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

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:

$\mathrm{Viability\%}=\frac{\mathrm{A\; sample}}{\mathrm{A\; negative\; control}}\times 100$

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.

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).

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%).

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.

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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