A Study of the Seasonal Variation in the Antimicrobial Constituents of the Leaves of Loranthus micranthus Sourced from Percia americana
A comparative study of the antimicrobial constituents of the leaves of Loranthus micranthus (parasitic on Percia americana) harvested at different seasons of the year, namely, January, April, July and November, was carried out. The air-dried and pulverized leaves harvested at the stated periods were extracted with petroleum ether and the extract subjected to antimicrobial screening and phytochemical investigation. Using various solvent treatments the powdered leaves harvested in April was fractionated into four fractions, A, B, C and D; each fraction was screened for antimicrobial activity and phytochemical constituents. Phytochemical analysis of the extracts showed presence of tannins, flavonoids, alkaloids, terpenoids and saponins with some of these constituents showing variations across the seasons. Broad spectrum antibacterial activity was observed for all the extracts. However, the activity against Bacillus subtillis and Salmonella kapemba was significantly (p< 0.001) lower for the extracts of the leaves harvested in January when compared with the extracts of the leaves harvested in the other months. Only the extracts of the leaves harvested in April showed antifungal activity. Fractions A, B and D showed antimicrobial activity comparable (p< 0.05) to standard antibiotic, chloramphenicol. Fraction A is rich in alkaloids, B in terpenoids, A and D in tannins. The presence of alkaloids only in April and July may explain the higher antimicrobial activity observed in these months. In conclusion, mistletoe used for herbal remedy of nonspecific infections may be preferentially harvested in April and July.
Loranthus micranthus Linn. (Loranthaceae) is the Eastern Nigerian species of the African mistletoe, which has been used widely in ethnomedicine as anti-diabetic, anticancer, anti-hypertensive, antimicrobial etc. (Oliver-Bever, 1986; Kafaru, 1993). Different research teams have worked on various species of the plant and demonstrated some pharmacological activities, which supported the claimed ethnomedicinal uses (Obatomi et al., 1994; Dalziel, 1955; Osadebe et al., 2004; Obatomi et al., 1996; Kuttan et al., 1990; Bolksman et al., 1982; Osadebe and Akabogu, 2006).
Studies have shown that the composition and activities of mistletoe are host
tree and harvesting period-dependent (Obatomi et al., 1994; Scheer et
al., 1992; Wagner et al., 1996; Osadebe et al., 2004). In
our earlier study (Osadebe and Ukwueze, 2004), we demonstrated a host tree variation
in the antimicrobial activity of Loranthus micranthus leaves. The plant
material sourced from Persea americana and Kola acuminate was
found to show relatively better spectrum of antibacterial activity when compared
with that sourced from the other host trees. The present study is aimed at investigating
the seasonal variation in the antimicrobial constituents of Loranthus micranthus
leaves sourced from Persea americana. Considering the recent popularity
of Loranthus micranthus as herbal remedy, such knowledge will ultimately
make for more economic and optimal use of the plant in alternative medicine.
MATERIALS AND METHODS
The leaves of Loranthus micranthus Linn. (Loranthaceae) parasitic
on Persea americana, were harvested in April and July (the onset and
peak of rainy season) and in November and January (the onset and peak of dry
season) 2004 from Obukpa in Nsukka Enugu State, Nigeria. These periods represent
the prominent seasons of the year in the tropics. The plant materials were authenticated
by Mr. Ozioko of Bioresources Development and Conservation Project, Nsukka,
Enugu State Nigeria.
Standard cultures of Staphylococcus aureus (NCTC 3761), Bacillus
substillis (NCTC 3610), Escherichia coli (NCTC 9001), Salmonella
kapemba (laboratory strain), Pseudomonas aeruginosa (NCTC 6750),
Candida albicans (laboratory strain) and Aspergillus niger (laboratory
strain) were collected from the Pharmaceutical Microbiology Unit of the Department
of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria.
Nutrient Agar, nutrient broth, Sabourands dextrose Agar and Sabourauds
Dextrose Broth (Merck) were used. The media were prepared according to the manufacturers
instruction. Analytical grade methanol, petroleum ether (40-60°C), chloroform
ethyl acetate (BDH) and freshly distilled water were used.
Preparation of Culture Media
Nutrient Agar (28 g) was dispersed in 1 L of deionized water and the mixture
allowed to soak for 10 min and then mixed. The dispersion was sterilized by
autoclaving for about 15 min at 121°C. It was allowed to cool to 40°C
before pouring into plates. Other media were similarly prepared.
Preparation of Plant Materials
Ten gram portion each of the powered leaves of Lorathanthus micranthus
(parasitic on Persea americana) harvested at the stated periods were
extracted with 100 mL petroleum ether (40-60°C) by cold maceration for 48
h. The extracts were concentrated in vacuo using rotary evaporator and
stored in the refrigerator at 4°C until use.
Another portion (100 g) of the powered leaves of Loranthus micranthus harvested in April was extracted with 1 L petroleum ether by cold maceration for 48 h. The resulting extract (A) was concentrated in vacuo (5.1%). The air-dried residue of (A) was repeatedly extracted (500 mL x 4) with 90% methanol for 4 days. The methanol extract (10.4%) was treated (washed) with chloroform and ethyl acetate to obtain the chloroform soluble, B (2.6%); ethyl acetate soluble, C (3.1%) and ethyl acetate insoluble, D (4.7%) fractions.
The sensitivities of the selected bacteria and fungi to extracts and fractions
of the leaves of Loranthus micranthus were evaluated by the cup plate
agar diffusion method (Lovian, 1987). Stock solutions (10 mg mL-1)
of the extracts and fractions were prepared in 10% Tween 20.
Molten nutrient agar and Sabourands dextrose Agar (20 mL each) were seeded with 0.2 mL of standardized broth cultures of bacteria and fungi, respectively. Cups, 8 mm in diameter were made in the agar using a sterile cork borer. Equal volumes (0.04 mL) of 10 mg mL-1 extracts and fractions were introduced into respective cups using sterile Pasteur pipettes. The dishes were allowed to stand for one hour at room temperature for proper diffusion of the drugs before incubating at 37°C for 24 h and at room temperature (25°C) for 48 h for bacteria and fungi respectively. The experiments were done in triplicates and the average of the diameter of zone of inhibition (IZD) measured at the end of incubation period taken. Similar experiments were run using standard drugs Chloramphenicol for bacteria and nistatin for fungi. Control experiments were also done using 10% Tween 20.
Phytochemical analysis on extracts and fractions was carried out using standard
methods (Harborne, 1998). In each case, small portions of the dried extracts/fractions
were extracted in a test tube and a given quantity of the reagent added. The
mixtures were shaken vigorously or gently and the presence or absence of saponins,
flavonoids, tannins, alkaloids etc observed.
The mean IZD of three replicate results were used. The statistical difference
between the activities of the extracts at different seasons was evaluated using
one way ANOVA (SPSS 11). p<0.05 and p<0.001 were regarded as significant
and highly significant respectively.
RESULTS AND DISCUSSION
The result did not show any particular trend in the distribution of phytochemical constituents across the seasons. However, judging from the intensity of the phytochemical reactions, the extract of the leaves harvested in January, April and July could be said to contain more tannins than that of the leaves harvested in November. Flavonoids are more in the extracts of the leaves harvested in July and November while alkaloids are detected only in April and July. The contents of saponins and terpenoids were similar in all the months (Table 1).
A broad spectrum antibacterial activity was observed for all the tested extracts.
Activity against Bacillus subtillis and Salmonella kapemba was,
however, significantly (p<0.001) lower for the extracts of the leaves harvested
in January than the extract of the leaves harvested in April, July and November.
Also, the extract of the leaves harvested in April showed significantly (p<0.05)
higher activity against Salmonella kapemba when compared to that harvested
in November, while that harvested in July showed significantly (p<0.05) higher
activity against Bacillus subtillis than that harvested in November.
Activity against Escherichia coli was significantly (p<0.05) higher
for the extracts of the leaves harvested in January and July than that harvested
in April and November. There was no significant (p<0.05) difference in the
activity against Staphylococcus aureus and Pseudomonas aeruginosa
for the extracts of the leaves harvested in all the seasons. Only the extracts
of the leaves harvested in April showed mild antifungal activity (Table
|| Result of the phytochemical tests of the extracts and fractions
of Loranthus micranthus leaves at different seasons
|+++ = Present in large quantity; ++ = Moderately present;
+ = Present in small quantity; - = Absent
|| Results of sensitivity of tests of 10 mg mL-1
petroleum ether extracts of Loranthus micrantus leaves
|Each value represents the Mean±SEM, n = 3, *: p<0.05,
**: p<0.01, ***: p<0.001 significantly lower when compared with values
obtained at the other months
|| Result of sensitivity of 10 mg mL-1 of different
solubility fractions of Laranthus micranthus leaves
|Each value represents the mean±SEM, n = 3, a:
p<0.05, b: p<0.01 significantly lower when compared with
the control drug, chloramphenicol and nistatin; A = Petroleum ether fraction,
B = Chloroform soluble fraction, C = Ethyl acetate soluble fraction, D =
Ethyl acetate insoluble, Chlor. = Chloramphenicol, Nist. = Nistatin
Plants are affected by changes in environmental factors and this is reflected in the relative differences often found in the pharmacological activities and phytochemical constituents of plant materials harvested at different seasons of the year (Longman and Jenik, 1987). Two major seasons namely rainy and dry seasons are recognized in the tropics. These periods are marked with sharp differences in environmental factors like temperature, rainfall, radiation, daylight, evaporation etc (Evans, 1993). Most likely, these environmental factors directly or indirectly affect the availability of certain precursors needed for the biosynthesis of plant secondary metabolites. The rate and/or the extent of these reactions thus vary from season to season. This could be a plausible explanation for the observed seasonal variations in the antimicrobial constituents of Loranthus micranthus. More so, since the different phytochemical constituents exhibit varying degrees of antimicrobial activity, any variation in kind or in proportion of these constituents across the season will likely reflect in the antimicrobial activity.
Attempt to associate the antimicrobial activity of the plant material to specific
phytochemical constituent led to the fractionation of the extracts into various
solubility groups as shown in Table 1. Fraction A contains
tannins, flavonoids, alkaloids, terpenoids, saponins and reducing sugar. Fraction
B and C contains only terpenoids and flavonoids respectively while fraction
D contains tannins, saponins and reducing sugar. The antibacterial activity
of A, B and D are comparable to that of chloramphenicol. Fraction C showed only
mild activity against Salmonella kapemba, but inactive against the other
microorganisms tested (Table 3). The antimicrobial activity
of Loranthus micranthus leaves may thus be attributed to the presence
of some phytoconstituents like tannins, terpenoids and alkaloids. These constituents
were shown in previous studies to be responsible for the antimicrobial activity
of Loranthus micranthus (Osadebe and Ukwueze, 2004; Osadebe and Akabogu,
2006). Moreover, tannins and alkaloids are plant secondary metabolites
frequently reported to be responsible for the antimicrobial properties of most
medicinal plants (Cowan, 1999; Khwaja et al., 1996; Esimone et al.,
2003). Though some of these constituents e.g., tannins and terpenoids did not
show variation across the seasons, the presence of alkaloids only in April and
July may explain the observed better antimicrobial activity of the extracts
of the leaves of Loranthus micranthus harvested at these months.
In conclusion, significant variation in antimicrobial activity was shown by the extracts of the leaves of Loranthus micranthus harvested at the different seasons of the year. Generally, better antimicrobial activity was shown by the extracts of the leaves harvested in April and July. Mistletoe used for the treatment of non-specific infections may thus, be preferentially harvested both at the onset and peak of the rainy season.
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