research article hypoglycemic effect of aqueous and...

Research ArticleHypoglycemic Effect of Aqueous and Methanolic Extract ofArtemisia afra on Alloxan Induced Diabetic Swiss Albino Mice

Idris Ahmed Issa1 and Mohammed Hussen Bule2

1Department of Physiology Faculty of Medicine Mekelle University Ethiopia2Department of Pharmacy College of Medicine and Health Sciences Ambo University Ethiopia

Correspondence should be addressed to Idris Ahmed Issa edrisahmed84gmailcomand Mohammed Hussen Bule gorawnayahoocom

Received 7 May 2015 Accepted 21 July 2015

Academic Editor Mark A Yorek

Copyright copy 2015 I A Issa and M Hussen BuleThis is an open access article distributed under theCreative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Diabetes mellitus is metabolic syndrome that causes disability early death and many other complications Currently insulin andmany synthetic drugs are used in diabetes treatment However these pharmaceutical drugs are too expensive particularly for sub-Saharan population in addition to their undesirable side effects The present study was aimed to evaluate antidiabetic effect andtoxicity level of Artemisia afra which was collected from its natural habitat in Bale Zone around Goba town 455 km southeastof Addis Ababa Air dried aerial parts of Artemisia afra were separately extracted with both distilled water and 95 methanolOral acute toxicity test was conducted on healthy Swiss albino mice Antidiabetic effect of the aqueous and methanolic extractsof Artemisia afra was separately evaluated on alloxan induced diabetic mice at doses of 500 750 and 1000mgKg body weightorally The results indicate that mean lethal dose (LD

50) for aqueous extract of Artemisia afra was 98334mgKg Blood glucose

level was significantly decreased by 24 (119901 lt 0005) and 569 (119901 lt 00004) in groups that received aqueous extract of Artemisiaafra at dose of 500mgKg and 750mgKg respectively The methanolic extract of Artemisia afra also significantly lowered bloodglucose by 498 (119901 lt 00001) at doses of 1000mgkg on the 5th hr Aqueous extract of Artemisia afra was regarded as nontoxicand safe since its LD

50was found above 5000mgKg Aqueous extract showed higher effect at relatively lower dose as compared to

methanolic extract The aqueous extract was screened positive for phytochemicals like flavonoids polyphenols and tannins thatwere reported to have antioxidant activity

1 Background

There is a global increase in the prevalence of diabetesmellitus predominantly related to life styles and the resultingsurge in obesity [1] Diabetes mellitus is a metabolic diseasecharacterized by disordered metabolism and abnormallyhigh levels of blood glucose It is a major public healthproblemaffecting 285million or 64of theworld populationfor the year 2010 [2] Approximately 90ndash95 of patients withdiabetes have type 2 diabetes (T2D) or non-insulin dependentdiabetes mellitus T2D is accounting for a combination ofinsulin resistance and an inadequate compensatory insulinsecretory response [3]

Management of diabetes without any side effect is still achallenge to the medical community The use of the drugsis restricted by their pharmacokinetic properties secondary

failure rates and accompanying side effects [4] Despiteconsiderable progress in the treatment of diabetes by oralhypoglycemic agents search for newer drugs continuesbecause the existing synthetic drugs have several limitations[5] The available therapies for diabetes include insulin andoral antidiabetic agents such as sulfonylureas biguanidesand 120572-glycosidase inhibitors Many of these oral antidiabeticagents have a number of serious adverse effects Thus themanagement of diabetes without any side effects is still achallenge [6] Therefore there is a growing interest in herbalremedies because of their effectiveness minimal side effectsin clinical experience and relatively low costs Herbal drugsor their extracts are prescribed widely even when theirbiological active compounds are unknown Even the WorldHealth Organization (WHO) approves the use of plant drugsfor different diseases including diabetes mellitus Traditional

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 752486 5 pageshttpdxdoiorg1011552015752486

2 Evidence-Based Complementary and Alternative Medicine

plant medicines are used throughout the world for treatmentof diabetes mellitus [7]

Artemisia afra is locally called ldquochikugnrdquo also knownas African wormwood It belongs to family Asteraceae It isone of the best known and widely used plants in traditionalmedicine Various preparations such as infusions decoctionsmolasses and alcohol extracts of Artemisia afra are used forthe treatment of coughs colds chills stomachache and drydyspepsia and as a purgative and for the cure of smallpox andmalaria The herbaceous leaves of this plant contain between03 and 14vwof a bluish-green essential oil with a strongfragrance [8] This study was designed to systematicallyevaluate hypoglycemic effect of aqueous and methanolicextracts of Artemisia afra in comparison with each other andstandard hypoglycemic agents like glibenclamide

2 Materials and Methods

21 Plant Material Fresh aerial parts of Artemisia afrawere collected during their flowering period when plantsgive highest yield [9] The plants were collected from theirnatural habitat in Bale Zone around Goba town near theRiver Togona (7∘01015840N 39∘91015840E) 2743 meters above sea levelAuthentication and taxonomic identification of the plantsamples were done by using standard botanical monographsat Addis Ababa University Science Faculty Herbarium

22 Plant Extraction

221 Aqueous Extraction Fresh aerial parts of Artemisiaafra were cleaned with distilled water and air dried at roomtemperature It was ground into powder using grinding millPowdered aerial part of Artemisia afra weighing 220 gm wassorely mixed with 450mL distilled water in flat bottom flaskon orbital shaker for 24 hours Distilled water was addeduntil completely wet and the dried plant powder was suffi-ciently covered After gentle maceration for 24 hours plantmaterials were filtered separately through mousseline clothinto 1000mL beakers The obtained filtrate was deep frozenat minus70∘C and lyophilized (freeze dried) using lyophilizerto collect crude aqueous extract [10] The aqueous extractswere stored in desecrator at room temperature until usedfor experiment The above aqueous extracted powders ofArtemisia afra provided yield of 21 gm (95 ww)

222 Methanolic Extraction Fresh aerial parts of Artemisiaafra were cleaned with distilled water and air dried at roomtemperature A dried and powdered aerial part of Artemisiaafra weighing 165 gm was sorely mixed with 400mL of95 methanol in flat bottom flask on orbital shaker Aftergentle maceration for 24 hours methanolic extracts werefiltered throughWhatman filter paper chartThe filtrate fromArtemisia afra was concentrated under reduced pressureusing rotary evaporator at 50 rpmand40∘Cbath temperatureFinally concentratedmethanolic extract was collected in vialsand placed on water bath at 40∘C to evaporate methanolcompletely The crude methanolic extract of Artemisia afrahad a yield of 13 gm (78 ww)

23 Inducing Experimental Diabetes Diabetic condition wasinduced to Swiss albino mice of both sexes weighing 25ndash35 gafter overnight fasting by intraperitoneal injection of freshlyprepared alloxan (200mgKg IP) dissolved in distilled water[11] Three days (72 hr) after the injection of alloxan theanimals were fasted for 16 hours and their blood glucoseconcentration was determined with glucometer Animalsshowing stabilized diabetes (FBG ge 250mgdL) on the thirdday after alloxan injection were selected for the experiment[12] Mice that did not show diabetic condition on the thirdday were excluded from the experiment

24 LaboratoryAnimals Healthy Swiss albinomiceweighing25 gndash35 gwere obtained fromEthiopianHealth andNutritionResearch Institute (EHNRI) animal unit and kept in labora-tory animal house of Drug Research Department during theexperiment The animals were provided standard pellet foodand tapwater ad libitumwith 12-hour lightdark photoperiodThe experiment was conducted according to the ethicalnorms approved by Animal Ethics Committee Guidelines ofthe EHNRI

25 ExperimentalDesign Alloxan induced experimental dia-betic Swiss albino mice (FGB ge 250mgdL) were dividedin to 9 test groups with 5 mice in each group that matchwith weight and fasting blood glucose level [13] Groups-1ndash3 included diabetic mice treated with crude aqueous extractof Artemisia afra at dose of 500mgKg 750mgKg and1000mgKg PO Groups-4ndash6 included diabetic mice treatedwith crude methanolic extract of Artemisia afra at doseof 500mgKg 750mgKg and 1000mgKg PO Group 7included diabetic mice treated with standard drug gliben-clamide (standard group) 10mgkgPO [14]Group 8 includeduntreated diabeticmice (diabetic control) given equal volumeof distilled water PO Group 9 included nondiabetic referencegroup (normal control) given equal volume of distilledwater PO Blood glucose level of these mice was measuredusing glucometer at interval of 1 hour after treatment forconsecutive 5 hr

26 Acute Toxicity Test in Mice The mice were divided into10 groups with 6 animals in each group matched for weightand sex These animals were housed 3 mice per sex per cagein a well-ventilated room with 12 hr cycle of day and nightlight conditions and temperature was maintained at around25∘C Food was withdrawn for the 12 hr fasting period beforeand for 24 hr fasting period after oral administration of thesingle doses crude aqueous extract [12 15] Aqueous extractof Artemisia afra was aseptically suspended in distilled waterand administered at a single doses of 0 1000 2000 30004000 5000 7500 10000 and 12000mgKg body weightby gavages perorally (PO) to the test groups The controlgroups were given an equal volume of water The change ingeneral behaviors of the mice was continuously monitored[16] The number of dead mice was recorded and used in thecalculation of mean lethal dose (LD

50) value All surviving

animals were euthanized with diethyl-ether at day 14 asdescribed by [17]

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Acute toxicity of Artemisia afra aqueous extract in Swiss albino mice (119899 = 6)

Dose of A afra aqueousextract in mgKg

Number of mice pergroup (119899 = 6)

Number of mice deadand percent dead Signs of toxicity

000mgKg Group-1 M = 3 03 0 NoneF = 3 03





5000mgKg Group-6 M = 3 03 0 Hypoactivity piloerectionF = 3 03


10000mgKg Group-8 M = 3 13 333 Hyperventilation hypoactivity and salivationF = 3 13

12000mgKg Group-9 M = 3 33 100 Convulsion hyperventilation hypoactivity and salivationF = 3 33

27 Phytochemical Screening Aqueous extract of Artemisiaafra aerial parts was tested qualitatively for presence ofmajor secondary metabolites The qualitative test was doneaccording to procedure described by [18] The method wasbased on observation of color formation that occurs dueto reaction of secondary metabolites with different standardreagents

28 Statistical Analysis All the results of blood glucose wereexpressed as mean plusmn standard deviation for (119899 = 5) animalsin each group The data was analyzed using Microsoft OfficeExcel 2007 by Studentrsquos 119905-test at 95 CI

3 Result and Discussion

31 Oral Acute Toxicity of Artemisia afra As shown in theTable 1 the absence of mortality in the test group follow-ing oral administration of Artemisia afra aqueous extractup to 5000mgkg dose indicates the median lethal dose(LD50) of the Artemisia afra aqueous extract is higher than

5000mgkg The expected median lethal dose (LD50) is

between 7500mgKg and 12000mgKg The actual medianlethal dose (LD

50) of Artemisia afra aqueous extract calcu-

lated from Table 1 was 98334mgKg which agrees with theexpected value Since its actual median lethal dose (LD

50) is

greater than 5000mgKg aqueous extract of Artemisia afrais nontoxic Substances with LD

50between 5000mgkg of

body weight and 15000mgkg of body weight are regardedas practically nontoxic [19]

32 Hypoglycemic Effect of Aqueous and Methanolic Extractsof Artemisia afra The aqueous extractof Artemisia afra was

administered to alloxan induced diabetic mice orally at singledoses of 500 750 and 1000mgkg Blood glucose level wassignificantly decreased in diabetic groups which receivedaqueous extract of Artemisia afra at dose of 500mgKg and750mgKg in hours after treatment relative to untreateddiabetic control with 119901 lt 001 and 119901 lt 0001 respectively(Table 2) Among the tested doses of Artemisia afra aqueousextract the maximal drop in blood glucose level of thediabetic mice was observed at dose of 750mgKg (569119901 lt 0001) A sharp decline in blood glucose level at doseof 750mgKg occurred in the first two hours after treatment

In contrast the methanolic extract of Artemisia afrashowed a remarkable blood glucose lowering effect in alloxaninduced diabetic mice at doses of 750 and 1000mgkg ascompared to the untreated diabetic control group (Table 2)Though the methanolic extract of Artemisia afra loweredblood glucose of diabetic mice by 257 and 498 at doselevels 750 and 1000mgkg respectively it brought statisticallysignificant blood glucose lowering effect only at dose level of1000mgkg (119901 lt 0001) as compared to the group receivingthe vehicle

Unlike the aqueous extract of Artemisia afra whichshowed maximal effect at 750mgkg dose level the methano-lic extract of Artemisia afra at this level did not significantlydecrease blood glucose at this dose level (119901 gt 005)Figure 1 Blood glucose level was initially increased in the1st hr after treatment and started decreasing steadily from2nd hr up to 5th hr of treatment in diabeticmice that receivedmethanolic extract of Artemisia afra at 750mgkg dose levelThus blood glucose level of diabetic mice did not come below2635mgdL throughout the 5 hr posttreatment period forthis dose


Table 2 Hypoglycemic effect of aqueous and methanolic extracts of Artemisia afra in alloxan induced diabetic mice (119899 = 5)

Treatment Mean change of blood glucose (mgdL) in hours after treatment (119899 = 5) reduction0 hr 1 hr 2 hr 3 hr 4 hr 5 hr

Diabetic control 322 plusmn 63 3855 plusmn 68 3922 plusmn 59 38225 plusmn 61 3847 plusmn 62 3772 plusmn 64 003AQ Artemisia 500 3505 plusmn 34 3497 plusmn 37 304 plusmn 32 31175 plusmn 40 280 plusmn 38lowast 26375 plusmn 36lowast 24AQ Artemisia 750 3303 plusmn 22 2743 plusmn 18 2093 plusmn 16 186 plusmn 18lowast 152 plusmn 15lowast 1423 plusmn 13lowast 569AQ Artemisia 1000 3517 plusmn 101 4057 plusmn 98 4027 plusmn 112 3807 plusmn 106 3495 plusmn 102 3245 plusmn 99 76Meth Artemisia 500 2932 plusmn 77 3467 plusmn 96 350 plusmn 52 3305 plusmn 64 329 plusmn 61 283 plusmn 72 34Meth Artemisia 750 35425 plusmn 38 3795 plusmn 40 3517 plusmn 52 30375 plusmn 46 28775 plusmn 44 2635 plusmn 35 257Meth Artemisia 1000 3195 plusmn 45 1998 plusmn 50 204 plusmn 48 1806 plusmn 22lowast 1665 plusmn 18lowast 1605 plusmn 22lowast 498Normal control 1105 plusmn 022 112 plusmn 021 114 plusmn 023 105 plusmn 028 9525 plusmn 031 101 plusmn 025 8lowast119901 lt 0005 as compared to diabetic control

0

50

100

150

200

250

300

350

400

450

Bloo

d gl

ucos

e lev

el (m

gdL

)

Hours after treatment

Diabetic controlNormal controlAlcho Artemisia 500Alcho Artemisia 1000

Alcho Artemisia 750AQ Artemisia 500AQ Artemisia 750AQ Artemisia 1000

0h 1h 2h 3h 4h 5h

Figure 1 Comparison of hypoglycemic effect of aqueous and methanolic extracts of Artemisia afra at dose of 500mgKg 750mgKg and1000mgKg

33 Phytochemical in Aqueous Extracts of Artemisia afraAerial Part The aqueous extract of Artemisia afra aerialpart was screened positive for presence of chromophoressaponins phosphosteroid and withanoids flavonoids tan-nins and anthraquinone but showed negative result forpresence of polyphenols cyanogenic glycoside anthranidescarotenoids phenolic glycoside cardiac glycoside and alka-loids (Table 3)

4 Conclusion

The cute toxicity study indicates that the aqueous extract ofArtemisia afra is found to be nontoxic through oral routesince the LD

50is greater than 5000mgKg Aqueous and

methanolic extracts of this medicinal plant have significantly

reduced blood glucose level in alloxan induced diabetic miceAlloxan is known to induce free radical production and causetissue injury and the pancreas is especially susceptible tothe action of alloxan induced free radical damage It hasbeen suggested that regeneration of islet beta cell followingdestruction by alloxan may be the primary mechanism of therecovery of alloxan-injected rats following drug administra-tion [1] Therefore the extract could be inducing pancreaticcell regeneration In addition aqueous extracts of these plantswere tested positive for presence of well-known antioxidantphytochemicals like flavonoids polyphenols and tanninsTherefore this medicinal plant can be good candidate to beused as alternative treatment of diabetes However intensiveinvestigations must be conducted on the fractions of theextracts to identify pharmacological active compound(s) to


Table 3 Results of preliminary phytochemical analysis in aqueous crude extracts of Artemisia afra aerial part

Secondary metabolite Reagents and method Indicators Artemisia afraChromophores 10mL distilled water + heat for 30min Yellow to red color +++Polyphenols 1 FeCl3 + 1mL of K3Fe (CN)6 Green blue color minus minus minus

Saponins 30mL distilled water + heat (5min) Formation of honey comb froth +++Phytosteroids and withanoids CHCl3 + conc H2SO4 Red reddish brown or violet color +++Flavonoids 5 drops of 2 lead acetate Yellow or orange color +++Tannins 3 drops of 1 K3[Fe(CN)6] + 3 drops of conc NH3 Formation of color +++Anthraquinone glycosides 2NHCL benzene 10 ammonia Red color +++

elucidate mechanism of action and to assure their safety andset appropriate dose These herbs reduce blood glucose andmay have beneficial effects on complications of diabetes

As compared to methanolic extracts of Artemisia afrathe aqueous extracts of Artemisia afra showed a higher bloodglucose lowering activity at relatively lower dose (750mgkg)than the methanolic extract at dose of 1000mgkg Thisfindingmanifests the presence of the active ingredients in theaqueous extract at higher concentration than in methanolicextract indicating the most active portions are polar

Conflict of Interests

The authors declare no conflict of interests

References

[1] P A Akah S U Uzodinma and C E Okolo ldquoAntidiabeticactivity of aqueous and methanol extract and fractions ofGongronema latifolium (asclepidaceae) leaves in alloxan dia-betic ratsrdquo Journal of Applied Pharmaceutical Science vol 1 no9 pp 99ndash102 2011

[2] C C June L H Wen H A Sani et al ldquoHypoglycemic effectsof Gynura procumbens fractions on streptozotocin- induceddiabetic rats involved phosphorylation of GSK3120573 (Ser-9) inliverrdquo Sains Malaysiana vol 41 no 8 pp 969ndash975 2012

[3] A Arya M A Abdullah B S Haerian and M A MohdldquoScreening for hypoglycemic activity on the leaf extracts of ninemedicinal plants in-vivo evaluationrdquo E-Journal of Chemistryvol 9 no 3 pp 1196ndash1205 2012

[4] C Stalin KVivekanandan andE Bhavya ldquoIn vitro antidiabeticactivity of Cardiospermum halicacabum leaves extractsrdquo GlobalJournal of Medical Research vol 13 no 7 pp 41ndash43 2013

[5] P O Osadebe P F Uzor E O Omeje M O Agbo and WO Obonga ldquoHypoglycemic activity of the extract and fractionsof Anthocleista vogelii (Planch) stem barkrdquo Tropical Journal ofPharmaceutical Research vol 13 no 9 pp 1437ndash1443 2014

[6] A A Shetti R D Sanakal and B B Kaliwal ldquoAntidiabeticeffect of ethanolic leaf extract of Phyllanthus amarus in alloxaninduced diabetic micerdquo Asian Journal of Plant Science ampResearch vol 2 no 1 pp 11ndash15 2012

[7] A Shetti and B B Kaliwal ldquoHypoglycemic activity of ethanolicleaf extract of Phyllanthus amarus in alloxan induced diabeticmicerdquo European Journal of Experimental Biology vol 5 no 1pp 26ndash29 2015

[8] M Burits K Asres and F Bucar ldquoThe antioxidant activity ofthe essential oils of Artemisia afra Artemisia abyssinica and

Juniperus procerardquo Phytotherapy Research vol 15 no 2 pp 103ndash108 2001

[9] A Kicel A Kurowska and D Kalemba ldquoComposition of theessential oil of Ocimum sanctum L Grown in Poland duringvegetationrdquo Journal of Essential Oil Research vol 17 no 2 pp217ndash219 2005

[10] G Sacchetti M Muzzoli G A Statti et al ldquoIntra-specific bio-diversity of Italianmyrtle (Myrtus communis) through chemicalmarkers profile and biological activities of leaf methanolicextractsrdquo Natural Product Research vol 21 no 2 pp 167ndash1792007

[11] H Richard T R Bell and J H Robert ldquoCurrent researchreview animal models of diabetes mellitusrdquo Journal of SurgicalResearch vol 35 pp 433ndash460 1983

[12] J E Hilaly and B Lyoussi ldquoHypoglycaemic effect of thelyophilised aqueous extract of Ajuga iva in normal and strep-tozotocin diabetic ratsrdquo Journal of Ethnopharmacology vol 80no 2-3 pp 109ndash113 2002

[13] M Kaleem M Asif Q U Ahmed and B Bano ldquoAntidi-abetic and antioxidant activity of Annona squamosa extractin streptozotocin-induced diabetic ratsrdquo Singapore MedicalJournal vol 47 no 8 pp 670ndash675 2006

[14] A N Nagappa P A Thakurdesai N V Rao and J SinghldquoAntidiabetic activity of Terminalia catappa Linn fruitsrdquo Journalof Ethnopharmacology vol 88 no 1 pp 45ndash50 2003

[15] A Sepici I Gurbuz C Cevik and E Yesilada ldquoHypoglycaemiceffects of myrtle oil in normal and alloxan-diabetic rabbitsrdquoJournal of Ethnopharmacology vol 93 no 2-3 pp 311ndash318 2004

[16] E J Hilaly Z H Israili and B Lyoussi ldquoAcute and chronictoxicological studies of Ajuga iva in experimental animalsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 43ndash50 2004

[17] J W Ogwal-Okeng C Obua and W W W AnokbonggoldquoAcute toxicity effects of the methanolic extract of Fagarazanthoxyloides (Lam) root-barkrdquo African Hhealth Sciences vol3 no 3 pp 124ndash126 2003

[18] A Debella Manual for Phytochmical Screening of MedicinalPlants Department of Drug Research Ethiopian Health andNutrition Research Institute Addis Ababa Ethiopia 2002

[19] T A Loomis and A W Hayes Loomisrsquos Essentials of ToxicologyAcademic Press San Diego Calif USA 4th edition 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


plant medicines are used throughout the world for treatmentof diabetes mellitus [7]

Artemisia afra is locally called ldquochikugnrdquo also knownas African wormwood It belongs to family Asteraceae It isone of the best known and widely used plants in traditionalmedicine Various preparations such as infusions decoctionsmolasses and alcohol extracts of Artemisia afra are used forthe treatment of coughs colds chills stomachache and drydyspepsia and as a purgative and for the cure of smallpox andmalaria The herbaceous leaves of this plant contain between03 and 14vwof a bluish-green essential oil with a strongfragrance [8] This study was designed to systematicallyevaluate hypoglycemic effect of aqueous and methanolicextracts of Artemisia afra in comparison with each other andstandard hypoglycemic agents like glibenclamide

2 Materials and Methods

21 Plant Material Fresh aerial parts of Artemisia afrawere collected during their flowering period when plantsgive highest yield [9] The plants were collected from theirnatural habitat in Bale Zone around Goba town near theRiver Togona (7∘01015840N 39∘91015840E) 2743 meters above sea levelAuthentication and taxonomic identification of the plantsamples were done by using standard botanical monographsat Addis Ababa University Science Faculty Herbarium

22 Plant Extraction

221 Aqueous Extraction Fresh aerial parts of Artemisiaafra were cleaned with distilled water and air dried at roomtemperature It was ground into powder using grinding millPowdered aerial part of Artemisia afra weighing 220 gm wassorely mixed with 450mL distilled water in flat bottom flaskon orbital shaker for 24 hours Distilled water was addeduntil completely wet and the dried plant powder was suffi-ciently covered After gentle maceration for 24 hours plantmaterials were filtered separately through mousseline clothinto 1000mL beakers The obtained filtrate was deep frozenat minus70∘C and lyophilized (freeze dried) using lyophilizerto collect crude aqueous extract [10] The aqueous extractswere stored in desecrator at room temperature until usedfor experiment The above aqueous extracted powders ofArtemisia afra provided yield of 21 gm (95 ww)

222 Methanolic Extraction Fresh aerial parts of Artemisiaafra were cleaned with distilled water and air dried at roomtemperature A dried and powdered aerial part of Artemisiaafra weighing 165 gm was sorely mixed with 400mL of95 methanol in flat bottom flask on orbital shaker Aftergentle maceration for 24 hours methanolic extracts werefiltered throughWhatman filter paper chartThe filtrate fromArtemisia afra was concentrated under reduced pressureusing rotary evaporator at 50 rpmand40∘Cbath temperatureFinally concentratedmethanolic extract was collected in vialsand placed on water bath at 40∘C to evaporate methanolcompletely The crude methanolic extract of Artemisia afrahad a yield of 13 gm (78 ww)

23 Inducing Experimental Diabetes Diabetic condition wasinduced to Swiss albino mice of both sexes weighing 25ndash35 gafter overnight fasting by intraperitoneal injection of freshlyprepared alloxan (200mgKg IP) dissolved in distilled water[11] Three days (72 hr) after the injection of alloxan theanimals were fasted for 16 hours and their blood glucoseconcentration was determined with glucometer Animalsshowing stabilized diabetes (FBG ge 250mgdL) on the thirdday after alloxan injection were selected for the experiment[12] Mice that did not show diabetic condition on the thirdday were excluded from the experiment

24 LaboratoryAnimals Healthy Swiss albinomiceweighing25 gndash35 gwere obtained fromEthiopianHealth andNutritionResearch Institute (EHNRI) animal unit and kept in labora-tory animal house of Drug Research Department during theexperiment The animals were provided standard pellet foodand tapwater ad libitumwith 12-hour lightdark photoperiodThe experiment was conducted according to the ethicalnorms approved by Animal Ethics Committee Guidelines ofthe EHNRI

25 ExperimentalDesign Alloxan induced experimental dia-betic Swiss albino mice (FGB ge 250mgdL) were dividedin to 9 test groups with 5 mice in each group that matchwith weight and fasting blood glucose level [13] Groups-1ndash3 included diabetic mice treated with crude aqueous extractof Artemisia afra at dose of 500mgKg 750mgKg and1000mgKg PO Groups-4ndash6 included diabetic mice treatedwith crude methanolic extract of Artemisia afra at doseof 500mgKg 750mgKg and 1000mgKg PO Group 7included diabetic mice treated with standard drug gliben-clamide (standard group) 10mgkgPO [14]Group 8 includeduntreated diabeticmice (diabetic control) given equal volumeof distilled water PO Group 9 included nondiabetic referencegroup (normal control) given equal volume of distilledwater PO Blood glucose level of these mice was measuredusing glucometer at interval of 1 hour after treatment forconsecutive 5 hr

26 Acute Toxicity Test in Mice The mice were divided into10 groups with 6 animals in each group matched for weightand sex These animals were housed 3 mice per sex per cagein a well-ventilated room with 12 hr cycle of day and nightlight conditions and temperature was maintained at around25∘C Food was withdrawn for the 12 hr fasting period beforeand for 24 hr fasting period after oral administration of thesingle doses crude aqueous extract [12 15] Aqueous extractof Artemisia afra was aseptically suspended in distilled waterand administered at a single doses of 0 1000 2000 30004000 5000 7500 10000 and 12000mgKg body weightby gavages perorally (PO) to the test groups The controlgroups were given an equal volume of water The change ingeneral behaviors of the mice was continuously monitored[16] The number of dead mice was recorded and used in thecalculation of mean lethal dose (LD

50) value All surviving

animals were euthanized with diethyl-ether at day 14 asdescribed by [17]























50) is












0

50

100

150

200

250

300

350

400

450

Bloo

d gl

ucos

e lev

el (m

gdL

)




0h 1h 2h 3h 4h 5h



4 Conclusion













References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































50) is












0

50

100

150

200

250

300

350

400

450

Bloo

d gl

ucos

e lev

el (m

gdL

)




0h 1h 2h 3h 4h 5h



4 Conclusion













References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















0

50

100

150

200

250

300

350

400

450

Bloo

d gl

ucos

e lev

el (m

gdL

)




0h 1h 2h 3h 4h 5h



4 Conclusion













References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article hypoglycemic effect of aqueous and...

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