Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2011, Article ID 620862, 9 pages
doi:10.1155/2011/620862

Research Article
Chemical Composition and, Cellular Evaluation of
the Antioxidant Activity of Desmodium adscendens Leaves

François Nsemi Muanda,1 Jaouad Bouayed,2 Abdelouaheb Djilani,1 Chunyan Yao,3

Rachid Soulimani,3 and Amadou Dicko1
1 Chemistry Laboratory and Methodology for the Environment, Paul-Verlaine University Metz, 1, boulevard Arago Technopole 2000,
57078 Metz, France
2 Environment and Agro-Biotechnolgies Department, CRP—Gabriel Lippmann, L-4422 Belvaux, Luxembourg
3 Laboratory of Neurotoxicology Alimentary and Bioactivity, Paul-Verlaine University Metz, BP 4102, 57040 Metz, France

Correspondence should be addressed to Amadou Dicko, dicko@univ-metz.fr

Received 12 February 2010; Revised 2 May 2010; Accepted 31 August 2010

Copyright © 2011 François Nsemi Muanda et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Desmodium adscendens plant is widely used as juice or tea in various parts of the world against a wide range of diseases.
This study determines the quality and the quantity of polyphenols, flavonoids, anthocyanins, and tannins in D. adscendens
leaves by UV-spectrophotometry and RP-HPLC methods. In addition, the antioxidant capacity of these phenolic compounds is
evaluated by ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic)), DPPH (2,2-diphenyl-1 picrylhydrazyl), and Cellular tests.
D. adscendens leaves are mainly composite of flavonoid compounds with 12.8 mg of catechin equivalent (CE)/g dw. The amounts of
total polyphenol compounds are 11.1 mg of gallic acid equivalent (GAE)/g dw. The quantity of total anthocyanin and total tannin
compounds is not considerable 0.0182 mg CgE/g dw and 0.39 mg CE/g dw, respectively. A direct correlation between phenolic
compounds and antioxidant activity is observed (R2 = 0.96). The RP-HPLC analyses reveal that the main phenolic compound
identified in the methanol-water extract is quercetrin dihydrat (2.11 mg/mL). According to the results, it is observed that D.
adscendens leaves possess a considerable scavenging antioxidant and antiradical capacity, therefore these antioxidant properties
might increase the therapeutic value of this medicinal plant.

1. Introduction which are rich in antioxidant activity [6, 7]. Epidemio-

logical studies have shown that many of these antioxidant
Medicinal plants are of great importance to the health of compounds possess anti-inflammatory, antiatherosclerotic,
individuals and communities; many people in the world antitumor, antimutagenic, anticarcinogenic, antibacterial, or
have difficulty in gaining access to modern medicine; they antiviral activities to a greater or lesser extent [8–10]. The
use traditional medicine, based on the use of medic- intake of natural antioxidants has been associated with
inal herbs and plants, as an alternative to a conven- reduced risks of cancer, cardiovascular disease, diabetes, and
tional treatment for their recovery. Several papers have diseases associated with ageing [10].
been published for pharmacological proprieties in medic- Phenolics inhibit carcinogenesis by affecting the molec-
inal plants or their isolated constituents like antioxidant, ular events in the initiation, promotion, and progression
antidiabetic, antibacterial, antiviral, and antiulcer activities stages [11]. They modulate the secretion of protein kinases
[1–5]. in tumor cell proliferation and induce the expression of
Plants (fruits, vegetables, medicinal herbs, etc.) contain anticarcinogenic enzymes or inhibit induction of cancer-
a wide variety of free radical scavenging molecules, such promoting enzymes [12–14].
as phenolic compounds (e.g., phenolic acids, flavonoids, Antioxidant activity is a fundamental important property
anthocyanins, and tannins), nitrogen compounds (alkaloids, for human life. Many of the biological functions, including
amines, and betalains), vitamins, terpenoids (including antimutagenicity, anticarcinogenicity, and antiaging, among
carotenoids), and some other endogenous metabolites, others, originate from this property [8, 11].
2 Evidence-Based Complementary and Alternative Medicine

Recently, Abu Bakar et al. [15] reported that phenolic Flow cytometry is used to separate the different immune
compounds have potentially beneficial effects on human cell populations according to size (forward light scatter, FSC)
health by reducing the occurrence of coronary heart disease, and relative granularity (side light scatter, SSC). FSC and
age-related eyes diseases, and artherogenic processes. These SSC were used after excitation of the immune cells with
compounds also have antioxidant and anti-free-radical prop- using 488 nm argon laser beams. The level of intracellular
erties that allow them to quench free radicals in the body ROS was measured in the granulocytes by monitoring the
[16]. emitted fluorescence of these cells (FACS-Scan, Becton-
Moreover, it was reported that antioxidants with ROS Dickinson, Immunofluorometry Systems, France). In addi-
scavenging ability have great relevance in the prevention tion, spectrophotometer analyses were carried out with UV-
of oxidative stress which is responsible for the majority of Vis spectrophotometer (Cary 50 scan).
diseases [17].
Desmodium adscendens is a vine, which grows wild in the 2.3. Animals. Swiss albino male mice (OF1) aged 9 weeks at
Amazon rainforest of Peru, in South American countries and the receipt from the breeder company (Charles River, France)
as well on the West Coast of Africa. Native people use this and weighing 40–45 g were used. They were maintained
plant as a juice or a tea. Also, D. adscendens is a medicinal under standard environmental conditions with free access to
plant which is widely used in popular medicine in different water and food (SD Dieted-France). All animal procedures
parts of the world. In the Brazilian traditional medicine, were carried out in accordance with the European Com-
the leaves of this plant treat leucorrhoea, body aches, pains, munities Council Directive of 24 November 1986 (86/609/
ovarian inflammations, excessive urination, gonorrhoea, and EEC).
diarrhoeas [18]. Its positive effect against hepatic infection
was also verified in vivo [19]. In African traditional medicine,
D. adscendens is extensively used to treat asthma and other 2.4. Plant Materials. Dried leaves of D. adscendens
diseases associated with smooth muscle contraction [20]. (Fabaceae) were obtained from Nigeria, and the biological
The Congolese healers treat fever, pain, and even epilepsy authentication was carried out by Professor Max Henry in
with aqueous extracts of the leaves of D. adscendens [21]. To the Botanic and Mycology Laboratory, Nancy University
our knowledge, there is still no report on the phytochemicals (France). All plant materials were dried at room temperature
and the antioxidant properties of this plant leaf. The present and were ground and sifted in a sieve (0.75 μm).
study is aimed to identify, to quantify the phenolic com-
pounds contained in the leaves of D. adscendens, and to eval- 2.5. Sample Preparation
uate their antioxidant capacity by ABTS, DPPH, and cellular
tests. 2.5.1. Total Phenolic, Flavonoid, and Anthocyanin Contents
and Antioxidant Capacity. Samples for total phenolic com-
pounds, total flavonoid compounds, total anthocyanin com-
2. Materials and Methods pounds, and antioxidant capacity assays were extracted from
2.1. Chemicals. All the chemicals used were of analytical the powders as described by Chitindingue et al. [16].
grade. 1,1-Diphenyl-2-picryl hydrazyl radical (DPPH), 2,2 - Two grams of powdered sample were extracted twice with
azino-bis (3-ethylbenzothiazoline-6-sulfonic) (ABTS), HBSS 10 mL of cold aqueous methanol solution (50%). The two
buffer, dioxygen water (H2 O2 ), 2,2 -azo-bis (2-amidino- volumes were combined, made up to 40 mL, centrifuged at
propane) dihydrochloride (AAPH), 2 ,7 -diacetate dichlo- 1536 × g for 20 minutes, transferred in small sample bottles,
rofluorescein (DCFH-DA), gallic acid, folin-Ciocalteu’s phe- and stored at +4◦ C in the dark until analysis.
nol reagent, alumin chloride, catechin, p-coumaric acid,
rutin, procatechiuc acid, vitamin C, caffeic acid, isovitexin, 2.6. Condensed Tannin (CT) Extraction. Samples for CT were
vitexin, chlorogenic acid, orientin, malvidin, homoori- extracted from the powders as described by Villarreal-Lozoya
entin, peonidin, catechin, quercetin, quercetin dihydrat, et al. [22]. One gram of powder was extracted twice with
quercetin-3-β-D-glucosyl, epicatechin, kuromanin chloride, 20 mL of n-hexane for 20 minutes and filtered, and the
and cyanidin chloride were purchased from Across organ- remaining powder was dried at 35◦ C under vacuum for 2 h.
ics (Geel, Belgium). Sodium carbonate, sodium nitrite, The powder was stored at +4◦ C until analyses.
chlorhydric acid, ethyl acetate sodium, sodium sulphate
anhydrous, ammonium phosphate, ferric ammonium sul-
2.7. Phenolic Compounds Extraction for RP-HPLC Analysis.
phate, acetonitrile, methanol, vanillin reagent, and n-
Polyphenols were extracted with water (WE) and with
hexane were obtained from Sigma and Roth (Strasbourg,
methanol-water (50-50, v-v) (MWE) as solvents according
France).
to the slightly modified method described by Sharma et al.
[23]. A sample (0.4 g) was extracted with 2 × 5 mL of solvent
2.2. Apparatus. The RP-HPLC analyses were performed with under intermittent shaking (2 minutes) on vortex mixer for
a Waters 600E pump coupled to a Waters 486 UV visible 30 minutes. The sample was centrifuged at 1536 × g for 20
tunable detector and equipped with a 20 μL injection loop minutes at 20◦ C. The supernatant was taken into a 10 mL
and an Alltech Intertsil ODS column (RP C18 column size volumetric flask. The extracts are stable for 24 h if stored at
4.6 mm × 150 mm; particle size, 5 μm). 4◦ C.
Evidence-Based Complementary and Alternative Medicine 3

2.8. Preparation of Samples for Cellular Tests. The extract 2.9.4. Condensed Tannins. The condensed tannin content
was prepared from the dried plant in the traditional way was estimated using the method described by Villarreal-
(Nigeria). A decoction was done for 1 h in distilled boiled Lozoya et al. [22] with some modifications. Briefly, an aliquot
(200 g/L) water. Then the resulting mixture was filtered and of 0.5 g of powder obtained after lixiviation (n-hexane) was
evaporated under reduced pressure and lyophilized by a placed in a centrifuge tube and 15 mL of 1% HCl in methanol
Lyophilizer (ALPHA 1-2 LD, Fisher Bio Block). The amount was added to each sample. Each tube was vortexed and
of lyophilized extract obtained was 24.2 g. placed in a water bath at 35◦ C with constant shaking for 20
minutes and vortexing every 5 minutes. After incubation, the
tubes were centrifuged (1536 × g), and the supernatants were
2.9. Dosage of Phenolic Compounds extracted. Aliquots of the supernatants (100 μL) were placed
in two separate assay tubes, one for the sample determination
2.9.1. Total Phenolic Compounds. The Folin-Ciocalteu meth- and the other for blank determination.
od was used to measure the total phenolic compounds Samples and blanks were incubated for exactly 20
(Dzingirai et al. [4]). To a sample (100 μL), distilled water minutes after adding 5 mL of vanillin reagent (0.5 g of reagent
was added to make 2 mL (Eppendorff tube), followed by and 200 mL of 4% HCl methanol) to samples and 4% HCl
1 mL of Folin Ciocalteu reagent 1 N and sodium carbonate in methanol to the blanks. After 20 minutes, absorbance
(20%). was read at 500 nm from each sample and blank using UV-
After 40 minutes at room temperature, absorbance at spectrophotometer Varian Cary 50. Samples absorbance was
725 nm was read on a Spectrophotometer against a blank rectified with the blank standard and compared against a
that contained methanol instead to sample. The results standard curve made with catechin. Results were expressed as
were compared to a gallic acid calibration curve, and the mg catechin equivalent (CE) of lixiviating sample. Analyses
total phenolic compounds were determined as gallic acid were triplicate.
equivalents (GAEs). The samples were analyzed at least three
replications.
2.10. RP-HPLC Analysis. The samples (WE) and (MWE)
were filtered through a 0.45 μm PTFE syringe tip filter
2.9.2. Total Flavonoid Compounds. The flavonoid contents and were analyzed using an RP-HPLC system equipped
were measured according to a colorimetric assay reported with a 20 μL injection loop, a waters UV-Visible tuneable
by Muanda et al. [24]. A 250 μL of standard solution of detector on a reverse phase (RP C18 ) column Alltech Interstsil
catechin at different concentrations or appropriately diluted ODS-5 μm × 4.6 mm × 150 mm. The flow rate was set
samples was added to 10 mL volumetric flask containing at 1 mL/minute at room temperature. To perform this
1 mL of distillate water. At the initial time, 75 μL of NaNO2 study, a gradient of three mobile phases was used; solvent
(5%) was added to the flask. After 5 minutes, 75 μL of A: 50 mM ammonium phosphate (NH4 H2 PO4 ) pH 2.6
AlCl3 (10%) was added and after 6 minutes, 500 μL of (adjusted with phosphoric acid), solvent B: (80 : 20 (v/v))
NaOH (1 N) was added to mixture. Immediately, the solution acetonitrile/solvent A, and solvent C: 200 mM of phosphoric
was diluted by adding 2.5 mL of distillate water and mixed acid pH 1.5 (pH adjusted with ammonium hydroxide).
thoroughly. Absorbance of the mixture, pink in colour, The solvents were filtered through a Whatman Maidstone
was determined at 510 nm versus the prepared blank. Total England paper N◦ 3 and placed in an ultrasonic apparatus
flavonoid compounds in medicinal plant were expressed as for 25 minutes. The gradient profile was linearly changed as
catechin equivalents (CEs). Samples were analysed in three follows (total 60 minutes): 100% solvent A at zero minutes,
replications. 92% A/8% B at 4 minutes, 14% B/86% C at 10 minutes, 16%
B/84% C at 22.5 minutes, 25% B/75% C at 27.5 minutes, 80%
B/20% C at 50 minutes, 100% solvent A at 55 minutes, and
2.9.3. Total Anthocyanin Compounds. The total anthocyanin
100% A at 60 minutes.
compound of the samples was estimated using a UV-
After each run, the system was reconditioned for 10
spectrophotometer by the pH-differential method [15, 25].
minutes before analysis of the next sample. Under these
Two buffer systems, potassium chloride buffer, pH 1.0
conditions, each sample analysis was done in triplicate.
(0.025 M) and sodium acetate buffer, pH 4.5 (0.4 M) were
Polyphenolic external standards were prepared by dissolving
used. Briefly, 400 μL of extract was mixed in 3.6 mL of
2 mg/mL and used as reference. In each sample, polyphenol
corresponding buffer solutions and read against a blank at
was identified by comparing its retention time with that of
510 and 700 nm. Absorbance (ΔA) was calculated as ΔA =
the corresponding external standard. Detection was done
(A510 − A700 )pH 1.0 − (A510 − A700 ) pH 4.5, monomeric
at 280 and 320 nm, and quantification was calculated by
anthocyanin pigment concentration in the extract was
comparing their peak areas.
calculated and expressed as equivalent cyaniding-3-glucoside
(mg/L): ΔA × Mw × Df × 1000/(Ma × 1), with ΔA: difference
of absorbance, Mw: molecular weight for cyaniding-3- 2.11. Antioxidant Activity Analyses
glucoside (449.2), Df: the dilution factor of the samples,
and Ma: molar absorptivity of cyaniding-3-glucoside (26 2.11.1. DPPH Radical Scavenging Test. The DPPH radical
900). Results were expressed as mg of Cyaniding-3-glucoside scavenging activity was evaluated according to the method
Equivalents (CgEs). slightly modified described by Marwah et al., Sharififar et al.,
4 Evidence-Based Complementary and Alternative Medicine

Adesengun et al., and Al-Zubairi et al. [26–29]. 1 mL of 12.84 mg

100 μM DPPH solution in methanol was mixed with 1 mL 11.15 mg CE/g dw
GAE/g dw
of plant extract. The reaction mixture was incubated in the
dark for 20 minutes, and the optical density was recorded
at 517 nm against the blank. For the control, 1 mL of DPPH
solution in methanol was mixed with 1 mL of methanol, and
optical density of solution was recorded after 20 minutes.
The decrease in optical density of DPPH in samples with
0.02 mg 0.39 mg
regard to control system was used to calculate the antioxidant
CgE/g dw CE/g dw
activity as a percentage inhibition (IP%) of DPPH radical,
IP% = ((Ato − At20 )/(Ato × 100)) were Ato : absorbance of TPC TFC TAC CT
the test sample after zero minutes and At2o : absorbance Figure 1: Phenolic compounds of leaves of D. adscendens. TPC:
of control after 20 minutes. Each assay was carried out in Total phenolic compounds; TFC: Total flavonoid Compounds; TAC:
triplicate. Total anthocyanin compounds; CT: Condensed tannins; dw: dry
From a plot of concentration against %IP, a linear weight; GAE: Gallic Acid Equivalent; CE: Catechin Equivalent; CgE:
regression analysis was performed to determine the IC50 Cyaniding-3-glycoside Equivalent.
value for each extract. The DPPH radical scavenging activity
of phenolic compounds was expressed as IC50 value in
terms of μg/mL of dry weight. To 2.90 mL of an aqueous
methanol solution (50%) of 100 μM of DPPH, 100 μL of the
plant extract solution was added. The mixture was shaken the granulocytes by monitoring of the emitted fluorescence
and stored at 20◦ C in the dark for 40 minutes. Then, the by these cells.
decrease of absorbance was measured; the resulting solution The ROS level was quantified using 2 ,7 -diacetate di-
was monitored at 517 nm. The DPPH radical scavenging chlorofluorescein (DCFH-DA). Nonpolar DCFH-DA crosses
activity of phenolic compounds was expressed in terms of the cellular membranes and is hydrolysed by intracellular
mg of Vitamin C Equivalents (VCEs) per g dw in 40 minutes. esterase to form the polar, nonfluorescent dichlorofluo-
The control solution was consisted of 100 μL of methanol rescein (DCFH). Then, the latter is oxidized to highly
and 2.90 mL of DPPH solution. The radical solution was fluorescent substance, the 2 ,7 -dichorofluorescein (DCF) by
prepared daily. intracellular ROS [31, 32].
The mice were anesthetized with the halothane and sacri-
ficed. The collected blood was heparinized and dispatched in
2.11.2. ABTS Radical Scavenging Test. The slightly modified
volumes of 100 μL in Eppendorff tubes. Erythrose was done
method developed by Djeridane et al. [30] was used in
using 2 mL of lysing solution in each tube; the whole was
this experiment. 1.0 mM of AAPH solution was mixed with
placed in darkness for 10 minutes. After centrifugation at
2.5 mM ABTS as diammonium salt in phosphate buffered
4◦ C (5 min; 1536 × g), the supernatant was eliminated, then
saline (PBS) solution 100 mM potassium phosphate buffered
2 mL cell wash solution was added to sediment containing
(pH 7.4) containing 150 mM NaCl. The mixture was heated
white cells, mixed, and followed by a new centrifugation in
in a water bath at 68◦ C for 20 minutes. The concentration
the same conditions. Supernatant was still eliminated.
of the resulting blue-green ABTS•+ (radical cation solution)
Three groups of cell belonging to the same blood were
was adjusted to an absorbance of 0.65 ± 0.02 at 734 nm.
used to assess the intracellular ROS level; the first one was a
The sample solution of 60 μL was added to 2.94 mL of
control without oxidative stress, and the second was a control
the resulting blue-green ABTS radical solution. The mixture,
with oxidative stress. The third group is served to evaluate the
protected from light, was incubated in a water bath at 37◦ C
antioxidant activity of the extract. 1 mL of HBSS buffer was
for 20 minutes.
added and 5 μL of DCFH-DA (50 μM) to white cells. For the
Then the decrease of the absorbance at 734 nm was
control (without oxidative stress), incubation was achieved
measured. The control solution was consisted of 60 μL of
in darkness during 30 minutes at 37◦ C. For the second
methanol and 2.94 mL of ABTS•+ solution. The stable ABTS
control (with oxidative stress), incubation was performed
radical cation scavenging activity of the phenolic compounds
in darkness during 15 min at 37◦ C, then is added 5 μL of
in the extract was expressed in terms of mg of Vitamin C
H2 O2 (89 mM) to provoke oxidative stress, the incubation
Equivalents (VCEs) per g dw in 20 minutes. All radical stock
continued for 15 minutes more.
solutions were prepared daily.
In the third group of cells, several concentrations of
the extract (D. adcendens) were used for the evaluation of
2.11.3. Cellular Test. A cellular assay was used for the in vitro its antioxidant activity. So, to this group, in addition to
antioxidant activity evaluation; flow cytometry technique HBSS buffer and DCFH-DA, 5 μL of D. adscendens extract
allows a separation of different immune cells population by were added. After 15 minutes of incubation in darkness, the
size (forward light scatter, FSC) and relative granularity (side oxidative stress was induced by addition of 5 μL of H2 O2
light scatter, ssc) parameters. FSC and SSC were used after (89 mM). It continued for 15 minutes at 37◦ C. As soon
excitation of the immune cells by the 488 nm laser beam as incubation times were completed, the ROS levels were
of argon. The level of intracellular ROS was measured in measured by the flow cytometry technique.
Evidence-Based Complementary and Alternative Medicine 5

×105 ×105
6 7 7
5
6
5 8
5 6
4 5
4 4

(μV)
3 3
3 2 6 2
(μV)

1 7 1
2 0
1 −1 10 15 20 25 30 35 40 45 50
(min)
0
5 15 25 35 45 (a)
−1
(min)
(a)
×104 ×105
7 14 7
50
45 12
40 8 10 8
35

(μV)
30 8
(μV)

6 6
25 6 5
4
20 4
3
15 2
10
5 0
0 10 15 20 25 30 35 40 45 50
−5 10 15 20 25 30 35 40 45 50 (min)
(min) (b)
(b)

Figure 2: (a) WE, HPLC chromatogram at 280 nm of the leaves Figure 3: (a) MWE, HPLC chromatogram at 280 nm of leaves
of D. adscendens 1: gallic acid; 2: protocatechuic acid; 4: rutin; 5: of D. adscendens 3: catechin; 6: quercetrin glucosyl; 7: quercetrin
quercetrin glucosyl; 6: quercetrin dihydrat; 7: cinnamic acid. (b) dihydrat; 8: cinnamic acid. (b) MWE, HPLC chromatogram at
WE, HPLC chromatogram at 320 nm of the leaves of D. adscendens 320 nm of leaves of D. adscendens 4: chlorogenic acid; 5: quercetrin
3: Catechin; 6: quercetrin glucosyl; 7: quercetrin dihydrat; 8: cinna- glycosyl; 6: quercetrin dihydrat; 7: cinnamic acid; 8: cinnamic
mic acid. acid.

In this test, oxidative stress is induced by addition of based on analysis of variance. Observed differences were
H2 O2 in the extracellular medium of the granulocytes. statistically considered significant at the level of P < .001.
Exogenous H2 O2 diffuses inside the cells, consequently their
DCFH oxidation increases (C) compared to granulocytes 3. Results
without oxidative stress (A). Under oxidative stress con-
ditions, D. adscendens extract led to the reduction of the 3.1. Total Phenolic Compounds. The results reveal that 1 g of
fluorescence intensity of treated cells (B). The fluorescence dry weight of D. adscendens leaves contains 11.15 mg GAE
intensity is expressed as decimal logarithm versus the cell of total phenolic compounds, 12.84 mg CE of total flavonoid
number. (A) and (B) are the controls corresponding to the compounds, and 0.0182 mg CgE of total anthocyanins, and
level of intracellular ROS without and with oxidative stress, the amount of condensed tannins observed is 0.39 mg CE
respectively. (C) is the treated granulocytes corresponding (Figure 1).
to ROS level in these cells incubated in the presence of
D. adscendens extract at 25 mg/mL then subjected to the 3.2. HPLC Analysis. The RP-HPLC profiles of the water
oxidative stress. extracts and the methanol-water extracts are shown in
The reduction percentage of ROS generated in granulo- Figures 2 and 3, respectively. Peaks were identified and
cytes by exogenous H2 O2 was calculated by the monitoring quantified on the basis of their retention time values and UV
of the emitted fluorescence intensity (Fi). The following spectra by comparison with those of the single compound
relation was used (Fito − Fit1 ) × 100/(Fito − Fit2 ) with Fito : in the standard solution. The retention time and the concen-
control with oxidative stress; Fit1 : treat cells; Fit2 : control tration of polyphenolic compounds contained in the extracts
without oxidative stress. are reported in Table 1. There were numerous peaks that were
not identified because of the lack of suitable standards. The
2.12. Statistical Analysis. Results are presented as mean ± samples were analyzed at least four replications at 280 and
standard Error; statistical analysis of experimental result was 320 nm.
6 Evidence-Based Complementary and Alternative Medicine

Table 1: Concentration of the phenolic compounds in the WE and the MWE.

Rt (min) Ref. Name of compounds WE (mg mL−1 ) MWE (mg mL−1 )

11.98 ± 0.22 [1] Gallic acid 0.12 ± 0.01 N.d
14.78 ± 0.92 [2] Protocatechiuc acid 0.11 ± 0.02 N.d
23.96 ± 0.23 [3] Catechin 0.06 ± 0.04 0.69 ± 0.04
25.80 ± 0.23 [4] Chlorogenic acid N.d 0.23 ± 0.06
37.02 ± 0.36 [5] Rutin 0.24 ± 0.01 0.28 ± 0.04
37.71 ± 0.18 [6] Quercitin glucosyl 0.46 ± 0.03 0.17 ± 0.01
39.47 ± 0.19 [7] Quercitin dihydrat 0.17 ± 0.02 2.11 ± 0.16
41.68 ± 0.65 [8] Cinnamic acid 0.14 ± 0.01 0.76 ± 0.07
WE: water extract; MWE: methanol water extract; Rt: Retention time in minute; N.d: not detected.

expressed as mg of VCE/g dw, are 12.83 mg (ABTS) and

8.47 mg DPPH.
Counts

(c)

0
100 101 102 103
(b)
3.3.2. Cellular Test. The ROS level was detected using a flu-
orescence probe, 2 ,7 -dichlorofluorescin diacetate (DCFH-
Counts

40
DA), which can be oxidized to highly fluorescent dichlo-
rofluorescein (DCF) by intracellular ROS [16]. In Figure 4,
0 a decrease of the fluorescence intensity (FI) was observed,
100 101 102 103
between (B) (log = 80) corresponding to control with
Counts

40 (a) oxidative stress and (C) (log = 35) representing the pretreated
cells, which was close to the level of the control of cells
0 without oxidative stress (A) (log = 30). This decrease is due
100 101 102 103 to the effect of the extract of D. adscendens on the ROS level
log. (FI) which is directly correlated to the FI.
Figure 4: The ROS levels were measured by flow cytometry. The The scavenging capacity of D. adscendens leaves extract
fluorescence intensity was expressed as decimal logarithm versus the on the ROS was highlighted. To a concentration of
cell number. (a) and (b) are the controls corresponding to the level 25 mg/mL, it is observed a reduction of 83.21 ± 6.21% of ROS
of intracellular ROS without and with oxidative stress, respectively. level generated by exogenous H2 O2 . Thus, pretreatment of
(c) is the pretreated granulocytes corresponding to ROS level in granulocytes with this extract appreciably inhibited the ROS
these cells incubated in the presence of Desmodium adscendens generation induced by H2 O2 .
extract at 25 mg/mL then subjected to the oxidative stress.

100
4. Discussion
90 R2 = 0.9614
(%R)/ROS exo. H2 O2

80 Among the phenolic compounds contained in the leaves of

70 D. adscendens, the major was the anthocyanins, and the least
60
50 was the condensed tannins.
40 Quantitative analyses carried out by RP-HPLC show
30
20 that water is not the best extract solvent for polyphenolic
10 compounds. This is in concordance with Marwah et al. [26],
0
who have reported that aqueous alcohols are the best solvents
0 10 20 30 40 50
for extracting polyphenolic compounds from plant materi-
CCE (mg/mL)
als. However, these results show that, in water, gallic acid
Figure 5: Concentration-response curve for reduction of ROS (0.12 mg/mL) and the protocatechuic acid (0.11 mg/mL) are
generated by exogenous H2 O2 . R (%) ROS exo H2 O2 , reduction more extracted than in the methanol-water medium because
(%) of ROS generated by exogenous H2 O2 ; CCE: Concentration of these acids can be transformed into their corresponding
extract. esters in hydroalcoholic medium [33].
The major compound identified in the WE was the
quercetrin glucosyl (0.46 mg/mL) and the least was catechin
3.3. Antioxidants Activities (0.06 mg/mL).
In contrast, it was found that, in the MWE, the quercetrin
3.3.1. ABTS and DPPH Tests. The ABTS test and the DPPH glucosyl (0.17 mg/mL) was the least. In this extract, the
test reveal that the extracts of D. adscendens leaves possess quercetrin dihydrat concentration (2.11 mg/mL) was the
a useful scavenging antioxidant activities, these results, highest followed by cinnamic acid (0.76 mg/mL).
Evidence-Based Complementary and Alternative Medicine 7

UV, radiation, ozone

Proinflammatory cytokines
(e.g, II-Iβ; TNF-α; IFN-γ)

Cigarette smoke; oxidants

Infections
Respiratory burst
O2 molecular oxygen
and
release of free radicals

MPO 1O
2 singlet oxygen

Desmodium
NADPH oxidase adscendens

Oo2− superoxide
anion radical

SOD

Enhancement
H2 O2 hydrogen
peroxide
GST SOD

Fe2+ ◦ OH hydroxyl GSH CAT

radical

ROS ROS
increasing decreasing

DNA damage
NF-κB
and mutation

Proteine damage
◦ DNA-repair protein Lipid peroxydation
◦ caspases Induction of various diseases

Figure 6: Role of D. adcendens in prevention of diseases caused by free radicals. Generation of ROS is initiated by respiratory burst, which
is set off by various physiological and environmental factors. The fabrication of an assortment of ROS from the molecular O2 , carried by
different enzymes such as MPO (myloperoxidase), NADPH oxidase, and SOD (superoxide dismutase), leads to diverse cellular phenomena,
namely, damage of DNA-repair proteins and caspases, lipid peroxydation, and DNA damage followed by mutation and NF-κB activation. All
these phenomena give rise to wide range of diseases. D. adscendens leaves extract inhibits the generations of the free radicals by scavenging
both the mother and the daughter products and also by inducing the increase of SOD, CAT, GST, and GSH, resulting in the obstruction of
various disease formation.

The antioxidant activities recorded both using the ABTS D. adscendens extract was 4.00 μg/mL; this result reveals
test and the DPPH test show that D. ascendens leaves possess that compared to Allophylus rubifolius (IC50 = 7.1 μg/mL)
useful antioxidant properties. To compare these activities [26], Phaulopsis fascisepala leaves (IC50 = 0.5 mg/mL) [28],
with those other medicinal plants previously described Anogeissus dhofarica (IC50 = 4.5 μg/mL), and Litchi seeds
in the literature such as Allophylus rubifolius, Phaulopsis (IC50 = 4.8 μg/mL) [34], D. adscendens leaves possess a
fascisepala, Anogeissus dhofarica, and Litchi seeds, the antiox- significant antioxidant activities, since more the value of IC50
idant activities were expressed as IC50 . The IC50 value of is low more the antioxidant activity is high.
8 Evidence-Based Complementary and Alternative Medicine

In order to confirm these results, the antioxidant capacity Acknowledgments

of D. adscendens was evaluated against ROS under biological
medium by using a cellular test. The DCFH-DA was used to This work was supported by a Grant from the Ministry of
evaluate the intracellular redox status [28]. Concentration- Scientific Research of Republic Democratic of Congo (no.
response curve in the ROS reduction reveals a linear and 132.49/060/KM/07). Mr. Frederic Desort (Neurotoxicology
positive relationship (R2 = 0.96) between the scavenger Alimentary and Bioactivity Laboratory, University P. Verlaine-
capacity and the concentration of D. adscendens extracts. Metz, BP 4102, 57040 Metz, France) is also acknowledged for
Then, this reduction is directly correlated to the ROS level technical assistance.
decreasing (Figure 5).
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