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Advances in Environmental Biology, 5(1): 188-193, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Chemical Composition, Brine Shrimp Toxicity and Free-radical Scavenging Activity of
Leaf Essential Oil of Acalypha Ornata (Hochst)
Patricia A. Onocha, Ganiyat K. Oloyede and Gbenga S. Olasunkanmi
1
Natural products/Medicinal Chemistry Unit, Department of Chemistry, University of Ibadan, Nigeria.
Patricia A. Onocha, Ganiyat K. Oloyede and Gbenga S. Olasunkanmi: Chemical Composition,
Brine Shrimp Toxicity and Free-radical Scavenging Activity of Leaf Essential Oil of Acalypha
Ornata (Hochst)
ABSTRACT
Essential oil from the leaves of Nigerian specie of Acalypha ornata of the family Euphorbiaceae
was obtained by distillation using a hydro-distiller (all-glass clavenger apparatus) and was found to
contain 100 compounds out of which 89 was successfully identified representing 54.64% of the total
oil composition when analysed by GC/GC-MS. Alcohols, aldehydes, esters and terpenoids are the
dominating group of compounds. The principal constituents are isopulegly acetate, valenchi, vividiflorene,
α –muurolene, 2-hexyne, 6-methyl – α – ionone, γ-elemene, (E) -2-methyl-4- undecene, ledol, cis-3hexynyl benzoate, 2-methyl -1- octadecene, apiole, oplopanone and γ-endesmol. Brine shrimp lethality
test was carried out to know the toxicity of the oils to living organisms (shrimps). LC50 value (µg/ml)
of 116.6 at 95% confidence level obtained showed that the essential oil of A. ornata leaves was toxic.
The antioxidant property of essential oil was investigated by using the UV/Visible spectrophotometer.
The essential oil of A. ornata scavenged 2, 2–diphenyl–1–picrylhydrazyl radical (DPPH) less effectively
than ascorbic acid and butylatedhydroxylanisole (BHA) but better than α-tocopherol at 517 nm. A.
ornata oil has weak ability to donate hydrogen. The absorption is however concetration dependent.
Key words: Free-radical, toxicity, 2, 2–diphenyl–1–picrylhydrazyl radical, ascorbic acid,
butylatedhydroxylanisole, α-tocopherol, Gas Chromatography/ Mass Spectroscopy.
Introduction
Naturally occurring antioxidants are substances
from plants or animals sources that can delay or
inhibit oxidation processes of free radicals.
Antioxidants like Vitamin C, Vitamin E,
carotenes, phenolic acids, polyphenols and
fl a v onoids scavenge free radica l s l i k e
hydroperoxide, peroxide, hydroxyl or lipid peroxyl
and thereby inhibit the oxidative mechanisms that
lead to degenerative diseases [3,4,27,20,23]. Search
for new antioxidant agents especially from plants
is therefore desirable.
Essential oil is any concentrated hydrophobic
containing volatile aroma compounds from plants.
Essential oils volatize on exposure to air at
normal temperature and find application in food,
confectioneries, perfumery industries and classical
medicine. Myrrh (Commiphora myrrha) containing
62% sesquiterpenes is used by Arabians for skin
conditions such as wrinkled, chapped and cracked
skin. Eugenol has antibacterial action; thymol is
used in mouth wash for its antiseptic principle,
chanlmoogra oil has curative action on leprosy
amongst others [8,37,38,6].
Acalypha ornata Hochst is a shrub which is
widespread across tropical Africa. It is claimed to
have several medicinal uses even though not
much is known about its chemistry and
pharmacology [5,34,3317]. Several species of the
family, A. indica, A. fruticosa, A. guatemalensis,
A. wilkesiana, A. siamensis, A. communis, A.
marginata have however been widely investigated.
They are found to contain condensed and
Corresponding Author: Patricia A. Onocha, Natural products/Medicinal Chemistry Unit, Department of Chemistry,
University of Ibadan, Nigeria.
E-mail: [email protected]
Telephone: +234 703 6015339
189
Adv. Environ. Biol., 5(1): 188-193, 2011
hydrolysable tannins as well flavonoid glycosides
[12,7, 2,31,18]. Antimicrobial activities and
anticancer potential of some Acalypha species
have also been reported [30,11].
The aim of this research work is to
determine the volatile oil constituent of the leaves
of Acalypha ornata which will be obtained by
h y d r o d i s t i llation a nd a na lyse d b y G a s
Chromatography/Gas Chromatography–Mass
Spectrometry (GC/GC-MS), to carry out in-vitro
antioxidant assays by determining the free radical
scavenging effect on 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH). DPPH radical gives strong
absorption at 517 nm (deep violet colour) in
visible spectroscopy. The absorption vanishes or
is decolorized as the electron becomes paired off
in the presence of a free radical scavenger.
Studies have revealed that essential oils serve as
powerful antioxidants that produce adverse
environment for damaging free radicals thus
preventing mutations and oxidants in cells [30,27].
A simple bench top bioassay for the
determination of elementary toxicity; Brine shrimp
lethality test (BST) was used to determine the
toxicity of the oil[9,8].
Materials and methods
Plant Materials:
Fresh samples of the A. ornata leaves (300
g) were collected in August 2009 at the
Botanical Gardens, University of Ibadan.
Specimens were identified at the Botany and
Microbiology department, University of Ibadan,
Oyo State, Nigeria.
The volatile oil was
immediately collected from the fresh plant
material by hydrodistillation.
Reagents:
Hexane and methanol (BDH chemicals),
ascorbic acid, butylatedhydroxyanisole (BHA), αtocopherol and 2, 2-diphenyl-1-picrylhydrazyl
radical (DPPH) were obtained from Sigma
Chemical Co (Germany).
accordance with the British pharmacopoeia
specifications [8]. The essential oil was collected
and stored at 4 0C until analysis. The oil yield
was calculated relative to the dry matter.
Analysis of the Essential Oils:
Gas Chromatography:
GC-MS analyses of the essential oil was
carried out on an Agilent Technologies 7890A
GC system coupled to a 5975C VLMSD mass
spectrometer with an injector 7683B series device.
An Agilent (9091)-413:325 0C HP-5 column (30
m x 320 µm x 0.25µm) was used with helium
as carrier gas at a flow rate of 3.3245ml/min.
The GC oven temperature was initially
programmed at 50 0C (hold for 1min) and finally
at 300 0C (hold for 5min) at a rate of 800C/min
while the trial temperature was 37.25 0C. The
column heater was set at 250 0C and was a
split less mode while the pressure was 10.153
psi with an average velocity of 66.45 cm/sec and
a hold-up time of 0.75245 min was recorded.
Mass spectrometry was run in the electron impact
mode (EI) at 70eV. The percentage compositions
were obtained from electronic integration
measurements using flame ionization detector
(FID) set at 250 oC. The peak numbers and
relative percentages of the characterized
components are given in Table 1.
Gas Chromatography–mass Spectrometry:
The essential oil was analysed by GC-MS on
an Agilent Technologies 7890A GC system
coupled to a 5975C VLMSD mass spectrometer
with an injector 7683B series device. An Agilent
(9091)-413:325 0C HP-5 column (30 m x 320
µm x 0.25 µm) was used with helium as carrier
gas at a flow rate of 3.3245 ml/min. GC oven
temperature and conditions were as described
above. The injector temperature was at 250 oC.
Mass spectra were recorded at 70 eV. Mass
range was from m/z 30 to 500.
Identification of Components:
UV-Visible Spectrophotometer (Unico1200 &
Perkin Elmer lambda 25 models), GC-Mass
spectrophotometer (Agilent Technologies), Hydro
distiller - Clavenger apparatus.
The individual constituents of the oil were
identified on the basis of their retention indices
determined with a reference to a homologous
series of n-alkanes and by comparison of their
mass spectral fragmentation patterns (NIST 08.L
database/chemstation data system) with data
previously reported in literature[1,14,19]
Isolation of Essential Oils:
Brine Shrimp Lethality Test:
The oil was obtained by hydro distillation on
a Clavenger type apparatus for 4 hours in
The brine shrimp lethality test (BST) was
used to predict the toxicity of the oils. The
Major Equipments Used:
Adv. Environ. Biol., 5(1): 188-193, 2011
shrimp’s eggs were hatched in sea water for 48h
at room temperature. The nauplii (harvested
shrimps) were attracted to one side of the vials
with a light source. Solutions of the extracts
were made in DMSO, at varying concentrations
(10000, 1000 and 100 ppm) and incubated in
triplicate vials with the brine shrimp larvae.
Ten brine shrimp larvae were placed in each
of the triplicate vials. Control brine shrimp larvae
were placed in a mixture of sea water and
DMSO only. After 24h the vials were examined
against a lighted background and the average
number of larvae that survived in each vial was
determined. The concentration killing fifty percent
of the larvae (LC50) was determined using the
Finney computer programme [21,10,16].
Free Radical Scavenging Activity:
Scavenging Effect on DPPH:
A
solution
of
0.5
mM
of
2,
2–diphenyl–1–picrylhydrazyl radical (DPPH) in
methanol was prepared and 3ml of this solution
was mixed with 1ml of the oil sample in
methanol. The free radical scavenging activity of
the oil at 10 and 20 µg/ml was determined in
the visible region of a UV-Visible
spectrophotometer. The decrease in absorption at
517nm of DPPH was measured after 10min of
incubation. The actual decrease in absorption was
measured against that of the control.
The same experiment was carried out on
ascorbic acid, butylatedhydroxylanisole (BHA) and
α-tocopherol which are known antioxidant agents.
All test and analysis were run in triplicates and
the result obtained was averaged [15,16,20,24].
The activities were determined as a function of
their %Inhibition which was calculated using the
formula;
% Inhibition 
A2  A1 100

A2
1
Where A1 is the absorbance sample and A2
is the absorbance of control.
Results and discussion
The yield of the 300 g hydrodistilled A.
ornata leaves oil was 0.60% (w/w). The essential
oil, colourless, with characteristic smell was
analyzed by GC and GC/MS systems using a
polar column, resulting in the identification of 89
constituents in the hydrodistilled sample,
representing 54.64% of the total essential oil. The
compounds present and their percentage
composition are reported in Table 1. Majority of
the compounds are less than 1-2% of the total
composition except the following compounds
isopulegyl acetate, valenche, viridiflorene, αmuurolene, 2-hexyne, 6-methyl-α-ionone, γ-elemene,
190
(E)-2-methyl-4-undecene, ledol, cis-3-hexenyl
benzoate, 2-methyl-1-octadecene, apiole,
oplopamone and γ-eudesmol. Monoterpenes
common in the essential oil of plants are found
present though in trace amount in the essential
oil of A. ornata.
The occurrence of different secondary
metabolites suggests a wide range of biological
application of the plant [35,36,29].
The result of brine shrimp lethality test as
analyzed by Finney probit computer programme
showed that the oil from the leaves of A.ornata
was found to be toxic having an LC50 value of
111.6 mg/ml with lower and upper confidence
limits of 26.38 μg/ml and 239.45
μg/ml
respectively therefore its use at higher
concentrations should be monitored.
The high toxicity can also be beneficial in
the therapy of some ailments involving cell or
tumour growth. It has been found out that
medicinally active natural products are most times
toxic to Artemia silina nauplii [26].
The DPPH assay method is used to evaluate
antioxidant potential of plant extracts, foods and
pure compounds [16].
The free radical scavenging activity of the
plant was evaluated by the decrease in absorption
of the stable radical 2, 2-diphenyl-1-picrylhydrazyl
radical (DPPH) at 517 nm. A ornata essential oil
decolorized DPPH due to its hydrogen donating
ability.
The activity of the essential oil of leaves of
A. ornata on the stable radical DPPH is
concentration dependent and possessed weak
scavenging activity as shown in Table 3.
The free radical scavenging activity was
compared with the activities of known
antioxidants; ascorbic acid, butylated
hydroxyanisole (BHA) and α –tocopherol. At 10
and 20 µg/ml, the oil gave a % inhibition of
20.5 and 14.8 respectively which were less than
that of ascorbic acid and BHA but higher than
α- tocopherol. Hence the oil of this plant has
weak ability in scavenging free radicals.
The absorption is stoichiometric with respect
to the number of electron taken up.
DPPH is known to be a stable free radical
and accepts an electron or hydrogen radical to
become a stable diamagnetic molecule [32].
A growing number of diseases are reported
to be caused or influenced by free radicals. Free
radicals are the toxic agents behind ageing and
cellular damage [4]. The results obtained from
this study are in agreement with other species of
Acalypha species [22,28].
191
Adv. Environ. Biol., 5(1): 188-193, 2011
Table 1: Chemical Composition of the volatile oil from A. ornata leaves by GC and GC/ MS analysis*
Peak No
Constituents
RT (sec)
%
1
2- hexenal
211.14
0.549
2
methyl tiglate
225.06
0.544
3
propyl butyrate
261.54
0.566
4
α – fenchene
335.10
0.553
menth -2-en-1-ol, (cis-para-)
559.08
0.761
6
3-octanyl acetate
690.42
0.532
7
p- cresyl acetate
797.64
0.430
8
4- Terpineol
820.92
0.468
9
Z-Ocimenone
960.66
0.433
10
Linalyl acetate
1024.38
0.509
11
Isopulegyl acetate
1066.44
1.275
12
Perilla alcohol
1129.02
0.704
13
δ-Elemene
1240.86
0.549
14
Benzyl butyrate
1257.00
0.999
15
Eugenol
1285.68
0.537
16
cis-Cariyl acetate
1297.26
0.655
17
Neryl acetate
1301.04
0.442
18
α – Ylangene
1321.2
0.712
19
α – copaene
1334.88
0.483
21
trans- methyl cinnamate
1340.28
0.636
23
Longifolene
1409.46
0.695
24
Caryophyllene
1444.5
0.478
25
α – Santalene
1445.7
0.608
26
Isobutyl salicylate
1452.3
0.874
27
Nopyl acetate
1456.26
0.438
31
β-Humulene
1494.54
0.532
32
trans-Cinnamyl acetate
1500.12
0.618
34
ethyl-Vanillin
1523.82
0.648
35
Geranyl acetate
1524.60
0.858
36
α –Humulene
1527.18
0.580
37
Cis-methyl isoenzenol
1530.6
0.691
38
(z), β-santalene
1547.52
0.439
39
Trans-ethyl cinnamate
1548.60
0.878
41
Furfuryl heptanoate
1560.48
0.997
42
γ – Gurjunene
1574.66
0.543
43
6-methyl-γ-Ionone
1590
0.558
45
β-selinene
1609.32
0.917
50
Valenche
1622.46
1.468
52
Viridiflorene
1628.22
2.658
53
Germacrene B
1632.84
0.608
54
α-Muurolene / 7-hexadecyne
1643.58
1.357
56
Cuparene
1650.18
0.605
57
2-hexyne
1679.88
1.770
58
6-methyl-α-Ionone
1686.24
0.747
61
Myristicin
1692.6
0.626
66
β-Sesquiphellandrene
1705.56
0.799
68
2-ethyl-1,1-dimethyl cyclopentane
1711.56
0.818
69
Trans-γ-bisabolene
1722.3
0.602
72
Thymo hydroquinine
1770.12
2.276
73
Elemicin
1772.34
0.790
74
γ-Elemene
1776.72
1.305
75
(E)-2-methyl-4-undecene
1780.68
1.506
77
Ledol
1798.02
1.610
78
cis-3-Hexenyl benzoate
1810.02
1.225
79
2-methyl-1-octadecene
1811.40
1.142
81
E,z-3, 13-octadecadien-1-ol, acetate
1820.16
1.325
86
cis-2-methyl-7-octadecene
1898.28
1.159
87
(Z)-2-methyl-4-undecene
1904.46
1.467
95
Apiole
2066.10
1.360
98
Oplopanone
2182.26
1.414
70
cis-Nerolidol
1728.90
1.074
89
γ-Eudesmol
1952.46
1.240
Total composition %
54.64%
*RT = Retention Time, T.P. = Total Percentage, (-)= Not quoted
DBS
0214
0228
0263
03375
0559
0690
0795
0820
09.57
1023
1067
1127
1236
1251
1279
1296
1303
1322
1334
1342
1404
1442
1447
1451
1455
1494
1500
1522
1525
1527
1532
1547
1548
1560
1575
1591
1808
1624
1628
1632
1643
1652
1688
1691
1702
1721
1770
1772
1777
1798
1809
2065
2183
1724
1951
192
Adv. Environ. Biol., 5(1): 188-193, 2011
Table 2: Brine shrimp lethality test of A. ornata leaves Essential Oils*
Sample
Concentration
----------------------------------------------------------------------------------------------------------------------10000ppm
1000ppm
100ppm
Control
Survivor
0
6
10
30
Dead
30
24
20
0
111.6
LC50(μg/ml)
*Upper confidence limit 239.45
Lower confidence limit
26.38
Table 3: Scavenging Effect of Essential Oils of A. ornata leaves at Absorbance517nm*
CONC. (μg/ml)
OIL SAMPLE
ASCORBIC ACID
BHA
10
0.785±0.016
0.0843±0.010
0.0370±0.006
20
0.842±0.031
0.2893±0.128
0.0460±0.008
*Absorbance of essential oil of A. ornata leaves, ascorbic Acid, BHA and α- Tocopherol at 517nm.
α- TOCOPHEROL
0.6800±0.029
0.7040±0.003
Table 4: %Inhibition of A. ornata Leaves Essential Oil and known Antioxidants on 2,2-dipheynl-1-picrylhydrazyl (DPPH)*
CONC. (μg/ml)
OIL SAMPLE
ASCORBIC ACID
BHA
α- TOCOPHEROL
10
20.50
90.90
95.42
15.4
20
14.80
78.71
94.31
12.4
* % inhibition of essential oil, ascorbic acid, BHA and α- Tocopherol measured at 517nm.
Absorbance of control = 0.988 ± 0.001
Conclusion:
The essential oil composition of the leaves of
A. ornata investigated revealed the presence of
89 compounds as determined by GC and GC/MS
analysis constituting 54.64% of the total oil
composition. Alcohols, aldehydes, esters and
terpenoids are the dominating group of
compounds. Brine shrimp lethality test was carried
out to know the toxicity of the oils to living
organisms (shrimps). LC50 value (µg/ml) of 111.6
obtained showed that the essential oil of leaves
of A. ornata is toxic. Photometric determination
of antioxidant activity of essential oils of A.
ornata revealed that the oil showed weak activity
as a radical scavenger in the experiment using 2,
2–diphenyl–1–picrylhydrazyl radical (DPPH)
indicating that A. ornata oil has very weak
ability to donate hydrogen when compared with
standards ascorbic acid and butylatedhydroxyanisole
(BHA). Thus the ability to scavenge free radicals
is an important property in order to minimize
oxidative damage to living cells.
References
1.
2.
3.
4.
Adams, R.P., 2001. Identification of essential
oils by ion trap mass spectrometry. Carol
Stream, IL: Allured Publishing Corporation.
Adesina, S.K, O. Idowu, A.O. Ogundaina, H.
Oladimeji, T.A. Olugbade, G.O. Onawunmi
and M. Pais, 2000. Antimicrobial constituents
of leaves of Acalypha wikesiana and
Acalypha hispida. Phytother 14: 371-374.
Allan, L. and N.D. Miller, 1996. Antioxidant
flavonoids, structure, function and clinical
Usage. Alternative Medicine., 1: 103-111.
Arouma, O., 1996. Characterization of drugs
as antioxidant prophylactics. Free Rad Bio
Med, 20(5): 657-705.
5.
Burkill, H.M., 1994. The Useful Plant of
West Tropical Africa. 2nd Ed. volume 2,
Families E- I. Royal Botanic Gardens, Kew,
Richmond, United Kingdom, 636-640.
6. Buttery, R.K., 1987. Common Compounds of
Essential Oils. Journal of Organic Chemistry,
15: 14-20.
7. Erute, M.O. and A.E. Oyibo, 2008. Effects
of three plants extracts (Ocimum gratissimum,
Acalypha wilkesiena and Acalypha
macrostachya) on post harvest pathogen of
Persea americana. Journal of Medicinal Plants
Research. 2(11): 311-314. (http:// www.
academic journals. Org /JMPR
8. Donald, L.P., M.L. Gary and K. George,
1980. Introduction to Organic Laboratory
Techniques. New York. Longman
Incorporation. pp: 112-116.
9. Dvorack, P., E. Sucman, D. Carvenakova, K.
Korinnek and R. Blechova, 1999. Utilization
of Bioassay with Artemia salina in
pharmacology. In Collection of abstracts from
9th Conference on Toxicity Biodegradability
of Waste and Chemicals Significant in
Aquatic Environment. Solan. Czech Republic,
13-15.
10. Falope, M.O., H. Ibrahim and Y. Takeda,
1993. Screening of higher plants reputed as
pesticides using the brine shrimp lethality
assay. International Journal of Pharmacology,
31(4): 250-254.
11. Gutierrez, L.,
M.P. Singh, W.M. Maiese,
and B. Timmermann, 2002. New
Antimicrobial cycloartane triterpenses from
Acalypha communis. Journal of Natural
products., 65: 872-875.
12. Iniagbe, O.M., O. Malomo, A. Adebayo,
2009. Proximate composition and
Phytochemical constituents of some Acalypha
spp. Journal of Nutrition. 8(3): 256- 258.
Adv. Environ. Biol., 5(1): 188-193, 2011
13. Jer-Min, Chun-Chung, L. Ming, L. Takashi,
F. and Atsushi, T 1995. Scavenging Effect of
Mallutus respandus on active oxygen species.
Journal of Ethnopharmacology. 46: 175-181.
14. Joulain, D. and W.A. Konig, 1998. The atlas
of Spectral Data of Sesquiterpenes
Hydrocarbons. E.B-Verlag Hamburg, Germany.
15. Koleva, I.I., T.A. Van- Beck, and A.
Evstaliva, 2002. Screening of plant for
antioxidant Activity. A comparative study on
three testing methods. Phytochem Anal. 13:
8-7.
16. Lugasi, A., P. Horvahorich, (deceased) and
Divorscherk, A. 1999. Additional Information
to the in-vitro anti Oxidant Activity of
Ginkgo biloba L. Phytotherapy Research, 13:
160-162.
17. Mahram, G., I. Shehata, M. Koheil, El-A.R.
Shabranyi and A. Al-Almady, 1993.
Phytochemical and Biological studies on two
Acalypha species. Journal of Pharmaceutical
Science, 2(2): 169-77.
18. Maienen, J.M., A.B.K. Godeliver, and H.M.
Zakaria, 2005. Plants used to treat epilepsy
by Tanzanian Traditional healers. Journal of
Ethropharmacology. 97: 327-336.
19. Mclafferty, F.W. and D.B. Stauffer, 1989.
The Willey/NBS Registry of Mass Spectral
Data. John Wiley & Sons, New York.
20. Mensor, L.L., F.S. Menezes, G.G. Leitão,
A.S. Reis,
C. Tereca,
C.S. Coube and
S.G. Leirao, 2001. Screening of Brazilian
Plant Extracts for Antioxidant Activity by
the use of DPPH Free Radical method.
Phytotherapy Research, 15: 127-130.
21. Meyer, B.N., R.N. Ferrign, J.E. Putnam, L.B.
Jacobson, D.E. Nicholas, and J.L.
McLaughlin, 1982. Brine shrimp: A
convenient general bioassay for active plant
constituents. Planta Medica, 45: 31-34.
22. Okorondu, S., T. Sokari, M. Okonrondu, E.
Chinakwe, 2009. Phytochemical and
antibacterial properties of Acalypha hispida
leaves. International J. of Nat. and Applied
Sci., 5(2): 38-45.
23. Okwu, D.E., 2004. Evaluation of the
chemical compositions of indigenous spices
and flavouring agents. Global Journal of Pure
and Applied Science, 176: 13-17.
24. Oloyede, G.K. and E.O. Farombi, 2010.
Antioxidant properties of Crinum ornatum
Bulb Extracts. World Journal of chemistry,
5(1): 32-36.
25. Oloyede, G.K., I.A. Oladosu and A.F. Shodia,
2010. Chemical Composition and Cytotoxicity
of the essential oils of Crinum ornatum (Ait)
Bury. African journal of Pure and Applied
Chemistry, 4(3): 35-37.
193
26. Onocha, P.A., 1995. Phytochemical
Investigations and Cytoxixity of Anthorocleista
djalonensis (A. Chev) and Newbouldia laevis
(Beaus) Seem. A. Ph.d Thesis in Organic
Chemistry, University of Ibadan, Nigeria.
27. Potterat, O., 1997. Antioxidants & Free
Radical Scavengers of Natural Origin. Current
Organic Chemistry, 1: 415-440.
28. Ramya, D.K., S. Karthikumar and Jegatheesan
2009. Isolation of potential Anti bacteria
and antioxidant compounds from Acalypha
indica and Ocimum basilicum. African Journal
of Plant Science, 4(5): 163-166.
29. Ramzi, A.A., S.A. Mothana, A.S. Faisal and
A. Lindquist, 2008. Antimicrobial, Antioxidant
and cytotoxic activities and phytochemical
screening of some Yemeni Medicinal plants.
Evidence–based Complementary and
Alternative Medicine., 7(3): 323-330.
30. Reed, J.D., 1995. Nutritional Toxicology of
tannins and related polyphenols in forage
legumes. Journal of Animal Science, 73:
1516-1528.
31. Sabina, K.C., M.H. Claude and H. Claudie,
2005. Ethnomedicinal and bioactive properties
of plants ingested by wild chimpanzees in
Uganda. Journal of Ethnopharmacology, 101:
1-15.
32. Soares, J., T. Dinnis, Cuncha and Almeida
1997. Anti-oxidant activity of some extracts
of Thymus zygis. Free Radical Res., 26: 469478.
33. Sofowora, A., 2008. Medicinal plants and
traditional Medicines in Africa. 3rd Edition
Spectrum Books. Ibadan, 190-193.
34. Swaminathan, M.S. and S.L. Kochhar, 1989.
Plant and Society. 1st ed. London Macmillan
Publishers., 312-320.
35. Tanrisever, N., H. Fischer, G.B. Williamson,
1988. Methofurans from Calamintha ashei:
Effects on Schizachyrium scoparium and
Lactuca sativa, Phytochemistry., 27(8): 25232526.
36. Tashiro, T., S. Sano and K. Mori, 2000.
Synthesis of Optically Active 3-methyl-Alphahimachelene, sex pheromone of Sandfly,
Lutzomyia longipalpis Nippon Kaggakai Koen
Yokoshu., 78(2): 900-908
37. Vairo, E.T., R.B. Lynn and J.E. Robbers,
1981. Pharmacognosy Ed. Lea & Febiger.,
103-107.
38. Visioli, F., G. Bellomo, G. Galli, 1988. Free
Radical scavenging of Olive oil polypheols.
Biochemical Biophysical Resources
Communications., 247(1): 60-64.