Chemical profiling and antioxidant activity of Equisetum ramosissimum Desf. stem extract, a potential traditional medicinal plant for urinary tract infections

Equisetum ramosissimum Desf. (E. ramosissimum) is a widely used traditional medicinal plant to treat urinary tract infections (UTIs) by ethnic people throughout the world. The utility of the plant in treating urinary-related disorders was evaluated against selected pathogenic bacteria which has major role in causing UTIs. Hence, the present study executed to extract phytochemicals like total phenolics and flavonoids, chemical profiling by GC–MS analysis and to test their antioxidant activity from stem extracts of E. ramosissimum. The extraction process was directed by petroleum ether, chloroform, ethyl acetate, methanol, and aqueous solvents. The GC–MS analysis yielded 24 phytoconstituents with linoleic acid, palmitic acid, nonacosane, hexahydrofarnesyl acetone, and octacosane as major compounds. Methanolic extract yielded maximum amount of phenolics (TPC) and flavonoids (TFC) with 600.02 ± 0.22 mg GAE/g and 631.38 ± 0.69 mg QE/g, respectively. Methanolic extract also exhibited notable free radical scavenging activity with an IC50 of 123.89 ± 0.73, 150.10 ± 1.02, 146.01 ± 0.54, and 63.73 ± 6.12 µg/mL for DPPH, FRAP, ABTS, and O2− assays, respectively. The minimum inhibitory concentration (MIC) required to inhibit the growth of tested pathogenic bacteria was observed in aqueous and methanolic extracts with the value being 31.25 µg/mL against R. equi and V. cholerae. As like, methanolic and petroleum ether extracts efficiently inhibited the growth of B. subtilis with the MIC of 31.25 µg/mL. It was concluded that the notable effect of methanolic and aqueous extracts against the uropathogenic bacteria reported in this study supported the traditional uses of this plant in treating UTIs. The results acquired from this investigation revealed that E. ramosissimum stem extract might be considered as an interesting candidate in the development of antibacterial agent against UTIs coupled with antioxidant properties.

species of Enterobacter, Enterococcus, Klebsiella, Proteus, Pseudomonas, and Staphylococcus also cause significant amount of UTIs in human. The UTIs caused by bacteria establish resistance by regular intake of antibiotics that also sometime less efficacious and cause side effects like neurotoxicity, nephrotoxicity, and hepatotoxicity [3]. Severe infections in the urinary tract may lead to various skin diseases including rashes and wounds [1]. Oral administration of renowned antibiotics such as trimethoprim, losporins, fluoroquinolone, and nitrofurantoin showed promising results as short time cure for UTIs [4].
Medicinal plants have been used as an alternative in managing infectious diseases with little or no adverse side effects [3]. To overcome the prevalent condition of UTIs, medicinal plants are widely used by the people since ancient times. The mechanism of herbal medicines used in treating UTIs is not well explained. But it was stated that secondary metabolites present in various parts of plants act as immunomodulators, boost/ regulate the body oxidant status by providing required amount of antioxidant compounds which in turn terminate the proliferation of microbes responsible for UTIs [5]. The major advantage in the use of medicinal plants and their bioactive constituents as potential herbal drugs is that the pathogenic bacteria have not developed resistance against them [6]. It may be due to the presence of rich amount of phytochemicals present in the herbal medicines.
While conducting a survey among the Malayali tribals in the Kolli hills of Eastern Ghats, it was noticed that they are frequently using the dried aerial parts of E. ramosissimum in the treatment of urinary-related diseases including stone formation in kidney, stomachache, skin diseases, bone fracture, joint pains, and rheumatism [6]. Likewise, the plant is broadly used by different ethnic people throughout the world in treating UTIs and related diseases like stone formation and kidney troubles [7,8]. Apart from its use in UTI infections, the plant has also used to treat inflammation, respiratory disorders, bone fracture, stomach problems, skin diseases (itching, scabies), gonorrhoea, rheumatism, female fertility, back pain, muscle pain, fever, sunstroke, and blood pressure [8].
Though the plant has much importance in traditional medicine with huge medicinal properties, there is a lacuna in chemical profiling of different extracts of the plant. With this backdrop information, the present investigation was envisioned to evaluate the GC-MS analysis, total phenolics content, total flavonoids content, and antioxidant activity of the E. ramosissimum stem using different solvents. The utility of the plant in treating urinary-related disorders was also evaluated against selected pathogenic bacteria which have a major role in causing UTIs.

Chemicals
The chemicals for the preparation of reagents (Folin-Ciocalteu reagent and Nash reagent) and buffers (phosphate buffer and acetate buffer) were of commercial grade (Sigma-Aldrich, Mumbai, India) and purchased from the local supplier, Techno Scientific, Thanjavur, 613 002, India. Other chemicals and standard drugs used in the study were also purchased from the same supplier with the commercial brand of Merck Life Science Pvt. Ltd. Mumbai, India.

Collection and preparation of plant extracts
The stem of E. ramosissimum used in this study was collected during August 2019 from Kolli hills of Namakkal district, India, at an altitude of 901-1100 m. The provisional identification of this specimen was performed using the standard flora 'Pteridophytic flora of the Western Ghats in South India' [12] and further authenticated by the taxonomists at the 'Malabar Botanical Garden and Institute for Plant Sciences' Kozhikode, Kerala, by submitting a preserved herbarium specimen. The wellprepared voucher specimen (SPCH1070) was submitted in the herbarium of host institution for future references.
The extraction was done using petroleum ether, chloroform, ethyl acetate, methanol, and aqueous solvents which have a wide range of polarity. Briefly, the powdered stem material was macerated to aforesaid solvents in the ratio of 1:5 and kept under shaker at 125 rpm for 8 h at about 40 °C. Filtration was done through muslin cloth and Whatman No. 1 filter paper. The excess solvent was removed by vacuum distillation to yield crude extract. The crude extracts were evaporated by reduced pressure at 40 °C in an evaporator except for aqueous extract.  7:192 Then, concentrated extracts were stored in tightly sealed glass containers at − 20 °C for further studies.

Phytochemical analysis Qualitative analysis of phytochemicals
The preliminary phytochemical screening of E. ramosissimum stem extract was done to find out the presence of different phytochemicals such as steroids (Salkowski test), triterpenoids (Liebermann-Burchard reaction), alkaloids (Hager's test), phenolic compounds (Lead acetate test), flavonoids (Alkaline reagent test), saponins (Frothing test), and tannins (Potassium hydroxide test) using standard methods [13].

GC-MS analysis of stem extract
Gas chromatography-mass spectrometry analysis was carried out in Shimadzu (QP-2020) interfaced to a mass spectrometer for the petroleum ether, ethyl acetate, and methanol extracts of E. ramosissimum stem. The TD20 was used to obtain the chromatogram using the AB-Innowax column. The initial temperature was programmed with 50 °C and extended up to 280 °C at 40 °C min −1 . Helium was used as carrier gas and the ion source temperature was maintained as 200 °C. Identification of detected compounds in the stem extracts was measured by computing the usual peak area to total area and electronic libraries [14].

Determination of total phenolics (TPC) and flavonoids (TFC) content
The quantitative estimation of TPC in various solvent extracts of E. ramosissimum stem was done by the Folin-Ciocalteu method [15]. About 0.5 mL of 1 N Folin-Ciocalteu reagent was added with 2.5 mL of 20% sodium carbonate solution to each tube of stem extract at different concentrations. The absorbance was recorded at 725 nm against blank which possess water alone. Gallic acid was used as standard and the results were expressed in terms of milligrams gallic acid equivalents per gram of extracts. The TFC was estimated by AlCl 3 method [15]. Briefly, 300 μL of various concentration of stem extract was added with 2 mL of distilled water followed by 150 μL of NaNO 2 . Then, 150 μL of AlCl 3 was added and incubated for 6 min. To this, 2 mL of 4% NaOH was added, vortexed, and kept at 37 °C. The absorbance was read at 510 nm against blank. Quercetin was used as standard and the results were expressed as milligrams quercetin equivalents per gram of extract.

In vitro antioxidant activity
The antioxidant potential of different solvent extracts of E. ramosissimum stem was determined by five in vitro assays. Absorbance was measured on 96 well microplate reader (Robonik India Pvt. Ltd., Thane, India). The DPPH free radical scavenging, FRAP, ABTS, O 2 − , and OH − free radical scavenging abilities of E. ramosissimum stem extracts were studied as defined in our earlier study [16]. Reference antioxidant used in all the assays was ascorbic acid. For each concentration, the percentage inhibition of standard and test samples was calculated and the graph was drawn between percentage inhibition and concentration. From the obtained graphs, the IC 50 was computed as the concentration at which 50% of inhibition observed against the concentration of E. ramosissimum stem extract.

Antibacterial activity Bacterial strains used
The stem extracts of E. ramosissimum were studied against four gram positive strains like

Broth microdilution method
The minimum inhibitory concentration required to inhibit the growth of pathogenic bacteria with E. ramosissimum stem extract was performed by microdilution method using 96 well plates [17]. For the experiment, 50 µL of stem extract at 500 to 0.488 µg/mL concentrations was obtained using serial dilution method. Fifty µL of Mueller-Hinton broth was poured in each well. Then, 50 µL of bacterial inoculum was added to each well and incubated for 24 h at 37 °C. Then, 20 µL of INT (p-iodonitro-tetrazolium chloride) was added and incubated for 30 min at 37 °C. Ampicillin and Deflox were (30-0.029 µg/mL) used as positive controls. The MIC of stem extract was observed by change in colour after the addition of INT dye. The bacterial growth was predicted by red-pink colour in wells and no colour change indicated the inhibition of bacterial growth with stem extract.

Statistical analysis
All the experimental parts were done in triplicate and the results were calculated as mean ± standard deviation. All the statistical analysis was considered highly significant with more than 95% confidence interval. The graphical representations were done with GraphPad Prism version 8.0.2 (GraphPad Software, Inc).  1a). Campesterol is one of the sterol groups of compound present in petroleum ether extract. In ethyl acetate extract, palmitic acid (34.4%), nonacosane (11.5%), and hexahydrofarnesyl acetone (9.4%) were identified as major constituents (Fig. 1b). As like, methanolic extract also contributes palmitic acid (44.3%) and hexahydrofarnesyl acetone (9.8%) as major constituents followed by nonacosane (7.8%) and n-tetracosane (6.9%) (Fig. 1c).
The measured levels of TPC and TFC are presented in Fig. 2. The gallic acid equivalent was computed from linear regression of standard calibration curve (y = 0.0113 + 0.204; R 2 = 0.9931) for TPC. The TPC was ranged from 58.67 ± 2.57 to 600.02 ± 0.22 mg GAE/g. The maximum amount of TPC was recorded in methanolic extract with 600.02 ± 0.22 mg GAE/g followed by petroleum ether and aqueous extracts with 393.48 ± 14.36 and 303.77 ± 3.84 mg GAE/g, respectively. The TFC of E. ramosissimum extract varied in the ranges of 47.93 ± 1.55 to 631.38 ± 0.69 mg QE/g. For quantification of TFC, quercetin equivalent was calculated from linear regression of standard calibration curve (y = 0.0117 + 0.1018; R 2 = 0.9907). The TFC was also higher in methanolic extract (631.38 ± 0.69 mg QE/g) followed by aqueous and petroleum ether extracts with 493.77 ± 0.96 and 405.14 ± 9.43 mg QE/g, respectively.

Antioxidant activity
In vitro antioxidant activity of E. ramosissimum stem extract was studied by DPPH, ABTS, O 2 − , OH − , and FRAP methods (Fig. 3). The lower the IC 50 values, higher the antioxidant activity of plant extract. The E. ramosissimum stem had comparable IC 50 values for DPPH free radical scavenging capacity in tested concentrations (2.5, 5, 10, 20, 40, 80, and 160 µg/mL). Methanolic extract at a concentration of 160 µg/mL showed highest free radical scavenging ability with an IC 50 of 123.89 ± 0.73 µg/ mL followed by ethyl acetate and aqueous extracts (146.46 ± 0.54 and 154.93 ± 0.54 µg/mL, respectively). It was observed that significant difference was found in petroleum ether (p ≤ 0.0001) and chloroform (p = 0.005) extracts. However, methanol and ethyl acetate extracts were showed no significance difference when compared to the standard. Similar to DPPH assay, methanolic extract of E. ramosissimum stem at 160 µg/mL concentration showed higher reducing power in FRAP method with an IC 50 of 150.10 ± 1.02 µg/mL and least activity was observed in petroleum ether extract (IC 50 of 248.12 ± 2.50 µg/mL). However, petroleum ether, chloroform, and ethyl acetate extracts are statistically different from the reference compound.
For the ABTS assay, highest free radical scavenging activity was recorded in methanolic and aqueous extracts at 160 µg/mL concentration with an IC 50 of 146.01 ± 0.54 and 162.42 ± 0.49 µg/mL, respectively. These extracts were observed with no significant difference to the standard. On the other hand, petroleum ether and chloroform  7:192 extracts were positively significant (p ≤ 0.0001) among the tested extracts. Likewise, aqueous and methanolic extracts at 160 µg/mL showed highest superoxide scavenging activity with an IC 50 of 63.73 ± 6.12 and 82.18 ± 2.39 µg/mL, respectively. The higher hydroxyl free radical scavenging activity was recorded at the concentration of 160 µg/mL in methanolic, aqueous, and chloroform extracts with an IC 50 of 122.91 ± 3.32, 144.87 ± 1.30, and 169.31 ± 1.76 µg/mL, respectively. In which, highest significance in difference was observed in petroleum ether (p ≤ 0.0001) and ethyl acetate extracts (p = 0.005).

Antibacterial activity
The results of the MIC value of the E. ramosissimum stem extracts are represented in Table 3, and inhibition images are provided as Additional files 1 and 2. Aqueous and methanolic extracts showed excellent MIC which requires just 31.25 µg/mL of plant extract to inhibit the growth of R. equi and V. cholerae. Methanolic and petroleum ether extracts efficiently inhibited the growth of B. subtilis at 31.25 µg/mL concentration. The growth of B. subtilitis is commendably controlled by all the tested solvent extracts at lower concentration of plant extracts (31.25-125 µg/mL). However, C. freundii is susceptible for most of the tested solvent extracts at even higher concentrations except aqueous (62.5 µg/mL) and ethyl acetate (125 µg/mL) extracts.

Discussion
The presence or absence of different phytochemicals in plant extract plays a major role in antioxidant and antibacterial activities of a specific plant part [18]. In the present study, qualitative phytochemical screening of E. ramosissimum revealed the presence of significant amount of secondary metabolites like phenolics, flavonoids, steroids, and tannins (Table 1). Previous studies also confirmed the presence of various phytochemicals in different parts of E. ramosissimum as reported in the present work [10,11]. To support our study, Chiu et al. [10] also identified sterol compounds from aerial parts of E. ramosissimum with chloroform-methanolic extract.  7:192 The phytochemical analysis of E. ramosissimum stem extract using GC-MS showed the presence of 24 compounds with palmitic acid (44. 3, 34.4, and 19.4% in methanolic, ethyl acetate, and petroleum ether extracts, respectively) and linoleic acid (41.4% in petroleum ether extract) as major constituents (Table 2). Palmitic acid is a saturated fatty acid (16-C long chain molecule), an essential component for human body and could be supplemented in diet or made endogenously from the other fatty acids, amino acids, and carbohydrates [19]. It has a major pathological role in metabolic syndromes, cancer, inflammation, cardiovascular, and neurodegenerative diseases and major proportion of saturated fatty acids found in human body cells and serum are comprising of this palmitic acid [20]. As reported in our study, Alebous et al. [11] also revealed the presence of linoleic acid (4.6%) in essential oil extracted from aerial parts of E. ramosissimum. Linoleic acid is one of the important polyunsaturated fatty acids which is necessary energy per day (at least 1-2%) for normal human growth and development process and considered as a precursor in the synthesis of arachidonic acid and eicosanoids [21]. The compound is most commonly found in the seeds, nuts, oils, and cereals of various plants and major vegetable oils like corn, olive, cotton seed, palm, sunflower, and coconut comprising about 1% of linoleic acid [22]. Linoleic acid is also involved in various health promoting properties like reducing the catabolic effects of immune stimulation, facilitates growth regulation and promotion, reduces body fat, anticarcinogenic and antiatherogenic activities [23].
Flavonoids are one of the major types of polyphenols and most of them have been reported to have antioxidant, anti-inflammatory, and antidiabetic activities [24]. The amount of TPC observed in the present study was noticeably higher than the earlier reports. Previously, methanolic extract of the stem of Equisetum telmateia (E. telmateia) was recorded with 262.7 ± 1.0 mg RU/g pf TPC [25]. Stajner et al. [26] reported 1.75 ± 0.09 mg/g of TFC in aerial parts of E. ramosissimum phosphate buffer extract. The results of this study revealed that the extracts of high polar solvents like methanol and aqueous showed highest amount of TPC and TFC in E. ramosissimum stem which showed highest extraction efficiency (Fig. 2). The polarity of solvent has a significant role in quantity of TPC and the efficiency of extraction for TPC and TFC is reduced with the decrease in polarity of solvents. It was well known that hydrophilic and hydrophobic features of phytochemicals recorded a high influence on their solubility, and hence, polarity of solvents used has a potential role in extracting efficiency of these phytocompounds [27].
Antioxidants can diminish the adverse effects of free radicals, viz., reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are generated during cellular metabolism. The ROS exert supportive effects on cellular redox signalling and immune functioning even at low concentrations. At higher concentrations, ROS and RNS deregulate several cellular functions and often lead to diverse pathological illnesses [28]. The use of plantbased natural antioxidants like flavonoids, phenolics, and tocopherols are biologically act at molecular level and reported to inhibit/reduce the formation of free radicals [29]. Antioxidant capacity of plant extracts could be due to the existence of polyphenolic compounds which have the ability to donate hydrogen atoms to their hydroxyl groups [17].
The DPPH was used as a source of free radicals, since it simulates ROS and RNS by in vitro that affect biological systems and involved in inhibiting the oxidative stressinduced cellular damage and lipid peroxidation [30]. To support our study, significant free radical scavenging effect with the DPPH method was reported by Paulsamy et al. [31] in methanolic extracts of E. ramosissimum whole plant with an IC 50 of 78.58 µg/mL. According to Li et al. [32], ethyl acetate extract of E. ramosissimum at 200 µg/mL showed comparable DPPH activity with an IC 50 of 43.41 ± 7.68 µg/mL. Previously, Stajner et al. [26] reported comparable antioxidant effect of aerial parts of  [35] reported the OH − radical scavenging effect of the ethyl acetate and aqueous extracts of E. arvense aerial parts with the EC 50 of 2.29 ± 0.11 and 3.29 ± 0.16 mg mL −1 , respectively. The values for antioxidant activity of methanolic and aqueous extracts of E. ramosissimum stem exhibited no significant changes in ABTS, hydroxyl radical, superoxide, and FRAP assays (Fig. 3).
The stem extracts of E. ramosissimum were effective against the growth of pathogenic bacteria (done by microdilution method) like S. epidermidis, B. subtilis, R. equi, MRSA, E. coli, V. cholerae, S. typhi, P. aeruginosa, K. oxytoca, and C. freundii which are recognized as wellknown causative agents of UTIs (Table 3 and

Conclusions
In the present study, we perceived for the first time that all the tested extracts derived from the E. ramosissimum stem possessed antioxidant and antibacterial properties. The GC-MS analysis of stem extract yielded 24 compounds with palmitic acid, linoleic acid, nonacosane, hexahydrofarnesyl acetone, and octacosane as prominent compounds. Our data suggest that the potency of tested biological activities is higher in polar solvents with considerable result in methanolic extract. The extracts recorded with significant amount of total phenolics and flavonoids exhibited significant antioxidant properties and antibacterial activities against the pathogenic bacteria. The notable effect of methanolic and aqueous extracts against the uropathogenic bacteria supported the traditional uses of this plant in treating UTIs. The isolation of pure bioactive compounds may have the potential usage to improve and manage novel drugs in treating UTIs by investigating in vivo methods. Further investigations still need to be conducted on toxicity of stem extracts to effectively maximize human health benefits. This study draws attention to methanolic extracts of the stem of E. ramosissimum as a potent source of active principles that can be further utilized as a prominent bioresources in drug discovery efforts.