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PII: S0731-7085(19)30088-3
DOI: https://doi.org/10.1016/j.jpba.2019.03.027
Reference: PBA 12540
Please cite this article as: Saleem H, Zengin G, Ahmad I, Lee JTB, Thet Htar T,
Mahomoodally FM, Naidu R, Ahemad N, Multidirectional insights into the biochemical
and toxicological properties of Bougainvillea glabra (Choisy.) aerial parts: A functional
approach for bioactive compounds, Journal of Pharmaceutical and Biomedical Analysis
(2019), https://doi.org/10.1016/j.jpba.2019.03.027
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bioactive compounds
Hammad Saleema,b, Gokhan Zenginc, Irshad Ahmadd, Joash Tan Ban Leee, Thet
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Thet Htara, Fawzi M. Mahomoodallyf, Rakesh Naidug, Nafees Ahemada,h,i*
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a
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan,
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47500 Bandar Sunway Selangor Darul Ehsan, Malaysia
b
Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal
Sciences (UVAS), Lahore-54000, Pakistan, U
N
c
Department of Biology, Faculty of Science, Selcuk University, Campus/Konya,
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Turkey
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d
Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan
e
School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 47500
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Mauritius
g
Jeffrey Cheah School of Medicine and Health Sciences, Monash University
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Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan,
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Malaysia.
h
Tropical Medicine and Biology Multidisciplinary Platform, Monash University
A
*
Corresponding author:
Dr. Nafees Ahemad (nafees.ahemad@monash.edu)
Highlights
Chemical fingerprinting of Bougainvillea glabra aerial extracts were evaluated by
UHPLC-MS.
Key enzyme inhibition and antioxidant propensities were determined via standard in
vitro bio assays.
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Flavonoids were the main components identified in the methanol extract.
B. glabra could be further studied to isolate novel bioactive compounds
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Abstract
The current research work was conducted in order to probe into the biochemical
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and toxicological characterisation of methanol and dichloromethane (DCM)
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extracts of Bougainvillea glabra (Choisy.) aerial parts. Biological fingerprints
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were assessed for in vitro antioxidant, key enzyme inhibitory and cytotoxicity
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evaluated via six different in vitro antioxidant assays namely DPPH, ABTS
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butyrylcholinesterase and urease inhibition. The DCM extract was most active
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extracts exhibited significant cytotoxic potential against five (MCF-7, MDA-
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MB-231, CaSki, DU-145, and SW-480) human carcinoma cell lines with half
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maximal inhibitory concentration values of 22.09 to 257.2 µg/mL. Results from
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the present study highlighted the potential of B. glabra aerial extracts to be
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further explored in an endeavour to discover novel phytotherapeutics as well as
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functional ingredients.
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bioactive compounds
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1. Introduction
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million deaths per year which is about 70% of all deaths worldwide [1]. NCD’s
are mostly linked to elevated levels of oxidative stress, which in turn is due to
levels in the body. As a part of normal body function, the free radicals are
produced all the times in the cells and are balanced by either internal
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scientific techniques have furnished impressive protocols for the isolation,
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purification and characterization of different bioactive compounds. This in turn,
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has provided key insights into their mechanism of action and therapeutics
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effects for humans [3]. The major focus of current research is more targeted on
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the biological activities of bioactive secondary metabolites from natural
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products and their health-related outcomes. Phenolic and flavonoids amongst
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the Garden”, is native to Southern America and has been used traditionally for
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spite of the considerable traditional importance, there have been only limited
in relation with its medicinal uses. Thus, we aimed to evaluate, in this paper, B.
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glabra aerial parts for their chemical composition (total phenolic and flavonoid
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cytotoxic activities were also evaluated using MTT assay against human breast,
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cervix, prostate and colon carcinoma cell lines. The observed finding would
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provide new intuitions for B. glabra plant species.
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2. Material and methods
Pakistan.
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After shade drying and grinding of the aerial parts of plant, its powder
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(DCM) and methanol. The pooled extracts were then filtered and dried under
vacuum at 40 ºC. The extracts were abbreviated as; BM (B. glabra aerial
Folin–Ciocalteu method [8] using gallic acid as a standard. The amount of total
phenolics was determined as milligrams of gallic acid equivalents per gram (mg
GAE/g extract). Moreover, the amount of flavonoids in all the extracts were
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flavonoids were noted as mg QE/g extract (milligrams of quercetin equivalents)
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using quercetin as standard. Similarly, the phytochemical composition of the
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methanol extract was evaluated by UHPLC Accurate-Mass Q-TOF (Agilent
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1290 Infinity LC system coupled to Agilent 6520) mass spectrometer with dual
reported earlier [8]. Total antioxidant capacity (TAC) of extracts was evaluated
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GAE/g extract). The results of ABTS and CUPRAC assays were expressed as
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trolox equivalents, while for metal chelating assay, EDTA was used as
reference standard.
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evaluated utilizing previously reported standard methods as reported previously
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[8]. Eserine was used as control for AChE and BChE, acarbose for α-
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glucosidase and kojic acid for urease. Inhibition percentage of the plant extracts
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at different concentrations was calculated as:
Control−Test U
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Inhibition (%) = ×100
Control
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The cytotoxicity was tested using MTT assay as reported earlier [8] against five
cancer), CaSki (cervical cancer), DU-145 (prostate cancer), and SW-480 (colon
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formula:
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means and these values are reported as the mean ± standard deviation (SD).
Moreover, the results were analysed via One way analysis of variance
(ANOVA) followed by Tukey’s test for the post hoc treatment using Statistical
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Package for Social Science (SPSS 24.0 for windows). The IC50 values were
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aerial parts, and the percentage extraction yield was also calculated. Both the
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extracts were screened for total bioactive composition to determine their total
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phenolic and flavonoid contents and the values are presented in Table 1. Total
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phenolic contents of methanol extract was significantly higher than DCM.
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Similarly, the highest flavonoid contents were obtained also from the methanol
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extract.
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using UHPLC-MS in negative ionization mode and its total ion chromatogram
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Two terpenoids (oleoside dimethyl ester and goyaglycoside h) and one alkaloid
(calystegin B2) were also identified. Overall, the B. glabra methanol extract
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was observed to have more flavonoids and this greater amount of flavonoid
as presented in Table 1.
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The free radical scavenging activity of B. glabra extracts were determined
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using DPPH and ABTS assays and the results are assembled in Table 3. The
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DPPH assay demonstrated that, BM exhibited the highest activity with IC50
excellent electron donors [10], they might be accountable for the observed
molybdenum (V) complex (green in color) [11] and the results are depicted in
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Table 3. The DCM extract (40.26 mg GAE/g extract) exhibited the higher total
extract). This assay also known as total antioxidant capacity assay, measures the
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the plant extracts. The present findings for the DCM extract by this method
researches [12] which had presented the DCM extracts to have higher total
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antioxidant capacities. Moreover, our results are further supported by some
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other reports presenting a weak correlation between phosphomolybdenum assay
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and total bioactive contents [8].
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3.2.2. Reducing power and metal chelating activity
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Ferric reducing antioxidant power (FRAP) and cupric reducing
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antioxidant capacity (CUPRAC) methods were utilized to measure the reduction
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capability of the studied plant samples and the results are shown in Table 3.
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Both assays measure the plant extracts potential for reducing ferric to ferrous
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and cupric to cuprous ions, respectively [13]. The methanol extract (FRAP:
scavenging results, phenolic and flavonoid rich BM extract was the most active
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ability to quench free radicals and reactive oxygen species [14]. Some
different plant extracts [15]. Moreover, the ferrous ion chelating capacity of
both extracts were determined by metal chelating ferrozine assay (Table 4).
be active for ferrous chelating activity with value of 20.48 mg EDTA/g extract,
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3.3. Enzyme inhibition activities
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There is an extravagant progress in the prevalence of Alzheimer's disease
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and diabetes mellitus and accordingly these are the burning challenges for
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public health. Likewise, according to recent reports, Alzheimer's disease had
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affected more than 50 million populations, and until 2050, this statistics is
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presumed to be increased by about three times more [16, 17]. Similarly, urease
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is one of the major responsible enzyme for killing Helicobacter pylori which
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exist in stomach and is the main cause for many gastrointestinal diseases, e.g.,
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gastric cancer, peptic, duodenal and gastritis ulcer, amongst others [18]. So,
problems have esteemed attentions [19]. Amid of the most efficacious options
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to overcome these disorders, the theory to inhibit the key enzymes involved in
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these pathologies is the one, well recognized approach [20]. Taking into
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glabra aerial extracts were tested against AChE, BChE, α-glucosidase and
urease, and the findings of these assays are illustrated in Table 4. Moreover,
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Both tested extracts showed least activity against AChE with IC50 of
recorded for BM with IC50 value of 0.28 mg/mL (Table 4). According to the
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photometric and chromatographic analyses, methanol extract was found to have
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higher phenolic and flavonoids. Therefore, it can be argued that these bioactive
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molecules may justify the observed BChE inhibitory activity. Indeed, previous
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studies have reported that phytochemicals, particularly phenolic and flavonoids,
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may exert powerful effects on cognitive functions [21]. Our results are also
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consistent with the findings of some previous reports [22], which also presented
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potential having IC50 value of 0.042 µg/mL. On the other hand, BM was least
active (Table 4). This higher α-glucosidase activity of DCM extract might be
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[23].
Table 4 indicate that BM (IC50; 0.24 mg/mL) extract displayed the higher anti-
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amounts of flavonoids in this extract as previously, Awllia et al. [24] also
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reported the activity of flavonoids as natural inhibitors of urease enzyme. This
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work is the foremost to investigate on such key enzyme inhibition potentials of
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aerial parts of B. glabra. Taken together, the findings from the current study can
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be a stimulus to develop natural enzyme inhibitors from this plant species and
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could open new horizons to design novel pharmaceuticals.
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3.4. Cytotoxicity
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glabra aerial parts were assessed against five different human cancer cell lines
(prostate cancer), and SW-480 (colon cancer). The percentage cell viability at
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Moreover, the IC50 values were also determined and expressed as mean of
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Both the extracts exhibited considerable toxicity against all cell lines with
IC50 values ranging from 22.09 to 257.2 µg/mL. BD was most active against
MCF-7 (IC50; 22.09 µg/mL) and CaSki (IC50; 91.88 µg/mL), whereas, BM
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extract showed high anti-proliferative activity for SW-480 (IC50; 65.59 µg/mL)
and MCF-7 (IC50; 109.1 µg/mL) cell lines. Previously, the ethanol extract of B.
glabra leaves has been reported for cytotoxic effects in HT-29, AGS, and BL-13
glabra stems and leaves reported the anti-proliferative activity against U373
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cells [26]. Similarly, Joshny (2012) reported the anticancer activity of hydro-
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alcoholic extract of B. glabra on Hela cells with IC50 value of 47.11 ug/mL [27].
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A recent study reported the isolated flavones from stem bark of B. spectabilis to
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show cytotoxicity against five cancer cell lines (KB, HeLa S-3, MCF-7, HT-29,
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and HepG2) [28]. The observed cytotoxic activity of B. glabra extracts might be
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linked to the flavonoids and terpenoids as identified by the UHPLC-MS
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analysis (Table 2), as these compounds classes are already reported for
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4. Conclusion
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antioxidant and enzyme inhibitory potential. The DCM extract showed potent
cervical, prostate and colon cancers. From the UHPLC-MS analysis, we found
conclude, this plant contain bioactive antioxidant and enzyme inhibitors which
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could be further utilized in drug designing, cosmetic applications and as food
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supplements. Nevertheless, isolation of potential bioactive molecules from this
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plants and their in vivo studies are required.
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Conflict of Interest Statement U
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No conflict of interest.
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Figure 1. Total ion chromatograms (TIC) of B. glabra aerial methanol extract.
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different superscript letters in the same column are significantly (p < 0.05)
different. GAE: gallic acid equivalent; QE: quercetin equivalent; BM: B. glabra
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aerial methanol extract, BD: B. glabra aerial DCM extract
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Table 2. UHPLC-MS analysis of B. glabra aerial methanol extract (negative ionization mode).
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Rt B. peak Compound Mol. Mol. Diff DB
S. No Proposed compounds
(min) (m/z) class formula mass (ppm)
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C7 H13 N 0.13
1 1.109 174.07 Calystegin B2 Alkaloid 175.08
A
O4
2 3.839 417.14 3,4-Dihydroxybenzoic acid Phenol C7 H6 O4 154.02 -3.14
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C18 H26 -2.35
3 7.511 417.14 Oleoside dimethyl ester Terpene 418.14
O11
ED C27 H32 -1.49
4 7.841 595.16 Neoeriocitrin Flavonoid 596.17
O15
C33 H40 0.48
5 8.389 755.20 Kaempferol 3-(2G-glucosylrutinoside) Flavonoid 756.21
O20
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O16
C28 H32 -4.81
10 8.689 639.15 Isorhamnetin 3-glucosyl-(1->6)-galactoside Flavonoid 640.16
O17
Kaempferol 3-neohesperidoside-7-(2''- C43 H48 -2.42
11 9.752 931.253 Flavonoid 932.26
ferulylglucoside) O23
C37 H38 -2.68
12 9.808 801.18 Quercetin 3-(2'''-feruloylsophoroside) Flavonoid 802.19
O20
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24
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Isovitexin 2''-O-(6'''-(E)-p-coumaroyl)glucoside 4'-O- C42 H46 -4.43
13 9.901 901.24 Flavonoid 902.25
glucoside O22
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Kaempferol 3-[2Gal-(6'''-feruloylglucosyl)- C43 H48 -2.96
14 9.904 931.25 Flavonoid 932.26
robinobioside] O23
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15 9.96 961.26 p-Salicylic acid Phenol C7 H6 O3 138.03 -4.43
C44 H50 -2.95
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16 9.99 901.24 Isovitexin 7-(6'''-sinapoylglucoside) 4'-glucoside Flavonoid 962.27
O24
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Kaempferol 3-rhamnoside-7-[6'''-ferulyglucosyl-(1- C43 H48 -3.75
17 10.10 915.25 Flavonoid 916.26
>3)-rhamnoside] O22
EDKaempferol 3-(6''-(E)-feruloylglucosyl)-(1->2)- C37 H38 -4.36
18 10.19 785.19 Flavonoid 786.20
galactoside O19
C42 H70 -1.73
19 11.47 813.46 Goyaglycoside h Terpenoid 814.47
O15
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Table 3. Antioxidant activities of B. glabra aerial extracts
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Total antioxidant Ferrous
Radical scavenging activity Reducing power
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capacity chelating
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Plant Code
ABTS CUPRAC Metal
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DPPH IC50 FRAP Phosphomolybdenum
(mgTE/g (mgTE/g chelating
(mg/mL) (mg GAE/g) (mg GAE/g)
ED extract) extract) (mgEDTA/g)
Ascorbic
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0.016±069 c nd nd nd nd nd
acid
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Data from three repetitions, with mean ± standard deviation; means with different superscript letters in the same column are
significantly (p < 0.05) different. FRAP: ferric reducing anti-oxidant power; CUPRAC: cupric reducing anti-oxidant power;
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IC50 (mg/mL)
Plant
Code AChE BChE α-glucosidase Urease
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Acarbose nd nd 37.45±0.16µM nd
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Kojic acid nd nd nd 21.25±0.15µM
Values are expressed as means ± S.D. of three replicates, nd: not determined,
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**The IC50 value was higher than 0.5 mg/mL. AChE: acetylcholinesterase;
BChE: butyrylcholinesterase
Eserine was control for AChE and BChE, Acarbose for α-glucosidase and
Kojic acid for urease; nd: not determined U
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Table 5. Cytotoxicity (IC50; µg/mL) of B. glabra aerial extracts.
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BM BD
IC50 value represents concentration that reduces cell viability to 50%. **The
IC50 value was higher than 500 µg/mL