Phytochemical Screening, Antioxidant and Antibacterial Properties of Alcoholic Extract of Saussurea costus Roots

Medicines of plant origin remain an important resource in the fight against serious diseases, especially in developing countries (Ansari et al., 2021). Traditional medicines are still used by 60-80% of the world’s population for the treatment of common diseases (Oyebode et al., 2016). Most  patients with arthritis and such long-standing impairments are reported to be using alternative medicines for the reduction of symptoms and relieving pain, with traditional Chinese medicines based on herbs and traditional formulas being the most commonly used (Mehta and Shah Shahista, 2021; Singh and Chahal, 2018). The evidence of the use of medicinal plants for the management of the symptoms of any disease can be witnessed from five thousand years back through the written documents of early civilizations in India, China and the Near East (Petri et al., 2015).

Although there is huge evidence of the benefits of plant-based medicines and their therapeutic effects, their use in the management of diseases and treatment had remained unexplored (Vijayalakshmi et al., 2022). In recent decades, there has been  increasing interest in studying natural products from various sources, especially from higher plants, to discover new antioxidant and antimicrobial agents such as flavonoids, tannins, alkaloids and terpenoids, which have been shown to have in vitro antimicrobial characteristics and properties (Deabes et al., 2021). In recent years, infectious diseases have increased dramatically and antibiotic resistance has become a growing therapeutic problem (Aslam et al., 2018). In the treatment of multiple infections such as urinary tract infec-tions, respiratory tract infections, gastric issues and typhoid fever the use of these plants as  therapeutic agents is extremely promising as it induces therapeutic effects and helps in curing the diseases (Mikawye et al., 2017).

However, the use of complex plants as a source of novel drugs remains unexplored. Plants are composed of an extensive range of phytochemicals, including tannins, terpenoids, alkaloids and flavonoids, which exhibit powerful antibacterial properties against multiple microorganisms including, bacteria, fungi and others (Kumar et al., 2024; Semwal et al., 2020). They are much safer and result in fewer adverse effects as compared to synthetic drugs (George, 2011). The search for antimicrobial ingredients has become increasingly important in recent years due to worldwide growing concerns about the rapidly increasing infections and diseases which are resulted from microorganisms which are antibiotic-resistant (Ashry, 2019; Kumar and Pundir, 2022). Many components of the plants have the potential to be powerful remedies for a variety of diseases (Gurib-Fakim, 2006).

Due to the presence of several compounds in Saus-surea costus, including Kaempferol, Cinnamic acid, Taxifolin, Naringenin, Ferulic acid, Vanillin, Coumaric acid, Ellagic acid, Rutin, Pyro catechol, Syringic acid, Coffeic acid, Methyl gallate, Catechin and Chlorogenic acid) with some of them have been proved as having biological activity against cabcer cell lines (Deabes et al., 2021). The objective of this study were to characterize the constituents present in the S. costus and assess their antimicrobial activities on common bacterial species in an attempt to overcome the problems of antimicrobial resistance through finding alternative remedies.

Sample collection: Roots of S. costus were purchased from the Mosul Herbarium Market, the plant authentication has been previously described in Mosul city (Iraq) (Jabori et al., 2023; Al-obaidy and Esmaeel, 2021). Four pathogenic microorganisms were kindly offered by Faculty of Science and University of Mosul: “Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Enterobacter faecium and Pseudomonas aeruginosa)”. This research was carried out at the “College of Pharmacy, University of Mosul.”
 
Preparation of extract
 
Dried S. costus roots were ground to a powder. A total of 25 g of the powder was added to absolute ethanol (150 ml, stirred for 24 h with a magnetic stirrer, room temperature). The mixture was then filtered (Berreghýoua and Ziane, 2024).
 
Phytochemical screening
 
Phytochemical screening of extracts from the roots of S. costus was carried out using standard methods (Fig 1, Table 1), including.




 
Alkaloid testing
 
A small part of the sample was pipetted into a test tube and one milliliter of Mayer’s reagent (mercury potassium iodide) was added and mixed. The presence of alkaloids was indicated by the formation of a yellow creamy precipitate (Thenmozhi et al., 2023).
 
Terpenoid testing
 
The terpenoids in the sample were confirmed by 1-3 drops of chloroform to a small sample followed by one millilitre of concentrated hydrochloric acid, which was heated for two minutes to form a reddish-brown colour (Berreghýoua and Ziane, 2024).
 
Phenol testing
 
Two millilitres of FeCl3 5% solution were added to a small amount of the specimen and incubated for 5 minutes at room temperature. The phenol content is denoted by the formation of a blackish blue colour (Akshaya et al., 2023).
 
Tannin testing
 
To do this test, two drops of 10% lead acetate were mixed with 1 ml of the plant extract leading to formation of yellow precipitate which indicates presence of tannic acid (Akshaya et al., 2023).
 
Carbohydrate testing (Molish test)
 
The extract was placed in test tube and the sulfuric acid added against test tube wall resulting in violent ring at the contact site which do indicated the presence of carbohy drates (Thenmozhi et al., 2023).
 
Saponins testing
 
To do this test, two drops of distilled water were mixed with 1 ml of plant extract, this has led to formation of foam which do indicates the presence of saponins (Berreghýoua and Ziane, 2024).

Coumarin glycosides
 
The formation of yellow precipitate following mixing 2 ml of diluted HNO₃ with equal amount of plant extract indicated the presence of Coumarin glycosides (Calimpang et al., 2023).
 
Flavonoid test
 
A few drops of  ferric chloride added to 1ml of the S. costus extract, the presence of flavonoids has vchanged the color of extract from green to black after addition of ferric chloride (Devi et al., 2023).
 
Quinine test
 
After 2 min of incubating equal amount of one milliliter of sodium hydroxide and plant extract solution it will lead to formation of a reddish-brown colour indicated quinine (Devi et al., 2023).
 
Protein analysis
 
In a test tube, small amount of sample was placed and a drop of concentrated HNO₃ was added. The light red colour formed indicates the presence of protein (Calimpang et al., 2023).
 
Detection of steroids (Salkowaski reaction)
 
After 5 min of incubating equal amount of one milliliter of chloroform, concentrated H₂SO₄ and plant extract solution it will lead to formation of a red color in the lower layer indicates the presence of steroids (Devi et al., 2023).
 
Radical scavenging effect of the extract on DPPH radical
 
The free radical scavenging activity of plant extracts was determined according to previous studies as follows (Afzal et al., 2021). A known volume of plant extracts was added to the test tubes and gently mixed and then (1.0 ml) of DPPH solution (0.2 mM of ethanol) was added to each tube, mixed well (incubated at room temperature, 30 min) to reach a known volume (1.0 ml). The same protocol was applied for the negative control group (a sample with no plant extract). A 0.03% (w/v) solution of ascorbic acid was used as an additional control. Absorbance (A) at 517 nm for samples and control was measured (Jenway 6300 spectrophotometer). The percentage of DPPH radical inhibition was calculated using the following formula:
   
Compound identification
 
Fourier transform infrared (FTIR) spectrophotometer was used to analyze the extracts of the plants using (Bruker-Alpha ATR-FTIR (Germany).

The roots of S. costus were studied against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Proteus spp. The growth of four bacteria obtained from the University was studied. The antibacterial effect of the drug was studied by the disc diffusion method, in which various types of microorganisms were cultured in nutrient broth and incubated at 37^oC for 24 hours. Then, the tested concentrations of different compounds, i.e. (200, 100, 50, 25, 12.5) mg/ml, were attended and one ml of each concentration was placed in test tubes containing 100 pieces of filter paper (Whatman No. 1) sterilized by autoclave with a diameter of (6) mm. Then, 0.1 ml of bacterial suspension was inoculated into several Petri dishes containing nutrient agar medium and sparing the surface of the nutrient agar, a disc was placed in each Petri dish for each concentration of the substance considered and the Petri dishes were placed in an incubator at 37^oC for 24 hours. The diameter of the zone of inhibition around the disc was  determined.
 
Minimum inhibitory concentration (MIC) assessment
 
The ethanolic extract of S. lappa roots was fixed at serial dilutions (25, 50, 100 and 200 mg/ml of supplement) followed by 5% dimethyl sulfoxide solution (DMSO). Then, 100L 106 cells/ml of four bacterial strains (S. aureus, P. aeruginosa, Escherichia coli and Proteus spp.) were cultured in a pre-arranged broth. After one day of incubation, the optical density (620 nm) of the cultured microorganisms was assessed using a spectrophotometer and the results were compared with negative control (broth medium without microorganisms) and positive control (microorganisms developed without any treatment). Then the serial dilution of the plant extract were used to prepare discs of different concentration. Once culture is ready these discs were incubated with bacteria in the cultured petri dishes, the zone of inhibition noticed after 2 days measured and calculated as zone of inhibition. For each tests a replicate of three tests were conducted and mean± standard deviation was calculated for each and compared to other concentations.
 
Statistical analysis
 
The collected data were grouped and represented as percen-tage of antioxidant activity. Diameter zone in millimeter for growth inhibition of affected bacteria caused by S. lappa roots extracts. Area under the curve considered for functional group characterization of the extract.

Study settings: The present study was conducted in College of Pharmacy at University of Mosul during the period January 2023 to December 2023. The bacterial isolates were previously characterized in the Department of Clinical Laboratory Sciences in College of Pharmacy at University of Mosul. The plant species was characterized and authenticated by the Department of Pharmacognosy and Medicinal plants in College of Pharmacy at University of Mosul.
 
Characterization of bioactive compounds
 
Chemical analysis revealed the composition of active chemicals and other unknown substances in the ethanolic extract of S. costus roots. Alkaloids, terpenoids, phenolics, tannins, coumarins, glycosides, flavonoids, quinines and steroids were found in the samples.
 
DPPH radical scavenging activity
 
Table 2 outlines the output of the DPPH scavenging activity of flavonoids in ethanolic extracts of the roots of S. costus and compares them with the ascorbic acid as a positive control. The free radical scavenging potential of serial dilutions of the root extracts of S. costus was 71.9% at 62.5 ppm, 87.3% at 125 ppm and 89.3% at 187 ppm, which is comparable to ascorbic acid.


 
FT-IR
 
Table 3 and Fig 2 show the FT-IR results. The solutions were analyzed separately using an FT-IR spectrophotometer (Bruker-Alpha ATR-FTIR, Germany). According to FT-IR studies, the molecule includes multifunctional groups such as phenolic -OH, carbonyl (-C=O) and olefin C=C, etc.

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Antibacterial activity
 
The antibacterial activity of S, lappa roots extract was tested against four different types of pathogens (Table 4 and Fig 3). The results showed that the extract had different antibac-terial activities against each of the tested strains. Using the disc diffusion method, S. lappa root extract showed the widest zone of inhibition against Staphylococcus aureus. Escherichia coli and Pseudomonas aeruginosa showed less inhibition, but S. pyogenes check whether it is pyoge-nes or aureus showed higher resistance to S. lappa roots extract (Table 5). Assumption of MIC values: The MIC values of S. lappa roots extract varied from 0.125 to 0.5 mg/ml against all the tested bacteria, with Proteus spp. showing considerable resistance to this extract.

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Over thousands of years, the effectiveness and bene-ficial effects of the plants have become known and plants have been considered one of the major sources of anti-biotics (Dhakal et al., 2021). The world’s population still relies primarily on conventional remedies such as herbs to obtain medicines (Gul, 2020). Plants can also be used as  alternatives to antibiotics and have fewer adverse effects than pharmaceutical drugs. The increase in antibiotic resistance and  adverse effects of synthetic drugs have stimulated interest in the discovery of new antimicrobial compounds from plants. S. lappa is a well-known essential medicinal plant that is widely used in many medicinal systems for curing and treating multiple different nature of diseases, including peptic ulcers, asthma and gastric disorders (Malik, 2020). The rhizomes and roots of S. lappa have bitter, astringent, purgative, anthelmintic, antioxi-dant, antitumor, digestive stimulant and stimulating properties.

Due to the active ingredients present in S. costus extract, with some of these biomolecules have been confirmed as having biological activity against cancer cell lines (Deabes et al., 2021; Kumar et al., 2024). S. costus extract has been demonstrated as having antimicrobial activity against Gram-positive (Streptococcus pyogenes and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli and P. aeruginosa) (Al-Zayadi  et al., 2023), this finding is highly critical to conquer these bacterial resistance which do have the capacity to persist in bleak surroundings (Pourhajibagher et al., 2016; Qi et al., 2016; Gheorghe-Barbu et al., 2022). Similarly, Al-Zayadi  et al., (2023) reported that their extract contents were congruent with compounds detected in the present study.

In this study, the bacterial inhibitory zone revealed weak or no impacts on proteus species and this result harmoni-zed with the studies conducted by Deabes et al., (2021) and Abdallah et al., (2017), who have shown a better antibacterial effects of S. costus extract on growth inhibition of Gram-positive (Streptococcus and Staphylococcus) versus Gram-negative bacteria (Salmonella and Escherichia coli) with detected presence of approximately similar active biomolecules.

Struvite renal stone is a renal lithiasis formed due to continuous successive renal tract infections with gram negative bacteria and is also called infectious lithiasis (Polat and Eral, 2022; Bichler et al., 2002). The dualistic mode of action of S. costus extract on struvite renal lithiassi which involve solubilization of the renal crystals alongside killing the causative agents of involved pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia) (Mammate et al., 2023). Furthermore, Sadki et al., (2017), reported that S. costus inhibited the formation of ammonium–magn-esium phosphate crystals which is the main component in renal lithiasis of struvite renal stone.

Several pharmacological experiments using in vitro and in vivo models have shown that S. lappa has anti-inflammatory, peptic ulcer, anticancer and hepatoprotective properties (Nadda et al., 2020; Sundar et al., 2018). The bioactive compounds extracted from this plant include dehydrocostusolide, Costunolide and Cynaropicrin. Given the impressive bioactivity of S. lappa and its compounds, it would be reasonable to develop them as drugs. The effect of ethanolic extract of S. lappa on the growth of Streptococcus mutans was examined in a dose-dependent manner (Nadda et al., 2020). S. lappa has been used in the management of dental diseases because it is effective against cariogenic Streptococcus mutans.

Saussurea can be used medicinally for oxidative stress or as an antibacterial drug. The outcome was based on the experimental identification of therapeutic effects of S. using laboratory tests. The effects could be further confirmed by clinical application or experimental animal studies.

The present study was registered and approved by the Department of Clinical Laboratory Science in the University of Mosul (Iraq).

The authors declare that there are no conflicts of interest regarding the publication of this article.