Accepted Manuscript

Title: Multidirectional insights into the biochemical and

toxicological properties of Bougainvillea glabra (Choisy.)
aerial parts: A functional approach for bioactive compounds

Authors: Hammad Saleem, Gokhan Zengin, Irshad Ahmad,

Joash Tan Ban Lee, Thet Thet Htar, Fawzi M. Mahomoodally,
Rakesh Naidu, Nafees Ahemad

PII: S0731-7085(19)30088-3
DOI: https://doi.org/10.1016/j.jpba.2019.03.027
Reference: PBA 12540

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 11 January 2019

Revised date: 12 March 2019
Accepted date: 13 March 2019

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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 1

Multidirectional insights into the biochemical and toxicological properties

of Bougainvillea glabra (Choisy.) aerial parts: A functional approach for

bioactive compounds

Hammad Saleema,b, Gokhan Zenginc, Irshad Ahmadd, Joash Tan Ban Leee, Thet

T
Thet Htara, Fawzi M. Mahomoodallyf, Rakesh Naidug, Nafees Ahemada,h,i*

R IP
a
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan,

SC
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,
A
Turkey
M

d
Department of Pharmacy, The Islamia University of Bahawalpur, Pakistan
e
School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 47500
ED

Bandar Sunway Selangor Darul Ehsan, Malaysia

f
Department of Health Sciences, Faculty of Science, University of Mauritius,
PT

Mauritius
g
Jeffrey Cheah School of Medicine and Health Sciences, Monash University
E

Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan,
CC

Malaysia.
h
Tropical Medicine and Biology Multidisciplinary Platform, Monash University
A

Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor, Darul

Ehsan, Malaysia
I
Global Asia in The 21st Century (GA21) Multidisciplinary Research Platform,
Monash University Malaysia
 2

*
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.


T
Flavonoids were the main components identified in the methanol extract.
 B. glabra could be further studied to isolate novel bioactive compounds

R IP
Abstract
The current research work was conducted in order to probe into the biochemical

SC
and toxicological characterisation of methanol and dichloromethane (DCM)

U
extracts of Bougainvillea glabra (Choisy.) aerial parts. Biological fingerprints
N
were assessed for in vitro antioxidant, key enzyme inhibitory and cytotoxicity
A

potential. Total bioactive contents were determined spectrophotometrically and

M

the secondary metabolite components of methanol extract was assessed by

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UHPLC mass spectrometric analysis. The antioxidant capabilities were

evaluated via six different in vitro antioxidant assays namely DPPH, ABTS
PT

(free radical scavenging), FRAP, CUPRAC (reducing antioxidant power),

E

phosphomolybdenum (total antioxidant capacity) and ferrous chelating activity.

CC

Inhibition potential against key enzymes urease, α-glucosidase and

A

cholinesterases were also determined. Methanol extract exhibited higher

phenolic (24.01 mg GAE/g extract) as well as flavonoid (41.51 mg GAE/g

extract) contents. Phytochemical profiling of methanol extract identified a total

of twenty secondary metabolites and the major compounds belonged to

 3

flavonoids, phenolics and alkaloid derivatives. The findings of antioxidant

assays revealed the methanol extract to exhibit stronger antioxidant (except

phosphomolybdenum) activities. Similarly, the methanol extract showed highest

butyrylcholinesterase and urease inhibition. The DCM extract was most active

for phosphomolybdenum and α-glucosidase inhibition assays. Moreover, both

T
extracts exhibited significant cytotoxic potential against five (MCF-7, MDA-

IP
MB-231, CaSki, DU-145, and SW-480) human carcinoma cell lines with half

R
maximal inhibitory concentration values of 22.09 to 257.2 µg/mL. Results from

SC
the present study highlighted the potential of B. glabra aerial extracts to be

U
further explored in an endeavour to discover novel phytotherapeutics as well as
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functional ingredients.
A
M

Keywords: Carcinoma cell lines; antioxidant; cytotoxicity; enzyme inhibitor;

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bioactive compounds
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1. Introduction
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Cancer, cardiovascular diseases, chronic respiratory disorders and

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diabetes, categorized as noncontagious diseases (NCD’s), are the primary cause

A

of deaths globally. These disorders are accountable for approximately 40

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

an imbalance between excessive free radical production and the antioxidant

 4

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

antioxidant defence system or by externally in the form of food. These free

radicals can be either oxygen derived or nitrogen derived molecules [2].

Currently, knowledge of ancient herbal medications and utilization of recent

T
scientific techniques have furnished impressive protocols for the isolation,

IP
purification and characterization of different bioactive compounds. This in turn,

R
has provided key insights into their mechanism of action and therapeutics

SC
effects for humans [3]. The major focus of current research is more targeted on

U
the biological activities of bioactive secondary metabolites from natural
N
products and their health-related outcomes. Phenolic and flavonoids amongst
A

the various classes of secondary metabolites, are considered as the major

M

compounds responsible for exhibiting various biological activities [4]. Keeping

ED

this in view, the above mentioned parameters, natural products or bioactive

phytochemicals are regarded as prospective materials.

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Bougainvillea glabra (Nyctaginaceae), commonly known as “Glory of

E

the Garden”, is native to Southern America and has been used traditionally for
CC

various medicinal purposes such as insecticidal, anti-inflammatory [5], anti-

A

diarrhoeal, anti-ulcer, anti-microbial [6] and anti-hyperglycaemic agent [7]. In

spite of the considerable traditional importance, there have been only limited

attempts to explore the chemical and pharmacological properties of this species

in relation with its medicinal uses. Thus, we aimed to evaluate, in this paper, B.
 5

glabra aerial parts for their chemical composition (total phenolic and flavonoid

contents and UHPLC-MS secondary metabolites profile), antioxidant

capabilities (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum and metal

chelation) and key enzyme inhibition potential against urease, α-glucosidase,

acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Moreover,

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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

2.1. Plant materials and extraction U

N
Aerial parts of B. glabra plant were collected from district, Muzaffar
A

Garh (Punjab), Pakistan and identified by Dr. Abdul Munsif, Department of

M

Botany, S.E. College, Bahawalpur. Furthermore, a voucher representative

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number (BG-AP-01-16-111) was also deposited in the herbarium of Department

of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur,

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Pakistan.
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After shade drying and grinding of the aerial parts of plant, its powder
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was extracted by maceration (72 hrs) successively with dichloromethane

A

(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

methanol extract) and BD (B. glabra aerial DCM extract).

2.2. Total bioactive contents and secondary metabolite profiling

 6

Total phenolic contents were determined using well-known standard

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

assessed utilizing standard aluminium chloride calorimetric method [8]. Total

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flavonoids were noted as mg QE/g extract (milligrams of quercetin equivalents)

IP
using quercetin as standard. Similarly, the phytochemical composition of the

R
methanol extract was evaluated by UHPLC Accurate-Mass Q-TOF (Agilent

SC
1290 Infinity LC system coupled to Agilent 6520) mass spectrometer with dual

ESI source as described previously [8]. U

N
2.3. Antioxidant assays
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1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay was performed as

M

described previously [8]. ABTS (2, 2-azino-bis (3-ethylbenzothiazoline-6-

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sulphonic acid) radical cation scavenging activity, CUPRAC (Cupric ion

reducing antioxidant capacity) and metal chelating activity were determined

PT

according to previous standard methods [9]. The FRAP (ferric reducing

E

antioxidant power) assay was performed according to standard method as

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reported earlier [8]. Total antioxidant capacity (TAC) of extracts was evaluated
A

by phosphomolybdenum method according to Prieto et al [8]. FRAP and

phosphomolybdenum activities were expressed as gallic acid equivalent (mg

GAE/g extract). The results of ABTS and CUPRAC assays were expressed as
 7

trolox equivalents, while for metal chelating assay, EDTA was used as

reference standard.

2.4. Enzyme inhibition assays

Cholinesterases (AChE: acetylcholinesterase and BChE:

butyrylcholinesterase), α-glucosidase and urease inhibitory potential was

T
evaluated utilizing previously reported standard methods as reported previously

IP
[8]. Eserine was used as control for AChE and BChE, acarbose for α-

R
glucosidase and kojic acid for urease. Inhibition percentage of the plant extracts

SC
at different concentrations was calculated as:

Control−Test U
N
Inhibition (%) = ×100
Control
A

2.5 Cytotoxicity assay

M

The cytotoxicity was tested using MTT assay as reported earlier [8] against five

different human carcinoma cell lines i.e., MDA-MB-231, MCF-7 (breast

ED

cancer), CaSki (cervical cancer), DU-145 (prostate cancer), and SW-480 (colon
PT

cancer). The percentage cell viability (%) was determined by following

formula:
E
CC

Cell viability (%) = Abss – Absc × 100

2.6. Statistical analysis

A

All the assays were performed in triplicates in order to determine the

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
 8

Package for Social Science (SPSS 24.0 for windows). The IC50 values were

determined utilizing Graph Pad Prism software.

3. Results and discussion

3.1. Phytochemical composition
Methanol and DCM solvents were used for the extraction of B. glabra

T
aerial parts, and the percentage extraction yield was also calculated. Both the

IP
extracts were screened for total bioactive composition to determine their total

R
phenolic and flavonoid contents and the values are presented in Table 1. Total

SC
phenolic contents of methanol extract was significantly higher than DCM.

U
Similarly, the highest flavonoid contents were obtained also from the methanol
N
extract.
A

Secondary metabolites profiling of the methanol extract was determined

M

using UHPLC-MS in negative ionization mode and its total ion chromatogram
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(TIC) is shown in Figure 1. The base peak analysis of the UHPLC–MS

chromatogram identified 20 different secondary metabolite compounds and are

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presented in Table 2. Most of these compounds were flavonoids derivatives

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including neoeriocitri, robinin, isorhamnetin 3-glucosyl-(1->6)-galactoside,

CC

kaempferol 3-neohesperidoside-7-(2''-ferulylglucoside), quercetin 3-(2'''-

A

feruloylsophoroside) and isovitexin 7-(6'''-sinapoylglucoside) 4'-glucoside.

Polyphenolics present were 3, 4-dihydroxybenzoic acid and p-salicylic acid.

Two terpenoids (oleoside dimethyl ester and goyaglycoside h) and one alkaloid

(calystegin B2) were also identified. Overall, the B. glabra methanol extract
 9

was observed to have more flavonoids and this greater amount of flavonoid

compounds in this extract is in accordance to its higher total flavonoid contents

as presented in Table 1.

3.2. Antioxidant properties

3.2.1. Radical scavenging and total antioxidant capacity

T
IP
The free radical scavenging activity of B. glabra extracts were determined

R
using DPPH and ABTS assays and the results are assembled in Table 3. The

SC
DPPH assay demonstrated that, BM exhibited the highest activity with IC50

values of 0.15 mg/mL. Similarly, in the ABTS assay, BM (111.29 mg TE/g

U
N
extract) had the highest radical scavenging activity. It is noted that, the total
A
bioactive contents of the tested extracts followed the same pattern as the
M

radicals scavenging capacities. As phenolic and flavonoid compounds are

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excellent electron donors [10], they might be accountable for the observed

DPPH and ABTS scavenging potential.

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Total antioxidant activity was determined via the phosphomolybenum

E

assay based on reduction of molybdenum (VI) to molybdenum (V) by the

CC

compounds having antioxidant capacity and the resultant formation of

molybdenum (V) complex (green in color) [11] and the results are depicted in
A

Table 3. The DCM extract (40.26 mg GAE/g extract) exhibited the higher total

antioxidant potential compared to methanol extract (29.14 mg GAE/g

extract). This assay also known as total antioxidant capacity assay, measures the
 10

antioxidant potential of both phenolic and non-phenolic compounds present in

the plant extracts. The present findings for the DCM extract by this method

might be correlated to the existence of some non-phenolic compounds, as

vitamin C or tocopherol as examples. This is also in line with the previous

researches [12] which had presented the DCM extracts to have higher total

T
antioxidant capacities. Moreover, our results are further supported by some

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other reports presenting a weak correlation between phosphomolybdenum assay

R
and total bioactive contents [8].

SC
3.2.2. Reducing power and metal chelating activity

U
Ferric reducing antioxidant power (FRAP) and cupric reducing
N
antioxidant capacity (CUPRAC) methods were utilized to measure the reduction
A

capability of the studied plant samples and the results are shown in Table 3.
M

Both assays measure the plant extracts potential for reducing ferric to ferrous
ED

and cupric to cuprous ions, respectively [13]. The methanol extract (FRAP:

52.13 mg GAE/g extract and CUPRAC: 130.41 mg TE/g extract) exhibited

PT

higher reducing power capacity as compared to DCM extract. Similar to radical

E

scavenging results, phenolic and flavonoid rich BM extract was the most active
CC

compounds in both reducing power assays. Phenolic and flavonoids are

A

regarded to be the most active anti-oxidative plant components because of their

ability to quench free radicals and reactive oxygen species [14]. Some

researchers have already reported a strong positive association between radical

scavenging potential, reducing capacities and total bioactive contents of

 11

different plant extracts [15]. Moreover, the ferrous ion chelating capacity of

both extracts were determined by metal chelating ferrozine assay (Table 4).

Interestingly, as observed in other antioxidant assay results, BM was found to

be active for ferrous chelating activity with value of 20.48 mg EDTA/g extract,

while the BD extract was inactive.

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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

R
and diabetes mellitus and accordingly these are the burning challenges for

SC
public health. Likewise, according to recent reports, Alzheimer's disease had

U
affected more than 50 million populations, and until 2050, this statistics is
N
presumed to be increased by about three times more [16, 17]. Similarly, urease
A

is one of the major responsible enzyme for killing Helicobacter pylori which
M

exist in stomach and is the main cause for many gastrointestinal diseases, e.g.,
ED

gastric cancer, peptic, duodenal and gastritis ulcer, amongst others [18]. So,

accordingly, new effective treatment strategies to manage these important health

PT

problems have esteemed attentions [19]. Amid of the most efficacious options
E

to overcome these disorders, the theory to inhibit the key enzymes involved in
CC

these pathologies is the one, well recognized approach [20]. Taking into
A

consideration the above stated parameters, the enzyme inhibitory ability of B.

glabra aerial extracts were tested against AChE, BChE, α-glucosidase and

urease, and the findings of these assays are illustrated in Table 4. Moreover,
 12

comparison of percentage enzyme inhibition of both extracts as compared to

standard drugs is presented in Figure 2.

Both tested extracts showed least activity against AChE with IC50 of

above 5 mg/mL. Although, a considerable BChE inhibition activity was

recorded for BM with IC50 value of 0.28 mg/mL (Table 4). According to the

T
photometric and chromatographic analyses, methanol extract was found to have

IP
higher phenolic and flavonoids. Therefore, it can be argued that these bioactive

R
molecules may justify the observed BChE inhibitory activity. Indeed, previous

SC
studies have reported that phytochemicals, particularly phenolic and flavonoids,

U
may exert powerful effects on cognitive functions [21]. Our results are also
N
consistent with the findings of some previous reports [22], which also presented
A

a linear association among bioactive contents and cholinesterases inhibition.

M

For α-glucosidase inhibition, BD presented the strongest inhibition

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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
PT

due to its significant phosphomolybdenum activity and the existence of some

E

non-phenolic compounds like ascorbic acid or tocopherols. Similarly, the

CC

observed α-glucosidase inhibition of B. glabra extracts can be correlated to its

A

higher flavonoid contents as previously, it has been reported that disaccharides

are targets of flavonoids in the regulation of glucose absorption and

consequently glucose homeostasis and some flavonoids, such as luteolin and

 13

kampferol have previously been reported for α-glucosidase inhibitory potentials

[23].

The urease percentage inhibition of the studied extracts as shown in

Table 4 indicate that BM (IC50; 0.24 mg/mL) extract displayed the higher anti-

urease activity. This observed anti-urease activity might be correlated to higher

T
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

R
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

U
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.
A

3.4. Cytotoxicity
M

In the present work, the cytotoxicity of methanol and DCM extracts of B.

ED

glabra aerial parts were assessed against five different human cancer cell lines

i.e., MCF-7, MDA-MB-231(breast cancer), CaSki (cervix cancer), DU-145

PT

(prostate cancer), and SW-480 (colon cancer). The percentage cell viability at
E

the concentrations ranging from 500-15.625 µg/mL is depicted in Figure 3.

CC

Moreover, the IC50 values were also determined and expressed as mean of
A

triplicates as given in Table 5.

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
 14

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

cell lines [25]. Another study conducted on different extracts from B.

glabra stems and leaves reported the anti-proliferative activity against U373

T
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].

R
A recent study reported the isolated flavones from stem bark of B. spectabilis to

SC
show cytotoxicity against five cancer cell lines (KB, HeLa S-3, MCF-7, HT-29,

U
and HepG2) [28]. The observed cytotoxic activity of B. glabra extracts might be
N
linked to the flavonoids and terpenoids as identified by the UHPLC-MS
A

analysis (Table 2), as these compounds classes are already reported for
M

anticancer and apoptosis inducing capabilities [29, 30]. As to earlier reports

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comparison, B. glabra revealed strong to moderate toxicity potential and as far

as literature search, this is the foremost outline regarding cytotoxicity of B.

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glabra aerial extracts against these cell lines.

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4. Conclusion
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In the present research, we have presented the biochemical profiles and

A

cell-toxicity potentials of B. glabra aerial parts. Our findings demonstrated the

methanol extract to be rich in bioactive compounds and have considerable

antioxidant and enzyme inhibitory potential. The DCM extract showed potent

activity for phosphomolybdenum and α-glucosidase inhibition assays.

 15

Moreover, both extracts showed varying cytotoxic potential against breast,

cervical, prostate and colon cancers. From the UHPLC-MS analysis, we found

that B. glabra contained maximum numbers of flavonoids compounds. These

secondary metabolites may justify the observed biological potentials. To

conclude, this plant contain bioactive antioxidant and enzyme inhibitors which

T
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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M
ED
E PT
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A
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ED
E PT
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A
 20

T
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Figure 1. Total ion chromatograms (TIC) of B. glabra aerial methanol extract.

R
SC
U
N
A
M
ED
PT

Figure 2. Comparison of percentage enzyme inhibition (0.5 mg/mL) of B.

glabra aerial extracts. Eserine was control for AChE and BChE, Acarbose for α-
glucosidase and Kojic acid for urease.
E
CC
A
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T
R IP
SC
U
N
A
M
ED
E PT
CC
A

Figure 3. Cytotoxicity of methanol and DCM extracts of B. glabra aerial

extracts.
**** indicates significant difference when compared with untreated (control)
cells (p value < 0.05)
 22

Tables and Figures.

Table 1. Extraction yield and total bioactive contents of B. glabra aerial extracts

Total phenolic Total flavonoid

Plant Code Yield (%) content (mg content (mg
GAE/g) QE/g)
a
BM 6.4 24.01±2.09 41.51±0.80 a
BD 2.8 15.49±2.26 b 18.68±0.74 b
Data from three repetitions, with mean ± standard deviation; means with

T
different superscript letters in the same column are significantly (p < 0.05)
different. GAE: gallic acid equivalent; QE: quercetin equivalent; BM: B. glabra

IP
aerial methanol extract, BD: B. glabra aerial DCM extract

R
SC
U
N
A
M
ED
E PT
CC
A
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23

SC
Table 2. UHPLC-MS analysis of B. glabra aerial methanol extract (negative ionization mode).

U
Rt B. peak Compound Mol. Mol. Diff DB
S. No Proposed compounds
(min) (m/z) class formula mass (ppm)

N
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

M
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
PT

Isorhamnetin 3-glucosyl-(1->2)-[rhamnosyl-(1->6)- C34 H42 -3.57

6 8.429 785.21 Flavonoid 786.22
galactoside] O21
C33 H40 0.89
E

7 8.614 739.20 Robinin Flavonoid 740.21

O19
CC

Kaempferol 4'-methyl ether 3-(2Glc- C34 H42 0.29

8 8.655 769.219 Flavonoid 770.22
glucosylrutinoside) O20
C27 H30 -2.06
9 8.655 609.14 Robinetin 3-rutinoside Flavonoid 610.15
A

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

SC
Isovitexin 2''-O-(6'''-(E)-p-coumaroyl)glucoside 4'-O- C42 H46 -4.43
13 9.901 901.24 Flavonoid 902.25
glucoside O22

U
Kaempferol 3-[2Gal-(6'''-feruloylglucosyl)- C43 H48 -2.96
14 9.904 931.25 Flavonoid 932.26
robinobioside] O23

N
15 9.96 961.26 p-Salicylic acid Phenol C7 H6 O3 138.03 -4.43
C44 H50 -2.95

A
16 9.99 901.24 Isovitexin 7-(6'''-sinapoylglucoside) 4'-glucoside Flavonoid 962.27
O24

M
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
PT

C38 H60 0.24

20 13.48 723.39 Alliospiroside C Steroid 724.40
O13
Rt: retention time; B. peak: base peak
E
CC
A
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25

SC
Table 3. Antioxidant activities of B. glabra aerial extracts

U
Total antioxidant Ferrous
Radical scavenging activity Reducing power

N
capacity chelating

A
Plant Code
ABTS CUPRAC Metal

M
DPPH IC50 FRAP Phosphomolybdenum
(mgTE/g (mgTE/g chelating
(mg/mL) (mg GAE/g) (mg GAE/g)
ED extract) extract) (mgEDTA/g)

BM 0.15±4.74 b 111.29±1.39 a 52.13±0.71 a 130.41±3.63 a 29.14±1.13 b 20.48±2.01 a

PT

BD 0.43±2.15 a 26.12±3.32 b 15.82±0.10 b 75.98±2.66 b 40.26±0.35 a na

Ascorbic
E

0.016±069 c nd nd nd nd nd
acid
CC

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;
A

nd: not determined; na: not active

 26

Table 4. Enzyme inhibition potential (IC50; mg/mL) of B. glabra aerial extracts

IC50 (mg/mL)
Plant
Code AChE BChE α-glucosidase Urease

BM >0.5** 0.28±0.36 >0.5** 0.24±0.26

BD >0.5** >0.5** 0.042±0.51 >0.5**

Eserine 0.04±0.01µM 0.85±0.01µM nd nd

T
IP
Acarbose nd nd 37.45±0.16µM nd

R
Kojic acid nd nd nd 21.25±0.15µM
Values are expressed as means ± S.D. of three replicates, nd: not determined,

SC
**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
N
A
Table 5. Cytotoxicity (IC50; µg/mL) of B. glabra aerial extracts.
M

Cell lines IC50 (µg/mL)

ED

BM BD

MCF-7 109.1 22.09

PT

MDA-MB-231 257.2 127.8

CaSki 176.7 91.88

E
CC

DU-145 >500** 195.3

SW-480 65.59 214.7

A

IC50 value represents concentration that reduces cell viability to 50%. **The
IC50 value was higher than 500 µg/mL