ISOLATION AND CHARACTERIZATION OF

COMPOUNDS WITH MEDICINAL VALUE

An Experimental Report Preente! to"
DEPARTMENT OF CHEMICAL AND PROCESS EN#INEERIN#
SCHOOL OF EN#INEERIN#
MOI UNIVERSIT$
In partial %&l%ilment o% t'e re(&irement %or t'e De)ree o% *a+'elor o% En)ineerin)
In
C'emi+al , Pro+e En)ineerin)
A*RAHAM -OVA .........../////
CPE01231024
MIRIAM DAN#ASU- ............/
CPE01221024
HARUNAN$ M/ SA*IR ............/
CPE056024
ISLAM HASSAN ............/
CPE01257027
LULE -HAMIS OMAR ............/
CPE01218024
MARAMI *EN9AMIN ............/
CPE011024
SUPERVISORS"
DR/ AM*ROSE -IPROP ............//
MR/ MILTON M:ARIMI ............//
52
t'
Mar+'; 3211

Moi University
ii
A<tra+t
Many plants accumulate organic substances in quantities sufficient to be economically
useful as chemical feedstock or raw materials for various scientific, technological, and
commercial applications. Economically useful organic substances serve as sources of
industrial oils, resins, tannins, saponins, natural rubber, gums, waxes, dyes,
pharmaceuticals, and many specialty products. lant chemicals are often classified as either
primary or secondary metabolites. rimary plant metabolites are substances widely
distributed in nature, occurring in one form or another in virtually all organisms. !econdary
plant metabolites are compounds biosynthetically derived from primary metabolites and are
more limited in distribution in the plant kingdom. !econdary metabolites are frequently
accumulated by plants in smaller quantities than are primary metabolites. !econdary plant
metabolites present a broad range of medicinal properties.
"he main ob#ective of our study is to extract then isolate and characteri$e compounds with
medicinal value. Annona squamosa is a plant which is grown locally and known as
matomoko and also known as sugar apple or sweet sop in English. "he experiment seeks to
extract, isolate and characteri$e the components present in Annona squamosa using
different analytical techniques. %mong them are& 'as chromatography(mass spectrometry
)'*(M!+ and ,ourier transform ( -nfra red ),"(-.+ spectroscopy.
i
Ta<le o% Content
Abstract.......................................................................................................................i
Table of Contents.......................................................................................................ii
List of Tables and Figures........................................................................................iv
Chapter 1: Introduction..............................................................................................1
1.1 Executive summar..........................................................................................1
1.! "b#ective..........................................................................................................!
Chapter !: Literature $evie%.....................................................................................&
!.1 'cientific classification.....................................................................................(
Chapter &: )ethodolog..........................................................................................1!
&.1 Extraction.......................................................................................................1!
3.1.1 Efficiency of the extraction of the active components of a plant......................16
3.1.2 Sampling of plant materials................................................................................16
3.1.3 Drying and crushing of the plant materials.......................................................16
3.1.4 Solvent extraction using Soxhlet apparatus.......................................................17
3.1. Solvent extraction using !.S.E...........................................................................17
3.1.6 Solvent recovery..................................................................................................1"
&.! Isolation..........................................................................................................!*
3.2.1 #hin $ayer %hromatography..............................................................................2&
3.2.2 %olumn %hromatography...................................................................................2
&.& Characteri+ation.............................................................................................!,
3.3.1 'nfrared spectroscopy..........................................................................................2"
Chapter (: $esults and -iscussions.......................................................................&1
(.1 Extraction.......................................................................................................&1
(.! Isolation..........................................................................................................&!
4.2.1 #hin layer chromatography (rocedure and )*servation..................................32
4.2.1.1 Discussions.......................................................................................................3
4.2.2 %olumn chromatography....................................................................................36
ii
(.& Characteri+ation.............................................................................................&.
4.3.1 !#+,-#'+ for !nnona s.uamosa leave/ dichloromethane 0D%12 extract.....36
Chapter /: Conclusion and $ecommendations......................................................&,
Appendix..................................................................................................................(*
Appendix 1: 'ome of the structures of Annona '0uamosa compounds.............(*
.................................................................................................................................(*
Appendix !: 1ictures of Analsis..........................................................................(!
.................................................................................................................................(!
iii
Lit o% Ta<le an! Fi)&re
Table !.1: 'cientific classification of sugar apple or s%eet sop................................(
Figure !.1: The fruits of Annona '0uamosa.............................................................(
Figure &.1: 1repared TLC plate..............................................................................!1
Figure &.!: -iagram sho%ing the plate after the solvent has moved half %a up it.
.................................................................................................................................!1
Figure &.&: TLC plate under 23 light.....................................................................!!
Figure &.(: Chemicall spraed chromatogram......................................................!&
Figure &./: 'ilica structure.......................................................................................!(
Figure &.. 1repared column for chromatograph...................................................!.
Figure &.4 A 'ample column...................................................................................!.
Figure &.5 Column chromatograph process..........................................................!4
Figure &., A multiple reflection AT$ sstem.........................................................&*
Figure (.1: TLC plate using solvent sstem of 1:16 methanol: hexane...................&&
................................................................................................................................&(
Figure (.!: 'pot 7 TLC sample on a !:1 hexane8 methanol sstem......................&(
................................................................................................................................&(
Figure (.&: 'pot A TLC sample on a 1:! hexane methanol sstem.......................&(
Figure (.(: A TLC sample on a chloroform sstem.................................................&/
Figure (./: A TLC sample on a Chloroform: 9exane sstem.................................&/
Figure (.. AT$:FTI$ spectrums for -C) crude Annona s0uamosa leave extract&5
iv
C'apter 1"/////////////////////////////////////////////////////////////////////////////////////Intro!&+tion
1.1 Executive summary
"he use of secondary metabolites to treat diseases such as cancer or human immune
deficiency virus )/-0+ has been impeded, in part, by the difficulty associated with
synthesi$ing secondary plant metabolites, using conventionally industrial chemical
techniques. 1ecause secondary plant metabolites often have highly complex structures with
many chiral centres that may impart biological activity, such complex compounds cannot
by synthesi$ed economically. %s a result, there is a need for an inexpensive, efficient, bulk
method for selectively extracting secondary metabolites from plants. .esearch reveals that
plants of the Annona genus have been used for medicinal purposes, including curing of
cancer, for thousands of years, but it is only in the last two decades that they have attracted
conventional researchers. Many folk remedies are based on the isolation and purification of
secondary metabolites from trees, shrubs, and flowers. .ecently, some plant secondary
metabolites have been found to exhibit cancer(inhibiting activity, or other activity related
to inhibiting diseases. ,or example, camptothecin, colchicine, docetaxel, etopside,
paclitaxel, podophyllotoxin, tetrahydrocannabinol, topotecan, vinblastine, vincristine,
vindesine, betulinc acid, as well as others, have been found to have anticancer activity.
-t is therefore incumbent to appreciate the relevant research work already carried out and
findings obtained on the present sub#ect. "he growing demand for herbal medicine has
reached an alarming rate in 2enya, with some herbalists claiming that they can cure even
terminal and chronic conditions like /-0(%-3!, ulcers and hypertension. "he 2enya
pharmaceutical and poisons board, and the 2E1! are responsible for regulating such
quacks. !uch herbal drugs that are known to cure the stated ailment or conditions should
have documented evidence from medical #ournals and reports. /erbal medicine has grown
to great lengths with even chartered universities offering degrees in herbal medicine. "he
%sian continent leads in such universities. Most medicines in the market are extracts from
plants or other living organism.
4
"he challenge lies in the identifying and isolation of the active healing ingredient from the
plant or organism. Most traditional herbal treatment has been researched on to later pave
the way for modern medicines. "he neem tree and aloe(vera plant extracts are examples of
traditional herbal treatments that have been extensively experimented on and documented.
Extracts of these herbs can now be found in soaps, lotions, food supplements and other
numerous products.
1.2 )*3ective
"he ob#ective of our study is to extract then isolate and characteri$e compounds with
medicinal value from the Annona squamosa plant, which is grown locally and also known
as matomoko and sugar apple or sweet sop in English. "he experiment seeks to extract,
isolate and characteri$e the components present in Annona squamosa using different
analytical techniques.
5
C'apter 3" Literat&re Re=ie>
"he most widely grown of all the species of Annona, is the sugar apple, A. squamosa.
Annona is a genus of flowering plants in the pawpaw6sugar apple family, %nnonaceae. -t is
the second largest genus in the family after 'uatteria, containing approximately 447
species of mostly 8eo(tropical and %fro(tropical trees and shrubs. Mainly because of its
abundance in the %ma$on rainforest and proximity to ma#or research institutions in the U!,
the %ma$onian variety has received the biggest share of academic research and commercial
attention. !everal annonaceaeous species have been found to contain acetogenins, a class of
natural compounds with a wide variety of biological activities ),eras et al., 499:). "hese
compounds are discussed later on.
0arious parts of this plant are utili$ed in various ways in different regions of the world. %
paste of the seed powder may be applied to the head to kill lice but must be kept away from
the eyes as it is highly irritating and can cause blindness. -f applied to the uterus, it induces
abortion. /eat(extracted oil from the seeds has been employed against agricultural pests. -n
Mexico, the leaves are rubbed on floors and put in hen;s nests to repel lice. Moreover, the
plant has been particularly effective for medicinal purposes )%ma$on /erb *ompany). -n
-ndia the crushed leaves are sniffed to overcome hysteria and fainting spells. "hey are also
applied on ulcers and wounds and a leaf decoction is taken in cases of dysentery.
"hroughout tropical %merica, a decoction of the leaves alone or with those of other plants
is imbibed as an emmenagogue, febrifuge, tonic, cold remedy, digestive, or clarifier for
urine. "he leaf decoction is also employed in baths to alleviate rheumatic pain. "he green
fruit, very astringent, is employed against diarrhoea in El !alvador. -n -ndia, the crushed
ripe fruit, mixed with salt, is applied on tumours. "he bark and roots are both highly
astringent. "he bark decoction is given as a tonic and to halt diarrhoea. "he root, because
of its strong purgative action, is administered as a drastic treatment for dysentery and other
ailments ('leye et al., 499<).
"he matomoko fruits that grow in the drier parts of the country are much smaller compared
to recent hybrids that grow in higher areas in parts of Murang=a and 2irinyaga in *entral
2enya.
<
2.1 Scientific classification
"he scientific classification of sugar apple or sweet sop as common known is as shown in
ta*le 2.1 below. "he fruits of the plant are as shown in figure 2.1 below.
2ingdom Plantae
>rder Magnoliales
,amily Annonaceae
'enus Annona
!pecies A. squamosa
1inomial name Annona Squamosa
#a*le 2.14 Scientific classification of sugar apple or s5eet sop
-igure 2.14 #he fruits of !nnona S.uamosa.
>ther species include? %temoya )A. cherimoya and A. squamosa+, *herimoya )A.
cherimoya+, !our sop )A. muricata+, *ustard apple )A. reticulata+, A. crassilora, A.
sal!mannii, Annona glabra.
@
Sweet sop is a small, upright evergreen tree, ABC m high, with large, glossy, dark green
leaves. -t produces a large, heart(shaped, edible fruit that is 4AB57 cm in diameter, is
yellow(green in colour, and has white flesh inside. "he fruit is mature and ready for
consumption when it feels slightly soft and is light green externally. "he skin is thin and is
covered with conical nibs. "he pulpy flesh, which contains #uice, is peppered with small
black inedible seeds, and has a pleasant, sweet(acidic taste. !ugar apple contains no
sodium, they are high in carbohydrates and rich in calcium, vitamin * and phosphorus, and
with a sugar content of about A7(A7 )glucose and sucrose+. "his fruit is also considered as a
good tonic in Ayurveda. -t enriches blood and is known to increase muscular strength.
)8=gouemo et al., 499D, ,eng et al., 49C5+.
"he plant components are used medicinally to treat a wide range of illnesses. "hese
include&
? "he seeds, which have emetic properties, can be used to stop vomiting.
? "he seeds when immersed in coconut oil are a traditional treatment for head and body
lice. "he seed is also crushed into powder and can be applied on head to kill lice in hair.
? "he leaves served as a purgative.
? 1ark decoction is used to stop diarrhoea.
? 3ecoction of the leaves and6or root is taken in cases of dysentery.
? 3ecoction of the leaves is good to cure diabetes.
? "he leaves are applied to abscesses and open wounds and used to cure skin itches.
? "he crushed fresh leaves can be applied on skin eruptions to promote healing.
? "he crushed leaves are sniffed to overcome fainting spells and hysteria.
? "he mashed, ripe fruit, mixed with salt, is applied on tumours.
? 3ecoction of the leaves is used to aid digestive problem, and to treat colds.
? 3ecoction of the leaves is employed in baths to alleviate rheumatic pain.
A
? 3ecoction of the leaves is used to clarify urine.
? "he leaf decoction is effective for head lice and bedbugs.
? "he #uice of the fruit can be taken orally as a remedy for urethritis, haematuria, liver
ailments and hypertension )high blood pressure+. "he #uice when taken when fasting, it is
believed to relieve liver ailments and leprosy.
? "o speed the healing of wounds, the flesh of the sour sop is applied as a poultice
unchanged for < days.
? % decoction of the young shoots or leaves is regarded as a remedy for gall bladder
trouble, as well as coughs, catarrh, diarrhoea, dysentery, fever and indigestion.
? Mashed leaves are used as a poultice to alleviate ec$ema and other skin problems and
rheumatism.
? "he root bark is used as an antidote for poisoning.
? !our sop flowers are believed to alleviate catarrh )United !tates atent+ )'leye et al.,
499<+.
Moreover, the plant has been known to have no definite side effects.
Many active compounds and chemicals have been found in 'raviola, as scientists have
been studying its properties since the 49@7s. Most of the present research on 'raviola
focuses on a set of chemicals called Annonaceous acetogenins. "he seeds of the !ugar
apple have been found to be acrid and poisonous. 1ark, leaves and seeds contain the
alkaloid, anonaine. !ix other aporphine alkaloids have been isolated from the leaves and
stems& corydine, roemerine, norcorydine, norisocarydine, isocorydine and glaucine.
%porphine, norlaureline and dienone may be present also. !tudies have shown that the
extract of the seeds have no residual toxicity after 5 days. /igh concentrations are potent
for 5 days and weaken steadily, all activity being lost after : days ),eras et al., 499:+.
C
%ccording to an extract from an article posted on the 3aily 8ation newspaper in the
previous year, a close relative of a well(known fruit tree found in Ukambani, "aita and 0oi
could hold the key to a cure for some of the world=s untreatable cancers. "he Annona
species has shown dramatic success in destroying lung, breast, prostate, colon, and liver,
ovarian, cervical, breast, bladder and skin cancerous cells. >ther members of the tree
family have been found to contain the chemical ingredient Annonaceous acetogenins,
which is responsible for anti(cancer activity. "his chemical ingredient has been shown in
laboratory tests to have strong anti(tumour properties even when administered in very small
quantities. !everal of the studies indicate that the active ingredient in the fruit, Annonaceus
acetogenins, must be administered in very small doses otherwise it could be dangerous.
!ome of the studies also warn that continued use of the compound could lead to the
development of early arkinson=s disease, a degenerative disorder of the central nervous
system. "his has been discovered to be particularly the case when people use the crushed
seed instead of the leaves, bark or the fruit pulp recommended by researchers )"he 3aily
8ation, !eptember @ 5779+.
"he principal interest in this plant is because of its strong anti(cancer effects. %lthough it is
effective for a number of medical conditions, it is its anti(tumour effect that is of most
interest. 1esides being a cancer remedy, "raviola is a broad spectrum antimicrobial agent
for both bacterial and fungal infections. -t is effective against internal parasites and worms,
lowers high blood pressure and is used for depression, stress and nervous disorders.
.esearch shows that with extracts from this tree it may be possible to&
? %ttack cancer safely and effectively with an all(natural therapy that does not cause
extreme nausea, weight loss and hair loss.
? rotect one=s immune system and avoid deadly infections.
? ,eel stronger and healthier throughout the course of the treatment.
(1oost energy and improve one=s outlook on life )adma et al., 5774+.
"he source of this information is from one of %merica;s largest drug manufacturers.
D
Extracts from the tree were shown to&
? Effectively target and kill malignant cells in 45 types of cancer, including colon, breast,
prostate, lung and pancreatic cancer.
? "he tree compounds proved to be up to 47,777 times stronger in slowing the growth of
cancer cells than %driamycin, a commonly used chemotherapeutic drug.
? Unlike chemotherapy, the compound extracted from the 'raviola tree selectively hunts
down and kills only cancer cells. -t does not harm healthy cells.
"he 8ational *ancer -nstitute of %merica performed the first scientific research in 49DC.
"he results showed that 'raviola;s leaves and stems were found effective in attacking and
destroying malignant cells. !ince 49DC, 'raviola has proven to be an immensely potent
cancer killer in 57 independent laboratory tests, yet no double(blind clinical trials were ever
initiated.
% study published in the Eournal of 8atural roducts? following a recent study conducted at
*atholic University of !outh 2orea stated that one chemical in 'raviola was found to
selectively kill colon cancer cells at 47,777 times the potency of %driamycin )the
commonly used chemotherapy drug+.
"he most significant part of the *atholic University of !outh 2orea report is that 'raviola
was shown to selectively target the cancer cells, leaving healthy cells untouched, unlike
chemotherapy, which indiscriminately targets all actively reproducing cells )such as
stomach and hair cells+, causing the often devastating side effects of nausea and hair loss in
cancer patients. (http#$$%%%.annieappleseedpro&ect.org$graviola.html)
% study at urdue University recently found that leaves from the 'raviola tree killed
cancer cells among six human cell lines and were especially effective against prostate,
pancreatic and lung cancers. -n 499D, urdue University published information with
promising news that several of the %nnonaceous acetogenins were not only effective in
killing tumours that have proven resistant to anti(cancer agents, but also seem to have a
special affinity for such resistant cells. ),eras et al., 499:+
:
urdue researchers reported that the acetogenins preferentially killed multi(drug(resistant
cancer cells by blocking the transfer of %", the chief source of cellular energy, into them.
% tumour cell needs energy to grow and reproduce, and a great deal more to run its pump
and expel attacking agents. 1y inhibiting energy to the cell, it can no longer run its pump.
Fhen acetogenins block %" to the tumour cell over time, the cell no longer has enough
energy to operate sustaining processes and it dies. 8ormal cells seldom develop such a
pump? therefore, they don;t require large amounts of energy to run a pump and, generally,
are not adversely affected by %" inhibitors. urdue researchers reported that 4@ different
acetogenins tested thus far demonstrate potent %"(blocking properties )including several
found only in graviola+. "hey also reported that 4< of these 4@ acetogenins tested were
more potent against multidrug resistance )M3.+ breast cancer cells than all three of the
standard drugs )adriamycin, vincristine, and vinblastine+ they used as controls.
3/3 Iolate! Compo&n!
%nnonaceous acetogenins, a rather new class of natural compounds only isolated from the
%nnonaceae, are usually *
<A
( *
<D
fatty acid derivatives connecting a variable number of
"etrahydrofuran )"/,+ or "etrahydropryan )"/+ rings and lactone terminal moiety.
!o far, more than three hundred compounds, most of which were steric isomers, have been
found and published, and their more biological activities, such as cytotoxic, antiparasitic,
insecticide and immunosuppressive activities, have been further proved )Eiang et al., 499D+.
*urrently, there have been more than forty annonaceous acetogenins isolated from the
stems, leaves and seeds of this plant. -n the previous study of annonaceous acetogenins
from Annona muricata )Gi;s et al., 499:+ three annonaceous acetogenins, muricatin %,
muricatin 1, and muricatin *, were found from the extract of the stem bark. -n those
annonaceous acetogenins, four known compounds, muricatetrocin %, muricatetrocin 1,
corossolin, and corossolone, show special selective cytotoxicities against hepatoma cell
lines, /ep '5
,
and 5,5,4A. "hese four compounds are discussed with seven newly
discovered annonaceous acetogenins in the detailed description in cytotoxicities of curing
hepatoma ),eras et al., 499:+.
9
*hromatography of a hexane extract of Annona muricata seeds yielded two new H(lactone
acetogenins, epomuricenins % and 1, which were the first examples of annonaceous
acetogenins having an epoxide and a double bond in the aliphatic chain in place of the
usual tetrahydrofuran moiety ).oblot et al., 499<+.
"ucupentol, a novel mono(tetrahydrofuranic acetogenin from %nnona montana, as a potent
inhibitor of mitochondrial complex - )*olom et al., 5779+. "en acetogenins, one of them
new, were isolated from leaves and twigs of a 1olivian collection of Annona montana. "he
new compound that was named tucupentol is a mono(tetrahydrofuran(pentahydroxy(
acetogenin. "he inhibitory potency of tucupentol on the mitochondrial complex - was
evaluated, and this activity was compared with that of the known acetogenins, annonacin(
%, cis(annonacin(47(one, aromin, and gigantetronenin, also isolated from this plant
material. "he mentioned acetogenins acted as selective inhibitors of mitochondrial complex
- in the 7.:(A.@(nM range ).ieser, 499C+.
!tructure(activity relationships of diverse annonaceous acetogenins against human tumour
cells has been investigated )*olom et al., 5779+.
"welve annonaceous acetogenins with different stereo(chemical structures and
configuration were selected to test for their inhibitions on the growth of /ela, !MM*(
DA@4, !'*(D974, M*,(D and %(A@7: tumour cell lines. "his was the first to
simultaneously investigate effects of structural factors of stereo(chemical structures and
configuration on cytotoxicities with structure(activity relationship. "he particular study
showed that cytotoxic selectivities of acetogenins with threo6trans6threo6trans6erythro
stereo(chemical arrangement were gently more active than those with
threo6trans6threo6trans6threo stereo(chemical arrangement, and %*'s with cis "/, ring
partly produced notable cytotoxic selectivities. ,urthermore, acetogenins with !
configuration at *(5@ exhibited gently more cytotoxic selectivities potency than those with
. configuration at *(5@ )United !tates atent+.
47
% methylene chloride extract of the pulp of Annona muricata '. was fractionated in search
for scarcely functionali$ed %nnonaceous acetogenins )type E+. reviously known *(<A and
*(<D mono(epoxy unsaturated compounds, epomuricenins(% and (1 )4I5+ and
epomusenins(% and (1 )<I@+, were obtained. "wo new mono(epoxy saturated *(<A
representatives, epomurinins(% and (1 )AIC+ were also isolated )Melot et al., 5779+.
,rom these researches, we can clearly state that most emphasis has been given on the
acetogenins due to their anti(cancer effect. *ancer cure has evaded medical researches only
analogous to the /-0(%-3! virus. !hould there be a ma#or breakthrough in the isolation of
the ma#or anticancer acetogenin, this will mean a new phase in the micro(biology
fraternity.
%part from the cloning invention, the microbiology world has been overshadowed with the
physics world.
44
C'apter 5" Met'o!olo)@
3.1 Extraction
Extraction, as the term is used pharmaceutically, involves separation of medicinally active
portions of plant or animal tissues from the inactive or inert components by using selective
solvents by appropriate extraction technology. "he products so obtained are relatively
impure liquids, semisolids or powder intended only for oral or external use. "here are
several methods of extraction that are normally used but the most common ones are&
i/ Giquid(liquid extraction
ii/ Giquid(solid extraction
"he most commonly used type of extraction is the liquid(solid extraction because the plant
material is solid while the solvent used to extract the %ctive -ngredients )%-+ is a liquid.
"he most common type of extraction is the solvent extraction. % Jsolvent extractionK is a
type of extraction wherein a mixture of components adsorbed from plant tissue are
separated utili$ing the differences in the solubilities and adsorption strengths of the
components that are separated.
,or the extraction of medicinal value extracts from plants, it is prudent to have knowledge
on whether the extract in question is acidic or basic. % method for selectively extracting
one or more non(acidic compounds from plant tissue in the presence of one or more acidic
compounds, the method comprises&
i/ *ontacting a mixture of a basic component and a first solvent with the plant tissue
to immobili$e the acidic compound as a salt on the plant tissue.
ii/ *ontacting the plant tissue with a second solvent suitable to remove the one or more
non(acidic compounds? thereby effectively providing a solution comprising the one
or more non(acidic compounds.
lant tissue contains both acidic and non(acidic compounds. "his complicates extraction
processes employed to isolate acidic compounds or basic compounds from plant tissue.
45
"herefore, the extraction method chosen should be capable of selectively extracting a wide(
range of plant materials.
"he following are some of the general methods of extraction of plant materials )samples+
with medicinal value&
aA Ma+eration" "he coarsely powdered plant sample is kept in contact with the solvent in
a stoppered container for a defined period with frequent agitation until soluble matter is
dissolved. "he mixture is strained, then the marc pressed and the combined liquid
filtered.
<A Di)etion"?"his is similar to maceration but gentle heat is used during the process of
extraction. "his method is employed when moderately elevated temperatures are not
ob#ectionable as the efficiency is increased.
+A Per+olation" ,or tinctures and fluid extracts. % percolator used is a narrow, cone
shaped vessel open at both ends. "he powdered sample is moistened with an
appropriate amount of the specified menstruum and allowed to stand for @ hrs in well
closed container. "he moistened mass loosely is packed in percolator and covered.
%dditional menstruum is added to give shallow layer above the mass and allowed to
macerate for 5@ hours. "he outlet of the percolator is opened and liquid allowed
dripping slowly while the additional menstruum is being added as required, until the
percolate measures about three quarter of the required volume. "he Marc is pressed,
liquid added to the percolate and sufficient menstruum is added to the required volume
and filtered.
!A In%&ion" -nfusion is dilute solution of the readily soluble constituents of the crude
sample. ,resh infusion is prepared by macerating the sample for a short period of time
with cold or hot )boiling+ water.
eA De+o+tion" owdered samples boiled in specified volume of water for defined time,
cooled and strained6filtered. !uitable for extracting water(soluble, heat stable
constituents.
4<
%A Co&nter +&rrent extra+tion BCCEA" .aw material in wet condition is pulverised using
toothed disc disintegrators to produce fine slurry. !lurry is moved in one direction
within a cylindrical extractor where it comes in contact with the extracting solvent
falling against it. "he further the starting material moves, the more concentrated the
extract becomes. *omplete extraction is possible with optimi$ation of quantities of
solvent and the material and their flow rates. "he process is efficient requiring least
time with no risk of high temperature.
)A S&per+riti+al %l&i! extra+tion" "he critical point of a pure substance is defined as the
highest pressure and temperature at which it exists in vapour(liquid equilibrium. %t
pressure and temperature above this point, single homogenous fluid which forms is said
to be supercritical. % substance in supercritical phase is neither a true liquid nor a true
gas and has some properties of each. !upercritical fluids can dissolve wide variety of
organic compounds and their solvent power can be raised near their critical points by
small pressure and temperature changes. %t high temperature and low pressure, the
density is low and the supercritical fluid behaves more like a gas. 1ut at low
temperature and high pressure, the density increases and it assumes the properties of a
liquid. !upercritical carbon dioxide )s*>
5
+ is considered to exist at pressure above
D<.:bar and temperatures above <4.5
7
*. "he process is simple where powdered sample
is kept in the extractor. *>
5
under high pressure passing through pre(heater enters the
extractor and passes through the sample. .eduction valve )back pressure regulator+
allows entry of the extracted material into the separator. .eduction in pressure converts
the liquid into *>
5
gas which through condenser enters the *>
5
reservoir from where,
it is recycled. "he extract is taken out of the separator.
'A Ultrao&n! extra+tion Boni+ationA" Use of ultrasound with frequencies ranging from
57 2/$ to 5777 2/$ increases permeability of cell wall to produce cavitations.
owdered crude sample is sonicated with an appropriate solvent to extract out solvent
soluble components )e.g. (a%olia serpentina extract+. 3eleterious effect of ultrasound
energy )more than 57,777 /$+ on the active constituents of the medicinal plant is
possible through formation of free radicals and consequently in the sample molecules.
4@
iA A(&eo& al+o'oli+ extra+tion <@ %ermentation" !oaking of the sample, either in
powder form or dried aqueous extract L)asayaK for defined period of time, during
which it undergoes fermentation generating alcohol in situ, thus facilitating extraction
of active constituents contained in the sample. %lcohol thus generated, also serves as a
preservative. ,ermentation is done in earthen vessels or porcelain #ars6steel tanks.
CA Hot Contin&o& Extra+tion BSox'letA" "his makes use of a soxhlet apparatus. %
finely powdered sample is placed in a porous bag )thimble+ placed in the chamber of
the soxhlet apparatus. *ondensed solvent drips into the thimble containing sample,
extracting it by contact. "he process is continued until a drop of solvent from the
siphon tube, when evaporated, does not leave a residue.
-t is the most cost efficient method in terms of the use of the solvent. -ts advantages
include&(
( Garge amount of sample can be extracted with much smaller quantity of solvent,
( "remendous economy in terms of time, energy and ultimately financial inputs,
( !mall scale use a batch(process,
( 1ecomes more economical when converted into continuous extraction procedure on large
scale.
-n our experimental work, we used solid(liquid extraction. Fe used different methods of
extraction, one being the hot continuous extraction method using a soxhlet extractor which
we decided to start with. "he present extraction procedure is therefore advantageous for
many commercial industries, including pharmaceutical industries. ,or example,
undesirable acidic components may be present in natural extracts along with desirable non(
acidic compounds. "hese acidic components may not only have little or no therapeutic
utility, but many mammals )e.g., humans+ may have adverse reactions to these undesirable
acidic components. "he acidic compounds, such as betulinic acid, may also be very
desirable. "he method of the present invention can be used to selectively extract non(acidic
and acidic compounds from plant tissue, wherein the non(acidic and acidic compounds are
essentially free of acidic and non(acidic compounds respectively.
4A
5/1/1 E%%i+ien+@ o% t'e extra+tion o% t'e a+ti=e +omponent o% a plant
0ariation of constituents in extracts can result from non(standardi$ed processes of
extraction. Extraction process affects physical as well as internal composition of essential
oils/ External appearance can result in re#ection of the batch even if analytical results are
within acceptable limits/ "he process of extraction determination produce batches with
quality as consistent as possible )within narrowest possible range+. Essential oils are
evaluated by their olfactory response in the hands of experienced perfumers, which
supersedes its analytical results.
5/1/3 Samplin) o% plant material
lant materials were collected from 2enya %gricultural .esearch -nstitute courtesy of the
8#oro station in 8akuru *ounty. "he collected plant materials were& leaves, fruits and bark.
"he sampling is categori$ed as either young or old. ,or our case we used samples from a
tree that was A years old. Fe harvested the plant materials from different parts of the plant
apple and we concentrated on the young parts of the plant. "he main reason is because
most of cell activity is high at the young parts of the tree to encourage the growth of the
tree. "his would suggest that most of the active components are in the young parts of the
tree. Fe did the harvesting with utmost care to prevent damaging, hurting or killing of the
tree because we would want to do experiments on them once they are older then compare
the results from both the young and old tree thus determining when the active ingredients
are more. "his would make future works easier either by the same extractions or any
developments. "he sampling of the plant materials is the same for all the extraction
procedures. %fter harvesting, the plant materials are prepared for transportation. "his is
basically wrapping them to avoid any U0(radiation into the samples.
5/1/5 Dr@in) an! +r&'in) o% t'e plant material
"his determines the process to be used. "here are two processes normally used either the
dry process or the wet process for extraction. Fet process involves dipping6soaking the
plant material into the solvent of extraction. "his is not a very efficient method of
extraction because the constituents of the %ctive -ngredients )%-s+ in the plant samples are
very minute and requires precision and time.
4C
,or most plant extracts the most efficient process is the dry process. -t involves drying of
the plant samples then crushing them and proceeding with the extraction procedure. ,or our
case, the plant materials are then dried, away from direct sunlight but in ambient conditions
to prevent any further photosynthesis from happening and kept away any U0 rays from
coming into contact with the plant materials. %fter drying, the plant materials were then
crushed. article reduction is done so as to increase the surface area for extraction. "he
crushed particles were then sieved through a 5mm mesh and the bigger particles re(crashed
again. "he crushed samples are then ready for extraction.
5/1/8 Sol=ent extra+tion &in) Sox'let apparat&
"he extraction process requires care and precision to prevent contamination of the extract
because of the very low contents of the %-s in the sample. "he choice of the solvent is also
very important. 3etermination of whether a solvent is polar or non(polar also affects the
analysis of the process. ,or our case we chose to use hexane as a solvent. "his is due to its
availability and its non(polar nature. "his solvent extraction is basically the method of
using a soxhlet extractor. "he finely powdered sample was placed in a thimble and placed
in the chamber of the soxhlet apparatus while the solvent was placed into the flask at the
bottom flask then heated. *ondensed solvent dripped into the thimble containing the
crushed plant sample, and extracted it by contact. "he process was continued until a drop
of solvent from the siphon tube, when evaporated, did not leave a residue. -t is a batch wise
procedure and we managed to extract two batches.
5/1/6 Sol=ent extra+tion &in) A/S/E
%.!.E is %ccelerated !olvent Extraction. "he aim of any extraction technique in analytical
chemistry is to effectively separate the anylate from its matrix quantitatively, rapidly and
with minimum solvent usage. %.!.E has been applied to quantitative extraction of a
selected list of semi(volatiles, which include polycyclic aromatic hydrocarbons )%/!+,
phenols, *1 )olychlorinated 1iphenyls+ and total petroleum hydrocarbons. -n the
preparation for the %!E experiment, a cell is usually set up. "he cell is mainly a dionex
%!E instrument with a 55ml stainless extraction cell. "he si$e of the cell should be at least
double the mass of the sample.
4D
,or instance, if the mass is 47g, we make use of a 55ml cell. "he cell is then packed with
the following from top to bottom respectively&(
i/ % filter? cellulose or glass(fibre filter.
ii/ !and
iii/ !ample
i=/ !ilica gel
=/ !and
=i/ % filter? cellulose filter paper or glass(fibre filters.
"he extracting solvent is then passed through the cell which is then brought to elevated
temperature. ressure is also applied to maintain the solvent in the liquid state. "he solvent
used in the previous extraction can be used. /owever, there should be no use of mineral
acids in the %!E system? this is because acids may etch the stainless steel and tubing
system of the %!E set(up. *onsequently, strong bases should not be used as they will
destroy the %!E pump components leading to its failure. %fter extraction, the extract
containing the target anylate is purged from the cell using nitrogen into a collection vial for
analysis. "he efficiency of the %!E system depends on the operating parameters such as
extraction temperature, extraction pressure and static extraction time. "o achieve an
optimum in the extraction process, a study on the effects of each of the parameters should
be conducted. -n general, elevated temperatures result into better extraction. "his is
because an increase in extraction temperature results into a faster desorption of anylates
from matrix adsorbent sites as transportation of anylates from remote places in the matrix
of the bulk of organic solvent in the solvent in the extraction cell increases. Moreover,
anylates solubility in the solvent is also improved at high temperatures. "he amount of
extract increases with an elevation of pressure. %n increase in pressure leads to an increase
in desorption kinetics of the anylates from the sample. *onsequently, high pressure also
allows the cells to fill and flush faster. % shorter extraction time will reduce the cost of an
analysis. Fhen developing a method for the %!E system, there are many parameters to
consider. ,irst, pre(treat the sample if required to ensure proper contact with the extracting
4:
solvent. !ample pre(treatment can be established in many ways, such as removing excess
water and properly dispensing the sample.
!econdly, determine the best %!E solvent to extract the target anylate. "he solvent that was
used for manual extraction method is usually a good starting point. "hirdly, optimi$e the
%!E method parameters accordingly. !tandard %!E *onditions )!%*+ are typically a good
starting point when developing a new %!E method.
,inally, determine if any post extraction treatment is required, such as removing any
unwanted water from the extract prior to analysis.
5/1/4 Sol=ent re+o=er@
"his is basically a method of concentrating the extract by recovering the solvent used.
>nce the first batch from the soxhlet was ready, the extract was put into a rotary
evaporator, to recover the hexane we used while concentrating the %-s in the extract. "he
rotary flask is immersed into a hot bath to heat up the extract and evaporate the hexane
used. "he vapours are condensed and then collected into a collection flask. "he solvent
recovery is done until all solvent is evaporated. %fter the process is done the sample is
taken for analysis to determine, isolate and characteri$e the %ctive -ngredients.
Methylene chloride has been previously used with success in the extraction of the active
ingredients from the sample. -n the isolation of the olyketides from the solvent, we will
employ thin layer chromatography )"G*+ so as to know how many compounds are in the
extract. %fter this analysis, we will use silica gel open column chromatography for
isolation of the secondary metabolites. >nce compounds have been extracted and isolated,
further analysis will be carried out using the following analytical techniques? 'as
chromatography(mass spectrometry )'*(M!+, and ,ourier transform B -nfra red
spectroscopy ),"(-.+.
49
3.2 'solation
-solation is done to determine the number of compounds in a sample of crude extract. "his
is mainly done using thin layer chromatography )"G*+ and column chromatography which
are explained in details below.
5/3/1 T'in La@er C'romato)rap'@
*hromatography is used to separate mixtures of substances into their components. %ll
forms of chromatography work on the same principle. "hey all have a stationary phase )a
solid, or a liquid supported on a solid."his consists of thin layer of silica gel coated on
aluminium plate )7.5Amm+, binder for example gypsum mixed with this phase to make the
latter stick better to the plate, fluorescence+ and a mobile phase )a liquid or a gas which
consists of either a polar or non polar solvent which is used to make the solvent system+.
"he mobile phase flows through the stationary phase and carries the components of the
mixture with it. 3ifferent components travel at different rates. "hin layer chromatography
is done using a thin, uniform layer of silica gel or alumina coated onto a piece of glass,
metal or rigid plastic. "he silica gel )or the alumina+ is the stationary phase. "he stationary
phase for thin layer chromatography also often contains a substance which fluoresces in
U0 light. "he mobile phase is a suitable liquid solvent or mixture of solvents.
5/3/1/1 Pro!&+tion o% +'romato)ram
aA % pencil line is drawn near the bottom of the plate and a small drop of a solution of
the sample mixture is placed on it.
<A "here should be labelling on the plate to show the original position of the drop must
also be in pencil. -f any of this was done in ink, dyes from the ink would also move
as the chromatogram developed.
+A Fhen the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a
covered beaker. -t is important that the solvent level is below the line with the spot
on it. "he reason for covering the beaker is to make sure that the atmosphere in the
beaker is saturated with solvent vapour.
57
"o help this, the beaker is often lined with some filter paper soaked in solvent. !aturating
the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up
the plate.
-igure 3.14 (repared #$% plate
!A %s the solvent slowly travels up the plate, the different components of the dye
mixture travel at different rates and the mixture is separated into different coloured
spots.
-igure 3.24 Diagram sho5ing the plate after the solvent has moved half 5ay up it.
54
eA "he solvent is allowed to rise until it almost reaches the top of the plate. "hat will
give the maximum separation of the sample components for this particular
combination of solvent and stationary phase.
5/3/1/3 Mea&rin) R
%
=al&e
"o know how many different components make up the mixture, it is ok to stop there.
/owever, measurements are often taken from the plate in order to help identify the
compounds present. "hese measurements are the distance travelled by the solvent, and the
distance travelled by individual spots. Fhen the solvent front gets close to the top of the
plate, the plate is removed from the beaker and the position of the solvent is marked with
another line before it has a chance to evaporate. "hese measurements are then taken&
"he .
f
value for each dye is then worked out using the formula&
6)#E4 -f the substance is colourless, the following ways are used?
i/ 7sing fluorescence4, "he thin layer plate often has a substance added to it which
will fluoresce when exposed to U0 light. -f U0 light shines on it, it will glow. "hat
glow is masked at the position where the spots are on the final chromatogram ( even
if those spots are invisible to the eye. "hat means that if you shine U0 light on the
plate, it will all glow apart from where the spots are. "he spots show up as darker
patches.
-igure 3.34 #$% plate under 78 light
55
Fhile the U0 is still shining on the plate, you mark the positions of the spots by drawing a
pencil circle around them. %s soon as you switch off the U0 source, the spots will
disappear again.
ii. Sho5ing the spots up chemically
-n some cases, it may be possible to make the spots visible by reacting them with
something which produces a coloured product. % good example of this is in
chromatograms produced from amino acid mixtures. "he chromatogram is allowed to dry
and is then sprayed with a solution of 6inhydrin. 8inhydrin reacts with amino acids to
give coloured compounds, mainly brown or purple.
-igure 3.44 %hemically sprayed chromatogram
-n another method, the chromatogram is again allowed to dry and then placed in an
enclosed container )such as another beaker covered with a watch glass+ along with a few
iodine crystals. "he iodine vapour in the container may either react with the spots on the
chromatogram, or simply stick more to the spots than to the rest of the plate. Either way,
the substances one is interested in may show up as brownish spots.
5<
5/3/1/5 WorDin) o% t'in la@er +'romato)rap'@
T'e tationar@ p'ae ? ili+a )el
!ilica gel is a form of silicon dioxide )silica+. "he silicon atoms are #oined via oxygen
atoms in a giant covalent structure. /owever, at the surface of the silica gel, the silicon
atoms are attached to (>/ groups. !o, at the surface of the silica gel you have !i(>(/
bonds instead of !i(>(!i bonds. "he diagram shows a small part of the silica surface.
-igure 3.4 Silica structure.
"he surface of the silica gel is very polar and, because of the (>/ groups, can form
hydrogen bonds with suitable compounds around it as well as van der Faals dispersion
forces and dipole(dipole attractions. "he other commonly used stationary phase is alumina
( aluminium oxide. "he aluminium atoms on the surface of this also have (>/ groups
attached. "his also applies equally to alumina.
Separation o% t'e +ompo&n! a a +'romato)ram !e=elop
%s the solvent begins to soak up the plate, it first dissolves the compounds in the spot that
you have put on the base line. "he compounds present will then tend to get carried up the
chromatography plate as the solvent continues to move upwards. /ow fast the compounds
get carried up the plate depends on two things&
/ow soluble the compound is in the solvent. "his will depend on how much
attraction there is between the molecules of the compound and those of the solvent.
/ow much the compound sticks to the stationary phase )the silica gel+. "his will
depend on how much attraction there is between the molecules of the compound
and the silica gel.
5@
!upposing the original spot contained two compounds ( one of which can form hydrogen
bonds, and one of which can only take part in weaker van der Faals interactions. "he one
which can hydrogen bond will stick to the surface of the silica gel more firmly than the
other one. Fe say that one is adsor*ed more strongly than the other. %dsorption is the
name given to one substance forming some sort of bonds to the surface of another one.
%dsorption is not permanent? there is a constant movement of a molecule between being
adsorbed onto the silica gel surface and going back into solution in the solvent. >bviously
the compound can only travel up the plate during the time that it is dissolved in the solvent.
Fhile it is adsorbed on the silica gel, it is temporarily stopped ( the solvent is moving on
without it. "hat means that the more strongly a compound is adsorbed, the less distance it
can travel up the plate.
-n the extract, the compound which contains hydrogen bond will adsorb more strongly than
the one dependent on van der Faals interactions, and so won;t travel so far up the plate. -t
is very unlikely that both will hydrogen bond and be soluble in the solvent to exactly the
same extent. %ttractions between the compound and the solvent are also important as they
will affect how easily the compound is pulled back into solution away from the surface of
the silica. /owever, it may be that the compounds don;t separate out very well when you
make the chromatogram. -n that case, changing the solvent may well help including
perhaps changing the p/ of the solvent. "his is to some extent #ust a matter of trial and
error ( if one solvent or solvent mixture doesn;t work very well, you try another one.
5/3/3 Col&mn C'romato)rap'@
-n thin layer chromatography, the stationary phase is a thin layer of silica gel or alumina on
a glass, metal or plastic plate. *olumn chromatography works on a much larger scale by
packing the same materials into a vertical glass column. 0arious si$es of chromatography
columns are used and it is often convenient to use an ordinary burette as a chromatography
column.
5A
-igure 3.6 (repared column for chromatography
5/3/3/1 Pro+e!&re
i/ Make a concentrated solution of the mixture preferably in the solvent used in the
column.
ii/ >pen the tap to allow the solvent already in the column to drain so that it is level
with the top of the packing material,
iii/ %dd the solution carefully to the top of the column. "hen you open the tap again so
that the coloured mixture is all absorbed into the top of the packing material, so that
it might look like figure <.D.
-igure 3.7 ! Sample column
5C
i=/ 8ext you add fresh solvent to the top of the column, trying to disturb the packing
material as little as possible.
=/ >pen the tap so that the solvent can flow down through the column, collecting it in
a beaker or flask at the bottom.
=i/ %s the solvent runs through, you keep adding fresh solvent to the top so that the
column never dries out.
"he next set of diagrams shows what might happen over a period of time.
-igure 3.9 %olumn chromatography process
"he blue compound is obviously more polar than the yellow one ( it perhaps even has the
ability to hydrogen bond because the blue compound doesn;t travel through the column
very quickly.
"hat means that it must adsorb more strongly to the silica gel or alumina than the yellow
one. "he less polar yellow one spends more of its time in the solvent and therefore washes
through the column much faster. "he process of washing a compound through a column
using a solvent is known as elution. "he solvent is sometimes known as the eluent.
5D
-t takes a very long time to wash the blue compound through at the rate it is travelling at
the moment. /owever, there is no reason why you can;t change the solvent during elution
to use a moe polar solvent once the yellow one has all been collected. "hat will have two
effects, both of which will speed the blue compound through the column.
"he polar solvent will compete for space on the silica gel or alumina with the blue
compound. %ny space temporarily occupied by solvent molecules on the surface of
the stationary phase will not be available for blue molecules to stick to and this will
tend to keep them moving along in the solvent.
"here will be a greater attraction between the polar solvent molecules and the polar
blue molecules. "his will tend to attract any blue molecules sticking to the
stationary phase back into solution.
"he net effect is that with a more polar solvent, the blue compound spends more time in
solution, and so moves faster. 1ut this alternative solvent is not used because if both of the
compounds in the mixture travel quickly through the column right from the beginning there
will not be a good separation. Fhen using column chromatography to purify the product of
an organic preparation, it is quite likely that the product targeted will be colourless even if
one or more of the impurities is coloured. *onsidering that everything is colourless, there is
no quick and easy way of knowing when the substance required has reached the bottom of
the column. -f this happens, collect what comes out of the bottom of the column in a whole
series of labelled tubes. "he si$e of each sample will depend on how big the column is
maybe, 4 cm
<
samples or A cm
<
samples. "ake a drop from each solution and make a thin
layer chromatogram from it. lace the drop on the base line alongside a drop from a pure
sample of the compound to be made. 1y doing this repeatedly, identify which of the
samples collected at the bottom of the column contain the desired product, and only the
desired product. >nce done, combine all of the samples which contain the pure product,
and then remove the solvent.
5:
3.3 %haracteri:ation
"he analysis of the extract will be done. "he first process is the isolation process using the
column chromatography and the thin layer chromatography. %fter the isolation is done the
next process is to characteri$e the isolated compounds. "his is will be done using any of
the following analytical techniques&
aA /igh performance liquid chromatography )/G*+
<A 'as chromatography B Mass spectrometry )'*(M!+
+A yrolysis( 'as chromatography B Mass spectrometry )'*(M!+
!A -nfrared spectroscopy )-.+
eA ,ourier transform infrared spectrometry ),"-.+
%A 8uclear magnetic resonance spectrometry )8M.+
)A U0(visible spectroscopy
5/5/1 In%rare! pe+tro+op@
-nfrared )-.+ spectroscopy is an extremely reliable and well recogni$ed fingerprinting
method. -t has long been used for the characteri$ation of humic substances )!chnit$er et al.,
49A9? -shiwatari, 49D7? !tevenson and 'oh, 49D4+. "he technique of %ttenuated "otal
.eflectance )%".+ has in recent years revolutioni$ed solid and liquid sample analysis
because it combats the most challenging aspects of infrared analysis, namely sample
preparation and spectral reproducibility.
%". is an -. sampling technique that provides excellent quality data in con#unction with
the best possible reproducibility of any -. sampling technique. -t has revolutioni$ed -.
solid and liquid sampling through&
? ,aster sampling
? -mproving sample(to(sample reproducibility and
? Minimi$ing user to user spectral variation.
59
%n attenuated total reflection accessory operates by measuring the changes that occur in a
totally internally reflected infrared beam when the beam comes into contact with a sample
)see -igure 3."+. %n infrared beam is directed onto an optically dense crystal with a high
refractive index at a certain angle. "his internal reflectance creates an evanescent wave that
extends beyond the surface of the crystal into the sample held in contact with the crystal.
"his evanescent wave protrudes only a few microns )7.A M ( A M+ beyond the crystal surface
and into the sample. *onsequently, there must be good contact between the sample and the
crystal surface. -n regions of the infrared spectrum where the sample absorbs energy, the
evanescent wave will be attenuated or altered. "he attenuated energy from each evanescent
wave is passed back to the -. beam, which then exits the opposite end of the crystal and is
passed to the detector in the -. spectrometer. "he system then generates an infrared
spectrum. %".(,"-. spectra in the region between @777(CA7 cm
(4
were obtained using
erkinElmer -nstruments, !pectrum >ne, and ,"(-. !pectrometer. "he scanner velocity
was 47 k/$, with the resolutions of @ cm
(4
. .eference is atmosphere for solids and pure
water for aqueous solutions. %". spectra are shown with absorbance scale corresponding
to log ).
reference
6.
sample
+, where . is the internal reflectance of the device.
-igure 3." ! multiple reflection !#+ system.
<7
C'apter 8" Re&lt an! Di+&ion
4.1 Extraction
3ue to the following setbacks, we were not able to extract the entire plant sample we had.
"his was mainly because&
( "here was a limited time for all the samples to be done therefore we only managed
to extract only the leaves.
( "he plant samples had not dried to the required standards.
"he following data was recorded on the extraction of the leaves using soxhlet extraction
and hexane as the solvent.
For <at+' one"
"ime for the first batch to extract ( A hours
Feight of thimble ( @.Ag
Feight of thimble with samples ( 47.4g
%mount of hexane solvent used ( 45AmG
"emperature of heater ( C7*
For <at+' t>o"
"ime for the first batch to extract ( @ hours
Feight of thimble ( @.<g
Feight of thimble with samples ( 44.7g
%mount of hexane solvent used ( 45AmG
"emperature of heater ( C7*
Feight of extract from both batches ( 7.<g
,urther calculations will be done once all the results are determined.
<4
"he following results were recorded from the %!E extraction of the leaves extract using
dichloromethane as the solvent and cellulose as the filter.
>nly one batch was completed for this procedure which was done with the help of the
research centres.
Feight of samples ( 5<.7Cg
3*M solvent used ( <77mG
"ime for extraction ( < hours
"otal weight of extract ( 5.9Cg
ercentages of the extract to the samples for both extractions are as follows&
N of the weight extract for %!E extraction ( 45.:@N
N of the weight extract for soxhlet extraction ( 5.@@N
,rom these percentages it is evident that the accelerated solvent extraction was more
efficient than the soxhlet extraction method.
4.2 'solation
8/3/1 T'in la@er +'romato)rap'@ Pro+e!&re an! O<er=ation
"he prepared plate is then stained with the dissolved sample to make two spots. "he stained
plate was then dipped into a single solvent or prepared solvent system. "he solvent will rise
while eluting the compounds available in the samples to different levels depending in the
ability to be carried away by the solvent. "he spots on the plate separated to different
colours and located at different locations on the plate surface. "he distinct spots observed
indicated the different compounds in the sample.
ure hexane was used as the solvent. "wo non(distinct $ones were observed and detected
only by the U0 light. !olvent system was changed to methanol& hexane mixture at the ratio
4&4. %gitation and warming enhanced the dissolution of the crude sample.
<5
>n the "G* plate two spots were observed under a U0 light as shown below?


-igure 4.14 #$% plate using solvent system of 141; methanol4 hexane
.
f
% O 56: O 7.5A .
f
1 O D6:
,ollowing the observations, we checked the purity of the obtained compounds by scrapping
regions where the spots were located i.e. regions % and 1. "he scrapped regions containing
silica from the plate were dissolved in a methanol&hexane system, 4&4 ratio. -t dissolved and
was allowed to settle. "he obtained solution was then evaporated to concentrate it and "G*
analysis done using a solvent system of methanol&hexane at the ratio 5&4 respectively for
spot 1 and a 4&5 ratio for spot %. -t was noted that for spot %, we used more of methanol
than in spot 1. "his is because methanol is more polar hence would carry the stationary
face farther.
<<

'pot 7
'pot A
-igure 4.24 Spot < #$% sample on a 241 hexane/ methanol system.
-igure 4.34 Spot ! #$% sample on a 142 hexane methanol system.
3ue to limitation of resources, a further analysis on the compounds could not be done.
/owever, from the obtained data on the samples % and 1, there are still other compounds
present in the extract. Fe performed another "G* test using pure chloroform as solvent.
*hloroform was the most polar solvent we used and so we expected the stationary phase to
be carried farther. 'reen spots were observed. "hey were carried up to various distances as
shown in figure @.@?
<@
-igure 4.44 ! #$% sample on a chloroform system
.,
4
OA6:, .,
5
O @6: and .,
<
O <6: respectively.
Under the U0 light, a region had gone past the solvent front. "o counter this, we used a
solvent system of 4&4 chloroform& hexane. % "G* plate using this solvent system gave a
relatively better observation as shown below&
-igure 4.4 ! #$% sample on a %hloroform4 =exane system
.,
4
O 46: and .,
5
O @.A6:
"he first spot was a dark green spot. 'enerally the crude extract sample contained several
compounds. "his was evident from the various spots obtained in the "G* plates.
8/3/1/1 Di+&ion
"he chloroform hexane solvent system gave the best results. -t therefore formed the basis
for the column chromatography to be conducted. -t was clear that the extract contained
several compounds. More analyses should be done on the crude extract sample using more
advanced analytical techniques like ,"-., '*(M!, 8M. etc.
<A
,rom the solvent system used, it could be deduced that the compounds contained in the
extract were polar in nature. % polar solvent system carried the stationary phase a farther
distance.
8/3/3 Col&mn +'romato)rap'@
4.2.2.1 )*servations and Discussions
3ifferent compounds in the sample have different affinities for the two phases and energy
from the stationary phase at different times. *olumn chromatography was carried out on
the extract using a solvent system of chloroform& hexane )4&4+. % sample of the crude
extract )3*M+ from %!E was dissolved in a ratio of 4&4 mixture of methanol&hexane.
!haking and warming was done to enhance solubility. !ilica gel was used as stationary
phase. -t was mixed with a solvent system to yield thick white slurry.
"he column was packed with silica gel. 3ifferent colour bands were obtained on the
column. !ix distinct bands could be observed. Each band was collected into a beaker.
!eparation is based on polarity of the substance. !ilica and the solvent mixture form a polar
matrix. "hus, more polar substances in the sample are adsorbed more strongly to stationary
phase and eludes slowly from the corner. Gess polar substances were quickly pushed
through the column by mobile phase. 'reater attraction between polar solvents and polar
substances molecules resulted in molecules of polar substance which had been sticking to
stationary phase to enter solution and elute through. "he samples obtained from the column
were later on evaporated to concentrate them and see if there would be any compound
extracted. robably due to small quantity of the crude extract used the mass of the fractions
got were negligible in terms of mass. -n comparison to the "G* data that was obtained
previously is evident that column chromatography gave a relatively better result, deducing
that there are at least six compounds in the crude extract sample.
4.3 %haracteri:ation
8/5/1 ATR?FTIR %or !nnona s.uamosa lea=e; !i+'loromet'ane BDCMA extra+t
"he %".(,"-. spectrum of Annona squamosa leave, dichloromethane crude extract
)solid+ is presented in -igure 4.6 below. "his spectrum is characteri$ed by four groups of
absorption bands which appear in the following wave number ranges&
<C
? <A77 B 5<77 cm
(4
,
? 4:77 B 4777 cm
(4
and
? 4777 B CA7 cm
(4
.
i/ 5622 E 3522 +m
?1
" "his wave number range contains five distinct bands&
aA % P>/ stretching band of an alcohol >B/ functional group )<A77 B <<77 cm
(4
+
<A % P>/ stretching band )wide band+ of the associated >B/ group in a
carboxylate functional group )<@77 B 5A77 cm
(4
+
+A % P*/ stretching band of an aromatic *B/ bond )<7DA B <7<7 cm
(4
+
!A P
a
*/
<
and P
a
*/
5
antisymmetric stretching bands of alkanes )59D7 B 594A cm
(4
+
eA P
s
*/
<
and P
s
*/
5
symmetric stretching bands of alkanes )5::7 B 5:@A cm
(4
+
"he broadband in the region 5<A7(4:77 cm
(4
corresponds to the strong absorption of
diamond used in the %".(,"-. instrument.
ii/ 1F22 E 1222 +m
?1
? "he band at 4D4A cm
(4
is assigned to the P*O> stretching mode of four different
functional groups& ketones, aldehydes, aliphatic or aromatic esters, or dimers of
carboxylic acids )arker, 49D4? Fo#tkowiak and *habanel, 49DD+.
? "he bands at 4@97 and 4C7< cm
(4
are assigned to the P*O*.
? "he band at 4<@4 cm
(4
is assigned to Q>(/ bend deformation of hydroxyl groups.
"he weak broadband in the 9A7 B 47<5 cm
(4
region could be associated to the in(plane Q*B
/ bending mode of aromatics.
iii/ 1222 E 462 +m
?1
%bsorption bands in the low frequency region CD< B :9A cm
(4
could be resulting from the
out(of(plane Q*B/ bending of the aromatic ring *B/ bonds.
<D
4000 3500 3000 2500 2000 1500 1000 500
0.04
0.05
0.06
0.07
0.08
673
715
1224
1021
1341
1490
1603
1715
3500-2300
A
b
s
o
r
b
a
n
c
e
Wavenumber (cm
-1
)
Annona squamosa leave, !" e#$rac$ (sol%&)
-igure 4.6 !#+,-#'+ spectrums for D%1 crude !nnona s.uamosa leave extract
<:
C'apter 6" Con+l&ion an! Re+ommen!ation
"his chapter summari$es the conclusions made basing on the experiments that were
carried. "hereafter recommendations are made.
!oxhlet extraction and %ccelerated !olvent Extraction )%!E+ were very good for the
extraction of Annona squamosa. "he accelerated solvent extraction is much faster and
economises on the solvent. ,urthermore %!E yields higher extraction efficiency compared
to soxhlet extraction. "here are many other different ways of extracting these compounds
of medicinal value as mentioned earlier which are more reliable but not readily available.
"he various solvent systems used in the isolation were sufficient in isolating the various
compounds present in the crude extract though other solvent systems could be tried. "here
is need to perform the isolation procedures using different and a wide variety of solvents
ranging from the most to the least polar and also performing the same variation for non
polar solvents. *ombining the polar and non polar solvent of different ranges i.e. the most
polar with non polar and the least polar with non polar helps to attain the most applicable
solvent system for isolation of the contained compounds in the crude extract.
-t can be concluded that compounds isolated from the crude extract were polar as indicated
by the results on isolation, elution having been carried out using a polar solvent system.
"here are different ways of characteri$ing isolated compounds as seen earlier. "he main set
back is that the analytical apparatus were not available and thus it was hard to perform the
expected characteri$ation on the isolated compounds.
-t is therefore recommended that further characteri$ation using more analytical techniques
such as '*(M!, /G*, 8M., U0(0isible spectroscopy and Electrospray ioni$ation mass
spectrometry be carried out so as to fully characteri$e the compounds isolated from the
Annona squamosa.
<9
Appen!ix
!ppendix 14 Some o the structures o Annona Squamosa compounds.
@7
@4
!ppendix 24 (ictures of !nalysis
Picture *# +olumn +hromatography
Picture ,# Samples o ractions rom column
Picture -# .'+ Analysis
@5
Re%eren+e
%ma$on /erb *ompany, /ndependent 0istributor 1*1-,.
*hristophe 'leye, hilippe 3uret, %lain Gaurens, .eynald /ocquemiller, and %ndreR
*ave, )499<+ cis2Monotetrahydrouran Acetogenins rom the (oots o Annona muricata.
*olom %.>., 8eske %, *hahboune 8, Safra(olo M*, 1ardTn %. )5779+. .ucupentol, a
novel mono2tetrahydrouranic acetogenin rom Annona montana, as a potent inhibitor o
mitochondrial complex /. +hem 3iodivers.
3aily 8ation abstract ( ,riday, !eptember @ 5779 at 55&<7
3epartment of harmacy, 8an#ing University of *hinese Medicine, 8an#ing, *hina )%pril
4A 5779+ 3ioorg Med +hem 'ett Abstract.
,eng, . *., et al. )49C5+ 4Pharmacological screening o some 5est /ndian medicinal
plants.6 7. Pharm. Pharmacol.8 *9# 11:;:*.
,eras U. %lali, Viao(Vi Giu, and Eerry G. McGaughlin )!eptember 4:, 499:+ Annonaceous
Acetogenins# (ecent Progress.
-shiwatari .. )49D7+ !trucutural characteristics of humic substances in .ecent lake
sediments. -n Advances in <rganic "eochemistry *=:: )eds. '.3. /obson and '.*.
!peers+, pp. 5:< ( <44. ergamon ress.
Geslie "aylor, )577A+. .he healing po%er o rainorest herbs.
McMaster, M. and McMaster, *. "+$MS. A Practical >sers "uide, Filey(0*/ )499:+.
Melot %, ,all 3, 'leye *, *hampy )8ovember 5, 5779+ Abstract no. *9(**)#9-?@2 =1 on
by. +A(S >M( ?B@: 3io+/S, CacultD de Pharmacie Paris2Sud **, +hEtenay2Malabry,
Crance.
Mills, E. !. and Fhite, .. )499@+ .he <rganic +hemistry o Museum <b&ects, 1utterworth(
/einmann, >xford )499@+.
@<
8=gouemo, ., et al. )499D+ 4Fects o ethanol extract o Annona muricata on
pentylenetetra!ol2induced convulsive sei!ures in mice.6 Phytother. (es.8 **(-)# ,9-;91.
adma, ., et al. )5774+ 4Fect o Annona muricata and Polyalthia cerasoides on brain
neurotransmitters and en!yme monoamine oxidase ollo%ing cold immobili!ation stress.6
7. Aatural (emedies8 *(,)# *99;9:.
.ieser M.E. )499C+. Cive Aovel Mono2tetrahydrouran (ing Acetogenins rom the Seeds o
Annona muricata, 7. Aat. Prod., ", *BB2*B?.
.oblot ,., Gaugel "., GebWuf M., *avX %. and GaprXvote >. )499<+. "wo acetogenins from
Annona muricata seeds, .he /nternational 7ournal o Plant 3iochemistry, 58, 4, 5:4(5:A.
!chnit$er M., !herarer 3.%. and Fright E... )49A9+ % study in the infrared of high(
molecular weight organic matter extracted with various reagents from a od$olic 1
hori$on. Soil Science F7, 5A5(5AD.
!mith, .. M. and 1usch, 2. G. >nderstanding Mass Spectra 2 A 3asic Approach, Eohn
Filey and !ons )4999+.
!tevenson ,.E. and 'oh 2.M. )49D4+ -nfrared spectra of humic acids and related
substances. "eochimica et +osmochimica Acta 56, @D4(@:<.
United !tates atent %pplication ,B*BB,:B?@9 and ,BB-B*99-9?.
Shong Eiang, .uo(Yun *hen, Ying *hen, and 3e(Uuan Yu )Euly 5, 499D+ 0onnaienin, a
Ae% Acetogenin 3earing a Gydroxylated .etrahydrouran (ing.
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