output of 600–700 Watts (Ballard, et al
2010, 120, 1185–1192). Recently, advanced techniques have become available to reduce the loss
of bioactive compound without increasing the extraction time. Therefore, microwave-assisted
extraction is demonstrated to be a good technique in multiple fields, especially in the medicinal
plant area. Moreover, this technique reduced the losses of the biochemical compounds being
extracted (Kingston, H.M.; Jessie, L.B. , 1998.) Microwave-assisted extraction (MAE) has been
will be use as an alternative to conventional techniques for the extraction of antioxidants because
of its ability to reduce both time and extraction solvent volume (Chem. Eng. Trans. 2013, 32,
1783–1788). In fact, the main objective of using MAE is to heat the solvent and extract
antioxidants from plants with a lesser amount of these solvents (Kingston, H.M.; Jessie, L.B. ,
1998.). Li et al. reported that conventional methods using various solvents presented less
antioxidant activity and phenolic content than MAE (Food Chem. 2012, 130, 928–936)
Therefore, the finding confirmed that MAE was more effective at increasing antioxidant activity
by measuring ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity
(ORAC), and total phenolic content (TPC). The efficiency of the microwave extraction can be
changed through some factors such as extraction temperature, solvent composition, and
extraction time. The extraction temperature was usually studied more than other factors due to its
ability to increase the efficiency of the microwave extraction. Tsubaki et al. reported that 170 C
was the most effective temperature for extracting phenolic compounds from Chinese tea. In
addition, Plants 2017, 6, 42 3 of 23 increasing the extraction temperature beyond this point
resulted in a reduced extraction yield (Food Biochem. 2004, 28, 113–122). Recently,
Christophoridou et al. will be use a new microwave-assisted extraction (MAE) process, which
converts energy to heat, thereby cooperating with solvents in order to extract a specific
compound (Food Biochem. 2004, 28, 113–122). Williams et al. showed many advantages of
MAE, including lower solvent consumption, shorter extraction times, and higher sensitivity
towards target molecules (Food Biochem. 2004, 28, 113–122).

2.6. PREPARATION OF EXTRACTS

Five hundred grams of coarse powder of shade dried leaves of Justicia Adhatoda was extracted
successively with petroleum ether (60-80°C), chloroform, ethyl acetate and methanol in soxhlet
extractor for 48 h. dark green residues were obtained after concentrating the extract under
reduced pressure (Yield 8.20%, 3.80%, 2.15% and 14.30% respectively). The obtained
extracts were stored in desiccators for further phytochemical and antimicrobial
investigations. The dried material was tested for its constituents by standard methods ( Harborne
JB,1973, 279). The plant extracts were diluted with respective solvents to the final
concentration of 20 mg/ml. Microorganisms like Escherichia coli, Klebsiella pneumonia,
Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris and Candida albicans
were will be use for testing.
 CHAPTER THREE
3. Materials and Methods
3.1. STUDY AREA
The leaves pericarp of the Adhatoda schimperiana plant will be collected from mountains located
at East Ethiopia (Arsi Zone). The plant material will be use in this study will be collected in july,
2020 from Oromia Region Arsi Zone Dodota Woreda, particular place called Dilfakar
mountaion, which is 50 km East of Arsi Zone and, 11 km from Dodota Woreda.

Dodota Woreda far from Addis Ababa 115 to the East part of the Country, The altitude of this
woreda ranges from 1400-2500 meters above sea level. The plant sample will be identified by
the authors. The plant specimen will be given a voucher number of ASC-007 and deposited in
the Biological Science Laboratory of chemistry, Department of Chemistry, University of Gonder.

The plant organ will be cut into small pieces and shade dried at room temperature (20 0C) for
three weeks, finely powdered plant materials will be stored in airtight polythene bags protected
from Sunlight until use.
 3.2. EXTRACTION AND ISOLATION

3.1.1. Extraction
215 g powder of air-dried leaf of Adhatoda schimperiana will be first soaked with 1L n-Hexane
for 3 days. After filtration the extract will be evaporated under reduced pressure and temperature
of 40 0C using rotary evaporator. The dried marc will be extracted with ethanol after soaking for
3 days at room temperature. The extract will be concentrated in rotary vapor and afforded 7 g
dark green residue which will be collected with distilled water and added to a separator funnel
with 215 ml diethyl ether to separate organic part and the aqueous parts. The filtrates will be
evaporated under reduced pressure using rotary evaporator and afforded 3.1gm green solid and
2.5 gm yellow solid for organic part and aqueous part respectively. When TLC will be developed
for the crude extract for the organic part on solvent chloroform: ethyl acetate (7:3 had shown
four colored spots and for aqueous part ethyl acetate: ethanol (7:3) had showed 6 colored spots.

3.1.2. ISOLATION
110 gm of silica gel will be measured and mixed with 200 mL of n-Hexane. Then the mixture
will be packed into a column, 2 ml of n-Hexane will be added to the 3.1 gm dried sample of the
crude extract. This concentrated sample will be then applied on to the top of packed silica gel
using a dropper. Elution will be done with 100 mL pure n- Hexane, followed by 100 ml n-
Hexane: CF (1:1), 100 mL pure CF 100 mL CF: EtOAc (1:1), 100 ml pure EtOAc and then 100
ml EtOAc: MeOH the elution stopped when the darkest part at top reached the bottom. A total of
30 fractions will collected (Table 3). Similarly, 2.5 g crude extract of aqueous part will be be
subjected to column chromatography elution with solvents systems; Chloroform, Chloroform:
Ethyl acetate, Ethyl acetate: Ethanol and Ethanol and a total of 20 fractions (Table 4) will
collected. As follows:

3.2. FRACTIONATION
3.2.1. ORGANIC PART FRACTIONATION

The fractions collected other than 14 will discarded because their TLC results did not how spots.
Fractions 14 showed pure spots. The solvent system will be use for the TLC examination of 14
will be CF: EtOAc (70:30), the fraction 14 will be left in a hood for 12hrs. While pale yellow
solid will be observed containing fraction 14. Fraction 14 as labeled compound ASH-14, its
amount will be 30 mg. The IR, NMR (1H, 13C, DEPT) and UV- VIS spectra for compound
ASH-14 will VIS spectra for compound ASH-14

Fraction Solve system Ratio Volume Remark

1. n-He 100 ml ASH_1
2. n-He Purely n- hexane ASH_2
3. n-He ASH_3
4. n-He ASH_4
5. n-He ASH_5
6. n-He: CF 100 ml ASH_6
7. n-He: CF 1:1 ASH_7
8. n-He: CF ASH_8
9. n-He: CF ASH_9
10. n-He: CF ASH_10
11. CF 100 ml ASH_11
12. CF Purely ASH_12
13. CF chloroform ASH_13
14. CF ASH-14
15. CF ASH_15

16. CF: EtOAc 100 ml ASH_16

17. CF: :EtOAc 1:1 ASH_17
18. CF: EtOAc ASH_18
19. CF: EtOAc ASH_19
20. CF: EtOAc ASH_20
21. EtOAc 100 ml ASH_21
22. EtOAc Purely ASH_22
23. EtOAc Ethyl acetate ASH_23
24. EtOAc ASH_24
25. EtOAc ASH_25
26. EtOAc: MeOH 1:1 100 ml ASH_26
27. EtOAc: MeOH ASH_27
28. EtOAc: MeOH ASH_28
29. EtOAc: MeOH ASH_29
30. EtOAc: MeOH ASH_1

3.2.2. ISOLATION OF COMPOUND ASH-14

3.2.2.1. PHYSICAL DATA
Nature: Compound ASH-14 is pale yellow solid with, Rf 0.58 (CF: EtOAc, 3.5:1.5), mp 167-170
C0
3.2.2. SPECTROSCOPIC DATA
IR Vmax (4000 cm-1 (KBr) to an olefinic system (1458 cm-1), the methyl C-H stretching in this
compound is indicated by a sharp peak at 2920 cm-1.). Three peaks near to 1625 cm-1, 1625 cm-
1 and 1462 cm-1 are indicative of aromatic ring.

1H NMR δ (400MHz, CDCl3), (Appendix 3): 5.40(t, 1H, H-3), 2.30(t, 1H, H-21), .8(d1H, H- 9),
2.20-1.15(m, 22H), 1.03(s, 6H, C-23 Me & C- 26 Me), 0.87(s, 6H, C-24 Me, C -25Me),13C
22.65 (C-24 & C-25), 25.52(C-16), 27.17(C-23 and C-26), 27.13 (C-15), 29.10 (C-29), 29.21 (C-
27), 29.49(C-20), 29.67 (C-21), 29.73 (C-7), 31.87 (C-22) 33.44 (C-6), 35.82 (C-19), 49.40 (C-
14, quaternary), 49.62 (C-13, quaternary), 49.83 (C-8, quaternary), 48.98 (C-17, quaternary),
48.98 (C-18, quaternary), 63.75 (C- 9), 116.17 (C-11), 118.44 (C-12), 128.83 (C-5 and C-10,
quaternary), 129.19 (C-2 and C-4) 129.71 (C-3) and 130.01 (C-30)

3.3.4. PHYTOCHEMICAL SCREENING

Each extract of Adhatoda schimperiana will be screened for the presence of various secondary
metabolites (phytochemicals) such as alkaloids, Polyphenols, Flavonoids, tannins, glycosides,
phytosterols/withanoids, saponins, terpenoids, Quinones. The methods of analysis employed will
those described by (Teferi G. 2003) for the presence of various active components

3.3.5. TEST FOR ALKALOIDS:

Extract 300 mg will be digested with 2 M HCl, and the acidic filtrate will be mixed with:
a) Wagner’s reagent; formation of brown
b) Hager’s reagent, presence of alkaloids confirmed by the yellow colored precipitate.
3.3.6. TEST FOR POLYPHENOL:
100 mg of extract in the test tube will be treated with 3% ferric chloride. The deep blue color of
solution shows the presence of phenol

3.3.7. TEST FOR FLAVONOIDS

a) Alkaline reagent test:
Extract will be treated with 10% NaOH solution, formation of intense yellow colour indicates
presence of Flavonoid.

b) Mg turning test:
Extract will be treated with Mg turning and add conc. HCl to this solution add 5 ml of 95%
ethanol, formation of crimson red colour indicates Flavonoid.

3.3.8. TEST FOR TANNINS:

 To an aliquot of extract (dissolved in water) 2 ml of sodium chloride will be (dissolved in
water) 2 ml of sodium chloride will be (dissolved in water) 2 ml of sodium chloride will be
added, filtered and mixed with 5 ml 1% gelatin (dissolved in water) 2 ml of sodium chloride
will be tannings

3.3.9. GLYCOSIDES: KELLER-KILLANI TEST:

Extract 100 mg treated with 2 ml glacial acetic acid containing a drop of FeCl3. A brown colour
ring indicates the presence of positive test.

3.3.10. TEST FOR PHYTOSTEROL: SALKOWSKI’S TEST:

Extract (150 mg) will be treated with chloroform and filtered. The filtrate will be treated with
few and filtered standing, appearance of golden red indicates the positive test

3.3.11. TEST FOR SAPONINS:

Extract (300 mg) will be boiled with 5 ml water for two minutes; the mixture will be cooled and
mixed vigorously and left for three minutes. The formation of froindicates the presence of
saponins

3.3.12. TEST FOR TRITERPENS:

Extract (300 mg) will be mixed with 5 ml chloroform and warmed for 30 minutes. Few rops of
concentrated sulphuric acid will be added and mixed well. The appearance of red color indicates
the presence of triterpens

3.3.13. TEST FOR QUINONE:

Addition of 200 mg extract with 5 ml hydrochloric acid result in yellow colored precipitate
denoting the presence of quinone

4. Time schedule
No Description of the activities to be Duration in (months)
performed
Activities March April May Jun July Augus Septe October
t mber
1. Selection of the research topic

2. Preparation of the proposal

3. Submission of the proposal

No Description of the activities to be Duration in (months)
performed
4. Data collection

5. Data editing and coding

6. Analysis of the data

7. Conclusion and recommendation

8. Submission of first draft of the report

9. Submission of second draft of the

report
10. Submission of final draft of the report

11. Presentation of the report and defense

5. Budget schedule
No
Description Quantity Unit cost in Birr Total cost in Birr
Stationary material
Paper 1 pack 200 birr/ pack 200
Pen 5 10 birr/pen 50
Pencil 5 4 birr/pen 20
Stapler 1 120birr 120
CDWR 4 45birr 180
1
Calculator (scientific) 1 500birr 500
Note Book 2 20Birr 40
Document Holder 1 20Birr 20
Bag Elico 1 1500Birr 1500

Others writing materials Different Different birr 245

2
Telephone cost & Internate Cost 50 card 100 birr 5000
Proposal 0
Proposal draft 60pages*2*0.75 90 90
3
Photo copy cost of questionnaire 4500pages 1birr/ page 4500
Report development
4 First draft 120 page 3*2birr/ page 720
Second Draft 120 page 3*2birr/ page 720
Final Draft 120 page 5*2birr/ page 720
5 Personal costs 0
No
Description Quantity Unit cost in Birr Total cost in Birr
Transportation cost 3month Per/day 50 Birr 4500
Sub total 19,130.00
6 Contingency 10% 1,913.00
Total estimated budget 21,043.00

6. References
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3. Ayehu A, Abebe D. Medicinal plants and enigmatic health practices of Northern and
 East Ethiopia Addis Ababa, Ethiopia, 1993.
4. Abebe D, Ayehu A. Medicinal plants and enigmatic health practices of Northern and
East Ethiopia. Berhanina Selam Printing Enterprise, Addis Ababa, Ethiopia, 1993; 341.
5. Murthy PN, Moges G, Hymete A, Gebremariam T. Antimicrobial and phytochemical
screening of Justicia Schimperiana. Eth Pharm 1993; 11:47-53.
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Development, In Proceedings of the Workshop on Development and Utilization of
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2000, 107‐111.
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Analysis. Edn 3, Chapmnan & Hall, London, UK, 1998,60-66
11. Harborne JB, Phytochemical Methods: A guide to modern techniques of plant
analysis, Chapman and Hall, New York, 3rd Edition, 1973, 279
12. Koffi, E.; Sea, T.; Dodehe, Y.; Soro, S. Effect of solvent type on extraction of
polyphenols from twenty three
13. ivorian plants. J. Anim. Plant Sci. 2010, 5, 550–558.
14. 12. Anokwuru, C.P.; Anyasor, G.N.; Ajibaye, O.; Fakoya, O.; Okebugwu, P. Effect of
extraction solvents on
15. phenolic, flavonoid and antioxidant activities of three nigerian medicinal plants. Nat.
Sci. 2011, 9, 53–61.
16. 13. Ballard, T.S.; Mallikarjunan, P.; Zhou, K.; O’Keefe, S. Microwave-assisted
extraction of phenolic antioxidant
17. compounds from peanut skins. Food Chem. 2010, 120, 1185–1192. [CrossRef]
18. 14. Kingston, H.M.; Jessie, L.B. Introduction to Microwave Sample Preparation;
 American Chemical Society:
19. Washington, DC, USA, 1998.
20. 15. Suzara, S.; Costa, D.A.; Gariepyb, Y.; Rochaa, S.C.S.; Raghavanb, V. Spilanthol
extraction using microwave:
21. Calibration curve for gas chromatography. Chem. Eng. Trans. 2013, 32, 1783–1788.
22. 16. Li, H.; Deng, Z.; Wu, T.; Liu, R.; Loewen, S.; Tsao, R. Microwave-assisted
extraction of phenolics with
23. maximal antioxidant activities in tomatoes. Food Chem. 2012, 130, 928–936.