Subscribe Now Subscribe Today
Research Article
 

Comparative Effects of Ficus exasperata Aqueous Leaf Extract and Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat



A.K. Amonkan, A.B. Konan, M.N. Bleyere, B.M.L. Ahui, L.K. Kouakou, G.M.K. Bouafou and S. Kati-Coulibaly
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

The prevalence of hypertension is increasing in many parts of the world. Management of this disease requires the use of diuretics, ACE inhibitors and β-blockers. In many pharmacopoeias, the leaves of Ficus exasperata are used to treat hypertension. The hypotensive effects of leaves of this species have been shown. The purpose of this study was to evaluate the diuretic effect of F. exasperata aqueous leaf extract (FEFIX) comparing to those of furosemide in salt hypertensive rats treated with deoxycorticosterone acetate (DOCA). Animals treated with DOCA were divided into three groups which received intraperitoneally NaCl (9‰, saline solution), FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1 b.wt.), respectively. Urine output was collected for 24 h. At the end of the experiment, the blood was collected and sampled. FEFIX and furosemide increased urinary volume (EUV) to 153.32±6.89 and 105.71±9.37%, respectively. F. exasperata aqueous leaf extract and furosemide increased the urinary excretion of electrolytes (Na+, Cl- and Ca2+), urea and creatinine. However, excretions induced by the plant extract were greater than those induced by furosemide. FEFIX and furosemide decreased the rate of plasma electrolytes with a decrease in serum sodium greater for this extract. The studied extract decreased urea and plasma creatinine like furosemide. These results showed that the diuretic effects of F. exasperata aqueous leaf extract were similar to those of furosemide in hypertension due to salt overload. However, the diuretic effects of this plant extract were superior to those of furosemide.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

A.K. Amonkan, A.B. Konan, M.N. Bleyere, B.M.L. Ahui, L.K. Kouakou, G.M.K. Bouafou and S. Kati-Coulibaly, 2013. Comparative Effects of Ficus exasperata Aqueous Leaf Extract and Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat. Journal of Medical Sciences, 13: 385-390.

DOI: 10.3923/jms.2013.385.390

URL: https://scialert.net/abstract/?doi=jms.2013.385.390
 
Received: March 27, 2013; Accepted: April 18, 2013; Published: June 14, 2013



INTRODUCTION

Hypertension is an elevation in blood pressure leading to a systolic blood pressure greater than or equal to 140 mm Hg and a diastolic blood pressure greater than or equal to 90 mm Hg (Erdine et al., 2006). It is currently a public health problem worldwide due to its frequency and risk of cardiovascular and renal diseases which are attached. Kearney et al. (2005) indicate a prevalence of 1.6 billion hypertensive subjects in 2025. Indeed, the prevalence of this disease increase in the world in general and developing countries in particular (Whitworth et al., 2003, Kearney et al., 2004). Generally, the treatment of this disease requires the use of diuretics, ACE inhibitors and beta-blockers (Armario and Waeber, 2013). So many people in developing countries employ several herbals to treat this pathology (Abrogoua et al., 2012; Anwar et al., 2007). Ficus exasperata Vahl. 1805 (Moraceae) is one of the plants used in this field. Its leaves are often found in many beverages antihypertensive preparations. Previous studies showed that F. exasperata aqueous leaf extract decreased the blood pressure in a dose-dependent manner (Ayinde et al., 2007; Amonkan et al., 2010; Adewole et al., 2011). According to this disease etiology, the sodium intake via a diet is involved in the onset of the disease. In addition, the control of sodium balance is necessary in hypertensive subjects. Therefore reducing sodium intake and promote renal excretion of sodium are necessary to reduce the blood pressure (Forman et al., 2012; Karppanen and Mervaala, 2006). Thus, the aim of this work was to evaluate the diuretic effect of F. exasperata aqueous leaf extract comparing to those of furosemide in the case of hypertension induced by salt overload.

MATERIALS AND METHODS

Ethics: Experimental procedures and protocols used in this study were approved by Ethical Committee of Health Sciences, University Felix Houphouet-Boigny. These guidelines were in accordance with the internationally accepted principles for laboratory animals’ use and care (NRC, 1996; Mosihuzzaman and Choudhary, 2008).

F. exasperata Aqueous leaf extract: Fresh leaves of F. exasperata Vahl. 1805 (Moraceae) were collected in a forest of the Southern region of Côte d’Ivoire (Region des Lagunes). This plant was authenticated by a Botany expert, Prof. Ake-Assi Laurent of the “Centre National de Floristique”, UFR-Biosciences, Felix Houphouët-Boigny University, Abidjan, Côte d’Ivoire. Ficus exasperata aqueous leaf extract (FEFIX) preparation was previously described (Amonkan et al., 2010 and 2013). Fresh leaves of F. exasperata were washed and dried in an oven at a temperature of 40±2°C. They were pulverized to obtain a fine powder which was left to macerate in n-hexane at a rate of 10 g of powder in 100 mL of n-hexane for 24 h. After filtration, the residue was collected and dried to be subjected to further maceration in distilled water at a rate of 5 g per 100 mL of solvent. The filtrate was then collected and dried using a rotavapor (Buchi, France). A powder of F. exasperata aqueous leaf extract (FEFIX) was obtained with a yield of 14.27±3.26 %. FEFIX was stored at 4°C until experiments.

Animals: Male Wistar rats weighing 200-250 g were used for these experiments. From Pasteur Institute, Abidjan (Côte d'Ivoire), the animals were acclimatized in plexiglass cages for 14 days before experimentation. They were maintained at a temperature of 25±2°C with dark and light cycle (12/12 h). They have free access to standard dry pellet diet and water ad libitum. Animals were treated with deoxycorticosterone acetate (DOCA) for 4 weeks. They received DOCA subcutaneously twice a week (25 mg kg-1 b.wt.). They were normally fed with a drink of NaCl 1% and KCl 0.2% ad libitum (Fournie-Zaluski et al., 2004; Bodineau et al., 2008). After 4 weeks of treatment, the animals become hypertensive and exhibit hemodynamic parameters and kidney similar to those observed in the spontaneously hypertensive rat (Johnson et al., 2004). The day before the experiment, all animals were fasted overnight. At the end of the experiment, the animals were anesthetized with ether and blood rats were sampled from the inferior vena cava.

Evaluation of the diuretic: The day of the experiment, the animals were divided into three groups of six rats and placed individually in metabolic cages. Fluid overload was conducted at 50 mL kg-1 and the animals received immediately following substances according to the group: saline (NaCl 9‰, control) FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1 b.wt.). The urine were collected separately every two hours for 24 h and sampled. They were stored at -20°C prior to determination of the levels of electrolytes, creatinine and urea. Excreted urine volume (EUV) was determined from the ratio of the volume of urine excreted and the volume of fluid overload.

Determination of plasma and urinary electrolytes: Automatic analyzer (Hitachi 902, Roche) was used to determine plasma and urinary electrolytes, creatinine and urea. The determination of sodium and potassium in urine and plasma was performed by the technique of photometry. Levels of calcium, chlorine and creatinine were performed by the technique of colorimetry. The levels of urea were determined by the principle of kinetics.

Chemicals used: The following reference drugs were used: Furosemide (Lasilix®, Sanofi-Aventis, France), Deoxycorticosterone acetate (DOCA, Sigma ). FEFIX and Furosemide were dissolved and diluted in saline solution (NaCl 9‰). DOCA was dissolved in sesame oil on each day of our experiments.

Statistical analysis: The experimental results were expressed as the mean with standard error of mean (m±sem). Data were assessed by the method of analysis of ANOVA followed by Tukey-Kramer test with GraphPad Instat software (Microsoft, San Diego, California, USA). Graphical representations of data were performed by GraphPad Prism 5 software (Microsoft, San Diego, California, USA). The difference between the averages is considered statistically significant when p<0.05.

RESULTS

Volume of urine excreted: Urinary excretion induced by furosemide after 2 h was greater than that induced by F. exasperata aqueous leaf extract (FEFIX). The values obtained were respectively 32.04±6.87 and 20.04±3.84%. From the fourth hour, FEFIX achieved relatively high urinary volumes and higher than those induced by furosemide administration. After 24 hours, FEFIX caused urinary excretion of 153.32±6.89% while that measured with furosemide was 105.71±9.37% (Fig. 1).

Urinary excretion of electrolytes: After 24 h, FEFIX caused a urinary sodium excretion of 15.77±0.62 mEq (Fig. 2). Furosemide induced urinary excretion of sodium, which amounted to 12.78±0.69 mEq. Urinary potassium levels were 1.54±0.12 and 1.19±0.09 mEq, respectively for FEFIX and furosemide. The rate of chlorine and calcium excreted after 24 h under the action of FEFIX were relatively higher than those induced by furosemide. Concerning chlorine, excretions recorded were 12.35±0.62 and 08.27±0.59 mEq, respectively following FEFIX and furosemide treatments. Calcium excretions were 4.84±0.48 mEq (FEFIX) and 4.32±0.42 mEq (furosemide).

Plasma electrolytes: After 24 h, FEFIX and furosemide caused a decrease in plasma electrolytes (Fig. 3). The serum sodium measured were 115.17±6.06 and 127.50±6.59 mEq L-1, respectively for FEFIX and furosemide.

Image for - Comparative Effects of Ficus exasperata Aqueous Leaf Extract and 
  Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat
Fig. 1: Evolution of urinary excretion volume measured for three groups of DOCA salt hypertensive rats: Saline solution (NaCl 9 ‰, Control), FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1 b.wt.). Urine output was measured every two hours for 24 h. FEFIX: F. exasperata aqueous leaf extract, DOCA: deoxycorticosterone acetate, n = 6, M±SEM

Image for - Comparative Effects of Ficus exasperata Aqueous Leaf Extract and 
  Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat
Fig. 2: Rate of electrolyte urinary excretion in rats at 24 hours in three groups of DOCA salt hypertensive rats treated with Saline solution (NaCl 9 ‰, Control), FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1 b.wt.) respectively. Electrolytes were measured in all urine sampled for 24 h after treatment in each group of rats. FEFIX: F. exasperata aqueous leaf extract, DOCA: deoxycorticosterone acetate, n = 6, M±SEM, ***: p<0.001 , **: p<0.01, *p<0.05

The plasma chlorine obtained were 75.00±4.74 mEq L-1 (FEFIX) and 76.32±6.51 mEq L-1 (furosemide).

Table 1: Effects of FEFIX and furosemide on creatinine and urea in urine output and plasma in DOCA salt hypertensive rat
Image for - Comparative Effects of Ficus exasperata Aqueous Leaf Extract and 
  Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat
Saline solution (NaCl 9 ‰, Control), FEFIX (F. exasperata aqueous leaf extract, 100 mg kg-1 b.wt.): furosemide (10 mg kg-1 b.wt.). DOCA: deoxycorticosterone acetate, u: urine, p: plasma, n = 6, m±sem; ***p<0.001, **p<0.01, *p<0.05

Image for - Comparative Effects of Ficus exasperata Aqueous Leaf Extract and 
  Furosemide on Urinary Excretion in DOCA-salt Hypertensive Rat
Fig. 3: Plasma levels of electrolytes at 24 h after administration of Saline solution (NaCl 9‰, Control), FEFIX (100 mg kg-1 b.wt.) and furosemide (10 mg kg-1 b.wt.) in three groups of DOCA salt hypertensive rats respectively. The rate of electrolyte was measured on blood samples. FEFIX: F. exasperata aqueous leaf extract, DOCA: deoxycorticosterone acetate, n = 6, M±SEM, ***: p<0.001 , **: p<0.01, *p<0.05.

The serum calcium obtained were 16.50±3.64 mEq L-1 (FEFIX) and 17.42±3.14 mEq L-1 (furosemide). However, changes in serum potassium induced by furosemide and FEFIX showed no significant difference to that obtained with the control (p>0.05).

Urea and creatinine in urine and plasma: FEFIX and furosemide induced significant urinary excretion of creatinine and urea (Table 1). The creatinine level obtained under FEFIX treatment after 24 h was 0.40±0.05 mmol. That induced by furosemide was 0.36±0.03 mmol. The urea measured in urine were 290.21±17.48 mmol for FEFIX and 249.42±18.15 mmol for furosemide. In the plasma, FEFIX and furosemide also caused significant changes in creatinine and urea. The creatinine levels measured after 24 h were 0.41±0.05 and 0.44±0.08 mM, respectively for FEFIX and furosemide. Uremia obtained in presence of FEFIX (2.08±0.33 mM) was less than that obtained when furosemide (2.54±0.25 mM) was administrated to rats.

DISCUSSION

Ficus exasperata aqueous leaf extract (FEFIX) and furosemide induced urinary excretion volume (EUV) relatively large. The two substances had similar urinary excretion kinetics. However, the urinary excretion obtained with FEFIX was greater than that obtained with furosemide. The increase in urinary excretion induced by FEFIX could result from stimulation of renal excretory function. Similar results were reported in previous works concerning several plants used in Thai Pharmacopoeia (Sripanidkulchai et al., 2001). Urtica dioica aqueous leaf extract increased urinary excretion according to the dose. For infusions of 4-24 mg/kg/h, this extract increased urine volume by 11 to 84% (Tahri et al., 2000). In addition, crude ethanolic extract of leaves Melothria maderaspatana protects the kidney in salt hypertensive rats. This protection is manifested by the reduction in histological damage associated with hypertension (Veeramani et al., 2012).

Urinary excretion induced by FEFIX and furosemide were associated with significant loss of sodium, chloride and calcium. Electrolytes Urinary excretion caused by FEFIX were relatively higher than those obtained under furosemide treatment. As furosemide, F. exasperata aqueous leaf extract inhibited renal reabsorption of electrolytes. Indeed, Tribulus terrestris aqueous extract increased the excreted urinary volume. Diuresis induced by this extract was relatively higher than that induced by furosemide (Al-Ali et al., 2003). In addition, the infusion of Salvia scutellarioides caused high urinary excretion of electrolytes which increased dose-dependent manner (Ramirez et al., 2006).

Diuretic effects of FEFIX and furosemide altered the plasma electrolytes. Both substances decreased serum sodium, chloride and serum calcium without affecting significantly the plasma potassium. The decrease in plasma electrolytes could result from their significant urinary excretion. Indeed, previous studies had shown that administration of saponins from Herniara glabra decreased arterial blood pressure by reducing the reabsorption of sodium and water in the renal tubules. Saponins of H. glabra, thereby increasing urine flow and excretion of sodium and potassium (Rhiouani et al., 1999). Furosemide increased urine volume and urinary excretion of sodium. This increase of diuresis and natriuresis resulted from the inhibition of electrolytes cotransporter along the ascending limb of the nephron (Carmosino et al., 2001; Haque et al., 2011). In addition, the ethanolic extract of Tropaeolum majus and his purified fraction containing isoquercitrin increased diuresis with potassium-sparing. This effect observed in spontaneously hypertensive rats results from inhibition of angiotensin converting enzyme and activity of Na+/K+-ATPase (Gasparotto et al., 2012). In addition, the Flavangenol extracted from pine bark marine attenuated significantly renal lesions in salt hypertensive rats. This protective effect could be attributed to its antioxidant property which protects against endothelial dysfunction (Kwak et al., 2009; Ohkita et al., 2011).

CONCLUSION

In salt hypertension, F. exasperata aqueous leaf extract induced a diuretic effect relatively large and greater than that induced by furosemide. This diuresis was associated with a significant loss of electrolytes in the urine. These urine outputs of electrolytes, creatinine and urea decreased their levels in plasma.

ACKNOWLEDGMENTS

We are grateful to Pr YAO Datte Jacques and Pr OFFOUMOU Atte Michel for their critical suggestions and encouragement.

REFERENCES

  1. Abrogoua, D.P., D.S. Dano, P. Manda, A.J. Adepo, B.J. Kablan, N.B. Goze and K. Ehoule, 2012. Effect on blood pressure of a dietary supplement containing traditional medicinal plants of Cote d'Ivoire. J. Ethnopharmacol., 141: 840-847.
    CrossRef  |  PubMed  |  


  2. Adewole, S.O., T. Adenowo, T. Naicker and J.A. Ojewole, 2011. Hypoglycaemic and hypotensive effects of Ficus exasperata vahl. (Moraceae) leaf aqueous extract in rats. Afr. J. Tradit. Complement. Altern. Med., 8: 275-283.
    PubMed  |  


  3. Al-Ali, M., S. Wahbi, H. Twaij and A. Al-Badr, 2003. Tribulus terrestris: Preliminary study of its diuretic and contractile effects and comparison with Zea mays. J. Ethnopharmacol., 85: 257-260.
    Direct Link  |  


  4. Amonkan, K.A., A.B. Konan, K.L. Kouakou, K.G.M. Bouafou and N.M. Bleyere et al., 2010. Phytochemical screening and effects of aqueous extract of Ficus exasperata Vahl. 1805 leaves (Moraceae) on blood pressure and contractile activity of the heart in mammals. Int. J. Biol. Chem. Sci., 4: 681-691 [In French]..


  5. Amonkan, K.A., B.A. Konan, B.M.L. Ahui, M.N. Bleyere, L.K.Kouakou and G.M.K. Bouafou, 2013. Diuretic effects of aqueous extract of Ficus exasperata Vahl. leaves in rat. Pak. J. Biol. Sci., Vol. 16. (In Press).


  6. Anwar, F., S. Latif, M. Ashraf and A.H. Gilani, 2007. Moringa oleifera: A food plant with multiple medicinal uses. Phytother. Res., 21: 17-25.
    CrossRef  |  PubMed  |  Direct Link  |  


  7. Armario, P. and B. Waeber, 2013. Therapeutic strategies to improve control of hypertension. J. Hypertens., 31: S9-S12.
    CrossRef  |  PubMed  |  


  8. Ayinde, B.A., E.K. Omogbai and F.C. Amaechina, 2007. Pharmacognosy and hypotensive evaluation of Ficus exasperata Vahl (Moraceae) leaf. Acta Pol. Pharm., 64: 543-546.
    PubMed  |  


  9. Bodineau, L., A. Frugiere, Y. Marc, N. Inguimbert, C. Fassot, F. Balavoine, B. Roques and C. Llorens-Cortes, 2008. Orally active aminopeptidase A inhibitors reduce blood pressure: A new strategy for treating hypertension. Hypertension, 51: 1318-1325.
    CrossRef  |  PubMed  |  


  10. Carmosino, M., F. Rizzo, P. Ferrari, L. Torielli, M. Ferrandi, G. Bianchi, M. Svelto, G. Valenti, 2011. NKCC2 is activated in Milan hypertensive rats contributing to the maintenance of salt-sensitive hypertension. Pflugers Arch., 462: 281-291.
    CrossRef  |  PubMed  |  


  11. Erdine, S., O. Ari, A. Zanchetti, R. Cifkova and R. Fagard et al., 2006. ESH-ESC guidelines for the management of hypertension. Herz, 31: 331-338.
    PubMed  |  


  12. Forman, J.P., L. Scheven, P.E. de Jong, S.J. Bakker, G.C. Curhan and R.T. Gansevoort, 2012. Association between sodium intake and change in uric acid, urine albumin excretion and the risk of developing hypertension. Circulation, 125: 3108-3116.
    CrossRef  |  PubMed  |  


  13. Fournie-Zaluski, M.C., C. Fassot, B. Valentin, D. Djordjijevic and A. Reaux-Le Goazigo et al., 2004. Brain renin-angiotensin system blockade by systemically active aminopeptidase A inhibitors: A potential treatment of salt-dependent hypertension. Proc. Natl. Acad. Sci., 101: 7775-7780.
    CrossRef  |  PubMed  |  


  14. Gasparotto Jr., A., T.B.L. Prando, T.S.V. Leme, F.M. Gasparotto and E.L.B. Lourenco et al., 2012. Mechanisms underlying the diuretic effects of Tropaeolum majus L. extracts and its main component isoquercitrin. J. Ethnopharmacol., 141: 501-509.
    CrossRef  |  


  15. Haque, M.Z., G.R. Ares, P.S. Caceres and P.A. Ortiz, 2011. High salt differentially regulates surface NKCC2 expression in thick ascending limbs of Dahl salt-sensitive and salt-resistant rats. Am. J. Physiol. Renal Physiol., 300: F1096-F1104.
    CrossRef  |  PubMed  |  


  16. Johnson, F.K., W. Durante, K.J. Peyton and R.A. Johnson, 2004. Heme oxygenase-mediated endothelial dysfunction in DOCA-salt, but not in spontaneously hypertensive, rat arterioles. Am. J. Physiol. Heart Circ Physiol., 286: H1681-H1687.
    CrossRef  |  PubMed  |  


  17. Karppanen, H. and E. Mervaala, 2006. Sodium intake and hypertension. Prog. Cardiovas. Dis., 49: 59-75.
    PubMed  |  


  18. Kearney, P.M., M. Whelton, K. Reynolds, P. Munter, P.K. Whelton and J. He, 2005. Global burden of hypertension: Analysis of worldwide data. Lancet, 365: 217-223.
    CrossRef  |  PubMed  |  Direct Link  |  


  19. Kearney, P.M., M. Whelton, K. Reynolds, P.K. Whelton and J. He, 2004. Worldwide prevalence of hypertension: A systematic review. J. Hypertens., 22: 11-19.
    PubMed  |  


  20. Kwak, C.J., E. Kubo, K. Fujii, Y. Nishimura and S. Kobuchi et al., 2009. Antihypertensive effect of French maritime pine bark extract (Flavangenol): Possible involvement of endothelial nitric oxide-dependent vasorelaxation. J. Hypertens., 27: 92-101.
    CrossRef  |  


  21. Mosihuzzaman, M. and M.I. Choudhary, 2008. Protocols on safety, efficacy, standardization, and documentation of herbal medicine. Pure Appl. Chem., 80: 2195-2230.
    Direct Link  |  


  22. NRC, 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC., USA., ISBN-10: 0309053773, Pages: 140
    Direct Link  |  


  23. Ohkita, M., Y. Kiso and Y. Matsumura, 2011. Pharmacology in health foods: Improvement of vascular endothelial function by French maritime pine bark extract (Flavangenol). J. Pharmacol. Sci., 115: 461-465.
    CrossRef  |  


  24. Ramirez, J.H., M. Palacios and O. Gutierrez, 2006. Diuretic effect of an infusion of the herbal plant, Salvia scutellarioides, in rats. Biomedica, 26: 145-149.
    PubMed  |  


  25. Rhiouani, H., A. Settaf, B. Lyoussi, Y. Cherrah, M.A. Lacaille-Dubois and M. Hassar, 1999. Effects of saponins from Herniaria glabra on blood pressure and renal function in spontaneously hypertensive rats. Therapie, 54: 735-739.
    PubMed  |  Direct Link  |  


  26. Sripanidkulchai, B., V. Wonqpanich, P. Laupattarakasem, J. Suwansakri and D. Jirakulsomchok, 2001. Diuretic effects of selected Thai indigenous medicinal plants in rats. J. Ethnopharmacol., 75: 185-190.
    CrossRef  |  PubMed  |  Direct Link  |  


  27. Tahri, A., S. Yamani, A. Legssyer, M. Aziz, H. Mekhfi, M. Bnouham and A. Ziyyat, 2000. Acute diuretic, natriuretic and hypotensive effects of a continuous perfusion of aqueous extract of Urtica dioica in the rat. J. Ethnopharmacol., 73: 95-100.
    CrossRef  |  PubMed  |  Direct Link  |  


  28. Veeramani, C., K.S. Al-Numair, G. Chandramohan, M.A. Alsaif and K.V. Pugalendi, 2012. Antihyperlipidemic effect of Melothria maderaspatana leaf extracts on DOCA-Salt induced hypertensive rats. Asian Pacific J. Trop. Med., 5: 434-439.
    CrossRef  |  PubMed  |  Direct Link  |  


  29. Whitworth, J.A., WHO and ISHWG, 2003. World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. J. Hypertens., 21: 1983-1992.
    PubMed  |  


©  2022 Science Alert. All Rights Reserved