MATERIALS AND METHODS

Plant material: The plant was collected from Matta, District Swat, NWFP, Pakistan in the month of July 2001. The plant was identified by Mehboob-ur-Rehman, plant taxonomist Government College Matta, District Swat, NWFP, Pakistan and verified by Dr.Abdur-Rashid, Chairman Department of Botany, University of Peshawar, NWFP, Pakistan, where voucher specimens were deposited in their Herbarium.

Extraction and fractionation: The shade dried plant material was chopped into small pieces and finally pulverized into fine powder. The powdered plant material (10 Kg) was soaked in methanol (3 x 10 L), with occasional shaking, at room temperature. After 15 days, the methanol soluble materials were filtered off. The filtrate was concentrated under vacuum at low temperature (40°C) using rotary evaporator. A dark greenish crude extract (253 g) was obtained.

The crude methanolic extract (253 g) was suspended in distilled water (500 ml) and partitioned with n-hexane (3 x 500 ml), chloroform (3 x 500 ml), ethyl acetate (3 x 500 ml) and n-butanol (3 x 500 ml) to yield the n-hexane (24 g), chloroform (61 g), ethyl acetate (30 g), n-butanol (35 g) and aqueous (73 g) fractions, respectively.

Screening of extract and its various fractions for different pharmacological and biological activities: The crude methanolic extract and various fractions thereof including chloroform, ethyl acetate, n-butanol and aqueous fractions were screened for different pharmacological and biological activities such as antifungal, antibacterial, insecticidal, brine-shrimp cytotoxic and phytotoxic activities.

Antifungal activities: Antifungal activity of the crude extract and various fractions were evaluated by agar tube dilution method (Atta-ur-Rehman et al., 1991). The samples in the concentration of 24 mg ml^-1 were dissolved in sterile dimethyl sulfoxide (DMSO), which served as stock solution. Sabouraud dextrose agar (SDA) (4ml) was dispensed into screw cap tubes, which were autoclaved at 121°C for 15 min. and then cooled to 50°C. The non-solidified SDA media was poisoned with stock solution (66.6 μl), giving the final concentration of 400 μg of the extract/ml of SDA. Each tube was inoculated with a piece (4 mm diameter) of inoculum removed from a seven days old culture of fungi. For non-mycelial growth, an agar surface streak was employed. Inhibition of fungal growth was observed after 7 days of incubation at 28±1°C. A control experiment with test substance (medium supplemented with appropriate amount of DMSO) was carried out for verification of the fungal growth.

Antibacterial activities: The crude extract and its fractions were screened against various human pathogens including, Escherichia coli, Bacillus subtilis, Klebsiella pneumonia, Shigella flexenari, Staphylococcus aurous, Pseudomonas aeruginosa and Salmonella typhi, by agar well diffusion method (Atta-ur-Rehman et al., 1991).

In this method 1 loop full of 24 h old culture containing approximately 10⁴-10⁶ CFU was spread on the surface of Mueller-Hinton Agar plates. Wells were dug in the medium with the help of a sterile metallic cork borer. Stock solutions of the test samples in the concentration of 1mg ml^-1 were prepared in dimethyl sulfoxide (DMSO) and 100 μl and 200 μl of each dilution was added in their respective wells. Control well received only 100 μg μl^-1 and 200 μg μl^-1 of DMSO. Imipinem was used as the standard drug.

Insecticidal activities: Tribolium castaneum, Rhyzopertha dominica and Callosbruchus analis were used to determine the insecticidal activity of the crude extract and its various fractions (Naqvi and Parveen, 1991).

Phytotoxic activities: Phytotoxic activity of the crude extract and various fractions were carried out against the Lemna acquinoctialis welv. (McLaughlin et al., 1991). The medium was prepared by mixing various constituents in distilled water (100 ml) and the pH was adjusted (5.5 - 6.5) by adding KOH solution. The medium was then autoclaved at 121^oC for 15 min. The samples (30.0 mg) dissolved in ethanol (1.5 ml) served as stock solution. Nine sterilized flasks, three for each concentration, were inoculated with 1000 μl, 100 μl and 10 μl of the stock solution to give the final concentration of 1000, 100 and 10 μg ml^-1 respectively. The solvent was allowed to evaporate overnight under sterile conditions. To each flask, medium (20 ml) and plants (10), each containing a rosette of three fronds, of Lemna acquinoctialis welv., were added. One other flask supplemented with solvent and reference growth inhibitor (Paraquate), served as negative control. All flasks were plugged with cotton and kept in the growth cabinet for seven days. The number of fronds per flask were counted and recorded on day seven.

Brine-shrimp cytotoxicity: Artemia salina (brine-shrimp eggs) was used to determine the cytotoxic activity of the crude extract and various fractions thereof (Meyer et al., 1982).

RESULTS AND DISCUSSION

The shade dried plant material of whole plant of Chenopodium murale Linn. was macerated with methanol. The crude methanolic extract was suspended in water and partitioned with n-hexane, chloroform, ethyl acetate and n-butanol to yield the n-hexane, chloroform, ethyl acetate, n-butanol and aqueous fractions, respectively.

Antifungal activity of the crude extract and its fractions of Chenopodium murale were tested against Trichophyton longifusus, Candida albicans, Aspergilus flavus, Microsporum canis, Fusarium solani and Candida glaberata. Growth in the medium containing the extracts was determined by measuring the linear growth in mm and the % growth inhibition was calculated with reference to the negative control. The results (Table 1) indicate that the crude extract exhibited a good antifungal activity (70%) against Trichophyton longifusus while it displayed a weak inhibition (30%) against Microsporum canis. The crude extract was devoid of any antifungal inhibition against the rest of tested fungi. Table 1 also shows that CHCl₃ fraction showed good antifungal activities against Trichophyton longifusus (75%), Microsporum canis (65%) and Candida glaberata (57%) while it devoid of any activity against the rest of pathogens. The EtOAc fraction displayed an overall moderate antifungal activity with an excellent activity against Trichophyton longifusus (80%). However, this fraction did not show any activity against Aspergilus flavus. The n-BuOH fraction showed excellent activity against Trichophyton longifusus (90%) while having moderate antifungal activity against Candida albicans (55%) and Microsporum canis (45%), while a weak activity against Fusarium solani (10%) with no activity against Aspergilus flavus and Candida glaberata. The aqueous fraction of the extract of C. murale displayed good activity against Trichophyton longifusus (65%). However, the aqueous fraction did not show any activity against the rest of fungi. Table 1 shows that all the fractions have well to excellent (65–90%) antifungal activity against the Trichophyton longifusus. The same table also shows that none of fractions have displayed any activity against Aspergilus flavus.

The antibacterial activities of the crude extract as well the fractions thereof of C. murale were tested against the bacteria Escherichia coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi. The results are given in Table 2, which shows that the crude extract displayed moderate inhibitory activities against E. coli (50.00%), B. subtilis (58.06%) and P. aeruginosa (64.00%), while it was devoid of any antibacterial activities against the rest of the pathogens.

Fig. 1:	Phytotoxic activities of crude methanolic extract and fractions of C. murale

The Table 2 also indicates that CHCl₃ fraction showed good inhibition (60.00%) against E. coli and moderate inhibition (46.51%) against S. aureus, while it was devoid of any antibacterial inhibition against the rest of tested bacteria. The EtOAc fraction also displayed moderate inhibitory activities (46.66%) and (51.51%) against E. coli and S. flexenari respectively, while it was ineffective against the rest of the bacteria. The n-BuOH fraction exhibited weak inhibition (33.33%) against E. coli and moderate inhibitions (41.93%) and (48.00%) against B. subtilis and P. aeruginosa, respectively. However, this fraction did not display any inhibitory activities against the rest of the tested bacteria. The aqueous fraction exhibited good inhibition (63.63%) against S. flexenari and weak inhibition (20.90%) against S. aureus, while it was devoid of any inhibitory activities against the rest of the tested pathogens.

The Insecticidal activities were checked against Tribolium castaneum, Rhyzopertha dominica and Callosbruchus analis. Results in the Table 3 shows that CHCl₃ fraction displayed mild activity against Rhyzopertha dominica (25%) and Callosbruchus analis (20%). The EtOAc fraction showed moderate activity against Callosbruchus analis (50%). The n-BuOH fraction also exhibited mild activity against Tribolium castaneum (30%) and Callosbruchus analis (26%). While in rest of the cases no activities were observed.

Results of the phytotoxic activities of the crude extract and its fractions from C. murale were interpreted by analyzing the growth regulation in (%) calculated with reference to the negative control. Paraquate was used as standard inhibitor (0.902 μg ml^-1).

Table 1:	Antifungal activities of crude methanolic extract and fractions of C. murale

Table 2:	Antibacterial activities of crude methanolic extract and fractions of C. murale

Table 3:	Insecticidal activities of crude methanolic extract and fractions of C. murale

The negative values show the growth promotion of the plant. The results (Fig. 1) showed that the crude extract has remarkable phytotoxic activity against Lemna acquinoctialis welv. at highest concentration (1000 μg ml^-1) and caused complete (100%) inhibition of the plant growth. However, it was completely inactive (0.00%) at (100 μg ml^-1) concentration while at further lower concentration (10 μg ml^-1) it rather promoted the growth of Lemna acquinoctialis by 10%. The CHCl₃ extract also showed excellent inhibition (100%) of Lemna acquinoctialis at highest concentration (1000 μg ml^-1) however, it displayed a weak phytotoxic activity at lower concentrations (23.33% at 100 μg ml^-1) and (6.66% at 10 μg ml^-1). The EtOAc fraction of the crude extract of C. murale also displayed maximum inhibition (100%) of L. acquinoctialis at highest concentration (1000 μg ml^-1). The lower concentration (100 μg ml^-1) did not show remarkable phytotoxic activity and inhibited the growth of the L. acquinoctialis by only 16.66%. While at the lowest concentration tested (10 μg ml^-1) it promoted the growth of Lemna acquinoctialis by 26.66%. Following the same pattern of activity the n-BuOH fraction also showed maximum inhibition (100%) to Lemna acquinoctialis. The same fraction at lower concentration (100 μg ml^-1 and 10 μg ml^-1) did not show a significant phytotoxic activity and caused inhibition by only 23.33 and 3.33%, respectively. The aqueous fraction was unique in a sense that it promoted the growth of Lemna acquinoctialis at all tested concentrations. It promoted the growth of the plant by 3.33, 30.00 and 13.33%, respectively at concentrations of 1000, 100 and 10 μg ml^-1.

The crude extract and its fractions from the C. murale were also screened for Brine Shrimp cytotoxicity studies but did not display any significant activity in this bioassays.

It is concluded from this study that all the tested samples exhibited well to excellent (65–90%) antifungal activity against the Trichophyton longifusus, while none of them displayed any activity against Aspergilus flavus. In rest of the cases all samples showed good activity against different pathogens in antifungal bioassay.

The crude methanolic extract and its various fractions of C. murale also exhibited mild to moderate inhibitory activities against different bacteria, while they were devoid of any antibacterial activities against other pathogens in the same study.

During this study it was observed that no significant insecticidal and brine shrimp cytotoxic activities were displayed by the tested samples.

It was also observed that the crude extract and its various fractions (except aqueous) showed remarkable phytotoxic activity against Lemna acquinoctialis welv. at highest concentration (1000 μg ml^-1). However, they displayed weak phytotoxic activity at lower concentrations (100 μg ml^-1), while at lowest concentration (10 μg ml^-1) few of them rather promoted the growth of Lemna acquinoctialis welv. The aqueous fraction promoted the growth of Lemna acquinoctialis welv. at all tested concentrations.

ACKNOWLEDGMENT

We are thankful to Dr. Zafar Iqbal, Associate Professor, Department of Pharmacy, Dr. Abdur-Rashid, Chairman Department of Botany, University of Peshawar, Dr. Muhammad Iqbal Choudhry, Professor, HEJ Research Institute of Chemistry, University of Karachi and Mr. Mehboob-ur-Rehman, Plant Taxonomist, Govt. College Matta, District Swat for technical help.