GC-MS analysis of Taraxacum officinale flowers and investigation of antimicrobial,anti-pellicle & anti-biofilm activities

Abstract

Plants synthesize large amount of useful and complex products which have no obvious metabolic and growth functions. These complex materials are said to be as secondary metabolites—phytochemicals which are plants active compounds possessing the potential to inhibit diseases. The purpose of the recent study was to investigate the pharmaceutical values of the flowers of Taraxacum officinale, for antimicrobial, anti-pellicle and anti-biofilm properties. Metanolic extracts with chloroform and n-hexane fractions against selected different bacterial (E.coli, P.aeruginosa, S.aureus, S.typhi) and fungal (F.oxysporum, A.niger, A.alternata, A.Terreus) strains were tested and GC-MS, FTIR and HPLC techniques, for detection of various secondary metabolites which are responsible for these activities, were performed. In antimicrobial assay, the result of the methanolic extract and fractions of the flowers was found to be effective against the tested bacterial and fungal strains. The GC-MS and FTIR analysis of chloroform fractions of T. officinale flowers reported the presence of a wide range of phytochemicals and secondery metabolites liable for the biological activities that can be purified in future for the synthesis of noval improved and valuable pharmaceutical products.

List of abbreviations

T.officinale = Taraxacum officinale

E.coli = Escherichia coli

S. aureus = Staphylococcus aureus

P.aeruginosa = Pseudomonas aeruginosa

S.typhi = Salmonella typhi

A.niger = Aspergillus niger

F.oxysporum = Fusarium oxysporium

A.alternata = Alternaria alternate

A.Terreus = Alternaria teurus

1 Introduction

The use of plant-derived drugs has been in practice for a long time due to the high mortality caused by microbial infectious diseases [1] and its importance cannot be overemphasized with the recent trends in the high rate of multidrug resistance in today’s antibiotics [2]. For the cure of microbial infections in human, medicinal plant materials have been used successfully [3, 4]. Medicinal plants are of great value as millions of plants are present in the world and the pharmacological activities of maximum plants have not been explored yet [5, 6].

For many years different plants parts, herbs and spices have been used for the trearment of infectious diseases. Moreover, those plants have great importance whose antimicrobial properties are known[4, 7]. Renewed interest in plants having antimicrobial activities has been arisen through the last twenty years, but still a great number of plants having antimicrobial potential are not fully explored [8]. Plants having antimicrobial compounds inhibit microbial growth through different mechanism than the antibiotics, and therefore could be of scientific value in the treatment of different microbial diseases [9 –11]. Microbial ability to shows resistance against potent antimicrobial compounds is increasing surprisingly [12, 13]. Bioactive compounds extracted from plants having medicinal value facilitate pharmacological studies which lead to the synthesis of a more effective drugs with minimum side effects [14 –17]. It is believed that phytochemicals have the disease prevention ability because of their antioxidant effect [18 –20].

Taraxacum officinale is a herbaceous perennial plant commonly known as Dandelion. It belongs to the family Asteraceae. It grows in temperate region of the world, on roadsides, shores, in lawns, in moist soils, disturbed banks and shores of watery ways. These are the weedy species [21 –24]. Dandeline has been also called fairy clock in English [24 –26]. Herb, containing white milky juice; stems short and narrow; leaves oblong, spatulate or oblanceolate, 5–40 x 1–10 cm, nearly entire to pinnately lobed, stalkless, midrib hollow and winged at the base; inflorescence heads 4.5 cm across, on scapes to 30 cm high, outer involucral bracts bent sharply downward; flowers deep yellow; fruit (achenes) beaked, gray-brown to olive-brown; epigeal germination [27 –29]. Dandelion is used to treat stomach, Liver and pancreatic diseases. It is an antioxidant, analgesic, proebiotic, anti-carcinogenic, anti-inflammatory agent and as a tonic for the whole body [30 –32]. The high amount of potassium, vitamin A and vitamin C content of the leaves makes this edible [33]. The aims and objectives of the current study are to explore inhibition potential of Taraxacum officinale, flowers against various pathogenic bacteria and to study various phyto constituents using different phyto analysistechniques.

2 Methods and materials

2.1 Flowers collection

The flowers from the research plant were collected from different areas of Islamia College Peshawar in the month of March 2019. After confirm identification, the flowers were assigned voucher number i.e. ICP0000412 and placed in herbarium at Department of Botany, Islamia college Peshawar. The flowers were cleaned and kept in shade for about 20 days at room temperature. The dried flowers were grinded in electric grinder to get powder and make the extraction process more efficient.

2.2 Test microorganisms

Clinical isolates of Bacterial (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi) and fungal strains (Aspergillus niger, Fusarium oxysporum, Alternaria alterneta and Aspergillus terreus) were collected from Tertiary care hospitals and Agriculture University Peshawar.

2.3 Extract preperation

From grinded powder 100 gm was taken and soaked in methanol solvent in a ratio of 1 : 3 and placed for three days with random shaking. When the powder was totally dissolved in solvent, it was filtrated with what man filter paper no 01. To make its extracts the filtrate was dried in rotary vacuum evaporator at 60°C. For the evaporation of that methanol in which powder of the flowers is mixed, the solvent was recollected and concentrated solvent was dried in water bath at 55°C for 3–5 days to get crude extract.

2.4 Fractionation

10 g of crude extracts were dissolved in 300 ml of distilled water, and then add 100 ml of chloroform, kept it on shaker for 20 m for continuous shaking. After shaking, the solution was poured in separating funnel and allowed to stand in separating funnel for 10–15 min. when the two layers were clearly visible Chloroform layer was separated in beaker and in remaining solvent 100 ml of n-hexane was mixed and placed in separating funnel, after clear visibility of two layers, the n-hexane layer were separated and both the fractions were placed in water bath at 55°C for drying. These extracts were used for different types of activities.

2.5 Antimicrobial activity

Antibacterial activity of Taraxacum officinale was carried out by well diffusion method followed the protocol of Hsouna et al., [34] with slight modification. Bacterial inoculums were prepared by colony suspension method. After comparing with 0.5 Macfarland standered the bacterial strains were streaked three times on muller Hilton agar media plates with the help of cotton swab by rotating at an angle of 60 degree. After streaking wells were made with the help of cork borer. Stock solution of crude methanolic extract along with fractions (chloroform and n-hexane) of different concentrations i-e: 6, 12, 18 and 24μl/ml was loaded in wells. The plates were then placed in an incubator for 18–20 hrs at±37°C.

2.6 Antifungal activity

Antifungal activity of Taraxacum officinale was carried out by well diffusion method followed the protocol of Hsouna et al., [34] with slight modification. Fungal inoculums were streaked three times on potato dextrose agar media plates with the help of cotton swab by rotating at an angle of 60 degree. After streaking wells were made with the help of cork borer. Stock solution of crude methanolic extract along with fractions (chloroform and n-hexane) of different concentrations i-e: 6, 12, 18 and 24μl/ml was loaded in wells. The plates were then placed in an incubator for 48 hrs at±37°C.

2.7 Anti-biofilm activity

The anti-biofilm activity was performed by followeing the method of Brambiila et al., [35] with slight modifications.of crude methanolic extract and chloroform, n-hexane fractions obtained from flowers of Taraxacum officinale was carried out against different strains of bacteria i-e: Escherechia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi. Nutrient agar media was poured in each well of sterile 96-well polystyrene micro titer plate. For the preparation of stock solution 20 mg of each extracts were dissolved in DMSO. From this stock solution, different concentrations from the stock solution were taken i-e:–30, 60, 90 and 120μg/ml was taken. Crude methanol extract, chloroform and N-hexane extracts were added in concentrations of 30, 6, 90 and 120μg/ml having nutrient broth. From the overnight culture of bacterial species 20μl were added to the wells and incubated for 24 hours. After incubation the plates were washed twice with distilled water and were dried for 30 minutes. After complete drying the wells of micro plate were stained for 16 to 20 minutes with 0.1% crystal violet dye. Extra stain was removed by washing the plate twice with distilled water. Dye which was bound with the wall of plate were solubilized by using ethanol to each well and after 20 minutes the absorbance were measured by using micro plate reader at 630 nm.

2.8 Anti-pellicle activity

Anti-pellicle activity was carried out by following the procedure of Khan et al., [36] with slight modification. 5 ml of nutrient broth and 60μl of tested bacterial inoculums were added in test tubes followed by different concentrations 7.5, 10, 12.5 and 15 mg/ml of crude methanolic extracts and were incubated at 37°C for seven days without agitation. For control positive media along with bacterial inoculum was used. Media along with antibiotic ciprofloxacin was used as negative control. The results were compared with these controls by checking the strong, moderate and weak pellicle layers.

2.9 Phytochemical analysis

To find out the biologically active compounds in Taraxacum officinale flowers chloroform extracts were used and analyzed through FTIR and GC-MS.

2.10 FTIR

FTIR analysis was carried out using Fourier transform infrared spectrophotometer (PerkinElmer Spectrum Version 10.5.1). The chloroform fraction was used for the analysis. The functional groups were detected by scanning the sample at the range of 400 to 4000 cm^–1, so the different peaks detected shows diferent functional groups.

2.11 GC-MS

The chloroform extract of Taraxacum officinale flowers of the plant was subjected to the GC-MS (thermo scientific GC series) to detect different chemical compounds present in the sample. The total run time for the sample was 46.97 min. The molecular weight and structure of the compounds of the test materials were ascertained by interpreting them. The mass spectrum of the unknown components was compared with the spectrum of known components. By using the information name, molecular weight and structure of the components of the test material were ascertained.

2.12 Flavonoid isolation and identification (HPLC)

Isolation of flavonoids was carried out according to the already cited protocols with slight modifications. Powder of selected flowers were dissolved in methanol in 1 : 6 ratio and was kept for three days. When the powder was completely dissolve then filtered the solution. The filtrates were dried by keeping on water bath at 50°C. When the extract was dried, it was dissolved in 150 ml of distilled water and then put 100 ml of petroleum ether to that aqueous to remove the unnecessary compounds. After that put 100 ml of ethyl acetate to remove fatty contents.This procedure was done in separating funnel. The remaining solution was air dried and the crude flavonoids were prepared. HPLC analysis was carried out to find different flavonoids. The sample was run and compared with two standards i-e: Chlorogenic and vanillic acid. Different peaks were shown at different retention time. Through peaks different flavonoids were identified.

2.13 Statistical analysis

The data obtained by activities were analysed by MS excel 2010.The mean and standard deviation of the three replicates were done and one way anova of varience, tukey tests were performed using Graph pad Prism version 5 software to detect significance group differences and means were considered as significant value which have probability value of less than 0.05 at confidence interval of 95%.

3 Result

3.1 Antibacterial activity of taraxacum officinale flowers

Crude methanolic extract along with fractions (chloroform and n-hexane) of Taraxacum officinale flowers showed significant antibacterial activity against 4 tested pathogenic bacteria. The highest activity against E.coli was showed by n-hexane fraction of Taraxacum officinale flowers followed by chloroform fraction and the least activity was showed by crude methanolic extract. Antibacterial activity against Staphylococcus aureus was displaced by all the extracts. The maximum activity observed by n-hexane which was followed by methanolic crude extract while the minimum activity was showed by chloroform. Highest activity against Pseudmonas aeruginosa was showed by n-hexane fraction followed by crude methanolic extract and the lowest activity was showed by chloroform. In case of Salmonella typhi highest antibacterial activity against was observed by chloroform fraction followed by n-hexane fraction and the minimum inhibition was showed by crude methanolic extract. The overall results have been shown in Table 1.

Table 1
Antibacterial potential of all the extracts of Taraxacum officinale flowers

Treatments Concen: Used Escherichia coli % inhibition Staphylococcus aureus % inhibition Pseudomonas aeruginosa % inhibition Salmonella typhi % inhibition

Control Control Control Control

6μl/ml 18.67 23.00 28.00 32.00

12μl/ml 30.34 30.67 28.34 34.34

18μl/ml 30.34 30.67 32.67 35.00

24μl/ml 32.67 31.34 32.67 38.34

Teraxaxum flower crude 6μl/ml 10.00±1.00 53.56 12.33±0.58 53.60 11.33±1.15 40.46 9.33±0.58 29.15

12μl/ml 11.00±1.00 36.25 13.00±0.00 42.38 13.66±0.57 48.20 11.33±0.58 32.99

18μl/ml 11.66±1.15 38.4 12.67±1.15 41.31 14.00±1.00 42.85 10.67±2.52 30.48

24μl/ml 12.66±0.57 38.75 12.33±1.15 39.34 14.66±2.30 44.87 12.67±1.53 33.04

Choloroform 6μl/ml 11.33±1.52 60.68 10.00±0.00 43.47 12.00±1.00 42.85 11.33±0.58 35.40

12μl/ml 13.66±0.57 45.02 11.00±1.00 35.86 11.33±0.57 39.97 12.33±1.15 35.90

18μl/ml 13.00±1.73 42.84 10.67±0.58 34.78 13.33±1.52 40.80 14.33±1.53 40.94

24μl/ml 14.00±1.00 42.85 12.00±1.00 38.28 14.33±0.57 43.86 19.00±2.65 49.55

n-hexane 6μl/ml 9.00±1.00 48.20 11.67±1.53 50.73 12.00±1.00 42.85 12.33±1.53 38.53

12μl/ml 10.33±0.57 34.04 10.33±0.58 33.68 11.66±1.15 41.14 13.00±1.00 37.85

18μl/ml 11.00±1.00 36.25 11.33±1.53 36.94 14.33±1.52 43.86 14.00±1.73 40

24μl/ml 15.33±1.52 46.92 18.00±1.00 57.43 19.33±1.52 59.16 16.00±1.00 41.73

Treatments	Concen: Used	Escherichia coli	% inhibition	Staphylococcus aureus	% inhibition	Pseudomonas aeruginosa	% inhibition	Salmonella typhi	% inhibition
Teraxaxum flower crude	6μl/ml	10.00±1.00	53.56	12.33±0.58	53.60	11.33±1.15	40.46	9.33±0.58	29.15
	12μl/ml	11.00±1.00	36.25	13.00±0.00	42.38	13.66±0.57	48.20	11.33±0.58	32.99
	18μl/ml	11.66±1.15	38.4	12.67±1.15	41.31	14.00±1.00	42.85	10.67±2.52	30.48
	24μl/ml	12.66±0.57	38.75	12.33±1.15	39.34	14.66±2.30	44.87	12.67±1.53	33.04
Choloroform	6μl/ml	11.33±1.52	60.68	10.00±0.00	43.47	12.00±1.00	42.85	11.33±0.58	35.40
	12μl/ml	13.66±0.57	45.02	11.00±1.00	35.86	11.33±0.57	39.97	12.33±1.15	35.90
	18μl/ml	13.00±1.73	42.84	10.67±0.58	34.78	13.33±1.52	40.80	14.33±1.53	40.94
	24μl/ml	14.00±1.00	42.85	12.00±1.00	38.28	14.33±0.57	43.86	19.00±2.65	49.55
n-hexane	6μl/ml	9.00±1.00	48.20	11.67±1.53	50.73	12.00±1.00	42.85	12.33±1.53	38.53
	12μl/ml	10.33±0.57	34.04	10.33±0.58	33.68	11.66±1.15	41.14	13.00±1.00	37.85
	18μl/ml	11.00±1.00	36.25	11.33±1.53	36.94	14.33±1.52	43.86	14.00±1.73	40
	24μl/ml	15.33±1.52	46.92	18.00±1.00	57.43	19.33±1.52	59.16	16.00±1.00	41.73

3.2 Antifungal activity of taraxacum officinale flowers

Crude methanolic extracts along with chloroform and n-hexane fractions have antifungal properties. The highest activity displaced against Aspergillus niger by n-hexane followed by crude methanolic extract and chloroform. In case of Fusarium oxysporium the highest activity was displaced by chloroform followed by n-hexane and crude methanolic extract respectively. The highest activity against Alternaria alternata was displayed by n-hexane followed by crude methanolic extract while the minimum zone of inhibibition was showed by chloroform. For Alspergillus terreus the highest zone of inhibition was displaced by crude methanolic extract followed by n-hexane and the least activity was showed by chloroform. The detailed results have been explained in Table 2.

Table 2
Antibacterial potential of all the extracts of Taraxacum officinale flowers

Taraxacum flower Concen: used Aspergillus niger % inhibition Fusarium oxysporum % inhibition Alternaria alternata % inhibition Aspergillus terreus % inhibition

Control Control Control Control

6 45.34 6 27.34 6 38.00 6 30.34

12 49.00 12 32.67 12 40.00 12 37.67

18 49.34 18 40.00 18 42.00 18 43.00

24 56.34 24 44.00 24 47.67 24 49.34

Methanol 6μl/ml 0.00±0.00 0.00 0.00±0.00 0.00 0.00±0.00 0.00 11.33±0.58 37.34

12μl/ml 11.00±0.00 22.44 12.33±0.58 37.74 11.67±0.58 29.17 12.00±0.00 31.85

18μl/ml 12.66±0.58 25.65 12.67±0.58 31.67 12.33±0.58 29.35 12.33±0.58 28.67

24μl/ml 12.66±0.58 22.47 14.33±0.58 32.56 13.33±0.58 27.96 13.33±0.58 27.01

Choloroform 6μl/ml 8.33±0.58 18.37 10.33±0.58 37.78 11.00±0.00 28.94 10.67±0.58 35.16

12μl/ml 10.00±0.00 20.40 13.33±0.58 40.80 11.67±0.58 29.17 11.00±1.00 29.20

18μl/ml 11.33±0.58 22.96 14.00±1.00 35.00 11.67±0.58 27.78 11.00±0.00 25.58

24μl/ml 12.66±1.15 22.47 16.33±1.53 37.11 12.00±1.00 25.17 11.67±0.58 23.65

n-hexane 6μl/ml 10.67±0.58 23.53 10.67±0.58 39.02 11.00±1.00 28.94 11.00±0.00 36.25

12μl/ml 11.33±0.58 23.12 14.67±0.58 44.90 12.00±0.00 30.00 11.33±0.58 30.07

18μl/ml 14.67±0.58 29.73 15.00±0.00 37.50 12.33±0.58 29.35 11.67±0.58 27.13

24μl/ml 15.33±0.58 27.20 15.33±0.58 34.84 13.67±0.58 28.67 12.00±0.00 24.32

Taraxacum flower	Concen: used	Aspergillus niger	% inhibition	Fusarium oxysporum	% inhibition	Alternaria alternata	% inhibition	Aspergillus terreus	% inhibition
Methanol	6μl/ml	0.00±0.00	0.00	0.00±0.00	0.00	0.00±0.00	0.00	11.33±0.58	37.34
	12μl/ml	11.00±0.00	22.44	12.33±0.58	37.74	11.67±0.58	29.17	12.00±0.00	31.85
	18μl/ml	12.66±0.58	25.65	12.67±0.58	31.67	12.33±0.58	29.35	12.33±0.58	28.67
	24μl/ml	12.66±0.58	22.47	14.33±0.58	32.56	13.33±0.58	27.96	13.33±0.58	27.01
Choloroform	6μl/ml	8.33±0.58	18.37	10.33±0.58	37.78	11.00±0.00	28.94	10.67±0.58	35.16
	12μl/ml	10.00±0.00	20.40	13.33±0.58	40.80	11.67±0.58	29.17	11.00±1.00	29.20
	18μl/ml	11.33±0.58	22.96	14.00±1.00	35.00	11.67±0.58	27.78	11.00±0.00	25.58
	24μl/ml	12.66±1.15	22.47	16.33±1.53	37.11	12.00±1.00	25.17	11.67±0.58	23.65
n-hexane	6μl/ml	10.67±0.58	23.53	10.67±0.58	39.02	11.00±1.00	28.94	11.00±0.00	36.25
	12μl/ml	11.33±0.58	23.12	14.67±0.58	44.90	12.00±0.00	30.00	11.33±0.58	30.07
	18μl/ml	14.67±0.58	29.73	15.00±0.00	37.50	12.33±0.58	29.35	11.67±0.58	27.13
	24μl/ml	15.33±0.58	27.20	15.33±0.58	34.84	13.67±0.58	28.67	12.00±0.00	24.32

3.3 Antibiofilm activity of taraxacum officinale flowers

Extract along with chloroform and N-hexane fractions showed various inhibitory potential for biofilm formation against four isolated strains. The detail of the result was shown in the Table 3 respectively. According to SBF level the value of strong inhibition of biofilm formation is less than 0.5, weak inhibition value is equal to 0.5 and resistivity value is 1 or greater than 1.

Table 3
Antibiofilm activity of different extracts of the flowers of T.officinale L

Treatments Concen: used Escherichia coli SBF value Staphylococcus aureus SBF value Pseudomonas aeruginosa SBF value Salmonella typhi SBF value

OD OD OD OD

Cell free 0.03 0.03 0.03 0.03

Bacterial growth control 0.126 0.139 0.126 1.15

30μg/ml 0.067 0.08 0.082 0.111

60μg/ml 0.081 0.06 0.061 0.058

90μg/ml 0.085 0.078 0.093 0.076

120 0.097 0.059 0.06 0.068

Taraxacum flower crude 30μg/ml 0.11 0.634 0.112 0.589 0.115 0.674 0.141 0.74

60μg/ml 0.09 0.476 0.119 0.640 0.115 0.674 0.138 0.72

90μg/ml 0.1 0.555 0.107 0.553 0.077 0.373 0.119 0.593

120μg/ml 0.083 0.420 0.074 0.316 0.089 0.468 0.099 0.46

Choloroform 30μg/ml 0.096 0.523 0.118 0.633 0.097 0.531 0.113 0.553

60μg/ml 0.069 0.309 0.12 0.647 0.1 0.555 0.115 0.56

90μg/ml 0.069 0.309 0.122 0.661 0.099 0.547 0.106 0.506

120μg/ml 0.057 0.523 0.097 0.482 0.099 0.547 0.096 0.44

n-hexane 30μg/ml 0.12 0.714 0.129 0.712 0.11 0.634 0.14 0.733

60μg/ml 0.096 0.523 0.1 0.503 0.01 0.079 0.1 0.466

90μg/ml 0.076 0.365 0.099 0.496 0.099 0.547 0.099 0.46

120μg/ml 0.07 0.317 0.078 0.345 0.087 0.452 0.091 0.406

Treatments	Concen: used	Escherichia coli	SBF value	Staphylococcus aureus	SBF value	Pseudomonas aeruginosa	SBF value	Salmonella typhi	SBF value
Cell free		0.03		0.03		0.03		0.03
Bacterial growth control		0.126		0.139		0.126		1.15
	30μg/ml	0.067		0.08		0.082		0.111
	60μg/ml	0.081		0.06		0.061		0.058
	90μg/ml	0.085		0.078		0.093		0.076
	120	0.097		0.059		0.06		0.068
Taraxacum flower crude	30μg/ml	0.11	0.634	0.112	0.589	0.115	0.674	0.141	0.74
	60μg/ml	0.09	0.476	0.119	0.640	0.115	0.674	0.138	0.72
	90μg/ml	0.1	0.555	0.107	0.553	0.077	0.373	0.119	0.593
	120μg/ml	0.083	0.420	0.074	0.316	0.089	0.468	0.099	0.46
Choloroform	30μg/ml	0.096	0.523	0.118	0.633	0.097	0.531	0.113	0.553
	60μg/ml	0.069	0.309	0.12	0.647	0.1	0.555	0.115	0.56
	90μg/ml	0.069	0.309	0.122	0.661	0.099	0.547	0.106	0.506
	120μg/ml	0.057	0.523	0.097	0.482	0.099	0.547	0.096	0.44
n-hexane	30μg/ml	0.12	0.714	0.129	0.712	0.11	0.634	0.14	0.733
	60μg/ml	0.096	0.523	0.1	0.503	0.01	0.079	0.1	0.466
	90μg/ml	0.076	0.365	0.099	0.496	0.099	0.547	0.099	0.46
	120μg/ml	0.07	0.317	0.078	0.345	0.087	0.452	0.091	0.406

The susceptibility of E.coli was observed at different concentrations i.e.–30μg/ml, 60μg/ml, 90μg/ml and 120μg/ml of crude methanolic extract, chloroform and N-hexane. Among these concentrations of different extracts, the highest inhibition of biofilm formation was observed at concentration of 120μg/ml by n-hexane fraction, it’s SBF value is 0.317 and the SBF values of Methanol and chloroform at120μg/ml is 0.420, 0.523. Thus the result indicates that the SBF value calculated against E.coli showed strong and moderate biofilm inhibition. The inhibitory potential of biofilm formation against S.aureus was observed by crude methanolic extract, chloroform and n-hexane fractions at different concentrations i:e–30μg/ml, 60μg/ml, 90μg/ml and 120μg/ml. The highest inhibition of biofilm formation was showed by crude methanolic extract at the concentration of 120μg/ml. SBF values indicated that S.aureus is highly and moderately inhibited.In case of P.aeruginosa The highest biofilm inhibitory was observed at 60μg/ml of n-hexane, where the SBF value is 0.079. The SBF values indicated that the biofilm of P.aeruginosa is inhibited at moderate level except few values which showed strong biofilm inhibition. The highest biofilm inhibition was displayed by n-hexane against S.typhi and its SBF value at 120μg/ml is 0.406.

3.4 Antipellicle activity

The antipellicle activity was done to observe the effect of crude methanolic extracts of different flowers of T.officinale against four strains of bacteria. Different concentrations were used i-e: 7.5 mg/ml, 10 mg/ml, 12.5 mg/ml and 15 mg/ml. The disruption of pellicle layers were indicated by following signs i-e: strong (+++), moderate (++), weak (+) and no inhibition (–). The strong disruption of pellicle layer was recorded for methanolic crude extracts of T.officinale, against S.typhi at the concentrations of 15 mg/ml respectively, while moderate disruption showed for the other bacterial strains at the same concentration. S.aureus and S.typhi and P.aeruginosa were not inhibited at the concentratios of 7.5 mg/ml, 10 mg/ml and 12.5 mg/ml respectively. While moderate pellicle disruption was recorded against E.coli at all the different concentrations. The data is shown in Table 4.

Table 4
Antipellicle activity of T.officinale L., methanol extract against different bacterial strains after 7 days of incubation

Treatment (Methanol) Conc:used Salmonella typhi Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli

Taraxacum flowers 7.5 mg/ml ++ – – ++

10 mg/ml – ++ ++ ++

12.5 mg/m ++ – + ++

15 mg/ml +++ ++ ++ ++

Treatment (Methanol)	Conc:used	Salmonella typhi	Staphylococcus aureus	Pseudomonas aeruginosa	Escherichia coli
Taraxacum flowers	7.5 mg/ml	++	–	–	++
	10 mg/ml	–	++	++	++
	12.5 mg/m	++	–	+	++
	15 mg/ml	+++	++	++	++

KEY: (+++) indicate Strong pellicle disruption. (++) Moderate disruption. (+) Weak disruption. (–) No inhibition.

3.5 GC-MS analysis

Phytochemical components in chloroform fraction of Taraxacum officinale were reported by GC/MS. The GC/MS analysis revealed different compounds which are shown in the figure ... From the result, it was observed that Phenol, 2, 4, - bis(1, 1- dimethylethyl), 1-Hexadecanol,1-Tridecanol, Pentadecanoic acid, 14-methyl-, methyl Ester, Heneicosane, Eicosane, 1,2-Benzenedicarboxylic acid, diisooctyl ester 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester were present. These compounds were identified by RT value, molecular weight, molecular formula and are shown in the Table 5 and Fig. 1.

Table 5
Phytochemical componenets identified in the chloroform fraction of T.officinale flowers (GC-MS study)

S.No Retention time (RT) Compound name M.formula M.wt Area (%) Structure

1. 5.99 Phenol, 2, 4, - bis (1, 1- dimethylethyl), C14H22O 206 0.03

2. 6.74 1-Hexadecanol C16H34O 242 0.01

1-Tridecanol C13H28O 200

3. 12.49 Pentadecanoic acid, 14-methyl-, methyl ester C17H34O2, 270 0.01

4. 16.56 Heneicosane C21H44 296 0.06

Eicosane C20H42 282

5. 27.62 tara 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 0.05

1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester C16H22O4 278

S.No	Retention time (RT)	Compound name	M.formula	M.wt	Area (%)
1.	5.99	Phenol, 2, 4, - bis (1, 1- dimethylethyl),	C14H22O	206	0.03
2.	6.74	1-Hexadecanol	C16H34O	242	0.01
		1-Tridecanol	C13H28O	200
3.	12.49	Pentadecanoic acid, 14-methyl-, methyl ester	C17H34O2,	270	0.01
4.	16.56	Heneicosane	C21H44	296	0.06
		Eicosane	C20H42	282
5.	27.62 tara	1,2-Benzenedicarboxylic acid, diisooctyl ester	C24H38O4	390	0.05
		1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	C16H22O4	278

Fig. 1

GC-MS spectra of chloroform fraction of T.officinale flowers.

3.6 FTIR spectroscopic analysis

The Chlorofom fraction of taraxacum officinale flowers was passed into the FTIR, the functional groups in the sample were separated based on its peaks ratio. The region of IR radiation helps to identify the functional groups of biological active components present in the sample. The results of FTIR analysis of the Taraxacum officinale flowers confirm the presence of methylene, Amino, keto (carbonyl) and methyl functional groups. Major peaks were observed and are shown in the Fig. 2. Different functional groups present in the Chloroform fraction of Taraxacum officinale are shown in Table 6.

Fig. 2

FTIR spectra of chloroform fraction of T.officinale flower.

Table 6

FTIR spectral peak values and functional groups obtained for the chloroform fraction of the T.officinale L., flowers

Frequency range(cm^–1)	Chloroform fraction of T.officinale Peak wave No.(cm^–1)	Functional group
515–680
685–770	760	Methylene
800 –770
985–995
1300 –1200	1235
1460–1550	1472	Amino
1725 – 1700	1709	saturated(Carbonyl) Keto
2850–2960	2894
2500–3200	2973

3.7 HPLC analysis

HPLC analysis of T.officinale flowers has been done for identification of flavonoids. Crude flavonoid extracts were run at 260 nm wavelength. Two standards were also run on same wavelength. The peaks shows in the standard had retention time of 1.22 and 1.34 shown in Fig. 3a and 3b and the samples shoed different peaks at different retention time As it is clear from the chromatogram that these two flavonoids were absent in the samples. Besides these two some other peaks with different retention time shows unknown compounds Fig. 3c.

Fig. 3

a) Standard Ellegic acid b) Standard Vinilic acid c) Flavonoid extract of T.officinale flowers.

4 Discussion

For thousands of year’s nature have therapeutic values and a number of modern drugs have been derived from the natural sources. In the recent years interest has been developed in natural products that are the potential source for modern medicines. These modern drugs have been used to treat different diseases such as bacterial, funal and parasitic infections, cancers, gastric problems,alzhiemers etc [37].

In the present study, antibacterial activity of Taraxacum officinale flowers was evaluated against common bacterial isolates. Almost similar findings were reported by Asghari et al.,. They studied the antimicrobial potential of the seeds and flowers of Artemisia aucheri Boiss against five bacterial isolate. Dandelion (T. officinale) flowers extracts have a potential to suppress the reactive oxygenated compounds like Nitric oxide so inhibits lipid oxidation as per the results revealed by Kitts and Hu [39]. Crude methanolic extracts of Taraxacum officinale along with chloroform and N-hexane fractions have antifungal properties. Abdel-Rahim et al. [40] reported antifungal activity of flower extracts of Romman (Punica grantum L.) against A. niger and P. italicum but significant inhibition was observed only at the higher concentrations.

Crude methanolic extract and chloroform and n-hexane fraction of Taraxacum officinale flowers showed various inhibitory potential for biofilm formation against four isolated strains. Among all these the highest inhibition of biofilm formation were showed by crude N-hexane extract against E.coli, P.aeruginosa and S.typhi while against S.aureus the highest inhibition of biofilm formation were showed by crude methanolic extract. Namsivayam and Roy [41] confirmed that methanol extract from certain medicinal plants had potent Antibiofilm activity against S.aureus, E. coli, and P. aeruginosa. Our results are in accordance with the results obtained by Anita and Retna [42]. They studied antibiofilm activity of Aerva lanata against S.aureus, E. coli, B. subtilis, and P.vulgaris in which the methanol extract showed strong inhibitory effect.

All the floweres extracts showed pellicle inhibition at different concentrations. As compared to biofilm the pellicle is susceptible to the extracts. Some other researchers also work on antipellicle activities and use different extrcats against bacterial strains. Khan et al., [36] also reported the anti-pellicle activity of M. jilapa and A. bracteosa methanolic extracts against different strains of P.aeruginosa (P1, P2 and P3). By using different concentrations the weak pellicle of the bacterial strains were disrupted. They also reported that the extracts inhibition of bacterial cell attachment confirmed that prevention of bacterial attachment to surface is easier than mature biofilm. So it is well known that the bacterial biofilm show more resistant to antibacterial agents and is suggested that further study is needed to isolated antibacterial compounds in order to use against P.aeruginosa biofilm formation in bacterial infections.

To explore the medicinal value, the extract of the flowers of T.officinale were analyzed and different phytochemical constituents were identified using GC-MS technique. Elaiyaraja and Chandramohan,[43]used differend solvent extracts for the identification of biological componenets through GC-MS analysis. They identified the presence of Phenol, 2,4-bis(1,1- dimethylethyl)-,1,4-Dicyano-2- cyclohexylbenzene, Pentadecanoic acid, 13- methyl-, methyl ester, n-Hexadecanoic acid, 10-Octadecenoic acid, methyl ester, 3,5-Dicarbethoxy-1-methyl1,4,5,6,7,8-hexahydropyrrolo (2,3-b)azepin-4,7-dione and Butanoic acid, 3-methyl-, hexadecyl ester in chloroform extract of Indoneesiella echioides (L) Nees. They also reported that Phenol, 2,4-bis(1,1- dimethylethyl) play an important role in the antifungal, antibacterial, antioxidant and antimalarial activities.

By using FTIR spectrum of chloroform fraction of T.officinale flowers, different functional groups were identified. These functional groups indicate the presence of biological active constituents which justifies the antimicrobial potential of the extract and also indicate the qualities of medicinal materials. Ahmad and Ali [44] reported FTIR analysis of different extract including chloroform extrcats of plant which shows strong presence of hydroxyl group which is common in all phenolic compounds. Phenolic compounds derived from natural resources exhibit antifungal activity.

5 Conclusion

Flowers of T.officinale possess antimicrobial and anti-biofilm inhibition properties due to the presence of different types of phytochemical constituents identified by GC-MS and FTIR analysis. The antimicrobial inhibition property of selected flowers may help in finding and developing new chemical classes of antibiotic drugs that could aid as selective agents for infectious disease chemotherapy and control. The inhibitory potential of the folowers against pathogens was due to the presence of different phytochemicals such as tannins, alkaloids, triterpenes, carbohydrates and flavonoids. The inhibition of the biofilm might be because of the suppression of QS (quorum sensing) gene and that the plant extracts might have some active component that possess gene silencing properties. In future these bioactive compounds can be further exploited by isolating them from the extracts and detailed investigation of the mechanism of action of these active compounds can be studied further for the development of new therapeutic options.

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