4.1.1 Plant Material: Stem barks of Alstonia boone1were collected 1n February I March 1991. 1992, 1993, from the Department or Botany, University or lbadan The collection and Herbarium numbers were Lowe 2323 and u I H
13134, respectively The plant material was cut into small pieces and oven dried (35 ° C)
4.1.2 Extraction Procoduro:
4.1.2.1 Soxhlct oxtroct: Coarsely ground plant material was extracted in e Soxhlel apparatus with petroleum ether (PE, 10hr), Diethyl ether (DE, 10hr),
Ethyl acetate (ElOAC, 10hr) and Ethanol (EtOH, 10hr) The mare \Vas dried and refluxed ,n ,vater (AO, 3hr) The soxhlel fractions were concentrated under reduced pressure, ond subsequently lyoph11lzed olong wtlh aqueous extract
4.1.2.2 Cold Extract: In order to rule out any posstblhty that application or heat as 1 n soxhlel extraction effects the ac11v1ty or the extracts. the extracllon
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s carried out In \he cold so that the activity of soxhlet and cold extracts could be compared
Coarsely ground plant material was steeped in petroleum ether for 3 nights followed by filtration The residue which ,vas dried et room temperature was weighed end soaked 1n diethyl ether for 3 nights and filtered The mere
was dned, weighed and steeped 1n ethytaeetete ro� 3 nights and filtered The mare from ethylacetato was dned, weighed and steeped In ethanol ror 3 nights fol10'Ned by f11lratron Finally, the mare from alcohol extraction ,vas dried,
weighed and steeped ,n distilled water for 3 nights followed by flllratton The extracts of petroleum ether, diethyl ether ethyl acetate and alcohol were first concentrated l/1 vacuo They v.-ere later lyoph1hzed together with aqueous extract The e,ctracts of petroleum ether diethyl ether ethylacetete ethanol rad aqueous extract were referred to as cPE, cOE, cEtOAc, eEtOH and cAQ,
rnpec:1 r.-el</
4.1.3 Fractionation of EtOH extract The EtOH P.xtract was dissolved 1n did*>tome-.i-rane (CH J Cl,) end sub;Cded to hquld • liquid extreclton with
•••arSwr� The rr: ,...'.lfc wns ma�.on ror aboul 15 minutes afle< which two
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layers developed, namely, the lipophilic layer and the hydrophilic layer. Both layers were separated 1n round bottom nasks, and each was evaporated under reduced pressure and lyoph1hzed The concentrate from the llpophillc and hydrophilic layers were referred to as EtOH L and EIOH N . respectively ElOH h was
part1honed into MeOH soluble (Ms) and MeOH insoluble (Ml) subfracllons The Ms subfract,on later crystallized out on coollng (refrigeration) to give while Clystals The crystals, called Alston/a boonei -1 (AB-1 ), were separated from
the mixture by ,vash1ng ,vtth MeOH 1n a bucknner funnel under low pressure and thereafter dried The filtrate (Msf) and the MeOH insoluble subfractlon (Mi) were concentrated under reduced pressure and a stream of nitrogen followed by
4.1.4 Extract prepared by prolonged boiling
Ttn tost was carried out 1 n order 10 find out how the 1radlt1onal method or pr9Pa1mg the decOC:OOn of A ooonei using water can be ophm1zed Whole,
�OU'ld A boonei stmn bark was d1v!Cled 1n10 7 equal parts of 1g each One part .... steeped rn d:s!::!ed wntOf for 3 days Each of the roma1n1ng 6 parts was
� in en• 'Cd wa!a fOf varym9 tcmes (2-64 hrs) The start,ng volume of
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stilled water 1n all 6 parts was the same (2L). At each reflux, a constant starting olume was maintained All extracts were concentrated 1n vacuo and lyophihzed
4.1.5 Determination of the Purity of the Antimalarial Compound (AB-1)
In pharmaceutical preparations, natural products from higher plants are used either as pure compounds or as extracts (Phillipson, 1994) In traditional medicine, one
plant or several are combined 1n one prescription whereas in modem medicine a single active ingredient, (usually synthesized) 1s prescribed (Phillipson, 1994) Thus in
traditional medicine, unlike 1n modem medicine, the activity and clinical efficacy of prescriptions cannot be attributed to a single achve ingredient (Phillipson, 1994). When
plants are extracted and fractionated 1n order to obtain a single active ingredient, the purity of the constituents isolated can be checked by TLC HPLC and spectroscopy In 1h11 seclton TLC and HPLC melhods are used as described below •
4.1.8 HPLC analysts.
Apparatus: HPLC analysis was performed on a Kralos hqu1d chromatograph equipped Wllh 8 Kratos spoctronow 400 solvent delivery system type 5140
I (K Pp & Zn) and Rheodyne model 7125 iniector The
IO vent programmer 1
Injector was connected to a HP , 040A dfode-erray detector (Hewlett-Packard)
•
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With HP series 9000 workstahan (Purity determ,natton) or Ktatos spectroOow 757 absorbance detector with CRIB chromatopac data processor (shimadzu.
quantitative analysis) A chromspher St 200x3mm I D column (Chrompack Cal No. 28277) was used. Mixtures of EtOAcJMeoH = 70 30 served as eluent
(Oow rate O 5m1/mln)
Methods: Solutions or 100.ug/ml or AB-1 were prepared and aliquots or 20,uL were 1n1ected into the HPLC The mobile phase consisted or EtOAcJMeoH =
70 30 The spectrum v,as scanned from 220 to 320nm
4.1.7 Thin Layer Chromatography (TLC) of AB-1
TLC ls a chromatographic technique which effects the physical separation or two or more components 1n a mixture on a plane surface Separation 1s achtoved by the differences In adsorpllon of components or Iha mixture on the stationary phase (solid) and the solubility in the mobile phase ll can be used to detennine a suitable solvent or so!venl system for separallng comPonents of, ror example a plant extract II can also be used lo detenn1ne
the purity of an organic material
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TLC was performed on pre-coated plates (silica gel 60F 254 , Merck, Cat.
No 5729) with a thickness of O 25mm Volumes of Sul of the solution of the compound (4mg/ml) in MeoH were applied to the TLC plates in bands
The plates were developed with EtOAc/MeoH = 70:30 in a saturated chamber Spots were sprayed with vanillin - H 2 SO. (Vanillin 1 % ,n ethanol and H 2 SO., 5%
1n EtOH), fast blue (1mg/100ml of H 2 0) and Draggendolfs reagents A & B UV
detection was at 254 and 366nm
In document
Abstract Algebra: Theory and Applications
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