Organized medicinal systems like most of the developing countries have adopted traditional medical practice as an integral part of their culture. Historically, all medicinal preparations were derived from plants, whether in the simple form of raw plant materials or in the refined form of crude extracts, mixtures, etc.,. It was suggested that several thousands of plants have been known with medicinal applications in various cultures [1].

Spices and herbs have been used for thousands of centuries by many cultures to enhance the flavor and aroma of foods. Early, cultures also recognized the value of using spices and herbs in preserving foods and for their medicinal value. Scientific experiments since the late 19th century have documented the antimicrobial properties of some spices, herbs, and their components [2].

Bioactive principles isolated from plants appear to be one of the alternatives for the control of the antibiotic resistant plant and human pathogens. Though the literature is replete with information on the antimicrobial effect on human pathogenic bacteria, a similar investigation on plant pathogens has not been given an adequate attention. Acacia nilotica (Fabaceae) showed significant antibacterial activity [3].

Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine [4]. The beneficial medicinal effects of plant materials typically result from the combinations of secondary products present in the plant. In plants, these compounds are mostly secondary metabolites such as alkaloids, steroids, tannins, and phenol compounds, which are synthesized and deposited in specific parts or in all parts of the plant. These compounds are more complex and specific and are found in certain taxa such as family, genus and species, but heterogeneity of secondary compounds is found in wild species [5,6].

Experimental agents derived from natural products offer opportunities to evaluate not only totally new chemical classes of antitumor agents, but also novel and potentially relevant mechanisms of action [7]. Plants have been a main source of highly effective drugs for the treatment of many forms of tumor. In many instances, the actual compound isolated from the plant may not serve as the drug, but leads to the development of potential novel agents. The ability to attach agents to carrier molecules directed to specific tumors holds promise for the effective targeting of highly cytotoxic natural products to the tumors while avoiding their toxic side effects on normal healthy tissues [8].

Interest in ethnobotany has increased dramatically. Use of ethnobotanical information in medicinal plant research has gained considerable attention in segments of the scientific community [9]. Natural crude extracts and biologically active compounds isolated from plant species used in traditional medicine can be prolific resources for new drugs [10].

There are two main strategies for the selection of plants species in antitumor drug discovery. The First approach includes random screening and ethnomedical knowledge. The second approach includes plants used in organize traditional medical systems like herbalism and folklore [11].

Zygophyllin and quinovic acid exhibited anti-inflammatory activity, cortisone-like action, choleretic and antipyretic activities. The aqueous extract produced lowering in blood pressure, diuretic, antipyretic, local anesthetic and antihistaminic activities [12].

The aims of this study included investigating the antimicrobial larvicidal and cytotoxic properties of Z. coccineum collected from north Sinai, Egypt were evaluated.

Medicinal plants used in this study

Medicinal plant used in this study was collected from the North Sinai (Wadi El Maghara) Zygophyllum coccineum. Fresh areal parts leaves and stem of medicinal plants were stored in air tight container and maintained at 4°C until use to store and minimize the loss of volatile compounds.

Test organisms for Antimicrobial assay

Gram positive bacteria Staphylococcus aureus ATCC 29213, Methicillin-resistant, Staphylococcus aureus (MRSA) clinical isolate Bacillus subtilis NRRL B 543, Alcaligenes faecalis ATCC 29217 and Enterococcus faecalis ATCC 29212.

Gram negative bacteria were Enterobacter cloacae ATCC 13047, Klebsiella pneumonia ATCC 13883, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 10145.

Unicellular Fungi Candida albicans MTCC183, Candida glabrata clinical isolate and Candida parapsilosis clinical isolate.

Filamentous fungi such as Aspergillus niger NRRL 595, Trichoderma viride RCMB 017002, Trichoderma longibrachiatum RCMB 017015, Fusarium moniliforme RCMB 008002, Rhizopus stolonifer RCMB014001, Curvularia clavata RCMB 019003 and Penicillium aurantiacum RCMB 001025 (Figure 1).

Figure 1: Cytotoxic evaluation of Z. coccineum on HaLa cell line viability (Mean ± s.d., n=3).

Mammalian cell lines

Mammalian cell lines: HeLa (cervical carcinoma cell line) and MCF-7 (human breast adeno-carcinoma cell line) and Vero (African green monkey kidney cells) were purchased from VACSERA, Egypt.

Plant Identification: Identification of plant species in a given stand has been tentatively recorded in the field and the authentication of their identification with the help of the documented floristic workers [13,14].

Preparation of plant extracts for in vitro testing: Different organic solvent were used for extraction of antimicrobial and cytotoxic substances from medicinal plants. Twenty gram of the aerial parts (leaves and stem) of all dried medicinal plants in this study was macerated in mortar dissolved in 100 ml of different organic solvents (methanol, ethyl alcohol (absolute), acetone, petroleum ether and ethyl acetate). Also, aqueous extract were prepared by boiled distilled water for 5 minutes left and then filtered using No.1 filter paper. Medicinal plants extracts were evaporated till dryness under reduced pressure using rotary vacuum evaporator at 40°C stored in sterile screw capped vials in the refrigerator until needed. Each concentrated crude extract was separately sterilized by filtration and further dilutions were made from the stock (Figures 2 and 3).

Figure 2: Microphotographs characteristic showing the inhibitory effect of boiled water extract of Z. coccineum on HeLa cell growth under Phasecontrast microscopy (A) control HeLa cells at 24 h. (B) HeLa cells treated with 50 μl at 24 h. Magnification: × 40

Figure 3: Cytotoxic evaluation of Z. coccineum on MCF-7 cell line viability (Mean ± s.d., n=3).

Determination of antimicrobial activity by Agar Well diffusion method

The Agar well diffusion technique has been widely used to assay plant extract for antimicrobial activity based on the observation of inhibition of microbial growth on microbial nutrient media. The previous mentioned human pathogenic bacteria Gram-positive and Gram-negative bacteria as well as unicellular and filamentous fungi were used in this test. For examination of antimicrobial activity nutrient agar for bacteria, Malt agar for fungi and Sabouraud agar medium for unicellular fungi were prepared and seeded with one of the tested organisms (100 μl of bacterial culture in 20 ml medium and 100 μl of fungal spore suspension in 25 ml broth medium). After solidification holes in media were made by cork-borer then medicinal plants extracts 100 μl were loaded into holes. Petri dishes were kept in refrigerator for 2 h, for diffusion of tested substances before incubation at 37°C for 24 h for bacteria, yeast and fungi 48 h at 28°C to allow complete diffusion of tested substances inside culture media after growth of test organisms. The detection of a clear zone around the well on the inoculated plates is an indication of antimicrobial activities of the medicinal plants extracts under study [15] (Tables 1 and 2).

Table 1: Antibacterial evaluation of Z. coccineum extracts by using agar well diffusion method. The data are expressed as the mean in inhibition zone of diameter in mm ± standard deviation.

Table 2: Antifungal evaluation of Z. coccineum extracts by using by using agar well diffusion method.

Minimum inhibitory concentration (MIC) using dilution method

The series of double fold were performed in 96-well microtitre plates with U-shaped wells, using two-fold serial dilutions. One hundred μl of medicinal plants extracts were added to the first well and mixed. The series of douple-fold dilutions were done until the last well of the microwell plate and then 100 μl of bacterial suspension was added in respective wells and in control wells. The plates were sealed, placed in plastic bags and incubated at 37°C for 24 h. The MIC which is defined as the lowest concentration of extract that exhibited no growth by visual reading [16].

Cytopathic effect assay

The cytotoxicity medicinal plant extracts were measured by the cytopathic effect assay. The assay was carried out using 100 μl of cell suspension, containing 10,000 cells seeded in each well of a 96-well microtitre plate. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Then, serial two-fold dilutions of the medicinal plants extracts were added to confluent cell monolayer. The microtitre plates were incubated at 37°C in a humidified incubator with 5% CO₂ for a period of 48 h. Six wells were used for each concentration of the test sample. Control cells were incubated without test sample. After the incubation period, media were aspirated and the cells were fixed with 10% formalin solution for at least 20 min. The fixed cells were rinsed with phosphate buffer solution (PBS) then stained with a 1% crystal violet for 1 hour. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. The cell cultures were examined for evidence of the cytopathic effect that observed microscopically as detectable alterations. The stained cells were lysed by using glacial acetic acid solution and then plates were read on ELISA reader, using a test wave length under 490 nm [17].

Evaluation of the larvicidal activity against the mosquito vector Culex pipiens L.

Mosquito culture: The mosquito C. pipiens L.was obtained from Medical Entomology Research Center, Doqqi, Giza. The sample was reared for several generations in the Department of Zoology, Faculty of science Al-Azhar University, Madenit Nasr, Cairo under controlled conditions (27 ± 2°C, RH 70 ±10% and 12 light-dark regime). Adult mosquitoes were kept in (30 × 30 × 30 cm) wooden cages and daily provided with sponge pieces soaked in 10% sucrose solution for a period of 3-4 days after emergence. After this period the females were allowed to take a blood meal from a pigeon host. Plastic cup oviposition (15 × 15 cm) containing tap water was placed in the cage. The resulting egg rafts picked up from the plastic dish and transferred into plastic pans (25 × 30 × 15 cm) containing 3 liters of tap water left for 24 h. The hatching larvae were provided daily with fish food as a diet [18].

Larval treatment: In order to study the toxicity of the medicinal plant extracts, different concentrations of each extract were prepared in order to detect mortalities (200, 100, 50 and 25 ppm). All tested plant dried extracts were performed in 100 ml. of tap water contained in 200 ml plastic cups. Then, third instar larvae were put immediately into plastic cups contained different concentrations of extracts. At least three replicates were usually used for each tested concentration. All plastic cups were incubated under controlled conditions (27 ± 2°C, RH 70 ±10% and 12 light-dark regime). Control larvae not treated with any plants 100 ml water.

Mortality was recorded daily and the dead larvae. Abnormally formed pupae were removed daily and Placed in labeled glass vials containing 70% ethanol and one drop of glycerin for the photography under binocular microscope. Larval mortality was indicated by a failure to respond to mechanical stimulation [19].

Motility %=dead larva/ control × 100.

Separation and fractionation of the active compounds

Column chromatography: Prokaslo- England Column chromatography (20 × 2 cm). Ethyl acetate and acetone with different ratio were used as eluting solvent packed with Silica gel 60 GF 200 (MERK). Fractions were collected and assayed for their antimicrobial and cytotoxic activity. Finally the column eluted with methanol to wash and remove the remnant of biologically active compound (Figure 4).

Figure 4: The suggested chemical for the purified compound of ethyl acetate extract Z. coccineum from column chromatography Fraction No. 3 at TLC violet band at Rf 0.62 was suggested as 2-(3,4-Dihydroxyphenyl)-3,5,7- trihydroxy-6-methoxy-4-benzopyrone with molecular Formula C₁₆H₁₂O₈

Predicting the chemical structure: The spectroscopic data were employed to reach complete characterization of the chemical structure and molecular formula (Graph 1-3).

Graph 1: Ultra violet spectroscopy of the purified compound ethyl acetate extract Z. coccineum from column chromatography Fraction No.3 at TLC violet band at R_f 0.62.

Graph 2: Infra- red spectrum of the purified compound ethyl acetate extract Z. coccineum from column chromatography Fraction No.3 at TLC violet band at R_f 0.62.

Graph 3: Mass spectrum of the purified compound ethyl acetate extract Z. coccineum from column chromatography Fraction No.3 at TLC violet band at R_f 0.62.

Statistical analysis: The data were expressed as mean ± S.D. The statistical significance of the difference mean values was determined by Student’s unpaired t-test. Data were considered statistically significant at a significance level of P<0.05.

Medicinal plants of folk-origin are significant sources of synthetic and herbal drugs. In the commercial market, folk-medicinal plants were used as raw drugs, extracts or tinctures. Isolated active constituents are used for applied research for finding their bioactivity. For the last few decades, phytochemical examination has been making rapid progress and herbal products are becoming popular as sources of antitumor compounds [3].

Antimicrobial and cytotoxicity evaluation of Zygophyllum coccineum

Antimicrobial evaluation of Z. coccineum extracts showed that ethyl acetate extract was the most potent antimicrobial effect on bacterial and fungal test organisms. Antibacterial activities of Z. coccineum ethyl acetate extract against P. aeruginosa with inhibition zone 20 mm was the most sensitive test organism. Also ethyl acetate of Z. coccineum has antifungal activity against all fungal test organisms. Ethyl acetate extracts showed that F. moniliforme was the most sensitive organisms with inhibition zone 22 mm and 20 mm against C. albicans whereas boiled water extracts showed antifungal activity against Curvularia clavata with 20 mm inhibition zone.

Cytotoxicity evaluation of Z. coccineum on HeLa cell line viability indicated that acetone, petroleum ether and boiled water showed very strong cytotoxic activity with cell line viability 13.36%, 15.36% and 16.43% respectively. Cytotoxicity evaluation of Z. coccineum MCF7 cell line viability indicated that ethyl acetate, petroleum ether and methanol showed strong cytotoxic activity with cell line viability 48.15%, 67.59% and 35.19% respectively. Nevertheless, there is no scientific report on the in vitro and in vivo study the effect of Z. coccineum as anti-tumor. Thus, this work were studied to the antitumor effect of Z. coccineum also interesting to complete our study by the in vitro study.

Evaluation of the mortality effect of Z. coccineum extracts against the mosquito vector Culex pipiens L

The extracts of Z. coccineum were studied to evaluate the insecticidal activity with 200 ppm of each extract against 3^rd instar larvae of Culex pipiens L. Mortality was recorded daily.

Ethyl acetate extract of Z. coccineum possess and induced a potent insecticidal and toxic effect against 3^rd instar larvae of Culex pipiens L with 100% mortality after 24 h of treatment. So, the different concentrations of ethyl acetate of Z. coccineum were be studied to know the lowest conc. of ethyl acetate which possess toxic effect against 3^rd instar larvae of Culex pipiens L with 100% mortality after 24 h of treatment. Also, ethanol extract of Z. coccineum possess and induced a potent insecticidal and toxic effect against 3rd instar larvae of Culex pipiens L with 58.07% mortality after 24 h of treatment and possess 100% mortality the 5^th day of treatment as appeared in Table 3.

Table 3: Evaluation of the mortality effect of Z. coccineum extracts against the mosquito vector Culexpipiens L.

Different concentrations of ethyl acetate extract was selected to evaluate the lowest conc. used as insecticidal activity of Z. coccineum with 100, 50, and 25 ppm against 3^rd instar larvae of Culex pipiens L for 7 days. Significant differences were observed among concentrations and exposure time on mortality in each treatment. At 100 ppm showed a moderate insecticidal effect with 41.46% mortality after 24 h of treatment (Table 4).

Table 4: Evaluation of the different concentration of ethyl acetate crude extracts of Z. coccineum against the mosquito vector Culexpipiens L.

From the above results we observed that, ethyl acetate with 200 ppm conc. of Z. coccineum is the lowest conc. possess toxic effect against 3^rd instar larvae of Culex pipiens.

Minimum inhibitory concentration (mic) of ethyl acetate extracts of the tested medicinal plants

To determine the smallest amount of antimicrobial agents necessary to inhibit growth of the tested organisms. MIC crude extract of ethyl acetate extracts of the tested medicinal plants were studied against S .aureus, K. pneumonia, Enterobacter colaca and C. albicans. The results obtained in Table 5 showed that Z. coccineum were the most potent medicinal plants as antimicrobial effect with MIC 12.5 μg/ml whereas other medicinal plant extracts varied in its MIC activity. So, Z. coccineum were selected for purification and identification for their biological active compounds and also for more studies for their active compounds.

Table 5: Minimum inhibitory concentration of ethyl acetate crude extracts of the tested medicinal plants. The data is expressed in μg/ml.

Separation, fractionation and identification of the active compounds

As the above results of antimicrobial activity in this study and the results of MIC of all medicinal plants showed that ethyl acetate extract Z. coccineum were the most potent medicinal plants as antibacterial effect with MIC 12.5 μg/ml. Z. coccineum also showed significant antifungal activity so, purification and characterization of bioactive phytochemical compounds by column chromatography using different ratio of solvent system ethyl acetate and acetone and T.L.C. Toluene: Ethyl Acetate: Formic Acid (5:4:1) solvent system were used in this study.

From the results of column chromatography the antimicrobial activity of antimicrobial activity of Z. coccineum only fractions From 2 to 5 showed antimicrobial activity ranging from 13 to 22 mm of inhibition zone. The highest fraction with antimicrobial activity was fraction No 3 with inhibition zone 22 mm.

Minimum inhibitory concentration of the highest antimicrobial fractions were studied against S .aureus, K. pneumonia, Enterobacter colaca and C. albicans which showed that Fraction No 3 of Z. coccineum antibacterial effect with MIC ≤ 12.5 μg/ml (Tables 6 and 7).

Table 6: Anti-bacterial evaluation of the fractions obtained after silica gel column fraction of ethyl acetate extract of Z. coccineum against K. pneumonia.

Table 7: Minimum inhibitory concentrations (MIC) of active fractions of Z. coccineum. The data are expressed in μg/ml.

The chemical compound structure of ethyl acetate extract of Z. coccineum was identified as was suggested as 2-(3, 4-Dihydroxyphenyl)- 3,5,7-trihydroxy-6-methoxy-4- benzopyrone (Table 8).

Table 8: Nuclear magnetic resonance spectroscopy (H¹ NMR analysis).

This project was funded from Science & Technology Development Fund in Egypt Project No. 5520 Short term Fellowship.