The ricinoleate triglyceride was extracted from castor-oil seeds grown in Algeria and isolated by catalytically methanolyse to methyl ricinoleate. Six diazole and triazole derivatives of ricinoleic acid were synthesized and characterized: 1,3,4-oxadiazole-5-thione (4); 1,3,4-thiadiazole-5-thione (5); 4-N-amino-1,2,4-triazole-5-thiol (7); 1,2,4-triazole-5-thione (9); 5-amino-1,3,4-oxadiazole (10) and 5-amino-1,3,4-thiadiazole (11). The antibacterial and antifungal screening data of synthesized compounds showed appreciable inhibition and among them, 5, 7 and 8 showed more inhibition on Gram positive
Castor oil is a natural production of the castor plant (
The high percentage of ricinoleic acid residues in castor oil and their derivatives inhibit virus, bacteria or fungi (Ghosh
Heterocyclic-fatty acid hybrids such as oxadiazole, thiadiazole and triazole derivatives of vegetable oils are a new class of fatty acid derivatives with a wide range of biological activities and significance in the field of medicinal chemistry. They possess a broad spectrum of therapeutic uses such as analgesic, antimicrobial, anti HIV activity, antitumor, antimalarial, anticancer, anticonvulsant, anti-diabetic, antioxidant (Cao
This work is mainly concerned with the extraction of castor oil from seeds of the plant and the isolation of ricinoleic acid. The latter was subjected to synthetic modifications focused on the carboxylic group to ultimately give six diazole derivatives. The synthetic intermediates and final products were studied to determine the biological evaluation of their α-amylase inhibitory, antimicrobial and antioxidant activities.
All reactions were monitored by TLC, silica gel F254, made by Merck, Germany. Melting points (ºC) were measured in open glass capillaries using a Branstead 9001 Electrothermal melting point apparatus and were not corrected. The UV visible electron spectroscopy was recorded on an Optisen View 4.2 spectrometer. The IR spectra were recorded using KBr disks in a GENESISIIFTIR spectrophotometer, in
The castor oil (1) was extracted with a soxhlet extractor. Hexane (1L) and ground castor beans (200 g) were packed into a filter paper thimble. The extraction lasted 10 h. The crude oil was treated with hot water to remove gums, hydrates, phosphates and other impurities and then it was neutralized with 0.1N NaOH to remove free fatty acid and soap to give castor oil (1), (84 g, 40.2%) (Nakarmi
The extracted oil (1) (25g; 0.028 mol) was kept in three necked round-bottom flasks and heated to 65 ˚C. NaOH /MeOH (2,5g /20 mL) were added with the aid of stirring and the mixture was heated for three hours (Akhabue et al.,
Methyl ricinoleate (2), (2.11 g; 0.01mol), ethanol (50 mL) and hydrazine hydrate 64% (12 mL) were refluxed for 10 h. Ethanol and hydrazine were evaporated under reduced pressure, white solid was produced, recrystallized from acetone/cyclohexane to give ricinoleic hydrazide (3), 2.2g, 90%). M.p. (80–85 °C); UV (λmax) nm: 205; 262. IR (KBr), ν cm -1: 3323. 71 (OH, NH, NH2); 3008 (HC = C); 2849.31 and 2918.73 (alkyl groups); 1636. 3 (N-C = O). 1H NMR (400 MHZ, DMSO d6)
Ricinoleic acid hydrazide (3), (1g; 0.0032 mol) in ethanol (80 mL) was added to CS2 (30 mL) followed by (1.2 g) KOH, and the mixture was refluxed for 16 h until the release of H2S had ceased. The mixture was then cooled and acidified with dilute HCl. The crude compound was recrystallized from chloroform to give 5-Ricinoleyl-1,3,4-oxadiazole-2-thione-thiol (4), (1.36 g, 70%), Rf: 0.64 (cyclohexane /acetone 7/3); m.p. (117–120), UV (λmax) nm: 205; 252. IR (KBr), ν cm -1: 3418. 21 (OH); 3222. 47 (NH); (2957, 2868) (alkyl groups); 3046 (CH unsaturated); 2800 (S-H); 1594(C = N); 1423 (C = S); 1109 (COC). 1H NMR (400 MHZ, DMSO d6) σ (ppm): 14.38(s, 1H, SH); 5.75 (m, 1H, H10); 5.85 (m, 1H, H9); 3.42(s, 1H, OH); 3.33(s, 1H, OH); 2.69-1.21 (m, 32H, CH paraffinic); 0.84 (t, 3H, CH3 terminal). 13C NMR (400 MHZ, DMSO d6)
The ricinoleic acid hydrazide (3), (1 g, 0.0032 mole) was mixed with KOH (0.5 g) dissolved in absolute ethanol. A solution of CS2 (2.5 g) was added to the mixture with 15 mL of ethanol. The mixture was stirred for 1 h at room temperature and then refluxed for 24 hours. The final product was acidified with hydrochloric acid. The resulting solid residue was filtered and recrystallized from ethanol. The product was obtained in the form of yellowish crystals (5), (1.45g, 75%); RF: 0.35 (cyclohexane /acetone 6/4); m.p. 97 °C. UV (λmax) nm: 205; 285. IR (CCl4), ν cm-1: 3308.29(OH,NH); 2980.45(C = C); (2921.63,2849.31) (alkyl groups); 1589.06(C = N); 1462.74(C = S). 1H NMR (400 MHZ, DMSO d6) δ (ppm): 5.42 (s, 1H, NH); 5.33 (m, 1H, H10); 4. 75 (m, 1H, H9); 3.33 (s, 1H, OH); 3.32 (s, 1H, OH); 2.24-1.16 (m, 32H, CH paraffinic); 0.84 (t, 3H, CH3 terminal). 13C NMR (400 MHZ, DMSO d6) δ ppm: 174.9; 173.3; 130.8; 128.9; 70.0; 64.4; 60.3; 37.9; 34.3; 31.9; 29.5; 25.7; 24.9; 22.5; 14.5. MS: Molecular formula C19H34N2OS2: 364 m/z: 364 (M+, 100%).
Ricinoleic acid hydrazide (3), (1g; 0.0032 mol) and KOH (1.5 g) in absolute ethanol (10 mL) were mixed together until the solution became clear. CS2 (25 mL) was added. The solution was stirred for 10 h at room temperature, and then diethyl ether (20 mL) was added to form a precipitate ricinoyl-potassium thiocarbazinic acid (6). The intermediary product was mixed with (NH2NH2-H2O) (4 mL). The solution was refluxed for 15 h until the color of the solution became clear green. After cooling to room temperature, ice water (10 mL) was added to the reaction mixture, which was then neutralized with 3N HCl to form a precipitate. The precipitate was isolated by filtration and purified by recrystallization from ethanol/water to afford the desired product in the form of yellow crystal: 5-Ricinoleyl-4-amino-1, 2, 4-triazole-3-thiol (7), 1.2g, 65%); Rf: 0.61 (cyclohexane /acetone: 6/4); Mp. (132˚C) UV (λ max) nm: 205; 240. IR (KBr), ν cm -1: 3417.24 (OH); 3177 (NH2); 2953, 2868 (alkyl groups); 2700 (SH); 1617 (C = N). 1H NMR (400 MHZ, DMSO d6)
Ricinoleic acid hydrazide (3), 1g; 0.0032 mol) was dissolved in ethanol (20 mL) with stirring. Ammonium thiocyanate (0.58 g) and HCl (30%) were added, and the reaction mixture was refluxed for 6 h. Excess solvent was evaporated to almost dryness and recrystallized from methanol/petroleum ether to give N-Thiosemicarbazide ricinoleic (8), 0.97g, 65%) ; Rf : 0,45 (cyclohexane /acetone: 7/3) ; Mp (97 °C) ; UV (λmax) nm: 210; 265. IR (KBr) ν cm -1: 3373.85 (OH); (3271.64, 3172.33, 3106.76) (NH and NH2); (2851.24 and 2919.70) (alkyl groups); 1699 (C = O); 1621 (C = O-N); 1492 (C = S). 1H NMR (400 MHZ, DMSO d6) σ (ppm): 8.87 (s, 1H, NH); 8.44 (s, 1H, NH); 4.32 (d,2H, NH2); 5.36 (m, 1H, H10 and H9); 3.33 (s, 1H, OH); 2.68-1.21 (m, 32H, CH paraffinic); 0.84 (t, 3H, CH3 terminal). 13C NMR (400 MHZ, DMSO d6) δ ppm: 183.0; 178.4; 166.9; 164.7; 131.7; 127.5; 70.4; 69.9; 37.6; 29.3; 29.3; 28.6; 25.5; 25.4; 25.2; 14.5. MS: Molecular formula C19H37N3O2S: 371, m/z: 371 (M+, 100 %).
N-Thiosemicarbazide ricinoleic (8), (1.0 g; 0.0024 mol) in ethanol (15 mL) was added to an alcoholic solution of 10% NaOH (20 mL), and the reaction mixture was refluxed for 12 h. The mixture was cooled and acidified with dilute HCl to pH (5–6). The crude compound was recrystallized from ethanol to give white needle-like crystals: 5-Ricinoleyl-4H-1,2,4-triazole-3-thiol (9), (0.75 g, 75%) ; m.p. (95 °C) ; Rf: 0.46 (cyclohexane /acetone: 6/4) ; UV (λmax) nm: 210. IR (KBr) ν cm-1: 3433 (OH, NH); 3100(C = C); 2953 and 2868 (alkyl groups); 2722.05 (SH); 1636. 3 (C = N). 1H NMR (400 MHZ, DMSO d6)
N-Thiosemicarbazide ricinoleic (8); (1.0 g, 0.0026 mol) was dissolved in an alcoholic solution of NaOH (5N) with the aid of stirring. The mixture was refluxed at 80 °C, during which an aqueous iodine solution (KI / I2) was added gradually until the iodine color (pink-purple) persisted. Reflux was continued for 15h. Once the solution had cooled, it was filtered and then treated with a dilute sodium thiosulfate solution and finally washed with distilled water to give a straw-yellow solid which was recrystallized from ethanol to give 5-Ricinoleyl-2-amino-1,3,4-oxadiazole (10), (0.78g, 78%); m.p (128 °C); Rf : 0.56 (cyclohexane /acetone : 6/4) ; UV (λmax) nm: 210; 275. IR (KBr), ν cm-1: 3389 (OH, NH2); 2921.63 and 2850.27 (alkyl groups); 165.45 (O-C = N); 1119 ( = C-O-C = ). 1H NMR (400 MHZ, DMSO d6)
N-Thiosemicarbazide ricinoleic (
All the synthesized compounds were tested for their in vitro antimicrobial activity against the Gram positive bacteria
The DPPH solution was prepared in advance by dissolving 4 mg of DPPH in 100 mL of absolute methanol. 0.1 mL of each sample at different concentrations (65.5; 125; 250; 500; 1000) μg/mL were added to 3.9 mL of DPPH. Reference antioxidant solutions (ascorbic acid) were also prepared under the same conditions to serve as a positive control. The negative control consisted only of DPPH and methanol. The mixture was left in the dark for 30 min until discoloration. The presence of the DPPH radicals gave a dark purple color to the solution and which was absorbed rapidly at 517 nm when reduced. The color became pale yellow. During the reaction, the layer of this radical became saturated on contact with an antioxidant, which explained the disappearance of its coloring. This discoloration highlighted the trapping power of the free radical by the tested product. The percentage of the anti-free radical activity was estimated according to the equation below (Vijayalaxmi
PI % = (Abs control - Abs product /Abs control) *100
PI: Percentage inhibition Abs control: Absorbance at the 517 nm wavelength of the negative control (DPPH + methanol).
The α-amylase inhibitory activity of the synthetic compounds was determined using the chromogenic DNSA method with a few modifications (Adegboye
Inhibition % = 1- (Asamp / Acont) 100%).
Where Asamp and Acont were defined as absorbance of the sample and the control, respectively.
The ricinoleiate triglyceride (1) was catalytically transesterified with methanol to give methyl ricinoleiate (2) in quantitative yield (Kumar
Conversion of ricinoleic triglyceride (1) to ricinoleic hydrazide (3).
Synthetic pathways to diazole and triazole derivatives (
The second set of heterocycles (9-1)1 was prepared from N-thiosemicarbazide ricinoleic (8), which had already been prepared by treating hydrazide (3) with ammonium thiocyanide. 5-Ricinoleyl-4H-1,2,4-triazole-3-thiol (9) was obtained by the cyclization of 8 with KOH (Belkhadem et al.,
Characteristic signals in H1NMR of heterocyclic derivatives: 5-Ricinoleyl-1,3,4-oxadiazole-2-thione-thiol (4) ¸ 5-Ricinoleyl-1,3,4-thiadiazole-2-thione (5)¸ 5-Ricinoleyl-4-amino-1, 2, 4-triazole-3-thiol (7)¸ 5-Ricinoleyl-4H-1,2,4-triazole-3-thiol (9)¸ 5-Ricinoleyl-2-amino-1,3,4-oxadiazole (10)¸ 5-Ricinoleyl-2-amino-1,3,4-thiadiazole (11).
The results for antimicrobial activities are summarized in
Antibacterial activity of castor oil (1) and its synthesized derivatives (2-11).
Compound | Gram-positive bacteria | Gram-negative bacteria | ||||||
---|---|---|---|---|---|---|---|---|
S.a. | E.f. | B.c. | P.a. | E.c. | K.P. | Sal. | P.v. | |
Zone of inhibition in mm and MIC (minimum inhibitory concentration) in μg/mL | ||||||||
- | - | 7 (100) | 7 (25) | 7 (25) | 8 (50) | 10 (6.5) | 10 (25) | |
- | - | 7 (50) | 7 (25) | 8 (25) | 7 (50) | 12 (25) | 10 (25) | |
- | - | - | - | 10 (25) | 10 (25) | 15 (25) | - | |
14 (6.25) | 7 (50) | - | 7 (100) | 10 (12.5) | 10 (25) | 15 (12.5) | 8 (25) | |
10 (6.25) | 12 (25) | - | - | 8 (25) | 10 (25) | 10 (6.25) | 8 (25) | |
7 (25) | 9 (50) | 7 (25) | 7 (25) | - | 10 (25) | 10 (25) | - | |
25 (100) | 7 (100) | 7 (50) | 8 (25) | 10 (6.25) | 8 (50) | 10 (6.25) | 10 (100) | |
7 (25) | 7 (50) | - | 7 (50) | 7 (25) | 10 (25) | 7 (50) | 8 (25) | |
7 (50) | 7 (12.5) | 7 (50) | 7 (50) | - | 8 (25) | 10 (25) | - | |
8 (50) | 8 (50) | 7 (100) | 7 (100) | - | 10 (25) | 10 (25) | - | |
25 | 8 | 32 | 15 | 20 | 32 | 18 | 22 |
The figures in the table show the zone of inhibition (mm) and the corresponding MIC (μg/mL) values in brackets.
Amp: Ampicillin (10 μg/ disc).
Antifungal activity of castor oil (1) and its synthesized derivatives (2-11) at 100 μg/mL.
Compound | Fungi | |||||
---|---|---|---|---|---|---|
Mold | Yeast | |||||
Candida albicans | Trichosporon Sp | Aspergillus niger | Fusarium | Penicillium Sp | Altenaria | |
- | - | +++ | - | - | +++ | |
- | - | ++ | - | - | +++ | |
++ | ++ | - | + | +++ | - | |
++ | ++ | - | - | - | + | |
++ | ++ | - | - | - | + | |
+++ | - | - | - | - | ++ | |
- | +++ | - | +++ | +++ | - | |
- | - | - | - | - | + | |
++ | +++ | - | +++ | - | +++ | |
- | ++ | - | +++ | - | +++ | |
+++ | ++ | +++ | + | +++ | +++ |
Key to the inhibition zones activities: Highly active = (21-30 mm) +++; Moderately active = (16-20 mm) ++; Slightly active = (10-15 mm) +; Inactive = (˂ 10mm) -
Ref: amphotericin B (100 μg/disc.).
The gram-positive bacteria under consideration showed zones of inhibition inferior to those observed by the Gram-negative bacteria. In general, the first site of action of the products tested on bacterial cells was the plasma membrane. This was directly related to the amphiphilic nature of the tested products which facilitated their insertion between the membrane phospholipids and ensured their solubilization in the lipid bilayer (Soliman
From a methodological point of view, the free radical test, 2,2-diphenyl-1- picrylhydrazyl radical (DPPH) is recommended for compounds containing the SH, NH and OH (Barbuceanu
DPPH scavenging effect (%) of castor oil and its synthesized derivatives at different concentrations of: (A) Intermediate compounds: Castor oil (1) ¸ Methyl ricinoleate (2) ¸ Ricinoleic acid hydrazide (3) ¸ N-Thiosemicarbazide ricinoleic (8) and ascorbic acid as reference. (B) Heterocyclic synthetic products: 5-Ricinoleyl-1,3,4-oxadiazole-2-thione-thiol (4)¸ 5-Ricinoleyl-1,3,4-thiadiazole-2-thione (5)¸ 5-Ricinoleyl-4-amino-1, 2, 4-triazole-3-thiol (7)¸ 5-Ricinoleyl-4H-1,2,4-triazole-3-thiol (9)¸ 5-Ricinoleyl-2-amino-1,3,4-oxadiazole (10)¸ 5-Ricinoleyl-2-amino-1,3,4-thiadiazole (11) and Ascorbic acid as reference. Each point shows the average value of three replicates ± SD.
IC50 (Inhibitory concentration 50), also referred to as EC50 (Efficient Concentration 50), is the concentration of the test sample needed to reduce 50% of the DPPH radical. The IC50 are calculated graphically by percent inhibition as a function of different concentrations of the tested product. From the value shown in
IC50 values for castor oil 1 and its synthesized derivatives (2-11) with references of DPPH scavenging and α amylase inhibitory activity.
Compound | DPPH Scavenging Activity | Antiradical Power | α-amylase inhibitory activity |
---|---|---|---|
(IC50 (μg/mL) | (1/ IC50) | (IC50 (μg/mL)) | |
32,16 ± 0.58 | 0,031 | 130.42 |
|
44,45 ± 0.45 | 0,022 | 120.25 ± 1.17 | |
ND | ND | 400.64 ± 2.75 | |
56,74 ± 0.32 | 0,019 | 325.90 ± 2.75 | |
58,24 ± 0.30 | 0,020 | 552.80 ± 3.15 | |
50,15 ± 0.41 | 0,018 | 736.00 ± 5.66 | |
ND | ND | 125.00 ± 1.75 | |
57,49 ± 0.35 | 0,017 | 920.56 ± 8.15 | |
48,46 ± 0.28 | 0,001 | ND | |
52,48 ± 0.38 | 0,001 | ND | |
33,67 ± 0.45 | 0,030 | - | |
- | - | 85.65 ± 1.09 |
Each point shows the average value of three replicates
DPPH (2, 2-diphenyl-1- picrylhydrazyl radical); IC50 (Inhibitory concentration 50).
The digestive enzyme (α-amylase) was responsible for hydrolyzing dietary starch (maltose), which broke down into glucose prior to absorption. The inhibition of α-amylase led to a reduction in post prandial hyperglycemia under diabetic conditions (Yilmazer-Musa
Percentage of α- amylase inhibition versus different concentration of: (C) Intermediate products: Castor oil (1) ¸ Methyl ricinoleate (2), Ricinoleic acid hydrazide (3) ¸ N-Thiosemicarbazide ricinoleic (8) and Acarbose as reference. (D) Heterocyclic synthetic products: 5-Ricinoleyl-1,3,4-oxadiazole-2-thione-thiol (4) ¸ 5-Ricinoleyl-1,3,4-thiadiazole-2-thione (5)¸ 5-Ricinoleyl-4-amino-1, 2, 4-triazole-3-thiol (7)¸ 5-Ricinoleyl-4H-1,2,4-triazole-3-thiol (9)¸ 5-Ricinoleyl-2-amino-1,3,4-oxadiazole (10)¸ 5-Ricinoleyl-2-amino-1,3,4-thiadiazole (11) and Acarbose as reference. Each point shows the average value of three replicates ± SD.
Group B, comprised of compounds (3) and (4), which showed inhibition at moderately higher concentrations (400.64 ± 2.75, 325.90 ± 2.75) μg/mL. Group C consisted of compounds (5), (7), and (9) at corresponded to the highest concentrations (552.80 ± 3.15, 736.00 ± 5.66, 920.56 ± 8.15) μg/mL. Among the synthetic compounds (4), (5), (7), (9), (10) and (11), no significant inhibitory effect was detected for the heterocycles (10) and (11). Oxadiazole 4 showed the best α-amylase inhibition.
The oil extract (1) (ricinoleate triglyceride) reacted over 40% from original castor oil and was closest to highest percentage reported in the literature (45%) (Nakarmi
The authors declare no conflict of interest, financial or otherwise.