in vitro Evaluation of Cytotoxicity and Antibacterial Activities of Ribwort Plantain (Plantago Lanceolata L.) Root Fractions and Phytochemical Analysis by Gas Chromatography-Mass Spectrometry

Document Type : Original Articles

Authors

1 Department of Plant Production and Genetics, Faculty of Agriculture, University of Zanjan, Zanjan, Iran

2 Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

3 Department of Pharmaceutical Biotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran

Abstract

Ribwort plantain (Plantago lanceolata L.), which belongs to the Plantaginaceae family, has been widely used as a herbal plant in traditional medicine across the globe. The present study aimed to investigate the biologically active substances of P. lanceolata root fractions, as well as the cytotoxic and antibacterial activities of extracts. The cytotoxic activity of ethyl acetate, dichloromethane, and n-butanol extracts of P. lanceolata root was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The P.lanceolata root extracts were also evaluated on gram-positive and negative bacteria by disc diffusion and microtiter broth dilution methods. The phytochemical content was also examined by gas chromatography-mass spectrometry. The P.lanceolata root extracts were cytotoxic; IC50 values against HCT-116 at 72 h were 168.553 μg/mL, 167.458 μg/mL, and 205.004 μg/mL for ethyl acetate, dichloromethane, and n-butanol root extracts, respectively. The dichloromethane extract of P. lanceolata root had the highest inhibitory effect against S. paratyphi (14.00±1.0 mm) at the concentration of 100 mg/mL. The minimum MIC and MBC (5 and 15 mg/mL) were observed for dichloromethane extract of P. lanceolata root against S. paratyphi. The main composition of ethyl acetate extract was 1,2-Benzenedicarboxylic acid and mono(2-ethylhexyl) ester (60.93%). The major compositions in dichloromethane and n-butanol extracts were 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (60.64%) and 2-Methyl-1-butanol (.+/-.)- (17.85%). As evidenced by the results of the present research, P. lanceolata extracts are a significant source of bioactive metabolites. Therefore, they can play a prominent role in the production of pharmaceutical materials.

Keywords

Main Subjects


Introduction

Ribwort plantain (Plantago lanceolata L.) belongs to the Plantaginaceae family ( 1 ). This perennial medicinal herb which possesses about 275 species ( 2 ) is native to temperate regions of Asia and Europe; moreover, it can grow in moderate areas ( 3 ). Leaves, roots, and bark parts of P. lanceolata contain secondary metabolites with high therapeutic potentials ( 4 ). In general, P. lanceolata encompasses valuable secondary metabolites, such as mucilage, tannins, flavonoids, phenylpropanoid glycoside, acteoside, iridoid glycosides, phenyl carboxylic acid, silica, zinc, and potassium salts. Aucubin and catalpol are among the most common iridoid glycosides of P. lanceolata ( 5 ).

Numerous reports have pointed to the therapeutic features of P. lanceolate, including its anticancer, antibacterial, antifungal, anthelmintic, antiviral, and antioxidant activities, in addition to the effective treatment of bee bite, colic, malaria, diarrhea, dysentery, embolism, pneumonia, tuberculosis ( 6 , 7 ), and skin problems ( 8 ). Despite the growing interest in the medicinal effects of plants, there exists insufficient scientific data on the biological activities and the nature of the active compounds of herbal extracts.

P. lanceolata is a prominent source of active and beneficial metabolites, leading to its extensive use in traditional and modern medicine. Several recent studies have demonstrated that the crude extract of Plantago can exert cytotoxic effects on tumors ( 9 - 12 ). In light of the aforementioned issues, the present study aimed to examine the biologically active substances of P. lanceolata root fractions using gas chromatography-mass spectrometry (GC-MS). This study also investigated the cytotoxic and antibacterial effects of P. lanceolata root extract.

2. Materials and Methods

2.1. Herbal Materials

The P. lanceolata was collected from Zanjan, Iran (36°41'15.5"N 48°24'02.2"E) and then authenticated in the Department of Pharmacognosy, School of Pharmacy, Zanjan, Iran. The root part was cut into small pieces and dried in the shade and at room temperature for one week.

2.2. Plant Extraction

Firstly, 250 grams of P. lanceolata root was extracted with petroleum ether by the reflex method for 16 h. The extracts were then filtered with filter paper and washed with methanol like before. The methanolic extract was poured into the separating funnel using the liquid-liquid extraction method. The separation was performed by a funnel using ethyl acetate, dichloromethane, n-butanol, and aqueous phase ( 13 ) (Figure 1). The extracts were concentrated by a rotary evaporator and then located at room temperature to fully dry.

Figure 1. Flow chart of partitioning protocol of methanolic extract of P. lanceolata root

2.3. Cell Line Culture

Embryonic Kidney normal cell line (HEK-293) and Colorectal cancer cell line (HCT-116) were supplied from the Pasteur Institute of Iran, Tehran. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented by penicillin-streptomycin (1%) and 10% Fetal bovine serum (FBS) in an incubator (5% CO2 and 37 °C).

2.4. Viability Assay

The cytotoxicity of ethyl acetate, dichloromethane, and butanol extracts of P. lanceolata root was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay ( 14 ). The cells were seeded at the density of 7 × 103 cells/well. Cell attachment and growth continued overnight, followed by the dissolving of 25-400 μg/mL of the extracts. In brief, 10 mg of powdered extracts in 100 μL of Dimethyl sulfoxide (DMSO) and 900 μL of DMEM were used to make the first stock. The cells were treated by the extracts after their filtration by 0.45 μm membrane filters. Fluorouracil (5-FU) and DMSO served as positive and negative controls, respectively. After one to three days, 20 μL of MTT (5 mg/mL) was added and incubated for four h; subsequently, the medium was removed and 200 μL of DMSO was added to dissolve formazan. The absorbance of the samples was read at 570 and 690 nm using an ELISA plate reader (Tecan Infinite M200, Austria). The cell growth inhibition rates were determined by:

Viability%=A sampleA negative control×100

where A signifies the absorbance.

2.5. Microorganism Culture

Gram-positive bacteria of Bacillus cereus (ATCC 10702), as well as Gram-negative bacteria of Salmonella paratyphi (ATCC 5702) and Proteus vulgaris (PTCC 1182), were prepared from the Iranian Biological Resource Centre Culture collection of bacteria.

2.6. Antibacterial Assay

The sterile blotting paper discs (6 mm diameter) were soaked in ethyl acetate, dichloromethane, and butanol extracts of P. lanceolata root and left to fully dry. The dried discs were used for the disc diffusion method ( 15 ). The turbidity of inoculums was matched with the 0.5 McFarland standard (1.5×108 CFU/mL). The bacterial inoculums were uniformly spread onto the Mueller-Hinton agar (MHA) by a sterile cotton swab. The discs were then impregnated with 3µL of extract dissolved in DMSO to obtain a concentration of 100 mg/mL. Gentamicin (10 µg/mL) and DMSO-soaked discs were considered positive and negative controls, respectively. The plates were incubated at 37°C for 24 h. Finally, the antibacterial properties were reported based on the diameter of the inhibition zone (mm).

The microtiter broth dilution method was conducted to determine the minimum inhibitory concentration (MIC) ( 16 ). 20 µL of diluted extracts at 50-250 mg/mL concentration was introduced into a 96-well plate. Thereafter, 200µL of bacterial suspensions (108CFU/mL) was introduced into each well and incubated at 37°C for 24 h (5-25 mg/mL desired final concentration). The absorbance of wells was recorded at 600nm using an ELISA plate reader. The MBCs were determined by culturing extracts at 15-40 mg/mL concentrations on MHA for 24 h of incubation at 37°C.

2.7. Phytochemistry

The GC-MS analysis of phytocomponents in different extracts was performed using Agilent technologies 5975c, USA. In a typical measurement, 1 μL of the ethyl acetate, dichloromethane, and butanol extracts of P. lanceolata root was subjected to the GC-MS system equipped with a capillary column (30 m × 250 μm ×0.25 μm, Agilent). The flow rate of Helium was 1.0 mL/min. The temperatures of the injector and the interface were set to 350°C. The following temperature program was considered: the initial column temperature was set to 50°C for 2 min, followed by an increase to 230°C at the rate of 4°C/min for 2 min. The compositions were identified by comparing the mass spectral fragmentation patterns with MS databases (NIST08.L) ( 13 ). To make the working solutions at a 5mg/mL concentration for GC/MS analysis, dried extracts were dissolved in methanol (HPLC grade), and the solutions were then filtered by a 0.22 μm sterile filter.

2.8. Statistical Analysis

The experiments were entirely carried out in triplicates. Duncan’s multiple comparison test was implemented in SPSS software (version 21) (P<0.05) for group-wise comparison and statistical analysis. The IC50 values was calculated by ED50plus software (version 1.0).

3. Results and Discussion

3.1. Cytotoxicity Activity

The colorectal cancer cell line was incubated with different concentrations of ethyl acetate, dichloromethane, and butanol extract to assess the cytotoxicity of P. lanceolata root. The dichloromethane extract of P. lanceolata root exhibited higher antiproliferative properties on HCT-116 at 24 and 48 h, as compared to ethyl acetate and butanol extracts.

Nevertheless, the dichloromethane extract was more active on HEK-293 at 24 and 48 h at 200 and 400 μg/mL concentrations (Figures 2a and 2b). The proliferation inhibition activities of the cells occurred in a dose- and time-dependent manner. Three extracts of P. lanceolata root exhibited similar cytotoxic effects on cancer cell line at concentrations of 200 and 400 μg/mL at 72 h (Figure 2c). Moreover, 25 μg/mL of three extracts displayed no significant cytotoxicity against HCT-116 and HEK-293 (maximum 5%). These results confirmed remarkable antiproliferative properties of these three extracts of P. lanceolata root against HCT-116, compared to HEK-293.

Figure 2. The MTT result for the colorectal carcinoma cell lines (HCT-116) and embryonic kidney normal cell line (HEK-293) upon treatment with ethyl acetate, dichloromethane, and butanol extracts of P. lanceolata root for 24, 48, and 72 h(a-c). Values represent the mean of three replications±standard deviations. Duncan test was used for mean comparison (P<0.05). Charts with the same letters are not statistically significant

As illustrated in table 1, the IC50 values of the ethyl acetate and dichloromethane extracts of P. lanceolata root (168.553 μg/mL and 167.458 μg/mL) on HCT-116 were lower than those of butanol extract (205.004 μg/mL). However, the lowest IC50 for P. lanceolata root extracts on a normal cell line was related to dichloromethane extract on HEK-293 (269.937μg/mL). Similar results have been established by P. lanceolata extract on breast Adenocarcinoma cell line (MCF-7) with LC50 of 212 µg/mL ( 12 ). In a study by Beara, Lesjak ( 11 ), P. lanceolata exhibited a stronger cytotoxic effect on MCF-7, cervix epithelioid carcinoma (HeLa), colon adenocarcinoma (HT-29), and human fetal lung (MRC-5) cell lines with IC50 values of 142.8 μg/mL, 172.3 μg/mL, 405.5 μg/mL, and 551.7 mg/mL, respectively.

Extract(µg/mL)/Cell 24h 48h 72h
HCT-116 HEK-293 HCT-116 HEK-293 HCT-116 HEK-293
Dichloromethane 342.23±5.8 593.46±2.8 249.06±4.2 348.76±7.8 167.45±4.6 269.93±1.8
Ethyl acetate 605.43±7.2 911.1±8.2 324.25±5.5 575.58±6.5 168.55±8.2 328.02±3.4
n-Butanol 606.04±4.3 1077.8±6.2 356.44±3.6 518.62±2.8 205.00±2.4 413.66±6.6
IC50 values are the mean values of three replications±standard deviations at 24, 48, and 72 h. Values were calculated for 5-fluorouracil (IC50:4.136 µg/mL)
Table 1.IC50 of various extracts of Plantago lanceolata on HCT-116 and HEK-293 cell lines

Asadi-Samani, Rafieian-Kopaei ( 10 ) reported the cytotoxicity of P. lanceolata extracts against prostate cancer cell lines (Du-145 and PC-3) with IC50 values of 300 μg/mL. In this respect, Alsaraf, Mohammad ( 9 ) found IC50 values of 674.2 μg/mL and 23.71 μg/mL for the P. lanceolata extract against MCF-7 and triple-negative breast cancer cells (CAL-51), respectively. According to Rahamooz-Haghighi, Bagheri ( 17 ), IC50 values of methanolic, ethanolic, and acetonic extracts of P. major root were 470.16 μg/mL, 405.59 μg/mL, and 82.26 μg/mL, respectively, against HCT-116, while higher IC50 values were reported for methanolic, ethanolic, and acetonic extracts of P. major root toward HEK-293 (1563.04 μg/mL, 948.15 μg/mL and 125.89μg/mL, respectively). In the present research, the different extracts of P. lanceolata root were a proper choice for in vitro treatment of colon cancer cells at the concentration range of 100-400 µg/mL. In line with previous studies on P. lanceolata, the present research detected the cytotoxicity of extracts against cancer cells.

3.2. Antibacterial Activity

The ethyl acetate, dichloromethane, and butanol extracts of P. lanceolata root were evaluated on gram-positive and negative bacteria. The results indicated that the different fractions inhibited the visible growth of bacteria after 24 h (Table 2). The Dichloromethane extract of P. lanceolata root demonstrated the highest inhibitory activity against S. paratyphi (14.00±1.0 mm) at a concentration of 100 mg/mL. The dichloromethane root extract showed a similar inhibitory effect against S. paratyphi, as compared to gentamicin (14±1.1mm). The dichloromethane extract of P. lanceolata root exhibited the lowest MIC and MBC values (5 mg/mL and 15 mg/mL) against S. paratyphi (Table 3).

Extracts (100mg/mL) B.cereus (ATCC 11778) P.vulgaris (PTCC 1182) S.paratyphi (ATCC 5702)
Ethyl acetate 10.00±1.0 13.00±1.6 6.66±0.6
Dichloromethane - 11.00±1.0 14.00±1.0
n-Butanol 9.00±1.0 13.50±2.0 7.00±0.5
Gentamicin (10µg/mL) 31.00±2.0 26.00±1.3 14.00±1.1
Diameter of the inhibition zone (mm), no inhibition (–)
Table 2.Antimicrobial activities of Plantago lanceolata root extracts
Fractions (mg/mL) B.cereus (ATCC 11778) P.vulgari (PTCC 1182) S.paratyphi (ATCC 5702)
MIC MBC MIC MBC MIC MBC
Ethyl acetate 20 40 10 20 10 25
Dichloromethane 25 R 20 40 5 15
n-Butanol 25 R 15 35 15 30
R: MBCs are not determined in concentrations (15-40 mg/mL)
Table 3.MICs and MBCs of different fractions of Plantago lanceolata root

The researchers reported that P. lanceolata aqueous extract illustrated poor or moderate antimicrobial activities against S. aureus, S. epidermidis, P. vulgaris, and S. arcescens; nonetheless, P. lanceolata methanolic extract demonstrated no significant antibacterial activity against the mentioned bacteria ( 18 ). The sensitivity of the tested bacteria to methanol, 80% and 90% aqueous-methanol, pure petroleum ether, and pure chloroform extracts of P. lanceolata leaves were reported by Abate, Abebe ( 19 ).

P. lanceolata extracts displayed suitable antibacterial effects against P. vulgaris, B. cereus, and S. paratyphi. The antimicrobial activity of P. lanceolata root fractions was estimated in the current study. The fractionation of methanolic extract of P. lanceolata root improved to separate the active compositions in P. lanceolata and resulted in its proper antibacterial activity. The difference in the antimicrobial properties of specific species can be attributed to the geographical region of the plant growth, extraction method, and the presence of diverse antibacterial secondary metabolites.

3.3. Phytochemical Screening

The components of P. lanceolata extracts were analyzed by GC-MS according to the NIST08.L library. The major aromatic constituents of P. lanceolata root belonged to alcohols, aldehydes, alkanes, benzofurans, fatty acids, fatty esters, phenols, phytosterols, siloxanes, and terpenoids. The highest amount of fatty acids and esters pertained to the dichloromethane fraction, which could explain the higher cytotoxic effect of this extract. Phytosterols, ethers, and phenols were found in the butanol extract. Plant-derived terpenoids are the largest class of natural products. In the current study, terpenoids were only present in ethyl acetate extract.

Following the chemical grouping of compositions in P. lanceolata, many biological activities have been reported for these compounds. As one of the most prominent medicinal sources, fatty acids have exhibited numerous biological activities, such as antimicrobial and antifungal activities ( 20 ), as well as anticancer behavior ( 21 ). Previous studies have stated that phytosterols could act as cytotoxic and antioxidant agents ( 22 , 23 ). Phenolic compounds prevented the growth and spread of cancers in vitro and in vivo in cells and animals, respectively ( 24 ). Several studies pointed to numerous biological properties of terpenoids, including antiproliferative, antitumor, apoptotic, antimetastatic, and antiangiogenic activities ( 25 ).

The compositions of each extract of P. lanceolata root are presented in table 4. A number of 13 compounds were detected in ethyl acetate extract, the majority of which was 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester (60.93%). The dichloromethane extract included 24 compounds, mainly 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (60.64%), while n-butanol extracts encompassed 18 compounds, including 1-Butanol and 2-methyl-, (.+/-.)- (17.85%). In the present study, the common components in the three extracts of P. lanceolata root were n-Hexadecanoic acid (3.04%, 6.073%, and 6.495%), cycloheptasiloxane, tetradecamethyl- (8.89%, 1.66% and 4.83%), and Cyclohexasiloxane, dodecamethyl- (6.58%, 0.97% and 5.89%).

Library/ID. Plantago lanceolata root part RT (min) Ethyl acetate (%) RT (min) Dichloromethane (%) RT (min) n-Butanol (%)
n-Hexadecanoic acid 38.83 3.04 38.82 6.0734 38.74 6.4956
Hexanal 4.4 2.55 - - - -
Beta.-Sitosterol - - - - 48.15 9.07
Cycloheptasiloxane, tetradecamethyl- 26.04 8.89 26.22 1.66 26.21 4.83
Hexadecanoic acid, methyl ester 37.68 3.84 - - - -
Octadecanoic acid, methyl ester - - 42.35 0.82 - -
Eicosane - - 34.48 1.38 - -
Tetradecanal 32.28 1.2 - - - -
1-Butanol, 2-methyl- - - - - 3.59 5.52
Benzofuran - - - - 12.09 3.67
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- - - 41.76 1.77 - -
cis-Vaccenic acid - - 43.26 1.8 - -
Cyclohexasiloxane, dodecamethyl- 20.6 6.58 20.83 0.97 20.8 5.89
Linoleic acid ethyl ester 43.12 2.25 - - - -
Hexadecane - - - - 29.05 3.03
Cyclotetrasiloxane, octamethyl- - - 9.53 0.14 - -
Butane, 1,1',1''-[methylidynetris(oxy)]tris- - - - - 22.95 3.67
Hexadecanoic acid, ethyl ester 39.31 3.89 39.32 4.82 - -
Heptadecane - - 43.91 1.12 - -
Nonadecane - - 39.4 2.03 - -
Octadecanal - - 32.3 0.63 - -
trans-13-Octadecenoic acid - - 43.39 0.74 - -
9,12-Octadecadienoic acid, methyl ester 41.62 2.32 41.62 2.51 - -
Butanoic acid, hexyl ester - - - - 22.02 1.94
Benzofuran, 2-methyl- - - - - 15.55 4.04
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester 52.3 60.93 52.38 60.64 - -
Pentadecanoic acid, 14-methyl-, methyl ester - - 37.68 4.39 37.67 1.9
Butane, 1,1-dibutoxy- - - - - 18.41 2.15
9,12-Octadecadienoic acid, ethyl ester - - 43.12 2.36 - -
2-Methoxy-4-vinylphenol - - - - 20.65 4.36
1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester - - 38.62 1.34 - -
2-Hydroxy-4-hydroxylaminopyrimidine - - - - 25.68 9.54
1,2-Benzenedicarboxylic acid, diisooctyl ester - - - - 52.21 5.3
1-Butanol, 2-methyl-, (.+/-.)- - - - - 3.67 17.85
Benzeneacetic acid, .alpha.,3,4-tris[(trimethylsilyl)oxy]-, trimethylsilyl ester - - 31.07 0.49 - -
Heptadecane, 2,6,10,15-tetramethyl- - - 48.13 0.95 - -
Silane, [[4-[1,2-bis[(trimethylsilyl)oxy]ethyl]-1,2- phenylene]bis(oxy)]bis[trimethyl- 31.08 1.33 - - - -
7,10,13-Hexadecatrienoic acid, methyl ester 41.77 1.61 - - - -
O-Butylisourea - - - - - 8.54
N-(Trifluoroacetyl)-N,O,O',O''- tetrakis(trimethylsilyl)norepinephrine - - - - 31.07 2.12
11,13-Dimethyl-12-tetradecen-1-ol acetate - - 35.49 0.52 - -
2-Methyl-Z,Z-3,13-octadecadienol 43.26 1.51 - - - -
Toluene-4-sulfonic acid, 2,7-dioxatricyclo[4.3.1.0(3,8)]dec-10-yl ester - - 48.47 0.75 - -
Methyl 17-methyl-octadecanoate - - 43.83 0.86 - -
Ethyl 14-methyl-hexadecanoate - - 48.07 1.15 - -
Table 4.Identification of compounds obtained by fractionation of Plantago lanceolata root

The cytotoxicity and antibacterial activities of the extracts can be ascribed to the presence of benzofuran; Cyclohexasiloxane, dodecamethyl-; Cycloheptasiloxane, tetradecamethyl-; hexadecanoic acid, methyl ester; eicosane; octadecanoic acid, methyl ester; 9,12,15-Octadecatrienoic acid, methyl ester, (Z, Z, Z)-(Linolenic acid); Cis-Vaccenic acid; hexadecanoic acid, ethyl ester, (Palmitic acid ethyl ester); Cyclotetrasiloxane, octamethyl-; trans-13-Octadecenoic acid; 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester; 11,13-Dimethyl-12-tetradecen-1-ol acetate, and Methyl 17-methyl-octadecanoate (Table 5). The compounds in different extracts of P. lanceolata root displayed numerous medicinal properties, including antibacterial, anticancer, antifungal, anti-inflammatory, antioxidant, antiparasitic, anti-yeast, and antiviral features (Table 5). The antibacterial properties of P. lanceolata extracts may be attributed to the presence of antibacterial compounds as reported in GC/MS analysis.

Library/ID. Plantago lanceolata root part Formula MW (g/mol) Nature of Composition Biological activity Structure
n-Hexadecanoic acid (palmitic acid) C16H32O2 256.42 Fatty acid Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Antiandrogenic, Antioxidant, Antifibrinolytic, Hemolytic, Antialopecic, Antimicrobial, Antifungal
Hexanal C6H12O 100.16 Fatty aldehyde Antimicrobial, Fungicide
Beta.-Sitosterol C29H50O 414.7 Phytosterol As a sterol methyltransferase inhibitor, an anticholesteremic drug, an antioxidant, a plant metabolite
Cycloheptasiloxane, tetradecamethyl- C14H42O7Si7 519.07 Cyclic methyl siloxane Antifungal, Skin- Conditioning Agent, Fragrance, Antimicrobial, Antifouling, Immunomodulatory, Antitumor
Hexadecanoic acid, methyl ester C17H34O2 270.5 Fatty acid methyl ester(triterpenoid) Antioxidant, Nematicidal, Pesticidal, Hemolytic, Antiinflammatory, Cancer preventive, epatoprotective, Antihistaminic, Anticoronary, Antibacterial, Antifungal
Octadecanoic acid, methyl ester C19H38O2 298.5 Fatty acid methyl ester Antimicrobial, Anti-inflammatory, Anticancer
Eicosane C20H42 282.5 Aliphatic alkanes Antifungal, Antitumor, Anticancer, Antibacterial
Tetradecanal C14H28O 212.37 Fatty aldehyde Antibacterial
1-Butanol, 2-methyl- C5H12O 88.15 Alcohol Antimicrobial on phytopathogen
Benzofuran C8H6O 118.13 Ether Anti-inflammatory, Antimicrobial, Antifungal, Antihyperglycemic, Analgesic, Antiparasitic, Antitumor, Antidepressant, Anticancer, Antiviral, Antifungal, Antioxidant, Anti-psychotic
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-/ Linolenic acid C19H32O2 292.5 Fatty acid methyl ester Anticancer, Antibacterial, Antioxidant, Antipyretic, Cardioprotective, Neural function, Antiandrogenic (5- alpha reductase inhibitor), Antiarthritic
Cis-Vaccenic acid C18H34O2 282.5 Omega-7 Fatty acid Antibacterial, Hypolipidemic effect in rats
Cyclohexasiloxane, dodecamethyl- C12H36O6Si6 444.92 Cyclic methyl siloxane Antifungal, Emollient, Personal care products, Lubricant, de- foaming agent, Antimicrobial, Antioxidant
Linoleic acid ethyl ester C20H36O2 308.49 Fatty acid ethyl ester a plant metabolite, Anti- inflammatory
Hexadecane C16H34 226.44 Alkane a plant metabolite
Cyclotetrasiloxane, octamethyl- C8H24O4Si4 296.61 Cyclosiloxane Antimicrobial, Antiseptic, Hair conditioning agent, Skin conditioning agent- emollient
Butane, 1,1',1''- [methylidynetris(oxy)]tris- C13H28O3 232.36 Ether Not found
Hexadecanoic acid, ethyl ester/ Palmitic acid ethyl ester C18H36O2 284.47 Fatty acid ethyl ester Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Antiandrogenic flavor, Hemolytic, Alphareductase inhibitor, Pesticide, Lubricant, 5- Alpha reductase inhibitor, antipsychotic, Antifungal, Antitumour, Antibacterial
Heptadecane C17H36 240.5 Alkane Antibacterial
Nonadecane C19H40 268.5 Alkane Antibacterial
Octadecanal C18H36O 268.5 Alpha-CH2- containing aldehyde As the indicator of Sjogren- Larsson syndrome
trans-13-Octadecenoic acid C18H34O2 282.46 Fatty acid Antimicrobial
9,12-Octadecadienoic acid, methyl ester C19H34O2 294.5 Fatty acid methyl ester of linoleic acid. Hepatoprotective, Anti- histaminic, Antieczemic, Hypocholesterolemic, Antioxidant, Antimicrobial
Butanoic acid, hexyl ester C10H20O2 172.26 Fatty acid ester a potential biomarker for the consumption of these foods
Benzofuran, 2-methyl- C9H8O 132.16 Ether Not found
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester C16H22O4 278.34 Aromatic dicarboxylic ester Antimicrobial, Cytotoxicity, Antioxidant, Antiinflammatory, Antiviral
Pentadecanoic acid, 14-methyl-, methyl ester C17H34O2 270.5 Palmitic acid/ Fatty acid methyl ester Antifungal, Antimicrobial
Butane, 1,1-dibutoxy- C12H26O2 202.33 Ether Not found
9,12-Octadecadienoic acid, ethyl ester/Linolelaidic acid ethyl ester C20H36O2 308.49 Fatty acid ester Hypocholesterolemic, Nematicide, Antiarthritic, Hepatoprotective, Antiandrogenic, 5-α reductase inhibitor, Antihistaminic, Anticoronary, Insectifuge, Antieczemic, Antiacne, Antimicrobial.
2-Methoxy-4-vinylphenol C9H10O2 150.17 Phenol A pheromone, a flavoring agent, a plant metabolite
1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester C16H22O4 278.34 Ester Antiviral, Antimicrobial
2-Hydroxy-4- hydroxylaminopyrimidine C4H5N3O2 127.10 Not determined Not found
1,2-Benzenedicarboxylic acid, diisooctyl ester/ phthalate ester C24H38O4 390.6 Fatty acid ester, diester Antioxidant, Antimicrobial
1-Butanol, 2-methyl-, (.+/-.)- C5H12O 88.14 Alcohol Antimicrobial, Antiyeast
Benzeneacetic acid, .alpha.,3,4- tris[(trimethylsilyl)oxy]-, trimethylsilyl ester C20H40O5Si4 472.9 Ester Chronic oral toxicity study of erythritol in dogs
Heptadecane, 2,6,10,15-tetramethyl- C21H44 296.6 Alkane Antituberculous activity along with other pharmacological activities
Silane, [[4-[1,2- bis[(trimethylsilyl)oxy]ethyl]-1,2- phenylene]bis(oxy)]bis[trimethyl- C20H42O4Si4 458.9 Alkane Not found
7,10,13-Hexadecatrienoic acid, methyl ester C17H28O2 264.40 Fatty acid methyl ester Not found
O-Butylisourea - - Not determined Not found Not found
N-(Trifluoroacetyl)-N,O,O',O''- tetrakis(trimethylsilyl)norepinephrine C22H42F3NO4Si4 553.9 Not determined Not found Not found
11,13-Dimethyl-12-tetradecen-1-ol acetate C18H34O2 282.5 Alcohol Antioxidant, Antitumour
2-Methyl-Z,Z-3,13-octadecadienol C19H36O 280.5 Terpenoid Pesticide, Herbicide, Insecticide, Pheromone
Toluene-4-sulfonic acid, 2,7- dioxatricyclo[4.3.1.0(3,8)]dec-10-yl ester C15H18O5S 310.4 Not determined Not found
Methyl 17-methyl-octadecanoate C20H40O2 312.5 Ester Antimicrobial, Antioxidant, Antitumor
Ethyl 14-methyl-hexadecanoate C18H36O2 284.5 Ester Not found
Table 5.Compositions detected in Plantago lanceolata root extracts using GC/MS analysis

The study by Jamaluddin, Sharifa ( 13 ) exhibited various main constituents in P. major leaf extracts, including ethyl acetate extract (30.70% glycerin; 21.81% benzene and 16.22% dibuthyl phthalate) and n-butanol extract (24.62% phthalic acid; 16.83% benzene propanoic acid and 10.20% phenol group). In our previous study, GC-MS analysis detected octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13, 15,15-hexadecamethyl- (4.97%); cyclohexasiloxane, dodecamethyl- (6.35%); verbenone (6.96%); isoborneol (8.68%); tetradecamethylcycloheptasiloxane (9.74%); and n-hexadecanoic acid (13.8%) in the methanolic extract of P. major root ( 17 ).The results of the current study on the Iranian P. lanceolata demonstrated similarities and differences in the amounts and types of compounds observable in the extracts.

4. Conclusion

As evidenced by the results of the present research, P. lanceolata extracts are a significant source of bioactive metabolites. Therefore, they can play a prominent role in the production of pharmaceutical materials and the development of anticancer drugs. In general, the results of the current study highlight the potential use of various fractions of P. lanceolata as a source of cytotoxic agents. P. lanceolata extracts possess antibacterial properties and could be employed as a natural antibacterial agent to control pathogenic strains. These results are particularly important in the case of human pathogenic infections, such as P. vulgaris, S. typhi, and B. cereus.

Authors' Contribution

Project administration, Investigation, Formal analysis, and Writing ‒ original draft: S. R. H.

Funding, Supervision: Kh. B.

Funding, Supervision, Conceptualization: A. S. H.

English edit: N. M. P.

Ethics

The above-mentioned sampling/treatment protocols obtained approval from the University of Zanjan Research Ethics Committee, and Zanjan University of Medical Sciences, Zanjan, Iran (ethical code: A-12-848-35).

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

This study was supported by the University of Zanjan, Zanjan, Iran. The authors also would like to thank the authority of the School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran.

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