Contents

1. Introduction
2. Objectives
3. Materials and methods
4. Results
5. Conclusion
6. Summary
7. References
INTRODUCTI

ON
Medicinal plants are plants that contain beneficial compounds used to treat diseases and maintain health, playing a key role in
traditional medicine and serving as a source for modern phaemaceuticals.
 Nowadays, there is great concern about the use of herbal medicines worldwide. Additionally, laboratory studies have been
conducted to explore the pharmacological characteristics of bioactive compounds and their potential to treat a range of illnesses
in both animals and humans.
 The integration of traditional medicine and ethnopharmacology has facilitated the introduction of numerous novel drugs into
the global market (Shah et al., 2010).
 For several decades, herbal remedies have been used to treat and control a variety of diseases.
 Herbal medicines represent a potential substitute for traditional artificial treatments owing to the minimal side effects and are
considered safe and effective in treating human ailment (Ashraf et al., 2023).
 Every medicinal plant used in traditional medicine should be scientifically evaluated to reveal its active ingredient, which can
be used as a phytomedicine (Ali et al., 2022).
 Various herbs are claimed to improve human sexual dysfunction in this vast collection of phytoproducts (Changxing et al.,
2018).
 The entire T. terrestris plant’s phytochemical, as well as phytopharmacological activities, have been intensively explored,
including diuretic, anti-urolithiasis, anti-hypertensive, analgesic, anti-hyperlipidemic, immunomodulatory, antidiabetic,
anticancer, anti-helminthic, aphrodisiac, antibacterial, hepatoprotective, and anti-inflammatory properties (Yanala et al., 2016).
 T. terrestris is a creeping annual (occasionally perennial) herb, and the plant, especially the fruits, has traditionally been utilized
for sexual health advantages.
 T. terrestris can flourish in various soil types, but it particularly prospers in loose, arid, sandy soils found near dunes or in
loose, fertile soils along the borders of fields.
 Many different compounds with a variety of biological properties and chemical structures have been identified from T.
terrestris including steroidal saponins, flavonoids, glycosides, phytosterols, tannins, terpenoids, amide derivatives, amino acids,
and proteins. Among the different types of constituents, steroidal saponins and flavonoids are considered to be the most
important metabolites with various bioactivities (Wang et al., 1990).
 Gas Chromatography-Mass Spectroscopy (GC-MS) analysis for T. terrestris extract revealed that it contains many
compounds, most of which have been recognized for their biologically active functions like hexadecanoic acid,
octadecanoic acid, phytol, rhodoxanthin, α-Amyrin, cholestane, etc (Abirami & Rajendran 2011).
 T. terrestris was extracted with different solvents (methanol, petroleum ether, chloroform, and ethanol). Te results showed that
methanol extract has the highest inhibition zone for Bacillus cereus, Escherichia coli and Staphylococcus aureus. For
Staphylococcus aureus and Pseudomonas aeruginosa, WETT also had a certain inhibitory effect (Kiran et al., 2011).
 T. terrestris exhibits in-vitro anticancer activity by inducing apoptosis (programmed cell death) and inhibiting the growth of
various cancer cell lines, including breast and liver cancer cells. Its effects are attributed to its rich content of bioactive
compounds, such as saponins, flavonoids, phenols and steroids.

Considering all the aspects, the development of phytochemistry is one of the most exciting approaches for cancer
treatment using phytochemicals. Therefore, we carried out the present investigation with the following objectives.
 OBJECTIVES

1. To identify the preliminary phytochemicals in stem extract of

Tribulus terrestris.

2 . To analyze the GC-MS and antibacterial activity of Tribulus terrestris

stem extracts.

3. To investigate the in-vitro anti-cancer activity of Tribulus terrestris

against human colorectal carcinoma (HCT-116) cell line by MTT assay.
MATERIALS AND METHODS
Botanical description of Tribulus terrestris
Tribulus terrestris
Kingdom – Plantae
Division – Magnoliophyta
Class – Magnoliopsida
Order – Zygophyllales
Family – Zygophyllaceae
Genus – Tribulus
Species – terrestris L.
Tribulus terrestris L. is a plant that grows especially in South Africa, Australia, India, and Europe. It is part of the
Zygophyllaceae family, a widespread family with 25 genera and about 250 species. TT is a crawling herbal plant that generally
grows in arid climates and sandy soils and grows up to one meter high. The name Tribulus comes from the Greek name
“tribolos” which means spike fruit. The fruits are used in traditional Chinese medicine (TCM), in Ayurvedic medicine in India,
and traditional medicine in Bulgaria for the treatment of different conditions (Pokrywka et al., 2014).
Many compounds with a variety of biological properties and chemical structures have been identified in TT extract,
especially steroidal saponins, flavonoids, tannins, terpenoids, polyphenol carboxylic acids, and alkaloids. The composition of
TT extract depends on various factors such as the extraction method and whether roots, stem, leaves, or fruits have been used.
COLLECTION OF PLANT MATERIAL
Fresh plant material of Tribulus terrestris was collected from Chitradurga. Plant material washed under running tap water 2-3
times to remove soil and dust particles and dust. The plant material was shade dried for 15 days. After drying plant materials
(stem part) grinded into fine powder and then transfer into airtight container with proper labeling.
PREPARATIONS OF PLANT EXTRACTS
Powdered Tribulus terrestris stem extract of 50 grams were extracted using the Soxhlet extraction method with 500 mL organic
solvents including methanol, aqueous, and chloroform in a Soxhlet apparatus. The extraction duration was set to 40 cycles, and
the temperature was adjusted not beyond the boiling point of the solvents. Then, the obtained extracts were filtered and the
filtrates were concentrated under reduced pressure with a rotary evaporator at 80 ℃, prior to drying process in a freeze dryer.
The dried extract was stored at 4℃.
PHYTOCHEMICAL ANALYSIS
The aqueous, methanolic and chloroform extract of the leaves were assessed for the presence of various phytoconstituents. the
crude solvents extracts were re-suspended in their respective solvents at a concentration of 10 μg/mL and analyzed for
qualitative tests, including alkaloids, flavonoids, tannins, saponins, glycosides, steroids, phenols and xanthoprotein. (Poonam et
al., 2016).
Test for Alkaloids:
Mayer’s test) 2 mL of sample extract was treated with 2-3 drops of Mayer’s reagent. Formation of white or yellow precipitate
indicates the presence of alkaloids.
Test for Flavanoids: 2 mL of sample and to it 0.1N NaOH (Sodium hydroxide) and 0.1 N HCl (Hydrochloric acid) are added.
If there is formation of yellow or orange color, it indicates the presence of Flavanoids.
Test for Steroids: (Salkowaski test) :
2 mL of sample is taken and to it 1 mL of Chloroform and 1mL of concentrated H2SO4 (Sulphuric acid) are added.
Formation of bluish red on upper layer, lower layer with yellowish green color indicates the presence of Steroids.
Test for Saponins: (Froth test) :
2 mL of sample extract was treated with 10 mL of distilled water on vigorous shaking formation of froth indicates the
presence of saponins.
Test for Terpenoids: (Libermann Buchard’s test) :
2 mL of extract was taken and to it 0.5 mL of acetic acid , 1 mL of concentrated H2SO4 (Sulphuric acid) are added to the
sides of test tube .Formation of bluish green color indicates the presence of Terpenoids.
Test for Carbohydrates: (Benedict’s test):
To 1 mL of extract 5mL of Benedict’s reagent is added and boiled in hot water bath for 5 min. If there is formation of brick
red color precipitation, it indicates the presence of carbohydrates.
Test for Proteins: (Biuret test) :
To 2 mL of extract add few drops of Biuret reagent. If there is formation of violet red colour, it indicates the presence of
proteins.
GC-MS ANALYSIS
 Using a gas chromatography & mass spectrometer (GC-2010) with 200V electron ionization (EI) energy. The methanolic
extract was analyzed using GC-MS.

 A splitless injection was used for introduction & the split ratio was set to 50:0 with injection temperature of 260 ℃.

 By injecting 1 mL of solvent extract into ELITE C-5 column, at a constant flow rate of 1mL/min, helium gas was
employed as an inert carrier gas.

 Other GC-MS conditions are ion-source temperature 200 ℃, interface temperature 280, pressure 57 Kpa.

 The injector temperature of the oven was programmed to start at 60 ℃ & then the temperature was increased at a rate of
20 ℃ per min to 280 ℃ & maintained for 5 min, & the total run time for spectral analysis was 40 min.

 After comparing the obtained spectral configurations with available mass spectral database (Nation Institute of Standard
and Technology (NIST)), the compounds were identified. (Shashiraj et al., 2022).
ANTIBACTERIAL ACTIVITIY OF TRIBULUS TERRESTRIS STEM METHANOLIC
EXTRACT
After the inoculation of the inoculums, it is spread uniformly over the medium using L-shaped glass rod. 6 mm
diameter wells were bored on 4 mm thick NA (nutrient agar at pH 7.4) plates and approximately 25, 50, 70, and 100 µL
concentrations of Tribulus terrestris stem methanolic extract were poured into each well. The sterile water filled wells served
as a negative control for the microorganism. Streptomycin standard was utilized. The zones of inhibition (ZOI) were
evaluated after a 24 h incubation period at 37 °C.
 `ANTICANCER ACTIVITY
 Cell viability assessment was determined using 3-(4, 5 dimethyithiazol2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay according

assay according to process of Mosmann, (1983) on human colorectal carcinoma (HCT-116) cell line.

 The cell line was procured from the NCCS (National Centre for Cell Science) Pune, India and was used for in-vitro experimental purpose. The
cells were cultured on Dulbecco Modified Eagle medium with high glucose for cell proliferation at 37 ℃ and 5% CO2 atmosphere was
maintained.

 After successful proliferation the cells were seeded in a 96 well plate at density of 20,000 cells per well in 200 µL of medium.

 Several concentrations of Tribulus terrestris stem methanolic extract (12.5, 25, 50, 100, and 200 µL) were prepared in DMSO and the cancer
cells were treated separately followed by incubation at 37 ℃ for 24 h 5 µM/mL of Doxorubicin was used as standard and cells without Tribulus
terrestris stem methanolic extract were used as negative control for the completion of experiment.

 At the end of incubation period, freshly prepared 200 µL (0.5 mg/mL) of MTT was added to medium containing cells and incubated at 37 ℃
until formazan crystals were formed. After incubation, crystals were dissolved in 100 µL of dimethyl sulfoxide and viable cells were determined
at 570 nm and the IC50 value was also determined at the end.

 The total number of cell viability was calculated using the following equation: % of cell viability = (OD of treated sample ∕ OD of control
sample) × 100.
RESULTS
The results obtained during investigation of qualitative phytochemical analysis, GCMS profiling, antibacterial and
anticancer activity of Tribulus terrestris stem methanolic extract are given below.

Preliminary phyto-chemical analysis of different solvent stem extracts of Tribulus terrestris

The Phyto-chemical analysis had done by using various chemicals. The results of the preliminary phytochemical
analysis of Tribulus terrestris stem extract showed in Table 1.

 The methanolic extract (displayed in Figure 2) contains flavonoids, saponins, carbohydrates, and proteins.

 The chloroform extract (Figure 3) contains alkaloids, steroids, carbohydrates, and Terpenoids.

 The aqueous extract (Figure 4) contains alkaloids and saponins.

Table-1 : Preliminary phytochemical analysis of Tribulus terrestris
Sl Test Methanol Chloroform Aqueous
no extract extract extract
1 Alkaloids - + +

2 Flavanoids + - -

3 Steroids - + -

4 Saponins + - +

5 Carbohydrates + + -

6 Terpenoids - + -

7 Proteins + -

Note :” +” indicates ‘Present ‘ and “ –” indicates ‘ Absence’

 Phytochemical Analysis of
Figure 2 Methanol Extract of stem of
Tribulus terrestris

Phytochemical Analysis of
Figure 3 Chloroform Extract of stem
of Tribulus terrestris

Figure 4 Phytochemical Analysis of

Aqueous Extract of stem of
Tribulus terrestris
GC-MS analysis

The GC-MS analysis of Tribulus terrestris stem methanolic extract revealed a comprehensive overview of the
compounds. The chromatogram of methanol extracts were displayed in Figure 5 and tabulated in Table 2 respectively.

Figure 5: GC- MS chromatogram of methanolic stem extract of Tribulus terrestris

 Table-2: GC-MS profile of methanol extract of Tribulus terrestris stem
Retention Identified Probable Compound Name Peak Area (%) Molecular formula
Time
7.85 Methyl tridecanoate, methyl ester 2.30% C14H28O2
11.21 Phytol 4.96% C20H40O
12.64 Pectolinarigenin 5.09% C17H14O6
14.82 8,11-Octdecadienoic acid 11.20% C19H34O2
20.32 4- methylquinazoline 9.12% C9H8N2
20.95 Palmitic acid 7.45% C16H32O2
22.04 Octadecane, 1,1'-[1,3- 1.56% C39H80O2
propanediylbis(oxy)]bis
23.23 Estra-1,3,5[10]-trien-17 α-ol,3- methoxy-17- 4.47% C23H32O2
[2- mythylallyl]
24.41 Glycerol-1-palmitate 16.52% C19H38O4
25.05 23,28-Cyclostigmasta-5-en-3β-ol 3.44% C29H48O
25.51 Octadeca-9,12-dionic acid 8.48% C18H32O2
26.32 Heptadecanoic acid,16-methyl, methyl ester 1.12% C19C38O2
28.57 4,4-Dimethylcholestan-3-one 2.68% C29H50O
 The associated compounds are listed in Table 2, along with their molecular formulas and molecular weights.
The chromatogram revealed 14 compounds in methanolic extract. The GC-MS chromatogram revealed Glycerol-1-
palmitate (16.52%), n-Octadecanoic acid (12.84%), 8,11-Octadecadienoic acid (11.20%), and 4- methylquinazoline
(9.12%) are the major metabolic compounds present in the methanolic extract of the stem of Tribulus terrestris.
Antibacterial activity of Tribulus terrestris stem methanolic extract
 Antibacterial effect of Tribulus terrestris stem methanolic extract are shown in Figure 6, were tested against human
pathogens like Gram-positive S. aureus and B. subtilis, Gram-negative E. coli and S. flexneri.

 The anti-bacterial efficacy of Tribulus terrestris stem methanolic extract was assessed by agar well diffusion assay.
Results showed a significant growth reduction against human pathogenic bacteria at minimum doses.

 The bacterial colony reduction was observed against E. coli at 25, 50, 75, and 100 µL was 2, 7, 12 and 15 mm and for
S. flexneri it was 5, 8, 10, and 12 mm, respectively and for S. aureus at 25, 50, 75, and 100 µL was 3, 12, 15 and 17 mm and for
B. subtilis it was 8, 12, 14, and 16 mm, respectively.
 The highest bacterial colony reduction was observed against S.aureus and B. subtilis at 100 µL concentration of Tribulus
terrestris stem methanolic extract (17 mm and 16 mm). Increasing the concentration of Tribulus terrestris stem methanolic
extract enhances the bacterial zone of inhibition (Table 3). The results showed that lower doses result in a considerable amount
of bacterial zone of inhibition.

Table 3: Antibacterial assay results methanolic stem extract of Tribulus terrestris

Figure 6: Antibacterial activity of Tribulus terrestris stem methanolic extract against different
pathogens using agar well diffusion method: A) E. coli, B) S. flexneri, C) S. aureus, D) B. subtilis,
and
E) Graphical representation of the obtained results.
 In vitro anticancer activity of Tribulus terrestris stem methanolic extract by MTT assay
 The Tribulus terrestris stem methanolic extract demonstrated considerable cytotoxicity against human colorectal
carcinoma (HCT-116) cell line at various doses. In this study, there was a dose-dependent cytotoxic response
occurred with human colorectal carcinoma (HCT-116) cancer cells at higher concentrations (Figure 7A-G).

The complete mortality rate is 86.25% of cell death was observed in 200 µg/mL concentration of Tribulus
terrestris stem methanolic extract. The Tribulus terrestris stem methanolic extract exhibit noteworthy anticancer
properties, with an IC50 concentration of 147.51 µg/mL resulting in 50% cell death.

A reduction in the percentage of cell viability was observed in a dose-dependent manner when exposed to Tribulus
terrestris stem methanolic extract at concentrations of 12.5, 25, 50, and 100 µg/mL, resulting in values of 93.56%,
88.24%, 76.32%, and 59.63% respectively.

The percentage of cell viability further decreased to 14.75% upon treatment with 200 µg/mL of Tribulus terrestris
stem methanolic extract. The graphical representation of the aforementioned percentages can be observed in Figure
7(H).
Figure 7: Anticancer activity of Tribulus terrestris stem methanolic extract against HCT116 cell line
using MTT assay: A) Negative control, B) Standard control, C) 12.5 µg/mL, D) 25 µg/mL, E) 50
µg/mL, F) 100 µg/mL, G) 200 µg/mL, and H) Bar graph showing the comparative percentage of cell
viability. The images were captured at 40 X magnification.
Summary
 Since ancient times, Ayurveda, a traditional Indian medical system that utilises plant-based medicines, has proven to be
effective in protecting against or suppressing a wide range of ailments through the utilization of diverse therapeutic methods.
For thousands of years, physicians have relied on natural remedies, particularly plants, to cure a wide range of illnesses. The
purpose of this study was to investigate the Tribulus terrestris plant in order to determine its potential therapeutic applications.

 The plant Tribulus terrestris belonging to the Zygophyllaceae family, was evaluated for their phytochemical analysis, Gas
Chromatography -Mass Spectroscopy and its biological applications tests were carried out in current study.

 Phytochemical analysis stem extract of Tribulus terrestris was investigated. The stem extracts showed the presence of
alkaloids, and saponins, in the aqueous extract, the stem methanolic extract of Tribulus terrestris displayed the presence of
flavonoids, saponins, carbohydrates, and proteins. In addition, chloroform stem extract of Tribulus terrestris contains
alkaloids, steroids, carbohydrates, and terpenoids.

 In the present study, Tribulus terrestris stem extract were utilized alongside three solvent extracts. GC-MS analysis of
Tribulus terrestris stem methanolic extract exhibited the presence of bioactive compounds . The chromatogram revealed 14
 compounds in methanolic extract. The GC-MS chromatogram revealed Glycerol-1- palmitate (16.52%), n-Octadecanoic
acid (12.84%), 8, 11-Octadecadienoic acid (11.20%), and 4- methylquinazoline (9.12%) are the major metabolic compounds
present in the stem methanolic extract of Tribulus terrestris.

 The antimicrobial activity of Tribulus terrestris stem methanolic extract was evaluated on bacterial strain such as, S.
flexneri, E. coli, S. aureus and B. subtilis. The in vitro antibacterial activity was performed by agar well diffusion method.
The antibacterial activities of methanolic stem extract of Tribulus terrestris were tested against Gram negative and Gram
positive organisms. The methanol stem extract of Tribulus terrestris showed moderate antibacterial activity against tested
clinical pathogens.

 However, there are fewer reports on the anticancer effect of Tribulus terrestris stem extract human colorectal carcinoma
(HCT-116) cell line. In the present study, we investigated the effects of the methanolic stem extract of Tribulus terrestris on
the inhibition of cell growth and induction of colorectal carcinoma (HCT-116) cell line. The anticancer efficacy was
analyzed by MTT method. Methanol extract of Tribulus terrestris significantly inhibited cell viability in the colorectal
carcinoma cells in a concentration-dependent manner.

 These results suggest that methanolic stem extract of Tribulus terrestris can be used as a natural anticancer drug for the
treatment of colorectal carcinoma and also for the some bacterial diseases.
 CONCLUSION
The significance of medicinal plants as potential sources of therapeutic compounds and as sources of lead compounds
in drug development has led to significant pharmacological research in recent decades. These plants are the foundation of
traditional medicine. Three solvent extracts were employed in conjunction with Tribulus terrestris stem in the current
investigation. The presence of 14 compounds with antibacterial properties was demonstrated in the GC-MS analysis of the
methanolic extract of Tribulus terrestris stem. The MTT assay results indicate that Tribulus terrestris has an effective cytotoxic
potential against Human colorectal carcinoma (HCT-116) cells, as evidenced by its low IC 50 value. In order to assess the
mechanism of action responsible for the anticancer activity of Tribulus terrestris against the colorectal cancer cell line in in-
vivo conditions, additional mechanistic studies regarding the anticancer potential must be conducted. Due to the presence of
these phytochemical compounds and their biological applications, Tribulus terrestris may serve as a new potential source of
medications as a result of this study.
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