1 Introduction

Essential oils are notably significant within the secondary metabolites of medicinal plants, often recognized as a form of green technology [1]. An essential oil is a concentrated hydrophobic solution containing a blend of intricate volatile chemical constituents extracted from plants. These oils, derived from botanical sources, are characterized by their natural aroma and taste, preserving the distinctive fragrance and flavor of the original plant material [1]. Essential oils (EOs) are extracted from various parts of medicinal plants, including leaves, buds, flowers, shoots, peels, barks, twigs, fruits, seeds, and roots [2]. Essential oils have experienced a significant rise in popularity due to their cost-effective nature [3]. These botanical extracts offer a plethora of advantageous characteristics such as antioxidant, antibacterial, anti-inflammatory, antioxidant, antidiabetic, antiparasitic, and antifungal properties, as evidenced by scientific literature [4].

Calotropis gigantea, is commonly known as the giant milkweed and belongs to the Asclepiadaceae family [5]. It is a perennial shrub native to diverse regions encompassing tropical Africa and Asia [6]. This species is widely distributed across road sides, railway tracks and deserted land pockets and can be found in countries such as Bangladesh, Burma, China, India, Indonesia, Malaysia, Pakistan, the Philippines, Thailand, Nepal, and Sri Lanka, where it holds significant economic importance [7]. Calotropis gigantea is a large shrub or small tree, typically growing to heights between 3 and 4 m. Its robust branches emerge from the base, with stems reaching diameters of up to 20 cm [8]. The leaves are broadly elliptical to oblong-obovate, measuring 9 to 20 cm in length and 6 to 12.5 cm in width, and are almost sessile [9]. The extensive use of Calotropis gigantea in traditional medicine is attributed to its diverse medicinal properties, which are primarily derived from a range of active constituents. These constituents include alkaloids, steroids, terpenoids, flavonoids, saponins, proteins, and amino acids [8]. Calotropis gigantea is renowned for its diverse array of pharmacological activities. It is notably effective in countering toxins, supporting liver health, facilitating wound healing, combating oxidative stress and anti-inflammatory activity [10]. Moreover, the plant offers a multitude of other benefits including pain relief, antimicrobial action, cell regeneration, antipyretic effects, insecticidal properties, pregnancy-interceptive properties, laxative effects, and pro-coagulant activity [5]. Calotropis gigantea essential oil has been traditionally employed to address an extensive spectrum of health issues, encompassing conditions like syphilis, boils, inflammation, epilepsy, hysteria, fever, muscular spasms, warts, leprosy, gout, snakebites, and cancer [11]. To further explore the potential medicinal benefits of Calotropis gigantea essential oil, ongoing research aims to achieve several objectives. These objectives include conducting phytochemical analysis to determine the content of phenolic compounds, tannins, and flavonoids. Additionally, in vitro antioxidant assays were performed to assess free radical scavenging activity, nitric oxide scavenging activity, iron reducing power, and iron chelating ability. Furthermore, the research investigated the essential oil’s antibacterial activity and evaluate its anti-diabetic activity by determining its α-amylase inhibition and mode of action. These methods aim to provide a comprehensive understanding of the chemical composition and properties of Calotropis gigantea essential oil and its potential therapeutic effects.

2 Material and methods

2.1 Plant material

Fresh leaves were used to isolate essential oil from Calotropis gigantea (Linn.) Aiton f. (designated as CEO). Leaves were collected from plants growing wild in adjoining areas of Lyallpur Khalsa College, Jalandhar (located at 71°–31° east latitude and 30°–33° north longitude). Plants were validated by Dr Inderjeet Kaur and voucher bearing number BT 110 was deposited to biotechnology herbarium. CEO was extracted from fresh leaves by hydro-distillation method following Amrita et al. 2023 [12].

2.2 GC-FID analysis of CEO

The technique of gas chromatography (GC-FID) was employed to find bioactive compounds in CEO. Standard procedures as outlined in Amrita et al. 2023 [12], were followed with minor alterations utilizing the GC-FID, Chemtron 2045 system. For this, 10% OV-17 and an 80–100% mesh Chromosorb W (HP) stainless steel column was used. The carrier gas used in this experiment was nitrogen gas, flowing at a rate of 30 ml/min. The temperatures of the injector and detector were maintained at 250 °C and 200 °C, respectively. A 0.2 µl injection of pure oil was performed. A beginning temperature of 64 °C was maintained for the oven’s ramping conditions, and it was raised to 220 °C at a rate of 3 °C/min. By comparing the relative retention durations (RT) of the GC-FID spectra of CEO with genuine standards and literature data, bioactive components in CEO were found.

2.3 UV–Vis, FT-IR and fluorescent analysis

According to Amrita et al., 2023[12]; FT-IR, UV–VIS, and fluorescence spectroscopy of CEO were performed.

2.4 In-vitro evaluation of antioxidants assays

To evaluate in vitro antioxidant potential of CEO, different antioxidant assays as described in Amrita et al. 2023 [12] with minor modifications, various antioxidant content, such as total phenolic, total flavonoid, and total tannin, and associated antioxidant activities, such as iron reducing assay, iron chelating assay, and nitric oxide scavenging, were carried out. Briefly described as under:

2.4.1 Total phenolic content

Three ml of distilled water, five ml of CEO, and half a ml of Folin-Ciocalteu reagent were added. After a thorough shaking, the mixture was allowed to sit at room temperature for five minutes. Next, 2 ml of 20% (w/v) Na2CO3 was added, and the mixture was allowed to sit at room temperature for an hour while it was in the dark. At 650 nm, the mixture’s absorbance was measured, with distilled water serving as the blank. The results were represented as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DWT), with gallic acid serving as the benchmark.

2.4.2 Total flavonoid content

After adding 1 ml of methanol and 4 ml of distilled water to 0.5 ml of CEO, the mixture was allowed to sit at room temperature for five minutes. 0.3 ml of 5% (w/v) NaNO3 and 0.3 ml of AlCl3 were added after incubation, and the mixture was once again incubated for 15 min at room temperature in the dark. Rutin was used as the standard curve to measure absorbance at 510 nm. The findings were represented as milligrams of Rutin per gram of dry weight (mg RUF/g DWT).

2.4.3 Total condensed tannins

After thoroughly mixing 1.5 ml of concentrated HCl and 3 ml of 4% vanillin solution into 0.5 ml of the sample (CEO), it was allowed to sit at room temperature for 15 min. Following that, methanol was utilized as a blank to measure the absorbance at 500 nm. In milligrams of ascorbic acid per gram of dry weight (mg AA/g DW), the total tannin concentration was reported.

2.4.4 Iron chelating assay

In this experiment, 900 µl of FeSO4 (500 mM) was mixed with CEO (50 µl), and 78 µl of 1–10-phenanthroline (0.25% v/v) was added after that. Using water as a reference, the absorbance of the resulting solution was measured at 510 nm. The control was a solution of FeSO4. EDTA was used as the positive control. Cheating capacity (%): (A CONTROL − A SAMPLE)/A CONTROL × 100. where A SAMPLE is the absorbance of the reaction mixture when either the positive control (EDTA) or the sample (CEO) are present, and A CONTROL is the absorbance of FeSO4.

2.4.5 DPPH free radical scavenging activity

The ability of CEO to neutralize free radicals was evaluated using the DPPH test. Three ml of the DPPH Solution were combined with CEO (50 µl). The reaction mixture was allowed to sit at room temperature (between 25 and 30 °C) for an hour. Using an 82% methanol blank solution, absorbance was measured following incubation at 517 nm. A 3 ml DPPH solution control sample was employed. Ascorbic acid served as the positive control. DPPH radical scavenging activity (%) = (A0-A1)/A0 100 is the and A0 is the absorbance of the control. A1 is test sample/standard absorbance.

2.4.6 Nitric (NO) oxide radical scavenging activity

To CEO (50 µl), 3 ml of sodium nitroprusside solution (SNP, 10 mM in 1X PBS) was added. The reaction mixture was then incubated at 25 °C for 150 min. Following incubation, Griess reagent was combined with 0.5 ml of the previously indicated reaction mixture. SNP in 1X PBS (3 ml) was used as a control. Ascorbic acid was the standard/positive control that was employed. Nitric oxide radical scavenging activity (%): (A0-A1)/A0 × 100] is the where A0 is the SNP’s PBS absorbance. The absorbance of the test sample (CEO) is A1.

2.4.7 α-Amylase inhibition assay of CEO

To test tubes containing various aliquots of CEO (50–250 µl), 0.5 ml of sodium phosphate buffer (0.02 M, 6.9 pH) and 125 ml of α-amylase solution (5 mg/ml) were added. For ten minutes, the mixture was pre-incubated at 25 °C. 500 µl of a 2% W/V starch solution made in 0.03 M sodium phosphate (pH: 6.9) was added, and the mixture was then incubated at 25 °C for an additional 10 min. To stop the reaction, 0.5 ml of DNS reagent was introduced. The test tubes were removed after 5 min from the boiling water bath and cooled to room temperature. Acarbose as standard/positive control was employed. The samples were diluted with six ml of distilled water for testing after incubation. The α-amylase inhibitor’s activity was calculated using the formula below: The formula for percentage inhibition is ACONTROL-ASAMPLE/ACONTROL*100. ACONTROL is the reaction mixture’s absorbance without a sample or positive control. ASAMPLE: Absorbance when the sample or positive control is present.

2.4.8 Mode of α-amylase inhibition

The following procedure was used to block α-amylase using CEO. One set of tubes with 500 µl of 0.02 M sodium phosphate buffer (pH 6.9), 200 µl of α-amylase solution, and 75 µl of EAE underwent a 10-min pre-incubation period at 25 °C. In another set of tubes, 500 µl of 0.02 M sodium phosphate buffer (pH 6.9) pre-incubated with α-amylase was placed. The reaction was started by adding varying amounts of starch solution to both sets of tubes at increasing concentrations of 1–5 mg/ml. The mixture was allowed to incubate for 10 min at 25 °C before the reaction was stopped with the addition of 500 µl of the dinitrosalicyclic acid (DNS) reagent. The concentrations and amounts of released reducing sugars were measured using the maltose standard. Substrate concentration (S) vs velocity (V) (1/V versus 1/S) was shown as a double reciprocal plot. The Lineweaver–Burk plot revealed the Michaelis–Menten Kinetics approach of CEO inhibition on α-amylase activity.

2.4.9 In-vitro antibacterial activity

Utilizing the Agar Disc Diffusion Method, the antimicrobial activity of CEO was assessed against four test organisms: Gram-negative Escherichia coli (MTCC 40). The above microbes were obtained from the Institute of Microbial Technology, Chandigarh, or IMTECH. Fresh inoculums were made from cultures left for 12 h for each strain. A 0.1 ml swab of bacterial cultures was put over the LB-Agar plates and left for 30 min. The paper discs were positioned in the middle of the petri plates after being soaked with 50 µl of CEO. Following a 20-min room temperature incubation period to allow the extract from the disc to diffuse into the medium, the plates were then incubated for a full day at 37 °C. Ten mg discs of streptomycin were taken as a positive control. Following incubation, plates were seen under a transilluminator, and the radius of the zone of inhibition that developed around the disc was measured. This zone shows how effective the CEO is against bacteria.

2.5 Photostability analysis of CEO

The experiment was initiated to analyze the photostability of CEO under ultraviolet light and visible light. Two small glass beakers were taken, and each was filled with 5 ml of CEO. Subsequently, one beaker was subjected to UV light exposure (Philips 20 W), while the other was placed under visible light (Philips 40 W). Both beakers were positioned at a distance of approximately 10 cm from their respective light sources. During the one- hour duration, aliquots of the essential oil were collected from each beaker at 15-min intervals. These aliquots were subsequently subjected to spectroscopic analysis using a spectrophotometer (Labtronics MODEL LT-291). The spectrophotometer scanned the range of 200 nm to 400 nm to obtain the spectrum.

2.6 In-silico analysis of phytoconstituents against α-amylase

2.6.1 Preparation of ligands

Principal bioactive components viz; Phytol, Stearic acid and Cyclohexyl ketone were chosen as ligands for molecular docking studies. SMILES of all selected ligands were retrieved from the PubChem (https://pubchem.ncbi.nlm.nih.gov/). Acarbose was used as a standard drug or inhibitor. 3-D structures of ligands were prepared by retrieving SMILES from the NCBI-PubChem database and by using UCSF-chimera.

2.6.2 Target protein preparation

The crystal structures of the enzyme’s amylase (PDB ID: 3bai) served as the study’s targets. The RCSB-PDB database, which can be accessed at https://www.rcsb.org, provided the sources for their structures. Target enzyme receptors were built up for docking research using the Chimera dock prep system. In the optimization process known as dock prep, irregularities in atomic bond length, structure, and charge are corrected.

2.6.3 Molecular docking study

Molecular docking was done using Cb-dock2 (https://cadd.labshare.cn/cb-dock2/php/index.php) to study the binding mechanism of Phytol, Stearic acid and Cyclohexyl ketone with α-amylase. Docking was executed by uploading the ligand and target enzyme molecules in a.pdb file to the cb-dock2 tool. The best-generated model in the.pdb file is downloaded and saved. The enzyme-ligand 2-D interactions were predicted by using the Biovia 2020 and UCSF Chimera tools.

2.6.4 Drug-likeness

ADMET (Absorption, Metabolism, Toxicity and Excretion), drug likeness, physiochemical properties and pharmacokinetics of Phytol, Stearic acid and Cyclohexyl ketone were studied using SWISSADME tool (http://www.swissadme.ch/). SMILES of all selected ligands was used to find out ADSMET by SWISSADME online tool.

2.6.5 Investigation of molecular interaction between α-amylase and CEO

Interaction between EO’s and α-amylase using fluorescence quenching analysis was carried out following Anigboro et al. 2021[13] with minor modifications. Fluorescence quenching assay was performed at room temperature (20–25 °C) at excitation wavelength of 280 nm with slit band widths was set at 5 nm. Briefly, 3.5 ml of α-amylase (prepared in 0.5 mg/ml in sodium phosphate buffer, 0.02 M, pH 6.9 and 0.006 M NaCl) was mixed with varying concentration of CEO (50–250 µl) mixture was incubated for 10 min and fluorescence emission spectra were recorded from 250 to 500 nm (Perkin Elmer spectrophotometer, FL6500).

2.7 Statistical analysis

Data (anti-oxidant, anti-bacterial and amylase inhibition) obtained was represented by Mean ± SD of three determinations (n = 3). Each data was statistically validated by subjecting values to Tukey HSD test (SPSS 26.0). Briefly, in under antioxidant assays, CEO values were compared with positive control and significant values are denoted by * sign. It was significantly different only when p < 0.05. For anti-bacterial and amylase inhibition assays: values are represented by different alphabet letters (a-e) in bars at p < 0.05. Using https://www.aatbio.com/tools/ic50-calculator, the IC50 values of CEO offering 50% inhibition were determined. The IC50 (half-maximal inhibitory concentration) value is a measure of the concentration of a drug or compound required to inhibit a particular biological or biochemical process by 50%.

3 Results and discussion

Because they are safer than manufactured ones, essential oils are among a number of secondary metabolites that are known to be physiologically active and have high pharmacological characteristics. According to the current investigation, the estimated values of total flavonoids, total phenolics, and total condensed tannins were 154 µ/g RE, 90 µ/g AE, and 87 µ/g GAE, (Table 1), respectively. Through secondary metabolic pathways, plants produce polyphenolics in response to oxidative stress and photodamage [14]. The results of this investigation showed that CEO has a significant concentration of polyphenolic chemicals with strong antioxidant properties. Therefore, we believe that polyphenolics may play a role in Calotropis gigantea’s medical benefits. These results offered good pharmacological justification for the traditional and folk uses of Calotropis gigantea. The high polyphenolic content demonstrated CEO’s efficacy as a powerful source of bioactive chemicals derived from Calotropis gigantea. The results of this study provide insight into how essential oils, with their abundance of bioactive components, might lower the risk of several illnesses by scavenging free radicals in biological pathways.

Table 1 Phytochemical analysis of Calotropis gigantea essential oil
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3.1 Gas chromatography analysis

The chromatogram obtained by GC-FID was depicted in Fig. 1, the observed peaks and their retention time is also given in the Table 2. The GC-FID analysis of CEO extracted from Calotropis gigantea resulted in the identification of 30 compounds. CEO is endowed with major components like: Phytol (24%), Stearic acid (10%) and Cyclohexyl ketone (6%) along with other minor constituents. The major compound phytol, also known as phytosol or floral alcohol, is an acyclic hydrogenated diterpene alcohol used in fragrance manufacturing and as a precursor for the synthesis of synthetic variants of vitamins K and E [15]. Other commercial applications for it include detergents, shampoos, toilet soaps, and cosmetics [16]. It is also used as a flavoring or dilution in certain cannabis distillates. The bioactive substances found in the trace amounts may be responsible for the tiny peaks. It was discovered that the essential oil’s composition differed from earlier studies. CEO composition is influenced by a number of variables, including weather, extraction technique, soil humidity, harvesting season, distance between plants, harvest time, drying method, and storage conditions as well as genotype [14].

Fig. 1
figure 1

GC-FID profile of Calotropis gigantea essential oil

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Table 2 GC-FID components in Calotropis gigantea essential oil
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3.2 Fingerprint analysis

To completely comprehend the qualitative and quantitative contents of herbal derivatives and bioactive chemicals derived from plants, spectrophotometry can be employed. In this context, conventional procedures for phytocomponent detection include the use of UV–VIS, fluorescence, and FT-IR spectroscopy techniques, either alone or in combination [12]. The CEO’s qualitative UV spectroscopic profile, revealed several discrete, strong peaks from 240 to 360 nm with an absorbance of 2.0–3.0 (Fig. 2a). The presence of secondary metabolites such as flavonoids, tannins, and phenolics is indicated by these peaks. A study conducted by Donkor et al. 2019 [17], provided evidence for the presence of phytochemicals. It revealed absorption maxima in the UV–Vis spectra of plant extracts in the wavelength ranges of 202–423.0 nm, 203.0–379.0 nm, and 203.40–385.1 nm.

Fig. 2
figure 2

a UV–Vis Spectrum b FT-IR Spectra c Fluorescence Analysis of Calotropis gigantea essential oil; INT-intensity

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The CEO’s FT-IR peak values are displayed in Fig. 2b, Table 3. In the FT-IR spectra, the peaks at 2922, 2853, and 1462 showed C-H stretching caused by alkanes, whereas the peaks at 1743 and 1373 were caused by esters, aldehydes, anhydrides, phenols, ketones, carboxylic acids, respectively. Donkor et al. 2019 [17] also reported similar secondary metabolites found in Duranta erecta. The chemicals are all also classified as secondary plant metabolites, as per Paul et al. 2011 [18].

Table 3 FT-IR peak value analysis of Calotropis gigantea essential oil (CEO)
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These findings suggest that every CEO has a high concentration of secondary metabolites. The therapeutic qualities of CEO may be due to the presence of the aforementioned secondary metabolites. Fluorescence spectroscopy is an alternative method that is recommended for the precise and useful identification of plant bioactive substances. Figure 2c displays the fluorescence spectra of the CEO. Two prominent peaks were seen in nearly all spectra: a big peak at 679 nm in the Green Fluorescent Region (GFR) and a lesser peak at 519 nm in the Red Florescent Region (RFR). The bioactives in the 500–600 nm range (GFR) have been suggested to be polyphenolic chemicals such flavonoids, flavins, and terpenoids [19].

3.3 In-vitro antioxidant analysis of CEO

Evaluating an herb’s antioxidant capacity and associated properties is usually the first step in determining its therapeutic potential. According to Borrás-Linares et al. 2014 [20], essential oils may have high antioxidant qualities since they are rich in bioactive compounds. According to earlier studies, the antioxidant activity of compounds such phenolics, flavonoids, and tannins, among others, was responsible for the medicinal efficacy of the plants containing them [21]. According to the authors, natural antioxidants derived from aromatic plants are becoming more and more important in complementary and preventive medicine as well as the nutritional field (food stability and preservation).

The IC50 values for the various antioxidants showed that the CEO has strong antioxidant activity (Table 4). IC50 values of CEO were in as order: Iron chelating activity: 18.610, DPPH radical scavenging activity: 28.246, Iron reducing activity:37.682, Nitric oxide radical scavenging activity: 58.113. To examine the relationship between different antioxidant activities and total flavonoid, total tannin, and total phenolic content, a linear correlation analysis was also conducted. The findings are shown in Table 5. There was shown to be a significant relationship between the number of polyphenolics and various antioxidant activities. The R2 value for the total phenolic content and the iron-reducing activity was 0.99, whereas the R2 value for the flavonoid content and the iron-reducing activity was 0.99. The R2 values for the iron chelating activity, and total condensed tannin content was 0.99. Several studies have shown a favorable link between plant antioxidant activity and phenolic component content [22]. The results of the evaluation of antioxidant activity employing iron chelating activity, DPPH scavenging, and nitrous oxide scavenging (NOR) are shown in Fig. 3. The antioxidant activity of CEO at 50µl was measured in the current investigation. The data make it abundantly evident that the CEO had exceptional DPPH radical scavenging activity with 96% value. The reference substance, ascorbic acid (Vitamin C), a well-known strong antioxidant and free radical scavenger with 99% activity, was used for comparison. The CEO had a scavenging activity of 82% Nitric Oxide, which was similar to the positive control. Furthermore, the CEO showed 97% iron chelating activity. A statistical test revealed that the CEO’s chelating activity was substantially greater than that of the positive control. This was consistent with other research [22], that mentioned the antioxidant function of essential oils. Overall, these studies conclude that the inclusion of polyphenolics and major and minor bioactive components, such as Phytol, Stearic acid, and Cyclohexyl ketone bioactive components, in-toto, may be responsible for the antioxidant benefits of CEO. Antioxidant activity against DPPS were shown to be correlated with antioxidant content, chemical structures, and degree of polymerization, as reported by Khan et al. 2012 [23]. Furthermore, high molecular weight phenolics, such as tannins, have been shown to be more effective in scavenging free radicals.

Table 4 I \({\mathbf{C}}_{50}\) values for different activities of Calotropis gigantea essential oil (CEO)
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Table 5 \({\mathbf{R}}^{2}\) values for different activities of Calotropis gigantea essential oil
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Fig. 3
figure 3

DPPH scavenging, Nitric Oxide scavenging (NOR) and Iron chelating activity (ICA) of Calotropis gigantea essential oil. CEO: Calotropis gigantea essential oil, PC: ascorbic acid. Value with * indicates significant difference of CEO vs PC at P ≤ 0.05

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3.4 α-Amylase inhibition activity

The goal of alpha-amylase inhibition is to slow down the metabolism of carbohydrates using a variety of inhibitors to regulate blood sugar levels [24]. α-Amylase Inhibition potential of CEO was determined as shown in Fig. 4a. As indicated in Fig. 4 CEO blocked α-Amylase in dose -dependent way. At a higher amount of 250 µl, CEO inhibition was 63% which was comparable to compared with acarbose (a known inhibitor for α-amylase), which also showed 63% inhibition at 10 µg/ml concentration. Michaelis–Menten and Lineweaver–burk plot indicated that the mode of CEO inhibition for α-amylase was un-competitive (Fig. 4b, c). Uncompetitive inhibitors only recognize and interact with ES (Enzyme substrate complex) and subsequent downstream catalytic species with no binding to free enzyme. Thus, to exhibit enzyme binding, uncompetitive inhibitors require formation of ES and inhibition of enzyme activity is characterized by a decrease in both substrate Km and Vmax. Uncompetitive inhibitors do not bind to free enzyme; instead, they only identify and interact with ES and subsequent downstream catalytic species. Therefore, uncompetitive inhibitors require ES in order to display enzyme binding, and a drop in substrate Km and Vmax indicates that the enzyme is not functioning as intended. However, when the inhibitor attaches itself to the ES complex and stabilizes it, it increases the enzyme’s affinity for S, lowering substrate Km and making it more difficult for S to dissociate or be converted to product. Because the inhibitors bind to the enzyme target only when the target is active and substrate is present, this mechanism of action is appealing for drug design. The majority of the inhibitory action of plant extracts is attributed to phytochemicals like flavonoids, which have the ability to inhibit a-amylase activity, according to literature studies [25].

Fig. 4
figure 4

a α-amylase inhibition assay, b Michaelis–Menten Plot, c and Lineweaver–burk plot of α-Amylase activity of Calotropis gigantea oil. CEO:Calotropis gigantea essential oil. Mean values with different letters across the bars are considered significantly different at p < 0.05

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3.5 Antibacterial activity

The essential oil isolated from Calotropis gigantea was quantitatively evaluated for antibacterial activity using the agar disc diffusion technique against Gram-negative Escherichia coli (MTCC 40). According to the results of the current investigation, CEO had significant antibacterial action, as seen in Fig. 5. CEO exhibited a 0.5 cm inhibitory zone and significant antibacterial action against Escherichia coli (MTCC 40 G−). According to earlier research, anti-microbial activity was sometimes linked to the synergistic effects of major and minor chemical components rather than the high concentration of a single main chemical molecule [26]. Nejhad et al., 2023 [27], also reported antibacterial activity Calotropis procera leaf aqueous extract against E. coli, however ZOI was just 0.14 cm. Our study’s CEO revealed a noteworthy observation: high antimicrobial toxicity toward G-bacteria. This is noteworthy because previous research suggested that G− bacteria are more resistant than G+ bacteria due to their thick cell walls, which enable them to express resistance to toxic drugs and antibiotics in particular [28].

Fig. 5
figure 5

Anti-microbial Activity (zone of inhibition: ZOI) of Calotropis gigantea essential oil against MTCC40, PC: Positive control (Gentamycin 10 mg/disc). Mean values with different letters across the bars are considered significantly different at p < 0.05

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3.6 Photostability of CEO

Earlier research cited that essential oil are generally prone to photolysis when exposed to visible of UV light thus could be stored in dark bottles at cold conditions. CEO gets absorbed in the UV region displaying a peak around 240 nm, whose intensity retarded upon UV and VIS irradiation (Fig. 6). Notably, visible light did more photolysis than UV light. The reduction in intensity indicated photolysis of essential oil. Similar degradation of essential oil upon UV and VIS has been reported by Wadhwa et al. 2019 [29].

Fig. 6
figure 6

Photostability assay of Calotropis gigantea essential oil. CEO: Calotropis gigantea essential oil

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3.7 In silico analysis of bioactive substances with anti-α-amylase

Bioactive substances with anti-α-amylase activity are regarded as important therapeutic agents. Research showed that medications that target α-amylase may be therapeutic agents with promise for managing type 2 diabetes (T2DM) [30]. Since α-amylase is the primary enzyme involved in the digestion of carbohydrates, it is crucial to use it as a primary target for combating phytocompounds that can block α-amylase. The current study planned to dock primary bioactive compounds of CEO: phytol, stearic acid, and cyclohexyl ketone against α-amylase. Acarbose (a well-known inhibitor) was also studied as a vital enzyme inhibiting competitors in opposition to α-amylase. Docking analysis showed that all the bioactive compounds from CEO efficiently docked with alpha-amylase enzyme (Table 6). The largest potential for inhibition is indicated by bioactive substances with the lowest vina scores when used with enzymes. Among all the bioactive components, the most effective binding was observed with cyclohexyl ketone with a docking score of -6.6. The docking data for the α-amylase enzyme showed that acarbose, a common type-2 anti-diabetic medication, demonstrated docking with a vina score of − 7.9. Cyclohexyl ketone’s docking score was similar to that of acarbose. Figure 7 displays the molecular interactions and best docking poses of the ligand’s studied with α-amylase. It is evident from this that the essential oils include a wide range of phytocompounds that, used individually or in combination, reduced the activity of α-amylase as demonstrated by the in-vitro experimental α-amylase inhibition studies. A study on molecular interactions was carried out to explore the possible binding mechanism of our compounds to the active site residues of the α-amylase protein. The ligand-target interactions of bioactive substances CEO with α-amylase are illustrated in Table 7 involving hydrophobic interactions, hydrogen bonds, and other important details.

Table 6 Molecular docking of target enzymes with bioactive component of essential oil used as ligand
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Fig. 7
figure 7

3-D ribbon best fit models showing cavity (left panel) and 2-D models showing intercations of ligands with α-amylase (right panel)

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Table 7 Ligand-Enzyme interactions
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It was observed that most stable complex cyclohexyl ketone- α-amylase was stabilized by 2 Pi-alkyl, 9 Van der Waal interactions involving HIS:101,305; TRP:58; ASP:197,300; LEU:162,165; THR:163; GLN:63 and TYR:62; TRP:59 (Pi-alkyl) amino acid residues. Phytol illustrated 2 H-bond through GLU:233; ASP:300,1 Pi-sigma with TYR:62, 3 alkyl type interactions via ALA:198; LEU:162,165, 6 Van der Waal through ILE:235; GLN:63; TRP:58; ASP:197; HIS:101; ASN:298 and 3 Pi-alkyl bond interactions via HIS:201,299; TRP:59 amino acids. Stearic acid was docked to α-amylase enzyme via 10 Van der Waal 2 alkyl and 2 Pi- alkyl type interactions as given inn Table 7. Three particular amino acids have been identified by the X-ray crystal structure of α-amylase: ASP: 197, ASP: 300, and GLU: 233. These amino acids are known to contribute to the overall catalytic activity of starch hydrolytic enzymes [13]. The ligands used in the current study revealed binding to these amino acid residues. Phytol interacted with all three key enzyme residues GLU: 233, ASP: 197,300. Stearic acid also interacted with all the three key enzyme residues via Van der wall interactions. Cyclohexyl ketone was found to interact with two of the key enzyme residues. Overall, it was discovered that acarbose and the natural metabolites examined in this study interacted with the α-amylase active site through exact amino acid residues in native ligands, which are important residues of the enzyme. Our results suggest that the chemicals found in the CEO might be important metabolites that contribute to the suppression of the α-amylase enzyme. Our findings were consistent with those of Dalli et al. 2022 [31], reported using α-amylase to effectively analyze in silico the bioactive components of Nigella sativa essential oil, including α-Phellandrene, β-Cymene, 4-Caranol, Thymol, and β-Pinene.

For in vivo therapeutic applications, drug properties such as Prediction of Activity Spectra for Substances (PASS) and Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) are required [32]. The Lipinski Five Rule (RO5) identifies pharmacological similarity for all ligands, including phytol, stearic acid, and cyclohexyl ketone, which are all employed in molecular docking studies. As shown in Table 8, the oral activity of ligand compound is estimated by computing other chemical parameters like mlogP (partition coefficient) and TPSA (polar surface area). PASS and ADMET analyses demonstrated the low molecular weight of all three ligands. It has been shown that low-molecular-weight ligands have a higher probability of diffusing fast and transferring across biological membranes in comparison to high-molecular-weight ligands [33]. Topological Polar Surface Area readings for Phytol were 20.23 Å2, stearic acid was 37.30 Å2, and cyclohexyl ketone was 17.07 Å2. Wu et al. 2020 [32], state that TPSA is a helpful method for deciphering drug transfer characteristics such as enhanced absorption in the gut, increased bioavailability, and superior permeability. Since it is clear from the Table 4, the GI absorption (Gastrointestinal tract) of stearic acid and cyclohexyl ketone was high excluding phytol. Stearic acid and cyclohexyl ketone were not substrate to P-gp (P-glycoprotein) efflux transporter excluding phytol. Drug absorption into the lumen is decreased by the P-gp efflux carrier in the gut, which returns medicines [34]. These findings demonstrated that all ligands could readily interact with the α-amylase. Additionally, it confirmed the possibility of the binding interaction between bioactive components of essential oils and α-amylase, which was investigated and confirmed using the additional spectroscopic techniques and fluorescence quenching approach reported in this study.

Table 8 ADMET properties of bioactive compounds used as ligands
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3.8 Fluorescence quenching analysis

One of the most important methods for analyzing conformational changes in proteins caused by ligand interaction is fluorescent quenching [35]. In the present study, binding interaction of phytocomponents of CEO with α-amylase was also monitored. Previous research has indicated that the amino acids tryptophan, phenylalanine, and tyrosine may be implicated in the intrinsic endogenous fluorescence emission of an enzyme [36]. Changes to the microenvironments around these amino acid residues therefore affect the enzyme’s activity. The ligand–protein interaction-induced variations in the microenvironments of the enzyme’s chromophoric groups may be measured by detecting the corresponding changes in the protein’s maximum emission intrinsic fluorescence intensity [35, 36]. This work assessed the observed changes in the intrinsic fluorescence of the enzyme at different concentrations of CEO (50, 100, 150, 200, and 250 μL) and measured the α-amylase fluorescence emission intensities (Fig. 8). The fluorescence emission spectra show that the intrinsic fluorescence intensity of α-amylase gradually decreases with increasing concentration of CEO, suggesting the existence of molecular interactions between α-amylase and phytocomponents in CEO. These findings were consistent with a paper published by Anigboro et al. 2021 [13], which found that the presence of plant herbal extract in Justicia carnea extract inhibited the intrinsic fluorescence of the α-amylase enzyme.

Fig. 8
figure 8

Fluorescence Quenching analysis of CEO showing interaction with α-amylase

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4 Conclusion

The current study revealed high phenolics, tannins, and flavonoids content in essential oil (CEO) extracted from Calotropis gigantea. The UV–VIS, fluorescence and FT-IR spectrum of CEO discovered occurrence of polyphenolics in high concentrations. The GC-FID chromatogram discovered the presence of bioactive compounds like phytol and other minor components. High potential of CEO in performing, antioxidant, anti-diabetic activities was revealed in dose dependent manner. In silico analysis also validated the role of bioactive from CEO as antidiabetic agents. CEO performed well to mitigate growth of G− bacteria. This study suggests that CEO has high potential to be used as an important raw material in pharmaceutical, food, and cosmetic industries.