Saudi Journal of Biological Sciences 30 (2023) 103609

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Saudi Journal of Biological Sciences

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Original article

Antioxidant and antiproliferative activity of Basella alba against

colorectal cancer
Aliya Sheik a, Eunsu Kim a, Uma Adepelly b, Munirah Alhammadi a, Yun Suk Huh a,⇑
a
NanoBio High-Tech Materials Research Center, Biological Sciences and Bioengineering, Inha University, 100 Inha-ro, Incheon 22212, Republic of Korea
b
Centre for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India

a r t i c l e i n f o a b s t r a c t

Article history: Basella alba, a green leafy vegetable with remarkable nutraceutical potential is widely used since ancient
Received 23 October 2022 times to maintain a healthy colon. This plant has been investigated for its medicinal potential due to the
Revised 4 February 2023 increase in young adult cases of colorectal cancer each year. This study was accomplished to
Accepted 23 February 2023
investigate Basella alba methanolic extract (BaME) antioxidant and anticancer properties. BaME consisted
Available online 2 March 2023
of a substantial amount of both phenolic and flavonoid compounds which exhibited significant antioxi-
dant reactivity. Both colon cancer cell lines experienced a cell cycle arrest at the G0/G1 phase after receiv-
Keywords:
ing treatment with BaME, which inhibited pRb and cyclin D1 and raised p21 expression levels. This was
Antioxidant
Antiproliferative
associated with the survival pathway molecule inhibition and downregulation of E2F-1. The results of the
Basella alba current investigation confirm that BaME inhibits CRC cell survival and expansion. To conclude, the bioac-
Colorectal cancer cell lines tive principles in the extract act as potential antioxidants and antiproliferative agents against colorectal
E2F-1 cancer.
Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction women respectively (Sung et al., 2021). Colorectal cancer is consid-

ered to be a young person’s disease as every year 20% of people
Cancer has become the deadliest disease growing beyond between the age group 20–54 are diagnosed with this cancer
boundaries reckoning the death toll to nearly 10 million in 2020. (Deen et al., 2016). This alarming data of incidence (1.93 million
The Global Cancer Incidence report of a cancer agency (Interna- cases), low survival chance, and death rates of colon cancer cases
tional Agency for cancer research) of the world health organization have raised high concern over the efficiency of radio or chemother-
(WHO) has designated colon cancer to be leading cause of mortal- apy. The chemotherapeutic drugs utilized currently for cancer
ity in the world (935000 deaths in 2020). Globally colon cancer is treatment initiate several side effects and could lead to fatal condi-
reported to be about 10.6% and 9.4% of all cancers type in men and tions since they are not targeted drugs, as they fail to differentiate
healthy cells and cancer cells(Sung et al., 2021). People worldwide
have started adopting a traditional approach to medicine, an alter-
Abbreviations: BaME, Basella alba methanolic extract; CRC, colorectal cancer; native for successful management of various diseases including
ROS, Reactive oxygen species; WHO, world health organization; TPC, total phenol main cancer. A survey has reported that nearly 31% of the world
content; GC–MS, Gas chromatography - Mass spectrophotometer; QE, quercetin
Equivalents; DPPH, 2,20 - diphenyl-1-picrylhydrazyl; qRT-PCR, Quantitative real
population’s cancer patients have preferred traditional medicines
time-polymerase chain reaction; PMS-NADH, phenazine methosulfate - nicoti- before choosing conventional medicine (Kumar et al., 2016,
namide adenine dinucleotide; NBT, Nitroblue tetrazolium salt. Sharma et al., 2022). Therefore, the investigation of plant agents
⇑ Corresponding author. that have natural therapeutic characteristics and a plant extract
E-mail addresses: aliya@inha.ac.kr (A. Sheik), esk22212103@inha.edu (E. Kim), that shows cell cycle modulating properties for cancer patients is
vedavathi1@jntuh.ac.in (U. Adepelly), munirah@inha.edu (M. Alhammadi), yunsuk.
huh@inha.ac.kr (Y.S. Huh).
the high priority of the hour (See Scheme 1).
Peer review under responsibility of King Saud University. Reactive oxygen species (ROS) are a group of different types of
free radicals (superoxide anionic radicals, singlet oxygen,
hydroxyl-radicals) and non-free-radical (hydrogen peroxide) spe-
cies generated as byproducts of oxidation reactions. It is known
Production and hosting by Elsevier to exert both beneficial (at low concentration) as well as deleteri-

https://doi.org/10.1016/j.sjbs.2023.103609
1319-562X/Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

Scheme 1. A diagrammatic representation of general strategies applied for assaying methanolic extract from Basella alba for their anticancer activity.

ous (at high concentration) actions (Li et al., 2016). The cell has its 2.2. GC–MS spectral assessment
own antioxidant system to remove the radicals produced inter-
nally but its efficiency is altered during pathological infections. Gas chromatography-mass spectrophotometer (GC–MS) assess-
Even oxidative stress conditions cause an imbalance where excess ment was done using an Alltech EC-5 column in Agilent 6890 gas
ROS are produced which causes damage to the vital components of chromatography device.
the cells (biomolecules). This is mainly observed in degenerative
diseases such as cancer (Perillo et al., 2020). Recently, the investi- 2.3. Analysis of total phenol content (TPC) and total flavonoid content
gation of dietary natural antioxidants has been increasing to pre-
vent the overproduction of ROS. Plant sources that decrease the Gao et al., 2019 procedure was followed with minor alterations
cumulative effect of oxidative damage are the target of the to determine the total phenolic and flavonoid content in the
researchers (Santos-Sánchez et al., 2019). extract (Gao et al., 2019).
Basella alba L. which belongs to the Basellaceae family is a fast-
growing green succulent mucilage leafy vegetable with great
2.4. Analysis of in vitro antioxidant activity
medicinal potential. Well known as Malabar or Indian spinach in
India, Remayung in Malaysia or Pui shak in Bangladesh. This eth-
2.4.1. DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging
nomedicinally important plant has been used in the ancient Indian
activity
medicinal system to cure various diseases and ailments. The plant
Applying a slightly modified protocol of DPPH assay (Müller
has been reported to be rich in vitamins and minerals. It exhibits
et al., 2011), in vitro measurement of free radical scavenging activ-
androgenic potential, antiviral, antibacterial, antioxidant, antidia-
ity of the extracts’ was done. The detailed protocol is given in sup-
betic, anti-inflammatory, antidepressant, antiulcer (as it cures
plementary information.
aphtha- the mouth ulcers), wound healing, nephron- and hepato-
protective activity (Deshmukh and Gaikwad 2014). Despite having
numerous biological benefits, there is a paucity of information 2.4.2. Superoxide (SO) radical scavenging activity
about B. alba’s effect on cancer in the literature. Also, in view of A tad-modified Chun et al (2003) method (Chun et al., 2003)
the increasing use of B. alba by the traditional healers or praction- was used for the analysis of the ability of plant extracts, quercetin,
ers among different communities, who claim it to have aphro- and BHT to quench the generation of superoxide radicals.
disiac, virilizing or anabolizing potentials and prospective to
prevents catarrh and various diseases, it is highly necessary to sci- 2.4.3. Phosphomolybdate assay
entifically investigate its biological activities especially its cyto- The little modified phosphomolybdate method (Benzie and
toxic property which is less explored. Basella alba is used in the Szeto 1999) was used to analyze the total antioxidant capacity of
present study to investigate its methanolic extract role in render- the extracts.
ing effective therapeutic properties against colorectal cancer
(CRC) cell lines HT-29 and HCT-116.
2.4.4. Hydroxyl radical scavenging (HRS) activity
Hydroxyl-radical is the most potent ROS that reacts with the
cell membrane by abstracting hydrogen atoms from phospholipids
2. Material and methods (polyunsaturated fatty acid moieties) and leads to cellular damage
by peroxidic reaction. Its scavenging activity was analyzed follow-
2.1. Basella alba extract preparation ing a standard protocol (Chimi et al., 1991).

Fresh and healthy Basella alba, commonly called Indian spinach 2.5. Analysis of anticancer activity
plant material was collected, shade dried, grounded into a coarse
powder, and sieved. Stored at 4 °C in airtight containers for further 2.5.1. Antiproliferative activity of Basella alba methanol extract
analysis. The selected plant material was weighted and soaked in HT-29 and HCT-116 are two CRC human cell lines (procured
10 L of anhydrous methanol and was left for 10 days at RT with from ATCC, Manassas, VA) that were used. Cells were cultured
intermittent stirring. The methanolic extract was filtered and con- according to standard protocol (Ganji et al., 2013). Using XTT assay
centrated at temperature 40 °C on a rotary-evaporator. The extract (ATCC, Manassas, VA) cell viability was obtained. Absorbance at
yield was 20.8%. The extract was collected and stored in brown 475 nm with reference to 660 nm was read using a microplate
bottle at 20 °C until used. reader.
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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

2.5.2. Clonogenicity assay Ciocalteu reagent). The result of TPC was calculated from the
The above-mentioned two CRC cell lines were seeded at density regression equation of the standard plot (y = 0.0108x + 0.2665,
of 100 cells/well in 6 well plates. After 12 h. treatment with vehicle r2 = 0.9948). TPC in BaME was 57.9 ± 2.66 lg gallic acid equivalent
(DMSO), BaME at specified concentrations (12.5, 25 & 50 lg/ml) (GAE)/g of extract. The regression equation of the standard plot
maintained at different times (24, 48, and 72 h) was done. The (y = 0.0009x + 0.0569, r2 = 0.9826) was used to calculate flavonoid
media was replenished every 3 days. After the 12th day, crystal content and it is expressed as quercetin equivalents (QE). Total fla-
violet was used to stain cell colonies. The number of colonies vonoid content in BaME was 118.1 ± 0.79 lg/g quercetin equiva-
was analyzed visually and the colonies with > 50 cells were only lent per extract.
counted. All experiments were performed.
3.3. Antioxidant activity assessments
2.5.3. Flow cytometry
As described above, Cells treated with vehicle and BaME were The antioxidant activity of the BaME is shown in Tables 1 and 2.
harvested after 24 h, fixed for 1 h at 4 °C in 70% ethanol. Then, The radical scavenging activity and the total antioxidant capacity is
using Propidium iodide (Sigma-Aldrich, US) the cells were stained evaluated. Since the complex makeup of the phytochemicals
for 15 min in the dark. Beckman Coulter Cytoflex flow cytometer makes it impossible to test the antioxidant properties of plant
was used for FACS analysis. Assays were performed in triplicates. extracts using just one method, four distinct approaches have been
used.
2.5.4. Western blotting
Treated CRC cell lines were harvested, processed, and lysed 3.3.1. DPPH radical scavenging activity
(protease inhibitors and RIPA buffer). Sample protein estimation DPPH assay is the widely applied method to assess the ability of
was done using BCA protein assay kit. The western blotting tech- phytoconstituents to act as free radical scavengers or hydrogen
nique was used to analyze extracted proteins (Ganji et al., 2013). donors. The concentration of plant extract needed to reduce the
The nitrocellulose membrane was used to blot the proteins that initial concentration of DPPH by 50% is known as the IC50 value
had been resolved using SDS-PAGE. Following primary antibody of the plant extract. The results in Table 1 are showing the study
incubation, secondary antibody treatment was applied to the blot- of the scavenging property of methanolic plant extract on DPPH.
ted membranes. Chemiluminescence enabled for the visualization To our knowledge, DPPH activity of methanolic extract of Basella
of the attached antibodies. As a positive control and to confirm alba have not been reported earlier. The present study showed
equal loading, b-actin housekeeping proteins were used. Flow-Jo the DPPH radical scavenging potential of BaME ranging from
software was used to analyze the results. 20.06% to 93.9% at different concentrations (20–100 lg/ml) and
ascorbic acid ranging from 28.24% to 94.81% at different concentra-
tions (20 to 100 lg/ml) showed moderate to high scavenging abil-
2.5.5. Quantitative real-time-polymerase chain reaction (qRT-PCR)
ities respectively. The extracts showed dose-dependent scavenging
Total RNA was isolated from CRC cell lines treated with BaME
of DPPH radicals.
for 24 hrs using Trizol reagent. The reaction was performed with
Applied Biosystems, USA. Using specific primers (Table S1) qRT-
3.3.2. SO anion radical scavenging assay
PCR was done by following the methodological steps. Initial denat-
The SO radical scavenging activity of BaME was evaluated using
uration (95 °C, 3 min), followed by 30 cycles of denaturation (95 °C,
ascorbic acid as control. The extracts’ scavenging of radicals was in
1 min), primer annealing (57 °C, 30 sec), synthesis (72 °C, 1 min),
a dose dependent way. The highest scavenging effect towards SO
then followed by final extension (72 °C, 7 min) step. Melting curve
was observed at concentration of 100 lg/mL (70.11 ± 0.80 lg/m
analysis was done. The relative quantities was compared to b-actin
L). We can rank the samples’ SO scavenging activity in the order
quantity (Ganji et al., 2013).
of BaME (47.2 ± 1.2 lg/mL) and ascorbic acid (55.2 ± 2.10 lg/m
All the experimental data is presented as the means standard
L). When compared to ascorbic acid, BaME has significantly higher
deviation for three replications.
IC50 values (Table 1). The highest flavonoids and polyphenols con-
tent may probably would have contributed to the greater scaveng-
3. Results ing activity of the BaME. The findings thus strengthen BaME as a
potential source of SO scavenging activity.
3.1. GC–MS spectral assessments
3.3.3. Phosphomolybdate assay (Total antioxidant capacity)
The chromatogram of GC–MS analysis of the BaME exhibited BaME examined demonstrated overall antioxidant property, as
five peaks (Figure S1). The first peak showed to be 10-[Methoxycar shown in Table 2. The decrease in phosphomolybdate antioxidant
bonyl]-N-acetylcolchinol. The second peak was determined to be activity that we observed in this investigation suggests that it
Propanoic acid, 2- [3-acetoxy-4, 4, 14-trimethylandrost-8-en-17- occurs in a dose-dependent manner. The lowest percentage reduc-
yl]-. The next peaks were Hydroymethyl colchicines, Propanoic tion was exhibited by BaME (19.97%) at 200 lg/mL and highest
acid, 2- [3-acetoxy-4, 4, 14-trimethylandrost-8-en-17-yl]-, [22S]- (53.95%) at 1 mg/mL concentration. The lowest IC50 values were
21-Acetoy-6a, 11a-dihydroxy-16a, 17a-propylmethylenedioxy exhibited by BaME (918.5 lg/mL) when compared to ascorbic acid
pregna 1, 4-diene-3, 20- dione. The five biomolecules identified (124.3 ± 1.57 lg/mL) (Table 2). Compared to the extract the potent
were tabulated (Table S2) and their structures elucidated by mass antioxidant ascorbic acid exhibited significant total antioxidant
spectrum were depicted in Figure S2 (a-f). Different phytochemi- capacity. These results demonstrated that BaME has antioxidant
cals which contribute to the medicinal and bioactivities of the activity, but very high concentration is required to exhibit its
extract are shown in Table S2. potential.

3.2. Analysis of total phenol and flavonoid content 3.3.4. HRS activity
As shown in Table 2, the hydroxyl ion scavenging by BaME
Particularly, the study is focused on the biological activities increased in a dose-dependent manner. The lowest percentage of
owing to the functions of phenolic and flavonoid compounds. The reduction was exhibited by BaME compared to the commercial
TPC of the BaME was determined using the FCR method (Folin – ascorbic acid. The IC50 values of BaME and ascorbic acid are 380.
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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

Table 1
Antioxidant activity of BaME and ascorbic acid in DPPH and Superoxide radical scavenging assay.

Antioxidant assay Sample Percentage inhibition at different concentration of sample (lg/ml) * IC50 (lg/ml)
20 40 60 80 100
DPPH assay Basella alba 20.6 ± 1.9 39.4 ± 0.0 76.8 ± 0.02 82.6 ± 0.2 93.9 ± 1.2 46.6 ± 0.0
Ascorbic acid 28.24 ± 0.28 42.76 ± 0.43 62.27 ± 0.72 73.73 ± 0.37 94.81 ± 0.49 45.5 ± 0.4
Superoxide radical scavenging activity Basella alba 38.95 ± 0.39 47.21 ± 0.85 56.72 ± 0.64 68.90 ± 0.28 70.11 ± 0.80 47.2 ± 1.2
Ascorbic acid 38.46 ± 3.87 41.24 ± 0.02 53.59 ± 1.54 59.56 ± 2.11 63.77 ± 1.64 55.2 ± 2.10

* values represent means ±.S.D, n = 3; Means not sharing the same letter are significantly different (LSD) at P < 0.01 probability level in last column.

Table 2
Antioxidant activity of BaME and ascorbic acid in Phosphomolybdate and Hydroxyl radical scavenging assay.

Antioxidant assay Sample Percentage inhibition at different concentration of sample (lg/ml) * IC50 (lg/ml)
200 400 600 800 1000
Phosphomoly bdate assay Basella alba 19.97 ± 0.67 27.84 ± 0.86 37.83 ± 0.39 43.63 ± 0.58 53.95 ± 0.39 918.5 ± 0.1
Ascorbic acid 50.66 ± 8.4 63.66 ± 4.2 74.00 ± 7.2 84.00 ± 2.6 86.00 ± 5.3 124.3 ± 1.57
Hydroxyl radical scavenging activity Basella alba 24.8 ± 1.16 52.4 ± 2.06 68.5 ± 2.73 76.1 ± 2.36 79.3 ± 1.34 380.3 ± 0.9c
Ascorbic acid 24.0 ± 7.21 48.0 ± 5.29 62.6 ± 8.38 77.0 ± 2.65 94.6 ± 4.16 430.4 ± 0.6

* values represent means ±.S.D, n = 3; Means not sharing the same letter are significantly different (LSD) at P < 0.01 probability level in last column.

3 ± 0.9 lg/mL and 430.4 ± 0.6 lg/mL respectively, the HRS activity 3.4.2. Clonogenicity assay
can be ranked. All results showed that the potency and scavenging The descriptive images of the clonogenic assessment are repre-
capability of BaME increased with the amount of the sample at sented in Fig. 2a. When a cell can proliferate indefinitely and make
concentrations of 200–1000 g/mL (Table 2). a big colony it is called clonogenic. Clonogenic assay is regarded an
The antioxidant study shows remarkable findings that suggest in vitro gold standard to assess the sensitivity of the cell towards
that BaME could be a possible source of natural antioxidants with drug therapy. In other words, it studies the drug’s effect on cell sur-
significant therapeutic properties to inhibit or regulate degenera- vival and proliferation (Willers et al., 2021). After treatment, a cell
tive diseases related to oxidative stress. is deemed to be viable if it survives and can divide. Over time, in a
drug-free environment, it forms clones (a small colony of geneti-
cally identical cells). Fig. 2b is showing the results of the clonogenic
3.4. Anticancer activity assay. In both cell lines HT-29 and HCT-116, the formation of the
colony was significantly (***P < 0.001) inhibited in BaME compared
3.4.1. Antiproliferative activity to untreated cells. Therefore, the data reveals that after BaME treat-
To investigate BaME anti-proliferative effects on HT-29 and ment, the number of viable cells at IC50 values and the next higher
HCT-116 CRC cancer cell lines, we performed a cell proliferation doses reduced dramatically in both cell lines. It can be concluded
assay using XTT. The extract had the highest cytotoxicity against that when BaME initiates an irreversible cell arrest process or else
HT-29 at a concentration of 50 g/mL, inhibiting cell growth by exerts ‘cidal’ and not ‘static’ activity.
78.9% in 72 h. At 48 h, it was discovered that extract in HCT-116
and HT-29 had IC50 values of 51 lg/mL and 22 lg/mL, respec- 3.4.3. FACS analysis
tively, with 70% cell growth inhibition (Fig. 1 a and b respectively). First, using FACS analysis we evaluated DNA content in treated
These findings suggested that BaME, depending on dose and dura- and untreated CRC cells. The cells were stained with a DNA binding
tion, had an anti-proliferative effect on CRC cells. dye called propidium iodide which has an absorption maximum of

Fig. 1. Determination of BaME cytotoxicity on CRC cell lines using the XTT assay. Cytotoxic effect on (a) HCT-116 cell lines and (b) HT- 29 cell lines. Each value in the figure is
represented as mean ± SD (n = 6). *** are significantly different (P < 0.05).

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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

Fig. 2. Effect of BaME in colony formation in HCT116 and HT29 cell lines. (a) Descriptive images of the stained colonies and clonogenicity assessment. (b) Cell survival curves
of both CRC cell lines with different concentration of BaME treatment. (***P < 0.001). Each bar represents mean ± standard deviation, n = 3.

535 nm. The DNA content was depicted through fluorescence in the G0/G1 phase, indicating that BaME can arrest the cell cycle.
emission from the propidium iodide-DNA complex, which exhibits These cells will eventually undergo apoptosis (a desirable form of
a maximum absorption at 617 nm. The results showed that CRC cell death in cancer) if they are arrested at a specific stage of the
cells treated with BaME induced G0/G1 arrest (Figs. 3 & 4). Nearly cell cycle.
56.3 to 80.6% of HCT-116 cells were arrested at G0/G1 stage,
whereas 83.2 to 97% of HT-29 cells were arrested at the same stage. 3.4.4. Western blotting
Figs. 3 and 4 show that applying the IC50 concentration of BaME Further, we assessed the protein levels of cognate associates
caused an accumulation of cells in the G0/G1 stages in both CRC associated with the cell cycle in CRC cell lines (HT-29 and HCT-
cell lines, as seen by the existence of the sub G0 peak and a decline 116) treated with BaME. Compared to controls, the level of two
in the proportion of cells in the S phase as the extract concentra- proteins cyclin D1 and Cdk4 in BaME-treated CRC cells were noted
tion was raised. The cells were arrested within 24 h of treatment to be significantly decreased (Fig. 5). Gene p21 which is the tumor

Fig. 3. Cell cycle effects of BaME in colorectal cancer cell line HCT-116. T1, T2, T3 represent increase in concentration of the extract respectively.

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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

Fig. 4. Cell cycle effects of BaME in colorectal cancer cell line HT- 29. T1, T2, T3 represent increase in concentration of the extract respectively.

Fig. 5. Treatment with BaME influences regulatory molecules involved in (a),(b) survival, and (c),(d) cell cycle molecular pathways in HCT-116 and HT-29 cell lines
respectively representing the effect of the increase in the concentration of the extract on regulatory molecules expression.

suppressor has regulated the expression levels of Cdk4 and cyclin BaME therapy. The increase in p21 protein and mRNA expression
D1. Then, in BaME-treated versions of both CRC cell lines, we tested was accompanied by a reduction in pRb protein expression. Sche-
the protein and messenger expression of the cyclin-dependent matic representation of molecular action of BaME on cell cycle
kinase inhibitor, p21 and the protein retinoblastoma (pRB). The checkpoints and EGFR signaling pathway molecules is described
amount of p21 protein expression in cells increased following in Fig. 6. pRb is a downstream molecule of Cdk4 and Cyclin D1

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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

Fig. 6. Schematic representation of molecular action of BaME on cell cycle checkpoints and EGFR signaling pathway molecules.

and the condition of Rb phosphorylation is found to control cell

growth, proliferation, and different cellular activities. Overall, our
findings suggest that in BaME-treated CRC cell lines, increased
p21 expression is associated with decreased levels of Cdk4 and
cyclinD1. These molecular effects clearly exhibit the arrest of cells
at the G0/G1 stage. Because these pathways have been linked to
survival and tumor resistance in previous research, the effects of
BaME on the PI-3 k/Akt and MEK/ERK pathways were examined
in the current investigation.
We found that active biomolecules in BaME decreased the sig-
nal transduction proteins pERK PI-3 K and pAKT expression
(Fig. 5). BaME causes the G0/G1 cell cycle to be arrested by upreg-
ulating the expression of p21 and downregulating the expression
of cyclin D1, Cdk4, and also pRb in human colorectal cancer
(Fig. 6). Proliferative and cell cycle signaling pathways were mark-
edly reduced after treatment with BaME (p < 0.001). Cell cycle
molecules (E2F-1, pRb, cyclin D1, and Cdk4) as well as survival
molecules (PI3K, pERK, and pAkt) are inhibited in both cell lines.
Fig. 7. qRT- PCR analysis of signal molecules involved in survival and cell cycle.
The HT-29 cell line was significantly suppressed as compared to
Each value represents the mean ± standard deviation, *** p < 0.001.
HCT-116. It is worth noting that Cyclin D1/Cdk4 complexes are
studied extensively as cancer therapeutic targets since their role
discovery in human cancers. 4. Discussion

There is a wealth of research to support the idea that consump-

3.4.5. Quantitative RT-PCR. tion of fruits and vegetables regularly lowers the chance of devel-
Additionally, qRT-PCR analysis was done to verify the protein oping chronic illnesses like cancer (Aune et al., 2017) (Hartley
expression observed by western blotting at the messenger RNA et al., 2013). Fruits and vegetables include a wide range of phyto-
level, and similar results were obtained (Fig. 7). Cells treated for chemicals, such as phenols and flavonoids, which have been dis-
24 hrs with DMSO and BaME (50 nM) were harvested, and mRNA covered to have anticancer activity and to provide a number of
extraction was done for qRT-PCR analysis. Results from qRT-PCR health advantages (Bøhn et al., 2010) (Boye et al., 1990). There
were compared with amplified b-actin (normalized). Results have been reports of certain phytocompounds exhibiting both
revealed a decrease in cyclin D1, pRb, and E2F-1 levels and a sub- anti-diabetes mellitus (Venkatachalam et al., 2013) and anti-
stantial (p < 0.001) increase in p21 mRNA expression levels. This inflammatory activity (Albinjose et al., 2015). The green leafy veg-
expression pattern was shown in BaME-treated CRC cell lines com- etable Basella alba, an indigenous plant chosen for the present
pared to control. The results indicate that BaME inhibits cell sur- study, is not commercialized and less explored, should be encour-
vival and cell cycle factors controlling the development of aged for active farming as it exhibits good antioxidant and anti-
colorectal cancer. cancer activity.

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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

Plant-based compounds have enormous therapeutic potential the absorbance at 560 nm, which shows a dose-dependent rise in
with fewer side effects. Vimala and Keerthana, 2014 reported the superoxide scavenging activity. These outcomes are equivalent to
presence of 10-Undecen-1-ol, Heptanoic acid 2-ethyl-, Phytol, 1, those of the TPC. The relativity between TPC and the antioxidative
4-Cyclohexanedimethanol, 1, 2-Benzenedicarboxylic acid (di isooc- activity of fruits, plants, and vegetables is close which was reported
tyl ester), and Squalene in ethanolic extract of B.alba leaves (Vimala by many researchers (Paz et al., 2015). Most importantly, for the
and Keerthana 2014). Compounds like 10-[Methoxycarbonyl]-N-a antioxidant activity of an extract based on its phenolic content, it
cetylcolchinol, Hydroymethyl colchicines, and [22S]-21-Acetoy-6a needs identification, further isolation, and characterization of each
,11a-dihydroxy-16a,17a-propylmethylenedioxy pregna 1,4- phenolic compound (Hossain and Shah 2015).
diene-3,20-dione whose mass spectra is illustrated in Figure S1 a, Total antioxidant capacity includes both water- and oil-soluble
b, and c are demonstrated to be the cause of the extract’s anti- antioxidants that can neutralize ROS and guard against chronic ill-
inflammatory properties. It has been reported that several main nesses like diabetes, cancer, and arthritis. The ability of the extracts
molecules that have a prominent role in CRC progression and to act as antioxidants has been regularly assessed using the phos-
development are related to inflammation. So, the molecules also phomolybdate method (Sahreen et al., 2010). The effectiveness of
play a role in anticancer activity (Madka and Rao 2013). The TPC antioxidants is controlled by the structures and chemical composi-
of the methanolic extract of Arisaema jacquemontii Blume was tion of bioactive extract components (Valdés et al., 2015). In patho-
reported to be 45.17 ± 1.70 GAE/g (Baba and Malik 2015). The physiological processes, the hydroxyl radical is referred to as a
results demonstrated that the investigated sample in the present harmful ROS which can damage proteins, fatty acids, DNA, and
work contains a considerable content of phenol phytoconstituent practically every molecule in the biological system, and it pro-
(57.9 ± 2.66 lg GAE/g). Many reported studies have related the motes mutagenesis, cytotoxicity, and carcinogenesis (Sökmen
TPC of plants with their antioxidant activity and demonstrated that and Khan 2016). The antioxidant activity of BaME is directly corre-
extracts containing high amounts of phenolic exhibited higher lated with its HRS capacity. The maximum HRS activity was found
antioxidant activity (scavenging and reducing capacity) (Dileep in the seeds of C. occidentalis, followed by the stem and leaf
et al., 2012). extracts respectively (Arya et al., 2011). An earlier study reported
Flavonoids, the most widespread and extensively distributed the ethanolic extracts of Ixora coccinea to have an increase in NO
class of plant phenolic chemicals, are distinguished by a benzo-c- scavenging activity that was dose-dependent (Banerjee et al.,
pyrone chemical structure. It is present in vegetables and fruits. 2011). In another report, the methanolic extract of Limonia acidis-
Flavonoids that widely exist in plants are called natural biological sima Linn. leaves showed greater NO scavenging compared to its
modifiers and they belong polyphenolic compounds group. Flavo- petroleum ether and also chloroform extract (Attarde et al., 2011).
noids, as biological modifiers, modify the degradative effects of A previously study reported aqueous Basella alba extract with
carcinogens allergens, and viruses (Shan et al., 2019). It exhibits high content of phenolic compounds exhibited high antioxidant
anti-allergic, anti-inflammatory, anticancer, antioxidant, and activity (Kumar et al., 2018) with significant cytotoxic activity on
antimicrobial properties. Previously it was reported that higher Jurkat cell lines (Sushila et al., 2010), Hep G2 (hepatocellular carci-
consumption of flavonoids, decreased the risk of cancer and cardio- noma), A431 (epidermoid carcinoma), and MG63 (osteosarcoma)
vascular diseases (Kabra et al., 2019). Total flavonoid content in (Kumar et al., 2018) cell lines. According to literature review, cell
BaME was 118.1 ± 0.79 lg/g quercetin equivalent per extract. cycle molecules have been proved to regulate cell proliferation
While in the methanolic extract of Arisaema jacquemontii, it was and survival (Maddika et al., 2007). Every type of cancer has dis-
reported to be 35 ± 2.20 rutin equivalent/g (Baba and Malik tinct molecular alterations, such as the up- or down-regulation of
2015). This study was able to point out the potential flavonoid con- biological molecules in cancer cells (Son et al., 2022). Studies on
tent in BaME. cell signaling have shown that cancer cells with faulty checkpoints
Aqueous extract of B.alba was evaluated for antioxidant activity are more susceptible to anticancer drugs. (Gabrielli et al., 2012).
while methanol extracts of B.alba aerial plants are reported to con- Thus, compounds that interfere with cell cycle checkpoints can
tain terpenoids, a class of psychoactive compounds, which has a be effective anticancer agents. In the current investigation, the
central nervous system-suppressing effect that makes them effec- effects of IC-50 value of BaME on the PI-3 k/Akt and MEK/ERK path-
tive for treating some stress-related issues, such as getting sound ways were examined because previous research has reported the
refreshing sleep in today’s hectic lifestyle (Bamidele et al., 2020). link of these pathways with survival and tumor resistance
DPPH is a free radical nitrogen-centered molecule that reacts sim- (Steelman et al., 2011, Ye and She 2013). In preclinical studies
ilarly to peroxyl radical. The rate of the reaction directly correlates reported on CRC models, the activation of Epidermal growth factor
with antioxidant activity. The odd electron in the DPPH free radical receptor (EGFR), insulin-like growth factor receptor (IGFR), or their
(purple) corresponds to a significant maximum absorption at downstream signaling pathways involving proteins like Akt, Ras, or
517 nm. The reduced DPPH-H is created when the odd electron Raf resulted in increasing cancer proliferation and resistance to
from the DPPH radical combines with hydrogen from antioxidants therapy (Chen et al., 2010). While simultaneous suppression of
that have the ability to scavenge free radicals. This results in a shift both EGFR and IGFR-I receptors was linked to enhanced apoptosis
in color from purple to yellow and a reduction in the molar absorp- and decreased proliferation in CRC cells (Kaulfub et al., 2009). The
tivity of the DPPH radical at 517 nm. With regard to the quantity of current work documents a decrease in pAkt and EGFR expression
electrons caught, the resulting decolorizing is stoichiometric patterns, illuminating the function of BaME in regulating cell pro-
(Sánchez-Moreno et al., 1999). The current results shows that liferation. Normally, Rb is found complex with E2F-1. Activation of
BaME reacts with the hydrogen donors, reduces the radical to the c-myc and cyclin D1 is due to their response to growth-promoting
corresponding hydrazine in the antioxidant principles. Biological signals (Butt et al., 2005, Chen et al., 2010). This results in Rb phos-
reactions produce highly toxic radical species, a weak oxidant phorylation to form pRB, releasing E2F transcriptional factors (E2F
called superoxide anions. This ROS damages DNA and in turn dam- –TFs) bound to Rb. E2F-1 plays a crucial role in cell proliferation
ages cells leading to the generation of various diseases (Baba and and facilitates the transition of G1 phase cells into the S phase
Malik 2015). They are produced endogenously by flavoenzymes. (Ramana et al., 2010, Garcia-Jove Navarro et al., 2013). Also, it tran-
They serve as precursors for strong and harmful radicals (single scribes genes involved in cancer cell resistance and growth
oxygen as well as hydroxyl radicals) that cause oxidative stress (Stanelle et al., 2002). Impairment of the PI3K/Akt pathway causes
(Chanda and Dave 2009). The production of superoxide anion rad- a variety of human diseases, including diabetes, cardiovascular dis-
icals by the PMS/NADH-NBT combination results in a decrease in ease, neurological disorders, and cancer. While its elevated condi-
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A. Sheik, E. Kim, U. Adepelly et al. Saudi Journal of Biological Sciences 30 (2023) 103609

tion is considered to be a hallmark of cancer (Fruman et al., 2017). Appendix A. Supplementary material
In cancer biology research, Akt (a Protein Kinase B) has emerged as
a therapeutic target protein because of its major role in regulating Supplementary data to this article can be found online at
various cellular functions like transcription, protein synthesis, https://doi.org/10.1016/j.sjbs.2023.103609.
metabolism, growth (influences TSC1/TSC2 complex and mTOR
signaling) (Pham et al., 2021), cell migration, invasion, proliferation
(via phosphorylation of p21 and p27, the CDK inhibitors), and sur-
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