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In vitro antioxidant, brine shrimp lethality and antibacterial activities of
methanol extract of Trevesia palmata leaves
Article · December 2017
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Md. Tanvir Hossain
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1 Department of Applied Chemistry and Chemical Engineering, Noakhali Science and Technology University,
Noakhali!3814, Bangladesh.
2
Department of Microbiology, Noakhali Science and Technology University, Noakhali!3814, Bangladesh.
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& , , known as the Snowflake Aralia is found in Lawachara Forest Reserve of Bangladesh. The present study was carried out to assess the phytochemical and biological properties of methanol extract of . & , leaves (TPME). Preliminary phytochemical screening yielded the presence of paramount bioactive compounds having a high antioxidant capacity. The antioxidant activity of the extract was tenacious utilizing 2,2!diphenyl!1!picrylhydrazyl (DPPH) radical scavenging activity, reducing power assay, and total antioxidant activity (TAC). Antibacterial activity of extract was analyzed against & &
0 , and+ .TPME showed moderate antioxidant activity on DPPH and reducing power assay but good in TAC (394.5±7.8 AAE/g of extract). IC50 value (524.64±25.01
<g/mL) based on the DPPH showed higher than standard. TPME is active against both gram positive and gram negative bacteria. TPME showed very good brine shrimp lethality activity and membrane stability. LC50 value
of (0.57 <g/mL) against brine shrimp lethality and IC50 value of (491.26 =g/mL) against membrane stability
were observed for TPME. The plant has got a broad spectrum antibacterial and good antioxidant, cytotoxicity and membrane stability and has a promising potential for testing diseases.
. & , ; cytotoxicity; membrane stability; antioxidant; antibacterial
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Herbal medicines, botanical medicines or phytomedicines are generally known as traditional therapeutic plants insinuate to restorative items acquired from plant roots, stems, leaves, bark, seeds, and foods grown from the ground that can be utilized to advance general well being and treat diseases. These herbal materials might be utilized categorically as a component of a solution detailing or handled into different yare!to!use products (Tang and Halliwell, 2010). The medicinal value of plants is due to the substances that it contains which engender a physiological action on the human body. Some paradigm of these substances is alkaloids, essential oils, tannins, resins, and many others. Moreover, some negative effects obtained in the utilization of local plants as the source of medicine are fundamentally due to over! dosage and lack of adequate erudition of other pernicious byproducts contained in
some plants. Antioxidants postulate an indispensable part in ascertaining cell harm by responsive oxygen species. From many investigations, it can be endorsed that the plant source antioxidants with free radical scavenging properties may have awe! inspiring therapeutic paramountcy in free radical mediated diseases. Numerous synthetic antioxidant compounds have demonstrated poisonous or potentially mutagenic impacts; while generally plant predicated medicines give fewer side effects than the synthetic medicate in some instances (Tapsell et al., 2006).
Belonging to the Araliaceae family
& , (Roxb. ex Lindl.) Vis. is an
Arifin et al.
penis of children in Chittagong Hill Tracts. Paste prepared from roots and fruits are applied to snake!bites. It has been reported that lactone glycosides are present in the plant and the plant is utilized as a postpartum herbal bath recipe by the Mien (Yao) group in Laos use decoction of roasted stems and roots for postpartum instauration, perineal rejuvenating, retraction of the uterus, abdominal pain, and as a lactagogue (Lamxay et al., 2011). Phytochemical screening, antihyperglycemic, and analgesic potential have been reported for leaves (Rahman et al., 2014).
To the best of our knowledge, very few pharmacological studies have been reported so far on . & , leaves. As a part of the continuation of our research on bioactivity screening of Bangladeshi medicinal plants (Dev et al., 2015, Islam et al., 2016, 2017), present study was carried out to assess the phytochemical nature, antioxidant, cytotoxicity, membrane stability and antibacterial activity of TPME to scientifically evaluate the claimed biological activities.
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The plant leaves ( . & , ) were collected
from Lawachara Forest Reserve,
Maulvibazar, Bangladesh. The green leaves were plucked from a healthy plant and washed with distilled water followed by sun! drying for one week. Dried leaves were pulverized to minute powder and stored in an airtight container for further use.
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The powdered leaves (100 g) were macerated with methanol (500 mL) and the solution was filtered through Whatman filter papers. The filtrate was concentrated in a rotary
evaporator and dried in an oven at low temperature and stored for further use.
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The preliminary phytochemical screening of TPME was performed to detect the presence of active chemical constituents e.g. alkaloid, flavonoid, saponin, tannin, reducing sugar, phenolic compound, protein and amino acid as the methods described in Hossain (2015) and Afroza and Hossain (2015).
!2
The antioxidant properties of TPME was determined through DPPH scavenging activity, reducing power assay and total antioxidant capacity as the methods described in Islam et al. (2017, 2016) and Dev et al. (2015).
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The antibacterial activity of TPME against gram!positive as well as gram!negative bacterial strains was evaluated by disc diffusion method reported previously (Islam et al., 2017). The cultures were . were hatched in artificial seawater prepared from commercial sea salt 38 g/L. 250 <L of TPME solution (2, 20, 100 and 200 =g/mL) were taken in different test tube and ten brine shrimp nauplii in artificial seawater were added to each test tube. Finally, the volume of each test tube was adjusted to 5 mL by adding artificial seawater. All tests were performed at room temperature (~30o C), under a continuous light. After 24 h, the test tubes were inspected using a magnifying glass and the number of surviving nauplii in each test tube were counted. The lethal concentrations of TPME resulting in 50% mortality of the brine shrimp (LC50) from the
trend line fit linear regression analysis. The NSAIDs (Non Steroidal Anti!Inflammatory Drugs) for 2 weeks prior to the experiment and transferred to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of normal saline. The reaction mixture consisted of 1 mL test sample of different concentrations (250 ! 1000 =g/mL) and 1 mL of 10% RBCs suspension, instead of test sample only saline was added to the control test tube. Aspirin (250 ! 1000 =g/mL) was used as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in water bath at 56 ºC for 30 min. At the end of the incubation the tubes were cooled under running tap water and centrifuged at 2500 rpm for 5 min. The absorbance of the supernatants was taken at 560 nm and the percentage inhibition of haemolysis was calculated as follows: Percentage inhibition = [(Abscontrol –
Abssample)/ Abscontrol]×100
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All investigations were performed in triplicate, unless generally expressed. Data was expressed as mean ± SEM. Data
Preliminary phytochemical screening of TPME was qualitatively analyzed to observe the presence of secondary metabolites and found alkaloid, reducing sugar, flavonoid, saponin, tannin, and protein and amino acids. The phytochemical analysis is a measure of the bioactive compounds which can be used to treat chronic as well as infectious diseases. The presence of secondary metabolites for TPME is comparable to that reported by Rahman et al. (2014).
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Antioxidants are characterized as substances that even at low concentration significantly delay or avoid oxidation of simple oxidizable substrates. It works through preventing the generation of free radicals produced in the body or reducing/chelating the transition metal composition.
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The comparative quantitative DPPH radical scavenging activities (% inhibition) of TPME against standards are depicted in Figure 1. It is a kind of measure of antioxidant activity. Percentage inhibition by TPME was lower compared to standards. It is evident from the data that, the values of 50% scavenging activity (IC50 value) of TPME was
significantly higher (524.64±25.01 <g/mL) in contrast to standards (ascorbic acid 26.28±4.36 <g/mL and BHT 59.28±2.02 <g/mL). Lower IC50 value indicates higher
antioxidant activity. In general, TPME showed little or no activity. The DPPH radical scavenging ability of a plant extract is based on its ability to decolorize the deep purple color measured from changes in absorbance.
Figure 1. DPPH radical scavenging activity of TPME and standard antioxidants.
Arifin et al.
were able to decolourise. It has been found that cysteine, glutathione, ascorbic acid, tocopherol, polyhydroxy aromatic compounds (hydroquinone, pyrogallol, etc.), and aromatic amines (p!phenylene diamine, p!aminophenol etc.), reduce and decolourise DPPH by their hydrogen donating ability (Blois, 1958). It appears that the extracts from the . & , leaves process hydrogen donating capabilities to act as antioxidant.
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The reducing power abilities (absorbance at 700 nm) of TPME and BHT are shown graphically in Figure 2. The absorbance of TPME and standard is a function of their concentrations, and generally, increased linearly with the increase in concentration. The reducing power assay is based on single electron transfer reaction, which is a measure of the antioxidant capacity to reduce ferric ions to ferrous ions in a reaction mixture. The presence of reductants (i.e. antioxidant) causes the conversion of the Fe3+/ ferricyanide complex (Perl’s Prussian blue) to the ferrous form, where the intensity is dependent on the concentration of reductants (Islam et al., 2017). From the result, it is assumed that TPME has lower reducing power may be due to absence of phenolic contents.
Figure 2. Reducing power of TPME and standard antioxidant.
The reducing power assay is based on single electron transfer reaction, which is a measure of the antioxidant capacity to reduce ferric ions to ferrous ions in a reaction mixture. The presence of antioxidant causes the conversion of the Fe3+/ ferricyanide complex (Perl's Prussian blue) to the ferrous form, where the intensity is dependent on the
concentration of antioxidants (Amel et al., 2014). From the results, it is plausible that, the high antioxidant activity observed in the TPME due to its alkaloid(s) contents.
1! !1
The TAC of TPME was expressed quantitatively in ascorbic acid equivalents per gram of extract (AAE/g extract). TPME showed significantly high TAC (394.5±7.8 AAE/g). TAC mainly concentrates on the thermodynamic conversion and measures the number of electrons or free radicals donated or quenched by a given antioxidant molecule. It is based on the reduction of Mo (VI) to Mo (V) and subsequent formation of green phosphate/Mo (V) complex, which evaluates both water!soluble and fat!soluble antioxidant capacity. The present study reveals that TPME showed good TAC. Furthermore, it can be assumed that the good TAC of extract may be due to presence of alkaloids and flavinoids.
1!1
The antibacterial activities of TPME were observed against two!gram positive bacteria,
. . and two gram observed that TPME showed antibacterial activity against both gram positive and gram negative bacteria.
Figure 3. Zone of inhibition of the . & ,
1!2 $ cytotoxic activity exhibited by the solvent extract was promising and this clearly indicates the presence of potent bioactive compounds (Meyer et al., 1982).
1!3
The human red blood cell membrane stability was studied to establish the mechanism of anti!inflammatory action of TPME. The extract was effective in inhibiting the heat induced hemolysis at different concentrations of the sample. IC50 was observed at 491.26
=g/mL at correlation coefficient R2 of 0.989. Aspirin standard showed the inhibition of 86.5±2.2 =g/mL at 200 =g/mL.
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This study revealed the antioxidant, cytotoxicity and anti!inflammatory and antibacterial activity of TPME. The TPME reduced molybdenum (VI) to molybdenum (V) with capacity of 394.5±7.8 AAE/gm and exhibited DPPH radical scavenging activity with IC50 of 5.24.64±25.01 =g/mL. LC50
value of (0.57 <g/mL) against brine shrimp lethality and IC50 value of (491.26 =g/mL)
against membrane stability were observed for TPME. The presence of flavonoids and alkaloids may be responsible for the activity. Further studies are required to find the active component of the extract and to confirm the mechanism of action.
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-Authors would like to express gratitude to the authority of Noahkali Science and Technology University (NSTU) for their co! operation in this research. They are thankful to the Department of Applied Chemistry and Chemical Engineering and the Department
of Microbiology, NSTU for providing lab facilities during this research.
%-;-%-
&-Afroze, F. and Hossain, M. T. 2015. Proximate analysis, phytochemical screening and antioxidant activity of 0 , 4 Phytochemical analysis, antioxidant activity and hypotensive effect of algerian azarole (% 5 L.) leaves extracts. RJPBCS. 5(2): 286!305. Blois, M. S. 1958. Antioxidants determination by the use of a stable free radical. Nature 181: 1199–1200.
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