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Effect of Cinnamomum cassia Methanol Extract and Sildenafil on Arginase and Sexual Function of Young Male Wistar Rats Sumanta K. Goswami, PhD,* Mohammed N. Inamdar, PhD,* Rohitash Jamwal, M.Tech,† and Shekhar Dethe, PhD† *Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore, India; †Bioassay Lab, Research and Development Centre, Natural Remedies Private Limited, Bangalore, India DOI: 10.1111/jsm.12535
ABSTRACT
Introduction. Herbs have been used as an aphrodisiac since ages. Cinnamomum cassia is an important ingredient of many Ayurvedic formulations to treat male sexual disorder including erectile dysfunction (ED). Aim. The objective of the present study was to evaluate erectogenic and aphrodisiac activity of methanol extract of C. cassia bark in young male rats. Methods. Methanol extract of C. cassia was screened in vitro for arginase inhibition potential and IC50 was determined. Effect of the extract was observed in vitro on phenylephrine pre-contracted isolated rat corpus cavernosum smooth muscle (CCSM) at 0.1, 1, 10, and 100 μg/mL. Young male Wistar rats were dosed with extract at 100 mg/kg body weight for 28 days and its effects on sexual behavior and penile smooth muscle : collagen level were observed. Main Outcome Measure. Effect of C. cassia was studied on arginase activity in vitro and sexual behavior of young male rats. Results. C. cassia inhibited arginase activity in vitro with an IC50 of 61.72 ± 2.20 μg/mL. The extract relaxed phenylephrine pre-contracted isolated rat CCSM up to 43% and significantly increased (P < 0.05) sexual function of young male rats. Treatment with the extract also increased smooth muscle level and decreased collagen level in rat penile tissue. Conclusion. The study proves usefulness of methanol extract of C. cassia bark for increasing sexual function. Goswami SK, Inamdar MN, Jamwal R, and Dethe S. Effect of Cinnamomum cassia methanol extract and sildenafil on arginase and sexual function of young male Wistar rats. J Sex Med 2014;11:1475–1483. Key Words. Cinnamomum cassia; Arginase; Enzymes and Enzyme Inhibitors Regulating Corporal Smooth Muscle Relaxation; Sexual Function; Sildenafil; NOHA
Introduction
P
enile erection is an integral part of male sexual function, and many neurotransmitters, ion channels, and enzymes are involved in the control of erectile mechanisms, centrally and peripherally [1]. Cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 inhibitors (PDE5is) like sildenafil, vardenafil, and tadalafil are commonly used for the management of erectile dysfunction (ED), a common male sexual dysfunction [2,3]. PDE5is act by relaxing
© 2014 International Society for Sexual Medicine
corpus cavernosum smooth muscle (CCSM) (penile tissue smooth muscle) and increasing penile blood flow. Other enzymes and their inhibitors, which have been implicated in the management of ED, include arginase [4] and Rho kinase 2 (ROCK-II) [5]. Indian medicinal plants described as Vajikaran herbs/aphrodisiacs have been used for the management of male sexual disorders including ED [6–11]. Because of a lack of literature, it is difficult to comment on the mechanism of action of these plants. However, the efficacy of these extracts J Sex Med 2014;11:1475–1483
1476 might be attributed to inhibition of one or more of the above mentioned enzymes. One such plant with an unknown mechanism of action is Cinnamomum cassia. Methanol extract of the C. cassia (CCME) was previously reported by authors of this article to inhibit ROCK-II in vitro with an IC50 of 9.40 ± 1.93 μg/mL [12]. In this study, we evaluated the effect of CCME on in vitro arginase activity and isolated rat CCSM. Effect of the extract was also studied on sexual function of young male rats in addition to its effect on smooth muscle : collagen level in penile tissue of these rats.
Materials and Methods
Plant Material and Extraction Bark of C. cassia (500 g) was procured from a local market in Bangalore, India, during January 2011. The raw material was identified and authenticated by comparison with the crude drug reference standard by Dr. P. Santhan, Taxonomist, Natural Remedies Pvt. Ltd. The voucher specimen was stored in the natural product library of Natural Remedies Pvt. Ltd., and methanol extract of remaining dried bark was prepared as described elsewhere [12]. Chemicals and Materials L-Arginine, MnCl2, Nω-hydroxy-L-arginine (NOHA, an arginase inhibitor), and Sirius red (direct red 80) from Sigma-Aldrich, Co. (St. Louis, MO, USA), dimethylaminobenzaldehyde, bovine serum albumin (BSA), potassium sodium tartrate, picric acid, xylene, and Tween 20 from HiMedia Labs (Mumbai, India), and Folin–Ciocalteu reagent from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India) were collected. Diethylstilbestrol from Penta Pharmaceuticals (Mumbai, India) and progesterone from Sun Pharmaceutical Ind. Ltd. (Mumbai, India) were procured. Watson Pharma India Ltd. (Mumbai, India) provided sildenafil as a gift sample. All other reagents used in the study were of analytical grade. Animals The animal experimentation was performed after review and approval of the study protocol by our institutional animal ethics committee. Young Wistar rats of either sex weighing 200–250 g were fed with normal rat chow, had free access to drinking water, and were maintained in 12-hour light and dark cycles at 25°C. J Sex Med 2014;11:1475–1483
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Isolation of Arginase Enzyme Crude arginase enzyme was isolated from liver of rat as described by the method of Schimke [13] with some modifications. Briefly, rat weighing 250 g was anaesthetized and sacrificed by cervical dislocation. Thoracic cavity was opened up and liver was carefully removed. The liver was washed twice in 0.01 M Tris-HCl buffer containing 0.05 M MnCl2 (pH 7.5). Further, liver was cut into pieces and homogenized in three volumes of icecold buffer. The homogenate was centrifuged at 4°C, 15,000 g for 15 minutes, and supernatant was separated. To the supernatant, 1.5 volumes of chilled acetone (−10°C) was added, mixed, and centrifuged at −10°C, 15,000 g for 5 minutes. The precipitate was homogenized in five volumes of buffer and centrifuged at 4°C, 15,000 g for 10 minutes. The supernatant was collected and dialyzed for 20 minutes against buffer in an ice bath using a magnetic stirrer. The dialyzed solution was heated at 60°C for 20 minutes on water bath while continuously stirring it with glass rod. The resulting solution was cooled on ice bath and centrifuged again at 4°C, 15,000 g for 10 minutes. The supernatant was cooled to 0°C followed by addition of equal volume of chilled ethanol (−10°C) containing 0.05 M MnCl2. The mixture was centrifuged at −10°C, 15,000 g for 10 minutes. Three volumes of ethanol (4°C) were added to the above supernatant and centrifuged at 4°C, 15,000 g for 10 minutes. The precipitate was finally suspended and homogenized in 3 mL of 0.01 M Tris-HCl buffer containing 0.05 M MnCl2 (pH 7.5) and stored at −80°C till use. Arginase Inhibition Assay Protein content of crude arginase enzyme was estimated by the method of Lowry et al. using BSA as a standard [14]. In arginase (inhibition) assay, specific activity of arginase can be determined by the amount of arginine consumed or the amount of urea formed. We measured the amount of urea generated in arginase (inhibition) assay to establish the specific activity of arginase used. Absorbance of ureaEhlrich’s reagent (p-dimethylaminobenzaldehyde in 3.6 N H2SO4) complex of the assay was plotted against the standard curve of different concentration of urea and a fixed concentration of Ehlrich’s reagent [15]. Arginase inhibition assay was performed as per the method of Hagan and Dallam [16]. In brief, a solution containing crude enzyme (protein
Cinnamon Increases Sexual Function of Young Male Rats concentration of 30 μg/mL) and 50 μg/mL of herbal extract were pre-incubated for 5 minutes in 0.01 mM Tris-HCl buffer (pH 7.5) containing 0.05 mM MnCl2. The reaction was initiated by addition of 50 mM L-arginine followed by incubation at 37°C for 10 minutes. The reaction was stopped by an addition of 4 × 10−4 M Ehlrich’s reagent, and the microplate was read at an absorbance of 450 nm using a microplate reader (VERSAmax; Molecular Devices, Sunnyvale, CA, USA) after 20 minutes. The plant extract was prepared in 50% dimethyl sulfoxide (DMSO), wherein final concentration of DMSO in the assay was limited to 2.5%. NOHA was used as positive control in this study. IC50 was calculated and the study was performed in triplicate. Similarly, arginase inhibition potential of sildenafil at 50 μg/mL was also studied.
Effect on Isolated Rat CCSM As described previously [12], two corpus cavernosa (3 × 3 × 15 mm) were isolated from each young male Wistar rats weighing 200–230 g, anesthetized by intraperitoneal administration of ketamine (70 mg/kg) and xylazine (10 mg/kg). Tunica albuginea covering corpus cavernosum was partially removed, and the CCSMs were mounted in a four-channel organ bath containing modified Krebs–Henseleit (K-H) salt solution (composition [mM]: 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.5 CaCl2, 25 NaHCO3, 11 glucose, 0.01 Na2EDTA, and 0.006 indomethacin) maintained under standard conditions (temperature: 37°C; aeration with carbogen gas; replacing K-H solution every 15 minutes). Tissues were stretched up to 500 mg of tension by a steel hook at the bottom of organ bath and a force transducer (MLT0201; ADInstruments, Bella Vista, NSW, Australia) positioned at the top of organ bath, and connected to PowerLab/8SP data acquisition system (Chart 7.0; ADInstruments). After equilibration for 1 hour, relaxant effects of DMSO (2.5%), CCME, or sildenafil were recorded separately on phenylephrine (3 μM) pre-contracted rat CCSM. Percent relaxation of CCSM by the vehicle, extract, and sildenafil was calculated considering maximum contraction of tissue by phenylephrine as 100% response. Sexual Behavior Study Sexually experienced young (5 months, weighing 200–250 g) male Wistar albino rats were used for the study. Rats were divided into three groups with
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each containing six rats: group I (young rat treated with 1% Tween 20), group II (young rat treated with 100 mg/kg body weight CCME), and group III (young rat treated with 5 mg/kg body weight sildenafil). Treatment was continued for 28 days and sexual behavior of male rats was observed on 0 (before dosing), 14th, and 28th day of dosing [7,8,10]. Ovariectomized [17] female rats were brought to estrus phase by administration of diethylstilbestrol (1 mg/kg, p.o., administered 48 hours prior to study) and progesterone (5 mg/kg, s.c., administered 4 hours prior to the study). One hour after dosing, male rat was introduced to the observation chamber (45 × 50 × 35 cm) illuminated by red light and allowed to accustom to the environment for 5 minutes. Female rat in estrus phase was then introduced into the cage by lifting upper glass lid slowly, and a number of sexual behavior parameters were observed. Mount latency (ML) (time taken by the male rat up to the first mount on female rat) and intromission latency (IL) (time taken by the male rat up to the first intromission) were recorded. Ejaculation latency (EL) (time taken by the male rat from first intromission until ejaculation) was confirmed by presence of semen on vagina of female rat or vaginal plug. Mount frequency (MF) (number of mounting by a male rat on a female rat in a given sexual cycle), intromission frequency (IF) (number of intromission by a male rat in a given sexual cycle), and post-ejaculatory interval (PEI) (time from end of one sexual cycle to start of another consecutive sexual cycle) were also studied. The PEI was characterized by lethargy of the male rats.
Histopathology of Rat Penile Tissue Penile tissues were isolated from anesthetized rats, and histopathology of the tissues was performed as described earlier [18,19] with modification. Briefly, the excised shaft of the penile tissues were fixed over night in 7% formalin saline and then transferred to 70% ethanol till the tissues were further processed. The tissues samples were dehydrated in graded ethanol for 30 minutes, followed by clearing in chloroform twice for 30 minutes each. The tissues were then impregnated in paraffin block and cut into five micron thick in size for fixing to glass slides. For determining smooth muscle : collagen level in rat penile tissues, Masson’s trichrome staining was performed. The tissue were hydrated and dipped in Coplin staining jar containing Bouin’s J Sex Med 2014;11:1475–1483
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Goswami et al. IC50 of CCME and NOHA was found to be 61.72 ± 2.20 and 156.78 ± 7.76 μg/mL, respectively. Specific activity of arginase was found to be 490 μM urea/minute/mg of protein. Protein concentration of arginase stock solution (crude enzyme) was found to be 3.068 mg/mL, and 30 μg/mL of crude enzyme was used for assay. Sildenafil (a PDE5-specific inhibitor) did not inhibit in vitro arginase activity up to 250 μg/mL.
Percent Arginase inhibition
80 Cinnamomum cassia ME NOHA
60
40
20
34 2.
2. 04
2
74 1.
69 1.
33 1.
04 1.
0. 79
0
log concentration (mg) Figure 1 Effect of Cinnamomum cassia methanol extract and NOHA on arginase inhibition in vitro. ME = methanol extract; NOHA = Nω-hydroxy-L-arginine.
fluid for half a day at 27°C. The tissues were washed and rinsed with distilled water. The tissues were stained in Weigert’s iron hematoxylin (10 minutes) and Biebrich scarlet-acid fuchsin staining (5 minutes). The tissues were stained with aniline blue (3 minutes) after treating in phosphomolybdic-tungstic acid solution. The tissues were rinsed in dilute acetic acid (5 minutes). Tissues were washed and rinsed with distilled water between different staining procedures. Tissues were then dehydrated in alcohol, cleared in xylene, and mounted in dibutyl phathalate xylene resin. Tissue slides were studied under a light microscope (40×; Nikon, Otawara, Japan) and photographs of CCSM were taken with a Nikon digital camera (model number: DS-Ri1).
Statistical Methods Inhibition of enzyme activity by herbal extract and sildenafil is expressed as mean ± standard deviation. Effects of vehicle, herbal extract, and sildenafil on isolated rat CCSM, and sexual behavior are expressed as mean ± standard error of mean. Statistical significance with respect to vehicle was evaluated using one-way analysis of variance followed by Dunnett’s t-test using SPSS software version 17 (SPSS Inc., Chicago, IL, USA). Results
Arginase Inhibition Assay As shown in Figure 1, CCME was found to be a more potent arginase inhibitor than NOHA [20]. J Sex Med 2014;11:1475–1483
Effect on Isolated Rat CCSM Percent tissue relaxation by CCME and sildenafil was studied at four different concentrations i.e., 0.1, 1, 10, and 100 μg/mL. Both CCME and sildenafil relaxed CCSM at these concentrations in a dose-dependent manner. At highest concentration, sildenafil relaxed CCSM beyond 100% while CCME showed up to 43% relaxation (Figure 2). Sexual Behavior Study Sexual behavior increased significantly in young Wistar rats after C. cassia and sildenafil treatment as is evident by the decrease in ML, IL, and PEI and increase in MF, IF, and EL. ML and PEI are measures of sexual motivation whereas intromission depends on relaxation of penile smooth muscle [1,21]. Treatment with CCME decreased ML, IL, and PEI in young male rats while the treatment increased MF, IF, and EL. The treatment increased sexual function of rats in a timedependent manner. Effect of sildenafil was better than CCME (Figure 3).
Figure 2 Relaxant effect of Cinnamomum cassia (n = 5) and sildenafil (n = 6) on isolated rat corpus cavernosum smooth muscle (CCSM) vs. vehicle (DMSO, n = 6). The relaxant effect of C. cassia methanol extract and sildenafil was recorded at 0.1, 1, 10, and 100 μg/mL and compared with that of DMSO. Each bar represents mean ± standard error of mean. *P < 0.001. DMSO = dimethyl sulfoxide.
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A
B
C
Figure 3 Increase in sexual behavior of young rats treated with methanol extract of Cinnamomum cassia (CCME) and sildenafil (SIL) vs. vehicle (V). n = 6. 0, 14, and 28 denotes day(s). Values are expressed as mean ± standard error of mean. # $ P ≤ 0.05, P ≤ 0.01, *P ≤ 0.001. EL = ejaculation latency; IF = intromission frequency; IL = intromission latency; MF = mount frequency; ML = mount latency; PEI = postejaculatory interval.
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Figure 4 Microscopic image of rat corpus cavernosum smooth muscle (CCSM) treated with vehicle/methanol extract of Cinnamomum cassia (CCME)/sildenafil stained with Masson’s trichrome staining and imaged through light microscopy (40×). Smooth muscle (shown by arrows) was stained as red/pink, while collagen (shown by broken arrow) was stained as blue. Treatment with CCME and sildenafil increased smooth muscle and decreased collagen level.
Histopathology of Rat Penile Tissue Treatment with CCME and sildenafil increased the level of smooth muscle and decreased the level of collagen in the rat penile tissue (Figure 4). Discussion
In our previous study, we have reported ROCK-II inhibition potential of CCME [12]. This study further revealed arginase inhibition potential of the extract. CCME was more potent than NOHA, an endogenous arginase inhibitor. Pro-erectile effect of C. cassia was proved as it significantly relaxed (P < 0.001) phenylephrine-contracted, isolated rat CCSM. CCME also decreased ML and PEI and increased MF, IF and EL significantly. The increase in sexual function [21] justifies the role of CCME in Vajikaran chikitsa, a branch of Ayurveda for management of male sexual dysfunction [6]. Arginine/nitric oxide (NO)/cGMP pathway was found to relax CCSM, which is essential for penile erection [22]. Up-regulation of arginase in the penis of aged animal has been reported to deplete arginine level resulting in decreased erectile function [23]. L-arginine 5 g per day for 6 weeks increased sexual function of human participants [24]. Further, the relaxation of phenylephrinecontracted, isolated human corpus cavernosum has also been shown to increase after incubation with L-arginine for 6 to 10 hours [25]. Neuronal nitric oxide synthase (nNOS) utilizes arginine for production of NO that diffuses into CCSM and stimulates soluble guanylyl cyclase to generate cGMP from guanosine triphosphate. cGMP, the J Sex Med 2014;11:1475–1483
second messenger, relaxes the penile smooth muscle by opening potassium channel. Arginase competes with nNOS for arginine and decreases the level of cGMP, and arginase inhibition increases cGMP level in penile smooth muscle (Figure 5). Rho kinase was reported to be present in human, rabbit, and rat CCSM and its inhibition by Y-27632 dihydrochloride has been reported to relax CCSM [26,27]. In another study, Y-27632 improved sexual function in aged rat where elevated RhoA/Rhokinase activity had decreased intracavernosal pressure [28]. Recently, Toque et al. demonstrated that ROCK-II is associated with diabetes-induced increase in the level of arginase in mice penile smooth muscle. Corpora cavernosa (CC) from diabetic mice showed reduced relaxation response to acetylcholine in comparison with CC from normal mice, whereas CC from Rho-kinase knockout diabetic mice showed better relaxation response. Arginase level and activity were found to be increased in the CC of diabetic mice, whereas CC from Rhokinase knockout diabetic mice had reduced arginase level and activity [29]. Waldkirch et al. demonstrated the presence of Rho-kinase-related proteins in human cavernous arteries. Y-27632 was reported to relax norepinephrine pre-contracted cavernous arteries. This study confirmed the role of Rhokinase in penile blood flow and endothelial relaxation [30]. The erectile potential of apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), a phytochemical in diabetic rats, was reported to be due to Rho-kinase inhibition [31]. Because the increase in the amount of dephos-
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CCSM Relax
Cavernous nerve
GTP
NO
sGC cGMP nNOS Sildenafil
PDE5
L-Arginine
GMP CCME
Arginase Ornithine + Urea
Figure 5 A proposed mechanism of Cinnamomum cassia action for increasing corpus cavernosal smooth muscle function. Arrow represents stimulation, whereas dotted arrow represents inhibition. Arginine is a common substrate for nNOS and arginase for production of NO and urea, respectively. NO after diffusing to CCSM from nonadrenergic noncholinergic nerve terminal (cavernous nerve) stimulates sGC for converting GTP to cGMP that relaxes CCSM. Inhibition of arginase increases the level of arginine that ultimately leads to more production of cGMP. However, PDE5 can metabolize cGMP, decreasing relaxant effect of cGMP. Sildenafil relaxes CCSM by inhibiting PDE5. Both C. cassia and sildenafil works through arginine/ NO/cGMP pathway, but inhibit different enzymes. CCME = methanol extract of Cinnamomum cassia; CCSM = corpus cavernosum smooth muscle; cGMP = cyclic guanosine monophosphate; GTP = guanosine triphosphate; nNOS = neuronal nitric oxide synthase; NO = nitric oxide; PDE5 = phosphodiesterase type 5; sGC = soluble guanylyl cyclase
phorylated myosin light chain as a result of ROCK-II inhibition leads to relaxation of isolated rat CCSM, CCME-induced relaxation can be partly attributed to ROCK-II inhibition. The relaxation of rat CCSM and increase in IF by CCME might be due to stimulation of arginine/NO/cGMP pathway and inhibition of RhoA/Rho-kinase pathway. The level of smooth muscle was greater in the corpus cavernosum of rats treated with CCME when compared with rats treated with vehicle. This increase in smooth muscle level by CCME could be due to its cinnamic acid content. Ethanol extract of C. cassia and cinnamic acid (a marker compound for C. cassia) were reported to induce endothelial cell proliferation in vitro. The extract and cinnamic acid also increased the production of vascular endothelial growth factor (VEGF) and fetal liver kinase-1/kinase insert domain receptor, the VEGF receptor involved in proliferation of endothelial cells [32]. cGMP has been reported to
possess antifibrotic property [18]. Decrease of collagen level in penile tissue of rats treated with CCME and sildenafil could be due to increase in level of cGMP due to arginase and PDE5 inhibition, respectively. Conclusion
Erectogenic and aphrodisiac effects of CCME bark can be attributed to its potential to inhibit arginase activity and increase smooth muscle collagen ratio in the young rat penile tissue. Thus, the study supports aphrodisiac use of C. cassia in Ayurveda as sexual invigorator and suggests CCME as a potential lead for the management of ED. Further studies should be conducted to know the efficacy of C. cassia in age and diabetes-induced ED. Acknowledgments
This study was supported by research grant (R&D/KR03/2009–10-NMPB) to Al-Ameen College of PharJ Sex Med 2014;11:1475–1483
1482 macy, Bangalore and Natural Remedies Private Limited, Bangalore from National Medicinal Plants Board, New Delhi. We are thankful to Mr. Deepak Kumar Khajuria for ovariectomy. Corresponding Author: Sumanta Kumar Goswami, PhD, Department of Pharmacology, Al-Ameen College of Pharmacy, Near Lalbagh Main gate, Hosur road, Bangalore 560027, India. Tel: +91-080-2211-3861; Fax: +91-080-2222-5834; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. Statement of Authorship
Category 1 (a) Conception and Design Sumanta K. Goswami; Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar (b) Acquisition of Data Sumanta K. Goswami; Rohitash Jamwal (c) Analysis and Interpretation of Data Sumanta K. Goswami; Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar
Category 2 (a) Drafting the Article Sumanta K. Goswami (b) Revising It for Intellectual Content Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar
Category 3 (a) Final Approval of the Completed Article Sumanta K. Goswami; Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar References 1 Andersson KE. Pharmacology of penile erection. Pharmacol Rev 2001;53:417–50. 2 Burchardt M, Burchardt T, Anastasiadis AG, Kiss AJ, Shabsigh A, de La Taille A, Pawar RV, Baer L, Shabsigh R. Erectile dysfunction is a marker for cardiovascular complications and psychological functioning in men with hypertension. Int J Impot Res 2001;13:276–81. 3 Carson CC. Erectile dysfunction: Evaluation and new treatment options. Psychosom Med 2004;66:664–71. 4 Christianson DW. Arginase: Structure, mechanism, and physiological role arginase in male and female sexual arousal. Acc Chem Res 2005;38:191–201. 5 Wingard CJ, Johnson JA, Holmes A, Prikosh A. Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction. Am J Physiol Regul Integr Comp Physiol 2003;284:R1572–9. 6 Dalal PK, Tripathi A, Gupta SK. Vajikarana: Treatment of sexual dysfunctions based on Indian concepts. Indian J Psychiatry 2013;55:S273–6.
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Effect of Cinnamomum cassia Methanol Extract and Sildenafil on Arginase and Sexual Function of Young Male Wistar Rats Sumanta K. Goswami, PhD,* Mohammed N. Inamdar, PhD,* Rohitash Jamwal, M.Tech,† and Shekhar Dethe, PhD† *Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore, India; †Bioassay Lab, Research and Development Centre, Natural Remedies Private Limited, Bangalore, India DOI: 10.1111/jsm.12535
ABSTRACT
Introduction. Herbs have been used as an aphrodisiac since ages. Cinnamomum cassia is an important ingredient of many Ayurvedic formulations to treat male sexual disorder including erectile dysfunction (ED). Aim. The objective of the present study was to evaluate erectogenic and aphrodisiac activity of methanol extract of C. cassia bark in young male rats. Methods. Methanol extract of C. cassia was screened in vitro for arginase inhibition potential and IC50 was determined. Effect of the extract was observed in vitro on phenylephrine pre-contracted isolated rat corpus cavernosum smooth muscle (CCSM) at 0.1, 1, 10, and 100 μg/mL. Young male Wistar rats were dosed with extract at 100 mg/kg body weight for 28 days and its effects on sexual behavior and penile smooth muscle : collagen level were observed. Main Outcome Measure. Effect of C. cassia was studied on arginase activity in vitro and sexual behavior of young male rats. Results. C. cassia inhibited arginase activity in vitro with an IC50 of 61.72 ± 2.20 μg/mL. The extract relaxed phenylephrine pre-contracted isolated rat CCSM up to 43% and significantly increased (P < 0.05) sexual function of young male rats. Treatment with the extract also increased smooth muscle level and decreased collagen level in rat penile tissue. Conclusion. The study proves usefulness of methanol extract of C. cassia bark for increasing sexual function. Goswami SK, Inamdar MN, Jamwal R, and Dethe S. Effect of Cinnamomum cassia methanol extract and sildenafil on arginase and sexual function of young male Wistar rats. J Sex Med 2014;11:1475–1483. Key Words. Cinnamomum cassia; Arginase; Enzymes and Enzyme Inhibitors Regulating Corporal Smooth Muscle Relaxation; Sexual Function; Sildenafil; NOHA
Introduction
P
enile erection is an integral part of male sexual function, and many neurotransmitters, ion channels, and enzymes are involved in the control of erectile mechanisms, centrally and peripherally [1]. Cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 inhibitors (PDE5is) like sildenafil, vardenafil, and tadalafil are commonly used for the management of erectile dysfunction (ED), a common male sexual dysfunction [2,3]. PDE5is act by relaxing
© 2014 International Society for Sexual Medicine
corpus cavernosum smooth muscle (CCSM) (penile tissue smooth muscle) and increasing penile blood flow. Other enzymes and their inhibitors, which have been implicated in the management of ED, include arginase [4] and Rho kinase 2 (ROCK-II) [5]. Indian medicinal plants described as Vajikaran herbs/aphrodisiacs have been used for the management of male sexual disorders including ED [6–11]. Because of a lack of literature, it is difficult to comment on the mechanism of action of these plants. However, the efficacy of these extracts J Sex Med 2014;11:1475–1483
1476 might be attributed to inhibition of one or more of the above mentioned enzymes. One such plant with an unknown mechanism of action is Cinnamomum cassia. Methanol extract of the C. cassia (CCME) was previously reported by authors of this article to inhibit ROCK-II in vitro with an IC50 of 9.40 ± 1.93 μg/mL [12]. In this study, we evaluated the effect of CCME on in vitro arginase activity and isolated rat CCSM. Effect of the extract was also studied on sexual function of young male rats in addition to its effect on smooth muscle : collagen level in penile tissue of these rats.
Materials and Methods
Plant Material and Extraction Bark of C. cassia (500 g) was procured from a local market in Bangalore, India, during January 2011. The raw material was identified and authenticated by comparison with the crude drug reference standard by Dr. P. Santhan, Taxonomist, Natural Remedies Pvt. Ltd. The voucher specimen was stored in the natural product library of Natural Remedies Pvt. Ltd., and methanol extract of remaining dried bark was prepared as described elsewhere [12]. Chemicals and Materials L-Arginine, MnCl2, Nω-hydroxy-L-arginine (NOHA, an arginase inhibitor), and Sirius red (direct red 80) from Sigma-Aldrich, Co. (St. Louis, MO, USA), dimethylaminobenzaldehyde, bovine serum albumin (BSA), potassium sodium tartrate, picric acid, xylene, and Tween 20 from HiMedia Labs (Mumbai, India), and Folin–Ciocalteu reagent from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India) were collected. Diethylstilbestrol from Penta Pharmaceuticals (Mumbai, India) and progesterone from Sun Pharmaceutical Ind. Ltd. (Mumbai, India) were procured. Watson Pharma India Ltd. (Mumbai, India) provided sildenafil as a gift sample. All other reagents used in the study were of analytical grade. Animals The animal experimentation was performed after review and approval of the study protocol by our institutional animal ethics committee. Young Wistar rats of either sex weighing 200–250 g were fed with normal rat chow, had free access to drinking water, and were maintained in 12-hour light and dark cycles at 25°C. J Sex Med 2014;11:1475–1483
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Isolation of Arginase Enzyme Crude arginase enzyme was isolated from liver of rat as described by the method of Schimke [13] with some modifications. Briefly, rat weighing 250 g was anaesthetized and sacrificed by cervical dislocation. Thoracic cavity was opened up and liver was carefully removed. The liver was washed twice in 0.01 M Tris-HCl buffer containing 0.05 M MnCl2 (pH 7.5). Further, liver was cut into pieces and homogenized in three volumes of icecold buffer. The homogenate was centrifuged at 4°C, 15,000 g for 15 minutes, and supernatant was separated. To the supernatant, 1.5 volumes of chilled acetone (−10°C) was added, mixed, and centrifuged at −10°C, 15,000 g for 5 minutes. The precipitate was homogenized in five volumes of buffer and centrifuged at 4°C, 15,000 g for 10 minutes. The supernatant was collected and dialyzed for 20 minutes against buffer in an ice bath using a magnetic stirrer. The dialyzed solution was heated at 60°C for 20 minutes on water bath while continuously stirring it with glass rod. The resulting solution was cooled on ice bath and centrifuged again at 4°C, 15,000 g for 10 minutes. The supernatant was cooled to 0°C followed by addition of equal volume of chilled ethanol (−10°C) containing 0.05 M MnCl2. The mixture was centrifuged at −10°C, 15,000 g for 10 minutes. Three volumes of ethanol (4°C) were added to the above supernatant and centrifuged at 4°C, 15,000 g for 10 minutes. The precipitate was finally suspended and homogenized in 3 mL of 0.01 M Tris-HCl buffer containing 0.05 M MnCl2 (pH 7.5) and stored at −80°C till use. Arginase Inhibition Assay Protein content of crude arginase enzyme was estimated by the method of Lowry et al. using BSA as a standard [14]. In arginase (inhibition) assay, specific activity of arginase can be determined by the amount of arginine consumed or the amount of urea formed. We measured the amount of urea generated in arginase (inhibition) assay to establish the specific activity of arginase used. Absorbance of ureaEhlrich’s reagent (p-dimethylaminobenzaldehyde in 3.6 N H2SO4) complex of the assay was plotted against the standard curve of different concentration of urea and a fixed concentration of Ehlrich’s reagent [15]. Arginase inhibition assay was performed as per the method of Hagan and Dallam [16]. In brief, a solution containing crude enzyme (protein
Cinnamon Increases Sexual Function of Young Male Rats concentration of 30 μg/mL) and 50 μg/mL of herbal extract were pre-incubated for 5 minutes in 0.01 mM Tris-HCl buffer (pH 7.5) containing 0.05 mM MnCl2. The reaction was initiated by addition of 50 mM L-arginine followed by incubation at 37°C for 10 minutes. The reaction was stopped by an addition of 4 × 10−4 M Ehlrich’s reagent, and the microplate was read at an absorbance of 450 nm using a microplate reader (VERSAmax; Molecular Devices, Sunnyvale, CA, USA) after 20 minutes. The plant extract was prepared in 50% dimethyl sulfoxide (DMSO), wherein final concentration of DMSO in the assay was limited to 2.5%. NOHA was used as positive control in this study. IC50 was calculated and the study was performed in triplicate. Similarly, arginase inhibition potential of sildenafil at 50 μg/mL was also studied.
Effect on Isolated Rat CCSM As described previously [12], two corpus cavernosa (3 × 3 × 15 mm) were isolated from each young male Wistar rats weighing 200–230 g, anesthetized by intraperitoneal administration of ketamine (70 mg/kg) and xylazine (10 mg/kg). Tunica albuginea covering corpus cavernosum was partially removed, and the CCSMs were mounted in a four-channel organ bath containing modified Krebs–Henseleit (K-H) salt solution (composition [mM]: 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.5 CaCl2, 25 NaHCO3, 11 glucose, 0.01 Na2EDTA, and 0.006 indomethacin) maintained under standard conditions (temperature: 37°C; aeration with carbogen gas; replacing K-H solution every 15 minutes). Tissues were stretched up to 500 mg of tension by a steel hook at the bottom of organ bath and a force transducer (MLT0201; ADInstruments, Bella Vista, NSW, Australia) positioned at the top of organ bath, and connected to PowerLab/8SP data acquisition system (Chart 7.0; ADInstruments). After equilibration for 1 hour, relaxant effects of DMSO (2.5%), CCME, or sildenafil were recorded separately on phenylephrine (3 μM) pre-contracted rat CCSM. Percent relaxation of CCSM by the vehicle, extract, and sildenafil was calculated considering maximum contraction of tissue by phenylephrine as 100% response. Sexual Behavior Study Sexually experienced young (5 months, weighing 200–250 g) male Wistar albino rats were used for the study. Rats were divided into three groups with
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each containing six rats: group I (young rat treated with 1% Tween 20), group II (young rat treated with 100 mg/kg body weight CCME), and group III (young rat treated with 5 mg/kg body weight sildenafil). Treatment was continued for 28 days and sexual behavior of male rats was observed on 0 (before dosing), 14th, and 28th day of dosing [7,8,10]. Ovariectomized [17] female rats were brought to estrus phase by administration of diethylstilbestrol (1 mg/kg, p.o., administered 48 hours prior to study) and progesterone (5 mg/kg, s.c., administered 4 hours prior to the study). One hour after dosing, male rat was introduced to the observation chamber (45 × 50 × 35 cm) illuminated by red light and allowed to accustom to the environment for 5 minutes. Female rat in estrus phase was then introduced into the cage by lifting upper glass lid slowly, and a number of sexual behavior parameters were observed. Mount latency (ML) (time taken by the male rat up to the first mount on female rat) and intromission latency (IL) (time taken by the male rat up to the first intromission) were recorded. Ejaculation latency (EL) (time taken by the male rat from first intromission until ejaculation) was confirmed by presence of semen on vagina of female rat or vaginal plug. Mount frequency (MF) (number of mounting by a male rat on a female rat in a given sexual cycle), intromission frequency (IF) (number of intromission by a male rat in a given sexual cycle), and post-ejaculatory interval (PEI) (time from end of one sexual cycle to start of another consecutive sexual cycle) were also studied. The PEI was characterized by lethargy of the male rats.
Histopathology of Rat Penile Tissue Penile tissues were isolated from anesthetized rats, and histopathology of the tissues was performed as described earlier [18,19] with modification. Briefly, the excised shaft of the penile tissues were fixed over night in 7% formalin saline and then transferred to 70% ethanol till the tissues were further processed. The tissues samples were dehydrated in graded ethanol for 30 minutes, followed by clearing in chloroform twice for 30 minutes each. The tissues were then impregnated in paraffin block and cut into five micron thick in size for fixing to glass slides. For determining smooth muscle : collagen level in rat penile tissues, Masson’s trichrome staining was performed. The tissue were hydrated and dipped in Coplin staining jar containing Bouin’s J Sex Med 2014;11:1475–1483
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Goswami et al. IC50 of CCME and NOHA was found to be 61.72 ± 2.20 and 156.78 ± 7.76 μg/mL, respectively. Specific activity of arginase was found to be 490 μM urea/minute/mg of protein. Protein concentration of arginase stock solution (crude enzyme) was found to be 3.068 mg/mL, and 30 μg/mL of crude enzyme was used for assay. Sildenafil (a PDE5-specific inhibitor) did not inhibit in vitro arginase activity up to 250 μg/mL.
Percent Arginase inhibition
80 Cinnamomum cassia ME NOHA
60
40
20
34 2.
2. 04
2
74 1.
69 1.
33 1.
04 1.
0. 79
0
log concentration (mg) Figure 1 Effect of Cinnamomum cassia methanol extract and NOHA on arginase inhibition in vitro. ME = methanol extract; NOHA = Nω-hydroxy-L-arginine.
fluid for half a day at 27°C. The tissues were washed and rinsed with distilled water. The tissues were stained in Weigert’s iron hematoxylin (10 minutes) and Biebrich scarlet-acid fuchsin staining (5 minutes). The tissues were stained with aniline blue (3 minutes) after treating in phosphomolybdic-tungstic acid solution. The tissues were rinsed in dilute acetic acid (5 minutes). Tissues were washed and rinsed with distilled water between different staining procedures. Tissues were then dehydrated in alcohol, cleared in xylene, and mounted in dibutyl phathalate xylene resin. Tissue slides were studied under a light microscope (40×; Nikon, Otawara, Japan) and photographs of CCSM were taken with a Nikon digital camera (model number: DS-Ri1).
Statistical Methods Inhibition of enzyme activity by herbal extract and sildenafil is expressed as mean ± standard deviation. Effects of vehicle, herbal extract, and sildenafil on isolated rat CCSM, and sexual behavior are expressed as mean ± standard error of mean. Statistical significance with respect to vehicle was evaluated using one-way analysis of variance followed by Dunnett’s t-test using SPSS software version 17 (SPSS Inc., Chicago, IL, USA). Results
Arginase Inhibition Assay As shown in Figure 1, CCME was found to be a more potent arginase inhibitor than NOHA [20]. J Sex Med 2014;11:1475–1483
Effect on Isolated Rat CCSM Percent tissue relaxation by CCME and sildenafil was studied at four different concentrations i.e., 0.1, 1, 10, and 100 μg/mL. Both CCME and sildenafil relaxed CCSM at these concentrations in a dose-dependent manner. At highest concentration, sildenafil relaxed CCSM beyond 100% while CCME showed up to 43% relaxation (Figure 2). Sexual Behavior Study Sexual behavior increased significantly in young Wistar rats after C. cassia and sildenafil treatment as is evident by the decrease in ML, IL, and PEI and increase in MF, IF, and EL. ML and PEI are measures of sexual motivation whereas intromission depends on relaxation of penile smooth muscle [1,21]. Treatment with CCME decreased ML, IL, and PEI in young male rats while the treatment increased MF, IF, and EL. The treatment increased sexual function of rats in a timedependent manner. Effect of sildenafil was better than CCME (Figure 3).
Figure 2 Relaxant effect of Cinnamomum cassia (n = 5) and sildenafil (n = 6) on isolated rat corpus cavernosum smooth muscle (CCSM) vs. vehicle (DMSO, n = 6). The relaxant effect of C. cassia methanol extract and sildenafil was recorded at 0.1, 1, 10, and 100 μg/mL and compared with that of DMSO. Each bar represents mean ± standard error of mean. *P < 0.001. DMSO = dimethyl sulfoxide.
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A
B
C
Figure 3 Increase in sexual behavior of young rats treated with methanol extract of Cinnamomum cassia (CCME) and sildenafil (SIL) vs. vehicle (V). n = 6. 0, 14, and 28 denotes day(s). Values are expressed as mean ± standard error of mean. # $ P ≤ 0.05, P ≤ 0.01, *P ≤ 0.001. EL = ejaculation latency; IF = intromission frequency; IL = intromission latency; MF = mount frequency; ML = mount latency; PEI = postejaculatory interval.
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Figure 4 Microscopic image of rat corpus cavernosum smooth muscle (CCSM) treated with vehicle/methanol extract of Cinnamomum cassia (CCME)/sildenafil stained with Masson’s trichrome staining and imaged through light microscopy (40×). Smooth muscle (shown by arrows) was stained as red/pink, while collagen (shown by broken arrow) was stained as blue. Treatment with CCME and sildenafil increased smooth muscle and decreased collagen level.
Histopathology of Rat Penile Tissue Treatment with CCME and sildenafil increased the level of smooth muscle and decreased the level of collagen in the rat penile tissue (Figure 4). Discussion
In our previous study, we have reported ROCK-II inhibition potential of CCME [12]. This study further revealed arginase inhibition potential of the extract. CCME was more potent than NOHA, an endogenous arginase inhibitor. Pro-erectile effect of C. cassia was proved as it significantly relaxed (P < 0.001) phenylephrine-contracted, isolated rat CCSM. CCME also decreased ML and PEI and increased MF, IF and EL significantly. The increase in sexual function [21] justifies the role of CCME in Vajikaran chikitsa, a branch of Ayurveda for management of male sexual dysfunction [6]. Arginine/nitric oxide (NO)/cGMP pathway was found to relax CCSM, which is essential for penile erection [22]. Up-regulation of arginase in the penis of aged animal has been reported to deplete arginine level resulting in decreased erectile function [23]. L-arginine 5 g per day for 6 weeks increased sexual function of human participants [24]. Further, the relaxation of phenylephrinecontracted, isolated human corpus cavernosum has also been shown to increase after incubation with L-arginine for 6 to 10 hours [25]. Neuronal nitric oxide synthase (nNOS) utilizes arginine for production of NO that diffuses into CCSM and stimulates soluble guanylyl cyclase to generate cGMP from guanosine triphosphate. cGMP, the J Sex Med 2014;11:1475–1483
second messenger, relaxes the penile smooth muscle by opening potassium channel. Arginase competes with nNOS for arginine and decreases the level of cGMP, and arginase inhibition increases cGMP level in penile smooth muscle (Figure 5). Rho kinase was reported to be present in human, rabbit, and rat CCSM and its inhibition by Y-27632 dihydrochloride has been reported to relax CCSM [26,27]. In another study, Y-27632 improved sexual function in aged rat where elevated RhoA/Rhokinase activity had decreased intracavernosal pressure [28]. Recently, Toque et al. demonstrated that ROCK-II is associated with diabetes-induced increase in the level of arginase in mice penile smooth muscle. Corpora cavernosa (CC) from diabetic mice showed reduced relaxation response to acetylcholine in comparison with CC from normal mice, whereas CC from Rho-kinase knockout diabetic mice showed better relaxation response. Arginase level and activity were found to be increased in the CC of diabetic mice, whereas CC from Rhokinase knockout diabetic mice had reduced arginase level and activity [29]. Waldkirch et al. demonstrated the presence of Rho-kinase-related proteins in human cavernous arteries. Y-27632 was reported to relax norepinephrine pre-contracted cavernous arteries. This study confirmed the role of Rhokinase in penile blood flow and endothelial relaxation [30]. The erectile potential of apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), a phytochemical in diabetic rats, was reported to be due to Rho-kinase inhibition [31]. Because the increase in the amount of dephos-
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CCSM Relax
Cavernous nerve
GTP
NO
sGC cGMP nNOS Sildenafil
PDE5
L-Arginine
GMP CCME
Arginase Ornithine + Urea
Figure 5 A proposed mechanism of Cinnamomum cassia action for increasing corpus cavernosal smooth muscle function. Arrow represents stimulation, whereas dotted arrow represents inhibition. Arginine is a common substrate for nNOS and arginase for production of NO and urea, respectively. NO after diffusing to CCSM from nonadrenergic noncholinergic nerve terminal (cavernous nerve) stimulates sGC for converting GTP to cGMP that relaxes CCSM. Inhibition of arginase increases the level of arginine that ultimately leads to more production of cGMP. However, PDE5 can metabolize cGMP, decreasing relaxant effect of cGMP. Sildenafil relaxes CCSM by inhibiting PDE5. Both C. cassia and sildenafil works through arginine/ NO/cGMP pathway, but inhibit different enzymes. CCME = methanol extract of Cinnamomum cassia; CCSM = corpus cavernosum smooth muscle; cGMP = cyclic guanosine monophosphate; GTP = guanosine triphosphate; nNOS = neuronal nitric oxide synthase; NO = nitric oxide; PDE5 = phosphodiesterase type 5; sGC = soluble guanylyl cyclase
phorylated myosin light chain as a result of ROCK-II inhibition leads to relaxation of isolated rat CCSM, CCME-induced relaxation can be partly attributed to ROCK-II inhibition. The relaxation of rat CCSM and increase in IF by CCME might be due to stimulation of arginine/NO/cGMP pathway and inhibition of RhoA/Rho-kinase pathway. The level of smooth muscle was greater in the corpus cavernosum of rats treated with CCME when compared with rats treated with vehicle. This increase in smooth muscle level by CCME could be due to its cinnamic acid content. Ethanol extract of C. cassia and cinnamic acid (a marker compound for C. cassia) were reported to induce endothelial cell proliferation in vitro. The extract and cinnamic acid also increased the production of vascular endothelial growth factor (VEGF) and fetal liver kinase-1/kinase insert domain receptor, the VEGF receptor involved in proliferation of endothelial cells [32]. cGMP has been reported to
possess antifibrotic property [18]. Decrease of collagen level in penile tissue of rats treated with CCME and sildenafil could be due to increase in level of cGMP due to arginase and PDE5 inhibition, respectively. Conclusion
Erectogenic and aphrodisiac effects of CCME bark can be attributed to its potential to inhibit arginase activity and increase smooth muscle collagen ratio in the young rat penile tissue. Thus, the study supports aphrodisiac use of C. cassia in Ayurveda as sexual invigorator and suggests CCME as a potential lead for the management of ED. Further studies should be conducted to know the efficacy of C. cassia in age and diabetes-induced ED. Acknowledgments
This study was supported by research grant (R&D/KR03/2009–10-NMPB) to Al-Ameen College of PharJ Sex Med 2014;11:1475–1483
1482 macy, Bangalore and Natural Remedies Private Limited, Bangalore from National Medicinal Plants Board, New Delhi. We are thankful to Mr. Deepak Kumar Khajuria for ovariectomy. Corresponding Author: Sumanta Kumar Goswami, PhD, Department of Pharmacology, Al-Ameen College of Pharmacy, Near Lalbagh Main gate, Hosur road, Bangalore 560027, India. Tel: +91-080-2211-3861; Fax: +91-080-2222-5834; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. Statement of Authorship
Category 1 (a) Conception and Design Sumanta K. Goswami; Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar (b) Acquisition of Data Sumanta K. Goswami; Rohitash Jamwal (c) Analysis and Interpretation of Data Sumanta K. Goswami; Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar
Category 2 (a) Drafting the Article Sumanta K. Goswami (b) Revising It for Intellectual Content Rohitash Jamwal; Shekhar Dethe; Mohammed N. Inamdar
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