INTRODUCTION
Five macrohydrophytes representing the different forms of aquatic vegetation in the Nile Delta region have been selected for the present investigation. Bolboschoenus glaucus and Veronica anagallis-aquatica are belonging to emergent hydrophytes, Nymphaea lotus is belonging to rooted floating hydrophytes, Pistia stratiotes is belonging to free floating hydrophytes and Myriophyllum spicatum is belonging to rooted submerged hydrophytes.
B. glaucus is a small grass-like perennial sedge of saline to fresh watershores (Browning, 1998). It was able to bioaccumulate and phytostabilization of Cd and Pb in its roots (Almedia et al., 2006).
V. anagallis-aquatica is a perennial herb, often 4-angled towards the base, commonly spread in marshy ground, river-banks and irrigation channels (Boulos, 2002). Pandey and Sirvastava (1989), Harput et al. (2004) and Kupeli et al. (2005) mentioned that, Veronica, a semi-aquatic weed, is a potential source of leaf protein and iridoid glycosides.
N. lotus is herbaceous aquatic plant, whose leaves floats or submerged in water. It is a good phytoaccumulator and can selectively bioaccumulate heavy metals particularly zinc and lead (Khedr and Hegazy, 1998).
P. stratiotes is a free-floating stoloniferous herb commonly found in ponds and streams. Its leaves are obovate, light green in colour and have many prominent longitudinal veins (Arber, 1991). The oil extracted from Pistia is used in the treatment of worm infestations, tuberculosis and dysentery and is applied externally to treat skin diseases, inflammation, piles, ulcers and burns (Kirtikar and Basu, 2000). Pistia leaves possess antifungal properties that explain the use of this plant in folk medicine for the treatment of various diseases whose symptoms might involve fungal infections (Premkumar and Shyamsundar, 2005).
M. spicatum is a fresh water angiosperm that contains high concentrations of tannins and secondary metabolites known for their antimicrobial properties (Walenciak et al., 2002). Elisabeth et al. (1996) stated that aqueous acetone extract of the shoot of M. spicatum exhibit an inhibitory action against various coccoid and filamentous Cyanobacteria.
The present study aims at evaluation of the periodical changes in the vegetative yield, growth characteristics, chemical constituents and antimicrobial bioactivities of the investigated plants.
MATERIALS AND METHODS
The plant samples were collected monthly for one year from their natural habitats
using quadrates (50x50 cm).Samples were taken along two parallel transects located
in the central portion of the representative stand and new quadrate location
were selected so that production was not influenced by previous sampling (Clark
and Clay, 1984). Ten individuals of each species were randomly chosen and used
for measurement of the growth parameters; mean height of stems or length of
stolons, number of leaves and their areas. Plants of each quadrate were air-dried
and the biomass of the different plant parts were measured separately and expressed
as g dry wt. m-2 (Cochran, 1963; Polisetty et al., 1984).
Data of the successive estimation of the assimilating surface area and the biomass
were applied to estimate growth characteristics as described by Radford (1967),
Chapman (1976) and Coombs and Hall (1982). The growth characteristics measured
are: Relative Growth Rate (RGR), Relative Assimilating Surface Growth Rate (RASGR),
Net Assimilation Rate (NAR) and Leaf Area Ratio (LAR). For phytochemical analysis,
plant samples were collected, handly cleaned, air dried and ground to fine powder.
In each sample, moisture content, total ash, water soluble ash, acid insoluble
ash, total nitrogen, total protein, total lipid, crude fiber, total soluble
sugars, glucose, sucrose and polysaccharides were determined according to the
methods adopted by Ward and Johnson (1962), Handel (1968) and Thayumanavan and
Sadasivam (1984). Phytochemical screening was carried out using the powdered
samples and the alcoholic extracts to detect the active principles: Glycosides,
sterols, alkaloids, flavonoides, tannins, saponins and resins according to Claus
(1967), Harper (1975) and Markham (1982). For extraction of the different elements,
0.1 g of air-dried powder was digested by concentrated HNO3, heated
gently until the solution turned quite clear. The samples were made up to a
known volume by distilled water. Na, K and Ca were determined by flame photometer,
while Mg, Fe, Mn, Zn, Cu, Ni, Cd, Pb and As were estimated by atomic absorption
spectrometer (Allen et al., 1974). The elements were expressed as mg/100
g dry weight. For antimicrobial screening, methanolic extracts were prepared
using 100 g of each powdered samples and 400 mL of 80% methanol by refluxing
for 3 h. A stock solution of extract was prepared in dimethyl sulfoxide (DSMO)
and kept at -20°C for antimicrobial assay (Mehraban et al., 2005).
The bacterial strains that used as tested organisms are Bacillus subtilis,
Erwinia cartovora, Escherichia coli, Pseudomonas fluorescence
and Staphylococcus aureus, while the tested fungi are Alternaria
alternata, Aspergillus niger, Bibolaris oryza, Botrytis
faba, Fusarium oxysporium and Penicillium chrysogenum. The
extracts were screened for their inhibitory activities against the tested bacteria
and fungi using agar diffusion technique (Calvo et al., 1986; Deans and
Ritchie, 1987). After inoculation with constant inoculums, the plates were incubated
for 24 h for the bacterial strains and 3-4 days for fungi. Controls had solvent
(DSMO) without extracts of the tested plants. The antimicrobial bioactivity
was determined by the measuring the diameters of inhibition zones in cm.
RESULTS AND DISCUSSION
Vegetative yield: Records of the monthly variations in the assimilating
surface area (cm2 m-2) and biomass content (g dry wt.
m-2) of B. glaucus, M. spicatus, N. lotus,
P. stratiotes and V. anagallis-aquatica are demonstrated in Fig.
1-10. The maximum assimilating surface area of the five
species (4277.3, 94580.0, 66652.9, 89347.7 and 11774.2 cm2 m-2,
respectively) were attained in August except veronica in June. The phytomass
showed a similar trend, increasing gradually from February till reached its
peak in August (16.6, 39.8, 251.8, 80.2 and 57.9 g dry wt. m-2, respectively)
and coincided with the maximum leaf, stem and root biomass. At maturity stage,
which begins in September, it showed a gradual decline. The maximum necromass
was in January.
Growth characteristics: The monthly changes in the Relative Growth Rate
(RGR) of the studied species are presented in Fig. 11-15.
It is obvious that, the total RGR is generally higher at early vegetative stage
than at maturity (fruiting stage). The maximum RGR of B. glaucus and
M. spicatum (0.0081-0.0129 g g-1 day-1, respectively)
were recorded in winter (January-February), those of N. lotus and P.
stratiotes were 0.0408-0.0132 g g-1 day-1, respectively
in spring (March-May) and that of V. anagallis-aquatica was 0.0515 g
g-1day-1 during October. The results demonstrated in Fig.
16-20 indicated that, the highest Relative Assimilating
Surface Growth Rate (RASGR) of leaves and stem of B. glaucus were 0.015
and 0.03 cm2 (cm2)-1 day-1, respectively
then tend to decline and even became negative sign. The RASGR of M. spicatum
leaves (0.001-0.027 cm2 (cm2)-1 day-1)
and stem (0.001-0.017 cm2 (cm2)-1 day-1)
were increased at the vegetative stage and beginning of flowering. At fruiting,
RASGR as well as RGR suddenly became a negative sign. The monthly changes of
RASGR of leaf lamina of N. lotus fluctuated between 0.0396 and 0.0572
cm2 (cm2)-1 day-1. Those of
P. strariotes ranged between 0.007 and 0.033 cm2 (cm2)-1
day-1 and V. anagallis-aquatica had (0.002-0.065 cm2
(cm2)-1 day-1).
|
| Fig. 1: |
Monthly variation in the assimilating surface area of Bolboschoenus
glaucus |
|
| Fig. 2: |
Monthly variation in the biomass of B. glaucus |
|
| Fig. 3: |
Monthly variation in the assimilating surface area of Myriophyllum
spicatum |
|
| Fig. 4: |
Monthly variation in the biomass of Myriophyllum spicatum |
|
| Fig. 5: |
Monthly variation in the assimilating surface area of Nymphaea
lotus |
|
| Fig. 6: |
Monthly variation in the biomass of Nymphaea lotus |
|
| Fig. 7: |
Monthly variation in the assimilating surface area of Pistia
stratiotes |
|
| Fig. 8: |
Monthly variation in the biomass of Pistia stratiotes |
|
| Fig. 9: |
Monthly variation in the assimilating surface area of Veronica
anagallis-aquatica |
|
| Fig. 10: |
Monthly variation in the biomass of Veronica anagallis-aquatica |
|
| Fig. 11: |
Monthly variation in RGR of Bolboschoenus glaucus |
|
| Fig. 12: |
Monthly variation in RGR of Myriophyllum spicatum |
|
| Fig. 13: |
Monthly variation in RGR of Nymphaea lotus |
|
| Fig. 14: |
Monthly variation in RGR of Pistia stratiotes |
|
| Fig. 15: |
Monthly variation in RGR of RGR of Veronica anagallis-aquatica |
|
| Fig. 16: |
Monthly variation in RASGR of Bolboschoenus glaucus |
As illustrated in Fig. 21-25, the highest
values of Net Assimilation Rate (NAR) were recorded in October for B. glaucus
(0.00004 g (cm2)-1 day-1), in February for
M. spicatum (0.0108 g (cm2)-1 day-1),
in July for N. lotus (0.1332 g (cm2)-1 day-1)
and in September for P. stratiotes (0.015 g (cm2)-1
day-1) and in December for V. anagallis-aquatica. The Leaf
Area Ratio (LAR) of the studied plants showed gradual increase from July to
October then decline during November and December (Fig. 26-30).
|
| Fig. 17: |
Monthly variation in RASGR of Myriophyllum spicatum |
|
| Fig. 18: |
Monthly variation in RASGR of Nymphaea lotus |
|
| Fig. 19: |
Monthly variation in RASGR of Pistia stratiotes |
|
| Fig. 20: |
Monthly variation in RASGR of Veronica anagallis-aquatica |
From the above results it can be concluded that, the assimilating surface area
and the biomass content increased gradually with advanced age then declined
at the beginning of fruiting stage. Sometimes these two growth parameters elevated
again due to appearance of new branches (El-Habibi et al., 1988).
|
| Fig. 21: |
Monthly variation in NAR of Bolboschoenus glaucus |
|
| Fig. 22: |
Monthly variation in NAR of Myriophyllum spicatum |
|
| Fig. 23: |
Monthly variation in NAR of Nymphaea lotus |
|
| Fig. 24: |
Monthly variation in NAR of Pistia stratiotes |
|
| Fig. 25: |
Monthly variation in NAR Veronica anagallis-aquatica |
|
| Fig. 26: |
Monthly variation in LAR of Bolboschoenus glaucus |
|
| Fig. 27: |
Monthly variation in LAR of Myriophyllum spicatum |
The relative assimilating surface growth rate showed the same trend of relative
growth rate of these plants. The periodical fluctuation in the growth characteristics
may be attributed to temperature changes. These findings are in accordance with
those of Parsons (1980) and Papchenkov (1985). Abo El-Lil (1987) stated that,
at the period of maturity, the dehydrated nutrient substances accumulated in
the ripening seeds and with fruiting the lower leaves are about to fall. The
reduction of weight may be related to these senescence phenomena in addition
to competition.
|
| Fig. 28: |
Monthly variation in LAR of Nymphaea lotus |
|
| Fig. 29: |
Monthly variation in LAR of Pistia stratiotes |
|
| Fig. 30: |
Monthly variation in LAR of Veronica anagallis-aquatica |
Phytochemical analyses
Chemical constituents: The obtained data in Table 1
indicated that, the highest values of moisture content (13.64%), crude fiber
(40.0%), total soluble salts (339.3 mg g-1 dry wt.), glucose (11.6
mg g-1 dry wt.), sucrose (353.6 mg g-1 dry wt.), polysaccharides
(456.4 mg g-1 dry wt.) and total carbohydrates contents (1160.9 mg
g-1 dry wt.) recorded in B. glaucus while those of total nitrogen
(230.0 mg g-1 dry wt.) total protein (120.5 mg g-1 dry
wt.), total lipid (1.82 mg g-1 dry wt.) and water soluble ash (9.46%)
contents were recorded in N. lotus. M. spicatum is attained the highest
total ash (37.58%) and acid insoluble ash (17.8%) contents.
| Table 1: |
Mean values of different metabolic products of the studied
plants |
|
| TSS = Total Soluble Sugars, Polysac. = Polysaccharides and
T. carbohyd. = Total carbohydrates |
| Table 2: |
Concentrations of elements (expressed as mg/100 g dry wt.)
in the investigated plants |
|
| (-) sign = Undetectable value |
| Table 3: |
The inhibitory activity of the plant extracts against the
tested bacteria as demonstrated by diameters of inhibition zones |
|
|
| Table 4: |
The inhibitory activity of the plant extracts against the
tested fungi as demonstrated by diameters of inhibition zones |
|
|
|
| Plate 1: |
The inhibitory activity of the methanolic extracts of the
N. lotus (A) and V. anagallis-aquatica (B) against different bacterial strains.
(1) Bacillus subtilis, (2) Erwinia carotovora carotovora, (3) Escherichia
coli, (4) Pseudomonas fluorescence and (5) Staphylococcus aureus |
|
| Plate 2: |
The inhibitory activity of the methanolic extracts of the
N. lotus (A) and V. anagallis-aquatica (B) against different fungal sspecies.
(1) Alternaria alternata, (2) Aspergillus niger, (3) Bibolaris oryza, (4)
Botrytis faba and (5) Penicillium chrysogenum |
In general, all the studied plants showed a relatively high concentration
of carbohydrates.
The preliminary phytochemical screening revealed the presence of sterols, alkaloids, flavonoides, tannins, chlorides and sulphates in the studied plants. Resins were detected in B. spicatus and N. lotus, while saponins were absent in B. spicatus.
Elementary analysis: The highest value of sodium ion concentration was
recorded in N. lotus (4920.0 mg/100 g dry wt.) and the lowest value
was recorded in P. stratiotes (2040.0 mg/100 g dry wt.). Potassium ion
concentration ranged between 319.0 mg/100 g dry wt. in M. spicatum
and 2111.2 mg/100 g dry wt. in P. stratiotes. M. spicatum attained the
highest value of calcium ion content (2764.0 mg/100 g dry wt.) while N. lotus
attained the lowest value (580.0 mg/100 g dry wt.).
It is clear that, mg++ content of B. glaucus (1980.0 mg/100
g dry wt.) is relatively higher than that of other investigated plants. Its
minimum value was that of M. spicatum being 500.0 mg/100 g dry wt. The
results in Table 2 showed the obvious ability of these plants
to absorb and accumulate heavy metals from the interstitial water. V. anagallis-aquatica
has the highest value of ferric ion content (5674.0 mg/100 g dry wt.). The
maximum values of manganese, zinc, nickel and arsenic (121.6, 10.2, 0.145 and
7.95 mg/100 g dry wt., respectively) were recorded in M. spicatum. The
highest concentrations of both cadmium and lead ions were 0.023 and 0.086 mg/100
g dry wt., respectively in N. lotus, B. spicatus accumulated the
highest copper ion content (4.8 mg/100 g dry wt.). The minimum concentrations
of Fe, Mn and Cd were 224.2, 27.6 and 0.01 mg/100 g dry wt., respectively in
P. stratiotes, those of Zn and Cu were 5.8 and 1.4 mg/100 g dry wt.,
respectively in N. lotus, Pb and As were 0.015 and 0.649 mg/100 g dry
wt., respectively in N. anagallis-aquatica and that of nickel was 0.038
mg/100 g dry wt. B. glaucus and P. stratiotes showed undetectable
values of arsenic.
The macro-nutrients (Na, K, Ca and Mg) were detected with relatively high concentrations. Sodium appeared to be an essential accumulatent in the investigated plants as compared with K, Ca and Mg. These results are coinciding with those obtained by Polisetty et al. (1984).
With respect heavy metals accumulation, M. spicatum appeared to be the major accumulator among the studied plants, while P. stratiotes appeared to be the minor one. According to the toxicological evaluations of the contaminants and naturally occurring toxicants carried out by the joint FAO/WHO (Food and Agricultural Organization/World Health Organization) expert committee on food additives for human consumption, the maximum permissible concentrations of the studied heavy metals: Fe, Zn, Cu, Cd, Pb and As are 0.8, 0.3-1.0, 0.05-0.5, 0.007, 0.025 and 0.015 mg kg-1 body weight, respectively (WHO, 1993, 1997). Consequently, the concentrations of all estimated heavy metals are obviously higher than the permissible levels and appeared to be harmful for human and therefore, the studied plants are not recommended as a fodder for animals consumption. The obtained data indicated that, these plants could be used as bioindicator for water pollution. Also, they appeared to have high potentiality for significant metals accumulation.
Antimicrobial potentialities
Antibacterial assay: The methanol extracts exhibited inhibitory activities
against the tested bacterial strains with different degrees as demonstrated
by measuring the diameters of inhibition zones (Table 3).
The extracts of N. lotus and V. anagallis-aquatica showed the
highest activity against the tested bacteria (Plate 1), while
the extracts of B. glaucus and M. spicatum showed the lowest activity.
The extract of P. stratiotes exhibited moderate range of antibacterial
activity.
Antifungal assay: The antifungal activity of methanol extracts of the
five plants presented in Table 4 and Plate 2.
The extracts of N. lotus and V. anagallis-aquatica also showed
the highest inhibitory activity against the tested fungi. In contrast, the extract
of M. spicatum showed the lowest antifungal activity.
It is apparent that, the methanolic extract of N. lotus is found to have the most effective antimicrobial activities and showed a wider inhibition zone than the extract of other plants. From the results of both the present and the previous studies (Walenciak et al., 2002; Premkumar and Shuamsundar, 2005), it may be concluded the therapeutic possibilities of these plants. In this respect, Kupeli et al. (2005) found that V. anagallis-aquatica contained iridoid glycosides with antinociceptive and anti-inflammatory activities.