Lilium philadelphicum Flower as a Novel Source of Antimicrobial Agents: A Study of Bioactivity, Phytochemical Analysis, and Partial Identiﬁcation of Antimicrobial Metabolites

: The members of the Liliaceae family are considered an excellent source of biologically active compounds. However, work on antimicrobial potential and characterization of the bioactive fractions of the Lilium philadelphicum ﬂower is limited and needs to be explored. The present study reports the antimicrobial potential of the bioactive fraction extracted from the ﬂower of L. philadelphicum (red lily) and partial characterization of the bioactive compound(s). The antimicrobial activity was tested against nine different Gram-positive and Gram-negative bacterial strains. The minimum inhibitory concentration (MIC) values of methanolic extract of the L. philadelphicum ﬂower against Acinetobacter bouvetii , Achromobacter xylosoxidans , Bacillus subtilis MTCC 121, Candida albicans MTCC 183, Klebsiella pneumoniae MTCC 3384, and Salmonella typhi MTCC 537 were 25, 50, 12.5, 50, 100, and 50 µ g/mL, respectively. The phytochemical analysis of the extract revealed the presence of phenols, ﬂavonoids, tannins, terpenoids, glycosides, coumarins, and quinones. The cytotoxicity of the partially puriﬁed compound against the HepG2 cell line using MTT assay demonstrated up to 90% cell viability with a bioactive compound concentration of 50 µ g/mL. However, the increase in the bioactive compound’s concentration up to 1000 µ g/mL resulted in nearly 80% cell viability. This minor decline in cell viability suggests the importance and suitability of the bioactive compound for therapeutic applications. Spectroscopic studies of the bioactive compound by UV-visible spectroscopy, FT-infrared spectroscopy, gas chromatography-mass spectrometry (GC-MS), as well as phytochemical analysis, suggested the presence of a terpenoid moiety, which may be responsible for the antimicrobial property of the L. philadelphicum ﬂower.


Introduction
Plants with the most diversity in volatile compounds comprise a substantial portion of biogenic hydrocarbons in the environment [1]. These volatile compounds, such as terpenes, phenolics, benzenoids, and fatty acid derivatives, along with nitrogen and sulfur-containing compounds produced as secondary metabolites, possess antimicrobial

Purification and Characterization of Bioactive Compounds from the Red Lily Flower
L. philadelphicum flowers collected from the college campus (Institute of Engineering and Technology, Lucknow, Uttar Pradesh, India) were used in this study. Flowers were dried under shade at 30-40 • C (Supplementary Information: Figure S1a) and subsequently powdered. The crude powder was extracted (Supplementary Information: Figure S1b) with various solvents viz. hexane, chloroform, methanol, and water in a ratio of 1:10 (crude powder:solvent ratio) at 28 • C. The extracts were filtered with Whatman filter paper, concentrated under vacuum, and stored at 4 • C until further use. The extraction yields of different solvents were determined using the following formula: where, W1 is the net weight of the flower powder in gm after extraction, and W2 is the total weight of the flower powder in gm taken for extraction.

Evaluating Antimicrobial Activity of the Red Lily Flower Extract
The antimicrobial activity of the crude methanolic extract of the lily flower was determined using the agar well diffusion method against A. xylosoxidans, A. bouvetii, B. pumilus MTCC 1607, B. subtilis MTCC 121, C. albicans MTCC183, E. coli MTCC 1304, K. pneumoniae MTCC 3384, S. typhi MTCC 537, and S. aureus MTCC 96 (Supplementary Information: Table S1; Figure S2). The 24 h old bacterial and fungal test cultures were grown on nutrient agar (NA) (Hi-Media, Mumbai, India) and potato dextrose agar (PDA) (Hi-Media, Mumbai, India), respectively. The wells of 9 mm diameter were bored and filled with 200 µL of the solvent extracts of the red lily flower, and the culture plates were incubated at 37 • C for 24 h for bacteria and 72 h for fungus. After the incubation, the plates were observed for the inhibition of the growth of the test organisms, and the diameter of the zone of inhibition from each plate was measured [10].

Phytochemical Screening
The methanolic extract of the L. philadelphicum flower was screened to examine the presence of chemical groups and active compounds, such as carbohydrates, saponins, phenols and tannins, coumarins, flavonoids, amino acids, glycosides, terpenoids, and quinones (Supplementary Information: Figure S3).
To detect the presence of carbohydrates, the flower extract was dissolved in 5 mL distilled water and filtered. The filtrate was hydrolyzed with dilute HCl and further neutralized with alkali and subsequently heated with Fehling's solution A and B and observed for the formation of a red precipitate of reducing sugars [11]. The examination of saponin was performed by the foam test. The flower extract (0.5 g) was vigorously mixed with 2 mL of water and observed for foam formation for more than 10 min as an indication of the presence of saponin [11]. The presence of phenols and tannins was detected by performing the ferric chloride test. Ferric chloride (0.5%) solution was added drop by drop to 2 mL of flower extract and observed for the formation of a bluish-black precipitate of phenols and tannins [11]. About 0.5 g of the moistened flower extract was placed into a test tube. The mouth of the test tube was covered with filter paper treated with 1 N NaOH solution. The treated test tube was placed in boiling water for a few minutes and examined for the formation of yellow color as an indication of the presence of coumarins [12]. To test the presence of flavonoids in the flower extract, a 10% lead acetate solution was added to the extract. The formation of yellow precipitate confirmed the presence of flavonoids [11]. The presence of amino acids in L. philadelphicum flowers was checked by employing a ninhydrin test. A few drops of ninhydrin solution were added to the flower extract, and the appearance of blue color indicated the presence of amino acid [13]. For the identification of glycosides, 1 mL of glacial acetic acid, a few drops of ferric chloride solution, and concentrated H 2 SO 4 (mixed slowly through the sides of the test tube) were added to the flower extract and observed for the appearance of a reddish-brown ring of de-oxy sugars at the junction of the liquids [11]. For terpenoids identification in the flower extract, 2 mL of chloroform was added to 5 mL of the flower extract, and thereafter 3 mL of concentrated H 2 SO 4 was added slowly and observed for the appearance of the reddish-brown color of terpenoids [11]. The flower extract was treated with a few drops of concentrated H 2 SO 4 and observed for the formation of yellow color as an indication of the presence of quinones compound(s) [11]. The partial purification of the crude extract of the L. philadelphicum flower was performed by column chromatography using silica gel (mesh size 230-400) as a matrix. The column was eluted successively with an increasing gradient of methanol and chloroform. Further, the fractions were collected and examined by thinlayer chromatography (TLC) using a silica plate (TLC silica gel 60 F254) with methanol: chloroform (0.2% to 5%) as the mobile phase. Afterward, the TLC plate was removed from the solvent chamber, dried, and observed in the iodine chamber. The fractions having the same retention factor (R f ) values were pooled together and subjected to the bioassay [11].

Spectroscopic Analyses of Active Fraction
The chemical characterization of the active fraction containing volatile compounds was carried out with the help of UV-visible spectroscopy (PerkinElmer UV WinLab 5.2.0.0646/Lambda 25 spectrophotometer) and Fourier transform infrared spectroscopy IR (FT-IR PerkinElmer Model RX-1 spectrometer). Gas chromatography-mass spectroscopy (TSQ Quantum XLS) was performed to analyze the volatile compounds present in the active fraction.

Estimating Minimum Inhibitory Concentration (MIC) of the Partially Purified Compound
The MIC values of the partially purified compound were checked against Grampositive and Gram-negative bacterial strains and fungus with erythromycin (E15) as a standard antibacterial drug according to standard protocol [14]. A 24 h old culture of each bacterial test strain (5 × 10 −5 cells/mL) was grown in on NB; 200 µg/mL of the compound was taken as an initial concentration in the first test tube and was serially diluted. NB with and without erythromycin served as a negative and positive control, respectively. Another control, i.e., pure solvent (DMSO) only, was also included to observe the effect of the solvent on microbial growth. All the tubes were incubated at 28 • C overnight, and thereafter bacterial growth was observed [14].

Analyzing Cytotoxicity Using MTT Assay
In vitro cytotoxicity of the active compound against the HepG2 liver cancer cell line was analyzed by MTT assay as described by Mosmann [15] with minor modifications. A total of 100 µL of HepG2 cells suspension in a 96-well microtitre plate was incubated overnight at 37 • C in a CO 2 incubator. After 24 h, the medium was replaced by 100 µL of the fresh medium and treated with varying concentrations of the flower extract (0-1000 µg/mL). The microtitre plate was re-incubated at 37 • C under 5% CO 2 in the air for 24 h. The wells without flower extract served as a positive control. Further, MTT at 0.5 mg/mL concentration was added to the cell culture, and the plate was further incubated at 37 • C for 4 h. After the incubation, the culture supernatant was removed, and the cell layer was dissolved in DMSO (200 µL) and analyzed in a microplate reader (BioTek Instruments Inc, Winooski, VT, USA) at a test wavelength of 550 nm and a reference wavelength of 660 nm.

Antimicrobial and Phytochemical Screening of Solvent Extracts
The chemical investigation of the reddish-orange bioactive fraction extracted from L. philadelphicum revealed the presence of polar compounds. The methanolic and aqueous extract had greater extraction yields of 23.12 and 23.54%, respectively. Among the four solvents used during the extraction process, the yields obtained from chloroform and hexane extracts were 2.25 and 10.29%, respectively. The flower extract exhibited antimicrobial activity against S. typhi MTCC 537, A. bouvetii, B. subtilis MTCC 121, A. xylosoxidans, K. pnemoniae MTCC 3384, and C. albicans MTCC 183. However, the methanolic extract was not effective against S. aureus MTCC 96, B. pumilus MTCC 1607, and E. coli MTCC 1304. The aqueous extract of the L. philadelphicum flower exhibited antimicrobial activity against B. subtilis, B. pumilus, K. pneumoniae, A. bouvetti, and C. albicans. However, chloroform and hexane extracts were found slightly effective only against K. pneumoniae MTCC 3384 and A. xylosoxidans.
In most of the developing countries, including India, various infectious diseases are still creating challenges in the healthcare sector. To counteract such challenges, a variety of antimicrobials have been discovered to fight against the pathogens responsible for various diseases. These antimicrobials can be obtained as secondary metabolites from microbes, animals, or plants or can be synthesized chemically [16,17]. Due to the occurrence of wide-spectrum and multiple drug resistance (MDR) in pathogens towards existing antibiotics and the unpleasant side effects of the currently used antibiotics and synthetic Sustainability 2021, 13, 8471 5 of 10 drugs, investigation of other sources, such as medicinal plants, for their antimicrobial properties is gaining importance [18]. The phytochemical analysis of the L. philadelphicum flower revealed the presence of flavonoids, tannins, glycosides, phenols, coumarins, and terpenoids (Table 1).
Several bioactive constituents have been reported from plants such as Azardirachta indica [19], Senna alata [20], and Terminalia bellerica [21]. In their efforts to search for potential antimicrobial compounds, Dontha et al. [22] reported the isolation of active constituents of Ixora Javanica DC flower extract and its phytochemical characterization. Similarly, in our study, the flower of L. philadelphicum was investigated, and the reddish-orange bioactive compound was isolated from the methanolic extract. The flower extract was found to be active against some Gram-negative/-positive bacteria and Candida albicans. Soliman et al. [17] reported the antifungal activity of a range of plants and suggested that they can be used as anti-candida agents after more in vivo investigations and suggested studies regarding the use of the nanostructured lipid system [23]. Following the encouraging results of the antimicrobial activities of the lily flower, we proceeded to the phytochemical analysis, purification, and identification of the bioactive compound.

Purification and Chemical Characterization of Bioactive Compound
Phytochemical analysis revealed that the L. philadelphicum flower is rich in phenols, flavonoids, quinones, tannins, glycosides, coumarins, and terpenoids. Spectroscopic and phytochemical analyses of the bioactive fraction from the lily flower suggest the presence of terpenoids. The flower's crude methanolic extract purified on silica column showed the presence of forty-eight fractions; these fractions were separately collected, their Rf values were determined, and all the fractions were subjected to antimicrobial bioassay. The initial fractions obtained from the silica column were more active against all the test organisms; however, fractions eluted later were active against one or two microbes only (Figure 1). The most active fraction was selected and subjected to partial characterization by UV-visible, FTIR, and GC-MS spectrum. The active fraction absorbed in the range of 290 to 360 nm and the pattern of the absorption suggested the presence of a double bond in the conjugation, which in turn reduces the possibility of the presence of saturated compounds (Figure 2).
The presence of strong absorption bands in the ultraviolet (UV)/visual (VIS) absorption spectrum is possibly due to p→p * or n→p * transitions [24]. In the performed experiments, the pattern of absorbance observed during the analysis suggests the presence of conjugation in the structure [24]. The IR spectrum explains different types of bonds, i.e., single, double, or triple among carbon, hydrogen, nitrogen, and oxygen atoms, which have diverse vibrational frequencies [25].   The presence of strong absorption bands in the ultraviolet (UV)/visual (VIS) absorption spectrum is possibly due to p→p * or n→p * transitions [24]. In the performed experiments, the pattern of absorbance observed during the analysis suggests the presence of conjugation in the structure [24]. The IR spectrum explains different types of bonds, i.e., single, double, or triple among carbon, hydrogen, nitrogen, and oxygen atoms, which have diverse vibrational frequencies [25].   The presence of strong absorption bands in the ultraviolet (UV)/visual (VIS) absorption spectrum is possibly due to p→p * or n→p * transitions [24]. In the performed experiments, the pattern of absorbance observed during the analysis suggests the presence of conjugation in the structure [24]. The IR spectrum explains different types of bonds, i.e., single, double, or triple among carbon, hydrogen, nitrogen, and oxygen atoms, which have diverse vibrational frequencies [25].  The FTIR spectrum of the samples exhibits bands at 2994.75 and 2911.83 cm −1 , which confirms the CH stretching ( Figure 3). The band at 1435.83 cm −1 indicates the aromatic or heteroatomic C-C stretching vibrations. The absorption band at 1309.05 cm −1 corresponds to the C=O moiety in the compound. The frequencies were identified at 951.52, 696.04, and 666.66 cm −1 corresponds to =C-H bending vibrations.  Further, GC-MS of the sample was recorded on TSQ Quantum XLS for the identification of the bioactive component(s) of the flower. The generated spectrum was processed for major ideal peaks found during the analysis and compared with the mass spectrum from the library standards available in the database (NIST library). The GC-MS spectrum shows nine peaks at different retention times (RT) with different probability factors (PF), which in turn reflects the presence of nine different compounds. These compounds are The FTIR spectrum of the samples exhibits bands at 2994.75 and 2911.83 cm −1 , which confirms the CH stretching (Figure 3). The band at 1435.83 cm −1 indicates the aromatic or heteroatomic C-C stretching vibrations. The absorption band at 1309.05 cm −1 corresponds to the C=O moiety in the compound. The frequencies were identified at 951.52, 696.04, and 666.66 cm −1 corresponds to =C-H bending vibrations.  Further, GC-MS of the sample was recorded on TSQ Quantum XLS for the identification of the bioactive component(s) of the flower. The generated spectrum was processed for major ideal peaks found during the analysis and compared with the mass spectrum from the library standards available in the database (NIST library). The GC-MS spectrum shows nine peaks at different retention times (RT) with different probability factors (PF), which in turn reflects the presence of nine different compounds. These compounds are

Estimating Minimum Inhibitory Concentration (MIC) and Cytotoxicity of the Active Fraction
The MIC of the partially purified compound against Candida albicans MTCC 183, Achromobacter xylosoxidans, and Salmonella typhi MTCC 537 was 50 µg/mL. The purified compound was found to be less active (shows MIC 100 µg/mL) against Klebsiella pneumoniae MTCC 3384. However, it was found to be more effective against Acinetobacter bouvetii and

Estimating Minimum Inhibitory Concentration (MIC) and Cytotoxicity of the Active Fraction
The MIC of the partially purified compound against Candida albicans MTCC 183, Achromobacter xylosoxidans, and Salmonella typhi MTCC 537 was 50 μg/mL. The purified compound was found to be less active (shows MIC 100 μg/mL) against Klebsiella pneumoniae MTCC 3384. However, it was found to be more effective against Acinetobacter bouvetii and Bacillus subtilis MTCC 121 with MIC values of 25 and 12.5 μg/m, respectively.
Further, the cytotoxicity analysis of the bioactive compound on the HepG2 cancer cell line employing MTT assay demonstrated approximately 9.7% cell inhibition at 50 μg/m concentration and approximately 20% cell inhibition at 1000 μg/mL concentration ( Figure 5). The phytochemical analysis of the L. philadelphicum flower is in line with earlier findings, where it has been reported that carbonyl compounds such as terpenoids, especially monoterpenes [C10], sesquiterpenes [C15], alcohols, aldehydes, acyclic esters or lactones, and exceptionally nitrogen and sulfur-containing compounds, coumarins, and homologs of phenylpropanoids, exhibit a wide spectrum of biological activity [26]. The active fraction may contain terpenoids, as evidenced by the spectroscopic and phytochemical analyses. The terpenoids in the active fractions might be responsible for the antimicrobial properties of the L. philadelphicum flower. To the best of our knowledge, this is the first study deciphering the antimicrobial activity of the L. philadelphicum flower.

Conclusions
The potent and broad-spectrum antimicrobial properties of the L. philadelphicum (red lily) flower investigated in the present study suggest its therapeutic potential against Gram-positive and Gram-negative pathogens. The phytochemical analyses reveal that the polar organic solvents are more efficient in the extraction process and thus suggest the polar nature of the metabolites present in the flower. Further, the cytotoxicity assay concludes that approximately 90% of cells are viable up to 50 μg/mL concentration of the The phytochemical analysis of the L. philadelphicum flower is in line with earlier findings, where it has been reported that carbonyl compounds such as terpenoids, especially monoterpenes [C10], sesquiterpenes [C15], alcohols, aldehydes, acyclic esters or lactones, and exceptionally nitrogen and sulfur-containing compounds, coumarins, and homologs of phenylpropanoids, exhibit a wide spectrum of biological activity [26]. The active fraction may contain terpenoids, as evidenced by the spectroscopic and phytochemical analyses. The terpenoids in the active fractions might be responsible for the antimicrobial properties of the L. philadelphicum flower. To the best of our knowledge, this is the first study deciphering the antimicrobial activity of the L. philadelphicum flower.

Conclusions
The potent and broad-spectrum antimicrobial properties of the L. philadelphicum (red lily) flower investigated in the present study suggest its therapeutic potential against Grampositive and Gram-negative pathogens. The phytochemical analyses reveal that the polar organic solvents are more efficient in the extraction process and thus suggest the polar nature of the metabolites present in the flower. Further, the cytotoxicity assay concludes that approximately 90% of cells are viable up to 50 µg/mL concentration of the partially purified compound from the L. philadelphicum flower. In conclusion, the antimicrobial activity of the L. philadelphicum flower, along with cytotoxicity results, suggests the future application of this flower for antimicrobial purposes in the treatment of various infectious diseases. Further research in the direction of the complete characterization of the active compound and in vivo mechanistic studies in animal models may provide better insight into the understanding of the identification and development of suitable bioactive agents to treat various infectious diseases.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/su13158471/s1, Table S1. Antimicrobial activity of the extracts of L. philadelphicum tested against different microorganisms: Zone of Inhibition obtained using various solvent extracts. Figure  S1. (a) Dried flowers of lily; (b) example of crude extract of lily flower. Figure S2. Some examples of Zone of Inhibition (ZOI) obtained from various solvents (methanol/hexane/aqueous/chloroform) extract of the flower. Figure S3. Phytochemical analysis of the methanolic extract of red lily flower.