1. INTRODUCTION
Mistletoe, a common name used generally for woody shoot parasites, belongs to the order Santalales, it includes Loranthaceae, Santalaceae, and Misodendraceae families [1]. Historically, the word mistletoe originated from the Celtic word which means “all-heal” and it was used to treat several ailments [2]. There are more than 1500 species of mistletoe documented globally [3]. They have been widely utilized in ethno medicine for a variety of purposes, including antihypertensive, anticancer, antispasmodic, and antidiabetic, as well as therapy for epilepsy, headache, infertility, menopausal syndrome, and rheumatism. Due to its many traditional uses, mistletoe has been referred to as “an all-purpose herb” [4]. In northern America, mistletoe was used as an abortifacient, by veterinarians and formers for “clearing cattle” [5]. Mistletoe is used to treat a wide range of stomach issues in Africa, including diarrhea, diabetes, schizophrenia, and hypertension. In addition, it was utilized to strengthen the immunological system. Similarly, Argentina and ancient Greeks used these mistletoes to treat menstrual and spleen ailments. In Japanese traditional medicine, mistletoe (Taxillus kaempferi) was used as a remedy for hypotension, while some other members of mistletoes (Loranthus parasiticus, Loranthus yadoriki, and Viscus coloratum) were used in folk Chinese medicine to treat hypertension, rheumatic pain, and spasms of heart and locally to treat frostbite. In India, tea is prepared from mistletoe tree leaves and is traditionally used to treat diabetes [6].
Medicinal plants may constitute a reservoir of new bioactive compounds which seem to be new antimicrobial compounds such compounds are yet to be discovered. To defend themselves against a broad range of pathogens, plants are known to generate a variety of chemicals [7]. The emergence and spread of antimicrobial resistance in microorganisms need greater efforts in the discovery of novel antibiotics. Even though several plants with antimicrobial properties have been found, many remain unidentified. Many plants are being used as antibacterial or fungicidal agents [8]. Wound healing is an important biological process for preventing infections [9]. Over time, the search for new biological agents to treat wound infections has attracted interest. There have been numerous studies on the antibacterial activities of medicinal plants on pathogenic bacterial strains that cause skin infections [10].
Elytranthe parasitica (L.) Danser is a hemiparasitic shrub popularly known as Macrosolen parasiticus and belongs to the Loranthaceae family [11]. In Karnataka, known as Bandanekke or Baranike, it has been used in traditional veterinary treatment and as a leaf paste to eradicate ticks [12]. It grows extensively in the Western Ghats regions of India [13]. The previous studies on E. parasitica have suggested that it has antioxidant properties [14,15] and cytotoxic properties against different cancer cell lines [16-19]. However, the antibacterial and anticancer properties of this plant on prostate and pancreatic cancer cell lines have not been investigated. Hence, an attempt was made to check its phytochemical constituents, antibacterial potential, and cytotoxic activity against prostate and pancreatic cancer cells.
2. MATERIALS AND METHODS
2.1. Plant Samples Collection and Identification
The fresh stem samples of E. parasitica (L.) Danser were collected from the Western Ghats region of Karnataka, India. The taxonomical identification of the test plant was done using the Flora (Gamble, 1935), and a voucher specimen is deposited with a sample code (KU/AB/RN/KPS-001).
2.2. Preparation of Plant Samples and Extraction
The collected plant samples were washed under running tap water, shade dried for about 25-28 days, and mechanically pulverized. The powdered samples were subjected to Soxhlet extraction, with petroleum ether, chloroform, and methanol solvents. All the obtained crude extracts were concentrated in a rotary flash evaporator with reduced pressure and controlled temperature. Stored at 4°C in airtight glass vials.
2.3. Preliminary Phytochemical Profiling
Preliminary profiling of phytochemicals to identify the variety of phytoconstituents, which includes, tannins, alkaloids, saponins, flavonoids, phenols, steroids, and glycosides using standard methods [20,21].
2.4. Gas chromatography and Mass Spectroscopic Profiling
Gas chromatography and mass spectroscopic profiling of the methanolic stem extracts of E. parasitica was performed using the equipment Thermo GC-Trace Ultra Version: 5.0, Thermo MS DSQ II. The equipment has DB 35 – MS Capillary Standard non-polar column with 30 mm × 0.25 mm ID × 0.25 μm film dimensions. The carrier gas used is helium, and the flow detector’s temperature was set at 250°C with a flow rate of 1.0 mL/min. The temperature of the oven was programmed as follows: 60°C for 15 min, then gradually increased to 280°C at 3 min. The components of extract and fractions were identified based on the spectra of the unidentified constituent matched with the spectra of the identified component stored in the National Institute Standard and Technology-based Automated mass spectral deconvolution and identification software V 2.69 software. The name, retention time, percentage area, and molecular weight of the constituents of the test sample were determined.
2.5. Antibacterial Activity
The agar well diffusion method was used to test the methanolic stem extract of E. parasitica for its antibacterial activities. The crude methanolic extract (10 mg) was dissolved in 1000 μL of dimethyl sulfoxide (DMSO) and diluted to 100, 50, and 25% concentrations. 20 μL of the extract was poured into each well of the agar plate. Triplicates of the test were performed. Amoxicillin and DMSO, respectively, were employed as positive and negative controls. The test bacterial strains included five human pathogenic bacterial strains which include Escherichia coli (MTCC-1599); Klebsiella pneumonia (MTCC-7028); Staphylococcus aureus (MTCC-4734); Salmonella typhi (MTCC-734); and Enterococcus faecallis (MTCC 439) and one plant pathogenic bacterial strain Xanthomonas campestris (MTCC-228). The plates were inoculated and incubated at 35–37°C overnight to determine the inhibition zone [22].
2.6. Cytotoxic Activity
2.6.1. Culturing of cell lines
Prostate and pancreatic cancer cell lines were purchased from National Center for Cell Science, Pune, India. In tissue culture flasks, the cells were subcultured in Dulbecco Modified Eagle Medium with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% non-essential amino acids. The flasks were, then, incubated in a condition with 95% humidity and a 5% carbon dioxide incubator. After trypsinization, the cells were counted and the viability of the cells was determined. To perform, an 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, a known quantity of cells (20,000 cells per well in 200 μL cell suspension), was placed onto 96-well plates.
2.6.2. Treatment groups
Prostate and pancreatic cancer cells were treated with methanolic stem extract of E. parasitica. Before the experiment, the preferred concentrations of test samples were prepared in DMSO. The reactant mixtures were diluted with medium, and cells were treated with various concentration ranges (31.25–500 μg/mL) of the extract and incubated for 24 h. The effect of induced was compared with a standard drug used, namely, curcumin. The study has established the following test groups. Negative control: Cancer cells only; Positive control: Cancer cells + curcumin (10 μM); and Treatment group: Cancer cells + methanolic extracts. The same treatment group was used for the normal cell lines MEF-L239 (mice embryo fibroblast).
2.6.3. MTT cell viability assay
After 24 h of incubation, remove the plates from the incubator, discard the used medium, and then add the MTT reagent at a final level of 0.5 mg/mL of the overall amount. Plates should be placed back in the incubator and incubated for 3 h. Following incubation, 100 μL of DMSO solution was added to solubilize the formazan that had been generated. The suspension was placed on a gyratory shaker for 5 min; then, the absorbance was measured at 570 nm and 630 nm by an Enzyme-Linked Immunosorbent Assay reader. The inhibitory concentration at 50% growth (IC50) was determined.
3. RESULTS
3.1. Extracts Yield of E. parasitica Stem with Different Solvents
The obtained extracts yield of E. parasitica stem using petroleum ether, chloroform, and methanol were 18.24 g, 21.52 g, and 58.46 g, (weight), respectively, concerning the shade-dried plant material of about 750 g.
3.2. Preliminary Qualitative Screening of Phytoconstituents
The qualitative screening of secondary metabolites using different solvent extracts revealed the presence of a variety of phytoconstituents [Table 1]. The methanolic stem extract shows the existence of tannins, phenols, glycosides, flavonols, sterols, saponins, terpenoids, carbohydrates, oil, and fats. Whereas, the presence of glycosides, terpenoids, and sterols was detected in chloroform extract. Petroleum ether extracts reveal the presence of glycosides and sterols. Methanolic extracts revealed the occurrence of a maximum number of phytoconstituents. Hence, we used methanolic extract for further pharmacological research.
Table 1: Preliminary qualitative screening of phytoconstituents of Elytranthe parasitica stem extracts.
Secondary metabolites | Name of the test | Petroleum ether | Chloroform | Methanol |
---|---|---|---|---|
Alkaloids | Mayer’s test | − | − | − |
Wagner’s test | − | − | − | |
Tannin and Phenolic compounds | Ferric chloride test | − | − | + |
Gelatin test | − | − | + | |
Lead acetate | − | − | − | |
Salkowski’s Test | − | − | + | |
Glycosides | Keller-killiani’s test | + | + | + |
Legal’s test | + | + | + | |
Flavonoids | Ferric chloride test | − | − | + |
Shinoda test | − | − | + | |
Sterols | Libermann Burchard’s Test | + | + | + |
Saponins | Foam test | − | − | + |
Terpenoids | Libermann Burchard’s Test | − | + | + |
Carbohydrates | Benedict’s Test | − | − | + |
Fehling’s Test | − | − | + | |
Fixed oils and fats. | Saponification Test | − | − | + |
−: Negative result; +: Positive results
3.3. Quantitative Gas Chromatography-Mass Spectroscopy (GC-MS) Profiling of Stem Methanol Extract of E. parasitica
GC-MS profiling of the stem methanolic extract of E. parasitica reveals the presence of eleven bioactive chemical constituents [Figure 1]. The name, retention time, percentage area, molecular weight, molecular formula, and properties of the phytoconstituents are represented in Table 2. The identified compounds include Octadecenoic acid,4-hydroxybutyl ester (22.95%); 2H-1-Benzopyran-7-ol, 3,4-dihydro-5-methoxy-2-phenyl- (21.90%); Oleic acid (14.18%); 9-Hexadecenoic acid (10.59%); Hexadecanoic acid, 2,3-dihydroxypropyl ester (8.15%); Z-8-Methyl-9-tetradecenoic acid (8.05%); b-D-Glucopyranose, 1,6-anhydro-(6.54%); n-Hexadecanoic acid (2.86%); 1-Nitro-beta-d-arabinofuranose, tetraacetate (1.87%); Pentadecanoic acid (1.62%); and Ethyl iso-allocholate (1.20%).
Figure 1: GC-MS chromatogram of methanolic stem extracts of Elytranthe parasitica. [Click here to view] |
Table 2: List of identified phytochemicals in methanolic stem extract of Elytranthe parasitica (L.) Danser by GC-MS analysis.
Retention time | Average percentage | Chemical compound present | Molecular formula | Molecular weight | Properties |
---|---|---|---|---|---|
8.42 | 1.87 | 1-Nitro-beta-d-arabinofuranose, tetraacetate | C13H17NO11 | 363.27 | No significant report. |
14.07 | 1.20 | Ethyl iso-allocholate | C26H44O5 | 436.6 | Antimicrobial, diuretic, anti-inflammatory, antiasthma. |
14.71 | 6.54 | b-D-Glucopyranose, 1,6-anhydro- | C6H10O5 | 162.14 | Anti-bacterial and antioxidant activity. |
20.97 | 2.86 | n-Hexadecanoic acid | C16H32O2 | 256.42 | Antioxidant, hypocholesterolemic, nematicidal, pesticide, hemolytic, antiandrogenic, hemolytic, 5-alpha reductase inhibitor, anti-inflammatory activity. |
22.84 | 22.95 | Octadecenoic acid, 4-hydroxybutyl ester | C22H44O3 | 356.6 | No significant report |
22.94 | 3.61 | Oleic acid | C18H34O2 | 282.5 | Antimicrobial, antioxidant, and apoptotic activity. |
23.65 | 10.59 | 9-Hexadecenoic acid | C16H30O2 | 254.41 | Anti-inflammatory protective effects on hepatic steatosis and insulin signaling in murine. |
23.98 | 1.62 | Pentadecanoic acid | C15H30O2 | 242.4 | Antimicrobial. |
28.83 | 8.15 | Hexadecanoic acid, 2,3-dihydroxypropyl ester | C19H38O4 | 330.5 | Palmitate-induced inflammatory effect on microphage |
29.24 | 21.90 | 2H-1-Benzopyran-7-ol, 3,4-dihydro-5-methoxy-2-phenyl- | C16H16O3 | 256.30 | Anti-inflammatory, anti-allergic agent. |
31.43 | 2.35 | Z-8-Methyl-9-tetradecenoic acid | C15H28O2 | 240.38 | No significant report. |
32.86 | 2.73 | Oleic acid | C18H34O2 | 282.5 | Antimicrobial, antioxidant apoptotic activity. |
33.10 | 0.98 | Z-8-Methyl-9-tetradecenoic acid | C15H28O2 | 240.38 | No significant report. |
34.46 | 1.54 | Z-8-Methyl-9-tetradecenoic acid | C15H28O2 | 240.38 | No significant report. |
36.29 | 1.49 | Z-8-Methyl-9-tetradecenoic acid | C15H28O2 | 240.38 | No significant report. |
38.67 | 1.69 | Z-8-Methyl-9-tetradecenoic acid | C15H28O2 | 240.38 | No significant report. |
41.68 | 7.84 | Oleic acid | C18H34O2 | 282.50 | Antimicrobial, antioxidant, and apoptotic activity. |
GC-MS: Gas Chromatography-Mass Spectroscopy.
3.4. Antibacterial Screening of Stem Methanolic Extract of E. parasitica against Selected Bacterial Pathogens
The antibacterial activity of E. parasitica (L.) Danser. methanolic stem extract against selected bacterial pathogens exhibited a concentration-dependent zone of inhibition. The methanolic stem extract exhibited considerable antibacterial activity and showed a maximum inhibition zone of 19.83 ± 0.44 mm against X. campestris followed by S. Typhi (15.50±0.28 mm) and E. faecallis (15.50±0.28 mm) and the minimum inhibition zone against E. coli (12.66 ± 0.33 mm). The results of the experiment were triple-checked, and they were presented as mean ± standard error of the mean. The inhibition zone is measured in millimeters [Table 3 and Figure 2].
Table 3: Antibacterial activity of the stem methanolic extract of Elytranthe parasitica (L.) Danser against selected pathogenic bacterial strains.
S. No. | Name of the bacterial strains | Inhibition zone in mm (Mean±Standard Error of Mean) | ||||
---|---|---|---|---|---|---|
Concentration in percentage | Standard (Amoxylin) | Control (Dimethyl sulfoxide) | ||||
100% | 50% | 25% | ||||
1. | Escherichia coli | 12.66±0.33 | 9.50±0.28 | 8.16±0.16 | 20.33±0.88 | 00 |
2. | Staphylococcus aureus | 14.16±0.44 | 8.33±0.33 | 6.50±0.28 | 26.00±0.57 | 00 |
3. | Salmonella typhi | 15.50±0.28 | 9.00±0.57 | 6.83±0.16 | 24.66±0.66 | 00 |
4. | Klebsiella pneumonia | 13.16±0.44 | 11.33±0.33 | 7.83±0.16 | 15.50±0.28 | 00 |
5. | Enterococcus faecallis | 15.50±0.28 | 12.83±0.44 | 6.66±0.33 | 22.66±0.33 | 00 |
6. | Xanthomonas campestris | 19.83±0.44 | 13.50±0.28 | 11.66±0.33 | 24.16±0.60 | 00 |
Figure 2: Antibacterial activity of the stem methanolic extract of E. parasitica against selected pathogenic bacterial strains. [Click here to view] |
3.5. Effect of E. parasitica Methanolic Stem Extracts on Prostate Cancer Cell Lines (PC-3) and Pancreatic Cancer Cell Line (PANC-1) Cancer Cells
In vitro cytotoxic effect of E. parasitica, methanolic stem extract was evaluated against human prostate and pancreatic cancer cells using MTT assay, at various concentrations (31.25, 62.5, 125, 250, and 500 μg/mL) and 24 h of incubation time. The results of the MTT assay revealed dose-dependent cytotoxic activity on both the cancer cells, but to a different extent. The methanolic stem extract showed more cytotoxic activity against PC-3 cancer cell lines when compared with the PANC-1 cancer cells and it exhibited no cytotoxic activity against normal cell lines (MEF-L929 mice embryo blast) [Table 4 and Figure 3]. The results of the cytotoxic effects of methanolic stem extract are comparable to those of conventional chemotherapic drugs like curcumin, which is frequently used to treat cancer. The IC50 values of methanolic stem extracts against PC-3 and PANC-1 cell lines are 372.27 ± 22.07 μg/mL and 443.33 ± 17.85 μg/mL at higher concentrations.
Table 4: Cytotoxicity of Elytranthe parasitica methanolic stem extracts against prostate and pancreatic cancer cell lines.
S. No. | Concentration (mg/mL) | PC-3 cell lines (Mean±Standard Error of Mean) | PANC-1 cell lines (Mean±Standard Error of Mean) | ||||
---|---|---|---|---|---|---|---|
% of cell viability | IC50 value (mg/mL) | Standard (curcumin 10 mM) % of cell viability | % of cell viability | IC50 value (mg/mL) | Standard (curcumin 10 mM) % of cell viability | ||
1. | Untreated | 100±0.00 | 372.27±22.07 | 47.95±0.36 | 100±0.00 | 443.33±17.85 | 49.03±0.24 |
2. | 31.25 | 88.57±0.24 | 95.51±1.59 | ||||
3. | 62.5 | 78.29±2.36 | 85.32±1.20 | ||||
4. | 125 | 67.35±0.75 | 76.16±0.27 | ||||
5. | 250 | 58.99±0.46 | 61.82±0.34 | ||||
6. | 500 | 40.17±3.03 | 48.30±2.18 |
PC-3: Prostate cancer cell lines, PANC-1: Pancreatic cancer cell line
Figure 3: Cytotoxicity of Elytranthe parasitica methanol stem extracts against PC-3 and PANC-1 cells. PC-3: Prostate cancer cell lines and PANC-1: Pancreatic cancer cell line. [Click here to view] |