Tenzin C, Jeyanthi P, Kumar A, Sujesh S and Ramalingam C*
Nano-Food Research Group, Instrumental and Food Analysis Laboratory, School of Biosciences and Technology, VIT University, Tamil Nadu, India
Received date: April 06, 2017; Accepted date: April 26, 2017; Published date: May 02, 2017
Citation: Tenzin C, Jeyanthi P, Kumar A, Sujesh S, Ramalingam C (2017) Estimation of Bioactive Compounds from Saccharum munja Extract for the Evaluation of Anti-oxidants and Anti-bacterial Activities. J Food Process Technol 8:671. doi: 10.4172/2157-7110.1000671
Copyright: © 2017 Tenzin C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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From time memorial, humans have used many plants as a treatment for many diseases. About two third of plant species are estimated to have a medicinal value. Plants synthesize phytochemicals which have anti-inflammatory, anti-oxidative and anti-microbial microbial properties thus making them important therapeutic sources. In this study, stem and leaf extract of Saccharum munja was prepared in ethanol and was screened for the presence of important phytochemical compounds. The different constituents present in the extract were identified by using GC-MS analysis. The extract was also found to possess potential antibacterial property and anti-oxidative activities. Also, an attempt was made to compare the effectiveness of stem and leaf extract of the plant. Stem extract was found to have better anti-oxidative activity than leaf extract.
Saccharum munja; Anti-oxidant; DPPH; GC-MS; Phytochemical screening
Throughout the human history, many Medicinal plants have been identified and used to treat many diseases. Plants synthesize tonnes of chemical compounds that are for biological functions, including defence against insects, and micro-organisms. At least 12,000 such compounds have been isolated so far . Saccharum munja commonly known as munja and is a grass found along river banks. The common name of the plant is Kana, sarkanda, Moonja and is distributed from north and North West India to Pakistan. The plant belongs to the family Gramineae. The plant is a large tufted grass and is of little account as fodder plant as cattle doesn’t eat them . Saccharum munja was believed to have a medicinal value and are employed to treat diseases. Not much of the research was done to evaluate the effect of Saccharum munja. The Saccharum munja extracts are having many beneficial and advantages in fields like biological, medicinal and nanotechnology. Ethanol is highly employed to prepare a plant extract since most of the phyto compounds get easily dissolved in ethanol . The plant is used to reduce burning sensation, thirst, dyspraxia, erysipelas and urinary complaints.
The Ayurveda Pharmacopoeia of India recommends the use of the root in dysuria, giddiness and vertigo . The stem is a good source of furfural (yield 5.67%, dry basis). When digested with sulphuric acid it yields 19.5% (on dry weight) of reducing sugars. Glucose, galactose and rhamnose have been identified in the hydrolysate which contains 34.5% fermentable sugars. It can be used as a potential source of alcohol . In Kerala, Saccharum arundinaceum Retz. is used as Shara for dysuria, diseases due to vitiated blood, erysipelas, leucorrhoea and piles. The grass is known as Raamshara in North India the plant is known as Raamshara. The plant can also be used for the production of furfural (yield 5.1% dry basis) and yields 24.1% of reducing sugars when digested with sulphuric acid. The hydrolysate contains 65% of fermentable sugars, viz. glucose, xylose, galactose and rhamnose. Saccharum munja are abundantly found in most places and thus could be used as potential sources of bioactive compounds and metabolites [6,7].
Most of the neurodegenerative diseases, AIDS etc. involve free radicals. Free radicals attacks important macromolecules like carbohydrate, lipids, and nucleic acids etc. thereby causing damage to the cell and disrupts body homeostasis. Antioxidants produced from the plants due to their scavenging property can neutralize the effect of free radicals .
Collection of plant sample
Saccharum munja plant sample was collected from the field of Vellore, Tamil Nadu. The plant was washed thoroughly with water to remove dust particles.
Preparation of the extract
The leaves and stem of Saccharum munja was considered for the experiment. The plant collected was allowed to dry. The dried leaf and stems were then powdered using machine grinder to get a uniform size for the extraction. About 5 g of leaf and stem dried powder was ta ken into 2 separate clean conical flasks and 50 mL of ethanol was added into the flask. This is ethanol extract of Saccharum munja. The extract was covered tightly to avoid evaporation of ethanol since bioactive compounds gets dissolved in the ethanol .
Phytochemical screening of the different chemical groups by preliminary phytochemical analysis
Mayer's test: 1.358 g of HgCl2 mixture was added in 60 mL H2O and 5 g of KI in 10 mL of H2O are mixed to prepare Mayer’s reagent. About 2 mL of this reagent was added to 1 mL of extract followed by little HCl and was heated for few minutes. The appearance of yellow precipitate indicates presence of alkaloids.
Wagner's test: Wagner's reagent was prepared with Iodine and KI. 1 mL of Wagner's reagent was added to the plant extract followed by 2 mL of HCl. This was then heated for few minutes. Presence of alkaloids is confirmed with the appearance of brown or reddish colour precipitate [8-10].
Ferric chloride test: Few drops of Ferric chloride solution were added to plant extract in a clean test tube. The formation of intense green colour indicates the presence of phenol.
Molisch's test: About 2 mL of Molisch's reagent was added to the plant extract and was heated for 3 minutes. The presence of carbohydrates was marked by the appearance of violet ring in the test mixture.
Borntrager's test: About 1 mL of plant extract was acidified with dil. HCl and was allowed to heat for 1 minute. The plant extract mixture was then added with little amount of ferric chloride solution and boiled for 5 min. The mixture was left to cool down to normal temperature. After cooling, 1 mL of benzene was added which results in the appearance of benzene layer. Sufficient amount of ammonia solution was then added to the benzene layer. The presence of glycosides was marked by the appearance of light pink colour.
Legal test: Few drops of sodium hydroxide and sodium nitroprusside hydroxide were added to the plant extract. Change of colour from pink to blood red colour confirms the presence of cardiac glycosides.
Keller killani test: About 1 mL of plant extract was added to equal volume of glacial acetic acid. Some 5 mL of Con. H2SO4 was added slowly with proper care into the plant extract mixture. The positive test showed appearance of Blue in the acetic acid layer.
Ferric chloride test: 1 mL of plant extract was subjected to addition of about 5 drops of ferric chloride solution in a clean test tube. The presence of tannins was confirmed with the appearance of purple colour.
Alkaline reagent test: About 1 mL of sodium hydroxide solution was added to the plant extract drop by drop. The mixture turns into dark yellow colour. Addition of HCl to the extract mixture forms a colourless solution. The formation of intense yellow colour indicates the presence of Flavonoids.
Shinoda test: The few fragments of magnesium ribbon were added to the plant extract followed by 1 mL of Con. HCl drop wise slowly. The test was confirmed positive with the appearance of crimson red colour.
GC–MS Analysis of plant extract
Both the stem and leaf ethanol crude extract was subjected to Gas chromatography–Mass spectrometry (GC–MS). GC-MS was analyzed using ionization energy of electron (70 ev). Helium gas was used for the analysis with constant flow rate of 1 mL/min. The mass transfer line and injector temperature was set at 280°C and 250°C. The spectrum of the components of the plant extract formed was compared with the stored database known components spectrum in the GC-MS library (NIST).
Anti-bacterial activity of Munja extract
The anti-bacterial activity assay of the ethanol extract of both steam and leaf was evaluated using well diffusion method. The most commonly available bacteria, Escherichia coli (E. coli) were isolated from the sludge and were inoculated in nutrient agar plate. The inoculum was made which had OD value of 0.5. This E. coli inoculum was streaked uniformly over Mueller-Hinton agar plate using sterile cotton swab. 4 wells were punched using well borer with 7 mm diameter. About 25 mL of both stem and leaf extract was inoculated into the punch well. Metronidazole and ampicillin was used as positive control whereas distilled water was used as negative control. The plates were incubated for 24 hours at room temperature .
Anti-oxidant activity of plant extract
DPPH solution (0.1 mmol/L) was prepared by mixing 2 mg of DPPH in 100 mL of methanol. 1 mL of the DPPH was added to each tube, containing 20 μL, 40 μL, 60 μL, 80 μL and 100 μL of both leaf and plant extracts separately. The mixtures were kept in the dark at room temperature for 30 min and absorbance value for each volume of the stem and plant extracts was measured at 517 nm against a blank. The concentration of stock solution of both stem and plant extracts were (1 mg/mL). After taking the OD for each volume of both stem and leaf extracts, we calculated the percentage of radical scavenging activity by taking control as Ascorbic acid which has an OD value of 0.684. After that we plotted 2 separate graphs for stem and leaf by taking “volume of the stem in μalong Y axis against “OD at 517 nm” along X-axis .
Preliminary phytochemical analysis of plant extract
Phytochemical results were shown in Table 1. The Saccharum munja plant extract was found to contain alkaloids, flavonoids, glycosides and Tannins. The presence of these bioactive compounds confers certain attributes to plant. For instances, presence of alkaloids indicates its role in plant metabolism. Also, the plant extract might possess cardiovascular and anti-inflammatory property because of the presence of cardiac glycosides. Flavonoids are known to play an important role in free radical scavenging activity. Since the plant extracts has tannins, it has a potential to confer cytotoxic activity against micro-organisms. As the plant extract has these important bioactive compounds, therefore could be an important source of therapeutic compounds (Figures 1 and 2).
|Ferric Chloride test||Negative|
|Keller Killani test||Negative|
|Ferric Chloride test||Positive|
|Alkaline Reagent test||Positive|
Table 1: Phytochemical analysis of the extract.
GC–MS analysis of bioactive compounds from plant extract
The GC-MS performance of leaf extract of Saccharum munja was recorded in Table 2. Out of many, 3 compounds are found to show high peaks, i.e., high concentration. They are 2,6-Pyrazinediamine with 20.39 RT, 18-Nonadecen-1-Ol with 20.99 RT and Cyclotrisiloxane, Hexamethyl- at RT 27.70 and 30.01.
|Sl. no.||Compound name||RT||Mol.
Table 2: GC-MS analysis of leaf extract.
Cyclotrisiloxane, Hexamethyl- was observed in highest concentration.
GC-MS performance of stem extract of Saccharum munja was recorded in Table 3. Four compounds are found in excess amount as they showed high peaks as compared to other compounds. They are Pentanoic Acid, 2-(Aminooxy) - at RT 17.79, Propanoic Acid, 2-(Aminooxy) - at RT 19.86, 2,6-Pyrazinediamine at RT 21.04 and Cyclotrisiloxane, Hexamethyl - at RT 21.10, 27.75, 29.20, 30.05. Cyclotrisiloxane, Hexamethyl- was found in excess amount as compared to others (Figures 3 and 4).
|Sl. No.||Compound name||RT||Mol. Weight||Formula||Structure|
|1.||Pentanoic acid, 2-(Aminooxy)-||17.79||133||C5H11O3N|
|2.||Propanoic acid, 2- (Aminooxy)-||19.86||105||C3H7O3N|
Table 3: GC-MS analysis of stem extract.
Anti-bacterial activity of plant extract
Figures 1 and 2 respectively shows the antibacterial activity of leaf and stem extract of Saccharum munja against gram negative E. coli. Both the leaf and stem extract shows much larger zone of inhibition than positive control i.e., ampicillin. This result shows that this plant extract has antibacterial property and hence could be potential antibacterial therapeutic source. The free radicals produced by the bioactive compounds present in the plant extract might have destroyed the bacterial cell membrane. Also, the compounds that possess negative charge can interfere with bacterial metabolic pathway and hence causes cell death (Figures 5-7).
Anti-oxidant activity of plant extract
The antioxidant activity of the extracts was evaluated based on their ability to trap DPPH radical. DPPH stable free radical method is most sensitive ways to estimate the antioxidant activity of plant. Percentage of radical scavenging activity of stem extract was found to be 42.66% whereas that of leaf was 16.79%. Since stem extract of Saccharum munja has more free radical scavenging activity than leaf extract and hence stem extract has more anti-oxidative property.
Large amount of resources are employed in synthesizing chemical compounds which requires lot of money and time. Therefore, there’s a need to look for alternative approach which are equally effective but cost efficient. In this study, Saccharum munja extract was found to possess antibacterial and anti-oxidative property. It was found that stem extract was found to have better free radical scavenging activity than leaf extract. This may be due to the presence of more bioactive compounds in stem extract than in leaf as shown in GC-MS analysis. Possibly those extra compounds present in the stem acts as a good radical scavenging.
We would like to thank VIT University for supporting the work.