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© 2020 Adama Science & Technology University. All rights reserved
Ethiopian Journal of Science and Sustainable Development
e-ISSN 2663-3205 Volume 7 (1), 2020
Journal Home Page: www.ejssd.astu.edu.et ASTU
Research Paper
Antibacterial Steroids from Roots of Bersama Abyssinica
Fitsum Lemilemu, Solomon Girmay, Kebede Shenkute, Milkyas Endale
Department of Applied Chemistry, School of Applied Natural Science, Adama Science and Technology, P. O. Box 1888, Adama,
Ethiopia
Article Info Abstract
Keywords:
Bersama abyssinica
steroids
phytochemical
screening
antibacterial activity
Bersama abyssinica is one of the medicinal plants used traditionally to treat various diseases such
as leprosy, wound, diarrhea, fever, eye disease, rabies and tumor/cancer. Phytochemical screening
test of dichloromethane/methanol (1:1) and methanol extracts revealed the presence of glycosides,
alkaloids, tannins, flavanoids, saponins, terpenoids, steroids and phytosterols. Silica gel column
chromatography separation of dichloromethane/methanol (1:1) root extracts afforded β-sitosterol
(1), 7-hydroxysitosterol (2) and 2-methylamino-butyric acid (3) of which the latter is isolated for the
first time from natural source. The crude extracts and isolated compounds were screened for in vitro
antibacterial activity against strains of Salmonella thphimurium, Escherichia coli, Bacillus subtilis
and Staphylococcus aureus. Dichloromethane/methanol (1:1) extract, methanol extract and β-
sitosterol (1) showed moderate activity against E. coli and S. aureus (zone of inhibition 13±0, 13±2
and 12.6±0.48, respectively) and (zone of inhibition 13.6±0.55, 12±2, and 12.5±0.5 mm,
respectively) compared to ciprofloxacin (28.6±1.25 and 26±5.1 mm) at 0.5 mg/mL. The structures
of compounds were determined by spectroscopic techniques (IR and NMR) and comparison with
literature report.
1. Introduction
The use of traditional medicine for treating human
diseases still remains widespread in low income
countries with a wide range of biological and
pharmacological activities (Ajayi et al., 2011; Ejele
2010). Bersama abyssinica (Melianthaceae) is an ever
green shrub to small tree up to 18 m tall and its bark,
leaf and root decoctions are widely taken as a purgative
to treat a range of stomach disorders, such as abdominal
pain, colic, diarrhea, cholera, intestinal worms,
dysentery, and also for the treatment of rabies, tumour,
syphilis, gonorrhea, malaria, rheumatism, aphrodisiac
and snake bites (Djemgou et al., 2010; Lather et al.,
2010; Kuete et al., 2008; Teklehaymanot et al.,
2007). B. abyssinica is known in Ethiopia as Azamer
(Amharic) and Lolchissa (Afan Oromo) (Verdcourt,
1989). It is also distributed in Democratic Republic of
Congo, Tanzania, Mozambique, Zambia, Zimbabwe,
Angola, Nigeria, Ethiopia, Kenya, Sudan and Uganda
(Mikkelsen and Seberg, 2001). Previous phytochemical
works reported compounds including 3,11,15-
tetramethyl-2-hexadecen-ol, 7,8-epoxyanostan-11-ol,3-
acetoxy, pyrogallol, capric ether, 2,3-dimethylfumaricacid,
5-methyl-2-furancarboxyaldhyde, β-sitigmasterol and
ethyl iso-allocholate (Zekeya et al., 2014; Kuete et al.,
2008). This study report the isolation, spectroscopic
identification, and antibacterial analysis of the roots of
B. abyssinica.
Corresponding author, e-mail: milkyas.endale@astu.edu.et
https://doi.org/10.20372/ejssdastu:v7.i1.2020.156
http://www.ejssd.astu.edu/
mailto:milkyas.endale@astu.edu.et
https://doi.org/10.20372/ejssdastu:v7.i1.xxxx.xx
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
28
2. Experimental section
2.1. General
TLC was performed using precoated aluminum
backed supported silica gel 60 F254 (0.2 mm thickness)
and glass supported silica gel 60 F254 (1.0 mm
thickness), respectively. Phytosterols were detected on
TLC stained with the Salkowiski reagent. Column
chromatography was carried out using silica gel 60-120
mesh. 1H and 13C NMR data were obtained in CDCl3 on
a Bruker Avance 400 MHz.
2.2. Plant material collection and identification
The roots of B. abyssinica were collected in
December, 2018 from Oromia Region Arsi zone, Asela
town, Tiyo woreda which is 175 km far from Addis
Ababa, the capital of Ethiopia. The plant was identified
with the help of botanist and voucher specimen was
deposited (FL 001) at National Herbarium of Ethiopia,
Addis Ababa University. The roots were cut into small
pieces, air-dried and ground into a fine powder.
2.3. Extraction and isolation
Air-dried root powder (300 g) was extracted
exhaustively with dichloromethane/methanol (1:1) (2 L)
for 72 h at room temperature. The marc left was further
extracted with methanol (2 L) soaked for 72 h at room
temperature. The extracts were evaporated under
reduced pressure at 40ºC using Rotary evaporator to
afford 23.68 g (7.89%) and 30.08 g (10.03%) crude
extracts, respectively. The crude dichloromethane/methanol
(1:1) crude extract (15 g) was adsorbed on 15 g silica
gel and subjected to silica gel (160 g) column
chromatography separation. Elution was carried out
with increasing gradient of ethyl acetate in n-hexane
followed by increasing gradient of methanol in
dichloromethane. A total of 145 fractions were collected
each concentrated under reduced pressure to dryness.
Fractions that showed similar Rf values and the same
characteristic color on TLC were combined. Fraction
33-38 afforded single spot (β-sitosterol (1, 10.8 mg))
derivative of β-stigmasterol (EtOAc/n-hexane 1:1 as
eluent on TLC with Rf value of 0.64). Fraction 22-26
afforded single spot (7-hydroxy-β-sitosterol (2, 9.3 mg,
EtOAc/n-hexane 3:7 as eluent on TLC with Rf value
0.85). Fractions 42-79 afforded 2-methylbutyric acid (3,
26.5 mg, EtOAc/n-hexane 8:2 as eluent on TLC with Rf
value of 0.6).
2.4. Phytochemical screening test
2.4.1. Test for flavonoids: To DCM/MeOH (1:1)
and MeOH crude extracts (0.5 g), 10 mL of ethyl
acetate was added and heated for 3 min using steam
bath. The mixture was filtered, and mixed with 1
mL of dilute ammonia solution. Formation of
intense yellow color ratifies the presence of
flavonoids (Sofowora and Debiyi, 1978).
2.4.2. Test for saponins: To DCM/MeOH (1:1) and
MeOH crude extracts (0.5 g), 5 mL of distilled water
was added and shaken while heating to boil. Frothing
showed the presence of saponins (Evans and Trease,
1989).
2.4.3. Test for phenols: To DCM/MeOH (1:1) and
MeOH crude extracts (0.5 g), 5 drops of 2 % of FeCl3
were added and formation of bluish green to black color
indicates the presence of phenols (Roopashree et al.,
2008).
2.4.4. Test for tannins: The crude extracts (DCM:
MeOH (1:1) and MeOH extract) (0.5 g each) was boiled
in 10 mL of water in a test tube and filtered. To the
filtrate, 5 drops of 0.1 % FeCl3 were added to give a
brownish green or a blue-black color which confirms the
presence of tannins (Ayoola et al., 2008).
2.4.5. Test for terpenoids (Salkowski test):
DCM/MeOH (1:1) and MeOH crude extracts (0.5 g)
were mixed with 2 mL of chloroform and 3 mL
concentrated H2SO4 carefully to form a layer. A reddish
brown coloration of the interface was formed to show
positive results for the presence of terpenoids
(Ugochukwu et al., 2013).
2.4.6. Test for steroids: To DCM/MeOH (1:1) and
MeOH crude extracts (0.5 g), 10 mL of chloroform and
10 mL of concentrated H2SO4 were added by sides of
the test tube. The upper layer turns red and H2SO4 layer
showed yellow with green fluorescence indicating the
presence of steroids (Alhadi et al., 2015).
2.4.7. Alkaloids (Wagner’s test): DCM/MeOH (1:1)
and MeOH crude extracts (0.5 g each) were dissolved
individually in wagner’s reagent (Iodine in Potassium
Iodide). Formation of brown/reddish precipitate was
examined (Sadoon et al., 2014).
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
29
2.4.8. Detection of phytosterols (Salkowski’s test):
To DCM/MeOH (1:1) and MeOH crude extracts (0.5 g
each), 10 mL of chloroform was added and filtered. To
the filtrate, 5 drops of concentrated H2SO4 was added,
shaken and examined for the appearance of the golden
yellow color (Roopashree et al., 2008).
2.4.9. Test for anthraquinones (Borntrager's test):
DCM/MeOH (1:1) and MeOH crude extracts (0.5 g
each) were boiled with concentrated hydrochloric acid
for few minutes in water bath and filtered. The filtrate
was allowed to cool and equal volume of CHCl3 was
added to it. Few drops of ammonia were added to the
mixture and heated in water bath. Formation of rose-
pink color was inspected (Roopashree et al., 2008).
2.5. Antibacterial testing
2.5.1. Preparation of discs containing extracts
The same concentrations of 0.5 mg/mL were
prepared from the extract, isolated pure compounds and
the standard. The concentration was incorporated into
sterile agar-disc diffusion and dried at 37°C. The agar
disc was weighed carefully to confirm the exact amount
of the extract and isolated pure compounds being
incorporated (compared to preweighed blank discs).
2.5.2. Bacterial culture
Escherichia coli was isolated from stool specimens
collected from clinic and identified according to routine
cultural properties and biochemical tests. Four strains of
each were included in the study. A few colonies from
the overnight culture of Eosin Methylene Blue (EMB)
agar was transferred into approximately 4-5 mL
Tripticase soy broth (TSB) medium. The broth was
incubated at 37 °C for 3-4 h, and the turbidity of
suspension was adjusted to that of 0.5 McFarland
barium sulfate standards. The standard suspension was
used for both qualitative and quantitative antibacterial
assays.
2.5.3. Bacterial susceptibility testing
Standardized inoculums (0.5 mg/mL) were
introduced on to the surface of sterile agar plates, and a
sterile glass spreader was used for even distribution of
the inoculums. Sterile agar-disc diffusion previously
soaked in a known concentration of extract or pure
compound (0.5 mg/mL per disc) was carefully placed at
the center of the labeled seeded plate. The same
procedure was used for all the MRSA strains used. The
plates were incubated aerobically at 37°C and examined
for zones of inhibition after 24 hr. The inhibition zones
were measured with a ruler and compared with the
control disc (disc containing only physiological saline).
Strains of human pathogen microorganisms used in this
study were as follows: two Gram-negative bacteria,
Escherichia coli, Salmonella thphimurium and two
Gram-positive bacteria Staphylococcus aureus and
Bacillus subtilis. The bacterial stock cultures were
incubated for 24 h at 37°C on nutrient agar medium
(Adama Science and Technology University,
Department of Applied Biology, Adama). The bacterial
strains were grown in the Mueller–Hinton agar (MHA)
plates at 37°C. The agar was melted (50°C), and the
microorganism cultures were then added aseptically to
the agar medium at 45°C in plates and poured into sterile
petri dishes to give a solid plate. All these experiments
were performed in triplicate. The plates were incubated
for 24-48 h at 37°C for bacteria. The inhibition zones
produced by the plant extracts were compared with the
inhibition zones produced by commercial standard
antibiotics (ciprofloxacin). One dilution (0.5 mg/mL) of
B. abyssinica extract, pure compound, and standard
drugs was prepared in DMSO using nutrient agar tubes.
Mueller–Hinton sterile agar plates were seeded with
indicator bacterial strains (1.3 x 108 cfu/mL) and
allowed to stay at 37°C for 3 h. Control experiments
were carried out under similar conditions by using
ciprofloxacin for antibacterial activity as a standard
drug. The zones of growth inhibition around the disks
were measured after 24 h of incubation at 37°C for
bacteria. The sensitivities of the microorganism species
to the plant extract and isolated pure compounds were
determined by measuring the sizes of inhibitory zones
(including the diameter of disk) on the agar surface
around the disks, and values < 6 mm were considered as
not active against microorganisms. DMSO used as
negative control during the whole test on bacteria. The
results were expressed as mean value ± standard
deviation (SD) (Murai et al., 1995). The results are
calculated as averages of triplicate tests. The zone of
inhibitions in all cases were includes the diameter of the
wells.
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
30
3. Result and Discussion
3.1. Phytochemical screening
Phytochemical screening test of dichloromethane/
methanol (1:1) and methanol roots extracts revealed the
presence of alkaloids, flavonoids, phytosterols, phenols,
steroids, tannins, terpenoids, coumarins, anthraquinones,
terpenes and saponins in both extracts, whereas,
saponins were found to be absent in methanol extract
(Table 1). The presence of these secondary metabolites
may be attributed to the traditional use of the plant to
treat various diseases.
3.2. Characterization of compounds
Compound 1 was isolated as white solid with melting
point 134-136°C and Rf value of 0.64 (50% EtOAc in n-
hexane as eluent). The 1H NMR spectrum (Table 2)
showed a series of proton signal at δ 1.0-1.8 due to
overlapping of methylenes and methines, a
characteristic frame work of steroid. Oxygenated sp3
methine proton was observed at δ 3.68 (m, 1H, H-3)
which is a characteristic of steroids with hydroxyl group
at C-3 position. Olefinic proton was observed at δ 5.3
suggesting that the proton is next to methylene. The
presence of six methyl groups at δ 0.68, 0.93, 0.83, 0.81,
0.84 and 1.01 is also in agreement with the steroidal
nucleus. The 13C NMR spectrum (Table 2) revealed the
presence of twenty nine carbon signals which is a
characteristic feature of triterpenes. The 13C NMR and
DEPT-135 spectra displayed the presence of six methyl
carbon signals which resonated at δC 11.9, 14.0 18.8,
19.2, 19.4 and 19.8. Eleven methylene carbon signals
were observed at δC 21.1, 23.1, 25.9, 27.9, 28.3, 31.9,
32.0, 35.5, 37.3, 39.8 and 42.3 in (Table 2). Presence of
five methine carbons (δC 48.0, 48.30, 50.5, 55.3 and
38.1), two olefinic carbons (δC 143.8 and 121.2), of
which the former suggests sp2 quaternary carbon, and
two sp3 quaternary carbon signals (at δC 36.2 and 42.8)
were also confirmed. Oxygenated sp3 methine was
observed at δC 76.8 (C-3), in agreement with
oxygenation pattern of steroids at C-3. Thus, based on
the above spectral data and comparison with literature,
the structure of the compound was identified as β-
sitosterol (1) (Chaturvedula and Prakash, 2012; Anjoo
et al., 2011; Pateh et al., 2008).
HO
1
12
7
19
26
27
23
24
20
16
1328
1
Compound 2 was isolated as white solid (mp: 135-
137°C) with Rf value of 0.6 (50% EtOAc in n-hexane as
eluent). The 1H NMR and 13C NMR spectra displayed
comparable spectral feature to that of β-sitosterol except
additional peak observed at δC 79.1 suggesting the
presence of additional sp3 oxygenated methine.
Comparison with literature reports and with NMR
features of β-sitosterol, compound 2 was identified as 7-
hydroxy-β-sitosterol (2).
Table 1: Phytochemical screening tests of crude extracts of DCM: MeOH (1:1) and MeOH (100%)
Key: DCM=Dichoromethane and MeOH=Methanol
Phytochemical screening Test DCM:MeOH(1:1) extract MeOH extract
Alkaloids Wagner’s test + +
Flavonoids Ammonia test + +
Phytosterols Salkowski’s test + +
Steroids Salkowski’s test + +
Phenols Ferric Chloride test + +
Tannins Gelatin Test + +
Terpenoids Salkowski’s test + +
Anthraquinones Borntrager's test + +
Terpenes Salkowski test + +
Saponins Froth test + -
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
31
Table 2: 1H (CDCl3, 400 MHz) and
13C NMR (CDCl3, 100 MHz) spectral data of β-sitosterol (1)
Position δH (δ in ppm) δC Chaturvedula and Prakash, 2012
1 37.3 37.2
2 31.9 31.6
3 3.18 (1H, t) 71.4 3.53 (dd, 1H, J = 4.5, 4.2, 3.8 Hz)
71.7
4 42.3 42.2
5 143.8
140.9
6 5.38 (1H, t) 121.2 5.36 (t, 1H) 121.9
7 32.0 32.1
8 33.1 32.1
9 50.4 50.3
10 36.2 36.7
11 21.1 21.3
12 39.8 39.9
13 42.8 42.6
14 56.8 56.9
15 25.9 26.3
16 28.3 28.5
17 56.1 56.3
18 36.2 36.3
19 0.91 (d, 3H, J = 6 Hz) 19.4 0.93 (d, 3H, J = 6.5 Hz) 19.2
20 35.5 34.2
21 27.9 28.4
22 45.9 46.1
23 23.1 23.3
24 0.85 (t, 3H) 14.0 0.84 (t, 3H)
12.2
25 2.06 (m, 1H) 29.2 29.4
26 0.96 (d, 3H, J = 6 Hz) 19.8 0.83 (d, 3H, J = 6.4 Hz)
20.1
27 0.97 (d, 3H, J = 6 Hz) 19.2 0.81 (d, 3H, J = 6.4 Hz) 19.6
28 0.65 (s, 3H) 18.8 0.68 (s, 3H)
19.0
29 0.99 (s, 3H) 11.9 1.01 (s, 3H)
12.0
HO
1
12
7
19
26
27
23
24
20
16
13
10
28
OH
2
Compound 3 was isolated as a white solid with Rf value
of 0.7 (n-hexane/EtOAc (8/2) as eluent. The 1H NMR
spectrum showed (Table 3) the presence of one terminal
methyl protons at δ 1.27 (3H, t) suggesting it is adjacent
to methylene. The spectrum also displayed a multiplet
methine signal at δH 3.5 (q, 1H), methylene at δH 2.36
(2H, t) and methyl at δH 2.8 (3H, m) where the former
suggests a methine protons next to a carbonyl of
carboxylic acid and also connected to hetroatom
whereas the later suggests methyl attached to hetroatom.
The 13C NMR spectrum with the help of DEPT-135
(Table 3) revealed the presence of five well resolved
carbon signals of which one carbonyl carbon (δc 179.2),
methyl (δc 17.6), methine (δc 48.7), one methylene (δc
29.0) and methyl (δc 30.7). Its DEPT-135 spectrum
displayed that signal at δc 29.0 pointing down attributed
to methylene signal (C-3). Thus, based on the above
spectral data the compound was found to be 2-
methylamino-butyric acid (3) isolated for the first time
from a natural source.
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
32
Table 3: 1H and 13C NMR spectral data of compound 3
HO
O
HN
1 2
3
3
Table 4: Zone of bacterial growth inhibition diameter (mm)
Sample name Zone of inhibition (mm) Mean ± standard deviation
E. coli S. thyphimerium S. aureus B. subtlis
DCM/MeOH (1:1) extract 13±0.00 11.6±0.48 13±2.00 12.3±1.25
MeOH extract 13.6±0.55 12±0.00 12±2.00 11.6±0.48
β-sitosterol (1) 11.6±0.55 11.6±0.48 12.5±0.50 11±0.00
7-Hydroxy-β-sitosterol (2) 12.5±0.48 10.6±0.48 12.5±0.50 11±0.82
2-methylamino-butyric acid (3) 11.3±0.48 11± 0.82 11.5±0.50 11±0.00
Ciprofloxacin 28.6±1.25 28.6±0.94 26±5.1.00 34.3±0.94
3.3. Antibacterial activity
The antibacterial activity of the crude extracts of
DCM:MeOH(1:1), MeOH and isolated compound were
examined at a concentration of 0.5mg/mL against four
pathogenic bacterial strains. Promising antibacterial
activity was observed for DCM:MeOH (1:1) and
methanol extracts against E. coli, S. thyphimerium, S.
aureus and B. subtlis with zone of inhibition of 13±0,
11.6±0.48, 13±2, and 12.3±1.25, respectively, for
DCM: MeOH extract and 13.6±0.55, 12±0, 12±2 and
11.6±0.48, respectively, for methanol extract. 7-
hydroxy-β-sitosterol (2) showed promising antibacterial
activity against E. coli and S.aureus with zone of
inhibition of 12.6±0.48 and 12.5±0.5, respectively,
compared to ciprofloxacin 28.6±1.25 and 26±5.1 (Table
4).
4. Conclusion
For decades traditional medicines have been used
and continued to be an alternative approach on treatment
for various diseases caused by protozoan, bacteria,
fungi, viruses and helminthes. Currently, the growing
interest of consumers in substances of natural origin in
association with the increasing concern of potentially
harmful infectious disease has directed to a rising
interest in the use of plant extracts as functional
ingredients in many pharmaceutical products. B.
abyssinica is one of these medicinal plants used
traditionally to heal various infectious diseases. The
phytochemical screening tests showed that crude
extracts of root barks B. abyssinica plants are rich in
alkaloids, flavonoids, saponins, phenols, tannins,
terpenoids, steroids, phytosterols and glycosides. Silica
Position δH (multiplicity)
13C NMR (δC in ppm) DEPT-135 (δC in ppm)
1 175.1 -
2 3.5 (1H, m) 49.4 49.4
3 2.3 (2H, m). 29.0 29.0
4 1.27 (3H, t). 17.6 17.6
5 2.8 (3H, s) 30.7 30.7
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
33
gel column chromatography separation of the
DCM:MeOH (1:1) crude extract furnished two
triterpenoids named β-sitosterol (1), 7-hydroxy-β-
sitosterol (2) and 2-methylamino-butyric acid (3). The
extracts and isolated compounds were evaluated in vitro
for antibacterial activity using the disc diffusion method
against E coli, S. aureus, S. thphimurium and B. subtilis.
Moderate antibacterial activity was observed for
DCM/MeOH (1:1) and methanol extracts against E.
coli, S. thyphimerium, S. aureus and B. subtlis with zone
of inhibition of 13±0, 11.6±0.48, 13±2, and 12.3±1.25,
respectively, for DCM/MeOH (1:1) extract and
13.6±0.55, 12±0, 12±2 and 11.6±0.48, respectively, for
methanol extract. 7-Hydroxy-β-sitosterol (2) showed
moderate antibacterial activity against E. coli and S.
aureus with zone of inhibition of 12.6±0.48 and
12.5±0.5, respectively, compared to ciprofloxacin
28.6±1.25 and 26±5.1.
Acknowledgment
The authors acknowledge Adama Science and
Technology University, Adama, Ethiopia for funding
part of the work. Temesgen Asefa (staff of Department
of Applied Biology, Adama Science and Technology
University) is duly acknowledged for his assistance
during antibacterial assay.
Reference
Ajayi, I.A., Ajibade, O., Oderinde, R.A., (2011). Preliminary phytochemical analysis of some plant seed. Research Journal of
chemical sciences, 1(3):58-62.
Alhadi, E.A., Khalid, H.S., Alhassan, M.S., Kabbashi, A.S., Noor, O.M. (2015). Antimicrobial and phytochemical screening of
Cordia africana in Sudan, World journal of pharmaceutical research, 4(3): 257-269.
Anjoo, K., Ajay, Kumar, S., (2011). Isolation of stigmasterol and β-sitosterol from petroleum ether extract of aerial parts of
Ageratum conyzoides (Asteraceae), International journal of pharmacy and pharmaceutical sciences, 3: 94-96.
Ayoola, G.A, Coker, H.A.B., Adesegun, S.A., Adepoju-Bello, A.A., K., Obaweya, (2008). Phytochemical screening and
antioxidant activities of some selected medicinal plants used for malaria therapy in southwestern Nigeria, Tropical journal
of pharmacological research, 7:1019-1024.
Chaturvedula, P., Prakash, I.( 2012) .Isolation of Stigmasterol and β-Sitosterol from the dichloromethane extract of Rubus
suavissimus. International current pharmaceutical journal, 1(9): 239-242.
Djemgou, P.C., Hussien, T.A., Hegazy, M.E.F., Ngandeu, F., Neguim, G., Tane, P. (2010). C-Glucosidexanthone from the stem
bark extract of Bersama engleriana, Pharmacognosy research, 2:229.
Ejele, A. E. (2010). Effects of secondary metabolites of Cajanus cajan extract on sickling and gelation of human HbSS
erythrocytes. Nigerian journal of biochemistry and molecular biology, 25(2):10-16.
Evans, W.C., Trease G. E., (1989). Phenols and phenolic glycosides,” in Textbook of Pharmacognosy, 12:343 -383, Balliese,
Tindall and Co Publishers, London, UK.
Kuete, V., Mbaveng, A.T., Tsaffack, M., Beng, V.P., Etoa, F.X, Nkengfack, A.E., (2008). Antitumor, antioxidant and
antimicrobial activities of Bersama engleriana (Melianthaceae). Journal of ethnopharmacology, 115:494-84.
Lather, V., Gupata, V., Tyagi, V., Kumar, S.G., (2010). Phytochemistry and pharmacological activities of Bersama engleriana
Guerke. International research journal of pharamacy, 1: 89-94
Mikkelsen, K., Seberg, O., (2001). Morphometric analysis of the Bersama abyssinica Fresen. Complex (Melianthaceae) in East
Africa. Plant systematics and evolution, 227:157-182.
Murai, M., Tamayam, Y., Nishibe, S., (1995). Phenylethanoids in the herb of Plantago lanceolata and inhibitory effects on
arachidonic acid-induced mouse ear edema. Planta medica, 61: 479.
Pateh, U.U., Haruna, A. K., Garba, M., Iliya, I., Sule, I. M., Abubakar, M. S., Ambi, A.A., (2008). Isolation of stigmasterol , β-
sitosterol and 2-Hydroxyhexadecanoic acid methyl ester from the Rhizomes of Stylochiton lancifolius, Nigerian journal
of pharmaceutical sciences, 7:19 -25.
Pollock, J.R.A, Stevem, R.S. (ed.), (1965). Dictionary of organic compounds. 4th ed., vol. 5 Eyre and spottiswoode (Publishers)
Ltd.
Roopashree, T. S., Dang, R.., Rani, R. H., Narendra S., (2008). Antibacterial activity of antipsoriatic herbs: Cassia tora,
Momordica charantia and Calendula officinalis, International journal of applied research in natural products, 1(3):20-28.
Sadoon, A.H., Liu, X., Zhang, J., (2014). Extraction of alkaloids from C. komarovii. Journal of animal and veterinary advances,
13(15): 905-907.
Sofowora, F.A., Debiyi, O.O., (1978). Phytochemical screening of Nigerian medical plants II, Lloydia, 41: 234-246.
Teklehaymanot, T., Giday, M., Medhin, G., Mekonnen, Y., (2007). Knowledge and use of medicinal plants b y p eo p le ar o u nd
Deb r e Lib a no s mo na s ter y in Et hio p ia. Jo u rn a l o f E th n o p h a rma co lo g y . 1 1 1 :2 7 1 -2 8 3 .
Ugochukwu, S., Uche, I., Ifeanyi, O., (2013). Preliminary phytochemical screening of different solvent extracts of stem, bark
and roots of Dennetia tripetala G., Baker”, Asian journal of plant science research, 3:10-13.
Fitsum Lemilemu et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020
34
Verdcourt, B., (1989). The National Herbarium, Addis Ababa University, Ethiopia and the Department of Systematic Botany,
Uppsala University, Sweden, In: Hedberg I, and Edwards S eds, Flora of Ethiopia, 511-512.
Zekeya N., Chacha M., Shahada F., Kidukuli A., (2014). Analysis of phytochemical composition of Bersama abyssinica by gas
chromatography-mass spectrometry. J Pharmacognosy Phytochemistry. 3(4): 246-52.