Rhizophora mangle L. is a vegetal species with high disponibility in Cuba for guarantying economical factibility by drug development. Preview studies showed promissory pharmacological results by the use of this plant in the treatment of gastroduodenal ulcers. The present work involved another evaluation of the active extract important by the development of a new drug. It was evaluated by intestinal absorption in vitro and in situ models and anti-diarrheic activity in rats. The polyphenolic fractions were separated from the aqueous extract and its cytoprotective activity was studied on gastric ulceration induced by ethanol plus hydrochloric acid in rats. Some toxicological studies were carried out. We made a validation of HPLC method by the analysis of one mark in the tablet development. The aqueous extract showed high absorption, and high anti-diarrheic effect. The low-molecular weight polyphenolic fraction showed the highest level of gastric protection. No toxicological signs were obtained by it. HPLC method evidenced linearity, precision, sensitivity and accuracy.
Rhizophora mangle L. was widely distributed in Cuba and other Caribbean countries [
In previous works, we reported the cytoprotective effect of freeze dried aqueous extract from red mangrove bark on gastric ulceration induced by ethanol-hydrochloric acid in rats [
Other preview studied performance with this extract; it had shown the presence polyphenolic structures (54.78%) and other structural components (45.22%). Polymeric tannins were the major polyphenolic component (80%) and hydrolysable tannins were 20%. Epicatechin, catechin, chlorogenic acid, gallic acid and ellagic acid were monomeric structures determined in this extract. Phytosterols (0.0285%): stigmasterol, b-sitosterol and likewise campesterol were presented too [
This vegetal specie with pharmacological activity as the antiulcer in gastroduodenal tract was processed by analyzing your possible toxic effect in acute and sub-acuteoral toxicology in rats. Any toxicological sign was shown in both studies [
For all preview studies, it is possible to consider this plant as a very good leader or source by the development of a phytomedicament. However other studies will be necessary by it, for example compounds present in total extract are responsible of the activity obtaining with the extract; studies of the security and secondary adverse effects. Regulatory requirements for drugs development request different levels of genotoxicological testing and the effect on other system in the organism.
Other aspect necessary is to determinate the quality control and the stability from new formulation. In this sense it will be proceeding to define first a chemical marker or active substance which could determinate in the end formulation, for example tablets. After defined analysis method to determinate at this substance, it is a requirement to validate a method by its sensibility and reproducibility.
The objective of the present work was to complete the study of this plant by its use in human medicine for treating gastroduodenal ulcers. These studies include completing the study of the possible adverse effects in the administration of vegetal drugs, and its effects on other biological systems: two models of intestinal absorption in vitro and in situ in rats. The activity of this extract was to test on diarrhea produced by ricin oil in rats. Other proposal of the present work was to evaluate the cytoprotective activity of the major compounds, polyphenols and the possible synergic activity of low- and high-molecular weigh polyphenolic fractions; genotoxicological tests: spermatozoa head assay and Comet’s test. HLPC analysis methods to catechin determination in tablet validation were included.
R. mangle L. was collect from the western zones of Cuba in 2011. The identity of the plant was authentifique by a botanist and a voucher specimen has been deposite in National Botanical Garden’s Herbarium. The extract was prepared for the decoction of the bark in distillate water. The proportion of vegetal matter: water was 1:7; the decoction was make for 20 min at 90˚C in lab reactor with 2 L of capacity. The plant material was separate by centrifugation and the aqueous extract was concentrated and freeze-dried to preserve it.
In the experimental models of intestinal absorption in vitro were used small intestine segments from three animals.
The administration of aqueous freeze dried of R. mangle was make as it was representing in
The selections of these doses were determinate by therapeutically answer as antiulcer effect. The concentration of tannin insert in each segment of small intestine was 30.74 mg of tannins in 250 mg/Kg (39.68% of tannins in the freeze dried extract by Spray-Drier). The tannin determination was make by precipitation of protein methods [
The absorption model was to asses as a tool for describe passive absorption mechanism and to go deeply into the structures of the physiological absorbing barriers.
The methodology consists of establishing correlations between absorption rate constants and lipophilicity for a several homologous series of xenobiotics.
Part of small intestine | Segment (cm) | Doses (mg/Kg) |
---|---|---|
DUODENUM | 6 | 250 |
12 | 250 | |
18 | 250 | |
24 | 250 | |
JEJUNE | 30 | 500 |
36 | 500 | |
42 | 500 | |
48 | 500 | |
54 | 250 | |
ILEUM | 60 | 250 |
66 | 250 |
In this experiment were used Wistar rats weighing 150 - 180 g, with 12 hours faster. Three groups were made with 5 rats by group. First group was administered with 1 ml of vehicle after 30 minutes was administer 2 ml of ricin oil by oral administration. Groups 2 and 3 were treatment with 125 and 250 mg/Kg b.w. of extract from R. mangle (1 ml), after were administered 2 ml of ricin oil. After 30 minutes the rats were sacrificed. The small intestines were isolated by pyloric esfinger to ileocecal valvule and it were weight with intraluminal content, after the content of intestine were separated and it were weight another. The longitudes of intestines were measured. Intraluminal (enteropooling) fluid was calculated in mg/cm with the following (W1 − W2)/L, where W1 is the weight of small intestine, W2 is the weight of empty small intestine with intraluminal content and L is the intestine longitude.
The evaluation of gastric protection from polyphenolic fractions was make two experiments. One experiment we used the following doses: negative control with distilled water; one group with 500 mg/kg b.w. of freeze-dried aqueous extract from R. mangle; a group with 100 mg/kg of high molecular weight polyphenols fraction; a group with 100 mg/kg of low molecular weight polyphenols fraction and one group using ranitidine (100 mg/kg b.w.) as positive control. In the second experiment, we used the following groups: distillated water as negative control, two groups treated by 200 and 400 mg/Kg b.w. from high molecular weight polyphenols (HMWP); two groups treated by 200 and 400 mg/Kg b.w. from low molecular weight polyphenols (LMWP); one group with 200 mg/Kg b.w. of HMWP and 60 mg/Kg b.w. of LMWP and one group treated with 500 mg/Kg b.w. of Rhizophora mangle L. (freeze-dried). In all cases were given orally (gavage).
a) Gastric protection from polyphenolic compounds fractions
The high and low molecular weight polyphenolic fractions separation of the freeze dried aqueous extract from Rhizophora mangle L bark were employed the scheme described in the
b) Gastric lesions induced by necrotizing agent
Animals in testing groups were given 1 ml of necrotic agent (60 ml ethanol + 1.7 ml hydrochloric acid + 38.3 ml water). Freeze dried R. mangle extract, polyphenolic fractions and ranitidine were given 30 min before the necrotic agent. Animals were killed by cervical dislocation 1 h after treatment with the ulcerogenic agent. The
stomach of each animal was excised and opened along the greater curvature. After washing with the normal saline, the gastric lesions were quantified using a binocular magnifier, the sum of length (in mm) to alls lesions in each animal was used as a lesion index. The number of erosions per stomach was assessed for the severity according to, 1) absence of lesion, vasodilatation or up to three pinpoint ulcers; 2) more than three pinpoint ulcers; 3) from one to five small ulcers (<2 mm); 4) more than five small ulcers (<2 mm); 5) one or more giantulcers (≥2 mm).
c) Statistical analysis
The data are expressed as means ± S.E.M. (standard deviation of the means). Student’s t-test was used for statistical analysis, P values < 0.05 were considered significant. Non-parametric data were analyzed by Wilcoxon Score test.
The high-performance liquid chromatography method validation to the quantitative assessment of cathechin as a marker substance in tablets obtained from the bark of Rhizophora mangle L. dry extract used in gastroduodenal ulcers treatment. Considering that this method as such is classified to quantitative assessment of the major compound or active ingredient in formulae or raw material, the following parameters were assessed: linearity, accuracy, sensitivity and precision expressed in its two ways: repetition and intermediate precision.
Reference substance catechin was of Sigma. Phytodrugs in tablets forms were manufactured in PHARMALAB Enterprise, Guatemala.
All reactive used were pure quality by analysis.
Analytical method by High Resolution Liquid Chromatography was made with isocratic bomb Kanauer, detector UV Knauer with the following chromatographic conditions:
Column: LiChrosorb, RP-18, 5 µm, 125 × 4 mm d.i.; mobile phase: Methanol: acetic acid 2% (20:80 v/v), Detector: variable ultraviolet at 254 nm; Flow velocity: 0.8 ml/min; Injector volume: 20 µL. Detection System coupled at BIOCHROM program for the analysis of retention time and area of the picks.
The reference solution was made with catechin in methanol at 1 mg/ml concentration.
The fraction of ethyl acetate was obtained with 20 tablets with the addition of 100 ml of distilled water and heated at 60˚C. It was treated with 36 g of sodium chloride for the precipitation of polyphenols of major molecular weight. It was centrifuge at 3500 rpm during 15 minute at the supernatant was extracted successively with ethyl acetate (3 extractions). Ethyl acetate fraction was dried and it was dissolved in 10 ml of methanol by the injection in HPLC.
Experiments were performed using 8 - 12 weeks, Cenp: NMRI out bred, male mice, provided by Centro para la Producción de Animales de Laboratorio (CENPALAB), Havana, Cuba. Animals were kept at room temperature and room relative humidity and exposed to the natural light-dark cycle. They were randomly distributed in groups of 6 animals per dose in each treatment, and the standard rodent diet and tap water were ad libitum. All the animal procedures reported here, were carried out in accordance with the Cuban regulations on the protection of animals. The experimental protocol was also revised by ethical committee and conducted humanely.
1) Experimental design
Three different experimental series (referred as I, II and III) were designed to evaluate the possible genotoxic effect of the aqueous extract of R. mangle. In all cases, the extract was dissolved in saline solution (NaCl 0.9%) and given by oral administration using 3 different dose levels (500, 1000 and 2000 mg of total solids/kg b.w.). An equal volume of saline solution was considered as a negative control group in all experimental series. In series I, the extract was administered during 3 days within 24 hours-time interval and the animals were humanely sacrificed on day 4 after starting the administrations. In series II, the extract was also given once per day, but the treatments lasted 5 days and the animals were sacrificed on day 21. In series III, the administrations of the extract were conducted as in series II, but the animals were sacrificed on day 35.
2) Sperm morphology test
The test was performed according to the method described by Wyrobek and Bruce, 1978 [
3) Statistical analysis
Results of sperm count and morphology are presented as the mean ± standard deviation (SD). Data of normal distribution and variance homogeneity were studied by Kolmogorov-Smirnov and Bartlet tests, respectively. Treatments were compared using ANOVA and Dunney’s post-test. p < 0.05 was considered significant.
Mice NMRI were used of both sexes. It was distributed in three experimental groups (10 animals/group): once as negative control treated with distillated water, the second group treated with 2000 mg/Kg b.w. of extract from R. mangle. It these case the treatment were administered for 14 days and the third group was considered as positive control and it was treated with methylmethanosulfonate (MMS) during five days before test’s conclusion. The administration at negative and treatment groups were made by oral and in the case of positive group the administration was intra peritoneal. Animals were sacrificed by euthanasia 2 to 6 hours after the last administration. By the hepatocytes isolate each animal were extracted the kidney and immediately it were put in Hanks solution. It was cold on ice during 20 minutes. Portion of principal hepatic lobules was put on cold port object, courting in rectangular pieces. It was washed with 15 ul of Sorensen buffer ( 50 mM , pH 7.2). They were maintained at ambient temperature 1 - 2 minutes before low fusion point of agarose addition (BPF). Alkaline electrophoresis test * (Comet Test) were made. For also, 15 - 30 ul of cellular suspension and 140 ul 0f low fusion point agarose 0.5% were extended in sheets preview prepared with normal fusion point 0.5%. They were submerged in lisis solution (NaCL 2.5 M, EDTA 100 mM and Tris 10 mM, 1% Triton, 10%) during 1 hour at 4˚C and after they were submerged 30 minutes in electrophoresis regulator solution (3% NaOH 10 N, 0.5% EDTa 200 mM, pH > 13). Electrophoresis was made at 300 mA and 0.7 - 1 V/cm during 20 - 30 minutes. It was made 3 washes with neutralization regulator solution (Tris 0.4 M, pH 7.5) and it was cleared with distillated water. The tin was made with argent nitrate (0.05%) and the nucleoids’ colored were evaluated employed an optic microscopy. By the recount on induced injury sheets were observe to optic microscopy with 40× of increase and it were classified as 50 comets/gel. Each comet was classified accord to category 0 grade of injury correspond to DNA between 0 and 4. Injury magnitude in DNA was expressed in arbitraries unity (UA) according with Collins [
TCG0 = Total cells grade 0 (not injury cells); TCG1 = Total cells grade 1 (minimum frequency of lesions in DNA); TCG2 = Total cells grade 2 (low injury, with frequency low of lesions in DNA); TCG3 = Total cells grade 3 (high injury, with high frequency of lesions in DNA); TCG4 = Total cells grade 4 (cells totally injury).
Statistical analysis was made with Stat soft program by Window version 6.1. Significant was 0.05. It were used Kolmogorov-Smirnov by normality and variance homogeneity (Levene or Bartlett). Student t Test were used by comparison between media inter groups.
In the
Parte of intestine | Segment | Doses | Animal 1 Content of residual tannins (mg/ml) | Animal 2 Content of residual tannins (mg/ml) | Animal 3 Content of residual tannins (mg/ml) | Media ± D.S. Content of residual tannins (mg/ml) |
---|---|---|---|---|---|---|
DUODENUM | 6 | 250 | 0.309 | 0.287 | 0.283 | 0.293 ± 0.014 |
YEYUNE | 12 | 250 | 0.290 | 0.291 | 0.283 | 0.288 ± 0.004 |
18 | 250 | 0.289 | 0.311 | 0.281 | 0.294 ± 0.015 | |
24 | 250 | 0.3093 | 0.291 | 0.283 | 0.294 ± 0.013 | |
30 | 500 | 0.286 | 0.292 | 0.273 | 0.284 ± 0.01 | |
36 | 500 | 0.281 | 0.288 | 0.277 | 0.282 ± 0.005 | |
42 | 500 | 0.294 | 0.286 | 0.277 | 0.286 ± 0.008 | |
48 | 500 | 0.293 | 0.286 | 0.277 | 0.285 ± 0.008 | |
ILLEUM | 54 | 250 | 0.283 | 0.293 | 0.288 | 0.288 ± 0.005 |
60 | 250 | 0.289 | 0.288 | 0.286 | 0.288 ± 0.001 | |
66 | 250 | 0.294 | - | - | 0.294 |
that the content of residual tannins in 250 mg/kg b.w. is similar in duodenum, jejune and ileum. These results suggest that the absorption of tannins not depend of intestinal segment. The absorption in gut of tannins in the freeze dried extract of R. mangle is superiority at 97%. These results suggest that the polyphenols present in the extract are polyphenols with high absorption, for also these compounds are structures with relative low molecular weigh structure (monomer, dimer or trimer) and don’t present polymeric compounds.
In situ study to intestinal absorption of freeze dried extract of R. mangle in total segment of small intestine.
The parameter representative of absorption process using was the constant apparent of absorption rate (kap) because it represents an index of the absorption rate intrinsic off solute.
This constant was determinate within recirculation evading the aqueous layer of adjacent diffusion to the membrane.
It in situ method was choice because it plows considered in general reproducible with it lives reliable the absorption process whom occur in vivo. The perfumed volume stay all time to testing (30 minutes) in contact with intestinal mucosa, for also the absorption yield is high than that the obtain by other techniques, where the contact fluid-mucosa is intermittent. In this method is it maintains blood flow and tisular integrity similar at in vivo models.
In the
The accumulation of fluid in small intestine in rats is quick, qualitative and predictive of diarrhea and it is useful to research agents that block this effect. The accumulation of fluid in small intestine in named “enteropooling”. This accumulation is a sum of fluid secreted from blood to lumen and a portion of fluid of lumen that absorption is inhibit by prostaglandin or other laxante agents as ricin oil hypertonic’s solution that cause enteropooling too.
Ricin oil increase a volume of intestinal fluid, this effect is maximum to 30 minutes, which it reduces the fluid accumulation. The
First, we made a preliminary test of the cytoprotective effect of freeze dried aqueous extract of Rhizophora mangle L. in a acute ulcer model induced by ethanol plus hydrocloride acid (6). But, we not defined what compounds was responsible of this activity. This extract was different structural compounds: fatty acids, phytosterols, volatile compounds and polyphenols as major secondary metabolites.
Extract 250 mg/Kg b.w. C0 = 0.8285 mg/ml | ||||||
---|---|---|---|---|---|---|
Time (min) | Remnant concentration in the lumen = C = mg/ml | |||||
Experimental Animals | ||||||
1 | 2 | 3 | 4 | 5 | 6 | |
5 | 0.5528 | 0.1744 | 0.2449 | 0.2162 | 0.2945 | 0.6389 |
10 | 0.37014 | 0.1379 | 0.2053 | 0.2018 | 0.1444 | 0.5267 |
15 | 0.3023 | 0.1366 | 0.1679 | 0.1705 | 0.1340 | 0.5254 |
20 | 0.2632 | 0.1314 | 0.1666 | 0.1575 | 0.1131 | 0.4850 |
25 | 0.241 | 0.1275 | 0.1601 | 0.1366 | 0.0792 | 0.3988 |
30 | 0.1835 | 0.1066 | 0.1249 | 0.0909 | 0.074 | 0.1797 |
Kap (h-1): 2.03100775; D.E.: 0.76895328. Kap: constant of apparent rate of absorption.
In the first experiment with simple doses the low and high molecular weight polyphenols fractions (100 mg/ kg b.w.) we found a decreasing in lesion index respect to negative control but it was not statically significant (
In the study of gastric protection effect by low and high molecular weight polyphenolic fractions using different doses level (experiment 2) was shown that low molecular weight polyphenolic fraction had the major cytoprotector activity (
It was found a doses dependent decreasing in the lesion score in both cases (treatment with HMWP and LMWP). There is not additive effect between both fractions. It was confirmed in the group treated with 200 mg/kg HMWP more 60 mg/kg LMWP, where the action or LMWP was capable to decrease the lesion score.
Animal group treated by freeze dried aqueous extract of R. mangle (500 mg/Kg) was best answer than a group treated with a mixing of both fractions in the same proportion of calculated concentration of these substances in total extract. For also, we could to consider that the gastric protection activity of total aqueous extract was influence by other compounds present in this extract. These other compounds increase the gastric activity and this activity is lost in the fractionation process.
Results obtained showed that this method is reliable allowing the compound assessment in presence of other substances, including excipients and auxilliary substances and to detect the presence of degradation products. Also, the statistical processing of results evidenced the linearity, precision, sensitivity and accuracy of this method.
The
Lesion Index | |
---|---|
Treatment | (Media ± E.S.M.) (mm) |
Negative Control | 42.4 ± 11.9 |
Freeze dried red mangrove 500 mg/Kg b.w. | 13.2 ± 8.0** |
HMWP 100 mg/Kg b.w. | 37.5 ± 23.1 |
LMWP 100 mg/Kg b.w. | 25.4 ± 17.8 |
Ranitidine 100 mg/Kg b.w. | 10.8 ± 9.1** |
Lesion Score | |
---|---|
Treatment | (Media ± E.S.M.) (mm) |
Negative Control | 4.2 ± 0.84 |
HMWP 200 mg/Kg b.w. | 4 ± 0 |
HMWP 400 mg/Kg b.w. | 3.4 ± 0.55 |
LMWP 200 mg/Kg b.w. | 3.2 ± 0.84 |
LMWP 400 mg/Kg b.w. | 1.8 ± 0.45** |
HMWP 200 mg/Kg + LMWP 60 mg/Kg b.w. | 3.5 ± 1.00 |
Freeze dried red mangrove 500 mg/Kg b.w. | 2.75 ± 0.50* |
Catechin patron was not discomposed by the action of Hydrogen peroxide (H2O2). The concentration of catechin calculated after oxidation process was 1.5 mg/ml, for also it guarantees not oxidation itself. In case of filmic placebo appear a new peak at Rt = 11.90, it was an interference in the catechin determination.
In chromatogram from filmic tablets after acid hydrolysis appearance catechin peak and a new peak to Rt = 15.70 minutes possibly fluroglucinol.
In case of basic hydrolysis to catechin patron appearance equal to acid hydrolysis a new peak at Rt = 14.86, for also it produced decomposition product that not interference with mark determination.
The calibration curve resulted lineal in the analysis concentration ranges, the correlations coefficient valor and determination were more than 0.99. Student’s t test application with t calc > t tab were shown lineal correlation between x and y valor. Variation coefficient of the pendant was minor or equal that this parameter limit (<2%).
Precision study, variation coefficient for the repeat was 2.81%, less than 5%.
Precision in intermediate conditions shown no significant difference between the results obtain by analysts because to made Fisher test was obtain F exp < F tab for proportionality of 0.05. Student’s t Test shown that calculated valor was minor to tabulate valor for 95% of confidante. For also it was shown significant difference between analyzer’s media.
The method’s robust is adequate because is this not significant differences between media of areas to chance conditions as lot of mobile phase, test day and column.
The method showed a good exactly as placebo and tablets as matrix with recuperate major 80%.
For also, HPLC method proposal present a detection limit of 0.002376 mg/tab and quantity limit of 0.01364 mg/tab. It method was sensible, by determination of marker in tablets.
The effect of the oral administration of the aqueous extract of R. mangle in terms of germ cells viability is presented in
Results of the sperm morphology test, conducted in the three different experimental series are shown in
On the other hand, in series II and III we appreciated that none of the doses of the aqueous extract (500, 1000 and 2000 mg/kg b.w.) induced variations in the percentage of appearance of anomalous sperms when it is compared with the values in the control group of mice treated with saline solution (NaCl 0.9%).
In daily clinical inspection any animals showed toxic signs.
In
TRATAMIENTOS | CONCENTRACIÓN (106/ML) | ||
---|---|---|---|
Series I (4 DÍAS) | Serie II (21 DÍAS) | Serie III (35 DÍAS) | |
NaCl 0.9% | 15.65 ± 3.90 | 13.43 ± 4.36 | 14.14 ± 2.02 |
R. mangle 500 | 13.84 ± 2.02 | 13. 51 ± 1.82 | 12.17 ± 1.51 |
R. mangle 1000 | 16.11 ± 4.53 | 13.86 ± 3.34 | 11.95 ± 3.25 |
R. mangle 2000 | 14.53 ± 4.46 | 12.75 ± 2.86 | 9.70 ± 4.13 |
Treatments | Percentage of Abnormal Spermatozoa (% ± SD) | ||
---|---|---|---|
Series I | Series II | Series III | |
Control | 2.9 ± 0.8 | 3.1 ± 0.3 | 2.9 ± 0.4 |
R. mangle 500 | 2.9 ± 0.5 | 2.4 ± 0.8 | 3.2 ± 0.7 |
R. mangle 1000 | 3.3 ± 0.9 | 3.3 ± 1.0 | 3.6 ± 0.8 |
R. mangle 2000 | 4.8 ± 1.03* | 2.7 ± 0.6 | 3.5 ± 0.5 |
Values represent means ± standard deviation (SD) of the experiments (n = 6/doses). ANOVA and Dunney post test (p < 0.05).
Doses | Sex | (X ± DE) |
---|---|---|
Sterile Water | M | 33.8 ± 3.05 |
H | 31.3 ± 6.44 | |
2000 mg/Kg p.c. | M | 44.3 ± 10.7 |
H | 37.25 ± 11.7 | |
Methylmethanosulfonate | M | 150.5 ± 5.73* |
H | 144.8 ± 12.1* |
*p = 0.05.
freeze-dried extract. Not statistical significant difference was shown between treated group and negative control group in both sexes. The injuries index in DNA in major Comets observed in experimental groups were 0 and 1 (low injury level). Only in group treated with metylmethanosulfonate shown a migration of DNA classified in 3 or 4 level (high or extreme injury).
The major active principles of the red mangrove are polyphenols, represented in their majority by polymerictannins (80%) and hydrolysable tannins (20%) and special emphasis has been given to the presence of epicathechin, catechin, chlorogenic, gallic and elagicacids, as well as galotannins, elagitannins and condensed tannins [
Tannins or polyphenols have a number of physical and chemical properties in common, which underlie their physiological and pharmacological actions: their antioxidant and radical scavenging activities and their ability to complex with other molecules such as proteins and polysaccharides [
Vegetable polyphenols are known to inhibit lipid peroxidation in vitro and there is evidence about their ability to scavenge radicals such as hydroxyl, superoxide, and peroxyl, which are important in cellular prooxidant states. Incidentally, it has been shown that chlorogenic acid has antioxidant effect as high as DL-tocopherol [
On the other hand, tannins may prevent ulcer development due to their protein precipitating and vasoconstriction effect [
The topical action of the aqueous extract of R. mangle in accelerating wound healing has been explained by several mechanisms, such as coating the wound, forming complexes with proteins of microorganism cell wall, chelating free radicals and reactive oxygen species, stimulating the contraction of the wound and increasing the formation of new capillaries and fibroblasts [
The presence of fatty acids in Rhizophora genera was not reported before [
Essential oils, phytosterols and glycosides of Marsclemiacondurango are effectiveness in ulcer gastriccarcinoma and bleeding. Phytosterols have antiulcer effect, with protective action against Helicobacter pylori.
In recent study phytosterols esters of an herb of Dolichus genera was protective in a model of pyloric ligation ulcer [
Fatty acids have protective effect against pepticulce ration in several experimental models [
For also, we could be inferred that the presence of fatty acids and other functional chemical groups present in this aqueous extract as semivolatile compounds, phytosterols, carbohydrate joint at the major functional group, tannins [
The aqueous extract of R. mangle not has toxic effect in acute and sub-acute toxicity study [
In the last decade the antimutagenic study had been associated to modulation of enzymes from xenobiotic’s metabolism. In this case the plants had shown antimutagenic capacity by its heterogeneous compounds. Between them have a high potential antimutagenic the polyphenols of green tea. Epigallocatechin 3-gallate is an inhibitor of some enzymes of fase I as the citocrom P450, CYP1A, 2B1 and 2E1 and the ellagic acid as inductor of detoxification enzyme (fase II) as the GSTS and NAD (P) Hquininereductase. These polyphenol decrease significantly the incidence of carcinoma produced by the exposition at aromatic polycyclic hydrocarbons because they reduce the activity of benzo (a) pirenehidroxilase [
When evaluating the effect of the aqueous extract of R. mangle over the germinal cells viability it was shown that it does not induce cytotoxicity on the sperm tides that are in differentiation roads to sperms (spermatogenesis phase) or on the sperms that are already formed at 4, 21 or 35 days, corresponding to experimental series I, II and III respectively. Altogether, these results reveal that the plant extract does not affect neither the viability of the primary and secondary spermatocytes (meiosis phase) nor the viability of the spermatogonium (mitosis phase).
The absence of toxicity observed in germinal cells after exposure to the aqueous extract of the bark of R. mangle can be explained on the basis of its chemical components. No toxic effects on in vivo models have been attributed to condensed and hydrolysable tannins, phytosterols, semi volatile compounds and fatty acids, which are constituents of R. mangle aqueous extract [
However, a significant increase in abnormal spermatozoa was observed in the group administered with the higher dose of the plant extract within the experimental series I, pointing out to a genotoxic effect on the germ cells, particularly at the spermatogenesis phase. The finding could be related to certain components of the extract such as hydrolysable tannins (e.g. gallic and ellagic acids) or its metabolites able of exert toxicity particularly during the spermatogenesis process.
Researchers have documented the in vitro cytotoxicity and genotoxicity induced by ellagic and gallic acid (15 - 240 mM concentration range) as measured in culture of B14 cell line [
On the other hand, the mutagenic effect of the extract could be also related to the lacking of effective cell repair mechanisms at this stage or to the nature of the damage, as in some cases no endogenous mechanism exists for its reparation. In any case, the values of anomalous sperms observed at the higher dose of 2000 mg/kg fall below the values reported in similar studies [
Current results show biggest susceptibility in the sperm tides cells, in disagreement with previous approaches in which the late spermatogonial cells and/or early primary spermatocytes appear to be the most susceptible [
The exposure to R. mangle aqueous extract lasting 21 and 35 days did not increase the frequency of appearance of anomalous sperms, according to the data in experimental series II and III. One line of thoughts, guided by results of Wyrobek and Bruce, 1978 [
R. mangle (red mangrove) represents an ethno botanically relevant plant in Cuba, traditionally used for different biomedical applications [
Nair et al., 2008 [
Absorption is a complex problem; for also many works had been made to prediction of absorption potentiality in candidate to drugs, for this important in the biodisponibility of itself. It is necessary ground in the knowledge of absorption mechanism and metabolism of substance clinically active.
In vitro model have high complex and it not evaluated many factors in the absorption. In situ models in general reproduce with more dependability the absorption process that had been place in vivo [
Polyphenols-flavonoids, phenolic acids and tannins―are contained in several foods of plant origin (fruit and vegetables). Much evidence has been provided in recent years supporting a role for these compounds as protective agents against cardiovascular disease and certain forms of cancer, including breast, esophageal, gastrointestinal, lung and skin cancer. Increasing evidence for the possible effects of plant polyphenols on human health have been obtained in in vitro and in vivo systems. However, data on their absorption from the gastrointestinal tract are still scarce and, often, contradictory, although it is likely that the different biological effects of polyphenols are related to their absorption and bioavailability for target organs and tissues. Polyphenol bioavailability was evaluated using segments of the small intestine of rats. Two polyphenolic compounds, tannic acid and catechin, were tested in our model system as representatives of high and low molecular weight polyphenols with gallic acid and flavonoid structures, respectively [
There have been several human studies that have investigated the absorption and bioavailability of flavonoids. Initially, absorption of flavonoids from the diet was believed negligible, given that the majority of food flavonoids are bound to glycosides. It was expected that the aglycones only could pass freely into the bloodstream from the gut wall, because no enzymes are secreted in the gut that could cleave the glycosidic bonds. Recent studies, however, have demonstrated that he bioavailability of specific flavonoids is much higher than previously believed [
The bioavailability of polyphenols is reviewed, with particular focus on intestinal absorption and the influence of chemical structure (e.g., glycosylation, esterification, and polymerization), food matrix, and excretion back into the intestinal lumen. Information on the role of microflora in the catabolism of polyphenols and the production of someactive metabolites is presented. Mechanisms of intestinal and hepatic conjugation (methylation, glucuronidation, and sulfation), plasma transport, and elimination in bile and urine are also described [
Proanthocyanidins differ from most other plantpolyphenols because of their polymeric nature and highmolecular weight. This particular feature should limit their absorption through the gut barrier, and oligomers larger than trimers are unlikely to be absorbed in the small intestine in their native forms. In vitro experiments using single layers of Caco-2 cells as a model of absorption in the small intestine showed that only the dimers and trimmers of flavanols are able to cross the intestinal epithelium. Procyanidin B2 is very poorly absorbed in rats, whereas procyanid in B3 is not absorbed. The possibility that procyanidin oligomers are hydrolyzed to mixtures of flavanol monomers and dimers in acidic conditions was suggested by Spencer et al. [
The freeze dried extract of R. mangle as active principle in antiulcer effect has more than 50% of tannins identify principally proanthocyanidins (monomer and oligomer). For also the results obtain in this report about it absorptionby in vitro and in situ models could be verify the report of other author in the field of absorption and bioavailability of polyphenols in animals and in human. Our found coincide by other report about the antiulcerogenic activity for some polyphenols [
The freeze-dried aqueous extract of R. mangle is with a high concentration of high molecular weigh polyphenols. We demonstrated that the cytoprotective effect of this extract in peptic ulcer is a synergic effect of polyphenolic structures more than other secondary metabolites present in the aqueous extract. The extract possessed antidiarrheic activity and it had a high absorption in in-vitro and in-vivo models which warrant the biodisponibility of this extract in the body. The extract does not have secondary effect in genotoxicological test. Catechin was validated as a good chemical mark in the development of phytodrugs with this plant.
Sánchez Perera Luz María,Janet Piloto,Deyanira Canelsota,Liliam Pelzer,Betty Mancebo, (2016) Further Pharmacological Evidence Supporting the Development of an Antiulcerogenic Drug Based on Rhizophora mangle L. Aqueous Extract. HPLC Method Proposed for Determinating a Chemical Marker. Open Access Library Journal,03,1-16. doi: 10.4236/oalib.1101625