In vitro antioxidant and antimicrobial potential of Sterculia urens Roxb. root extract and its bioactive phytoconstituents evaluation

The plant Sterculia urens Roxb. of Malvaceae family is comparatively understudied. Genus Sterculia is widely recognized by its phytomedicinal and ethnomedicinal attributes. The study is aimed to evaluate the qualitative analysis, Fourier transform infrared (FT-IR), thin layer chromatography (TLC), total phenolic content (TPC), antioxidant and antimicrobial activities of the crude hydro-methanolic extract of S. urens root. The antioxidant activity, antimicrobial assay for clinical isolates, and TPC were measured by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity, agar well diffusion method, and Folin–Ciocalteu assay respectively. Hydro-methanolic extract confirmed the presence of alkaloids, flavonoids, tannins, phenols, saponins, steroids, and glycosides as primary and secondary metabolites, which was later confirmed by TLC. FT-IR spectroscopy revealed the presence of alkanes, alkenes, alkyl halides, halogen compounds, primary alcohol, tertiary alcohol, aldehyde, aromatic amine, secondary amines, amide group, and carboxylic acid. The crude extract was composed of a significant quantity of total phenolic content with 705 ± 0.40 mg GAE/g. Synergistically, the IC50 value of the crude extract and ascorbic acid was found to be 27.055 μg/ml and 37.244 μg/ml, respectively, which suggests that root extract possesses strong antioxidant properties. The majority of the microbial strains exhibited varying degrees of sensitivity to the root extract with a notable inhibitory effect against Escherichia coli, Klebsiella pneumoniae, and Penicillium glaucum. The findings of this analysis suggest that the hydro-methanolic extract from S. urens root exhibit antioxidant activity quantified by its ability to scavenge DPPH; antimicrobial activity displayed appreciable microbial sensitivity. These properties are associated with the presence of high phenolic content, different secondary metabolites, and their functional groups. The results are suggestive that S. urens root is rich in bioactive compounds, which serve as a novel natural source for potential therapeutic applications.

saponins, terpenes, alkaloids, lactones, oleoresins, glycosides, resins, and essential or fixed oils [1]. For all living organisms, metabolism is necessary, and plants have two types called primary metabolites and secondary metabolites [2,3]. Although secondary metabolites are reported lower than primary metabolites, they have considerable biological activities [4]. For that reason, the most promising source of novel biologically active substances that protect human from various diseases is medicinal plants [5].
Moreover, concerns about the adverse health effects are growing due to the industrial use of synthetic products. It has been shown that some synthetically manufactured antioxidants, including dibutylhydroxytoluene and butylated hydroxyanisol, promote carcinogenic activity [6,7], while naturally obtained phenolic antioxidants are shown to manifest a variety of healthpromoting biological activities. Since the last few years, FT-IR has played a crucial role in scientific research; most recently, spectroscopy has made considerable advances in the area of clinical assessment and arose as one of the key tools for biomedical claims. Research on many different natural tissues utilizing spectroscopic techniques, such as FT-IR spectroscopy, has been adapted [8].
Natural products derived from plants such as terpenoids, alkaloids, and flavonoids have gained significant exposure for the past few years due to their various pharmacological properties, including antimicrobial, antioxidant, and anticancer activities [9]. Antioxidants are natural or artificial substances capable of preventing or limiting certain kinds of cell injuries. Antioxidants often are found in several plants produces, especially vegetables and fruits, and also readily available as food supplements [10]. Adequate intake of dietary antioxidants can boost defence against free radicals, as the plant-based natural antioxidants can scavenge nocuous free radicals generated in the human body. It is also suggested that the intake of antioxidant-rich foods plays a vital and beneficial role in preventing or postponing the onset of degenerative diseases [11]. Recently, high levels of bioactive compounds with good antioxidant potential have been found in the roots [12,13].
The discovery of novel drugs from plants was encouraged by the resistance developed in pathogenic microbes against conventional antibiotics and oxidative stress produced by free radicals. A significant number of plants are recorded to possess antimicrobial and antioxidant properties [14]. Since ancient times, plants have been used to produce a range of chemical substances with medicinal value, including antidiabetic, anticancer, antimicrobial, antioxidant, and anti-inflammatory. The World Health Organization (WHO) also considers plants to be the ultimate source of numerous drugs and advocates the continued use of these herbal medicines to treat various microbial and non-microbial diseases [15].
Moreover, various plant genera are extensively explored for phytochemicals with regard to their ethnomedicinal properties. One of those genera includes Sterculia representing 200 species, predominantly distributed across tropical and subtropical regions. Plants which belong to this genus contain significant to the low amounts of flavonoids, terpenoids, phenolic acids, phenylpropanoids, alkaloids, and other metabolites, including carbohydrates, lipids, lignans, and lignin [16]. One such species of these genera is Sterculia urens Roxb. (Fig. 1) commonly known as 'gum karaya'; it is one of the critical commercial trees of India [17]. Earlier, it was kept under the family Sterculiaceae but currently belongs to the subfamily Sterculioideae and classified under family Malvaceae [16,18]. In India, it is distributed across the sub-Himalayan range, Rajasthan, Madhya Pradesh, Andhra Pradesh, Maharashtra, Uttar Pradesh, Kerala, and Gujarat [19,20]. It offers numerous applications in food, dairy, beverages, cosmetic, textile, oil, and pharmaceutical sectors [18]. Therefore, this experiment is aimed to investigate the phytochemical profiles, including total phenolic content, thin-layer chromatography assay, and FTIR screening, along with antioxidant and antimicrobial activities of the S. urens root extract.

Collection and authentication
Sterculia urens Roxb. (gum karaya) was collected by the first author from Kansarvid, Banaskantha district, Gujarat, India, in August 2019 (latitude 24.26°N and longitude 72.51°E); immediately, its identification and pre-treatment were carried out. The plant material was authenticated, and the herbarium specimen was deposited with reference ID (GUSU-01).

Plant material
The root samples appeared reddish within the cortex region ( Fig. 2) and washed twice using deionized water (Milli-Q Millipore, 0.054 mS/cm) to clean soil and dirt. The root sample was slightly scraped with a scalpel blade (No. 24), cut into pieces with 2.5 cm length each, sun-dried for 7 days, ground to a fine powder, and then stored for further analysis at − 20°C.

Preparation of plant extract
The Soxhlet extraction method was used to prepare plant extract; 90% hydro-methanolic (1:9 v/v) was used as an extracting solvent. This fresh solvent and solid residues (solid-to-solvent ratio, 1:10 g/ml) were placed in a 22 mm × 80 mm extraction thimble [DWK Life Sciences (Wheaton), Mumbai, India] and extracted at 65°C with 90 ml methanol and 10 ml distilled water (D. W.) for 18 h with 4-5 cycles/h. The collected extracts were filtered with Whatman No. 1 filter paper, and the extra solvent was evaporated by rotary evaporator at reduced pressure for 30 min (IKA-RV10 Digital V, Karnataka, India) at a controlled temperature of 40°C and stored for further analyses at 4°C. Finally, the yield value of crude hydromethanolic extract was calculated by the given standard formula [21]. Yield % ð Þ ¼ Final weight of extract obtained Initial weight of extract Â 100

Reagents and chemicals
The DPPH and the Folin-Ciocalteu phenol reagent were purchased from Sigma-Aldrich Co., Germany. Sodium carbonate (Na 2 CO 3 ) ethanol and gallic acid and were obtained from Himedia, India. The majority of the chemicals (AR: analytical grade) sourced in the present study were procured locally (India).

Preliminary phytochemical screening
Qualitative phytochemical analysis of crude extract determines the presence of compounds like sterols, glycosides, saponins, tannins, flavonoids, phenols, resins, and alkaloids using the standard protocol. The qualitative phytochemical screening of crude extracts was done using the methods described by the various workers [22,23].

Fourier-transformed infrared spectra
For FTIR analysis, lyophilized extracts were used, because it increases the intensity of spectral bands as it removes water and methanol interference. FTIR spectra were recorded on Bruker alpha eco atr, Optics, Germany, in conjunction with an attenuated total reflectance (ATR), incorporated with a reflection crystal (ZnSe). The spectra were achieved at 20°C with an average of 128 scans, ranging frequency from 3500 to 500 cm −1 , processing software OPUS (v.5.5, Bruker Optics, Germany) for a quick scan. For background, the air spectrum was recorded and subtracted spontaneously with suitable software [24].

Total phenolic content
For measuring total phenolic content (TPC) of samples, the Folin-Ciocalteu assay was followed. At this method, solutions of 7.5% w/v, Na 2 CO 3 , and 10% v/v Folin-Ciocalteu reagent (Sigma-Aldrich Co., Germany) were formulated. Then, 0.75 ml of Folin-Ciocalteu reagent was added to 100 μl extract and was stored in the dark (5 min, 20°C). Next, 7.5 w/v of 0.75 ml Na 2 CO 3 solution was mixed and kept in the dark (90 min, 20°C) and mixed randomly until a homogenous solution was achieved. Lastly, using a spectrophotometer (Thermo Fisher EVO300PC, USA) at 765 nm, the absorbance was estimated and compared with the calibration curve, which was formulated applying known quantities of gallic acid. The findings are measured as milligrams of gallic acid equivalent (GAE)/100 g [25].

DPPH radical scavenging assay
Antioxidant activity was measured by DPPH radicalscavenging assay given by Brand-Williams [26] with several alterations. The fresh stock solution 0.01 mM of DPPH (Sigma-Aldrich Co., Germany) was prepared by dissolving in methanol (SRL, Mumbai, India). 2.7 ml of prepared DPPH was added to various concentrations of samples (20-200 μg/ml), which was then incubated in pitch-dark for 15 min at ambient temperature (20°C). A gradual decline in absorbance was determined at 517 nm in a spectrophotometer (Thermo Fisher EVO 300PC, USA). Ascorbic acid (SRL, Mumbai, India) solution of the same concentration can be used as a positive control for the free radical scavenging assay. The test was conducted in three replicates. The findings were expressed in IC 50 value, ascertained by plotting the percentage of scavenging activity against concentration.
The potential ability to scavenge DPPH free radical was estimated by the below equation: %inhibition ratio ¼ Absorbance of the control − Absorbance of the tested compound Absorbance of the control Â 100 • Absorbance of control = absorbance of solution that contain all reagents except sample • Absorbance of compound = absorbance of sample or standard

Antimicrobial test Antibacterial test
The antibacterial activity of crude extract of S. urens roots was checked against bacterial strains Escherichia coli, Bacillus subtilis, and Klebsiella pneumoniae by employing agar well diffusion assay [27]. Obtained bacterial cultures were maintained at 37°C in Mueller Hinton nutrient broth (Himedia, Mumbai, India). The bacterial cell suspension culture was prepared in sterile saline water, and it was optimized to 0.5 McFarland turbidity standards with the help of a spectrophotometer to achieve the desired cell density (1.5 × 108 cfu/ml). Each bacterial culture uniformly streaked on Mueller Hinton agar plates, and 100 μl hydro-methanolic root extract was filled in the wells with 200 and 300 μg/ml concentration. Bacterial plates were kept at temperature 37°C and incubated for about 24 h. Later, antimicrobial activity was reckoned by measuring the inhibition zone.

Antifungal test
The experiment of diffusion in agar well was performed in compliance with the Clinical Laboratory Standard Institute document M51-A2 [25], with several adaptations. Initially, species were cultured in Sabouraud's agar medium (Himedia, Mumbai, India), which was kept at 30°C temperature for 7 days of incubation. The fungal spore suspension culture of Penicillium glaucum was prepared using sterile PBS (phosphate-buffered saline). Spore concentrations were standardized using haemocytometer to 105 spores/ml limit. The root extract (50 μl) was added with a concentration of 150 and 250 μg/ml, after punching the agar plates with a sterilized cork borer (2 cm in diameter). The plates were then kept at 30°C temperature for 48 h immediately followed by the measurement of inhibition halos.

Statistical analysis
Each value obtained is an aggregate of three replicas, and it was denoted as mean ± standard deviation. All the recorded data were analysed with the help of the statistical tool of the Window's statistical software package SPSS (release 8.0). The test of significance was conducted by oneway analysis of variance (ANOVA; P < 0.05).

Yield value and extract properties
The present study highlights yield value to quantify the number of active constituents relative to the amount of the crude drug material which was found co-related. Hydro-methanolic root extract recorded significantly high yield by 5.68%, along with the physical properties like the color (dark red), stickiness (non-sticky), and appearance (dry solid). The formula used for calculating the yield value of crude methanol extract follows:

%yield ¼
The final weight of extract obtained The initial weight of extract Â 100

Preliminary phytochemical screening
Hydro-methanolic root extract exhibited the presence of tannins, flavonoids, alkaloids, phenols, saponins, glycosides, steroids, and proteins and the absence of carbohydrate or sugar. Besides, methanol efficiently extracted phytochemical constituents indicating maximum polar molecules like phenolics (Table 1).

Thin-layer chromatography
The number of compounds in the extracts was calculated using the TLC method of analysis. The chromatographic spots which were representative of compounds in the various extracts were observed, and their Rf values determined ( Table 2). The extract exhibited the separation of phenolics with a yellowish-brown spot having the highest Rf value of 0.92, whereas separation of alkaloids revealed a yellowish spot was having Rf value 0.72 in visible light, red and blue at UV365. Further, flavonoids resulted in a brown spot having an Rf value of 0.84 in visible light, orange, red, and green at UV365. TLC assay separated steroids with purplish spot having Rf value 0.74 in visible light, red and blue at UV365. The analysis showed the presence of tannins with a greenishbrown spot (visible), red and blue at UV365, having the lowest Rf value of 0.52. Rf values denote relative migration, while absolute values vary by position based on different parameters, including humidity and temperature.

Fourier-transformed infrared (FT-IR) spectra analysis
Hydro-methanolic dried root extract was measured sourcing ATR-FTIR (Fig. 3) to analyze functional groups and to identify the peaks characteristic of each species. The possible functional groups and peak value interpretation (obtained by FTIR analysis) are shown in Table 3. Eighteen significant bands were spotted between 500 and 4000 cm −1 ; the transmission was converted into absorbance, and evaluation was generated. Band (A) at 529 cm −1 , (B) at 546 cm −1 , and (C) at 576 cm −1 appeared as three small peaks, which could indicate the C-halogen (Out-of-plane-ring bending). Fourth band (D) was distinguished at 631 cm −1 , representing skeletal vibration of C-Cl, an alkyl aldehyde stretches, and at 763 cm At 3356 cm −1 (R), a broad intensity peak was detected, which is specifying OH vibration of alcohol, aldehyde, or carboxylic acid.

Total phenolic content analysis
The 90% root extract consisted of a significant amount of total phenolic content with 705 ± 0.40 mg GAE/g DE. The calibrated curve equation is described below.
The current study investigated and confirmed that the hydro-methanolic extract of S. urens root represents high phenolic content as well as antioxidant activity.

Antioxidant activity
The DPPH assay is often used to test the antioxidant potential of the natural compounds. It is known as one of the standards and an effective and rapid method for colorimetric evaluation of antioxidant properties of biological samples. The basic principle of the assay is that the DPPH is a stable molecule that tends to absorb at 515 nm and gives purple colour in methanol. The reduction of DPPH by the antioxidant agent has resulted in a change from purple to yellow colour with a consequent decline in absorbance at 515 nm, which is monitored by spectrophotometer [28]. In the present investigation, the DPPH radicals were used for the determination of the prospective antioxidant properties of S. urens root. Ascorbic acid was chosen for positive control. Findings were expressed as 50% inhibition concentration (IC 50 ) computed from the linear regression equation obtained through a plot of concentration against the percentage of inhibition (Fig. 4). The lower IC 50 value of the compound indicates its stronger antioxidant properties [29]. In this study, the IC 50 value of a hydro-methanolic plant extract and ascorbic acid was estimated to be 27.055 μg/ ml and 37.244 μg/ml, respectively, which suggests that root extract possesses strong antioxidant properties.

Antimicrobial activity
The present investigation of antimicrobial potency of hydro-methanolic extract of S. urens roots was tested against two strains of gram-negative bacteria Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), one gram-positive bacteria strain Bacillus subtilis (B. subtilis), and one fungal species Penicillium glaucum (P. glaucum) by measuring the zone of inhibition (Fig. 5). For E. coli, the result obtained from the current investigation shows a higher zone of inhibition, 30 mm and 50 mm for 200 μg/ml and 300 μg/ml concentration of root extract, respectively, which indicates good antimicrobial activity against E. coli bacteria.
The results of the current study revealed that root extract with a concentration of 300 μg/ml shows significant    In this study, we found that root extract was ineffective for B. subtilis strain. Penicillium is the most familiar and widespread fungus; several members of the genus produce the antibiotic penicillin while others are used to make cheese [30]. Hydro-methanolic extract of S. urens root with concentrations of 200 μg/ml and 300 μg/ml showed 45 mm and 25 mm zone of inhibition, respectively, which indicates unusual antifungal activity against P. glaucum.

Discussion
At present, research in herbal medicines and their natural substances is substantially focused. S. urens has its traditional significance in treating different human diseases. Moreover, ethnomedicinally, its roots are used for facilitating childbirth, relieving constipation, and reducing body swelling [31]. For utilizing this plant in modern therapeutic systems, rigorous experimental evidence, toxicity analyses, and human clinical trials are needed.
Henceforth, the main purpose of this research was to investigate and elucidate the medicinal values of S. urens. This is a first study that we are reporting since there is less prior literature on the phytochemical characterization of the root. Consequently, Sterculia urens Roxb. was selected and standardized on their physicochemical parameters such as extractive values, phytochemical study, qualitative estimation, TPC, TLC along with FTIR analysis, antioxidant activity, and antimicrobial activity for the examination.
Yield value of S. urens root extract is noteworthy which suggests that the plant contains a variable number of active constituents or relative amounts of soluble compounds in methanol extract. There will be common secondary metabolites found in two distinct species of a similar genus. It has been claimed that the genus Sterculia includes phenolics, steroids, terpenes, alkaloids, and flavonoids, but literature on S. urens root chemistry is not yet documented. It poses a challenge when it comes to discovering new constituents from S. urens root. Moreover, several biological activities in genus Sterculia like Sterculia tavia-anti-proliferative activity, Sterculia villosa-antidiabetic activity, Sterculia guttata-larvicidal activity, Sterculia foetida-anti-fertility and anti-inflammatory activity,  and Sterculia quadrifida-antioxidant activity are studied [32]. Despite a successful investigation into the genus Sterculia, S. urens was underexplored for its chemical constituents and biological activities. Further, experiments are underway to isolate and classify bioactive substances obtained from S. urens. Later, bioactive phytoconstituents were confirmed using TLC. S. urens displayed notable concentration of TPC which is supported by various studies in the same genera. S. quadrifida root is traditionally known to cure diabetes and cancer-like ailments. The findings obtained in our analysis for TPC are higher than the results published by Lulan et al. [32], who reported a maximum of 116.84 ± 1.22 mg of GAE/100 g S. quadrifida extracted with methanol. Few researchers stated total phenolic content values of S. oblongata bark ranges from 100 to 600 mg GAE/g with chloroform, ethyl acetate, and methanol extracts which supports the phenolic content of the current study [33]. Phenolic compounds act as antioxidant because of their redox properties. According to Annapandian and Rajagopal [34], TPC substantially impacts its antioxidant activity, which is crucial for radical scavenging. Several studies claimed to have phenolics as crucial substances showing different biological activity and medicinal properties.
The synergistic role of the phytochemicals can serve as a natural cure for oxidative damage resulting in chronic inflammation. Corresponding outcomes were reported by Haque et al. [35], in crude methanolic extract of S. villosa bark (IC 50 value 27.28 μg/ml), and Dillak et al. [36], in the ethanolic extract of S. quadrifida root (IC 50 value 20.55 ± 0.42). The result derived from this analysis was in good agreement towards earlier reports suggesting the use of methanol extract for the radical scavenging activity analysis [37]. Different metabolic processes of the living system produce reactive species called free radicals; they spread throughout the body and damage important biomolecules of the body by disrupting their structure and interfering with their function, leading to cellular dysfunction and cell death. This can result in serious health problems such as cancer and ageing, which can be prevented by radical scavenging potential of flavonoid and phenolics [38].
Since the last few decades, multiple antibiotic drug resistance has been acquired by pathogenic bacteria. For that, alternative strategies are required to combat antibiotic resistance. Escherichia coli is a widely occurring bacterium in human's intestine and homeothermic organisms. The vast majority of E. coli strains are innocuous. Nonetheless, a few other strains, including enterohemorrhagic E. coli, may cause severe foodborne diseases. In general, K. pneumoniae is an encapsulated, non-motile opportunistic pathogen that is found in the environment and targets principally people with compromised immune systems and causes nosocomial infection. K. pneumoniae is also responsible for various diseases like urinary tract, bloodstream, and septic infection [39,40]. The presence of considerate phytoconstituents like phenolics, alkaloids, flavonoids, saponins, terpenoids, glycosides, tannins, and steroids in root extract might be the reason behind its significant in vitro antioxidant and antimicrobial activity. Such results offer a comparable bioactivity study and serve as an example of their future application in the pharmaceutical and food industries.

Conclusion
The principal objective of the study is to identify the presence of different secondary metabolites, their functional groups, antioxidant and antimicrobial activity of Sterculia urens root. The presence of these bioactive metabolites implies that the hydro-methanolic root extract of S. urens has quite a few pharmaceutical products that would have quite several medicinal applications. It showed high phenolic content, which is a valuable reference for antioxidant properties. The extract of root exhibited high DPPH radical scavenging activity, indicating S. urens as a potential candidate for antioxidant study for further studies. Additionally, isolation, purification, and characterization of the phytochemicals will make remarkable studies. This primary information will simplify in leading further studies on the discovery of bioactive ingredients, resolve their efficacy by in vivo studies, and demonstrate their safety and effectiveness in clinical trials. The study suggests that crude extract possesses promising antimicrobial and antioxidant activity; thus, organic antioxidant and antimicrobial medicines can be generated for possible therapeutic applications. This is the fundamental collective report on phytochemical screening, TLC, TPC, FTIR, antioxidant, and antimicrobial activity on the roots of Sterculia urens Roxb.