Chemical compositions, yield and antioxidant activity of the essential oil of hyssop (Hyssopus officinalis L.) under intercropping with fenugreek (Trigonella foenum-graecum L.)

Abstract The effect of intercropping of hyssop (Hyssopus officinalis L.) from Lamiaceae with a medicinal legume, that is, fenugreek (Trigonella foenum-graecum L.) on the hyssop essential oil was investigated. The highest herbage and essential oil yields of hyssop were produced from sole cropping and also intercropped hyssop with fenugreek (3:1 ratio); however, the highest antioxidant activity was obtained from the intercropped hyssop with fenugreek (1:3 ratio). The highest contents of the major constituents in the hyssop essential oil such as cis- and trans-pinocamphone and β-pinene were obtained from sole cropped of hyssop and the intercropping of hyssop with fenugreek (3:1 ratio). In conclusion, the intercropping of hyssop with fenugreek can increase biodiversity and efficiency of using resources. Therefore, this strategy could be considered in order to recommend in simultaneous production of secondary metabolites in the herbs like hyssop in an agricultural land. Graphical Abstract


Introduction
Intercropping system of a legume plant like fenugreek with a species of medicinal and aromatic plants as an essential element of agricultural sustainability has shown a significant potential for the improvement of quality and productivity of secondary metabolites and active substances Machiani et al. 2019). Fenugreek (Trigonella foenum-graecum L.) known as 'Shanbalileh' in Farsi, belonging to the family Leguminosae, is an annual plant which is extensively cultivated in most regions. The aerial parts and seeds of fenugreek are consumed for medicinal and nutritional purposes (Salehi et al. 2018). Fenugreek can be a very useful legume crop for intercropping with crops (Salehi et al. 2018). Hyssop (Hyssopus officinalis L.) known as 'Zoofa' in Farsi is an herbaceous plant widely used in Iran for its medicinal and aromatic properties. In folkloric medicine, the aerial of parts of hyssop are used as carminative, antiseptic, antimicrobial, and antispasmodic and also to relieve and treat catarrh, asthma, bronchitis and cough, ulcers and wounds . The hyssop essential oil as an important final product is broadly used in the cosmetics, pharmaceutical, and food industries . The essential oil from the aerial parts of various species of Hyssopus is used in the food industry as a condiment and spice and it is also valued in treatment of various diseases especially of the respiratory tract (Venditti et al. 2015;Shomirzoeva et al. 2019). The study of the impact of ecological and management factors and their interaction effects on the quantity and quality of the natural essential oils like hyssop can be important (Kong et al. 2019).
In our knowledge, there is no study on the effect of the planting patterns on the essential oil from hyssop (H. officinalis L.) with fenugreek. Therefore, the aim of this study was to examine the influence of the intercropping on quantity and quality traits of the hyssop essential oil. In general, if the amount of dry matter yield or quantity and quality (the main compounds) of volatile oil like land equivalent ratio (LER) as the important index of the evaluation of investigations on the mixed and intercropping systems is equal to or greater than monoculture condition, we reach important goals in intercropping system.

Results and discussion
The contents of the clear yellow essential oil extracted from hyssop range from 0.3% to 0.5% v/w (Supplementary material, Table S1), in agreement with the values reported by other investigators (Venditti et al. 2015;Aghaei et al. 2019;Ghasemi Pirbalouti et al. 2019). Results analysis of variance indicated that the cropping pattern had significant influences on the essential oil content (p 0.05) and the essential oil yield or essential oil per area unit, that is, hectare (p 0.01). The highest values of the essential oil content and yield were obtained from the sole cropped of hyssop and the intercropping of hyssop þ fenugreek (3:1 ratio) (Table S1). Sole cropping yielded more the essential oil than did other planting pattern due to lack of interspecific competition, lack of shade, and having sufficient space for extending vegetative organs in order to take advantage of available resources (Kordi et al. 2020). The essential oil is a mixture of secondary metabolites and its production is related to leaf oil gland population and monoterpenes and sesquiterpenes biosynthesis. Increased the level of photosynthesis activity and biomass production in hyssop (Table S1) due to increased availability of nutrients in sole cropped system resulting in higher monoterpenes biosynthesis and essential oil production . The essential oil is mostly composed of terpenoid compounds. The isoprenoids, biosynthetic units, making up these metabolites need Acetyl-CoA, NADPH, and ATP, whose synthesis depends on the availability of elements such as nitrogen (Rezaei-Chiyaneh et al. 2020).
The results indicated that the intercropping had significant influences on dry herbal yields in hyssop and fenugreek (Table S1). The maximum values dry herbal yield were obtained from sole cropped of hyssop and the intercropping of hyssop with fenugreek of 3:1 (Table S1). Nasar et al. (2020) reported that the intercropping system by increasing the use efficiency of resources (water, light, and soil nutrients) improves the yield of crops compared to sole cropping. However, in this research the intercropping reduced the herbal weight of fenugreek (Table S1). This might be due the hyssop higher competition than fenugreek for available resources such as nutrients, water, and solar energy (Nasar et al. 2020;Raza et al. 2020).
Based on GC-FID and GC/MS analysis results of the hyssop essential oil, six major compounds were identified (Supplementary material, Table S2 and Figure S1). Trans and cis-pinocamphone and b-pinene were the main components (Table S2). In general, oxygenated monoterpenes (54-60%) and hydrocarbons monoterpenes (18.6-21.3%) were the main class of the chemical components in the hyssop essential oils. The results of the this study are in agreement with those of Ghasemi , who underlined that trans and cis-pinocamphone and b-pinene in the volatile oil of H. officinalis are abundant constituents. Results indicated that the planting pattern had not significantly influence on some major compounds of the hyssop essential oil (Table S2). The concentrations of trans and cis-pinocamphone as important components of hyssop in monoculture and intercropping have not significant difference, which this result can be a desirable outcome. If the concentrations of the main compounds of the volatile oil in the intercropping and monoculture conditions do not differ, we reached the goal of intercropping; however, the planting pattern had significant (p 0.05) effect on the percentages of b-pinene and myrtenol (Table S2). Probably, the intercropping was effective in absorbing nutrients from the soil and receiving light and water, and there was competition for these resources, which ultimately affected the biosynthetic pathways of the two compounds (Brooker et al. 2015;Vafadar-Yengeje et al. 2019;Nasar et al. 2020). The highest amount of b-pinene was achieved in the intercropping of hyssop with fenugreek in 3:1 ratio. Probably due to the competition between these two plants and perhaps the positive effect of fenugreek with biological fixation of nitrogen and soil fertility, intercropping had positive effects on the aromatic composition of b-pinene .
Due to the competition between the two plants, we expected the amounts of trans and cis-pinocamphone and the essential oil yield to decrease, whereas no significant difference between sole cropped and intercropping was observed and these quality traits were maintained under the intercropping conditions. The increase of volatile compounds in intercropping systems could be explained by the improvement in nutrient use due to the better temporal, spatial and chemical complementarity (Duchene et al. 2017). In general, the results of the current study and previous investigations indicate that the cropping pattern can increase or decrease the chemical compositions of the essential oils from the herbs. For instance, the intercropping dragonhead (Dracocephalum moldavica) and soybean (Glycine max) decreased the content of major components in the essential oil dragonhead (Geranial, geranyl acetate, and neral) ) and the intercropping of dill and common bean decreased the content of main components such as dill ether, dill apiole, and p-cymene (Weisani et al. 2015). The meaningful decrease of the content of some major components in the herbs essential oil under the intercropping system may be attributed to the higher competitive ability of medicinal plants with legumes due to the reduced complementary and the facilitative interaction of the two plants species (Rezaei-Chiyaneh et al. 2020). Results of previous investigation  indicated that the intercropping of dill þ common bean or soybean enhanced the amounts of a-phellandrene and b-phellandrene in the dill essential oil. Generally, the intercropping system of the medicinal plants with legumes has shown a significant potential for the improvement of quality and productivity of active substance like essential oil and secondary metabolites (Machiani et al. 2019). This could be explained by higher efficiency in using available environmental resources and improved nitrogen transfer from legume species to companion crops (Machiani et al. 2019). Plant secondary metabolites are often referred to as compounds that have no fundamental role in the maintenance of life processes in the plants, but they are important for the plant to interact with its environment for adaptation (Thakur et al. 2019). In current study, these variations on the major constituents of the essential oil and especially phytocomplex as the main compounds for the biological activity are most likely associated with changes in the biosynthesis pathways and the role of them in plant physiology and metabolism (Vafadar-Yengeje et al. 2019). Generally, the intercropping system has been shown to be more productive and land use efficiency than monoculture. Intercropping of the medicinal and aromatic plants with legumes has shown a significant potential for the improvement of quality and productivity of secondary metabolites and active substances.
The finding of this study indicates that the antioxidant activity of the hyssop essential oil was significantly affected by cropping pattern (p 0.05) ( Table S1). The highest antioxidant activity was obtained from the intercropping of hyssop þ fenugreek (1:3 ratio). The variation in antioxidant activity among the treatments could be attributed to differences in their polyphenolic compounds (Ghasemi ).

Conclusions
The results indicated that the highest quantity and quality yields of hyssop (H. officinalis L.) were produced from sole cropping and also intercropped hyssop with fenugreek (3:1 ratio); while, the maximum antioxidant capacity was obtained from the intercropped hyssop with fenugreek (1:3 ratio). In addition, the sole cropped of fenugreek was produced the highest dry herbage. The concentrations of some main constituents detected in the hyssop essential oil were affected by the planting pattern; the highest amounts of (cis-and trans-) pinocamphone as predominant compound were obtained from sole cropped of hyssop and the intercropping of 75% hyssop with 25% fenugreek. Oxygenated monoterpenes (ketones) like cis-pinocamphone and trans-pinocamphone in the hyssop essential oil have biological activities such as antioxidant effect and antimicrobial activity against many food-borne pathogens. In addition, the highest concentration of b-pinene, as a volatile compound was detected in the essential oil obtained from the intercropping of hyssop and fenugreek. Finally, it can be stated that some characteristics of the intercropping hyssop with fenugreek were higher than the monoculture and the intercropping system of hyssop and a legume with an increase biodiversity and the efficiency of using resources.