Abstract
From previous studies, Calotropis procera showed the presence of cardiac glycosides, sterols, hydrocarbons, fatty acids, flavonoids. The abundant constituents are cardiac glycosides and lipids including sterols, hydrocarbons and fatty acids that encourage the author in this work to study them quantitatively. The distinct odour of the plant paid the attention for studying the essential oils of the plant organs. In addition, cardiac glycosides were estimated, after a rapid purification method, with two reagents for comparison and to judge the reliability. The methods can be used for industrial purposes.
Introduction
Calotropis procera is one of the traditional medicine in Africa and Asia. The milky juice of the plant is used as a purgative, a stomachic and a carminative. In addioon, the plant is used for skin diseases and enlargement of abdominal viscera [1, 2]. The plant was used as arrow poison in India and Africa [3]. Cardiac glycosides are a class of secondary metabolites. They comprise a large family of naturally derived compounds. All member of this family share a common structure skeleton. The core structure consists of a steroidal part, which considered a pharmacophoric moiety responsible for the activity [4]. The steroidal moiety is connected to unsaturated lactone ring at position 17 and a sugar portion at position 3. The structure of cardenolids, the common cardiac glucosides in plant kingdom, has a five-memberd lactone ring. Whereas, the structure of bufadienolids, the common cardiac glycosides in animal kingdom, have a six- membered lactone ring. Large number of cardiac glycosides were isolated from C. procera, which reflect how much this plant contains cardiac glycosides [5–17]. Cardiac glycosides are used to increase the contractile force of heart in patients with cardiac arrhythmias and congestive heart failure [18]. Recently, an interesting work showed the increasing of susceptibility of cancer cells to cardiac glycosides, which supports their use as a cancer therapies [19]. The analytical methods for cardiac glycosides depends on two groups. The first is the classical methods of photometry and chromatography which have an established place in the pharmacopoeias and very widely employed in control laboratories for quantitative determination of the content and purity of glycoside preparations [20]. The second was for pharmacokinetic investigations. The available methods for this purpose require greater expenditure on apparatus. They comprise the isotope technique, Gas chromatography coupled to a mass spectrometer (GCMS) or a well-developed analytical method by coupling a chromatographic unit, GC or HPLC, to a sensitive detector (MS or fluorescence detector). Such method affords reliable measurements in the ng range [20] but it is very sophisticated and need more time and effort with very high expenses. Whereas the classical methods required preliminary purification, usually by chromatography that needs more time to prevent the interference with other plant constituents. In the classical methods, direct measurement by UV leads to the absorption maxima for cardenolides at 217 nm (ɛmol = 16,595). For qualitative and quantitative determination of the cardiac glycosides, they must therefore be converted into colored derivatives. It can be converted into colored derivatives by reaction with polynitroaromatic derivatives in alkaline solution, Keller-Kiliani or xanthydrol in acidic medium or by treatment with strong acids (Fig. 1) [20]. The products of these reactions can be measured by conventional photometers or fluorimeter [21–23]. Polynitroaromatic reagents in alkaline solutions, Baljet’s and Kedde’s reagent, react with the unsaturated lactone ring which is very unique and characteristic for cardiac glycosides and decrease of the interference possibilities of the plant constituents that give a good way for accurate determination of the total cardiac glycosides in plants. Karawya et al. [24] determined the total cardenolides in different organs of Acokanthra spectabilis using Baljet’s reagent (picric acid). The total cardenolides were calculated as acovenoside A, acovenoside C, acobioside A and ouabain. The absorption was measured at λmax 495 nm. On the other hand, Negm and Radwan [25] determined the total cardiac glycosides in seeds of Corchorus olitorius using Kedde’s reagent (3,5 dinitrobenzoic acid).
From the previous work [1, 9], C. procera showed the presence of nonpolar material like fatty acids, sterols and hydrocarbon and polar materials like cardiac glycosides as the major constituents. In this work, the author estimated quantitatively the most abundant constituents in the plant. Cardiac glycosides is one of the characteristic constituents in C. procera and they gave the plant its importance [3–8]. Consequently, determination of cardiac glycosides with reliable and rapid methods that make it is possible to measure the glycosides directly in the presence of other biological materials is necessary task. This work was based on the fact that all cardiac glycosides form the same type of Meisenheimer compounds (Fig. 2) of the cyclohexadienate type with polynitroaromatic compounds in alkaline medium [20, 27]. Furthermore, the absorption maximum of Meisenheimer compound in its UV- measurement depends on the type of polynitroaromatic compounds used in the assay and the amount of cardiac glycosides regardless of the structure of the cardiac glycosides under investigation.
Experimental
Materials
Calotropis procera R. Br. was collected from Khulais, Mecca, Kingdom of Saudi Arabia, in May 2014. The plant was identified in King Abdulaziz University, Saudi Arabia. Voucher specimens is deposited at the Herbarium of King Abdulaziz University. Gitoxigenin was obtained from Fluka Company. β-Sitosterol and stigmasterol were obtained from E. Merck, Darmstadt, Germany. All the chemical used are analytical grade. UV Spectra were measured on a Shimadzu UV-visible recording spectrophotometer (UV- 2401 PC). Gas liquid chromatography analysis of the unsaponifiable matter, fatty acids and essential oils were performed on Pye Unicam PU 4450 equipped by flame ionization detector. Flow rate of nitrogen: 30 mL/min, hydrogen: 33 ml/min, air: 330 mL/min. The analysis of unsaponifiable matter was conducted under the following condition: column: OV-17, column temperature: 70–270C in rate of: 10C/min, injection at 250C and detection at 300C. The fatty acids and essential oils were done with column P5 GA % 10 (1.5×4 mm), column temperature: 70–190C in rate of 8C/min and 4C/min for fatty acids and essential oil, respectively, injection at 250C and detection at 300C.
Methods
Cardiac glycosides estimation
2.2.1.1. Plant extract. Two grams of each of the dried fruit, stems, leaves, flowers roots and latex residue of C. procera were percolated with 25 mL 70% methanol three times (macerating 24 hours). The percolated extract was filtered and transferred to 100 mL volumetric flask with 80% methanol and kept tightly in refrigerator.
Five mL of the extract was added to 10 mL distilled water then 5 mL of freshly prepared lead acetate (12.5%) was added. The volume was completed to 25 mL with distilled water and filtered. Ten ml of the filtrate were taken in a 25 mL-volumetric flask and then 5 mL of disodium hydrogen phosphate (4.7%) were added. The content of the flask was completed to 25 mL with distilled water. Then, it was shaken and filtered [24, 29].
2.2.1.2. Preparation of standard curve of gitoxigenin using Baljet’s reagent. Gitoxigenin (25 mg) was dissolved in 25 mL spectroscopic methanol in a volumetric flask. The following volumes: 0.025, 0.05, 0.10, 0.20, 1.00, 1.40, 2.00, 3.00, 4.00, 5.00 and 6.00 mL were withdrawn to volumetric flasks and each one was completed to 10 mL with spectroscopic methanol. These were equivalent to the corresponding concentrations: 0.025, 0.05, 0.10, 0.20, 1.00, 1.40, 2.00, 3.00, 4.00, 5.00 and 6.00 mg/10 mL. Ten mL of freshly prepared Baljet’s reagent was added to each one. The mixture was kept at room temperature for 30 min followed by measuring its absorbance at 490 nm against a blank of 10 mL of Baljet’s reagent mixed with 10 mL of spectroscopic methanol.
2.2.1.3. Determination of the total cardenolides referring to gitoxigenin using Baljet’s reagent. Two mL of the purified extract of each organ and two ml of freshly prepared Baljet’s reagent were mixed and the absorbance of each one were measured after 30 min at 490 nm [24].
2.2.1.4. Preparation of standard curve of gitoxigenin using Kedde’s reagent. The same method mentioned before was applied using Kedde’s reagent instead of Baljet’s reagent. The absorbance was measured after 2.5 min at 560 nm.
2.2.1.5. Determination of the total cardenolides referring to gitoxigenin using Kedde’s reagent. Two mL of the purified extract of each organ and 1 mL of 2% methanolic 3,5-dinritrobenzoic acid and 1 mL of 1 N methanolic solution of potassium hydroxide were mixed together. The absorbance of each one was measured after 2.5 min at 560 nm [25].
Extractions of fatty acids, sterols and hydrocarbons of Calotropis procera
The plant sample were obtained following the conventional method by extraction with petroleum ether. It was fractionated to unsaponifiable matter (sterol and hydocarbons) and fatty acids [30–32]. The dried powdered plant (100 g) of each plant organs (stems, leaves, flowers, fruit and roots) was exhaustively extracted with petroleum ether (60–80 °C) at room temperature to afford an oily extracts. Each one of the oily extracts was separately treated with 50 mL of 10% alcoholic potassium hydroxide for 2 hours. Each of the saponified solution was treated with 50 mL water (equal volume) then exhaustively extracted with chloroform. The combined chloroform extract was washed with water until it becomes free from alkali, dried over anhydrous sodium sulphate and the solvent distilled off under vacuum to give the unsaponifiable fraction as an oily matter. The identification of the sterols and hydrocarbon contents were achieved by comparing the retention time of their peaks with those of authentic samples.
The aqueous alkaline solution (mother liquor), left after separation of the unsaponifiable matter, was acidified by diluted HCl. The liberated fatty acids were then exhaustively extracted with chloroform several times, and then the combined chloroform extract was washed with water until the washing was neutral to litmus paper. The chloroform layer dried over anhydrous sodium sulphate and the solvent distilled off to give the fatty acid fractions.
Preparation of fatty acid methyl esters
The fatty acids isolated were methylated by refluxing with absolute methanol (15 mL) containing 5% sulphuric acid (0.5 mL) for about 1 hour on a water bath. After cooling the solution, it was diluted with water (until become 100 mL) and was exhaustively extracted with chloroform. The chloroform layer was dried over anhydrous sodium sulphate and then the solvent distilled off [33]. The residue was then subjected to GLC analysis.
Essential oils of Calotropis procera
Flowers, leaves and fruit of Calotropis procera (50 g) was extracted three times with n-pentane/methylene chloride (2 : 1) for two hours at room temperature [34–36]. The extract was dried over anhydrous sodium sulphate and concentrated to 1 mL at 40 °C. The prepared oil was subjected to GLC analysis.
Results and discussion
Quantitative estimation of cardiac glycosides of Calotropis procera
Cardiac glycoside contents in each organ of C. procera was determined using both of Baljet’s and Kedde’s reagents for comparison. The total cardiac glycosides were calculated with referring to gitoxigenin and the absorption were measured at 495 and 560 nm to establish a general method for the total quantitative estimation of unknown cardiac glycosides in a plant sources.
The percentage of cardiac glycoside content calculated from Baljet’s and Kedde’s reagents are approximately the same as shown in Table 1 and Fig. 3. It was remarked that the highest concentration of cardenolides was present in the latex and the lowest concentration was present in the stems.
Study of sterols, hydrocarbons and fatty acids
It has been reported that the plant contains sterols [1], hydrocarbon [37] and fatty acids [38, 39]. This induced the author to carry out a complete comparative study of the fatty acids, phytosterols and hydrocarbons in the lipid contents of each organ of C. procera.
Sterols and hydrocarbons
The total sterol and hydrocarbon contents were found to be 0.08, 1.33, 3.00, 3.09 and 0.33% (dry weight bases) in stems, leaves, fruit, flowers and roots of C. procera (Table 2 and Fig. 3). GLC of the unsaponifiable fractions showed that stigmasterol was found in stems and roots. In addition, fruit and roots, while β-sitosterol was found in flowers and roots. On the other hand, cholesterol was found only in the roots (Table 2). Examination of hydrocarbon contents showed that dodecane is the major hydrocarbon in stems and roots, while tricontane is the major one in flowers and fruit. In addition, squalene was found in the leaves and fruit (Table 2).
Fatty acid fractions
The total fatty acids were found to be 0.03, 0.04, 1.92, 0.84 and 0.02% (dry weight bases) in the organs of plant samples; stems, leaves flowers, fruit and roots respectively (Table 2). The fatty acids of stems, leaves flowers, fruit and roots were composed approximately of saturated fatty acids (62.94, 38.14, 77.69, 30.41 and 91.14%, respectively) and unsaturated fatty acids (37.06, 61.86, 22.3, 69.59 and 8.86%, respectively). Stearic acid (43.69%) and linoleic acid (37.06%) were present as the major fatty acids in stems while palmatic acid (35.33%), oleic acid (30.63%) and linoleic acid (31.23%) were the major compounds in leaves. Flowers and roots contain palmatic acid in a higher percentage (57.75 and 54.43%, respectively). On the other hand, linoleic acid (29.92%), palmatic acid (27.08%) and oleic acid (26.03%) are the major compounds in fruit (Table 2).
Study of the essential oils
Since essential oils have marked pharmacological activity, it was deemed desirable to investigate the nature of its existence in the flowers, leaves and fruit of C. procera and it is worthy to mention that the essential oil had not been previously investigated. Investigation of the GLC of flowers, leaves and fruit of C. procera showed that they contains thirty three, thirty two and twelve compounds, respectively. Seven compounds were identified in both flowers and leaves of the plant and only two compounds in its fruit (Table 3). Terpinyl acetate and β-ionone were identified in the three organs, while caryphyllene, geraniol, methyl anthranilate and diethyl phthalate were common in the flowers and leaves. Cineole was identified only in the leaves. Terpinyl acetate (24.46%) represented the major compounds in the fruit (Table 3).
Conclusions
Calotropis procera is a reach plant with cardiac glycosides, lipid soluble materials such as sterols, hydrocarbons, and fatty acids. The flowers and leaves of C. procera are rich with mono- and sesquiterpene and aroma compounds. Cardiac Glycoside content in C. procera is very large as shown from the quantitative measurements and it contains a large number of individual compounds, which revealed from the number of isolated compounds. Recently, the importance of cardiac glycosides was shown with their medical use in treatment of several diseases. This study showed desirable methods for quantitative estimation of the total contents of cardiac glycosides in C. procera extract with very simple and fast purification method that save time, expenses and effort. These methods are suggested to be suitable for quality control and industrial application.
Footnotes
Acknowledgments
The author is thankful to Dr. Radwa Moustafa Elfaiomy for her support and encouragement during this work.