Underutilized Plant Cymbopogan martinii Derived Essential Oil Is Excellent Source of Bioactives with Diverse Biological Activities

Abstract

Cymbopogan martinii, also known as Palmarosa, is an underutilized plant of tropical region. Due to outstanding antioxidant potential it has been used as a part of conventional medicine and beauty product. Regardless of its importance, complete pharmacological and phytochemical studies are still in its early stages. In the current study, Palmarosa essential oil (PRO) was extracted from Cymbopogan martinii and was evaluated for its phytochemicals, antimicrobial and antifungal, anti-inflammatory and anti-diabetic and protection from UV rays. Oil from fresh leaves was extracted and analysed for presence of phytochemicals (Tannin, Flavonoids, and Phenolics). Various antioxidant activities like DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulphonic acid), Nitric oxide radical, Hydroxyl radical, iron reducing, iron cheating activity were performed. Antibacterial, anti-inflammatory, Antidiabetic, membrane integrity assay, and UV-absorption assay was also performed. Antifungal activity against “Aspergillosis” and “Mucormycosis” causing fungal strains was also evaluated. High concentration of polyphenolics like Tannin, Flavonoid, phenolics were revealed through phytochemical analysis. GC-FID revealed the presence of Geraniol, major component of Palmarosa oil and other bioactive compound in PRO. PRO showed high anti-inflammatory and anti-diabetic potential and can be used as an Antidiabetic agent due to inhibitory effect on α-amylase activity. Further study revealed that PRO inhibits α-amylase in competitive manner. Hence from the results obtained it is confirmed that the PRO possesses considerable amount of bioactive compounds and can be used in pharmaceutical, food and cosmetic industries.

INTRODUCTION

Underutilized aromatic plants are used as source of medicine in the field of Herbal Biotechnology [1].Natural products from plants are rich source of compounds with molecular and biological diversity, which play a vital role in the process of discovery of drug and its development [2]. Since prehistoric times; plentiful cultivated and wild plants have been used as a therapeutic agent. Recently medicinal plants have gained a lot of importance in the field of cosmetic industry as a natural ingredient, despite being an herbal medicine [3]. Nowadays, scientist around the world are attracted towards the contribution of therapeutic plants in phytomedicine and its utilization [4]. Phenols, Tannins, Flavonoids, Saponin are major bioactive compounds present in the medicinal plants and are used in therapeutic drugs [5].

Essential oils are fragrant liquids which are extracted from aromatic plants [6]. Essential oils are highly condensed, hydrophobic, volatile and scented compounds. Essential oils can be extracted from various parts of plants such as: roots, flower, buds, twigs, leaves, seeds, bark, wood and fruit [7]. Essential oils are secondary metabolites of flora derived from shikimate pathway. Steroid metabolites and a big variety of terpenoids incorporate: terpenoids crucial oil, saponin and caretenoids from deoxyxylulose and mevalonote pathway [8]. Essential oils have common aroma and taste, and very own organic activity, and are extensively utilized in healthcare and aromatherapy. Moreover, several industries for example: insecticides and repellents, cosmetics, perfume and flavoring, over and above natural drinks use critical oils as a substrate [7].

PALMAROSA (Cymbopogan martinii) is a tall perennial grass which includes a candy smelling oil of rose like odour. It is a tropical plant and grows in heat humid regions and is used in perfumery, meals flavouring and medicinal pharmaceutical industry. Palmarosa EO has been broadly utilized in fragrance based treatment as a skin tonic due to its antibacterial also, antiviral properties and gentle mitigating quality. It has additionally been utilized in Ayurvedic medication for skin issues and to alleviate nerve pain [9]. Moreover it is a hardy crop that can be grown on numerous sorts of soil and climatic conditions. It can even tolerate drought and should be grown on marginal waste lands which include saline/sodic/alkaline soils. Geraniol is the major component of the PRO and found to have anticancer, anti-inflammatory, anti-oxidant, HMG-CoA reductase activity, Neuroprotectivity and Hepatoprotectivity [10]. By virtue of having sweet rose like aroma, PRO is used as insecticides, in cosmetics such as perfumes, deodorants, in aromatherapy, in culinary, and soaps. Literature studies reveal that the Cymbopogan Spp are used in the treatment of sore throat, has insecticidal, anti-protozoan, anticancer, anti-HIV, antiinflammatory and anti-diabetes effects, used as hypotensive agent for rheumatism. C. martinii has stood out for scientists of late in light of its natural properties, like antimicrobial, antigenotoxic and cancer prevention agent ones [9]. Cymbopogan martinii is an underlined plant crop and is not grown extensively by reason of appropriate research on its agro-economic, pharmacological and medicinal value. The mechanism of action of PRO is still in its early stages; however it has been reported for its use in traditional medicines. Hence, in the present study the objectives were: Essential oil extraction from Cymbopogan martinii, phytochemical determination, evaluation of Antifungal, Antibacterial, Anti-inflammatory and anti-diabetic activity.

MATERIALS AND METHODS

Extraction of Palmarosa Essential Oil

From CSIR-AROMA nursery fresh leaves of Cymbopogan martinii were collected from naturally growing plants. The palmarosa essential oil was extracted through steam distillation method as described by Sharma et al. [11].

UV-VIS, FT-IR and Fluorescence Analysis

The UV-VIS spectral examination of pure Palmarosa essential oil (PRO) extracted from Cymbopogan martinii was led by utilizing UV-VIS spectrophotometer (Labtronics) with slit width of 2 nm, involving 10 mm cell at room temperature in the scale of 200 to 400 nm. The FT-IR (Fourier Transfer Infra-Red) Spectroscopy was utilized for identification of various functional groups and their particular bioactive compounds present in PRO. About 10–20 μL of PRO was taken and placed on the sample cup of diffuse reflectance accessory and analyzed by means of FT-IR spectrophotometer (Perkin Elmer, USA spectrophotometer in the scale 4000–400 cm^–1. The fluorescence spectrum of PRO was recorded utilizing Perkin Elmer spectrophotometer (FL6500).

In Vitro Anti-Oxidant Examination

Since single Antioxidant activity can never confirms absolute antioxidant contents present in any sample. Consequently, the inspection of antioxidant contents in PRO were performed utilizing different Antioxidant assays. Tests were performed by following Sharma et al. [11].

Nitric (NO) Oxide Radical Scavenging Activity

To various volume of PRO (10–50 μL) 3 mL of SNP solution was added. The reaction mixture was then incubated at 25°C for 150 min. After incubation, 0.5 mL of Griess Reagent was added to the 0.5 mL of the above reaction mixture. The absorbance was noted at 546 nm against PBS as blank. SNP in 1X PBS (3 mL) was assumed as control. Ascorbic Acid was taken as standard/positive control. The examine was led in sets of three. The nitric oxide radical scavenging activity of PRO was articulated as far as %scavenging action by utilizing the accompanying formula: % Searching Activity = A₀ – A₁/A₁ × 100. While: A₀ = Absorbance of SNP in PBS. A₁ = Absorbance of test sample (PRO).

DPPH Radical Scavenging Activity

The free radical scavenging capacity of PRO was assessed using the DPPH test. Add 3 mL of DPPH solution to different volumes of PRO (10–50 μL). Incubate the reaction mixture for 1 hour at room temperature (25–30°C) protected from light. Absorbance was measured after incubation at 517 nm with 82% blank methanol. Take 3 mL of DPPH in methanol as a control. Ascorbic acid served as a standard/positive control. The test was performed in triplicates. The DPPH free radical scavenging activity for PRO is expressed as a percentage of the scavenging activity using the following formula: Scavenging Activity % = A₀ – A₁/A₀ × 100. Where, A₀ = absorbance of DPPH solution. A₁ = Absorbance in the presence of the sample to be tested.

ABTS Radical Scavenging Activity

Generation of ABTS cation. Mix 50 mL of ABTS (7 mM) with 50 ml of Potassium Persulfate (2.45 mM) and leave in the dark at room temperature for 15–16 h until the completion of the reaction and stable absorbance. Measure the absorbance of the generated stable ABTS+ and equilibrated to 0.7 (±0.05) with ethanol. To 3 mL of the generated ABTS radicals, various amounts of PRO (10–50 μl) were added. The absorbance was measured at 734 nm against ethanol as blank after incubation for 6 min at room temperature. Ascorbic acid was taken as positive control/standard. Pure ABTS radical solution was taken as control. The examine was held in triplicates. The ABTS radical scavenging activity of PRO was articulated in terms of % Scavenging activity by applying the following formula given: % Scavenging Activity = A₀ – A₁/A₀ × 100. Where, A₀ = Absorbance of the ABTS radical in the absence of test sample/positive control. A₁ = Absorbance in the presence of test sample (PRO)/standard (Ascorbic Acid).

Hydroxyl Radical Scavenging Activity

Generation of Hydroxyl radical. The reaction mixture for generation of hydroxyl radical consists of 1 mL of Phenanthroline (0.75 mM), 2 mL Sodium Phosphate Buffer (0.2 M), 1 mL of FeSO₄ (0.75 mM), and 1 mL of H₂O₂ (0.01%). To this reaction mixture add PRO in increasing volume (10–50 μL). Then incubate for 30 min at 37°C. After the completion of the incubation time, absorbance was recorded at 532 nm against de-ionized water as blank solution, while ascorbic acid was used as standard/positive control. The experiment was conducted in the set of three. The % age Hydroxyl Radical Scavenging Activity was calculated by using the given formula: Scavenging Activity (%) = (Abs of sample – Abs of blank)/(Abs of control – Abs of blank) × 100. Whereas; Abs of control = Absorbance of water in the reaction mixture in place of H₂O₂ and sample (PRO).

Iron Reducing Assay

The Iron Reducing Assay of PRO was determined by following the procedure of Obafemi et al. [12]. The reaction mixture consists of 1.25 mL of Sodium Phosphate Buffer (200 mM; 6.6 pH), 1.25 mL of Potassium Ferricyanide. Then PRO was added in increasing volume (10–50 μL). Incubate it for 20 min at 50°C. 2.5 mL of TCA (10%) was added and then centrifuged at 3000 rpm for 10 min. To the 1.5 mL of upper layer add 1.5 mL of H₂O add 100 μL of FeCl₂. After 10–15 min noted the O.D at 700 nm against distilled water as blank solution whereas Ascorbic acid was in use as standard/Positive control. The experiment was held in triplicates. Increasing absorbance indicates, increasing reducing capacity.

Iron Chelating Activity

To 900 μL of aq. FeSO₄ (500 μM) add PRO in increasing volume (10–50 μL). Add 78 μL of 1.10-phenanthroline (0.25%). Measure the absorbance of the resulting reaction mixture at 510nm against distilled water as blank. FeSO₄ was taken as control, while EDTA was taken as positive control/standard. The chelating activity of Fe²⁺ by PRO was calculated using the formula given: Chelating ability (%) = (A_control – A_sample)/A_control × 100. Where, A_control = Absorbance of FeSO₄. A_SAMPLE = Absorbance of the reaction mixture in the presence of sample (PRO) or positive control (EDTA).

Total Phenolic Content

Different measurements of PRO (10–50 μL) were taken and final volume was made up to 3 mL with distilled H₂O. Then 0.5 mL of 1:10 FC reagent was added. Incubate the reaction mixture for 5 min at room temperature. After incubation add 2 mL of 20% Na₂CO₃. The resulting reaction mixture was then incubated in dark conditions for 1 h. Record the absorbance at 650 nm against distilled water in place of PRO as blank. For calibration curve Gallic Acid was used as standard/positive control. Results were expressed as Gallic Acid Equivalent (GAE)/g PRO by using standard equation y = 0.001x + 0.105, R² = 0.995. All samples were investigated in triplicates.

Total Condensed Tannins

To 3 mL solution of vanillin (4%) in methanol, PRO was added in various amounts (10–50 μL). Then 1.5 mL of conc. HCl was added, followed by incubation for 15 min at room temperature. The absorbance was noted down at 500 nm using methanol as blank solution. Ascorbic Acid was used as standard/positive control for calibration curve. The results were articulated as μg of Ascorbic acid equivalent (AAE)/g PRO using standard curve equation y = 0.000x + 0.011, R² = 0.996. All samples were operated in triplicates.

Total Flavonoid Content

Add 1 mL of methanol and 4mL of distilled H₂O to various volumes of PRO (10–50 μL). Incubate the mixture at room temperature for 5 min, following the addition of 0.3 mL NaNO₃ (5%). To this resulting mixture, 0.3 mL of AlCl₃ (10%) was added, and then again incubated at room temperature for 6 min. Add 2 mL of NaOH (1 M), and made the final volume of reaction mixture up to 10 mL by using distilled water. Incubate the resulting solution in dark (R.T) for 15 min. After incubation measure the absorbance at 510 nm. For calibration curve, Rutin (1 mg/mL) was used. The results were articulated as mg of equivalent of Rutin (RuE)/g of PRO by means of standard curve equation y = 0.002x + 0.094; R² = 0.991.The test was conducted in sets of three.

Anti-Inflammatory Activity (Protein Denaturation) of Palmarosa Essential Oil

By using the method described by Sharma et al. [11], by fluorescent assay the anti-inflammatory activity of PRO was determined. The reaction mixture involves 400 μL BSA (1%) and 4.78 mL PBS (1X; pH 6.4); to this reaction mixture PRO (10–50 μL) was added. Then incubate at 37°C for 15 min in water bath. Heat it at 70°C for 5 min. Immediaetly cool down the reaction mixture after incubation. Then the reaction mixture (1 mL) was exposed to Perkin Elmer Fluorescence Spectrophotometer (FL6500) for Fluorescence Analysis. The excitation wavelength was 280 nm and noted the fluorescence emission spectrum at range of 300–400 nm wavelength. All assays were performed at room temperature (~30°C).

Anti-Diabetic Activity (Alpha-Amylase Inhibition Assay)

To different volumes of PRO (50–250 μL) set in test tubes, add 125 μL of α-amylase solution (5 mg/mL) prepared in Sodium Phosphate buffer (0.02 M, 6.9 pH). 0.5 mL of Sodium phosphate buffer (0.02 M, 6.9 pH) was added. Pre-incubation was given to the solution for 10 min at 25°C. Then add 500 μl of Starch solution (2%) prepared in 0.03 M Sodium Phosphate (6.9 pH) and further incubate at 25°C for 10 min. 0.5 mL of DNS reagent was added to stop the reaction. The test tubes were again incubated for 5 min in boiling water bath and later cooled down to room temperature. Acarbose was taken as positive control. Sample was replaced with water for control. After incubation 6mL distilled water was added to dilute the samples and measure O.D at 540 nm. Calculate the α-amylase inhibitor activity according to the formula given: % age Inhibition = Abs_CONTROL – Abs_SAMPLE/Abs_CONTROL × 100.

Mode of α-Amylase Inhibition

Determination of mode of α-amylase Inhibition was performed according to the following protocol: In first set of test tubes 75 μL of PRO was pre-incubated with 0.2 mL of α-amylase solution (prepared in 0.02 M Sodium phosphate buffer) and 0.5 mL of 0.02 M Sodium Phosphate buffer (6.9 pH) at 25°C for 10 min. In second set of test tubes 0.5 mL of sodium phosphate buffer (6.9 pH, 0.02 M) was pre-incubated with α-amylase solution at 25°C for 10 min. To initiate the reaction, various measurements of starch solution in increasing concentration (1–5 mg/mL) were added to both sets of test tubes. Incubation was given to the resulting reaction mixture at 25°C for 10 min. To terminate the reaction 0.5 mL of DNS reagent was added followed by incubation at 100°C for 5 min. Maltose standard curve was used to determine the quantity of released reducing sugars and their conversion to reaction velocities. A double reciprocal plot (1/V vs. 1/S) between substrate concentration (S) and velocity (V). By analyzing Lineweaver-burk plot and Michaelis-Menten kinetics mode of inhibition of PRO on α-amylase activity was recognized.

In-Vitro Anti-Bacterial Activity

Agar Disc Diffusion Method was used to determine the anti-microbial activity of PRO against four test organisms, Gram-negative Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424) and Gram-positive Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 121). Microorganisms were bought from IMTECH (Institute of Microbial Technology, Chandigarh). Sterilized paper disc (5 mm in diameter) were impregnated with various amounts of PRO. From 12 h old cultures fresh inoculums were prepared for all microbial strains. A swab of bacterial suspension was spread onto the LB-Agar plates and allowed to dry for 30 min. The paper discs impregnated with PRO were then placed in the centre of the petri plates. The plates were left at room temperature for 20 min for diffusion of oil from disc to media followed by incubation for 24 h at 37°C. Streptomycin (10 mg) was taken as Positive control. After incubation visualize the plates under transilluminator and note down the radius of zone of inhibition formed around the disc, which indicates antibacterial activity of PRO.

Anti-Fungal Activity

The food Poisoning method was also used to determine antifungal activity of PRO by following the method described by Agnish et al. 2022 [1]. In this method, petri plates containing 25 mL of sterile Potato dextrose agar media and different volumes of PRO (5–200 μL/mL) were prepared. Mycelial agar disc (10 mm in diameter) were cut from actively budding zone of 7-day old pure culture of Aspergillus flavus (MT9CC 277), Aspergillus fumigatum (MTCC343), and Mucor spp. (MTCC 3373) by using a sterilized cork borer. Aseptically inoculate the fungal discs in the middle of the earlier prepared petri plates. Negative control/blank plates were also made for each fungal strain which contain water in place of PRO. Ketoconazole, Fluconazole, and Itraconazole were taken as positive control. All plates were incubated at 25°C. The plates were observed for 15 days and the results were noted down. The antifungal activity of PRO against all fungal strains was articulated in the form of percent inhibition of mycelial growth by applying the formula described by Agnish et al. 2022 [1]. Inhibition of mycelial growth = d_C – d_T/d_C × 100. Whereas, d_C = mean diameter of the colony in the negative control. d_T = mean diameter of colony in the treated sample (PRO, positive control).

The antifungal activity of PRO against Aspergillus fumigatus (MTCC 343), Aspergillus flavus (MTCC 277) and Mucor sp. (MTCC 3373) was determined according to the protocol given by Agnish et al. [1]. 10 mycelial agar discs (10 mm in diameter) were taken from edge of actively budding 7-day old pure culture of all the three fungal strains and suspended in 20 mL of autoclaved distilled water. 0.1 mL of this fungal suspension was spread onto the petri plates containing Potato Dextrose Agar (PDA) medium. Keep the plates at room temperature for 15–20 min. Sterile paper discs impregnated with different volumes of PRO 5, 200 μL were placed in the middle of the each petri plates. Negative control plate contained autoclaved distilled water in place of PRO. Antifungal drug Ketoconazole (25 μg/mL) was taken as positive control. All plates were incubated at 25°C. Observe the plates for minimum 15 days and note down the zone of inhibition. The antifungal activity was expressed as zone of inhibition (cm).

Gas Chromatography Analysis

The Gas Chromatography (GC-FID) study of PRO was performed by using a Chemtron 2045 gas chromatograph coupled with flame ionization detector. A stainless steel column (2 m long) filled with 10% OV-17 on 80–100% mesh Chromosorb W (HP) was used. Nitrogen gas was used as carrier gas a with flow rate of 30 mL/min. The detector and injector temperature were kept at 200 and 250°C. 0.2 μL of sample was injected. Ramping conditions for oven were: 110°C maintained initially then ramped to 200°C at rate of 2°C/min. Bioactive compounds were identified by comparing the relative retention time with known standards or with data published in the literature and by matching their mass GC-FID spectra with spectra obtained from authentic samples or the NIST spectral libraries spectra and literature data.

Membrane Integrity Assay

The effect of PRO on membrane integrity was studied against bacterial strains, Pseudomonas aeruginosa (MTCC 424), Escherichia coli (MTCC 40), Staphylococcus aureus (MTCC 3160), and Bacillus subtilis (MTCC 121) by following the method described by [13]. The bacterial cells at 1 × 10⁸ CFU/mL were treated with 200 μl of pure PRO. Bacterial cells treated with 200 μL of Triton X-100 were taken as positive control. Untreated cells were considered as Negative control. The treated cells were incubated in orbital shaker incubator (from REMI instruments) at 37°C and 150 rpm and then studied for 12 h. 1 mL of sample was withdrawn from each flask of all bacterial strains after equal interval of time. The cells were then centrifuged at 6000 rpm for 10 min in order to extract the Cytoplasmic contents. The supernatant obtained was collected and measured at 260 nm by using single beam UV-Spectrophotometer (Labtronics). The Cytoplasmic content released (CCR) was articulated as percentage (%) units of UV absorbing components leaked out of the bacterial cells. The percentage Cytoplasmic content release was calculated using the formula given: CCR (%) = A₁/A₀ × 100.

UV-Absorption Assay

The UV-absorption assay was also performed with PRO to determine the protection against UV radiations. In this method, PRO in different concentrations were measured spectrophotometricaly in the UV region (200–400 nm) and its UV-absorption capacity was compared with the sun protection creams (SPF 30 and SPF 45). Different concentrations of PRO and sun protection creams were taken and dissolved in methanol as solvent. Then the UV-spectra was recorded at wavelength ranging from 200–400 nm. From the spectra, wavelength at which the PRO has shown maximum absorption was known and readings were noted down.

Statistical Analysis

MS-Excel software was used to determine p values (P < 0.05 was significant).

RESULTS AND DISCUSSIONS

Essential oils are one between many secondary metabolites which are known to active biologically and hold potent pharmacological activities; hence they are preferred over synthetic ones because of safety concerns [1]. In the present study the total phenolics, total flavonoids, and total condensed tannins were estimated to be 2.91 mg/g GAE, 9.29 mg/g RE, and 3.78 mg/g VAE respectively. The contents of flavonoids were higher than phenolics. Literature studies revealed that essential oils from Cymbopogan spp. are rich in flavonoid content [14]. Essential oils can diminish lower the risk of various diseases by scavenging free radicals in biological mechanisms due to their richness in bioactive compounds [1, 11].

Fingerprint Analysis

UV-VIS spectroscopy measures the scattering and absorption of light passing through the sample. UV-VIS, FT-IR and Fluorescent spectroscopy methods provide a very broad conception about the quantitative and qualitative formulation of bioactive compounds of plant origin [15]. The UV profile of Palmarosa revealed a sharp peak at 230nm with the absorbance of 3.0 (Fig. 1a), signifying presence of secondary metabolites [1, 11]. Various small peaks were observed in the spectra in the range of 250–290 nm, indicating presence of flavonoids and phenolic compounds in the oil [16]. A further method Fluorescent spectroscopy is more reliable and sensible for detection of plant bioactives. The fluorescent spectrum of PRO is given in Fig. 1b. On the basis of excitation wavelength all spectra’s are grouped together in one. Notably two peaks, one minor in Red Florescent region (RFR) at 779 nm and one major in Green Fluorescent region (GFR) at 428 nm were detected. It was suggested that the bioactives near 500 nm (GFR) are Polyphenolics such as, Flavonoids, flavins and terpenoids [17]. A different set of bioactive components (peak in Red Fluorescent Region) was also detected, possibly due to other supplementary fluorescent compounds such as phenolics, alkaloids, anthocyanins, aromatic benzeoids [11]. Peaks of different intensities were observed with respect to their excitation wavelength. As excitation wavelength increased the intensity of major peak increase initially, at 410 nm maximum peak intensity was observed. With the increase in the wavelength there is drastic decrease in the intensity of the minor peak. These differences in the fluorescence intensities might be due to chemical composition of PRO. For analysis of functional groups present in PRO FT-IR spectroscopy was also followed. It is the most remarkable, fast, non damaging strategy to fingerprint plant extracts and for discovery of functional groups [15]. The FT-IR spectrum of PRO is shown in the Fig. 1c and Table 1. In FT-IR spectrum peak at 3325 cm^–1 indicates C–H stretching due to Alkynes, peak at 2943 cm^–1 indicated C–H stretching due to Alkane, peak at 2832.40 cm^–1 indicates C–H stretching due to Aldehydes, peak at 1666.5 cm^–1 indicates C=O stretching due to presence of conjugated ketones, peak at 1448.9 represents Bending of CH₂, peak at 1111.55 cm^–1 indicates C–O–H stretching and peak at 1033.36 denotes C–F stretching due to presence of Fluoro compound.

Fig. 1.

UV-VIS Spectra (a), Fluorescent (b), FT-IR (c) spectra of PRO.

Table 1.

FT-IR analysis of PRO

Peak no.	X, cm–1	Y, %T	Vibration mode	Functional group	Intensity
1	3325.0	75	C–H, stretching	Alkyne	Strong, sharp
2	2943.80	81	C–H, stretching	Alkane	Medium
3	2832.40	84	C–H, stretching	Aldehyde	Medium
4	1666.5	95	C=O, stretching	Conj. Ketones	Strong
5	1448.99	83	CH2, Bending		Medium
6	1111.55	89	C–O–H, stretching		Strong
7	1033.36	34	C–F, stretching	Fluoro compounds	Strong

Antioxidant Analysis

Prior to the coming of present day medication spices and natural items were the backbones of medicines all over the planet. The phytochemicals present in usually devoured plant food varieties are ordinarily non-harmful and have the potential for forestalling ongoing illnesses. The plant essential oil envelop high amount of flavonoids and phenolic compounds. As powerful cancer prevention agents, flavonoids are particularly significant for insurance against human sicknesses. The numerous properties of these phytochemicals have made them more alluring, as they can adjust different perspectives of illnesses like lipid peroxidation associated with atherogenesis, apoplexy, carcinogenesis, hepatotoxicity and an assortment of sickness conditions. The assessment of the antioxidant activity of medicinal plants has turned into a critical part of the cycles utilized in unwinding the therapeutic potential of such plants. Analysts believed that no single assay can precisely mirrors the mechanism of activity of every single radical source or all antioxidants in a complex framework [18], and that at least two techniques or assays ought to be utilized to assess the absolute antioxidant activity [19], because of different oxidative cycles.

In present study antioxidant activity of essential oil extracted from Cymbopogan martinii were determined. A number of assays like ABTS, DPPH, OH^– scavenging, NO scavenging assays, iron reducing, iron chelation, and anti-diabetic assay were evaluated. The IC₅₀ value of PRO for various assays is given in Table 2. Performance wise the antioxidant potential of PRO was observed in the following order: DPPH (9.4637), ABTS (32.2306), iron chelating (35.1876), NO (37.9576), α-amylase inhibition (89.7599), iron reducing (79.937) and OH^– (93.914).

Table 2.

IC₅₀ values for different anti-oxidant assays of PRO

Activity	IC50 value
DPPH radical scavenging activity	9.4637
Hydroxyl radical scavenging activity	93.914
ABTS radical scavenging activity	32.2608
Nitric oxide radical scavenging activity	37.9576
Iron chelating activity	35.1876
Alpha-amylase inhibition	89.7599

The linear correlation analysis was also followed to check correlation between various antioxidant activities and total flavonoid content, total tannin content, total phenolic content, results are given in Table 3. A strong correlation between polyphenolics content and different antioxidant activities was observed. For instance: R² value for Flavonoid content and DPPH activity is 0.945, and R² value for flavonoid content and ABTS is 0.99.

Table 3.

R² values for different activities of PRO

Activity	DPPH activity	Hydroxyl radical scavenging activity	Nitricoxide radical scavenging activity	ABTS radical scavenging activity	Alpha-amylase inhibition	Iron reducing power	Iron chelating activity
Total flavonoid	0.945226	0.94996	0.95389	0.991824	0.929566	0.97212084	0.998857193
Total phenolic	0.77112	0.970041	0.939756	0.86333	0.821056	0.87958322	0.833336923
Total tannin	0.981515	0.808975	0.879851	0.824574	0.973659	0.919885277	0.880617418

DPPH is a stable free radical which has ability to decolorize in the presence of antioxidants. It is direct, reliable and most widely used method for determination of the antioxidant activity. DPPH radical consists of an odd electron which is in charge of absorbance at 517 nm and for noticeable deep purple color. By gaining an electron from an antioxidant it decolorizes and can be measured in decrease in absorbance [20]. In the present study PRO at different concentrations was used measure DPPH radical scavenging activity, as shown in Fig. 2. It is clear from the results, that the PRO shows tremendous DPPH radical scavenging activity as 84.7% activity at its higher concentration used (50 μL) was observed, whereas 78.9% activity was shown by ascorbic acid at concentration 10 μg/mL. Scavenging activity of DPPH radical was found to be increasing as the volume increases.

Fig. 2.

DPPH scavenging, ABTS scavenging, Nitric Oxide scavenging, and Hydroxyl radical scavenging activity of PRO.

ABTS^+• radical is a blue colored chromophore formed by the reaction between Potassium Persulfate and ABTS. In the presence of antioxidants (PRO or ascorbic acid) preformed ABTS^+• radical gets reduced and decolorizes, and the reaction with antioxidant compound was then quantified [12]. In the present study as presented in Fig. 2, PRO showed dose-dependent increase in the scavenging activity at 50 μl which was the highest concentration of PRO tested showed 35.1% scavenging activity. Ascorbic acid which was used as standard showed 94.4% scavenging activity at concentration of 10 μg/mL.

Nitric oxide (NO^•) is a free radical which is produced in mammalian cells, and is engaged with the regulation of different physiological cycles including neurotransmission, vascular homeostasis, and antimicrobial and antitumor activities. In any case, abundance production of NO^• is related with a few diseases. It would be fascinating to build up strong and particular inhibitors of NO^• for effective curative use [21]. In the present study PRO at different concentrations was used to measure NO^• scavenging activity as shown in Fig. 2. It is clear from the results that PRO showed remarkable NO^• scavenging activity.

At 50 μL which was the highest concentration of PRO showed 92.7% scavenging activity while ascorbic acid showed 83.8% scavenging activity at concentration of 10 μg/mL.

Hydroxyl radical (OH^–) is one of the vastly reactive and damaging species in free radical, able to damage all molecules formed in a cellular system [12]. It induces severe damage to biomolecules, but also causes breakage of DNA strands and Denaturation of DNA which leads to carcinogenesis, cytokinesis and mutagenesis [22]. The current study was designed to reveal the OH^– radical scavenging activity of PRO. The results are shown in Fig. 2. PRO showed dose dependent Hydroxyl Radical Scavenging Activity, with 91.9% activity at its highest concentration 50 μL. Ascorbic acid which was used as Standard showed 84.2% scavenging activity at concentration 10 μg/mL. The reducing potential is mainly coupled with the existence of reductones and their antioxidant potential arises due to breaking of the free radical chain by accepting a Hydrogen atom from antioxidant content present in the sample [23].

Iron reducing assay of PRO was also determined in the present study results shown in Fig. 3. Various fractions of PRO were taken and reduction potential. PRO shows high reduction potential in comparison to ascorbic acid. The higher reducing potential of PRO might have happened due to the presence of the Polyphenols and flavonoids which are performing in same way as reductones. In the present study, the metal chelating tendencies of PRO was also estimated to determine the iron chelating activity of PRO. Figure 3 shows chelating activity of PRO at different fractions. A commercially used metal chelating agent EDTA was used as standard/positive control. From results obtained it is clear that PRO showed higher metal chelating activity 74% at its higher concentration 50 μL as compared to EDTA which showed 18.4% activity at concentration of 10ug/mL. Hence, these studies conclude that the antioxidant properties of PRO may appear due to presence of phenolic compounds, flavonoids in higher concentration [24]. A bioactive complex with plentiful antioxidant properties gives its confirmation for its use as a drug/food supplement for the control of biomolecules damage by inhibiting the production of free radicals in the biological system and therefore rejuvenation of the body functions [1].

Fig. 3.

Iron reducing power and Iron Chelating activity of PRO.

GC-FID Analysis of Bioactive Compounds in PRO

The chromatogram obtained by GC-FID was depicted in Fig. 4a the observed peaks and their retention time is also given in the figure. The GC-FID analysis of palmarosa essential oil extracted from Cymbopogan martinii exposed 13 compounds of total 100%. All identified compounds were: Terpinolene, 6‑Methylhept-5-en-2-one, 2-Norbornaneacetic acid, Citronellyl Acetate, Geraniol, Borneol, Nerol, Epi-α-cadinol, δ-cadinol, Linalool, Fenchyl Alcohol. GC-FID chromatogram contained three major peaks along with many small peaks indicative of presence of major compounds. The major compounds were Geraniol (45.8%), Geranial (16.9%) and 6-methylhept-5-en-2-one (7.1%). The small peaks may be credited to the bioactive compounds present in the minor concentrations. Literature studies also reveal the presence of geraniol and linalool in palmarosa oil extracted from Cymbopogan spp. [25].

Fig. 4.

GC-FID analysis of PRO (a), Anti-inflammatory (Protein denaturation) activity (b) of PRO.

Anti-Inflammatory (Protein Denaturation) Activity

Inflammation is a typical defensive reaction prompted by tissue injury or disease and capabilities to battle trespassers in the body (microorganisms and non-self cells) and to eliminate dead or harmed host cells. In the inflammatory reaction there is an increment of porousness of endothelial covering of cells and convergences of blood leukocytes into the interstitium, oxidative burst, and arrival of cytokines [interleukins and cancer rot factor-α (TNF-α)]. Simultaneously, there is additionally an induction of the action of a few catalysts (oxygenases, nitric oxide synthases, and peroxidases) as well as the arachidonic corrosive digestion. In the provocative cycle there is additionally the outflow of cell grip atoms, like intercellular bond particle (ICAM) and vascular cell attachment particle (VCAM) [26].

Protein Denaturation is the major reason behind the tissue inflammation that results in loss of function, swelling, redness and release of heat around the affected areas. It was suggested that bioactives that are capable to inhibit the production of heat induced protein Denaturation can be used as a therapeutic anti-inflammatory drugs [1]. The protein Denaturation inhibitory action of Palmarosa essential oil was determined in the present study by the fluorescent technique. Characteristic fluorescent spectra of denatured BSA (Bovine Serum Albumin) and BSA + PRO at different concentrations are shown in Fig. 4b. A notable increase in the inhibition of protein Denaturation was shown by PRO, as there is decrease in fluorescent intensity to a large extent afterwards the addition of the PRO to BSA. These results grant strong verification for the potential of the PRO as an anti-inflammatory agent. The high protein denaturation inhibition ability of PRO might be credited to complex compounds having synergistics associations of numerous compounds or involvement of minor components. Literature studies reveal that the use of Cymbopogan spp. essential oil and its major component Geraniol diminished the levels of inflammatory markers induced by pamidronate stimuli [9]. Previous studies revealed that bioactive compounds of essential oils are important component in anti-inflammatory ointments, creams and spray [1]. The defensive component Geraniol of essential oils of Cymbopogan spp. has fundamentally ascribed to the regulation of the inflammatory cytokines (IL-1β, IL-6, and TNF-α) and oxidative variables (GSH-Px, MDA, and OH^–) combined with improvement of the inflammatory changes in the lung. Hence, geraniol has a promising potential as feed added substances to be created and applied for the counteraction of MRSA contamination [27].

Anti-Diabetic (α-Amylase Inhibition) Activity

The Alpha-amylase inhibition activity is aimed to delay the metabolism of carbohydrate by various inhibitors to control the blood sugar level [28]. α‑Amylase Inhibition potential of PRO was determined as shown in Fig. 5a. It is clear from the figure that PRO showed inhibition in dose dependent and showed 90.7% inhibition of α-amylase at its higher concentration of 250 μL as compared with Acarbose (a known inhibitor for α-amylase), showed 86.3% inhibition at 10 μg/mL concentration. Inhibition of carbohydrate digesting enzymes such as α-amylase to cure diabetes mellitus is one of the strategies used to reduce the gastrointestinal glucose absorption thereby lowering the glucose level in the body [28]. This is an endeavor to look for elective medications from therapeutic plants with expanded power and lesser antagonistic impacts than existing medications [29]. The mode of inhibition of PRO was determined by Lineweaver-burk plot and Michaelis-Menten likewise showed Figs. 5b, 5c that displays PRO inhibits α-amylase competitively. This proposes that the active components in the PRO contend with the substrate for binding to the active site of the enzyme accordingly forestalling the breakdown of oligosaccharides to disaccharides. Literature studies revealed the inhibitory activity of plant extracts majorly belongs to phytochemicals like flavonoids that contain ability to inhibit a-amylase activity [29].

Fig. 5.

α-amylase inhibition assay (a), Michaelis-Menten Plot (b), and Lineweaver-burk plot (c) showing competitive inhibition of α-Amylase activity by PRO.

Antibacterial Activity

Essential oil extracted from Cymbopogan martinii was assessed quantitatively for determination of in-vitro antibacterial activity against Gram-negative Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424) and Gram-positive Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 121) by Agar disc diffusion method. The present study reveals that Palmarosa essential oil has shown strong antibacterial activity than antibacterial drug. The results are shown in Fig. 6. PRO showed substantial antibacterial activity against E. coli (MTCC 40 Gram –ve) while strong inhibition against S. aureus (MTCC 3160 Gram +ve), and moderate activity against B. subtilis (MTCC 121 Gram +ve) and P. aeruginosa (MTCC 424 Gram –ve). The difference in antibacterial activity of PRO against bacterial strains could be due to incidence of multiple targets or single target for their activity. Staphylococcus aureus causes different suppurative diseases in people. It causes shallow skin sores; more serious diseases like pneumonia, meningitis, also, urinary plot diseases; food contamination by delivering enterotoxins into food and poisonous shock disorder by arrival of superantigens into the blood stream [30]. Literature studies also revealed antibacterial activity of essential oils against methicilin-resistant S. aureus (MRSA) derived from medicinal plants [27, 31]. Escherichia coli is ordinarily present in humandigestive system yet at last reason for food poisoning [30]. Geraniol, major component of Palmarosa essential oil is a lipophilic compound which inhibits microbial growth by adhering and interacting with the lipids present in the cell membrane of the bacteria, thus making it more permeable and binds with the intracellular sites to destroy the structure [32].

Fig. 6.

Antibacterial Activity of PRO against bacterial strains.

Anti-Fungal Activity

The antifungal activity of PRO was quantitatively assessed against Aspergillum flavus (MTCC 277), Aspergillus fumigatus (MTCC 343) and Mucor spp. (MTCC 3373) which is responsible for causing COVID-19 associated infections “Aspergillosis” and “Mucormycosis”. In the present study two strategies were followed to determine the antifungal activity of PRO i.e. Agar disc Diffusion Fig. 7b, Table 4 and Food Poisoning Method the results are given in Fig. 7a and Table 5. High antifungal activity of PRO was seen in both the strategies. In case of Agar disc diffusion method total inhibition was seen in higher concentration (200 μL) as compared to lower concentration (5 μL). In food poisoning method, 50 μl of PRO showed 100% inhibition of Mycelial Growth in MTCC 277 whereas for MTCC 343 it showed 100% mycelial growth inhibition at 10 μl and 100% mycelial growth inhibition at 15 μL in case of MTCC 3373. With the increase in the PRO volume, decrease in the rate of growth of fungal mycelia was seen. Literature studies reveal that, major constituent of Palmarosa essential oil Geraniol, a monoterpene contribute to antifungal activity of PRO in addition to other components such as Geranial, Linalool, Nerol etc. The mechanism of activity of essential oil incorporate lipid peroxidation, hindrance of ergosterol biosynthesis and increase of reactive oxygen species (ROS), which cause porousness of cell membrane which leads to loss of essential molecules and influencing crucial cycles that trigger the course of cell death by apoptosis [33]. Linalool a terpene inhibit proliferation of yeast cell by disrupting biosynthesis of ergosterol a steroid involved in maintaining flow through fungal cell membrane [34].

Fig. 7.

Anti-fungal activity of PRO against fungal strains by Food Poisoning method (a), and Agar disc diffusion method (b). Where NC: –ve control; PC (K): Positive control (Ketoconazole 25 μg/mL).

Table 4.

Antifungal activity of PRO against different fungal strains (by Agar Disc Diffusion Method)

Fungal culture	Sample/control name	Sample/control volume, μL/25 mL	Growth diameter (cm) day 3
MTCC 277	NC	0	NI
	PRO	5	NI
	PRO	200	CI
	PC	Ketoconazole (25 μg/25 mL)	NI
MTCC 343	NC	0	NI
	PRO	5	NI
	PRO	200	CI
	PC	Ketoconazole (25 μg/25 mL)	NI
MTCC 3373	NC	0	NI
	PRO	5	NI
	PRO	200	CI
	PC	Ketoconazole (25 μg/25 mL)	NI

Table 5.

Antifungal activity of PRO against Fungal strains (by Food Poisoning Method)

Fungal culture	Sample/Control volume, μL/25 mL	Mycelial growth inhibition, %
MTCC 343	0	NI
	5	38.6
	10	81.8
	15	84.0
	20	84
	50	65.9
	100	77.2
	200	77.2
	Itraconazole (25 μg)	4.5
	Ketoconazole (25 μg)	4.5
	Fluconazole (25 μg)	4.5
MTCC 277	–	NI
	5	6.6
	10	13.3
	15	46.6
	20	84.4
	50	NI
	100	NI
	200	NI
	Itraconazole (25 μg)	68.8
	Ketoconazole (25 μg)	2.2
	Fluconazole (25 μg)	4.4
MTCC 3373	0	NI
	5	0
	10	11.6
	15	76.7
	20	100
	50	100
	100	100
	200	100
	Itraconazole (25 μg)	5.34
	Ketoconazole (25 μg)	0
	Fluconazole (25 μg)	2.32

Membrane Integrity Assay

The membrane integrity assay was further performed to confirm antibacterial activity of PRO. The integrity of cell membrane in MTCC 40, MTCC 424, MTCC 121 and MTCC 3160 after exposing them to 200 μL of PRO. Then the Cytoplasmic content released (% age) from PRO treated cells was compared with the untreated ones. The PRO treated cells showed high Cytoplasmic content released than the untreated one as shown in Fig. 8a. In case of MTCC 40 PRO treated cells 123.74% of the Cytoplasmic content when compared to control 104.92%. 180.51% Cytoplasmic content released from treated cells as compared to control 62.1% (MTCC 424), 99.29% release from treated cells as compared to control 66.92% (MTCC 3160) and 100.56% release from treated cells as compared to control 51.97% (MTCC 121). Literature studies of membrane cell disruption by essential oil from various medicinal plants indicated that Natural oils and their parts have action against different targets, especially the cell membrane and cytoplasm, and at times, they totally change the morphology of the cells [35]. The bacterial cell film is accountable for significant capabilities like osmoregulation, transport, lipid blend, and peptidoglycan cross-connecting. The integrity of the bacterial cell membrane is essential for endurance, and its interruption can straightforwardly or in an oblique way lead to cell demise [36].

Fig. 8.

Showing Percentage Cytoplasmic Content Release (CCR) from different bacterial strains treated with pure PRO (a), UV-absorption assay (b) of PRO.

UV-Absorption Assay

The PRO shows strong UV- absorption in the UC-C and UV-B region as shown in Fig. 8b. The utilization of healthy skin items enhanced with a few successful specialists managing various pathways related to the utilization of sunscreens might be a compelling methodology for lessening UV-B-produced ROS-intervened photo aging [38]. Literature studies also revealed the ability of essential oil to block UV rays coming from sun [39].

CONCLUSIONS

The current study revealed high phenolics, tannins, and flavonoids content in Palmarosa essential oil (PRO) extracted from Cymbopogan martinii. The UV-VIS, fluorescence and FT-IR spectrum of PRO revealed presence of, polyphenolics in high concentrations. The GC-FID chromatogram revealed bioactive compounds like geraniol, Linalool, etc. High potential of PRO in performing anti-bacterial, anti-inflammatory, sun-protection, anti-diabetic activities was revealed in dose dependent manner.

High Antifungal activity against fatal fungal disease like “Aspergillosis” and “Mucormycosis” was revealed. This study suggests that PRO has high potential to be used as an important raw material in pharmaceutical, food, and cosmetic industries.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.