Abstract
Phytochemical and biological activity screening are the primary steps for isolation of new biologically active compounds, which lead to the discovery of new drugs. Calotropis procera (Ait). R. Br. (Asclepiadaceae) are widely used in traditional medicine to treat various diseases. To provide a scientific basis for traditional uses of Calotropis procera, the crude methanol extract of plant organs were investigated phytochemically. The plant contains various classes of bioactive secondary metabolites such as terpenoids, flavonoids, saponins, steroids and cardiac glycosides. The antimicrobial activities of the crude extracts of various plant organs were investigated too. The results were sufficiently positive to encourage further investigation.
Introduction
Research has explored the nature of the secondary metabolites that in various medicinal plants. Medicinal plants represents a precious, renewable source for new drugs. Around 500,000 plant spices were estimated but only a small amount has been investigated phytochemically. More than 130 drugs in the world’s markets were extracted from higher plants or modified synthetically [1–5]. Although hundreds of plants were tested for antifungal and antibacterial properties, the majority of them have not been adequately evaluated [6].
The available drugs to treat fungal infections have been restricted. Furthermore, there are some problems with dose limiting nephrotoxicity, the rapid development of resistance, drug interactions and fungistatic mechanism of action. Thus, there is a need for the development of more efficacious antifungal agents with fewer limitations and less side effects [7, 8]. Infections from resistant bacteria are now very common, and some pathogens have even become resistant to multiple types or classes of antibiotics. Efforts to prevent such threats build on the searching to find new compounds with potent antimicrobial activities with less side effects especially from natural products [9, 10].
Plants are valuable source for the discovery of novel pharmacologically active compounds. This is because of broader degree of chemical diversity and novelty of molecules found in natural products than that from any other source. Many drugs are derived directly or indirectly from plants [11].C. procera belong to family Asclepiadaceae and it is a large broadleaf evergreen plant with a strong odour, abundant in the tropical regions of Asia and Africa, which is commonly known as Milkweed.C. procera is used as a folk medicine for the treatment of various diseases. It has been reported that the plant possess potential anthelmintic, antimicrobial, anticancer, anticoagulant, analgesic, anti-inflammatory, purgative and antipyretic properties and is also used in the treatment of leprosy, leucoderma, liver and abdomen [12]. C. procera flowers causes temporary paralysis of red stomach worm in sheep and notably reduces egg count percent of gastrointestinal nematodesin naturally infected sheep [13]. The latex of C. procera plants has important indigenous medicinal uses becauseof its purgative, antisyphilitic and antiodontalgic action [14]. Dry latex of C. procera has potential anti-cancer properties due to its differentiable targets and non-interference with regular pathway of apoptosis [15]. The pharmacological properties of C. procera could be interesting for the pharmaceutical industry to develop new drugs [16]. Many reports studied the phytochemical screening of C. procera [17–21] but they did not include all the organs of the plant and their results do not coincide with each other. This encourage the authors to study the phytochemical constituents of all the plant organs comprehensively with the most reliable methods. The current work demonstrated the chemical constituents, antimicrobial properties of C. procera in the Middle East region.
Experimental
Plant materials
Calotropis procera R. Br. was collected from Khulais, Kingdom of Saudi Arabia, in May 2012.Dr. Emad Al Sherif in King Abdulaziz University, Saudi Arabia, identified the plant. Voucher specimens is deposited at the Herbarium of King Abdulaziz University.
Chemicals
All the chemical used are analytical grade.
Methods
Phytochemical screening
The extractions and all the qualitative methods have been done according to the most common and reliable methods [22–26].
Extraction of plant samples
Fresh plant samples of leaves, fruit, stems, flowers and roots (500 g) was homogenized in 70% methanol and then refluxed for 30 min. After cooling, the liquid was filtered and the plant material was washed with methanol. The combined extracts and washing with methanol were made up to 500 mL.
Detection of alkaloids
The residue obtained from the evaporation of 50 mL of the alcoholic extract was titrated with 20 mL of dilute hydrochloric acid and 0.5 g of sodium chloride and filtered. The filtrate was rendered alkaline with ammonium hydroxide and then extracted with successive portions of chloroform. The combined chloroform extract evaporated to dryness, the residues dissolved in 2 mL hydrochloric acid and tested with silicotungestic acid and Mayer’s reagents. The formed precipitate was, in each case, indicates the presence of primary, secondary and tertiary alkaloids. The aqueous alkaline layer was acidified with hydrochloric acid and tested with silicotungestic acid and Mayer’s reagents. A precipitate was formed indicates the presence of quaternary alkaloids.
Detection of flavonoids and anthocyanidins
About 50 mL of alcoholic extract was evaporated to dryness. The residue was titrated with 15 mL of petroleum ether (60–80°C) while warming. The residue was filtered and re-extracted again in the same manner. The defatted residue was titrated with 50 mL of 80% methanol while warming then filtered. To 2 mL of the filtrate, 0.5 mL of hydrochloric acid was added and the mixture was warmed on a steam bath for 5 min, a red-violet colour, indicates the presence of leucoanthocyanidins. To another 2 mL of the filtrate Shinoda test for flavonoids was applied. A red colour observed within 10 min indicates the presence of flavonoid compounds.
Detection of unsaturated sterols and triterpenoids
The petroleum ether extracts, which were collected from flavonoid test, were evaporated to dryness. The residue was dissolved in 10 mL choroform, dried over anhydrous sodium sulfate and filtered. The filtrate was divided into three portions. The first portion was subjected to Liebermann-Burchard test; a blue green colour indicated a positive test for sterols while red-pink or violet indicate a positive test for triterpenoids. The second portion was subjected to Salkowski test; a red colour indicates a positive test for sterol. The third portion was used as control for colour changes.
Detection of saponins
About 1 g of the dried powdered organs was macerated with 4 mL of water, filtered, and the filtrate was shaken vigorously. A persisting froth for about 30 min was formed, indicating the possible presence of saponins. Five mL of the alcoholic extract was evaporated to dryness under vacuum and the residue was dissolved in 10 mL of normal saline. To 8 mL of this solution 2 mL of defibrinated blood in normal saline (1 : 40) were added and left for 24 hours. Blood haemolysis was noticed, indicating the presence of saponins.
Detection of coumarins
About 50 mL of the hydroalcoholic extract was concentrated to 5 mL and then treated with 25 mL 10% alcoholic potassium hydroxide at room temperature. After standing for 30 minutes with occasional shaking, it was extracted with chloroform (4×25 mL). The aqueous layer was acidified with 10 mL of 10% hydrochloric acid and refluxed for 1 hour. After cooling, it was extracted with chloroform, washed with water and dried over anhydrous sodium sulphate. The chloroform extract was evaporated to dryness. The residue was sublimed at 100°C for 1 hour under vacuum. The sublimate was dissolved in 10 mL spectroscopic alcohol and measured in the UV region 250–350 nm. The absorption in this region was taken as an evidence for presence of coumarin.
Detection of anthraqinones
The alcoholic extract corresponding to 5 g of each plant material was shaken with 10 mL benzene and filtered. About 5 mL of 10% ammonium hydroxide solution was added to the filtrate, shaken and allowed to stand until the two layers were separated. The development of pink, to violet colour in the ammonical phase indicates the presence of free anthraquinones. About 5 g of each plant extract was boiled with 10 mL of 1% aqueous sulphuric acid and filtered while hot, the filtrate was shaken with 5 mL benzene. The benzene layer was removed and shaken with 5 mL of 10% ammonia solution. The presence of a pink to violet colour in the ammonical phase indicates the presence of combined anthraquinones.
Detection of tannins
About 20 mL of alcoholic extract was evaporated under vacuum (temperature not more than 35°C). The residue was stirred with 10 mL of distilled water and filtered. On the addition of ferric chloride reagent to a portion of the filtrate, the formation of a green blue to bluish black colour or precipitate may indicate the presence of tannins.
Detection of cardiac glycosides
The 80% alcoholic extract remaining after the flavonoid test was divided to three portions. 5 mL of the solution were placed in small porcelain evaporating dish, 5 mL of kedde’s reagent, and 5 mL of 2 N sodium hydroxide solution were added. The appearance of purple colour indicates a positive test for cardiac glycosides. Another 10 mL of the solution were evaporated to dryness, the residue was triturated with 3 mL of ferric chloride solution and filtered. The filtrate was transferred to a test tube and 1 mL of concentrated sulphuric acid was added slowly down the side of the test tube. The appearance of purple ring indicates presence of cardiac glycosides (desoxysugar). If the above two tests are positive, 5 mL of the solution were evaporated to dryness. The residue was dissolved in 2 mL chloroform and transferred to a small test tube. Acetic anhydride (0.3 mL) was added and mixed gently, then, a drop of concentrated sulphuric acid was added. The appearance of blue-green colour, observed during 60 min, indicates presence of cardiac glycosides (as steroids).
Antimecrobial activity of Calotropis procera
The 70% methanol–water extract of the stems fruit, leaves and flowers of C. procera and its n-hexane, ether, chloroform and water fraction were investigated for the Antimicrobial and Antipathogenic Activities. The in vitro qualitative screening of the antimicrobial activity was carried out by an adapted agar diffusion technique using a bacterial suspension of 0.5 McFarland density obtained from 24 hours cultures [27]. The antimicrobial activity of the plant fractions was tested against Klebsiella Pneumonia for antibacterial activity as well as Aspergillus Niger for antifungal activity. The microbial strains identification was confirmed by aid of VITEK II automatic system. VITEK cards for identification and susceptibility testing (GNS-522) were inoculated and incubated according to the manufacturer’s recommendations. The extracts were tested starting with a concentration of 10 mg/mL. A volume of 10 μL of each tested extract sample of 10 mg/mL concentration was spotted on Muller Hinton agar for bacteria and Yeast Peptone Glucose (YPG) agar for fungi, previously seeded with the microbial inocula of 0.5 MacFarland density. The inoculated plates were incubated for 24 hours at 37°C. The antimicrobial activity was assessed by measuring the growth inhibition zones diameters expressed in mm [27].
Results and discussion
Phytochemical screening of Calotropis procera
The methodology of screening of fresh stems, leaves, fruit, flowers and roots of C. procera in the present work depends on our previous experiences with the plant [28–31]. All the results obtained from the phytochemical screening tests were recorded in Table 1 according to the intensity of colour or precipitates produced in terms of scores of 0, +, ++, +++ using standardized values based on prepared plant extracts originally proven to be free from sterols, triterpenoids, flavonoids, alkaloids, cardiac glycosides, tannins and anthraquinones and to which the calculated amounts of typical models of these products were added. A score of (+) corresponds to 0.01–0.1%, a score of (++) corresponds to 0.1–0.3% and a score of (+++) corresponds to more than 0.3% of any of these types of products (dry weight basis of plant material). As it was indicated from the previous tests, stems, leaves, fruit, flowers and roots of C. procera gave positive response for the presence of sterols, triterpenes, cardiac glycosides and saponins (Table 1). On the other hand, flavonoid glycosides were detected only in leaves and flowers while tannins were found in the leaves, fruit and flowers. A low quantity of alkaloids was found in stems and roots. Anthraquinones (combined) were only detected in the flowers. The presence of cardiac glycosides in high quantity in the different organs of C. procera prompts the author to study it quantitatively in the future.
Antimicrobial activity of Calotropis procera
All the fractions showed antibacterial activities with Klebsiella pneumonia as shown in Table 2. However, they differ significantly in their action according to the organ of the plant and the fraction of the extract. The most potent fractions is n-hexane and ether fractions of flower extract. Fractions of n-hexane, ether and chloroform showed potent activities of all plant organs under investigations. It is noted that the most potent activities is showed in nonpolar fractions, which may be attributed to less polar compounds. This finding should be investigatedin details in the future work. Fractions of n-hexaneand ether of C. procera extracts showed considerable antifungal activity against Aspergillus niger as shown in Table 2 and Fig. 1.
Conclusions
Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi. It is an increasingly serious threat to global public health that requires action through the world’s society. Antimicrobial resistance invaded all parts of the world. Since then, further increases in resistance to first-line treatment drugs were reported, which might require using more drugs that are expensive in the near future. Thus, it is a great challenge faces scientists to find new drugs with antifungal or antibacterial activities. Some of the primarily results obtained from this work are promising. The future work should include examinations against various fungi and bacteria strains with bioassay-guided fractionation. Isolation of the pure active compounds will consequently assist to study the mechanism of action.
Footnotes
Acknowledgments
This paper was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (94-130-D1432). The authors, therefore, acknowledge with thanks DSR technical and financial support.