Research Articles

2023  |  Vol: 8(5)  |  Issue: 5(September-October) | https://doi.org/10.31024/apj.2023.8.5.4

Evaluation of Cytotoxic, Antibacterial and Antioxidant activities of leaf extract of Coleus ambionicus

Amisha Murugesan Angel, W. A. Manjusha*, Briscilla C.

Department of Biotechnology, Malankara Catholic College Mariagiri, Affiliated to Manonmaniam Sundaranar University, Tirunelveli, 627012, Tamil Nadu, India

*Address for Corresponding author

Dr. W. A. Manjusha

Head and Assistant Professor,

Department of Biotechnology, Malankara Catholic College Mariagiri, Tamil Nadu, India

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Abstract

Objective: Coleus ambionicus has therapeutic properties attributed to its natural bio active compounds which are highly valued in the pharmacological industry. The present study evaluated the antibacterial, antioxidant & anticancer activity of coleus ambionicus leaf extract. Material and methods: The organic solvents such as ethanol, methanol, ethyl acetate and chloroform were used for the extraction of bioactive compounds. The antibacterial activity was screened by disc diffusion method and antioxidant activity was determined by reducing power method. Results and conclusion: The ethanol extract showed high antibacterial activity against Klebsiella pneumonia and the same extract was showed highest antioxidant activity. So, the ethanol extract of Coleus amboinicus was subjected to anticancer activity against A549, the lung cancer cell line and it showed dose dependent inhibition of lung cancer cell line. For the identification of bioactive compounds, the ethanol extract was subjected to column chromatography and Gas Chromatography-Mass Spectroscopy (GC-MS) analysis. GC-MS analysis showed the presence of three bio active compounds viz Phenol 2-methyl-5-(1-methylethyl), 2,4-Di-tert-butylphenol and Bis(2-ethylhexyl) phthalate and these bioactive compounds are responsible for the pharmacological properties of Coleus amboinicus.

Keywords: Coleus amboinicus, pharmacological activity, bioactive compounds, GC-MS

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Introduction

Coleus amboinicus (Mexican mint) (Karpuravalli) is a large succulent aromatic perennial herbal well-known plant which belongs to the Lamiaceae family and it is found in almost all over India. This plant is effective in wound healing with very less side effects, so this herb is pretty impressive in this sense. Pharmacological uses of P. amboinicus gives impact effective in the treatment of famous diseases such as cephalgia, otalgia, anorexia, dyspepsia bloating, colic, diarrhea, cholera, gums, seizures, asthma, cough, chronic bronchitis, kidney calculi, vesicle calculi, hiccough, strangury, hepatopathy, fever, and malaria (Saraswati et al., 2016).

In this modern world, there are many commercial antibiotic drugs which act against microbial activity but indiscriminative for many infectious diseases and also cause several side effects on the host like immune suppression and allergenic reaction, so scientist searched for new and effective drugs from medicinal plants as alternatives (Uddin et al., 2021). Certainly, natural products are considered as an important key factor in medicinal sector and extensively used in pharmaceutical industries. This has been led towards the increased global demand for medicinal plants in the modern era of natural medicine, leading to exploration and exploitation of new plant sources for their medicinal properties (Swamy et al., 2015). The Lamiaceae members of plant species belonging to commercially important genera, such as Plectranthus, Salvia, Ocimum and Mentha, are attributed with a rich diversity of ethnobotanical benefits. This herb is most commonly used by indigenous people in tropical rain forests. This is mainly due to its natural production of an essential oil with high amounts of bioactive compounds such as Carvacrol (Castillo et al., 1999), Thymol (Singh et al., 2002)  β-Caryophyllene, α-Humulene, γ-Terpinene, p-Cymene, α-Terpineol and β-Selinene, identified in the oil component of its leaves (Senthilkumar et al., 2010). These biochemical components exhibit various biological properties (Bhatt et al., 2012) and are widely used as a medicine to treat conditions like cold, asthma, constipation, headache, cough, fever and skin diseases. Antibacterial agents can be further subdivided into bactericidal agents, which kill bacteria, and bacteriostatic agents, which slow down or stall bacterial growth. Antioxidants which occurs from the plants called natural antioxidant, which increases the antioxidants capacity and help to reduce the risk of certain disease such as cancer, heart disease and stroke (Prior et al., 2016). The secondary metabolic produced from plant are phenolic compounds and flavonoid, which are reported to be free radical scavenger (Zeeshan et al., 2022).

Cancer is a health problem of global concern (Graidist et al., 2015). Chemotherapy, the most common mode of intervention in cancer, is associated with adverse effects, from nausea to bone marrow failure. In addition, development of drug resistance is also a concern (Raguz and Yague, 2008). Therefore, the search for effective therapeutics for cancer is an ongoing process (Fadeyi et al., 2013). medicines such as Ayurveda are playing an important role in the treatment of cancer. Hence isolation of bioactive products from medicinal plants and herbs is gaining importance too. Eleven percent of the basic drugs are considered as essential by World Health Organization (WHO), which are plant derived and modified  products.

The main objective of the present study was to analyze the phytochemicals present in coleus amboinicus plant extract and to investigate its pharmacological activities such as antibacterial, antioxidant and anticancer activity against lung cancer cell line. To identify the bioactive compounds responsible for the anticancer activity is analyzed by GC-MS.

Materials and Methods

Collection of plant sample

Fresh Coleus amboinicus (Karpuravalli) leaves were collected from Parthivapuram, Kanyakumari district, India. The leaves were cleaned with running tap water and  shade dried for one month to get dry sample and directly subjected to  solvent extraction.

Preparation of plant leaf extract

The plant extract was prepared based on the method followed by Khalel Nabih et al. (2014).  The prepared dry leaf sample was finely grounded into powder and 8 grams of dried leaf powder and 30 grams of fresh leaf paste was extracted using 100ml of solvents such as ethanol, methanol, ethyl acetate and chloroform and stored in dark place at room temperature for one week. Then it was filtered and stored at room temperature for further use (Nikhal et al., 2011).

Screening of antibacterial activity of leaf extract

Antibacterial activity of Coleus amboinicus leaf extract was screened based on the disc diffusion method (Kirby-Bauer Method). The pathogenic bacterial test organisms was obtained from Microbial Type culture Collection (MTCC), Institute of Microbial technology, Chandigarh. The bacterial strains used are Klebsiella pneumoniae (MTCC 4030), Escherichia coli (MTCC 40), Enterobacter aerogenes (MTCC 3906), Streptococcus pyogenes (MTCC 928) and Pseudomonas aeruginosa (MTCC 741). These bacterial cultures were sub cultured in nutrient agar medium. The Whatman filter paper disc of uniform size (6mm) was impregnated with the plant extracts was seeded on nutrient agar plates cultured with test organisms. Then the plates were incubated at 37°C for 24 hours (Bauer et al., 1966).

Screening of Antioxidant activity

The evaluation of antioxidant activity for different plant extracts was done by reducing power method. The plant extract (diluted in 1ml of distilled water) of different concentration (25ul, 50ul, 100ul and 200ul) was mixed with 1.5ml of 0.2M sodium phosphate buffer (pH 6.6) and 1.5ml of potassium ferric cyanide (1%) and the reaction mixture was incubated at 50°C for 20 minutes. After incubation, 1.5 ml of tri chloro acetic acid (TCA) was added to the reaction mixture and then centrifuged at 5000 rpm for 10 minutes at 27°C. Then  1ml of supernatant obtained was treated with 100ul of 0.1% ferric chloride and 400ul of distilled water and the absorbance was measured at 700nm (Abubakar et al., 2020).

Isolation and purification of bioactive compounds

The fresh and dry leaf ethanol extract of Coleus amboinicus which showed higher activity in antibacterial and antioxidant activity was subjected to Column chromatography. The silica gel column was prepared with Chloroform and methanol in the ratio of 99:1. The extract of 10ml was chromatographed over silica gel column (100-200 mesh) using ethanol as solvent with increased polarity. The eluents were collected and subjected to antibacterial activity against Klebsiella pneumonia. The eluent which showed higher activity was subjected to further analysis (Gini et al., 2018). The collected eluents were subjected to antibacterial activity against Klebsilla pneumonia & the highly active eluent was utilized for further analysis.

Screening of anticancer activity against lung cancer

The Human lung cancer cell line (A549) was procured from (NCCS), Pune, India and maintained in Dulbecco’s modified Eagles medium, DMEM. The cultured cell lines was incubated at 37ºC in a humidified 5% CO₂ incubator (NBS Eppendorf, Germany). The viability of cells were evaluated by direct observation by Inverted phase contrast microscope and followed by MTT assay. Then the two days old confluent monolayer of cells were trypsinized and suspended in 10% growth medium and then 100µl of cell suspension (5x10³ cells/well) was seeded in 96 well tissue culture plate and incubated at 37ºC in a humidified 5% CO₂ incubator (Raj et al., 2023).

For the preparation of sample extract 1ml of highly active eluent was dissolved in 1mL DMEM using a cyclomixer. The sample solution was filtered through 0.22 µm Millipore syringe filter to ensure the sterility. Then the entire plate was observed after 24 hours of treatment in an inverted phase contrast tissue culture microscope (Olympus CKX41 with Optika Pro5 CCD camera) and microscopic observation was recorded as images.  After 24 hours the growth medium was removed and freshly prepared sample in DMEM were five times serially diluted by two fold dilution (100µg, 50µg, 25µg, 12.5µg, 6.25µg in 500µl of DMEM) and each concentration of 100µl are added in triplicates to the respective wells and incubated at 37ºC in a humidified 5% CO₂ incubator. Untreated cell was maintained as control.

Anticancer Assay by MTT Method:

After 24 hours of incubation period, the sample content in wells were removed and 30µl of reconstituted MTT solution (15mg MTT in 3ml PBS) was added to all test and control wells. The plates were gently shaken well, then incubated at 37ºC in a humidified 5% CO₂ incubator for 4 hours. After the incubation period, the supernatant was removed and 100µl of MTT solubilization Solution (Dimethyl sulphoxide, DMSO, Sigma Aldrich, USA) was added and the wells were mixed to solubilize the formazan crystals. The absorbance values were measured by microplate reader at a wavelength of 540 nm (Thomas et al., 2017).

The percentage of growth inhibition was calculated using the formula:

Identification of bioactive compound by GC-MS analysis

The GC–MS analysis of ethanol extract (highly active eluents) of Coleus amboinicus was carried out using GC–MS QP2010 Ultra (Shimadzu) with Rxi-5Sil MS fused-silica capillary column of 30 m length, 0.25 mm diameter, and 0.25 mm film thickness. An electron ionization system with ionization energy of 70 eV was used for analysis. Helium gas (99.99%) was used as the carrier gas at the constant flow-rate of 8.9 ml/min. The temperatures of the injector and mass transfer line were set at 250°C and 240°C, respectively. The oven temperature was programmed from 80°C to 200°C at 3°C/min, and finally increased to 260°C at 10°C/min. Aliquots of 1 ml of the diluted samples were injected in split mode with a split ratio of 1:10 and with a mass scan range of 45–900 AMU. The total running time of the GC–MS analysis was 51 min.

Results

Antibacterial activity of C. amboinicus leaf extract

In dry leaf extract, the ethanol extract shows highest activity against Streptococcus pyogenes (2.13±0.094) and in fresh leaf extract, the ethanol extract shows highest activity against Klebsiella pneumoniae (2.26±0.057) and Streptococcus pyogenes (2.13±0.05) moderate activity was noticed in other tested pathogens. The antibacterial activity of both leaf extract showed that the ethanol extract is the best one for the antibacterial activity.

Table 1: Antibacterial activity of dry and fresh leaf extract

Pathogenic bacteria	Dry leaf				Fresh leaf
	Ethanol	methanol	Ethyl acetate	Chloroform	Ethanol	methanol	Ethyl acetate	Chloroform
Klebsiella pneumoniae	1.73±0.205	0.03±0.047	0.3±0.081	0.36±0.047	2.26±0.057	1.367±0.047	1.57±0.05	-
Escherichia coli	-	0.23±0.047	0.67±0.047	0.36±0.047	1.5±0.1	0.967±0.047	2.33±1.1	0.97±0.5
Pseudomonas aeruginosa	1.1±141	0.63±0.047	0.03±0.047	0.36±0.047	-	0.63±0.047	0.97±0.05	-
Streptococcus pyogenes	2.13±0.094	1.1±0.141	-	0.16±0.047	2.13±0.05	1.13±0.047	0.97±0.05	-
Enterobacter aerogenes	1.567±0.047	0.367±0.047	-	0.16±0.047	0.6±0.057	1.167±0.047	0.37±0.05	0.37±0.11

Antioxidant activity of C. amboinicus leaf extract

The antioxidant activity of different extracts of C. amboinicus was measured by reducing power assay method. This method used to compare the anti-oxidant activity of dry and fresh extracts (Ethanol, Methanol, Ethyl acetate, Chloroform). In dry leaf extract, ethanol extract exhibited highest reducing power ability of 62.3±0.27 at a concentration of 200µg/ml and the lowest activity was noticed as 10.61±0.30 at 25 µg concentration. All other extract showed moderate antioxidant activity and  in fresh  leaf extracts ethanol extract exhibited highest reducing power ability of 49.9±0.18 at a concentration of 200µg/ml and  lowest activity was noticed as 10.61±0.30 at 25 µg concentration. All other extract showed moderate antioxidant activity. The extracts showed good reducing power ability in a dose dependent manner. When compared with fresh leaf extract, the dry leaf ethanol extract showed highest anti-oxidant activity. The extracts showed good reducing power ability in a dose dependent manner showed in table 2. Reducing power of the extracts increases with the increase in concentration The result suggests that the ethanol extract possess antioxidants which can be used in pharmaceutical industry.

Table   2. Antioxidant activity of different extracts by reducing power method for dry leaf and fresh leaf samples

Conc. µg/ml	Ascorbic acid	Dry leaf				Fresh leaf
		Ethanol	Methanol	Ethyl acetate	chloroform	Ethanol	methanol	Ethyl acetate	Chloroform
25	19.53±0.29	16.6±0.27	11.33±0.21	19.51±0.43	13.33±0.34	19.5±0.43	12.32±0.49	12.09±0.41	14.24±0.11
50	37.4±0.18	28.8±0.16	14.24±0.11	12.32±0.49	10.61±0.30	24.4±0.18	16.39±0.06	10.61±0.30	12.09±0.41
100	88.8±0.13	61.1±0.14	16.39±0.06	10.60±0.30	12.09±0.41	36.15±0.17	16.6±0.27	16.39±0.06	13.33±0.34
200	97.13±0.17	62.3±0.27	13.59±0.36	15.31±0.32	16.39±0.06	49.9±0.18	15.31±0.32	14.24±0.11	11.33±0.21

Purification of C. amboinicus leaf extract by Column chromatography

Column Chromatography was performed for the purification of bioactive compound both in dry and fresh leaf extract. Totally 8 fractions were collected from both  dry and fresh leaf extract with an increased polarity of solvents. The collected fractions were subjected to antibacterial screening against Klebsiella pneumoniae for the selection of best fraction and the concentration of inhibition (cm) of dry and fresh leaf extract of C. ambionicus was shown in table 3. The 4^th fraction of dry leaf extract showed the best antibacterial activity (2.1±2.1 cm) against Klebsiella pneumoniae so it was chosen for further analysis.

Table 3. Purification of C. amboinicus leaf extract by Column chromatography of ethanol dry and fresh leaf extract                                                          

Eluted fraction	Klebsiella pneumoniae (Zone of Inhibition)
Control	Ethanol Dry leaf extract	Ethanol Fresh leaf extract
1	-	-
2	1±1	0.2±0.02
3	1.1±0.06	0.5±0.5
4	2.1±.0.21	0.96±
5	1.6±0.06	0.6±0
6	1.3±0.21	0.2±0.02
7	0.5±0.5	0.1±0.01
8	-	-

Screening of Anticancer activity of C. amboinicus dry leaf extract

Anticancer activity of C. amboinicus dry leaf extract (4^th fraction) was done on Human Lung Cancer Cell line (A549) using MTT cell viability assay method. The anticancer result showed dose dependent inhibition of cancer cell line. The 80% of cell viability was noticed at 6.25 concentrations, 75% cell viability at 12.5 µg/ml concentrations, 69% cell viability at 25µg/ml concentration, 63% cell viability at 50 µg/ml concentrations and 55% cell viability at 100µlg/ml. The dry leaf extract of C. amboinicus showed a high activity against the lung cancer Cell line (A549) as it killed 45% of Lung cancer cell  at 100µg/ml concentration (figure 1).

Figure 1. Anticancer activity of dry leaf extract of C. amboinicus on Lung cancer cell lines A549

Analysis of bioactive compounds by GC-MS

GC-MS analysis of ethanol extract (4^th fraction) of C. amboinicus dry leaf extract showed three peaks which indicated the presence of three bioactive compounds (figure 2A). On comparison of the spectra with Pub Chem database, the identified compounds are identified such as Phenol,2-mehyl-5-(1-methyl ethyl) molecular weight 150 (figure 2B), 2,4-Di-tert-butylphenol (C₁₄H₂₂O) molecular weight of 206 (figure 2C), Bis(2-ethylhexyl) phthalate (C₂₄H₃₈O₄) molecular weight of 390 (figure 2D). Among these bioactive compounds the most prevailing compound was Bis (2-ethylhexyl) phthalate.

Figure 2. Analysis of bioactive compounds by GC-MS

Table 4. Characterization of bioactive compounds

Peak	R Time	Area	Area%	Name
1	9.721	2017999	44.78	Phenol, 2-methyl-5-(1-methylethyl)-
2	17.004	451715	10.02	2,4-Di-tert-butylphenol
3	43.640	2036918 4506632	45.20 100.00	Bis(2-ethylhexyl) phthalate

Discussion

Since from the prehistoric times, plants have been used for medical purposes⁵and used as a important ailments human therapeutic aid. Nowadays, due to the high  demand for the treatment of various disease increases,  herbal plants has been widely explored globally (Khandelwal et al., 2011). Antibacterial properties of Coleus amboinicus have been reported from different parts of the world. Bhatt et al. (2017) studied that the acetone extract of C. amboinicus leaves shows the effective antibacterial with least MIC values against the tested food born  bacteria namely Stapylococcus aures, Bacillus cereus, Escherichia coli and Yersinia entereolitica as the leaf acetone extract had the second highest polyphenolic content. Mallappa Kumara Swamy et.al. evaluated in their study that methanol extract shows highest antibacterial activity all the tested pathogen includes Bacillus subtilis, Methicillin- Staphylococcus aureus, Pseudomonas aeruginosa, escherichia coli and Candida albicans, but other two extract which include hexane and acetone failed to inhibit E. coli, S.aureus and C. albicans were more susceptible to all the extracts.

In this study, the dry and fresh leaf extract with different solvents including ethanol, methanol, ethyl acetate and chloroform were tested against different bacteria. The ethanolic extract of C. aromaticus shows high impact of antibacterial activity against E. coli, Bacillus sp, Pseudomonas sp, Staphylococcus and Klebsiella sp (Malini, 2013). Various kinds of solvent extract of C. aromaticus as well as its essential oils have demonstrated high antimicrobial activity on both Gram positive and Gram negative bacteria. It is also found to be quite effective against drug resistant microorganisms as well as the phytopathogenic microorganisms (Rashmi  et al., 2011). The extracted oil of C. aromaticus showed great antibacterial activity against S. aureus, E. coli, and K. pneumonia with mild activity shown against P. aeruginosa (Afaf Mohammed et al., 2011). Ethanolic extract has been proven to have impressive antibacterial activity against S. aureus and P. aeruginosa which increase the opportunities for C. aromaticus to act as an important source of herbal antibacterial agents (Amar et al., 2012). The ethanol extract of dry leaf shows highest activity compared to other solvents against Klebsiella pneumonia, Pseudomonas aeruginosa, Streptococcus pyogenes and Enterobacter aerogenes, however it failed to inhibit the activity of Escherichia coli. Similarly, in fresh leaf extracts, the ethanol extract of C. amboinicus shows highest activity against all the five different tested pathogens compared to other extracts. Bhatt et al. (2017) studies that the ethyl acetate extract of C. amboinicus leaves shows the highest appreciable DPPH radical scavenging and total antioxidant capacity as it had highest polyphenolic content compared to other extract obtained from solvent including hexane, acetone, methanol, hydro alcohol and freeze dried form. Patel et al. (2010) evaluated that the total phenolic compared in C. amboinicus contributes in anti-oxidant property. The reducing power of ethanolic and aqueous extracts and antioxidant potential increases with increasing concentration of extract. The most important biomolecules in the body get damaged due to the bio chemical reaction which produces reactive oxygen species which causes several disease conditions. The antioxidant prevents the harmful action of free radicals by blocking them and scavenging and it corrects their damaging effect on the cellular constituents. Rai et.al., (2016) investigates the cytotoxicity of two different plants fresh leaf extract with solvents including ethanol and chloroform on two breast cancer cell lines of different grads, MCF-7 and MDA-231 by MTT assay and reported that chloroform extracts of P. heyneanus and ethanol extract of C. amboinicus were effective against MCF-7 cells. Likewise, Rosidah et al., (2014) studied the cytotoxic activity of C. amboinicus extract using different solvents include n-hexane, ethyl acetate and ethanol against HeLa and Vera cell lines using MTT assay. Here n-hexane and ethanol extract were found selective to HeLa cells.

In this study, the anticancer activity of dry leaf ethanol extract of C. amboinicus was done on Human Lung cancer cell line (A549) using MTT cell viability assay method. The dry leaf ethanol extract from 8^th fraction of Column chromatographed sample which is used to kill the cells and it showed the highest against the cell line (A549) as it killed 45% of carcinoma cells with just 100µl/ml concentration of extracts. Gas chromatography Mass Spectrometry can be used to analyze the bioactive components presents in plant extract. Savina et al. (2014) evaluated the GCMS analysis revealed the presence of 11 chemical compounds. C. amboinicus dry leaf ethanol extract on GC-MS analysis shows three peaks which indicate the presence of three bio compounds namely Phenol,2-methyl-5-(1-mthyl(ethyl), 2,4-Di-butyl phenol and Bis (2-ethyl hexyl) phthalate. Thus it can be concluded that the biological activities of Coleus amboinicus of dry and fresh leaf extract can be utilized for the development of medicinal product for human and animal uses.

Conclusion

In conclusion, the present study demonstrates the bioactive compound identification and screening of pharmacological activities such as antimicrobial activity, antioxidant activity and anticancer activity of C. amboinicus. The ethanol extract of dry leaf possesses high antibacterial against Klebsiella pneumoniae and shows moderate activity against other pathogens. All other extracts also showed moderate activity against most of the pathogens. In antioxidant assay also ethanol extract exhibited highest reducing power ability in a dose dependent manner. The result suggests that the ethanol extract possess antioxidants which can be used in pharmaceutical industry. For the further studies from the ethanol extract the bioactive compounds were purified  and the eluents were screened to antibacterial activity of Klebsiella pneumoniae. The highest active fraction was subjected to screening of anticancer activity using Human Lung Cancer Cell line (A549). The anticancer result showed dose dependent inhibition of cancer cell line. The extract showed potent anti-n cancer activity against lung cancer cells. The result obtained from GC MS analysis indicated the existences of many valuable bioactive compounds that would be contributed to pharmacological properties of Coleus amboinicus. Coleus amboinicus (Lour) shows potential as chemotherapeutic agents for cancer and might become an ingredient for health-beneficial foods to prevent cancer.

Acknowledgment

Authors grateful to Malankara Catholic College, Kanyakumari, India

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