Extracts and isolated compounds from <i>Cakile arabica </i>for treatment of ulcer

Abstract

The present invention relates to extracts, compounds isolated from Cakile arabica for use in the treatment of ulcer and to pharmaceutical compositions thereof.

Claims

1. A method for isolating tetracosanoic acid and 4-hydroxy-5-n-butoxy-5-oxopentanoic acid from Cakile arabica, the method comprising the steps: a. extracting a plant material of Cakile arabica to obtain an extract; b. concentrating the extract to obtain a concentrate; c. chromatographing the concentrate into fractions; and d. isolating fractionated compounds tetracosanoic acid and 4-hydroxy-5n-butoxy-5-oxopentanoic acid.

2. The method according to claim 1, wherein the plant material comprises the aerial parts of Cakile arabica.

3. The method according to claim 1, wherein extracting is carried out by using at least one organic solvent, selected from the group consisting of petroleum ether, heptane, hexane, ethanol, isopropanol, methanol and mixtures thereof, preferably is ethanol.

4. The method according to claim 1, wherein chromatographing is carried out by means of thin layer chromatography (TLC) and/or column chromatography.

5. The method according to claim 4, wherein said chromatographing is carried out with an eluent selected from the group consisting of petroleum ether, heptane, ethanol, methanol, benzene, diethyl ether, chloroform, dichloromethane, water and/or mixtures thereof.

6. The method according to claim 4, wherein said chromatographing is carried out with a stationary phase selected from the group consisting of silica gel and aluminum oxide, or mixtures thereof.

Description

EXAMPLES

(1) Materials and Methods:

(2) Plant Material:

(3) Arial parts of Cakile arabica were collected during flowering stage in March 2012 from Riyadh territory, the sample was identified by Dr. Jacob Thomas; assistant professor of taxonomy, Botany and Microbiology Department, Faculty of Science, King Saud University, and specimen were kept in the herbarium of Chemistry Department.

(4) Samples of the aerial parts were air dried in shade, reduced to fine powder and kept for phytochemical and biological investigation.

(5) Apparatus:

(6) Melting points were determined on a Kofler hot-stage apparatus and are uncorrected.

(7) Mass spectra (Electrospray negative ion) were taken from samples dissolved in acetonitrile with a Micromass Quattro spectrometer.

(8) .sup.1H- and .sup.13C-NMR spectra, using external electronic referencing through the deuterium resonance frequency of the solvent, were determined at 600.17 or 150.91 MHz respectively with a JEOL ECA600 NMR spectrometer fitted with an auto-tune 5 mm X/H probe. Carbon atom types were established in the .sup.13CNMR spectrum by employing a combination of broad-band proton-decoupled and DEPT (90 and 135) experiments. [.sup.1J.sub.C—H], [.sup.2J.sub.C—H] and [.sup.3J.sub.C—H] .sup.1H-.sup.13C correlations were determined by using HMQC and HMBC pulse sequences. .sup.1H-.sup.1H correlations were determined by double quantum filtered COSY.

(9) Pye Unicam pu 8800 spectrophotometer was used for UV spectral analysis.

(10) Amino acid analysis was carried out using the amino acid analyzer (Eppendorf-LC 3000).

(11) IR spectra were taken with a Shimadzu—IR-435 infrared spectrophotometer.

(12) Phytockemical Screening:

(13) Powdered samples of the aerial parts of E. granulata were subjected to preliminary phytochemical screening according to the published methods (Awaad, 2009).

Example 1

(14) Extraction and Isolation:

(15) Extraction of Plant Material:

(16) Air-dried powder (1 kg) of Cakile arabica (aerial parts) was extracted by percolation in ethanol 95 for two days and filtered off (this process was repeated for three times). The combined alcohol portions were concentrated under reduced pressure at a temperature not exceeding 25° C. to yield a dry extract of 170 g.

(17) Isolation:

(18) An amount of 10 g dry TAE was dissolved in methanol and mixed with alumina for column chromatography. The solvent was evaporated on steam bath with continuous stirring to form a free flowing powder. The powder was then applied on the top of glass column (150×2.5 cm) packed with alumina (300 g). Elution was performed using a stop-gradient system with benzene-ethyl acetate. 100 fractions were collected (50 ml each) and reduced to three sub-groups after chromatographic examination using solvent system-a: benzene-ethyl acetate (86:14).

(19) Sub-fraction I (2.50 g) contained two spots with R.sub.f-values=0.66 and 0.60 (system a, TLC). It was fractionated on an alumina column (120 g, 1×90 cm) for separation of these two compounds that eluted with benzene-ethyl acetate 9:1 v/v. 20 fractions were collected and concentrated using rotary evaporator to obtain one compound which re-purified by re-crystallization from methanol to get compound 1.

(20) Sub-fraction II (3.5 g) contained one spot with R.sub.f-value=0.51 (system a, TLC). The solvent was evaporated using high pressure at low temperature to obtain 2.

Tetracosanoic acid (1)

(21) It was isolated as white amorphous powder (220 mg); mp 78-79° C.; R.sub.f-value=0.56 (system: benzene-ethyl acetate, 90:10 v/v). IR ν.sub.max (KBr, cm.sup.−1) 3415, 2918, 2849, 1709, 1463, 720; .sup.1H NMR (500 MHz, CDCl.sub.3): δ ppm 2.32 (2H, t, J=7 Hz, H-2), 1.61 (2H, pentet-like, J=7 Hz, H-3), 1.23 (40H, br s, H-4 to H-23), 0.86 (3H, t, J=6.5 Hz, H-24); .sup.13C NMR (125 MHz, CDCl.sub.3): δ ppm 177.0 (C-1), 34.0 (C-2), 31.9 (C-3), 29.7 (C-4-16), 29.6 (C-17), 29.5 (C-18), 29.4 (C-19), 29.3 (C-20), 29.1 (C-21), 24.7 (C-22), 22.7 (C-23), 14.2 (C-24).

(22) The structure of 1 was established as tetracosanoic acid according to its chemical and physicochemical data (IR, .sup.1H and .sup.13C NMR) given above and confirmed by DEPT and H,H-COSY, HSQC and HMBC 2D-NMR correlation experiments.

4-Hydroxy-5-n-butoxy-5-oxopentanoic acid (2)

(23) It was isolated as white crystals (320 mg); R.sub.f-value=0.45 (system: chloroform-methanol 90:10 v/v, TLC); bp 99-100° C.; IR ν.sub.max (KBr, cm.sup.−1): 3415, 2918, 2849, 1709, 1463, 720; .sup.1H NMR (500 MHz, CDCl.sub.3): δ ppm 4.21 (1H, dd, J=7.8, 5 Hz, H-4), 4.10 (1H, t, J=6.25, 5 Hz, H-1′), 2.45 (1H, m, H-3), 2.31 (1H, m, H-2), 2.16 (1H, m, H-3′), 1.58 (2H, pentet-like, J=7.1 Hz, H-2′), 1.33 (2H, sixtet-like, J=7.3 Hz, H-3′), 0.88 (3H, t, J=7.3 Hz, H-4′); .sup.13C NMR (125 MHz, CDCl.sub.3): δ ppm 178.5 (C-1), 172.3 (C-5), 65.3 (C-1′), 55.6 (C-4), 30.5 (C-2′), 29.3 (C-2), 24.8 (C-3), 19.0 (C-3′), 13.6 (C-4′). The structure of 2 was established by chemical and physicochemical data (IR, .sup.1H and .sup.13C NMR) given above and confirmed by DEPT and H,H-COSY, HSQC and HMBC 2D-NMR correlation experiments.

(24) The isolated compounds were identified using different physical and spectral methods; melting point, UV and IR spectra, .sup.1H-NMR, .sup.13C-NMR, DEPT and correlation 2D NMR as 1 and 2.

(25) ##STR00001##

Example 2

(26) Pharmacological Study

(27) Animals:

(28) Swiss albino mice of both sex (26-30 g) and male Wistar rats (180-200 g) were supplied by the animal house of King Saud University, KSA. Animals were housed in standard polypropylene cages with wire mesh top and maintained under standard conditions (temperature 23±1.0° C., humidity 55±10%, 12 h light/12 h dark cycle). They fed with a standard pellet diet with water ad libitum and were allowed to adapt to the laboratory environment for one week before experimentation.

(29) Pharmacological Activities

(30) 1. Preparation of the TAE

(31) Dried aerial parts of Cakile arabica (100 g) were extracted by percolation in 90% ethanol at room temperature for two days. The ethanol extract was filtered and the residues were re-percolated for four times. The total ethanol extract was concentrated under reduced pressure at a temperature not exceeding 35° C. to yield a dry extract of 25 g. The dried plant extract was freshly suspended in distilled water just before administration by the aid of Tween 80.

(32) 2. Determination of Median Lethal Dose (LD.sub.50).

(33) The oral median lethal dose (LD.sub.50) of the TAE determined as described by Lorke Arch. Toxicology, 1983, 54, 251-287. Swiss albino mice in groups of six, received one of 500, 1000, 2000, or 4000 mg/kg doses of the tested TAE. Control animals were received the vehicle and kept under the same conditions. Signs of acute toxicity and number of deaths per dose within 24 h were recorded.

(34) 3. Anti-Ulcerogenic Activity:

(35) Evaluation of the anti-ulcerogenic activity was carried out using absolute ethanol-induced ulcer model as described by Muthu et al. Int. J. Pharm. Pharm. Sci. 2013, 5, Suppl1, 269-272.

(36) About 30 Wistar rats were used, they divided into 5 groups each of 6 rats. Group 1 received the vehicle and served as control group, group 2 received ranitidine (100 mg/kg) and served as standard group, groups 3 received the total alcohol extract of the plant under investigation at a dose of 1000 mg/kg. groups 4 and 5 received the isolated compounds 1 and 2 at dose 50 mg/kg respectively.

(37) Rats of all groups were fasted for 24 h then all medications were administered orally. One hour after treatment, the animals received an oral dose of absolute ethanol (1 ml/200 g) and then sacrificed one hour later, by ether inhalation, the stomachs were rapidly removed, opened along their greater curvature and gently rinsed under running tap water.

(38) Number of lesions in the glandular part of the stomach were measured under an illuminated magnifying microscope (10×). Long lesions were counted and their lengths were measured. Petechial lesions were counted, and then each five petechial lesions were taken as 1 mm of ulcer.

(39) The lesion scores: the mucosal lesions were quantified by the scoring system (0-5) 0=no damage, 1=Local edema and inflammation without ulcers; 2=One ulcer without inflammation; 3=one to two ulcers with inflammation & lesion diameter <1 cm; 4=More than two ulcers with lesion diameter 1-2 cm; 5=Sever ulceration with lesion diameter >2 cm.

(40) Ulcer index To calculate the ulcer index (mm), the sum of the total length of long ulcers and petechial lesions in each group of rats was divided by its number. The curative ratio was determined according to the formula:
% Protection of control ulcer=Control UI−Test UI/Control UI×100

(41) 4. Effect on Liver and Kidney Functions:

(42) Male Wister rats were divided into 2 equal groups each of 10 rats. The 1.sup.st group was left as a control and administrated water orally, while the 2.sup.nd group was orally given the TAE in a dose of 1000 mg/kg for 15 days. Blood samples were collected from the orbital plexus of rats, 6 hr after the last dose. Samples were left to clot at room temperature for 30 min then centrifuged at 1000 rpm for 20 min.

(43) The collected sera were used for determination of the activity of both (AST) aspirate aminotransferase and (ALT) alanine aminotransferase as a liver markers. In addition, levels of blood urea, serum creatinine were also estimated as a kidney markers (Awaad et al., Phytother. Res. 2013, 27, 126-130).

(44) Biological Activities

(45) 1. Determination of Median Lethal Dose (LD.sub.50).

(46) The total alcohol extract Cakile arabica did not produce any behavioral changes or mortality in treated mice in doses up to 4000 mg/kg. Therefore, the tested plant can be categorized as highly safe since substances possessing LD.sub.50 higher than 50 mg/kg are non-toxic (Soliman, Pharmaceutical Biology, 2012, 50(1) 105-112).

(47) 2. Anti-Ulcerogenic Activity.

(48) The present results showed that the TAE at dose levels of 500 and 1000 mg/kg possessed a potent anti-ulcerogenic activity against ulcer-induced by absolute alcohol. It produced a percent protection of control ulcer by 44.6% and 98.5% respectively. The isolated compounds 1 and 2 (50 mg/kg), showed significant anti-ulcerogenic activity they produced percent protection of control ulcer by 80 & 55.3% respectively which are more effective than ranitidine which produce 44.6%. 1 was the most effective compound in the present study, it produced percent protection of control ulcer by 80% and it was mainly responsible for the activity of the investigated plant. (Table 1).

(49) TABLE-US-00001 TABLE 1 Antiulcerogenic effect of TAE and isolates 1 and 2 from Cakile arabica Dose No. of % pro- Groups mg/kg score ulcers ulcer index tection Control 4 14 ± 2.35 .sup.  13 ± 2.24 .sup.  Ranitidine 100 2.2 7.2 ± 0.84***  7 ± 1.58** 46.2 TAE-1000 1000 0.2 0.2 ± 0.45*** 0.2 ± 0.45*** 98.5 1 50 1 .sup. 2 ± 2.74*** 2.6 ± 3.58*** 80 2 50 2  3.6± 2.97*** 5.8 ± 3.42**  55.3 Data are expressed as mean ± SD, n = 6, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

(50) 3. Liver and Kidney Functions:

(51) Both liver and kidney functions were not affected after treatment of TAE (1000 mg/kg) for 2 weeks, as there is no significant difference between control and test group in all experiments, at the 0.05 level of probability (Table 2).

(52) TABLE-US-00002 TABLE 2 Effect of TAE on liver and kidney functions of rats. Serum Blood Urea Creatinine Groups ALT(U/l) AST(U/l) (mg/dl) (mg/dl) Control 4.8 ± 0.37 5.2 ± 0.37 45.50 ± 1.36 0.82 ± 0.02 TAE-1000 5.1 ± 0.22 5.3 ± 0.39 46.00 ± 1.9  0.84 ± 0.02 Data are expressed as mean ± SD, n = 10

(53) These results showed that, the alcohol extract of the investigated plant didn't reveal hepatotoxic manifestation. In addition, no apparent nephrotoxic manifestations were recorded; this indicated that no side effects were obtained the alcohol extract of Cakile Arabica.

(54) The features disclosed in the foregoing description and claims may both separately and in any combination be material for realizing the invention in diverse forms thereof.