Extraction and Demonstration of Uterotonic Activity from the Root of Steganotaenia Araliacea Hochst

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1 Original Article Medical Jurnal f Zambia, Vl. 44, N.3: (2017) Extractin and Demnstratin f Utertnic Activity frm the Rt f Stegantaenia Araliacea Hchst 1* FM Gma, C Ezeala, J Nyirenda, D Chuba, L Prashar, N Simfukwe, C Lengwe 1 Department f Physilgical Sciences, Schl f Medicine; 2 Department f Chemistry, Schl f Natural Sciences; 3 Department f Bilgical Sciences, University f Zambia, Lusaka, Zambia ABSTRACT Backgrund: The rt f Stegantaenia araliacea is used fr assisting labur in flk medicine. Recent reprts indicate that the rt culd pssess utertnic substances. Objectives: The study aimed t evaluate three methds fr the extractin f utertnic principles frm the rt f S. araliacea grwing in Zambia. Methds: Rts f the plant were cllected frm Chngwe District f Zambia. The air-dried rts were size-reduced, and the pwdered material extracted with ht ethanl, ht distilled water, and cld distilled water. The slvent extracts were 0 cncentrated and dried at 110 C. Slutins f the ht aqueus and cld aqueus extracts were prepared in distilled water and used fr rgan bath experiments t demnstrate utertnic activities using strips f pregnant rat uterus. The frequencies and amplitude f cntractile frces were recrded. The amplitudes were pltted against lg cncentratin f extract with GraphPad Prism sftware, and the EC50 values determined. Results: The percentage yields were 31.3 % fr the ht aqueus extract, 8.15 % fr the ethanlic extract, and 3.27 % fr the cld aqueus extract. The cld *Crrespnding Authr Dr. Fastne M Gma PhD, MBChB Department f Physilgical Sciences, Schl f Medicine, University f Zambia, Ridgeway Campus, Lusaka 10101, Zambia; gmafm@unza.zm gmafm@yah.c.uk aqueus extract shwed higher ptency (EC50 f 0.54 mg/ml) cmpared t the ht aqueus extract (EC50 f 2.09 mg/ml). Cnclusin: Rt extracts f S. araliacea pssess demnstrable utertnic effects. Extractin f the rts fr this purpse culd benefit frm preliminary defatting with rganic slvents, fllwed by successive extractin with ht and cld water. INTRODUCTION The demand fr revitalizatin f health care is ever n increase, parallel t this is the demand f services f Traditinal Medicines. The use f Traditinal Medicines all ver the wrld is demnstrating t be phenmenal. Traditinal medicine has a great ptential f therapeutic benefits in its cntributin t mdern medicine. Presently mre than 30% f marketed mdern medicines are derived directly r 1 indirectly frm medicinal plants. WHO estimates that 80% f the ppulatin in develping cuntries depends n traditinal 2 medicines fr their primary health care. The wrld market fr Traditinal Medicines has reached US$60 millin, with annual grwth rates between 5 and 15%. Traditinal medicinal plants and prducts can be targeted fr patent claims since they have becme f great interest t the glbally rganized 3 herbal drug and csmetic industries. Zambia is a member f Wrld Trade Organizatin since Keywrds: Childbirth, extractin methds, medicinal plant, ptency, Stegantaenia araliacea, utertnic effect 125

2 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) Under Article 27 f Trade Related Intellectual Prperty Rights Agreement; members will prvide fr the prtectin f plant varieties either by patents r by an effective sui generis system r by any 4 cmbinatin theref. With the Dha declaratin n TRIPS agreement and Public Health, it becmes imprtant fr us t prtect ur Intellectual Prperty Rights in the frm f Indigenus Knwledge 5 Systems (IKS). IKS generally refers t age-ld lng-standing traditins and practices invlving wisdm, knwledge and teachings f cmmunities and 6 encmpassing traditinal technlgies. Fr the mst part, the knwledge is passed dwn thrugh generatins, usually frm designated persns t thers using varius mdes f cmmunicatin like stry-telling, rituals, sngs r laws. This framewrk enables grwth and sustainability f cmmunities which invariably includes sustaining the health f cmmunity members. It can, therefre, be said that a prtin f what indigenus knwledge imparts is traditinal 7 medicine knwledge. These therapeutic practices have been in existence fr hundreds f years befre the develpment f cnventinal scientific medicines e.g. mrphine frm Papever smniferum, quinine frm Cinchna fficinalis, strychnine frm Srychnx nuxvmica and ephedrine 8 frm Ephedra sinica. T preserve these knwledge systems, the reginal ffice in Brazzaville, respnsible fr African regin has facilitated activities leading t the present trend f recgnitin and develpment f herbal medicines in Africa by varius internatinal rganisatins. This calls fr psitive respnse frm all the medical and pharmaceutical research scientists f African rigin. The decade was tagged as 'Decade st f Traditinal Medicine in Africa and 31 August is celebrated every year as the, 'Traditinal Medicine 8 Day'. Traditinal health practitiners (THPs) have used herbal plants and ther remedies fr beneficial effects during pregnancy, t induce labur, in the remval f retained placenta and management f 9 pst-partum bleeding. The effects f these medicines have been reprted infrmally, mstly in circumstances where their effects have been rather excessive causing injury t the uterus (rupture) r death by peri partum heamrrhage. These exaggerated respnses are suspected t be related t verdsing since there are n regulated traditinal measurements. This study cmpared utertnic activities in extracts f Stegantaenia araliacea Hchst btained using three different slvents and cnditins. The purpse was t ptimise the extractin prcess fr utertnic principles frm the rt. METHODS A. Plant cllectin and identificatin The investigatrs cnsulted with lcal peple in Chngwe District t help lcate the plant. Rt samples f the plant were cllected frm the Chngwe District f Zambia using standard prcedures. The samples were immediately placed in a black plastic bag t prevent any pssible slar degradatin f active cmpunds. Fr taxnmic verificatin, specimens f the plant were dried by placing them in a plant press and sun drying fr abut three days while changing bltting papers each day. The plant was identified t species level in the field using prir knwledge and later cnfirmed by physical cmparisn with specimens in the herbarium at University f Zambia (UNZA) and by cnsulting the taxnmic literature. Vucher specimens were depsited in the UNZA herbarium. B. Prcessing and extractin f the rts Preliminary Treatment The rts were freed f sil and debris, and size reduced using a clean labratry axe and then airdried in the shade fr 14 days under bltting paper. Misture cntent was mnitred t a cnstant level 126

3 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) f 6 % (wt/wt), as a functin f weight lss. A labratry blender (Warring Cmmercial, Christisn Particle Technlgies, UK) was used t reduce the samples further, and the pwdered material stred in Ziplc plastic bag. Extractin The pwdered sample was subjected t three types f extractin; sxhlet, ht and cld water extractin. Sxhlet extractin using ethanl (distilled) Apprximately, 302 g f the pwdered sample was extracted with abut 2600 ml f distilled ethanl fr abut 10 hrs using sxhlet apparatus. This was fllwed by slvent evapratin using a rtary evapratr (Rtavapr R11, BUCHI, Switzerland) at 45 C. The resultant glue-like residue was further dried in an ven at 80 C fr 48 hurs. The resulting dry extract was stred in the refrigeratr at 4 C. Ht water extractin Fifty grams (50.0 g) f the pwdered sample was extracted with 800 ml f biling distilled water fr 10 min. The extract was cled t rm temperature, and then centrifuged at 4000 rpm fr 10 min. The supernatant was decanted and evaprated t near dryness using a ht plate, and further dried in an ven at 110 C. The dry extract was stred in the refrigeratr at 4 C until required fr analysis. Cld water extractin Apprximately, g f the pwdered sample was saked in 1,750 ml cld distilled water vernight. The mixture was centrifuged (4000 rpm fr 10 min) and decanted t btain a clear slutin. The decanted supernatant was evaprated t near dryness with a ht plate, and then dried in an ven at 110 C. The extract was then stred in the refrigeratr at 4 C. C. Habituatin f the animals and islatin f uterine muscle Pregnant Female Wistar albin rats aged 7 weeks and weighing between 180 t 200 g were btained frm the Schl f Veterinary Sciences f the University f Zambia, Lusaka. They were hused in standard metal cages, maintained at rm temperature (20-24 C), 12 hurs light/dark cycles, and allwed free access t water and rdent pellets (btained frm Tiger Animal Feeds Ltd, Lusaka). D. Demnstratin f utertnic activity n islated uterine muscle th On the 10 day f gestatin, the rats were sacrificed by cervical dislcatin, immediately dissected and the uteri extracted. Any adherent fat, cnnective tissues, and the fetuses were remved. The uteri were placed in De Jaln's physilgical slutin at rm temperature, and lngitudinal strips (abut 2 cm lng) were cut ut frm the cleaned uteri and munted in the rgan baths (AD Instruments) cntaining abut 25 ml De Jaln's slutin and maintained at abut 32 C. The frce transducers were cnnected t PwerLab and LabTutr (AD Instruments). Baseline spntaneus cntractins were recrded fr 5 minutes. Thereafter, slutins f the cld aqueus extract were added t give final bath cncentratins varying frm mg/ml t mg/ml with 3 washes between additins f each cncentratin. The cntractins prduced by each cncentratin were recrded n the Lab Tutr fr 5 minutes. Using the same tissue, the abve prcedure was repeated fr the ht aqueus extract. The prcedure was nt carried ut fr the alchlic extract as it failed t disslve in water r saline. Oxytcin was used as psitive cntrl; it als served t demnstrate sustained viability f the islated uterine tissues during the experiments. E. Data Analysis The cntractins f the uterine muscle prduced by graded dses f the ht and cld crude aqueus extracts were analysed t determine the frequency (number f cntractins per minute) and magnitude f the frce f cntractin (the difference between the peak and baseline). Prism GraphPad sftware (versin 7) was used t plt the respnse against the lgarithm f cncentratins f the extract. The EC 50 values and Hill Slpe factrs were determined. 127

4 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) RESULTS Descriptin f the extracts The alchlic extract yielded a reddish-brwn ily viscus residue. This culd nt disslve in water r nrmal saline and was nt analysed further. The cld-water extract gave a brwnish-black gummy residue that was readily sluble in water. This was recnstituted with cld distilled water and used fr the islated rgan experiments. The ht extract was a semi-slid dark-brwn residue that was als sluble in water. After recnstitutin with distilled water, it was als used fr the rgan bath studies. Figure 1: Physical appearance f: a. Htethanlic extract, b. Ht-water extract, and c. Cld-water extract a. b. Table 1: Percentage yield f crude extracts f rt f Stegantaenia araliaceais Hchst Mass f pwder (g) Mass f extract (g) Organ Bath Studies Sxhlet Yield (%) 8.15 Ht water Cld water The cld aqueus extract markedly increased the amplitude and frequencies f cntractins f the uterine muscles in a cncentratin dependent manner, with the lwest tested dse, mg/ml, prducing demnstrable increase in cntractin frequency. The ht aqueus extract prduced bservable increases in the frequencies and amplitudes f cntractins f the uterine smth muscle, but the effects were smaller than the effects f similar cncentratins f the cld extract. Oxytcin prduced a much higher effect. Figure 2: Baseline spntaneus rhythmic cntractins f the rat uterine smth muscle c. The Percentage Yield Ht water extract f rt exhibited highest yield (31.3%) fllwed by ethanlic Sxhlet extract (8.15%). The cld water extract shwed the lwest yield f 3.27%. 128

5 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) Figure 3: Cntractins induced by different cncentratins f the cld aqueus extract (Final bath cncentratins f the cld aqueus extract were a) mg/ml, b) mg/ml, c) mg/ml, d) mg/ml, e) mg/ml and f) mg/ml, respectively). Figure 4: Cntractins induced by different cncentratins f the ht aqueus extract ( T h e f i n a l b a t h cncentratins f the ht aqueus extract were a) mg/ml, b) mg/ml, c) mg/ml and d) mg/ml, respectively). 129

6 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) Figure 5: Cntractins induced by 5 iu/ml f xytcin (Lg (agnist) vs. respnse -- Variable slpe (fur parameters), EC50 and Hill Slpe factr fr cld extract were and 2.09, respectively) Figure 7: Mdified cncentratin-respnse curve fr the ht extract Frce Cncentratin (Dse) - Effect Analysis Quantitative analysis f the cncentratin respnse relatinships shwed that the cld extract was mre ptent than the ht extract with a lwer EC f 0.58mg/ml cmpared t EC f 1.06 mg/ml fr the ht extract. The semi-lg plt f the cld extract was cmpletely sigmidal. Plt f the ht extract was remarkably sigmid; hwever, utlying pints were remved in rder t cnstruct a mre representative curve frm which the EC was 50 calculated. Figure 6: Cncentratin (Dse) -respnse curves fr the ht and cld extracts. Frce Lg (DseX100) (Lg (agnist) vs. respnse -- Variable slpe (fur parameters) reanalysed mitting utlying pints, EC 50 and Hill Slpe factr, fr ht extract, were 1.06 and 0.76, respectively) DISCUSSION Lg (DseX100) Extractin f S. araliacea rt with ethanl, ht and cld water resulted in the ht extract having the highest percentage yield f the extract. The ethanlic extract, with a mderate percentage yield (8.15 %) was ily, suggesting that the rt cntains a reasnable quantity f fatty material. Defatting f the pwdered rt with rganic slvents as a preliminary step in the prcessing f the rt fr analysis f its biactivity is recmmended,. Furthermre, the il frm the rt f this plant culd pssess prperties that may be useful medicinally and/r industrially. Plant ils are widely used in medicine, csmetlgy, armatherapy, bifuel prductin, and ther 12, 13, 14, 15, 16 industrial applicatins. The study therefre recmmends further extractin and distillatin f the rt ils and investigatin f their ptential usefulness in these areas. Cld Ext Ht Ext 130

7 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) The ht aqueus extract shwed the highest percentage yield, but, utertnic biactivity was higher with the cld aqueus extract, which demnstrated a much higher ptency in the rgan bath studies. This finding prbably suggests that ht water extractin remved significant quantities f substances that have n utertnic activities 17 (artefacts), leaving a purer cld aqueus extract. Based n these bservatins n the percentage yields and the biactivities f the extracts, the study further emphasizes that extractin prcesses fr the recvery f utertnic principles frm the rt f S. araliacea culd benefit frm preliminary defatting f the pwdered material with rganic slvents. Ht water extractin t remve artefacts culd fllw this step, befre cld aqueus extractin f the bilgically active materials frm the rt. The study cnfirms the presence f utertnic principles in the rt extract. This bservatin is 18 cnsistent with previus reprt. Imprtantly, it has prvided scientific justificatin fr the use f the rt by traditinal practitiners in maternal healthcare and specifically fr assisting labur. Furthermre, the extract culd prvide a lead fr the develpment f an rally active drug that culd serve in bstetrics fr assisting wmen with prlnged labur. Its further develpment shuld include purificatin, identificatin, and chemical characterizatin f the active principles. These steps shuld precede preclinical txiclgy, safety pharmaclgy, and clinical tests in human and/r large animal subjects. The material can als be useful fr medical abrtin fr pregnant wmen at 19, 20 risk. Inductin f uterine smth muscle cntractin invlves cmplex interplay f different signalling mechanisms that include neurnal, endcrine, mechanical, and metablic prcesses. Current evidence shws that pathways mediated by acetylchline, xytcin, histamine, and prstaglandins may be invlved, while calcium and ther divalent catins and insitl triphsphate play 21, 22 rles as secnd messengers. The exact mlecular mechanism by which the cnstituents f S. araliacea interact with these physilgical prcesses t prduce uterine cntractin is nt bvius. Chemical characterizatin f the extract and in depth pharmaclgical studies t elucidate these physilgical interactins is in prcess. In cnclusin, extractin f pwdered rt f S. araliacea with ethanl, ht water and cld water yielded crude extracts with significant utertnic activities. The cld aqueus extract demnstrated higher utertnic ptency than the ht water extract. This article recmmends preliminary defatting f the pwder with rganic slvents t remve the ils, fllwed by ht-water extractin t remve artefacts that are nt utertnic, befre final cldwater extractin f the active ingredients. The prduct btained by this prcess shwed great ptential fr develpment int a clinically useful drug fr bstetric use. This is wrk in prgress, and much mre wrk is nging t islate and chemically characterise the active principles and pssibly translate this prduct t the bench side. ACKNOWLEDGEMENT This wrk was supprted by a research grant frm Natinal Science & Technlgy Cuncil (NSTC), thrugh the Ministry f Science &Technlgy f Zambia. DECLARATION OF INTEREST The authrs declare n cnflict f interest assciated with this wrk and have n relevant affiliatins r financial invlvement with any rganizatin r entity with a financial interest in r financial cnflict with the subject matter r materials discussed in this write-up. This includes emplyers, cnsultancies, hnraria, patents (received r pending), expert testimny, stck wnership, r ryalties. 131

8 Medical Jurnal f Zambia, Vl. 44, N.3: (2017) REFERENCES 1. WHO Traditinal Medicine Strategy ; W H O A v a i l a b l e f r m : 1/ _eng.pdf/ [Accessed n th 19 August, 2017]. 2. Kasil Ossy, M.J., Trapsida,J.M., Mwikisa, C.N., Dikassa Lusamba, P.S. An Overview f the Traditinal Medicine Situatin in the African Regin (WHO African Health Observatry). Available Frm: https: //www. ah. afr.wh.int/sites/default/files/ahm/reprts/32/ a h m s p e c i a l - i s s u e - p a g e s - th 7t15.pdf/[Accessed n 19 August, 2017]. 3. Kartal, M. Intellectual prperty prtectin in the natural prduct drug discvery, traditinal herbal medicine and herbal medicinal prducts. Phytther Res. 2007; 21: TRIPS and pharmaceutical patents, WTO OMC Fact Sheets. September Available Frm: ipsfactsheet_pharma_2006_e.pdf/ [Accessed rd n 23 August, 2017]. 5. DOHA declaratin: Wrld Trade Organizatin Available frm: wt.rg/english/res_e/bksp_e/ddec_e.pdf/ th [Accessed n 25 August, 2017]. 6. Gruber, C.W., O'Brien, M. Utertnic plants and their biactive cnstituents. Planta Med. 2011; 77: Egeland, G.M., Harrisn, G.G. Health disparities prmting Indigenus Peples' health thrugh traditinal fd systems and selfdeterminatin. In Kuhnlein, H.V., Erasmus, B, Spigelaski, D., Burlingame, B., editrs. Indigenus peples' fd systems and wellbeing interventins and plicies fr healthy cmmunities. ISBN Elujba, A.A., Odeleye, O.M., Ogunyemi, C.M. Traditinal medicine develpment fr medical and dental primary health care delivery system in Africa. Afr. J. Trad. CAM 2005; 2: Gruber, C.W., O'Brien M. Utertnic plants and their biactive cnstituents. Planta Med 2011; 77: Jnes, W. P., & Kinghrn, A. D. Extractin f plant secndary metablites. Natural prducts islatin 2012; 864: Misra, L., & Wagner, H. Extractin f biactive principles frm Mucuna pruriens seeds. Indian Jurnal f Bichemistry and Biphysics 2007; 44: Asif, M. Health effects f mega-3, 6, 9 fatty acids: Perilla frutescens is a gd example f plant ils. Oriental Pharmacy & Experimental Medicine 2011; 11: Chhetri, A. B., Tang, M. S., Budge, S. M., Watts, K. C., & Islam, M. R. Nn-edible plant ils as new surces fr bidiesel prductin. Internatinal Jurnal f Mlecular Sciences 2008; 9: Isman, M. B. Plant essential ils fr pest and disease management. Crp Prtectin 2000; 19: Martini, N. and Schell, J. S. Plant ils as fuels: present state f science and future develpments: Springer Science & Business Media Setzer, W. N. Essential ils and anxilytic a r m a t h e r a p y. N a t u r a l P r d u c t Cmmunicatins 2009; 4: Azmir, J., Zaidul, I., Rahman, M., et al. Techniques fr extractin f biactive cmpunds frm plant materials: a review. Jurnal f Fd Engineering 2013; 117: Lwiindi, L., Gma, F., Mushabati, F., Prashar, L., & Chng, K. Jurnal f Med Sc & Tech 2015; 4: ACOG. Practice bulletin n. 143: Medical Management f First-trimester Abrtin. Obstetrics and Gyneclgy 2014; 123: Kulier, R., Kapp, N., Gülmezglu, A. M., Hfmeyr, G. J., Cheng, L., and Campana, A. Medical methds fr first trimester abrtin. The Cchrane Data base Cyst Rev 2011;11:CD Aguilar, H. N., and Mitchell, B. Physilgical pathways and mlecular mechanisms regulating uterine cntractility. Human Reprductin Update 2010; 16: Carsten, M. E., and Miller, J. D. A new lk at uterine muscle cntractin. American Jurnal f Obstetrics and Gyneclgy 1987; 157: