In vitro evaluation of immunomodulatory, anti-diabetic, and anti-cancer molecular mechanisms of Tribulus terrestris extracts

Abstract

Dampened immunity and impaired wound healing in diabetic patients may lead to diabetic foot ulcer disease, which is the leading cause of limb amputations and hospitalization. On the other hand, cancer is the most significant cause of mortality globally, accounting for over 10 million fatalities in 2020, or nearly one in every six deaths. Plants and herbs have been used to treat chronic diseases due to their essential pharmaceutical attributes, such as mitigating drug resistance, ameliorating systemic toxicities, reducing the need for synthetic chemotherapeutic agents,and strengthening the immune system. The present study has been designed to evaluate the effects of Tribulus terrestris on wound healing, cytotoxic and anti-inflammatory responses against HepG-2 liver cancer cell line. Two solvents (methanol and ethanol) were used for root extraction of T. terrestris. The wound healing potential of the extracts was studied on diabetic cell culture line by scratch assay. The anti-oxidant and cytotoxic potentials were evaluated by in vitro assays against HepG2 cell line. The methanolic root extract resulted in the coverage of robust radical scavenging or maximum inhibition of 66.72%,potent cytotoxic activity or reduced cell viability of 40.98%, and anti-diabetic activity having mighty α-glucosidase inhibition of 50.16% at a concentration of 80 μg/ml. Significant reduction in the levels of LDH leakage (56.38%), substantial ROS (48.45%) and SOD (72.13%) activities were recorededMoreover, gene expression analysis demonstrated the down-regulation of inflammatory markers (TNF-α, MMP-9, Bcl-2, and AFP) in HepG-2 cells when treated with T. terresteris methanolic extract as compared to stress. Furthermore, the down-regulation of inflammatory markers was validated through ELISA-mediated protein estimation of IL-1β and TNF-α. It is expected that this study will lay a foundation and lead to the development of efficient but low-cost, natural herbs extract-based dressing/ointment for diabetic patients and identify potential drug metabolites to treat out-of-whack inflammatory responses involved in cancer onset, progression, and metastasis.

Full text

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In vitro evaluation
of immunomodulatory,
anti‑diabetic, and anti‑cancer
molecular mechanisms of Tribulus
terrestris extracts
Abdullah Khalid
1, Tariq Nadeem
2, Muhammad Asaf Khan
3*, Qurban Ali
4* &
Muhammad Zubair
1
Dampened immunity and impaired wound healing in diabetic patients may lead to diabetic foot
ulcer disease, which is the leading cause of limb amputations and hospitalization. On the other hand,
cancer is the most signicant cause of mortality globally, accounting for over 10 million fatalities in
2020, or nearly one in every six deaths. Plants and herbs have been used to treat chronic diseases
due to their essential pharmaceutical attributes, such as mitigating drug resistance, ameliorating
systemic toxicities, reducing the need for synthetic chemotherapeutic agents,and strengthening the
immune system. The present study has been designed to evaluate the eects of Tribulus terrestris
on wound healing, cytotoxic and anti‑inammatory responses against HepG‑2 liver cancer cell
line. Two solvents (methanol and ethanol) were used for root extraction of T. terrestris. The wound
healing potential of the extracts was studied on diabetic cell culture line by scratch assay. The anti‑
oxidant and cytotoxic potentials were evaluated by in vitro assays against HepG2 cell line. The
methanolic root extract resulted in the coverage of robust radical scavenging or maximum inhibition
of 66.72%,potent cytotoxic activity or reduced cell viability of 40.98%, and anti‑diabetic activity
having mighty α‑glucosidase inhibition of 50.16% at a concentration of 80 μg/ml. Signicant reduction
in the levels of LDH leakage (56.38%), substantial ROS (48.45%) and SOD (72.13%) activities were
recorededMoreover, gene expression analysis demonstrated the down‑regulation of inammatory
markers (TNF‑α, MMP‑9, Bcl‑2, and AFP) in HepG‑2 cells when treated with T. terresteris methanolic
extract as compared to stress. Furthermore, the down‑regulation of inammatory markers was
validated through ELISA‑mediated protein estimation of IL‑1β and TNF‑α. It is expected that this
study will lay a foundation and lead to the development of ecient but low‑cost, natural herbs
extract‑based dressing/ointment for diabetic patients and identify potential drug metabolites to treat
out‑of‑whack inammatory responses involved in cancer onset, progression, and metastasis.
Cancer and diabetes are the leading causes of death worldwide, and the development of novel anticancer and
antidiabetic drugs has become the most pressing necessity in recent years1. Although there are dierent types of
treatment, such as immunotherapy, chemotherapy, radiation, and hormonal therapy, they all possess substantial
shortcomings, including severe toxicities and drug resistance2. e limitations of targeted therapy become appar-
ent aer years in practice. e vast majority of malignancies are not targetable, because they evolve adaptive
mechanisms and multiple oncogenic pathways3.
erefore, the limitations mentioned above drive up the demand for plant-based natural products possess-
ing therapeutic ecacy for diseases ranging from the common cold to cancer and diabetes4,5. Herbal remedies
rarely cause serious adverse eects, and reports of deaths or hospitalizations as a result of these remedies are
OPEN
1Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad,
Pakistan. 2Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. 3Institute of Plant
Breeding and Biotechnology, MNS-University of Agriculture, Multan, Pakistan. 4Department of Plant Breeding
and Genetics, Faculty of Agricultural Sciences, University of the Punjab Lahore, Lahore, Pakistan. *email:
muhammad.zubair1751@gmail.com; saim1692@gmail.com
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exceptionally rare. e United States National Poison Control Center does not even have a section for herb-
related adverse eects in its database. e healing process can be generally divided into four phases: the hemo-
static, inammatory, proliferative, andremodeling phase, ultimatelydetermining the strength and manifestation
of the healing tissue6.
e genus Tribulus, belonging to the Zygophyllaceae family, contains about 20 dierent species, of which three
species Tribuluscistoides, Tribulusalatus, and Tribulusterrestris, are widely found in India7. T.terrestris extract has
been shown to inhibit cell proliferation and promote apoptosis in liver cancer cells. Its biological activity has
been related to steroidal saponinsby inducing an apoptotic pathway in breast cancer cells. Trans-N-feruloyl-
3-hydroxytyramine and trans-N-feruloyl-3-ethoxytyramine, have been shown to trigger apoptosis in leukemic
cancer cells8–10.
Plant anti-oxidant compounds are commonly isolated by solvent extraction technique. However, the extract
yields and anti-oxidant activities of the plant materials are mainly dependent on the nature of the solvent, polari-
ties, and presence of various anti-oxidant compounds of diverse characteristics of chemical which may or may
not be soluble in a specic solvent11,12. Polar solvents are normally used to recover polyphenols from the plant
material. e most appropriate of these solvents are aqueous mixtures in Methanol, ethanol, acetone, and ethyl
acetate. Ethanol and Methanol have been widely used to extract anti-oxidant compounds from plants and plant-
based foods like plum, pomegranate, rosemary, rice bran, wheat grain, citrus peel, and many other fruit peels13,14.
Tribulusterrestris extract containsalkaloids, resins, avonoid oil, and nitrates, which have antihypertensive,
anti-inammatory, hypolipidemic, and anti-diabetic activities15. e ethanolicextract of T. terrestris has been
reported topossesssubstantial anti-oxidant activity against STZ-induced diabetic rats, signicantly reducingout-
of-whack serum glucose,triglycerides, and cholesterol levels. Furthermore,serum superoxide dismutase (SOD)
activity was enhanced in alloxan-induced diabetic mice with subsequent inhibition of gluconeogenesis16–18.
To evaluate the eectiveness of T. terresteris plant extract, we studied the anti-diabetic, cytotoxic, and anti-
inammatory properties of methanol and ethanol solvent-based extracts. We used HepG-2 liver cancer cell line
to study the cytotoxic activity response of root extract.
Materials and methods
Extract preparation. Fresh samples of the genotype of T. terrestris were obtained in April 2019 from the
University of Agriculture, Faisalabad, Pakistan. It has been conrmed that the experimental sample plants (T.
terrestris), including the collection of plants, complied with relevant institutional, national, and international
guidelines and legislation with appropriate permission from the Department of Bioinformatics and Biotechnol-
ogy, Government College University Faisalabad, Pakistan. A specimen voucher was put forward to the her-
barium department. Roots were washed, dried at 37°C, and ground to get a ne powder. Ethanol and methanol
solvents were used for extraction purposes. ese solutions were placed in an incubator for two days for shaking
purpose. Each solution was ltered with Whatman No. 1 lter paper. e solvents were evaporated with the help
of a rotary evaporator, and extracts were dissolved in phosphate buer saline (PBS) for use in future studies.
Antidiabetic activity: α‑glucosidase assay. e anti-diabetic activity of Tribulus terrestris (ethanol and
methanol-based extracts of the samples dried at 37˚C) was assessed using an α-glucosidase assay. A reaction
mixture having 12.5 μL of a T. terrestris extract, 40 μL of α-glucosidase (5μ/mL),and 140 μL of phosphate buer
saline 1X (pH 7.4) was added to 96-well plates. Incubation was done at 37˚C for 5min, and 40 μL 4-nitrophenyl-
β-D glucopyranoside (PNPG 5mM) was added to each well. Acarbose was used as a positive control with acon-
centration of 3mg/ml and PBS as a negative control. An ELISA plate reader was used to measure optical density
at 405nm. Percent inhibition of α-glucosidase was calculated as [(Ac–As)/(Ac)]*100, where Ac and As shows the
absorbance of the control and sample, respectively.
Wound healing: scratch assay. e retinal pigment epithelial (RPE) cell line was cultured at the rate
of 2 × 105 cell/ ml in Dulbecco’s modiedEagle’s medium (DMEM) containing 10% fetal bovine serum (FBS)
and 1% penicillin/streptomycin for 24h on 12- well cell culture plates at 37°C and 5% CO2 in an incubator to
produce 80–100% convergent cell monolayer. A sterile pipette tip of 200 μL was used to create a linear wound.
e wells were washed two times with PBS by removing the medium. Each well was added with fresh medium
containing ethanol and methanol-based extracts of Tribulus terrestris. DMEM containing 1ml with 20 μLof PBS
was used asa negative control. e platelet-derived growth factor (PDGF) at the rate of 5ng/ml has been used as
a positive control. Digital images of the wound on the cell monolayer wound were taken at 0h, 4h, 8h and 16h.
Evaluation of these images at 1712 × 1368 pixels in Image J 1.440 soware. e measureof wound healing from
time to time compared with the initial width at 0h to determine the percentage of cell migration and increase
induced by the secretion of Tribulusterrestris extracts.
Scratch assay. e assessment of migration potential ofT. terrestris extract in RPE was tested by cell scratch
assay. In short, 8 × 104 cells were cultured on a 6-well plate and le to form a monolayer. e monolayer was then
scratched using sterile 200μLpipette tips aer 80% conuency. e cells were then washed to remove the non-
adhesive cells, and the medium was replaced. e images were taken in a phase contrast microscope abruptly
aer the scratch creation, followed by microscopy aer 4h, 8h, 16h, and 18h.
Cytotoxic activity: MTT assay. Cytotoxicity was assessed using human liver cancer cell line HepG2 cell
line using T. terrestris extracts. e same medium described above was used in 96-well culture plates with an ini-
tial concentration of 2 × 104 cells per well. Each cell culture plate was treated with ethanol and methanol solvent-
based T. terrestris extracts with a concentration of 0.1mg/ml. Phosphate buer saline (PBS) was used for nega-
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tive control. Aer 2days of incubation, 10 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) was added to each well in a nal concentration of 5mg/ml. ese plates were again incubated for 4h.
Optical density was measured at 490nm aer adding 150 μLof 0.1% dimethyl sulfoxide (DMSO). In the case of
HepG2, percent inhibition was calculated as [(Ac–As)/(Ac)] *100, where Ac and As show the absorbance of the
control and sample, respectively.
Anti‑oxidant activity determination. e plant extract was also assessed for anti-oxidant activity
through DPPH assay. e DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay was used to determine the free
radical scavenging activity released by T. terrestris extracts in ethanol and methanol solvents at various concen-
trations (5μg/ml,10μg/ml,20μg/ml,40μg/ml,80μg/ml and 160μg/ml). e stock solution (0.3mM) DPPH
was prepared in Methanol and ethanol with 10 µL of root sample was assorted with 190 µL of DPPH in 96 well
plate or gallic acid taken as standard with consequent incubation period of 30min in dark. Microplate reader
(ELx808IU Biotek USA) was used to measure absorbance value at 517nm and percentage inhibition of DPPH
solution was calculated by their measurements.
Cytotoxicity assessment through LDH assay.. LDH activity in media samples was measured as stated
by the manufacturer’s instructions (Roche Diagnostics, Cat No. 04744926001). In short, a mixture of lactate
dehydrogenase was prepared by adding the same volume of LDH co-factor, dye, and substrate solution. Approxi-
mately 50 μL of media samples and 100 µL of LDH test compounds were added to each well in a 96-well plate.
e incubation period of the plate was 20–30min at room temperature. e stop solution was used to stop the
reaction, and a microtiter plate reader measured the measurement of absorption at 490nm and a reference
absorption at 690nm.
Superoxide dismutase (SOD) assay. As the manufacturer’s guidelines directed, the SOD assay kit deter-
mined the SOD activity (Abcam USA, Cat No. ab65354). e addition of 20 μL/well supernatant of dierent
groups was followed by 200 μL of WST solution in each well, which included blanks 1, 2, and 3. e working
solution of an enzyme (20 μL) was added to all wells of the samples and blank 1. Slightly stirred the plate for mix-
ing, and the incubation period was 20min at 37 °C. e absorption was measured at 450nm using a microtiter
plate reader.
Determination of ROS generated by HepG2 cells. e ROS assay determined the number of reactive
oxygen species produced by cells. e cell capability can be decreased, which enhances oxidative stress damage
by increasing ROS production. ROS activity was measured with the help of the Cellular ROS Detection Assay Kit
(Abcam, ab113851DCFDA) through the manufacturer’s protocol. HepG2 cells (25 × 104) were added to 96 well
plate at the p3 stage. e cells were washed three times with 1X PBS and 100 μLof DCF-DA working solution for
1h at 37°C. DCF-DA media was taken out, and cells were washed with 1X PBS. In each well, PBS 100 μL was
added, and a spectrophotometer was used for uorescence readings at 485nm and 535nm.
Estimation of inammatory cytokines concentration through ELISA. According to the manufac-
turer’s protocol, the concentration of IL-1β and TNF-α was determined by using an ELISA kit. e cells were
cultured at a density of 1 × 104 cells/well into 96-well plates for 24h at 37°C and then exposed to a methanolic
extract of Tribulus terrestris. Untreated cells were used as a negative control. e collected material was tested
with IL-1β (Invitrogen ELISA kit Catalog # BMS224-2) and TNF-α (Invitrogen ELISA kit Catalog # KHC3011)
as per the protocol recommendations. A microplate ELISA reader determined the nal absorbance at 405nm.
Real‑time polymerase chain reaction for gene expression analysis. e trizol technique was
employed to purify total RNA from the control, stress, and methanolic plant extract-treated HepG-2 cell line
groups. A Nanodrop spectrophotometer was used to estimate extracted RNA. RNA was reverse transcribed into
cDNA by RevertAidTM rst-strand synthesis kit (qRT-PCR) according to the manufacturer’s instructions. e
expression of the identied genes involved in HCC was investigated, and qPCR was performed using a gener-
ated cDNA template. Table1 highlights the sequence of the primers. All real-time PCR tests were performed
in triplicate, and GAPDH mRNA levels were measured to normalize the AFP, MMP-9, TNF alpha, and Bcl-2
mRNA expression values.
Percentage inhibition
=

control Absorbance −sample Absorbance

/control Absorbance ∗
100.
Table 1. Sequence of the primers.
Genes Forward Primer Reverse Primer Reference
AFP GCA GAG GAG ATG TGC TGG ATTG CGT GGT CAG TTT GCA GCA TTCTG www. orige ne. com
MMP-9 GCC ACT ACT GTG CCT TTG AGTC CCC TCA GAG AAT CGC CAG TACT www. orige ne. com
TNF-αCTC TTC TGC CTG CTG CAC TTTG ATG GGC TAC AGG CTT GTC ACTC www. orige ne. com
Bcl-2 ATC GCC CTG TGG ATG ACT GAGT GCC AGG AGA AAT CAA ACA GAGGC www. orige ne. com
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Statistical analyses. All experiments were performed in triplicates (n = 3), and data were expressed as
mean ± standard deviation. Statistical analysiswas carried out using Minitab 16 soware. Analysis of variance
and Tukey’s post hoc tests were performed to compare dierent treatment groups. Graphs were plotted to show
percentage scratch coverage aer dierent treatments for the scratch assay data. Graphs were made through
GraphPad Prism soware (version 5.00 for Windows, GraphPad Soware, USA).One-way ANOVA with Bon-
ferroni’s test for signicant dierences between the groups was determined, and the p-value less than 0.05 was
considered statistically signicant.
Results
Anti‑diabetic activity. e protective eect against diabetes was evaluated by quantifying the inhibition of
α-glucosidase using plant root extracts of T. terrestris (Fig.1). Ethanol and Methanol were used for comparing
potent solvent activity at various concentrations (5μg/ml, 10μg/ml, 20μg/ml, 40μg/ml, 80μg/ml and 160μg/
ml). Methanol showed the best activity as compared to ethanol. Solvent exhibited a signicant eect (P < 0.0001).
Root extracts demonstrated the maximum inhibition activity of 58.16% at the concentration of 80µg/ml dis-
solved in methanol solvent at 37°C and minimum inhibition activity of 41.87% at 5µg/ml when compared with
negative control PBS. Acarbose used as a standard drug produced only 60.91% inhibition.
Wound healing. Retinal epithelial cells were used in a cell scratch assay to study T. terrestris extract for the
proliferation or migration of invitro cells. e cells treated with T. Terrestris extracts covered the scratch area
more rapidly than the cells of negative control, but even the methanolic extracts was more ecacious than the
PDGF, which was the positive control. Tukey’s post hoc test and variance analysis were conducted indepen-
dently, illuminating signicant dierences (P < 0.05) among the tested extracts. e scratch area expresses the
data as a percentage (Figs.2 and 3). ere is a decrease in the percentage of scratch area with an increase in the
concentration of T. terrestris extract.
Cytotoxic activity. e cytotoxic activity activity of T. terrestris extracts against the HepG-2 cell line was
assessed aer 48h. All the tested extracts showed signicant cytotoxic activity activity compared with the con-
trol (Fig.4). Me methanol-based root extract showed reduced cell viability of liver cancer cells (40.98%) at the
concentration of 80μg/ml.M Extracts in the case of Methanol showed more inhibition and followed the same
trend as ethanol but in a more potent manner.
Anti‑oxidant activity. A comparison of root extracts of plants in two dierent solvents, i.e., ethanol and
Methanol, aer 30min of incubation showed anti-oxidant activity at dierent concentrations (5μg/ml, 10μg/ml,
20μg/ml, 40μg/ml, 80μg/ml and 160μg/ml) (Fig.5). Gallic acid was used as positive control. Methanolic extract
showed maximum inhibition of 66.72% at 80μg/ml concentration compared to ethanol, which was 63.56%.
Scavenging activity increases with concentration but decreases aer 80μg/ml.
LDH assay. e release of LDH is directly proportional to the impairment of the plasma membrane and
the high quantity of damage/death to the cell membrane. However, drastically low LDH was noticed in IL-1β-
Figure1. Eects of T. terrestris root extracts on α-glucosidase inhibition. Plant part root of T. terrestriswere
prepared in two dierent solvents. Extracts were tested at a concentration of (5μg/ml, 10μg/ml, 20μg/
ml, 40μg/ml, 80μg/ml and 160μg/ml). Phosphate buer saline was used as negative control. Acarbose at a
concentration of 3mg/ml was used as a positive control. Bars represent the mean (based on 3 replications of
each treatment).
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% of scratch
Methanol
Ethanol
0
20
40
60
80
Control
5 µg/ml
10 µg/ml
20 µg/ml
40 µg/ml
80 µg/ml
160 µg/m
l
Figure2. Eects of T. terrestris extracts of root on cell proliferation of retinal pigment epithelial (RPE) cells. T.
terrestris extracts were prepared in two dierent solvents (ethanol and Methanol) and dried at 37°C temperature
and. Phosphate buer saline was used as negative control and PDGF was used as positive control. Evaluations
were conducted aer 16h of incubation. Bars showed the mean ± standard deviation (based on 3 replications of
each treatment and 100 observations of scratch width for each replication).
Figure3. Eects of T. terrestris extracts of root on cell proliferation of retinal pigment epithelial (RPE) cells. T.
terrestris extracts were prepared in two dierent solvents (ethanol and Methanol) and dried at 37°C temperature
and. Migration of cells have been noticed aer 0, 4, 8 and 16h of incubation.
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induced Hep2 liver cancer cells preconditioned with T. terrestris extract concentrations compared to stress
(Fig.6).
Superoxide dismutase assay. Superoxide Dismutase (SOD) is a critical anti-oxidative enzyme in cells
that can reduce superoxide ions into less detrimental products (Fig.7). SOD activity is higher in normal cells
than in cells that respond to oxidative stress. We observed higher SOD activity in T. terrestris preconditioned
HepG2 cells compared with IL-1β induced stress group.
Reactive oxygen species assay. e ROS assay determined the percentage of reactive oxygen species
produced by cells. e cell capability decreases with enhanced oxidative stress damage due to increased ROS
production (Fig.8). ROS activity was measured with the help of the Cellular ROS Detection Assay Kit (Abcam,
ab113851DCFDA) according to the manufacturer’s protocol. HepG2 cells at the rate of 25 × 104 were seeded to
a 96-well plate at the p3 stage. e cells were washed three times with 1X PBS and 100μl of DCF-DA working
Figure4. Eects of T. terrestris extract on HepG2 cell line.. T. terrestris extracts were prepared in two dierent
solvents (ethanol and Methanol) and dried at 37°C temperature and. Migration of cells have been noticed
aer 0, 4, 8 and 16h of incubation. Extracts were tested at a concentration of 0.1mg/mL. (5μg/ml, 10μg/ml,
20μg/ml, 40μg/ml, 80μg/ml and 160μg/ml). Bars represent the mean values (based on 3 replications of each
treatment).
% scavenging activity
Methanol
Ethanol
0
20
40
60
80
Positive Control
5 µg/ml
10 µg/ml
20 µg/ml
40 µg/ml
80 µg/ml
160 µg/ml
Figure5. DPPH radical scavenging activity of root extracts in ethanol and zethanol of T. terrestrisaer 30min
of incubation.Bars with dierent alphabets indicate signicant dierences between treatment means at p < 0.05
based on Tukey’s post hoc test.
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solution, which was used for the staining for 1h at 37°C. e DCF-DA media was removed, and the cells were
washed with 1X PBS. A spectrophotometer was used for uorescence readings at 485nm and 535nm.
Protein concentration studies of inammatory cytokines through ELISA. Two dierent con-
centrations (20μg/ml and 40μg/ml) of Tribulusterrestris methanolic plant extract were subjected to assess the
protein concentration of TNF-α and IL-1β through ELISA in the HepG-2 liver cancer cell line (Fig.10A,B). e
ELISA result shows that the methanolic plant extract decreases the protein concentration of IL-1 β at a 40μg/
Figure6. Cytotoxicity decreased by preconditioning of T.terrestris. e release of LDH is directly proportional
to the impairment of the plasma membrane and high quantity damage/death the cell membrane. However,
drastically low LDH was noticed in IL-1β induced HepG2 preconditioned with 40μg/ml and 60μg/ml T.T
concentrations as compared to stress control.
% SOD activity
Ethanol
Methanol
0
20
40
60
80
Control
Stress
5 µg/ml
10 µg/ml
20 µg/ml
40 µg/ml
80 µg/ml
160 µg/m
l
Figure7. Superoxide Dismutase (SOD) is signicant anti-oxidative enzyme present in cells that can reduce
superoxide ions into less detrimental products. SOD activity is higher in normal cells compared to cells that
respond to oxidative stress. We have seen higher SOD activity in T. terrestris preconditioned HepG2 when
compared with IL-1β stimulate HepG2.
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mL concentration. Contrary to this, TNF-α protein expression increases when exposed to 40ug/ml methanolic
extract compared to 20μg/ml.
Downregulation of inammatory marker genes by methanolic plant extract in HepG‑2 liver
cancer cells. In order to evaluate the mRNA expression levels of inammation and apoptosis-associated
genes, including tumor necrosis factor-alpha (TNF-α), matric metalloproteinase-9 (MMP-9), alpha-fetoprotein
(AFP), and B-cell leukemia and lymphoma 2 (Bcl-2), RT-PCR study was performedusing T.terrestrismethanolic
extract treated HepG2 cells line along with GAPDH, which was tested as a housekeeping gene. Methanolic plant
extract of T. terrestris signicantly downregulated the mRNA expression of anti-apoptotic and inammatory
genes in the liver cancer cell line. Figure9 shows the expression variation of TNF-α, MMP-9, AFP, and Bcl-2.
Methanolic plant extract of Tribulusterrestris diminished the expression of tumor-mediated genes in a dose-
dependent manner, particularly at a concentration of 20μg/ml.
Discussion
e concentration of bioactive compounds in plant extract is aected by factors like drying conditions, extraction
protocol, chemical nature, storage conditions, and duration. e solvent used in the extraction of plant material
plays a vital role in the recovery of bioactive compounds, which aects the health benets19,20. Methanol and
ethanol are more commonly used solvents; their boiling points are 64 ℃ and 78.4 ℃, respectively. erefore, in
Figure8. T.T preconditioned enhance the scavenging capacity of reactive oxygen species. Results showed that
the cells preconditioned with 40μg/ml and 60μg/ml T.terrestris concentrations help in decreasing reactive
oxygen species in IL-1β induced HepG2 cells as compared to stress condition.
Figure9. Cytokine concentrations (n = 3) of control, stress and methanolic plant extract treated HepG-2 cells
(a) indicates protein concentration of IL-1β (b) represents the protein concentration of TNF-α. Bars represent
standard devi ation. Moreover, * , * * and * * * indicate the signicance level at p < 0.5, p < 0:01, p < and p < 0:001.
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the case of Methanol, there is a low-temperature requirement to evaporate the solvent in the rotavapor, so there
is less harm to plant extract metabolites than ethanol. In literature, most polar compounds like phenolics, alka-
loids, and terpenes have shown potent anti-diabetic and cytotoxic activity activity when dissolved in methanol
and ethanol solvents13,21,22.
Inhibition of α-glucosidase activity was studied by adding T. terrestris extracts dissolved in ethanol and
Methanol. We tested the extracts prepared in two solvents (Methanol and ethanol) to check the enzymatic
inhibitory response against α-glucosidase (Fig.1). MM Methanol-based root extract showed robust activity at
80μg/ml concentration compared to ethanol.ExtractiMng polyphenols using solvents like Methanol, ethanol,
and acetone are more procient alternatives than organic solvents23. Every solvent has a unique property that
may vary among solvents. While on the other hand, ethanol and Methanol are more impactful for extracting
polyphenols from Hibiscus sabdaria24. According to our results, Methanol provided the highest extraction yield.
Polarity and chemical nature may be the source of variation in phenolic compounds in dierent organic solvents.
In the case of organic solvents ethanol and Methanol, the addition of water creates a more polar medium which
can enhance the yield of phenolic compounds even more, but the maximum concentration of polyphenols was
gained by using pure solvents from the C. citratus25.
e cells treated with the T. Terrestris extracts covered the scratch area more quickly than the negative control
cells. Even the best extracts were more procient than the positive control. Coverage of scratch area by cell prolif-
eration expresses the data as a percentage. Root showed covering of scratch area maximum in case of Methanol
compared to ethanol (Figs.2 and 3). In the case of wound healing, the essential stage is cell proliferation, and
a variety of plant species have been found to study the coverage of wound healing by using the scratch test26.
Our research showed magnicent results on wound healing by migration and proliferation of cells by using the
extract of T. terrestris at a rapid rate, even higher than any positive control. In earlier studies, the optimal time
aer adding extracts treatment was 12–72h to estimate the cell proliferation, though the time of cell proliferation
may vary depending on the cell type, plant extract, and width of scratch27.
In the present study, the optimum time for cell proliferation was 0–16h aer treating cells with the extract.
Moreover, roots used for the Mmethanol extraction had meaningful conclusive results on cell proliferation. A
compelling study of cell proliferation of freeze-dry leaves of Plantago major showed that the optimum concen-
tration was 1.0mg/mL on a dry weight basis, and in contrast, 0.1mg/mL was better than 10mg/mL28. It is also
noted that ethanol-based extracts with a higher concentration (10mg/mL)had a deleterious impact on cell mul-
tiplication and migration. In our research, concentration was conned to 0.1mg/mL in dierent parts of plant
extracts. Plant extracts with the most optimum concentration of particular compounds may obstruct or retard
cell migration and proliferation29. On the other hand, extract taken from the plant of legume genus Astragalus
and Simmondsia jojoba seeds (jojoba liquid wax; 5% v/v) induced slow migration and proliferation of cells, while
compared to the extracts of lower concentration (1ng/mL and 1%v/v) respectively30.
e investigation of medicinal plants for the potential cytotoxic activity eect is assessed by evaluating their
ability to neutralize cancer cells invitro. In the present study, extracts of T. terrestris reduced the viability of a
HepG2 cancer cell line (Fig.4), conrming the previous results of cytotoxic activity9 M31,32.
In literature, potent co-relationships have been reported between cell cytotoxicity evaluated by the LDH assay
and anti-oxidant avonoids and total phenolic compounds assessed through DPPH assay measuring radical
scavenging activity. e main components of T. terrestris species have avonoids and phenylethanoids possessing
anti-oxidant activity. eyare responsible for protecting the cell membrane from oxidative damage, which may
improve cell viability and reduce cytotoxicity was later conrmed by the DPPH assay33,34 (Fig.5). In the current
study, an improvement in cell survival was detected in the LDH assay (Fig.6). LDH assay was used to monitor
the amount of lysed cells by any damage in the plasma membrane, and the results showed indirect detection of
necrosis linked to the leakage of the cytosolic enzyme due to loss of control of membrane permeability. In the
case of DPPH assay, the study highlighted the signicant anti-oxidant potential of polyphenol-rich T. terrestris
extract. rough its anti-oxidant capacity, its pretreatment inhibited mitochondrial alterations and necrosis
caused by ischemia. Anti-oxidant metabolites inhibit and remedy oxidative stress-related diseases like diabetes,
cancer, Alzheimer’s disease, atherosclerosis, and stroke35,36. Moreover, the study of the anti-oxidant activity of
our plant extract exhibited potent activity to scavenge the free radical DPPH.
Most diseases and disorders are mainly associated with oxidative stress due to free radicals. Free radicals are
the base of any biochemical process and represent an essential part of metabolism and aerobic life37. Oxidative
stress describes the presence of free radicals and reactive oxygen species, which are produced under physiological
conditions but become more unsafe and deleterious when not removed by the endogenous system38. However,
oxidative stress will dierentiate endogenous anti-oxidant and reactive oxygen species. ROS results in various
diseases like cardiovascular disease, cancer, Alzheimer’s disease, cytotoxic, and causes tissue injuries39 Anti-
oxidantssupply electrons to the free radicals and thus reduce the adverse eects by stabilizing them. Naturally,
anti-oxidants in leafy vegetables and seeds such as vitamin E, Vitamin C, and phenolic compounds can decrease
the oxidative damage linked with many diseases, including diabetes, immune-decient arthritis, cancer, cardio-
vascular and ageing40,41. Phenolics and avonoids have been reported to hold robust anti-oxidant properties.
e anti-oxidant activity of polyphenols helps prevent and cure diseases that are majorly associated with free
radicals42. In the current study, Methanol and ethanol-basedplant root extractMshowed anti-oxidant activity
aer 30min of incubation. Methanol has more inhibition with increasing concentrations up to 80μg/ml (Fig.7).
e TNF-α and IL-1β markers were also examined at the protein level using ELISA, which demonstrated a
substantial decrease in methanolic plant extract treated groups (Fig.9). ese ndings are consistent with earlier
studies43. e current investigation found a signicant drop in TNF-α and IL-1β protein levels, resulting in the
downregulation of pro-inammatory markers, implying that plant extract has an inhibitory inuence on these
markers.
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e methanol extract of the Tribulusterrestris plant showed superior activity to its counterpart ethanol-based
extract in various assays. As a result, we investigated the gene expression proling of inammatory marker genes
in the HepG-2 liver cancer cell line aer treatment with methanol extract. Among cancer patients, those with
diabetes have a higher occurrence of all-cause mortality than those without diabetes. Probable metabolic links
between diabetes mellitus (DM) and cancer development are hyperglycemia, hyperinsulinemia, and chronic
inammation.e biochemical process of insulin signaling proceeds by insulin receptor autophosphorylation
itself or by directly maintaining the substrate by activating multiple pathways including lipid kinase PI3K/Akt,
Bax/Bcl-2, and mTOR leading to carcinogenesis by abnormal cell proliferation and retardation of apoptosis44,45.
IL-1β was used to trigger various inammatory signal transduction pathways. erefore, we utilized it to
simulate oxidative stress and inammatory conditions invitro. It is widely understood that numerous signal-
ing pathways are involved in the inammatory process, which impairshomeostasis46. When methanolic plant
extract treated HepG-2 cells were compared to their counterpart IL-1β treated stress group, there was a sub-
stantial decrease in mRNA expression of TNF-α, AFP, Bcl-2, and MMP-9 (Fig.10). MMP-9 (matrix metallo-
proteinase-13) is a metalloproteinase enzyme involved in the breakdown of the extracellular matrix. MMPs are
overexpressed in inammatory situations. MMP-9, despite its involvement in dierentiation, aids in activating
TNF pro-inammatory markers by cleaving pro-TNF into bioactive TNF47,48. One study reported that silencing
of LPA1 notably attenuated LPA-induced MMP-9 expression and HCC cell invasion. e induction of MMP-9
through coordinated activation of PI3K and p38 MPAK signaling cascades provides novel biomarkers and prob-
able therapeutic targets for HCC.
On the other hand, increased MMP9 activity has been shown to play a role in stimulating endothelial cell
apoptosis and dysfunction in diabetic mice. MMP-9 knockout in diabetic mice prominently decreased the
nephropathy changes. MMP-9 disrupted podocyte cell integrity, promoting podocyte monolayer permeability
to albumin and extracellular matrix protein synthesis. In diabetic patients, urinary MMP-9 concentrations were
upregulated before the onset of microalbuminuria. us, MMP-9 appears to play a role in the development of
diabetic nephropathy49–51.
Compared to control cells, methanolic plant extract of T.terrestris induced a signicant drop in Bcl-2 levels
in the current investigation. It implies that the plant extract promotes a pro-apoptotic tendency in HepG2 cells.
is activity has been reported in previous studies in the case of MCF-7 breast cancer cell lines52. e Bcl-2
gene produces an apoptosis-blocking protein in the mitochondrial membrane. It is over-expressed in a variety
of malignancies53. High levels of AFP can indicate liver cancer, ovarian or testicular cancer, and non-cancerous
liver disorders such as cirrhosis and hepatitis. AFP levels may be raised due to tumor development or regenerated
hepatocytes. Many patients with HCC have elevated AFP serum expression levels, and a continuously elevated
AFP level is a risk factor for HCC development54,55. Many studies have shown that AFP acts as an immune sup-
pressor, promotes malignant transformation during HCC development, and may be involved in the MDR process
Figure10. e mRNA expression level of anti-apoptotic and inammatory markers in HepG-2 liver cancer
cell line treated with methanolic plant extract of T.terrestris(a) indicates mRNA expression of AFP, (b) reveals
mRNA levels of Bcl-2, (c) fold change of MMP-9, (d) eect on TNF-α mRNA expression. Bars highlight
standard devi ation. * , * * and * * * indicate the signicance level at p < 0.5, p < 0:01, p < and p < 0:001.
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in patients with liver cancer56. In the current study, mRNA expression was signicantly downregulated in the
HepG-2 cancer cell line aer treatment with plant extract.
Previous studies have reported that matrix metalloproteinases can activate IL-1β via proteolytic processing.
is study revealed that IL-1β-induced HepG-2 cells exhibited higher MMP-9 expression than their untreated
group. Previous research has also shown that MMP-9 is down-regulated in human HNSCC, HN22, HSC-3, and
RAW 264.7 cell lines when treated with various plant extracts57,58.
TNF-α and IL-1β marker expression levels were also measured to determine gene regulation of these
cytokines. IL-1β binds to its receptor leading to the activation of macrophages, intratumoral assembly of immu-
nosuppressive myeloid cells, invasiveness, tumor growth, metastasis, and angiogenesis. is master inam-
matory cytokine has also been reported to contribute to the failure of β-cell in the pancreas46. Various studies
have reported that therapeutic interventions targeting IL-1β have ameliorated DM disease severity, albeit with
variegated results in numerous clinical trials. TNF-α is an adipocytokine involved in developing insulin resistance
and the pathogenesis of T2DM by disrupting insulin signaling through serine phosphorylation. e binding of
TNF-α to TNFRII leads to activation of the NF-κB, JNK, p38 MAPK, ERK, and PI3K pathways. It induces tissue-
specic inammation by association with ROS generation59. e literature has reported that necrosis of DEN-
exposed hepatocytes activated neighboring myeloid cells in the liver to produce hepatic mitogens that endorsed
compensatory explosion of surviving and mutated hepatocytes; TNF-α was one of the signicant liver mitogens.
One study demonstratedthe deletion of IKKβ in hepatic Kuper cells, which resulted in decreased TNF-α, IL-6,
and HGF and reduced liver tumorigenesis60,61. Our ndings show a decrease in TNF-α mRNA expression and
protein levels of TNF-α and IL-1β in T.terrestris treated methanolic plant extract. Likewise, down-regulation of
IL-1β and TNF-α at the mRNA and protein levels has recently been described in Lipopolysaccharide-Induced
Human Monocyte-derived Macrophagesyy62,63. erefore, we can conclude that inhibiting these molecular moie-
ties can dampen the progression of T2DM and malignancy. In this regard, T. terrestris plant extract exhibited
robust anti-diabetic and cytotoxic activity activities, which can bestow potent drug candidates.
Conclusion
e current study reports anti-diabetic, anti-inammatory, anti-oxidant, and anti-proliferative properties of T.
terrestris plant extract treated HepG2 liver cancer cell line. T. terrestrismethanolic plant extract alleviated the
adverse eects of IL-1β induced stress invitro via activating the inammatory pathways. e methanolic plant
extract of T. terrestris reduced the expression of inammatory mediators and cytokines in a dose-dependent
manner, which has a favorable eect on various inammatory disorders, including diabetes and cancer. e
ndings could help with the therapeutic applications T.terrestris by identifying potential drug metabolites to
treat inammatory diseases.
Data availability
All data generated or analyzed during this study are included in the manuscript.
Received: 30 March 2022; Accepted: 20 December 2022
References
1. Tomic, D., Shaw, J. E. & Magliano, D. J. e burden and risks of emerging complications of diabetes mellitus. Nat. Rev. Endocrinol.
18, 525–539. https:// doi. org/ 10. 1038/ s41574- 022- 00690-7 (2022).
2. Debela, D. T. et al. New approaches and procedures for cancer treatment: Current perspectives. SAGE Open Med. 9,
20503121211034370. https:// doi. org/ 10. 1177/ 20503 12121 10343 66 (2021).
3. Curigliano, G. & Criscitiello, C. Successes and limitations of targeted cancer therapy in breast cancer. Progress in Tumor Res. 41,
15–35. https:// doi. org/ 10. 1159/ 00035 5896 (2014).
4. Khan, T. et al. Anticancer plants: A review of the active phytochemicals, applications in animal models, and regulatory aspects.
Biomolecules https:// doi. org/ 10. 3390/ biom1 00100 47 (2019).
5. Kumar Srivastava, A. in Synthesis of Medicinal Agents from Plants (eds Ashish Tewari & Supriya Tiwari) 1–24 (Elsevier, 2018).
6. Ozgok Kangal, M. K. & Regan, J. P. in StatPearls (StatPearls Publishing Copyright © 2022, StatPearls Publishing LLC., 2022).
7. Chhatre, S., Nesari, T., Somani, G., Kanchan, D. & Sathaye, S. Phytopharmacological overview of Tribulus terrestris. Pharmacogn.
Rev. 8, 45–51. https:// doi. org/ 10. 4103/ 0973- 7847. 125530 (2014).
8. Han, R., Yang, H., Lu, L. & Lin, L. Tiliroside as a CAXII inhibitor suppresses liver cancer development and modulates E2Fs/
Caspase-3 axis. Sci. Rep. 11, 8626. https:// doi. org/ 10. 1038/ s41598- 021- 88133-7 (2021).
9. Yonbawi, A. R. & Abdallah, H. M. Anti-proliferative, cytotoxic and antioxidant properties of the methanolic extracts of ve saudi
arabian ora with folkloric medicinal use: aizoon canariense, citrullus colocynthis, maerua crassifolia. Rhazya stricta and Tribulus
macropterus. https:// doi. org/ 10. 3390/ plant s1010 2073 (2021).
10. Khalid, A. et al. Phytochemical, cytotoxic, and antimicrobial evaluation of Tribulus terrestris L., Typha domingensis Pers., and
Ricinus communis L.: Scientic evidences for folkloric uses. Evidence-Based Complement. Altern. Med.: eCAM 2022, 6519712.
https:// doi. org/ 10. 1155/ 2022/ 65197 12 (2022).
11. Abubakar, A. R. & Haque, M. Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental
purposes. J. Pharm. Bioall. Sci. 12, 1–10. https:// doi. org/ 10. 4103/ jpbs. JPBS_ 175_ 19 (2020).
12. Onyebuchi, C. & Kavaz, D. Eect of extraction temperature and solvent type on the bioactive potential of Ocimum gratissimum
L. extracts. Sci. Rep. 10, 21760. https:// doi. org/ 10. 1038/ s41598- 020- 78847-5 (2020).
13. Ma, Y. L. et al. Solvent eect on phenolics and antioxidant activity of Huangshan Gongju (Dendranthema morifolium (Ramat)
Tzvel. cv. Gongju) extract. Food and Chem. Toxicol.: An Int. J. Publis. British Indus. Biol. Res. Assoc. 147, 111875, https:// doi. org/
10. 1016/j. fct. 2020. 111875 (2021).
14. Omar, G. & Abdallah, L. Invitro haemostatic ecacy of aqueous, methanol and ethanol plant extracts of three medicinal plant
species in Palestine. Braz. J. Biol. 80, 763–768, https:// doi. org/ 10. 1590/ 1519- 6984. 219186 (2020).
15. Sun, X. C. et al. Terrestrosin D, a spirostanol saponin from Tribulus terrestris L. with potential hepatorenal toxicity. J. Ethnophar-
macol. 283, 114716, https:// doi. org/ 10. 1016/j. jep. 2021. 114716 (2022).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

12
Vol:.(1234567890)
Scientic Reports | (2022) 12:22478 | https://doi.org/10.1038/s41598-022-26742-6
www.nature.com/scientificreports/
16. Figueiredo, C. C. M. et al. Antiglycation and antitumoral activity of Tribulus terrestris dry extract. Avicenna J. Phytomed. 11,
224–237 (2021).
17. Misiakiewicz-Has, K. et al. e obscure eect of tribulus terrestris saponins plus inulin on liver morphology, liver fatty acids,
plasma glucose, and lipid prole in SD rats with and without induced type 2 diabetes mellitus. Int. J. Mol. Sci. https:// doi. org/ 10.
3390/ ijms2 21686 80 (2021).
18. Figueiredo, C. C. M. et al. Antiglycation and antioxidant activities of the crude extract and saponin fraction of Tribulus terrestris
before and aer microcapsule release. J. Int. Med. 20, 153–162. https:// doi. org/ 10. 1016/j. joim. 2021. 12. 003 (2022).
19. Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K. M. & Yoga Latha, L. Extraction, isolation and characterization of bioactive
compounds from plants’ extracts. Afr. J. Tradit. Complement Altern. Med. 8, 1–10 (2011).
20. Dawurung, C. J. et al. Isolation of bioactive compounds from medicinal plants used in traditional medicine: Rautandiol B, a
potential lead compound against Plasmodium falciparum. BMC Complement. Med. erap. 21, 231. https:// doi. org/ 10. 1186/
s12906- 021- 03406-y (2021).
21. Onyebuchi, C. & Kavaz, D. Eect of extraction temperature and solvent type on the bioactive potential of Ocimum gratissimum
L. extracts. Sci. Rep. 10, 21760, https:// doi. org/ 10. 1038/ s41598- 020- 78847-5 (2020).
22. Seow, L. J., Beh, H. K., Umar, M. I., Sadikun, A. & Asmawi, M. Z. Anti-inammatory and antioxidant activities of the methanol
extract of Gynura segetum leaf. Int. Immunopharmacol. 23, 186–191. https:// doi. org/ 10. 1016/j. intimp. 2014. 08. 020 (2014).
23. Iloki-Assanga, S. B. et al. Solvent eects on phytochemical constituent proles and antioxidant activities, using four dierent
extraction formulations for analysis of Bucida buceras L. and Phoradendron californicum. BMC Res. Notes 8, 396, https:// doi. org/
10. 1186/ s13104- 015- 1388-1 (2015).
24. Singh, M., rimawithana, T., Shukla, R. & Adhikari, B. Extraction and characterization of polyphenolic compounds and potas-
sium hydroxycitrate from Hibiscus sabdaria. Future Foods 4, 100087, https:// doi. org/ 10. 1016/j. fufo. 2021. 100087 (2021).
25. Muala, W. C. B., Desobgo, Z. S. C. & Jong, N. E. Optimization of extraction conditions of phenolic compounds from Cymbopogon
citratus and evaluation of phenolics and aroma proles of extract. Heliyon 7, e06744–e06744. https:// doi. org/ 10. 1016/j. heliy on.
2021. e06744 (2021).
26. Shedoeva, A., Leavesley, D., Upton, Z. & Fan, C. Wound Healing and the Use of Medicinal Plants. Evid.-Based Complement. Alternat.
Med. https:// doi. org/ 10. 1155/ 2019/ 26841 08 (2019).
27. Saud, B., Malla, R. & Shrestha, K. A review on the eect of plant extract on mesenchymal stem cell proliferation and dierentiation.
Stem Cells Int. 2019, 7513404. https:// doi. org/ 10. 1155/ 2019/ 75134 04 (2019).
28. Kartini, Piyaviriyakul, S., ongpraditchote, S., Siripong, P. & Vallisuta, O. Eects of Plantago major Extracts and Its Chemical
Compounds on Proliferation of Cancer Cells and Cytokines Production of Lipopolysaccharide-activated THP-1 Macrophages.
Pharmacognosy Magaz. 13, 393–399, https:// doi. org/ 10. 4103/ pm. pm_ 406_ 16 (2017).
29. Khan, M., Khan, M., Adil, S. F. & Alkhathlan, H. Z. Screening of potential cytotoxic activities of some medicinal plants of Saudi
Arabia. Saudi J. Biol. Sci. 29, 1801–1807. https:// doi. org/ 10. 1016/j. sjbs. 2021. 10. 045 (2022).
30. Al-Obaidi, J. R. et al. A review on plant importance, biotechnological aspects, and cultivation challenges of jojoba plant. Biol. Res.
50, 25. https:// doi. org/ 10. 1186/ s40659- 017- 0131-x (2017).
31. Zhu, F., Du, B. & Xu, B. Anti-inammatory eects of phytochemicals from fruits, vegetables, and food legumes: A review. Crit.
Rev. Food Sci. Nutr. 58, 1260–1270. https:// doi. org/ 10. 1080/ 10408 398. 2016. 12513 90 (2018).
32. Tasneem, S., Liu, B., Li, B., Choudhary, M. I. & Wang, W. Molecular pharmacology of inammation: Medicinal plants as anti-
inammatory agents. Pharmacol. Res. 139, 126–140. https:// doi. org/ 10. 1016/j. phrs. 2018. 11. 001 (2019).
33. Jing, L., Ma, H., Fan, P., Gao, R. & Jia, Z. Antioxidant potential, total phenolic and total avonoid contents of Rhododendron
anthopogonoides and its protective eect on hypoxia-induced injury in PC12 cells. BMC Complement. Altern. Med. 15, 287. https://
doi. org/ 10. 1186/ s12906- 015- 0820-3 (2015).
34. Zhu, W., Du, Y., Meng, H., Dong, Y. & Li, L. A review of traditional pharmacological uses, phytochemistry, and pharmacological
activities of Tribulus terrestris. Chem. Cent. J. 11, 60. https:// doi. org/ 10. 1186/ s13065- 017- 0289-x (2017).
35. Apostolova, N. & Victor, V. M. Molecular strategies for targeting antioxidants to mitochondria: therapeutic implications. Antioxid.
Redox Signal. 22, 686–729. https:// doi. org/ 10. 1089/ ars. 2014. 5952 (2015).
36. Banerjee, J., Das, A., Sinha, M. & Saha, S. Biological ecacy of medicinal plant extracts in preventing oxidative damage. Oxidat.
Med. Cell. Longevity https:// doi. org/ 10. 1155/ 2018/ 79043 49 (2018).
37. Unsal, V., Cicek, M. & Sabancilar, I. Toxicity of carbon tetrachloride, free radicals and role of antioxidants. Rev. Environ. Health
https:// doi. org/ 10. 1515/ reveh- 2020- 0048 (2021).
38. Liguori, I. et al. Oxidative stress, aging, and diseases. Clin. Interv. Aging 13, 757–772. https:// doi. org/ 10. 2147/ cia. s1585 13 (2018).
39. Yang, S. & Lian, G. ROS and diseases: Role in metabolism and energy supply. Mol. Cell. Biochem. 467, 1–12. https:// doi. org/ 10.
1007/ s11010- 019- 03667-9 (2020).
40. Aiello, P. et al. Medicinal plants in the treatment and prevention of colon cancer. Oxidative Med. Cell. Longev 2019, 2075614.
https:// doi. org/ 10. 1155/ 2019/ 20756 14 (2019).
41. Chen, J. T. Phytochemical omics in medicinal plants. Biomolecules https:// doi. org/ 10. 3390/ biom1 00609 36 (2020).
42. Alkadi, H. A review on free radicals and antioxidants. Infect. Disord. Drug Targets 20, 16–26. https:// doi. org/ 10. 2174/ 18715 26518
66618 06281 24323 (2020).
43. Lee, H. H., Ahn, E.-K., Hong, S.-S. & Oh, J. S. Anti-inammatory eect of tribulusamide D isolated from Tribulus terrestris in
lipopolysaccharide-stimulated RAW264.7 macrophages. Mol. Med. Rep. 16, 4421–4428, https:// doi. org/ 10. 3892/ mmr. 2017. 7208
(2017).
44. Zhu, B. & Qu, S. e relationship between diabetes mellitus and cancers and its underlying mechanisms. Front. Endocrinol. 13,
800995, https:// doi. org/ 10. 3389/ fendo. 2022. 800995 (2022).
45. Toda, G. et al. Insulin- and lipopolysaccharide-mediated signaling in adipose tissue macrophages regulates postprandial glycemia
through Akt-mTOR activation. Mol. Cell 79, 43-53.e44. https:// doi. org/ 10. 1016/j. molcel. 2020. 04. 033 (2020).
46. Bent, R., Moll, L., Grabbe, S. & Bros, M. Interleukin-1 Beta-A Friend or Foe in Malignancies?. Int. J. Mol. Sci. https:// doi. org/ 10.
3390/ ijms1 90821 55 (2018).
47. Joseph, C. et al. Elevated MMP9 expression in breast cancer is a predictor of shorter patient survival. Breast Cancer Res. Treat. 182,
267–282. https:// doi. org/ 10. 1007/ s10549- 020- 05670-x (2020).
48. Mondal, S., Adhikari, N., Banerjee, S., Amin, S. A. & Jha, T. Matrix metalloproteinase-9 (MMP-9) and its inhibitors in cancer: A
minireview. Eur. J. Med. Chem. 194, 112260, https:// doi. org/ 10. 1016/j. ejmech. 2020. 112260 (2020).
49. Shi, W. et al. CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis. Annals
Trans. Med. 9, 42, https:// doi. org/ 10. 21037/ atm- 20- 1013 (2021).
50. Augo, K., Hryniewicz-Jankowska, A., Tabola, R. & Stach, K. MMP9: A Tough Target for Targeted erapy for Cancer. Cancers
14, https:// doi. org/ 10. 3390/ cance rs140 71847 (2022).
51. Scheau, C. et al. e role of matrix metalloproteinases in the epithelial-mesenchymal transition of hepatocellular carcinoma. Anal.
Cell. Pathol. https:// doi. org/ 10. 1155/ 2019/ 94239 07 (2019).
52. Patel, A., Soni, A., Siddiqi, N. J. & Sharma, P. An insight into the anticancer mechanism of Tribulus terrestris extracts on human
breast cancer cells. 3 Biotech 9, 58–58, https:// doi. org/ 10. 1007/ s13205- 019- 1585-z (2019).
53. Knight, T., Luedtke, D., Edwards, H., Taub, J. W. & Ge, Y. A delicate balance - e BCL-2 family and its role in apoptosis, onco-
genesis, and cancer therapeutics. Biochem. Pharmacol. 162, 250–261. https:// doi. org/ 10. 1016/j. bcp. 2019. 01. 015 (2019).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

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Vol.:(0123456789)
Scientic Reports | (2022) 12:22478 | https://doi.org/10.1038/s41598-022-26742-6
www.nature.com/scientificreports/
54. Wang, X. & Wang, Q. Alpha-fetoprotein and hepatocellular carcinoma immunity. Can. J. Gastroenterol. Hepatol. 2018, 9049252.
https:// doi. org/ 10. 1155/ 2018/ 90492 52 (2018).
55. Liu, S. et al. Diagnostic value of serum glypican-3 alone and in combination with AFP as an aid in the diagnosis of liver cancer.
Clin. Biochem. 79, 54–60. https:// doi. org/ 10. 1016/j. clinb iochem. 2020. 02. 009 (2020).
56. Li, W., Liu, K., Chen, Y., Zhu, M. & Li, M. Role of alpha-fetoprotein in hepatocellular carcinoma drug resistance. Curr. Med. Chem.
28, 1126–1142. https:// doi. org/ 10. 2174/ 09298 67327 99920 07291 51247 (2021).
57. Alsaraf, K. M., Mohammad, M. H., Al-Shammari, A. M. & Abbas, I. S. Selective cytotoxic eect of Plantago lanceolata L. against
breast cancer cells. J. Egypt. Nat. Cancer Inst. 31, 10, https:// doi. org/ 10. 1186/ s43046- 019- 0010-3 (2019).
58. Khan, Y. H. et al. Potential role of phytochemicals against matrix metalloproteinase induced breast cancer; an explanatory review.
Front. Chem. 8 (2021).
59. Sabio, G. & Davis, R. J. TNF and MAP kinase signalling pathways. Semin. Immunol. 26, 237–245. https:// doi. org/ 10. 1016/j. smim.
2014. 02. 009 (2014).
60. Jing, Y. et al. Tumor necrosis factor-α promotes hepatocellular carcinogenesis through the activation of hepatic progenitor cells.
Cancer Lett. 434, 22–32. https:// doi. org/ 10. 1016/j. canlet. 2018. 07. 001 (2018).
61. Yang, J. et al. Myeloid IKKβ promotes antitumor immunity by modulating CCL11 and the innate immune response. Cancer Res
74, 7274–7284. https:// doi. org/ 10. 1158/ 0008- 5472. can- 14- 1091 (2014).
62. Fereydouni, Z., Amirinezhad Fard, E., Mansouri, K., Mohammadi Motlagh, H.-R. & Mostafaie, A. Saponins from Tribulus terrestris
L. Extract Down-regulate the Expression of ICAM-1, VCAM-1 and E-selectin in Human Endothelial Cell Lines. Int. J. Mol. Cell
Med. 9, 73–83, https:// doi. org/ 10. 22088/ IJMCM. BUMS.9. 1. 73 (2020).
63. Zhao, W.-R. et al. Tribulus terrestris L. Extract Protects against Lipopolysaccharide-Induced Inammation in RAW 264.7 Mac-
rophage and Zebrash via Inhibition of Akt/MAPKs and NF-κB/iNOS-NO Signaling Pathways. Evid.-Based Complement. Alternat.
Med.: eCAM 2021, 6628561–6628561, https:// doi. org/ 10. 1155/ 2021/ 66285 61 (2021).
Author contributions
A.K. conducted research. T.N. and M.A.K. write up the initial dra of the manuscript. Q.A., and M.Z. helped in
the analysis of data and nal corrections in the manuscript. All authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to M.A.K.orQ.A.
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