




































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: swagh.gene@gmail.com; 
 
 
 

Asian Journal of Immunology 
 
2(1): 78-88, 2019; Article no.AJI.53611 
 

 
 

 

 

Phytochemical Analysis of Simarouba glauca Dc. 
and Comparison of its Bioactivity 

 
Kiran R. Pawar1, Ganesh B. Janavale1, Sopan G. Wagh1*, Archana N. Panche1,  

A. Abhijit Daspute2, Manoj B. Pohare3 and Sanjay N. Harke1 
 

1
Mahatma Gandhi Mission’s Institute of Biosciences and Technology, N-6 CIDCO, Main Campus, 

Aurangabad, Maharashtra 431003, India. 
2
Laboratory of Plant Cell Technology, Faculty of Applied Biological Sciences, Gifu University,  

Gifu 5011193, Japan. 
3
Department of Infection, Immunity and Cardiovascular Diseases, University of Sheffield, Beech Hill 

Road, Sheffield, S10 2RX, United Kingdom. 
 

Authors’ contributions 
 

This work was carried out in collaboration among all authors. Authors KRP, SNH and GBJ designed 
the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the 

manuscript. Authors ANP, SGW and AAD managed the analyses of the study. Author MBP managed 
the literature searches. All authors read and approved the final manuscript. 

 
Article Information 

 
Editor(s): 

(1) Dr. Darko Nozic, Professor, Higher Medical School in Belgrade, University of Belgrade, Serbia. 
Reviewers: 

(1) Pipat Chooto, Prince of Songkla University, Thailand.  
(2) Oluwatosin Johnson, University of Lagos, Nigeria. 

Complete Peer review History: http://www.sdiarticle4.com/review-history/53611 

 
 
 
 

Received 20 October 2019  
Accepted 25 December 2019 
Published 26 December 2019 

 
 

ABSTRACT 
 

Medicinal implications of plants are well known to us since the time immemorial and are well 
documented in the ancient literature. According to WHO report, 80% of world populations count on 
herbal medicines to treat themselves in case of illness. And the main reason behind this could be 
the side effects caused by the drugs which are recommended now a days by the general 
practitioners. Herbal medicines are known to produce negligible amount of side effects and that is 
why they are among the favorite candidates for health care. In the present study, we have 
attempted to rediscover the medical potential of the Simarouba glauca. We have performed 
phytochemical analysis of Simarouba glauca and compared its bioactivities (antibacterial, 
antifungal, anti-diabetic, hemolytic, antioxidant, and antimicrobial)with synthesized zinc oxide 
nanoparticles. 

Original Research Article 



 
 
 
 

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79 

 

Keywords: Simarouba glauca; antibacterial; antifungal; anti-diabetic; antioxidant. 
 

1. INTRODUCTION 
 

The potential of plant to serve human beings in 
wellness of health and overall immunity and 
range of aspects has been well documented 
since former times [1]. According to World Health 
Organization, 80% of world’spopulation mainly 
counts on traditional medicines for health care. 
Plant products have been a part of 
phytomedicines since time immemorial [1,2]. 
There are about 2000 species of plants that are 
used for medicinal purposes and detected as 
having a therapeutic significance. Many of these 
medicinal plants, which are known to the humans 
since ancient time, either are on the verge of 
extinction or already listed as endangered. Lots 
of efforts are already in progress in the different 
corners of the world to conserve these plants 
and/or to enhancetheir ability to produce extra 
amount of medicinal compounds they are 
producing either by plant tissue culture 
techniques [3-7] or by the use of recently 
developed advanced techniques like Clustered 
Regularly Interspaced Short Palindromic 
Repeats with CRISPR associated protein 
9(CRISPR/Cas9) [8,9]. Medicinal plants play a 
vital role in preventing various diseases [10] and 
their medicinal value lies in some bioactive 
constituents (like flavonoids and phenols) that 
produce a definite physiological action on the 
human body [1,11]. It has been estimated that 14 
- 28% of higher plant species are used 
medicinally and that 74% of pharmacologically 
active plant derived components were 
discovered [12]. Out of the several hundred 
thousand medicinal plant species around the 
globe, only a small portion has been investigated 
so far both phytochemically and 
pharmacologically [12]. 
 

Plants are enriched with various phytochemicals 
such as vitamins, terpenoids, phenolic acids, 
lignins, stilbenes, tannins, flavonoids, quinones, 
coumarins, alkaloids, amines, betalains and 
other metabolites, which are rich in antioxidant 
activity [13]. Studies have shown that many of 
these antioxidant compounds possess anti-
inflammatory, antiatherosclerotic, antitumor, 
antimutagenic, anticarcinogenic, antibacterial, 
and antiviral activities [14].  
 

Phytochemicals are chemicals, which are 
naturally present in plants. Recently phyto 
chemicals have become more popular due to 
their countless medicinal uses. Phytochemicals 
play a vital role against number of diseases such 

as asthma, arthritis, cancer etc. unlike 
pharmaceutical chemicals these phytochemicals 
do not have any side effects. Phytochemicals are 
derived from different parts of plant such as 
leaves, flowers, roots, pulps, seeds, etc. [15]. 
Phytochemicals are also known as “Man friendly 
medicines” as they cure diseases without 
causing any harm to human beings [15]. To 
produce synthetic molecular analogs, a large 
proportion of plant based compounds are used 
as lead molecules in drug discovery which 
implies that phytochemicals play a crucial role in 
diversity oriented synthesis of natural product like 
pharma compounds [16]. It is a rain fed waste 
land evergreen edible oil tree. Simarouba glauca 
belongs to the family Simaroubaceae. It contains 
various groups of chemical components [17].  
Quassinoids is the main active group of 
chemicals in Simarouba glauca which belongs to 
triterpine family [16]. Pharmacological properties 
of Simarouba glauca are haemostatic, 
antihelmenthic, antiparasitic, antidysentric, 
antipyretic and anticancerous. Simarouba glauca 
is a native species to Florida in the United 
States, southern Florida, South America, and the 
Lesser Antilles [18]. Simarouba glauca is 
indigenous to the rainforest and other tropical 
areas in Mexico, Cuba, Haita and Central 
America. Its cultivation depends on rainfall 
distribution, water holding capacity of the soil and 
sub-soil moisture.  Simarouba glauca is found in 
several cultivated parts of India and was 
introduced in India during 1960s by plant genetic 
resources in the research station at Amravati in 
Maharashtra and University of agriculture 
science Banglore in 1986 [19]. Simarouba glauca 
is suited for temperature range of 10 to 50°C. It 
can grow at elevations from sea level to 1,000 m 
(3,300 ft). It grows 40 to 50 ft (12 to 15 m) tall 
and has a span of 25 to 30 ft (7.6 to 9.1 m) [18]. 
It has bright green leaves, small white flowers 
and small red fruits [16]. Leaves of Simarouba 
glauca are bluish oily green and smooth at apex 
and are pinnately compound. It has 3-21 leaflets. 
Simarouba leaf decoction when taken in limited 
amounts can raise the immunity, reduce appetite 
loss and increase quality of life in cancer patients 
[18]. Simarouba glauca has very good anti-
bacterial, anti-tumorous properties so it is very 
effective in reducing the size of tumors and 
secondary infections in cancer patients and in 
curing cancer of first/second stages, whereas in 
later stages it can considerably increase the 
quality of life [18].Nanotechnology involves the 
use of materials having nanoscale dimensions in 



 
 
 
 

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80 

 

the range of 1–100 nm [20]. Nanomaterials are 
particles having nanoscale dimension, and 
nanoparticles are very small sized particles with 
enhanced catalytic reactivity, thermal 
conductivity, non-linear optical performance and 
chemical steadiness owing to its large surface 
area to volume ratio [21,22].  
 

The nanomaterials can be synthesized by 
different methods including chemical, physical, 
irradiation and biological methods. The 
development of new chemical or physical 
methods has resulted in environmental 
contaminations, since the chemical procedures 
involved in the synthesis of nanomaterials 
generate a large amount of hazardous 
byproducts [23,24]. Hence the green synthesis 
methods for the nanoparticle synthesis are being 
used now a day’s which are clean, safe, eco-
friendly and environmentally nontoxic. Moreover, 
in this method there is no need to use high 
pressure, energy, temperature and toxic 
chemicals [24,25,26]. Nanoparticles possess a 
wide array of application in the different fields’ 
viz., medicine, electronics, and therapeutics               
and as diagnostic agents [24]. Nano-particles 
have started being considered as nano-antibiotic 
because of their antimicrobial activity [22]. They 
also have potential application in the field of 
medicine like drug discovery, biological activities 
such as anti-microbial, antioxidant etc. and 
diagnosis of diseases [20]. 
 

The Zinc oxide nanoparticles have found 
fabulous applications in bimolecular, diagnostics 
and microelectronics [24], electromagnetic 
coupled sensors [20]. The Zinc oxide nano 
particles have been used to remove arsenic and 
sulphur from water even through bulk zinc               
oxide cannot absorb arsenic. It is because 
nanoparticles have much larger surface areas 
than bulk particles [27]. They have fascinating 
properties and extensive applications. Bio-
inspired synthesis of the zinc oxide nanoparticles 
has been achieved using environmentally and 
eco-friendly accepted systems. 
 

In this research work, we have performed 
phytochemical analysis of Simarouba glauca and 
compared its bioactivities with synthesized zinc 
oxide nanoparticles. 
 

2. MATERIALS AND METHODS 
 

2.1 Collection and Preparation of Plant 
Material 

 

Simarouba glauca leaves were collected from 
local areas of Aurangabad, MS, India. After 

collection of leaves they are cleanedwith distil 
waterto remove all the impurities and unwanted 
materials. Dried to remove all the moisture 
content present in leaves so that it can be stored 
for further analysis. Complete drying of leaves is 
followed by grinding to convert them in powder 
form for further use. 
 

2.2 Preparation of Plant Extract  
 

Methanol extract: For the methanol extract, 200 
ml of methanol and 20 gm of dry powder placed 
in soxhlet extractor. Extraction was carried out 
for 3hrs and 9 cycles were performed. Dark 
green colour extract was obtained. 
 
Ethyl acetate extract: 200 ml of ethyl acetate 
and 20 gm of dry powder were placed in soxhlet 
extractor. Extraction was carried out for 3 hrs and 
9 cycles were performed. Dark green color 
extract was obtained. Methanol and ethyl acetate 
extract were evaporated using rotary evaporator 
and powder obtained was collected for further 
analysis. The powder was then kept in sterile 
bottles, under refrigerated conditions, until further 
use. 
 
Preliminary phytochemical analysis: The 
extracts were subjected to preliminary 
phytochemical testing to detect for the presence 
of different chemicals or compounds. S. glauca 
leaves extract were screened for the presence of 
alkaloids, flavonoids, carbohydrates, glycosides, 
tannins, terpenoids, saponins and steroids. 
 
Test for alkaloids: Mayer’s test: In 1 ml of 
extract add 1 ml of Mayer’s reagent. Appearance 
of creamy white precipitation indicates the 
presence of alkaloids. 
 

Test for flavonoids: In 5 mg of extract 
powdered 10 ml of sodium hydroxide. Yellow 
fluorescence indicates the presence of 
flavonoids. 
 

Test for terpenoids: Salkowski’s test: In 5 ml of 
extract add 2 ml of chloroform and 3 ml of 
concentrated sulphuric acid. Reddish brown 
colour indicates the presence of Terpenoids. 
 

Test for steroids: In 5 mg of extract add 2 ml of 
acetic acid unhydride and 2 ml of sulphuric     
acid. Green colour indicates the presence of 
steroids. 
 

Test for saponins: 1 mg of extract was mixed 
with 5 ml of distilled water and shaken well. 
Appearance of creamy white miss bubbles 
indicates the presence of saponins. 



 
 
 
 

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81 

 

Test for tanins: 5 mg of extract was mixed with 
3 ml of distilled water and heated on a water 
bath. The mixture was filtered and add 5 mg of 
ferric chloride to the filtrate. Dark green colour 
indicates the presence of tanins. 

 
Test for carbohydrates and glycosides: 
Benedict’s test: In 2 ml of extract add 2 ml 
Benedict’s reagent, heat in boiling water bath for 
5 min, appearance of precipitation indicates the 
presence of glycosides. 

 
2.3 Qualitative Analysis of Phyto-

chemicals by Thin Layer Chromato-
graphy 

 
In the TLC analysis, 1 cm from the edge of the 
thin layer plate for a straight line across the plate.  
Spot the extract in the line with the capillary tube 
to dry the spot and place the plate in a beaker 
with a mobile phase. Layer the beaker when    
the solvent reaches more than 1 cm from the top 
of the plate. Remove the beaker from the solvent 
line before the Rf values have been calculated. 

 
2.4 Synthesis of Zinc Oxide Nanoparticles 
 
For the synthesis of zinc oxide nanoparticles, 
prepare 1 mM of Zinc acetate in 50 ml of sterile 
distilled water.   20 ml of 0.1 M sodium hydroxide 
and 25 ml plant leaf extract was then addedto the 
1 mM Zinc acetate solution. Thissolution was 
then kept incubated on magnetic stirrer for 3 hrs. 
Appearance of yellow color indicates the 
presence of Zinc oxide nanoparticles. This 
mixture was then centrifuged to separate 
supernatant and pellete. Supernatantwas 
discarded and pellete was dried in the hot air 
oven. Dried powder was then preserved in an 
airtight bottle for further studies. Characterization 
of zinc oxide nanoparticles was carried out by 
using UV visible spectrophotometry. 

 
2.5 Antibacterial Activity 
 
In vitro antibacterial activities were examined for 
methanol, ethyl acetate extract and zinc oxide 
nanoparticles. Antibacterial activities of plant leaf 
extracts and zinc oxide nanoparticles against 
three pathogenic bacteria (E. coli, S. auerus, and 
Bacillus subtilis) were investigated by the well 
diffusion method. The petriplateswere poured 
with approximately 25 ml autoclaved nutrient 
agar media each.  Agar plates were inoculated 
with three different bacterial colonies and    
spread with sterile swab. Four wells were 

punched on each plate using a sterile cork borer 
of 8 mm diameter. 100 μL of each extract was 
pipetted into respective wells. Standard leaf 
extract was used as a control.The Petri       
plates were incubated overnight at 37°C and the 
anti-bacterial activity was measured after 18     
hrs of incubationby measuring the zone of 
inhibition. 
 

2.6 Antifungal Activity 
 
In vitro antifungal activities were examined for 
methanol, ethyl acetate extract and zinc oxide 
nanoparticles. Antifungal activities of plant leaf 
extracts and zinc oxide nanoparticles against 
pathogenic fungus (Asperillusflavus) were 
investigated by the well diffusion method. All 
plates were autoclaved with PDA agar media of 
approx. 25 ml.  Aspergillus flavus colonies were 
inoculated with agar plates and sterile swab 
spread. Each plate was hit with four wells using a 
sterile 8 mm diameter cork borer. The pipetting 
into respective wells was 100 μL of each extract. 
Standard leaf extract was used as a control. The 
Petri plates were incubated overnight at 37°C 
and the antifungal activity was measured after 48 
hrs of incubationby measuring the zone of 
inhibition.  
 

2.7 Haemolytic Assay 
 

The haemolytic activity of the extract was 
determined using agar diffusion technique on 
blood agar plate. Blood agar was prepared and 
well measuring 8 mm were made on the agar 
using cork borer. The wells were filled with 100 
μL of different concentration of plant extracts 
solution and zinc oxide nanoparticles solution. 
Standard leaf extract was used as a control. The 
plates were then incubated at 37ºC for 24 hrs 
and the haemolytic activity was measured after 
24 hrs of incubationby measuring the zone of 
inhibition. 
 

2.8 Antioxidant Activity 
 
Scavenging of H2O2 radicals: The ability of the 
extracts to scavenge H2O2 was determined 
according to the reported method (reference). A 
solution of H2O2 (40 mM) was prepared in 
phosphate buffer (pH 7.4). Extract of selected 
concentrations in distilled water were added to 
H2O2 solution (0.6 mL, 40 mM). The absorbance 
of H2O2 at 230 nm was determined after 10 
minutes against a blank solution containing 
phosphate buffer without H2O2. Ascorbic                  
acid was used as a standard antioxidant. The % 



 
 
 
 

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82 

 

of H2O2 scavenging of both extracts and 
standard compounds were calculated. % 
scavenged [H2O2] = [(A−A)/A] x 100                  
where A was the absorbance of the control and A 
was the absorbance in the presence of the 
sample of extract (rewrite this formula correctly 
again). 

 
DPPH (1,1-Diphenyl -2-picryl hydarzil) free 
radical scavenging activity: 0.1 mM solution of 
DPPH in ethanol was prepared. 500 µg of 
sample was added in 1 ml of solution. The 
mixture was shaken vigorously and allowed to 
stand room temperature for 30 min. Then the 
absorbance was measured at 517 nm by using 
UV visible spectrophotometer. Lower absorbance 
of reaction mixture indicates higher free radical 
scavenging activity. The % DPPH scavenging 
effect was calculated using following 
equation.The capability to scavenge the DPPH 
radical was calculated using the following 
equation: DPPH scavenging effect (%) = [(A0 – 
A1)/A0) × 100], where A0 is the absorbance of 
the control reaction, and A1 is the absorbance of 
presence of all of the extract samples and 
standard. 

 
Antidiabetic activity: The antidiabetic activity of 
the extract was determined using starch agar 
plates. Starch agar plates were prepared by 
using 0.1% starch and 1% nutrient agar. After 
that 5wells measuring 8 mm were made on the 
agar plates using cork borer. The wells were 
filled with given concentrationsof phosphate 
buffer and amylase enzyme and sample as 
indicated below. 
 

1
st
 well – 40 ul amylase + 60ul phosphate buffer 

= 100 ul 
 

2
nd

 well – 100 ul buffer 
 
3

rd
 well – 40 ul amylase + 40 ul phosphate buffer 

+ 20 ul sample = 100 ul 
 

4
th
 well – 40 ul amylase + 20 ul phosphate buffer 

+ 40 ul sample = 100 ul 
 

5
th
 well – 40 ul amylase + 60 ul sample = 100 ul 

 

3. RESULTS AND DISCUSSION 
 

3.1 Preliminary Phytochemical Analysis 
 

Phytochemical screening is a preliminary step in 
the characterization of plant products. 
Preliminary screening for presence or absence of 
a particular class of compound is qualitative in 

nature and form a robust base for the 
quantitative estimation of bioactive components. 
Phytochemicals were screened for Alkaloids, 
Carbohydrates, Glycosides, Coumarins, Proteins 
& Amino acids, Phenolic compounds, Tannins, 
Flavonoids, Terpenoids.  
 
Qualitative phytochemical investigation of extract 
of Simarouba glauca leaves showed the 
presence of saponins, tannins, flavonoids, 
glycosides, alkaloids, steroids, terpenoids. 
Methanol and ethyl acetate extract of Simarouba 
glauca leaves contain maximum number of 
phytoconstituents. The results of qualitative 
phytochemical analysis areshowed in the Tables 
1, 2, 3. 
 

Table 1. Preliminary phytochemical analysis 
of methanolic and ethyl acetate extracts 

 

Sr 
No. 

Phytochemicals Methanol 
extract 

Ethyl 
acetate 
extract 

1 Alkaloids + + 
2 Glycosides + + 
3 Flavonoids + + 
4 Terpenoids + + 
5 Steroids + + 
6 Saponins - + 
7 Tannins + + 

 

3.2 Qualitative Determination of Bioactive 
Compounds by Thin Layer Chromato-
graphy 

 

Methanol and Ethyl acetate extract obtained from 
S. glauca leaves was used to perform thin layer 
chromatography in order to establish the purity 
and composition of materials. The purified 
components of Methanolic and Ethyl acetate 
extract of S. glauca leaves were identified by 
iodine test. For different components different 
bands were observed under ultraviolet light (Figs. 
1, 2). 

 
3.3 Synthesis of Zinc Oxide Nanoparticles 
 
In visual observation colour of zinc acetate 
solution was changed to yellow after addition of 
simarouba leaf extract and yellowish precipitate 
indicates the formation of zinc oxide 
nanoparticles. For further confirmation UV visible 
spectroscopy was carried out. 

 
UV-visible spectroscopy is usually conducted to 
confirm the synthesis of zinc oxide nanoparticles. 
The absorption spectrum was recorded for the 



 
 
 
 

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83 

 

sample in the range of 200 – 400 nm. The 
spectrum showed the absorbance peak at 310 
nm corresponding to the characteristic band of 
zinc oxide nanoparticles. 
 

 
 

Fig. 1. TLC for ethyl acetate, showing the 
separation of alkaloids, Teroenoids and 

flavonoids 
 

 
 

Fig. 2. TLC for methanol, showing the 
separation of alkaloids, teroenoids and 

flavonoids 

 
3.4 Antibacterial Activity 
 
The results of antimicrobial activity of methanol, 
ethyl acetate extracts and zinc oxide 
nanoparticles of E. coli, S. auerus, and Bacillus 
subtilis are given in Table 4. From the results it 
was found that zinc oxide nanoparticles have 
widerinhibitory zone as compared tomethanol 
and ethyl acetate extracts. Ethyl acetate extract 
have the least inhibitory zone.  These results 
clearly indicate that zinc oxide nanoparticles 
have enhancedantibacterial activity as compared 
to methanol and ethyl acetate extracts(Tables. 3, 
4) (Figs. 3, 4). 

 
3.5 Antifungal Activity 
 
The antifungal assay showed that Simarouba 
glauca has antifungal property against the tested 

fungi Aspergillus Niger. In this analysis, zinc 
oxide nanoparticles were found to be more 
effective as compared to methanolic and ethyl 
acetate extracts against the growth of fungi. The 
results of antifungal activity of methanolic, ethyl 
acetate extracts and zinc oxide nanoparticles of 
Aspergillus flavusis given in Table 5. 

 
3.6 Haemolytic Assay 
 
The zone of haemolysis was directly proportional 
to concentration of the extract. From the results it 
was found that zinc oxide nanoparticles (Table. 
6) have enhancedzone of haemolysisas 
compared tomethanol and ethyl acetate extracts 
(Fig. 5). 

 
3.7 Antioxidant Activity 
 
Antioxidant acitivity of the zinc oxide 
nanoparticles, methanolic and ethyl acetate 
extracts from the plant leaf SSimarouba glauca 
was carried out with hydroxyl radical scavenging 
assay and DPPH scavenging activity. 

 
3.8 Antidiabetic Activity 
 

Amylase inhibition detection was carried out 
using starch agar plates technique. The amylase 
activity inhibition zone appeared around the wells 
having zinc oxide nanoparticles, methanolic and 
ethyl acetate extract (Table. 7) (Fig. 6). 
 

A large number of biologically active plant 
constituents have been found in the Simarouba 
glauca extract in the phytochemical analysis. The 
result showed variations in the number of 
phytochemicals obtained from both extracts. 
More than 124 metabolites were detected in the 
ethyl acetate extract compared to both solvent 
extracts. This may be due to the high solubility of 
the active compounds of Simarouba glauca with 
ethyl acetate by extraction compared to the 
petroleum ether solvent. 
 

In addition to these results, it is also documented 
that different light intensities have various effects 
on the efficiency of the metabolites produced by 
the plants as seen in case of Glechoma longituba 
[28]. Zang et al. (2015) explained, chloroplasts 
produces different metabolites, and chloroplasts 
development mainly depends on the different 
light intensities [29,30,31]. Similar kind of effects 
can also be possible in case of Simarouba 
glauca and appropriate light intensity 
management might be feasible to obtain higher 
yields of secondary metabolites in it, which might 



 
 
 
 

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84 

 

be helpful in improving its either antibacterial, 
antifungal, anti-diabetic, hemolytic, antioxidant, 
and antimicrobial. Moreover,S. glaucaand similar 
kind of medicinal plants’ specific antimicrobial 
activity isnowwell established, so one can also 
make the efforts to do the molecular studies, 
trace back the proteins and genes mainly 
responsible for these effects and clone them with 

easy, cheapand quick methods of cloning [32]   
for further studies by considering its 
commercialization in mind. Producing these 
antimicrobial components in bacteria is easy    
and quick and additionally we do not have to 
destroy plants for this, which will be environment 
friendly and is the absolute need of the current 
time. 

 
Table 2. Ethyl acetate 

 

Parameters  Stationary 
phase  

Mobile phase  No. of 
spots 

Rf value 

Alkaloids  Silica gel  Benzene : ethanol(45:5) 10 0.04, 0.22, 0.27, 0.31, 0.36, 0.47, 
0.54, 0.61, 0.75, 1 

Glycosides  Silica gel  Ethyl acetate : butanol : 
water(20:20:15) 

5 0.16, 0.30, 0.66, 0.76, 0.95 

Flavonoids  Silica gel  Ethyl acetate : formic acid : 
glacial acetic acid : water 

(20:2:2:5) 

 

4 

0.42, 0.81, 0.87, 0.93 

Terpenoids Silica gel  Toluene : ethyl acetate 

 

11 0.65, 0.09, 0.13, 0.21, 0.34, 0.44, 
0.50, 0.59, 0.63, 0.73, 0.98 

 
Table 3. Methanol 

 

Parameters  Stationary 
phase  

Mobile phase  No. of 
spots 

Rf value 

Alkaloids  Silica gel  Benzene : ethanol(45:5) 7 0.29, 0.37, 0.72, 
0.95, 0.78, 0.81, 0.89 

Glycosides  Silica gel  Ethyl acetate : butanol : water 
(20:20:15) 

2 0.35, 0.91 

Flavonoids  Silica gel  Ethyl acetate : formic acid : glacial 
acetic acid : water 

(20:2:2:5) 

5 0.10, 0.40, 0.75, 
0.89, 0.95 

Terpenoids Silica gel  Toluene : ethyl acetate 

(9:1) 

7 0.27, 0.33, 0.41, 
0.49, 0.56, 0.66, 1 

 
Table 4. Antibacterial activity and zone of inhibition for respective sample 

 

Sr. no. Extracts Conc. In mg/ml 

 

Diameter of zone of inhibition in cm 

E. coli S. auerus B. subtilis 

1 

 

Methanol 1 1.1 1.1 1.1 

2 1.2 1.1 1.2 

3 1.2 1.2 1.3 

4 1.2 1.2 1.3 

2 Ethyl acetate 1 1.1 1.1 1.1 

2 1.1 1.1 1.1 

3 1.1 1.1 1.1 

4 1.1 1.1 1.1 

3 

 

 

Zinc oxide 
nanoparticles 

1 1.2 1.1 1.3 

2 1.3 1.3 1.4 

3 1.4 1.5 1.5 

4 1.5 1.5 1.5 

 

 



 
 
 
 

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85 

 

 
 

Fig. 3. Antibacterial activity, in against from left zinc oxide nanoparticals, methanol, ethyl 
acetate 

 

Table 5. Antifungal activity and zone of inhibition for respective sample 
 

Sr. no. Extracts Conc. In mg/ml 
 

Diameter of zone of inhibition in cm 

Aspergillus flavus 

1 
 

Methanol 1 1.1 
2 1.2 
3 1.2 

2 Ethyl acetate 1 1.1 
2 1.1 
3 1.1 

3 Zinc oxide nanoparticles 1 1.2 
2 1.3 
3 1.5 

 

 
 

Fig. 4. Antifungal activity at different concentration level 
 

 
 
Fig. 5. Haemolytic activity in against from left zinc oxide nanoparticals, methanol, ethyl acetate 



 
 
 
 

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86 

 

Table 6. Zones of haemolysis for respective sample 
 

Extracts Zones of haemolysis in mm 

Conc. of extracts in mg/ml 

 1 mg/ml 2 mg/ml 3 mg/ml 

Methanol 1.2 1.3 1.4 
Ethyl acetate 1.2 1.2 1.4 
Zinc oxide nanoparticles 1.3 1.3 1.4 

 
Table 7. Zones of antidiabetic activity in against from left zinc oxide nanoparticals, methanol, 

and ethyl acetate 
 

Extracts Zones in cm 

Well no. 

 1 2 3 4 5 
Methanol 1.2 - 1.1 1 - 
Ethyl acetate 1.5 - 1.2 1.2 - 
Zinc oxide nanoparticles 1.2 - 1.2 1.1 - 

 

 
 

Fig. 6. Antidiabetic activity in against from left zinc oxide nanoparticals, methanol, and ethyl 
acetate 

 
The present study revealed that the S. glauca 
leaf posess antimicrobial activity, which is big 
plus of this study. Currently the world is facing 
the problem of antimicrobial resistance and 
pathogenic bacteria are developing resistance to 
the commercially available antibiotics. Therefore, 
it is an absolute necessity to keep looking for 
new antimicrobial solutions. And who know this 
Simarouba glaucaextract might be a solution to 
nullify the effect of antibiotic resistance in 
bacteria and could become a most favored 
medicine in the near future. 
 

4. CONCLUSION 
 

Based on our findings we conclude that 
Simarouba glauca plant leaves have significant 
amount of phytochemical compounds. Zinc oxide 
nanoparticles of Simarouba glauca have shown 
enhanced in vitro antioxidant, antibacterial 
activity, haemolytic activity, Antifungal activity 
compared to methanolic and ethyl acetate 
extracts of Simarouba glauca. The present study 
revealed that the S. glauca leaf 

possessantimicrobial activity, which is big plus of 
this study. Currently the world is facing the 
problem of antimicrobial resistance and 
pathogenic bacteria are developing resistance to 
the commercially available antibiotics. Therefore, 
it is an absolute necessity to keep looking for 
new antimicrobial solutions. And who know this 
Simarouba glaucaextract might be a solution to 
nullify the effect of antibiotic resistance in 
bacteria and could become a most favored 
medicine in the near future. 
 

CONSENT 
 

It is not applicable. 
 

ETHICAL APPROVAL 
 

It is not applicable. 
 

ACKNOWLEDGEMENT 
 
We are grateful to Meander Kumar Gakula for 
checking the English in the manuscript.  



 
 
 
 

Pawar et al.; AJI, 2(1): 78-88, 2019; Article no.AJI.53611 
 

 

 
87 

 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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