Presently, the interest in traditional medicine from plant sources has been increasing. Therefore, it’s a necessity to carry out a screening of these plant in order to support their use in folk medicine and discover new active components with the possibility of action against multi-resistant bacteria and also to tray decreasing toxicity of commercial antibiotics^[1],[2],[3]. Where Pathogenic Microbes are always trying to develop resistance to the different antimicrobial commercial antibiotic drugs^[4]. Adverse side effects and high cost of commercial antibiotic are commonly associated with popular synthetic antibiotics (such as allergic reactions, hypersensitivity, immunosuppression, etc.)^[5]. Compared to commercial antibiotics, natural phytochemicals are found to be more effective with fewer side effects that is why they are used as an alternative remedy for the treatment of various diseases.

Bioactive compounds like alkaloids, tannins, flavonoids, phenols, etc., showed antimicrobial activity in the area of food preservation, pharmaceutics, etc.^[4],[6]. The strong plant components can compete with human and plant pathogenic bacteria, viruses, and fungi without side effects and environmental dangers^[7],[8].

The majority of diseases or disturbances are mainly related to oxidative stress due to free radicals^[9],[10]. Free radicals are fundamental to any biochemical process and represent an essential part of metabolism and aerobic life^[10]. Phytochemicals occurring antioxidants such as ascorbic acid, vitamin E, flavonoids and phenols compounds possess the ability to reduce the oxidative damage associated with many diseases including cardiovascular disease, atherosclerosis, cancer, cataract, immune deficiency diseases, diabetes, arthritis, and aging^[11]. Djeridane et al, and Wong et al^[12],[13], reported that the antioxidant potential of medicinal plants may be correlated to the concentration of their phenolic compounds which include flavonoids, phenolic acids, tannins, and anthocyanin.

Tribulus terrestris L. (the common name in Hindi Gokharu) belongs to the Zygophyllaceae family, an annual plant and worldwide distribution. Commonly found throughout India, it mostly grows wild especially in Maharashtra, West Rajasthan, Uttar Pradesh, and Gujarat. The genus Tribulus contains about 20 species which increase as bushes around 10-60 cm high.^[9][14],[15]. In India, there are three species of common occurrence Tribulus, Tribulus terrestris, Tribulus alatus, and Tribulus cistoides^[16]. This plant is used in folk medicine in many countries against various diseases such as eye trouble, edema, skin disorders, urinary troubles, kidney stones, painful micturition and aphrodisiac^[17].

Due to the role of plant extracts in the treatment of many infectious diseases, we study in this work, the Antibacterial and Antioxidant activity of methanol and aqueous extracts of Tribulus terrestris L. (fruits) on some Pathogenic bacteria in Aurangabad city (India).

MATERIALS AND METHODS:

Plant Materials:

Tribulus terrestris (fruits) were purchased from dealers of a traditional medicinal product at Aurangabad city, India. It was authenticated by Dr. Rafiuddin Nasser, Assistant professor, Department of Botany, Maulana Azad College, Aurangabad.

Preparation of the extracts:

Fruits of T. terrestris were crushed to powder form. Methanol and Aqueous extracts were accomplished according to established protocols^[18].

Methanol extract:

50g of plant fruits powder were extracted with 500ml methanol (50–55°C) using a Soxhlet apparatus. After extraction, the solvent was evaporated by a rotary evaporator, then leaving it in an incubator at a temperature of 40°C for 24 h to get a dried form. It was weighed and kept in a refrigerator.

Aqueous extract:

50g of dry plant powder was taken in a beaker, 500ml of distilled water was added and the mixture was stirred by a magnetic stirrer for 24 h. After that, it’s filtered by Whatman paper No. 1 and filtrate was centrifuged at speed of 3000rpm for 10 minutes. The supernatant extract was evaporated by leaving it in an incubator at a temperature of 60°C for 24 h. Finally, the extract was weighed and kept in a refrigerator in sterilized and dark glass containers.

Phytochemical tests:

Extracts were subjected to phytochemical tests to investigate the presence of the following phytochemicals: alkaloids, tannins, glycosides, saponins, flavonoids, sterols, and Phenolic^[18],[19].

Bacterial Strains:

The bacterial strains used for this study were Gram-positive, Staphylococcus aureus (clinical sample) and Methicillin-resistant Staphylococcus aureus MRSA (ATCC 43300) and Gram-negative, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae (clinical samples), and Escherichia coli, (ATCC 25922). All bacterial strains were obtained from Government Medical College, Aurangabad. India.

Antibacterial assay:

Bacterial suspensions were prepared and adjusted to 0.5 McFarland turbidity stander to get a final density of 1.5 x 106 CFM/ml. Agar well diffusion method is used to evaluate the antibacterial activity of plant extracts according to^[20]. Methanol and water extracts were dissolved in 10% dimethyl sulphoxide (DMSO) and in distilled water to final concentrations of (100, 200, 300, 400mg/ml). Nutrient agar plates were prepared, allowed to solidify and 50µl of bacterial suspensions were seeded on the surfaces of these plates using cotton swabs. Wells of 8mm in diameter were bored on the agar using a sterile cork borer. 100µl of extracts were then introduced into the wells (10, 20, 30, 40mg/well). Antibiotic Streptomycin (100μg/well) was used as a positive control and 10% DMSO or distilled water as a negative control. Then the plates were incubated at 37℃ for 18-24 h. The experiment was performed in triplicate and the mean of the diameter of the inhibition zones was measured.

Minimum Inhibitory Concentration (MIC):

MIC was evaluated by the classical double dilution method in Nutrient broth as described by^[21]. A series of different concentrations for methanol and water extracts were prepared (ranging from 100mg/ml to 3.125mg/ml). The MIC knows as the lowest concentration of extract that resulted in no apparent growth after 24 h incubation at 37°C.

DPPH free radical scavenging assay:

Antioxidant activity of T. terrestris methanolic and aqueous extracts were determined using 1, 1-Diphenyl-2-picryl hydrazyl radical method with minor modification^[22].

0.05mL of the extracts dissolved in methanol were diluted to 1.0ml using (ethanol) to attain the concentrations 1-200μg/ ml, and were added to DPPH (Final concentration 200μM, in 95% ethanol). The absorbance of the resulting solution was read at 515 nm after 20 min using a spectrophotometer. The tests were performed in triplicate and the percentage inhibition was calculated by using the following formula:

% Inhibition = [(Abs_control - Abs_sample) / Abs_control] × 100

Where Abs_control is the absorbance of DPPH radical solution without extract, Abs_sample is the absorbance of DPPH radical solution mixed with the extract.

IC₅₀ values were calculated using Linear regression by plotting scavenging activity against sample concentrations using Microsoft Excel software. Ascorbic acid was Stander.

STATISTICAL ANALYSIS:

Experimental data are expressed as means ± Stander error. Statistical analyses were performed by one-way ANOVA and t-value. The difference was considered to be statistically significant when p < 0.05. This analysis was done using SPSS ver. 20.0 software.

RESULTS AND DISCUSSION:

The present study showed phytochemical analysis, antibacterial and antioxidant activity of the methanolic and aqueous extracts of T. terrestris fruits. The weight and percent (%) of methanol and distilled water extracts were 4.64 g (9.28%) and 7g (14%), respectively.

Phytochemical analysis:

Phytochemical screening of aqueous and methanol extracts of the Tribulus terrestris (fruits) plants revealed the presence of alkaloids, tannins, glycosides, flavonoids, Phenols, are found in both extracts and are shown in (Table1). Glycosides and saponin were also found in methanol extract and absent in aqueous extract. Steroids was absent in each extract. Phytochemicals such as alkaloids, tannins, flavonoids and several other which are naturally happening in most plants, are known to have medicinal importance, which can be used in realizing a defense mechanism against many microorganisms^[23]. The presence of secondary metabolite compounds in the plants is affected by several environmental factors such as light intensity, drought stress, temperature, salinity^[24].

Table 1: Phytochemical screening of methanolic and aqueous extracts of Tribulus terrestris L. fruits.

		Extracts
Phytochemical	Test	Methanol	Aqueous
Alkaloids	-Mayer -Wagner	+ +	+ -
Tannins	-Lead acetate -5% Fecl₃	+ +	+ +
Glycosides	-Keller-killianit	+	-
Saponin	-Foam test	+	-
Flavonoids	- Lead acetate -Sulphuric acid -5%FeCl3	+ - +	+ - +
Steroids	-Salkowski	-	-
Phenolic	-5%FeCl₃	+	+

Note: (+) Presence and (^_) Absence.

Antibacterial activity:

In this study methanol and aqueous extracts of the fruit of Tribulus terrestris plant were tested for their antibacterial activity. As the results showed a positive effect in inhibiting the growth of bacterial strains, the results were different depending on the extract used, the type of bacteria, and concentrations. (Table 1 and Fig 1 and 2).

Methanolic extract, with a concentration of 40mg/Well, showed the best inhibitory effect against S. aureus, S. aureus (MRSA), K. pneumonia, P. aeruginosa and P. mirabilis with inhibition zones of 26mm, 24mm, 22.2 mm, 16.7mm and 15.7mm respectively. No activity was observed at concentrations of 10 and 20mg/well for P. mirabilis (Table 2 and Fig 1). Methanolic extract showed no inhibitory effect on E. coli.

Aqueous extract at a concentration of (40mg/well) showed more potent activity against K. pneumonia and P. mirabilis in which the diameter of inhibition zone were 18.3mm and 16mm, respectively. No activity was observed at concentrations of 10mg/well for K. pneumonia. The aqueous extract has no activity against S. aureus, MRSA, P. aeruginosa and E. coli (Table 2 and Fig 2).

MIC was applied only on bacteria that was sensitive to extracts at concentration 10mg and had an inhibition zone of 10mm and more. MIC value of the methanolic extract of T. terrestris fruits against S. aureus, MRSA, K. pneumonia and P. aerunginosa was 6.25mg/ml. MIC value of the aqueous extract of T. terrestris fruits against P. mirabilis was 6.25mg/ml (Table 3).

Methanolic extract showed higher activity than aqueous extract (Table 2). According to Sonam Pandey^[25], ethanolic extract showed a better inhibition than aqueous extract and this is maybe attributed to the phytochemical compounds less solubility in water. Methanolic extract of T. terrestris fruit in this study revealed close results to the methanolic extract of T. terrestris leave against most of the bacteria under study with MIC value 6.25 in Nigeria^[26]. Similar results also were reported by Kianbakht, and Jahaniani^[27], in which MIC value was 2 mg/mL for methanolic extracts of Iranian T. terrestris fruits, leaves and stems against all bacteria. Another study revealed that all extracts from different parts of The Iraqi T. terrestris showed antimicrobial activity against most tested bacteria^[28]. Also, in one study, the Indian T. terrestris were active exclusively against, E. coli, K. pneumonia, and P. mirabilis; among ten bacterial were studied^[29]. In another finding, ethanolic extract of Yemeni T. terrestris had no detectable antibacterial activity against any bacterial used^[30]. Different results concerning the antibacterial activity of T. terrestris might be due to different types of strains used and different geographic sources of the plant used.

Table 2: Antibacterial activity of methanol and aqueous extracts of T. terrestris (Fruit) against bacterial strains.

Extracts Organism	Methanol					Aqueous
	Conc. Of the extract (mg/well)
	10	20	30	40	St	10	20	30	40	St
	Zone of inhibition (mm) (Mean ± SE)
Gram-positive:
S. aureus	20.1±1.3	22.0 ±0.57	24.3 ±0.37	26.0 ±1.2	34.7 ±0.60	-	-	-	-	-
S. aureus (ATCC)	18.0 ±1.2	18.0 ±0.67	23.0 ±0.58	24.0 ±0.58	29.2 ±0.44	-	-	-	-	-
Gram-negative:
P. aerunginosa	10.0 ±0.29	13.0 ±0.58	14.8 ±0.44	16.7 ±0.88	21.5 ±0.76	-	-	-	-	-
P. mirabilis	-	-	12.3 ±0.64	15.7 ±0.33	28.0 ±0.0	10.3 ±0.88	12.0 ±1.15	13.0 ±0.0	16.0 ±1.0	27.3±0.33
K. pneumonia	15.2 ±0.44	16.3 ±0.88	20.3 ±0.33	22.2 ±0.44	29.0 ±0.58	-	10.7 ±0.33	16.3 ±0.88	18.3 ± 0.33	28.3 ±0.60
E. coli (ATCC)	-	-	-	-	-	-	-	-	-	-

St: Streptomycin (positive control 100μg /well), - : No zone of inhibition. All values are expressed as Mean ± SE (n=3).

Figure 1: Growth inhibition activity of methanolic extract of T. terrestris against: S. aureus (SA), MRSA (SA*), P. aeruginosa (PA), P. mirabilis (PM), and K. pneumonia (KP) at concentrations 10 (A), 20 (B), 30 (C), and 40 (D) mg/well, negative control 10% v/v DMSO (DM) and the positive control 100 μg /well Streptomycin (St).

Figure 2: Growth inhibition activity of aqueous extract of T. terrestris against P. mirabilis (PM), and K. pneumonia at concentrations 10(A), 20(B), 30(C), and 40(D) mg/well, negative control distilled water (DW) and the positive control 100 μg /well Streptomycin (St).

Table 3. MIC values of T. terrestris fruits extracts (mg/mL).

Bacteria

Extracts

S. aureus

MRSA

P. aerunginosa

P. mirabilis

K .pneumonia

E. coli

Methanolic

6.25

Aqueous

6.25

NT: Not tested

DPPH free radical scavenging activity:

Antioxidant activity of methanolic and aqueous extracts of T. terrestris fruits were determined by DPPH radical scavenging assay and both extracts showed antioxidant activity in a concentration-dependent manner. Methanolic extract showed a higher scavenging ability with IC₅₀ value of 3.94µg/ml and aqueous showed medium scavenging ability with IC₅₀ of 22.45µg/ml. The IC₅₀ value for the standard (ascorbic acid) was 3.35 μg/ml.

A study conducted by Lokhande, et al,^[31] using T. terrestris leaves showed that aqueous and alcoholic extracts have a strong antioxidant activity in which IC₅₀ were 43.96 and 41.66μg/ml, respectively.

In another study, among a total alcoholic, ethyl acetate fractions, rutin, and quercetin extracts of T. terrestris fruits, the maximum scavenging ability was recorded in rutin and quercetin (IC₅₀=12.1 and 11.2μg/ml respectively), when compared with ascorbic acid (10.2 μg/ml)^[32]. On the other hand, antioxidant activity from acetone, hexane, isopropanol, and aqueous extracts of T. terrestris fruits, the strongest free radical scavenging activity was showed in hexane extract^[33]. Similar results highlighting the scavenging ability of ethanolic extracts of T. terrestris fruits, with IC₅₀ 142.27μg/ml^[34].

According to Hetty Manurung,^[24] there are potent relationships between secondary metabolite compounds such as phenolic, alkaloids, and flavonoids and antioxidants. And it has been shown that the scavenging effects of the DPPH radical is a dose-dependent in both samples and standards^[33],[34]. T. terrestris is a possible new powerful natural source of antioxidants^{[15], [31], [32]}.

CONCLUSION:

Methanolic extract of Tribulus terrestri fruits have more phytochemical compounds, higher antibacterial and a strong antioxidant activity than aqueous extract. Further studies are recommended to isolate and identify antioxidant components in T. terrestris.

ACKNOWLEDGEMENTS:

Authors are thankful to the dean of Faculty of Pharmacy, and Dr. Sabur Khan assistant professor, Dept. of Pharmacognosy, pharmacy college, Maulana Azad College, Aurangabad, India for providing laboratory facilities to do this work.

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Received on 05.01.2020 Modified on 08.03.2020

Research J. Pharm. and Tech. 2021; 14(1):331-336.

DOI: 10.5958/0974-360X.2021.00061.5