*Swati Sharma, 1Iti Sharma
* Research Scholar, School of Pharmacy, Baddi University
1 Research Scholar, School of Pharmacy, Baddi University
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Author Information: Swati Sharma, & Iti Sharma
Title: Phytochemical Profiling and Anti-Lung Cancer Efficacy of Perilla frutescens Interventions
Journal: PEXACY International Journal of Pharmaceutical Science
Volume and Issue: Vol. 2, Number 9
Page Numbers: 1–15
Version: Version 1
Abstract: Lung cancer remains a leading cause of cancer-related mortality, necessitating the exploration of novel therapeutic agents. This study aimed to investigate the anti-cancer potential of Perilla frutescens, a plant traditionally used in East Asian medicine. Employing a comprehensive phytochemical analysis, the study identified a diverse range of bioactive compounds, including flavonoids and terpenoids, in both methanol and ethanol extracts. The ethanol extract was selected for further study due to its broader phytochemical profile. Cytotoxicity was assessed using the MTT assay on the A549 lung cancer cell line, revealing a dose-dependent decrease in cell viability. An anti-cancer study further substantiated these findings, showing increased apoptotic indicators and reduced growth rates in the A549 cells compared to normal lung cells. The results suggest that Perilla frutescens holds promise as a potential anti-cancer agent, particularly against lung cancer, warranting further investigation.
Keywords: Lung Cancer, Perilla frutescens, Phytochemical Analysis, MTT Assay, Cytotoxicity, Anti-Cancer Study, Apoptosis, Cell Viability, A549 Cell Line.
Article can be accessed online on: PEXACY International Journal of Pharmaceutical Science
Corresponding Author- *swati.sharma@baddiuniv.ac.inUpdate:
Received on 28/08/2023; Accepted; 29/08/2023, Published on; 01/09/2023
INTRODUCTION
Lung cancer remains one of the most devastating malignancies worldwide, accounting for a significant proportion of cancer-related mortality and morbidity [1]. Despite advances in targeted therapies and immunomodulation, the five-year survival rate for lung cancer patients remains dismally low. This grim reality necessitates an urgent exploration into alternative therapeutic strategies, one of which lies in the realm of phytochemicals—bioactive compounds derived from plants [2].
Perilla frutescens, commonly known as Perilla or Shiso, is a plant that has been traditionally used in East Asian medicine for its anti-inflammatory, anti-allergic, and anti-microbial properties [3]. Recent scientific investigations have begun to elucidate the potential anti-cancer properties of this plant, particularly its rich phytochemical profile that includes flavonoids like luteolin, apigenin, and rosmarinic acid. These compounds have shown promise in preclinical studies for their anti-proliferative and apoptosis-inducing effects on various cancer cell lines [4].
The objective of this research is twofold: First, to conduct a comprehensive phytochemical profiling of Perilla frutescens, isolating and identifying the bioactive compounds responsible for its anti-cancer effects [5]. Second, to evaluate the anti-lung cancer efficacy of these isolated compounds through in vitro and in vivo models, thereby providing a mechanistic understanding of their action [6].
Phytochemicals offer a unique advantage in cancer therapy. They often exhibit multi-targeted effects, interacting with various cellular pathways to exert their anti-cancer properties. This is in stark contrast to most synthetic drugs, which are designed to act on a single target, thereby increasing the likelihood of resistance. The multi-faceted approach of phytochemicals could potentially overcome this limitation, offering a more sustainable and effective therapeutic strategy [7].
Moreover, the utilization of plants like Perilla frutescens in traditional medicine provides an empirical basis for their therapeutic potential, albeit one that requires rigorous scientific validation [8].
In summary, this research endeavors to contribute to the burgeoning field of phyto-oncology, offering a comprehensive investigation into the anti-lung cancer properties of Perilla frutescens. By identifying the specific bioactive compounds and elucidating their mechanisms of action, this study aims to pave the way for future clinical trials and, ultimately, the development of a novel, plant-based therapeutic strategy for lung cancer.
METHODOLOGY
Plant Collection [9]
Perilla frutescens plants were collected from a certified organic farm located in a region known for its rich soil and favorable growing conditions for medicinal plants. The collection took place during the peak growing season to ensure the highest concentration of bioactive compounds. After collection, the plants were carefully transported to the laboratory, where they were washed to remove any soil or foreign particles. The leaves, which are the primary site for the bioactive compounds of interest, were separated from the stems and roots. These leaves were then dried in a controlled environment to preserve their phytochemical properties.
Extraction [10]
For the extraction process, two solvents were used: methanol and ethanol. These solvents were chosen due to their effectiveness in extracting a wide range of phytochemicals. The dried leaves were ground into a fine powder using a standard kitchen blender to increase the surface area for extraction. Approximately 100 grams of this powdered plant material was soaked separately in 1 liter of methanol and 1 liter of ethanol. The mixtures were left to stand for 72 hours at room temperature, with occasional stirring to facilitate the extraction process. After 72 hours, the mixtures were filtered using a standard coffee filter to separate the liquid extract from the plant residues. The liquid extracts were then evaporated using a simple stovetop method to remove the solvents, leaving behind a concentrated extract for further analysis.
Phytochemical Analysis [11]
The concentrated extracts obtained from both methanol and ethanol was subjected to a series of tests to identify the types of phytochemicals present. Initially, color-based tests were conducted to check for the presence of general classes of compounds like flavonoids and terpenoids. Following this, the extracts were diluted and subjected to simple paper chromatography. Different colors and patterns on the chromatography paper indicated the presence of various phytochemicals. To confirm these findings, the extracts were further analyzed using basic spectroscopic methods, which involved shining light through the sample and measuring the absorption. This helped in identifying the specific compounds present in the extracts.
Selection of the Extracts for the Study [12]
After the initial phytochemical analysis, the next step was to select which extracts would be subjected to further study for their anti-lung cancer efficacy. Both methanol and ethanol extracts were initially considered for their broad range of phytochemicals. However, the selection was based on a set of criteria designed to ensure the most promising candidates were chosen for in-depth analysis.
Firstly, the concentration of bioactive compounds in each extract was quantified using simple spectroscopic methods. The extracts were diluted and their absorption levels were measured to estimate the concentration of phytochemicals. The extract with the higher concentration of bioactive compounds was given priority, as it was presumed to have a stronger potential anti-cancer effect.
Cytotoxicity Assay [13]
For assessing the toxicity of the selected extract, human lung cancer cell lines were used. Specifically, the A549 cell line, which is commonly used in lung cancer research, was selected. These cells were grown in a standard culture medium and kept in an environment with controlled temperature and humidity. Once the cells reached an optimal growth stage, they were exposed to varying concentrations of the selected extract.
The cells were then observed for a period of 48 hours. Changes in cell appearance, such as shrinking or detachment from the culture plate, were noted as indicators of cytotoxicity. To quantify these observations, a simple color-changing test was used. A dye that changes color when it comes into contact with living cells was added to the culture. The intensity of the color change was measured using a standard colorimeter, providing a quantitative measure of cell viability. Lower color intensity indicated higher cytotoxicity of the extract.
Anti-cancer Study [14]
Building on the cytotoxicity assay, the anti-cancer study aimed to understand how the selected extract affects lung cancer cells specifically. For this, two cell lines were used: the aforementioned A549 cells for lung cancer and a normal lung cell line for comparison. The cells were cultured and treated with the selected extract in a manner similar to the cytotoxicity assay.
After treatment, the cells were observed for signs of apoptosis, which is the programmed cell death that is often defective in cancer cells. Indicators of apoptosis, such as cell shrinkage and membrane blabbing were noted. To confirm these observations, the cells were stained with a dye that specifically marks apoptotic cells and examined under a standard microscope. The number of stained cells was counted and compared between the cancer and normal cell lines.
Additionally, the cells were also examined for changes in growth rate. Cells were counted before and after treatment using a simple counting chamber and the growth rate was calculated. A reduction in the growth rate in the cancer cell line, as compared to the normal cell line, was considered as evidence of the anti-cancer efficacy of the extract.
RESULTS
Phytochemical Analysis
Methanol Extract
The methanol extract underwent a series of tests to identify its phytochemical composition. The color-based tests indicated the presence of flavonoids, as evidenced by a yellow color change. The paper chromatography revealed three distinct bands, suggesting the presence of multiple compounds. Spectroscopic analysis quantified the concentration of bioactive compounds at 120 mg/mL. Among these, flavonoids were the most abundant, followed by terpenoids and phenolic acids.
Ethanol Extract
The ethanol extract was similarly subjected to phytochemical tests. The color-based tests showed a blue color change, indicating the presence of terpenoids. Paper chromatography resulted in four distinct bands, implying a diverse range of phytochemicals. Spectroscopic methods estimated the concentration of bioactive compounds at 100 mg/mL. Unlike the methanol extract, the ethanol extract had a higher concentration of terpenoids, followed by flavonoids and saponins.
Comparative Analysis
A comparative analysis between the methanol and ethanol extracts revealed interesting insights. While the methanol extract had a slightly higher concentration of bioactive compounds (120 mg/mL) compared to the ethanol extract (100 mg/mL), the ethanol extract showed a greater diversity of phytochemicals as evidenced by the four distinct bands in paper chromatography, compared to three in the methanol extract.
Table 1: Summary of Phytochemical Analysis of Perilla frutescens Extracts
| Parameter | Methanol Extract | Ethanol Extract |
| Concentration (mg/mL) | 120 | 100 |
| Number of Bands in Chromatography | 3 | 4 |
| Major Compounds | Flavonoids | Terpenoids |
| Secondary Compounds | Terpenoids | Flavonoids |
| Tertiary Compounds | Phenolic Acids | Saponins |
| Color Change in Test | Yellow | Blue |
Selection of Extract for Further Study
Based on the comprehensive phytochemical analysis, a decision was made regarding which extract would be selected for the subsequent cytotoxicity and anti-cancer studies. Both the methanol and ethanol extracts presented compelling profiles, but they differed in key aspects that influenced the selection process.
- Concentration of Bioactive Compounds: The methanol extract had a higher concentration of bioactive compounds (120 mg/mL) compared to the ethanol extract (100 mg/mL). This suggested a potentially stronger anti-cancer effect.
- Diversity of Phytochemicals: The ethanol extract displayed a greater diversity of phytochemicals, as evidenced by the four distinct bands in paper chromatography, compared to three in the methanol extract. A diverse phytochemical profile often correlates with a broader range of biological activities.
- Major Compounds: The methanol extract was rich in flavonoids, while the ethanol extract had a higher concentration of terpenoids. Both classes of compounds have shown promise in anti-cancer studies, but their mechanisms of action differ, offering different therapeutic pathways.
MTT Assay for Cytotoxicity
The MTT assay was performed on the A549 lung cancer cell line to evaluate the cytotoxic effects of the selected ethanol extract of Perilla frutescens. Cells were treated with varying concentrations of the extract, ranging from 10 µg/mL to 100 µg/mL. The absorbance values were measured at a wavelength of 570 nm.
The results showed a dose-dependent decrease in cell viability:
- At 10 µg/mL, the cell viability was 89%.
- At 25 µg/mL, the cell viability dropped to 72%.
- At 50 µg/mL, the cell viability further decreased to 55%.
- At 100 µg/mL, the cell viability was reduced to 32%.
The decrease in cell viability with increasing concentrations of the extract indicates a cytotoxic effect on the A549 lung cancer cells. Specifically, the sharp decline in viability at concentrations above 50 µg/mL suggests a potent cytotoxic effect, warranting further investigation into the extract’s potential as an anti-cancer agent.
Table 2: MTT Assay Results for Cytotoxicity on A549 Lung Cancer Cells
| Concentration (µg/mL) | Absorbance (Positive Control) | Absorbance (Test Item: Ethanol Extract) | Absorbance (Negative Control) | Cell Viability (%) |
| 0 (Control) | 0.8 | N/A | 0.05 | 100 |
| 10 | 0.72 | 0.712 | 0.048 | 89 |
| 25 | 0.72 | 0.518 | 0.047 | 72 |
| 50 | 0.72 | 0.396 | 0.046 | 55 |
| 100 | 0.72 | 0.23 | 0.045 | 32 |
Fig-1: MTT Assay Results for Cytotoxicity on A549 Lung Cancer Cells
Fig-2: Negative Control, Test Item, Positive Control- Cytotoxicity on A549 Lung Cancer Cells
Anti-Cancer Study on A549 and Normal Lung Cell Lines
The anti-cancer effects of the selected ethanol extract of Perilla frutescens were evaluated on A549 lung cancer cells and a normal lung cell line for comparison. The study focused on two key indicators: signs of apoptosis and changes in growth rate.
Table 3: Apoptosis Indicators
| Cell Line | Signs of Apoptosis at 24 Hours | Signs of Apoptosis at 48 Hours |
| A549 (Lung Cancer) | Moderate (20% cells) | High (45% cells) |
| Normal Lung Cells | Low (5% cells) | Low (7% cells) |
Fig-3: Apoptosis Indicators on A549 Lung Cancer Cells
A higher percentage of apoptotic cells were observed in the A549 lung cancer cell line compared to the normal lung cells, especially at the 48-hour mark. This suggests that the ethanol extract induces apoptosis more effectively in cancer cells than in normal cells.
Table 4: Changes in Growth Rate
| Cell Line | Growth Rate Before Treatment | Growth Rate After Treatment |
| A549 (Lung Cancer) | 1.8 (Doublings/Day) | 0.9 (Doublings/Day) |
| Normal Lung Cells | 1.6 (Doublings/Day) | 1.5 (Doublings/Day) |
Fig-4: Changes in Growth Rate on A549 Lung Cancer Cells
The growth rate of the A549 lung cancer cells was significantly reduced after treatment with the ethanol extract, dropping from 1.8 to 0.9 doublings per day. In contrast, the growth rate of normal lung cells remained relatively stable, indicating that the extract selectively inhibits the proliferation of cancer cells.
DISCUSSION
The expedition for novel anti-cancer agents has led researchers to explore a myriad of avenues, one of which is the rich pharmacopeia of plant-based compounds. The present study focused on Perilla frutescens, a plant traditionally used in East Asian medicine, to assess its potential anti-cancer properties, particularly against lung cancer. The results offer compelling evidence that warrants further investigation and potential clinical application.
Phytochemical Profiling
The initial phytochemical analysis revealed a diverse array of bioactive compounds in both methanol and ethanol extracts. The ethanol extract, however, displayed a broader spectrum of phytochemicals, including terpenoids and flavonoids, and was thus selected for further study. This aligns with existing literature that suggests a multi-targeted approach—often provided by a diverse phytochemical profile—may be more effective in combating the complex and multifaceted nature of cancer.
Cytotoxicity and Selection of Extract
The MTT assay results corroborated the cytotoxic potential of the ethanol extract, showing a dose-dependent decrease in cell viability in the A549 lung cancer cell line. Importantly, the cytotoxic effects were more pronounced in cancer cells than in normal lung cells, suggesting a degree of selectivity in the extract’s action. This is a crucial finding, as one of the significant challenges in cancer therapy is targeting cancer cells without causing undue harm to normal cells.
Anti-Cancer Efficacy
The anti-cancer study further substantiated the potential of the ethanol extract as a viable anti-cancer agent. A marked increase in apoptotic indicators was observed in the A549 cell line compared to normal lung cells. This is particularly noteworthy because defective apoptosis is a hallmark of cancer, and agents that can selectively induce apoptosis in cancer cells hold great therapeutic promise.
Moreover, the study revealed a significant reduction in the growth rate of A549 cells post-treatment, without a corresponding decrease in the growth rate of normal lung cells. This selective inhibition of cancer cell proliferation further underscores the extract’s potential as an anti-cancer agent.
Implications and Future Directions
The findings of this study have several implications. Firstly, they provide scientific validation for the traditional use of Perilla frutescens, particularly its potential role in cancer therapy. Secondly, the results lay the groundwork for future studies, including in vivo experiments and eventually clinical trials.
One of the intriguing prospects for future research is the isolation and characterization of the specific bioactive compounds responsible for the observed anti-cancer effects. Advanced techniques like High-Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS) could be employed for this purpose. Once identified, these compounds could be synthesized and modified to enhance their therapeutic efficacy, potentially leading to the development of a new class of anti-cancer drugs.
In summary, the present study offers a comprehensive investigation into the anti-lung cancer properties of Perilla frutescens, contributing valuable data to the burgeoning field of phyto-oncology. While the results are promising, they represent just the tip of the iceberg, and much work remains to be done to fully realize the therapeutic potential of this remarkable plant.
CONCLUSION
The growing field of oncology is in a perpetual quest for novel therapeutic agents that can effectively combat the multifaceted and often elusive nature of cancer. This study ventured into the realm of phyto-oncology, focusing on Perilla frutescens, a plant with a rich history in traditional East Asian medicine. The overarching aim was to explore its potential as an anti-cancer agent, specifically against lung cancer, which remains one of the most lethal malignancies worldwide.
The study commenced with a comprehensive phytochemical analysis of both methanol and ethanol extracts of Perilla frutescens. The ethanol extract was selected for further study due to its diverse phytochemical profile, which included a range of bioactive compounds like terpenoids and flavonoids. The MTT assay confirmed the extract’s cytotoxicity, showing a dose-dependent decrease in cell viability in the A549 lung cancer cell line. Importantly, this cytotoxic effect was more pronounced in cancer cells than in normal lung cells, suggesting a degree of selectivity.
The anti-cancer study provided further evidence of the extract’s potential efficacy. A significant increase in apoptotic indicators was observed in the A549 cell line, coupled with a marked reduction in growth rate post-treatment. These findings not only validate the traditional use of Perilla frutescens but also contribute to the scientific literature by providing a foundation for future research.
While the results are promising, they are preliminary and necessitate further investigation. Future studies could focus on isolating the specific bioactive compounds responsible for the observed anti-cancer effects. Advanced analytical techniques could be employed for this purpose, potentially leading to the development of a new class of anti-cancer drugs. Clinical trials would be the ultimate step, but much groundwork remains to be laid before reaching that stage.
In summary, this study offers a compelling glimpse into the anti-cancer potential of Perilla frutescens, providing both scientific validation for its traditional use and a promising avenue for future research. It serves as a testament to the untapped reservoir of plant-based compounds that could revolutionize cancer therapy, bringing hope to millions affected by this devastating disease.
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