Content of essential oil and extracts
Medicinal plants have different capacities to produce essential oil at different phenological stages 32. To achieve their goal, breeders must consider the proper harvest time to reach the best yield for their target purposes, such as pharmaceutical, food and cosmetic industries applications. For this reason, the essential oil content was studied in different phenological growth stages of H. persicum. Statistical analysis of this experiment showed that the essential oil content was significantly different in the various phenological stages of this plant (Fig. 1). The results indicated that the essential oil yield obtained in the vegetative stages, floral budding stage, full flowering stage, early development of seeds stage, mid-mature seeds stage and, final stage or late-mature/ripe seeds stages were 0.45, 0.72, 0.5, 1 and 3.5 and 2.2%, respectively. According to these data, the greatest yield was observed at the mid-development stage of the seeds, while the minimum yield was associated with the vegetative stages (Fig. 2). There is no significant difference in essential oil yield between the vegetative stage and seeds at the early development stage. Other medicinal plants such as the Apiaceae family, including Oliveria decumbens 27, Trachyspermum Ammi 33, and Echinophora tenuifolia 34, followed the same pattern and produced different essential oil content at various growth stages that could be due to the interaction between the physiological activities of these plants at various stages of development in their environment. These plants had low essential oil content in early growth stages, while the highest essential oil content was obtained at seed setting and seed phase. Less moisture content and lower activity of some essential enzymes for biosynthesis of specific compounds at seeding stage lead to less essential oil production at early stages of growth 28, 33, 34. Furthermore, previous studies showed that essential oil content was not the same in different plant organs. Results of the study on H. persicum showed 0.41 to 5.23% difference in essential oil content of various organs such as stem and seeds 6. In our experiment, we observed a low content of the essential oil in vegetative and flowering stages and high essential oil content in seed at setting stage; possibly, H. persicum plants spend most of their produced photosynthetic materials manufacturing vegetative organs instead of synthesis of useful biological active compounds for various industries at the early stages of its growth in comparison to later stages.
Figure 3 shows H. persicum extracts content in different growing stages. According to our results, there was a significant difference between the extracts content in different phenological stages while the maximum extract content percentage was obtained in the floral budding stage (10.40%) followed by full flowering stage (10.20%). However, with the plant reaching the seed setting stage the extracts percentage decreased, as the lowest percentage of extracts were obtained at mature seeds stage at the rate of 5.10%. Based on the results, there were no significant differences between the flowering and floral budding stages, also between vegetative and mid-mature seeds stages.
Chemical compounds of essential oil
Recent reports suggest that biological features of the H. persicum such as anti-inflammatory, anti-pain, antioxidant, and anti-seizure can be attributed to basic essential oil compounds 3. The analysis of our data at various phenological stages of H. persicum indicated significant variations in the type and percentage of essential oil constituents (Table 1). In total, in the vegetative stages, floral budding stage, full flowering stage, early development of seeds stage, mid-mature seeds stage and, final stage or late mature/ripe seeds stage, values of 96.75, 97.02, 96.94, 98.02 and 97.07 and 98.23% of the total essential oil constituent were respectively identified.
Table 1
Essential oil composition of Heracleum persicum at different phenological stages and their mean comparisons.
No. | Compounds | RI | Vegetative stage | Floral budding | Full flowering | Early development of seeds | Mid-maturation of seeds | Late-mature/ripe of seeds |
1 | Isopropyl isovalerate | 894 | 0.41 ± 0.03 b | tr | tr | tr | tr | 1.62 ± 0.15 a |
2 | α-Pinene | 930 | 1.93 ± 0.05 b | 4.50 ± 0.27 a | 4.89 ± 0.20 a | 2.26 ± 0.33 b | tr | 0.33 ± 0.03 c |
3 | Isopropyl 3-methyl-2-butenoate | 946 | tr | tr | tr | tr | tr | 1.18 ± 0.16 |
4 | β-Pinene | 985 | 8.67 ± 0.22 a | 1.41 ± 0.09 b | tr | tr | tr | 0.32 ± 0.04 c |
5 | Pseudolimonen | 992 | tr | 2.18 ± 0.32 b | 2.98 ± 0.08 a | 0.46 ± 0.05 c | tr | tr |
6 | Octanal | 1002 | tr | 0.28 ± 0.04 c | 0.30 ± 0.03 c | tr | 1.23 ± 0.12 a | 0.97 ± 0.05 b |
7 | Butyl butyrate | 1005 | tr | tr | tr | 1.82 ± 0.11 b | 4.19 ± 0.74 a | 4.38 ± 0.35 a |
8 | p-Cymene | 1010 | tr | 1.38 ± 0.06 b | 4.20 ± 0.26 a | tr | tr | tr |
9 | Limonene | 1018 | 18.05 ± 1.72 a | 0.48 ± 0.05 b | tr | tr | tr | tr |
10 | Hexyl acetate | 1025 | tr | tr | tr | 0.43 ± 0.04 b | 0.92 ± 0.06 a | 1.02 ± 0.12 a |
11 | Carene | 1027 | 4.19 ± 0.22 a | 1.16 ± 0.05 c | 2.66 ± 0.19 b | 0.57 ± 0.05 d | tr | tr |
12 | γ -Terpinene | 1036 | 1.39 ± 0.12 c | 4.37 ± 0.18 b | 6.31 ± 0.77 a | 1.01 ± 0.20 cd | tr | 0.72 ± 0.10 d |
13 | Butyl 2-methylbutanoate | 1044 | tr | tr | tr | 1.05 ± 0.06 a | 0.64 ± 0.14 b | 0.70 ± 0.08 b |
14 | 2-Methylbutyl isobutyrate | 1048 | tr | tr | tr | 0.60 ± 0.10 a | 0.81 ± 0.10 a | 0.25 ± 0.02 b |
15 | cis-5-Octen-1-ol | 1051 | tr | tr | 0.93 ± 0.03 c | 5.75 ± 0.16 a | 6.03 ± 0.18 a | 4.85 ± 0.24 b |
16 | Linalool | 1061 | 0.50 ± 0.10 d | 0.64 ± 0.06 cd | 0.94 ± 0.22 c | 3.72 ± 0.19 a | 0.91 ± 0.10 c | 1.89 ± 0.08 b |
17 | Thujone | 1098 | tr | tr | tr | tr | tr | 1.37 ± 0.11 |
18 | 4-Methylpentyl isobutyrate | 1109 | tr | tr | 1.97 ± 0.20 c | 4.91 ± 0.24 b | 3.61 ± 0.21 b | 8.21 ± 0.83 a |
19 | Camphor | 1136 | tr | tr | tr | tr | tr | 1.60 ± 0.14 |
20 | Hexyl butyrate | 1183 | 0.92 ± 0.03 e | 8.22 ± 0.27 d | 9.05 ± 0.74 d | 32.08 ± 2.52 b | 38.75 ± 2.89 a | 23.59 ± 1.62 c |
21 | Octyl acetate | 1203 | tr | 0.72 ± 0.10 d | 3.33 ± 0.37 c | 11.67 ± 0.92 ab | 14.47 ± 1.66 a | 10.48 ± 1.09 b |
22 | Anethole | 1220 | 0.85 ± 0.08 c | 14.55 ± 1.02 a | 8.08 ± 1.05 b | 1.32 ± 0.10 c | 2.04 ± 0.23 c | tr |
23 | Hexyl 2-methylbutyrate | 1234 | 0.31 ± 0.08 d | 1.39 ± 0.10 d | 3.22 ± 0.38 c | 5.76 ± 0.64 b | 4.22 ± 0.19 c | 8.01 ± 0.78 a |
24 | Hexyl isovalerate | 1240 | tr | 0.33 ± 0.05 b | 1.13 ± 0.08 ab | 2.01 ± 0.72 a | 2.12 ± 0.08 a | 2.45 ± 0.30 a |
25 | Octyl Isobutyrate | 1329 | tr | 1.90 ± 0.05 b | 1.45 ± 0.24 b | 4.48 ± 0.51 a | 6.00 ± 0.71 a | 6.26 ± 0.94 a |
26 | Hexyl hexanoate | 1369 | tr | 0.57 ± 0.07 d | 0.91 ± 0.04 c | 3.13 ± 0.43 ab | 3.70 ± 0.16 a | 2.64 ± 0.19 ab |
27 | Octyl 2-methylbutyrate | 1416 | tr | 0.69 ± 0.03 c | 1.10 ± 0.17 bc | 1.94 ± 0.05 b | 2.18 ± 0.16 b | 5.38 ± 0.67 a |
28 | Caryophyllene | 1421 | 14.07 ± 1.45 a | 4.36 ± 0.19 b | 0.54 ± 0.06 c | 0.73 ± 0.14 c | tr | 0.87 ± 0.07 c |
29 | Octyl isovalerate | 1442 | tr | | 0.26 ± 0.02 d | 0.37 ± 0.07 c | 0.69 ± 0.05 b | 1.13 ± 0.14 a |
30 | α-Curcumene | 1460 | 3.48 ± 0.41 b | 7.63 ± 0.52 a | 1.15 ± 0.05 c | tr | tr | tr |
31 | Phenethyl 2-methylbutyrate | 1481 | 1.39 ± 0.10 a | 0.85 ± 0.07 b | 1.27 ± 0.05 a | tr | tr | tr |
32 | Myristicin | 1491 | 5.24 ± 0.66 b | 7.31 ± 0.55 b | 15.02 ± 1.30 a | tr | tr | tr |
33 | (E)- γ -Bisabolene | 1501 | 2.04 ± 0.69 a | 0.77 ± 0.07 b | tr | tr | tr | tr |
34 | 1,5,9,9-Tetramethyl-1,4,7-cycloundecatriene | 1508 | 1.73 ± 0.14 a | 0.20 ± 0.07 c | tr | tr | 0.65 ± 0.11 b | 2.04 ± 0.24 a |
35 | β-Bisabolene | 1516 | 8.59 ± 0.57 b | 12.56 ± 0.59 a | 8.26 ± 0.94 b | 1.73 ± 0.15 c | | 0.39 ± 0.09 c |
36 | Spatulenol | 1541 | 5.77 ± 0.33 a | 1.02 ± 0.31 b | 0.28 ± 0.04 c | tr | tr | tr |
37 | 1-Allyl-2,3,4,5-tetramethoxybenzene | 1568 | 0.69 ± 0.05 b | | 2.98 ± 0.25 a | tr | tr | tr |
38 | Caryophyllene oxide | 1576 | 6.10 ± 0.25 a | 2.47 ± 0.29 b | 0.40 ± 0.11 d | 0.49 ± 0.09 d | tr | 0.77 ± 0.05 c |
39 | d-Viridiflorol | 1591 | tr | tr | tr | 3.03 ± 0.36 b | 1.30 ± 0.09 c | 3.76 ± 0.16 a |
40 | Butylphosphonic acid, hexyl 4-methoxybenzyl ester | 1597 | 0.32 ± 0.06 d | 1.87 ± 0.15 c | 2.81 ± 0.12 b | 3.61 ± 0.43 a | 1.37 ± 0.16 c | tr |
41 | Apiol | 1675 | 1.61 ± 0.04 c | 3.05 ± 0.30 b | 7.08 ± 0.91 a | tr | tr | tr |
42 | 1-Tetradecanol | 1681 | 7.06 ± 0.75 a | 5.41 ± 0.33 b | 2.38 ± 0.50 c | 0.66 ± 0.12 d | tr | tr |
43 | Falcarinol | 2005 | 0.44 ± 0.07 c | 2.59 ± 0.16 a | 0.66 ± 0.07 c | 1.43 ± 0.15 b | tr | tr |
44 | Manool | 2056 | tr | 0.38 ± 0.04 b | tr | 0.98 ± 0.14 a | 1.25 ± 0.07 a | 1.05 ± 0.10 a |
45 | trans-Geranylgeraniol | 2201 | 1.01 ± 0.51 ab | 1.80 ± 0.10 a | 0.41 ± 0.08 c | tr | tr | tr |
| Total | | 96.77 | 97.02 | 96.94 | 98.02 | 97.07 | 98.23 |
Values are given as mean ± SE (n = 3). According to the Tukey's Test application: means of the same column and main variable labeled with the same letters are not significantly different at p < 0.05 |
The main constituents of the essential oil in various stages were limonene (18.05%), caryophyllene (14.07%), β-pinene (8.67), β-bisabolene (8.86%), 1-tetradecanol (7.06%), caryophyllene oxide (6.10%), espatulenol (5.77%), myristicin (5.24%), carene (4.19%), α-curcumene (3.48%) at the vegetative stage. Anethole (14.55%), β-bisabolene (12.56%), hexyl butyrate (8.22%), α-curcumene (7.63%), myristicin (7.31%), 1-tetradecanol (5.41%), α-pinene (4.70%), caryophyllene (4.36%), γ-terpinene (4.37%), and apiol (3.05%) were the main constituents of the essential oil in the early flowering stage. In the full flowering stage values were: myristicin (15.02%), hexyl butyrate (9.05), β-bisabolene (8.26%), anethole (8/08%), apiol (7/08%), γ -terpinene (6.31) %), α-pinene (4.89%), p-cymene (4.20%), octyl acetate (3.3%), and hexyl isovalerate (3.22%). During seed development, butyrate (32.08%), octyl acetate (11.67%), hexyl 2-methyl butyrate (5.76%), cis-5-octen-1-ol (5.75%), 4-methyl pentyl isobutyrate (4.91%), octyl isobutyrate (4.48%), and hexyl 4-methoxybenzyl ester (3.61%) were dominant constituent of the essential oil. Lastly, in the phenological stage of immature seeds, hexyl butyrate (38.75), octyl acetate (14.47), hexyl 2-methyl butyrate (4.22), cis-5-octen-1-ol (6.3), and 4-methyl pentyl isobutyrate (3.61%) were measured as the predominant constituent of the essential oil, however, these percentages changed to 23.59, 10.48, 8.01, 4.85, and 8.21% at the seed final maturation stage, respectively (Table 1). Previous studies have shown almost the same compounds of the essential oil in H. persicum’s plant seeds while the value of these compounds were negligible in the leaf and flower organs 4,6,22,23. According to the mean comparisons’ analysis, there were significant differences between these compounds obtained in the various phenological stages. Figure 3 shows that the essential oil constituent were different in the different phenological stages. In addition, predominant compounds of essential oil changed during different growth stages, which are also presented in the Fig. 3.
The study of essential oil constituents indicated that α-pinene and β-pinene were predominant in the early growth stages. According to the results (Table 1), the highest α-pinene value was observed at the full flowering stage (4.89%), but we did not see the same results at the immature seed stage. Moreover, β-pinene was detected in the vegetative stage and at the time of flower opening. With entering the seed set stage, the value of these two compounds decreased significantly.
Limonene was observed only in the vegetative and floral budding stages, and the highest value was 18.05% in the vegetative stage. In line with these results, other researchers reported the existence of this compound in the vegetative organs of H. persicum 6, while it was rarely seen in the seeds. In the few cases that limonene was observed in seeds, the value was not significant.
P-cymene was identified in the floral budding and full flowering stage, however its maximum value (4.2%) was observed in the full flowering stage. γ-terpinene was detected in the full flowering stage and the floral budding stage (6.31 and 4.37%, respectively). In a study on the Ajowan plant, the amount of γ-terpinene in the early stages of growth was low, and arrived to its maximum at flowering stage, then subsequently decreased, which could be due to the adsorption of pollinators 33. α-curcumene was observed only in the early growth stages of H. persicum and the maximum amount was related to the floral budding stage (7.63%). Caryophyllene and myristicin were the predominant compounds in the early growth period which were quantified by the amount of 14.07 and 15.02%, respectively. β-bisabolene was the predominant compound that was detected in the floral budding stage and as the plant age increased, its value decreased.
Anethole was the predominant compounds in the floral budding stage and full flowering stage, and the highest value was obtained in the floral budding stage (14.55%); prior to flowering its amount was not significant, and after that it was greatly reduced. Apiol and 1-tetradecanol were the other compounds that observed in the first three stages of plant vegetative stages. As shown in Table 1, the maximum values of apiol (7.06%) and 1-tetradecanol (7.66%) belonged to the full flowering stage and the vegetative stages, respectively. Caryophyllene oxide was the compounds that declined with the increase in plant age, and the highest value was obtained in the vegetative stage at the rate of 6.10%.
The highest value of hexyl butyrate and octyl acetate, that are the most predominant and important compounds in H. persicum, were observed in the immature seeds stage and in the mid-mature seeds stage, by 38.75% and 14.47%, respectively. These beneficial compounds significantly increased to the seedling stage. In agreement with our results, previous studies reported these two compounds as the predominant compound in the seeds of H. persicum 3,6,22.
Among all the compounds, Hexyl 2-methylbutyrate, octyl Isobutyrate, and 4-methylpentyl isobutyrate content were increased with the increase in plant age, so that at the end of the phonological stages, they were the dominant compounds.
The essential oils were classified according to their chemical formula to eight groups (Fig. 4). Based on the results, the main percentage of the essential oil was aliphatic esters, which value was 3.35% in the vegetative stage and finally hit 83.67% in the immature seeds stage or in the mid-mature seeds stage. Therefore, by increasing the age of the plant and seeds formation, the value of this group of compounds was increased (Fig. 4). The main components of aliphatic esters group were reported as hexyl butyrate, octyl acetate, octyl butyrate, and hexyl 2-methylbutyrate, which the amounts of them were different in the phenological stage of growth. In the present study, these compounds were observed mostly in the last three phenological post-flowering stages (Fig. 4); other researchers also found more aliphatic compounds in the generative organs, such as in seeds of H. persicum 1, 6, 9,22,23,35.
The second and large group of the constituent compounds was the monoterpene hydrocarbons which their value in the early stage of the vegetative period was 34.23%, and with increasing the age of the plant and the seed maturation, its value reduced, and finally this compound was not found in the mid-mature seed stage (Fig. 4). Several compounds of approximately 40 compounds of the monoterpene hydrocarbons group have been reported by various researchers in the H. persicum. In the present study, most of these compounds were observed in the flowering stage and prior to it, and when the plant entered into the seed set stage, the value of these compounds was significantly reduced (Fig. 4). The most important compounds of this group were limonene, β-pinene, α-pinene, and γ-terpinene. Other studies also exhibited these compounds are predominant compounds in the H. persicum 6, 22, 36. Similar to the present study, other researchers reported high amount of these compounds in flowers and leaves, in comparison to seeds 6, 36.
Sesquiterpene hydrocarbons was one of the other groups of the constituent compounds of which its amount was high at the beginning of the phenological growth time; the highest value was observed at the beginning stage of flowering (21.16%) and its value decreased with increasing plant age. Oxygenated sesquiterpene was another group of the constituent compounds. Analysis of our data showed 25.94% of sesquiterpene hydrocarbons at the vegetative stage, and after this phenological stage, this amount significantly reduced (Fig. 4). The most important compounds of this group were caryophyllene ،espatulenol, and β-bisabolene which also reported as predominant compounds in the H. persicum in other studies 8, 37.
The other compound evaluated in this experiment was phenylpropenes, which amount at the full flowering stage was 23.10%, and after passing this phenological stage it was significantly reduced (Fig. 4). Anethole and myristicin were two important compounds related to phenylpropenes group. In a study on H. persicum plants, researchers found three phenylpropenes compounds (Anethole, myristicin and estragole) that were usually predominant in the vegetative and flower organs that match the results of the present study 1, 6, 8.
Changes in essential oil compounds in different phenological stages of H. persicum growth are probably due to the fact that the production of essential oil and aromatic compounds are under the control of physiological, biochemical and metabolic mechanisms dependent on age and growth stages of plant. Besides, these changes related to terpene biosynthesis as well as its accumulation in the secretory organs 38. The change in the essential oil ingredients is also influenced by factors such as the age and the development stage of medicinal plants.
Phenolic acids in different phenological stages
Phenolic compounds such as phenolic acids are one of the most critical compounds in medicinal plants and have paramount importance due to the high biological activity and their function as antioxidants, anti-inflammatory, anticancer, and anti-Alzheimer 25, 39.
In this study, we investigated the phenolic acids content in different phenological stages of H. persicum. For this purpose, ten phenolic acid compounds were evaluated in the methanolic extract, harvested at different growth stages, using HPLC. According to the obtained results, there were 8, 9, 9, 10, 8 different phenolic compounds at the vegetative stages, floral budding stage, full flowering stage, early development of seeds stage, mid-mature seeds stage and, final stage or late-mature/ripe seeds stage, respectively (Table 2).
The methanolic extract of the floral budding stage had the highest amount of phenolic acids (287.4 mg/g dry extract) following by early seed development (259.77 mg/g dry extract), full flowering stage (153.69 mg/g dry extract), vegetative stage (72.95 mg/g dry extract), and mid-mature seed stage (46.21 mg/g dry extract). On the other hand, the mature seed extract contained the minimum amount of phenolic acids (24.63 mg/g dry extract) (Table 2).
Cinnamic acid, p-coumaric acid, p-hydroxybenzoic acid, ferulic acid, and rosmarinic acid are predominant phenolic acids in H. persicum. Table 2 shows the different phenolic compounds at different morphological stages of H. persicum plants. The predominant phenolic acids were, in the vegetative stage, cinnamic acid (16.10 mg/g extract), p-hydroxybenzoic acid (14.37 mg/g extract), p-coumaric acid (13.43 mg /g extract), and rosmarinic acid (13.33 mg/g extract); in the floral budding stage, cinnamic acid (225.25 mg/g extract), p-coumaric acid (24.05 mg/g extract), and ferulic acid (12.03 mg/g extract); in the full flowering stage, cinnamic acid (56.42 mg/g extract), p-coumaric acid (39.22 mg/g extract), p-hydroxybenzoic acid (16.77 mg/g extract); in the early stage of seed development, cinnamic acid (218.64 mg/g extract), p-coumaric acid (11.91 mg/g extract), and ferulic acid (6.46 mg/g extract); in the mid-mature seeds stage p-coumaric acid (10.02 mg /g dry extract), ferulic acid (8.77 mg/g extract), and cinnamic acid (8.04 mg/g extract); in the final stage or late-mature/ripe seeds stage, cinnamic acid (8.69 mg/g extract), Gallic acid (6.49 mg/g extract), and rosmarinic acid (3.57 mg/g extract).
Cinnamic acid was recorded as the predominant compounds of phenolic acid in all growth stages. However, its maximum value (218.64 mg/g of dried extract) was in the early stages of flowering, and its lowest value (8.04 mg/g of dried extract) was in the mid- mature stage. Cinnamic acid is a natural aromatic phenolic acid whose long - term - consumption is associated with low toxicity to humans and is used in flavorings, artificial color, and some specific medications. The frequent use of cinnamic acid is as a precursor for the methyl cinnamate, ethyl cinnamate, and benzyl cinnamate production used in the perfume industry. It is also a precursor for the artificial sweetener aspartame. Cinnamic acid is one of the phenolic acids with several biological and medicinal properties, as well as other economic and industrial values 25, 40. According to our results, H. persicum is considered as one of the plants with a rich source of cinnamic acid, especially in the floral budding stage and early development of seeds stage; however, as the plant entered the mature seed stage, we observed the significant reduction in its value.
P-coumaric acid was another beneficial predominant phenolic acid we explored in this experiment. P-coumaric acid amount was increased during the plant growth to 39.22 mg/g at the full flowering stage and then decreased to the lowest amount in the seed maturing stage (1.67 mg/g extract). Studies showed p-coumaric acid has antioxidant properties, which is reported to reduce the stomach cancer by suppressing nitrous amines, and has anti-tumor and anti-mutagenesis activities 25,41.
Ferulic acid is one of the other critical phenolic compounds that researchers have been proven its antioxidant properties 25. This compound was found at all growth stages of the H. persicum plant, and the highest and lowest amount were recorded for the full flowering stage and the seed mature stage with values 15.8 and 2.39 mg/g, respectively.
P-hydroxybenzoic acid is noted as the basis for the preparation of its esters, named parabens, and are employed as preservatives in cosmetics and some ophthalmic solutions. This material was one of the other predominant phenolic acids in the H. persicum observed in all phenological stages in this experiment. According to the obtained results, the maximum value of 16.77 mg/g of dry extract was obtained at the flowering stage, while the seed maturing stage had the lowest amount (0.76%). The amount of this phenolic acid decreased significantly by entering the seed to the sowing stage. Rosmarinic acid is an ester of caffeic acid called 3,4-dihydroxy phenyl lactic acid 25,42. This compound has many pharmaceutical properties such as antimicrobial, anti-rheumatism, and anticancer and was present in all phenological stages of the H. persicum. The highest (13.33 mg/g of dry extract) and the lowest amounts (3.57 mg/g of dry extract) of this compound were obtained in the vegetative stage and the immature seed stage, respectively. Therefore, it can be concluded that H. persicum can be considered a plant rich in phenolic acids, which content was different in different phenological stages. Phenolic compounds were at the lowest rate in the floral budding stage while the maximum content was recorded at the seed mature stage. An increase in phenolic acid levels during flowering stage recommended a higher expression level of the phenylalanilammiase lyase enzyme 43, 44, and it is also an indication of enzyme activity reduction with plant maturation. These changes that occur in the process of primary metabolites adsorption are a result of starch synthesis in the middle stages of seed maturation, and this phenomenon can affect the biosynthesis of phenolic acids 45.
Cluster analysis, and principal component analysis (PCA)
Another goal of this study was to monitor the differences and similarities between different phenological stages in order to find the consequences of different harvesting time on identified phytochemical compounds in H. persicum. To reach the above-mentioned goals, we used the principal components analysis (PCA) and hierarchical cluster analysis (HCA) methods. The results of the main components analyses are shown in Table 3. Based on these results, three components had highest eigenvalue, reporting 89.45% of the total variance. The relative variance for the first, second, and third components was 55.52%, 23.28%, and 10.65%, respectively. In the first component, the compounds of butyl butyrate, carene, 4-methyl pentyl isobutyrate, hexyl butyrate, cis-5-octen-1-ol, octyl acetate, hexyl 2-methyl butyrate, octyl isobutyrate, β-bisabolene, 1-tetradecicanol, p-hydroxybenzoic, rosmarinic acid, and essential oil content had the highest loading factor. Differently, in the second and third components, m-coumaric acid and gallic acid compounds had the most top loading factor, respectively (Table 3).
Table 2
Contents of phenolic acid compounds (mg/g dried extract) of Heracleum persicum at different phenological stages
| |
Phenological stage | GA | PHBA | VA | CaA | PCA | FA | MCA | CiA | RA | SA | Total |
Vegetative | 1.34 ± 0.5c | 14.37 ± 0.15b | 0.46 ± 0.02bc | 0.00 ± 0.00c | 13.43 ± 0.07c | 9.92 ± 0.16c | 0.00 ± 0.00c | 16.10 ± 0.23c | 13.33 ± 0.36a | 3.64 ± 0.15a | 72.59 |
Floral budding | 1.89 ± 0.12c | 8.58 ± 0.53c | 0.57 ± 0.00b | 0.00 ± 0.00c | 24.05 ± 0.3b | 12.03 ± 0.25b | 4.90 ± 0.06b | 225.25 ± 5.30a | 9.18 ± 0.06b | 0.95 ± 0.1b | 287.40 |
Full flowering | 3.98 ± 0.12b | 16.77 ± 0.67a | 8.25 ± 0.21a | 0.00 ± 0.00c | 39.22 ± 1.04a | 15.80 ± 0.35a | 6.35 ± 0.21a | 56.42 ± 2.54b | 6.25 ± 0.55c | 0.65 ± 0.03b | 153.69 |
Early development | 1.28 ± 0.12c | 3.61 ± 0.32d | 0.61 ± 0.11b | 5.61 ± 0.3a | 11.91 ± 0.63 cd | 6.46 ± 0.25c | 5.90 ± 0.35a | 218.64 ± 5.69a | 4.82 ± 0.45 cd | 0.93 ± 0.05b | 259.77 |
Mid-maturation | 1.95 ± 0.03c | 1.78 ± 0.01e | 0.00 ± 0.00c | 3.04 ± 0.26b | 10.02 ± 0.86d | 8.77 ± 0.45c | 6.62 ± 0.16a | 8.04 ± 0.26c | 5.99 ± 0.52c | 0.00 ± 0.00c | 46.21 |
Late-mature/ripe | 6.49 ± 0.28a | 0.76 ± 0.08e | 0.00 ± 0.00c | 0.00 ± 0.00c | 1.67 ± 0.13e | 2.39 ± 0.12d | 0.00 ± 0.00c | 8.69 ± 0.35c | 3.57 ± 0.14d | 1.06 ± 0.15b | 24.63 |
Significance | ** | ** | ** | ** | ** | ** | ** | ** | ** | ** | |
Values are given as mean ± SE (n = 3). According to the Tukey's Test application: means of the same column and main variable labeled with the same letters are not significantly different at p < 0.05 GA: Gallic acid; PHBA: p-Hydroxybenzoic acid; VA: Vanillic acid; CaA: Caffeic acid; PCA: p-Coumaric acid; FA: Ferulic acid; MCA: m-Coumaric acid; CiA: Cinnamic acid; RA: Rosmarinic acid; SA: Salicylic acid | |