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ORIGINAL ARTICLE DIURETIC EFFECT FROM THE LEAVES OF MAYTENUS MACRO-
CARPA "CHUCHUHUASI" IN ALBINE RATS
Bastidas Garcia Fanny 1,2,a Huaccho Rojas Juan Jesús 1,2,a Chambi Torres Javier 1,2,a Padil a Alexander Antonio 1,2,a Aguirre Tipismana Luis 1,2,a Salazar Granara Alberto 1,b Castañeda Castañeda Benjamín 1,b 1 Faculty of Medical Sciences, University of San Martin de Porres, Lima. Peru 2 Scientific Society of Medical Students, University of San Martin de Porres. a Medical Student Objective: To evaluate the diuretic activity of the aqueous extract obtained from the leaves of Maytenus macrocarpa "Chuchuhuasi" in albino rats. Material and Methods: 68 male albino rats were used, with average weight of 250 g. The Lipschitz method was used, recording the urine volume per hour and the total volume at the sixth hour. The experimental groups were: control (saline 0.9%), chuchuhuasi 1 (250mg / kg), chuchuhuasi 2 (500 mg / kg), chuchuhuasi 3 (750mg / kg), chuchuhuasi leaves 4 (1000 mg / kg), furosemide 1 (10mg / kg) and furosemide 2 (20mg / kg). For statistical validation Shapiro-Wilk, Turkey and Dunns tests were Results: The largest total volume obtained during the first 24 hours was from the chuchuhuasi 1000 mg/ kg group with 5,17 ml. This was an even greater result than the one obtained with furosemide 20 mg/kg. On the other hand, chuchuhuasi 250 mg/kg group (2.32 ml) achieved less diuretic action than the control group (2,55 ml). Tukey's test indicated no significant difference. Nevertheless, the Xi squared test's results from the biochemical analysis got a p< 0.05 for pH. Conclusion: A positive diuretic response it's demonstrated in all dose levels tested using ethanolic extract of Maytenus macrocarpa: 250mg/kg, 500mg/kg, 750mg/kg and a greater effect of 1000 mg/kg compared with the control group and furosemide. Keywords: Diuresis, Furosemide, Traditional Medicine, Ethnopharmacology. gical properties of the plant.
Even though Maytenus macrocarpa is used as a diuretic in po- According to the World Health Organization (WHO) and pular medicine, no studies have been conducted to demons- the Food and Agriculture Organization of the United Na- trate this ethnopharmacological statement. This is the reason tions (FAO), two thirds of the worldwide population 4 billion why the objective of this work was to evaluate the diuretic ac- people use plants for medical intent (1,2).
tivity of the aqueous extract obtained from the leaves of Ma- Chuchuhuasi, scientifical y called Maytenus macrocarpa ac- ytenus macrocarpa "Chuchuhuasi" in albino rats.
cording to Briquet, it is a large tree that grows in Peru, especia- l y in the Amazon jungle, that has particular botanical charac- MATERIALS AND METHODS:
teristics such as: whorled roots, entire and coriaceous leaves, oblong seeds with white aril, etc. (3). Study Design: A quasi-experimental preclinical, longitudinal
Plants related to the Maytenus (Celastraceae) genus, are used and prospective double-blind study was conducted during in popular medicine for their therapeutic properties; some of the months of March to October 2014.
their effects are well known, but there are many others that re- Samples: main unknown and may cause adverse reactions, intoxication - Plant Sample: Maytenus macrocarpa leaves collected
and even death. Chuchuhuasi use is known for the treatment in Pucal pa (Ucayali, Peru) were used. The taxonomic certifi- of a variety of diseases including rheumatism, arthritis, lower cation was done under the Cerrate, E.1969 criteria. The etha- back pain, colds, bronchitis, diarrheas, hemorrhoids, infertili- nolic extract was extracted from the dried and then ground ty and others, being the relieving of gastric disorders its most leaves of Maytenus macrocarpa. It was macerated in 70% common use (4,5). Studies were made in order to find the ethanol for a week, then the mixture was filtered and the re- photochemical components of the plants; two eterpenoids, sidue obtained was dried in an oven for 48 hours. The sample flavonoid glycosides (6), saponins, steroids, phenolic deriva- obtained was ground in a mortar to a fine powder that was tives, vitamins, starch, maytenine, Proanthocyanidin dimers then stored in airtight containers and refrigerated until use.
and friedelane triterpene were found (7); in addition, the anti- - Animal Sample: 68 male albino rats Rattus norvegicus,
oxidant and analgesic activity of this plant was demonstrated whose weights ranged between 200 and 300 grams, from the (8-10). The previous data confirms the multiple pharmacolo- animal facility of the National Institute of Health were used. CIMEL 2016, Volume 21, Number 1 The animals were used by the ethical codes of International was completed for each possible paired combination.
Guiding Principles for Biomedical Research Involving Ani- Ethical aspects: This study was approved by the Ethics Com-
mit ee of the Universidad de San Martin de Porres after being Chemical Sample: Furosemide in ampoules, batch evaluated by the Centro de Investigación de Medicina Tradicio-
071011, sanitary registration (RS) GBE 10008-03-07 (expira- nal y Farmacológica de la Universidad de San Martin de Porres, tion date 08/14).
whose members constantly monitored the research process.
Diuresis measurement: The animals were kept for 3 days in
adaptation to the experimental conditions with a temperature RESULTS
of 27 ± 1 ° C, relative humidity of 60 ± 5% and 12-hour light/ dark cycles.
They were housed in aluminum cages with metal grid at 3 The largest total volume obtained during the first 24 hours animals per cage. They were deprived of food 24 hours before was 5,17ml for Chuchuhuasi of 1000 mg / kg, getting to have starting the experiment and drinking water an hour before. even greater value than furosemide 20 mg / kg. Furthermo- The rats were placed in metabolic cages and it was proceeded re, Chuchuhuasi of 250 mg / kg (2.32) group resulted obtai- to record the volume of urine excreted at 1, 2, 3, 4, 5, 6 and 24 ning lower diuretic action than the control (2.55 ml) group, as hours post administration using 2 micropipettes of 5 and 10 shown in Table No. 1 ml, depending on the volume of urine to be measured.
The Shapiro-Wilk test showed a non-Gaussian distribution, Biochemical determinations: Biochemical analysis was per- where quantitative analysis did not show statistical differences formed after a greater volume than 0.5 ml (minimum volume by Dunns test, This result was verified by performing the Tuc- required for the biochemical analysis) was obtained from each key test assuming a Gaussian distribution. However,the graph rat. Urine reagent test strips were used for this purpose, testing No. 1 shows some differences between the progress of urine for the presence and levels of bilirubin, urobilinogen, ketones, volume in 24 hours.
glucose, proteins, hemoglobin, nitrites, pH and leucocytes in the urine samples.
Table 1. Comparison of average volumes of urine at 24
Group 2: Furosemide 10 mg/kg (11) Group 3: Furosemide 20mg/kg (12) Group 4: Chuchuhuasi (Maytenus macrocarpa) 250 mg/kg Group 5: Chuchuhuasi (Maytenus macrocarpa) 500 mg/kg Group 6: Chuchuhuasi (Maytenus macrocarpa) 750 mg/kg Group 7: Chuchuhuasi (Maytenus macrocarpa) 1000 mg/kg A database in Microsoft Excel was created, including the urine volumes measured and the results of the biochemical analysis.
Statistical analysis was performed using the Statistical Packa- ges GraphPrism 5.0 and the Statistical Packages for the Social Sciences (SPSS) for Windows (version 12.0) Quantitative variables were expressed as mean and standard deviation, in order to observe the volume variation according to the hour the samples were obtained. Additional y, only urine volumes obtained 24 hours after administration were taken in account for the analysis of the variance and correla- * The Shapiro Wilk test showed no Gaussian distribution tion, using Shapiro-Wilk's test (to know if the results were in ** The Tukey test showed no statistical difference in any relation.
Gaussian distribution) to compare the means using the ap- propriate test later.
For the results of the biochemical determinations a qualitative The chi-square test using table in biochemical results gave analysis via contingency tables was performed. Chi square test us a p <0.05 value for the pH between the control group and CIMEL 2016, Volume 21, Number 1 ORIGINAL ARTICLE the Chuchuhuasi group of 750 mg / kg (p = 0.036), and the bition in the epithelial cel s, inflammatory cel s or cholinergic group of furosemide 20 mg / kg and Chuchuhuasi 750 mg / neural pathways (9). Furosemide is subtype specific noncom- kg (p = 0.027). Also obtained by the same test, p <0.05 value petitive blocker for the GABA-A receptor that gets through a for leukocytes from the groups: furosemide 10 mg / kg and process of glucuronization and is a reversible antagonist of the Chuchuhuasi 250 mg / kg (p = 0.036), furosemide 10 mg / kg evoked streams of GABA- a62. (16) and Chuchuhuasi 500 mg / kg (p = 0.030), furosemide 10 mg It had been recognized that the main active metabolites pre- / kg and Chuchuhuasi 1000 mg / kg (p = 0.009).
sent in medicinal plants that may have diuretic effect are: es- sential oil, saponoside, potassium salts, and flavonoids, the lat- ters were found in Maytenus macrocarpa in previous studies. Their main site of action is the glomerulus, rather than the In the present study a dose-response diuretic effect of Furose- tubuli. This causes an increase of the renal circulation and an mide between 10 and 20mg/kg was evidenced, as was a supe- increased glomerular filtration rate and urine primary forma- rior diuretic action compared to the one obtained in the con- tion. It is also considered that cardiotonic heterosides could be trol group, which only received saline solution (ClNa0.9%). involved in the diuretic activity. (1-9) These results confer validity to the methodology used as wel About the biochemical analysis, the significant difference in as our results, because it shows normal physiological condi- pH indicates an acidification effect on urine, this is explained tions in the animals studied.
by the Furosemide inhibitory effect on the Na-K-2Cl sym- Among the results of Maytenus macrocarpa, the 1000 mg/ porter in the thick ascending limb of the loop of Henle, and kg dose resulted in greater diuresis at 24 hours than the one it would also improve the H+ and K+ secretion in the cortical obtained with the 250mg/kg dose, 500mg/kg dose and even collecting ducts by producing high luminal electronegativity with the 750mg/kg dose, which gives an outline of a possible (17,18).
range of peak response. We must emphasize that the effect of The means obtained from groups administrated with Chu- this dose also exceeded the effect given by Furosemide 10 mg/ chuwasi with doses of 250, 500, 750 and 1000 mg/kg also kg and Furosemide 20 mg/kg in 24 hours.
indicate an effect of urinary acidification. The mechanism of Furosemide has a half-life of 30 minutes, an effect duration this effect may be related to the inhibitory property of Mayte- of 8 hours depending on the dose and a 50% bioavailability nus macrocarpa on prostaglandins (19), given it has been des- (5). A similar effect had Xanthiumstru marium L. at 400mg/ cribed to be many receptors for these located in kidneys (20). kg dose in relation to Furosemide at 5mg/kg dose, which re- However, until now there are no direct studies that show the ached a superior diuretic effect of the drug since the 1st hour effect of Maytenus macrocarpa on urinary pH.
of application (13). Unlike C. alata and P.americana, which We suggest that this scientific evidence can be taken into ac- managed to equal the Furosemide's diuretic power at 5 hours count and applied to medical praxis, this is explained by the of being applied (14). Another study concludes that Maytenus fact that a large amount of population consumes phytodrugs krukovii at a 1000 mg/kg dose also has a hypotensive activity, believing they are free of adverse effects or drug interaction, giving evidence of a possible diuretic effect of this plant, which thus exposing themselves to renal insufficiency and hypoten- belongs to the same genre of the one studied in this work. (15) sion due to sodium depletion (7) and the consequent hypo- We can notice that the 1000mg/kg dose of Maytenus macro- volemia, caused by ACE inhibitor interaction, digitalis intoxi- carpa has a long term diuretic activity, similar to Furosemide cation by digoxin interaction, ototoxicity and nephrotoxicity which could indicate that the mechanism of action also has a by the use of amphotericin B and other otologic drugs which would have a potentiated effect, amidst others (21, 22).
The Furosemide's main mechanism of action is blocking the From our results we suggest to continue the preclinic research Na+/K+/2Cl- symporter pump in the thick ascending limb of this plant. More studies aimed to determine the mecha- of the loop of Henle, which alters the flow of ions and water nisms of action, the interactions of this plant with commonly across the cell membranes, this process increases the sodium, used drugs and the isolation of active principles of Maytenus potassium and chloride excretion and concomitantly genera- macrocarpa(13,14) are required.
tes water excretion, which explains the diuretic effect.
At the end of the investigation we could conclude that the diu- Additional y, there is a change in the electrical potential of retic effect from the ethanolic extract of Maytenus macrocar- the cell which disturbs the dynamics of the Ca 2+ and Mg 2+ pa is evidenced in this research with positive diuretic outcome transportation (8), it is also thought that it produces the Na+/ in all levels of doses applied: 250, 500, 750 and 1000mg/kg in K+/2Cl- symporter inhibition or the carbonic anhydrase inhi- comparison to the control and furosemide groups.
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