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Impacts of harvest time and water stress on the growth and essential oil components of horehound (Marrubium vulgare)
Scientia Horticulturae 232 (2018) 139–144
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Impacts of harvest time and water stress on the growth and essential oil components of horehound (Marrubium vulgare)
T
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Abeer A. Mahmouda, A.S.H. Gendyb, H.A.H. Said-Al Ahlc, D. Grulovad, Tess Astatkiee, , T.M. Abdelrazikc a
Department of Botany (Plant Physiology Section), Faculty of Agriculture, Cairo University, Egypt Department of Horticulture, Faculty of Agriculture, Zagazig University, Egypt c Medicinal and Aromatic Plants Researches Department, National Research Centre,33 El-Bohouth St., Dokki, Giza, 12622, Egypt d Department of Ecology, Faculty of Humanities and Natural Sciences, University of Prešov, Prešov, Slovak Republic e Dalhousie University, Faculty of Agriculture, Truro, NS, Canada b
A R T I C L E I N F O
A B S T R A C T
Keywords: Marrubium vulgare Irrigation Harvesting frequency Essential oil Thymol Carvacrol
The study investigated the effect of water stress and harvesting frequency on the growth and essential oil (EO) content and components of Marrubium vulgare grown under Egyptian conditions. Two-month old seedlings of Marrubium vulgare were transplanted into pots and irrigation treatments (4, 8 and 12 days) were applied one month after transplanting. Growth and chemical component response variables were determined at flowering stage using plants harvested at 80, 150 and 210 days after transplanting. Essential oil was obtained by hydrodistillation and expressed as ml 100 g−1 fresh herb. Chemical composition of the EO was determined using liquid chromatography linked to mass spectrometry (GC–MS). The results showed that Marrubium plants irrigated every 4 days and harvested at 210 days gave the highest plant height, number of branches and fresh weight, whereas their lowest values were obtained when harvested at 210 days and irrigated every 12 days. On the other hand, the EO content of Marrubium plants irrigated every 12 days and harvested at 80 days was the highest, and the lowest was obtained when harvested at 210 days and irrigated every 4 days. Eighty compounds were identified in the essential oil of Marrubium vulgare. Thymol (29.6–60.7%), carvacrol (0.5–19.3%), m-cymene (1.0–14.2%), γ-terpinene (1.1–12.1%), thymol methyl ether (0.4–10.4%) and α-himachalene (0.0–10.3%) were the major marker compounds, whereas 14 compounds were minor and 60 compounds were considered as traces. In summary, water stress and harvesting frequency affected growth and caused quantitative changes in the essential oil components. It can be concluded that irrigating Marrubium vulgare plants every 4 days and harvesting at 210 days from the transplanting date is essential to maximize production, whereas irrigating every 12 days and harvesting 80 days after transplanting is recommended to maximize essential oil content.
1. Introduction
antioxidants (Vergara-Galicia et al., 2012) and other biological activities. Plant production and essential oil content can be influenced independently by changes in harvesting time or by environmental factors (Hay and Waterman, 1993). Environmental stresses are among the factors most limiting to plant productivity. The environmental factors can also influence the growth and biosynthesis of secondary metabolites in medicinal and aromatic plants. Essential oil yields have been affected by osmotic stress (Charles et al., 1990). Water deficit in plants may lead to physiological disorders, such as a reduction in photosynthesis and transpiration (Sarker et al., 2005). Water stress in plants influences many metabolic processes, and the extent of its effects depends on drought severity. The optimization of irrigation for the
Marrubium vulgare L. (horehound, white horehound) belongs to the Lamiaceae family. It is a perennial, herbaceous medicinal plant native to Europe, northern Africa, and southwestern and central Asia. This plant was frequently employed as a folk medicine to treat a variety of ailments related to upper respiratory tract infections. Nowadays, the plant is widely used as an herbal medicine to treat liver diseases, biliary tract disorders, bronchial asthma and nonproductive cough (Verma et al., 2012). It possesses tonic, stimulant, expectorant, antispasmodic, antidiabetic, diaphoretic, and diuretic properties (Boudjelal et al., 2012; Vergara-Galicia et al., 2012). Essential oils are appreciated for their bioactive efficacy as fungicides, bactericides (Zarai et al., 2011),
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Corresponding author. E-mail address: [email protected] (T. Astatkie).
https://doi.org/10.1016/j.scienta.2018.01.004 Received 2 October 2017; Received in revised form 28 December 2017; Accepted 4 January 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
Scientia Horticulturae 232 (2018) 139–144
A.A. Mahmoud et al.
stage. Plants from each water stress level were harvested at three times; 80, 150 and 210 days after transplanting. The essential oil percentage was determined in the fresh herb using modified Clevenger apparatus (Guenther, 1961. Essential oil (%) was expressed as (ml 100 g−1 fresh herb). The resulted essential oil of each experimental unit was collected and dehydrated over anhydrous sodium sulphate and kept in refrigerator until GC–MS analyses.
production of fresh herbs and essential oils is important, since water is a major component of the fresh produce and affects both weight and quality (Jones and Tardien, 1998). In aromatic plants, soil moisture may cause significant changes in the yield and composition of essential oils; for example, water deficit decreased the oil yield of rosemary (Rosmarinus officinalis L.) (Singh and Ramesh, 2000) and anise (Pimpinella anisum L.) (Zehtab-Salmasi et al., 2001). By contrast, water stress caused a significant increase in oil yield of citronella grass (Cymbopogen winterianus Jowitt.) expressed on the basis of plant fresh mass (Fatima et al., 2000) with the severity of the water stress response varying with cultivar and plant density in parsley (Petroselinum crispumL.) (Petropoulos et al., 2008). Said-Al Ahl and Abdou (2009) on dragonhead and Said-Al Ahl et al. (2009a, 2009b) showed that both essential oil content (%) and yield significantly decreased with increasing water stress levels of Origanum vulgare and Melissa officinalis, respectively. In most perennial aromatic plants, reduction in biomass yield due to repeated harvesting affects essential oil yield. Repeated harvests can either be beneficial or detrimental to oil production, depending on other environmental factors. For example, herbage yield is usually high at first harvest, then becomes constant, and then declines with repeated harvests (Murtagh, 1996; Weiss, 1997). Kothari et al. (2004) found that biomass yield was higher in the first harvest and gradually declined in the subsequent harvests of Ocimum tenuiflorum. Contrary to the decrease in biomass yield, essential oil content was lower in the first harvest and increased gradually in subsequent harvests to reach maximum in the fourth harvest (Kothari et al., 2004). The essential oil in geranium is mostly contained in the leaves; therefore, the higher the proportion of leaves in the harvested produce the better the yield of oil is (Rao et al., 1990). Determination of the correct harvesting time is extremely important both for maximizing yield and oil quality as it exerts remarkable influences on the oil yield of essential oil content of crops (Weiss, 1997). Doimo et al. (1999) reported that not only the harvest time affected oil yield, but that the geographic area where these crops were grown also influenced yield. The chemical composition of the essential oil was also observed to be influenced by environmental changes (i.e., different soil water content, temperature and photoperiod). These environmental conditions may increase or decrease different terpenoids in the crop. However, studies on irrigation intervals and harvesting frequency on yield and essential oil of Marrubium vulgare have not been investigated. Therefore, this study aimed to evaluate the effect of irrigation intervals on the fresh herb yield and essential oil content and their main constituents of Marrubium vulgare L. harvested at three times.
2.2. GC–MS analyses and identification of components GC–MS analyses were carried out on a Varian 450-GC connected with a Varian 220-MS. Separation was achieved using a Factor Four TM capillary column VF 5 ms (30m × 0.25 mm i.d., 0.25 μM film thickness). Injector type 1177 was heated to a temperature of 220 °C. Injection mode was splitless (1 μL of a 1:1000 n-hexane solution). Helium was used as a carrier gas at a constant column flow rate of 1.2 mL min−1. Column temperature was programmed: initial temperature was 50 °C for 10 min, then increased to 100 °C at 3 °C min−1, maintained isothermal for 5 min and then increased to 150 °C at 10 °C min−1. The total time for analysis was 46.7 min. The mass spectrometer trap was heated to 200 °C, manifold 50 °C and transfer line 270 °C. Mass spectra were scanned every 1 s in the range 40–650m/z. Components were identified by comparison of their mass spectra with those stored in NIST 02 (software library) or with mass spectra from the literature (Jennings and Shibamoto, 1980; Adams, 2007), as well as by comparison of their retention indices with standards. 2.3. Statistical analysis Repeated Measures Analysis (RMA) of a Randomized Blocks Design with the factor of interest being Irrigation (3 levels: 4, 8, and 12 days interval), and harvesting repeatedly at three time points (Day 80,150, and 210 after transplanting) was completed to determine the effect of irrigation on height, number of branches, weight, and volatile oil of Marrubium, and how this effect evolved over the harvesting times. Since the whole experiment was conducted during two seasons (2014 and 2015), Season was used as a blocking factor. Since the response measurements were measured at different harvest times from the same experimental unit (pot), the values measured repeatedly are expected to be dependent on each other. The most appropriate covariance structure (dependence) for each response variable was determined using the Akaike Information Criterion (Littell et al., 1998). The validity of model assumptions (normal distribution and constant variance of the error terms) were verified by examining the residuals as described in Montgomery (2013). All analyses were completed using the Mixed Procedure of SAS (SAS Institute Inc., 2014). For significant (pvalue < 0.05) or marginally significant (0.05 < p-value < 0.1) effects, multiple means comparisons were completed by comparing the least squares means of the corresponding treatment combinations. Letter groupings were generated using a 5% level of significance for the main effects and using a 1% level of significance for interaction effects to protect Type I experimentwise error rate from over inflation.
2. Materials and methods 2.1. Plant material Seeds of Marrubium vulgare were obtained from the HEM ZADEN B.V Venhuizen − The Netherlands. Seeds were sown in the nursery on March 15 of 2014 and 2015. Two months after seed sowing, the seedlings were transplanted into pots (30 cm diameter, 50 cm depth) filled with 10 kg of air dried soil on May 15 of each year. The pots contained three seedlings each and were placed in full sun light. The experiment was carried out under the natural conditions of the greenhouse of the National Research Center, Dokki, Giza, Egypt. The soil texture was sandy loam, having a physical composition of: 45.00% sand, 28.25% silt, 26.75% clay and 0.85% organic matter. Chemical analyses of the soil showed: pH = 8.40; E.C. = 0.79 dsm−1; total nitrogen = 0.13%; available phosphorus = 2.18 mg/100gram; potassium = 0.02 mg/ 100gram. All properties were determined according to the standard methods (Jackson, 1973). Irrigation interval treatments (4, 8 and 12 days), of two liters of water were applied per pot a month after transplanting. Growth characters (plant height [cm], number of branches/plant, herb fresh weight [g/plant]) and chemical constituents were determined at the flowering
3. Results and discussion 3.1. Growth parameters The interaction effect of Irrigation interval and Harvest day was significant on plant height, number of branches, and herb fresh weight of Marrubium vulgare (Table 1), which suggests that the effect of irrigation interval was not consistent at all harvest days after transplanting. The plants irrigated every 4 days gave the highest height, number of branches, and weight at 210 days after transplanting (Fig. 1). The lowest mean height, number of branches, and weight was obtained from those irrigated every 12 days and harvested on day 80 after transplanting (Fig. 1). However, at 80 days after transplanting, 140
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resulted in higher absorption of nutrients and water leading to production of higher vegetative biomass (Singh et al., 1997). Increasing levels of water stress reduces growth and yield due to reduction in photosynthesis and plant biomass because photosynthesis would be limited by low CO2 availability due to reduced stomatal and mesophyll conductance. Drought stress is associated with stomatal closure that leads to reduced CO2 fixation. However, irrigation repeated in every 4 days seemed to be optimal and produced the highest production of fresh biomass in other plants as well (Srivastava and Srivastava, 2007). The superiority of the plants that received the highest rate of irrigation treatments in producing the heaviest total plant fresh weight was in agreement with that in literature (Said-Al Ahl et al., 2009c; Moeini Alishah et al., 2006).
Table 1 ANOVA p-values that show the main and interaction effects of Irrigation (Irr) and Days to harvesting (DH) on height, number of branches, weight, and volatile oil of Marrubium. Significant effects that require multiple means comparison are shown in bold. Effect
Height
Number of branches
Weight
Volatile oil
Block Irr DH Irr*DH
0.003 0.001 0.001 0.001
0.032 0.001 0.001 0.001
0.234 0.001 0.001 0.001
0.151 0.001 0.001 0.025
irrigation intervals 4 and 8 days did not give significantly different height and number of branches, but only weight, with 4 days interval giving higher weight than 8 days interval (Fig. 1). Although there was no significant difference between the mean heights of 4 and 8 days irrigation interval when harvested at 150 days after transplanting, 4 days irrigation interval gave significantly higher number of branches and weight (Fig. 1). These results are in agreement with those reported by Said-Al Ahl et al. (2009a, b, c) who concluded that the highest herb yield of oregano were recorded in plants that received the highest amount of water (irrigated every 3 days). The pronounced effect of increased irrigation on fresh herb yield may be attributed to the availability of sufficient moisture around the root concentrated and thus a greater proliferation of root biomass. This
3.2. Essential oil content Irrigation intervals and harvest frequency interacted on the percentage of essential oils (volatile oil) of Marrubium as well (Table 1). However, the effect of these factors on volatile oil reversed from that on the growth parameters. The highest volatile oil was obtained from irrigation intervals of 8 days and 12 days harvested at 80 days after transplanting and the lowest volatile oil was obtained from those irrigated every 4 days and 8 days harvested at 150 days and 210 days after transplanting (Fig. 1). Although there was no significant difference
Fig. 1. Mean A) height (cm), B) number of branches, C) weight (g/plant), and D) volatile oil (%) of Marrubium obtained from the three irrigation intervals and harvested at the three days after transplanting. Within each plot, means sharing the same letter are not significantly different from each other.
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camphene (0.0–1.0%), 2-cyclohexen-1-ol (0.0-1.0%), and cis-α-bisabolene (0.0–1.0%) were the minor compounds (Table 2). The remaining 60 compounds, namely β-Phellandrene, (−)-β-Pinene, (−)-Limonene, trans-β-Ocimene, cis-β-Ocimene, cis-β-Terpineol, Terpinolene, L-transPinocarveol, (−)Camphor, Menthone, Isogeraniol, 1-Terpinen-4-ol, αTerpineol, p-Menth-8-en-2-one, trans, d-Verbenol, (+)Carvone, Thymoquinone, cis-Geraniol, Piperitone, Carvenone, α-Citral, β-Citronellal, Bornyl acetate, Thymol acetate, Cedrene, Copaene, Isobornyl propionate, β-Bourbonene, Phenol, 2-methyl-5-(1-methylethyl)-acetate, Aromadendrene, trans-β-Bergamotene, β-Cadinene, Neoclovene/β-Chamigrene, β-Cubebene, α-Bergamotene, Valencene, Isoledene, αCaryophyllene, (+)-epi-bicyclosesquiphellandrene, α-Amorphene, δElemene, γ-Muurolene, Germacrene D, δ-Salinene, β-Eudesmene, αCubebene, β-Gurjunene, α-Muurolene, Isoleden, δ-Cadinene, Ledol, αCopaene, α-Calacorene, Calacorene epoxide, (−)-Spathulenol, Caryophyllene oxide, β-Guaiene, β-Vatirene, γ-Eudesmol, τ-Cadinol, Seychellene, Aromadendrene oxide, τ-Muurolol, Globulol, Isoaromadendrene epoxide, Longipinocarveol, α-Bisabolol, and Cubenol were considered as traces. The chemical composition of Marrubium vulgare essential oil from various origins has been the subject of many studies. The literature reveals the occurrence of several chemotypes. In Egypt, Salama et al. (2012) reported that thymol and γ-cadinene are major components; ELLeithy et al. (2013) reported β-caryophyllene and germacrene-D as major components; and Said-Al Ahl et al. (2015) reported carvacrol, βphellandrene, carvyl acetate as the major components. In Lithuania, (Z)-β-farnesene, β-caryophyllene, (E)-2-hexenal, α-humulene and germacrene-D were the main components of M. vulgare essential oil (Weel et al., 1999). Such differences were also observed across the world in various chemotypes of M. vulgare and their main constituents (Belhattaba et al., 2006; Hamdaoui et al., 2013; Khanavi et al., 2005; Mahnaz et al., 2005; Morteza-Semnani et al., 2008; Saleh and Glombitza, 1989; Zawiślak, 2012). The results shown in Table 2 indicate that thymol and carvacrol compounds represent a percentage ranging from 42 to 61% of the total oil components. It was also observed that increasing the irrigation interval increases the percentage of thymol, but decreases that of carvacrol content. Specifically, plants that were irrigated with low level of water (12 days) gave the highest percentage of thymol (60.7%) and the lowest percentage of carvacrol (0.5%), while plants irrigated with medium level of water (8 days) and harvested after 150 days from transplanting (second harvest) gave the highest percentage of carvacrol (19.3%) and the lowest percentage of thymol (29.6%). For the other major compounds, plants irrigated with medium level of water (8 days) gave the highest percentages of both thymol methyl ether (10.4%) and α-himachalene (10.3%). Plants irrigated with the highest water amount (4 days) gave the highest percentage of m-cymene (14.2%). Similar to thymol behavior, γ-terpinene compound was the highest in plants that were irrigated with low level of water (12 days). Previous studies have shown that changes in the oil composition of oregano as a result of water stress and harvest date (Letchamo et al., 2004; Rodrigues et al., 2013; Said-Al Ahl and Abdou, 2009; Said-Al Ahl et al., 2009a, b, c).
between mean volatile oil irrigated every 8 and 12 days when harvested at 80 days after transplanting, the mean volatile oil obtained from 12 days irritation interval was significantly higher than that of 8 days irrigation interval when harvested at 150 and 210 days after transplanting. Similar results were reported by Said-Al Ahl et al. (2009c); Simon et al. (1992). These results are also in agreement with those reported by Baher et al. (2002) who concluded that the accumulation of oil increased significantly under severe water stress in Satureja hortensis. Although a limited supply of water or water stress has a negative effect on the development of the plant and yield, when focusing on the biosynthesis of secondary metabolites, water stress is not always completely detrimental (Murtagh, 1996; Yaniv and Palevitch, 1982). Photosynthetic activity is known to be reduced in crops subjected to low light levels and water deficit conditions. Letchamo and Xu (1996) hypothesized that dry matter formation and accumulation of essential oil in thyme was closely related to photosynthesis, and limitations in the net CO2 assimilation rates had a direct or indirect effect on shoot growth and production of the volatile oil. Doimo et al. (1999) reported that the months of harvest affected oil yield. The month of harvesting was also observed to exert remarkable influences on the oil yield of essential oil crops. For example, Weiss (1997) reported that the highest oil content of geranium was observed in July and lowest in February in southern India. Hegazy et al. (2016) indicated that three medicinal Lamiaceae plants harvested in the second cut were much superior in plant height, number of branches, herb fresh weight, and essential oil percentages compared with those in the first cut. It has been reported that yield and its components in plants in general is primarily related to their genetic, climate, edaphic, and their interaction (Rahimmalek et al., 2009; Basu et al., 2009). Also, Said-Al Ahl and Mahmoud, (2010) reported that Ocimum basilicum plants harvested in the second harvest gave the highest plant height, number of branches, fresh matter yield as well as essential oil than those in the first cut. Also Said-Al Ahl et al. (2009a, 2009c) in two experiments on oregano plants reported that harvesting in the second cut gives the best result of herb fresh weight and essential oil content compared to in the first cut. Also, a study on comparing two cutting times indicated that dragonhead performed better in second than in first cutting (Said-Al Ahl et al., 2010). This may have resulted from suitable climatic conditions and state of maturity during the second cutting period. The yield and quality of essential oil produced depends on various intrinsic and extrinsic factors. Intrinsic factors comprise all internal hereditary characters. Extrinsic factors affecting the production of essential oil in plants are soil, nutrition and water supply, climate (light/temperature), maturity, postharvest treatment and distillation method (Varshney, 1991). Penka (1978) showed that the formation and accumulation of essential oil in plants was caused by the action of environmental factors. It might be claimed that the formation and accumulation of essential oil was directly dependent on growth and development of the plants producing oils. 3.3. GC/MS analysis The results of the GC/MS analysis of essential oils of the Marrubium vulgare in the second (2015) season revealed that there are 6 major and 14 minor compounds, and 60 trace compounds. The compounds were categorized as major if the largest percentage measured is more than 10%, as minor if it is between 1 and 10%, and as trace if it is less than 1%. Accordingly, thymol (29.6–60.7%), carvacrol (0.5–19.3%), mcymene (1.0–14.2%), γ-terpinene (1.1–12.1%), thymol methyl ether (0.4–10.4%), and α-himachalene (0.0-10.3%) were major compounds (Table 2); and isoborneol (0.7–9.4%), benzene 1-methoxy-4 methyl2(1-methylethyl) (0.0–9.2%), linalyl anthranilate (0.8–7.0%), γ-cadinene (tr-2.9%), α-terpinene (1.4–2.0%), β-caryophyllene (0.6–1.9%), βmyrcene (0.0–1.8%), eucalyptol/cineole (0.6–1.5%), α-phellandrene (0.4–1.4%), 1-octen-3-ol (0.0–1.2%), 1R-α-pinene (0.0–1.1%),
4. Conclusion The current study showed that water deficiency (irrigated every 12 days) and harvesting frequency influence the growth and essential oil accumulation of Marrubium vulgare plants. Increasing irrigation levels increased the herb fresh weight of Marrubium, but decreased its essential oil content. The highest yields were obtained from plants harvested after 210 days from transplanting date and irrigated every 4 days. But, essential oil content behaved in reverse as the highest values of essential oil was obtained from plants harvested after 80 days from transplanting date and irrigated every 12 days.
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Table 2 Mean values of the major and minor compounds of Marrubium vulgare L obtained from the three irrigation intervals (I-4, I-8, and I-12 Irrigation every 4, 8, and 12 days, respectively) and harvested after 80 days (H1), after 150 days (H2), and after 150 days (H3) from transplanting during the 2015 season. Compound
Major compounds Thymol Carvacrol m-Cymene γ-Terpinene Thymol methyl ether α-Himachalene Minor compounds Isoborneol Benzene 1-methoxy-4 methyl-2(1-methylethyl) Linalyl anthranilate γ-Cadinene α-Terpinene β-Caryophyllene β-Myrcene Eucalyptol/Cineole α-Phellandrene 1-Octen-3-ol 1R-α-Pinene Camphene 2-Cyclohexen-1-ol cis-α-Bisabolene
Rt
RI
I-4
I-8
I-12
Identification
H1
H2
H3
H1
H2
H3
H1
H2
H3
32.4 32.8 16.1 18.2 28.4 38.4
1289 1298 1020 1054 1231 1505
35.6 15.4 14.2 11.5 1.4 0.2
46.1 18.8 8.4 8.9 0.4 nd
54.6 5.9 12.0 1.1 1.4 0.3
31.5 10.7 7.8 7.8 10.4 10.3
29.6 19.3 9.1 9.0 2.4 tr
47.8 8.2 10.2 10.3 3.1 0.4
60.7 0.5 1.4 12.1 1.6 Nd
46.3 3.3 8.3 7.5 5.0 1.6
51.7 4.2 1.0 8.1 4.3 1.5
RI, RI, RI, RI, RI, RI,
MS, Co-I MS, Co-I MS MS, Co-I MS MS
24.8 27.9 21.0 38.6 15.5 36.3 13.8 16.5 9.1 13.4 9.6 10.8 19.1 39.3
1155 1216 1114 1511 1014 1413 988 1026 930 977 932 946 1097 1527
1.8 1.7 2.0 0.1 1.9 1.2 1.7 0.9 1.4 0.7 1.1 0.5 nd 0.2
0.7 0.2 1.2 2.9 1.5 1.9 nd 1.3 0.4 nd nd nd 0.4 –
1.7 3.1 2.6 0.2 1.6 1.7 1.3 1.1 1.0 0.8 0.7 0.3 1.0 0.1
4.7 nd 1.3 0.2 1.4 0.6 1.3 1.5 1.1 0.7 1.1 1.0 nd –
9.4 nd 7.0 0.2 1.4 1.5 1.4 0.8 1.0 0.5 0.7 0.3 nd tr
1.3 nd 2.1 0.3 1.4 1.6 1.2 0.6 0.8 0.8 0.5 0.2 0.9 0.2
1.0 2.8 2.5 0.2 2.0 1.9 1.8 1.0 1.4 0.8 1.0 0.4 nd –
3.0 9.2 0.8 tr 1.4 0.9 1.2 0.6 0.9 1.2 0.7 0.5 nd 1.0
4.7 6.3 1.1 0.2 1.5 1.1 1.3 0.8 1.1 0.9 0.9 0.8 0.7 tr
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS, MS MS MS MS MS, MS, MS MS, MS MS, MS, MS MS
Co-I
Co-I Co-I Co-I Co-I Co-I
Rt = retention time; RI = Kovats retention index determined relative to the series of n-alcanes (C10-C35) on VF-5 MS Capillary Column; Identification method: RI = comparison on Kovats retention indices with published data, MS = comparison of mass spectra with those listed in the NIST 02 library and with published data, Co-I = coinjection with authentic compound; nd = not detetected; tr = traces (˂0.1%).
References
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Said-Al Ahl, H.A.H., Hussein, M.S., Abd El-Kader, A.A., 2010. Effect of nitrogen fertilizer and/or some foliar application on growth, herb yield, essential oil and chemical composition of dragonhead. J. Medicinal Food Plants 2, 12–28. Said-Al Ahl, H.A.H., Gendy, A.S.H., Mahmoud, A.A., Mohamed, H.F.Y., 2015. Essential oil composition of marrubium vulgare L. cultivated in Egypt. Int. J. Plant Res. 1, 138–141. Said-Al Ahl, H.A.H., Mahmoud, A.A., 2010. Effect of zinc and/or iron foliar application on growth and essential oil of sweet basil (Ocimum basilicum L.) under salt stress. Ozean J. Appl. Sci. 3, 97–111. Salama, M.M., Taher, E.E., El-Bahy, M.M., 2012. Molluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus Hoff. Et Link. and Marrubium vulgare L. Rev. Inst. Med. Trop. Sao Paulo. 54, 281–286. Saleh, M.M., Glombitza, K.W., 1989. Volatile oil of Marrubium vulgare and its antischistosomal activity. Planta Med. 55, 105–108. Sarker, B.C., Hara, M., Uemura, M., 2005. Proline synthesis, physiological responses and biomass: yield of eggplants during and after repetitive soil moisture stress. Sci. Hortic. 103, 387–402. Simon, J.E., Reiss-Bubenheim, D., Joly, R.J., Charles, D.J., 1992. Water stress-induced alterations in essential oil content and composition of sweet basil. J. Essent. Oil Res. 4, 71–75. Singh, M., Ramesh, S., 2000. Effect of irrigation and nitrogen on herbage, oil yield and water- use efficiency in rosemary grown under semi- arid tropical conditions. J. Med. Aromatic Plant Sci. 22, 659–662. Singh, M., Ganesha Rao, R.S., Ramesh, S., 1997. Irrigation and nitrogen requirement of lemongrass (Cymbopogon flexuosus (Sleud) Wats) on a red sandy loam soil under semiarid tropical conditions. J. Essent. Oil Res. 9, 569–574. Srivastava, N.K., Srivastava, A.K., 2007. Influence of gibberellic acid on 14 CO2 metabolism, growth and production of alkaloids in Catheranthus roseus. Photosynthetica
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Impacts of harvest time and water stress on the growth and essential oil components of horehound (Marrubium vulgare)
T
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Abeer A. Mahmouda, A.S.H. Gendyb, H.A.H. Said-Al Ahlc, D. Grulovad, Tess Astatkiee, , T.M. Abdelrazikc a
Department of Botany (Plant Physiology Section), Faculty of Agriculture, Cairo University, Egypt Department of Horticulture, Faculty of Agriculture, Zagazig University, Egypt c Medicinal and Aromatic Plants Researches Department, National Research Centre,33 El-Bohouth St., Dokki, Giza, 12622, Egypt d Department of Ecology, Faculty of Humanities and Natural Sciences, University of Prešov, Prešov, Slovak Republic e Dalhousie University, Faculty of Agriculture, Truro, NS, Canada b
A R T I C L E I N F O
A B S T R A C T
Keywords: Marrubium vulgare Irrigation Harvesting frequency Essential oil Thymol Carvacrol
The study investigated the effect of water stress and harvesting frequency on the growth and essential oil (EO) content and components of Marrubium vulgare grown under Egyptian conditions. Two-month old seedlings of Marrubium vulgare were transplanted into pots and irrigation treatments (4, 8 and 12 days) were applied one month after transplanting. Growth and chemical component response variables were determined at flowering stage using plants harvested at 80, 150 and 210 days after transplanting. Essential oil was obtained by hydrodistillation and expressed as ml 100 g−1 fresh herb. Chemical composition of the EO was determined using liquid chromatography linked to mass spectrometry (GC–MS). The results showed that Marrubium plants irrigated every 4 days and harvested at 210 days gave the highest plant height, number of branches and fresh weight, whereas their lowest values were obtained when harvested at 210 days and irrigated every 12 days. On the other hand, the EO content of Marrubium plants irrigated every 12 days and harvested at 80 days was the highest, and the lowest was obtained when harvested at 210 days and irrigated every 4 days. Eighty compounds were identified in the essential oil of Marrubium vulgare. Thymol (29.6–60.7%), carvacrol (0.5–19.3%), m-cymene (1.0–14.2%), γ-terpinene (1.1–12.1%), thymol methyl ether (0.4–10.4%) and α-himachalene (0.0–10.3%) were the major marker compounds, whereas 14 compounds were minor and 60 compounds were considered as traces. In summary, water stress and harvesting frequency affected growth and caused quantitative changes in the essential oil components. It can be concluded that irrigating Marrubium vulgare plants every 4 days and harvesting at 210 days from the transplanting date is essential to maximize production, whereas irrigating every 12 days and harvesting 80 days after transplanting is recommended to maximize essential oil content.
1. Introduction
antioxidants (Vergara-Galicia et al., 2012) and other biological activities. Plant production and essential oil content can be influenced independently by changes in harvesting time or by environmental factors (Hay and Waterman, 1993). Environmental stresses are among the factors most limiting to plant productivity. The environmental factors can also influence the growth and biosynthesis of secondary metabolites in medicinal and aromatic plants. Essential oil yields have been affected by osmotic stress (Charles et al., 1990). Water deficit in plants may lead to physiological disorders, such as a reduction in photosynthesis and transpiration (Sarker et al., 2005). Water stress in plants influences many metabolic processes, and the extent of its effects depends on drought severity. The optimization of irrigation for the
Marrubium vulgare L. (horehound, white horehound) belongs to the Lamiaceae family. It is a perennial, herbaceous medicinal plant native to Europe, northern Africa, and southwestern and central Asia. This plant was frequently employed as a folk medicine to treat a variety of ailments related to upper respiratory tract infections. Nowadays, the plant is widely used as an herbal medicine to treat liver diseases, biliary tract disorders, bronchial asthma and nonproductive cough (Verma et al., 2012). It possesses tonic, stimulant, expectorant, antispasmodic, antidiabetic, diaphoretic, and diuretic properties (Boudjelal et al., 2012; Vergara-Galicia et al., 2012). Essential oils are appreciated for their bioactive efficacy as fungicides, bactericides (Zarai et al., 2011),
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Corresponding author. E-mail address: [email protected] (T. Astatkie).
https://doi.org/10.1016/j.scienta.2018.01.004 Received 2 October 2017; Received in revised form 28 December 2017; Accepted 4 January 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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stage. Plants from each water stress level were harvested at three times; 80, 150 and 210 days after transplanting. The essential oil percentage was determined in the fresh herb using modified Clevenger apparatus (Guenther, 1961. Essential oil (%) was expressed as (ml 100 g−1 fresh herb). The resulted essential oil of each experimental unit was collected and dehydrated over anhydrous sodium sulphate and kept in refrigerator until GC–MS analyses.
production of fresh herbs and essential oils is important, since water is a major component of the fresh produce and affects both weight and quality (Jones and Tardien, 1998). In aromatic plants, soil moisture may cause significant changes in the yield and composition of essential oils; for example, water deficit decreased the oil yield of rosemary (Rosmarinus officinalis L.) (Singh and Ramesh, 2000) and anise (Pimpinella anisum L.) (Zehtab-Salmasi et al., 2001). By contrast, water stress caused a significant increase in oil yield of citronella grass (Cymbopogen winterianus Jowitt.) expressed on the basis of plant fresh mass (Fatima et al., 2000) with the severity of the water stress response varying with cultivar and plant density in parsley (Petroselinum crispumL.) (Petropoulos et al., 2008). Said-Al Ahl and Abdou (2009) on dragonhead and Said-Al Ahl et al. (2009a, 2009b) showed that both essential oil content (%) and yield significantly decreased with increasing water stress levels of Origanum vulgare and Melissa officinalis, respectively. In most perennial aromatic plants, reduction in biomass yield due to repeated harvesting affects essential oil yield. Repeated harvests can either be beneficial or detrimental to oil production, depending on other environmental factors. For example, herbage yield is usually high at first harvest, then becomes constant, and then declines with repeated harvests (Murtagh, 1996; Weiss, 1997). Kothari et al. (2004) found that biomass yield was higher in the first harvest and gradually declined in the subsequent harvests of Ocimum tenuiflorum. Contrary to the decrease in biomass yield, essential oil content was lower in the first harvest and increased gradually in subsequent harvests to reach maximum in the fourth harvest (Kothari et al., 2004). The essential oil in geranium is mostly contained in the leaves; therefore, the higher the proportion of leaves in the harvested produce the better the yield of oil is (Rao et al., 1990). Determination of the correct harvesting time is extremely important both for maximizing yield and oil quality as it exerts remarkable influences on the oil yield of essential oil content of crops (Weiss, 1997). Doimo et al. (1999) reported that not only the harvest time affected oil yield, but that the geographic area where these crops were grown also influenced yield. The chemical composition of the essential oil was also observed to be influenced by environmental changes (i.e., different soil water content, temperature and photoperiod). These environmental conditions may increase or decrease different terpenoids in the crop. However, studies on irrigation intervals and harvesting frequency on yield and essential oil of Marrubium vulgare have not been investigated. Therefore, this study aimed to evaluate the effect of irrigation intervals on the fresh herb yield and essential oil content and their main constituents of Marrubium vulgare L. harvested at three times.
2.2. GC–MS analyses and identification of components GC–MS analyses were carried out on a Varian 450-GC connected with a Varian 220-MS. Separation was achieved using a Factor Four TM capillary column VF 5 ms (30m × 0.25 mm i.d., 0.25 μM film thickness). Injector type 1177 was heated to a temperature of 220 °C. Injection mode was splitless (1 μL of a 1:1000 n-hexane solution). Helium was used as a carrier gas at a constant column flow rate of 1.2 mL min−1. Column temperature was programmed: initial temperature was 50 °C for 10 min, then increased to 100 °C at 3 °C min−1, maintained isothermal for 5 min and then increased to 150 °C at 10 °C min−1. The total time for analysis was 46.7 min. The mass spectrometer trap was heated to 200 °C, manifold 50 °C and transfer line 270 °C. Mass spectra were scanned every 1 s in the range 40–650m/z. Components were identified by comparison of their mass spectra with those stored in NIST 02 (software library) or with mass spectra from the literature (Jennings and Shibamoto, 1980; Adams, 2007), as well as by comparison of their retention indices with standards. 2.3. Statistical analysis Repeated Measures Analysis (RMA) of a Randomized Blocks Design with the factor of interest being Irrigation (3 levels: 4, 8, and 12 days interval), and harvesting repeatedly at three time points (Day 80,150, and 210 after transplanting) was completed to determine the effect of irrigation on height, number of branches, weight, and volatile oil of Marrubium, and how this effect evolved over the harvesting times. Since the whole experiment was conducted during two seasons (2014 and 2015), Season was used as a blocking factor. Since the response measurements were measured at different harvest times from the same experimental unit (pot), the values measured repeatedly are expected to be dependent on each other. The most appropriate covariance structure (dependence) for each response variable was determined using the Akaike Information Criterion (Littell et al., 1998). The validity of model assumptions (normal distribution and constant variance of the error terms) were verified by examining the residuals as described in Montgomery (2013). All analyses were completed using the Mixed Procedure of SAS (SAS Institute Inc., 2014). For significant (pvalue < 0.05) or marginally significant (0.05 < p-value < 0.1) effects, multiple means comparisons were completed by comparing the least squares means of the corresponding treatment combinations. Letter groupings were generated using a 5% level of significance for the main effects and using a 1% level of significance for interaction effects to protect Type I experimentwise error rate from over inflation.
2. Materials and methods 2.1. Plant material Seeds of Marrubium vulgare were obtained from the HEM ZADEN B.V Venhuizen − The Netherlands. Seeds were sown in the nursery on March 15 of 2014 and 2015. Two months after seed sowing, the seedlings were transplanted into pots (30 cm diameter, 50 cm depth) filled with 10 kg of air dried soil on May 15 of each year. The pots contained three seedlings each and were placed in full sun light. The experiment was carried out under the natural conditions of the greenhouse of the National Research Center, Dokki, Giza, Egypt. The soil texture was sandy loam, having a physical composition of: 45.00% sand, 28.25% silt, 26.75% clay and 0.85% organic matter. Chemical analyses of the soil showed: pH = 8.40; E.C. = 0.79 dsm−1; total nitrogen = 0.13%; available phosphorus = 2.18 mg/100gram; potassium = 0.02 mg/ 100gram. All properties were determined according to the standard methods (Jackson, 1973). Irrigation interval treatments (4, 8 and 12 days), of two liters of water were applied per pot a month after transplanting. Growth characters (plant height [cm], number of branches/plant, herb fresh weight [g/plant]) and chemical constituents were determined at the flowering
3. Results and discussion 3.1. Growth parameters The interaction effect of Irrigation interval and Harvest day was significant on plant height, number of branches, and herb fresh weight of Marrubium vulgare (Table 1), which suggests that the effect of irrigation interval was not consistent at all harvest days after transplanting. The plants irrigated every 4 days gave the highest height, number of branches, and weight at 210 days after transplanting (Fig. 1). The lowest mean height, number of branches, and weight was obtained from those irrigated every 12 days and harvested on day 80 after transplanting (Fig. 1). However, at 80 days after transplanting, 140
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resulted in higher absorption of nutrients and water leading to production of higher vegetative biomass (Singh et al., 1997). Increasing levels of water stress reduces growth and yield due to reduction in photosynthesis and plant biomass because photosynthesis would be limited by low CO2 availability due to reduced stomatal and mesophyll conductance. Drought stress is associated with stomatal closure that leads to reduced CO2 fixation. However, irrigation repeated in every 4 days seemed to be optimal and produced the highest production of fresh biomass in other plants as well (Srivastava and Srivastava, 2007). The superiority of the plants that received the highest rate of irrigation treatments in producing the heaviest total plant fresh weight was in agreement with that in literature (Said-Al Ahl et al., 2009c; Moeini Alishah et al., 2006).
Table 1 ANOVA p-values that show the main and interaction effects of Irrigation (Irr) and Days to harvesting (DH) on height, number of branches, weight, and volatile oil of Marrubium. Significant effects that require multiple means comparison are shown in bold. Effect
Height
Number of branches
Weight
Volatile oil
Block Irr DH Irr*DH
0.003 0.001 0.001 0.001
0.032 0.001 0.001 0.001
0.234 0.001 0.001 0.001
0.151 0.001 0.001 0.025
irrigation intervals 4 and 8 days did not give significantly different height and number of branches, but only weight, with 4 days interval giving higher weight than 8 days interval (Fig. 1). Although there was no significant difference between the mean heights of 4 and 8 days irrigation interval when harvested at 150 days after transplanting, 4 days irrigation interval gave significantly higher number of branches and weight (Fig. 1). These results are in agreement with those reported by Said-Al Ahl et al. (2009a, b, c) who concluded that the highest herb yield of oregano were recorded in plants that received the highest amount of water (irrigated every 3 days). The pronounced effect of increased irrigation on fresh herb yield may be attributed to the availability of sufficient moisture around the root concentrated and thus a greater proliferation of root biomass. This
3.2. Essential oil content Irrigation intervals and harvest frequency interacted on the percentage of essential oils (volatile oil) of Marrubium as well (Table 1). However, the effect of these factors on volatile oil reversed from that on the growth parameters. The highest volatile oil was obtained from irrigation intervals of 8 days and 12 days harvested at 80 days after transplanting and the lowest volatile oil was obtained from those irrigated every 4 days and 8 days harvested at 150 days and 210 days after transplanting (Fig. 1). Although there was no significant difference
Fig. 1. Mean A) height (cm), B) number of branches, C) weight (g/plant), and D) volatile oil (%) of Marrubium obtained from the three irrigation intervals and harvested at the three days after transplanting. Within each plot, means sharing the same letter are not significantly different from each other.
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camphene (0.0–1.0%), 2-cyclohexen-1-ol (0.0-1.0%), and cis-α-bisabolene (0.0–1.0%) were the minor compounds (Table 2). The remaining 60 compounds, namely β-Phellandrene, (−)-β-Pinene, (−)-Limonene, trans-β-Ocimene, cis-β-Ocimene, cis-β-Terpineol, Terpinolene, L-transPinocarveol, (−)Camphor, Menthone, Isogeraniol, 1-Terpinen-4-ol, αTerpineol, p-Menth-8-en-2-one, trans, d-Verbenol, (+)Carvone, Thymoquinone, cis-Geraniol, Piperitone, Carvenone, α-Citral, β-Citronellal, Bornyl acetate, Thymol acetate, Cedrene, Copaene, Isobornyl propionate, β-Bourbonene, Phenol, 2-methyl-5-(1-methylethyl)-acetate, Aromadendrene, trans-β-Bergamotene, β-Cadinene, Neoclovene/β-Chamigrene, β-Cubebene, α-Bergamotene, Valencene, Isoledene, αCaryophyllene, (+)-epi-bicyclosesquiphellandrene, α-Amorphene, δElemene, γ-Muurolene, Germacrene D, δ-Salinene, β-Eudesmene, αCubebene, β-Gurjunene, α-Muurolene, Isoleden, δ-Cadinene, Ledol, αCopaene, α-Calacorene, Calacorene epoxide, (−)-Spathulenol, Caryophyllene oxide, β-Guaiene, β-Vatirene, γ-Eudesmol, τ-Cadinol, Seychellene, Aromadendrene oxide, τ-Muurolol, Globulol, Isoaromadendrene epoxide, Longipinocarveol, α-Bisabolol, and Cubenol were considered as traces. The chemical composition of Marrubium vulgare essential oil from various origins has been the subject of many studies. The literature reveals the occurrence of several chemotypes. In Egypt, Salama et al. (2012) reported that thymol and γ-cadinene are major components; ELLeithy et al. (2013) reported β-caryophyllene and germacrene-D as major components; and Said-Al Ahl et al. (2015) reported carvacrol, βphellandrene, carvyl acetate as the major components. In Lithuania, (Z)-β-farnesene, β-caryophyllene, (E)-2-hexenal, α-humulene and germacrene-D were the main components of M. vulgare essential oil (Weel et al., 1999). Such differences were also observed across the world in various chemotypes of M. vulgare and their main constituents (Belhattaba et al., 2006; Hamdaoui et al., 2013; Khanavi et al., 2005; Mahnaz et al., 2005; Morteza-Semnani et al., 2008; Saleh and Glombitza, 1989; Zawiślak, 2012). The results shown in Table 2 indicate that thymol and carvacrol compounds represent a percentage ranging from 42 to 61% of the total oil components. It was also observed that increasing the irrigation interval increases the percentage of thymol, but decreases that of carvacrol content. Specifically, plants that were irrigated with low level of water (12 days) gave the highest percentage of thymol (60.7%) and the lowest percentage of carvacrol (0.5%), while plants irrigated with medium level of water (8 days) and harvested after 150 days from transplanting (second harvest) gave the highest percentage of carvacrol (19.3%) and the lowest percentage of thymol (29.6%). For the other major compounds, plants irrigated with medium level of water (8 days) gave the highest percentages of both thymol methyl ether (10.4%) and α-himachalene (10.3%). Plants irrigated with the highest water amount (4 days) gave the highest percentage of m-cymene (14.2%). Similar to thymol behavior, γ-terpinene compound was the highest in plants that were irrigated with low level of water (12 days). Previous studies have shown that changes in the oil composition of oregano as a result of water stress and harvest date (Letchamo et al., 2004; Rodrigues et al., 2013; Said-Al Ahl and Abdou, 2009; Said-Al Ahl et al., 2009a, b, c).
between mean volatile oil irrigated every 8 and 12 days when harvested at 80 days after transplanting, the mean volatile oil obtained from 12 days irritation interval was significantly higher than that of 8 days irrigation interval when harvested at 150 and 210 days after transplanting. Similar results were reported by Said-Al Ahl et al. (2009c); Simon et al. (1992). These results are also in agreement with those reported by Baher et al. (2002) who concluded that the accumulation of oil increased significantly under severe water stress in Satureja hortensis. Although a limited supply of water or water stress has a negative effect on the development of the plant and yield, when focusing on the biosynthesis of secondary metabolites, water stress is not always completely detrimental (Murtagh, 1996; Yaniv and Palevitch, 1982). Photosynthetic activity is known to be reduced in crops subjected to low light levels and water deficit conditions. Letchamo and Xu (1996) hypothesized that dry matter formation and accumulation of essential oil in thyme was closely related to photosynthesis, and limitations in the net CO2 assimilation rates had a direct or indirect effect on shoot growth and production of the volatile oil. Doimo et al. (1999) reported that the months of harvest affected oil yield. The month of harvesting was also observed to exert remarkable influences on the oil yield of essential oil crops. For example, Weiss (1997) reported that the highest oil content of geranium was observed in July and lowest in February in southern India. Hegazy et al. (2016) indicated that three medicinal Lamiaceae plants harvested in the second cut were much superior in plant height, number of branches, herb fresh weight, and essential oil percentages compared with those in the first cut. It has been reported that yield and its components in plants in general is primarily related to their genetic, climate, edaphic, and their interaction (Rahimmalek et al., 2009; Basu et al., 2009). Also, Said-Al Ahl and Mahmoud, (2010) reported that Ocimum basilicum plants harvested in the second harvest gave the highest plant height, number of branches, fresh matter yield as well as essential oil than those in the first cut. Also Said-Al Ahl et al. (2009a, 2009c) in two experiments on oregano plants reported that harvesting in the second cut gives the best result of herb fresh weight and essential oil content compared to in the first cut. Also, a study on comparing two cutting times indicated that dragonhead performed better in second than in first cutting (Said-Al Ahl et al., 2010). This may have resulted from suitable climatic conditions and state of maturity during the second cutting period. The yield and quality of essential oil produced depends on various intrinsic and extrinsic factors. Intrinsic factors comprise all internal hereditary characters. Extrinsic factors affecting the production of essential oil in plants are soil, nutrition and water supply, climate (light/temperature), maturity, postharvest treatment and distillation method (Varshney, 1991). Penka (1978) showed that the formation and accumulation of essential oil in plants was caused by the action of environmental factors. It might be claimed that the formation and accumulation of essential oil was directly dependent on growth and development of the plants producing oils. 3.3. GC/MS analysis The results of the GC/MS analysis of essential oils of the Marrubium vulgare in the second (2015) season revealed that there are 6 major and 14 minor compounds, and 60 trace compounds. The compounds were categorized as major if the largest percentage measured is more than 10%, as minor if it is between 1 and 10%, and as trace if it is less than 1%. Accordingly, thymol (29.6–60.7%), carvacrol (0.5–19.3%), mcymene (1.0–14.2%), γ-terpinene (1.1–12.1%), thymol methyl ether (0.4–10.4%), and α-himachalene (0.0-10.3%) were major compounds (Table 2); and isoborneol (0.7–9.4%), benzene 1-methoxy-4 methyl2(1-methylethyl) (0.0–9.2%), linalyl anthranilate (0.8–7.0%), γ-cadinene (tr-2.9%), α-terpinene (1.4–2.0%), β-caryophyllene (0.6–1.9%), βmyrcene (0.0–1.8%), eucalyptol/cineole (0.6–1.5%), α-phellandrene (0.4–1.4%), 1-octen-3-ol (0.0–1.2%), 1R-α-pinene (0.0–1.1%),
4. Conclusion The current study showed that water deficiency (irrigated every 12 days) and harvesting frequency influence the growth and essential oil accumulation of Marrubium vulgare plants. Increasing irrigation levels increased the herb fresh weight of Marrubium, but decreased its essential oil content. The highest yields were obtained from plants harvested after 210 days from transplanting date and irrigated every 4 days. But, essential oil content behaved in reverse as the highest values of essential oil was obtained from plants harvested after 80 days from transplanting date and irrigated every 12 days.
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Table 2 Mean values of the major and minor compounds of Marrubium vulgare L obtained from the three irrigation intervals (I-4, I-8, and I-12 Irrigation every 4, 8, and 12 days, respectively) and harvested after 80 days (H1), after 150 days (H2), and after 150 days (H3) from transplanting during the 2015 season. Compound
Major compounds Thymol Carvacrol m-Cymene γ-Terpinene Thymol methyl ether α-Himachalene Minor compounds Isoborneol Benzene 1-methoxy-4 methyl-2(1-methylethyl) Linalyl anthranilate γ-Cadinene α-Terpinene β-Caryophyllene β-Myrcene Eucalyptol/Cineole α-Phellandrene 1-Octen-3-ol 1R-α-Pinene Camphene 2-Cyclohexen-1-ol cis-α-Bisabolene
Rt
RI
I-4
I-8
I-12
Identification
H1
H2
H3
H1
H2
H3
H1
H2
H3
32.4 32.8 16.1 18.2 28.4 38.4
1289 1298 1020 1054 1231 1505
35.6 15.4 14.2 11.5 1.4 0.2
46.1 18.8 8.4 8.9 0.4 nd
54.6 5.9 12.0 1.1 1.4 0.3
31.5 10.7 7.8 7.8 10.4 10.3
29.6 19.3 9.1 9.0 2.4 tr
47.8 8.2 10.2 10.3 3.1 0.4
60.7 0.5 1.4 12.1 1.6 Nd
46.3 3.3 8.3 7.5 5.0 1.6
51.7 4.2 1.0 8.1 4.3 1.5
RI, RI, RI, RI, RI, RI,
MS, Co-I MS, Co-I MS MS, Co-I MS MS
24.8 27.9 21.0 38.6 15.5 36.3 13.8 16.5 9.1 13.4 9.6 10.8 19.1 39.3
1155 1216 1114 1511 1014 1413 988 1026 930 977 932 946 1097 1527
1.8 1.7 2.0 0.1 1.9 1.2 1.7 0.9 1.4 0.7 1.1 0.5 nd 0.2
0.7 0.2 1.2 2.9 1.5 1.9 nd 1.3 0.4 nd nd nd 0.4 –
1.7 3.1 2.6 0.2 1.6 1.7 1.3 1.1 1.0 0.8 0.7 0.3 1.0 0.1
4.7 nd 1.3 0.2 1.4 0.6 1.3 1.5 1.1 0.7 1.1 1.0 nd –
9.4 nd 7.0 0.2 1.4 1.5 1.4 0.8 1.0 0.5 0.7 0.3 nd tr
1.3 nd 2.1 0.3 1.4 1.6 1.2 0.6 0.8 0.8 0.5 0.2 0.9 0.2
1.0 2.8 2.5 0.2 2.0 1.9 1.8 1.0 1.4 0.8 1.0 0.4 nd –
3.0 9.2 0.8 tr 1.4 0.9 1.2 0.6 0.9 1.2 0.7 0.5 nd 1.0
4.7 6.3 1.1 0.2 1.5 1.1 1.3 0.8 1.1 0.9 0.9 0.8 0.7 tr
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS, MS MS MS MS MS, MS, MS MS, MS MS, MS, MS MS
Co-I
Co-I Co-I Co-I Co-I Co-I
Rt = retention time; RI = Kovats retention index determined relative to the series of n-alcanes (C10-C35) on VF-5 MS Capillary Column; Identification method: RI = comparison on Kovats retention indices with published data, MS = comparison of mass spectra with those listed in the NIST 02 library and with published data, Co-I = coinjection with authentic compound; nd = not detetected; tr = traces (˂0.1%).
References
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