Author's Accepted Manuscript

Evaluation of the diuretic activity in two

Mexican medicinal species: Selaginella notho-
hybrida and S. lepidophylla and its effects with
ciclooxigenases inhibitors
María I. Aguilar, Wendy V. Benítez, Arturo
Colín, Robert Bye, Ramiro Ríos-Gómez,
Fernando Calzada

www.elsevier.com/locate/jep

PII: S0378-8741(15)00046-X
DOI: http://dx.doi.org/10.1016/j.jep.2015.01.031
Reference: JEP9278

To appear in: Journal of Ethnopharmacology

Received date: 6 November 2014

Revised date: 18 January 2015
Accepted date: 21 January 20152 inhib

Cite this article as: María I. Aguilar, Wendy V. Benítez, Arturo Colín, Robert
Bye, Ramiro Ríos-Gómez, Fernando Calzada, Evaluation of the diuretic activity
in two Mexican medicinal species: Selaginella nothohybrida and S. lepidophylla
and its effects with ciclooxigenases inhibitors, Journal of Ethnopharmacology,
http://dx.doi.org/10.1016/j.jep.2015.01.031

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Evaluation of the diuretic activity in two Mexican medicinal species: Selaginella

nothohybrida and S. lepidophylla and its effects with ciclooxigenases inhibitors

María I. Aguilara*, Wendy V. Beníteza, Arturo Colína, Robert Byeb Ramiro Ríos-

Gómezc, and Fernando Calzadad

a
Facultad de Química; bJardín Botánico, Instituto de Biología; cUnidad de Investigación

en Sistemática Vegetal y Suelo, FES Zaragoza. Universidad Nacional Autónoma de

México, México D. F., 04510, México

d
Unidad de Investigación Médica en Farmacología UMAE, Hospital de Especialidades-

CORCE, 2° piso, Centro Médico Nacional Siglo XXI, IMSS, Av. Cuauhtémoc 330,

Col. Doctores, México, D.F., 06725, México

Corresponding author

*To whom correspondence should be addressed. Tel.: 525 55622 5281; Fax: +52 55

5622 5329. E-mail: ilaurents@hotmail.com

1
ABSTRACT

Ethnopharmacological relevance: Doradilla is a plant that has a long history in the

Mexican traditional system of medicine for gall and renal stones, diuresis, stomach and

liver inflammation among other diseases.

Major components isolated from these plants include biflavonoids as amentoflavone (1),

robustaflavone (2) and (S)-2,3-dihydrorobustaflavone (3) and the carbohydrate trehalose

(4). The aim of this study was to evaluate the diuretic effect of the decoction of S.

nothohybrida Valdespino and S. lepidophylla (Hook & Grev) Spring (Selaginellaceae),

and compounds 1 – 4. We also explored the probable mode of action comparing the

effects when using nonspecific and specific COX’s inhibitors.

Materials and methods: Three biflavonoids (1 – 3) were isolated from the ethyl acetate

extraction of the aqueous decoction and the carbohydrate trehalose (4) from the aqueous

phase. The structures of all compounds were elucidated by spectroscopic methods and

comparisons were made against published data. The diuretic activity was assessed in

mice by oral administration of the decoctions in doses of 1000 and 2000 mg/kg and

biflavonoids 1 – 3 and trehalose (4) in a dose range of 10 mg/kg using furosemide as a

standard drug. Inhibitors of COX’s such as acetyl salicylic acid, sodium naproxen,

indomethacin and Celebrex were also assayed to analyze the involvement of renal

prostaglandins in diuresis. Water excretion rate, pH, density, conductivity, and contents

of Na+ and K+ were measured in the urine of mice.

Results: Decoction of S. lepidophylla showed lower effect in the urine output at doses of

1000 and 2000 mg/kg, while decoction of S. nothohybrida produced an increase at 2000

mg/kg (P<0.05). Urinary electrolytes excretion was also affected by this last extract and

pure compounds: decoction diminished urinary excretion of sodium and potassium ions,

so as compounds 1 and 4; compounds 2 and 3 observed just a natriuretic effect.

2
Pretreated mice with COX’s inhibitors and then with test compounds 1, 2, 4 and

decoction showed inhibition of diuresis in all cases exception for treatment with

trehalose (4); natriuretic effect was observed in all cases except for biflavonoid

robustaflavone (2) which behaved as the reference compound furosemide. S.

nothohybrida decoction behaved similarly to COX-2 inhibitor Celebrex (8), inhibiting

diuresis.

Conclusions: S. nothohybrida presents a moderate diuretic effect, which appears to be in

partly mediated by the presence of biflavonoids and trehalose. Renal prostaglandins

may be involved in the mechanism of diuresis. The present results provide a

quantitative basis explaining the traditional folk medicine use of S. nothohybrida as a

diuretic agent by Mexican population.

Keywords: Diuretic; Selaginella lepidophylla; Selaginella nothohybrida; biflavonoids,

prostaglandins.

Chemical compounds studied in this article:

Robustaflavone (PubChem CID: 5281694)

Amentoflavone (PubChem CID: 5281600)

2,3-Dihydrorobustaflavone (PubChem CID: 76315513)

Trehalose (PubChem CID: 7427)

3
1. Introduction

Medicinal plants are very commonly used to treat renal diseases; two examples of

species traditionally reported to possess significant diuretic activity are Equisetum

giganteum (Equicetaceae) and Zea mays (Poaceae) (Farmacopea Herbolaria, 2013;

Biblioteca Digital de la Medicina Tradicional Mexicana, 2009). Many plants as diuretic

agents used in ethnomedicine have been studied and activity confirmed in experimental

animals (Lahlou et al., 2007; Nedi et al., 2004; Abdala et al., 2008). In this context,

“doradilla” is the popular name given to a group of plants used in Mexican traditional

medicine which belong to different Selaginella species, with similar macroscopic

botanical characteristics and they are expended without distinction (Aguilar et al.,

2013). Two species are commonly used as diuretics: Selaginella nothohybrida

Valdespino and S. lepidophylla Hook et Grev., which often represent commercially and

medicinally adulterated samples. However, both have different microscopic

characteristics (Miquel and Valdespino, 1992). These species have the ability to resist

drought periods due to poikilohydric properties (Miquel and Valdespino, 1992) and

their high trehalose (4) content provides a protection mechanism against drought

(Richards et al., 2001). Under dry conditions, their branches curl inward to appear like

golden balls of about 10 cm in diameter; in humid surroundings, their branches extend

and turn green again (Korall et al., 1999; Korall and Kenrick, 2002), so “doradillas” are

characterized by their rosette-like appearance. Both, Selaginella nothohybrida and S.

lepidophylla grow in the Mexican states of Guerrero, Oaxaca and Veracruz. Other

common names that these plants have are “resurrection ferns” or “resurrection plants”,

“much-kok”, “texochitl yamanqui” and “flor de piedra” (Argueta et al., 1994; Martínez,

1989). Traditional treatments with “doradilla” include urinary obstruction, cystitis, renal

4
calculus, kidneys inflammation, and for waist and back pains. Most of these

preparations are based in the decoction of the whole plant. Other uses include treatment

of digestive problems, as eupeptic as well as for cough, bronchitis and parasitic

infections (Argueta, et al., 1994; Márquez, et al., 1999; Martínez, 1989). A previous

ethnomedical study performed by Vázquez-Ramírez et al. (2005) highlights the

importance of this plant as diuretic in Mexican traditional medicine. Several reported

chemical studies have identified biflavonoids such as amentoflavone (1), robustaflavone

(2), (S)-2,3-dihydrorobustaflavone (3) (Figure 1), (S)-2,3-dihydro-5-methoxy-

robustaflavone, isocriptomerin, heveaflavone, hinoquiflavone trimetil eter and 5',5''-

dihydroxy-7,7'',4',4'''-tetramethoxy-amentoflavone as main components of these

medicinal plants (Quasim, et al., 1985; Aguilar, et al., 2008).

Up to date there are not pharmacological reports of the diuretic effect of doradilla, so,

the purpose of this work is to evaluate the diuretic activities of S. nothohybrida and S.

lepidophylla to justify their common traditional use and to test a probable mechanism of

diuretic action.

2. Materials and methods

2.1. Plants materials and preparation of the extracts

S. nothohybrida was purchased in the Mercado de Sonora, México in May 2005; S.

lepidophylla, was collected by R. Ríos-Gómez in Cerro Pájaro, Oaxaca in November

2009. Both plants were identified by M. Sc. A. Reyes-García and voucher specimens

were deposited at the National Herbarium of this University under numbers SN-0805

and SL-0909-03 respectively.

5
2.2. Extracts preparation

Five hundred grams of each complete vegetal species were decocted with water (ten

min). The decoction was filtered, frozen at -20 °C and lyophilized to give 22 g of dry

extract of S. nothohybrida and 19.8 g of S. lepidophylla. Then, 5 g of each extract were

independently suspended in water and partitioned with ethyl acetate, obtaining the

respective ethyl acetate extracts and the residual aqueous phases. The two lyophilized

products were dissolved in water before administration at the calculated doses.

2.3. Isolation of biflavonoids and trehalose

The total extract of S. nothohybrida was obtained by maceration of 350 g in CHCl3-

CH3OH (1:1). Partitions with n-hexane, CHCl3 and ethyl acetate gave the corresponding

fractions. The ethyl acetate fraction with the biflavonoids contents, was applied in a

Sephadex LH-20 (Sigma) column chromatography, eluting with methanol, and

amentoflavone (1), robustaflavone (2) and (S)-2,3-dihydrorobustaflavone (3) were

isolated. Their identities were confirmed by spectroscopic methods (Aguilar et al.,

2008; Aguilar et al., 2013). Trehalose (4) was directly obtained by crystallization from

the decoction.

6
2.4. Animals

Male albino mice (25-30 g) obtained from bioterio Centro Médico Nacional Siglo XXI

were used in all experiments. Procedures involving animals and their care were

conducted according to the Mexican Official Norm for Animal Care and Handling

(NOM-062-ZOO-1999) in accordance with the international accepted principles for

laboratory animal use and care.

2.5. Drugs

Furosemide (Moléculas Finas de México, S.A. de C.V.) was used as the reference

diuretic agent. It was dissolved in water prior to administration. The following

compounds were used as inhibitors of COX-1 and COX-2: acetylsalicylic acid (5) (Pisa

Farmacéutica, México), sodium naproxen (6) (Moléculas Finas de México, S.A. de

C.V., México), indomethacin (7) (Kener, S. A. de C. V., México) and Celebrex (8)

(Pfizer, S. A. de C. V., México)

2.6. Diuretic test

The methods of Mazid et al. (2009) were used, with modifications, for the

determination of the diuretic effect. In all cases, samples were administered through a

gastric catheter. Animals were divided into eight groups (n=6); water (1.0 mL) was

orally administered to each one of the animals in the control group (I). Seven groups

were independently treated with furosemide (II, 10 mg/kg), with S. nothohybrida (III)

and S. lepidophylla (IV) decoctions (1 and 2 g/kg) and groups V, VI, VII and VIII with

7
compounds 1 - 4 (10 mg/kg). In a parallel experiment, with the exception of group VII

with compound 3 which did not observed diuretic activity in the previous experiment,

groups I, II, III, V, VI and VIII (n = 6), were independently administered with a) acetyl

salicylic acid (5), b) sodium naproxen (6), c) indomethacin (7), and d) Celebrex (8), in

concentrations of 10 mg/kg, 30 min after the administration of test compounds or

extract, for the assessment of the role of these last ones on acute diuretic activity. Urine

samples were taken at two and four hours after administration in all cases. The animals

had free access to standard commercial diet and water ad libitum in a 12-h light/12-h

dark cycle at 22 °C. The mice were observed for symptoms of toxicity for 2 months in

terms of weight loss, and autonomic and neurobehavioral alterations. On the 60th day,

the animals were sacrificed and their vital organs were individually observed for overt

pathology.

2.7. Diuretic activity

Diuretic activity of all compounds was compared with control group, so as with the

groups which were administered with representative compounds (5, 6, 7 and 8) as

inhibitors of COX-1 and COX-2 enzymes. The percentage of urine excreted during 4 h

for each animal in the group gives a measure of urinary excretion independent of the

animal weigh.

2.8. Analytical procedures

Urinary Na+ and K+ contents were analyzed by atomic absoption using a Jenway Corp.

model PF30 flame photometer. The instrument was calibrated with standard solutions

8
containing different concentrations of Na+ and K+, pH and conductivity were directly

determined on fresh urine samples using a Conductronic pH 120 apparatus. Density

estimation was made by weighing with an Ohaus Analytical Plus (±0.1 mg) analytical

balance on urine volume measured with 50 ȝL Hamilton syringes.

2.9. Statistical analysis

Results are expressed as the mean values ± S.E.M. (standard error of mean). The

statistical evaluation was carried out by analysis of variance (ANOVA) followed by

Dunett and Tukey test for multiple comparisons. When comparing with control groups,

values of P less than 0.05 were considered significant. Graphs were drawn and

statistical analysis was carried out using Graphpad prism version 5.0 for windows

(GraphPad software. San Diego. CA, USA).

3. Results

3.1. Diuretic activity of the plants extracts and drugs

3.1.1. Effect on urine volume

Urinary excretion was measured at 2, 4 and 6 h after administration of S. nothohybrida

and S. lepidophylla aqueous extracts at doses of 1 and 2 g/kg. S. nothohybrida produced

increased excretion levels ranging from 41.3 % to 47.8 % of those measured in the

untreated controls. In the case of S. lepidophylla, values ranged from 34.7 % to 32.6 %

(Table 1).

9
3.1.2. Effect on the excretion of electrolytes and urine volume, four hours after oral

treatment of infusion and compounds 1 - 4.

Different parameters were analyzed for possible diuretic action in a) infusion and b) test

compounds 1 - 4 and c) furosemide (reference drug) such as the urinary electrolytes

(Na+ and K+) (mmol/L) contents and the urinary volume on each case compared versus

control (water) and furosemide (reference drug) (Table 2). Amentoflavone (1) induced

significant excretion value for water (36.95%), but values of excretion of Na+ and K+

were 26.7% and 41.0% lower when compared with the untreated control group (Table

2). Robustaflavone (2) induced significant excretion for water of 45.6%, and 28.3%

major excretion of Na+, but lower elimination of K+ (36.9%) when compared with the

untreated control group (Table 2). Trehalose (4) induced the most significant excretion

value for water of 58.69% (14.5% for the excretion of Na+ and 9.0% for K+, being both

values lower, to those of the untreated control group) (Table 2). The 2 g/kg dose of the

aqueous extract of S. nothoybrida induced excretion values for water (19.5%), and

14.3% and 28.3% for the excretion of Na+ and K+ which were lower values than the

control (Table 2). (S)-2,3-dihydrorobustaflavone (3) induced less diuretic effect than

water (73.91%).

3.1.3. Effects on the excretion of electrolytes and urine volume 4 h after oral treatment

with compounds 1, 2, 4 and 5-8 and study of involvement of these compounds on

inhibition of prostaglandins.

Mice were pretreated with ASA (5) (10 mg/kg) and independently administered with an

oral single dose of compounds 1, 2, 4 and decoction of S. nothohybrida, comparing the

10
results with the diuretic values of the tested compounds without ASA. Results showed

diuresis inhibition provoked by ASA in all cases (Fig. 2). Furosemide plus ASA had

29.4% of diuresis inhibition; in the case of the tested compounds, it was observed that

robustaflavone (2) plus ASA had the higher inhibition percentage (53.4%). Compound 1

showed 16.3% of inhibition while compound 4 induced an increase of diuresis in 13.0%

and decoction presented the lowest value (10% inhibition) (Fig. 2).

There was an important increase in the urine conductivity of the mice treated with

robustaflavone (2) (-064) versus compound 1 (amentoflavone) which displayed the

same effect as the control (-042). In the case of compounds 3, 4 and the aqueous extract,

values were inferior in relation to the control group (-032, -036, -031). The pH values

were higher in the case of biflavonoids 1 and 2 (5.41, 5.17), and lower in compounds 3,

4 (4.17, 4.23) and in aqueous extract (4.15) and there was no statistically significant

differences in urine density among treated and control values (Table 2).

4. DISCUSSION

In the present study, the diuretic effect of two doses of aqueous extracts of Selaginella

nothohybrida and S. lepidophylla (doradillas) was evaluated in mice. Oral

administration was chosen as how these plants are consumed in Mexican traditional

medicine.

Treatment with a dose of 2 g/kg of S. nothohybrida extract increased diuresis as

compared to a dose of 1 g/kg and also with respect to the control group at 4 h after the

dose. Treatment with a dose of 1 g/kg of S. lepidophylla increased diuresis to a lower

extent compared to S. nothohybrida, the effect was rapid but not time constant; at a dose

of 2 g/kg, diuresis was even lower. Considering that S. lepidophylla and S. nothohybrida

11
contain mostly the same biflavonoids (Aguilar et al., 2013), S. lepidophylla decoction

was not further studied.

Our results were closely similar with the values reported in the literature on the diuretic

effect of furosemide at a dose of 10 mg/kg (Lahlou, et al., 2007), when compared with

the untreated control group. In the other hand, furosemide showed its maximum effect

at 4 h, consequently experiments with extracts and pure compounds were developed at

this time. All tested compounds presented different diuresis percentage values for each

experiment and were lower in all cases compared with furosemide.

The effect of increased diuresis induced by the aqueous extract was not reflected in

terms of ionic excretion when compared with controls since excretion of Na+ and K+

were lower with respect to control group. The specific conductivity, which is an indirect

measure of the ionic content of the urine and the pH, also decreased in the extract when

compared with the controls, probably related with its poor ionic excretion.

It is a known fact that compounds of the type of biflavonoids increase renal blood flow

and the glomerular filtration rate, which promotes a rise in urine formation (Loew,

1991). Of all tested biflavonoids, robustaflavone (2) induced the highest diuretic action

followed by amentoflavone (1) (Table 2). In contrast with that, urinary excretion of

mice treated with (S)-dihydrorobustaflavone (3) was lower than the exerted by the

control group. Consequently, this compound was not further studied. In this last case

saluretic values were the highest of all tested compounds, as urine was concentrated

(Table 2) but conductivity and pH values were the lowest of all and density was

augmented, as the result of the low water loss. The increase in diuresis promoted by

amentoflavone (1) showed a lower saluretic effect than that of control group in contrast

to robustaflavone (2) that induces an important Na+ excretion even that K+ excretion is

lower. Analysis of these values suggests that although these two isomeric biflavonoids

12
possess a similar structure, their mechanism of diuresis is different. We hypothesize that

robustaflavone (2) acts in a similar mechanism as furosemide, having both natriuretic

effects. In general, physicochemical characteristics of urine did not show important

changes compared to controls when furosemide, amentoflavone (1) and robustaflavone

(2) were administered, however, pH values increased. To this, it is reported that diuretic

compounds of furosemide type produce alkalosis (Abdala et al., 2008). Density values

in excreted urine of mice remained very close to 1 g/mL.

Of all tested compounds and infusion, the highest percentage of diuresis was observed

by trehalose (4) whose ionic excretion effect diminished. This in turn induced a

concomitant low conductivity and pH values compared to the control group (Table 2),

having an additional kaluretic value similar to furosemide. According to this, trehalose

(4) appears to induce diuresis of the osmotic type. This behavior has been observed in

related substances such as mannitol, other polisacarides and glucose (Segarra, 2003).

Aqueous extract in the other hand promoted diuresis in a less extent than pure

compounds. This could be owed to the presence of additional compounds with

antagonist or no diuretic effect in the extract. Dunnet comparative analysis (data not

shown) indicated that all treatments were statistically different to control administered,

this means that all tested compounds have diuretic activity with the exception of (S)-

2,3-dihydrorobustaflavone (3), which behaves as antidiuretic. Tuckey multiple

comparative analysis results showed that besides furosemide, the most active compound

is trehalose, followed by robustaflavone (2), amentoflavone (1), aqueous extract and

(S)-2,3-dihydrorobustaflavone (3) which was considered non active. Differences among

treatments with amentoflavone (1) and robustaflavone (2) were not significant, so was

the case of amentoflavone (1) versus aqueous extract, which indicates that

13
amentoflavone (1) activity could be comparative to robustaflavone (2) and to aqueous

extract activities.

According to Jackson (1996), diuresis occurs in two events: increase in water excretion

(urine volume) and a net loss of solutes (i.e. electrolytes). These processes result from

suppression of renal tubular reabsorption of water and electrolytes into the blood

stream. The reference drug, furosemide, increases urine output and urinary excretion of

sodium by inhibiting Na+/K+/2Cl- symporter (co-transporter system) in the thick

ascending limb of the loop of Henley (Jackson, 1996; Lahlou et al., 2007).

Once diuretic effects were observed, it was interesting to assess the probable

involvement of prostaglandins in the mechanism of action by which diuresis was

induced by these metabolites and the extract, evaluating the variation of excretion of

urinary volumes so as physicochemical characteristics of urine in mice previously

administered with unspecific COX’s inhibitors such as ASA (5), sodium naproxen (6)

and indomethacin (7) and then with test compounds and aqueous extract. The results

showed an important inhibition of diuretic action in all cases (Fig. 2). Administration of

ciclooxigenase inhibitors is commonly associated with Na+ retention, edema and/or

hyperkalemia resulting from loss of intrarenal prostaglandin synthesis (Breyer, 2000).

Physicochemical properties of urine excreted after treatment with COX’s inhibitors

were not significantly modified, as with the exception of trehalose (4) whose pH

diminished when it was treated with sodium naproxen (6); the pH turned slightly more

acidic in all cases, probably for the rise in Cl- elimination. Salicilates act over renal

tubules affecting uric acid reabsorption that induces a uricosuric effect potentially

explaining the acidification of urine, while a possible deficit in reabsorption of filtered

H2CO3 could have occurred. Density of urine kept values of 1 g/mL and conductivity

varied according to electrolytes concentration.

14
When mice were previously treated with compound 5 (ASA) and then with furosemide,

an expected inhibition of diuresis was observed (Gasparotto et al., 2009). Under the

same conditions, with the exception of trehalose (4), inhibition of diuresis was observed

with all tested compounds, but with amentoflavone (1), a marked saluretic effect was

shown giving an idea that mechanism of action is different to that of furosemide.

Trehalose (4) observed the opposite effect, increasing diuresis when it was administered

after ASA (5) (Fig. 2). With respect to the participation of indomethacin (7), all tested

compounds and extract observed higher inhibition of diuresis than furosemide (Fig. 2),

having trehalose (4), amentoflavone (1) and the extract the highest values, contrary to

the results obtained with ASA (5). Assaying test compounds, extract and furosemide

with mice previously administered with sodium naproxen (6) showed similar results to

treatment with ASA and indomethacin (Fig. 2), which inhibited diuresis. Mice treated

with naproxen (6) and then with aqueous extract showed the highest inhibition of

diuresis than the rest of the tested compounds (1, 2 and 4).

In treatment with Celebrex, aqueous extract presented the lowest inhibition of diuresis

compared to the rest of tested compounds (1, 2 and 4). This can be attributable to the

presence of additional compounds contained in the extract, which could have antagonist

effects to diuresis that compete with tested compounds effects. However, diuresis is the

net effect of the aqueous extract. When this specific COX-2 inhibitor (8) was

administered, diuretic activities of tested compounds (1, 2 and 4) diminished up to 50%

(Table 2). This situation could have been related to the loss of synthesis of

prostaglandins present in kidneys acting in the diuretic process and also because of

reduction of glomerular filtration rate, renal flux and renal circulation promoted by

biflavonoids (Lote, 1981). When the COX-1 inhibitor (a nonspecific inhibitor, sodium

15
naproxen, 6) was administered, inhibition of diuresis was also visualized, but in a lower

extent than with the COX-2 inhibitor (8), as prostaglandins synthesis was less affected.

Based on these results, we conclude that the different involved molecules may be acting

through different mechanisms. For instance, the two isomeric biflavonoids (1 and 2)

showed diuresis. When administered to mice with nonspecific COX’s inhibitors, they

had opposite activities, and same situation was observed after treatment with the

specific COX-2 inhibitor. This can be explained in terms of differential structural

arrangements in space of the two isomeric biflavonoids (1, 2) (Fig. 1).

5. CONCLUSION

S. nothohybrida presents a moderate diuretic effect, which appears to be in partly

mediated by the presence of biflavonoids and trehalose. Renal prostaglandins may be

involved in the mechanism of diuresis. The present results provide a quantitative basis

explaining the traditional folk medicine use of S. nothohybrida as a diuretic agent by

Mexican population.

Acknowledgements

Authors want to thank to A. Reyes-García of UNAM for identification of vegetal

samples, so as to A. Tovar, M. I. Chávez and A. Acosta of UNAM for assistance in the

atomic absorption and spectroscopic experiments and to DGAPA-UNAM (IN217914).

We thank Dr. Manuel Gutiérrez for critically reading the manuscript. W. Benítez thanks

to CONACyT for the M. Sc. grant.

16
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 Group Doses (g/kg) Urine volume (mL) Urinary effect

Control (water) 0.46 ± 0.05 1%

S. lepidophylla 1 0.62 ± 0.17 34.7 %
S. lepidophylla 2 0.61 ± 0.25 32.6 %
S. nothohybrida 1 0.65 ± 0.08 41.3 %
S. nothohybrida 2 0.68 ± 0.23 47.8 %
Results show mean values ± mean standard error (M.S.E.).

21
Table 2

Table 2. Effect of oral administration of the aqueous extract and compounds 1-4 on
urinary volume and electrolyte excretion and urinary excretion with simultaneous

Electrolyte
Urine
Dose Density Diuretic concentration
Treatment pH Conductivity volume
(mg/kg) (g/mL) index Na+ K+
(mL n=1)
(mmol/L) (mmol/L)
Control (water) 1 mL 4.9 1.06 -041 0.46±0.03 1 87.0±5.4 280.8±6.1
Furosemide 10 5.3 1.01 -077 0.95±0.01 2.06 132.2±5.9 315.5±5.0
Amentoflavone 10 5.4 1.03 -042 0.62±0.02 1.34 69.8±5.7 161.5±2.5
Robustaflavone 10 5.1 1.00 -064 0.67±0.03 1.45 122.3±5.7 181.9±8.5
(S)-2,3-dihydro
10 4.1 1.10 -032 0.34±0.02 0.76 139.0±7.4 377.2±8.8
Robustaflavone
Trehalose 10 4.2 1.02 -036 0.73±0.03 1.58 81.4±4.2 246.5±7.6
Aqueous
2000 4.1 1.04 -031 0.54±0.02 1.19 84.0±2.3 195.6±3.4
extract
Furosemide +
10 4.4 1.02 -037 0.82±0.03 1.7 125.2±1.6 186.7±9.5
NS
Amentoflavone
10 4.4 1.01 -037 0.52±0.01 1.13 97.0±1.7 510.6±4.6
+ NS
Robustaflavone
10 4.5 1.03 -045 0.47±0.02 1.02 84.9±4.1 214.7±3.0
+ NS
Trehalose + NS 10 4.4 0.96 -048 0.61±0.02 1.32 94.3±4.9 166.3±9.4
Aqueous
2000 4.2 1.02 -040 0.32±0.03 0.69 139.8±7.9 285.0±8.6
Extract + NS
Furosemide + C 10 4.2 1.02 -040 0.63±0.02 1.36 58.8±9.4 40.1±4.2
Amentoflavone
10 4.3 1.03 -046 0.25±0.02 0.54 102.7±1.8 190.4±3.7
+C
Robustaflavone
10 4.1 1.06 -036 0.39±0.03 0.84 48.3±3.2 114.4±0.5
+C
Trehalose + C 10 4.3 1.07 -045 0.28±0.03 0.60 87.0±5.4 175.4±5.0
Aqueous 0.43±0-
2000 4.0 1.02 -028 0.93 85.3±10.8 106.8±6.9
Extract + C 01
administration of COX’s inhibitors.

The results show the mean values ± S.E.M., NS= naproxen sodium, C= Celebrex, (-)
antidiuretic effect. Diuretic index= (volume problem group/volume control group)

22
Captions

Fig. 1. Structures of amentoflavone (1), robustaflavone (2),

(S)-2,3,dihydrorobustaflavone (3) and trehalose (4)

Fig. 2. Urinary excretion induced by compounds 1, 2, 4 and aqueous extract (Aq. E.) in
mice administered with COX’s inhibitors

Table 1. Effect of two oral doses of the aqueous extract of Selaginella nothohybrida
and S. lepidophylla on urinary volume (4h).

Table 2. Effect of oral administration of the aqueous extract and compounds 1-4 on
urinary volume and electrolyte excretion and urinary excretion with simultaneous
administration of COX’s inhibitors.

23
Figure

(1) (2)

(3) (4)
Fig.1.
 Figure

Volume (mL) Volume (mL)

0.0
0.2
0.4
0.6
0.8
1.0

0.0
0.2
0.4
0.6
AS

Figure 2
A
I+ +
(1 (1
) )
AS
A
I+ +
(2
(2 )
)
AS
A
+
I+ (4
(4 )
) AS
A
+
I+ Aq
Aq .E
.E

Vol ume (mL)

Volume (mL)
0.0
0.2
0.4
0.6
0.8

0.0
0.2
0.4
0.6

NS
C +
+ (1
)
(1
)
NS
C +
+ (2
(2 )
)
NS
C +
+ (4
(4 )
)
NS
C +
+ Aq
Aq
.E .E
*Graphical Abstract (for review)