Variability in phenolic compounds, antioxidant capacity and essential oil profile in different tissues of Ferula assa-foetida L. populations in Iran: an opportunity for industrial products

Abstract

Plant responses to multiple environmental conditions in the habitat are characterized by intra-species variety. Ferula assa-foetida L. is a native medical plant in Iran, which has a wide range of therapeutic and pharmacological properties. The objective of the current study was to identify changes in phenolic content, antioxidant capacity and essential oil (EO) composition in different tissues (leaf, flower and fruit) of three populations of F. assa-foetida L. (Sarbisheh, Nehbandan, located in South Khorasan province of Iran). The results showed that total phenolic content (TPC), total flavonoid content (TFC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity in the leaves of Sarbisheh were higher than in other treatments. The amount of TPC, TFC and DPPH scavenging activity in leaves was higher than in other tissues in the Sarbisheh population, increasing by 31 and 70%, 41 and 20%, 8 and 13%, respectively, over that of flowers and fruits. The main EO compounds of leaves were α-pinene (11.86–13.56%), β-pinene (6.80–7.99%), bornyl acetate (15.80–17.33%). The main EO constitutes of flowers were Z-propenyl-sec-butyldisulfide (10.12–12.4%), eco-fenchyl acetate (10.87–12.4%), myrcisticin (13.43–15.87%) and α-bisabolol (11.23–13.76%). The primary compounds of fruit EO were β-pinene (9.32–11.26%), (Z)-1-propenyl sec butyldisulf (17.54–18.87%) and (E)-1-propenyl sec butyldisulf (28.32–30.02%). The agglomerative hierarchical clustering showed that three distinguished clusters were identified for population, where Nehbandan and Qaen had the higher similarity and Sarbisheh completely differed from the two ones. Different plant tissues contain varying types of chemicals, with leaves exhibiting higher levels of phenolic content and antioxidant capacity. This characteristic can be beneficial for industries such as food and pharmaceuticals.

Introduction

Environmental elements including species (genetic), climate, edaphic, elevation and geography, as well as genetic factors, have an impact on the growth and development of plants in ecosystems (Li et al., 2020; Zare Hoseini et al., 2022). The amount of natural compounds in plants can be greatly influenced by these environmental agents (Bajalan et al., 2018). There are a variety of bioactive substances found in plants, most frequently in the form of secondary metabolites, which are more efficient than primary metabolites in improving a plant's ability to survive and deal with environmental problems (Stanković et al., 2017; Hassanabadi et al., 2019). Because of their considerable capacity to produce secondary metabolites, medicinal plants all over the world are valuable resources for novel medications (Ali-Arab et al., 2022). Today, due to increased demand for medicinal herbs, their harvesting is a global concern, particularly when plants are harvested improperly (Hassanabadi et al., 2022). It not only results in the extinction of generations of species, but it also poses a significant risk to the biodiversity of the region and the world. As a result, it is vital to safeguard plants both inside and outside of their ecosystem, which necessitates botanical knowledge in terms of phytochemical properties of plant species (Ali-Arab et al., 2022).

Primary and secondary metabolites are the two categories for phytochemicals in medicinal plants, with primary components consisting of carbohydrates, amino acids, proteins and chlorophyll, and secondary metabolites consisting primarily of alkaloids and terpenoids (Aharoni and Galili, 2011; Fang et al., 2019). Secondary metabolites do not directly participate in the developmental or reproductive stages of living organisms, but they do play a significant role in plant defence against various biotic and abiotic challenges (Jha and Mohamed, 2022). Among these materials, phenolic compounds have been linked to improved human health by scavenging free radicals (Khosropour et al., 2021; Ali-Arab et al., 2022). Moreover, essential oils (EOs) are a mixture of volatile oily compounds that are made as a secondary metabolite in medicinal plants, which their chemical composition is remarkably different based on plant, environment and extraction method (Perczak et al., 2019; Li et al., 2020; Ali-Arab et al., 2022; Vozhdehnazari et al., 2022). Therefore, the viability of secondary metabolites can be observed among the plant populations in different sites (Bajalan et al., 2018; Vozhdehnazari et al., 2022). As a result, the populations of a given plant species can have different EO compounds depending on their adaptation to the corresponding environment. Secondary metabolites play an important role in plant defence in the face of environmental (Vozhdehnazari et al., 2022).

Ferula assa-foetida L., the well-known species of Ferula in the Apiaceae family, is noteworthy for its application in pharmacology, food and industry. It is an herbaceous, downy, monocarpic plant that is perennial and straight, meaty and dense in its root system (Akbarian et al., 2021). Different parts of the plant like flowers and leaves contain a notable EO, with different medicinal applications. Due to the climatic conditions of South Khorasan province, Iran, the effective materials of medicinal plants in this province have high quality (Niazmand and Razavizadeh, 2021). Accordingly, F. assa-foetida L. is one of the native medicinal plants of this province with food and medicinal consumption, which grows in different geographical locations.

There is an increasing interest to obtain the optimum secondary metabolites among different populations of medicinal plants. South Khorasan province due to its wide-range climate is capable of creating different populations of medicinal plants. The current study's hypothesis is that various plant tissues differ in their phenolic content, antioxidant activity and EO compound concentrations. In several research, the changes in biochemical attributes of phenolic content and EO composition of F. assa-foetida L. have been reported. Dastan et al. (2022) represented the variability in antioxidant capacity of different F. pseudalliacea L. tissues, with IC50 in a range of 35.4–39.1 mg mL⁻¹. Hassanabadi et al. (2022) showed that the main EO compounds of fruits in different accession were (E)-1-propenyl sec-butyl disulfide, β-pinene, (Z)-1-propenyl sec-butyl disulfide, α-pinene, thiophene and thiourea have been addressed. However, there is little information on different plant tissues of F. assa-foetida L. concerning secondary metabolites in different habitats. The key research questions are which F. assa-foetida L. tissue has a higher phenolic content and antioxidant capacity, and how EO compounds are distributed in various tissues. Therefore, the goal of the present experiment was to evaluate different tissues (leaf, flower and fruit) in terms of their phenolic content, antioxidant capacity and EO profile in three populations of F. assa-foetida L. in South Khorasan.

Materials and methods

Plant materials and site description

The three different populations of F. assa-foetida L. were sampled from their natural habitats South Khorasan of Iran. Sample identification was done by expert botanists at Birjand University. Meteorological data were also obtained from the nearest meteorological station. The mean precipitation for accessions of Sarbisheh (32°34′42″E; 59°47′44″N), Qaen (33°44′8″E; 59°10′49″N) and Nehbandan (31°32′20″E; 60°2′21″N) was 176.7, 155.9 and 62.2 mm and mean annual temperature was 13.8, 16 and 20.9 °C, respectively. The developed leaves, flowers and mature fruits were harvested at April 10–12, May 5–8 and June 1–3, respectively, in 2019 and 2020. The experiment was done based on completely randomized design at three replicates for biochemical assays. For subsequent analyses, all samples were subjected to a drying process under shaded conditions, utilizing natural air flow and maintaining an ambient temperature.

Total phenolic content (TPC) measurement

The TPC was measured using a Folin–Ciocalteu reagent according to the method described by Slinkard and Singleton (1977). To obtain the extract, 200 mg of plant samples were dissolved in 8 mL ethanol (80%) and centrifuged at 1300 rpm for 15 min. Subsequently, 2.5 mL of 0.2 N Folin–Ciocalteu reagent was added to and after 5 min, 2 mL of 75 g L⁻¹ sodium carbonate solution was added. After 2 h, the absorption of the mixture was read at 760 nm by a spectrophotometer (JENWAY 6305, JENWAY, UK). Gallic acid (GA) was used as the standard to draw the calibration curve and TPC was expressed as mg GA per g of dry weight (DW).

Total flavonoid content (TFC)

The TFC was measured using an aluminium chloride reagent. In total, 1.5 mL of methanol, 0.1 mL of 10% aluminium chloride solution, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water were added to 0.5 mL of the extract. The absorption of the mixture was read at 415 nm after 30 min at room temperature. Quercetin (QE) was used as the standard for drawing the calibration curve. The amount of flavonoids was reported based on the equivalent of mg of QE per g DW (Shraim et al., 2021).

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The tissue extracts’ antioxidant activity was assessed using the DPPH scavenging test (Mensor et al., 2001). The DPPH is a stable radical that absorbs at 517 nm, is purple in colour and changes to yellow when reduced. The well containing the various extract concentrations (0.02, 0.2, 2, 20, 50 and 80 μg mL⁻¹) was filled with 50 μL of DPPH solution (0.004%), which was then left to sit for 30 min at room temperature. In total, 517 nm was used to quantify absorbance. Ascorbic acid was served as the positive control. This is how the percentage of DPPH inhibition was determined:

$${\rm Inhibition\ }( {\rm \% } ) {\rm} = [ {( {{\rm A}_{{\rm con}} \hbox{-}{\rm \ }{\rm A}_{{\rm sam}}} ) {\rm /\ }{\rm A}_{{\rm con}}} ] {\rm \times 100, \;}$$

where A_con is the absorbance of negative control and A_sam is the absorbance of the sample.

EO extraction

Plant samples were shade-dried and 100 g was used in a Clevenger device for 3 h to extract the EO content. In preparation for further examination, the extracted EOs dried with anhydrous sodium sulfate and kept at −4 °C (Sefidkon et al., 2006).

Gas chromatography/mass spectrometry (GC/MS) and compound identification

The EO was first diluted in hexane (1 mg mL⁻¹), and 1 μL was then injected into an Agilent 6890 GC equipped with a BPX5 capillary column 30 m × 0.25 mm with a film thickness of 0.25 m. During 5 min, the column's temperature was set at 50 °C. From there, it was raised by 3 °C per minute to 240 °C and then 15 °C per minute to 300 °C. With a split ratio of one-third, the injector and detector were adjusted to respective temperatures of 240 and 290 °C. At a 0.5 mL min⁻¹ flow rate, helium was used as the carrier gas. The GC/MS (Agilent 5973N mass detector) followed the same flow pattern as the GC in terms of oven and column temperature as well as helium flow rate. The ion source's temperature was set to 220 °C, and the ionization voltage was set at 70 eV.

Retention time and mass spectra of the compounds were compared with data from the Wiley library and the literature, as well as with those of real compounds, in order to identify the components. The components were further identified and their retention indices were calculated using a mixture of n-alkanes (C8–C25).

Statistical analysis

The data were analysed by SAS software version 9.2 and LSD test was used to compare the means. XLSTAT version 2021.5 (Addinsoft, France) was applied to prepare agglomerative hierarchical clustering (AHC) based on Ward variance method and principal component analysis (PCA).

Results

TPC and TFC

The findings demonstrated that plant tissues and populations differed significantly (P ≤ 0.05) in terms of plant TPC. When compared to other tissues, the leaf had the highest concentration of TPC. In particular, fruits and flowers in the Sarbisheh population showed declines of 41 and 23%, respectively, relative to leaves. The TPC responded to ecological conditions differently, with Sarbisheh's leaf TPC exhibiting a greater amount by 47 and 113% relative to Qaen and Nehbandadn, respectively (Fig. 1a). The TFC represented different responses to plant tissues and populations. The highest TFC was obtained in leaves of Sarbisheh population as 27.9 mg QE g⁻¹ DW. In Qaen population, 25 and 34% declines in TFC of flowers and fruits as compared with leaves. The maximum TFC in all tissues were obtained in Sarbisheh and followed by Qaen and Nehbandan. In particular, Sarbisheh showed significant increases of 60 and 220% in fruit TFC compared to Qaen and Nehbandan, respectively (Fig. 1b).

Figure 1. Total phenolic content (TPC, a), total flavonoid content (TFC, b) and DPPH scavenging activity (c) in plant tissues of Ferula assa-foetida L. populations.

DPPH scavenging activity

The DPPH scavenging activity had different amounts among the plant tissues and populations, and the effect of environmental conditions was greater than tissues in variability of antioxidant capacity of F. assa-foetida populations. DPPH scavenging activity differed from 31.4% in the fruits of Nehbandan to 55.4% in the leaves of Sarbisheh. Flower antioxidant capacity in Sarbisheh was 27 and 42% greater than Qaen and Nehbandan, respectively (Fig. 1c).

EO profile

The GC and GC-MS analysis showed that 27 compounds were identified in leaf EO, where the main EOs were α-pinene, β-pinene and bornyl acetate. α-pinene ranged from 11.86% in Nehbandan to 13.56% in Sarbisheh. β-pinene was obtained in a range of 6.8–7.99% with the maximum amount in Sarbisheh (Table 1). The main constitutes of EOs of the flowers were Z-propenyl-sec-butyldisulfide, eco-fenchyl acetate, myrcisticin and α-bisabolol. Although Z-propenyl-sec-butyldisulfide and eco-fenchyl acetate in Sarbisheh were higher, myrcisticin and α-bisabolol represented the maximum amounts in Nehbandan (Table 2). The primary compounds of EO fruits were β-pinene, (Z)-1-propenyl-sec-butyldisulfide and (E)-1-propenyl-sec-butyldisulfide. β-pinene in Sarbisheh was higher and (Z)-1-propenyl-sec-butyldisulfide and (E)-1-propenyl-sec-butyldisulfide were lower (Table 3).

Table 1. Essential oil composition in leaves of Ferula assa-foetida L. populations

Table 2. Essential oil composition in flowers of Ferula assa-foetida L. populations

Table 3. Essential oil composition in fruits of Ferula assa-foetida L. populations

Multivariate analysis

The PCA revealed that the predominant alterations can be attributed to two primary factors, F1 and F2. These factors were found to account for the majority of the observed changes. F1 mainly justified all leaf parameters among the F. assa-foetida populations. Nehbandan and Sarbisheh were explained by F1, and represented the negative correlation based on leaf EO profile and biochemical attributes (Fig. 2a). However, Qaen was determined by F2. For flower compounds, F1 justified 97% of the changes. The interesting results were that myrcisticin and α-bisabolol negatively correlated with other traits (Fig. 2b). Like leaf and flower tissues, fruits were mainly justified by F1, and the negative collation was obtained for Sarbisheh with Qaen and Nehbandan (Fig. 2c).

Figure 2. Principal component analysis (PCA) for leaves (a), flowers (b) and fruits (c) of Ferula assa-foetida L. populations. TPC, total phenolic content; TFC, total flavonoid content; DPPH, 2-diphenyl-1-picrylhydrazyl.

Based on the AHC of leaves (Fig. 3a), flowers (Fig. 3b) and fruits (Fig. 3c), three distinguished clusters were identified for population, where Nehbandan and Qaen had the higher similarity and Sarbisheh were completely differed from the tow ones.

Figure 3. Agglomerative hierarchical clustering (AHC) for leaves (a), flowers (b) and fruits (c) of Ferula assa-foetida L. populations.

Discussion

In three distinct populations of F. assa-foetida, the alterations in TPC and TFC were noted. The anti-oxidant properties of medicinal plants are closely tied to the phenolic chemicals. Many populations of medicinal plants have reported similar alterations in phenolic chemicals to findings of the present work. Hassanabadi et al. (2019) represented that variability in TPC (5.2–6.5 mg GA g⁻¹ DW) and TFC (3.1–3.8 mg rutin g⁻¹ DW) in five Pulicaria gnaphaloides (Vent.) Boiss populations. These changes can be due to the type of extract, genetics and environment. According to Narimani et al. (2021), the populations of Ferula cupularis showed significant variations in terms of TPC (250–387 mg GA 100 g⁻¹) and TFC (34.4–41.1 mg QE 100 g⁻¹). In populations and plant tissues, the relationship between TPC and TFC was generally similar and has previously been approved for Coriandrum sativum (Afshari et al., 2021), Pulicaria gnaphalodes (2019) and Berberis integerrima bunge (Khosropour et al., 2021). Changes in TPC and TFC are caused by environmental variables such as sunshine, temperature and rainfall (Bibi et al., 2022). Sarbisheh's considerably higher elevation and more frequent rainfall may result in higher concentrations of TPC and TFC. The sun's direct impact on plant metabolism and the accumulation of more phenolic chemicals, which increase the potential for antioxidants, may explain why plants at higher elevations produce more phenols (Ralepele et al., 2021). The intensity of light can be different due to different elevation of studied sites. Light intensity plays a crucial role in regulating phenolic compound synthesis. Plants exposed to high light intensity often produce higher levels of phenolic compounds compared to those grown under low light conditions. This response is attributed to the activation of light-dependent enzymes involved in the phenylpropanoid pathway, leading to increased phenolic production (Narimani et al., 2021). In similar results, Hassanabadi et al. (2019) represented the higher TPC and TFC in populations from higher elevations. Ali-Arab et al. (2022) showed the modification in secondary metabolism in Thymus daenensis Celak. populations, which mainly can be due to the elevation and temperature. Hence, TPC and TFC are the secondary metabolites in plants that are susceptible to variations in environmental factors. Among the tissues, the leaf showed higher TPC and TFC relative to the flower and fruit. Leaves are exposed to environmental changes for a longer period of time than flowers or fruits, which could explain why they contain more TPC and TFC (Ralepele et al., 2021). It was previously discovered by Niazmand and Razavizadeh (2021) that the TPC and TFC in F. assa-foetida leaves were higher than those in their gums.

An efficient way to describe the antioxidant capability of medicinal plants is through their DPPH scavenging activity. The rate of hydrogen transfers to free radicals and the inhibitory strength are both accelerated by the high concentration of phenolic substances. Sarbisheh has stronger DPPH scavenging capacity than other populations, as evidenced by the higher DPPH scavenging activity in its leaves. The high TPC and TFC at all populations, which confirmed the potent antioxidant activity of these chemicals as previously suggested by Hassanabadi et al. (2019), may be responsible for the significant DPPH scavenging activity of F. assa-foetida populations extract.

The changes in EO composition were reported under environmental status. Hassanabadi et al. (2022) showed that elevation was the main factor affecting the EO profile of assa-foetida accessions, which has a close correlation with EO and phenolic compounds. The α-pinene has a close relationship with TPC and TFC among populations, which can be due to the phenolic attributes of the chemicals (Shih et al., 2020). The variability in EO profile among plant tissues can be due to the fact that each organ in the plant plays a distinguished role as a sink and source of chemical compounds (Dodoš et al., 2019; Lima et al., 2021). Dastan et al. (2022) showed that α-pinene was the main compound in all tissues of Ferula pseudalliacea Rech. However, in this study, each tissue represented different EO composition, as previously reported by Niazmand and Razavizadeh (2021), who indicated that leaf extract constituted greater concentration of carvacrol and α-bisabolol, while gum extract had high amounts of (Z)-b-ocimene (20.91%) and (E)-1-propenyl-sec-butyl-disulfide. The variability of EO compounds in the tissues can be due to the different metabolic pathways of chemicals in each part of plants (Altyar et al., 2020). Accordingly, Marzoug et al. (2011) have represented different amounts of EO compounds in stem, leaf, flower and fruit of Eucalyptus oleosa. In addition, the changes in leaf and fruit have been previously reported in some ferula species like Dastan et al. (2022), who reported the different compounds of EOs from leaves, flowers and fruits of F. pseudalliacea Rech. f.

Conclusions

The current study illustrated how F. assa-foetida L. populations differed in terms of phenolic content, antioxidant activity and EO profile. Higher antioxidant activity and phenolic content were more prevalent in the population inhabiting at higher elevations. The tissues reflected several chemical types that were separate from populations. Although the types of chemicals in the various plant tissues varied, the results showed that leaves had higher phenolic content and antioxidant capability, which can be advantageous for the food and pharmaceutical industries. The results of this study emphasize how crucial it is to take into account population diversity in F. assa-foetida L. when developing novel and highly valuable industrial products. By recognizing and utilizing the diversity in phenolic compounds, antioxidant capacity and EO profiles, industries can capitalize on the potential of F. assa-foetida L. as a valuable resource for various applications. Further research and exploration are needed to fully unlock the industrial potential of F. assa-foetida L. populations in Iran and pave the way for the creation of novel and sustainable products.