Abstract: The present research demonstrates the biological activity of Dipterygium glaucum since extensive literature survey has shown no documented biological activity of this plant. Ethanol (80%) extract (A) of the plant was subfractioned by hexane (B), ethyl acetate (C) and butanol (D). These fractions (1 mg mL-1) of D. glaucum showed 45 to 100% phytotoxicity as determined by the inhibition of Lemna minor plant growth but showed no cytotoxicity by brine shrimp lethality assay. DPPH radical scavenging activity of fraction `A` was 87% whilst other fractions had antioxidant activity below 35%. When fraction `A` was tested for antispasmodic activity, spontaneous contractions were recorded at 0.1-3.0 mg mL-1 concentration in isolated rabbit jejunum preparations. It also inhibited K+ induced contractions to 60% at 1-3 mg mL-1 level, suggesting a calcium channel blockade activity. The n-hexane (B), ethyl acetate (C) and n-butanol (D) fractions exhibited no or little antibacterial, antifungal, antileishmanial and insecticidal activities as compared with their respective controls. This is the first report on the biological activity of D. glaucum.
INTRODUCTION
Dipterygium glaucum belongs to Capparidaceae family and is a monotypic genus with only one species D. glaucum that is found in Egypt, Arabia, Sudan and Pakistan. D. glaucum is branched undershrub, upto 60 cm tall and woody at the base (Jafri, 1973). Extensive literature survey has shown no reported biological activity of this plant. The present work therefore describes the biological activity of the various fractions of the crude 80% ethanolic extract of the whole plant.
MATERIALS AND METHODS
D. glaucum was collected from Cholistan desert and identified by Dr. M. Arshad (Cholistan Institute of Desert Studies, where a specimen is deposited). Plant was dried in shade and soaked in 80% ethanol for 2-3 weeks. After solvent evaporation, residue (A) was suspended in water and extracted with n-hexane (B), ethyl acetate (C) and n-butanol (D) and its various dilutions were made for the determination of the biological activity as described (Atta-ur-Rehman et al., 2001). Antispasmodic activity was determined as described earlier (Gilani et al., 1994). Briefly, abdomen of rabbits of local breed was cut open and jejunum was taken out. Segments of 2 cm length were suspended in Tyrode’s solution aerated with a mixture of 95% oxygen and 5% carbon dioxide at 37°C. Tissues were allowed to equilibrate for 30 min before the addition of test fractions. The spontaneous rhythmic movements were recorded isotonically using BioScience transducers and an oscillograph.
RESULTS AND DISCUSSION
D. glaucum ethanol (80%) extract was studied for its antispasmodic activity in rabbit jejunum (Table 1). It exhibited inhibition of spontaneous contractions of jejunum at 0.1-3.0 mg mL–1. To test whether this effect is mediated through the blockade of Ca2+ influx, a high dose of K+ (50 mM) was used to depolarize the tissue. Addition of test sample caused 60% inhibition of the K+ pre-contracted jejunum as compared to the control verapamil (Table 1). It is therefore said that spasmolytic (antispasmodic) activity may be due to any active constituent found in the extract that should be isolated and identified.
Antioxidant activity of extract was found maximum (87%) at 200 μg mL–1 as compared with the stndared (92%) as determined by DPPH radical scavenging method whilst other fractions exhibited low levels of antioxidant activity (Table 2).
| Table 1: | Antispasmotic activity of the crude ethanolic extract |
| Table 2: | Antioxidant and antileishmanial activity of various fractions |
Antioxidant activity was determined by DPPH radical
scavenging method using propyl gallate as standard at 200 μg/mL levels.
Antileishmanial activity was determined by 96-well serial dilution protocol
and data expressed in terms of IC50 values (μg mL–1).
Amphotericin B at 0.19 μg mL–1 gave 100% mortality of parasites
(n = 3) |
|
| Table 3: | Phytotoxicity and cytotoxic activity of various fractions |
Phytotoxicity was determined by the inhibition
of growth of Lemna minor plant using paraquat as standard drug (0.9025
μg mL–1). Cytotoxicity was measured by brine shrimp method
and number of larvae survived after addition of various concentrations of
test sample was determined. Permethrin (236 μg cm–3) was
used as standard. Results are expressed in terms of percent (n = 3) |
|
| Table 4: | Insecticidal activity was measured by contact toxicity method
against insects |
Test sample (1572.7 μg cm–2) was applied to
filter paper in petri dish and placed at room temperature. The number of
insects survived at 1 day were counted. Permethrin (236 μg cm–2)
was used as standard that gave 100% mortality |
|
| Table 5: | Antibacterial activity of various fractions |
Test samples, 3 mg mL–1 in DMSO, were placed in
wells of 6 mm diameter against standard drug, 10 μg disc–1.
Zone of inhibition of standard drugs was 27-33 mm and ‘+’ indicates
zone of inhibition in 10-14 mm (n = 3).Symbols ‘++++’ indicate
27-33 mm zone of inhibition, ‘+’ is 10-14 mm zone of inhibition
and ‘-’sign means no activity |
|
| Table 6: | In vitro antifungal activity of various fractions
by agar tube dilution protocol |
| Standard drug miconazole showed 70-98% inhibition of fungal growth. Test samples (0.4 mg mL–1 in DMSO) were added to growing fungi at 27°C for 7-days and %inhibition of linear fungal growth measured in mm (n = 3) | |
As far as the antileishmanial activity is concerned, no such activity was found in in vitro assays as compared with the control (Table 2).
All fractions exhibited phytotoxic activity against Lemna plant in dose-dependent manner (Table 3) where in n-hexane (B) and ethyl acetate (C) fractions showed 100% phytotoxicity at 1 mg mL–1 followed by 70% mortality of plant by n-butanol fraction (D) as compared with the control. It is assumed that some phytotoxic constituent(s) are present in the fractions responsible for the activity. However, these fractions demonstrated little (fraction B) or no (fraction C or D) insecticidal or cytotoxicity (Table 3 and 4). Similar profiles have been seen when these fractions showed no antibacterial or antifungal activity (Table 5 and 6).
In summary, the 80% ethanol extract of this plant demonstrated antioxidant and antispasmodic activities with low phytotoxicity levels whilst n-hexane, ethyl acetate and butanol fractions showed considerable high levels of phytotoxicity. These fractions were devoid of any antileishmanial, insecticidal, cytoxicity, antibacterial or antifungal activities as determined by the given experimental methods. Further work is ongoing on the isolation and identification of chemical constituents of this plant.
ACKNOWLEDGMENTS
Thanks are due to Prof .Anwar H. Gilani (Agha Khan University, Karachi) and Prof. Iqbal A. Choudhary (HEJ Research Institute of Chemistry, Karachi) for help in conducting some of the assays in their labs.