2.1. Identification of Scent Components
Thirty-nine VOCs were identified, representing more than 99% of the total emission of the flowers. These volatiles grouped by their biochemical synthesis pathways [10
] were described in Table 1
. A total of 18 same volatile compounds were shared at four different stages of flower development of L. pinceana
. Within these compounds, the main aroma-active one was paeonol followed by (E,E
)-α-farnesene, cyclosativene, and δ-cadinene. These compounds might dominate the flavor for L. pinceana
. For instance, paeonol has a specific odour; (E
)-α-farnesene has a woody and sweet odour; δ-Cadinene gives thyme, medicine and wood odour [11
]. As one of these components, γ-muurolene has a smell of herb, wood and spice; methyl salicylate has a flavor with peppermint aroma. The two compounds ranked second in relative content of VOCs at the full-flowering stage and the end-flowering stage separately, so they could also influence the floral aroma.
The idea that L. pinceana
has medicinal properties goes back hundreds of years in China [12
]. Some of volatile compounds from flowers are pharmacologically active compounds. For example, paeonol has several interesting biological activities, and it has been used as an anti-inflammatory, analgesic, antioxidant, antidiabetic, and acaricidal agent [13
]; cyclosativene demonstrates strong anti-inflammatory, expectorant, antifungal effect [15
]; γ-muurolene and δ-cadinene have antifungal properties. Despite the fact that essential oils are seldom encountered in the Rubiaceae [16
], L. pinceana
would have a lot of potential for essential oil extraction according to the L. pinceana
solid phase microextraction results. Not only that, the essential oil of L. pinceana
flowers might have special therapeutic qualities in view of the above active ingredients among the volatile compounds.
Benzenoids were the most abundant amongst floral scent compounds, which content reached at least 51%. The same scenario was noted in the floral essential oil of Randia matudae
] compared to other species of different genera in the family Rubiaceae. In contrast, the quantity and amount of predominant compounds in the floral scent of Posoqueria latifolia
], the leaf essential oil of Rustia formosa
and the essential oil from aerial parts of Anthospermum emirnense
and A. perrieri
were sesquiterpenes [16
]; the floral scent of Cephalanthus occidentalis
, Warszewiczia coccinea
and Gardenia jasminoides
were monoterpenes [20
]; the floral scent of Coffea Arabica
were aliphatics [22
]. It has been reported that the floral scent composition probably significantly varied amongst closely related species, and our results partly support this view [10
2.2. Dynamic Changes of Scent Emission in Different Development Floral Stages
flowers were selected on the basis of their botanical characteristics to evaluate the dynamic changes and diversity of floral volatiles according to different development stages: bud stage, initial-flowering stage, full-flowering stage, and end-flowering stage (Figure 1
). Table 1
and Figure 2
show the distinct changes in scent composition and concentration across flowering stages. Scent components were drastically emitted at the initial-flowering stage, and then declined gradually at the full-flowering stage. The amount of VOCs at the bud stage and the end-flowering stage was obviously lower than that at the initial-flowering stage. The emission pattern of L. pinceana
flowering stages was different from Cananga odorata
Mimi Palmer [24
], and Hosta
] of which the fragrance ingredients were drastically emitted at the full-flowering stage and decreased greatly afterwards. These results showed that the emissions at different flower stages evidently differed. Investigation of the spatial and temporal patterns of gene expression has provided new information on the factors regulating the emission of plant volatile compounds [26
As for the bud stage, 26 volatile compounds belonging to different chemical classes were identified: benzenoids (51.61%), sesquiterpenes (44.41%), aliphatics (2.46%), and monoterpenes (1.48%). The most abundant compound was paeonol, accounting for about 52% of the total GC peak area, followed by δ-cadinene (10.98%), cubebol (5.48%), isoledene (5.45%), and cyclosativene (4.96%). By contrast, relative content of (E)-β-ocimene (0.87%), (−)-β-cadinene (2.34%), cubebol (5.48%), and cubenol (1.02%) at the bud stage were higher than that at the other stages of flower development.
As for the initial-flowering stage, 26 volatile compounds belonging to different chemical classes were identified: benzenoids (85.88%), sesquiterpenes (13.37%), aliphatics (0.53%), and monoterpenes (0.15%). The most abundant compound was paeonol, accounting for about 83% of the total GC peak area, followed by (E,E)-α-farnesene (3.89%), methyl salicylate (2.85%), and δ-cadinene (2.61%). On the other hand, relative content of nonanoic acid, ethyl ester (0.01%) and methyl salicylate (2.85%) in the initial-flowering stage were significantly lower than that in the end-flowering stage, but was higher than that in the bud stage and the full-flowering stage. Relative content of hexyl caprylate was significantly lower than that in the other flower development stages.
As for the full-flowering stage, 32 volatile compounds belonging to different chemical classes were identified: benzenoids (80.05%), sesquiterpenes (16.79%), monoterpenes (1.63%), and aliphatics (1.46%). The most abundant compound was paeonol, accounting for about 80% of the total GC peak area, followed by γ-muurolene (5.86%), (E,E)-α-farnesene (4.90%), and cyclosativene (2.07%). In addition, relative content of β-ylangene (0.09%) and γ-muurolene (5.86%) in the full-flowering stage were higher than that in the other stages of flower development.
As for the end-flowering stage, 32 volatile compounds belonging to different chemical classes were identified: benzenoids (75.38%), sesquiterpenes (13.91%), monoterpenes (7.91%), and aliphatics (2.81%). The most abundant compound was paeonol, accounting for about 70% of the total GC peak area, followed by methyl salicylate (4.81%), (E,E)-α-farnesene (4.75%), and 3-carene (3.73%). By contrast, relative content of monoterpenes in the end-flowering stage were significantly higher than that in the other stages of flower development, including (1S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene (0.45%), α-pinene (2.11%), 3-carene (3.73%), and α-santoline alcohol (0.32%). Relative content of nonanoic acid, ethyl ester (0.02%) and benzyl benzoate (0.78%) in the end-flowering stage were significantly higher than that in the other stages of flower development.
The Bray-Curtis similarity index is a statistic used for comparing the similarity of two samples [27
]. The mean Bray-Curtis similarity index was 74.57% ± 11.53% (range: 61.81%~90.23%, n
= 12, Table 2
). The initial-flowering stage was more similar to the full-flowering stage (BCS
= 90.23%) than to the end-flowering stage (BCS
= 83.59%), and was largely dissimilar to the bud stage (BCS
= 62.77%). Across all the flower-life stages, the bud stage was distinctly dissimilar to the full-flowering stage (BCS
= 61.81%). Variations of the volatile compositions were apparently involved in the maturity stages of flower. The same phenomena are also observed in other plants, such as the flowers of Ocimum citriodorum
], Penstemon digitalis
], and Cananga odorata
]. In this study, the highest diversity of floral volatiles was detected at the third and later periods of the flower development. Meanwhile, the richness of volatile compounds showed an unimodal pattern between the number of VOCs and times of flower development.
To identify which volatiles contributed the most to the differences among the four flower stages and to display the differences in a more visually appealing manner, the data on 39 volatile compounds identified in L. pinceana
at a full life-flower scale were analyzed by using principal component analysis (PCA). The first three components of PCA explained 37.43%, 26.34%, and 23.15% of the variation, explaining ~87% of combined variance (Figure 3
). Hereinto, volatiles that had high positive scores on PC 1 included (−)-β-cadinene, δ-cadinene, cadine-1,4-diene, cubenol, cubebol, isoledene, caryophyllene and α-cubebene, which were highly positively related to the bud stage and the initial-flowering stage. Volatiles with high positive scores on PC 2 comprised β-ylangene, γ-muurolene, unknown-1, unknown-2 and perilla alcohol, which were positively correlated with the full-flowering stage and initial-flowering stage. Volatiles with high positive scores on PC 3 included cis
-verbenol, α-acorenol, (3E
)-1,3,5-undecatriene and cedrol, which were negatively correlated with the full-flowering stage. The remaining 22 volatiles were composed of common components, megastigma-4,6(E
)-triene, cyclosativene and 4-epi
-cubebol. The principal component plots did not overlap amongst the four flower developmental stages indicated that the composition and its relative content of floral scent differed throughout the whole floral development, and the initial-flowering stage was recommended the best harvesting time when the high level of VOCs and essential oil are a concern.