Verbascum Thapsus (Mullein) Versatile Polarity Extracts: GC-MS Analysis, Phytochemical Profiling, Anti-bacterial Potential and Anti-oxidant Activity

Cure of microbial ailments via plant extracts is in practice for centuries1 deals with infectious diseases because of their antibiotic and antioxidant potential. Verbascum thapsus also known as common mullein is utilized as traditional medicine and tea rich in antioxidants and antibacterial plants for long time1,2. More than one hundred accepted names are given to large genus Verbascum belonging from Scrophulariaceae family in plant databases3. Leaves and flowers of V. Thapsus have expectorant and anti-inflammatory potential in herbal medicines4. V. thapsus reduces the severity of respiratory conditions including bronchitis and asthma along with reported effectiveness against haemorrhoids, fungal infections, and diarrhea5. Yet, the detailed phytochemical spectrum in versatile solvents remains relatively understudied. Plants from Verbascum genus have exhibited inhibitory activity against murine lymphocytic leukaemia, several strains of influenza viruses, Trichomonas vaginalis, etc.2. Plants of Verbascum species also have potential anticarcinogenic properties5. Verbascum Thapsus has been used as herbal treatment of several disorders, but no FDA approved drug is yet available. Prominent medicinal uses include treatment for Parkinson’s disease, diabetes, bronchitis, asthma, joints and stomach pain, and skin issues etc3. Flower extracts of V. Thapsus also show promising concentrations of Terpenoids. Iridoid glycosides, Lignan glycosides, phenylethanoid glycosides, sterones and saponins etc3,6. Verbascum thapsus is extensively grown in temperate areas of Pakistan5 and its medicinal importance prompted us to study it comprehensively. The phytochemistry of Verbascum thapsus is usually studied in a single solvent i.e., methanol, in the past decade7 and not a single study discusses it in all five range of solvents. Therefore, in continuation of prior studies, the present study aims to explore multiple aspects i.e., phytochemical analyses of Verbascum thapsus and quantitative/qualitative investigations via GCMS and its antibacterial activities in five different solvents (Figure 1).


INTRODUCTION
Cure of microbial ailments via plant extracts is in practice for centuries 1 deals with infectious diseases because of their antibiotic and antioxidant potential. Verbascum thapsus also known as common mullein is utilized as traditional medicine and tea rich in antioxidants and antibacterial plants for long time 1,2 . More than one hundred accepted names are given to large genus Verbascum belonging from Scrophulariaceae family in plant databases 3 . Leaves and flowers of V. Thapsus have expectorant and anti-inflammatory potential in herbal medicines 4 . V. thapsus reduces the severity of respiratory conditions including bronchitis and asthma along with reported effectiveness against haemorrhoids, fungal infections, and diarrhea 5 . Yet, the detailed phytochemical spectrum in versatile solvents remains relatively understudied. Plants from Verbascum genus have exhibited inhibitory activity against murine lymphocytic leukaemia, several strains of influenza viruses, Trichomonas vaginalis, etc. 2 . Plants of Verbascum species also have potential anticarcinogenic properties 5 . Verbascum Thapsus has been used as herbal treatment of several disorders, but no FDA approved drug is yet available. Prominent medicinal uses include treatment for Parkinson's disease, diabetes, bronchitis, asthma, joints and stomach pain, and skin issues etc 3 . Flower extracts of V. Thapsus also show promising concentrations of Terpenoids. Iridoid glycosides, Lignan glycosides, phenylethanoid glycosides, sterones and saponins etc 3,6 . Verbascum thapsus is extensively grown in temperate areas of Pakistan 5 and its medicinal importance prompted us to study it comprehensively. The phytochemistry of Verbascum thapsus is usually studied in a single solvent i.e., methanol, in the past decade 7 and not a single study discusses it in all five range of solvents. Therefore, in continuation of prior studies, the present study aims to explore multiple aspects i.e., phytochemical analyses of Verbascum thapsus and quantitative/qualitative investigations via GCMS and its antibacterial activities in five different solvents (Figure 1).

RESULTS AND DISCUSSION
Verbascum thapsus is used in the treatment of pulmonary problems, inflammatory diseases, asthma, spasmodic coughs, diarrhoea, tuberculosis, and migraine headaches, it also possesses antiviral, anti-cancer, and antibacterial potential 8,10 . Numerous bioactive agents have been extracted from oils and other extracts of Verbascum species 11 .

Phytochemical analysis
The phytochemical profiling of Verbascum thapsus extracts in methanol, ethanol, water, acetone, and hexane was performed to reveal phenol, alkaloids, tannins, saponins, cardiac glycosides, flavonoids, water-soluble phenols, water-insoluble phenols, triterpenoids, free anthraquinones, and combined anthraquinones (Table 1). Alkaloids and phenols were present in most extracts while saponins and combined anthraquinones were only detected in water-based solvents. The highest flavonoid content was present in acetone extract (3.1969 ug/ ml) and the highest phenol content (2.770 ug/ ml) was present in methanol extract ( Figure 2).  Phenolic and flavonoid compounds enhance the antioxidant activity of plants and are pharmaceutically important. Enormous antimicrobial, antioxidant, antitumorigenic, cardiovascular protective, immuneboosting, and anti-inflammatory effects 12 are highlighted in earlier studies. Sufficient phenol and flavonoid percentage may contribute to prior mentioned pharmaceutical benefits 13 . Several Verbascum species like V. phlomoides, V. pestalozzae, V. detersile, V. densiflorum contain important phytochemicals in the phenol range that makes them pharmacologically important 3,14,16 .

GCMS analysis
Methanolic extract of Verbascum thapsus showed the presence of 25 phytochemicals, while 41 phytochemicals were detected in ethanolic extracts. In the water extract, 15 phytochemicals were detected while 29 phytochemicals were detected in acetone extracts ( Figure  3, Supplementary Table 2). Fifteen phytochemicals were detected in hexane extract. Mass spectrometric analysis of V. thapsus extracts revealed the presence of many important compounds. Ethanolic extract, alone, contains 41 bioactive compounds. However, the least number of metabolites were present in the water and hexanebased extract of V. thapsus. Some prominent compounds include 2-Methoxy-4-vinylphenol (antioxidant, flavouring agent, antiinflammatory effect), Phosphonic acid (medical imaging, pro-drug), Stigmasterol and Hexa-decanoic acid, methyl es (antibacterial activity), n-Hexa-decanoic acid (cosmetics) and Vanillic acid (flavouring agent), 2(5H)-Furanone (appetite suppressant), 3-Hydroxy-.beta.-damascone (anti-inflammatory properties), Mequinol (skin depigmentation), Fluoroacetic acid, dodecyl ester (pesticide), Squalene (antiaging, antiinflammatory, acne, eczema), Vitamin E (antioxidant), Hydroquinone (hyperpigmentation) and Phytol (fragrance agent, transcription modulator). We would like to report many chemicals that did not match any reference molecule in GCMS library. These may have potential antioxidant, antimicrobial or any other activities. Therefore, it is of vital importance to identify and separate the unmatched compounds (Supplementary Table 2) via further analytical techniques like Mass spectrometry and flow cytometry. Further experiments could be conducted to evaluate the pharmacological potential of these unknown compounds present in Verbascum Thapsus extracts.
Plants use metabolites for their defence against pathogenic microbes using various strategies. These metabolites contribute to the intervention of microbe invasion and repel herbivores from potential harm they may pose to plant survival and activity 17,18 . Furthermore, new antibiotics are demanded worldwide to answer the question arise by antibiotic resistant variants 3,19 . Pure plants and herbal extracts are used universally to design chemicals of pharmaceutical importance.

Antibacterial activity
Antibacterial experiments (well plate method, Kirby's disc diffusion method, and resazurin absorption method) were performed on ten bacterial strains. Gram-positive strains included: Bacillus subtilis (ATCC_6051), Micrococcus luteus (ATCC_4698), Staphylococcus aureus (ATCC_25923), Lactococcus lactis (ATCC_LMO230), Listeria monocytogenes (ATCC_LM21) while the gram-negative bacterial strains included: Shigella sonnei (ATCC_25931), Salmonella enterica (ATCC_14028), Escherichia coli (ATCC_25922) and Klebsiella oxytoca (ATCC_43863). Overall maximum antimicrobial activity was displayed by ethanol and methanol-based extracts of V. Thapsus against most of the bacterial strains. Maximum percent growth inhibition of S. sonnei was produced by ethanol extract at 1000ug concentration. However, both ethanol and methanol extracts at 1000ug and 500ug concentration showed growth inhibition of L. lactis and C. freundii. Acetone based extract inhibited the growth of L. monocytogenes, L. lactis, and B. subtilis. On the other hand, water-based extracts of V. thapsus showed the least bacterial growth inhibition against all the bacterial strains.
Kirby disk diffusion method was used to study the bacterial growth inhibition by plant extract in various solvents. Ethanol, methanol, and acetone-based extracts showed significant antimicrobial activity against most of the bacterial strains while water and hexane-based extracts showed least antibacterial activity as compared to control treatment. Maximum inhibition was observed against C. freundii, M. luteus, S. sonnei, and K. oxytoca (Supplementary Table 3). The average diameter (mm) of inhibition zone in culture plates was recorded in triplicate and the mean value ± standard deviation of diameter has been presented in Supplementary files as table 3. Verbascum thapsus total extract showed amazing MIC in terms of percentage growth inhibition against L. lactis, E. coli, and K. oxytoca. (Figure 4). More than seventy percent of bacterial growth of L lactis was inhibited via V. thapsus extract at 100ul concentration.
Well plate method and Kirby's disc diffusion method indicated maximum antimicrobial activity of ethanol, methanol, and acetone extracts of V. thapsus ( Figure 5). Our results support previous findings 18,20,21 where ethanolic and methanolic extracts performed better in antibacterial action as compared to aqueous extracts. One way ANOVA results indicate a significant difference (p-value: <0.05) in the antibacterial activity of Verbascum extracts ( Figure 6).
Another experiment of DMSO mediated solubilization was conducted. Studies report prominent antimicrobial action of V. thapsus extracts against S. epidermidis, S. aureus, K. pneumonia, and E coli strains 18 . The present study explored the promising antibacterial activity of V. thapsus against L. lactis, E. coli, and K. oxytoca. Our results not only validate the research findings of the antibacterial properties of V. Thapsus 22 but also coincide with the other Verbascum species i.e., Verbascum macrurum 23 . It is therefore indicated that significant antimicrobial potential in Verbascum species ranks them high as medicinal plants 18,24 .

DPPH antioxidant activity
The use of DPPH to evaluate antioxidant activity in plant extracts. The highest antioxidant activity was recorded in acetone extract of V. thapsus (651.03125umol/L) followed by water-based extracts ( Figure  7). The antioxidant order of V. thapsus extract was acetone > water > hexane > ethanol > methanol. The lowest IC50 value shows the highest antioxidant potential of extracts. This indicates the presence of free radical scavenging active metabolites and makes this plant a good antioxidant 25,26 . On the contrary, prior studies indicated the maximum antioxidant potential of Verbascum plant species in water and methanol extracts 27,28 . For long, Verbascum species have been used in several regions across the globe for their antitumorigenic, anti-inflammatory, and anti-spasmodic effects along with diminishing migraine symptoms and wound healing 12,23 . Several other investigations also highlighted the antioxidant activity of Verbascum species in hydrophilic solvents 26,29 . Therefore, the antioxidant activity of V. thapsus revealed from our study is supported by other studies. The presence of phenolic compounds and antioxidants may provide advantageous health and medicinal benefits by Verbascum consumption in either raw or processed form after intensive tests and trials 25 .

Preliminary qualitative analysis
The plant extracts prepared in different solvents were subjected to preliminary qualitative analysis by adopting well-established procedures 7 . All the phytochemical tests were performed to confirm the presence of saponin, phenolic compounds, water-soluble phenol, water-insoluble phenol, flavonoids, polysteroids, terpenoids, cardiac glycosides, free anthraquinones, combined anthraquinones, tannins, and alkaloids in the tested plant samples.

Phenols
Phenols were quantified by following the standard procedures 7 . Briefly, 75 µL deionized distilled water (ddH2O) was added to each well of 96-well plate. 25 µL Folin C (F-C reagent, Sigma-Aldrich) was also added to each well of a plate (diluted 1:1 v/v with ddH2O) and left it to stand for 6min. 100 µL of Na2CO3 (75 g/L) was added to each well. After thorough mixing, plates were put in dark for 90 min. Each of the samples was repeated in triplicate. A spectrophotometric microplate reader (SPECTRA MAX M2e) was used to take readings at 765 nm. Gallic acid was used as a standard at 12.5-400 µg/mL and a standard calibration curve were generated. Phenols were determined as μg of gallic acid equivalents / mL, which was calculated via formula, y = 0.6053 x − 0.0567, where y is the absorbance at 765 nm and x is the amount of gallic acid equivalent in μg/mL 30 .

Flavonoids
Flavonoids were quantified by following the standard procedures 6 . 100 µL ddH2O was dispensed in each of the 96 wells of the well plate. 10 µL of NaNO2 (50 g/L) and 25 µL of standard or plant extract sample solution were added to these wells followed by incubation for 5 minutes at room temperature. 15 µL of AlCl3 (100 g/L) was added to the mixture and left for 6 minutes. Later, 50 µL of NaOH (1 mol/L) and 50 µL of ddH2O were added to each well, and the plate was shaken for 30 sec, and absorbance was measured at 510 nm using SPECTRA MAX M2e plate reader. Catechin was used as a standard at 5-500 µg/mL to generate calibration curve and flavonoids content of plant extract was expressed in µg of Catechin equivalents / mL and were calculated by the formula, y = 0.5377 x + 0.316, where y is the absorbance at 510 nm and x is the amount of Catechin equivalent in μg/mL 30 .

DPPH Antioxidant assay
DPPH radical scavenging assay by 31 Bersuder, Hole and Smith 1998 was used to determine the antioxidant activity of plant extract. 25uLof Plant extracts were left to react with free DPPH (200 uL) prepared in ethanol for 6 hrs in dark in all 96 wells. Ascorbic acid was used as a standard at 50-1000 µmol/L concentrations to generate a calibration curve. It was dissolved with DMSO alone to act as a negative control for percentage calculation of plant extract radical scavenging activity. Absorbance was measured at 517 nm 32 .

Gas Chromatography-Mass Spectrometry (GC/MS) Analysis
The phytochemical investigation of plant crude extract was performed using GC-MS equipment (Bruker Scion 456 GC, EVOQ triple quadrupole GC-MS/MS) following the standard procedures 33 . The experimental conditions of the GC-MS system were as follows: Column: 15m, 0.25mm inner diameter, 0.25mm film thickness. The flow rate of the mobile phase (carrier gas: He) was set at 1.5mL/min. In the gas chromatography part, temperature-programmed (oven temperature) was 45°C hold 3 min-raised to 250°C at 8°C/min hold 10 min and injection volume of 1 uL using the varying split ratio (
All these bacteria were separately grown on Tryptic Soy Broth (TSB) medium (Thermo Fisher Scientific, USA) and incubated at 26 ± 2 °C for twenty-four hours. All the pure bacterial cultures were stored at -4 °C till further use in experimentation. Bacterial inoculum was prepared by suspending the pure bacterial colonies in 25 mL capacity lid vials containing sterilized nutrient broth medium followed by incubation at 26 ± 2 °C on a rotary shaker for 24 hours. The concentration of each of the bacterial inoculum was maintained at 10 5 to 10 6 CFU/mL.

Determination of Minimum Inhibitory Concentrations (MIC) and percentage inhibition / antibacterial activity
The MIC of Verbascum thapsus was investigated by following three methodologies including 96 well test, Kirby-Buyer disk diffusion and resazurin-based well plate microdilution method. 96 well sterile microtiter tray assay 96 well sterile microtiter tray assay was reformed by adopting the standard operating procedure 12 . 100 µL TSB medium,100 µL of plant extract fractions at 5 dilution levels (1000 µg, 500 µg, 250 µg, 125 µg, and 62.5 µg) and 50 µL of each bacterial culture (105 to 106 CFU/ mL concentration) were loaded in the wells 34 . A well-containing TSB medium only (lacks Plant extract and culture) was maintained as a double negative well to check the sterility of the medium. Another wellcontained TSB medium and bacterial inoculation (lacks Plant extract) to check average bacterial growth trends as single negative control wells. Plates were covered, sealed, and incubated at room temperature for 24 hours. Standard readings for MIC were taken by recording the absorbance at 570 nm on Elx 800 plate reader. The following formula was used for the calculation of bacterial inhibition:

Kirby-Bauer Disk Diffusion Method
The Paper disc diffusion method proposed by Kirby 13 was used to evaluate the antimicrobial potency of all plant extracts. In this assay, solidified agar plates were swabbed with 24 hours old bacterial inoculum (106CFU/mL) under aseptic conditions. Paper discs (10mm) were soaked in 20ul of prepared plant extract at all concentrations (i.e.,1000 µg, 500 µg, 250 µg, 125 µg, and 62.5 µg) and were placed on to the solidified culture media under aseptic conditions followed by incubation at 26 ± 2 °C for 24 hours. The negative control contained paper discs dipped in distilled water. All the experimental treatments were repeated five times. The zone of inhibition (mm) was measured to confirm the susceptibility of bacterial agents 35 .

Resazurin based Well Plate Microdilution Method
The resazurin-based Well Plate Microdilution test was performed by adopting the standard procedure 14 . Before the test, Resazurin (7-Hydroxy-3H-phe-noxazin-3-one 10-oxide) solution was prepared by adding 121.5 mg of resazurin powder in 18 mL ddH2O and was homogenized on a vortex mixer for 1 hour. Phosphate Buffer Saline (PBS) was used to adjust the pH 7.4 of the solution. The total plant extract was prepared by mixing all the five extracts in Dimethyl sulfoxide (DMSO) to yield a homogenous mixture. 100 µL of TSB liquid medium and total plant extract was added to the wells 36 24 hours old bacterial inoculum (106 CFU/mL) of each bacterium were added to these wells. To ensure media sterility Double negative control wells were maintained that contain TSB medium only. Single negative control wells contained TSB, bacterial culture, and Resazurin. After overnight incubation at 26 ± 2 °C, 20 µL of Resazurin dye was added to each well and again incubated for 2 -4 h for the development of color change. Absorption readings of resazurin developed color intensity were taken at 550-590 nm wavelength by using the SPECTRA MAX M2e plate reader 36 .

Statistical analysis
Results or antimicrobial activity were statistically analysed using GraphPad prism version 8.0 and Microsoft office excel 2010 version. Means, Standard Deviations, and a One-way analysis of variance (ANOVA) test was performed for statistically testing the zone of inhibition of bacterial strains presented by Kirby's disc diffusion method to evaluate the antibacterial activity of plant extracts. The significance threshold was kept at 0.05 probability.

CONCLUSION
Important phytochemicals including phenols, flavonoids, alkaloids, tannins, and glycosides were indicated in Verbascum thapsus extracts via phytochemical analyses, antimicrobial activity, antioxidant assays, and GCMS analyses. The antioxidant and antimicrobial activity were observed against most bacterial strains and has the potential for development as a bactericide and medicinally important drug.