Identification of volatile compounds and their bioactivities from unpolar fraction of Alpinia oxyphylla Miq. and mining key genes of nootkatone biosynthesis

Abstract In this study, analysis of the chemical constituents and bioactivities of the unpolar fractions [petroleum ether (PE) and chloroform (C)] of fruits and leaves of Alpinia oxyphylla Miq. were carried out, as well as the bioactivities of the main compounds nootkatone and valencene. From PE and C fractions of the fruits, and PE fraction of the leaves, 95.80%, 59.30%, and 82.11% of the chemical constituents respectively were identified by GC-MS. Among these identified compounds, nootkatone was the main compound in all of three fractions, while valencene was the second main compound in the PE fractions of the fruits and leaves. The bioactivities results showed that all of the fractions and the major compound nootkatone showed tyrosinase inhibitory, as well as inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. While valencene only presented inhibitory activity on NO production in RAW264.7 cells. The critical genes involved in nootkatone biosynthesis in A. oxyphylla were identified from the public transcriptome datasets, and protein sequences were preliminarily analyzed. Our studies develop the usage of the unpolar fractions of A. oxyphylla, especially its leaves as the waste during its production, and meanwhile provide the gene resources for nootkatone biosynthesis. Graphical Abstract


Introduction
Alpinia oxyphylla miq.which is a member of the family of Zingiberaceae grows mainly in the south of China (Zhao et al. 2016) and some of them are also found in subtropical and tropical rain forests of asia, pacific islands and australia.their fruits have long been useful in traditional Chinese medicine because of their high therapeutic value and ability to regulate the immune system (Li et al. 2013).However, the leaves are usually discarded after the fruits have been harvested.Because of the multipurpose ability of A. oxyphylla, such as anti-inflammatory, anti-depressant, anti-bacteria (Xie et al. 2018;Zhang et al. 2018), more and more attention from researcher has been drawn to studies on its chemical components and their bioactivities.
previous studies have reported the different polar chemical constituents among fruits, leaves and roots (Ying et al. 2021).However, the unpolar chemical composition of different parts are still not clear.the seeds, as the medical part, the unpolar chemical compositions play an important role in the pharmaceutical usage, and Chinese pharmacopeia (2020 edition) describes that the content of volatile oil of the seed should be higher than 1.0% (ml/g), while standard substance in volatile oil has not been confirmed yet.Based on above, this study focused on analysis of the chemical compositions of the unpolar fractions[petroleum ether (pE) and chloroform (C)] of fruits and leaves, as well as their biological activities.meanwhile, the bioactivities of the main compounds nootkatone and valencene was carried out, and the gene resources of nootkatone were also provided.

Results and discussion
in this study, the fruits and leaves of A. oxyphylla were extracted with ethanol.then the concentrated ethanol extract was suspended in water and successively partitioned with petroleum ether (pE) and chloroform (C). the obtained fractions were analyzed using Gas Chromatography-mass Spectrometry (GC-mS) and tested for bioactivities.the fractions from the fruits have a very sharp aromatic odor, while the fractions from the leaves have lesser aromatic smell.

GC-MS analysis
the results from GC-mS analysis of different fractions showed that 95.80% constituents were identified from pE fraction of the fruits, including 83.57% sesquiterpenes, 2.74% diterpenes, 2.99% monoterpenes, 2.82% fatty acids, 0.88% phenols, and 2.80% other compounds; 59.03% constituents were identified from C fraction of the fruits, including 49.76% sesquiterpenes, 0.99% monoterpenes, 1.76% fatty acids, and 6.52% phenols; and 82.11% constituents were identified from pE fraction of the leaves, including 57.11% sesquiterpenes, 16.52% phenols, 4.76% fatty acids, 1.24% diterpenes, and 2.48% other compounds.While the constituents of C fraction of the leaves could not be identified.the percentage composition, retention time and relative contents were shown in tables S1-S3.
the results from the analysis of the fruits showed that sesquiterpenes were the major compounds identified from both pE and C fractions of the fruits and pE fraction of the leaves.nootkatone was the main sesquiterpene with the highest relative content in all of these three fractions with values of 16.03%, 19.88% and 16.90% respectively, while valencene was the second most abundant in pE fractions of the fruits and leaves (tables S1, S2).
Further comparison of the compounds identified, found that the compounds identified from three fractions had some similarity. in the identified 15 compounds in the C fraction of the fruits, there were 8 compounds that were the same with those found in pE fraction of the fruits.in the identified 26 compounds in the pE fraction of the leaves, 18 compounds could also be found in pE fraction of the fruits, and 1 could be found in the C fraction of the fruits, so totally 19 compounds were the same with those in the fruits, which had the proportion of 73%.However, the presence of butylated hydroxytoluene in the C fraction of fruits and pE fraction of leaves in this study is considered a possible contaminant, which may be derived from solvent as a stabilizer (Venditti 2020).

Tyrosinase inhibitory activity
the results from the tyrosinase inhibitory activity showed that all of the tested fractions showed inhibitory activity, while pE fraction of the fruits showed highest inhibition with inhibition value of 89.7% at the concentration of 200 μg/ml, and C fraction of the leaves showed lowest inhibition with inhibition value of 53.7% at the same concentration (table S4).
nootkatone and valencene were also tested for the tyrosinase inhibitory activity, and it presented that nootkatone showed a significant inhibitory activity with inhibitory value of 85.4% at the concentration of 200 μg/ml, while valencene almost had no inhibitory effect (table S4).
according to the chemical analysis results, nootkatone was the major compound in pE and C fractions of fruits, and pE fraction of leaves, and all of these fractions presented a certain inhibitory activity on tyrosinase.therefore, nootkatone should be one of the main compounds responsible for the tyrosinase inhibition in these fractions.

Anti-inflammatory activity
the anti-inflammatory results showed that all of the fractions presented inhibitory effects on no production in LpS-stimulated RaW264.7 cells (table S4).C fraction of the fruits showed strongest activity with inhibitory value of 92.9% at the concentration of 100 μg/ml.moreover, C fractions of fruits and leaves showed stronger cytotoxicity on RaW264.7%than their pE fractions.
Both nootkatone and valencene showed significant inhibitory effects on no production in LpS-stimulated RaW264.7 cells with the inhibitory values of 75.4% and 80.5% at the concentration of 100 μg/ml respectively without cytotoxicity (table S4).therefore, both nootkatone and valencene contributed anti-inflammatory effects to A. oxyphylla extract.
the results of cytotoxicities on five human cancer cell lines showed that all the fractions except the pE fraction of the fruits showed moderately cytotoxic activities.Comparison the effects on different cancer cells, K562 cells was more sensitive than other cell lines for all of the fractions (table S5).
Both nootkatone and valencene did not show cytotoxicity on the five human cancer cell lines at the tested concentration.

The gene related to nootkatone biosynthesis in A. oxyphylla
as nootkatone is the major compound identified from A. oxyphylla, as well as present siginificant biological activities including tyrosinase inhibitory activity and anti-inflammatory activity.therefore, we proposed biosynthesis pathway of nootkatone in A. oxyphyllaas in Figure 1a.three gene or gene families involved in plant nootkatone biosynthesis, including valencene synthase (VS), valencene oxidase (Vo) and dehydrogenase (Figure 1a). the closest homologue of aoVS is valencene synthase from Zingiber officinale with 83.1% sequence identity.in addition, aoVS is 42.4% similar with the longifolene synthase from Pichia abies.But all the valencene synthase contained the conservative residues for cyclization reaction (Figure 1B; Figure S4).as a typical type of p450, aoVo is lower similarity with Vo from other species although conservative motifs are all identified in these proteins, even the aoVo is more distantly related to ZoVo, with only 37.6% identity on amino acids level (Figure 1C; Figure S5). the gene encoding dehydrogenase, which is involved in the final step of nootkatone biosynthesis, showed highest sequences identity on amino acids residues (Figure 1d).

Conclusion
GC-mS was used to analysis of the chemical costituents of the unploar fractions of fruits and leaves of A. oxyphylla, and nootkatone was found as the main compound.the biological activities results showed that all the tested fractions and nootkatone presented tyrosinase inhibitory, as well as inhibitory effect on no production in LpS-stimulated RaW264.7 cells.moreorver, the critical genes involved in nootkatone biosynthesis in A. oxyphylla were identified from the public transcriptome datasets, and protein sequences were preliminarily analyzed.

Disclosure statement
no potential conflict of interest was reported by the authors.

Funding
this research was financially supported by natural Science Foundation of Hainan (220rc702), and China agriculture Research System of moF and maRa (CaRS-21).

Figure 1 .
Figure 1. the proposed biosynthesis pathway of nootkatone in plant and protein sequences analysis of valencene synthase (Vs), valencene oxidase (Vo) and dehydrogenase.(a) the potential pathway of nootkatone biosynthesis in plant; (B) representative part of the alignment between aoVs and other Vs. the red box marked are the important residues for cyclization reaction; (c) representative part of the alignment between aoVo and other Vo; (d) representative alignment between dehydrogenase in A. oxyphylla and other species.