Journal of Health Sciences.

2013;3(11):051-062
The journal has had 5 points in Ministry of Science and Higher Education of Poland parametric evaluation.
Part B item 1107. (17.12.2013)
TheAuthor (s) 2013;
This article is published with open access at Licensee Open Journal Systems of Radom University in Radom, Poland
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Conflict of interest: None. Received: 15.10.2013. Revised: 24.12.2013. Accepted: 24.12.2013.

Pharmacological, Phytochemical and Physicochemical

properties of methanol extracts of Erioglossum rubiginosum
barks
Santanu Barua1, S. M. Masud Rana1*, Md. Mustahsan Billah1,
Zannatul Naim2, Golam Sarwar1
1

Department of Pharmacy, Noakhali Science and Technology University, Sonapur,

Noakhali - 3814, Bangladesh
2
Department of Pharmacy, Atish Dipankar University of Science and Technology, Banani,
Dhaka, Bangladesh
*

Corresponding author
S. M. Masud Rana
Department of Pharmacy
Noakhali Science and Technology University, Sonapur, Noakhali- 3814, Bangladesh
Cell: +8801683788782
E- mail: kaktarua.r@gmail.com
Abstracts
Objectives: To evaluate the Pharmacological (cytotoxic and antimicrobial activities),
phytochemical properties and physicochemical properties of Erioglossum rubiginossum barks,
an evergreen plant, belonging to the family Sapindaceae.
Methods: Powdered barks of the plant were treated with methanol using hot extraction method
and the extract has been investigated for its biological activities. Crude methanol extracts of the
bark of E. rubiginosum were used for brine shrimp lethality bioassay. Vincristine sulphate was
used as standard. The crude methanol extract of E. rubiginosum was screened for their
antibacterial activity against a wide range of bacteria (both gram-positive and gram-negative)
by disc diffusion method, antifungal activity by modified poisoned food technique.

51

Phytochemical evaluation was by qualitative analysis and Physicochemical properties was

determined according to method (Anonymous 1968) described in British Pharmacopeia.
Results: The LC50 values of methanol extract were found to be 29.47g/ml compared to
standard Vincristine sulphate (0.451g/ml). From these results, it can be well predicted that the
barks of Erioglossum rubiginossum possess very low cytotoxic property. The crude methanol
extract showed significant antibacterial activity against gram-positive Staphylococcus aureus
(17.020.11mm), gram negative Salmonella typhi (7.050.03mm) and Shigella dysenteriae
(5.030.04mm) compared to Cephradine. Interestingly, gram- positive Bacillus cereus was
found resistant towards this extract. Again this methanol extract exhibited mild antifungal
activity against Aspergillus niger and Candida albicans. The phytochemical evaluation showed
the presence of alkaloids, flavonoids, phenols, saponins and carbohydrate. Various
physicochemical parameters like moisture content, total cash value, acid soluble ash value,
water soluble ash value, alcohol soluble extractive value and water soluble extractive value
were to be found 8.07%, 18.00%, 21.00%, 9.50%, 1.00% and 0.50% respectively.
Conclusion: Further investigations are required for isolating active compound that are
responsible for antimicrobial activity and low cytotoxicity and finding out the mechanism
behind these effects.
Keywords Erioglossum rubiginosum, Brine shrimp lethality, Antibacterial, Antifungal, Zone
of inhibition, Phytochemicals.

1. Introduction
The plant under investigation Erioglossum rubiginosum belongs to the family Sapindaceae. The
common and local name of the plant is Kalayo and Boro Harina respectively. Erioglossum
rubiginosum is distributed throughout the Philippines, also occurs from northern India to IndoChina and Thailand, through Malaya to tropical Australia1. Commonly using part of the plant is
seeds, roots, barks and leaves. Different study had shown that major components of flower
essential oil were nerolidol (34.8%), palmitic acid (13.2%), and farnesol (10.0%). Fruit
essential oil yielded palmitic acid (66.1%), myristic acid (10.0%), and linolenic acid (5.5%)2.
Methanolic fraction isolated a tetrasaccharide derivative of farnesol named rubiginoside along
with known triterpenoid saponins3.
The plant commonly used for the treatment of leprosy4. This plant is extensively used as
folkloric medicine such as roots are used as astringent, leaves and fruits are used for the
treatment of fever and poultice5, 1. Recent investigation had shown the leaves of the plant could
be used as a natural source of membrane stabilizers6. Another recent investigation had proved
that the leaves of the plant possess antioxidant, thrombolytic and cytotoxic activity7. CNS
depressant activity was found significantly by the plant8.
According to literature survey, the study, investigation and information of barks of Erioglossum
rubiginosum is still insufficient. So, the present study was undertaken for crude methanol
extract of barks of Erioglossum rubiginisum for investigation of cytotoxic, antibacterial,
antifungal activities and phytochemical properties.

52

2. Materials and Methods

2.1 Plant material and extraction
The barks of Eriglossum rubiginosum was collected from National Botanical Garden, Mirpur,
Dhaka. The plant was identified by the taxonomist of Bangladesh National Herbarium, Mirpur,
Dhaka, Bangladesh and a voucher specimen has been deposited in the herbarium unit
(Accession no DACB 38566).
The barks were sun dried for several days and then oven dried for 24 hours at considerably low
temperature (not more than 400o C) for better grinding. The dried barks were then ground to a
coarse powder using high capacity grinding machine in the Phytochemical Research
Laboratory, Faculty of Pharmacy, University of Dhaka, Bangladesh. The current study used hot
extraction method. The powdered material (500gm) was taken in a cleaned, ambered color
reagent bottle (2.5 liters) and soaked in 1.5 L of 99% methanol. The container with its content
was sealed by bottle cap and kept for a period of 20 days accompanying occasional shaking and
stirring. The whole mixtures were then filtered through a fresh cotton plug and finally with a
Whatman No.1 filter paper separately in two separate beakers. The volume of the filtrate was
then allowed to evaporate at Rotary evaporator at 40 degree Celsius temp until approximately
80% solvent was evaporated. It rendered a blackish-brown gummy concentrated extract. The
blakish-brown extract concentrated was designated as crude extract of methanol.
2.2 Test Microorganisms
Authentic pure cultures of human pathogenic gram positive bacteria (Bacillus cereus ATCC
10876, Staphylococcus aureus ATCC-29740), gram negative bacteria (Salmonella typhi ATCC14028, Shigella dysenteriae ATCC-9027) and fungi (Aspergillus niger ATCC-16404, Candida
albicans ATCC-10231) were obtained from Poultry Research & Training Center (PRTC),
Chittagong Veterinary and Animal Sciences University, Chittagong, Bangladesh.
2.3 Brine shrimp lethality bioassay
Brine shrimp lethality bioassay was used for probable cytotoxic action according to
McLaughlin (1998)9 and Persoone (1980)10.
According to this method, natural product extracts, the pure compounds and fractions can be
tested for their biological activity. In this method, in vivo lethality in a simple zoological
organism (Brine shrimp nauplii, Artemis salina) was used as a favorable monitor for screening
and fractionation in the discovery of new bioactive natural products. Vincristine sulphate was
used as a positive control group whereas DMSO was used as negative control group according
to Tyler and Brady (1988)11; McKey (1994)12 and Lewis et al., (2005)13.
2.4 Antibacterial screening
Antibacterial screening was determined by the disc diffusion method14 against gram positive
(Bacillus cereus, Staphylococcus aureus) and gram negative bacteria (Shigella dysenteriae,
Salmonella typhi). Cephradin was used as standard.
2.5 Antifungal Screening
Antifungal screening was determined against Aspergillus niger and Candida albicans by the
poisoned food technique15 with slight modification. Briefly, 1st Plate preparation: Preparing
potato dextrose agar > Placing it in Petridis > Mixing extract with media > Drying up for 30

53

minutes. 2nd Test suspension preparation: Selection of a few colonies of fungus > Placing it
in in savored agar media > Incubation at room temperature for 3 days > Match with standard.
3rd Streking the plates: Strek all plates with test organism > Dry up for 30 minutes >
Incubation at room temperature for 3 days > Recording zone of growth > Calculating
percentage of inhibition by following formula:
Here,
C= Diameter of control = 08 cm
T= Diameter of zone of growth
I= Percentage of inhibition
2.6 Phytochemical screening
The methanol extracts of barks of Erioglossum rubiginosum were subjected to qualitative tests
for the identification of various phytochemicalconstituents.16, 17, 18, 19, 20.
2.7 Physicochemical Profile21-26
2.7.1 Ash value
Total ash, acid insoluble ash and water soluble ash were determined as reported in the
Anonymous (1968)21 and MHFW (1999)22. Briefly, total ash was determined using 2 g of the
air-dried powdered sample. The total ash was boiled for 5 minutes with 25 ml of distilled water;
the insoluble matter was collected on an ashless filter paper, washed with hot distilled water,
and ignited for 15 minutes at a temperature not exceeding 450oC. The weight of the insoluble
matter was subtracted from the weight of the total ash; the difference in weight represents the
water-soluble ash. The percentage of the water-soluble ash was calculated with reference to the
air-dried powdered plant sample.
2.7.2 Extractive values and Moisture content
Extracts of the plant samples were prepared with different solvents for the study of extractive
values. For present study alcohol and water were used as solvent for the study of extractives
value

3. Result
3.1 Brine shrimp lethality bioassay
In case of brine shrimp lethality bioassay, the methanol extracts of bark of Erioglossum
rubiginosum demonstrated very low cytotoxic potentiality against Artemis salina with LC50
value of 29.47g/ml as compared to Vincristine sulphate (0.451g/ml) Table 1 and Table 2.
3.2 Antibacterial screening
The antibacterial activity of Erioglossum rubiginosum test samples (400g/disc) was evaluated
against two gram positive and two gram negative bacteria and the results were found significant
compared with standard, Cephradin. The test samples of Erioglossum rubiginosum revealed
antibacterial activity with zone of inhibition ranging from 5.030.04 mm to 17.020.11 mm.
The highest zone of inhibition 17.020.11 mm and 7.050.03 mm was shown against gram

54

positive bacteria (Staphylococcus aureus) and gram negative bacteria (Salmonella typhi)
respectively. But, gram- positive Bacillus cereus was found resistant towards this extract
Table 3.
3.4 Antifungal screening
The antifungal activities of methanol extracts of Erioglossum rubiginosum (100 l/disc) and
standard Griseofulvin (30 l/disc) were determined against two pathogenic fungi (Aspergillus
niger, Candida albicans) where, 25% and 30.5% of inhibition was found against Aspergillus
niger and Candida albicans respectively in comparison to Griseufulvine (100% and 98.5%)
Table 4
3.5 Phytochemical screening
In preliminary phytochemical screening, the methanol extract of Erioglossum rubiginosum
confirmed the presence of alkaloids, saponins, phenolic compound and carbohydrate Table 5.
3.6 Physicochemical screening
3.6.1 Ash value
Results of total ash value, acid insoluble ash and water soluble ash value has shown in Table 6.
Total ash value was found to be 9.50 %), acid soluble ash value 1.00% and water soluble ash
value was found to be 0.50%.
3.6.2 Extractive value and Moisture content
Result of the extractive value has shown in Table 6. Alcohol soluble and water soluble
extractive value were found to be 18.00 % and 21.00% respectively. Moisture content to found
8.07%

4. Discussion
Plants may offer a new source of cytotoxic, antibacterial, antifungal and antiviral agents with
significant activity27, 28. Brine shrimp cytotoxicity assay has been considered as prescribing
assay for antibacterial, anti-fungal, insecticidal, anti-parasitological and various pharmacologic
activities29. Several studies have shown that brine shrimp bioassay has been an excellent
method to screen the cytotoxic property of medicinal plants and for the isolation of a great
variety of biologically active compounds30.
In present brine shrimp lethality bioassay, ten different concentrations (0, 0.78, 1.56, 3.13, 6.25,
12.5, 25, 50, 100, 200, 400 g/ml) of E. rubiginosum extract were used to determine its
cytotoxicity by brine shrimp lethality bioassay (Table 2, Figure 1). The test samples showed
different mortality rates at different concentrations where the percentage of mortality increased
with an increase in concentration. The variation in results may be due to the difference in the
amount and kind of cytotoxic substances (e.g. tannins, flavonoids, triterpenoids, or coumarins
etc.) present in the crude extracts. The LC50 value and Chi- square was found 29.47g/ml and
0.783 respectively (Table 1). From these results, it can be well predicted that the barks of
Erioglossum rubiginossum possess very low cytotoxic property which may be due to little
presence of cytotoxic substances.
In antibacterial screening, significant zone of inhibition was observed against Staphylococcus
aureus and both gram negative bacteria (Salmonella typhi, Shigella dysenteriae) as well.
Though Bacillus cereus is gram positive, but it revealed maximum resistance against the test
sample. It was also observed that the test samples showed most significant and best activity
against Shigella dysenteriae than the standard Cephradin.

55

In general, gram-negative bacteria are more resistant to antibiotics than gram-positive

bacteria31, 32. The resistance is due to the differences in their cell wall composition. In gramnegative bacteria the outer membrane acts as a great barrier to many environmental substances
including antibiotics33. Presence of thick murine layer in the cell wall prevents the entry of the
entry of the inhibitors34. The ability of the extracts to exhibit antibacterial activity against the
bacteria suggested the presence of hydrophilic and hydrophobic antibacterial compounds such
as luteolin, apigenin etc35.
But the present study revealed a controversy report that gram-negative bacteria were more
susceptible to the crude extracts than gram-positive bacteria. It may be due to the presence of
broad spectrum of antibacterial compounds in the bark of E. rubiginosum.
The antifungal activities of methanol extracts of Erioglossum rubiginosum was expressed in
little amount against Aspergillus niger and Candida albicans due to the presence of antifungal
constituents in less quantity.
From several previous study we know that, e.g. presence of alkaloids, which may be used as
antimicrobial activity36, phenolic compounds and flavonoids, which can be referred to as
natures biological response modifiers, have shown antimicrobial and anticancer (cytotoxic)
activities37. Again flavonoids and polyphenolic compounds are considered as one of the major
chemical constituents responsible for cytotoxic and antimicrobial activity38. Our present study
had shown the presence of these biological response modifiers such as phenolic compounds,
saponins, alkaloids and flavonoids. So we can say that the presence of these chemical
constituents may be the reasons for cytotoxic and antimicrobial activity.
Physicochemical parameters of the plants prove that it possess good quantitative properties. Its
dried form is expected to have a long shelf-life with reduced chance of microbial growth due to
its relatively low moisture content of 8.07%. Total ash value of 9.50% indicates low inorganic
components in the herbal plant. Acid insoluble ash value of 1.00% indicates high digestibility
when the plant is consumed. Water soluble ash value of 0.50% is indicative of negligible level
of water-soluble minerals absorption from the plant when it is consumed. The alcohol-soluble
extractive value of 18.00% and water-soluble extractive value of 21.00% shows that both
solvents would be good for extraction of this potential drug-plant.

5. Conclusion
From the present study, we can say that the methanol extracts of bark of E. rubiginosum can be
used as significant antibacterial agent having mild anti-fungal and less cytotoxic potentiality.
So, further chemical and pharmacological investigations to isolate and identify chemical
constituents responsible for these potential bioactivities should be suggested.

Acknowledgment
The authors are grateful to Bangladesh National Herbarium, Bangladesh to identify the plant
and Chittagong Veterinary and Animal Sciences University, Bangladesh to supply the
microorganism. Authors are also thankful to Department of Pharmacy, Noakhali Science and
Technology University; Poultry Research and Training Center, Bangladesh for providing the
laboratory facilities and technical support.

Conflict of interest statement

We declare that we have no conflict of interest.

56

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58

Table 1: LC50 values of the methanol extracts E. rubiginosum barks

Test
Regression line
R2
LC50 (g/ml)
samples
VS
y = 30.8x + 60.64
0.972
0.451
ME
y = 24.88x + 13.44
0.783
29.47
VS
= Vincristine sulphate
ME = Methanol extract of E. rubiginosum bark
Table 2: Effect of crude methanol extract (ME) of barks of E. rubiginosum on shrimp
nauplii
Conc. (g/ml)

Log10 conc.

0
0.78
1.56
3.13
6.25
12.5
25
50
100
200
400

0
-1.1072
0.19382
0.49485
0.79588
1.09691
1.39794
1.69897
2
2.30103
2.60206

% of mortality

0
10
10
20
20
30
40
40
70
80
100

LC50 (g/ml)

29.47

Table 3: Antibacterial activity of methanol extracts of Erioglossum rubiginosum barks

Test
Diameter of zone of inhibition (mm)
microorganisms
Test sample
Cephradin
(400g/disc)
(30 g/disc)
Gram positive bacteria
Bacillus cereus

00.000.00

14.010.03

Staphylococcus aureus

17.020.11

30.000.06

Gram negative bacteria

7.050.03
10.010.07
Salmonella typhi
5.030.04
2.00.05
Shigella dysenteriae
Values are the mean of three replicates standard error. P<0.05
59

Table 4: Antifungal activity of methanol extracts of Erioglossum rubiginosum barks

Test microorganisms
Percentage of inhibition (
)

Test sample
(100 l/disc)

Griseofulvin
(30 l/disc)

Aspergillus niger

25%

100%

Candida albicans

30.5%

98.5%

Table 5: Phytochemical constituents identified in the methanol extracts of Erioglossum

rubiginosum barks
Plant constituents test / Reagent used
Methanol extracts
Alkaloids
+
a) Mayers Test
+
b) Wagners Test
+
c) Hagers Test
Phenolic compound
a) 5% FeCl3 Solution
b) Reaction with lead acetate
c) Drug + K3Fe (CN)6 + NH3
Saponin

+
+
+

a) Foam test
Carbohydrate

a) Molischs Test
b) Fehlings Test
c) Benedicts Test

+
+
+

Phytosterols
a) Libermanns Test

Flavonoids
a) Fluorescence Test
b) Reaction with lead acetate

+
+

60

Table 6: Physicochemical parameter of the plant of Erioglossum rubiginosum

Parameter
Value (%)
Moisture content
Alcohol-soluble extractive
value
Water-soluble extractive value
Total ash
Acid-insoluble ash
Water-soluble ash

8.07
18.00
21.00
9.50
1.00
0.50

Effect of Vincristine sulphate (positive control) on

shrimp nauplii
120

% mortality

100
80
60
y = 30.8x + 60.645
R2 = 0.9729

40
20
0
-2

-1

Log. concentration

Figure 1: Plot of % mortality and predicted regression line of Vincristine sulphate

61

Effect of Vincristine sulphate (positive control) on

shrimp nauplii
120
100

% mortality

80
60
40
y = 24.883x + 13.449
R2 = 0.7835

20
0
-2

-1

-20

Log. concentration
Figure 2: Plot of % mortality and predicted regression line of methanol extract

62