PHYTOCHEMICAL SCREENING, ANTIMICROBIAL, AND ANTIOXIDANT ACTIVITIES OF ROOT AND LEAF EXTRACTS OF LEUCAS ASPERA

Objective: The present study was designed to evaluate the phytochemical screening, antimicrobial, and antioxidant activities from the roots and leaves of Leucas aspera. Methods: The phytochemical screening and Fourier transform infrared (FTIR) analysis of root and leaf extracts were studied using standard methods. The disk diffusion method was performed to analyze the antimicrobial activity of aqueous extract, methanol extract and hexane extract of root and leaf against to selected bacterial and fungal strains. Antibiotics, streptomycin and nystatin were used as standards for bacteria and fungi, respectively. The antioxidant activity of the extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and scavenging of H2O2 free radicals. Results: Preliminary phytochemical screening of extracts exhibited the presence of carbohydrates, proteins, amino acids, steroids flavonoids, terpenoids, saponins, alkaloids, tannins, and phlobatannins. Leaf extracts exhibited effective antibacterial and antifungal activities compared with root extracts against all the tested bacteria and fungi. ME of the leaves exhibited highest antibacterial activity against Staphylococcus aureus (12.8±0.31 mm), followed by Bacillus subtilis (11.4±0.3 mm), Escherichia coli (9.8±0.21 mm), and Pseudomonas aeruginosa (7.3±0.29 mm). Leaf extracts of L. aspera showed effective scavenging activity compared with root extracts. ME of the leaves showed maximum scavenging activities of 38.39 and 36.85%, respectively, against DPPH and H2O2 free radicals with half maximal inhibitory concentration values of 136.17 and 142.42 μg/ml. Conclusion: Phytochemical analysis and FTIR spectrum revealed that different plant secondary metabolites particularly alkaloids, terpenoids, and flavonoids could be responsible for antimicrobial and antioxidant activities of L. aspera leaf extracts.


INTRODUCTION
Traditional medicine is an integral part of the primary health-care system in many countries such as India, China, and Sri Lanka. Plants are good examples and a number of plant species are known to have medicinal value. India is known for its incredible source of medicinal plants, and large body of evidence has been accumulated to highlight the potential use of medicinal plants against the harmful diseases. Over the past 100 years, development of chemotherapy and technology for the production of synthetic drugs changed the world scenario in medicine. Usage of synthetic drugs against a range of pathogenic microbes is becoming complicated due to non-response/modification of microbes. Moreover, the wide usage of commercial antimicrobial drugs lead to side effects, and most notably, development of drug resistance in the majority of the pathogenic microbes is an another major problem. To minimize the synthetic antimicrobial drugs and to reduce side effects, screening of plants with antimicrobial properties is growing day-by-day.
It is well known that several medicinal plants have been used to treat microbial infections from 1000 years [1,2]. A number of studies reported the antimicrobial properties of plants [3][4][5][6][7][8][9]. In addition to medicinal value, there is an increasing interest toward natural antioxidants present in plants. Antioxidant molecules have the capability to quench free radicals and remediate the effects caused by reactive oxygen species and have roles in the prevention of degenerative diseases which caused by oxidative stress [10]. Medicinal plants are rich sources of antioxidant molecules such as flavonoids, triterpenoids, tannins, coumarins, quinones, vitamins, and polyphenolic compounds and it was reported by several studies [11][12][13].
Numerous studies have investigated the presence of pharmacological properties from several medicinal plants. Since of more importance to the individual compounds, the recent research is in much focus to isolate and identify the specific compounds using various methods of recent technology. The most popular methods such as high-performance liquid chromatography, gas chromatography-mass spectrometry (MS), matrix-assisted laser desorption ionization time-of-flight, MS-MS, and Fourier transform infrared (FTIR) are in use to isolate the pure compounds from various parts of the plant [14][15][16].
Based on the medicinal properties of L. aspera, the present study was aimed to evaluate the antimicrobial activity of root and leaf extracts (aqueous extract [AE], methanol extract [ME], and hexane extract [HE]) of L. aspera against a range of selected bacterial and fungal strains. The active compounds were also analyzed by FTIR screening and

FTIR analysis of leaf and root extracts
The dried powders of different extracts were made as pellet by taking 2 mg of the sample mixed with 200 mg KBr (FT-IR grade). The sample pellet kept into the sample holder and recorded the spectra range between 4000 and 450 cm -1 in FTIR spectroscopy [22].

Microorganisms
To screen of antimicrobial activity of L. aspera roots and leaves, Grampositive, Gram-negative bacteria such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa and fungi Candida albicans and Aspergillus niger were used. All the cultures were obtained from the Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, Tirupati.

Antibacterial activity
Antibacterial activity of the different extracts of roots and leaves of L. aspera was evaluated against selected bacteria according to the Kirby-Bauer disk diffusion method [23]. Actively growing bacterial culture of 200 µl was evenly spread on the surface of nutrient agar plates using the sterile glass rod. The extracts were prepared with the concentrations of 25, 50, 75, and 100 μg/ml and antibacterial activity was evaluated by placing the discs of different concentrations on the nutrient agar plates with standard antibiotic, streptomycin and plates were kept for incubation at 37°C for 24 h. The diameters of the inhibitory zones were recorded after incubation.

Antifungal activity
Antifungal activity of the different extracts of roots and leaves of L. aspera was evaluated against C. albicans and Aspergillus niger using disk diffusion method [24]. 200 µl of actively growing fungal inoculum was swabbed on the surface of potato dextrose agar (PDA) plates. The antibiotic discs prepared with 25, 50, 75, and 100 μg/ml concentrations were placed on the PDA plates with standard antibiotic, nystatin. The PDA plates were incubated at 24°C for 72 h. After incubation, the diameters of zone of inhibitions were measured.

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay
In vitro, antioxidant activity of the root and leaf extracts was evaluated by DPPH free radical scavenging assay [25]. 1 ml of each extract at selected concentrations (25,50,75, and 100 μg/ml) was mixed with 2 ml of DPPH solution. The reaction mixture was incubated for 30 min at room temperature, and absorbance was recorded at 517 nm. Ascorbic acid was used as a standard. The DPPH scavenging activity was calculated using the following formula: The amount of sample necessary to decrease the absorbance of DPPH by 50% (half maximal inhibitory concentration [IC 50 ]) was calculated graphically.

H 2 O 2 radical scavenging assay
Antioxidant activity of the different extracts of L. aspera was further proved by H 2 O 2 radical scavenging assay [26]. 2 ml of H 2 O 2 solution prepared in 40 mM phosphate buffer (pH 7.4) was added to 1 ml of different concentrations, 25, 50, 75, and 100 μg/ml of extracts separately and kept in the dark at room temperature for 20 min. After incubation, absorbance was measured at 230 nm for each test sample. Ascorbic acid was used as a standard. The percentage of radical scavenging activity (RSA) was calculated using the equation.

RESULTS
The present study reports the phytochemical screening, FTIR analysis, antimicrobial, and antioxidant activities of different extracts of roots and leaves of L. aspera.

Phytochemical screening
Phytochemical screening of the L. aspera suggests the presence of major phytochemicals in the root and leaf extracts (Tables 1 and 2). ME and HE of roots and leaves showed the presence of carbohydrates, cholesterols, proteins, amino acids, steroids, saponins, flavonoids, alkaloids, tannins, and phlobatannins, whereas, AEs of both root and  leaf also showed the presence of all phytochemicals except cholesterols and steroids (Tables 1 and 2).

FTIR analysis of the plant sample
FTIR analysis of leaf and root extracts of L. aspera was carried out to reveal the functional groups present in the leaves and roots. FTIR spectrum of different extracts leaves of the L. aspera (Fig. 1a-

Antibacterial activity
Antibacterial activity of the AE, ME, and HE of roots and leaves of the L. aspera was evaluated, and zone of inhibition values of all the extracts was presented in Table 3. All the extracts of roots and leaves showed dose-dependent inhibitory activity against all the tested bacteria. All the extracts showed maximum zone of inhibition at the highest concentration of 100 µg/ml tested. All the prepared extracts of roots and leaves of L. aspera displayed the highest inhibitory activity against Gram-positive bacteria compared to Gram-negative bacteria. ME of leaves formed the maximum inhibition zone of 11

Antifungal activity
Antifungal activity of the AE, ME, and HE of roots and leaves of the L. aspera was evaluated against C. albicans and A. niger; results are represented in Table 4. All the extracts of leaves and roots showed concentration-dependent inhibitory activity against the fungal species.
All the extracts of roots and leaves of L. aspera showed effective antifungal activity against A. niger compared with C. albicans. ME of the leaves showed highest antifungal activity compared with HE and AE of leaves. ME of leaves formed the inhibition zones of 10.3±0.27 and

DISCUSSION
In the present study, the phytochemical screening and FTIR analysis of root and leaf extracts of L. aspera revealed that the presence of   carbohydrates, cholesterols, steroids, proteins, amino acids, saponins, flavonoids, alkaloids, terpenoids, tannins, and phlobatannins in ME and HE in AE except cholesterols and steroids remaining all were detected. In earlier studies, the presence of different phytochemicals was reported in L. aspera whole plant in different extracts. In that series Mangathayaru et al. [27] identified the alkaloids and nicotine in methanolic extract of L.aspera whole plant (without roots). The presence of alkaloids, glycosides, saponins, tannins, terpenoids, and flavonoids were reported in ethanol, hexane, and ethyl acetate extracts [28]. High amounts of glycosides, tannins, and flavonoids observed in ethanolic extract, while the other two extracts contain moderate amount of the chemical constituents [28]. In ethanol extract of the whole plant reported with all major phytochemicals [29]. In another study, except flavonoids and tannins, others phytochemicals were reported in the whole plant of L. aspera in MEs [30]. The presence of different phytochemicals in the same plant is due to solvents which were used to extract preparations and plant parts selected. The occurrence of these secondary metabolites suggests that the plant might be of medicinal importance. It was reported that the presence of phenols, tannins, and flavonoids exhibits different pharmacological activities such as antimicrobial [31,32], antioxidant [33,34], anti-inflammatory [35,36], and anticancer [33,37].
Antimicrobial activity of L. aspera leaf and root extracts observed in this study, antibacterial studies of L. aspera showed that leaf extracts exhibited effective activity compared with root extracts. Among the different extracts, MEs showed potential activity compared with HE and AE. Among the different bacteria used in this study, Gram-positive bacteria were more susceptible than the Gram-negative bacteria to L. aspera extracts. The results of the antibacterial activity are very effective and are in line with many earlier antimicrobial studies of plant extracts. Earlier ME of leaf showed potent antibacterial activity against to E. coli, Klebsiella, Pseudomonas, and Staphylococcus aureus [38]. Similarly, ME of root, leaf, stem, and flower showed 7.0-11.0 nm zone of inhibition against selected bacteria [39]. Among all parts, root showed high antibacterial activity against E. coli, S. aureus, S. choleraesuis, S. typhimurium, S. flexneri, and P. aeruginosa and leaf extract showed against to S. aureus, S. choleraesuis, S. typhimurium, and S. flexneri [39]. In another study, whole plant ethyl acetate and MEs of L. aspera showed moderate to high and concentration-dependent antibacterial activity against all the tested bacterial strains [30]. Similarly, whole plant ME and ethanol showed antibacterial activity against to B. cereus, B. megaterium, B. subtilis, P. aeruginosa, S. paratyphi, S. typhi, S. dysenteriae, S. sonnei, and S. aureusi [40]. Leaf extract of L. aspera from methanol, ethyl acetate, and petroleum ether showed potential antibacterial activity at 1mg/disc in other study [41]. Many scientists tried to elucidate the plausible mechanism of action of the antimicrobial activity of plant extracts. Triterpenoids or saponins present in the extracts could form pore-like structures and cause the dissipation of membrane electrical potential or membrane proton motive force, and thus membrane destruction occurs [42].
MEs of roots and leaves of L. aspera showed highest inhibitory activity against selected fungi than HE and AE. The present results agreement with the earlier study, methanol, ethyl acetate, and petroleum ether leaf extract of L. aspera showed potential antifungal activity at 1 mg/disc against to Aspergillus fumigates, Botryodiplodia theobromae, Colletotrichum corchori, Curvularia lunata, and Fusarium equiseti but not to C. albicans and Saccharomyces cerevisiae [41]. Similar to this in another study, methanol and ethanol of whole plant extract showed potent antifungal activity against to F. equiseti, B. theobromae, and C. corchori [40]. FTIR analysis and phytochemical screening revealed that plant secondary metabolites present in the extracts can perturb the fungal cell wall and also caused the release of cellular components such as ions and intracellular proteins which, in turn, halt the growth of fungi. It had long been documented that plants metabolites saponins, tannins, and alkaloids are known for antimicrobial activity [43].
Antioxidants or radical scavengers are the compounds of bio or synthetic origin that protect the cells from various damaging effects and diseases caused by reactive oxygen species. In addition, naturally occurring antioxidants can be formulated to give nutraceuticals, which can help to prevent oxidative damage from occurring in the body. Assays associated with lipid peroxidations is a common method for estimation of antioxidant activity; however, estimation of stable free radical DPPH is other method used in plants other natural sources [44,45]. In the present study, free radical scavenger levels of H 2 O 2 and DPPH were observed in ME, AE, and HE leaf and root extract of L. aspera. The levels of free radical scavengers increased with dose-dependent in all extracts and ME of leaf showed high antioxidant activity compared with root extracts. Antioxidant or RSA of the different extracts is mainly due to the presence of hydrogen-donating molecules such as proteins, flavonoids, and terpenoids and other biomolecules are present in the extracts. Antioxidant activity of L. aspera with different extracts produced in this study is consistent with earlier reports [28,38,39,46]. Apart L. aspera, numerous plant components have proven to show antioxidants activity [28,47]. Previous reports emphasized that tannins and flavonoids are natural antioxidants that could play the main role in the antioxidant activity of the plant extracts. Besides, these alkaloids are natural antioxidants used for medicinal and commercial needs [48]. The presence of alkaloids, terpenoids, and flavonoids could be responsible for the antioxidant activities of the L. aspera extracts.

CONCLUSION
All the extracts showed antibacterial and antifungal activity with maximum inhibition against to selected microorganisms. MEs of leaf exhibited maximum antibacterial, antifungal, and highest RSA. Phytochemical screening and FTIR analysis revealed that alkaloids, triterpenoids, and flavonoids could be responsible for the antimicrobial and antioxidant activities, respectively, of the L. aspera extracts. From the present results, it is concluded that compounds of L. aspera can be used as antimicrobial agents and ingredients in the antioxidant formulations in different food and pharmaceutical fields.