Abstract: The antibacterial potential of Nyctanthes arbor-tristis was evaluated on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacteria. The dried leaf, flower, fruits and seed extracts prepared in ethyl acetate and chloroform were used to assess their antibacterial potential in terms of zone of inhibition of bacterial growth. Both ethyl acetate and chloroform extract of plant parts have shown the significant antibacterial activity against gram-negative bacteria. Fresh plant materials however, had pronounced antibacterial activity as compared to that of dried plant parts. Synergetic effect of combined extract (leaf, flowers, fruit and seeds) on bacteria was less effective as compared to those of individual extract. Furthermore, the antibacterial potential of the extracts were found to be dose dependent. These activities of plant parts were due to the presence of various plant secondary metabolites viz., glycosides and phenolics. The glycosidic and phenolic content measured spectrophotometrically fluctuates markedly among different plant parts.
INTRODUCTION
Nyctanthes arbor-tristis (family: Oleaceae) commonly known as Night jasmine or coral jasmine, mainly characterized by the presence of phenylethanoid derivatives and iridoid glycosides (Jensen et al., 2002). It is used in traditional medicine as stomachic, carminative, intestinal astringent, expectorant, in biliousness, piles, various skin diseases and as hair tonic (Khatune et al., 2001). The decoction of leaves is widely used in ayurvedic medicine for the treatment of sciatica and arthritis (Kirtikar and Basu, 1935; Shri Gulabkumaverba Ayurvedic Society, 1949; Chopra et al., 1958; Nadkarni, 1976). It has also been reported to possess hepatoprotective, anti-leishmanial, anti-viral and anti-fungal activities (Puri et al., 1994) and analgesic, antipyretic and ulcerogenic activities (Saxena et al., 1987). Roots are used for emaciation and stem bark of this plant is taken to cure dysentery, ulcer of palate and internal injuries (Gupta et al., 2006). Saxena et al. (2002) have reported the tranquilizing, antihistaminic and purgative activity of Nyctanthes arbor-tristis leaf extract.
The N. arbor-tristis demonstrate diverse pharmacological and biological activities like anti-inflammatory (Saxena et al., 1984), analgesic, anti-pyretic along with ulcerogenic (Saxena et al., 1987) activities. The plant also possess anti-allergic (Gupta et al., 1993), anti-malarial (Badam et al., 1988; Misra et al., 1991), leishmanicidal (Singh et al., 1992; Tandon et al., 1991), amoebicidal (Chitravanshi et al., 1992), antihelminthic (Lal et al., 1976) activities and recently reported hepatoprotective (Hukkeri Kusum et al., 2006), anti spermatogenic (Gupta et al., 2006) and antioxidant activities (Rathee et al., 2007).
The present study was undertaken to investigate the antibacterial activity of N. arbor-tristis in view of its diverse pharmacological application in ancient and modern system of medicine. Flowers, leaves, seeds and fruits ethyl acetate and chloroform extracts investigated for the antibacterial screening. The seed and fruit extracts, however investigated for the first time.
MATERIALS AND METHODS
Plant
Nyctanthes arbor-tristis fresh flowers, leaves, fruits and seeds
were collected from Arcot in Vellore district of Tamil Nadu during the month
of December (2006) to February (2007). The materials were immediately brought
to School of Biotechnology, Chemical and Biomedical engineering, VIT University,
Vellore-632 014, India, where the experiments conducted.
Preparation of the Extract
Fresh and dried flowers, leaves, fruits and seeds were collected, weighed
(4 g each), washed and macerated in 10 mL of ethyl acetate and chloroform separately.
The mixtures were kept for 6 h at room temperature. The mixtures were then filtered
through sterile Whatmann filter paper No. 1. Filtrate, thus obtained were centrifuged
at 5000 rpm for 5 min. The supernatants were collected in the beaker and the
solvents were evaporated to dryness, the residue left over was stored at 4°C
in refrigerator. At the time of antibacterial assays the residue was dissolved
in 1-3 mL of DMSO.
Preparation of Microorganisms
The organisms used in this study were Escheichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 9027), Klebsiella pneumoniae (ATCC
2719) and Staphylococcus aureus (ATCC 25923). The strains were maintained
on nutrient agar slants at 4°C. A loopful of each bacterial strain was inoculated
into 50 mL of sterile nutrient broth in 100 mL conical flask. The flasks were
incubated on a rotary shaker for 24 h to activate the strain. Mueller Hinton
Agar medium was used as bacterial culture medium in the antibacterial assays.
Antibacterial Activity
The antibacterial activity of Nyctanthes arbor-tristis ethyl acetate
and chloroform extracts were evaluated by agar well diffusion method (Bauer
et al., 1966). Twenty four hours broth cultures of the bacteria used
for the assay. A sterile cotton swab was dipped into the bacterial suspension
and evenly streaked over the entire surface of sterile Mueller Hinton agar plate
to obtain uniform inoculum. Wells were punched on the seeded plates using sterile
borer (8 mm). The plates were allowed to dry for 5 min. Ethyl acetate and chloroform
extracts (100, 200 and 300 μL) were dispensed into each well using sterile
micropipette. Streptomycin (10 μL) was used as positive control. The plates
were incubated overnight at 37°C. Antibacterial activity was determined
by measuring the diameter of zone of inhibition (mm).
Extraction and Analysis of Glycosides and Phenolics
The glycosides were extracted according to Ganjewala et al. (2000).
The dried leaves, fruits and seeds powder (1 g each) first extracted three times
with warm hexane (20 mL) for removal of pigments. The hexane extract was centrifuged
at 5000 rpm for 5 min and the pellets were extracted twice with warm methanol
(20 mL) and centrifuged at 5000 rpm for 5 min after each methanol treatment.
The supernatant was collected in a conical flask and methanol was evaporated
to dryness. The residues contained glycosides were analyzed by thin layer chromatographic
procedure. Thin layer chromatography was performed using pre-coated silica gel
60 F254 TLC plate. The mobile phase contained ethyl acetate: methanol:
water (60:14:10, v/v/v). The spots were visualized after spraying the developed
TLC plate with vanillin sulfuric acid reagent and incubating the plates at 110°C
for 5 min.
Phenolic content of the dried leaves, fruits and seeds (1 g each) were extracted using 10 mL of 0.3 M methanolic HCl. The supernatant obtained after centrifuging the mixture at 5000 rpm for 5 min was collected and evaporated to dryness. The residues were dissolved in 5 mL of distilled water. Aliquots (0.1 mL) of the extracts were transferred to the test tubes and made upto 7.0 mL with distilled water and stirred well. The Folin-phenol reagent (0.5 mL) was added to the solution in test tubes and shaken vigorously. After 3 min, 1 mL of 35% sodium carbonate solution was added. The mixtures were shaken and allowed to stand for 1 h. The absorbance was recorded at 630 nm.
Qualitative Analysis of Phytochemicals
Specific qualitative tests were performed for the presence or absence of
phyto-chemicals viz., alkaloids, saponins, phytosterols, tannins, phlobatannins,
flavonoids, terpenoids and cardiac glycosides in leaf, fruit and seed extract.
RESULTS
Antibacterial Activities of Different Plant Parts
Antibacterial potential of flower, leaf, seed and fruit ethyl acetate and
chloroform extract was assessed in terms of zone of inhibition of bacterial
growth. The results of the antibacterial activities are presented in Table
1 and 2. 100, 200 and 300 μL of each extract was
used for antimicrobial screening. The antibacterial activity of extract increased
linearly with increase in volume of extract (μL). Both, ethyl acetate and
chloroform extract (volume 300 μL) have shown significant antibacterial
activity against the microorganisms tested. The results revealed that the gram
negative (Pseudomonas aeruginosa and Klebsiella pneumoniae) bacteria
were more sensitive as compared to those of gram positive bacteria to all the
extracts. The growth inhibition zone measured ranged from 15 to 25 mm for all
the sensitive bacteria.
| Table 1: | Antibacterial activities of Nyctanthes arbor-tristis fresh flower, leaves, seeds and fruits extracts |
| R = Resistant | |
| Table 2: | Antibacterial activities of Nyctanthes arbor-tristis dried leaves, seeds and fruits extracts |
| R = Resistant | |
| Fig. 1: | Nyctanthes arbor-tristis glycosidic profile in thin layer chromatographic plate. (1) leaves (2) seeds and (3) fruits |
Flower ethyl acetate and seed chloroform extract have shown significant broad spectrum antibacterial activity against gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) as well gram-positive (Staphylococcus aureus) bacteria. Leaf extract showed antibacterial activity against all the gram negative bacteria tested. Fruit and seed ethyl acetate and flower and seeds chloroform extracts have shown the inhibitory effect against only two of the gram negative bacteria (Pseudomonas aeruginosa and Klebsiella pneumoniae). E. coli was resistant to fresh fruit and seed ethyl acetate extract. E. coli and Pseudomonas, however, were most sensitive to the fresh fruit and seed chloroform extracts, respectively. In general, it was observed that fresh plant parts had more antibacterial activity as compared to that of the dried plant parts. Dried leaves, fruits and seeds chloroform extract only had strongest antibacterial activity against gram negative E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae, while that of dried leaves and seed extract in ethyl acetate had broad spectrum antibacterial activity.
Antibacterial activity of combined extracts containing equal amount of leaf, fruit and seed ethyl acetate and chloroform extracts have also been evaluated. The combined extract showed less antibacterial activities as compared to antibacterial activity of extract in individual. Similar to individual extracts, combined ethyl acetate extract had more antibacterial activity against gram negative bacteria. However, combined chloroform extract has shown less effect against gram positive S. aureus.
Phytochemical Analysis
Presence of some important secondary metabolite viz., alkaloids,
phenolics, flavanoids, glycosides, saponins and tannins in leaves, fruits and
seeds extract were confirmed after performing specific qualitative tests (Table
3). Alkaloids, however not present in leaf extract. Moreover, Phenolic content
was measured spectrophotometrically and glycosides were analyzed by thin layer
chromatographic procedure. Phenolic content (mg g-1 dry weight) was
found to be maximum in seeds and minimum in fruits. Seed’s phenolic content
(mg g-1 dry weight) was almost double than leaf while four fold higher
to that of fruit’s phenolic content. Thin layer chromatographic analysis
have shown the similarity between glycosidic profile of leaf and seed while
that of fruit did not match with either of leaf and seed glycoside profiles
(Fig. 1).
| Table 3: | Phytochemical composition of Nyctanthes arbor-tristis leaves, seeds and fruits |
| + = Present; - = Absent | |
DISCUSSION
The interest in medicinal and aromatic plants has been shown all over the world because of safe and effective constituents of plant products and in particularly the presence of active principles of medicinal plants. In the present study antibacterial properties of N. arbor-tristis fresh and dried flowers, leaves, fruits and seeds have been investigated. Both of ethyl acetate and chloroform extracts possess significant antibacterial activity against gram-negative and gram-positive bacteria. The results presented are consistent to those of earlier reported in N. arbor-tristis (Khatune et al., 2001, 2005). These results, however did not match with those described by Nair et al. (2005). Nair et al. (2005) used whole plant water and methanol extracts which was less effective against bacteria tested. The antibacterial activity of N. arbor-trsitis seed and fruits (fresh and dried) ethyl acetate and chloroform extract reported here is for the first time hence in addition to those reported earlier. The seed and fruits have significant antibacterial activity against the microorganisms tested.
The results of the present studies indicate that the fresh plant material extracts is more effective as compared to those of dried plant extracts. The differences in the antibacterial effects of plant materials used are expected due to the differences in their phyto-chemical compositions. Moreover, the fresh plant part’s extracts are more effective as compared to the dried ones. The drying may have caused conformational changes to occur in some of the chemical constituents present in these plant parts (Nair et al., 2005).
Also, the results indicate that both ethyl acetate and chloroform extracts are more effective against gram negative as compared to those of gram positive bacteria. The reason for the different sensitivity between gram-positive and gram-negative bacteria may be attributed to the morphological differences between these microorganisms. Cell wall of gram-negative bacteria is consisted of phospholipids and lipopolysaccharide, hence impermeable to lipophilic solutes (Nikaido and Vaara, 1985). In spite of this permeability barrier the ethyl acetate and chloroform extract exert strong inhibition on gram-negative bacteria. Gram-positive bacteria should be more susceptible because of only an outer layer of peptidoglycan, however, they are resistant against both ethyl acetate and chloroform extract. Only flower, leaf and seed extracts exerts some inhibition on gram-positive bacteria and have broader spectrum of inhibitory activity than the other (fruit) extract.
Phytochemical analysis of leaf, fruit and seeds of N. arbor-tristis revealed the presence of phytosterols, phenolics, tannins, flavonoids, glycosides and saponins. The secondary metabolites present in N. arbor-tristis are known to be biologically active. Tannins have been found to form irreversible complexes with proline rich proteins, resulting in the inhibition of the cell protein synthesis. This activity was exhibited against test organisms with all the plant extracts. Tannins have important roles such as stable and potent antioxidant (Trease et al., 1983), astringent and for treating diarrhea and dysentery (Dharmananda, 2003). The alkaloids the largest groups of chemicals produced by plants have many biological activities. Therefore the antibacterial activities of extracts are expected. Flavonoids are phenolic structures containing one carbonyl group complexes with extra-cellular and soluble protein and with bacterial cell wall. Thus, exhibits antibacterial activity (Cowan, 1999).
ACKNOWLEDGMENT
The authors are thankful to the Chancellor, VIT-University, Vellore and Dean, School of Biotechnology, Chemical and Biomedical Engineering for providing the necessary facilities and support.