Introduction

Herbal treatments are gaining popularity due to their efficiency and either no or very low side effects. Approximately 80% of the world’s residents currently rely on herbal remedies to treat their medical conditions. Generally, herbs and spices are safe and are useful in treating several illnesses1. The use of medicinal and aromatic plants has both health and financial advantages for mankind2. Zingiberaceae is regarded as the “spice family” and cardamom, ginger, turmeric, etc. are some of the medicinally important plants of the family. There are over 1,500 species in this family, which contains 52 genera, and they are used in the food, cosmetics, and medicine industries. The encyclopedia of Ayurvedic science, Charak Samhita, describes the use of these medicinal plants3.

The species H. spicatum is a traditional Indian medicine and a perennial rhizomatous herb from the Zingiberaceae family, also known as Kapoor Kachri, Van-Haldi, and Ginger lily4. It is found in the subtropical states of India such as Assam, Arunachal Pradesh, Andhra Pradesh, Himachal Pradesh, Manipur, Karnataka, Sikkim, Meghalaya, Nagaland, Mizoram, Orissa, and Uttarakhand at an elevation of 1000 to 3000 m. Besides India, this plant is widely distributed throughout the world in countries like Bhutan, Japan, China, Nepal, Myanmar, Pakistan, Mauritius, Thailand, Seychelles, and Madagascar5. The species is a tall perennial stout herb with a thick straight stem and broad leaves3.

It has been utilized in several conventional treatments for conditions like bronchitis, indigestion, eye illness, inflammation, and diarrhea. In Ayurvedic and traditional Chinese medicine, the decoctions and infusions prepared from the leaves, flowers, and rhizomes of this herb are advised for their stomachic, blood-purifying, and carminative effects5. This herb shows a wide range of properties beneficial for health, such as antioxidant, anti-inflammatory, anti-allergic, anti-asthmatic, and analgesic. It is also useful in ulcer protection, lowering blood pressure, and exhibiting hepatoprotective properties. It contains antihyperglycemic, memory-restoring, cytotoxic, anticancerous, hair growth-promoting, tranquilizing, radical-scavenging, anthelmintic, and antimicrobial properties3,4,6. It is widely used in the making of ‘Abir’, an aromatic colored powder used during the Holi festivities in India7. This aromatic medicinal plant is more readily available, less expensive, and quite effective in treating several diseases. Ayurveda, Unani, Siddha, and other traditional Indian healthcare systems are still employed to offer primary care, especially in rural regions8. The essential oil of H. spicatum contains 1,8-cineole, α-terpinene, β-phellandrene, limonene, p-cymene, α-terpineol, and linalool6.

Today, the food sector struggles to maintain the high quality of processed fruit and vegetable products. Due to their efficiency and either no or minimal side effects, herbal remedies are currently becoming more and more popular. Thus, for preserving the medicinal herbs for long periods, drying could be a better alternative. The dried H. spicatum rhizomes are used for the curing asthma. Similarly, ginger lily is widely used as a spice in the majority of curries. Some previous investigations have shown the influence of drying on the chemical composition of Zingiberaceae plants8,9,10,11. Until now, there has been no record found in the literature on the effect of different drying conditions on the essential oil content and essential oil composition of H. spicatum. So, the present study aims to determine the influence of drying conditions on the essential oil profile and antioxidant potential of H. spicatum rhizomes.

Materials and methods

Collection and identification of the plant

In October 2022, H. spicatum rhizomes were collected from Ramgarh, Nainital district of Uttarakhand, at an elevation of 2100 m (29.418375° N, 79.56459° E). The plant specimen was identified at Regional Ayurveda Research Institute (Central Council for Research in Ayurvedic Sciences), Ranikhet (Thapla), Almora, Uttarakhand, India (Voucher No. 29958) by Dr. Deepshikha Arya.

Isolation of essential oil

To help in the essential oil release from the rhizomes, they were first submerged in water for an hour. Then they were rinsed under running water. The rhizomes were separated into six batches and cut into 0.5*0.5*4 cm-wide slices before being dried under various temperatures (fresh: HSF; sun: HSS; blower: HSB, shade: HSSH; oven drying at 30 °C: HSOV30; oven drying at 50ºC: HSOV50). The rhizomes were dried until a constant weight was detected. The Clevenger apparatus was used for 5 h to extract the essential oil (EO) from the fresh and dried H. spicatum rhizomes. These samples were hydrodistilled thrice and dried over anhydrous sodium sulphate.

Identification of individual compounds

Gas chromatography-flame ionization detector (GC-FID)

The six essential oil samples of H. spicatum were run on a Shimadzu 2010 GC with an RXi-5 (30 m*0.25 mm*0.25 μm) column, programmed at an oven temperature of 50 °C to 280 °C equipped with FID. The temperature programming of the column was 50 °C for 2 min, 210 °C for 2 min, and 280 °C for 6 min. The injector temperature was set to 260 °C, and the detector temperature was 280 °C, with a split ratio of 100.0. As a carrier gas, nitrogen was used. The injector flow rate of N2 was 16.3 mL/min and the pressure was 89.8 kPa.

Gas chromatography-mass spectrometry

The GC-MS analysis of all six H. spicatum oil samples was done using a Shimadzu 2010 GC-MS equipped with an RXi-5 fused silica column. The same temperature programming was used as in GC. Both the ion source temperature and the interface temperature were set at 220 °C and 270 °C respectively. The mass range (m/z) was 40–600, the start time was 3 min and the end time was 74.98 min with a scan speed of 2000.

Retention index and library matching

The identification of compounds was possible by analyzing the retention index (RI) determined using homologous series of n-alkanes C9-C33, and verified by combining mass spectrum, data given in the literature, NIST (Version 2.1), and Wiley (7th edition) library12.

The calculation of retention index was calculated by the following formula:

$$\:RI=\left[\left\{\frac{({\text{R}}_{\text{d}\text{e}\text{r}\text{i}\text{v}\text{e}\text{d}}\--\:{\text{R}}_{\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}})}{{\text{R}}_{\text{a}\text{f}\text{t}\text{e}\text{r}}-{\text{R}}_{\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}}}\right\}+N\:\right]\times\:100$$

N = Carbon number of Retention index of the carbon which is less than the derived RI. Rderived = Retention Index of the compound for which Retention Index is being calculated. Rbefore = Retention Index of the carbon number which is lower than the derived Retention Index. Rafter= Retention Index of the carbon number which is higher than the derived Retention Index.

Antioxidant activity

Several in vitro tests were conducted to assess the essential oil’s antioxidant activity, and the results were presented as the mean ± standard deviation (SD) of three experimental data.

DPPH free radical scavenging activity

The methods employed by Bhatt et al. (2019)13 and Rani et al. (2022)14 were followed to test the antioxidant potential of DPPH (2,2’- diphenyl-1- picrylhydrazyl). For this, 5 mL of DPPH in a 0.004% methanol solution was applied to each oil sample to reach the final concentration in the 5–25 µg/mL range. As an antioxidant standard, butylated hydroxytoluene (BHT) was examined at similar concentrations. After 30 min in the dark at room temperature, the absorbance at 517 nm was measured in triplicate using a UV-visible spectrophotometer (Thermo Scientific Evolution-201). The following equation was used to calculate how much DPPH was inhibited:

$$\:\text{\%}\:\text{D}\text{P}\text{P}\text{H}\:\text{r}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l}\:\text{s}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:{IC}_{50}=\left[1-\frac{\text{A}\text{t}}{\text{A}\text{o}}\right]\times\:100$$

Ao stands for absorbance of the control, At for the sample or standard absorbance at 517 nm, and IC for inhibitory concentration. Inhibition percentages were plotted against concentration to determine the IC50 (half-maximal inhibitory concentration) values.

Iron metal chelating activity

The metal chelating activity was examined using the technique outlined by Bhatt et al. (2019)13. Different test sample contents were combined with 0.1 mL of 2mM FeCl2, 0.2 mL of 5mM ferrozine, and 4.7 mL of methanol to produce a final volume of 5mL and final concentrations of 5–25 µg/mL. The solution was then incubated for 10 min at room temperature. A UV visible spectrophotometer (Thermo Scientific, Evolution-201) was used to test the solution’s absorbance at 562 nm after shaking and 30 min of room temperature incubation. Ascorbic acid was used as a common antioxidant within the concentration range of 5–25 µg/mL13,14. The following equation was used to calculate the test sample’s ability to chelate Fe2+ ions:

$$\:\text{\%}\:{Fe}^{2+}\:\text{m}\text{e}\text{t}\text{a}\text{l}\:\text{c}\text{h}\text{e}\text{l}\text{a}\text{t}\text{i}\text{n}\text{g}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:{IC}_{50}=\left[1-\frac{\text{A}\text{t}}{\text{A}\text{o}}\right]\times\:100$$

Where Ao stands for absorbance of the control, At for the sample or standard absorbance at 562 nm, and IC for inhibitory concentration. Inhibition percentages were plotted against concentration to determine the IC50 (half-maximal inhibitory concentration) values.

Hydrogen peroxide radical scavenging activity

Using the previously described methods by Bhatt et al. (2019)13 and Rani et al. (2022)14, the H2O2 radical scavenging activity of the studied materials was measured. The test solution contained 0.4 mL of methanolic solution, 0.6 mL of H2O2 solution (40 mM) in phosphate-buffered saline (PBS; 0.1 M; pH 7.4), and different amounts of essential oils or standard (BHT) to prepare final concentrations in the range of 5–25 µg/mL. The solution was incubated for ten minutes at room temperature. The absorbance at 230 nm was measured in comparison to methanol, which was used as a blank. The formula to determine the H2O2 scavenging is as follows:

$$\:\text{\%}\:{H}_{2}{O}_{2}\text{ a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:{IC}_{50}=\left[1-\frac{\text{A}\text{t}}{\text{A}\text{o}}\right]\times\:100$$

Where, Ao stands for absorbance of the control, At for the absorbance value of the essential oil sample or standards, and IC for inhibitory concentration. Inhibition percentages were plotted against concentration to determine the IC50 (half-maximal inhibitory concentration) values.

Molecular docking

A molecular docking study was conducted to determine the binding modes of key components in essential oils (1,8-cineole, elemol, germacrene-4-ol, α-muurolol, and α-cadinol) with the NADPH oxidase (NOX) receptor, known for producing reactive oxygen species (ROS), following the methodology outlined by Karakoti et al. (2023)15. The NO protein structure was sourced from the Protein Data Bank (PDB) using PDB ID: 2CDU. This structure was prepared for docking by removing water molecules and co-crystallized ligands and adding polar hydrogens, using Discovery Studio to convert it into PDBQT format. The 3D structures of the selected volatile compounds were obtained from the PUBCHEM database in SDF file format. Docking was carried out using PyRx software, where the ligands were imported into Open Babel within PyRx, and energy minimization was performed by adding charges and optimizing using the universal force field. The ligands were then saved in AutoDock Ligand format (PDBQT). The binding energy values for the docked ligand structures were recorded in -kcal/mol. The BIOVIA Discovery Studio visualizer (v21.1.0.20298) was utilized to visualize the PDB files and to generate 2D interaction images of the docking results, as described by Dallakyan and Olson (2015)16.

Ethics approval

The plant experiments were in accordance with relevant institutional, national, and international guidelines and legislation. Additionally, experiments do not violate the IUCN policy statement on Research Involving Species at Risk of Extinction and Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Statistical analysis

The one-way ANOVA was applied using SPSS 16.0 software, and the data on the percentage variation in the constituents of essential oils was statistically evaluated. Using MS-Excel 2019, the results were calculated and presented as the mean value and standard deviation (SD). The agglomerative hierarchical cluster study using the Ward method (XLSTAT, free version) was carried out to understand the effect of drying conditions on the essential oil composition as well as to find out the relationships between the applied drying conditions. The BIOVIA Discovery Studio visualizer (v21.1.0.20298) was used for molecular docking.

Results and discussion

The present study focused on the effect of different drying conditions on the essential oil composition and antioxidant potential of H. spicatum. Effects on oil yield and moisture content were also determined.

Essential oil yield

The oil content present in the fresh, sun, blower, shade, and oven-dried samples at 30 °C and 50˚C was 0.18%, 0.68%, 1.00%, 0.96%, 1.31%, and 1.23%, respectively (Fig. 1). A substantial increase in oil yield was observed in H. spicatum rhizomes following different drying conditions. Raina and Negi (2015)17 reported an oil yield range of 0.84–1.45% for shade dried H. spicatum samples collected from the Nainital district. Furthermore, in a study from Uttarakhand, Bhardwaj et al. (2019)18 observed that 1.16–1.64% oil content was extracted from H. spicatum. According to Negi et al. (2014)19, the oil yield of H. spicatum was found to be 6.12% when the oil was extracted from rhizomes that were dried at 40 °C. The findings of the present research were found to be identical to the results of a study carried out by Aabha et al. (2022)11 in which the oven-dried Zingiber officinale rhizomes at 30 °C showed the highest oil yield. The results showed that as a result of drying, the oil yield of H. spicatum increased significantly.

Fig. 1
figure 1

Effect of drying conditions on the essential oil yield of H. spicatum.

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Essential oil composition

The essential oil composition of fresh H. spicatum rhizomes

The essential oil of fresh H. spicatum rhizomes showed the existence of 51 compounds, out of which 35 were known for representing 92.66% of the total oil. The fresh oil had an 85% moisture content. The oil contained α-cadinol (20.33%), elemol (20.03%), 1,8-cineole (14.62%), germacrene-4-ol (11.26%), and α-muurolol (7.21%) as major constituents. The minor constituents were δ-cadinene (3.97%) and γ-eudesmol (2.78%) (Table 1; Fig. 2). Prakash et al. (2010)6 from Uttarakhand showed the presence of 1,8-cineole (17.60%), γ-10-epi-eudesmol (9.70%), δ-cadinene (7.50%), germacrene-D-4-ol (6.80%), and γ-cadinene (5.40%) as major components. However, in the essential oil of H. spicatum, a significant percentage of 1,8-cineole (64%) was discovered by Verma and Padalia (2010)5. Furthermore, 1,8-cineole (4.3–42.8%), terpinen-4-ol (0.9–19.5%), 10-epi-γ-eudesmol (1.2–12.4%), β-selinene (1.3–6.8%), linalool (0.6–6.4%), β-pinene (4.0-5.9%), α-thujene (0.1–5.8%) and α-pinene (1.8–5.7%) were observed as the major components of the oil20.

Table 1 Percentage of compounds present in the essential oil of H. spicatum under different drying conditions.
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Fig. 2
figure 2

GC Chromatogram of fresh rhizomes oil sample.

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According to Koundal et al. (2015)21, the predominant compounds of H. spicatum oil were 1,8- cineole (52.9–58.20%), β-eudesmol (4.0-11.7%) and 10-epi-γ-eudesmol (1.8–7.1%). In another study, 1,8-cineole (30.84%), β-eudesmol (14.50%), β-pinene (9.24%), α-pinene (9.12%), elemol (6.42%), and linalool (5.29%) were observed to be the predominant compounds in the oil of fresh H. spicatum22. An investigation by Mishra et al. (2016)23 showed the presence of β-eudesmol (0.00-26.57%), 1,8-cineole (5.00-25.78%), hedycaryol (1.10-22.38%), spathulenol (1.67–13.83%), γ-eudesmol (0.00-12.35%), cubenol (1.65–8.85%), β-cadinene (1.74–6.77%), and γ-muurolol (0.00-6.63%) as the predominant components in the oil. Compounds such as 1,8-cineole (21.81–21.18%) and δ-cadinol (18.43–17.49%) were the main compounds present in summer and rainy season collection from Uttarakhand17. Rawat et al. (2018)3 observed 1,8-cineole (38.69%), α-eudesmol (15.81%), β-eudesmol (8.21%), cadin-4-en-10-ol (7.87%), elemol (6.92%) and linalool (5.69%) as the predominant components of the oil. Additionally, a study by Rawat et al. (2021)4 identified the primary constituents of the essential oil as 1,8-cineole (40.5–59.9%), elemol (2.9-13.82%), and β-eudesmol (5.1–10.4%), confirming their presence as major compounds.

The essential oil composition of sun-dried H. spicatum rhizomes

Twenty-eight compounds out of 40 were identified, which represent 94.83% The oil contained α-cadinol (25.06%), 1,8-cineole (23.68%), elemol (15.00%), and α-muurolol (7.74%) as major components, along with germacrene-4-ol (4.95%), γ-eudesmol (3.57%) and δ-cadinene (3.37%) in smaller amounts (< 5.00%) (Table 1; Fig. 3). It took 38 h to get the constant weight and the moisture content was 8.64%.

Fig. 3
figure 3

GC Chromatogram of sun dried rhizomes oil sample.

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The essential oil composition of blower dried rhizomes of H. spicatum

In the blower-dried rhizome oil sample, a total of 68 compounds were present, among which 35 compounds representing 95.68% were identified. The dominant compounds of the oil were 1,8-cineole (40.85%), α-cadinol (14.71%), elemol (12.28%), and germacrene-4-ol (5.13%) (Table 1; Fig. 4). The other constituents present in minor amounts were α-muurolol (4.61%), linalool (3.32%), γ-eudesmol (2.04%), and δ-cadinene (2.00%) (Table 1). It took 17 h to get the constant weight.

Fig. 4
figure 4

GC Chromatogram of blower dried rhizomes oil sample.

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The essential oil composition of shade dried H. spicatum rhizomes

Thirty-three compounds out of fifty-three compounds were recognized in the shade-dried sample. Therefore, the total identified percentage was 96.85. The oil contained 1,8-cineole (53.87%), α-cadinol (12.15%), and elemol (6.78%) in considerable amounts, while germacrene-4-ol (3.73%), α-muurolol (3.68%), and linalool (3.41%) were observed as minor constituents (Table 1; Fig. 5). It has taken 13 days to get the constant weight.

Fig. 5
figure 5

GC Chromatogram of shade dried rhizomes oil sample.

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The essential oil composition of oven dried H. spicatum rhizomes at 30 °C

There were 64 compounds in the essential oil of oven-dried rhizomes of H. spicatum at 30˚C, out of which 34 compounds were identified, representing 97.10% of the entire oil. This type of drying required 53 h in an oven at 30 °C to achieve a constant weight. The highest oil yield (1.31%) was observed for this drying condition. The oil contained 1,8-cineole (53.14%), α-cadinol (10.62%), elemol (6.56%), and germacrene-4-ol (6.16%) as the major compounds, while linalool (3.56%), α-muurolol (3.32%), and β-pinene (2.10%), were found as the minor components of the oil (Table 1; Fig. 6).

Fig. 6
figure 6

GC Chromatogram of oven dried rhizomes at 30ºC oil sample.

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The essential oil composition of oven dried H. spicatum rhizomes at 50 °C

Thirty-four compounds corresponding to 93.22% were identified in the essential oil extracted from H. spicatum oven dried rhizomes at 50 °C. The predominant compounds of the oil were 1,8-cineole (37.28%), α-cadinol (14.9%), elemol (10.31%), and germacrene-4-ol (6.92%). However, the minor components were α-muurolol (4.63%), linalool (3.85%), δ-cadinene (2.82%) and γ-eudesmol (2.29%) (Table 1; Fig. 7). It took 45 h to reach the constant weight.

Fig. 7
figure 7

GC Chromatogram of oven dried rhizomes at 50ºC oil sample.

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Variation in the essential oil components as a result of drying

The results showed that the amount of 1,8-cineole increased by all types of drying conditions. The percentage of 1,8-cineole under all drying conditions followed the order: HSSH > HSOV30 > HSB > HSOV50 > HSS (Fig. 8; Table 1). The highest amount was present in shade dried rhizomes (53.87%) while the lowest amount was observed in the sun-dried material (23.68%). Bhatt et al. (2015)13 reported a significant increase in the amount of 1,8-cineole (from 9.14 to 17.86%) in O. americanum under shade drying conditions. Drying also resulted in a decrease in the percentage of certain major components.The highest percentage of elemol was present in the fresh H. spicatum rhizomes (20.03%) and observed to decrease under all drying conditions in the following sequence: HSS > HSB > HSOV50 > HSSH > HSOV30. Further, the content of germacrene-4-ol was observed to decrease from 11.26% in HSF (fresh) to 6.92% in HSOV50, 6.16% in HSOV30, 5.13% in HSB, 4.95% in HSS and 3.73% in shade dried (HSSH) material. The percentage of two important major compounds (α-cadinol and α-muurolol) decreased upon drying except for sun dried material, where the content increased from 20.33% (fresh) to 25.0% (HSS) for α-cadinol and 7.21% (fresh) to 7.74% (HSS) for α-muurolol (Fig. 8). The percentage of linalool increased by all the drying conditions and the highest percentage was obtained by HSOV50 (3.85%) and followed by HSOV30 (3.56%) and HSSH (3.41%). Six compounds namely 2-nonanone, α-copaene, (E)-9-epi-caryophyllene, γ-murolene, germacrene D, and α-muurolene were absent in the rhizomes dried under sunny conditions while only one compound i.e., germacra-4(5), 5(10) trien-1-α-ol was absent in the essential oil of H. spicatum dried under shade.

Fig. 8
figure 8

Major compounds present in H. spicatum under different drying conditions.

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Class of compounds

The highest percentage of oxygenated sesquiterpenes (OS) (66.27%) and sesquiterpene hydrocarbons (SH) (7.17%) was detected for the fresh H. spicatum oil. However, the percentage of OS and SH was observed to decrease under all drying conditions, and the highest decrease was noted for HSSH (30.74 and 2.55% respectively). Furthermore, the highest percentages of monoterpene hydrocarbons (MH) (56.59%) and oxygenated monoterpenes (OM) (6.76%) were observed for HSSH (Fig. 9). According to Sabulal et al. (2007)24, 2.5% monoterpene hydrocarbons and 20.5% oxygenated monoterpenes were found in H. spicatum oil. However, 23.3% sesquiterpene hydrocarbons and 41.0% oxygenated hydrocarbons existed in the fresh rhizome essential oil. According to Prakash et al. (2010)6, the fresh oil of H. spicatum revealed a significant presence of monoterpene hydrocarbons (16.21%) along with 2.88% oxygenated hydrocarbons. Koundal et al. (2015)21 indicated the presence of a different class of compounds in the following order: OM (57.6–68.0%) > OS (9.9–25.3%) > MH (6.7–10.8%) > SH (2.7–5.9%) in the essential oil of fresh rhizomes of H. spicatum.

Fig. 9
figure 9

The effect of drying condition on the chemical classes composition of H. spicatum rhizomes EO.

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Cluster analysis

A hierarchical cluster approach (HCA) was adopted to explore the impact of drying conditions on the essential oil constituents and separate the identical groups of drying conditions based on their essential oil constituents. The HCA of H. spicatum essential oils obtained from fresh samples and those subjected to different drying conditions revealed a distinct clustering pattern, segregating the oils into two main clusters. Cluster I included two drying conditions: fresh and sun drying however Cluster II contained the essential oils obtained under blower, shade, and oven drying at 30 °C and oven drying at 50 °C (Fig. 10).

  • Cluster I (C1): 1,8-Cineole (14.62–23.68%), α-cadinol (20.33–25.06%), elemol (15.00-20.03%), germacrene-4-ol (4.95–11.26%), α-muurolol (7.21–7.74%).

  • Cluster II (C2): 1,8-Cineole (37.28–53.87%), α-cadinol (10.62–14.90%), elemol (6.56–12.28%), germacrene-4-ol (3.73–6.92%), α-muurolol (5.00%).

The analysis suggested that sun drying retained the bioactive components of H. spicatum as in Cluster II, fresh and sun drying conditions grouped. A previous investigation by Aabha et al. (2022)11 suggested the presence of three clusters in Z. officinale (a Zingiberaceae family plant): Cluster I included blower drying, sun drying, and oven drying at 30 °C and 50 °C conditions while Cluster II contained essential oils extracted from fresh plants. However, cluster III was formed by the oil obtained from shade dried plant material.

Fig. 10
figure 10

Cluster analysis of the essential oils of H. spicatum under different drying conditions.

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Principle component analysis

The multivariate analysis PCA (principal component analysis) applied to the major compounds for different drying conditions is shown in Fig. 11 where PC1 explains 82.36%, and PC2 explains 16.29% of the variations. In addition, Fig. 11 shows the compounds that each characteristic group formed in the multivariate analysis, group I, which comprises HSF and HSS conditions having germacrene-4-ol, elemol, α-cadinol, and α-muurolol. On the other hand, group II is formed by HSOV50, HSOV30 HSB, and HSSH conditions and the compound 1,8-cineole.

Fig. 11
figure 11

Principal component analysis of the essential oils processed under different drying conditions on the basis of their major compounds.

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Compounds germacrene-4-ol and elemol contributed positively towards HSF to PC2, α-cadinol, and α-muurolol contributed negatively towards HSS to PC2. Group II comprised HSOV50, HSOV30 HSB, and HSSH conditions towards1,8-cineole. 1,8-Cineole contributed positively to HSOV50, HSOV30 to PC2 and negatively towards HSB and HSSH to PC2.

Antioxidant activity

Since all of the antioxidant features cannot be expressed by a single process, the in-vitro antioxidant activity was evaluated using three separate techniques: DPPH, reducing power, and H2O2 assay. To express the essential oil’s ability to inhibit DPPH, reducing power, and H2O2, the 50% scavenging activity, or IC50 (µg/mL) values were calculated. Ascorbic acid was used as the standard for metal chelating activity, whereas BHT served as the reference for DPPH and H2O2 assays.

According to DPPH assay, fresh oil sample (HSF) exhibited the highest antioxidant activity (IC50 = 2.19 µg/mL), followed by HSOV30 (IC50 = 4.58 µg/mL), HSSH (IC50 = 4.70 µg/mL), HSOV50 (IC50 = 4.73 µg/mL), HSS (IC50 = 4.80 µg/mL) and HSB (IC50 = 5.68 µg/mL) samples. For DPPH, metal chelating activity, and H2O2 assays, the IC50 values for the oils were lower than the respective standard (Table 2). The standard had an IC50 value of 8.38 µg/mL which indicated that the six oil samples had higher antioxidant activity than the standard. Germacrene-4-ol (r = − 0.879, p < 0.05) exhibited a significant negative correlation with the DPPH assay IC50 value, indicating a direct relationship between germacrene-4-ol and antioxidant activity as measured by DPPH assay (Table 3). Rawat et al. (2022) found the IC50 to be 10.00 µg/mL and 25.40 µg/mL for H. spicatum rhizome essential oils collected from Nainital and Himachal Pradesh sites25.

Table 2 Antioxidant activities of H. spicatum drying under different drying conditions.
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Table 3 Pearson correlation coefficient (r) between major components of the essential oils, class of compounds (MH, OM, SH, OS) and IC50 of antioxidant activities by DPPH radical scavenging, metal chelating and H2O2 scavenging assays.
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For metal chelating assay, the order of activity was as follows: HSF (IC50 = 1.60 µg/mL) > HSSOV30 (IC50 = 4.29 µg/mL) > HSSH (IC50 = 4.81 µg/mL) > HSOV50 (IC50 = 4.98 µg/mL) > HSS (IC50 = 5.09 µg/mL) > HSB (IC50 = 5.12 µg/mL). The IC50 value for ascorbic acid was 20.66 µg/mL which was higher than all the tested essential oil samples (Table 2). For metal chelating activity, the IC50 values of the essential oils of H. spicatum oil collected from Nainital and Himachal Pradesh sites were 5.79 and 3.10 µg/mLrespectively25.

Germacrene-4-ol (r = − 0.854, p < 0.05) and elemol (r = − 0.875, p < 0.05) showed a strong and significant negative correlation with the IC50 value, suggesting a direct effect of these components on the antioxidant potential of H. spicatum oil (Table 3).

H2O2 assay showed the lowest value of minimum inhibitory concentration for fresh oil (IC50 = 4.28 µg/mL), followed by HSOV30 (IC50 = 4.65 µg/mL), HSSH (IC50 = 5.64 µg/mL), HSOV50 (IC50 = 5.66 µg/mL), HSS (IC50 = 5.79 µg/mL) and HSB (IC50 = 5.99 µg/mL) samples. BHT had the highest IC50 value of 13.05 µg/mL (Table 2). In a previous investigation on H2O2 assay by Rawat et al. (2022), the IC50 values of the essential oil of H. spicatum rhizomes collected from Nainital and Himanchal Pradesh were detected to be 1.45 μg/mL and 18.62 µg/mL respectively25. The correlation Table 3 indicated that elemol (r = − 0.875, p < 0.05), muurolol (r = − 0.892, p < 0.05), and α-cadinol (r = − 0.826, p < 0.05) were observed to directly affect the activity which was further supported by the significant negative correlation of OS with H2O2 assay IC50.

Ray et al. (2018) analyzed the antioxidant activity of H. coronarium essential oils and found that the essential oils demonstrated significant antioxidant activity, with IC50 values of 0.39–1.66 mg/mL (reducing power) and 0.57–2.19 mg/ml (DPPH)26. Hedychium griffithianum is also known to have significant antioxidant potential27.

The rhizome oil of H. spicatum exhibits considerable antioxidant potential, likely due to the its high content of oxygenated terpenoids, particularly sesquiterpenes. The antioxidation properties of theses oxygenated compounds can be usually noted because of the presence of free electrons or/and hydroxl functional groups, making them more biologically active than non-oxygenated compounds28,29,30. The predominant components of the oil inclusive elemol, germacrene-4-ol, muurolol and α-cadinol ought to act as inhibitors of free radicals and prevent oxidative stress either independently or synergistical31. Geramcrene-4-ol helps in curing skin health because of its possible anti-inflammatory properties. It might also contribute to a plant’s defence against diseases and pests. Because of its adaptability, it is useful in a variety of applications, including food preservation and cosmetics32. Elemol, because of its antibacterial qualities, it is used in natural scents and preservatives. Additionally, elemol also have anti-inflammatory properties that make it useful in skincare products. Its application in perfumery and aromatherapy is facilitated by its pleasing scent33. Muurolol finds application in formulations for natural preservatives. Additionally, it has anti-inflammatory properties due to which it is used in skin care products. The chemical enhances the usage of essential oils in aromatherapy and perfumery by adding to their secent34.

The strong antioxidant effects, including radical scavenging, metal ion chelating, and reducing characteristics, have also been seen in essential oils extracted from H. spicatum rhizomes21,28. Three distinct collections of H. spicatum essential oils exhibited remarkable variability in their composition. Specifically, 1,8-cineole and 10-epi-γ-eudesmol were identified as key markers for two collections, whereas terpin-4-ol and sabinene predominated in one collection from Uttarakhand35. According to Rawat et al. (2011)36, there is a substantial (p < 0.05) association between ABTS, DPPH, and FRAP and total phenolic chemicals. Caryopteris odorata leaf oils were found to be dominated by sesquiterpene hydrocarbons and showed good reducing power and DPPH radical scavenging activity (IC50 = 1.1 ± 0.8)37.

Molecular docking

Molecular modeling techniques have been employed to explore the interactions between natural compounds and pharmacologically relevant molecular targets. To support the experimental findings of antioxidant activity, a molecular docking study was conducted on the protein with PDB ID 2CDU. The binding energies of the primary constituents from the oil ranged from − 6.9 to -5.1 kcal/mol, suggesting moderate to good activity. Both α-muurolol and α-cadinol demonstrated strong binding affinity within the enzyme’s active site. The optimal docked conformation of α-muurolol showed pi-sigma and pi-alkyl interactions with the Tyr71 residue, pi-alkyl interaction with the Leu299 residue, and alkyl interaction with the Ile160 residue. Similarly, α-cadinol exhibited pi-alkyl interaction with the Tyr188 residue and alkyl interactions with the Leu299 and Tyr159 residues. The study indicated that the major constituents engaged favorably with the receptor, predominantly through hydrophobic interactions, suggesting that these compounds could be effective antioxidant agents (Fig. 12; Table 4).

Fig. 12
figure 12

Binding pocket of the interaction of compounds with NADPH oxidase; Molecular interactions established by [A] 1,8-cineole, [B] Elemol, [C] Germacrene-4-ol [D] α-muurolol [E] α-cadinol with the active site NADPH oxidase.

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Table 4 Binding energy (–kcal/mol) of selected major compounds interaction with the protein.
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Conclusions

In the present work, different drying conditions were applied, for preserving the high-quality essential oil, which positively affected the essential oil yield and the content of 1,8-cineole in H. spicatum essential oil. Thus, drying is a good option for keeping therapeutic herbs over time. This study suggested that oven drying at 30 °C might be a better method for obtaining a high essential oil yield and 1,8-cineole content. Among the oils of dried rhizomes of H. spicatum, HSOV30 exhibited the maximum antioxidant activity which could be used for many respiratory diseases like bronchitis rhinosinusitis, asthma, and pneumonia. α-Cadinol, elemol, 1,8-cineole, and germacrene-4-ol were the major compounds present in all the samples. Molecular docking simulations showed that α-muurolol and α-cadinol exhibited promising binding affinities, characterized by extensive hydrophobic interactions with key residues within the enzyme’s active site, suggesting potent inhibitory activity. The present study showed that monoterpene content increased upon drying.

This study provides an in-depth examination of drying techniques’ effects on essential oils’ composition and antioxidant properties of H. spicatum. Molecular docking reveals potential therapeutic applications, but limitations include restricted drying conditions and the need for in vivo studies to confirm biological significance. While the research makes significant contributions, further investigation is needed to explore the broader implications of its findings.