Abstract
GC-MS analysis of isolated alkaloids from aerial parts of Leptadenia pyrotechnica (Forsk.) Decne (Asclepiadaceae) was performed. Twenty-four alkaloids and six simple amines were detected for the first time in this plant. Almost all of the alkaloids belonged to pyridine, pyrrole, pyrazine, and indole types. The acute LC50 of the total alkaloids and alcohol extracts estimated by means of brine shrimp toxicity test were 63.09 and 11.89 ppm, respectively. The antitumor activities of these extracts, using potato disc screen, showed good activity represented by −33.6% and −49.3%, respectively.
Introduction
Plants belonging to Asclepiadaceae are frequently used in traditional medicine and have been reported to be rich in steroidal glycosides, cardenolides, flavonoids, triterpenes and polyoxypregnane derivatives (CitationBazzaz et al., 2003; CitationPaulo & Houghton, 2003; CitationAtta & Mouneir, 2005; CitationCioffi et al., 2006; CitationKhanna & Kannabiran, 2007). The plants of this family are known to contain cytotoxic and tumoricidal C/D-cis-polyoxypregnane esters and glycosides. Leptadenia pyrotechnica (Forsk.) Decne (Asclepiadaceae) is a wild plant occurring in Sharm El-Sheikh region, southern Sinai, Egypt. This plant is used as an antispasmodic, anti-inflammatory, antihistaminic, antibacterial, diuretic, expectorant, and to expel uroliths (CitationCioffi et al., 2006; CitationAquino et al., 1996; CitationPanwara & Tarafdarb, 2006).
About 70 species of Asclepiadaceae plants are reported to contain alkaloids. Indole alkaloids, phenanthroindolizidine, pyridine, simple amine groups, tylophorine and naphthoindolizidine derivatives have been detected from this family. The two later compounds have been targets of synthetic modification because of their profound cytotoxic antitumor activity (CitationLee et al., 1989; CitationGao et al., 2007; CitationMichael, 2004, Citation2008).
Among the various techniques used for the rapid identification of alkaloids, gas chromatography coupled with mass spectrometry (GC-MS) is very efficient because it allows the simultaneous separation and identification of trace alkaloids in a complex mixture (CitationPhilipov & Berkov, 2002; CitationZocoler et al., 2005).
To our knowledge, little data regarding the alkaloids in the genus Leptadenia are found in the current literature (CitationEl-Hassan et al., 2003; CitationVaghasiya & Chanda, 2007; CitationLavhale & Mishra, 2007; CitationParabia et al., 2007). Therefore, this paper deals with the isolation and identification of alkaloids in L. pyrotechnica and also evaluates their antitumor activity and toxicity using potato disc assay and the brine shrimp method.
Materials and methods
Plant material
Fresh aerial parts of L. pyrotechnica were collected in September, during the flowering stage, from Wadi Khashab and Wadi Matzos, Sharm El-Sheikh to El-Tur road, southern Sinai, Egypt. The identity was established by Samia Heneidak, Department of Botany, Faculty of Science, Suez Canal University. A voucher specimen (Number AMYM-1004) has been deposited in the herbarium of the Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.
General methods
Total alkaloids were investigated by GC-MS on a Hewlett Packard gas chromatograph 5890 Series II Plus linked to a Hewlett Packard 5972 mass spectrometer system equipped with a 30 m length, 0.25 mm ID, 0.25 μm film thickness HP5-MS capillary column, coated with cross-linked 5% phenyl substituted methyl polysiloxane. Ionization energy was 70 eV, ionization current 300 μA, detector voltage 1000 V. Helium was used as a carrier gas with a constant flow rate at 1 mL min−1, and linear velocity 36.2 cm sec−1. The injection mode was splitless. The injection and detector temperatures were adjusted at 200°C and 280°C respectively. The column temperature was programmed to rise from 50°C to 220°C at 5°C min−1, from 220°C to 260°C at 20°C min−1 and isothermal at 260°C for 5min. The identification of the chemical constituents was based on comparisons of their relative retention times and mass spectra with those obtained from authentic samples and/or the National Bureau of Standards (NBS) and Wiley Library of Spectra (CitationMassada, 1976) as well as by comparison of their retention indices with literature data (CitationAromdee & Sriubolmas, 2006; CitationViña & Murillo, 2003; CitationBisio et al., 1998).
The plant samples were dried by freeze-dryer (Labconco Company, USA) under 133 × 10−3 Mbar vacuum at -50°C for 24 h. The alkaloid constituents were analyzed by thin layer chromatography (TLC) plates (0.2 mm thickness, silica gel 60 F254, Sigma-Aldrich and Merck aluminum oxide G plates). Also, silica gel G plates impregnated with sodium hydroxide were used. The elution system was performed with (S1) chloroform-acetone-diethylamine (85:15:1), silica gel, (S2) benzene-ethyl acetate-diethylamine (70:20:10), silica gel, (S3) toluene-dichloromethane (9:1), silica gel, (S4) ethyl acetate-n-hexane (80:20) + 3 drops of ammonia, alumina, (S5) chloroform-acetone (85:15), alumina, (S6) benzene-methanol (90:10) + 3 drops of ammonia, alumina and (S7) n-butanol-acetic acid-water (80:3:17), cellulose. The plates were developed separately under ultraviolet light (254; 366 nm) and with Dragendorff’s and iodoplatinic acid reagents. The detection sensitivity could be increased appreciably by subsequent spraying with 10% sulfuric acid (CitationMacek, 1972; CitationBalbaa et al., 1976; CitationZocoler et al., 2005).
Vacuum liquid chromatography (VLC) was performed using 42.5 mm ID centered glass Pyrex Buckner filled with silica gel or alumina covered with celite.
Extraction of alkaloids
The phytochemical screening was performed in accordance with CitationAOAC (Association of Official Agricultural Chemists) (1990). About 1 kg of the aerial parts of L. pyrotechnica was shock-frozen with liquid nitrogen, lyophilized, pulverized, and then percolated with methanol. The residue, 445 g (44.5%) remaining after concentration to dryness under reduced pressure, was defatted with petroleum ether (40°-60°C) to give 400 g (40%), then acidified with 1M hydrochloric acid, filtered, and the acid aqueous solution was then basified with concentrated ammonium hydroxide and extracted with dichloromethane. The organic phase was filtered, dried with anhydrous sodium sulfate, filtered again and finally concentrated in vacuo. The acid-base purification procedure was repeated three times to give 5.68 g of a yellowish brown semi-solid alkaloid extract, which represented 0.57% from total powdered dry plant.
TLC of the alkaloid constituents was carried out using different elution systems and adsorbents. Plates coated with silica gel G and developed with S1, S2, S3 and S4 elution systems gave the best resolution and the results obtained revealed the presence of nine spots as the major alkaloid components (Rf; 0.07, 0.11, 0.14, 0.17, 0.24, 0.29, 0.44, 0.5, and 0.81, S2) beside others as minors (Rf; 0.39, 0.56, 0.7, 0.87, 0.9, and 0.97, S2).
About 1.5 g of the alkaloid extract was applied onto VLC packed with silica gel. Elution was firstly performed with toluene-dichloromethane (90:10) with increasing ratios of the latter to 100%, then with dichloromethane-methanol (95:5) with increasing ratios of the latter. Three fractions (A, B and C) were obtained.
The alkaloid constituents of fractions A (0.528 g), B (0.644 g) and C (0.188 g) were applied for further isolation by VLC, using silica gel and/or aluminum oxide. Elution was carried out with cyclohexane, dichloromethane, and acetone with different ratios. VLC fractionation of the three fractions A, B and C give nine subfractions: four (I-IV), three (I-III) and two (I-II) for A, B and C, respectively.
Brine shrimp lethality bioassay
The cytotoxic effect of total alkaloids, methanol and defatted methanol extracts of L. pyrotechnica were evaluated by LC50 values of the brine shrimp lethality test (CitationKrishnaraju et al., 2006; CitationPoli et al., 2006; CitationHo et al., 2005; CitationPisutthanana et al., 2004). The eggs of brine shrimp were obtained from San Francisco Bay Brand, Newark, CA. The tested samples were dissolved in methanol, and three graded doses, 10, 100 and 1000 μg/mL, respectively, were used for 5 mL of seawater containing 10 brine shrimp nauplii in each group. The number of survivors was counted in each well after 6 h. Counting of the chronic LC50 was begun after 24 h from starting of the test. LC50 was determined by probit analysis as described elsewhere (CitationMeyer et al., 1982). The experiment was carried out in five replicates, and mean LC50 values were measured. Control discs were prepared using only methanol and five replicates were prepared for each dose level. The negative control solution was simply the same saline solution used to prepare the stock test sample solution. Potassium dichromate was used as standard toxicant and dissolved in artificial seawater, to obtain concentrations of 1000, 100 and 10 ppm.
Antitumor screening: Potato disc assay
The potato disc bioassay for the plant extracts were carried out as described elsewhere (CitationFerrigni et al., 1982; CitationFerrigni & McLughlin, 1984; CitationAnderson et al., 1988, Citation1992; CitationBryant et al., 1994). Tumors were initiated on potato discs (usually Pontiac red or red russet variations). Fresh, disease-free potato tubers were obtained from local markets. Agrobacterium tumefaciens strain B6 was maintained on solid slants under refrigeration. Subcultures were grown in 0.8% nutrient broth (Difco) supplemented with 0.5% sucrose and 0.1% yeast extract. Controls were made in the following way: 0.5 mL of DMSO was filtered through Millipore filters (0.22 μm) into 1.5 mL of sterile distilled water and added to tubes containing 2 mL of the same A. tumefaciens strain B6. A standard solution of camptothecin was made as follow: 8 mg of camptothecin was dissolved in 2 mL of DMSO. This solution was filtered through a 0.22 μm Millipore filter into a sterile tube. From this solution 0.5 mL was added to 1.5 mL of sterile water and 2 mL of the same broth culture of A. tumefaciens strain B6. A blank solution was made in the following way: 0.5 mL of DMSO was added to 1.5 mL of sterile water. Using a sterile disposable pipette, 1 drop (0.05 mL) from these tubes was used to inoculate each potato disc, spreading it over the disc surface. The medium was solidified as required with 1.5% agar (Difco). The results were expressed as + or - percentages versus the number of tumors on the control discs; inhibition was expressed as a negative percentage and stimulation was expressed as a positive percentage. Significant activity was indicated when two or more independent assays gave consistent negative values of 20% or greater inhibition.
Results and discussion
In order to identify individual substances the extract was examined by means of GC-MS. As seen from the chromatograms indicate a wide variety of compounds present in the alkaloid extract. Twenty-four alkaloid compounds were represented in . Among them, ten were identified as pyridines (C-2, 5, 7, 10, 16, 18, 20, 21, 23, 24); three pyrazines (C-9, 11, 13); four pyrroles (C-4, 14, 15, 22); two pyrimidines (C-1, 12); two piperidines (C-6, 17); two indoles (C-3, 19) and one purine (C-8), in addition, six simple amine compounds could be identified in the alkaloid extract as shown in .
Table 1. GC-MS experimental data of alkaloid mixture from fractions A(I-IV), B(I-III) and C(I-II).
Table 2. GC-MS experimental data of simple amine constituents of different fractions from alkaloid extract.
Figure 1. GC-MS chromatogram of the alkaloid components of fraction AI.
Figure 2. GC-MS chromatogram of the alkaloid components of fraction AII.
Figure 3. GC-MS chromatogram of the alkaloid components of fraction AIII.
Figure 4. GC-MS chromatogram of the alkaloid components of fraction AIV.
Figure 5. GC-MS chromatogram of the alkaloid components of fraction BI.
Figure 6. GC-MS chromatogram of the alkaloid components of fraction BII.
Figure 7. GC-MS chromatogram of the alkaloid components of fraction BIII.
Figure 8. GC-MS chromatogram of the alkaloid components of fraction C1.
Figure 9. GC-MS chromatogram of the alkaloid components of fraction CII.
With the exception of indole, it is noted that the total percentages of pyridine alkaloids represented the highest ratio in all fractions, while the other classes of alkaloids were detected in small amounts compared with pyridine alkaloids in these fractions. The detected pyridine and indole alkaloids and simple amine compounds of this plant were close to those reported in the alkaloid constituents of the Asclepiadaceae (CitationAbe et al., 2001). While the phenanthroindolizidine alkaloids which represented the third class of alkaloids in this family not detected in our study. The classes’ pyrazine, pyrrole, pyrimidine, piperidine and purine were detected in our study on this plant but not detected in Asclepiadaceae.
LC50 determinations
The data of mortality rates point out that with 1000 ppm concentration the methanol extracts exhibited high mortality, 100%, while the other extracts, defatted methanol and ethyl acetate (alkaloids), represented 98.91% and 93.26%, respectively. Moreover, with 100 ppm concentration, the methanol and defatted methanol extracts exhibited high mortality, and represented 88%, and 82.35%, respectively. Alkaloids represented a lower percentage, 58.21%. On the other hand, with 10 ppm concentration, alkaloids represented the lowest percentage, 14.10%, but the other extracts were not detected. The estimated LC50 and its 95% confidence limits of alkaloids were 63.09 (34.38-115.79).
Potato disc assay
The results obtained from the potato disc assay showed that the methanol and alkaloid extracts have the most activity as antitumor agents, which represented −49.30% and −33.60%, respectively.
We can conclude that gas chromatography coupled to mass spectrometry (GC-MS) proved to be a viable tool for alkaloid analysis of L. pyrotechnica. Substances that occur in L. pyrotechnica alkaloid extract in higher amounts were identified for the first time from this plant. A total of 24 alkaloids and 6 simple amine compounds could be quickly and easily identified in the alkaloid extract of the studied plant. The acute LC50 of the total alkaloid and alcohol extracts by means of brine shrimp toxicity test were 63.09 and 11.89 ppm, respectively. The antitumor activities of these extracts, using potato disc screen, showed good influences. The relatively antitumor activity of the L. pyrotechnica plant could be attributed mainly to its alkaloid content.
Acknowledgements
AMYM is grateful to the Egyptian Ministry of Higher Education, and the Environmental Chemistry and Toxicology Laboratory, Texas Southern University, Houston, TX, USA for the Fellowship it has provided her to undertake this work. This work was supported by RCMI grant number R003045-17A and NASA/URC grant number NCC 9.165.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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