Antihypertensive effect of Heterophragma adenophyllum Wall. ex G.Don in dexamethasone-induced hypertensive Wistar rats

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1 Reserch Article Antihypertensive effect of Heterophrgm denophyllum Wll. ex G.Don in dexmethsone-induced hypertensive Wistr rts Mohmmd Fizn Bht 1 *, Nrender Goel 1, Mushir Hussin Msoodi 2, Mohmmd Akr Dr 2 ABSTRACT Bckground: Hypertension is one of the most common crdiovsculr diseses in the world, nd often, the tretment is limited y huge side effects due to llopthic medicine regimens. Ojective: The purpose of this study ws to investigte the ntihypertensive properties nd effects on iochemicl prmeters of Heterophrgm denophyllum Wll. ex G.Don on dexmethsone (DEX)- induced hypertensive Wistr rts. Methods: H. denophyllum ethnolic extrct (HAE) ws orlly dministered to DEX-induced hypertensive Wistr rts, nd their lood pressures were mesured y til-cuff method. Plsm level of insulin, lood glucose, nd lipid profile ws mesured y the enzymtic method, nd lipid peroxidtion (LPO), ctlse (CAT), nd nitrite (NO) levels were mesured y mlondildehyde (MDA), Beers, nd Griese methods, respectively. Results: HAE could lower the lood pressure of Wistr rts dose-dependently y periorl dministrtion. Menwhile, the plsm levels of glucose nd insulin were decresed y HAE dministrtion, nd there ws slight llevition in lipid profile. Furthermore, CAT ctivity ws significntly improved with HAE while LPO ctivity nd NO ctivity significntly reduced. Finlly, crdic muscle firosis cused due to DEX ws lso normlized. Conclusion: HAE my e effective in lowering lood pressure, nd this cn perhps e ttriuted to its positive effect on underlying iochemicl nd histologicl mechnisms, which need further investigtions. KEY WORDS: Heterophrgm Adenophyllum, Hypertension, Phrmcognosy, In vivo ntihypertension evlution, Antioxidnt evlution INTRODUCTION Crdiovsculr diseses ccount for nerly out 17 million deths yer, which roughly trnsltes into one-third of the totl deths worldwide. [1] Of these, complictions rising from hypertension ccount for 9.4 million deths every yer. [2] Of the two min types of hypertension, primry hypertension nd the type of hypertension for which no medicl cuse cn e found ccounts for out 90% of the totl cuse of hypertension. [3] Persistent hypertension is one of the risk fctors for strokes, hert ttcks, hert filures, nd rteril neurysm nd is leding cuse of chronic kidney filure. [4] All this gruesome dt unequivoclly point to the fct tht hypertension is Access this rticle online Wesite: jprsolutions.info ISSN: disese tht demnds to e tken cre of, without discriminting etween the system of medicine, e it llopthic or trditionl plnt-sed ntihypertensive drugs. Plenty of Indin plnts hve een proved to show ntihypertensive properties, mong which the one tht needs specil mention include, Cssi sus (fmily: Ceslpinicee), [5] Blond psyllium (fmily: Plntgincee), [6] nd Lumnitzer rcemos (fmily: Comretcee). [7] Heterophrgm denophyllum (HA) (fmily: Bignonicee) is tree commonly found in Indi, minly spred in Gujrt, Andmn Nicor, Assm, Punj, Tmil Ndu, Mhrshtr, nd Rjsthn. It hs vrious phrmcologicl properties including ntifungl ctivity, ntimicroil, leishmnicidl, ntivirl, nd crdiovsculr ctivity. [8-10] In ddition to tht, lpchol, mjor component of HA, hs een modified, nd vriety of its 1 Deprtment of Phrmceuticl Engineering nd Technology, Indin Institute of Technology, Bnrs Hindu University, Vrnsi, Uttr Prdesh, Indi, 2 Deprtment of Phrmceuticl Science, University of Kshmir, Sringr, Jmmu nd Kshmir , Indi *Corresponding uthor: Mohmmd Fizn Bht, Indin Institute of Technology, Bnrs Hindu University, Vrnsi , Uttr Prdesh, Indi. Phone: E-mil: mfizn.ht.phe14@ithu.c.in Received on: ; Revised on: ; Accepted on: These uthors contriuted eqully to this work This study ws supported y Indin Institute of Technology, Bnrs Hindu University, Indi. 1306

2 properties hve een elucidted, including ut not limited to nti-scess, ntiulcer, ntileishmnil, ntiedemic, nticrcinogenic, nti-inflmmtory, ntiseptic, ntitumor, ntimlril, ntivirl, ctericidl, fungicidl, pesticide, nd respirtory depressnt. [11] HA hs een trditionlly used s n ntihypertensive, [12] nd lso, the ethnolic extrct of the eril prts of the plnts hs een used trditionlly s hypotensive. [13] Mny in vivo nd in vitro studies hve proved tht some extrcts nd components from EU hve ntihypertensive ction. Vrious components of HA tht hve shown ntihypertensive properties hve een tested, some of which include ursolic cid, [14] olenolic cid, [15] nd lpchol. [16] Ech of these isolted compounds hs their reported mechnism of ction. [15,17-19] The gol of our reserch ws to evlute the ntihypertensive properties of HA extrct nd to compre it with the effectiveness of other stndrd drugs tht re ville for the sme purpose. MATERIALS AND METHODS Plnt Mterils nd Extrct Rw H. denophyllum Wll. ex G.Don leves (1.2 kg) were collected from Botnicl Grden of M.G Science College, Ahmedd, Gujrt, Indi, from June to Septemer Two methods were used for the extrction. In the hot extrction method, 4 g of corsely pulverized ir-dried leves ws plced in glssstoppered conicl flsk. 100 ml ethnol ws dded to the flsk, nd fter vigorous shking, the solution ws left to stnd for 1 h. After 1 h, the flsk ws ttched to reflux condenser nd oiled gently for 1 h nd then grdully cooled nd weighed. The solution ws then filtered through dry filter, nd 25 ml of the filtrte ws trnsferred to tred flt-ottomed dish nd vporized to dryness on wter th. The content of the extrctle mteril in mg/g of ir-dried mteril ws clculted. In the cold extrction method, 4 g of mteril ws plced in conicl flsk, nd 100 ml ethnol ws dded nd left for mcertion for 6 h. This ws followed my timely shking nd stnding for 18 h. The solution ws then filtered through dry filter, nd 25 ml of the filtrte ws trnsferred to tred flt-ottom dish nd llowed to dry over hot wter th. The resulting extrct ws then dried t 105 C for 6 h nd cooled in desicctor for 30 min nd weighed. The content of the extrctle mtter in mg/g of ir-dried mteril ws clculted. Animls nd Animl Housing Helthy mle Wistr rts (8-week-old, 230 ± 30 g) were otined from the Centrl Animl House of Institute of Medicl Sciences, Bnrs Hindu University, Vrnsi (Uttr Prdesh, Indi). The rts were cged in propylene cges t n mient temperture (25 ± 2 C) nd reltive humidity 45-55% with 12 h light/drk cycle (lights on 7:00 to 19:00) nd fed with commercil food pellets (Institute of Medicl Sciences, Bnrs Hindu University, Uttr Prdesh, Indi). Wter ws freely ville. All rts were cclimed for t lest 1 week to lortory conditions efore experiments were conducted. All protocols relted to nimls were followed s pproved y the Animl Ethics Committee, Institute of Medicl Sciences, Bnrs Hindu University (Letter No. Den/2016/CAEC/1653). Effect of HA on Blood Pressure of Wistr Rts y Periorl (p.o) Administrtion A totl of 36 Wistr rts were rndomly ssigned to 6 groups (n = 6): Group 1 (croxymethyl cellulose [CMC], norml control), group 2 (dexmethsone [DEX], toxic control), group 3 (cptopril [CAP], negtive control), group 4 (H. denophyllum ethnolic extrct [HAE], 100 mg/kg), group 5 (HAE, 200 mg/kg), nd group 6 (HAE, 300 mg/kg). The drugs or CMC were dministeredorlly, nd lood pressure ws mesured using non-invsive til-cuff method (BIOPAC System, Inc., USA,) every other dy. [20] To ssess the glucocorticoid effect of HAE, thymus glnd ws extricted nd weighed t the end of the experiment nd expressed in reltion to ody weight (mg/100 g) of ody weight. [21] Determintion of HAE Acute Orl Toxicity OECD Guidelines 425 were employed for the determintion of cute orl toxicity. 6 femle Wistr rts were used. After proper cclimtiztion of the nimls, the overnight-fsted rts were given incresing single doses of ethnolic lef extrct of HA (500, 100, nd 2000 mg/kg p.o). The nimls were oserved every 30 min for ny signs of toxicity or normlities for the durtion of 48 h which ws further extended to 14 dys. The numer of nimls tht survived t the end ws recorded nd ny discrepncies in ody or orgn weights were clculted, in ddition to tht, iochemicl nd histopthologicl chrcters were lso mesured. Mesurement of Blood Glucose, Plsm Insulin, Lipid Profile, Lipid Peroxidtion (LPO), Ctlse (CAT), nd Nitrite (NO) Level Blood glucose levels were mesured y enzymtic methods using commercil kits otined from Spn Dignostics, Pvt, Ltd., Indi, nd expressed s mg/dl. Plsm insulin levels were mesured using insulin enzyme-linked immune sorent ssy Kit otined from ThermoFisher Scientific, Indi, nd expressed s µu/ml. Plsm lipid prmeters such s totl cholesterol, triglycerides, nd highdensity lipoprotein cholesterol (HDL-C) levels 1307

3 were determined y enzymtic methods using commercil kits otined from Spn Dignostics, Indi. The low-density lipoprotein-cholesterol (LDL-C) nd very low-density lipoprotein-cholesterol (VLDL-C) were lso determined [22] nd expressed s mmol/l. Lipid peroxidse level ws estimted regrding mlondildehyde (MDA) nd expressed s nmol MDA/g. [23] Beers nd Sizer method ws used for determintion of CAT ctivity nd expressed t CAT ctivity unit/g of tissue. [24] Finlly, NO levels in liver were estimted clorimetriclly using Greiss regent nd expressed s nmol/mg of protein. [25] Sttisticl Methods All the vlues of the experimentl results re expressed s men ± stndrd error of men. Multiple comprisons evluted y one-wy nd two-wy nlysis of vrince, followed y Student Newmn-Keuls post-hoc test nd Bonferroni post-hoc test, respectively. GrphPd Prism 5 hs een used for sttisticl nlysis. Vlues of P < 0.05 were considered sttisticlly significnt. RESULTS Effect of HAE on Hypertension of Wistr Rts y Orl Administrtion Orl dministrtion of HAE for 7 dys showed decrese in hypertension in DEX-induced hypertension (Figure 1). The dministrtion of DEX significntly incresed lood pressure from ± 1.86 to ± 2.8 mmhg (P < 0.05) compred to the norml control group ( ± 1.81 mmhg). While the D.B.P. ws lso lowered from ± 3.10 mmhg to ± 7.00 (P < 0.05) in HAE-treted rts compred to toxic control ( ± 6.2) (Figure 1). Acute Orl Toxicity Results Orl dministrtion of HAE t doses 500, 1000, nd 2000 mg/kg, p.o, did not produce ny symptoms of Tle 1: Effect of HAE on ody weight Groups Body weight (g) Dy 1 Dy 7 Dy 14 Control ± ± ± mg/kg 168.5± ± ± mg/kg ± ± ± mg/kg ± ± ±0.98 toxicity nd ehviorl chnges t 48 h nd up to 14 dys. Finl ody nd orgn weights nd other iochemicl nd histopthologicl prmeters were lso checked nd found to e norml. Henceforth, the drug ws found to e sfe up to 2000 mg/kg, p.o. A triple dose optimiztion of 100, 200, nd 400 mg/kg, p.o ws selected for the evlution of the ctivity (Tles 1-3). Effect of HAE on Body nd Thymus Weight DEX dministrtion cused significnt decrese in the thymus glnd weight in hypertensive rts (F [5, 30] = 8.638, P < 0.05). Orl dministrtion of HAE nd CAP hd no significnt effect on thymus weight loss (Figure 2). Injection of DEX significntly decresed the ody weight in hypertensive rts when compred with the norml control group (F [5, 240] = 19.84, P < 0.05), time (F [7, 240] = 9.435, P < 0.05), nd interction (F [35, 240] = 4.888, P < 0.05). Orl dministrtion of HAE nd stndrd CAP could not prevent ody weight chnges in rts, nd lso, the ody weight loss ws more thn tht of DEX group (P < 0.05) (Figure 2). Effect of HAE on Insulin, Blood Glucose, Lipid Profile, LPO, CAT, nd NO Level Chronic dministrtion of DEX incresed the plsm insulin nd glucose level in ll the tested group compred with the norml control (F [5, 30] = 6.178, P < 0.05) nd (F [5, 30] = 7.192, P < 0.05). Orl dministrtion of HAE extrct s well s stndrd CAP decresed the insulin nd glucose level compred to toxic control (P < 0.05) (Figure 3 nd ). DEX injection incresed the LDL-C nd VLDL-C levels while HDL-C decresed ( P < 0.05 compred to control nd P < 0.05 compred to toxic control) (Figure 4-d) nd HAE incresed HDL-C (Good Cholestrol) ut did not show significnt effect on VLDL-C nd LDL-C. Effect of HAE on ntioxidnt enzymes is presented in Tle 4, nd sttisticl nlysis reveled significnt differences etween the groups CAT (F [5, 30] = 19.49, P < 0.05), MDA (F [5, 30] = 26.98, P < 0.05), nd NO (F [5, 30] = 26.98, P < 0.05) levels. The posthoc test reveled tht DEX injection significntly decresed CAT ctivity nd incresed LPO ctivity nd incresed NO level s compred with the norml control group. CAT ctivity ws significntly improved with HAE. Moreover, LPO ctivity ws Tle 2: Effect of HAE on orgn weight Groups Hert (g) Brin (g) Liver (g) Kidney Lungs (g) Stomch (g) Right (g) Left (g) Control 0.32± ± ± ± ± ± ± mg/kg 0.33± ± ± ± ± ± ± mg/kg 0.33± ± ± ± ± ± ± mg/kg 0.34± ± ± ± ± ± ±0.05 HAE: Heterophrgm denophyllum ethnolic extrct 1308

4 Figure 1: () Effects of Heterophrgm denophyllum ethnolic extrct (HAE) (100, 200, nd 400 mg/kg) nd cptopril (CAP) (40 mg/kg) on systolic lood pressure in dexmethsone (DEX) induced hypertension. () Effects of HAE (100, 200, nd 400 mg/kg) nd CAP (40 mg/kg) on distolic lood pressure in DEX induced hypertension Tle 3: Effect of HAE on iochemicl prmeters Enzymes/ Biochemicl prmeters Control 500 mg/kg 1000 mg/kg 2000 mg/kg Totl protein (g/l) 5.96± ± ± ±0.19 SGOT (U/L) 70.5± ± ± ±0.61 SGPT (U/L) 18.43± ± ± ±0.42 Alkline phosphtse (U/L) 65.26± ± ± ±0.64 Ure (mg/dl) 14.39± ± ± ±0.34 Cretinine (mg/dl) 0.41± ± ± ±0.04 Sodium (meq/l) ± ± ± ±0.61 Potssium (meq/l) 4.043± ± ± ±0.06 Cholesterol (mmol/l) 2.78± ± ± ±0.12 Triglycerides (mmol/l) 0.82± ± ± ±0.006 HDL (mmol/l) 1.31± ± ± ±0.033 LDL (mmol/l) 0.92± ± ± ±0.007 VLDL (mmol/l) 0.41± ± ± ±0.007 SGOT: Serum glutmic oxlocetic trnsminse, SGPT: Serum glutmic pyruvic trnsminse, HDL: High density lipoprotein, LDL: Low density lipoprotein, VLDL: Very low density lipoprotein, HAE: Heterophrgm denophyllum ethnolic extrct 1309

5 Tle 4: Effect of HAE on DEX induced ltertions in ntioxidnt enzymes Groups Figure 2: () Effects of Heterophrgm denophyllum ethnolic extrct (HAE) (100, 200, nd 400 mg/kg) nd cptopril (CAP) (40 mg/kg) on thymus weight in dexmethsone (DEX) induced hypertension. () Effects of HAE (100, 200, nd 400 mg/kg) nd CAP (40 mg/kg) on ody weight in DEX induced hypertension CAT ctivity (µg/min/mg protein) LPO level (nm MDA/mg protein) NO level (nm NO/mg protein) Norml control 23.02± ± ±0.011 Toxic control 13.91± ± ±0.023 Positive control 20.83± ± ±0.012 HAE 100 mg/kg 16.73±0.559,,c 0.86±0.037,c 0.51±0.032,,c HAE 200 mg/kg 19.58±0.881,,d 0.56±0.019,d 0.38±0.017,d HAE 400 mg/kg 21.12±0.986,d 0.54±0.028,d 0.36±0.016,d HAE: Heterophrgm denophyllum ethnolic extrct, CAT: Ctlse, DEX: Dexmethsone, LPO: Lipid peroxidse, MDA: Mlondildehyde, NO: Nitrite. All vlues re men±sem (n=6). p<0.05 compred to norml control; p<0.05 compred to toxic control; c p<0.05 compred to positive control; d p<0.05 compred to HAE 100 mg/kg (one-wy ANOVA followed y Student Newmn Keuls Test). 1310

6 Figure 3: () Effects of Heterophrgm denophyllum ethnolic extrct (HAE) (100, 200, nd 400 mg/kg, periorl [p.o]) nd cptopril (40 mg/kg, p.o) on insulin level in dexmethsone (DEX) induced hypertension. () Effects of HAE (100, 200, nd 400 mg/kg, p.o) nd cptopril (40 mg/kg, p.o) on glucose level in DEX induced hypertension significntly reduced y HAE (200 nd 400 mg/kg). NO ctivity ws significntly reduced y HAE (200 nd 400 mg/kg), nd 100 mg/kg ws not found to e significntly different when compred with the toxic group. Effect of HAE on Histopthology of Myocrdium The histopthologicl studies of myocrdium showed the intct rrngement of the crdic muscle fiers in the norml control group, wheres in the DEX (30 µ/kg, s.c)-induced group, myocrdium showed 1311

7 c d Figure 4: () Effects of Heterophrgm denophyllum ethnolic extrct (HAE) (100, 200, nd 400 mg/kg, periorl [p.o]) nd cptopril (40 mg/kg, p.o) on triglycerides levels in dexmethsone (DEX) induced hypertension. () Effects of HAE (100, 200, nd 400 mg/kg, p.o) nd cptopril (40 mg/kg, p.o) on high-density lipoprotein cholesterol level in DEX induced hypertension. (c) Effects of HAE (100, 200, nd 400 mg/kg, p.o) nd cptopril (40 mg/kg, p.o) on low-density lipoprotein cholesterol level in DEX induced hypertension. (d) Effects of HAE (100, 200, nd 400 mg/kg, p.o) nd cptopril (40 mg/kg, p.o) on very lowdensity lipoprotein cholesterol level in DEX induced hypertension prtilly hphzrd rrngement of hert muscle fiers. This cn e ttriuted to incresed worklod during chronic hypertension tht cuses myocyte hypertrophy. HAE extrct (100, 200, nd 400 mg/kg, p.o) nd stndrd CAP (40 mg/kg, p.o) showed the restortion to norml level s compred to norml control group (Figure 5). c d e f DISCUSSIONS The mjor findings in the present study were (i) to perform effective extrction of the ctive chemicl constituents of HA nd (ii) to check for ntihypertensive properties of HA extrct nd lso check for its effect on different iochemicl nd histopthologicl properties. Hypertension hs een treted with HA for long time in history, round the gloe; however, its use hs not een yet ccepted interntionlly ecuse of lck of proper 1312 Figure 5: Histopthology report of myocrdium. In the histologicl report for the myocrdium; () Control group, the myocrdium showed the intct rrngement of the crdic muscle fiers, () Induced group showed prtilly hphzrd rrngement of the crdic muscle fiers, (c) Cptopril (40 mg\kg) treted, showed the intct rrngement of the crdic muscle fiers, which showed its restortion to norml. (d-f) Heterophrgm denophyllum ethnolic extrct treted (100, 200, nd 400 mg/kg, peri orl) group showed dosedependent restortion effect on myocrdium

8 detiled scientific study to ccess ntihypertensive property of this plnt. Severl components of HA hve shown ntihypertensive properties nd hve een tested, some of which include ursolic cid, [14] olenolic cid, [15] nd lpchol. [16] Extrct of the leves of this plnt hs not een tested for ntihypertensive properties nywhere in the literture, so we chose the ethnolic extrct for our study. In our experiment, we used cold nd hot extrction of the leves of HA. We studied the effect of HAE on DEX-induced hypertension in Wistr rts y orl dministrtion. We found tht HAE could dose dependently decrese lood pressure in Wistr rts. This experiment provides sustnce to the ntihypertensive property clims of HA. In the present experiment, we further investigted the iochemicl nd histopthologicl prmeters tht hve een linked to hypertension. Studies on severl ntioxidnt nd herl medicines to prevent hypertension in experimentl models hve een crried out [26,27] so LPO, CAT, nd NO levels were estimted. Mny investigtors employed glucose tolernce test to report tht ptients with essentil hypertension hve n elevtion in insulin nd glucose concentrtion compred with normotensive control. [28,29] Furthermore, insulin-resistnt sttes such s essentil hypertension nd type-ii dietes re ssocited with ccelerted therosclerosis, which is lso n underlying cuse of hypertension, so tht led us to study glucose nd insulin levels of the nimls too. Furthermore, the mechnism underlying glucocorticoid dyslipidemi my e n impired ctolism of LDL, increse in the ctivity of lipoprotein lipse, nd increse in the VLDL levels due to incresed plsm insulin [20] which unequivoclly pointed us towrd lipid profile estimtion. Due to DEX-induced chronic hypertension myocyte hypertrophy, crdic remodeling nd crdic muscle firosis hve een reported. Hence, HAE ws ssessed for restortive properties on crdic muscle firosis, nd the results were promising. 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