Abstract
On the bases of ethnomedicinal and random plant collection approaches for searching out new pharmacologically bioactive agents, 45 Egyptian plant species belonging to 25 families were collected, and methanol extracts (52) were assayed in vitro for their antischistosomal and antimicrobial activities. Only extracts of Curcuma longa L. (Zingiberaceae) and Nerium oleander L. (Apocynaceae) were lethal to Schistosoma mansoni worms after a 24-h incubation period in a culture medium at concentration up to 100 μ g/mL. The different successive organic extracts of C. longa showed variable activities and the greatest activity was exhibited by the chloroform extract (EC50 = 28.92 and 31.58 μ g/mL against male and female worms, respectively). On the other hand, the antimicrobial potency of the methanol extracts were tested using the disk diffusion method against three bacteria and two fungi species: Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Aspergillus niger, and Candida albicans. Three plants, Chamomilla recutita L. (Asteraceae), Buddleja hybrida Lour. (Buddlejaceae), and Glinus lotoides L. (Molluginaceae), showed the highest antimicrobial potency. Also, antimicrobial screening of the different organic solvent extracts of these three plants was investigated by measuring the diameter of inhibition zone. Finally, the active extracts were subjected to preliminary phytochemical analysis using chemical tests and thin-layer and paper chromatography to explore the major classes of natural products that may be responsible for their activity.
Introduction
Plant extracts have been used for a wide variety of medicinal purposes for many centuries because they contain numerous components of therapeutic value. These purposes vary from region to region according to the prevalence of certain health problems. Therefore, special care had been focused on medicinal plants as they are known to have effective pharmaceutical compounds including those with antimicrobial and antischistosomal activity (CitationHammer et al., 1999; CitationJiwajinda et al., 2002).
Schistosomiasis is a tropical disease caused by the blood flukes Schistosoma spp. and is widespread in developing countries including Egypt. One of the most effective drug treatments of schistosomiasis is praziquantel. This is expensive, often out of reach for the population in the third world, and possible resistance may develop (CitationIsmail et al., 1999; CitationJiwajinda et al., 2002). Traditional medicinal plants are still widely used for the treatment of various diseases in Africa (CitationKaterere et al., 2003) and may be seen as an important alternative or supplement to praziquantel. A few researches have dealt with the efficiency of traditional plants to treat schistosomiasis in some African countries such as South Africa (CitationSparg et al., 2000) and Zimbabwea (CitationNadamba et al., 1994; CitationMolgaard et al., 2001).
On the other hand, the development of microbial resistance to available antibiotics due to random selection (CitationMckane & Kandel, 1996), and possible side effects, have led some authors to investigate the antimicrobial activity of indigenous medicinal plants in many parts of the world, such as Qatar (CitationMahasneh, 2002), Siberia (CitationKokoska et al., 2002), Peru (CitationRojas et al., 2003), Lebanon (CitationBarbour et al., 2004), Brazil (CitationDe Souza et al., 2004), and Malaysia (CitationWiart et al., 2004).
In the discovery of new bioactive compounds from plants, several approaches to the selection of plants for pharmacologic screening are known. First, the random approach, which involves the collection of all plants found in the study area. Second, phytochemical targeting, which entails the collection of all members of a plant family known to be rich in bioactive compounds, and finally, the ethno-directed sampling approach, based on traditional uses of plant (CitationHeinrich et al., 2004).
However, screening the biological activity of plant extracts against schistosomiasis worms and microorganisms in Egypt is poorly investigated (CitationEassa & Beih, 2002; CitationDiab et al., 2005). Therefore, there is a need to explore and search for more plants in Egypt as a source for new drugs for these two purposes.
In view of all the above considerations, the main aim of this study was to test a large number of plant extracts (52) (the plants collected randomly and on the basis of ethnomedicinal approach in plant collection) against a diverse range of organisms comprising Schistosoma mansoni worms, Gram-negative and Gram-positive bacteria, and fungi, using the in vitro bioassay to conclude direct antischistosomal and antimicrobial data. Different organic fractions of the most active extracts would be further subjected to bioassay followed by phytochemical analysis of the active extract to determine the major phytochemical classes that may be responsible for activity.
Materials and Methods
Plant materials
The plants under investigation () were collected from different regions in Egypt between May and September 2005. Other plants, such as Curcuma longa L. (Zingiberaceae), whole tubers, were purchased from the Egyptian herbal market. The collected and the purchased plants were kindly identified by Dr. Abdel-Halim Abdel-Motagaly, Horticulture Department, Agriculture Research Center, and Mrs. Traes Labib, general manager and head of specialists of Plant Taxonomy at El-Orman Botanical Garden, Giza, Egypt. Voucher specimens (given numbers Schi.1–Schi.47) were deposited at the Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Egypt. The plants were shade-dried and powdered using an electric mill. Known weights of each powdered plant were used for the extraction process, and the remainder was kept in dark closed bottles.
Table 1 Preliminary in vitro antischistosomal and antimicrobial screening of 52 methanol plant extracts.
Preparation of extracts
A known weight (100 g) of each of the collected plants was soaked in 95% methanol (500 mL) for 1 week at room temperature followed by filtration using filter paper Whatmann No. 1. The procedure was repeated three times. The filtrates were evaporated using a rotatory evaporator under reduced pressure at a temperature lower than 40°C and yielded a known weight of dried extract of each plant. Yields of extracts in terms of dry starting materials are listed in . The dried extracts were kept protected from light until bioassay investigations.
Later on the most active plant extracts were subjected to further extraction (fractionation) process using different successive organic solvents with increasing polarities in the following order: petroleum ether, chloroform, ethyl acetate, and n-butanol.
Schistosoma mansoni worms and culture media
Adult Schistosoma mansoni worms were freshly obtained from Schistosoma Biological Supply Center (SBSC), Theodor Bilharz Research Institute, Egypt. The culture media used consists of 4.2 mL RPMI 1640 (Gibco, USA) supplemented with 0.3 mL sterilizing antibiotic (streptomycin 100 μ g/mL + penicillin 100 μ g/mL) and 0.5 mL fetal calf serum as nourishing agent.
Test microorganisms
The antimicrobial activity was determined using the following species of bacteria; two Gram-positive (Bacillus subtilis andStaphylococcus aureus) and one Gram-negative (Escherichia coli). The fungus used wasAspergillus niger and the yeast-like fungus was Candida albicans. Identification and maintenance of microorganism cultures was performed at the Department of Agricultural Microbiology, National Research Center, Egypt.
In vitro antischistosomal screening
The testing agents were prepared by dissolving a known weight of each extract in a minimum amount of DMSO to obtain a stock solution. From the stock solution, the desired concentrations (200 and 100 μ g/mL) in duplicate were prepared. A negative control with DMSO only was also prepared. Under sterilized conditions, 10 adult S. mansoni worms (of mixed sexes) were added to the culture media of the tested extracts and the negative control and then incubated for 24 h at 37°C. After the incubation period, the viability and mortality of worms were recorded in comparison with negative control (CitationJiwajinda et al., 2002).
The same procedure was followed on gradual concentrations prepared from different organic extracts of Curcuma longa to determine the EC50 and EC84, which were evaluated using the program Pharm/PCS version 4.2 (pharmacological calculation system) based on Litchfield and Wilcoxon methods.
Antimicrobial activity
The antimicrobial activities of the methanol extracts were evaluated by the disk diffusion method using sterile Whatman No. 1 filter paper disks (6 mm) (CitationLoo et al., 1945). Each extract was dissolved in 95% methanol and sterilized by membrane filtration (pore size 0.45 μ m). Disks were moistened with exactly 25 μ L of the extracts (equivalent to 1 mg/disk of the dried extract) and allowed to dry at room temperature before being placed at equidistance onto the surface of an agar plate previously seeded with one of the test organisms. Test plates were prepared by pouring 10 mL penassay agar medium (CitationDifco, 1969), and after solidification, 5 mL of seeded penassay broth medium (8X 107 CFU/mL) was distributed on the surface of the prepared agar. All the plates were then incubated for 24 h at 37°C for bacteria and for 48 h at room temperature for fungi. The tests were carried out in triplicate and in parallel a negative control test was carried out in every experiment by applying 25 μ L of 95% methanol per disk. For comparison, ampicillin (10 μ g/disk) and nystatin (20 μ g/disk) were included in the assay as positive controls. After incubation, the antimicrobial activity was evaluated by measuring the diameter of the growth inhibition zones including the diameter of the filter paper disk (6 mm).
Phytochemical analysis
The three active methanol extracts and their active successive fractions of Chamomilla recutita L. (Asteraceae), Buddleja hybrida Lour. (Buddlejaceae), and Glinus lotoides L. (Molluginaceae) (showing antimicrobial potency) were selected for preliminary phytochemical analysis to explore the major classes of natural products (which may be responsible for activity) in these plants as follows.
By chemical tests. Tests for sterols/triterpenes, phenolic compounds, carbohydrates/glycosides, saponins, and alkaloids were carried out according to the previously reported methods (CitationHarborne, 1973; CitationShellard, 1957; CitationHostettmann & Marston, 1995).
By thin layer and paper chromatography.Precoated aluminum silica gel plates were used (GF254, Merck) using different developing systems (CH2Cl2:MeOH, 19:1; CHCl3:MeOH, 9:1 and 8:2; CHCl3:MeOH:H2O, 65:35:5; n-BuOH:MeOH:H2O, 5:1:1). The components were visualized under visible and UV light (254 and 366 nm) and sprayed with the following reagents in order to reveal spots of different groups: Dragendorff's reagent for alkaloids, methanol KOH for coumarins, aluminum and ferric chloride for phenolic compounds, anisaldhyde/sulfuric acid for steroids/triterpenes, and 40% methanol sulfuric acid for saponins (CitationKrebs et al., 1969; CitationHostettmann & Marston, 1995). Also, two-dimensional paper chromatography (TDPC) was performed on Whatman No. 1 (57 × 46 cm) using BAW solvent system (n-BuOH:AcOH:H2O, 4:1:5 organic layer) for the first dimension followed by 15% AcOH/H2O as the second dimension solvent system. After drying, the change of spot color on the chromatograms was detected by exposing to ammonia vapor or spraying with 1% methanol AlCl3 or FeCl3 (CitationMabry et al., 1970).
Results and Discussion
In Egypt, like in other developing countries, medicinal plants still represent one of the main therapeutic tools in traditional medicine. The Egyptian flora offers great possibilities for the discovery of new compounds with various medicinal activities (CitationHamed & El-Emary, 1999). In this study, the investigated extracts were tested in an in vitro experiment as in vivo is very costly, complicated, and disputable on ethical grounds (CitationMolgaard et al., 2001). A total of 52 methanol extracts from 47 Egyptian plants (belonging to 25 different families) were investigated for their antischistosomal and antimicrobial potency using in vitro system.
As antischistosomal
Results () revealed that among these extracts only those of Nerium oleander L. (Apocynaceae) and Curcuma longa (Zingiberaceae) demonstrated strong antischistosomal potency or 100% worm mortality at lower concentration (up to 100 μ g/mL) after a 24 h incubation period. However, N. oleander extract was excluded from further study because previous investigations reported the extreme toxicity of this plant (CitationAslani et al., 2004; CitationGechtman et al., 2006).
The antischistosomal activities of different successive organic solvent extracts (petroleum ether, chloroform, ethyl acetate, and n-butanol) () revealed that the chloroform fraction showed the highest activity, and the EC84 values were 42.59 and 47.07 μ g/mL against male and female worms, respectively. Also, the ethyl acetate extract demonstrated high activity, whreras the methanol extract had moderate antischistosomal potency, and the petroleum ether and n-butanol fractions were inactive up to 100 μ g/mL.
Table 2 In vitro effective concentrations of some successive organic extracts of Curcuma longa against S. mansoni worms.
Meanwhile, C. longa, an herb cultivated widely throughout India, China, and tropical regions of Asia, has been used as a daily dietary therapy for centuries and is added to most foodstuffs in these countries (CitationMills & Bone, 2000). It proved to have a wide range of therapeutic effects. It has cytoprotective (CitationFang et al., 2005; CitationPal et al., 2005), strong antioxidant (CitationTilak et al., 2004; CitationOsawa & Kato, 2005), antiinflammatory (CitationSong et al., 2001), and anticancer potency (CitationPadmaja & Raju, 2004, Citation2005). However, reviewing the current literature showed that no previous reports concerning the antischistosomal activity of these two plants were recorded.
As to the chemical constituents of C. longa, many phytochemical works were done on it and revealed that the major constituents of C. longa are curcuminoid compounds, “analogues of diarylheptanoids.” Curcumin and two derivatives, demethoxy curcumin and bisdemethoxy curcumin, were the major three compounds, in addition to other minor curcuminoid derivatives. The observation of high potency of this plant in the current study was in good agreement with the work of CitationKiuchi et al. (1993), who found that the methanol and chloroform extracts of C. longa have activity against Taxocara canis, whereas the isolated curcuminoid compounds did not show activity when applied independently, but the activity was observed when they were mixed, suggesting a synergetic action between these compounds.
As antimicrobial
also summarizes the antimicrobial data of the investigated extracts. They were tested for antimicrobial activity against three bacteria, one fungus, and one yeast using the disk diffusion method. Results showed that five (9.6%) of the extracts showed some degree of activity against at least one of the microorganisms. The plants that exhibited significant antimicrobial activity, defined as a perfectly clear zone with a diameter greater than 7 mm, were Chamomilla recutita (Asteraceae), Buddleja hybrida (Buddlejaceae), and Glinus lotoides (Molluginaceae), and the latter extract was most active. Therefore, these three potent extracts were subjected to partial fractionation with successive organic solvents.
Data in concerning the antimicrobial potency of different organic extracts proved that the methanol extract of G. lotoides showed a potent effect against all tested microorganisms, and it was especially active against the dermatophytic fungus Aspergillus niger Inhibition Zone (I.Z) = 18 mm). The most susceptible microorganisms were Escherichia coli (the Gram-negative bacterium), which were easily inhibited by six (40%) of the extracts. On the other hand, out of all extracts, two extracts only of G. lotoides inhibited A. niger growth. The greatest zone of inhibition (22 mm) was displayed by the n-butanol extract of G. lotoides against A. niger, while B. hybrida methanol and n-butanol extracts showed significant inhibitory activity against bacteria and not against fungi. The 95% methanol acted as negative control and showed no inhibitory effect against the tested microorganisms, whereas the positive control showed inhibition diameters ranging from 14 to 20 mm (ampicillin) against bacteria and from 11 to 14 mm (nystatin) against fungi.
Table 3 Antimicrobial activity (inhibition zone diameter) of some successive organic extracts of Chamomilla recutita, Buddleja hybrida, and Glinus lotoides against different microorganisms.
Chamomilla recutita flower (chamomile) is one of the most popular herbal teas that has been used traditionally for medicinal purposes. It has moderate in vitro antioxidant and antimicrobial activity against other microorganisms. In animal models it has potent anti-inflammatory, antimutagenic, antispasmodic, and cholesterol-lowering effects (CitationBol Shakova et al., 1998; CitationMcKay & Blumberg, 2006). Also, the in vitro anti–Helicobacter pylori activity of the plant extract has recently been shown (CitationStamatis et al., 2003). Many of these activities were attributed to the presence of flavonoids such as apigenin (CitationAvallone et al., 2000; CitationSvehlikova et al., 2004). The results of the study supported the folkloric use of this plant.
Glinus lotoides methanol extract displayed the broadest spectrum of activity, being active against all the microorganisms tested. However, despite many published reports dealing with the bioactivity of extracts from G. lotoides and Buddleja spp. (CitationBiswas et al., 2006; CitationCortes et al., 2006; CitationLee et al., 2006; CitationReid et al., 2006), nothing was known about their antimicrobial activity against some pathogens prior to our investigation. Thus, to the best of our knowledge, this work is the first report of most of the active plants. However, the antifungal activity of terpenoids from other Buddleja spp. were described against the soil fungi Fusarium culmorum and Sordari fimicola (CitationHoughton et al., 2003). Also, the phytochemical analysis carried out in the current investigation confirms the finding by CitationNostro et al. (2000) that high terpene and flavonoid contents in the plant extracts are responsible for their antimicrobial activity.
Plant extracts differ significantly in their activities against the tested microorganisms, yet the following results showed that E. coli (Gram-negative bacteria) is more sensitive toward many of these extracts than are Gram-positive bacteria. This is in accord with other reports (CitationEassa & Beih, 2002). However, CitationNostro et al. (2000) concluded that the activity of some extracts was more pronounced against Gram-positive and fungal organisms than against Gram-negative bacteria. The authors attributed these differences in susceptibility between various microorganisms to the difference in the structure of their cell membrane, which may be permeable to some chemical compounds and not permeable to the other compounds.
Phytochemical screening
The phytochemical study (using chemical tests, TLC, and PC) of the active fractions obtained from successive fractionation of the four methanol extracts were carried out. This may give a preliminary idea about the relationship between activity and phytochemicals present in each active extract (). In C. recutita, it was found that the major constituents are phenolic compounds, especially flavonoids and coumarins, which are found in the chloroform extract as aglycone and in the ethyl acetate as aglycone and/or monoglycosides. The n-BuOH fraction of B. hybrida was found to contain a moderate amount of phenolic compounds and large contents of saponins. The EtOAc and n-BuOH active extracts of G. lotoides contained moderate amount of phenolic compounds, whereas a great amount of saponin compounds was found only in the n-BuOH fraction. Curcuma longa is a familiar species so it was not subjected to phytochemical analysis in this study. However, TLC (solvent system CH2Cl2:MeOH, 19:1) of the four different successive extracts pet. ether, CHCl3, EtOAc, and n-BuOH, with the crude methanol extract of C. longa, showed the presence of three yellow major spots in CHCl3 and MeOH extracts, with a moderate amount in EtOAc and absent in both pet. ether and n-BuOH. On spraying with methanol KOH, a red-brown color appeared, especially for the three spots, which indicated that these compounds are curcuminoids derivatives. This may be the reason for presence of activity in CHCl3 and EtOAc and the disappearance of activity in pet. ether and n-BuOH extracts.
Table 4 Phytochemical screening of active successive organic extracts of Chamomilla recutita, Buddleja hybrida, and Glinus lotoides.
Based on these data, it can be finally recommended that future investigations would be focused on the in vivo antimicrobial and antischistosomal activities and on chemical identification of the potent ingredients of these plants for further exploration of their potential efficacy.
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