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Phytochemical and antimicrobial investigations of methanolic extract & ethyl

acetate extract of rice husk (ORYZA SATIVA)

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Archives • 2014 • vol.3 •74-84

PHYTOCHEMICAL AND ANTIMICROBIAL INVESTIGATIONS

OF METHANOLIC EXTRACT & ETHYL ACETATE EXTRACT OF
RICE HUSK (ORYZA SATIVA)

Repon Kumer Saha*, Farhana Hossain , Nabila Tabassum Amin, Shakhawat Hossain Bhuiyan

Department of Pharmacy, East West University, Dhaka, Bangladesh.

*reponsaha@yahoo.com; drks@ewubd.edu

Abstract

The crude methanolic extract of rice husk (Oryza Sativa) was investigated for possible phytochemical,
antimicrobial activities. Thin layer chromatography and ultra-violet spectroscopy were used to detect the
presence of various types of compound in methanolic extract of rice husk. The methanolic extract showed
antibacterial activities. The crude methanolic extract was fractionated by vacuum liquid chromatography by
differents seven polar solvent. The 7 fractions were identified by performing TLC and later Disc diffusion
assay of the primary seven fractions were performed to show the antibacterial effect using gram positive,
gram negative strains of bacteria and fungi. Among the fractions n-butanol demonstrated antimicrobial
activity. Other six fractions did not demonstrate antimicrobial activity. The n-butanol fraction was further
exploited VLC to isolate the active principle which exhibited the antimicrobial activity. Dichloromethane
fraction showed good anti-microbial activity than other six fractions. DCM fraction also showed good result
in the MIC and MBC using bacteria and fungi. Lethality index and Receptor binding activities of the DCM
fraction was investigated by hemagglutination inhibition assay. DCM fraction showed the negligible amount
of toxicity effect and showed moderate hemagglutination inhibition activities.

Key words: Oryza sativa, antioxidant, antibacterial, TLC, hemagglutination assay

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Introduction TLC Analysis

Rice is the seed of plants (Oryza sativa). Its one of The extracts were analyzed by performing TLC to
the important foods of the world. Particularly, determine the composition of extract. TLC was done
countries in Asia are popular eating rice daily and using three solvent systems. The best result was
main food rather than in order regions of the world. obtained from solvent system-2 (chloroform: ethyl
Rice is sources of many bioactive non-nutrient acetate: formic acid- 5:4:1) [7]. After development of
compounds, known as phytochemicals [1]. TLC plates, they were exposed to UV light. For
Globally, approximately 600 million tons of rice charring the plates were sprayed with 10% sulphuric
paddy is produced each year. The total production acid solution, dried and then heated to 80-90ºC. This
of 120 million tones of rice husk is get on average allowed the spots to be visible. For detection of
20% from rice paddy [2]. In majority of rice flavanoids the plates were dipped into 0.04% DPPH
producing countries most of the husk produced solution and dried while keeping in a dark place. For
from processing of rice paddy which is used either detection of polyphenols the plates were washed
burnt or dumped as waste. The chemical and with Folin-ciocalteu reagent and dried.
functional components in different parts rice seed
(Oryza sativa ) before and after germination. Rice Preliminary Phytochemical Investigation
was separated into hull, then analysed for crude The ethyl acetate extract of husk Oryza sativa was
protein, crude lipid, free sugars, fatty acids, phytic qualitatively tested for the detection of any kind of
acid, vitamin E, γ-oryzanol and γ-aminobutyric acid secondary metabolites like alkaloid (Wagner Test),
(GABA). Before germination, the crude protein anthraquinone (Borntrager’s Test), cardiac glycosides
content of rough rice was 97.28 mg/g, whereas (Keller-kiliani Test), flavanoids (NaOH Test), steroids
after germination, it increased to 105.14 mg/g [3]. and terpenoids (Liebermann- Burchardt Test) [8].
The chemical compositions of RHA especially silica
content, is not high (in the range of 85% to 95%). All Chemical analysis by UV spectroscopy
the other constituents of RHA, Al2O3 , CaO , K2O Ultraviolet (UV) spectroscopy scanning[9]. of the
except potassium and magnesium, are available in a ethyl acetate extract of rice husk was performed
very small range, i.e., less than 1%[4]. Rice can be within 200nm to 400nm using a Lambda UV
used to treat skin conditions. The rice is boiled, spectrometer (Shimadzu, Japan). (Shimadzu,Japan).
drained and allowed to cool and mashed. The rice is
made into a paste or moulded into balls and these DPPH Free Radical Scavenging Assay
can be applied to boils, sores, swellings and skin The DPPH radical scavenging method was used for
blemishes. Other herbs are sometimes added to the the determination of the antioxidant capacity of the
rice balls to increase their medicinal effects. Sticky sample [10]. Different concentrations of the ethyl
glutinous rice is often taken to treat stomach acetate extract of husk Oryza sativa (10, 20, 30, 40 &
upsets, heart-burn and indigestion[5]. 50μg/ml, in water) was prepared and 100 µl of DPPH
Extracts from brown rice have been used to treat solution was added. Different concentrations of L-
breast and stomach cancer and warts. They have Ascorbic acid (10-50 µg/ml) were used as the
also been used to treat indigestion, nausea and standard antioxidant. After 30 min at room
diarrhea[6]. temperature, the absorbance values were measured
at 517 nm on a spectrophotometer and expressed
Materials and Methods: into percentage of antioxidant activity using the
Plant collection and identification: following equation: DPPH antiradical scavenging
The fresh Rice and plant sample were collected capacity (%) = (Absorbance of sample – Absorbance
from Comilla, under Chittagong division on 15th of blank) × 100/Absorbance of blank. DPPH solution
June, 2013 and identified by the taxonomist of the plus water was used as a control. IC50values denote
Bangladesh National Herbarium, Mirpur, Dhaka as the concentration of the sample required to
Oryza sativa. A voucher specimen of the plant has scavenge 50% of DPPH radicals. The results were
been deposited (Accession No.: 39530) in the expressed as mean ± standard deviations.
herbarium for further reference
Determination of total phenolic content
Extraction of the plant material The total phenolic content of ethyl acetate extract
700g of testa powder was measured using an was determined using Folin-Ciocalteu method using
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salicylic acid as standard [11] with some used to carry this assay. Dried and sterilized filter
modifications. The samples were oxidized with 10% paper discs (6 mm diameter), dried ethyl acetate
Folin-Ciocalteu reagent and were neutralized with extract of the husk, a stock solution of 60mg/ml was
700 mM sodium carbonate solution. The made in normalsaline. The plant samples were two
absorbance of the sample was measured at 765 nm fold serially diluted in eppendorf tubes to get
after 60 minutes. A calibration curve of salicylic acid different concentrations were prepared and discs
was prepared. The total phenolic content was were soaked with each solutions of 10μl of test
calculated as salicylic acid equivalent by the samples were placed gently on the previously
following equation: T = C x V /M, where, T is the marked zones in the agar plates. After incubation at
total phenolic content in mg·g –1 of the extracts as 370C for 24 hours, the antimicrobial activity of the
SAE, C is the concentration of salicylic acid test agents was determined by measuring the
established from the calibration curve in mg/ml, V is diameter of zone of inhibition expressed in mm. The
the volume of the extract solution in ml and M is results were expressed as mean ± standard
the weight of the extract in g. The estimation of the deviations.
phenolic compounds was carried out in triplicate.
The results were expressed as mean ± standard Minimum Bacterial Concentration of ethyl acetate
deviations. extract of Rice Husk
The minimum bactericidal concentration (MBC) is the
Antimicrobial Screening of ethyl acetate Extract of lowest concentration of an antibacterial agent
husk Oryza sativa required to kill a particular bacterium [15]. 100μl of
The antibacterial activity was carried out by the disc sample solution was placed on petridish. Nutrient
diffusion method [12] using 100μL of suspension agar was prepared by dissolving the appropriate
containing ~103 CFU/mL of microorganism spread amount of agar in distilled water in an agar bottle. All
on nutrient agar medium (Himedia, India). Four the necessary equipment and the nutrient agar were
different bacterial strains of gram positive, Eight autoclaved at 121ºC for 45 minutes to ensure
different strains of gram negative bacteria and complete sterilization. After this all the equipment
three different strains of fungi were used to carry were shifted to the laminar airflow hood to prevent
out this assay. Dried and sterilized filter paper discs any contaminations. The sample of bacteria 1ml was
(6 mm diameter), ethyl acetate extract of husk, a taken in in an eppendrof tube containing 600µg
stock solution of 10mg/ml was prepared and discs ethyl acetate extract and then five times dilution in
was soaked with solutions of 10μl of test samples the five eppendrof tube to get different
and dried placed[13]. Standard disc of ciprofloxacin concentrations. The each tube were taken into
(30 μg/disc) was used as positive control. After vortex to ensure that the bacteria and ethyl acetate
incubation at 370C for 24 hours, the antimicrobial extract mixture out evenly. The agar was poured into
activity of the test agents was determined by the petridishes and left to cool and solidify. Using a
measuring the diameter of zone of inhibition sterile spreader the 100µl bacteria solution were
expressed in mm. The results were expressed as taken and carefully swabbed on the agar plate so
mean ± standard deviations. that the bacteria were uniformly distributed
everywhere. The plates were left to rest for some
Minimum Inhibitory concentration of ethyl time and then place in an inverted position in an
acetate Rice Husk incubator at 37ºC for 24 hrs[16]. After the prescribed
The minimal inhibitory concentration (MIC) values, incubation time the plates were removed from the
which represent the lowest extract concentration incubator and the counting of the bacteria colony
that completely inhibits the growth of formation. Each experiment was carried out 2 times
microorganisms. The sample in question is first and was correlated against the controls.
prepared by producing a standard stock solution
then subsequently diluting it to obtain different Brine shrimp lethality test
concentration[14]. Minimum Inhibitory Brine shrimp lethality test [17] was carried out to
concentration is carried out by using 100μL of investigate the possible cytotoxic effect of the ethyl
suspension containing ~103 CFU/mL of acetate extract of husk Oryza sativa. Brine shrimp
microorganism spread on nutrient agar medium eggs were hatched in a shallow rectangular dish filled
(Himedia, India). S. aureus and S.cerevisiae were with artificial seawater provided with light and
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aeration. We allowed 2 days (48h) for the eggs to presence of alkaloid and Cardiac Glycoside in the
hatch and mature as nauplii. Samples were ethyl acetate extract of husk Oryza sativa. Other
prepared by dissolving 20 mg of the husk ethyl phytochemicals were absent.
acetate extract in the normalsaline Dilution of this
stock solution gives the series of concentrations UV-Visible Spectrophotometric Sanning of ethyl
required for testing. 6 vials for each sample were acetate extract of Oryza sativa.
taken. The no.6 vial was used as control (nauplii The spectrum of wavelength vs. absorbance for ethyl
without sample). To each sample vial ten shrimps acetate extract of the husk was
were transferred using a Pasteur pipette, and obtained from the UV-Visible spectrophotometer
artificial seawater was added to make a total and the values for the absorbance of the fraction was
volume of 5 ml. The nauplii were counted against a recorded. The graph is shown below.
lighted background. Counting for the chronic LC50
began 24 hour after initiation of tests [18]. Nauplii DPPH Free Radical Scavenging Assay
were considered dead if they were lying immobile From the analyses of Figure-2, we can conclude that
at the bottom of the vials and the percentage of the scavenging effect of ethyl acetate extract and
deaths at each concentration and at the control ascorbic acid are very near to each other.
were determined.
Total phenolic content assay of ethyl acetate extract
Hemagglutination Inhibition Assay In case of polyphenolic content a standard curve was
Hemagglutination activity of ethyl acetate extract of used where the equation is y = 0.044x + 0.024, R2 =
rice husk was tested against human erythrocyte 0.972, From the standard curve, the total phenolic
blood group A+ (positive) with some compounds as Salicylic acid equivalent (SAE) of the
modifications[19]. Stock solution of the test ethyl acetate extract was 40.9mg/g.
samples was prepared at concentration of 10
mg/ml and each solution was serially diluted. Fresh Antimicrobial Screening of ethyl acetate Extract
blood was collected from healthy persons, From the test conducted these results it is observed
centrifuged and the erythrocytes were separated. the husk of Oryza sativa possess antibacterial and
2% erythrocyte suspension was prepared in antifungal activities.
phosphate buffer (pH 7.4). 100μl of sample was
placed in the first well and then this was Minimum Inhibitory Concentration of Ethyl Acetate
subsequently diluted two fold up to the 8th well. Extract of Rice Husk.
100μl of the RBC suspension was added to all the The husk ethyl acetate extract was tested upon the
wells and was incubated for one hour at 4°C. After Gram positive S. aureus and fungi S.cerevisiae the
incubation, the results were noted. Smooth button zones of inhibition were recorded. The results of this
formation in bottom indicated negative activity, experiment are as follows
while a rough granular deposition at bottom
showed positive activity. The intensity of Minimum Bacterial Concentration of ethyl acetate
hemagglutination was determined from the extent Extract
of deposition. The husk ethyl acetate extract was tested upon the
Gram positive S.aureus the minimum bacterial
Results concentration was recorded. The results of this
TLC Analysis experiment are as follows.
TLC analysis was done as described in materials and
methods. The plate was observed under UV light Brine shrimp lethality test
(indicated as 1). After charring of the TLC plate with In brine shrimp lethality bioassay, percentage of
sulfuric acid (indicated as 2) has visualized the three mortality increased gradual with the increase in
spots. After being soaked into DPPH and FC concentration of the test samples. Using the linear
solution, plate 3 and 4 showed moderate yellow regression equation, y = -12.28x + 104.6 where R2 =
color. 0.984 the LC50 values of ethyl acetate extract was
determined. From the results, it was revealed that,
Preliminary Phytochemical Investigation LC50 values was found to be 4.4.
Preliminary phytochemical screening showed the
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Hemagglutination Inhibition Assay In antimicrobial screening, ethyl acetate extract husk

Ethyl acetate extract exhibited hemagglutination Oryza sativa showed good result against antibacterial
inhibition activity potentially from highest and antifungal activities. The positive control was
concentration 4 mg/ml to 0.5 mg/ml i.e. it has used Ciprofloxacin. Ethyl acetate extract was showed
potential binding capacity with human erythrocytes zone of inhibition up to 20mm antibacterial activity
at the concentrations used against S. aureus, A.
Discussions niger, B. subtilis, Saprophyti than other strain. A
Single chromatogram observed under UV light in previous study showed that the oils possessing
TLC indicated the presence of different compounds antimicrobial activity can be employed against
in that sample. Spraying of DPPH solution on the human pathogens[22]. The intake of rice bran oil
TLC plate have shown significant formation of plate might promote human health by preventing bacterial
yellow color. This provides us a preliminary idea of pathogenesis The result found in our study might
the various types of compounds that may be have correlation with this. Methanol extract of Rice
present in the ethyl acetate extract of the husk of husk of Oryza sativa was further exploited in an
Oryza sativa. From the preliminary Phytochemical attempt to isolate the active principle which
screening it was concluded that the ethyl acetate exhibited the different antimicrobial activity. Due to
extract might be alkaloid and cardiac glycosides the good result minimum inhibitory concentration
because it gave positive result for the alkaloid test test was carried out with ethyl acetate extract
only. against the Gram positive S. aureus and fungi
The spectrum of wavelength vs. absorbance for S.cerevisiae From the results we see that the MIC of
ethyl acetate extract of the husk was obtained from the ethyl acetate extract of the husk of Oryza sativa
the UV-Visible spectrophotometer and the value for for S. aureus and S.cerevisiae is 3.75μg/disk.
the absorbance of the extract was recorded. Therefore, the dichloromethane fraction of
232nm, 285nm & 316 nm were found to be the λ methanolic extract of Oryza sativa may be
max for the husk ethyl acetate extract of Oryza considered as a useful source for discovering a safe
sativa. and novel antimicrobial compound.
To evaluate the antioxidant activities of ethyl The MBC test determines the lowest concentration
acetate extract of husk DPPH Free Radical at which an antimicrobial agent will kill a particular
Scavenging Assay was used. The results show that microorganism. The MBC test was carried out with
the IC50% of ascorbic acid is 36.38 µg/µl and husk the ethyl acetate extract of rice husk since it
ethyl acetate extract is 45.99 µg/µl. These results exhibited good antimicrobial activity against
show that the husk of Oryza sativa possess microbes. The concentration of 600µg, 300µg and
antioxidant free radical scavenging activity. This 150µg husk ethyl acetate extract exhibited good
indicates that there are many compounds present result against B .cereus. A study showed that, 500
in them that have antioxidant potential. A previous μg/ml methanolic crude extracts of Anabaena BT2
study showed that, antioxidant activity measured in produced 72-78 % growth inhibition of test bacteria
ethanolic extract of Rice paddy (µmol Ascorbic acid [23].
equivalent/g fresh mass) were 67.09 and 15.55 as The brine shrimp lethality assay was used for the
determind by DPPH radical –scavenging acitivity evaluation of cytotoxic effects of the ethyl acetate
respectively [20] . So the isolated compound could extract of husk Oryza sativa. The LC50 values of ethyl
be a future drug interest for antioxidant activity. acetate extract was determined respectively. The
A study reported that the total polyphenolic LC50 values for ethyl acetate extract was 6.3 mg/ml.
content found in methanolic extract of boro and Hemagglutination inhibition assay was performed to
amon ,compared to boro rice and amon rice investigate the receptor binding affinity of the
contained more phenolic compounds. Aman rice compounds present in the husk ethyl acetate extract
TPC ranged between 13.58 mg/g eq (BR22) to 25.30 on human erythrocytes. It was observed that the
mg/g eq and BRRI dhan37 had the TPC value of extract has different binding affinity to the different
21.14 mg/g[21]. In our study showed that the receptors of erythrocytes and prevent agglutination.
phenolic content of ethyl acetate extract of rice Hence the results showed a possible benefits of
husk was 40.09 mg/g eq. of salicylic acid which was Oryza sativa Ethyl acetate extract as an antiviral
higher than reported the phenolic content of therapeutics.
methanolic extract extract of aman_______________________________________
and boro rice.
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ISSN: 1827-8620
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Malaysian Rice Husk Ash Improving the Durability and Concentrations of Boron Compounds against Several
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5. Huang, Xuehui, Kurata, Nori, Wei, Xinghua, Wang, Zi-Xuan, 16. Koochak, H., Seyyednejad, M.S., Motamedi, H. Preliminary
Wang, Ahong, Zhao, Qiang, Zhao, Yan, Liu, Kunyan et al. A study on the antibacterial activity of some medicinal plants
map of rice genome variation reveals the origin of of Khuzestan (Iran). Asian Pacific Journal of Tropical
cultivated rice. Nature.2012; 490 (7421): 497–501. Medicine 2010; 23(3): 180-184
6. Vaughan, et al., Lu, B., Tomooka, N. The evolving story of 17. Zarai,Z., Kadri, A. The in-vitro evaluation of antibacterial,
rice evolution. Plant Science 2008; 174 (4): 394–408. antifungal and cytotoxicproperties of Marrubium vulgareL.
7. Malbaša, R.V., Lončar, E.S., Kolarov L. A. TLC Analysis of essential oil grown in Tunisia. Lipids in Health and Disease
Some Phenolic Compounds in Kombucha Beverage. Aceta 2011; 10 (161): 1-8.
Periodica Technologica. 2004; 35: 199-205. 18. Tawaha, K.A. Cytotoxicity Evaluation of Jordanian Wild
8. Sashidharan, S., Chen, Y., Saravanan, D., Sundram, K.M., Plants using Brine Shrimp Lethality Test. Journal of Applied
Latha L.Y. Extraction, Isolation and Characterization of Sciences 2006; 8(1): 12-17.
Bioactive Compounds from Plants Extracts. African Journal 19. Saha, R.K., Takahashi, T., Suzuki, T. Glucosyl hesperidin
of Traditional, Complementary and Alternative Medicine. prevents influenza a virus replication in vitro by inhibition of
2011; 8(1):1-10. viral Sialidase. Biological & pharmaceutical bulletin. 2009;
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SerumAsian Journal of Microbiology, Biotechnology and 20. Dutta K.A., Gope S.P., Banik S., Makhnoon S., Siddique A.
Environmental Science. 2003; 5(4): 581-582. M., Kabir Y.Antioxidant properties of ten high yielding rice
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and Antioxidant Activity of Different Grades of Turkish Biomedicine2012; (99): 103.
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11. Blainski, A., Lopes, G.C., de Mello J.C.P. Application and 5(2): 19.
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Determination of the Total Phenolic Content from Some Cyanobacteria. International Journal Of Pharmacy
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Figure 1:

1 2 3 4

Table1. Results of preliminary Phytochemical

Investigation of secondary metabolites
Phytochemical Test Result
Alkaloid +
Anthra-quinone -
Cardiac Glycoside -
Flavonoid -
Steroid & Terpenoid -

Figure 2. The graph of Wavelength vs. Absorbance for husk ethyl acetate
extract of Oryza sativa

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Table2. Ethyl acetate results of the Total Phenolic

Content Assay

Total phenolic content

Sample Name
(mg/g SAE)

Ethyl acetate 40.9 ±2.0

Table 3: Antibacterial Activity of ethyl acetate extract on

the microorganisms test

Zone of inhibition (mm)

Ethyl
acetate
extract of Negative
Species Ciprofloxacin
rice control
husk(10m
g/ml)
Vibriomimicus 15.5±0.7 20.5±0.7 -
S. cerevisae 18±2.8 22.5 ±2.1 -
S. pyrogen 20±0.0 22.5±3.5 -
Shigella boydii 20±0.0 23±1.4 -
S. typhi 16.5±2.1 19.5±2.1 -
C. albicans 17±0.0 19±1.4 -
Saprophyti 21±2.8 23.5±2.1 -
B. subtilis 22.5±2.1 26.5±2.1 -
S. aureus 23±1.4 23.5±2.1 -
S. dysentry 10.5±0.7 17.5±0.7 -
A.niger 22±1.4 23±1.4 -
Klebshiella 17.5±0.7 20±2.8 -
E.coli 17.5±2.1 21.5±3.5 -
Beta Hemolyte 17±1.4 22±0.7 -

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Figure 3. Zone of inhibition for Ethyl acetate extract of rice husk. The values were
expressed as mean ± standard error of mean. Blue bars represent values of Ethyl
acetate extract; red bars represent values of positive control ciprofloxacin.

Table 4. Zones of Inhibition of S. aureus and S.cerevisiae when

tested with the husk Ethyl acetate extract

Minimum Zones of Inhibition (mm)

Species
30µg 15µg 7.5µg 3.75µg 1.88µg

S. aureus 17±1.4 13.5±0.7 13±1.4 10 ±0.0 6.5 ±0.7

S.cerevisiae 15.5±0.7 12 ±1.4 9.5 ±2.1 7.5 ±2.1 -

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Figure 4. Graph of zones of inhibition of S. aureus and

S.cerevisiae when tested with the husk Ethyl acetate extract.

Table5. Minimum bacterial concentration of S.aureus

when tested with the husk Ethyl acetate extract.

Minimum bacterial count (CFU)

Species
600µg 300µg 150µg 75µg Control

S.aureus 4±4.24 4 ±2.83 7±4.24 169±4.24 177.5±10.61

Figure 5. Graph of the Minimum bacterial concentration of

S.aureus tested with the husk ethyl acetate extract

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Figure 6. Concentration vs. percentage of lethality graph of Ethyl

acetate extract

Table 6. Hemagglutination Inhibition, Test for ethyl acetate extract

Sample concentrations (mg/ml)

Sample
Name
4 2 1 0.5 0.25 0.125 0.0625 0.03125

Ethyl
acetate +++ +++ +++ +++ - - - -
extract

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