Evaluation of Tinospora cordifolia stem extract bioactive content and anticancer activity against breast cancer cells

Abstract

Breast cancer remains a major public health challenge, highlighting the importance of finding new therapeutic approaches, including the use of traditional plants with unstudied medicinal properties. This study aims to explore the bioactive potential of Tinospora cordifolia stem extract by investigating the content of flavonoids, phenols, terpenoids and alkaloids as well as the cytotoxic and apoptotic effects on T47D breast cancer cells. Analysis of bioactive compound content revealed a total flavonoid content of 29.7 ± 0.30 mg QE/g extract, a total phenol content of 120.4 ± 4.25 mg GAE/g extract, a total terpenoid content of 2.41 ± 2.18 mg LE/g extract and a total alkaloid content of 2.55 ± 0.27 mg LE/g extract. Cytotoxicity tests on T47D cells using the WST-1 assay showed a dosedependent decrease in cell viability with an IC50 of 571.3 ± 33.41 µg/ml. Furthermore, at a concentration of 2IC50, T. cordifolia stem extract can induce apoptosis by 36.7 ± 4.19%. These results suggest that T. cordifolia has considerable anticancer activity, although further studies are needed to understand its mechanism of action and evaluate its potential in breast cancer therapy.

Full text

Evaluation of Tinospora cordifolia stem extract
bioactive content and anticancer activity against
breast cancer cells
Yuslinda Annisa1, Nuraini Rosyadah1, Fairuz Sarah Kamila1, Siti Mariyah Ulfa2, Sri
Rahayu Lestari3, and Nashi Widodo1
*
1Department of Biology, Faculty Mathematics and Natural Science, Universitas Brawijaya, Jl.
Veteran, Ketawanggede, Malang, 65145, Indonesia
2Department of Chemistry, Faculty Mathematics and Natural Science, Universitas Brawijaya, Jl.
Veteran, Ketawanggede, Malang, 65145, Indonesia
3Department of Biology, Faculty Mathematics and Natural Science, Universitas Negeri Malang, Jl.
Semarang No.5, Malang, 65145, Indonesia
Abstract. Breast cancer remains a major public health challenge,
highlighting the importance of finding new therapeutic approaches,
including the use of traditional plants with unstudied medicinal properties.
This study aims to explore the bioactive potential of Tinospora cordifolia
stem extract by investigating the content of flavonoids, phenols, terpenoids
and alkaloids as well as the cytotoxic and apoptotic effects on T47D breast
cancer cells. Analysis of bioactive compound content revealed a total
flavonoid content of 29.7 ± 0.30 mg QE/g extract, a total phenol content of
120.4 ± 4.25 mg GAE/g extract, a total terpenoid content of 2.41 ± 2.18 mg
LE/g extract and a total alkaloid content of 2.55 ± 0.27 mg LE/g extract.
Cytotoxicity tests on T47D cells using the WST-1 assay showed a dose-
dependent decrease in cell viability with an IC50 of 571.3 ± 33.41 µg/ml.
Furthermore, at a concentration of 2IC50, T. cordifolia stem extract can
induce apoptosis by 36.7 ± 4.19%. These results suggest that T. cordifolia
has considerable anticancer activity, although further studies are needed to
understand its mechanism of action and evaluate its potential in breast cancer
therapy.
1 Introduction
Breast cancer is the most common cancer among women, with a prevalence of 16% of all
cancers in women [1]. In 2020, 684,996 deaths from breast cancer were reported. There will
be an estimated 2.2 million new cases of cancer, of which 1 in 10 will be breast cancer [2].
Breast cancer is often potentially curable if treated in early stages, but metastases are almost
always fatal due to the development of therapeutic resistance during the treatment process.
[3]. In most cancers, single-target drug development does not provide optimal therapeutic
outcomes or tends to develop resistance even in the initial response to treatment [4].
*
Corresponding author: widodo@ub.ac.id
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/).
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Currently, the utilisation of herbs is one of the promising alternatives for the development
of cancer therapy and shows real advantages in dealing with the complexity of cancer
treatment due to its multi-target mechanism [4–6]. One of the herbs that has been utilised
empirically for the prevention and treatment of various diseases is Tinospora cordifolia [7].
T. cordifolia, belonging to the Menispermaceae family, is an important medicinal plant in
Ayurvedic medicine and is included in the Indian System of Medicine (ISM) due to its
bioactive constituents and various therapeutic properties. In Hindu mythology, the plant is
called ‘Guduchi’, which means a celestial herb that saved a celestial being from old age and
kept him young [8–10]. In Indonesia, this plant is known by the local name ‘Brotowali’,
which has traditionally been used by the community as Jamu Pahitan [11]. T. cordifolia has
various potentials as antioxidant, antiallergic, anti-inflammatory, antimicrobial, antiviral,
antidote, antitumor, antileprotic, antispasmodic, and antidiabetic, and has been used
extensively in traditional medicine, especially the stem [12,13].
Bioactive compound content in T. cordifolia is dominated by glycosides, terpenoid
steroids, phenolics, polysaccharides, aliphatic compounds, and alkaloids [14–16]. These
compounds are known to have anticancer activity through various apoptotic pathway
mechanisms, including reactive oxygen species (ROS) enhancement, cell cycle inhibition,
caspase pathway activation, and cell proliferation inhibition [10,16,17]. T. cordifolia shows
great potential to be developed as an anti-breast cancer herbal candidate. The objectives of
this study were to evaluate the content of bioactive compounds in the ethanol extract of T.
cordifolia stem and test its effectiveness on Breast cancer cell line T47D in vitro.
2 Materials and methods
2.1 Extraction of T. cordifolia stems
T. cordifolia stem simplisia was obtained from UPT Materia Medica Herbal Laboratory, Batu
city, East Java, Indonesia, with batch number 220215.BRT.B.MMB.001. Simplisia and 96%
ethanol in a proportion of 1:10 (w:v) were placed in a microwave-assisted extraction (MAE)
vessel (Anton Paar, Austria). The following settings were used for the MAE: holding
temperature, 50°C; 5 min heat-up, 50°C; holding time, 15 min; 5 min cool-down; power,
1500 W. The filtrate was filtered with filter paper, and the solvent was evaporated with a
Rotary Evaporator Hei-VAP Expert (Heidolph, Korea) at 50 rpm and 50°C. The storage
temperature of the extract was 4°C [18].
2.2 Total flavonoid determination
The aluminium chloride (AlCl₃) colourimetric method was used to measure the total
flavonoid content of T. cordifolia stem extract according to the method of Nurcholis et al.
with modification [19]. A total of 50 μL of extract (1 mg/mL) and quercetin standard solution
(1.5625 - 400 μg/mL) was mixed with 150 μL of 96% ethanol and 10 μL of 10% AlCl₃ in a
96-well plate. The mixture was then incubated for 40 minutes in the dark at room temperature
with the addition of 10 µL of 1M sodium acetate (CH₃COONa). Absorbance was measured
at a wavelength of 405 nm using a Multiskan SkyHigh Microplate Spectrophotometer
(Thermo Fisher Scientific, USA). The total amount of flavonoids in the extracts was
expressed as milligrams of quercetin equivalent per gram of extract (mg QE/g) [19].
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2.3 Total phenolic determination
The Folin-Ciocalteu method was used to determine the total phenolic content with a modified
protocol adapted from Molole et al. [20]. In a 96-well plate, 10 μL of extract (1 mg/mL) and
gallic acid standard solution (1.5625 - 400 μg/mL) were mixed with 100 μL of Folin-
Ciocalteu reagent in dH2O (1:10, v/v). Then, 1 µL of 7.5% sodium carbonate solution
(Na₂CO₃) was added. The mixture was incubated for 90 minutes in the dark at room
temperature. Sample absorbance was measured at a wavelength of 725 nm using a Multiskan
SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, USA). The total phenolic
content of T. cordifolia stem extract is expressed as milligrams of gallic acid equivalents per
gram extract (mg GE/g) [20].
2.4 Total terpenoid determination
The total terpenoid content of the extracts was determined according to the modified method
of Łukowski et al. (2022). Total terpenoid content was expressed as mg linalool equivalent
per gram of extract (mg LE/g). Standard solutions (0 - 63.66 μg/mL) and T. cordifolia stem
extract (3 mg/mL) of 1 mL were put in a test tube, then 3 mL of chloroform was added and
homogenised. Then 500 μL of concentrated sulphuric acid (H₂SO₄) was slowly added to the
wall of the test tube and the mixture was incubated for 4 hours in the dark at room
temperature. A reddish-brown precipitate formed at the bottom of the tube after incubation.
The precipitate formed was retained, and added with 500 μL of 96% methanol and mixed
until homogeneous. A total of 150 μL of the sample mixture was transferred to a 96-well
plate. The absorbance was measured at 538 nm using a Multiskan SkyHigh Microplate
Spectrophotometer (Thermo Fisher Scientific, USA) [21].
2.5 Total alkaloid determination
The total alkaloid content of T. cordifolia stem extract was determined using a modification
of Tan's method (2018). A total of 69.8 g of bromocresol green (BCG) was dissolved in 3
mL of 2N NaOH and 5 mL of distilled water. The solution was then heated to complete
dissolution. The extract (2 mg/mL) was dissolved in 2N HCl, filtered, and pH adjusted to
neutral using 0.1N NaOH. 1 mL of extract solution and atropine standard solution (0-16,670
μg/mL) were added to 5 mL of BCG solution and 5 mL of phosphate buffer. The mixture
was homogenised. An equal volume of chloroform was added to the mixture, followed by
vigorous shaking until a yellow layer was formed at the bottom of the solution. A total of 150
µL of the yellow layer was transferred to a 96-well plate. The absorbance was measured at
470 nm using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher
Scientific, USA). Total alkaloids were expressed as milligrams of atropine equivalent per
gram extract (mg AE/g) [22].
2.6 Cell culture preparation and cell viability assay
Breast cancer cell line T47D was obtained from the Laboratory of Animal Physiology,
Structure, and Development, Biology Department, Faculty of Mathematics and Natural
Sciences, Universitas Brawijaya. T47D cells were cultivated in RPMI 1640 medium (Gibco,
USA) with an additional 10% fetal bovine serum (Gibco, USA) and 1% penicillin-
streptomycin (Gibco, USA). The cells were grown in 96-well plates at a density of 7,500
cells per well and incubated for 24 hours at 37°C in a 5% CO2. Cells were then treated with
various graded concentrations of T. cordifolia stem extract (0, 160, 320, 640, and 1280
μg/mL) for 24 hours. The treated medium was replaced with fresh medium containing 5%
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WST-1 reagent (Sigma-Aldrich, USA), and incubated for 30 minutes. This was followed by
a measurement of the absorbance at 450 nm using a Multiskan SkyHigh microplate
spectrophotometer (Thermo Fisher Scientific, USA) [23]. Percentage cell viability for T47D
was calculated, and IC₅₀ was determined using a linear regression equation between
percentage cell viability and extract concentration.
2.7 Apoptosis assay
T47D cells were grown at a density of 50,000 cells in 500 μL of culture medium per well in
a 24-well plate. The cells were incubated for 24 hours. Cells were exposed to T. cordifolia
stem extract at concentrations of ½IC₅₀, IC₅₀, and 2IC₅₀ for 24 hours at 37°C with 5% CO₂.
Treated T47D cells were harvested by the trypsinisation method and stained with FITC-
Annexin V/propidium iodide (PI) (BioLegend, USA) according to the method of Worsley et
al. with modifications. The cells were incubated for 20 minutes at 4°C in the dark. Stained
cell suspensions were analysed using FACS-Calibur flow cytometry (BD FACS Calibur,
USA) and data were analysed using Cell Quest software (BF Bioscience, USA) [24].
2.8 Statistical analysis
One-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test
(DMRT) for post hoc comparisons was used for statistical analysis of apoptosis assay results.
Statistical significance was set at a probability level of p < 0.05. All data are presented as
mean ± standard deviation (SD), with each value representing the average of three replicates.
3 Results and discussions
3.1 Content of flavonoids, phenolics, terpenoids, and alkaloids in the T.
Cordifolia stem extract
T. cordifolia possesses a wide range of important bioactive chemical constituents such as
steroids, alkaloids, glycosides, tannins, sesquiterpenoids, flavonoids, phenols,
polysaccharides, essential oils, aliphatic compounds and fatty acid combinations that have
been previously investigated [10,25]. T. cordifolia extraction process and solvent selection
are important steps in the concentration and targeting of plant bioactive compounds [26]. The
existence of bioactive compounds in the stem extract of T. cordifolia was determined by
measuring the total content of flavonoids, phenols, terpenoids and alkaloids. The most
abundant among these compounds was found to be phenolic, with a concentration of 120.4
± 4.25 mg GAE/g (Table 1). This indicates that phenolics are the dominant class of bioactive
compounds in the extract.
Table 1. Total flavonoids, phenolics, terpenoids, and alkaloids content in T. cordifolia stem extract.
Total Bioactive Content
T. cordifolia stem extract
Total flavonoid content (mgQE/g extract)
29.7 ± 0.30
Total phenolic content (mgGAE/g extract)
120.4 ± 4.25
Total terpenoid content (mgLE/g extract)
2.41 ± 2.18
Total alkaloid content (mgAE/g extract)
2.55 ± 0.27
Note: Data are presented as mean ± SD.
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Phenolic compounds in T. cordifolia play an important role in its bioactivity in free radical
scavenging due to the availability of hydroxyl groups and can act as reducing agents, metal
chelators, and hydrogen donors [27,28]. Previous studies have shown that the ethanolic
extract of T. cordifolia stem contains a large amount of phenolics, which play a role in
antioxidant activity, as well as significant proliferation inhibition on the HeLa cervical cancer
cell line [27]. This study evaluated the anticancer potential of the bioactive compounds of T.
cordifolia stem extract on the breast cancer cell line T47D based on cytotoxicity and
apoptosis induction.
3.2 Cytotoxicity of T. cordifolia stem extract on T47D breast cancer cells
The toxicity of medicinal plants is closely linked to the presence of bioactive compounds in
the plant material and the toxic potential of these compounds [29]. The cytotoxicity test of T.
cordifolia stem extract showed toxicity to T47D cells that increased with increasing extract
concentration with an IC50 value of 571.3 ± 33.41 µg/mL (Figure 1). T. cordifolia has shown
anticancer activity in several types of hepatocellular carcinoma, lymphoma and glioblastoma
[30–32].
Fig. 1. Cytotoxicity of the stem extract of T. cordifolia on T47D cells and its IC50 value. Data are
expressed as mean ± SD.
The results of another study showed that the hexane fraction of T. cordifolia has potent
anticancer activity on breast cancer cells MCF-7 and MDA-MB-231 with IC50 values of 37.2
± 2.77 and 47.5 ± 2.53 µg/mL, respectively [33]. While the results of a related study using
50% ethanol solvent showed anticancer activity on U87MG, HeLa and C6 cell lines with
higher IC50 values at a concentration of 200 µg/mL [32]. Differences in the bioactivity of T.
cordifolia are due to differences in extraction methods and types of solvents used, which
allow differences in the content of bioactive compounds contained in the extracts.
3.3 T. cordifolia stem extract induced apoptosis of T47D cells
The induction of apoptosis in T47D cells was evaluated using propidium iodide (PI) and
annexin V to detect the relative number of live, apoptotic and necrotic cells. Annexin V can
bind to phosphatidylserine, which translocates to the outside of the plasma membrane during
apoptosis, whereas PI binds to DNA in necrotic cells [33, 34]. T. cordifolia stem extract was
potent in inducing apoptosis in T47D cells, which increased significantly with increasing
extract concentration (Figure 2B). The highest increase in apoptosis was 36.7 ± 4.12% at an
extract concentration of 1142.6 µg/mL (Figure 2A-2B).
T. cordifolia has been reported to contain a variety of phytochemical components capable
of inducing anticancer effects through mitochondrial-mediated apoptosis, triggering reactive
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oxygen species, mutagenic activity, and decreased expression of genes that regulate the cell
cycle [10,14,17,33]. The mechanism of anticancer action of T. cordifolia depends on its
phytochemical components. Ethanolic extract of T. cordifolia has been reported to be capable
of inducing apoptosis by enhancing the sub-G0 phase without altering the cell cycle [14].
Fig. 2. Apoptosis induction in T47D cells by T. cordifolia stem extract. (A-B) Annexin V/FITC-PI
assay showing the induction of apoptosis in T47D cells. Different letters on the graph indicate
statistically significant differences between groups (p < 0.05). Data are expressed as mean ±
SD.
The alkaloid content of T. cordifolia extract plays a role in the anticancer activity in
addition to the phenolic compounds. Alkaloid compounds in the extract have been widely
reported to have potent anticancer activity against various cancers such as prostate cancer,
liver cancer and leukaemia due to their antioxidant activity. They reduce cancer-causing
ROS, induce apoptosis by increasing the expression of caspase-8, caspase-9 and caspase-3,
and arrest the cancer cell cycle in the G1 phase [12,32,36].
4 Conclusion
T. cordifolia stem extract contains bioactive compounds with the highest concentration of
phenolics, followed by flavonoids, alkaloids and terpenoids. T. cordifolia stem extract has
toxicity to T47D cells and can induce apoptosis, which increases with increasing extract
concentration. T. cordifolia has considerable potential as an anticancer agent, but further
research is necessary to understand the mechanism of action and potential compounds in the
anti-breast cancer pathway.
This research was granted by the Directorate of Research, Technology and Community Service,
Ministry of Education, Culture, Research and Technology, Republic of Indonesia, National
Competition Research - Doctoral Dissertation Research with contract number
1133.2/UN10.C10/TU/2023.
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