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Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension
Life Sciences 70 (2002) 2599 – 2609
Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension Dae Gill Kanga, Tae Young Hurb, Geon Mok Leeb, Hyuncheol Oha, Tae Oh Kwonc, Eun Jin Sohna, Ho Sub Leea,* a
Department of Herbal Resources, Professional Graduate School of Oriental Medicine, and Medicinal Resources Research Centre, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea b Third Medicine, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea c College of Life Sciences and Natural Resources, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea Received 29 March 2001; accepted 12 July 2001
Abstract A pharmacological inhibition of nitric oxide synthase (NOS) in rats for 4 – 6 weeks produces renal vasoconstriction, renal dysfunction, and severe hypertension. The present study was aimed at investigating whether Cudrania tricuspidata (C. tricuspidata) water extract ameliorates NG-NitroL-arginine methylester (L-NAME)-induced hypertension. Treatment of L-NAME (60 mg/L drinking water, 4 weeks) causes a sustained increase in systolic blood pressure (SBP). The concentration of plasma NO metabolites and NO/cGMP productions in the vascular tissues of the L-NAME-treated group were significantly reduced as compared with those in the control. C. tricuspidata water extract blocked increase of SBP in the L-NAME-treated group and restored SBP to normal level. Futhermore, C. tricuspidata water extract was able to preserve the vascular NO/cGMP production and plasma NO metabolites concentration. However, there are no changes in the expression of ecNOS and iNOS of thoracic aorta among the rats of control, L-NAME-treated group, and L-NAME and C. tricuspidata water extract co-treated group. The urinary sodium level, urine volume, and creatinine clearance were significantly higher in rats co-treated with C. tricuspidata water extract and L-NAME than in
* Corresponding author. Tel.: +82-63-850-6841; fax: +82-63-850-7324. E-mail address: [email protected] (H.S. Lee). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 4 7 - 3
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L-NAME-treated group. Taken together, these results suggest that C. tricuspidata water extract prevents the increase of SBP in the L-NAME-induced hypertension that may have been caused by enhanced generation of vascular NO/cGMP. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cudrania tricuspidata; L-NAME; Hypertension; NO/cGMP
Introduction Endothelium-dependent relaxations are impaired in hypertension and the ability of nitric oxide (NO) to maintain vascular tone is also deficient in hypertensive condition [1]. Pharmacological long-term blockade of NO synthesis by the chronic administration of NG-nitro-L-arginne methyl ester (L-NAME), an inhibitor of NO synthesis, produces systemic arterial hypertension, vascular structural change, renal dysfunction [1,2]. Recently, many studies have been performed to directly measure nitric oxide release from vascular endothelium elicited by a plant extract and found that the extract’s vasodilating effect is related to stimulation of nitric oxide release from vascular tissues [3–12]. C. tricuspidata is a deciduous tree inhabitated over Korea, China, and Japan. The stems of this species have been used as a traditional medicine for curing neuritis and inflammation in the Orient and a folk remedy for gastritis, liver damage, and hypertension in Korea. From the stem or root bark of C. tricuspidata, flavonoids and benzonoids have been isolated. They were identified as 6,8-di-p-hydroxybenzyltaxifolin, 8-p-hydroxybenzyltaxifolin and 6-p-hydroxybenzyltaxifolin, respectively, by means of spectral studies [13]. These compounds have the cytotoxic effects on human tumor cell lines [13]. However, other pharmacological actions of C. tricuspidata extract are not well established, yet. Thus, the present study was aimed at investigating whether C. tricuspidata water extract ameliorates L-NAME-induced hypertension in rats.
Materials and Methods Plant material and Extraction The extract of C. tricuspidata was prepared from 1200 g of fresh stem collected at the Botanical Garden of Wonkwang University, Korea in the month of September 2000. Dr. Kyu Kwan Jang at the Botanical Garden of Wokwang University identified plant material. The stems were air-dried at room temperature and reduced to fine powder by milling. The resulting powder was subjected to extraction with 1 L of methanol, three times, 24 hours each. The methanol extract was concentrated (76.5 g) suspended in H2O, and sequentially partitioned with n-hexane, dichloromethane, ethylacetate. The resulting aqueous portion was concentrated (27.2 g) and used in this study.
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Experimental animals Male Sprague–Dawley rats ( 200 g) were purchased from a commercial source (Korean Experimental Animal, Korea). The rats were divided into four groups: The control group was allowed access to tap water for four weeks, the second group was allowed to drinking water containing L-NAME (60 mg/L drinking water), the third group was co-administrated with a drinking water containing both L-NAME and 0.72 g/kg/day of C. tricuspidata water extract during the last 2 weeks of L-NAME-treatment, while C tricuspidata water extract was supplemented with L-NAME during the whole period of experiments in the fourth group. Systolic blood pressure (SBP) was measured in conscious rats by the tail-cuff plethysmography. On the experiment day, the trunk blood was collected by decapitation without anesthesia in a prechilled tube containing 1 mg/mL ethylenediaminetetraacetic acid (EDTA) for the determination of plasma NO metabolites. The thoracic aorta was removed and immediately frozen in liquid nitrogen, and stored at 72 C until assayed. Methods and experimental procedures Colorimetric assay of nitrite/nitrate Nitrite/nitrate in the thoracic aorta and plasma samples were measured with colorimetric assay kit (Oxford, MI, USA). A microplate was used to perform enzyme reaction in vitro. For spectrophotometric assay of nitrites with Griess reagent, 50 mmol/L 3-[N-Morpholino]propanesulfonic acid (MOPS)/EDTA (1 mmol/L) buffer and samples were added to wells in duplicates. Nitrate reductase (0.01 U) and 2 mmol/L NADH were added to the reaction mixture, and the plate was shaken for 20 minutes at room temperature. Color reagents comprised with 1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride were added and absorbance values at 540 nm were read in a microplate reader (Bio–Rad, Hercules, CA, USA). Protein preparation and Western blot analysis The thoracic aorta were homogenized with Polytron homogenizer at 3,000 rpm in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmo/L phenylmethylsulfonyl fluoride and 20 mmol/L potassium phosphate buffer, at pH 7.6. Large tissue debris and nuclear fragments were removed by two low speed spins in succession (1,000 g, 5 min; 10,000 g 10 min) at 4 C. Supernatants from these low speed spins were ultracentrifuged at 100,000 g for 1 hour at 4 C. The pellet was resuspended for protein blotting of endothelial nitric oxide synthase (ecNOS), and the supernatant was used for blotting of inducible nitric oxide synthase (iNOS). The protein concentration was determined by the method of Bradford [14] with bovine serum albumin as a standard. Protein samples (80 mg) were electrophoretically fractionated with a discontinuous system consisting of a 7.5% polyacrylamide resolving gel and 5% stacking gel, followed by transfer to a nitrocellulose membrane at 20 V and 100 mA (current constant) overnight. The membrane was washed, blocked, incubated with a 1:2000 dilution of mouse monoclonal anti-NOS antibodies (Transduction Laboratories, Lexington, KY, USA). The bound horseradish peroxidase-conjugated secondary antibody
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was detected by enhanced chemiluminescence (Amersham, Buckingghamshire, England) procedure. The protein expression levels of NOS were detemined by analysing the signals captured on Hyperfilm (Amersham, Buckinghamshire, England) using an image analyzer (Imager III, Bioneer, Korea). cGMP measurement The tissue homogenate resuspended in 6% trichloroacetic acid was centrifuged at 1,000 g for 10 min at 4 C. The supernatant was extracted four times with 1 mL of water-saturated ether, and the extract was dried and assayed for cGMP using a radioimmunoassay described by Kim et. al. [15]. In brief, standards or samples were introduced in a final volume of 100 ml of 50 mM sodium acetate buffer (pH 4.8). Then, 100 ml of diluted cGMP antiserum (Calbiochem–Novabiochem Co., Sandiego, CA, USA) and iodinated cGMP (10,000 cpm/ 100 ml, specific activity 2,200 Ci mM1, Dupont-New England Nuclear, Wilmington, DE, USA) were added in succession and incubated for 24 h at 4 C. The bound form was separated from the free form by charcoal suspension. Results were expressed as picomoles of cGMP generated per milligram protein per minute (pmole/min/mg protein). Drugs and statistics All drugs were purchased from the Sigma Chemical Company (St Louis, MO, USA), unless stated otherwise. Results were expressed as means ± SEM. The statistical significance of difference between the groups means were determined using one-way ANOVA and Student’s t–test.
Results Effects of C. tricuspidata water extract on body weight Table 1 shows the body weight changes in rats by administration of L-NAME or coadministration of L-NAME and C. tricuspidata water extract for 4 weeks. At the starting point
Table 1 Effects of Cudrania tricuspidata water extract on rats body weight in different experimental groups Body weight (g) 1 week Control L-NAME CT-1 CT-2
240 249 234 244
± ± ± ±
0.66 2.95 6.25 3.99
2 week 289 270 284 286
± ± ± ±
3.57 19.7 5.40 3.03
3 week 319 318 315 312
± ± ± ±
4.07 4.33 5.69 2.03
4 week 341 334 331 335
± ± ± ±
3.99 8.68 4.76 3.31
L-NAME; L-NAME-treated group, CT-1; Cudrania tricuspidata water extract was supplemented with L-NAME during the last 2 weeks, and CT-2; ditto except treated for the whole period of experiments. Values are means ± S.E. (n=7, each group).
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of experiment, the body weight in all groups is approximately 200 g. In the whole experimental period, there was no difference of body weight among the four experimental groups. Effects of C. tricuspidata water extract on SBP Fig. 1 shows effects on the SBP during the four weeks period of blockade of NO synthesis with L-NAME and those of C. tricuspidata water extract. At the starting point of experiment, the SBP in all groups is approximately 116–120 mmHg. Oral administration of L-NAME increased SBP to 148 ± 4 mmHg at first 1 week and this increased SBP was sustained for the whole experiment period (p < 0.01 vs control). This increased SBP was reversed to normotension by co-administration of C. tricuspidata water extract during the last 2 weeks (131 ± 3 mmHg, p < 0.05). Furthermore, the supplement with C. tricuspidata water extract during whole experiment prevented the increase in SBP by the oral administration of L-NAME in rats (p < 0.01 vs L-NAME). Effects of C. tricuspidata water extracts on the NOx/cGMP production As shown in Fig. 2, there were no changes in the expression of ecNOS and iNOS in the aortic tissue from L-NAME administrated rats for 4 weeks compared to control group. The expression of these NOS isoforms in the aortic tissue did not change by administration of C. tricuspidata water extract, either. However, the aortic production of NO metabolites was decreased by L-NAME administration (control: 3.91 ± 0.40, L-NAME: 2.51 ± 0.18 nmol/mg protein, p < 0.05, Fig. 3A). The supplementation with C. tricuspidata water extract during the
Fig. 1. Effects of Cudrania tricuspidata water extract on rats systolic blood pressures in different experimental groups. CON; control group, L-NAME; L-NAME-treated group, CT-1; Cudrania tricuspidata water extract was supplemented with L-NAME during the last 2 weeks, and CT-2; ditto except treated for the whole period of experiments. There were seven experiments in each group. **p < 0.01 compared with control; #p < 0.01, *p < 0.05 compared with L-NAME-treated group.
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Fig. 2. Representative western blots and densitometric analyses showing ecNOS and iNOS expression in the thoracic aorta of obtained from different experimental groups. Upper panel: 1, 2, 3, 4 denote Control, L-NAME, CT-1, and CT-2, respectively. Legends are the same as in Fig. 1. There were four experiments in each group.
last 2 weeks reversed the decreased release of aortic NO metabolites by L-NAME-treatment (3.29 ± 0.30 nmol/mg protein, p < 0.05) toward normal. The supplement with C. tricuspidata water extract during whole experiment prevented the decrease in NO metabolites induced by L-NAME (3.28 nmol/mg protein). Plasma concentration of NO metabolites was paralleled aortic NO production. Plasma concentration of NO metabolites was also decreased by L-NAME administration, which was reversed and prevented by co-administration with C. tricuspidata water extracts (Fig. 3B). Fig. 4 show that the blockade of NO synthesis by oral administration of L-NAME significantly inhibited aortic cGMP production (control: 1.64 ± 0.12, L-NAME: 0.52 ± 0.16 nmol/mg protein, p < 0.01). These inhibitions were partially reversed by supplementation with C. tricuspidata water extract during the last 2 weeks and partially prevented by supplementation during the whole experimental period (CT-1: 1.02 ± 0.08, CT-2: 0.96 ± 0.07 nmol/mg protein, p < 0.05 vs L-NAME, respectively).
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Fig. 3. Effects of Cudrania tricuspidata water extract on the vascular tissue and plasma nitrite/nitrate levels in the different experimental groups. See Fig. 1 for legends. There were seven experiments in each group. *p < 0.05, compared with control; # p < 0.05, compared with L-NAME-treated group.
Fig. 4. Effects of Cudrania tricuspidata water extract on the vascular tissue cGMP levels in the different experimental groups. See Fig. 1 for legends. There were seven experiments in each group. *p < 0.05, compared with control; # p < 0.05, compared with L-NAME-treated group.
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Effects of C. tricuspidata water extracts on renal functions Administration of L-NAME for 4 weeks significantly lowered renal creatinine clearance (p < 0.05 vs control, Fig. 5A). Renal sodium excretion (UNaV) and solute-free water reabsorption (TcH2O) also decreased in the 4-weeks L-NAME administrated rats (p < 0.05 vs control, respectively, Fig. 5B and C). These inhibitions were fully reversed by supplementation with C. tricuspidata water extract during the last 2 weeks and prevented by supplemen-
Fig. 5. Effects of Cudrania tricuspidata water extract on renal creatinine clearance (Ccr), urinary sodium excretion (UNaV), and solute-free water reabsorption (TcH2O) in the different experimental groups. The legends are the same as in Fig. 1. There were seven experiments in each group. *p < 0.05, compared with control; # p < 0.01, compared with L-NAME-treated group.
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tation for the whole experiment period of four weeks (p < 0.01 vs L-NAME, Fig. 5A, B, C). Moreover, two renal functional parameters (Ccr and TcH2O) were larger than respective value in control rats (p < 0.05 vs control).
Discussion NOS is inhibited by L-arginine analogues such as NG-monomethyl-L-arginine and L-NAME. When they are infused into animals or humans, NO production is inhibited and leads to vascular smooth muscle contraction, renal dysfunction, and increased blood pressures [1,2]. Various models of hypertension have been associated with a decreased release of NO. In hypertension mediated by volume overload, such as deoxycorticosterone acetate (DOCA)salt and Dahl-salt sensitive hypertensive models, the renal and aortic NO levels are decreased [1,16]. We also observed in the present study that chronic treatment with L-NAME increased the blood pressure in association with decreases plasma NO and aortic tissue NO/cGMP levels. However, L-NAME had no discernible effects on NOS isoforms expression profiles in the aorta. These findings most likely reflect the diminished vasodilator action of endogenous NO, where L-NAME may have interfered with enzyme activity of NOS as opposed to quantitative modification of NOS isoforms expression. These results are in accordance with previously study [17]. After administration of L-NAME for 2 weeks, the SBP was declined by co-administration with C. tricuspidata water extract. When the co-administration of L-NAME with C. tricuspidata water extract in rats, there were no changes in SBP. The plasma NO and aortic NO/cGMP were reversed by co-administration of C. tricuspidata water extract during the last 2 weeks. Furthermore, there were also no decreases in plasma NO and aortic NO/cGMP level by the co-administration with C. tricuspidata water extract during the whole experiment. The pharmacological action of C. tricuspidata extract has seldom been examined. Lee et. al. isolated three new dihydroflavonols from stem bark of Cudrania tricuspidata, gericudranins A–C, had a cytotoxic effects on human tumor cell lines [13]. Recently, many studies have been performed to find more suitable antihypertensives from other natural sources. Among them, extracts of hawthorn [5,6], Caesalpine sappa [7], Uncaria rhynchophylla [5,9], Cordyceps sinensis [3], Gynostemma pentaphyllum [11], Salviae miltiorrhizae [10] had a hypotensive effects due to endothelium-dependent vasorelaxation or direct stimulation of NO release. Moreover, garlic extract has a antihypertensive effect on L-NAME-induced hypertension [8]. According to our study, C. tricuspidata water extract may also have an antihypertensive effect via stimulation of NO/cGMP system in the vascular cells. It is well known that NO is an important regulator of renal hemodynamics and sodium handling. In the normal rats, nitric oxide level increases as an adaptive response to increased dietary salt intake, perhaps facilitating natriuresis and thus blood pressure homeostasis [18]. NO is synthesized directly in the renal vasculature. Immunohistochemical studies have shown expression of constitutive NOS on glomerular capillaries, afferent and efferent arteriols, medullary vasa recta, and intrarenal arteries [19]. With the use of PCR techniques, expression
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of inducible NOS has also been suggested in renal vessel [20]. Inhibition of nitric oxide synthesis by administration of nitro-L-arginine or L-NAME leads to a decrease in urinary sodium excretion (UNaV) along with the decrease renal blood flow, urine flow rates, glomerular filtration rates (GFR), and free-water clearance [2,18,21]. Animals treated chronically with NOS inhibitors develop hypertension, with a resetting of the pressure– natriuresis relation toward higher blood pressure. Similar results were obtained in the present study. Where administration with L-NAME decreased the creatinine clearance (Ccr), sodium excretion (UNaV), and free-water reabsorption (TcH2O), which was prevented and reversed by co-administration with C. tricuspidata water extract. Therefore, the effects of C. tricuspidata water extract may be attributable to amelioration of renal function in the L-NAME-induced hypertension via activation of NO/cGMP system. In summary, the inactivation of NO/cGMP system was prevented and reversed along with SBP normalization in the L-NAME-induced hypertension by co-administration with C. tricuspidata water extract without significant changes in NOS isoproteins expression in the thoracic aorta of a rat model. Furthermore, C. tricuspidata water extract ameliorated renal functional parameters in the L-NAME-induced hypertensive rats. These results suggest that C. tricuspidata water extract attenuated the increase in SBP in the L-NAMEinduced hypertension by enhancing the generation of vascular NO/cGMP and amelioration of renal functions.
Acknowledgments This study was supported by the Brain Korea 21 Project (2001) and grant of the Oriental Medicine R and D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-CO03-0003). We were grateful to Kyung Woo Cho, PhD., MD. for his advise in the course of this study.
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8. Pedraza-Chaverri J, Tapia E, Medina-Campos O, de Los Angeles Granados M, Franco M. Galic prevents hypertension induced by chronic inhibition of nitric oxide synthesis. Life Sciences 1998;62(6):PL 71 – 7. 9. Goto H, Sakakibara I, Shimada Y, Kasahara Y, Terasawa K. Vasodilator effect of extract prepared from Uncariae ramulus on isolated rat aorta. American Journal of Chinese Medicine 2000;28(2):197 – 203. 10. Kamata K, Noguchi M, Nagai M. Hypotensive effects of lithospermic acid B isolated from the extract of Salviae miltiorrhiza radix in the rat. General Pharmacology 1994;25(1):69 – 73. 11. Tanner MA, Bu X, Steimle JA, Myers PR. The direct release of nitric oxide by gypenosides derived from the herb Gynostemma pentaphyllum. Nitric Oxide 1999;3(5):359 – 65. 12. Li CP, Yung KH, Chiu KW. Hypotensive action of Salvia miltiorrhiza cell culture extract. American Journal of Chinese Medicine 1990;18:157 – 66. 13. Lee IK, Kim CJ, Song KS, Kim HM, Koshino H, Uramoto M, You ID. Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata. Phytochemistry 1996;41(1):213 – 6. 14. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976;72:248 – 54. 15. Kim SZ, Kim SH, Park JK, Koh GY, Cho KW. Presence and biological activity of C-type natriuretic peptidedependent guanylate cyclase-coupled receptor in the penile corpus cavernosum. Journal of Urology 1998;159 (5):1741 – 6. 16. Hirata Y, Hayakawq H, Suzuki E, Kimura K, Kikuchi K, Nagano T, Horobe M, Omata M. Direct measurements of endothelium-derived nitric oxide release by stimulation of endothelin receptors in rat kidney and its alteration in salt-induced hypertension. Circulation 1995;91:1229 – 36. 17. Lee J, Lee SH, Kang DG, Kim SW. Effects of nitric oxide synthesis on the atrial natriuretic peptide system. Pharmocological Research 2000;42(3):275 – 9. 18. Bech JN, Nielsen CB, Ivarsen P, Jensen KT, Pedersen EB. Dietary sodium affects systemic and renal hemodynamic response to NO inhibition in healthy humans. American Journal of Physiology 1998;274(43): F914 – 23. 19. Bachmann SH, Bosse HM, Mundel P, Madsen KM. Topology of nitric oxide synthesis by localizing constitutive NO synthases in mammarian kidney. American Journal of Physiology 1995;268(37):F855 – 98. 20. Mohaupt MG, Elzie JL, Ahn KY, Clapp WL, Wilcox CS, Kone BC. Differential expression and induction of mRNA encoding two inducible nitric oxide synthases in rat kidney. Kidney International 1994;46:653 – 65. 21. Kang DG, Kim JW, Lee J. Effects of nitric oxide synthesis inhibition on the Na, K-ATPase activity in the kidney. Pharmacological Research 2000;41(1):123 – 7.
Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension Dae Gill Kanga, Tae Young Hurb, Geon Mok Leeb, Hyuncheol Oha, Tae Oh Kwonc, Eun Jin Sohna, Ho Sub Leea,* a
Department of Herbal Resources, Professional Graduate School of Oriental Medicine, and Medicinal Resources Research Centre, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea b Third Medicine, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea c College of Life Sciences and Natural Resources, Wonkwang University, Iksan, Jeonbuk, 570-749, Republic of Korea Received 29 March 2001; accepted 12 July 2001
Abstract A pharmacological inhibition of nitric oxide synthase (NOS) in rats for 4 – 6 weeks produces renal vasoconstriction, renal dysfunction, and severe hypertension. The present study was aimed at investigating whether Cudrania tricuspidata (C. tricuspidata) water extract ameliorates NG-NitroL-arginine methylester (L-NAME)-induced hypertension. Treatment of L-NAME (60 mg/L drinking water, 4 weeks) causes a sustained increase in systolic blood pressure (SBP). The concentration of plasma NO metabolites and NO/cGMP productions in the vascular tissues of the L-NAME-treated group were significantly reduced as compared with those in the control. C. tricuspidata water extract blocked increase of SBP in the L-NAME-treated group and restored SBP to normal level. Futhermore, C. tricuspidata water extract was able to preserve the vascular NO/cGMP production and plasma NO metabolites concentration. However, there are no changes in the expression of ecNOS and iNOS of thoracic aorta among the rats of control, L-NAME-treated group, and L-NAME and C. tricuspidata water extract co-treated group. The urinary sodium level, urine volume, and creatinine clearance were significantly higher in rats co-treated with C. tricuspidata water extract and L-NAME than in
* Corresponding author. Tel.: +82-63-850-6841; fax: +82-63-850-7324. E-mail address: [email protected] (H.S. Lee). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 4 7 - 3
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L-NAME-treated group. Taken together, these results suggest that C. tricuspidata water extract prevents the increase of SBP in the L-NAME-induced hypertension that may have been caused by enhanced generation of vascular NO/cGMP. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Cudrania tricuspidata; L-NAME; Hypertension; NO/cGMP
Introduction Endothelium-dependent relaxations are impaired in hypertension and the ability of nitric oxide (NO) to maintain vascular tone is also deficient in hypertensive condition [1]. Pharmacological long-term blockade of NO synthesis by the chronic administration of NG-nitro-L-arginne methyl ester (L-NAME), an inhibitor of NO synthesis, produces systemic arterial hypertension, vascular structural change, renal dysfunction [1,2]. Recently, many studies have been performed to directly measure nitric oxide release from vascular endothelium elicited by a plant extract and found that the extract’s vasodilating effect is related to stimulation of nitric oxide release from vascular tissues [3–12]. C. tricuspidata is a deciduous tree inhabitated over Korea, China, and Japan. The stems of this species have been used as a traditional medicine for curing neuritis and inflammation in the Orient and a folk remedy for gastritis, liver damage, and hypertension in Korea. From the stem or root bark of C. tricuspidata, flavonoids and benzonoids have been isolated. They were identified as 6,8-di-p-hydroxybenzyltaxifolin, 8-p-hydroxybenzyltaxifolin and 6-p-hydroxybenzyltaxifolin, respectively, by means of spectral studies [13]. These compounds have the cytotoxic effects on human tumor cell lines [13]. However, other pharmacological actions of C. tricuspidata extract are not well established, yet. Thus, the present study was aimed at investigating whether C. tricuspidata water extract ameliorates L-NAME-induced hypertension in rats.
Materials and Methods Plant material and Extraction The extract of C. tricuspidata was prepared from 1200 g of fresh stem collected at the Botanical Garden of Wonkwang University, Korea in the month of September 2000. Dr. Kyu Kwan Jang at the Botanical Garden of Wokwang University identified plant material. The stems were air-dried at room temperature and reduced to fine powder by milling. The resulting powder was subjected to extraction with 1 L of methanol, three times, 24 hours each. The methanol extract was concentrated (76.5 g) suspended in H2O, and sequentially partitioned with n-hexane, dichloromethane, ethylacetate. The resulting aqueous portion was concentrated (27.2 g) and used in this study.
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Experimental animals Male Sprague–Dawley rats ( 200 g) were purchased from a commercial source (Korean Experimental Animal, Korea). The rats were divided into four groups: The control group was allowed access to tap water for four weeks, the second group was allowed to drinking water containing L-NAME (60 mg/L drinking water), the third group was co-administrated with a drinking water containing both L-NAME and 0.72 g/kg/day of C. tricuspidata water extract during the last 2 weeks of L-NAME-treatment, while C tricuspidata water extract was supplemented with L-NAME during the whole period of experiments in the fourth group. Systolic blood pressure (SBP) was measured in conscious rats by the tail-cuff plethysmography. On the experiment day, the trunk blood was collected by decapitation without anesthesia in a prechilled tube containing 1 mg/mL ethylenediaminetetraacetic acid (EDTA) for the determination of plasma NO metabolites. The thoracic aorta was removed and immediately frozen in liquid nitrogen, and stored at 72 C until assayed. Methods and experimental procedures Colorimetric assay of nitrite/nitrate Nitrite/nitrate in the thoracic aorta and plasma samples were measured with colorimetric assay kit (Oxford, MI, USA). A microplate was used to perform enzyme reaction in vitro. For spectrophotometric assay of nitrites with Griess reagent, 50 mmol/L 3-[N-Morpholino]propanesulfonic acid (MOPS)/EDTA (1 mmol/L) buffer and samples were added to wells in duplicates. Nitrate reductase (0.01 U) and 2 mmol/L NADH were added to the reaction mixture, and the plate was shaken for 20 minutes at room temperature. Color reagents comprised with 1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride were added and absorbance values at 540 nm were read in a microplate reader (Bio–Rad, Hercules, CA, USA). Protein preparation and Western blot analysis The thoracic aorta were homogenized with Polytron homogenizer at 3,000 rpm in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmo/L phenylmethylsulfonyl fluoride and 20 mmol/L potassium phosphate buffer, at pH 7.6. Large tissue debris and nuclear fragments were removed by two low speed spins in succession (1,000 g, 5 min; 10,000 g 10 min) at 4 C. Supernatants from these low speed spins were ultracentrifuged at 100,000 g for 1 hour at 4 C. The pellet was resuspended for protein blotting of endothelial nitric oxide synthase (ecNOS), and the supernatant was used for blotting of inducible nitric oxide synthase (iNOS). The protein concentration was determined by the method of Bradford [14] with bovine serum albumin as a standard. Protein samples (80 mg) were electrophoretically fractionated with a discontinuous system consisting of a 7.5% polyacrylamide resolving gel and 5% stacking gel, followed by transfer to a nitrocellulose membrane at 20 V and 100 mA (current constant) overnight. The membrane was washed, blocked, incubated with a 1:2000 dilution of mouse monoclonal anti-NOS antibodies (Transduction Laboratories, Lexington, KY, USA). The bound horseradish peroxidase-conjugated secondary antibody
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was detected by enhanced chemiluminescence (Amersham, Buckingghamshire, England) procedure. The protein expression levels of NOS were detemined by analysing the signals captured on Hyperfilm (Amersham, Buckinghamshire, England) using an image analyzer (Imager III, Bioneer, Korea). cGMP measurement The tissue homogenate resuspended in 6% trichloroacetic acid was centrifuged at 1,000 g for 10 min at 4 C. The supernatant was extracted four times with 1 mL of water-saturated ether, and the extract was dried and assayed for cGMP using a radioimmunoassay described by Kim et. al. [15]. In brief, standards or samples were introduced in a final volume of 100 ml of 50 mM sodium acetate buffer (pH 4.8). Then, 100 ml of diluted cGMP antiserum (Calbiochem–Novabiochem Co., Sandiego, CA, USA) and iodinated cGMP (10,000 cpm/ 100 ml, specific activity 2,200 Ci mM1, Dupont-New England Nuclear, Wilmington, DE, USA) were added in succession and incubated for 24 h at 4 C. The bound form was separated from the free form by charcoal suspension. Results were expressed as picomoles of cGMP generated per milligram protein per minute (pmole/min/mg protein). Drugs and statistics All drugs were purchased from the Sigma Chemical Company (St Louis, MO, USA), unless stated otherwise. Results were expressed as means ± SEM. The statistical significance of difference between the groups means were determined using one-way ANOVA and Student’s t–test.
Results Effects of C. tricuspidata water extract on body weight Table 1 shows the body weight changes in rats by administration of L-NAME or coadministration of L-NAME and C. tricuspidata water extract for 4 weeks. At the starting point
Table 1 Effects of Cudrania tricuspidata water extract on rats body weight in different experimental groups Body weight (g) 1 week Control L-NAME CT-1 CT-2
240 249 234 244
± ± ± ±
0.66 2.95 6.25 3.99
2 week 289 270 284 286
± ± ± ±
3.57 19.7 5.40 3.03
3 week 319 318 315 312
± ± ± ±
4.07 4.33 5.69 2.03
4 week 341 334 331 335
± ± ± ±
3.99 8.68 4.76 3.31
L-NAME; L-NAME-treated group, CT-1; Cudrania tricuspidata water extract was supplemented with L-NAME during the last 2 weeks, and CT-2; ditto except treated for the whole period of experiments. Values are means ± S.E. (n=7, each group).
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of experiment, the body weight in all groups is approximately 200 g. In the whole experimental period, there was no difference of body weight among the four experimental groups. Effects of C. tricuspidata water extract on SBP Fig. 1 shows effects on the SBP during the four weeks period of blockade of NO synthesis with L-NAME and those of C. tricuspidata water extract. At the starting point of experiment, the SBP in all groups is approximately 116–120 mmHg. Oral administration of L-NAME increased SBP to 148 ± 4 mmHg at first 1 week and this increased SBP was sustained for the whole experiment period (p < 0.01 vs control). This increased SBP was reversed to normotension by co-administration of C. tricuspidata water extract during the last 2 weeks (131 ± 3 mmHg, p < 0.05). Furthermore, the supplement with C. tricuspidata water extract during whole experiment prevented the increase in SBP by the oral administration of L-NAME in rats (p < 0.01 vs L-NAME). Effects of C. tricuspidata water extracts on the NOx/cGMP production As shown in Fig. 2, there were no changes in the expression of ecNOS and iNOS in the aortic tissue from L-NAME administrated rats for 4 weeks compared to control group. The expression of these NOS isoforms in the aortic tissue did not change by administration of C. tricuspidata water extract, either. However, the aortic production of NO metabolites was decreased by L-NAME administration (control: 3.91 ± 0.40, L-NAME: 2.51 ± 0.18 nmol/mg protein, p < 0.05, Fig. 3A). The supplementation with C. tricuspidata water extract during the
Fig. 1. Effects of Cudrania tricuspidata water extract on rats systolic blood pressures in different experimental groups. CON; control group, L-NAME; L-NAME-treated group, CT-1; Cudrania tricuspidata water extract was supplemented with L-NAME during the last 2 weeks, and CT-2; ditto except treated for the whole period of experiments. There were seven experiments in each group. **p < 0.01 compared with control; #p < 0.01, *p < 0.05 compared with L-NAME-treated group.
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Fig. 2. Representative western blots and densitometric analyses showing ecNOS and iNOS expression in the thoracic aorta of obtained from different experimental groups. Upper panel: 1, 2, 3, 4 denote Control, L-NAME, CT-1, and CT-2, respectively. Legends are the same as in Fig. 1. There were four experiments in each group.
last 2 weeks reversed the decreased release of aortic NO metabolites by L-NAME-treatment (3.29 ± 0.30 nmol/mg protein, p < 0.05) toward normal. The supplement with C. tricuspidata water extract during whole experiment prevented the decrease in NO metabolites induced by L-NAME (3.28 nmol/mg protein). Plasma concentration of NO metabolites was paralleled aortic NO production. Plasma concentration of NO metabolites was also decreased by L-NAME administration, which was reversed and prevented by co-administration with C. tricuspidata water extracts (Fig. 3B). Fig. 4 show that the blockade of NO synthesis by oral administration of L-NAME significantly inhibited aortic cGMP production (control: 1.64 ± 0.12, L-NAME: 0.52 ± 0.16 nmol/mg protein, p < 0.01). These inhibitions were partially reversed by supplementation with C. tricuspidata water extract during the last 2 weeks and partially prevented by supplementation during the whole experimental period (CT-1: 1.02 ± 0.08, CT-2: 0.96 ± 0.07 nmol/mg protein, p < 0.05 vs L-NAME, respectively).
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Fig. 3. Effects of Cudrania tricuspidata water extract on the vascular tissue and plasma nitrite/nitrate levels in the different experimental groups. See Fig. 1 for legends. There were seven experiments in each group. *p < 0.05, compared with control; # p < 0.05, compared with L-NAME-treated group.
Fig. 4. Effects of Cudrania tricuspidata water extract on the vascular tissue cGMP levels in the different experimental groups. See Fig. 1 for legends. There were seven experiments in each group. *p < 0.05, compared with control; # p < 0.05, compared with L-NAME-treated group.
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Effects of C. tricuspidata water extracts on renal functions Administration of L-NAME for 4 weeks significantly lowered renal creatinine clearance (p < 0.05 vs control, Fig. 5A). Renal sodium excretion (UNaV) and solute-free water reabsorption (TcH2O) also decreased in the 4-weeks L-NAME administrated rats (p < 0.05 vs control, respectively, Fig. 5B and C). These inhibitions were fully reversed by supplementation with C. tricuspidata water extract during the last 2 weeks and prevented by supplemen-
Fig. 5. Effects of Cudrania tricuspidata water extract on renal creatinine clearance (Ccr), urinary sodium excretion (UNaV), and solute-free water reabsorption (TcH2O) in the different experimental groups. The legends are the same as in Fig. 1. There were seven experiments in each group. *p < 0.05, compared with control; # p < 0.01, compared with L-NAME-treated group.
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tation for the whole experiment period of four weeks (p < 0.01 vs L-NAME, Fig. 5A, B, C). Moreover, two renal functional parameters (Ccr and TcH2O) were larger than respective value in control rats (p < 0.05 vs control).
Discussion NOS is inhibited by L-arginine analogues such as NG-monomethyl-L-arginine and L-NAME. When they are infused into animals or humans, NO production is inhibited and leads to vascular smooth muscle contraction, renal dysfunction, and increased blood pressures [1,2]. Various models of hypertension have been associated with a decreased release of NO. In hypertension mediated by volume overload, such as deoxycorticosterone acetate (DOCA)salt and Dahl-salt sensitive hypertensive models, the renal and aortic NO levels are decreased [1,16]. We also observed in the present study that chronic treatment with L-NAME increased the blood pressure in association with decreases plasma NO and aortic tissue NO/cGMP levels. However, L-NAME had no discernible effects on NOS isoforms expression profiles in the aorta. These findings most likely reflect the diminished vasodilator action of endogenous NO, where L-NAME may have interfered with enzyme activity of NOS as opposed to quantitative modification of NOS isoforms expression. These results are in accordance with previously study [17]. After administration of L-NAME for 2 weeks, the SBP was declined by co-administration with C. tricuspidata water extract. When the co-administration of L-NAME with C. tricuspidata water extract in rats, there were no changes in SBP. The plasma NO and aortic NO/cGMP were reversed by co-administration of C. tricuspidata water extract during the last 2 weeks. Furthermore, there were also no decreases in plasma NO and aortic NO/cGMP level by the co-administration with C. tricuspidata water extract during the whole experiment. The pharmacological action of C. tricuspidata extract has seldom been examined. Lee et. al. isolated three new dihydroflavonols from stem bark of Cudrania tricuspidata, gericudranins A–C, had a cytotoxic effects on human tumor cell lines [13]. Recently, many studies have been performed to find more suitable antihypertensives from other natural sources. Among them, extracts of hawthorn [5,6], Caesalpine sappa [7], Uncaria rhynchophylla [5,9], Cordyceps sinensis [3], Gynostemma pentaphyllum [11], Salviae miltiorrhizae [10] had a hypotensive effects due to endothelium-dependent vasorelaxation or direct stimulation of NO release. Moreover, garlic extract has a antihypertensive effect on L-NAME-induced hypertension [8]. According to our study, C. tricuspidata water extract may also have an antihypertensive effect via stimulation of NO/cGMP system in the vascular cells. It is well known that NO is an important regulator of renal hemodynamics and sodium handling. In the normal rats, nitric oxide level increases as an adaptive response to increased dietary salt intake, perhaps facilitating natriuresis and thus blood pressure homeostasis [18]. NO is synthesized directly in the renal vasculature. Immunohistochemical studies have shown expression of constitutive NOS on glomerular capillaries, afferent and efferent arteriols, medullary vasa recta, and intrarenal arteries [19]. With the use of PCR techniques, expression
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of inducible NOS has also been suggested in renal vessel [20]. Inhibition of nitric oxide synthesis by administration of nitro-L-arginine or L-NAME leads to a decrease in urinary sodium excretion (UNaV) along with the decrease renal blood flow, urine flow rates, glomerular filtration rates (GFR), and free-water clearance [2,18,21]. Animals treated chronically with NOS inhibitors develop hypertension, with a resetting of the pressure– natriuresis relation toward higher blood pressure. Similar results were obtained in the present study. Where administration with L-NAME decreased the creatinine clearance (Ccr), sodium excretion (UNaV), and free-water reabsorption (TcH2O), which was prevented and reversed by co-administration with C. tricuspidata water extract. Therefore, the effects of C. tricuspidata water extract may be attributable to amelioration of renal function in the L-NAME-induced hypertension via activation of NO/cGMP system. In summary, the inactivation of NO/cGMP system was prevented and reversed along with SBP normalization in the L-NAME-induced hypertension by co-administration with C. tricuspidata water extract without significant changes in NOS isoproteins expression in the thoracic aorta of a rat model. Furthermore, C. tricuspidata water extract ameliorated renal functional parameters in the L-NAME-induced hypertensive rats. These results suggest that C. tricuspidata water extract attenuated the increase in SBP in the L-NAMEinduced hypertension by enhancing the generation of vascular NO/cGMP and amelioration of renal functions.
Acknowledgments This study was supported by the Brain Korea 21 Project (2001) and grant of the Oriental Medicine R and D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-CO03-0003). We were grateful to Kyung Woo Cho, PhD., MD. for his advise in the course of this study.
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