First Report of Anthracnose Caused by Colletotrichum ciggaro on Lonicera macranthoides Hand.-Mazz. in China.

Lonicera macranthoides Hand.-Mazz. is a traditional medicinal plant that is cultivated in Hunan, Yunnan, and Guizhou Provinces in China. In June 2020, a new leaf spot disease was observed on this plant in Longhui County, Shaoyang City, Hunan Province, China, where 14,000 hm2 of L. macranthoides had been planted. About 20% of the total cultivated area exhibited symptoms. Brown spots appeared on the leaves during the early stage and gradually expanded into irregular lesions, which became necrotic and dry. The whole plant withered and died in severe cases. To isolate the pathogen, the infected leaves were collected from different fields and washed with flowing sterile water. The small lesions were then cut and surface sterilized with 75% alcohol for 45 s followed by a 3 min treatment in 3% sodium hypochlorite. The lesions were rinsed five times in sterile water, incubated on potato dextrose agar (PDA) plates and cultured for 3-5 d at 28℃. In total, eleven isolates were obtained, and eight of them were Colletotrichum (isolation frequency 73%). Three representative isolates (JYH1, JYH2, and JYH5) were selected for further study. The fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were aseptate, transparent, cylindric, and thin walled, which measured 11.54 to 22.64 × 3.55 to 4.75 µm (n=100). Six genetic regions were amplified and sequenced to further confirm the identity of fungus. They included ß-tubulin (TUB2), the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS), calmodulin (CAL) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences were submitted to GenBank (ITS: OQ746331; ON954583; OQ746334; TUB2: OQ772278; ON960155; OQ772279; CHS: OQ772280; ON960156; OQ772281; ACT: OQ772282; ON960157; OQ772283; CAL: OQ772284; ON960158; OQ772285; GAPDH: OQ772286; ON960159; OQ772287). The construction of a 6-gene joint phylogenetic tree analysis showed that the three isolates unambiguously clustered with Colletotrichum kahawae subsp. ciggaro strains C022-1 (GenBank: KJ001120.1, KJ001124.1, KJ001109.1, KJ001102.1, KJ001106.1, KJ001113.1) and R019 (GenBank: JN715847.1, KC860023.1, KC859980.1, KC859954.1, KC859972.1, KC859997.1), which was recently reclassified as C. cigarro (Cabral et al. 2020). Three representative isolates were used for the pathogenicity test on the young leaves of the whole plant. A sterile pin was used to prick the leaf epidermis, and 6 × 6 mm mycelial blocks that had been cultured on PDA for 7 d were placed on the leaf wounds. The controls were treated in the same manner except that sterile blocks of PDA were used. There were three replicates per treatment. All the plants used in the experiment were maintained at 28°C in a climate chamber. There was a 12 h photoperiod, and the chamber was kept at 80% relative humidity. Dark brown spots appeared at the sites of inoculation on the plants after 5 days. All the strains that were re-isolated from the lesions shared the same morphological characteristics and had the same type of colonies as the pathogen Colletotrichum ciggaro. Thus, Koch's postulates were fulfilled. C. ciggaro had been shown to cause anthracnose on Olea europaea L. (Weir et al. 2012), Mangifera indica L. (Ismail et al. 2015), Citrus reticulata L. (Perrone et al. 2016) and Areca catechu L. (Zhang et al. 2020). To our knowledge, this is the first report of C. ciggaro causing anthracnose on L. macranthoides in China and worldwide. This research provides a basis for further research to control epidemics of this disease.

[Mining and identification of members of MYB transcription factor family in Lonicera macranthoides].

As a large family of transcription factors, the MYB family plays a vital role in regulating flower development. We studied the MYB family members in Lonicera macranthoides for the first time and identified three sequences of 1R-MYB, 47 sequences of R2R3-MYB, two sequences of 3R-MYB, and one sequence of 4R-MYB from the transcriptome data. Further, their physicochemical properties, conserved domains, phylogenetic relationship, protein structure, functional information, and expression were analyzed. The results show that the 53 MYB transcription factors had different conserved motifs, physicochemical properties, structures, and functions in wild type and 'Xianglei' cultivar of L. macranthoides, indicating their conservation and diversity in evolution. The transcript level of LmMYB was significantly different between the wild type and 'Xianglei' cultivar as well as between flowers and leaves, and some genes were specifically expressed. Forty-three out of 53 LmMYB sequences were expressed in both flowers and leaves, and 9 of the LmMYB members showed significantly different transcript levels between the wild type and 'Xianglei' cultivar, which were up-regulated in the wild type. The results provide a theoretical basis for further studying the specific functional mechanism of the MYB family.

Light plays a critical role in the accumulation of chlorogenic acid in Lonicera macranthoides Hand.-Mazz.

Light has important effects on plant metabolism. However, the relationship between the chlorogenic acid (CGA) content and light in plants remains unclear. Here, we investigated the effects of shading treatment on gene expression and CGA content in Lonicera macranthoides Hand.-Mazz. (LM), a widely used medicinal plant. A total of 1891 differentially expressed genes (DEGs) were obtained in flower buds and 819 in leaves in response to light in shading treatment compared to the control sample by RNA-Seq. After shading treatment, the content of CGA in LM leaves decreased significantly by 1.78-fold, the carotenoid content increased, and the soluble sugar and starch contents significantly decreased. WGCNA and the expression of related genes verified by qRT‒PCR revealed that CGA synthesis pathway enzyme genes form a co-expression network with genes for carbohydrate synthesis, photosynthesis, light signalling elements, and transcription factor genes (TFs) that affect the accumulation of CGA. Through a virus-induced gene silencing (VIGS) system and CGA assay in Nicotiana benthamiana (NB), we determined that downregulation of NbHY5 expression decreased the CGA content in NB leaves. In this study, we found that light provides energy and material for the accumulation of CGA in LM, and light affects the expression of CGA accumulation-related genes. Our results show that different light intensities have multiple effects on leaves and flower buds in LM and are able to coregulate LmHY5 expression and CGA synthesis.

Comparative transcriptome analysis to reveal key ethylene genes involved in a Lonicera macranthoides mutant.

BACKGROUND: Lonicera macranthoides Hand.-Mazz. is an important medicinal plant. Xianglei-type (XL) L. macranthoides was formed after many years of cultivation by researchers on the basis of the natural mutant. The corolla of L. macranthoides XL remains unexpanded and its flowering period is nearly three times longer than that of wild-type (WT) plants. However, the molecular mechanism behind this desirable trait remains a mystery. OBJECTIVE: To understand the floral phenotype differences between L. macranthoides and L. macranthoides XL at the molecular level. METHODS: Transcriptome analysis was performed on L. macranthoides XL and WT. One DEG was cloned by RT-PCR amplification and selected for qRT-PCR analysis. RESULTS: Transcriptome analysis showed that there were 5603 differentially expressed genes (DEGs) in XL vs. WT. Enrichment analysis of DEGs showed that pathways related to plant hormone signal transduction were significantly enriched. We identified 23 key genes in ethylene biosynthesis and signal transduction pathways. The most abundant were the ethylene biosynthesis DEGs. In addition, the open reading frames (ORFs) of WT and XL ETR2 were successfully cloned and named LM-ETR2 (GenBank: MW334978) and LM-XL-ETR2 (GenBank: MW334978), respectively. qRT-PCR at different flowering stages suggesting that ETR2 acts in the whole stage of flower development of WT and XL. CONCLUSIONS: This study provides new insight into the molecular mechanism that regulates the development of special traits in the flowers of L. macranthoides XL. The plant hormone ethylene plays an important role in flower development and flowering duration prolongation in L. macranthoides. The ethylene synthesis gene could be more responsible for the flower phenotype of XL. The genes identified here can be used for breeding and improvement of other flowering plants after functional verification.

[Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides].

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

[Different development phase of transcription proteomics and metabolomics of flower of Lonicera macranthoides].

In order to study the regulation mechanism of secondary metabolites biosynthesis in Lonicera macranthoides, the key genes involved in the regulation of biosynthesis and the mechanism of differential metabolites were explored. In this study, high-throughput sequencing technology was used for transcriptome sequencing of L. macranthoides at different development stages. By using Liquid chromatography-tandem mass spectrometry(LC-MS/MS) technology, the laws of qualitative, quantitative and synthetic accumulation of its metabolites were studied, and the key enzyme genes for the biosynthesis of phenolic acid and flavonoids were screened out according to the differentially expressed genes. A total of 111 differentially accumulate metabolites(DAM) and 6 653 differentially expressed genes(DGE) were obtained by metabonomics and transcriptomics analysis. The metabolites and key enzyme genes in the Erqing(KE) were significantly different from those in the Dabai(KD) and Yinhua(KY) stages. In the phenylalanine biosynthesis pathway, the ion abundance of chlorogenic acid, naringin, quercetin, rutin, coniferol and other metabolites decreased with the development of flowers, while the ion abundance of ferulic acid, coumarin and syringoside increased with the development of flowers. Key enzyme genes such as CHS, HCT, CCR, FLS and COMT positively regulate the downstream metabolites, while PAL, C4H and 4CL negatively regulate the downstream metabolites. This study provides candidate genes and theoretical basis for the further exploration of key enzymes in the biosynthesis of secondary metabolites and for the regulation of the accumulation of secondary metabolites in L. macranthoides by molecular biological methods.

A R2R3-MYB transcriptional activator LmMYB15 regulates chlorogenic acid biosynthesis and phenylpropanoid metabolism in Lonicera macranthoides.

Lonicera macranthoides Hand-Mazz is an important medicinal plant widely distributed in southern China that has long been used in Chinese traditional medicines. Chlorogenic acid (CGA, 3-caffeoylquinic acid) is the major biologically active ingredient in L. macranthoides. Although key CGA biosynthetic genes have been well documented, their transcriptional regulation remains largely unknown. In this study, we observed that a R2R3 MYB transcription factor LmMYB15 showed a significant correlation with CGA content, indicating its potential role in CGA biosynthesis. A yeast two-hybrid assay suggested that LmMYB15 functions as a transcriptional activator. Overexpression of LmMYB15 in tobacco led to increased accumulation of CGA compared to those in wild-type leaves. To elucidate its functional mechanism, genome-wide DAP-seq was employed and identified the conserved binding motifs of LmMYB15, that is [(C/T) (C/T) (C/T) ACCTA(C/A) (C/T) (A/T)], as well as its direct downstream target genes, including 4CL, MYB3, MYB4, KNAT6/7, IAA26, and ETR2. Subsequently, yeast one-hybrid and dual-luciferase reporter assays verified that LmMYB15 could bind and activate the promoters of 4CL, MYB3 and MYB4, thereby facilitating CGA biosynthesis and phenylpropanoid metabolism. Our findings provide a new track for breeding strategies aiming to enhance CGA content in L. macranthoides that can significantly contribute to better mechanical properties.

Prenyleudesmanes and A Hexanorlanostane from the Roots of Lonicera macranthoides.

Three previously undescribed compounds, two prenyleudesmanes (1 and 2), and one hexanorlanostane (3), were isolated from the roots of Lonicera macranthoides. Their structures were established based on 1D and 2D nuclear magnetic resonance (NMR) spectra and high-resolution electrospray ionization mass spectral (HR-ESI-MS) data. The absolute configurations of 1 and 3 were determined by X-ray diffraction. To the best of our knowledge, this is the first time that the absolute configuration of a prenyleudesmane with a trans-decalin system and a hexanorlanostane have been unambiguously confirmed by single-crystal X-ray diffraction with Cu Kα radiation. Thecompounds were tested for their antiproliferative activity on the cancer cell lines (HepG2 and HeLa). The compounds 1-3 exhibited moderate inhibitory effects against two human cancer cell lines.

Comparison of Free, Esterified, and Insoluble-Bound Phenolics and Their Bioactivities in Three Organs of Lonicera japonica and L. macranthoides.

Dried flower buds of Lonicera japonica and L. macranthoides have long been used as herbs in numerous Chinese traditional medicines. Comparisons of three phenolic fractions (i.e., free, esterified, and insoluble-bound phenolics) in three different organs (i.e., flower, leaf, and stem) of the two species revealed that the free phenolics were the highest in terms of total phenol and total flavonoid content, composed of the most numerous phenolics and flavonoids; thus, they exhibited the most excellent antioxidant activities (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and oxygen radical absorbance capacity (ORAC)), as well as protective effects on DNA damage induced by free radicals. In identical free and esterified phenolics of a same organ, higher contents and bioactivities were observed in L. macranthoides than in L. japonica. Phenolics identified by ultra-performance liquid chromatography with a diode array detector, alongside tandem mass spectrometry coupled with a quadrupole time-of-flight mass spectrometer (UPLC-DAD⁻QTOF-MS/MS) mainly included chlorogenic acid and its five derivatives, three flavonoids that were only found in the free phenolic fraction and closely correlated with its bioactivity, and caffeic acid that was the major contributor to antioxidant activity of the esterified and insoluble-bound phenolic fractions. It was, thus, concluded that, like L. japonica, L. macranthoides, which was underestimated since being separately listed by the 2010 edition of the Chinese Pharmacopoeia, is also a good (and better) herbal medicine.

Two new ursane-type nortriterpenes from Lonicera macranthoides and their iNOS-inhibitory activities.

The flower buds of Lonicera macranthoides (Shan Yin-Hua), represent an important traditional Chinese medicine and food ingredient. A phytochemical investigation of the 70% EtOH extract of the flower buds of L. macranthoides resulted in the isolation of 12 triterpenoids (1-12), including two new ursane-type nortriterpenes, 2α, 24-dihydroxy-23-nor-ursolic acid (1) and 2α, 4α-dihydroxy-23-nor-ursolic acid (2). Their structures were established by multiple spectroscopic methods and comparison with literature data. All isolated compounds were evaluated for their anti-inflammatory effects in LPS-activated RAW264.7 cells. Compounds 1 and 2 exhibited inhibitory effects on iNOS at the concentration of 30 µmol·L-1.

Target discovery of chlorogenic acid derivatives from the flower buds of Lonicera macranthoides and their MAO B inhibitory mechanism.

Chlorogenic acids (CGAs), exhibiting health benefits in many foods, also played an important role for their broad bioactive properties in nature. Obtaining more diverse CGAs was helpful to discover their potential edible and medical value. In this study, 11 CGAs, including four new (1-4) and seven known compounds (5-11), were obtained from the flower buds of Lonicera macranthoides Miq.-Hazz. The possible targets of all isolated CGAs were predicted using the ligand-based reverse screening and compound-target network, suggesting that MAO B (monoamine oxidase B) was the primary target of these CGAs. Subsequently, 7 out of 11 CGAs were confirmed to possess inhibitory effects by in vitro assay. The detailed interaction mechanism between compound and MAO B was also announced by molecular docking and molecular dynamics simulation.

Two new ursane-type nortriterpenes from Lonicera macranthoides and their iNOS-inhibitory activities / 中国天然药物

The flower buds of Lonicera macranthoides (Shan Yin-Hua), represent an important traditional Chinese medicine and food ingredient. A phytochemical investigation of the 70% EtOH extract of the flower buds of L. macranthoides resulted in the isolation of 12 triterpenoids (1-12), including two new ursane-type nortriterpenes, 2α, 24-dihydroxy-23-nor-ursolic acid (1) and 2α, 4α-dihydroxy-23-nor-ursolic acid (2). Their structures were established by multiple spectroscopic methods and comparison with literature data. All isolated compounds were evaluated for their anti-inflammatory effects in LPS-activated RAW264.7 cells. Compounds 1 and 2 exhibited inhibitory effects on iNOS at the concentration of 30 μmol·L.

Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides.

Lonicera macranthoides is an important medicinal plant widely used in traditional Chinese medicine. Luteoloside is a critical bioactive compound in L. macranthoides. To date, the molecular mechanisms underlying luteoloside biosynthesis are still largely unknown. In this work, high performance liquid chromatography (HPLC) was employed to determine the luteoloside contents in leaves, stems, and flowers at different developmental stages. Results showed that senescing leaves can accumulate large amounts of luteoloside, extremely higher than that in young and semi-lignified leaves and other tissues. RNA-Seq analysis identified that twenty-four differentially expressed unigenes (DEGs) associated with luteoloside biosynthesis were significantly up-regulated in senescing leaves, which are positively correlated with luteoloside accumulation. These DEGs include phenylalanine ammonia lyase 2, cinnamate 4-hydroxylase 2, thirteen 4-coumarate-CoA ligases, chalcone synthase 2, six flavonoid 3'-monooxygenase (F3'H) and two flavone 7-O-ß-glucosyltransferase (UFGT) genes. Further analysis demonstrated that two F3'Hs (CL11828.Contig1 and CL11828.Contig2) and two UFGTs (Unigene2918 and Unigene97915) might play vital roles in luteoloside generation. Furthermore, several transcription factors (TFs) related to flavonoid biosynthesis including MYB, bHLH and WD40, were differentially expressed during leaf senescence. Among these TFs, MYB12, MYB75, bHLH113 and TTG1 were considered to be key factors involved in the regulation of luteoloside biosynthesis. These findings provide insights for elucidating the molecular signatures of luteoloside accumulation in L. macranthoides.

Characterization of the complete chloroplast genome of Lonicera macranthoides.

Lonicera macranthoides is an important Chinese traditional medicine plant which is endemic in southwest China. The complete chloroplast genome from L. macranthoides is determined in this study. The genome size was 154,897 bp, containing a large single copy (LSC) region of 88,692 bp and a small single copy (SSC) region of 18,623 bp, which were separated by a pair of 23,786 bp inverted repeat (IR) regions. The plastome contained 130 genes, including 81 protein-coding genes (77 PCG species), eight ribosomal RNA genes (4 rRNA species), 39 tRNA genes (29 tRNA species) and two pseudognes. The most of gene species occurred as a single copy, while 13 gene species occurred in double copies, including all rRNA species, six tRNA species and three PCG species, and two tRNA genes occur in treble copies. Finally, phylogenetic analysis demonstrated that L. macranthoides is closely related to L. japonica, and genus Lonicera shows a closer relationship with Triostrum for the current data.

Determination and Isolation of Four Anti-tumour Saponins from Lonicera macranthoides by HPLC-ESI-QTOF/MS and HSCCC.

BACKGROUND: Lonicera macranthoides is a Chinese herb that contains a large number of bioactive spanions possessing important pharmacological activities, such as anti-tumour activity. However, detailed information about their anti-tumor activity and bioactive compounds is limited. METHODS: In order to evaluate the scientific basis, the method of high-speed counter-current chromatography (HSCCC) combined with high performance liquid chromatography mass spectrometry (HPLC-ESI-QTOF/MS) has been developed to separate, purify and analyze saponins from Lonicera macranthoides. Four main saponins, Macranthoidin B (I), Macranthoidin A (II), Macranthoides B (III) and Akebia saponin D (IV) were separated by HSCCC with the solvent systems of ethyl acetate-nbutanol- water (3:2:5) and n-butanol-water-methanol-ethyl acetate (1:6:0.5:4). The purities of these four bioactive ingredients (I-IV) identified and detected by HPLC-ESI-QTOF/MS were 95.1%, 92.7%, 91.8% and 96.3%, respectively. The separated saponins were evaluated for their cytotoxic activities against six tumor cell lines (MCF-7, Hela, A549, HepG2, HT29 and Eca109). RESULTS: Results show that compounds I-IV exhibited particular significant anti-tumor activities against human mammary adenocarcinoma MCF-7 cell with IC50 values ranging from 12.7 to 30.8 µM. CONCLUSION: It was demonstrated that the combinative method using HPLC-ESI-QTOF/MS and HSCCC was suitable for rapid screening and isolating saponins of Lonicera macranthoides and the isolated compounds have great potential for the development of new antitumor drugs.

[Study on influence factors of seed germination and seeding growth of Lonicera macranthoides].

In order to improve reproductive efficiency and quality standard, the influence factors of seed germination and seeding growth of Lonicera macranthoides werew studied. The fruit and seed morphological characteristics of L. macranthoides were observed, the seed water absorbing capacity was determined, and different wet sand stratification time, temperature and germination bed treatment were set up. The effects of the parameters on seed germination and seedling growth were analysed. There was no obstacles of water absorption on L. macranthoides seed, quantity for 22 h water absorption was close to saturation. In the first 80 d, with the increase of the stratification time, seed initial germination time was shortened, germination rate and germination potential was improved. Stratification for 100 d, germination rate decreased. At 15 ℃, seed germination and seedling growth indicators were the best. The seedling cotyledon width in light was significantly higher than that in dark. Seeds on the top of paper and top of sand germination rate, germination potential, and germination index was significantly higher than that of other germination bed and mildew rate is low. The optimal conditions of seeds germination test was stratified in 4 ℃ wet sand for 80 d, 15 ℃ illuminate culture on the top of paper or top of sand. The first seeding counting time was the 4th day after beginning the test, the final time was the 23th day. The germination potential statistical time was the 13th day after beginning the test.

[Study on seed quality test and quality standard of Lonicera macranthoides].

Referring to the rules for agricultural seed testing (GB/T 3543-1995) issued by China, the test of sampling, purity, thousand seed weight, moisture, viability, relative conductivity and germination rate had been studied for seed quality test methods of Lonicera macranthoides. The seed quality from 38 different collection areas was measured to establish quality classification standard by K-means clustering. The results showed that at least 7.5 g seeds should be sampled, and passed 20-mesh sieve for purity analysis.The 500-seed method used to measure thousand seed weight. The moisture was determined by crushed seeds dried in high temperature (130±2) ℃ for 3 h.The viability determined by 25 ℃ 0.1% TTC stained 5h in dark. 1.0 g seeds soaked in 50 ml ultra pure water in 25 ℃ for 12 hours to determine the relative conductivity. The seed by 4 ℃stratification for 80 days were cultured on paper at 15 ℃. Quality of the seeds from different areas was divided into three grades. The primary seed quality classification standard was established.The I grade and II grade were recommend use in production.

A comparison of volatile fractions obtained from Lonicera macranthoides via different extraction processes: ultrasound, microwave, Soxhlet extraction, hydrodistillation, and cold maceration.

BACKGROUND: Hydrodistillation has been traditionally used to extract volatile fraction in traditional Chinese medicine. However, with the development of Soxhlet extraction (SE), microwave (MW), ultrasound (US), and cold maceration (CM), hydrodistillation (HD) is being replaced to meet some practical requirements. In this study, we investigated the effect of the five methods on the volatile fraction extract of Lonicera macranthoides. METHODS: Volatile fraction from the flower buds of Lonicera macranthoides was obtained by using different extraction methods, HD, SE, MW, US, and CM. The compositions of volatile fraction were analyzed by gas chromatography-mass spectrometric and further compared among extraction methods. RESULTS: Extracts obtained by the five methods reveal the qualitative and quantitative diversity in their compositions, especially for the low-content compositions. According to the results, SE shows the great value in the research where the high molecular-mass compound is of primary interest, and MW offers a way for the isolation of specific compound like octadecadienoic acid and hexadecanoic acid. HD, US, and CM have the advantage over SE and MW for the integrity of the constituents, whereas the phenomenon of compound degradation seems not so serious in solvent extraction methods such as US or CM as HD. Additionally, US and CM show superiority over time or material saving and diversity of the constituent. CONCLUSION: HD is still the best choice for the pure volatile fraction without organic solvent pollution. However, when it comes to some specifically actual demands, it can be replaced by the four methods for the volatile fraction extraction process, especially for production of certain compound groups.