Introduction

Oxidative stress is a concern in between free radicals and cancer prevention agents. More radicals present in our body can destroy because cancer prevention agents, the free radicals then start to harming fatty tissue, DNA and proteins. This results in numerous diseases and disorders such as aging, cancer, atherosclerosis, cirrhosis ad cataracts (El-Baroty, 2010). Though the synthetic antioxidants are in-expensive compared to natural anti-oxidants, their usage has shown to have several side effects, including their deposition in adipose tissues while the natural anti-oxidants could be completely metabolized (Ramana, 2018). This has paved the way for may researches focusing on the secondary metabolites of medicinal plant's majority essential oils as novel anti-oxidants source. On the other hand, anti-fungal treatment is a focal segment of patient administration for acute and chronic mycoses. However, treatment decisions are limited due to the meagre number of anti-fungal medication classes. Clinical administration of fungal illnesses is additionally undermined by the rise of anti-fungal drug resistance, which disposes of accessible medication classes as treatment alternatives. When thought about an uncommon event, against anti-fungal medication, the opposition is on the ascent in some high-hazard clinical focuses. Most concerning is the advancement of multidrug resistance hard-headed to a few unique classes of against anti-fungal agents, particularly among common Candida species and dandruff-causing fungi - Malassezia species. Among the bioactive compounds containing medicinal plants, we found an essential oil which exhibited various bio-activities such as anti-microbial, anti-oxidant, anti-tumor or an anti-inflammatory effect.

As per research survey, these effects are certainly due to the presence of terpenoids bio-active compounds in essential oils (Christaki, 2012). Coleus zeylanicus is considered as aromatic therapeutic application plant that belongs to the Lamiaceae family. This plant contains several medicinally important components such as alkaloids, volatile compounds, sesquiterpene and flavonoids. Most of these compounds has been used for the anti-hepatotoxic, anti-ulcerogenic, anti-inflammatory, appetizer, digestive, stomachic, liver tonic, cardiotonic (Duke, 2002; Rajkumar & Malathi, 2016). Recent pharmacological investigation of the leaves and root extract of genus Coleus revealed its immunomodulator, antimicrobial, anticancer, anti inflammatory, antioxidant, antiallergic property (Ramalakshmi, 2014; Saraswathy & Lavanya, 2013).

The literature survey reveals that Coleus species have much pharmacological importance. It is considered as best medicine against urinary tract infection and also to increase the digestion. An opportunistic contagious microbe Candida albicans is answerable for candidiasis in human hosts. C. albicans can grow in a few distinctive morphological, structural features with cellular equal side divider (Nithya, 2013). Similarly, dandruff is a scalp disorder which can be easily treated with specialized shampoos. There is, however, no true cure (Turkington & Dover, 2007). The current system for treating dandruff with against anti-dandruff items and most are synthetic compounds, excessively rough for everyday use. Other than the chemical constituents, there is a wide scope of home-grown fixings like pepper remove, basil extricate, neem extract, rosemary oil, basil oil, clove oil, coleus oil, tea tree oil which have been reported to have great anti-dandruff activity (Nithya, 2013).

Homegrown herbal medication, presently as days are picking up significance for treating numerous infections because of their huge impact and lesser side effects when contrasted with allopathic meds. The essential oil of Coleus species is not been largely studied. This study proposed to determine the chemical composition of Coleus zeylanicus essential oil and evaluate their anti-fungal and anti-oxidant property. To our knowledge, there are no reports available on this plant. Hence the current study is designed to investigate and to profile the chemical composition of Coleus zeylanicus essential oil and evaluate the anti-fungal and anti-oxidant properties.

Materials and Methods

Sampling and Essential oil extraction

The leaves of Coleus zeylanicus were collected and the samples were air-dried at room temperature. The extraction of essential oil was obtained by hydro distillation method using Clevenger type apparatus. The obtained oil was dried, weighed and stored at 4˚C for further use. The oil obtained was strongly aromatic, pale yellowish in colour and liquid at room temperature. The extracted oil had a pleasant smell of the plant.

Fourier Transform Infrared Spectroscopy (FT-IR) analysis

The FT-IR spectrum of essential oil sample was profiled. The IR spectra were set up in % transmittance. The wave number region was 4000-400 cm^-1 for scanning the functional group's presence in the extract (Adinew, 2014).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The volatile components of Coleus zeylanicus essential oil were analyzed by GC-MS. Compounds were identified by their retention index were compared with standards. The unknown compounds spectrum in essential oil was compared with the spectrum of the known components reported in the Wiley and NIST Library (Adams, 2007; McLafferty & Stauffer, 1989). The atlas of spectral data of sesquiterpene hydrocarbons was also used for reference for terpenoids and related compounds (Joulain, 1998; Joulain, 2001).

Antioxidant activity

DPPH is a method for the determination of free radical scavenging reaction of C. zeylanicus essential oil. DPPH (200 µm) was prepared in methanol solution, 0.05ml of test samples were added at 20, 40, 100, 125 and 250 µg/ml in water and to that equal amount of water was added and served as a control.

After then, 20 minutes of incubation, the samples were read at 517nm. The percentage of inhibition was predicted by using the standard formula:

DPPH scavenging activity(%)=[(A_c-A_t)/A_c]x100

A_c is the absorbance of the control (without oil), and A_t is the absorbance of the sample. Butylated hydroxytoluene (BHT) was used as a positive control and EO concentration was calculated by plotting the inhibition degrees against the sample concentrations and shown 50% inhibition (IC₅₀).

Anti-fungal activity

Fungal strains

The anti-fungal effects of Coleus zeylanicus essential oil were evaluated against Candida albicans (MTCC 227) and Malassezia furfur (MTCC 1374). The culture was retrieved from the Microbial Type Culture Collection, Chandigarh, India.

Strains were stored in agar slant at 4˚C before use the fungal cultures were retrieved by two sub-cultures in an appropriate medium, Potato dextrose agar at 30˚C for 18 – 24 hrs.

For the test final inoculum of 10⁶ CFU/ml fungi were used.

In vitro antifungal activity

The antifungal activity was studied by agar-well diffusion methods (Holder & Boyce, 1994). The essential oil sample was loaded on to each well with various concentrations (10 ml, 20 ml, 30 ml and 40 ml). The clotrimazole added well was taken as a positive control.

All test samples were incubated at 30˚C for 24-48 hours. After incubation, the anti-fungal activity was studied by measuring the zone of inhibition measured in millimetres. All the fungal growth inhibition assays were performed in triplicates.

Statistical analysis

The percentage of response, anti-fungal activity indicated by a zone of growth inhibition was taken as growth parameters. According to New Duncan’s Multiple Range Test, three independent experiments data were represented by triplicates from each experiment and they were subjected to statistical analysis (mean ± SE) (Gomez & Gomez, 1984).

Results and Discussion

Determination of the functional groups presents using FT-IR.

FT-IR analysis is commonly used for the identification of functional groups. The sample spectrum graph retrieved after FTIR analysis were used to identify the functional groups Figure 1 and Table 1.

GC-MS analysis

GC-MS analysis of the leaf essential oil resulted in a total of 14 volatile organic compounds Figure 2. Among the identified compounds, the highest percentage of a compound is α-Gurjunene (62.91%), followed by α-Bisabolol (10.82%), γ-Selinene (4.26%) and α-Cadinol (4.17%) Table 2.

Antioxidant activity

To evaluate the antioxidant effects of Coleus zeylanicus EO, DPPH scavenging assay was used and the results of the screening are shown in figure as comparable Figure 3 with known antioxidant BHT was expressed as IC₅₀ values. Active ingredients can be detected at low concentration effectively by DPPH scavenging assay.

Due to scavenging of radical by hydrogen donor, there is a decrease in absorbance of DPPH radical caused by antioxidants. A color change from purple to yellow indicates the scavenging activity. Aqueous phase extract of Coleus zeylanicus showed scavenging activity in a concentration-dependent manner, with the correlation coefficient values [r] of 0.758 and IC50 value of 59.78 µg/ml Table 3 and Figure 4. The antioxidant activity of the essential oil exhibited a high degree of activity of more than 50%. The activity of the raw extracts was comparatively higher than that of commercial antioxidant BHT, the reference antioxidant Table 4 and Figure 5.

Anti-fungal activity

The anti-fungal activity of Coleus zeylanicus EO was evaluated against two pathogenic fungal strains using agar well diffusion assay. The obtained results were expressed in terms of the zone of inhibition developed around the wells Table 5. EO of Coleus zeylanicus showed a higher antifungal effect against Malassezia furfur than Candida albicans with remarkable inhibition of 32.00±0.50 mm and 15.00±1.25 mm respectively. The effects were higher than those obtained with the positive control Clotrimazole. Clotrimazole was selected as a positive control. Values mentioned the results obtained from mean±SD for triplicate experiments. Diameter of inhibition zone included a well diameter of 6 mm, by the agar well diffusion method at different concentrations of oil/ well. Final fungal density was around 10⁶ CFU/ml. Essentials oils have a function of significance for many years as parts of herbal medicines for an assortment of ailments in humans and animals to cure. There are many confirmations of plant-derived aromatic compounds, being utilized by practically all old civic establishments (Chang & Cheng, 2002).

Essential oils have been evidences to possess analgesic, anesthetic, ant acne, antidiarrheal, antineuralgic, antiseptic, antispasmodic, Central Nervous System stimulant, anti-cancer, cosmetic, biopesticide, anti asthmatic, anti-bacterial, anti-inflammatory, sedative and its pleasant odor is used in perfume (Edris, 2007; Saraswathy et al., 2013). The application of essential oil for the treatment of various infectious ailments and its prevention has been gained more views within the research field over the many decades (Selvakumar, 2012). Coleus is an ornamental plant with many medicinal applications and therapeutic properties. It is a major compound in medicinal plants biodiversity. It has been investigated to study colic, heart failure, hypertension, insomnia, convulsions, eczema, painful urination and respiratory disorders (Khan, 2012). FT-IR spectroscopy is a very effective method for categorizing the class of compounds of functional groups identification in it. The essential oil components of Coleus zeylanicus was fractioned and reported in this research study for the first time. The GC-MS analysis revealed the volatile constituents’ presence in the essential oil of the plant leaf.

Out of the 14 volatile compounds analyzed, the activity of the essential oil can be associated with the presence of a-gurjunene (62.91%), a-bisabolol (10.82%), γ-selinene (4.26%), cadinol (4.17%) and lemonol (3.46%). The a-gurjunene which is of importance in the perfume industry is reported in a lesser % in essential oil in aerial parts of Anaphalis nubigena DC. var. monocephala (DC.) (6.0%) (Pande, 2008).

Thus, it is comparatively 10 times greater in our Coleus zeylanicus plant. a-Bisabolol is known for anti-inflammatory, anti-irritant and anti-microbial properties. This compound is also reported to have the percutaneous absorption of specific molecules. a-Bisabolol is reported to be in a concentration of 7% in Coleus vettiveroides (Saraswathy et al., 2013) while it is found in a higher concentration (10.87%) in our Coleus zeylanicus plant. The increased concentration of a- isabolol can be exploited medicinally, as a liver and glandular stimulant and anti-allergen (Cavalieri, 2011; Kamatou & Viljoen, 2010). Similar GC-MS analysis of essential oil has been reported in different species of Coleus plant and was found to exhibit antimicrobial activity. One such species of Coleus plant, Coleus aromaticus shows thymol (41.3), carvacrol (13.3), 1, 8- cineole (5.5), eugenol (4.4), β-caryophyllene (4.2) which shows good anti-inflammatory activity and low anti-microbial activity (Khare, 2011).

Coleus amboinicus (Lour.) another aromatic shrub with ethno botanical applications was analyzed by GC-FID and GC-MS. 15 components were identified by GC-MS and carvacrol (65.2%) was found to be the major constituent. The essential oil showed antibacterial activity against important enteric pathogens (Salmonella sp., Shigella sp., Escherichia coli and Vibrio sp.) (Velasco, 2009).

better source for the manufacturer of perfumery derived products and as a constituent in anti-fungal agents. This research work on a phytochemical analysis of Coleus zeylanicus and its anti-dandruff activity is firs (Singh, 2011) of its kind. The phytochemical components identified belong to sesquiterpenes which are characterized by their strong odour and are thu (Thoppil & Jose, 1995) efficient antimicrobial and anti-inflammatory agents. The essential oil showed an inhibitory zone of 23 mm and proved it as an effective antifungal agent. However, the anti-dandruff activity of the essential oil was already reported and found to have a similar response against Malassezia furfur studied using Coleus amboinicus (Selvakumar, 2012).

The evaluation of antioxidant activity revealed that the IC50 values obtained with the DPPH activity were lower than those obtained with other species of Coleus where the antioxidant activity of it is reported. The oxygenated monoterpenes found in this essential oil may act as anti-oxidant agents. The anti-radical activity of Coleus zeylanicus EO using DPPH assay (IC50 = 59.78 µg/ml) was comparatively higher than that of synthetic antioxidant BHT (IC50 = 39.86 µg/ml). The anti-oxidant properties of essential oil of Coleus zeylanicus are due to the phenolic compounds present in the essential oil which are able to neutralize the free radicals via different mechanisms. Anti-oxidant activity of leaf of Coleus blumei was found to be lowest with percentage scavenging (IC50 = 0.261 mg/ml) which is comparatively lower than the DPPH scavenging activity of Coleus zeylanicus. Controversially another species of genus Coleus - Coleus aromaticus has been reported to have a greater IC50 value of 97.78 µg/ml which is higher than that shown Coleus zeylanicus. The main purpose of the anti-oxidant study is to reveal free radicals scavenging property. Anti-oxidant compounds scavenge free radicals such as peroxide, hydrogen peroxide and thus inhibit the oxidation mechanism that leads to cell damage and degenerative ailments (Rout & Mishra, 2012).

Conclusions

In this study, the analysis of the chemical component fractioned showed various components that contribute to the aroma of the plant and also proving it as a potent medicinal plant. The antioxidant effects and anti-fungal property shown by Coleus zeylanicus leaf essential oil approves the usage of the essential oil in food and drug industry. However, the future research of the work could be to purify and isolate the compound that is responsible for the inhibition of dandruff-causing organism and develop it into a potential herbal product.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that they have no conflict of interest for this study.