|Year : 2021 | Volume
| Issue : 6 | Page : 162-171
Bioprospecting of Lobelia nicotianifolia Roth. plant parts for antioxidant and cytotoxic activity and its phytoconstituents
Rupali Mukesh Kolap1, Kailash D Datkhile2, Saurabha Bhimrao Zimare1
1 Naoroji Godrej Centre for Plant Research, Lawkim Motor Campus, Shirwal, Maharashtra, India
2 Molecular and Genetic Laboratory, Krishna Institute of Medical Sciences University, Satara, Maharashtra, India
|Date of Submission||02-Sep-2020|
|Date of Decision||29-Dec-2020|
|Date of Acceptance||31-May-2021|
|Date of Web Publication||15-Sep-2021|
Saurabha Bhimrao Zimare
Naoroji Godrej Centre for Plant Research, Lawkim Motor Campus, Shindewadi, Post-Shirwal, Khandala, Satara - 412 801, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Though the Lobelia nicotianifolia Roth. is ethnobotanically important plant of India and Sri Lanka its phytoconstituents, antioxidant, and anticancer potential was not yet reported. Objective: The objective of this study is to analyze the phytoconstituents of plant parts of L. nicotianifolia and to determine its antioxidant and cytotoxic potential. Materials and Methods: The plant parts of L. nicotianifolia were extracted with different solvents and qualitative analysis revealed the presence of different phytoconstituents. Total phenolic content (TPC) and total flavonoid content (TFC) were recorded in all plant parts. The extracts were subjected to the antioxidant assays and the potent methanolic extracts were used for cytotoxicity study and further characterized by Fourier–transform infrared spectroscopy and liquid chromatography with a high resolution mass spectrometer (LC–HRMS). Results: The qualitative analysis showed the presence of a wide array of phytoconstituents in L. nicotianifolia plant parts. A significantly higher TPC, TFC, and antioxidant activities were seen in methanolic stem extract. Stem extract showed maximum cytotoxicity against human breast adenocarcinoma (MCF–7) and human cervical adenocarcinoma (HeLa) cell lines whereas, root extract had higher cytotoxicity against human colon adenocarcinoma (HCT–15) cells. The results of cell viability indicated that the methanolic extracts of L. nicotianifolia plant parts exhibited a range of cytotoxic activity in a concentration and time dependent manner against selected cancer cell lines. The LC–HRMS showed the presence of cytotoxic compounds comparatively higher in stem. Conclusion: The study confirms the antioxidant and cytotoxic potential of L. nicotianifolia. To understand the detailed mechanism of cytotoxicity of L. nicotianifolia, it is necessary to study the molecular mechanism involved in this study.
Keywords: Antioxidant, cytotoxic, HCT–15, HeLa, Lobelia nicotianifolia, MCF–7
|How to cite this article:|
Kolap RM, Datkhile KD, Zimare SB. Bioprospecting of Lobelia nicotianifolia Roth. plant parts for antioxidant and cytotoxic activity and its phytoconstituents. Phcog Mag 2021;17, Suppl S2:162-71
|How to cite this URL:|
Kolap RM, Datkhile KD, Zimare SB. Bioprospecting of Lobelia nicotianifolia Roth. plant parts for antioxidant and cytotoxic activity and its phytoconstituents. Phcog Mag [serial online] 2021 [cited 2022 Aug 14];17, Suppl S2:162-71. Available from: http://www.phcog.com/text.asp?2021/17/6/162/326022
- The phytoconstituents, antioxidant, and anticancer potential of Lobelia nicotianifolia Roth. plant parts were analysed
- Methanolic extracts of stem showed significantly higher phenolic compounds and antioxidant activity
- Stem and root extracts has higher cytotoxicity against MCF–7, HeLa and MCF-7 cell lines respectively
- Cytotoxicity of L. nicotianifolia plant parts were depending on concentrations of extracts and treatment duration
- Chemical characterization of stem by LC–HRMS showed 23 cytotoxic compounds
Abbreviations used: TPC: Total phenolic content; TFC: Total flavonoid content; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); RSA: Radical scavenging activity; HeLa: Human cervical carcinoma; MCF–7: Human breast carcinoma; HCT–15: Human colon adenocarcinoma; ATR–FTIR: Attenuated total reflectance Fourier–transform infrared spectroscopy; LC–HRMS: Liquid chromatography with high-resolution mass spectrometer; HPLC: High-performance liquid chromatography; DMSO: Dimethyl sulfoxide; RT: Room temperature; TAE: Tannic acid equivalent; QE: Quercetin equivalent; MEM: Minimum essential medium; FBS: Fetal bovine serum; AlCl3: Aluminum chloride, CO2: Carbon dioxide; Abs: Absorbance; RPMI 1640: Roswell Park Memorial Institute media.
| Introduction|| |
The morbidity and mortality in humans are mainly because of four main non-communicable diseases such as cardiovascular disease, chronic respiratory disease, cancer, and diabetes. In 2018, mortality because of cancer was 9.6 million and increased up to 18 million by 2020., There are different types of life-threatening cancers of which cervical (19.6%), breast (12.5%), and colon (23.4%) cancer are most common. Based on the type of cancer and its progression, it is treated with different therapies such as hormone therapy, chemotherapy, and radiotherapy. Among all these, extensively employed approach is chemotherapy. The use of chemotherapy for the management of cancer is found to be insubstantial because of the development of multidrug resistance in the cancer cells and other severe adverse effects as reviewed by Aslam et al. To overcome the limitations more attention has been paid to the alternatives and complementary plant-based natural products., Medicinal plants are known for their wide array of phytoconstituents of which phenolics are recognized for their free radical scavenging and anticancer property.,, In most of the cases, cancerous conditions are related to the overproduction of free radicals in cells. Cells are capable of neutralizing the consequences of free radicals by producing antioxidants. Based on origin, the antioxidants are categorized as endogenous and exogenous. Endogenous antioxidants are produced within the cells while exogenous sources are mainly obtained primarily from different plants. The exogenous antioxidants are in limelight due to lower side effects and their cost. The consumption of different antioxidants in an appropriate amount reduces the chances of morbidity and mortality due to cancer.
Campanulaceae is one of the important family of the tropical and warm temperate regions which is known for its bioactive alkaloids and phenolic compounds. The genus Lobelia is known to have numerous bioactivities like antitumor, immunomodulatory, anti-inflammatory, antioxidant, antiviral, antipyretic, and antidiabetic activities. Campanulaceae consist of approximately 300 taxa of which Lobelia chinensis and Lobelia inflata are native to China and northern America respectively and are well explored for its phenolic compounds and anticancer activity., Flavonoids such as apigenin and luteolin are reported from L. chinensis which are well associated with the antioxidant and anticancer property. Nevertheless, such a scientific study has not yet been documented for Lobelia nicotianifolia Roth. It is a common plant of Indo-Malayan region and the ethnobotanical studies revealed that it is used in the treatment of numerous diseases and disorders. With the help of this ethnobotanical data researchers have documented analgesic, antimicrobial, antioxidants, and antiepileptic activities. Considering the antioxidant and anticancer potential of other over-exploited Lobelia species there is scope to have similar bioactivities in L. nicotianifolia which can be used as a substitute.
Considering the medicinal value of L. nicotianifolia, in the present study plant parts were extracted with different solvents and preliminary phytochemical analysis was reported to perceive different secondary metabolites. The quantitative analysis was done for the total phenolic content (TPC) and total flavonoid content (TFC). Ultimately our study aimed at determining the in vitro antioxidant potential using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The study was further extended to elaborate the potential cell growth inhibitory effects of crude extracts of L. nicotianifolia on different human cancer cell lines such as human cervical carcinoma (HeLa), human breast carcinoma (MCF–7), and human colon adenocarcinoma (HCT–15). The promising cytotoxic effects were tested against cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the potent extract was characterized with Fourier–transform infrared (FTIR) spectroscopy and liquid chromatography with high-resolution mass spectrometer (LC–HRMS).
| Materials and Methods|| |
Plant material and authentication
L. nicotianifolia was collected around the Kas lake area of Satara district and was identified and authenticated using Flora of Maharashtra state, India, and the herbarium specimen (NGCPR–1904) was deposited to NGCPR, Shirwal.
Chemicals, reagents, and standard
HPLC and analytical reagent grade organic solvents and chemicals used for extraction were procured from Himedia, India. Tannic acid (GRM7541-100G) and quercetin (RM6191-100G) from Himedia, India, were used as standard for quantification of total phenolics and flavonoids. The HeLa, MCF–7, and HCT–15 cell lines were obtained from National Centre for Cell Sciences, Pune, India. Minimum essential Medium (10370-021), Fetal Bovine Serum (2614079), Roswell Park Memorial Institute (RPMI1640) (11875-085), penicillin and streptomycin (15140-122), dimethyl sulfoxide (D2650) were purchased from Sigma Aldrich. MTT (M6494) was procured from Invitrogen.
Extraction of plant parts
The dried leaf, stem, and root powder (10 g) were extracted with Soxhlet extractor with 100 mL of different solvents. The selection of solvents was based on the polarity where nonpolar (petroleum ether), mid–polar (chloroform), and polar (methanol) solvents were used. The obtained extracts were filtered through Whatman filter paper 1 and concentrated on rotary evaporator under reduced pressure. Obtained viscous extracts were stored at-20°C till further analysis.
Phytochemical tests for phenolics, flavonoids, anthraquinones, coumarin, terpenes, saponins, and alkaloids (Dragendroff's) were carried out for plant part extracts of L. nicotianifolia with some minor modifications.
Quantitative analysis of phenolics and flavonoids
The TPC and TFC in L. nicotianifolia extracts were determined using a modified Folin–Ciocalteu method and Aluminum chloride (AlCl3) methods respectively. For TPC 50 microliter (μL) (equivalent to 100 μg) extracts were added to 2 N Folin–Ciocalteu (200 μL) and 1 mL of sodium carbonate followed by incubation of 30 min (min) at 25°C. TFC of different extracts was determined by the addition of an equal volume of extracts with 2% AlCl3. This reaction mixture was incubated for 60 min at room temperature (RT). A UV–Visible spectrophotometer (Shimadzu UV-1900 UV–VIS) was used for the quantification of TPC and TFC at 765 and 420 nm respectively. A graph of absorbance against concentration (0–250 μg/mL) was plotted to obtain the calibration curve and TPC and TFC were expressed as milligrams of tannic acid equivalent (TAE) and quercetin equivalent (QE) per gram extract respectively.
Determination of antioxidant activity
2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity
The percentage radical scavenging activity (% RSA) of L. nicotianifolia extracts against DPPH radical was evaluated as described by Zheleva-Dimitrova et al. with minor modifications. In brief, 1 ml (mL) of extracts (100-500 μg/mL) was mixed with 4 mL methanolic DPPH (0.2 mM) solution and vortexed thoroughly. This reaction mixture was incubated in the dark for 30 min at RT and the absorbance was measured by spectrophotometer at 517 nm.
The percentage DPPH RSA was calculated using the following equation:
Percentage inhibition = (A0-A1)/A0) ×100 equation 1
2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay
Two stock solutions were prepared (A) ABTS (7 mM) and (B) potassium persulfate (2.4 mM). The ABTS solution was prepared by mixing equal volumes of stock A and B and allowed them to react in dark for 14 h at RT. One mL ABTS solution was further diluted with 60 mL methanol to attain a specific absorbance of 0.706 ± 0.01 at 734 nm by a spectrophotometric method. L. nicotianifolia extracts (1 mL) was reacted with 1 mL of the ABTS solution and the absorbance was recorded spectrophotometrically at 734 nm after 7 min. The percentage ABTS scavenging activity of the extract was calculated by the following formula:
Percentage inhibition = (A0–A1)/A0) ×100 equation 2
In equation 1 and 2: A0 is the absorbance of control and A1 absorbance of test. The results were compared with Ascorbic acid as reference standard.
In vitro cytotoxicity
Cell line and cell culture
The in vitro cytotoxicity of L. nicotianifolia extracts was studied against HeLa), MCF–7, and HCT–15 cell lines. The HeLa and MCF–7 cell lines were maintained in T-25 flasks with minimum essential medium (MEM), while HCT–15 cells were maintained in RPMI-1640 media with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL and 100 μg/mL penicillin and streptomycin, respectively. These cell lines were maintained under an atmosphere of 5% Carbon dioxide (CO2) and 95% humidity at 37°C until further study.
Cell proliferation assay (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay)
The in vitro cell viability of different human cancer cell lines was determined by MTT colorimetric assay. In total, 1 × 104 cells in 200 μL of the respective medium per well were seeded in a 96–well plate and incubated at 37°C under 5% CO2. After 24 h (h) of incubation, the confluent cells were exposed to different concentrations of plant extracts in their respective culture media without FBS and incubated at 37°C under 5% CO2. After completion of the treatment at 48 h, the medium was removed and the cells were washed with Hanks' balanced salt solution. Thereafter, 10 μL/well of 5 mg/mL concentration of MTT was added to the cells, and the cells were incubated for another 4 h at 37°C under 5% CO2. Then, the MTT containing media was removed through aspiration and replaced with 200 μL of dimethyl sulfoxide, which was added to each well to dissolve the formazan crystals. The absorbance of the developed purple color was measured at 560 nm wavelength by using the spectrophotometer (Shimadzu, UV–Vis 1800). The results of cell viability were expressed as the percentage growth inhibition of treated and untreated cells, using the following formula:
Percentage inhibition = 100-(A0 ÷ A1) ×100 equation 3
In equation 3: A0 is the absorbance of treated cells at 560 nm and A1 absorbance of untreated (control) cells at 560 nm.
Selected cells were seeded at 1 × 106 cells/well in a 6-well culture plate and incubated for 24 h at 37°C under 5% CO2. Later, the cells were treated with different concentrations of plant extracts in their respective culture media without FBS and further incubated up to 48 h. The cytomorphology of the cells was then observed under phase contrast microscope (Primovert Carl Zeiss).
Characterization of extract
Analysis of functional groups by Fourier–transform infrared
The viscous extract was loaded in Attenuated total reflectance Fourier–transform infrared spectroscopy (ATR–FTIR) spectroscope (Shimadzu IRAffinity-1S 00466, Serial No. A221354), with a scan range from 500 to 4000 cm-1 with a resolution of 4 cm− 1. Prior to every scan, the ATR plate was carefully cleaned with 70% acetone. Further, the obtained results were processed through IR solution software.
Liquid chromatography with high-resolution mass spectrometer analysis of methanolic extracts
The chromatographic system (Agilent, USA) consisted of a binary LC pump (G1312B) with an autosampler (G1329B) and HRMS (G6540B). Extracts were injected onto RPC18 Zorbax (2.1 mm × 50 mm, 1.8-μm) column where the mobile phase for this analysis was two MS grade solvents (A) 0.1% Formic acid in water (95%) and (B) acetonitrile (5%) which was delivered at 0.4 mL/min. The ion source was Dual AJS ESI performing in positive or negative mode with a gas flow set at 8 L/min, spray voltage set at 3500 V, and auxiliary gas and capillary temperatures set at 325°C and 300°C, respectively. The stepwise gradient (A and B) was delivered for a different time duration which was 5% of phase A and 95% of phase B for 0–18 min and 25 min. Whereas, for 0–25 min and 30 min it was 95% of phase A and 5% of phase B. The injection volume was 10 μL and the HRMS full scans were acquired from m/z 60–1600 Da with a scanning rate of 2 scans/s. Mass calibration was done before the analysis using the Agilent Q–TOF ESI calibration mix.
All experiments were performed in triplicates and the values were expressed as Mean ± standard deviation. One-way analysis of variance followed by Duncan's new multiple range test to evaluate the significance at P ≤ 0.05. The IC50 values of extracts were calculated using the Statistical Package for the Social Sciences – 11 (SPSS 11, IBM, USA) at 95% confidence level.
| Results and Discussion|| |
Phytochemical analysis, total phenolic content, and total flavonoid content
In the present study leaf, stem, and root when extracted with nonpolar, mid-polar, and polar solvents showed the presence of phenolics, flavonoids, anthraquinones, and coumarin [Table 1]. It has been reported that the solubility of different phenolics is governed by the polarity of extracting solvents. Previous investigations of L. chinensis and L. erinus revealed the presence of phenolics and flavonoids. Anthraquinones are active phytoconstituents of various plants and have been reported for the first time for Lobelia species [Table 1]. In the present investigation, methanolic extracts of leaf, stem, and root showed the presence of anthraquinones. Another important polyphenol, i.e. coumarin was detected in chloroform and methanolic extracts of leaf, stem, and root [Table 1]. The presence of coumarin in L. nicotianifolia has shown a concurrence with the previous study of L. chinensis. In our study, nonpolar and polar extracts showed the presence of terpenoids while saponins were recorded in polar extracts only [Table 1]. Terpenoids have a broad range of chemical properties and can be detected in polar and nonpolar solvents whereas saponins are more soluble in polar solvents., The occurrences of terpenoids and saponins have been reported from L. chinensis and L. sessilifolia., Alkaloids were detected using Dragendorff's reagent in chloroform and methanolic extracts of leaf, stem, and root. Extensive research has been carried out on alkaloids of Lobelia and 46.05% species are known to produce pharmaceutically important alkaloids. Plant phenolics and flavonoids are the important secondary metabolites known for their bioactivities., In this study, higher TPC and TFC were seen in methanolic extracts of the stem as compared to leaves and root but with no significant difference among the plant parts used [Table 2]. The efficiency of extraction of TPC (mg TAE/g extract) was in following order methanol (13.46) > chloroform (9.57) > petroleum ether (3.22). For the extraction of TFC, the maximum content was seen in methanol extract (11.66 mg QE/g extract) followed by chloroform (8.05 mg QE/g extract) and petroleum ether (2.72 mg QE/g extract) and a similar pattern was observed for leaf and root of L. nicotianifolia. The results represented in [Table 2] express that the increase in solvent polarity linked with increased contents of TPC and TFC which showed the concurrence with previous studies. A wide variety of phenolic compounds showed the presence of polysaccharides, proteins, terpenes, chlorophyll, inorganic compounds that dissolve in polar solvents. Hence, in the present study, a higher amount of TPC and TFC were seen in methanol as compared to chloroform and petroleum ether [Table 2]. The variations of TPC and TFC in different plant parts of L. nicotianifolia may be related to the function of these phenolic compounds in plant species, life cycle, and the growth phase as previously reported for different plant species.,
|Table 1: Presence of phytochemicals in leaf, stem, and root of Lobelia nicotianifolia|
Click here to view
|Table 2: Concentrations of total phenolic content and total flavonoid content in different plant part extracts (leaf, stem, and root) of Lobelia nicotianifolia|
Click here to view
In vitro antioxidant assays and correlation with total phenolic content and total flavonoid content
DPPH and ABTS % RSA of plant part extracts of L. nicotianifolia were evaluated in the present investigation and are shown in [Figure 1]. The DPPH % RSA was seen in the range of 32.87%–88.08% based on plant parts and extracting solvents [Figure 1]a. The DPPH % RSA of methanolic stem extract (88.08%) was higher as compared to ascorbic acid (85.21%) and other studied extracts. The plant parts extracted with chloroform and petroleum ether has intermediate and lower DPPH % RSA respectively [Figure 1]a. A similar pattern was seen for ABTS % RSA for different plant parts of L. nicotianifolia with superiority for methanolic extracts of the stem [Figure 1]b. The stem extract of L. nicotianifolia quenches a maximum of 85.35% ABTS radicals which was higher as compared to leaf and root extracts. DPPH and ABTS are the most employed antioxidant assays to define the antioxidant potential of food sources and medicinal plant extracts. These assays are based on the capacity to test extracts to donate hydrogen which works as a chain-breaker. The stem of L. nicotianifolia extracted with methanol has higher RSA [Figure 1] which might be associated with the array of secondary metabolites [Table 1] and higher phenolics and flavonoids content [Table 2]. Phenolic compounds have hydroxyl groups at the ortho- and para-positions, which contribute to antioxidant activity. We have also studied the correlation between TPC and TFC with DPPH and ABTS assays by using the Pearson correlation coefficient which revealed a significant positive correlation [Table 3]. A significant correlation between antioxidant assays and of the test extracts is associated with the hydroxyl groups and hydrogen atoms of phenolic compounds which helps in the quenching of free radicals and end the chain formation. This study has indicated that the polarity of extracting solvents and the plant parts influences the antioxidant potential of L. nicotianifolia [Figure 1]a and [Figure 1]b which showed a concurrence with the previous study.
|Figure 1: Representative histogram showing radical scavenging activity of Lobelia nicotianifolia plant parts (leaf, stem, and root) in (a) 2,2 diphenyl 1 picrylhydrazyl and (b) 2,2' azino bis (3 ethylbenzothiazoline 6 sulfonic acid) assays at 1 mg/mL. The results represent the means of three independent experiments ± standard deviation and the different letters as superscripts are significantly different from each other at P≤0.05. PE: Petroleum ether; CH: Chloroform; ME: Methanol|
Click here to view
|Table 3: Correlation analysis between total phenolic content and total flavonoid content of leaf, stem, and root of Lobelia nicotianifolia with 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays|
Click here to view
In vitro cytotoxicity and characterization of extracts
Preliminary phytochemical analysis [Table 1], higher phenolic and flavonoid contents [Table 2], and in vitro antioxidant activity [Figure 1] has revealed the superiority of methanolic extracts of L. nicotianifolia hence, the extract was used for cytotoxicity study. The cytotoxicity of methanolic extracts of leaf, stem, and root was tested for 24 and 48 h against HeLa, MCF–7, and HCT–15 cells by MTT assay. The cytotoxicity against HeLa cell lines is represented in [Figure 2] which revealed that methanolic stem extract at 200 μg/mL has higher inhibition (67.59% and 92.05%). The cell viability for MCF–7 indicated that a higher concentration (200 μg/mL) of methanolic stem extract was lethal and showed 74.28% and 92.77% inhibition at 24 and 48 h, respectively [Figure 3]. In contrast to these cell lines, HCT–15 cells were inhibited by methanolic root extract which was 43.70% for 24 h and 65.33% for 48 h [Figure 4]. Similarly, the IC50 values (μg/mL) were also calculated in this study which indicates that the stem and root extracts have potent cytotoxicity against HeLA and MCF–7 cell lines [Table 4]. A lower IC50 value (66.05 μg/mL) was seen for stem extract against breast cancer cell line (MCF-7) followed by root extract (73.38 μg/mL). Whereas, the higher IC50 was recorded for the leaf extract of L. nicotianifolia against HeLA, MCF–7, and HCT–15 cell lines for study duration [Table 4]. Thus, the present observations revealed that the cytotoxicity of L. nicotianifolia is specific to plant parts, their concentrations, and study duration. This observation is in agreement with Mazumder et al. who has reviewed 99 plants belonging to 57 families and concluded that plant parts and their chemical constituents played a crucial role for a potent cytotoxicity against various cancer cell lines. Further, the cell morphology of HeLa [Figure 5]a, MCF–7 [Figure 5]d, and HCT–15 [Figure 5]g cells were altered and showed shrunken appearance because of loss of membrane integrity and cytoplasm condensation when treated with higher concentration (100 and 200 μg/mL) [Figure 5]b, [Figure 5]c, [Figure 5]e, [Figure 5]f, [Figure 5]h and [Figure 5]i. The morphological alterations were because of abnormal accumulation of substances in the cytoplasm and depend on the array of phytoconstituents of plant extracts. For the identification of functional groups and phytoconstituents in the plant parts of L. nicotianifolia potent methanolic extracts were characterized with ATR–FTIR, and LC–HRMS.
|Figure 2: Representative histogram showing in vitro concentration dependent cytotoxicity of methanolic extracts Lobelia nicotianifolia plant parts (leaf, stem, and root) on HeLa cells after (a) 24 h and (b) 48 h treatments. The results represent the means of three independent experiments, and error bars represent the standard deviation of the mean|
Click here to view
|Figure 3: Representative histogram showing in vitro concentration dependent cytotoxicity of methanolic extracts Lobelia nicotianifolia plant parts (leaf, stem, and root) on MCF–7 cells after (a) 24 h and (b) 48 h treatments. The results represent the means of three independent experiments, and error bars represent the standard deviation of the mean|
Click here to view
|Figure 4: Representative histogram showing in vitro concentration dependent cytotoxicity of methanolic extracts Lobelia nicotianifolia plant parts (leaf, stem, and root) on HCT–15 cells after (a) 24 h and (b) 48 h treatments. The results represent the means of three independent experiments, and error bars represent the standard deviation of the mean|
Click here to view
|Table 4: The putative identification of cytotoxic compounds in different plant parts (leaf, stem, and root) Lobelia nicotianifolia by liquid chromatography with high resolution mass spectrometer|
Click here to view
|Figure 5: Morphological changes in HeLA cells (a): Control, (b): 100 μg/mL methanolic leaf extract, and(c): 200 μg/mL methanolic leaf extract), MCF–7 (d): Control, (e): 100 μg/mL methanolic leaf extract, and (f): 200 μg/mL methanolic leaf extract), and human colon adenocarcinoma: 15 (g): Control, h: 100 μg/mL methanolic root extract, and (i): 200 μg/mL methanolic root extract) at 48 h exposure. All images are captured at × 20 magnification with phase contrast microscope where a scale bars represent 100 μm|
Click here to view
ATR–FTIR spectroscopy is a rapid, non-invasive, and cost-effective method employed for the analysis of functional groups in crude extracts. The methanolic extract of stem has shown maximum peaks (14) followed by leaf (11) and root (9) [Figure 6]. These variations state the chemical profiles of the plant parts and could be related to the cytotoxicity in this study. The present analysis has revealed the presence of polysaccharides, proteins, lipids amide, alcohols, phenols, alkanes, carboxylic acids, aldehydes, ketones, alkenes, primary amines, aromatics, esters, ethers, alkyl halides, in different plant parts of L. nicotianifolia. The characterization of cytotoxic phytoconstituents in methanolic plant part extracts of L. nicotianifolia were done by LC–HRMS. In this technique, a LC along with HRMS was used for the characterization of volatile and nonvolatile phytoconstituents in the complex plant extracts. The methanolic stem extract of L. nicotianifolia has maximum 23 cytotoxic compounds followed by leaf (21) and root (6) [Table 4] and could be a reason for higher cytotoxicity of stem extract for MCF–7 and HeLa cells. These compounds are categorized into 21 distinct classes [Table 5] which are well known for its cytotoxicity.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
|Figure 6: Representative attenuated total reflectance Fourier–transform infrared spectroscopy spectra of observed peaks in methanolic leaf (a), stem (b), and root (c) extracts of Lobelia nicotianifolia in the 4000-500 cm− 1 range|
Click here to view
|Table 5: Analysis of inhibitory concentration50 (IC50) values of Lobelia nicotianifolia plant part extracts against human cervical adenocarcinoma, human breast adenocarcinoma, and human colon adenocarcinoma at 24 and 48 h|
Click here to view
| Conclusion|| |
The present study reveals the phytochemical profiling, in vitro antioxidant and anticancer activity of L. nicotianifolia plant parts. Based on the results it can be concluded that the extracts prepared in higher polarity solvents were significant radical scavengers than those prepared in less polar solvents. Methanolic extracts showed a large array of different phytochemicals and phenolic compounds. Extracts with higher phenolic and flavonoid contents also had higher antioxidant and anticancer activity (percent inhibition and IC50). However, further studies are needed to understand detailed molecular mechanism responsible for antioxidant and anticancer activity in this plant. Thus, the obtained results could form a good basis for further investigation in the potential discovery of new natural bioactive compounds and molecular mechanism involved in those bioactive compounds from this traditional plant with medicinal value.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Nethan S, Sinha D, Mehrotra R. Non communicable disease risk factors and their trends in India. Asian Pac J Cancer Prev 2017;18:2005-10.
Weir HK, Thompson TD, Soman A, Møller B, Leadbetter S, White MC. Meeting the healthy people 2020 objectives to reduce cancer mortality. Prev Chronic Dis 2015;12:1-11.
Dilshad E, Ismail H, Khan MA, Cusido RM, Mirza B. Metabolite profiling of Artemisia carvifolia Buch transgenic plants and estimation of their anticancer and antidiabetic potential. Biocatal Agric Biotechnol 2020;101539:1-24.
Arruebo M, Vilaboa N, Sáez-Gutierrez B, Lambea J, Tres A, Valladares M, et al.
Assessment of the evolution of cancer treatment therapies. Cancers (Basel) 2011;3:3279-330.
Tinoush B, Shirdel I, Wink M. Phytochemicals: Potential Lead Molecules for MDR Reversal. Front Pharmacol 2020;11:832.
Aslam MS, Naveed S, Ahmed A, Abbas Z, Gull I, Athar MA. Side effects of chemotherapy in cancer patients and evaluation of patients opinion about starvation based differential chemotherapy. J Cancer Res Ther 2014;5:817-22.
Seca AM, Pinto DC. Plant secondary metabolites as anticancer agents: Successes in clinical trials and therapeutic application. Int J Mol Sci 2018;19:1-22.
Dai J, Mumper RJ. Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 2010;15:7313-52.
Wink M. Modes of action of herbal medicines and plant secondary metabolites. Medicines (Basel) 2015;2:251-86.
Ghosh S, Derle A, Ahire M, More P, Jagtap S, Phadatare SD, et al.
Phytochemical analysis and free radical scavenging activity of medicinal plants Gnidia glauca and Dioscorea bulbifera. PLoS One 2013;8:e82529.
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44-84.
Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev 2010;4:118-26.
Singh K, Bhori M, Kasu YA, Bhat G, Marar T. Antioxidants as precision weapons in war against cancer chemotherapy induced toxicity – Exploring the armoury of obscurity. Saudi Pharm J 2018;26:177-90.
Mabberley DJ. Mabberley's Plant-Book: A Portable Dictionary of Plants, their Classification and Uses. 4th
ed. Cambridge University Press; Cambridge, United Kingdom 2018.
Stolom S, Oyemitan IA, Matewu R, Oyedeji OO, Oluwafemi SO, Nkeh-Chungag BN, et al.
Chemical and biological studies of Lobelia flaccida
(C. Presl) A. DC leaf: A medicinal plant used by traditional healers in Eastern Cape, South Africa. Trop J Pharm Res 2016;15:1715-21.
Folquitto DG, Swiech JN, Pereira CB, Bobek VB, Halila Possagno GC, Farago PV,et al.
Biological activity, phytochemistry and traditional uses of genus Lobelia (Campanulaceae):
A systematic review. Fitoterapia 2019;134:23-38.
Chen MW, Chen WR, Zhang JM, Long XY, Wang YT. Lobelia chinensis
: Chemical constituents and anticancer activity perspective. Chin J Nat Med 2014;12:103-7.
József VV, Péter B, Ákos M, László K, Éva S. Increasing the anti-Addictive piperidine alkaloid production of in vitro
micropropagated Indian tobacco by nitrate treatments. J Plant Biochem Physiol 2017;5:1-6.
Tamboli A, Khan I, Bhutkar K, Rub R. Systematic review on phytochemical and pharmacological profile of Lobelia nicotianaefolia
Roth E and S. Pharmacologyonline 2011;3:7-22.
Vigneshwaran V, Somegowda M, Pramod SN. Pharmacological evaluation of analgesic and antivenom potential from the leaves of folk medicinal plant Lobelia nicotianaefolia
. AJPCT 2014;2:1404-15.
Kalaimathi SK, Muthu G, Manjula K. Antibacterial activity of Lobelia nicotianifolia
against various bacterial strains. Int J Life Sci Pharma Res 2015;5:19-25.
Murthy NK, Pushpalatha KC, Joshi CG. Antioxidant activity and phytochemical analysis of endophytic fungi isolated from Lobelia nicotianifolia
. J Chem Pharm Res 2011;3:218-25.
Tamboli AM, Rub RA, Ghosh P, Bodhankar SL. Antiepileptic activity of lobeline isolated from the leaf of Lobelia nicotianaefolia
and its effect on brain GABA level in mice. Asian Pac J Trop Biomed 2012;2:537-42.
Londhe AN. Lobeliaceae. In: Singh, NP, Karthikeyan S, editors. Flora of Maharashtra state (Dicptyledones). Culcutta: Botanical Survey of India; 2001. p. 277.
Pochapski MT, Fosquiera EC, Esmerino LA, Dos Santos EB, Farago PV, Santos FA, et al.
Phytochemical screening, antioxidant, and antimicrobial activities of the crude leaves' extract from Ipomoea batatas
(L.) Lam. Pharmacogn Mag 2011;7:165-70.
Aryal S, Baniya MK, Danekhu K, Kunwar P, Gurung R, Koirala N. Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from Western Nepal. Plants (Basel) 2019;8:1-2.
Zheleva-Dimitrova D, Nedialkov P, Kitanov G. Radical scavenging and antioxidant activities of methanolic extracts from Hypericum species growing in Bulgaria. Pharmacogn Mag 2010;6:74-8.
Li KC, Ho YL, Huang GJ, Chang YS. Anti-oxidative and anti-inflammatory effects of Lobelia chinensis in vitro
and in vivo.
Am J Chin Med 2015;43:269-87.
Wang PP, Luo J, Yang MH, Kong LY. Chemical constituents of Lobelia chinensis
. Chin Tradit Herb Drug 2013;44:794-7.
Jiang Y, Shi R, Liu B, Wang Q, Dai Y. Studies on chemical components of Lobelia chinensis
. Zhongguo Zhong Yao Za Zhi 2009;34:294-7.
Chua LS, Lau CH, Chew CY, Dawood DA. Solvent fractionation and acetone precipitation for crude saponins from Eurycoma longifolia
extract. Molecules 2019;24:1-10.
Sun J, Wang X, Zhang H, Yang J. A new triterpenoid ester from Lobelia sessilifolia
. Chem Nat Compd 2012;48:416-8.
Iloki-Assanga SB, Lewis-Luján LM, Lara-Espinoza CL, Gil-Salido AA, Fernandez-Angulo D, Rubio-Pino JL,et al.
Solvent effects on phytochemical constituent profiles and antioxidant activities, using four different extraction formulations for analysis of Bucida buceras
L. and Phoradendron californicum
. BMC Res Notes 2015;8:396.
Guimaraes KC, Salgado DL, Carvalho EE. Evaluation of different methodologies for the determination of phenolic compounds in tropical fruits. Braz J Food Technol 2020;23:1-7.
Akar Z, Küçük M, Doğan H. A new colorimetric DPPH•
scavenging activity method with no need for a spectrophotometer applied on synthetic and natural antioxidants and medicinal herbs. J Enzyme Inhib Med Chem 2017;32:640-7.
Pisoschi AM, Pop A, Cimpeanu C, Predoi G. Antioxidant capacity determination in plants and plant-derived products: A review. Oxid Med Cell Longev 2016;2016:1-36.
Bendary E, Francis RR, Ali HM, Sarwat MI, El Hady S. Antioxidant and structure-activity relationships (SARs) of some phenolic and anilines compounds. Ann Agric Sci 2013;58:173-81.
Nawaz H, Shad MA, Rehman N, Andaleeb H, Ullah N. Effect of solvent polarity on extraction yield and antioxidant properties of phytochemicals from bean (Phaseolus vulgaris
) seeds. Brazilian J Pharm Sci 2020;56:1-9.
Mazumder K, Biswas B, Raja IM, Fukase K. A review of cytotoxic plants of the Indian subcontinent and a broad-spectrum analysis of their bioactive compounds. Molecules 2020;25:1-40.
Saleh KA, Albinhassan TH, Elbehairi SE, Alshehry MA, Alfaifi MY, Al-Ghazzawi AM, et al.
Cell cycle arrest in different cancer cell lines (Liver, Breast, and Colon) induces apoptosis under the influence of the chemical content of Aeluropus lagopoides
leaf extracts. Molecules 2019;24:507-19.
Durak T, Depciuch J. Effect of plant sample preparation and measuring methods on ATR–FTIR spectra results. Environ Exp Bot 2020;169:1-3.
Baker MJ, Trevisan J, Bassan P, Bhargava R, Butler HJ, Dorling KM, et al.
Using Fourier transform IR spectroscopy to analyze biological materials. Nat Protoc 2014;9:1771-91.
Beccaria M, Cabooter D. Current developments in LC-MS for pharmaceutical analysis. Analyst 2020;145:1129-57.
Kogure K, Manabe S, Suzuki I, Tokumura A, Fukuzawa K. Cytotoxicity of alpha-tocopheryl succinate, malonate and oxalate in normal and cancer cells in vitro
and their anti-cancer effects on mouse melanoma in vivo
. J Nutr Sci Vitaminol (Tokyo) 2005;51:392-7.
Islam MT, Ali ES, de Carvalho RM, Paz MA, Braga AL, de Lima RA, et al.
Phytanic acid, a daily consumed chlorophyll-yielded phytol bio-metabolite: A comprehensive review. Afr J Pharm Pharmacol 2016;10:1025-33.
Venepally V, Nethi SK, Pallavi K, Patra CR, Jala RC. Synthesis and cytotoxic studies of undecenoic acid-based Schiffs base derivatives bearing 1, 2, 4-Triazole moiety. Indian J Pharm Sci 2019;81:737-46.
Li X, Wang H, Wang J, Chen Y, Yin X, Shi G, et al.
Emodin enhances cisplatin-induced cytotoxicity in human bladder cancer cells through ROS elevation and MRP1 downregulation. BMC Cancer 2016;16:578.
Bai LY, Chiu CF, Chiu SJ, Chen YW, Hu JL, Wu CY, et al.
Alphitolic acid, an anti-inflammatory triterpene, induces apoptosis and autophagy in oral squamous cell carcinoma cells, in part, through a p53-dependent pathway. J Funct Foods 2015;18:368-78.
Peng-Jun YI, Jun-Song WA, Peng-Ran WA, Ling-Yi KO. Sesquiterpenes and lignans from the fruits of Illicium simonsii and their cytotoxicities. Chin J Nat Med 2012;10:383-7.
Yan X, Qi M, Li P, Zhan Y, Shao H. Apigenin in cancer therapy: Anti-cancer effects and mechanisms of action. Cell Biosci 2017;7:50.
Chang KC, Duh CY, Chen IS, Tsai IL. A cytotoxic butenolide, two new dolabellane diterpenoids, a chroman and a benzoquinol derivative formosan Casearia membranacea. Planta Med 2003;69:667-72.
Zhang J, Fang C, Qu M, Wu H, Wang X, Zhang H, et al.
CD13 inhibition enhances cytotoxic effect of chemotherapy agents. Front Pharmacol 2018;9:1042.
Shabbits JA, Mayer LD. Intracellular delivery of ceramide lipids via liposomes enhances apoptosis in vitro.
Biochim Biophys Acta 2003;1612:98-106.
Juang YP, Liang PH. Biological and pharmacological effects of synthetic saponins. Molecules 2020;25:1-23.
Bulle S, Reddyvari H, Nallanchakravarthula V, Vaddi DR. Therapeutic potential of Pterocarpus santalinus
L.: An update. Pharmacogn Rev 2016;10:43-9.
Lu X, Qiu H, Yang L, Zhang J, Ma S, Zhen L. Anti-proliferation effects, efficacy of cyasterone in vitro
and in vivo
and its mechanism. Biomed Pharmacother 2016;84:330-9.
Macur K, Grzenkowicz-Wydra J, Konieczna L, Bigda J, Temporini C, Tengattini S, et al.
A proteomic-based approach to study the mechanism of cytotoxicity induced by interleukin-1α and cycloheximide. Chromatographia 2018;81:47-56.
Wada K, Yamashita H. Cytotoxic effects of diterpenoid alkaloids against human cancer cells. Molecules 2019;24:1-22.
Hernández-Tiedra S, Fabriàs G, Dávila D, Salanueva ÍJ, Casas J, Montes LR, et al.
Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization. Autophagy 2016;12:2213-29.
Ahamed A, Panneerselvam A, Alaklabi A, Arif IA, Ambikapathy V, Thajuddin N. Molecular perspective and anticancer activity of medicinal plants. Saudi J Biol Sci 2020;27:666-75.
Ko JH, Lee SG, Yang WM, Um JY, Sethi G, Mishra S, et al.
The application of embelin for cancer prevention and therapy. Molecules 2018;23:1-4.
Chen H, Wang Z, Yang L. Analysis of euphornin in Euphorbia helioscopia
L. and its cytotoxicity to mice lung adenocarcinoma cells (LA795). Nat Prod Res 2012;26:2112-6.
Sak K. Cytotoxicity of dietary flavonoids on different human cancer types. Pharmacogn Rev 2014;8:122-46.
Law BY, Mok SW, Chan WK, Xu SW, Wu AG, Yao XJ, et al.
Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers. Oncotarget 2016;7:8090-104.
Han M, Gao H, Ju P, Gao MQ, Yuan YP, Chen XH, et al.
Hispidulin inhibits hepatocellular carcinoma growth and metastasis through AMPK and ERK signaling mediated activation of PPARγ. Biomed Pharmacother 2018;103:272-83.
Tang M, Hu X, Wang Y, Yao X, Zhang W, Yu C, et al.
Ivermectin, a potential anticancer drug derived from an antiparasitic drug. Pharmacol Res 2021;163:105207.
Dailey OD Jr, Wang X, Chen F, Huang G. Anticancer activity of branched-chain derivatives of oleic acid. Anticancer Res 2011;31:3165-9.
Yoo YC, Shin BH, Hong JH, Lee J, Chee HY, Song KS, et al.
Isolation of fatty acids with anticancer activity from Protaetia brevitarsis
larva. Arch Pharm Res 2007;30:361-5.
Ashmawy AM, Ayoub IM, Eldahshan OA. Chemical composition, cytotoxicity and molecular profiling of Cordia africana
Lam. on human breast cancer cell line. Nat Prod Res 2020:1-6.
Harada H, Yamashita U, Kurihara H, Fukushi E, Kawabata J, Kamei Y. Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red alga. Anticancer Res 2002;22:2587-90.
Zerbini LF, Bhasin MK, de Vasconcellos JF, Paccez JD, Gu X, Kung AL, et al.
Computational repositioning and preclinical validation of pentamidine for renal cell cancer. Mol Cancer Ther 2014;13:1929-41.
Alizadeh F, Bolhassani A. In vitro
cytotoxicity of Iranian saffron and two main components as a potential anti-cancer drug. SM J Pharmac Ther 2015;1:1-5.
Dobhal MP, Li G, Gryshuk A, Graham A, Bhatanager AK, Khaja SD, et al.
Structural modifications of plumieride isolated from Plumeria bicolor and the effect of these modifications on in vitro
anticancer activity. J Org Chem 2004;69:6165-72.
Montenegro I, Tomasoni G, Bosio C, Quiñones N, Madrid A, Carrasco H, et al.
Study on the cytotoxic activity of drimane sesquiterpenes and nordrimane compounds against cancer cell lines. Molecules 2014;19:18993-9006.
Denicolaï E, Baeza-Kallee N, Tchoghandjian A, Carré M, Colin C, Jiglaire CJ, et al.
Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo
. Oncotarget 2014;5:10934-48.
Sharma V. A polyphenolic compound rottlerin demonstrates significant in vitro
cytotoxicity against human cancer cell lines: Isolation and characterization from the fruits of Mallotus philippinensis
. J Plant Biochem Biot 2011;20:190-5.
Mortaz E, Rad MV, Johnson M, Raats D, Nijkamp FP, Folkerts G. Salmeterol with fluticasone enhances the suppression of IL-8 release and increases the translocation of glucocorticoid receptor by human neutrophils stimulated with cigarette smoke. J Mol Med (Berl) 2008;86:1045-56.
Avato P, Migoni D, Argentieri M, Fanizzi FP, Tava A. Activity of saponins from medicago species against HeLa and MCF-7 cell lines and their capacity to potentiate cisplatin effect. Anticancer Agents Med Chem 2017;17:1508-18.
Khan AA, Alanazi AM, Jabeen M, Chauhan A, Abdelhameed AS. Design, synthesis and in vitro
anticancer evaluation of a stearic acid-based ester conjugate. Anticancer Res 2013;33:2517-24.
Tsimplouli C, Demetzos C, Hadzopoulou-Cladaras M, Pantazis P, Dimas K. In vitro
activity of dietary flavonol congeners against human cancer cell lines. Eur J Nutr 2012;51:181-90.
Kumar R, Rai D, Lown JW. Synthesis and in vitro
cytotoxicity studies of novel L-tryptophan-polyamide conjugates and L-tryptophan dimers linked with aliphatic chains and polyamides. Oncol Res 2004;14:247-65.
Feng XM, Su XL. Anticancer effect of ursolic acid via mitochondria-dependent pathways. Oncol Lett 2019;17:4761-7.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]